AU2021105025A4 - Method for efficiently preparing food-derived ace inhibitory peptides by dual-frequency ultrasonic coupling enzymolysis - Google Patents
Method for efficiently preparing food-derived ace inhibitory peptides by dual-frequency ultrasonic coupling enzymolysis Download PDFInfo
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- 101000984728 Chiropsoides quadrigatus Angiotensin-converting enzyme inhibitory peptide Proteins 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 27
- 235000013305 food Nutrition 0.000 title claims abstract description 22
- 230000008878 coupling Effects 0.000 title claims abstract description 15
- 238000010168 coupling process Methods 0.000 title claims abstract description 15
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 15
- 229920002494 Zein Polymers 0.000 claims abstract description 117
- 239000005019 zein Substances 0.000 claims abstract description 117
- 229940093612 zein Drugs 0.000 claims abstract description 117
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 27
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 26
- 238000010408 sweeping Methods 0.000 claims abstract description 24
- 239000004365 Protease Substances 0.000 claims abstract description 17
- 108091005804 Peptidases Proteins 0.000 claims abstract description 12
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract 5
- 239000000725 suspension Substances 0.000 claims description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 238000000108 ultra-filtration Methods 0.000 claims description 26
- 238000009210 therapy by ultrasound Methods 0.000 claims description 25
- 239000000413 hydrolysate Substances 0.000 claims description 24
- 239000000843 powder Substances 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 239000012153 distilled water Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- 108091005658 Basic proteases Proteins 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 229940088598 enzyme Drugs 0.000 claims description 13
- 230000009977 dual effect Effects 0.000 claims description 10
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 8
- 235000019419 proteases Nutrition 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 108090000526 Papain Proteins 0.000 claims description 5
- 102000057297 Pepsin A Human genes 0.000 claims description 5
- 108090000284 Pepsin A Proteins 0.000 claims description 5
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 229940055729 papain Drugs 0.000 claims description 5
- 235000019834 papain Nutrition 0.000 claims description 5
- 229940111202 pepsin Drugs 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 229920006395 saturated elastomer Polymers 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 36
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- 230000002401 inhibitory effect Effects 0.000 abstract description 26
- 238000002360 preparation method Methods 0.000 abstract description 14
- 230000003276 anti-hypertensive effect Effects 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000036541 health Effects 0.000 abstract description 3
- 108010055615 Zein Proteins 0.000 description 112
- 230000007062 hydrolysis Effects 0.000 description 27
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 102000005158 Subtilisins Human genes 0.000 description 14
- 108010056079 Subtilisins Proteins 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
- 238000004506 ultrasonic cleaning Methods 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 9
- 102000035195 Peptidases Human genes 0.000 description 7
- 240000008042 Zea mays Species 0.000 description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 7
- 235000005822 corn Nutrition 0.000 description 7
- 238000009835 boiling Methods 0.000 description 6
- 230000003301 hydrolyzing effect Effects 0.000 description 6
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- 108010068370 Glutens Proteins 0.000 description 5
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- 239000012460 protein solution Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- ZDLZKMDMBBMJLI-FDMDGMSGSA-N 2-[[2-[[(2s)-2-[[(e)-3-(furan-2-yl)prop-2-enoyl]amino]-3-phenylpropanoyl]amino]acetyl]amino]acetic acid Chemical compound C([C@@H](C(=O)NCC(=O)NCC(=O)O)NC(=O)\C=C\C=1OC=CC=1)C1=CC=CC=C1 ZDLZKMDMBBMJLI-FDMDGMSGSA-N 0.000 description 1
- 108010048632 2-furanacryloyl-phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 102000004881 Angiotensinogen Human genes 0.000 description 1
- 108090001067 Angiotensinogen Proteins 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004531 blood pressure lowering effect Effects 0.000 description 1
- 235000021245 dietary protein Nutrition 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000003754 machining Methods 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000029865 regulation of blood pressure Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P9/12—Antihypertensives
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- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses a method for efficiently preparing food-derived ACE inhibitory peptides
by dual-frequency ultrasonic coupling enzymolysis, which belongs to the technical field of
preparing a food-derived ACE inhibitory peptide. The method of the invention for preparing
foodbome ACE inhibitory peptide by sweeping ultrasound coupled enzyme-hydrolysis is that
zein is processed by sweeping ultrasound, and then the protease is hydrolyzed. The final
prepared food-bome ACE inhibitory peptide had a high ACE inhibitory activity, up to 81.77%;
Compared with traditional enzymatic hydrolysis, ACE inhibitory peptide activity increased by
42.35% , and compared with traditional ultrasound-assisted enzymatic hydrolysis, ACE
inhibitory peptide activity increased by 26.20% . Therefore, the foodbome ACE inhibitory
peptide prepared by the method of the invention has a wide application prospect in the
preparation of drugs, health products, food or preparation with antihypertensive effect.
Description
[0001] 1.Technical Field
[0002] The invention belongs to the technical field of preparing food-borne ACE inhibitory peptides, in particular to a method for preparing food-bome ACE inhibitory peptides by dual frequency ultrasonic coupling enzymolysis.
[0003] 2. Description of Related Art
[0004] Angiotensinogen converting enzyme inhibitory peptide (ACE inhibitory peptide) is a key peptide for blood pressure regulation. Most of the clinically used ACE inhibitory peptides are chemically synthesized. The extraction of ACE inhibitory peptides from food proteins to reduce the side effects of chemically synthesized ACE inhibitory peptides has attracted more and more attention of researchers. Compared with the existing chemical synthesis of ACE inhibitory peptides, food-derived ACE inhibitory peptides have the advantages of mild blood pressure lowering effect, safety and no side effect, and no effect on normal blood pressure, etc. It is an ideal natural blood pressure lowering functional component .Zein contains Pro and Leu, and Pro content is high, so it is a good source of ACE inhibitory peptides.
[0005] The traditional enzymolysis method has some problems such as low product conversion rate, long enzymolysis time and low product activity. The intrinsic properties of zein structure hinder the release of functional ACE inhibitory peptides, reduce the utilization of zein. It is urgent to develop more effective technologies to solve this problem. Therefore, if ACE inhibitory peptides can be efficiently released from Zein, it is of great theoretical and practical significance to improve the comprehensive value of Zein.
[0006] Many studies have shown that ultrasound can significantly promote enzymatic hydrolysis. Swept frequency ultrasound is a high efficient ultrasonic machining technology developed in recent years. The frequency of the ultrasonic wave fluctuates up and down in a certain scanning period within a set range near the central frequency. Previous studies have found that compared with the constant frequency of ultrasound, scan ultrasound can stimulate the protein solution to produce resonance frequency matching its natural frequency, to achieve better protein pretreatment effect. For example, Ren et al. (Ren X , Ma H , Mao S , et al. Effects of sweeping frequency ultrasound treatment on enzymatic preparations of ACE-inhibitory peptides from zein[J]. European Food Research & Technology, 2014, 238(3):435-442.) The zein hydrolysate prepared by 40 2 kHz scan ultrasound (SFU) has 42.9% ACE inhibitory activity, it increased by 116.7% compared with the control. Mao Shuyun et al. (Mao Shuyun, Ma Haile, Ren Xiaofeng, etc. . Effect of scanning ultrasound treatment on enzymatic hydrolysis of zein
[ J ] . Food Industry Science and technology, 2013,34(014) : 142-147,155.) Using dual frequency sweep mode (40 2) kHz/(68 2) kHz in combination with enzymatic hydrolysis, the ACE inhibition rate of zein was 48.48% .At present, although the treatment of zein with scanning ultrasound or dual-frequency scanning ultrasound combined with enzymatic hydrolysis has significantly improved the ACE inhibition rate in the preparation of ACE inhibitory peptide, the inhibition rate is still at a low level. In order to promote the research value of zein in the preparation of blood pressure drugs and health care products, Methods for efficient release of ACE inhibitory peptides from zein are urgently needed.
[0007] In view of the problems existing in the prior art, the invention aims to provide a method for preparing a food-derived ACE inhibitory peptide by sweeping ultrasound coupled with enzymatic hydrolysis.
[0008] In order to achieve the above purposes, the invention adopts the following technical scheme:
[0009] The invention relates to a method for preparing a food-derived ACE inhibitory peptide by sweeping ultrasound coupled with enzymatic hydrolysis, which comprises the following steps: (1) Dispersing zein into distilled water to form zein suspension; (2) Performing sweeping ultrasonic treatment on the zein suspension in step (1); (3) Adding protease to the zein suspension after sweeping ultrasonic treatment in step (2) , and adjusting the pH value to the appropriate pH of the protease; Adjust the temperature of enzymatic hydrolysis to 40-50°C for enzymatic hydrolysis, and the reaction tends to stop when the reaction is saturated, pH will be adjusted to neutral, killing enzyme;
(4) the hydrolysate of step (3) is cooled to room temperature and centrifuged to precipitate, then the supernatant is ultrafiltrated by the ultrafiltration membrane with a molecular retention of 5 kda; (5) a freeze-dried powder of hydrolysate rich in ACE inhibitory peptide is prepared after vacuum freeze-drying the material liquid rich in ACE inhibitory peptide obtained in step (4)
[0010] In one embodiment, the zein suspension in step (1) is obtained by adding the zein to distilled water in a ratio of 1:25(g/mL) and stirring sufficiently to uniformly disperse the zein.
[0011] Dual-frequency ultrasound processing frequency is one of 25/33 khz, 25/40 khz, 25/59 khz, 33/40 khz, 33/59 khz, 40/59 khz. The two frequencies cycle repeatedly, and the frequency sweep cycle period is 4min.
[0012] The protease in the step (3) is one of trypsin, papain, alkaline protease and pepsin, and the optimum pH for enzymatic hydrolysis is 8.0,6.0,9.0 and 1.8, respectively Alkaline protease is preferred.
[0013] In several embodiments, the amount of protease added in step (3) is 2800 to 3600 U/G Zein.
[0014] In several specific embodiments, the step (3) has an optimal enzymolysis time of 40 to 60 minutes and the enzymolysis reaction stops when it approaches saturation.
[0015] In order to carry out the enzymolysis reaction better, the enzymolysis in step (3) can match the stirring speed of 600-700 r/min.
[0016] In one embodiment, the centrifugation in step (4) is 3000r/min for 15min, and the centrifugation temperature is room temperature.
[0017] In one embodiment, the vacuum freeze drying in the step (5) is precooled at an ultra-low temperature of-80 °C for 4 hours and further vacuum freeze drying for 3 days.
[0018] Compared with the traditional enzymolysis method, the activity of ACE inhibitory peptides prepared by the above-mentioned method increased by 42.35% , and reached 81.77%
. Compared with traditional ultrasound-assisted enzyme-lysis method, the inhibitory activity of ACE was increased by 26.20%. Therefore, the food-derived ACE inhibitory peptides prepared by this method have a broad application prospect in the preparation of drugs, health products, foods or preparations with antihypertensive effect.
[0019] Compared with the prior art, the invention has the following advantages and beneficial effects: The invention makes full use of the advantages of sweep ultrasound, which can stimulate protein solutions to produce resonance frequencies that match their natural frequencies, thus achieving better protein pretreatment and dispersion effect. At the same time, Zein is soluble in alkaline solution and is hydrolyzed into soluble polypeptide by alkaline protease under alkaline condition. In addition, sweeping ultrasound can accelerate the change of the internal structure of protein, exposing the active part of the protein and increasing the binding site of alcalase, in the course of enzymolysis, the binding of protein to enzyme and the release of ACE inhibitory peptide were promoted, and the activity of ACE inhibitory peptide was increased remarkably, up to 81.77% . Compared with the traditional enzymatic hydrolysis, the activity of ACE inhibitory peptide was increased by 42.35% , and compared with the traditional ultrasound-assisted enzymatic hydrolysis, the activity of ACE inhibitory peptide was increased by 26.20% .It effectively solved the problems of low extraction rate of food-borne ACE inhibitory peptide and low ACE inhibitory activity.
[0020] The technical proposal of the invention has important theoretical and practical significance for developing novel food-borne antihypertensive peptides and improving the high value utilization of zein; The obtained hydrolysate has definite antihypertensive effect and better market prospect.
[0021] In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the accompanying drawings required in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention, and a person of ordinary skill in the art can still derive other drawings according to these accompanying drawings without creative efforts.
[0022] Fig. 1 effect of different proteases on degree of hydrolysis of zein;
[0023] Fig. 2 effect of different proteases on ACE inhibitory activity of zein hydrolysate
[0024] Fig. 3 effect of substrate concentration on hydrolysis degree of zein;
[0025] Fig. 4 effect of single-frequency ultrasound frequency on hydrolysis degree of zein;
[0026] Fig. 5 effect of dual-frequency ultrasound on ACE inhibitory activity of zein hydrolysate
[0027] Fig. 6 effect of three-frequency and four-frequency ultrasound treatment on ACE inhibitory activity of zein hydrolysate
[0028] Fig. 7 electron micrograph of the effect of different ultrasonic treatment conditions on protein structure.
[0029] The terms used in the present invention, unless otherwise specified, generally have a meaning commonly understood by ordinary technical personnel in the field.
[0030] The present invention is described in further detail in conjunction with a specific embodiment and with reference to data. The following embodiments are intended to illustrate the invention only and do not in any way limit the scope of the invention.
[0031] Used in the following embodiments and test methods: Zein, Purchased from Yifa Biotechnology Co. Ltd. , Cas No. : 9010-66-6; Trypsin, From Solebo NO. T8150; Papain, G8430; Pepsin, From BBI Life Scirnces 9001-75-6; Alkaline protease, Bought from Sorrebo No. 1008S022.
Implementation Case 1
[0032] The invention relates to a method for efficiently preparing food-derived ACE inhibitory peptides by dual-frequency ultrasonic coupling enzymolysis, which comprises the following steps:
[0033] (1) Configuration of zein suspension: 4g Zein was dissolved in100mL distilled water, and was uniformly dispersed by stirring to obtain zein powder suspension.
[0034] (2) Sweeping ultrasonic treatment: setting ultrasonic temperature 25 °C, placing zein suspension in sweeping ultrasonic cleaning machine, treating zein suspension at 25/33 kHz, the ultrasonic time is 30 min, the cycle time of sweep frequency is 4 min.
[0035] (3) Enzymolysis: adding alcalase to the sonicated suspension of zein, the addition amount of zein was 2800U/g, the activity of alcalase was 20000u/g, and the reaction was stirred at a speed of 700r/min in a water bath at 50°C. The sample solution was titrated with 0.1 mol/L NaOH to keep the pH value at 9.0 and the reaction was stopped after 60 min of enzymatic hydrolysis. When the pH value was adjusted to 7.0 and the enzyme was eliminated by boiling water bath for 10mmin, the hydrolysate with high ACE inhibitory activity was obtained. The degree of hydrolysis was 25.8%.
[0036] (4) The hydrolysate is cooled to room temperature, the centrifugal temperature is set to room temperature, the centrifugal temperature is 3000r/min for 15min, and the supernatant is retained. Then the ultrafiltration membrane with molecular interception of 5kDa was used for ultrafiltration, the ultrafiltration time was lh, and the permeation liquid was collected, and the feed containing ACE inhibitory peptide was obtained. The activity of ACE inhibitory peptide was 78.1 %.
[0037] (5) Preparation of freeze-dried powder of hydrolysates rich in ACE inhibitory peptides: The material liquid rich in ACE inhibitory peptide obtained by ultrafiltration was precooled at °C for 4 hours.Further vacuum freeze drying for 3 days was performed to obtain lyophilized hydrolytic powder rich in ACE inhibitory peptide.
Implementation Case 2
[0038] The invention relates to a method for efficiently preparing food-derived ACE inhibitory peptides by dual-frequency ultrasonic coupling enzymolysis, which comprises the following steps:
[0039] (1)Configuration of zein suspension: 4g Zein was dissolved in 100mL distilled water, and was uniformly dispersed by stirring to obtain zein powder suspension.
[0040] (2) Sweeping ultrasonic treatment: setting ultrasonic temperature 25 °C, placing zein suspension in sweeping ultrasonic cleaning machine, treating zein suspension at 25/40kHz, the ultrasonic time is 30 min, the cycle time of sweep frequency is 4 min.
[0041] (3) Enzymolysis: adding alcalase to the sonicated suspension of zein, the addition amount of zein was 3000U/g, the activity of alcalase was 20000u/g, and the reaction was stirred at a speed of 680r/min in a water bath at 48°C. The sample solution was titrated with 0.1 mol/L NaOH to keep the pH value at 9.0 and the reaction was stopped after 55 min of enzymatic hydrolysis. When the pH value was adjusted to 7.0 and the enzyme was eliminated by boiling water bath for 10mmin, the hydrolysate with high ACE inhibitory activity was obtained. The degree of hydrolysis was 25.6%.
[0042] (4) The hydrolysate is cooled to room temperature, the centrifugal temperature is set to room temperature, the centrifugal temperature is 3000r/min for 15min, and the supernatant is retained. Then the ultrafiltration membrane with molecular interception of 5kDa was used for ultrafiltration, the ultrafiltration time was lh, and the permeation liquid was collected, and the feed containing ACE inhibitory peptide was obtained. The activity of ACE inhibitory peptide was 80.11%.
[0043] (5) Preparation of freeze-dried powder of hydrolysates rich in ACE inhibitory peptides: The material liquid rich in ACE inhibitory peptide obtained by ultrafiltration was precooled at °C for 4 hours.Further vacuum freeze drying for 3 days was performed to obtain lyophilized hydrolytic powder rich in ACE inhibitory peptide.
Implementation Case 3
[0044] The invention relates to a method for efficiently preparing food-derived ACE inhibitory peptides by dual-frequency ultrasonic coupling enzymolysis, which comprises the following steps:
[0045] (1) Configuration of zein suspension: 4g Zein was dissolved in100mL distilled water, and was uniformly dispersed by stirring to obtain zein powder suspension.
[0046] (2) Sweeping ultrasonic treatment: setting ultrasonic temperature 25 °C, placing zein suspension in sweeping ultrasonic cleaning machine, treating zein suspension at 33/40kHz, the ultrasonic time is 30 min, the cycle time of sweep frequency is 4 min.
[0047] (3) Enzymolysis: adding alcalase to the sonicated suspension of zein, the addition amount of zein was 3200U/g, the activity of alcalase was 20000u/g, and the reaction was stirred at a speed of 660r/min in a water bath at 46°C. The sample solution was titrated with 0.1 mol/L NaOH to keep the pH value at 9.0 and the reaction was stopped after 50min of enzymatic hydrolysis. When the pH value was adjusted to 7.0 and the enzyme was eliminated by boiling water bath for 10mmin, the hydrolysate with high ACE inhibitory activity was obtained. The degree of hydrolysis was 25.22%.
[0048] (4) The hydrolysate is cooled to room temperature, the centrifugal temperature is set to room temperature, the centrifugal temperature is 30OOr/min for 15min, and the supernatant is retained. Then the ultrafiltration membrane with molecular interception of 5kDa was used for ultrafiltration, the ultrafiltration time was lh, and the permeation liquid was collected, and the feed containing ACE inhibitory peptide was obtained. The activity of ACE inhibitory peptide was 79.56%.
[0049] (5) Preparation of freeze-dried powder of hydrolysates rich in ACE inhibitory peptides: The material liquid rich in ACE inhibitory peptide obtained by ultrafiltration was precooled at °C for 4 hours.Further vacuum freeze drying for 3 days was performed to obtain lyophilized hydrolytic powder rich in ACE inhibitory peptide.
Implementation Case 4
[0050] The invention relates to a method for efficiently preparing food-derived ACE inhibitory peptides by dual-frequency ultrasonic coupling enzymolysis, which comprises the following steps:
[0051] (1)Configuration of zein suspension: 4g Zein was dissolved in 100mL distilled water, and was uniformly dispersed by stirring to obtain zein powder suspension.
[0052] (2) Sweeping ultrasonic treatment: setting ultrasonic temperature 25 °C, placing zein suspension in sweeping ultrasonic cleaning machine, treating zein suspension at 33/59kHz, the ultrasonic time is 30 min, the cycle time of sweep frequency is 4 min.
[0053] (3) Enzymolysis: adding alcalase to the sonicated suspension of zein, the addition amount of zein was 3400U/g, the activity of alcalase was 20000u/g, and the reaction was stirred at a speed of 640r/min in a water bath at 44°C. The sample solution was titrated with 0.1 mol/L NaOH to keep the pH value at 9.0 and the reaction was stopped after 45min of enzymatic hydrolysis. When the pH value was adjusted to 7.0 and the enzyme was eliminated by boiling water bath for 10mmin, the hydrolysate with high ACE inhibitory activity was obtained. The degree of hydrolysis was 24.3%.
[0054] (4) The hydrolysate is cooled to room temperature, the centrifugal temperature is set to room temperature, the centrifugal temperature is 30OOr/min for 15min, and the supernatant is retained. Then the ultrafiltration membrane with molecular interception of 5kDa was used for ultrafiltration, the ultrafiltration time was lh, and the permeation liquid was collected, and the feed containing ACE inhibitory peptide was obtained. The activity of ACE inhibitory peptide was 78.75%.
[0055] (5) Preparation of freeze-dried powder of hydrolysates rich in ACE inhibitory peptides: The material liquid rich in ACE inhibitory peptide obtained by ultrafiltration was precooled at °C for 4 hours.Further vacuum freeze drying for 3 days was performed to obtain lyophilized hydrolytic powder rich in ACE inhibitory peptide.
Implementation Case 5
[0056] The invention relates to a method for efficiently preparing food-derived ACE inhibitory peptides by dual-frequency ultrasonic coupling enzymolysis, which comprises the following steps:
[0057] (1)Configuration of zein suspension: 4g Zein was dissolved in OOmL distilled water, and was uniformly dispersed by stirring to obtain zein powder suspension.
[0058] (2) Sweeping ultrasonic treatment: setting ultrasonic temperature 25 °C, placing zein suspension in sweeping ultrasonic cleaning machine, treating zein suspension at 40/59kHz, the ultrasonic time is 30 min, the cycle time of sweep frequency is 4 min.
[0059] (3) Enzymolysis: adding alcalase to the sonicated suspension of zein, the addition amount of zein was 3600U/g, the activity of alcalase was 20000u/g, and the reaction was stirred at a speed of 620r/min in a water bath at 42°C. The sample solution was titrated with 0.1 mol/L NaOH to keep the pH value at 9.0 and the reaction was stopped after 40min of enzymatic hydrolysis. When the pH value was adjusted to 7.0 and the enzyme was eliminated by boiling water bath for 10mmin, the hydrolysate with high ACE inhibitory activity was obtained. The degree of hydrolysis was 24.3%.
[0060] (4) The hydrolysate is cooled to room temperature, the centrifugal temperature is set to room temperature, the centrifugal temperature is 30OOr/min for 15min, and the supernatant is retained. Then the ultrafiltration membrane with molecular interception of 5kDa was used for ultrafiltration, the ultrafiltration time was lh, and the permeation liquid was collected, and the feed containing ACE inhibitory peptide was obtained. The activity of ACE inhibitory peptide was 78.36%.
[0061] (5) Preparation of freeze-dried powder of hydrolysates rich in ACE inhibitory peptides: The material liquid rich in ACE inhibitory peptide obtained by ultrafiltration was precooled at °C for 4 hours.Further vacuum freeze drying for 3 days was performed to obtain lyophilized hydrolytic powder rich in ACE inhibitory peptide.
Implementation Case 6
[0062] The invention relates to a method for efficiently preparing food-derived ACE inhibitory peptides by dual-frequency ultrasonic coupling enzymolysis, which comprises the following steps:
[0063] (1)Configuration of zein suspension: 4g Zein was dissolved in 100mL distilled water, and was uniformly dispersed by stirring to obtain zein powder suspension.
[0064] (2) Sweeping ultrasonic treatment: setting ultrasonic temperature 25 °C, placing zein suspension in sweeping ultrasonic cleaning machine, treating zein suspension at 25/40kHz, the ultrasonic time is 30 min, the cycle time of sweep frequency is 4 min.
[0065] (3) Enzymolysis: adding alcalase to the sonicated suspension of zein, the addition amount of zein was 2800U/g, the activity of alcalase was 20000u/g, and the reaction was stirred at a speed of 700r/min in a water bath at 45°C. The sample solution was titrated with 0.1 mol/L NaOH to keep the pH value at 9.0 and the reaction was stopped after 60min of enzymatic hydrolysis. When the pH value was adjusted to 7.0 and the enzyme was eliminated by boiling water bath for 10mmin, the hydrolysate with high ACE inhibitory activity was obtained. The degree of hydrolysis was 25.93%.
[0066] (4) The hydrolysate is cooled to room temperature, the centrifugal temperature is set to room temperature, the centrifugal temperature is 30OOr/min for 15min, and the supernatant is retained. Then the ultrafiltration membrane with molecular interception of 5kDa was used for ultrafiltration, the ultrafiltration time was lh, and the permeation liquid was collected, and the feed containing ACE inhibitory peptide was obtained. The activity of ACE inhibitory peptide was 81.77%.
[0067] (5) Preparation of freeze-dried powder of hydrolysates rich in ACE inhibitory peptides: The material liquid rich in ACE inhibitory peptide obtained by ultrafiltration was precooled at °C for 4 hours. Further vacuum freeze drying for 3 days was performed to obtain lyophilized hydrolytic powder rich in ACE inhibitory peptide.
Effects of different proteases on hydrolysis degree and ACE inhibitory activity of Zein
[0068] The suspension of corn gluten meal was prepared by dissolving 4g zein in100ML distilled water, stirring it to disperse it evenly, and then under the condition of single frequency kHz, 25 °C, 30min after ultrasonic treatment, Adding the same amount of trypsin, papain, alkaline protease and pepsin (3200U/g zein),the optimum pH of the enzyme was 8.0 for trypsin, 6.0 for Papain, 9.0 for alcalase and 1.8 for pepsin, The hydrolysis temperature was 41°C, the hydrolysis time was 60min, and the speed was 640 r/min. Ph-start method and FAPGG substrate method were used to determine the hydrolysis degree (Figure 1) and ACE inhibitory activity (Figure 2) of zein hydrolysates.The results showed that the hydrolysis degree and ACE inhibitory activity of alkaline protease were significantly higher than those of the other three enzymes. Therefore, alkaline protease is the best enzyme in the preparation of ACE inhibitory peptides from zein.
The effect of substrate concentration on the degree of hydrolysis of zein
[0069] Zein suspensions with different concentrations (g/mL: 1%, 2%, 3%, 4%) were prepared by dissoluble zein in distilled water in different amounts. Under the condition of single frequency 40kHz ultrasound, ultrasonic treatment was performed at 25°C for 30min. After the completion of ultrasonic treatment, alkaline protease was added to perform enzymatic hydrolysis of zein suspensions with different concentrations. The added amount was 3200U/g zein, the pH was 9.0, the hydrolysis temperature was 40°C, the hydrolysis time was 60min, and the rotation speed was 600 r/min. Finally, by measuring the hydrolysis degree of zein hydrolysate (as shown in FIG. 3), it can be seen that the hydrolysis degree of 3% (3:1OOg/mL) and 4% (1:25g/mL) is significantly higher than other substrate concentrations, and 4% (1:25g/mL) is the optimal hydrolysis degree, which can be used as the optimal substrate concentration of zein suspension.
Effects of sweeping ultrasound on hydrolysis degree and ACE inhibitory activity of Zein
[0070] (1)single frequency ultrasonic pretreatment
[0071] The corn gluten meal suspension was prepared by dissolving 4g zein in100ML distilled water, stirring it thoroughly to disperse it evenly, and setting the ultrasonic temperature at 25 °C, the suspension of corn protein powder was treated with ultrasound at 25 kHz, 33 kHz, 40 kHz and 59 kHz respectively. The temperature of ultrasound was 25 °C, the time of ultrasound was 30 min, and the suspension of corn protein powder without ultrasound treatment was used as control group After ultrasonic treatment, an alkaline protease was added to the above-mentioned samples in the proportion of 2800U/G Zein, the pH was 9.0, the temperature was 45 °C, the time was min, the speed was 700R/min, by measuring the degree of hydrolysis of zein hydrolysate (fig. 4) ,the degree of hydrolysis of samples treated with 25kHz and 40kHz ultrasonic frequencies was significantly higher than that of other samples.
[0072] 2) Dual-frequency ultrasonic pretreatment
[0073] 4g zein was dissolved in 1OOmL distilled water, and fully stirred to disperse it evenly to obtain suspension of corn gluten meal. The ultrasonic temperature was set at 25 °C, and the zein suspension was placed in a frequency sweep ultrasonic cleaning machine. The zein suspension was pretreated at six dual-frequency ultrasonic frequencies of 25/33 kHz, 25/40 kHz, 25/59 kHz, 33/40 kHz, 33/59 kHz and 40/59 kHz, respectively. The ultrasonic time was 30min, the two frequencies were repeated, and the frequency sweep cycle was 4min. At the same time, non ultrasonic pretreatment and single-frequency ultrasonic pretreatment (40kHz) were used as double control.
[0074] After ultrasonic treatment, alkaline protease was added to the above samples for enzymatic hydrolysis according to the proportion of 2800U/g zein. The pH was adjusted to 9.0, the hydrolysis temperature was 45°C, the hydrolysis time was 60min, and the rotation speed was 700 r/min. The ACE inhibition rate was determined, as shown in Figure 5.
[0075] The ACE inhibitory activities of zein hydrolysates with different dual-frequency ultrasound enzyme-hydrolysates were measured respectively (Figure 5). The results showed that the ACE inhibitory activities of zein hydrolysates with single-frequency and dual-frequency ultrasound pretreatment were significantly higher than that of zein hydrolysates without ultrasonic pretreatment. The ACE inhibitory activity of zein hydrolysates pretreated with dual frequency ultrasound at 25/33 kHz, 25/40 kHz, 33/40 kHz, 33/59 kHz and 40/59 kHz was significantly higher than that of zein hydrolysates pretreated with single-frequency ultrasound and 25/59 kHz. The inhibitory activity of ACE was 75.6% ~ 81.77%.
[0076] 3) Three-frequency and four-frequency ultrasonic pretreatment
[0077] 4g zein was dissolved in 100mL distilled water, and fully stirred to disperse it evenly to obtain suspension of corn gluten meal. The ultrasonic temperature was set at 25 °C, and the zein suspension was placed in a frequency sweeping ultrasonic cleaning machine. The ultrasonic cleaning machine was set at 25/33/40 kHz, 25/33/59 kHz, 25/40/59 kHz, 25/40/59 kHz, Zein suspension was pretreated with four three-frequency ultrasonic frequencies and one four frequency ultrasonic frequency of 25/33/40/59 kHz. The ultrasonic time was 30min. In the three frequency treatment, three frequencies were repeatedly cycled, and the frequency sweep cycle was 6 min. In the four-frequency ultrasonic treatment, the four frequencies circulate repeatedly, and the sweep cycle period is 8min. At the same time, non-ultrasonic pretreatment and single frequency ultrasonic pretreatment (40kHz) were used as double control.
[0078] After ultrasonic treatment, alkaline protease was added to the above samples for enzymatic hydrolysis according to the proportion of 2800U/g zein. The pH was adjusted to 9.0, the hydrolysis temperature was 45°C, the hydrolysis time was 60min, and the rotation speed was 700 r/min. The ACE inhibition rate was determined, as shown in Figure 6.
[0079] The ACE inhibitory activities of zein hydrolysate hydrolyzed by different three-frequency and four-frequency ultrasound were measured (Fig. 6) , the results showed that the ACE inhibitory activities of four kinds of tri-frequency ultrasound pretreatment zein hydrolysates were significantly higher than those of the non-and four-frequency ultrasound pretreatment zein hydrolysates. The ACE inhibitory activity of four kinds of tri-frequency ultrasound pretreated zein hydrolysates was 64.5% -66.1% , but significantly lower than that of the five kinds of
dual-frequency ultrasound pretreated zein hydrolysates. The influence of scanning ultrasonic treatment on the characteristics of zein
[0080] 4g zein was dissolved in 100mL distilled water, and fully stirred to disperse it evenly to obtain suspension of corn gluten meal. The ultrasonic temperature was set at 25 °C, and the zein suspension was placed in a frequency-scanning ultrasonic cleaning machine, treated with dual frequency ultrasound at 25/40khz, and the zein suspension without ultrasonic treatment was used as the control.
[0081] The above ultrasonic suspension of zein (without enzymatic hydrolysis) and the control group were centrifuged at 11000r/min for10min at room temperature. The precipitated material was freeze-dried, pre-cooled at -80°C for 4 hours, and further vacuum freeze-dried for 3 days to obtain zein freeze-dried powder, which was stored in a dryer and used to analyze the protein properties (electron microscope figure 7).
[0082] The effect of different treatments on the microstructure of zein can be clearly reflected by Sem. It can be seen from Fig. 7 that zein particles without ultrasonic treatment (fig. A) have a lumpy structure and a smooth surface under SEM, the surface of zein particles treated by scanning ultrasound (fig. B) became rough and uniform, and the surface of zein particles appeared small depressions and folds. The double-frequency ultrasound made the protein more dispersed, increased the binding sites with the alkaline protease, promoted the enzymatic hydrolysis reaction and the release of ACE inhibitory peptides.
[0083] The above is only a better embodiment of the invention and is not a restriction of the invention in any other form, any technician who is familiar with the profession may use the technical content disclosed above to modify or adapt it to an equivalent embodiment of the change. However, any simple modification, equivalent change and modification of the above embodiment according to the technical essence of the invention, which is not separated from the content of the technical proposal of the invention, still belongs to the protection scope of the technical proposal of the invention.
Claims (5)
1. A method for efficiently preparing food-derived ACE inhibitory peptides by dual frequency ultrasonic coupling enzymolysis is characterized by the following steps: (1) dispersing zein into distilled water to form zein suspension; (2) performing sweeping ultrasonic treatment on the zein suspension in step (1); (3) adding protease to the zein suspension after sweeping ultrasonic treatment in step (2), and adjusting the pH value to the appropriate pH of the protease; adjust the temperature of enzymatic hydrolysis to 40-50°C for enzymatic hydrolysis, and the reaction tends to stop when the reaction is saturated, pH will be adjusted to neutral, killing enzyme; (4) the hydrolysate of step (3) is cooled to room temperature and centrifuged to precipitate, then the supernatant is ultrafiltrated by the ultrafiltration membrane with a molecular retention of kda; (5) a freeze-dried powder of hydrolysate rich in ACE inhibitory peptide is prepared after vacuum freeze-drying the material liquid rich in ACE inhibitory peptide obtained in step (4).
2. The method for efficiently preparing food-derived ACE inhibitory peptides by dual frequency ultrasonic coupling enzymolysis according to claim 1, the frequency of the dual frequency ultrasonic processing is one of 25/33 kHz, 25/40 kHz, 25/59 kHz, 33/40 kHz, 33/59 kHz and 40/59 kHz, the cycle time of sweep frequency is 4 min.
3. The method for efficiently preparing food-derived ACE inhibitory peptides by dual frequency ultrasonic coupling enzymolysis according to claim 1, the protease in step (3) is anyone of trypsin, papain, alkaline protease and pepsin.
4. The method for efficiently preparing food-derived ACE inhibitory peptides by dual frequency ultrasonic coupling enzymolysis according to claim 1, the amount of protease added in the step (3) is 2800 ~ 3600U/g,
5. The method for efficiently preparing food-derived ACE inhibitory peptides by dual frequency ultrasonic coupling enzymolysis according to claim 1,the time of enzymatic hydrolysis is 40 ~ 60min.
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