Grifola frondosa antioxidant peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antioxidant peptides, and particularly relates to a grifola frondosa antioxidant peptide as well as a preparation method and application thereof. The amino acid sequence of the grifola frondosa antioxidant peptide comprises at least one of EPYPLP, FDWFK, DWFK and WDYH, and the amino acid sequences are respectively shown as SEQ ID NO. 1-4. According to the method, the novel antioxidant peptide is efficiently screened and identified from grifola frondosa protein by utilizing an LC-MS / MS technology in an extraction mode of performing enzymolysis on grifola frondosa and then performing ultrafiltration, molecular docking is performed on the novel antioxidant peptide and a protein receptor Keap1-Nrf2, and the best docking energy is-6.32 kcal / mol,-5.79 kcal / mol,-5.68 kcal / mol and-5.49 kcal / mol in sequence. Peptides with the lowest docking energy are chemically synthesized, and through cell experiments and antioxidant enzyme activity detection, the screened four antioxidant peptides have high comprehensive antioxidant activity. The screened four antioxidant peptides have wide prospects in the fields of food, cosmetics and the like, and an efficient way is provided for development of grifola frondosa functional polypeptide products.

Description

Technical Field

[0001] The present invention belongs to the technical field of antioxidant peptides, and particularly relates to a Grifola frondosa antioxidant peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Antioxidant peptides are a class of bioactive peptides with low molecular weight, and have attracted much attention due to their wide sources, high activity, and easy absorbability. As natural antioxidants, antioxidant peptides have a relatively simple structure, are easy to absorb, have good stability, and have no immunoreactivity. Compared with synthetic food antioxidants, natural antioxidant peptides are safer and more reliable. Therefore, the preparation of natural antioxidant peptides from agricultural and sideline products to replace synthetic antioxidants has increasingly attracted people's interest. Antioxidant peptides help prevent or slow down the risk of cell damage and degenerative diseases caused by oxidative stress.

[0003] The aging of the human body and the occurrence of many diseases are closely related to free radicals generated by the oxidation of substances in the body. More and more evidence shows that oxidative stress is defined as an imbalance between oxidants and antioxidants. Under normal circumstances, the defense system composed of antioxidant enzymes and endogenous antioxidants in the body can effectively scavenge various free radicals generated during metabolism, thereby maintaining the dynamic balance of free radicals in the body. However, when the body is stimulated by the outside world, the dynamic balance is broken, which will lead to oxidative stress and cause oxidative damage. Research shows that by supplementing dietary antioxidants exogenously, the level of free radicals in the body can be significantly reduced, the body's own antioxidant capacity can be enhanced, and the effects of preventing diseases and maintaining the health of the body can be achieved.

[0004] Grifola frondosa, also known as chestnut mushroom or Polyporus frondosus, is a rare edible and medicinal fungus, rich in various active ingredients such as amino acids, polysaccharides, and trace elements. In 100 g of dry weight of Grifola frondosa, there are 25.2 g of protein and 33.7 g of dietary fiber, and it includes 18 kinds of essential amino acids for the human body. Modern pharmacological research shows that Grifola frondosa polysaccharide has functions such as antiviral, inhibiting tumor growth, regulating immunity, treating diabetes, and reducing cholesterol. Therefore, domestic and foreign scholars have mostly studied the antioxidant properties of Grifola frondosa polysaccharide, and it has been confirmed that Grifola frondosa polysaccharide has certain antioxidant activity. However, there are still few reports on the extraction of Grifola frondosa polypeptides and the further development and utilization of antioxidant peptides. A high-efficiency polypeptide extraction method for Grifola frondosa and its application in improving the activity of antioxidant peptides, as well as a method for evaluating the biological activity and antioxidant performance of Grifola frondosa polypeptides through a Caco-2 cell oxidative damage model, remain to be further studied and developed. Summary of the Invention

[0005] The purpose of the present invention is to provide a Grifola frondosa antioxidant peptide, a preparation method thereof, and an application thereof. The antioxidant peptide is prepared and screened from Grifola frondosa and has significant antioxidant activity.

[0006] A Grifola frondosa antioxidant peptide, the amino acid sequence of the Grifola frondosa antioxidant peptide includes at least one of EPYPLP, FDWFK, DWFK, and WDYH, as shown in SEQ ID NO. 1-4 respectively.

[0007] A preparation method of a Grifola frondosa antioxidant peptide, comprising the following steps: S1. Prepare a crude extract of Grifola frondosa polypeptide S1-1. Raw material pretreatment: Weigh 1 g of Grifola frondosa powder, add 15-35 mL of ultrapure water and homogenize; S1-2. Enzymolysis: Add 20000 U / g of cellulase to the Grifola frondosa powder homogenate obtained in the above step S1-1. The enzyme addition amount is 0.5-2.5% of the mass of the Grifola frondosa powder homogenate. Adjust the pH to 4.0-5.5, and enzymolyze at 50 °C for 2-4 h, then boil to inactivate the enzyme; after cooling, add 20000 U / g of bromelain. The enzyme addition amount is 0.1-0.5% of the mass of the Grifola frondosa powder homogenate after adding cellulase. Adjust the pH to 6-8, and enzymolyze at 55 °C for 2-4 h, then boil at 100 °C to inactivate the enzyme; centrifuge at 8000-12000 r / min for 14-16 min, and take the supernatant; S1-3. Ultrafiltration: Use a 3 kDa ultrafiltration tube to ultrafilter the supernatant obtained in the above step S1-2, freeze-dry the obtained component, and store it at -20 °C for later use; detect the antioxidant activity of this component, and the results include DPPH free radical scavenging ability, ABTS+ free radical scavenging ability and reducing power; S2. Perform polypeptide sequence analysis by LC-MS / MS Use LC-MS / MS to perform polypeptide sequence analysis on the component obtained in the above step S1-3. The whole set of systems is an electrospray-combined ion trap mass spectrometer; the mass spectrometer operates in data-dependent acquisition mode and automatically switches between MS and MS / MS acquisitions; after obtaining the mass spectrometry data, perform comparison and analysis through a database; S3. Assist in screening Grifola frondosa antioxidant peptides with bioinformatics tools S3-1. Predict the antioxidant activity of active peptides for the sequences screened in the above step S2 through the Peptide Ranker tool. Sequences with a score exceeding 0.5 have potential activity and are subjected to subsequent screening; S3-2. Predict the water solubility of the sequences screened in the above step S3-1 through the Innovagen tool, predict the potential allergenicity of the sequences screened in the above step S3-1 through the Aller TOPv.2.0 tool, and predict the potential toxicity and physicochemical properties of the sequences screened in the above step S3-1 through the ToxinPred tool. Sequences without potential allergenicity and toxicity are subjected to subsequent molecular docking, synthesis and verification; S3-3. Collect the key target protein structures of the Keap1-Nrf2 protein through the PDB database. Use Pymol software to optimize the key target protein structures of the Keap1-Nrf2 protein by removing water molecules and small molecule ligands. Then use AutoDock Tools to perform hydrogenation and charge processing on the optimized key target protein structures of the Keap1-Nrf2 protein and save them in the pdbqt format. Use the processed Keap1-Nrf2 protein as the receptor and the sequences screened in step S3-2 as the ligands. Use AutoDock Tools software to perform molecular docking, calculate the binding energy, and output the result file. Finally, use PyMol software to perform visualization processing on the above output result file. Through visual analysis, the amino acid sequences of 4 antioxidant peptides are obtained, including EPYPLP, FDWFK, DWFK, and WDYH.

[0008] In step S1-1, weigh 1 g of Grifola frondosa powder, add 30 mL of ultrapure water and homogenize. Add 20000 U / g of cellulase to the Grifola frondosa powder homogenate obtained in the above step S1-1. The enzyme addition amount is 0.6% of the mass of the Grifola frondosa powder homogenate. Adjust the pH to 4.8 and enzymatically hydrolyze at 50 °C for 3 h, then boil to inactivate the enzyme. After cooling, add 20000 U / g of bromelain. The enzyme addition amount is 0.3% of the mass of the Grifola frondosa powder homogenate after adding cellulase. Adjust the pH to 7 and enzymatically hydrolyze at 55 °C for 3 h, then boil at 100 °C to inactivate the enzyme. Centrifuge at 10000 r / min for 15 min.

[0009] In step S2, the mass spectrometry database retrieval software used for database comparison and analysis is PEAKS Studio10.6 Denovo, and the existing polypeptide database is used to perform in silico analysis on the identified peptide sequences.

[0010] An application of Grifola frondosa antioxidant peptides, which is the application of Grifola frondosa antioxidant peptides in food or cosmetics.

[0011] Grifola frondosa antioxidant peptides are applied to functional foods.

[0012] The beneficial effects of the present invention are as follows: In the present invention, an extraction method of first enzymatically hydrolyzing and then ultrafiltering Grifola frondosa is adopted to efficiently screen and identify novel antioxidant peptides from Grifola frondosa proteins by using LC-MS / MS technology. Through molecular docking with the protein receptor Keap1-Nrf2, the best docking energies are -6.32 kcal / mol, -5.79 kcal / mol, -5.68 kcal / mol, and -5.49 kcal / mol in sequence. The peptide with the lowest docking energy is chemically synthesized, and through cell experiments and antioxidant enzyme activity detection, the four screened antioxidant peptides have strong comprehensive antioxidant activities.

[0013] The four antioxidant peptides screened in the present invention have broad prospects in the fields of food, cosmetics, etc., providing an efficient way for the development of Grifola frondosa functional polypeptide products. Brief Description of the Drawings

[0014] Figure 1 It is a graph showing the DPPH and ABTS·+ results of the antioxidant activity of the crude extract of Grifola frondosa polypeptide obtained after ultrafiltration in Example 1.

[0015] Figure 2 It is a graph showing the reducing power results of the antioxidant activity of the crude extract of Grifola frondosa polypeptide obtained after ultrafiltration in Example 1.

[0016] Figure 3 It is a total ion current chromatogram obtained by LC-MS / MS in step 1 of Example 1.

[0017] Figure 4 It is a molecular docking result graph of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 1 and the protein receptor Keap1-Nrf2 in Example 1.

[0018] Figure 5 It is a molecular docking result graph of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 2 and the protein receptor Keap1-Nrf2 in Example 1.

[0019] Figure 6 It is a molecular docking result graph of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 3 and the protein receptor Keap1-Nrf2 in Example 1.

[0020] Figure 7 It is a molecular docking result graph of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 4 and the protein receptor Keap1-Nrf2 in Example 1.

[0021] Figure 8 It is a graph showing the detection results of the cytotoxicity of the polypeptide to Caco-2 cells in Example 1.

[0022] Figure 9 is H in Example 1 2 O 2 Result graph of cell viability of Caco-2 cells induced by oxidative damage after intervention with polypeptide.

[0023] Figure 10 Result graph of determination of SOD activity of antioxidant enzyme of synthetic polypeptide in Example 1.

[0024] Figure 11 Result graph of determination of CAT activity of antioxidant enzyme of synthetic polypeptide in Example 1.

[0025] Figure 12 Result graph of determination of MDA activity of antioxidant enzyme of synthetic polypeptide in Example 1. Detailed implementation manners

[0026] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations for the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0027] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific examples.

[0028] The materials, reagents, etc. used in the following examples can be obtained from commercial channels without special instructions.

[0029] In the detailed implementation manners, Grifola frondosa is purchased from Zhejiang Huayu Food Co., Ltd.

[0030] In the detailed implementation manners, cellulase, bromelain, flavor protease and pepsin are purchased from Wanbang Biology.

[0031] In the detailed implementation manners, the detection methods for antioxidant activity are as follows: The method for detecting the ability to scavenge DPPH free radicals refers to the first method DPPH method for determining the antioxidant properties of polypeptides in GB / T 39100-2020; the method for detecting the ability to scavenge ABTS·+ free radicals refers to the second method ABTS method for determining the antioxidant properties of polypeptides in GB / T 39100-2020.

[0032] Example 1 I. Preparation of Grifola frondosa polypeptide crude extract 1. Raw material pretreatment: Weigh 1 g of Grifola frondosa powder, add 30 mL of ultrapure water and homogenize; 2. Enzymatic hydrolysis: Add cellulase at 20000 U / g to the Grifola frondosa powder homogenate obtained in step 1. The enzyme addition amount is 0.6% (w / w), that is, the addition amount of cellulase is 0.6% of the mass of the Grifola frondosa powder homogenate. Adjust the pH to 4.8 and carry out enzymatic hydrolysis at 50 °C for 3 h, then inactivate the enzyme by boiling; after cooling, add bromelain, flavor enzyme, pepsin and alkaline protease at 20000 U / g respectively. The enzyme addition amount is 0.3% (w / w), that is, the addition amount of bromelain is 0.3% of the mass of the Grifola frondosa powder homogenate after adding cellulase. Adjust to the optimal pH value and carry out enzymatic hydrolysis for 3 h, then inactivate the enzyme by boiling at 100 °C; centrifuge at 10000 r / min for 15 min and take the supernatant; The working conditions of the above enzymatic hydrolysis are preferably as follows: The enzyme activity of bromelain is 20000 U / g, the enzymatic hydrolysis temperature is 55 °C, and the pH is 7.0; the enzyme activity of flavor enzyme is 20000 U / g, the enzymatic hydrolysis temperature is 50 °C, and the pH is 5.0; the enzyme activity of pepsin is 20000 U / g, the enzymatic hydrolysis temperature is 50 °C, and the pH is 2.0; the enzymatic hydrolysis temperature of alkaline protease is 50 °C, and the pH is 9.0; 3. Ultrafiltration: Use a 3 kDa ultrafiltration tube for ultrafiltration. Freeze-dry the fraction less than 3 kDa obtained and store it at -20 °C for later use. Detect the antioxidant activity of this fraction. The results (including DPPH radical scavenging ability, ABTS+ radical scavenging ability and reducing power) are shown in Figure 1 and Figure 2 ; 1) Determination method of DPPH radical scavenging rate: Mix 2 mL of the sample solution with 2 mL of 0.1 mmol / L DPPH absolute ethanol solution, and after standing in the dark at room temperature for 20 min, detect the absorbance of the mixture at 517 nm. The calculation formula for the DPPH scavenging rate is as follows: DPPH scavenging rate (%) = [1 - (A1 - A2) / A0] × 100% In the formula, A0 is the mixture of 2 mL of ultrapure water and 2 mL of DPPH solution, A1 is the mixture of 2 mL of the sample solution and 2 mL of DPPH solution, and A2 is the mixture of 2 mL of the sample solution and 2 mL of absolute ethanol solution.

[0033] Preparation of 4 mmol / L DPPH absolute ethanol solution: Take 3.94 mg of DPPH and dissolve it in 100 mL of absolute ethanol in the dark.

[0034] 2) Determination method of ABTS+ radical scavenging rate: Take 100 μL of the sample solution with an appropriate concentration and add it to a test tube. Then add 3.9 mL of ABTS+ solution. Use absolute ethanol to replace the sample solution in the blank tube, and use absolute ethanol to replace the ABTS+ working solution in the control tube. Place it in the dark at room temperature for 6 min, and measure its absorbance at a wavelength of 734 nm. The calculation formula for the ABTS+ scavenging rate is as follows: ABTS+ scavenging rate (%) = [1 - (A1 - A2) / A0] × 100% In the formula, A0 is the mixture of 0.1 mL of absolute ethanol and 3.9 mL of ABTS+ solution, A1 is the mixture of 0.1 mL of sample solution and 3.9 mL of ABTS+ solution, and A2 is the mixture of 0.1 mL of sample solution and 3.9 mL of absolute ethanol solution.

[0035] 3) Determination method of reducing power: Take 2 mL of the sample solution respectively, and then add 2 mL of 0.2 mol / L phosphate buffer (pH 6.6) and 2 mL of 1% potassium ferricyanide solution in sequence. Let it stand in a water bath at 50 °C for 20 min, then add 2 mL of 10% trifluoroacetic acid, and centrifuge at 3000 r / min for 10 min. Take 2 mL of the supernatant and mix it evenly with 2 mL of ultrapure water and 0.4 mL of 0.1% FeCl 3 solution. Let it stand in a water bath at 50 °C for 10 min, and detect the absorbance value of the reaction solution at 700 nm.

[0036] The results show that the DPPH scavenging rate of the bromelain hydrolysate is the highest, the ABTS+ scavenging rate is second only to that of flavor protease, and the reducing power is second only to that of pepsin. Therefore, considering comprehensively, the crude peptides obtained by bromelain hydrolysis are selected for subsequent experiments.

[0037] II. Polypeptide sequence analysis by LC-MS / MS Perform polypeptide sequence analysis on the above-obtained components using LC-MS / MS. The whole set of systems is a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, an electrospray-combined ion trap Orbitrap mass spectrometer (Thermo Fisher Scientific, MA, USA). Analytical column: 150 μm i.d. × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100 Å; separate the sample with a gradient of 66 min, control the column flow rate at 600 nL / min, the column temperature is 40 °C, and the electrospray voltage is 2 kV; the gradient starts from 4% of phase B (80% acetonitrile, 0.1% formic acid) and increases to 95% in a non-linear gradient at 56 minutes.

[0038] The mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisitions. The mass spectrometry parameters are set as follows: (1) MS: scan range (m / z): 100 - 1500; resolution: 70000; AGC target: 3e6; maximum injection time: 100 ms; (2) HCD-MS / MS: resolution: 17500; AGC target: 1e5; maximum injection time: 50 ms; TOPN: 20; NCE / stepped NCE: 28; (3) After obtaining the mass spectrometry data (MS and MS / MS data), it is analyzed by comparison through a database. The mass spectrometry database retrieval software is PEAKS Studio10.6 De novo, and the identified peptide sequences are analyzed in silico using an existing polypeptide database.

[0039] III. Screening of Grifola frondosa antioxidant peptides assisted by bioinformatics tools 1. The antioxidant activity of the sequences screened in Step 2 is predicted by the Peptide Ranker tool. Sequences with a score exceeding 0.5 have potential activity and are subjected to subsequent screening.

[0040] 2. The water solubility of the peptide is predicted by the Innovagen tool, the potential allergenicity of the peptide is predicted by the Aller TOP v.2.0 tool, and the potential toxicity and physicochemical properties of the peptide are predicted by the Toxin Pred tool. Sequences without potential allergenicity and toxicity are subjected to subsequent molecular docking, synthesis, and verification.

[0041] 3. The key target protein structure of the Keap1-Nrf2 protein (PDB ID: 2FLU) is collected from the PDB database. The key target protein structure of the Keap1-Nrf2 protein is optimized by removing water molecules and small molecule ligands using Pymol software, and the optimized key target protein structure of the Keap1-Nrf2 protein is hydrogenated and charged using AutoDock Tools and saved as a pdbqt format; using the processed Keap1-Nrf2 protein as the receptor and the sequences screened in Step (III)-2 above as ligands, molecular docking is performed using AutoDock Tools software to calculate the binding energy and output the result file; finally, the above output result file is visualized using PyMol software; among them, the Affinity (kcal / mol) value represents the binding ability of the two, and the lower the binding ability, the more stable the binding of the ligand to the receptor. Through visual analysis, four antioxidant polypeptides are obtained, and their amino acid sequences are shown as SEQ ID NO. 1 - 4 respectively; The amino acid sequence is from the C-terminus to the N-terminus as: Glu-Pro-Tyr-Pro-Leu-Pro (SEQ ID NO. 1); Phe-Asp-Try-Phe-Lys (SEQ ID NO. 2); Asp-Try-Phe-Lys (SEQ ID NO. 3); Try-Asp-Tyr-His (SEQ ID NO. 4).

[0042] The amino acid sequences of each antioxidant peptide and the corresponding docking energies are shown in Table 1. The predicted antioxidant activity scores, potential allergenicity, potential toxicity, and physicochemical properties of each antioxidant peptide are shown in Table 2.

[0043] Table 1 Amino acid sequences of antioxidant peptides and docking energies Table 2 Prediction of antioxidant peptide properties It can be seen from Tables 1-2 that all four peptides are non-allergenic, non-toxic, and soluble in water, indicating their stability, suggesting that they have good processing performance and high safety, and the binding energy is less than -5 kcal / mol, indicating that they have good binding ability with the Keap1-Nrf2 protein.

[0044] The molecular docking results of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 1 and the receptor protein Keap1-Nrf2 are shown in Figure 4 The molecular docking results of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 2 and the receptor protein Keap1-Nrf2 are shown in Figure 5 The molecular docking results of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 3 and the receptor protein Keap1-Nrf2 are shown in Figure 6 The molecular docking results of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 4 and the receptor protein Keap1-Nrf2 are shown in Figure 7 .

[0045] Combined Figures 4 - 7It can be concluded that from the binding modes and binding sites of the four antioxidant peptides with the receptor protein Keap1-Nrf2, the binding of the antioxidant peptides to Keap1-Nrf2 is mainly through hydrogen bond interactions and hydrophobic interactions with the receptor amino acid residues. Among them, the hydrogen bond binding sites formed by the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 1 and Keap1-Nrf2 are Asn469, Gly 371, and Val 369, 370, 420; the hydrogen bond binding sites formed by the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 2 and Keap1-Nrf2 are Val 369, Val608, Val 467, and Asn 517; the hydrophobic interaction pi-alkyl binding site is Val420; the hydrogen bond binding sites formed by the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 3 and Keap1-Nrf2 are Val 561, Ile 559, Val467, and Asn 517; the hydrophobic interaction pi-alkyl binding site is Val 514; the hydrogen bond binding sites formed by the antioxidant peptide with the amino acid sequence shown in SEQ ID NO. 4 and Keap1-Nrf2 are Asn517, Val 465, and Arg 326, 470; the hydrophobic interaction pi-alkyl binding site is Ala 466.

[0046] It can be thus concluded that such tight binding may more effectively activate the Keap1-Nrf2 signaling pathway, resulting in a higher expression level of antioxidant enzymes, and thus exhibiting strong antioxidant activity.

[0047] IV. Synthesis of the Amino Acid Sequences of Grifola frondosa Antioxidant Peptides The amino acid sequences of the Grifola frondosa antioxidant peptides prepared above were entrusted to Shanghai Hongtai Biotechnology Co., Ltd. for chemical synthesis.

[0048] V. Toxicity Assay of the Synthesized Polypeptides on Caco-2 Cells Caco-2 cells were inoculated into 96-well plates at a concentration of 1×10 5 cells / well and cultured. A blank group and experimental groups were set up. 100 μL of DMEM complete medium was added to the blank group, and 100 μL of the diluted cell suspension was added to the experimental groups. They were statically cultured for 12 h; The old culture medium in each well was aspirated. 90 μL of DMEM medium and 10 μL of sterile PBS were added to the blank group, and 90 μL of DMEM medium and 10 μL of the prepared peptide samples at each concentration gradient were added to the experimental groups. The final working concentration of the peptides in the experimental groups was 5 μg / mL, 50 μg / mL, 150 μg / mL, 300 μg / mL, 500 μg / mL, and they were cultured for 24 h; The blank group and the experimental group were respectively added with 100 μL of complete DMEM medium, and then 10 μL of CCK-8 reagent was added. After incubation for 2 h, the absorbance values of each well were measured at the ultraviolet absorption wavelength of 450 nm ( Figure 8 ).

[0049] VI. Determination of cell viability of H 2 O 2 induced oxidative damage Caco-2 cells after intervention with antioxidant peptides H 2 O 2 100 µL of DMEM medium was added to each well of the induced oxidative damage group, and 100 µL of DMEM growth medium containing the polypeptide at the final mass concentrations of 0.15 mg / mL, 0.3 mg / mL, and 0.5 mg / mL was added to each well of the polypeptide intervention group. After incubation for 24 h; After the incubation, H 2 O 2 DMEM growth medium containing H 2 O 2 at a final concentration of 800 µmol / L was added to each well of the induced oxidative damage group and the polypeptide intervention protection group, and incubation was continued for 2 h; The liquid in each well of the cell culture plate was aspirated, 100 μL of complete DMEM medium and 10 μL of CCK-8 reagent were added, and then incubated for 2 h. The absorbance values of each well were measured at the ultraviolet absorption wavelength of 450 nm ( Figure 9 ).

[0050] The calculation formula for the above cell survival rate is as follows: Cell survival rate (%) = absorbance value of the experimental group / absorbance value of the control group × 100 VII. Determination of cell-related antioxidant indices The Caco-2 cell suspension was inoculated into a 6-well plate at a concentration of 2×10 5 cells / well and cultured for 24 h; 2 mL of DMEM growth medium containing the polypeptide at the final mass concentrations of 0.15 mg / mL, 0.3 mg / mL, and 0.5 mg / mL was added and incubation was continued for 24 h; DMEM growth medium containing H 2 O 2 at a final concentration of 800 µmol / L was added to each well and cultured for 2 h; 200 μL of RIPA cell lysate was added to each well, and the cells were lysed on ice for 30 min; the cell lysate was collected; Using the kit provided by Nanjing Jiancheng Bioengineering Institute, the collected cell lysate was measured for the contents of SOD, CAT, and MDA ( Figures 10 - 12 ).

[0051] The formulation of the above DMEM growth medium is as follows: high-glucose DMEM contains 20% fetal bovine serum, 1% penicillin, and streptomycin; the culture conditions are in a 37 °C, 5% CO 2 incubator for incubation.

[0052] The results showed that the antioxidant peptides with amino acid sequences as SEQ ID NO. 1 - 4 were non-toxic to Caco-2 cells, and their cell viability values were all greater than 90%; after H 2 O 2 induced oxidative damage to cells, when the concentration of the antioxidant peptide was 0.5 mg / mL, the cell viability was the highest. Compared with the damage group, the cell viability increased by 51.98%, 75.04%, 50.00%, and 56.37% respectively. For the antioxidant peptides with amino acid sequences as SEQ ID NO. 1 - 4, their SOD activity and CAT activity were significantly improved compared with the damage group; relative to the damage group, they could significantly reduce the MDA content in cells.

[0053] In summary, the antioxidant peptides EPYPLP, FDWFK, DWFK, and WDYH (SEQ ID NO. 1 - 4) were non-toxic to Caco-2 cells and had a protective effect on H 2 O 2 induced damage to Caco-2 cells. The analysis of antioxidant indicators at the cell level showed that the antioxidant peptides could reduce oxidative stress damage to cells.

[0054] Example 2 To prepare a crude extract of Grifola frondosa polypeptide, the steps are as follows: 1. Raw material pretreatment: Weigh 1 g of Grifola frondosa powder, add 15 mL of ultrapure water and homogenize; 2. Enzymolysis: Add 20000 U / g of cellulase to the homogenate of Grifola frondosa powder obtained in step 1, the enzyme addition amount is 0.5% (w / w), that is, the addition amount of cellulase is 0.5% of the mass of the Grifola frondosa powder homogenate. Adjust the pH to 4.0 and enzymolyze at 50 °C for 2 h, then boil to inactivate the enzyme; after cooling, add 20000 U / g of bromelain, flavor enzyme, pepsin, and alkaline protease respectively, the enzyme addition amount is 0.1% (w / w), that is, the addition amount of bromelain is 0.1% of the mass of the Grifola frondosa powder homogenate after adding cellulase. Adjust to the optimal pH value and enzymolyze for 2 h, then boil at 100 °C to inactivate the enzyme; centrifuge at 8000 r / min for 14 min and take the supernatant; The working conditions of the above enzymatic hydrolysis are preferably as follows: the enzyme activity of bromelain is 20000 U / g, the enzymatic hydrolysis temperature is 55 °C, and the pH is 6.5; the enzyme activity of flavor protease is 20000 U / g, the enzymatic hydrolysis temperature is 50 °C, and the pH is 4.0; the enzyme activity of pepsin is 20000 U / g, the enzymatic hydrolysis temperature is 50 °C, and the pH is 1.5; the enzymatic hydrolysis temperature of alkaline protease is 50 °C, and the pH is 8.0; 3. Ultrafiltration: Use a 3 kDa ultrafiltration tube for ultrafiltration. Freeze-dry the fraction less than 3 kDa obtained and store it at -20 °C for later use. Detect the antioxidant activity of this fraction.

[0055] Example 3 Prepare a crude extract of Grifola frondosa polypeptide, and the steps are as follows: 1. Raw material pretreatment: Weigh 1 g of Grifola frondosa powder, add 35 mL of ultrapure water and homogenize; 2. Enzymatic hydrolysis: Add 20000 U / g of cellulase to the Grifola frondosa powder homogenate obtained in step 1. The enzyme addition amount is 2.5% (w / w), that is, the addition amount of cellulase is 2.5% of the mass of the Grifola frondosa powder homogenate. Adjust the pH to 5.5 and carry out enzymatic hydrolysis at 50 °C for 4 h, then boil to inactivate the enzyme; after cooling, add 20000 U / g of bromelain, flavor enzyme, pepsin and alkaline protease respectively. The enzyme addition amount is 0.5% (w / w), that is, the addition amount of bromelain is 0.5% of the mass of the Grifola frondosa powder homogenate after adding cellulase. Adjust to the optimal pH value, carry out enzymatic hydrolysis for 4 h, and boil at 100 °C to inactivate the enzyme; centrifuge at 12000 r / min for 16 min and take the supernatant; The working conditions of the above enzymatic hydrolysis are preferably as follows: the enzyme activity of bromelain is 20000 U / g, the enzymatic hydrolysis temperature is 55 °C, and the pH is 7.5; the enzyme activity of flavor protease is 20000 U / g, the enzymatic hydrolysis temperature is 50 °C, and the pH is 5.5; the enzyme activity of pepsin is 20000 U / g, the enzymatic hydrolysis temperature is 50 °C, and the pH is 2.5; the enzymatic hydrolysis temperature of alkaline protease is 50 °C, and the pH is 9.5; 4. Ultrafiltration: Use a 3 kDa ultrafiltration tube for ultrafiltration. Freeze-dry the fraction less than 3 kDa obtained and store it at -20 °C for later use. Detect the antioxidant activity of this fraction.

[0056] The present invention uses an extraction method of first enzymatically hydrolyzing and then ultrafiltering Grifola frondosa, and uses LC-MS / MS technology to efficiently screen and identify novel antioxidant peptides from Grifola frondosa proteins. Through molecular docking with the protein receptor Keap1-Nrf2, the best docking energies are -6.32 kcal / mol, -5.79 kcal / mol, -5.68 kcal / mol, and -5.49 kcal / mol in sequence. Chemically synthesize the peptide with the lowest docking energy. Through cell experiments and antioxidant enzyme activity detection, the four screened antioxidant peptides have strong comprehensive antioxidant activities.

[0057] Four antioxidant peptides with antioxidant activities extracted from Grifola frondosa can be added to various foods as natural antioxidants, such as beverages, health products, baked foods, etc., which helps to extend the shelf life of foods, maintain the freshness and nutritional value of foods. For example, adding Grifola frondosa antioxidant peptides to fruit juice beverages can effectively prevent the oxidative loss of nutrients such as vitamin C, and at the same time can also enhance the antioxidant function of the beverage, meeting the needs of consumers for healthy beverages; it can also be developed into an independent nutritional supplement product, provided to consumers in the form of capsules, tablets, powders, etc., to provide antioxidant support for the human body, enhance the antioxidant capacity of the body, and prevent and relieve various health problems caused by oxidative stress, such as aging, chronic diseases, etc.; moreover, it can also be added to skin care products, such as essence, cream, mask, etc., to use its antioxidant properties to fight skin free radicals, reduce the formation of wrinkles and spots, delay skin aging, and improve the elasticity and gloss of the skin. Therefore, the four antioxidant peptides screened by the present invention have broad prospects in the fields of food, cosmetics, etc., providing an efficient way for the development of Grifola frondosa functional polypeptide products.

[0058] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Grifola frondosa antioxidant peptide, characterized in that: The amino acid sequence of the Grifola frondosa antioxidant peptide includes at least one of EPYPLP, ​​FDWFK, DWFK, and WDYH, as shown in SEQ ID NOs. 1 to 4, respectively.

2. A method for preparing the Grifola frondosa antioxidant peptide according to claim 1, characterized in that: The steps include: S1. Preparation of Grifola frondosa polypeptide crude extract S1-1. Raw material pretreatment: Weigh 1 g of Grifola frondosa powder, add 15-35 mL of ultrapure water and homogenize; S1-2. Enzymolysis: Add 20,000 U / g of cellulase to the slurry of foetida powder obtained in step S1-1 above, the amount of enzyme added is 0.5-2.5% of the mass of the slurry of foetida powder, adjust the pH to 4.0-5.5, enzymolysis at 50 ° C for 2-4 h, and boil to inactivate the enzyme; after cooling, add 20,000 U / g of bromelain, the amount of enzyme added is 0.1-0.5% of the mass of the slurry of foetida powder after the addition of cellulase, adjust the pH to 6-8, enzymolysis at 55 ° C for 2-4 h, and boil at 100 ° C to inactivate the enzyme; centrifuge at 8000-12000 r / min for 14-16 min, and take the supernatant; S1-3. Ultrafiltration: The supernatant obtained in step S1-2 is ultrafiltered using a 3 kDa ultrafiltration tube, and the obtained component is freeze-dried and stored at -20 °C for later use; the antioxidant activity of the component is detected, and the results include DPPH free radical scavenging ability, ABTS+ free radical scavenging ability and reducing power; S2. Peptide sequence analysis by LC-MS / MS The components obtained in the above step S1-3 are subjected to peptide sequence analysis using LC-MS / MS, and the entire system is an electrospray-combined ion trap mass spectrometer; the mass spectrometer operates in a data-dependent acquisition mode and automatically switches between MS and MS / MS acquisition; after obtaining the mass spectrometry data, comparison analysis is performed through a database; S3. Bioinformatics tools assisted screening of antioxidant peptides from Grifola frondosa S3-1. The antioxidant activity of active peptides was predicted for the sequences screened in step S2 using the Peptide Ranker tool. Sequences with a score of more than 0.5 were considered to have potential activity and were subsequently screened; S3-2. The water solubility of the sequences screened out in step S3-1 is predicted by the Innovagen tool, the potential allergenicity of the sequences screened out in step S3-1 is predicted by the Aller TOPv.2.0 tool, and the potential toxicity and physicochemical properties of the sequences screened out in step S3-1 are predicted by the ToxinPred tool. Sequences without potential allergenicity and toxicity are subsequently subjected to molecular docking, synthesis and verification; S3-3. The key target protein structures of Keap1-Nrf2 protein were collected through the PDB database, and the key target protein structures of Keap1-Nrf2 protein were optimized by removing water molecules and small molecule ligands using Pymol software. The optimized key target protein structures of Keap1-Nrf2 protein were hydrogenated and charged using AutoDock Tools and saved as pdbqt format. The treated Keap1-Nrf2 protein was used as the receptor, and the sequence screened in the above step S3-2 was used as the ligand. Molecular docking was performed using AutoDock Tools software, and the binding energy was calculated and the result file was output. Finally, the output result file was visualized using PyMol software. After visualization analysis, the amino acid sequences of 4 antioxidant peptides were obtained, including EPYPLP, ​​FDWFK, DWFK, and WDYH.

3. The method for preparing the Grifola frondosa antioxidant peptide according to claim 2, characterized in that: In step S1-1, 1 g of Grifola frondosa powder is weighed, 30 mL of ultrapure water is added and homogenized; 20,000 U / g of cellulase is added to the Grifola frondosa powder homogenate obtained in the above step S1-1, the enzyme amount is 0.6% of the mass of the Grifola frondosa powder homogenate, the pH is adjusted to 4.8, the enzyme is hydrolyzed at 50°C for 3 hours, and the enzyme is boiled to inactivate the enzyme; after cooling, 20,000 U / g of bromelain is added, the enzyme amount is 0.3% of the mass of the Grifola frondosa powder homogenate after the cellulase is added, the pH is adjusted to 7, the enzyme is hydrolyzed at 55°C for 3 hours, and the enzyme is boiled at 100°C to inactivate the enzyme; centrifuge at 10,000 r / min for 15 minutes.

4. The method for preparing the Grifola frondosa antioxidant peptide according to claim 3, characterized in that: The mass spectrometry database retrieval software used for database comparison analysis in step S2 is PEAKS Studio 10.6 De novo, and the identified peptide sequences are analyzed in silico using the existing peptide database.

5. A use of the Grifola frondosa antioxidant peptide as claimed in claim 1, characterized in that: Application of Grifola frondosa antioxidant peptides in food or cosmetics.

6. The use of Grifola frondosa antioxidant peptide according to claim 5, characterized in that: Antioxidant peptides from Grifola frondosa are used in functional foods.

Images