CN1884574A - Angiotensin converting enzyme inhibitory peptide production method - Google Patents
Angiotensin converting enzyme inhibitory peptide production method Download PDFInfo
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- CN1884574A CN1884574A CN 200610050845 CN200610050845A CN1884574A CN 1884574 A CN1884574 A CN 1884574A CN 200610050845 CN200610050845 CN 200610050845 CN 200610050845 A CN200610050845 A CN 200610050845A CN 1884574 A CN1884574 A CN 1884574A
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
This invention exposed the production method of angiotensin-converting enzyme inhibitory petides. The steps are as follow: 1) mix the rice dregs and distiller in container by the proportion of 1:4 - 4.5, then adjust the pH value to 7.9 - 8.1 with alkali solutions of 2.0 - 3.0M density; 2) add trypsin in container then put it in constant temperature water bath to react; the temperature of water bath is 35 - 40DEG C, the time of reaction is 3.5 - 4.5 hours. The amount of trypsin is as much as 1.4 - 1.6% of the weight of rice dregs; 3) then put the container in boiling water to stop the reaction; separating the obtained product by centrifugal, then condensing, freezing and drying the supernatant, what obtained is angiotensin-converting enzyme inhibitory petides. This finally obtained angiotensin-converting enzyme inhibitory petides is safe, economy and effective.
Description
Technical field
The present invention relates to a kind of production method of angiotensin converting enzyme inhibitory peptide.
Background technology
Rice is one of main food in the world, and the whole world is over half, and the population of China more than 2/3rds is staple food with the rice.China's cultivated area occupies the 2nd in the world, and ultimate production ranks first in the world, and annual production is 1.85 hundred million tons, accounts for about 36% of world's paddy ultimate production.These rice are except that part supply people's diet, and major part all is used for industrial use, as gourmet powder fermenting, glucose industry etc.Produce a large amount of byproducts in the industrial production that with the rice is raw material, it is poor for example all can to produce a large amount of rice in Gourmet Powder Factory, grape sugar refinery and other fermentation plants.Rice is poor to be that rice produces through after the saccharification, and protein degradation wherein is few, and major part has all kept.Protein content during rice is poor can reach in 40%～65% (butt) scope.Rice is poor as the byproduct after the rice processing, if can not fully utilize, certainly will cause the wasting of resources, environmental pollution, financial loss.If can further fully utilize, will receive considerable economical, societal benefits.
Biologically active peptides (bioactivity peptides) is meant that those have the peptide class of special physiological function, not only easily digest and assimilate, but also have multiple body metabolism and physiological regulation function, as promote immunity, hormone regulation, antibiotic, antiviral, hypotensive, promote effect such as mineral substance absorption.The bioactive peptide that can be divided into naturally occurring bioactive peptide, protein digestion bioactive peptide and chemosynthesis by its source.Wherein, naturally occurring bioactive peptide comprises peptide antibiotics, hormone and the bioactive peptide that exists in various tissues, bone, muscle, immunity system, Digestive tract, cental system; The shortcoming of these bioactive peptides is that content is few or extraction is relatively more difficult.Chemistry synthetic bioactive peptide is wasted time and energy the cost costliness.Therefore adopting the method for enzymolysis protein to produce the thing bioactive peptide next life is a kind of more rational approach.In the method for enzymolysis protein, biologically active peptides generally obtains from the hydrolysis of bacterioprotein, plant protein and animal protein, is generally molecular weight ratio smaller polypeptides fragment.
Angiotensin i-converting enzyme can change into Angiotensin II to angiotensin I, and Angiotensin II is well-known strong vasoconstrictor substance, in addition, and all right deactivation bradykinin, rising blood pressure.This enzyme also has the physiological action of other endogenous peptide levels of adjusting, for example enkephalin, P material.Therefore, Zinc metallopeptidase Zace1 (ACE) suppresses the activity of bioactive peptide by the inhibition Angiotensin II, thereby improves the level of enkephalin, P material, finally brings high blood pressure down.The active inhibiting peptide of ACE generally prepares by following three kinds of modes.A kind of is directly to extract from animal vegetable tissue; Second kind is by chemosynthesis; The third is that mode by enzymolysis protein matter prepares.From food, obtain biologically active peptides and mainly utilize enzyme process and microbial method; Enzyme process owing to efficient, protein nutritive value destroyed advantage such as little, free from extraneous odour and is widely adopted.The biologically active peptides that enzymolysis is produced from food protein is all safe and reliable, has no side effect.The researchist separates the bioactive peptide that has obtained having hypotensive activity with the method for protease hydrolysis from raw materials such as gel, dairy protein, fish protein, garlic, egg, soybean, corn, wheat germ, vinasse, wakame.
Enzymolysis process prepares Zinc metallopeptidase Zace1 (ACE) inhibition bioactive peptide process will pass through many steps, three processes such as extraction separation of the selection of rough selection that can be divided into protein raw materials and processing, proteolytic enzyme and hydrolysis, biologically active peptides.In the chosen process of protein raw materials, because the proteinic amino acid The Nomenclature Composition and Structure of Complexes of different material also is not quite similar, therefore, the 26S Proteasome Structure and Function of the small peptide that obtains after hydrolysis under the effect of proteolytic enzyme is also different.Discover, in structure with highly active ACE inhibition polypeptide, C end generally has proline(Pro) or hydrophobic amino acid residue, if therefore proline rich or hydrophobic amino acid residue in the protein raw materials might obtain highly active ACE by enzymolysis and suppress polypeptide.Contain rich in protein during rice is poor, its content can reach 65%, is higher than soybean protein, and therefore rice is poor is to obtain the good raw material that ACE suppresses bioactive peptide.
The kind of the enzyme that relates in food proteins hydrolysis research is a lot, comprise plant protease, animal protease, microbial protease etc., because the enzymolysis product of different proteolytic enzyme is not quite similar, the process of therefore correct selection proteolytic enzyme also is an important link.Have report to propose microbial protease and have higher selectivity, but simple interest is produced bioactive peptide with the microorganism enzymolysis and also do not appeared in the newspapers.Aspect the selection of proteolytic enzyme, some researchist selects single proteolytic enzyme, also has the researchist to select mixing protease, and its effect has nothing in common with each other.Someone utilizes 12 kinds of protease hydrolysis soybean proteins to produce blood pressure lowering peptide, find all to have certain inhibition activity in 12 kinds of zymolytes, but 12 kinds of mutual composite degradations of enzyme does not obtain better result.These show the degradation mechanism that exists complexity in the enzymolysis process.
The proteolysis process can be divided into two processes, and a process is the polypeptide forming process, and the another one process is the polypeptide degradation process.Protein zymolyte polypeptide composition depends on some process parameters, for example proteolytic enzyme kind, proteolytic enzyme amount, hydrolysis time, hydrolysis pH, hydrolysis temperature and solid-liquid ratio.Many results of study show that most important factor is pH in the proteolysis reaction, temperature, proteolytic enzyme amount (E/S), solid-liquid ratio (W/R) and reaction times.
Summary of the invention
At the deficiencies in the prior art part, the invention provides the poor method of producing angiotensin converting enzyme inhibitory peptide of a kind of utilization rice with low cost.
The present invention is to realize by such technical scheme: a kind of production method of angiotensin converting enzyme inhibitory peptide is provided, may further comprise the steps for reaching above purpose:
1), with rice poor and distilled water according to 1: 4～4.5 weight ratio in container mixed after, be alkaline solution adjusting pH value to 7.9～8.1 of 2.0～3.0M with concentration;
2), putting into water bath with thermostatic control behind the adding trypsinase in container reacts; Described bath temperature is 35～40 ℃, and the reaction times is 3.5～4.5 hours, and tryptic consumption is 1.4～1.6% of the poor weight of rice;
3), container being put into boiling water boils and came termination reaction in 8～12 minutes again; Products therefrom carries out centrifugal, and the supernatant liquor of centrifugal gained promptly gets angiotensin converting enzyme inhibitory peptide through concentrated, lyophilize.
A kind of improvement as the production method of angiotensin converting enzyme inhibitory peptide of the present invention: in the step 1), a rice weight ratio poor and distilled water is 1: 4.13, and selecting concentration for use is that the sodium hydroxide of 2.5M is regulated pH value to 8.02; Step 2) in, bath temperature is 37 ℃, and the reaction times is 4 hours, and tryptic consumption is 1.5% of the poor weight of rice; In the step 3), container is put into boiling water and is boiled and came termination reaction in 10 minutes.
In order to prove the inhibiting rate of the angiotensin converting enzyme inhibitory peptide that the inventive method is produced, adopt following method to detect:
Contain 5mM Hip-His-Leu as reaction substrate in the step 1:50 μ l reaction mixture, 0.3MNaCl and 5mU ACE and 50mM borate buffer solution (pH8.3).The angiotensin converting enzyme inhibitory peptide that the inventive method of 50 μ l is produced joins in the above-mentioned reaction mixture as sample then, after 37 ℃ are reacted 3 hours down, adds the 1.0N HCl enzymolysis reaction of 250 μ l.In reaction system, add the 1.5mL ethyl acetate again.After shaking 2min in the vortex oscillator, centrifugal 15min under 2500 * g, after leaving standstill 10min, draw 1.0mL ethyl acetate layer liquid in other test tube with pipettor, dry 60min in loft drier, add 3.0mL distilled water again it is dissolved, measure the light absorption value of this solution at the 228nm place, institute's value is decided to be B.
Step 2: do not add sample, directly react with reaction mixture, all the other are with step 1, and the light absorption value of gained is set at A.
Step 3: do not add sample, and the content of the ACE in the cancellation reaction mixture, directly to react with this reaction mixture, all the other are with step 1, and the light absorption value of gained is set at C.
Calculation formula is as follows:
Adopt above-mentioned detection method to learn, the inhibiting rate of the angiotensin converting enzyme inhibitory peptide that the inventive method is produced reaches as high as 84.95%.
The present inventor has done many experiments in order to obtain the present invention:
Experiment one, to the screening of different single protease hydrolysis agent:
Selecting 6 kinds of proteolytic enzyme for use is hydrolysising protease, compound protease, stomach en-, trypsinase, aspartic protease and neutral protease, and the characteristic of various proteolytic enzyme is as shown in table 1.Under the optimum reaction condition of each proteolytic enzyme, carried out enzymolysis to rice is poor, enzymolysis time is 4 hours; And the zymolyte of each proteolytic enzyme has been measured its ACE suppressed active, detection method is the same, and enzymatic hydrolysis condition and experimental result are as shown in table 2.
The various protease preparation characteristics of table 1 (being food grade)
| The proteolytic enzyme kind | pH | Proteinase activity | Temperature (℃) | Production unit |
| Hydrolysising protease | 8.0 | 3.0AU/g | 65.0 | Denmark Novozymes Company |
| Compound protease | 6.0 | 500LAPU/g | 50.0 | Denmark Novozymes Company |
| Stomach en- | 1.8 | 10,000 u/mg | 37.0 | Chinese Medicine group |
| Trypsinase | 8.0 | 2500u/mg | 37.0 | Chinese Medicine group |
| Aspartic protease | 3.0 | 200,000 units/g | 40.0 | Denmark Novozymes Company |
| Neutral protease | 6.5 | 1.5AU/g | 45.0 | Denmark Novozymes Company |
The different single protease hydrolyzed product of table 2 suppresses activity influence to ACE
| Numbering | The proteolytic enzyme kind | Reaction conditions | ACE suppresses active (%) |
| 1 | Hydrolysising protease | PH8.0. temperature is 65 ℃ | 19.4±1.715 |
| 2 | Compound protease | PH6.0. temperature is 50 ℃ | 42.3±2.000 |
| 3 | Stomach en- | PH1.8. temperature is 37 ℃ | 7.40±0.665 |
| 4 | Trypsinase | PH8.0. temperature is 37 ℃ | 81.2±0.907 |
| 5 | Aspartic protease | PH3.0. temperature is 40 ℃ | 59.9±0.637 |
| 6 | Neutral protease | PH6.5. temperature is 45 ℃ | 77.6±2.193 |
Learn from table 2, the ACE of various proteolytic enzyme suppress active by strong be trypsinase＞neutral protease＞aspartic protease＞compound protease＞stomach en-＞hydrolysising protease to weak order, wherein tryptic enzymolysis product ACE suppresses activity and has been up to 81.2%, and the enzymolysis product ACE of hydrolysising protease suppress active minimum be 7.4%.These explanations, poor its ACE of the enzymolysis product that produces suppresses the active bigger difference that exists with different protease hydrolyzed rice, correctly selects proteolytic enzyme that enzymolysis product ACE is suppressed activity and has extremely important meaning.
Test two, different combined protein enzymic hydrolysis agent screened:
With trypsinase is the center, hydrolysising protease+trypsinase, neutral protease+trypsinase, stomach en-+trypsinase, four kinds of proteolytic enzyme combinations of aspartic protease+trypsinase have been designed, promptly successively respectively carried out enzymolysis 4 hours to rice is poor with above-mentioned two kinds of proteolytic enzyme, then final zymolyte is measured its ACE and suppress active, detection method is the same, and enzymatic hydrolysis condition and experimental result are as shown in table 3.With neutral protease+trypsinase is example, promptly earlier with neutral protease reaction 4 hours under the condition of 45 ℃ of pH6.5 and temperature; Be adjusted to the condition of 37 ℃ of pH8.0 and temperature then, under tryptic effect; Reacted again 4 hours.
Table 3 proteolytic enzyme combination enzymolysis suppresses active influence to product A CE
| Numbering | The proteolytic enzyme kind | Reaction conditions | ACE suppresses active (%) |
| 1 | Hydrolysising protease+trypsinase | 65 ℃ of pH8.0 temperature, 37 ℃ of pH8.0 temperature | 10.4±1.778 |
| 2 | Neutral protease+trypsinase | 37 ℃ of 45 ℃ of pH8.0 temperature of pH6.5 temperature | 60.1±1.601 |
| 3 | Stomach en-+trypsinase | 37 ℃ of 37 ℃ of pH8.0 temperature of pH1.8 temperature | 46.9±1.442 |
| 4 | Aspartic protease+trypsinase | 37 ℃ of 40 ℃ of pH8.0 temperature of pH3.0 temperature | 38.8±1.701 |
It is active that the ACE of the single proteolytic enzyme of synopsis 2 gained suppresses, and as can be known: the ACE of single protease hydrolyzed product suppresses the active ACE that is higher than proteolytic enzyme combination enzymolysis product on the whole and suppresses active.
Experiment three, employing response surface analysis method (RSM) are optimized the enzyme digestion reaction parameter:
Optimizing process is divided into 3 steps, i.e. part factor design (FFD), climbing experimental design, center combination experimental design (CCD).Producing the optimum enzymatic hydrolysis condition that ACE suppresses bioactive peptide at the white enzyme digestion reaction of rice Egg preserved in wine that utilizes test design method to obtain is: pH=8.02,37.0 ℃ of temperature, proteolytic enzyme amount [E]/[S]=1.5%, solid-liquid ratio (W/R) are 4.13, hydrolysis time 4.0h, and it is 84.95% that the ACE of the enzyme digestion reaction thing that obtains under this condition suppresses activity.
The production method of angiotensin converting enzyme inhibitory peptide of the present invention, its advantage is as follows:
1, owing to select for use rice poor, therefore reduced production cost as raw material; Wide, the safety of selecting for use in trypsinase source is easy to extensive use;
2, the final ACE that obtains suppresses bioactive peptide safety, economy, effective, and its ACE suppresses activity and reached more than 84.95%, can be used as medicine and food grade additives.
Embodiment
The production method of embodiment 1, a kind of angiotensin converting enzyme inhibitory peptide, carry out following steps successively:
1), with rice poor and distilled water according to 1: 4.13 weight ratio in container mixed after, be the sodium hydroxide solution adjusting pH value to 8.02 of 2.5M with concentration;
2), in container, put into water bath with thermostatic control behind the adding trypsinase and carry out enzyme digestion reaction; Described bath temperature is 37 ℃, and the reaction times is 4 hours, and tryptic consumption is 1.5% of the poor weight of rice;
3), container being put into boiling water boils and stopped hydrolysis reaction in 10 minutes again; Products therefrom carries out centrifugal, and the supernatant liquor of centrifugal gained promptly gets angiotensin converting enzyme inhibitory peptide through concentrated ,-45 ℃ of-30 ℃ of following vacuum freezings (vacuum tightness 40-44Pa) dryings.
The angiotensin converting enzyme inhibitory peptide of gained, its ACE inhibition activity is 84.95% after testing.
The production method of embodiment 2, a kind of angiotensin converting enzyme inhibitory peptide, carry out following steps successively:
1), with rice poor and distilled water according to 1: 4 weight ratio in container mixed after, be the sodium hydroxide solution adjusting pH value to 7.9 of 2.0M with concentration;
2), in container, put into water bath with thermostatic control behind the adding trypsinase and carry out enzyme digestion reaction; Described bath temperature is 35 ℃, and the reaction times is 4.5 hours, and tryptic consumption is 1.6% of the poor weight of rice;
3), container being put into boiling water boils and stopped hydrolysis reaction in 12 minutes again; Products therefrom carries out centrifugal, and the supernatant liquor of centrifugal gained promptly gets angiotensin converting enzyme inhibitory peptide through concentrated ,-45 ℃ of-30 ℃ of following vacuum freezings (vacuum tightness 40-44Pa) dryings.
The angiotensin converting enzyme inhibitory peptide of gained, its ACE inhibition activity is 83.52% after testing.
The production method of embodiment 3, a kind of angiotensin converting enzyme inhibitory peptide, carry out following steps successively:
1), with rice poor and distilled water according to 1: 4.5 weight ratio in container mixed after, be the sodium hydroxide solution adjusting pH value to 8.1 of 3.0M with concentration;
2), in container, put into water bath with thermostatic control behind the adding trypsinase and carry out enzyme digestion reaction; Described bath temperature is 40 ℃, and the reaction times is 3.5 hours, and tryptic consumption is 1.4% of the poor weight of rice;
3), container being put into boiling water boils and stopped hydrolysis reaction in 8 minutes again; Products therefrom carries out centrifugal, and the supernatant liquor of centrifugal gained promptly gets angiotensin converting enzyme inhibitory peptide through concentrated ,-45 ℃ of-30 ℃ of following vacuum freezings (vacuum tightness 40-44Pa) dryings.
The angiotensin converting enzyme inhibitory peptide of gained, its ACE inhibition activity is 76.55% after testing.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (2)
1, a kind of production method of angiotensin converting enzyme inhibitory peptide is characterized in that may further comprise the steps:
1), with rice poor and distilled water according to 1: 4～4.5 weight ratio in container mixed after, be alkaline solution adjusting pH value to 7.9～8.1 of 2.0～3.0M with concentration;
2), putting into water bath with thermostatic control behind the adding trypsinase in container reacts; Described bath temperature is 35～40 ℃, and the reaction times is 3.5～4.5 hours, and tryptic consumption is 1.4～1.6% of the poor weight of rice;
3), container being put into boiling water boils and came termination reaction in 8～12 minutes again; Products therefrom carries out centrifugal, and the supernatant liquor of centrifugal gained promptly gets angiotensin converting enzyme inhibitory peptide through concentrated, lyophilize.
2, the production method of angiotensin converting enzyme inhibitory peptide according to claim 1 is characterized in that: in the described step 1), rice weight ratio poor and distilled water is 1: 4.13, and selecting concentration for use is the sodium hydroxide adjusting pH value to 8.02 of 2.5M; Described step 2) in, bath temperature is 37 ℃, and the reaction times is 4 hours, and tryptic consumption is 1.5% of the poor weight of rice; In the described step 3), container is put into boiling water and is boiled and came termination reaction in 10 minutes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101904404A (en) * | 2010-08-04 | 2010-12-08 | 浙江工商大学 | Method for producing rice peptone with rice dregs by biological enzyme hydrolysis and application of rice peptone |
| CN101570568B (en) * | 2009-06-15 | 2012-05-30 | 东北农业大学 | ACE inhibitory peptide in fermented milk and preparation method thereof |
| CN102864200A (en) * | 2012-09-17 | 2013-01-09 | 武汉工业学院 | Method for preparing ACE (Angiotensin Converting Enzyme) inhibitory peptide by hydrolyzing rice protein isolate with complex enzyme |
| CN111484545A (en) * | 2020-03-31 | 2020-08-04 | 华南农业大学 | Blood pressure lowering oligopeptide from rice wine lees and preparation method and application thereof |
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2006
- 2006-05-22 CN CN 200610050845 patent/CN1884574A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570568B (en) * | 2009-06-15 | 2012-05-30 | 东北农业大学 | ACE inhibitory peptide in fermented milk and preparation method thereof |
| CN101904404A (en) * | 2010-08-04 | 2010-12-08 | 浙江工商大学 | Method for producing rice peptone with rice dregs by biological enzyme hydrolysis and application of rice peptone |
| CN102864200A (en) * | 2012-09-17 | 2013-01-09 | 武汉工业学院 | Method for preparing ACE (Angiotensin Converting Enzyme) inhibitory peptide by hydrolyzing rice protein isolate with complex enzyme |
| CN111484545A (en) * | 2020-03-31 | 2020-08-04 | 华南农业大学 | Blood pressure lowering oligopeptide from rice wine lees and preparation method and application thereof |
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