Walnut-derived DPP-IV inhibitory peptide as well as preparation and screening method and application thereof

Abstract

The invention discloses a walnut source DPP-IV inhibitory peptide as well as a preparation and screening method and application thereof. The walnut source DPP-IV inhibitory peptide comprises FPAG and / or LPSYQPTP, the preparation and screening method comprises the following steps: (1) adding walnut protein powder into deionized water, and fully dissolving to obtain a walnut protein solution; (2) adding hydrolyzed compound protease into the walnut protein solution for enzymolysis to obtain walnut protein hydrolysate freeze-dried powder; (3) re-suspending the walnut protein hydrolysate freeze-dried powder in deionized water, and performing ultrafiltration separation by using an ultrafiltration membrane with the molecular weight cutoff of 1kDa to obtain a component F1; step (4), performing peptide fragment sequence identification on the obtained F1 component through peptidomics; and step (5), screening from peptide fragment sequences obtained by identification through a bioinformatics technology. The walnut source DPP-IV inhibitory peptide screened by the invention can be used for preparing a medicine or food with a DPP-IV inhibition function, so that the problems that the existing walnut source DPP-IV inhibitor has many side effects and the like are solved.

Description

Technical Field

[0001] This invention relates to the field of DPP-IV inhibitory peptide preparation technology. Specifically, it relates to a walnut-derived DPP-IV inhibitory peptide, its preparation and screening method, and its applications. Background Technology

[0002] Diabetes mellitus, especially type 2 diabetes mellitus (T2DM), involves insufficient insulin secretion or insulin resistance, leading to chronically elevated blood glucose levels and various complications. Dipeptidyl peptidase-IV (DPP-IV) plays a crucial role in regulating glycemic homeostasis. It weakens the physiological functions of incretins, such as promoting insulin secretion and inhibiting glucagon release, by rapidly degrading glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). Therefore, inhibiting DPP-IV activity has become one of the effective strategies for the clinical treatment of T2DM. Currently, several chemically synthesized DPP-IV inhibitors (such as sitagliptin and vildagliptin) are on the market and widely used in clinical practice. Although their efficacy is clear, long-term use may be accompanied by side effects such as gastrointestinal discomfort, increased risk of infection, and potential cardiovascular adverse events. Compared with the potential side effects of chemically synthesized drugs, the discovery of natural and safe DPP-IV inhibitory peptides from dietary proteins has become an important direction for the development of functional foods and drugs.

[0003] Bioactive peptides are considered ideal candidate molecules for developing functional foods and adjuvant therapeutics due to their natural origin, high biocompatibility, low metabolic burden, and often multi-target regulatory potential. In recent years, the enzymatic hydrolysis of food proteins to obtain peptides with DPP-IV inhibitory activity has become a research hotspot in the scientific field. Studies have confirmed that peptide sequences with significant DPP-IV inhibitory activity can be isolated from various protein sources, including dairy products, marine organisms, legumes, and grains.

[0004] Walnut (Juglans regia L.) is an important woody oilseed crop. In 2023, my country's walnut planting area reached 7.105 million hectares, with an annual output exceeding 5.93 million tons. The oilseed cake byproduct produced during walnut oil pressing is rich in high-quality protein (40-60%), containing all eight essential amino acids in a balanced ratio, making it an ideal substrate for developing bioactive peptides. In recent years, studies have identified multiple peptide sequences with DPP-IV inhibitory activity from walnut protein using different technical approaches, confirming its hypoglycemic potential. For example, one study identified nine novel DPP-IV inhibitory peptides from walnut protein using alkaline protease hydrolysis, ultrafiltration, and ion-exchange chromatography combined with mass spectrometry. Another study identified three highly active DPP-IV inhibitory peptides—LPFA, VPFWA, and WGLP—using trypsin digestion, chromatographic separation, and mass spectrometry analysis, with IC50 values ​​of [missing information].50 The values ​​were 267.9 ± 7.2 μM, 325.0 ± 8.4 μM, and 350.9 ± 8.3 μM, respectively. Despite successful precedents, existing techniques still have limitations, hindering the efficient and precise discovery and application of walnut-derived DPP-IV inhibitory peptides. Traditional preparation, separation, and purification methods are cumbersome, time-consuming, and expensive, making rapid screening of large-scale peptide libraries difficult. Existing studies have reported a limited number of active peptide sequences, and the vast sequence space of walnut proteins remains largely unexplored. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a walnut-derived DPP-IV inhibitory peptide, its preparation and screening method and application, so as to solve the problems of multiple side effects of existing walnut-derived DPP-IV inhibitors and the cumbersome, costly and low-throughput separation and purification process of DPP-IV inhibitory peptides.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] A walnut-derived DPP-IV inhibitory peptide, comprising FPAG and / or LPSYQPTP;

[0008] The amino acid sequence of FPAG is phenylalanine-proline-alanine-glycine, as shown in SQE.ID.NO.1; SQE.ID.NO.1: Phe-Pro-Ala-Gly;

[0009] The amino acid sequence of LPSYQPTP is leucine-proline-serine-tyrosine-glutamine-proline-threonine-proline, as shown in SQE.ID.NO.2; SQE.ID.NO.2: Leu-Pro-Ser-Tyr-Gln-Pro-Thre-Pro.

[0010] A method for preparing and screening walnut-derived DPP-IV inhibitory peptides includes the following steps:

[0011] Step (1): Add walnut protein powder to deionized water and treat with intermittent ultrasound to fully dissolve the walnut protein powder to obtain a walnut protein solution;

[0012] Step (2): Add hydrolytic complex protease to the walnut protein solution for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme by boiling water bath and cool to room temperature to adjust the pH to neutral. Then centrifuge and freeze-dry the solid product obtained by centrifugation to obtain walnut protein hydrolysate freeze-dried powder.

[0013] Step (3): Resuspend the lyophilized walnut protein hydrolysate in deionized water to obtain a lyophilized powder resuspension; use an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to perform ultrafiltration separation on the lyophilized powder resuspension, and denote the component separated by the ultrafiltration membrane as the F1 component, and the remaining walnut protein hydrolysate as the WPH component.

[0014] Step (4): Identify the peptide sequences of the obtained F1 fraction using peptidomics.

[0015] Step (5): The above-mentioned walnut-derived DPP-IV inhibitory peptide was obtained by screening from the identified peptide sequences using bioinformatics techniques.

[0016] In the above-mentioned method for preparing and screening DPP-IV inhibitory peptides from walnuts, in step (2), the hydrolytic complex protease is a mixture of neutral protease and flavor protease at an enzyme activity ratio of 1:1; the conditions for enzymatic hydrolysis are: enzyme-to-substrate ratio of 7000-9000 U / g (enzyme-to-substrate ratio is the ratio of enzyme activity to substrate mass), hydrolysis time of 3-5 h, hydrolysis temperature of 45-55 ℃, hydrolysis pH of 6-8; boiling water bath inactivation time of 8-12 min; and centrifugation conditions are: centrifugation rate of 9000-11000 r / min, centrifugation time of 25-35 min.

[0017] In the above method for preparing and screening walnut-derived DPP-IV inhibitory peptides, step (4) involves using an Easy-nLC 1200 / Q-Exactive system for peptide sequence identification and analysis. The analytical column is a Reprosil-Pur 120 C18-AQ 1.9 μm column with an inner diameter of 150 μm and a length of 170 mm. The pre-column is a Reprosil-Pur 120 C18-AQ 3 μm column with an inner diameter of 150 μm and a length of 50 mm. Mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is a 0.1% formic acid solution in 80% acetonitrile. The method for preparing an aqueous solution of formic acid is as follows: take 1 mL of formic acid and dilute to 1000 mL with ultrapure water; the method for preparing an 80% acetonitrile solution of 0.1% formic acid is as follows: first take 200 mL of acetonitrile and dilute to 1000 mL with ultrapure water to obtain an 80% acetonitrile solution; then take 1 mL of formic acid and dilute to 1000 mL with the 80% acetonitrile solution.

[0018] For identification and analysis: the sample was first diluted with 0.1% formic acid aqueous solution to a final concentration of 0.5 mg / mL, and then filtered through a 0.22 μm microfiltration membrane before injection; the injection volume was 5.0 μL, and the elution rate was 0.6 mL / min; the gradient elution program was: 0~2 min, 4%~8% B; 2~35 min, 8%~28% B; 35~55 min, 28~40% B; 55~56 min, 40%~95% B; 56~66 min, 95% B (in the gradient elution of this invention, the proportion of mobile phase B is expressed as a volume percentage); the full scan range of mass spectrometry was 300-1800 m / z, the first-stage mass spectrometry resolution was 70,000, and the second-stage MS resolution was 17,500, obtaining the raw mass spectrometry file; the raw mass spectrometry file was analyzed for peptide sequence resolution using the PEAKS De novo method.

[0019] In the above method for preparing and screening walnut-derived DPP-IV inhibitory peptides, step (5) first screens peptides with a De novo score exceeding 95%, a peptide chain length less than or equal to 10 amino acids, and a peak area greater than 1×10⁻⁶. 7 The peptides were selected; then, potential bioactive peptides with a score greater than 0.5 were screened using PeptideRanker, and toxicity and sensitization were screened using ToxinPred 3.0 and AllerCatPro2.0, respectively; next, DPP-IV inhibitory activity was predicted using BIOPEP-UWM and StackDPP-IV, and potential DPP-IV inhibitory peptides with predicted activities greater than 0.70 were screened; finally, the screened bioactive peptides were molecularly docked using Autodock-vina software, and bioactive peptides with a binding energy to DPP-IV molecules less than or equal to −7 kcal / mol were identified as walnut-derived DPP-IV inhibitory peptides.

[0020] The above-mentioned method for preparing and screening DPP-IV inhibitory peptides from walnuts, in step (1), the protein content of the walnut protein powder is greater than or equal to 80wt%; the conditions for intermittent ultrasonic treatment are: ultrasonic power 200-300W, working frequency 15-25kHz, pulse duration 15-25min, pulse on for 5s and off for 5s; the mass fraction of walnut protein in the walnut protein solution is 3-8wt%.

[0021] The above-mentioned method for preparing and screening walnut-derived DPP-IV inhibitory peptides includes the following steps in step (1):

[0022] Step (1-1): Add defatted walnut meal to distilled water at a mass-to-volume ratio of 1g:(14-18)mL and stir until well mixed. Then, dialyze the mixture using a dialysis membrane with a molecular weight cutoff of 3kDa to obtain a walnut meal dispersion. The purpose of dialyzing using a dialysis membrane with a molecular weight cutoff of 3kDa is to remove free phenolic small molecule compounds. Since the activity of polyphenols themselves can affect the subsequent screening of active peptides, it is necessary to remove free phenolic small molecule compounds first. In addition, during the walnut protein extraction process of this invention, excessive pH changes can easily cause walnut protein to bind with polyphenols and become difficult to remove. Therefore, this invention chooses to perform dephenolization treatment before walnut protein extraction, which can avoid the influence of polyphenols on the extraction of walnut protein and also avoid their influence on the subsequent screening of active peptides.

[0023] Steps (1-2): Adjust the pH of the walnut meal dispersion to 9-11 using a 0.8-1.2 mol / L sodium hydroxide solution, heat it to 40-55℃ and stir for 1-2 hours, then centrifuge it at 4500-5500 r / min for 10-20 minutes at 3-5℃ and collect the supernatant.

[0024] Steps (1-3): Adjust the pH of the supernatant to 4.4-4.6 with 0.8-1.2 mol / L hydrochloric acid solution, and then carry out the precipitation reaction at 3-5℃ for 50-70 min. After the precipitation reaction is completed, centrifuge at 3500-4500 r / min for 15-25 min at 3-5℃. Wash the protein precipitate obtained by centrifugation with deionized water until neutral, and then freeze-dry to obtain walnut protein powder.

[0025] In the above method for preparing and screening walnut-derived DPP-IV inhibitory peptides, in step (3), the mass concentration of walnut protein hydrolysate lyophilized powder in the lyophilized powder resuspension is 40-60 mg / mL.

[0026] The preparation and screening method of the above-mentioned walnut-derived DPP-IV inhibitory peptide, in step (1), the intermittent ultrasonic treatment conditions are: ultrasonic power 250W, working frequency 20kHz, pulse duration 20min, pulse on for 5s and off for 5s; the mass fraction of walnut protein in the walnut protein solution is 5wt%; the preparation and screening method of walnut protein powder includes the following steps:

[0027] In step (1-1), defatted walnut meal is added to distilled water at a mass-to-volume ratio of 1g:15mL and stirred until well mixed. Then, it is dialyzed using a dialysis membrane with a molecular weight cutoff of 3kDa to obtain a walnut meal dispersion.

[0028] In steps (1-2), the pH of the walnut meal dispersion was adjusted to 10 using a 1.0 mol / L sodium hydroxide solution, and then heated to 50°C and stirred for 1.5 h. After that, it was centrifuged at 5000 r / min for 15 min at 4°C, and the supernatant was collected.

[0029] In steps (1-3), the pH of the supernatant was adjusted to 4.5 using 1.0 mol / L hydrochloric acid solution, and the precipitation reaction was carried out at 4℃ for 60 min. After the precipitation reaction was completed, the supernatant was centrifuged at 4000 r / min for 20 min at 4℃. The protein precipitate obtained by centrifugation was washed with deionized water until neutral and then freeze-dried to obtain walnut protein powder.

[0030] In step (2), the hydrolytic complex protease is a mixture of neutral protease and flavor protease at an enzyme activity ratio of 1:1; the conditions for enzymatic hydrolysis are: enzyme-to-protein ratio of 8500 U / g, hydrolysis time of 4 h, hydrolysis temperature of 50 °C, and hydrolysis pH of 7; the enzyme inactivation time in boiling water bath is 10 min; the conditions for centrifugation are: centrifugation speed of 10000 r / min and centrifugation time of 30 min.

[0031] In step (3), the mass concentration of lyophilized walnut protein hydrolysate in the lyophilized powder resuspension is 50 mg / mL.

[0032] An application of a walnut-derived DPP-IV inhibitory peptide: the above-mentioned walnut-derived DPP-IV inhibitory peptide is used to prepare drugs, dietary supplements or foods with DPP-IV inhibitory function.

[0033] The technical solution of the present invention achieves the following beneficial technical effects:

[0034] This invention provides a method for preparing and screening walnut-derived DPP-IV inhibitory peptides. By integrating peptidomics and bioinformatics screening methods, it overcomes the limitations of traditional experimental procedures, such as cumbersome procedures, high costs, and low throughput, enabling the efficient discovery of novel DPP-IV inhibitory peptides from walnut protein. The method yields walnut-derived DPP-IV inhibitory peptides FPAG and LPSYQPTP, which possess high inhibitory activity and novel structures. The walnut-derived DPP-IV inhibitory peptides obtained by this invention can be applied to the development of functional foods, dietary supplements, or pharmaceutical compositions for the prevention / adjunctive treatment of type 2 diabetes, providing new core raw materials and solutions for developing natural and safe blood glucose management products. Attached Figure Description

[0035] Figure 1 Curves showing the change in degree of hydrolysis of different proteases over time in embodiments of the present invention;

[0036] Figure 2 Comparison of peptide yield and DPP-IV inhibition rate with different protease ratios in the embodiments of the present invention;

[0037] Figure 3 Secondary spectra of FPAG of active peptide sequences selected in the sieve in this embodiment of the invention;

[0038] Figure 4 Secondary spectra of the active peptide sequence LPSYQPTP selected in the sieve in this embodiment of the invention;

[0039] Figure 5 The docking diagram of the active peptide sequences FPAG and LPSYQPTP selected in the sieve of this invention with the DPP-IV molecule;

[0040] Figure 6 Lineweaver-Burk double reciprocal plot of the active peptide sequence FPAG selected in the embodiment of the present invention;

[0041] Figure 7 The Lineweaver-Burk double reciprocal plot of the active peptide sequence LPSYQPTP selected in the sieve in this embodiment of the invention;

[0042] Figure 8 IC50 of the active peptide sequences FPG and LPSYQPTP selected in the screening of this invention 50 value;

[0043] Figure 9 A diagram showing the gastrointestinal digestion simulation results of the active peptide sequences FPAG and LPSYQPTP selected in the screening of this invention.

[0044] Figure 10 The cell activity test results of the active peptide sequence FPAG selected in the screening of this embodiment of the invention are shown in the figure.

[0045] Figure 11 A graph showing the cell viability test results of the LPSYQPTP active peptide sequence selected from the screening in this embodiment of the invention;

[0046] Figure 12 The following is a graph showing the results of the activity test of the active peptide sequences FPAG and LPSYQPTP selected in the screening of this invention to inhibit the secretion of DPP-IV by STC-1 cells;

[0047] Figure 13 The figure shows the test results of the active peptide sequences FPAG and LPSYQPTP selected in the screening of this invention on the GLP-1 content in the STC-1 cell model. Detailed Implementation

[0048] 1. Walnut protein extraction

[0049] Defatted walnut meal was weighed and added to distilled water at a feed-to-liquid ratio of 1:15 (w / v). After mixing, the mixture was dialyzed using a 3 kDa dialysis membrane. After dialysis, the pH was adjusted to 10.0 with 1 mol / L NaOH solution, and the mixture was stirred at 50 °C for 1.5 h. The mixture was then centrifuged (5000 r / min, 4 °C) for 15 min. The supernatant was adjusted to pH 4.5 with 1 mol / L HCl solution, and then chilled at 4 °C for 1 h before centrifugation (4000 r / min, 4 °C) for 20 min. The precipitate was washed with deionized water until neutral. After freeze-drying, walnut protein powder was obtained. The protein content of the walnut protein powder was determined to be 84.14% using the Kjeldahl method.

[0050] 2. Optimization of the preparation process of walnut protein hydrolysate

[0051] 2.1 Walnut protein hydrolysis process

[0052] Walnut protein powder was redissolved in deionized water to obtain a 5% (w / v) walnut protein solution. Intermittent ultrasound was performed using a probe sonicator (pulse duration 20 min, pulse on for 5 s, pulse off for 5 s, ultrasound power 250 W, working frequency 20 kHz) to unfold the protein structure, which is beneficial to increase the contact area of ​​the protease during the hydrolysis process.

[0053] 2.1 Box-Behnken Experimental Design

[0054] according to Figure 1 Changes in degree of hydrolysis and Figure 2 The peptide yield and DPP-IV inhibitory activity were measured to screen neutral protease and flavor protease (enzyme activity ratio = 1:1) as the hydrolytic complex protease. Based on the recommended optimal reaction conditions, four key variables—enzyme-to-protein ratio, hydrolysis time, hydrolysis temperature, pH, and their preliminary ranges—were determined. A four-factor, three-level optimization experiment was conducted using a Box-Behnken design (Table 1), resulting in 29 experiments. The combined score of peptide yield and DPP-IV inhibition rate was used as the response index.

[0055] Table 1. Box-Behnken Experimental Factor Levels

[0056]

[0057] 3. Ultrafiltration separation of walnut protein hydrolysate

[0058] Hydrolysis was performed according to the optimal process determined by response surface methodology. The enzyme-to-protein ratio (i.e., the ratio of active protease to walnut protein) was 8500 U / g, the hydrolysis temperature was 50℃, the pH was 7.5, and the hydrolysis time was 4 h. After hydrolysis, the enzyme was inactivated by boiling water for 10 min. After cooling, the pH was adjusted to 7.0, and the mixture was centrifuged (10000 rpm, 30 min) and lyophilized to obtain walnut protein hydrolysate. The lyophilized walnut protein hydrolysate powder was resuspended in deionized water (50 mg / mL) and ultrafiltered using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The ultrafiltered fraction was collected and designated as fraction F1 for sequence identification; the remaining fraction was designated as "WPH fraction" and left untreated.

[0059] 4. Identification of bioactive peptide sequences and bioinformatics screening

[0060] Peptide sequences of the F1 fraction in walnut protein hydrolysate were identified using peptidomics. Analysis was performed using an Easy-nLC1200 / Q-Exactive system (Thermo Fisher Scientific, USA). The chromatographic column was a Reprosil-Pur 120 C18-AQ 1.9 μm (150 μm id × 170 mm) pre-column with a Reprosil-Pur 120C18-AQ 3 μm (150 μm id × 50 mm). The column was diluted to a final concentration of 0.5 mg / mL with 0.1% formic acid aqueous solution and filtered through a 0.22 μm microfiltration membrane before injection. The elution rate was 0.6 mL / min, and the injection volume was 5.0 μL. Mobile phase A and mobile phase B were 0.1% formic acid aqueous solution and 0.1% formic acid in 80% acetonitrile solution, respectively. Gradient elution was performed as follows: 0–2 min, 4%–8% B; 2–35 min, 8%–28% B; 35–55 min, 28–40% B; 55–56 min, 40%–95% B; 56–66 min, 95% B (the proportion of B in the mobile phase during gradient elution is by volume). The full scan range of mass spectrometry was 300–1800 m / z, with a first-stage mass spectrometry resolution of 70,000 and a second-stage MS resolution of 17,500, yielding raw MS files. Peptide sequences were resolved using the PEAKS Denovo method from the raw mass spectrometry files.

[0061] In this embodiment, the method for preparing a 0.1% formic acid aqueous solution is as follows: take 1 mL of formic acid and dilute it to 1000 mL with ultrapure water; the method for preparing an 80% acetonitrile solution of 0.1% formic acid is as follows: first take 20 mL of acetonitrile and dilute it to 100 mL with ultrapure water to obtain an 80% acetonitrile solution; then take 1 mL of formic acid and dilute it to 1000 mL with the 80% acetonitrile solution.

[0062] Using bioinformatics techniques, we screened peptides with a De novo score of over 95%, a peptide chain length of ≤10 amino acids, and a peak area >1×10⁻⁶. 7 Peptides were selected. Potentially bioactive peptides with a score >0.5 were screened using the Peptide Ranker assay, and toxicity and sensitization were screened using ToxinPred 3.0 and AllerCatPro 2.0, respectively. Finally, DPP-IV inhibitory activity was predicted using BIOPEP-UWM and StackDPP-IV, and potential DPP-IV inhibitory peptides with predicted activities >0.70 were screened.

[0063] Table 2

[0064]

[0065] The selected active peptides were subjected to molecular docking using Autodock-Vina software. The 3D structures of the peptides were constructed using PyMOL software. The DPP-IV PDB file (PDB ID: 1WCY) was downloaded from the PDB database (https: / / www.rcsb.org / ). Water molecules, hydrogen atoms, and charges were removed from the 1WCY protein, and the proligands in the structure were extracted and docked with the peptides for verification. The DPP-IV binding site was defined as a sphere containing protein residues within 20 Å. The final selected active peptide sequences were FPG and LPSYQPTP (…). Figure 3 and Figure 4 The binding energies of the molecule to DPP-IV were -8.1 and -8.7 kcal / mol, respectively. Both binding energies were less than -7 kcal / mol, indicating strong binding ability. Figure 5 The amino acid sequence of FPAG is SQE.ID.NO.1: Phe-Pro-Ala-Gly; the amino acid sequence of LPSYQPTP is SQE.ID.NO.2: Leu-Pro-Ser-Tyr-Gln-Pro-Thr-Pro.

[0066] Table 3 Molecular docking binding energy and active sites

[0067]

[0068] 5. Solid-phase synthesis of bioactive peptides and verification of DPP-IV inhibitory activity.

[0069] FPAG and LPSYQPTP, screened for molecular docking, were synthesized using Fmoc solid-phase synthesis, and their DPP-IV inhibitory activity was verified. The DPP-IV activity was measured using the luminescent substrate glycine-alanine-aminomethylcoumarin (AMC). Upon hydrolysis of the peptide bonds in DPP, free AMC groups are released, generating a fluorescence signal that can be analyzed at excitation wavelengths of 360 nm and emission wavelengths of 460 nm. The specific procedure was as follows: 30 µL of Tris-HCl buffer, 10 µL of DPP-IV, and 10 µL of the test sample (10 mg / mL) or solvent (as a blank control) were added to a microplate. Then, 50 µL of AMC was added to initiate the reaction. The microplate was incubated at 37 °C for 30 min, and data were read using a microplate reader at excitation and emission wavelengths of 360 nm and 460 nm, respectively. Tris-HCl buffer was used as a control group. The inhibition rate was calculated using the following formula.

[0070] (1);

[0071] In equation (1), , , The absorbance values ​​are for the control group, blank group, and sample group, respectively.

[0072] 6. Inhibition Dynamics

[0073] The reaction rate between the peptide and DPP-IV was measured using different concentrations of AMC as substrates. The inhibition type of the peptide on DPP-IV was evaluated by constructing a Lineweaver-Burk double reciprocal plot. The intercept (1 / Vmax) and slope (Km / Vmax) in the Lineweaver-Burk equation reflect the variation of the maximum initial velocity (Vmax) and Michaelis constant (Km) with peptide concentrations (0, 40, 80, 120 μM).

[0074] The Lineweaver-Burk equation lines all intersect in the second quadrant, indicating that FPG (Frontier-Female-Girl-Girl) equations all intersect in the second quadrant. Figure 6 ) and LPSYQPTP ( Figure 7 The inhibition mode of FPAG and LPSYQPTP is a mixed inhibition mode, primarily competitive inhibition. As the concentrations of FPAG and LPSYQPTP increase from 0 to 120 μM, Km increases while Vmax decreases. These two peptides mainly inhibit enzyme activity by binding to the active site of DPP-IV and competing with the substrate for binding sites. Furthermore, as mixed competitive inhibitors, FPAG and LPSYQPTP can also bind to the enzyme-substrate complex, reducing the enzyme's catalytic activity and enhancing its inhibitory activity by altering the conformation of DPP-IV or preventing product formation. Figure 8As shown, the ICs for FPAG and LPSYQPTP 50 The values ​​were 105.6±1.893 and 132.4±2.877 μM, respectively.

[0075] 7. In vitro digestion simulation

[0076] Simulated gastric juice stock solution (6.9 mM KCl, 0.9 mM KH2PO4, 25 mM NaHCO3, 47.2 mM NaCl, 0.1 mM MgCl2, 0.5 mM (NH4)2CO3). Simulated intestinal stock solution (6.8 mM KCl, 0.8 mM KH2PO4, 85 mM NaHCO3, 38.4 mM NaCl, 0.33 mM MgCl2), and 5 mL of trypsin solution (800 U mL⁻¹). The samples underwent simulated gastrointestinal digestion as follows: the samples were first dissolved in simulated gastric juice (SGF), and the pH was adjusted to 3.0. Then, pepsin was added to a final concentration of 2000 U / mL, and the mixture was incubated in a 37°C shaking water bath for 2 h to complete the gastric digestion stage. The pH was then adjusted to 7.5 to initiate the intestinal phase, during which trypsin (final concentration 2000 U / mL) and bile salts (10 mM) were added, and incubation continued at 37°C for 2 h. The enzymatic reaction was terminated by heating at 95°C for 10 min. The resulting digest was centrifuged at 10,000 × g for 10 min, and the supernatant was collected for the determination of DPP-IV inhibitory activity.

[0077] FPAG and LPSYQPTP (both at 0.2 mM) were subjected to simulated gastrointestinal digestion to assess their stability under gastrointestinal conditions, followed by determination of their DPP-IV inhibitory activity. Figure 9 As shown, despite a decrease in activity after digestion, FPAG and LPSYQPTP retained more than 50% of their DPP-IV inhibition rate, demonstrating significant inhibitory effects. These results indicate that FPAG and LPSYQPTP retain their DPP-IV inhibitory function after digestion, supporting their potential for oral administration.

[0078] 8. In vitro cell experiments

[0079] STC-1 (mouse intestinal endocrine tumor cell line) cells were cultured in Dulbecco modified Eagle medium (DMEM) complete medium supplemented with 1% penicillin (10,000 U / mL penicillin, 10 mg / mL streptomycin) and 10% fetal bovine serum, and incubated in a humidified incubator at 37°C and 5.0% CO2. When the cell confluence reached 80-90%, the cells were passaged by trypsin digestion.

[0080] 8.1 Cell viability

[0081] The effect of peptides on STC-1 cell viability was assessed using the CCK-8 assay. Cells were cultured at a density of 1 × 10⁶ cells. 5 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The culture medium was then removed, and 100 μL of DMEM containing different concentrations (0.05, 0.1, 0.2, 0.4, 0.8, 1.0, 2.0 mM) of FPAG and LPSYQPTP were added, followed by incubation for another 24 h. The culture plates were washed once with PBS buffer, and then 100 μL of DMEM containing 10% CCK-8 solution was added, followed by incubation for 2 h. The absorbance was measured at 450 nm. Cell viability was calculated using the following formula.

[0082]

[0083] In equation (2), The absorbance of samples with different concentrations. The absorbance is the control absorbance without peptides. The absorbance is the blank absorbance for cell-free cells.

[0084] 8.2 Assay of DPP-IV activity in STC-1 cells

[0085] Cells (3 × 10) 5 Cells (per well) were seeded in 24-well plates and incubated at 37°C in a humidified incubator with 5% CO2 for 12 h. The cells were then treated with culture media containing different concentrations of peptides or positive controls and cultured for another 24 h at 37°C in 5% CO2. The culture medium was collected, and the supernatant was collected by centrifugation. Cells were lysed on ice using cell lysis buffer, and the lysate was collected and centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was collected, and the protein content was determined using a BCA protein quantification kit. 50 μL of each sample was mixed with an equal volume of 100 μM Gly-Pro-AMC substrate in a 96-well plate and incubated at 37°C for 30 min. Fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The AMC concentration in the samples was calculated based on a standard curve showing the release of AMCs after incubation of different concentrations of Gly-Pro-AMC with recombinant human DPP-IV at 37°C for 30 min. The DPP-IV activity of the cells was calculated using Equation 3.

[0086] (3).

[0087] 8.3 GLP-1 secretion

[0088] STC-1 cells were seeded in 24-well plates (3.0 × 10⁻⁶ cells / well). 5Cells were cultured in complete DMEM medium (1 cell / well) at 37°C and 5% CO2. When cell confluence reached 80-90%, the medium was discarded, and the cells were washed twice with PBS. Then, Krebs-Ringer buffer containing peptides or a positive control was added to stimulate GLP-1 secretion. The cells were incubated at 37°C and 5% CO2 for 2 h. The GLP-1 stimulation buffer was recovered, and the cells were centrifuged at 3500 rpm for 10 min at 4°C. The supernatant was collected and stored at -80°C. The protein content in the cell lysate was determined using a BCA kit for GLP-1 quantification.

[0089] GLP-1 levels were determined using a GLP-1 enzyme-linked immunosorbent assay (ELISA) kit. 50 μL of diluted stimulation buffer was added to a 96-well plate coated with GLP-1 antibody. Then, 50 μL of Biotin-antibody working solution was added to each well, and the plate was incubated at 37°C for 1 h. The plate was then washed three times with washing buffer. Next, 100 μL of HRP conjugate working solution was added to each well, and the plate was incubated at 37°C for 30 min. The plate was washed five times with washing buffer. 90 μL of substrate (TMB) was added to each well, and the plate was incubated at 37°C in the dark for 15 min. Finally, 50 μL of stop solution was added, and the OD value of each well was immediately measured at 450 nm. A standard curve was constructed using the kit standards to calculate the GLP-1 level. The level of GLP-1 secreted by cells was calculated using the following formula.

[0090] (4).

[0091] 8.4 Results Analysis:

[0092] (1) Cell viability of FPG and LPSYQPTP cells was assessed using the CCK-8 assay kit. Figure 10 and Figure 11 The results showed that after treating cells with FPAG and LPSYQPTP at concentrations ranging from 50 to 1000 μM for 24 h, the cell survival rate was over 95%, indicating that neither FPAG nor LPSYQPTP had significant toxicity.

[0093] (2) The inhibitory activity of FPAG and LPSYQPTP on DPP-IV secretion by STC-1 cells was evaluated by monitoring the release of the fluorescent compound AMC from the Gly-Pro-AMC substrate. Figure 12The results showed that, compared with the blank control group, treatment with FPAG and LPSYQPTP at concentrations of 100, 200, and 400 μM significantly reduced the DPP-IV activity of STC-1 cells (p < 0.05). Specifically, treatment with 100 μM FPAG and LPSYQPTP reduced the DPP-IV activity of STC-1 cells by 44.6% and 30.37%, respectively. This indicates that low concentrations of FPAG and LPSYQPTP can inhibit endogenous DPP-IV in STC-1 cells.

[0094] (3) To determine the final retention level of GLP-1 after DPP-IV inhibition, the effects of FPG, LPSYQPTP, and the positive control sitagliptin on GLP-1 levels in the STC-1 cell model were evaluated. Figure 13 The results showed that GLP-1 levels were significantly increased in the 200 μM FPAG treatment group (p < 0.05), reaching 1.58-fold; and significantly increased in the LPSYQPTP treatment group (p < 0.05), reaching 1.44-fold. Furthermore, except for the 100 μM treatment group, the total GLP-1 levels in all other treatment groups were significantly higher than those in the control group (p < 0.05). This effect was mainly attributed to the inhibition of DPP-IV, which prevented the degradation of endogenous active GLP-1 secreted by STC-1 cells. DPP-IV inhibition is a key factor in increasing circulating endogenous active GLP-1 levels, which can prolong its half-life and further stimulate β-cell insulin release.

Claims

1. A walnut-derived DPP-IV inhibitory peptide, characterized in that, Including FPG and / or LPSYQPTP; The amino acid sequence of FPAG is phenylalanine-proline-alanine-glycine, as shown in SQE.ID.NO.1; The amino acid sequence of LPSYQPTP is leucine-proline-serine-tyrosine-glutamine-proline-threonine-proline, as shown in SQE.ID.NO.

2.

2. A method for preparing and screening walnut-derived DPP-IV inhibitory peptides, characterized in that, Includes the following steps: Step (1): Add walnut protein powder to deionized water and treat with intermittent ultrasound to fully dissolve the walnut protein powder to obtain a walnut protein solution; Step (2): Add hydrolytic complex protease to the walnut protein solution for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme by boiling water bath and cool to room temperature to adjust the pH to neutral. Then centrifuge and freeze-dry the solid product obtained by centrifugation to obtain walnut protein hydrolysate freeze-dried powder. Step (3): Resuspend the lyophilized walnut protein hydrolysate in deionized water to obtain a lyophilized powder resuspension; use an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to perform ultrafiltration separation on the lyophilized powder resuspension, and define the component separated by the ultrafiltration membrane as the F1 component. Step (4): Identify the peptide sequences of the obtained F1 fraction using peptidomics. Step (5): The walnut-derived DPP-IV inhibitory peptide as described in claim 1 is obtained by screening from the identified peptide sequences using bioinformatics techniques.

3. The method for preparing and screening walnut-derived DPP-IV inhibitory peptides according to claim 2, characterized in that, In step (2), the hydrolyzed complex protease is a mixture of neutral protease and flavor protease at an enzyme activity ratio of 1:1; the conditions for enzymatic hydrolysis are: enzyme-to-protein ratio of 7000-9000 U / g, hydrolysis time of 3-5 h, hydrolysis temperature of 45-55 ℃, and hydrolysis pH of 6-8; the enzyme inactivation time in boiling water bath is 8-12 min; the conditions for centrifugation are: centrifugation speed of 9000-11000 r / min and centrifugation time of 25-35 min.

4. The method for preparing and screening walnut-derived DPP-IV inhibitory peptides according to claim 2, characterized in that, In step (4), the peptide sequence was identified and analyzed using an Easy-nLC 1200 / Q-Exactive system; the analytical column was a Reprosil-Pur 120 C18-AQ 1.9 μm column with an inner diameter of 150 μm and a length of 170 mm, and the pre-column was a Reprosil-Pur 120 C18-AQ 3 μm column with an inner diameter of 150 μm and a length of 50 mm; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid in 80% acetonitrile solution; 0.1% The method for preparing an aqueous solution of formic acid is as follows: take 1 mL of formic acid and dilute to 1000 mL with ultrapure water; the method for preparing an 80% acetonitrile solution of 0.1% formic acid is as follows: first take 200 mL of acetonitrile and dilute to 1000 mL with ultrapure water to obtain an 80% acetonitrile solution; then take 1 mL of formic acid and dilute to 1000 mL with the 80% acetonitrile solution. For identification and analysis: F1 fraction was first diluted with 0.1% formic acid aqueous solution to a final concentration of 0.5 mg / mL, and filtered through a 0.22 μm microfiltration membrane before injection; the injection volume was 5.0 μL, the elution rate was 0.6 mL / min; the gradient elution program was: 0~2 min, 4%~8% B; 2~35 min, 8%~28% B; 35~55 min, 28~40% B; 55~56 min, 40%~95% B; The mass spectrometry scan ranged from 56 to 66 minutes, with 95% B as the mobile phase (B percentage is by volume). The full scan range was 300–1800 m / z, with a first-stage mass spectrometry resolution of 70,000 and a second-stage MS resolution of 17,500, yielding the raw mass spectrometry file. The raw mass spectrometry file was then analyzed for peptide sequences using the PEAKSDe novo method.

5. The method for preparing and screening walnut-derived DPP-IV inhibitory peptides according to claim 2, characterized in that, In step (5), the first step is to screen for peptides with a De novo score of over 95%, a peptide chain length of less than or equal to 10 amino acids, and a peak area greater than 1×10⁻⁶. 7 The peptides were selected; then, potential bioactive peptides with a score greater than 0.5 were screened using Peptide Ranker, and toxicity and sensitization were screened using ToxinPred 3.0 and AllerCatPro 2.0, respectively; next, DPP-IV inhibitory activity was predicted using BIOPEP-UWM and StackDPP-IV, and potential DPP-IV inhibitory peptides with predicted activities greater than 0.70 were screened; finally, the screened bioactive peptides were molecularly docked using Autodock-vina software, and bioactive peptides with a binding energy to DPP-IV molecules less than or equal to −7 kcal / mol were identified as walnut-derived DPP-IV inhibitory peptides.

6. The method for preparing and screening walnut-derived DPP-IV inhibitory peptides according to claim 2, characterized in that, The walnut protein powder in step (1) contains a protein content greater than or equal to 80wt%; the conditions for intermittent ultrasonic treatment are: ultrasonic power 200-300W, working frequency 15-25kHz, pulse duration 15-25min, pulse on for 5s and off for 5s; the mass fraction of walnut protein in the walnut protein solution is 3-8wt%.

7. The method for preparing and screening walnut-derived DPP-IV inhibitory peptides according to claim 6, characterized in that, In step (1), the preparation and screening method for walnut protein powder includes the following steps: Step (1-1): Add defatted walnut meal to distilled water at a mass-to-volume ratio of 1g:(14-18)mL and stir until well mixed. Then, dialyze the mixture using a dialysis membrane with a molecular weight cutoff of 3kDa to obtain a walnut meal dispersion. Steps (1-2): Adjust the pH of the walnut meal dispersion to 9-11 using a 0.8-1.2 mol / L sodium hydroxide solution, heat it to 40-55℃ and stir for 1-2 hours, then centrifuge it at 4500-5500 r / min for 10-20 minutes at 3-5℃ and collect the supernatant. Steps (1-3): Adjust the pH of the supernatant to 4.4-4.6 with 0.8-1.2 mol / L hydrochloric acid solution, and then carry out the precipitation reaction at 3-5℃ for 50-70 min; after the precipitation reaction is completed, centrifuge at 3500-4500 r / min for 15-25 min at 3-5℃. The protein precipitate obtained by centrifugation was washed with deionized water until neutral and then freeze-dried to obtain walnut protein powder.

8. The method for preparing and screening walnut-derived DPP-IV inhibitory peptides according to claim 2, characterized in that, In step (3), the mass concentration of lyophilized walnut protein hydrolysate in the lyophilized powder resuspension is 40-60 mg / mL.

9. The method for preparing and screening walnut-derived DPP-IV inhibitory peptides according to any one of claims 2-8, characterized in that, In step (1), the conditions for intermittent ultrasonic treatment are: ultrasonic power 250W, working frequency 20kHz, pulse duration 20min, pulse on for 5s and off for 5s; the mass fraction of walnut protein in the walnut protein solution is 5wt%; the preparation and screening method of walnut protein powder includes the following steps: In step (1-1), defatted walnut meal is added to distilled water at a mass-to-volume ratio of 1g:15mL and stirred until well mixed. Then, it is dialyzed using a dialysis membrane with a molecular weight cutoff of 3kDa to obtain a walnut meal dispersion. In steps (1-2), the pH of the walnut meal dispersion was adjusted to 10 using a 1.0 mol / L sodium hydroxide solution, and then heated to 50°C and stirred for 1.5 h. After that, it was centrifuged at 5000 r / min for 15 min at 4°C, and the supernatant was collected. In steps (1-3), the pH of the supernatant was adjusted to 4.5 using a 1.0 mol / L hydrochloric acid solution, and the precipitation reaction was carried out at 4℃ for 60 min. After the precipitation reaction was completed, the supernatant was centrifuged at 4000 r / min for 20 min at 4℃. The protein precipitate obtained by centrifugation was washed with deionized water until neutral and then freeze-dried to obtain walnut protein powder. In step (2), the hydrolytic complex protease is a mixture of neutral protease and flavor protease at an enzyme activity ratio of 1:1; the conditions for enzymatic hydrolysis are: enzyme-to-protein ratio of 8500 U / g, hydrolysis time of 4 h, hydrolysis temperature of 50 °C, and hydrolysis pH of 7; the enzyme inactivation time in boiling water bath is 10 min; the conditions for centrifugation are: centrifugation speed of 10000 r / min and centrifugation time of 30 min. In step (3), the mass concentration of the lyophilized walnut protein hydrolysate in the lyophilized powder resuspension is 50 mg / mL.

10. The application of a walnut-derived DPP-IV inhibitory peptide, characterized in that, The walnut-derived DPP-IV inhibitory peptide as described in claim 1 can be used to prepare drugs, dietary supplements, or foods with DPP-IV inhibitory function.

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