CN114057833B - Mussel polypeptide and polypeptide powder based on melanin precipitation reducing effect, and preparation method and application thereof - Google Patents
Mussel polypeptide and polypeptide powder based on melanin precipitation reducing effect, and preparation method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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
-
- 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
- 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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- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Dermatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
The invention relates to the technical field of polypeptides, and discloses mussel polypeptides and polypeptide powder with melanin reducing effect, and a preparation method and application thereof, wherein the mussel polypeptides are short peptides or a mixture of at least two short peptides; the short peptide is one of AEELGIQ, GANSIEL, VTHCNVK, TSGFSDSGY, WTGFSFM, KHSVITG, EVDDAGG, LDVSWASD, FCENVCPY, TAGSTTCLD, DNMSIMV; the mussel polypeptide is obtained by sequentially carrying out pepsin and trypsin sectional enzymolysis, centrifugation, filtering and intercepting 500-1000 Da molecules, and then separating and purifying on the whole mussel tissue. The invention hydrolyzes mussel protein by using gastrointestinal homologous protease to obtain polypeptide with melanin reducing effect, and better original integrity is maintained.
Description
Technical Field
The invention relates to the technical field of polypeptides, in particular to mussel polypeptides and polypeptide powder with melanin reducing effect, and a preparation method and application thereof.
Background
Polypeptides are a class of compounds that bind to a target with greater specificity in vivo, making their biological activity more efficient and better applicable, and their unrelated side effects more limited. Peptides have smaller molecular sizes than proteins, and sophisticated methods of polypeptide chemical synthesis provide more and more economical yields. Furthermore, the shorter length of peptides suggests that their use induces bone growth stimulation not only in systemic injection but also orally for medical clinical convenience tablets. Mussels are high in nutrient content and contain 8 essential amino acids such as valine and leucine, which are essential to the human body. The protein and amino acid in the components are far higher than those of raw eggs, chickens, ducks, fish, shrimps and meat products. According to scientific research reports, mussel fat also contains oleic acid which is vital to human body. The saturated fat component of the sea chicken is lower than foods such as pigs, beef, mutton, milk and the like, and the unsaturated fat component is relatively more, so that mussels have the reputation of eggs in the sea.
Disclosure of Invention
To develop mussel protein resources, the invention aims at: providing mussel polypeptides having melanin-reducing efficacy, and polypeptide powders containing mussel polypeptides; preparation methods of mussel polypeptides and polypeptide powders; application of mussel polypeptide and polypeptide powder in preparing melanin-reducing face cream is provided.
The invention provides the following technical scheme:
in one aspect, the invention provides a mussel polypeptide with melanin reducing effect, wherein the mussel polypeptide is a short peptide or a mixture of at least two short peptides;
the short peptide is one of AEELGIQ, GANSIEL, VTHCNVK, TSGFSDSGY, WTGFSFM, KHSVITG, EVDDAGG, LDVSWASD, FCENVCPY, TAGSTTCLD, DNMSIMV;
the amino acid sequence of each short peptide is:
AEELGIQ：Ala-Glu-Glu-Leu-Gly-Ile-Gln；
GANSIEL：Gly-Ala-Asn-Ser-Ile-Glu-Leu；
VTHCNVK：Val-Thr-His-Cys-Asn-Val-Lys；
TSGFSDSGY：Thr-Ser-Gly-Phe-Ser-Asp-Ser-Gly-Tyr；
WTGFSFM：Trp-Thr-Gly-Phe-Ser-Phe-Met；
KHSVITG：Lys-His-Ser-Val-Ile-Thr-Gly；
EVDDAGG：Glu-Val-Asp-Asp-Ala-Gly-Gly；
LDVSWASD：Leu-Asp-Val-Ser-Trp-Ala-Ser-Asp；
FCENVCPY：Phe-Cys-Glu-Asn-Val-Cys-Pro-Tyr；
TAGSTTCLD：Thr-Ala-Gly-Ser-Thr-Thr-Cys-Leu-Asp；
DNMSIMV：Asp-Asn-Met-Ser-Ile-Met-Val。
the invention also provides a preparation method of the mussel polypeptide, which comprises the following steps:
(1) Removing shell of mussel, collecting whole tissue, cleaning, and homogenizing;
(2) Sequentially adding pepsin and trypsin into the homogenate of mussels for segmented enzymolysis;
(3) Centrifuging 10000g of the enzymolysis liquid obtained in the step (2), and then taking supernatant;
(4) Filtering the supernatant obtained in the step (3), intercepting molecules with the molecular weight of 500-1000 Da, and freeze-drying to obtain polypeptide powder
(5) And (3) separating and purifying the polypeptide powder to obtain the mussel polypeptide.
As the preference of the method, in the step (2), the pH value of the homogenate is kept to be 1-3 during the enzymolysis, the enzymolysis time is 2-4 hours, the enzymolysis temperature is 35-38 ℃, the pepsin addition amount is 2-4% of the weight of the protein in the homogenate, and the activity of the pepsin is more than or equal to 250U/mg.
As the preference of the method, when trypsin is added in the step (2), the pH value of the homogenate is kept at 7.0-8.6, the enzymolysis time is 1-2 hours, the enzymolysis temperature is 35-38 ℃, the adding amount of the trypsin is 1-3% of the weight of protein in the homogenate, and the activity of the trypsin is 10000U/mg.
The invention also provides polypeptide powder containing the mussel polypeptide.
The preparation method of the polypeptide powder comprises the following steps:
(1) Removing shell of mussel, collecting whole tissue, cleaning, and homogenizing;
(2) Sequentially adding pepsin and trypsin into the homogenate of mussels for segmented enzymolysis;
(3) Centrifuging the enzymolysis liquid obtained after enzymolysis in the step (2), and then taking supernatant;
(4) Filtering the supernatant obtained in the step (3), intercepting molecules with the molecular weight of 500-1000 Da, and freeze-drying to obtain polypeptide powder.
Preferably, the polypeptide powder obtained in the step (4) is a mixture of a plurality of short peptides, and the polypeptide powder obtained in the step (4) can be further separated and purified to obtain a single short peptide or a mixture of a plurality of short peptides.
As the preference of the method, in the step (2), the pH value of the homogenate is kept to be 1-3 during the enzymolysis, the enzymolysis time is 2-4 hours, the enzymolysis temperature is 35-38 ℃, the pepsin addition amount is 2-4% of the weight of the protein in the homogenate, and the activity of the pepsin is more than or equal to 250U/mg.
As the preference of the method, when trypsin is added in the step (2), the pH value of the homogenate is kept at 7.0-8.6, the enzymolysis time is 1-2 hours, the enzymolysis temperature is 35-38 ℃, the adding amount of the trypsin is 1-3% of the weight of protein in the homogenate, and the activity of the trypsin is 10000U/mg.
The invention also provides application of the mussel polypeptide or polypeptide powder in preparing melanin-reducing face cream.
The common enzymolysis methods of edible polypeptides are mainly alkaline protease, neutral protease, flavourzyme and the like, however, peptides obtained by hydrolyzing the enzymes have some defects in exerting biological effects in vivo, because the polypeptide of the human heterologous digestive enzyme can be hydrolyzed by protease in gastrointestinal tract for two or more times after being eaten, and the biological activity of the original polypeptide is difficult to ensure. The effect of many polypeptides in vitro activity assays is reduced in vivo. The polypeptide hydrolyzed by the gastrointestinal tract homologous protease can better reduce the possibility of re-hydrolysis, is beneficial to preserving the original integrity and prevents the activity change or reduction caused by the gastrointestinal tract enzyme in vivo.
The beneficial effects of the invention are as follows: the invention hydrolyzes mussel protein by using gastrointestinal homologous protease to obtain polypeptide with melanin reducing effect, and better original integrity is maintained.
Drawings
FIG. 1 is a LC-MS spectrum of polypeptide isolation and identification;
FIG. 2 shows the tyrosinase inhibition rates of enzymatically hydrolyzed polypeptides to the major active polypeptides.
Detailed Description
The following is a further description of embodiments of the invention.
Unless otherwise indicated, all starting materials used in the present invention are commercially available or are commonly used in the art, and unless otherwise indicated, the methods in the examples below are all conventional in the art.
Example 1
Mussel polypeptide with melanin reducing effect is prepared by the following steps:
(1) Removing shell of mussel, collecting whole tissue, cleaning, and homogenizing;
(2) Sequentially adding pepsin and trypsin into the homogenate of mussels for segmented enzymolysis;
(3) Centrifuging 10000g of the enzymolysis liquid obtained in the step (2), and then taking supernatant;
(4) Filtering the supernatant obtained in the step (3), intercepting molecules with the molecular weight of 500-1000 Da, and freeze-drying to obtain polypeptide powder;
(5) Polypeptide powder identification sequences are respectively short peptides: AEELGIQ, GANSIEL, VTHCNVK, TSGFSDSGY, WTGFSFM, KHSVITG, EVDDAGG, LDVSWASD, FCENVCPY, TAGSTTCLD, DNMSIMV.
In the step (2), when pepsin is subjected to enzymolysis, the pH value of the homogenate is kept at 1-3, the enzymolysis time is 2-4 hours, the enzymolysis temperature is 35-38 ℃, the added amount of the pepsin is 2-4% of the protein amount in the homogenate, and the activity of the pepsin is more than or equal to 250U/mg;
in the preferred implementation method of the invention, when pepsin is hydrolyzed, the pH value of the homogenate is kept to be 2.0, the enzymolysis time is 2 hours, the enzymolysis temperature is 37 ℃, the added amount of the pepsin is 2% of the mass of the homogenate, and the activity of the pepsin is more than or equal to 250U/mg.
And (3) when trypsin is added for enzymolysis in the step (2), the pH value of the homogenate is kept at 7.0-8.6, the enzymolysis time is 1-2 hours, the enzymolysis temperature is 35-38 ℃, the adding amount of the trypsin is 1-3% of the protein amount in the homogenate, and the activity of the trypsin is 10000U/mg. In the preferred embodiment of the invention, when trypsin is added for enzymolysis, the pH value of the homogenate is kept at 8.6, the enzymolysis time is 2 hours, the enzymolysis temperature is 37 ℃, the adding amount of the trypsin is 2 percent of the protein amount in the homogenate, and the activity of the trypsin is 10000U/mg
Example 2
Mussel polypeptides with melanin reducing effect, which are a mixture of at least two of the short peptides AEELGIQ, GANSIEL, VTHCNVK, TSGFSDSGY, WTGFSFM, KHSVITG, EVDDAGG, LDVSWASD, FCENVCPY, TAGSTTCLD, DNMSIMV prepared in example 1, can be obtained by controlling the separation and purification conditions or directly compounded by adopting the short peptides.
Example 3
Mussel polypeptide powder with melanin reducing effect is obtained by the steps (1) to (4) of the example 1.
Example 4
The cream with melanin reducing effect is prepared by adding mussel polypeptide or mussel polypeptide powder in example 1, example 2 or example 3 into cosmetic base cream formula.
Performance testing
1. Results of the simulation of tyrosinase against the docking molecule by each of the short peptides of example 1
| CDOCKER ENERGY | CDOCKER INTERACTION ENERGY | ||
| 1 | AEELGIQ | 117.898 | 64.8148 |
| 2 | GANSIEL | 117.412 | 73.535 |
| 3 | VTHCNVK | 116.755 | 67.9029 |
| 4 | TSGFSDSGY | 116.09 | 74.1901 |
| 5 | WTGFSFM | 116.081 | 80.0388 |
| 6 | KHSVITG | 115.435 | 94.7525 |
| 7 | EVDDAGG | 115.014 | 63.0653 |
| 8 | LDVSWASD | 113.487 | 88.8423 |
| 9 | FCENVCPY | 113.341 | 79.1719 |
| 10 | TAGSTTCLD | 113.021 | 79.4192 |
| 11 | DNMSIMV | 112.823 | 80.4715 |
| 12 | Kojic acid | 112.339 | 63.0521 |
As can be seen from the above table, the above short peptides have higher scores than positive control kojic acid, have potential certain tyrosinase inhibition effects, and have different inhibition effects, wherein AEELGIQ, GANSIEL, VTHCNVK, TSGFSDSGY, WTGFSFM and other effects are good, so that melanin reduction effect is shown
2. Melanin reduction efficacy test of each short peptide.
(1) Test conditions and test data.
1. Solution preparation
1) 50mM phosphate buffer or PBS solution, pH 6.4;
2) 500 μ M L-DOPA in PBS (first dissolved in DMSO containing 1% by volume);
3) Tyrosinase (sigma) was dissolved in PBS solution to a final concentration of 1000U/ml.
4) Experimental group polypeptide solutions: the polypeptide was dissolved in PBS at a concentration of 1mg/ml.
5) Positive control kojic acid solution: the tretinoin solution was dissolved in PBS solution at a concentration of 500. Mu.M.
2. Experimental procedure
1) 160 μ l L-DOPA in 96 well plates, three in parallel;
2) 20. Mu.L of polypeptide solution was added to a 96-well plate, blank was added to 20. Mu.L of PBS solution, and positive control was added to 20. Mu.L of kojic acid solution.
3) Each well was added with 20. Mu.L of tyrosinase at a final concentration of 1000U/mL, incubated at 37℃for 10min, and after 20min, absorbance was measured at 492nm, respectively.
3. Inhibition rate calculation
S0=sample self absorbance sa=absorbance after reaction of polypeptide sample c0=floor absorbance ca=absorbance after reaction of only substrate and enzyme
(2) Melanin-lowering effects of polypeptides
FIG. 1 is a LC-MS spectrum of polypeptide isolation and identification. The figure shows the total ion current signal detected by mass spectrometry after separation of the polypeptide by liquid phase (TIC, total lon chromatograms);
FIG. 2 shows the tyrosinase inhibition rates of enzymatically hydrolyzed polypeptides to the major active polypeptides. Wherein, the enzymolysis method of alkaline protease comprises the following steps: after mussel homogenate, the pH is adjusted to 9.0, the temperature is 50 ℃, the enzymolysis time is 2 hours, and the enzyme amount is added: 2% (ratio of the amount of protein in solution); the enzymolysis method of neutral protease comprises the following steps: after mussel homogenate, the pH is regulated to 7.0, the temperature is 40 ℃, the enzymolysis time is 2 hours, and the enzyme amount is added: 2% (ratio of the amount of protein in solution). The tyrosinase inhibition rate of the method is about 75%, is slightly lower than that of positive control kojic acid and is higher than that of the enzymatic hydrolysis polypeptides by other methods. All the predicted active polypeptides in the invention are synthesized (the predicted active polypeptides can also be obtained by separating and purifying the polypeptide powder) and then are subjected to tyrosinase inhibition rate tests (each active polypeptide is dissolved in PBS solution, the concentration is 1mg/ml, and the test steps are the same as above), so that the sequences KHSVITG and EVDDAGG have obvious tyrosinase inhibition activity.
Claims (6)
1. Mussel polypeptide having melanin reducing effect, characterized in that the mussel polypeptide is a short peptide or a mixture of two short peptides;
the short peptide is KHSVITG or EVDDAGG
The amino acid sequence of each short peptide is:
KHSVITG：Lys-His-Ser-Val-Ile-Thr-Gly；
EVDDAGG：Glu-Val-Asp-Asp-Ala-Gly-Gly。
2. a polypeptide powder comprising a mussel polypeptide according to claim 1.
3. The method for preparing polypeptide powder as claimed in claim 2, comprising the steps of:
(1) Removing shell of mussel, collecting whole tissue, cleaning, and homogenizing;
(2) Sequentially adding pepsin and trypsin into the homogenate of mussels for segmented enzymolysis;
(3) Centrifuging the enzymolysis liquid obtained after enzymolysis in the step (2), and then taking supernatant;
(4) Filtering the supernatant obtained in the step (3), intercepting molecules with the molecular weight of 500-1000 Da, and freeze-drying to obtain polypeptide powder.
4. The preparation method according to claim 3, wherein in the step (2), the pH value of the homogenate is kept at 1-3 during the enzymolysis of pepsin, the enzymolysis time is 2-4 hours, the enzymolysis temperature is 35-38 ℃, the added amount of pepsin is 2-4% of the protein amount in the homogenate, and the activity of pepsin is more than or equal to 250U/mg.
5. The preparation method according to claim 3, wherein in the step (2), trypsin is added for enzymolysis, the pH value of the homogenate is kept at 7.0-8.6, the enzymolysis time is 1-2 hours, the enzymolysis temperature is 35-38 ℃, the adding amount of the trypsin is 1-3% of the protein amount in the homogenate, and the activity of the trypsin is 10000U/mg.
6. Use of a mussel polypeptide according to claim 1 or a polypeptide powder according to claim 2 for the preparation of a reduced melanin facial cream.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017011983A1 (en) * | 2015-07-20 | 2017-01-26 | 江阴市本特塞缪森生命科学研究院有限公司 | Uses of mussel adhesive protein in protection of skin and accessory organs of skin |
| KR102064602B1 (en) * | 2018-07-24 | 2020-01-09 | 주식회사 코리아나화장품 | Cosmetic Composition for Whitening of the Skin Comprising the Extract of Fermented Perna Canaliculus |
| CN112625088A (en) * | 2021-03-15 | 2021-04-09 | 烟台市华昕生物医药科技有限公司 | Preparation method and application of mussel ACE inhibitory peptide |
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| AU2015402833B2 (en) * | 2015-07-20 | 2022-01-06 | Jiangyin Bengt I. Samuelsson Institute Of Life Science Co., Ltd. | Applications of mussel adhesive protein product in treatment and prevention of diseases related to melanin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017011983A1 (en) * | 2015-07-20 | 2017-01-26 | 江阴市本特塞缪森生命科学研究院有限公司 | Uses of mussel adhesive protein in protection of skin and accessory organs of skin |
| KR102064602B1 (en) * | 2018-07-24 | 2020-01-09 | 주식회사 코리아나화장품 | Cosmetic Composition for Whitening of the Skin Comprising the Extract of Fermented Perna Canaliculus |
| CN112625088A (en) * | 2021-03-15 | 2021-04-09 | 烟台市华昕生物医药科技有限公司 | Preparation method and application of mussel ACE inhibitory peptide |
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