Wheat ACE inhibitory peptide, preparation method and application thereof, and pharmaceutical composition

Abstract

The present invention relates to the technical field of bioactive peptides, and specifically to wheat ACE inhibitory peptides, preparation methods and applications thereof, and pharmaceutical compositions. The present invention uses gluten powder as a raw material to separate and identify ACE inhibitory peptides, which are LDLPGCPR, LSMLLPR, and VSLLLPR, respectively, and the ACE inhibition rates reach 85.61%, 87.95%, and 70.94%, respectively, which are higher than the biological activity of most existing ACE inhibitory peptides; and in a specific embodiment of the present invention, the ACE inhibitory peptide separated and identified by the present invention is verified by in vitro cell tests to have biological activity of lowering blood pressure, and can be used as a pharmaceutical active ingredient for the preparation of drugs for the prevention / treatment of cardiovascular diseases such as hypertension; the present invention only requires one-step enzymatic hydrolysis, and combines molecular weight screening separation, gel column chromatography, and liquid-mass spectrometry technology to separate and identify small molecule peptides with high inhibitory activity on ACE, which is simple to operate and easy to promote and apply industrially.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and in particular to a wheat ACE inhibitory peptide, a preparation method and application thereof, and a pharmaceutical composition. Background Art

[0002] Hypertension is one of the most common chronic diseases and the most important risk factor for the onset and mortality of heart disease, stroke, and kidney disease. Angiotensin converting enzyme (ACE), also known as kininase II or peptidyl-carboxypeptidase, is an enzyme bound to the endothelial cell membrane. ACE indirectly increases blood pressure by causing vasoconstriction. ACE, angiotensin I and angiotensin II are part of the renin-angiotensin system (RAS), which controls blood pressure by regulating the volume of fluid in the body. It has two main functions: first, it catalyzes the conversion of angiotensin I to angiotensin II, which is an effective vasoconstrictor; second, it degrades bradykinin, an effective vasodilator, and other vasoactive peptides. These two effects make ACE inhibition an important way to treat diseases such as hypertension, heart failure, and type 2 diabetes. ACE inhibitors reduce the formation of angiotensin II and the metabolism of bradykinin, thereby causing systemic dilation of arteries and veins and lowering arterial blood pressure.

[0003] The mechanism of action of synthetic ACE inhibitors such as lisinopril, which are widely used to treat cardiovascular diseases, is to bind to ACE as a competitive inhibitor and interfere with the synthesis of angiotensin II. However, these treatments are accompanied by side effects such as dizziness, dry cough, and inflammatory reactions. Therefore, finding safer, less toxic, milder, and long-term drugs for the treatment of cardiovascular diseases has become a current research hotspot. Many studies have shown that bioactive peptides derived from plant proteins have lower induced resistance than traditional drugs and have a variety of physiological regulatory activities, including antibacterial activity, antithrombotic activity, hypotensive activity, antioxidant activity, and immunomodulatory activity. They are considered to be alternatives to traditional drugs, and many bioactive peptides have been approved for use in therapeutic drugs. Therefore, ACE inhibitory peptides from natural sources have also become one of the current research hotspots, especially obtaining bioactive peptides from industrial and agricultural by-products has become the core research direction for separating and screening bioactive peptides.

[0004] As a byproduct of the wheat starch industry, wheat gluten is rich in protein. Due to its wide source and low cost, it is a high-quality source of active peptides. Through the analysis of the amino acid sequence of wheat protein, it was found that its sequence is rich in a variety of bioactive peptides, and it has great application potential in bioactive peptides. At present, many kinds of bioactive peptides have been identified from the hydrolyzate of wheat gluten, including ACE inhibitory peptides. The separation of ACE inhibitory peptides from wheat gluten reported so far still has certain limitations in industrial application. On the one hand, the types of ACE inhibitory peptides that have been separated and identified are relatively small, and the inhibitory activity on ACE is not high enough, mostly 50% to 60%, which cannot meet the application requirements of drug research and development; on the other hand, it requires complex and cumbersome compound enzyme hydrolysis or multi-step hydrolysis or complex ultrasonic / heat treatment steps, and the preparation and purification steps are cumbersome, which is not conducive to industrial application. Summary of the invention

[0005] In order to overcome the defects of the prior art, one of the purposes of the present invention is to provide a wheat ACE inhibitory peptide with a high ACE inhibition rate and a pharmaceutical value for treating cardiovascular diseases.

[0006] The second object of the present invention is to provide a method for preparing wheat ACE inhibitory peptide, which is simple to operate and suitable for industrial application.

[0007] The third object of the present invention is to provide a pharmaceutical composition for preventing / treating hypertension, which uses the ACE inhibitory peptide provided by the present invention as a pharmaceutical active ingredient and has a pharmaceutical effect of lowering blood pressure.

[0008] A fourth object of the present invention is to provide an application of wheat ACE inhibitory peptide in the preparation of drugs for preventing / treating cardiovascular diseases.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The wheat ACE inhibitory peptide comprises one or a mixture of more than one of the following three small molecule peptides: small molecule peptide 1: its amino acid sequence is LDLPGCPR, as shown in the sequence list SEQ ID NO.1; small molecule peptide 2: its amino acid sequence is LSMLLPR, as shown in the sequence list SEQ ID NO.2; small molecule peptide 3: its amino acid sequence is VSLLLPR, as shown in the sequence list SEQID NO.3.

[0011] The preparation method of the wheat ACE inhibitory peptide comprises the following steps:

[0012] 1) taking gluten powder for enzymatic hydrolysis to prepare enzymatic hydrolyzate;

[0013] 2) purifying the enzymatic hydrolysate prepared in step 1) using a filter membrane with a molecular weight cutoff of 3 kDa to obtain an enzymatic hydrolysate 1 with a molecular weight ≤ 3 kDa;

[0014] 3) The enzymatic hydrolysate 1 prepared in step 2) is sequentially separated by column chromatography and / or liquid chromatography-mass spectrometry to separate and identify small molecule peptides with high ACE inhibition rate.

[0015] The specific method of the enzymatic hydrolysis treatment in the above step 1) is: adding gluten powder to deionized water to dissolve and stir to prepare a gluten powder solution with a mass content of 3-8%, 200-500W ultrasound for 10-20min, adjusting pH=8.5, heating to 60-65°C, adding alkaline protease in an amount of 3000-4000U / g, enzymolysis at 55-60°C for 1-3h, inactivating the enzyme in a boiling water bath for 10-15min, centrifuging at a speed of 4000-5000r / min for 10-15min after cooling, taking the supernatant to obtain an enzymatic hydrolyzate.

[0016] The specific method of purifying the enzymatic hydrolysate prepared in step 1) using a filter membrane with a molecular weight cutoff of 3 kDa in the above step 2) is: processing pressure 0.8-2.0 MPa, processing volume 1-5 L / h, collecting the retentate, vacuum freeze-drying, and obtaining enzymatic hydrolysate 1.

[0017] In the above step 3), the hydrolysate 1 is re-dissolved with deionized water to a sample solution with a concentration of 20 to 100 mg / mL and then purified again. Sephadex G-15 glucose gel column filtration chromatography is used, and the eluent is continuously collected in the order of outflow to obtain component 1, component 2, and component 3, a total of 3 different components. The 3 components are vacuum freeze-dried and their ACE inhibition rates are determined respectively. The component with the highest ACE inhibition rate is used as column chromatography hydrolysate 2 for subsequent processing.

[0018] Optionally, the specific conditions of Sephadex G-15 glucose gel column filtration chromatography are: 0.2-0.3 μm filter membrane for impurity filtration, ultrapure water as eluent, flow rate set at 0.5-2 mL / min, wavelength set at 280 nm, and collection in continuous tubes according to the order in which the eluent flows out, 4 mL per tube, collection for 4 min, and determination of absorbance of the obtained components by spectrophotometer to obtain three components, component I, component II and component III, and component II with the highest absorbance is determined to have the highest ACE inhibition rate, which is used as column chromatography hydrolysate 2 for subsequent treatment.

[0019] Optionally, the hydrolysate 2 is separated and identified by liquid chromatography-mass spectrometry. Specifically, the LC-MS / MS detection liquid chromatography conditions are as follows: pre-column: 150 μm idx 5 cm, packing: Reprosil-Pur 120C18-AQ 3 μm; analytical column: 100 μm i.dx 180 mm, packing: Reprosil-Pur 120C18-AQ 3 μm; mobile phase A: 0.1% formic acid aqueous solution by volume; mobile phase B: 80% acetonitrile / 0.1% formic acid aqueous solution by volume; flow rate: 300 nL / min; analysis time for each component: 60 min;

[0020] A total of three peptides were detected and identified, namely, small molecule peptide 1: its amino acid sequence is LDLPGCPR, as shown in the sequence list SEQ NO.1; small molecule peptide 2: its amino acid sequence is LSMLLPR, as shown in the sequence list SEQ NO.2; small molecule peptide 3: its amino acid sequence is VSLLLPR, as shown in the sequence list SEQ NO.3.

[0021] A pharmaceutical composition for preventing / treating hypertension, wherein the active ingredient comprises the above-mentioned ACE inhibitory peptide and / or the enzymatic hydrolysate 2 prepared by the above-mentioned preparation method.

[0022] The ACE inhibitory peptide and the enzymatic hydrolysate 2 prepared by the preparation method can be used to prepare drugs or health foods for preventing / treating cardiovascular diseases. The prepared drugs have the efficacy of lowering blood pressure.

[0023] Beneficial effects of the present invention:

[0024] 1) The present invention uses gluten powder as a raw material to separate and identify ACE inhibitory peptides, namely LDLPGCPR, LSMLLPR, and VSLLLPR, with ACE inhibition rates of 85.61%, 87.95%, and 70.94%, respectively, solving the problem of low biological activity of existing ACE inhibitory peptides; and in a specific embodiment of the present invention, an in vitro cell test verifies that the ACE inhibitory peptides separated and identified by the present invention have biological activity of lowering blood pressure, and can be used as a pharmaceutical active ingredient for preparing drugs for preventing / treating cardiovascular diseases such as hypertension;

[0025] 2) The preparation method of the present invention only requires one step of enzymatic hydrolysis, and combines molecular weight screening separation, gel column chromatography and liquid chromatography-mass spectrometry technology to separate and identify small molecule peptides with high inhibitory activity against ACE, thereby overcoming the defects of the prior art that requires multiple enzymatic hydrolysis and complex temperature and ultrasonic processes, and is simple to operate and easy to promote and apply in industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the ACE inhibition rate test results of the enzymatic hydrolysate components with different molecular weights in Example 1;

[0027] Figure 2 This is the elution curve of the enzymatic hydrolysate gel column chromatography in Example 1;

[0028] Figure 3 This is a schematic diagram of the ACE inhibition rate test results of different enzymatic hydrolysate gel column chromatography fractions collected in Example 1;

[0029] Figure 4 This is a schematic diagram of the ACE inhibition rate test results of different small molecule peptides separated and identified in Example 1;

[0030] Figure 5 The secondary mass spectrum of the small molecule peptide LDLPGCPR separated and identified in Example 1;

[0031] Figure 6 is the secondary mass spectrum of the small molecule peptide LSMLLPR separated and identified in Example 1;

[0032] Figure 7 is the secondary mass spectrum of the small molecule peptide VSLLLPR separated and identified in Example 1;

[0033] Figure 8 The two-dimensional structure (left) and three-dimensional structure (right) of the small molecule peptide LDLPGCPR binding to the ACE enzyme;

[0034] Fig. 9 The two-dimensional structure (left) and three-dimensional structure (right) of the small molecule peptide LSMLLPR binding to the ACE enzyme;

[0035] Fig.10 The two-dimensional structure (left) and three-dimensional structure (right) of the small molecule peptide VSLLLPR binding to the ACE enzyme;

[0036] Fig.11 This is a schematic diagram of the test results of the effects of each group in Example 3 on the secretion of ET-1 by HUVECs cells. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with specific embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and test example can be obtained from commercial sources.

[0038] The ACE inhibition rate test method in the following embodiments and test examples is as follows:

[0039] ACE inhibitory activity was determined by UV spectrophotometry:

[0040] Prepare the reaction reagents:

[0041] Preparation of borate buffer solution: weigh 0.6183g boric acid and 1.7532g sodium chloride respectively, dissolve in appropriate amount of pure water, make up to 100mL, and prepare 0.1mol / L boric acid solution; weigh 0.9534g sodium tetraborate and 1.7532g sodium chloride respectively, dissolve in appropriate amount of pure water, make up to 100mL, and prepare 0.025mol / L borax solution; gradually and slowly add the prepared borax solution into the boric acid solution, stir magnetically, and adjust the pH value to 8.3.

[0042] Preparation of 0.1U / mL ACE solution: Dissolve ACE in 1mL of the above borate buffer solution to a concentration of 0.1U / mL.

[0043] Preparation of HHL (hippuryl histidyl leucine) solution: weigh 4 mg of HHL and dissolve it in 1.864 mL of the above borate buffer solution.

[0044] The detection method is shown in Table 1:

[0045] Table 1 In vitro detection method of ACE inhibition rate

[0046]

[0047]

[0048] After adding ethyl acetate, shake for 2 minutes, centrifuge at 4000r / min for 10 minutes, collect 800μl supernatant, evaporate in a boiling water bath at 100℃ for 30 minutes, add 1ml ultrapure water to the evaporated residue, mix well, and measure the absorbance at 228nm. Repeat three times for one sample, and calculate the ACE inhibitory activity of the sample to be tested according to the following formula. The calculation method is as follows:

[0049] ACE inhibition rate = (BS) / (BK) × 100%

[0050] Wherein: B is the absorbance of the group without inhibitor (sample to be tested); S is the absorbance of the sample group with inhibitor (sample to be tested); K is the absorbance of the blank group.

[0051] Example 1

[0052] This example provides a method for preparing wheat ACE inhibitory peptide, and the specific steps are as follows:

[0053] 1) Enzymatic hydrolysis:

[0054] Add gluten powder into deionized water, dissolve and stir to prepare a gluten powder solution with a mass concentration of 5%, ultrasonic power 300W for 17 minutes, adjust the pH to 8.5, heat to 60°C, add 3600U / g alkaline protease according to the mass of the substrate, hydrolyze at 60°C for 2 hours, inactivate the enzyme in a boiling water bath for 10 minutes, centrifuge at 4000r / min for 15 minutes after cooling, take the supernatant to obtain the enzymatic solution.

[0055] 2) Preliminary purification treatment:

[0056] The enzymatic hydrolysate prepared in step 1) was initially purified by using filter membranes with a molecular weight cutoff of 5 kDa and 3 kDa, respectively, at a processing pressure of 1.0 MPa and a processing volume of 4 L / h, and the retentates with molecular weights greater than 5 kDa, 3 to 5 kDa, and ≤3 kDa were collected, respectively, and after vacuum freeze-drying, the ACE inhibition rates were detected according to the detection method provided by the present invention. The results are as follows: Figure 1 As shown, it was shown that the hydrolysate with a molecular weight of ≤3 kDa had the highest ACE inhibition rate and was further purified as hydrolysate 1;

[0057] 3) Gel column chromatography to further separate and purify the enzymatic hydrolysate 1:

[0058] The hydrolysate 1 was re-dissolved in deionized water to a sample solution with a concentration of 60 mg / mL and purified again. Sephadex G-15 glucose gel column filtration chromatography was used. 0.2 μm filter membrane was used to filter and remove impurities. The eluent was ultrapure water. The flow rate was set to 1 mL / min, the sample volume was 5 mL, the wavelength was set to 280 nm, and the eluent was collected in succession according to the order in which the eluent flowed out. Each tube had 4 mL and was collected for 4 minutes. The gel column chromatography elution curves of the collected components in each tube are shown in the following figure. Figure 2 After the components of each tube were freeze-dried in vacuum, the ACE inhibitory activity of each tube component was determined, as shown in FIG. Figure 3 As shown, component II with the highest ACE inhibition rate is column chromatography hydrolyzate 2, which is used to further separate and identify small molecule peptides.

[0059] 4) Identification of amino acid sequence of wheat ACE inhibitory peptide:

[0060] The hydrolysate 2 was analyzed and identified by liquid chromatography-mass spectrometry:

[0061] Sample pretreatment: Accurately weigh 6.6 mg of sample into a centrifuge tube and use a pipette to accurately transfer 660 μL of 50 mmol / L NH 4 HCO 3 In a centrifuge tube, prepare sample solution 1. Use a pipette to take 30 μL of sample solution 1 into a centrifuge tube, and then add 70 μL of 50 mmol / L NH 4 HCO 3In a centrifuge tube, add DTT (dithiothreitol) solution to a final concentration of 10mmol / L, reduce in a 56℃ water bath for 1h, add iodoacetamide solution to a final concentration of 50mmol / L, and react in the dark for 40min. Desalt using a self-filled desalting column, and evaporate the solvent in a 45℃ vacuum centrifugal concentrator.

[0062] Liquid chromatography conditions for LC-MS / MS detection: pre-column: 150μm idx 5cm, packing: Reprosil-Pur120C18-AQ 3μm; analytical column: 100μm idx 180mm, packing: Reprosil-Pur 120C18-AQ 3μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 80% acetonitrile / 0.1% formic acid aqueous solution; flow rate: 300nL / min; analysis time for each component: 60min.

[0063] The mass spectrometry raw files were analyzed for peptide sequences using the software PEAKS Studio10.6 De novo method, and the inhibition rate of each peptide segment screened was detected, such as Figure 4 As shown, among them, small molecule peptide 1: LDLPGCPR (as shown in the sequence list SEQ ID NO.1), small molecule peptide 2: LSMLLPR (as shown in the sequence list SEQ ID NO.2), small molecule peptide 3: VSLLLPR (as shown in the sequence list SEQ ID NO.3), these three ACE inhibitory peptides have a higher ACE inhibition rate, among which the secondary mass spectra of small molecule peptide 1, small molecule peptide 2, and small molecule peptide 3 are shown as follows Figure 5 , Figure 6 and Figure 7 shown.

[0064] It should be noted that, in the preparation process of the ACE inhibitory peptide of the present invention, the parameters in the enzymatic hydrolysis step can be adjusted within a certain range, and the ACE inhibitory peptide of the present invention can also be separated and identified. The specific mass concentration of the gluten powder solution is 3%-8%; the ultrasonic treatment condition is 200-500W ultrasonic treatment for 10-20min; the temperature, enzyme dosage and enzymatic hydrolysis time and other parameter conditions are: after heating to 60-65°C, alkaline protease is added at an amount of 3000-4000U / g, and enzymatic hydrolysis is carried out at 55-60°C for 1-3h; the enzyme inactivation and centrifugal separation parameter conditions are: inactivating the enzyme in a boiling water bath for 10-15min, and centrifuging at a speed of 4000-5000r / min for 10-15min after cooling.

[0065] Example 2 Evaluation of ACE Inhibitory Activity

[0066] The biological activity of the ACE inhibitory peptide isolated and identified by the present invention was preliminarily characterized by molecular docking simulation of the peptide:

[0067] According to the crystal structure of angiotensin converting enzyme protein (PDB ID: 1O8A), a complete angiotensin converting enzyme protein model was obtained using SWISS-MODEL homology modeling. The protein-protein docking program in ROSETTA software was used to carry out molecular docking of three peptide molecules with angiotensin converting enzyme. The molecular docking of ACE and peptide molecules adopted the Localdocking mode. The position of the docking system was manually adjusted to place the peptide molecule near the catalytic center of the receptor protein (ACE) ( and set the ligand to be able to After 500 random dockings, the system with the highest score (lowest score) was selected as the most suitable docking conformation.

[0068] like Figure 8 As shown, LDLPGCPR (small molecule peptide 1) interacts with 9 amino acid residues at the catalytic center of ACE, including: Gln124, Asp128, Asn131, Ala134, Thr135, Asn249, Asp252, Thr266, and Asn338. There are 2 hydrogen bonds and 2 salt bridges between the protein and the polypeptide.

[0069] like Fig. 9 As shown, LSMLLPR (small molecule peptide 2) interacts with five amino acid residues at the catalytic center of ACE, including Asp128, Asn131, Asp252, Asp264, and Thr266. There are two hydrogen bonds and two salt bridges between the protein and the polypeptide.

[0070] like Fig.10 As shown, VSLLLPR (small molecule peptide 3) interacts with 8 amino acid residues at the catalytic center of ACE, including: Asn31, His117, Thr135, Asp141, Trp144, Glu267, Leu270, and Gly271. There is one hydrogen bond and four salt bridges between the protein and the polypeptide.

[0071] The free binding energy values ​​of the three small molecule peptides separated and identified by the present invention are all less than -8.00 kcal / mol, especially VSLLLPR (small molecule peptide 3) has the lowest binding free energy (-9.50 kcal / mol), indicating that these three small molecule peptides can interact well with ACE and can form stable complexes.

[0072] Example 3 In vitro cell assay to further characterize the activity of the ACE inhibitory peptide isolated and identified by the present invention

[0073] Principle of in vitro cell test design: ET-1 is mainly produced by vascular smooth muscle cells, endothelial cells, cardiomyocytes and other cells, which can cause vasoconstriction and have a pressor effect. The ACE inhibitory peptide isolated and identified by the present invention is used to treat HUVECs cells, and the changes in ET-1 levels in the cell culture medium can be detected to reflect the hypotensive effect of the ACE inhibitory peptide isolated and identified by the present invention.

[0074] Experimental method: Commercially available HUVECs cells were used as experimental subjects. They were first cultured with high-glucose DMEM complete medium for 24 hours, and the culture medium was discarded to serve as the experimental cells for the following groups;

[0075] The blank control group was cultured for 48 h with 0.2 mL of serum-free culture medium;

[0076] The model control group was added with 0.2 mL of 1000 μmol / L H 2 O 2 Culture for 48 hours;

[0077] The experimental group was first added with 0.2 mL of 1000 μmol / L H 2 O 2 After culturing for 24 hours, the culture medium was discarded, and 0.2 mL of serum-free culture medium containing a sample at a concentration of (0.4 (low), 0.8 (medium), 1.2 mg / mL (high)) was added and cultured for 24 hours; wherein the sample was the ACE inhibitory peptide VSLLLPR separated and identified by the present invention;

[0078] The positive control group was treated with 0.2 mL of 1000 μmol / L H 2 O 2 After culturing for 24 h, 0.2 mL of serum-free culture medium containing 1.2 mg / mL captopril was added and cultured for another 24 h;

[0079] Each group had 6 wells and was tested using an endothelin-1 (ET-1) kit.

[0080] The results are as follows Fig.11 As shown, the wheat ACE inhibitory peptide VSLLLPR isolated and identified in the present invention has a certain inhibitory effect on the secretion of ET-1 in HUVECs cells, indicating that the ACE inhibitory peptide isolated and identified in the present invention has biological activity of lowering blood pressure and can be used as a pharmaceutical active ingredient for preparing drugs for preventing / treating cardiovascular diseases such as hypertension.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Wheat ACE inhibitory peptide, characterized in that It comprises one or more of the following three small molecule peptides as a mixture: small molecule peptide 1: its amino acid sequence is LDLPGCPR, as shown in the sequence list SEQ ID NO.1; small molecule peptide 2: its amino acid sequence is LSMLLPR, as shown in the sequence list SEQ ID NO.2; small molecule peptide 3: its amino acid sequence is VSLLLPR, as shown in the sequence list SEQ ID NO.

3.

2. A method for preparing wheat ACE inhibitory peptide, characterized in that: The steps include: 1) taking gluten powder for enzymatic hydrolysis to prepare enzymatic hydrolyzate; 2) purifying the enzymatic hydrolysate prepared in step 1) using a filter membrane with a molecular weight cutoff of 3 kDa to obtain an enzymatic hydrolysate 1 with a molecular weight ≤ 3 kDa; 3) The enzymatic hydrolysate 1 prepared in step 2) is subjected to column chromatography separation and liquid chromatography-mass spectrometry separation to identify small molecule peptides with high ACE inhibition rate.

3. The method for preparing the wheat ACE inhibitory peptide according to claim 2, characterized in that: The specific method of enzymatic hydrolysis in step 1) is as follows: adding gluten powder to deionized water, dissolving and stirring to prepare a gluten powder solution with a mass concentration of 3-8%, ultrasonicating at 200-500W for 10-20min, adjusting the pH to 8.5, heating to 60-65°C, adding alkaline protease at a dosage of 3000-4000U / g, enzymolyzing at 55-60°C for 1-3h, inactivating the enzyme in a boiling water bath for 10-15min, centrifuging at a speed of 4000-5000r / min for 10-15min after cooling, taking the supernatant to obtain an enzymatic hydrolyzate.

4. The method for preparing the wheat ACE inhibitory peptide according to claim 3, characterized in that: The specific method of purifying the enzymatic hydrolysate prepared in step 1) using a filter membrane with a molecular weight cutoff of 3 kDa in step 2) is as follows: treatment pressure 0.8-2.0 MPa, treatment volume 1-5 L / h, collecting the retentate, vacuum freeze-drying, and obtaining enzymatic hydrolysate 1.

5. The method for preparing the wheat ACE inhibitory peptide according to claim 4, characterized in that: In step 3), the hydrolysate 1 is re-dissolved in deionized water to a sample solution with a concentration of 20 to 100 mg / mL and then purified again. Sephadex G-15 glucose gel column filtration chromatography is used, and the eluent is continuously collected in the order of outflow to obtain component 1, component 2, and component 3, a total of 3 different components. The 3 components are vacuum freeze-dried and their ACE inhibition rates are determined respectively. The component with the highest ACE inhibition rate is used as column chromatography hydrolysate 2 for subsequent processing.

6. The method for preparing the wheat ACE inhibitory peptide according to claim 5, characterized in that: The specific conditions of Sephadex G-15 glucose gel column filtration chromatography are as follows: 0.2-0.3 μm filter membrane for impurity removal, ultrapure water as eluent, flow rate set at 0.5-2 mL / min, wavelength set at 280 nm, and collection in continuous tubes according to the order in which the eluent flows out, 4 mL per tube, collection for 4 min, and the absorbance of the obtained components is measured by a spectrophotometer to obtain three components, component I, component II and component III, and component II with the highest absorbance is determined to have the highest ACE inhibition rate, which is used as column chromatography hydrolysate 2 for subsequent treatment.

7. The method for preparing the wheat ACE inhibitory peptide according to claim 6, characterized in that: The hydrolysate 2 was separated and identified by liquid chromatography-mass spectrometry. Specifically, the LC-MS / MS detection liquid chromatography conditions were as follows: pre-column: 150 μm i.dx 5cm, packing: Reprosil-Pur 120C18-AQ 3μm; analytical column: 100μm idx 180mm, packing: Reprosil-Pur 120C18-AQ 3μm; mobile phase A: 0.1% formic acid aqueous solution by volume; mobile phase B: 80% acetonitrile / 0.1% formic acid aqueous solution by volume; flow rate: 300nL / min; analysis time for each component: 60min; A total of three peptides were detected and identified, namely, small molecule peptide 1: its amino acid sequence is LDLPGCPR, as shown in the sequence list SEQ NO.1; small molecule peptide 2: its amino acid sequence is LSMLLPR, as shown in the sequence list SEQ NO.2; small molecule peptide 3: its amino acid sequence is VSLLLPR, as shown in the sequence list SEQ NO.

3.

8. A pharmaceutical composition for preventing / treating hypertension, characterized in that: The active ingredients thereof include the ACE inhibitory peptide according to claim 1, and / or the enzymatic hydrolysate 2 prepared by the preparation method according to any one of claims 1 to 6.

9. Use of the ACE inhibitory peptide according to claim 1 in the preparation of drugs or health foods for preventing / treating cardiovascular diseases, characterized in that: The drug has a blood pressure lowering effect.

10. Use of the enzymolysate 2 prepared by the preparation method according to any one of claims 1 to 6 in preparing drugs or health foods for preventing / treating cardiovascular diseases, characterized in that: The drug has a blood pressure lowering effect.

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