A Cu-MOF material for specifically enriching antioxidant peptides and its preparation and application method

Abstract

The present invention discloses a Cu-MOF material for specifically enriching antioxidant peptides and its preparation and application method. The Cu-MOF is an organic metal framework material with Cu as the metal atom and a specific surface area of ​​1300 to 1500 m 2 / g, pore diameter is #imgabs0#, pore volume is 0.6~0.8cm 3 / g, the Cu metal atoms of the Cu-MOF and the antioxidant peptides specifically and selectively adsorb each other through chelation, and the huge specific surface area and micropore volume significantly increase the contact area of ​​the chelation, greatly improving the purification efficiency of the antioxidant peptides, greatly simplifying the subsequent chromatographic screening procedure, and reducing the manual operation of the staff while maintaining the purity and quantity of the obtained antioxidant peptides.

Description

Technical Field

[0001] The present invention relates to the field of peptide screening technology, and in particular to a Cu-MOF material for specifically enriching antioxidant peptides and a preparation and application method thereof. Background Art

[0002] Free radicals are harmful compounds produced by oxidative reactions in the body. They are highly oxidizing and can damage cells and tissues. Consuming foods rich in antioxidants is an effective way to maintain an appropriate antioxidant status. Food-derived antioxidant peptides offer advantages such as high safety, strong antioxidant activity, and ease of absorption. However, the peptide content in food is complex, and separation and purification steps are complex and time-consuming. Furthermore, selective isolation of target active peptides is difficult. Therefore, developing a new and efficient method for screening food-derived antioxidant peptides has become a pressing task.

[0003] Chinese Patent 202010370897.2 discloses a method for the preparation, separation, purification, and identification of pumpkin seed antioxidant peptides. Pumpkin seeds are subjected to multiple enzymatic hydrolysis steps and then centrifugal freeze-dried to obtain a mixture of pumpkin seed antioxidant peptides. Components of varying molecular weights are then collected by ultrafiltration, and the component with the strongest antioxidant capacity is selected and dissolved in water. The components are further separated by gel chromatography, and the functional peptide segments in the pumpkin seed antioxidant peptides are gradually purified and enriched using the hydroxyl radical scavenging rate as an indicator. The resulting components are then subjected to structural identification. This method aims to obtain antioxidant peptides with the strongest antioxidant properties. However, the purification process can easily lead to the loss of antioxidant peptides and waste of raw materials. Furthermore, the process is cumbersome, the antioxidant peptides are not highly targeted, and the purity of the antioxidant peptides cannot be guaranteed. Summary of the Invention

[0004] In order to solve the problems in the prior art that the purification rate of food-derived antioxidant peptides is not high enough and raw materials are easily wasted, the present invention provides a Cu-MOF material for specifically enriching antioxidant peptides, which has specific selectivity for antioxidant peptides in food-derived peptides, reduces the loss of antioxidant peptides and obtains antioxidant peptides with high purity; the present invention also provides a preparation method of the Cu-MOF material for specifically enriching antioxidant peptides, and the prepared Cu-MOF material has specific selectivity for antioxidant peptides in food-derived peptides, reduces the loss of antioxidant peptides and obtains antioxidant peptides with high purity; the present invention also provides an application method of the Cu-MOF material for specifically enriching antioxidant peptides, which is used to specifically enrich antioxidant peptides, has the functions of reducing the loss of antioxidant peptides and purifying antioxidant peptides with high precision.

[0005] The present invention is achieved by the following technical solutions:

[0006] A Cu-MOF material for specifically enriching antioxidant peptides, wherein the Cu-MOF is an organic metal framework material with Cu as the metal atom and a specific surface area of ​​1300 to 1500 m 2 / g, pore size Pore ​​volume is 0.6~0.8cm 3 / g, the Cu metal atoms of the Cu-MOF and the antioxidant peptides are specifically and selectively adsorbed to each other through chelation.

[0007] Cu-MOF significantly increases the contact area of ​​chelation with its huge specific surface area and micropore volume, and specifically and selectively adsorbs antioxidant peptides from mixed peptides extracted from natural products, greatly improving the purification efficiency of antioxidant peptides and greatly simplifying the subsequent chromatographic screening procedures. While maintaining the purity and quantity of the obtained antioxidant peptides, it reduces the manual operation workload of staff.

[0008] Preferably, the Cu-MOF adsorbed with the antioxidant peptide is desorbed by a desorbent, wherein the desorbent is a uniformly mixed solution of ACN, H2O, 0.1 mol / L TFA and 1 mol / L NaCl in a ratio of 3.5 to 4.5:4:1:1 by volume.

[0009] The desorbent formula is designed based on the molecular structure characteristics and interaction affinity of Cu-MOF adsorbed antioxidants. The desorption is efficient and accurate, and there is no waste of antioxidants.

[0010] Preferably, the Cu-MOF is a granular organic metal framework structure formed by two pyramidal tetrahedron bottom surfaces connected in pairs.

[0011] A method for preparing a Cu-MOF material for specifically enriching antioxidant peptides, characterized by comprising the following steps:

[0012] 1) dissolving trimesic acid and a soluble copper salt in an ethanol aqueous solution, stirring uniformly at room temperature, and heating to react to obtain a reaction solution;

[0013] 2) The reaction solution obtained in step 1) was centrifuged to remove the supernatant, and ethanol was added to the remaining precipitate. After multiple centrifugation and washing, the precipitate was dried overnight to obtain Cu-MOF.

[0014] Preferably, in step 1), the mixing ratio of trimesic acid, the Cu element in the soluble copper salt, and the ethanol aqueous solution is 1 mol: 1.8-2.2 mol: 10-12 L, the soluble copper salt includes one or more of copper nitrate, copper sulfate, copper chloride, copper acetate, and hydrates thereof, and the volume ratio of ethanol to water in the ethanol aqueous solution is 1: 0.8-1.2; the mixing and stirring time is 30 minutes, the heating temperature is 110-130° C., and the heating time is 12 hours.

[0015] Preferably, in step 2), the centrifugal speed is 7000-9000 r / min, the time is 5 min, the centrifugal washing is performed 2-4 times, and the drying process is first dried in air at room temperature overnight, and then dried in vacuum at 75-85° C. for several hours.

[0016] An application of the Cu-MOF material or the Cu-MOF material prepared by the above-mentioned preparation method in the specific and selective adsorption of antioxidant peptides.

[0017] A method for using the above-mentioned Cu-MOF material for enriching antioxidant peptides or the Cu-MOF material prepared according to the above-mentioned preparation method for specifically and selectively adsorbing antioxidant peptides is characterized by comprising the following steps:

[0018] (1) A mixed peptide solution extracted from natural products was prepared, and the PPH components with different molecular weights were separated by ultrafiltration. The component with the highest free radical scavenging ability, PPH-I, was determined by testing and comparison. Cu-MOF was added, mixed and shaken at 35-38°C, and the precipitate was collected by centrifugation;

[0019] (2) mixing the precipitate prepared in step (1) with a desorbent, shaking, and centrifuging, taking the supernatant, and removing the organic phase with nitrogen blowdown to obtain a crude purified antioxidant peptide PPH-I-Cu;

[0020] (3) The PPH-I-Cu prepared in step (2) is sequentially subjected to gel filtration chromatography and reverse-phase high performance liquid chromatography, and elution fractions are collected and separated multiple times, and the antioxidant peptides in the elution fractions are structurally identified.

[0021] Preferably, the concentration of the mixed peptide solution in step (1) is 0.4-0.6 mg / mL, the mass ratio of the mixed peptide to Cu-MOF is 1:1, the oscillation temperature is 35-38°C, the oscillation time is 40-80 min, the centrifugal speed is 5000 r / min, and the centrifugal time is 5 min; in step (2), the oscillation temperature is 35-38°C, the oscillation time is 10-14 h, the centrifugal speed is 5000 r / min, and the centrifugal time is 5 min.

[0022] Preferably, the gel filtration chromatography purification process of the PPH-I-Cu in step (3) is as follows: the chromatographic column is fixed vertically on the bracket, the Sephadex G-10 gel is placed in a suction flask for vacuum degassing, the excess liquid is poured out, and the Sephadex G-10 and water are mixed in a ratio of 3:1, slowly poured into the chromatographic column along the inner wall of the column, and the upper end interface is tightened; the column is equilibrated with 2 to 3 column volumes of eluent, and then the sample is loaded; the PPH-I-Cu is filtered and loaded, and deionized water is used for elution at a flow rate of 2 mL / min, and the detection wavelength is 220 nm; the eluted fractions are collected and lyophilized, which is the PPH-I-Cu gel filtration chromatography separation fraction PPH-I-Cu-A;

[0023] The process of RP-HPLC purification of PPH-I-Cu-A in step (3) is as follows:

[0024] The chromatograph is a Water e2695 chromatograph, equipped with a 2998 UV detector and an Empower workstation, the chromatographic column is a SunFireTMC18, 5_μm (4.6×250mm), the detection wavelength is 220nm, the sample amount is 100_μL, the column temperature is 30°C, the elution flow rate is 1mL / min, and the elution pattern is 0-20min: 100-0% A, 0-100% B; 20-22min: 0-100% A, 100-0% B; 22-28min: 100% A; the mobile phase A liquid is a 5% acetonitrile solution containing 0.05% TFA, and the mobile phase B liquid is a 40% acetonitrile solution containing 0.05% TFA; each sample is repeatedly injected, and after collecting the elution fractions multiple times, the organic phase in the solvent is removed by nitrogen blowing, which is the pea antioxidant peptide reversed-phase high performance liquid chromatography purification fraction PPH-I-Cu-AB;

[0025] The structural identification process of the antioxidant peptide PPH-I-Cu-AB in different elution fractions in step (3) is as follows:

[0026] The ions to be sequenced were selected by mass spectrometry, and the parent ions of the PPH-I-Cu-AB ions to be sequenced were collided into an ordered series of ions by adjusting the collision gas energy. The ion fragments in the mass spectrum were processed into single-point charge stick diagrams. Finally, the sequence information of the peptides was analyzed using MaxEnt3 and PepSequencing software.

[0027] Beneficial effects of the present invention:

[0028] (1) The present invention uses Cu-MOF materials to selectively adsorb food-derived antioxidant peptides through chelation, which can finely select antioxidant peptides from complex food-derived peptides, preventing the waste of antioxidant peptides during the extraction and purification process, and the obtained antioxidant peptides are high in purity and strong in antioxidant activity.

[0029] (2) The desorbent used in the present invention can fully desorb the antioxidant peptides adsorbed on Cu-MOF in a targeted manner, thereby preventing the waste of antioxidant peptides.

[0030] (3) The present invention uses Cu-MOF for the screening and adsorption of food-derived antioxidant peptides, and then finely purifies the desorbed antioxidant peptides using chromatography. The process is simple and quick to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a scanning electron microscope image of Cu-MOF.

[0032] Figure 2 This is a diagram of the PPH-I-Cu Sephadex G-10 purification.

[0033] Figure 3 This is a reverse-phase high-performance liquid chromatography purification diagram of PPH-I-Cu-A.

[0034] Figure 4 The mass spectrum and peptide sequence of the antioxidant peptide LPSLL.

[0035] Figure 5 This is the molecular structure diagram of pea antioxidant peptide LPSLL.

[0036] Figure 6 Molecular docking diagram of pea antioxidant peptide LPSLL and Keap1 protein.

[0037] Figure 7 is the hydroxyl radical scavenging ability of each component at different mass concentrations after ultrafiltration.

[0038] Figure 8 is the superoxide anion free radical scavenging ability of each component at different mass concentrations after ultrafiltration.

[0039] Figure 9 The ABTS free radical scavenging ability of each component with different mass concentrations after ultrafiltration.

[0040] Figure 10 The scavenging ability of PPH-I-Cu-AB at different mass concentrations on hydroxyl radicals, superoxide anion radicals and ABTS radicals. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the 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; in the embodiments, unless otherwise specified, the means used are conventional means in the art; the terms "comprising", "including" or any other variations thereof used herein are intended to cover non-exclusive inclusions; for example, a composition, step, method, product or apparatus comprising the listed elements is not necessarily limited to those elements, but may include other elements not explicitly listed or elements inherent to such a composition, step, method, product or apparatus; in addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other; the experimental raw materials used in the embodiments of the present invention and the comparative examples are all commercially available products.

[0042] Example 1

[0043] A preparation method and application method of a Cu-MOF material for specifically enriching antioxidant peptides, comprising the following steps:

[0044] (1) Dissolve 0.21 g of 1,3,5-trimethylbenzenecarboxylic acid and 0.43 g of Cu(NO3)2·H2O in 12 mL of a solution (ethanol: water, V:V = 1:1). Mix and stir at room temperature (20 ± 1 °C) for 30 min, heat at 120 °C for 12 h, pour the liquid into a centrifuge tube, centrifuge at 8000 r / min for 5 min, remove the supernatant, and retain the solid in the centrifuge tube. Add 5 mL of ethanol to the centrifuge tube, wash for 5 min, centrifuge at 8000 r / min for 5 min, remove the supernatant, repeat this process three times, dry at room temperature overnight, and then vacuum dry at 80 °C to obtain Cu-MOF.

[0045] (2) Separating pea peptides using ultrafiltration membranes with different molecular weight cutoff ranges to obtain three molecular weight components of <1 kDa, 1-3 kDa and >3 kDa, which specifically includes: soaking the ultrafiltration membrane in pure water for 4 to 6 hours, changing the water and washing it 3 times; placing the ultrafiltration cup on a magnetic stirrer at room temperature, controlling the pressure of the nitrogen bottle to about 0.1 MPa, and maintaining a vacuum state; fractionating 10 mg / mL pea peptide (PPH) solution using 3 kDa and 1 kDa ultrafiltration membranes in turn; collecting the separated components, lyophilizing them for later use, and respectively including <1 kDa component (PPH-I), 1-3 kDa component (PPH-II), and >3 kDa component (PPH-III);

[0046] (3) Detecting the scavenging ability of the components obtained in step (2) for hydroxyl radicals, superoxide anion radicals, and ABTS radicals, comprehensively evaluating the antioxidant properties of each component, and selecting the component with higher antioxidant properties, as follows:

[0047] S1 hydroxyl radical scavenging ability

[0048] The measuring tube was rinsed with a small amount of borax buffer solution, and sample reagents were added to the measuring tube, in the order of 50 μL of sample, 50 μL of o-phenanthroline, 50 μL of copper sulfate solution, and 20 μL of ascorbic acid solution. Then, 780 μL of borax buffer solution was dropped into the measuring tube. After flicking the measuring tube, 50 μL of 30% H2O2 solution was added and the tube was immediately placed in the reaction pool. The measuring tube was placed in the reaction pool, and the luminescence intensity was measured every 0.1 s and recorded. The peak value of the recorded luminescence intensity was taken as the average value of three measurements. Ultrapure water was used as a blank sample solution to calculate the clearance rate, and a fitting curve was drawn based on the clearance rates of samples with different concentrations to calculate the half inhibition concentration IC. 50 , GSH is the positive control:

[0049] Hydroxyl radical scavenging rate (%) = [(Δ blank - Δ sample) / Δ blank] × 100%.

[0050] S2 superoxide anion free radical scavenging ability

[0051] Rinse the measuring tube with a small amount of luminol buffer solution. Add sample reagents to the measuring tube in the following order: 100 μL of sample, 100 μL of pyrogallol, and 800 μL of luminol buffer solution. After flicking the measuring tube, immediately place it in the reaction cell. Measure the luminescence intensity every 0.1 seconds. The peak luminescence intensity is taken as the average of three measurements. Use ultrapure water as a blank solution to calculate the clearance rate. Plot a fitting curve based on the clearance rates of samples at different concentrations to calculate the half-inhibitory concentration (IC). 50 , GSH is the positive control:

[0052] Superoxide anion radical scavenging rate (%) = [(Δ blank - Δ sample) / Δ blank] × 100%.

[0053] S3ABTS free radical scavenging ability

[0054] Weigh 200.0 mg ABTS and 34.4 mg potassium persulfate in a 50 mL volumetric flask, dilute to volume, and place in the dark at room temperature for 24 h to prepare the ABTS stock solution. Take an appropriate amount of the stock solution and dilute it with 95% ethanol until the absorbance is within 0.70 ± 0.02 to prepare the ABTS test solution. Prepare and use it immediately. Add 20 μL of sample solution and 180 μL of ABTS test solution to a 96-well plate in sequence, mix well, incubate at room temperature for 10 min, and then add 20 μL of sample solution and 180 μL of ABTS test solution to a 96-well plate. 734 The absorbance value was measured at 400 nm, and ultrapure water was used as blank sample to calculate the clearance rate. The half inhibition concentration IC was calculated by drawing a fitting curve based on the clearance rate of samples with different concentrations. 50 , GSH is the positive control:

[0055] ABTS free radical scavenging rate (%) = [(A 空白 -A 样品 ) / A 空白 ]×100%

[0056] Scavenging rates of hydroxyl radicals, superoxide anion radicals, and ABTS radicals, as well as fitting curve equations and IC 50 , as shown in Tables 1 to 3.

[0057] (4) The component with higher antioxidant activity obtained in step (3) was enriched by the metal organic framework material Cu-MOF, and the Cu-MOF enriched material containing antioxidant peptides was obtained by centrifugation; the Cu-MOF material saturated with PPH-I adsorption was taken into a 50 mL centrifuge tube, 10 mL of desorbent (ACN: H2O: TFA: NaCl = 4:4:1:1) was added, and constant temperature oscillation was performed for 12 h (190 r / min) at a temperature of 37 ° C. The mixture was centrifuged at 5000 r / min for 5 min, and the supernatant was taken. The organic phase in the supernatant was removed by nitrogen blowing to obtain the pea antioxidant peptide Cu-MOF enriched component (PPH-I-Cu), with a yield of 10.5%;

[0058] (5) Gel filtration chromatography separation and purification of PPH-I-Cu: Fix the chromatographic column with a specification of 1.6cm×100cm vertically on the bracket, place Sephadex G-10 gel in a suction flask and vacuum degas for 20 minutes, pour out the excess liquid, mix the sedimentation medium and the supernatant in a ratio of 3:1, use a glass rod to drain the chromatographic medium slowly along the inner wall of the column into the chromatographic column, prevent the generation of bubbles in this process, and tighten the upper end interface. Use 2 to 3 column volumes of eluent to balance the column, and load the sample after the machine stabilizes. 10 mg / mL PPH-I-Cu is filtered through a 0.45μm microporous filter membrane and loaded, and eluted with deionized water at a flow rate of 2mL / min. The detection wavelength is 220nm. The eluted fractions are collected and freeze-dried, which is the PPH-I-Cu gel filtration chromatography separation fraction PPH-I-Cu-A;

[0059] (6) Reverse-phase high performance liquid chromatography purification of PPH-I-Cu-A: RP-HPLC was used to separate the eluents of each component to obtain a single peak. The chromatograph was a Watere 2695 chromatograph (equipped with a 2998 UV detector and an Empower workstation), the chromatographic column was a SunFireTM C18, 5 μm (4.6×250 mm), the detection wavelength was 220 nm, the sample load was 100 μL, the column temperature was 30°C, the elution flow rate was 1 mL / min, and the elution pattern was 0-20 min: 100-0% A, 0-100% B; 20-22 min: 0-100% A, 100-0% B; 22-28 min: 100% A; the mobile phase A was a 5% acetonitrile solution containing 0.05% TFA, and the mobile phase B was a 40% acetonitrile solution containing 0.05% TFA. Each sample was injected repeatedly, and the eluted fractions were collected multiple times and then the organic phase in the solvent was removed by nitrogen blowing, which was the pea antioxidant peptide reverse phase high performance liquid chromatography purified fraction PPH-I-Cu-AB;

[0060] (7) Structural identification: PPH-I-Cu-AB was structurally identified. The ions to be sequenced were selected by primary mass spectrometry. The parent ions of the ions to be sequenced were collided into an ordered series of ions by adjusting the collision gas energy. The ion fragments in the mass spectrum were processed into single-point charge stick diagrams. Finally, the sequence information of the peptide was analyzed using MaxEnt3 and PepSequencing software.

[0061] Table 1 Hydroxyl radical fitting curve equation

[0062]

[0063] GSH is glutathione, PPH is non-ultrafiltered pea peptide, PPH-I is pea peptide with a molecular weight of less than 1k after ultrafiltration, PPH-I-Cu-AB is the final purified pea peptide, PPH-II is pea peptide with a molecular weight of 1-3k after ultrafiltration, and PPH-III is pea peptide with a molecular weight greater than 3k after ultrafiltration.

[0064] Table 2 Superoxide anion radical scavenging rate fitting curve equation

[0065]

[0066] GsH is glutathione, PPH is non-ultrafiltered pea peptide, PPH-I is pea peptide with a molecular weight of less than 1k after ultrafiltration, PPH-I-Cu-AB is the final purified pea peptide, PPH-II is pea peptide with a molecular weight of 1-3k after ultrafiltration, and PPH-III is pea peptide with a molecular weight greater than 3k after ultrafiltration.

[0067] Table 3 ABTS + Free radical scavenging rate fitting curve equation

[0068]

[0069] GSH is glutathione, PPH is non-ultrafiltered pea peptide, PPH-I is pea peptide with a molecular weight of less than 1k after ultrafiltration, PPH-I-Cu-AB is the final purified pea peptide, PPH-II is pea peptide with a molecular weight of 1-3k after ultrafiltration, and PPH-III is pea peptide with a molecular weight greater than 3k after ultrafiltration.

[0070] As shown in Tables 1 to 3, GSH is a strong antioxidant, glutathione is used as the control group, IC 50 The closer it is to GSH, the stronger its antioxidant activity is. The IC values ​​of PPH-I in all sample groups are 50 It is closest to GSH, so it is concluded that PPH-I is the second strongest antioxidant group among all samples and is selected for further purification by Cu-MOF. The final purified product PPH-I-Cu-AB has the highest scavenging rate for hydroxyl radicals, superoxide anion radicals and ABTS radicals; Figures 7-10 As shown in the figure, the scavenging rates of all samples increase with the concentration, and the lower the concentration but the higher the scavenging rate, the better the antioxidant property. It can also be seen from the figure that the antioxidant property of PPH-I-Cu-AB is the best compared with other samples.

[0071] like Figure 1 The microscopic shape of Cu-MOF is a particle formed by two pyramid bases connected together. The total specific surface area is huge, which can provide a large number of chelating sites for pea peptides, maintaining specific selectivity while maintaining the amount of antioxidant peptides. Figures 2-3 The chromatogram of antioxidant peptides is shown in Figure 1. The chromatographic retention time of the peaks is concentrated in a smaller range, indicating that the antioxidant peptides have similar structures and high purity. Figure 4 The following is the mass spectrum of antioxidant peptides. The structural formulas of some of the antioxidant peptides analyzed are as follows: Figure 5 As shown, Figure 6 This is a simulation diagram of the molecular docking of antioxidant peptides and Keap1 protein. LPSLL forms hydrogen bonds with amino acid residues VAL418, VAL465, THR560, and VAL608 in Keap1 protein.

[0072] The above-mentioned embodiments only express several implementation methods of the present invention. The description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention patent.

[0073] For ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention should be based on the attached claims.

Claims

1. Application of a Cu-MOF material in the specific enrichment of antioxidant peptides, characterized in that: The Cu-MOF is an organic metal framework material with Cu as the metal atom and a specific surface area of ​​1300 to 1500 m 2 / g, pore size Pore ​​volume is 0.6~0.8cm 3 / g, the Cu metal atoms of the Cu-MOF and the antioxidant peptides are selectively adsorbed to each other through chelation, and the antioxidant peptides can be specifically selected from the food-derived peptides; The preparation method of the Cu-MOF comprises the following steps: 1) dissolving trimesic acid and a soluble copper salt in an ethanol aqueous solution, stirring uniformly at room temperature, and heating to react to obtain a reaction solution; 2) The reaction solution obtained in step 1) was centrifuged to remove the supernatant, and ethanol was added to the remaining precipitate. After multiple centrifugation and washing, the precipitate was dried overnight to obtain Cu-MOF.

2. The use of a Cu-MOF material for specifically enriching antioxidant peptides according to claim 1, characterized in that: The Cu-MOF adsorbed with the antioxidant peptide is desorbed by a desorbent, wherein the desorbent is a solution of ACN, H2O, 0.1 mol / L TFA and 1 mol / L NaCl uniformly mixed in a volume ratio of 3.5-4.5:4:1:

1.

3. The use of a Cu-MOF material for specifically enriching antioxidant peptides according to claim 1, characterized in that: The Cu-MOF is a granular organic metal framework structure formed by two pyramidal tetrahedron bottom surfaces connected in pairs.

4. The use according to claim 1, characterized in that In step 1), the mixing ratio of trimesic acid, the Cu element in the soluble copper salt, and the ethanol aqueous solution is 1 mol:1.8-2.2 mol:10-12 L, the soluble copper salt includes one or more of copper nitrate, copper sulfate, copper chloride, copper acetate, and hydrates thereof, and the volume ratio of ethanol to water in the ethanol aqueous solution is 1:0.8-1.2; the mixing and stirring time is 30 minutes, the heating temperature is 110-130° C., and the heating time is 12 hours.

5. The use according to claim 1, characterized in that In step 2), the centrifugal speed is 7000-9000 r / min, the time is 5 minutes, and the centrifugal washing is performed 2-4 times. The drying process is first dried in air at room temperature overnight, and then dried in vacuum at 75-85° C. for several hours.

6. The use according to any one of claims 1 to 5, characterized in that: The application method includes the following steps: (1) A mixed peptide solution extracted from natural products was prepared, and the PPH components with different molecular weights were separated by ultrafiltration. The component with the highest free radical scavenging ability, PPH-I, was determined by testing and comparison. Cu-MOF was added, mixed and shaken at 35-38°C, and the precipitate was collected by centrifugation; (2) mixing the precipitate prepared in step (1) with a desorbent, shaking, and centrifuging, taking the supernatant, and removing the organic phase with nitrogen blowdown to obtain a crude purified antioxidant peptide PPH-I-Cu; (3) The PPH-I-Cu prepared in step (2) is sequentially subjected to gel filtration chromatography and reverse-phase high performance liquid chromatography, and elution fractions are collected and separated multiple times, and the antioxidant peptides in the elution fractions are structurally identified.

7. The use according to claim 6, characterized in that The concentration of the mixed peptide solution in step (1) is 0.4-0.6 mg / mL, the mass ratio of the mixed peptide to Cu-MOF is 1:1, the oscillation temperature is 35-38°C, the oscillation time is 40-80 min, the centrifugal speed is 5000 r / min, and the centrifugal time is 5 min; in step (2), the oscillation temperature is 35-38°C, the oscillation time is 10-14 h, the centrifugal speed is 5000 r / min, and the centrifugal time is 5 min.

8. The use according to claim 6, characterized in that The gel filtration chromatography purification process of the PPH-I-Cu in step (3) is as follows: The chromatographic column was fixed vertically on a bracket, and Sephadex G-10 gel was placed in a suction flask for vacuum degassing. Excess liquid was poured out, and Sephadex G-10 and water were mixed in a ratio of 3:

1. The mixture was slowly poured into the chromatographic column along the inner wall, and the upper end interface was tightened. The column was equilibrated with 2 to 3 column volumes of eluent, and then the sample was loaded. PPH-I-Cu was filtered and loaded, and deionized water was used for elution at a flow rate of 2 mL / min, and the detection wavelength was 220 nm. The eluted fractions were collected and lyophilized, which was the PPH-I-Cu gel filtration chromatography separation fraction PPH-I-Cu-A. The process of purifying PPH-I-Cu-A by reversed-phase high performance liquid chromatography in step (3) is as follows: Chromatograph is Water An e2695 chromatograph was equipped with a 2998 UV detector and an Empower workstation. The chromatographic column was SunFireTMC18, 4.6×250 mm, 5 μm. The detection wavelength was 220 nm, the sample load was 100 μL, the column temperature was 30° C., the elution flow rate was 1 mL / min, and the elution pattern was 0-20 min: 100-0% mobile phase A, 0-100% mobile phase B; 20-22 min: 0-100% mobile phase A, 100-0% mobile phase B; 22-28 min: 100% mobile phase A. Mobile phase A was a 5% acetonitrile solution containing 0.05% TFA, and mobile phase B was a 40% acetonitrile solution containing 0.05% TFA. Each sample was injected repeatedly, and the eluted fractions were collected multiple times. After the organic phase in the solvent was removed by nitrogen blowing, the pea antioxidant peptide reversed-phase high performance liquid chromatography purified fraction PPH-I-Cu-AB was obtained.

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