CN101849606B - Method for preparing oat antihypertensive peptides by ultrasound auxiliary enzymolysis - Google Patents
Method for preparing oat antihypertensive peptides by ultrasound auxiliary enzymolysis Download PDFInfo
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Abstract
The invention relates to an ultrasound auxiliary enzymolysis preparation method of antihypertensive peptides, in particular to a method for preparing oat antihypertensive peptides by ultrasound auxiliary enzymolysis, which belongs to the technical field of oat deep processing. The method is carried out according to the following steps: (1) crushing raw materials; (2) extracting protein by an alkali extraction and acid precipitation method; and (3) preparing the obtained protein into a protein solution with certain mass concentration, adding alkali protease, exerting ultrasound under the condition of maintaining the optimal pH and temperature of the alkali protease of a reaction system, carrying out ultrasound promoted enzymolysis treatment, closing the ultrasound after a certain time, converting the enzymolysis into conventional enzymolysis, maintaining unchanged pH and temperature in the enzymolysis process, killing enzymes at 100 DEG C after a certain time of the enzymolysis, carrying out centrifugation to collect supernatant fluid, and obtaining extract liquid of the antihypertensive peptides. After the ultrasound treatment in the preparation process of the oat antihypertensive peptides by the invention, the enzymolysis time is shortened, the enzymolysis effect is improved, and the activity of the enzymolysis products is improved.
Description
Technical field
A kind of ultrasonic assistant preparation method of avenin blood pressure lowering peptide belongs to oat deep process technology field.
Background technology
Though oat is long at the cultivation history of China, be one type of important standing grain cereals food crop.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 preparation.Along with people's is to the healthy pay attention to day by day of self diet, the sustainable growth of global oat consumption, and the world market is in great demand to avenaceous.Yet China's oat deep process technology is relatively backward at present; Most oat does not pass through deep processing, and purposes is single, and oat based food kind is single on the market; This situation has not only caused the significant wastage of resource, and has limited further developing of China's oat industry.Oat not only nutritive value is abundant, can satisfy human body requirements, and existing research shows that avenin can obtain high hypotensive activity polypeptide through enzymolysis, so avenin has good development prospect aspect the exploitation antihypertensive function food.
Hypertension has great harm to human body, is the one of the main reasons that causes cardiovascular disorder, whole world every year because of the dead number of hypertension up to 1,200 ten thousand.Prevention and treatment hyperpietic are the important topics that current society faces.In recent years, reported many small peptides with hypotensive activity that derive from food proteins both at home and abroad, these bioactive peptides have efficiently, at a low price and the characteristics of low toxicity, can play prevention and treat hypertensive effect.Therefore making full use of the peculiar effect of avenaceous carries out the development of oat antihypertensive peptides, not only can realize the comprehensive utilization of oat resource, can also develop a kind of active antihypertensive peptide with food proteins source of good efficacy.
Traditional method for preparing avenin polypeptide or ace inhibitory peptide generally is to adopt from oat bran, to extract avenin, and then adds proteolytic enzyme and carry out enzymolysis, carries out separation and purification at last again and obtains avenin polypeptide fragment or mixture.But traditional enzyme solution ubiquity reaction efficiency is not high, the utilization ratio of proteolytic enzyme is low wait not enough, problem such as therefore cause the consumption of long reaction time, enzyme big, its lytic activity is low.Ultrasonic in recent years more and more wider in Application in Food Processing, ultrasonic technology can also significantly improve the activity of blood pressure lowering polypeptide when quickening proteolysis.But the relevant article of ultrasonic assistance enzymolysis technology preparation oat step-down peptide that utilizes does not also appear in the newspapers.
Summary of the invention
The object of the invention is exactly in order to overcome the deficiency of traditional enzyme solution in the above-mentioned prior art, a kind of ultrasonic assistant preparation method of oat antihypertensive peptides to be provided.
Technical scheme of the present invention: elder generation's enzymolysis certain hour under ultrasonic field, close the ultrasonic conventional enzymolysis that changes over to again.Concrete technical scheme is: oat is pulverized in (1), crosses 40 mesh sieves, the oatmeal of system; (2) extraction of avenin: with the oatmeal is raw material, through pulverizing, adds water at 1: 8 by solid-liquid ratio, transfers pH=10 lixiviate 90min; With vat liquor 10000r/min, centrifugal 20min gets supernatant again, regulates pH to 4.0; 10000r/min then, centrifugal 20min dissolves the centrifugal throw out that obtains once more, processes solution; And carry out spraying drying, wherein inlet temperature is 180 ℃, 90 ℃ of temperature outs; Obtain avenin; (3) ultrasonic assistance enzymolysis: with the above-mentioned avenin that makes; Being mixed with mass concentration is the avenin solution of 3% (W/W), adds 2% (E/S) Sumizyme MP, keeps reaction system under 50 ℃ of the best pH9.0 of Sumizyme MP and temperature; Apply ultrasonic; Carry out ultrasonic promotion enzymolysis processing, wherein ultrasonic power is 250-1250W/100mL, ultrasonic time 5-10min; Ultrasonic intermittence is than being 2s-6s/2s; Close ultrasonicly behind the certain hour, change conventional enzymolysis over to, promptly keep pH9.0 in the enzymolysis process, 50 ℃ of temperature, enzymolysis 60min, the enzyme 10min that goes out under 100 ℃, centrifugal 20min under the 10000r/min collects supernatant, can obtain the extracting solution of oat antihypertensive peptides.
Technical scheme of the present invention also can be: earlier protein solution is carried out ultrasonic pretreatment, carry out enzyme digestion reaction again.Concrete technical scheme is: prepare avenin with reference to above-mentioned steps (1), (2); (3) ultrasonic pretreatment: with the above-mentioned avenin that makes, being mixed with mass concentration is the avenin solution of 3% (W/W), applies supersound process, and wherein ultrasonic power is 250-1250W//100mL, ultrasonic time 5-20min; Ultrasonic intermittence is than being 2s-6s/2s; (4) add protease hydrolyzed: add 2% (E/S) Sumizyme MP and carry out enzymolysis, keep pH9.0 in the enzymolysis process, 50 ℃ of temperature; Enzymolysis 90min, the enzyme 10min that goes out under 100 ℃, centrifugal 20min under the 10000r/min; Collect supernatant, can obtain the extracting solution of oat antihypertensive peptides.
Technical scheme of the present invention can also be: earlier avenin solution is carried out ultrasonic pretreatment; In ultrasonic field, avenin is carried out ultrasonic assistance enzymolysis again; Close ultrasonicly behind the certain hour, change conventional enzymolysis then over to.Concrete technical scheme is: (1) is mixed with the avenin solution that mass concentration is 3% (W/W) with avenin, carries out impulse ultrasound pre-treatment 5-20min, and wherein ultrasonic power is that 250-1250W/100mL, ultrasonic intermittence are than being 2s-6s/2s; (2) in the avenin solution that is processed; Add 2% (E/S) Sumizyme MP; Keep reaction system under 50 ℃ of the best pH9.0 of Sumizyme MP and temperature; In ultrasonic field, carry out enzyme digestion reaction 5-10min, wherein ultrasonic power is that 250-1250W/100mL, ultrasonic intermittence are than being 2s-6s/2s; (3) close ultrasonicly behind the certain hour, change conventional enzymolysis over to, promptly keep 50 ℃ of pH9.0, temperature in the enzymolysis process, enzymolysis 60min, the enzyme 10min that goes out under 100 ℃, centrifugal 20min under the 10000r/min collects supernatant, can obtain the extracting solution of oat antihypertensive peptides.
The present invention adopts the ultrasonic pretreatment material protein, in enzymolysis process, applies ultrasonic and not only adopts the ultrasonic pretreatment material protein but also in enzymolysis process, apply ultrasonic three kinds of modes, and the ACE inhibition activity of enzymolysis product after the ultrasonication is studied.Through after the supersound process, shortened enzymolysis time, improved hydrolysis result, improved the activity of enzymolysis product.
Embodiment
Weigh the reaction efficiency of entire reaction among the present invention with degree of hydrolysis (DH), the mensuration of degree of hydrolysis (DH) adopts the pH-stat method; Too much angiotensin-converting enzyme (ACE) is one of major reason that causes elevation of the blood pressure in the human body simultaneously.Therefore the present invention estimates its hypotensive activity with enzymolysis product to the inhibiting rate of ACE.Enzymolysis product ACE suppress active mensuration according to document " Liu Zhiguo; Wu Qiong, Lv Lingxiao etc. enzymolysis rice bran protein separation and Extraction ace inhibitory peptide and structural research [J] thereof. Food science, 2007; 28 (3): 223～227. " method carry out; As the catalytic substrate of ACE, study the variation of its absorbancy with N-[3-(2-furyl) acryloyl]-L-phenylalanyl glycylglycine (FAPGG), utilize enzyme scalar quantity appearance to measure.
Reference examples: conventional enzymolysis
(1) oat is pulverized, cross 40 mesh sieves, the oatmeal of system; (2) extraction of avenin: with the oatmeal is raw material, through pulverizing, adds water at 1: 8 by solid-liquid ratio, transfers pH=10 lixiviate 90min; With vat liquor 10000r/min, centrifugal 20min gets supernatant again, regulates pH to 4.0; 10000r/min then, centrifugal 20min dissolves the centrifugal throw out that obtains once more, processes solution; And carry out spraying drying, wherein inlet temperature is 180 ℃, 90 ℃ of temperature outs; Obtain avenin; (3) conventional enzymolysis (both do not had ultrasonic pretreatment albumen, and do not had ultrasonic promoted proteolysis again): the conventional enzymolysis through parameter optimization obtains is operated as follows: the avenin solution 100mL of preparation 3% (W/W), put into the thermostat water bath of being with rotor; Add 2% (E/S) Sumizyme MP and carry out enzymolysis, keep pH9.0 in the enzymolysis process, 50 ℃ of temperature; Enzymolysis 90min; The enzyme 10min that goes out under 100 ℃ afterwards, centrifugal 20min under the 10000r/min collects supernatant; Measure ACE after diluting 40 times and suppress active, and calculate degree of hydrolysis (DH).See table 1.
Embodiment 1
(1) oat is pulverized, cross 40 mesh sieves, the oatmeal of system; (2) extraction of avenin: with the oatmeal is raw material, through pulverizing, adds water at 1: 8 by solid-liquid ratio, transfers pH=10 lixiviate 90min; With vat liquor 10000r/min, centrifugal 20min gets supernatant again, regulates pH to 4.0; 10000r/min then, centrifugal 20min dissolves the centrifugal throw out that obtains once more, processes solution; And carry out spraying drying, wherein inlet temperature is 180 ℃, 90 ℃ of temperature outs; Obtain avenin; (3) ultrasonic assistance enzymolysis: the avenin solution 100mL of preparation 3% (W/W), pour in the beaker that diameter is 4cm, add 2% (E/S) Sumizyme MP; With 1～2cm position under the ultrasound probe insertion reaction liquid; Remain under 50 ℃ of recommendation pH9.0 and the temperature of Sumizyme MP, carry out ultrasonic promotion enzymolysis processing, ultrasonic power is 750W; Ultrasonic intermittence is than being 2s/2s, ultrasonic time 20min.Close ultrasonicly behind the certain hour, change conventional enzymolysis over to, promptly keep pH9.0 in the enzymolysis process, 50 ℃ of temperature.Enzymolysis 60min, the enzyme 10min that goes out under 100 ℃, centrifugal (10000r/min, 20min) supernatant is collected in the back, and measuring ACE after dilute 40 times suppresses active, and calculating degree of hydrolysis (DH).See table 1.
Embodiment 2
The test treating processes is with embodiment 1, and its difference is that wherein ultrasonic power is 500W, ultrasonic time 20min; Ultrasonic intermittence is than being 2s/2s.See table 1.
Embodiment 3
The test treating processes is with embodiment 1, and its difference is that wherein ultrasonic power is 1250W, ultrasonic time 20min; Ultrasonic intermittence is than being 2s/2s.See table 1.
Embodiment 4
The test treating processes is with embodiment 1, and its difference is that wherein ultrasonic power is 750W, ultrasonic time 5min; Ultrasonic intermittence is than being 2s/2s.See table 1.
Embodiment 5
The test treating processes is with embodiment 1, and its difference is that wherein ultrasonic power is 750W, ultrasonic time 50min; Ultrasonic intermittence is than being 2s/2s.See table 1.
Embodiment 6
The test treating processes is with embodiment 1, and its difference is that wherein ultrasonic power is 750W, ultrasonic time 20min; Ultrasonic intermittence is than being 4s/2s.See table 1.
Embodiment 7
(1) oat is pulverized, cross 40 mesh sieves, the oatmeal of system; (2) extraction of avenin: with the oatmeal is raw material, through pulverizing, adds water at 1: 8 by solid-liquid ratio, transfers pH=10 lixiviate 90min; With vat liquor 10000r/min, centrifugal 20min gets supernatant again, regulates pH to 4.0; 10000r/min then, centrifugal 20min dissolves the centrifugal throw out that obtains once more, processes solution; And carry out spraying drying, wherein inlet temperature is 180 ℃, 90 ℃ of temperature outs; Obtain avenin; (3) ultrasonic preparatory place separates: with the above-mentioned avenin that makes, being mixed with mass concentration is the avenin solution 100mL of 3% (W/W), carries out ultrasonic pretreatment, and wherein ultrasonic power is 500W, ultrasonic time 10min; Ultrasonic intermittence is than being 2s/2s; (4) add protease hydrolyzed: add 2% (E/S) Sumizyme MP and carry out enzymolysis, keep pH9.0 in the enzymolysis process, 50 ℃ of temperature; Enzymolysis 90min; The enzyme 10min that goes out under 100 ℃, centrifugal (10000r/min, 20min) supernatant is collected in the back; Measure ACE after diluting 40 times and suppress active, and calculate degree of hydrolysis (DH).See table 1.
Embodiment 8
The test treating processes is with embodiment 7, and its difference is that wherein ultrasonic power is 1250W, ultrasonic time 10min; Ultrasonic intermittence is than being 2s/2s.See table 1.
Embodiment 9
The test treating processes is with embodiment 7, and its difference is that wherein ultrasonic power is 500W, ultrasonic time 20min; Ultrasonic intermittence is than being 2s/2s.See table 1.
Embodiment 10
The test treating processes is with embodiment 4, and its difference is that wherein ultrasonic power is 500W, ultrasonic time 10min; Ultrasonic intermittence is than being 4s/2s.See table 1.
Embodiment 11
(1) oat is pulverized, cross 40 mesh sieves, the oatmeal of system; (2) extraction of avenin: with the oatmeal is raw material, through pulverizing, adds water at 1: 8 by solid-liquid ratio, transfers pH=10 lixiviate 90min; With vat liquor 10000r/min, centrifugal 20min gets supernatant again, regulates pH to 4.0; 10000r/min then, centrifugal 20min dissolves the centrifugal throw out that obtains once more, processes solution; And carry out spraying drying, wherein inlet temperature is 180 ℃, 90 ℃ of temperature outs; Obtain avenin; (3) avenin is mixed with the avenin solution 100mL that mass concentration is 3% (W/W), carries out the impulse ultrasound pre-treatment, wherein ultrasonic power is 250W, ultrasonic time 10min, and ultrasonic intermittence is than being 2s/2s; (4) in the avenin solution that is processed; Add 2% (E/S) Sumizyme MP, keep reaction system under 50 ℃ of the best pH9.0 of Sumizyme MP and temperature, in ultrasonic field, carry out enzyme digestion reaction; Reaction times 5min, wherein ultrasonic power is that 250W, ultrasonic intermittence are than being 2s/2s; (5) close ultrasonicly behind the certain hour, change conventional enzymolysis over to, promptly keep 50 ℃ of pH9.0, temperature in the enzymolysis process; Enzymolysis 60min, the enzyme 10min that goes out under 100 ℃, centrifugal 20min under the 10000r/min; Collect supernatant, measuring ACE after dilute 40 times suppresses active, and calculating degree of hydrolysis (DH).See table 1.
Embodiment 12
The test treating processes is with embodiment 11, and its difference is that wherein ultrasonic power is 1250W in the ultrasonic pretreatment, ultrasonic time 20min; Ultrasonic intermittence is than being 6s/2s; When in ultrasonic field, carrying out enzyme digestion reaction, reaction times 10min, wherein ultrasonic power is that 1250W, ultrasonic intermittence are than being 6s/2s.See table 1.
The ACE of the different embodiment enzymolysis products of table 1 suppresses the result of active and degree of hydrolysis
The different treatment mode is to the comparative analysis of hydrolysis result
Following three kinds of processing modes are made an experiment under the same conditions, with the 503nhibiting concentration (IC of final enzymolysis product to ACE
50) be index, compare analysis.
Table 2 different treatment mode suppresses active comparative analysis to the ACE of enzymolysis product
Can find out that by table 2 it is active that supersound process can improve the inhibition of enzymolysis product, wherein with behind ultrasonic pretreatment material protein and the ultrasonic pretreatment material protein and the ACE that in enzymolysis process, applies the enzymolysis product of ultrasonic gained to suppress activity best.
Claims (2)
1. ultrasonic assistance enzymolysis method for preparing oat antihypertensive peptides is characterized in that in ultrasonic field, avenin being carried out ultrasonic assistance enzymolysis earlier, closes ultrasonicly behind the certain hour, changes conventional enzymolysis again over to; Specifically carry out according to following step: (1) is mixed with mass concentration with avenin is 3% avenin solution; Add 2% Sumizyme MP according to E/S; Keep reaction system under 50 ℃ of the best pH9.0 of Sumizyme MP and temperature, apply ultrasonicly, carry out ultrasonic promotion enzymolysis processing; Wherein ultrasonic power is 250-1250W/100mL, ultrasonic time 5-10min; Ultrasonic intermittence is than being 2s-6s/2s; (2) close ultrasonicly behind the certain hour, change conventional enzymolysis over to, promptly keep pH9.0 in the enzymolysis process, 50 ℃ of temperature, enzymolysis 60min, the enzyme 10min that goes out under 100 ℃, centrifugal 20min under the 10000r/min collects supernatant, promptly obtains the extracting solution of oat antihypertensive peptides.
2. a ultrasonic assistance enzymolysis method for preparing oat antihypertensive peptides is characterized in that (1) elder generation carries out ultrasonic pretreatment to avenin solution; (2) in ultrasonic field, avenin is carried out ultrasonic assistance enzymolysis again; (3) close ultrasonicly behind the certain hour, change conventional enzymolysis then over to; Specifically carry out according to following step: (1) is mixed with mass concentration with avenin is 3% avenin solution, carries out impulse ultrasound pre-treatment 5-20min, and wherein ultrasonic power is that 250-1250W/100mL, ultrasonic intermittence are than being 2s-6s/2s; (2) in the avenin solution that is processed; Add 2% Sumizyme MP according to E/S; Keep reaction system under 50 ℃ of the best pH9.0 of Sumizyme MP and temperature; In ultrasonic field, carry out enzyme digestion reaction 5-10min, wherein ultrasonic power is that 250-1250W/100mL, ultrasonic intermittence are than being 2s-6s/2s; (3) close ultrasonicly behind the certain hour, change conventional enzymolysis over to, promptly keep 50 ℃ of pH9.0, temperature in the enzymolysis process, enzymolysis 60min, the enzyme 10min that goes out under 100 ℃, centrifugal 20min under the 10000r/min collects supernatant, promptly obtains the extracting solution of oat antihypertensive peptides.
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| CN103125989B (en) * | 2012-03-31 | 2014-08-06 | 大连工业大学 | Method of extracting fish cerebrol and fish brain peptide |
| CN104131056A (en) * | 2014-06-18 | 2014-11-05 | 江苏大学 | Sesame cake ACE inhibitory peptide preparation method based on microwave and ultrasonic wave technology and application |
| US20180023110A1 (en) * | 2015-01-30 | 2018-01-25 | Jiangsu University | Method for preparing functional polypeptide through multimode ultrasonic enhancing enzymolysis |
| CN104846046B (en) * | 2015-01-30 | 2018-06-01 | 江苏大学 | A kind of method that gluten protein blood pressure lowering peptide is prepared based on the ultrasound-enhanced enzymolysis of order |
| CN104774893A (en) * | 2015-03-27 | 2015-07-15 | 江苏大学 | Method of gluten enzymolysis through countercurrent ultrasound treatment |
| CN104762355A (en) * | 2015-03-04 | 2015-07-08 | 江苏大学 | Preparation method for fish scale collagen protein active peptide |
| CN108410937A (en) * | 2018-04-24 | 2018-08-17 | 烟台金蕊女性用品有限公司 | Herba Saussureae Involueratae peptide molecule extraction process |
| FI129490B (en) | 2018-07-30 | 2022-03-15 | Fazer Ab Oy Karl | Method of preparing liquid oat base |
| CN111513340A (en) * | 2020-06-12 | 2020-08-11 | 通化天肽生物科技有限公司 | Oat polypeptide extraction method and nutritional product |
| 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 |
| CN115197285A (en) * | 2022-06-24 | 2022-10-18 | 北京小仙炖生物科技有限公司 | Method for extracting cubilose acid by ultrasonic wave combined enzymolysis |
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Application publication date: 20101006 Assignee: Nanjing Biological Engineering Co. Ltd., lamps and candles of a myriad families Assignor: Jiangsu University Contract record no.: 2017320000144 Denomination of invention: Method for preparing oat antihypertensive peptides by ultrasound auxiliary enzymolysis Granted publication date: 20120815 License type: Common License Record date: 20170608 |
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