Protein peptide with ACE and DPP-IV inhibitory activity and screening method thereof

Abstract

The invention discloses a protein peptide with ACE (Angiotensin Converting Enzyme) and DPP-IV (Dipeptidyl Peptidase-IV) inhibitory activity and a screening method thereof, and belongs to the technical field of food-borne bioactive peptide development. The difunctional peptide with ACE and DPP-IV inhibitory activity is identified from potato protein for the first time, the amino acid sequence of the difunctional peptide is LRWI, FGPK, FNL, IPF, TYF or VPFY, the difunctional peptide plays a role in relieving hypertension and type 2 diabetes at the same time, and the difunctional peptide has application value in preparation of products with the efficacy of reducing blood pressure and blood sugar due to the difunctionality of the difunctional peptide.

Description

Technical Field

[0001] This invention relates to a protein peptide with ACE and DPP-IV inhibitory activity and a screening method thereof, belonging to the field of food-derived bioactive peptide development technology. Background Technology

[0002] Hypertension and type 2 diabetes mellitus (T2DM) are two common chronic diseases that pose a significant threat and burden to global health. The incidence of cardiovascular disease in diabetic patients is 2-3 times higher than in the general population. Traditional treatments typically rely on combinations of medications to control blood pressure and blood sugar levels. Chemically synthesized ACE inhibitors (such as captopril and enalapril) and DPP-IV inhibitors (such as sitagliptin and vildagliptin) have shown good antihypertensive and hypoglycemic effects clinically; however, their therapeutic effects are often accompanied by significant side effects, and they frequently lack multifunctional efficacy. Inhibitors with dual antihypertensive and hypoglycemic effects can effectively mitigate the impact of complications. Therefore, the discovery of natural bioactive compounds that can simultaneously treat hypertension and hyperglycemia has received increasing attention.

[0003] In the renin-angiotensin system (RAS), a system that regulates blood pressure, ACE enzymes catalyze the conversion of angiotensin I to angiotensin II and also regulate the kallikrein-kinin system (KKS), thereby increasing blood pressure. Therefore, ACE inhibitors have a blood pressure-lowering effect. DPP-IV inhibitors, on the other hand, exert their hypoglycemic effect by inhibiting the inactivation of hormones such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). GLP-1 and GIP effectively stimulate insulin secretion; however, they are readily degraded by DPP-IV and rapidly inactivated, thus losing their function in regulating blood glucose. Therefore, inhibiting DPP-IV activity is beneficial for the treatment of type 2 diabetes (T2D).

[0004] Potatoes are a globally important food crop. Potato protein itself has a balanced amino acid composition, making it a potential high-quality plant protein source. Hydrolyzing potato protein into peptides using enzymatic techniques has revealed significant ACE inhibitory activity and some DPP-IV inhibitory activity due to the presence of specific amino acid sequences (such as rich hydrophobic amino acids, proline, or alanine residues). However, the specific active components in potato protein hydrolysates and their mechanisms of action remain unknown. Therefore, this study aims to establish an efficient screening and identification method to identify, isolate, and purify highly active peptides with these dual activities from potato protein hydrolysates. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention aims to provide a peptide with ACE and DPP-IV inhibitory activity, which can be used in the application development of products for lowering blood pressure and blood sugar.

[0006] The first technical solution provided by this invention is a method for screening ACE and DPP-IV inhibitory peptides, which mainly includes the following steps:

[0007] (1) Extracting tuber plant protein from tuber plants using the alkali dissolution and acid precipitation method.

[0008] After adding water to the tuber plants and pulping them, the pH was adjusted to 8.0-10.0. The mixture was stirred continuously at room temperature for 3-4 hours and then centrifuged (8000 r / min, 15-20 min). The supernatant was collected and stored. The pH of the supernatant was adjusted to 4.5 and centrifuged again (8000 r / min, 15-20 min). The precipitate was reconstituted and the pH was adjusted to 7.0. After dialyzing, the mixture was freeze-dried to obtain tuber plant protein powder.

[0009] (2) Producing tuber plant protein peptides by hydrolyzing tuber plant protein with proteases.

[0010] Mix the tuber protein powder obtained in step (1) with water in a certain proportion, then add protease to hydrolyze the tuber protein. After hydrolysis, boil the tuber protein hydrolysate in a water bath to inactivate the enzyme, cool it to room temperature, adjust the pH to 7.0, centrifuge, take the supernatant and freeze dry to obtain tuber protein hydrolysate powder, and store it at -20℃ for later use. The protease is: alkaline protease, neutral protease, trypsin, flavor protease, complex protease or papain.

[0011] (3) Identification and screening

[0012] The peptide sequences of the potato plant protein hydrolysate obtained in step (2) were identified by peptidomics, and potential novel bifunctional ACE and DPP-IV inhibitory peptides were screened from the potato plant peptides.

[0013] (4) Synthesis verification

[0014] The intrinsic mechanism by which it inhibits ACE and DPP-IV was explored through molecular docking.

[0015] In one embodiment, in step (1), the following implementation conditions are adopted: after adding water to the tuber plants and pulping them, the pH is adjusted to 8.0-10.0, and the mixture is stirred continuously at room temperature for 3-4 hours and then centrifuged (8000 r / min, 15-20 min). The supernatant is collected and stored. The pH of the supernatant is adjusted to 4.5 and then centrifuged again (8000 r / min, 15-20 min). The precipitate is re-dissolved and the pH is adjusted to 7.0. After dialysis, the mixture is freeze-dried to obtain tuber plant protein powder. During the hydrolysis process, 1 mol / L NaOH or HCl is used to maintain a constant pH value of the solution.

[0016] In one embodiment, in step (2), when the protease is an alkaline protease, the hydrolysis conditions are: temperature 55°C, pH 8.5, and hydrolysis time 4h; when the protease is a neutral protease, the hydrolysis conditions are: temperature 50°C, pH 7.0, and hydrolysis time 4h; when the protease is trypsin, the hydrolysis conditions are: temperature 37°C, pH 8.5, and hydrolysis time 3h; when the protease is a flavor protease, the hydrolysis conditions are: temperature 50°C, pH 7.0, and hydrolysis time 2.5h; when the protease is a complex protease, the hydrolysis conditions are: temperature 55°C, pH 8.0, and hydrolysis time 4h; and when the protease is papain, the hydrolysis conditions are: temperature 55°C, pH 7.0, and hydrolysis time 2h.

[0017] In one embodiment, step (2) is carried out under the following conditions: potato plant protein powder and water are mixed at a ratio of 1:20 (w:v) to prepare a protein solution with a substrate concentration of 5%. After mixing, the pH and temperature are adjusted to the optimal pH and temperature conditions for the enzyme used. Protease is added for hydrolysis at an enzyme-to-substrate ratio of 10000 U / g protein. The mixture is stirred and extracted at room temperature for 2-4 hours. After hydrolysis to a constant pH, the enzyme is inactivated by boiling in a water bath for 10 minutes. After cooling to room temperature, the pH is adjusted to 7.0. The mixture is centrifuged at 8000 r / min for 20 minutes. The supernatant is collected, dialyzed, and then freeze-dried to obtain potato plant protein hydrolysate powder under different protease hydrolysis conditions.

[0018] In one implementation, step (3) may specifically take the following steps:

[0019] ① Preliminary screening criteria: The amino acid sequences obtained from sequencing were analyzed using PeptideRanker software, protein databases, Toxinpred software, and AllerTOP software to predict potential biological activity, protein origin, toxicity, and sensitization. Peptides from the sequenced peptides that were of protein origin, not modified by functional groups, and had a peak area >1.00 × 10⁻⁶ were selected. 6 The peptide sequence contains 3-12 amino acids and has an activity score ≥0.6.

[0020] ② Rapid screening conditions: Peptide sequences containing hydrophobic amino acids at the C-terminus or Ile, Leu, or Val at the N-terminus have potential ACE inhibitory activity; peptides containing highly hydrophobic amino acids (Leu or Ile) at the N-terminus or Pro or Ala at the second / third position of the N-terminus have potential DPP-IV inhibitory activity.

[0021] In one implementation, step (4) may specifically take the following steps:

[0022] ① Obtaining small molecule ligands: Construct 2D and 3D structures of the peptide sequences to be docked using ChemDraw (version ChemBioDraw 22.0.0) and Chem3D (version ChemBioDraw 22.0.0) software.

[0023] ②Preparation of PDB files for ACE and DPP-IV: Retrieve the protein crystal structures of ACE and DPP-IV (1O86 and 1WCY, respectively) from the protein database, and remove excess ligands and water molecules in order to facilitate subsequent docking with peptides.

[0024] ③ Molecular docking: In AutoDockTools 1.5.6, the ligand and receptor formats were converted to PDBQT. Molecular docking simulation experiments were performed using AutoDockVina software. The output was the predicted binding energy; a lower binding energy indicates a stronger binding between the ligand and the protein pocket. ACE and DPP-IV repressive peptides with publicly available sequences were excluded, and the remaining peptides were identified as potential bifunctional ACE and DPP-IV repressive peptides.

[0025] In some embodiments, the tuber plant includes potatoes and cassava.

[0026] The second technical solution provided by the present invention is a bifunctional peptide with ACE and DPP-IV inhibitory activities, the amino acid sequence of which is LRWI, FGPK, FNL, IPF, TYF or VPFY.

[0027] The third technical solution provided by the present invention is a product containing the bifunctional peptide described in the second technical solution.

[0028] In one implementation, the product includes food, medicine, or health products.

[0029] The fourth technical solution provided by this invention is the application of the bifunctional peptide described in the first technical solution in the preparation of drugs for lowering blood pressure and / or lowering blood sugar.

[0030] In one embodiment, the drug has both angiotensinase inhibitory activity and dipeptidyl peptidase-4 inhibitory activity.

[0031] The fifth technical solution provided by this invention is the application of the bifunctional peptide described in the second technical solution in the preparation of ACE and / or DPP-IV inhibitors.

[0032] The sixth technical solution provided by the present invention is a tuber plant protease hydrolysate containing the bifunctional peptides described in the second technical solution, wherein the tuber plant protease hydrolysate contains protein peptides of LRWI, FGPK, FNL, IPF, TYF and VPFY.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] This invention is the first to identify a bifunctional peptide from potato protein that has both ACE and DPP-IV inhibitory activities. Its amino acid sequence is LRWI, FGPK, FNL, IPF, TYF or VPFY. It can simultaneously alleviate hypertension and type 2 diabetes. Its bifunctionality makes it valuable for the preparation of products with antihypertensive and hypoglycemic effects. Attached Figure Description

[0035] Figure 1 The effect of enzyme type on the inhibition rate of potato protein peptides ACE and DPP-IV;

[0036] Figure 2 The inhibition rate of potato protein peptides produced by neutral protease on ACE(A) and DPP-IV(B);

[0037] Figure 3 The molecular structure of LRWI;

[0038] Figure 4 This is a molecular docking diagram of LRWI and ACE;

[0039] Figure 5 This is a diagram showing the molecular docking of LRWI and DPP-IV. Detailed Implementation

[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0041] Test method:

[0042] The following examples illustrate a substrate chemistry method for determining the inhibition rates of ACE and DPP-IV:

[0043] (1) Detection method of ACE inhibition rate

[0044] Add 40 μL of HEPES buffer (pH 8.2, 80 mmol / L, containing 0.3 mol / L NaCl) and a certain concentration of captopril (or the sample solution to be tested) to a 96-well plate, then add 50 μL of FAPPGG (1 mmol / L). After preheating at 37 °C for 10 min, pipette 10 μL of 0.1 U / mL ACE into the 96-well plate. Measure the initial absorbance values ​​(A1 and B1) at 340 nm, and then incubate at 37 °C in the dark for 30 min before measuring the endpoint absorbance (A2 and B2). Captopril is used as a positive control.

[0045] The ACE inhibition rate is calculated as follows:

[0046] ACE inhibition rate (%) = [(A1-A2)-(B1-B2)] / (A1-A2)×100%;

[0047] Wherein, A1-A2: absorbance change of the blank group within 30 minutes; B1-B2: absorbance change of the sample group within 30 minutes.

[0048] (2) Detection method of DPP-IV inhibition rate

[0049] Add 25 μL of sample solution and 25 μL of Gly-Pro-pNA (1.6 mM, 25 μL) to a 96-well microplate with shaking. Incubate at 37°C for 10 min with shaking. Add 50 μL of DPP-IV (10 U / L) and incubate at 37°C for another 60 min with shaking to allow the reaction to proceed. After the reaction, add 100 μL of HAC-NaAC (pH 4.0, 1 mol / L) buffer and measure the absorbance at 405 nm. DiprotinA (IPI) is used as the positive control. Tris-HCl (0.1 mol / L, pH 8.0) buffer is used for solution preparation.

[0050] The method for calculating the DPP-IV inhibition rate is as follows:

[0051] DPP-IV inhibition rate (%) = [1-(A 样品 -A 背景 ) / (A 阴性对照 -A 空白 )]×100%;

[0052] Among them, A 样品 For sample group A 背景 To avoid adding a background group for DPP-IV, A 阴性对照 As a negative control group without sample solution, A 空白 This is the blank group without samples and DPP-IV.

[0053] In the following embodiments, the amino acid sequence of the LRWI is Leu-Arg-Trp-Ile, the amino acid sequence of the FGPK is Phe-Gly-Pro-Lys, the amino acid sequence of the FNL is Phe-Asn-Leu, the amino acid sequence of the IPF is Ile-Pro-Phe, the amino acid sequence of the TYF is Thr-Tyr-Phe, and the amino acid sequence of the VPF is Val-Pro-Phe-Tyr.

[0054] Example 1: Method for obtaining potato protein hydrolysate from potato protein

[0055] (1) Extraction of potato protein from potatoes using the alkali dissolution and acid precipitation method.

[0056] Potatoes were blended with water to form a pulp, and the pH was adjusted to 8.0-10.0. The pulp was stirred continuously at room temperature for 3-4 hours, then centrifuged (8000 rpm, 15-20 min). The supernatant was collected and stored. The pH of the supernatant was adjusted to 4.5, and the pulp was centrifuged again (8000 rpm, 15-20 min). The precipitate was reconstituted, and the pH was adjusted to 7.0. After dialysis, the pulp was freeze-dried to obtain potato protein powder.

[0057] (2) Producing potato peptides by hydrolyzing potato protein with proteases.

[0058] Potato protein powder and water were mixed at a ratio of 1:20 (w:v) to prepare a protein solution with a substrate concentration of 5%. After mixing, the pH and temperature were adjusted to the optimal pH and temperature conditions for the enzyme used. Different proteases were added according to an enzyme-to-substrate ratio of 10000 U / g protein for hydrolysis. Hydrolysis was carried out by continuous stirring at room temperature until the pH was constant. The hydrolysis conditions are shown in Table 1.

[0059] Table 1 Hydrolysis conditions for different proteases

[0060]

[0061] During hydrolysis, 1 mol / L NaOH or HCl is used to maintain a constant pH value of the solution.

[0062] After hydrolysis, the potato protein hydrolysate was boiled for 10 minutes to inactivate the enzymes. After cooling to room temperature, the pH was adjusted to 7.0, and the solution was centrifuged at 8000 rpm for 20 minutes. The supernatant was collected, dialyzed, and then freeze-dried to obtain potato protein hydrolysate powder under different protease hydrolysis conditions.

[0063] (3) The ACE and DPP-IV inhibitory activities of potato protein hydrolysate obtained under different protease hydrolysis conditions were determined. Figure 1 Neutral protease hydrolysates with high inhibitory activity were screened out. The results showed that the ACE and DPP-IV inhibition rates of the neutral protease hydrolysates were 67.66±5.21% and 62.88±1.45%, respectively, and the IC50 values ​​for ACE and DPP-IV inhibitory activities were 0.179 mg / mL and 1.142 mg / mL, respectively. Figure 2 Then proceed to the next step of sequencing.

[0064] Example 2: Method for isolating and identifying ACE and DPP-IV repressive peptides from potato protein hydrolysates

[0065] The specific steps are as follows:

[0066] The potato protein hydrolysate obtained by neutral protease digestion in Example 1 was selected for further identification.

[0067] (1) Identification of peptide sequences in potato protein hydrolysates using peptidomics

[0068] Potato protein hydrolysate samples were first desalted using a C18 desalting column, and then analyzed by LC-MS / MS equipped with an online nanospray ionization source. The entire system was an Orbitrap Explorise mass spectrometer with a UltiMate 3000 system in series. A total of 1 μL of sample (C18 column: 20 cm × 75 μm·d, 1.9 μm particle size) was loaded, and the sample was separated by a gradient at a rate of 60 min. The column flow rate was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, the gradient started from 2.2% B phase, increased non-linearly to 44% at 45 min, increased to 90% within 3 min, and maintained for 12 min.

[0069] The mass spectrometer operates in data-dependent acquisition mode and automatically switches between MS and MS / MS acquisition. The mass spectrometry parameters are set as follows: (1) MS: Scan range (m / z): 200-1200; Resolution: 60,000; AGCtarget: 4e5; Maximum injection time: 50ms; (2) HCD-MS / MS: Resolution: 15,000; AGCtarget: 5e4; Maximum injection time: 22ms; Collision energy: 30%; Dynamic exclusion time: 30s.

[0070] Tandem mass spectrometry analysis was performed using PEAKS Studio version 10.6. The uniprot-Solanum_tuberosum (version 2025, 63500 entries) database was searched via PEAKSDB. Peptide card values ​​were: -10lgP ≥ 20, and at least one specific peptide was required.

[0071] A total of 5071 peptides were identified after sequencing potato protein hydrolysate using peptidomics.

[0072] (2) Highly active ACE and DPP-IV inhibitory peptides were screened from the 5071 peptides identified in (1) using various peptide screening conditions (Table 2). The main steps included:

[0073] ① Preliminary screening criteria: Potential biological activity, protein origin, toxicity, and sensitization were predicted using PeptideRanker, protein databases, Toxinpred, and AllerTOP software, respectively. Peptides from the sequenced samples were selected that were of protein origin, not modified by functional groups, and had a peak area >1.00 × 10⁻⁶. 6The peptide sequence contains 3-12 amino acids and has an activity score ≥0.6.

[0074] ② Rapid screening conditions: steric hindrance ≤ 0.65; peptide sequences containing hydrophobic amino acids at the C-terminus or Ile, Leu, or Val at the N-terminus, peptides with these characteristics have potential ACE inhibitory activity; peptides containing highly hydrophobic amino acids (Leu or Ile) at the N-terminus or Pro or Ala at the second / third position of the N-terminus, peptides with these characteristics have potential DPP-IV inhibitory activity.

[0075] Table 2 Activity prediction of potential peptide sequences

[0076]

[0077]

[0078]

[0079]

[0080] (3) Use molecular docking pairs to screen and verify the peptides obtained from (2).

[0081] The specific steps are as follows:

[0082] ① Obtaining small molecule ligands: Construct 2D and 3D structures of the peptide sequences to be docked using ChemDraw (version ChemBioDraw 22.0.0) and Chem3D (version ChemBioDraw 22.0.0) software.

[0083] ②Preparation of PDB files for ACE and DPP-IV: Retrieve the protein crystal structures of ACE and DPP-IV (1O86 and 1WCY, respectively) from the protein database, and remove excess ligands and water molecules in order to facilitate subsequent docking with peptides.

[0084] ③ Molecular docking: In AutoDockTools 1.5.6, the ligand and receptor formats were converted to PDBQT. Molecular docking simulation experiments were performed using AutoDockVina software. The output was the predicted binding energy; a lower binding energy indicates a stronger binding between the ligand and the protein pocket. ACE and DPP-IV repressive peptides with publicly available sequences were excluded, and the remaining peptides were identified as potential ACE and DPP-IV repressive peptides.

[0085] Specifically, a total of 6 peptides were screened, and their binding energies to ACE and DPP-IV were all less than -6.8 kcal / mol (Table 3).

[0086] Table 3. Binding energies of peptides to ACE and DPP-IV molecules.

[0087]

[0088] Example 3: LRWI, an ACE and DPP-IV inhibitory peptide

[0089] (1) Structural analysis of LRWI

[0090] like Figure 3 As shown, the molecular structure of LRWI was obtained from ChemDraw and AutoDock.

[0091] (2) LRWI docking with ACE and DPP-IV inhibitory molecules

[0092] like Figure 4 As shown, LRWI forms hydrogen bonds with seven amino acid residues in the ACE active pocket: GLU-162, ASN-374, ASN-277, GLN-281, LYS-511, TYR-520, and ASP-377. Figure 5 As shown, LRWI forms hydrogen bonds with four amino acid residues, ASP-709, ASN-710, HIS-740, and ILE-3, in the active pocket of DPP-IV.

[0093] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A bifunctional peptide with ACE and DPP-IV inhibitory activities, characterized in that, Its amino acid sequence is LRWI, FGPK, FNL, IPF, TYF or VPFY.

2. A method for screening bifunctional peptides according to claim 1, characterized in that, The main steps include: (1) Extracting tuber plant protein from tuber plants using the alkali dissolution and acid precipitation method; (2) Producing tuber protein peptides by hydrolyzing tuber plant proteins with proteases: Mix the tuber protein powder obtained in step (1) with water, add protease to hydrolyze the tuber protein, after hydrolysis, boil the tuber protein hydrolysate in a water bath to inactivate the enzyme, cool to room temperature and adjust the pH to 7.0, centrifuge, take the supernatant and freeze dry to obtain tuber protein hydrolysate powder, store at -20℃ for later use, the protease is: alkaline protease, neutral protease, trypsin, flavor protease, complex protease or papain; (3) Identification and screening: The peptide sequences of the potato plant protein hydrolysate obtained in step (2) were identified by peptidomics, and potential novel ACE and DPP-IV bifunctional inhibitory peptides were screened from potato peptides. (4) Synthesis verification: The intrinsic mechanism of the inhibition of ACE and DPP-IV by the potential novel bifunctional inhibitory peptides of ACE and DPP-IV in step (3) was explored through molecular docking.

3. The method according to claim 2, characterized in that, The specific steps in step (1) are as follows: After adding water to the tuber plants and pulping them, adjust the pH to 8.0-10.0, stir continuously at room temperature for 3-4 hours, centrifuge, collect the supernatant and store it, adjust the pH of the supernatant to 4.5 and centrifuge again, redissolve the precipitate, adjust the pH to 7.0, dialyze and freeze dry to obtain tuber plant protein powder; The tuber plants mentioned include potatoes and cassava.

4. The method according to claim 2, characterized in that, In step (2), potato plant protein powder and water are mixed at a ratio of 1:20 (w:v) to prepare a protein solution with a substrate concentration of 5%; protease is added at an enzyme-to-substrate ratio of 10000 U / g protein for hydrolysis. The hydrolysis conditions for alkaline protease were: 55℃, pH 8.5, and 4h; for neutral protease, 50℃, pH 7.0, and 4h; for trypsin, 37℃, pH 8.5, and 3h; for flavor protease, 50℃, pH 7.0, and 2.5h; for complex protease, 55℃, pH 8.0, and 4h; and for papain, 55℃, pH 7.0, and 2h.

5. The method according to claim 2, characterized in that, Step (3) specifically adopts the following steps: ① Preliminary screening criteria: The amino acid sequences obtained from sequencing were analyzed using PeptideRanker software, protein databases, Toxinpred software, and AllerTOP software to predict potential biological activity, protein origin, toxicity, and sensitization. Peptides from the sequenced peptides that were of protein origin, not modified by functional groups, and had a peak area >1.00 × 10⁻⁶ were selected. 6 Peptide sequences containing 3-12 amino acids with an activity score ≥0.6; ② Rapid screening conditions: Peptide sequences containing hydrophobic amino acids at the C-terminus or Ile, Leu, or Val at the N-terminus have potential ACE inhibitory activity; peptides containing highly hydrophobic amino acids (Leu or Ile) at the N-terminus or Pro or Ala at the second / third position of the N-terminus have potential DPP-IV inhibitory activity.

6. The method according to claim 1, characterized in that, Step (4) specifically adopts the following steps: ① Obtaining small molecule ligands: Constructing 2D and 3D structures of the peptide sequences to be docked using ChemDraw and Chem3D software; ②Preparation of PDB files for ACE and DPP-IV: Retrieve the protein crystal structures of ACE and DPP-IV from the protein database and remove excess ligands and water molecules in order to facilitate subsequent docking with peptides. ③ Molecular docking: In AutoDockTools 1.5.6, the format of ligands and receptors was converted to PDBQT. Molecular docking simulation experiments were performed using AutoDockVina software. The results were output as predicted binding energies. The lower the binding energy, the stronger the binding between the ligand and the protein pocket. Bifunctional repressive peptides of ACE and DPP-IV were screened out.

7. A product containing the bifunctional peptide of claim 1, characterized in that, The products include food, medicine, or health products.

8. The use of the bifunctional peptide according to claim 1 in the preparation of drugs for lowering blood pressure and / or lowering blood sugar, characterized in that, The drug has both angiotensinase inhibitory activity and dipeptidyl peptidase-4 inhibitory activity.

9. The use of the bifunctional peptide according to claim 1 in the preparation of ACE and / or DPP-IV inhibitors.

10. A tuber plant protease hydrolysate containing the bifunctional peptide of claim 1, characterized in that, The components of the tuber plant protease hydrolysate contain protein peptides of LRWI, FGPK, FNL, IPF, TYF, and VPFY.

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