A method for preparing a soy peptide having ACE and renin inhibitory activity
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-28
- Publication Date
- 2026-08-11
AI Technical Summary
水开菲尔粒的发酵可以使蛋白质降解产生生物活性肽；然而，目前尚未见利用水开菲尔粒发酵脱脂大豆粉得到具有高 ACE 和肾素抑制活性的大豆肽研究
本发明的大豆肽由水开菲尔粒发酵得到，目前国内的水开菲尔产品市场基本空白。
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Figure CN122541518A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioactive peptides, and more particularly to a method for preparing soybean peptides with ACE and renin inhibitory activity. Background Technology
[0002] Hypertension, a common global cardiovascular disease, is a significant risk factor for a variety of serious complications such as stroke, heart failure, kidney failure, and atherosclerosis. In the face of this major public health issue, the safe and effective prevention and treatment of hypertension has received widespread attention.
[0003] The renin-angiotensin-aldosterone system (RAAS) is an important endocrine system that regulates blood pressure and fluid homeostasis. Angiotensin-converting enzyme (ACE) and renin are two key enzymes in this system. Renin first catalyzes the conversion of angiotensinogen into angiotensin I (Ang I), and then ACE converts Ang I into angiotensin II (Ang II). When the RAAS is overactivated, Ang II can directly constrict vascular smooth muscle, increase peripheral vascular resistance, and at the same time stimulate aldosterone secretion, promoting the reabsorption of sodium and water by the kidneys, which in turn leads to an increase in extracellular fluid volume and blood volume, ultimately causing elevated blood pressure. Therefore, inhibiting the activity of ACE and renin is considered an effective strategy for the prevention and treatment of hypertension.
[0004] The search for safe and efficient dietary ACE and renin dual inhibitory peptides from natural food proteins has become a research hotspot; however, compared with ACE inhibitory peptides, there are still relatively few renin inhibitory peptides.
[0005] Soy protein is a high-quality plant protein source, and its hydrolysis products have yielded various bioactive peptides with in vitro hypotensive effects. Water kefir grains are naturally occurring complex microbial cultures, primarily composed of lactic acid bacteria, yeast, and acetic acid bacteria. The lactic acid bacteria produce extracellular polysaccharides that encapsulate the various microorganisms, forming irregular, gelatinous particles. Fermentation of water kefir grains can degrade proteins to produce bioactive peptides; however, there is currently no research on using water kefir grains to ferment defatted soybean flour to obtain soybean peptides with high ACE and renin inhibitory activity. Summary of the Invention
[0006] The purpose of this invention is to provide a method for preparing soybean peptides with ACE and renin inhibitory activities, which utilizes water kefir grain fermentation to defatted soybean flour to obtain soybean peptides with ACE and renin inhibitory activities.
[0007] To achieve the above objectives, the technical solution adopted in this invention is: a method for preparing soybean peptides with ACE and renin inhibitory activity, comprising the following steps: Step 1: Activation of water kefir grains. Prepare brown sugar water, inoculate with water kefir grains to activate them, and filter the activated water kefir grains using a sterile sieve for fermentation. Step 2: Preparation of fermentation products. The activated water kefir grains from Step 1 are inoculated into the fermentation broth prepared with soybean flour and brown sugar for fermentation to obtain a mixed fermentation product containing the target peptide. Step 3: Identification of peptide sequences in fermentation products. The lyophilized fermentation product powder was dissolved, ultrafiltered using a 10 kDa ultrafiltration tube, desalted using a Waters SEP-PAK C18 solid-phase extraction column, and lyophilized. The product was then reconstituted with formic acid solution, centrifuged, and the supernatant was collected. The peptide sequences in the sample were identified using LC-MS / MS (liquid chromatography-tandem mass spectrometry). The physicochemical properties and functions of the identified peptide sequences were predicted using computer software and an online website, and three potential ACE and renin dual-inhibitory peptides were screened. Step 4: Synthesis and activity verification of peptides. Potential inhibitory peptides were synthesized in solid phase using the Fmoc / t-Bu method, with a purity >95%. Their ACE and renin inhibition rates were measured, and the half-inhibitory concentration (IC50) was calculated. 50 Soybean peptides with ACE and renin inhibitory activities were screened out.
[0008] Preferably, step one specifically involves: preparing 10% brown sugar water, stirring to dissolve, autoclaving at 121℃ for 20 minutes, dispensing into 500 mL reagent bottles, cooling to room temperature, inoculating with 6% (w / v) water kefir granules, and sealing and culturing at 25℃ for 24 hours, changing the brown sugar solution every 24 hours; when dense small bubbles are continuously produced in the brown sugar water, it indicates that its activation is successful; filtering with a sterile sieve to separate the activated water kefir granules for fermentation.
[0009] Preferably, step two specifically involves: passing dried defatted soybean flour through a 100-mesh sieve; weighing 6% (m / v) soybean flour and 8% (m / v) brown sugar separately and dissolving them in 200 mL of sterile distilled water cooled to 60-65℃, mixing thoroughly, pasteurizing, cooling to room temperature, inoculating with 5% (m / v) water kefir grains, and anaerobic fermenting in a 25℃ constant temperature incubator for 6 h; after fermentation, separating the water kefir grains with a sterile sieve, pasteurizing the fermentation product, and freeze-drying to obtain a mixed fermentation product containing the target peptide; the pasteurization temperature is 63℃ for 30 min.
[0010] Preferably, the liquid chromatography separation conditions in step three are as follows: solution A is a 0.1% formic acid aqueous solution, solution B is a 0.1% formic acid acetonitrile aqueous solution, and acetonitrile content is 80%; the chromatographic column is equilibrated with 92% solution A, the injection volume is 1 μL, and the gradient is set as follows: 0-98 min, solution B from 8% to 28%; 98-113 min, solution B from 28% to 37%; 113-117 min, solution B from 37% to 100%; 117-120 min, solution B is maintained at 100%.
[0011] Preferred mass spectrometry identification conditions: After separation by capillary high-performance liquid chromatography, mass spectrometry analysis was performed using a Thermo QE HF mass spectrometer for 120 min; detection mode: positive ion; the mass-charge ratio of peptides and peptide fragments was collected as follows: 20 fragment spectra were collected after each full scan, with a scan range of 400-1800, a primary resolution of 60000, a secondary resolution of 15000, and a collision energy of CE 28 eV. The raw mass spectrometry test files were retrieved from the relevant database using ProteomeDiscoverer 2.5 software to obtain the peptide identification results.
[0012] Preferably, the physicochemical properties and functions of the identified peptide sequences are predicted using computer software and online websites. First, peptides with 2-15 amino acids are selected, and potential ACE and renin dual inhibitory peptides are further screened using computer-aided methods. The average hydrophilicity coefficient (GRAVY) of the peptides is calculated using the Expasy online platform. The renin and ACE inhibitory activities of the peptides are predicted using the BIOPEP-UWM database. Toxicity assessment is performed using the ToxinPred online platform. AllerTOP is used to predict sensitization, and peptides with potential toxicity and sensitization are removed. Then, the peptides screened in the above steps are subjected to molecular docking. The three-dimensional crystal structures of the protein receptors ACE (PDB ID: 1O86) and renin (PDB ID: 2V0Z) are obtained from the protein database. PyMOL software is used to prepare them for docking, removing water molecules and small molecule ligands. The Zn in the ACE center is removed. 2+ The data needs to be retained; then, hydrogenation and charge processing are performed using AutoDock Tools software, and the data is saved in pdbqt format; the three-dimensional structure of the peptide is constructed using Tinker, and energy minimization processing is performed; finally, molecular docking is performed using AutoDock Vina to obtain the binding energy, and peptides with molecular docking binding energies ≤-9 kcal / mol with both ACE and renin are selected, thereby screening out 3 potential ACE and renin dual repressor peptides.
[0013] Preferably, the specific steps for determining the ACE inhibitory activity of the peptide in step four are as follows: Prepare a 100 mmol / L borate buffer solution with pH 8.3 containing 0.3 mol / L NaCl; determine the ACE inhibition rate of the synthetic peptide at different concentrations; vortex mix 30 μL of 2.5 mmol / L HCl with 10 μL of sample, preheat in a 37℃ water bath for 5 min, then add 20 μL of 100 mU / mL ACE, mix well, and react in a 37℃ water bath for 60 min. Terminate the reaction by adding 60 μL of 1 mol / L HCl; the borate buffer solution serves as a blank control. Detection was performed using HPLC with isocratic elution. The chromatographic column was a C-18 column (150 × 4.6 mm, 5 μm); mobile phase A consisted of 78% (0.05% trifluoroacetic acid), and mobile phase B consisted of 22% (HPLC-grade acetonitrile); the flow rate was 0.5 mL / min; the elution time was 25 min; the column temperature was 25℃; the detector was a DAD detector with a detection wavelength of 228 nm; the ACE inhibition rate was calculated using the following formula: Where A1 is the peak area of hippuric acid in the sample group; A2 is the peak area of hippuric acid in the sample control group; A3 is the peak area of hippuric acid in the blank group; and A4 is the peak area of hippuric acid in the reagent blank group.
[0014] Preferably, the renin inhibitory activity assay of the peptide is performed using a 96-well microplate as the reaction vessel. The blank control group is prepared with 20 μL substrate, 160 μL buffer, and 10 μL solvent; the blank control group is prepared with 20 μL substrate, 150 μL buffer, and 10 μL solvent; and the sample group is prepared with 20 μL substrate, 150 μL buffer, and 10 μL sample solution. After preheating to 37°C, 10 μL of renin is added to both the blank control and sample wells to initiate the reaction. After mixing, the reaction is incubated at 37°C for 15 min. The fluorescence intensity is measured using an excitation wavelength of 335 nm and an emission wavelength of 485 nm. The renin inhibition rate of the sample is calculated using the following formula: Wherein, F0 is the fluorescence intensity of the blank group; F1 is the fluorescence intensity of the blank control group; and F2 is the fluorescence intensity of the sample group.
[0015] The technical effects of this invention are as follows: The soybean peptides of this invention are obtained by fermenting water kefir grains, and the domestic market for water kefir products is currently largely untapped.
[0016] The soybean peptide DTNRHHGTTGVY of this invention exhibits good ACE and renin inhibitory activity (IC50).50 The concentrations were 1.37 mmol / L and 2.79 mmol / L, respectively. Existing research on the antihypertensive activity of soybean peptides mainly focuses on ACE inhibition activity, with less research on renin inhibition activity. This invention fills this gap and achieves inhibition of both ACE and renin.
[0017] This invention is applicable to the addition of antihypertensive peptide biological agents and can also be used in the field of special dietary and medical foods. Attached Figure Description
[0018] Figure 1 The total ion chromatogram shows the identification results of the polypeptide sequences in the fermentation products of Example 1. Figure 2 The IC50 of peptide DTNRHHGTTGVY against ACE in Example 1 50 value; Figure 3 The IC50 of the polypeptide DTNRHHGTTGVY against renin in Example 1 is shown. 50 value; Figure 4 This is a secondary mass spectrum of the soybean peptide DTNRHHGTTGVY, which has high ACE and renin inhibitory activity, as described in Example 1. Detailed Implementation
[0019] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to the accompanying drawings. The description in this part is only exemplary and explanatory, and should not be used to limit the scope of protection of the present invention in any way. Example
[0020] This embodiment is a soybean peptide with high ACE and renin inhibitory activity; The method for preparing the soybean peptide with high ACE and renin dual inhibitory activity is as follows: Step 1: Activation of water kefir particles Prepare a 10% brown sugar solution, stir thoroughly to dissolve, autoclave at 121℃ for 20 min, dispense into 500 mL reagent bottles, cool to room temperature, inoculate with 6% (w / v) water kefir granules, and incubate at 25℃ for 24 h. Change the brown sugar solution every 24 h. The activation is successful when dense, fine bubbles continuously appear in the brown sugar solution. Filter the activated water kefir granules using a sterile sieve for fermentation.
[0021] Step 2: Preparation of fermentation products The dried, defatted soybean flour was passed through a 100-mesh sieve. 6% (m / v) soybean flour and 8% (m / v) brown sugar were weighed and dissolved in 200 mL of sterile distilled water cooled to 63°C. The mixture was thoroughly mixed, pasteurized (63°C, 30 min), and then rapidly cooled to room temperature. 5% (m / v) water kefir grains were inoculated and anaerobic fermented in a 25°C incubator for 6 h. After fermentation, the water kefir grains were separated using a sterile sieve. The fermentation product was pasteurized (63°C, 30 min) and then freeze-dried to obtain the mixed fermentation product containing the target peptide.
[0022] Step 3: Identification of polypeptide sequences in fermentation products The lyophilized fermentation product powder was dissolved and ultrafiltered using a 10 kDa ultrafiltration tube. Desalting was performed using a Waters SEP-PAKC18 solid-phase extraction column, followed by lyophilization, reconstitution with formic acid solution, centrifugation, and analysis of the supernatant.
[0023] Liquid chromatography separation conditions: Solution A was a 0.1% formic acid aqueous solution, and Solution B was a 0.1% formic acid-acetonitrile aqueous solution (acetonitrile content 80%). The chromatographic column (50 μm × 150 mm, Acclaim PepMap™ RSLC, Thermo Scientific Technology Inc.) was equilibrated with 92% Solution A, with an injection volume of 1 μL. The gradient settings were as follows: 0–98 min, Solution B from 8% to 28%; 98–113 min, Solution B from 28% to 37%; 113–117 min, Solution B from 37% to 100%; 117–120 min, Solution B maintained at 100%.
[0024] Mass spectrometry identification conditions: After separation by capillary high-performance liquid chromatography (HPLC), mass spectrometry analysis was performed using a Thermo QE HF mass spectrometer (ThermoFisher). Analysis time: 120 min; detection mode: positive ion. The mass-charge ratio of the peptide and peptide fragments was acquired as follows: 20 fragment spectra were acquired after each full scan (MS2 scan). Scan range: 400-1800 μm; primary resolution: 60,000 μm; secondary resolution: 15,000 μm; collision energy: CE 28 eV.
[0025] The raw mass spectrometry test files were searched in the relevant database using Proteome Discoverer 2.5 software to obtain the peptide identification results. The database used for the database search was uniprot-taxonomy_3847 (Glycine max), and the total ion chromatogram is shown in Figure 1.
[0026] The physicochemical properties and functions of the identified peptide sequences were predicted using computer software and online websites. First, peptides with 2-15 amino acids were selected, and potential ACE and renin dual-inhibitory peptides were further screened using computer-aided methods. The average hydrophilicity coefficient (GRAVY) of the peptides was calculated using the Expasy online platform, which also provides the theoretical isoelectric point and the total number of positively charged residues. The renin and ACE inhibitory activities of the peptides were predicted using the BIOPEP-UWM database. Toxicity assessment was performed using the ToxinPred online platform. Peptides with potential toxicity and sensitization were removed based on sensitization prediction. Next, molecular docking was performed on the peptides screened in the above steps. The three-dimensional crystal structures of the protein receptors ACE (PDB ID: 1O86) and renin (PDB ID: 2V0Z) were obtained from protein databases. Pre-doping preparations were performed using PyMOL software to remove water molecules and small molecule ligands, including the Zn group at the ACE center. 2+ The data needs to be retained; subsequently, hydrogenation and charge processing were performed using AutoDock Tools software, and the data was saved in pdbqt format. The three-dimensional structure of the peptide (ligand) was constructed using Tinker, and energy minimization was performed. Finally, molecular docking was performed using AutoDock Vina to obtain the binding energy. Peptides with molecular docking binding energies ≤-9 kcal / mol with both ACE and renin were selected, thus identifying three potential ACE and renin dual repressor peptides. Their sequences and characteristic parameters are shown in the table below.
[0027] Step 4: Polypeptide Synthesis The above-mentioned potential inhibitory peptides were synthesized in solid phase using the Fmoc / t-Bu method, and the purity of all peptides was >95%.
[0028] Step 5: Assay of the ACE inhibitory activity of the peptide Prepare a 100 mmol / L borate buffer solution (pH 8.3, containing 0.3 mol / L NaCl); determine the ACE inhibition rate of different concentrations of the synthesized peptides; vortex 30 μL of 2.5 mmol / L HCl with 10 μL of sample, preheat in a 37℃ water bath for 5 min, then add 20 μL of 100 mU / mL ACE, mix well, and react in a 37℃ water bath for 60 min; terminate the reaction by adding 60 μL of 1 mol / L HCl. The borate buffer solution serves as a blank control.
[0029] Detection was performed using HPLC with isocratic elution. The chromatographic column was a C-18 column (150 × 4.6 mm, 5 μm); mobile phase A consisted of 78% (0.05% trifluoroacetic acid), and mobile phase B consisted of 22% (HPLC-grade acetonitrile); the flow rate was 0.5 mL / min; the elution time was 25 min; the column temperature was 25℃; and the detector was a DAD detector with a detection wavelength of 228 nm. The ACE inhibition rate was calculated using the following formula: Where A1 is the peak area of hippuric acid in the sample group; A2 is the peak area of hippuric acid in the sample control group (ACE is replaced by buffer solution); A3 is the peak area of hippuric acid in the blank group (sample is replaced by buffer solution); A4 is the peak area of hippuric acid in the reagent blank group (both sample and ACE are replaced by buffer solution).
[0030] The three synthetic peptides GHVRVLQ, GVVGSHPIGTN, and DTNRHHGTTGVY have different IC50 values for ACE. 50 The values were 3.06 mmol / L, 6.23 mmol / L, and 1.37 mmol / L, respectively. Among them, the peptide DTNRHHGTTGVY showed the best ACE inhibitory activity (Figure 2).
[0031] Step Six: Determination of the renin-inhibiting activity of the peptide The renin inhibitor screening assay was performed according to the instructions of the kit. A 96-well microplate was used as the reaction vessel. For the blank control group, 20 μL of substrate, 160 μL of buffer, and 10 μL of solvent were added; for the blank control group, 20 μL of substrate, 150 μL of buffer, and 10 μL of solvent were added; and for the sample group, 20 μL of substrate, 150 μL of buffer, and 10 μL of sample solution were added. After preheating to 37°C, 10 μL of renin was added to both the blank control and sample wells to initiate the reaction. After mixing, the reaction was incubated at 37°C for 15 min. Fluorescence intensity was measured using an excitation wavelength of 335 nm and an emission wavelength of 485 nm. The renin inhibition rate of the sample was calculated using the following formula: Wherein, F0 is the fluorescence intensity of the blank group; F1 is the fluorescence intensity of the blank control group; and F2 is the fluorescence intensity of the sample group.
[0032] The IC50 of three synthetic peptides GHVRVLQ, GVVGSHPIGTN, and DTNRHHGTTGVY on renin 50The values were 6.40 mmol / L, 16.07 mmol / L, and 2.79 mmol / L, respectively. Among them, the peptide DTNRHHGTTGVY showed the best renin inhibitory activity (Figure 3).
[0033] Thus, the soybean peptide DTNRHHGTTGVY with high ACE and renin inhibitory activity was screened out, and its secondary mass spectrum is shown in Figure 4.
[0034] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0035] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only for the purpose of helping to understand the method and core ideas of the present invention. The above descriptions are only preferred embodiments of the present invention. It should be noted that due to the limitations of textual expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of the present invention.
Claims
1. A method for preparing soybean peptides with ACE and renin inhibitory activity, characterized in that, Includes the following steps: Step 1: Activation of water kefir grains. Prepare brown sugar water, inoculate with water kefir grains to activate them, and filter the activated water kefir grains using a sterile sieve for fermentation. Step 2: Preparation of fermentation products. The activated water kefir grains from Step 1 are inoculated into the fermentation broth prepared with soybean flour and brown sugar for fermentation to obtain a mixed fermentation product containing the target peptide. Step 3: Identification of peptide sequences in fermentation products. The lyophilized fermentation product powder was dissolved, ultrafiltered using a 10 kDa ultrafiltration tube, desalted using a Waters SEP-PAK C18 solid-phase extraction column, and lyophilized. The product was then reconstituted with formic acid solution, centrifuged, and the supernatant was collected. The peptide sequences in the sample were identified using LC-MS / MS (liquid chromatography-tandem mass spectrometry). The physicochemical properties and functions of the identified peptide sequences were predicted using computer software and an online website, and three potential ACE and renin dual-inhibitory peptides were screened. Step four: synthesis of polypeptide and verification of activity, Fmoc / t-Bu method was used for solid-phase synthesis of potential inhibitory peptides, the purity was all >95%, the ACE and renin inhibition rates were determined and the half-inhibitory concentration IC 50 was calculated, and soybean peptides with ACE and renin inhibition activity were screened.
2. The method for preparing soybean peptides with ACE and renin inhibitory activity according to claim 1, characterized in that, Step one is as follows: Prepare 10% brown sugar water, stir to dissolve, autoclave at 121℃ for 20 min, dispense into 500 mL reagent bottles, cool to room temperature, inoculate with 6% (w / v) water kefir granules, and incubate at 25℃ for 24 h, changing the brown sugar solution every 24 h; when dense small bubbles are continuously produced in the brown sugar water, it indicates that the activation is successful; filter with a sterile sieve to separate the activated water kefir granules for fermentation.
3. The method for preparing soybean peptides with ACE and renin inhibitory activity according to claim 2, characterized in that, Step two is as follows: The dried defatted soybean flour is passed through a 100-mesh sieve; 6% (m / v) soybean flour and 8% (m / v) brown sugar are weighed and dissolved in 200 mL of sterile distilled water cooled to 60-65℃, mixed thoroughly, pasteurized, cooled to room temperature, inoculated with 5% (m / v) water kefir grains, and anaerobic fermented in a 25℃ constant temperature incubator for 6 h. After fermentation, the water kefir grains are separated using a sterile sieve. The fermentation product is pasteurized and freeze-dried to obtain a mixed fermentation product containing the target peptide; the pasteurization temperature is 63℃ for 30 min.
4. The method for preparing soybean peptides with ACE and renin inhibitory activity according to claim 1, characterized in that, The liquid chromatography separation conditions in step three are as follows: Solution A is a 0.1% formic acid aqueous solution, and Solution B is a 0.1% formic acid acetonitrile aqueous solution with acetonitrile content of 80%. The chromatographic column is equilibrated with 92% Solution A, with an injection volume of 1 μL. The gradient settings are as follows: 0-98 min, Solution B from 8% to 28%; 98-113 min, Solution B from 28% to 37%; 113-117 min, Solution B from 37% to 100%; 117-120 min, Solution B is maintained at 100%.
5. The method for preparing soybean peptides with ACE and renin inhibitory activity according to claim 4, characterized in that, Mass spectrometry identification conditions: After separation by capillary high performance liquid chromatography, mass spectrometry analysis was performed using a Thermo QE HF mass spectrometer. Analysis time: 120 min; Detection mode: positive ion; The mass-charge ratio of peptides and peptide fragments was collected as follows: 20 fragment spectra were collected after each full scan, with a scan range of 400-1800, a primary resolution of 60000, a secondary resolution of 15000, and a collision energy of CE 28 eV. The raw mass spectrometry test files were retrieved from the relevant database using Proteome Discoverer 2.5 software to obtain the peptide identification results.
6. The method for preparing soybean peptides with ACE and renin inhibitory activity according to claim 5, characterized in that, The physicochemical properties and functions of the identified peptide sequences were predicted using computer software and online websites. First, peptides with 2-15 amino acids were selected, and potential ACE and renin dual-inhibitory peptides were further screened using computer-aided methods. The average hydrophilicity coefficient (GRAVY) of the peptides was calculated using the Expasy online platform. The renin and ACE inhibitory activities of the peptides were predicted using the BIOPEP-UWM database. Toxicity assessment was performed using the ToxinPred online platform. AllerTOP was used to predict sensitization, and peptides with potential toxicity and sensitization were removed. Then, molecular docking was performed on the peptides screened in the above steps. The three-dimensional crystal structures of the protein receptors ACE (PDB ID: 1O86) and renin (PDB ID: 2V0Z) were obtained from protein databases. PyMOL software was used for pre-docking preparation, removing water molecules and small molecule ligands. The Zn at the center of ACE was removed. 2+ The data needs to be retained; then, hydrogenation and charge processing are performed using AutoDock Tools software, and the data is saved in pdbqt format; the three-dimensional structure of the peptide is constructed using Tinker, and energy minimization processing is performed; finally, molecular docking is performed using AutoDock Vina to obtain the binding energy, and peptides with molecular docking binding energies ≤-9 kcal / mol with both ACE and renin are selected, thereby screening out 3 potential ACE and renin dual repressor peptides.
7. The method for preparing soybean peptides with ACE and renin inhibitory activity according to claim 6, characterized in that, The specific steps for determining the ACE inhibitory activity of the peptide in step four are as follows: Prepare a 100 mmol / L borate buffer solution with pH 8.3 containing 0.3 mol / L NaCl; determine the ACE inhibition rate of the synthetic peptide at different concentrations; vortex 30 μL of 2.5 mmol / L HCl with 10 μL of sample, preheat in a 37℃ water bath for 5 min, then add 20 μL of 100 mU / mL ACE, mix well, and react in a 37℃ water bath for 60 min. Stop the reaction by adding 60 μL of 1 mol / L HCl; the borate buffer solution serves as a blank control. Detection was performed using HPLC with isocratic elution. The chromatographic column was a C-18 column (150 × 4.6 mm, 5 μm); mobile phase A was 78% (0.05% trifluoroacetic acid), and mobile phase B was 22% (HPLC-grade acetonitrile); the flow rate was 0.5 mL / min; the elution time was 25 min; and the column temperature was 25℃. Detector: DAD detector, detection wavelength 228 nm; ACE inhibition rate is calculated by the following formula: Where A1 is the peak area of hippuric acid in the sample group; A2 is the peak area of hippuric acid in the sample control group; A3 is the peak area of hippuric acid in the blank group; and A4 is the peak area of hippuric acid in the reagent blank group.
8. The method for preparing soybean peptides with ACE and renin inhibitory activity according to claim 7, characterized in that, The renin inhibitory activity assay of the peptides was performed using a 96-well ELISA plate as the reaction vessel. For the blank control group, 20 μL of substrate, 160 μL of buffer, and 10 μL of solvent were added; for the blank control group, 20 μL of substrate, 150 μL of buffer, and 10 μL of solvent were added; and for the sample group, 20 μL of substrate, 150 μL of buffer, and 10 μL of sample solution were added. After preheating to 37°C, 10 μL of renin was added to both the blank control and sample wells to initiate the reaction. After mixing, the reaction was incubated at 37°C for 15 min. Fluorescence intensity was measured using an excitation wavelength of 335 nm and an emission wavelength of 485 nm. The renin inhibition rate of the sample was calculated using the following formula: Wherein, F0 is the fluorescence intensity of the blank group; F1 is the fluorescence intensity of the blank control group; and F2 is the fluorescence intensity of the sample group.