CN114129712A - Whitening collagen peptide freeze-dried powder and preparation method thereof - Google Patents
Whitening collagen peptide freeze-dried powder and preparation method thereof Download PDFInfo
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- CN114129712A CN114129712A CN202111547757.9A CN202111547757A CN114129712A CN 114129712 A CN114129712 A CN 114129712A CN 202111547757 A CN202111547757 A CN 202111547757A CN 114129712 A CN114129712 A CN 114129712A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 56
- 108010035532 Collagen Proteins 0.000 title claims abstract description 56
- 229920001436 collagen Polymers 0.000 title claims abstract description 56
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 51
- 239000000843 powder Substances 0.000 title claims abstract description 37
- 230000002087 whitening effect Effects 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 21
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims abstract description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
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- 229940088598 enzyme Drugs 0.000 claims description 22
- 238000003756 stirring Methods 0.000 claims description 22
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- 239000004365 Protease Substances 0.000 claims description 19
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 17
- 238000000108 ultra-filtration Methods 0.000 claims description 15
- 230000000415 inactivating effect Effects 0.000 claims description 14
- 239000012528 membrane Substances 0.000 claims description 14
- 108091005658 Basic proteases Proteins 0.000 claims description 10
- 102000057297 Pepsin A Human genes 0.000 claims description 10
- 108090000284 Pepsin A Proteins 0.000 claims description 10
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- 229960002376 chymotrypsin Drugs 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 229940111202 pepsin Drugs 0.000 claims description 10
- 108090000526 Papain Proteins 0.000 claims description 9
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 9
- 229940055729 papain Drugs 0.000 claims description 9
- 235000019834 papain Nutrition 0.000 claims description 9
- 235000019419 proteases Nutrition 0.000 claims description 9
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- 108090000631 Trypsin Proteins 0.000 claims description 2
- 102000004142 Trypsin Human genes 0.000 claims description 2
- 239000012588 trypsin Substances 0.000 claims description 2
- 102000003425 Tyrosinase Human genes 0.000 abstract description 8
- 108060008724 Tyrosinase Proteins 0.000 abstract description 8
- 201000001441 melanoma Diseases 0.000 abstract description 7
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- 241000283690 Bos taurus Species 0.000 description 2
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- 238000010521 absorption reaction Methods 0.000 description 2
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- 210000000434 stratum corneum Anatomy 0.000 description 2
- 206010008570 Chloasma Diseases 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
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- 239000000413 hydrolysate Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
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- 208000030159 metabolic disease Diseases 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 230000019612 pigmentation Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
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- 210000002435 tendon Anatomy 0.000 description 1
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0216—Solid or semisolid forms
- A61K8/022—Powders; Compacted Powders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- 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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- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
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Abstract
The invention belongs to the field of skin-care external drugs, and particularly relates to a whitening collagen peptide freeze-dried powder and a preparation method thereof. The content of protein in the whitening collagen peptide freeze-dried powder is more than or equal to 95 wt.%; wherein the content of protein peptide with molecular weight of 500-1000 in the protein is more than or equal to 95 wt.%; the content of short peptide containing tyrosine residue in the protein peptide is more than or equal to 50 wt.%. The invention provides a freeze-dried powder of a whitening collagen peptide, which has tyrosinase inhibition activity of over 50 percent and can reduce melanin content of melanoma cells by over 40 percent.
Description
Technical Field
The invention belongs to the field of skin-care external drugs, and particularly relates to a whitening collagen peptide freeze-dried powder and a preparation method thereof.
Background
The process of pigmentation of human skin is important, not only in determining the colour of the skin, eyes and hair, but also as a primary barrier to protect the skin from ultraviolet radiation. However, melanin metabolism disorder can cause pigmentation disorders such as stain, freckle, chloasma and melanoma. The melanin metabolism process includes the production of melanin, the transport of melanin from melanocytes to keratinocytes, and the distribution and degradation of melanin within keratinocytes. The simple process is as follows: first, under the control of genes, a structure of melanosome is formed, and tyrosinase reaches golgi body after synthesis of endoplasmic reticulum to perform saccharification processing, and then reaches stage ii melanosome carried by the transport vesicle. In melanosome, in vivo tyrosine is catalyzed by proteases such as tyrosinase and the like to synthesize melanin through a series of biochemical reaction processes, melanin particles are transported to surrounding keratinocytes through dendritic projections, and after the distribution of the keratinocytes, the melanin particles move to the stratum corneum along with the growth of epidermis, and finally the melanin particles are replaced and fall off along with the period of the stratum corneum.
Collagen is a natural protein, widely exists in skin, bone, cartilage, teeth, tendon ligament and blood vessel of animals, is the most abundant and widely distributed protein in the mammal body, is an important structural protein of connective tissue, plays the functions of supporting organs and protecting the body, and has strong biological activity and biological function. Collagen is a macromolecular protein and cannot be directly absorbed by the human body. Collagen peptide is a hydrolysate of collagen, contains all amino acids of collagen, and has good absorption, solubility, water absorption and the like which are not comparable to collagen.
At present, the variety of collagen peptide products is many, but products with good whitening effect are lacked.
Disclosure of Invention
Aiming at the problem of lack of the existing whitening collagen peptide product, the invention provides the whitening collagen peptide freeze-dried powder, the tyrosinase inhibition activity of the collagen peptide freeze-dried powder can reach more than 50%, and the melanin content of melanoma cells can be reduced by more than 40%.
The invention also provides a preparation method of the collagen peptide freeze-dried powder, the preparation method can utilize collagen from various sources, and the prepared collagen peptide freeze-dried powder has high whitening activity, simple process and easy operation and can be industrially produced in a large scale through twice complex enzyme enzymolysis.
In order to realize the purpose, the invention adopts the following technical scheme:
a freeze-dried powder of whitening collagen peptide, wherein the content of protein in the freeze-dried powder is more than or equal to 95 wt.%; wherein the content of protein peptide with molecular weight of 500-1000 in the protein is more than or equal to 95 wt.%; the content of short peptide containing tyrosine residue in the protein peptide is more than or equal to 50 wt.%.
A preparation method of the whitening collagen peptide freeze-dried powder comprises the following steps:
(1) preparing raw materials: adding collagen into water, adjusting pH and stirring to prepare a solution;
(2) primary enzymolysis: adding pepsin and papain into the solution obtained in the step (1), adjusting pH, stirring, controlling temperature for enzymolysis, inactivating enzyme, and adjusting pH to be neutral;
(3) membrane separation: carrying out ultrafiltration treatment on the enzymolysis liquid obtained in the step (2), and collecting trapped liquid;
(4) secondary enzymolysis: adding the trapped fluid obtained in the step (3) into trypsin/chymotrypsin composite protease and alkaline protease, adjusting pH and stirring, controlling temperature for enzymolysis, and inactivating enzyme; the molecular weight of the collagen peptide is reduced through secondary enzymolysis, so that the collagen peptide is convenient to absorb;
(5) vacuum freeze drying: and (4) carrying out vacuum freeze drying on the enzymolysis liquid obtained in the step (4), wherein the dried powder is the whitening collagen peptide freeze-dried powder.
Preferably, the collagen of step (1) is derived from animal skin, bone or fish scale; the pH value in the step (1) is 3-4.
Preferably, step (1) employs acetic acid to adjust the pH.
Preferably, the adding amount of the pepsin and the papain in the step (2) is 0.3-1.0% of the mass of the collagen in the step (1), and the adding amount of the pepsin: the papain is 1:1-1:3, the pH is 1.5-2.5, the enzymolysis temperature is 30-40 ℃, and the enzymolysis time is 0.5-2 hours.
Preferably, the ultrafiltration membrane in the step (3) has the cut-off molecular weight of 1500Da, and short peptides released by primary enzymolysis are removed by ultrafiltration, so that peptide fragments containing Tyr residues are enriched.
The adding amount of the trypsin-chymotrypsin complex protease and the alkaline protease in the step (4) is 1.0-2.0% of the mass of the collagen in the step (1), and the adding amount of the trypsin-chymotrypsin complex protease is as follows: the alkaline protease is 3:1-2:1, the pH is 7.5-8.5, the enzymolysis temperature is 30-40 ℃, and the enzymolysis time is 1-3 hours.
In the present invention, the vacuum freeze-drying is a conventional technique in the art, and a person skilled in the art can select conditions according to needs, and is not particularly limited herein.
Compared with the prior art, the invention has at least the following beneficial effects: the preparation method can prepare the freeze-dried powder of the whitening collagen peptide, can utilize collagen from various sources, has high whitening activity, can reduce the melanin content of melanoma cells by more than 40 percent, and has simple process, easy operation and industrialized large-scale production, and the tyrosinase inhibition activity can reach more than 50 percent.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention and all modifications or alterations to the methods, procedures or conditions of the present invention which do not depart from the spirit and substance of the invention are deemed to be within the scope of the invention.
Example 1
A preparation method of a collagen peptide freeze-dried powder for whitening skin comprises the following steps:
(1) preparing raw materials: adding 100g of pigskin collagen into water, adjusting the pH value to 3.0 by using acetic acid, and stirring to prepare a solution;
(2) primary enzymolysis: adding 0.15g of pepsin and 0.15g of papain into the solution obtained in the step (1), adjusting the pH value to 1.5, stirring, controlling the temperature to 30 ℃ for enzymolysis for 0.5 hour, inactivating enzyme, and adjusting the pH value to be neutral;
(3) membrane separation: carrying out ultrafiltration treatment on the enzymolysis liquid obtained in the step (2) by using an ultrafiltration membrane with the molecular weight cutoff of 1500Da, and collecting the cutoff liquid;
(4) secondary enzymolysis: adding 0.75g of trypsin-chymotrypsin complex protease and 0.25g of alkaline protease into the trapped fluid obtained in the step (3), adjusting the pH to 7.5, stirring, controlling the temperature to 30 ℃, performing enzymolysis for 1 hour, and inactivating the enzyme;
(5) vacuum freeze drying: and (4) carrying out vacuum freeze drying on the enzymolysis liquid obtained in the step (4), wherein the dried powder is the whitening collagen peptide freeze-dried powder.
Through detection, the content of the protein in the prepared collagen peptide freeze-dried powder is 95.4 wt.%, wherein the content of the protein peptide with the molecular weight of 500-1000 is 96.1 wt.%, the content of the short peptide containing tyrosine residue is 51.3 wt.%, the tyrosinase inhibitory activity is 52.4%, and the melanin content of melanoma cells can be reduced by 41.3%.
Example 2
A preparation method of a collagen peptide freeze-dried powder for whitening skin comprises the following steps:
(1) preparing raw materials: adding 100g bovine bone collagen into water, adjusting pH to 4.0 with acetic acid, and stirring to prepare a solution;
(2) primary enzymolysis: adding 0.2g of pepsin and 0.4g of papain into the solution obtained in the step (1), adjusting the pH to 2.5, stirring, controlling the temperature to be 40 ℃ for enzymolysis for 2 hours, inactivating enzymes, and adjusting the pH to be neutral;
(3) membrane separation: carrying out ultrafiltration treatment on the enzymolysis liquid obtained in the step (2) by using an ultrafiltration membrane with the molecular weight cutoff of 1500Da, and collecting the cutoff liquid;
(4) secondary enzymolysis: adding 1.5g of trypsin-chymotrypsin complex protease and 0.5g of alkaline protease into the trapped fluid obtained in the step (3), adjusting the pH to 8.5, stirring, controlling the temperature to be 40 ℃, performing enzymolysis for 3 hours, and inactivating the enzyme;
(5) vacuum freeze drying: and (4) carrying out vacuum freeze drying on the enzymolysis liquid obtained in the step (4), wherein the dried powder is the whitening collagen peptide freeze-dried powder.
Through detection, the content of the protein in the prepared collagen peptide freeze-dried powder is 96.7 wt.%, wherein the content of the protein peptide with the molecular weight of 500-1000 is 98.3 wt.%, the content of the short peptide containing tyrosine residue is 50.8 wt.%, the tyrosinase inhibitory activity is 51.2%, and the melanin content of melanoma cells can be reduced by 40.7%.
Example 3
A preparation method of a collagen peptide freeze-dried powder for whitening skin comprises the following steps:
(1) preparing raw materials: adding 100g of fish scale collagen into water, adjusting the pH value to 3.5 by using acetic acid, and stirring to prepare a solution;
(2) primary enzymolysis: adding 0.35g of pepsin and 0.35g of papain into the solution obtained in the step (1), adjusting the pH to 2.0, stirring, controlling the temperature to 35 ℃ for enzymolysis for 1 hour, inactivating enzyme, and adjusting the pH to be neutral;
(3) membrane separation: carrying out ultrafiltration treatment on the enzymolysis liquid obtained in the step (2) by using an ultrafiltration membrane with the molecular weight cutoff of 1500Da, and collecting the cutoff liquid;
(4) secondary enzymolysis: adding 1.0g of trypsin-chymotrypsin complex protease and 0.5g of alkaline protease into the trapped fluid obtained in the step (3), adjusting the pH to 8.0, stirring, controlling the temperature to 35 ℃ for enzymolysis for 2 hours, and inactivating the enzyme;
(5) vacuum freeze drying: and (4) carrying out vacuum freeze drying on the enzymolysis liquid obtained in the step (4), wherein the dried powder is the whitening collagen peptide freeze-dried powder.
Through detection, the content of the protein in the prepared collagen peptide freeze-dried powder is 97.3 wt.%, wherein the content of the protein peptide with the molecular weight of 500-1000 is 98.1 wt.%, the content of the short peptide containing tyrosine residue is 53.4 wt.%, the tyrosinase inhibitory activity is 55.1%, and the melanin content of melanoma cells can be reduced by 42.7%.
Comparative example 1
(1) Adding 100g of pigskin collagen into water, adjusting the pH value to 3.0 by using acetic acid, and stirring to prepare a solution;
(2) enzymolysis: adding 0.15g of pepsin and 0.15g of papain into the solution obtained in the step (1), adjusting the pH value to 1.5, stirring, controlling the temperature to 30 ℃ for enzymolysis for 0.5 hour, inactivating enzyme, and adjusting the pH value to be neutral;
(3) membrane separation: carrying out ultrafiltration treatment on the enzymolysis liquid obtained in the step (2) by using an ultrafiltration membrane with the molecular weight cutoff of 1500Da, and collecting the cutoff liquid;
(4) vacuum freeze drying: and (4) carrying out vacuum freeze drying on the trapped fluid obtained in the step (3), wherein the dried powder is the collagen peptide freeze-dried powder.
Comparative example 2
(1) Adding 100g bovine bone collagen into water, adjusting pH to 3.0 with acetic acid, and stirring to prepare a solution;
(2) enzymolysis: adding 0.75g of trypsin-chymotrypsin complex protease and 0.25g of alkaline protease into the solution obtained in the step (1), adjusting the pH to 7.5, stirring, controlling the temperature to 30 ℃ for enzymolysis for 1 hour, and inactivating the enzyme;
(3) membrane separation: carrying out ultrafiltration treatment on the enzymolysis liquid obtained in the step (2) by using an ultrafiltration membrane with the molecular weight cutoff of 1500Da, and collecting the cutoff liquid;
(4) vacuum freeze drying: and (4) carrying out vacuum freeze drying on the trapped fluid obtained in the step (3), wherein the dried powder is the collagen peptide freeze-dried powder.
Comparative example 3
(1) Adding 100g of fish scale collagen into water, adjusting the pH value to 3.0 by using acetic acid, and stirring to prepare a solution;
(2) primary enzymolysis: adding 0.3g of pepsin into the solution obtained in the step (1), adjusting the pH value to 1.5, stirring, controlling the temperature to be 30 ℃, performing enzymolysis for 0.5 hour, inactivating enzyme, and adjusting the pH value to be neutral;
(3) secondary enzymolysis: adding 0.75g of trypsin-chymotrypsin composite protease and 0.25g of alkaline protease into the enzymolysis liquid obtained in the step (2), adjusting the pH to 7.5, stirring, controlling the temperature to be 30 ℃ for enzymolysis for 1 hour, and inactivating the enzyme;
(5) vacuum freeze drying: and (4) carrying out vacuum freeze drying on the enzymolysis liquid obtained in the step (4), wherein the dried powder is the collagen peptide freeze-dried powder.
Table 1 comparison of collagen peptide lyophilized powder results table
Claims (7)
1. The lyophilized powder of whitening collagen peptide is characterized in that the content of protein in the lyophilized powder is more than or equal to 95 wt.%; wherein the content of protein peptide with molecular weight of 500-1000 in the protein is more than or equal to 95 wt.%; the content of short peptide containing tyrosine residue in the protein peptide is more than or equal to 50 wt.%.
2. The preparation method of the collagen whitening peptide freeze-dried powder of claim 1, characterized by comprising the following steps:
(1) preparing raw materials: adding collagen into water, adjusting pH and stirring to prepare a solution;
(2) primary enzymolysis: adding mixed enzyme of pepsin and papain into the solution obtained in the step (1), adjusting pH, stirring, controlling temperature for enzymolysis, inactivating enzyme, and adjusting pH to be neutral;
(3) membrane separation: carrying out ultrafiltration treatment on the enzymolysis liquid obtained in the step (2), and collecting trapped liquid;
(4) secondary enzymolysis: adding the trapped fluid obtained in the step (3) into mixed enzyme of trypsin-chymotrypsin composite protease and alkaline protease, adjusting pH and stirring, controlling temperature for enzymolysis, and inactivating enzyme;
(5) vacuum freeze drying: and (4) carrying out vacuum freeze drying on the enzymolysis liquid obtained in the step (4), wherein the dried powder is the whitening collagen peptide freeze-dried powder.
3. The method according to claim 2, wherein the collagen of step (1) is derived from animal skin, bone or fish scales; the pH value in the step (1) is 3-4.
4. The method according to claim 2, wherein the pH in the step (1) is adjusted with acetic acid.
5. The preparation method according to claim 2, wherein the addition amount of the mixed enzyme in the step (2) is 0.3-1.0% of the mass of the collagen in the step (1), the mass ratio of pepsin to papain in the mixed enzyme is 1:1-1:3, the pH is 1.5-2.5, the enzymolysis temperature is 30-40 ℃, and the enzymolysis time is 0.5-2 hours.
6. The method according to claim 2, wherein the ultrafiltration membrane of step (3) has a molecular weight cut-off of 1500 Da.
7. The preparation method of claim 2, wherein the addition amount of the mixed enzyme in the step (4) is 1.0-2.0% of the mass of the collagen in the step (1), the mass ratio of the trypsin/chymotrypsin complex enzyme to the alkaline protease in the mixed enzyme is 3:1-2:1, the pH value is 7.5-8.5, the enzymolysis temperature is 30-40 ℃, and the enzymolysis time is 1-3 hours.
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| CN114671937A (en) * | 2022-03-18 | 2022-06-28 | 大连工业大学 | Polypeptide with tyrosinase inhibitory activity and preparation method thereof |
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