CN101117642B - Polypeptide capable of lowering blood pressure and method for preparing same - Google Patents
Polypeptide capable of lowering blood pressure and method for preparing same Download PDFInfo
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- CN101117642B CN101117642B CN2007101197712A CN200710119771A CN101117642B CN 101117642 B CN101117642 B CN 101117642B CN 2007101197712 A CN2007101197712 A CN 2007101197712A CN 200710119771 A CN200710119771 A CN 200710119771A CN 101117642 B CN101117642 B CN 101117642B
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Abstract
The present invention discloses a polypeptide which can decrease the blood pressure and a method for preparing the polypeptide. The method includes the following steps: firstly, oat bran is suspended in the water, PH is adjusted between 8.5 and 9.5, and the oat bran is heat treated under the temperature of 50 to 60 DEG C; secondly, the pretreated liquid is added with alkaline protease, and then is agitated at 50 to 60 DEG C for enzymolysis for 3 to 5 hours; thirdly, the obtained enzymolysis liquid in the second step is centrifuged, and the supernatant is collected to be added with Beta-glucanase, medium temperature amylase and glucoamylase, after reacting for 2 to 4 hours, the supernatant is filtered, and after the filtrate big hole absorbs resin, de-ionized water is used to flush sugarand salt, then ethanol solution whose volume percentage is 75 percent is used to the elution, thus attaining polypeptide which can decrease the blood pressure. The method of the present invention hasthe advantages that the operation is simple, the manufacturing cost is low, the prepared product has higher purity, and the productive rate is high. The oat polypeptide prepared by the method has stronger activity of restraining ACE, and can be widely used to the fields of antihypertensive health food, etc.
Description
Technical field
The present invention relates to a kind of polypeptide that can be hypotensive and preparation method thereof, particularly a kind of active oat polypeptide of ACE and preparation method thereof that suppresses.
Background technology
Oat, Gramineae oat family Avena, annual or per nnial herb.Cereals nutrient is worth abundant and comprehensive, mineral element content such as its protein, fat, vitamin-E, food fibre and calcium, magnesium, iron, phosphorus all are higher than other cereal crop, have multiple bioactive functions such as reducing blood-fat, reducing cholesterol, hypotensive, hypoglycemic and oxidation-resistance.
(angiotensin I-converting enzyme, ACE are a kind of zinc dipeptides carboxylic acids that contains EC3.4.15.1) to angiotensin-converting enzyme, are distributed widely in the mammalian tissues and to its blood pressure regulation to play critical effect.Therefore reaching the hypertensive purpose of treatment by ACE activity in the inhibition body is a very important approach.
In recent years, some have the biologically active peptides of specific physiologically active, have caused people's very big concern as ace inhibitory peptide, opioid peptides, immunomodulatory peptides etc., and it is safe that these peptides have, and have no side effect, advantage such as wide material sources.Vegetable-protein becomes the main source that people study bioactive peptide with its irreplaceable advantage.
Summary of the invention
The purpose of this invention is to provide a kind of polypeptide that can be hypotensive and preparation method thereof.
Preparation provided by the present invention can be hypotensive the method for polypeptide, comprise the steps:
1) pre-treatment: oat bran is suspended in water, regulate pH to 8.5-9.5,50-60 ℃ of heat treated;
2) enzyme is handled: adding Sumizyme MP in through the pretreated liquid of step 1), is 8.0-9.5 in the pH value, and temperature is 50-60 ℃ and stirred enzymolysis 3-5 hours down;
3) purifying: the enzymolysis solution that step 2 obtains is centrifugal, get supernatant liquor, add beta-glucanase, in warm amylase and saccharifying enzyme reaction 2-4 hour, filter the footpath and be the filtration of 0.5-1.2 μ m, with filtrate with absorption with macroporous adsorbent resin after, usefulness deionized water rinsing desaccharification and salt, be the ethanolic soln wash-out of 25-75% with volumn concentration again, obtain can be hypotensive polypeptide.
In the described method, described step 2) also being included in in the liquid behind the Sumizyme MP enzymolysis and adding flavor protease, is 6.5-7.5 in the pH value, and temperature is 50-60 ℃ and stirred enzymolysis 1-2 hours down; The addition of described flavor protease is 500,000-1,500,000 U/g oat brans, is preferably 1,000,000 U/g oat brans.
In the described method, described macroporous resin is DA201-C polymeric adsorbent, HZ-816 or XAD1180.
In the described method, described step 2) in, the addition of described Sumizyme MP is 500,000-1,500,000 U/g oat brans, is preferably 1,000,000 U/g oat brans;
In the described method, in the described step 1), the time of described heat treated is 1-5 hours.
In order to improve extraction yield, in the described method, in the described step 1), the granular size of described oat bran is the 20-60 order.
In the described method, in the described step 1), the ratio of weight and number of described oat bran and water is 1:8-12.
In the described method, in the described step 3), described water is deionized water; The concentration expressed in percentage by volume of described ethanolic soln is preferably 75%.
In the described method, in the described step 3), the addition of described beta-glucanase is 50 ten thousand-150 ten thousand U/g oat brans, is preferably 1,000,000 U/g oat brans; Warm diastatic addition is 50 ten thousand-150 ten thousand U/g oat brans in described, is preferably 1,000,000 U/g oat brans; The addition of described saccharifying enzyme is 50 ten thousand-150 ten thousand U/g oat brans, is preferably 1,000,000 U/g oat brans.
Comprise also in the described method that the elutriant that contains polypeptide that can be hypotensive that described ethanolic soln wash-out is obtained concentrates, lyophilize obtain can be hypotensive the polypeptide powder.
The polypeptide that can hypotensive polypeptide comprise above-mentioned either party's method preparation provided by the present invention.
Of the present invention can hypotensive oat polypeptide be to use enzymic degradation oat bran albumen, makes by membrane filtration, absorption with macroporous adsorbent resin etc., and 85% polypeptide molecular weight is below 2000Da.Experiment showed, and of the present inventionly can have the activity (concentration is that the inhibiting rate of the oat polypeptide of 0.6mg/ml is more than 85%) of very strong inhibition ACE, the 503nhibiting concentration (IC of ACE inhibitor by hypotensive oat polypeptide
50) reach 0.391mg/ml, can be widely used in fields such as antihypertensive health care food.
The inventive method is simple to operate, has higher purity and yield.The inventive method is simple to operate, has higher purity and yield.
Description of drawings
Fig. 1 prepares the process flow sheet of oat polypeptide for the present invention;
Fig. 2 is the distribution of oat polypeptide relative molecular weight;
Fig. 3 is the elution peak curve of 75% ethanol elution purifying oat polypeptide.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Percentage composition described in the following embodiment if no special instructions, is the quality percentage composition.
Embodiment 1, the preparation of oat polypeptide and blood pressure lowering effect experiment thereof that can be hypotensive
One, the preparation of oat polypeptide that can be hypotensive
The process flow sheet that extracts oat polypeptide as shown in Figure 1, concrete steps are as follows:
1, oat bran is handled:
For more acquisition oat polypeptides, oat bran is crushed to the particle of 20 orders to 60 order sizes, Kjeldahl determination measures wherein that the protein mass percentage composition is 20%.
2, pre-treatment:
Above-mentioned oat bran of 80g and deionized water are mixed and made into oat bran suspension by weight 1:8, regulate the pH value to 8.5-9.5, be heated to 50-60 ℃, evenly stirred pre-treatment 1 hour with NaOH.
3, enzyme is handled:
With the pretreated suspension of step 2, measure the pH value, the result shows that (the pH value of enzymolysis should be 8.0-9.5 to pH value 8.0-8.5, if the pH of suspension value is not in this scope, regulate the pH of suspension value with NaOH), adding 3.5g is the Sumizyme MP of 1,000,000 U/g than vigor, and temperature 50-60 ℃ is continued stirring and heated 3-5 hour; Adding 1g then is the flavor protease of 1,000,000 U/g than vigor, and 55 ℃ are continued stirring and heated 1 hour.
4,, under 4000rpm, got supernatant liquor in centrifugal 10 minutes with the liquid behind step 3 enzymolysis; Add 1,000,000 U beta-glucanases in this supernatant liquor, the reaction of warm amylase and 1,000,000 U saccharifying enzyme is 3 hours among 1,000,000 U, utilizes flame filter press to carry out membrane filtration under the filter footpath of 0.8 μ m, adds 20g diatomite and helps filter, obtains the brown clarified liq of 500ml.
5, the clarified liq that step 2 is obtained is that the ratio (usually the corresponding 200ml enzymolysis solution of 200ml resin) of 1:1 joins in the adsorption column (φ 2.5*60cm) of DA201-C polymeric adsorbent according to liquid and resin volume ratio, does not have sugar and salt (reflecting by measuring sugared content and conductivity value) with deionized water rinsing to elutriant.Use 75% ethanol elution again, effluent volume is 2 times of column volume, under 220nm, utilize ultraviolet spectrophotometer to measure the changing conditions of absorbance, make absorbance-elution volume curve determination polymeric adsorbent elution curve, the result as shown in Figure 3, the elutriant that obtains be can be hypotensive the oat polypeptide extracting solution.
It is about 20% that oat polypeptide extracting solution rotary evaporation that can be hypotensive is concentrated into the quality percentage composition of solid substance, lyophilize (vacuum freeze: U.S. VIRTIS company; Condition :-40 ℃, 70Pa) obtain can be hypotensive the oat polypeptide powder.
The molecular weight analyse of oat polypeptide powder that two, can be hypotensive
With the oat polypeptide powder of above-mentioned acquisition, get 5mg and be dissolved in the 1ml distilled water, adopt high performance liquid chromatography (HPLC) method to measure its relative molecular weight and distribute; Measure chromatographic condition: An Jielun high performance liquid chromatograph 1100; Chromatographic column is TSKge12000 SWXL Size Exclusion Chromatograph SEC post (300mm * 7.8mm); Moving phase is the mixed solution of acetonitrile, Glacial acetic acid and trifluoroacetic acid, acetonitrile/Glacial acetic acid/trifluoroacetic acid=45/55/0.1 (volume ratio); Detect UV220nm; Flow velocity 0.5ml/min; 30 ℃ of column temperatures.The result as shown in Figure 2, the polypeptide molecular weight of gained oat step-down peptide 85% is below 2000Da.
Three, the step-down oat polypeptide of step 1 preparation suppresses the active effect experiment of ACE
1) angiotensin-converting enzyme (ACE) suppresses active mensuration with reference to (Hayakari M such as Cushman, KondoY, Izumi H.A rapid and simple spetrophotometric assay of angiotensin-converting enzyme[J] .Anal Biochem, 1978,84:361-369.) method, concrete grammar is as described below:
ACE substrate Hip-His-Leu (available from U.S. SIGMA) is dissolved in 0.1mol/L contains in the borate buffer solution of 0.3mol/L sodium-chlor (pH=8.3), being mixed with concentration is the Hip-His-Leu solution of 5mmol/l.Get 100 μ l 5mmol/l the preparation of Hip-His-Leu solution and 40 μ l step 1 can be hypotensive oat polypeptide solution (0.6mg/ml) mixes, behind 37 ℃ of water-bath preheating 5min, adding 0.1U/ml ACE solution 10 μ L continue 37 ℃ of water-bath 60min.Add 1mol/l HCl solution 1500 μ L termination reactions subsequently.Add 1.2ml ethyl acetate uniform mixing again, with the urobenzoic acid in the extractive reaction system.Take out 0.9ml ester layer solution behind centrifugal (1000g) 10min and change in another test tube, 80 ℃ of oven dry, and be dissolved in again in the 3ml deionized water.Simultaneously, be provided with one group do not add step 1 preparation can be hypotensive oat polypeptide solution, the experiment that other steps are the same and one group do not add ACE and step 1 preparation can be hypotensive oat polypeptide solution, the experiment that other steps are the same, in contrast.The reaction solution that above-mentioned three groups of experiments obtain is all measured light absorption value at the 228nm place.The ACE inhibiting rate is as shown in the formula calculating:
ACE inhibiting rate (%)=(ODb-ODa)/(ODb-ODc) * 100
In the formula:
The preparation of ODa---ACE and step 1 can be hypotensive oat polypeptide solution absorbancy under the existence condition all;
ODb---do not add step 1 preparation can be hypotensive the oat polypeptide solution absorbency;
The preparation of ODc---ACE and step 1 can be hypotensive oat polypeptide solution absorbancy of added-time not.
The result show step 1 preparation can be hypotensive the ACE inhibiting rate (%) of oat polypeptide be 85%.
2) step 1 preparation can be hypotensive 503nhibiting concentration (the IC of oat polypeptide
50) mensuration
IC
50Be the active strong and weak common counter of objective evaluation ace inhibitory peptide, concrete grammar is as described below:
Preparation different concns (0.2mg/mL, 0.5mg/mL, 0.8mg/mL, 1.2mg/mL, 1.5mg/mL, 2.0mg/mL, the preparation of 3.0mg/mL) step 1 can be hypotensive oat polypeptide solution, the described method of step 1) is measured the ACE inhibiting rate of oat polypeptide solution that can be hypotensive set by step, then, with ACE inhibiting rate (%) is ordinate zou, and log (inhibitor concentration) is the X-coordinate curve plotting, and the line retrace analysis of going forward side by side draws regression equation y=15.31-64.04x-19.29x
2(R
2=0.9999)
According to the preparation of this regression equation calculation step 1 can be hypotensive 503nhibiting concentration (the IC of oat polypeptide
50).
The result shows, the step 1 preparation can be hypotensive 503nhibiting concentration (the IC of oat polypeptide
50) is 0.391mg/ml.
Other food proteins source ACE inhibitor peptides of having reported is as Ox blood plasma albumen (IC5
0=0.56mg/ml), shrimp protein (IC
50=0.98mg/ml), beef protein (IC
50=0.12mg/ml), mushroom albumen (IC
50=0.31mg/ml), soybean protein (IC
50=0.28mg/ml), garbanzo albumen (IC
50=0.18mg/ml) wait and to compare, step 1 preparation be can hypotensive oat polypeptide having stronger ACE and suppressing active of raw material with oat bran albumen.
Above-mentioned experiment shows that what the present invention prepared can have very strong ACE inhibition activity by hypotensive oat polypeptide, and what this explanation the invention described above prepared can have hypotensive ability by hypotensive oat polypeptide.
Claims (11)
- One kind can be hypotensive the preparation method of polypeptide, comprise the steps:1) pre-treatment: oat bran is suspended in water, regulate pH to 8.5-9.5,50-60 ℃ of heat treated;2) enzyme is handled: adding Sumizyme MP in through the pretreated liquid of step 1), is 8.0-9.5 in the pH value, and temperature is 50-60 ℃ and stirred enzymolysis 3-5 hour down; Adding flavor protease in the liquid behind the Sumizyme MP enzymolysis, is 6.5-7.5 in the pH value, and temperature is to stir enzymolysis 1-2 hour under 50-60 ℃ the condition;3) purifying: the enzymolysis solution that step 2 obtains is centrifugal, get supernatant liquor, add beta-glucanase, in warm amylase and saccharifying enzyme reaction 2-4 hour, filter the footpath and be the filtration of 0.5-1.2 μ m, with filtrate with absorption with macroporous adsorbent resin after, usefulness deionized water rinsing desaccharification and salt, be the ethanolic soln wash-out of 25-75% with volumn concentration again, obtain can be hypotensive polypeptide.
- 2. method according to claim 1 is characterized in that: in the described step 3), described macroporous resin is DA201-C polymeric adsorbent, HZ-816 or XAD1180.
- 3. method according to claim 2 is characterized in that: described step 2), the addition of described Sumizyme MP is 500,000-1,500,000 U/g oat brans; The addition of described flavor protease is 500,000-1,500,000 U/g oat brans.
- 4. method according to claim 3 is characterized in that: described step 2), the addition of described Sumizyme MP is 1,000,000 U/g oat brans; The addition of described flavor protease is 1,000,000 U/g oat brans.
- 5. according to claim 3 or 4 described methods, it is characterized in that: in the described step 1), the time of described heat treated is 1-5 hour.
- 6. method according to claim 5 is characterized in that: in the described step 1), the granular size of described oat bran is the 20-60 order.
- 7. method according to claim 6 is characterized in that: in the described step 1), the ratio of weight and number of described oat bran and water is 1: 8-12.
- 8. method according to claim 7 is characterized in that: in the described step 3), described water is deionized water; The concentration expressed in percentage by volume of described ethanolic soln is 75%.
- 9. method according to claim 8 is characterized in that: in the described step 3), the addition of described beta-glucanase is 500,000-1,500,000 U/g oat brans; Warm diastatic addition is 500,000-1,500,000 U/g oat brans in described; The addition of described saccharifying enzyme is 500,000-1,500,000 U/g oat brans.
- 10. method according to claim 9 is characterized in that: in the described step 3), the addition of described beta-glucanase is 1,000,000 U/g oat brans; Warm diastatic addition is 1,000,000 U/g oat brans in described; The addition of described saccharifying enzyme is 1,000,000 U/g oat brans.
- 11., it is characterized in that according to claim 9 or 10 described methods: comprise also in the described method that the elutriant that contains polypeptide that can be hypotensive that described ethanolic soln wash-out is obtained concentrates, lyophilize obtain can be hypotensive the polypeptide powder.
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| CN101875956A (en) * | 2010-05-14 | 2010-11-03 | 北京工商大学 | Hair-care coix seed polypeptide and preparation method thereof |
| CN102212599B (en) * | 2011-04-29 | 2013-07-17 | 北京工商大学 | Method for preparing oat ACE (Angiotensin Converting Enzyme) inhibitory peptide by using enzymatic membrane reactor |
| CN102605030A (en) * | 2012-03-22 | 2012-07-25 | 无锡德冠生物科技有限公司 | Enzymatic extraction method for oat peptide |
| CN103461643A (en) * | 2013-09-06 | 2013-12-25 | 江苏丘陵地区镇江农业科学研究所 | Method for extracting bran polypeptide and bran estrogen through wet ball milling in combination with enzymic method |
| CN106036420A (en) * | 2016-06-24 | 2016-10-26 | 甘洛县彝家山寨农牧科技有限公司 | Tartary buckwheat health care food applicable to diabetics and preparation method thereof |
| CN110734947A (en) * | 2019-09-17 | 2020-01-31 | 江苏大学 | preparation method of oat blood pressure lowering polypeptide |
| CN111187763A (en) * | 2019-11-22 | 2020-05-22 | 武汉新华扬生物股份有限公司 | Complex enzyme preparation for oat hydrolysis and application thereof |
| CN111926051A (en) * | 2020-07-10 | 2020-11-13 | 内蒙古燕谷坊全谷物产业发展有限责任公司 | Oat peptide powder and preparation method thereof |
| CN114438156A (en) * | 2022-03-04 | 2022-05-06 | 北京如慧健康管理有限公司 | Oat peptide extraction method |
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| CN1875737A (en) * | 2006-06-30 | 2006-12-13 | 北京工商大学 | An oat peptide and extraction method thereof |
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