Lotus seed protein peptide with ACE inhibitory activity and application thereof

Abstract

The invention discloses preparation and application of lotus seed protein peptide with ACE (angiotensin converting enzyme) inhibitory activity, and belongs to the technical field of food-borne active peptide development. According to the ACE inhibitory peptide WVPRYFPF and the preparation method thereof, the ACE inhibitory peptide is rapidly screened by utilizing a polypeptiomics and molecular docking technology, the defects of time consumption and labor consumption caused by step-by-step separation and purification are avoided, the obtained ACE inhibitory peptide WVPRYFPF has extremely high ACE inhibitory activity, IC50 of the ACE inhibitory peptide WVPRYFPF is 16.7 mu M, and the ACE inhibitory peptide WVPRYFPF can be used for assisting or replacing hypertension drugs. The lotus seed protein peptide disclosed by the invention has the characteristic of inhibiting ACE enzyme activity in vitro, and animal experiments prove that the protein peptide can also play a role in lowering blood pressure in a spontaneous hypertension rat (SHR).

Description

Technical Field

[0001] The invention belongs to the technical field of development of food-derived bioactive peptides, and specifically relates to a lotus seed protein peptide with ACE inhibitory activity and application thereof. Background Art

[0002] Hypertension is a common disease. It usually develops slowly. Typical symptoms include headache, fatigue or restlessness, arrhythmia, palpitations and tinnitus. Improper treatment can cause other health problems such as stroke, myocardial infarction, and renal failure. Excessive systolic blood pressure can cause more than 10 million deaths worldwide each year. The types of antihypertensive drugs used clinically are divided into angiotensin-converting enzyme inhibitors (ACEI), β-receptor blockers, calcium channel blockers, diuretics, etc., mostly ACEI; so far, although synthetic antihypertensive drugs such as captopril and lisinopril have a good antihypertensive effect, long-term use will have certain side effects, such as sinus tachycardia, inferior myocardial infarction, cough, dizziness, etc. It is an urgent need to find safe and effective bioactive substances that can exert ACE inhibitory effects to achieve the effect of lowering blood pressure.

[0003] The regulation mechanism of blood pressure is relatively complex, including the renin-angiotensin system (RAAS system) and the kallikrein-kinin system (KKS system). In the RAAS system, angiotensin I (AngI) released by the liver will be further hydrolyzed by angiotensinase (ACE enzyme) into angiotensin II (AngII) with vasoconstriction function, thereby increasing the body's blood pressure. At the same time, AngII binds to receptor AT1 to release aldosterone (ALD), promoting the reabsorption of water and sodium ions, causing an increase in sodium concentration and blood volume, resulting in an increase in the body's blood pressure.

[0004] Many food-derived proteins themselves do not have antihypertensive activity, but through enzymatic hydrolysis or fermentation, the proteins therein are decomposed and peptides with antihypertensive activity are released. At present, a large number of highly active ACE inhibitory peptides have been isolated from a variety of food-derived proteins, showing good antihypertensive effects. For example, the patent with publication number CN115947781A discloses the production of antihypertensive peptides using fermented wheat as raw material. For another example, the patent with publication number CN117247425A discloses a method for producing antihypertensive peptides using corn germ protein as raw material. This natural peptide segment can avoid the side effects of synthetic drugs, but the types of antihypertensive peptides from plant sources currently reported are limited, and new plant antihypertensive peptides are urgently needed to be developed. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a protein peptide having ACE inhibitory activity.

[0008] To solve the above technical problems, the present invention provides the following technical solution: a protein peptide with ACE inhibitory activity, characterized in that the amino acid sequence of the protein peptide is WVPRYFPF, i.e. Trp-Val-Pro-Arg-Tyr-Phe-Pro-Phe.

[0009] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of a protein peptide in the preparation of a blood pressure-lowering product.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for screening and identifying protein peptides having ACE inhibitory activity, characterized in that: comprising:

[0011] Lotus seed protein was extracted from lotus seed powder by alkali dissolution and acid precipitation.

[0012] Using protease to hydrolyze lotus seed protein to produce lotus seed peptide;

[0013] Ultrafiltration was used for separation and purification, and the fraction <3 kDa with an activity almost twice that before ultrafiltration was selected for sequencing;

[0014] The lotus seed peptides <3 kDa were sequenced by peptidomics;

[0015] Screening for potential highly active ACE inhibitory peptides;

[0016] The peptides obtained by screening were verified using molecular docking technology;

[0017] Computer simulation of gastrointestinal digestion;

[0018] In vitro solid phase synthesis and verification of ACE inhibitory activity.

[0019] As a preferred embodiment of the method of the present invention, the lotus seed protein is extracted from the lotus seed powder by alkali dissolution and acid precipitation, wherein the solid-liquid ratio of lotus seed powder to water is 1:10 to 1:25.

[0020] As a preferred embodiment of the method of the present invention, the lotus seed protein is hydrolyzed by protease to produce lotus seed peptides, wherein the hydrolysis temperature is 45-60° C. and the pH is 6.5-8.5.

[0021] As a preferred embodiment of the method of the present invention, the screening of potential highly active ACE inhibitory peptides includes: predicting and scoring the biological activity of peptides in PeptideRanker; querying the protein source of the peptides in the protein database; predicting toxicity through Toxinpred; predicting sensitization through AllerTOP; selecting from the sequenced peptides those of protein origin, not modified by functional groups, with a peak area greater than 1.00×107, an activity score>0.8, 3-12 amino acid sequences, non-toxic and non-sensitizing sequences that have not been disclosed, and simultaneously screening peptides with hydrophobic amino acids at both the C-terminus and the N-terminus.

[0022] As a preferred embodiment of the method of the present invention, the verification of the screened peptide segments using molecular docking technology includes:

[0023] Preparation of ACE enzyme PDB file: Retrieve the protein crystal structure of ACE enzyme from the protein database, remove redundant ligands and H2O from 1O86 to facilitate subsequent docking experiments with peptides;

[0024] The screened peptide sequences were docked with 1O86 to obtain binding energy, and the potential ACE inhibitory activity was evaluated based on the size of the binding energy. The lower the molecular docking binding energy, the stronger the potential ACE inhibitory activity.

[0025] As a preferred embodiment of the method of the present invention, the molecular docking binding energies of the peptide segments obtained by screening are all less than -9.4 kcal / mol.

[0026] As a preferred embodiment of the method of the present invention, the computer simulation of gastrointestinal digestion includes: enzymatic cleavage of peptides by the BIOPEP-UWM:ANALYSIS function in the BIOPEP database; and selection of chymotrypsin A, trypsin, and pepsin for simulated enzymatic cleavage.

[0027] Beneficial effects of the present invention:

[0028] (1) The lotus seed peptide extracted by the present invention has higher in vitro ACE inhibitory activity compared with the other ten plant-derived ACE inhibitory peptides.

[0029] (2) The present invention effectively identified the highly active ACE inhibitory peptide WVPRYFPF from lotus seed protein peptides, and its IC 50The molecular docking results showed that the docking binding energy was -12.1 kcal / mol, and WVPRYFPF formed pocket hydrogen bonds with two residues His353 and His513 in the ACE active pocket, non-pocket hydrogen bonds with six residues Asn66, Arg124, Trp220, Tyr360, Ser517, and Arg522, and hydrophobic interactions with nine residues Tyr62, Glu123, Arg124, Glu143, Ile204, Ala207, Trp357, Phe512, and Val518.

[0030] (3) The present invention is the first to separate a lotus seed protein peptide with ACE inhibitory effect from lotus seed protein. The polypeptide has been verified by spontaneously hypertensive rats (SHR) experiments and has good characteristics of reducing blood pressure in SHR rats. After 2 weeks of lotus seed peptide intervention, it begins to show a good blood pressure lowering effect. Therefore, the lotus seed peptide provided by the present invention can be used to inhibit ACE enzyme activity in vitro and prepare antihypertensive drugs, enriching the types of antihypertensive drugs and greatly improving the medicinal value of lotus seed protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0032] Figure 1 This is a comparison chart of the activity of ACE inhibitory peptides derived from plants;

[0033] Figure 2 It is a schematic diagram of the molecular structure of WVPRYFPF;

[0034] Figure 3 The protein crystal structure of ACE;

[0035] Figure 4 This is the molecular docking and interaction force analysis diagram between WVPRYFPF and ACE;

[0036] Figure 5 It is the ACE inhibition rate determination of WVPRYFPF;

[0037] Figure 6 is the standard curve of Ang II concentration in rat serum;

[0038] Figure 7 This is the effect of lotus seed peptide on the concentration of Ang II in the serum of spontaneously hypertensive rats. DETAILED DESCRIPTION

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0040] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0041] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0042] Experimental materials and reagents used in the examples of the present invention: raw material lotus seed powder, Amway (Shanghai) Technology Development Co., Ltd.; composite protease (15000U / g), Novozymes (China) Biotechnology Co., Ltd.; angiotensin converting enzyme (ACE), N-[3-(2-furyl) acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG), Sigma-Aldrich, USA; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, Shanghai Yuanye Biotechnology Co., Ltd.; rat angiotensin II (AngII) ELISA kit, Shanghai ELISA Biotechnology Co., Ltd.

[0043] Instruments used in the embodiments of the present invention: SW22 electric constant temperature water bath, JULABO, Germany; LXJ-IIB centrifuge, Shanghai Anting Instrument Factory; FE20K precision pH meter, AL104 electronic analytical balance, Shanghai Mettler Instrument Company; LGJ-10E freeze dryer, Ningbo Senz Biotechnology Co., Ltd.; Muliskan GO full wavelength microplate reader, Thermo Fisher Scientific, China; rat non-invasive sphygmomanometer, Kent, USA.

[0044] Example 1

[0045] This example is a comparison of the activities of ACE inhibitory peptides from various plant sources. By consulting domestic and foreign literature, the IC values ​​of dozens of common ACE inhibitory peptides from plant sources were summarized. 50 The result is Figure 1 As shown in the figure, by comparison, it can be found that the in vitro ACE inhibitory activity of lotus seed peptide is at a medium to high level, IC 50The value is relatively low, which has great research value. Lotus seeds, as mature seeds of the Nymphaeaceae plant, are widely used as food. Lotus seeds are rich in protein, about 19.0-25.0%. Compared with the phytochemicals (starch, phenolic substances) in lotus seeds, the bioactive substances of lotus seed protein have not been widely studied. Therefore, the lotus seed ACE inhibitory peptide was further studied.

[0046] Example 2

[0047] This embodiment provides a method for obtaining lotus seed protein hydrolysate from lotus seed protein and separating and identifying ACE inhibitory peptides, comprising the following steps:

[0048] (1) Extracting lotus seed protein from lotus seed powder by alkali dissolution and acid precipitation

[0049] After the lotus seed powder is sieved through an 80-mesh sieve, it is dissolved in water at a solid-liquid ratio of 1:10-1:25 (w / v), the pH is adjusted to 9.5-12.0 with NaOH, and the mixture is extracted by stirring at room temperature for 3.0-6.0 hours, centrifuged at 8000-10000 r / min for 10-30 minutes, the supernatant is taken, the pH is adjusted to an isoelectric point of 3.2-4.0 with HCl, the mixture is allowed to stand for 20-40 minutes, centrifuged at 8000-10000 r / min for 10-30 minutes, the supernatant is discarded, the precipitate is taken, deionized water is added for re-dissolution, the pH is adjusted to a neutral 7.0, the mixture is dialyzed at 4°C in a dialysis bag for 2-3 days, and finally freeze-dried to obtain lotus seed protein.

[0050] (2) Using protease to hydrolyze lotus seed protein to produce lotus seed peptide

[0051] Weigh an appropriate amount of lotus seed protein powder to prepare a protein solution with a substrate concentration of 4-10%, preheat at 80-100° C. for 10-30 min to ensure that the lotus seed protein is thermally denatured, adjust the temperature to 45-60° C. and pH 6.5-8.5 to achieve the optimal enzymolysis conditions of the composite protease, add protease according to the enzyme-substrate ratio (6000-12000 U / g protein), stir continuously during the reaction, and use NaOH to maintain the pH of the reaction system until it is constant. After the hydrolysis is completed, stop heating and stirring, place in a boiling water bath for 10-30 min to inactivate the enzyme, adjust the pH to 7.0 with HCl, then centrifuge at 8000-10000 r / min for 10-30 min, collect the supernatant into a dialysis bag, dialyze at 4° C. for 2-3 days, obtain lotus seed peptide after freeze drying, and store at -20° C. for standby use.

[0052] (3) Ultrafiltration for separation and purification, and selection of highly active components <3 kDa for the next step of sequencing

[0053] Take a 50mL 3kDa ultrafiltration tube for ultrafiltration, dissolve the lotus seed peptide in step (2) in water and place it in the ultrafiltration tube, centrifuge at 3000-4000r / min for 5-10min, repeat the operation, take the component <3kDa in the lower layer of the ultrafiltration tube, freeze-dry it for later use.

[0054] (4) Sequencing of lotus seed peptides <3 kDa by peptideomics

[0055] The lotus seed peptide sample <3 kDa obtained in step (3) was analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was a Q ExactiveTM mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200. A total of 5 μL lotus seed protease hydrolyzed sample (analytical column: Acclaim PepMap C18, 75 μm x25 cm) was loaded, and the sample was separated by a gradient of 120 min. The column flow was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 2% B phase, and increased to 35% in 110 minutes with a nonlinear gradient, and increased to 100% within 1 minute, and maintained for 9 minutes. The mass spectrometer was operated in data-dependent acquisition mode and automatically switched between MS and MS / MS acquisition.

[0056] The mass spectrometry parameters were set as follows:

[0057] ①MS: scan range (m / z): 200-1800; resolution: 70000; AGC target: 3e6; maximum injection time: 60ms; ②HCD-MS / MS: resolution: 17500; AGC target: 5e4; maximum injection time: 50ms; collision energy: 27; dynamic exclusion time: 20s. The tandem mass spectra were analyzed by PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). PEAKSDB searched the uniprot_Cicer arietinum (version 202112, 24812 entries) database and set None enzyme digestion. The search parameters are as follows: fragment ion mass tolerance: 0.02Da, parent ion mass tolerance: 7ppm, variable modification: Oxidation (M) 15.99, Deamidation (NQ) 0.98, Acetylation (Protein N-term) 42.01. Protein card value: -10lgP ≥ 0, at least 1 unique peptide; peptide card value: -10lgP ≥ 20.

[0058] A total of 6534 peptides were identified after sequencing the components of lotus seed protein hydrolysate <3kDa using peptideomics.

[0059] (5) Using multiple screening conditions to screen for potential high-activity ACE inhibitory peptides from the 6534 peptides identified in (4), the main steps include:

[0060] ① Use PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) to predict the bioactivity of peptides;

[0061] ②Query the protein source of the peptide in the protein database (https: / / www.uniprot.org / );

[0062] ③ Toxicity prediction through Toxinpred (https: / / crdd.osdd.net / raghava / toxinpred / );

[0063] ④ Prediction of sensitization through AllerTOP (https: / / www.ddg-pharmfac.net / AllerTOP / );

[0064] ⑤ Select the peptides from the sequenced peptides that are of protein origin, not modified by functional groups, and have a peak area greater than 1.00×10 7 , activity score>0.8, 3-12 amino acid sequences, non-toxic and non-sensitizing and unpublished sequences, and peptides with hydrophobic amino acids at both the C-terminus and the N-terminus.

[0065] (6) Using molecular docking technology to verify the peptide obtained in step (5), mainly including the following steps:

[0066] ① Preparation of ACE enzyme PDB file: Retrieve the protein crystal structure of ACE enzyme (code: 1O86) from the protein database, remove redundant ligands and H2O from 1O86 for subsequent docking experiments with peptides;

[0067] ② The selected peptide sequences were docked with 1O86 to obtain binding energy, and the potential ACE inhibitory activity was evaluated based on the size of the binding energy. The lower the molecular docking binding energy, the stronger the potential ACE inhibitory activity.

[0068] (7) Computer simulation of gastrointestinal digestion

[0069] The peptides with low molecular docking binding energy screened out in step (6) are subjected to computer simulated gastrointestinal digestion to check whether the screened peptides have the potential to resist gastrointestinal digestion. The specific steps are as follows:

[0070] ① Enzyme digestion of peptides was performed using the BIOPEP-UWM:ANALYSIS function in the BIOPEP database (https: / / biochemia.uwm.edu.pl);

[0071] ② Select chymotrypsin A (chymotrypsin A), trypsin (trysin), and pepsin (pH 1.3) for simulated enzymatic cleavage, and choose peptides that can resist digestion or have potential activity after digestion for subsequent solid-phase synthesis.

[0072] (8) In vitro solid phase synthesis and verification of ACE inhibitory activity.

[0073] Specifically, 6 peptides were screened out, and their molecular docking binding energies were all less than -9.4 kcal / mol. The ACE inhibitory activities of the 6 peptides were verified after in vitro solid phase synthesis, as shown in Table 1.

[0074] Table 1 Docking binding energy of 6 peptides with ACE molecule

[0075]

[0076] The results showed that the lotus seed protein-derived ACE inhibitory peptides were obtained by the method of the present invention, and the amino acid sequences thereof were WVPRYFPF, GFEWISF, GDDGFEWISF, FALP, IFSGF, and FLPVLL, respectively.

[0077] Example 3

[0078] This embodiment is a method for detecting the activity of ACE inhibitory peptides, comprising the following steps:

[0079] Add 40 μL HEPES buffer (pH 8.2, 80 mmol / L, containing 0.3 mol / L NaCl) and a certain concentration of captopril (or the sample solution to be tested) to a 96-well plate, then add 50 μL FAPGG (1 mmol / L), preheat at 37°C for 10 min, and then draw 10 μL 0.1 U / mL ACE into the 96-well plate. Measure the initial absorbance value (A1 and B1) at 340 nm, and then measure the end point absorbance (A2 and B2) after incubation at 37°C in the dark for 30 min.

[0080] Captopril was used as a positive control.

[0081] The ACE inhibition rate was calculated as follows:

[0082]

[0083] Among them, A1-A2: absorbance change of blank group within 30 minutes; B1-B2: absorbance change of sample group within 30 minutes.

[0084] The results of ACE inhibitory activity detection are shown in Tables 2 and 3.

[0085] Table 2 Comparison of ACE inhibition rate before and after ultrafiltration

[0086]

[0087] The results showed that within a certain concentration range, the ACE inhibitory activity of lotus seed peptide <3kDa after ultrafiltration was significantly better than that of lotus seed peptide before ultrafiltration, IC 50 It also dropped from 0.276±0.019 mg / mL before ultrafiltration to 0.122±0.014 mg / mL, so the component <3 kDa after ultrafiltration was selected for sequencing.

[0088] Table 3 IC of 6 synthetic peptides 50 Test results

[0089]

[0090] The results showed that three peptides (WVPRYFPF, GFEWISF, and GDDGFEWISF) had strong ACE inhibitory activity, with IC 50 All of them were less than 200 μM, among which WVPRYFPF had the strongest ACE inhibitory activity (IC 50 =16.7 μM).

[0091] Example 4

[0092] This example is an analysis of the ACE inhibitory peptide WVPRYFPF, specifically:

[0093] (1) Structure of WVPRYFPF and ACE

[0094] like Figure 2 As shown, the molecular structure of WVPRYFPF was obtained by Chemdraw and Autodock.

[0095] like Figure 3 Shown is the protein crystal structure of ACE(1O86).

[0096] (2) Molecular docking of WVPRYFPF and ACE

[0097] like Figure 4As shown, WVPRYFPF forms pocket hydrogen bonds with two residues of ACE, His353 and His513, forms non-pocket hydrogen bonds with six residues, Asn66, Arg124, Trp220, Tyr360, Ser517, and Arg522, and forms hydrophobic interactions with nine residues, Tyr62, Glu123, Arg124, Glu143, Ile204, Ala207, Trp357, Phe512, and Val518.

[0098] (3) ACE IC of WVPRYFPF 50

[0099] According to the method of Example 3, the peptide sample WVPRYFPF was prepared into solutions of different concentrations to determine its ACE inhibition rate and plot the results to calculate its ACE half inhibition concentration (IC 50 ), which is the peptide concentration required to achieve 50% inhibition of ACE activity.

[0100] The results are as follows Figure 4 As shown, the IC of peptide sample WVPRYFPF 50 The value is 16.7 μM, which has a relatively low IC value compared with thousands of ACE inhibitory peptides reported in the BIOPEP-UWM database. 50 value..

[0101] Example 5

[0102] This example is an experiment on the effect of lotus seed ACE inhibitory peptide on spontaneously hypertensive rats, comprising the following steps:

[0103] Spontaneously hypertensive rats (SHR), male, 12 weeks old, weighing 200±20g, a total of 18 rats, were individually housed in stainless steel cages before the experiment, with light and darkness controlled for 12 hours each day, temperature 24°C, and standard feed used in the experiment, adaptive feeding for one week, during which adaptive blood pressure measurements were performed to reduce stress response during the experiment. Before each measurement, the rats were placed on a 37°C heating plate to preheat for 5-10 minutes, and then the measurement was performed. One measurement was set to 15 cycles, and at least 5 sets of valid data were collected for each rat.

[0104] 18 rats were randomly divided into 3 groups, 6 rats in each group, and oral administration was used to reduce the harm to the rats. The 3 groups were a control group of sterile tap water, a positive control group of captopril, and an experimental group intervened by lotus seed peptide. Among them, the dose of captopril was 0.05mg / mL, the concentration of lotus seed peptide was 15mg / mL, and the drug was administered once a day for 6 consecutive weeks. The blood pressure of the rats was measured every two weeks, and the blood pressure was measured by the rat tail artery systolic pressure method.

[0105] During the experiment, the blood pressure of the blank group control mice increased slightly with age, from an initial 215 mmHg at 12 weeks of age to around 220 mmHg at 14 weeks of age, after which the blood pressure stabilized and no longer increased; the blood pressure of the positive control group and the lotus seed peptide group began to decline significantly after 2 weeks, with a decrease of 30 to 40 mmHg, and remained stable for 3 to 6 weeks, with no obvious upward trend.

[0106] like Figure 6 , 7 As shown in the figure, after 6 weeks of intervention, the content of angiotensin II (Ang II) in the lotus seed peptide group was significantly lower than that in the blank control group and was equivalent to that in the positive control group (p < 0.05). The experimental results show that the lotus seed protein peptide of the present invention reduces the generation of angiotensin II (Ang II) by inhibiting the activity of ACE enzyme and regulating the RAAS system, thereby lowering blood pressure.

[0107] The above results show that the protein peptide of the present invention also has a good antihypertensive effect in vivo, can be used as a raw ingredient for developing antihypertensive drugs and functional foods, and has great development prospects.

[0108] 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 preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A protein peptide having ACE inhibitory activity, characterized in that The amino acid sequence of the protein peptide is WVPRYFPF, i.e., Trp-Val-Pro-Arg-Tyr-Phe-Pro-Phe.

2. Use of the protein peptide as claimed in claim 1 in the preparation of antihypertensive products.

3. The use according to claim 2, characterized in that The product is a health food, medicine or chemical.

4. The method for screening and identifying protein peptides with ACE inhibitory activity according to claim 1, characterized in that: include, Lotus seed protein was extracted from lotus seed powder by alkali dissolution and acid precipitation. Using protease to hydrolyze lotus seed protein to produce lotus seed peptide; Ultrafiltration was used for separation and purification, and the fraction <3 kDa with an activity almost twice that before ultrafiltration was selected for sequencing; The lotus seed peptides <3 kDa were sequenced by peptidomics; Screening for potential highly active ACE inhibitory peptides; The peptides obtained by screening were verified using molecular docking technology; Computer simulation of gastrointestinal digestion; In vitro solid phase synthesis and verification of ACE inhibitory activity.

5. The method according to claim 4, characterized in that: The lotus seed protein is extracted from the lotus seed powder by using an alkali dissolution and acid precipitation method, wherein the solid-liquid ratio of the lotus seed powder to water is 1:10-1:

25.

6. The method according to claim 4, characterized in that: The lotus seed peptide is produced by hydrolyzing lotus seed protein with protease, wherein the hydrolysis temperature is 45-60° C. and the pH is 6.5-8.

5.

7. The method according to claim 4, characterized in that: The screening of potential highly active ACE inhibitory peptides includes: predicting and scoring the biological activity of peptides in PeptideRanker; querying the protein source of peptides in a protein database; predicting toxicity through Toxinpred; predicting sensitization through AllerTOP; selecting protein-derived peptides from sequenced peptides that are not modified by functional groups and have a peak area greater than 1.00×10 7 , activity score>0.8, 3-12 amino acid sequences, non-toxic and non-sensitizing and unpublished sequences, and peptides with hydrophobic amino acids at both the C-terminus and the N-terminus.

8. The method according to claim 4, characterized in that: The method of verifying the peptide segments obtained by screening by using molecular docking technology includes: Preparation of ACE enzyme PDB file: Retrieve the protein crystal structure of ACE enzyme from the protein database, remove redundant ligands and H2O from 1O86 to facilitate subsequent docking experiments with peptides; The screened peptide sequences were docked with 1O86 to obtain binding energy, and the potential ACE inhibitory activity was evaluated based on the size of the binding energy. The lower the molecular docking binding energy, the stronger the potential ACE inhibitory activity.

9. The method according to claim 8, characterized in that: The molecular docking binding energies of the peptides obtained by the screening are all less than -9.4 kcal / mol.

10. The method according to claim 4, characterized in that: The computer simulation of gastrointestinal digestion includes: enzymatically cutting the peptide segments through the BIOPEP-UWM:ANALYSIS function in the BIOPEP database; and selecting chymotrypsin A, trypsin, and pepsin for simulated enzymatic cutting.

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