CN112608380A - Preparation method of collagen with molecular weight within certain range from pigskin - Google Patents
Preparation method of collagen with molecular weight within certain range from pigskin Download PDFInfo
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- CN112608380A CN112608380A CN202011410186.XA CN202011410186A CN112608380A CN 112608380 A CN112608380 A CN 112608380A CN 202011410186 A CN202011410186 A CN 202011410186A CN 112608380 A CN112608380 A CN 112608380A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 88
- 108010035532 Collagen Proteins 0.000 title claims abstract description 88
- 229920001436 collagen Polymers 0.000 title claims abstract description 88
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000012528 membrane Substances 0.000 claims abstract description 53
- 239000000843 powder Substances 0.000 claims abstract description 26
- 238000004108 freeze drying Methods 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 15
- 238000004332 deodorization Methods 0.000 claims abstract description 13
- 238000000926 separation method Methods 0.000 claims abstract description 10
- 230000001877 deodorizing effect Effects 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 53
- 230000002572 peristaltic effect Effects 0.000 claims description 35
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 29
- 239000000203 mixture Substances 0.000 claims description 28
- 210000003491 skin Anatomy 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 25
- 108090000145 Bacillolysin Proteins 0.000 claims description 15
- 108010059892 Cellulase Proteins 0.000 claims description 15
- 229920001661 Chitosan Polymers 0.000 claims description 15
- 108091005507 Neutral proteases Proteins 0.000 claims description 15
- 102000035092 Neutral proteases Human genes 0.000 claims description 15
- 102000004139 alpha-Amylases Human genes 0.000 claims description 15
- 108090000637 alpha-Amylases Proteins 0.000 claims description 15
- 229940024171 alpha-amylase Drugs 0.000 claims description 15
- 229940106157 cellulase Drugs 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000012510 hollow fiber Substances 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 108091005658 Basic proteases Proteins 0.000 claims description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 10
- 108090000526 Papain Proteins 0.000 claims description 10
- 239000004365 Protease Substances 0.000 claims description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 10
- 229910052799 carbon Inorganic materials 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 10
- 235000019834 papain Nutrition 0.000 claims description 10
- 229940055729 papain Drugs 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 9
- 210000002615 epidermis Anatomy 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000000227 grinding Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 9
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 7
- 229920001184 polypeptide Polymers 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 241000251539 Vertebrata <Metazoa> Species 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract description 3
- 230000036772 blood pressure Effects 0.000 abstract description 3
- 230000003647 oxidation Effects 0.000 abstract description 3
- 238000007254 oxidation reaction Methods 0.000 abstract description 3
- 230000001766 physiological effect Effects 0.000 abstract description 3
- 108010010803 Gelatin Proteins 0.000 abstract description 2
- 229920000159 gelatin Polymers 0.000 abstract description 2
- 239000008273 gelatin Substances 0.000 abstract description 2
- 235000019322 gelatine Nutrition 0.000 abstract description 2
- 235000011852 gelatine desserts Nutrition 0.000 abstract description 2
- 230000009471 action Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 229940127554 medical product Drugs 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 235000015277 pork Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000037384 skin absorption Effects 0.000 description 2
- 231100000274 skin absorption Toxicity 0.000 description 2
- 230000037394 skin elasticity Effects 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- -1 tissue engineering Substances 0.000 description 1
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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]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention relates to the technical field of donkey-hide gelatin production, in particular to a preparation method of collagen with a molecular weight in a certain range from pigskin, which comprises the following steps: pretreating pigskin, circularly separating by an enzymolysis membrane, decoloring, deodorizing and freeze-drying. The invention carries out pigskin pretreatment, enzymolysis membrane circular separation, decoloration and deodorization treatment and freeze drying through specific combined process steps and parameter conditions to obtain collagen powder with molecular weight of 2-5KD, and the collagen powder has the following characteristics: the three-strand helical structure for preparing the pigskin collagen is not damaged, and the original biological activity of the pigskin collagen is still kept; the main component is 2-5KD collagen powder; the pigskin collagen is derived from the collagen of vertebrates, and has good homology and compatibility with human bodies and skins; the collagen polypeptide with molecular weight less than 5kDa has various physiological activities such as lowering blood pressure and resisting oxidation.
Description
Technical Field
The invention relates to the technical field of donkey-hide gelatin production, in particular to a preparation method of collagen with a molecular weight in a certain range from pigskin.
Background
Collagen is a biological macromolecule, a main component in the connective tissue of animals, and is also a functional protein with the largest content and the widest distribution in mammals, accounting for 25-30 percent of the total protein, and even reaching more than 80 percent of certain organisms. Collagen has good biocompatibility, biodegradability and bioactivity, and thus is widely used in the fields of food, medicine, tissue engineering, cosmetics, and the like. The pigskin is a by-product of the pork production process, and the pigskin is subjected to heat treatment in the pork processing process of a meat product factory, and cannot be used for leather production. The protein content is up to more than 33%, the collagen content is about 88%, and the nutritive value is considerable. The molecular weight of the collagen reaches 300KD due to the special complex spatial structure, and the collagen is difficult to be absorbed by human bodies compared with collagen peptide, so that the collagen is hydrolyzed into small molecular polypeptide substances by a biological means, and the product beneficial to the human bodies is developed, and has wide prospects.
Disclosure of Invention
The invention aims to provide a method for preparing collagen with a molecular weight in a certain range from pigskin, so as to solve the problem that the pigskin collagen with an overlarge molecular weight is difficult to be absorbed by a human body in the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of collagen with a molecular weight within a certain range from pigskin comprises the following specific steps:
s1: pretreating pigskin;
s2: performing enzymolysis membrane circulation separation;
s3: decoloring and deodorizing;
s4: and (5) freeze drying.
Preferably, the specific steps of the pigskin pretreatment are as follows:
s11: removing hair from fresh pigskin, cleaning to remove epidermis, and removing fat layer;
s12: adding H according to the proportion of 1:202O2NaOH solution of 10-25 deg.CStirring and soaking for 1h, washing with distilled water to neutrality, and draining;
s13: adding liquid nitrogen into the treated pigskin fragments and grinding the pigskin fragments into powder.
Preferably, H in step S122O2The addition amount of (B) is 0.8% -1.2%.
Preferably, the concentration of the NaOH solution in step S12 is 0.01-0.02 mol/L.
Preferably, the enzymolysis membrane circulation separation comprises the following specific steps:
s21: preparing a reaction tank A and a reaction tank B, mixing pigskin powder with water according to a ratio of 1:2, adding the mixture into the reaction tank A, adding a mixture of immobilized neutral protease, alpha amylase and cellulase which take chitosan as a carrier into the reaction tank A, and adjusting the reaction temperature to 48-52 ℃ and the pH to 6.5-7.0;
s22: after the reaction tank A reacts for 35-40min, the mixed solution is sequentially added into a peristaltic pump A, B, C, the flow rate of the peristaltic pump A is adjusted to be 200mL/min, the flow rate of the peristaltic pump B, C is adjusted to be 100mL/min, the mixed solution passes through a hollow fiber membrane A, the molecular interception size is 20KD, and the filtration volume/membrane area ratio is 450mL/m2Pressurizing the filter membrane to 0.01MPa, adjusting the reaction temperature to 37-40 ℃ and the PH to 7.0-7.5;
s23: adding 2.8-3.3% of chitosan immobilized alkaline protease and papain mixture into the reaction tank B, and carrying out secondary enzymolysis at 50-55 ℃ and pH of 8.5-9.0;
s24: conveying the mixture 20min after the peristaltic pump C is started into a peristaltic pump D, E, F, adjusting the flow rate of the peristaltic pump D to be 100mL/min, adjusting the flow rate of the peristaltic pump E, F to be 50mL/min, passing the mixture through a hollow fiber membrane B, wherein the molecular interception size is 5KD, the filtration volume/membrane area ratio is 350mL/m2, pressurizing a filter membrane to be 0.01MPa, adjusting the reaction temperature to be 37-40 ℃ and the PH to be 7.0-7.5;
s25: passing through hollow fiber membrane C, the molecular interception size is 3KD, and the filtration volume/membrane area ratio is 300mL/m2Setting the pressure to be 0.01MPa, adjusting the reaction temperature to be 37-40 ℃ and the PH to be 7.0-7.5;
s26: intercepting collagen with target molecular weight of 2-5KD, and performing subsequent treatment.
Preferably, the amount of the mixture of immobilized neutral protease, alpha-amylase and cellulase added with chitosan as a carrier in step S21 is 4.0-4.3%.
Preferably, the ratio of neutral protease, alpha amylase and cellulase in step S21 is 5: 1.5: 1.
preferably, the ratio of the alkaline protease to the papain added in step S23 is 1: 1.2.
preferably, the specific steps of the decoloring and deodorization treatment are as follows:
s31: adjusting pH to 4.0, adding 0.55% active carbon, controlling temperature at 40 deg.C, and decolorizing for 35-40 min;
s32: ultrafiltering to remove active carbon, adding 1-1.2% yeast, pH4.5, and fermenting at 35 deg.C for 30-35 min;
s33: sterilizing at high temperature, and microfiltering to remove residual yeast to obtain collagen solution.
Preferably, the freeze-drying comprises the following specific steps: and (3) carrying out freeze drying treatment on the collagen solution obtained by decoloring and deodorization treatment to obtain collagen powder with the molecular weight of 2-5 KD.
Compared with the prior art, the invention has the beneficial effects that:
1. in the preparation method of the collagen with the molecular weight within a certain range of the pigskin, the pigskin pretreatment, the enzymolysis membrane circulation separation, the decoloration and deodorization treatment and the freeze drying are carried out through the specific combined process steps and parameter conditions, so as to obtain the collagen powder with the molecular weight of 2-5KD, and the collagen powder has the following characteristics: the three-strand helical structure for preparing the pigskin collagen is not damaged, and the original biological activity of the pigskin collagen is still kept; the main component is 2-5KD collagen powder, the water solubility is good, and the collagen powder can be diluted by water in any proportion; the pigskin collagen is derived from vertebrate collagen, has good homology and compatibility with human body and skin, and has similar molecular weight and structure; the collagen polypeptide with molecular weight less than 5kDa has various physiological activities, such as lowering blood pressure and resisting oxidation.
2. The pigskin collagen prepared by the preparation method of the pigskin collagen with a molecular weight within a certain range has the denaturation temperature of about 36.5 ℃, can be biodegraded into small molecular polypeptides and single peptides under the action of the normal body temperature and in vivo collagenase after being absorbed by human bodies and skins, can be continuously decomposed into more than twenty kinds of amino acids under the action of other enzymes, is easy to absorb and continuously exerts the nutrition function; the pigskin collagen can enrich blood, improve skin elasticity, and improve skin moisture and oil content. The human body feeding test result shows that the skin moisture is averagely increased by 20 percent and the oil content is increased by 10 percent after the pig skin collagen is taken for 45 days, the conditions of the skin moisture and the oil content are improved, the total effective rate of a test group reaches 60 percent, and the pig skin collagen can be used for preparing surgical implants and tissue engineering medical products, developing medicines, medical reagents, health care products and the like.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides the following technical scheme:
example 1
A preparation method of collagen with a molecular weight within a certain range from pigskin comprises the following specific steps:
s1: pretreating pigskin;
s2: performing enzymolysis membrane circulation separation;
s3: decoloring and deodorizing;
s4: and (5) freeze drying.
The specific steps of the pigskin pretreatment in this example are as follows:
s11: removing hair from fresh pigskin, cleaning to remove epidermis, and removing fat layer;
s12: adding H according to the proportion of 1:202O2Stirring and soaking the NaOH solution at the temperature of 20 ℃ for 1 hour, washing the solution to be neutral by distilled water, and draining;
s13: adding liquid nitrogen into the treated pigskin fragments and grinding the pigskin fragments into powder.
Specifically, step S12 is H2O2The amount of (B) was 0.8%, and the concentration of the NaOH solution in step S12 was 0.01 mol/L.
The enzymolysis membrane in this embodiment is separated in a circulating manner, and comprises the following specific steps:
s21: preparing a reaction tank A and a reaction tank B, mixing pigskin powder with water according to a ratio of 1:2, adding the mixture into the reaction tank A, adding a mixture of immobilized neutral protease, alpha amylase and cellulase which take chitosan as a carrier into the reaction tank A, and adjusting the reaction temperature to 48 ℃ and the pH to 6.5;
s22: after the reaction tank A reacts for 35-40min, the mixed solution is sequentially added into a peristaltic pump A, B, C, the flow rate of the peristaltic pump A is adjusted to be 200mL/min, the flow rate of the peristaltic pump B, C is adjusted to be 100mL/min, the mixed solution passes through a hollow fiber membrane A, the molecular interception size is 20KD, and the filtration volume/membrane area ratio is 450mL/m2Pressurizing the filter membrane by 0.01MPa, adjusting the reaction temperature to 37 ℃ and the PH value to 7.0;
s23: adding 2.8% of a mixture of chitosan immobilized alkaline protease and papain into the reaction tank B, and carrying out secondary enzymolysis at the reaction temperature of 50 ℃ and the pH of 8.5;
s24: conveying the mixture 20min after the peristaltic pump C is started into the peristaltic pump D, E, F, adjusting the flow rate of the peristaltic pump D to be 100mL/min, the flow rate of the peristaltic pump E, F to be 50mL/min, passing through the hollow fiber membrane B, and keeping the molecular interception size to be 5KD, and the filtration volume/membrane area ratio to be 350mL/m2Pressurizing the filter membrane by 0.01MPa, adjusting the reaction temperature to 37 ℃ and the PH value to 7.0;
s25: passing through hollow fiber membrane C, the molecular interception size is 3KD, and the filtration volume/membrane area ratio is 300mL/m2Setting the pressure to be 0.01MPa, adjusting the reaction temperature to be 37 ℃ and the PH to be 7.0;
s26: intercepting collagen with target molecular weight of 2-5KD, and performing subsequent treatment.
Specifically, the amount of the mixture of immobilized neutral protease, alpha amylase and cellulase added with chitosan as a carrier in step S21 is 4.0%, and the ratio of neutral protease, alpha amylase and cellulase in step S21 is 5: 1.5: 1, the ratio of the alkaline protease to the papain added in the step S23 is 1: 1.2.
the specific steps of the decoloring and deodorization treatment in this embodiment are as follows:
s31: adjusting pH to 4.0, adding 0.55% active carbon, controlling temperature at 40 deg.C, and decolorizing for 35 min;
s32: ultrafiltering to remove active carbon, adding 1% yeast, pH4.5, and fermenting at 35 deg.C for 30 min;
s33: sterilizing at high temperature, and microfiltering to remove residual yeast to obtain collagen solution.
The freeze-drying in this example specifically comprises the steps of: and (3) carrying out freeze drying treatment on the collagen solution obtained by decoloring and deodorization treatment to obtain collagen powder with the molecular weight of 2-5 KD.
Example 2
A preparation method of collagen with a molecular weight within a certain range of pigskin, which is a preparation method of collagen with a molecular weight within a certain range of pigskin, comprises the following steps:
s1: pretreating pigskin;
s2: performing enzymolysis membrane circulation separation;
s3: decoloring and deodorizing;
s4: and (5) freeze drying.
The specific steps of the pigskin pretreatment in this example are as follows:
s11: removing hair from fresh pigskin, cleaning to remove epidermis, and removing fat layer;
s12: adding H according to the proportion of 1:202O2Stirring and soaking the NaOH solution at 15 ℃ for 1h, washing the solution to be neutral by distilled water, and draining;
s13: adding liquid nitrogen into the treated pigskin fragments and grinding the pigskin fragments into powder.
Specifically, step S12 is H2O2The amount of (B) was 1%, and the concentration of the NaOH solution in step S12 was 0.01 mol/L.
The enzymolysis membrane in this embodiment is separated in a circulating manner, and comprises the following specific steps:
s21: preparing a reaction tank A and a reaction tank B, mixing pigskin powder with water according to a ratio of 1:2, adding the mixture into the reaction tank A, adding a mixture of immobilized neutral protease, alpha amylase and cellulase which take chitosan as a carrier into the reaction tank A, and adjusting the reaction temperature to be 50 ℃ and the pH value to be 6.6;
s22: after the reaction tank A reacts for 40min, the mixed solution is sequentially added into a peristaltic pump A, B, C, the flow rate of the peristaltic pump A is adjusted to 200mL/min, the flow rate of the peristaltic pump B, C is adjusted to 100mL/min, the mixed solution passes through a hollow fiber membrane A, the molecular interception size is 20KD, and the filtration volume/membrane area ratio is 450mL/m2Pressurizing the filter membrane by 0.01MPa, adjusting the reaction temperature to 38 ℃ and the PH to 7.2;
s23: adding a mixture of chitosan immobilized alkaline protease and papain in an amount of 3% into the reaction tank B, and carrying out secondary enzymolysis at a reaction temperature of 52 ℃ and a pH of 8.6;
s24: conveying the mixture 20min after the peristaltic pump C is started into the peristaltic pump D, E, F, adjusting the flow rate of the peristaltic pump D to be 100mL/min, the flow rate of the peristaltic pump E, F to be 50mL/min, passing through the hollow fiber membrane B, and keeping the molecular interception size to be 5KD, and the filtration volume/membrane area ratio to be 350mL/m2Pressurizing the filter membrane by 0.01MPa, adjusting the reaction temperature to 38 ℃ and the PH to 7.2;
s25: passing through hollow fiber membrane C, the molecular interception size is 3KD, and the filtration volume/membrane area ratio is 300mL/m2Setting the pressure to be 0.01MPa, adjusting the reaction temperature to be 38 ℃ and the PH to be 7.2;
s26: intercepting collagen with target molecular weight of 2-5KD, and performing subsequent treatment.
Specifically, the amount of the mixture of immobilized neutral protease, alpha amylase and cellulase added with chitosan as a carrier in step S21 is 4.2%, and the ratio of neutral protease, alpha amylase and cellulase in step S21 is 5: 1.5: 1, the ratio of the alkaline protease to the papain added in the step S23 is 1: 1.2.
the specific steps of the decoloring and deodorization treatment in this embodiment are as follows:
s31: adjusting pH to 4.0, adding 0.55% active carbon, controlling temperature at 40 deg.C, and decolorizing for 35-40 min;
s32: ultrafiltering to remove active carbon, adding 1.1% yeast, pH4.5, and fermenting at 35 deg.C for 32 min;
s33: sterilizing at high temperature, and microfiltering to remove residual yeast to obtain collagen solution.
The freeze-drying in this example specifically comprises the steps of: and (3) carrying out freeze drying treatment on the collagen solution obtained by decoloring and deodorization treatment to obtain collagen powder with the molecular weight of 2-5 KD.
Example 3
A preparation method of collagen with a molecular weight within a certain range from pigskin comprises the following specific steps:
s1: pretreating pigskin;
s2: performing enzymolysis membrane circulation separation;
s3: decoloring and deodorizing;
s4: and (5) freeze drying.
The specific steps of the pigskin pretreatment in this example are as follows:
s11: removing hair from fresh pigskin, cleaning to remove epidermis, and removing fat layer;
s12: adding H according to the proportion of 1:202O2Stirring and soaking the NaOH solution at the temperature of 20 ℃ for 1 hour, washing the solution to be neutral by distilled water, and draining;
s13: adding liquid nitrogen into the treated pigskin fragments and grinding the pigskin fragments into powder.
Specifically, step S12 is H2O2The amount of (B) was 1.2%, and the concentration of the NaOH solution in step S12 was 0.02 mol/L.
The enzymolysis membrane in this embodiment is separated in a circulating manner, and comprises the following specific steps:
s21: preparing a reaction tank A and a reaction tank B, mixing pigskin powder with water according to a ratio of 1:2, adding the mixture into the reaction tank A, adding a mixture of immobilized neutral protease, alpha amylase and cellulase which take chitosan as a carrier into the reaction tank A, and adjusting the reaction temperature to be 50 ℃ and the pH value to be 6.6;
s22: after the reaction tank A reacts for 40min, the mixed solution is sequentially added into a peristaltic pump A, B, C, the flow rate of the peristaltic pump A is adjusted to 200mL/min, the flow rate of the peristaltic pump B, C is adjusted to 100mL/min, the mixed solution passes through a hollow fiber membrane A, the molecular interception size is 20KD, and the filtration volume/membrane area ratio is 450mL/m2Pressurizing the filter membrane by 0.01MPa, adjusting the reaction temperature to 40 ℃ and the PH to 7.5;
s23: adding a mixture of chitosan immobilized alkaline protease and papain in an amount of 3% into the reaction tank B, and carrying out secondary enzymolysis at a reaction temperature of 52 ℃ and a pH of 8.6;
s24: conveying the mixture 20min after the peristaltic pump C is started into the peristaltic pump D, E, F, adjusting the flow rate of the peristaltic pump D to be 100mL/min, the flow rate of the peristaltic pump E, F to be 50mL/min, passing through the hollow fiber membrane B, and keeping the molecular interception size to be 5KD, and the filtration volume/membrane area ratio to be 350mL/m2Pressurizing the filter membrane by 0.01MPa, adjusting the reaction temperature to 38 ℃ and the PH to 7.2;
s25: passing through hollow fiber membrane C, the molecular interception size is 3KD, and the filtration volume/membrane area ratio is 300mL/m2Setting the pressure to be 0.01MPa, adjusting the reaction temperature to be 38 ℃ and the PH to be 7.2;
s26: intercepting collagen with target molecular weight of 2-5KD, and performing subsequent treatment.
Specifically, the amount of the mixture of immobilized neutral protease, alpha amylase and cellulase added with chitosan as a carrier in step S21 is 4.2%, and the ratio of neutral protease, alpha amylase and cellulase in step S21 is 5: 1.5: 1, the ratio of the alkaline protease to the papain added in the step S23 is 1: 1.2.
the specific steps of the decoloring and deodorization treatment in this embodiment are as follows:
s31: adjusting pH to 4.0, adding 0.55% active carbon, controlling temperature at 40 deg.C, and decolorizing for 35-40 min;
s32: ultrafiltering to remove active carbon, adding 1.2% yeast, pH4.5, and fermenting at 32 deg.C for 35 min;
s33: sterilizing at high temperature, and microfiltering to remove residual yeast to obtain collagen solution.
The freeze-drying in this example specifically comprises the steps of: and (3) carrying out freeze drying treatment on the collagen solution obtained by decoloring and deodorization treatment to obtain collagen powder with the molecular weight of 2-5 KD.
The skin moisture, oil and skin absorption rates of the pigskin collagen produced by the preparation method of the pigskin collagen with a certain range of molecular weight of the three examples of the invention and the existing collagen after 45 days of administration are shown in the following table.
| Contrast parameter | Increase in skin moisture content/%) | Oil content increase/%) | Skin absorption rate/%) |
| Example 1 | 18.36-23.58 | 9.75-10.69 | 87.67-91.53 |
| Example 2 | 19.68-22.79 | 9.46-10.36 | 88.63-90.75 |
| Example 3 | 18.56-21.97 | 9.97-11.23 | 87.68-92.01 |
| Existing | 6.36-7.25 | 3.95-4.65 | 39.85-41.23 |
As can be seen from the above table, the preparation method of collagen with molecular weight in a certain range of pigskin of the invention carries out pigskin pretreatment, enzymolysis membrane circular separation, decoloration and deodorization treatment and freeze drying through specific combined process steps and parameter conditions to obtain collagen powder with molecular weight of 2-5KD, and the collagen powder has the following characteristics: the three-strand helical structure for preparing the pigskin collagen is not damaged, and the original biological activity of the pigskin collagen is still kept; the main component is 2-5KD collagen powder, the water solubility is good, the collagen powder can be diluted by water in any proportion, and the collagen polypeptide with the molecular weight lower than 5KDA has various physiological activities, such as blood pressure reduction and oxidation resistance; the collagen from the vertebrate has good homology and compatibility with human body and skin, and the molecular weight and the structure are similar; after being applied to the skin, the product can be quickly dissolved into the epidermis; the denaturation temperature of the pigskin collagen is about 36.5 ℃, after being absorbed by human body and skin, the pigskin collagen can be biodegraded into small molecular polypeptide and single peptide under the action of normal body temperature and in vivo collagenase, can be continuously decomposed into more than twenty kinds of amino acid under the action of other enzymes, is easy to be absorbed, and continuously plays a role in nutrition; the pigskin collagen can enrich blood, improve skin elasticity, and improve skin moisture and oil content. The human body feeding test result shows that the skin moisture is averagely increased by 20 percent and the oil content is increased by 10 percent after the pig skin collagen is taken for 45 days, the conditions of the skin moisture and the oil content are improved, the total effective rate of a test group reaches 60 percent, and the pig skin collagen can be used for preparing surgical implants and tissue engineering medical products, developing medicines, medical reagents, health care products and the like.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A preparation method of collagen with a molecular weight within a certain range from pigskin is characterized by comprising the following steps: the method comprises the following specific steps:
s1: pretreating pigskin;
s2: performing enzymolysis membrane circulation separation;
s3: decoloring and deodorizing;
s4: and (5) freeze drying.
2. The method of claim 1, wherein the collagen is derived from a range of pig skin molecular weights: the specific steps of the pigskin pretreatment are as follows:
s11: removing hair from fresh pigskin, cleaning to remove epidermis, and removing fat layer;
s12: adding H according to the proportion of 1:202O2Stirring and soaking the NaOH solution at the temperature of between 10 and 25 ℃ for 1 hour, washing the solution to be neutral by distilled water, and draining the water;
s13: adding liquid nitrogen into the treated pigskin fragments and grinding the pigskin fragments into powder.
3. The method of claim 1, wherein the collagen is derived from a range of pig skin molecular weights: h in step S122O2The addition amount of (B) is 0.8% -1.2%.
4. The method of claim 1, wherein the collagen is derived from a range of pig skin molecular weights: the concentration of the NaOH solution in the step S12 is 0.01-0.02 mol/L.
5. The method of claim 1, wherein the collagen is derived from a range of pig skin molecular weights: the enzymolysis membrane circulation separation comprises the following specific steps:
s21: preparing a reaction tank A and a reaction tank B, mixing pigskin powder with water according to a ratio of 1:2, adding the mixture into the reaction tank A, adding a mixture of immobilized neutral protease, alpha amylase and cellulase which take chitosan as a carrier into the reaction tank A, and adjusting the reaction temperature to 48-52 ℃ and the pH to 6.5-7.0;
s22: after the reaction tank A reacts for 35-40min, the mixed solution is sequentially added into a peristaltic pump A, B, C, the flow rate of the peristaltic pump A is adjusted to be 200mL/min, the flow rate of the peristaltic pump B, C is adjusted to be 100mL/min, and the mixture passes through hollow fibersMembrane A, molecular cut-off size 20KD, filtration volume/membrane area ratio 450mL/m2Pressurizing the filter membrane to 0.01MPa, adjusting the reaction temperature to 37-40 ℃ and the PH to 7.0-7.5;
s23: adding 2.8-3.3% of chitosan immobilized alkaline protease and papain mixture into the reaction tank B, and carrying out secondary enzymolysis at 50-55 ℃ and pH of 8.5-9.0;
s24: conveying the mixture 20min after the peristaltic pump C is started into a peristaltic pump D, E, F, adjusting the flow rate of the peristaltic pump D to be 100mL/min, adjusting the flow rate of the peristaltic pump E, F to be 50mL/min, passing the mixture through a hollow fiber membrane B, wherein the molecular interception size is 5KD, the filtration volume/membrane area ratio is 350mL/m2, pressurizing a filter membrane to be 0.01MPa, adjusting the reaction temperature to be 37-40 ℃ and the PH to be 7.0-7.5;
s25: passing through hollow fiber membrane C, the molecular interception size is 3KD, and the filtration volume/membrane area ratio is 300mL/m2Setting the pressure to be 0.01MPa, adjusting the reaction temperature to be 37-40 ℃ and the PH to be 7.0-7.5;
s26: intercepting collagen with target molecular weight of 2-5KD, and performing subsequent treatment.
6. The method of claim 5, wherein the collagen is derived from pig skin over a range of molecular weights, and wherein: the amount of the mixture of immobilized neutral protease, alpha amylase and cellulase added with chitosan as a carrier in the step S21 is 4.0-4.3%.
7. The method of claim 5, wherein the collagen is derived from pig skin over a range of molecular weights, and wherein: the ratio of neutral protease, alpha amylase and cellulase in step S21 is 5: 1.5: 1.
8. the method of claim 5, wherein the collagen is derived from pig skin over a range of molecular weights, and wherein: the ratio of the alkaline protease to the papain added in step S23 is 1: 1.2.
9. the method of claim 1, wherein the collagen is derived from a range of pig skin molecular weights: the specific steps of the decoloring and deodorization treatment are as follows:
s31: adjusting pH to 4.0, adding 0.55% active carbon, controlling temperature at 40 deg.C, and decolorizing for 35-40 min;
s32: ultrafiltering to remove active carbon, adding 1-1.2% yeast, pH4.5, and fermenting at 35 deg.C for 30-35 min;
s33: sterilizing at high temperature, and microfiltering to remove residual yeast to obtain collagen solution.
10. The method of claim 9, wherein the collagen is derived from a range of pig skin molecular weights: the freeze drying comprises the following specific steps: and (3) carrying out freeze drying treatment on the collagen solution obtained by decoloring and deodorization treatment to obtain collagen powder with the molecular weight of 2-5 KD.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317661A (en) * | 2022-02-15 | 2022-04-12 | 无锡贝迪生物工程股份有限公司 | Method for preparing multiple active collagen |
| WO2023115912A1 (en) * | 2021-12-24 | 2023-06-29 | 杭州华迈医疗科技有限公司 | Preparation method for decellularized matrix biomaterial |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367461A (en) * | 2016-08-26 | 2017-02-01 | 湖州佳美生物化学制品有限公司 | Method for extracting low molecular weight active collagen peptide from pigskins |
| CN108545835A (en) * | 2018-04-16 | 2018-09-18 | 成都大学 | A method of taking off protein in yellow serofluid using enzyme mebrane reactor ultrafiltration |
| CN109627325A (en) * | 2019-01-04 | 2019-04-16 | 广东海洋大学 | A kind of preparation method and applications of gradient collagen and collagen polypeptide molecule |
| CN110151566A (en) * | 2019-05-20 | 2019-08-23 | 上海应用技术大学 | A kind of skin care freeze-dried powder of the polypeptide containing pigskin collagen and its preparation |
| US20200222585A1 (en) * | 2019-01-11 | 2020-07-16 | Infinitus (China) Company Ltd. | Composition of collagen peptide and elastin peptide, method of producing the same and use thereof |
-
2020
- 2020-12-03 CN CN202011410186.XA patent/CN112608380A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367461A (en) * | 2016-08-26 | 2017-02-01 | 湖州佳美生物化学制品有限公司 | Method for extracting low molecular weight active collagen peptide from pigskins |
| CN108545835A (en) * | 2018-04-16 | 2018-09-18 | 成都大学 | A method of taking off protein in yellow serofluid using enzyme mebrane reactor ultrafiltration |
| CN109627325A (en) * | 2019-01-04 | 2019-04-16 | 广东海洋大学 | A kind of preparation method and applications of gradient collagen and collagen polypeptide molecule |
| US20200222585A1 (en) * | 2019-01-11 | 2020-07-16 | Infinitus (China) Company Ltd. | Composition of collagen peptide and elastin peptide, method of producing the same and use thereof |
| CN110151566A (en) * | 2019-05-20 | 2019-08-23 | 上海应用技术大学 | A kind of skin care freeze-dried powder of the polypeptide containing pigskin collagen and its preparation |
Non-Patent Citations (1)
| Title |
|---|
| 王芳等: "猪皮胶原蛋白多肽膜的制备及性能测定", 《海峡药学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023115912A1 (en) * | 2021-12-24 | 2023-06-29 | 杭州华迈医疗科技有限公司 | Preparation method for decellularized matrix biomaterial |
| CN114317661A (en) * | 2022-02-15 | 2022-04-12 | 无锡贝迪生物工程股份有限公司 | Method for preparing multiple active collagen |
| CN114317661B (en) * | 2022-02-15 | 2024-04-09 | 无锡贝迪生物工程股份有限公司 | Method for preparing multiple active collagen |
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Inventor after: Niu Haitao Inventor after: Liang Ye Inventor after: Wang Yonghua Inventor after: Li Dan Inventor before: Niu Haitao Inventor before: Liang Ye Inventor before: Wang Yonghua Inventor before: Li Dan |
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