CN104342473A - Preparation and purification method of amaranth seed antihypertensive bioactive peptide - Google Patents
Preparation and purification method of amaranth seed antihypertensive bioactive peptide Download PDFInfo
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- CN104342473A CN104342473A CN201310339637.9A CN201310339637A CN104342473A CN 104342473 A CN104342473 A CN 104342473A CN 201310339637 A CN201310339637 A CN 201310339637A CN 104342473 A CN104342473 A CN 104342473A
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Abstract
The invention relates to a preparation and purification method of an amaranth seed antihypertensive bioactive peptide. The method is as below: preparing an amaranth seed crude protein by using alkali-solution and acid-isolation, then acquiring antihypertensive bioactive peptide by using alkaline protease enzymolysis, and finally separating and purifying by macroporous resin adsorption, gel chromatography and high performance liquid chromatography to obtain the antihypertensive bioactive peptide. The method extracts peptide from plants, has high edible safety, little side effect, and the function of reducing blood pressure for patients with hypertension but no antihypertensive effect to the normal blood pressure. Therefore, the invention has the good prospects for development.
Description
Technical field
The present invention relates to the preparation of a kind of amaranth seed active antihypertensive peptide and separation purification method.
Background technology
Amaranth seed contains the physiologically active substances such as rich in protein, amino acid, unsaturated fatty acids and vitamin-E, vitamins C, para-insulin and squalene, be a kind of good quality and high output, efficient multi-functional rare species, there is very high comprehensive development and utilization and be worth.Amaranth seed crude protein content 14 ~ 18%, is significantly higher than general cereal.Amaranth seed albumen forms mainly white protein, sphaeroprotein and gluten three kinds of caustic solubility albumen, and the amaranth seed albumen composition of different varieties is slightly different, but content of prolamine is all very low, is about 1.6% ~ 2.0%, and the content of gluten is the highest is about 60% ~ 73%.Therefore, amaranth seed albumen had certain future as a kind of new plant protein resource exploitation.
In recent years, research finds that natural plant protein matter is after external enzymolysis or the digested enzymolysis of body, can produce the bioactive peptide with special physiological function.This kind of peptide edible safety is high, and side effect is little, can play hypotensive effect to hyperpietic, to normal arterial pressure then without hypotensive effect.The biologically active peptides deriving from food proteins at present more and more causes the attention of knowledgeable people, and the polypeptide particularly with hypotensive activity becomes one of focus of biologically active peptides research.
It is raw material that the present invention intends with amaranth seed, and separation and purification goes out a kind of polypeptide with blood pressure reduction effect, and this polypeptide security of extracting from plant is good, will have good development prospect.
Summary of the invention
The object of this invention is to provide the preparation of a kind of amaranth seed active antihypertensive peptide and separation purification method, with amaranth seed for raw material, optimization amaranth seed protein extraction technique, protease hydrolysis amaranth seed albumen are prepared active antihypertensive peptide technique and are purified into highly active active antihypertensive peptide by technology separation such as macroporous resin adsorption, gel chromatography and half preparative high-performance liquid chromatographics (HPLC), and the exploitation be intended to for amaranth seed active antihypertensive peptide provide theoretical foundation.The technical scheme of invention is summarized as follows:
(1) preparation of amaranth seed albumen
Be that the NaOH solution of 0.018% is by weight for 1:35 prepares feed liquid by amaranth powder of seeds and mass concentration, constant temperature oscillation shaking table is warming up to 35 DEG C, mechanical shaking extraction 30min, adjusts pH to make precipitate soluble proteins to iso-electric point 4.6 reacted feed liquid 1mol/LHCl solution; The centrifugal 20min of 6000r/min, abandons supernatant liquor, gets precipitation afterwards;-55 DEG C of vacuum lyophilization 16h will be deposited in, i.e. obtained amaranth seed albumen after centrifugal;
(2) preparation of amaranth seed active antihypertensive peptide
Get amaranth seed albumen in beaker, being mixed with mass concentration is 4%, in 90 DEG C of thermal treatment 10min, then take out and be cooled to 54.4 DEG C, pH=8.78 is regulated with 1moL/LNaOH, add the Sumizyme MP Novi letter of E/S=3.5%, in 54.4 DEG C, vibrate in 160r/min shaking bath 3h, and with 1moL/LNaOH maintain reaction system pH=8.78 ± 0.05 between, take out at 100 DEG C, 10min goes out after enzyme and cools rapidly, at 10000r/min, 4 DEG C of centrifugal 20min, get supernatant liquor lyophilize and namely obtain thick amaranth seed ace inhibitory peptide;
(3) separation of amaranth seed active antihypertensive peptide
In the chromatography column of 3.2 × 100cm, fill the DA201-C macroporous resin that pre-treatment is good, wet method upper prop, allow its natural sedimentation obtain high 87cm, the resin fixed bed of bed volume (BV) 700mL; Be 10mg/ml by mass concentration, pH=5, volume is that the enzymolysis solution of 1BV flows through chromatography column with the speed of 6mL/min, the albumen do not adsorbed and the impurity sticked on resin are rinsed well by the distilled water having adsorbed rear 1BV, again the ethanolic soln of the volume fraction 75% of elution volume 4BV is flowed through chromatography column with the speed of 5mL/min and carry out wash-out, to stop wash-out during A280nm≤0.05, rotary evaporation concentrates postlyophilization, obtains amaranth seed ace inhibitory peptide;
(4) purifying of amaranth seed active antihypertensive peptide
First adopt gel chromatography, by sephadex G 25 3 ~ 5 times of volume water expansion 3h, with water cleaning until supernatant liquor does not have impurity, slowly put into 10 × 100(internal diameter mm × length cm) in chromatography column, can not bubble be had in pipe, otherwise again fill post; To install after pillar vertical natural sedimentation a whole night, make post bed closely homogeneous; Loading is, the water level in pipe is first allowed to be down to gel top layer 2 ~ 3mm, adding 2mL mass concentration is amaranth seed ace inhibitory peptide solution after the macroporous resin purification of 50mg/mL, when peptide solution is left to gel top layer 2 ~ 3mm, the water adding 3 ~ 4mL rinses post jamb, connects constant flow pump, with pure water with the flow velocity wash-out of 0.4ml/min, every 5min collects a pipe, rinses, balances pillar after collecting with same flow velocity;
Secondly half preparative high-performance liquid chromatographic is adopted: by the sample collected after using gel chromatography again through more than half preparative high-performance liquid chromatographic purifying, its liquid-phase condition: Varian ProStar218 system, VarianC18(21.2*150mm) chromatographic column, flow velocity 10ml/min, determined wavelength 220nm, sample size 10mg/ml-1ml, column temperature 25 DEG C, elution requirement: 0 ~ 40min, acetonitrile mass ratio is 5 ~ 45%, and trifluoroacetic acid mass ratio is 0.1%.The component of collecting 6 ~ 9min gained both obtained amaranth seed active antihypertensive peptide.
Present method with amaranth seed for raw material, optimize the extraction process of amaranth seed albumen, utilize protease hydrolysis amaranth seed albumen to prepare active antihypertensive peptide technique, be purified into highly active active antihypertensive peptide finally by technology separation such as macroporous resin adsorption, gel chromatography and half preparative high-performance liquid chromatographics (HPLC).This polypeptide security of extracting from plant is good, will have good development prospect.
A kind of amaranth seed active antihypertensive peptide preparation of the present invention and separation purification method, with amaranth seed for raw material, optimization amaranth seed protein extraction technique, protease hydrolysis amaranth seed albumen are prepared active antihypertensive peptide technique and are purified into highly active active antihypertensive peptide by technology separation such as macroporous resin adsorption, gel chromatography and half preparative high-performance liquid chromatographics (HPLC), this polypeptide edible safety extracted from plant is high, side effect is little, hypotensive effect can be played, to normal arterial pressure then without hypotensive effect to hyperpietic.
Claims (2)
1. the preparation of amaranth seed active antihypertensive peptide and a separation purification method, is characterized in that:
(1) preparation of amaranth seed albumen
By amaranth powder of seeds and mass concentration be the NaOH solution of 0.018% by weight preparing feed liquid for 1:35, constant temperature oscillation shaking table is warming up to 35 DEG C, mechanical shaking extraction 30min; PH is adjusted to make precipitate soluble proteins to iso-electric point 4.6 reacted feed liquid 1mol/LHCl solution; The centrifugal 20min of 6000r/min, abandons supernatant liquor, gets precipitation afterwards;-55 DEG C of vacuum lyophilization 16h will be deposited in, i.e. obtained amaranth seed albumen after centrifugal;
(2) preparation of amaranth seed active antihypertensive peptide
Get amaranth seed albumen in beaker, being mixed with mass concentration is 4%, in 90 DEG C of thermal treatment 10min, then take out and be cooled to 54.4 DEG C, pH=8.78 is regulated with 1moL/LNaOH, add the Sumizyme MP Novi letter of E/S=3.5%, in 54.4 DEG C, vibrate in 160r/min shaking bath 3h, and with 1moL/LNaOH maintain reaction system pH=8.78 ± 0.05 between, take out at 100 DEG C, 10min goes out after enzyme and cools rapidly, at 10000r/min, 4 DEG C of centrifugal 20min, get supernatant liquor lyophilize and namely obtain thick amaranth seed ace inhibitory peptide;
(3) separation of amaranth seed active antihypertensive peptide
The DA201-C macroporous resin that pre-treatment is good is filled in 3.2 × 100cm chromatography column, wet method upper prop, its natural sedimentation is allowed to obtain high 87cm, the resin fixed bed of bed volume (BV) 700mL, be 10mg/ml by mass concentration, pH=5, volume is that the enzymolysis solution of 1BV flows through chromatography column with the speed of 6mL/min, the albumen do not adsorbed and the impurity sticked on resin are rinsed well by the distilled water having adsorbed rear 1BV, again the ethanolic soln of the volume fraction 75% of elution volume 4BV is flowed through chromatography column with the speed of 5mL/min and carry out wash-out, to stop wash-out during A280nm≤0.05, rotary evaporation concentrates postlyophilization, obtain amaranth seed ace inhibitory peptide,
(4) purifying of amaranth seed active antihypertensive peptide
First gel chromatography is adopted, by sephadex G 25 3 ~ 5 times of volume water expansion 3h, with water cleaning until supernatant liquor does not have impurity, slow threading 10 × 100(internal diameter mm × length cm) in chromatography column, bubble can not be had in pipe, otherwise again fill post, to install after pillar vertical natural sedimentation a whole night, make post bed closely homogeneous; Loading is, the water level in pipe is first allowed to be down to gel top layer 2 ~ 3mm, adding 2mL mass concentration is amaranth seed ace inhibitory peptide solution after the macroporous resin purification of 50mg/mL, when peptide solution is left to gel top layer 2 ~ 3mm, the water adding 3 ~ 4mL rinses post jamb, connect constant flow pump, with pure water with the flow velocity wash-out of 0.4ml/min, every 5min collects a pipe; Rinse with same flow velocity after collecting, balance pillar;
Then half preparative high-performance liquid chromatographic is adopted: will the sample collected after gel chromatography be used again through more than half preparative high-performance liquid chromatographic purifying, its liquid-phase condition: Varian ProStar218 system, VarianC18(21.2*150mm) chromatographic column, flow velocity 10ml/min, determined wavelength 220nm, sample size 10mg/ml-1ml, column temperature 25 DEG C, elution requirement: 0 ~ 40min, acetonitrile mass ratio is 5 ~ 45%, and trifluoroacetic acid mass ratio is 0.1%.The component of collecting 6 ~ 9min gained both obtained amaranth seed active antihypertensive peptide.
2. an amaranth seed active antihypertensive peptide, is characterized in that: preparation method according to claim 1 obtains.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003828A (en) * | 2007-01-23 | 2007-07-25 | 南京师范大学 | Method of producing bioactive peptide for anti high blood pressure by using protease of Switzerland lactobacillus to hydrolyze lactoprotein |
| CN101007841A (en) * | 2007-01-29 | 2007-08-01 | 浙江大学 | Method for separating and purifying ACE inhibition peptide from rice draff and active peptide obtained therefor |
| CN101906135A (en) * | 2010-07-27 | 2010-12-08 | 鲁军 | Novel spirulina source antihypertensive peptide and preparation method thereof |
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- 2013-08-06 CN CN201310339637.9A patent/CN104342473A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003828A (en) * | 2007-01-23 | 2007-07-25 | 南京师范大学 | Method of producing bioactive peptide for anti high blood pressure by using protease of Switzerland lactobacillus to hydrolyze lactoprotein |
| CN101007841A (en) * | 2007-01-29 | 2007-08-01 | 浙江大学 | Method for separating and purifying ACE inhibition peptide from rice draff and active peptide obtained therefor |
| CN101906135A (en) * | 2010-07-27 | 2010-12-08 | 鲁军 | Novel spirulina source antihypertensive peptide and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 陈飞平 等: "苋籽ACE抑制肽的大孔树脂分离纯化", 《食品工业科技》 * |
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