Angiotensin converting enzyme inhibiting peptides, screening method and use thereof

Abstract

The present application belongs to the field of bioactive peptides. The present application provides an angiotensin converting enzyme inhibitory peptide and a screening method and application thereof, and the amino acid sequence of the inhibitory peptide is GKGLW and / or GDGLKW. The screening method of the inhibitory peptide of the present application uses soybean protein as raw material, adopts targeted alkaline protease and chymotrypsin double enzymolysis technology for preparation and screening, realizes rapid screening of ACE inhibitory peptides, and provides experimental basis for structure-activity relationship. The inhibitory peptide has strong blood pressure lowering activity, high safety and no toxic side effects. The determination of the stability of the digestive tract shows that the inhibitory peptide has good gastrointestinal stability in the gastrointestinal digestion, and can still maintain good activity after entering the blood.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptides, specifically relating to an angiotensin-converting enzyme inhibitory peptide, its screening method, and its application. Background Technology

[0002] Bioactive peptides are organic substances composed of a specific arrangement of several to a dozen amino acids linked by covalent bonds of amide or peptide bonds. Research on peptides in disease began in 1922 when insulin, extracted from animal pancreas, was first applied to the medical field, revolutionizing the treatment of type 1 diabetes. Subsequently, peptides have been widely used in the treatment of various diseases, such as cardiovascular diseases, antibacterial agents, immune modulation, and cancer. Therefore, peptides play an important role in human physiology and pathology as therapeutic lead compounds, and have the potential to replace chemical drugs.

[0003] Hypertension, as one of the major diseases affecting human health, poses a significant challenge to global healthcare systems. Angiotensin-converting enzyme (ACE) is a key mediator in the pathogenesis of hypertension. In the renin-angiotensin system (RAS), ACE catalyzes the conversion of Ang I to Ang II. Ang II has a strong vasoconstrictive effect, leading to aldosterone secretion and elevated blood pressure. This suggests that inhibiting ACE activity is an effective strategy to prevent elevated blood pressure. Currently, several ACE inhibitors (such as lisinopril, captopril, and enalapril) have been developed for the treatment of hypertension; however, the use of chemical drugs is often accompanied by numerous side effects, such as cough, headache, and loss of taste. In contrast, food-derived ACE inhibitory peptides (ACEIPs) are safe, have a mild effect, and are easily absorbed, and are increasingly being promoted as an alternative therapy for hypertension.

[0004] Soybeans, due to their abundant protein and economical and environmentally friendly characteristics, are considered an excellent source for developing ACE inhibitory peptides. For example, the soybean protein-derived peptide LSW, obtained by digestion with thermophilic bacteria protease, has anti-inflammatory effects on vascular smooth muscle cells; the bioactive peptide VHVV, obtained by enzymatic hydrolysis of soybean protease using flavor protease, has had its ameliorative effect on hypertensive kidney injury verified using the SHRs model.

[0005] Two-step enzymatic hydrolysis offers significant advantages in improving hydrolysis efficiency, enhancing peptide stability, and shortening hydrolysis time. For example, a combination of neutral and alkaline proteases was used to prepare tuna ACE-inhibiting peptides. Results showed that two-step hydrolysis was superior to single-step hydrolysis in terms of degree of hydrolysis, amino acid content, and ACE inhibition.

[0006] Despite numerous studies on soybean ACE inhibitory peptides, the two-step enzymatic hydrolysis method for preparing soybean ACEIPs has not yet been widely applied. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides angiotensin-converting enzyme (ACE) inhibitory peptides, their screening method, and applications. The screening method uses soybean protein as raw material and employs a targeted alkaline protease and chymotrypsin dual-enzymatic digestion technique for preparation and screening, achieving rapid screening of ACE inhibitory peptides and providing experimental evidence for their structure-activity relationship. The angiotensin-converting enzyme inhibitory peptides of this invention are GKGLW, GDGLKW, and IC. 50 The concentrations were 34.96 μM and 33.98 μM, respectively, indicating strong antihypertensive activity, high safety, and no toxic side effects. Gastrointestinal stability assays showed that GKGLW and GDGLKW exhibited good gastrointestinal stability during digestion and maintained good activity after entering the bloodstream.

[0008] The purpose of this invention is to provide an angiotensin-converting enzyme inhibitory peptide, wherein the amino acid sequence of the inhibitory peptide is GKGLW or GDGLKW.

[0009] Another object of the present invention is to provide a composition comprising the above-mentioned inhibitory peptide and pharmaceutically or health-product-acceptable excipients.

[0010] The present invention also aims to provide the application of the above-mentioned angiotensin-converting enzyme inhibitory peptide or composition in the preparation of antihypertensive drugs or adjuvant antihypertensive health products.

[0011] The purpose of this invention is to provide a method for screening angiotensin-converting enzyme inhibitory peptides, comprising the following steps:

[0012] (1) Soy protein was hydrolyzed stepwise using alkaline protease and chymotrypsin to obtain a protein peptide solution;

[0013] (2) The above protein peptide solution was subjected to ultrafiltration to obtain components with different molecular weights, and peptides with molecular weight <3kDa were screened out.

[0014] (3) The peptides obtained in step (2) were enriched and purified by gel chromatography column, the components were collected and the ACE inhibitory activity of different components was measured, and the component with the strongest ACE inhibitory ability was screened.

[0015] (4) The components obtained in step (3) were identified by LC-MS / MS, and peptides with ALC > 90% were screened out. PeptideRanker (with a scoring threshold of > 0.5) and BIOPEP-UWM (with a scoring threshold of > 0.5) were used to further screen out peptides with potential ACE inhibitory effects. Seqlogo was used to analyze the amino acid distribution of the peptides and to screen peptides with W and F residues at the C-terminus.

[0016] (5) Among the peptides with W and F residues at the C-terminus obtained in step (4), select peptides that are water-soluble, non-toxic, and non-sensitizing, and determine the in vitro ACE inhibitory activity of the peptides. Screen out the peptides with the strongest ACE inhibitory ability, GKGLW and GDGLKW.

[0017] Furthermore, the peptide sequences obtained from the above screening were subjected to virtual verification and evaluation. The stability of the binding conformation of the peptides to the ACE molecules was evaluated using MOE 2019 software for molecular docking.

[0018] Furthermore, molecular dynamics simulations were performed on the peptide sequences obtained from the screening and the ACE receptor. Based on the Rg value, RMSD, RMSF, binding free energy, and Gibbs free energy data, it was verified that the selected peptide sequences GKGLW and GDGLKW form a more stable complex with ACE.

[0019] Different functional peptides possess unique amino acid sequence characteristics due to their specific biological activities. However, there are currently no standards for selecting proteases to produce hydrolysates with specific biological activities. Therefore, a deep understanding of the correlation between peptide sequences and the biological activity of ACE inhibitors is crucial for developing efficient and rapid methods to produce novel antihypertensive peptides.

[0020] This invention systematically evaluated the ACE-inhibiting effects of 168,000 tripeptides and tetrapeptides based on molecular dynamics simulations. It was found that ACE-inhibiting peptides exhibit a greater preference for aromatic amino acids at their ends, particularly when the C-terminus contains tryptophan, resulting in the best inhibitory effect. Therefore, utilizing specific enzymes to cleave and modify the ends of peptides to ensure that the C-terminal amino acid residues are predominantly w / f, can help improve the efficiency of preparing ACE-inhibiting peptides.

[0021] This invention establishes a novel targeted dual-enzyme hydrolysis technology. Using soybean protein as raw material, it analyzes the different enzymatic preferences of five proteases and the differences in the amino acid composition of the hydrolysates, determining the optimal enzymatic combination of chymotrypsin and alkaline protease. Soybean protein hydrolysates were prepared by targeting these two proteases, achieving an ACE inhibition rate of 75.98%. Subsequently, through ultrafiltration, gel chromatography purification, LC-MS / MS identification, and computer screening, several novel C-terminal tryptophan-containing ACE-inhibiting peptides were identified, and the peptide sequence with the best activity was identified. Furthermore, molecular simulation was used to explore the ACE inhibition mechanism from the perspective of binding structure. The peptides mainly interact with key residues in the ACE active pocket through hydrogen bonds, demonstrating that hydrophobic and cyclic amino acids are beneficial to the ACE inhibitory activity of the peptides. Finally, the gastrointestinal digestibility of these peptides was evaluated, providing useful information on enzyme selection and mechanism of action for the development and application of soybean antihypertensive peptides.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) This invention discloses for the first time two angiotensin-converting enzyme (ACE) inhibitory peptides and their screening method. The two ACE inhibitory peptides obtained have structures GKGLW and GDGLKW, respectively, and their corresponding IC50 values ​​are as follows: 50 The values ​​were 34.96 μM and 33.98 μM, respectively, showing good ACE inhibition ability, indicating that they are strong inhibitors of ACE;

[0024] (2) This invention uses soybean protein as raw material and adopts a two-step enzymatic hydrolysis method of alkaline protease and chymotrypsin to screen the enzymatic hydrolysis products to obtain ACEIPs. The targeted double enzyme hydrolysis technology of this invention realizes the efficient preparation of soybean highly active ACE inhibitory peptides, and provides experimental basis for the rapid screening and structure-activity relationship of ACE inhibitory peptides.

[0025] (3) The angiotensin-converting enzyme inhibitory peptide of the present invention has strong antihypertensive activity, high safety, no toxic side effects, small molecular weight and easy absorption by the human body, and has a wide range of sources, and has important market value and application prospects. Attached Figure Description

[0026] Figure 1 The amino acid composition of peptides prepared from different proteases;

[0027] Figure 2 The inhibitory effects of different single-enzyme hydrolysis and tandem hydrolysis with alkaline protease on ACE;

[0028] Figure 3 The ACE inhibition capacity and IC50 of different ultrafiltration components 50 value;

[0029] Figure 4 This is a schematic diagram of partial purification of Sephadex G-25 gel;

[0030] Figure 5 The graph shows the activity distribution, peptide number, and peak area of ​​the G3 component.

[0031] Figure 6 This is a diagram showing the amino acid distribution of peptides 4-9.

[0032] Figure 7 This is a diagram showing the docking results of potential bioactive peptides with ACE receptors.

[0033] Figure 8 A two-dimensional diagram of molecular docking between GKGLW and ACE molecules;

[0034] Figure 9 A two-dimensional diagram of molecular docking between GDGLKW and ACE molecules;

[0035] Figure 10A two-dimensional diagram of molecular docking between EWEGF and ACE molecules;

[0036] Figure 11 Molecular dynamics analysis of the interactions between GKGLW, EWEGF, and GDGLKW with the ACE receptor;

[0037] Figure 12 For ACE-GKGLW Gibbs free energy diagram and ACE-GDGLKW Gibbs free energy diagram (2D, 3D);

[0038] Figure 13 The flowchart of the in vitro digestion method for food is used as a reference, and the comparison chart of the digestive tract stability test data of GKGLW and GDGLKW is shown. Detailed Implementation

[0039] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1 Preparation of protease hydrolysate

[0041] Defatted soybeans (80 mesh, sourced from Northeast China by Qingmei Group, variety Heihe 43) were mixed with deionized water (1 g / 15 ml), and the pH was adjusted to 9.0 with 1.0 M NaOH. The resulting mixture was stirred with a magnetic stirrer at 45°C for 1 h, then centrifuged three times at 4500 × g for 10 min each time. The supernatant was adjusted to pH 4.5 with 1.0 M HCl, and then centrifuged at 4500 × g at 25°C for 10 min using a 5804R centrifuge (epppendorf, Germany). The resulting precipitate was redispersed in deionized water and adjusted to pH 7.0. The protein solution was freeze-dried and stored at -20°C.

[0042] Soy protein hydrolysate was prepared by suspending SPI (soy protein isolate) in deionized water (5%, w / w). Five different proteases were used to hydrolyze the soy protein hydrolysate. The enzymes were introduced at a ratio of 2000 U / g (E / S) into alkaline protease (pH 9.0, 50 ℃), flavor protease (pH 7.0, 50 ℃), papain (pH 7.0, 50 ℃), trypsin (pH 8.0, 37 ℃), and chymotrypsin (pH 7.0, 37 ℃) for hydrolysis. The enzymes were inactivated by heating at 90 ℃ for 10 min, thus terminating the reaction. The resulting hydrolysate was centrifuged at 8000 × g for 15 min. The supernatant was collected and stored at -20 ℃ until further use.

[0043] Example 2: Determination of the amino acid composition of peptides after treatment with different enzymes

[0044] The preparation of highly efficient ACE-inhibiting peptides is closely related to the specific cleavage sites of enzymes and the amino acid composition of substrate proteins. To investigate the cleavage preference of each protease, the amino acid composition and content of peptide solutions after treatment with different enzymes were determined.

[0045] Methods for the determination of amino acids

[0046] 0.01 g of sample was dissolved in 10 mL of HCl (6 M) and hydrolyzed at 110 °C for 23 h (purged with nitrogen). The solution was then diluted to 50 mL in a volumetric flask, followed by a 5-fold dilution with 0.02 M HCl and filtered (using a 0.22 μm filter membrane). The solution was then analyzed using an automated amino acid analyzer (L-8900). Tryptophan content was determined using an alkaline hydrolysis method.

[0047] The amino acid composition of peptides prepared from different proteases was determined as follows: Figure 1 As shown, the results indicate that the content of hydrophobic amino acids in the alkaline protease hydrolysate is 311.42 mg / g, higher than other hydrolysates (286.36-301.12 mg / g), especially Leu, which accounts for 20.94% of the total hydrophobic amino acids. Previous studies have shown that when the content of hydrophobic amino acids in the ACE-I peptide chain reaches 19.8%, the ACE inhibition effect can reach 65.09%. Therefore, alkaline protease may help improve ACE inhibition activity by specifically cleaving hydrophobic amino acids. The Trp tryptophan content in the chymotrypsin hydrolysate is 138 mg / 100 g, higher than other hydrolysates (27-91 mg / 100 g), indicating that the cleavage site of chymotrypsin may be related to Trp. Furthermore, the steric hindrance brought by the indole group in tryptophan can effectively increase ACE activity and Zn. 2+ The binding force.

[0048] Example 3 Stepwise Enzymatic Digestion of Different Proteases and Alkaline Proteases

[0049] A high-performance alkaline protease was combined with other proteases in a stepwise enzymatic hydrolysis method. In the two-step hydrolysis, an alkaline protease (E / S = 1000 U / g) was first added and incubated at 50 °C for 1 h. The enzyme was then inactivated by heating at 90 °C for 10 min. Next, other proteases (E / S = 1000 U / g) were added separately for further hydrolysis for 1 h. Finally, the reaction was terminated by heating at 90 °C for 10 min again. The resulting hydrolysate was centrifuged at 8000 × g for 15 min, and the supernatant was collected and stored at −20 °C. The ACE inhibitory activity of the combined hydrolysate and the single-enzyme hydrolysate prepared in Example 1 was measured and compared.

[0050] Methods for determining ACE inhibitory activity

[0051] ACE and HHL were dissolved in 0.1M sodium borate buffer (pH 8.3) for assay. The synthetic peptide was dissolved in distilled water to six concentration levels. The test peptide (20 μL) was mixed thoroughly with 2 mM HHL (20 μL), preheated at 37 °C for 5 min, then 20 μL of ACE (0.1 U) was added to initiate the reaction. The reaction was maintained at 37 °C for 30 min, and finally terminated by adding 50 μL of 1 M HCl. The resulting reaction solution was analyzed by HPLC. The analytical conditions were: C18 HPLC column (4.6 mm × 250 mm × 5 μm), column temperature 35 °C, mobile phase (acetonitrile / water = 1:3 (v / v), containing 0.1% trifluoroacetic acid), isocratic elution at a flow rate of 1 mL / min, and detection wavelength 228 nm. The ACE inhibition activity was calculated as follows:

[0052] ACE inhibitory activity(%)=(A control -A inhibitor ) / A control

[0053] Among them, A inhibitor The relative areas of the HA peaks obtained from the reaction of ACE and HHL in the presence of inhibitors, A control The relative areas of the HA peak obtained from the uninhibited reaction of ACE and HHL are shown in Figure 1. 50 The value is defined as the peptide concentration that can inhibit half of the ACE activity.

[0054] The alkaline protease was combined with other proteases for stepwise enzymatic digestion, and the results of the ACE inhibitory activity assay are as follows: Figure 2As shown, stepwise hydrolysis with flavor protease, trypsin, and papain, along with alkaline protease, increased the ACE inhibition rate by 6.94%, 10.14%, and 11.41%, respectively, compared to single-enzyme hydrolysis. The combination of alkaline protease and chymotrypsin showed the greatest improvement, increasing the ACE inhibition rate from 48.32% to 67.98%. Therefore, using the Alcalas-Chymotrypsin two-step hydrolysis method to progressively degrade proteins based on the specific preferences of different enzymes can yield peptides with greater antihypertensive activity.

[0055] Example 4: Ultrafiltration and gel chromatography purification of soybean peptides

[0056] To further analyze the ACE inhibitory activity of peptides with different molecular weight ranges, we used ultrafiltration to further separate the protein hydrolysates. The enzymatic hydrolysate after two-step hydrolysis with alkaline protease and chymotrypsin was separated into different fractions via ultrafiltration: <3 kDa, 3-10 kDa, 10-30 kDa, and >30 kDa. The ACE inhibitory activity of peptides with different molecular weight ranges was measured, and the results are shown below. Figure 3 As shown, components with different molecular weights exhibit different ACE inhibitory activities. When the sample concentration is 50 μg / mL, the ACE inhibitory activity gradually increases with decreasing molecular weight of the hydrolyzed components. When the peptide molecular weight is 3-10 kDa and 10-30 kDa, the ACE inhibitory activities are 65.72% (IC50). 50 =40.98 ± 1.40 μg / mL) and 50.89% (IC 50 =65.91±1.51 μg / mL), the inhibition rate of the <3 kDa fraction was the highest, at 72.57% (IC50). 50 =32.59 ±1.22 μg / mL). After separation by ultrafiltration, these low molecular weight peptides exhibited stronger ACE inhibitory activity, confirming that peptides with a molecular weight <3kDa are an ideal source of ACE inhibitory peptides.

[0057] Subsequently, further enrichment and purification were performed using a Sephadex G-25 gel chromatography column. Gel chromatography separates peptides based on size exclusion; during elution, low molecular weight peptides elute more slowly due to the longer flow path into the gel pores, while high molecular weight peptides flow through the gaps, resulting in a shorter and faster elution path. Figure 4 As shown, three components, G1, G2, and G3, were collected within a retention time of 50-200 min. The ACE inhibitory activity of these three components was determined. G3 exhibited the best ACE inhibitory activity (69.65 ± 0.62%), with an IC50 of [missing value]. 50 The value was 33.73 ± 0.89 μg / mL. Therefore, LC-MS / MS was performed on fraction G3 to identify ACEIPs in SPI.

[0058] Example 5: LC-MS / MS identification and distribution characteristics analysis of G3 component, and peptide screening.

[0059] To further elucidate the ACE-inhibiting effect of the G3 component, its molecular weight and amino acid sequence were identified using UHPLC-Q-Orbitrap-MS / MS and Peaks Studio 7.5, respectively. The G3 component was identified by UHPLC-Q-Orbitrap-MS / MS, and 991 peptides with ALC > 90% were screened. The peptide number and peak area are shown below. Figure 5 As shown in the figure, the outer pie chart represents the MS region of the peptide, and the inner pie chart represents the content of the peptide number. Figure 5 It was found that tetrapeptides to nonapeptides were distributed among these peptides, with heptapeptides and hexapeptides having relatively high MS peak areas, accounting for 28.4% and 27.6% of the total MS area, respectively. Hexapeptides had the largest number of peptides, with 288, accounting for 29.1% of the total number of peptides. Based on this, 252 peptides with potential ACE inhibitory effects were screened using PeptideRanker (with a scoring threshold > 0.5) and BIOPEP-UWM (with a scoring threshold > 0.5).

[0060] To further analyze the characteristics of these peptides, Seqlogo analysis was used to analyze the amino acid distribution of the peptides. The analysis results of the amino acid distribution of peptides 4-9 are as follows: Figure 6 As shown, the amino acid character represents the proportion; the larger the amino acid character, the higher the proportion. The results show that aromatic amino acids (F) and hydrophobic amino acids (L, P) appear most frequently at the C-terminus and N-terminus, polar uncharged amino acids (S, G) have a higher proportion at the C-terminus, while polar positively charged amino acids (R, H, K) have a higher proportion at the N-terminus. With peptide chain elongation, the proportion of acidic amino acids (D, E) at the C-terminus and the second C-terminal position increases. Furthermore, aromatic and cyclic amino acids (such as TrpW and PheF) are the most common C-terminal residues in the structures of highly active ACE inhibitors. For example, the dipeptide VW (IC) from yeast protein hydrolysate... 50 = 0.1 mg / mL), VF (IC 50 = 0.135 mg / mL). The hexapeptide VVCVPW (IC50) in the enzymatic hydrolysis products of Clam protein... 50 =4.1μM).

[0061] Based on this, the screening criterion is peptides containing W and F residues at the C-terminus. The prediction results for peptides with a peptide ranking score > 0.5 and their correlation with allergens, toxicity, and water solubility are shown in the table below:

[0062]

[0063] Based on the water solubility, toxicity, and sensitization properties shown in the table, four peptides with C-terminal Trp and one with C-terminal Phe were finally selected and synthesized, namely DHPPASW, GDGLKW, GKGLW, EWEGF, and LGKW.

[0064] By measuring the in vitro ACE inhibitory activity, the IC50 values ​​of these peptides ranged from 33.98 μM to 78.23 μM. Among them, GKGLW and GDGLKW had the highest IC50 values. 50 The values ​​were 34.96 and 33.98 μM, respectively, indicating that they are strong inhibitors of ACE. The PeptideRanker score and IC50 of the synthetic peptides were also analyzed. 50 The molecular docking affinity values ​​of ACE and other molecules are shown in the table below:

[0065]

[0066] Example 6: Analysis of the molecular interaction mechanism between peptides and ACE

[0067] To investigate the interaction mechanism between these five bioactive peptides and the ACE receptor, the stability of the binding conformation was assessed using molecular docking with MOE 2019 software. Molecular docking method for potential bioactive peptides with the ACE receptor: Using the MOE 2019 molecular docking tool, the three-dimensional structures of the screened potential bioactive peptides were successfully constructed, and energy minimization was performed on them. ACE (PDB ID: 1086) crystal structure data were obtained from the RCSB protein database (https: / / www.rcsb.org). The protein receptor binding cavity and site of ACE were predicted using the DoGSiteScorer online tool (https: / / proteins.plus / ). The three-dimensional diagrams of the interactions between the five peptides and the ACE receptor are shown below. Figure 7 As shown in Figure A, B is a 3D diagram of molecular docking between GKGLW and ACE molecules, C is a 3D diagram of molecular docking between GDGLKW and ACE molecules, and D is a 3D diagram of molecular docking between EWEGF and ACE molecules. Figure 8 This is a two-dimensional diagram of the molecular docking between GKGLW and ACE molecules. Figure 9 This is a two-dimensional diagram of the molecular docking between GDGLKW and ACE molecules. Figure 10 This is a two-dimensional diagram of the molecular docking between EWEGF and ACE molecules.

[0068] The docking results are shown in Table 1, which displays the molecular docking affinity values ​​between the synthesized peptides and ACE. All five peptides could enter the ACE binding pocket, with docking binding energies ranging from -10.91 kcal / mol to -12.31 kcal / mol, and an average binding energy of -11.36 kcal / mol. The stability of the receptor-ligand complex can be determined by the docking energy; the lower the docking energy, the more stable the binding. Based on the binding energy, GKGLW and GDGLKW exhibited good ACE inhibitory activity, consistent with the ACE inhibition experiments.

[0069] 3D of peptide-ACE complex ( Figure 7 BD and 2D Figure 8 , 9 10) The structural diagram shows that the peptide interacts with ACE residues through multiple mechanisms, including van der Waals forces, hydrophobicity, and electrostatic forces. Hydrogen bonds are the main interaction force for forming a stable peptide-ACE complex. GKGLW (-12.31 kcal / mol), with the lowest binding energy, forms five hydrogen bonds with ACE residues Ala354, Tyr523, His387, Ser284, and Gln281. GKGLW forms two hydrogen bonds with the S1 pocket (Ala354, Tyr523) and one hydrogen bond with the S2 pocket (Gln281). GDGLKW (-11.04 kcal / mol) forms four hydrogen bonds with ACE, involving residues Gln403, Arg522, His353, and His387. EWEGF (-10.91 kcal / mol) forms only one hydrogen bond with the ACE residue His387.

[0070] The number of hydrogen bonds directly reflects or determines the affinity and stability between the ACE receptor and the peptide, explaining the strong inhibitory effect of GKGLW and GDGLKW on ACE. Furthermore, GKGLW and GDGLKW also form non-bonded interactions with the metal ion Zn2+ (an important component of the ACE active catalytic site), interfering with the binding of ACE to Zn2+ and thus inhibiting its biological activity. Docking results show that GKGLW and GDGLKW mainly achieve their strong ACE inhibitory activity by forming hydrogen bonds with the active pocket of ACE; the C-terminus being tryptophan is more conducive to binding to ACE, thereby enhancing their inhibitory activity. Therefore, the ACE inhibitory activity of these peptides may be due to two factors: firstly, they compete with the substrate by completely embedding themselves in the ACE catalytic cavity; secondly, they hinder substrate entry by occupying the substrate recognition surface.

[0071] Example 7: Molecular dynamics simulation of peptides and ACE receptors

[0072] To further validate ligand-receptor binding at the atomic level, three parallel 100 ns molecular dynamics simulations were performed on GKGLW, GDGLKW, and EWEGF. Specifically, protein parameters were generated using an AMBER14SB force field, sodium ions were added to the solvent environment of the TIP3P container, and the pH was maintained neutral with 12 Å buffer. While ensuring the normal structure of the system, the fastest descent method and the conjugate gradient method were used to minimize the energy of the solvent and solute structures. The entire system was then heated to 300 K under a constraint of 10 kcal / (mol∙Ų). All chemical bonds involving hydrogen atoms were subjected to the SHAKE algorithm. After the system reached energy and density equilibrium, 100 ns molecular dynamics simulations were performed on the ACE receptor complexes of the potentially bioactive peptides.

[0073] Simulation results are as follows Figure 11 As shown in the figure, A is the radius of rotatability (Rg) curve, B is the root mean square deviation (RMSD) plot, C is the root mean square fluctuation (RMSF) plot, and D is the binding free energy plot of peptide to ACE calculated by the MMPBSA method. The Rg value can be used to assess the structural compactness of protein systems. Simulation results are shown in the figure. Figure 11 As shown in Figure A, the average Rg values ​​of GKGLW, GDGLKW, and EWEGF are 23.80, 23.78, and 23.95 Å, respectively, indicating that GKGLW and GDGLKW bind more tightly to ACE. The root mean square deviation (RMSD) is an important indicator for evaluating the stability of the ACE-peptide system, reflecting the degree of deviation of the protein molecule from its initial structure during dynamic simulation. Generally, peptides with higher activity are usually accompanied by higher RMSD values, as shown in the simulation results. Figure 11 As shown in Figure B, the average RMSD of the GKGLW-ACE (1.98 Å) and GDGLKW-ACE complexes (2.14 Å) is higher than that of the EWEGF-ACE complex (1.64 Å), indicating that the GKGLW-ACE and GDGLKW-ACE complexes undergo more conformational changes, resulting in higher ACE inhibitory activity. Root mean square fluctuation (RMSF) is an indicator used to describe the average amplitude of motion of a single amino acid residue within a specific time range. The simulation results are shown in Figure B. Figure 11 As shown in Figure C. The results indicate that the average RMSF value of the GKGLW-ACE complex (0.86 Å) is lower than that of the EWEGF-ACE complex (0.93 Å), suggesting that the GKGLW-ACE complex structure is relatively stable. Binding energy reflects the strength of the interaction between the ligand and the protein. The binding free energies of these three peptides were calculated using the MMPBSA method, and the results are shown in Figure C. Figure 11As shown in Figure D, compared with the binding free energy of EWEGF (-43.4832 kJ / mol), the binding energies of GKGLW (-100.3606 kJ / mol) and GDGLKW (-93.6422 kJ / mol) are more favorable for binding to ACE. MMPBSA results indicate that GKGLW and GDGLKW have higher ACE inhibitory activity, consistent with previous studies on ACE inhibitory activity.

[0074] Finally, the Gibbs free energy was calculated based on the RMSD and Rg values, and the conformational stability of the complex was characterized. The results are as follows: Figure 12 As shown in Figures E and F, where E represents the Gibbs free energy diagram of ACE-GKGLW (2D, 3D) and F represents the Gibbs free energy diagram of ACE-GDGLKW (2D, 3D), it can be seen that both GKGLW and GDGLKW exhibit only one energy well in their two-dimensional free energy landscapes. This indicates that the system exists in a single stable conformation and is unlikely to undergo significant conformational changes or polymorphisms under given conditions. These findings provide further supporting evidence for the binding mechanism of peptide-protein interactions, suggesting that both peptides can rapidly bind to ACE to form a stable and favorable complex structure.

[0075] Example 8: Stability test of two peptides during gastrointestinal digestion

[0076] Before bioactive peptides can cross the small intestinal epithelium and exert their blood pressure-lowering effect, they must resist gastrointestinal digestion and maintain their integrity. Although digestive enzymes break down large molecules into smaller molecules that are more easily absorbed by the body, these breakdown products may lose their original biological activity. Methods that simulate gastrointestinal digestion in vitro can mimic the fate of peptides under oral administration conditions, referring to methods such as the INFOKE digestion assay (e.g.,...). Figure 13 (Flowchart of in vitro digestion method for food shown in Figure A) Simulated saliva (SSF), gastric juice (SGF), and simulated intestinal juice (SIF) were prepared. SSF consisted of salivary amylase (75 U / mL) and 1.5 mM CaCl2. SGF consisted of pepsin (2000 U / mL), gastric lipase (60 U / mL), and 0.15 mM CaCl2 at pH 3.0. SIF, in 0.6 mM CaCl2, consisted of trypsin (100 U / mL) and 10 mM bile salts. Digestion was performed at 37°C with continuous shaking. In vitro digestion was performed with pepsin at pH 1.2 for 1 h and with trypsin and chymotrypsin at pH 7.8 for 3.5 h. Gastric digests were collected after 0.5 and 1 h, respectively, and intestinal digests were collected after 1.5, 2.5, and 4 h, respectively. Enzymes were inactivated by heating at 90°C.

[0077] The results of the gastrointestinal stability assays for GKGLW and GDGLKW, such as Figure 13 As shown in Figure B, the IC at the end of GKGLW digestion 50 The value changed from 34.96 μM to 35.08 μM, and the IC50 value at the end of GDGLKW digestion also changed. 50 The value also increased, from 33.98 μM to 34.21 μM. There was no significant difference in ACE inhibition between the untreated and treated peptides; therefore, GKGLW and GDGLKW exhibit good gastrointestinal stability during digestion and maintain good activity after entering the bloodstream.

[0078] In summary, this invention provides a novel targeted dual-enzymatic hydrolysis technique for preparing soybean ACE-inhibiting peptides with characteristic structures. Soybean protease hydrolysates were prepared by targeting alkaline protease and chymotrypsin, enabling rapid screening of ACE-inhibiting peptides and providing experimental evidence for structure-activity relationships. The inhibitory peptide GKGLW(IC) provided by this invention... 50 =34.96 μM) and GDGLKW (IC 50 (33.98 μM) showed good ACE inhibition ability, high safety, no toxic side effects, and good gastrointestinal stability during gastrointestinal digestion, and has good market value and application prospects.

[0079] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention and should be defined by the claims.

Claims

1. An angiotensin-converting enzyme inhibitory peptide, characterized in that, Its amino acid sequence is GKGLW or GDGLKW.

2. A composition of angiotensin-converting enzyme inhibitory peptides, characterized in that, The composition comprises the angiotensin-converting enzyme inhibitory peptide of claim 1 and pharmaceutically or health-product-acceptable excipients.

3. The use of the angiotensin-converting enzyme inhibitory peptide of claim 1 or the composition of claim 2 in the preparation of antihypertensive drugs or adjuvant antihypertensive health products.

4. A method for screening angiotensin-converting enzyme inhibitory peptides as described in claim 1, characterized in that, Includes the following steps: (1) Soy protein was hydrolyzed stepwise using alkaline protease and chymotrypsin to obtain a protein peptide solution. First, alkaline protease was added at an amount of E / S = 1000 U / g, and the mixture was incubated at 50 °C for 1 h. Then, the enzyme was inactivated by heating at 90 °C for 10 min. Next, chymotrypsin was added at an amount of E / S = 1000 U / g, and the mixture was further hydrolyzed for 1 h. Finally, the reaction was terminated by heating at 90 °C for 10 min. (2) The protein peptide solution was subjected to ultrafiltration to obtain components with different molecular weights, and peptides with molecular weight <3 kDa were screened out. (3) The peptides obtained in step (2) were enriched and purified by gel chromatography column, the components were collected and the ACE inhibitory activity of different components was measured, and the component with the strongest ACE inhibitory ability was screened. (4) The components obtained in step (3) were identified by LC-MS / MS, and peptides with ALC > 90% were screened. PeptideRanker and BIOPEP-UWM were used to further screen peptides with potential ACE inhibitory effects. Seqlogo was used to analyze the amino acid distribution of peptides and to screen peptides with W and F residues at the C-terminus. (5) Among the peptides with W and F residues at the C-terminus obtained in step (4), select peptides that are water-soluble, non-toxic, and non-sensitizing, and determine the ACE inhibitory activity of the peptides to screen out the peptides with the strongest ACE inhibitory ability, GKGLW and GDGLKW.

5. The screening method according to claim 4, characterized in that, The selected peptide sequences were subjected to virtual validation and evaluation. The stability of the binding conformation of the peptides to the ACE molecules was evaluated using MOE 2019 software for molecular docking.

6. The screening method according to claim 4, characterized in that, The selected peptide sequences were subjected to molecular dynamics simulations with the ACE receptor. Based on the Rg value, RMSD, RMSF, binding free energy, and Gibbs free energy data, it was verified that GKGLW and GDGLKW form more stable complexes with ACE.

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