Tyrosinase inhibitory peptide from kelp as well as preparation method and application of tyrosinase inhibitory peptide

Abstract

The invention discloses a tyrosinase inhibitory peptide from kelp as well as a preparation method and application of the tyrosinase inhibitory peptide. The preparation method comprises the following steps: pretreating kelp, fermenting the kelp by using lactobacillus plantarum, carrying out centrifugation, ultrafiltration and component identification on the obtained fermentation liquor to obtain the composition of kelp polypeptide, screening the tyrosinase inhibitory peptide through molecular docking, and finally carrying out solid-phase synthesis to obtain the tyrosinase inhibitory peptide prepared by the invention. The lactobacillus plantarum fermentation technology is simple and feasible, the production cost is low, equipment is easy to obtain, active ingredients in kelp can be released to the maximum extent, and the prepared polypeptide has the effect of inhibiting the activity of tyrosinase and can be used for developing a tyrosinase inhibitor.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a tyrosinase inhibitory peptide derived from kelp, and a preparation method and application thereof. Background Art

[0002] Tyrosinase is a vital enzyme in living organisms, widely present in plants, animals, and microorganisms. It plays a key role in the synthesis of melanin in the human body. Excessive melanin production can lead to skin pigmentation, causing skin problems such as chloasma and freckles, affecting the skin's appearance and health. This is one of the challenges that the beauty and skincare industry has long been committed to solving. Furthermore, during some food processing and preservation processes, the enzymatic browning reaction catalyzed by tyrosinase can darken the color of food and reduce its quality, limiting its shelf life and commercial value. Therefore, inhibiting tyrosinase activity is also of great significance in the food industry.

[0003] Traditionally, chemically synthesized tyrosinase inhibitors, while effective at inhibiting enzyme activity to a certain extent, often carry safety risks, such as the potential for skin allergies and irritation. Long-term use can also pose potential health risks to humans. In food applications, the use of chemical additives is also subject to increasingly stringent regulations, leading to an increasing consumer demand for natural, safe, and non-toxic inhibitors.

[0004] Therefore, the development of a new tyrosinase inhibitor that is widely available, low in cost, safe, natural, and highly effective has become an urgent need in many fields such as beauty and skin care, and food preservation. Summary of the Invention

[0005] The purpose of the present invention is to provide a tyrosinase inhibitory peptide derived from kelp and a preparation method and application thereof.

[0006] One of the technical solutions adopted by the present invention to solve the technical problem is: providing a tyrosinase inhibitory peptide 1 derived from Laminaria japonica, whose amino acid sequence is YRL, as shown in SEQ ID NO: 1.

[0007] The molecular weight of tyrosinase inhibitory peptide 1 is 450.53 Da, and its charge is +1.

[0008] Preferably, the above tyrosinase inhibitory peptide 1 is used in the preparation of a tyrosinase inhibitor.

[0009] The second technical solution adopted by the present invention to solve the technical problem is: providing a tyrosinase inhibitory peptide 2 derived from Laminaria japonica, whose amino acid sequence is LPD, as shown in SEQ ID NO: 2.

[0010] The molecular weight of tyrosinase inhibitory peptide 2 is 343.38 Da, and its charge is +1.

[0011] Preferably, the above tyrosinase inhibitory peptide 2 is used in the preparation of tyrosinase inhibitors.

[0012] The third technical solution adopted by the present invention to solve the technical problem is: to provide a food, the active ingredient of which includes the above-mentioned tyrosinase inhibitory peptide 1.

[0013] The fourth technical solution adopted by the present invention to solve the technical problem is: to provide a food, the effective ingredient of which includes the above-mentioned tyrosinase inhibitory peptide 2.

[0014] The fifth technical solution adopted by the present invention to solve its technical problem is: a cosmetic, the active ingredient of which includes the above-mentioned tyrosinase inhibitory peptide 1.

[0015] The sixth technical solution adopted by the present invention to solve its technical problem is: a cosmetic, the active ingredient of which includes the above-mentioned tyrosinase inhibitory peptide 2.

[0016] The seventh technical solution adopted by the present invention to solve the technical problem is to provide a method for preparing a tyrosinase inhibitory peptide derived from kelp, comprising the following steps:

[0017] S1. Select intact and undamaged kelp, soak, dry, crush and sieve to obtain kelp powder, prepare liquid fermentation raw materials, and sterilize and cool;

[0018] S2 activated Lactobacillus plantarum seed solution, inoculated into the liquid fermentation raw material sterilized and cooled in step S1 for fermentation to obtain a fermentation product, the fermentation product was centrifuged to obtain a supernatant;

[0019] S3. ultrafiltration of the supernatant in step S2 to obtain polypeptides of different molecular weights, and determination of their tyrosinase inhibitory activity;

[0020] S4. Structural identification of the peptide with inhibitory activity in step S3 using LC-MS / MS;

[0021] S5. The polypeptide identified in step S4 is subjected to molecular docking with tyrosinase to screen polypeptides capable of binding to tyrosinase, and the screened polypeptides are subjected to solid phase synthesis to obtain tyrosine inhibitory peptides derived from kelp.

[0022] In summary, the principle of preparing tyrosinase inhibitory peptides by fermenting kelp with Lactobacillus plantarum is mainly based on the biotransformation effect during microbial fermentation, which can be specifically explained from the following aspects:

[0023] 1. Preparation of kelp raw materials:

[0024] Kelp, a rich marine biological resource, contains a variety of proteins, polysaccharides, and other bioactive substances. By selecting intact, undamaged kelp and then soaking, drying, crushing, and screening it, we obtain kelp powder, which provides a suitable substrate for subsequent fermentation.

[0025] 2. Activation and inoculation of Lactobacillus plantarum:

[0026] Lactobacillus plantarum is a beneficial lactic acid bacterium with multiple biological functions, including fermentation, acid production, and enzyme production. By activating Lactobacillus plantarum seed liquid and inoculating it proportionally into sterilized, cooled kelp powder fermentation raw materials, Lactobacillus plantarum begins to grow and multiply in the kelp powder, utilizing the nutrients in the kelp for metabolic activities.

[0027] 3. Biotransformation during fermentation:

[0028] During the fermentation process, Lactobacillus plantarum degrades and transforms the proteins in kelp through its own enzymes (such as proteases and peptidases). These enzymes can cleave peptide bonds in proteins, releasing small peptides and amino acids. Simultaneously, the metabolic activity of Lactobacillus plantarum may also produce other bioactive substances, such as organic acids and bacteriocins, which may affect the structure and activity of peptides.

[0029] 4. Production and screening of tyrosinase inhibitory peptides:

[0030] During the fermentation process, the proteases of Lactobacillus plantarum degrade the proteins in kelp into a mixture of peptides of varying molecular weights. These peptides may contain peptides with tyrosinase inhibitory activity. Through centrifugation and ultrafiltration, peptides of varying molecular weights can be separated and their tyrosinase inhibitory activity measured.

[0031] Using modern biotechnological methods such as LC-MS / MS, we can identify the structure of the most inhibitory peptides and determine their amino acid sequences. Then, through molecular docking technology, we can screen for peptides that can bind to tyrosinase. These peptides are the target tyrosinase inhibitory peptides.

[0032] 5. Solid phase synthesis and product acquisition:

[0033] The peptides screened for tyrosinase binding can be synthesized using solid-phase synthesis technology to obtain high-purity tyrosinase inhibitory peptides. These inhibitory peptides have broad application prospects as potential raw materials in cosmetics, pharmaceuticals, or food additives.

[0034] The implementation of the present invention has the following beneficial effects:

[0035] The present invention utilizes Lactobacillus plantarum fermentation technology, which is simple and feasible, has low production costs, readily available equipment, and can release the active ingredients in kelp to the greatest extent. The active peptides 1 and 2 obtained by the present invention have strong tyrosinase inhibitory activity, a reasonable amino acid composition, are non-allergenic, safe and reliable, and can be added to foods and cosmetics as tyrosinase inhibitors, thus having good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a graph showing the results of investigating the tyrosinase inhibitory activity of polypeptides of different molecular weights according to Example 1 of the present invention.

[0037] Figure 2 This is a graph showing the experimental results of measuring the copper ion chelating ability of the kelp polypeptide according to Example 2 of the present invention.

[0038] Figure 3 This is a graph showing the experimental results of fluorescence quenching of tyrosinase treated with kelp polypeptides in Example 3 of the present invention.

[0039] Figure 4 This is a diagram showing the changes in shrimp treated with kelp polypeptide during refrigeration according to Example 4 of the present invention.

[0040] Figure 5 This is a graph showing the color difference changes of shrimp treated with kelp polypeptide during refrigeration according to Example 4 of the present invention.

[0041] Figure 6 This is a heat map of polypeptide abundance before and after kelp fermentation in Example 5 of the present invention.

[0042] Figure 7 This is a structural diagram of tyrosinase inhibitory peptide 1 according to Example 5 of the present invention.

[0043] Figure 8 This is a visualization diagram of the docking results of the tyrosinase inhibitory peptide 1 of Example 5 of the present invention to the tyrosinase crystal.

[0044] Figure 9 This is a structural diagram of tyrosinase inhibitory peptide 2 according to Example 5 of the present invention.

[0045] Figure 10 This is a visualization diagram of the docking results of the tyrosinase inhibitory peptide 2 of Example 5 of the present invention to the tyrosinase crystal.

[0046] Figure 11 This is the liquid chromatogram of tyrosinase inhibitory peptide 1 of Example 6 of the present invention.

[0047] Figure 12 This is the mass spectrum of tyrosinase inhibitory peptide 1 of Example 6 of the present invention.

[0048] Figure 13This is a graph showing the results of investigating the inhibitory activity of tyrosinase inhibitory peptide 1 according to Example 6 of the present invention.

[0049] Figure 14 This is the liquid chromatogram of tyrosinase inhibitory peptide 2 of Example 7 of the present invention.

[0050] Figure 15 This is the mass spectrum of tyrosinase inhibitory peptide 2 of Example 7 of the present invention.

[0051] Figure 16 This is a graph showing the results of investigating the inhibitory activity of tyrosinase inhibitory peptide 2 according to Example 7 of the present invention. DETAILED DESCRIPTION

[0052] In order to better understand the present invention, the present invention is further described in detail below with reference to the embodiments and drawings. However, those skilled in the art will understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] Example 1 Preparation of Laminaria polypeptide

[0056] The method of preparing kelp tyrosinase inhibitory peptide by fermentation with Lactobacillus plantarum described in this embodiment comprises the following steps:

[0057] Select intact kelp, add 10 times the volume of water to soak for 2 hours, remove the kelp after soaking, dry it at 60℃ for 8 hours, crush it with a high-speed grinder, pass it through a 60-mesh sieve to obtain kelp powder; prepare the fermentation raw material according to the ratio of 3g kelp powder and 100g pure water, then sterilize it at 121℃ for 15min, and cool it to room temperature to obtain the fermentation raw material; take glycerol-preserved Lactobacillus plantarum and inoculate it into MRS liquid culture medium with a sterile pipette at a ratio of 1:100, and incubate it at 37℃ and 180rpm. The fermentation product was obtained by culturing the fermentation material at a constant temperature for 24 hours under the following conditions: activating the fermentation material by adding the activated seed solution at a 7% inoculum ratio, placing the fermentation material at 37°C and shaking the flask at 180 rpm for 24 hours; the fermentation material and the fermentation product were centrifuged at 6000×g for 10 minutes, and the supernatant was ultrafiltered using 3kDa and 10kDa ultrafiltration tubes, respectively, to obtain polypeptides with molecular weights of <3kDa, 3-10kDa, and >10kDa, and their tyrosinase inhibitory activity was determined. The specific experimental method is as follows:

[0058] The experimental method for determining the tyrosinase inhibitory activity of different components is as follows: tyrosinase was diluted to 200 U / mL with PBS buffer (50 mM, pH = 6.8), 50 μL was added to a 96-well plate, and then 100 μL of sample solution was added. Then, 50 μL of levodopa was added simultaneously using a multichannel pipette. After shaking, the plate was incubated at room temperature for 20 minutes, and the absorbance at 475 nm was measured, with distilled water as the control.

[0059]

[0060] Where: A1 is the absorbance of the sample at 475 nm; A0 is the absorbance of distilled water instead of the sample at 475 nm; Ablank is the absorbance of distilled water instead of levodopa at 475 nm.

[0061] The results are as follows Figure 1 As shown, peptides with a molecular weight of less than 3 kDa have the highest inhibitory effect on tyrosinase activity.

[0062] Example 2 Determination of Copper Ion Chelating Ability of Kelp Polypeptides

[0063] To investigate the inhibitory mechanism of kelp polypeptides on tyrosinase, their ability to chelate copper ions was determined. The specific method was as follows: 0.2M HAc-NaAc buffer (pH = 3.75) was prepared, and CuCl2 solution and 2mM murexide solution were dissolved and prepared in the buffer. 0, 0.2, 0.4, 0.6, and 0.8mM CuCl2 solutions were added to a 96-well plate, shaken and mixed, and then 200μL of 2mM murexide solution was added. The absorbance was measured at 480nm and 540nm, and a copper ion concentration standard curve was plotted with A480 / A540 as the ordinate and copper ion concentration as the abscissa.

[0064] The specific detection method for the sample is as follows: a sample of a certain concentration is mixed with an equal volume of 2mmol / L CuCl2 solution and incubated at room temperature for 1 hour. 50μL of the reaction solution and 50μL of buffer are added to a 96-well plate and mixed thoroughly. Then, 200μL of 2mM murexide solution is added. After oscillation, the absorbance at 480nm and 540nm is measured. The equivalent copper ion concentration is expressed as A480 / A540, and the copper ion chelation rate is calculated using the following formula:

[0065]

[0066] Where: [Cu 2+ ] is the total copper ion concentration; [Cu free 2+ ] is the free copper ion concentration.

[0067] Copper ion chelation experiments confirmed that kelp polypeptide chelates Cu 2+Concentration-dependent, such as Figure 2 As shown, it shows that kelp polypeptide can directly destroy the copper ion cluster structure in the active center of tyrosinase, thereby causing tyrosinase to lose activity.

[0068] Example 3 Fluorescence Quenching Effect of Laminaria Japonica Polypeptide on Tyrosinase

[0069] Add 400 μL of 250 U / mL tyrosinase to a centrifuge tube, followed by 0, 40, 80, 120, 160, and 200 μL of the 50 mg / mL fermentation sample, sequentially. Vortex to mix thoroughly, then add PBS to bring the total reaction volume to 1 mL. Vortex to mix thoroughly, then incubate at room temperature for 30 minutes. Excite fluorescence at 280 nm, and scan the emission wavelength from 360 to 420 nm with a 2 nm step size.

[0070] Fluorescence quenching experiments showed that Figure 3 As shown, tyrosinase exhibits a maximum absorption peak at 378nm. The intrinsic fluorescence of tyrosinase originates from chromophore residues such as tryptophan in its molecular structure. When the hydrophobicity or conformation of tyrosinase changes, it will affect the microenvironment in which these chromophores are located, resulting in a decrease in fluorescence intensity or a red-shift or blue-shift in the maximum absorption peak. As the concentration of kelp polypeptide increases, the intrinsic fluorescence intensity of tyrosinase gradually decreases, suggesting that kelp polypeptide is likely to interact with the fluorescent chromophore of tyrosinase. At the same time, the maximum absorption peak was observed to be red-shifted to 380nm, indicating that the addition of kelp polypeptide has changed the electronic structure and spectral characteristics of tyrosinase. This change may be caused by multiple interactions between kelp polypeptide and tyrosinase. For example, kelp polypeptide may bind to tyrosinase through hydrophobic interactions and hydrogen bonds, and may also bind to Cu in tyrosinase. 2+ The results of the copper ion chelation experiments above show that these interactions, combined, alter the charge distribution within the tyrosinase molecule, thereby affecting its electronic transitions and ultimately leading to changes in its spectral properties, manifested as a red shift in the maximum absorption peak.

[0071] Example 4 Application of Kelp Polypeptide in the Anti-Melanosis Process of Shrimp during Refrigeration

[0072] Fresh prawns were randomly divided into three groups and immersed in deionized water (control) and kelp peptides for 15 minutes, respectively. After immersion, the prawns were drained, placed in sterile plastic bags, and stored at 4°C for 6 days. The prawns were photographed daily. The yellow-blue intensity (b*) of the prawn abdomen was measured using an automatic colorimeter (WSC-S, Shanghai Precision Scientific Instrument Co., Ltd.). This value correlates well with the freshness of the prawns. Three samples were taken from each group for measurement.

[0073] like Figure 4 、 5As shown, compared with the control group, kelp polypeptide-treated shrimps can inhibit the melanization of shrimps and delay the melanization of shrimps to a certain extent.

[0074] Example 5 Identification and Screening of Tyrosinase Inhibitory Peptides

[0075] LC-MS / MS was used to structurally identify polypeptides <3 kDa. The specific identification process was as follows: the polypeptides <3 kDa were loaded into a Nano Aquity UPLC system (Waters Corp) and eluted through an analytical column C18 reverse-phase column (2 μm, 75 μm × 25 cm). The elution procedure was: solution A 0.1% formic acid / water, solution B 0.1% formic acid / acetonitrile, instrument column temperature: 40°C; flow rate: 0.4 mL / min; injection volume: 4 μL. Gradient elution program: 0–2 min, 95%–80% A; 2–5 min, 80%–40% A; 5–6 min, 40%–1% A; 6–7.5 min, 1% A; 7.5–7.6 min, 1%–95% A; and 7.6–10 min, 95% A. Mass spectrometry was performed using a ThermoFisher Q Exactive system (ThermoFisher, USA) coupled with a nanospray NanoFlex ion source (ThermoFisher, USA). The spray voltage was 1.9 kV, and the ion transfer tube was heated at 275°C. The molecular mass and amino acid sequence of the peptides were determined by primary and secondary mass spectrometry, respectively. The MS / MS raw files were analyzed using the pFind search engine. The primary structure of the peptides was determined by searching the UniProt protein database and matching the peptide sequences. In order to more intuitively display the characteristics and changing patterns of peptide-related data as well as the differences in relative abundance of peptides before and after fermentation, we normalized the data to relative content (Z-score) and used heatmaps, an effective data analysis and visualization method. Figure 6 As shown in the figure, after fermentation, the contents of various polypeptides including YRL and LPD increased significantly.

[0076] Molecular docking was performed using the open-source software AutoDock Vina to screen for tyrosinase inhibitory peptides. The specific process was as follows: First, the crystal structure of tyrosinase (PDB ID: 2y9x) was downloaded from the PDB database. The A chain and the two copper ions in the active center were retained. All water molecules and other ligands were deleted using PyMOL software, and the structure was saved in pdbqt format after hydrogenation and other operations. Then, the molecular structure of the peptide was drawn using Chemdraw 22.0 software, converted into a 3D structure using Chem3D (MM2 force field optimization), hydrogenated using AutoDock Tools software, and the torsion center and torsion bond were detected, and the structure was saved in pdb format. Finally, molecular docking simulations were performed using Autodock Vina software: the docking center coordinates were set to (-7.456, -23.653, -32.538) (x, y, z), the box size was set to 60 × 60 × 70, the grid spacing was set to 1 Å, and other parameters were kept as default values. The interactions were displayed using PyMOL and LigPlus software.

[0077] After molecular docking, two peptides were screened. One is a peptide with the amino acid sequence YRL, named tyrosinase inhibitory peptide 1, and its structure is as follows Figure 7 The docking results are shown in Figure 8 As shown, its binding energy with tyrosinase is -6.7kcal / mol, its molecular weight is 450.53Da, and its charge is +1; the other is a polypeptide with an amino acid sequence of LPD, named tyrosinase inhibitory peptide 2, its amino acid sequence is LPD, and its structure is as shown Figure 9 The docking results are shown in Figure 10 As shown, its binding energy with tyrosinase is -6.2 kcal / mol, its molecular weight is 343.38 Da, and its charge is +1. This indicates that the two polypeptides have a good binding effect with tyrosinase (2y9x), theoretically indicating that the peptide has the ability to tightly bind to the active center of tyrosinase.

[0078] Example 6 Synthesis and Activity Verification of Tyrosinase Inhibitory Peptide 1

[0079] 1. Synthesis of Tyrosinase Inhibitory Peptide 1

[0080] The peptides screened were synthesized by solid phase synthesis. Shanghai Jier Biochemical Co., Ltd. synthesized the peptides and purified them by high performance liquid chromatography to obtain a peptide with a purity of more than 95%. The peptides were identified by LC-MS / MS. Figure 11 、 12 shown.

[0081] 2. Activity Evaluation of Tyrosinase Inhibitory Peptide 1

[0082] According to the aforementioned tyrosinase inhibition activity test, the tyrosinase inhibition activity of the synthesized peptides was evaluated. The peptide concentration at which the inhibition rate was 50% was calculated as IC 50 Value. Figure 13 As shown, the IC of tyrosinase inhibitory peptide 1 50 The value was 1.427 mg / mL, indicating that it had a strong inhibitory effect on tyrosinase activity.

[0083] Example 7 Synthesis and Activity Verification of Tyrosinase Inhibitory Peptide 2

[0084] 1. Synthesis of tyrosinase inhibitory peptides

[0085] The peptides screened were synthesized by solid phase synthesis. Shanghai Jier Biochemical Co., Ltd. synthesized the peptides and purified them by high performance liquid chromatography to obtain a peptide with a purity of more than 95%. The peptides were identified by LC-MS / MS. Figure 14 、 15 shown.

[0086] 2. Activity Evaluation of Tyrosinase Inhibitory Peptides

[0087] According to the aforementioned tyrosinase inhibition activity test, the tyrosinase inhibition activity of the synthesized peptides was evaluated. The peptide concentration at which the inhibition rate was 50% was calculated as IC 50 Value. Figure 16 As shown, the IC of tyrosinase inhibitory peptide 2 50 The value was 0.106 mg / mL, indicating that it had a strong inhibitory effect on tyrosinase activity.

[0088] In summary, the present invention pre-treats kelp and then ferments it using Lactobacillus plantarum. The resulting fermented liquid is subjected to centrifugation, ultrafiltration, and component identification to obtain the composition of kelp polypeptides. Tyrosinase inhibitory peptides are then screened by molecular docking, and finally synthesized by solid phase synthesis to obtain the tyrosinase inhibitory peptides obtained by the present invention. The present invention uses Lactobacillus plantarum fermentation technology, which is simple and feasible, has low production cost, is easy to obtain equipment, and can release the active ingredients in kelp to the greatest extent. The obtained polypeptide has the effect of inhibiting tyrosinase activity and can be used to develop tyrosinase inhibitors.

[0089] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A tyrosinase inhibitory peptide 1 derived from Laminaria japonica, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A tyrosinase inhibitory peptide 2 derived from Laminaria japonica, the amino acid sequence of which is shown in SEQ ID NO:

2.

3. Use of the tyrosinase inhibitory peptide 1 according to claim 1 in the preparation of a tyrosinase inhibitor.

4. Use of the tyrosinase inhibitory peptide 2 according to claim 2 in the preparation of a tyrosinase inhibitor.

5. A food, characterized in that: The active ingredient comprises the tyrosinase inhibitory peptide 1 according to claim 1.

6. A food, characterized in that: The effective ingredient includes the tyrosinase inhibitory peptide 2 according to claim 2.

7. A cosmetic, characterized in that: The active ingredient comprises the tyrosinase inhibitory peptide 1 according to claim 1.

8. A cosmetic, characterized in that: The effective ingredient includes the tyrosinase inhibitory peptide 2 according to claim 2.

9. A method for preparing a tyrosinase inhibitory peptide derived from Laminaria japonica, characterized in that: The steps include: S1. Select intact and undamaged kelp, soak, dry, crush and sieve to obtain kelp powder, prepare liquid fermentation raw materials, and sterilize and cool; S2 activated Lactobacillus plantarum seed solution, inoculated into the liquid fermentation raw material sterilized and cooled in step S1 for fermentation to obtain a fermentation product, the fermentation product was centrifuged to obtain a supernatant; S3. ultrafiltration of the supernatant in step S2 to obtain polypeptides of different molecular weights, and determination of their tyrosinase inhibitory activity; S4. Structural identification of the peptide with inhibitory activity in step S3 using LC-MS / MS; S5. The polypeptide identified in step S4 is subjected to molecular docking with tyrosinase to screen polypeptides capable of binding to tyrosinase, and the screened polypeptides are subjected to solid phase synthesis to obtain tyrosine inhibitory peptides derived from kelp.

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