Small molecule recombinant collagen peptide and preparation method thereof

Abstract

The present invention discloses a small-molecule recombinant collagen peptide and a method for preparing the same. The method involves placing an aqueous solution of recombinant collagen with a molecular weight of 55 kD into a reactor and heat-treating the reactor to a temperature of 121-125°C using steam heating and pressure control to produce a small-molecule recombinant collagen peptide with an average molecular weight of 1000 Da and a molecular weight of 4000 Da. While maintaining the good biological activity of the original recombinant collagen, the small-molecule recombinant collagen peptide produced by the present invention exhibits improved transdermal absorption, a short onset of action, and rapid efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetic preparations and relates to a small molecule recombinant collagen peptide and a preparation method thereof. Background Art

[0002] Collagen is a biopolymer, a fibrous protein, the primary component of animal connective tissue and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of total protein. It is closely involved in tissue formation and maturation, intercellular communication, joint lubrication, wound healing, calcification, blood coagulation, and aging. Collagen is one of the most critical raw materials in the biotechnology industry, with extensive applications in medical materials, cosmetics, and the food industry. Collagen companies source their products from two sources: animal (including pig, cattle, and chicken) and fish collagen; and recombinant collagen obtained through genetic engineering. Recombinant collagen, due to its excellent biocompatibility, lack of viral hazards, and high biological activity, is now widely used in daily cosmetics, medical plastic surgery, and medical devices.

[0003] According to the "Technical Guidelines for Cosmetic Safety Assessment (2021 Edition)" and "Cosmetic Dermatology: Products and Methods," the minimum permeability requirement for skincare products is, in most cases, a molecular weight of 1,000 Daltons. Currently, the molecular weight of recombinant collagen is generally greater than 10,000 Daltons, which affects its transdermal absorption and its effectiveness within the skin. Summary of the Invention

[0004] The purpose of the present invention is to provide a small molecule recombinant collagen peptide and a preparation method thereof.

[0005] The technical solutions for achieving the purpose of the present invention are as follows:

[0006] The preparation method of small molecule recombinant collagen peptide comprises the following steps:

[0007] The aqueous solution of recombinant collagen is added to a reactor, and the reactor temperature is controlled at 121-125° C. by steam heating and pressure, and heat treated for 260-545 minutes to obtain a small molecule recombinant collagen peptide with an average molecular weight of 1000D < 4000D. The recombinant collagen is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No. 5021.

[0008] Preferably, the concentration of the aqueous solution of recombinant collagen is 59 to 64 g / L. In a specific embodiment of the present invention, the concentration of the aqueous solution of recombinant collagen is 59.38 to 63.09 g / L.

[0009] Preferably, the reactor temperature is controlled at 125° C., the pressure is 0.16-0.18 MPa, and the heat treatment time is 315-325 min to obtain a small molecule recombinant collagen peptide with an average molecular weight of 1000D < 2000D.

[0010] Preferably, the reactor temperature is controlled at 121° C., the pressure is controlled at 0.10-0.12 MPa, and the heat treatment time is 525-545 min to obtain a small molecule recombinant collagen peptide with an average molecular weight of 1000D < 2000D.

[0011] Preferably, the reactor temperature is controlled at 125° C., the pressure is 0.16-0.18 MPa, and the heat treatment time is 260-300 min to obtain a small molecule recombinant collagen peptide with an average molecular weight of 2000D < 4000D.

[0012] Preferably, the reactor temperature is controlled at 125° C., the pressure is 0.16-0.18 MPa, and the heat treatment time is 260-270 min to obtain a small molecule recombinant collagen peptide with an average molecular weight of 3000D < 4000D.

[0013] Preferably, the reactor temperature is controlled at 121° C., the pressure is controlled at 0.10-0.12 MPa, and the heat treatment time is 420-500 min to obtain a small molecule recombinant collagen peptide with an average molecular weight of 2000D < 4000D.

[0014] Preferably, the reactor temperature is controlled at 121° C., the pressure is controlled at 0.10-0.12 MPa, and the heat treatment time is 420-450 min to obtain a small molecule recombinant collagen peptide with an average molecular weight of 3000D < 4000D.

[0015] Furthermore, the present invention also provides a small molecule recombinant collagen peptide with an average molecular weight of 1000D < 4000D prepared by the above preparation method.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The present invention uses highly water-soluble recombinant collagen produced by fermentation of Pichia pastoris, deposited with CGMCC No. 5021, as raw material. By controlling the heat treatment temperature, pressure, and time, small-molecule recombinant collagen peptides of varying molecular weights are obtained. Compared to the original recombinant collagen, the small-molecule recombinant collagen peptides prepared by the present invention, with an average molecular weight of 1000D < 4000D, have better transdermal absorption when used as skin care products while maintaining good biological activity. Therefore, they have the advantages of rapid onset, can rapidly reduce pore volume, and rapidly tighten and remove wrinkles. They can also rapidly improve texture roughness and reduce bulge volume, thereby repairing the skin. They can also rapidly increase hemoglobin, thereby reducing redness. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a logarithmic curve of peak time-relative molecular weight of small molecule recombinant collagen peptide.

[0019] Figure 2 The pore volume changes of each group are shown in Figure 4.

[0020] Figure 3 The graph shows the changes in overall wrinkle size in each group.

[0021] Figure 4 Figure 3 is a graph showing the changes in skin protrusion volume in each group.

[0022] Figure 5 The texture roughness change diagram of each group.

[0023] Figure 6 The graph shows the changes in hemoglobin in each group. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0025] The recombinant collagen used in the present invention is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No. 5021, has a molecular weight of 55 kD, and has been fully disclosed in Chinese Patent No. 201110327865.5.

[0026] Example 1

[0027] 5L of an aqueous solution of recombinant collagen with a concentration of 59.38g / L was put into a 10L reactor, heated by steam to a reactor temperature of 125°C, the reactor pressure was controlled at 0.16-0.18MPa, and heat treated for 325min. After the heat treatment, it was cooled to room temperature to obtain a small molecule recombinant collagen peptide with an average molecular weight of 1701D.

[0028] The molecular weight of the small molecule recombinant collagen peptide was detected by HPLC using a TSKgel G2000 SWXL column with a mobile phase of acetonitrile: water: trifluoroacetic acid = 40:60:0.05. A standard curve was drawn using cytochrome C (12355D), bacillus enzyme (1422D), tyrosine-tyrosine-arginine (451D), tyrosine-tyrosine-tyrosine (189D), and BSA (66000D) of known molecular weight (the logarithm of the relative molecular mass was plotted against the retention time to obtain the relative molecular mass calibration curve and equation). During the implementation process, the sample chromatogram and its data were calculated and processed according to the relative molecular mass calibration curve equation to obtain the relative molecular mass size and distribution range of the small molecule recombinant collagen peptide obtained after different process treatments.

[0029] Examples 2-3

[0030] The heat treatment temperature and pressure of Examples 2 to 3 are the same as those of Example 1. The specific experimental parameters are shown in Table 1.

[0031] Example 4

[0032] 5L of an aqueous solution of recombinant collagen with a concentration of 62.28g / L was put into a 10L reactor, heated by steam to a reactor temperature of 121°C, the reactor pressure was controlled at 0.10-0.12MPa, and heat treated for 545min. After the heat treatment, it was cooled to room temperature to obtain a small molecule recombinant collagen peptide with an average molecular weight of 1726D.

[0033] Examples 5-6

[0034] The heat treatment temperature and pressure of Examples 5 to 6 are the same as those of Example 4. The specific experimental parameters are shown in Table 1.

[0035] Table 1 Experimental conditions of Examples 1 to 6 and average molecular weight of the obtained small molecule recombinant collagen peptides

[0036]

[0037] Example 7

[0038] 5L of an aqueous solution of recombinant collagen with a concentration of 61.42g / L was put into a 10L reactor, heated by steam to a reactor temperature of 125°C, the reactor pressure was controlled at 0.16-0.18MPa, and heat treated for 270min. After the heat treatment, it was cooled to room temperature to obtain a small molecule recombinant collagen peptide with an average molecular weight of 3452D.

[0039] Examples 8-9

[0040] The heat treatment temperature and pressure of Examples 2 to 3 are the same as those of Example 7. The specific experimental parameters are shown in Table 2.

[0041] Example 10

[0042] 5L of an aqueous solution of recombinant collagen with a concentration of 63.09g / L was put into a 10L reactor, heated by steam to a reactor temperature of 121°C, the reactor pressure was controlled at 0.10-0.12MPa, and heat treated for 545min. After the heat treatment, it was cooled to room temperature to obtain a small molecule recombinant collagen peptide with an average molecular weight of 3369D.

[0043] Examples 11-12

[0044] The heat treatment temperature and pressure of Examples 11 to 12 are the same as those of Example 10. The specific experimental parameters are shown in Table 2.

[0045] Table 2 Experimental conditions of Examples 7 to 12 and average molecular weight of the obtained small molecule recombinant collagen peptides

[0046]

[0047] As shown in the examples, controlling the heat treatment temperature at 125°C significantly reduces the reaction time, but the equipment operating pressure must reach 0.16-0.18 MPa, which places high demands on the equipment. In specific implementations, appropriate temperature and pressure can be selected based on the equipment conditions to control the average molecular weight of the small molecule recombinant collagen peptide.

[0048] Comparative Example 1

[0049] The heat treatment temperature and pressure of Comparative Example 1 were the same as those of Example 7, and the specific experimental parameters are shown in Table 3. A small molecule recombinant collagen peptide with an average molecular weight of 14458D was obtained.

[0050] Comparative Example 2

[0051] The heat treatment temperature and pressure of Comparative Example 2 were the same as those of Example 7, and the specific experimental parameters are shown in Table 3. A small molecule recombinant collagen peptide with an average molecular weight of 823D was obtained.

[0052] Table 3 Experimental conditions of Comparative Examples 1-2 and average molecular weight of the obtained small molecule recombinant collagen peptides

[0053]

[0054] Application Examples

[0055] Volunteer trials compared the efficacy of recombinant collagen (RHC) before and after treatment (small molecule recombinant collagen peptides). The formula of the skincare product used in the volunteer test is shown in Table 4. Besides RHC and four small molecule recombinant collagen peptides of varying average molecular weights, no other active ingredients were present in the formula. For the four small molecule recombinant collagen peptides of varying average molecular weights, those with <1000D represent Comparative Example 2, <2000D represent Example 1, <4000D represent Example 7, and <20000D represent Comparative Example 1.

[0056] Table 4 Formulations of skin care products used in the test

[0057]

[0058] The present invention has five groups of test volunteers, each group has 20 people, and the test lasts for 4 weeks. The relevant data are collected every week, mainly from the aspects of firming (change in pore volume), wrinkle removal (wrinkle size), repair (texture roughness, bulge volume change) and hemoglobin change. The weekly data are compared with the pre-test (0W). The specific results are as follows Figures 2 to 6 shown.

[0059] from Figures 2 to 6 It has been shown that recombinant collagen, processed into small-molecule recombinant collagen peptides with molecular weights of 1000D < average molecular weight < 2000D and 3000D < average molecular weight < 4000D, can rapidly reduce pore volume and wrinkle size, tightening and anti-wrinkle, quickly improve texture roughness and reduce bulge volume, repairing the skin, and increasing hemoglobin, reducing redness. While recombinant collagen can also improve these aspects, the overall effect is slower, likely due to the absorption rate affected by its molecular weight.

[0060] However, the small molecule recombinant collagen peptides with an average molecular weight of less than 1000D and 10000D < average molecular weight < 20000D in Comparative Examples 1 and 2 did not have the effects of Examples 1 and 7. The possible reasons are: for an average molecular weight of less than 1000D, although the absorbability increased, the molecular weight was too small, resulting in partial loss of the efficacy of the recombinant collagen, and therefore it was inferior to the untreated recombinant collagen; for an average molecular weight of 10000D < 20000D, most of the recombinant collagen peptides had a molecular weight that could not achieve rapid transdermal absorption. Therefore, although the effect was better than that of the untreated recombinant collagen, it was inferior to the small molecule recombinant collagen peptides with an average molecular weight of 1000D < 2000D and 3000D < 4000D. In summary, only by processing recombinant collagen into small molecule recombinant collagen peptides with an average molecular weight of 1000D < 4000D according to the process of the embodiment can the purpose of increasing transdermal absorption and rapid onset of effect be achieved, and the small molecule recombinant collagen peptides with an average molecular weight of 1000D < 2000D have the best efficacy.

Claims

1. A method for preparing small molecule recombinant collagen peptide, characterized in that: The following steps are involved: The aqueous solution of recombinant collagen was put into a reactor, and the reactor temperature was controlled to 125°C, the pressure was 0.16-0.18 MPa, and the heat treatment time was 315-325 minutes to obtain a small molecule recombinant collagen peptide with an average molecular weight of 1000D < 2000D; Alternatively, the aqueous solution of recombinant collagen is put into a reactor, and the reactor temperature is controlled to 121°C and the pressure is controlled to 0.10-0.12 MPa by steam heating and pressure treatment for 525-545 minutes to obtain a small molecule recombinant collagen peptide with an average molecular weight of 1000D < 2000D; Alternatively, the aqueous solution of recombinant collagen is put into a reactor, and the reactor temperature is controlled to 125°C, the pressure is 0.16-0.18 MPa, and the heat treatment time is 260-300 minutes by steam heating and pressure control to obtain a small molecule recombinant collagen peptide with an average molecular weight of 2000D < 4000D; Alternatively, the aqueous solution of recombinant collagen is put into a reactor, and the reactor temperature is controlled to 125°C, the pressure is 0.16-0.18 MPa, and the heat treatment time is 260-270 minutes by steam heating and pressure control to obtain a small molecule recombinant collagen peptide with an average molecular weight of 3000D < 4000D; Alternatively, the aqueous solution of recombinant collagen is put into a reactor, and the reactor temperature is controlled to 121°C and the pressure is controlled to 0.10-0.12 MPa by steam heating and pressure treatment for 420-500 minutes to obtain a small molecule recombinant collagen peptide with an average molecular weight of 2000D < 4000D; Alternatively, the aqueous solution of recombinant collagen is put into a reactor, and the reactor temperature is controlled to 121°C and the pressure is controlled to 0.10-0.12 MPa by steam heating and pressure treatment for 420-450 minutes to obtain a small molecule recombinant collagen peptide with an average molecular weight of 3000D < 4000D; The recombinant collagen is prepared from Pichia pastoris with a deposit number of CGMCC No.5021. Pichia pastoris Produced by fermentation.

2. The preparation method according to claim 1, characterized in that The concentration of the recombinant collagen aqueous solution is 59~64g / L.

3. The preparation method according to claim 1, characterized in that The concentration of the aqueous solution of recombinant collagen is 59.38~63.09g / L.

4. A small molecule recombinant collagen peptide with an average molecular weight of 1000D < 4000D obtained by the preparation method according to any one of claims 1 to 3.

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