A kind of sea cucumber active polypeptide and its preparation method and application

Abstract

The present invention relates to a sea cucumber active polypeptide and a preparation method and application thereof. The amino acid sequence of the sea cucumber active polypeptide is shown in SEQ ID NO: 1. The total protein is extracted by ammonium sulfate precipitation method, and then the sea cucumber polypeptide is obtained by enzymatic hydrolysis and gel chromatography separation and purification method. The separated and purified product is identified by high performance liquid phase technology, and is identified by mass spectrometry to prepare the sea cucumber active polypeptide. It is tested that the sea cucumber active polypeptide has good whitening, anti-wrinkle and anti-oxidation effects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more specifically to a sea cucumber active polypeptide with whitening, anti-oxidation and anti-wrinkle effects and a preparation method thereof. Background Art

[0002] Sea cucumber is an echinoderm and an important marine food. It is high in protein, low in fat and sugar, rich in various essential amino acids and fatty acids, vitamins and trace elements, etc., and has high nutritional value. Sea cucumber is rich in protein and is a good source for preparing bioactive peptides. In recent years, more and more bioactive sea cucumber peptides have been discovered, which have antioxidant, anti-aging, anti-inflammatory, anti-tumor, immunomodulatory, and blood sugar-lowering effects.

[0003] During the process of hydrolysis of sea cucumber protein to form peptides, the peptide bonds are degraded, which will increase the number of dissociable groups and thus increase its hydrophilicity; the degradation of peptide bonds will also change its molecular structure, exposing the hydrophobic groups inside the protein. In addition, the number of amino acids and the relative molecular mass of the peptide will also change. These changes improve the solubility and stability of sea cucumber polypeptides. In addition, compared with amino acids, sea cucumber peptides have lower osmotic pressure and higher biological safety. Summary of the invention

[0004] The purpose of the present application is to provide a preparation method of sea cucumber active polypeptide and its application, aiming to solve the problems existing in the prior art.

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

[0006] The invention utilizes an ammonium sulfate precipitation method to extract total protein, an enzymatic hydrolysis method and a gel chromatography separation and purification method to obtain a sea cucumber polypeptide, and a mass spectrometry method to determine the amino acid sequence of the sea cucumber polypeptide, thereby separating and identifying a sea cucumber polypeptide.

[0007] The following steps are involved:

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

[0009] Step 2: Dissolve 100 mg of the prepared sea cucumber total protein sample in 10 mM PBS buffer solution of pH 7.5, place it in a cellulose acetate dialysis bag with a molecular weight of 2000-5000Da, and dialyze it against a 5 mM ammonium acetate buffer solution of pH 6.0; after dialysis, place the protein solution in a 1KD ultrafiltration column for desalting and concentration, and after concentration, add alkaline protease with a concentration of 5000U / g to the concentrated solution, enzymolyze the protein at pH 8 and temperature 40°C, and then freeze-dry it in a vacuum freeze dryer to obtain a crude sea cucumber polypeptide;

[0010] Step 3: The crude sea cucumber polypeptide was separated and purified by Sephadex G-15 gel column chromatography, 2 ml of the column was taken, eluted with distilled water, the flow rate was set to 0.5 ml / min, the detection wavelength was 280 nm, the elution peak was collected, the components of the same absorption peak were repeatedly collected, and concentrated, and freeze-dried in a vacuum freeze dryer to obtain the separated and purified sea cucumber polypeptide;

[0011] Step 4: The purified product of the sea cucumber polypeptide separation was detected by high performance liquid chromatography column, using a mobile phase of 20 mM phosphate buffer solution, a flow rate of 0.5 ml / min, and a detection wavelength of 280 nm; the purified product with a single peak detected by high performance liquid chromatography was a sea cucumber polypeptide monomer;

[0012] Step 5: Use mass spectrometry to identify the amino acid sequence of the sea cucumber polypeptide monomer to obtain the amino acid sequence shown in SEQ ID NO: 1.

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

[0014] The method of extracting total protein by ammonium sulfate precipitation method in the present invention has been widely used, and then the sea cucumber polypeptide is obtained by enzymatic hydrolysis and gel chromatography separation and purification method, the separated and purified product is identified by high performance liquid phase technology, and the amino acid sequence of the sea cucumber polypeptide is identified by mass spectrometry to prepare the sea cucumber active polypeptide.

[0015] The effect of sea cucumber active polypeptide on the cell viability of mouse melanoma cells (B16-F10) was detected by MTT method. It was found that sea cucumber polypeptide had no toxicity to cells when acting alone. After B16-F10 cells were treated with sea cucumber polypeptide for 24 hours, the melanin content and tyrosinase activity level in the cells were significantly reduced. The experimental results showed that the sea cucumber polypeptide can inhibit the activity of tyrosinase in cells and reduce the synthesis of melanin, reflecting the biological activity of whitening.

[0016] Using in vitro experimental determination method, after the sea cucumber polypeptide treated porcine pancreatic elastase, it reacted with specific substrate and found that the product was reduced. The experimental results showed that the sea cucumber polypeptide can significantly inhibit the activity of elastase, reflecting that the sea cucumber polypeptide has strong anti-wrinkle activity.

[0017] The in vitro assay was used to detect the effects of sea cucumber peptides on -OH free radicals, DPPH free radicals, O2 - The free radical scavenging ability of sea cucumber polypeptides was shown by the experimental results of Fenton reaction to have a strong scavenging ability for -OH free radicals; the DPPH experimental results showed that sea cucumber polypeptides had a strong scavenging ability for DPPH free radicals; the O2 - The experimental results of free radical scavenging rate show that sea cucumber polypeptide has strong O2 - The free radical scavenging ability shows that sea cucumber polypeptide has strong antioxidant activity.

[0018] The beneficial effects of the present invention are as follows: the present invention provides a sea cucumber active polypeptide, which has good whitening, anti-wrinkle and anti-oxidation effects.

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

[0020] 2. The sea cucumber active polypeptide can inhibit the activity of elastase, which is a key enzyme that hydrolyzes key connective tissue matrix proteins. The inhibition of elastase activity can prevent the damage of elastic fibers by ultraviolet rays or free radicals by protecting the skin from aging, thereby playing an anti-wrinkle role.

[0021] 3. The sea cucumber active peptide can effectively remove -OH free radicals, DDPH free radicals and O2 - Free radicals. -OH free radical, DDPH free radical, O2 - Free radicals have the strongest oxidative activity in the body and can bind to biomacromolecules such as amino acids and DNA, causing oxidative damage to cells and scavenging -OH, DDPH and O2 - The ability of free radicals can reflect good antioxidant activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the Sephadex G-15 gel column chromatography separation diagram for the separation and purification of sea cucumber polypeptides.

[0023] Figure 2 This is the identification spectrum of the sea cucumber polypeptide separation and purification peak in high performance liquid chromatography.

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

[0025] Figure 4 This is the secondary spectrum of the amino acid structure of sea cucumber polypeptides identified by mass spectrometry.

[0026] Figure 5 This is a graph of melanin content in B16-F10 cells after treatment with different concentrations of sea cucumber polypeptides.

[0027] Figure 6 This is the tyrosinase activity spectrum of B16-F10 cells after being treated with sea cucumber polypeptides at different concentrations.

[0028] Figure 7 This is a graph showing the results of detecting the activity of pig pancreatic elastase after treatment with sea cucumber polypeptides at different concentrations.

[0029] Figure 8 This is a graph showing the test results of the -OH free radical scavenging ability of sea cucumber polypeptides at different concentrations.

[0030] Fig. 9 This is a graph showing the test results of the DPPH free radical scavenging ability of sea cucumber polypeptides at different concentrations.

[0031] Fig.10 The effects of different concentrations of sea cucumber peptides on O2 - Graph of free radical scavenging ability test results. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0033] 1. Preparation of Sea Cucumber Peptides

[0034] 1. Extraction of total protein from sea cucumber

[0035] Clean the fresh sea cucumber and remove impurities. Cut the processed sea cucumber into small pieces and add an appropriate amount of PBS buffer solution for homogenization. After homogenization, add 20 mM pH 7.0 Tris-HCl buffer at a volume ratio of 1:20 and stir evenly. Stir and extract at 4°C for 24 hours. Then centrifuge at 10000 r / min for 30 minutes and collect the supernatant. Then add solid (NH4)2SO4 to a final concentration of 80%, stir evenly, then stand at 4°C overnight, centrifuge at 10000 r / min for 30 minutes, dialyze in water, and freeze-dry to obtain total sea cucumber protein.

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

[0037] 100 mg of the prepared sea cucumber total protein sample was dissolved in 10 mM pH7.5 PBS buffer solution, placed in a cellulose acetate dialysis bag with a molecular weight of 2000-5000Da, and dialyzed against 5mM pH6.0 ammonium acetate buffer solution. After dialysis, the protein solution was placed in a 1KD ultrafiltration column for desalting and concentration. After concentration, alkaline protease (Novo protease was used in this study) with a concentration of 5000U / g was added to the concentrate, and the protein was enzymatically hydrolyzed for 24 hours at pH 8 and temperature 40°C, and then freeze-dried in a vacuum freeze dryer to obtain the crude sea cucumber polypeptide.

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

[0039] 3. High performance liquid chromatography detection

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

[0041] 4. Identification of amino acid sequences of sea cucumber polypeptides

[0042] Mass spectrometry detection: The peptides were dissolved in a dissolving solution (0.1% formic acid, 2% acetonitrile), vortexed, and centrifuged at 10,000 r / min for 10 min at 4°C. The supernatant was taken for mass spectrometry identification. The separated peptides were sequentially detected by the primary mass spectrometer and the secondary mass spectrometer. The primary mass spectrometer parameters were Resolution, 80,000; AGC target, 3e6; Maximum IT, 50 ms; Scan range, 50-3000 m / z; the secondary mass spectrometer parameters were: Resolution, 20,000; AGC target, 1e5; Maximum IT, 50 ms; TopN, 20NCE / stepped NCE 28. The primary mass spectrometer and the secondary mass spectrometer are shown in Figure 2. Figure 3 and Figure 4 shown.

[0043] Database search: The original mass spectrometry file was processed and converted by MS Convert software to obtain an MGF format file, and then MaxQuant was used to search for the amino acid sequence of the sea cucumber polypeptide, which was CPPLTCPG (SEQ ID NO: 1), in which several amino acids were 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 sequences in secondary mass spectrometry

[0045] N-terminal number N-terminal amino acid sequence C-terminal amino acid sequence C-terminal sequence number b1 C PPLTCPG y7 b2 CP PLTCPG y6 b3 CPP LTCPG y5 b4 CPPL TCPG y4 b5 CPPLT CPG y3 b6 CPPLTC PG y2 b7 CPPLTCP G y1

[0046] 2. Study on the whitening activity of sea cucumber polypeptides: Effects of sea cucumber polypeptides 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 in a constant temperature incubator at 37°C and 5% CO2.

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

[0049] Take B16-F10 cells with good growth status and add 2×10 3The cells were seeded in a 96-well plate and incubated for 24 hours. After that, the cells were treated with different concentrations of sea cucumber polypeptides (10 μM, 20 μM, 30 μM, dissolved in water) for 24 hours (6 replicate wells were set for each concentration). The culture medium was discarded, 20 μL of MTT solution was added to each well, and the cells were cultured at 37°C for 4 hours. The MTT solution was carefully discarded, 150 μL of DMSO solution was added, and the cells were shaken on an oscillator for 5 minutes. The absorbance (OD value) was detected at a wavelength of 490 nm on an ELISA reader.

[0050] After treatment with different concentrations of sea cucumber polypeptides, the viability of B16-F10 cells was not affected. The results are shown in Table 2 below.

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

[0052] Group Cell viability (%) Statistical results compared with the blank control group Blank control group 98.0±1.20 Sea cucumber peptide (10 μM) 97.8±2.13 Sea cucumber peptide (20 μM) 98.3±1.63 >0.05 Sea cucumber peptide (30 μM) 97.7±1.25 >0.05

[0053] After being treated with different concentrations of sea cucumber polypeptides, the viability of B16-F10 cells was not affected, indicating that sea cucumber polypeptides were non-toxic to cells and had no effect on cell proliferation.

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

[0055] The content of intracellular melanin was determined by NaOH lysis method. After B16-F10 cells were counted, 5×10 8 / mL was inoculated in a 6-well plate, 2 mL per well, and cultured for 24 hours. According to the experimental groups, 0.2μM α-melanocyte stimulating hormone (α-MSH) was added for induction (except the blank control group), and an α-MSH-induced melanin high expression cell model was constructed, and then different concentrations of sea cucumber polypeptides were added, where the final concentrations of sea cucumber polypeptides were 10, 20, and 30μM, and 6 replicates were set up in each group. In addition, 2 mL of culture medium was added to the blank control group and the model group, respectively. After 24 hours of culture, the supernatant was discarded, and after washing with PBS three times, 200μL of NaOH solution (containing 10% DMSO) was added to each well, and the cells were fully lysed at 80℃ for 1 hour, and the absorbance value (OD value) of each well was measured at a wavelength of 470nm. The protein content in the solution was determined by the BCA method. According to the same method, using melanin as the standard, a standard curve between OD value (Y) and mass concentration (X) was drawn: Y=0.0099X+0.00008 (R 2 =0.995), and the content of intracellular melanin was calculated by the standard curve. The relative content of intracellular melanin was calculated according to the following formula: relative content of intracellular melanin (%) = melanin content per gram of protein in the experimental group / melanin content per gram of protein in the α-MSH model group × 100%.

[0056] The experimental results showed that compared with the blank group, the melanin content in the α-MSH-induced model group increased significantly (p<0.05). When compared with the α-MSH-induced model group, each concentration of sea cucumber polypeptide could significantly inhibit the melanin content in B16-F10 cells (p<0.05), and showed obvious dose dependence. When the concentration of sea cucumber polypeptide was 30μM, it could completely inhibit the melanin produced by α-MSH. Figure 5 shown.

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

[0058] The activity of intracellular tyrosinase was determined by L-Dopa oxidation method. After B16-F10 cells were counted, 5×10 8 / mL was inoculated in a 6-well plate, 2 mL per well, and cultured for 24 hours. According to the experimental grouping, 0.2μM α-MSH was added for induction (except the blank control group) to construct an α-MSH-induced melanin high-expression cell model, and then different concentrations of sea cucumber polypeptides were added, where the final concentration of sea cucumber polypeptides was 10, 20, and 30μM, and 6 replicates 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 3 times with PBS, and 1 mL of PBS buffer containing 1% TritonX-100 was added to each well, placed in a -80℃ refrigerator for 1 hour, then thawed at room temperature, and the cell lysate was placed at 4℃, centrifuged at 12000r / min for 20 min, and the supernatant was collected. Take 60 μL of the supernatant in a 96-well plate, add 140μL of 5 mM L-Dopa, incubate at 37℃ for 1 hour, and measure the absorbance (OD value) of each well at a wavelength of 475 nm. The protein content in the solution was determined by the Bradford method. The relative activity of intracellular tyrosinase was calculated according to the following formula: relative activity of intracellular tyrosinase (%) = tyrosinase activity per gram of protein in the experimental group / tyrosinase activity per gram of protein in the α-MSH model group × 100%.

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

[0060] 3. Study on the anti-wrinkle activity of sea cucumber polypeptides: Effect of sea cucumber polypeptides on the activity of porcine pancreatic elastase

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

[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), and incubate on a shaker at 37 °C and 400 r / min for 30 min. Then add 2 mL of 0.5 M phosphate buffer at pH 6 to the prepared elastin solution, vortex mix, incubate on a shaker at 37 °C and 400 r / min for 30 min, add 2 mL of 0.5 M phosphate buffer at pH 6, take an appropriate amount of the mixed solution into a 2 mL centrifuge tube, centrifuge at 10000 r / min for 10 min, draw 200 μL of the supernatant into a 96-well plate, and measure the absorbance at a wavelength of 495 nm. The substrate plus enzyme solution was used as the blank control group, the substrate plus enzyme and sea cucumber polypeptide solution was used as the enzyme inhibition group, and the substrate plus sea cucumber polypeptide without enzyme solution was used as the background subtraction. Each group has 6 replicate wells. Inhibition rate (%) = [1–(An–An′) / (A0–A0′)] × 100%, where A0 is the absorbance of the solution with enzyme but no sea cucumber polypeptide, A0′ is the absorbance of the solution with substrate but no sea cucumber polypeptide and enzyme, An is the absorbance of the solution with only sea cucumber polypeptide, and An′ is the absorbance of the solution with sea cucumber polypeptide and enzyme.

[0063] The experimental results showed that sea cucumber polypeptides at various concentrations could inhibit the activity of porcine pancreatic elastase to varying degrees, and exhibited a dose-dependent inhibition. When the concentration of sea cucumber polypeptides was 30 μM, the inhibition rate of elastase reached 90%. Figure 7 shown.

[0064] 4. Study on the antioxidant activity of sea cucumber polypeptides: sea cucumber polypeptides against -OH free radicals, DPPH free radicals and O2 - Effect of free radical scavenging ability

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

[0066] Utilize H2O2 / Fe 2+ Produce -OH, o-phenanthroline -Fe through Fenton reaction 2+ The solution is oxidized by -OH to phenanthroline-Fe 3+ . 2-Phenanthroline-Fe 2+ The degree of fading of (orange-red) can be used to measure the amount of -OH radical scavenging. Sea cucumber polypeptides were prepared into 10, 20, and 30 μM solutions with pure water. 1 mL of each sea cucumber polypeptide solution of different concentrations 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 to it, 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. Six replicate wells were set up in each group, and the absorbance value (OD value) was measured at 510 nm. -OH radical scavenging rate (%) = [1-(Ai-Aj) / A0] × 100%, where 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. Its antioxidant capacity is calculated by the -OH radical scavenging effect.

[0067] The experimental results showed that all concentrations of sea cucumber polypeptides had the activity of scavenging -OH free radicals, and showed a dose-dependent inhibition. When the concentration of sea cucumber polypeptide was 30 μM, the scavenging rate of -OH free radicals reached 90%, which was consistent with the scavenging rate of vitamin C, and had strong antioxidant activity. Figure 8 shown.

[0068] 2. Effect of sea cucumber polypeptide on DPPH free radicals

[0069] DPPH free radical is a stable nitrogen-centered free radical with maximum absorption at a wavelength of 517 nm. The DPPH solution is purple, and its concentration is linearly related to absorbance. After adding sea cucumber polypeptide to the DPPH solution, the sea cucumber polypeptide can bind to or replace DPPH, reducing the amount of DPPH, and the color of the solution becomes lighter, showing that the absorbance value decreases continuously until it stabilizes. The antioxidant activity was calculated by the scavenging effect of DPPH free radicals. 1 mL of sea cucumber polypeptides of different concentrations (10, 20, 30 μM) was added to 3 mL of 0.1 mM DPPH solution, and the mixture was kept at room temperature and away from light for 30 min. Pure water was used as the reference solution, and 10 μM vitamin C was used as the control. Six replicate wells were set up in each group, and the absorbance value (OD value) was measured at 517 nm. DPPH free radical scavenging rate (%) = [1-(Ai-Aj) / A0] × 100%, where 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 is calculated by the DPPH free radical scavenging effect.

[0070] The experimental results showed that all concentrations of sea cucumber polypeptides had the activity of scavenging DPPH free radicals, and showed a dose-dependent inhibition. When the concentration of sea cucumber polypeptide was 30 μM, the scavenging rate of -OH free radicals reached 85%, which was consistent with the scavenging rate of vitamin C, and had strong antioxidant activity. Fig. 9 shown.

[0071] 3. Effect of sea cucumber polypeptide on O2 - The impact of free radicals

[0072] Pyrogallol auto-oxidizes under alkaline conditions, continuously releasing O2 - , O2 - It can also promote self-oxidation. The colored product has strong light absorption at 320 nm. Since the self-oxidation rate depends on O2 - concentration, remove O2 - The self-oxidation reaction is inhibited and the accumulation of products is prevented, thereby evaluating the ability of sea cucumber polypeptides to remove O2 - Capacity. Take 2 ml of 50 mM Tris-HCl buffer preheated at 25 °C, pH 8.2, and place it in a 10 ml test tube. Add different concentrations of sea cucumber polypeptide solutions (10, 20, 30 μM), then add 80 μL of 45 mM pyrogallol preheated at 25 °C, mix quickly, react for 5 min, and then add 20 μL of 10 M HCl to terminate the reaction. Pure water was used as the reference solution and 10 μM vitamin C was used as the control. Six replicate wells were set up in each group, and the absorbance value (OD value) was measured at 320 nm. O2 -Free radical scavenging rate (%) = [1-(Ai-Aj) / A0] × 100%, 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 was calculated based on the free radical scavenging effect.

[0073] The experimental results showed that all concentrations of sea cucumber peptides could remove O2 - The activity of free radicals was inhibited in a dose-dependent manner. When the concentration of sea cucumber polypeptide was 30 μM, it had no significant effect on O2 - The free radical scavenging rate reaches 90%, which is consistent with the scavenging rate of vitamin C and has strong antioxidant activity. Fig.10 shown.

[0074] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered as exemplary and non-restrictive in all respects.

Claims

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

1.

2. A method for preparing the sea cucumber active polypeptide according to claim 1, characterized in that: The preparation method comprises the following steps: Step 1: clean the fresh sea cucumber and cut it into small pieces, add an appropriate amount of PBS buffer solution for homogenization; after homogenization, add 20 mM pH 7.0 Tris-HCl buffer at a volume ratio of 1:20 and stir evenly, stir and extract at 4°C for 24 hours; centrifuge at a speed of 10000 r / min for 30 minutes, collect the supernatant; extract the total protein of the sea cucumber from the supernatant by ammonium sulfate precipitation method; Step 2: Dissolve 100 mg of the prepared sea cucumber total protein sample in 10 mM PBS buffer solution of pH 7.5, place it in a cellulose acetate dialysis bag with a molecular weight of 2000-5000Da, and dialyze it against a 5 mM ammonium acetate buffer solution of pH 6.0; after dialysis, place the protein solution in a 1KD ultrafiltration column for desalting and concentration, and after concentration, add alkaline protease with a concentration of 5000U / g to the concentrated solution, enzymolyze the protein at pH 8 and temperature 40°C, and then freeze-dry it in a vacuum freeze dryer to obtain a crude sea cucumber polypeptide; Step 3: The crude sea cucumber polypeptide was separated and purified by Sephadex G-15 gel column chromatography, 2 ml of the column was taken, eluted with distilled water, the flow rate was set to 0.5 ml / min, the detection wavelength was 280 nm, the elution peak was collected, the components of the same absorption peak were repeatedly collected, and concentrated, and freeze-dried in a vacuum freeze dryer to obtain the separated and purified sea cucumber polypeptide; Step 4: The purified product of the sea cucumber polypeptide separation was detected by high performance liquid chromatography column, using a mobile phase of 20 mM phosphate buffer solution, a flow rate of 0.5 ml / min, and a detection wavelength of 280 nm; the purified product with a single peak detected by high performance liquid chromatography was a sea cucumber polypeptide monomer; Step 5: Use mass spectrometry to identify the amino acid sequence of the sea cucumber polypeptide monomer to obtain the amino acid sequence shown in SEQ ID NO:

1.

3. The method for preparing the sea cucumber active polypeptide according to claim 2, characterized in that: The ammonium sulfate precipitation method is to add solid (NH4)2SO4 to the supernatant to a final concentration of 80%, stir evenly, then stand at 4°C overnight, centrifuge at a speed of 10000 r / min for 30 minutes, fully dialyze in water, and freeze-dry.

4. An application of the sea cucumber active polypeptide as claimed in claim 1, characterized in that: Application of the sea cucumber active polypeptide in the preparation of whitening efficacy products.

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

6. A use of the sea cucumber active polypeptide as claimed in claim 1, characterized in that: Application of the sea cucumber active polypeptide in the preparation of anti-wrinkle products.

7. The use of the sea cucumber active polypeptide according to claim 6, characterized in that: Application of the sea cucumber active polypeptide in inhibiting the activity of elastase.

8. An application of the sea cucumber active polypeptide according to claim 1, characterized in that: Application of the sea cucumber active polypeptide in the preparation of products with antioxidant efficacy.

9. The use of the sea cucumber active polypeptide according to claim 8, characterized in that: The sea cucumber active polypeptide can remove -OH free radicals, DDPH free radicals and O2 - Application in free radical activity.

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