A novel walnut antioxidant peptide and its preparation method and application

Abstract

The present invention relates to a novel walnut antioxidant peptide and a preparation method and application thereof. The oxidized walnut meal source polypeptide comprises polypeptide 1, polypeptide 2, polypeptide 3, polypeptide 4 or a combination thereof, and the amino acid sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively. Compared with other preparation technologies of fermentation and hydrolysis of proteins, the preparation method of the walnut meal antioxidant peptide of the present invention can effectively reduce the demand for equipment and technology in the production process, and can be industrially produced on a large scale. The prepared walnut antioxidant peptide has strong activity and high safety, and can meet the use requirements of various health food industries.

Description

Technical Field

[0001] The invention belongs to the field of active peptide preparation and relates to a novel walnut antioxidant peptide and a preparation method and application thereof. Background Art

[0002] Walnut is one of the four major woody oil crops. At present, my country's walnut resources rank first in the world in terms of cultivation and output, accounting for more than 50% of the global total output, with an output of more than 1.5 million tons. After walnut oil is pressed, a large amount of walnut meal resources will be produced, with a total amount of 400,000 tons, which are rich in protein (more than 40%), dietary fiber and phenolic substances. At present, walnut meal is mostly used in simple and extensive ways such as animal feed or fertilizer, and its high-value utilization is low. The nutritional active substances in high-quality walnut meal have not been effectively utilized, which seriously restricts the high-quality development of the walnut industry.

[0003] At present, most defatted walnut meal is exposed to a natural aerobic environment. The presence of reactive oxygen makes walnut meal prone to oxidative deterioration, which often leads to structural changes in proteins, the production of soluble or insoluble protein aggregates, and affects protein function and nutritional properties. Previous studies have found that in different oxidation processes, the antioxidant capacity of walnut meal, such as free radical scavenging capacity (DPPH) and cationic free radical scavenging capacity (ABTS), showed a trend of first increasing and then decreasing. Among them, on the fourth day of oxidation, DPPH and ABTS in walnut meal reached peak values ​​of 48.63% and 73.54, respectively, which were significantly higher than the activity of DPPH (30.47%) and ABTS (58.21%) in unoxidized walnut meal. This may be because under natural aerobic conditions, the protein in walnut meal will be subjected to aerobic stress, which will induce it to form polypeptides with antioxidant activity and be used to remove self-accumulated ROS.

[0004] In recent years, the research and development of natural antioxidant peptides has been a hot topic in the field of functional active substances, and many aspects of the preparation of antioxidant peptides and their related biological activity have been explored. In view of the excellent characteristics of natural antioxidant peptides, such as biological functional activity, easy absorption and no side effects, they have been used as important additives in plant-based foods, health foods, and special medical foods, and have great potential for the development of functional foods. However, there are many unresolved issues in the preparation, purification, and identification of antioxidant peptides from walnut meal, which seriously hinder the production and industrial application of walnut antioxidant peptides. Therefore, by separating and purifying peptides from walnut meal at different oxidation stages and combining new technologies based on computer-assisted analysis, such as computer screening, safety assessment, in vivo simulated transport and absorption, faster and lower-cost screening of walnut meal active peptides, prediction and clarification of the potential activity and mechanism of peptides can be achieved, and their application in the development of healthy foods that repair oxidative damage has good prospects. Summary of the invention

[0005] The invention provides a novel walnut antioxidant peptide and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A novel walnut antioxidant peptide and a preparation method and application thereof, characterized in that the walnut antioxidant peptide comprises polypeptide 1, polypeptide 2, polypeptide 3, polypeptides or a combination thereof, polypeptide 1 is the amino acid sequence (YWSPNDEQFR) shown in SEQ ID NO.1; polypeptide 2 is the amino acid sequence (YRYL) shown in SEQ ID NO.2; polypeptide 3 is the amino acid sequence (RNHPQF) shown in SEQ ID NO.3; and polypeptide 4 is the amino acid sequence (PGHFE) shown in SEQ ID NO.4.

[0008] Preferably, the method comprises: extracting, separating and purifying the defatted walnut meal after oxidative stress.

[0009] Further preferably, the specific method of oxidative stress is: using walnut defatted meal as raw material, storing it at 55-65° C. for 3-5 days.

[0010] More preferably, the specific method of extraction is: after the walnut defatted meal after oxidative stress is mixed with phosphate buffer in a mass ratio of 1:(3-6), stirred for 1.5-2.5 hours, homogenized in an ice water bath, and then centrifuged to remove the supernatant to obtain the walnut meal polypeptide extract.

[0011] Further preferably, the separation and purification specifically comprises the following steps:

[0012] 1) Separating and purifying the walnut meal polypeptide extract by gel filtration: using a tangential ultrafiltration system to separate polypeptides with a molecular weight of ≤3Kda, and using a G-25 gel filtration column to separate and elute to obtain eluted components; the eluted components are dried in a vacuum freeze dryer;

[0013] 2) Separation and purification of oxidative stress walnut meal polypeptide by fast protein liquid chromatography (FPLC): The components obtained in step 1) are further separated by FPLC, dissolved in ultrapure water after freeze-drying, injected into the FPLC system, and gradient eluted by reverse phase LXMS-15 chromatographic column;

[0014] Obtain new walnut antioxidant peptides.

[0015] Still more preferably, in the step 1), the separation conditions of the G-25 gel filtration column are: the eluent is ultrapure water, and the flow rate is 1.0 mL / min.

[0016] Step 2) gradient elution: flow rate 1.0 mL / min, mobile phase A is 0.1% trifluoroacetic acid acetonitrile solution, mobile phase B is 0.1% acetonitrile solution; mobile gradient: 0-20 min, 100% A, 20-40 min, 0-50% B; 40 min, 50% A

[0017] A novel walnut antioxidant peptide and a preparation method and application thereof. The novel walnut antioxidant peptide or an oxidized walnut meal-derived polypeptide obtained by the preparation method and capable of repairing active oxygen-induced oxidative damage is screened. The screening and identification method is to use liquid chromatography-tandem mass spectrometry to screen and analyze the polypeptides with the activity of repairing active oxygen-induced oxidative damage obtained by separation and purification, and identify the structure of the active peptides.

[0018] Further preferably, the identification and screening method specifically comprises the following steps:

[0019] A: The isolated and purified peptide components that can repair the oxidative damage induced by reactive oxygen species are freeze-dried and dissolved in ultrapure water, injected into the FPLC system, and gradient eluted using a reversed-phase LXMS-15 column; the peaks corresponding to the peptides are divided into six parts, namely P-1-1, P-1-2, P-1-3, P-1-4, P-1-5, and P-1-6, and freeze-dried; and the antioxidant activity of the purified fractions is evaluated;

[0020] B: Easy-nLC high performance liquid chromatography system was used to separate the peptide peak P-1-4 with the most active repair of reactive oxygen species-induced oxidative damage using RPLC C18 chromatographic column; gradient elution: mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 600nL / min; mobile gradient: 0-20min, 100% A, 20-40min, 0-50% B; 40min, 50% A; the mass range of precursor ion recording was m / z=300-4000; Byonic was used to search the target protein database, analyze the mass spectrum information, and screen out peptide 1, peptide 2, peptide 3 and peptide 4 respectively;

[0021] C: Screening: Computer simulation technology is used to perform virtual screening of peptides with the greatest activity in repairing reactive oxygen species-induced oxidative damage. The functional activity of peptides with the greatest activity in repairing reactive oxygen species-induced oxidative damage is analyzed based on the virtual screening results.

[0022] Further preferably, in the step C, the virtual screening method is: using Autodock software to study the size of the binding energy between the polypeptide and Keap1, hydrogen bonds and intermolecular forces; using Gromacs software, the peptide-Keap1 complex is subjected to molecular dynamics simulation through the CHARMM36 force field, and the molecular dynamics simulation conditions are as follows: using the steepest descent algorithm to minimize the energy, and continuing until the maximum force is <600.0 kJ / mol / nm, the step size is 0.001; using the frog leaping algorithm to simulate for 1 ns, the step size is 2 fs, the temperature and pressure are set to 200 K and 1 bar, respectively, the simulation time is 50 ns, and the root mean square deviation, the number of hydrogen bonds, the radius of gyration, and the surface solvent accessible area are calculated to analyze the stability of the peptide-Keap1 complex; using Gaussian 09 for density functional theory calculations, using the semi-empirical AM1 method for configuration optimization, and in the Gaussian 09 program, using B3LYP / 6-31 to obtain the geometry to analyze the active site, and calculating the energy index and structural index of the polypeptide.

[0023] Application of the novel walnut antioxidant peptide or the novel walnut antioxidant peptide obtained by the preparation method or the screening method in antioxidant health products, medicines and foods.

[0024] Beneficial effects of the present invention:

[0025] 1. Compared with other preparation technologies of fermented and hydrolyzed proteins, the preparation method of walnut meal antioxidant peptides of the present invention can effectively reduce the demand for equipment and technology in the production process, and can be produced on a large scale industrially. The walnut antioxidant peptides prepared by this method are highly active and safe, and can meet the use requirements of various health food industries.

[0026] 2. The polypeptide for repairing oxidative damage induced by active oxygen derived from oxidized walnut meal of the present invention has potential medical value.

[0027] 3. Compared with other screening technologies, the present invention can screen, predict and clarify the potential activity and mechanism of peptides faster and at a lower cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the polypeptide content in walnut meal at different oxidation times;

[0029] Figure 2The scavenging abilities of polypeptides DPPH, ferrous ions, OH- and O2- in walnut meal with different molecular weights, including: (a) scavenging abilities of polypeptides DPPH and ferrous ions in walnut meal with molecular weight greater than 10Kda; (b) scavenging abilities of polypeptides OH- and O2- in walnut meal with molecular weight greater than 10Kda; (c) scavenging abilities of polypeptides DPPH and ferrous ions in walnut meal with molecular weight between 3Kda and 10Kda; (d) scavenging abilities of polypeptides OH- and O2- in walnut meal with molecular weight between 3Kda and 10Kda; (e) scavenging abilities of polypeptides DPPH and ferrous ions in walnut meal with molecular weight less than 3Kda; (f) scavenging abilities of polypeptides OH- and O2- in walnut meal with molecular weight less than 3Kda.

[0030] Figure 3 The figures are the gel chromatogram of a polypeptide of less than 3Kda and the scavenging abilities of its different components for DPPH, ferrous ions, OH- and O2-, wherein: (a) the gel chromatogram of a polypeptide of less than 3Kda; (b) the scavenging abilities of the elution peak components of P1, P2 and P3 in the polypeptide of less than 3Kda and DPPH and ferrous ions; (c) the scavenging abilities of the elution peak components of P1, P2 and P3 in the polypeptide of less than 3Kda and OH- and O2-.

[0031] Figure 4 The FPLC chromatogram of the P-1 component and the scavenging abilities of its different components for DPPH, ferrous ions, OH- and O2-, including: (a) FPLC chromatogram of the P-1 component; (b) DPPH and ferrous ion scavenging abilities of the six elution peak components from P-1-1 to P-1-6 in the P-1 polypeptide component; (c) OH- and O2- scavenging abilities of the six elution peak components from P-1-1 to P-1-6 in the P-1 polypeptide component.

[0032] Figure 5 These are the mass spectra (MS) of four active peptides, including: (a) mass spectrum of active peptide YRYL; (b) mass spectrum of active peptide YWSPNDEQFR; (c) mass spectrum of active peptide RNHPQF; and (d) mass spectrum of active peptide PGHFE.

[0033] Figure 6 The diagrams show the docking results of four active peptide molecules, including: (a) the docking binding site, binding energy and intermolecular force type of Keap1 and Nrf2; (b) the docking binding site, binding energy and intermolecular force type of Keap1 and PGHFE; (c) the docking binding site, binding energy and intermolecular force type of Keap1 and RNHPQF; (d) the docking binding site, binding energy and intermolecular force type of Keap1 and YRYL; (e) the docking binding site, binding energy and intermolecular force type of Keap1 and YWSPNDEQFR.

[0034] Figure 7The figures show the results of molecular dynamics simulation of four active peptides, including: (a) root mean square deviation (RMSD) distribution of four active peptides during molecular dynamics simulation; (b) radius of gyration (Rg) distribution of four active peptides during molecular dynamics simulation; (c) number of hydrogen bonds distribution of four active peptides during molecular dynamics simulation; (d) surface solvent accessible area (SASA) distribution of four active peptides during molecular dynamics simulation; (e) root mean square fluctuation (RMSF) distribution of 54 active peptides during molecular dynamics simulation; (f) differences in average values ​​of RMSD, Rg, number of hydrogen bonds, SASA, and RMSF during molecular dynamics simulation of four active peptides.

[0035] Figure 8 The active sites and optimal structure diagrams of 4 active peptides were simulated based on quantum chemistry;

[0036] Fig. 9 The docking results of four active peptides with DPPH and ABTS molecules;

[0037] Fig.10 The effects of four active peptides on DPPH and ABTS in vitro and on cell viability, ROS content, SOD and CAT activities, including: (a) the difference in the in vitro ABTS scavenging ability of the four active peptides; (b) the difference in the in vitro DPPH scavenging ability of the four active peptides; (c) the effect of different concentrations of AAPH on the degree of oxidative damage of HepG2 cells; (d) the effect of different concentrations of four active peptides on the viability of HepG2 cells; (e) the effect of different concentrations of four active peptides on the cell viability after AAPH-induced HepG2 cell damage; (f) the effect of different concentrations of four active peptides on the ROS concentration in cells after AAPH-induced HepG2 cell damage; (g) the effect of different concentrations of four active peptides on the CAT enzyme activity in cells after AAPH-induced HepG2 cell damage; (h) the effect of different concentrations of four active peptides on the SOD enzyme activity in cells after AAPH-induced HepG2 cell damage;

[0038] Fig.11 The effect of YR-10 on ROS. (a) ROS fluorescence image of YR-10 on HepG2 cells after AAPH-induced oxidative damage; (b) average fluorescence intensity value of ROS image; (c) DCP fluorescence intensity value in ROS image;

[0039] Fig.12The effects of YR-10 on cellular oxidative stress response, including: (a) apoptosis of HepG2 cells in the control group; (b) apoptosis of HepG2 cells in the AAPH model group; (c) the effect of YR-10 on apoptosis of HepG2 cells; (d) the effect of low concentration (100 μg / mL YR-10) on apoptosis of HepG2 cells induced by AAPH; (e) the effect of medium concentration (200 μg / mL YR-10) on apoptosis of HepG2 cells induced by AAPH; (f) the effect of high concentration (400 μg / mL YR-10) on apoptosis of HepG2 cells induced by AAPH;

[0040] Fig.13 Effects of YR-10 on key proteins in the cellular antioxidant metabolic pathway, including: (a) immunoblotting of key proteins in HepG2 cells induced by AAPH after YR-10-induced oxidative damage; (b) effect of YR-10 on the expression of Keap1 protein in HepG2 cells induced by AAPH after oxidative damage; (c) effect of YR-10 on the expression of HO-1 protein in HepG2 cells induced by AAPH after oxidative damage; (d) effect of YR-10 on the expression of SOD1 protein in HepG2 cells induced by AAPH after oxidative damage. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific embodiments. The present invention is further described in detail, but is not limited to these embodiments.

[0042] Example 1

[0043] A novel walnut antioxidant peptide and its preparation method and application

[0044] Production method and steps:

[0045] 1. Preparation of peptide extracts using walnut meal oxidized for 0, 2, 4, 6, 8, 10 and 12 days as raw materials

[0046] 50 g of walnut meal was mixed with 200 mL of phosphate buffer (0.2 mmol / L, pH 7.0), stirred at room temperature for 2 hours, and homogenized using a homogenizer in an ice-water bath for 3 times, each time for 10 seconds, with a homogenizer speed of 20,000 rpm, followed by centrifugation at 10,000 rpm for 15 minutes at 4°C, and the supernatant was removed to obtain a walnut meal polypeptide extract.

[0047] 2. Purification of walnut meal polypeptide extract

[0048] The collected supernatant was ultrafiltered by a tangential ultrafiltration system equipped with a 3Kda membrane, and the ultrafiltrate was collected, and then separated using a G-25 gel filtration column. The elution conditions were as follows: the eluent was ultrapure water, the flow rate was 1.0 mL / min, and a 280 nm ultraviolet detector was used to detect the eluted components, and 2 mL of the eluted components in each tube were collected by an automatic collector; the eluted components were dried in a vacuum freeze dryer for 48 hours; after freeze-drying, 2 mg was weighed and dissolved in 1 mL of ultrapure water, injected into the FPLC system, and gradient elution was performed using a reversed-phase LXMS-15 chromatographic column. Gradient elution: flow rate 1.0 mL / min, mobile phase A was 0.1% trifluoroacetic acid acetonitrile solution, and mobile phase B was 0.1% acetonitrile solution; the mobile gradient was: 0-20 min, 100% A, 20-40 min, 0-50% B; 40 min, 50% A; the elution peak was detected at 280 nm UV wavelength; the peak corresponding to the polypeptide was divided into six parts, freeze-dried; and the antioxidant activity of the purified fractions was evaluated.

[0049] 3. Identification of peptide structures that can repair reactive oxygen species-induced oxidative damage

[0050] Liquid chromatography-tandem mass spectrometry was used to screen and analyze the polypeptides with the strongest activity in repairing reactive oxygen species-induced oxidative damage isolated from S3, and the structures of the active peptides were identified, and a peptide sequence with the activity in repairing reactive oxygen species-induced oxidative damage was obtained: Tyr-Typ-Ser-Pro-Asn-Asp-Glu-Gln-Phe-Arg (YR-10); the specific identification method included: using an Easy-nLC high performance liquid chromatography system and a RPLC C18 chromatographic column to separate the polypeptide peak with the strongest activity in repairing reactive oxygen species-induced oxidative damage; gradient elution: mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 600nL / min; mobile gradient: 0-20min, 100% A, 20-40min, 0-50% B; 40min, 50% A; the mass range of the precursor ion record was m / z=300-4000; and Byonic was used to search the target protein database and analyze the mass spectrum information.

[0051] 4. Screening of peptides that can repair oxidative damage induced by reactive oxygen species

[0052] Computer simulation technology was used to virtually screen the peptides with the greatest activity in repairing reactive oxygen species-induced oxidative damage. Autodock software was used to study the binding energy, hydrogen bonds and intermolecular forces between the peptide (receptor) and Keap1 (ligand molecule). Gromacs software was used to perform molecular dynamics simulation of the peptide-Keap1 complex through the CHARMM36 force field. The molecular dynamics simulation conditions were as follows: energy minimization was performed using the steepest descent algorithm and continued until the maximum force was <600.0 kJ / mol / nm with a step size of 0.001. The frog leaping algorithm was used for 1 ns simulation with a step size of 2 fs. The temperature and pressure were set to 200 K and 1 bar, respectively. The simulation time was 50 ns. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), number of hydrogen bonds, radius of gyration (Rg), and surface solvent accessible area (SASA) were calculated to analyze the stability of the peptide-Keap1 complex. Density functional theory calculations were performed using Gaussian 09, and the semi-empirical AM1 method was used for configuration optimization. In the Gaussian 09 program, B3LYP / 6-31 was used to obtain the geometry to analyze the active site and calculate the energy index of the peptide (highest electron occupied orbital (E HOMO ), the lowest unoccupied electron orbital (E LOMO The energy level of the peptide was calculated. The comprehensive virtual screening results were used to analyze the functional activity of the peptide with the best activity in repairing reactive oxygen species-induced oxidative damage.

[0053] 5. Artificial synthesis of active peptides and screening of active peptides with the greatest antioxidant activity

[0054] The four active peptides screened out in step 4 of the artificial synthesis are as follows:

[0055] Weigh 1mmmol resin resin (2-chlorotriacyl chloride resin) and swell in dichloromethane (DCM) for 30min. Dissolve the first amino acid (3bei) in a solution of 4mL DCM, 3mL dimethylformamide (DMF) and 1mL N,N-diisopropylethylamine (DIPEA) and let stand overnight. Perform a methanol end-capping after the first amino acid coupling. Use a DMF solution containing 20% ​​piperidine to remove the amino protecting group (Fmoc) (repeat twice, once for 5min and once for 10min). The amino acid is then activated with hexafluorophosphate benzotriazole-1-yl-oxytripyrrolidinophosphine (PyBOP) for coupling, which contains 3 equivalents of Fmoc-amino acid, 3 equivalents of PyBOP in peptide-grade DMF and 6 equivalents of DIPEA. The coupling reaction is carried out at 70°C under microwave for 5min. Repeat the above steps until all amino acids are inserted. After the synthesis is complete, the resin is dried and treated on an orbital shaker at room temperature for 3 hours with a cleavage mixture consisting of 95% trifluoroacetic acid, 2.5% triisopropylsilane and 2.5% ethanedithiol. The crude peptide is dropped into ice-cold ether for precipitation, and the mixture is then centrifuged (3000 g, 10 min) and excess ether is decanted to give a solid precipitate of the crude peptide.

[0056] For purification, the crude peptide was dissolved in ultrapure water and HPLC grade acetonitrile (1:1 ratio) and injected onto a BEH C18 column (20 mm inner diameter, 150 mm length, 5 μm particle size) at a certain flow rate. The flow rate was 5 mL / min. The elution solvent system (0.1% v / v formic acid was used as an ion pairing agent in all cases) had a linear gradient of 20% (v / v) acetonitrile in water for 5 min, gradually increasing to 80% (v / v) acetonitrile in water for 35 min. This concentration was kept constant until 40 min, when the gradient was reduced to 20% (v / v) acetonitrile in water at 42 min. Elution was determined by UV detection at 280 nm. The purified peptide was collected from the HPLC fractions, combined and lyophilized to obtain a fluffy white polypeptide solid.

[0057] The polypeptide of the invention, which is derived from oxidized walnut meal and can repair the active oxygen-induced oxidative damage activity, has the functions of regulating the metabolic pathway of cellular oxidative damage and directly chelating free radicals, and has no cytotoxicity and can be widely used as a key ingredient in the development of healthy foods that repair cellular oxidative damage.

[0058] The content and antioxidant capacity of peptides in walnut meal at different oxidation stages have an important influence on the repair of cellular oxidative damage. Figure 1 It can be found that the content of polypeptides in the walnut meal oxidized for 4 days (86.74%) is significantly higher than that of other components. Therefore, the polypeptides in the walnut meal oxidized for 4 days are determined to be used for the separation of active peptides.

[0059] The peptides in walnut meal after oxidation for 4 days were separated into three peptide fractions (molecular weight <3Kda, molecular weight 3Kda-10Kda and molecular weight >10Kda) by ultrafiltration. In addition, the scavenging capacity of DPPH, ferrous ion, OH- and O2- of samples with different molecular weights was also determined. Figure 2 The results showed that the DPPH, ferrous ion, OH- and O2- scavenging abilities of the fractions with a molecular weight of <3Kda in different concentration ranges (0.1mg / mL-25mg / mL) were higher than those of the fractions with a molecular weight of 3Kda-10Kda and the fractions with a molecular weight of >10Kda, indicating that small molecular weight polypeptides had the strongest antioxidant activity. Therefore, the fractions with a molecular weight of <3Kda were selected for further purification.

[0060] After gel chromatography, three elution peaks (P1, P2 and P3) were obtained, and the eluents of the peaks were collected and freeze-dried. The elution curves and antioxidant activities of each component are shown in Figure 3 As shown in Figure 2, the antioxidant capacity of P1 was higher than that of components P2 and P3. Specifically, the scavenging capacity of DPPH, ferrous ions, OH-, and O2- of P1 was higher than that of components P2 and P3. Therefore, P1 was selected for further separation by FPLC.

[0061] The P1 fraction was further purified by FPLC using a reverse phase LXMS-15 column. A total of 6 fractions were isolated from P-1-1 to P-1-6. The purification results and antioxidant activity of each peak are shown in Figure 4 As shown, the separated fractions were lyophilized and their antioxidant activities were determined. P-1-4 showed significantly stronger free radical scavenging activity than the other peaks.

[0062] Easy-nLC high performance liquid chromatography system was used to separate P-1-5 by mass spectrometry using RPLC C18 column; gradient elution: mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 600nL / min; mobile gradient: 0-20min, 100% A, 20-40min, 0-50% B; 40min, 50% A; the mass range of precursor ion recording was m / z=300-4000; Byonic was used to search the target protein database and analyze the mass spectrum information, and four polypeptides including PGHFE, YWSPNDEQFR, YRYL, and RNHPQF were found ( Figure 5 ).

[0063] In order to determine the activity of the four peptides and their repair effects on reactive oxygen species-induced cell damage, computer simulation technology was first used to virtually screen the peptides with the greatest activity in repairing reactive oxygen species-induced oxidative damage, and Autodock software was used to study the binding energy, hydrogen bonds, and intermolecular forces between the peptide (receptor) and Keap1 (ligand molecule); Gromacs software was used to perform molecular dynamics simulation of the peptide-Keap1 complex through the CHARMM36 force field, and the molecular dynamics simulation conditions were as follows: energy minimization was performed using the steepest descent algorithm, and continued until the maximum force was <600.0 kJ / mol / nm, with a step size of 0.001. The frog leaping algorithm was used for 1 ns simulation with a step size of 2 fs. The temperature and pressure were set to 200 K and 1 bar, respectively. The simulation time was 50 ns. The root mean square deviation (RMSD), number of hydrogen bonds, radius of gyration (Rg), and surface solvent accessible area (SASA) were calculated to analyze the stability of the peptide-Keap1 complex. Density functional theory calculations were performed using Gaussian 09, and the semi-empirical AM1 method was used for configuration optimization. In the Gaussian 09 program, B3LYP / 6-31 was used to obtain the geometry to analyze the active site and calculate the energy index of the peptide (total energy, highest electron occupied orbital (E HOMO ), the lowest unoccupied electron orbital (E LOMO ) energy level) and structural index (highest electron occupied orbital, atomic net charge distribution, bond length) were calculated. The functional activity of the peptides with the most active repair of reactive oxygen species induced oxidative damage was analyzed by comprehensive virtual screening results. The repair effect of the four peptides was explored by inducing HepG2 cell model with azobisisobutylamidine hydrochloride (AAPH), and the levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione oxidase (GSH-Px) in cells were detected. It was found that the peptides repaired the effects of reactive oxygen species induced oxidative damage by chelating free radicals and regulating key enzymes in the antioxidant metabolic pathway.

[0064] like Figure 6 As shown in the figure, the four peptides can bind to Keap1 through hydrogen bonds, hydrophobic interactions and π-π bonds, and the binding energy is PGHFE>YWSPNDEQFR>YRYL>RNHPQF. The stability difference of peptide-Keap1 complexes was calculated by molecular dynamics, and the stability is PGHFE>YWSPNDEQFR>YRYL>RNHPQF( Figure 7 ), which is consistent with the molecular docking results. The structure-activity relationship of active peptides was explored by quantum chemistry and the energy index of the peptides was analyzed, which showed that the antioxidant activity of the peptides was in the order of YWSPNDEQFR>YRYL>RNHPQF>PGHFE( Figure 8). All four peptides can directly chelate free radicals to scavenge free radicals. By analyzing the binding energy of ABTS and DPPH, it was found that YWSPNDEQFR had the greatest chelating ability ( Fig. 9 ).like Fig.10 As shown in the results of CCK-8 analysis, the four peptides showed no cytotoxic effects on HepG2 cells and even promoted cell proliferation. Cell experiments showed that at a concentration of 0.05 mg / mL, the viability of HepG2 cells treated with the four peptides increased significantly from 74.95% to 96.50%, 95.21%, 94.88%, 95.73% and 93.73%. Secondly, compared with the model group, the ROS content in the five peptide-treated groups (at a concentration of 0.05 mg / mL) was significantly reduced by 81.45%, 84.49%, 71.02%, 64.56% and 55.01%, respectively. All four peptides can increase the activity of SOD and CAT to the level of normal cells and have significant antioxidant activity.

[0065] Example 2: A novel walnut antioxidant peptide and its preparation method and application

[0066] Production method and steps:

[0067] 1. Preparation of peptide extract using 4-day oxidized walnut meal as raw material

[0068] 50 g of walnut meal was mixed with 200 mL of phosphate buffer (0.2 mmol / L, pH 7.0), stirred at room temperature for 2 hours, and homogenized using a homogenizer in an ice-water bath for 3 times, each time for 10 seconds, with a homogenizer speed of 20,000 rpm, followed by centrifugation at 10,000 rpm for 15 minutes at 4°C, and the supernatant was removed to obtain a walnut meal polypeptide extract.

[0069] 2. Purification of walnut meal polypeptide extract

[0070] The collected supernatant was ultrafiltered by a tangential ultrafiltration system equipped with a 3Kda membrane, and the ultrafiltrate was collected, and then separated using a G-25 gel filtration column. The elution conditions were as follows: the eluent was ultrapure water, the flow rate was 1.0 mL / min, and a 280 nm ultraviolet detector was used to detect the eluted components, and 2 mL of the eluted components in each tube were collected by an automatic collector; the eluted components were dried in a vacuum freeze dryer for 48 hours; after freeze-drying, 2 mg was weighed and dissolved in 1 mL of ultrapure water, injected into the FPLC system, and gradient elution was performed using a reversed-phase LXMS-15 chromatographic column. Gradient elution: flow rate 1.0 mL / min, mobile phase A was 0.1% trifluoroacetic acid acetonitrile solution, and mobile phase B was 0.1% acetonitrile solution; the mobile gradient was: 0-20 min, 100% A, 20-40 min, 0-50% B; 40 min, 50% A; the elution peak was detected at 280 nm UV wavelength; the peak corresponding to the polypeptide was divided into six parts, freeze-dried; and the antioxidant activity of the purified fractions was evaluated.

[0071] 3. Identification of peptide structures that can repair reactive oxygen species-induced oxidative damage

[0072] Liquid chromatography-tandem mass spectrometry was used to screen and analyze the polypeptides with the strongest activity in repairing reactive oxygen species-induced oxidative damage isolated from S3, and the structures of the active peptides were identified, and a peptide sequence with the activity in repairing reactive oxygen species-induced oxidative damage was obtained: Tyr-Typ-Ser-Pro-Asn-Asp-Glu-Gln-Phe-Arg (YR-10); the specific identification method included: using an Easy-nLC high performance liquid chromatography system and a RPLC C18 chromatographic column to separate the polypeptide peak with the strongest activity in repairing reactive oxygen species-induced oxidative damage; gradient elution: mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 600nL / min; mobile gradient: 0-20min, 100% A, 20-40min, 0-50% B; 40min, 50% A; the mass range of the precursor ion record was m / z=300-4000; and Byonic was used to search the target protein database and analyze the mass spectrum information.

[0073] The decapeptide of the present invention has excellent solubility, safety and stability. YR-10 exhibits good antioxidant activity in extracellular assays (41.57 μmol TE / g and 29.41% in DPPH and ABTS assays, respectively), and has a protective effect on AAPH-induced oxidative damage in HepG2 cells, and exhibits a significant dose-dependency. Fluorescence and flow cytometry revealed that YR-10 effectively reduced the content of ROS in cells and reduced the occurrence of apoptosis ( Fig.11 and Fig.12In addition, Western blot and molecular docking results showed that YR-10 achieved antioxidant capacity by occupying the binding sites of Nrf2 and Keap1, activating the Keap1-Nrf2 signaling pathway, and upregulating the expression of antioxidant enzymes and Nrf2 / HO-1 / SOD1 ( Fig.13 ).

[0074] The above embodiments are only preferred technical solutions of the present invention and should not be regarded as limitations of the present invention. The embodiments and features in the embodiments of the present application can be arbitrarily combined with each other without conflict. The protection scope of the present invention shall be the technical solutions recorded in the claims, including the equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. A polypeptide having antioxidant and repairing activity of reactive oxygen species-induced oxidative damage, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.1; the oxidized walnut meal-derived polypeptide has both antioxidant and repairing effects on oxidative damage induced by active oxygen.

2. A polypeptide composition having antioxidant and repairing activity of reactive oxygen species-induced oxidative damage, characterized in that: The polypeptide composition comprises polypeptide 1, polypeptide 2, polypeptide 3 and polypeptide 4, wherein polypeptide 1 is the amino acid sequence shown in SEQ ID NO.1; polypeptide 2 is the amino acid sequence shown in SEQ ID NO.2; polypeptide 3 is the amino acid sequence shown in SEQ ID NO.3; polypeptide 4 is the amino acid sequence shown in SEQ ID NO.4; the oxidized walnut meal-derived polypeptide has both antioxidant and repairing effects on active oxygen-induced oxidative damage.

3. A method for preparing the polypeptide according to claim 1 or the polypeptide composition according to claim 2, characterized in that: The method comprises the following steps: extracting, separating and purifying the walnut defatted meal after oxidation stress.

4. The preparation method according to claim 3, characterized in that: The oxidative stress is as follows: using defatted walnut meal as raw material, storing it at 55-65° C. for 3-5 days.

5. The preparation method according to claim 3, characterized in that: The extraction method comprises the following steps: mixing the defatted walnut meal subjected to oxidative stress with a phosphate buffer solution at a mass ratio of 1:(3-6), stirring for 1.5-2.5 hours, homogenizing in an ice water bath, and then centrifuging to remove the supernatant to obtain a walnut meal polypeptide extract.

6. The preparation method according to claim 3, characterized in that: The separation and purification comprises the following steps: 1) Gel filtration separation and purification of walnut meal polypeptide extract: Use a tangential ultrafiltration system to separate polypeptides with a molecular weight of ≤3Kda, and use a G-25 gel filtration column to separate and elute to obtain eluted components; the eluted components are dried in a vacuum freeze dryer; 2) Separation and purification of oxidative stress walnut meal polypeptide by fast protein liquid chromatography (FPLC): The components obtained in step 1) were further separated by FPLC, dissolved in ultrapure water after freeze-drying, injected into the FPLC system, and gradient eluted by reverse phase LXMS-15 chromatographic column; Get walnut antioxidant peptides.

7. The preparation method according to claim 6, characterized in that: In the step 1), the separation and elution conditions of the G-25 gel filtration column are as follows: the eluent is ultrapure water, and the flow rate is 1.0 mL / min; Step 2) gradient elution: flow rate 1.0 mL / min, mobile phase A is 0.1% trifluoroacetic acid in acetonitrile solution, mobile phase B is 0.1% acetonitrile solution; mobile gradient is: 0-20 min, 100% mobile phase A, 20-40 min, 0-50% mobile phase B; 40 min, 50% mobile phase A.

8. A method for identifying and screening polypeptides having antioxidant and repairing activities of reactive oxygen species-induced oxidative damage, characterized in that: The polypeptide according to claim 1, the polypeptide composition according to claim 2, or the walnut antioxidant peptide obtained by the preparation method according to any one of claims 3 to 7 is screened, and the identification and screening method is to use liquid chromatography tandem mass spectrometry to screen and analyze the walnut antioxidant peptides obtained by separation and purification to identify the structure of the active peptide.

9. The identification and screening method according to claim 8, characterized in that: The identification and screening method comprises the following steps: A: The purified peptide components that can repair the oxidative damage induced by reactive oxygen species are freeze-dried and dissolved in ultrapure water, injected into the FPLC system, and gradient eluted using a reversed-phase LXMS-15 column; the peaks corresponding to the peptides are divided into six parts, namely P-1-1, P-1-2, P-1-3, P-1-4, P-1-5, and P-1-6, and freeze-dried; and the antioxidant activity of the purified fractions is evaluated; B: Easy-nLC high performance liquid chromatography system was used to separate the peptide peak P-1-4 with the most active repair of reactive oxygen species-induced oxidative damage using RPLC C18 column; gradient elution: mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 600 nL / min; mobile gradient: 0-20 min, 100% mobile phase A, 20-40min, 0-50% mobile phase B; 40 min, 50% mobile phase A; the mass range of precursor ion recording was m / z=300-4000; Byonic was used to search the target protein database and analyze the mass spectrum information to screen out peptide 1, peptide 2, peptide 3 and peptide 4 respectively; C: Screening: Computer simulation technology was used to virtually screen the peptides with the best activity in repairing oxidative damage induced by reactive oxygen species, and the functional activity of the peptides with the best activity in repairing oxidative damage induced by reactive oxygen species was analyzed based on the virtual screening results; in step C, the virtual screening method was as follows: Autodock software was used to study the binding energy between the peptide and Keap1, hydrogen bonds and intermolecular forces; Gromacs software was used to perform molecular dynamics simulation of the peptide-Keap1 complex through the CHARMM36 force field, and the molecular dynamics simulation conditions were as follows: the steepest descent algorithm was used for energy minimization, and the step size was 0.001 until the maximum force was <600.0 kJ / mol / nm; the frog leaping algorithm was used for 1 ns simulation, the step size was 2 fs, the temperature and pressure were set to 200 K and 1 bar, respectively, and the simulation time was 50 ns. The root mean square deviation, the number of hydrogen bonds, the radius of gyration, and the surface solvent accessible area were calculated to analyze the stability of the peptide-Keap1 complex; Gaussian 09 was used for density functional theory calculations, the semi-empirical AM1 method was used for configuration optimization, and the Gaussian 09 program, the active sites were analyzed using B3LYP / 6-31 to obtain the geometry, and the energy index and structural index of the peptide were calculated.

10. Use of the polypeptide according to claim 1, the polypeptide composition according to claim 2, or the polypeptide having antioxidant and repairing activity of oxidative damage induced by active oxygen obtained by the preparation method according to any one of claims 3 to 7, or the polypeptide having antioxidant and repairing activity of oxidative damage induced by active oxygen obtained by the identification and screening method according to any one of claims 8 to 9 in the preparation of antioxidant drugs.

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