Preparation method and application of sea cucumber egg-derived peptide compound

Abstract

The invention relates to a preparation method and application of a sea cucumber egg-derived peptide compound. The preparation method of the sea cucumber egg source peptide compound comprises the following steps: performing synchronous enzymolysis on sea cucumber egg powder by using alkaline protease-trypsin; the sea cucumber ovum source active peptide is obtained by further carrying out ultrafiltration, sephadex chromatography and high performance liquid chromatography separation and adopting liquid chromatography-mass spectrometry technology analysis, a computer-aided screening technology and in-vitro verification, and the amino acid sequence of the sea cucumber ovum source active peptide comprises one or more of WGN, WI GG and GFPVG (Fibroblast Growth Factor Peptide Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth). The sea cucumber ovum-derived peptide compound and the sea cucumber ovum-derived active peptide provided by the invention have double activities of resisting oxidation and inhibiting tyrosinase, can reduce the melanin content, and have the advantages of good water solubility, good safety and the like.

Description

Technical Field

[0001] The invention belongs to the field of biological products, and in particular relates to a preparation method and application of a sea cucumber egg-derived peptide complex. Background Art

[0002] Based on the free radical theory of aging, the intake of exogenous antioxidants has become one of the important ways to fight aging. For skin aging, the accumulation of melanin is also an important manifestation. Melanin is an indole polymer widely present in the skin. When the skin is exposed to ultraviolet radiation, the body's melanocytes will produce melanin and accumulate in melanosomes to protect the skin. However, excessive melanin deposition can lead to a series of skin problems such as chloasma, freckles, melanosis, seborrheic keratosis, etc., which has become a major problem for beauty lovers. Tyrosinase is the rate-limiting enzyme that controls the production of cellular melanin, and can catalyze the production of melanin through redox reactions. Its activity directly affects the amount of melanin produced, and finding substances with tyrosinase inhibitory activity is the key to reducing the body's melanin. In addition, a large number of studies have confirmed that the production of melanin and the body's oxidative stress interact with each other. Specifically, the process of melanin production will produce a large number of reactive oxygen free radicals, and excessive free radicals will further damage melanocytes, promote pigment accumulation, and increase the degree of aging. Therefore, reducing the body's oxidative stress and reducing tyrosinase activity are important for reducing pigment deposition and avoiding skin aging. However, most of the current research on skin anti-aging focuses only on antioxidants, and lacks the evaluation of its tyrosinase inhibitory activity. It is of great significance to find and prepare substances with both antioxidant and tyrosinase inhibitory activities.

[0003] Bioactive peptides are gaining more and more attention due to their high efficiency and high stability. Traditional preparation of bioactive peptides mainly uses enzymatic hydrolysis technology to obtain peptide mixtures and mainly uses single enzymatic hydrolysis technology. In actual experiments, the inventors found that the preparation of bioactive peptides by single enzyme preparations has the disadvantages of enzymatic hydrolysis dead corners, long time consumption, uneven action, low stability, and poor reproducibility. The complex peptide components and the presence of peptides with no / low bioactivity in the mixture may affect its activity efficiency. On the basis of the mixture, purification technology can be used to remove some peptide segments in the mixture to obtain peptide segments with known sequences. It is of great significance to achieve efficient and rapid preparation by chemical methods to complete its large-scale industrial precision application and related mechanism analysis. Traditional single peptides are usually obtained by separation and purification technology, which is cumbersome and time-consuming. With the development of bioinformatics, computer-assisted technology has gradually emerged in the screening of bioactive peptides. Its high-throughput analysis method can save peptide screening time and improve efficiency, but single computer screening may have the phenomenon that the screening results are inconsistent with the actual activity.

[0004] Sea cucumber eggs are a byproduct of sea cucumber processing and contain a large amount of nutrients such as protein. With the increase in consumer demand for sea cucumbers, the number of sea cucumber eggs has increased dramatically and is usually discarded as waste, resulting in a huge waste of protein resources. The low deep processing rate of sea cucumber egg protein is mainly related to its amino acid composition. The protein in sea cucumber eggs is mainly collagen, which is composed of glycine, arginine and other basic amino acids. The single enzymatic method for preparing bioactive peptides is mainly through enzymatic cleavage of the outer amino acids of the protein. After the outer amino acid chain disintegrates, the protein is then enzymatically hydrolyzed internally. The enzymatic hydrolysis time is long, the efficiency is low, and the biological activity is low.

[0005] Therefore, it is urgent to provide a method for preparing a dual-effect sea cucumber egg-derived peptide that is simple to operate, efficient, and rapid. At present, there is no report on the use of alkaline protease-trypsin in combination with sea cucumber eggs to prepare active peptides with antioxidant, tyrosinase inhibitory and melanin-reducing properties. Summary of the invention

[0006] In view of the low utilization rate and single activity of existing sea cucumber eggs, the present invention provides a preparation method and application of a sea cucumber egg-derived peptide complex with dual activities (antioxidation and tyrosine inhibition) treated with a composite enzyme. On this basis, the present invention uses the sea cucumber egg-derived peptide complex as a raw material, and realizes the preparation of a bioactive peptide that can reduce the melanin content and has dual activities of antioxidant and tyrosinase inhibition through traditional separation and purification technology combined with computer-aided technology.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The invention provides a method for preparing a sea cucumber double-functional sea cucumber egg-derived peptide complex treated with a composite enzyme, comprising the following steps: simultaneously enzymolyzing sea cucumber egg powder with alkaline protease-trypsin.

[0009] The beneficial effects of adopting the above technical solution include:

[0010] The low degree of green processing of industrial byproducts of sea cucumber processing is currently a bottleneck restricting the development of the industry. The abandonment of a large number of sea cucumber eggs has caused huge economic and resource waste. The present invention prepares a sea cucumber egg bifunctional peptide complex with antioxidant and tyrosinase inhibitory activity, which can promote the extension of the sea cucumber industry chain and achieve the improvement of the deep processing degree of sea cucumber industrial byproducts. The preparation method of the sea cucumber egg-derived composite peptide of the present invention has the characteristics of high efficiency, good reproducibility, can reduce melanin content, good antioxidant effect, high tyrosinase inhibitory activity, and small peptide molecular weight.

[0011] During the preparation process, the present invention uses alkaline protease and trypsin for simultaneous enzymolysis, and uses trypsin to specifically cut the peptide bonds of basic amino acids on the carboxyl side, further exposing the internal hydrophobic amino acids and aromatic amino acid cleavage sites. At the same time, the alkaline protease is accurately identified and cut, and the "layer-by-layer" enzymatic cleavage of sea cucumber protein is achieved, the number of small molecule peptides with hydrophobic amino acids and aromatic amino acids as the ends in the complex is increased, the antioxidant and tyrosinase inhibitory activities are improved, and the preparation of a bifunctional active peptide complex is achieved.

[0012] Furthermore, the sea cucumber egg powder is defatted sea cucumber egg powder.

[0013] Furthermore, the method further comprises the step of ultrafiltering the enzymatic hydrolysis product: passing the enzymatic hydrolysis product through ultrafiltration membranes with molecular weights of 10 kDa and 3 kDa in sequence, and collecting components with molecular weights greater than 10 kDa, components with molecular weights between 3 kDa and 10 kDa, and components less than 3 kDa.

[0014] Furthermore, the method also includes a step of separating components smaller than 3 kDa using a dextran gel resin Sephadex G-15, combining tubes 25 to 50 as the SCP-1 component, combining tubes 58 to 77 as the SCP-2 component, and combining tubes 85 to 103 as the SCP-3 component.

[0015] Furthermore, it also includes subjecting the SCP-2 components to high performance liquid chromatography separation, collecting components with retention times of 2.099 min, 3.148 min, 4.833 min, 5.352 min, 13.533 min, 14.992 min, and 20.99 min, and naming them SCP-2-1 to SCP-2-7, respectively.

[0016] Furthermore, the enzymatic hydrolysis conditions include: adjusting the pH of the aqueous solution of sea cucumber spawning powder to 7.5, adding a mixed enzyme, the ratio of the enzyme activity of the mixed enzyme to the substrate mass is 2000U / g, the enzyme activity ratio of alkaline protease to trypsin in the mixed enzyme is 1:1, and enzymatic hydrolysis is carried out at 25°C for 3 hours; then the enzyme is inactivated.

[0017] Furthermore, the enzyme inactivation conditions include: heating in a boiling water bath for 20 minutes.

[0018] The invention provides a sea cucumber egg-derived peptide complex, which is prepared by the method.

[0019] Furthermore, the sea cucumber egg-derived peptide complex includes one or a combination of WGN, WIGG, and GFPVG.

[0020] Furthermore, the present invention provides a sea cucumber egg-derived active peptide having dual activities of anti-oxidation and inhibition of tyrosinase, wherein the amino acid sequence of the sea cucumber egg-derived peptide comprises one or more of WGN, WIGG, and GFPVG.

[0021] Preferably, the amino acid sequence of the sea cucumber egg-derived peptide is one or more of WGN, WIGG, and GFPVG.

[0022] The beneficial effects of adopting the above technical scheme include: the sea cucumber egg-derived active peptides provided by the present invention can increase the added value of sea cucumber by-products, reduce the waste of resources and extend the industrial chain of sea cucumber by-products. It has been experimentally verified that WGN, WIGG and GFPVG screened by the present invention can effectively remove DPPH free radicals, ABTS free radicals and inhibit tyrosinase activity. The molecular weight of the sea cucumber egg-derived active peptides is less than 650KDa, and the small molecule peptides are easily digested and absorbed by the human body and act on the target. The sea cucumber egg-derived active peptides provided by the present invention have good solubility and are non-toxic to cells.

[0023] The present invention provides a method for preparing the sea cucumber egg-derived active peptide, comprising the following steps: solid phase synthesis of the sea cucumber egg-derived peptide.

[0024] The present invention provides a method for screening the above-mentioned sea cucumber egg-derived active peptides, comprising the following steps:

[0025] (1) hydrolyzing sea cucumber eggs with alkaline protease-trypsin mixed enzymolysis to obtain sea cucumber egg enzymolysis solution;

[0026] (2) subjecting the sea cucumber egg hydrolysate obtained in step (1) to three-step separation by ultrafiltration, dextran gel chromatography, and high performance liquid chromatography, and freeze drying to obtain active peptide freeze-dried powder;

[0027] (3) analyzing the active peptide lyophilized powder by liquid chromatography-mass spectrometry to obtain the peptide amino acid sequence;

[0028] (4) using computer-assisted screening technology to screen the peptides with known amino acid sequences obtained in step (3);

[0029] (5) Solid phase synthesis and screening of peptides according to the screening results of step (4) and in vitro verification, for example, antioxidant activity, tyrosinase inhibitory activity, melanin content, etc.

[0030] The beneficial effects of adopting the above technical solution include: the method provided by the present invention is an efficient, rapid and accurate peptide acquisition method, which uses a combination of traditional separation and purification technology and computer-aided technology to save the screening time of active peptides, improve efficiency, obtain components with high antioxidant peptide content, and thus improve the accuracy of peptides. The obtained peptides are screened using computer-aided technology, thereby improving the screening time, efficiency and accuracy.

[0031] Alkaline protease-trypsin mixed enzymatic hydrolysis is used. Trypsin and alkaline protease can respectively cut the peptide bonds on the carboxyl side of alkaline amino acids and hydrophobic amino acids. During the enzymatic hydrolysis, trypsin first recognizes and acts on the surface amino acids of sea cucumber egg proteins to loosen the protein structure. At the same time, alkaline protease accurately cuts hydrophobic and aromatic amino acids to improve the antioxidant and tyrosinase inhibitory activity of peptides.

[0032] Furthermore, the specific operation process of step (1) may include: weighing sea cucumber spawning powder, dissolving it in 10 times the volume of deionized water, adjusting the pH to 7.5, adding a mixed enzyme, the enzyme activity of the mixed enzyme to the substrate mass ratio is 2000U / g, and the enzyme activity ratio of alkaline protease to trypsin in the mixed enzyme is 1:1; enzymolysis at 25°C for 3h, heating in a boiling water bath for 20min to inactivate the enzyme, centrifuging at 10000r / min for 20min, and retaining the supernatant as the sea cucumber egg enzymolysis solution.

[0033] Further, the specific operation process of step (2) may include: classifying the sea cucumber egg hydrolysate through 10 kDa and 3 kDa ultrafiltration membranes in sequence, and collecting components less than 3 kDa; separating the components less than 3 kDa through dextran gel chromatography, collecting 2.0 mL of components in each tube, and collecting the components combined from tubes 58 to 77 as SCP-2 components; separating the SCP-2 components through high performance liquid chromatography, and collecting the components corresponding to the retention time of 5.352 min as SCP-2-4 components.

[0034] Furthermore, the specific operation process of step (3) may include: using liquid chromatography-mass spectrometry (LC-MS / MS) to identify the amino acid sequence of the SCP-2-4 component; mobile phase A is 2.0% acetonitrile-0.1% formic acid-water (V / V / V); mobile phase B is 0.1% formic acid-acetonitrile solution (V / V); the gradient starts from 2% (volume percentage) of phase B, increases to 35% (volume percentage) in 47 minutes with a nonlinear gradient, increases to 100% (volume percentage) within 1 minute, and is maintained for 12 minutes; the sample volume is 1.5 μL, and the column flow rate is 300 nL / min.

[0035] Furthermore, the specific operation process of step (4) may include: preliminary screening of active peptides identified by LC-MS / MS based on peptide chain length and hydrophobicity; predicting their biological activity values ​​through the online tool PeptideRanker, and retaining peptides with predicted biological activity values ​​greater than 0.84; predicting the water solubility and toxicity of the peptides through the online tools Innovagen and ToxinPred, retaining active peptide sequences that show good water solubility and non-toxicity for molecular docking; using the CDOCK mode in Discovery Studio 2019Client to perform molecular docking with the Keap1 receptor, and screening peptides based on binding energy.

[0036] Furthermore, the specific operation process of step (5) may include: synthesizing the screened peptide using a solid phase synthesis method, and verifying its biological activity through DPPH free radical scavenging ability, ABTS free radical scavenging ability, and tyrosinase inhibition rate.

[0037] The present invention provides the use of the sea cucumber egg-derived peptide complex in any one or more of (1) to (3);

[0038] (1) Preparing antioxidant products;

[0039] (2) preparing products for inhibiting tyrosinase activity;

[0040] (3) Reduce melanin content.

[0041] The present invention provides the use of the above sea cucumber egg-derived active peptide in any one or more of (1) to (3);

[0042] (1) Preparing antioxidant products;

[0043] (2) preparing products for inhibiting tyrosinase activity;

[0044] (3) Reduce melanin content.

[0045] The products include but are not limited to food, health products, cosmetics, medicines, etc.

[0046] The sea cucumber egg-derived active peptide provided by the invention has good free radical scavenging and antioxidant capabilities, has excellent tyrosinase inhibition activity, and can reduce the content of melanin.

[0047] The beneficial effects of adopting the above technical scheme include: the sequences of the sea cucumber egg-derived active peptides separated by the present invention are WGN, WIGG, and GFPVG, which have high application development value and can be used as functional ingredients in food, health products, cosmetics and other fields. WGN, WIGG, and GFPVG can effectively remove DPPH free radicals, ABTS free radicals and inhibit tyrosinase activity, and their DPPH free radical removal rates are 55.49±1.70%, 41.28±1.18%, and 42.55±0.87%, respectively; their ABTS free radical removal rates are 85.41±1.78%, 73.40±1.54%, and 52.09±0.93%, respectively. The tyrosinase inhibition rates of WGN, WIGG, and GFPVG are 90.04±1.06%, 84.92±1.34%, and 89.10±1.79%, respectively. WGN, WIGG, and GFPVG can effectively reduce the content of melanin. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 These are the results of molecular weight distribution measurement of sea cucumber egg-derived peptide complexes, where 1 represents the sample of sea cucumber egg protein hydrolyzed by alkaline protease-trypsin complex enzymatic hydrolysis, 2 represents the sample of sea cucumber egg protein hydrolyzed by alkaline protease, and 3 represents the sample of sea cucumber egg protein hydrolyzed by trypsin.

[0049] Figure 2 These are the results of the determination of the hydrophobicity of the composite surface of sea cucumber egg-derived peptides, wherein 1 represents the sample of sea cucumber egg protein hydrolyzed by alkaline protease-trypsin complex enzymatic hydrolysis, 2 represents the sample of sea cucumber egg protein hydrolyzed by alkaline protease, and 3 represents the sample of sea cucumber egg protein hydrolyzed by trypsin.

[0050] Figure 3 This is the elution curve of the G-15 glucan gel of the present invention, wherein the abscissa is the number of elution tubes and the ordinate is the absorbance at 280 nm.

[0051] Figure 4 It is the high performance liquid chromatography (RP-HPLC) of the present invention, wherein the abscissa is the retention time (min) and the ordinate is the absorbance value at 214 nm.

[0052] Figure 5 In the figure, the upper figure shows the result of the interaction between WGN and Keap1, and the lower figure shows the result of the interaction between WGN and tyrosinase (2Y9X).

[0053] Figure 6 In the figure, the upper figure shows the result of the interaction between WIGG and Keap1, and the lower figure shows the result of the interaction between WIGG and tyrosinase (2Y9X).

[0054] Figure 7In the figure, the upper figure shows the result of the interaction between GFPVG and Keap1, and the lower figure shows the result of the interaction between GFPVG and tyrosinase (2Y9X).

[0055] Figure 8 It is the effect of synthetic peptides on the scavenging rate of DPPH free radicals, wherein the horizontal axis is the type of synthetic peptides, and the vertical axis is the scavenging rate of DPPH free radicals.

[0056] Fig. 9 It is the effect of synthetic peptides on the scavenging rate of ABTS free radicals, wherein the abscissa is the type of synthetic peptides, and the ordinate is the scavenging rate of ABTS free radicals.

[0057] Fig.10 It is the effect of synthetic peptides on the tyrosinase inhibition rate, wherein the horizontal axis is the type of synthetic peptides, and the vertical axis is the tyrosinase inhibition rate.

[0058] Fig.11 The effects of different concentrations of WGN, WIGG and GFPVG on the survival rate of HepG2 cells, where A is the effect of different concentrations of WGN on the survival rate of cells, B is the effect of different concentrations of WIGG on the survival rate of cells, and C is the effect of different concentrations of GFPVG on the survival rate of cells.

[0059] Fig.12 This is the effect of WGN on tyrosinase activity in B16-F10 cells.

[0060] Fig.13 This is the effect of WGN on melanin production in B16-F10 cells.

[0061] Fig.14 This is the effect of WIGG on tyrosinase activity in B16-F10 cells.

[0062] Fig.15 This is the effect of WIGG on melanin production in B16-F10 cells.

[0063] Fig.16 This is the effect of GFPVG on tyrosinase activity in B16-F10 cells.

[0064] Fig.17 This is the effect of GFPVG on melanin production in B16-F10 cells. DETAILED DESCRIPTION

[0065] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0066] The invention provides a method for preparing a sea cucumber egg-derived peptide complex with dual activities of sea cucumber egg-derived antioxidant and tyrosinase inhibition based on alkaline protease-trypsin treatment.

[0067] The preparation method of the sea cucumber egg-derived peptide complex may include the following steps: using alkaline protease-trypsin mixed enzymolysis to obtain a sea cucumber egg hydrolysate. The obtained sea cucumber egg hydrolysate may also be subjected to three-step separation and purification by ultrafiltration-dextran gel chromatography-high performance liquid chromatography, determining the DPPH free radical scavenging rate and ABTS scavenging rate of each separated component, and freeze-drying to obtain a high antioxidant peptide composite powder.

[0068] Further, the preparation of the sea cucumber egg source peptide mixed solution may include the following steps: using sea cucumber eggs as raw materials, selecting alkaline protease and trypsin for composite synchronous enzymolysis, and the enzymolysis process includes: the ratio of the enzymatic activity of the mixed enzyme to the substrate mass is 2000U / g, the mixed enzyme includes alkaline protease and trypsin, the enzymatic activity ratio of alkaline protease to trypsin is 1:1, the enzymolysis time is 3h, the enzymolysis temperature is 25°C, and the pH is 7.5. After the enzymolysis is completed, the enzyme is heated in a boiling water bath for 20min to inactivate the enzyme, centrifuged at 10000r / min for 20min, and the supernatant is taken to obtain the sea cucumber egg enzymolysis solution.

[0069] Furthermore, the separation of sea cucumber egg-derived antioxidant peptides may include the following steps: the sea cucumber egg hydrolysate is sequentially graded through 10 kDa and 3 kDa ultrafiltration membranes, the DPPH free radical scavenging ability of different components is determined, the eluted components with high DPPH scavenging ability are collected, and freeze-dried. The high antioxidant components obtained by ultrafiltration are re-dissolved and separated through dextran gel chromatography, the eluted components with the strongest DPPH free radical scavenging ability are collected, and freeze-dried. The high antioxidant components obtained by dextran gel chromatography are re-dissolved and separated in the third step through high performance liquid chromatography, the components are collected for DPPH free radical scavenging ability determination, the eluted components with the strongest antioxidant activity are retained, and freeze-dried to obtain a high antioxidant peptide composite powder.

[0070] The invention provides a sea cucumber egg-derived peptide complex, which is prepared by the method.

[0071] The present invention provides the use of the sea cucumber egg-derived peptide complex in preparing products for anti-oxidation and / or inhibiting tyrosinase activity.

[0072] The invention provides sea cucumber egg-derived active peptides having dual activities of anti-oxidation and inhibition of tyrosinase, a preparation method, a screening method and applications thereof.

[0073] The present invention provides three sea cucumber egg-derived active peptides with antioxidant and tyrosinase inhibitory activities, wherein the amino acid sequences of the sea cucumber egg-derived active peptides are WGN, WIGG, and GFPVG. The sea cucumber egg-derived active peptides can reduce the content of melanin, and are also dual-functional sea cucumber egg-derived active peptides, having antioxidant and tyrosinase inhibitory effects.

[0074] The method for preparing the sea cucumber egg-derived active peptide comprises the following steps: solid phase synthesis of the sea cucumber egg-derived active peptide having the above amino acid sequence.

[0075] The present invention provides a method for screening the above-mentioned sea cucumber egg-derived active peptides, comprising the following steps:

[0076] (a) The highly antioxidant peptide complex obtained above was used to identify the peptide amino acid sequence using liquid chromatography-mass spectrometry.

[0077] (b) Using computer-assisted screening technology to screen the peptides with known amino acid sequences obtained in step (a) to obtain peptides with potential high antioxidant activity and high tyrosinase inhibition activity.

[0078] (c) Solid phase synthesis and screening of peptides were performed, and their antioxidant and tyrosinase inhibitory activities and effects on melanin content were verified in vitro.

[0079] Further, the identification of sea cucumber egg-derived antioxidant peptides may include the following steps: using liquid chromatography-mass spectrometry (LC-MS / MS) to identify the amino acid sequence of the components separated by reversed-phase high performance liquid chromatography. Mobile phase A is 2.0% acetonitrile-0.1% formic acid-water (V / V / V); mobile phase B is 0.1% formic acid-acetonitrile solution (V / V); the gradient starts from 2% (volume percentage) of phase B, and the nonlinear gradient phase B increases to 35% (volume percentage) in 47 minutes, and then the phase B increases to 100% (volume percentage) within 1 minute, and is maintained for 12 minutes; the sample volume is 1.5 μL, and the column flow rate is 300 nL / min.

[0080] Furthermore, the screening of bifunctional peptides from sea cucumber eggs may include the following steps: preliminary screening of active peptide segments identified by LC-MS / MS based on peptide chain length and hydrophobicity. Prediction of the biological activity value of the polypeptide using the online tool PeptideRanker (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.php). Prediction of the water solubility and toxicity of the polypeptide using the online tools Innovagen (www.innovagen.com / proteomicstools) and ToxinPred (http: / / crdd.osdd.net / raghava / / toxinpred), retaining active peptide sequences with good water solubility and non-toxicity for molecular docking.

[0081] Furthermore, the activity of the sea cucumber egg-derived bifunctional peptide can be verified experimentally, including the following steps: synthesizing the screened peptide using a solid phase synthesis method, and verifying its biological activity through DPPH free radical scavenging ability, ABTS free radical scavenging ability and tyrosinase inhibition rate experiments.

[0082] The present invention aims at the phenomenon that the utilization rate of existing sea cucumber intestines is low and the activity is single, and provides a method for obtaining bifunctional sea cucumber egg-derived active peptides based on alkaline protease-trypsin simultaneous enzymolysis, separation and purification-computer screening in sequence. The present invention uses ultrafiltration, gel chromatography, high performance liquid chromatography separation technology, with the help of protein database and online software, and uses properties such as DPPH clearance rate, ABTS clearance rate, water solubility and biological toxicity for multiple screening; combined with molecular docking to explore the interaction mode between active peptides and receptor Keap1 and tyrosinase (2Y9X), and finally solid phase synthesis and verification of the antioxidant activity and inhibition of tyrosinase activity of the polypeptide. The results show that the three active peptides screened by the present invention have the advantages of good water solubility, good safety, good antioxidant activity, excellent ability to inhibit tyrosinase, and can reduce melanin content.

[0083] The experimental methods used in the following examples are conventional methods in the art unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and those skilled in the art can obtain the solutions used in the experiments through commercial channels or prepare them by conventional methods. The solutions involved in the present invention, unless otherwise specified, are all made of water.

[0084] Sea cucumber spawning powder was purchased from Linghai Dalian Sea Treasures Breeding Co., Ltd.; dextran gel resin Sephadex G-15 was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; HepG-2 cells were purchased from China Center for Type Culture Collection (CCTCC); PBS was purchased from Beijing Bio-Toda Technology Co., Ltd.; L-dopa was purchased from Shanghai Aladdin Reagent Co., Ltd.; CCK-8 kit was purchased from Biyuntian Biotechnology Co., Ltd.; MEM culture medium was purchased from Wuhan Pronosai Life Science Technology Co., Ltd. B16-F10 mouse melanoma cells were purchased from Wuhan Pronosai Life Science Technology Co., Ltd., fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd., and 1% double-antibody RPMI-1640 culture medium was purchased from Wuhan Pronosai Life Science Technology Co., Ltd., containing penicillin G sodium salt and streptomycin sulfate, the content of penicillin G sodium salt was 1% (mass percentage), and the content of streptomycin sulfate was 1% (mass percentage).

[0085] The following is an introduction through specific embodiments.

[0086] Example 1

[0087] The method for preparing a sea cucumber egg-derived peptide enzymatic hydrolysate comprises the following steps: weighing defatted sea cucumber spawning powder, dissolving it in 10 times the volume of deionized water, adjusting the pH to 7.5, adding a mixed enzyme, wherein the mass ratio of the enzymatic activity of the mixed enzyme to the substrate is 2000U / g, the mixed enzyme comprises alkaline protease and trypsin, and the enzymatic activity ratio of the alkaline protease to the trypsin in the mixed enzyme is 1:1, for example, for every 1g of substrate, the alkaline protease is added in an amount of 1000U, the trypsin is added in an amount of 1000U, and the enzymatic hydrolysis is performed at 25°C for 3h. After heating in a boiling water bath for 20min to inactivate the enzyme, the mixture is centrifuged at 10000r / min for 20min, and the supernatant is retained as the sea cucumber egg enzymatic hydrolysate.

[0088] Comparative Example 1

[0089] On the basis of Example 1, in the enzymatic hydrolysis process of Comparative Example 1, alkaline protease was used to replace the mixed enzyme (alkaline protease-trypsin) in Example 1, and the reaction was carried out under the optimal enzymatic hydrolysis conditions.

[0090] The specific operations in comparative example 1 include: adjusting pH=10, temperature 45°C, adding 2000U / g enzyme, and enzymolysis time 3h. After heating in a boiling water bath for 20min to inactivate the enzyme, centrifuging at 10000r / min for 20min, retaining the supernatant as the sea cucumber egg enzymolysis liquid. The rest is the same as in Example 1.

[0091] Comparative Example 2

[0092] On the basis of Example 1, in the enzymatic hydrolysis process of Comparative Example 2, trypsin was used to replace the mixed enzyme (alkaline protease-trypsin) in Example 1, and the reaction was carried out under its optimal enzymatic hydrolysis conditions.

[0093] The specific operations in comparative example 1 include: adjusting pH=7.2, temperature 37°C, adding 2000U / g enzyme, and enzymolysis time 3h. After heating in a boiling water bath for 20min to inactivate the enzyme, centrifuging at 10000r / min for 20min, and retaining the supernatant as the sea cucumber egg enzymolysis liquid. The rest is the same as in Example 1.

[0094] Effect Example 1 Detection of the biological activity of sea cucumber egg hydrolysate

[0095] The sea cucumber egg enzymatic hydrolysates of Example 1, Comparative Example 1 and Comparative Example 2 were freeze-dried to obtain a sea cucumber egg peptide complex, and the antioxidant activity (DPPH clearance rate and ABTS clearance rate) and tyrosinase inhibitory activity of the sea cucumber egg peptide complex were detected.

[0096] (1) Detecting the DPPH scavenging rate includes the following steps: setting up a sample group, a control group, and a blank group. Sample group: 4.0 mL of the sample solution to be tested was mixed with an equal volume of DPPH solution; control group: 4.0 mL of the sample solution to be tested was mixed with an equal volume of anhydrous ethanol; blank group: DPPH solution. In the above groups, the solvent in the DPPH solution was anhydrous ethanol, and the DPPH concentration was 0.1 mmol / L; in the sample solution to be tested, the solvent was water, and the polypeptide concentration was 2.0 mg / mL. After each group was reacted in the dark at room temperature for 30 minutes, the absorbance at 517 nm was measured using an ultraviolet spectrophotometer. The DPPH free radical scavenging ability (i.e., DPPH scavenging rate) was calculated according to the following formula.

[0097]

[0098] In the formula, A 样品组 A is the absorbance of the sample group at 517 nm; 对照组 A is the absorbance of the control group at 517 nm; 空白组 The absorbance of the blank group was 517 nm.

[0099] (2) Detecting ABTS clearance rate, including the following steps:

[0100] ①Prepare working solution: Mix ABTS solution and potassium persulfate solution of equal volume, the concentration of ABTS in ABTS solution is 7.0mmol / L, and the concentration of potassium persulfate in potassium persulfate solution is 2.45mmol / L. Place at 25℃ for 12-16h, and react in dark conditions to obtain ABTS mother solution. Dilute ABTS with 80% ethanol solution as working solution, so that the absorbance of the working solution at 734nm reaches 0.7±0.1.

[0101] ② Prepare sample group, control group and blank group. Sample group: Mix 0.5mL 2.0mg / mL of the sample to be tested with 3.0mL of working solution; Control group: Mix 0.5mL 2.0mg / mL of the sample to be tested with 3.0mL of distilled water; Blank group: Evenly mix 0.5mL of distilled water with 3.0mL of working solution. Incubate each group in dark conditions for 10min and measure the absorbance at 734nm. Determine the ABTS clearance rate according to the following formula.

[0102]

[0103] In the formula, A 样品组 is the absorbance value of the sample group at 734 nm; A 对照组 is the absorbance value of the control group at 734 nm; A 空白组 The absorbance of the blank group was measured at 734 nm.

[0104] (3) Detecting tyrosinase inhibitory activity, including the following steps: setting up a blank group, a negative group, a background group and a sample group, and preparing solutions for each group according to Table 1.

[0105] Table 1 Reaction solution grouping and dosage

[0106]

[0107] Each group was incubated in a 37°C water bath for 30 min, the absorbance was measured at 475 nm, and the inhibition rate of tyrosinase by different concentrations of active substances was calculated according to the following formula.

[0108]

[0109] In the above formula, A 阴性 A is the absorbance of the negative group at 475 nm; 空白 is the absorbance of the blank group at 475 nm; A 样品 is the absorbance of the sample group at 475 nm; A 背景 is the absorbance of the background group at 475 nm.

[0110] (4) Detecting the relative melanin content of cells, comprising the following steps:

[0111] Cell culture: B16-F10 mouse melanoma cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% double antibody, and cultured in a cell culture incubator at 37°C and 5% CO2.

[0112] The experimental method was slightly modified from the following literature: Liu JZ, Xu XH, Jiang RJ, et al. Vanillic acid in Panax ginseng root extract inhibits melanogenesis in B16F10 cells via inhibition of the NO / PKG signaling pathway [J]. Bioscience, Biotechnology, and Biochemistry, 2019, 83 (7): 1205-1215. The control group was cells cultured normally, and the experimental group was cells treated with 2 mg / mL of different samples. The cells were resuspended in PBS, 1 mol / L NaOH was added, incubated at 80 °C for 2 h, and vortexed to dissolve. Centrifuged at 10000 rpm for 15 min, 100.0 μL of the supernatant was taken in a 96-well plate, and the absorbance was measured at 405 nm.

[0113]

[0114] In the above formula, A 实验组 A is the absorbance of cells in the experimental group at 405 nm; 对照 is the absorbance of the control group at 405 nm.

[0115] Experimental results:

[0116] The results of the antioxidant and tyrosinase inhibitory activity tests are shown in Table 2. It can be seen from Table 2 that the antioxidant activity, tyrosinase inhibitory activity and melanin relative content of the sea cucumber egg peptide complex prepared by the method of Example 1 of the present invention are better than those of the sample prepared by a single enzyme.

[0117] Table 2 Evaluation of the biological activity of peptide complexes

[0118]

[0119] Effect Example 2: Detecting whether proteins are enzymatically hydrolyzed into small peptides

[0120] The molecular weight of the sea cucumber egg-derived peptide complex obtained by the methods of Example 1, Comparative Example 1 and Comparative Example 2 was measured.

[0121] Figure 1 This is the molecular weight distribution diagram of sea cucumber egg-derived peptide complex. Figure 1 In the figure, 1 represents a sea cucumber egg-derived peptide complex sample prepared using alkaline protease-trypsin (i.e., a sea cucumber egg-derived peptide complex prepared using the method of Example 1), 2 represents a sea cucumber egg-derived peptide complex prepared using alkaline protease (i.e., a sea cucumber egg-derived peptide complex prepared using the method of Comparative Example 1), and 3 represents a sea cucumber egg-derived peptide complex prepared using trypsin (i.e., a sea cucumber egg-derived peptide complex prepared using the method of Comparative Example 2). Figure 1 It can be seen that the molecular weight of the peptide samples prepared by alkaline protease-trypsin composite enzymolysis is mainly concentrated between 0-1500Da, and the peptides in the alkaline protease treatment group complex are mainly peptides with a molecular weight greater than 3000Da. The trypsin treatment group complex is mainly composed of peptides with a molecular weight greater than 3000Da and less than 5000Da. Alkaline protease-trypsin treatment can obtain peptides with a smaller molecular weight. The inventor speculates that it is mainly due to the fact that the enzyme cleavage sites of the composite enzyme are more than those of comparative examples 1 and 2, which can make the protein fully enzymatically cleaved. Arginine, glycine, and alkaline amino acids are mainly used in sea cucumbers. Alkaline protease can cut hydrophobic amino acids (glycine), and arginine and other alkaline amino acids are trypsin cleavage sites, so the number of small molecular weight peptides in the trypsin enzymolysis composite peptide is higher than that in the alkaline protease treatment group. In addition, the high proportion of low molecular weight peptides is also one of the important reasons why the sea cucumber egg-derived peptide complex prepared by the method of Example 1 has high biological activity.

[0122] Effect Example 3 Detecting whether there are more hydrophobic amino acids exposed in each peptide complex

[0123] The surface hydrophobicity of the peptide complex prepared by the methods of Example 1, Comparative Example 1 and Comparative Example 2 was evaluated using the 8-aniline-1-naphthalenesulfonic acid (ANS) fluorescent probe method.

[0124] Each enzymatic hydrolysate was prepared into a 0.2 mg / mL liquid as a sample solution. ANS was dissolved in 10 mmol / L PBS to obtain an ANS solution, and the concentration of ANS in the ANS solution was 8 mmol / L. 4 mL of the sample solution was placed in a 5 mL centrifuge tube, 20 μL of the ANS solution was added, and the mixture was shaken for 30 s and allowed to stand for 2 min. The fluorescence spectrophotometer was used for detection, and the excitation wavelength was set to 395 nm, the emission wavelength was fixed to 400-600 nm, and the slit width was 5 nm.

[0125] Figure 2 The results of surface hydrophobicity determination in Example 1 and different comparative examples are shown in the figure. The horizontal axis is the sea cucumber egg-derived peptide complex prepared by different proteases, and the vertical axis is the fluorescence intensity, which can reflect the hydrophobicity. Figure 2 In the figure, 1 represents a sea cucumber egg-derived peptide complex sample prepared using alkaline protease-trypsin (i.e., the sea cucumber egg-derived peptide complex prepared in Example 1), 2 represents a sea cucumber egg-derived peptide complex prepared using alkaline protease (i.e., the sea cucumber egg-derived peptide complex prepared in Comparative Example 1), and 3 represents a sea cucumber egg-derived peptide complex prepared using trypsin (i.e., the sea cucumber egg-derived peptide complex prepared in Comparative Example 2). Figure 2 It can be seen that the surface hydrophobicity of the peptide sample prepared by alkaline protease-trypsin combined enzymatic hydrolysis is significantly better than that of the peptide complex sample prepared by a single enzyme.

[0126] Example 2 Separation and Purification

[0127] (1) Ultrafiltration: The sea cucumber egg hydrolysate obtained in Example 1 was prepared into a sea cucumber egg hydrolysate with a concentration of 20 mg / mL using ultrapure water, and passed through ultrafiltration membranes with molecular weights of 10 kDa and 3 kDa in sequence to collect components with molecular weights greater than 10 kDa, 3 kDa-10 kDa, and less than 3 kDa. The DPPH clearance rate, ABTS clearance rate, tyrosinase inhibitory activity, and melanin content of each collected component were determined, and the detection method was referred to Effect Example 1.

[0128] According to the above screening results, the components with higher clearance rate were selected and freeze-dried for later use. The screening results are shown in Table 3. It can be seen that the DPPH clearance rate, ABTS clearance rate, tyrosinase inhibitory activity and melanin relative content of the components less than 3 kDa are significantly better than those of other components. Therefore, the components less than 3 kDa were selected for subsequent gel chromatography separation experiments.

[0129] Table 3 Antioxidant and tyrosinase inhibitory activities of ultrafiltration fractions

[0130]

[0131] (2) Gel chromatography: The fraction less than 3 kDa obtained in step (1) is further separated using Sephadex G-15, a dextran gel resin.

[0132] The column (2.6×60.0 cm) was balanced with distilled water, and the injection volume was 5 mL; the sample (components less than 3 kDa) concentration was 10 mg / mL. The UV detection wavelength was 280 nm. 2.0 mL of the fraction was collected in each tube and freeze-dried.

[0133] According to the G-15 dextran gel elution curve ( Figure 3 ), it can be seen that SCP-1 (tubes 25 to 50 combined), SCP-2 (tubes 58 to 77 combined), and SCP-3 (tubes 85 to 103 combined) have obvious absorbance values ​​at 280nm.

[0134] The DPPH scavenging rate, ABTS scavenging rate and tyrosinase inhibitory activity of SCP-1 component, SCP-2 component and SCP-3 component were tested respectively, and the detection method was referred to Example 1. According to the results of DPPH free radical scavenging rate and ABTS scavenging rate, the component with the strongest antioxidant activity was screened out, and according to the results of tyrosinase inhibitory activity and melanin relative content, the component with the strongest tyrosinase inhibitory ability and the lowest melanin content was screened out. According to Table 4, it can be seen that the DPPH free radical scavenging rate, ABTS scavenging rate and tyrosinase inhibitory activity of component SCP-2 were significantly higher than those of other components, and the relative content of melanin was low. Therefore, component SCP-2 was selected for HPLC separation.

[0135] Table 4 Antioxidant properties and tyrosinase inhibitory activity of each fraction of G-15 gel chromatography

[0136]

[0137] (3) HPLC separation: Agilent C18 column (250.0×4.6 mm, 5.0 μm) was used to further separate the components with high antioxidant activity from the SCP-2 components. Mobile phase A was acetonitrile and mobile phase B was 0.1% (volume percentage) trifluoroacetic acid. Isocratic elution (10% A, 90% B, both volume percentages) was performed at a flow rate of 1.0 mL / min and a UV detection wavelength of 214 nm. The components were collected according to the results of the HPLC chromatogram, as shown in FIG. Figure 4As shown, the corresponding retention times of SCP-2-1, SCP-2-2, SCP-2-3, SCP-2-4, SCP-2-5, SCP-2-6 and SCP-2-7 are 2.099min, 3.148min, 4.833min, 5.352min, 13.533min, 14.992min and 20.99min respectively.

[0138] After that, each component was freeze-dried, and the scavenging rate of each component on DPPH and ABTS, the tyrosinase inhibitory activity and the relative content of melanin were measured by the method of Example 1. As can be seen from Table 5, the scavenging rate and tyrosinase inhibitory activity of the SCP-2-4 component on DPPH and ABTS were significantly higher than those of the other components, and the relative content of melanin was also low. Therefore, the SCP-2-4 component was selected for the identification of the amino acid sequence.

[0139] Table 5 Antioxidant and tyrosinase inhibitory activity of each HPLC component

[0140]

[0141] Example 3 Identification of amino acid sequence of sea cucumber egg antioxidant peptide

[0142] The amino acid sequence identification was carried out by Nanjing Positive Function Biotechnology Co., Ltd., including the following steps: liquid chromatography-mass spectrometry (LS-MS / MS) was used to perform secondary mass spectrometry analysis on the component SCP-2-4 separated by high performance liquid phase, wherein the mobile phase A was 2.0% acetonitrile-0.1% formic acid-water (V / V / V); the mobile phase B was 0.1% formic acid-acetonitrile solution (V / V), and the gradient started from 2% B phase, and the B phase was increased to 35% in 47 minutes with a nonlinear gradient, and then the B phase was increased to 100% within 1 minute and maintained for 12 minutes; the sample volume was 1.5 μL, and the column flow rate was 300 nL / min.

[0143] By comparing with the protein database, the amino acid sequences of 35 novel active peptides were obtained (Table 6).

[0144] Table 6 Identification of peptide sequences in SCP-2-4 by LC-MS / MS

[0145]

[0146]

[0147] Example 4 Screening and molecular docking of bifunctional peptides from sea cucumber eggs

[0148] First, the peptide sequences were screened according to molecular weight and number of amino acids, and the peptides with molecular weight less than 500 were selected.

[0149] Peptides with Da and amino acid number ≤5 were screened subsequently.

[0150] The biological activity values ​​of the peptides were predicted using the online tool PeptideRanker (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.php), and peptides with predicted biological activity values ​​greater than 0.84 were retained.

[0151] The water solubility and biological toxicity of the peptides were screened and predicted using the online tools Innovagen (www.innovagen.com / proteomicstools) and ToxinPred (http: / / crdd.osdd.net / raghava / / toxinpred), and the peptides that were shown by the software results to be "well soluble" and non-toxic were retained.

[0152] The CDOCK mode in Discovery Studio 2019 Client was used to perform preliminary molecular docking screening (LibDock) with the Keap1 (PBD ID: 2ZMX) receptor. The peptide sequence docked with Keap1 was further docked with tyrosinase (PBD ID: 2Y9X) using Maestro12.8 software, and the LibDock score and binding score (Dockingscore) were recorded. The smaller the energy, the tighter the binding). Finally, three new peptides with potential good biological activity, good water solubility and no toxicity were screened (Table 7).

[0153] Table 7 Prediction of biological activity, toxicity, solubility and other characteristics of active peptides from sea cucumber eggs

[0154]

[0155]

[0156] Note: “-” in the table means that simultaneous docking is not possible and no docking score is given.

[0157] The molecular docking results of WGN, WIGG, and GFPVG are shown in Figure 5 , Figure 6 and Figure 7As shown. WGN binds most tightly to the two receptors, indicating that WGN may have potential antioxidant activity and tyrosinase inhibition ability. The active peptide WGN is mainly connected to the ILE416, GLY364, LEU365, VAL418, LEU557, and VAL604 residues of Keap1 through hydrogen bonds; the amino acid residue that produces alkyl / pi-alkyl interactions is ALA366; the amino acid that produces electrostatic interactions is ARG415. The aspartic acid (N) on the active peptide WGN interacts with GLU 256, tryptophan (W) and VAL 283 on tyrosinase through hydrogen bonds; the tryptophan (W) on the polypeptide interacts with the residue PHE 264 through π-π bonds. The active peptide WIGG can generate alkyl / pi-alkyl interactions with the VAL512, VAL465, ALA366, and CYS513 residues of the Keap1 protein, can be connected to the GLY367, VAL606, LEU365, and ILE559 residues of Keap1 through hydrogen bonds, and generate electrostatic interactions with the ARG415 residue. In the interaction with tyrosinase, it mainly occupies the tyrosinase site through hydrogen bonds with the ASN260 and HIS85 residues. The active peptide GFPVG interacts with the ALA607, CYS368, and VAL606 residues of Keap1 mainly through alkyl / pi-alkyl interactions, can form hydrogen bonds and electrostatic interactions with VAL465 and ARG415, respectively, and occupies the Nrf2 site. The active peptide GFPVG interacts with the tyrosinase ASN260 and HIS85 through hydrogen bonds.

[0158] Example 5 In vitro activity verification of active peptides WGN, WIGG, and GFPVG

[0159] The three active peptides selected were entrusted to Sangon Biotech (Shanghai) Co., Ltd. for solid phase synthesis, and the purity was over 98%. The three active peptides were verified for in vitro DPPH clearance rate, ABTS clearance rate and tyrosinase inhibition rate, and their safety was evaluated.

[0160] The detection methods of DPPH clearance rate, ABTS clearance rate and tyrosinase inhibition rate refer to effect example 1. The test results are as follows Figure 8 , Fig. 9 , Fig.10As shown in the results, WGN, WIGG and GFPVG can effectively scavenge DPPH free radicals and ABTS free radicals and have excellent ability to inhibit tyrosinase activity. The DPPH free radical scavenging rates of the three active peptides WGN, WIGG and GFPVG are 55.49±1.70%, 41.28±1.18% and 42.55±0.87%, respectively; the ABTS free radical scavenging rates of the three active peptides WGN, WIGG and GFPVG are 85.41±1.78%, 73.40±1.54% and 52.09±0.93%, respectively. The tyrosinase inhibition rates of WGN, WIGG and GFPVG are 90.04±1.06%, 84.92±1.34% and 89.10±1.79%, respectively.

[0161] Compared with the data of effect example 1, the tyrosinase inhibition rate of sea cucumber egg-derived active peptides WGN, WIGG, and GFPVG is significantly higher than that of the sea cucumber egg-derived peptide complex. The antioxidant activity of the sea cucumber egg-derived peptide complex is better than that of the sea cucumber egg-derived active peptides WGN, WIGG, and GFPVG. The inventors speculate that the components of the sea cucumber egg-derived peptide complex have a synergistic effect in antioxidant activity. By further purifying and identifying the sea cucumber egg-derived peptide complex, a single peptide with a high tyrosinase inhibition rate can be obtained.

[0162] Example 6 Biosafety Assessment of Active Peptides WGN, WIGG, and GFPVG

[0163] The biosafety of the peptides was evaluated using the CCK-8 method. Cells were cultured in MEM at a concentration of 1×10 4 Density of cells / well HepG2 cells in the logarithmic growth phase were plated in a 96-well plate, and 100.0 μL of MEM medium containing cells was added to each well, and 6 parallel groups were set up. After 24 hours of culture in the incubator, the medium was aspirated. The cells were divided into a control group and an experimental group. 100.0 μL of peptide samples of different concentrations (0.02, 0.05, 0.10, 0.20, 0.50, 1.0, 1.5, 2.0, 5.0 mg / mL, dissolved in MEM) were added to the experimental group, 100.0 μL of medium without peptide samples was added to the control group, and 100.0 μL of medium without HepG2 cells was added to the blank group. Then 10.0 μL of CCK-8 solution (Biyuntian Biotechnology Co., Ltd., C0037) was added to each well, incubated at 37°C for 1 hour, the absorbance was measured at 450 nm, and the cell survival rate was calculated according to the following formula.

[0164]

[0165] The results are as follows Fig.11As shown, different concentrations of WGN, WIGG, and GFPVG had no effect on the survival rate of HepG-2 cells, indicating that the polypeptide prepared by the present invention has no toxic side effects.

[0166] Example 7 Detection of the inhibitory effect of peptides on tyrosinase activity and melanin production in B16-F10 cells

[0167] Cell culture: B16-F10 mouse melanoma cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% double antibody, and cultured in a cell culture incubator at 37°C and 5% CO2.

[0168] The experimental method for determining the cellular tyrosinase inhibitory activity was slightly modified from the following literature: Rodboon T, Okada S, Suwannalert P. Germinated riceberry rice enhanced protocatechuic acid and vanillic acid to suppress melanogenesis through cellular oxidant-related tyrosinase activity in B16 cells [J]. Antioxidants, 2020, 9 (3): 247. B16-F10 melanoma cells in the logarithmic growth phase were plated on a 6-well plate and cultured at 37 °C for 24 h. They were set as the control group and the experimental group, and three parallel groups were set up. The control group was added with RPMI-1640 culture medium, and the experimental group was added with different concentrations of WGN, WIGG and GFPVG (50.0, 100.0, 200.0, 300.0, 500.0 μmol / L) and cultured for 48 h, then washed twice with PBS, centrifuged at 1100 rpm for 10 min, and the cell pellet was retained. 500.0 μL of 1% TritonX-100 (dissolved in PBS) was added to each well for lysis, and the cells were placed at -80°C for 30 min. After the cells were completely dissolved at 4°C and centrifuged, 80 μL of the supernatant was taken and 20.0 μL of L-dopa was added, and the cells were incubated for 1 h, and the absorbance was measured at 475 nm.

[0169]

[0170] The results are as follows Fig.12 , Fig.14 , Fig.16As shown in the figure, all three peptides can effectively inhibit the tyrosinase activity in B16-F10 cells. The three peptides showed obvious inhibitory activity at a concentration of 200 μmol / L. Compared with the control group, the tyrosinase activity of the three peptides WGN, WIGG, and GFPVG decreased by 30.93%, 20.00%, and 27.31%, respectively. And the inhibitory ability on tyrosinase activity increased significantly with the increase of concentration.

[0171] The method for detecting the inhibition of melanin production in cells is as shown in Example 1. Fig.13 , Fig.15 , Fig.17 As shown in the figure, after the cells were treated with peptides, the intracellular melanin content gradually decreased with the increase of concentration. When the peptide concentration was 200 μmol / L, all experimental groups showed significant inhibitory effects, and the intracellular melanin content decreased to 83.23±2.34 (WGN), 85.88±3.76 (WIGG), and 73±2.64 (GFPVG), respectively. The experimental results show that the activity of tyrosinase in B16-F10 cells is closely related to the production of melanin. Reducing tyrosinase activity will affect the production of melanin, thereby affecting the intracellular melanin content.

[0172] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a sea cucumber egg-derived peptide complex, characterized in that: The following steps are involved: Sea cucumber egg powder was hydrolyzed by alkaline protease-trypsin.

2. The method for preparing the sea cucumber egg-derived peptide complex according to claim 1, characterized in that: The method also includes the step of ultrafiltering the enzymatic hydrolysis product, wherein the enzymatic hydrolysis product is sequentially passed through ultrafiltration membranes with molecular weights of 10 kDa and 3 kDa to collect components with molecular weights greater than 10 kDa, 3 kDa-10 kDa components, and less than 3 kDa components.

3. The method for preparing the sea cucumber egg-derived peptide complex according to claim 2, characterized in that: It also includes the step of separating components smaller than 3 kDa using dextran gel resin Sephadex G-15, combining tubes 25 to 50 as the SCP-1 component, combining tubes 58 to 77 as the SCP-2 component, and combining tubes 85 to 103 as the SCP-3 component.

4. The method for preparing the sea cucumber egg-derived peptide complex according to claim 3, characterized in that: It also includes separating the SCP-2 components by high performance liquid chromatography, and collecting components with retention times of 2.099 min, 3.148 min, 4.833 min, 5.352 min, 13.533 min, 14.992 min, and 20.99 min, respectively.

5. The method for preparing the sea cucumber egg-derived peptide complex according to any one of claims 1 to 4, characterized in that: The enzymatic hydrolysis conditions include: the mass ratio of the mixed enzyme activity to the substrate is 2000U / g, the enzymatic activity ratio of alkaline protease to trypsin in the mixed enzyme is 1:1, and the enzymatic hydrolysis is carried out at 25°C for 3 hours; then the enzyme is inactivated.

6. A sea cucumber egg-derived peptide complex, characterized in that: Prepared by the method according to any one of claims 1 to 5.

7. Sea cucumber egg-derived active peptide, characterized in that: The amino acid sequence of the sea cucumber egg-derived peptide includes one or more of WGN, WIGG, and GFPVG.

8. The method for preparing the sea cucumber egg-derived active peptide according to claim 7, characterized in that: The following steps are involved: Solid phase synthesis of the sea cucumber egg-derived peptide according to claim 7.

9. The method for screening active peptides from sea cucumber eggs according to claim 7, characterized in that: The following steps are involved: (1) hydrolyzing sea cucumber eggs with alkaline protease-trypsin mixed enzymolysis to obtain sea cucumber egg enzymolysis solution; (2) subjecting the sea cucumber egg hydrolysate obtained in step (1) to three-step separation by ultrafiltration, polysaccharide gel chromatography, and high performance liquid chromatography, and freeze-drying to obtain a freeze-dried active peptide powder; (3) analyzing the active peptide lyophilized powder by liquid chromatography-mass spectrometry to obtain the peptide amino acid sequence; (4) screening the peptides of the amino acid sequence obtained in step (3) using computer-assisted screening technology; (5) Based on the screening results of step (4), solid phase synthesis and screening peptides were performed and verified in vitro.

10. Use of the sea cucumber egg-derived peptide complex of claim 6 or the sea cucumber egg-derived active peptide of claim 7 in any one or more of (1) to (3); (1) Preparing antioxidant products; (2) preparing products for inhibiting tyrosinase activity; (3) Reduce melanin content.

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