Recombinant collagen peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cosmetics, and particularly discloses a recombinant collagen peptide as well as a preparation method and application thereof. The preparation method of the recombinant collagen peptide comprises the following steps: S1, reacting recombinant collagen with modified trehalose to form a recombinant collagen solution; s2, putting the recombinant collagen solution into a reaction kettle, and performing heat treatment to obtain recombinant collagen peptide; the modified trehalose is prepared by the following steps: firstly grafting maleic anhydride on trehalose to form a graft, compounding the graft with 4-sulfocalixarene, and then cross-linking with gelatin and xylitol. The recombinant collagen peptide prepared by the invention has good stability and high transdermal property, can be used for preparing skin care products, effectively enhances moisturizing and moisturizing properties and increases skin elasticity, and has important application value.

Description

Technical Field

[0001] The present application relates to the field of cosmetic technology, and more specifically, to a recombinant collagen peptide and a preparation method and application thereof. Background Art

[0002] Collagen is a fibrous protein that is mainly distributed in the extracellular matrix. It is the most abundant protein in the human body and is widely present in human skin, bones, muscles, cartilage and other tissues. It plays important roles in support, repair, and protection, and plays an important role in the growth and renewal of skin cells.

[0003] Recombinant collagen is a type of collagen produced through genetic engineering technology. Compared with traditional animal-derived collagen, it has higher safety, better biological activity and biocompatibility. Recombinant collagen peptides are smaller fragments cut from recombinant collagen by enzymatic hydrolysis or other techniques. They are usually composed of several to dozens of amino acids, have good water solubility and permeability, can act directly on the skin, replenish collagen for the skin, and achieve various cosmetic effects such as anti-wrinkle, moisturizing, and repair. They are widely used in the preparation of skin care products. However, the stability and activity of recombinant collagen peptides during use are still insufficient, and the transdermal effect has limitations. With the continuous improvement of consumers' awareness of health and beauty, and the pursuit of natural, safe, and efficient products, there is an urgent need to develop a recombinant collagen peptide with high transdermal permeability, good stability and high activity. Therefore, this application proposes a recombinant collagen peptide and its preparation method and application. Summary of the Invention

[0004] In order to solve the problems of poor stability, low activity and insufficient transdermal performance of recombinant collagen in the prior art, the present application provides a recombinant collagen peptide and a preparation method and application thereof.

[0005] In a first aspect, the present application provides a method for preparing recombinant collagen peptides, which specifically comprises the following steps: S1. Stir and mix recombinant collagen, modified trehalose, and phosphate buffer, add sodium ascorbate, continue stirring and mixing, heat the reaction, cool down to terminate the reaction, and then place the reaction solution into a dialysis bag for dialysis with a molecular weight cut-off of 1-3 kDa to obtain a recombinant collagen solution; S2. Place the recombinant collagen solution into a reactor and heat treat for 3-5 hours to obtain recombinant collagen peptides.

[0006] Preferably, the method for preparing the recombinant collagen peptide specifically comprises the following steps: S1. Stir and mix recombinant collagen, modified trehalose, and phosphate buffer for 1-2 hours, add sodium ascorbate, continue stirring and mixing for 8-10 hours, control the temperature to 40-60°C, cool down to terminate the reaction, and then place the reaction solution into a dialysis bag for dialysis with a molecular weight cutoff of 1-3 kDa to obtain a recombinant collagen solution; S2. Place the recombinant collagen solution into a reactor and heat treat for 3-5 hours to obtain recombinant collagen peptides.

[0007] Preferably, in step S1, the mass ratio of recombinant collagen, modified trehalose, phosphate buffer, and sodium ascorbate is 1:1-3:20:0.2.

[0008] Preferably, in step S2, the temperature of the reactor is 100-120° C., and the pressure of the reactor is 0.11-0.15 MPa.

[0009] Preferably, the modified trehalose in step S1 is prepared by the following method: (1) Stir and mix trehalose, maleic anhydride and anhydrous ethanol, add p-toluenesulfonic acid, control the temperature at 70-80°C under nitrogen protection, continue stirring and mixing for 4-6 hours, centrifuge, wash with water, and dry at 40-50°C to obtain the grafted product; (2) Stirring the grafted product, 4-sulfonic acid calixarene and ethanol solution for 1-2 hours at a temperature of 40-50°C to form a composite solution; (3) The composite solution and gelatin solution were stirred and mixed for 30 minutes, and then xylitol was added and the mixture was stirred and mixed. The mixture was kept at 50°C for 4-6 hours, and then centrifuged and dried to obtain modified trehalose.

[0010] Preferably, in step (1), the mass ratio of trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid is 1:1-3:20:0.03.

[0011] Preferably, in step (2), the mass ratio of the grafted product, 4-sulfonic acid calixarene and ethanol solution is 6-8:2-4:50.

[0012] Preferably, the mass concentration of the ethanol solution in step (2) is 30%.

[0013] Preferably, in step (3), the mass ratio of the composite solution, gelatin solution and xylitol is 3-5:1-2:0.5.

[0014] Preferably, the gelatin solution in step (3) is prepared by the following method: gelatin and water are stirred and mixed at 40° C. at a mass ratio of gelatin to water of 1:10.

[0015] In a second aspect, the present application provides a recombinant collagen peptide prepared by the method described above.

[0016] Preferably, the recombinant collagen peptide is used to prepare skin care products.

[0017] In summary, this application has the following beneficial effects: 1. This application adopts a reasonable combination of raw materials and preparation process, and the recombinant collagen peptide prepared has good stability and transdermal properties, which can significantly improve the stability and activity of the recombinant collagen peptide during use, and effectively promote the transdermal absorption of the recombinant collagen peptide, and has broad application prospects in the cosmetics field.

[0018] 2. The present application reacts recombinant collagen with modified trehalose during the preparation process of recombinant collagen peptides, which not only forms a protective film to inhibit the hydrolysis of protein peptides and improve the structural stability and thermal stability of recombinant collagen peptides, but also increases the water content of the skin, keeps the skin stratum corneum in a hydrated state, and promotes the transdermal absorption of recombinant collagen peptides. Modified trehalose is prepared by first grafting maleic anhydride onto trehalose to form a graft, and then compounding the graft with 4-sulfonic acid calixarene and then cross-linking with gelatin and xylitol. Grafting maleic anhydride onto trehalose can, on the one hand, increase the reactive sites, improve the intermolecular forces, and promote the subsequent interaction with 4-sulfonic acid calixarene; on the other hand, it can provide cross-linking reaction groups for the subsequent cross-linking reaction, making the cross-linked network structure tighter and more stable, thereby forming a larger steric hindrance, enhancing its ability to resist changes in the external environment, and improving stability. The grafted compound is then cross-linked with 4-sulfonic acid calixarene to form a three-dimensional network structure that encapsulates the recombinant collagen peptide. It also improves the biocompatibility and skin affinity of the modified trehalose, promoting the recombinant collagen peptide's penetration through the stratum corneum and other barrier layers of the skin, thereby enhancing transdermal permeability. The addition of xylitol not only acts as a bridge, making the cross-linking structure more uniform and stable, but also introduces more hydrophilic groups, increasing the hydrophilicity and moisturizing properties of the modified trehalose, further enhancing the activity and stability of the recombinant collagen peptide. DETAILED DESCRIPTION

[0019] The present application is further described in detail below with reference to the embodiments.

[0020] Examples 1-5 provide a recombinant collagen peptide, a preparation method, and an application thereof.

[0021] The recombinant collagen used in the examples and comparative examples of the present application (item number: 102001, purchased from Wuhan Xinxin Jiali Biotechnology Co., Ltd.), trehalose (CAS: 99-20-7, purchased from Anhui Youtai Bioengineering Co., Ltd.), maleic anhydride (CAS: 108-31-6, purchased from Jinan Jinhao Chemical Co., Ltd.), 4-sulfonic acid calixarene (CAS: 137407-62-6, purchased from Hubei Chenghai Chemical Co., Ltd.), gelatin (CAS: 9000-70-8, purchased from Anhui Runtai Biotechnology Co., Ltd.), xylitol (CAS: 87-99-0, purchased from Jinhua Shihai Biotechnology Co., Ltd.), and sodium ascorbate (CAS: 134-03-2, purchased from Hubei Xinkang Pharmaceutical Chemical Co., Ltd.)

[0022] Example 1 A method for preparing recombinant collagen peptides comprises the following steps: S1. According to the mass ratio of recombinant collagen, modified trehalose, phosphate buffer, and sodium ascorbate of 1:1:20:0.2, the recombinant collagen, modified trehalose, and phosphate buffer (0.1 M, pH 7.0) were stirred at 400 r / min for 1 hour, sodium ascorbate was added, and stirring was continued at 400 r / min for 8 hours. The temperature was controlled at 40°C, and the temperature was lowered to terminate the reaction. The reaction solution was then placed in a dialysis bag for dialysis with a molecular weight cutoff of 1 kDa to obtain a recombinant collagen solution; S2. Place the recombinant collagen solution into a reactor, control the reactor temperature to 100° C., the reactor pressure to 0.11 MPa, and heat treat for 3 hours to obtain recombinant collagen peptide.

[0023] The modified trehalose in step S1 is prepared by the following method: (1) Trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid were stirred and mixed at 600 r / min for 30 min in a mass ratio of trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid of 1:1:20:0.03. P-toluenesulfonic acid was added. Under nitrogen protection, the temperature was controlled at 70°C, and the mixture was stirred and mixed at 400 r / min for 4 h. The mixture was centrifuged at 10,000 r / min for 20 min, washed with water, and dried at 40°C for 1 h to obtain a grafted product. (2) The grafted product, 4-sulfonic acid calixarene and ethanol solution were mixed at a mass ratio of 6:2:50, and the grafted product, 4-sulfonic acid calixarene and 30% ethanol solution were stirred at 500 r / min for 1 h and the temperature was controlled at 40°C to form a composite solution; (3) The composite solution was mixed with gelatin solution (prepared by mixing gelatin and water at a mass ratio of 1:10 at 40°C at 600 r / min for 40 min) at a mass ratio of 3:1:0.5 among the composite solution, gelatin solution and xylitol. The mixture was stirred at 400 r / min for 30 min, and xylitol was added. The mixture was stirred at 600 r / min for 30 min, kept warm at 50°C for 4 h, and then centrifuged at 12000 r / min for 10 min. The mixture was dried at 40°C for 1 h to obtain modified trehalose.

[0024] Example 2 A method for preparing recombinant collagen peptides comprises the following steps: S1. According to the mass ratio of recombinant collagen, modified trehalose, phosphate buffer and sodium ascorbate of 1:1.5:20:0.2, the recombinant collagen, modified trehalose and phosphate buffer (0.1M, pH 7.0) were stirred at 400 r / min for 1.2 h, sodium ascorbate was added, and stirring was continued at 400 r / min for 8.5 h. The temperature was controlled at 45°C, and the temperature was lowered to terminate the reaction. The reaction solution was then placed in a dialysis bag for dialysis with a molecular weight cutoff of 1.5 kDa to obtain a recombinant collagen solution; S2. Place the recombinant collagen solution into a reactor, control the reactor temperature to 105° C., the reactor pressure to 0.12 MPa, and heat treat for 3.5 hours to obtain recombinant collagen peptide.

[0025] The modified trehalose in step S1 is prepared by the following method: (1) Trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid were stirred and mixed at 600 r / min for 30 min in a mass ratio of trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid of 1:1.5:20:0.03. P-toluenesulfonic acid was added. Under nitrogen protection, the temperature was controlled at 75°C and the stirring was continued at 400 r / min for 4.5 h. The mixture was centrifuged at 10,000 r / min for 20 min, washed with water and dried at 45°C for 1.2 h to obtain a grafted product. (2) The grafted product, 4-sulfonic acid calixarene and ethanol solution were mixed at a mass ratio of 6.5:2.5:50, and the grafted product, 4-sulfonic acid calixarene and 30% ethanol solution were stirred at 500 r / min for 1.2 h and the temperature was controlled at 42°C to form a composite solution; (3) The composite solution and gelatin solution (prepared by mixing gelatin and water at a mass ratio of 1:10 at 40°C at 600 r / min for 40 min) were stirred at 400-600 r / min for 30 min, with the mass ratio of composite solution, gelatin solution and xylitol being 3.5:1.2:0.5. Xylitol was then added and the mixture was stirred at 600 r / min for 30 min. The mixture was kept warm at 50°C for 4.5 h, and then centrifuged at 12,000 r / min for 10 min. The mixture was dried at 42°C for 1 h to obtain modified trehalose.

[0026] Example 3 A method for preparing recombinant collagen peptides comprises the following steps: S1. According to the mass ratio of recombinant collagen, modified trehalose, phosphate buffer and sodium ascorbate of 1:2:20:0.2, the recombinant collagen, modified trehalose and phosphate buffer (0.1M, pH 7.0) were stirred at 400 r / min for 1.5 h, sodium ascorbate was added, and stirring was continued at 400 r / min for 9 h. The temperature was controlled at 50°C, and the temperature was lowered to terminate the reaction. The reaction solution was then placed in a dialysis bag for dialysis with a molecular weight cutoff of 2 kDa to obtain a recombinant collagen solution; S2. Place the recombinant collagen solution into a reactor, control the reactor temperature to 110° C., the reactor pressure to 0.13 MPa, and heat treat for 4 hours to obtain recombinant collagen peptide.

[0027] The modified trehalose in step S1 is prepared by the following method: (1) Trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid were stirred and mixed at 600 r / min for 30 min in a mass ratio of trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid of 1:2:20:0.03. P-toluenesulfonic acid was added. Under nitrogen protection, the temperature was controlled at 75°C and the stirring was continued at 400 r / min for 5 h. The mixture was centrifuged at 10,000 r / min for 20 min, washed with water and dried at 45°C for 1.5 h to obtain a grafted product. (2) The grafted product, 4-sulfonic acid calixarene and ethanol solution were mixed at a mass ratio of 7:3:50, and the grafted product, 4-sulfonic acid calixarene and 30% ethanol solution were stirred at 500 r / min for 1.5 h and the temperature was controlled at 45°C to form a composite solution; (3) The composite solution was mixed with gelatin solution (prepared by mixing gelatin and water at a mass ratio of 1:10 at 40°C at 600 r / min for 40 min) at a stirring rate of 500 r / min for 30 min, with the mass ratio of composite solution, gelatin solution and xylitol being 4:1.5:0.5. Xylitol was then added and the mixture was stirred at 600 r / min for 30 min. The mixture was kept warm at 50°C for 5 h, and then centrifuged at 12,000 r / min for 10 min. The mixture was dried at 45°C for 1 h to obtain modified trehalose.

[0028] Example 4 A method for preparing recombinant collagen peptides comprises the following steps: S1. According to the mass ratio of recombinant collagen, modified trehalose, phosphate buffer and sodium ascorbate of 1:2.5:20:0.2, the recombinant collagen, modified trehalose and phosphate buffer (0.1M, pH 7.0) were stirred at 400 r / min for 1.8 h, sodium ascorbate was added, and stirring was continued at 400 r / min for 9.5 h. The temperature was controlled at 55°C, and the temperature was lowered to terminate the reaction. The reaction solution was then placed in a dialysis bag for dialysis with a molecular weight cutoff of 2.5 kDa to obtain a recombinant collagen solution; S2. Place the recombinant collagen solution into a reactor, control the reactor temperature to 115° C., the reactor pressure to 0.14 MPa, and heat treat for 4.5 hours to obtain recombinant collagen peptide.

[0029] The modified trehalose in step S1 is prepared by the following method: (1) Trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid were stirred and mixed at 600 r / min for 30 min in a mass ratio of trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid of 1:2.5:20:0.03. P-toluenesulfonic acid was added. Under nitrogen protection, the temperature was controlled at 78°C. The mixture was stirred and mixed at 400 r / min for 5.5 h. The mixture was centrifuged at 10,000 r / min for 20 min, washed with water and dried at 48°C for 1.8 h to obtain a grafted product. (2) The grafted product, 4-sulfonic acid calixarene and ethanol solution were mixed at a mass ratio of 7.5:3.5:50, and the grafted product, 4-sulfonic acid calixarene and 30% ethanol solution were stirred at 500 r / min for 1.8 h and the temperature was controlled at 48°C to form a composite solution; (3) The composite solution was mixed with gelatin solution (prepared by mixing gelatin and water at a mass ratio of 1:10 at 40°C at 600 r / min for 40 min) at a stirring rate of 550 r / min for 30 min, with the mass ratio of composite solution, gelatin solution and xylitol being 4.5:1.8:0.5. Xylitol was then added and the mixture was stirred at 600 r / min for 30 min. The mixture was kept warm at 50°C for 5.5 h, centrifuged at 12,000 r / min for 10 min, and dried at 48°C for 1 h to obtain modified trehalose.

[0030] Example 5 A method for preparing recombinant collagen peptides comprises the following steps: S1. According to the mass ratio of recombinant collagen, modified trehalose, phosphate buffer, and sodium ascorbate of 1:3:20:0.2, the recombinant collagen, modified trehalose, and phosphate buffer (0.1 M, pH 7.0) were stirred at 400 r / min for 2 h, sodium ascorbate was added, and stirring was continued at 400 r / min for 10 h. The temperature was controlled at 60°C, and the temperature was lowered to terminate the reaction. The reaction solution was then placed in a dialysis bag for dialysis with a molecular weight cut-off of 3 kDa to obtain a recombinant collagen solution; S2. Place the recombinant collagen solution into a reactor, control the reactor temperature to 120° C., the reactor pressure to 0.15 MPa, and heat treat for 5 hours to obtain recombinant collagen peptide.

[0031] The modified trehalose in step S1 is prepared by the following method: (1) Trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid were stirred and mixed at 600 r / min for 30 min in a mass ratio of trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid of 1:3:20:0.03. P-toluenesulfonic acid was added. Under nitrogen protection, the temperature was controlled at 80°C, and the stirring was continued at 400 r / min for 6 h. The mixture was centrifuged at 10,000 r / min for 20 min, washed with water, and dried at 50°C for 2 h to obtain a grafted product. (2) The grafted product, 4-sulfonic acid calixarene and ethanol solution were mixed at a mass ratio of 8:4:50, and the grafted product, 4-sulfonic acid calixarene and 30% ethanol solution were stirred at 500 r / min for 2 h and the temperature was controlled at 50°C to form a composite solution; (3) The composite solution was mixed with gelatin solution (prepared by mixing gelatin and water at a mass ratio of 1:10 at 40°C at 600 r / min for 40 min) at a mass ratio of 5:2:0.5. Xylitol was then added and the mixture was stirred at 600 r / min for 30 min. The mixture was kept warm at 50°C for 6 h, centrifuged at 12,000 r / min for 10 min, and dried at 50°C for 1 h to obtain modified trehalose.

[0032] Comparative Example 1 Comparative Example 1 is the same as Example 1, except that the trehalose is not modified, as shown below: A method for preparing recombinant collagen peptides comprises the following steps: S1. According to the mass ratio of recombinant collagen, trehalose, phosphate buffer, and sodium ascorbate of 1:1:20:0.2, the recombinant collagen, trehalose, and phosphate buffer (0.1M, pH 7.0) were stirred at 400 r / min for 1 hour, sodium ascorbate was added, and stirring was continued at 400 r / min for 8 hours. The temperature was controlled at 40°C, and the temperature was lowered to terminate the reaction. The reaction solution was then placed in a dialysis bag for dialysis with a molecular weight cutoff of 1 kDa to obtain a recombinant collagen solution; S2. Place the recombinant collagen solution into a reactor, control the reactor temperature to 100° C., the reactor pressure to 0.11 MPa, and heat treat for 3 hours to obtain recombinant collagen peptide.

[0033] Comparative Example 2 Comparative Example 2 is the same as Example 1, except that maleic anhydride is not grafted onto the modified trehalose during its preparation, as shown below: The modified trehalose in step S1 is prepared by the following method: (1) Trehalose, 4-sulfonic acid calixarene and ethanol solution were mixed at a mass ratio of 6:2:50, and trehalose, 4-sulfonic acid calixarene and 30% ethanol solution were stirred at 500 r / min for 1 h, and the temperature was controlled at 40°C to form a composite solution; (2) The composite solution was mixed with gelatin solution (prepared by mixing gelatin and water at a mass ratio of 1:10 at 40°C at 600 r / min for 40 min) at a mass ratio of 3:1:0.5 among the composite solution, gelatin solution and xylitol. The mixture was stirred at 400 r / min for 30 min, and xylitol was added. The mixture was stirred at 600 r / min for 30 min, kept warm at 50°C for 4 h, and then centrifuged at 12000 r / min for 10 min. The mixture was dried at 40°C for 1 h to obtain modified trehalose.

[0034] Comparative Example 3 Comparative Example 3 is the same as Example 1, except that when preparing the modified trehalose, the trehalose is first compounded with 4-sulfonic acid calixarene and then grafted with maleic anhydride, as shown below: The modified trehalose in step S1 is prepared by the following method: (1) Trehalose, 4-sulfonic acid calixarene and ethanol solution were mixed at a mass ratio of 6:2:50, and trehalose, 4-sulfonic acid calixarene and 30% ethanol solution were stirred at 500 r / min for 1 h, the temperature was controlled at 40°C, and then dried at 40°C for 1 h to obtain a composite; (2) The composite, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid were stirred at 600 r / min for 30 min in a mass ratio of 1:1:20:0.03. p-toluenesulfonic acid was added. Under nitrogen protection, the temperature was controlled at 70°C and the mixture was stirred at 400 r / min for 4 h to obtain a grafted solution. (3) The graft solution and gelatin solution (prepared by mixing gelatin and water at a mass ratio of 1:10 at 40°C at 600 r / min for 40 min) were stirred at 400 r / min for 30 min, with the graft solution, gelatin solution and xylitol being 3:1:0.5. Xylitol was then added and the mixture was stirred at 600 r / min for 30 min. The mixture was kept warm at 50°C for 4 h, and then centrifuged at 12,000 r / min for 10 min. The mixture was dried at 40°C for 1 h to obtain modified trehalose.

[0035] Comparative Example 4 Comparative Example 4 is the same as Example 1, except that the composite solution, gelatin solution, and xylitol react simultaneously during the preparation of the modified trehalose, as shown below: The modified trehalose in step S1 is prepared by the following method: (1) Trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid were stirred and mixed at 600 r / min for 30 min in a mass ratio of trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid of 1:1:20:0.03. P-toluenesulfonic acid was added. Under nitrogen protection, the temperature was controlled at 70°C, and the mixture was stirred and mixed at 400 r / min for 4 h. The mixture was centrifuged at 10,000 r / min for 20 min, washed with water, and dried at 40°C for 1 h to obtain a grafted product. (2) The grafted product, 4-sulfonic acid calixarene and ethanol solution were mixed at a mass ratio of 6:2:50, and the grafted product, 4-sulfonic acid calixarene and 30% ethanol solution were stirred at 500 r / min for 1 h and the temperature was controlled at 40°C to form a composite solution; (3) The composite solution, gelatin solution and xylitol were mixed at a mass ratio of 3:1:0.5, and the composite solution, gelatin solution (prepared by mixing gelatin and water at a mass ratio of 1:10 at 40°C at 600 r / min for 40 min) and xylitol were stirred at 400 r / min for 30 min, kept at 50°C for 4 h, and then centrifuged at 12000 r / min for 10 min, and dried at 40°C for 1 h to obtain modified trehalose.

[0036] Comparative Example 5 Comparative Example 5 is the same as Example 1, except that when preparing the modified trehalose, the graft is first cross-linked and then compounded with 4-sulfonic acid calixarene, as shown below: The modified trehalose in step S1 is prepared by the following method: (1) Trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid were stirred and mixed at 600 r / min for 30 min in a mass ratio of trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid of 1:1:20:0.03. P-toluenesulfonic acid was added. Under nitrogen protection, the temperature was controlled at 70°C, and the mixture was stirred and mixed at 400 r / min for 4 h. The mixture was centrifuged at 10,000 r / min for 20 min, washed with water, and dried at 40°C for 1 h to obtain a grafted product. (2) According to the mass ratio of graft solution, gelatin solution and xylitol being 3:1:0.5, the graft solution (graft and anhydrous ethanol at a mass ratio of 1:10 were mixed at 600 r / min for 30 min) was mixed with gelatin solution (gelatin and water at a mass ratio of 1:10 were mixed at 400 r / min for 40 min) at 400 r / min, and then xylitol was added. The mixture was stirred at 600 r / min for 30 min, kept warm at 50°C for 4 h, and then centrifuged at 12000 r / min for 10 min. The functional material was dried at 40°C for 1 h. (3) The functional substance, 4-sulfonic acid calixarene and ethanol solution were mixed at a mass ratio of 6:2:50, and the functional substance, 4-sulfonic acid calixarene and 30% ethanol solution were stirred at 500 r / min for 1 hour, the temperature was controlled at 40°C, and then dried at 40°C for 1 hour to obtain modified trehalose.

[0037] Performance testing Skin care products were prepared using the recombinant collagen peptides prepared in Examples 1-5 and Comparative Examples 1-5, and comprehensive performance tests were performed.

[0038] Skin care product ingredients: 10% recombinant collagen peptide, 5% glycerin, 2% ethylene glycol, 5% grape seed oil, 2% olive oil, 0.2% phenoxyethanol, 0.15% polysorbate, 0.5% green tea extract, 1.5% vitamin C, and the rest water.

[0039] 1. Moisturizing performance test Several subjects aged 40-55 were randomly divided into 9 groups, each consisting of 10 subjects (5 males and 5 females). The subjects cleansed and dried their faces, then applied approximately 1.5g of the above cosmetics to their faces. The test was then conducted in the same indoor environment.

[0040] The skin moisture content test method is: use the German CK company's skin moisture tester (model: CM825-MDD) to measure the facial skin moisture content before using skin care products and 5 hours and 10 hours after use, and record the results. Calculate the increase rate of skin moisture content. The results are shown in Table 1.

[0041] Skin moisture increase rate = 100% × (skin moisture after use - skin moisture before use) / skin moisture before use.

[0042] 2. Skin elasticity test Several subjects aged 40-55 were randomly divided into nine groups, each consisting of 10 participants (five males and five females). Each participant applied approximately 1.5 g of the aforementioned cosmetics daily to a test area of facial skin. A skin elasticity tester (model: MPA580) from German company CK was used to measure the increase in skin elasticity before use and after one and three months of use. The results are recorded and shown in Table 2.

[0043] Skin elasticity increase rate = 100% × (skin elasticity value after use - skin elasticity value before use) / skin elasticity value before use.

[0044] Table 1 Table 2 As shown in Tables 1 and 2, the skin care products prepared using the recombinant collagen peptides of Examples 1-5 showed significantly higher increases in skin moisture content and skin elasticity than the skin care products prepared using the recombinant collagen peptides of Comparative Examples 1-5. Comparative Examples 1-5, when compared with Example 1, demonstrate that changes in the raw materials and preparation process of modified trehalose can reduce the stability and transdermal efficacy of the recombinant collagen peptides. The recombinant collagen peptides prepared in this application exhibit excellent moisturizing, hydration, and skin elasticity-enhancing effects.

[0045] 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 non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing recombinant collagen peptide, characterized in that: The specific steps include the following: S1. Stir and mix recombinant collagen, modified trehalose, and phosphate buffer, add sodium ascorbate, continue stirring and mixing, heat the reaction, cool down to terminate the reaction, and then place the reaction solution into a dialysis bag for dialysis with a molecular weight cut-off of 1-3 kDa to obtain a recombinant collagen solution; S2. Place the recombinant collagen solution into a reactor and heat treat for 3-5 hours to obtain recombinant collagen peptides.

2. The method for preparing a recombinant collagen peptide according to claim 1, characterized in that: In step S1, the mass ratio of recombinant collagen, modified trehalose, phosphate buffer, and sodium ascorbate is 1:1-3:20:0.

2.

3. The method for preparing a recombinant collagen peptide according to claim 1, characterized in that: In step S2, the temperature of the reactor is 100-120° C., and the pressure of the reactor is 0.11-0.15 MPa.

4. The method for preparing a recombinant collagen peptide according to claim 1, characterized in that: The modified trehalose is prepared by the following method: (1) Stir and mix trehalose, maleic anhydride and anhydrous ethanol, add p-toluenesulfonic acid, control the temperature at 70-80°C under nitrogen protection, continue stirring and mixing for 4-6 hours, centrifuge, wash with water, and dry to obtain the grafted product; (2) Stirring the grafted product, 4-sulfonic acid calixarene and ethanol solution for 1-2 hours at a temperature of 40-50°C to form a composite solution; (3) The composite solution and gelatin solution were stirred and mixed, and then xylitol was added, and the stirring was continued, and the mixture was kept warm for 4-6 hours, and then centrifuged and dried to obtain modified trehalose.

5. The method for preparing a recombinant collagen peptide according to claim 4, characterized in that: In the step (1), the mass ratio of trehalose, maleic anhydride, anhydrous ethanol and p-toluenesulfonic acid is 1:1-3:20:0.

03.

6. The method for preparing a recombinant collagen peptide according to claim 4, characterized in that: In the step (2), the mass ratio of the grafted product, 4-sulfonic acid calixarene and the ethanol solution is 6-8:2-4:

50.

7. The method for preparing a recombinant collagen peptide according to claim 4, characterized in that: In step (3), the mass ratio of the composite solution, gelatin solution and xylitol is 3-5:1-2:0.

5.

8. The method for preparing a recombinant collagen peptide according to claim 4, characterized in that: The gelatin solution in step (3) is prepared by the following method: gelatin and water are stirred and mixed at a mass ratio of gelatin to water of 1:

10.

9. A recombinant collagen peptide, characterized in that: The recombinant collagen peptide is prepared by the preparation method of any one of claims 1 to 8.

10. A use of the recombinant collagen peptide according to claim 9, characterized in that: The recombinant collagen peptide is used for preparing skin care products.

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