CN104846046A - Method for preparing gluten antihypertensive peptide based on sequential ultrasonic enhanced enzymolysis - Google Patents
Method for preparing gluten antihypertensive peptide based on sequential ultrasonic enhanced enzymolysis Download PDFInfo
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Abstract
The invention provides a method for preparing gluten antihypertensive peptide based on sequential ultrasonic enzymolysis, which belongs to the technical field of deep processing of agricultural and sideline products. The method comprises the following steps: carrying out ultrasonic treatment on gluten suspension, wherein frequencies of sequential tri-frequency ultrasonic waves are 20 KHz, 28 KHz and 35 KHz, ultrasonic power is 16.67 W/L and pretreatment time is 20 to 50 min; after pretreatment, adding protease for enzymolysis, wherein during enzymolysis, a pH value is maintained to be 9.0, temperature is maintained to be 50 DEG C, and enzymolysis time is 30 min; and carrying out enzyme deactivation at 100 DEG C for 10 min, then carrying out centrifugation at 12000 r/min for 10 min and collecting supernatant. According to the invention, the gluten suspension is pretreated by using tri-frequency sequential ultrasonic waves, so enzymolysis efficiency is improved; and compared with other ultrasonic pretreatment manners, the ACE inhibition rate of enzymatic hydrolysate is increased by 13 to 20% and energy consumption is reduced by 3 to 53.75 times.
Description
technical field:
The present invention relates to bioengineering field, refer in particular to employing order Three-frequency ultrasonic pre-treatment gluten protein raw material, recycling enzyme solution prepares a kind of technology of gluten protein blood pressure lowering peptide.
technical background:
Wheat gluten protein is the by product of wheat starch processing, and its protein content is up to 75-80%, and amino acid composition is balanced complete, is nutritious, inexpensive vegetalitas high protein polymkeric substance.About more than 60 ten thousand tons of the wheat gluten protein annual production of China, industrial for food and feed mainly as flour quality improver, feed adhesive, nutritional additive etc. at present.Current research finds, gluten protein is as a kind of very potential plant protein source preparing biologically active peptides, and its enzymolysis product by suppressing the activity of Zinc metallopeptidase Zace1 (ACE) significantly, and reaches the object reduced blood pressure.But because gluten protein hydrophobic amino acid content (mainly higher containing Pro, Gln, Leu, Ala content) solvability is poor, bad dispersibility, cause gluten protein can not be combined fully with enzyme, enzymolysis efficiency is lower.The functional property of protein is primarily of its structures shape, and modification is the effective ways improving or strengthen some food proteins functional property.Ultrasonic pretreatment protein substrate is by changing Secondary structure, protein molecule particles size, and the tightness degree combined between molecule and improve the enzymolysis property of gluten protein raw material, improves Product Activity.But sonication treatment time and operating mode directly have influence on the intensity of variation of protein structure, improvement and the bioactive raising of this change and its enzymolysis property are closely related.
Research finds, double frequency or multiple frequency ultrasonic combination good fortune are penetrated has obvious reinforcing effect to the raising of phonochemical reaction yield, and the conjunction effect of double frequency or multiple frequency ultrasonic radiation simultaneously is all obviously greater than the independent radiation effect sum of each frequency ultrasound.Such as, the double-frequency ultrasound combination radiation that the double-frequency ultrasound that 28 kHz and 1.7 MHz combine combines, is greater than 3 W/cm when getting 1.7 MHz ultrasound intensities
2time, it closes 1.5 times that effect is about individual effect sum; When 1.7 MHz change 1.06 MHz or 0.87 MHz into, it closes effect can close to 2 of individual effect sum times.The very plinth of double frequency orthogonal radiation adds a branch of low-frequency ultrasonic formation three orthogonal irradiation frequently, its ultrasonic chemical disposal can be made to get a greater increase, three closes 1.4-1.8 times that exercising result is approximately three's difference irradiation yield sum." modern food science and technology " (2014, " the ultrasonic impact on silver carp fribrillin structure of different frequency " one 30(3): 23-26) delivered is civilian, take silver carp as raw material, adopt single-frequency, double frequency and triple-frequency ultrasonic to process silver carp fribrillin respectively, research different frequency supersound process is on the impact of fribrillin surface hydrophobicity and surperficial sulfhydryl content.Find to contrast with not ultrasonic sample, after triple-frequency ultrasonic process 10 min, single-frequency supersound process 15 min, the surface hydrophobicity of albumen improves 65.34%, 56.88% respectively, and protein surface sulfhydryl content improves 13.32% respectively, and 11.19%.Double-frequency ultrasound to the influence of protein surface hydrophobicity and surperficial sulfydryl between single-frequency and triple-frequency ultrasonic process.This illustrates that triple-frequency ultrasonic also just uses obvious advantage in protein-modified.
Existing document is with under the operating mode of synchronous multifrequency to the research of multiple frequency ultrasonic mostly, and the effect utilizing multi-frequency ultrasonic peak-to-peak to superpose, improves hyperacoustic treatment effect.This effect be the ultrasonic generator of multiple frequency work simultaneously send hyperacoustic situation simultaneously under occur, the synthesis wave spectrum of formation is very complicated, can along with the prolongation in treatment time, and the peak-to-peak produced in various degree is added and disappears mutually with peak-to-peak.
summary of the invention:
The object of the invention is this patent adopts the order triple-frequency ultrasonic equipment of lower-wattage to carry out pre-treatment to gluten protein, is intended to the hypotensive activity of the enzymolysis product improving gluten protein, shortens enzymolysis time, and reduce energy consumption.Develop a kind of good efficacy that has, safety, the active antihypertensive peptide in the food proteins source of reasonable price.Above-mentioned purpose of the present invention is realized by following technique means:
First ultrasonic pretreatment is carried out to protein solution, then carry out enzyme digestion reaction.Concrete technical scheme is a kind of method based on the ultrasound-enhanced enzyme-squash techniqued gluten protein blood pressure lowering peptide of order, carry out according to following step: gluten powder is mixed with the suspension that mass concentration is 3% (W/W) by (1), apply supersound process, wherein order triple-frequency ultrasonic frequency is: 20 KHz, 28 KHz, 35 KHz; The triple-frequency ultrasonic time is respectively 20s, 20s, 20s; Ultrasonic power is 16.67 W/L, pretreatment time 10-60 min.(2) protease hydrolyzed is added: add 3800 U/g Sumizyme MPs and carry out enzymolysis, in enzymolysis process, keep PH 9.0, temperature 50 DEG C.Enzymolysis time 30 min, go out at 100 DEG C enzyme 10 min, centrifugal 10 min under 12000 r/min, collects supernatant liquor, can obtain the extracting solution of protein blood pressure-reducing peptide.
the present invention is advantageously:
The present invention adopts ultrasonic pretreatment material protein to be studied the ACE inhibitory activity of enzymolysis product after ultrasonication.After triple-frequency ultrasonic pre-treatment, improve hydrolysis result, improve the activity of enzymolysis product, and reduce energy consumption compared with original ultrasound treatment patterns.The ACE inhibiting rate activity of enzymolysis solution improves 13% ~ 20%.Energy consumption reduces 3 ~ 53.75 times.
Below by drawings and Examples, technical scheme of the present invention is described in further detail.
accompanying drawing illustrates:
Fig. 1 is the equipment drawing of the order triple-frequency ultrasonic pre-treatment gluten protein that the present invention uses.
Embodiment
Fig. 1 is the equipment drawing of the order triple-frequency ultrasonic pre-treatment gluten protein that the present invention uses, and is Jiangsu University's independent development.Ultra-sonic generator 9,10,11 can send the ultrasonic wave of 20,28,35 kHz, and separate unit ultrasonic generator power is 100 w.In ultrasonic pond 4, inwall vertically places ultrasonic wave vibration plate, controls ultrasonic wave vibration plate 1,2,3 respectively by ultrasonic generator 9,10,11; After computer controller 8 sets each parameter of ultrasonic wave, control ultrasonic generator 9,10,11, send satisfactory ultrasonic wave; The 5 thermostat(t)ed water bathing pools that are equipment in the present invention, by thermometer 6 monitoring temperature in real time, and regulate medium temperature according to need of work; Need be pumped in ultrasonic pond 4 by peristaltic pump 7 and carry out ultrasonication by liquid raw material to be processed.
Angiotensin-converting enzyme (ACE) too much in human body is one of major reason caused elevation of the blood pressure.Therefore the present invention evaluates its hypotensive activity with the inhibiting rate of enzymolysis product to ACE.The mensuration of enzymolysis product ACE inhibitory activity is carried out according to the method for document " J. Jia; H. Ma; W. Zhao; The use of ultrasound for enzymatic preparation of ACE-inhibitory peptides from wheat germ protein; Food Chemistry. 119 (2010) 336-342. ", with hippuryl histidyl-leucine (Hippuryl-His-Leu, HHL) as the substrate of ACE catalysis, study the change of its absorbancy, utilize high effective liquid chromatography for measuring.
reference examples:
(1) prepare 2%(W/W) gluten protein suspension 500 mL, the 30 degrees Celsius of thermostat water baths putting into band rotor stir 30min, add 3800 U/g (E/S) Sumizyme MP and carry out enzymolysis, pH 9.0 is kept, temperature 50 C, enzymolysis 30 min in enzymolysis process, boiling water bath goes out enzyme 10min afterwards, centrifugal 10 min under 12000 r/min, collect supernatant liquor, measure ACE inhibitory activity after diluting 2 times.Through measuring, the ACE inhibiting rate of gluten protein enzymolysis product diluent is 64.957%.
(2) prepare 3%(W/W) gluten protein suspension 500 mL, the 20 degrees Celsius of thermostat water baths putting into band rotor stir 30min, add 3800 U/g (E/S) Sumizyme MP and carry out enzymolysis, pH 9.0 is kept, temperature 50 C, enzymolysis 30 min in enzymolysis process, boiling water bath goes out enzyme 10min afterwards, centrifugal 10 min under 12000 r/min, collect supernatant liquor, measure ACE inhibitory activity after diluting 3 times.Through measuring, the ACE inhibiting rate of gluten protein enzymolysis product diluent is 66.012%.
(3) gluten protein suspension 500 mL of 3% (W/W) is prepared, the 20 DEG C of thermostat water baths putting into band rotor stir 50min, add 3800 U/g (E/S) Sumizyme MP and carry out enzymolysis, pH 9.0 is kept, temperature 50 C, enzymolysis 30 min in enzymolysis process, boiling water bath goes out enzyme 10min afterwards, centrifugal 10 min under 12000 r/min, collect supernatant liquor, measure ACE inhibitory activity after diluting 3 times.Through measuring, the ACE inhibiting rate of gluten protein enzymolysis product diluent is 64.761%.
(4) gluten protein suspension 500 mL of 2% (W/W) is prepared, adopt single-frequency probe type ultrasonic process gluten protein suspension, ultrasonic initial temperature 30 DEG C, power 896 W/L, burst length 4 s, intermittent time 3 s, add 3800 U/g (E/S) Sumizyme MP after the ultrasonic end of total sonication time 30 min. and carry out enzymolysis, pH 9.0 is kept, temperature 50 C, enzymolysis time 30 min in enzymolysis process, boiling water bath goes out enzyme 10 min afterwards, centrifugal 10 min under 12000 r/min, collect supernatant liquor, measure ACE inhibitory activity after diluting 2 times.Through measuring, the ACE inhibiting rate of gluten protein enzymolysis product diluent is 70.770%.
(5) prepare gluten protein suspension 6 L of 2% (W/W), under churned mechanically condition, adopt frequency to be 20 kHz, 28kHz, 35kHz, the synchronizing ultrasound time is 10 s, the intermittent time is 5 s; Ultrasonic power is 50 W/L, and initial temperature is divergence expression synchronous Three-frequency ultrasonic process 30 min of 30 DEG C; Add Sumizyme MP with the ratio of 3800 U/g and carry out enzymolysis, the temperature of enzymolysis is 50 DEG C, and pH is 9.0, enzymolysis 30min; After enzymolysis terminates, enzymolysis solution is boiled 10 min and to go out enzyme, centrifugal 10 min under 12000 r/min, collect supernatant liquor, the extracting solution of protein blood pressure-reducing peptide can be obtained.ACE inhibitory activity is measured after gluten protein blood pressure lowering peptide extracting solution dilutes 2 times.Through measuring, the ACE inhibiting rate of gluten protein enzymolysis product diluent is 70.07%.
embodiment 1.gluten protein suspension 6 L of preparation 2%, under churned mechanically condition, adopt frequency to be 20 kHz, 28 kHz, 35 kHz, the triple-frequency ultrasonic time is 20s, 20s, 20s; Ultrasonic power is 16.67 W/L, and initial temperature is divergence expression ultrasonication 30 min of 30 degree; Add Sumizyme MP with the ratio of 3800 U/g and carry out enzymolysis, the temperature of enzymolysis is 50 DEG C, and pH is 9.0, enzymolysis 30min; After enzymolysis terminates, enzymolysis solution is boiled 10 min and to go out enzyme, centrifugal 10 min under 12000 r/min, collect supernatant liquor, the extracting solution of protein blood pressure-reducing peptide can be obtained.ACE inhibitory activity is measured after gluten protein blood pressure lowering peptide extracting solution dilutes 2 times.
Through measuring, have the ACE inhibiting rate of the enzymolysis product diluent of ultrasonication to be 73.80%, compared with contrast (1), hypotensive activity improves 13.62%.Hypotensive activity improves 4.29% compared with reference examples (4), and power density reduces 53.75 times.Hypotensive activity improves 5.32% compared with reference examples (5), and power density reduces 3 times.
embodiment 2.gluten protein suspension 6 L of preparation 3%, under churned mechanically condition, adopt frequency to be 20kHz, 28kHz, 35kHz, the triple-frequency ultrasonic time is 20s, 20s, 20s; Ultrasonic power is 16.67 W/L, and initial temperature is divergence expression ultrasonication 30 min of 20 degree; Add Sumizyme MP with the ratio of 3800 U/g and carry out enzymolysis, the temperature of enzymolysis is 50 DEG C, and pH is 9.0, enzymolysis 30 min; After enzymolysis terminates, enzymolysis solution is boiled 10 min and to go out enzyme, centrifugal 10 min under 12000 r/min, collect supernatant liquor, the extracting solution of protein blood pressure-reducing peptide can be obtained.ACE inhibitory activity is measured after gluten protein blood pressure lowering peptide extracting solution dilutes 3 times.
Through measuring, have the ACE inhibiting rate of the enzymolysis product diluent of ultrasonication to be 77.73%, hypotensive activity improves 17.75% compared with reference examples (2).
embodiment 3.gluten protein suspension 6 L of preparation 3%, under churned mechanically condition, adopt frequency to be 20 kHz, 28 kHz, 35 kHz, single-frequency ultrasonic time is 20s, 20s, 20s; Ultrasonic power is 16.67 W/L, and initial temperature is divergence expression ultrasonication 50 min of 20 DEG C; Add Sumizyme MP with the ratio of 3800 U/g and carry out enzymolysis, the temperature of enzymolysis is 50 DEG C, and pH is 9.0, enzymolysis 30 min; After enzymolysis terminates, enzymolysis solution is boiled 10 min and to go out enzyme, centrifugal 10 min under 12000 r/min, collect supernatant liquor, the extracting solution of protein blood pressure-reducing peptide can be obtained.ACE inhibitory activity is measured after gluten protein blood pressure lowering peptide extracting solution dilutes 3 times.
Through measuring, have the ACE inhibiting rate of the enzymolysis product diluent of ultrasonication to be 76.76%, hypotensive activity improves 18.53% compared with reference examples (3).
Claims (5)
1. based on a method for the ultrasound-enhanced enzyme-squash techniqued gluten protein blood pressure lowering peptide of order, it is characterized in that carrying out according to following step: gluten powder is mixed with the suspension that mass concentration is 3% (W/W) by (1), the process of applying order triple-frequency ultrasonic; (2) protease hydrolyzed is added: add Sumizyme MP and carry out enzymolysis, in enzymolysis process, keep PH 9.0, temperature 50 DEG C;
Enzymolysis time 30 min, go out at 100 DEG C enzyme 10 min, centrifugal 10 min under 12000 r/min, collects supernatant liquor, can obtain the extracting solution of protein blood pressure-reducing peptide.
2. a kind of method based on the ultrasound-enhanced enzyme-squash techniqued gluten protein blood pressure lowering peptide of order according to claim 1, is characterized in that wherein order triple-frequency ultrasonic frequency is: 20 KHz, 28 KHz, 35 KHz.
3. a kind of method based on the ultrasound-enhanced enzyme-squash techniqued gluten protein blood pressure lowering peptide of order according to claim 1, is characterized in that the triple-frequency ultrasonic time is respectively 20s, 20s, 20s.
4. a kind of method based on the ultrasound-enhanced enzyme-squash techniqued gluten protein blood pressure lowering peptide of order according to claim 1, is characterized in that ultrasonic power is 16.67 W/L, pretreatment time 10-60 min.
5. a kind of method based on the ultrasound-enhanced enzyme-squash techniqued gluten protein blood pressure lowering peptide of order according to claim 1, the ratio that to it is characterized in that according to albumen enzyme-to-substrate gluten powder be 3800 U/g adds Sumizyme MP and carries out enzymolysis.
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| CN201510048637.2A CN104846046B (en) | 2015-01-30 | 2015-01-30 | A kind of method that gluten protein blood pressure lowering peptide is prepared based on the ultrasound-enhanced enzymolysis of order |
| PCT/CN2016/071590 WO2016119629A1 (en) | 2015-01-30 | 2016-01-21 | Method for preparing functional polypeptide through multimode ultrasonic enhancing enzymolysis |
| US15/547,458 US20180023110A1 (en) | 2015-01-30 | 2016-01-21 | Method for preparing functional polypeptide through multimode ultrasonic enhancing enzymolysis |
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Cited By (5)
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| WO2016119629A1 (en) * | 2015-01-30 | 2016-08-04 | 江苏大学 | Method for preparing functional polypeptide through multimode ultrasonic enhancing enzymolysis |
| CN106119329A (en) * | 2016-07-08 | 2016-11-16 | 江苏大学 | A kind of synchronize the method that multiple frequency ultrasonic assisted immobilization enzyme enzymolysis prepares Semen Maydis polypeptide |
| CN106858342A (en) * | 2017-01-17 | 2017-06-20 | 江苏大学 | Wheat embryo zymolyte compound, preparation method and the purposes as health food |
| CN108785329A (en) * | 2017-04-27 | 2018-11-13 | 天津科技大学 | A kind of processing method of functionality leech powder |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016119629A1 (en) * | 2015-01-30 | 2016-08-04 | 江苏大学 | Method for preparing functional polypeptide through multimode ultrasonic enhancing enzymolysis |
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| CN105628644B (en) * | 2015-12-21 | 2019-08-27 | 江苏大学 | Device and method based on real time spectrum in situ on-line monitoring protease solution preocess |
| CN106119329A (en) * | 2016-07-08 | 2016-11-16 | 江苏大学 | A kind of synchronize the method that multiple frequency ultrasonic assisted immobilization enzyme enzymolysis prepares Semen Maydis polypeptide |
| CN106858342A (en) * | 2017-01-17 | 2017-06-20 | 江苏大学 | Wheat embryo zymolyte compound, preparation method and the purposes as health food |
| CN108785329A (en) * | 2017-04-27 | 2018-11-13 | 天津科技大学 | A kind of processing method of functionality leech powder |
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