Specific affinity enrichment and preparation method of Chinese torreya protease hydrolysate ACE (Angiotensin Converting Enzyme) inhibitory peptide

Abstract

The invention relates to the technical field of biochemistry, in particular to a specific affinity enrichment and preparation method of a torreya grandis protease hydrolysate ACE inhibitory peptide, which comprises the following steps: step 1, preparing and characterizing magnetic aminated Fe3O4 nanoparticles; step 2, preparing and characterizing the immobilized trypsin; 3, preparing and characterizing the immobilized ACE, and optimizing reaction conditions; step 4, determining the activity of the immobilized ACE; step 5, then preparing and specifically enriching the Chinese torreya antihypertensive peptide; step 6, then, carrying out molecular docking through AutoDock Vina; according to the method, genipin is adopted as a cross-linking agent, magnetic Fe3O4 nanoparticles with the ACE inhibitory peptide specific affinity recognition function are constructed, specific enrichment of the ACE inhibitory peptide from the torreya grandis cake protein hydrolysate is achieved, and specific separation is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry technology, in particular to a method for specific affinity enrichment and preparation of ACE inhibitory peptides from the protease hydrolysis products of Torreya grandis Fort. Background Art

[0002] ACE inhibitory peptides are well-known for their ability to significantly inhibit the activity of angiotensin-converting enzyme (ACE) and have attracted much attention in drug research. Researchers have successfully isolated such active peptides from various plant proteins (such as wheat germ, nut powder, etc.). Among them, Torreya grandis Fort., as a unique rare economic nut, has the characteristics of both medicine and food homology and is rich in various active factors beneficial to health. These factors can not only enhance human immunity but also play a preventive role in cardiovascular diseases. According to research reports, the average protein content of Torreya grandis Fort. is about 10%, and more than 30 ACE inhibitory peptides have been identified in its protein hydrolysis products. Therefore, Torreya grandis Fort. protein can be used as a potential resource for mining ACE inhibitory peptides.

[0003] At present, the preparation process and purification technology for isolating and extracting ACE inhibitory peptides from food-derived proteins have been relatively mature. However, most of these separation and purification processes are based on the differences in molecular polarity or molecular weight, resulting in a large amount of inactive substances still remaining in the purified products and unable to achieve specific separation. Summary of the Invention

[0004] Aiming at the technical problem in the prior art that most of the separation and purification processes are based on the differences in molecular polarity or molecular weight, resulting in a large amount of inactive substances still remaining in the purified products and unable to achieve specific separation, the present invention provides a method for specific affinity enrichment and preparation of ACE inhibitory peptides from the protease hydrolysis products of Torreya grandis Fort.

[0005] The technical solution adopted by the present invention is: a method for specific affinity enrichment and preparation of ACE inhibitory peptides from the protease hydrolysis products of Torreya grandis Fort., specifically including the following steps:

[0006] Step 1, preparation and characterization of magnetic aminated Fe3O4 nanoparticles;

[0007] Step 2, preparation and characterization of immobilized trypsin;

[0008] Step 3, preparation, characterization and optimization of reaction conditions of immobilized ACE;

[0009] Step 4, determination of the activity of immobilized ACE;

[0010] Step 5, then preparation and specific enrichment of hypotensive peptides from Torreya grandis Fort.;

[0011] Step 6, then, through AutoDockVina molecular docking;

[0012] Step 7. Finally, determine the content of torreya grandis ACE inhibitory peptide.

[0013] In one of the embodiments, in Step 1, prepare and characterize magnetic aminated Fe3O4 nanoparticles. The specific method is as follows:

[0014] Weigh 2.7 g of ferric chloride hexahydrate (FeCl3·6H2O) and 7.2 g of sodium acetate (CH3COONa), add 100 mL of ethylene glycol, mix well by magnetic stirring, seal, and react in a 200 mL high-pressure reactor at 200 °C for 8 hours;

[0015] Wait until it cools to room temperature, wash 6 times with ethanol, and dry in vacuum at 60 °C for 12 hours to obtain magnetic Fe3O4 nanoparticles;

[0016] Weigh 150 mg of Fe3O4 nanoparticles and disperse them in 100 mL of ethanol. Ultrasonically treat for 0.5 hour, add 25 mL of deionized water containing 1.2 mL of concentrated ammonia water, continuously stir, and then slowly dropwise add 0.5 mL of ethanol solution containing 100 μL of tetraethyl orthosilicate (TEOS), and react for 9 hours;

[0017] Collect the product by magnetic separation, wash it alternately with ethanol and deionized water multiple times, and dry at room temperature;

[0018] Disperse the dried product in 120 mL of isopropanol by ultrasonic treatment, add 0.5 mL of (3-aminopropyl)triethoxysilane (APTES), and stir and react for 9 hours;

[0019] After magnetic separation, wash with ethanol and deionized water, and dry in vacuum overnight to obtain aminated magnetic Fe3O4@NH2 nanoparticles;

[0020] Use an X-ray diffractometer (XRD) to analyze the crystal structures of magnetic Fe3O4 and Fe3O4@NH2;

[0021] Observe its morphological characteristics with a scanning electron microscope (SEM);

[0022] Use a thermogravimetric analyzer (TGA) to determine the thermal stability of the material;

[0023] Characterize the functional groups with Fourier transform infrared spectroscopy (FTIR);

[0024] Test the magnetic properties of the material with a vibrating sample magnetometer (VSM).

[0025] In one of the embodiments, in Step 2, prepare and characterize immobilized trypsin. The specific method is as follows:

[0026] Weigh 2.0 g of Fe3O4@NH2 nanoparticles and place them in a 50 mL stoppered conical flask. Add 16 mL of a 0.6 g / L aqueous solution of genipin and crosslink at 55 °C for 8 hours;

[0027] After the reaction is completed, wash the unreacted genipin thoroughly with distilled water. The resulting product is stored refrigerated at 4 °C for later use;

[0028] Weigh 1 g of the genipin-crosslinked Fe3O4@NH2 nanoparticles and transfer them to a 50 mL stoppered conical flask. Add 20 mL of a 12.5 μg / mL trypsin solution and incubate statically at 25 °C and pH = 6.0 for 16 hours;

[0029] After incubation, collect the immobilized enzyme by magnetic separation and wash it 6 times with distilled water to remove unbound trypsin and impurities. Finally, store it refrigerated at 4 °C;

[0030] Characterize the surface functional groups of the immobilized enzyme using Fourier transform infrared spectroscopy (FTIR).

[0031] In one of the embodiments, in step three, the preparation, characterization, and reaction condition optimization of immobilized ACE are as follows:

[0032] Weigh Fe3O4@NH2 nanoparticles and ultrasonically disperse them in an ACE phosphate buffer solution;

[0033] Use EDC as a crosslinking agent, slowly oscillate the reaction mixture, and slowly oscillate the reaction for a certain time under constant temperature conditions (37 °C), then magnetically separate the ACE-immobilized enzyme;

[0034] Collect the supernatant and measure the content of ACE in it to determine the solid loading amount of ACE;

[0035] Wash the magnetic particles containing immobilized ACE several times with deionized water to ensure complete removal of free ACE;

[0036] Finally, freeze-dry the particles and store them for further use;

[0037] Through XRD, SEM, TGA, and FTIR analyses, the crystal structure, morphology, thermal stability, and functional groups of the magnetically loaded Fe3O4@NH2@ACE nanoparticles were determined;

[0038] Study its magnetic field-assisted characteristics using a vibrating sample magnetometry (VSM);

[0039] By changing the pH value of the system (5.0, 6.0, 7.0, 8.0, and 9.0), plot a change curve with the fixed load as the ordinate and the pH value as the abscissa, and select the optimal pH value;

[0040] By adjusting the initial concentration of the ACE buffer solution (0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 mmol / L), under the condition of a fixed load, a change curve is plotted with the initial concentration as the abscissa to select the optimal initial concentration;

[0041] By changing the reaction time (5, 10, 15, 20, 25, 30, 35, and 40 min), a change curve is plotted with the fixed load as the ordinate and the reaction time as the abscissa to select the optimal reaction time.

[0042] In one of the embodiments, in step four, for the determination of the activity of immobilized ACE, the specific method is as follows:

[0043] The activity of ACE is evaluated by measuring the amount of hippuric acid (HA) generated. The experimental mixture (0.3 mL) consists of the following three parts: 130 μL of BBS buffer solution (pH 8.3, 0.1 mmol·L -1 , containing 0.3 mol·L -1 NaCl), 40 μL of HHL solution (5 mmolL -1 ), as well as 100 μL of free enzyme and 100 μL of immobilized enzyme ACE;

[0044] After the mixture is incubated at a constant temperature of 37 °C for 10 minutes, 0.1 mL of HCl (1 mol·L -1 ) is added to terminate the incubation;

[0045] Subsequently, high-performance liquid chromatography (RP-HPLC) (Agilent 1260) equipped with a DAD detector is used to detect the content of HA at 228 nm;

[0046] The definition of the unit activity (U) of ACE is: under the experimental conditions, the amount of enzyme required to catalyze the formation of 1 μmol·L -1 HA of HHL per minute; the relative ACE activity can be calculated by the following formula:

[0047]

[0048] where A1 is the peak area of HA in the control group and A2 is the peak area of HA of the immobilized ACE.

[0049] In one of the embodiments, in step five, for the preparation and specific enrichment of torreya grandis antihypertensive peptide, the specific method is as follows:

[0050] Mix 2.0 g of torreya grandis seed cake powder with 40 mL of 0.01 mol / L PBS buffer solution (pH = 7.0);

[0051] Subsequently, centrifugation was carried out at 11000 r / min for 15 minutes to obtain the supernatant for the preparation of torreya grandis protein solution;

[0052] Subsequently, 1.0 g of immobilized trypsin was added to the protein solution, and the mixture was incubated at a constant temperature of 45 °C (pH = 6.0) for 4 hours to allow hydrolysis to occur;

[0053] Then, the immobilized enzyme was separated magnetically and centrifuged at a speed of 7200 r / min for 15 minutes;

[0054] The supernatant was taken and immobilized ACE was added, and the hydrolysate containing the target active peptide was obtained by magnetic separation; then, immobilized ACE was added to the solution to adsorb the ACE inhibitory peptide;

[0055] After enrichment, SDS was used to elute the ACE inhibitory peptide; finally, liquid chromatography - mass spectrometry (LC - MS / MS) was used to analyze the sample under the following conditions: the mobile phase was 0.1% formic acid aqueous solution (A) and 0.1% formic acid - 80% acetonitrile mixture (B);

[0056] The chromatographic column used was a Diamonsil C18 reversed - phase chromatographic column (150 μm × 150 mm, 1.9 μm), the flow rate was 300 nL / min, and the elution time was 30 minutes; a mass spectrometer was used to analyze the oligopeptide sequence.

[0057] In one of the embodiments, in step six, through molecular docking with AutoDockVina, the specific method is as follows:

[0058] The ACE crystal structure (UniProtKB:P12821) was obtained through UniProt;

[0059] To prepare the receptor, hydrogen atoms were added and water molecules were removed using AutoDockTools 1.5.7;

[0060] Before docking, the three - dimensional conformations of the peptide chains CGDDGY, ANPCKYE, AAELLPGS, PRGKKEY, and TAFLGMFLEHW were predicted by AlphaFold3;

[0061] Subsequently, these structures were converted into PDB files with the help of PYMOL software;

[0062] Then, molecular docking and screening of potential inhibitory peptides were carried out using AutodockVina;

[0063] The docking was set at the coordinates (x: - 4.144, y: 32.153, z: - 18.399), the search space was 126×126×126 Å, and the resolution was set to

[0064] The traversal parameter was set to 8, the docking of the peptide with the ACE receptor protein was completed, and the interaction strength and binding energy between different ligand conformations and the ACE crystal structure were evaluated. The lower the binding energy, the higher the score, indicating a tighter binding and stable interaction between the ligand and the ACE receptor protein, thus having potential inhibitory activity.

[0065] In one of the embodiments, in step seven, the determination of the content of Torreya grandis ACE inhibitory peptide is carried out as follows:

[0066] When measuring the ACE inhibition rate, the sample is used to replace the BBS buffer solution and calculated according to the following formula:

[0067]

[0068] Where A1 is the peak area of HA in the control group, and A2 is the peak area of HA and Torreya grandis ACE inhibitory peptide.

[0069] The beneficial effects of the present invention are as follows: Compared with the prior art, in the present invention, genipin is used as a cross-linking agent to construct magnetic Fe3O4 nanoparticles with specific affinity recognition function for ACE inhibitory peptides, realizing the specific enrichment of ACE inhibitory peptides from Torreya grandis cake meal protein hydrolysate and achieving specific separation. Through the immobilized enzyme technology, 61 ACE inhibitory peptides were successfully enriched from Torreya grandis cake meal protein hydrolysate. Among them, the molecular docking binding energies of 5 peptide segments such as CGDDGY, ANPCKYE, AAELLPGS, PRGKKEY, and TAFLGMFLEHW with ACE are lower than -9 kcal / mol. In this study, magnetic Fe3O4 nanoparticles with an affinity recognition functional layer for ACE inhibitory peptides were constructed using genipin as a cross-linking agent. A total of 61 peptide segments were co-enriched using immobilized ACE. Through molecular docking, it was found that these peptide segments all have a certain binding energy with ACE, and the binding energies of 5 peptide segments are less than -9. Among them, the inhibition rate of CGDDGY is the highest (about 35% at 1 μmol / L), verifying the potential application value of this peptide segment in the treatment of hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 a of which is the scanning electron microscope image of Fe3O4;

[0071] Figure 1 b of which is the scanning electron microscope image of Fe3O4@SiO2;

[0072] Figure 1 c of which is the scanning electron microscope image of Fe3O4@NH2;

[0073] Figure 1d is the scanning electron microscope image of Fe3O4@NH2@ACE, with a scale of 500 nm;

[0074] Figure 1 e is the scanning electron microscope image of Fe3O4@NH2@Trypsin;

[0075] Figure 1 f is the scanning electron microscope image of Fe3O4@NH2@ACE, with a scale of 100 nm;

[0076] Figure 1 g is the Fourier transform infrared spectroscopy (FTIR) result graph (scale: 100 mm);

[0077] Figure 1 h is the X-ray diffractometer (XRD) analysis data graph;

[0078] Figure 1 i is the thermogravimetric analysis (TGA) result graph;

[0079] Figure 1 j is the vibrating sample magnetometer analysis (VSM) graph;

[0080] Figure 2 In a, it is the schematic diagram of buffer solution pH value analysis;

[0081] Figure 2 In b, it is the schematic diagram of immobilization time analysis;

[0082] Figure 2 In c, it is the schematic diagram of initial enzyme concentration analysis;

[0083] Figure 2 In d, it is the broken line statistical distribution graph;

[0084] Figure 3 In a and d, they are both the schematic diagrams of initial concentration and pH value;

[0085] Figure 3 In b and e, they are both the schematic diagrams of pH value and reaction time;

[0086] Figure 3 In c and f, they are both the schematic diagrams of reaction time and initial concentration;

[0087] Figure 4 In a, it is the 3D and 2D images of CGDDGY;

[0088] Figure 4 In b, it is the 3D and 2D images of ANPCKYE;

[0089] Figure 4 In c, it is the 3D and 2D images of AAELLPGS;

[0090] Figure 4 Among them, d is the 3D and 2D images of PRGKKEY;

[0091] Figure 5 It is a schematic diagram of the ACE inhibitory activities of GDDGY, ANPCKYE, AAELLPGS, PRGKKEY, and TAFLGMFLEHWLT. Specific implementation manners

[0092] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "front", "upper", "lower", "left", "right", "vertical", "horizontal", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0093] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection, or an indirect connection through an intermediate medium, and can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0094] The following further describes the present invention in conjunction with the attached Figures 1 - 5 drawings.

[0095] The materials selected in the present invention are as follows:

[0096] Ferric chloride hexahydrate (FeCl3·6H2O, 99%), 3-aminopropyltriethoxysilane (APTES, 99%), genipin (98%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 98.5%), and ammonium bicarbonate (NH4HCO3, analytical pure) were purchased from Macklin (Shanghai Macklin Biochemical Co., Ltd.). Sodium acetate (CH3COONa, analytical pure), ethylene glycol (analytical pure), ethanol (analytical pure), and isopropyl alcohol (analytical pure) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing). Bovine serum albumin (BSA) was purchased from Xiya Chemical Reagents (Shandong). Tetraethyl orthosilicate (TEOS, 98%) and N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE, 98%) were purchased from Aladdin (Shanghai Aladdin Biochemical Technology Co., Ltd.). Angiotensin-converting enzyme (ACE, 90%) was purchased from Yingxin Laboratory (Shanghai). Hippuryl-histidyl-leucine (HHL, 98%) was purchased from Yuanye Bio-Technology (Shanghai). Borate buffer was purchased from Feijing Bio (Fuzhou). Torreya grandis fruits were collected from Lin'an City, Zhejiang Province, China in September 2024.

[0097] To solve the problems existing in the background art, the present application proposes the following technical solutions: a method for specific affinity enrichment and preparation of ACE inhibitory peptides from Torreya grandis protease hydrolysates, which specifically includes the following steps:

[0098] Step 1, prepare and characterize magnetic aminated Fe3O4 nanoparticles;

[0099] Among them, in Step 1, the preparation and characterization of magnetic aminated Fe3O4 nanoparticles are carried out according to the following specific method:

[0100] Weigh 2.7 g of ferric chloride hexahydrate (FeCl3·6H2O) and 7.2 g of sodium acetate (CH3COONa), add 100 mL of ethylene glycol, mix well by magnetic stirring, seal, and react in a 200 mL high-pressure reactor at 200 °C for 8 hours;

[0101] Wait until it cools to room temperature, wash 6 times with ethanol, and dry in vacuum at 60 °C for 12 hours to obtain magnetic Fe3O4 nanoparticles;

[0102] Weigh 150 mg of Fe3O4 nanoparticles and disperse them in 100 mL of ethanol. Ultrasonically treat for 0.5 hour, add 25 mL of deionized water containing 1.2 mL of concentrated ammonia water, continuously stir, and then slowly dropwise add 0.5 mL of ethanol solution containing 100 μL of tetraethyl orthosilicate (TEOS), and react for 9 hours;

[0103] Collect the product by magnetic separation, wash it alternately with ethanol and deionized water for multiple times, and dry at room temperature;

[0104] Disperse the dried product ultrasonically in 120 mL of isopropanol, add 0.5 mL of (3-aminopropyl)triethoxysilane (APTES), and stir and react for 9 hours;

[0105] After magnetic separation, wash with ethanol and deionized water, and dry in vacuum overnight to obtain aminated magnetic Fe3O4@NH2 nanoparticles;

[0106] Analyze the crystal structures of magnetic Fe3O4 and Fe3O4@NH2 by using an X-ray diffractometer (XRD);

[0107] Observe its morphological characteristics with a scanning electron microscope (SEM);

[0108] Determine the thermal stability of the material with a thermogravimetric analyzer (TGA);

[0109] Characterize the functional groups by Fourier transform infrared spectroscopy (FTIR);

[0110] Test the magnetic properties of the material with a vibrating sample magnetometer (VSM).

[0111] Step 2: Preparation and characterization of immobilized trypsin;

[0112] Among them, in Step 2, for the preparation and characterization of immobilized trypsin, the specific method is as follows:

[0113] Weigh 2.0 g of Fe3O4@NH2 nanoparticles and place them in a 50 mL stoppered conical flask. Add 16 mL of 0.6 g / L genipin aqueous solution and crosslink at 55 °C for 8 hours;

[0114] After the reaction is completed, wash the unreacted genipin thoroughly with distilled water, and store the obtained product at 4 °C for later use;

[0115] Weigh 1 g of genipin-crosslinked Fe3O4@NH2 nanoparticles and transfer them to a 50 mL stoppered conical flask. Add 20 mL of 12.5 μg / mL trypsin solution and incubate statically at 25 °C and pH = 6.0 for 16 hours;

[0116] After incubation, collect the immobilized enzyme by magnetic separation, wash it 6 times with distilled water to remove unbound trypsin and impurities, and finally store it at 4 °C;

[0117] Characterize the surface functional groups of the immobilized enzyme by Fourier transform infrared spectroscopy (FTIR).

[0118] Step 3: Preparation, characterization and reaction condition optimization of immobilized ACE;

[0119] Among them, in Step 3, for the preparation, characterization and reaction condition optimization of immobilized ACE, the specific method is as follows:

[0120] Weigh Fe3O4@NH2 nanoparticles and ultrasonically disperse them in ACE phosphate buffer;

[0121] Use EDC as a crosslinking agent, slowly oscillate the reaction mixture, and slowly oscillate the reaction for a certain time under constant temperature conditions (37 °C), then magnetically separate the ACE immobilized enzyme;

[0122] Collect the supernatant, measure the content of ACE in it, and determine the solid loading amount of ACE;

[0123] Wash the magnetic particles containing immobilized ACE several times with deionized water to ensure complete removal of free ACE;

[0124] Finally, freeze-dry the particles and store them for further use;

[0125] Through XRD, SEM, TGA and FTIR analyses, the crystal structure, morphology, thermal stability and functional groups of the magnetically loaded Fe3O4@NH2@ACE nanoparticles were determined;

[0126] The magnetic field-assisted properties were studied by vibrating sample magnetometry (VSM);

[0127] By changing the pH value of the system (5.0, 6.0, 7.0, 8.0, and 9.0), a change curve was plotted with the fixed load as the ordinate and the pH value as the abscissa to select the optimal pH value;

[0128] By adjusting the initial concentration of the ACE buffer (0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 mmol / L), a change curve was plotted with the initial concentration as the abscissa under the condition of fixed load to select the optimal initial concentration;

[0129] By changing the reaction time (5, 10, 15, 20, 25, 30, 35, and 40 min), a change curve was plotted with the fixed load as the ordinate and the reaction time as the abscissa to select the optimal reaction time.

[0130] Step four, perform the determination of the activity of immobilized ACE;

[0131] Among them, in step four, for the determination of the activity of immobilized ACE, the specific method is as follows:

[0132] The ACE activity was evaluated by measuring the amount of hippuric acid (HA) generated. The experimental mixture (0.3 mL) consisted of the following three parts: 130 μL of BBS buffer (pH 8.3, 0.1 mmol·L -1 , containing 0.3 mol·L -1 NaCl), 40 μL of HHL solution (5 mmolL -1 ), as well as 100 μL of free enzyme and 100 μL of immobilized enzyme ACE;

[0133] After the mixture was incubated at a constant temperature of 37 °C for 10 minutes, 0.1 mL of HCl (1 mol·L -1 ) was added to terminate the incubation;

[0134] Subsequently, the content of HA was detected at 228 nm using a high-performance liquid chromatography (RP-HPLC) (Agilent 1260) equipped with a DAD detector;

[0135] The definition of the unit activity (U) of ACE is: under the experimental conditions, the amount of enzyme required to catalyze the formation of 1 μmol·L -1 HA of HHL per minute; the relative ACE activity can be calculated by the following formula:

[0136]

[0137] where A1 is the peak area of HA in the control group and A2 is the peak area of HA of the immobilized ACE.

[0138] Step 5, then prepare and specifically enrich Torreya grandis seed peptide with antihypertensive effect;

[0139] Among them, in Step 5, the preparation and specific enrichment of Torreya grandis seed peptide with antihypertensive effect are carried out as follows:

[0140] Mix 2.0 g of Torreya grandis seed cake powder with 40 mL of 0.01 mol / L PBS buffer (pH = 7.0);

[0141] Subsequently, centrifuge at 11000 r / min for 15 minutes to obtain the supernatant for preparing Torreya grandis seed protein solution;

[0142] Subsequently, add 1.0 g of immobilized trypsin to the protein solution and incubate at a constant temperature of 45 °C (pH = 6.0) for 4 hours to cause hydrolysis reaction;

[0143] Then, magnetically separate the immobilized enzyme and centrifuge at a speed of 7200 r / min for 15 minutes;

[0144] Take the supernatant and add immobilized ACE, and obtain the hydrolyzate containing the target active peptide through magnetic separation; then add immobilized ACE to the solution to adsorb ACE inhibitory peptide;

[0145] After enrichment, use SDS to elute ACE inhibitory peptide; finally, use liquid chromatography - mass spectrometry (LC - MS / MS) to analyze the sample under the following conditions: the mobile phase is 0.1% formic acid aqueous solution (A) and 0.1% formic acid - 80% acetonitrile mixture (B);

[0146] Select a Diamonsil C18 reverse - phase chromatography column (150 μm×150 mm, 1.9 μm) for the chromatographic column, the flow rate is 300 nL / min, and the elution time is 30 minutes; use a mass spectrometer to analyze the oligopeptide sequence.

[0147] Step 6, then perform molecular docking through AutoDockVina;

[0148] Among them, in Step 6, the molecular docking through AutoDockVina is carried out as follows:

[0149] The ACE crystal structure (UniProtKB:P12821) is obtained through UniProt;

[0150] To prepare the receptor, use AutoDockTools 1.5.7 to add hydrogen atoms and remove water molecules;

[0151] Before docking, the three-dimensional conformations of the peptide chains CGDDGY, ANPCKYE, AAELLPGS, PRGKKEY, and TAFLGMFLEHW were predicted by AlphaFold3;

[0152] Subsequently, these structures were converted into PDB files with the aid of PYMOL software;

[0153] Then, molecular docking and screening of potential inhibitory peptides were performed using AutodockVina;

[0154] The docking settings were at coordinates (x: -4.144, y: 32.153, z: -18.399), the search space was 126×126×126 Å, and the resolution was set to

[0155] The traversal parameter was set to 8. The docking of the peptide with the ACE receptor protein was completed, and the interaction strength and binding energy between different ligand conformations and the ACE crystal structure were evaluated. The lower the binding energy, the higher the score, indicating a tighter binding and more stable interaction between the ligand and the ACE receptor protein, and thus potential inhibitory activity.

[0156] Step seven, finally, the determination of the content of Torreya grandis ACE inhibitory peptide was carried out.

[0157] In step seven, for the determination of the content of Torreya grandis ACE inhibitory peptide, the specific method is as follows:

[0158] The ACE inhibition rate was measured by the following method. The sample was used to replace the BBS buffer, and the calculation was carried out according to the following formula:

[0159]

[0160] where A1 is the peak area of HA in the control group, and A2 is the peak area of HA and Torreya grandis ACE inhibitory peptide.

[0161] The method is as follows:

[0162] 0.3 mL of the experimental mixture consisted of the following three parts: 130 μL of the sample solution, 40 μL of 5 mmol L-1 HHL solution, 100 μL of free enzyme, and 100 μL of immobilized enzyme ACE. After the mixture was incubated at 37 °C for 10 minutes, 0.1 mL of 1 mol·L-1 HCl was added to terminate the incubation. Subsequently, high-performance liquid chromatography was used, equipped with a DAD detector, to detect the content of HA at 228 nm and calculate according to the following formula:

[0163]

[0164] where A1 is the peak area of HA in the control group, and A2 is the peak area of HA and Torreya grandis ACE inhibitory peptide.

[0165] In summary:

[0166] The process for preparing Fe3O4@NH2@ACE nanoparticles is as follows: First, magnetic Fe3O4 nanoparticles are prepared using the solvothermal method. Subsequently, silica is coated on the surface of Fe3O4 to obtain SiO2-coated Fe3O4@SiO2, which is then modified by APTES to obtain Fe3O4@NH2. Finally, ACE or trypsin is cross-linked with the aminated Fe3O4 to prepare Fe3O4@NH2@ACE or Fe3O4@NH2@Trypsin.

[0167] The morphologies of Fe3O4, Fe3O4@SiO2, Fe3O4@NH2, Fe3O4@NH2@ACE, and Fe3O4@NH2@Trypsin were confirmed by scanning electron microscope (SEM) images (see Figure 1 a- Figure 1 f). As can be seen from Figure 1 b, the surface of Fe3O4@SiO2 is smoother than that of unmodified Fe3O4 and shows a dense and uniform spherical structure of approximately 120 nm. As shown in Figure 1 c, Fe3O4@NH2 after APTES modification presents a typical core-shell structure. As seen in Figure 1 d- Figure 1 f, the SEM images of Fe3O4@NH2, Fe3O4@NH2@ACE, and Fe3O4@NH2@Trypsin show the same surface morphology, indicating that grafting ACE or trypsin onto the surface of Fe3O4@NH2 does not affect its morphology and size.

[0168] The functional groups of Fe3O4, Fe3O4@SiO2, Fe3O4@NH2, Fe3O4@NH2@ACE, and Fe3O4@NH2@Trypsin were analyzed by Fourier transform infrared spectroscopy (FTIR) (see Figure 1 g). The signals at 580 cm -1 and 1100 cm -1 are consistent with the characteristic bands of Fe-O and Si-O. For Fe3O4@NH2 particles, the peaks at 3200 - 3500 cm -1 and 1600 cm -1 can be attributed to the stretching vibration and bending vibration of N-H, respectively, indicating that the modification has a significant effect on the surface functional group structure of Fe3O4. In addition, the demagnetization and remanent magnetic moment of all samples are close to zero, indicating that the samples have good recyclability and dispersibility after demagnetization treatment.

[0169] The crystal structures of Fe3O4, Fe3O4@SiO2, Fe3O4@NH2, Fe3O4@NH2@ACE, and Fe3O4@NH2@Trypsin were analyzed by X-ray diffraction (XRD) (see Figure 1 h). The results of the crystal morphology analysis showed that both Fe3O4@NH2@ACE and Fe3O4@NH2@Trypsin nanoparticles exhibited typical Fe3O4 structural characteristics, with the main diffraction peaks located at approximately 30°, 35°, and 43°, corresponding to the crystal plane indices (220), (311), and (400), respectively, demonstrating the good quality of the Fe3O4 crystal structure after modification.

[0170] The thermal property characteristics of Fe3O4, Fe3O4@SiO2, Fe3O4@NH2, Fe3O4@NH2@ACE, and Fe3O4@NH2@Trypsin were analyzed by thermogravimetric analysis (TGA) (see Figure 1 j). An initial weight loss occurred at approximately 190 °C, which may be related to the residual solvent in the pores of the Fe3O4@NH2 structure. The weight loss in the range of 190 - 300 °C may be related to the decomposition of part of APTES on Fe3O4@NH2@ACE or Fe3O4@NH2@Trypsin. When the temperature rises from 300 °C to 800 °C, the weight loss phenomenon may be related to the structural collapse of Fe3O4@NH2@ACE or Fe3O4@NH2@Trypsin.

[0171] The magnetic properties of the samples were characterized by vibrating sample magnetometry (VSM) (see Figure 1 j). The saturation magnetization of the pure Fe3O4 sample was the largest (approximately 80 emu / g), indicating its strong magnetism, while the saturation magnetization of the surface-modified samples gradually decreased, and the saturation magnetization of Fe3O4@NH2, Fe3O4@NH2@ACE, and Fe3O4@NH2@Trypsin decreased to approximately 50 emu / g. In addition, the demagnetization and remanent magnetic moment of all samples were close to zero, indicating that the samples did not retain magnetism after the removal of the magnetic field and had good recyclability and dispersibility, suitable for applications under dynamic magnetic field conditions.

[0172] Among them, in order to achieve the maximum activity of immobilized ACE enzyme, three key parameters were optimized: buffer pH value, immobilization time, and initial enzyme mass concentration. First, with the initial enzyme mass concentration maintained at 1.0 μg / mL and the immobilization time of 30 minutes, the effect of buffer pH on the relative activity of immobilized ACE was studied (see Figure 2 a). The results showed that the pH of the buffer significantly affected the relative activity of immobilized ACE, and its highest value increased by 83.92%, occurring at pH 8.0.

[0173] Next, under the conditions of an initial enzyme mass concentration of 1.0 μg / mL and a buffer pH maintained at 8.0, the effect of the immobilization time on ACE activity was investigated (see Figure 2 b). The results showed that the enzyme activity reached its peak at 30 minutes after immobilization, increasing by 96.41%. Insufficient or excessive immobilization time led to a decrease in enzyme activity, and the possible reasons included enzyme inactivation, binding site saturation, non-specific adsorption, and disruption of the immobilization equilibrium.

[0174] Finally, under the conditions of an initial enzyme mass concentration of 2.0 μg / mL, a buffer pH maintained at 8.0, and an immobilization time of 30 minutes, the effect of different initial enzyme mass concentrations on ACE activity was evaluated (see Figure 2 c). The results showed that the highest enzyme activity increase of 80.17% occurred at an initial enzyme mass concentration of 2.0 μg / mL. Deviating from this optimal concentration might lead to a decrease in enzyme activity, and the possible reasons included non-specific adsorption, steric exclusion effects, and saturation of the binding sites on the carrier surface.

[0175] Based on the above screening results, the Box-Behnken design was used to determine the optimal conditions for the ACE enzyme immobilization reaction. The extraction conditions were optimized according to the actual activity of the ACE immobilized enzyme, including the initial enzyme concentration (A: 1.0, 1.5, 2.0, 2.5, and 3.0 μg / mL), the buffer pH value (B: 5, 6, 7, 8, and 9), and the immobilization reaction time (C: 10, 20, 30, 40, and 50 min). The specific results are shown in Table 1. Through multiple regression analysis of the actual experimental data, the relationship between the three variables and the activity of the ACE immobilized enzymatic reaction was determined, and the following quadratic polynomial equation was obtained:

[0176] Relative activity = 97.37 - 0.67×A – 3.61×B + 1.85×C – 6.69×A×B – 24.77×A×C – 5.15×B×C – 15.76×A 2 - 9.65×B 2 - 21.49×C 2

[0177] The fitting results of the model showed that the regression was significantly meaningful (p = 0.0211), effectively capturing the relationship between the experimental conditions and the response value. The analysis results indicated that the quadratic term of the reaction time (C, p = 0.0064) and the interaction between the initial concentration and the reaction time (AC, p = 0.0030) had a significant impact on the relative activity. In contrast, other interaction terms (such as AB, BC) and the linear terms did not reach a significant level (p > 0.05).

[0178] Further examination of the response surface and contour plots highlighted the crucial importance of the interaction between the initial concentration and reaction time, which showed a non-linear trend ( Figure 3 a-f). Additionally, the quadratic effect of the reaction time was significant, highlighting its importance in optimizing the relative activity. By analyzing the conditions near the midpoint of the experimental range, the optimal conditions were determined: an initial concentration of 3 μg / mL, a pH value of 7, and a reaction time of 30 min. Under these conditions, the relative activity predicted by the model was close to 100%.

[0179] Furthermore, by screening three key factors - initial concentration (A), pH value (B), and reaction time (C), and using the Plackett-Burman test to evaluate their importance and impact on the relative activity (%), the results showed that the initial concentration was the most influential positive factor (with an effect value of 15.08 and a coefficient of 7.54), and the P-value was 0.0894, close to the significant level, so it was determined to be a key factor worthy of further optimization. The influence of the reaction time was second only to the initial concentration, with an effect value of 5.79 and a coefficient of 2.90, and its P-value was 0.4793, showing a moderate but not significant positive effect. In contrast, the pH value had the least impact on the relative activity, with an effect value of -4.10 and a coefficient of -2.05, and its P-value was 0.6133, indicating that its impact on the relative activity could be ignored and was regarded as a secondary factor. Therefore, it was decided to focus on the initial concentration in future optimization work. At the same time, to simplify the experimental process and improve the optimization efficiency, the pH value and reaction time would be maintained at appropriate levels.

[0180] Table 1 Variables and levels in the Box-Behnken response surface analysis design.

[0181]

[0182] Table 2 Plackett-Burman experimental design and corresponding values

[0183]

[0184]

[0185] Table 3 Estimated effects, coefficients, and significance tests of relative activity

[0186] Time Effect Coefficient Standard Error T - value P - value Constant 68.79 3.90 17.63 0.000 A: Initial Concentration 15.08 7.54 3.90 1.93 0.089 B: pH Value -4.10 -2.05 3.90 -0.53 0.613 C: Reaction Time 5.79 2.90 3.90 0.74 0.479

[0187] To evaluate the effect of peptide enrichment by immobilized enzyme on ACE inhibitory activity, molecular docking technology ( Figure 4) was used to study the interaction between ACEI peptides and ACE receptors and to determine their low-energy binding conformations. Based on the molecular docking analysis of four active peptides (CGDDGY, ANPCKYE, AAELLPGS, PRGKKEY) with ACE receptors, the binding energy and specific binding affinity predicted that these active peptides had better inhibitory effects.

[0188] CGDDGY exhibited the strongest binding affinity (BE: -10.2 kcal / mol), forming a large number of hydrogen bonds, hydrophobic interactions, and electrostatic interactions at the binding site, ensuring the stability of its binding. The binding energy of ANPCKYE was -9.9 kcal / mol. Although its binding affinity was slightly inferior to that of CGDDGY, strong interactions, including hydrogen bonds and hydrophobic interactions, were still formed at its binding site, indicating its high inhibitory potential. The binding energies of AAELLPGS and PRGKKEY were -9.2 kcal / mol. Compared with the former two, fewer hydrogen bonds were formed at the binding site, resulting in slightly weaker binding affinity. The binding results of the four peptides with ACE receptors verified the feasibility of the inhibitory activity of the active peptides against ACE.

[0189] In addition, based on molecular docking technology, the inhibitory activity of the peptides against ACE was further evaluated by measuring the amount of hippuric acid (HA) generated ( Figure 5 ). The experimental results showed that when the concentration was 1 μmol / L, all five active peptides exhibited good inhibitory effects. Among them, CGDDGY showed the best inhibitory activity, which was completely consistent with the binding energy predicted by molecular docking technology. Due to some uncontrollable factors in the actual operation, the inhibitory activities of the other four peptides deviated. Specifically, under the same experimental conditions, the inhibition rate of CGDDGY reached approximately 35%, which was the active peptide with the strongest inhibitory activity. The inhibition rate of ANPCKYE was 32%, showing significant inhibitory activity.

[0190] The inhibition rates of AAELLPGS, PRGKKEY, and TAFLGMFLEHWLT were 25%, 30%, and 23% respectively. Although the inhibition rates of these active peptides were slightly lower than those of CGDDGY and ANPCKYE, they still had certain inhibitory activities. As a control group, the activity of ACE in the blank control group without adding any peptides was basically not affected, verifying the effectiveness of the experiment. These results further supported the predictive analysis of molecular docking technology, indicating that these active peptides had the potential to be candidate molecules for ACE inhibitors.

[0191] By optimizing the synthesis conditions of the ACE immobilized enzyme and adopting orthogonal design, the present invention constructs a functional magnetic Fe3O4 composite nanoparticle with functional groups for recognizing and binding ACE inhibitory antihypertensive peptides on its surface. Under the conditions of buffer pH 8.0, initial enzyme concentration 1.0 μg / mL, and immobilization time 30 min, uniformly spherical Fe3O4@NH2@ACE nanoparticles with a size of about 120 nm are successfully prepared. Using this immobilized enzyme to enrich and screen peptides in the protein hydrolysate of torreya grandis cake meal, a total of 61 ACE inhibitory antihypertensive peptides are obtained, and 5 ACE inhibitory antihypertensive peptides CGDDGY, ANPCKYE, AAELLPGS, PRGKKEY, and TAFLGMFLEHW with a binding energy less than -9 kcal / mol are screened out by molecular docking method. The activity evaluation of the selected 5 peptides inhibiting ACE activity shows that at a concentration of 1 μg / mL, the ACE inhibitory activity can reach 35%. The experimental results show that the novel active ACE inhibitory antihypertensive peptide separation and enrichment material helps to efficiently screen and identify active peptides, providing a powerful tool for the design and synthesis of new antihypertensive drugs.

[0192] In summary, the present invention uses genipin as a cross-linking agent to construct magnetic Fe3O4 nanoparticles with specific affinity recognition function for ACE inhibitory peptides, realizing the specific enrichment of ACE inhibitory peptides from the protein hydrolysate of torreya grandis cake meal. Through the immobilized enzyme technology, 61 ACE inhibitory peptides are successfully enriched in the protein hydrolysate of torreya grandis cake meal, and the molecular docking binding energies of 5 peptide segments such as CGDDGY, ANPCKYE, AAELLPGS, PRGKKEY, and TAFLGMFLEHW with ACE are lower than -9 kcal / mol. This technical solution provides a new method and technical support for the preparation of food-derived hypertension inhibitors and the development of food functional factors.

[0193] The standard parts used in the present invention can all be purchased from the market. The special-shaped parts can be customized according to the description in the specification and the drawings. The specific connection methods of each part all adopt conventional means such as bolts, rivets, and welding that are mature in the prior art. The machines, parts, and equipment all adopt conventional models in the prior art, and the circuit connection adopts the conventional connection method in the prior art, which will not be elaborated here. The content not described in detail in this specification belongs to the prior art well-known to those skilled in the art.

[0194] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for specific affinity enrichment and preparation of ACE inhibitory peptides from Torreya grandis protease hydrolysate, characterized in that: The specific steps include: Step 1, preparation and characterization of magnetic aminated Fe3O4 nanoparticles; Step 2, preparation and characterization of immobilized trypsin; Step 3, preparation, characterization and reaction condition optimization of immobilized ACE; Step 4, performing immobilized ACE activity assay; Step 5, then the preparation and specific enrichment of Torreya grandis antihypertensive peptides are carried out; Step 6, then, molecular docking was performed by AutoDock Vina; Step seven, finally, the content of ACE inhibitory peptide in Torreya grandis is determined.

2. The method for specific affinity enrichment and preparation of ACE inhibitory peptides from Torreya grandis protease hydrolysate according to claim 1, characterized in that: In step 1, the preparation and characterization of magnetic aminated Fe3O4 nanoparticles are carried out, and the specific method is as follows: Weigh 2.7 g of ferric chloride hexahydrate and 7.2 g of sodium acetate, add 100 mL of ethylene glycol, stir magnetically to mix, seal, and react in a 200 mL autoclave at 200°C for 8 hours; After cooling to room temperature, the mixture was washed with ethanol for 6 times and dried under vacuum at 60°C for 12 hours to obtain magnetic Fe3O4 nanoparticles; 150 mg of Fe3O4 nanoparticles were dispersed in 100 mL of ethanol and ultrasonically treated for 0.5 h. 25 mL of deionized water containing 1.2 mL of concentrated ammonia was added. After continuous stirring, 0.5 mL of ethanol solution containing 100 μL of ethyl orthosilicate was slowly added dropwise and reacted for 9 h. The product was collected by magnetic separation, washed alternately with ethanol and deionized water, and then dried at room temperature; The dried product was ultrasonically dispersed in 120 mL of isopropanol, 0.5 mL of triethoxysilane was added, and the reaction was stirred for 9 hours; After magnetic separation, the mixture was washed with ethanol and deionized water and dried overnight in vacuum to obtain aminated magnetic Fe3O4@NH2 nanoparticles; The crystal structure of magnetic Fe3O4 and Fe3O4@NH2 was analyzed by X-ray diffractometer; Scanning electron microscope was used to observe its morphological characteristics; Thermogravimetric analyzer was used to determine the thermal stability of materials; Fourier transform infrared spectroscopy to characterize functional groups; Vibrating sample magnetometers test the magnetic properties of materials.

3. The method for specific affinity enrichment and preparation of ACE inhibitory peptides from Torreya grandis protease hydrolysate according to claim 1, characterized in that: In step 2, the immobilized trypsin is prepared and characterized, and the specific method is as follows: 2.0 g of Fe3O4@NH2 nanoparticles were weighed and placed in a 50 mL stoppered conical flask, and 16 mL of 0.6 g / L genipin aqueous solution was added, and cross-linked at 55 °C for 8 h; After the reaction was completed, the unreacted genipin was fully washed with distilled water, and the obtained product was refrigerated at 4°C for later use; Weigh 1 g of Fe3O4@NH2 nanoparticles cross-linked with genipin and transfer them to a 50 mL stoppered conical flask. Add 20 mL of 12.5 μg / mL trypsin solution and incubate at 25 °C and pH = 6.0 for 16 h. After the incubation, the immobilized enzyme was collected by magnetic separation, washed six times with distilled water to remove unbound trypsin and impurities, and finally stored in a refrigerator at 4°C; Fourier transform infrared spectroscopy was used to characterize the surface functional groups of the immobilized enzyme.

4. The method for specific affinity enrichment and preparation of ACE inhibitory peptides from Torreya grandis protease hydrolysate according to claim 1, characterized in that: In step 3, the preparation, characterization and reaction condition optimization of immobilized ACE are as follows: Fe3O4@NH2 nanoparticles were weighed and dispersed in ACE phosphate buffer by ultrasonication; Using EDC as a cross-linking agent, the reaction mixture was slowly shaken at 37°C, followed by magnetic separation of the ACE-immobilized enzyme; collecting the supernatant, measuring the content of ACE therein, and determining the solid loading amount of ACE; The magnetic particles containing immobilized ACE were washed with deionized water to ensure complete removal of free ACE; Finally, the pellets were freeze-dried and stored for further use; The crystal structure, morphology, thermal stability and functional groups of the magnetically loaded Fe3O4@NH2@ACE nanoparticles were determined by XRD, SEM, TGA and FTIR analyses; The magnetic field-assisted characteristics were studied using vibrating sample magnetic analysis. By changing the pH value of the system, a change curve is drawn with fixed load as the ordinate and pH value as the abscissa to select the optimal pH value; By adjusting the initial concentration of ACE buffer, a change curve was drawn with the initial concentration as the horizontal axis under fixed load conditions to select the optimal initial concentration; By changing the reaction time, a change curve is drawn with fixed load as the vertical axis and reaction time as the horizontal axis to select the optimal reaction time.

5. The method for specific affinity enrichment and preparation of ACE inhibitory peptides from Torreya grandis protease hydrolysate according to claim 1, characterized in that: In step 4, the activity of immobilized ACE is determined, and the specific method is as follows: ACE activity is assessed by measuring the amount of hippuric acid produced. The test mixture consists of three parts: 130 μL of BBS buffer with 100 μL of free enzyme and 100 μL of immobilized enzyme ACE; After the mixture was incubated at 37°C for 10 min, 0.1 mL of HCl was added to terminate the incubation; Subsequently, HPLC was used with a DAD detector to detect the HA content at 228 nm; The definition of ACE unit activity is: under experimental conditions, the ACE unit activity can catalyze the production of 1 µmol·L HHL per minute. -1 The amount of enzyme required for HA; the relative ACE activity can be calculated by the following formula: ； Where A1 is the peak area of ​​HA in the control group, and A2 is the peak area of ​​HA with fixed ACE.

6. The method for specific affinity enrichment and preparation of ACE inhibitory peptides from Torreya grandis protease hydrolysate according to claim 1, characterized in that: In step 5, the preparation and specific enrichment of Torreya grandis antihypertensive peptides are as follows: Mix 2.0 g Torreya grandis cake powder with 40 mL 0.01 mol / L PBS buffer (pH=7.0); Then, the mixture was centrifuged at 11000 r / min for 15 minutes to obtain the supernatant for preparing Torreya grandis protein solution; Subsequently, 1.0 g of immobilized trypsin was added to the protein solution and kept at a constant temperature of 45°C for 4 hours to allow the hydrolysis reaction to occur; Then, the immobilized enzyme was separated magnetically and centrifuged at 7200 r / min for 15 min; The supernatant is taken and immobilized ACE is added, and a hydrolyzate containing the target active peptide is obtained by magnetic separation; then, immobilized ACE is added to the solution to adsorb the ACE inhibitory peptide; After enrichment, ACE inhibitory peptides were eluted with SDS; finally, the samples were analyzed by liquid chromatography-mass spectrometry under the following conditions: the mobile phase was 0.1% formic acid aqueous solution and 0.1% formic acid-80% acetonitrile mixture; The chromatographic column used was a Diamonsil C18 reverse phase column with a flow rate of 300 nL / min and an elution time of 30 minutes. The oligopeptide sequence was analyzed by mass spectrometry.

7. The method for specific affinity enrichment and preparation of ACE inhibitory peptides from Torreya grandis protease hydrolysate according to claim 1, characterized in that: In step 6, molecular docking is performed by AutoDock Vina. The specific method is as follows: The ACE crystal structure was obtained through UniProt; To prepare the receptors, hydrogen atoms were added and water molecules were eliminated using AutoDockTools 1.5.7; Before docking, the three-dimensional conformations of the peptide chains CGDDGY, ANPCKYE, AAELLPGS, PRGKKEY, and TAFLGMFLEHW were predicted by AlphaFold3; These structures were then converted into PDB files with the help of PYMOL software; Next, Autodock Vina was used for molecular docking and screening of potential inhibitory peptides; The docking was set at coordinates, x: -4.144, y:32.153, z:-18.399, the search space was 126 × 126 × 126A, and the resolution was set to 1 Å; The traversal parameter was set to 8, the docking of the peptide with the ACE receptor protein was completed, and the interaction strength and binding energy between different ligand conformations and the ACE crystal structure were evaluated. The lower the binding energy, the higher the score, indicating a tighter binding and stable interaction between the ligand and the ACE receptor protein, thus having potential inhibitory activity.

8. The method for specific affinity enrichment and preparation of ACE inhibitory peptides from Torreya grandis protease hydrolysate according to claim 1, characterized in that: In step 7, the content of ACE inhibitory peptide in Torreya grandis is determined by the following method: The ACE inhibition rate was measured by using the sample instead of the BBS buffer and calculated according to the following formula: ； Among them, A1 is the peak area of ​​HA in the control group, and A2 is the peak area of ​​HA and Torreya grandis ACE inhibitory peptide.

Images