Carya illinoensis derived antioxidant active peptide as well as screening method and application thereof

Abstract

The invention discloses an apocarya source antioxidant active peptide as well as a screening method and application thereof. Three unreported antioxidant active peptides WDPFE, FDPFP and WDPNY with antioxidant activity are screened from carya illinoensis protein zymolyte for the first time, the carya illinoensis antioxidant peptides can improve the activity of human epithelial cells Caco-2 under the oxidative stress condition, the levels of antioxidant enzymes SOD and CAT in the cells are remarkably improved, and the antioxidant activity of the carya illinoensis is remarkably improved. And the compound is safe, non-toxic, good in water solubility and high in biological activity, can be widely applied to research and development of functional foods, medicines and health products, and has important economic value and social significance.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology and relates to anti-oxidative active peptides derived from walnut shells and a screening method and application thereof. Background Art

[0002] Antioxidant peptides are a class of functional molecules that reduce oxidative stress in the body by scavenging free radicals and inhibiting oxidation reactions. In recent years, with the increasing demand for antioxidants and the focus on natural, safe and efficient functional food ingredients, the research and development of antioxidant peptides has received widespread attention. The unique advantages of bioactive peptides, including small molecular weight, high absorption and utilization rate, low toxicity and side effects, and strong stability, make them have great potential for application in functional foods and drugs.

[0003] Studies have shown that antioxidant peptides can play a role through the following mechanisms: first, by providing hydrogen atoms or electrons through amino acid residues to remove free radicals; second, by chelating metal ions to inhibit their participation in oxidation reactions; third, by enhancing the activity of endogenous antioxidant enzymes (such as superoxide dismutase SOD and catalase CAT) to reduce cellular oxidative damage. The antioxidant properties of peptides are usually closely related to their molecular structure and amino acid composition. Aromatic amino acids (such as tyrosine and tryptophan) and amino acids with hydrophobic side chains are considered to be the key factors in their antioxidant capacity.

[0004] Carya illinoinensis is a nut with high economic value. Its kernel is not only rich in high-quality lipids and proteins, but also contains a variety of bioactive ingredients. Although the nutritional value of Carya illinoinensis has been widely studied, the high-value utilization of its protein, especially its application potential in the field of functional foods, has not been fully developed. At present, there are relatively few studies on the protein hydrolysates of Carya illinoinensis and their antioxidant functions, and it is urgent to explore its potential bioactive ingredients.

[0005] In response to this demand, the present invention successfully screened and identified a variety of small molecule peptides with significant antioxidant properties by enzymatic hydrolysis of thin-shell pecan protein. These peptides have good water solubility and stability, can significantly scavenge free radicals, increase the antioxidant enzyme activity of cells, and effectively alleviate oxidative stress damage, providing a new idea for the efficient utilization of thin-shell pecan protein, and at the same time providing a scientific basis for the development of natural, non-toxic antioxidant functional foods and drugs. Summary of the invention

[0006] In order to solve the above problems, the present invention provides thin-shell pecan source antioxidant active peptides and screening methods and applications thereof.

[0007] In one aspect, the present invention provides an antioxidant active peptide derived from walnuts, the antioxidant active peptide comprising at least one of the following amino acid sequences: WDPFE, FDPFP and / or WDPNY, wherein the amino acid sequence of WDPFE is SEQ ID No. 1, the amino acid sequence of FDPFP is SEQ ID No. 2, and the amino acid sequence of WDPNY is SEQ ID No. 3. Specifically, the amino acid sequence of WDPFE is Trp-Asp-Pro-Phe-Glu, the amino acid sequence of FDPFP is Phe-Asp-Pro-Phe-Pro, and the amino acid sequence of WDPNY is Trp-Asp-Pro-Asn-Tyr.

[0008] In one embodiment, the above-mentioned antioxidant active peptides are derived from pecan protein.

[0009] In one embodiment, the above antioxidant active peptides can improve the activity of human epithelial cells Caco-2 under oxidative stress conditions and significantly increase the levels of intracellular antioxidant enzymes SOD and CAT.

[0010] In one embodiment, the above-mentioned pecan-derived antioxidant active peptide has the ability to alleviate oxidative stress.

[0011] In another aspect, the present invention provides a method for screening anti-oxidative active peptides derived from walnut shells, the method comprising the following steps:

[0012] (1) Enzymatic hydrolysis: using neutral protease and alkaline protease to perform double enzymatic hydrolysis on the thin-shell pecan protein to obtain protein hydrolysate;

[0013] (2) ultrafiltration: ultrafiltration of the protein hydrolysate using a 3 kDa ultrafiltration membrane to collect components with a molecular weight less than 3 kDa;

[0014] (3) Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: The components with a molecular weight less than 3 kDa were subjected to reduction alkylation and desalting, and then the treated samples were analyzed by LC-MS / MS to obtain a raw file of the original mass spectrometry results. After PEAKSStudio10.6 De novo analysis, the peptide sequence analysis results of the pecan small molecule peptides were obtained;

[0015] (4) Screening: Virtual screening of the above peptide sequences is performed, and peptides with no toxicity, high biological activity and antioxidant function are screened according to bioinformatics prediction scores;

[0016] (5) In vitro assay: The antioxidant activity of the peptides in (4) was evaluated by ABTS and hydroxyl radical scavenging ability tests, as well as iron reducing power assay, and H 2 O 2The induced human colon cancer epithelial cell Caco-2 oxidative stress model was used to further verify the antioxidant activity of the candidate peptides, and the above-mentioned thin-shell pecan-derived antioxidant active peptides were screened and obtained.

[0017] In one embodiment, the enzymatic hydrolysis conditions of the neutral protease in the above double enzymatic hydrolysis process are pH 7.2 and temperature 53°C, and the enzymatic hydrolysis conditions of the alkaline protease are pH 8.4 and temperature 56°C.

[0018] On the other hand, the present invention also provides an application of an antioxidant active peptide derived from a pecan nut in the preparation of a functional food, wherein the functional food is intended to remove free radicals, relieve oxidative stress and improve the antioxidant capacity of the human body through antioxidant activity.

[0019] On the other hand, the present invention also provides a use of an antioxidant active peptide derived from walnut shells in the preparation of an antioxidant drug, wherein the antioxidant drug is used to prevent or treat diseases caused by oxidative stress.

[0020] In one embodiment, the active ingredient in the functional food or antioxidant drug is the above-mentioned antioxidant active peptide.

[0021] On the other hand, the present invention also provides a composition containing walnut-derived antioxidant active peptides, and the composition also includes food-grade auxiliary materials or pharmaceutical excipients.

[0022] This application is the first to screen out three previously unreported antioxidant active peptides WDPFE, FDPFP and WDPNY with antioxidant activity from thin-shelled pecan protein hydrolysate. The thin-shelled pecan antioxidant peptides can improve the activity of human epithelial cells Caco-2 under oxidative stress conditions and significantly increase the levels of intracellular antioxidant enzymes SOD and CAT. They are safe, non-toxic, have good water solubility and high biological activity. They can be widely used in the research and development of functional foods, medicines and health products, and have important economic value and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Control is a blank group, Model is a model control group, GSH is a positive control group (glutathione), WDPNY (WY5), WDPFE (WE5) and FDPFP (FP5) are peptide treatment groups. The different letters above each bar graph are significant differences between different treatments (p<0.05). Figure 5 The * on the bar graph indicates a significant difference compared with the Model group (p<0.05).

[0024] Figure 1It is the mass spectrometry analysis diagram of the antioxidant active peptides WDPNY, WDPFE and FDPFP of the present invention.

[0025] Figure 2 These are the test results of the iron reducing power index of the antioxidant active peptides WDPFE, FDPFP and WDPNY of the present invention.

[0026] Figure 3 The results are the test results of the ABTS free radical scavenging ability of the antioxidant active peptides WDPNY, WDPFE and FDPFP of the present invention.

[0027] Figure 4 This is to verify the toxicity of the antioxidant active peptides WDPNY, WDPFE and FDPFP of the present invention to Caco-2 cells.

[0028] Figure 5 The antioxidant activity of the peptides WDPNY, WDPFE and FDPFP of the present invention is 2 O 2 Improvement of oxidative stress induced in Caco-2 cells.

[0029] Figure 6 The antioxidant activity of the peptides WDPNY, WDPFE and FDPFP of the present invention is 2 O 2 Effects of ROS levels on oxidative stress in Caco-2 cells.

[0030] Figure 7 The antioxidant activity of the peptides WDPNY, WDPFE and FDPFP of the present invention is 2 O 2 Effect of MDA on malondialdehyde content in Caco-2 cells under oxidative stress induction.

[0031] Figure 8 The antioxidant activity of the peptides WDPNY, WDPFE and FDPFP of the present invention is 2 O 2 Effects of induced oxidative stress on SOD levels in Caco-2 cells.

[0032] Fig. 9 The present invention relates to the effects of the antioxidant active peptides WDPNY, WDPFE and FDPFP on the CAT catalase level under H2O2-induced oxidative stress in Caco-2 cells. DETAILED DESCRIPTION

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] Example 1

[0037] To illustrate the extraction of pecan protein, the steps are as follows:

[0038] 1. Raw material preparation: Select defatted walnut meal from thin shells, add it into distilled water at a ratio of 1:50 (w / v), and disperse it fully.

[0039] 2. Dissolution and pH adjustment: After heating the mixture to 55°C, adjust the pH to 11.0 using 1 mol / L sodium hydroxide solution and continue stirring for 90 minutes to ensure that the protein is completely dissolved.

[0040] 3. Centrifugation: Centrifuge at 8000 rpm for 10 minutes, collect the supernatant and remove impurities.

[0041] 4. Precipitate protein: Use 1 mol / L hydrochloric acid to adjust the pH of the supernatant to 4.0, let it stand and precipitate, and finally take the precipitate, which is the thin-shell pecan protein.

[0042] Example 2

[0043] To illustrate the preparation of thin-shell pecan peptide, the steps are as follows:

[0044] Disperse the thin-shell pecan protein in distilled water at a ratio of 1:50 (w / v), heat to 90°C for 15 minutes. Cool to the enzymatic temperature of 53°C, adjust the protein solution to pH 7.2 with 1.0mol / L NaOH, add 9000U / g neutral protease, and enzymolyze for 120 minutes. During the process, adjust the pH value to 7.2 with NaOH continuously. Put it in a 90°C water bath for 15 minutes to inactivate the enzyme. Cool to the enzymatic temperature of 56°C, adjust the protein solution to pH 8.4 with 1.0mol / L NaOH, add 7000U / g alkaline protease, and enzymolyze for 180 minutes. During the process, adjust the pH value to 8.4 with NaOH continuously. Put it in a 90°C water bath for 15 minutes to inactivate the enzyme, adjust the pH value to 7.0, 8000r / min, centrifuge at 4°C for 10 minutes, and the supernatant is the thin-shell pecan peptide.

[0045] The above-mentioned thin-shell pecan peptide liquid was subjected to ultrafiltration separation using a 3kDa ultrafiltration membrane and a peristaltic pump at 120rpm. The ultrafiltration fractions with molecular weight <3kDa in the filtrate were collected and freeze-dried in a vacuum to prepare thin-shell pecan peptide dry powder.

[0046] Example 3

[0047] To illustrate the structural identification of pecan protein peptide, the steps are as follows:

[0048] (1) Sample pretreatment: Weigh 1 mg of the thin-shelled pecan peptide powder obtained in Example 2, add an appropriate amount of 50 mmol / L ammonium bicarbonate solution to dissolve the sample, add dithiothreitol solution to make the final concentration 10 mmol / L, and reduce it in a 56°C water bath for 1 h. Add iodoacetamide solution to make the final concentration 55 mmol / L, react in the dark for 40 min, desalt using a self-filled desalting column, and evaporate the solvent in a 45°C vacuum centrifugal concentrator.

[0049] (2) LC-MS / MS detection: Liquid chromatography conditions: pre-column: 150 μm idx 5 cm, filler: Reprosil-Pur120C18-AQ 3 μm; analytical column: 100 μm idx 180 mm, filler: Reprosil-Pur 120 C18-AQ 3 μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 80% ACN / 0.1% formic acid aqueous solution; flow rate: 600 nL / min; analysis time for each component: 60 min.

[0050] Mass spectrometry conditions: primary mass spectrometry parameters: resolution: 70,000; AGC target: 3e6; Maximum IT: 60 ms; scan range: 100 to 1500 m / z. secondary mass spectrometry parameters: resolution: 17,500; AGC target: 5e4; Maximum IT: 80 ms; NCE / stepped NCE: 27.

[0051] (3) Database search: The mass spectrometry raw files were analyzed for peptide sequences using the PEAKS Studio 10.6 De novo method. The search parameters were as follows: fixed modifications; variable modifications; enzyme; maximum missed cleavages; primary mass spectrometry error; fragment mass tolerance. The mass spectrometry analysis results of WDPNY, WDPFE, and FDPFP were identified by LC-MS / MS. Figure 1 , which is the antioxidant active peptide of the present invention (attached Figure 1 A: WDPFE, attached Figure 1 B: FDPFP, attached Figure 1 C: WDPNY).

[0052] Example 4

[0053] The steps of bioinformatics test for describing the antioxidant activity of pecan peptides WDPFE, FDPFP and WDPNY are as follows:

[0054] (1) Prediction of potential biological activity of peptides: The potential biological activity of the obtained peptide sequences was analyzed using the PeptideRanker online platform (http: / / distilldeep.ucd). Peptides were ranked according to their predicted probability of biological activity. The preset threshold of the PeptideRanker prediction model was 0.5, and a threshold greater than 0.5 was considered to have biological activity. The predicted values ​​of the new peptides WDPFE, FDPFP, and WDPNY screened in this application were 0.907842, 0.983751, and 0.840305, respectively. The results of bioinformatics prediction analysis showed that WDPFE, FDPFP, and WDPNY had high potential biological activity.

[0055] (2) Prediction of potential toxicity of peptides: using the online tool Toxinpred

[0056] (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) platform, the potential toxicity of new peptides was predicted based on the SVM (Swiss-Port) algorithm. After potential toxicity analysis, the peptides WDPFE, FDPFP and WDPNY were Non-Toxin (non-toxic).

[0057] (3) Prediction of potential antioxidant activity of peptides: using the online website tool AnOxPePred

[0058] (https: / / services.healthtech.dtu.dk / services / AnOxPePred-1.0 / ) platform, based on the peptide pattern algorithm to predict the potential of peptides to scavenge free radicals and ion chelate to evaluate their antioxidant capacity. After potential antioxidant activity analysis, the free radical scavenging ability scores of peptides WDPFE, FDPFP and WDPNY were 0.48352, 0.47303 and 0.52326, respectively, and the ion chelating ability scores were 0.2497, 0.28511 and 0.26513, respectively, showing potential antioxidant activity.

[0059] Example 5

[0060] The steps used to illustrate the chemical synthesis of the antioxidant active peptides WDPFE, FDPFP and WDPNY from walnut shells and their in vitro chemical antioxidant and cellular antioxidant capacity detection are as follows:

[0061] (1) Peptide synthesis: The small peptides WDPFE (Trp-Asp-Pro-Phe-Glu), FDPFP (Phe-Asp-Pro-Phe-Pro) and WDPNY (Trp-Asp-Pro-Asn-Tyr) were chemically synthesized using amino acid solid phase synthesis technology. The purity of the synthetic peptides produced by Jiangsu Jitai Peptide Technology Co., Ltd. was >95%.

[0062] (2) Verification of the in vitro chemical antioxidant function of WDPFE, FDPFP and WDPNY peptides: Ferric reducing ability FRAP (Ferric Reducing Ability of Plasma) and ABTS free radical scavenging ability were used to detect in vitro chemical antioxidant indicators:

[0063] i. Iron reducing power index of peptide

[0064] The total antioxidant capacity detection kit (FRAP method) of Beyotime Biotech was used for detection, and the standard curve was prepared according to the instructions, and the antioxidant capacity of the peptide was obtained by substituting it into the calculation formula.

[0065] According to the experimental results, the absorbance of the treatment groups WDPNY, WDPFE and FDPFP after mixing with the FRAP working solution at 37 degrees Celsius for 5 minutes was measured at a wavelength of 593 nm. Substituting into the standard curve, it can be seen that the absorbance values ​​of the 5 mM peptide solution corresponding to the FeSO 4 The concentrations are 1.24mM, 1.31mM and 1.27mM respectively. According to the calculation, we can get Figure 2As shown in the figure, the iron reducing power of the relative antioxidant capacity of WDPNY, WDPFE and FDPFP are 0.247, 0.262 and 0.254 respectively. Compared with the relative antioxidant capacity of the positive control GSH of 0.608, although the iron reducing power of the peptides is not as good as the positive control glutathione, it still has a certain antioxidant capacity.

[0066] ii. ABTS free radical scavenging ability

[0067] The total antioxidant capacity detection kit (ABTS method) of Beyotime Biotechnology Company was used for detection, and the standard curve was prepared according to the instructions, and the antioxidant capacity of the peptide was obtained by substituting it into the calculation formula.

[0068] According to the experimental results, the absorbance of the treatment groups WDPNY, WDPFE and FDPFP mixed with ABTS working solution and incubated at room temperature for 6 minutes was measured at a wavelength of 734 nm. Substituting into the standard curve, it can be seen that the absorbance values ​​of 1.0 mM peptide solution correspond to Trolox concentrations of 1.592 mM, 1.259 mM and 0.202 mM, respectively. According to the calculation, the following can be obtained: Figure 3 As shown, the iron reducing power of the relative antioxidant capacity of WDPNY, WDPFE and FDPFP were 1.592mM, 1.259mM and 0.202mM respectively. Compared with the relative antioxidant capacity of the positive control GSH of 0.893mM, the ABTS free radical scavenging ability of WDPNY and WDPFE peptides was significantly higher than that of the positive control glutathione, but the scavenging ability of FDPFP on ABTS free radicals was weaker.

[0069] (3) In vitro cellular antioxidant function verification of WDPFE, FDPFP and WDPNY peptides

[0070] The Caco-2 cells in the logarithmic phase were collected and passaged using DMEM complete medium containing 20% ​​fetal bovine serum + 1% penicillin / streptomycin. 2 , 37°C incubator. For 96-well plate culture, 1×10 4 Inoculate at a density of 100 cells / well in a 5% CO 2 , 37°C incubator. Add 100 μL DMEM complete medium to 6 wells of the 96-well plate, and replace with 100 μL DMEM basal medium after the cells adhere to the wall after 24 h of culture, as the blank group (Control). Add 100 μL DMEM complete medium to 6 wells of the 96-well plate, and replace with 100 μL DMEM basal medium after the cells adhere to the wall after 24 h of culture, as the blank group (Control). 2 O 2The cells were treated with DMEM basal medium for 2 h as the model group (Model). DMEM complete solution of peptides (WDPNY, WDPFE and FDPFP) with gradient concentrations of 25-3200 μM was added to the 96-well plate (6 wells for each concentration) and replaced with 100 μL of 2.0 mM PBS after culturing for 24 h. 2 O 2 DMEM basal medium was used for 2 h as the peptide sample group. For 6-well plate culture, 1×10 6 Inoculate at a density of 100 cells / well in a 5% CO 2 , 37°C incubator. Add 2 mL of DMEM complete medium to each well of the 6-well plate, and replace with 2 mL of new DMEM basal medium after the cells adhere to the wall after 24 h of culture, as the blank group (Control). Add 2 mL of DMEM complete medium to each well of another 6-well plate, and replace with 2 mL of 2.0 mM H 2 O 2 DMEM basal medium was used for 2 h as the model group (Model). DMEM complete solution containing 400 μM peptides (WDPNY, WDPFE and FDPFP) was added to each well of another 6-well plate and replaced with 2 mL of 2.0 mM H 2 O 2 DMEM basal medium was used for 2 h as the peptide sample group. For 24-well plate culture, 1×10 5 Inoculate at a density of 100 cells / well in a 5% CO 2 , 37°C incubator. Add 0.5 mL of DMEM complete medium to 3 wells of a 24-well plate, and replace with 0.5 mL of new DMEM basal medium after the cells adhere to the wall after 24 h of culture, as a blank group (Control). Add 0.5 mL of DMEM complete medium to 3 wells of a 24-well plate, and replace with 0.5 mL of 2.0 mM H 2 O 2 The cells were treated with DMEM basal medium for 2 h as the model group (Model). DMEM complete solution containing 400 μM peptides (WDPNY, WDPFE and FDPFP) was added to 3 wells of a 24-well plate and replaced with 0.5 mL of 2.0 mM H 2 O 2 The cells were treated with DMEM basal medium for 2 h as the peptide sample group.

[0071] i. Toxicity of peptides to Caco-2 cells

[0072] After the blank group (Control) and peptide sample group (25-3200 μM) were treated in the 96-well plate, the culture medium was completely aspirated and washed twice with 0.01M pH 7.4 PBS. 100 μL of 10% CCK-8 was added to each well and placed in a 5% CO 2 After 2 hours in a 37°C incubator, the absorbance of each well was measured at 450 nm using an enzyme marker, and the average value of 6 wells in each group was taken. Cell viability was calculated according to the formula. Reference formula: Cell viability % = absorbance of peptide sample group / absorbance of blank group × 100%.

[0073] from Figure 4 It can be seen that under the treatment of WDPNY, WDPFE and FDPFP peptides at concentrations of 25-3200 μM, the viability of Caco-2 cells was above 90%, which means that WDPNY, WDPFE and FDPFP peptides had no toxic effect on cells under these concentration gradients.

[0074] ii. Effects of WDPNY, WDPFE and FDPFP peptides on H 2 O 2 Improvement of oxidative stress induced in Caco-2 cells

[0075] After the blank group (Control), model group (Model) and peptide sample group (400 and 800 μM) were treated in the 96-well plate, the culture medium was completely aspirated and washed twice with 0.01M pH 7.4 PBS. 100 μL of 10% CCK-8 was added to each well and placed in a 5% CO 2 After 2 hours in a 37°C incubator, the absorbance of each well was measured at 450 nm using an enzyme marker, and the average value of 6 wells in each group was taken. Cell viability was calculated according to the formula. Reference formula: Cell viability % = absorbance of model group or peptide sample group / absorbance of blank group × 100%.

[0076] from Figure 5 It can be seen that under the action of 400 and 800 μM concentrations of WDPNY, WDPFE and FDPFP peptides, H 2 O 2 The viability of Caco-2 cells after induction was significantly higher than that of the Model group, which means that the peptide at this concentration had no significant effect on H 2 O 2 Inducing oxidative stress in Caco-2 cells has a certain improvement effect.

[0077] iii. Effects of WDPNY, WDPFE and FDPFP peptides on H 2 O 2 Effect of ROS levels on oxidative stress in Caco-2 cells

[0078] After the blank group, model group and peptide sample group were treated in the 24-well plate, the culture medium was aspirated and washed twice with 0.01M pH 7.4 PBS. 500 μL of basal DMEM medium solution containing 10 μmol / L DCFH-DA was added to each well and placed in 5% CO 2 , incubated at 37°C for 30 minutes. Wash the cells three times with serum-free DMEM cell culture medium to fully remove the DCFH-DA that has not entered the cells. Take out the cell slides and observe and photograph them with a fluorescence microscope. Use black and white mode and green pseudocolor to calculate the gray value of the fixed cell area and the fluorescence value.

[0079] like Figure 6 As shown, WDPNY, WDPFE and FDPFP peptides can reduce the level of reactive oxygen species in Caco-2 cells, thereby improving the degree of cellular oxidative stress and achieving antioxidant effects, especially WDPNY and FDPFP peptides have stronger effects.

[0080] iv. Effect of WDPNY, WDPFE and FDPFP peptides on H 2 O 2 Effect of MDA malondialdehyde content on Caco-2 cells induced by oxidative stress

[0081] After the blank group, model group and peptide sample group were treated in a 6-well plate, all the culture medium was aspirated and washed twice with 0.01M pH7.4 PBS. Collect the cells, centrifuge at 1000rpm for 3 minutes, and discard the supernatant. Add 2mL of 0.01M pH7.4 PBS and gently blow to mix, centrifuge again at 1000rpm for 3 minutes, discard the supernatant, and repeat twice. Add 1mL of MDA extract in the MDA kit, and ultrasonically disrupt the cells on ice (power 200W, ultrasound 3 seconds, interval 10 seconds, repeat 30 times), centrifuge at 8000g at 4°C for 10 minutes, take the supernatant and place it on ice for testing. The extract was tested according to the Solebao malondialdehyde (MDA) content detection kit.

[0082] like Figure 7 As shown in the figure, WDPNY, WDPFE and FDPFP peptides can significantly reduce the level of malondialdehyde (MDA) in Caco-2 cells, thereby improving the degree of oxidative stress in cells and achieving antioxidant effects, especially WDPNY and WDPFE have stronger effects. Malondialdehyde is a hallmark product of lipid peroxidation, and its increased level usually reflects the degree of damage to cells caused by oxidative stress. The intervention of WDPNY, WDPFE and FDPFP peptides significantly reduced the generation of MDA, indicating that WDPNY, WDPFE and FDPFP peptides can effectively inhibit lipid peroxidation and protect cells from oxidative damage.

[0083] v. Effects of WDPNY, WDPFE and FDPFP peptides on H2 O 2 Effect of superoxide dismutase level on oxidative stress in Caco-2 cells

[0084] The operation is the same as step iv above. The extract is tested using the Solebol superoxide dismutase (SOD) activity test kit (WST-1 method).

[0085] like Figure 8 As shown in the figure, WDPNY, WDPFE and FDPFP peptides can significantly increase the activity of superoxide dismutase (SOD) in Caco-2 cells, thereby effectively alleviating the oxidative stress state of cells and exerting significant antioxidant effects. SOD is an important antioxidant enzyme in cells, which is mainly responsible for catalyzing the superoxide anion free radical (O 2 - ) to generate relatively low-toxic hydrogen peroxide and oxygen, thereby reducing the damage of free radicals to cells. The experimental results show that WDPNY, WDPFE and FDPFP peptides can activate the antioxidant defense system in cells and enhance the antioxidant capacity of cells by increasing SOD activity.

[0086] vi. Effects of WDPNY, WDPFE and FDPFP peptides on H 2 O 2 Effect of CAT on catalase levels under oxidative stress in Caco-2 cells

[0087] The operation is the same as step iv above. The extract is tested using the Solebol Catalase (CAT) Activity Detection Kit.

[0088] like Fig. 9 As shown in the figure, the WDPFE peptide treatment did not significantly increase the activity of catalase (CAT) in Caco-2 cells, while the WDPNY and FDPFP peptides significantly increased the activity of catalase (CAT) in the cells, effectively alleviating the oxidative stress state and showing significant antioxidant effect. CAT is one of the important antioxidant enzymes in cells, which can quickly decompose hydrogen peroxide (H 2 O 2 ) generates water and oxygen, thereby reducing oxidative damage to cells.

[0089] The test results of the above multiple antioxidant indicators show that the thin-shell pecan-derived peptides WDPNY, WDPFE and FDPFP have antioxidant activity and can be used in the development of foods and drugs related to antioxidant efficacy.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antioxidant active peptide derived from walnut shell, characterized in that: The antioxidant active peptide comprises at least one of the following amino acid sequences: WDPFE, FDPFP and / or WDPNY, wherein the amino acid sequence of WDPFE is SEQ ID No.1, the amino acid sequence of FDPFP is SEQ ID No.2, and the amino acid sequence of WDPNY is SEQ ID No.

3.

2. The antioxidant peptide according to claim 1, characterized in that The antioxidant active peptide is derived from pecan protein.

3. The antioxidant active peptide according to claim 1, characterized in that The antioxidant active peptide can improve the activity of human epithelial cells Caco-2 under oxidative stress conditions and significantly increase the levels of intracellular antioxidant enzymes SOD and CAT.

4. The antioxidant active peptide according to claim 1, characterized in that The antioxidant active peptide has the ability to alleviate oxidative stress.

5. A method for screening anti-oxidative active peptides derived from walnut shells, characterized in that: The method comprises the following steps: (1) Enzymatic hydrolysis: using neutral protease and alkaline protease to perform double enzymatic hydrolysis on the thin-shell pecan protein to obtain protein hydrolysate; (2) ultrafiltration: ultrafiltration of the protein hydrolysate using a 3 kDa ultrafiltration membrane to collect components with a molecular weight less than 3 kDa; (3) Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: The components with a molecular weight less than 3 kDa were subjected to reduction alkylation and desalting, and then the treated samples were analyzed by LC-MS / MS to obtain a raw file of the original mass spectrometry results. After PEAKSStudio10.6 De novo analysis, the peptide sequence analysis results of the pecan small molecule peptides were obtained; (4) Screening: Virtual screening of the peptide sequences is performed, and peptides with no toxicity, high biological activity and antioxidant function are screened according to bioinformatics prediction and scoring; (5) In vitro assay: The antioxidant properties of the polypeptide described in (4) were evaluated by ABTS and hydroxyl radical scavenging ability tests, as well as iron reducing power assay methods, and the antioxidant activity of the candidate peptides was further verified using the H2O2-induced human colon cancer epithelial cell Caco-2 oxidative stress model, thereby screening and obtaining the thin-shell pecan source antioxidant active peptide described in claim 1.

6. The method according to claim 5, characterized in that The enzymatic hydrolysis conditions of the neutral protease in the double enzymatic hydrolysis process are pH 7.2 and temperature 53°C, and the enzymatic hydrolysis conditions of the alkaline protease are pH 8.4 and temperature 56°C.

7. An application of an antioxidant active peptide derived from walnut shell in the preparation of functional food, characterized in that: The functional food is intended to scavenge free radicals, relieve oxidative stress and enhance the body's antioxidant capacity through antioxidant activity.

8. Use of an antioxidant active peptide derived from walnut shell in the preparation of antioxidant drugs, characterized in that: The antioxidant drug is used for preventing or treating diseases caused by oxidative stress.

9. The use according to claim 7 or 8, characterized in that: The active ingredient in the functional food or antioxidant drug is the antioxidant active peptide according to claim 1.

10. A composition comprising an antioxidant active peptide derived from walnut shell, characterized in that: The composition further comprises food grade auxiliary materials or pharmaceutical excipients.

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