阅读说明：本技术 一种大米抗氧化活性肽的制备方法 (Preparation method of rice antioxidant active peptide ) 是由 梁盈 崔校基 林亲录 陈忠旭 吴钇心 吴伟 于 2020-12-01 设计创作，主要内容包括：本发明涉及一种大米抗氧化活性肽的制备方法,所述大米抗氧化活性肽为KHNRGDEF,对米糠谷蛋白酶解液依次进行微滤处理、超滤处理,获得多肽溶液；以多肽溶液为电解液,活性炭或外表包裹有活性炭层的导电材料作为电极,将电解液的pH值调节至7.86-7.90,然后在2个电极之间通直流电,使得等电点不在7.88的多肽被电极吸附,获得高纯多肽溶液；采用改性PC膜作为超滤膜,对S3获得的高纯多肽溶液进行超滤处理后,喷雾干燥,获得大米抗氧化活性肽粉末。本发明的制备方法可以使大米抗氧化活性肽的纯度提高的90%以上,具有工业化应用前景。(The invention relates to a preparation method of a rice antioxidant active peptide, wherein the rice antioxidant active peptide is KHNRGDEF, and rice bran gluten enzymatic hydrolysate is sequentially subjected to microfiltration treatment and ultrafiltration treatment to obtain a polypeptide solution; taking the polypeptide solution as electrolyte, taking active carbon or a conductive material coated with an active carbon layer on the surface as an electrode, adjusting the pH value of the electrolyte to 7.86-7.90, and then introducing direct current between 2 electrodes to ensure that the polypeptide with the isoelectric point not at 7.88 is adsorbed by the electrode to obtain a high-purity polypeptide solution; and (3) carrying out ultrafiltration treatment on the high-purity polypeptide solution obtained in the step S3 by using a modified PC membrane as an ultrafiltration membrane, and then carrying out spray drying to obtain rice antioxidant active peptide powder. The preparation method can improve the purity of the rice antioxidant active peptide by over 90 percent, and has industrial application prospect.)

1. A preparation method of rice antioxidant active peptide is KHNRGDEF, and is characterized by comprising the following steps:

s1, providing rice bran and glutelin enzymolysis liquid;

s2, sequentially carrying out microfiltration treatment and ultrafiltration treatment on the rice bran gluten enzymatic hydrolysate to obtain a polypeptide solution;

wherein the molecular weight of the polypeptide in the polypeptide solution is 900-1200 Da;

s3, taking the polypeptide solution obtained in S2 as electrolyte, taking activated carbon or conductive materials coated with activated carbon layers as electrodes, adjusting the pH value of the electrolyte to 7.86-7.90, and then, introducing direct current between 2 electrodes to enable the polypeptide with the isoelectric point not being 7.88 to be adsorbed by the electrodes, thereby obtaining a high-purity polypeptide solution;

s4, carrying out ultrafiltration treatment on the high-purity polypeptide solution obtained in the step S3 by adopting a modified PC membrane as an ultrafiltration membrane, and carrying out spray drying to obtain rice antioxidant active peptide powder;

the preparation method of the modified PC film comprises the following steps: swelling the PC membrane in acetone for 3-8s, dip-coating in a dip-coating solution for 2-4h, taking out, air drying, soaking in distilled water for 45-90min, taking out, and drying to obtain the final product;

the dip-coating liquid is Zn (NO)₃)₂、NH₃·H₂Mixed solution of O and water, Zn (NO)₃)₂The concentration of (A) is 0.4-0.8wt%, and the pH value of the dip-coating liquid is 9-11.

2. The method according to claim 1, wherein the rice bran gluten enzymatic hydrolysate is prepared by a method comprising the steps of:

(1) providing defatted rice bran, and sequentially cleaning, drying, crushing and sieving the defatted rice bran to obtain defatted rice bran powder;

(2) uniformly mixing the defatted rice bran powder obtained in the step (1) with water according to the mass ratio of 1:8-12, adjusting the pH value to 8.5-9.0, heating to 35-45 ℃, stirring for 2-3h, and then centrifuging at the rotating speed of 7000-8000r/min for 15-30min to obtain a supernatant;

(3) adjusting the pH value of the supernatant obtained in the step (2) to 4-4.5, standing for 30-60min, and performing centrifugal separation at the rotating speed of 6000-;

(4) re-dissolving the rice bran gluten solid phase substance obtained in the step (3) in water, adjusting the pH value to 7, and stirring for 2-3h to obtain a rice bran gluten solution;

(5) adjusting the rice bran gluten concentration of the rice bran gluten solution in the step (4) to 6-8wt%, keeping standing at 37 ℃ for 20-40min, and adjusting the pH value to 8.5-10.0 to obtain an alkaline rice bran gluten solution;

(6) and (4) sequentially carrying out enzymolysis and centrifugation treatment on the alkaline rice bran gluten solution obtained in the step (5) to obtain rice bran gluten enzymolysis liquid.

3. The method according to claim 2, wherein the oil content of the defatted rice bran in the step (1) is less than 6 wt%.

4. The method according to claim 2, wherein in the steps (2) and (5), the pH is adjusted by sodium bicarbonate.

5. The method according to claim 2, wherein in the step (6), the rice bran gluten enzymatic hydrolysate is obtained by adding neutral protease to the alkaline rice bran gluten solution at a [ E ]/[ S ] ratio of 1.5 to 1.8%, hydrolyzing at 45 to 55 ℃ for 4 to 5 hours, and then centrifuging at 6000-.

6. The method as claimed in claim 1, wherein the molecular weight of the polypeptide in the polypeptide solution in S2 is 1000-1100 Da.

7. The method according to claim 1, wherein in S3, Na is used₂HPO₄•2H₂O, adjusting the pH value of the electrolyte; preferably, 100-120V direct current is conducted among 2 electrodes; more preferably, the energization time is 20-40 min.

8. The method according to claim 1, wherein in S3, the activated carbon is a biological activated carbon.

9. The method according to claim 1, wherein the dip-coating liquid has a pH of 10 at S4.

10. The method according to claim 1, wherein in S4, the spray drying process conditions are as follows: the air inlet temperature is 180-200 ℃, the air outlet temperature is 70-90 ℃, and the flow rate of the material liquid is 20-25 r/min.

Technical Field

The invention relates to a preparation method of a rice antioxidant active peptide, in particular to an industrial preparation method of the rice antioxidant active peptide, and belongs to the technical field of deep processing of rice byproducts.

Background

As a large-scale food country in China, rice has huge yield, wherein rice with unhulled rice is taken as staple food which is usually eaten by Chinese people, while rice bran is a main byproduct in a rice production factory, the composition of protein amino acid of the rice is similar to that of protein amino acid in the rice, the composition of the amino acid is reasonable, and the rice has higher nutritional value. Most of the industrially produced rice bran is not effectively utilized, so that the rice bran protein needs to be deeply processed to improve the added value thereof and realize the full utilization of rice resources. The nutritive value of protein of the rice bran can be greatly exerted by carrying out proteolysis on peptide fragments in the rice bran, and various active peptides such as antioxidant active peptide, opioid antagonist peptide, immunological activity regulating peptide and the like are found at present. The rice antioxidant active peptide found in the rice antioxidant peptide has good free radical scavenging capacity, has strong scavenging effect on free radicals induced by oxidative damage, can protect oxidative stress of various somatic cells of human bodies or animals, and also has the effects of resisting inflammation and aging.

Through research, in the processing of rice byproducts, the industrial production of oil extraction is realized by utilizing the high oil content of rice bran, but the nutritional value of rice bran protein is neglected, and the rice bran after oil extraction still has high utilization value. With the recent rise of various protein production industries, many active peptide production plants have been produced, including processes such as raw material extraction, microbial fermentation and chemical synthesis. The applicant successfully separates KHNRGDEF peptide segment (molecular weight: 1002.4 Da, named KF-8) with antioxidant effect from rice bran (see CN 106636274A), which has proved to have physiological functions of cell protection, anti-aging and the like, but needs to be separated and purified by RP-HPLC and other technologies, and the large-scale preparation of the active peptide is difficult to realize. In recent years, the industrial production of rice bran bioactive peptides is always a blank, so how to fully utilize rice bran raw materials to produce protein products and how to efficiently obtain high-purity and high-activity rice bran bioactive peptide industrial products are technical problems to be solved.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a preparation method of rice antioxidant active peptide so as to obtain high-purity rice antioxidant active peptide.

The technical scheme of the invention is as follows:

a preparation method of rice antioxidant active peptide is KHNRGDEF, and comprises the following steps:

s1, providing rice bran and glutelin enzymolysis liquid;

s2, sequentially carrying out microfiltration treatment and ultrafiltration treatment on the rice bran gluten enzymatic hydrolysate to obtain a polypeptide solution;

wherein the molecular weight of the polypeptide in the polypeptide solution is 900-1200 Da;

s3, taking the polypeptide solution obtained in S2 as electrolyte, taking activated carbon or conductive materials coated with an activated carbon layer as electrodes, adjusting the pH value of the electrolyte to 7.86-7.90, and then, introducing direct current between 2 electrodes to enable the polypeptide with the isoelectric point not at 7.88 to be adsorbed by the electrodes to obtain a high-purity polypeptide solution;

s4, adopting a modified PC membrane (modified polycarbonate membrane) as an ultrafiltration membrane, carrying out ultrafiltration treatment on the high-purity polypeptide solution obtained in the step S3, and carrying out spray drying to obtain rice antioxidant active peptide powder;

the preparation method of the modified PC film comprises the following steps: swelling the PC membrane in acetone for 3-8s, dip-coating in a dip-coating solution for 2-4h, taking out, air drying, soaking in distilled water for 45-90min to remove unstable polymer, taking out, and drying to obtain the final product;

the dip-coating liquid is Zn (NO)₃)₂、NH₃·H₂Mixed solution of O and water, Zn (NO)₃)₂The concentration of (A) is 0.4-0.8wt%, and the pH value of the dip-coating liquid is 9-11.

Further, the preparation method of the rice bran gluten enzymatic hydrolysate comprises the following steps:

(1) providing defatted rice bran, and sequentially cleaning, drying, crushing and sieving the defatted rice bran to obtain defatted rice bran powder;

(2) uniformly mixing the defatted rice bran powder obtained in the step (1) with water according to the mass ratio of 1:8-12 (preferably 1: 10), adjusting the pH value to 8.5-9.0, heating to 35-45 ℃ (preferably 38-42 ℃, further preferably 40 ℃) and stirring for 2-3h, then centrifuging for 15-30min at the rotating speed of 6000-8000r/min (preferably 6500-7500 r/min), and removing precipitates to obtain a supernatant;

(3) adjusting the pH value of the supernatant obtained in the step (2) to 4-4.5 (preferably, hydrochloric acid is used for adjusting the pH value, and more preferably, the hydrochloric acid is food-grade hydrochloric acid), standing for 30-60min, and then centrifuging at the rotating speed of 6000-8000r/min (preferably 6500-7500 r/min) to obtain a rice bran gluten solid phase;

(4) re-dissolving the rice bran gluten solid phase substance obtained in the step (3) in water, adjusting the pH value to 7, and stirring for 2-3h to obtain a rice bran gluten solution;

(5) adjusting the rice bran gluten concentration of the rice bran gluten solution in the step (4) to 6-8wt%, further 6-7wt%, keeping standing at 37 ℃ for 20-40min, and adjusting the pH value to 8.5-10.0 to obtain an alkaline rice bran gluten solution;

(6) and (4) sequentially carrying out enzymolysis and centrifugation treatment on the alkaline rice bran gluten solution obtained in the step (5) to obtain rice bran gluten enzymolysis liquid.

Further, the preparation method of the defatted rice bran comprises the following steps: squeezing testa oryzae to remove oil, cleaning to remove insoluble substances and particulate matter, drying, pulverizing, and sieving to obtain 45-55 mesh defatted testa oryzae powder. Preferably, the defatted rice bran is washed with fresh water while stirring to allow the particulate matter and insoluble matter to settle, optionally stirring for 2-4 hours, during which time the water in the wash tank is changed every 30 min. Preferably, drying is carried out by adopting a dryer, the temperature is controlled to be 50-60 ℃, preferably 55 ℃, drying is carried out for 4-8 hours, and the rice bran in the dryer is finished every 2 hours to fully evaporate the water.

Further, the oil content of the defatted rice bran is less than 6 wt%.

Further, in the step (2) and the step (5), sodium bicarbonate or a sodium bicarbonate solution is adopted to adjust the pH value. Preferably, the sodium bicarbonate is food grade sodium bicarbonate.

Further, in the step (3), the centrifugation time is 15-20 min.

Further, in the step (6), neutral protease is added into the alkaline rice bran gluten solution according to the ratio of [ E ]/[ S ] of 1.5-1.8%, after hydrolysis is carried out for 4-5h at the temperature of 45-55 ℃, the solution is centrifuged for 10-20min at the rotating speed of 6000-8000r/min (preferably 6500-7500 r/min), and the rice bran gluten enzymolysis solution is obtained. Preferably, the hydrolysis is carried out at 50 ℃.

Further comprising the step of carrying out enzyme deactivation and sterilization treatment on the rice bran gluten enzymatic hydrolysate, namely controlling the temperature of the rice bran gluten enzymatic hydrolysate at 85-90 ℃ and keeping the temperature for 8-12 min. Alternatively, the enzyme deactivation and sterilization treatment may be performed between the enzymolysis and centrifugation in step (6), or may be performed after step (6).

Further, in S2, polypeptide solution is obtained by ultrafiltration fractionation in steps of high to low molecular weight.

Preferably, in S2, the molecular weight of the polypeptide in the polypeptide solution is 1000-1100 Da.

Further, in S3, Na was used₂HPO₄•2H₂O, adjusting the pH value of the electrolyte; preferably, 100-120V direct current is conducted among 2 electrodes; more preferably, the energization time is 20-40 min.

Further, in S3, the activated carbon is Biological Activated Carbon (BAC), and the BAC has good adsorption rate and adsorption capacity, so as to improve the stability of the bioactive peptide product; furthermore, the aperture of the biological activated carbon is 3-7nm, and the specific surface area is 1000-²(ii)/g; preferably, the pore size of the biological activated carbon is 5nm,specific surface area is 1100m²Is/g and.

Further, in S4, the dip-coating liquid has a pH of 10.

Further, in S4, the process conditions of spray drying are as follows: the air inlet temperature is 180-200 ℃, the air outlet temperature is 70-90 ℃, and the flow rate of the material liquid is 20-25 r/min.

Optionally, the unmodified PC membrane has a pore size of 0.04-0.06 μm, a smooth texture, high temperature resistance, a thickness of 9-11 μm, and a calculated pore size density: 3X 10⁸-5×10⁸pore/cm²。

Optionally, in the invention, the rice bran gluten enzymolysis solution passes through a microfiltration machine to filter out enzyme, bacteria and organic substances with large molecular weight, then the microfiltration enzymolysis solution is ultrafiltered through a food ultrafiltration machine, the ultrafiltration is carried out in a grading manner according to the molecular weight from large to small, an ultrafiltration membrane from 900Da to 1200Da can be selected, active peptides with the molecular weight more than 1200Da and the molecular weight less than 900Da in the solution are filtered out, and a polypeptide solution from 900Da to 1200Da is reserved. Further, the pH value of the gluten enzymolysis solution can be changed to reach the isoelectric point of KF-8 by food grade hydrochloric acid and food grade sodium bicarbonate solution, other peptide substances in the solution can move directionally in the solution by the action of electric field force and are adsorbed by activated carbon, so that the KF-8 and other peptide substances can be further separated, and the high-purity polypeptide solution can be obtained. And then, carrying out further ultrafiltration on the high-purity polypeptide solution by using a modified PC membrane, and carrying out spray drying to obtain a high-purity KF-8 product.

At present, KF-8 active peptide does not realize industrialized production, which is different from a laboratory preparation method, the separation condition of an ultrafiltration method is milder, the damage to the biological function of the active peptide is less, and the stability and the yield are improved. In the process of separating and treating the ultrafiltration product of the active peptide, the Polycarbonate (Polycarbonate) film is modified by using an electro-adsorption and swelling anchoring method of the biological activated carbon to intercept KF-8 molecules, so that the purity of the antioxidant active peptide of rice can be improved by more than 90 percent, and the purity of the KF-8 product is greatly improved.

Because the genes of the rice seed coat glutelin are quite conservative, mutants are quite rare in nature, so that the common rice hulls all contain KF-8, and the rice bran is prepared from common rice hulls, such as common rice bran prepared from rice hulls of indica rice and japonica rice.

Compared with the prior art, the invention has the following advantages:

(1) the method of the invention is different from common protein separation, has extremely high specific separation capacity to polypeptide KF-8, can achieve extraction purity of more than 90%, and obtains high extraction rate.

(2) The preparation method provided by the invention has mild conditions, can keep the nutritive value of the rice antioxidant active peptide as far as possible, and has good industrial application prospects.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise.

FIG. 1 is a flow chart of a conventional preparation method of a rice antioxidant peptide.

FIG. 2 is a graph showing the purity of KF-8 produced by different filtration methods.

FIG. 3 is a schematic diagram of the electrolytic adsorption of the present invention.

FIG. 4 is a graph showing the effect of different preparation methods on the KF-8 extraction rate, wherein BAC indicates that only electrolytic adsorption means is used for further treatment after ultrafiltration; PC (Zn (NO)₃)₂) Showing that the ultrafiltration is further processed by adopting a modified PC membrane; BAC + PC (Zn (NO)₃)₂) Showing that after the ultrafiltration, the further treatment is carried out by adopting an electrolytic adsorption means and a modified PC membrane in sequence.

FIG. 5 shows the extraction rate and purity of KF-8 product obtained in example 1.

FIG. 6 is a graph showing the effect of pH of the dip coating solution on KF-8 extraction efficiency in the preparation of a modified PC film.

FIG. 7 is a graph showing the effect of dip coating time on KF-8 extraction efficiency in the preparation of modified PC films.

FIG. 8 is a graph of KF-8 extraction yield as a function of dip coating time.

FIG. 9 is a diagram showing the quality of KF-8 product obtained by filtration using an unmodified ultrafiltration membrane.

Detailed Description

The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

Comparative example 1

Squeezing testa oryzae, defatting to obtain defatted testa oryzae with oil content of less than 6wt%, and using defatted testa oryzae as raw material can skip the above steps. Adding clear water into the cleaning pool, adding defatted rice bran, slowly stirring the defatted rice bran in the cleaning machine until the clear water completely submerges the defatted rice bran to achieve the purpose of cleaning, discharging water and impurities in the cleaning machine every 30min of stirring, adding new clear water, and cleaning for 3 h. And (3) putting the cleaned defatted rice bran into a dryer for drying, setting the drying temperature to be 55 ℃, and turning over the defatted rice bran in the dryer every 2 hours for drying for 6 hours in total. Taking out the dried defatted rice bran from the dryer, putting the defatted rice bran into a powdering machine to be powdered, wherein the particle size of the powder can pass through a 50-mesh sieve, and fully collecting the defatted rice bran powder.

Adding defatted rice bran powder and deionized water (mass ratio of 1: 10) into a centrifugal stirrer, dispersing the defatted rice bran powder in the deionized water, and starting a stirring paddle of the centrifugal stirrer to fully mix. Subsequently, food grade NaHCO was again added to the centrifugal stirrer₃The pH of the solution was adjusted to 9.0 and the temperature of the centrifuge stirrer was set to 40 ℃ and stirred for 2 hours. Adjusting the rotation speed of the centrifugal stirrer to 8000rpm, and centrifuging for 15 min. The defatted rice bran solution in the centrifugal mixer is separated into two phases by solid-liquid separation, the precipitate is discharged from a slag outlet and removed, and the supernatant is retained. And (3) adjusting the pH of the supernatant to 4.5 by food-grade hydrochloric acid, and standing for 1 h. Starting the centrifugal stirrer again, adjusting the rotation speed to 8000rpm to precipitate rice bran gluten, collecting the rice bran gluten solid phase from the slag outlet of the centrifugal stirrer, putting into the stirrer, injecting deionized water into the stirrer,so that the rice bran gluten solid-phase precipitate is redissolved in deionized water. Adding food-grade acid-base regulator into the stirrer to regulate pH of the solution to 7.0, and stirring at 37 deg.C for 3 hr.

Adjusting the protein concentration of the rice bran gluten solution to 7wt%, keeping at 37 deg.C, standing for 30min, and using food grade NaHCO₃Adjusting pH of the solution to 10.0, and feeding alkaline rice bran gluten solution into food enzymolysis tank to obtain [ E ]]/[S]Adding neutral protease (food grade neutral protease from Corne Biotech Ltd. of Jinan), adjusting the temperature of the enzymolysis tank to 50 deg.C, and hydrolyzing for 5 hr. Then, enzyme deactivation and sterilization treatment are carried out on the enzymolysis solution by a high-temperature enzyme deactivation program of the enzymolysis tank, namely enzyme deactivation is carried out for 10min at 85 ℃. Inputting the gluten hydrolysate subjected to enzyme deactivation and sterilization into a centrifuge, adjusting the rotation speed of the centrifuge to 8000rpm, centrifuging for 15min to obtain rice bran gluten enzymolysis solution, and discharging the precipitate from a slag outlet of the centrifuge.

Injecting the rice bran gluten enzymolysis solution into a micro-filter by a booster pump for micro-filtration treatment (the aperture of the micro-filtration membrane is 0.2 mu m, and the pressure difference is 0.1 Mpa), and discharging macromolecular organic substances in the rice bran gluten enzymolysis solution. And (2) conveying the treated filtrate into a food-grade ultrafiltration machine from a water outlet pipe of the microfiltration machine, carrying out ultrafiltration classification step by step according to the molecular weight of the polypeptide in the rice bran gluten enzymolysis solution from high to low, removing the polypeptide with higher molecular weight by adopting an ultrafiltration membrane of 1005Da, removing the polypeptide with lower molecular weight by adopting an ultrafiltration membrane of 1000Da, and finally enriching to obtain the active peptide solution rich in the polypeptide KHNRGDEF (molecular weight: 1002.4 Da). And finally, conveying the obtained active peptide solution into a spray dryer for drying treatment. The technological conditions of spray drying are that the air inlet temperature is 180 ℃, the air outlet temperature is 80 ℃, and the flow rate of the material liquid is 20 rpm. Collecting the rice antioxidant peptide powder at the nozzle.

Detection shows that the purity of KF-8 in the rice antioxidant peptide powder is 77.52wt%, and the extraction rate is 37.91%.

Comparative example 2

The raw material is defatted rice bran powder or rice bran protein powder. Adding defatted rice bran powder into centrifugal stirrer and removingDispersing the degreased rice bran powder in deionized water (the mass ratio is 1: 10), and starting a stirring paddle of a centrifugal stirrer to fully mix. Subsequently, food grade NaHCO was again added to the centrifugal stirrer₃The pH of the solution was adjusted to 9.0, and the solution was stirred for 2 hours at 40 ℃ with the centrifugal stirrer set. Adjusting the rotation speed of the centrifugal stirrer to 8000rpm, and centrifuging for 15 min. The defatted rice bran solution in the centrifugal mixer is separated into two phases by solid-liquid separation, the precipitate is discharged from a slag outlet and removed, and the supernatant is retained. And (3) regulating the pH of the degreased rice bran protein supernatant in the centrifugal stirrer to 4.5 by food-grade hydrochloric acid, and standing for 1 h. And starting the centrifugal stirrer again, adjusting the rotating speed of the centrifugal stirrer to 8000rpm to separate out the rice bran gluten precipitate, collecting the solid-phase precipitate from a slag outlet of the centrifugal stirrer, putting the solid-phase precipitate into the stirrer, and injecting deionized water into the stirrer simultaneously to redissolve the rice bran gluten solid-phase precipitate in the deionized water. Adding food-grade acid-base regulator into the stirrer to regulate pH of the solution to 7.0, and stirring at 37 deg.C for 3 hr.

Adjusting the protein concentration of the rice bran gluten solution to 7wt%, keeping at 37 deg.C, standing for 30min, and using food grade NaHCO₃Adjusting pH of the solution to 10.0, and feeding alkaline rice bran gluten solution into food enzymolysis tank to obtain [ E ]]/[S]Adding neutral protease into the enzymolysis tank at a ratio of 1.8%, adjusting the temperature of the enzymolysis tank to 50 deg.C, and hydrolyzing for 5 hr. Then, enzyme deactivation and sterilization treatment are carried out on the enzymolysis solution by a high-temperature enzyme deactivation program of the enzymolysis tank, namely enzyme deactivation is carried out for 10min at 85 ℃. Inputting the gluten hydrolysate subjected to enzyme deactivation and sterilization into a centrifuge, adjusting the rotation speed of the centrifuge to 8000rpm, centrifuging for 15min to obtain rice bran gluten enzymolysis solution, and discharging the precipitate from a slag outlet of the centrifuge.

Injecting the rice bran gluten enzymolysis solution into a micro-filter through a booster pump for micro-filtration treatment, and discharging macromolecular organic substances in the rice bran gluten enzymolysis solution. And (2) conveying the treated filtrate into a food ultrafilter from a water outlet pipe of the microfilter, carrying out ultrafiltration classification step by step according to the molecular weight of the polypeptide in the rice bran gluten enzymolysis solution from high to low, removing the peptide with larger molecular weight by adopting an ultrafiltration membrane of 1200Da, removing the peptide with smaller molecular weight by adopting an ultrafiltration membrane of 1000Da, and finally enriching to obtain the active peptide solution rich in the polypeptide KHNRGDEF (molecular weight: 1002.4 Da). And finally, conveying the obtained active peptide solution into a spray dryer for drying treatment. The technological conditions of spray drying are that the air inlet temperature is 180 ℃, the air outlet temperature is 80 ℃, and the flow rate of the material liquid is 20 rpm. Collecting the rice antioxidant peptide powder at the nozzle.

Detection shows that the purity of KF-8 in the rice antioxidant peptide powder is 53.76wt%, and the extraction rate is 44.26%.

The method can improve the yield of KF-8 on the basis of the embodiment 1. Comparing examples 1 and 2, it was readily found that the method of ultrafiltration and the difference in molecular weight of ultrafiltration resulted in a wide variation in the purity of KF-8 and that most of the KF-8 was wasted.

Comparative example 3

Preparing rice bran gluten enzymatic hydrolysate according to the method of comparative example 1; and injecting the rice bran and glutelin enzymolysis solution into a micro-filter through a booster pump for micro-filtration treatment, and discharging macromolecular organic substances in the rice bran and glutelin enzymolysis solution. And (3) conveying the treated filtrate into a food-grade ultrafiltration machine from a water outlet pipe of the microfiltration machine, carrying out ultrafiltration classification step by step according to the molecular weight of the polypeptide in the rice bran and glutelin enzymolysis solution from high to low, and carrying out spray drying according to the method of the comparative example 1. The polypeptide KF-8 (molecular weight: 1002.4 Da) is easily enriched in the range of 900 Da-1200 Da by an ultrafiltration membrane, and if the ultrafiltration membrane is enriched in the range of 1000 Da-1200 Da, the purity of the finally obtained KF-8 product is 53.76 +/-1.62%; if ultrafiltration membranes are selected to be enriched in a range of 1000 Da-1100 Da, the purity of the KF-8 of the final KF-8 product is 68.21 +/-2.97%; if the ultrafiltration membrane is selected to be enriched in the range of 900 Da-1100 Da, the purity of the final KF-8 product is 47.43 +/-0.85% (see FIG. 2).

In order to improve the purity of the KF-8 product obtained by ultrafiltration to more than 90%, the solution obtained by ultrafiltration enrichment needs to be subjected to adsorption filtration. Further carrying out ultrafiltration (enriched in the range of 1000 Da-1100 Da) on the obtained active peptide solution for 1h by adopting a modified PC membrane, desorbing for 4h, collecting the solution in an ultrafiltration machine, and carrying out spray drying to obtain a KF-8 product with the purity of 91.24 +/-3.16 wt%, wherein the KF-8 product is shown in figure 2; the extraction rate of KF-8 can reach more than 80% (see FIG. 5). The method can greatly improve the purity of the rice antioxidant active peptide.

The applicant researches and discovers that in the active peptide KF-8, the amino acid at the second position is heterocyclic amino acid histidine, and metal zinc has special adsorption capacity on the amino acid histidine, so that a complex with weak adsorption can be formed, and the adsorption capacity is strongest when the pH value is 10 under alkaline conditions. In addition, the PC membrane has good hydrophobicity, can permeate salt ions in a solution and intercept protein molecules, has excellent physical and mechanical properties, particularly excellent impact resistance, high tensile strength, bending strength and compressive strength, and is suitable for an ultrafiltration machine. The invention adopts the swelling-anchoring method to fix Zn (NO) on the PC film in the alkaline environment₃)₂The PC membrane has the optimal adsorption capacity for KF-8 in alkaline environment to retain KF-8 in solution, salt ions in the solution pass through the semipermeable membrane, and then the pH value in the solution is reduced to slowly reach neutral Zn (NO)₃)₂The adsorption ability to TF-8 gradually deteriorated and the membrane was released. The specific implementation method comprises the following steps:

zn (NO) at pH 10₃)₂·6H₂O and NH₃H₂The mass concentration ratio of O is 1: the hydrophobic effect is best when 1 is used. The PC film was swelled in acetone for 5s and immediately placed with 0.6% by mass of Zn (NO)₃)₂In solution and with NH₃·H₂Adjusting pH to 10 to obtain a solution, dip-coating at room temperature for 2 hr, taking out the membrane, air drying, soaking in distilled water for 1 hr to remove weak Zn (NO)₃)₂And drying the polymer to obtain the modified PC film.

The original PC membrane has the aperture of 0.05 μm, smooth texture, high temperature resistance and the thickness of 10 μm, and the calculated aperture density is as follows: 4X 10⁸pore/cm²。

Comparative example 3 was repeated, changing only the pH of the dip-coating solution to 7, 8, 9, 11, 12, 13, 14, respectively. As can be seen from FIG. 7, when the pH was controlled to 9 to 11, a good index of extraction rate was obtained.

Comparative example 3 was repeated, and the dip coating time was changed to 1h and 3h, respectively, only in the preparation of the modified PC film. As can be seen from FIG. 8, the dip coating time was controlled to be 2 hours or more, and a good index of extraction rate was obtained, with 2 hours being the most preferred.

Comparative example 4

The research of the applicant finds that KF-8 is a polypeptide with an isoelectric point of 7.88 and good hydrophilicity. Therefore, by utilizing the physical property of KF-8, the pH of the gluten enzymolysis solution is changed to reach the isoelectric point of KF-8, other peptide substances in the solution are directionally moved in the solution by the action of an electric field force and are adsorbed by bioactive carbon (peptide segment pI is less than 7.88 and is adsorbed by a positive pole, peptide segment pI is more than 7.88 and is adsorbed by a negative pole, and peptide segment pI =7.88 cannot be adsorbed by electrophoresis), KF-8 is kept in the solution, and the solution is electrified to electrolyze water so as to improve the concentration of KF-8 in the solution.

Introducing the active peptide solution enriched in the range of 1000 Da-1100 Da obtained by the method of the comparative example 3 into an electrolytic tank, and using Na₂HPO₄•2H₂And O, adjusting the pH value of the enzymolysis solution to 7.88 +/-0.02 to form a pH buffer system, and switching on 110V industrial direct current to enable the polypeptide with the isoelectric point not being 7.88 to be adsorbed by the bioactive carbon wrapped outside the electrode under the action of electric field force. After electrifying for 30min, cutting off the power, and spray drying the obtained high-purity active peptide solution to prepare KF-8 powder, wherein the extraction rate of KF-8 is about 90% (see figure 4).

The phosphate is used for preparing the phosphate buffer solution, so that the change of pH caused by electrolysis of water in the solution can be prevented. Because water is electrolyzed during electrophoresis, the water content and pH change of the solution are monitored in real time during long-time electrophoresis, and water is added in time. The bioactive carbon used for polypeptide adsorption needs to be replaced after multiple times of adsorption. The gas generated by electrolysis needs to be removed in time so as to avoid potential safety hazard.

The operation principle of the adsorption filtration and electrolysis tank is shown in figure 5.

Example 1

A highly pure active peptide solution was prepared by the method of comparative example 4, the modified PC membrane prepared by the method described in comparative example 3 was loaded into an ultrafilter, and the highly pure active peptide solution was treated with NaHCO₃Adjusting pH to 10, and filtering the high-purity active peptide solution by an ultrafilter. The zinc on the modified PC membrane can adsorb KF-8 in the high-purity active peptide solution on the membrane, and salt ions can be separated through the membrane. Then slow downThe pH of the solution passing through the ultrafilter was slowly lowered to neutral, and the KF-8 solution was further purified while the KF-8 was slowly released from the modified PC membrane and redissolved in the solution. Adsorbing and filtering for 1h by using a modified PC membrane, desorbing for 4h, collecting the solution in an ultrafilter, and spray-drying to obtain a high-purity KF-8 product with the purity of 92.5 +/-0.43 wt%, wherein the extraction rate of KF-8 in the raw material can reach 98.62%, see FIGS. 4 and 6. Therefore, the KF-8 product with high purity can be obtained by using the modified PC membrane to adsorb the KF-8 after the biological activated carbon is used for electric adsorption, and the high extraction rate is obtained.

Comparative example 5

Example 1 was repeated with the only difference that: carrying out ultrafiltration on the high-purity active peptide solution by adopting an unmodified PC membrane.

Preparing a high-purity active peptide solution according to the method of the comparative example 4, removing the polypeptide with larger molecular weight by adopting an ultrafiltration membrane of 1005Da, removing the polypeptide with smaller molecular weight by adopting an ultrafiltration membrane of 1000Da, and spray-drying to obtain a KF-8 product with the purity of 86.31wt%, wherein the extraction rate of KF-8 in the raw material reaches 84.7%, and the KF-8 is filtered by using an unmodified ultrafiltration membrane, so that the quality of the product cannot reach the standard of using the modified ultrafiltration membrane. (FIG. 9)

The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.