Anti-photoaging damage peptide and preparation method thereof

Abstract

The invention belongs to the technical field of protein, and relates to an anti-photoaging damage peptide and a preparation method thereof. The amino acid sequence of the peptide is KSYELPDGQVITI, and the key active fragment of the peptide is ELPDGQVIT. The peptide is obtained by performing enzymolysis on degreased maggot powder with alkaline protease, trypsin, papain, neutral protease and flavourzyme. The peptide can remove ROS free radicals after UVA irradiation, improve SOD activity and inhibit MDA generation, and finally alleviate oxidative stress caused by UVA.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein, and relates to an anti-photoaging damage peptide and a preparation method thereof. BACKGROUND

[0002] Skin, as the largest organ of the human body, can affect the body's dynamic balance, and also plays a role in protecting the internal environment from harmful external factors. Studies have shown that long-term exposure to ultraviolet (UV) radiation can cause skin photoaging, which is an important cause of skin damage. It can reduce the skin's defense ability, which is not conducive to the skin's resistance to harmful external stimuli. It can also cause various skin diseases, including solar keratosis, photoelastic fiberization, melanoma, basal cell carcinoma, etc. This chronic skin damage not only affects the appearance of the human body, causing a certain psychological burden, but also has an adverse effect on physical health.

[0003] A large number of reactive oxygen species (ROS) free radicals induced by ultraviolet radiation have been proven to be the most critical factor causing skin photoaging. It can undergo peroxidation with unsaturated fatty acids in the cell membrane to produce malondialdehyde (MDA), further damaging nucleic acids, proteins, phospholipids, and other macromolecular substances, changing the permeability of the cell membrane, and causing cell apoptosis. At the same time, ROS free radicals can also attack endogenous antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and the synthesis-related signal pathway Kelch-like ECH-associated protein 1 (Keap1) / nuclear factor erythroid 2-related factor 2 (Nrf2), thereby inhibiting the activity and production of antioxidant enzymes, causing an imbalance in the body's oxidation / antioxidation defense system, oxidative stress, and serious oxidative damage to the body.

[0004] Studies have shown that specific protein hydrolysates and peptide substances, such as bovine milk peptides, Hupu pearl oyster peptides, and Zhanjiang chrysomonas peptides, have been proven to be able to scavenge ROS free radicals, promote the synthesis and activity of antioxidant enzymes, and play a role in relieving oxidative stress and inhibiting skin photoaging. Therefore, in-depth research and development of peptide substances that can inhibit the production of ROS free radicals and MDA, improve SOD activity, and activate the Keap1 / Nrf2 signal pathway may provide a new strategy for reducing oxidative stress, which in turn helps to reduce skin photoaging.

[0005] In the clinical practice of alleviating skin photoaging, drug therapy is still the main way, mainly including topical application of vitamin A, 5-fluorouracil cream and ointment containing antioxidants or alpha-hydroxy acid. These drugs have been proved to have good effect in alleviating skin photoaging, but at the same time, they are accompanied by certain side effects, such as skin dryness, peeling, erythema, itching, increased skin photosensitivity, etc. If long-term or improper use, it may aggravate these adverse reactions, including skin irritation symptoms, pigment changes, telangiectasia, increased risk of secondary infection, etc. In view of the potential risks of drug and surgical treatment, nutritional intervention as a complementary treatment strategy is more and more favored because of its lower side effects and higher patient compliance. In the field of nutritional intervention, food-derived bioactive peptides have become the focus of research due to their potential benefits in dietary supplements. Five grain worms are traditional Chinese medicines, which have the effect of inhibiting skin ulceration. However, the research on five grain worms in anti-photoaging is still unclear, and there is a lack of effective preparation process and screening method to identify peptides with anti-photoaging potential, which limits its application in improving skin photoaging after oral administration. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a peptide with anti-photoaging damage effect and its preparation method, to provide a choice for alleviating skin photoaging.

[0007] In one aspect, the present application provides an anti-photoaging damage peptide, the amino acid sequence of which is KSYELPDGQVITI.

[0008] In an embodiment of the present application, the key active fragment of the anti-photoaging damage peptide is ELPDGQVIT.

[0009] In another aspect, the present application provides an anti-photoaging damage composition comprising the anti-photoaging damage peptide KSYELPDGQVITI.

[0010] In a third aspect, the present application provides the use of the anti-photoaging damage peptide in the preparation of an anti-photoaging damage drug.

[0011] In a fourth aspect, the present application provides a method for preparing the anti-photoaging damage peptide KSYELPDGQVITI, comprising the following steps:

[0012] 1) Defatted five grain worm powder is dissolved in water, and incubated at 80-90℃ for 10-20min;

[0013] 2) The pH of the defatted five grain worm powder solution obtained in step 1) is adjusted to 8.0, alkaline protease is added and hydrolyzed at 45-55℃ for 1.5-2.5h to obtain an enzyme hydrolysate;

[0014] 3) adjusting the pH of the enzymatic solution of step 2) to 8.0, adding trypsin and enzymolysis at 35-42°C for 1 h; or adjusting the pH of the enzymatic solution to 7.5, adding papain, and enzymolysis at 45-55°C for 0.5-1.5 h; or adjusting the pH of the enzymatic solution to 7.0, adding neutral protease, and enzymolysis at 45-55°C for 0.5-1.5 h;

[0015] 4) adjusting the pH of the enzymatic solution obtained in step 3) to 7.5, adding flavor protease and enzymolysis at 45-55°C for 0.5-1.5 h;

[0016] 5) vacuum freeze-drying the enzymatic solution obtained in step 4) to obtain a powdered enzymatic product, and then identifying the peptides in the enzymatic product to obtain the anti-photoaging damage peptide KSYELPDGQVITI.

[0017] In step 1), the incubation temperature can be 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, or 80°C, and the incubation time can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min.

[0018] In step 2), the enzymolysis temperature can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C, and the enzymolysis time can be 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2.0 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, or 2.5 h.

[0019] In step 3), for trypsin, the enzymolysis temperature can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C; for papain or neutral protease, the enzymolysis temperature can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C; and the enzymolysis time can be 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, or 1.5 h.

[0020] In step 4), the enzymolysis temperature can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C, and the enzymolysis time can be 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, or 1.5 h.

[0021] In an embodiment of the present application, the mass ratio of defatted five-grain worm powder to alkaline protease is 50:1.

[0022] In an embodiment of the present application, the mass ratio of defatted five-grain worm powder to trypsin is 125:1.

[0023] In an embodiment of the present application, the mass ratio of defatted five-grain worm powder to papain is 390:1.

[0024] In an embodiment of the present application, the mass ratio of defatted five-grain worm powder to neutral protease is 50:1.

[0025] In an embodiment of the present application, the mass ratio of defatted five-grain worm powder to flavor protease is 10:1.

[0026] In an embodiment of the present application, after each enzymatic reaction is completed, a step of heating the enzymatic solution in boiling water to terminate the reaction can be included. In a specific embodiment, the enzymatic reaction can be terminated by boiling the enzymatic solution in boiling water.

[0027] The method of the fourth aspect of the present application can be referred to as a step-by-step enzymatic method.

[0028] In a fifth aspect, the present application provides a method for preparing an anti-photoaging damage peptide, comprising the following steps:

[0029] 1) defatted five-grain worm powder is dissolved in water and incubated at 80-90°C for 10-20 min;

[0030] 2) the pH of the solution obtained in step 1) is adjusted to 7.5, and alkaline protease, trypsin, papain or neutral protease, and flavor protease are added, and enzymatic reaction is carried out at 40-50°C for 3-5 h;

[0031] 3) the enzymatic solution obtained in step 2) is subjected to vacuum freeze-drying treatment to obtain a powdered enzymatic product, and then the peptides in the enzymatic product are identified to obtain the anti-photoaging damage peptide KSYELPDGQVITI.

[0032] In an embodiment of the present application, in step 1), the incubation temperature can be 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or 80°C, and the incubation time can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0033] In an embodiment of the present application, in step 2), the enzymolysis temperature can be 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃ or 50℃, and the enzymolysis time can be 3h, 3.1h, 3.2h, 3.5h, 3.6h, 3.7h, 3.8h, 3.9h, 4.0h, 4.1h, 4.2h, 4.3h, 4.4h, 4.5h, 4.6h, 4.7h, 4.8h, 4.9h or 5.0h.

[0034] In an embodiment of the present application, the mass ratio of defatted maggot powder to alkaline protease is 50:1, and the mass ratio to trypsin, papain and neutral protease is 125:1, 390:1 and 50:1 respectively, and the mass ratio to flavor protease is 10:1.

[0035] Alternatively, in an embodiment of the present application, the mass ratio of alkaline protease:trypsin:flavor protease is 5:1:12.5, the mass ratio of alkaline protease:papain:flavor protease is 15.625:1:39.0625, and the mass ratio of alkaline protease:neutral protease:flavor protease is 2:1:5.

[0036] In an embodiment of the present application, after step 2), a step of heating the enzymolysis solution in boiling water to terminate the reaction can be included.

[0037] The method of the fifth aspect of the present application can be referred to as simultaneous enzymolysis method.

[0038] The anti-photoaging ability of the peptide KSYELPDGQVITI of the present application can be derived from the active fragment ELPDGQVIT, and its efficacy mainly manifests in that it can clear ROS free radicals in L929 cells after UVA irradiation, improve SOD activity and inhibit MDA production, and at the same time, it can spontaneously bind to the Keap1 receptor to form a compact and stable conformation, which is conducive to the activation of the Keap1 / Nrf2 signaling pathway and the promotion of SOD synthesis, and ultimately relieves the oxidative stress caused by UVA. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Process flow diagrams of the step-by-step enzymolysis method (A) and the simultaneous enzymolysis method (B) are shown.

[0040] Figure 2 The types and contents of amino acids constituting the maggot anti-photoaging peptide are shown. Figure 2 A represents the total content of different characteristic amino acids in the peptide chain; Figure 2 B represents the content of different characteristic amino acids at the 1st and 2nd positions at the C-terminal and the 1st and 2nd positions at the N-terminal of the peptide chain; Figure 2C represents the content of 20 kinds of amino acid residues at the first and second positions of the C-terminal of the peptide chain and the first and second positions of the N-terminal of the peptide chain.

[0041] Figure 3 The figure shows the affinity energy diagram of different peptide segments docking with the Keap1 receptor molecule.

[0042] Figure 4 The figure shows the effect of the A→T→F group of anti-photoaging peptides (A), P1 peptide segment (B) and P2 peptide segment (C) on the viability of L929 cells.

[0043] Figure 5 The figure shows the effect of the A→T→F group of anti-photoaging peptides, P1 peptide segment and P2 peptide segment on the ROS free radical level, SOD activity and MDA content in L929 cells. Figure 5 A represents the ROS free radical content in cells; Figure 5 B represents the SOD activity in cells; Figure 5 C represents the MDA content in cells.

[0044] Figure 6 The figure shows the effect of P1 and P2 peptide segments on the protein expression level of the Keap1 / Nrf2 signaling pathway in L929 cells. Figure 6 A represents the Western Blot visualization figure of the Keap1 / Nrf2 signaling pathway and the internal reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Figure 6 B represents the protein expression level of Keap1; Figure 6 C represents the protein expression level of Nrf2.

[0045] Figure 7 The figure shows the visualization figure of the results of P1 peptide segment (A) and P2 peptide segment (B) after molecular docking with the Keap1 receptor protein.

[0046] Figure 8 The figure shows the result figure of P1 and P2 peptide segments during the molecular dynamics simulation binding process with the Keap1 receptor protein. Figure 8 A represents the root mean square deviation of the Keap1 receptor protein, Keap1-P1 and Keap1-P2 complex; Figure 8 B represents the root mean square fluctuation of the Keap1 receptor protein, Keap1-P1 and Keap1-P2 complex; Figure 8 C represents the gyration radius of the Keap1-P1 and Keap1-P2 complex; Figure 8 D represents the solvent accessible surface area of the Keap1-P1 and Keap1-P2 complex. DETAILED DESCRIPTION

[0047] The application will be further described below in conjunction with the drawings and specific embodiments. It should be noted that the following described embodiments or technical features can be combined in any manner to form new embodiments without conflict.

[0048] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained through commercial channels unless otherwise specified.

[0049] The medium formula used in the following examples is as follows:

[0050] 1) Basic medium: purchased from Gibco Company, USA.

[0051] 2) Complete medium: 10 mL of heat-inactivated horse serum, 5 mL of penicillin-streptomycin, 5 mL of non-essential amino acids, 5 mL of sodium pyruvate and 475 mL of basic medium were thoroughly mixed together to obtain the complete medium. The reagents used in the complete medium were purchased from Gibco Company, USA.

[0052] 3) Medium containing A→T→F group, A→P→F group, A→N→F group, A+T+F group, A+P+F group and A+N+F group of five insect peptides: 5 mg of five insect peptides was weighed in a 10 mL centrifuge tube, 5 mL of basic medium was added thereto and the five insect peptides were thoroughly dissolved to obtain the medium containing the five insect peptides.

[0053] 4) Medium containing peptide segments P1 and P2: 2 mg of peptide segments P1 and P2 was weighed in a 5 mL centrifuge tube, 4 mL of basic medium was added thereto and the peptide segments P1 and P2 were thoroughly dissolved to obtain the medium containing the peptide segments P1 and P2.

[0054] Example 1. Preparation method of anti-photoaging damage peptide KSYELPDGQVITI (stepwise enzymatic hydrolysis method)

[0055] This example uses a stepwise enzymatic hydrolysis method to prepare an anti-photoaging damage peptide, and the specific operation is as follows:

[0056] In the step-by-step enzymatic hydrolysis method, the sample was divided into 3 groups, each group weighed 3.4 g of defatted mealworm powder (the protein content in the defatted mealworm powder was about 2.5 g), and 50 mL of distilled water was added. The solution was incubated at 85°C for 15 min. Then, the pH of the solution was adjusted to 8.0, 0.025 g of alkaline protease was added, and the solution was incubated at 50°C for 2 h. After the treatment, the enzymatic solution was heated in boiling water for 10 min to terminate the enzymatic reaction. Then, the pH of the solution was adjusted to 8.0, 7.5, and 7.0, respectively, 0.005 g of trypsin, 0.0016 g of papain, and 0.0125 g of neutral protease were added, respectively, and the solution was enzymatically hydrolyzed at 37°C, 50°C, and 50°C, respectively, for 1 h. After that, the enzymatic solution was heated in boiling water for 10 min to terminate the reaction. Subsequently, the pH of the solution was adjusted to 7.5, 0.0625 g of flavor protease was added, and the solution was reacted at 50°C for 1 h. After the reaction, the enzymatic solution was heated in boiling water for 10 min to terminate the reaction. Finally, the enzymatic solution was placed at -40°C and vacuum freeze-dried for 48 h to obtain the mealworm peptides prepared by the step-by-step enzymatic hydrolysis method. They were step-by-step enzymatic hydrolysis group 1: alkaline protease→trypsin→flavor protease (A→T→F); step-by-step enzymatic hydrolysis group 2: alkaline protease→papain→flavor protease (A→P→F); and step-by-step enzymatic hydrolysis group 3: alkaline protease→neutral protease→flavor protease (A→N→F). The proteases used in this method were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China, Shanghai), and the enzyme activities of the five proteases were as follows: the enzyme activity of alkaline protease was 2×10 5 U / g (S10154), the enzyme activity of trypsin was 2.5×10 5 U / g (S10032), the enzyme activity of papain was 8×10 5 U / g (S10011), the enzyme activity of neutral protease was 1×10 5 U / g (S10013), and the enzyme activity of flavor protease was 2×10 4 U / g (S10153).

[0057] The specific process is shown as A in Figure 1 .

[0058] Example 2. Preparation method of anti-photoaging damage peptide KSYELPDGQVITI (simultaneous enzymatic hydrolysis method)

[0059] In this embodiment, the simultaneous enzymatic hydrolysis method was used to prepare the anti-photoaging damage peptide, and the specific operation was as follows:

[0060] In the simultaneous enzymatic hydrolysis method, the sample was divided into 3 groups, 3.4 g of defatted mealworm powder (protein content in defatted mealworm powder is about 2.5 g) was weighed, 50 mL of distilled water was added, and the solution was incubated at 85°C for 15 min. Then, the pH of the solution was adjusted to 7.5, and 0.025 g of alkaline protease, 0.005 g of trypsin, 0.0625 g of flavor protease; 0.025 g of alkaline protease, 0.0016 g of papain, 0.0625 g of flavor protease; 0.025 g of alkaline protease, 0.0125 g of neutral protease, 0.0625 g of flavor protease were added to each group, respectively, and enzymatic hydrolysis was carried out at 45°C for 4 h. After incubation was completed, the enzymatic hydrolysate was heated in boiling water for 10 min to terminate the reaction. Finally, the enzymatic hydrolysate was placed at -40°C and vacuum freeze-dried for 48 h to obtain the mealworm peptides by simultaneous enzymatic hydrolysis. They were simultaneous enzymatic hydrolysis group 1: alkaline protease + trypsin + flavor protease (A + T + F); simultaneous enzymatic hydrolysis group 2: alkaline protease + papain + flavor protease (A + P + F); simultaneous enzymatic hydrolysis group 3: alkaline protease + neutral protease + flavor protease (A + N + F). The proteases used in this method were purchased from Shanghai Yuan Ye Biological Technology Co., Ltd. (China, Shanghai), and the enzyme activities of the 5 proteases were as follows: the enzyme activity of alkaline protease was 2 x 10 5 U / g (S10154), the enzyme activity of trypsin was 2.5 x 10 5 U / g (S10032), the enzyme activity of papain was 8 x 10 5 U / g (S10011), the enzyme activity of neutral protease was 1 x 10 5 U / g (S10013), and the enzyme activity of flavor protease was 2 x 10 4 U / g (S10153).

[0061] The specific process is shown in B of Figure 1 .

[0062] Example 3. Screening of Anti-photoaging Peptides and Key Active Fragments

[0063] In this example, anti-photoaging peptides and their key active fragments were screened by a photoaged L929 cell model. The L929 mouse fibroblasts used in this example were provided by the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (China, Beijing).

[0064] In this example, the process of screening anti-photoaging peptides is as follows:

[0065] First, the effect of mealworm peptides on the migration ability of photoaged cells was determined according to the method of Ribeiro et al. [1] . L929 cells were seeded at 1.9 x 10 5The cells were inoculated in a 6-well plate containing complete medium (containing 10% heat-inactivated horse serum (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), 1% non-essential amino acids (Gibco, USA), 1% sodium pyruvate (Gibco, USA), and 87% basal medium (Gibco, USA)) at a density of 1.8 x 10

[0066] Then, the effect of the Wuguying peptide on the intracellular ROS free radical level was determined using the fluorescent probe 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beijing Gaoke Bridge Biological Technology Co., Ltd.). The L929 cells were inoculated in a 6-well plate containing complete medium at a density of 1.8 x 10 5 The cells were inoculated in a 6-well plate containing complete medium at a density of 1.8 x 10

[0067] Then, the effect of the Wuguying peptide on the intracellular ROS free radical level was determined using the fluorescent probe 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beijing Gaoke Bridge Biological Technology Co., Ltd.). The L929 cells were inoculated in a 6-well plate containing complete medium at a density of 1.8 x 10 5The cells were seeded at a density of 100 cells / mL in a 6-well plate containing complete medium and incubated in an incubator for 24 hours. When the cell density reached 80%, the complete medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 minutes. After irradiation, different groups were added with basal medium and basal medium containing five-grain insect peptides and incubated for another 24 hours. After the incubation was completed, the solution was removed and 4°C RIPA lysis buffer (Beijing Solebold Technology Co., Ltd.) was added to lyse the cells at 4°C for 1 hour. After the end, the cells were centrifuged at 12,000 g for 5 minutes at 4°C and the supernatant was collected. Finally, the protein concentration was determined using a BCA protein concentration detection kit (Beijing Solebold Technology Co., Ltd.), and the SOD activity and MDA content were determined using a SOD assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) and an MDA assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.).

[0068] Finally, referring to Liu et al. [2] The effect of five-grain insect peptide on the collagen metabolism level of L929 cells was determined by the method of 5 The cells were seeded at a density of 10 cells / mL in a 6-well plate containing complete medium and incubated in an incubator for 24 hours. When the cell density reached 80%, the complete medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 minutes. After irradiation, the basal medium and the basal medium containing five-grain insect peptide were added to the different groups and incubated for another 24 hours. Subsequently, the method of the literature was followed. [2]The sample solution was collected and the protein concentration was unified. Then, the protein (25 μg) in the sample solution was separated on a 10% separation gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The PVDF membrane was blocked with 5% skim milk (BD-Difco, USA) at room temperature for 2 h, and then incubated with diluted specific first antibodies, including matrix metalloproteinases (MMP) 1 / 3 / 9, collagen type I (COL1) and GAPDH overnight at 4°C (MMP1 rabbit polyclonal antibody and MMP9 rabbit polyclonal antibody were purchased from Wuhan Synergy Biotech Co., Ltd.; MMP3 rabbit polyclonal antibody, COL1A1 rabbit polyclonal antibody and GAPDH monoclonal antibody were purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.). The volume ratio of MMP1 to Western primary antibody diluent (Shanghai Biyun Tian Biotechnology Co., Ltd.) was 1:4000; the volume ratio of MMP3 to Western primary antibody diluent was 1:2000; the volume ratio of MMP9 to Western primary antibody diluent was 1:3000; the volume ratio of COL1 to Western primary antibody diluent was 1:1000; and the volume ratio of GAPDH to Western primary antibody diluent was 1:5000. Then, the PVDF membrane was incubated with diluted secondary antibody horseradish peroxidase-labeled goat anti-rabbit (IgG (H+L)) at room temperature for 2 h. The volume ratio of IgG (H+L) to Western secondary antibody diluent (Shanghai Biyun Tian Biotechnology Co., Ltd.) was 1:2000. Finally, the method in the literature was referred to [2] The expression amount of the target protein was determined.

[0069] It was found that the A→T→F group of the stepwise enzymatic hydrolysis group had a strong potential for anti-photoaging. It showed the strongest effect in promoting L929 cell migration and scavenging ROS free radicals, and also had a strong effect in inhibiting MDA production, MMP1 / 3 / 9 expression and promoting COL1 expression and SOD activity. Therefore, the A→N→F group was selected as the anti-photoaging peptide of the five insects for peptide identification.

[0070] From Figure 2 A-C, it can be seen that the C-terminal and N-terminal of the A→N→F group of the anti-photoaging peptide of the five insects are rich in hydrophobic and charged amino acids, respectively. Studies have shown that [3] such amino acid distribution characteristics are the key to the anti-photoaging effect of bioactive peptides. Therefore, this structural feature of the anti-photoaging peptide of the five insects may also provide it with high biological activity.

[0071] Next, the key active fragments screened from the anti-photoaging peptides of the five grain insects were identified by mass spectrometry. As shown in Table 1, among the identified peptide segments, a repeatedly occurring parent peptide segment ELPDGQVIT was found, and its amino acid composition and distribution characteristics were consistent with the structural characteristics of the anti-photoaging peptides. Therefore, it was considered that the peptide ELPDGQVIT might be a key parent fragment for exerting anti-photoaging activity. The identified peptide segment was subjected to molecular docking with the Keap1 receptor, the structure of Keap1 (PDB ID: 2FLU) was derived from the RCSB PDB protein database (https: / / www.rcsb.org / ), and the structure of the peptide was derived from the PEP-FOLD4 polypeptide structure prediction website (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD4 / ). The AutoDockTools 1.5.6 was used for water removal and hydrogenation treatment of Keap1 and the peptide segment. The center coordinates of Keap1 were set as: x = 8.5, y = 10.1, z = 1.8; and the size of the docking box was: Subsequently, molecular docking was performed using AutoDockVina to obtain the binding energies of different peptide segments with Keap1, and the results are shown in Figure 3 The affinity energies of the 17 peptide segments with the Keap1 receptor were all negative values, indicating that the two could spontaneously bind. Among them, the affinity energies of YLPGSAPCR, GIPPAPR, VDSVLDVVRK, TEAPLNPK, SYELPDGQVITIG, and KSYELPDGQVITI were lower, being -7.4, -7.1, -6.8, -6.7, -6.7, and -6.5 kcal / mol, respectively. At the same time, the molecular weight, isoelectric point, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, and total average hydrophilic / hydrophobicity (GRAVY) of the peptide segments were analyzed using the ExpasyProtParam online database (https: / / web.expasy.org / protparam / ). The results are shown in Table 1. Finally, the peptide segments were screened in combination with the physicochemical properties, structural characteristics, affinity energy size, and content, and the peptides KSYELPDGQVITI (referred to as P1) and the potential active fragment ELPDGQVIT (referred to as P2) appearing multiple times were selected for subsequent verification.

[0072] Table 1. Analysis of physicochemical properties of different peptide segments

[0073]

[0074]

[0075] Example 4. Activity evaluation of the anti-photoaging damage peptide KSYELPDGQVITI

[0076] 4.1 Evaluation of the effects of A→T→F group of five insects anti-photoaging peptides and P1, P2 peptide segments on L929 cell viability

[0077] In this embodiment, the effects of A→T→F group of five insects anti-photoaging peptides and P1, P2 peptide segments on L929 cell viability will be evaluated by cell counting kit-8 (CCK-8). CCK-8 was purchased from Beijing High Bridge Biotechnology Co., Ltd.

[0078] 100 μL of L929 cells (provided by the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were inoculated in a 96-well plate containing complete culture medium (containing 10% heat-inactivated horse serum, 1% penicillin-streptomycin, 1% non-essential amino acids, 1% sodium pyruvate and 87% basal medium) at a density of 1.5 x 10 5 The cells were incubated in an incubator for 24 h. When the cell density was 80%, the complete culture medium was discarded and different concentrations of basal medium containing A→T→F group of five insects anti-photoaging peptides (0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL) or P1, P2 peptide segments (0.03125 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL) were added for further incubation for 24 h. After incubation, the basal medium containing A→T→F group of five insects anti-photoaging peptides, P1 peptide segment and P2 peptide segment was removed and the cells were washed with PBS solution, 100 μL of 10% CCK-8 solution was added, incubated for 1 h and then the absorbance was measured at 450 nm using a microplate reader. The results are shown in A-C of Figure 4 As shown in A-C of the results, A→T→F group of five insects anti-photoaging peptides had no significant damage to L929 cells in the range of 0.1-10 mg / mL, and the cell viability was greater than 80%. When the concentration of A→T→F group of anti-photoaging peptides was 1 mg / mL, the cell viability was higher than that of the blank group (P>0.05), indicating that the anti-photoaging peptides at this concentration had a certain promoting effect on cell proliferation. Therefore, 1 mg / mL of A→T→F group of anti-photoaging peptides was selected for subsequent experiments. For P1 and P2 peptide segments, in the range of 0.03125-1 mg / mL, they had no toxicity to L929 cells, while when the concentration was 0.5 mg / mL, the cell viability of P1 and P2 groups was higher than that of the blank group (P<0.05), indicating that this concentration promoted cell growth. Moreover, the cell viability of each group was close to the maximum value at this concentration. Therefore, 0.5 mg / mL of P1 and P2 peptide segments were selected for subsequent experiments.

[0079] 4.2. The fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to determine the effects of three groups of anti-photoaging peptides on the level of ROS free radicals in UVA-induced L929 cells

[0080] L929 cells were cultured at 1.8 × 10 5 Cells were seeded at a density of 10 cells / mL in a 6-well plate containing complete medium and incubated in an incubator for 24 hours. When the cell density reached 80%, the complete medium was removed and 1 mL of PBS solution was added. The cells were then irradiated with UVA for 50 minutes. After irradiation, different groups were added with basal medium and basal medium containing anti-photoaging peptides, respectively, and incubated for a further 24 hours. A blank group (no UVA irradiation, basal medium added), a model group (UVA irradiation, basal medium added), an A→T→F group (UVA irradiation, basal medium containing A→T→F group anti-photoaging peptides added), a P1 group (UVA irradiation, basal medium containing peptide P1 added), and a P2 group (UVA irradiation, basal medium containing peptide P2 added). After incubation, the solution was discarded and 1 mL of diluted DCFH-DA was added (DCFH-DA:basal medium = 1:1000). The cells were then incubated in an incubator for a further 30 minutes. After completion, the cells were washed three times with basal medium and then passaged with 0.05% trypsin-EDTA (Gibco, USA) at a passage ratio of 1:2. The cells were then resuspended in PBS and added to a black 96-well plate. The excitation wavelength was set at 488 nm and the emission wavelength was set at 525 nm to measure the fluorescence intensity of each group. Figure 5 As shown in Figure A. Compared with the blank group, the ROS free radical content in the model group was significantly increased (P < 0.05), indicating that the cells had already suffered severe oxidative damage. After treatment with the anti-photoaging peptides, the ROS free radical levels in all three groups of cells were significantly reduced (P < 0.05). Peptide P1 showed the strongest inhibitory activity, with ROS free radical content reduced by 43.11% compared to the model group. This indicates that the peptide KSYELPDGQVITI not only alleviates UVA-induced oxidative stress but also restores ROS free radical content in cells to the normal physiological range, confirming its strong free radical scavenging ability.

[0081] 4.3 The effects of the three groups of anti-photoaging peptides on SOD activity and MDA content in UVA-induced L929 cells were determined using the SOD assay kit (WST-1 method) and the MDA assay kit (TBA method).

[0082] L929 cells were cultured at 1.8 × 10 5The L929 cells were seeded in 6-well plates at a density of 1.0 x 105cells / mL in complete medium and incubated in an incubator for 24 h. When the cell density reached 80%, the complete medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, the different groups were incubated for another 24 h in basal medium and basal medium containing anti-photoaging peptides, respectively. After incubation, the solution was removed and RIPA lysis buffer (Beijing Solyle Bioscience Co., Ltd.) at 4°C was added to lyse the cells for 1 h at 4°C. After lysis, the supernatant was collected by centrifugation at 12,000 g for 5 min at 4°C. Finally, the protein concentration in the supernatant was determined using a BCA protein concentration assay kit (Beijing Solyle Bioscience Co., Ltd.), and the SOD activity and MDA content were determined using an SOD assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) and an MDA assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), respectively. The results are shown in Figures 6A and 6B. Figure 5 As shown in Figures 6A and 6B, after UVA irradiation, the SOD activity in the L929 cells decreased significantly (P < 0.05), and the MDA content increased significantly (P < 0.05), indicating that the antioxidant and oxidative systems in the cells were imbalanced at this time, causing serious oxidative damage to the cells. After treatment with anti-photoaging peptides, the SOD activity and MDA content of all test groups were significantly improved. In terms of SOD activity, the P2 treatment group had the highest activity of 13.41 ± 0.07 U / mg protein, which was 38.39% higher than that of the model group. Next were the A→T→F group and the P1 treatment group, with SOD activities of 12.78 ± 0.02 U / mg protein and 12.56 ± 0.07 U / mg protein, respectively. Although the SOD activities of the two groups were close, there was still a significant difference (P < 0.05). In terms of MDA content, the P2 treatment group was again the best, with a decrease of 74.99% compared to the model group and no significant difference from the blank group (P > 0.05). Next were the A→T→F group and the P1 treatment group, but there was no significant difference in MDA content between the two groups (P > 0.05). This indicates that when the active fragment P2 is released by enzymatic hydrolysis, it has a stronger ability to improve antioxidant enzyme activity. In summary, the peptide KSYELPDGQVITI has a strong ability to scavenge ROS free radicals and improve antioxidant enzyme activity, and the peptide ELPDGQVIT as the parent fragment provides the ability to alleviate photoaging.

[0083] 4.4 Western Blot (WB) test for detecting the protein expression levels of Keap1 and Nrf2

[0084] The WB test was performed according to the method of Liu et al. [2] The L929 cells were seeded in 6-well plates at a density of 1.8 x 105cells / mL in complete medium and incubated in an incubator for 24 h. When the cell density reached 80%, the complete medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, the different groups were incubated for another 24 h in basal medium and basal medium containing anti-photoaging peptides, respectively. After incubation, the solution was removed and RIPA lysis buffer (Beijing Solyle Bioscience Co., Ltd.) at 4°C was added to lyse the cells for 1 h at 4°C. After lysis, the supernatant was collected by centrifugation at 12,000 g for 5 min at 4°C. Finally, the protein concentration in the supernatant was determined using a BCA protein concentration assay kit (Beijing Solyle Bioscience Co., Ltd.), and the SOD activity and MDA content were determined using an SOD assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) and an MDA assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), respectively. The results are shown in Figures 6A and 6B. 5The cells were inoculated at a density of 1 x 104cells / mL in 6-well plates containing complete medium and incubated in an incubator for 24 h. When the cell density reached 80%, the complete medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, different groups were added with basal medium and basal medium containing anti-photoaging peptides, respectively, and incubated for 24 h. After incubation, the solution was removed and RIPA lysis buffer (Beijing Solyle Bioscience Co., Ltd.) at 4°C was added to lyse the cells at 4°C for 1 h. After completion, centrifugation was performed at 4°C at 12000 g for 5 min, and the supernatant was collected. The protein concentration in the supernatant was determined by a BCA protein concentration detection kit (Beijing Solyle Bioscience Co., Ltd.), and then the protein concentration of the supernatant was unified to 2 mg / mL. The proteins (25 μg) in the supernatant were separated on a 10% separating gel and then transferred to a PVDF membrane (Millipore, USA). The PVDF membrane was blocked with 5% skim milk (BD-Difco, USA) at room temperature for 2 h, and then incubated with diluted specific primary antibodies Keap1 (Chengdu Zhengneng Biological Technology Co., Ltd.), Nrf2 (Bi Yun Tian Biological Technology Co., Ltd., Shanghai) and GAPDH (Bi Yun Tian Biological Technology Co., Ltd., Shanghai) at 4°C overnight. Among them, the volume ratio of Keap1 to Western primary antibody diluent (Bi Yun Tian Biological Technology Co., Ltd., Shanghai) was 1:1000; the volume ratio of Nrf2 to Western primary antibody diluent was 1:2000; and the volume ratio of GAPDH to Western primary antibody diluent was 1:5000. Then the PVDF membrane was incubated with diluted secondary antibody IgG (H+L) at room temperature for 2 h. The volume ratio of IgG (H+L) to Western secondary antibody diluent (Bi Yun Tian Biological Technology Co., Ltd., Shanghai) was 1:2000. Finally, the ECL kit (Bi Yun Tian Biological Technology Co., Ltd., Shanghai) was used to detect the protein bands, ImageJ 1.8.0 software was used to analyze the band gray scale, and the relative expression amount of the target protein was calculated. The results are as follows Figure 6The expression levels of Keap1 and Nrf2 in the cells were normal before UVA irradiation. After UVA stimulation, the expression level of Nrf2 in the nucleus showed an upward trend. It is suggested that under the condition of continuous oxidative stress, the cell itself activates the antioxidant gene by releasing Nrf2 into the nucleus to cope with oxidative damage. After incubation with anti-photoaging peptide segments, the Nrf2 level of the P1 treatment group was further increased compared with the model group, indicating that the peptide KSYELPDGQVITI can further promote the synthesis of intracellular antioxidant enzymes, thereby alleviating the damage caused by oxidative stress. In addition, the expression level of Keap1 in the cells after UVA treatment was also observed to increase (P < 0.05). This directly inhibits the synthesis of endogenous antioxidant enzymes, reduces the ability of cells to remove ROS free radicals, and leads to an increase in oxidative stress damage to cells. After P1 and P2 peptide segment treatment, the expression level of Keap1 in the cells was significantly lower than that of the model group and returned to a level close to that of the normal group, indicating that the peptides KSYELPDGQVITI and active fragment ELPDGQVIT can restore the normal antioxidant function of cells.

[0085] 4.5 Analysis of the binding mode of peptide segments P1 and P2 with Keap1 receptor protein by molecular docking

[0086] The docking results of peptides P1 and P2 with Keap1 receptor were imported into Pymol for visualization, and the results are shown in Figure 7A and B in the above formula. Then, the complex formed by the two was uploaded to Protein-Ligand Interaction Profiler (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index) to analyze the interaction between the peptide segment and the Keap1 receptor protein (including the binding mode, binding site of the receptor protein, etc.). The results are shown in Table 2. The affinity energy of P1, P2 and the Kelch domain of Keap1 is less than 0, indicating that the peptide segments P1 and P2 can spontaneously and stably bind to Keap1. In addition, the peptide segments P1 and P2 interact with the target protein through hydrophobic interaction, hydrogen bond, π-π stacking and salt bridge, among which hydrophobic interaction and hydrogen bond are the two main binding modes. Among the intermolecular interactions, hydrogen bond is one of the stronger types of non-covalent interactions, and its number has an important influence on the stability of the complex. P1 forms the most hydrogen bonds (12) with the Kelch domain, and the number of hydrogen bonds between P2 and Keap1 is 9. Therefore, P1 may have a stronger interaction with Keap1, thereby affecting the protein expression of Nrf2 in the nucleus. When the peptide segments P1 and P2 interact with the domain of the Keap1 receptor protein, they can compete with the binding site of Nrf2, thereby stimulating its dissociation from Keap1 and entering the nucleus to bind to ARE, ultimately stimulating the synthesis of antioxidant enzymes and promoting the dynamic balance of cell oxidation and reduction.

[0087] Table 2 Analysis of the interaction of P1 and P2 with Keap1 receptor

[0088]

[0089] 4.6. Molecular dynamics simulation of the binding process of peptide segments P1 and P2 with Keap1 receptor protein

[0090] The P1-Keap1 complex, P2-Keap1 complex and Keap1 receptor protein obtained by molecular docking were placed in a dodecahedron box as the initial structure using Gromacs 2023, ensuring that the distance between the complex and the box boundary was at least 1 nm. Then, water molecules (solvation) were added to the box, and Na + and Cl - were added to make the total charge of the system 0. When constructing the simulation system, the Amber99SB force field and TIP3P water molecule model were selected. Referring to An et al. [4]method, the initial simulation system was subjected to two-step energy minimization. The first step was performed using the steepest descent method for 10,000 times, and the second step was performed using the conjugate gradient for 5,000 times. After energy optimization, 200ps of NVT temperature control simulation and NPT constant pressure simulation were performed, and the temperature and pressure were balanced to 310K and below 1 bar, respectively. Then, a formal simulation of 100ns was performed, and the conformation was saved every 10ps. The temperature control algorithm used in this process was V-rescale, and the pressure control algorithm was parrinello-rahman. Finally, Gromacs related commands were used to analyze the obtained simulation results. The stability of the system was evaluated by root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg) and solvent accessible surface area (SASA). By Figure 8 As shown in Figure A, within 100 ns, the RMSD values ​​fluctuated within the range of 0.12-0.30, indicating that the complexes formed by different peptides with Keap1 all reached a stable equilibrium state. Furthermore, the average RMSD values ​​of Keap1, Keap1-P1, and Keap1-P2 were 0.267, 0.227, and 0.142, respectively. Among them, the average RMSD values ​​of the peptide-target protein complexes were all less than 0.267 (reference conformation), indicating that the binding of P1 and P2 enhances the stability of the Keap1 receptor, and that the Keap1-P2 complex has the best stability. Figure 8 Figure B shows the RMSF changes of Keap1 and the Keap1-peptide complex. Key amino acid residues near the binding sites of Keap1-P1 and Keap1-P2 (such as Arg380, Arg415, Ala556, Ser363, Ser555, Asn382, Tyr334, Ser602, Arg483, Ser508, and Gln530) exhibit both increasing and decreasing RMSF trends. This suggests that the peptide directly binds to some sites of Keap1 through hydrophobic interactions or hydrogen bonds, restricting the degree of freedom in these regions and leading to structural stability. Meanwhile, for non-direct binding regions near the binding site, this may be related to allosteric effects induced by peptide binding—peptide binding triggers conformational changes, resulting in increased flexibility in these regions. However, the overall fluctuation trends of the two complexes with Keap1 are similar, indicating that peptide binding does not significantly alter the overall dynamic properties of Keap1 and enhances the stability of the complexes. Figure 8The C in the above formula represents the change of the two complexes in Rg, and the average Rg of the Keap1-P1 and Keap1-P2 complexes is 1.820 and 1.826, respectively. The Rg values of the two systems tend to be stable after 60 ns, indicating that the complexes have formed stable conformations during the simulation process. Among them, the Rg value of the Keap1-P1 complex is smaller, indicating that it has a more compact and stable structure. Figure 8 The D in the above formula represents the change of the surface area of the two complexes that can directly contact the solvent during the simulation process. After 60 ns, the SASA value of the complex fluctuates, indicating that the conformation of the complex tends to be stable at this time. The average SASA of Keap1-P1 and Keap1-P2 is 129.302 and 130.659, respectively, both of which are within the range of 125-137 nm 2 This result is consistent with the analysis of the RMSF value. The above results show that the two anti-photoaging peptides can form stable complexes with Keap1, and the structure of Keap1-P1 is more compact, while the conformation of Keap1-P2 is the most stable. Both of them ultimately inhibit the interaction between Nrf2 and Keap1 through competitive inhibition, thereby activating the Keap1 / Nrf2 signaling pathway to a greater extent.

[0091] The above embodiments are only preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application. Any non-essential changes and substitutions made by those skilled in the art based on the present application are within the scope of the present application.

[0092] References

[0093] [1] RIBEIRO F M, DE OLIVEIRA M M, SINGH S, et al. Ceria Nanoparticles Decrease UVA-Induced Fibroblast Death Through Cell Redox Regulation Leading to Cell Survival, [2] RIBEIRO F M, DE OLIVEIRA M M, SINGH S, et al. Ceria Nanoparticles Decrease UVA-Induced Fibroblast Death Through Cell Redox Regulation Leading to Cell Survival,

[0094] [3] RIBEIRO F M, DE OLIVEIRA M M, SINGH S, et al. Ceria Nanoparticles Decrease UVA-Induced Fibroblast Death Through Cell Redox Regulation Leading to Cell Survival, [4] RIBEIRO F M, DE OLIVEIRA M M, SINGH S, et al. Ceria Nanoparticles Decrease UVA-Induced Fibroblast Death Through Cell Redox Regulation Leading to Cell Survival,

[0095] [2] LIU W, YU S, HAN Y, et al. Systematic sequence characterization of enzymatic-derived soybean peptides for precision enhancement of anti-inflammatory properties [J]. Food Bioscience, 2024, 60.

[0096] [3] LIU R, HE L, CHEN L, et al. Skin’s New Shield: Food-Derived Bioactive Peptides in Combating Photoaging—An Investigation Into Inhibitory Mechanisms and Structure-Activity Relationships [J]. Journal of Food Biochemistry, 2025, 2025 (1).

[0097] [4] AN J, WANG M, LIU W, et al. Unraveling amino acid sequence features that confer high calcium-binding capacity to soybean peptides and improve absorption in caco-2 cells [J].

[0098] Journal of Functional Foods, 2025, 130.

Claims

1. Anti-photoaging damage peptide, characterized in that, The amino acid sequence of the peptide is KSYELPDGQVITI.

2. The peptide according to claim 1, characterized in that The key active fragment of the peptide is ELPDGQVIT.

3. An anti-photoaging damage composition comprising the peptide according to any one of claims 1-2.

4. Use of the anti-photoaging damage peptide according to claim 1 or 2 in the preparation of an anti-photoaging damage drug.

5. A method for preparing the anti-photoaging damage peptide according to claim 1 or 2, characterized in that: The following steps are involved: 1) Dissolve defatted grain insect powder in water and incubate at 80-90°C for 10-20 minutes; 2) adjusting the pH of the defatted grain insect powder solution obtained in step 1) to 8.0, adding alkaline protease and performing enzymatic hydrolysis at 45-55° C. for 1.5-2.5 hours to obtain an enzymatic hydrolyzate; 3) adjusting the pH of the enzymatic hydrolysate from step 2) to 8.0, adding trypsin, and performing enzymatic hydrolysis at 35-42° C. for 1 hour; or adjusting the pH of the enzymatic hydrolysate to 7.5, adding papain, and performing enzymatic hydrolysis at 45-55° C. for 0.5-1.5 hours; or adjusting the pH of the enzymatic hydrolysate to 7.0, adding neutral protease, and performing enzymatic hydrolysis at 45-55° C. for 0.5-1.5 hours; 4) adjusting the pH of the enzymatic hydrolyzate obtained in step 3) to 7.5, adding flavor protease, and performing enzymatic hydrolysis at 45-55° C. for 0.5-1.5 h; 5) The enzymatic hydrolysate obtained in step 4) is subjected to vacuum freeze-drying treatment to obtain a powdered enzymatic hydrolysate, and then the peptides in the enzymatic hydrolysate are identified to obtain the anti-photoaging damage peptide KSYELPDGQVITI.

6. The method according to claim 5, characterized in that After each enzymatic hydrolysis reaction is completed, the method may include placing the enzymatic hydrolysis solution in boiling water to heat the solution to terminate the reaction.

7. A method for preparing the anti-photoaging damage peptide according to claim 1 or 2, characterized in that: The following steps are involved: 1) Dissolve defatted grain insect powder in water and incubate at 80-90°C for 10-20 minutes; 2) adjusting the pH of the solution obtained in step 1) to 7.5, adding alkaline protease, trypsin, papain or neutral protease, and flavor protease, and performing enzymatic hydrolysis at 40-50° C. for 3-5 hours; 3) The enzymatic hydrolysate obtained in step 2) is subjected to vacuum freeze-drying treatment to obtain a powdered enzymatic hydrolysate, and then the peptides in the enzymatic hydrolysate are identified to obtain the anti-photoaging damage peptide KSYELPDGQVITI.

8. The method according to claim 7, characterized in that After step 2), the enzymatic hydrolyzate may be placed in boiling water and heated to terminate the reaction.

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