ACE inhibitory peptide VR8, and application and preparation method thereof

Abstract

The invention discloses ACE (angiotensin converting enzyme) inhibitory peptide VR8 as well as application and a preparation method thereof, and relates to the technical field of biology, and the key point of the technical scheme is that the amino acid sequence of the ACE inhibitory peptide VR8 is VPNSAEER. The invention also discloses application of the ACE inhibitory peptide VR8 in functional food for assisting in lowering blood pressure. The ACE inhibitory peptide VR8 obtained by the invention is extracted from bovine cheese protein, controls blood pressure while effectively inhibiting in-vitro ACE activity, and has the advantages of high specificity, small toxic and side effects and good curative effect, so that daily use and health care requirements are met in a mode of increasing the dosage.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to an ACE inhibitory peptide VR8, and an application and a preparation method thereof. Background Art

[0002] Hypertension is a chronic cardiovascular disease. Angiotensin converting enzyme (ACE) has the effect of constricting blood vessels and increasing blood pressure. Antihypertensive drugs can control blood pressure by inhibiting ACE activity, but long-term use will produce certain side effects. Natural bioactive peptides derived from dairy products have the effect of inhibiting ACE activity and lowering blood pressure. At the same time, given that it is derived from food-derived protein, it has high specificity, low toxicity and side effects, and good efficacy. A larger dose can be used during use to meet the needs of daily use and health care.

[0003] ACE inhibitory peptides are inactive in the original protein, but can be released from the original protein by enzymatic hydrolysis. The Chinese patent with the announcement number CN100398661C discloses a casein hydrolyzate and its production process and application, which uses an enzyme group containing extracellular enzymes from Aspergillus oryzae to hydrolyze casein, with XP and XPP as the core component preparation method. In addition, Zhong Yuwang et al. reported the process of preparing ACE inhibition in buffalo cheese using moringa seed rennet in "Application of Moringa Seed Rennet in the Preparation of Buffalo Cheese and Analysis of ACE Inhibitory Peptides"; Tang Haixia et al. reported the process of producing ACE inhibitory peptides in sheep milk casein using alkaline protease in "Process Optimization and Inhibitory Mechanism of Enzymatic Preparation of Sheep Milk Casein ACE Inhibitory Peptides". However, how to prepare ACE inhibitory peptides using enzyme groups and trypsin mixed complex enzymes is still a difficult technical problem to solve.

[0004] The patent with announcement number CN117551170B discloses an ACE inhibitory peptide LTFSY and STHPHF from tuna, with IC50 values ​​of 20.72 and 30.71 μg / mL, respectively. Sun Meiling et al. isolated two ACE inhibitory peptides in the article "Separation and Purification of ACE Inhibitory Peptides from Sea Cucumber Boiled Liquid", with IC50 values ​​of 0.74 and 1.77 mg / mL, respectively. However, the composition of casein hydrolysis products is complex, and the chain length and amino acid composition of different ACE inhibitory peptides vary greatly. There is no unified rule, and it is impossible to obtain them by simple inference through the existing ACE inhibitory peptide structure. Summary of the invention

[0005] In view of this, the first object of the present application is to provide an ACE inhibitory peptide VR8 to achieve the purpose of having in vitro ACE inhibitory activity and blood pressure lowering function. The specific scheme is as follows:

[0006] An ACE inhibitory peptide VR8, the amino acid sequence of the ACE inhibitory peptide VR8 is: VPNSAEER.

[0007] Preferably: the molecular weight of the ACE inhibitory peptide VR8 is 900.4301Da, and the in vitro ACE inhibition rate under the condition of 1 mg / ml is greater than 70%.

[0008] The second object of the present application is to provide an application of ACE inhibitory peptide VR8, comprising using the ACE inhibitory peptide VR8 as described above and applying it to functional foods that assist in lowering blood pressure.

[0009] The third object of the present application is to provide a method for preparing the above ACE inhibitory peptide VR8, comprising the following steps:

[0010] Step 1, taking cow casein, adding protease for enzymolysis, controlling the enzymolysis temperature to 37°C-55°C, the enzyme addition amount to 2%-6%, the pH to be greater than 7, the enzymolysis time to be 3h-12h, and obtaining an enzymolysis product;

[0011] Step 2: Filter the enzymatic hydrolysis product to remove the residue to obtain a clarified polypeptide enzymatic hydrolysate, and freeze-dry the polypeptide enzymatic hydrolysate to obtain a polypeptide freeze-dried powder.

[0012] Preferably, the added amount of casein in the milk casein is 2%.

[0013] Preferably: the enzymatic hydrolysis pH is controlled at 8.0, the enzymatic hydrolysis temperature is 37° C., the enzyme addition amount is 5%, and the protease is composed of trypsin and enzyme group X in a ratio of 3:1, and the enzymatic hydrolysis time is 6 hours.

[0014] Preferably, the method further comprises screening out the ACE inhibitory peptide VR8 portion from the polypeptide lyophilized powder.

[0015] Preferably, the method of screening out the ACE inhibitory peptide VR8 part from the polypeptide freeze-dried powder comprises the following steps:

[0016] Step 3: using LC-MS / MS to perform mass spectrometry on the polypeptide freeze-dried powder obtained in step 2, and analyzing the results of the mass spectrometry using mass spectrometry analysis software to obtain several polypeptide sequences;

[0017] Step 4: Molecular docking of the polypeptide sequence with the ACE protein is performed by software. Before docking, the 2D structure of the polypeptide is converted into a 3D structure by energy minimization, and polypeptide sequences with strong binding ability to the ACE protein are screened;

[0018] Step 5: Solid phase synthesize the peptide with the highest prediction score and detect its in vitro ACE inhibition rate.

[0019] Preferably: in step 3, the conditions for mass spectrometry are as follows:

[0020] ①, in the liquid phase method, the chromatographic column is C18, 3μm, 250mm X 75μm, phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300n l / min, the injection volume is 4μl, the chromatographic gradient is 60min, and the specific elution gradient is: 0-48min: phase A is uniformly reduced from 95% to 60%; 48-55min: phase A is uniformly reduced from 60% to 30%; 55-56min: phase A is uniformly reduced from 30% to 0; 56-60min: maintain 0% phase A;

[0021] ②Mass spectrometry method: Orbitrap Experiment 480, positive ion detection mode, primary resolution 120000, AGC setting 300, scanning range 200-1600 m / z;

[0022] ③. Set MIPS mode to peptide ide, select valence states 1-5, secondary resolution 15000, and separation window 1.6m / z.

[0023] Preferably: in step 5, the in vitro ACE inhibition rate of the ACE inhibitory peptide VR8 at a concentration of 1 mg / ml is detected to be 70.12%.

[0024] It can be seen from the above scheme that the present application provides an ACE inhibitory peptide VR8, its application and preparation method, and the ACE inhibitory peptide VR8, its application and preparation method have the following beneficial effects: the ACE inhibitory peptide VR8 obtained by the preparation method of the present invention has the effect of effectively inhibiting the activity of ACE in vitro, thereby achieving the purpose of blood pressure control, and can be applied to the development of functional foods that assist in lowering blood pressure. At the same time, because the ACE inhibitory peptide VR8 is extracted from milk casein, it has the advantages of high specificity, low toxicity and side effects, and good efficacy, so as to meet daily use and health care needs in a way that increases the dosage of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the effect of different enzymes on ACE inhibition rate in this example;

[0026] Figure 2 This is the effect of different enzyme mixing ratios on the ACE inhibition rate when the enzymatic hydrolysis time is 3 hours in this embodiment;

[0027] Figure 3This is the effect of different enzyme mixing ratios on the ACE inhibition rate when the enzymatic hydrolysis time is 5 hours in this embodiment;

[0028] Figure 4 This is the effect of different enzyme addition amounts on ACE inhibition rate in this example;

[0029] Figure 5 It is the effect of different enzymolysis time on ACE inhibition rate in this example;

[0030] Figure 6 It is a schematic diagram of the overall structure of the interaction between VPNSAEER and ACE in this embodiment;

[0031] Figure 7 It is a schematic diagram of the local structure of the interaction between VPNSAEER and ACE in this embodiment;

[0032] Figure 8 It is a schematic diagram of the two-dimensional structure of the amino acid residues of the binding site between VPNSAEER and ACE in this example. DETAILED DESCRIPTION

[0033] In order to make the technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0034] An ACE inhibitory peptide VR8, whose amino acid sequence is: VPNSAEER. The molecular weight of the ACE inhibitory peptide VR8 is 900.4301Da. According to the test, the in vitro ACE inhibition rate under the condition of 1mg / ml is greater than 70%.

[0035] A method for preparing the above ACE inhibitory peptide VR8 comprises the following steps:

[0036] Step 1, taking cow casein, adding protease for enzymolysis, controlling the enzymolysis temperature to 37°C-55°C, the enzyme addition amount to 2%-6%, the pH to be greater than 7, the enzymolysis time to be 3h-12h, and obtaining an enzymolysis product;

[0037] Step 2, filtering the enzymatic hydrolysis product, removing the residue to obtain a clarified polypeptide enzymatic hydrolysate, and freeze-drying the polypeptide enzymatic hydrolysate to obtain a polypeptide freeze-dried powder;

[0038] Step 3: using LC-MS / MS to perform mass spectrometry on the polypeptide freeze-dried powder obtained in step 2, and analyzing the results of the mass spectrometry using mass spectrometry analysis software to obtain several polypeptide sequences;

[0039] Step 4: Molecular docking of the polypeptide sequence with the ACE protein is performed by software. Before docking, the 2D structure of the polypeptide is converted into a 3D structure by energy minimization, and polypeptide sequences with strong binding ability to the ACE protein are screened;

[0040] Step 5: Solid phase synthesize the peptide with the highest prediction score and detect its ACE inhibition rate.

[0041] The conditions for mass spectrometry are as follows:

[0042] ①, in the liquid phase method, the chromatographic column is C18, 3μm, 250mm X 75μm, phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300n l / min, the injection volume is 4μl, the chromatographic gradient is 60min, and the specific elution gradient is: 0-48min: phase A is uniformly reduced from 95% to 60%; 48-55min: phase A is uniformly reduced from 60% to 30%; 55-56min: phase A is uniformly reduced from 30% to 0; 56-60min: maintain 0% phase A;

[0043] ②Mass spectrometry method: Orbitrap Experiment 480, positive ion detection mode, primary resolution 120000, AGC setting 300, scanning range 200-1600 m / z;

[0044] ③. Set MIPS mode to peptide ide, select valence states 1-5, secondary resolution 15000, and separation window 1.6m / z.

[0045] In order to improve the in vitro ACE inhibition rate, the casein added to the milk casein was 2%. During the enzymatic hydrolysis, the pH was controlled to be 8.0, the enzymatic hydrolysis temperature was 37°C, the enzyme addition amount was 5%, and the protease was composed of trypsin and enzyme group X in a ratio of 3:1, and the enzymatic hydrolysis time was 6h.

[0046] In the examples of the present application, the ACE inhibition rate was used as an assessment index, and experiments were conducted under different mixing ratios, enzyme addition amounts and enzymolysis times according to different selected proteases to examine the effects of various factors on the ACE inhibition rate. The specific experimental results are described in detail below.

[0047] (1) Effects of different protease types on ACE inhibition activity

[0048] like Figure 1As shown, four proteases with relatively compatible enzymatic hydrolysis conditions were selected, namely trypsin (37°C, pH8), alkaline protease (55°C, pH8), papain (55°C, pH7) and flavor protease (55°C, pH7), and single enzyme hydrolysis was performed for 3 hours. The ACE inhibitory activity was detected after the enzymatic hydrolysis samples were freeze-dried. The results showed that trypsin hydrolysis had the most significant improvement in ACE inhibitory activity, with the highest ACE inhibition rate reaching 60.7% (1 mg / ml), followed by papain (38.9%) and alkaline protease (32.75%), while flavor protease had the worst effect (23.7%).

[0049] (2) Effects of different enzyme mixing ratios on ACE inhibition activity

[0050] like Figure 2 , Figure 3 As shown, trypsin was selected as the main hydrolyzing protease, and enzyme group X was added for further optimization according to the actual situation. Under the conditions of controlling the enzyme addition amount of 2%, temperature of 37°C and pH 7-8, the ratio of trypsin and enzyme group X was changed from 1:3 to 3:1, and the ACE inhibition rate of the samples hydrolyzed for 3h and 5h was detected. The results showed that as the proportion of enzyme group X decreased, the inhibitory activity of ACE gradually increased; within the tested range, when the ratio of trypsin and enzyme group X was 3:1, the ACE inhibition rate of the hydrolyzed sample was the highest.

[0051] (3) Effects of different enzyme addition amounts on ACE inhibition activity

[0052] like Figure 4 As shown in the figure, when the enzyme addition amount increases from 2% to 5%, the ACE inhibition rate shows a gradual upward trend. However, when the enzyme addition amount increases to 6%, the ACE inhibition rate reverses and decreases. It is speculated that the addition of a large amount of enzyme will cause the active peptide to fragment and destroy its ability to bind to ACE. Therefore, 5% is a relatively ideal enzyme addition amount under this condition.

[0053] (4) Effects of different enzymatic hydrolysis times on ACE inhibition activity

[0054] like Figure 5 As shown in the figure, as the hydrolysis time increases, the ACE inhibition rate of the product gradually increases; when the hydrolysis time reaches 6h, the ACE inhibition rate reaches the maximum. When the enzymatic hydrolysis time is further increased to 9h, the ACE inhibition rate begins to decrease. Therefore, 6h is a relatively ideal enzymatic hydrolysis time under this condition.

[0055] (5) Determination and verification of the optimal enzymatic hydrolysis process

[0056] The optimal process parameters for hydrolyzing milk casein are pH 8.0, substrate addition 2%, enzymatic hydrolysis temperature 37°C, enzyme addition 5% (based on substrate concentration), the mixing ratio of the two enzymes is trypsin: enzyme group X = 3:1, and the enzymatic hydrolysis time is 6h.

[0057] The above conditions were used to prepare milk casein hydrolysate. The ACE inhibition rates of the products hydrolyzed for 3h, 6h and 9h at a concentration of 1mg / ml were 71.89%, 72.21% and 59.75%, respectively, while the ACE inhibition rate of the imported raw material was 58.12%.

[0058] In the embodiment of the present application, the specific conditions of mass spectrometry are as follows: the chromatographic column in the liquid phase method is C18, 3μm, 250mmX 75μm (Eksigent), phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300n l / min, the injection volume is 4μl, 60min chromatographic gradient, and the specific elution gradient is: 0-48min: phase A is uniformly reduced from 95% to 60%; 48-55min: phase A is uniformly reduced from 60% to 30%; 55-56min: phase A is uniformly reduced from 30% to 0; 56-60min: maintain 0% phase A. Mass spectrometry method: Orbitrap Exp l oris 480 (Thermofisher), positive ion detection mode, primary resolution is 120000, AGC is set to 300, and the scanning range is 200-1600m / z. The MIPS mode was peptide ide, valence states 1-5 were selected, the secondary resolution was 15000, and the separation window was 1.6 m / z. As shown in Table 1, by comparing the protein database, 19 peptide sequences were obtained by screening high-abundance active peptides with peak areas ≥ 5×107 and active peptide chain lengths ≤ 8 amino acids.

[0059] Table 1. Composition of high-abundance active peptides in casein hydrolysate

[0060]

[0061]

[0062] In step 4, the peptide sequence was molecularly docked with the ACE protein using the Discovery Studio software, and the 3D structure of the protein ACE was downloaded from the RCSB protein database (PDB ID: 108A). The results of molecular docking are shown in Table 2 below. The greater the interaction energy, the stronger the binding ability of the peptide to the ACE enzyme, and the more likely it is to inhibit the activity of the ACE enzyme.

[0063] Table 2. Prediction of interaction between high-abundance active peptides and ACE

[0064]

[0065]

[0066] like Figure 6 , Figure 7 , Figure 8 As shown in Table 3, the interaction energy between VPNSAEER and ACE is the largest. The specific situation of molecular docking is shown in Table 3 below.

[0067] Table 3. Detailed information of VPNSAEER and ACE molecular docking

[0068]

[0069]

[0070] Meanwhile, in step 5, the ACE inhibition rate is tested as follows:

[0071] The samples and hydrolyzed samples were diluted 9-fold with distilled water to a final concentration of 1 mg / ml.

[0072] 0.1 mM boric acid-borax buffer: weigh 12.37 g of boric acid and dilute to 1000 mL with distilled water.

[0073] Weigh 19.07 g of sodium tetraborate and dilute to 1000 mL with distilled water.

[0074] Take 175 mL of sodium tetraborate solution and 325 mL of boric acid solution, adjust the pH to 8.3 with hydrochloric acid or sodium oxide, and finally add 17.532 g of sodium chloride, dissolve and adjust the volume to 1000 mL.

[0075] 5 mM HHL solution: Weigh 2 mg HHL and dissolve it in 920 μL of 0.1 mol / L borate buffer (pH 8.3) containing 0.3 mol / L sodium chloride.

[0076] 0.1U / mL ACE solution: The whole bottle of ACE contains 0.25U, which is dissolved in 125μL of 0.01M potassium phosphate buffer (pH7.0) containing 0.5M sodium chloride, stored at -20℃, and diluted 20 times with 0.1M borate buffer (pH8.3) containing 0.3M sodium chloride before use.

[0077] 1M hydrochloric acid solution: Slowly add 9 mL of hydrochloric acid along the wall into 100 mL of distilled water.

[0078] 1 mg / mL hippuric acid standard solution: weigh 10 mg hippuric acid and dilute to 10 mL with distilled water. When using, dilute with distilled water to make 0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL standard solutions.

[0079] Reaction conditions: Take a 2mL centrifuge tube, add 80μL HHL solution (5mM) and 20μL sample, shake and mix thoroughly, keep warm at 37℃ for 5min, then add 20μL ACE solution (0.1U / mL), mix and keep warm at 37℃ for 30min. Add 200μL 1M hydrochloric acid solution to terminate the reaction. Filter with 0.22μm filter membrane and use HPLC to determine the content of hippuric acid. In the control experiment, use 20μL distilled water instead of sample.

[0080] Liquid phase conditions: Chromatographic column: Agilent ZORBAX SB-C18 chromatographic column (4.6×250mm, 5μm). Mobile phase: The volume ratio of acetonitrile and water is 25:75, and 0.05% trifluoroacetic acid is added to the water phase. Detection parameters: Column oven temperature 30℃, injection volume 20μL, flow rate 0.5mL / min, detection wavelength 228nm.

[0081] The calculation formula of ACE inhibition rate is: ACE inhibition rate (%) = (BA) x 100 / B, wherein A is the hippuric acid concentration in the sample; B is the hippuric acid concentration in the blank control.

[0082] Finally, the in vitro ACE inhibition rate of VPNSAEER was 70.12% at a concentration of 1 mg / ml.

[0083] It has been tested that the ACE inhibitory peptide VR8 obtained through the preparation method of the present invention through comprehensive enzymatic hydrolysis, screening, and solid-phase synthesis interacts with the ACE enzyme, thereby effectively inhibiting the activity of ACE, so that the ACE inhibitory peptide VR8 can be applied to functional foods that assist in lowering blood pressure.

[0084] In summary, the present application provides an ACE inhibitory peptide VR8, its application and preparation method. The ACE inhibitory peptide VR8 obtained by the preparation method of the ACE inhibitory peptide VR8 has the effect of effectively inhibiting the activity of ACE in vitro, thereby achieving the purpose of blood pressure control, and can be applied to the development of functional foods that assist in lowering blood pressure. At the same time, since the ACE inhibitory peptide VR8 is extracted from milk casein, it has the advantages of high specificity, low toxicity and side effects, and good efficacy, so as to meet daily use and health care needs in a way that increases the dosage of the drug.

[0085] The "first", "second", "third", "fourth", etc. (if any) referred to in this application are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods or devices.

[0086] It should be noted that the descriptions involving "first", "second", etc. in this application are only for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0087] Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. An ACE inhibitory peptide VR8, characterized in that: The amino acid sequence of the ACE inhibitory peptide VR8 is: VPNSAEER.

2. The ACE inhibitory peptide VR8 according to claim 1, characterized in that: The molecular weight of the ACE inhibitory peptide VR8 is 900.4301Da, and the in vitro ACE inhibition rate is greater than 70% under the condition of 1 mg / ml.

3. An application of ACE inhibitory peptide VR8, characterized in that: The invention comprises using the ACE inhibitory peptide VR8 as claimed in claim 1 or 2 and applying it to functional food for assisting in lowering blood pressure.

4. A method for preparing the ACE inhibitory peptide VR8 according to claim 1 or 2, characterized in that: The steps include: Step 1: Take cow casein, add protease for enzymolysis, control the enzymolysis temperature to 37°C-55°C, add enzyme amount to 2%-6%, pH greater than 7, enzymolysis time to 3h-12h, and obtain enzymolysis product; Step 2: Filter the enzymatic hydrolysis product to remove the residue to obtain a clarified polypeptide enzymatic hydrolysate, and freeze-dry the polypeptide enzymatic hydrolysate to obtain a polypeptide freeze-dried powder.

5. The method for preparing the ACE inhibitory peptide VR8 according to claim 4, characterized in that: The added amount of casein in the milk casein is 2%.

6. The method for preparing the ACE inhibitory peptide VR8 according to claim 4, characterized in that: The enzymatic hydrolysis pH is controlled at 8.0, the enzymatic hydrolysis temperature is 37° C., the enzyme addition amount is 5%, and the protease is composed of trypsin and enzyme group X in a ratio of 3:1, and the enzymatic hydrolysis time is 6 hours.

7. The method for preparing the ACE inhibitory peptide VR8 according to claim 4, characterized in that: The method also includes screening out the ACE inhibitory peptide VR8 portion from the polypeptide freeze-dried powder.

8. The method for preparing the ACE inhibitory peptide VR8 according to claim 7, characterized in that: The method of screening out the ACE inhibitory peptide VR8 part from the polypeptide freeze-dried powder comprises the following steps: Step 3: using LC-MS / MS to perform mass spectrometry on the polypeptide freeze-dried powder obtained in step 2, and analyzing the results of the mass spectrometry using mass spectrometry analysis software to obtain several polypeptide sequences; Step 4: Molecular docking of the polypeptide sequence with the ACE protein is performed by software. Before docking, the 2D structure of the polypeptide is converted into a 3D structure by energy minimization, and polypeptide sequences with strong binding ability to the ACE protein are screened; Step 5: Solid phase synthesize the peptide with the highest prediction score and detect its in vitro ACE inhibition rate.

9. The method for preparing the ACE inhibitory peptide VR8 according to claim 8, characterized in that: In step 3, the conditions for mass spectrometry are as follows: ①. In the liquid phase method, the chromatographic column is C18, 3μm, 250mm X 75μm, phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300nl / min, the injection volume is 4μl, and the chromatographic gradient is 60min. The specific elution gradient is: 0-48min: phase A is uniformly reduced from 95% to 60%; 48-55min: phase A is uniformly reduced from 60% to 30%; 55-56min: phase A is uniformly reduced from 30% to 0; 56-60min: maintain 0% phase A; ②Mass spectrometry method: Orbitrap Exploris 480, positive ion detection mode, primary resolution 120000, AGC setting 300, scanning range 200-1600 m / z; ③. Set MIPS mode to peptide, select valence states 1-5, secondary resolution to 15000, and separation window to 1.6m / z.

10. The method for preparing the ACE inhibitory peptide VR8 according to claim 8, characterized in that: In step 5, the in vitro ACE inhibition rate of the ACE inhibitory peptide VR8 at a concentration of 1 mg / ml was detected to be 70.12%.

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