CN112813123A - Preparation method of copper-containing complex - Google Patents

Preparation method of copper-containing complex Download PDF

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CN112813123A
CN112813123A CN202011406585.9A CN202011406585A CN112813123A CN 112813123 A CN112813123 A CN 112813123A CN 202011406585 A CN202011406585 A CN 202011406585A CN 112813123 A CN112813123 A CN 112813123A
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glue solution
adjusting
filter cloth
stirring
copper
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邹国栋
郑红云
凌天阳
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Suzhou Aibomaier New Material Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

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.

Description

Preparation method of copper-containing complex
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.

Claims (6)

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.
CN202011406585.9A 2020-12-04 2020-12-04 Preparation method of copper-containing complex Pending CN112813123A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102260727A (en) * 2011-06-24 2011-11-30 申铉日 Preparation method of low-molecule aquatic collagen peptide
CN105567771A (en) * 2016-01-14 2016-05-11 安徽生物肽产业研究院有限公司 Pigskin small peptide chelate and preparation method and application thereof
CN105695548A (en) * 2016-03-30 2016-06-22 蔡庭守 Preparation method of donkey-hide gelatin small molecular peptide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102260727A (en) * 2011-06-24 2011-11-30 申铉日 Preparation method of low-molecule aquatic collagen peptide
CN105567771A (en) * 2016-01-14 2016-05-11 安徽生物肽产业研究院有限公司 Pigskin small peptide chelate and preparation method and application thereof
CN105695548A (en) * 2016-03-30 2016-06-22 蔡庭守 Preparation method of donkey-hide gelatin small molecular peptide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CRISTINA MEGÍAS, ET AL.: "Production of copper-chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin", LWT - FOOD SCIENCE AND TECHNOLOGY, vol. 41, pages 1973 - 1977, XP024528229, DOI: 10.1016/j.lwt.2007.11.010 *
刘温等: "多肽金属元素螯合物研究进展", 食品与发酵工业, vol. 40, no. 4, pages 142 - 146 *

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