Preparation method of antioxidant tartary buckwheat polypeptide

Abstract

The invention provides a preparation method of antioxidant tartary buckwheat polypeptide, which comprises the following steps: extracting tartary buckwheat protein powder, dissolving the tartary buckwheat protein powder in distilled water, adding activated carbon for adsorption and decoloration to obtain a pretreated tartary buckwheat protein solution, adding alkaline protease into the pretreated tartary buckwheat protein solution for enzymolysis, and inactivating to obtain tartary buckwheat protein enzymatic hydrolysate; carrying out fractional separation on the enzymatic hydrolysate by using ultrafiltration membranes with the molecular weight cutoff of 3 kDa and 10 kDa, collecting polypeptide components with the molecular weight less than 3 kDa, carrying out gel filtration chromatography on the polypeptide components, collecting eluted components, carrying out freeze drying, and then carrying out desalination and liquid chromatography-tandem mass spectrometry identification, so as to obtain the antioxidant tartary buckwheat polypeptide. Activated carbon adsorption decolorization pretreatment is carried out before tartary buckwheat proteolysis, pigments and part of impurities can be effectively removed, and it is proved that the prepared antioxidant tartary buckwheat polypeptide has good antioxidant capacity in organisms through in-vitro and cell antioxidant combined evaluation and ROS detection.

Description

Technical Field

[0001] This invention belongs to the field of natural active polypeptide preparation and functional evaluation technology, specifically relating to a method for preparing an antioxidant tartary buckwheat polypeptide. Background Technology

[0002] Tartary buckwheat (Fagopyrum tataricum) is rich in high-quality plant protein, and its enzymatic hydrolysis products can form a variety of bioactive peptides. Naturally derived antioxidant peptides have broad application prospects in functional foods and biomedicine due to their high safety and good biocompatibility.

[0003] Existing research indicates that enzymatic hydrolysis of buckwheat protein can produce certain antioxidant substances, but the following shortcomings still exist: (1) Lack of a systematic integrated process of "pretreatment-enzymatic hydrolysis-separation-evaluation"; (2) Insufficient removal of impurities and pigments before enzymatic hydrolysis affects the accuracy of subsequent activity evaluation; (3) The differences in activity between polypeptide components of different molecular weights are not sufficiently studied; (4) Most studies focus on in vitro antioxidant activity, lacking validation at the cellular level; (5) Lack of further purification and structural analysis of active peptides.

[0004] In addition, buckwheat contains polyphenols and pigments, which may interfere with ultraviolet detection and antioxidant experiments. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing antioxidant tartary buckwheat peptides in response to the shortcomings of the prior art. The method involves pretreatment with activated carbon adsorption and decolorization before tartary buckwheat protein hydrolysis, which can effectively remove pigments and some impurities. In vitro and cellular antioxidant combined evaluation and ROS detection have proved that the antioxidant tartary buckwheat peptides prepared by this method have good antioxidant capacity in organisms and have broad application prospects.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing antioxidant buckwheat polypeptides, the method being as follows: S1. Extraction of buckwheat protein powder: S101. The dried buckwheat seeds are crushed and passed through a 100-mesh sieve to obtain buckwheat flour. Petroleum ether is added to the buckwheat flour to defatt it. After centrifugation and drying, defatted buckwheat flour is obtained. S102. Add distilled water to the defatted buckwheat powder obtained in S101, and adjust the pH value to 10 with a 1 M sodium hydroxide solution. Extract, centrifuge, take the supernatant, adjust the pH value of the supernatant to 4.5 with a 1 M hydrochloric acid solution, centrifuge, collect the precipitate, freeze-dry at -55℃ for 12 h to obtain buckwheat protein powder. S2, Activated carbon decolorization pretreatment: Distilled water is added to the buckwheat protein powder obtained in S102 to dissolve it into a buckwheat protein solution. Activated carbon is then added to the buckwheat protein solution for adsorption and decolorization at a pH of 7.9 for 53 min. After standing for 30 min, a pretreated buckwheat protein solution is obtained. S3, Buckwheat protein hydrolysis: Alkaline protease was added to the pretreated buckwheat protein solution obtained in S2, and the solution was hydrolyzed for 3 h at a temperature of 50℃ and a pH of 9.5. The protease was then inactivated at a temperature of 95℃ for 10 min to obtain buckwheat protein hydrolysate. S4. Ultrafiltration fractionation to screen out active components <3 kDa: The buckwheat protein hydrolysate obtained in S3 was separated by ultrafiltration using ultrafiltration membranes with molecular cutoffs of 3 kDa and 10 kDa to obtain three polypeptide components with molecular weights >10 kDa, 10 kDa ≥ molecular weight ≥3 kDa, and molecular weight <3 kDa, which were named polypeptide component 1, polypeptide component 2, and polypeptide component 3, respectively. S5. Gel chromatography purification: The peptide fraction 3 with the best antioxidant activity in S4 was subjected to gel filtration chromatography. Ultrapure water was used as the mobile phase for isocratic elution at a flow rate of 1.3 mL / min. The elution curve was monitored at a wavelength of 280 nm. Each elution fraction was collected, and two elution fractions were obtained, which were named elution fraction B1 and elution fraction B2, respectively. Both were freeze-dried at -55℃ for 12 h. S6. Peptide Structure Identification: Select elution fractions B1 and B2 obtained in S5, and first use C... 18 Desalting was performed using stage-tips, followed by vacuum drying at 45°C to obtain the desalted eluent. The sequence of the desalted eluent was identified by liquid chromatography-tandem mass spectrometry, yielding antioxidant tartary buckwheat peptides.

[0007] Preferably, the ratio of buckwheat flour to petroleum ether in S101 is 1 g: 5 mL; the centrifugation conditions are: centrifugation at 7000 rpm for 10 min.

[0008] Preferably, the ratio of defatted buckwheat powder to distilled water in S102 is 1 g: 10 mL; the extraction conditions are: extraction for 30 min at a power of 1000 W and a temperature of 45℃; and the centrifugation conditions are: centrifugation for 20 min at a speed of 7000 rpm.

[0009] Preferably, the ratio of the amount of buckwheat protein solution to activated carbon in S2 is 100 mL: 1.6 g.

[0010] Preferably, the conditions for liquid chromatography-tandem mass spectrometry in S6 are: a pre-column size of 150 μm·d. × 50 mm, packed with Reprosil-Pur 120 C. 18 -AQ3 μm; the analytical column size is 150 μm·d. × 170 mm, filled with Reprosil-Pur 120 °C. 18 -AQ 1.9 μm; mobile phase A is 0.1 v / v% formic acid aqueous solution; mobile phase B is 80 v / v% acetonitrile solution containing 0.1 v / v% formic acid; flow rate for both is 600 nL / min.

[0011] Preferably, the number of antioxidant tartary buckwheat peptides in S6 is seven, named antioxidant tartary buckwheat peptide 1, antioxidant tartary buckwheat peptide 2, antioxidant tartary buckwheat peptide 3, antioxidant tartary buckwheat peptide 4, antioxidant tartary buckwheat peptide 5, antioxidant tartary buckwheat peptide 6, and antioxidant tartary buckwheat peptide 7, respectively; the amino acid sequence of antioxidant tartary buckwheat peptide 1 is GFY, and its molar mass is 385.45 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 2 is as shown in SEQ ID No:1, and its molar mass is 567.69 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 3 is as shown in SEQ ID No:2, and its molar mass is 800.01 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 4 is as shown in SEQ ID No:3, and its molar mass is 690.86 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 5 is as shown in SEQ ID No:4, and its molar mass is 764.89 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 6 is as shown in SEQ ID No:5, and its molar mass is 600.77 g / mol. g / mol; the amino acid sequence of the antioxidant tartary buckwheat polypeptide 7 is shown in SEQ ID No:6, and the molar mass is 512.6 g / mol.

[0012] Compared with the prior art, the present invention has the following advantages: 1. Buckwheat contains polyphenols and pigments, which can easily interfere with UV detection and antioxidant experiments. Therefore, this invention performs activated carbon adsorption decolorization pretreatment before enzymatic hydrolysis to effectively remove pigments and some impurities, reducing interference with subsequent UV detection and antioxidant evaluation.

[0013] 2. This invention establishes a systematic method including decolorization pretreatment, enzymatic hydrolysis, graded screening and cell function verification to obtain highly active tartary buckwheat polypeptides.

[0014] 3. This invention uses "ultrafiltration fractionation + activity screening" to identify components with a value less than 3 kDa as the main antioxidant active components. Then, Sephadex G-25 gel chromatography column is used to separate the screened antioxidant active peptide components. Based on the difference in molecular weight, the peptides are further purified to improve the purity of the target active peptides.

[0015] 4. The antioxidant buckwheat peptides prepared in this invention, through in vitro and cellular antioxidant combined evaluation and ROS detection, can more realistically reflect the good antioxidant capacity of the prepared antioxidant buckwheat peptides in organisms, and have broad application prospects.

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is an interaction diagram of activated carbon addition amount and decolorization time in Example 2 of the present invention, showing the effect of the interaction of two factors on the decolorization rate.

[0018] Figure 2 This is a graph showing the interaction between activated carbon addition and pH in Example 2 of the present invention, illustrating the effect of the interaction between two factors on the decolorization rate.

[0019] Figure 3 This is a graph showing the interaction between decolorization time and pH in Example 2 of the present invention, illustrating the effect of the interaction between two factors on the decolorization rate.

[0020] Figure 4 This is a graph showing the interaction between the amount of activated carbon added and the decolorization time in Example 2 of the present invention, which illustrates the effect of the interaction between two factors on protein retention rate.

[0021] Figure 5 This is a graph showing the interaction between activated carbon addition and pH in Example 2 of the present invention, illustrating the effect of the interaction between two factors on protein retention rate.

[0022] Figure 6 This is a graph showing the interaction between decolorization time and pH in Example 2 of the present invention, illustrating the effect of the interaction between two factors on protein retention rate.

[0023] Figure 7 This is a graph showing the in vitro antioxidant capacity of different molecular weight tartary buckwheat peptides in Example 3 of the present invention.

[0024] Figure 8 This is the chromatogram of dextran gel separation in Example 4 of the present invention.

[0025] Figure 9 This is a comparison chart of the in vitro antioxidant activities of elution components B1 and B2 in Example 4 of the present invention.

[0026] Figure 10 This is a graph showing the ROS level of Heap1-6 cells in Example 5 of the present invention.

[0027] Figure 11 This is a visualization of molecular docking in Embodiment 6 of the present invention. Detailed Implementation

[0028] Example 1

[0029] This embodiment describes the preparation of antioxidant buckwheat peptides, using the following method: S1. Extraction of buckwheat protein powder: S101. The dried buckwheat seeds are crushed and passed through a 100-mesh sieve to obtain buckwheat flour. Petroleum ether is added to the buckwheat flour and mixed and stirred to defatt it. The ratio of buckwheat flour to petroleum ether is 1 g: 5 mL. After full extraction, the solvent is removed by centrifugation at 7000 rpm for 10 min. After evaporation in a fume hood, defatted buckwheat flour is obtained. S102. Add distilled water to the defatted buckwheat powder obtained in S101. The ratio of defatted buckwheat powder to distilled water is 1 g: 10 mL. Adjust the pH value to 10 with 1 M sodium hydroxide solution. Extract for 30 min at 1000 W and 45℃. Centrifuge for 20 min at 7000 rpm. Take the supernatant. Adjust the pH value of the supernatant to 4.5 with 1 M hydrochloric acid solution. Let stand for 30 min. Centrifuge for 20 min at 7000 rpm. Collect the precipitate and wash with water. Freeze dry at -55℃ for 12 h to obtain buckwheat protein powder. S2, Activated carbon decolorization pretreatment: Weigh 20 mg of the buckwheat protein powder obtained in S102 and dissolve it in 10 mL of distilled water to prepare a buckwheat protein solution of 20 mg / mL. Then add activated carbon to the buckwheat protein solution. The ratio of buckwheat protein solution to activated carbon is 100 mL: 1.6 g. After adsorption and decolorization for 53 min at a pH of 7.9, a pretreated buckwheat protein solution is obtained. S3. Tartary buckwheat protein hydrolysis: Alkaline protease (B8360, Solarbio Science & Technology Co., Ltd., Beijing) was added to the pretreated tartary buckwheat protein solution obtained in S2. The solution was hydrolyzed for 3 h at a temperature of 50℃ and a pH of 9.5. The amount of enzyme added was 14000 U / g. The protease was then inactivated at a temperature of 95℃ for 10 min to obtain tartary buckwheat protein hydrolysate. S4. Ultrafiltration fractionation to screen out active components <3 kDa: The buckwheat protein hydrolysate obtained in S3 was subjected to ultrafiltration fractionation using ultrafiltration membranes with molecular weight cutoffs of 3 kDa and 10 kDa to obtain three polypeptide components with molecular weights >10 kDa, 10 kDa ≥ molecular weight ≥3 kDa, and molecular weight <3 kDa, which were named polypeptide component 1, polypeptide component 2, and polypeptide component 3, respectively. S5. Gel Chromatography Purification: The peptide fraction 3 with the best antioxidant activity in S4 was separated using a Sephadex G-25 gel chromatography column. The specific steps were as follows: the chromatography system (including tubing and column) was thoroughly rinsed and equilibrated with ultrapure water; equilibration was considered complete when the baseline was stable and there was no absorption peak at 280 nm wavelength, as monitored by a UV detector; the sample was loaded, and the flow rate was controlled at 1.3 mL / min. Isocratic elution was performed using ultrapure water as the mobile phase. The elution curve was monitored at 280 nm wavelength, and fractions were collected according to the absorption peaks; each elution peak was collected to obtain two elution fractions, named B1 and B2, respectively, and both were freeze-dried at -55℃ for 12 h. S6. Peptide Structure Identification: Select two elution fractions B1 and B2 obtained in S5, and first use C... 18 Desalting was performed using stage-tips, followed by vacuum drying at 45°C. The final concentration was determined using a micro spectrophotometer to obtain the desalted eluent. The desalted eluent was then sequenced using liquid chromatography-tandem mass spectrometry (LC-MS / MS) on an Easy-nLC 1200 / QExactive system. The LC conditions were as follows: a pre-column size of 150 μm·d × 50 mm, packed with Reprosil-Pur 120 C. 18 -AQ3 μm; the analytical column size is 150 μm·d. × 170 mm, filled with Reprosil-Pur 120 °C. 18-AQ 1.9 μm; Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 80% acetonitrile solution containing 0.1% formic acid, using gradient elution at a constant flow rate of 600 nL / min; Mass spectrometry acquisition was performed in data-dependent acquisition mode (DDA). The primary mass spectrometry parameters were set as follows: resolution 70000, AGC target value 3e6, maximum injection time 100 ms, scan range 300–1800 m / z; the secondary mass spectrometry parameters were set as follows: resolution 17500, AGC target value 1e5, maximum injection time 50 ms. The top 20 most abundant precursor ions were selected for fragmentation in each cycle (TopN: 20), with a fragmentation energy of 28. The obtained raw mass spectrometry data were used for database searching using PEAKS Studio software. The search parameters were: fixed modification as carboxymethylation of cysteine ​​(Carbamidomethyl(C)), variable modification as oxidation of methionine (Oxidation(M)) and N-terminal acetylation of peptides (Acetyl(Peptide)). The enzyme digestion method was non-specific, the database was uniprotkb_taxonomy_id_62330_2025_12_10.fasta, and the mass deviation tolerance of the first and second stage mass spectrometry was set to 20 ppm and 0.02 Da, respectively. Finally, the antioxidant buckwheat peptide was obtained. The number of antioxidant tartary buckwheat peptides is seven, named antioxidant tartary buckwheat peptide 1, antioxidant tartary buckwheat peptide 2, antioxidant tartary buckwheat peptide 3, antioxidant tartary buckwheat peptide 4, antioxidant tartary buckwheat peptide 5, antioxidant tartary buckwheat peptide 6, and antioxidant tartary buckwheat peptide 7. The amino acid sequence of antioxidant tartary buckwheat peptide 1 is GFY, and its molar mass is 385.45 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 2 is SWLY (SEQ ID No:1), and its molar mass is 567.69 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 3 is AQLWPW (SEQ ID No:2), and its molar mass is 800.01 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 4 is LFWEP (SEQ ID No:33), and its molar mass is 690.86 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 5 is YWSYF (SEQ ID No:4), and its molar mass is 764.89 g / mol; the amino acid sequence of antioxidant tartary buckwheat peptide 6 is LWPW (SEQ ID No:33), and its molar mass is 764.89 g / mol. The amino acid sequence of the antioxidant buckwheat polypeptide 7 is SFPY (SEQ ID No: 5), with a molar mass of 600.77 g / mol; the molar mass of the antioxidant buckwheat polypeptide 7 is 512.6 g / mol. Example 2

[0030] This embodiment is an optimization of the activated carbon decolorization conditions in step S2 of the method for preparing antioxidant tartary buckwheat peptides in Example 1.

[0031] Using decolorization rate as the evaluation index, the effects of activated carbon addition, solution pH, and decolorization time on the decolorization effect were investigated. Based on single-factor experiments, activated carbon addition, pH, and decolorization time were selected as independent variables, and response surface methodology was used to optimize the decolorization conditions.

[0032] Experimental results are as follows Figures 1-6 As shown, where, Figures 1-3 The effect of the interaction of two factors on the decolorization rate, Figure 1 The interaction between activated carbon addition amount and decolorization time Figure 2 The interaction between activated carbon addition amount and pH Figure 3 The decolorization time and pH interact; Figures 4-6 The effect of the interaction of two factors on protein retention rate, Figure 4 The interaction between activated carbon addition amount and decolorization time Figure 5 The interaction between activated carbon addition amount and pH Figure 6 The decolorization time and pH interacted; each factor had a certain influence on the decolorization effect and there was an interaction. Through response surface methodology, the optimal decolorization process conditions were obtained as follows: activated carbon addition of 1.6%, pH of 7.9, and decolorization time of 53 min. Under these conditions, the decolorization effect of the buckwheat protein solution reached the best, with a decolorization rate of 81.8% and minimal impact on protein loss. Example 3

[0033] This embodiment compares the antioxidant activity of different molecular weight tartary buckwheat polypeptide components in step S4 of the method for preparing antioxidant tartary buckwheat polypeptides in Example 1.

[0034] Solutions of peptide component 1 (molecular weight > 10 kDa), peptide component 2 (3 kDa ~ 10 kDa), and peptide component 3 (molecular weight < 3 kDa) with concentrations of 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL were prepared respectively. Their in vitro antioxidant activities were measured, including DPPH free radical scavenging assay, total antioxidant capacity assay, and superoxide anion free radical scavenging assay.

[0035] Experimental results are as follows Figure 7 As shown, peptide components of different molecular weights all exhibited certain antioxidant activities, but the activities varied considerably. Among them, the components with molecular weights less than 3 kDa showed the strongest antioxidant capacity in all free radical scavenging experiments, followed by the 3 kDa to 10 kDa components, while the components with molecular weights greater than 10 kDa showed the lowest activity. These results indicate that small molecule peptides, due to their smaller molecular weight and relatively simpler structure, are more likely to react with free radicals, thus exhibiting stronger antioxidant capabilities.

[0036] Therefore, by using the method of "ultrafiltration fractionation + activity screening", target components with high antioxidant activity can be effectively screened out, among which tartary buckwheat peptides with less than 3 kDa are the preferred active components of this invention. Example 4

[0037] This embodiment compares the antioxidant activity of elution fractions B1 and B2 in step S5 of the method for preparing antioxidant tartary buckwheat peptides in Example 1.

[0038] After collecting the eluent, an elution curve was plotted based on the absorbance change. The sample was then separated into two components, B1 and B2, based on the elution peaks. Figure 8 As shown, the in vitro antioxidant activities of elution fractions B1 and B2 were determined, including DPPH, superoxide anion radical scavenging capacity, and total antioxidant capacity.

[0039] Experimental results are as follows Figure 9 As shown, the antioxidant activities of the peptide components corresponding to different elution peaks are different, among which elution component B2 shows a strong free radical scavenging ability. Example 5

[0040] This example illustrates the effect of the antioxidant tartary buckwheat peptide prepared in Example 1 on intracellular reactive oxygen species levels.

[0041] The antioxidant tartary buckwheat peptide obtained in Example 1 was used to evaluate its cellular antioxidant activity. Specifically, Hepa1-6 cells were seeded in culture plates and cultured at 37°C and 5% CO2 until the logarithmic growth phase. The cells were divided into a control group, a model group, and an antioxidant tartary buckwheat peptide treatment group (experimental group). The control group was not treated in any way. The model group was given 2 μg / mL of oxidative stress inducer (H2O2) to establish an oxidative damage model. The experimental group was pretreated with 0.5 mg / mL of antioxidant tartary buckwheat peptide before the addition of oxidative stress inducer.

[0042] After culturing for 4 h, the culture medium was discarded, and the DCFH-DA fluorescent probe was added. The cells were incubated in the dark for 30 min to allow the probe to enter the cells and be hydrolyzed into DCFH. Subsequently, under the action of reactive oxygen species (ROS), DCFH was oxidized into DCF with fluorescent properties. The intracellular ROS level was measured by fluorescence detection method.

[0043] Experimental results are as follows Figure 10 As shown in the figure, the scale bar is 200 μm. Compared with the control group, the intracellular ROS level in the model group was significantly increased, indicating that the oxidative stress model was successfully constructed. Compared with the model group, the intracellular ROS level in the experimental group was significantly decreased.

[0044] The above results demonstrate that the buckwheat polypeptide prepared in this invention can effectively reduce the level of reactive oxygen species in cells and alleviate oxidative stress damage, thereby exerting an antioxidant effect at the cellular level. Example 6

[0045] This embodiment is the identification and virtual screening of the amino acid sequence of the antioxidant tartary buckwheat prepared in Example 1.

[0046] Sequencing results yielded 10,272 peptides. To further screen for tartary buckwheat peptides with high antioxidant activity and save time, a computer-based virtual screening method was used to assess the bioactivity, toxicity, and antioxidant activity of each peptide. The PeptideRanker server (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict peptide bioactivity. PeptideRanker was trained with a threshold of 0.5, meaning any peptide with a prediction score exceeding 0.5 was labeled as a bioactive peptide; higher scores indicated stronger bioactivity. To reduce false positives, a threshold of 0.8 was chosen, meaning peptides with a prediction score exceeding 0.8 were considered bioactive. Based on predictions, 1379 peptides were highly likely to possess biological activity. ToxinPred was used to predict the toxicity and physicochemical properties of the peptides. All peptides were predicted to be non-toxic. However, considering that strong non-specific binding between highly positively charged peptides and negatively charged cell membranes could lead to cytotoxicity, and that peptides with strong hydrophobicity and a molar mass >1000 are not easily absorbed, 175 peptides remained after screening. These 175 peptides were then placed in the BIOPEP database to predict their potential activity, and previously reported peptides were removed. Most peptides were predicted to have multiple biological activities, including inhibition of dipeptidyl peptidase (DPP), inhibition of angiotensin-converting enzyme (ACE), antioxidant activity, and inhibition of α-glucosidase. Peptides with antioxidant activity were selected and scored on the AnOxPePred server, with scores ranging from 0 to 1. Generally, higher antioxidant activity corresponds to a higher score. Then, 24 peptides with an FRS score greater than 0.5 were selected for molecular docking. The safety assessment, activity prediction scores, and physicochemical properties of these 24 peptides are shown in Table 1.

[0047] Table 1. Predicted scores and physicochemical properties of antioxidant buckwheat peptides. Molecular docking of antioxidant buckwheat peptides with Keap1 protein: Table 2 shows the molecular docking scores of potential antioxidant peptides binding to Keap1 protein, based on molecular docking scoring. Lower docking scores indicate lower required binding energy and more stable binding. Seven peptides were found to have scores less than -7: GFY, SWLY, AQLWPW, LFWEP, YWSYF, LWPW, and SFPY. Molecular docking visualizations are shown below. Figure 11 As shown, a is AQLWPW, b is GFY, c is LFWEP, d is LWPW, e is SFPY, f is SWLY, and g is YWSYF. Through analysis of these seven Keap1-peptide docking models, all Keap1-side residues (such as R483, S602, R415, R380, Y525, and Y572) are located within the Kelch domain of Keap1, and most are consistent with the known key residues of Keap1-Nrf2 interaction. Therefore, the binding regions of these peptides all fall within the classic Kelch domain of Keap1-Nrf2 interaction, enabling them to act as competitive inhibitors to block this protein-protein interaction (PPI). Among them, R483 and S602 are the most frequently occurring core residues (each appearing 5 times), while the presence of arginine residues such as R415 and R380 further confirms the authenticity and reliability of the binding pocket. In summary, the binding of Keap1-LFWEP (containing R483, R415, R380, and S602, and the LFWEP peptide contains glutamate E4, which can form a strong salt bridge, mimicking the ETGE motif of natural Nrf2) and Keap1-SWLY... Figure 11 It can be seen that the binding site is associated with up to 6 key residues, which are linked by hydrogen bonds and may form an extensive hydrogen bond and hydrophobic network, thus making it a high-potential inhibitor.

[0048] Table 2. Docking score between potential antioxidant buckwheat peptides and Keap1 protein. In summary, the seven peptides with the strongest antioxidant capacity identified in this invention from antioxidant buckwheat peptides—GFY, SWLY, AQLWPW, LFWEP, YWSYF, LWPW, and SFPY—exhibit excellent free radical scavenging capabilities. These peptides can be used as natural antioxidant active ingredients in food, health products, pharmaceuticals, and cosmetics to improve the antioxidant properties of products, enhance nutritional value, or alleviate oxidative stress-related damage. Furthermore, these peptides exhibit high safety and can serve as a partial alternative source of synthetic antioxidants, demonstrating promising application prospects.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing an antioxidant buckwheat polypeptide, characterized in that, The method is as follows: S1. Extraction of buckwheat protein powder: S101. The dried buckwheat seeds are crushed and passed through a 100-mesh sieve to obtain buckwheat flour. Petroleum ether is added to the buckwheat flour to defatt it. After centrifugation and drying, defatted buckwheat flour is obtained. S102. Add distilled water to the defatted buckwheat powder obtained in S101, and adjust the pH value to 10 with a 1 M sodium hydroxide solution. Extract, centrifuge, take the supernatant, adjust the pH value of the supernatant to 4.5 with a 1 M hydrochloric acid solution, centrifuge, collect the precipitate, freeze-dry at -55℃ for 12 h to obtain buckwheat protein powder. S2, Activated carbon decolorization pretreatment: Distilled water is added to the buckwheat protein powder obtained in S102 to dissolve it into a buckwheat protein solution. Activated carbon is then added to the buckwheat protein solution for adsorption and decolorization at a pH of 7.9 for 53 min. After standing for 30 min, a pretreated buckwheat protein solution is obtained. S3, Buckwheat protein hydrolysis: Alkaline protease was added to the pretreated buckwheat protein solution obtained in S2, and the solution was hydrolyzed for 3 h at a temperature of 50℃ and a pH of 9.

5. The protease was then inactivated at a temperature of 95℃ for 10 min to obtain buckwheat protein hydrolysate. S4. Ultrafiltration fractionation to screen out active components <3 kDa: The buckwheat protein hydrolysate obtained in S3 was subjected to ultrafiltration fractionation using ultrafiltration membranes with molecular weight cutoffs of 3 kDa and 10 kDa to obtain three polypeptide components with molecular weights >10 kDa, 10 kDa ≥ molecular weight ≥3 kDa, and molecular weight <3 kDa, which were named polypeptide component 1, polypeptide component 2, and polypeptide component 3, respectively. S5. Gel chromatography purification: The peptide fraction 3 with the best antioxidant activity in S4 was subjected to gel filtration chromatography. Ultrapure water was used as the mobile phase for isocratic elution at a flow rate of 1.3 mL / min. The elution curve was monitored at a wavelength of 280 nm. Each elution fraction was collected, and two elution fractions were obtained, which were named elution fraction B1 and elution fraction B2, respectively. Both were freeze-dried at -55℃ for 12 h. S6. Peptide Structure Identification: Select elution fractions B1 and B2 obtained in S5, and first use C... 18 Desalting was performed using stage-tips, followed by vacuum drying at 45°C to obtain the desalted eluent. The sequence of the desalted eluent was identified by liquid chromatography-tandem mass spectrometry, yielding antioxidant tartary buckwheat peptides.

2. The method for preparing an antioxidant tartary buckwheat polypeptide according to claim 1, characterized in that, The ratio of buckwheat flour to petroleum ether in S101 is 1 g: 5 mL; the centrifugation conditions are: centrifugation at 7000 rpm for 10 min.

3. The method for preparing an antioxidant tartary buckwheat polypeptide according to claim 1, characterized in that, The ratio of defatted buckwheat powder to distilled water in S102 is 1 g: 10 mL; the extraction conditions are: extraction for 30 min at a power of 1000 W and a temperature of 45℃; the centrifugation conditions are: centrifugation for 20 min at a speed of 7000 rpm.

4. The method for preparing an antioxidant tartary buckwheat polypeptide according to claim 1, characterized in that, The ratio of buckwheat protein solution to activated carbon in S2 is 100 mL: 1.6 g.

5. The method for preparing an antioxidant tartary buckwheat polypeptide according to claim 1, characterized in that, The liquid chromatography-tandem mass spectrometry conditions described in S6 are as follows: the pre-column size is 150 μm·d. × 50 mm, and it is packed with Reprosil-Pur 120 C. 18 -AQ3 μm; the analytical column size is 150 μm·d. × 170 mm, filled with Reprosil-Pur 120 °C. 18 -AQ 1.9 μm; mobile phase A is a 0.1 v / v% aqueous solution of formic acid; mobile phase B is an 80 v / v% acetonitrile solution containing 0.1 v / v% formic acid; the flow rate for both is 600 nL / min.

6. The method for preparing an antioxidant buckwheat polypeptide according to claim 1, characterized in that, S6 describes seven antioxidant buckwheat peptides, named antioxidant buckwheat peptide 1, antioxidant buckwheat peptide 2, antioxidant buckwheat peptide 3, antioxidant buckwheat peptide 4, antioxidant buckwheat peptide 5, antioxidant buckwheat peptide 6, and antioxidant buckwheat peptide 7. The amino acid sequence of antioxidant buckwheat peptide 1 is GFY, and its molar mass is 385.45 g / mol. The amino acid sequence of antioxidant buckwheat peptide 2 is shown in SEQ ID No:1, and its molar mass is 567.69 g / mol. The amino acid sequence of antioxidant buckwheat peptide 3 is shown in SEQ ID No:2, and its molar mass is 800.01 g / mol. The amino acid sequence of antioxidant buckwheat peptide 4 is shown in SEQ ID No:3, and its molar mass is 690.86 g / mol. The amino acid sequence of antioxidant buckwheat peptide 5 is shown in SEQ ID No:4, and its molar mass is 764.89 g / mol. The amino acid sequence of antioxidant buckwheat peptide 6 is shown in SEQ ID No:5, and its molar mass is 600.77 g / mol. g / mol; the amino acid sequence of the antioxidant tartary buckwheat polypeptide 7 is shown in SEQ ID No:6, and the molar mass is 512.6 g / mol.

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