Thermally Stable Recombinant Collagen Gel and Its Preparation Process

Abstract

The present application discloses a thermally stable recombinant collagen gel and its preparation process, which relates to the technical field of recombinant collagen. The raw materials include, by mass percentage, 10 - 20% water, 40 - 50% modified recombinant collagen, 13 - 20% traditional Chinese medicine preparation, 8 - 16% sodium hyaluronate, 2 - 4% composite heat stabilizer, 1 - 2% preservative, and the balance is glycerol; the modified recombinant collagen is thiolated modified recombinant collagen; the composite heat stabilizer includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4 - 0.8. The present application significantly improves the thermal stability of the recombinant collagen gel by thiolating and modifying the recombinant collagen and adding a composite heat stabilizer to the gel components.

Description

Technical Field

[0001] This application relates to the field of recombinant collagen technology, in particular to a thermostable recombinant collagen gel and its preparation process. Background Art

[0002] Collagen is the main component of the extracellular matrix, playing an important role in supporting cell adhesion, proliferation, and differentiation, and has a wide range of applications in the fields of wound repair, bone tissue engineering, and nerve regeneration.

[0003] Recombinant humanized collagen is prepared by genetic engineering based on human collagen and further expressed in Pichia pastoris. Recombinant humanized collagen has characteristics and stability similar to those of natural human collagen, and has advantages such as easy availability, good water solubility, low immunogenicity, no hidden virus risk, and a stable production process. Recombinant collagen gel dressings accelerate the wound healing process by forming a protective layer on the wound surface.

[0004] However, due to its unique triple-helix structure, collagen is heat-sensitive and prone to thermal denaturation when heated, resulting in the destruction of the triple-helix structure, which easily leads to the loss of protein biological activity function. Therefore, in order to improve the stability and efficacy of recombinant collagen gel dressings, it is urgent to improve the thermal stability of recombinant collagen gels. Summary of the Invention

[0005] In order to improve the thermal stability of recombinant collagen gels, this application provides a thermostable recombinant collagen gel and its preparation process.

[0006] The thermostable recombinant collagen gel provided by this application adopts the following technical scheme:

[0007] For the thermostable recombinant collagen gel, the raw materials by mass percentage include 10 - 20% water, 40 - 50% modified recombinant collagen, 13 - 20% traditional Chinese medicine preparation, 8 - 16% sodium hyaluronate, 2 - 4% composite heat stabilizer, 1 - 2% preservative, and the balance is glycerol; the modified recombinant collagen is thiolated modified recombinant collagen.

[0008] By adopting the above technical scheme, this application conducts thiolation modification on the recombinant collagen gel, introducing thiol groups on the surface of the recombinant collagen, significantly improving the thermal stability of the recombinant collagen, and preventing the problem of denaturation and instability of the recombinant collagen during the processing; and adding a composite heat stabilizer to the raw materials to further improve the high-temperature resistance performance of the recombinant collagen gel, ensuring the use effect of the product.

[0009] Preferably, the raw materials include, by mass percentage, 12 - 17% water, 42 - 48% modified recombinant collagen, 14 - 18% traditional Chinese medicine preparation, 10 - 14% sodium hyaluronate, 2.5 - 3.5% compound heat stabilizer, 1.3 - 1.7% preservative, and the balance is glycerol.

[0010] Preferably, the raw materials include, by mass percentage, 15% water, 45% modified recombinant collagen, 16% traditional Chinese medicine preparation, 12% sodium hyaluronate, 3% compound heat stabilizer, 1.5% preservative, and the balance is glycerol.

[0011] Preferably, the compound heat stabilizer includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4 - 0.8.

[0012] By adopting the above technical solution, trehalose and pomegranate peel polyphenols are compounded as the heat stabilizer of the recombinant collagen gel. The trehalose molecule contains multiple free hydroxyl groups and has a strong ability to form hydrogen bonds. These hydroxyl groups can form hydrogen bonds with the polar groups on the surface of collagen, replacing the bound water lost around the polar groups of collagen, thereby stabilizing the secondary structure of collagen. Under the combined action with pomegranate peel polyphenols, it can have a synergistic effect and further improve the thermal stability of the recombinant collagen gel, ensuring the high-temperature resistance performance of the recombinant collagen gel.

[0013] Preferably, the raw materials of the traditional Chinese medicine preparation include paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica sinensis extract, honeysuckle extract, isatis root extract, aloe extract, and the mass ratio is 2:2:5:5:2:1:1.

[0014] Preferably, the preservative is one or a mixture of two of nipagin ester and phenoxyethanol.

[0015] Preferably, the preparation method of the mercapto-modified recombinant collagen includes the following steps:

[0016] T1. Add recombinant humanized collagen into acetic acid solution, stir and dissolve to obtain a collagen solution; transfer the collagen solution into a dialysis bag and dialyze at 4 - 6°C. The dialysis solution is an alkaline phosphate buffer solution, and dialyze until the pH is 9 - 10 to obtain a collagen / phosphate mixed solution;

[0017] T2. Add DL-N-acetylhomocysteine thiolactone to the collagen / phosphate mixed solution, stir evenly, and add ethylenediaminetetraacetic acid and dithiothreitol; under a nitrogen atmosphere, carry out a mercapto reaction on the reaction mixture at 4 - 6°C; after the reaction is completed, transfer the reaction system into a dialysis bag and dialyze with a neutral phosphate buffer solution at 4 - 6°C for 3 - 4 days to obtain the mercapto-modified recombinant collagen.

[0018] By adopting the above technical solution, in this application, N-acetylhomocysteine thiolactone is used as a mercapto group-introducing reagent, and the mercapto group is introduced by aminolysis of the thiolactone bond of N-acetylhomocysteine thiolactone with the free amino group on the side chain of collagen, thereby synthesizing mercapto group-modified recombinant collagen, effectively improving the mechanical properties and thermal stability of recombinant human collagen, effectively preventing the problem of denaturation and instability of recombinant collagen during subsequent processing, and effectively maintaining the performance of the product.

[0019] Preferably, the mass ratio of the raw material recombinant humanized collagen, acetic acid solution, DL-N-acetylhomocysteine thiolactone, ethylenediaminetetraacetic acid, and dithiothreitol is 0.2-0.4: 100-200: 0.3-0.6: 0.02-0.04: 0.01-0.02.

[0020] Preferably, the concentration of the acetic acid solution is 0.4-0.6 M.

[0021] Preferably, the pH of the alkaline phosphate buffer solution is 9-10.

[0022] The heat-stable recombinant collagen gel provided by this application and its preparation process adopt the following technical solution:

[0023] The preparation process of the heat-stable recombinant collagen gel includes the following steps:

[0024] S0. Weigh the raw materials of water, modified recombinant collagen, traditional Chinese medicine preparation, sodium hyaluronate, composite heat stabilizer, preservative, and glycerol according to mass percentages;

[0025] S1. Stir and mix the modified recombinant collagen with water to obtain a modified recombinant collagen stock solution; filter the collagen stock solution to remove undissolved modified recombinant collagen;

[0026] S2. Put the filtered collagen solution into a reaction kettle for emulsification reaction, and after the reaction, perform centrifugal precipitation. After removing the supernatant, a modified recombinant collagen solution is obtained;

[0027] S3. Mix and stir the modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer, and preservative, then add the traditional Chinese medicine preparation and stir evenly, and then add glycerol and stir evenly to obtain the heat-stable recombinant collagen gel.

[0028] Preferably, the temperature of the emulsification reaction is 30-40 °C and the pressure is 1-1.5 MPa.

[0029] In summary, this application includes at least one of the following beneficial technical effects:

[0030] 1. This application uses the compound of trehalose and pomegranate peel polyphenols as the thermal stabilizer of the recombinant collagen gel. The trehalose molecule contains multiple free hydroxyl groups and has a strong ability to form hydrogen bonds. These hydroxyl groups can form hydrogen bonds with the polar groups of collagen, replacing the lost bound water around the polar groups of collagen, thereby stabilizing the secondary structure of collagen. Under the combined action with pomegranate peel polyphenols, it can have a synergistic effect and further improve the thermal stability of the recombinant collagen gel;

[0031] 2. This application uses N-acetylhomocysteine thiolactone as the mercapto group-introducing reagent, and realizes the introduction of the mercapto group by aminolyzing the thiolactone bond of N-acetylhomocysteine thiolactone through the free amino group on the side chain of collagen, thereby synthesizing mercapto group-modified recombinant collagen, effectively improving the mechanical properties and thermal stability of recombinant human collagen, effectively preventing the problem of denaturation and instability that may occur during the subsequent processing of recombinant collagen, and effectively maintaining the performance of the product. Detailed implementation manners

[0032] The following further elaborates on this application in combination with examples.

[0033] The chemical reagents used in the preparation examples, examples and comparative examples provided by the present invention are all commercially available products, and their trademarks and manufacturers are as follows:

[0034] Recombinant type III humanized collagen, Shanxi Jinbo Biopharmaceutical Co., Ltd.;

[0035] DL-N-acetylhomocysteine thiolactone, Shanghai Merck Chemical Technology Co., Ltd.;

[0036] DL-dithiothreitol, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0037] Salvia miltiorrhiza extract, Xi'an Hongsheng Biotechnology Co., Ltd.;

[0038] Angelica sinensis extract, Parni (Shaanxi) Industry Co., Ltd.;

[0039] Paeonol, Hubei Changao Pharmaceutical Co., Ltd.;

[0040] Panax notoginseng extract, Chengdu 06 Biotechnology Co., Ltd.;

[0041] Honeysuckle extract, Shaanxi Parni Biotechnology Co., Ltd.;

[0042] Isatis root extract, Hubei Widely Chemical Technology Co., Ltd.;

[0043] Aloe extract, Shaanxi Jianchuang Biotechnology Co., Ltd.;

[0044] Trehalose, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0045] Pomegranate peel polyphenols, Fufeng Sinuote Biotechnology Co., Ltd.;

[0046] Sodium hyaluronate, Nanjing Luomaimei Biotechnology Co., Ltd.;

[0047] Glycerol, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0048] Paraben, Hebei Jijie Biotechnology Co., Ltd.;

[0049] Phenoxyethanol, Wuhan Xinli Biotechnology Co., Ltd.

[0050] Preparation Example 1: T1. Add 0.2 g of recombinant type III humanized collagen into 100 g of 0.5 M acetic acid solution, stir to dissolve to obtain a collagen solution; transfer the collagen solution into a dialysis bag (MWCO 8000 - 14000 kDa) and dialyze at 4°C, the dialysis solution is an alkaline phosphate buffer solution with a pH of 9, dialyze until the pH is 9 to obtain a collagen / phosphate mixed solution;

[0051] T2. Add 0.3 g of DL-N-acetylhomocysteine thiolactone to the collagen / phosphate mixed solution, and add 0.02 g of ethylenediaminetetraacetic acid and 0.01 g of DL-dithiothreitol; under a nitrogen atmosphere, carry out a thiolation reaction on the reaction mixture at 4°C for 30 h; after the reaction is completed, transfer the reaction system into a dialysis bag and dialyze with a neutral phosphate buffer solution with a pH of 7 at 4°C for 3 days, changing the dialysis solution every 4 h to obtain thiolated modified recombinant collagen.

[0052] Preparation Example 2: T1. Add 0.3 g of recombinant type III humanized collagen into 150 g of 0.5 M acetic acid solution, stir to dissolve to obtain a collagen solution; transfer the collagen solution into a dialysis bag (MWCO 8000 - 14000 kDa) and dialyze at 5°C, the dialysis solution is an alkaline phosphate buffer solution with a pH of 9, dialyze until the pH is 9 to obtain a collagen / phosphate mixed solution;

[0053] T2. Add 0.45 g of DL-N-acetylhomocysteine thiolactone to the collagen / phosphate mixed solution, and add 0.03 g of ethylenediaminetetraacetic acid and 0.015 g of DL-dithiothreitol; under a nitrogen atmosphere, carry out a thiolation reaction on the reaction mixture at 5°C for 40 h; after the reaction is completed, transfer the reaction system into a dialysis bag and dialyze with a neutral phosphate buffer solution with a pH of 7 at 4°C for 3.5 days, changing the dialysis solution every 4 h to obtain thiolated modified recombinant collagen.

[0054] Preparation Example 3: T1. Add 0.4 g of recombinant humanized type III collagen into 200 g of 0.5 M acetic acid solution, stir to dissolve to obtain a collagen solution; transfer the collagen solution into a dialysis bag (MWCO 8000 - 14000 kDa) and dialyze at 6 °C, the dialysis solution is an alkaline phosphate buffer solution with a pH of 9, dialyze until the pH is 9 to obtain a collagen / phosphate mixed solution;

[0055] T2. Add 0.6 g of DL-N-acetylhomocysteine thiolactone into the collagen / phosphate mixed solution, and add 0.04 g of ethylenediaminetetraacetic acid and 0.02 g of DL-dithiothreitol; under a nitrogen atmosphere, carry out a thiolation reaction on the reaction mixture at 6 °C for 50 h; after the reaction is completed, transfer the reaction system into a dialysis bag and dialyze with a neutral phosphate buffer solution with a pH of 7 at 6 °C for 4 days, change the dialysis solution every 4 h to obtain thiolated modified recombinant collagen.

[0056] Preparation Example 4: The difference between Preparation Example 4 and Preparation Example 1 is that the concentration of the acetic acid solution used in Preparation Example 4 is 0.4 M.

[0057] Preparation Example 5: The difference between Preparation Example 5 and Preparation Example 1 is that the concentration of the acetic acid solution used in Preparation Example 5 is 0.6 M.

[0058] Preparation Example 6: The difference between Preparation Example 6 and Preparation Example 1 is that the concentration of the acetic acid solution used in Preparation Example 6 is 0.2 M.

[0059] Preparation Example 7: The difference between Preparation Example 7 and Preparation Example 1 is that the concentration of the acetic acid solution used in Preparation Example 7 is 0.8 M.

[0060] Preparation Example 8: The difference between Preparation Example 8 and Preparation Example 1 is that the pH of the alkaline phosphate buffer solution used in Preparation Example 8 is 10.

[0061] Preparation Example 9: The difference between Preparation Example 9 and Preparation Example 1 is that the pH of the alkaline phosphate buffer solution used in Preparation Example 9 is 8.

[0062] Preparation Example 10: The difference between Preparation Example 10 and Preparation Example 1 is that the pH of the alkaline phosphate buffer solution used in Preparation Example 10 is 12.

[0063] Example 1: S0. Weigh 10 g of water, 40 g of the modified recombinant collagen prepared in Preparation Example 1, 20 g of a traditional Chinese medicine preparation, 16 g of sodium hyaluronate, 2 g of a composite heat stabilizer, 1 g of a preservative, and 11 g of glycerol by mass percentage;

[0064] In this embodiment, the traditional Chinese medicine preparation includes paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica extract, honeysuckle extract, isatis root extract, and aloe extract, with a mass ratio of 2:2:5:5:2:1:1;

[0065] In this embodiment, the composite heat stabilizer includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4;

[0066] In this embodiment, the preservative is nipagin ester;

[0067] S1. Stir and mix the modified recombinant collagen with water at 600 rpm to obtain the modified recombinant collagen stock solution; filter the collagen stock solution to remove the undissolved modified recombinant collagen;

[0068] S2. Put the filtered collagen solution into a reaction kettle, carry out an emulsification reaction at a temperature of 30 °C and a pressure of 1 MPa for 30 minutes, and after the reaction, carry out centrifugal precipitation. After removing the supernatant, a modified recombinant collagen solution is obtained;

[0069] S3. Mix and stir the modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer, and preservative at 600 rpm, then add the traditional Chinese medicine preparation and stir evenly, and then add glycerol and stir evenly to obtain a heat-stable recombinant collagen gel.

[0070] Example 2: Weigh 12 g of water, 42 g of the modified recombinant collagen prepared in Preparation Example 1, 18 g of the traditional Chinese medicine preparation, 14 g of sodium hyaluronate, 2.5 g of the composite heat stabilizer, 1.3 g of the preservative, and 10.2 g of glycerol by mass percentage;

[0071] In this embodiment, the traditional Chinese medicine preparation includes paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica extract, honeysuckle extract, isatis root extract, and aloe extract, with a mass ratio of 2:2:5:5:2:1:1;

[0072] In this embodiment, the composite heat stabilizer includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4;

[0073] In this embodiment, the preservative is nipagin ester;

[0074] S1. Stir and mix the modified recombinant collagen with water at 600 rpm to obtain the modified recombinant collagen stock solution; filter the collagen stock solution to remove the undissolved modified recombinant collagen;

[0075] S2. Put the filtered collagen solution into a reaction kettle, carry out an emulsification reaction at a temperature of 30 °C and a pressure of 1 MPa for 30 min, carry out centrifugal precipitation after the reaction, and obtain a modified recombinant collagen solution after removing the supernatant;

[0076] S3. Mix and stir the modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer, and preservative at 600 rpm, then add the traditional Chinese medicine preparation and stir evenly, and then add glycerol and stir evenly to obtain a heat-stable recombinant collagen gel.

[0077] Example 3: S0. Weigh 15 g of water, 45 g of the modified recombinant collagen prepared in Preparation Example 1, 16 g of the traditional Chinese medicine preparation, 12 g of sodium hyaluronate, 3 g of the composite heat stabilizer, 1.5 g of the preservative, and 7.5 g of glycerol by mass percentage;

[0078] In this example, the traditional Chinese medicine preparation includes paeonol, notoginseng extract, salvia extract, angelica extract, honeysuckle extract, isatis root extract, and aloe extract, and the mass ratio is 2:2:5:5:2:1:1;

[0079] In this example, the composite heat stabilizer includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4;

[0080] In this example, the preservative is nipagin ester;

[0081] S1. Stir and mix the modified recombinant collagen and water at 600 rpm to obtain a modified recombinant collagen stock solution; filter the collagen stock solution to remove undissolved modified recombinant collagen;

[0082] S2. Put the filtered collagen solution into a reaction kettle, carry out an emulsification reaction at a temperature of 30 °C and a pressure of 1 MPa for 30 min, carry out centrifugal precipitation after the reaction, and obtain a modified recombinant collagen solution after removing the supernatant;

[0083] S3. Mix and stir the modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer, and preservative at 600 rpm, then add the traditional Chinese medicine preparation and stir evenly, and then add glycerol and stir evenly to obtain a heat-stable recombinant collagen gel.

[0084] Example 4: S0. Weigh 17 g of water, 48 g of the modified recombinant collagen prepared in Preparation Example 1, 14 g of the traditional Chinese medicine preparation, 10 g of sodium hyaluronate, 3.5 g of the composite heat stabilizer, 1.7 g of the preservative, and 5.8 g of glycerol by mass percentage;

[0085] In this embodiment, the traditional Chinese medicine preparation includes paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica sinensis extract, honeysuckle extract, isatis root extract, and aloe extract, with a mass ratio of 2:2:5:5:2:1:1;

[0086] In this embodiment, the composite heat stabilizer includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4;

[0087] In this embodiment, the preservative is nipagin ester;

[0088] S1. Stir and mix the modified recombinant collagen with water at 600 rpm to obtain the modified recombinant collagen stock solution; filter the collagen stock solution to remove the undissolved modified recombinant collagen;

[0089] S2. Put the filtered collagen solution into a reaction kettle, carry out an emulsification reaction at a temperature of 30 °C and a pressure of 1 MPa for 30 min, perform centrifugal precipitation after the reaction, and obtain the modified recombinant collagen solution after removing the supernatant;

[0090] S3. Mix and stir the modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer, and preservative at 600 rpm, then add the traditional Chinese medicine preparation and stir evenly, and then add glycerol and stir evenly to obtain the heat-stable recombinant collagen gel.

[0091] Example 5: S0. Weigh 20 g of water, 50 g of the modified recombinant collagen prepared in Preparation Example 1, 13 g of the traditional Chinese medicine preparation, 8 g of sodium hyaluronate, 4 g of the composite heat stabilizer, 2 g of the preservative, and 3 g of glycerol by mass percentage;

[0092] In this embodiment, the traditional Chinese medicine preparation includes paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica sinensis extract, honeysuckle extract, isatis root extract, and aloe extract, with a mass ratio of 2:2:5:5:2:1:1;

[0093] In this embodiment, the composite heat stabilizer includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4;

[0094] In this embodiment, the preservative is nipagin ester;

[0095] S1. Stir and mix the modified recombinant collagen with water at 600 rpm to obtain the modified recombinant collagen stock solution; filter the collagen stock solution to remove the undissolved modified recombinant collagen;

[0096] S2. Put the filtered collagen solution into a reaction kettle, carry out an emulsification reaction at a temperature of 30 °C and a pressure of 1 MPa for 30 min, perform centrifugal precipitation after the reaction, and obtain the modified recombinant collagen solution after removing the supernatant;

[0097] S3. Mix and stir the modified recombinant collagen solution, sodium hyaluronate, compound heat stabilizer, and preservative at 600 rpm. Then add the traditional Chinese medicine preparation and stir evenly, and then add glycerol and stir evenly to obtain a heat-stable recombinant collagen gel.

[0098] Example 6: The difference between Example 6 and Example 1 is that the mass ratio of trehalose and pomegranate peel polyphenols, which are used as the compound heat stabilizer in Example 6, is 1:0.6.

[0099] Example 7: The difference between Example 7 and Example 1 is that the mass ratio of trehalose and pomegranate peel polyphenols, which are used as the compound heat stabilizer in Example 7, is 1:0.8.

[0100] Example 8: The difference between Example 8 and Example 1 is that the mass ratio of trehalose and pomegranate peel polyphenols, which are used as the compound heat stabilizer in Example 8, is 1:0.2.

[0101] Example 9: The difference between Example 9 and Example 1 is that the mass ratio of trehalose and pomegranate peel polyphenols, which are used as the compound heat stabilizer in Example 9, is 1:1.

[0102] Example 10: The difference between Example 10 and Example 1 is that the preservative used in Example 10 is a mixture of parabens and phenoxyethanol, and the mass ratio of the two is 1:1.

[0103] Example 11: The difference between Example 11 and Example 1 is that the modified recombinant collagen used in Example 11 is from Preparation Example 2.

[0104] Example 12: The difference between Example 12 and Example 1 is that the modified recombinant collagen used in Example 12 is from Preparation Example 3.

[0105] Example 13: The difference between Example 13 and Example 1 is that the modified recombinant collagen used in Example 13 is from Preparation Example 4.

[0106] Example 14: The difference between Example 14 and Example 1 is that the modified recombinant collagen used in Example 14 is from Preparation Example 5.

[0107] Example 15: The difference between Example 15 and Example 1 is that the modified recombinant collagen used in Example 15 is from Preparation Example 6.

[0108] Example 16: The difference between Example 16 and Example 1 is that the modified recombinant collagen used in Example 16 is from Preparation Example 7.

[0109] Example 17: The difference between Example 17 and Example 1 is that the modified recombinant collagen used in Example 17 is from Preparation Example 8.

[0110] Example 18: The difference between Example 18 and Example 1 is that the modified recombinant collagen used in Example 18 is from Preparation Example 9.

[0111] Example 19: The difference between Example 19 and Example 1 is that the modified recombinant collagen used in Example 19 is from Preparation Example 10.

[0112] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that no composite heat stabilizer is added in Comparative Example 1, and it is replaced by glycerol.

[0113] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that the heat stabilizer used in Comparative Example 2 is a single trehalose.

[0114] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that the heat stabilizer used in Comparative Example 3 is a single pomegranate peel polyphenol.

[0115] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that the recombinant collagen used in Comparative Example 4 is unmodified recombinant type III humanized collagen.

[0116] Performance Detection Test 1: Thermal Stability Test: After culturing HaCaT cells in good growth state in a 24-well plate for 17 h, replace the medium with serum-free basal medium and culture for 7 h, then discard the old medium. Use a 10 μL pipette tip to scratch the cells, and then add the medium containing the recombinant collagen gel prepared from Examples 1-19 and Comparative Examples 1-4 (the concentration of the recombinant collagen gel in the medium is 2 μg / mL). The medium in the blank group does not contain the recombinant collagen gel but contains 20 mM phosphate buffer. Take pictures and count the cells at the beginning of the culture and after 48 h of culture to obtain the effect of the test samples on the migration of HaCaT cells. After accelerating the destruction of the recombinant collagen gel at 48 °C for 30 min, 4 h, 24 h, and 7 d, then detect the cell migration rate after 48 h of culture. The results are shown in Table 1.

[0117] Determination of cell migration distance: Use Image J software to measure the area of each fixed-point scratch.

[0118]

[0119] The specific detection results are as follows:

[0120]

[0121]

[0122] As can be seen from the detection results in Table 1, the recombinant collagen gel provided by this application has excellent thermal stability. After heating for 7 days, it still maintains a high cell migration rate.

[0123] This specific embodiment is only an explanation of this application and does not limit this application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of this application, it is protected by the patent law.

Claims

1. A heat-stable recombinant collagen gel, characterized in that: The raw materials by mass percentage include 10 - 20% water, 40 - 50% modified recombinant collagen, 13 - 20% traditional Chinese medicine preparation, 8 - 16% sodium hyaluronate, 2 - 4% compound heat stabilizer, 1 - 2% preservative, and the balance is glycerol; the modified recombinant collagen is mercapto - modified recombinant collagen; The compound heat stabilizer includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4 - 0.

8.

2. The thermally stable recombinant collagen gel according to claim 1, wherein: The raw materials by mass percentage include 12 - 17% water, 42 - 48% modified recombinant collagen, 14 - 18% traditional Chinese medicine preparation, 10 - 14% sodium hyaluronate, 2.5 - 3.5% compound heat stabilizer, 1.3 - 1.7% preservative, and the balance is glycerol.

3. The heat-stable recombinant collagen gel according to claim 2, wherein: The raw materials by mass percentage include 15% water, 45% modified recombinant collagen, 16% traditional Chinese medicine preparation, 12% sodium hyaluronate, 3% compound heat stabilizer, 1.5% preservative, and the balance is glycerol.

4. The thermally stable recombinant collagen gel according to claim 1 or 2 or 3, characterized in that: The preparation method of the mercapto - modified recombinant collagen includes the following steps: T1. Add recombinant humanized collagen into acetic acid solution, stir and dissolve to obtain a collagen solution; transfer the collagen solution into a dialysis bag and dialyze at 4 - 6°C. The dialysis solution is alkaline phosphate buffer solution, and dialyze until the pH is 9 - 10 to obtain a collagen / phosphate mixed solution; T2. Add DL - N - acetylhomocysteine thiolactone to the collagen / phosphate mixed solution and stir evenly, then add ethylenediaminetetraacetic acid and dithiothreitol; under a nitrogen atmosphere, carry out a mercapto - reaction on the reaction mixture at 4 - 6°C; after the reaction is completed, transfer the reaction system into a dialysis bag and dialyze with neutral phosphate buffer solution at 4 - 6°C for 3 - 4 days to obtain mercapto - modified recombinant collagen.

5. The thermally stable recombinant collagen gel according to claim 4, wherein: The mass ratio of the raw materials recombinant humanized collagen, acetic acid solution, DL - N - acetylhomocysteine thiolactone, ethylenediaminetetraacetic acid, and dithiothreitol is 0.2 - 0.4:100 - 200:0.3 - 0.6:0.02 - 0.04:0.01 - 0.

02.

6. The heat-stable recombinant collagen gel according to claim 5, wherein: The concentration of the acetic acid solution is 0.4 - 0.6M.

7. The heat-stable recombinant collagen gel according to claim 4, wherein: The pH of the alkaline phosphate buffer solution is 9 - 10.

8. The preparation process of the thermally stable recombinant collagen gel according to any one of claims 1-7, characterized in that: It includes the following steps: S0. Weigh the raw materials water, modified recombinant collagen, traditional Chinese medicine preparation, sodium hyaluronate, compound heat stabilizer, preservative, and glycerol by mass percentage; S1. Stir - mix the modified recombinant collagen and water to obtain a modified recombinant collagen stock solution; filter the collagen stock solution to remove undissolved modified recombinant collagen; S2. Put the filtered collagen solution into a reaction kettle for emulsification reaction, and after the reaction, carry out centrifugal precipitation. After removing the supernatant, a modified recombinant collagen solution is obtained; S3. Mix and stir the modified recombinant collagen solution, sodium hyaluronate, compound heat stabilizer, and preservative, then add the traditional Chinese medicine preparation and stir evenly, and then add glycerol and stir evenly to obtain a heat - stable recombinant collagen gel.

9. The preparation process of the thermally stable recombinant collagen gel according to claim 8, characterized in that: The temperature of the emulsification reaction is 30 - 40°C, and the pressure is 1 - 1.5MPa.

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