A chickpea protein-derived DPP-IV inhibitory peptide and its application

Abstract

The present invention discloses a chickpea protein-derived DPP-IV inhibitory peptide and its application, belonging to the technical field of development of food-derived bioactive peptides. The present invention uses proteomics, multiple screening conditions and molecular docking technology to rapidly screen DPP-IV inhibitory peptides, avoiding the time-consuming and laborious disadvantages caused by step-by-step separation and purification. The present invention effectively obtains chickpea protein hydrolysates with high DPP-IV inhibitory activity, antioxidant activity and Fe<supgt;2+< / supgt; chelating activity from chickpea protein by enzymatic hydrolysis with neutral protease, obtains chickpea protein hydrolysates with cholesterol micelle solubility inhibitory activity by enzymatic hydrolysis with alkaline protease, and obtains chickpea protein hydrolysates with high ACE inhibitory activity by enzymatic hydrolysis with papain. The mode of action of the highly active DPP-IV inhibitory peptide IAIPPGIPYW of the present invention with DPP-IV is competitive-non-competitive mixed inhibition, and it has extremely high DPP-IV inhibitory activity, with an IC<subgt;50< / subgt> of 12.43 μM (14.00 μg / ml).

Description

Technical Field

[0001] The present invention relates to a chickpea protein-derived DPP-IV inhibitory peptide and its application, belonging to the technical field of development of food-derived bioactive peptides. Background Art

[0002] Data released by the International Diabetes Federation (IDF) show that in 2021, there were as many as 537 million adult diabetes patients globally. The medical and health expenditures caused by this disease globally accounted for 9% of the total medical and health expenditures, bringing a heavy economic burden. China is the country with the largest number of adult diabetes patients, and it is predicted that this number will reach 174.4 million by 2045. The cause of type 2 diabetes (T2D) is insufficient insulin secretion by the pancreas. DPP-IV makes glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) lose their insulinotropic activities. By inhibiting DPP-IV, the concentration of endogenous GLP-1 in the human body can be increased, and the purpose of lowering blood sugar can be achieved. In modern medicine, diabetes treatment drugs focusing on insulin secretion and insulin sensitization have emerged continuously. There are already DPP-IV inhibitors such as sitagliptin and vildagliptin as anti-type 2 diabetes drugs, but these drugs have certain side effects and are expensive. Therefore, there is an urgent need to develop new natural DPP-IV inhibitors with no toxicity, higher safety, and fewer side effects, such as food-derived DPP-IV inhibitory peptides.

[0003] In terms of its own nutritional value and protein content, chickpea protein is considered an ideal raw material for bioactive peptides, being non-toxic, harmless, and having no side effects, and is suitable for food processing applications. At present, a large number of highly active DPP-IV inhibitory peptides have been obtained from various food proteins, all of which show good blood sugar-lowering effects. And a large number of studies have shown that chickpea peptides obtained by hydrolyzing chickpea protein have various bioactive functions such as antioxidant, blood pressure-lowering, blood sugar-lowering, and blood lipid-lowering. However, so far, there has been no report on the efficient and rapid identification of chickpea protein-derived DPP-IV inhibitory peptides.

[0004] The traditional method for identifying DPP-IV inhibitory peptides is to obtain peptide segments with higher purity through step-by-step purification such as ultrafiltration and chromatographic columns, then identify their structures by mass spectrometry, and finally verify their DPP-IV inhibitory activities through in vitro synthesis. However, this method has the disadvantages of complex operation, time-consuming, and high cost. Therefore, there is an urgent need to develop a rapid, convenient, and efficient method for identifying DPP-IV inhibitory peptides. Summary of the Invention

[0005] The present invention provides a method for screening DPP-IV inhibitory peptides from chickpea protein, which uses proteomics, multiple screening conditions, and molecular docking technology to rapidly screen chickpea protein-derived DPP-IV inhibitory peptides.

[0006] The present invention provides a chickpea protease hydrolyzate powder. The composition of the chickpea protease hydrolyzate powder contains protein peptides IAIPPGIPYW, AAWPGHPEF, LAFP, and PPGIPYW. The peak areas of the LC-MS / MS tandem mass spectra of the protein peptides are successively: 4.35×10 9 、1.49×10 7 、1.54×10 8 and 1.91×10 8 .

[0007] In one embodiment of the present invention, the chickpea protease hydrolyzate powder is prepared by the following method:

[0008] (1) Extract chickpea protein from chickpeas by the alkali dissolution and acid precipitation method

[0009] Mix chickpea powder and water in proportion, adjust the pH to 10.0 - 12.0, continuously stir at room temperature and then centrifuge. Take the supernatant for preservation. Mix the precipitate and water in proportion and continue to operate according to the above alkali extraction method 1 - 4 times; Mix the supernatants obtained from three times of alkali extraction, adjust the pH to 3.0 - 5.0 and then centrifuge. Redissolve the precipitate, dialyze and then freeze-dry to obtain chickpea protein powder;

[0010] (2) Hydrolyze chickpea protein with protease to produce chickpea peptides

[0011] Mix chickpea protein powder and water in proportion, preheat to denature the chickpea protein, and then add protease to hydrolyze the chickpea protein. After the hydrolysis is completed, adjust the pH of the chickpea protein hydrolysis solution to 7.0, then heat to inactivate the enzyme, then cool and centrifuge. Take the supernatant, freeze-dry and store it at -20°C for standby; The protease is: neutral protease, alkaline protease, papain, and compound protease.

[0012] In one embodiment of the present invention, when the protease is neutral protease, the hydrolysis conditions are: pH is: 6.5 - 7.5, temperature is: 40 - 50°C, and the time to constant pH is: 100 - 200 min;

[0013] In one embodiment of the present invention, when the protease is alkaline protease, the hydrolysis conditions are: pH is: 8.5 - 9.5, temperature is: 50 - 60°C, and the time to constant pH is: 150 - 250 min;

[0014] In one embodiment of the present invention, when the protease is papain, the hydrolysis conditions are: pH is: 6.5 - 7.5, temperature is: 50 - 60°C, and the time to constant pH is: 100 - 200 min;

[0015] In one embodiment of the present invention, when the protease is a compound protease, the hydrolysis conditions are as follows: the pH is 7.5 - 8.5, the temperature is 50 - 60 °C, and the time until the pH becomes constant is 150 - 250 min;

[0016] In one embodiment of the present invention, when the protease is an alkaline protease, the hydrolysis conditions are as follows: the pH is 8.5, the temperature is 55 °C, and the time until the pH becomes constant is 180 min;

[0017] When the protease is papain, the hydrolysis conditions are as follows: the pH is 7.0, the temperature is 55 °C, and the time until the pH becomes constant is 120 min;

[0018] When the protease is a compound protease, the hydrolysis conditions are as follows: the pH is 8.0, the temperature is 55 °C, and the time until the pH becomes constant is 180 min.

[0019] In one embodiment of the present invention, the chickpea powder refers to the dry powder obtained by pulverizing chickpeas and passing through a 40 - mesh sieve.

[0020] In one embodiment of the present invention, the chickpea protein powder refers to the dry protein powder after removing starch and fat.

[0021] In one embodiment of the present invention, the protease is a neutral protease, with mild action and relatively complete cleavage.

[0022] In one embodiment of the present invention, step (1) is as follows: Mix chickpea powder and water at a ratio of 1:10 - 1:15 (w:v), adjust the pH to 10.0 - 12.0, stir and extract at room temperature for 1.5 - 3.0 h, centrifuge at 4000 - 6000 r / min for 20 - 40 min, and then save the supernatant. After mixing the precipitate and water at a ratio of 1:2 - 1:8 (w:v), perform the above alkali extraction method 1 - 4 times; Concentrate the supernatant from the three - time alkali extraction, adjust the pH to 3.0 - 5.0, centrifuge again, redissolve the precipitate, and obtain chickpea protein powder after dialysis and freeze - drying.

[0023] In one embodiment of the present invention, step (2) can adopt the following implementation conditions: Mix chickpea protein powder and distilled water at a ratio of 1:15 - 1:30 (w:v), preheat at 80 - 100 °C for 10 - 20 minutes, and continuously stir during this period to ensure that the chickpea protein is completely heat - denatured; After the chickpea protein solution cools to room temperature, add protease for hydrolysis according to the enzyme - substrate ratio of 6000 - 10000 U / g protein;

[0024] During the hydrolysis process, 0.5 - 1 mol / L of NaOH or HCl was used to maintain the constant pH value of the solution; after the hydrolysis was completed, the chickpea protein hydrolysate was boiled for 10 - 30 minutes to terminate the hydrolysis reaction; after cooling to room temperature, it was centrifuged at 6000 - 10000 g for 20 - 40 min, and the supernatant was freeze-dried and stored at -20 °C to prepare chickpea protein hydrolysates under different protease hydrolysis conditions. The supernatant was freeze-dried and stored at -20 °C for the next sequencing.

[0025] The present invention provides chickpea-derived DPP-IV inhibitory peptides: (1) The peptide sequences of the above-mentioned chickpea protein hydrolysates were identified by peptidomics;

[0026] (2) Using a variety of screening conditions and molecular docking techniques, DPP-IV inhibitory peptides were screened from the chickpea peptides obtained by sequencing in step (1), and their DPP-IV inhibitory activities were verified by in vitro solid-phase synthesis. The amino acid sequences of the inhibitory peptides are AAWPGHPEF, LAFP, IAIPPGIPYW, or PPGIPYW.

[0027] In one embodiment of the present invention, step (2) can specifically adopt the following steps:

[0028] ① A variety of screening conditions: Predict whether the peptide segment has the potential to inhibit DPP-IV activity or has been reported in the BIOPEP database (https: / / biochemia.uwm.edu.pl / ); Perform a bioactivity prediction score on the peptide segment in PeptideRanker

[0029] (http: / / distilldeep.ucd.ie / PeptideRanker / ); Query the protein source of the peptide segment in the protein database (https: / / www.uniprot.org / ); Perform toxicity prediction through Toxinpred

[0030] (https: / / crdd.osdd.net / raghava / toxinpred / ); Perform allergenicity prediction through AllerTOP

[0031] (https: / / www.ddg-pharmfac.net / AllerTOP / ). Select sequences from the sequenced peptide segments that are protein-derived, not modified by functional groups, with a peptide chain length < 15, peak area > 1.00×10 7 , activity score > 0.8, without toxicity and allergenicity and not yet published. At the same time, screen for peptides with Pro or Ala at the second position from the NH2 terminus and Leu or Ile at the NH2 terminus of the peptide. Peptides with such characteristics have higher DPP-IV inhibitory activity.

[0032] ② Preparation of DPP-IV PDB file: Retrieve the protein crystal structure of DPP-IV (code: 5J3J) from the Protein Data Bank, and remove the redundant ligands and H2O from 5J3J for subsequent docking experiments with peptides.

[0033] ③ After docking the selected peptide sequences with 5J3J, the binding energy is obtained. The DPP-IV binding site is defined as a sphere containing 15 or fewer protein residues.

[0034] ④ Determine whether the selected peptide sequences are potential DPP-IV inhibitory peptides based on whether the binding energy is negative, and verify their DPP-IV inhibitory activity through in vitro solid-phase synthesis. The lower the value of the binding energy, the stronger the DPP-IV inhibitory activity of the corresponding polypeptide. The in vitro solid-phase synthesis is specifically the polypeptide chemical synthesis method (Fmoc solid-phase synthesis method). The verification of DPP-IV inhibitory activity is specifically that after the selected polypeptide is synthesized, the substrate chemical method is used to determine the strength of its DPP-IV inhibitory activity to verify the screening results.

[0035] The present invention provides chickpea-derived DPP-IV inhibitory peptides, and their amino acid sequences are AAWPGHPEF, LAFP, IAIPPGIPYW, and PPGIPYW respectively. Preferably, it is IAIPPGIPYW. The IAIPPGIPYW can inhibit DPP-IV activity at an extremely low concentration, and its IC 50 is 12.43 μM, lower than most currently identified DPP-IV inhibitory peptides, and even close to the inhibitory activity (IC 50 : 6.8 μM) of the positive control diprotinA.

[0036] The present invention provides a DPP-IV inhibitory peptide, and the amino acid sequence of the inhibitory peptide is AAWPGHPEF, LAFP, IAIPPGIPYW, or PPGIPYW.

[0037] The present invention also provides the application of the above DPP-IV inhibitory peptide in the preparation of hangover products, antioxidant products, cholesterol-lowering products, or products for relieving diabetes symptoms.

[0038] In one embodiment of the present invention, the product is a health food, a drug, or a chemical.

[0039] In one embodiment of the present invention, the health food contains the above DPP-IV inhibitory peptide, a carrier, and / or excipients.

[0040] In one embodiment of the present invention, the drug contains the above DPP-IV inhibitory peptide, a drug carrier, and / or pharmaceutical excipients.

[0041] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, and oral liquids.

[0042] In one embodiment of the present invention, the pharmaceutical carrier is one or more of a filler, a binder, a wetting agent, a disintegrant, a lubricant, and a flavoring agent.

[0043] The present invention also provides a product containing the above DPP-IV inhibitory peptide, and the product is a health food, a drug, or a chemical.

[0044] In one embodiment of the present invention, the product includes, but is not limited to, hangover products, antioxidant products, cholesterol-lowering products, products for relieving diabetes symptoms, ACE inhibitors, and DPP-IV inhibitors.

[0045] In one embodiment of the present invention, the health food contains the above DPP-IV inhibitory peptide, a carrier, and / or excipients.

[0046] In one embodiment of the present invention, the drug contains the above DPP-IV inhibitory peptide, a pharmaceutical carrier, and / or medicinal excipients.

[0047] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, and oral liquids.

[0048] In one embodiment of the present invention, the pharmaceutical carrier is one or more of a filler, a binder, a wetting agent, a disintegrant, a lubricant, and a flavoring agent.

[0049] The present invention also provides the use of the above DPP-IV inhibitory peptide in the preparation of an ACE inhibitor or a DPP-IV inhibitor.

[0050] Beneficial effects

[0051] (1) The present invention quickly and efficiently identifies highly active DPP-IV inhibitory peptides from chickpea protein through a variety of screening conditions and a screening method of molecular docking.

[0052] (2) The present invention creatively combines proteomics, multiple screening conditions and molecular docking to construct a method for rapidly screening DPP-IV inhibitory peptides, avoiding the time-consuming and laborious disadvantages caused by step-by-step separation and purification. First, high-throughput sequencing is performed on the peptide segments contained in the chickpea protein hydrolysate through proteomics to obtain the sequences of all peptide segments. Then, multiple screening conditions are used to screen the peptide sequences, and the screened peptide segments are docked with DPP-IV using molecular docking technology to obtain the binding energy. Theoretically, the lower the energy, the stronger the DPP-IV inhibitory activity of the peptide segment. Finally, the peptide segments obtained from the previous screening are synthesized in vitro and their activities are verified.

[0053] (3) The present invention effectively obtains chickpea protein hydrolysates with high DPP IV inhibitory activity, antioxidant activity (scavenging ABTS free radicals), and Fe 2+ chelating activity by enzymatic hydrolysis of chickpea protein with neutral protease, chickpea protein hydrolysates with cholesterol micelle solubility inhibitory activity by enzymatic hydrolysis with alkaline protease, and chickpea protein hydrolysates with high ACE inhibitory activity by enzymatic hydrolysis with papain.

[0054] (4) The present invention effectively identifies the highly active DPP IV inhibitory peptide IAIPPGIPYW from chickpea protein. The mode of action of IAIPPGIPYW with DPP IV is competitive-noncompetitive mixed inhibition, and it has extremely high DPP-IV inhibitory activity, with its IC 50 being 12.43 μM (14.00 μg / ml). Description of the Drawings

[0055] Figure 1 : Standard curve of cholesterol content.

[0056] Figure 2 : Schematic diagram of the molecular structure of IAIPPGIPYW.

[0057] Figure 3 : Analysis diagram of the molecular docking of IAIPPGIPYW with DPP-IV and their interaction forces.

[0058] Figure 4 : Inhibitory rate of DPP-IV activity of IAIPPGIPYW. Detailed Embodiments

[0059] The chickpeas used in the following examples were purchased from Amway (Shanghai) Technology Development Co., Ltd., the neutral protease used was purchased from Novozymes (China) Biotechnology Co., Ltd., the measured enzyme activity was 29,634 U, the alkaline protease used was purchased from Novozymes (China) Biotechnology Co., Ltd., the measured enzyme activity was 141,510 U, the papain used was purchased from Nanning Pangbo Bioengineering Co., Ltd., the measured enzyme activity was 46,771 U, and the compound protease used was purchased from Novozymes (China) Biotechnology Co., Ltd., the measured enzyme activity was 1,012,450 U.

[0060] The chickpea protein powder used in the following examples is a dry protein powder after removing starch and fat.

[0061] Example 1: Method for obtaining chickpea protease hydrolysate from chickpea protein

[0062] (1) Extract chickpea protein from chickpeas by the alkali-solution acid-precipitation method

[0063] Mix chickpea powder and water at a ratio of 1:12 (w:v), adjust the pH to 11.0 - 12.0, stir and extract at room temperature for 1.5 - 2 h, centrifuge at 4000 r / min for 20 min, and then save the supernatant. Mix the precipitate and water at a ratio of 1:5 (w:v) and perform the above alkali extraction method twice.

[0064] After concentrating the supernatants obtained from the three alkali extractions above, adjust the pH to 3.8 - 4.2, centrifuge again, redissolve the precipitate, dialyze, and then freeze-dry to obtain chickpea protein powder.

[0065] (2) Hydrolyze chickpea protein with protease to produce chickpea peptides

[0066] Mix chickpea protein powder and distilled water at a ratio of 1:25 (w:v), preheat at 80 - 90 °C for 10 minutes, and continuously stir during this period to ensure that the chickpea protein is completely heat-denatured. After the chickpea protein solution cools to room temperature, add protease (the ratio of enzyme to substrate addition is: 8000 U / g protein) and enzymatically hydrolyze until the pH is constant at the optimal pH and temperature. The hydrolysis conditions are shown in Table 1.

[0067] Table 1 Hydrolysis conditions of different proteases

[0068]

[0069] During the hydrolysis process, 1 mol / L NaOH or HCl is used to maintain the pH value of the solution constant.

[0070] After the hydrolysis was completed, the chickpea protein hydrolysate was boiled for 10 minutes to terminate the hydrolysis reaction. After cooling to 4°C, it was centrifuged at 8000 g for 20 min. The supernatant was taken and freeze-dried, and then stored at -20°C to prepare chickpea protein hydrolysates under different protease hydrolysis conditions. Example 2: Application of chickpea protein hydrolysates prepared by different proteases

[0071] 1. Application of chickpea protein hydrolysate in DPP-IV activity inhibition

[0072] The specific steps are as follows:

[0073] (1) Dissolve the chickpea protein hydrolysates prepared in Example 1 in 100 mmol / L Tris-HCl buffer at pH 8.0, and the actual reaction concentration of the sample is 1.0 mg / mL.

[0074] (2) Preparation of chickpea protein hydrolysate samples:

[0075] In a 96-well microplate, mix 25 μL of the chickpea protein hydrolysate sample with 25 μL of Gly-Pro-PNA (1.6 mmol / L) respectively, incubate at 37°C for 10 min, add 50 μL of DPP-IV (8 U / L) solution to initiate the reaction, react at 37°C for 60 min, and then add 100 μL of sodium acetate buffer (1 mol / L, pH 4.0) to terminate the reaction. Measure the absorbance at 405 nm.

[0076] (3) Use Tris-HCl buffer instead of DPP-IV solution as the blank group;

[0077] (4) Use Tris-HCl buffer instead of the chickpea protein hydrolysate sample as the control group.

[0078] The calculation formula for the DPP-IV inhibition rate is:

[0079]

[0080] where Ac, Ab, and As are the absorbances of the control group, blank group, and sample group respectively.

[0081] The results of the DPP-IV activity inhibition assay are shown in Table 2.

[0082] Table 2 Results of the DPP-IV activity inhibition assay

[0083]

[0084] The results showed that: at the same concentration, the inhibition rate of the chickpea protease hydrolysate obtained by hydrolysis with neutral protease was the highest for DPP-IV activity, that is, neutral protease was the optimal enzyme for DPP-IV activity inhibition. The enzyme hydrolysis conditions were selected for subsequent DPP-IV activity inhibition experiments.

[0085] 2. Application of chickpea protease hydrolysate in antioxidant (ABTS radical scavenging)

[0086] The specific steps are as follows:

[0087] (1) Mix 5 mL of 7 mmol / L ABTS solution and 88 μL of 140 mmol / L potassium persulfate solution (final concentration), and then react in the dark at room temperature for 12 - 16 h to generate ABTS radical cation (ABTS·+). Before use, dilute the ABTS·+ solution with 10 mmol / L phosphate buffer (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm.

[0088] (2) Preparation of chickpea protease hydrolysate samples:

[0089] Dissolve the chickpea protease hydrolysate powder prepared in Example 1 in 10 mmol / L phosphate buffer with pH 8.0, and the actual reaction concentration of the sample is 0.5 mg / mL.

[0090] (3) Combine 50 μL of chickpea protease hydrolysate samples with 150 μL of ABTS·+ solution respectively, react in the dark for 30 min, and then measure the absorbance at 734 nm.

[0091] Control group: Replace the sample with 10 mmol / L phosphate buffer.

[0092] The calculation method of ABTS radical scavenging activity is as follows:

[0093] ABTS radical scavenging activity (%) = [1 - (A Sample / A Blank )] × 100%

[0094] In the formula: A sample is the absorbance of the sample; A blank is the absorbance of the control.

[0095] The results of ABTS radical scavenging activity determination are shown in Table 3.

[0096] Table 3 Results of ABTS radical scavenging activity determination

[0097]

[0098] The results showed that at the same concentration, the ABTS radical scavenging rate of the chickpea protease hydrolysis sample obtained by neutral protease hydrolysis was the highest, that is, neutral protease was the optimal enzyme for ABTS radical scavenging activity. The enzyme hydrolysis conditions were selected for the subsequent ABTS radical scavenging activity experiment.

[0099] 3. Application of chickpea protease hydrolysis powder in reducing cholesterol

[0100] The specific steps are as follows:

[0101] (1) Cholesterol content standard curve

[0102] Prepare cholesterol standard solution 0.1mg / mL: Accurately weigh 0.1g of cholesterol standard product and dissolve it in glacial acetic acid, and make up the volume to 100mL. Pipette 10mL of the above solution and make up the volume to 100mL with glacial acetic acid.

[0103] Prepare ferric alum color reagent: Accurately weigh 4.4630g of ammonium ferric sulfate and dissolve it in 100mL of 80% phosphoric acid. Pipette 20mL of this solution and make up the volume to 250mL with sulfuric acid, and store it in a silica gel desiccator.

[0104] Take 6 stoppered colorimetric tubes and prepare solutions according to the dosages shown in Table 4. Shake well, and within 30 - 40 min, measure the absorbance value at a wavelength of 560nm, draw the standard curve, and establish the regression equation at the same time.

[0105] Table 4 Preparation plan of cholesterol content standard curve solution

[0106]

[0107] The curve is as Figure 1 shown.

[0108] (2) Determination method of inhibition rate of cholesterol micelle solubility

[0109] Preparation of chickpea protease hydrolysis sample:

[0110] Dissolve the chickpea protease hydrolysis powder prepared in Example 1 in pH 8.0, 100mmol / L Tris-HCl buffer solution respectively, and the actual reaction concentration of the sample is 5mg / mL.

[0111] Prepare 1 mL of simulated bile micelle solution containing: 0.002 mol / L cholesterol, 0.01 mol / L sodium taurocholate, 0.005 mol / L oleic acid, 0.132 mol / L sodium chloride, phosphate buffer solution with pH 7.4, and chickpea protease hydrolysate sample with a mass concentration of 5 mg / mL; it is prepared by emulsifying the above mixture with ultrasonic waves (power 400 W, frequency 20 kHz, for 20 min), then culturing at 37 °C for 24 h, centrifuging at 100,000×g for 60 min, and taking the supernatant to measure the cholesterol concentration.

[0112] The cholesterol concentration in the supernatant is the solubility of cholesterol micelles. Taking the micelle solution without peptide as the blank, the inhibition rate of the peptide on the solubility of cholesterol in the simulated bile micelle solution is calculated according to the following formula:

[0113] Inhibition rate = (M - N) / M × 100%;

[0114] In the formula: M is the cholesterol solubility (mol / L) of the blank control solution;

[0115] N is the cholesterol solubility (mol / L) of the sample solution.

[0116] The standard curve of cholesterol content is shown in Figure 1 , and the results of the determination of the inhibitory activity of cholesterol micelle solubility are shown in Table 5.

[0117] Table 5 Results of the determination of the inhibitory activity of cholesterol micelle solubility

[0118]

[0119] The results show that: at the same concentration, the inhibitory rate of the chickpea protease hydrolysate sample obtained by alkaline protease hydrolysis on cholesterol micelle solubility is the highest, that is, alkaline protease is the optimal enzyme for the inhibitory activity of cholesterol micelle solubility. The enzyme hydrolysis conditions are selected for the subsequent experiments on the inhibitory activity of cholesterol micelle solubility.

[0120] 4. Application of chickpea protease hydrolysate in ACE inhibition

[0121] The specific steps are as follows:

[0122] (1) Preparation of chickpea protease hydrolysate sample:

[0123] Dissolve the chickpea protease hydrolysate powder prepared in Example 1 in 80 mmol / L HEPES buffer solution (pH 8.2, containing 0.3 mol / L NaCl) respectively, and the actual reaction concentration of the sample is 0.4 mg / mL;

[0124] Add 40 μL of HEPES buffer solution or the same volume of chickpea protease hydrolysate sample to the control well and the sample well respectively;

[0125] (2) Then, add 50 μL of 1 mmol / L FAPGG (N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine) to each well. After preheating at 37 °C for 10 min, pipette 10 μL of 0.1 U / mL ACE into the 96-well plate.

[0126] Measure the initial absorbance of the control well and the sample wells at a wavelength of 340 nm (i.e., a1 and b1 respectively), and then incubate them in the dark at 37 °C for 30 min and measure the absorbance again (i.e., a2 and b2 respectively). Each sample is measured 3 times.

[0127] The decrease in absorbance value of the control well A = a1 - a2, and the decrease in absorbance value of the sample well B = b1 - b2.

[0128] The calculation of the ACE inhibition rate of the sample is calculated according to the following formula.

[0129]

[0130] The results of the ACE inhibition activity assay are shown in Table 6.

[0131] Table 6 Results of ACE inhibition activity assay

[0132]

[0133] The results show that: at the same concentration, the ACE activity inhibition rate of the chickpea protease hydrolysate obtained by papain hydrolysis is the highest, that is, papain is the optimal enzyme for ACE inhibition activity. The enzyme hydrolysis conditions are selected for the subsequent ACE inhibition activity experiment.

[0134] 5. Role of chickpea protease hydrolysate in Fe 2+ Chelation

[0135] The specific steps are as follows:

[0136] Preparation of chickpea protease hydrolysate samples:

[0137] Dissolve the chickpea protease hydrolysate powder prepared in Example 1 in ultrapure water respectively, and the actual reaction concentrations of the samples are 0.8 mg / mL;

[0138] In 50 μL of the sample, add 25 μL of FeCl2 (2 mmol / L) in sequence, let it stand for 10 min, add 200 μL of Ferrozine (5 mmol / L), mix well, let it stand at room temperature for 10 min, take out 200 μL from each sample and add it to the 96-well plate, and measure the OD562nm value.

[0139] Use ultrapure water instead of the sample for the control;

[0140] Calculate the Fe of the sample according to the following formula 2+ Chelating ability.

[0141] Sample Fe 2+ The chelation rate is calculated according to the following formula.

[0142]

[0143] Among them, Ac, Ab, and As are the absorbance of the control group, blank group, and sample group, respectively.

[0144] Fe 2+ The results of the chelating activity assay are shown in Table 7.

[0145] Table 7 Fe 2+ Chelation activity assay results

[0146]

[0147] The results showed that at the same concentration, the Fe content of chickpea protein hydrolyzed samples obtained by neutral protease hydrolysis was 2+ The chelation rate is the highest, that is, the neutral protease is Fe 2+ The best enzyme for chelating activity. 2+ This enzymatic hydrolysis condition was selected for the chelation activity experiment.

[0148] Example 3: Method for Isolating and Identifying DPP IV Inhibitory Peptides from Chickpea Protein Hydrolysate

[0149] The specific steps are as follows:

[0150] The specific implementation method is the same as Example 1, except that the protease is adjusted to neutral protease to prepare chickpea protein hydrolysate powder.

[0151] (1) Identification of peptide sequences from chickpea protein hydrolyzed powder by peptidomics

[0152] Chickpea protein digest samples were analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system is a Q Exactive LC-MS / MS system connected to an EASY-nanoLC 1200. TM A mass spectrometer (Thermo Fisher Scientific, MA, USA) was used. A total of 4 μL of chickpea protein digest sample was loaded onto an Acclaim PepMap C18 analytical column, 75 μm x 25 cm. The sample was separated using a 120-min gradient with a flow rate of 300 nL / min, a column temperature of 40°C, and an electrospray voltage of 2 kV. The gradient started with 2% phase B and increased nonlinearly to 35% over 110 minutes, then to 100% over 1 minute, and then held for 9 minutes. The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition.

[0153] The mass spectrometry parameters are set as follows: (1) MS: scanning range (m / z): 200 - 1800; resolution: 70000; AGC target: 3e6; maximum injection time: 60 ms; (2) HCD-MS / MS: resolution: 17500; AGC target: 5e4; maximum injection time: 50 ms; collision energy: 27; dynamic exclusion time: 20 s. The tandem mass spectrometry map was analyzed by PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). PEAKS DB was used to search the uniprot_Cicer arietinum (version 202112, 24812 entries) database, and none enzymatic digestion was set. The search parameters for fragment ion mass tolerance: 0.02 Da, precursor ion mass tolerance: 7 ppm, variable modifications: Oxidation(M) 15.99, Deamidation(NQ) 0.98, Acetylation(Protein N-term) 42.01. The protein cut-off value is: -10lgP ≥ 0, with at least 1 unique peptide; the peptide cut-off value is: -10lgP ≥ 20.

[0154] A total of 4847 polypeptides were identified after sequencing the chickpea protein hydrolysate by polypeptideomics.

[0155] (2) Screening for highly active DPP-IV inhibitory peptides from the 4847 polypeptides identified in (1) using multiple peptide screening conditions

[0156] Using multiple screening conditions: predicting whether the peptide has the potential to inhibit DPP-IV activity or has been reported in the BIOPEP database (https: / / biochemia.uwm.edu.pl / ); performing a bioactivity prediction score for the peptide in PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ); querying the protein source of the peptide in the protein database (https: / / www.uniprot.org / ); performing toxicity prediction through Toxinpred (https: / / crdd.osdd.net / raghava / toxinpred / ); performing allergenicity prediction through AllerTOP (https: / / www.ddg-pharmfac.net / AllerTOP / ). Selecting from the sequenced peptides those that are protein-derived, not modified by functional groups, with a peptide chain length < 15, and a peak area > 1.00×107 Sequences with an activity score > 0.8, no toxicity and no allergenicity, and not yet published.

[0157] At the same time, peptides with Pro or Ala at the second position of the NH2 terminus and Leu or Ile at the NH2 terminus of the peptide are screened. Peptides with such characteristics have high DPP-IV inhibitory activity.

[0158] (3) Use molecular docking technology to verify the peptide segments obtained from condition (2), which mainly includes the following steps:

[0159] ① Preparation of the DPP-IV enzyme PDB file: Retrieve the protein crystal structure of DPP-IV (code: 5J3J) from the Protein Data Bank, and remove the redundant ligands and H2O from 5J3J for subsequent docking experiments with the peptide segments;

[0160] ② After the screened peptide sequences are docked with 5J3J, the binding energy is obtained. The DPP-IV binding site is defined as a sphere containing protein residues within 15 or less.

[0161] ③ Determine whether the screened peptide sequences are potential DPP IV inhibitory peptides according to whether the binding energy is negative, and verify their DPP IV inhibitory activity through in vitro solid-phase synthesis.

[0162] Specifically, a total of 4 polypeptides are screened, and the binding energies are all less than -5.61; the DPP IV inhibitory activities of the 4 polypeptides are verified after in vitro solid-phase synthesis, as shown in Table 8 specifically.

[0163] Table 8 Binding energies of peptides docked with DPP-IV

[0164]

[0165] The results show that chickpea-derived DPP-IV inhibitory peptides are obtained by the method of the present invention, and their amino acid sequences are AAWPGHPEF, LAFP, IAIPPGIPYW, and PPGIPYW respectively.

[0166] Example 4: Method for detecting the activity of DPP IV inhibitory peptides

[0167] The specific steps are as follows:

[0168] Preparation of DPP-IV inhibitory peptide samples:

[0169] Dissolve the DPP-IV inhibitory peptides prepared in Example 3 in a pH 8.0, 100 mmol / L Tris-HCl buffer respectively;

[0170] In a 96-well microplate, 25 μL of the DPP-IV inhibitory peptide sample was mixed with 25 μL of Gly-Pro-PNA (1.6 mmol / L) respectively, incubated at 37 °C for 10 min, 50 μL of DPP-IV (8 U / L) solution was added to initiate the reaction, reacted at 37 °C for 60 min, and then 100 μL of sodium acetate buffer (1 mol / L, pH 4.0) was added to terminate the reaction, and the absorbance was measured at 405 nm.

[0171] Tris-HCl buffer was used instead of the DPP-IV solution as the blank group;

[0172] Tris-HCl buffer was used instead of the DPP-IV inhibitory peptide sample as the control group.

[0173] The calculation formula for the DPP-IV inhibition rate is:

[0174]

[0175] where Ac, Ab, and As are the absorbances of the control group, blank group, and sample group respectively.

[0176] Its DPP-IV half-inhibitory concentration (IC 50 ) was calculated by fitting, that is, the polypeptide concentration required when the DPP-IV activity inhibition rate reached 50%. The IC 50 detection results of the DPP-IV inhibitory peptide are shown in Table 9.

[0177] Table 9 Detection results of the IC 50 of the DPP-IV inhibitory peptide

[0178]

[0179] The results showed that three polypeptides (AAWPGHPEF, IAIPPGIPYW, and PPGIPYW) had strong DPP-IV inhibitory activities, and their IC 50 were all less than 100 μM. Among them, IAIPPGIPYW had the strongest DPP-IV inhibitory activity (IC 50 : 12.43 μM), which was comparable to the positive control diprotin A (IC 50 : 6.8 μM).

[0180] Example 5: Analysis of the DPP-IV inhibitory peptide IAIPPGIPYW

[0181] (1) Structural analysis of IAIPPGIPYW

[0182] As Figure 2 shown, the molecular structural formula of IAIPPGIPYW was obtained by ChemDraw and AutoDock.

[0183] (2) Docking of IAIPPGIPYW with DPP-IV molecule

[0184] As Figure 3 shown, IAIPPGIPYW forms hydrogen bonds with six residues, namely LYS554, ASP545, TYR547, ARG125, SER209, and TYR585, in the active pocket of DPP IV. Among them, the hydrogen bond interactions formed by IAIPPGIPYW with TYR547 in the S1 pocket, and ARG125 and SER209 in the S2 pocket are the key to its strong inhibitory ability. For subsequent screening and verification using this method, key attention can be paid to the hydrogen bonds formed by the polypeptide with the S1 or S2 pocket in the DPP-IV structure to predict its DPP-IV inhibitory activity.

[0185] (3) DPP-IV IC of IAIPPGIPYW 50

[0186] According to the method of Example 4, the chickpea protease hydrolysis sample IAIPPGIPYW was prepared into solutions with different actual reaction concentrations, and its DPP-IV inhibition rate was measured and plotted for analysis. By fitting, its DPP-IV half-inhibitory concentration (IC 50 ) was calculated, that is, the polypeptide concentration required when the DPP-IV activity inhibition rate reached 50%. The results are shown in Table 10 and Figure 4 as follows.

[0187] Table 10 DPP-IV activity inhibition results of different concentrations of IAIPPGIPYW

[0188]

[0189] As Figure 4 shown, the DPP-IV IC 50 value of the chickpea protease hydrolysis sample IAIPPGIPYW is 12.43 μM (14.00 μg / mL).

[0190] Example 6: Preparation of a drug containing a DPP-IV inhibitory peptide

[0191] (1) Weigh the prescribed amount of preservative and isotonic regulator and add them to the batching tank; (2) Add an appropriate amount of water for injection to completely dissolve; (3) Take an appropriate amount of water for injection to completely dissolve the prescribed amount of DPP-4 inhibitor and add it to the above batching tank; (4) Make up the volume with water for injection; (5) Adjust to the target pH value; (6) Filter and sterilize with a 0.22 μm filter membrane, and divide into portions to prepare an injection solution. The whole process is carried out in a sterile environment (for the specific method, see: the Chinese invention patent text with the publication number CN104548096B).

[0192] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A DPP-IV inhibitory peptide, characterized in that, The amino acid sequences of the inhibitory peptides are IAIPPGIPYW, AAWPGHPEF, LAFP or PPGIPYW.

2. A product containing the DPP-IV inhibitory peptide according to claim 1, characterized in that, The product is a health food.

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