CN1782091A - Method for producing angiotonin converzyme inhibiting peptide by whey proteinase method - Google Patents
Method for producing angiotonin converzyme inhibiting peptide by whey proteinase method Download PDFInfo
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- CN1782091A CN1782091A CN 200410009910 CN200410009910A CN1782091A CN 1782091 A CN1782091 A CN 1782091A CN 200410009910 CN200410009910 CN 200410009910 CN 200410009910 A CN200410009910 A CN 200410009910A CN 1782091 A CN1782091 A CN 1782091A
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Abstract
The present invention relates to whey protein enzyme method of producing angiotonin converting enzyme (ACE) inhibiting piptide. The whey protein enzyme method includes the main steps of: hydrating whey protein, heating, lowering to required temperature, regulating pH value, adding enzyme, hydrolysis, heating to deactivate enzyme, desalting, and drying to obtain the product.
Description
[technical field]
The present invention relates to a kind of method of utilizing whey-protein Production by Enzymes angiotensin-converting enzyme (ACE) inhibiting peptide, belong to biological technical field.
[background technology]
Hypertension is that latent period is long to the great a kind of disease of human health risk.Hypertensive commitment does not generally have significant discomfort disease, causes death until clinical indication heart attack, rupture of blood vessel in brain take place.According to national hypertension research centre recent statistics, China hyperpietic has reached 1.2 hundred million people, and its harm is only second to tumour.Because there is side effect in pharmacotherapy, the development and use of the antihypertensive function factor in the natural food will become the integral part of hypertension non-drug therapy from now on.
Whey-protein is a by product of producing cheese, accounts for about 20% of milk protein, and its biological value is high and contain abundant indispensable amino acid.Whey-protein includes multiple biological activity sequence, with suitable protease hydrolysis and after discharging from whey-protein, becomes peptide section (Aubois etc., 1991 with physiologically active; European patent 4745506).This class biologically active peptides described in a large amount of articles, for example, has the ace inhibitory peptide of possesses antihypertensive properties in the whey-protein, under certain conditions, and can be through the processing of enzyme by effective (Mullally etc., 1997 of discharging; Amhar etc., 1998; Pihlanto etc., 2000; Cornelly etc., 2002).The ace inhibitory peptide research to milk protein sources abroad has certain report, and especially Japan has carried out more research to the ace inhibitory peptide that derives from whey-protein, casein and the sour milk, and part has obtained bigger progress.Domestic research of producing ace inhibitory peptide about the enzymolysis whey-protein does not appear in the newspapers.From the ace inhibitory peptide of milk-protein, safe, have no side effect, experimentation on animals confirms spontaneous hypertensive rat is brought high blood pressure down.Studies show that: can reach the hypertensive effect of prevention, inhibition, alleviation and assisting therapy by taking ace inhibitory peptide for a long time.Therefore, develop protective foods and have most important theories meaning and using value with blood pressure regulation effect.
[summary of the invention]
Main purpose of the present invention is to produce ace inhibitory peptide by enzymolysis process, and the selection of this enzyme wherein is crucial, because ace inhibitory peptide none fixed structure should be screened from numerous proteolytic enzyme according to the specificity of enzyme.
Whey isolate protein (WPI9000) can be hydrolyzed in the scope of the about 5%-8% solid matter of concentration, and preferred concentration of substrate is 6%.
WPI9000 with trypsin treatment can be hydrolyzed to pH9.0 at pH7.5, preferably pH8.0.
The WPI9000 that handles with neutral protease (neutrase) can be hydrolyzed to pH8.0 at pH5.0, preferably pH7.0.
The WPI9000 that handles with Sumizyme MP (alcalase) can be hydrolyzed to pH9.0 at pH7.0, preferably pH8.5.
WPI9000 with pepsin can be hydrolyzed to pH4 at pH2, preferably pH3.
The WPI9000 that handles with papoid can be hydrolyzed to pH7 at pH6, preferably pH6.2.
Used enzyme all is that Novozymes company produces
Proteolysis can be at temperature range 25-65 ℃.
The enzyme amount that adds can be 1.5% to 4.5%.
Using various protease hydrolysis whey-proteins respectively, is 6% at concentration of substrate, and E/S is hydrolysis 12 hours under 3% condition, and reaction is carried out lyophilize after finishing, and carries out ACE after the drying and suppresses active vitro detection, measurement result such as table 1
It is active that the ACE of the various hydrolyzate of protease of table 1 suppresses
| Used proteolytic enzyme | ACE suppresses active (%) |
| Trypsinase | 71.51±2.02 |
| Neutral protease | 82.74±0.93 |
| Sumizyme MP | 90.68±2.83 |
| Stomach en- | 36.27±1.89 |
| Papoid | 66.68±0.36 |
Suppressing activity with hydrolyzate ACE is index, filters out Sumizyme MP as the hydrolysis enzyme by table 1.
The present invention further utilizes orthogonal experiment and experiment of single factor that the hydrolysising condition of Sumizyme MP is optimized, the ratio of pH, hydrolysis temperature and enzyme and substrate and the relation between the ACE inhibition activity have been set up, the optimum hydrolysising condition that draws Sumizyme MP is pH8.5, concentration of substrate 6%, 55 ℃ of temperature, enzyme amount 3%, and hydrolysis time is 6 hours.Then hydrolyzate is made with extra care, obtained having ACE by drying at last and suppress active lactalbumin hydrolysate by desalination.
Advantage of the present invention: the hydrolyzate that 1, the present invention produced is only can play hypotensive effect to the hyperpietic, the normotensive is not had hypotensive effect, thereby can not produce step-down over-drastic problem.2, these hydrolyzates obtain under mild conditions through the food grade enzyme, and its security is high, has no side effect.3, the active diversity of hydrolyzate, except that buck functionality, often simultaneously tool immunological enhancement, decreasing cholesterol, easily digest and assimilate, function such as antitumor.
[description of drawings]
Fig. 1 is a production scheme of the present invention
[specific embodiment]
Embodiment one,
Get 6g whey isolate protein (WPI9000), be dissolved in the 100ml water, then at 90 ℃ of heating 10min, be cooled to 37 ℃ and regulate pH to 8.0, then add 0.16g trypsinase, hydrolysis 12h in 37 ℃ of water bath with thermostatic control vibrators, hydrolytic process is constantly added 0.1NNaOH to keep pH 8.0 (differ up and down and be no more than 0.1), heat the 20min deactivating enzymes at 85 ℃ after hydrolysis is intact, then cool off postlyophilization, get the dry thing of part and carry out ACE and suppress active vitro detection, it is 71.51% ± 2.02% that the ACE that draws hydrolyzate suppresses activity
Embodiment two,
Get 6g whey isolate protein (WPI9000), be dissolved in the 100ml water, then at 90 ℃ of heating 10min, be cooled to 50 ℃ and regulate pH to 7.0, then add the 0.16g neutral protease, hydrolysis 12h in 50 ℃ of water bath with thermostatic control vibrators, hydrolytic process is constantly added 0.1NNaOH to keep pH 7.0 (differ up and down and be no more than 0.1), heat the 20min deactivating enzymes at 85 ℃ after hydrolysis is intact, then cool off postlyophilization, get the dry thing of part and carry out ACE and suppress active vitro detection, it is 82.74% ± 0.93% that the ACE that draws hydrolyzate suppresses activity.
Embodiment three,
Get 6g whey isolate protein (WPI9000), be dissolved in the 100ml water, then at 90 ℃ of heating 10min, be cooled to 60 ℃ and regulate pH to 8.5, then add the 0.16g Sumizyme MP, hydrolysis 12h in 60 ℃ of water bath with thermostatic control vibrators, hydrolytic process is constantly added 0.1NNaOH to keep pH 8.5 (differ up and down and be no more than 0.1), heat the 20min deactivating enzymes at 85 ℃ after hydrolysis is intact, then cool off postlyophilization, get the dry thing of part and carry out ACE and suppress active vitro detection, it is 90.68% ± 2.83% that the ACE that draws hydrolyzate suppresses activity.
Embodiment four
Get 6g whey isolate protein (WPI9000), be dissolved in the 100ml water, then at 90 ℃ of heating 10min, be cooled to 60 ℃ and regulate pH to 3, then add the 0.16g stomach en-, hydrolysis 12h in 60 ℃ of water bath with thermostatic control vibrators, hydrolytic process is constantly added 0.1N hydrochloric acid to keep pH 3 (differ up and down and be no more than 0.1), heat the 20min deactivating enzymes at 85 ℃ after hydrolysis is intact, then cool off postlyophilization, get the dry thing of part and carry out ACE and suppress active vitro detection, it is 36.27% ± 1.89% that the ACE that draws hydrolyzate suppresses activity.
Embodiment five
Get 6g whey isolate protein (WPI9000), be dissolved in the 100ml water, then at 90 ℃ of heating 10min, be cooled to 60 ℃ and regulate pH to 6.2, then add the 0.16g papoid, hydrolysis 12h in 60 ℃ of water bath with thermostatic control vibrators, hydrolytic process is constantly added 0.1N hydrochloric acid to keep pH 6.2 (differ up and down and be no more than 0.1), heat the 20min deactivating enzymes at 85 ℃ after hydrolysis is intact, then cool off postlyophilization, get the dry thing of part and carry out ACE and suppress active vitro detection, it is 66.68% ± 0.36% that the ACE that draws hydrolyzate suppresses activity.
Experimental example one,
With the Sumizyme MP is the hydrolysis enzyme, is experimental factor with consumption, pH, the temperature of concentration of substrate, enzyme, adopts L
9(3)
4Orthogonal experimental design, setting hydrolysis time is 12 hours, the hydrolysising condition that screening is best.Have experimental result as can be known, under the level that this test is selected, each factor is the ratio of pH＞concentration of substrate＞temperature＞enzyme and substrate to the primary and secondary that ACE suppresses active influence in proper order.And having the result can draw the hydrolysis by novo whey-protein, to produce the best of breed of ace inhibitory peptide be pH8.5+6% concentration of substrate+55 ℃ temperature+3% enzyme amount.
Experimental example two,
(pH8.5+6% concentration of substrate+55 ℃ temperature+3% enzyme amount) hydrolyzing lactoalbumin under the Sumizyme MP optimum hydrolysising condition is respectively 1,2,3,6,9, sampling in 12,24 hours, fermentoid carries out lyophilize later on, then carry out ACE and suppress active mensuration, in hydrolytic process, ACE suppresses active prolongation along with the time and constantly raises, when arriving 6 hours, ACE suppresses activity and reaches more than 90%, along with the prolongation ACE inhibition activity change of hydrolysis time is little.Therefore, suppress activity from ACE, hydrolysis 6 hours is just passable.In addition the variation of degree of hydrolysis in the hydrolytic process is measured, the result shows: in initial one hour of reaction, react very fast, degree of hydrolysis sharply rises.Show that protein is degraded rapidly by proteolytic enzyme when the reaction beginning, degree of hydrolysis increases obviously, when reaction reaches 6h, degree of hydrolysis reaches 16.92%, and along with the continuation increase of hydrolysis time, degree of hydrolysis slowly increases, therefore, from the angle of degree of hydrolysis, hydrolysis 6h is also proper.
Experimental example three,
Get 6g whey isolate protein (WPI9000), be dissolved in the 100ml water, then at 90 ℃ of heating 10min, be cooled to 60 ℃ and regulate pH to 8.5, then add the 0.16g Sumizyme MP, hydrolysis 6h in 60 ℃ of water bath with thermostatic control vibrators, hydrolytic process is constantly added 0.1NNaOH to keep pH 8.5 (differ up and down and be no more than 0.1), heat the 20min deactivating enzymes at 85 ℃ after hydrolysis is intact, then carry out desalination, obtain having ACE after the drying and suppress active lactalbumin hydrolysate by anion-cation exchange resin.Suppress active lactalbumin hydrolysate basal component and analyze having ACE, the result shows: protein content is greater than 96%, and ash content is less than 1.5%.
Claims (4)
1, a kind of method of utilizing the whey-protein enzyme to produce inhibiting peptide of tonin, the hydrolysising condition that it is characterized in that adding behind the proteolytic enzyme is: pH2.0-9.0, concentration of substrate 5%-8%, 20 ℃-65 ℃ of temperature, enzyme amount 1.5%-4.5%, hydrolysis time is 3h-8h.
2, the method for production inhibiting peptide of tonin as claimed in claim 1, the hydrolysising condition that it is characterized in that adding behind the basonuclin enzyme is: pH8.5, concentration of substrate 6%, 55 ℃ of temperature, enzyme amount 3%, hydrolysis time is 6 hours.
3, the method for production inhibiting peptide of tonin as claimed in claim 1 or 2 is characterized in that described proteolytic enzyme is: trypsinase, neutral protease (Neutrase), Sumizyme MP (Alcalase), stomach en-, papoid.
4, the method for production inhibiting peptide of tonin as claimed in claim 3 is characterized in that described proteolytic enzyme is the enzyme of thermolability, can make its inactivation, the carrying out that stops hydrolysis reaction by heating.
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| CNB2004100099102A CN100415768C (en) | 2004-11-29 | 2004-11-29 | Method for producing angiotonin converzyme inhibiting peptide by whey proteinase method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003828B (en) * | 2007-01-23 | 2010-05-19 | 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 |
| CN101906455A (en) * | 2010-07-22 | 2010-12-08 | 东北农业大学 | Preparation method of egg white protein assistant antihypertensive peptides |
| CN102618608A (en) * | 2012-03-23 | 2012-08-01 | 华东理工大学 | Application of amygdalus comnnis in preparation of angiotensin converting enzyme (ACE) inhibitor |
| CN101724555B (en) * | 2009-12-18 | 2013-07-17 | 中国农业大学 | Whey protein antihypertensive peptide prepared by utilizing continuous enzyme membrane reactor and special device thereof |
| CN104877007A (en) * | 2014-10-16 | 2015-09-02 | 任发政 | Yak milk lactalbumin-source ACE inhibitory peptides and preparation method thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101736067B (en) * | 2010-01-08 | 2012-04-18 | 中国农业大学 | Whey protein antihypertensive peptide, and preparation method and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3091772B2 (en) * | 1991-03-12 | 2000-09-25 | カルピス株式会社 | Angiotensin converting enzyme inhibiting peptide composition |
| DE69920219T2 (en) * | 1998-06-17 | 2005-09-22 | New Zealand Dairy Board | BIOACTIVE WHEY BULB HYDROLYSATES |
| US6630320B1 (en) * | 2000-05-08 | 2003-10-07 | Devisco Foods International, Inc. | Treatment of hypertension in mammals with hydrolyzed whey proteins |
| NZ506866A (en) * | 2000-09-11 | 2003-05-30 | New Zealand Dairy Board | Bioactive whey protein hydrolysate free of bitter flavours wherein the enzyme used is a heat labile protease |
-
2004
- 2004-11-29 CN CNB2004100099102A patent/CN100415768C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003828B (en) * | 2007-01-23 | 2010-05-19 | 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 |
| CN101724555B (en) * | 2009-12-18 | 2013-07-17 | 中国农业大学 | Whey protein antihypertensive peptide prepared by utilizing continuous enzyme membrane reactor and special device thereof |
| CN101906455A (en) * | 2010-07-22 | 2010-12-08 | 东北农业大学 | Preparation method of egg white protein assistant antihypertensive peptides |
| CN101906455B (en) * | 2010-07-22 | 2013-07-31 | 东北农业大学 | Preparation method of egg white protein assistant antihypertensive peptides |
| CN102618608A (en) * | 2012-03-23 | 2012-08-01 | 华东理工大学 | Application of amygdalus comnnis in preparation of angiotensin converting enzyme (ACE) inhibitor |
| CN104877007A (en) * | 2014-10-16 | 2015-09-02 | 任发政 | Yak milk lactalbumin-source ACE inhibitory peptides and preparation method thereof |
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| CN100415768C (en) | 2008-09-03 |
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