Method for extracting holboellia latifolia seed protein and application thereof
Through microwave-assisted alkali extraction, pulsed electric field ultrasonic enzymatic decomposition and salting out steps, the problem of low extraction rate and purity of August melon seed protein was solved, and efficient and purified protein extraction was achieved, which enhanced its application potential in the fields of medicine and health care.
Patent Information
- Application Number
- CN202510383842.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-07-04
AI Technical Summary
The lack of an effective August melon seed protein extraction process in the prior art has resulted in a low protein extraction rate and purity, limiting its application in the fields of medicine and health care.
The protein in August melon seeds is gradually isolated and purified, including degreasing, microwave treatment, enzymatic extraction, centrifugation, and dialysis, etc., and combined with specific parameters and reagents, the protein in August melon seeds is gradually isolated and purified, including degreasing, microwave treatment, enzymatic extraction, salting, centrifugation, dialysis and other steps to ensure efficient extraction and purification of proteins.
It significantly improves the extraction rate and purity of August melon seed protein, improves the antioxidant ability of proteins, and provides high-purity protein products for the preparation of antioxidants.
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Figure CN120249425A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of protein extraction, and particularly relates to a method for extracting protein from Akebia trifoliata seeds. Background Art
[0002] Akebia trifoliata, also known as Akebia trifoliata fruit, golden kidney fruit, August explosion, wild banana, cow kidney fruit, etc., its fruit is edible and has significant medicinal value and health care effects, and is a typical resource for both medicine and food. Akebia trifoliata seeds account for more than 20% of the fresh weight of the fruit and are one of the main by-products in the processing and utilization of Akebia trifoliata. Akebia trifoliata seeds are rich in oil, vitamins, protein and carbohydrates, among which the protein content is about 35.5%, and it is a good source of plant protein. At present, there is no relevant research on the extraction process of Akebia trifoliata seed protein, and it has great development prospects. Summary of the Invention
[0003] The present invention aims to provide a method for extracting protein from Akebia trifoliata seeds to improve the extraction rate and purity of Akebia trifoliata seed protein.
[0004] A method for extracting protein from Akebia trifoliata seeds in this solution includes the following steps:
[0005] Step 1: Crush Akebia trifoliata seeds, and perform stirring degreasing treatment with n-hexane or petroleum ether at 40-45 °C. After removing the oil, dry to obtain Akebia trifoliata seed powder.
[0006] Step 2: Put the Akebia trifoliata seed powder into a microwave reaction container, and then add a sodium hydroxide solution with a concentration of 0.1-0.12 mol / L for microwave-assisted alkali extraction. The ratio of Akebia trifoliata seed powder to sodium hydroxide solution is 1 g: 10-15 ml; during microwave-assisted alkali extraction, the microwave power is 300-350 W, and the microwave treatment time is 15-20 min.
[0007] Step 3: Cool the reaction solution of microwave-assisted alkali extraction to room temperature, and then transfer it to the reaction chamber of a pulsed electric field treatment device for pulsed electric field-assisted ultrasonic-assisted enzymatic hydrolysis. After treatment, add neutral protease to the reaction solution, and then put it into an ultrasonic cleaner for ultrasonic-assisted enzymatic hydrolysis.
[0008] Step 4: After the enzymatic hydrolysis in Step 3 is completed, cool the reaction solution to room temperature; slowly add ammonium sulfate powder to the reaction solution for salting-out precipitation.
[0009] Step 5: Centrifuge the salted-out mixture at a rotation speed of 3500-4500 r / min for 10-20 min; collect the supernatant.
[0010] Step 6: Pass the supernatant collected in Step 5 through an affinity chromatography column pre-equilibrated with pH 7.0 phosphate buffer, controlling the flow rate at 1 - 1.2 mL / min; then rinse the chromatography column with pH 7.0 phosphate buffer to remove unbound impurities; then use an eluent containing 1 - 1.2 mol / L sodium chloride to elute the protein of Hovenia acerba seeds bound to the chromatography column, and collect the eluent;
[0011] Step 7: Add a 0.9 - 1.2 mol / L sodium hydroxide solution to the eluent collected in Step 6, adjust the pH value to 10.5 - 11.5, and stir and extract at 40 - 45 °C for 1 - 2 h; after the extraction is completed, centrifuge again to collect the supernatant; slowly add a 0.9 - 1.2 mol / L hydrochloric acid solution to the supernatant to adjust the pH value of the supernatant to 5, then centrifuge and collect the precipitate;
[0012] Step 8: Dissolve the precipitate collected in Step 7 with pH 7.0 phosphate buffer, put it into a dialysis bag and dialyze it in deionized water at 3 - 5 °C for 20 - 30 h, changing the dialysis solution 3 - 4 times during the period; after the dialysis is completed, take out the solution, place it in an oven, and dry it to a constant weight at 40 - 50 °C to obtain the protein of Hovenia acerba seeds.
[0013] Beneficial effects of this scheme: This scheme first uses n-hexane or petroleum ether to stir and degrease the crushed Hovenia acerba seeds at 40 - 45 °C to remove oil interference and lay a foundation for subsequent steps; then, using a sodium hydroxide solution with a specific concentration, under precise control of microwave power and time, the protein is fully dissolved, greatly improving the release efficiency of the protein from Hovenia acerba seed powder. The subsequent pulsed electric field-assisted ultrasonic-assisted enzymatic hydrolysis and ultrasonic-assisted enzymatic hydrolysis processes further decompose and dissociate protein molecules. The reaction solution after enzymatic hydrolysis provides a suitable system for salting-out precipitation, enabling the protein to be preliminarily separated by salting out.
[0014] The mixture after salting out is centrifuged to collect the supernatant, and the supernatant enters a pre-equilibrated affinity chromatography column. The affinity chromatography column uses the binding characteristics of a specific ligand and the protein of Hovenia acerba seeds to separate the protein from impurities under the control of the flow rate, and then precisely elutes the target protein with a specific eluent, greatly improving the protein purity. The eluted protein solution is further purified by adjusting the pH value for extraction, precipitation and other operations to remove residual impurities. The finally collected precipitate is dissolved with phosphate buffer, dialyzed and dried to obtain high-purity protein of Hovenia acerba seeds.
[0015] Further, in Step 1, the ratio of the crushed Augustea seeds to the solvent is 1 g : 8 - 10 ml, and the degreasing treatment is carried out 2 - 3 times, each time for 1.5 - 2 h; the stirring speed is 200 - 300 rpm. Multiple degreasing treatments ensure a lower residual oil content, reduce its interference with processes such as enzymatic hydrolysis and separation in the subsequent protein extraction process, create a more favorable environment for the efficient extraction of subsequent proteins, and further improve the protein extraction rate. A stirring speed of 200 - 300 rpm can not only ensure sufficient contact between the solvent and the Augustea seed powder but also prevent the destruction of the protein structure due to overly intense stirring.
[0016] Further, in Step 3, when using pulsed electric field-assisted ultrasonic-assisted enzymatic hydrolysis, the electric field strength is 18 - 22 kV / cm, the pulse width is 90 - 110 μs, the pulse frequency is 9 - 10 Hz, and the treatment is carried out for 2 - 5 min. This parameter combination can effectively utilize the synergistic effect of the pulsed electric field and ultrasound to further accelerate the decomposition and dissociation of protein molecules. An appropriate electric field strength can change the structure of protein molecules, increasing their sensitivity to enzymatic hydrolysis, while the cavitation effect of ultrasound can further promote the contact between the enzyme and the protein substrate. The two work together to achieve a better enzymatic hydrolysis effect in a shorter time, improve the protein extraction efficiency, reduce the enzymatic hydrolysis time and the amount of enzyme used, and lower the cost.
[0017] Further, in Step 3, the mass ratio of neutral protease to the substrate is 1:90 - 120. At this ratio, the enzyme can fully act on the protein substrate, neither resulting in incomplete enzymatic hydrolysis due to insufficient enzyme amount nor causing waste due to excessive enzyme amount.
[0018] Further, in Step 3, the parameters for ultrasonic-assisted enzymatic hydrolysis are: ultrasonic power 180 - 220 W, frequency 30 - 50 kHz, and ultrasonic-assisted enzymatic hydrolysis is carried out at 40 - 45 °C for 1 - 2 h. This ultrasonic power and frequency can, without destroying the protein structure, further promote the interaction between the enzyme and the protein through the mechanical effect and thermal effect of ultrasound, accelerating the enzymatic hydrolysis reaction. The temperature condition of 40 - 45 °C not only conforms to the active temperature range of neutral protease but also synergizes with the ultrasonic effect to achieve the best enzymatic hydrolysis effect, further improving the protein extraction efficiency and quality.
[0019] Further, in Step 4, when adding ammonium sulfate powder, it is added while stirring to make its saturation reach 50%, and after adding, continue to stir for 30 - 50 min for salting-out precipitation. This helps to ensure the uniform dispersion of ammonium sulfate in the reaction solution, makes the salting-out process more complete and stable, and helps the protein to precipitate fully, improving the effect of salting-out precipitation and providing a better basis for the subsequent separation and purification of proteins.
[0020] Further, in Step 6, the stationary column of the affinity chromatography column is selected as a dextran gel column.
[0021] Further, in step six, the flow rate during the elution of the Hovenia acerba seed protein bound to the chromatography column is 0.5 - 0.8 mL / min. This elution flow rate enables the eluent to fully interact with the protein bound to the chromatography column, ensuring that the target protein can be completely eluted, while avoiding incomplete protein elution due to too fast a flow rate or affecting production efficiency due to too slow a flow rate.
[0022] Further, in step seven, the rotation speed for the centrifugation treatment is 3500 - 4500 r / min, and the centrifugation treatment time is 10 - 20 min. This rotation speed and time setting can ensure complete precipitation of the protein while avoiding damage to the protein structure caused by too high a rotation speed or too long a centrifugation time.
[0023] Further, in step eight, during the dialysis treatment, dialysis is carried out in deionized water at 3 - 5 °C for 20 - 30 h, and the dialysis solution is changed 3 - 4 times during this period. Low-temperature dialysis can reduce protein degradation and maintain the activity and structural integrity of the protein.
[0024] Experiments have proved that the above-mentioned Hovenia acerba seed protein has obvious antioxidant ability. Therefore, this application also claims the protection of the application of Hovenia acerba seed protein in the preparation of antioxidants. Description of the Drawings
[0025] Figure 1 It is a graph showing the determination of the DPPH radical scavenging ability of the Hovenia acerba seed protein prepared in Example 1 of the present invention.
[0026] Figure 2 It is a graph showing the determination of the ABTS radical scavenging ability of the Hovenia acerba seed protein prepared in Example 1 of the present invention.
[0027] Figure 3 It is a graph showing the determination of the hydroxyl radical scavenging ability of the Hovenia acerba seed protein prepared in Example 1 of the present invention.
[0028] Figure 4 It is the IC of the scavenging rates of the Hovenia acerba seed protein prepared in Example 1 of the present invention on DPPH, ABTS, and OH radicals 50 . Detailed Description of the Embodiments
[0029] The following is a more detailed description through specific embodiments:
[0030] Example 1: A method for extracting Hovenia acerba seed protein, comprising the following steps:
[0031] Step 1: Crush the Hovenia acerba seeds, carry out stirring and degreasing treatment with n-hexane at 45 °C for 2 times, 1.5 h each time; the stirring speed is 200 rpm, and the ratio of the crushed Hovenia acerba seeds to the solvent is 1 g:8 ml; after removing the oil, dry to obtain Hovenia acerba seed powder, that is, defatted Hovenia acerba seed powder;
[0032] Step 2: Put the powder of Akebia trifoliata seeds into a microwave reaction vessel, and then add 0.1 mol / L sodium hydroxide solution for microwave-assisted alkali extraction. The ratio of the powder of Akebia trifoliata seeds to the sodium hydroxide solution is 1 g:10 ml. When performing microwave-assisted alkali extraction, the microwave power is 300 W and the microwave treatment time is 15 min.
[0033] Step 3: Cool the reaction solution of microwave-assisted alkali extraction to room temperature, and then transfer it to the reaction chamber of a pulsed electric field treatment device for pulsed electric field-assisted ultrasonic-assisted enzymatic hydrolysis. The electric field strength is 20 kV / cm, the pulse width is 100 μs, the pulse frequency is 10 Hz, and the treatment time is 3 min. After the treatment, add neutral protease to the reaction solution. The mass ratio of neutral protease to the substrate is 1:100, and then put it into an ultrasonic cleaner for ultrasonic-assisted enzymatic hydrolysis. The ultrasonic power is 200 W, the frequency is 40 kHz, and the ultrasonic-assisted enzymatic hydrolysis is carried out at 45 °C for 1.5 h.
[0034] Step 4: After the enzymatic hydrolysis in Step 3 is completed, cool the reaction solution to room temperature. Slowly add ammonium sulfate powder to the reaction solution. When adding ammonium sulfate powder, stir while adding to make its saturation reach 50%. After adding, continue to stir for 30 min for salting-out precipitation.
[0035] Step 5: Centrifuge the salted-out mixture at a rotation speed of 4000 r / min for 15 min; collect the supernatant.
[0036] Step 6: Pass the supernatant collected in Step 5 through an affinity chromatography column pre-equilibrated with pH 7.0 phosphate buffer. The fixed column uses an affinity chromatography medium Sephadex column, and the flow rate is controlled at 1 mL / min. Then rinse the chromatography column with pH 7.0 phosphate buffer to remove unbound impurities. Then use an eluent containing 1 mol / L sodium chloride to elute the Akebia trifoliata seed protein bound to the chromatography column at a flow rate of 0.6 mL / min, and collect the eluent.
[0037] Step 7: Add 1 mol / L sodium hydroxide solution to the eluent collected in Step 6 to adjust the pH value to 11, and stir and extract at 45 °C for 1.5 h. After the extraction is completed, centrifuge again to collect the supernatant. The rotation speed of the centrifugation treatment is 4000 r / min, and the centrifugation treatment time is 15 min. Slowly drop 1 mol / L hydrochloric acid solution into the supernatant to adjust the pH value of the supernatant to 5, and then centrifuge and collect the precipitate. The rotation speed of the centrifugation treatment is 4000 r / min, and the centrifugation treatment time is 15 min.
[0038] Step 8: Dissolve the precipitate collected in Step 7 with phosphate buffer solution at pH 7.0, load it into a dialysis bag for dialysis treatment. During dialysis treatment, dialyze it in deionized water at 4°C for 24 h, and change the dialysis fluid 3 times during this period. After dialysis, take out the solution, place it in an oven, and dry it to constant weight at 45°C to obtain the protein of Hovenia acerba seeds.
[0039] Example 2: A method for extracting the protein of Hovenia acerba seeds, comprising the following steps:
[0040] Step 1: Crush the Hovenia acerba seeds, and carry out stirring degreasing treatment with n-hexane at 43°C for 3 times, 2 h each time; the stirring speed is 200 rpm, and the ratio of the crushed Hovenia acerba seeds to the solvent is 1 g:10 ml; after removing the grease, dry it to obtain the powder of Hovenia acerba seeds.
[0041] Step 2: Put the powder of Hovenia acerba seeds into a microwave reaction container, and then add 0.12 mol / L sodium hydroxide solution for microwave-assisted alkali extraction. The ratio of the powder of Hovenia acerba seeds to the sodium hydroxide solution is 1 g:10 ml; during microwave-assisted alkali extraction, the microwave power is 350 W, and the microwave treatment time is 15 min.
[0042] Step 3: Cool the reaction solution of microwave-assisted alkali extraction to room temperature, and then transfer it to the reaction chamber of a pulsed electric field treatment device for pulsed electric field-assisted ultrasonic-assisted enzymatic hydrolysis. The electric field strength is 18 kV / cm, the pulse width is 90 μs, the pulse frequency is 9 Hz, and the treatment time is 5 min. After treatment, add neutral protease to the reaction solution, and the mass ratio of neutral protease to substrate is 1:120. Then put it into an ultrasonic cleaner for ultrasonic-assisted enzymatic hydrolysis. The ultrasonic power is 200 W, the frequency is 40 kHz, and the ultrasonic-assisted enzymatic hydrolysis is carried out at 40°C for 2 h.
[0043] Step 4: After the enzymatic hydrolysis in Step 3 is completed, cool the reaction solution to room temperature; slowly add ammonium sulfate powder to the reaction solution. When adding ammonium sulfate powder, stir while adding to make its saturation reach 50%, and continue to stir for 50 min after adding for salting-out precipitation.
[0044] Step 5: Centrifuge the salted-out mixture at a speed of 4500 r / min for 10 min; collect the supernatant.
[0045] Step 6: Pass the supernatant collected in Step 5 through an affinity chromatography column pre-equilibrated with phosphate buffer solution at pH 7.0. The stationary column is selected as Sephadex G-50 dextran gel column, and the flow rate is controlled at 1.2 mL / min; then rinse the chromatography column with phosphate buffer solution at pH 7.0 to remove the unbound impurities; then use an eluent containing 1.2 mol / L sodium chloride to elute the protein of Hovenia acerba seeds bound to the chromatography column at a flow rate of 0.6 mL / min, and collect the eluent.
[0046] Step 7: Add 0.9 mol / L sodium hydroxide solution to the eluate collected in Step 6, adjust the pH value to 10.5, and stir and extract at 40 °C for 2 h; after the extraction is completed, centrifuge again to collect the supernatant, with the centrifugation speed being 4000 r / min and the centrifugation time being 15 min; slowly dropwise add 1 mol / L hydrochloric acid solution to the supernatant, adjust the pH value of the supernatant to 5, then centrifuge and collect the precipitate, with the centrifugation speed being 4000 r / min and the centrifugation time being 15 min;
[0047] Step 8: Dissolve the precipitate collected in Step 7 with phosphate buffer solution at pH 7.0, put it into a dialysis bag for dialysis treatment. During dialysis, dialyze in deionized water at 4 °C for 24 h, and change the dialysis solution 3 times during this period; after the dialysis is completed, take out the solution, place it in an oven, and dry it to a constant weight at 45 °C to obtain the protein of Hovenia acerba seeds.
[0048] Experimental verification:
[0049] I. Protein extraction rate of Hovenia acerba seeds
[0050] Protein extraction rate / % = (protein content in the extract × volume of the extract) / (protein content in the defatted Hovenia acerba seed powder × sampling amount of the Hovenia acerba seed powder); the protein content of the defatted Hovenia acerba seed protein powder is determined by the micro-Kjeldahl method, and the protein content in the extract is determined by the Coomassie brilliant blue colorimetric method. Through measurement, the protein extraction rates of the methods in Example 1 and Example 2 are 78.2%3 and 76.35% respectively. The purity of the Hovenia acerba seed protein obtained in Example 1 and Example 2 is detected by SDS-PAGE electrophoresis method, and the purities are 93.65% and 91.37% respectively.
[0051] II. In vitro antioxidant determination of Hovenia acerba seed protein
[0052] 1. Determination of DPPH free radical scavenging ability
[0053] Referring to the method of Xin Yue et al. (Xin Yue, Song Xiaoxiao, Wang Yuxiao, et al. Comparison of polysaccharide structure characteristics and antioxidant properties of different parts of cowpea [J]. Food Science, 2022, 43(16): 61-67), determine the DPPH free radical scavenging ability of the Hovenia acerba seed protein prepared in Example 1. Prepare solutions of the Hovenia acerba seed protein with different concentrations for standby; take 100 μL of the Hovenia acerba seed protein and 100 μL of 0.2 mM DPPH solution (anhydrous ethanol) in a 96-well enzyme-linked immunosorbent assay plate, and let it stand in the dark at room temperature for 30 min, and measure the absorbance value at 510 nm. Calculate the DPPH free radical scavenging rate:
[0054] DPPH radical scavenging rate / % = (A0 - (A - A1)) / A0, where: A0 is the absorbance of the blank control; A1 is the absorbance of the sample background; A is the absorbance of the sample solution.
[0055] The results are as follows Figure 1 shown Figure 1 showing that: when the concentration of seed protein increases from 0.2 mg / ml to 1 mg / ml, the DPPH radical scavenging rates are 24.3%, 33.8%, 41.7%, 49.1%, 60.81% respectively, that is, when the concentration of seed protein increases from 0.2 mg / ml to 1 mg / ml, the DPPH radical scavenging rate of Augustea triflora seed protein increases from 24.3% to 60.81%.
[0056] 2. ABTS cation radical scavenging ability
[0057] Referring to the determination method of Zong Xinyan et al. (
[20] Zong Xinyan, Xu Dechang, Nie Shaoping, et al. Isolation, purification, physicochemical properties and antioxidant function of polygonatum odoratum polysaccharide [J]. Journal of Nanchang University (Natural Science Edition), 2019, 43(01): 70 - 75), take 7.4 mM ABTS diammonium salt and 2.6 mM potassium persulfate solution, mix them in a 1:1 (v) ratio and react in the dark for 12 h, then dilute 20 times with distilled water. Take 100 μL of AP - SDF solution and 100 μL of ABTS working solution and place them in a 96 - well microplate and let stand for 6 min, then measure the absorbance at 734 nm. Calculate the ABTS radical scavenging rate:
[0058] ABTS radical scavenging rate / % = (A0 - (A - A1)) / A0, where: A0 is the absorbance of the blank control; A1 is the absorbance of the sample background; A is the absorbance of the sample solution.
[0059] The results are as follows Figure 2 shown Figure 2 showing that: when the concentration of seed protein increases from 0.2 mg / ml to 1 mg / ml, the ABTS radical scavenging rates are 10.94%, 22.27%, 32.81%, 41.15%, 47.14% respectively. When the concentration of seed protein increases from 0.2 mg / ml to 1 mg / ml, the ABTS radical scavenging rate of Augustea triflora seed protein increases from 10.94% to 47.14%.
[0060] 3. Hydroxyl radical scavenging ability
[0061] Referring to the method of Bai Ranran et al. in
[21] (
[21] Bai Ranran, Song Mingfa, Zhang Qian, et al. Study on the modification of insoluble dietary fiber from ginger residue by compound enzyme method and its antioxidant activity [J]. China Condiment, 2023, 48(01): 87-92), 50 μL of AP-SDF, 50 μL of 9 mM FeSO4 solution, 50 μL of 9 mM salicylic acid-ethanol solution, and 50 μL of 8 mM H2O2 solution were taken in a 96-well microplate, shaken and mixed evenly, incubated at 37 °C for 30 min, and the absorbance was measured at 510 nm. Calculate the hydroxyl radical scavenging rate:
[0062] Hydroxyl radical scavenging rate / % = (A0 - (A - A1)) / A0, where: A0 is the absorbance of the blank control; A1 is the absorbance of distilled water instead of H2O2; A is the absorbance of the sample solution.
[0063] The results are as shown in the appendix Figure 3 as follows: Figure 3 It shows that when the concentration of seed protein increases from 0.2 mg / ml to 1 mg / ml, the hydroxyl radical scavenging rates are 29.45%, 37.85%, 43.08%, 47.68%, and 56.98% respectively. When the concentration of Actinidia latifolia seed protein increases from 0.2 mg / ml to 1 mg / ml, the hydroxyl radical scavenging rate of Actinidia latifolia seed protein increases from 29.45% to 56.98%.
[0064] 4. IC 50 comparison
[0065] By calculating the IC 50 values of the scavenging rates of Actinidia latifolia seed protein against DPPH, ABTS, and OH free radicals, the antioxidant activities were further compared. The results are as shown in Figure 4 the following. The scavenging ability of Actinidia latifolia seed protein against free radicals is in the order of ABTS > OH > DPPH. The IC 50 of ABTS free radical = 0.5128 mg / mL, the IC 50 of hydroxyl free radical = 0.5710 mg / mL, and the IC 50 of DPPH free radical = 0.5899 mg / mL. Among them, the scavenging ability against ABTS free radical is the strongest. It may be due to the difference in the types of free radicals and the different mechanisms of its scavenging activity against different free radicals, resulting in differences in the scavenging rates of different free radicals.
[0066] The above are only embodiments of the present invention, and common knowledge such as specific structures and characteristics known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the structure of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicability of the patent. The protection scope required by this application shall be subject to the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.
Claims
1. A method for extracting Augustea trifoliata seed protein, characterized in that: It includes the following steps: Step 1: Crush the seeds of Akebia trifoliata var. australis, and carry out stirring degreasing treatment with n-hexane or petroleum ether at 40-45°C. After removing the grease, dry it to obtain Akebia trifoliata var. australis seed powder; Step 2: Put the Akebia trifoliata var. australis seed powder into a microwave reaction container, and then add a sodium hydroxide solution with a concentration of 0.1-0.12 mol / L for microwave-assisted alkali extraction. The ratio of Akebia trifoliata var. australis seed powder to sodium hydroxide solution is 1 g: 10-15 ml; When performing microwave-assisted alkali extraction, the microwave power is 300-350 W, and the microwave treatment time is 15-20 min; Step 3: Cool the reaction solution of microwave-assisted alkali extraction to room temperature, and then transfer it to the reaction chamber of a pulsed electric field treatment device for pulsed electric field-assisted ultrasonic-assisted enzymatic hydrolysis. After treatment, add neutral protease to the reaction solution, and then put it into an ultrasonic cleaner for ultrasonic-assisted enzymatic hydrolysis; Step 4: After the enzymatic hydrolysis in Step 3 is completed, cool the reaction solution to room temperature; Slowly add ammonium sulfate powder to the reaction solution for salting-out precipitation; Step 5: Centrifuge the salted-out mixture at a speed of 3500-4500 r / min for 10-20 min; Collect the supernatant; Step 6: Pass the supernatant collected in Step 5 through an affinity chromatography column pre-equilibrated with a pH 7.0 phosphate buffer solution, and control the flow rate at 1-1.2 mL / min; Then rinse the chromatography column with a pH 7.0 phosphate buffer solution to remove unbound impurities; Then use an eluent containing 1-1.2 mol / L sodium chloride to elute the Akebia trifoliata var. australis seed protein bound to the chromatography column, and collect the eluent; Step 7: Add a sodium hydroxide solution with a concentration of 0.9-1.2 mol / L to the eluent collected in Step 6, adjust the pH value to 10.5-11.5, and stir and extract at 40-45°C for 1-2 h; After the extraction is completed, centrifuge again to collect the supernatant; Slowly drop a 0.9-1.2 mol / L hydrochloric acid solution into the supernatant to adjust the pH value of the supernatant to 5, and then centrifuge and collect the precipitate; Step 8: Dissolve the precipitate collected in Step 7 with a pH 7.0 phosphate buffer solution, put it into a dialysis bag and dialyze it in deionized water at 3-5°C for 20-30 h, and change the dialysis solution 3-4 times during this period; After dialysis is completed, take out the solution, place it in an oven, and dry it to constant weight at 40-50°C to obtain Akebia trifoliata var. australis seed protein.
2. The method for extracting Augustea trifoliata seed protein according to claim 1, wherein: In Step 1, the ratio of the crushed Akebia trifoliata var. australis seeds to the solvent is 1 g: 8-10 ml, and the degreasing treatment is carried out 2-3 times, each time for 1.5-2 h; The stirring speed is 200-300 rpm.
3. The method for extracting Augustea trifoliata seed protein according to claim 2, wherein: In Step 3, when performing pulsed electric field-assisted ultrasonic-assisted enzymatic hydrolysis, the electric field strength is 18-22 kV / cm, the pulse width is 90-110 μs, the pulse frequency is 9-10 Hz, and the treatment time is 2-5 min.
4. The method for extracting Augustea trifoliata seed protein according to claim 3, characterized in that: In Step 3, the mass ratio of neutral protease to substrate is 1:90-120.
5. The method for extracting Augustea trifoliata seed protein according to claim 4, wherein: In Step 3, the parameters of ultrasonic-assisted enzymatic hydrolysis are: ultrasonic power 180-220 W, frequency 30-50 kHz, and ultrasonic-assisted enzymatic hydrolysis at 40-45°C for 1-2 h.
6. The method for extracting Augustea trifoliata seed protein according to claim 5, characterized in that: In Step 4, when adding ammonium sulfate powder, stir while adding to make its saturation reach 50%, and continue stirring for 30 - 50 min after adding for salting - out precipitation.
7. The method for extracting Augustea trifoliata seed protein according to claim 6, wherein: In Step 6, the stationary column of the affinity chromatography column is selected as a dextran gel column.
8. The method for extracting Augustea trifoliata seed protein according to claim 7, characterized in that: In Step 6, the flow rate for eluting the protein of Schisandra propinqua var. sinensis seeds bound to the chromatography column is 0.5 - 0.8 mL / min.
9. The method for extracting Augustea trifoliata seed protein according to claim 8, wherein: In Step 7, the rotation speed for centrifugation is 3500 - 4500 r / min, and the centrifugation time is 10 - 20 min.
10. Use of the protein of Schisandra propinqua var. sinensis seeds obtained by extracting according to any one of Claims 1 - 9 in the preparation of an antioxidant.