Preparation method of sea cucumber egg-derived peptide complex and application thereof

Abstract

The present application relates to a preparation method of sea cucumber egg-derived peptide complex and application thereof.The preparation method of sea cucumber egg-derived peptide complex comprises the following steps: sea cucumber egg powder is subjected to synchronous enzymolysis by using alkaline protease and trypsin.Further separation is carried out through ultrafiltration, dextran gel chromatography and high performance liquid chromatography, sea cucumber egg-derived active peptides are obtained by using liquid chromatography-mass spectrometry technology, computer-aided screening technology and in-vitro verification, and the amino acid sequence of the sea cucumber egg-derived active peptides comprises one or several of WGN, WIGG and GFPVG.The sea cucumber egg-derived peptide complex and the sea cucumber egg-derived active peptides provided by the present application both have double activities of antioxidant and tyrosinase inhibition, can reduce melanin content, and have the advantages of good water solubility, good safety and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological products, and particularly relates to a preparation method of sea cucumber egg-derived peptide complex and application thereof. BACKGROUND

[0002] Based on the theory of aging free radicals, the intake of exogenous antioxidants has become one of the important ways to resist 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 melanin bodies to protect the skin. However, excessive melanin deposition can cause a series of skin problems such as chloasma, freckles, melanosis, and seborrheic keratosis, which has become a major problem for beauty lovers. Tyrosinase is a 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 are interrelated. Specifically, the process of melanin production generates a large amount of active oxygen free radicals, and excessive free radicals can further damage melanocytes, promote pigment accumulation, and increase the degree of aging. Therefore, reducing the body's oxidative stress and reducing the activity of tyrosinase play an important role in reducing pigment deposition and preventing skin aging. However, most current studies on skin anti-aging only focus on antioxidant activity and lack evaluation of tyrosinase inhibitory activity, so it is of great significance to find and prepare substances with antioxidant and tyrosinase inhibitory activity.

[0003] Bioactive peptides are increasingly attracting attention due to their high efficiency and stability. Traditional bioactive peptide preparation mainly obtains peptide mixtures through enzymatic hydrolysis technology, and single enzymatic hydrolysis technology is mainly used. In actual experiments, the inventors found that single enzyme preparation for bioactive peptide preparation has drawbacks such as enzyme hydrolysis dead angle, long time consumption, uneven action, low stability, and poor reproducibility. The presence of complex peptide components and non / low bioactive peptides in the mixture may affect the activity efficiency. Based on the mixture, purification technology can be used to remove part of the peptide segments in the mixture to obtain peptides with known sequences. It is of great significance to use chemical methods to achieve efficient and rapid preparation for large-scale industrialization and related mechanism analysis. Traditional single peptides are usually obtained by separation and purification technology, which is time-consuming and laborious. With the development of bioinformatics, computer-aided technology has gradually emerged in bioactive peptide screening. Its high-throughput analysis method can save peptide screening time and improve efficiency, but single computer screening may not match the actual activity of the screening results.

[0004] Sea cucumber eggs are by-products of sea cucumber processing industry, containing a large amount of protein and other nutrients. With the increasing demand of consumers for sea cucumber, the quantity of sea cucumber eggs has increased rapidly, and they are usually discarded as waste, causing 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 main method for preparing bioactive peptides by single enzyme hydrolysis is to hydrolyze the outer layer of amino acids of the protein, and then hydrolyze the inside of the protein after the outer layer of amino acids is broken down. The enzyme hydrolysis time is long, the efficiency is low, and the bioactivity is low.

[0005] Therefore, it is urgent to provide a method for preparing double-activity sea cucumber egg peptide with simple operation, high efficiency and rapid implementation. At present, there is no report on the use of alkaline protease and trypsin in combination for sea cucumber eggs to prepare active peptides with antioxidant, tyrosinase inhibitory and reduced melanin content. SUMMARY

[0006] In view of the low utilization rate and single activity of existing sea cucumber eggs, the present application provides a method for preparing a double-activity (antioxidant and tyrosine inhibitory) sea cucumber egg peptide complex treated by a composite enzyme, and application thereof. On this basis, the present application uses sea cucumber egg peptide complex as raw material, and realizes the preparation of bioactive peptides with antioxidant and tyrosinase inhibitory double activity and reduced melanin content by combining traditional separation and purification technology with computer aided technology.

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

[0008] The present application provides a method for preparing a double-activity sea cucumber egg peptide complex treated by a composite enzyme, comprising the following steps: synchronously hydrolyzing sea cucumber egg powder with alkaline protease and trypsin.

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

[0010] The low degree of green processing of sea cucumber processing industry by-products is currently the bottleneck limiting the development of the industry, and the waste of a large amount of sea cucumber eggs causes huge economic and resource waste. The double-functional sea cucumber egg peptide complex prepared by the present application has antioxidant and tyrosinase inhibitory activity, which can promote the extension of the sea cucumber industry chain and improve the deep processing degree of sea cucumber industry by-products. The sea cucumber egg peptide preparation method of the present application has the characteristics of high efficiency, good reproducibility, reduced melanin content, good antioxidant effect, high tyrosinase inhibitory activity, small peptide molecular weight, etc.

[0011] In the preparation process, the alkaline protease and trypsin are used for synchronous enzymolysis, the trypsin is used for cutting the peptide bond with basic amino acid on the carboxyl side, the internal hydrophobic amino acid and aromatic amino acid enzyme cutting site are further exposed, the accurate recognition and cutting of the alkaline protease are completed, the 'layer by layer' enzyme cutting of the sea cucumber protein is realized, the number of small molecules with hydrophobic amino acid and aromatic amino acid as the terminal in the complex is increased, the antioxidant and tyrosinase enzyme inhibition activity are improved, and the preparation of the double-functional active peptide complex is realized.

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

[0013] Further, the step of ultrafiltrating the product after the enzymolysis is further included, the enzymolysis product is sequentially subjected to the ultrafiltration membranes with molecular weights of 10 kDa and 3 kDa, and the components with molecular weights greater than 10 kDa, the components of 3 kDa-10 kDa and the components less than 3 kDa are collected.

[0014] Further, the step of separating the components less than 3 kDa by using the Sephadex gel resin Sephadex G-15 is further included, the tubes 25 to 50 are combined together as the SCP-1 component, the tubes 58 to 77 are combined together as the SCP-2 component, and the tubes 85 to 103 are combined together as the SCP-3 component.

[0015] Further, the step of separating the SCP-2 component by using the high performance liquid chromatography is further included, the 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 are collected, and are sequentially named as SCP-2-1 to SCP-2-7.

[0016] Further, the enzymolysis condition includes that the water solution of the sea cucumber spawning powder is adjusted to pH 7.5, the mixed enzyme is added, the enzyme activity of the mixed enzyme is 2000 U / g to the mass of the substrate, the enzyme activity ratio of the alkaline protease to the trypsin in the mixed enzyme is 1:1, the enzymolysis is performed at 25 DEG C for 3 h, and then the enzyme is inactivated.

[0017] Further, the enzyme inactivation condition includes that the boiling water bath is heated for 20 min.

[0018] The sea cucumber egg source peptide complex is prepared by using the above method.

[0019] Further, the sea cucumber egg source peptide complex includes one or a combination of several of WGN, WIGG and GFPVG.

[0020] Further, the application provides sea cucumber egg-derived active peptides with dual activities of antioxidant and tyrosinase inhibition, and the amino acid sequences of the sea cucumber egg-derived peptides include one or more of WGN, WIGG and GFPVG.

[0021] Preferably, the amino acid sequences of the sea cucumber egg-derived peptides are one or more of WGN, WIGG and GFPVG.

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

[0023] The application provides a preparation method of the sea cucumber egg-derived active peptides, including the following steps: solid-phase synthesis of the sea cucumber egg-derived peptides.

[0024] The application provides a screening method of the sea cucumber egg-derived active peptides, including the following steps:

[0025] (1) sea cucumber eggs are subjected to mixed enzymolysis by using alkaline protease and trypsin to obtain sea cucumber egg enzymolysis liquid;

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

[0027] (3) the active peptide freeze-dried powder is subjected to liquid chromatography-mass spectrometry analysis to obtain peptide amino acid sequences;

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

[0029] (5) the screening peptides are synthesized by solid phase according to the screening results of step (4), and in-vitro verification is performed, for example, antioxidant activity, tyrosinase inhibition activity, melanin content, etc.

[0030] The beneficial effects of the above technical solutions include that the method provided by the application is an efficient, rapid and accurate peptide acquisition method, traditional separation and purification technology is combined with computer-aided technology, the screening time of active peptides is saved, the efficiency is improved, components with high antioxidant peptide content are obtained, and the accuracy of the peptides is improved. The obtained peptides are screened by using computer-aided technology, and the screening time, efficiency and accuracy are improved.

[0031] The alkaline protease-trypsin mixed enzyme hydrolysis can realize the cleavage of the peptide bond on the carboxyl side of the basic amino acid and the hydrophobic amino acid, respectively. The trypsin can recognize and act on the surface amino acid of the sea cucumber egg protein to loosen the protein structure. At the same time, the alkaline protease can accurately cut the hydrophobic and aromatic amino acids to improve the antioxidant and tyrosinase inhibitory activity of the peptide.

[0032] Further, the specific operation process of step (1) can include: weighing the sea cucumber spawning powder, dissolving in 10 times the volume of deionized water, adjusting pH = 7.5, adding mixed enzymes, the enzyme activity of the mixed enzymes to the substrate mass ratio is 2000 U / g, and the enzyme activity ratio of alkaline protease to trypsin in the mixed enzymes is 1:1; enzyme hydrolysis at 25℃ for 3h, boiling water bath heating for 20min to inactivate the enzyme, 10000r / min centrifugation for 20min, and the supernatant is reserved as the sea cucumber egg hydrolysate.

[0033] Further, the specific operation process of step (2) can include: sequentially grading the sea cucumber egg hydrolysate through 10kDa and 3kDa ultrafiltration membranes, and collecting the component less than 3kDa; separating the component less than 3kDa through dextran gel chromatography, collecting 2.0mL of the component per tube, and collecting the components of the 58th tube to the 77th tube together as the SCP-2 component; separating the SCP-2 component through high performance liquid chromatography, and collecting the component corresponding to the retention time of 5.352min as the SCP-2-4 component.

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

[0035] Further, the specific operation process of step (4) can include: screening the active peptide segments identified by LC-MS / MS according to the length of the peptide chain and the hydrophobic nature; predicting the biological activity value of the polypeptide by the online tool PeptideRanker, and retaining the polypeptide with a biological activity prediction value greater than 0.84; predicting the water solubility and toxicity of the peptide by the online tools Innovagen and ToxinPred, and retaining the active peptide sequence with good water solubility and no toxicity for molecular docking; using the CDOCK mode in Discovery Studio 2019Client to perform molecular docking with the Keap1 receptor, and screening the polypeptide according to the binding energy.

[0036] Further, the specific operation process of step (5) can include: synthesizing the screened peptide by solid-phase synthesis, and verifying the biological activity by DPPH free radical scavenging capacity, ABTS free radical scavenging capacity, and tyrosinase inhibition rate.

[0037] The application provides application of the sea cucumber egg-derived peptide compound in any one or any number of (1) to (3).

[0038] (1) preparation of an antioxidant product;

[0039] (2) preparation of a product for inhibiting tyrosinase activity;

[0040] (3) reduction of melanin content.

[0041] The application provides application of the sea cucumber egg-derived active peptide in any one or any number of (1) to (3).

[0042] (1) preparation of an antioxidant product;

[0043] (2) preparation of a product for inhibiting tyrosinase activity;

[0044] (3) reduction of melanin content.

[0045] The product includes but is not limited to food, health care products, cosmetics, pharmaceuticals and the like.

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

[0047] The beneficial effects of the above technical solutions include: the sea cucumber egg-derived active peptide sequences WGN, WIGG and GFPVG obtained by the application have high application development value and can be applied to the fields of food, health care products, cosmetics and the like as functional ingredients. The WGN, WIGG and GFPVG can effectively scavenge DPPH free radicals, ABTS free radicals and inhibit tyrosinase activity, and the DPPH free radical scavenging rates thereof are 55.49±1.70%, 41.28±1.18% and 42.55±0.87% respectively; the ABTS free radical scavenging rates thereof are 85.41±1.78%, 73.40±1.54% and 52.09±0.93% respectively. The tyrosinase inhibition rates of the WGN, WIGG and GFPVG are 90.04±1.06%, 84.92±1.34% and 89.10±1.79% respectively. The WGN, WIGG and GFPVG can effectively reduce the melanin content. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Figure 1 is a molecular weight distribution determination result of a sea cucumber egg-derived peptide complex, wherein 1 represents a sample of sea cucumber egg protein digested by a basic protease-trypsin complex enzyme, 2 represents a sample of sea cucumber egg protein digested by a basic protease, and 3 represents a sample of sea cucumber egg protein digested by trypsin.

[0049] Figure 2 Figure 2 is a surface hydrophobicity determination result of a sea cucumber egg-derived peptide complex, wherein 1 represents a sample of sea cucumber egg protein digested by a basic protease-trypsin complex enzyme, 2 represents a sample of sea cucumber egg protein digested by a basic protease, and 3 represents a sample of sea cucumber egg protein digested by trypsin.

[0050] Figure 3 Figure 3 is an elution curve of G-15 dextran gel of the application, wherein the abscissa represents the number of elution tubes, and the ordinate represents the absorbance at 280 nm.

[0051] Figure 4 Figure 4 is a high performance liquid chromatogram (RP-HPLC) of the application, wherein the abscissa represents the retention time (min), and the ordinate represents the absorbance at 214 nm.

[0052] Figure 5 Figure 5 is a result graph of the interaction between WGN and Keap1, and a result graph of the interaction between WGN and tyrosinase (2Y9X).

[0053] Figure 6 Figure 6 is a result graph of the interaction between WIGG and Keap1, and a result graph of the interaction between WIGG and tyrosinase (2Y9X).

[0054] Figure 7Fig. 1 is a result chart of GFPVG interacting with Keap1, and Fig. 2 is a result chart of GFPVG interacting with tyrosinase (2Y9X).

[0055] Figure 8 Fig. 3 is an effect of synthetic peptides on DPPH radical scavenging rate, wherein the abscissa is the type of synthetic peptide, and the ordinate is the DPPH radical scavenging rate.

[0056] Figure 9 Fig. 4 is an effect of synthetic peptides on ABTS radical scavenging rate, wherein the abscissa is the type of synthetic peptide, and the ordinate is the ABTS radical scavenging rate.

[0057] Figure 10 Fig. 5 is an effect of synthetic peptides on tyrosinase inhibition rate, wherein the abscissa is the type of synthetic peptide, and the ordinate is the tyrosinase inhibition rate.

[0058] Figure 11 Fig. 6 is an effect of different concentrations of WGN, WIGG and GFPVG on the survival rate of HepG2 cells, wherein 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] Figure 12 Fig. 7 is an effect of WGN on the tyrosinase activity in B16-F10 cells.

[0060] Figure 13 Fig. 8 is an effect of WGN on the melanin production in B16-F10 cells.

[0061] Figure 14 Fig. 9 is an effect of WIGG on the tyrosinase activity in B16-F10 cells.

[0062] Figure 15 Fig. 10 is an effect of WIGG on the melanin production in B16-F10 cells.

[0063] Figure 16 Fig. 11 is an effect of GFPVG on the tyrosinase activity in B16-F10 cells.

[0064] Figure 17 Fig. 12 is an effect of GFPVG on the melanin production in B16-F10 cells. DETAILED DESCRIPTION

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

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

[0067] The preparation method of the sea cucumber egg-derived peptide complex can comprise the following steps: obtaining a sea cucumber egg enzymatic hydrolysate by using a mixed alkaline protease-trypsin to carry out enzymolysis; and further subjecting the obtained sea cucumber egg enzymatic hydrolysate to three-step separation and purification of 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 complex powder.

[0068] Further, the preparation of the sea cucumber egg-derived peptide mixture can comprise the following steps: using sea cucumber eggs as raw materials, and simultaneously carrying out enzymolysis by using a mixed alkaline protease and trypsin, wherein the enzymolysis process comprises: the ratio of the enzyme activity of the mixed enzyme to the mass of the substrate is 2000 U / g, the mixed enzyme comprises alkaline protease and trypsin, the enzyme activity ratio of the alkaline protease to the trypsin is 1:1, the enzymolysis time is 3 h, the enzymolysis temperature is 25 DEG C, and pH is 7.5; after the enzymolysis is completed, enzyme inactivation treatment is carried out by heating in a boiling water bath for 20 min, centrifugation is carried out at 10 000 r / min for 20 min, and the supernatant is taken to obtain a sea cucumber egg enzymatic hydrolysate.

[0069] Further, the separation of the sea cucumber egg-derived antioxidant peptide can comprise the following steps: subjecting the sea cucumber egg enzymatic hydrolysate to grading through 10 kDa and 3 kDa ultrafiltration membranes in sequence, determining the DPPH free radical scavenging capacity of different components, collecting the elution components with high DPPH scavenging capacity, and freeze-drying; redissolving the high-antioxidant components obtained by ultrafiltration, and subjecting the redissolved components to separation by dextran gel chromatography, collecting the elution components with the strongest DPPH free radical scavenging capacity, and freeze-drying; redissolving the high-antioxidant components obtained by dextran gel chromatography separation, and subjecting the redissolved components to third-step separation by high performance liquid chromatography, collecting the components for DPPH free radical scavenging capacity determination, retaining the elution components with the strongest antioxidant capacity, and freeze-drying to obtain a high-antioxidant peptide complex powder.

[0070] The present application provides a sea cucumber egg-derived peptide complex prepared by the above method.

[0071] The present application provides the use of the above sea cucumber egg-derived peptide complex in the preparation of antioxidant and / or tyrosinase activity inhibiting products.

[0072] The present application provides a sea cucumber egg-derived active peptide with antioxidant and tyrosinase activity inhibiting dual activities, a preparation method, a screening method and applications thereof.

[0073] The present application provides three sea cucumber egg-derived active peptides with antioxidant and tyrosinase activity inhibiting activities, and the amino acid sequences of the sea cucumber egg-derived active peptides are WGN, WIGG and GFPVG. The above sea cucumber egg-derived active peptides can reduce melanin content, and are also dual-function sea cucumber egg-derived active peptides with antioxidant and tyrosinase activity inhibiting activities.

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

[0075] The application provides a screening method of the sea cucumber egg-derived active peptide, comprising the following steps:

[0076] (a) The obtained high-antioxidant peptide complex is subjected to liquid chromatography-mass spectrometry (LC-MS / MS) to identify the amino acid sequence of the peptide.

[0077] (b) The peptide with the known amino acid sequence obtained in step (a) is screened by using computer-aided screening technology, so as to obtain a peptide with potential high antioxidant activity and high tyrosinase inhibition activity.

[0078] (c) The screened peptide is synthesized by solid phase, and the antioxidant activity, tyrosinase inhibition activity and influence on melanin content of the peptide are verified in vitro.

[0079] Further, the identification of the sea cucumber egg-derived antioxidant peptide can comprise the following steps: the components separated by reverse-phase high-performance liquid chromatography are subjected to amino acid sequence identification by using liquid chromatography-mass spectrometry (LC-MS / MS). The mobile phase A is 2.0% acetonitrile-0.1% formic acid-water (V / V / V); the mobile phase B is 0.1% formic acid-acetonitrile solution (V / V); the gradient is started from 2% (volume percent) of B phase, and is increased to 35% (volume percent) in 47 minutes in a non-linear gradient, and then is increased to 100% (volume percent) in 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] Further, the screening of the sea cucumber egg-derived bifunctional peptide can comprise the following steps: the active peptide segments identified by LC-MS / MS are preliminarily screened according to the length of the peptide chain and the hydrophobic property. The biological activity value of the polypeptide is predicted by using an online tool PeptideRanker (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.php). The water solubility and toxicity of the polypeptide are predicted by using online tools Innovagen (www.innovagen.com / proteomicstools) and ToxinPred (http: / / crdd.osdd.net / raghava / / toxinpred), and the active peptide sequence with good water solubility and no toxicity is reserved for molecular docking.

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

[0082] The present application aims at the phenomenon that the utilization rate of the existing sea cucumber intestine is low and the activity is single, and provides a method for obtaining bifunctional sea cucumber egg-derived active peptides based on alkaline protease-pancreatic trypsin synchronous enzymolysis, separation and purification, and computer screening. The present application uses ultrafiltration, gel chromatography, and high-performance liquid chromatography separation technology, with the aid of protein databases and online software, and uses DPPH clearance rate, ABTS clearance rate, water solubility, and biological toxicity for multiple screening; the active peptides are combined with molecular docking to explore the interaction mode of the active peptides and the receptors Keap1 and tyrosinase (2Y9X), and finally the polypeptides are synthesized by solid-phase synthesis and the antioxidant activity and tyrosinase inhibition activity of the polypeptides are verified. The results show that the three active peptides screened by the present application all have the advantages of good water solubility, good safety, good antioxidant activity, excellent tyrosinase inhibition capacity, and the ability to 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 can be obtained by commercial channels or prepared by conventional methods. The solutions involved in the present application are water unless otherwise specified.

[0084] The sea cucumber spawning powder was purchased from Linghai City Dalian Seafood Aquaculture Co., Ltd.; the dextran gel resin Sephadex G-15 was purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.; the HepG-2 cells were purchased from the China Center for Type Culture Collection (CCTCC); the PBS was purchased from Beijing Boao Tuoda Technology Co., Ltd.; the L-dopa was purchased from Shanghai Aladdin Reagent Co., Ltd.; the CCK-8 kit was purchased from Biyun Tian Biotechnology Co., Ltd.; the MEM medium was purchased from Wuhan Punsai Life Science and Technology Co., Ltd. The B16-F10 mouse melanoma cells were purchased from Wuhan Punsai Life Science and Technology Co., Ltd., the fetal bovine serum was purchased from Zhejiang Tianhang Biological Technology Co., Ltd., the RPMI-1640 culture medium containing 1% double antibody was purchased from Wuhan Punsai Life Science and Technology Co., Ltd., and contained penicillin G sodium salt and streptomycin sulfate, the content of penicillin G sodium salt was 1% (mass percent), and the content of streptomycin sulfate was 1% (mass percent).

[0085] The following will be introduced through specific examples.

[0086] Example 1

[0087] The sea cucumber egg source peptide enzymatic hydrolysate is prepared by the following steps: weighing the defatted sea cucumber spawning powder, dissolving in 10 times the volume of deionized water, adjusting pH to 7.5, adding mixed enzymes, and the enzyme activity of the mixed enzymes to the mass ratio of the substrate is 2000 U / g. The mixed enzymes include alkaline protease and trypsin, and the enzyme activity ratio of alkaline protease to trypsin in the mixed enzymes is 1:1. For example, for every 1 g of substrate, the amount of alkaline protease added is 1000 U, and the amount of trypsin added is 1000 U. Enzymatic hydrolysis is carried out at 25℃ for 3h. After boiling water bath heating for 20min to inactivate the enzyme, centrifugation at 10000r / min for 20min, the supernatant is reserved as the sea cucumber egg enzymatic hydrolysate.

[0088] Comparative Example 1

[0089] Based on Example 1, in the enzymatic hydrolysis process of Comparative Example 1, alkaline protease is used to replace the mixed enzymes (alkaline protease-trypsin) in Example 1, and the reaction is carried out under the optimum enzymatic hydrolysis conditions thereof.

[0090] The specific operation in Comparative Example 1 includes: adjusting pH to 10, temperature to 45℃, enzyme addition amount to 2000 U / g, and enzymatic hydrolysis time to 3h. After boiling water bath heating for 20min to inactivate the enzyme, centrifugation at 10000r / min for 20min, the supernatant is reserved as the sea cucumber egg enzymatic hydrolysate. The rest is the same as Example 1.

[0091] Comparative Example 2

[0092] Based on Example 1, in the enzymatic hydrolysis process of Comparative Example 2, trypsin is used to replace the mixed enzymes (alkaline protease-trypsin) in Example 1, and the reaction is carried out under the optimum enzymatic hydrolysis conditions thereof.

[0093] The specific operation in Comparative Example 1 includes: adjusting pH to 10, temperature to 45℃, enzyme addition amount to 2000 U / g, and enzymatic hydrolysis time to 3h. After boiling water bath heating for 20min to inactivate the enzyme, centrifugation at 10000r / min for 20min, the supernatant is reserved as the sea cucumber egg enzymatic hydrolysate. The rest is the same as Example 1.

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

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

[0096] (1) DPPH scavenging rate detection, including the following steps: setting sample group, control group and blank group. Sample group: 4.0 mL of sample solution to be tested is mixed with equal volume of DPPH solution; control group: 4.0 mL of sample solution to be tested is mixed with equal volume of anhydrous ethanol; blank group: DPPH solution. In each group, the solvent in DPPH solution is anhydrous ethanol, and the concentration of DPPH is 0.1 mmol / L; in sample solution to be tested, the solvent is water, and the concentration of polypeptide is 2.0 mg / mL. After each group is dark reacted at room temperature for 30 min, the absorbance at 517 nm is determined by using ultraviolet spectrophotometer. The DPPH free radical scavenging capacity (i.e. DPPH scavenging rate) is calculated according to the following formula.

[0097]

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

[0099] (2) ABTS scavenging rate detection, including the following steps:

[0100] ① Preparation of working solution: ABTS solution and equal volume of potassium persulfate solution are mixed, the concentration of ABTS in ABTS solution is 7.0 mmol / L, and the concentration of potassium persulfate in potassium persulfate solution is 2.45 mmol / L. The ABTS mother liquor is obtained by reacting under the condition of avoiding light for 12-16 h at 25℃. ABTS is diluted with 80% ethanol solution to obtain working solution, so that the absorbance of working solution at 734 nm reaches 0.7±0.1.

[0101] ② Configuration of sample group, control group and blank group. Sample group: 0.5 mL of 2.0 mg / mL sample to be tested is mixed with 3.0 mL of working solution; control group: 0.5 mL of 2.0 mg / mL sample to be tested is mixed with 3.0 mL of distilled water; blank group: 0.5 mL of distilled water is uniformly mixed with 3.0 mL of working solution. Each group is incubated under the condition of avoiding light for 10 min, and the absorbance at 734 nm is determined. The ABTS scavenging rate is determined according to the following formula.

[0102]

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

[0104] (3) Detection of tyrosinase inhibitory activity, including the following steps: setting blank group, negative group, background group and sample group, preparing solutions of 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 active substances of different concentrations on tyrosinase was calculated according to the following formula.

[0108]

[0109] In the above formula, A 阴性 is the absorbance of the negative group at 475 nm; A 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) Detection of relative melanin content of cells, including 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. Cultured in a 37°C, 5% CO2 cell incubator.

[0112] The experimental method is based on the method of the following literature with slight modification: Liu J Z, Xu X H, 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 is the normally cultured cells, and the experimental group is the cells treated with 2 mg / mL different samples. Resuspend the cells with PBS, add 1 mol / L NaOH, incubate at 80°C for 2 h, and shake to dissolve. Centrifuge at 10000 rpm for 15 min, take 100.0 μL of supernatant to 96-well plate, and measure the absorbance at 405 nm.

[0113]

[0114] In the above formula, A 实验组 is the absorbance of the experimental group cells at 405 nm; A 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 detection are shown in Table 2. As can be seen from Table 2, the antioxidant activity, tyrosinase inhibitory activity and relative content of melanin of the sea cucumber egg peptide complex prepared by the method of Example 1 of the present application are all better than those of the sample prepared by a single enzyme.

[0117] Table 2 Evaluation of biological activity of peptide complex

[0118]

[0119] Effect Example 2 Detection of whether the protein is enzymatically hydrolyzed into small molecule peptides

[0120] The sea cucumber egg-derived peptide complexes obtained by the methods of Example 1, Comparative Example 1 and Comparative Example 2 were subjected to molecular weight determination.

[0121] Figure 1 is the molecular weight distribution graph of the sea cucumber egg-derived peptide complex. Figure 1 In the above formula, 1 represents the sea cucumber egg-derived peptide complex sample prepared using alkaline protease-trypsin (i.e., the sea cucumber egg-derived peptide complex prepared by the method of Example 1), 2 represents the sea cucumber egg-derived peptide complex prepared using alkaline protease (i.e., the sea cucumber egg-derived peptide complex prepared by the method of Comparative Example 1), and 3 represents the sea cucumber egg-derived peptide complex prepared using trypsin (i.e., the sea cucumber egg-derived peptide complex prepared by the method of Comparative Example 2). As can be seen from the above formula, Figure 1 It can be seen that the molecular weight of the peptide sample prepared by alkaline protease-trypsin complex enzymolysis is mainly concentrated between 0-1500 Da, the peptides in the complex treated by alkaline protease are mainly peptides with a molecular weight greater than 3000 Da. The complex treated by trypsin is mainly composed of peptides with a molecular weight greater than 3000 Da and less than 5000 Da. Alkaline protease-trypsin treatment can obtain smaller peptides, and the inventors guess that this is mainly due to the fact that the enzyme cutting site of the complex enzyme is more than that of Comparative Example 1 and Comparative Example 2, which can fully hydrolyze the protein. Arginine, glycine and basic amino acids are mainly present in sea cucumbers, and alkaline protease can cut hydrophobic amino acids (glycine), arginine and other basic amino acids are all trypsin enzyme cutting sites, so the number of small molecular weight peptides in the trypsin enzymolysis complex is higher than that of the alkaline protease treatment group. In addition, the high proportion of low molecular weight peptides is 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 Detection of whether more hydrophobic amino acids are exposed in each peptide complex

[0123] The peptide complex surface hydrophobicity was evaluated using the 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescent probe method with the complexes prepared by the method of Example 1, Comparative Example 1, Comparative Example 2.

[0124] Each enzyme hydrolysate was configured as a 0.2 mg / mL liquid as a sample solution. The 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 taken into a 5 mL centrifuge tube, 20 μL of the ANS solution was added, shaken for 30 s, and allowed to stand for 2 min. The detection was performed using a fluorescence spectrophotometer, 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 is the surface hydrophobicity determination result in Example 1 and different comparative examples, the abscissa is the sea cucumber egg-derived peptide complex prepared by different proteases, and the ordinate is the fluorescence intensity, which can reflect the hydrophobicity; Figure 2 In Table 1, 1 represents the sea cucumber egg-derived peptide complex sample prepared by using alkaline protease-trypsin (i.e., the sea cucumber egg-derived peptide complex prepared in Example 1), 2 represents the sea cucumber egg-derived peptide complex prepared by using alkaline protease (i.e., the sea cucumber egg-derived peptide complex prepared in Comparative Example 1), and 3 represents the sea cucumber egg-derived peptide complex prepared by using trypsin (i.e., the sea cucumber egg-derived peptide complex prepared in Comparative Example 2). From Figure 2 It can be seen that the surface hydrophobicity of the peptide sample prepared by using alkaline protease-trypsin complex enzymolysis is significantly better than that of the peptide complex sample prepared by using a single enzyme.

[0126] Example 2 Separation and purification

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

[0128] According to the above screening results, the component with a higher clearance rate was 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 inhibition activity, and relative content of melanin of the component less than 3 kDa are significantly better than those of other components. Therefore, the component less than 3 kDa was 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) was further separated using Sephadex G-15 resin.

[0132] A distillation level balance column (2.6 x 60.0 cm) was used with a sample volume of 5 mL; the sample (fraction less than 3 kDa) concentration was 10 mg / mL. The UV detection wavelength was 280 nm. Fractions were collected at 2.0 mL per tube and freeze-dried.

[0133] According to the G-15 Sephadex elution curve ( Figure 3 ), it can be seen that SCP-1 (pooled from tube 25 to tube 50), SCP-2 (pooled from tube 58 to tube 77), and SCP-3 (pooled from tube 85 to tube 103) have significant absorbance values at 280 nm.

[0134] The DPPH clearance rate, ABTS clearance rate, and tyrosinase inhibitory activity of the SCP-1 fraction, SCP-2 fraction, and SCP-3 fraction were detected, respectively, according to the detection method of Effect Example 1. According to the DPPH free radical clearance rate and ABTS clearance rate results, the fraction with the strongest antioxidant activity was screened out, and according to the tyrosinase inhibitory activity and relative melanin content results, the fraction with the strongest tyrosinase inhibitory capacity and the least melanin content was screened out. According to Table 4, it can be seen that the DPPH free radical clearance rate, ABTS clearance rate, and tyrosinase inhibitory activity of the fraction SCP-2 are significantly higher than those of other fractions, and the relative melanin content is less. Therefore, the fraction SCP-2 was selected for high-performance liquid chromatography separation.

[0135] Table 4 Antioxidant and tyrosinase inhibitory activities of G-15 gel chromatography fractions

[0136]

[0137] (3) High-performance liquid chromatography separation: An Agilent C18 chromatographic column (250.0 x 4.6 mm, 5.0 μm) was used to further separate the fraction with high antioxidant activity from the SCP-2 fraction. The mobile phase A was acetonitrile, and the mobile phase B was 0.1% (volume percent) trifluoroacetic acid. Isocratic elution (10% A, 90% B, both by volume), flow rate 1.0 mL / min, UV detection wavelength 214 nm. According to the results of the high-performance liquid chromatogram, the fractions were collected, such as Figure 4As shown, the corresponding retention time of SCP-2-1, SCP-2-2, SCP-2-3, SCP-2-4, SCP-2-5, SCP-2-6, SCP-2-7 is 2.099 min, 3.148 min, 4.833 min, 5.352 min, 13.533 min, 14.992 min, 20.99 min, respectively.

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

[0139] Table 5 Antioxidant activity and tyrosinase inhibitory activity of each component by high performance liquid chromatography

[0140]

[0141] Example 3 Amino acid sequence identification of sea cucumber egg antioxidant peptide

[0142] Amino acid sequence identification was performed by Nanjing Zheng Function Biotechnology Co., Ltd., including the following steps: using liquid chromatography mass spectrometry (LC-MS / MS) to analyze the secondary mass spectrum of the component SCP-2-4 separated by high performance liquid chromatography, mobile phase A was 2.0% acetonitrile-0.1% formic acid-water (V / V / V); mobile phase B was 0.1% formic acid-acetonitrile solution (V / V), with a gradient starting from 2% B phase, rising to 35% in 47 minutes, then rising to 100% in 1 minute, and maintaining 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, 35 new active peptide amino acid sequences were obtained (Table 6).

[0144] Table 6 Peptide sequences in SCP-2-4 identified by LC-MS / MS method

[0145]

[0146]

[0147] Example 4 Screening and molecular docking of sea cucumber egg bifunctional peptide

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

[0149] Peptides with Da and amino acid number ≤ 5 were selected for subsequent screening.

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

[0151] The water solubility and biological toxicity of the polypeptides were screened and predicted by the online tools Innovagen (www.innovagen.com / proteomicstools) and ToxinPred (http: / / crdd.osdd.net / raghava / / toxinpred), and polypeptides with "good solubility" and no toxicity were retained.

[0152] Molecular docking was performed using the CDOCK mode in Discovery Studio 2019 Client with the Keap1 (PBD ID: 2ZMX) receptor for preliminary screening (LibDock), and the polypeptide sequence after docking with Keap1 was further subjected to molecular docking with tyrosinase (PBD ID: 2Y9X) using Maestro 12.8 software. The LibDock score and docking score (the smaller the energy, the tighter the binding) were recorded. Finally, 3 novel peptide segments with potential good biological activity, good water solubility and no toxicity were screened (Table 7).

[0153] Table 7 Biological activity prediction, toxicity, solubility and other characteristics of sea cucumber egg-derived active peptides

[0154]

[0155]

[0156] Note: "-" in the table indicates that it cannot be docked at the same time, and no docking score is given.

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

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

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

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

[0161] Compared with the data of Example 1, the tyrosinase inhibition rates of sea cucumber egg-derived active peptides WGN, WIGG, GFPVG are significantly higher than those of sea cucumber egg-derived peptide complex. The antioxidant activity of sea cucumber egg-derived peptide complex is better than that of sea cucumber egg-derived active peptides WGN, WIGG, GFPVG. The inventors speculate that the components of sea cucumber egg-derived peptide complex have a synergistic effect on antioxidant activity. Through further purification and identification of sea cucumber egg-derived peptide complex, a single peptide with high tyrosinase inhibition rate can be obtained.

[0162] Example 6 Biological safety evaluation of active peptides WGN, WIGG, GFPVG

[0163] The CCK-8 method was used to evaluate the biological safety of the peptides. Cells were cultured in MEM, and 1×10 4 HepG2 cells in logarithmic growth phase were plated in a 96-well plate at a density of 1×10

[0164]

[0165] The results are shown in Table 1. Figure 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 in this 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% penicillin-dextrose antibody. The culture was carried out at 37°C in a 5% CO2 cell culture incubator.

[0168] The experimental method for determining cellular tyrosinase inhibitory activity was based on the method described in the following literature with slight modifications: 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 logarithmic growth phase were seeded into 6-well plates and cultured at 37℃ for 24 h. Three parallel groups were set up as a control group and an experimental group. The control group was cultured in RPMI-1640 medium, while the experimental groups were cultured in different concentrations of WGN, WIGG, and GFPVG (50.0, 100.0, 200.0, 300.0, 500.0 μmol / L) for 48 h. The cells were then washed twice with PBS, centrifuged at 1100 rpm for 10 min, and the cell pellet was retained. Add 500.0 μL of 1% Triton X-100 (dissolved in PBS) to each well for lysis and incubate at -80°C for 30 min. Then, allow the cells to completely lyse at 4°C, centrifuge, and take 80 μL of the supernatant. Add 20.0 μL of L-dopa and incubate for 1 h. Measure the absorbance at 475 nm.

[0169]

[0170] The results are as follows Figure 12 , Figure 14 , Figure 16As shown, the three peptides can effectively inhibit the tyrosinase activity in B16-F10 cells. The three peptides have shown obvious inhibitory activity at a concentration of 200 μmol / L, and the tyrosinase activity of WGN, WIGG and GFPVG is reduced by 30.93%, 20.00% and 27.31% respectively compared with the control group. And the inhibitory ability of tyrosinase activity is significantly improved with the increase of concentration.

[0171] The melanin production inhibition detection method refers to the method of Example 1, and the results are shown in Figure 13 、 Figure 15 、 Figure 17 As shown, the melanin content in the cells gradually decreases with the increase of concentration after the cells are treated with the peptides. When the peptide concentration is 200 μmol / L, all the experimental groups show significant inhibitory effect, and the melanin content in the cells is reduced 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 the activity of tyrosinase will affect the production of melanin, thereby affecting the melanin content in the cells.

[0172] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Sea cucumber egg-derived bioactive peptides, characterized in that, The amino acid sequence of sea cucumber egg-derived active peptides is one or more of WGN, WIGG, and GFPVG.

2. The method for preparing the sea cucumber egg-derived active peptide according to claim 1, characterized in that, Includes the following steps: Solid-phase synthesis of the sea cucumber egg-derived active peptide of claim 1.

3. The use of the sea cucumber egg-derived active peptide according to claim 1 in any one or more of (1) to (3); (1) Preparation of antioxidant products; (2) Preparation of products that inhibit tyrosinase activity; (3) Reduce melanin content.

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