Sea cucumber active polypeptide, and preparation method therefor and use thereof

Abstract

Provided in the present invention are a sea cucumber active polypeptide, and a preparation method therefor and the use thereof. The preparation method comprises extracting the total protein by using ammonium sulfate precipitation, then performing separation and purification by means of enzymatic hydrolysis and gel chromatography, and then performing analysis and identification to obtain the sea cucumber active polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.

Description

A sea cucumber active polypeptide and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and more particularly to a sea cucumber active polypeptide with whitening, antioxidant and anti-wrinkle effects and a preparation method thereof. BACKGROUND

[0002] Sea cucumber is an echinoderm and an important marine food, which is high in protein, low in fat and sugar, rich in various amino acids, fatty acids, vitamins and trace elements necessary for the human body, and has high nutritional value. Sea cucumber is rich in protein and is a good source for preparing bioactive polypeptides. In recent years, more and more sea cucumber polypeptides with biological activities have been discovered, which have antioxidant, anti-aging, anti-inflammatory, anti-tumor, immunoregulatory, hypoglycemic and other effects.

[0003] During the hydrolysis of sea cucumber protein to form peptides, the peptide bonds are degraded, which increases the number of dissociable groups and thus increases the hydrophilicity. The degradation of the peptide bonds also changes the molecular structure, exposing the hydrophobic groups inside the protein. In addition, the number of amino acids and the relative molecular mass of the peptides are also changed, which improves the solubility of the sea cucumber polypeptides and increases their stability. In addition, the osmotic pressure of the sea cucumber peptides is lower than that of amino acids, and the biological safety is high. SUMMARY

[0004] The present application aims to provide a sea cucumber active polypeptide and its use, and aims to solve the problems in the prior art.

[0005] The present application provides a sea cucumber active polypeptide, and the amino acid sequence of the sea cucumber active polypeptide is shown in SEQ ID NO: 1.

[0006] The present application uses ammonium sulfate precipitation method to extract total protein, enzyme hydrolysis method and gel chromatography separation and purification method to obtain sea cucumber polypeptides, and mass spectrometry to determine the amino acid sequence of the sea cucumber polypeptides, and separates and identifies a sea cucumber polypeptide.

[0007] The method comprises the following steps:

[0008] Step one, wash the fresh sea cucumber clean and cut into small pieces, add appropriate amount of PBS buffer solution for homogenization; after homogenization, add 20 mM pH 7.0 Tris-HCl buffer solution at a volume ratio of 1:20, stir uniformly, and stir and extract at 4℃ for 24 hours; centrifuge at a speed of 10000 r / min for 30 minutes, collect the supernatant; use ammonium sulfate precipitation method to extract sea cucumber total protein from the supernatant;

[0009] Step two, the prepared sea cucumber total protein sample 100 mg, 10 mM pH7.5 PBS buffer solution is dissolved, placed in the molecular weight size 2000-5000Da cellulose acetate dialysis bag, ammonium acetate buffer solution in 5 mM pH 6.0 dialysis;After dialysis, the protein solution is placed in a 1KD ultrafiltration column to remove salt and concentrate, after concentration, the concentration of 5000U / g of alkaline protease is added to the concentrated solution, and the protein is enzymolyzed at pH 8 and 40℃, and then vacuum freeze-dried to obtain sea cucumber polypeptide crude product;

[0010] Step three, the sea cucumber polypeptide crude product is separated and purified by Sephadex G-15 gel column chromatography, 2 ml is taken for column, and distilled water is used for elution, the flow rate is set to 0.5 ml / min, and the detection wavelength is 280 nm;The same absorption peak components are repeatedly collected and concentrated, and vacuum freeze-dried to obtain sea cucumber polypeptide separation and purification product;

[0011] Step four, the sea cucumber polypeptide separation and purification product is detected by high performance liquid chromatography column, the mobile phase is 20 mM phosphate buffer solution, the flow rate is 0.5 ml / min, and the detection wavelength is 280 nm;The purified product with a single peak in high performance liquid chromatography is a sea cucumber polypeptide monomer;

[0012] Step five, the amino acid sequence of the sea cucumber polypeptide monomer is identified by mass spectrometry, and the amino acid sequence shown as SEQ ID NO:1 is obtained.

[0013] The ammonium sulfate precipitation method is to add solid (NH4)2SO4 to the supernatant to a final concentration of 80%, stir uniformly, then stand at 4℃ overnight, centrifuge at a speed of 10000 r / min for 30 minutes, dialyze in water, and freeze-dry.

[0014] The method for extracting total protein by ammonium sulfate precipitation in the application has been widely used, then the sea cucumber polypeptide is obtained by enzymolysis and gel chromatography separation and purification method, the separation and purification product is identified by high performance liquid chromatography technology, and the amino acid sequence of the sea cucumber polypeptide is identified by mass spectrometry, and the sea cucumber active polypeptide is prepared.

[0015] The influence of the sea cucumber active polypeptide on the activity of mouse melanoma cells (B16-F10) is detected by thiazolyl blue (MTT) method. It is found that the sea cucumber polypeptide alone has no toxicity to the cells. After the B16-F10 cells are treated by the sea cucumber polypeptide for 24 hours, the melanin content and tyrosinase activity in the cells are both obviously reduced, and the experimental results show that the sea cucumber polypeptide can inhibit the activity of tyrosinase in the cells and reduce the synthesis of melanin, and embodies the biological activity of whitening.

[0016] The sea cucumber polypeptide is detected by an in-vitro experiment determination method, and the product is found to be reduced after reacting with a specific substrate after the sea cucumber polypeptide is used to treat porcine pancreatic elastase, and the experimental result shows that the sea cucumber polypeptide can obviously inhibit the activity of elastase, and the sea cucumber polypeptide has strong anti-wrinkle activity.

[0017] The sea cucumber polypeptide is detected by an in-vitro experiment determination method, and the product is found to be reduced after reacting with a specific substrate after the sea cucumber polypeptide is used to treat porcine pancreatic elastase, and the experimental result shows that the sea cucumber polypeptide can obviously inhibit the activity of elastase, and the sea cucumber polypeptide has strong anti-wrinkle activity.

[0018] The sea cucumber polypeptide is detected by an in-vitro experiment determination method, and the product is found to be reduced after reacting with a specific substrate after the sea cucumber polypeptide is used to treat porcine pancreatic elastase, and the experimental result shows that the sea cucumber polypeptide can obviously inhibit the activity of elastase, and the sea cucumber polypeptide has strong anti-wrinkle activity.

[0019] 1、The active polypeptide can inhibit the activity of tyrosinase in mouse melanoma cells (B16-F10), thereby inhibiting the synthesis of melanin and reducing the content of melanin, and the active polypeptide has good whitening effect.

[0020] 2、The sea cucumber active polypeptide can inhibit the activity of elastase, and elastase is a key enzyme for hydrolyzing key connective tissue matrix proteins, and the inhibition of elastase activity can prevent ultraviolet rays or free radicals from damaging elastic fibers, thereby playing an anti-wrinkle role.

[0021] 3、The sea cucumber active polypeptide can effectively scavenge -OH free radicals, DDPH free radicals and O2- free radicals. -OH free radicals, DDPH free radicals and O2- free radicals have the strongest oxidative activity in the body, can combine with amino acids and DNA and other biological macromolecules, and cause oxidative damage to cells, and the ability to scavenge -OH, DDPH and O2- free radicals can reflect good antioxidant activity. BRIEF DESCRIPTION OF DRAWINGS

[0022] Fig. 1 is a Sephadex G-15 gel column chromatography separation spectrum of sea cucumber polypeptide separation and purification.

[0023] Fig. 2 is an identification spectrum of sea cucumber polypeptide separation and purification peaks in high performance liquid chromatography.

[0024] Fig. 3 is a primary spectrum of the amino acid structure of the sea cucumber polypeptide detected by mass spectrometry.

[0025] Fig. 4 is a secondary spectrum of the amino acid structure of the sea cucumber polypeptide identified by mass spectrometry.

[0026] Figure 5 is a graph of the melanin content in B16-F10 cells treated with different concentrations of sea cucumber polypeptide.

[0027] Figure 6 is a graph of the tyrosinase activity in B16-F10 cells treated with different concentrations of sea cucumber polypeptide.

[0028] Figure 7 is a graph of the elastase activity in pig pancreas treated with different concentrations of sea cucumber polypeptide.

[0029] Figure 8 is a graph of the detection results of the -OH radical scavenging capacity of different concentrations of sea cucumber polypeptide.

[0030] Figure 9 is a graph of the detection results of the DPPH radical scavenging capacity of different concentrations of sea cucumber polypeptide.

[0031] Figure 10 is a graph of the detection results of the O2- radical scavenging capacity of different concentrations of sea cucumber polypeptide. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0033] I. Preparation of sea cucumber polypeptide

[0034] 1. Extraction of sea cucumber total protein

[0035] Fresh sea cucumbers were washed clean and impurities were removed. The treated sea cucumbers were cut into small pieces and added with appropriate PBS buffer solution for homogenization. After homogenization, 20 mM pH 7.0 Tris-HCl buffer solution was added at a volume ratio of 1:20, and stirred uniformly. The mixture was stirred and extracted at 4°C for 24 hours. Then, centrifugation was performed at a speed of 10000 r / min for 30 minutes, and the supernatant was collected. Then, solid (NH4)2SO4 was added to a final concentration of 80%, and the mixture was stirred uniformly. Then, the mixture was left to stand at 4°C overnight, and centrifugation was performed at a speed of 10000 r / min for 30 minutes. The mixture was fully dialyzed in water, and freeze-dried to obtain sea cucumber total protein.

[0036] 2. Isolation and purification of sea cucumber polypeptide

[0037] The prepared sea cucumber total protein sample 100 mg, 10 mM pH7.5 PBS buffer solution was dissolved, placed in a cellulose acetate dialysis bag with a molecular weight of 2000-5000 Da, and dialyzed against 5 mM pH6.0 ammonium acetate buffer solution. After dialysis, the protein solution was desalted and concentrated on a 1KD ultrafiltration column, and after concentration, 5000U / g of alkaline protease (Novo protease was used in this study) was added to the concentrated solution, and the protein was enzymatically degraded at pH8 and 40℃ for 24h, and then vacuum freeze-dried to obtain sea cucumber polypeptide crude product.

[0038] The sea cucumber polypeptide crude product was dissolved in 15 mM pH7.5 PBS buffer solution, filtered with a 0.22μm filter membrane, and separated and purified by Sephadex G-15 gel column chromatography. 2ml was taken for column chromatography, eluted with distilled water, the flow rate was set at 0.5ml / min, the detection wavelength was 280nm, the elution peak was collected, the same absorption peak components were repeatedly collected and concentrated, and vacuum freeze-dried to obtain sea cucumber polypeptide separation and purification product. The elution peak spectrum is shown in Figure 1.

[0039] 3. High performance liquid chromatography detection

[0040] The sea cucumber polypeptide separation and purification product was dissolved in 10 mM pH7.5 PBS buffer solution, filtered with a 0.22μm filter membrane, and detected by high performance liquid C18 column. The mobile phase was 20mM phosphate buffer solution, the flow rate was 0.5ml / min, and the detection wavelength was 280nm. The high performance liquid chromatography detection of the purified product with a single peak was sea cucumber polypeptide monomer, and the results are shown in Figure 2.

[0041] 4. Identification of sea cucumber polypeptide amino acid sequence

[0042] Mass spectrometry detection: The peptide segment was dissolved in a dissolution solution (0.1% formic acid, 2% acetonitrile), vortexed, centrifuged at 10000r / min for 10min at 4℃, and the supernatant was subjected to mass spectrometry identification. The separated peptide segments were sequentially subjected to primary mass spectrometry and secondary mass spectrometry detection. The primary mass spectrometry parameters were Resolution, 80000; AGC target, 3e6; Maximum IT, 50 ms; Scan range, 50-3000m / z; and the secondary mass spectrometry parameters were Resolution, 20000; AGC target, 1e5; Maximum IT, 50 ms; TopN, 20NCE / stepped NCE 28. The primary mass spectrometry spectrum and the secondary mass spectrometry spectrum are shown in Figures 3 and 4.

[0043] Database retrieval: The mass spectrum raw file was processed and converted by MS Convert software to obtain MGF format file, and then MaxQuant was used to retrieve to obtain the amino acid sequence of sea cucumber polypeptide as CPPLTCPG (SEQ ID NO: 1), wherein several amino acids are cysteine (Cys, C), proline (Pro, P), leucine (Leu, L), threonine (Thr, T), and glycine (Gly, G). The ion fragments corresponding to the amino acid sequence in the secondary mass spectrum are shown in Table 1.

[0044] Table 1 Distribution of corresponding amino acid sequence in secondary mass spectrum

[0045]

[0046] II. Whitening activity research of sea cucumber polypeptide: Effect of sea cucumber polypeptide on melanin content and tyrosinase activity in mouse melanoma cells (B16-F10)

[0047] 1. Cell culture: B16-F10 cells were cultured in DMEM medium containing 10% fetal bovine serum, and the incubation conditions of the constant temperature incubator were 37℃ and 5% CO2.

[0048] 2. Effect of sea cucumber polypeptide on B16-F10 cell activity

[0049] Take well-grown B16-F10 cells, and seed 2×103 cells per well in a 96-well plate. After 24 hours of incubation, the cells were treated with different concentrations of sea cucumber polypeptide (10 μM, 20 μM, 30 μM, dissolved in water) for 24 hours (6 replicates for each concentration), and then the culture medium was discarded. Add 20 μL of MTT solution to each well, and continue to incubate at 37℃ for 4 hours. Carefully aspirate the MTT solution, add 150 μL of DMSO solution, and shake on a shaker for 5 minutes. Detect the absorbance value (OD value) at 490 nm wavelength on a microplate reader.

[0050] After treatment with different concentrations of sea cucumber polypeptide, the activity of B16-F10 cells was not affected, and the results are shown in Table 2.

[0051] Table 2 Effect of sea cucumber polypeptide on B16-F10 cell activity (mean ± SD, n=6)

[0052]

[0053] After treatment with different concentrations of sea cucumber polypeptide, the activity of B16-F10 cells was not affected, and the results are shown in Table 2.

[0054] 3. Effect of sea cucumber polypeptide on melanin content in B16-F10 cells

[0055] Intracellular melanin content was determined using the NaOH lysis method. After counting B16-F10 cells, 5 × 10⁸ cells / mL were seeded into 6-well plates, 2 mL per well, and cultured for 24 hours. α-Melanocyte-stimulating hormone (α-MSH) was added to each experimental group (except for the blank control group) to induce melanin overexpression, thus constructing an α-MSH-induced melanin-overexpressing cell model. Different concentrations of sea cucumber polypeptide (10, 20, and 30 μM) were then added, with 6 replicates per group. Additionally, 2 mL of culture medium was added to both the blank control and model groups. After 24 hours of culture, the supernatant was discarded, and the cells were washed three times with PBS. 200 μL of NaOH solution (containing 10% DMSO) was added to each well, and the cells were lysed at 80°C for 1 hour. The absorbance (OD) of each well was measured at 470 nm. Protein content in the solution was determined using the BCA method. Following the same method, using melanin as a standard, a standard curve was plotted between OD value (Y) and mass concentration (X): Y = 0.0099X + 0.00008 (R² = 0.995). The intracellular melanin content was calculated using the standard curve. The relative intracellular melanin content was calculated using the following formula: Relative intracellular melanin content (%) = Melanin content per gram of protein in the experimental group / Melanin content per gram of protein in the α-MSH model group × 100%.

[0056] Experimental results showed that, compared with the control group, the melanin content in the α-MSH-induced model group was significantly increased (p<0.05). Compared with the α-MSH-induced model group, all concentrations of sea cucumber polypeptide could significantly inhibit the melanin content in B16-F10 cells (p<0.05), and showed a clear dose-dependent effect. When the concentration of sea cucumber polypeptide was 30 μM, it could completely inhibit the melanin produced by α-MSH, as shown in Figure 5.

[0057] 4. Effects of sea cucumber polypeptides on tyrosinase activity in B16-F10 cells

[0058] The activity of tyrosinase in cells was determined by L-Dopa oxidation method. After counting B16-F10 cells, 5x108 / mL was inoculated in 6-well plates, 2 mL per well, and cultured for 24 hours. According to the experimental grouping, 0.2 μM of α-MSH was added for induction (except the blank control group), and the α-MSH-induced melanin high-expression cell model was constructed. Then different concentrations of sea cucumber polypeptide were added, and the final concentration of sea cucumber polypeptide was 10, 20, 30 μM, and 6 replicate wells were set in each group. In addition, 2 mL of culture medium was added to the blank control group and the model group. After 24 hours of culture, the supernatant was discarded, washed with PBS for 3 times, 1 mL of PBS buffer containing 1% Triton X-100 was added to each well, and it was frozen in a-80℃ refrigerator for 1 hour. Then it was thawed at room temperature, and the cell lysate was centrifuged at 12000 r / min for 20 min at 4℃. The supernatant was collected. 60 μL of supernatant was taken to a 96-well plate, 140 μL of 5 mM L-Dopa was added, and it was incubated at 37℃ for 1 hour. The absorbance (OD value) of each well was measured at 475 nm wavelength. The protein content in the solution was determined by Bradford method. The relative activity of tyrosinase in cells was calculated according to the following formula: relative activity of tyrosinase in cells (%) = tyrosinase activity in each gram of protein in the experimental group / tyrosinase activity in each gram of protein in the α-MSH model group x 100%.

[0059] The experimental results showed that compared with the blank group, the activity of tyrosinase in the α-MSH-induced model group was significantly increased (p<0.05). When compared with the α-MSH-induced model group, each concentration of sea cucumber polypeptide could significantly inhibit the activity of tyrosinase in B16-F10 cells (p<0.05), and showed a significant dose-dependent manner. When the concentration of sea cucumber polypeptide was 30 μM, the activity of tyrosinase induced by α-MSH could be completely inhibited, as shown in Figure 6.

[0060] III. Anti-wrinkle activity of sea cucumber polypeptide: effect of sea cucumber polypeptide on porcine pancreatic elastase activity

[0061] Take 5 mg elastin into a 10 mL centrifuge tube, add 2 mL of 37°C preheated 0.2 M borate buffer (pH 8.8), mix well, water bath for 20 min, add 1 mL of different concentrations of porcine pancreatic elastase solution prepared with borate buffer (concentration is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / mL) preheated at 37°C, shake at 400 r / min for 30 min, immediately add 2.5 mL of 0.5 M phosphate buffer with pH 6, take an appropriate amount of mixed solution into a 2 mL centrifuge tube, centrifuge at 10000 r / min for 10 min, take 200 μL of supernatant into a 96-well plate, measure the absorbance at wavelength 495 nm. Take the concentration of porcine pancreatic elastase as the abscissa and the absorbance as the ordinate to draw the standard curve.

[0062] Take 1 mL of 2 mg / mL porcine pancreatic elastase solution, add 1 mL of different concentrations of sea cucumber polypeptide (10, 20, 30 μM), shake and incubate at 37°C and 400 r / min for 30 min. Then add 2 mL of 0.5 M phosphate buffer with pH 6 in the prepared elastin solution, vortex mix, shake and incubate at 37°C and 400 r / min for 30 min, add 2 mL of 0.5 M phosphate buffer with pH 6, take an appropriate amount of mixed solution into a 2 mL centrifuge tube, centrifuge at 10000 r / min for 10 min, take 200 μL of supernatant into a 96-well plate, measure the absorbance at wavelength 495 nm. Take the substrate and enzyme solution as the blank control group, the substrate, enzyme and sea cucumber polypeptide solution as the enzyme inhibition group, and the substrate and sea cucumber polypeptide without enzyme solution as the background deduction. Each group has 6 replicates. Inhibition rate (%) = [1 - (An - An') / (A0 - A0')] × 100%, where A0 is the absorbance of enzyme without sea cucumber polypeptide, A0' is the absorbance of only substrate without sea cucumber polypeptide and enzyme, An is the absorbance of only sea cucumber polypeptide solution, and An' is the absorbance of sea cucumber polypeptide and enzyme.

[0063] The experimental results show that sea cucumber polypeptide of each concentration can inhibit the activity of porcine pancreatic elastase to different degrees, and shows a dose-dependent inhibition. When the concentration of sea cucumber polypeptide is 30 μM, the inhibition rate of elastase reaches 90%, as shown in Figure 7.

[0064] Four, antioxidant activity of sea cucumber polypeptide: effect of sea cucumber polypeptide on clearance of -OH free radical, DPPH free radical and O2- free radical

[0065] 1. Effect of sea cucumber polypeptide on -OH free radical

[0066] H2O2 / Fe2+ was used to generate -OH by Fenton reaction, and the solution of phenanthroline-Fe2+ was oxidized to phenanthroline-Fe3+ by -OH. The degree of fading of phenanthroline-Fe2+ (orange red) can be used to measure the amount of -OH radical scavenging. Sea cucumber polypeptide was prepared into 10, 20, 30 μM solution with pure water. 1 mL of each different concentration of sea cucumber polypeptide solution was taken, and 1 mL of 9 mM FeSO4 solution, 1 mL of 9 mM salicylic acid solution and 1 mL of 8.8 mM H2O2 solution were added, and mixed thoroughly, and reacted in a 37 ℃ water bath for 30 min. Deionized water was used as the reference solution, and 10 μM vitamin C was used as the control, and 6 replicate wells were set for each group, and the absorbance value (OD value) was measured at 510 nm. The -OH radical scavenging rate (%) = [1-(Ai-Aj) / A0]x100%, in the formula, A0 is the absorbance value of the blank control; Ai is the absorbance value after adding the sample; Aj is the absorbance value of the sample itself. The antioxidant capacity was calculated by the -OH radical scavenging effect.

[0067] The experimental results show that sea cucumber polypeptide of each concentration has -OH radical scavenging activity, and shows dose-dependent inhibition, and when the concentration of sea cucumber polypeptide is 30 μM, the -OH radical scavenging rate reaches 90%, which is consistent with the scavenging rate of vitamin C, and has strong antioxidant activity. As shown in Figure 8.

[0068] 2, Effect of sea cucumber polypeptide on DPPH radical

[0069] DPPH radical is a stable nitrogen-centered free radical with maximum absorption at 517 nm wavelength. The DPPH solution is purple, and its concentration is linearly related to the absorbance. After adding sea cucumber polypeptide to the DPPH solution, sea cucumber polypeptide can bind or replace DPPH, reducing the number of DPPH and making the solution color lighter, which shows a continuously decreasing absorbance value until stable. The antioxidant activity was calculated by the DPPH radical scavenging effect. 1 mL of different concentrations of sea cucumber polypeptide (10, 20, 30 μM) was added to 3 mL of 0.1 mM DPPH solution, and the solution was incubated at room temperature for 30 min in the dark. Pure water was used as the reference solution, and 10 μM vitamin C was used as the control, and 6 replicate wells were set for each group, and the absorbance value (OD value) was measured at 517 nm. The DPPH radical scavenging rate (%) = [1-(Ai-Aj) / A0]x100%, in the formula, A0 is the absorbance value of the blank control; Ai is the absorbance value after adding the sample; Aj is the absorbance value of the sample itself. The antioxidant capacity was calculated by the DPPH radical scavenging effect.

[0070] The experimental results show that the sea cucumber polypeptides of various concentrations have the activity of scavenging DPPH free radicals, and present the dose-dependent inhibition, when the concentration of the sea cucumber polypeptides is 30 μM, the clearance rate of ·OH free radicals reaches 85%, which is consistent with the clearance rate of vitamin C, and the sea cucumber polypeptides have strong antioxidant activity. As shown in Figure 9.

[0071] 3. Influence of sea cucumber polypeptides on O2- free radicals

[0072] Pyrogallol is automatically oxidized under alkaline conditions, and continuously releases O2-, and O2- can promote autoxidation. The colored product has strong light absorption at 320 nm. Since the autoxidation rate depends on the concentration of O2-, the scavenging of O2- inhibits the autoxidation reaction, prevents the accumulation of products, and thus evaluates the ability of the sea cucumber polypeptides to scavenge O2-. 2 ml of Tris-HCl buffer with a concentration of 50 mM, preheated at 25 ℃ and pH 8.2, is placed in a 10 ml test tube, and different concentrations of sea cucumber polypeptide solutions (10, 20, 30 μM) are added, then 80 μL of 45 mM pyrogallol preheated at 25 ℃ is added, mixed quickly, and after 5 min, 20 μL of 10 M HCl is added to terminate the reaction. Pure water is used as a reference solution, and 10 μM vitamin C is used as a control. Each group has 6 replicate wells, and the absorbance value (OD value) is measured at 320 nm. The O2- free radical clearance rate (%) = [1-(Ai-Aj) / A0]x100%, where A0 is the absorbance value of the blank control; Ai is the absorbance value after adding the sample; and Aj is the absorbance value of the sample itself. The antioxidant capacity is calculated by the O2- free radical scavenging effect.

[0073] The experimental results show that the sea cucumber polypeptides of various concentrations have the activity of scavenging O2- free radicals, and present the dose-dependent inhibition, when the concentration of the sea cucumber polypeptides is 30 μM, the clearance rate of O2- free radicals reaches 90%, which is consistent with the clearance rate of vitamin C, and the sea cucumber polypeptides have strong antioxidant activity. As shown in Figure 10.

[0074] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting from any point of view.

Claims

1. A sea cucumber active polypeptide, characterized in that, The amino acid sequence of the sea cucumber active polypeptide is shown as SEQ ID NO:

1.

2. The sea cucumber active polypeptide according to claim 1, characterized in that, The preparation method comprises the following steps: Step one, wash the fresh sea cucumber and cut it into small pieces, add appropriate amount of PBS buffer solution for homogenization; after homogenization, add 20 mM pH 7.0 Tris-HCl buffer solution in a proportion of 1:20, stir uniformly, and stir and extract at 4℃ for 24 hours; centrifuge at a speed of 10000 r / min for 30 minutes, and collect the supernatant; the supernatant is extracted by ammonium sulfate precipitation method to obtain sea cucumber total protein; Step two, dissolve 100 mg of the prepared sea cucumber total protein sample in 10 mM pH 7.5 PBS buffer solution, place it in a cellulose acetate dialysis bag with a molecular weight of 2000-5000 Da, and dialyze it in 5 mM pH 6.0 ammonium acetate buffer solution; after dialysis, desalt and concentrate the protein solution in a 1KD ultrafiltration column, add 5000 U / g of alkaline protease to the concentrated solution, and then freeze-dry the solution by a vacuum freeze-dryer to obtain sea cucumber polypeptide crude product; Step three, the sea cucumber polypeptide crude product is separated and purified by Sephadex G-15 gel column chromatography, 2 ml is taken for column, eluted with distilled water, the flow rate is set to 0.5 ml / min, the detection wavelength is 280 nm, the elution peak is collected, the same absorption peak components are repeatedly collected and concentrated, and vacuum freeze-drying is carried out to obtain sea cucumber polypeptide separation and purification product; Step four, the sea cucumber polypeptide separation and purification product is detected by high performance liquid chromatography column, the mobile phase is 20 mM phosphate buffer solution, the flow rate is 0.5 ml / min, and the detection wavelength is 280 nm; the purified product with a single peak by high performance liquid chromatography is sea cucumber polypeptide monomer; Step five, the amino acid sequence of the sea cucumber polypeptide monomer is identified by mass spectrometry, and the amino acid sequence shown as SEQ ID NO: 1 is obtained.

3. The novel flow meter of claim 2, wherein, The ammonium sulfate precipitation method is to add solid (NH4)2SO4 to the supernatant to a final concentration of 80%, stir uniformly, then stand at 4℃ overnight, centrifuge at a speed of 10000 r / min for 30 minutes, dialyze in water, and freeze-dry.

4. The sea cucumber active polypeptide according to any one of claims 1-3, characterized in that, The sea cucumber active polypeptide is applied in whitening efficacy products.

5. The sea cucumber active polypeptide according to claim 4, characterized in that, The sea cucumber active polypeptide is applied in inhibiting the tyrosinase activity of melanoma cells.

6. The sea cucumber active polypeptide according to any one of claims 1-3, characterized in that, The sea cucumber active polypeptide is applied in anti-wrinkle efficacy products.

7. The sea cucumber active polypeptide according to claim 6, characterized in that, The sea cucumber active polypeptide is applied in inhibiting elastase activity.

8. The sea cucumber active polypeptide according to any one of claims 1-3, characterized in that, The sea cucumber active polypeptide is applied in antioxidant efficacy products.

9. The sea cucumber active polypeptide according to claim 8, characterized in that, The sea cucumber active polypeptide is applied to scavenging -OH free radical, DDPH free radical and O2 - radical activity.

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