A2 beta-casein-based ace inhibitory peptide and preparation method and application thereof

Abstract

An ACE inhibitory peptide based on A2 beta-casein and a preparation method and application thereof belong to the technical field of bioactive peptides. To solve the technical problem that ACE inhibitor drugs in the prior art have many adverse reactions and are not suitable for long-term continuous use, a preparation method of an ACE inhibitory peptide based on A2 beta-casein is provided. The A2 beta-casein is subjected to enzymolysis by pepsin and trypsin, and through ultrafiltration, desalting, liquid chromatography tandem mass spectrometer analysis and molecular docking analysis, an ACE inhibitory peptide with an amino acid sequence shown in SEQ ID No. 1 is finally screened and obtained. The ACE inhibitory peptide can effectively increase the release amount of NO of human umbilical vein endothelial cells and significantly inhibit the secretion of ET1, and has no toxic side effects on human umbilical vein endothelial cells. The ACE inhibitory peptide provided by the application can be applied to the preparation of a drug for preventing or reducing blood pressure.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, and relates to an ACE inhibitory peptide, its preparation method, and its application. Background Technology

[0002] Hypertension is considered a major cause of cardiovascular disease, and its prevalence is rapidly increasing worldwide. (Regarding renin...)

[0003] In the angiotensin system (RAS), angiotensin I converting enzyme (ACE) can cleave angiotensin I into angiotensin II, which can cause vasoconstriction and lead to hypertension. This indicates that inhibiting ACE activity can prevent the rise in blood pressure. Currently, the main treatment for hypertension is ACE inhibitors, such as captopril, enalapril, and lisinopril. However, long-term use of these drugs can easily lead to many adverse reactions and is not suitable for long-term continuous use.

[0004] Bioactive peptides are defined as specific protein fragments that have a positive impact on bodily functions or conditions, possessing hormone- or drug-like activities such as antibacterial, antithrombotic, immunomodulatory, antioxidant, and lipid-lowering effects. They are widely used in disease prevention and are considered a new generation of bioactive regulators. Currently, numerous studies have shown that food-derived bioactive peptides with ACE-inhibiting activity, such as mung bean peptides, sea cucumber peptides, moringa peptides, large yellow croaker peptides, and sorghum peptides, have significant blood pressure-lowering effects. Compared to existing ACE inhibitor drugs, bioactive peptides obtained from natural foods are greener and safer, becoming a research hotspot in recent years.

[0005] Casein accounts for 80% of the total protein in milk. Some polypeptides obtained from the enzymatic hydrolysis of bovine casein possess biological activity, including antioxidant, antibacterial, antithrombotic, and antihypertensive effects. Hydrolysates or bioactive peptides derived from casein are widely used as functional ingredients in food. β-casein is the main component of bovine milk casein and has high nutritional value. However, due to differences in amino acid sequences, β-casein exists in various variants. The A2 variant is considered the oldest and most primitive, containing proline (Pro) at position 67 of the β-casein chain. Genetic variations primarily lead to AA exchanges or deletions within the coding sequence, thereby affecting the function of the encoded protein and resulting in differences in the structure, digestibility, and functional characteristics of various variant proteins.

[0006] Therefore, those skilled in the art have long desired to develop an ACE inhibitory peptide based on A2β-casein that has the effect of preventing hypertension and lowering blood pressure, so as to provide new ideas for the preparation of natural blood pressure lowering products. Summary of the Invention

[0007] The application provides an ACE inhibiting peptide based on A2 beta-casein and a preparation method and application thereof to solve the technical problem that ACE inhibitors in the prior art have many adverse reactions and are not suitable for long-term continuous use.

[0008] One of the purposes of the application is to provide a method for preparing an ACE inhibiting peptide based on A2 beta-casein, which comprises the following steps:

[0009] S1: uniformly mixing A2 beta-casein with distilled water at a certain ratio to prepare a hydrolysate, performing water bath enzymolysis reaction using pepsin first, high-temperature enzyme inactivation, and then performing digestion enzymolysis reaction using trypsin to obtain an enzymolysis product;

[0010] S2: performing ultrafiltration treatment on the enzymolysis product obtained in S1, performing desalination treatment using a C 18 column, and then obtaining a polypeptide by centrifugal concentration after drying, and storing the polypeptide in a freezer at -20 DEG C;

[0011] S3: performing liquid chromatography tandem mass spectrometry analysis on the polypeptide obtained in S2 to obtain a peptide segment, and artificially synthesizing an ACE inhibiting peptide through solid-phase synthesis.

[0012] In one preferred embodiment of the application, the mixing ratio of the A2 beta-casein to the distilled water in S1 is mixing according to a substrate concentration of 4%; the temperature of the high-temperature enzyme inactivation is 95 DEG C, and the time is 10 min.

[0013] In one preferred embodiment of the application, the concentration of the pepsin in S1 is 2000 U / mg, the enzymolysis reaction temperature of the pepsin is 37 DEG C, the time is 2 h, and the pH is 2.5; the concentration of the trypsin in S1 is 120 U / mg, the enzymolysis reaction temperature of the trypsin is 37 DEG C, the time is 3 h, and the pH is 7.0.

[0014] In one preferred embodiment of the application, the ultrafiltration treatment in S2 is filtering the enzymolysis product using a 10KD ultrafiltration tube to obtain a component with a molecular weight of less than 10KDa;

[0015] In one preferred embodiment of the application, the liquid chromatography condition in S3 is: the liquid chromatography is an UltiMate3000RSLCnano nanoliter liquid chromatography equipped with a C 18 chromatography column; the mobile phase A is 0.1% formic acid / 3% DMSO / 97% H2O; the mobile phase B is 0.1% formic acid / 3% DMSO / 97% ACN; and the flow rate is 300 nL / min.

[0016] In a preferred embodiment of the present application, the mass spectrometry conditions in S3 are: using a Q Exactive HF mass spectrometer; MS1 is resolution 60K@200m / z; the scan range is 350-1500m / z; the maximum injection time is 30ms; MS2 is resolution 15K@200m / z; AGC target is 1E5, the maximum injection time is 50ms; the dynamic exclusion time is 30s.

[0017] In a preferred embodiment of the present application, the amino acid sequence of the ACE inhibitory peptide in S3 is shown in SEQ ID No. 1.

[0018] The second object of the present application is to provide an ACE inhibitory peptide based on A2β-casein, which is obtained by the above preparation method.

[0019] In a preferred embodiment of the present application, the ACE inhibitor is an ACE competitive inhibitor.

[0020] The third object of the present application is to provide the use of the above ACE inhibitory peptide in the preparation of a drug for preventing or reducing blood pressure.

[0021] The present application provides an ACE inhibitory peptide based on A2β-casein, a preparation method and application thereof.

[0022] The ACE inhibitory peptide prepared by the present application has an IC 50 of 0.1760mg / mL, which can effectively increase the release amount of NO and significantly inhibit the secretion of ET 1 of human umbilical vein endothelial cells when the concentration of the ACE inhibitory peptide is 1.0mg / mL, and has no toxic side effects on human umbilical vein endothelial cells, similar blood pressure lowering mechanism and ability to the classic ACE inhibitor captopril, but does not have the problem of more adverse reactions of ACE inhibitors and is not suitable for long-term continuous use.

[0023] The preparation method of the ACE inhibitory peptide provided by the present application has the characteristics of simple operation, safety and easy production, and the ACE inhibitory peptide can be applied to the preparation of a drug for preventing or reducing blood pressure, and has a broad market prospect as a functional ingredient in the fields of food, health care products, medicine and the like. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1Figure of molecular docking result in Example 1; A is 3D structure surface conformation of ACE inhibitory peptide complex, B is 2D detail figure of ACE inhibitory peptide complex action;

[0025] Figure 2 Figure of effect of different concentrations of ACE inhibitory peptide on HUVEC cell viability in Example 2; abscissa is ACE inhibitory peptide concentration;

[0026] Figure 3 Figure of effect of different concentrations of ACE inhibitory peptide on HUVEC cell NO release amount in Example 2;

[0027] Figure 4 Figure of effect of different concentrations of ACE inhibitory peptide on HUVEC cell ET 1 content in Example 2. DETAILED DESCRIPTION

[0028] Those skilled in the art can improve the process parameters according to the content herein. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0029] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with specific embodiments and the drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0030] Angiotensin converting enzyme: Angiotensin I converting Enzyme, ACE.

[0031] Example 1: Preparation of an ACE inhibitory peptide based on A2β-casein

[0032] (1) Preparation of A2β-casein enzymatic hydrolysate

[0033] In this embodiment, A2β-casein was mixed with distilled water at a substrate concentration of 4% to prepare a hydrolysate. First, 2000 U / mg of pepsin was used for enzymatic hydrolysis at 37℃ and pH 2.5 for 2h. After adding an equal volume of simulated intestinal fluid, the pH value was adjusted to 7.0, and 120 U / mg of trypsin was used for enzymatic digestion at 37℃ for 3h. The enzyme was inactivated at 95℃ for 10min to obtain the enzymatic hydrolysate.

[0034] (2) Identification, screening and synthesis of ACE inhibitory peptides

[0035] The enzymatic product obtained in step (1) was dissolved in water, centrifuged at 12000 x g for 10 min, and the supernatant was filtered using a 10 KD ultrafiltration tube. The filtered solution was desalted using a C 18 column (Acclaim PepMap (75 μm*2 cm, C18, 3 μm, 120 A, Thermo) and the desalted solution was dried by centrifugal concentration and stored at -20°C for subsequent detection.

[0036] The peptides were separated on an UltiMate 3000 RSLCnano nanoliter liquid chromatograph coupled with a Q Exactive HF mass spectrometer (Waltham, USA) equipped with a C18 column; an analysis gradient was established using mobile phase A (0.1% formic acid / 3% DMSO / 97% H2O) and mobile phase B (0.1% formic acid / 3% DMSO / 97% ACN) at a flow rate of 300 nL / min, MS1: resolution: 60K@200m / z, scan range: 350-1500m / z, AGC target: 3E6, maximum IT: 30 ms; MS2 settings: resolution: 15K@200m / z, AGC target: 1E5, maximum IT: 50 ms; the raw mass spectrometry data were analyzed using MaxQuant (V1.6.6.0) software; the MS1 mass tolerance was set to 20 ppm and 4.5 ppm, respectively; the MS2 mass tolerance was set to 20 ppm, and the above search results were filtered by 1% FDR to obtain the peptide segments.

[0037] The biological activity of the above peptide segments was predicted using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), and those with an activity score >0.5 were marked as having biological activity. The peptide segments with an activity score >0.5 were pasted into the Tox inPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / ) and Innovagen (http: / / www.innovagen.com / proteomics-tools / ) and AllergenFP (http: / / www.innovagen.com / proteomics tools) websites to predict toxicity, solubility and allergenicity, and to screen for peptides that were non-toxic, had good solubility and no potential allergenicity.

[0038] ​To analyze the interaction between ACE inhibitory peptides and angiotensin converting enzyme at the molecular level, molecular docking was simulated using Discovery Studio 2019 client software. The 3D crystal structure of ACE (PDB ID: 1O86) was retrieved from the PDB database (https: / / www.rcsb.org / ). Before docking, water molecules in the conformation were removed, leaving the cofactors zinc and chlorine atoms, and hydrogen was added; the CDOCKER program was selected for semi-flexible molecular docking, and the CDOCKER ENERGY score and interaction force type with ACE were used to evaluate the results of molecular docking to obtain the peptide segments analyzed by molecular docking.

[0039] In this embodiment, the above peptide segments analyzed by molecular docking were sent to Shenguo Bioengineering Co., Ltd. (Shanghai, China) for synthesis of ACE inhibitory peptides by solid-phase synthesis method, with a purity of more than 95%.

[0040] Effect experiment:

[0041] (1) In this embodiment, UltiMate 3000 RSLCnano nanoliter liquid chromatography coupled with Q Exactive HF mass spectrometer was used for peptide identification of components less than 10 kDa, and MaxQuant (V1.6.6.0) software was used for analysis. The results are shown in Table 1. A total of 10 peptide segments were identified. The above identified peptide segments were searched and compared in the database (BIOPEP and PeptideRanker), and it was found that only 5 peptide segments had ACE inhibitory activity, and their predicted biological activity scores were all >0.5.

[0042] Table 1

[0043] SEQ ID No. AA sequence Molecular weight Length Biological activity score SEQ ID No. 1 AVPYPQR 829.94 6 0.57 SEQ ID No. 2 IEKF 535.63 4 0.30 SEQ ID No. 3 AQTASL 646.69 6 0.12 SEQ ID No. 4 HKEMPF 787.92 4 0.68 SEQ ID No. 5 VKEAMAPK 873.07 8 0.14 SEQ ID No. 6 TQTPVVVPPF 1084.26 8 0.21 SEQ ID No. 7 PPLLL 551.72 6 0.87 SEQ ID No. 8 EMPFPK 747.9 6 0.77 SEQ ID No. 9 PQNIPPL 777.9 6 0.81 SEQ ID No. 10 YQQPVLGPVR 1156.33 7 0.40

[0044] In this embodiment, the physicochemical properties of the above 5 peptide segments with ACE inhibitory activity retrieved from the database were analyzed, and the results are shown in Table 2. The peptide segment with the amino acid sequence of SEQ ID No. 1 (AVPYPQR) had good water solubility and no allergenicity; the peptide segment with the amino acid sequence of SEQ ID No. 8 (EMPFPK) and the peptide segment with the amino acid sequence of SEQ ID No. 4 (HKEMPF) had good water solubility, but had potential allergenicity; the peptide segment with the amino acid sequence of SEQ ID No. 7 (PPLLL) and the peptide segment with the amino acid sequence of SEQ ID No. 9 (PQNIPPL) had poor water solubility, had no allergenicity, and the above 5 peptide segments had no toxicity and could be used for subsequent cell experiments or animal experiments.

[0045] Table 2 Physicochemical properties of five polypeptides

[0046] SEQ ID No. AA sequence Toxicity Water solubility Sensitization SEQ ID No. 1 AVPYPQR None Good No sensitization SEQ ID No. 8 EMPFPK None Good Potential sensitization SEQ ID No. 4 HKEMPF None Good Potential sensitization SEQ ID No. 7 PPLLL None Poor No sensitization SEQ ID No. 9 PQNIPPL None Poor No sensitization

[0047] The present example evaluates the affinity of the receptor and the ligand by the values of "-CDOCKER Energy" and "-CDOCKER Interaction Energy", and the higher the relative value, the tighter the peptide segment binds to ACE; the reason why the peptide segment has higher ACE inhibitory activity is that the peptide segment forms more hydrogen bonds, hydrophobic forces and electrostatic forces with the active site of ACE, and the interaction of multiple hydrogen bonds significantly improves the stability of the binding between ACE and the peptide segment. As shown in the following table, the "-CDOCKER Energy" value and the "-CDOCKER Interaction Energy" value of the ACE-peptide segment complex with the amino acid sequence of SEQ ID No. 1 (AVPYPQR) are 107.316 and 123.436, respectively, and a total of 12 hydrogen bonds, 6 hydrophobic forces and 3 electrostatic forces are formed; it can be seen that the peptide segment with the amino acid sequence of SEQ ID No. 1 (AVPYPQR) has the most stable binding force with ACE among the above-mentioned 5 peptide segments. Figure 1

[0048] The ACE inhibitory activity of the ACE inhibitory peptide (with the amino acid sequence of SEQ ID No. 1) obtained by the above-mentioned screening and identification is determined by colorimetry in the present example: 40 μL of the peptide segment solution (1.0 mg / mL) obtained by screening in Example 1 (dissolved using PBS at a concentration of 1.0 mg / mL), 50 μL of FAPGG buffer (1.0 mmol / L) and 10 μL of ACE solution (0.1 U / mL) (Beijing Solabio Technology Co., Ltd.) are mixed, wherein 40 μL of PBS buffer (0.1 mol / L) is used instead of the above-mentioned ACE solution as a control group; immediately determine the absorbance at 340 nm wavelength using an enzyme marker, and after incubation at 37°C in the dark for 30 min, determine the absorbance at 340 nm wavelength again, and the ACE inhibitory activity is:

[0049]

[0050] In the formula, A1 and A2 represent the absorbance values before and after the reaction of the sample with the substrate and the enzyme; B1 and B2 represent the absorbance values before and after the reaction of the buffer with the substrate and the enzyme.

[0051] The ACE inhibitory activity of the ACE inhibitory peptide with the amino acid sequence of SEQ ID No. 1 is determined in the present example, and the ACE inhibition rate IC 50 value of the ACE inhibitory peptide with the amino acid sequence of SEQ ID No. 1 is 0.1760 mg / mL.

[0052] Example 2: Effect of an ACE inhibitory peptide based on A2β-casein on HUVEC cells ​

[0053] (1) Cytotoxic effects

[0054] This embodiment refers to the CCK-8 reagent kit (Lanjieke Technology Co., Ltd.) instructions for cytotoxicity assay, including the following steps: HUVEC cells were transferred to 96-well plates, and DMEM high-glucose medium (90% DMEM, 10% fetal bovine serum, 1% penicillin / streptomycin solution) was added to prepare a single-cell suspension. 200 μL of the single-cell suspension was added to each well, and the plates were incubated at 5 × 10⁻⁶ cells / well. 3 Cells / well were cultured until adhesion reached 80% or higher. Then, a solution of the ACE inhibitory peptide identified in Example 1 (amino acid sequence shown in SEQ ID No. 1), pre-sterilized by filtration through a sterile membrane, was added at concentrations of 0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL. The cells were then cultured in a CO2 incubator for 24 h. 20 μL of CCK-8 solution was added to each well, and the cells were incubated for 30 min. The absorbance at 450 nm was measured using a microplate reader. A single-cell suspension was used as the control group instead of the ACE inhibitory peptide sample. The blank group contained only a single-cell suspension. Cell viability was:

[0055]

[0056] In the formula, OD (加药) The absorbance values ​​of the wells containing cells, culture medium, CCK-8 solution, and ACE inhibitory peptide solution; OD (空白) The absorbance values ​​are for the wells containing the culture medium and CCK-8 solution; OD (0加药) : Absorbance values ​​for cells, culture medium, and CCK-8 solution wells.

[0057] The results are as follows Figure 2 As shown, when the concentration of the ACE inhibitory peptide solution (amino acid sequence shown in SEQ ID No. 1) obtained in Example 1 was 0-1.0 mg / mL, the ACE inhibitory peptide had no obvious toxic effect on the viability of HUVEC cells, and the cell survival rate was above 97%.

[0058] (2) Effect of ACE inhibitory peptide on NO release in HUVEC cells

[0059] NO is a substance secreted by endothelial cells with the ability to expand vascular tension, which can cause vasodilation by relaxing vascular smooth muscle cells, thereby regulating blood pressure. In this example, the ACE inhibitory peptide solution (amino acid sequence shown in SEQ ID No. 1) identified in Example 1 was used to treat damaged HUVEC cells in different dose groups (0.25, 0.5, 1.0 mg / mL), and the amount of NO released after 24 h of culture was measured. Captopril was used as a positive control, and angiotensin II (AngII) was used as a negative control (Shanghai Aladdin Biochem Technology Co., Ltd.). The specific steps for measuring the content of NO were performed according to the instructions of the NO kit (Bi Yun Tian Biological Technology Co., Ltd.). The cells were lysed using a cell and tissue lysis solution (Bi Yun Tian Biological Technology Co., Ltd.), and the supernatant was obtained by centrifugation at 4°C. The absorbance of the supernatant was measured at a wavelength of 550 nm using a microplate reader, and each group was repeated 3 times.

[0060] The results are shown in Table 1. Figure 3 As shown in Table 1, the content of NO measured in the ACE inhibitory peptide solution (amino acid sequence shown in SEQ ID No. 1) at a concentration of 0.25, 0.5, 1.0 mg / mL was significantly higher than that of the negative control group (19.45 ± 0.23 μmol / L) (P < 0.05). Among them, when the concentration of ACE inhibitory peptide solution was 1.0 mg / mL, the content of released NO was the highest, and the content of NO was 20.24 ± 0.38 μmol / L, but slightly lower than that of the captopril positive control group, and the content of NO was 22.51 ± 0.47 μmol / L. It can be seen that ACE inhibitory peptide (amino acid sequence shown in SEQ ID No. 1) at a certain concentration can significantly promote the release of NO by cells (P < 0.05), and has potential antihypertensive activity.

[0061] (3) Effect of ACE inhibitory peptide on the content of endothelin (ET1) in HUVEC cells

[0062] ET-1 is a factor with strong vasoconstriction effect, and plays an important role in maintaining blood pressure balance. In this embodiment, the ACE inhibitory peptide solution (amino acid sequence shown in SEQ ID No. 1) obtained in Example 1 was treated with different dose groups (0.25, 0.5, 1.0 mg / mL) of the ACE inhibitory peptide solution, and the intracellular ET-1 content was determined after 24 h of culture. Captopril was used as a positive control, and angiotensin II (Ang II) was used as a negative control. The ET-1 content determination method was according to the instructions of the enzyme-linked immunosorbent assay kit (Nanjing Jiancheng Biological Engineering Institute). In this embodiment, the ACE inhibitory peptide solution (amino acid sequence shown in SEQ ID No. 1) obtained in Example 1 was diluted to concentrations of 320 pg / mL, 160 pg / mL, 80 pg / mL, 40 pg / mL, 20 pg / mL and 0 pg / mL, and a standard curve was plotted with the concentration as the abscissa and the determined OD value as the ordinate; the absorbance value was determined at a wavelength of 450 nm using an enzyme marker, and each group had 3 replicates.

[0063] The results are shown in Table 1. Figure 4 As shown in Table 1, compared with the negative control group, the ACE inhibitory peptide solution (amino acid sequence shown in SEQ ID No. 1) at different concentrations and the positive control captopril group had a significant ability to reduce the ET-1 content in HUVEC cells (P<0.05), and as the concentration of the ACE inhibitory peptide solution increased, the ET-1 content secreted by the cells decreased. When the concentration of the ACE inhibitory peptide solution was 1.0 mg / mL, the intracellular ET-1 content was reduced to 17.02±0.80 pg / mL, which was not significantly different from the positive control captopril group (14.42±1.97 pg / mL) (P>0.05).

[0064] In summary, the ACE inhibitory peptide solution (amino acid sequence shown in SEQ ID No. 1) provided by the present application has a similar antihypertensive mechanism and ability as the classic ACE inhibitor captopril, and can enhance vasodilation by down-regulating ET-1 in Ang II-induced HUVEC cells, thereby achieving the effect of reducing blood pressure.

[0065] The content not described in detail in the specification of the present application is known to those skilled in the art. Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing ACE inhibitory peptides based on A2β-casein, characterized in that, The method includes the following steps: S1: A2β-casein and distilled water are mixed evenly in a certain proportion to prepare hydrolysate. First, pepsin is used for water bath enzymatic hydrolysis, and the enzyme is inactivated by high temperature. Then, trypsin is used for digestion enzymatic hydrolysis to obtain enzymatic hydrolysate. S2: The enzymatic hydrolysis products obtained in S1 are subjected to ultrafiltration using C 18 The column was desalted, and then the peptide was obtained by centrifugation and concentration. It was then stored at -20°C. S3: The polypeptide obtained in S2 was analyzed by liquid chromatography-tandem mass spectrometry to obtain the ACE inhibitory peptide, and the ACE inhibitory peptide was artificially synthesized by solid-phase synthesis. The mixing ratio of A2β-casein and distilled water in S1 is 4% substrate concentration; the high-temperature enzyme inactivation temperature is 95℃ and the time is 10 min; The concentration of pepsin in S1 is 2000 U / mg, and the enzymatic hydrolysis reaction of pepsin is carried out at a temperature of 37°C for 2 hours and a pH of 2.5; the concentration of trypsin in S1 is 120 U / mg, and the enzymatic hydrolysis reaction of trypsin is carried out at a temperature of 37°C for 3 hours and a pH of 7.

0. The conditions for liquid chromatography described in S3 are: equipped with C 18 The chromatographic column was an UltiMate 3000 RSLC nano liquid chromatography column; mobile phase A was 0.1% formic acid / 3% DMSO / 97% H2O; mobile phase B was 0.1% formic acid / 3% DMSO / 97% ACN; the flow rate was 300 nL / min. The mass spectrometry conditions described in S3 are as follows: a Q Exactive HF mass spectrometer is used; MS1 has a resolution of 60K@200 m / z; the scan range is 350-1500 m / z; the maximum injection time is 30 ms; MS2 has a resolution of 15K@200 m / z; the AGC target is 1E5; the maximum injection time is 50 ms; and the dynamic exclusion time is 30 s. The amino acid sequence of the ACE inhibitory peptide described in S3 is shown in SEQ ID No.

1.

2. The method according to claim 1, characterized in that, The ultrafiltration process described in S2 involves using a 10 KD ultrafiltration tube to filter the enzymatic hydrolysis products to obtain components with a density of <10 KDa.

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