阅读说明：本技术 一种含铜络合物的制备方法 (Preparation method of copper-containing complex ) 是由 邹国栋 郑红云 凌天阳 于 2020-12-04 设计创作，主要内容包括：本发明通过将胶原蛋白水解产物与氢氧化铜反应得到一种含铜络合物,制备步骤如下：以氢氧化钠水溶液溶解明胶,滤布滤去不溶物；用盐酸调节pH,加碱性蛋白酶反应；将胶液升温反应；用盐酸调至pH,加中性蛋白酶反应；用盐酸将胶液调至pH,加入酸性蛋白酶反应；用氢氧化钠将胶液调至pH,加入氢氧化铜,搅拌后用滤布除去不溶物；加无水乙醇,搅拌后用滤布除去不溶物；继续加入无水乙醇,搅拌后用滤布过滤收集不溶物,用乙醇冲洗后真空干燥；将干燥产物溶于水,减压旋干得到产品。这种络合物具有和GHK-Cu类似的生理活性,且制造成本更低,可应用于多种皮肤和毛发护理产品中。(The invention obtains a copper-containing complex by reacting collagen hydrolysate with copper hydroxide, and the preparation steps are as follows: dissolving gelatin with sodium hydroxide water solution, filtering insoluble substances with filter cloth; adjusting pH with hydrochloric acid, and adding alkaline protease for reaction; heating the glue solution for reaction; adjusting pH with hydrochloric acid, and adding neutral protease for reaction; adjusting the pH value of the glue solution to be hydrochloric acid, and adding acid protease for reaction; adjusting the pH value of the glue solution to be the same with sodium hydroxide, adding copper hydroxide, stirring, and removing insoluble substances by using filter cloth; adding anhydrous ethanol, stirring, and removing insoluble substances with filter cloth; adding anhydrous ethanol, stirring, filtering with filter cloth, collecting insoluble substance, washing with ethanol, and vacuum drying; dissolving the dried product in water, and carrying out rotary drying under reduced pressure to obtain the product. The complex has similar physiological activity with GHK-Cu, has lower manufacturing cost, and can be applied to various skin and hair care products.)

1. A preparation method of a copper-containing complex is characterized by comprising the following steps:

(1) adding edible gelatin into 1% sodium hydroxide water solution at 85 deg.C, stirring for 24 hr, filtering out insoluble substances with filter cloth, and cooling the filtrate to 50 deg.C;

(2) adjusting the pH value of the glue solution to 9.5 by using hydrochloric acid, adding alkaline protease, and reacting for 4 hours at 60 ℃;

(3) heating the glue solution to 90 ℃, preserving the heat for 1h, and then cooling to 40 ℃;

(4) adjusting the pH value of the glue solution to 7.8 by using hydrochloric acid, adding neutral protease, and reacting for 4 hours at 40 ℃;

(5) adjusting the pH value of the glue solution to 3.0 by using hydrochloric acid, adding acid protease, and reacting for 4 hours at 40 ℃;

(6) adjusting the pH of the glue solution to 7.0 with sodium hydroxide, adding copper hydroxide, stirring for 8h, and removing insoluble substances with filter cloth;

(7) adding absolute ethyl alcohol according to the volume of 1:4 of the glue solution, stirring for 1h, and removing insoluble substances by using filter cloth;

(8) continuously adding absolute ethyl alcohol until the volume of the ethyl alcohol and the glue solution reaches 1:1, stirring for 1h, filtering by using filter cloth to collect insoluble substances, washing by using ethyl alcohol, and then carrying out vacuum drying;

(9) dissolving the dried product in water, and carrying out decompression spin-drying at the temperature of 60 ℃ to obtain the product.

2. The method according to claim 1, wherein the edible gelatin is added in an amount of 3-5% by mass in step (1).

3. The method for preparing copper-containing complex according to claim 1, wherein in the step (2), the pH of the glue solution is in the range of 8.0-11.0, preferably, the pH is in the range of 9.0-10.0, and the mass percent of the alkaline protease added is 0.1-0.3%.

4. The method for preparing copper-containing complex according to claim 1, wherein in the step (4), the pH of the glue solution is in the range of 6.5-8.5, preferably, the pH is in the range of 7.5-8.0, and the mass percent of the neutral protease is 0.2-0.35%.

5. The method for preparing copper-containing complex according to claim 1, wherein in the step (5), the pH of the glue solution is in the range of 2.0-4.0, preferably, the pH is in the range of 2.5-3.5, and the mass percent of the acid protease added is 0.2-0.4%.

6. The method according to claim 1, wherein in step (6), the copper hydroxide is added in an amount of 1-2% by weight based on the total weight of the binder solution.

Technical Field

The invention belongs to the field of biological materials, and relates to a preparation method of a copper-containing complex.

Background

Copper is a trace element necessary for human bodies, is an accessory factor of a plurality of enzymes, is also a necessary condition for synthesizing collagen and elastin, and can tighten skin, resist damage of free radicals and even play a repairing role in cooperation with vitamin E. Free copper ions are difficult to enter the skin through external coating to take effect, and people mainly play a role by compounding copper and biomass molecules. For example, GHK-Cu is a complex product of copper and tripeptide, and experiments show that the combination of copper and tripeptide can play a plurality of roles of stimulating the formation of collagen and elastin, increasing the generation of intercellular skin mucus, assisting antioxidant enzyme SOD, expanding hair follicles and accelerating hair growth, and the like.

At present, GHK-Cu is mainly prepared by a manual synthesis method, and the existing method has higher cost and limits the application of the GHK-Cu in beauty products. Experiments show that GHK-Cu mainly realizes the functions by inducing fibroblast to generate Matrix Metalloenzymes (MMPs), and other materials used as copper ionophores can also realize similar effects.

The invention provides a method for generating small molecular peptide by collagen hydrolysis and further complexing with copper ions, which produces the small molecular peptide with the molecular weight of 500-2000 at relatively low cost, has high bioavailability of the small molecular peptide in the molecular weight region, and can replace the existing synthetic small peptide as a copper ion carrier.

Disclosure of Invention

The technical scheme of the invention is as follows:

a preparation method of a copper-containing complex comprises the following steps:

(1) adding edible gelatin into 1% sodium hydroxide water solution at 85 deg.C, stirring for 24 hr, filtering out insoluble substances with filter cloth, and cooling the filtrate to 50 deg.C;

(2) adjusting the pH value of the glue solution to 9.5 by using hydrochloric acid, adding alkaline protease, and reacting for 4 hours at 60 ℃;

(3) heating the glue solution to 90 ℃, preserving the heat for 1h, and then cooling to 40 ℃;

(4) adjusting the pH value of the glue solution to 7.8 by using hydrochloric acid, adding neutral protease, and reacting for 4 hours at 40 ℃;

(5) adjusting the pH value of the glue solution to 3.0 by using hydrochloric acid, adding acid protease, and reacting for 4 hours at 40 ℃;

(6) adjusting the pH of the glue solution to 7.0 with sodium hydroxide, adding copper hydroxide, stirring for 8h, and removing insoluble substances with filter cloth;

(7) adding absolute ethyl alcohol according to the volume of 1:4 of the glue solution, stirring for 1h, and removing insoluble substances by using filter cloth;

(8) adding anhydrous ethanol until the volume of ethanol and the glue solution reaches 1:1, stirring for 1h, filtering with filter cloth to collect insoluble substances,

washing with ethanol, and vacuum drying;

(9) dissolving the dried product in water, and carrying out decompression spin-drying at the temperature of 60 ℃ to obtain the product.

Further, in the step (1), the edible gelatin is added in a mass percentage of 3-5%.

Further, in the step (2), the pH range of the glue solution is 8.0-11.0, preferably, the pH range is 9.0-10.0, and the adding mass percentage of the alkaline protease is 0.1-0.3%.

Further, in the step (4), the pH range of the glue solution is 6.5-8.5, preferably, the pH range is 7.5-8.0, and the mass percent of the neutral protease is 0.2-0.35%.

Further, in the step (5), the pH range of the glue solution is 2.0-4.0, preferably, the pH range is 2.5-3.5, and the adding mass percentage of the acid protease is 0.2-0.4%.

Further, in the step (6), the adding amount of the copper hydroxide is 1-2% of the total weight of the glue solution.

Drawings

FIG. 1 shows a comparison of copper ions (10) containing the same equivalent weight^-9mol/L) of copper acetate, GHK-Cu and the product of experiment 1 on the release of Matrix Metalloenzymes (MMPs) from fibroblasts.

Detailed Description

The invention is further illustrated by the following examples.

Example 1

(1) Adding 500g edible gelatin into 10L 1% sodium hydroxide water solution at 85 deg.C, stirring for 24 hr, filtering out insoluble substances with filter cloth, and cooling the filtrate to 60 deg.C;

(2) adjusting pH of the gel to 9.0 with hydrochloric acid, adding 30g alkaline protease (Bacillus licheniformis), and reacting at 60 deg.C for 4 hr; heating the glue solution to 90 ℃, preserving the heat for 1h, and then cooling to 40 ℃;

(3) adjusting the pH value of the glue solution to 7.8 by using hydrochloric acid, adding 35g of neutral protease (bacillus subtilis), and reacting for 4 hours at 40 ℃;

(4) adjusting pH of the gel to 3.0 with hydrochloric acid, adding 40 acid protease (Aspergillus niger), and reacting at 40 deg.C for 4 hr;

(5) adjusting the pH of the glue solution to 7.0 with sodium hydroxide, adding 100g of copper hydroxide, stirring for 8h, and removing insoluble substances with a filter cloth;

(6) adding 2.5L anhydrous ethanol, stirring for 1 hr, and removing insoluble substances with filter cloth;

(7) continuously adding 7.5L of absolute ethyl alcohol, stirring for 1h, filtering with filter cloth, collecting insoluble substances, washing with ethanol, and vacuum drying;

(8) dissolving the dried product in water, and carrying out decompression spin-drying at the temperature of 60 ℃ to obtain the product.

Example 2

(1) Adding 500g edible gelatin into 10L 1% sodium hydroxide water solution at 85 deg.C, stirring for 24 hr, filtering out insoluble substances with filter cloth, and cooling the filtrate to 60 deg.C;

(2) adjusting pH of the gel to 9.0 with hydrochloric acid, adding 30g alkaline protease (Bacillus licheniformis), and reacting at 60 deg.C for 4 hr;

(3) heating the glue solution to 90 ℃, preserving the heat for 1h, and then cooling to 40 ℃;

(4) adjusting pH of the gel to 7.0 with hydrochloric acid, adding 35g neutral protease (papain), and reacting at 40 deg.C for 4 hr;

(5) adjusting pH of the gel to 3.0 with hydrochloric acid, adding 40 acid protease (Aspergillus niger), and reacting at 40 deg.C for 4 hr;

(6) adjusting the pH of the glue solution to 7.0 with sodium hydroxide, adding 100g of copper hydroxide, stirring for 8h, and removing insoluble substances with a filter cloth;

(7) adding 2.5L anhydrous ethanol, stirring for 1 hr, and removing insoluble substances with filter cloth;

(8) continuously adding 7.5L of absolute ethyl alcohol, stirring for 1h, filtering with filter cloth, collecting insoluble substances, washing with ethanol, and vacuum drying;

(9) dissolving the dried product in water, and carrying out decompression spin-drying at the temperature of 60 ℃ to obtain the product.

Example 3

(1) Adding 500g edible gelatin into 10L 1% sodium hydroxide water solution at 85 deg.C, stirring for 24 hr, filtering out insoluble substances with filter cloth, and cooling the filtrate to 60 deg.C;

(2) adjusting pH of the gel to 9.0 with hydrochloric acid, adding 30g alkaline protease (Bacillus licheniformis), and performing microwave treatment at 60 deg.C for 15 min;

(3) heating the glue solution to 90 ℃, preserving the heat for 1h, and then cooling to 40 ℃;

(4) adjusting pH of the gel to 7.8 with hydrochloric acid, adding 35g neutral protease (Bacillus subtilis), and performing microwave action at 40 deg.C for 15 min;

(5) adjusting pH of the gel to 3.0 with hydrochloric acid, adding 40 acidic protease (Aspergillus niger), and microwave treating at 40 deg.C for 15 min;

(6) adjusting the pH of the glue solution to 7.0 with sodium hydroxide, adding 200g of copper hydroxide, stirring for 8h, and removing insoluble substances with a filter cloth;

(7) adding 2.5L anhydrous ethanol, stirring for 1 hr, and removing insoluble substances with filter cloth;

(8) continuously adding 7.5L of absolute ethyl alcohol, stirring for 1h, filtering with filter cloth, collecting insoluble substances, washing with ethanol, and vacuum drying;

(9) dissolving the dried product in water, and carrying out decompression spin-drying at the temperature of 60 ℃ to obtain the product.

Comparative example 1

(1) Adding 500g edible gelatin into 10L 1% sodium hydroxide water solution at 85 deg.C, stirring for 24 hr, filtering out insoluble substances with filter cloth, and cooling the filtrate to 60 deg.C;

(2) adjusting pH of the gel to 9.0 with hydrochloric acid, adding 30g alkaline protease (Bacillus licheniformis), and reacting at 60 deg.C for 4 hr;

(3) heating the glue solution to 90 ℃, preserving the heat for 1h, and then cooling to 40 ℃;

(4) adjusting the pH value of the glue solution to 7.8 by using hydrochloric acid, adding 35g of neutral protease (bacillus subtilis), and reacting for 4 hours at 40 ℃;

(5) adjusting the pH of the gel to 7.0 with hydrochloric acid, adding 100g of copper hydroxide, stirring for 8h, and removing insoluble substances with filter cloth;

(6) adding 2.5L anhydrous ethanol, stirring for 1 hr, and removing insoluble substances with filter cloth;

(7) continuously adding 7.5L of absolute ethyl alcohol, stirring for 1h, filtering with filter cloth, collecting insoluble substances, washing with ethanol, and vacuum drying;

(8) dissolving the dried product in water, and carrying out decompression spin-drying at the temperature of 60 ℃ to obtain the product.

Comparative example 2

(1) Adding 500g edible gelatin into 10L 85 deg.C water, stirring for 24 hr, and cooling the filtrate to 60 deg.C;

(2) adjusting pH of the gel to 9.0 with sodium hydroxide, adding 30g alkaline protease (Bacillus licheniformis), and reacting at 60 deg.C for 4 hr;

(3) heating the glue solution to 90 ℃, preserving the heat for 1h, and then cooling to 40 ℃;

(4) adjusting the pH value of the glue solution to 7.8 by using hydrochloric acid, adding 35g of neutral protease (bacillus subtilis), and reacting for 4 hours at 40 ℃;

(5) adjusting pH of the gel to 3.0 with hydrochloric acid, adding 40 acid protease (Aspergillus niger), and reacting at 40 deg.C for 4 hr;

(6) adjusting the pH of the glue solution to 7.0 with sodium hydroxide, adding 100g of copper hydroxide, stirring for 8h, and removing insoluble substances with a filter cloth;

(7) adding 2.5L anhydrous ethanol, stirring for 1 hr, and removing insoluble substances with filter cloth;

(8) continuously adding 7.5L of absolute ethyl alcohol, stirring for 1h, filtering with filter cloth, collecting insoluble substances, washing with ethanol, and vacuum drying;

(9) dissolving the dried product in water, and carrying out decompression spin-drying at the temperature of 60 ℃ to obtain the product.

Comparative example 3

(1) Adding 500g edible gelatin into 10L 1% sodium hydroxide water solution at 85 deg.C, stirring for 24 hr, filtering out insoluble substances with filter cloth, and cooling the filtrate to 60 deg.C;

(2) adjusting the pH value of the glue solution to 7.8 by using hydrochloric acid, adding 35g of neutral protease (bacillus subtilis), and reacting for 4 hours at 40 ℃;

(3) heating the glue solution to 90 ℃, preserving the heat for 1h, and then cooling to 40 ℃;

(4) adjusting pH of the gel to 3.0 with hydrochloric acid, adding 40 acid protease (Aspergillus niger), and reacting at 40 deg.C for 4 hr;

(5) adjusting pH of the gel to 9.0 with sodium hydroxide, adding 30g alkaline protease (Bacillus licheniformis), and reacting at 60 deg.C for 4 hr;

(6) adjusting the pH of the gel to 7.0 with hydrochloric acid, adding 100g of copper hydroxide, stirring for 8h, and removing insoluble substances with filter cloth;

(7) adding 2.5L anhydrous ethanol, stirring for 1 hr, and removing insoluble substances with filter cloth;

(8) continuously adding 7.5L of absolute ethyl alcohol, stirring for 1h, filtering with filter cloth, collecting insoluble substances, washing with ethanol, and vacuum drying;

(9) dissolving the dried product in water, and carrying out decompression spin-drying at the temperature of 60 ℃ to obtain the product.

The experimental results are as follows:

1. the yields of the different molecular weight products obtained in the above experimental examples and comparative examples are as follows:

molecular weight region

Experimental example 1

Experimental example 2

Experimental example 3

Comparative example 1

Comparative example 2

Comparative example 3

Mw＝690

3.83％

3.21％

4.64％

-

0.14％

1.73％

Mw＝940

5.15％

4.90％

6.23％

0.47％

2.39％

3.54％

Mw＝1170

9.51％

8.62％

10.85％

1.81％

5.03％

7.21％

Mw＝1390

10.24％

11.44％

11.37％

5.12％

7.90％

9.51％

Mw＝1710

9.12％

11.67％

12.06％

8.28％

8.61％

8.63％

Mw＝1890

7.77％

9.38％

10.23％

8.81％

7.97％

8.15％

Mw>2000

12.63％

15.29％

17.05％

15.36％

16.07％

13.45％

2. Comparison of copper ions containing the same equivalent weight (10)^-9mol/L) of copper acetate, GHK-Cu and the product of experiment 1, the results are shown in FIG. 1.