Thermostable recombinant collagen gel and preparation process thereof

Abstract

The invention discloses thermally stable recombinant collagen gel and a preparation process thereof, and relates to the technical field of recombinant collagen, the thermally stable recombinant collagen gel comprises the following raw materials by mass: 10-20% of water, 40-50% of modified recombinant collagen, 13-20% of a traditional Chinese medicine preparation, 8-16% of sodium hyaluronate, 2-4% of a composite thermal stabilizer, 1-2% of a preservative, and the balance of glycerin; the modified recombinant collagen is a sulfhydrylation modified recombinant collagen; the composite heat stabilizer comprises trehalose and pomegranate peel polyphenol, and the mass ratio of the trehalose to the pomegranate peel polyphenol is 1: (0.4-0.8). The recombinant collagen is subjected to sulfhydrylation modification, and the composite heat stabilizer is added into the gel components, so that the heat stability of the recombinant collagen gel is remarkably improved.

Description

Technical Field

[0001] The present application relates to the field of recombinant collagen technology, and in particular to heat-stable recombinant collagen gel and its preparation process. Background Art

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

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

[0004] However, collagen is sensitive to heat due to its unique triple helix structure. It is easy to undergo thermal denaturation when heated, which causes the triple helix structure to be destroyed, and thus easily leads to the loss of the protein's biological activity. 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 gel. Summary of the invention

[0005] In order to improve the thermal stability of recombinant collagen gel, the present application provides a thermally stable recombinant collagen gel and a preparation process thereof.

[0006] The heat-stable recombinant collagen gel provided in the present application adopts the following technical solution: The heat-stable recombinant collagen gel comprises raw materials in percentage by mass including 10-20% water, 40-50% modified recombinant collagen, 13-20% 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 thiol-modified recombinant collagen.

[0007] By adopting the above-mentioned technical scheme, the present application significantly improves the thermal stability of the recombinant collagen by thiol-modifying the recombinant collagen gel and introducing thiol groups on the surface of the recombinant collagen, thereby preventing the recombinant collagen from denaturing and becoming unstable during processing; and adds a composite thermal stabilizer to the raw materials to further improve the high temperature resistance of the recombinant collagen gel and ensure the use effect of the product.

[0008] 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% composite thermal stabilizer, 1.3-1.7% preservative, and the balance is glycerol.

[0009] Preferably, the raw materials include, by mass percentage, 15% water, 45% modified recombinant collagen, 16% traditional Chinese medicine preparation, 12% sodium hyaluronate, 3% composite thermal stabilizer, 1.5% preservative, and the balance is glycerol.

[0010] Preferably, the composite thermal stabilizer comprises trehalose and pomegranate peel polyphenol, and the mass ratio of the two is 1:0.4-0.8.

[0011] By adopting the above technical scheme, trehalose and pomegranate peel polyphenols are used as the thermal stabilizer of recombinant collagen gel. Trehalose molecules contain multiple free hydroxyl groups and have a strong ability to form hydrogen bonds. These hydroxyl groups can form hydrogen bonds with polar groups on the surface of collagen to replace the bound water lost around the polar groups of collagen, thereby stabilizing the secondary structure of collagen. Under the action of the compound with pomegranate peel polyphenols, they can synergistically enhance the thermal stability of the recombinant collagen gel and ensure the high temperature resistance of the recombinant collagen gel.

[0012] Preferably, the raw materials of the traditional Chinese medicine preparation include paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica extract, honeysuckle extract, isatis root extract, and aloe extract, and the mass ratio is 2:2:5:5:2:1:1.

[0013] Preferably, the preservative is one of paraben and phenoxyethanol or a mixture of the two.

[0014] Preferably, the method for preparing the thiol-modified recombinant collagen comprises the following steps: T1. Add recombinant humanized collagen to acetic acid solution, stir and dissolve to obtain collagen solution; transfer the collagen solution to a dialysis bag and dialyze at 4-6°C, the dialysate is an alkaline phosphate buffer solution, dialyze to a pH of 9-10, and 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; in a nitrogen atmosphere, subject the reaction mixture to a thiol reaction at 4-6°C; after the reaction, transfer the reaction system to a dialysis bag, and dialyze it with a neutral phosphate buffer solution at 4-6°C for 3-4 days to obtain thiol-modified recombinant collagen.

[0015] By adopting the above technical scheme, the present application adopts N-acetylhomocysteine ​​thiolactone as a thiolating agent, and aminolyzes the thiolactone bond of N-acetylhomocysteine ​​thiolactone through the free amino group of the collagen side chain to introduce a thiol group, thereby synthesizing thiol-modified recombinant collagen, effectively improving the mechanical properties and thermal stability of recombinant human collagen, and effectively preventing the recombinant collagen from denaturing and becoming unstable during subsequent processing, thereby effectively maintaining the performance of the product.

[0016] Preferably, 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.

[0017] Preferably, the concentration of the acetic acid solution is 0.4-0.6M.

[0018] Preferably, the pH of the alkaline phosphate buffer solution is 9-10.

[0019] The heat-stable recombinant collagen gel and its preparation process provided in the present application adopt the following technical scheme: The preparation process of heat-stable recombinant collagen gel comprises the following steps: S0. Weigh the raw material water, modified recombinant collagen, Chinese medicine preparation, sodium hyaluronate, composite heat stabilizer, preservative, and glycerin according to mass percentage; S1. The modified recombinant collagen is stirred and mixed with water to obtain a modified recombinant collagen stock solution; the collagen stock solution is filtered to remove undissolved modified recombinant collagen; S2. The filtered collagen solution is placed in a reactor for emulsification reaction, and after the reaction, centrifugal precipitation is performed, and the supernatant is removed to obtain a modified recombinant collagen solution; S3. Mix the modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer and preservative, then add the traditional Chinese medicine preparation and stir evenly, then add glycerol and stir evenly to obtain a heat-stable recombinant collagen gel.

[0020] Preferably, the temperature of the emulsification reaction is 30-40° C. and the pressure is 1-1.5 MPa.

[0021] In summary, the present application includes at least one of the following beneficial technical effects: 1. The present application uses a compound of trehalose and pomegranate peel polyphenol as a thermal stabilizer for recombinant collagen gel. Trehalose molecules contain multiple free hydroxyl groups and have a strong ability to form hydrogen bonds. These hydroxyl groups can form hydrogen bonds with the polar groups of collagen to replace the bound water lost around the polar groups of collagen, thereby stabilizing the secondary structure of collagen. Under the action of compounding with pomegranate peel polyphenol, they can synergistically enhance the thermal stability of recombinant collagen gel. 2. The present application adopts N-acetylhomocysteine ​​thiolactone as a thiolating agent, and aminolyzes the thiolactone bond of N-acetylhomocysteine ​​thiolactone through the free amino group of the collagen side chain to introduce a thiol group, thereby synthesizing thiol-modified recombinant collagen, which effectively improves the mechanical properties and thermal stability of recombinant human collagen, can effectively prevent the recombinant collagen from denaturing and becoming unstable during subsequent processing, and effectively maintains the performance of the product. DETAILED DESCRIPTION

[0022] The present application is further described in detail below with reference to the embodiments.

[0023] The chemical reagents used in the preparation examples, embodiments and comparative examples provided by the present invention are all commercially available products, and their brands and manufacturers are as follows: Recombinant humanized type III collagen, Shanxi Jinbo Biopharmaceutical Co., Ltd.; DL-N-acetylhomocysteine ​​thiolactone, Shanghai Myrel Biochemical Technology Co., Ltd.; DL-dithiothreitol, Shanghai Aladdin Biochemical Technology Co., Ltd.; Salvia miltiorrhiza extract, Xi'an Hongsheng Biotechnology Co., Ltd.; Angelica extract, Panier (Shaanxi) Industrial Co., Ltd.; Paeonol, Hubei Changao Pharmaceutical Co., Ltd.; Panax notoginseng extract, Chengdu Zero Six Biotechnology Co., Ltd.; Honeysuckle extract, Shaanxi Panier Biotechnology Co., Ltd.; Radix Isatidis extract, Hubei Weideli Chemical Technology Co., Ltd.; Aloe vera extract, Shaanxi Jianchuang Biotechnology Co., Ltd.; Trehalose, Shanghai Aladdin Biochemical Technology Co., Ltd.; Pomegranate peel polyphenols, Fufeng Snot Biotechnology Co., Ltd.; Sodium hyaluronate, Nanjing Romai Biotechnology Co., Ltd.; Glycerol, Shanghai Aladdin Biochemical Technology Co., Ltd.; Parabens, Hebei Jijie Biotechnology Co., Ltd.; Phenoxyethanol, Wuhan Xinli Biotechnology Co., Ltd.

[0024] Preparation Example 1: T1. Add 0.2 g of recombinant type III humanized collagen to 100 g of 0.5 M acetic acid solution, stir and dissolve to obtain a collagen solution; transfer the collagen solution to a dialysis bag (MWCO 8000-14000 kDa) and dialyze at 4° C., the dialysate is an alkaline phosphate buffer solution with a pH of 9, and dialyze to a pH of 9 to obtain a collagen / phosphate mixed solution; T2. Add 0.3 g of DL-N-acetylhomocysteine ​​thiolactone, 0.02 g of ethylenediaminetetraacetic acid and 0.01 g of DL-dithiothreitol to the collagen / phosphate mixed solution; under a nitrogen atmosphere, carry out thiol reaction of the reaction mixture at 4°C for 30 hours; after the reaction, transfer the reaction system to a dialysis bag, dialyze with a neutral phosphate buffer solution of pH 7 at 4°C for 3 days, and change the dialysate every 4 hours to obtain thiol-modified recombinant collagen.

[0025] Preparation Example 2: T1. Add 0.3 g of recombinant type III humanized collagen to 150 g of 0.5 M acetic acid solution, stir and dissolve to obtain a collagen solution; transfer the collagen solution to a dialysis bag (MWCO 8000-14000 kDa) and dialyze at 5° C., the dialysate is an alkaline phosphate buffer solution with a pH of 9, and dialyze to a pH of 9 to obtain a collagen / phosphate mixed solution; T2. Add 0.45 g of DL-N-acetylhomocysteine ​​thiolactone, 0.03 g of ethylenediaminetetraacetic acid and 0.015 g of DL-dithiothreitol to the collagen / phosphate mixed solution; under a nitrogen atmosphere, carry out thiol reaction of the reaction mixture at 5°C for 40 hours; after the reaction, transfer the reaction system to a dialysis bag, dialyze with a neutral phosphate buffer solution of pH 7 at 4°C for 3.5 days, and change the dialysate every 4 hours to obtain thiol-modified recombinant collagen.

[0026] Preparation Example 3: T1. Add 0.4 g of recombinant type III humanized collagen to 200 g of 0.5 M acetic acid solution, stir and dissolve to obtain a collagen solution; transfer the collagen solution to a dialysis bag (MWCO 8000-14000 kDa) and dialyze at 6° C., the dialysate is an alkaline phosphate buffer solution with a pH of 9, and dialyze to a pH of 9 to obtain a collagen / phosphate mixed solution; T2. Add 0.6 g of DL-N-acetylhomocysteine ​​thiolactone, 0.04 g of ethylenediaminetetraacetic acid and 0.02 g of DL-dithiothreitol to the collagen / phosphate mixed solution; under a nitrogen atmosphere, carry out thiol reaction of the reaction mixture at 6°C for 50 hours; after the reaction, transfer the reaction system to a dialysis bag, dialyze with a neutral phosphate buffer solution of pH 7 at 6°C for 4 days, and change the dialysate every 4 hours to obtain thiol-modified recombinant collagen.

[0027] 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 0.4M.

[0028] 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 0.6M.

[0029] 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 0.2M.

[0030] 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 0.8M.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Example 1: S0. Weigh 10 g of water, 40 g of the modified recombinant collagen prepared in Preparation Example 1, 20 g of the Chinese medicine preparation, 16 g of sodium hyaluronate, 2 g of the composite thermal stabilizer, 1 g of the preservative, and 11 g of glycerol according to mass percentage; The Chinese medicine preparation described in this embodiment includes paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica extract, honeysuckle extract, isatis root extract, and aloe extract, and the mass ratio is 2:2:5:5:2:1:1; The composite thermal stabilizer in this embodiment includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4; The preservative in this embodiment is paraben; S1. The modified recombinant collagen and water were stirred and mixed at 600 rpm to obtain a modified recombinant collagen stock solution; the collagen stock solution was filtered to remove undissolved modified recombinant collagen; S2. The filtered collagen solution was placed in a reactor, and an emulsification reaction was performed at a temperature of 30°C and a pressure of 1 MPa for 30 min. After the reaction, the solution was centrifuged and the supernatant was removed to obtain a modified recombinant collagen solution. S3. The modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer and preservative are mixed and stirred at 600 rpm, and then the traditional Chinese medicine preparation is added and stirred evenly, and then glycerol is added and stirred evenly to obtain a heat-stable recombinant collagen gel.

[0035] Example 2: S0. Weigh 12 g of water, 42 g of the modified recombinant collagen prepared in Preparation Example 1, 18 g of the Chinese medicine preparation, 14 g of sodium hyaluronate, 2.5 g of the composite thermal stabilizer, 1.3 g of the preservative, and 10.2 g of glycerol according to mass percentage; The Chinese medicine preparation described in this embodiment includes paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica extract, honeysuckle extract, isatis root extract, and aloe extract, and the mass ratio is 2:2:5:5:2:1:1; The composite thermal stabilizer in this embodiment includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4; The preservative in this embodiment is paraben; S1. The modified recombinant collagen and water were stirred and mixed at 600 rpm to obtain a modified recombinant collagen stock solution; the collagen stock solution was filtered to remove undissolved modified recombinant collagen; S2. The filtered collagen solution was placed in a reactor, and an emulsification reaction was performed at a temperature of 30°C and a pressure of 1 MPa for 30 min. After the reaction, the solution was centrifuged and the supernatant was removed to obtain a modified recombinant collagen solution. S3. The modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer and preservative are mixed and stirred at 600 rpm, and then the traditional Chinese medicine preparation is added and stirred evenly, and then glycerol is added and stirred evenly to obtain a heat-stable recombinant collagen gel.

[0036] Example 3: S0. Weigh 15 g of water, 45 g of the modified recombinant collagen prepared in Preparation Example 1, 16 g of the Chinese medicine preparation, 12 g of sodium hyaluronate, 3 g of the composite thermal stabilizer, 1.5 g of the preservative, and 7.5 g of glycerol according to mass percentage; The Chinese medicine preparation described in this embodiment includes paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica extract, honeysuckle extract, isatis root extract, and aloe extract, and the mass ratio is 2:2:5:5:2:1:1; The composite thermal stabilizer in this embodiment includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4; The preservative in this embodiment is paraben; S1. The modified recombinant collagen and water were stirred and mixed at 600 rpm to obtain a modified recombinant collagen stock solution; the collagen stock solution was filtered to remove undissolved modified recombinant collagen; S2. The filtered collagen solution was placed in a reactor, and an emulsification reaction was performed at a temperature of 30°C and a pressure of 1 MPa for 30 min. After the reaction, the solution was centrifuged and the supernatant was removed to obtain a modified recombinant collagen solution. S3. The modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer and preservative are mixed and stirred at 600 rpm, and then the traditional Chinese medicine preparation is added and stirred evenly, and then glycerol is added and stirred evenly to obtain a heat-stable recombinant collagen gel.

[0037] Example 4: S0. Weigh 17 g of water, 48 g of the modified recombinant collagen prepared in Preparation Example 1, 14 g of the Chinese medicine preparation, 10 g of sodium hyaluronate, 3.5 g of the composite thermal stabilizer, 1.7 g of the preservative, and 5.8 g of glycerol according to mass percentage; The Chinese medicine preparation described in this embodiment includes paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica extract, honeysuckle extract, isatis root extract, and aloe extract, and the mass ratio is 2:2:5:5:2:1:1; The composite thermal stabilizer in this embodiment includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4; The preservative in this embodiment is paraben; S1. The modified recombinant collagen and water were stirred and mixed at 600 rpm to obtain a modified recombinant collagen stock solution; the collagen stock solution was filtered to remove undissolved modified recombinant collagen; S2. The filtered collagen solution was placed in a reactor, and an emulsification reaction was performed at a temperature of 30°C and a pressure of 1 MPa for 30 min. After the reaction, the solution was centrifuged and the supernatant was removed to obtain a modified recombinant collagen solution. S3. The modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer and preservative are mixed and stirred at 600 rpm, and then the traditional Chinese medicine preparation is added and stirred evenly, and then glycerol is added and stirred evenly to obtain a heat-stable recombinant collagen gel.

[0038] Example 5: S0. Weigh 20 g of water, 50 g of the modified recombinant collagen prepared in Preparation Example 1, 13 g of the Chinese medicine preparation, 8 g of sodium hyaluronate, 4 g of the composite thermal stabilizer, 2 g of the preservative, and 3 g of glycerol according to mass percentage; The Chinese medicine preparation described in this embodiment includes paeonol, notoginseng extract, salvia miltiorrhiza extract, angelica extract, honeysuckle extract, isatis root extract, and aloe extract, and the mass ratio is 2:2:5:5:2:1:1; The composite thermal stabilizer in this embodiment includes trehalose and pomegranate peel polyphenols, and the mass ratio of the two is 1:0.4; The preservative in this embodiment is paraben; S1. The modified recombinant collagen and water were stirred and mixed at 600 rpm to obtain a modified recombinant collagen stock solution; the collagen stock solution was filtered to remove undissolved modified recombinant collagen; S2. The filtered collagen solution was placed in a reactor, and an emulsification reaction was performed at a temperature of 30°C and a pressure of 1 MPa for 30 min. After the reaction, the solution was centrifuged and the supernatant was removed to obtain a modified recombinant collagen solution. S3. The modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer and preservative are mixed and stirred at 600 rpm, and then the traditional Chinese medicine preparation is added and stirred evenly, and then glycerol is added and stirred evenly to obtain a heat-stable recombinant collagen gel.

[0039] Example 6: The difference between Example 6 and Example 1 is that the mass ratio of the composite thermal stabilizer trehalose and pomegranate peel polyphenols used in Example 6 is 1:0.6.

[0040] Example 7: The difference between Example 7 and Example 1 is that the mass ratio of the composite thermal stabilizer trehalose and pomegranate peel polyphenol used in Example 7 is 1:0.8.

[0041] Example 8: The difference between Example 8 and Example 1 is that the mass ratio of the composite thermal stabilizer trehalose and pomegranate peel polyphenols used in Example 8 is 1:0.2.

[0042] Example 9: The difference between Example 9 and Example 1 is that the mass ratio of the composite thermal stabilizer trehalose and pomegranate peel polyphenols used in Example 9 is 1:1.

[0043] Example 10: The difference between Example 10 and Example 1 is that the preservative used in Example 10 is a mixture of paraben and phenoxyethanol, and the mass ratio of the two is 1:1.

[0044] Example 11: The difference between Example 11 and Example 1 is that the modified recombinant collagen used in Example 11 comes from Preparation Example 2.

[0045] Example 12: The difference between Example 12 and Example 1 is that the modified recombinant collagen used in Example 12 comes from Preparation Example 3.

[0046] Example 13: The difference between Example 13 and Example 1 is that the modified recombinant collagen used in Example 13 comes from Preparation Example 4.

[0047] Example 14: The difference between Example 14 and Example 1 is that the modified recombinant collagen used in Example 14 comes from Preparation Example 5.

[0048] Example 15: The difference between Example 15 and Example 1 is that the modified recombinant collagen used in Example 15 comes from Preparation Example 6.

[0049] Example 16: The difference between Example 16 and Example 1 is that the modified recombinant collagen used in Example 16 comes from Preparation Example 7.

[0050] Example 17: The difference between Example 17 and Example 1 is that the modified recombinant collagen used in Example 17 comes from Preparation Example 8.

[0051] Example 18: The difference between Example 18 and Example 1 is that the modified recombinant collagen used in Example 18 comes from Preparation Example 9.

[0052] Example 19: The difference between Example 19 and Example 1 is that the modified recombinant collagen used in Example 19 comes from Preparation Example 10.

[0053] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that the composite heat stabilizer is not added in Comparative Example 1, and is replaced by glycerol.

[0054] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that the thermal stabilizer used in Comparative Example 2 is single trehalose.

[0055] 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.

[0056] 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.

[0057] Performance test 1: thermal stability test: HaCaT cells in good growth state were plated on a 24-well plate and cultured for 17 hours, then replaced with serum-free basal medium and cultured for 7 hours, the old medium was discarded, a 10 μL gun tip was used to draw a line, and then a medium containing the recombinant collagen gel prepared in Example 1-19 and Comparative Example 1-4 was added (the concentration of the recombinant collagen gel in the medium was 2 μg / mL). The culture medium of the blank group did not contain recombinant collagen gel, but contained 20 mM phosphate buffer. Photos were taken and counted at the beginning of culture and after 48 hours of culture, respectively, to obtain the effect of the test sample on the migration of HaCaT cells, and the recombinant collagen gel was accelerated at 48°C for 30 minutes, 4 hours, 24 hours, and 7 days, and then the cell migration rate after 48 hours of culture was detected. The results are shown in Table 1.

[0058] Determination of cell migration distance: Image J software was used to measure the scratch area at each fixed point.

[0059]

[0060] The specific test results are as follows:

[0061] It can be seen from the test results in Table 1 that the recombinant collagen gel provided by the present application has excellent thermal stability and still maintains a high cell migration rate after being heated for 7 days.

[0062] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A heat-stable recombinant collagen gel, characterized in that: The raw materials 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 by mass percentage; the modified recombinant collagen is thiol-modified recombinant collagen.

2. The heat-stable recombinant collagen gel according to claim 1, characterized in that: 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% composite heat stabilizer, 1.3-1.7% preservative, and the balance is glycerol.

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

4. The heat-stable recombinant collagen gel according to claim 1, 2 or 3, characterized in that: The composite heat stabilizer comprises trehalose and pomegranate peel polyphenol, and the mass ratio of the two is 1:0.4-0.

8.

5. The heat-stable recombinant collagen gel according to claim 1, 2 or 3, characterized in that: The method for preparing the thiol-modified recombinant collagen comprises the following steps: T1. Add recombinant humanized collagen to acetic acid solution, stir and dissolve to obtain collagen solution; transfer the collagen solution to a dialysis bag and dialyze at 4-6°C, the dialysate is an alkaline phosphate buffer solution, dialyze to a pH of 9-10, and 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; in a nitrogen atmosphere, subject the reaction mixture to a thiol reaction at 4-6°C; after the reaction, transfer the reaction system to a dialysis bag, and dialyze it with a neutral phosphate buffer solution at 4-6°C for 3-4 days to obtain thiol-modified recombinant collagen.

6. The heat-stable recombinant collagen gel according to claim 5, characterized in that: 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.

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

8. The heat-stable recombinant collagen gel according to claim 5, characterized in that: The pH of the alkaline phosphate buffer solution is 9-10.

9. The process for preparing the heat-stable recombinant collagen gel according to any one of claims 1 to 8, characterized in that: The following steps are involved: S0. Weigh the raw material water, modified recombinant collagen, Chinese medicine preparation, sodium hyaluronate, composite heat stabilizer, preservative, and glycerin according to mass percentage; S1. The modified recombinant collagen is stirred and mixed with water to obtain a modified recombinant collagen stock solution; the collagen stock solution is filtered to remove undissolved modified recombinant collagen; S2. The filtered collagen solution is placed in a reactor for emulsification reaction, and after the reaction, centrifugal precipitation is performed, and the supernatant is removed to obtain a modified recombinant collagen solution; S3. Mix the modified recombinant collagen solution, sodium hyaluronate, composite heat stabilizer and preservative, then add the traditional Chinese medicine preparation and stir evenly, then add glycerol and stir evenly to obtain a heat-stable recombinant collagen gel.

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

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