CN112625122B - Collagen fiber extraction method - Google Patents
Collagen fiber extraction method Download PDFInfo
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- 238000000605 extraction Methods 0.000 title claims abstract description 19
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- 235000020661 alpha-linolenic acid Nutrition 0.000 claims description 4
- 229960004488 linolenic acid Drugs 0.000 claims description 4
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 claims description 4
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a collagen fiber extraction method, which comprises the following steps: s1, taking animal tissues rich in collagen fibers, cutting the animal tissues into slices, and cleaning the slices; s2, digesting the slices with protease digestive juice at 35-37 ℃ for 16-20 hours; s3, digesting for 22-26 hours by using a neutral protease solution, and washing by using a phosphate buffer solution; s4, swelling for 18-22 hours by using an acid solution, uniformly stirring, filtering and collecting a supernatant; s5, neutralizing the supernatant with an alkaline solution for 10-14 hours, and dialyzing to separate out crude collagen fibers; s6, soaking the crude collagen fiber in unsaturated fatty acid for 4-6 hours, and cleaning; s7, soaking the crude collagen fibers in hydrogen peroxide solution for 12-16 hours, and cleaning; soaking in acetone or isopropanol for 6-8 hr, and cleaning; s8, soaking the fabric in a sodium chloride solution for 12-16 hours, cleaning and draining; soaking in 95% ethanol for 10-14 hr to precipitate collagen fiber, and freezing for storage. The extraction method has high extraction rate, and the extracted collagen fiber has low immunogenicity.
Description
Technical Field
The invention relates to a collagen fiber extraction method, and belongs to the technical field of collagen extraction.
Background
Collagen is a functional protein with the highest content and the widest distribution in animal connective tissues, accounts for 25 to 30 percent of the total protein, and even reaches more than 80 percent of certain organisms. The collagen fiber is a fiber formed by collagen, has a triple helix structure, plays an important role in physiological processes of tissue development, intercellular information transmission, cell proliferation, differentiation and the like, has better histocompatibility, cell adaptability and the function of promoting cell proliferation, has biodegradation performance, is gradually degraded into low molecular oligopeptides or amino acids under the action of in vivo collagenase, and is collectively absorbed or discharged out of a body. Due to the special biological advantages of the collagen fibers, the collagen fibers are widely applied to the fields of cosmetics, biological materials and the like.
At present, the methods for extracting collagen fibers mainly include an acid extraction method, an enzyme extraction method, a neutral salt extraction method, an alkali extraction method, and the like. However, for various reasons, these methods extract collagen fibers with high immunogenicity and low extraction rates.
Disclosure of Invention
The invention provides a collagen fiber extraction method, which can effectively solve the problems.
The invention is realized by the following steps:
a collagen fiber extraction method comprises the following steps:
s1, taking animal tissues rich in collagen fibers, cutting the animal tissues into slices, and cleaning the slices;
s2, digesting the slices with alkaline or acidic protease digestion solution at 35-37 ℃ for 16-20 hours;
s3, digesting for 22-26 hours by using a neutral protease solution, and washing by using a phosphate buffer solution;
s4, swelling for 18-22 hours by using an acid solution, uniformly stirring, filtering and collecting a supernatant;
s5, neutralizing the supernatant with an alkaline solution for 10-14 hours, and dialyzing to separate out crude collagen fibers;
s6, soaking the crude collagen fibers in unsaturated fatty acid for 4-6 hours, and cleaning;
s7, soaking the crude collagen fibers in hydrogen peroxide solution for 12-16 hours, and cleaning; soaking in acetone or isopropanol for 6-8 hr, and cleaning;
s8, soaking the fabric in a sodium chloride solution for 12-16 hours, cleaning and draining; soaking in 95% ethanol for 10-14 hr to precipitate collagen fiber, and freezing for storage.
As a further improvement, the alkaline or acidic protease digestion solution comprises, by mass, 0.5-1% of alkaline or acidic protease, 1-2% of glutamic acid, 0.01-0.05% of sodium dehydroacetate, 0.01-0.05% of disodium ethylenediaminetetraacetate and 0.01-0.05% of tetrasodium ethylenediaminetetraacetate.
As a further refinement, the alkaline or acidic protease is trypsin, ficin or pepsin.
As a further improvement, the neutral protease is DispaseII.
As a further improvement, the final concentration of the neutral protease solution is 2.5U/mL.
As a further improvement, the acid is malonic acid, citric acid or acetic acid.
As a further improvement, the acid solution comprises 0.1-0.5% of acid and 0.01-0.05% of sodium dehydroacetate in percentage by mass.
As a further improvement, the alkali solution is 0.5-1mol/L sodium hydroxide solution.
As a further improvement, the final concentration of the hydrogen peroxide solution is 3-5wt%.
As a further refinement, the unsaturated fatty acid is selected from peanut oil, corn oil, soybean oil, linolenic acid, or linoleic acid.
The invention has the beneficial effects that:
the collagen fiber extracted by the method for extracting the collagen fiber has low immunogenicity.
The method for extracting the collagen fiber has the advantages of less protease consumption, high extraction rate of the collagen fiber and low production cost.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a photograph of extracted collagen fibers provided in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive efforts based on the embodiments of the present invention, are within the scope of protection of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
The invention provides a collagen fiber extraction method, which comprises the following steps:
s1, taking animal tissues rich in collagen fibers, cutting the animal tissues into slices, and cleaning the slices;
s2, digesting the slices with alkaline or acidic protease digestion solution at 35-37 ℃ for 16-20 hours;
s3, digesting for 22-26 hours by using a neutral protease solution, and washing by using a phosphate buffer solution;
s4, swelling for 18-22 hours by using an acid solution, uniformly stirring, filtering and collecting a supernatant;
s5, neutralizing the supernatant with an alkaline solution for 10-14 hours, and dialyzing to separate out crude collagen fibers;
s6, soaking the crude collagen fiber in unsaturated fatty acid for 4-6 hours, and cleaning; the immunogenicity of the collagen fibers can be eliminated by soaking the collagen fibers in unsaturated fatty acid;
s7, soaking the crude collagen fibers in hydrogen peroxide solution for 12-16 hours, and cleaning; soaking in acetone or isopropanol for 6-8 hr, and cleaning; removing redundant lipid substances by using hydrogen peroxide;
s8, soaking the fabric in a sodium chloride solution for 12-16 hours, cleaning and draining; soaking in 95% ethanol for 10-14 hr to precipitate collagen fiber, and freezing for storage.
As a further improvement, the alkaline or acidic protease digestion solution comprises, by mass, 0.5-1% of alkaline or acidic protease, 1-2% of glutamic acid, 0.01-0.05% of sodium dehydroacetate, 0.01-0.05% of disodium ethylene diamine tetraacetate and 0.01-0.05% of tetrasodium ethylene diamine tetraacetate. The glutamic acid improves the catalytic efficiency of the alkaline or acidic protease, thereby reducing the dosage of the alkaline or acidic protease and reducing the production cost under the condition of ensuring the same catalytic effect.
As a further refinement, the alkaline or acidic protease is trypsin, ficin or pepsin.
As a further improvement, the neutral protease is DispaseII.
As a further improvement, the final concentration of the neutral protease solution is 2.5U/mL.
As a further improvement, the acid is malonic acid, citric acid or acetic acid.
As a further improvement, the acid solution comprises 0.1-0.5% of acid and 0.01-0.05% of sodium dehydroacetate in percentage by mass.
As a further improvement, the alkali solution is 0.5-1mol/L sodium hydroxide solution.
As a further improvement, the final concentration of the hydrogen peroxide solution is 3-5wt%.
As a further refinement, the unsaturated fatty acid is selected from peanut oil, corn oil, soybean oil, linolenic acid, or linoleic acid.
Example 1
1. Cutting Corii Sus Domestica into slices with thickness of 1-2mm.
2. Soaking the raw materials in a digestive juice containing 0.5wt% of trypsin, 1wt% of glutamic acid, 0.03wt% of sodium dehydroacetate, 0.04wt% of disodium ethylene diamine tetraacetate and 0.02wt% of tetrasodium ethylene diamine tetraacetate, and preserving the raw materials in an incubator at 35-37 ℃ for 18 hours.
3. Soaking and digesting the mixture for 24 hours by using a DispaseII solution (2.5U/ml), and washing the mixture by using a phosphate buffer solution; the sliced tissue was soaked in TritonX-IOO solution (0.5%), soaked for 24 hours, further removed of dermal cell components, and then washed in phosphate buffered saline.
4. Soaking in solution containing 0.2wt% of malonic acid and 0.03wt% of sodium dehydroacetate, swelling for 20 hr, stirring, and filtering to obtain supernatant.
5. Neutralizing for 12 hours by using 0.6mol/L sodium hydroxide solution, cleaning and dialyzing to separate out crude collagen fibers.
6. Soaking the crude collagen fiber in peanut oil for 5 hours, and cleaning.
6. Soaking in 5% hydrogen peroxide solution for 12 hr, and cleaning.
7. Soaking in 5% acetone for 6 hr, and cleaning.
8. Soaking in 0.5mol/L sodium chloride solution for 12 hours, cleaning and draining.
9. Soaking in 95% ethanol for 12 hr to precipitate collagen fiber, and freezing for storage.
Example 2
1. Cutting pigskin into slices with a thickness of 1-2mm.
2. Soaking in digestive juice containing ficin 0.5wt%, glutamic acid 2wt%, sodium dehydroacetate 0.05wt%, disodium edetate 0.05wt% and tetrasodium edetate 0.05wt%, and preserving in incubator at 35-37 deg.C for 16-20 hr.
3. Soaking and digesting the mixture for 24 hours by using a DispaseII solution (2.5U/ml), and washing the mixture by using a phosphate buffer solution; the sliced tissue was soaked in TritonX-IOO solution (0.5%), soaked for 24 hours, further removed of dermal cell components, and then washed in phosphate buffered saline.
4. Swelling with acid solution containing citric acid 0.1wt% and sodium dehydroacetate 0.01wt% for 20 hr, stirring, and filtering to obtain supernatant.
5. Neutralizing the supernatant with 0.5mol/L sodium hydroxide solution for 12 hours, washing, dialyzing and precipitating crude collagen fibers.
6. Soaking the crude collagen fiber in corn oil for 4-6 hr, and cleaning.
7. Soaking in 5% hydrogen peroxide solution for 12H, and cleaning.
8. Soaking in 10% acetone or isopropanol for 8H, and cleaning.
9. Soaking in 0.3mol/L sodium chloride solution for 16H, cleaning, and draining.
10. Soaking in 95% alcohol for 12 hr, precipitating collagen fiber, and freezing for storage.
Example 3
1. Cutting pigskin into slices with the thickness of 1-2mm;
2. the mixture is preserved in an incubator containing 1wt% of pepsin, 1wt% of glutamic acid, 0.05wt% of sodium dehydroacetate, 0.01wt% of disodium ethylene diamine tetraacetate and 0.05wt% of tetrasodium ethylene diamine tetraacetate and at the temperature of 35-37 ℃ for 16-20 hours.
3. Soaking and digesting the dispaseII solution (2.5U/ml) for 24 hours, and washing the solution by phosphate buffer solution; the sliced tissue was soaked in TritonX-IOO solution (0.5%), soaked for 24 hours, further removed of dermal cell components, and then washed in phosphate buffered saline.
4. Swelling with 0.1wt% of malonic acid solution and 0.03wt% of sodium dehydroacetate for 20 hours, stirring, and filtering to obtain a supernatant.
5. Neutralizing for 12 hours by using 1mol/L sodium hydroxide solution, cleaning and dialyzing to separate out crude collagen fibers.
6. Soaking the crude collagen fiber in corn oil for 4-6 hr, and cleaning.
7. Soaking in 3% hydrogen peroxide solution for 12H, and cleaning.
8. Soaking in 10% acetone or isopropanol for 8H, and cleaning.
9. Soaking in 0.5mol/L sodium chloride solution for 12H, cleaning, and draining.
10. Soaking in 95% alcohol for 12 hr, precipitating collagen fiber, and freezing for storage.
Example 4
1. Cutting pigskin into slices with thickness of 1-2mm.
2. Using a digestive juice containing 0.5wt% of trypsin, 1wt% of glutamic acid, 0.02wt% of sodium dehydroacetate, 0.01wt% of disodium edetate and 0.05wt% of tetrasodium edetate, and preserving in an incubator at 35-37 ℃ for 16-20 hours.
3. Soaking and digesting the mixture for 24 hours by using DispaseII solution (2.5U/ml), and washing the mixture by using phosphate buffer solution; the sliced tissue was soaked in TritonX-IOO solution (0.5%), soaked for 24 hours, further removed of dermal cell components, and then washed in phosphate buffered saline.
4. 0.5wt% of acetic acid solution and 0.05wt% of sodium dehydroacetate, swelling for 20 hours, stirring, filtering and taking supernatant.
5. Neutralizing for 12 hours by using 1mol/L sodium hydroxide solution, cleaning and dialyzing to separate out crude collagen fibers.
6. Soaking crude collagen fiber in linolenic acid for 6 hr, and washing.
7. Soaking in 3% hydrogen peroxide solution for 12H, and cleaning.
8. Soaking in 10% acetone or isopropanol for 8H, and cleaning.
9. Soaking in 0.3mol/L sodium chloride solution for 16H, cleaning, and draining.
10. Soaking in 95% alcohol for 12 hr, precipitating collagen fiber, and freezing for storage.
Example 5
1. Cutting pigskin into slices with thickness of 1-2mm.
2. 0.5wt% of ficin, 1wt% of glutamic acid, 0.05wt% of sodium dehydroacetate, 0.02wt% of disodium ethylenediamine tetraacetic acid and 0.03wt% of tetrasodium ethylenediamine tetraacetic acid are adopted, and the mixture is preserved in an incubator at 35-37 ℃ for 16-20 hours.
3. Soaking and digesting the dispaseII solution (2.5U/ml) for 24 hours, and washing the solution by phosphate buffer solution; the sliced tissue was soaked in TritonX-IOO solution (0.5%), soaked for 24 hours, further removed of dermal cell components, and then washed in phosphate buffered saline.
4. 0.4wt% of malonic acid solution and 0.02wt% of sodium dehydroacetate, swelling for 20 hours, stirring, filtering and taking supernatant.
5. Neutralizing for 12 hours by using 0.5mol/L sodium hydroxide solution, cleaning and dialyzing to separate out crude collagen fibers.
6. Soaking crude collagen fiber in corn oil for 5 hr, and cleaning.
7. Soaking in 4% hydrogen peroxide solution for 15H, and cleaning.
8. Soaking in 8% acetone or isopropanol for 8H, and cleaning.
9. Soaking in 0.5mol/L sodium chloride solution for 12H, cleaning, and draining.
10. Soaking in 95% alcohol for 12 hr, precipitating collagen fiber, and freezing for storage.
Comparative example 1
The procedure of example 1 was otherwise the same without soaking in unsaturated fatty acid.
Comparative example 2
The digestion with DispaseII solution was not performed, but the procedure was the same as in example 1.
Comparative example 3
The operation was the same as in example 1 except that the immersion in the hydrogen peroxide solution was not carried out.
Comparative example 4
The procedure of example 1 was otherwise the same as that of example 1 without acetone immersion.
The immunogenicity assay was as follows: according to YY/T1465.2-2016 section 2 of the method for evaluating immunogenicity of medical devices: the method of serum immunoglobulin and complement component determination comprises setting positive control group (BSA), negative control group and test sample group, each group comprises 10 mice. The positive control group adopts BAS as a positive control, 3mg BSA is uniformly mixed with 9mL PBS (pH7.4), and then is uniformly mixed with CFA according to the volume of 1:1 to form emulsion, and each mouse is injected with 0.12mL subcutaneously on the back and once a week. In the test sample group, a certain amount of collagen fiber freeze-dried products are taken and implanted into subcutaneous tissues on the back of a mouse. The negative control group was sham-operated without using a sample. After 2 weeks, mouse serum was taken and the contents of serum immunoglobulin and serum complement components were measured using a mouse immunoglobulin G (IgG) ELISA kit and a mouse complement component C3a (C3 a) ELISA detection kit, respectively. Higher levels of IgG and C3a indicate a stronger immune response.
The extraction rate test method is as follows: respectively carrying out low-temperature freeze drying on a certain amount of pigskin slices and extracted collagen fibers to remove water, taking the freeze-dried pigskin and the collagen fibers, hydrolyzing for 24 hours by hydrochloric acid under a high-temperature condition, measuring the content of hydroxyproline by using a bergarn and Loxley method, determining the content of collagen, and calculating the extraction rate according to the ratio of the collagen fibers to the content of collagen in the pigskin.
The test results are shown in the following table:
the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A collagen fiber extraction method is characterized by comprising the following steps:
s1, taking animal tissues rich in collagen fibers, cutting the animal tissues into slices, and cleaning the slices;
s2, digesting the slices with alkaline or acidic protease digestion solution at 35-37 ℃ for 16-20 hours;
s3, digesting for 22-26 hours by using a neutral protease solution, and washing by using a phosphate buffer solution; the neutral protease is DispaseII;
s4, swelling for 18-22 hours by using an acid solution, uniformly stirring, filtering and collecting a supernatant;
s5, neutralizing the supernatant with an alkaline solution for 10-14 hours, and dialyzing to separate out crude collagen fibers;
s6, soaking the crude collagen fibers in unsaturated fatty acid for 4-6 hours, and cleaning; the unsaturated fatty acid is selected from peanut oil, corn oil, soybean oil, linolenic acid or linoleic acid;
s7, soaking the crude collagen fibers in hydrogen peroxide solution for 12-16 hours, and cleaning; soaking in acetone or isopropanol for 6-8 hr, and cleaning; the final concentration of the hydrogen peroxide solution is 3-5wt%;
s8, soaking the fabric in a sodium chloride solution for 12-16 hours, cleaning and draining; soaking in 95% ethanol for 10-14 hr to precipitate collagen fiber, and freezing for storage.
2. The method for extracting collagen fiber according to claim 1, wherein the alkaline or acidic protease digestion solution comprises, by mass, 0.5 to 1% of alkaline or acidic protease, 1 to 2% of glutamic acid, 0.01 to 0.05% of sodium dehydroacetate, 0.01 to 0.05% of disodium ethylenediaminetetraacetate, and 0.01 to 0.05% of tetrasodium ethylenediaminetetraacetate.
3. The method for extracting collagen fiber according to claim 2, wherein said alkaline or acidic protease is trypsin, ficin or pepsin.
4. The method for extracting collagen fiber according to claim 1, wherein the final concentration of said neutral protease solution is 2.5U/mL.
5. The method for extracting collagen fiber according to claim 1, wherein said acid is malonic acid, citric acid or acetic acid.
6. The method for extracting collagen fiber according to claim 5, wherein said acid solution comprises 0.1-0.5% acid and 0.01-0.05% sodium dehydroacetate by mass.
7. The method for extracting collagen fiber according to claim 1, wherein said alkali solution is 0.5-1mol/L sodium hydroxide solution.
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