Walnut meal active peptide and its preparation method and application

Abstract

The present invention belongs to the technical field of food-derived active peptides and discloses a walnut meal active peptide, wherein the amino acid sequence of the walnut meal active peptide is any one of SEQ ID NO. 1 and SEQ ID NO. 2. The walnut meal active peptide has excellent antioxidant activity, expands the application of walnut meal in industrial production, and provides a new technical basis and ideas.

Description

Technical Field

[0001] The invention belongs to the technical field of food-derived active peptides, and particularly relates to a walnut meal active peptide and a preparation method and application thereof. Background Art

[0002] In recent years, food-derived proteins have been extensively studied as a major source of functional peptides. These peptides are derived from food proteins and possess specific functional activities, typically with specific amino acid sequences. These peptides are released during protein degradation and exhibit high biological activities, such as immunomodulatory, antioxidant, antihypertensive, anti-inflammatory, and antimicrobial properties.

[0003] During normal aerobic metabolism, organisms produce numerous free radicals. Excessive free radical accumulation within human cells can cause oxidative stress, disrupting the body's original redox balance and contributing to aging and disease. As a potential source of natural antioxidants, food-derived antioxidant peptides not only provide nutrients but also exert antioxidant effects by inactivating reactive oxygen species (ROS) and chelating transition metals to prevent free radical formation. Against this backdrop, the development of food-derived antioxidant peptides has become a global research hotspot.

[0004] Walnuts are one of my country's major economic tree species, widely distributed in 21 provinces and municipalities, including Liaoning, Hunan, Sichuan, and Yunnan. Their cultivated area and annual output rank first in the world. Oil extraction is the primary way to utilize walnuts, but the resulting walnut meal is currently underutilized, resulting in significant resource waste. Summary of the Invention

[0005] The present invention aims to provide a walnut meal active peptide and a preparation method thereof. The walnut meal active peptide has excellent antioxidant activity.

[0006] The above-mentioned objectives are achieved by the following technical solutions.

[0007] A first aspect of the present invention provides a walnut meal active peptide, wherein the amino acid sequence of the walnut meal active peptide is any one of SEQ ID NO.1 and SEQ ID NO.2.

[0008] In some embodiments, the amino acid sequence of the walnut meal active peptide is shown as SEQ ID NO.2.

[0009] The second aspect of the present invention provides a use of the above-mentioned walnut meal active peptide in the preparation of an antioxidant.

[0010] A third aspect of the present invention provides a use of the walnut meal active peptide described above in the preparation of a drug for preventing and / or treating oxidative damage in zebrafish embryos.

[0011] A fourth aspect of the present invention provides an antioxidant, characterized in that the antioxidant comprises the walnut meal active peptide as described above.

[0012] A fifth aspect of the present invention provides a method for preparing the above-mentioned walnut meal active peptide, comprising the following steps:

[0013] walnut meal is subjected to a defatting pretreatment to obtain defatted walnut meal;

[0014] Extracting the defatted walnut meal powder using a fractionation method to obtain walnut meal complete protein;

[0015] Protease is added to the walnut meal whole protein for enzymolysis to obtain the walnut meal active peptides.

[0016] In some embodiments, the protease is alkaline protease.

[0017] In some embodiments, the fractionation method comprises the steps of:

[0018] Adding the defatted walnut meal powder into water to dissolve it and then centrifuging it to obtain an albumin solution and a precipitate;

[0019] Add NaCl solution to the precipitate 1, stir and centrifuge to obtain globulin solution and precipitate 2;

[0020] Add ethanol solution to the second precipitate, stir and centrifuge to obtain a supernatant and a third precipitate;

[0021] The supernatant was placed in a water bath at 55°C to 65°C and stirred until the ethanol evaporated to obtain alcohol-soluble protein solution. Sodium hydroxide solution was added to the precipitate 3, stirred and centrifuged to obtain gluten solution.

[0022] Adjust the albumin solution, globulin solution and gluten solution to the corresponding isoelectric points;

[0023] The albumin solution, globulin solution, gluten solution and alcohol-soluble protein solution adjusted to the isoelectric point are centrifuged to obtain precipitates, and the precipitates are mixed to obtain the walnut meal complete protein.

[0024] In some embodiments, the defatting pretreatment comprises the following steps: adding n-hexane to walnut meal, stirring to obtain a precipitate, and air-drying to obtain the defatted walnut meal; the solid-liquid ratio of the walnut meal to the n-hexane is 1:2 to 4.

[0025] In some embodiments, the added amount of the protease is 5900 U / g to 6100 U / g.

[0026] In the present invention, walnut meal is used as raw material, walnut whole protein is extracted from the walnut meal by a fractionation method, and then enzymatic hydrolysis products are obtained by using a specific alkaline protease. Finally, walnut meal active peptides are screened and found to be able to bind to the active part of the Keap1 protein. The binding is stable. In vivo experiments on zebrafish have found that the walnut meal active peptides have a strong protective effect on zebrafish embryos treated with hydrogen peroxide damage and can significantly improve the survival rate of zebrafish embryos. Therefore, the walnut meal active peptides have excellent antioxidant activity, expand the application of walnut meal in industrial production, and provide a new technical basis and ideas. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is a graph showing the results of the determination of the hydrolysis degree of five enzymes (data are expressed as mean ± standard deviation (n = 3), different letters indicate significant differences (P < 0.05)).

[0028] Figure 2 is the ABTS free radical scavenging rate of five enzymatic hydrolysis products; AH: alkaline protease hydrolysate; TH: trypsin hydrolysate; DH: neutral protease hydrolysate; FPH: flavor protease hydrolysate; PH: papain hydrolysate; GSH: glutathione (data are expressed as mean ± standard deviation (n = 9), different letters indicate significant differences (P < 0.05)).

[0029] Figure 3 is the DPPH free radical scavenging rate of the five enzymatic hydrolysis products (data are expressed as mean ± standard deviation (n = 9), different letters indicate significant differences (P < 0.05)).

[0030] Figure 4 is the hydroxyl radical scavenging rate of the five enzymatic hydrolysis products (data are expressed as mean ± standard deviation (n = 9), different letters indicate significant differences (P < 0.05)).

[0031] Figure 5 is the total reducing power of the five enzymatic hydrolysis products (data are expressed as mean ± standard deviation (n = 9), different letters indicate significant differences (P < 0.05)).

[0032] Figure 6 (A) is the hydroxyl radical scavenging rate of walnut protein alkaline protease hydrolysates at different concentrations; Figure 6 (B) is the DPPH clearance rate; Figure 6 (C) is the ABTS clearance rate (different letters indicate significant differences (P<0.05)).

[0033] Figure 7 (A) Effect of H2O2 concentration on the survival rate of zebrafish embryos; Figure 7(B) is the survival rate of zebrafish embryos treated with different concentrations of H2O2 for 96 h (different letters indicate significant differences (P<0.05)).

[0034] Figure 8 (A) Effects of different concentrations of enzymatic hydrolysates on the survival rate of zebrafish embryos; Figure 8 (B) Effects of different concentrations of enzymatic hydrolysate protection groups on the survival rate of zebrafish embryos.

[0035] Figure 9 is the MS / MS identification spectrum of the peptide sequence (Note: the letters ah correspond to the amino acid sequences listed in Table 2).

[0036] Figure 10 (A) is the amino acid interaction site between ALWPF and Keap1 receptor. Figure 10 (B) is the interaction diagram of ALWPF and Keap1 receptor; Figure 10 (C) is the amino acid interaction site between PLRWPF and Keap1 receptor; Figure 10 (D) is a diagram of the interaction between PLRWPF and Keap1 receptor.

[0037] Figure 11 Effects of different concentrations of ALWPF (A) and PLRWPF (B) on the survival rate of zebrafish embryos; effects of different concentrations of ALWPF (C) and PLRWPF (D) on the survival rate of zebrafish embryos after pre-protection.

[0038] Figure 12 The deformity of zebrafish embryos treated with different concentrations of ALWPF and PLRWPF; (Note: AF control group, H2O2 damage group, low concentration walnut meal active peptide ALWPF protection group, low concentration walnut meal active peptide PLRWPF protection group, high concentration ALWPF protection group, high concentration PLRWPF protection group). DETAILED DESCRIPTION

[0039] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0040] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0042] Example 1

[0043] 1 Method

[0044] 1.1 Walnut meal defatting pretreatment

[0045] Weigh a certain amount of walnut meal powder and add n-hexane at a material-liquid ratio of 1:3. Stir at room temperature for 2 hours and let it stand. After obvious stratification, pour out the supernatant. Repeat the above steps once, and place the lower precipitate in a fume hood to air-dry the solvent to obtain defatted walnut meal powder.

[0046] 1.2 Extraction of walnut protein

[0047] Weigh defatted walnut flour and add deionized water at a material-liquid ratio of 1:10. Stir at room temperature for 2 hours and centrifuge at 3600 rpm for 30 minutes. The supernatant is albumin. Add 5% NaCl solution at a material-liquid ratio of 1:10 to the remaining precipitate. Stir at room temperature for 2 hours and centrifuge at 3600 rpm for 30 minutes. The supernatant is globulin. Add 70% ethanol at a material-liquid ratio of 1:10 to the remaining precipitate. Stir at room temperature for 2 hours and centrifuge at 3600 rpm for 30 minutes. Place the supernatant in a 60°C waterbath and stir until the ethanol completely evaporates. This yields alcohol-soluble protein. Finally, add 0.1 mol / L NaOH solution at a material-liquid ratio of 1:20 to the remaining precipitate. Stir at room temperature for 2 hours and centrifuge at 3600 rpm for 30 minutes. The supernatant is gluten. The protein solution, excluding alcohol-soluble proteins, was adjusted to the corresponding isoelectric points (i.e., pH 4.1 for albumin, pH 4.3 for globulin, and pH 4.5 for gluten). The solution was centrifuged at 3600 rpm for 30 minutes to collect the precipitate. The protein precipitates were mixed and freeze-dried to obtain the complete walnut protein, which was then ground uniformly and stored at -80°C.

[0048] 1.3 Enzymatic hydrolysis process

[0049] Weigh a certain amount of walnut protein and add deionized water at a material-liquid ratio of 1:20. Hydrate for 2 hours, with three replicates for each sample. Enzymatically hydrolyze the protein for 3 hours according to the temperature, pH, and enzyme dosage shown in Table 1. Add 0.2 mol / L NaOH during the hydrolysis process to maintain the pH of the hydrolyzate within the set range, and record the volume of NaOH added. After the hydrolysis is completed, immediately place the solution in a 95°C water bath for 10 minutes to inactivate the enzyme. After the hydrolyzate cools to room temperature, adjust the pH to neutral, centrifuge at 10,000 rpm for 20 minutes, and freeze-dry the supernatant and store in a -20°C refrigerator for later use.

[0050] Table 1 Reaction conditions of different proteases

[0051] Enzyme type Enzymatic hydrolysis temperature pH Enzyme dosage (U / g) Alkaline protease 50 10.0 6000 Trypsin 37 7.0 6000 Flavor protease 50 6.5 6000 Papain 65 6.5 6000 Neutral protease 50 7.0 6000

[0052] 1.4 Determination of hydrolysis degree

[0053] The degree of hydrolysis was determined according to the pH-state method.

[0054]

[0055] B: volume of NaOH consumed (mL);

[0056] α: dissociation degree of α-NH2, The pK value is 7;

[0057] N b : molar concentration of NaOH (mol / L);

[0058] M p :Mass of protein (g)

[0059] h tot :7.35mmol / g

[0060] 1.3.5 Determination of antioxidant activity

[0061] (1) Analysis of ABTS free radical scavenging ability

[0062] Weigh 200 mg of ABTS solid and 34.4 mg of K₂S₂O₄ in water, then dilute to 50 mL. Incubate in a dark place for 24 hours to react. This will serve as the ABTS stock solution. Mix the stock solution with 95% ethanol in a ratio such that the absorbance at 734 nm remains around 0.7 ± 0.02. This will serve as the ABTS assay solution (prepare immediately for use).

[0063] Weigh different enzymatic hydrolysis products to prepare a 0.25 mg / mL test solution. Add 0.4 mL of the enzymatic hydrolysis solution and 3.6 mL of ABTS test solution to a centrifuge tube as the sample group; replace the enzymatic hydrolysis solution with deionized water as the blank group. After thorough mixing, place in a dark place away from light and react for 5 minutes. Use the blank group to adjust to zero and measure the absorbance at 734 nm. Calculate the ABTS free radical scavenging rate according to the following formula:

[0064]

[0065] (2) Analysis of DPPH free radical scavenging ability

[0066] Weigh different enzymatic hydrolysis products separately to prepare 2 mg / mL test solution. Add 1 mL of enzymatic hydrolysis solution and 3 mL of 50 μg / mL DPPH solution (anhydrous ethanol as solvent) into a centrifuge tube as the sample group; replace the DPPH solution with anhydrous ethanol as the control group; replace the test solution with deionized water as the blank group; after thorough mixing, immediately place in the dark to avoid light and react for 30 minutes. Measure its absorbance at a wavelength of 517 nm. Its DPPH free radical scavenging rate is calculated according to the following formula:

[0067]

[0068] Where:

[0069] A s ——Absorbance of the mixture of the test solution and DPPH solution

[0070] A c ——Absorbance of the mixture of the test solution and anhydrous ethanol

[0071] A b ——Absorbance of the mixture of DPPH solution and sample solvent solution

[0072] (3) Analysis of hydroxyl radical scavenging ability

[0073] Weigh different enzymatic hydrolysates to prepare a 1 mg / mL test solution. Before measurement, rinse the measuring tube with a borax-boric acid buffer solution at pH 7.5, and add 50 μL of the polypeptide test solution, 50 μL of 1.5 mmol / L o-phenanthroline, 50 μL of 1.25 mmol / LCuSO4, 20 μL of 0.25 mmol / L ascorbic acid, and 780 μL of a borax-boric acid buffer solution at pH 7.5 in sequence. Tap the measuring tube, add 50 μL of 30% hydrogen peroxide, and immediately place it in the reaction cell. Cover the lid and measure the luminescence intensity. Set the measurement program to a reaction time of 3000 s, measure the luminescence intensity every 0.1 s, stop the measurement after a clear peak appears, and record the peak value. Replace the polypeptide test solution with deionized water, measure its luminescence intensity, and record the peak value as the blank; its hydroxyl radical scavenging rate is calculated according to the following formula

[0074]

[0075] (4) Total reducing capacity analysis

[0076] Weigh different enzymatic hydrolysis products to prepare a 0.5 mg / mL test solution. Take 1 mL of the test solution, add 2.5 mL of 0.2 mol / L phosphate buffer (pH 6.6) and 2.5 mL of 1.0% K3[Fe(CN)6]. Mix thoroughly, and place in a 50°C water bath for 25 minutes. Immediately add 0.5 mL of 10% trichloroacetic acid solution to stop the reaction. Centrifuge at 10,000 rpm for 5 minutes. Take 2.5 mL of the supernatant, add 2.5 mL of deionized water and 0.5 mL of FeCl3, respectively, and react at room temperature for 10 minutes. Adjust the concentration to zero with deionized water, and measure the absorbance of the solution at 700 nm. The absorbance is positively correlated with the total reducing capacity.

[0077] In all the above experiments, GSH was used as a positive control, and three parallel experiments were set up.

[0078] 1.5 LC-MS / MS sequencing analysis

[0079] (1) Peptide desalting

[0080] The alkaline protein hydrolysate was dissolved in a washing solution (containing 0.1% FA and 2% ACN) and then transferred to a 10KD ultrafiltration centrifuge tube. The solution was centrifuged at 12,000 × g in a refrigerated centrifuge for 10 min. The ultrafiltration solution was desalted using a C18 desalting column and then eluted with an eluent (containing 0.1% FA and 60% ACN). The eluted solution was transferred to a new EP tube, and the eluted polypeptide sample was centrifuged, concentrated, and dried.

[0081] (2) LC-MS / MS analysis

[0082] After desalting, the peptide sample was centrifuged and dried, redissolved in 100 μL Nano-LC mobile phase A (0.1% formic acid / water), and loaded into a bottle for online LC / MS analysis. 2 μL of the dissolved sample was loaded onto a Nano Viper C18 pre-column (3 μm, ), 20 μL volume was used for desalting. High-performance liquid chromatography (HPLC) analysis was performed using the Easy nLC 1200 nanoliter HPLC system. The sample was desalted and retained on a pre-column before being separated on an analytical column. The analytical column specifications were a C18 reversed-phase column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm ), the mobile phase B (80% acetonitrile, 0.1% formic acid) was increased from 5% to 38% within 60 min. The amino acid sequence was analyzed using a ThermoFisher Q Exactive mass spectrometer system combined with a nanospray NanoFlex ion source. The spray voltage was 1.9 kV, and the ion transfer tube heating temperature was 275 ° C. The mass spectrometer scanning mode was data dependent analysis (DDA), the primary mass spectrometer scanning resolution was 70000, the scanning range was 100-1500 m / z, and the maximum injection time was 100 ms. A maximum of 20 charge 1 was collected in each DDA cycle. + to 3 + The maximum injection time for the secondary mass spectrometry ion was 50 ms. The collision cell energy was set to 28 eV for all precursor ions, and the dynamic exclusion time was set to 6 s.

[0083] 1.6 Screening of antioxidant peptides and simulated molecular docking

[0084] Peptide sequences were submitted to the PeptideRanker database (http: / / distilldeep.ucd.ie / PeptideRanker / ) for bioactive peptide prediction. The probability of a peptide possessing bioactivity is determined based on its amino acid composition and sequence, charge, and other properties (ranging from 0 to 1). A probability closer to 1 indicates a greater probability of bioactivity. A parameter threshold was manually set to 0.5, and all peptide sequences with a score above 0.5 were saved to an Excel spreadsheet.

[0085] The predicted bioactive peptide sequences were submitted to the BIOPEP-UWM database (http: / /

[0086] The antioxidant activity of the peptides was predicted by comparing the homology of the submitted peptides with those in the database. The peptides and amino acid sites with potential antioxidant activity were screened.

[0087] Molecular docking is widely used to study protein-peptide interactions and binding affinities. The Keap1 protein molecule was dehydrated and hydrogenated using AutoDock 1.5.6. The Keap1 protein served as the receptor for docking with the walnut meal active peptide. Nine docking runs were performed using the built-in VINA program, and the docked small molecule was exported in the "pbdqt" format. Contact and detail maps of the walnut meal active peptide and Keap1 protein with the highest binding energy were plotted using PyMOL 2.3.0. The bond parameters of the binding site were displayed using Discovery Studio 4.5. The 3D model was converted to a 2D plot using LigPlot+ v 2.2.8.

[0088] 1.7 Zebrafish culture and antioxidant model construction

[0089] Zebrafish were maintained under a 14h / 10h light / dark cycle and a water temperature of 28.0±0.5°C. They were fed shelled brine shrimp eggs three times daily (8:00, 12:00, and 17:00). Adult zebrafish were separated into incubation tanks separated by partitions at a male-to-female ratio of 1:2. The partitions were removed on the second day, and females were stimulated to spawn by light exposure, and embryos were harvested.

[0090] A H2O2-induced zebrafish embryo model was established. Experimental and control groups were treated with 1, 2, 3, 4, and 5 mM H2O2 solutions prepared in 60 μg / L sea salt solution, respectively. All groups were cultured in a 28.5°C incubator, with six replicates per group. The number of surviving embryos was recorded and the medium was replaced every 24 hours. Observation was continued for 96 hours, and the median lethal concentration (LD50) was calculated at 96 hours.

[0091] 1.8 Determination of antioxidant activity of walnut enzymatic hydrolysates in vivo

[0092] The control was set at 60 μg / L sea salt solution. Experimental groups were treated with 0.2, 2, 10, 20, and 200 mg / mL of the enzymatic hydrolysate prepared in 60 μg / L sea salt solution. The cells were cultured in a 28.5°C incubator, with six replicates per group. The number of surviving embryos was recorded and the medium was changed every 24 hours, with observation continuing for 96 hours.

[0093] The experimental group, hydrogen peroxide-damaged group, and control group were treated with a mixture of 1 mL of 40 mg / mL enzymatic hydrolysate and 4 mM hydrogen peroxide (2 mL total) in 60 μg / L sea salt solution, a 2 mM H₂O₂ solution, and a 60 μg / L sea salt solution, respectively. The embryos were cultured in a 28.5°C incubator, with six replicates per group. The number of surviving embryos was recorded and the medium was changed every 24 hours, and the embryos were observed continuously for 96 hours.

[0094] 1.9 Determination of the antioxidant activity of walnut meal active peptides in vivo

[0095] The control and experimental groups were treated with 10, 30, 50, 70, and 90 μg / mL walnut meal active peptide solutions prepared in 60 μg / L sea salt solution, and 60 μg / L sea salt solution, respectively. Three replicates were set up for each group. The number of surviving embryos was recorded after 24 hours.

[0096] 1.10 Protective effect of walnut meal active peptides on zebrafish embryos damaged by hydrogen peroxide

[0097] Experimental, hydrogen peroxide-damaged, and control groups were treated with a safe concentration of walnut meal active peptides for 24 hours, followed by treatment with a half-lethal concentration of hydrogen peroxide, a half-lethal concentration of hydrogen peroxide, and a 60 μg / L sea salt solution. All embryos were cultured in a 28.5°C incubator, with six replicates per group. The number of surviving embryos was recorded and the medium was changed every 24 hours, with observation continuing for 96 hours.

[0098] 1.11 Statistical Analysis

[0099] SPSS 27.0 software was used for statistical analysis, with a minimum significance level of 0.05. Duncan multiple comparisons were performed on the data from different treatments, and data are presented as mean ± standard deviation.

[0100] 2 Results and Analysis

[0101] 2.1 Analysis of hydrolysis degree of different proteases

[0102] Studies have shown that peptides with a molecular weight of <3KD have stronger antioxidant activity, and the higher the degree of hydrolysis, the greater the possibility of obtaining small molecule peptides. Figure 1 As shown, the hydrolysis degree of alkaline protease was 33.74% ± 0.21%, the hydrolysis degree of trypsin was 8.47% ± 0.34%, the hydrolysis degree of flavor protease was 22.40% ± 0.36%, the hydrolysis degree of papain was 16.86% ± 0.36%, and the hydrolysis degree of neutral protease was 11.25% ± 0.30%.

[0103] 2.2 Analysis of antioxidant activity of five enzymatic protein hydrolysates

[0104] 2.2.1 ABTS free radical scavenging rate

[0105] The method of judging the antioxidant activity of peptides by ABTS free radical scavenging rate is currently widely used. Figure 2 As shown, the ABTS free radical scavenging rates of the five hydrolysates are, from high to low, trypsin hydrolysate (TH, 68.59% ± 1.05%), alcalase hydrolysate (AH, 67.06% ± 0.36%), dispase hydrolysate (DH, 56.66% ± 1.82%), flavorzyme hydrolysate (FPH, 54.11% ± 0.45%), and papain hydrolysate (PH, 54.11% ± 1.69%). In this example, the free radical scavenging rate is close to 69.21 ± 7.66% when the concentration of the protease hydrolysate is only 0.25 mg / mL. Compared with the existing research in which the peptide was obtained by hydrolyzing walnut protein with trypsin, when the peptide concentration was 0.5 mg / mL, its ABTS free radical scavenging rate reached 69.21±7.66%.

[0106] 2.2.2DPPH free radical scavenging rate

[0107] DPPH radical is a stable free radical that can be used to evaluate the antioxidant activity of natural compounds. Figure 3 As shown, the scavenging rates of the enzymatic hydrolysates for DPPH radicals ranged from 55.32% to 73.82%, with the highest being FPH, TH, PH, NPH, and APH. The positive control, GSH, had a scavenging rate of 88.71% ± 0.29%. In this example, all five protease products, at a concentration of 2 mg / mL, exhibited high DPPH radical scavenging rates.

[0108] 2.2.3 Hydroxyl radical scavenging rate

[0109] Hydroxyl radical is the most active free radical and can 2+ or Fe 3+ It is formed by the reaction of superoxide anion and H2O2 in the presence of Figure 4 As shown, the hydrolysates of the five proteases all had high hydroxyl radical scavenging rates. Among them, the flavor protease hydrolysate had the highest hydroxyl radical scavenging rate, at 85.54% ± 1.48%. The positive control, GSH, had a scavenging rate of 99.12% ± 1.39%.

[0110] 2.2.4 Total reduction capacity

[0111] Studies have shown that antioxidant activity is directly related to reducing power. Figure 5 As shown, the enzymatic hydrolysates of the five proteases all exhibited very low reducing abilities. The trypsin hydrolysate had the highest reducing ability, with an absorbance of 0.124±0.014, while the positive control, GSH, had an absorbance of 1.046±0.003, much higher than the hydrolysate itself. Furthermore, in this example, the concentration of the protease hydrolysates used was only 0.5 mg / mL, yet all exhibited a certain degree of reducing ability.

[0112] As shown above, all five enzymatic hydrolysis products exhibit strong antioxidant activity. Walnut protein releases peptides with antioxidant activity after enzymatic hydrolysis. However, due to the different cleavage sites of different proteases, the relative molecular weights and amino acid sequences of the resulting peptides vary, leading to differences in their antioxidant activity. Among them, the alkaline protease hydrolysis products exhibited the highest antioxidant activity both in vivo and in vitro. Therefore, we conducted an in-depth peptidomic analysis of the walnut protein alkaline protease hydrolysate and identified all peptide sequences in the hydrolysis mixture.

[0113] 2.3 In vitro antioxidant assay of walnut protein alkaline protease hydrolysate

[0114] The hydroxyl radical scavenging rate of 2mg / mL walnut protein alkaline protease hydrolysate was significantly higher than that of GSH, and it had a strong hydroxyl radical scavenging ability; Figure 6 As shown, the hydroxyl radical scavenging rate of 5 mg / mL walnut protein alkaline protease hydrolysate was 98.63±0.06%; the DPPH radical scavenging rate of 0.5 mg / mL walnut protein alkaline protease hydrolysate was 71.56±0.75%; and the ABTS radical scavenging rate of 0.25 mg / mL walnut protein alkaline protease hydrolysate was 60.63±0.45%, all of which were higher than GSH.

[0115] 2.4 Establishment of hydrogen peroxide-induced oxidative stress injury model in zebrafish

[0116] Oxidative stress caused by hydrogen peroxide can cause an imbalance between the oxidative and antioxidant systems in the body, resulting in excessive production of highly active molecules in the body, which can cause tissue damage, such as Figure 7 As shown in the figure, compared to the survival rate of zebrafish embryos in the control group, the survival rate of zebrafish embryos treated with H2O2 at various concentrations within 48 hours was similar. However, the survival rate of zebrafish embryos treated with H2O2 at concentrations greater than 2mM decreased significantly after 48 hours. The experiment determined that the 50% lethal concentration (LC50) of H2O2 treated zebrafish embryos was approximately 2mM through linear interpolation.

[0117] 2.5 Toxicity test of walnut protein alkaline protease hydrolysate

[0118] The safe concentration range of walnut protein alkaline protease hydrolysate for treating zebrafish embryos is 2-200 mg / mL; walnut protein alkaline protease hydrolysate at concentrations of 2 mg / mL and 20 mg / mL can significantly improve embryo survival rate ( Figure 8 A); 20 mg / mL walnut protein alkaline protease hydrolysate has a protective effect on zebrafish embryos damaged by hydrogen peroxide, and the survival rate can be restored to the same level as the blank group ( Figure 8 B).

[0119] 2.6 Sequence identification and bioinformatics prediction of antioxidant peptides

[0120] Walnut protein alkaline protease hydrolysate was subjected to peptidomics analysis using LC-MS / MS and bioinformatics techniques, resulting in a total of 1,391 peptide sequences. Bioactive peptides and antioxidant peptides were screened using the PeptideRanker and BIOPEP-UWM databases, yielding 217 potential antioxidant peptides, representing 15.60% of the identified peptides. Of these, 70.07% were less than 10 amino acids in length. The top eight predicted antioxidant activity sequences are listed in Table 2, and their corresponding MS / MS spectra are shown in Table 2. Figure 9 .

[0121] Table 2 Sequence information of potential antioxidant peptide TOP8

[0122]

[0123]

[0124] As shown in Table 2, antioxidant peptides range in length from 2 to 10 amino acids, and the 2 to 3 core amino acids are often responsible for their activity. Peptidomics and bioinformatics predictive analysis confirm the presence of antioxidant peptides in alkaline protein hydrolysates, explaining the antioxidant activity of alkaline protein hydrolysates in the aforementioned experiments. This suggests that the analytical strategy combining protein enzymatic hydrolysis with peptidomics can uncover potential bioactive peptides in walnut processing byproducts. Further research on these potential antioxidant peptides is expected to enrich the natural antioxidant pool.

[0125] Further molecular simulation docking analysis was performed on the two peptides with the highest scores in Table 2, ALWPF and PLRWPF. Molecular docking is a theoretical simulation method to study the interaction between molecules and predict their binding mode and affinity. The docking of walnut meal active peptides (ALWPF, PLRWPF) with Keap1 protein molecules revealed that the two bind and interact mainly through hydrogen bonds and hydrophobic interactions ( Figure 10 A and Figure 10 C), with binding energies of -9.2 and -9.6 kJ / mol, respectively. Figure 10 B indicates that ALWPF forms hydrogen bonds with Thr560, Gly367, Val512, Val324, and Val608 of Keap1, with bond lengths of 2.91, 3.13, 2.99, 2.80, It has hydrophobic interactions with Ala466, Cys513, Val369, Val467, Val561, Cys368, Val420, Ile559, Ala607, Val418, Gly417, Ala366, and Ile416. Figure 10 D showed that PLRWPF formed hydrogen bonds with Val514, Val561, Gly367, Val418, and Ile416 of Keap1 protein, with bond lengths of 2.97, 3.13, 2.87, 2.99, It has hydrophobic interaction with Val604, Gly364, Ala366, Val463, Arg415, Val512, Leu365, Gly464, Val606, Val465, Cys513, Ile559, Val467, Gly417, Ala466, Val420, Thr560, Ala607, Cys368, and Val608.

[0126] 2.7 Toxicity test results of walnut meal active peptides

[0127] Compared with the control group, the survival rate of zebrafish embryos treated with walnut meal active peptides ALWPF and PLRWPF at a concentration of 10 μg / mL was significantly increased. As the concentration of walnut meal active peptides increased, the survival rate of zebrafish embryos decreased; among them, the safe concentration range of walnut meal active peptide ALWPF is 0-30 μg / mL, and the safe concentration range of PLRWPF is 0-50 μg / mL ( Figure 11 A and Figure 11 B).

[0128] 2.8 Protective effect of walnut meal active peptides (ALWPF, PLRWPF) on zebrafish embryos damaged by hydrogen peroxide

[0129] Compared with the blank group and the injury group, the survival rate of zebrafish embryos in the high concentration (30 μg / mL) ALWPF, PLRWPF protection group and the low concentration (10 μg / mL) PLRWPF protection group was significantly improved; after 48 hours, the survival rate of zebrafish embryos in the low concentration ALWPF protection group was similar to that in the injury group, and the protection effect was poor ( Figure 11 C and Figure 11 D); and at this time, a large number of scoliosis deformities appeared in the injury group, while the surviving zebrafish embryos in the protection group had no deformities ( Figure 12The results showed that high concentrations of ALWPF, PLRWPF and low concentrations of PLRWPF had a strong protective effect on zebrafish embryos damaged by hydrogen peroxide; while low concentrations of ALWPF had a weaker protective effect.

[0130] In summary, walnut meal in this embodiment is used as raw material, and walnut whole protein is extracted from walnut meal by fractionation method, and the antioxidant activity of alkaline protease, neutral protease, flavor protease, trypsin and papain enzymatic hydrolysis products is determined. It is found that the enzymatic hydrolysis products of walnut meal protein have high antioxidant activity, among which the alkaline protease hydrolyzate has the best comprehensive effect of antioxidant activity in vitro and in vivo. The alkaline protease hydrolyzate was subjected to LC-MS / MS analysis, and the obtained polypeptide sequence was subjected to antioxidant activity prediction. It was found that short peptides had higher potential antioxidant activity than longer peptides. A total of 217 potential antioxidant active peptides were screened out, accounting for 15.60%, proving that the alkaline protease enzymatic hydrolysis strategy has the potential to prepare walnut antioxidant peptides.

[0131] Oxidants cause the production of large amounts of free radicals within cells, damaging organelles and even chromosome telomeres, thereby accelerating cellular aging and ultimately leading to organ failure and aging. Exploring antioxidant mechanisms is of great significance to human health research. This study investigated the in vitro antioxidant activity of alkaline protease hydrolysates of walnut protein and found that the hydrolysates had a strong scavenging effect on DPPH, ABTS, and hydroxyl radicals. Furthermore, a 20 mg / mL hydrolysate protected zebrafish embryos from H2O2 damage, restoring embryonic survival to normal levels after exposure to a half-lethal concentration of H2O2. To identify peptides with antioxidant activity in the hydrolysates, LC-MS / MS identification and activity prediction were performed using the PeptideRanker and BIOPEP-UWM databases. The results showed that the most active peptides were ALWPF and PLRWPF.

[0132] The Keap1-Nfr2-ARE pathway is an important antioxidant pathway in the body; studies have shown that this pathway can induce the expression of genes related to nearly 500 proteins or enzymes such as oxidative balance factors, detoxification enzymes, emergency response proteins, and metabolic enzymes. Therefore, in this embodiment, Keap1 protein is used as a receptor protein to perform molecular docking with ALWPF and PLRWPF. The results show that they can all bind to the active site of Keap1, with binding energies of -9.2 and -9.6 kJ / mol, respectively. Low binding energy indicates that the molecular binding is stable. Through the zebrafish model, it was found that walnut meal active peptides (ALWPF, PLRWPF) have a protective effect on zebrafish embryos within a safe range, and as the concentration of walnut meal active peptides increases, their protective ability increases.

[0133] In summary, the screened walnut meal active peptides have good antioxidant activity, which broadens the application of walnut meal in industrial production and provides a new technical basis and ideas.

[0134] The results of this study can provide theoretical guidance for the deep processing of walnut processing by-products and lay the foundation for the research and development of new antioxidants.

[0135] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0136] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A walnut meal active peptide, characterized in that: The amino acid sequence of the walnut meal active peptide is shown in SEQ ID NO.

2.

2. Use of the walnut meal active peptide according to claim 1 in the preparation of an antioxidant.

3. An antioxidant, characterized in that The antioxidant comprises the walnut meal active peptide according to claim 1.

4. A method for preparing the walnut meal active peptide according to claim 1, characterized in that: The steps include: walnut meal is subjected to a defatting pretreatment to obtain defatted walnut meal; Extracting the defatted walnut meal powder using a fractionation method to obtain walnut meal complete protein; Alkaline protease is added to the whole protein of the walnut meal to perform enzymatic hydrolysis to obtain the walnut meal active peptide; The fractionation method comprises the following steps: The defatted walnut meal powder was added to water at a material-liquid ratio of 1:10, stirred at room temperature for 2 h, and centrifuged at 3600 r / min for 30 min to obtain an albumin solution and a precipitate. Add 5% NaCl solution to the precipitate 1 at a material-liquid ratio of 1:10, stir at room temperature for 2 h, and centrifuge at 3600 r / min for 30 min to obtain globulin solution and precipitate 2; Add 70% ethanol solution to the precipitate 2 at a material-liquid ratio of 1:10, stir at room temperature for 2 h, and centrifuge at 3600 r / min for 30 min to obtain the supernatant and precipitate 3; The supernatant was placed in a water bath at 55°C to 65°C and stirred until the ethanol evaporated to obtain alcohol-soluble protein solution. 0.1 mol / L sodium hydroxide solution was added to the precipitate at a material-liquid ratio of 1:

20. The mixture was stirred at room temperature for 2 h and centrifuged at 3600 rpm for 30 min to obtain gluten solution. Adjust the albumin solution, globulin solution and gluten solution to the corresponding isoelectric points; The albumin solution, globulin solution, gluten solution and alcohol-soluble protein solution adjusted to the isoelectric point are centrifuged to obtain precipitates, and the precipitates are mixed to obtain the walnut meal complete protein.

5. The preparation method according to claim 4, wherein The defatting pretreatment comprises the following steps: adding n-hexane to walnut meal powder, stirring to obtain a precipitate, and air-drying to obtain the defatted walnut meal powder; the material-liquid ratio of the walnut meal powder to the n-hexane is 1:2-4.

6. The preparation method according to claim 4, wherein The added amount of the protease is 5900U / g~6100U / g.

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