CN100526471C

CN100526471C - Method for preparing avenin ACE inhibiting peptide

Info

Publication number: CN100526471C
Application number: CNB2006100972013A
Authority: CN
Inventors: 姚惠源; 张晖; 管骁; 郭晓娜; 王立
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2006-10-24
Filing date: 2006-10-24
Publication date: 2009-08-12
Anticipated expiration: 2026-10-24
Also published as: CN1944663A

Abstract

The present invention is process of preparing avenin ACE inhibiting peptide (ancovenin), and belongs to the field of oat deeply processing technology. The present invention prepares four kinds of avenin ancovenins, avenin ancovenin A, avenin ancovenin B, avenin ancovenin C and avenin ancovenin D, with oat bran as material, and through crushing, enzyme pre-treatment, alkali extraction, centrifugal separation, isoelectric point supernatant precipitation, water adding homogenizing of the precipitate, alkali proteinase adding hydrolysis, centrifuging to obtain supernatant and chromatographic separation. These avenin ancovenins have their structures identified and the semi-inhibiting rate IC50 measured. The process is reasonable and possesses importance in comprehensive utilization of oat resource.

Description

A kind of preparation method of avenin ACE inhibiting peptide

Technical field

A kind of preparation method of avenin ACE inhibiting peptide belongs to oat deep process technology field.

Background technology

Oat is the important cereal class food crop of a class.Compare with other all kinds of cereal, oat has higher nutritive value and nourishing function, and being described as is one of full price food best in the cereal food.Along with the pay attention to day by day of people to self diet health, the sustainable growth of global oat consumption, the world market is in great demand to avenaceous.Yet China's oat deep process technology is relatively backward at present, oat based food kind is single on the market, cause 4% of the not enough ultimate production of China's oat human consumption part, this situation has not only caused the significant wastage of resource, and has limited further developing of China's oat industry.

Avenin not only nutritive value is abundant, can satisfy human body requirements, has the potential antihypertensive function in addition, therefore the present invention is raw material with the oat bran, the peculiar effect that makes full use of avenin is carried out the development of avenin ACE (angiotensin-converting enzyme) inhibiting peptide, and make an addition in the food as a kind of novel food ingredients, reach the dual purpose of Nutrition and health care with prestige, realize the comprehensive utilization of oat resource simultaneously.

Summary of the invention

The preparation method who the purpose of this invention is to provide a kind of avenin ACE inhibiting peptide, products obtained therefrom has the superiority of nutrition and health care two aspects.

Technical scheme of the present invention: with the oat bran is raw material, through pulverizing, after adding water and complex polysaccharide enzyme ViscozymeL and carrying out the enzyme process pre-treatment, regulate extracting solution to alkaline environment and extract oat bran albumen, centrifugation, the supernatant liquor isoelectric precipitation, after the gained precipitation adds water homogenisation, add the Sumizyme MP reaction that is hydrolyzed again, centrifugation, peptide composition in the supernatant liquor carries out separation and purification through chromatographic technique, finally obtains four kinds of single avenin ACE inhibiting peptide components, is respectively avenin ACE inhibiting peptide A, B, C and D.

(1) pulverizing of raw material

Requirement is pulverized the raw material oat bran, and crosses 40 eye mesh screens.The raw material pulverizing effect is good more, helps proteic extraction in the oat bran more.

(2) the enzyme process pre-treatment of oat bran

Solid-liquid ratio

The mass ratio of oat bran and water is 1:10, reduces water consumption and can cause the avenin extraction yield on the low side, increases water consumption and can cause cost too high again, produces load and strengthens.Therefore relatively more suitable solid-liquid ratio is 1:10.

The temperature and time of enzyme reaction

The material, enzyme method preprocessing process adopts complex polysaccharide enzyme Viscozyme L (120FBG/mL), enzyme concentration 300FBG/100g oat bran, and regulating pH is 4.5～5.0,40～45 ℃ of the optimal reactive temperatures of enzyme.Prolong the enzyme pretreatment time and help reacting completely, but long meeting of reaction times increases the production cycle, so the enzyme reaction time of the present invention is 2 hours.

(3) the proteic extraction of oat bran

Help next step proteic stripping after the pre-treatment of raw material oat bran enzyme process.Oat bran albumen leaching condition preferably is under alkaline condition and the higher temperature.But pH is unsuitable too high, otherwise causes toxic substance to generate easily; Simultaneous temperature can not be too high, otherwise cause starch pasting to influence the albumen stripping easily.Therefore suitable protein extraction condition is 9.5,50 ℃ of following stirring and leaching 30min of pH value.

(4) centrifugation

Adopt link-suspended basket centrifuge protein extract effectively can be separated, centrifugal condition is 3000r/min, 20min, gets supernatant liquor.

(5) supernatant liquor is handled

The iso-electric point of avenin is about pH4.5, and therefore the protein extract pH value with the previous step gained transfers to the precipitation that can realize avenin after 4.5 to the full extent.Transfer the pH process to adopt HCl solution, the very fast change of extracting solution is muddy, and suspension liquid centrifugation (3000r/min, 20min) must precipitate.

(6) precipitation adds homogeneous behind the water mixing

Ratio in 1:20 adds water mixing and homogeneous in precipitation, homogenizing process helps the dispersion of protein molecular, helps next step enzymolysis process;

(7) proteolysis

Proteolytic enzyme the best that oat bran proteolysis production ace inhibitory peptide process is used is Sumizyme MP (Alcalase 3.0T enzyme is lived and is 3.0AU/g), the best use of temperature and the pH of this enzyme are respectively 60 ℃ and 7.5, suppress activity than the ACE of effect 90min products therefrom under the E/S=1.33% condition at the bottom of the enzyme and can reach the best.After enzyme digestion reaction finishes, in order to stop the further effect of proteolytic enzyme, the residual enzyme activity enzyme that goes out is handled, enzymolysis solution is warming up to 90 ℃, and keep the 10min enzyme that goes out.

(8) centrifugation

It is active that the little peptide composition of solubility has very strong ACE inhibition in the enzymolysis solution, for reaching the enrichment of active ingredient, carry out centrifugation to enzymolysis product.Enzymolysis solution adopts link-suspended basket centrifuge centrifugation (3000r/min, 30min) to reclaim supernatant liquor after being cooled to room temperature;

(9) separation and purification of active ingredient in the supernatant liquor

Ion-exchange chromatography separates: the gained supernatant liquor is directly gone up ion-exchange chromatography and is carried out the first step separation, and chromatographic condition is: separating medium DEAE Sepharose Fast Flow; Initial damping fluid 0.02mol/L, pH4.0HAc-NaAc damping fluid contain the NaCl buffered soln linear gradient elution of 0～1mol/L;

Gel filtration chromatography is separated: gel filtration chromatography is carried out the separation of second step on the separating obtained high ACE inhibition active ingredient of collection of ions exchange chromatography, and chromatographic condition is: separating medium Sephadex G-25; The distilled water wash-out;

RPLC separates: reversed-phase liquid chromatography carries out the separation of the 3rd step on the separating obtained high ACE inhibition active ingredient of collection gel filtration chromatography: chromatographic condition is: Lichrospher C18 reversed-phase column, elution requirement, 0～10min, 0～20%B; 10～20min, 20～35%B; 20～30min, 35～100%B; 30～34min, 100～0%B; 34～40min, 100%A; (A:5% acetonitrile+0.05% trifluoroacetic acid and all the other are water; B:80% acetonitrile+0.05% trifluoroacetic acid and all the other are water).

Behind three step purifying process, obtain four kinds of single high ACE altogether and suppress the bioactive peptide component, be respectively avenin ACE inhibiting peptide A, B, C and D.

(10) the peptide fragment structure is identified

Utilize MALDI-TOF/TOF to carry out structure to four kinds of newfound peptide sections and identify that aminoacid sequence is respectively AYKPIRSK, APRGF, GGDIIRPG and EGGYR, its half inhibiting rate IC ₅₀Value is respectively 10.6 μ M, 31.6 μ M, 113.8 μ M and 77.3 μ M.

Beneficial effect of the present invention: adopt the avenin ACE inhibiting peptide of the inventive method preparation active strong, can play hypotensive effect effectively, can give play to the effect of nutrition and health care two aspects simultaneously.

Description of drawings

Fig. 1 avenin ACE inhibiting peptide preparation technology schematic flow sheet.

Embodiment

Embodiment 1

The pulverizing of learning from else's experience, cross the 15kg oat bran of 40 mesh sieves, add 150L water stirring and evenly mixing, be heated to 45 ℃, transfer pH to 4.5～5.0, add complex polysaccharide enzyme Viscozyme L 0.375L, enzyme digestion reaction 2h is warming up to 50 ℃, and accent pH to 9.5, after continuing to stir 30min, centrifugation (3000r/min, 20min), collect supernatant liquor and transfer pH to 4.5, centrifugation (3000r/min, 20min) must precipitate 1.35kg, add homogeneous behind the 27L water mixing, temperature adjustment degree and pH add Sumizyme MP Alcalase3.0T18g reaction 90min respectively to 60 ℃ and 7.5, be warming up to 90 ℃, and keep the 10min enzyme that goes out, be cooled to room temperature after, centrifugation (3000r/min, 30min) get supernatant liquor, go up ion-exchange chromatography successively, gel filtration chromatography is separated with RPLC, obtains four kinds of strong ACE altogether and suppresses active peptide composition.

Embodiment 2

The pulverizing of learning from else's experience, cross the 10kg oat bran of 40 mesh sieves, add 100L water stirring and evenly mixing, be heated to 40 ℃, transfer pH to 4.5～5.0, add complex polysaccharide enzyme Viscozyme L 0.25L, enzyme digestion reaction 2h, be warming up to 50 ℃, and accent pH to 9.5, centrifugation (3000r/min behind the continuation stirring 30min, 20min), collect supernatant liquor and transfer pH to 4.5, centrifugation (3000r/min, 20min), must precipitate 0.9kg, add homogeneous behind the 18L water mixing, temperature adjustment degree and pH are respectively to 60 ℃ and 7.5, add Sumizyme MP Alcalase3.0T12g reaction 90min, be warming up to 90 ℃, and keep the 10min enzyme that goes out, be cooled to room temperature after, centrifugation (3000r/min, 30min), get supernatant liquor, go up ion-exchange chromatography successively, gel filtration chromatography is separated with RPLC, obtains four kinds of strong ACE altogether and suppresses active peptide composition.

Claims

1, a kind of preparation method of avenin ACE inhibiting peptide is characterized in that with the oat bran being raw material, through pulverizing, after adding water and complex polysaccharide enzyme Viscozyme L and carrying out the enzyme process pre-treatment, regulate extracting solution to alkaline environment and extract avenin, centrifugation, supernatant liquor isoelectric precipitation, after the gained precipitation adds water homogenisation, add the Sumizyme MP reaction that is hydrolyzed again, centrifugation, the gained supernatant liquor is through chromatographic separation technology, obtain highly active avenin ACE inhibiting peptide, technology is:

(1) raw material pulverizing: the raw material oat bran is pulverized, and crosses 40 eye mesh screens;

(2) enzyme process pre-treatment: the oat bran after the pulverizing adds water by the mass ratio of 1:10, regulate 40～45 ℃ of pH value to 4.5～5.0, temperature after, adding complex polysaccharide enzyme Viscozyme L, enzyme concentration 300FBG/100g oat bran, enzymolysis time are 2h;

(3) alkaline environment extracts avenin: regulate the pH to 9.5 of extracting solution after the enzyme process pre-treatment, 50 ℃ of following stirring and leaching 30 minutes;

(4) centrifugation: will react feed liquid through 3000 rev/mins, centrifugation 20 minutes, and get supernatant liquor;

(5) supernatant liquor isoelectric precipitation: supernatant liquor is with behind the HCl adjust pH to 4.5, and the gained suspension liquid through 3000 rev/mins, centrifugation 20 minutes, must be precipitated;

(6) precipitation adds water homogenisation: the ratio in 1:20 adds water mixing and homogeneous in precipitation;

(7) add protease hydrolysis: the solution adjust pH 7.5 behind the homogeneous, 60 ℃ of temperature are in adding Sumizyme MP Alcalase 3.0T effect 90 minutes than the ratio of E/S=1.33% at the bottom of the enzyme; Enzymolysis is warming up to 90 ℃, the 10 minutes enzymes that go out after finishing;

(8) centrifugation: enzymolysis solution is cooled to after the room temperature 3000 rev/mins, and supernatant liquor is got in centrifugation 30 minutes;

(9) purifying: centrifugal back supernatant liquor adopts ion-exchange chromatography, gel filtration chromatography and RPLC separation and purification to go out four kinds of single avenin ACE inhibiting peptide components;

Ion-exchange chromatography separates: the gained supernatant liquor is directly gone up ion-exchange chromatography and is carried out the first step separation, and chromatographic condition is: separating medium DEAE Sepharose Fast Flow; Initial damping fluid 0.02mol/L, pH4.0HAc-NaAc damping fluid; The NaCl buffered soln linear gradient elution that contains 0～1mol/L;

RPLC separates: reversed-phase liquid chromatography carries out the separation of the 3rd step on the separating obtained high ACE inhibition active ingredient of collection gel filtration chromatography: chromatographic condition is: Lichrospher C18 reversed-phase column; Elution requirement, 0～10min, 0～20%b; 10～20min, 20%～35%b; 20～30min, 35%～100%b; 30～34min, 100%～0%b; 34～40min, 100%a; A liquid is: 5% acetonitrile+0.05% trifluoroacetic acid and all the other are water; B liquid is: 80% acetonitrile+0.05% trifluoroacetic acid and all the other are water;

Behind three step purifying process, obtain four kinds of single high ACE altogether and suppress the bioactive peptide component, be respectively avenin ACE inhibiting peptide A, B, C and D; Its half inhibiting rate IC ₅₀Value is respectively 10.6 μ M, 31.6 μ M, 113.8 μ M and 77.3 μ M;

(10) the peptide fragment structure is identified

Utilize MALDI-TOF/TOF to carry out structure to avenin ACE inhibiting peptide A, B, C and four kinds of peptide sections of D of being purified into and identify that aminoacid sequence is respectively AYKPIRSK, APRGF, GGDIIRPG and EGGYR.

2, according to the preparation method of the described avenin ACE inhibiting peptide of claim 1, it is characterized in that the used complex polysaccharide enzyme Viscozyme L enzyme 120FBG/mL of being alive in the process, Sumizyme MP Alcalase 3.0T enzyme is lived and is 3.0AU/g.