Sea cucumber ovum-derived peptide compound and application thereof in antioxidation and melanin reduction

Abstract

The invention relates to a sea cucumber ovum-derived peptide compound and application thereof in antioxidation and melanin reduction. 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, WIGG and GFPVG. The sea cucumber ovum-derived peptide compound and the sea cucumber ovum-derived active peptide provided by the invention have relatively high application and development values, and can be applied to the fields of foods, health-care products, cosmetics and the like as functional components.

Description

[0001] This application is a divisional application with application number 202411847026.X, application date December 16, 2024, and the invention title at the time of application was "Preparation method of sea cucumber egg-derived peptide complex and its application". Technical Field

[0002] This invention belongs to the field of biological products, and particularly relates to a sea cucumber egg-derived peptide complex and its application in anti-oxidation and melanin reduction. Background Technology

[0003] Based on the free radical theory of aging, the intake of exogenous antioxidants has become one of the important ways to combat aging. For skin aging, melanin accumulation is also a significant manifestation. Melanin is an indole polymer widely present in the skin. When the skin is exposed to ultraviolet radiation, the body's melanocytes produce melanin, which accumulates in melanosomes to protect the skin. However, excessive melanin deposition can lead to a series of skin problems such as melasma, freckles, melanosis, and seborrheic keratosis, becoming a major concern for those concerned about their appearance. Tyrosinase is the rate-limiting enzyme controlling melanin production in cells, catalyzing melanin production through redox reactions. Its activity directly affects the amount of melanin produced; therefore, finding substances with tyrosinase inhibitory activity is key to reducing melanin production in the body. Furthermore, numerous studies have confirmed the interaction between melanin production and oxidative stress in the body. Specifically, the process of melanin production generates a large number of reactive oxygen species (ROS), and excessive free radicals can further damage melanocytes, promote pigment accumulation, and increase the rate of aging. Therefore, reducing oxidative stress and lowering tyrosinase activity play an important role in reducing pigment deposition and preventing skin aging. However, current research on skin anti-aging mostly focuses on antioxidant activity and lacks evaluation of its tyrosinase inhibitory activity. Therefore, finding and preparing substances with both antioxidant and tyrosinase inhibitory activities is of great significance.

[0004] Bioactive peptides are attracting increasing attention due to their high efficiency and stability. Traditional preparation methods primarily rely on enzymatic hydrolysis to obtain peptide mixtures, mainly using single-enzyme hydrolysis techniques. In practical experiments, the inventors discovered that the preparation of bioactive peptides using single-enzyme preparations suffers from drawbacks such as enzymatic dead zones, long processing times, uneven action, low stability, and poor reproducibility. The complex peptide components in the mixture and the presence of no / low-biologically active peptides may affect its activity efficiency. Based on the mixture, purification techniques can be used to selectively remove certain peptide fragments to obtain peptides with known sequences. This is significant for achieving efficient and rapid preparation using chemical methods for large-scale industrial precision applications and for elucidating related mechanisms. Traditional single peptides are typically obtained through separation and purification techniques, which are cumbersome and time-consuming. With the development of bioinformatics, computer-aided technology is gradually emerging in the screening of bioactive peptides. Its high-throughput analysis methods can save screening time and improve efficiency; however, computer-aided screening alone may result in discrepancies between the screening results and the actual activity.

[0005] Sea cucumber eggs are a byproduct of sea cucumber processing, containing abundant protein and other nutrients. With increasing consumer demand for sea cucumbers, the number of sea cucumber eggs has surged, and they are often discarded as waste, resulting in a significant waste of protein resources. The low processing efficiency of sea cucumber egg protein is mainly related to its amino acid composition. Sea cucumber egg protein is primarily composed of collagen, consisting of glycine, arginine, and other basic amino acids. The single enzymatic hydrolysis method for preparing bioactive peptides mainly involves enzymatic cleavage of the outer amino acid chain, followed by internal enzymatic hydrolysis. This method is time-consuming, inefficient, and results in low bioactivity.

[0006] Therefore, it is urgent to provide a simple, efficient, and rapid method for preparing dual-functional sea cucumber egg-derived peptides. Currently, there are no reports of using alkaline protease-trypsin combination on sea cucumber eggs to prepare bioactive peptides with antioxidant, tyrosinase-inhibiting, and melanin-reducing properties. Summary of the Invention

[0007] To address the low utilization rate and limited activity of existing sea cucumber eggs, this invention provides a sea cucumber egg-derived peptide complex and its application in anti-oxidation and melanin reduction. Based on this, this invention uses the sea cucumber egg-derived peptide complex as a raw material and combines traditional separation and purification techniques with computer-aided technology to prepare bioactive peptides that can reduce melanin content and possess both antioxidant and tyrosinase-inhibiting activities.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0009] This invention provides a method for preparing a dual-functional sea cucumber egg peptide complex treated with a compound enzyme, comprising the following steps: simultaneously hydrolyzing sea cucumber egg powder using alkaline protease and trypsin.

[0010] The beneficial effects of adopting the above technical solution include:

[0011] The low level of green processing of sea cucumber industrial by-products is currently a bottleneck restricting the industry's development. The large-scale waste of sea cucumber eggs results in significant economic and resource losses. This invention prepares a bifunctional peptide complex of sea cucumber eggs with antioxidant and tyrosinase inhibitory activities, which can promote the extension of the sea cucumber industry chain and improve the intensive processing of sea cucumber industrial by-products. The sea cucumber egg-derived complex peptide preparation method of this invention is characterized by high efficiency, good reproducibility, ability to reduce melanin content, good antioxidant effect, high tyrosinase inhibitory activity, and small peptide molecular weight.

[0012] In the preparation process, this invention uses alkaline protease and trypsin for simultaneous enzymatic hydrolysis. Trypsin specifically cleaves peptide bonds with alkaline amino acids at the carboxyl side, further exposing the cleavage sites of internal hydrophobic and aromatic amino acids. Simultaneously, alkaline protease precisely recognizes and cleaves the proteins, achieving layer-by-layer enzymatic cleavage of sea cucumber proteins. This increases the number of small peptides in the complex terminated with hydrophobic and aromatic amino acids, enhancing antioxidant and tyrosinase inhibitory activities, and realizing the preparation of a bifunctional active peptide complex.

[0013] Furthermore, the sea cucumber egg powder is defatted sea cucumber egg powder.

[0014] Furthermore, it also includes the step of ultrafiltration of the enzymatic hydrolysis products: passing the enzymatic hydrolysis products sequentially through ultrafiltration membranes with molecular weights of 10kDa and 3kDa, and collecting the components with molecular weights greater than 10kDa, the components with molecular weights between 3kDa and 10kDa, and the components with molecular weights less than 3kDa.

[0015] Furthermore, it also includes the step of separating components smaller than 3 kDa using Sephadex G-15 dextran gel resin, with tubes 25 to 50 combined as SCP-1 component, tubes 58 to 77 combined as SCP-2 component, and tubes 85 to 103 combined as SCP-3 component.

[0016] Furthermore, it also includes separating the SCP-2 components by high performance liquid chromatography and collecting 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, respectively, and naming them SCP-2-1 to SCP-2-7.

[0017] 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 with an enzyme activity to substrate mass ratio of 2000 U / g, and in the mixed enzyme, the enzyme activity ratio of alkaline protease to trypsin being 1:1, hydrolyzing at 25°C for 3 hours; and then inactivating the enzyme.

[0018] Furthermore, the enzyme inactivation conditions include: heating in a boiling water bath for 20 minutes.

[0019] This invention provides a sea cucumber egg-derived peptide complex, which is prepared using the method described above.

[0020] Furthermore, the sea cucumber egg-derived peptide complex includes one or a combination of several of WGN, WIGG, and GFPVG.

[0021] Furthermore, the present invention provides a sea cucumber egg-derived active peptide with dual antioxidant and tyrosinase inhibitory activities, wherein the amino acid sequence of the sea cucumber egg-derived peptide includes one or more of WGN, WIGG, and GFPVG.

[0022] Preferably, the amino acid sequence of the sea cucumber egg-derived peptide is one or more of WGN, WIGG, and GFPVG.

[0023] The beneficial effects of adopting the above technical solution include: the sea cucumber egg-derived active peptides provided by this invention can increase the added value of sea cucumber by-products, reduce resource waste, and extend the industrial chain of sea cucumber by-products. Experimental verification shows that WGN, WIGG, and GFPVG screened by this invention 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; these small peptides are easily digested and absorbed by the human body and act on their target sites. The sea cucumber egg-derived active peptides provided by this invention all have good solubility and are non-toxic to cells.

[0024] The present invention provides a method for preparing the above-mentioned sea cucumber egg-derived active peptide, comprising the following steps: solid-phase synthesis of the above-mentioned sea cucumber egg-derived peptide.

[0025] This invention provides a method for screening the above-mentioned sea cucumber egg-derived bioactive peptides, comprising the following steps:

[0026] (1) Sea cucumber eggs were hydrolyzed using a mixture of alkaline protease and trypsin to obtain sea cucumber egg hydrolysate;

[0027] (2) The sea cucumber egg enzymatic hydrolysate obtained in step (1) was separated in three steps by ultrafiltration, dextran gel chromatography and high performance liquid chromatography, and then freeze-dried to obtain active peptide freeze-dried powder.

[0028] (3) The active peptide lyophilized powder was analyzed by liquid chromatography-mass spectrometry to obtain the peptide amino acid sequence;

[0029] (4) Use computer-aided screening technology to screen peptides with known amino acid sequences obtained in step (3);

[0030] (5) Based on the screening results of step (4), solid-phase synthesis of the screened peptides is performed and in vitro verification is conducted, such as: antioxidant activity, tyrosinase inhibitory activity, melanin content, etc.

[0031] The beneficial effects of adopting the above technical solution include: the method provided by this invention is an efficient, rapid, and accurate peptide acquisition method. It combines traditional separation and purification techniques with computer-aided technology, saving screening time for active peptides, improving efficiency, obtaining components with high antioxidant peptide content, and thus improving peptide accuracy. Using computer-aided technology to screen the obtained peptides further improves the screening time, efficiency, and accuracy.

[0032] A mixed enzymatic hydrolysis method using alkaline protease and trypsin was employed. Trypsin and alkaline protease can respectively cleave the peptide bonds on the carboxyl side of basic amino acids and hydrophobic amino acids. During hydrolysis, trypsin first recognizes and acts on the amino acids on the surface of sea cucumber egg protein, loosening its protein structure. Simultaneously, alkaline protease precisely cleaves hydrophobic and aromatic amino acids, enhancing the peptide's antioxidant and tyrosinase inhibitory activities.

[0033] 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 mixed enzymes, the enzyme activity to substrate mass ratio of the mixed enzymes is 2000 U / g, and the enzyme activity ratio of alkaline protease to trypsin in the mixed enzymes is 1:1; enzymatic hydrolysis at 25℃ for 3 hours, after heating in a boiling water bath for 20 minutes to inactivate the enzymes, centrifuging at 10000 r / min for 20 minutes, and retaining the supernatant as sea cucumber egg enzymatic hydrolysate.

[0034] Furthermore, the specific operation process of step (2) may include: fractionating the sea cucumber egg enzymatic hydrolysate sequentially through 10kDa and 3kDa ultrafiltration membranes, and collecting the fractions smaller than 3kDa; separating the fractions smaller than 3kDa by dextran gel chromatography, collecting 2.0mL of each fraction from each tube, and collecting the fractions from tubes 58 to 77 together as SCP-2 fraction; separating the SCP-2 fraction by high performance liquid chromatography, and collecting the fraction corresponding to the retention time of 5.352min as SCP-2-4 fraction.

[0035] Furthermore, the specific operation process of step (3) may include: identifying the amino acid sequence of the SCP-2-4 component using liquid chromatography-mass spectrometry (LC-MS / MS); 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 a non-linear gradient over 47 minutes, increases to 100% (volume percentage) within 1 minute, and is maintained for 12 minutes; the sample loading volume is 1.5 μL, and the column flow rate is 300 nL / min.

[0036] 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 hydrophobic properties; prediction of their bioactivity values ​​using the online tool Peptid eRanker, retaining peptides with predicted bioactivity values ​​greater than 0.84; prediction of the water solubility and toxicity of peptides using the online tools Innovagen and ToxinPred, retaining active peptide sequences that show good water solubility and non-toxicity for molecular docking; molecular docking with the Keap1 receptor using the CDOCK mode in Discovery Studio 2019 Client, and screening peptides based on binding energy.

[0037] Furthermore, the specific operation process of step (5) may include: synthesizing the screened peptides using solid-phase synthesis, and verifying their biological activity through DPPH free radical scavenging ability, ABTS free radical scavenging ability, and tyrosinase inhibition rate.

[0038] The present invention provides the application of the above-mentioned sea cucumber egg-derived peptide complex in any one or more of (1) to (3);

[0039] (1) Preparation of antioxidant products;

[0040] (2) Preparation of products that inhibit tyrosinase activity;

[0041] (3) Reduce melanin content.

[0042] The present invention provides the application of the above-mentioned sea cucumber egg-derived active peptide in any one or more of (1) to (3);

[0043] (1) Preparation of antioxidant products;

[0044] (2) Preparation of products that inhibit tyrosinase activity;

[0045] (3) Reduce melanin content.

[0046] The products mentioned include, but are not limited to, food, health products, cosmetics, and pharmaceuticals.

[0047] The sea cucumber egg-derived active peptides provided by this invention have good free radical scavenging and antioxidant capabilities, excellent inhibition of tyrosinase activity, and can reduce melanin content.

[0048] The beneficial effects of adopting the above technical solution include: the active peptide sequences of sea cucumber eggs isolated by this 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 scavenge DPPH free radicals and ABTS free radicals and inhibit tyrosinase activity. Their DPPH free radical scavenging rates are 55.49±1.70%, 41.28±1.18%, and 42.55±0.87%, respectively; their ABTS free radical scavenging 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 all effectively reduce melanin content. Attached Figure Description

[0049] Figure 1 The results show the molecular weight distribution of sea cucumber egg-derived peptide complex. In this sample, 1 represents the sea cucumber egg protein sample hydrolyzed with alkaline protease-trypsin complex, 2 represents the sea cucumber egg protein sample hydrolyzed with alkaline protease, and 3 represents the sea cucumber egg protein sample hydrolyzed with trypsin.

[0050] Figure 2 The results show the hydrophobicity of the complex surface of sea cucumber egg peptide. In this sample, 1 represents the sea cucumber egg protein sample hydrolyzed by alkaline protease-trypsin complex, 2 represents the sea cucumber egg protein sample hydrolyzed by alkaline protease, and 3 represents the sea cucumber egg protein sample hydrolyzed by trypsin.

[0051] Figure 3 The elution curve of the G-15 dextran gel of the present invention is shown, where the horizontal axis represents the number of elution tubes and the vertical axis represents the absorbance at 280 nm.

[0052] Figure 4 This is the high performance liquid chromatogram (RP-HPLC) of the present invention, wherein the horizontal axis represents the retention time (min) and the vertical axis represents the absorbance at 214 nm.

[0053] Figure 5 The top figure shows the results of the interaction between WGN and Keap1, and the bottom figure shows the results of the interaction between WGN and tyrosinase (2Y9X).

[0054] Figure 6The top image shows the interaction between WIGG and Keap1, and the bottom image shows the interaction between WIGG and tyrosinase (2Y9X).

[0055] Figure 7 The top image shows the interaction between GFPVG and Keap1, and the bottom image shows the interaction between GFPVG and tyrosinase (2Y9X).

[0056] Figure 8 The effect of synthetic peptides on DPPH free radical scavenging rate is shown, where the horizontal axis represents the type of synthetic peptide and the vertical axis represents the DPPH free radical scavenging rate.

[0057] Figure 9 The effect of synthetic peptides on ABTS free radical scavenging rate is shown, where the horizontal axis represents the type of synthetic peptide and the vertical axis represents the ABTS free radical scavenging rate.

[0058] Figure 10 The effect of synthetic peptides on tyrosinase inhibition rate is shown, where the horizontal axis represents the type of synthetic peptide and the vertical axis represents the tyrosinase inhibition rate.

[0059] Figure 11 The effects of different concentrations of WGN, WIGG, and GFPVG on the survival rate of HepG2 cells were investigated. In this study, A represents the effect of different concentrations of WGN on cell survival rate, B represents the effect of different concentrations of WIGG on cell survival rate, and C represents the effect of different concentrations of GFPVG on cell survival rate.

[0060] Figure 12 The effect of WGN on intracellular tyrosinase activity in B16-F10 cells.

[0061] Figure 13 The effect of WGN on melanin production in B16-F10 cells.

[0062] Figure 14 The effect of WIGG on the activity of intracellular tyrosinase in B16-F10 cells.

[0063] Figure 15 The effect of WIGG on melanin production in B16-F10 cells.

[0064] Figure 16 The effect of GFPVG on the activity of tyrosinase in B16-F10 cells.

[0065] Figure 17 The effect of GFPVG on melanin production in B16-F10 cells. Detailed Implementation

[0066] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0067] This invention provides a method for preparing a sea cucumber egg-derived peptide complex with dual activities of antioxidant and tyrosinase inhibition based on alkaline protease-trypsin treatment.

[0068] The preparation method of sea cucumber egg-derived peptide complex may include the following steps: sea cucumber egg hydrolysate is obtained by enzymatic hydrolysis using a mixture of alkaline protease and trypsin. Alternatively, the obtained sea cucumber egg hydrolysate can be purified by a three-step separation process of ultrafiltration-dextran gel chromatography-high performance liquid chromatography, and the DPPH free radical scavenging rate and ABTS scavenging rate of each separated component are determined. The final product is then freeze-dried to obtain a high-antioxidant peptide complex powder.

[0069] Furthermore, the preparation of the sea cucumber egg-derived peptide mixture may include the following steps: using sea cucumber eggs as raw material, a combination of alkaline protease and trypsin is used for simultaneous enzymatic hydrolysis. The enzymatic hydrolysis process includes: the ratio of enzyme activity to substrate mass of the mixed enzyme is 2000 U / g; the mixed enzyme includes alkaline protease and trypsin; the ratio of alkaline protease to trypsin activity is 1:1; the hydrolysis time is 3 hours; the hydrolysis temperature is 25℃; and the pH is 7.5. After the enzymatic hydrolysis is completed, the mixture is heated in a boiling water bath for 20 minutes to inactivate the enzyme; centrifuged at 10000 r / min for 20 minutes; and the supernatant is collected to obtain the sea cucumber egg enzymatic hydrolysate.

[0070] Further, the separation of antioxidant peptides derived from sea cucumber eggs may include the following steps: Sea cucumber egg enzymatic hydrolysate is sequentially fractionated using 10kDa and 3kDa ultrafiltration membranes; the DPPH free radical scavenging capacity of different components is determined; the eluent fraction with high DPPH scavenging capacity is collected and freeze-dried. The highly antioxidant component obtained from ultrafiltration is reconstituted and separated by dextran gel chromatography; the eluent fraction with the strongest DPPH free radical scavenging capacity is collected and freeze-dried. The highly antioxidant component obtained from dextran gel chromatography is reconstituted and separated by high-performance liquid chromatography (HPLC) in a third step; the DPPH free radical scavenging capacity of the collected components is determined; the eluent fraction with the strongest antioxidant activity is retained and freeze-dried to obtain a highly antioxidant peptide composite powder.

[0071] This invention provides a sea cucumber egg-derived peptide complex, which is prepared using the method described above.

[0072] This invention provides the application of the above-mentioned sea cucumber egg-derived peptide complex in the preparation of antioxidant and / or tyrosinase-inhibiting products.

[0073] This invention provides sea cucumber egg-derived bioactive peptides with dual antioxidant and tyrosinase-inhibiting activities, their preparation methods, screening methods, and applications.

[0074] This invention provides three sea cucumber egg-derived bioactive peptides with antioxidant and tyrosinase-inhibiting properties, wherein the amino acid sequences of the sea cucumber egg-derived bioactive peptides are WGN, WIGG, and GFPVG. These sea cucumber egg-derived bioactive peptides can reduce melanin content and are also bifunctional sea cucumber egg-derived bioactive peptides, possessing both antioxidant and tyrosinase-inhibiting effects.

[0075] The preparation method of the above-mentioned sea cucumber egg-derived bioactive peptide includes the following steps: solid-phase synthesis of sea cucumber egg-derived bioactive peptide having the above-mentioned amino acid sequence.

[0076] This invention provides a method for screening the above-mentioned sea cucumber egg-derived bioactive peptides, comprising the following steps:

[0077] (a) The amino acid sequence of the high antioxidant peptide complex obtained above was identified by liquid chromatography-mass spectrometry.

[0078] (b) Using computer-aided screening technology, peptides with known amino acid sequences obtained in step (a) are screened to obtain peptides with potential high antioxidant activity and high tyrosinase inhibitory activity.

[0079] (c) Screen peptides by solid-phase synthesis and in vitro verification of their antioxidant, tyrosinase inhibitory activities and effects on melanin content.

[0080] Furthermore, the identification of sea cucumber egg-derived antioxidant peptides may include the following steps: Amino acid sequence identification of the components separated by reversed-phase high-performance liquid chromatography (LC-MS / MS) is performed. 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 at 2% (v / v) of phase B, increases to 35% (v / v) in a non-linear gradient over 47 minutes, then increases to 100% (v / v) within 1 minute and maintains this gradient for 12 minutes; the sample loading volume is 1.5 μL, and the column flow rate is 300 nL / min.

[0081] Furthermore, screening bifunctional peptides derived from sea cucumber eggs may include the following steps: Preliminary screening of active peptides identified by LC-MS / MS based on peptide chain length and hydrophobicity. Prediction of peptide bioactivity values ​​using the online tool PeptideRanker (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.php). Prediction of peptide water solubility and toxicity using the online tools Innovagen (www.innovagen.com / proteomicstools) and ToxinPred (http: / / crdd.osdd.net / raghava / / toxinpred), retaining the water-soluble and non-toxic active peptide sequences for molecular docking.

[0082] Furthermore, the activity of bifunctional peptides derived from sea cucumber eggs can be verified through experiments, including the following steps: synthesizing the screened peptides using solid-phase synthesis, and verifying their bioactivity through experiments on DPPH free radical scavenging ability, ABTS free radical scavenging ability, and tyrosinase inhibition rate.

[0083] This invention addresses the low utilization rate and limited activity of existing sea cucumber intestines by providing a method for obtaining bifunctional sea cucumber egg-derived bioactive peptides through simultaneous alkaline protease-trypsin enzymatic hydrolysis, separation and purification, and sequential computer screening. This invention utilizes ultrafiltration, gel chromatography, and high-performance liquid chromatography separation techniques, leveraging protein databases and online software, and employing multiple screening methods based on DPPH scavenging rate, ABTS scavenging rate, water solubility, and biotoxicity. Molecular docking is used to explore the interaction mechanisms between the bioactive peptides and the receptor Keap1 and tyrosinase (2Y9X). Finally, solid-phase synthesis is used to verify the antioxidant activity and tyrosinase inhibition activity of the peptides. Results show that the three bioactive peptides obtained by this invention possess advantages such as good water solubility, good safety, good antioxidant activity, excellent tyrosinase inhibition ability, and the ability to reduce melanin content.

[0084] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. The materials, reagents, methods, and instruments used, unless otherwise specified, are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially or prepared using conventional methods by those skilled in the art. Unless otherwise specified, the solvent for the solutions involved in this invention is water.

[0085] Sea cucumber spawning powder was purchased from Linghai Dalian Marine Products Aquaculture Co., Ltd.; Sephadex G-15 dextran gel resin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; HepG-2 cells were purchased from the China Center for Type Culture Collection (CCTCC); PBS was purchased from Beijing Bio-Top Technology Co., Ltd.; L-dopa was purchased from Shanghai Aladdin Reagent Co., Ltd.; CCK-8 kit was purchased from Beyotime Biotechnology Co., Ltd.; MEM medium was purchased from Wuhan Pronosai Life Science Co., Ltd. B16-F10 mouse melanoma cells were purchased from Wuhan Pronosai Life Science Co., Ltd.; fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; and RPMI-1640 medium containing 1% penicillin G sodium salt and streptomycin sulfate was purchased from Wuhan Pronosai Life Science Co., Ltd., with the content of penicillin G sodium salt being 1% (mass percentage) and the content of streptomycin sulfate being 1% (mass percentage).

[0086] The following is a description through specific embodiments.

[0087] Example 1

[0088] The preparation of sea cucumber egg-derived peptide enzymatic hydrolysate includes the following steps: Weigh defatted sea cucumber spawning powder, dissolve it in 10 times its volume of deionized water, adjust the pH to 7.5, add a mixed enzyme with an enzyme activity to substrate mass ratio of 2000 U / g. The mixed enzyme includes alkaline protease and trypsin, with an alkaline protease to trypsin activity ratio of 1:1. For example, for every 1g of substrate, the amount of alkaline protease added is 1000U, and the amount of trypsin added is 1000U. Enzymatic hydrolysis is performed at 25℃ for 3 hours. After inactivating the enzyme by heating in a boiling water bath for 20 minutes, centrifuge at 10000 rpm for 20 minutes, and retain the supernatant as the sea cucumber egg enzymatic hydrolysate.

[0089] Comparative Example 1

[0090] Based on 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 its optimal enzymatic hydrolysis conditions.

[0091] The specific procedures in Comparative Example 1 included: adjusting the pH to 10, setting the temperature to 45°C, adding an enzyme dosage of 2000 U / g, and hydrolyzing for 3 hours. After inactivating the enzyme by heating in a boiling water bath for 20 minutes, the mixture was centrifuged at 10000 rpm for 20 minutes, and the supernatant was retained as the sea cucumber egg hydrolysate. All other steps were the same as in Example 1.

[0092] Comparative Example 2

[0093] Based on 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.

[0094] The specific procedures in Comparative Example 1 included: adjusting the pH to 7.2, setting the temperature to 37°C, adding an enzyme dosage of 2000 U / g, and hydrolyzing for 3 hours. After inactivating the enzyme by heating in a boiling water bath for 20 minutes, centrifuging at 10000 rpm for 20 minutes, and retaining the supernatant as the sea cucumber egg hydrolysate. All other steps were the same as in Example 1.

[0095] Example 1: Detection of the bioactivity of sea cucumber egg enzymatic hydrolysate

[0096] Sea cucumber egg enzymatic hydrolysates from Examples 1, 1, and 2 were freeze-dried to obtain sea cucumber egg peptide complexes. The antioxidant properties (DPPH scavenging rate and ABTS scavenging rate) and tyrosinase inhibitory activity of the sea cucumber egg peptide complexes were then tested.

[0097] (1) The DPPH scavenging rate was determined by the following steps: A sample group, a control group, and a blank group were set up. Sample group: 4.0 mL of the test sample solution was mixed with an equal volume of DPPH solution; Control group: 4.0 mL of the test sample solution was mixed with an equal volume of anhydrous ethanol; Blank group: DPPH solution. In all groups, the solvent in the DPPH solution was anhydrous ethanol, and the DPPH concentration was 0.1 mmol / L; the solvent in the test sample solution was water, and the peptide concentration was 2.0 mg / mL. After reacting in the dark at room temperature for 30 min, the absorbance at 517 nm was measured using a UV spectrophotometer. The DPPH free radical scavenging capacity (i.e., DPPH scavenging rate) was calculated using the following formula.

[0098]

[0099] In the formula, A 样品组 A represents the absorbance of the sample group at 517 nm. 对照组 The absorbance at 517 nm is the control group; A 空白组 The absorbance of the blank group at 517 nm is given.

[0100] (2) Detecting ABTS clearance rate includes the following steps:

[0101] ① Preparation of working solution: Mix ABTS solution and an equal volume of potassium persulfate solution. The concentration of ABTS in the ABTS solution is 7.0 mmol / L, and the concentration of potassium persulfate in the potassium persulfate solution is 2.45 mmol / L. Let the mixture stand at 25℃ for 12-16 h, and react under light-protected conditions to obtain the ABTS mother liquor. Dilute ABTS with 80% ethanol solution to obtain the working solution, so that the absorbance of the working solution at 734 nm reaches 0.7 ± 0.1.

[0102] ② Prepare sample, control, and blank groups. Sample group: Mix 0.5 mL of the 2.0 mg / mL test sample with 3.0 mL of working solution; Control group: Mix 0.5 mL of the 2.0 mg / mL test sample with 3.0 mL of distilled water; Blank group: Mix 0.5 mL of distilled water with 3.0 mL of working solution. Incubate each group in the dark for 10 min and measure the absorbance at 734 nm. Determine the ABTS clearance rate using the following formula.

[0103]

[0104] In the formula, A 样品组 A represents the absorbance value of the sample group at 734 nm. 对照组 The absorbance value of the control group at 734 nm; A 空白组 The absorbance value of the blank group was measured at 734 nm.

[0105] (3) Detection of tyrosinase inhibitory activity includes the following steps: set up blank group, negative group, background group and sample group, and prepare solutions for each group according to Table 1.

[0106] Table 1. Grouping and Dosage of Reaction Solution

[0107]

[0108] Each group was incubated in a 37°C water bath for 30 minutes. The absorbance was measured at 475 nm, and the inhibition rate of different concentrations of active ingredients on tyrosinase was calculated according to the following formula.

[0109]

[0110] In the above formula, A 阴性 The absorbance of the negative group at 475 nm; A 空白 A represents the absorbance of the blank group at 475 nm. 样品 A represents the absorbance of the sample group at 475 nm. 背景 The absorbance of the background group at 475 nm is given.

[0111] (4) Detecting the relative melanin content of cells, including the following steps:

[0112] 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.

[0113] The experimental method was based on the following literature with slight modifications: 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 consisted of normally cultured cells, while the experimental group consisted of cells treated with different samples at 2 mg / mL. Cells were resuspended in PBS, and 1 mol / L NaOH was added. The cells were incubated at 80℃ for 2 h and then thawed. After centrifugation at 10000 rpm for 15 min, 100.0 μL of the supernatant was collected into a 96-well plate, and the absorbance was measured at 405 nm.

[0114]

[0115] In the above formula, A 实验组 A represents the absorbance of the experimental group cells at 405 nm. 对照 The absorbance at 405 nm represents the control group.

[0116] Experimental results:

[0117] The results of the antioxidant and tyrosinase inhibitory activity tests are shown in Table 2. As can be seen from Table 2, the antioxidant activity, tyrosinase inhibitory activity and relative melanin content of the sea cucumber oocyte peptide complex prepared by the method of Example 1 of the present invention are all better than those of the sample prepared by a single enzyme.

[0118] Table 2. Evaluation of the bioactivity of peptide complexes

[0119]

[0120] Example 2: Detection of whether proteins are enzymatically broken down into small peptides

[0121] The molecular weight of the sea cucumber egg peptide complex obtained by the methods of Example 1, Comparative Example 1 and Comparative Example 2 was determined.

[0122] Figure 1 This is a molecular weight distribution diagram of the sea cucumber egg-derived peptide complex. Figure 1In the table, 1 represents the sea cucumber oviduct peptide complex sample prepared using alkaline protease-trypsin (i.e., the sea cucumber oviduct peptide complex prepared using the method of Example 1), 2 represents the sea cucumber oviduct peptide complex prepared using alkaline protease (i.e., the sea cucumber oviduct peptide complex prepared using the method of Comparative Example 1), and 3 represents the sea cucumber oviduct peptide complex prepared using trypsin (i.e., the sea cucumber oviduct peptide complex prepared using the method of Comparative Example 2). Figure 1 It can be seen that the peptide samples prepared by alkaline protease-trypsin complex hydrolysis mainly have molecular weights concentrated between 0-1500 Da, while the peptides in the alkaline protease-treated group complex are mainly peptides with molecular weights greater than 3000 Da. The trypsin-treated group complex is mainly composed of peptides with molecular weights greater than 3000 Da and less than 5000 Da. Alkaline protease-trypsin treatment can obtain peptides with smaller molecular weights. The inventors speculate that this is mainly because the complex enzyme has more cleavage sites than Comparative Examples 1 and 2, which can fully cleave the protein. Sea cucumbers are mainly composed of arginine, glycine, and basic amino acids. Alkaline protease can cleave hydrophobic amino acids (glycine), and arginine and other basic amino acids are all trypsin cleavage sites. Therefore, the number of small molecular weight peptides in the trypsin-hydrolyzed complex peptides is higher than that in the alkaline protease-treated group. In addition, the higher 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.

[0123] Example 3: Detection of excessive leakage of hydrophobic amino acids in various peptide complexes.

[0124] The surface hydrophobicity of the peptide complexes prepared using 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.

[0125] Each enzyme digest was prepared to a concentration of 0.2 mg / mL as the sample solution. ANS was dissolved in 10 mmol / L PBS to obtain an ANS solution with a concentration of 8 mmol / L. 4 mL of the sample solution was placed in a 5 mL centrifuge tube, and 20 μL of the ANS solution was added. The tube was shaken for 30 seconds, allowed to stand for 2 minutes, and then detected using a fluorescence spectrophotometer. The excitation wavelength was set to 395 nm, the emission wavelength was fixed at 400-600 nm, and the slit width was 5 nm.

[0126] Figure 2 These are the surface hydrophobicity measurement results in Example 1 and different comparative examples. The horizontal axis represents the sea cucumber egg-derived peptide complexes prepared by different proteases, and the vertical axis represents the fluorescence intensity. Fluorescence intensity can reflect hydrophobicity. Figure 2In the table, 1 represents the sea cucumber oviduct peptide complex sample prepared using alkaline protease-trypsin (i.e., the sea cucumber oviduct peptide complex prepared in Example 1), 2 represents the sea cucumber oviduct peptide complex prepared using alkaline protease (i.e., the sea cucumber oviduct peptide complex prepared in Comparative Example 1), and 3 represents the sea cucumber oviduct peptide complex prepared using trypsin (i.e., the sea cucumber oviduct peptide complex prepared in Comparative Example 2). Figure 2 It can be seen that the surface hydrophobicity of peptide samples prepared by alkaline protease-trypsin complex hydrolysis is significantly better than that of peptide complex samples prepared by a single enzyme.

[0127] Example 2: Separation and Purification

[0128] (1) Ultrafiltration: The sea cucumber egg enzymatic hydrolysate obtained in Example 1 was prepared with ultrapure water to a concentration of 20 mg / mL. The hydrolysate was then passed sequentially through ultrafiltration membranes with molecular weights of 10 kDa and 3 kDa. Fractions with molecular weights greater than 10 kDa, 3 kDa-10 kDa, and less than 3 kDa were collected. The DPPH scavenging rate, ABTS scavenging rate, tyrosinase inhibitory activity, and melanin content of each collected fraction were determined using the same methods as in Example 1.

[0129] Based on the screening results above, the components with higher clearance rates were selected and freeze-dried for later use. The screening results are shown in Table 3. It can be seen that the components with a clearance rate less than 3 kDa showed significantly better DPPH clearance rate, ABTS clearance rate, tyrosinase inhibitory activity, and relative melanin content than other components. Therefore, components with a clearance rate less than 3 kDa were selected for subsequent gel chromatography separation experiments.

[0130] Table 3 Antioxidant and tyrosinase inhibitory activities of each component in ultrafiltration

[0131]

[0132] (2) Gel chromatography: The fractions less than 3 kDa obtained in step (1) were further separated using Sephadex G-15 dextran gel resin.

[0133] The column (2.6 × 60.0 cm) was equilibrated with distilled water, and the injection volume was 5 mL; the sample concentration (components less than 3 kDa) was 10 mg / mL. UV detection was performed at 280 nm. 2.0 mL of the sample was collected from each tube and lyophilized.

[0134] According to the elution curve of G-15 dextran gel ( 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 significantly higher absorbance values ​​at 280 nm.

[0135] The DPPH scavenging rate, ABTS scavenging rate, and tyrosinase inhibitory activity of SCP-1, SCP-2, and SCP-3 were measured separately, using the same methods as in Example 1. The component with the strongest antioxidant activity was selected based on the DPPH free radical scavenging rate and ABTS scavenging rate results. The component with the strongest tyrosinase inhibitory ability and the lowest melanin content was selected based on the tyrosinase inhibitory activity and the relative melanin content results. As shown in Table 4, component SCP-2 exhibited significantly higher DPPH free radical scavenging rate, ABTS scavenging rate, and tyrosinase inhibitory activity than the other components, while having a relatively lower melanin content. Therefore, component SCP-2 was selected for high-performance liquid chromatography (HPLC) separation.

[0136] Table 4. Antioxidant properties and tyrosinase inhibitory activity of each component in G-15 gel chromatography.

[0137]

[0138] (3) High-performance liquid chromatography (HPLC) separation: Agilent C18 column (250.0 × 4.6 mm, 5.0 μm) was used to further separate the highly antioxidant components from the SCP-2 fraction. Mobile phase A was acetonitrile, and mobile phase B was 0.1% (v / v) trifluoroacetic acid. Isocratic elution (10% A, 90% B, both v / v) was performed at a flow rate of 1.0 mL / min, and UV detection was performed at 214 nm. Components were collected based on the HPLC results, such as... Figure 4 As shown, the 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 2.099 min, 3.148 min, 4.833 min, 5.352 min, 13.533 min, 14.992 min, and 20.99 min, respectively.

[0139] The components were then freeze-dried, and the scavenging rates of DPPH and ABTS, tyrosinase inhibitory activity, and relative melanin content of each component were determined using the method described in Example 1. Table 5 shows that the SCP-2-4 component exhibited significantly higher scavenging rates of DPPH and ABTS and higher tyrosinase inhibitory activity than the other components, while also having a lower relative melanin content. Therefore, the SCP-2-4 component was selected for amino acid sequence identification.

[0140] Table 5. Antioxidant properties and tyrosinase inhibitory activity of each component in high performance liquid chromatography.

[0141]

[0142] Example 3: Identification of the amino acid sequence of antioxidant peptides from sea cucumber eggs

[0143] The amino acid sequence was identified by Nanjing Zhengfu Function Biotechnology Co., Ltd., including the following steps: HPLC-MS / MS was used to perform secondary mass spectrometry analysis on the fraction SCP-2-4 separated by HPLC. 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). The gradient started at 2% of phase B, increased to 35% of phase B in a non-linear gradient over 47 minutes, and then increased to 100% of phase B within 1 minute, maintaining this gradient for 12 minutes. The sample loading volume was 1.5 μL, and the column flow rate was 300 nL / min.

[0144] By comparing with protein databases, the amino acid sequences of 35 novel bioactive peptides were obtained (Table 6).

[0145] Table 6. Peptide sequences identified in SCP-2-4 by LC-MS / MS.

[0146]

[0147]

[0148] Example 4: Screening and Molecular Docking of Bifunctional Peptides from Sea Cucumber Eggs

[0149] First, peptide sequences are initially screened based on molecular weight and amino acid number, selecting those with a molecular weight less than 500.

[0150] Peptides with ≤5 amino acids were selected for further screening.

[0151] The bioactivity values ​​of peptides were predicted using the online tool PeptideRanker (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.php), and peptides with predicted bioactivity values ​​greater than 0.84 were retained.

[0152] The water solubility and biotoxicity of peptides were screened and predicted using the online tools Innovagen (www.innovagen.com / proteomicstools) and ToxinPred (http: / / crdd.osdd.net / raghava / / toxinpred). Peptides that were shown as having "good solubility" and being non-toxic by the software results were retained.

[0153] Preliminary molecular docking screening (LibDock) was performed using the CDOCK mode in Discovery Studio 2019 Client with the Keap1 (PBDID: 2ZMX) receptor. The peptide sequences docked with Keap1 were then further docked with tyrosinase (PBD ID: 2Y9X) using Maestro 12.8 software. LibDock scores and binding scores (Docking score, where lower energy indicates tighter binding) were recorded. Finally, three novel peptides with potentially good biological activity, good water solubility, and no toxicity were obtained (Table 7).

[0154] Table 7. Bioactivity prediction, toxicity, solubility and other properties of bioactive peptides derived from sea cucumber eggs.

[0155]

[0156]

[0157] Note: "-" in the table indicates that simultaneous docking is not possible, and no docking score is given.

[0158] The molecular docking results for WGN, WIGG, and GFPVG are as follows: Figure 5 , Figure 6 and Figure 7As shown, WGN binds most tightly to both receptors, suggesting that WGN may possess potentially good antioxidant activity and tyrosinase inhibitory capabilities. The bioactive peptide WGN is mainly linked to the ILE416, GLY364, LEU365, VAL418, LEU557, and VAL604 residues of Keap1 via hydrogen bonds; ALA366 is the amino acid residue generating alkyl / pi-alkyl interactions; and ARG415 is the amino acid generating electrostatic interactions. The aspartic acid (N) on the bioactive peptide WGN interacts with GLU 256 on tyrosinase, and tryptophan (W) interacts with VAL 283 via hydrogen bonds; the tryptophan (W) on the polypeptide interacts with the residue PHE 264 via π-π bonds. The bioactive peptide WIGG can form alkyl / pi-alkyl interactions with VAL512, VAL465, ALA366, and CYS513 residues of the Keap1 protein. It can also connect with GLY367, VAL606, LEU365, and ILE559 residues of Keap1 via hydrogen bonds and exhibit electrostatic interactions with ARG415 residues. In its interaction with tyrosinases, it primarily occupies tyrosinase sites through hydrogen bonding with ASN260 and HIS85 residues. The bioactive peptide GFPVG interacts with ALA607, CYS368, and VAL606 residues of Keap1 mainly through alkyl / pi-alkyl interactions. It can form hydrogen bonds and electrostatic interactions with VAL465 and ARG415, respectively, occupying the Nrf2 site. GFPVG interacts with tyrosinases ASN260 and HIS85 via hydrogen bonds.

[0159] Example 5: In vitro activity verification of active peptides WGN, WIGG, and GFPVG

[0160] The three selected bioactive peptides were synthesized in solid phase by Sangon Biotech (Shanghai) Co., Ltd., with a purity exceeding 98%. The in vitro DPPH scavenging rate, ABTS scavenging rate, and tyrosinase inhibition rate of the three bioactive peptides were verified, and their safety was assessed.

[0161] The detection methods for DPPH clearance rate, ABTS clearance rate, and tyrosinase inhibition rate are the same as in Example 1. The detection results are as follows: Figure 8 , Figure 9 , Figure 10As shown, WGN, WIGG, and GFPVG can effectively scavenge DPPH 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 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.

[0162] Compared with the data from Example 1, the tyrosinase inhibition rates of sea cucumber egg-derived active peptides WGN, WIGG, and GFPVG were significantly higher than those of the sea cucumber egg-derived peptide complex. The antioxidant activity of the sea cucumber egg-derived peptide complex was superior to that of the individual sea cucumber egg-derived active peptides WGN, WIGG, and GFPVG. The inventors hypothesize that the components of the sea cucumber egg-derived peptide complex have a synergistic effect in terms of antioxidant activity. Further purification and identification of the sea cucumber egg-derived peptide complex can yield single peptides with high tyrosinase inhibition rates.

[0163] Example 6: Biosafety assessment of active peptides WGN, WIGG, and GFPVG

[0164] The biosafety of the peptides was assessed using the CCK-8 assay. Cells were cultured in MEM at a concentration of 1 × 10⁻⁶. 4 Cell / well density: Logarithmic growth phase HepG2 cells were seeded in 96-well plates, with 100.0 μL of MEM medium containing cells added to each well, and six replicates were set up. After 24 h of incubation, the medium was removed. Cells were then divided into control and experimental groups. 100.0 μL of peptide samples at 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 (Beyotime Biotechnology Co., Ltd., C0037) was added to each well, and the cells were incubated at 37°C for 1 h. The absorbance was measured at 450 nm, and cell viability was calculated using the following formula.

[0165]

[0166] The results are as follows 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.

[0167] Example 7: Detection of the inhibitory effect of peptides on tyrosinase activity and melanin production in B16-F10 cells.

[0168] 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.

[0169] 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.

[0170]

[0171] The results are as follows Figure 12 , Figure 14 , Figure 16As shown, all three peptides effectively inhibited tyrosinase activity in B16-F10 cells. At a concentration of 200 μmol / L, all three peptides exhibited significant inhibitory activity; compared to the control group, the tyrosinase activities of WGN, WIGG, and GFPVG peptides decreased by 30.93%, 20.00%, and 27.31%, respectively. Furthermore, the inhibitory ability on tyrosinase activity significantly increased with increasing concentration.

[0172] Example 1: Detection Method for Inhibition of Cellular Melanin Production (Referencing Effect) ， The results are as follows Figure 13 , Figure 15 , Figure 17 As shown, after peptide treatment, the intracellular melanin content gradually decreased with increasing peptide concentration. At a peptide concentration of 200 μmol / L, all experimental groups showed a significant inhibitory effect, with intracellular melanin content decreasing to 83.23±2.34 (WGN), 85.88±3.76 (WIGG), and 73±2.64 (GFPVG), respectively. These results indicate that the activity of tyrosinase in B16-F10 cells is closely related to melanin production. Reducing tyrosinase activity affects melanin production, thereby influencing intracellular melanin content.

[0173] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A sea cucumber egg-derived peptide complex, characterized in that, The amino acid sequence includes one or more of WGN, WIGG, and GFPVG.

2. The sea cucumber egg-derived peptide complex according to claim 1, characterized in that, The preparation method includes, (1) using alkaline protease-trypsin mixed enzymatic hydrolysis to obtain sea cucumber egg hydrolysate; (2) The sea cucumber egg enzymatic hydrolysate obtained in step (1) is separated in three steps by ultrafiltration, dextran gel chromatography and high performance liquid chromatography, and then freeze-dried to obtain active peptide freeze-dried powder.

3. The sea cucumber egg-derived peptide complex according to claim 2, characterized in that, The preparation method also includes: (3) The active peptide lyophilized powder was analyzed by liquid chromatography-mass spectrometry to obtain the peptide amino acid sequence; (4) Use computer-aided screening technology to screen the peptides with amino acid sequences obtained in step (3); (5) Based on the screening results of step (4), solid-phase synthesis of screened peptides was performed and verified in vitro.

4. The use of the sea cucumber egg-derived peptide complex 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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