CN106636274B - Separation and preparation method of rice bran antioxidant active peptide - Google Patents

Separation and preparation method of rice bran antioxidant active peptide Download PDF

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CN106636274B
CN106636274B CN201611071570.5A CN201611071570A CN106636274B CN 106636274 B CN106636274 B CN 106636274B CN 201611071570 A CN201611071570 A CN 201611071570A CN 106636274 B CN106636274 B CN 106636274B
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rice bran
gluten
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梁盈
林亲录
王玉倩
李佳佳
黄萍
张琳
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Changsha Feichuang Biotechnology Co ltd
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Central South University of Forestry and Technology
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Abstract

The invention provides a rice bran gluten-derived bioactive peptide, the sequence of which is KHNRGDEF; the active peptide has antioxidant activity and can be used for preparing food and health care products with antioxidant effect. The invention separates and obtains a polypeptide with in vitro antioxidant activity from the enzymolysis products of rice bran gluten, crude gluten is extracted by an alkali extraction and acid precipitation method, then the crude gluten is extracted by classification, then the rice bran gluten is separated and purified by ion exchange chromatography and gel chromatography, then the obtained rice bran gluten is hydrolyzed by alkaline protease, centrifuged, concentrated in vacuum and freeze-dried to obtain an antioxidant active peptide product, the enzymolysis conditions are [ E ]/[ S ] 1.5-1.8%, pH is 10.0, the temperature is 50 ℃, the time is 4.2h, and then the required antioxidant active peptide product is separated and purified by ion exchange chromatography, gel chromatography, RP-HPLC preparation and analytical RP-HPLC to obtain the required antioxidant pure peptide. The invention has the advantages of low production cost, simple and convenient process, environmental protection, reasonable amino acid composition of the obtained rice bran active peptide, low irritability, safety, reliability and the like, and has better free radical scavenging capacity and good antioxidant effect.

Description

Separation and preparation method of rice bran antioxidant active peptide
Technical Field
The invention relates to a separation preparation method of rice bran antioxidant active peptide.
Background
The rice bran is a byproduct generated in a rice production factory, the protein amino acid composition of the rice bran is similar to that of the rice, and the amino acid composition is reasonable and is high-quality protein. A large amount of rice bran is produced in industrial production every year in China, and most of the rice bran is not effectively utilized, so that deep processing of rice bran protein is needed to improve the additional value of the rice bran protein, and full utilization of resources is realized. The protein solubility in rice and rice bran is poor, and the application of the rice and rice bran is seriously influenced. The peptide segments with smaller molecular weight in the rice bran have better solubility, and different peptide segments have different efficacies, such as oxidation resistance, blood pressure reduction, immunological activity and the like. The rice antioxidant active peptide has good free radical scavenging capacity and great development potential in the aspect of preventing cardiovascular and cerebrovascular diseases induced by free radicals. The rice bioactive peptide has good effect of eliminating free radicals induced by oxidative damage, and can improve the oxidation resistance of human cells.
Through research, no relevant literature report that scholars in China use neutral protease to extract antioxidant active peptide from rice bran gluten is found, so the research field of extracting antioxidant active peptide from rice bran gluten by neutral protease is in a blank stage. The research finds that the rice active peptide extracted from the rice bran protein by using the neutral protease has the effects of removing free radicals and inhibiting cell oxidation, effectively improves the added value of the rice bran, and realizes high-value utilization of the rice bran.
Disclosure of Invention
The invention aims to provide a separation and preparation method of rice bran antioxidant peptide, which takes rice bran as a raw material and utilizes neutral protease to prepare a peptide product with antioxidant activity so as to realize high-value utilization of the rice bran.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the separation and preparation method of the rice bran antioxidant active peptide comprises the steps of extracting degreased rice bran by an alkali extraction and acid precipitation method and an Osborne fractional extraction method to obtain rice bran gluten, then separating and purifying the rice bran gluten by ion exchange and gel chromatography technologies, hydrolyzing the separated and purified rice bran gluten by neutral protease, centrifuging, vacuum concentrating, freezing and drying, and then separating and purifying by using ion exchange chromatography, gel chromatography, preparative RP-HPLC and analytical RP-HPLC to obtain the antioxidant active peptide, wherein the sequence of the antioxidant active peptide is shown as SEQ ID NO. 1.
Preferably, the neutral protease hydrolysis conditions are [ E ]/[ S ]1.5% -1.8%, pH10.0, temperature 50 ℃, reaction time 4.2 h.
Preferably, the method specifically comprises the following steps:
(1) and (3) extraction of gluten: defatted rice bran → water is added and the pH value is adjusted to be 9.0, the material-liquid ratio is 1:10, the unit of the material-liquid ratio is mL/g, water bath at 40 ℃ is carried out for 4h → centrifugation → supernatant fluid is taken → acid precipitation under the condition of pH value being 4.0 → centrifugation → precipitation → water washing, the pH value is adjusted to be 7.0 → freeze drying → alkali extraction of rice bran gluten → Osborn fractional extraction, and the rice bran gluten is obtained;
(2) na with pH value of 10.0 and concentration of 0.1 mol/L is selected2CO3-NaHCO3Performing primary separation on rice gluten as a buffer solution; at a pH value of 12.0 and a concentration of 0.05 mol/L Na2HPO4Dissolving the alkali-extracted rice bran gluten in NaOH buffer solution, and further separating and purifying the alkali-extracted rice bran gluten by using DEAE-52 anion exchange chromatography medium;
(3) preparing rice bran protein suspension, adding neutral protease according to the mass ratio of the neutral protease to the gluten of 1.5-1.8%, adjusting the pH value to 10.0 → dissolving in an ultra-grade constant-temperature water bath enzyme reactor at 50 ℃ for 30min → performing enzymolysis for 4.2h → killing enzyme → centrifuging → taking supernatant → vacuum concentration → freeze drying to obtain rice gluten peptide;
(4) separating and purifying the rice gluten peptide to obtain the antioxidant active peptide.
The invention is further illustrated below:
the preparation method of the rice active peptide comprises the following steps:
(1) and (3) extraction of gluten: extracting gluten by an alkali extraction and acid precipitation method, and separating and purifying the rice gluten by ion exchange and gel chromatography technologies;
(2) hydrolysis of rice bran gluten: 5% (w/v, g/mL) rice bran gluten suspension → pH value adjustment → dissolution in a super constant temperature water bath enzyme reactor for 30min under certain temperature → enzymolysis (constant pH titrator stabilizes system pH) according to certain substrate concentration, enzyme addition amount, pH value and temperature → 85 ℃, 10min enzyme deactivation → 3500r/min centrifugation for 15min → supernatant → vacuum concentration → freeze drying → rice bran gluten peptide;
(3) separation and purification of rice bran gluten peptide
Using SP-Sephadex G-25 as an ion exchange chromatography medium, using 0.02mol/L HAc-Na Ac as an initial buffer solution with the pH value of 4.0 and the flow rate of 2mL/min, performing gradient elution by using an equilibrium buffer solution, an equilibrium buffer solution containing 0.5mol/L NaCl and an equilibrium buffer solution containing 1mol/L NaCl, and selecting a component with the strongest free radical scavenging capacity in a plurality of peptide components of rice bran gluten hydrolysate.
Separating the selected components into several components by Sephadex G-15 gel chromatography, and selecting the component with the strongest free radical scavenging ability.
Further separating the components selected in the last step by using preparative RP-HPLC, selecting a plurality of main components, screening the components with stronger antioxidant activity by measuring the free radical scavenging capacity of each main component, and analyzing whether the components are relatively single peaks by RP-HPLC.
(4) And measuring the relative molecular weight of the extracted rice active peptide by using a MALDI-TOF/TOF MS mass spectrometry. And combining TOF-MS/MS tandem mass spectrum to obtain the amino acid composition and the arrangement sequence thereof.
Compared with the prior art, the rice bran active peptide prepared by the method more effectively utilizes rice bran, improves the utilization of byproducts in rice production, saves production cost, and has important significance for realizing the comprehensive utilization of rice bran protein and improving the added value of the rice bran protein.
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FIG. 1 is a graph showing the results of TOF-MS/MS tandem mass spectrometry;
FIG. 2 shows ABTS of PA-PG fraction isolated by ion exchange chromatography+Free radical scavenging ability; component with the highest clearance andsignificant differences exist between other components;
FIG. 3 shows the fraction PF obtained by gel chromatography1-PF4ABTS of+Free radical scavenging ability; indicates that there is a significant difference between the fraction with the highest clearance and the other fractions;
FIG. 4 shows PF fraction isolated by RP-HPLC3-a to F3-gamma ABTS ·+Free radical scavenging ability; indicates that there was a significant difference between the fraction with the highest clearance and the other fractions.
Detailed Description
Example 1
The separation and preparation method of the rice bran antioxidant active peptide comprises the following steps:
(1) the process flow for extracting the rice bran gluten by the alkali extraction and acid precipitation method comprises the following steps: adding water into defatted rice bran (passing through a 60-mesh sieve) → and adjusting the pH value to 9.0 (the material-liquid ratio is 1:10, the unit of the material-liquid ratio is mL/g, water bath: 40 ℃, 4 h) → centrifugation (8000 r/min, 15 min) → taking supernatant → acid precipitation (pH 4.0) → centrifugation (8000 r/min, 15 min) → taking precipitate → washing with water for three times, adjusting the pH value to 7.0 → freeze drying (48 h) → alkali extracting rice bran gluten (4 ℃ sealed storage + silica gel) → Osborne fractional extraction, and obtaining purer gluten;
(2) selecting Na2CO3-NaHCO3(pH 10.0, 0.1 mol/L) as a buffer to perform a preliminary separation of rice gluten. At 0.05 mol/L Na2HPO4Dissolving the alkali-extracted rice bran gluten in NaOH (pH 12.0) buffer solution, further separating and purifying the alkali-extracted rice bran gluten by using DEAE-52 anion exchange chromatography medium, eluting the alkali-extracted rice gluten by using 0.15mol/L, 0.25mol/L, 0.35mol/L, 0.45mol/L and 0.55mol/L saline buffer solutions in sequence, and separating to obtain five elution peaks R1、R2、R3、R4、R5By HPLC on R3The fractions were analyzed for purity, which was 71.55% pure. Collecting R3Further purifying by Sephadex G-75 gel filtration chromatography to obtain R3-α、R3-β、R3-gamma three elution peaks by HPLCDetection of where R3The molecular weight of the gamma elution peak is 37530.34, and the purity reaches 93.45%;
(3) 5% (w/v, g/mL) of the rice bran gluten suspension, adding neutral protease according to the proportion that the neutral protease/gluten is 1.5% -1.8% (g/g), adjusting the pH value to 10.0 → dissolving in an ultra-grade constant temperature water bath enzyme reactor at 50 ℃ for 30min → carrying out enzymolysis for 4.2h (stabilizing the pH of the system by using a pH titrator) → 85 ℃, 10min inactivating enzyme → 3, centrifuging at 500r/min for 15min → supernatant → vacuum concentration → freeze drying → rice gluten peptide;
(4) separation and purification of rice gluten peptide
Using SP-Sephadex C-25 as ion exchange chromatography medium, using 0.02mol/L HAc-NaAc as initial buffer solution with pH4.0 and flow rate of 2m L/min, performing gradient elution with equilibrium buffer solution, equilibrium buffer solution containing 0.5mol/L NaCl and equilibrium buffer solution containing 1mol/L NaCl, separating seven components (PA-PG) from gluten neutral protease hydrolysate mixed peptide component, and separating ABTS of component PF+The radical scavenging capacity was strongest (71.09%, FIG. 2).
The Sephadex G-15 gel chromatography is utilized to take ultrapure water as a buffer solution, the sample injection concentration is 15mg/mL, and the component PF is divided into 4 components PF1、PF2、PF3And PF4. The component PF is detected3ABTS of+The radical scavenging ability was strongest and reached 69.24% (FIG. 3).
PF by preparative RP-HPLC3Further dividing into 5 main components, and determining free radical scavenging ability of each component to screen out PF as the component with the strongest antioxidant activity3- γ (FIG. 4), ABTS · thereof+The scavenging capacity of free radicals reaches 78.59%, and the scavenging capacity is relatively single peak through RP-HPLC analysis.
(5) Measuring antioxidant active peptide PF by MALDI-TOF MS mass spectrometry3The relative molecular weight of- γ is 1002.06 (FIG. 1).
Obtaining by combining TOF-MS/MS tandem mass spectrum; PF (particle Filter)3- γ is composed of 8 amino acids arranged in the order Lys-His-Asn-Arg-Gly-Asp-Glu-Phe (KHNRGDEF). Found by searching, the active peptide obtained by the invention has antioxidationActive novel active peptide sequences.
Example 2 detection of antioxidant Activity of Rice gluten-active peptides in vivo
Selecting cells in logarithmic growth phase, digesting with EDTA-free pancreatin to obtain cell suspension, measuring cell concentration in the suspension with a counting plate, inoculating 5000 cells per well into a 96-well plate, inoculating 100 μ L cells per well, setting 6 multiple wells per group, and culturing at 37 deg.C with 5% CO2After 6 hours of culture in an incubator, the cells were divided into normal group H2O2Treatment group H2O2+ the protection group of glutelin active peptide, the original culture medium was aspirated, 80. mu.L of culture medium was added to all groups, the normal group, H2O2The treatment groups were added 10. mu.L of serum-free medium, H2O2The protection group of the glutelin active peptide is respectively added with the equivalent-volume glutelin active peptide with corresponding concentration, so that the final concentration of the medicine is respectively 100, 150 and 200 mu mol/L, and 5 percent CO is carried out at 37 DEG C2After the incubator is pre-protected for 4H, H2O2Treatment groups and H2O2+ 10. mu. L H per well of the protection group of the glutelin-active peptide2O2The final concentration was 100. mu.ml/L, and 10. mu.L of serum-free medium was added to the normal control group at 37 ℃ in 5% CO2After 24h incubation in the incubator, the cell viability was determined by the MTS method (see table 1). Comparison with the Normal group, H2O2The cell survival rate of the injured group is obviously reduced; and H2O2Treatment group comparison, high dose group (200. mu. mol/L) of glutelin-active peptide versus H2O2The induced cell activity is obviously improved.
TABLE 1 Rice gluten active peptide pairs H2O2Effect of cell viability of injured endothelial progenitor cells
Figure DEST_PATH_IMAGE002

Claims (1)

1. A separation preparation method of a rice bran antioxidant active peptide is characterized in that the rice bran gluten is obtained by alkali extraction and acid precipitation and Osborne fractional extraction of defatted rice bran, then ion exchange and gel chromatography technologies are selected to separate and purify the rice bran gluten, the separated and purified rice bran gluten is hydrolyzed by neutral protease, centrifuged, concentrated in vacuum and freeze-dried, and then the active peptide antioxidant active peptide is obtained by ion exchange chromatography, gel chromatography, preparation RP-HPLC and analytical RP-HPLC separation and purification, wherein the sequence of the antioxidant active peptide is KHNRGDEF;
the hydrolysis condition of the neutral protease is 1.5% -1.8%, the pH value is 10.0, the temperature is 50 ℃, and the reaction time is 4.2 h; the method specifically comprises the following steps:
(1) and (3) extraction of gluten: defatted rice bran → water is added and the pH value is adjusted to be 9.0, the material-liquid ratio is 1:10, the unit of the material-liquid ratio is mL/g, water bath at 40 ℃ is carried out for 4h → centrifugation → supernatant fluid is taken → acid precipitation under the condition of pH value being 4.0 → centrifugation → precipitation → water washing, the pH value is adjusted to be 7.0 → freeze drying → alkali extraction of rice bran gluten → Osborn fractional extraction, and the rice bran gluten is obtained;
(2) na with pH value of 10.0 and concentration of 0.1 mol/L is selected2CO3-NaHCO3Performing primary separation on rice bran gluten as a buffer solution; at a pH value of 12.0 and a concentration of 0.05 mol/L Na2HPO4Dissolving the alkali-extracted rice bran gluten in NaOH buffer solution, and further separating and purifying the alkali-extracted rice bran gluten by using DEAE-52 anion exchange chromatography medium;
(3) preparing rice bran protein suspension, adding neutral protease according to the mass ratio of the neutral protease to the gluten of 1.5-1.8%, adjusting the pH value to 10.0 → dissolving in an ultra-grade constant temperature water bath enzyme reactor at 50 ℃ for 30min → performing enzymolysis for 4.2h → killing enzyme → centrifuging → taking supernatant → vacuum concentration → freeze drying to obtain rice bran gluten peptide;
(4) separating and purifying rice bran gluten peptide to obtain the antioxidant active peptide.
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CN112522354B (en) * 2020-12-01 2023-01-24 中南林业科技大学 Preparation method of rice antioxidant active peptide
CN113616773B (en) * 2021-08-25 2023-11-14 中南林业科技大学 Application of rice bran active peptide in intervention of caenorhabditis elegans aging or muscle injury
CN114150033B (en) * 2021-12-06 2023-05-19 哈尔滨商业大学 Preparation method of rice bran antioxidant peptide compound treated by peak alpha amylase
CN114558106B (en) * 2022-03-23 2024-02-23 长沙飞创生物技术有限责任公司 Application of rice bran active peptide in preventing or treating lipotoxicity related diseases
CN114478702B (en) * 2022-04-06 2023-12-12 中南林业科技大学 Rice bran-derived short peptide and application thereof in treating skin injury

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大米蛋白抗氧化肽的制备、分离纯化和结构鉴定;张君慧;《中国博士学位论文全文数据库（电子期刊）工程科技I辑》;20101215(第12期);摘要、第1-73页 *

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