CN114045322B - Method for extracting type II collagen from chicken breast cartilage and product thereof - Google Patents
Method for extracting type II collagen from chicken breast cartilage and product thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 23
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- 239000003792 electrolyte Substances 0.000 claims description 32
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/18—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing inorganic materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/425—Porous materials, e.g. foams or sponges
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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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Abstract
The invention discloses a method for extracting type II collagen from chicken breast cartilage and a product thereof, and relates to a collagen extraction process. When the type II collagen is extracted, the type II collagen is prepared by degreasing, grinding, acidolysis, enzymolysis, electrophoresis dialysis, secondary grinding and sterilization of chicken breast cartilage serving as a raw material, and then the type II collagen is prepared by preparing a carrier film, carrying out secondary electrophoresis and vacuum sealing. The collagen dressing film product prepared by the invention has good wound healing effect.
Description
Technical Field
The invention relates to a collagen extraction process, in particular to a method for extracting type II collagen by using chicken breast cartilage and a product thereof.
Background
The chicken breast cartilage is white cartilage on chicken bones and near the chicken breast, has a crisp taste, wherein the type II collagen is the most main organic component in cartilage matrix, has a compact fiber structure, and is a characteristic protein of cartilage tissues. The chicken breast cartilage is low in price and easy to obtain, the type II collagen content is high, and the chicken breast cartilage is a good extraction source of the type II collagen. The II-type collagen can be extracted from chicken breast cartilage to prepare various nutrition and skin care products, so that the value increase and the diversified utilization of resources are realized.
The molecular weight of the type II collagen is very high, about 30 kilodaltons, and the digestion and absorption rate is low, so that the degradation of the type II collagen into type II collagen hydrolysate with the molecular weight of about thousands to tens of thousands of daltons is very necessary. The molecular weight distribution of the type II collagen product in the current market is uneven, and the absorption efficiency is low. The type II collagen prepared by the invention is small molecular polypeptide with hydrolysis molecular weight of about thousands and is easy to be absorbed, and the prepared dressing film product can provide the collagen polypeptide required by cells and promote the proliferation and differentiation of the cells so as to achieve the effect of wound repair.
Disclosure of Invention
The invention aims to provide a method for extracting II-type collagen by using chicken breast cartilage and a product thereof, which are used for solving the problems in the prior art.
A method for extracting type II collagen by using chicken breast cartilage mainly comprises the following preparation steps: degreasing, grinding, acidolysis, enzymolysis, electrophoresis dialysis, secondary grinding and sterilization.
As optimization, the preparation prepared by the type II collagen further comprises the following preparation steps: and (3) preparing a carrier film, carrying out secondary electrophoresis, and sealing in vacuum.
As optimization, the preparation method of the type II collagen mainly comprises the following preparation steps:
(1) Degreasing: immersing fresh chicken breast cartilage without bones and meat into a sodium hydroxide solution with the mass fraction of 0.3-0.5%, performing ultrasonic vibration for 20-30 min at 20-30 ℃ and 30-40 kHz, washing to be neutral by pure water, and drying for 4-6 h at the temperature of minus 10-minus 5 ℃ and the pressure of 5-10 Pa to obtain defatted chicken breast cartilage;
(2) Grinding: firstly crushing defatted chicken breast cartilage to particles smaller than 9mm at the ambient temperature of minus 10 to minus 5 ℃, then placing the chicken breast cartilage into a pulverizer, and grinding the chicken breast cartilage to particles smaller than 1mm at the ambient temperature of minus 10 to minus 5 ℃ to prepare chicken breast cartilage powder;
(3) Acidolysis: mixing chicken breast cartilage powder with 0.3-0.5% hydrochloric acid solution according to a mass ratio of 1:10, mixing uniformly, standing for 20-24 h at 1-5 ℃, and washing to be neutral by pure water to prepare a precursor;
(4) Enzymolysis: precursor and pure water are mixed according to the mass ratio of 1:10, adding acetic acid with the mass of 0.03-0.05 times of the precursor and pepsin with the mass of 0.01-0.02 times of the precursor, stirring for 20-24 h at the temperature of 1-5 ℃ at the rotating speed of 1000-2000 r/min, centrifuging for 20-30 min at the temperature of 1-5 ℃ at the rotating speed of 12000-15000 r/min, and removing the centrifugal solid precipitate to obtain enzymolysis supernatant;
(5) Electrophoresis dialysis: adding the enzymolysis supernatant into the anode side of electrolyte with the mass 3-5 times of that of the enzymolysis supernatant, separating the anode and the cathode by using a dialysis membrane with the mass 3000-5000 Da, carrying out electrophoresis for 30-40 min at the temperature of 1-5 ℃ under the voltage of 30-36V, regulating the catholyte to be neutral by using sodium hydroxide solution with the mass fraction of 0.1%, filtering, and washing for 3-5 times by using pure water to prepare the type II collagen colloid;
(6) Secondary grinding: grinding the type II collagen colloid into slurry by a colloid mill, drying for 4-6 hours at the temperature of minus 10 ℃ to minus 5 ℃ and the pressure of 5-10 Pa, and grinding by a superfine grinder until the particle size is smaller than 0.01mm to obtain the type II collagen;
(7) Sterilizing: placing the type II collagen in a high-voltage pulse electric field, and treating for 20-30 s at the temperature of 5-10 ℃;
(8) Preparing a carrier film: the modified graphene and diethyl ether are mixed according to the mass ratio of 1:10, uniformly mixing, reacting in a reaction kettle taking polytetrafluoroethylene as a substrate at 120-150 ℃ for 12-15 hours, filtering, washing with absolute ethyl alcohol for 3-5 times, and drying at the temperature of minus 10-minus 5 ℃ and the pressure of 5-10 Pa for 6 hours to prepare a carrier film;
(9) And (3) secondary electrophoresis: taking the carrier film as a cathode, carrying out electrophoresis on a collagen electrolyte with the mass 100-200 times of that of the carrier film, carrying out electrophoresis for 8-10 min at the temperature of 1-5 ℃ under the voltage of 30-36V, taking out the carrier film with the II-type collagen attached on the surface, forming 45 DEG with the horizontal plane, rinsing the surface with pure water at the speed of 2mL/s for 3-5 min, and drying for 6-8 h at the temperature of 1-10 ℃ under the pressure of 5-10 Pa to prepare the collagen dressing film;
(10) Vacuum sealing: and vacuum sealing and packaging the collagen dressing film by taking polyethylene as a packaging material to prepare the product.
Preferably, the pepsin in step (4) has international system number EC3.4.23.1.
Preferably, the electrolyte in the step (5) is a sodium chloride solution with the mass fraction of 0.1%, and the pH is adjusted to 4-5 by hydrochloric acid with the mass fraction of 0.1%.
As optimization, the high-voltage pulse electric field parameters in the step (7) are as follows: the field intensity is 30-50 kV/cm, and the pulse frequency is 200-400 Hz.
As optimization, the preparation method of the modified graphene in the step (8) comprises the following steps: mixing graphene and 98% concentrated sulfuric acid according to a mass ratio of 1:10, adding potassium permanganate with the mass of 1 time of graphene, reacting for 4-6 hours under the condition of ultrasonic oscillation at 50-60 ℃ and 30-40 kHz, adding hydrogen peroxide with the mass of 0.1-0.3 time of graphene under the condition of controlling the reaction liquid to be 1-5 ℃, stirring for 10-15 minutes at the rotating speed of 800-1000 r/min, then putting into a centrifuge, centrifuging at the rotating speed of 8000-10000 r/min to obtain graphene oxide, washing the graphene oxide to be neutral by pure water, mixing with pure water with the mass of 100 times of the graphene oxide uniformly, adding sodium hydroxide with the mass of 20-30 times of the graphene oxide and monochloroacetic acid with the mass of 15-25 times of the graphene oxide, reacting for 3-4 hours under the condition of ultrasonic oscillation at 50-60 ℃ and 30-40 kHz, filtering at 60-70 ℃, and then mixing with calcium hydroxide according to the mass ratio of 1:0.1:20 are evenly mixed, stirred for 1-2 hours at 70-80 ℃ at a rotating speed of 2000-3000 r/min, filtered at 70-80 ℃, washed to be neutral by pure water, and dried for 4-6 hours at a temperature of minus 10 to minus 5 ℃ and a pressure of 5-10 Pa.
As optimization, the preparation method of the collagen electrolyte in the step (9) comprises the following steps: mixing type II collagen with pure water according to a mass ratio of 1:100, adding sodium chloride with the mass of 0.1-0.3 times of that of the II-type collagen, and regulating the pH value to 4-5 by acetic acid.
Compared with the prior art, the invention has the following beneficial effects:
when the type II collagen is extracted, the type II collagen is prepared by degreasing, grinding, acidolysis, enzymolysis, electrophoresis dialysis, secondary grinding and sterilization of chicken breast cartilage serving as a raw material, and then the type II collagen is prepared by preparing a carrier film, carrying out secondary electrophoresis and vacuum sealing.
Firstly, carrying out acidolysis treatment on chicken breast cartilage powder, wherein the capability of forming hydrogen bonds between acidolysis dissociated hydrogen ions and II-type collagen in the chicken breast cartilage powder is stronger, so that the intramolecular and intermolecular hydrogen bonds of the II-type collagen are broken, thereby unwinding the triple helix structure of the II-type collagen, improving the solubility of the II-type collagen and promoting the subsequent steps; then pepsin is used for carrying out enzymolysis on the chicken breast cartilage powder after acidolysis, and II-type collagen in the chicken breast cartilage powder after acidolysis is extracted and hydrolyzed into small molecular polypeptides, so that the II-type collagen is easier to be absorbed and utilized by human bodies; and then carrying out electrophoresis dialysis on the enzymolysis liquid to ensure that micromolecular polypeptide in the enzymolysis liquid is deposited on an electrode to remove macromolecular enzyme and solid impurities, and simultaneously, the high-purity type II collagen can be obtained quickly.
Secondly, carrying out secondary electrophoresis on the type II collagen by taking the carrier film as an electrode after the type II collagen is prepared, so that the type II collagen is uniformly deposited on the carrier film, and the prepared product is a collagen dressing film; oxidizing graphene, reacting with monochloroacetic acid to generate micropores on the surface of the graphene, introducing oxygen-containing groups such as carboxyl, hydroxyl, aldehyde groups and the like, reacting with calcium hydroxide to obtain modified graphene, and performing crosslinking reaction on the oxygen-containing functional groups between the modified graphene to obtain the carrier film. When the collagen dressing film is used for treating wounds, the collagen dressing film can well absorb moisture in blood exuded from the wounds, so that the local blood concentration is increased, the blood coagulation is accelerated, meanwhile, calcium carboxylate groups on the collagen dressing film can dissociate to remove calcium ions, the coagulation process is promoted, the pH value of the blood exuded from the wounds is improved after the calcium carboxylate groups are dissociated, the coagulation is further promoted, type II collagen on the collagen dressing film can be absorbed by cells, the proliferation and differentiation of the cells are promoted, the generation of epidermal cells is accelerated, and the repair of the wounds is promoted.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
For a clearer description of the method provided by the present invention, the following examples are provided for the detailed description, and the methods for testing the indexes of type II collagen products prepared in the following examples are as follows:
wound healing effect: male mice for experiments are taken for the same weight and age, the mice are dehaired, a temperature control scald apparatus is enabled to cause the same area scald at the same position of the mice after cleaning and disinfection, physiological saline is used for cleaning, the collagen dressing film products obtained in each embodiment are taken for the same mass and size as those of the comparative example materials to completely cover the scald, cleaning and replacing are carried out every day, the wound area and the healing area are measured after the same days, and the wound healing rate = healed area/initial wound area is calculated.
Example 1
A method for extracting type II collagen from chicken breast cartilage and a product thereof, wherein the preparation method of the type II collagen and the product thereof mainly comprises the following preparation steps:
(1) Degreasing: immersing fresh chicken breast cartilage without bones into 0.3% sodium hydroxide solution, performing ultrasonic vibration at 20 ℃ and 30kHz for 30min, washing with pure water to neutrality, and drying at-10 ℃ and 5Pa pressure for 6h to obtain defatted chicken breast cartilage;
(2) Grinding: firstly crushing defatted chicken breast cartilage to particles smaller than 9mm at the ambient temperature of minus 10 ℃, then placing the chicken breast cartilage into a pulverizer, and grinding the chicken breast cartilage to particles smaller than 1mm at the ambient temperature of minus 10 ℃ to obtain chicken breast cartilage powder;
(3) Acidolysis: mixing chicken breast cartilage powder with 0.3% hydrochloric acid solution according to a mass ratio of 1:10, uniformly mixing, standing for 24 hours at the temperature of 1 ℃, and washing to be neutral by pure water to prepare a precursor;
(4) Enzymolysis: precursor and pure water are mixed according to the mass ratio of 1:10, adding acetic acid with the mass of 0.03 times of the precursor and pepsin with the mass of 0.01 times of the precursor, stirring at the temperature of 1 ℃ for 24 hours at the rotating speed of 1000r/min, centrifuging at the temperature of 1 ℃ for 30 minutes at the rotating speed of 12000r/min, and removing the centrifugal solid precipitate to obtain enzymolysis supernatant;
(5) Electrophoresis dialysis: adding the enzymolysis supernatant into the anode side of electrolyte with the mass 3 times of that of the enzymolysis supernatant, separating the anode and the cathode by using a dialysis membrane with the mass 3000Da, carrying out electrophoresis at the temperature of 5 ℃ and the voltage of 30V for 40min, adjusting the catholyte to be neutral by using sodium hydroxide solution with the mass fraction of 0.1%, filtering, and washing 3 times by using pure water to obtain type II collagen colloid;
(6) Secondary grinding: grinding the type II collagen colloid into slurry by a colloid mill, drying for 6 hours at the temperature of minus 10 ℃ and the pressure of 5Pa, and grinding by a superfine grinder until the particle size is smaller than 0.01mm to obtain the type II collagen;
(7) Sterilizing: placing the type II collagen in a high-voltage pulse electric field, and treating for 30s at the temperature of 5 ℃;
(8) Preparing a carrier film: the modified graphene and diethyl ether are mixed according to the mass ratio of 1:10, uniformly mixing, reacting in a reaction kettle taking polytetrafluoroethylene as a substrate at 120 ℃ for 15 hours, filtering, washing with absolute ethyl alcohol for 3 times, and drying at the temperature of-10 ℃ and the pressure of 5Pa for 6 hours to prepare a carrier film;
(9) And (3) secondary electrophoresis: taking the carrier film as a cathode, carrying out electrophoresis on a collagen electrolyte with the mass 100 times of that of the carrier film, carrying out electrophoresis for 10min at the temperature of 1 ℃ under the voltage of 30V, taking out the carrier film with the type II collagen attached on the surface, wetting the surface with pure water for 3min at the speed of 2mL/s at the angle of 45 DEG with the horizontal plane, and drying for 8h at the temperature of 1 ℃ under the pressure of 5Pa to obtain a collagen dressing film;
(10) Vacuum sealing: and vacuum sealing and packaging the collagen dressing film by taking polyethylene as a packaging material to prepare the product.
Preferably, the pepsin in step (4) has international system number EC3.4.23.1.
Preferably, the electrolyte in the step (5) is a sodium chloride solution with the mass fraction of 0.1%, and the pH is adjusted to 4 by hydrochloric acid with the mass fraction of 0.1%.
As optimization, the high-voltage pulse electric field parameters in the step (7) are as follows: the field strength is 30kV/cm, and the pulse frequency is 400Hz.
As optimization, the preparation method of the modified graphene in the step (8) comprises the following steps: mixing graphene and 98% concentrated sulfuric acid according to a mass ratio of 1:10, adding potassium permanganate with the mass of 1 time of graphene, reacting for 6 hours under the condition of ultrasonic oscillation at 50 ℃ and 30kHz, adding hydrogen peroxide with the mass of 0.1 time of graphene into the reaction solution under the condition of controlling the temperature to be 1 ℃, stirring for 10 minutes at the rotating speed of 800r/min, putting into a centrifuge, centrifuging at the rotating speed of 8000r/min to obtain graphene oxide, washing the graphene oxide to be neutral by pure water, uniformly mixing the graphene oxide with the pure water with the mass of 100 times of the graphene oxide, adding sodium hydroxide with the mass of 20 times of the graphene oxide and monochloroacetic acid with the mass of 15 times of the graphene oxide, reacting for 4 hours under the condition of ultrasonic oscillation at 50 ℃ and 30kHz, filtering at 60 ℃, and mixing the graphene oxide with calcium hydroxide according to the mass ratio of 1:0.1:20 are evenly mixed, stirred for 2 hours at 70 ℃ at a rotating speed of 2000r/min, filtered at 70 ℃, washed to be neutral by pure water, and dried for 6 hours at the temperature of minus 10 ℃ and the pressure of 5 Pa.
As optimization, the preparation method of the collagen electrolyte in the step (9) comprises the following steps: mixing type II collagen with pure water according to a mass ratio of 1:100, adding sodium chloride with the mass of 0.1 times of that of the II-type collagen, and regulating the pH to 5 by using acetic acid.
Example 2
A method for extracting type II collagen from chicken breast cartilage and a product thereof, wherein the preparation method of the type II collagen and the product thereof mainly comprises the following preparation steps:
(1) Degreasing: immersing fresh chicken breast cartilage without bones into 0.4% sodium hydroxide solution, performing ultrasonic vibration at 25 ℃ and 35kHz for 25min, washing with pure water to neutrality, and drying at-8 ℃ and 8Pa pressure for 5h to obtain defatted chicken breast cartilage;
(2) Grinding: firstly crushing defatted chicken breast cartilage to particles smaller than 9mm at the ambient temperature of-8 ℃, then placing the chicken breast cartilage into a pulverizer, and grinding the chicken breast cartilage into particles smaller than 1mm at the ambient temperature of-8 ℃ to obtain chicken breast cartilage powder;
(3) Acidolysis: mixing chicken breast cartilage powder with 0.4% hydrochloric acid solution according to a mass ratio of 1:10, standing for 22 hours at 3 ℃, and washing to be neutral by pure water to prepare a precursor;
(4) Enzymolysis: precursor and pure water are mixed according to the mass ratio of 1:10, adding acetic acid with the mass of 0.04 times of the precursor and pepsin with the mass of 0.01 times of the precursor, stirring at the temperature of 3 ℃ for 22 hours at the rotating speed of 1200r/min, centrifuging at the temperature of 3 ℃ for 25 minutes at the rotating speed of 13000r/min, and removing the centrifugal solid precipitate to obtain enzymolysis supernatant;
(5) Electrophoresis dialysis: adding the enzymolysis supernatant into the anode side of electrolyte with the mass 4 times of that of the enzymolysis supernatant, separating the anode and the cathode by using a 4000Da dialysis membrane, carrying out electrophoresis at 33V voltage at 3 ℃ for 35min, adjusting the catholyte to be neutral by using sodium hydroxide solution with the mass fraction of 0.1%, filtering, and washing for 4 times by using pure water to obtain type II collagen colloid;
(6) Secondary grinding: grinding the type II collagen colloid into slurry by a colloid mill, drying for 5 hours at the temperature of minus 8 ℃ and the pressure of 8Pa, and grinding by a superfine grinder until the particle size is smaller than 0.01mm to obtain the type II collagen;
(7) Sterilizing: placing the type II collagen in a high-voltage pulse electric field, and treating for 25s at the temperature of 8 ℃;
(8) Preparing a carrier film: the modified graphene and diethyl ether are mixed according to the mass ratio of 1:10, uniformly mixing, reacting in a reaction kettle with polytetrafluoroethylene as a substrate at 130 ℃ for 13 hours, filtering, washing with absolute ethyl alcohol for 4 times, and drying at-8 ℃ and 8Pa pressure for 6 hours to prepare a carrier film;
(9) And (3) secondary electrophoresis: taking the carrier film as a cathode, carrying out electrophoresis on collagen electrolyte with the mass 150 times of that of the carrier film, carrying out electrophoresis for 9min at the temperature of 3 ℃ under the voltage of 33V, taking out the carrier film with the type II collagen attached on the surface, wetting the surface with pure water for 4min at the speed of 2mL/s at the angle of 45 DEG with the horizontal plane, and drying for 7h at the temperature of 5 ℃ under the pressure of 8Pa to obtain the collagen dressing film;
(10) Vacuum sealing: and vacuum sealing and packaging the collagen dressing film by taking polyethylene as a packaging material to prepare the product.
Preferably, the pepsin in step (4) has international system number EC3.4.23.1.
Preferably, the electrolyte in the step (5) is a sodium chloride solution with the mass fraction of 0.1%, and the pH is adjusted to 4 by hydrochloric acid with the mass fraction of 0.1%.
As optimization, the high-voltage pulse electric field parameters in the step (7) are as follows: the field strength is 40kV/cm, and the pulse frequency is 300Hz.
As optimization, the preparation method of the modified graphene in the step (8) comprises the following steps: mixing graphene and 98% concentrated sulfuric acid according to a mass ratio of 1:10, adding potassium permanganate with the mass of 1 time of graphene, reacting for 5 hours under the condition of ultrasonic oscillation at 55 ℃ and 35kHz, adding hydrogen peroxide with the mass of 0.2 time of graphene into the reaction solution under the condition of controlling the temperature to be 3 ℃, stirring for 12 minutes at the rotating speed of 900r/min, putting into a centrifugal machine, centrifuging at the rotating speed of 9000r/min to obtain graphene oxide, washing the graphene oxide to be neutral by pure water, uniformly mixing the graphene oxide with the pure water with the mass of 100 times of the graphene oxide, adding sodium hydroxide with the mass of 25 times of the graphene oxide and monochloroacetic acid with the mass of 10 times of the graphene oxide, reacting for 3 hours under the condition of ultrasonic oscillation at 55 ℃ and 35kHz, filtering at 65 ℃, and mixing the graphene oxide with calcium hydroxide and water according to the mass ratio of 1:0.1:20 are evenly mixed, stirred for 2 hours at the temperature of 75 ℃ at the rotating speed of 2500r/min, filtered at the temperature of 75 ℃, washed to be neutral by pure water, and dried for 5 hours at the temperature of-8 ℃ and the pressure of 8 Pa.
As optimization, the preparation method of the collagen electrolyte in the step (9) comprises the following steps: mixing type II collagen with pure water according to a mass ratio of 1:100, adding sodium chloride with the mass of 0.2 times of that of the II-type collagen, and regulating the pH to 5 by using acetic acid.
Example 3
A method for extracting type II collagen from chicken breast cartilage and a product thereof, wherein the preparation method of the type II collagen and the product thereof mainly comprises the following preparation steps:
(1) Degreasing: immersing fresh chicken breast cartilage without bones into 0.5% sodium hydroxide solution, performing ultrasonic vibration at 30 ℃ and 40kHz for 20min, washing with pure water to neutrality, and drying at-5 ℃ and 10Pa pressure for 4h to obtain defatted chicken breast cartilage;
(2) Grinding: firstly crushing defatted chicken breast cartilage to particles smaller than 9mm at the ambient temperature of minus 5 ℃, then placing the chicken breast cartilage into a pulverizer, and grinding the chicken breast cartilage to particles smaller than 1mm at the ambient temperature of minus 5 ℃ to obtain chicken breast cartilage powder;
(3) Acidolysis: mixing chicken breast cartilage powder with 0.5% hydrochloric acid solution according to a mass ratio of 1:10, uniformly mixing, standing for 20 hours at 5 ℃, and washing to be neutral by pure water to prepare a precursor;
(4) Enzymolysis: precursor and pure water are mixed according to the mass ratio of 1:10, adding acetic acid with the mass of 0.05 times of the precursor and pepsin with the mass of 0.02 times of the precursor, stirring at the temperature of 5 ℃ for 20 hours at the rotating speed of 2000r/min, centrifuging at the temperature of 5 ℃ for 20 minutes at the rotating speed of 15000r/min, and removing the centrifugal solid precipitate to obtain enzymolysis supernatant;
(5) Electrophoresis dialysis: adding the enzymolysis supernatant into the anode side of electrolyte with the mass of 5 times of that of the enzymolysis supernatant, separating the anode and the cathode by using a dialysis membrane with the mass of 5000Da, carrying out electrophoresis at the temperature of 5 ℃ and the voltage of 36V for 30min, adjusting the catholyte to be neutral by using sodium hydroxide solution with the mass fraction of 0.1%, filtering, and washing for 5 times by using pure water to obtain type II collagen colloid;
(6) Secondary grinding: grinding the type II collagen colloid into slurry by a colloid mill, drying for 6 hours at the temperature of minus 5 ℃ and the pressure of 10Pa, and grinding by a superfine grinder until the particle size is smaller than 0.01mm to obtain the type II collagen;
(7) Sterilizing: placing the type II collagen in a high-voltage pulse electric field, and treating for 20s at the temperature of 10 ℃;
(8) Preparing a carrier film: the modified graphene and diethyl ether are mixed according to the mass ratio of 1:10, uniformly mixing, reacting in a reaction kettle with polytetrafluoroethylene as a substrate at 150 ℃ for 12 hours, filtering, washing with absolute ethyl alcohol for 5 times, and drying at-5 ℃ and 10Pa pressure for 6 hours to prepare a carrier film;
(9) And (3) secondary electrophoresis: taking the carrier film as a cathode, carrying out electrophoresis on collagen electrolyte with the mass 200 times of that of the carrier film, carrying out electrophoresis for 8min at the temperature of 5 ℃ under the voltage of 36V, taking out the carrier film with the type II collagen attached on the surface, wetting the surface with pure water at the speed of 2mL/s for 5min at the angle of 45 DEG with the horizontal plane, and drying for 6h at the temperature of 10 ℃ under the pressure of 10Pa to obtain the collagen dressing film;
(10) Vacuum sealing: and vacuum sealing and packaging the collagen dressing film by taking polyethylene as a packaging material to prepare the product.
Preferably, the pepsin in step (4) has international system number EC3.4.23.1.
Preferably, the electrolyte in the step (5) is a sodium chloride solution with the mass fraction of 0.1%, and the pH is adjusted to 4 by hydrochloric acid with the mass fraction of 0.1%.
As optimization, the high-voltage pulse electric field parameters in the step (7) are as follows: the field strength is 50kV/cm, and the pulse frequency is 200Hz.
As optimization, the preparation method of the modified graphene in the step (8) comprises the following steps: mixing graphene and 98% concentrated sulfuric acid according to a mass ratio of 1:10, adding potassium permanganate with the mass of 1 time of graphene, reacting for 4 hours under the condition of ultrasonic oscillation at 60 ℃ and 40kHz, adding hydrogen peroxide with the mass of 0.3 time of graphene into the reaction solution under the condition of controlling the temperature to be 5 ℃, stirring for 10 minutes at the rotating speed of 1000r/min, putting into a centrifugal machine, centrifuging at the rotating speed of 10000r/min to obtain graphene oxide, washing the graphene oxide to be neutral by pure water, uniformly mixing the graphene oxide with the pure water with the mass of 100 times of the graphene oxide, adding sodium hydroxide with the mass of 30 times of the graphene oxide and monochloroacetic acid with the mass of 25 times of the graphene oxide, reacting for 3 hours under the condition of ultrasonic oscillation at 60 ℃ and 40kHz, filtering at 70 ℃, and mixing the graphene oxide with calcium hydroxide according to the mass ratio of 1:0.1:20 are evenly mixed, stirred for 1h at 80 ℃ and a rotating speed of 3000r/min, filtered at 80 ℃, washed to be neutral by pure water, and dried for 4h at-5 ℃ and a pressure of 10 Pa.
As optimization, the preparation method of the collagen electrolyte in the step (9) comprises the following steps: mixing type II collagen with pure water according to a mass ratio of 1:100, adding sodium chloride with the mass of 0.3 times of that of the II-type collagen, and regulating the pH to 5 by using acetic acid.
Comparative example 1
A method for extracting type II collagen from chicken breast cartilage and a product thereof, wherein the preparation method of the type II collagen and the product thereof mainly comprises the following preparation steps:
(1) Degreasing: immersing fresh chicken breast cartilage without bones into 0.3% sodium hydroxide solution, performing ultrasonic vibration at 20 ℃ and 30kHz for 30min, washing with pure water to neutrality, and drying at-10 ℃ and 5Pa pressure for 6h to obtain defatted chicken breast cartilage;
(2) Grinding: firstly crushing defatted chicken breast cartilage to particles smaller than 9mm at the ambient temperature of minus 10 ℃, then placing the chicken breast cartilage into a pulverizer, and grinding the chicken breast cartilage to particles smaller than 1mm at the ambient temperature of minus 10 ℃ to obtain chicken breast cartilage powder;
(3) Acidolysis: mixing chicken breast cartilage powder with 0.3% hydrochloric acid solution according to a mass ratio of 1:10, uniformly mixing, standing for 24 hours at the temperature of 1 ℃, and washing to be neutral by pure water to prepare a precursor;
(4) Enzymolysis: precursor and pure water are mixed according to the mass ratio of 1:10, adding acetic acid with the mass of 0.03 times of the precursor and pepsin with the mass of 0.01 times of the precursor, stirring at the temperature of 1 ℃ for 24 hours at the rotating speed of 1000r/min, centrifuging at the temperature of 1 ℃ for 30 minutes at the rotating speed of 12000r/min, and removing the centrifugal solid precipitate to obtain enzymolysis supernatant;
(5) Electrophoresis dialysis: adding the enzymolysis supernatant into the anode side of electrolyte with the mass 3 times of that of the enzymolysis supernatant, separating the anode and the cathode by using a dialysis membrane with the mass 3000Da, carrying out electrophoresis at the temperature of 5 ℃ and the voltage of 30V for 40min, adjusting the catholyte to be neutral by using sodium hydroxide solution with the mass fraction of 0.1%, filtering, and washing 3 times by using pure water to obtain type II collagen colloid;
(6) Secondary grinding: grinding the type II collagen colloid into slurry by a colloid mill, drying for 6 hours at the temperature of minus 10 ℃ and the pressure of 5Pa, and grinding by a superfine grinder until the particle size is smaller than 0.01mm to obtain the type II collagen;
(7) Sterilizing: placing the type II collagen in a high-voltage pulse electric field, and treating for 30s at the temperature of 5 ℃;
(8) Preparing a carrier film: graphene and diethyl ether are mixed according to a mass ratio of 1:10, uniformly mixing, reacting in a reaction kettle taking polytetrafluoroethylene as a substrate at 120 ℃ for 15 hours, filtering, washing with absolute ethyl alcohol for 3 times, and drying at the temperature of-10 ℃ and the pressure of 5Pa for 6 hours to prepare a carrier film;
(9) Coating: mixing type II collagen with pure water according to a mass ratio of 1:1, uniformly mixing, coating on a carrier film with the mass 1 time of that of the II-type collagen, and drying for 8 hours at 1 ℃ under the pressure of 5Pa to prepare a collagen dressing film;
(10) Vacuum sealing: and vacuum sealing and packaging the collagen dressing film by taking polyethylene as a packaging material to prepare the product.
Preferably, the pepsin in step (4) has international system number EC3.4.23.1.
Preferably, the electrolyte in the step (5) is a sodium chloride solution with the mass fraction of 0.1%, and the pH is adjusted to 4 by hydrochloric acid with the mass fraction of 0.1%.
As optimization, the high-voltage pulse electric field parameters in the step (7) are as follows: the field strength is 30kV/cm, and the pulse frequency is 400Hz.
Comparative example 2
A method for extracting type II collagen from chicken breast cartilage and a product thereof, wherein the preparation method of the type II collagen and the product thereof mainly comprises the following preparation steps:
(1) Degreasing: immersing fresh chicken breast cartilage without bones into 0.3% sodium hydroxide solution, performing ultrasonic vibration at 20 ℃ and 30kHz for 30min, washing with pure water to neutrality, and drying at-10 ℃ and 5Pa pressure for 6h to obtain defatted chicken breast cartilage;
(2) Grinding: firstly crushing defatted chicken breast cartilage to particles smaller than 9mm at the ambient temperature of minus 10 ℃, then placing the chicken breast cartilage into a pulverizer, and grinding the chicken breast cartilage to particles smaller than 1mm at the ambient temperature of minus 10 ℃ to obtain chicken breast cartilage powder;
(3) Acidolysis: mixing chicken breast cartilage powder with 0.3% hydrochloric acid solution according to a mass ratio of 1:10, uniformly mixing, standing for 24 hours at the temperature of 1 ℃, and washing to be neutral by pure water to prepare a precursor;
(4) Enzymolysis: precursor and pure water are mixed according to the mass ratio of 1:10, adding acetic acid with the mass of 0.03 times of the precursor and pepsin with the mass of 0.01 times of the precursor, stirring at the temperature of 1 ℃ for 24 hours at the rotating speed of 1000r/min, centrifuging at the temperature of 1 ℃ for 30 minutes at the rotating speed of 12000r/min, and removing the centrifugal solid precipitate to obtain enzymolysis supernatant;
(5) Electrophoresis dialysis: adding the enzymolysis supernatant into the anode side of electrolyte with the mass 3 times of that of the enzymolysis supernatant, separating the anode and the cathode by using a dialysis membrane with the mass 3000Da, carrying out electrophoresis at the temperature of 5 ℃ and the voltage of 30V for 40min, adjusting the catholyte to be neutral by using sodium hydroxide solution with the mass fraction of 0.1%, filtering, and washing 3 times by using pure water to obtain type II collagen colloid;
(6) Secondary grinding: grinding the type II collagen colloid into slurry by a colloid mill, drying for 6 hours at the temperature of minus 10 ℃ and the pressure of 5Pa, and grinding by a superfine grinder until the particle size is smaller than 0.01mm to obtain the type II collagen;
(7) Sterilizing: placing the type II collagen in a high-voltage pulse electric field, and treating for 30s at the temperature of 5 ℃;
(8) Preparing a carrier film: graphene and diethyl ether are mixed according to a mass ratio of 1:10, uniformly mixing, reacting in a reaction kettle taking polytetrafluoroethylene as a substrate at 120 ℃ for 15 hours, filtering, washing with absolute ethyl alcohol for 3 times, and drying at the temperature of-10 ℃ and the pressure of 5Pa for 6 hours to prepare a carrier film;
(9) And (3) secondary electrophoresis: taking the carrier film as a cathode, carrying out electrophoresis on a collagen electrolyte with the mass 100 times of that of the carrier film, carrying out electrophoresis for 10min at the temperature of 1 ℃ under the voltage of 30V, taking out the carrier film with the type II collagen attached on the surface, wetting the surface with pure water for 3min at the speed of 2mL/s at the angle of 45 DEG with the horizontal plane, and drying for 8h at the temperature of 1 ℃ under the pressure of 5Pa to obtain a collagen dressing film;
(10) Vacuum sealing: and vacuum sealing and packaging the collagen dressing film by taking polyethylene as a packaging material to prepare the product.
Preferably, the pepsin in step (4) has international system number EC3.4.23.1.
Preferably, the electrolyte in the step (5) is a sodium chloride solution with the mass fraction of 0.1%, and the pH is adjusted to 4 by hydrochloric acid with the mass fraction of 0.1%.
As optimization, the high-voltage pulse electric field parameters in the step (7) are as follows: the field strength is 30kV/cm, and the pulse frequency is 400Hz.
As optimization, the preparation method of the collagen electrolyte in the step (9) comprises the following steps: mixing type II collagen with pure water according to a mass ratio of 1:100, adding sodium chloride with the mass of 0.1 times of that of the II-type collagen, and regulating the pH to 5 by using acetic acid.
Comparative example 3
A method for extracting type II collagen from chicken breast cartilage and a product thereof, wherein the preparation method of the type II collagen and the product thereof mainly comprises the following preparation steps:
(1) Degreasing: immersing fresh chicken breast cartilage without bones into 0.3% sodium hydroxide solution, performing ultrasonic vibration at 20 ℃ and 30kHz for 30min, washing with pure water to neutrality, and drying at-10 ℃ and 5Pa pressure for 6h to obtain defatted chicken breast cartilage;
(2) Grinding: firstly crushing defatted chicken breast cartilage to particles smaller than 9mm at the ambient temperature of minus 10 ℃, then placing the chicken breast cartilage into a pulverizer, and grinding the chicken breast cartilage to particles smaller than 1mm at the ambient temperature of minus 10 ℃ to obtain chicken breast cartilage powder;
(3) Acidolysis: mixing chicken breast cartilage powder with 0.3% hydrochloric acid solution according to a mass ratio of 1:10, uniformly mixing, standing for 24 hours at the temperature of 1 ℃, and washing to be neutral by pure water to prepare a precursor;
(4) Enzymolysis: precursor and pure water are mixed according to the mass ratio of 1:10, adding acetic acid with the mass of 0.03 times of the precursor and pepsin with the mass of 0.01 times of the precursor, stirring at the temperature of 1 ℃ for 24 hours at the rotating speed of 1000r/min, centrifuging at the temperature of 1 ℃ for 30 minutes at the rotating speed of 12000r/min, and removing the centrifugal solid precipitate to obtain enzymolysis supernatant;
(5) Electrophoresis dialysis: adding the enzymolysis supernatant into the anode side of electrolyte with the mass 3 times of that of the enzymolysis supernatant, separating the anode and the cathode by using a dialysis membrane with the mass 3000Da, carrying out electrophoresis at the temperature of 5 ℃ and the voltage of 30V for 40min, adjusting the catholyte to be neutral by using sodium hydroxide solution with the mass fraction of 0.1%, filtering, and washing 3 times by using pure water to obtain type II collagen colloid;
(6) Secondary grinding: grinding the type II collagen colloid into slurry by a colloid mill, drying for 6 hours at the temperature of minus 10 ℃ and the pressure of 5Pa, and grinding by a superfine grinder until the particle size is smaller than 0.01mm to obtain the type II collagen;
(7) Sterilizing: placing the type II collagen in a high-voltage pulse electric field, and treating for 30s at the temperature of 5 ℃;
(8) Preparing a carrier film: the modified graphene and diethyl ether are mixed according to the mass ratio of 1:10, uniformly mixing, reacting in a reaction kettle taking polytetrafluoroethylene as a substrate at 120 ℃ for 15 hours, filtering, washing with absolute ethyl alcohol for 3 times, and drying at the temperature of-10 ℃ and the pressure of 5Pa for 6 hours to prepare a carrier film;
(9) And (3) secondary electrophoresis: taking the carrier film as a cathode, carrying out electrophoresis on a collagen electrolyte with the mass 100 times of that of the carrier film, carrying out electrophoresis for 10min at the temperature of 1 ℃ under the voltage of 30V, taking out the carrier film with the type II collagen attached on the surface, wetting the surface with pure water for 3min at the speed of 2mL/s at the angle of 45 DEG with the horizontal plane, and drying for 8h at the temperature of 1 ℃ under the pressure of 5Pa to obtain a collagen dressing film;
(10) Vacuum sealing: and vacuum sealing and packaging the collagen dressing film by taking polyethylene as a packaging material to prepare the product.
Preferably, the pepsin in step (4) has international system number EC3.4.23.1.
Preferably, the electrolyte in the step (5) is a sodium chloride solution with the mass fraction of 0.1%, and the pH is adjusted to 4 by hydrochloric acid with the mass fraction of 0.1%.
As optimization, the high-voltage pulse electric field parameters in the step (7) are as follows: the field strength is 30kV/cm, and the pulse frequency is 400Hz.
As optimization, the preparation method of the modified graphene in the step (8) comprises the following steps: mixing graphene and 98% concentrated sulfuric acid according to a mass ratio of 1:10, adding potassium permanganate with the mass of 1 time of graphene, reacting for 6 hours under the condition of ultrasonic oscillation at 50 ℃ and 30kHz, adding hydrogen peroxide with the mass of 0.1 time of graphene into the reaction solution under the condition of controlling the temperature to be 1 ℃, stirring for 10 minutes at the rotating speed of 800r/min, putting into a centrifuge, centrifuging at the rotating speed of 8000r/min, separating to obtain graphene oxide, washing the graphene oxide to be neutral by pure water, and drying for 6 hours under the temperature of-10 ℃ and the pressure of 5 Pa.
As optimization, the preparation method of the collagen electrolyte in the step (9) comprises the following steps: mixing type II collagen with pure water according to a mass ratio of 1:100, adding sodium chloride with the mass of 0.1 times of that of the II-type collagen, and regulating the pH to 5 by using acetic acid.
Comparative example 4
A method for extracting type II collagen from chicken breast cartilage and a product thereof, wherein the preparation method of the type II collagen and the product thereof mainly comprises the following preparation steps:
(1) Degreasing: immersing fresh chicken breast cartilage without bones into 0.3% sodium hydroxide solution, performing ultrasonic vibration at 20 ℃ and 30kHz for 30min, washing with pure water to neutrality, and drying at-10 ℃ and 5Pa pressure for 6h to obtain defatted chicken breast cartilage;
(2) Grinding: firstly crushing defatted chicken breast cartilage to particles smaller than 9mm at the ambient temperature of minus 10 ℃, then placing the chicken breast cartilage into a pulverizer, and grinding the chicken breast cartilage to particles smaller than 1mm at the ambient temperature of minus 10 ℃ to obtain chicken breast cartilage powder;
(3) Acidolysis: mixing chicken breast cartilage powder with 0.3% hydrochloric acid solution according to a mass ratio of 1:10, uniformly mixing, standing for 24 hours at the temperature of 1 ℃, and washing to be neutral by pure water to prepare a precursor;
(4) Enzymolysis: precursor and pure water are mixed according to the mass ratio of 1:10, adding acetic acid with the mass of 0.03 times of the precursor and pepsin with the mass of 0.01 times of the precursor, stirring at the temperature of 1 ℃ for 24 hours at the rotating speed of 1000r/min, centrifuging at the temperature of 1 ℃ for 30 minutes at the rotating speed of 12000r/min, and removing the centrifugal solid precipitate to obtain enzymolysis supernatant;
(5) Electrophoresis dialysis: adding the enzymolysis supernatant into the anode side of electrolyte with the mass 3 times of that of the enzymolysis supernatant, separating the anode and the cathode by using a dialysis membrane with the mass 3000Da, carrying out electrophoresis at the temperature of 5 ℃ and the voltage of 30V for 40min, adjusting the catholyte to be neutral by using sodium hydroxide solution with the mass fraction of 0.1%, filtering, and washing 3 times by using pure water to obtain type II collagen colloid;
(6) Secondary grinding: grinding the type II collagen colloid into slurry by a colloid mill, drying for 6 hours at the temperature of minus 10 ℃ and the pressure of 5Pa, and grinding by a superfine grinder until the particle size is smaller than 0.01mm to obtain the type II collagen;
(7) Sterilizing: placing the type II collagen in a high-voltage pulse electric field, and treating for 30s at the temperature of 5 ℃;
(8) Preparing a carrier film: the modified graphene and diethyl ether are mixed according to the mass ratio of 1:10, uniformly mixing, reacting in a reaction kettle taking polytetrafluoroethylene as a substrate at 120 ℃ for 15 hours, filtering, washing with absolute ethyl alcohol for 3 times, and drying at the temperature of-10 ℃ and the pressure of 5Pa for 6 hours to prepare a carrier film;
(9) Coating: mixing type II collagen with pure water according to a mass ratio of 1:1, uniformly mixing, coating on a carrier film with the mass 1 time of that of the II-type collagen, and drying for 8 hours at 1 ℃ under the pressure of 5Pa to prepare a collagen dressing film;
(10) Vacuum sealing: and vacuum sealing and packaging the collagen dressing film by taking polyethylene as a packaging material to prepare the product.
Preferably, the pepsin in step (4) has international system number EC3.4.23.1.
Preferably, the electrolyte in the step (5) is a sodium chloride solution with the mass fraction of 0.1%, and the pH is adjusted to 4 by hydrochloric acid with the mass fraction of 0.1%.
As optimization, the high-voltage pulse electric field parameters in the step (7) are as follows: the field strength is 30kV/cm, and the pulse frequency is 400Hz.
As optimization, the preparation method of the modified graphene in the step (8) comprises the following steps: mixing graphene and 98% concentrated sulfuric acid according to a mass ratio of 1:10, adding potassium permanganate with the mass of 1 time of graphene, reacting for 6 hours under the condition of ultrasonic oscillation at 50 ℃ and 30kHz, adding hydrogen peroxide with the mass of 0.1 time of graphene into the reaction solution under the condition of controlling the temperature to be 1 ℃, stirring for 10 minutes at the rotating speed of 800r/min, putting into a centrifuge, centrifuging at the rotating speed of 8000r/min to obtain graphene oxide, washing the graphene oxide to be neutral by pure water, uniformly mixing the graphene oxide with the pure water with the mass of 100 times of the graphene oxide, adding sodium hydroxide with the mass of 20 times of the graphene oxide and monochloroacetic acid with the mass of 15 times of the graphene oxide, reacting for 4 hours under the condition of ultrasonic oscillation at 50 ℃ and 30kHz, filtering at 60 ℃, and mixing the graphene oxide with calcium hydroxide according to the mass ratio of 1:0.1:20 are evenly mixed, stirred for 2 hours at 70 ℃ at a rotating speed of 2000r/min, filtered at 70 ℃, washed to be neutral by pure water, and dried for 6 hours at the temperature of minus 10 ℃ and the pressure of 5 Pa.
Effect example
The following table 1 gives the analysis results of wound healing effects of the type II collagen preparations of examples 1 to 3 and comparative examples 1 to 4 according to the present invention.
TABLE 1
| Wound healing rate | Wound healing rate | ||
| Example 1 | 92.3％ | Comparative example 1 | 55.7％ |
| Example 2 | 93.5％ | Comparative example 2 | 63.6％ |
| Example 3 | 92.7％ | Comparative example 3 | 82.2％ |
| Comparative example 4 | 81.3％ |
From comparison of experimental data of example 1, example 2 and example 3 in table 1, it can be found that the wound healing rates of example 1, example 2 and example 3 are basically unchanged, which indicates that the change of temperature, pressure, reaction time and the like in the process parameter range of the preparation method of the invention has little influence on the wound healing rate of the final product, and good wound healing effect can be achieved; from comparison of experimental data of example 1 and comparative example 1, it can be found that the wound healing rate of example 1, comparative example 1, is increased, which illustrates that the modified graphene and the product prepared by the secondary electrophoresis has better healing effect than the product prepared by directly coating without modification; from comparison of experimental data of the embodiment 1 and the comparative example 2, it can be found that the wound healing rate of the comparative example 2 in the embodiment 1 is increased, after the graphene is modified, the surface of the modified graphene is increased with pores and can be crosslinked into a network lamellar structure, so that the loading effect on the type II collagen is improved, the proliferation differentiation of cells is promoted, meanwhile, calcium carboxylate groups on the modified graphene can be dissociated to remove calcium ions, the coagulation process is promoted, the pH value of exuded blood from the wound is increased after the calcium carboxylate groups are dissociated, the coagulation is further promoted, and the wound healing effect is further improved; as can be found from the comparison of the experimental data of the comparative example 3 in the example 1, the wound healing rate of the comparative example 3 is increased, which indicates that the graphene is further modified after being oxidized, the calcium carboxylate group on the modified graphene can dissociate to remove calcium ions, promote the coagulation process, and the pH value of exuded blood of the wound is improved after the calcium carboxylate group is dissociated, so that the coagulation is further promoted, and the wound healing effect is improved; from comparison of experimental data of example 1 versus comparative example 4, it can be found that the wound healing rate of example 1 versus comparative example 4 is increased, indicating that the product obtained by the second electrophoresis is more uniform and finer than the product obtained by the coating, thereby improving the wound healing effect.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (4)
1. The method for extracting the type II collagen by using the chicken breast cartilage is characterized by mainly comprising the following preparation steps of:
(1) Degreasing: immersing fresh chicken breast cartilage without bones and meat into a sodium hydroxide solution with the mass fraction of 0.3-0.5%, performing ultrasonic vibration for 20-30 min at 20-30 ℃ and 30-40 kHz, washing to be neutral by pure water, and drying for 4-6 h at the temperature of minus 10-minus 5 ℃ and the pressure of 5-10 Pa to obtain defatted chicken breast cartilage;
(2) Grinding: firstly crushing defatted chicken breast cartilage to particles smaller than 9mm at the ambient temperature of minus 10 to minus 5 ℃, then placing the chicken breast cartilage into a pulverizer, and grinding the chicken breast cartilage to particles smaller than 1mm at the ambient temperature of minus 10 to minus 5 ℃ to prepare chicken breast cartilage powder;
(3) Acidolysis: mixing chicken breast cartilage powder with 0.3-0.5% hydrochloric acid solution according to a mass ratio of 1:10, mixing uniformly, standing for 20-24 h at 1-5 ℃, and washing to be neutral by pure water to prepare a precursor;
(4) Enzymolysis: precursor and pure water are mixed according to the mass ratio of 1:10, adding acetic acid with the mass of 0.03-0.05 times of the precursor and pepsin with the mass of 0.01-0.02 times of the precursor, stirring for 20-24 h at the temperature of 1-5 ℃ at the rotating speed of 1000-2000 r/min, centrifuging for 20-30 min at the temperature of 1-5 ℃ at the rotating speed of 12000-15000 r/min, and removing the centrifugal solid precipitate to obtain enzymolysis supernatant;
(5) Electrophoresis dialysis: adding the enzymolysis supernatant into the anode side of electrolyte with the mass 3-5 times of that of the enzymolysis supernatant, separating the anode and the cathode by using a dialysis membrane with the mass 3000-5000 Da, carrying out electrophoresis for 30-40 min at the temperature of 1-5 ℃ under the voltage of 30-36V, regulating the catholyte to be neutral by using sodium hydroxide solution with the mass fraction of 0.1%, filtering, and washing for 3-5 times by using pure water to prepare the type II collagen colloid;
(6) Secondary grinding: grinding the type II collagen colloid into slurry by a colloid mill, drying for 4-6 hours at the temperature of minus 10 ℃ to minus 5 ℃ and the pressure of 5-10 Pa, and grinding by a superfine grinder until the particle size is smaller than 0.01mm to obtain the type II collagen;
(7) Sterilizing: placing the type II collagen in a high-voltage pulse electric field, and treating for 20-30 s at the temperature of 5-10 ℃;
the product prepared from the type II collagen mainly comprises the following preparation steps: preparing a carrier film, carrying out secondary electrophoresis, and vacuum sealing;
the preparation method of the product mainly comprises the following preparation steps:
(8) Preparing a carrier film: mixing graphene and 98% concentrated sulfuric acid according to a mass ratio of 1:10, adding potassium permanganate with the mass of 1 time of graphene, reacting for 4-6 hours under the condition of ultrasonic oscillation at 50-60 ℃ and 30-40 kHz, adding hydrogen peroxide with the mass of 0.1-0.3 time of graphene under the condition of controlling the reaction liquid to be 1-5 ℃, stirring for 10-15 minutes at the rotating speed of 800-1000 r/min, then putting into a centrifuge, centrifuging at the rotating speed of 8000-10000 r/min to obtain graphene oxide, washing the graphene oxide to be neutral by pure water, mixing with pure water with the mass of 100 times of the graphene oxide uniformly, adding sodium hydroxide with the mass of 20-30 times of the graphene oxide and monochloroacetic acid with the mass of 15-25 times of the graphene oxide, reacting for 3-4 hours under the condition of ultrasonic oscillation at 50-60 ℃ and 30-40 kHz, filtering at 60-70 ℃, and then mixing with calcium hydroxide according to the mass ratio of 1:0.1:20 are evenly mixed, stirred for 1-2 hours at 70-80 ℃ at a rotating speed of 2000-3000 r/min, filtered at 70-80 ℃, washed to be neutral by pure water, and dried for 4-6 hours at a temperature of minus 10 to minus 5 ℃ and a pressure of 5-10 Pa to prepare modified graphene; the modified graphene and diethyl ether are mixed according to the mass ratio of 1:10, uniformly mixing, reacting in a reaction kettle taking polytetrafluoroethylene as a substrate at 120-150 ℃ for 12-15 hours, filtering, washing with absolute ethyl alcohol for 3-5 times, and drying at the temperature of minus 10-minus 5 ℃ and the pressure of 5-10 Pa for 6 hours to prepare a carrier film;
(9) And (3) secondary electrophoresis: mixing type II collagen with pure water according to a mass ratio of 1:100, adding sodium chloride with the mass of 0.1-0.3 times of that of the II-type collagen, and regulating the pH value to 4-5 by using acetic acid to prepare a collagen electrolyte; taking the carrier film as a cathode, carrying out electrophoresis on the collagen electrolyte with the mass 100-200 times of that of the carrier film at the temperature of 1-5 ℃ for 8-10 min at the voltage of 30-36V, taking out the carrier film with the type II collagen attached on the surface, wetting the surface with pure water at the speed of 2mL/s for 3-5 min, and drying at the temperature of 1-10 ℃ and the pressure of 5-10 Pa for 6-8 h to prepare the collagen dressing film;
(10) Vacuum sealing: and vacuum sealing and packaging the collagen dressing film by taking polyethylene as a packaging material to prepare the product.
2. The method of claim 1, wherein the pepsin enzyme in step (4) is numbered EC3.4.23.1.
3. The method for extracting type II collagen from chicken breast cartilage according to claim 1, wherein the electrolyte in the step (5) is a sodium chloride solution with a mass fraction of 0.1%, and the pH is adjusted to 4 to 5 with hydrochloric acid with a mass fraction of 0.1%.
4. The method of claim 1, wherein the high voltage pulse electric field parameters of step (7) are: the field intensity is 30-50 kV/cm, and the pulse frequency is 200-400 Hz.
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