WO2016119629A1 - 多模式超声波强化酶解制备功能多肽的方法 - Google Patents

多模式超声波强化酶解制备功能多肽的方法 Download PDF

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WO2016119629A1
WO2016119629A1 PCT/CN2016/071590 CN2016071590W WO2016119629A1 WO 2016119629 A1 WO2016119629 A1 WO 2016119629A1 CN 2016071590 W CN2016071590 W CN 2016071590W WO 2016119629 A1 WO2016119629 A1 WO 2016119629A1
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ultrasonic
frequency
enzymatic hydrolysis
preparing
khz
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PCT/CN2016/071590
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English (en)
French (fr)
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马海乐
任晓锋
周存山
王振斌
何荣海
曲文娟
张艳艳
毛舒云
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江苏大学
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Priority claimed from CN201510052239.8A external-priority patent/CN104846047A/zh
Priority claimed from CN201510048637.2A external-priority patent/CN104846046B/zh
Priority claimed from CN201510138570.1A external-priority patent/CN104774893A/zh
Priority claimed from CN201510138613.6A external-priority patent/CN104774894A/zh
Application filed by 江苏大学 filed Critical 江苏大学
Priority to US15/547,458 priority Critical patent/US20180023110A1/en
Publication of WO2016119629A1 publication Critical patent/WO2016119629A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J19/10—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing sonic or ultrasonic vibrations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

Definitions

  • the invention relates to the technical field of functional polypeptide preparation, in particular to a technique for preparing a blood pressure lowering peptide by using multi-mode ultrasonic pretreatment of gluten protein and corn protein raw material, and then using an enzymatic hydrolysis method.
  • wheat gluten protein As a by-product of wheat starch processing, wheat gluten protein has a protein content of 75-80% and a balanced amino acid composition. It is a nutrient-rich, inexpensive and high-protein polymer. China's annual production of wheat gluten is about 600,000 tons. It is currently used as a flour quality improver, feed binder, nutritional supplements, etc. in the food and feed industry. Recent studies have found that gluten is a highly promising source of plant protein for the preparation of bioactive peptides, and its enzymatically produced products can lower blood pressure by significantly inhibiting the activity of angiotensin-converting enzyme (ACE).
  • ACE angiotensin-converting enzyme
  • the solubility is poor, and the dispersibility is poor, which causes the gluten protein to not fully bind with the enzyme, but is fully enzymatically digested.
  • Corn gluten meal is rich in protein, and contains 15 kinds of inorganic salts and yellow pigment unique to corn.
  • the protein contained in it lacks essential amino acids such as lysine and tryptophan, and the finished product has a special taste and color. Therefore, it is mainly used in the domestic preparation of roughage, making nutritious food additives, or directly treating emissions, which greatly wastes resources.
  • hydrolysis of proteins into active peptides there have been many studies on the hydrolysis of proteins into active peptides, and there are many reports on the preparation of corn bioactive peptides by protease hydrolysis of zein.
  • ACE inhibitory peptides have been found in the enzymatic hydrolysates of zein, which are also commonly referred to as antihypertensive peptides.
  • these ACE inhibitory peptides can exhibit a good antihypertensive effect on patients with spontaneous hypertension. For those with normal blood pressure, there is no antihypertensive effect.
  • zein contains a large number of hydrophobic groups, and its solubility is poor.
  • proteases are not easily accessible to the enzyme cleavage site, the hydrolysis rate is slow, the enzymatic hydrolysis efficiency and product conversion rate are low, and the product quality is unstable. .
  • Ultrasonic waves are longitudinal waves with a frequency of more than 20 kHz with high frequency, small wavelength, and strong penetrating power.
  • the interaction between the ultrasonic wave and the medium mass produces a cavitation effect, and the cavitation effect produces high temperature and high pressure, and simultaneously
  • the shock wave generates a large pressure in a small local area, which may destroy the spatial structure of the protein, expose a large number of amino acids, and expose the enzyme cleavage site, thereby facilitating the later enzymatic hydrolysis, thereby increasing the degree of hydrolysis of the hydrolyzed product.
  • Mao Shuyun and other studies have shown that before the zein is subjected to alkaline protease enzymatic hydrolysis, it is subjected to ultrasonic pretreatment.
  • the hydrolysis degree of zeaxanthin powder is increased from 12.5% to 45.6% compared with conventional enzymatic hydrolysis; Du Shuangkui et al.
  • the alkaline hydrolysis enzyme was added for enzymatic hydrolysis, which could increase the hydrolysis degree of chickpeas from 20.03% to 25.04%.
  • the Ma Haile working group studied probe-type ultrasonic pretreatment of oat protein. It indicated that the degree of hydrolysis of oat protein in the treatment group was 15.8% higher than that in the conventional enzymatic hydrolysis group.
  • Ultrasonic pretreatment of protein substrates can improve the enzymatic properties of gluten protein raw materials and improve product activity by changing the secondary structure of proteins, the size of protein molecules, and the tightness of binding between molecules.
  • the sonication time and working mode directly affect the degree of change in protein structure, which is closely related to the improvement of its enzymatic properties and the improvement of biological activity.
  • the combined effect of simultaneous dual-frequency or multi-frequency ultrasound is significantly greater than the sum of the individual radiation effects of each frequency.
  • the dual-frequency ultrasonic combined radiation of a dual-frequency ultrasonic combination of 28 kHz and 1.7 MHz when the ultrasonic intensity of 1.7 MHz is greater than 3 W/cm 2 , the combined effect is about 1.5 times the sum of the individual effects; when 1.7 MHz is changed At 1.06MHz or 0.87MHz, the combined effect can be close to twice the sum of the individual effects.
  • the surface hydrophobicity of the protein was increased by 65.34%, 56.88% and the surface sulfhydryl content of the protein increased by 13.32% and 11.19% respectively after three-frequency sonication for 10 min and single-frequency sonication for 15 min.
  • the effect of dual-frequency ultrasound on the surface hydrophobicity and surface sulfhydryl groups is between single-frequency and triple-frequency sonication. This shows that tri-frequency ultrasound also has obvious advantages in protein modification.
  • the object of the present invention is to overcome the above deficiencies of the prior art and provide a multi-mode ultrasonic enhanced enzymatic hydrolysis preparation work.
  • the method of polypeptide can be aimed at increasing the degree of hydrolysis of the protein, increasing the blood pressure lowering activity of the enzymatic hydrolysate of the protein, shortening the enzymatic hydrolysis time, and reducing the energy consumption.
  • the above object of the present invention is achieved by the following technical means:
  • a method for preparing a functional polypeptide by multi-mode ultrasonic enzymatic hydrolysis, and a method for preparing a glutenin antihypertensive peptide by sequential ultrasonic enhanced enzymatic hydrolysis is carried out according to the following steps: (1) preparing gluten powder into a suspension having a mass concentration of 3% Liquid, applying sequential tri-frequency ultrasound for pretreatment; (2) enzymatic hydrolysis for 30 min at pH 9.0, temperature 50 ° C, substrate concentration 3%, alkaline protease plus enzyme amount 4560 U/g, and 0.2 after enzymatic hydrolysis. The pH was adjusted to 7.0 by mol/L HCl, and the enzyme was deactivated in a boiling water bath for 10 min. After cooling, the mixture was centrifuged at 10000 g for 10 min, and the supernatant was collected to obtain an extract of the protein hypotensive peptide.
  • the sequential three-frequency ultrasonic frequency is: 20kHz, 28kHz, 35kHz
  • the three-frequency ultrasonic time is 20s, 20s, 20s
  • the ultrasonic power per unit volume is 16.67W/L
  • the pretreatment time is 10-60min.
  • Scheme 2 The invention separately designs three interfaces of solid-liquid, liquid-liquid and gas-liquid to form ultrasonic reverberation fields of different forms, and the solid-liquid interface is formed by using an ultrasonic plate and a water body which are not working, and the liquid-liquid interface It is formed by vegetable oil and water, and the gas-liquid interface is formed by air and water.
  • a method for preparing a functional polypeptide by multi-mode ultrasonic enzymatic hydrolysis, and a method for preparing a zein blood pressure lowering peptide by ultrasonic resonance enzymatic hydrolysis of a frequency-reverberant reverberation field is carried out according to the following steps: (1) grading corn gluten into a mass concentration of 4.5% Suspension, pretreatment with reverberation field ultrasonic wave; (2) enzymatic hydrolysis for 60 min at pH 9.0, temperature 50 ° C, substrate concentration 4.5%, alkaline protease plus enzyme amount 5616 U/g, after enzymatic hydrolysis 0.2 mol/L HCl was adjusted to pH 7.0, the enzyme was deactivated in a boiling water bath for 10 min, cooled and centrifuged at 10000 g for 10 min, and the supernatant was collected to obtain an extract of the protein hypotensive peptide.
  • the sweep frequency is 22 ⁇ 2 kHz, 28 ⁇ 2 kHz, 33 ⁇ 2 kHz, 40 ⁇ 2 kHz or 68 ⁇ 2 kHz.
  • the sweeping ultrasonic wave forms a reverberant field of different reflective media through three interfaces of solid-liquid, liquid-liquid and gas-liquid, and the solid-liquid interface is formed by a metal plate and a water body, and the vegetable oil at the liquid-liquid interface is used.
  • the water body is formed, and the gas-liquid interface is formed by air and water.
  • the ultrasonic reverberation field is preferably a solid-liquid reflective medium; the frequency of the swept ultrasonic reverberation field is: (40 ⁇ 2) kHz.
  • the ultrasonic pretreatment conditions are as follows: pulsed ultrasonic working time 10s and intermittent time 3s, ultrasonic frequency sweeping period 500ms, unit volume ultrasonic power 120W/L, initial temperature 18°C, and pretreatment time 30min.
  • the gluten protein was pretreated with a countercurrent multi-mode ultrasound device to increase the blood pressure lowering activity of the gluten protein enzymatic hydrolysate and shorten the enzymatic hydrolysis time.
  • a multi-mode ultrasonic enhanced enzymatic hydrolysis method for preparing a functional polypeptide, and a method for preparing a glutenin lowering blood pressure peptide by countercurrent double-frequency ultrasonic intensification enzymatic hydrolysis is carried out according to the following steps: (1) using gluten protein at a margin of 0.003 mol/L The alkali solution is formulated into a 3% gluten protein suspension, and subjected to countercurrent dual-frequency ultrasonic wave for pretreatment; (2) at pH 9.0, temperature 50 ° C, alkaline protease Alcalase plus enzyme amount 4560 U / g, substrate Enzymatic hydrolysis for 30 min at a concentration of 10 g/L.
  • the working mode of the ultrasonic wave is sequential dual frequency and synchronous dual frequency, the frequency combination is 20-28, 20-35, 20-40 and 20-50 kHz, and the frequency combination is 20-35 kHz in sequential operation, and the sequential working time is 5s and 5s; the frequency combination is 20-28kHz during synchronous operation, the pulse time is 10s, and the intermittent time is 5s.
  • the ultrasonic pretreatment conditions are as follows: the ultrasonic power per unit volume is 200 W/L, the initial temperature is 30 ° C, and the pretreatment time is 15 min.
  • the ACE inhibitory activity of the enzymatic hydrolysate after ultrasonic treatment is studied by ultrasonic pretreatment of the raw material protein. After three-frequency ultrasonic pretreatment, the enzymatic hydrolysis effect is improved, the activity of the enzymatic hydrolyzate product is improved, and the energy consumption is reduced compared with the original ultrasonic treatment method.
  • the ACE inhibition rate activity of the enzymatic hydrolysate is increased by 13% to 20%. Energy consumption has been reduced by 3 to 53.75 times.
  • the second embodiment of the invention adopts a solid-liquid sweeping ultrasonic reverberation field to pretreat the zein powder suspension, and the ACE inhibitory activity of the enzymatic hydrolyzed product after ultrasonic treatment is studied, and the ACE inhibition rate activity of the enzymatic hydrolysate is improved by 44.9%. DH increased by 37.6%.
  • the activity of the enzymatic hydrolyzed product is improved, and it is found that in the ultrasonic reverberation field formed at the liquid-liquid interface, significant emulsification is likely to occur, so it is not possible in the test and actual production.
  • the formation of such interfaces has certain value for instrumental equipment design and industrial production.
  • the third aspect of the invention employs a countercurrent multimode ultrasound device for gluten. After sequential dual-frequency ultrasonic pretreatment, the enzymatic hydrolysis effect is improved and the activity of the enzymatic hydrolysate is improved. Compared with the traditional enzymatic hydrolysis method, the ACE inhibition rate activity of the enzymatic hydrolysate was increased by 16.4% to 25.4%, and the ACE inhibition rate activity of the enzymatic hydrolysate was increased by 6.0% to 13.7% compared with the countercurrent single-frequency ultrasound.
  • Figure 1 is a diagram showing the apparatus for sequential prefraction pretreatment of gluten by the use of the present invention.
  • Figure 2 is an ultrasonic reaction liquid-liquid transmission medium
  • Figure 3 is an ultrasonic reaction gas-liquid transmission medium
  • Figure 4 is an ultrasonic reaction solid-liquid transmission medium.
  • Figure 5 is a diagram showing the apparatus for pretreatment of gluten by countercurrent multimode dual frequency ultrasound used in the present invention.
  • the reaction efficiency of the entire reaction is measured by the degree of hydrolysis (DH), and the degree of hydrolysis (DH) is measured by the pH-stat method.
  • DH degree of hydrolysis
  • ACE angiotensin-converting enzyme
  • the present invention evaluates its blood pressure lowering activity by the inhibition rate of ACE by the enzymatic hydrolysate.
  • the ACE inhibitory activity of the enzymatic hydrolysate was determined according to the literature "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 The method was carried out by using Hippuryl-His-Leu (HHL) as a substrate for ACE catalysis, and the change in absorbance was studied by high performance liquid chromatography.
  • HHL Hippuryl-His-Leu
  • FIG. 1 is a device diagram of sequential three-frequency ultrasonic pretreatment of gluten protein used in the present invention, which is independently developed by Jiangsu University.
  • the ultrasonic generators 9, 10, 11 can emit ultrasonic waves of 20, 28, 35 kHz, and the power of a single ultrasonic generator is 100 w.
  • the ultrasonic vibration plate is placed vertically on the inner wall of the ultrasonic pool 4, and the ultrasonic vibration plates 1, 2, and 3 are respectively controlled by the ultrasonic generators 9, 10, and 11; after the ultrasonic parameters are set by the computer controller 8, the ultrasonic generators 9 and 10 are controlled. 11.
  • Example 1 Preparation of 2% gluten protein suspension 6L, under mechanical stirring conditions, using frequency of 20 kHz, 28 kHz, 35 kHz, three-frequency ultrasonic time is 20s, 20s, 20s; ultrasonic power is 16.67W / L,
  • the initial temperature was 30 degrees of divergent ultrasonic treatment for 30 min; the alkaline protease was added to the ratio of 4560 U/g for enzymatic hydrolysis, the temperature of enzymatic hydrolysis was 50 ° C, the pH was 9.0, and the hydrolysis was carried out for 30 min; after the end of enzymatic hydrolysis, the enzymatic hydrolysis was carried out.
  • the solution was boiled for 10 minutes to kill the enzyme, centrifuged at 10,000 g for 10 min, and the supernatant was collected to obtain an extract of the protein hypotensive peptide.
  • the glutenin antihypertensive peptide extract was diluted 2-fold to determine the ACE inhibitory activity.
  • the ACE inhibition rate of the digested product dilution solution with the sonication process was 73.80%, compared with the control 1- (1) Compared with the blood pressure lowering activity, the increase was 13.62%. Compared with the control (4), the blood pressure lowering activity was increased by 4.29%, and the power density was reduced by 53.75 times. Compared with the control (5), the blood pressure lowering activity was increased by 5.32%, and the power density was reduced by three times.
  • Example 2 Preparation of 3% gluten protein suspension 6L, under mechanical stirring conditions, using frequencies of 20 kHz, 28 kHz, 35 kHz, three-frequency ultrasonic time of 20 s, 20 s, 20 s; ultrasonic power of 16.67 W / L,
  • the initial temperature was 20 degrees of divergent ultrasonic treatment for 30 min; the alkaline protease was added to the ratio of 4560 U/g for enzymatic hydrolysis, the temperature of enzymatic hydrolysis was 50 ° C, the pH was 9.0, and the hydrolysis was 30 min; after the end of enzymatic hydrolysis, the enzymatic hydrolysis was carried out.
  • the solution was boiled for 10 minutes to kill the enzyme, centrifuged at 10,000 g for 10 min, and the supernatant was collected to obtain an extract of the protein hypotensive peptide.
  • the glutenin antihypertensive peptide extract was diluted 3 times and the ACE inhibitory activity was measured.
  • Example 3 Preparation of 3% gluten protein suspension 6L, under the condition of mechanical agitation, using frequency of 20 kHz, 28 kHz, 35 kHz, single frequency ultrasonic time is 20 s, 20 s, 20 s; ultrasonic power is 16.67 W / L,
  • the initial temperature was 20 ° C for 50 min of ultrasonic treatment; the alkaline protease was added to the ratio of 4560 U / g for enzymatic hydrolysis, the temperature of enzymatic hydrolysis was 50 ° C, pH was 9.0, enzymatic hydrolysis for 30 min; after enzymatic hydrolysis, enzymatic hydrolysis
  • the solution was boiled for 10 minutes to kill the enzyme, centrifuged at 10,000 g for 10 min, and the supernatant was collected to obtain an extract of the protein hypotensive peptide.
  • the glutenin antihypertensive peptide extract was diluted 3 times and the ACE inhibitory activity was measured.
  • the ACE inhibition rate of the digested product dilution with sonication was determined to be 76.76%, and the blood pressure lowering activity was improved by 18.53% compared with the control Example 1-(3).
  • the reverberation field is designed according to the present invention: the reverberation field, that is, the ultrasonic wave having a frequency of 20 kHz to 50 kHz, has a small absorption in a liquid medium such as an aqueous solution, and it is subjected to the inner wall and the liquid surface of the container in the sonochemical reactor. Multiple reflections, although the sound waves whose amplitudes are the same but whose phase changes randomly meet at a certain point in space, the total sound intensity is equal to the sum of the intensity of each column of sound waves, that is, there is no interference between them. According to the definition, when different interfaces exist, reflection occurs and a reverberation field is formed.
  • the invention separately designs solid-liquid, liquid-liquid and gas-liquid interfaces to form ultrasonic reverberation fields of different forms, and the solid-liquid interface is formed by using an inoperative ultrasonic plate and a water body, and the liquid-liquid interface is made of vegetable oil and The water body is formed, and the gas-liquid interface is formed by air and water.
  • Figure 2 is an ultrasonic reverberation field of a liquid-liquid transmission medium used in the present invention.
  • 1 is an ultrasonic reaction cell
  • 2 is an oil phase (formed from vegetable oil)
  • 4 is an aqueous phase (formed by distilled water)
  • a liquid-liquid transmission interface is formed at the contact faces of 2 and 4.
  • 5 is an ultrasonic generating plate, which can emit ultrasonic waves of frequencies of 22 ⁇ 2 kHz, 28 ⁇ 2 kHz, 33 ⁇ 2 kHz, 40 ⁇ 2 kHz, and 68 ⁇ 2 kHz.
  • the ultrasonic mode is swept ultrasonic, and the power is 600 W.
  • FIG. 3 is a sample bag placed in the middle of 2 and 4, and subjected to ultrasonic pretreatment in a liquid-liquid ultrasonic reverberation field.
  • Figure 3 is a gas-liquid transmission medium ultrasonic wave used in the present invention Reverberation field.
  • 1 is an ultrasonic reaction cell
  • 2 is a gas phase (formed by air)
  • 4 is an aqueous phase (formed by distilled water)
  • a gas-liquid transmission interface is formed at the contact faces of 2 and 4.
  • 5 is an ultrasonic generating plate, which can emit ultrasonic waves of frequencies of 22 ⁇ 2 kHz, 28 ⁇ 2 kHz, 33 ⁇ 2 kHz, 40 ⁇ 2 kHz, and 68 ⁇ 2 kHz.
  • the ultrasonic mode is swept ultrasonic, and the power is 600 W.
  • 3 is a sample bag placed in the middle of 2 and 4, and subjected to ultrasonic pretreatment in a gas-liquid ultrasonic reverberation field.
  • Figure 4 is an ultrasonic reverberation field of a solid-liquid transmission medium used in the present invention.
  • 1 is an ultrasonic reaction cell
  • 2 is a solid phase (formed by an inoperative ultrasonic plate)
  • 4 is an aqueous phase (formed by distilled water)
  • a solid-liquid transmission interface is formed at the contact faces of 2 and 4.
  • ultrasonic generating plate which can emit ultrasonic waves of frequencies of 22 ⁇ 2 kHz, 28 ⁇ 2 kHz, 33 ⁇ 2 kHz, 40 ⁇ 2 kHz, and 68 ⁇ 2 kHz.
  • the ultrasonic mode is swept ultrasonic, and the power is 600 W.
  • 3 is a sample bag placed in the middle of 2 and 4, and subjected to ultrasonic pretreatment in a solid-liquid ultrasonic reverberation field.
  • Comparative Example 2 Conventional enzymatic hydrolysis: 180 mL of a zein suspension having a concentration of 45 g/L was prepared, stirred at a temperature of 18 ° C for 30 min, and an alkaline protease was added for enzymatic hydrolysis.
  • the reaction conditions were: an enzyme amount of 5,616 U/ g ([E] / [S]), enzymatic hydrolysis time 60 min, enzymatic hydrolysis temperature 50 ° C, enzymatic hydrolysis pH 9.0.
  • the pH was adjusted to 7.0 using 0.2 mol/L HCL, and the enzyme was deactivated in a boiling water bath for 10 min, cooled and centrifuged at 10000 g for 10 min, and the supernatant was adjusted to a volume of 800 mL, and then diluted 5 times to determine the ACE inhibitory activity. .
  • the pH was controlled by the pH-stat method during the test and the DH value was determined.
  • the ACE inhibition rate of the corn proteolytic product dilution was determined to be 19.81%.
  • the DH is 7.52%.
  • Example 4 Designing an ultrasonic reverberant field of a solid-liquid transmission medium, the solid-liquid interface was formed using an inoperative ultrasonic plate and a water body.
  • the ultrasonic mode is sweep mode (40 ⁇ 2) kHz.
  • the treatment conditions are: initial temperature 18 °C, pulsed ultrasonic working time 10s and intermittent time. 3s, ultrasonic sweep cycle 500ms, ultrasonic treatment time 30min, unit volume ultrasonic power 120W / L.
  • the sonicated solution was directly hydrolyzed by alkaline protease.
  • the reaction conditions were: substrate concentration 4.5%, enzyme amount 5616 U/g ([E]/[S]), enzymatic hydrolysis time 60 min, enzymatic hydrolysis temperature 50 ° C, enzymatic hydrolysis pH 9.0.
  • the pH was adjusted to 7.0 using 0.2 mol/L HCL, and the enzyme was deactivated in a boiling water bath for 10 min, cooled and centrifuged at 10000 g for 10 min, and the supernatant was adjusted to a volume of 800 mL, and then diluted 5 times to determine the ACE inhibitory activity. .
  • the ACE inhibition rate of the enzymatic supernatant was 25.68%, and the DH was 8.81%. Compared with the control, the ACE inhibition rate was increased by 29.63% and the DH was increased by 15.8%.
  • Example 5 Design of an ultrasonic reverberant field of a gas-liquid transmission medium formed with air and water.
  • a corn gluten suspension of 180 g/L was prepared at a concentration of 45 g/L for ultrasonic pretreatment, and the ultrasonication process and the enzymatic hydrolysis process were the same as in the example (1).
  • the ACE inhibition rate of the enzymatic supernatant was 23.96%, and the DH was 9.24%. Compared with the control, the ACE inhibition rate was increased by 20.94% and the DH was increased by 21.4%.
  • Example 6 Designing an ultrasonic reverberant field of a liquid-liquid transmission medium, the liquid-liquid interface is formed with oil and water.
  • the concentration is A 45 g/L zein suspension of 180 mL was subjected to ultrasonic pretreatment, and the ultrasonication process and the enzymatic hydrolysis process were the same as those of the example (1).
  • the ACE inhibition rate of the enzymatic supernatant was 16.03%, and the DH was 7.41%. Compared with the control, the ACE inhibition rate decreased by 19.08%, and the DH decreased by 2.6%.
  • the ultrasonic reverberation field formed at the liquid-liquid interface significant emulsification is easy to occur, so the formation of such interfaces should be avoided in the test and actual production, which has certain reference value for instrument equipment design and industrial production.
  • Example 7 180 mL of a zein suspension at a concentration of 45 g/L was prepared and sonicated for 30 min in a solid-liquid swept ultrasonic reverberant field.
  • the ultrasonic frequency is 22 ⁇ 2kHz
  • the initial temperature is 18°C
  • the pulsed ultrasonic working time is 10s
  • the intermittent time is 3s
  • the sweeping period is 500ms
  • the unit volume ultrasonic power is 120W/L.
  • the alkaline protease was added for enzymatic hydrolysis, and the reaction conditions were the same as those of the comparative examples.
  • the pH was adjusted to 7.0 using 0.2 mol/L HCL, and the enzyme was deactivated in a boiling water bath for 10 min, cooled and centrifuged at 10000 g for 10 min, and the supernatant was adjusted to a volume of 800 mL, and then diluted 5 times to determine the ACE inhibitory activity. .
  • the pH was controlled by the pH-stat method during the test and the DH value was determined. Three tests in parallel.
  • the ACE inhibition rate of the corn proteolytic product dilution was determined to be 41.38% and the DH was 10.22%.
  • Example 8 The experimental procedure was the same as in Example 7, except that the sweep frequency was 28 ⁇ 2 kHz.
  • the ACE inhibition rate of the corn proteolytic product dilution was determined to be 22.25%.
  • the DH is 10.15%.
  • Example 9 The experimental procedure was the same as in Example 7, except that the sweep frequency was 33 ⁇ 2 kHz.
  • the ACE inhibition rate of the corn proteolytic product dilution was determined to be 29.94%.
  • DH was 9.91%.
  • Example 10 The experimental procedure was the same as in Example 7, except that the sweep frequency was 40 ⁇ 2 kHz.
  • the ACE inhibition rate of the corn proteolytic product dilution was determined to be 42.93%.
  • the DH is 10.35%.
  • Example 11 The experimental procedure was the same as in Example 7, except that the sweep frequency was 68 ⁇ 2 kHz.
  • the ACE inhibition rate of the corn proteolytic product dilution was determined to be 32.24%.
  • the DH was 9.87%.
  • 1 is a multi-mode ultrasonic intelligent control panel, which can control ultrasonic waves for single-frequency operation, dual-frequency sequential operation, and dual-frequency simultaneous operation.
  • 10 is an ultrasonic generator with a base frequency of 20 kHz.
  • 9 ultrasonic generators of 28, 35, 40, 50 kHz, respectively, the power of a single ultrasonic generator is 100w.
  • 2 is an ultrasonic probe with a base frequency of 20 kHz, and 3 is another combined probe.
  • 7 is the material inlet
  • 5 is the material liquid outlet.
  • 6 is the temperature control water inlet
  • 8 is the temperature control water outlet.
  • 11 is the temperature control system.
  • the experimental procedure was the same as that of Comparative Example 4, except that the single frequency was 28 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 69.3%.
  • the experimental procedure was the same as that of Comparative Example 4, except that the single frequency was 35 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 71.5%.
  • Comparative Example 7 40 kHz single-frequency ultrasonic pretreatment assisted enzymatic hydrolysis
  • the experimental procedure was the same as that of Comparative Example 4, except that the single frequency was 40 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 68.1%.
  • Comparative Example 8 50 kHz single-frequency ultrasonic pretreatment assisted enzymatic hydrolysis
  • the experimental procedure was the same as that of Comparative Example 4, except that the single frequency was 50 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 68.8%.
  • a 3% (w/w) gluten protein suspension 1.0L was prepared at 0.003 mol/L and pretreated with a countercurrent sequential dual-frequency ultrasound with a combined frequency of 20-28 kHz.
  • the sequence time is 5 s, 5 s; the ultrasonic power is 200 W/L, the initial temperature is 30 ° C, and the pretreatment time is 15 min.
  • alkaline protease was added at a ratio of 4560 U/g for enzymatic hydrolysis.
  • the temperature of enzymatic hydrolysis was 50 ° C, pH was 9.0, and enzymatic hydrolysis was carried out for 30 min.
  • the enzymatic hydrolysate was boiled for 10 min to kill the enzyme, 10000 g. After centrifugation for 10 min, the supernatant was collected to obtain an extract of the protein hypotensive peptide.
  • the ACE inhibitory activity was determined after 3 fold dilution.
  • the ACE inhibition rate of the digested product dilution was determined to be 75.80%. Compared with Control 1, the ACE inhibition rate was increased by 18.6%, and the ACE inhibition rate was increased by 8.0% and 9.4%, respectively, compared with Comparative Examples 4 and 5.
  • the experimental procedure is the same as in Example 12, except that the frequency combination of the countercurrent sequential dual-frequency ultrasound is 20-35 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 80.1%. Compared with Control 1, the ACE inhibition rate was increased by 25.4%, and the ACE inhibition rate was increased by 14.1% and 12.0%, respectively, compared with Comparative Examples 4 and 6.
  • the experimental procedure is the same as in Example 12, except that the frequency combination of the countercurrent sequential dual-frequency ultrasound is 20-40 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 75.6%. Compared with Control 1, the ACE inhibition rate was increased by 18.3%, and the ACE inhibition rate was increased by 7.7% and 11.0%, respectively, compared with Comparative Examples 4 and 7.
  • the experimental procedure is the same as in Example 12, except that the frequency combination of the countercurrent sequential dual-frequency ultrasound is 20-50 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 74.4%. Compared with Control 1, the ACE inhibition rate was increased by 16.4%, and the ACE inhibition rate was increased by 6.0% and 8.1%, respectively, compared with Comparative Examples 4 and 8.
  • a 3% (w/w) gluten protein suspension 1.0L was prepared at 0.003 mol/L and pretreated with a countercurrent synchronous dual-frequency ultrasound with a combined frequency of 20-28 kHz.
  • the pulse time is 10 s and the pause time is 5 s.
  • the single ultrasonic power is 100 W/L and the total ultrasonic power is 200 W/L.
  • the initial temperature was 30 ° C and the pretreatment time was 15 min.
  • alkaline protease was added at a ratio of 4560 U/g for enzymatic hydrolysis.
  • the temperature of enzymatic hydrolysis was 50 ° C, pH was 9.0, and enzymatic hydrolysis was carried out for 30 min.
  • the enzymatic hydrolysate was boiled for 10 min to kill the enzyme, 10000 g. After centrifugation for 10 min, the supernatant was collected to obtain an extract of the protein hypotensive peptide.
  • the ACE inhibitory activity was determined after 3 fold dilution.
  • the ACE inhibition rate of the digested product dilution was determined to be 78.8%. Compared with Control 1, the blood pressure lowering activity was increased by 23.3%, and the ACE inhibition rate was increased by 12.3% and 13.7%, respectively, compared with Comparative Examples 2 and 3.
  • the experimental procedure is the same as in Example 16, except that the frequency combination of the countercurrent synchronous dual-frequency ultrasound is 20-35 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 76.6%. Compared with Control 1, the blood pressure lowering activity was increased by 19.9%, and the ACE inhibition rate was increased by 9.1% and 7.1%, respectively, compared with Comparative Examples 4 and 6.
  • the experimental procedure is the same as in Example 16, except that the frequency combination of the countercurrent sequential dual frequency ultrasound is 20-40 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 75.1%. Compared with Control 1, the blood pressure lowering activity was increased by 17.5%, and the ACE inhibition rate was increased by 7.0% and 10.2%, respectively, compared with Comparative Examples 4 and 7.
  • the experimental procedure is the same as in Example 12, except that the frequency combination of the countercurrent sequential dual-frequency ultrasound is 20-50 kHz.
  • the ACE inhibition rate of the gluten-proteolytic product dilution was determined to be 74.5%. Compared with Control 1, the blood pressure lowering activity was increased by 16.6%, and the ACE inhibition rate was increased by 6.1% and 8.3%, respectively, compared with Comparative Examples 4 and 8.

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Abstract

多模式超声波强化酶解制备功能多肽的方法，涉及功能多肽制备技术领域。采用顺序超声强化酶解制备谷朊蛋白降血压肽的方法，超声波混响场制备玉米蛋白降血压肽的方法，以及采用逆流式多模式双频超声预处理谷朊蛋白解制备功能多肽的方法，旨在提高蛋白的水解度，提高蛋白的酶解产物的降血压活性，缩短酶解时间，并且降低能耗。采用固—液扫频超声波预处理玉米蛋白粉悬浮液，提高了酶解效率，改善了酶解效果。采用多模式超声波预处理谷朊蛋白悬浮液，提高了酶解效率。与传统酶解方法相比酶解液的ACE抑制率活性提高16.4％—25.4％，与逆流式单频超声相比酶解液的ACE抑制率活性提高6.0％—13.7％。

Description

多模式超声波强化酶解制备功能多肽的方法 技术领域：
本发明涉及功能多肽制备技术领域，特指采用多模式超声波预处理谷朊蛋白和玉米蛋白原料，再利用酶解方法制备降血压肽的一种技术。
背景技术：
我国农产品加工业中每年约有9000万吨蛋白类副产物产生，因变性程度高、溶解度低、营养品质差，不能被充分利用造成蛋白资源浪费严重。据统计我国每年年产油菜籽粕939万吨；玉米黄粉(玉米蛋白粉)100多万吨，豆粕约2252万吨，将上述低值蛋白质酶解成活性肽可以有效解决蛋白资源浪费严重的问题。
小麦谷朊蛋白作为小麦淀粉加工的副产物，其蛋白质含量高达75-80％，氨基酸组成均衡齐全，是营养丰富、物美价廉的植物性高蛋白聚合物。我国的小麦谷朊蛋白年产量大约60多万吨，目前主要作为面粉品质改良剂、饲料粘结剂、营养添加剂等用于食品和饲料工业。最新研究发现，谷朊蛋白作为一种非常有潜力的制备生物活性肽的植物蛋白源，其酶解产物可通过显著地抑制血管紧张素转换酶(ACE)的活性，而达到降低血压的目的。然而，由于谷朊蛋白疏水性氨基酸含量(主要含Pro、Gln、Leu、Ala含量较高)溶解性较差，分散性差，造成谷朊蛋白不能与酶充分的结合，而被充分酶解。
我国玉米产量居世界第二位，年产量约占世界的25％。玉米蛋白粉含有丰富的蛋白质，还含有15种无机盐以及玉米独有的黄色素，但由于其所含的蛋白质缺少赖氨酸，色氨酸等人体必需氨基酸，且成品具有特殊的味道和色泽，因此国内主要将其用于粗饲料制备，制作营养食品添加剂，或者直接处理排放，极大地浪费了资源。近年来，有关蛋白质水解成活性肽的研究较多，其中有关玉米蛋白经蛋白酶水解制备玉米生物活性肽的报道也很多。目前，从玉米蛋白的酶解产物中发现了大量的ACE抑制肽，通常也被称为降血压肽，试验表明这些ACE抑制肽对自发性高血压患者能够表现出很好的降压作用，而对血压正常者则无降压作用。但是玉米蛋白含有大量的疏水基团，溶解性较差，在酶解过程中会出现蛋白酶不易接近酶切位点，水解速度缓慢，酶解效率及产物的转化率低，产品质量不稳定等问题。
超声波是一种具有频率高、波长小、穿透力强等特性的，频率超过20kHz的纵波。超声波与媒介质之间相互作用，产生空化效应，空化效应会产生高温、高压，同时产生 激波，并在局部微小的区域产生很大的压强，从而有可能破坏蛋白的空间结构，使大量氨基酸暴露，酶切位点裸露，从而有利于后期酶解，进而提高酶解产物的水解度。毛淑云等研究表明在玉米醇溶蛋白进行碱性蛋白酶酶解前，对其加扫频超声波预处理，结果是相比于常规酶解，玉米黄粉的水解度由12.5％提高到了45.6％；杜双奎等研究利用超声波清洗机预处理鹰嘴豆后，加入碱性蛋白酶酶进行酶解，可以使鹰嘴豆的水解度从20.03％增加到25.04％；马海乐工作组研究探头式超声波预处理燕麦蛋白，结果表明，处理组燕麦蛋白的水解度比常规酶解组高了15.8％。
超声预处理蛋白底物可通过改变蛋白质的二级结构、蛋白分子颗粒大小，以及分子之间结合的紧密程度而改善谷朊蛋白原料的酶解特性，提高产品活性。然而，超声处理时间和工作模式直接影响到蛋白结构的变化程度，这种变化与其酶解特性的改善和生物活性的提高密切相关。
研究发现，双频或者多频超声组合福射对声化学反应产额的提高具有明显的增强效应，双频或者多频超声同时辐射的合效应均明显大于各频率超声单独辐射效应之和。例如，28kHz与1.7MHz组合的双频超声组合的双频超声组合辐射,当取1.7MHz超声强度大于3W/cm2时，其合效应约为单独效应之和的1.5倍；当1.7MHz改为1.06MHz或0.87MHz时，其合效应可接近单独效应之和的2倍。在双频正交辐射的甚础上加入一束低频率的超声构成三频正交辐照，可使其声化学产额得到更大的提高,三者合作用结果大约为三者分别辐照产额之和的1.4-1.8倍。《现代食品科技》(2014，30(3)：23-26)发表的“不同频率超声对鲢鱼肌原纤维蛋白结构的影响”一文，以鲢鱼为原料，采用单频、双频和三频超声分别处理鲢鱼肌原纤维蛋白，研究不同频率超声处理对肌原纤维蛋白表面疏水性和表面巯基含量的影响。发现与未超声的样品对比，经三频超声处理10min、单频超声处理15min后蛋白的表面疏水性分别提高了65.34％，56.88％，蛋白表面巯基含量分别提高了13.32％，11.19％。双频超声对蛋白表面疏水性和表面巯基的影响作用介于单频和三频超声处理的之间。这说明三频超声在蛋白质改性方面也就用明显的优势。
现有文献对多频超声的研究大多是以同步多频的工作模式下，利用多频超声波峰峰叠加的效应，提高超声波的处理效果。这种效果是在多个频率的超声波发生器同时工作同时发出超声波的情况下发生的，构成的合成波谱非常复杂，会随着处理时间的延长，产生不同程度的峰峰相加和峰峰相消。
发明内容：
本发明的目的是克服上述现有技术的不足，提供一种多模式超声波强化酶解制备功 能多肽的方法，旨在提高蛋白的水解度，提高蛋白的酶解产物的降血压活性，缩短酶解时间，并且降低能耗。开发一种具有良好疗效的，安全的，价格合理的食物蛋白源的降血压活性肽。本发明上述目的是通过以下技术手段实现的：
具体技术方案：
方案一：
多模式超声波强化酶解制备功能多肽的方法，采用顺序超声强化酶解制备谷朊蛋白降血压肽的方法，按照下述步骤进行：(1)将谷朊粉配制成质量浓度为3％的悬浮液，施加顺序三频超声进行预处理；(2)在pH 9.0、温度50℃、底物浓度3％、碱性蛋白酶加酶量4560U/g的条件下酶解30min，酶解结束后使用0.2mol/L HCl调节pH至7.0，沸水浴中灭酶10min，冷却后于10000g下离心10min，收集上清液，即可得到蛋白降血压肽的提取液。
其中顺序三频超声频率为：20kHz、28kHz、35kHz，三频超声时间分别为20s、20s、20s，单位体积超声功率为16.67W/L，预处理时间10-60min。
方案二：本发明分别设计固-液、液-液、气-液三种界面，形成不同形态的超声混响场，固-液界面用不工作的超声平板和水体来形成，液-液界面用植物油和水体来形成，气-液界面用空气和水体来形成。
多模式超声波强化酶解制备功能多肽的方法，采用扫频混响场超声波强化酶解制备玉米蛋白降血压肽的方法，按照下述步骤进行：(1)将玉米蛋白配成质量浓度4.5％的悬浮液，施加混响场超声波进行预处理；(2)在pH 9.0、温度50℃、底物浓度4.5％、碱性蛋白酶加酶量5616U/g的条件下酶解60min，酶解结束后使用0.2mol/L HCl调节pH至7.0，在沸水浴中灭酶10min，冷却后于10000g离心10min，收集上清液，即可得到蛋白降血压肽的提取液。
其中所述的扫频频率为22±2kHz、28±2kHz、33±2kHz、40±2kHz或68±2kHz。
其中所述的扫频超声波通过固-液、液-液、气-液三种界面形成不同反射介质的混响场，固-液界面用金属平板和水体来形成、液-液界面用植物油和水体来形成、气-液界面用空气和水体来形成。
其中所述的超声波混响场优选固-液反射介质；扫频超声波混响场频率为：(40±2)kHz。
其中所述的超声预处理条件为：脉冲超声的工作时间10s和间歇时间3s、超声扫频周期500ms、单位体积超声功率120W/L、初始温度18℃、预处理时间30min。
方案三：
采用逆流式多模式超声设备对谷朊蛋白进行预处理，旨在提高谷朊蛋白的酶解产物的降血压活性，缩短酶解时间。本发明上述目的是通过以下技术手段实现的：
多模式超声波强化酶解制备功能多肽的方法，采用逆流式双频超声强化酶解制备谷朊蛋白降血压肽的方法，按下列步骤进行：(1)将谷朊蛋白用0.003mol/L的稀碱溶液配制成质量浓度为3％的谷朊蛋白悬浮液，施加逆流式双频超声波进行预处理；(2)在pH 9.0、温度50℃、碱性蛋白酶Alcalase加酶量4560U/g、底物浓度10g/L的条件下酶解30min，酶解结束后使用0.2mol/L HCl调节pH至7.0，在沸水浴中灭酶10min，冷却后于10000g离心10min，收集上清液，即可得到蛋白降血压肽的提取液。
其中所述的超声波的工作模式为顺序双频和同步双频，频率组合为20-28、20-35、20-40和20-50kHz，顺序工作时频率组合为20-35kHz，顺序工作时间为5s和5s；同步工作时频率组合为20-28kHz，脉冲时间为10s，间歇时间为5s。
其中所述的超声预处理条件为：单位体积超声功率为200W/L、初始温度30℃、预处理时间15min。
本发明所具有的优点是：
本发明方案一采用超声预处理原料蛋白对超声作用后酶解产物的ACE抑制活性进行了研究。经过三频超声预处理后，改善了酶解效果，提高了酶解产物的活性，并且与原有的超声处理方式相比降低了能耗。酶解液的ACE抑制率活性提高13％～20％。能耗降低了3～53.75倍。
本发明方案二采用固-液扫频超声波混响场预处理玉米蛋白粉悬浮液，对超声作用后酶解产物的ACE抑制活性进行了研究，酶解液的ACE抑制率活性提高了44.9％，DH提高了37.6％。与其他类别的超声波预处理方式相比，提高了酶解产物的活性，发现在液-液界面形成的超声混响场中，极易发生显著的乳化现象，所以在试验及实际生产中不可以形成此类界面，这对于仪器装备设计及工业生产有一定价值。
本发明方案三采用逆流式多模式超声设备对谷朊蛋白。经过顺序双频超声预处理后，改善了酶解效果，提高了酶解产物的活性。与传统酶解方法相比酶解液的ACE抑制率活性提高16.4％～25.4％，与逆流式单频超声相比酶解液的ACE抑制率活性提高6.0％～13.7％。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明：
图1为本发明使用的顺序三频超声预处理谷朊蛋白的设备图。
图2为超声波反应液‐液透射介质；
图3为超声波反应气‐液透射介质；
图4为超声波反应固‐液透射介质。
图5为本发明使用的逆流式多模式双频超声预处理谷朊蛋白的设备图。
具体实施方式
本发明中以水解度(DH)来衡量整个反应的反应效率，水解度(DH)的测定采用pH-stat法。人体中过多的血管紧张素转化酶(ACE)是引起血压升高的重要原因之一。因此本发明以酶解产物对ACE的抑制率评价其降血压活性。酶解产物ACE抑制活性的测定按照文献“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.”的方法进行，以马尿酰组氨酰亮氨酸(Hippuryl-His-Leu,HHL)作为ACE催化的底物，研究其吸光度的变化，利用高效液相色谱法测定。
方案一：图1为本发明使用的顺序三频超声预处理谷朊蛋白的设备图，为江苏大学自主研制。超声发生器9、10、11能发出20、28、35kHz的超声波，单台超声波发生器功率为100w。超声池4中内壁竖直放置超声波振板，通过超声波发生器9、10、11分别控制超声波振板1、2、3；电脑控制器8设定超声波各参数后，控制超声波发生器9、10、11，发出符合要求的超声波；5为本发明中设备的恒温水浴池，通过温度计6实时监控温度，并根据工作需要调节介质温度；将需要处理的液原料通过蠕动泵7泵入超声池4中进行超声波处理。
对照例1：
(1)配制2％(W/W)的谷朊蛋白悬浮液500mL，放入带转子的30摄氏度恒温水浴锅中搅拌30min，加入4560U/g(E/S)碱性蛋白酶进行酶解，酶解过程中保持pH 9.0，温度50℃，酶解30min，之后沸水浴灭酶10min，10000g下离心10min，收集上清液，稀释2倍后测定ACE抑制活性。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为 64.957％。
(2)配制3％(W/W)的谷朊蛋白悬浮液500mL，放入带转子的20摄氏度恒温水浴锅中搅拌30min，加入4560U/g(E/S)碱性蛋白酶进行酶解，酶解过程中保持pH 9.0，温度50℃，酶解30min，之后沸水浴灭酶10min，10000g下离心10min，收集上清液，稀释3倍后测定ACE抑制活性。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为66.012％。
(3)配制3％(W/W)的谷朊蛋白悬浮液500mL，放入带转子的20℃恒温水浴锅中搅拌50min，加入4560U/g(E/S)碱性蛋白酶进行酶解，酶解过程中保持pH 9.0，温度50℃，酶解30min，之后沸水浴灭酶10min，10000g下离心10min，收集上清液，稀释3倍后测定ACE抑制活性。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为64.761％。
(4)配制2％(W/W)的谷朊蛋白悬浮液500mL，采用单频探头式超声波处理谷朊蛋白悬浮液，超声初始温度30℃，功率896W/L，脉冲时间4s，间歇时间3s，超声总时间30min.超声结束后加入4560U/g(E/S)碱性蛋白酶进行酶解，酶解过程中保持pH 9.0，温度50℃，酶解时间30min，之后沸水浴灭酶10min，10000g下离心10min，收集上清液，稀释2倍后测定ACE抑制活性。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为70.770％。
(5)配制2％(W/W)的谷朊蛋白悬浮液6L，在机械搅拌的条件下，采用频率为20kHz、28kHz、35kHz，同步超声时间为10s、间歇时间为5s；超声功率为50W/L，初始温度为30℃的发散式同步三频超声波处理30min；以4560U/g的比例加入碱性蛋白酶进行酶解，酶解的温度为50℃，pH为9.0，酶解30min；酶解结束后，将酶解液煮沸10min灭酶，10000g下离心10min，收集上清液，即可得到蛋白降血压肽的提取液。谷朊蛋白降血压肽提取液稀释2倍后测定ACE抑制活性。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为70.07％。
实施例1.配制2％的谷朊蛋白悬浮液6L，在机械搅拌的条件下，采用频率为20kHz、28kHz、35kHz，三频超声时间为20s、20s、20s；超声功率为16.67W/L，初始温度为30度的发散式超声波处理30min；以4560U/g的比例加入碱性蛋白酶进行酶解，酶解的温度为50℃，pH为9.0，酶解30min；酶解结束后，将酶解液煮沸10min灭酶，10000g下离心10min，收集上清液，即可得到蛋白降血压肽的提取液。谷朊蛋白降血压肽提取液稀释2倍后测定ACE抑制活性。
经过测定，有超声处理过程的酶解产物稀释液的ACE抑制率为73.80％，与对照1- (1)相比，降血压活性提高13.62％。与对照例(4)相比降血压活性提高4.29％，功率密度缩小53.75倍。与对照例(5)相比降血压活性提高5.32％，功率密度缩小3倍。
实施例2.配制3％的谷朊蛋白悬浮液6L，在机械搅拌的条件下，采用频率为20kHz、28kHz、35kHz，三频超声时间为20s、20s、20s；超声功率为16.67W/L，初始温度为20度的发散式超声波处理30min；以4560U/g的比例加入碱性蛋白酶进行酶解，酶解的温度为50℃，pH为9.0，酶解30min；酶解结束后，将酶解液煮沸10min灭酶，10000g下离心10min，收集上清液，即可得到蛋白降血压肽的提取液。谷朊蛋白降血压肽提取液稀释3倍后测定ACE抑制活性。
经过测定，有超声处理过程的酶解产物稀释液的ACE抑制率为77.73％，与对照例1-(2)相比降血压活性提高了17.75％。
实施例3.配制3％的谷朊蛋白悬浮液6L，在机械搅拌的条件下，采用频率为20kHz、28kHz、35kHz，单频超声时间为20s、20s、20s；超声功率为16.67W/L，初始温度为20℃的发散式超声波处理50min；以4560U/g的比例加入碱性蛋白酶进行酶解，酶解的温度为50℃，pH为9.0，酶解30min；酶解结束后，将酶解液煮沸10min灭酶，10000g下离心10min，收集上清液，即可得到蛋白降血压肽的提取液。谷朊蛋白降血压肽提取液稀释3倍后测定ACE抑制活性。
经过测定，有超声处理过程的酶解产物稀释液的ACE抑制率为76.76％，与对照例1-(3)相比降血压活性提高了18.53％。
方案二：本发明设计混响场：混响场即对于频率为20kHz～50kHz的超声波，在水溶液一类的液体媒质中的吸收小，它在声化学反应器中要经历在容器内壁、液面的多次反射，这些频率虽然相同但相位无规变化的声波在空间某点相遇迭加的合成声场，其总的声强等于各列声波强度之和，即它们之间不发生干涉。根据定义得知，不同界面存在时，会发生反射，并形成混响场。本发明分别设计固-液、液-液、气-液三种界面，形成不同形态的超声混响场，固-液界面用不工作的超声平板和水体来形成，液-液界面用植物油和水体来形成，气-液界面用空气和水体来形成。
图2为本发明中使用的液-液透射介质超声波混响场。1为超声反应池，2为油相(由植物油形成)，4为水相(由蒸馏水形成)，在2与4的接触面形成液-液透射介面。5为超声波发生板，能发出22±2kHz、28±2kHz、33±2kHz、40±2kHz、68±2kHz频率的超声波，超声波模式为扫频超声，功率为600W。3为样品袋，置于2与4的中间区域，在液-液超声波混响场中进行超声波预处理。图3为本发明中使用的气-液透射介质超声波 混响场。1为超声反应池，2为气相(由空气形成)，4为水相(由蒸馏水形成)，在2与4的接触面形成气-液透射介面。5为超声波发生板，能发出22±2kHz、28±2kHz、33±2kHz、40±2kHz、68±2kHz频率的超声波，超声波模式为扫频超声，功率为600W。3为样品袋，置于2与4的中间区域，在气-液超声波混响场中进行超声波预处理。图4为本发明中使用的固-液透射介质超声波混响场。1为超声反应池，2为固相(由不工作的超声平板形成)，4为水相(由蒸馏水形成)，在2与4的接触面形成固-液透射介面。5为超声波发生板，能发出22±2kHz、28±2kHz、33±2kHz、40±2kHz、68±2kHz频率的超声波，超声波模式为扫频超声，功率为600W。3为样品袋，置于2与4的中间区域，在固-液超声波混响场中进行超声波预处理。
对照例2：常规酶解：配制浓度为45g/L的玉米蛋白悬浮液180mL，在温度为18℃的条件下搅拌30min，加入碱性蛋白酶进行酶解反应，反应条件为：加酶量5616U/g([E]/[S])、酶解时间60min、酶解温度50℃、酶解pH 9.0。酶解结束后，使用0.2mol/L HCL调节pH至7.0，并在沸水浴中灭酶10min，冷却后于10000g离心10min，取上清液定容至800mL，再稀释5倍，测定ACE抑制活性。试验过程中采用pH-stat法控制pH，并测定DH值。平行三次试验，经过测定，玉米蛋白酶解产物稀释液的ACE抑制率为19.81％。DH为7.52％。
实施例4：设计固-液透射介质的超声混响场，固-液界面用不工作的超声平板和水体来形成。配制浓度为45g/L的玉米蛋白悬浮液180mL，进行超声预处理,超声工作模式为扫频模式(40±2)kHz，处理条件为：初始温度18℃、脉冲超声的工作时间10s和间歇时间3s、超声扫频周期500ms、超声处理时间30min、单位体积超声功率120W/L。经超声处理后的料液用碱性蛋白酶直接进行水解反应，反应条件为：底物浓度4.5％，加酶量5616U/g([E]/[S])、酶解时间60min、酶解温度50℃、酶解pH 9.0。酶解结束后，使用0.2mol/L HCL调节pH至7.0，并在沸水浴中灭酶10min，冷却后于10000g离心10min，取上清液定容至800mL，再稀释5倍，测定ACE抑制活性。酶解上清液的ACE抑制率为25.68％，DH为8.81％。对照例相比，ACE抑制率提高了29.63％，DH提高了15.8％。
实施例5：设计气-液透射介质的超声混响场，气-液界面用空气和水体来形成。配制浓度为45g/L的玉米蛋白悬浮液180mL，进行超声预处理,超声过程及酶解过程同实例(1)。酶解上清液的ACE抑制率为23.96％，DH为9.24％。对照例相比，ACE抑制率提高了20.94％，DH提高了21.4％。
实施例6：设计液-液透射介质的超声混响场，液-液界面用油和水体来形成。配制浓度为 45g/L的玉米蛋白悬浮液180mL，进行超声预处理，超声过程及酶解过程同实例(1)酶解上清液的ACE抑制率为16.03％，DH为7.41％。与对照例相比，ACE抑制率下降了19.08％，DH下降了2.6％。在液-液界面形成的超声混响场中，极易发生显著的乳化现象，所以在试验及实际生产中应避免形成此类界面，这对于仪器装备设计及工业生产有一定的参考价值。
实施例7：配制浓度为45g/L的玉米蛋白悬浮液180mL，在固-液扫频超声波混响场中超声30min。超声频率为22±2kHz，初始温度18℃，脉冲超声的工作时间10s，间歇时间3s，扫频周期500ms，单位体积超声功率120W/L。加入碱性蛋白酶进行酶解反应，反应条件同对照例。酶解结束后，使用0.2mol/L HCL调节pH至7.0，并在沸水浴中灭酶10min，冷却后于10000g离心10min，取上清液定容至800mL，再稀释5倍，测定ACE抑制活性。试验过程中采用pH-stat法控制pH，并测定DH值。平行三次试验。经过测定，玉米蛋白酶解产物稀释液的ACE抑制率为41.38％，DH为10.22％。
实施例8：实验过程同实例7，其不同为扫频频率为28±2kHz。经过测定，玉米蛋白酶解产物稀释液的ACE抑制率为22.25％。DH为10.15％。
实施例9：实验过程同实例7，其不同为扫频频率为33±2kHz。经过测定，玉米蛋白酶解产物稀释液的ACE抑制率为29.94％。DH为9.91％。
实施例10：实验过程同实例7，其不同为扫频频率为40±2kHz。经过测定，玉米蛋白酶解产物稀释液的ACE抑制率为42.93％。DH为10.35％。
实施例11：实验过程同实例7，其不同为扫频频率为68±2kHz。经过测定，玉米蛋白酶解产物稀释液的ACE抑制率为32.24％。DH为9.87％。
方案三：图5中，1为多模式超声波智能控制面板，能控制超声波进行单频工作、双频顺序工作、双频同时工作。10为基础频率20kHz的超声波发生器。9分别为28、35、40、50kHz的超声波发生器，单台超声波发生器功率为100w。2为基础频率20kHz的超声波探头，3为另一组合探头。7为物料入口，5为料液出口。6为控温水入口，8为控温水出口。11为控温系统。
对照例3：传统酶解
用0.003mol/L稀碱溶液配制成3％(W/W)的谷朊蛋白悬浮液1.0L，放入带转子的30摄氏度恒温水浴锅中搅拌15min，加入4560U/g(E/S)碱性蛋白酶进行酶解，酶解过程中保持pH 9.0，温度50℃，酶解30min，之后沸水浴灭酶10min，10000g下离心10min， 收集上清液，稀释3倍后测定ACE抑制活性。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为63.9％。
对照例4：20kHz单频超声预处理辅助酶解
用0.003mol/L稀碱溶液配制成3％(W/W)的谷朊蛋白悬浮液1.0L，采用20kHz单频探头式超声波处理谷朊蛋白悬浮液，超声初始温度30℃，功率200W/L，脉冲时间10s，间歇时间5s，预处理时间15min。超声结束后加入4560U/g(E/S)碱性蛋白酶进行酶解，酶解过程中保持pH 9.0，温度50℃，酶解时间30min，之后沸水浴灭酶10min，10000g下离心10min，收集上清液，稀释3倍后测定ACE抑制活性。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为70.2％。
对照例5：28kHz单频超声预处理辅助酶解
实验过程同对照例4，其不同为单频频率为28kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为69.3％。
对照例6：35kHz单频超声预处理辅助酶解
实验过程同对照例4，其不同为单频频率为35kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为71.5％。
对照例7：40kHz单频超声预处理辅助酶解
实验过程同对照例4，其不同为单频频率为40kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为68.1％。
对照例8：50kHz单频超声预处理辅助酶解
实验过程同对照例4，其不同为单频频率为50kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为68.8％。
实施例12：
用0.003mol/L配制3％(W/W)的谷朊蛋白悬浮液1.0L，采用组合频率为20-28kHz的逆流式顺序双频超声对其进行预处理。顺序时间为5s，5s；超声功率为200W/L，初始温度为30℃，预处理时间为15min。超声预处理后，以4560U/g的比例加入碱性蛋白酶进行酶解，酶解的温度为50℃，pH为9.0，酶解30min；酶解结束后，将酶解液煮沸10min灭酶，10000g下离心10min，收集上清液，即可得到蛋白降血压肽的提取液。稀释3倍后测定ACE抑制活性。
经过测定，酶解产物稀释液的ACE抑制率为75.80％。与对照1相比，ACE抑制率提高18.6％，与对照例4、5相比ACE抑制率分别提高了8.0％、9.4％。
实施例13：
实验过程同实例12，其不同为逆流式顺序双频超声波的频率组合为20-35kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为80.1％。与对照1相比，ACE抑制率提高25.4％，与对照例4、6相比ACE抑制率分别提高了14.1％、12.0％。
实施例14：
实验过程同实例12，其不同为逆流式顺序双频超声波的频率组合为20-40kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为75.6％。与对照1相比，ACE抑制率提高18.3％，与对照例4、7相比ACE抑制率分别提高了7.7％、11.0％。
实施例15：
实验过程同实例12，其不同为逆流式顺序双频超声波的频率组合为20-50kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为74.4％。与对照1相比，ACE抑制率提高16.4％，与对照例4、8相比ACE抑制率分别提高了6.0％、8.1％。
实施例16：
用0.003mol/L配制3％(W/W)的谷朊蛋白悬浮液1.0L，采用组合频率为20-28kHz的逆流式同步双频超声对其进行预处理。脉冲时间为10s,间歇时间为5s。单个超声功率为100W/L，总的超声功率为200W/L。初始温度为30℃，预处理时间为15min。超声预处理后，以4560U/g的比例加入碱性蛋白酶进行酶解，酶解的温度为50℃，pH为9.0，酶解30min；酶解结束后，将酶解液煮沸10min灭酶，10000g下离心10min，收集上清液，即可得到蛋白降血压肽的提取液。稀释3倍后测定ACE抑制活性。
经过测定，酶解产物稀释液的ACE抑制率为78.8％。与对照1相比，降血压活性提高23.3％，与对照例2、3相比ACE抑制率分别提高了12.3％、13.7％。
实施例17：
实验过程同实例16，其不同为逆流式同步双频超声波的频率组合为20-35kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为76.6％。与对照1相比，降血压活性提高19.9％，与对照例4、6相比ACE抑制率分别提高了9.1％、7.1％。
实施例18：
实验过程同实例16，其不同为逆流式顺序双频超声波的频率组合为20-40kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为75.1％。与对照1相比，降血压活性提高17.5％，与对照例4、7相比ACE抑制率分别提高了7.0％、10.2％。
实施例19：
实验过程同实例12，其不同为逆流式顺序双频超声波的频率组合为20-50kHz。经过测定，谷朊蛋白酶解产物稀释液的ACE抑制率为74.5％。与对照1相比，降血压活性提高16.6％，与对照例4、8相比ACE抑制率分别提高了6.1％、8.3％。

Claims (10)

  1. 多模式超声波强化酶解制备功能多肽的方法，采用顺序超声强化酶解制备谷朊蛋白降血压肽的方法，其特征在于按照下述步骤进行：(1)将谷朊粉配制成质量浓度为3％的悬浮液，施加顺序三频超声进行预处理；(2)在pH 9.0、温度50℃、底物浓度3％、碱性蛋白酶加酶量4560U/g的条件下酶解30min，酶解结束后使用0.2mol/L HCl调节pH至7.0，沸水浴中灭酶10min，冷却后于10000g下离心10min，收集上清液，即可得到蛋白降血压肽的提取液。
  2. 根据权利要求1所述的多模式超声波强化酶解制备功能多肽的方法，其特征在于其中顺序三频超声频率为：20kHz、28kHz、35kHz，三频超声时间分别为20s、20s、20s，单位体积超声功率为16.67W/L，预处理时间10-60min。
  3. 多模式超声波强化酶解制备功能多肽的方法，采用扫频混响场超声波强化酶解制备玉米蛋白降血压肽的方法，其特征在于按照下述步骤进行：(1)将玉米蛋白配成质量浓度4.5％的悬浮液，施加混响场超声波进行预处理；(2)在pH 9.0、温度50℃、底物浓度4.5％、碱性蛋白酶加酶量5616U/g的条件下酶解60min，酶解结束后使用0.2mol/L HCl调节pH至7.0，在沸水浴中灭酶10min，冷却后于10000g离心10min，收集上清液，即可得到蛋白降血压肽的提取液。
  4. 根据权利要求3所述的多模式超声波强化酶解制备功能多肽的方法，其特征在于扫频频率为22±2kHz、28±2kHz、33±2kHz、40±2kHz或68±2kHz。
  5. 根据权利要求3所述的多模式超声波强化酶解制备功能多肽的方法，其特征在于扫频超声波通过固-液、液-液、气-液三种界面形成不同反射介质的混响场，固-液界面用金属平板和水体来形成、液-液界面用植物油和水体来形成、气-液界面用空气和水体来形成。
  6. 根据权利要求3所述的多模式超声波强化酶解制备功能多肽的方法，其特征在于超声波混响场优选固-液反射介质；扫频超声波混响场频率为：(40±2)kHz。
  7. 根据权利要求3所述的多模式超声波强化酶解制备功能多肽的方法，其特征在于超声预处理条件为：脉冲超声的工作时间10s和间歇时间3s、超声扫频周期500ms、单位体积超声功率120W/L、初始温度18℃、预处理时间30min。
  8. 多模式超声波强化酶解制备功能多肽的方法，采用逆流式双频超声强化酶解制备谷朊蛋白降血压肽的方法，其特征在于按下列步骤进行：(1)将谷朊蛋白用0.003mol/L的稀碱溶液配制成质量浓度为3％的谷朊蛋白悬浮液，施加逆流式双频超声波进行预处 理；(2)在pH 9.0、温度50℃、碱性蛋白酶Alcalase加酶量4560U/g、底物浓度10g/L的条件下酶解30min，酶解结束后使用0.2mol/L HCl调节pH至7.0，在沸水浴中灭酶10min，冷却后于10000g离心10min，收集上清液，即可得到蛋白降血压肽的提取液。
  9. 根据权利要求8所述的多模式超声波强化酶解制备功能多肽的方法，其特征在于超声波的工作模式为顺序双频和同步双频，频率组合为20-28、20-35、20-40和20-50kHz，顺序工作时频率组合为20-35kHz，顺序工作时间为5s和5s；同步工作时频率组合为20-28kHz，脉冲时间为10s，间歇时间为5s。
  10. 根据权利要求8所述的多模式超声波强化酶解制备功能多肽的方法，其特征在于超声预处理条件为：单位体积超声功率为200W/L、初始温度30℃、预处理时间15min。
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