A polypeptide and its preparation method and application in cosmetics

Abstract

The present invention discloses a polypeptide and a preparation method thereof and an application thereof in cosmetics, and belongs to the field of polypeptides and cosmetics. The present invention uses prawns as raw materials, and obtains polypeptides with whitening, anti-wrinkle and antioxidant functions through enzymolysis with alkaline protease and pepsin, and separation and purification, wherein the amino acid sequence thereof is shown in SEQ ID NO.1‑3. Compared with traditional chemical synthesis and extraction methods, the beauty polypeptide prepared by the present invention is more efficient, safe and environmentally friendly, and has good application prospects in the fields of medicine and cosmetics.

Description

Technical Field

[0001] The present application relates to the field of polypeptides and cosmetics, and specifically to polypeptides with cosmetic functions, preparation methods thereof, and applications in cosmetics. Background Art

[0002] Melanin generally plays an important role in protecting the human body from ultraviolet radiation. Ultraviolet rays can activate the activity of tyrosinase, and after oxidizing tyrosine or 3,4-dihydroxyphenylalanine, melanin is formed through a series of chemical reactions. After the formation of melanin, it can reduce the free radicals produced by ultraviolet rays and reduce the toxic effects of free radicals on normal cells. Excessive formation of melanin will make the skin darker, and excessive local production will cause chloasma and freckles, which are both skin diseases. Tyrosinase is a key enzyme in the process of synthesizing melanin. An effective way to block the production of melanin is to inhibit the activity of tyrosinase.

[0003] Common tyrosinase inhibitors in the market include kojic acid, arbutin, hydroquinone, glabridin, glutathione and kojic acid, etc. These compounds have good antioxidant and melanin production inhibition capabilities, but they are carcinogenic or cause melanocyte death when used extensively for a long time, and the skin will become darker after use. After being absorbed by the skin, they will be reduced to produce hydroquinone, which will produce many side effects. In addition, with the development of the chemical synthesis industry in the last century, chemically synthesized cosmetics have dominated the cosmetics market, but the insecurity and pollution of chemical products are worrying, and people prefer safe, non-irritating and pollution-free natural cosmetics.

[0004] Peptide is a small molecule protein. It is a class of compounds with a molecular structure between amino acids and proteins. It is a structural and functional fragment of protein. Small molecule peptides can pass through the skin barrier and be absorbed. Peptides with special physiological activities can also improve a series of skin problems in essence and have a good cosmetic effect. In the natural aging and care process of the skin, peptides play an important role, such as cell proliferation, cell migration, angiogenesis, pigmentation, protein synthesis and regulation. At present, a variety of peptides have been used in beauty products to achieve the functions of anti-free radical oxidation, anti-wrinkle, whitening, anti-edema, anti-inflammatory repair and promotion of collagen synthesis. Small molecule peptides provide a practical and promising solution to such problems.

[0005] At present, most of the domestic marine product processing remains in the primary processing stage. Due to the low resource utilization rate and the waste of scraps generated during the processing, resources are wasted and even environmental pollution occurs. Prawns contain a large amount of protein, and its composition is balanced. The use of modern biotechnology to deeply process the protein resources, the rational selection of enzymes, etc., and the optimization of the process will make the research of beauty peptides with beauty effects have a broader prospect. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a polypeptide and a preparation method thereof and application in cosmetics. To achieve the above objectives, the present application provides the following technical solutions:

[0007] The present invention provides a polypeptide with beauty function, the amino acid sequence of the polypeptide is shown as SEQ ID No.1-3.

[0008] The present invention provides a method for preparing a beauty functional polypeptide, comprising the following steps:

[0009] (1) Clean the fresh prawns, remove the heads and shells, wash and drain the prawn tails, and grind them into minced meat in a meat grinder to obtain minced prawn meat;

[0010] (2) adding 2 to 8 times of water to the obtained shrimp meat paste according to the weight ratio, and then keeping it in a water bath at 90 to 95° C. for 10 to 15 minutes, and then homogenizing it in a dispersing homogenizer for 3 to 5 minutes to obtain a shrimp meat homogenate;

[0011] (3) adjusting the pH of the shrimp meat homogenate obtained in step (2) to 6.5-7.5, boiling for 15-30 minutes, cooling to room temperature, adding a composite protease for enzymatic hydrolysis, and simultaneously performing ultrasonic treatment during the enzymatic hydrolysis;

[0012] (4) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis reaction solution is kept at 90-95°C for 5-20 minutes to inactivate the enzyme, and then cooled to room temperature;

[0013] (5) taking the room temperature enzymatic hydrolysis reaction solution of step (4) and centrifuging it at 5000-10000 rpm for 15-30 min, collecting the supernatant, filtering and freeze-drying to obtain the enzymatic hydrolysis product of shrimp;

[0014] (6) The enzymatic hydrolysate of the shrimp in (5) was separated by ultrafiltration using membrane filtration systems with a molecular weight cutoff of 10 kDa and 3 kDa, respectively. The enzymatic hydrolysate was separated using ultrafiltration membranes with different molecular weight cutoff ranges to obtain polypeptides with different molecular weights, including components ≥10,000 Da, 3,000-10,000 Da, and ≤3,000 Da.

[0015] (7) Collecting the polypeptides of different molecular weights in step (6), separating them by SP-650M ion exchange column, and then separating them by Sephadex G-50 gel chromatography, with the elution peak measured at 215 nm; further separating them by RP-HPLC, collecting the components, dialyzing for desalting, and freeze-drying for later use, thereby obtaining high-purity beauty polypeptides;

[0016] (8) The beauty peptides were subjected to alternating primary mass spectrometry and secondary mass spectrometry using a mass spectrometer. With the aid of software analysis, six peptide amino acid sequences, SEQ ID NO. 1-3, were obtained as shown in the following table.

[0017] Furthermore, in step (3), the composite protease is added to the shrimp meat homogenate at a mass ratio of composite protease to shrimp meat paste of 1:500-800.

[0018] Furthermore, the composite protease consists of alkaline protease and pepsin.

[0019] Furthermore, the mass ratio of alkaline protease to pepsin is 1-5:1-5.

[0020] Furthermore, the mass ratio of alkaline protease to pepsin is 2:3.

[0021] Furthermore, the enzymatic hydrolysis conditions of step (3) are: enzymatic hydrolysis temperature of 40° C. and enzymatic hydrolysis time of 4 h.

[0022] The present invention also provides a cosmetic comprising a beauty functional polypeptide.

[0023] The present invention provides the use of polypeptides in preparing cosmetics for whitening, wrinkle removal and anti-oxidation. The cosmetics also include a matrix acceptable in the cosmetics field.

[0024] Furthermore, the matrix includes at least one of an emulsifier, an emulsifier aid, a skin conditioner, a whitening agent, a colorant, a moisturizer, a solubilizer, a surfactant, a preservative, a fragrance, an emollient, an anti-acne agent, a film former, a thickener, a pH adjuster, a buffer, a stabilizer or a UV absorber.

[0025] Furthermore, the cosmetics include but are not limited to facial cleansers, toners, gels, lotions, creams, eye creams, facial masks, essences, aerosols or sprays.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: the polypeptide with beauty function and its preparation method and application involved in the present invention have both high-efficiency tyrosinase inhibition activity and the function of promoting fibroblast proliferation and anti-oxidation aging and protecting normal cells, and the short peptide sequence can be controlled at a low production cost and good biocompatibility. Compared with the traditional chemical synthesis and extraction methods, the biological method for preparing beauty peptides is more efficient, safe and environmentally friendly, and the polypeptide has good application prospects in the preparation of antioxidant, whitening, anti-wrinkle drugs and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The inhibitory effect of the polypeptide of this application on tyrosinase.

[0029] Figure 2 The polypeptide of the present application promotes the proliferation of fibroblasts. DETAILED DESCRIPTION

[0030] The following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1 Preparation of beauty functional polypeptide

[0032] (1) Clean the fresh prawns, remove the heads and shells, wash and drain the prawn tails, and grind them into minced meat in a meat grinder to obtain minced prawn meat;

[0033] (2) adding 5 times the amount of water to the obtained shrimp paste according to the weight ratio, and then keeping it in a 95°C water bath for 15 minutes, and then homogenizing it in a dispersing homogenizer for 5 minutes to obtain a shrimp meat homogenate;

[0034] (3) The pH of the shrimp meat homogenate obtained in step (2) is adjusted to 7.0, boiled for 20 minutes, cooled to room temperature, and then added with a composite protease in a ratio of 1:600 ​​to the shrimp meat paste, and enzymolyzed at 40° C. for 4 hours, while performing ultrasonic treatment during the enzymolysis process; the composite protease is composed of alkaline protease and pepsin in a mass ratio of 2:3.

[0035] (4) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis reaction solution is kept at 95°C for 100 minutes to inactivate the enzyme, and then cooled to room temperature;

[0036] (5) taking the room temperature enzymatic hydrolysis reaction solution of step (4), centrifuging it at 6000 rpm for 30 min, collecting the supernatant, filtering and freeze-drying it to obtain the enzymatic hydrolysis product of shrimp;

[0037] (6) The enzymatic hydrolysate of the shrimp in (5) was separated by ultrafiltration using membrane filtration systems with a molecular weight cutoff of 10 kDa and 3 kDa, respectively. The enzymatic hydrolysate was separated using ultrafiltration membranes with different molecular weight cutoff ranges to obtain polypeptides with different molecular weights, including components ≥10,000 Da, 3,000-10,000 Da, and ≤3,000 Da.

[0038] (7) Collecting the polypeptides of different molecular weights in step (6), separating them by SP-650M ion exchange column, and then separating them by Sephadex G-50 gel chromatography, with the elution peak measured at 215 nm; further separating them by RP-HPLC, collecting the components, dialyzing for desalting, and freeze-drying for later use, thereby obtaining high-purity beauty polypeptides;

[0039] (8) The beauty peptides were subjected to primary mass spectrometry and secondary mass spectrometry alternately using a mass spectrometer, and analyzed with the help of software to obtain three peptide amino acid sequences SEQ ID NO.1-3 as shown in the following table. The peptides were commissioned to Shanghai Bioengineering for synthesis and then used.

[0040]

[0041] Example 2 Antioxidant Effect

[0042] (1) Determination of DPPH free radical scavenging ability

[0043] The concentration was 1×10 -5 mol / L DPPH ethanol solution, store in the dark. Add 2mL of 0.1mM DPPH anhydrous ethanol solution to a clean test tube containing 2mL of three peptide solutions of 0mM, 0.5mM, 1.0mM, and 2.0mM concentrations, and mix well. After standing at room temperature for 30 minutes, measure the absorbance at 517nm. The smaller the absorbance value, the stronger the free radical scavenging ability.

[0044] Clearance rate (%) = [1 - (A i -A j ) / A0] × 100%, where A0 is 2 mL, 0.1 mM DPPH anhydrous ethanol solution + 2 mL sample solvent, blank control; A i2mL, 0.1mM DPPH anhydrous ethanol solution + 2mL polypeptide solution; A j 2 mL of anhydrous ethanol + 2 mL of sample.

[0045] (2) Determination of ABTS free radical scavenging activity

[0046] Dissolve ABTS in deionized water to a concentration of 7 mmol / L, and add potassium persulfate to a concentration of 2.45 mmol / L. Then place the solution in the dark at room temperature for 12 to 16 hours overnight. Dilute the generated ABTS free radical solution with phosphate buffer (PBS, 0.2 mol / L, pH 7.4) to an absorbance of 0.70 at 734 nm. Take 0.1 ml of 0 mM, 0.5 mM, 1.0 mM, and 2.0 mM peptide solutions and mix with 2.9 ml of ABTS free radical solution, shake well for 30 seconds, react in the dark for 10 minutes, and then measure the absorbance of the reaction solution at 734 nm. Use distilled water instead of the hydrolyzate as a blank.

[0047] Clearance rate (%) = (A0-A j ) / A0× 100%, where A0 is the absorbance of a mixture of 2.9 mL ABTS reagent and 0.1 mL distilled water; A j The absorbance value of the mixture of 2.9 mL ABTS reagent and 0.1 mL enzymatic solution is shown in the following table.

[0048]

[0049] From the results in the above table, it can be seen that the obtained polypeptides of SEQ ID NO. 1-3 all have significant ability to scavenge DPPH and ABTS free radicals.

[0050] Example 3 Melanin synthesis inhibitory effect

[0051] B16 cells in the logarithmic growth phase were inoculated into T25 cell culture flasks and cultured overnight. Three peptide solutions with concentrations of 0mM, 0.5mM, 1.0mM, and 2.0mM were added respectively, and the untreated group was used as the cell control group. After 48h of culture, the cells were washed once with PBS, placed in an 80℃ water bath for 30min, and the supernatant was taken to read the absorbance value at 475nm.

[0052] Relative melanin content = (measured absorbance value - blank control group absorbance value) / (cell control group absorbance value - blank control group absorbance value) × 100%. Figure 1 As shown, the obtained polypeptide solution has a good inhibitory effect on melanin, and the polypeptides of the present invention all have whitening effects.

[0053] Example 4 Effect of Promoting Fibroblast Proliferation

[0054] BALB / c 3T3 cells were inoculated into a 96-well cell culture plate (5000 cells / well) and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours; DMEM serum-free medium was used for continued culture for 12 hours; three polypeptide solutions with concentrations of 0 mM, 0.5 mM, 1.0 mM, and 2.0 mM and PBS (negative control group) were added respectively, and culture was continued for 48 to 72 hours; 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 2 hours before being taken out; the absorbance values ​​of the 96-well plate at 450 nm and 630 nm were read with an enzyme-labeled instrument, and the absorbance was measured at 450 nm with 630 nm as the reference wavelength, and the measurement results were recorded.

[0055] Promoting cell proliferation rate = (OD 450 nm-negative control group OD 450 nm) / negative control group OD 450 nm×100%. The results are as follows Figure 2 As shown, the polypeptide of the present application has obvious cell proliferation promoting activity and has obvious anti-wrinkle effect.

[0056] Example 5 Skin safety evaluation of polypeptides

[0057] Subjects: 30 females and 30 males aged 30-50 years old, a total of 60 people. Ten males and ten females were divided into three groups, and the safety of SEQ ID NO.1-3 was measured respectively. The subjects had healthy skin and no history of skin allergies, and met the voluntary inclusion criteria.

[0058] Test method: Select a qualified patch device and use the closed patch test method. Drop about 0.20ml (1.0mM) of the three polypeptide solutions into the patch device respectively, and cover the face of the subject with special adhesive tape. Remove the test product after 24 hours, and observe the skin reaction at 0.5, 6, 12, 24, and 48 hours after removal. Record the results according to the skin reaction grading standards in the "Cosmetic Hygiene Standards".

[0059] Test results: In this test, 60 subjects passed the patch test, and no adverse skin reactions or allergic reactions were observed within 48 hours, indicating that the beauty polypeptide of the present invention is safe for skin use.

[0060] Based on the above description, it will be appreciated by those skilled in the art that the present disclosure may be implemented in different specific forms without changing its technical spirit and essential features. Therefore, it should be understood that the above-mentioned embodiments are not restrictive in all aspects, but illustrative. The scope of the present disclosure is limited by the appended claims, rather than by the preceding description thereof, and therefore all changes and modifications fall within the boundaries and scope of the claims, or the equivalents of such boundaries and scope are therefore intended to be covered by the claims.

Claims

1. A polypeptide with beauty function, characterized in that: The amino acid sequence of the polypeptide is shown in any one of SEQ ID No. 1-3.

2. The method for preparing the polypeptide according to claim 1, characterized in that: The following steps are involved: (1) Clean the fresh prawns, remove the heads and shells, wash and drain the prawn tails, and grind them into minced meat in a meat grinder to obtain minced prawn meat; (2) adding 2 to 8 times of water to the obtained shrimp meat paste according to the weight ratio, and then keeping it in a water bath at 90 to 95° C. for 10 to 15 minutes, and then homogenizing it in a dispersing homogenizer for 3 to 5 minutes to obtain a shrimp meat homogenate; (3) adjusting the pH of the shrimp meat homogenate obtained in step (2) to 6.5-7.5, boiling for 15-30 minutes, cooling to room temperature, adding a composite protease for enzymatic hydrolysis, and simultaneously performing ultrasonic treatment during the enzymatic hydrolysis; (4) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis reaction solution is kept at 90-95°C for 5-20 minutes to inactivate the enzyme, and then cooled to room temperature; (5) taking the room temperature enzymatic hydrolysis reaction solution of step (4) and centrifuging it at 5000-10000 rpm for 15-30 min, collecting the supernatant, filtering and freeze-drying to obtain the enzymatic hydrolysis product of shrimp; (6) using a membrane filtration system with a molecular weight cutoff of 10 kDa and 3 kDa to perform ultrafiltration separation on the enzymatic hydrolysate of the shrimp in (5), and using ultrafiltration membranes with different molecular weight cutoff ranges to separate the enzymatic hydrolysate to obtain polypeptides with different molecular weights, including components ≥10000 Da, 3000-10000 Da, and ≤3000 Da; (7) Collecting the polypeptides of different molecular weights in step (6), separating them by SP-650M ion exchange column, and then separating them by Sephadex G-50 gel chromatography, with the elution peak measured at 215 nm; further separating them by RP-HPLC, collecting the components, dialyzing for desalting, and freeze-drying for later use, thereby obtaining high-purity beauty polypeptides; (8) Using a mass spectrometer to perform primary mass spectrometry and secondary mass spectrometry on the beauty peptide alternately, and using software analysis to obtain the polypeptide according to claim 1; Wherein, in step (3), a composite protease is added to the shrimp meat homogenate at a mass ratio of 1:500-800; the composite protease is composed of alkaline protease and pepsin, and the mass ratio of alkaline protease to pepsin is 2:3; the enzymatic hydrolysis conditions of step (3) are: enzymatic hydrolysis temperature 40°C, enzymatic hydrolysis time 4h.

3. A cosmetic, characterized in that: The cosmetic comprises any one of the polypeptides described in claim 1.

4. Use of the polypeptide according to claim 1 and the polypeptide obtained by the preparation method according to claim 2 in the preparation of whitening, wrinkle-removing and anti-oxidation cosmetics, wherein the cosmetics also include a matrix acceptable in the cosmetics field.

5. The use according to claim 4, characterized in that The matrix includes at least one of an emulsifier, a co-emulsifier, a skin conditioner, a whitening agent, a colorant, a humectant, a solubilizer, a surfactant, a preservative, a fragrance, an emollient, an anti-acne agent, a film former, a thickener, a pH adjuster, a buffer, a stabilizer or a UV absorber.

6. The use according to any one of claims 4 to 5, characterized in that: The cosmetics include facial cleansers, toners, gels, lotions, creams, eye creams, facial masks, essences, aerosols or sprays.

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