CN113215212B - Soybean protein peptide with antioxidant and ACE (angiotensin converting enzyme) inhibiting functions and preparation method thereof - Google Patents
Soybean protein peptide with antioxidant and ACE (angiotensin converting enzyme) inhibiting functions and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of plant food processing, in particular to a soybean protein peptide with antioxidant and ACE inhibiting functions and a preparation method thereof. The preparation method comprises the following steps: the method comprises the following steps of (1) carrying out thermal denaturation treatment on soybean protein isolate serving as a raw material, and carrying out two-step enzymolysis to obtain an zymolyte; in the two-step enzymolysis, the enzyme used in the first step is alkaline protease, the enzyme used in the second step is compound protease, and the compound protease is neutral protease and flavourzyme. The soybean protein peptide contains specific molecular weight and peptide composition, not only has better antioxidation effect, but also can efficiently inhibit the activity of Angiotensin Converting Enzyme (ACE), IC 50 The value is lower than 0.018mg/mL, and the product can be widely applied to medicines, foods or food additives with corresponding functional requirements.
Description
Technical Field
The invention relates to the field of plant food processing, in particular to a soybean protein peptide with antioxidant and ACE inhibiting functions and a preparation method thereof.
Background
The soybean is originated from China, is an important economic, grain and oil crop in China, contains higher protein, is an important plant protein resource, has relatively balanced amino acid composition, and has important nutritive value for maintaining the health of human bodies. The abundant proteins in soy are a potential source of bioactive peptides. The soybean protein has high nutritive value and functional properties. They not only provide good dietary proteins, but also inhibit diseases associated with oxidative stress, such as coronary heart disease, atherosclerosis, diabetes, etc. With the development of modern society and economy and the improvement of living standard of people, the research, development and application of soybean peptide become the hot in the fields of medicine and food. At present, a plurality of enterprises in China use soybean peptide as a main raw material to respectively develop series products with different functions and aiming at different people.
Disclosure of Invention
The invention aims to provide a soybean protein peptide which has good functions of resisting oxidation and inhibiting the activity of Angiotensin Converting Enzyme (ACE) at the same time. The invention also aims to provide a preparation method and application of the soybean protein peptide.
The invention aims to develop the soybean protein peptide with the functions of resisting oxidation and inhibiting ACE activity, and develops the process for producing the soybean protein peptide with the functions of resisting oxidation and inhibiting ACE activity by taking the soybean protein isolate as a raw material according to the characteristics of the raw material and the composition of the components of the soybean protein isolate. The polypeptide product of the soybean protein isolate after enzymolysis has very complex composition, and contains a large number of polypeptides with unknown sequences and unknown functions, while the active peptide with specific functions may contain a plurality of peptides with different amino acid compositions and different molecular weights, and is difficult to distinguish through certain common characteristics. These have caused great difficulties in developing soybean protein peptides having specific functions. In the development process, a plurality of single enzymolysis and compound enzymolysis methods are tried, and although peptides with smaller molecular weight can be obtained by a plurality of methods, the obtained peptides have no functions of resisting oxidation and inhibiting ACE activity or have poor functions. After a great deal of attempts, the invention finally determines a process for obtaining the soybean protein peptide with excellent antioxidant and ACE inhibiting functions by using the soybean protein isolate as a raw material.
Specifically, the invention firstly provides a preparation method of soybean protein peptide, which comprises the following steps: the method comprises the following steps of (1) carrying out thermal denaturation treatment on soybean protein isolate serving as a raw material, and carrying out two-step enzymolysis to obtain an zymolyte;
in the two-step enzymolysis, the enzyme used in the first step is alkaline protease with the enzyme activity of 50,000-650,000U/g, the enzyme used in the second step is composite protease, and the composite protease is neutral protease with the enzyme activity of 100,000-650,000U/g and flavourzyme with the enzyme activity of 80,000-250,000U/g.
The invention discovers that the content of the peptide with the functions of resisting oxidation and inhibiting the activity of angiotensin converting enzyme in the product can be obviously improved after the two steps of enzymolysis, and further the functions of resisting oxidation and inhibiting the activity of ACE of the product are greatly improved.
Preferably, in the compound protease, the mass ratio of the neutral protease to the flavourzyme is 1-2: 1. Under the proportion, the compound protease has better effect and can further improve the content of the required functional peptide.
In the first step of enzymolysis, the mass of the raw material protein is taken as a reference, the dosage of the alkaline protease is 0.5-1.0%, and the enzymolysis conditions are that the pH value is 8-10 and the enzymolysis is carried out for 30-60min at 40-70 ℃;
in the second step of enzymolysis, the mass of the raw material protein is taken as a reference, the dosage of the compound protease is 1.0-2.0%, and the enzymolysis condition is enzymolysis for 3-7 hours at 40-70 ℃.
By adopting the two-step enzymolysis method, the soybean protein peptide can be fully degraded into the small molecular peptide, and meanwhile, the small molecular peptide is ensured to have high content of required functional peptide, so that the soybean protein peptide with specific molecular weight and peptide composition is obtained. Through identification, the polypeptides with high-content amino acid sequences of KDLVHKA (Lys-Asp-Leu-Val-His-Lys-Ala), RQGET (Arg-Gln-Gly-Glu-Thr) and ISDRTLG (Ile-Ser-Asp-Arg-Thr-Leu-Gly) can be obtained through the enzymolysis, and the polypeptides are key functional peptides for ensuring the effects of resisting oxidation and inhibiting ACE activity.
Conventionally, the two steps of enzymolysis also include a step of enzyme deactivation, which is not further limited herein.
Preferably, the heat denaturation treatment comprises: mixing the isolated soy protein with water, and keeping the temperature at 90-110 ℃ for 30-60 min.
More preferably, the mass ratio of the soy protein isolate to water is 1:15 to 20.
Preferably, the preparation method of the present invention further comprises: separating protein peptide liquid with the molecular weight less than 2000 daltons from the zymolyte, and then separating target peptides;
the isolation of the target peptide comprises:
separating the protein peptide liquid with the molecular weight less than 2000 by SephadexG-15 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; then RP-HPLC reversed-phase high performance liquid chromatography is used for separation, and the separation conditions are as follows: using a C18 chromatographic column, taking an aqueous solution containing 0.1% TFA as a mobile phase A and an acetonitrile solution containing 0.1% TFA as a mobile phase B, and performing separation by gradient elution: 0-5min, 5% mobile phase B; 5-45min, 5-45% of mobile phase B; 45-55min, 45-5% of mobile phase B, and the flow rate is 1 mL/min; and collecting the separated protein peptide solution for 9-13 minutes.
In a preferred embodiment, the C18 chromatography column is a Cosmosil 5C18-PAQ column (4.6X 250mm, Nacalai Tesque, Kyoto, Japan).
The invention discovers that the content of the functional peptide can be further improved by the separation method, so that the antioxidant property of the product can be further improved, and the ACE activity can be inhibited.
More preferably, the peptide having the amino acid sequence isdrrlg is more abundant when the protein peptide solution separated for 9-10 minutes is collected; when the separated protein peptide solution is collected for 10-11.5 minutes, the content of the peptide with the amino acid sequence KDLVHKA is higher; the peptide with the amino acid sequence RQGET is higher when the protein peptide solution is separated for 12-13 minutes.
Further preferably, the protein peptide solution with the molecular weight of less than 2000 daltons is obtained by filtration, and the filtration specifically comprises: firstly, filtering the zymolyte by using a ceramic membrane to remove macromolecular impurities, then collecting filtrate, carrying out filtration treatment by using a nanofiltration membrane, carrying out spray drying on trapped fluid to obtain soybean protein peptide powder, finally dissolving the soybean protein peptide powder, and then sequentially carrying out ultrafiltration by using ultrafiltration membranes with the aperture of 5000 daltons and 2000 daltons to obtain protein peptide liquid with the molecular weight of less than 2000.
The filtering method can not only improve the yield and the content of the protein peptide with the molecular weight less than 2000, but also improve the taste and the flavor of the soybean protein peptide.
The above-described schemes can be combined by those skilled in the art to arrive at a preferred embodiment of the preparation method of the present invention.
As a preferred embodiment, the method for preparing the soy protein peptide comprises the steps of:
(1) and (3) thermal denaturation treatment: mixing soybean protein isolate and water according to a mass ratio of 1: 15-20, and keeping the temperature at 90-110 ℃ for 30-60 min;
(2) enzymolysis: adding alkaline protease accounting for 0.5-1% of the total mass of the protein into protein liquid, performing enzymolysis for 30-60min at the temperature of 40-70 ℃ at the pH value of 8-10, adding neutral protease accounting for 1.0-2.0% of the total mass of the protein and compound protease consisting of flavourzyme, wherein the mass ratio of the neutral protease to the flavourzyme is 1-2:1, performing enzymolysis for 3-7h at the temperature of 40-70 ℃, and performing enzyme deactivation to obtain an enzymolysis product;
(3) and (3) filtering: firstly filtering the zymolyte by using a ceramic membrane to remove macromolecular impurities, then collecting filtrate, carrying out nanofiltration membrane filtration treatment, carrying out spray drying on trapped fluid to obtain soybean protein peptide powder, finally dissolving the soybean protein peptide powder, firstly separating protein and polypeptide with the molecular weight of less than 5000 daltons, and then separating protein peptide with the molecular weight of less than 2000 daltons by using a membrane with the pore size of 2000 daltons;
(4) separation of target peptide: separating the protein peptide liquid with the molecular weight of less than 2000 daltons by SephadexG-15 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; obtaining the soybean protein peptide by concentration and freeze drying; then RP-HPLC reversed phase high performance liquid chromatography is used for separation, and the separation conditions are as follows: using a C18 chromatographic column, taking an aqueous solution containing 0.1% TFA as a mobile phase A and an acetonitrile solution containing 0.1% TFA as a mobile phase B, and performing separation by gradient elution: 0-5min, 5% mobile phase B; 5-45min, 5-45% of mobile phase B; 45-55min, 45-5% of mobile phase B, and the flow rate is 1 mL/min; collecting the separated protein peptide solution for 9-13 minutes;
(5) concentrating and freeze-drying the peptide solution obtained in the step (6) to obtain soybean protein peptide powder with a specific function; the main components of the protein peptide are measured by LC-MS/MS, and the content of the polypeptide with the amino acid sequences of KDLVHKA, RQGET and ISDRTLG is more than or equal to 50 wt%.
In the preparation method, the soybean protein isolate is used as a raw material, high-temperature thermal denaturation treatment is carried out on the soybean protein isolate according to the characteristics of the raw material of the soybean protein isolate powder, multiple proteases are used for carrying out fractional enzymolysis, and the soybean protein peptides with specific amino acid polypeptide sequences (KDLVHKA, RQGET and ISDRTLG) and with the functions of oxidation resistance and ACE inhibition are separated through membrane separation, gel separation and reversed-phase HPLC separation technologies, so that a set of simple and efficient preparation method of the multifunctional soybean protein peptides is established.
The invention also provides a soybean protein peptide which is prepared by the preparation method of the soybean protein peptide.
Preferably, the soybean protein peptide contains functional peptides with the mass percentage content of more than or equal to 50%, and the sequence of the functional peptides is shown in any one of SEQ ID No. 1-3.
The invention also provides a functional polypeptide which has an amino acid sequence shown in any one of SEQ ID NO. 1-3.
The invention also provides application of the soybean protein peptide or the functional polypeptide in preparation of medicines, foods or food additives.
Preferably, the pharmaceutical, food or food additive has a function of resisting oxidation and/or inhibiting angiotensin converting enzyme activity.
The invention also provides a medicine, food or food additive containing the soybean protein peptide or the functional polypeptide.
The medicine, food or food additive can contain the soybean protein peptide or the functional polypeptide and auxiliary materials allowed in the field of medicines or foods.
Based on the technical scheme, the invention has the following beneficial effects:
(1) the soybean protein peptide contains specific molecular weight and peptide composition, not only has better antioxidation effect, but also can efficiently inhibit the activity of Angiotensin Converting Enzyme (ACE), IC 50 The value is lower than 0.018mg/mL, and the product can be widely applied to medicines, foods or food additives with corresponding functional requirements.
(2) The proportion of peptides with molecular weight less than 2000 in the soybean protein peptide is more than 90 percent, and the soybean protein peptide is separated by RP-HPLC reversed phase high performance liquid chromatography to achieve the composition with more definite peptides, wherein the content of the peptides of KDLVHKA, RQGET and ISDRTLG is more than 50 percent.
(3) The aqueous solution of the soybean protein peptide is clear and transparent, can not form turbidity even if being stored in an acid solution, and can be added into food and drink with any pH range.
(4) The invention combines the biological enzymolysis technology, the membrane ultrafiltration technology, the nanofiltration technology and the like to prepare the soybean protein peptide, and the obtained product has good taste, small and concentrated molecular weight and obvious absorption effect. In addition, the used enzymes are food-grade proteases, so that the safety is good.
Drawings
FIG. 1 is a graph of the soybean peptide obtained in example 1.
FIG. 2 is a graph of the soybean peptide assay obtained in example 2.
FIG. 3 is a graph of the soybean peptide assay obtained in example 3.
FIG. 4 is a LC-MS chart of the soybean peptide obtained in example 1.
FIG. 5 is a LC-MS chart of the soybean peptide obtained in example 2.
FIG. 6 is a LC-MS diagram of the soybean peptide obtained in example 3.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the following examples, the separation conditions of reverse phase HPLC are specifically as follows:
liquid phase system: shimadzu LC-16
A detector: ultraviolet detector 220nm
Mobile phase: A-Water + 0.1% TFA B-acetonitrile + 0.1% TFA
Column: cosmosil 5C18-PAQ column (4.6X 250mm, Nacalai Tesque, Kyoto, Japan)
Flow rate: 1mL/min
Elution procedure:
0-5min，5％B；
5-45min，5-45％B；
45-55min，45-5％B。
the enzyme activity of the alkaline protease used in the following examples was 300,000U/g, the enzyme activity of the neutral protease was 350,000U/g, and the enzyme activity of the flavourzyme was 180,000U/g.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Example 1 preparation of Soybean protein peptide having antioxidant and ACE inhibitory effects
The embodiment provides a preparation method of soybean protein peptide with antioxidant and ACE inhibitory functions, which comprises the following specific steps:
(1) adding water into the soybean protein isolate, mixing the soybean protein isolate and the water at a material-water ratio of 1:15, heating to 100 ℃, and keeping the temperature for 60 min; adjusting the pH of the solution after the heat treatment modification to be 8, adding alkaline protease accounting for 0.6% of the protein mass to react for 30min at the temperature of 55 ℃, then adding neutral protease accounting for 0.6% of the protein mass and flavourzyme accounting for 0.4% of the protein mass to react for 4h, and inactivating enzymes to obtain a soybean protein isolate enzymolysis product;
(2) clarifying the soybean separation protease hydrolysate by using a coarse screen, firstly filtering by using a ceramic membrane to remove macromolecular impurities, and collecting filtrate;
(3) filtering the filtrate collected in the step (2) by nanofiltration equipment, and carrying out enrichment, desalination and debittering treatment;
(4) spray drying the trapped fluid in the step (3) to obtain soybean protein peptide powder;
(5) dissolving the soybean protein peptide powder obtained in the step (4), performing ultrafiltration by using an ultrafiltration membrane with the aperture of 5000 daltons, separating the protein and the polypeptide with the molecular weight of less than 5000 daltons, and separating the protein peptide with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(6) Taking protein peptide liquid with molecular weight less than 2000, and then carrying out SephadexG-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; obtaining soybean protein peptide by concentration and freeze drying; separating for 1 time by RP-HPLC reversed phase high performance liquid chromatography, eluting with acetonitrile solution, and collecting separated protein peptide solution 10-11.5 min;
(7) and (3) concentrating and freeze-drying the peptide solution obtained in the step (6) to obtain soybean protein peptide powder with a specific function, wherein the detection result is shown in figure 1, the main components of the protein peptide are determined by LC-MS/MS (figure 4), and the content of the peptide with the amino acid sequence KDLVHKA is 59.1%.
The present example also provides a soy protein peptide prepared using the above method.
Example 2 preparation of Soybean protein peptide having antioxidant and ACE inhibitory effects
The embodiment provides a preparation method of soybean protein peptide with antioxidant and ACE inhibitory functions, which comprises the following specific steps:
(1) adding water into the soybean protein isolate, mixing the soybean protein isolate and the water at a material-water ratio of 1:17, heating to 100 ℃, and keeping the temperature for 60 min; adjusting the pH of the solution after the heat treatment modification to 9, adding 0.9% alkaline protease for reacting for 45min at 55 ℃, then adding 0.7% neutral protease and 0.7% flavourzyme for reacting for 5h, and inactivating enzyme to obtain a soybean protein isolate enzymolysis product;
(2) clarifying the soybean separation protease hydrolysate by using a coarse screen, firstly filtering by using a ceramic membrane to remove macromolecular impurities, and collecting filtrate;
(3) filtering the filtrate collected in the step (2) by nanofiltration equipment, and carrying out enrichment, desalination and debittering treatment;
(4) spray drying the trapped fluid in the step (3) to obtain soybean protein peptide powder;
(5) dissolving the soybean protein peptide powder obtained in the step (4), performing ultrafiltration by using an ultrafiltration membrane with the aperture of 5000 daltons, separating the protein and the polypeptide with the molecular weight of less than 5000 daltons, and separating the protein peptide with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(6) Taking protein peptide liquid with molecular weight less than 2000, separating by SephadexG-15 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and collecting the 2 nd elution peak; obtaining soybean protein peptide by concentration and freeze drying; separating for 1 time by RP-HPLC reversed phase high performance liquid chromatography, and collecting separated protein peptide solution from 12-13 min;
(7) concentrating and freeze-drying the peptide solution obtained in the step (6) to obtain soybean protein peptide powder with specific functions, wherein the detection result is shown in figure 2, and the main components of the protein peptide are measured by LC-MS/MS (figure 5), and the peptide content of which the amino acid sequence is RQGET is 68.9%.
The present example also provides a soy protein peptide prepared using the above method.
Example 3 preparation of Soybean protein peptide having antioxidant and ACE inhibitory effects
The embodiment provides a preparation method of soybean protein peptide with antioxidant and ACE inhibitory functions, which comprises the following specific steps:
(1) adding water into the soybean protein isolate to mix, wherein the ratio of the materials to the water is 1: 20, heating to 100 ℃, and preserving the heat for 60 min; adjusting the pH of the solution after the heat treatment modification to 10, at the temperature of 50 ℃, adding 1% of alkaline protease for reaction for 60min, then adding 1% of neutral protease and 0.5% of flavourzyme for reaction for 7h, and inactivating enzyme to obtain a soybean protein isolate enzymolysis product;
(2) clarifying the soybean separation protease hydrolysate by using a coarse screen, firstly filtering by using a ceramic membrane to remove macromolecular impurities, and collecting filtrate;
(3) filtering the filtrate collected in the step (2) by nanofiltration equipment to perform enrichment, desalination and debitterizing treatment;
(4) spray drying the trapped fluid in the step (3) to obtain soybean protein peptide powder;
(5) dissolving the soybean protein peptide powder obtained in the step (4), performing ultrafiltration by using an ultrafiltration membrane with the aperture of 5000 daltons, separating the protein and the polypeptide with the molecular weight of less than 5000 daltons, and separating the protein peptide with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(6) Taking protein peptide liquid with molecular weight less than 2000, and then carrying out SephadexG-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; obtaining the soybean protein peptide by concentration and freeze drying; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, and collecting 9-10 parts of separated protein peptide solution;
(7) and (3) concentrating and freeze-drying the peptide solution obtained in the step (6) to obtain soybean protein peptide powder with a specific function, wherein the detection result is shown in figure 3, the main components of the protein peptide are determined by LC-MS/MS (figure 6), and the peptide content of the amino acid sequence ISDRTLG is 62.6%.
The present example also provides a soy protein peptide prepared using the above method.
Experimental example 1 measurement test of antioxidant Activity of Soybean protein peptide
Test samples: samples 1-3 were soybean protein peptide powders prepared according to examples 1-3, respectively, sample 4 was a raw soybean protein isolate powder, sample 5 was a soybean protein peptide powder obtained by freeze-drying the soybean protein isolate enzymatic hydrolysate obtained in step (1) of example 1, sample 6 was a soybean protein peptide powder obtained by freeze-drying the soybean protein solution obtained in step (5) of example 1, and samples KDLVHKA, RQGET, and isdrrlg were polypeptides artificially synthesized according to amino acid sequences KDLVHKA, RQGET, and isdrrlg, respectively.
The method comprises the following steps:
(1) measurement of hydroxyl radical (. OH) scavenging ability: 2.0mL of 150mmol/L Phosphate Buffer Solution (PBS) with pH of 7.4, 0.2mL of 7.5mmol/L o-diazaphenanthrene, 0.5 mmol/LFeSO40.2mL of samples with different concentrations, 0.4mL of distilled water and 1% of H2O20.4mL in volume fraction are precisely measured in test tubes, the test tubes are uniformly mixed to form a sample group, the test tubes are simultaneously placed in a thermostatic water bath at 37 ℃ for 60min, and the absorbance value is measured at the wavelength of 536 nm. Replacing the blank group with distilled water for the sample, and performing the other steps of the blank group with the sample group; the blank control group was prepared by replacing the sample and H2O2 with distilled water, and the sample was prepared by the same procedure as the sample group, and the hydroxyl radical scavenging ratio (E (OH)%) of the sample was calculated according to the following formula:
e (& OH) { a (sample) -a 0}/{ a (blank) -a 0} × 100%
In the formula: a (sample) is the absorbance value of the sample group at the wavelength of 536 nm;
a0 is the absorbance value of blank set at 536 nm;
a (blank) is the absorbance value of the blank at 536 nm.
(2) Removal of O 2 - The determination of (1): taking 4.5mL of 0.05mol/L Tris2HCl buffer solution and a proper amount of deionized water, uniformly mixing, placing in a water bath at 25 ℃ for heat preservation for 10min, then adding 0.2mL of soybean peptide samples with different concentrations, finally adding 0.3mmol/L pyrogallol solution with the concentration of 3mmol/L to the final concentration of 0.1mmol/L, uniformly mixing, preserving at 25 ℃ for 4min, immediately stopping the reaction with 8mol/LHCl solution, measuring the absorbance value at 322nm, and replacing the sample with the deionized water in a blank group. p.O 2 - The clearance of (E (. O) was calculated as follows 2 - ))：
E(·O 2 - )＝{A 0 -A (sample) }/A 0 ×100％
In the formula: a (sample) is the absorbance value of the sample group at the wavelength of 322 nm; a. the 0 The absorbance of the blank at a wavelength of 322nm was determined.
As can be seen from Table 1, the soybean protein peptide of the invention has better oxidation resistance, and under the condition of 2mg/mL, the capacity of eliminating hydroxyl radical (. OH) reaches more than 80 percent, and the capacity of eliminating. O 2 - The free radical capacity reaches more than 45 percent, and the peptide is a better antioxidant peptide.
TABLE 1 antioxidant Activity test results for the Soy protein peptides of the present invention
Experimental example 2 measurement of inhibitory Activity of Soybean protein peptide Angiotensin Converting Enzyme (ACE)
The ACE inhibitory capacity of the samples was determined as follows:
methods of ACE inhibition. And quantitatively detecting the amount of the released Hip by using high performance liquid chromatography at 228nm so as to calculate the ACE inhibition rate of the polypeptide.
1. Preparation of reagents
phosphate buffer solution at ph 8.3: preparing with ultrapure water, wherein the pH value is adjusted to 8.3, and the phosphate content is 50mmol/L and the NaCl content is 300 mmol/L;
ACE enzyme solution: 2mL of phosphate buffer was added to 1U of ACE so that the concentration became 0.5U/mL.
HHL solution: HHL was dissolved in phosphate buffer to a final concentration of 5 mmol/L.
Sample solution: an appropriate amount of sample (test sample same as in Experimental example 1) was weighed and dissolved in a phosphate buffer solution of the desired concentration.
2. Chromatographic conditions for ACE inhibition assay
Detection wavelength: 228 nm; the flow rate is 1 mL/min; mobile phase A is ultrapure water (containing 0.1% of trifluoroacetic acid), and mobile phase B is methanol (containing 0.1% of trifluoroacetic acid); the sample injection amount is 10 mu L, and manual sample injection is carried out;
3. method for determining ACE inhibitory activity
Taking 120 mu L of HHL substrate solution, adding 20 mu L of sample, mixing uniformly, and preserving heat in a constant-temperature water bath at 37 ℃ for 10 min. Then 10 mul of ACE enzyme solution is added to react for 30min in a thermostatic water bath at 37 ℃, and 150 mul of 1mol/L HCl is added to stop the reaction, thus obtaining reaction solution. The reaction solution was analyzed by HPLC, and a blank control group was set. The ACE inhibitory activity was calculated as follows:
ACE inhibitory Activity% ((M-N)/M.times.100)
Wherein M is the peak area of hippuric acid in the control group, and N is the peak area of hippuric acid in the added sample group.
4. Determination of the semi-inhibitory concentration
The inhibitory activity of the ACE inhibitory peptide is measured according to an in vitro detection method of the ACE inhibitory peptide, a smooth curve is drawn by taking the concentration as the abscissa and the ACE inhibitory rate as the ordinate, and the IC50 value is calculated from the curve. The results are shown in Table 2.
As can be seen from Table 2, the soybean protein peptide prepared by the present invention has excellent ACE inhibitory activity and IC thereof 50 The value is less than 0.018mg/mL, and the molecular weight of the soybean protein peptide is less than 2000 and reaches more than 90 percent. Different order of enzymatic hydrolysis, its ACE inhibitionThe preparation activity is greatly different.
TABLE 2 ACE inhibitory Activity of Soybean protein peptides test results
| Product protein peptide | ACE inhibitory activity (IC50 value) mg/mL |
| Sample 1 | 0.0175 |
| |
0.0172 |
| Sample 3 | 0.0177 |
| Sample No. 4 | 0.356 |
| Sample No. 5 | 0.191 |
| Sample No. 6 | 0.182 |
| Sample KDLVHKA | 0.0161 |
| Sample RQGET | 0.0158 |
| Sample ISDRTLG | 0.0165 |
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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<120> a soybean protein peptide with antioxidant and ACE inhibitory functions and a preparation method thereof
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Claims (13)
1. A soybean protein peptide is characterized in that the preparation method comprises the following steps: the method comprises the following steps of (1) carrying out thermal denaturation treatment on soybean protein isolate serving as a raw material, and carrying out two-step enzymolysis to obtain an zymolyte;
in the two-step enzymolysis, the enzyme used in the first step is alkaline protease with the enzyme activity of 50,000-650,000U/g, the enzyme used in the second step is composite protease, the composite protease is neutral protease with the enzyme activity of 100,000-650,000U/g and flavourzyme with the enzyme activity of 80,000-250,000U/g;
the soybean protein peptide contains functional peptides with the mass percentage content of more than or equal to 50%, and the sequence of the functional peptides is shown in any one of SEQ ID NO. 1-3.
2. The soybean protein peptide according to claim 1, wherein the mass ratio of the neutral protease to the flavourzyme in the complex protease is 1-2: 1.
3. The soybean protein peptide according to claim 1 or 2, wherein in the first step of enzymolysis, the amount of alkaline protease is 0.5-1.0% based on the mass of the raw material protein, and the enzymolysis condition is that the pH is 8-10 and the enzymolysis is carried out at 40-70 ℃ for 30-60 min;
in the second step of enzymolysis, the mass of the raw material protein is taken as a reference, the dosage of the compound protease is 1.0-2.0%, and the enzymolysis condition is enzymolysis for 3-7 hours at 40-70 ℃.
4. The soy protein peptide as claimed in claim 1 or 2, wherein said heat denaturation treatment comprises: mixing the isolated soy protein with water, and keeping the temperature at 90-110 ℃ for 30-60 min.
5. The soy protein peptide of claim 3, wherein said heat denaturation treatment comprises: mixing the isolated soy protein with water, and keeping the temperature at 90-110 ℃ for 30-60 min.
6. The soy protein peptide of any of claims 1, 2, 5, wherein the method of preparation further comprises: separating protein peptide liquid with molecular weight less than 2000 daltons from the zymolyte, and then separating target peptide;
the isolation of the target peptide comprises:
separating the protein peptide liquid with the molecular weight less than 2000 daltons by SephadexG-15 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; then RP-HPLC reversed-phase high performance liquid chromatography is used for separation, and the separation conditions are as follows: using a C18 chromatographic column, taking an aqueous solution containing 0.1% TFA as a mobile phase A and an acetonitrile solution containing 0.1% TFA as a mobile phase B, and performing separation by gradient elution: 0-5min, 5% mobile phase B; 5-45min, 5-45% of mobile phase B; 45-55min, and 45-5% of mobile phase B, wherein the flow rate is 1 mL/min; and collecting the separated protein peptide solution for 9-13 minutes.
7. The soy protein peptide of claim 3, wherein the method of preparation further comprises: separating protein peptide liquid with molecular weight less than 2000 daltons from the zymolyte, and then separating target peptide;
the isolation of the target peptide comprises:
separating the protein peptide liquid with the molecular weight of less than 2000 daltons by SephadexG-15 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; then RP-HPLC reversed-phase high performance liquid chromatography is used for separation, and the separation conditions are as follows: using a C18 chromatographic column, taking an aqueous solution containing 0.1% TFA as a mobile phase A and an acetonitrile solution containing 0.1% TFA as a mobile phase B, and performing separation by gradient elution: 0-5min, 5% mobile phase B; 5-45min, 5-45% of mobile phase B; 45-55min, and 45-5% of mobile phase B, wherein the flow rate is 1 mL/min; and collecting the separated protein peptide solution for 9-13 minutes.
8. The soy protein peptide of claim 4, wherein the method of preparation further comprises: separating protein peptide liquid with molecular weight less than 2000 daltons from the zymolyte, and then separating target peptide;
the isolation of the target peptide comprises:
separating the protein peptide liquid with the molecular weight of less than 2000 daltons by SephadexG-15 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; then RP-HPLC reversed phase high performance liquid chromatography is used for separation, and the separation conditions are as follows: using a C18 chromatographic column, taking an aqueous solution containing 0.1% TFA as a mobile phase A and an acetonitrile solution containing 0.1% TFA as a mobile phase B, and performing separation by gradient elution: 0-5min, 5% mobile phase B; 5-45min, 5-45% of mobile phase B; 45-55min, and 45-5% of mobile phase B, wherein the flow rate is 1 mL/min; and collecting the separated protein peptide solution for 9-13 minutes.
9. The soy protein peptide as claimed in claim 6, wherein said protein peptide solution having a molecular weight of less than 2000 daltons is obtained by filtration, said filtration comprising:
filtering the zymolyte by using a ceramic membrane to remove macromolecular impurities, collecting filtrate, filtering the filtrate by using a nanofiltration membrane, spray-drying trapped fluid to obtain soybean protein peptide powder, dissolving the soybean protein peptide powder, and sequentially performing ultrafiltration by using ultrafiltration membranes with the aperture of 5000 daltons and 2000 daltons to obtain protein peptide liquid with the molecular weight of less than 2000.
10. The soy protein peptide as claimed in claim 7 or 8, wherein said protein peptide solution having a molecular weight of less than 2000 daltons is obtained by filtration, said filtration comprising:
firstly, filtering the zymolyte by using a ceramic membrane to remove macromolecular impurities, then collecting filtrate, carrying out filtration treatment by using a nanofiltration membrane, carrying out spray drying on trapped fluid to obtain soybean protein peptide powder, finally dissolving the soybean protein peptide powder, and then sequentially carrying out ultrafiltration by using ultrafiltration membranes with the aperture of 5000 daltons and 2000 daltons to obtain protein peptide liquid with the molecular weight of less than 2000.
11. The functional polypeptide is characterized in that the amino acid sequence of the functional polypeptide is shown in any one of SEQ ID NO. 1-3.
12. Use of a soy protein peptide according to any of claims 1 to 10 or a functional polypeptide according to claim 11 for the preparation of a medicament;
the medicine has functions of resisting oxidation and/or inhibiting angiotensin converting enzyme activity.
13. A pharmaceutical, food or food additive comprising the soybean protein peptide according to any one of claims 1 to 10 or the functional polypeptide according to claim 11.
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