CN108977488B - Elastin for intensive skin repair and preparation method thereof - Google Patents
Elastin for intensive skin repair and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an elastin for intensive skin repair and a preparation method thereof, wherein the preparation method comprises the following steps: (1) pre-treating; (2) carrying out enzymolysis; (3) dialyzing; (4) and (5) drying. The elastin for intensive skin repair and the preparation method thereof have the advantages that the method is simple and easy to implement, environment-friendly and pollution-free, the enzymolysis efficiency and the purity of the elastin are improved by degreasing the surface of the bovine jugular ligament, the obtained elastin has the effects of oxidation resistance and moisture retention, hyaluronic acid and water content in the skin can be remarkably improved, the intensive repair of aged skin can be realized, and the elastin can be used for skin care products and health care products and has good application prospects.
Description
Technical Field
The invention relates to the technical field of biochemistry, and particularly relates to elastin for intensive skin repair and a preparation method thereof.
Background
Elastin is an important extracellular matrix protein, the major constituent of which is elastic fiber. It is abundantly present in tissues and organs of mammals, such as lungs, aorta and skin, which are often deformed by stress, and mainly functions to provide the tissues and organs with the ability to resist repeated compression and deformation. The content of elastin in tissue varies with the degree of elastic function required by the tissue, with the lowest content in skin being about 2-3%; the contents of lung and aorta are intermediate, 10-25% and 30-50% respectively; the highest content in ligaments is about 80%. Meanwhile, elastin is mainly produced by fibroblasts, aortic smooth muscle cells and chondrocytes, and the produced cells are different due to different organs.
Elastin contains a unique amino acid structure, is a triple helix structure formed by cross-linking two alpha chains and one beta chain, has good elasticity and ductility due to its compact structure and hydrophobicity, and can resist enzymolysis of most of hydrolytic enzymes. However, due to the structure of the elastin material, enzymatic processes are hindered, hetero-peptides may be generated, and the flavor of the product is affected.
Elastin is often rich in lipid substances, which affect the use effect of elastin. In the invention patent with the application number of 2013102946564, bovine neck is taken as a raw material to prepare elastin, which is soaked in acetone for primary degreasing and then boiled by oxalic acid solution to remove fat, but acetone is low in toxic substance and easy to volatilize, which is not beneficial to environmental protection in actual production, and part of elastin is lost even though oxalic acid is boiled to remove part of fat.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to solve the technical problem of providing the elastin for intensive skin repair and the preparation method thereof, the method is simple and easy to implement, is environment-friendly and pollution-free, the enzymolysis efficiency and the purity of the elastin are improved by degreasing the surface of the bovine jugular ligament, the obtained elastin has the effects of oxidation resistance and moisture preservation, and the hyaluronic acid and the water content in the skin can be remarkably improved.
The purpose of the invention is realized by the following technical scheme:
a preparation method of elastin for intensive skin repair comprises the following steps:
(1) pre-treating;
(2) carrying out enzymolysis;
(3) dialyzing;
(4) and (5) vacuum freeze drying.
A preparation method of elastin for intensive skin repair comprises the following steps:
(1) pretreatment: removing fat and connective tissues from fresh bovine neck ligaments, cutting into blocks of (1-4) mm x (1-4) mm, mixing 10-20 parts of the blocks with 40-80 parts of 3-6wt% sodium carbonate aqueous solution, stirring at 40-60 ℃ for 20-40 minutes at 100 rpm, filtering, washing with water until the pH value of the washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) enzymolysis: mixing the pretreatment raw material with 40-80 parts of water, stirring for 5-10 minutes at 300 revolutions per minute of 100-;
(3) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 36-72 hours, wherein the dialysis medium is distilled water, the volume of the dialysis medium is 8-12 times of that of the supernatant, and the dialysis medium is replaced every 10-15 hours to obtain dialysate;
(4) and (3) drying: and (3) carrying out vacuum freeze drying on the dialysate to obtain the elastin for skin dense repair.
A preparation method of elastin for intensive skin repair comprises the following steps:
(1) pretreatment: removing fat and connective tissues from fresh bovine neck ligaments, cutting into blocks of (1-4) mm x (1-4) mm, mixing 10-20 parts of the blocks with 40-80 parts of 3-6wt% sodium carbonate aqueous solution, stirring at 40-60 ℃ for 20-40 minutes at 100 rpm, filtering, washing with water until the pH value of the washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) degreasing: mixing the pretreated raw material with 40-80 parts of water, adding 0.01-0.05 part of lipase, 0.1-0.3 part of dispersant and 0.1-0.3 part of fatty acid neutralizer, adjusting the pH to 8-10 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring at 40-60 ℃ for 90-180 minutes at 300 r/min with 100-;
(3) enzymolysis: mixing the pure elastin material obtained in the step (2) with 40-80 parts of water, stirring for 5-10 minutes at 300 revolutions per minute of 100-;
(4) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 36-72 hours, wherein the dialysis medium is distilled water, the volume of the dialysis medium is 8-12 times of that of the supernatant, and the dialysis medium is replaced every 10-15 hours to obtain dialysate;
(5) and (3) drying: and (3) carrying out vacuum freeze drying on the dialysate to obtain the elastin for skin dense repair.
The vacuum freeze-drying conditions are as follows: the thickness of the material is 3-6mm, the pre-freezing temperature is set to be-20 to-25 ℃, the sample is kept for 1-2 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 5-15 ℃, the analysis temperature is 30-35 ℃, the vacuum degree is 10-30Pa, and the drying time is 10-20 hours.
The mixed enzyme is papain and flavor protease in a mass ratio of (1-3): (1-3).
The dispersing agent is hydroxypropyl-beta-cyclodextrin and/or sulfobutyl-beta-cyclodextrin.
Preferably, the dispersing agent is a mixture of hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin, and the mass ratio of the hydroxypropyl-beta-cyclodextrin to the sulfobutyl-beta-cyclodextrin is (1-5): 1.
the fatty acid neutralizer is sodium carbonate and/or sodium hydroxide.
An elastin for skin intensive repair is prepared by the method.
The elastin for intensive skin repair and the preparation method thereof have the advantages that the method is simple and easy to implement, environment-friendly and pollution-free, the enzymolysis efficiency and the purity of the elastin are improved by degreasing the surface of the bovine jugular ligament, the obtained elastin has the effects of oxidation resistance and moisture retention, hyaluronic acid and water content in the skin can be remarkably improved, the intensive repair of aged skin can be realized, and the elastin can be used for skin care products and health care products and has good application prospects.
Detailed Description
In the present invention, all the equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.
Dialysis bag, United states Combined carbonation dialysis bag, molecular weight cut-off 1000D.
Lipase, nanning pombo bioengineering, ltd, 5 ten thousand U/g, type: PB 26.
Trypsin, sienprevis bioengineering, ltd, 5 ten thousand U/g, type: prerens-1121.
Papain, Pengbo bioengineering Co., Ltd, enzyme activity 5 ten thousand U/g, type: PB 01.
Flavourzyme, Pengpo bioengineering Co., Ltd, 5 ten thousand U/g of enzyme activity, type: PB 03.
Hydroxypropyl- β -cyclodextrin, CAS No.: 128446-35-5, available from Wuhan Dongkong science and technology Limited.
Sulfobutyl- β -cyclodextrin, CAS No.: 25167-62-8, available from Wuhan Dongkong science and technology Co., Ltd.
Determination of DPPH radical scavenging capacity: the elastin for skin dense repair obtained in the examples was measured for removing DPPH radicals according to "antioxidant peptide prepared from super-processed rice protein" by jianjonge, mahalanobis, etc., and the DPPH radical removal rate was measured at an elastin concentration of 1.0g/L for skin dense repair.
Experiment on the influence of elastin on mouse aging model for intensive skin repair: KM mice, SPF level, male, weight of 18-22g, normal feeding and quarantine observation for 7 days, are randomly divided into 9 groups, each group comprises 10 mice, and the groups respectively comprise a normal group, a model group and an experimental group 1-7. The elastin proteins obtained in examples 1-7 were diluted to 5.0mg/mL with distilled water, respectively, to obtain test samples of experimental groups 1-7.
The model group and the experimental group are injected with 1.0g/kg of 5 percent D-galactose subcutaneously in the neck and the back every day, and the normal group is injected with equal volume of physiological saline subcutaneously in the neck and the back every day. Carrying out UV irradiation on the mice in the experimental group and the model group simultaneously, wherein the UV irradiation wavelength is 350-400nm, the irradiation time is 40 min/day, the distance between a light source and the mice is 40 cm at a vertical high position, continuously molding for 40 days, carrying out normal illumination feeding on the mice in the blank group, starting on the 11 th day of molding, respectively carrying out intragastric administration on the experimental group and the tested sample every day, the dose of the drug is 50mL/kg, and carrying out intragastric administration on distilled water with the same volume every day in the normal group and the model group and continuously feeding for 30 days.
Skin moisture content: killing the mouse, taking 2cm multiplied by 2cm back skin tissue, scraping off light yellow subcutaneous adipose tissue, and weighing to obtain m1Drying at 80 deg.C for 12 hr, weighing to obtain m2Then the skin moisture content is: (m)1-m2)/m1×100％。
After measuring the moisture content of the skin, pre-cooled normal saline which is 9 times of the weight of the tissue block is taken by a measuring cylinder and poured into a beaker filled with the tissue block, then the tissue block is cut as soon as possible by ophthalmic scissors and poured into a homogenate tube, and the tissue is homogenized by a homogenizer for 5 min. Centrifuging the prepared 10% homogenate for 30min at 5000r/min, and collecting supernatant and storing at 4 deg.C.
Determination of hyaluronic acid content in skin: the hyaluronic acid content in the skin of mice was determined using a hyaluronic acid ELISA kit (supplied by zilian biotechnology (shanghai) ltd) exactly according to the kit instructions.
Example 1
The preparation method of the elastin for skin dense repair comprises the following steps:
(1) pretreatment: removing fat and connective tissues from fresh cattle neck ligaments, cutting into blocks of 2mm × 2mm × 2mm, mixing 15 parts of the blocks with 60 parts of 5wt% sodium carbonate aqueous solution, stirring at 50 ℃ at 200 rpm for 30 minutes, filtering with 100-mesh filter cloth, washing with water until the pH of a washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) enzymolysis: mixing the pretreated raw material with 60 parts of water, stirring for 6 minutes at 200 revolutions per minute, adding 0.03 part of trypsin, adjusting the pH to 8.0 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring for 3 hours at 50 ℃ at 200 revolutions per minute, adding 0.06 part of mixed enzyme, adjusting the pH to 6.5 by using 10% citric acid, stirring for 5 hours at 55 ℃ at 200 revolutions per minute, then heating to 98 ℃, preserving the temperature for 3 minutes, naturally cooling to 25 ℃, centrifuging for 30 minutes at 5000 revolutions per minute, and collecting supernatant;
(3) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 48 hours, wherein a dialysis medium is distilled water, the volume of the dialysis medium is 10 times of that of the supernatant, and the dialysis medium is replaced every 12 hours to obtain dialysate;
(4) and (3) drying: and (3) carrying out vacuum freeze-drying on the dialysate to obtain the elastin for skin dense repair, wherein the vacuum freeze-drying conditions are as follows: the thickness of the material is 5mm, the pre-freezing temperature is set to be-25 ℃, the sample is kept for 1.5 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 10 ℃, the analysis temperature is 35 ℃, the vacuum degree is 20Pa, and the drying time is 15 hours.
The mixed enzyme is papain and flavor protease according to the mass ratio of 2: 1.
Example 2
The preparation method of the elastin for skin dense repair comprises the following steps:
(1) pretreatment: removing fat and connective tissues from fresh cattle neck ligaments, cutting into blocks of 2mm × 2mm × 2mm, mixing 15 parts of the blocks with 60 parts of 5wt% sodium carbonate aqueous solution, stirring at 50 ℃ at 200 rpm for 30 minutes, filtering with 100-mesh filter cloth, washing with water until the pH of a washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) degreasing: mixing the pretreated raw material with 60 parts of water, adding 0.03 part of lipase and 0.2 part of fatty acid neutralizer, adjusting the pH to 9 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring at 50 ℃ for 120 minutes at 200 rpm, maintaining the reaction pH to 9 in the reaction process, filtering by using 100-mesh filter cloth after the reaction is finished, washing a filter cake for 2 times by using 30 parts of 5wt% sodium carbonate aqueous solution, washing by using water until the pH of a washing solution is neutral, and draining the surface water to obtain a pure elastin material;
(3) enzymolysis: mixing the pure elastin material obtained in the step (2) with 60 parts of water, stirring for 6 minutes at 200 revolutions per minute, adding 0.03 part of trypsin, adjusting the pH to 8.0 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring for 3 hours at 50 ℃ at 200 revolutions per minute, adding 0.06 part of mixed enzyme, adjusting the pH to 6.5 by using citric acid with the mass fraction of 10%, stirring for 5 hours at 55 ℃ at 200 revolutions per minute, heating to 98 ℃, keeping the temperature for 3 minutes, naturally cooling to 25 ℃, separating for 30 minutes at 5000 revolutions per minute, and collecting supernatant;
(4) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 48 hours, wherein a dialysis medium is distilled water, the volume of the dialysis medium is 10 times of that of the supernatant, and the dialysis medium is replaced every 12 hours to obtain dialysate;
(5) and (3) drying: and (3) carrying out vacuum freeze-drying on the dialysate to obtain the elastin for skin dense repair, wherein the vacuum freeze-drying conditions are as follows: the thickness of the material is 5mm, the pre-freezing temperature is set to be-25 ℃, the sample is kept for 1.5 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 10 ℃, the analysis temperature is 35 ℃, the vacuum degree is 20Pa, and the drying time is 15 hours.
The mixed enzyme is papain and flavor protease according to the mass ratio of 2: 1.
The fatty acid neutralizer is sodium carbonate.
Example 3
The preparation method of the elastin for skin dense repair comprises the following steps:
(1) pretreatment: removing fat and connective tissues from fresh cattle neck ligaments, cutting into blocks of 2mm × 2mm × 2mm, mixing 15 parts of the blocks with 60 parts of 5wt% sodium carbonate aqueous solution, stirring at 50 ℃ at 200 rpm for 30 minutes, filtering with 100-mesh filter cloth, washing with water until the pH of a washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) degreasing: mixing the pretreated raw material with 60 parts of water, adding 0.03 part of lipase, 0.2 part of dispersant and 0.2 part of fatty acid neutralizer, adjusting the pH to 9 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring at 50 ℃ for 120 minutes at 200 rpm, maintaining the reaction pH to be 9 in the reaction process, filtering by using 100-mesh filter cloth after the reaction is finished, washing a filter cake for 2 times by using 30 parts of 5wt% sodium carbonate aqueous solution, washing by using water until the pH of a washing solution is neutral, and draining the surface water to obtain a pure elastin material;
(3) enzymolysis: mixing the pure elastin material obtained in the step (2) with 60 parts of water, stirring for 6 minutes at 200 revolutions per minute, adding 0.03 part of trypsin, adjusting the pH to 8.0 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring for 3 hours at 50 ℃ at 200 revolutions per minute, adding 0.06 part of mixed enzyme, adjusting the pH to 6.5 by using citric acid with the mass fraction of 10%, stirring for 5 hours at 55 ℃ at 200 revolutions per minute, heating to 98 ℃, keeping the temperature for 3 minutes, naturally cooling to 25 ℃, separating for 30 minutes at 5000 revolutions per minute, and collecting supernatant;
(4) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 48 hours, wherein a dialysis medium is distilled water, the volume of the dialysis medium is 10 times of that of the supernatant, and the dialysis medium is replaced every 12 hours to obtain dialysate;
(5) and (3) drying: and (3) carrying out vacuum freeze-drying on the dialysate to obtain the elastin for skin dense repair, wherein the vacuum freeze-drying conditions are as follows: the thickness of the material is 5mm, the pre-freezing temperature is set to be-25 ℃, the sample is kept for 1.5 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 10 ℃, the analysis temperature is 35 ℃, the vacuum degree is 20Pa, and the drying time is 15 hours.
The mixed enzyme is papain and flavor protease according to the mass ratio of 2: 1.
The dispersing agent is hydroxypropyl-beta-cyclodextrin.
The fatty acid neutralizer is sodium carbonate.
Example 4
The preparation method of the elastin for skin dense repair comprises the following steps:
(1) pretreatment: removing fat and connective tissues from fresh cattle neck ligaments, cutting into blocks of 2mm × 2mm × 2mm, mixing 15 parts of the blocks with 60 parts of 5wt% sodium carbonate aqueous solution, stirring at 50 ℃ at 200 rpm for 30 minutes, filtering with 100-mesh filter cloth, washing with water until the pH of a washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) degreasing: mixing the pretreated raw material with 60 parts of water, adding 0.03 part of lipase, 0.2 part of dispersant and 0.2 part of fatty acid neutralizer, adjusting the pH to 9 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring at 50 ℃ for 120 minutes at 200 rpm, maintaining the reaction pH to be 9 in the reaction process, filtering by using 100-mesh filter cloth after the reaction is finished, washing a filter cake for 2 times by using 30 parts of 5wt% sodium carbonate aqueous solution, washing by using water until the pH of a washing solution is neutral, and draining the surface water to obtain a pure elastin material;
(3) enzymolysis: mixing the pure elastin material obtained in the step (2) with 60 parts of water, stirring for 6 minutes at 200 revolutions per minute, adding 0.09 part of trypsin, adjusting the pH to 8.0 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring for 8 hours at 40 ℃ at 200 revolutions per minute, heating to 98 ℃, preserving heat for 3 minutes, naturally cooling to 25 ℃, separating for 30 minutes at 5000 revolutions per minute, and collecting supernatant;
(4) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 48 hours, wherein a dialysis medium is distilled water, the volume of the dialysis medium is 10 times of that of the supernatant, and the dialysis medium is replaced every 12 hours to obtain dialysate;
(5) and (3) drying: and (3) carrying out vacuum freeze-drying on the dialysate to obtain the elastin for skin dense repair, wherein the vacuum freeze-drying conditions are as follows: the thickness of the material is 5mm, the pre-freezing temperature is set to be-25 ℃, the sample is kept for 1.5 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 10 ℃, the analysis temperature is 35 ℃, the vacuum degree is 20Pa, and the drying time is 15 hours.
The dispersing agent is hydroxypropyl-beta-cyclodextrin.
The fatty acid neutralizer is sodium carbonate.
Example 5
The preparation method of the elastin for skin dense repair comprises the following steps:
(1) pretreatment: removing fat and connective tissues from fresh cattle neck ligaments, cutting into blocks of 2mm × 2mm × 2mm, mixing 15 parts of the blocks with 60 parts of 5wt% sodium carbonate aqueous solution, stirring at 50 ℃ at 200 rpm for 30 minutes, filtering with 100-mesh filter cloth, washing with water until the pH of a washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) degreasing: mixing the pretreated raw material with 60 parts of water, adding 0.03 part of lipase, 0.2 part of dispersant and 0.2 part of fatty acid neutralizer, adjusting the pH to 9 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring at 50 ℃ for 120 minutes at 200 rpm, maintaining the reaction pH to be 9 in the reaction process, filtering by using 100-mesh filter cloth after the reaction is finished, washing a filter cake for 2 times by using 30 parts of 5wt% sodium carbonate aqueous solution, washing by using water until the pH of a washing solution is neutral, and draining the surface water to obtain a pure elastin material;
(3) enzymolysis: mixing the pure elastin material obtained in the step (2) with 60 parts of water, stirring for 6 minutes at 200 revolutions per minute, adding 0.09 part of mixed enzyme, adjusting the pH to 6.5 by using 10% citric acid by mass fraction, stirring for 8 hours at 55 ℃ at 200 revolutions per minute, heating to 98 ℃, keeping the temperature for 3 minutes, naturally cooling to 25 ℃, separating for 30 minutes at 5000 revolutions per minute, and collecting supernatant;
(4) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 48 hours, wherein a dialysis medium is distilled water, the volume of the dialysis medium is 10 times of that of the supernatant, and the dialysis medium is replaced every 12 hours to obtain dialysate;
(5) and (3) drying: and (3) carrying out vacuum freeze-drying on the dialysate to obtain the elastin for skin dense repair, wherein the vacuum freeze-drying conditions are as follows: the thickness of the material is 5mm, the pre-freezing temperature is set to be-25 ℃, the sample is kept for 1.5 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 10 ℃, the analysis temperature is 35 ℃, the vacuum degree is 20Pa, and the drying time is 15 hours.
The mixed enzyme is papain and flavor protease according to the mass ratio of 2: 1.
The dispersing agent is hydroxypropyl-beta-cyclodextrin.
The fatty acid neutralizer is sodium carbonate.
Example 6
The preparation method of the elastin for skin dense repair comprises the following steps:
(1) pretreatment: removing fat and connective tissues from fresh cattle neck ligaments, cutting into blocks of 2mm × 2mm × 2mm, mixing 15 parts of the blocks with 60 parts of 5wt% sodium carbonate aqueous solution, stirring at 50 ℃ at 200 rpm for 30 minutes, filtering with 100-mesh filter cloth, washing with water until the pH of a washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) degreasing: mixing the pretreated raw material with 60 parts of water, adding 0.03 part of lipase, 0.2 part of dispersant and 0.2 part of fatty acid neutralizer, adjusting the pH to 9 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring at 50 ℃ for 120 minutes at 200 rpm, maintaining the reaction pH to be 9 in the reaction process, filtering by using 100-mesh filter cloth after the reaction is finished, washing a filter cake for 2 times by using 30 parts of 5wt% sodium carbonate aqueous solution, washing by using water until the pH of a washing solution is neutral, and draining the surface water to obtain a pure elastin material;
(3) enzymolysis: mixing the pure elastin material obtained in the step (2) with 60 parts of water, stirring for 6 minutes at 200 revolutions per minute, adding 0.03 part of trypsin, adjusting the pH to 8.0 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring for 3 hours at 50 ℃ at 200 revolutions per minute, adding 0.06 part of mixed enzyme, adjusting the pH to 6.5 by using citric acid with the mass fraction of 10%, stirring for 5 hours at 55 ℃ at 200 revolutions per minute, heating to 98 ℃, keeping the temperature for 3 minutes, naturally cooling to 25 ℃, separating for 30 minutes at 5000 revolutions per minute, and collecting supernatant;
(4) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 48 hours, wherein a dialysis medium is distilled water, the volume of the dialysis medium is 10 times of that of the supernatant, and the dialysis medium is replaced every 12 hours to obtain dialysate;
(5) and (3) drying: and (3) carrying out vacuum freeze-drying on the dialysate to obtain the elastin for skin dense repair, wherein the vacuum freeze-drying conditions are as follows: the thickness of the material is 5mm, the pre-freezing temperature is set to be-25 ℃, the sample is kept for 1.5 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 10 ℃, the analysis temperature is 35 ℃, the vacuum degree is 20Pa, and the drying time is 15 hours.
The mixed enzyme is papain and flavor protease according to the mass ratio of 2: 1.
The dispersant is sulfobutyl-beta-cyclodextrin.
The fatty acid neutralizer is sodium carbonate.
Example 7
The preparation method of the elastin for skin dense repair comprises the following steps:
(1) pretreatment: removing fat and connective tissues from fresh cattle neck ligaments, cutting into blocks of 2mm × 2mm × 2mm, mixing 15 parts of the blocks with 60 parts of 5wt% sodium carbonate aqueous solution, stirring at 50 ℃ at 200 rpm for 30 minutes, filtering with 100-mesh filter cloth, washing with water until the pH of a washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) degreasing: mixing the pretreated raw material with 60 parts of water, adding 0.03 part of lipase, 0.2 part of dispersant and 0.2 part of fatty acid neutralizer, adjusting the pH to 9 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring at 50 ℃ for 120 minutes at 200 rpm, maintaining the reaction pH to be 9 in the reaction process, filtering by using 100-mesh filter cloth after the reaction is finished, washing a filter cake for 2 times by using 30 parts of 5wt% sodium carbonate aqueous solution, washing by using water until the pH of a washing solution is neutral, and draining the surface water to obtain a pure elastin material;
(3) enzymolysis: mixing the pure elastin material obtained in the step (2) with 60 parts of water, stirring for 6 minutes at 200 revolutions per minute, adding 0.03 part of trypsin, adjusting the pH to 8.0 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring for 3 hours at 50 ℃ at 200 revolutions per minute, adding 0.06 part of mixed enzyme, adjusting the pH to 6.5 by using citric acid with the mass fraction of 10%, stirring for 5 hours at 55 ℃ at 200 revolutions per minute, heating to 98 ℃, keeping the temperature for 3 minutes, naturally cooling to 25 ℃, separating for 30 minutes at 5000 revolutions per minute, and collecting supernatant;
(4) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 48 hours, wherein a dialysis medium is distilled water, the volume of the dialysis medium is 10 times of that of the supernatant, and the dialysis medium is replaced every 12 hours to obtain dialysate;
(5) and (3) drying: and (3) carrying out vacuum freeze-drying on the dialysate to obtain the elastin for skin dense repair, wherein the vacuum freeze-drying conditions are as follows: the thickness of the material is 5mm, the pre-freezing temperature is set to be-25 ℃, the sample is kept for 1.5 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 10 ℃, the analysis temperature is 35 ℃, the vacuum degree is 20Pa, and the drying time is 15 hours.
The mixed enzyme is papain and flavor protease according to the mass ratio of 2: 1.
The dispersing agent is a mixture of hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin, and the mass ratio of the hydroxypropyl-beta-cyclodextrin to the sulfobutyl-beta-cyclodextrin is 3: 1.
the fatty acid neutralizer is sodium carbonate.
Test example 1
The DPPH removing ability of the elastin for skin dense repair prepared in the example was tested, and the specific test results are shown in Table 1.
TABLE 1 Table of results of DPPH-eliminating ability test of elastin in skin intensive repair
| Removing DPPH. ability% | |
| Example 1 | 47.6 |
| Example 2 | 58.3 |
| Example 3 | 67.1 |
| Example 4 | 54.4 |
| Example 5 | 59.2 |
| Example 6 | 65.3 |
| Example 7 | 73.8 |
Test example 2
In the experiment of the influence of elastin for intensive skin repair on the mouse aging model, the test results of the moisture content and hyaluronic acid content of the mouse skin are shown in tables 2-3. The data difference between groups is compared by adopting a t test method, and the difference with P <0.05 has statistical significance. Experimental group data P <0.05 compared to model group, and model group data P <0.05 compared to blank group.
Table 2 skin moisture content test results table
Table 3 hyaluronic acid content test results table
Test example 3
The skin-improving effect of the elastin for skin dense repair prepared in the example was tested, and the specific test results are shown in table 4.
The test method comprises the following steps: 210 healthy women 30-40 years old, whose skins were dry and randomly divided into 7 groups, were administered with each of 30 persons, 3g of the elastin for skin dense repair of examples 1-7 once a day dissolved in warm water at 80 ℃ for 90 days.
And (4) testing standard:
the effect is shown: the skin is obviously improved, fine wrinkles are obviously reduced, and the skin is kept moist.
The method has the following advantages: the skin is improved, fine wrinkles are reduced, and the skin is kept relatively moist.
And (4) invalidation: the skin condition did not improve at all.
Table 4 test results of skin improvement
Claims (3)
1. The preparation method of the elastin for skin intensive repair is characterized by comprising the following steps of:
(1) pretreatment: removing fat and connective tissues from fresh cattle neck ligaments, cutting into blocks of (1-4) mm x (1-4) mm, mixing 10-20 parts of the blocks with 40-80 parts of 3-6wt% sodium carbonate aqueous solution, stirring at 40-60 ℃ for 20-40 minutes, filtering, washing with water until the pH of a washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) degreasing: mixing the pretreated raw material with 40-80 parts of water, adding 0.01-0.05 part of lipase, 0.1-0.3 part of dispersant and 0.1-0.3 part of fatty acid neutralizer, adjusting the pH to 8-10 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring at 40-60 ℃ for 90-180 minutes, maintaining the reaction pH to 8-10 in the reaction process, filtering after the reaction is finished, washing a filter cake for 1-3 times by using 25-35 parts of 3-6% wt sodium carbonate aqueous solution, washing with water until the pH of a washing liquid is neutral, draining the surface moisture to obtain a pure elastin material;
(3) enzymolysis: mixing the pure elastin material obtained in the step (2) with 40-80 parts of water, stirring for 5-10 minutes at 100-300 r/min, adding 0.01-0.1 part of trypsin, adjusting the pH to 7.0-8.5 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring for 2-4 hours at 45-55 ℃, adding 0.02-0.2 part of mixed enzyme, adjusting the pH to 6.0-7.0 by using 10% of citric acid, stirring for 4-6 hours at 50-60 ℃, heating to 90-100 ℃, keeping the temperature for 2-5 minutes, naturally cooling to 15-30 ℃, separating for 20-30 minutes at 4000-6000 r/min, and collecting supernatant;
(4) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 36-72 hours, wherein the dialysis medium is distilled water, the volume of the dialysis medium is 8-12 times of that of the supernatant, and the dialysis medium is replaced every 10-15 hours to obtain dialysate;
(5) and (3) drying: carrying out vacuum freeze drying on the dialysate to obtain elastin for skin dense repair; the dispersing agent is a mixture of hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin, and the mass ratio of the hydroxypropyl-beta-cyclodextrin to the sulfobutyl-beta-cyclodextrin is (1-5): 1;
the vacuum freeze-drying conditions are as follows: the thickness of the material is 3-6mm, the pre-freezing temperature is set to be-20 to-25 ℃, the sample is kept for 1-2 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 5-15 ℃, the analysis temperature is 30-35 ℃, the vacuum degree is 10-30Pa, and the drying time is 10-20 hours; the mixed enzyme is papain and flavor protease in a mass ratio of (1-3): the mixture of (1-3); the fatty acid neutralizer is sodium carbonate.
2. The method for preparing elastin for skin intensive repair according to claim 1, comprising the following steps, the following parts being by weight:
(1) pretreatment: removing fat and connective tissues from fresh cattle neck ligaments, cutting into blocks of 2mm × 2mm × 2mm, mixing 15 parts of the blocks with 60 parts of 5wt% sodium carbonate aqueous solution, stirring at 50 ℃ at 200 rpm for 30 minutes, filtering with 100-mesh filter cloth, washing with water until the pH of a washing solution is neutral, and draining off surface water to obtain a pretreatment raw material;
(2) degreasing: mixing the pretreated raw material with 60 parts of water, adding 0.03 part of lipase, 0.2 part of dispersant and 0.2 part of fatty acid neutralizer, adjusting the pH to 9 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring at 50 ℃ for 120 minutes at 200 rpm, maintaining the reaction pH to be 9 in the reaction process, filtering by using 100-mesh filter cloth after the reaction is finished, washing a filter cake for 2 times by using 30 parts of 5wt% sodium carbonate aqueous solution, washing by using water until the pH of a washing solution is neutral, and draining the surface water to obtain a pure elastin material;
(3) enzymolysis: mixing the pure elastin material obtained in the step (2) with 60 parts of water, stirring for 6 minutes at 200 revolutions per minute, adding 0.03 part of trypsin, adjusting the pH to 8.0 by using a sodium carbonate aqueous solution with the mass fraction of 5wt%, stirring for 3 hours at 50 ℃ at 200 revolutions per minute, adding 0.06 part of mixed enzyme, adjusting the pH to 6.5 by using citric acid with the mass fraction of 10%, stirring for 5 hours at 55 ℃ at 200 revolutions per minute, heating to 98 ℃, keeping the temperature for 3 minutes, naturally cooling to 25 ℃, separating for 30 minutes at 5000 revolutions per minute, and collecting supernatant;
(4) and (3) dialysis: dialyzing the supernatant in a dialysis bag for 48 hours, wherein a dialysis medium is distilled water, the volume of the dialysis medium is 10 times of that of the supernatant, and the dialysis medium is replaced every 12 hours to obtain dialysate;
(5) and (3) drying: and (3) carrying out vacuum freeze-drying on the dialysate to obtain the elastin for skin dense repair, wherein the vacuum freeze-drying conditions are as follows: the thickness of the material is 5mm, the pre-freezing temperature is set to be-25 ℃, the sample is kept for 1.5 hours after the temperature is reduced to the set temperature, the sublimation temperature is set to be 10 ℃, the analysis temperature is 35 ℃, the vacuum degree is 20Pa, and the drying time is 15 hours;
the mixed enzyme is papain and flavor protease according to the mass ratio of 2: 1;
the dispersing agent is a mixture of hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin, and the mass ratio of the hydroxypropyl-beta-cyclodextrin to the sulfobutyl-beta-cyclodextrin is 3: 1;
the fatty acid neutralizer is sodium carbonate.
3. An elastin for skin intensive repair, comprising the process of claim 1 or 2.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2092071C1 (en) * | 1995-10-06 | 1997-10-10 | Алла Ионовна Сапожникова | Method of elastin preparing |
| CN105411987A (en) * | 2015-12-30 | 2016-03-23 | 苏州威尔德工贸有限公司 | Silk peptide moisture retention skin care product suitable for babies and preparation method therefor |
| CN106381323A (en) * | 2016-10-26 | 2017-02-08 | 无限极（中国）有限公司 | Elastin peptide as well as preparation method and application thereof |
| CN106701879A (en) * | 2017-03-29 | 2017-05-24 | 武汉医佳宝生物材料有限公司 | Method for extracting type I collagen |
| CN107126555A (en) * | 2017-04-28 | 2017-09-05 | 安徽生物肽产业研究院有限公司 | A kind of pigskin collagen small peptide active component composition of wound healing |
| CN107312805A (en) * | 2017-08-07 | 2017-11-03 | 中国农业大学 | A kind of preparation and its application of the polyunsaturated fatty acid rich in CLA |
| CN107362076A (en) * | 2017-08-24 | 2017-11-21 | 广州聚注通用技术研究院有限公司 | A kind of Firm additive and its application |
-
2018
- 2018-09-03 CN CN201811020273.7A patent/CN108977488B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2092071C1 (en) * | 1995-10-06 | 1997-10-10 | Алла Ионовна Сапожникова | Method of elastin preparing |
| CN105411987A (en) * | 2015-12-30 | 2016-03-23 | 苏州威尔德工贸有限公司 | Silk peptide moisture retention skin care product suitable for babies and preparation method therefor |
| CN106381323A (en) * | 2016-10-26 | 2017-02-08 | 无限极（中国）有限公司 | Elastin peptide as well as preparation method and application thereof |
| CN106701879A (en) * | 2017-03-29 | 2017-05-24 | 武汉医佳宝生物材料有限公司 | Method for extracting type I collagen |
| CN107126555A (en) * | 2017-04-28 | 2017-09-05 | 安徽生物肽产业研究院有限公司 | A kind of pigskin collagen small peptide active component composition of wound healing |
| CN107312805A (en) * | 2017-08-07 | 2017-11-03 | 中国农业大学 | A kind of preparation and its application of the polyunsaturated fatty acid rich in CLA |
| CN107362076A (en) * | 2017-08-24 | 2017-11-21 | 广州聚注通用技术研究院有限公司 | A kind of Firm additive and its application |
Non-Patent Citations (1)
| Title |
|---|
| 鸡皮胶原蛋白肽的制备及其富肽饮品的开发;曾庆冉;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20140215(第2期);摘要，第8-16页第二章第2-6小节 * |
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