Xanthine oxidase inhibitory peptide derived from goat milk protein and application of xanthine oxidase inhibitory peptide
By screening and validating the FAWP peptide in goat milk protein, the problems of adverse reactions and unclear mechanisms of existing XOD inhibitors have been solved, achieving effective inhibition of XOD and demonstrating significant potential for regulating uric acid metabolism. This approach is suitable for food and nutritional health products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-29
- Publication Date
- 2026-04-17
AI Technical Summary
Existing XOD inhibitors have adverse effects with long-term use, and the screening and mechanism of action of specific short peptides derived from goat milk protein are still unclear, which limits their application in the regulation of hyperuricemia.
Using a combination of bioinformatics and machine learning, a xanthine oxidase inhibitory peptide with the amino acid sequence FAWP was screened from sheep milk protein. The peptide was prepared using a solid-phase peptide synthesis method, and its inhibitory effect on XOD was verified by molecular docking and molecular dynamics simulation.
This peptide exhibits significant inhibitory effects on XOD, with an IC50 value of 12.99 mM. It is stably bound to the catalytic active site, reducing uric acid production. Furthermore, it is safe in origin and has a well-defined structure, making it suitable for use in the food and nutrition fields.
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Figure CN121873166A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine and bioactive peptide technology, specifically relating to a xanthine oxidase inhibitory peptide derived from goat milk protein and its application. Background Technology
[0002] Hyperuricemia is a metabolic state caused by abnormal uric acid metabolism, leading to elevated uric acid levels. Long-term elevated uric acid levels may induce health problems such as gout and kidney stones. Xanthine oxidase (XOD) is a key rate-limiting enzyme in purine metabolism, catalyzing the conversion of xanthine and hypoxanthine into uric acid. Therefore, inhibiting XOD activity is considered one of the important ways to regulate uric acid levels in the body.
[0003] Currently, commonly used XOD inhibitors in clinical practice include allopurinol and febuxostat. Although these drugs are effective in lowering uric acid levels, long-term use may be accompanied by certain adverse reactions, such as skin reactions, abnormal liver function, or gastrointestinal discomfort, limiting their application in some populations.
[0004] In recent years, bioactive peptides derived from natural food proteins have gradually attracted attention due to their safe origin, well-defined structure, and ease of digestion and absorption. Existing studies have shown that some peptides derived from milk proteins have the potential to inhibit XOD activity. However, systematic studies on whether specific short peptides derived from goat milk proteins have XOD-inhibiting activity are limited, and the screening and mechanism of action of these peptides remain unclear. Based on this, this invention screens and identifies novel XOD-inhibiting peptides from goat milk proteins, providing a new technical solution for developing safe and well-defined XOD-regulating active substances. Summary of the Invention
[0005] The purpose of this invention is to address the limited sources of XOD inhibitors in existing technologies. It proposes a xanthine oxidase inhibitory peptide derived from goat milk protein and its applications, providing a safe and structurally well-defined XOD-regulating active peptide. This xanthine oxidase inhibitory peptide (XOD inhibitory peptide) can be applied in functional foods or nutritional interventions, providing a safe and natural active substance basis for dietary regulation of hyperuricemia and related metabolic diseases.
[0006] The technical solution of this invention is:
[0007] This invention provides a xanthine oxidase inhibitory peptide derived from goat milk protein, the amino acid sequence of which is FAWP (SEQ ID NO.1).
[0008] This xanthine oxidase inhibitory peptide is a short peptide obtained by screening the amino acid sequence of sheep milk protein (especially sheep milk protein). It is a tetrapeptide sequence with the amino acid sequence FAWP, and is named xanthine oxidase inhibitory peptide.
[0009] Furthermore, the xanthine oxidase inhibitory peptide is a water-soluble polypeptide that can dissolve in water and buffer systems.
[0010] Furthermore, the molecular weight of the xanthine oxidase inhibitory peptide is 519.6 Da.
[0011] Furthermore, the xanthine oxidase inhibitory peptide is prepared by solid-phase peptide synthesis.
[0012] The present invention also provides a composition wherein the active ingredient of the composition comprises the xanthine oxidase inhibitory peptide described above.
[0013] This invention provides the use of the xanthine oxidase inhibitory peptide or the composition thereof in the preparation of products that inhibit xanthine oxidase.
[0014] Furthermore, the products include xanthine oxidase inhibitors, pharmaceuticals, food, and beverages.
[0015] Furthermore, the xanthine oxidase inhibitory peptide or the composition is used to prepare an XOD inhibitor.
[0016] The present invention also provides the use of the xanthine oxidase inhibitory peptide or the composition thereof in the preparation of products for regulating uric acid levels in vivo.
[0017] Furthermore, the products include pharmaceuticals, beverages, food, functional foods, nutritional supplements, and food additives.
[0018] Furthermore, the xanthine oxidase inhibitory peptide or the composition can be used as a food additive or beverage additive for dietary regulation in people with high uric acid levels.
[0019] Furthermore, when the product is a pharmaceutical product, its dosage form includes powder, tablets, capsules, soft capsules, granules, pills, gel candies, oral liquids, or drops.
[0020] Furthermore, when the product is food or beverage, it includes dairy products, dairy beverages, solid beverages, and protein beverages.
[0021] The beneficial effects of this invention are:
[0022] (1) This invention utilizes the combined techniques of bioinformatics and machine learning to screen xanthine oxidase inhibitory peptide FAWP from sheep milk protein through targeted screening methods such as feature encoding, model prediction, molecular docking, and molecular dynamics simulation. This peptide has a significant inhibitory effect on xanthine oxidase and is derived from sheep milk protein, making it safe and structurally clear.
[0023] (2) The results show that the XOD inhibitory peptide screened in this invention can stably bind to the XOD catalytic active site and inhibit XOD activity. The IC50 value of the peptide was determined to be 12.99 mM by enzyme activity inhibition experiment, which significantly inhibits its enzyme activity and reduces uric acid production by inhibiting the activity of XOD enzyme, thus having potential application value in the regulation of uric acid metabolism. At the same time, the peptide has good binding stability and specificity at the molecular level.
[0024] (3) The XOD inhibitory peptide has a small molecular weight and a mature synthesis process, and has good application feasibility; it can be extended to food and nutrition health related fields, and has good industrial application prospects. Attached Figure Description
[0025] Figure 1 A schematic diagram of the process for screening and identifying the xanthine oxidase inhibitory peptide FAWP from goat milk protein.
[0026] Figure 2 This is a schematic diagram of the molecular docking model between xanthine oxidase inhibitory peptide and xanthine oxidase.
[0027] Figure 3 The figure shows the inhibitory effect of xanthine oxidase inhibitory peptide on xanthine oxidase activity.
[0028] Figure 4 This is a schematic diagram comparing the changes in the secondary structure of xanthine oxidase before and after the xanthine oxidase inhibitory peptide binds to xanthine oxidase.
[0029] Figure 5 This is a schematic diagram of the binding interface between the xanthine oxidase inhibitory peptide and xanthine oxidase, and the interaction of key amino acid residues, based on molecular dynamics simulations of the stable conformation. Detailed Implementation
[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0031] To further understand the present invention, it will be further described in conjunction with the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods, detection methods, etc., involved in the following embodiments are all conventional methods already existing in the prior art, performed according to the techniques and conditions described in the literature in this field, or according to the product instructions; the materials, reagents, etc., used in the following embodiments are all commercially available unless otherwise specified.
[0032] like Figure 1 As shown, this embodiment of the invention provides a method for screening sheep milk peptides from sheep milk, combining the amino acid sequences of key proteins in sheep milk retrieved from a database, performing high-throughput screening of peptides, and finally identifying the xanthine oxidase inhibitory peptide FAWP; then verifying the peptide activity and analyzing its inhibition mechanism.
[0033] Example 1: Identification of natural peptides in goat milk
[0034] Take 200 μL of sheep milk, filter it through a 3 kDa ultrafiltration membrane, centrifuge it at 12000 r / min for 10 minutes at 4℃, and then measure the peptide concentration at 205 nm.
[0035] 2 μL of 1 mol / L dithiothreitol (DTT, final concentration 10 mmol / L) was added to the peptide solution, and reduction was carried out at 56 °C for 1 h. Then, 4 μL of 1 mol / L iodoacetamide (IAM, final concentration 20 mmol / L) was added, and alkylation was carried out at room temperature for 40 min. Excess IAM was neutralized with DTT, and the system was acidified with trifluoroacetic acid (TFA). The peptide was desalted using a C18 solid-phase extraction column (Stage-Tips), followed by elution with 50% acetonitrile / 0.1% trifluoroacetic acid solution.
[0036] Nano-liquid chromatography-tandem mass spectrometry (Nano LC-MS / MS) was used to identify naturally occurring peptides in goat milk, providing fundamental data for subsequent screening of bioactive peptides.
[0037] Nano LC-MS / MS analysis was performed using a C18 reversed-phase chromatography system. Mobile phase B (acetonitrile solution containing 0.1% formic acid) was used with a gradient elution range of 0–95%, an elution time of 66 min, and a flow rate of 600 nL / min. Data acquisition was performed in data-dependent acquisition (DDA) mode, with a full-scan mass spectrometry resolution of 70,000 and a secondary mass spectrometry resolution of 17,500.
[0038] De novo peptide sequencing was performed using PEAKS software. During the sequencing process, modification parameters, peptide quality tolerance, and fragment ion quality tolerance were set, and a total of 9918 peptides were identified.
[0039] Example 2: Simulated enzymatic hydrolysis of goat milk protein
[0040] The amino acid sequences of key proteins from goat milk were retrieved from the UniProt database, including four caseins (κ-casein, αs1-casein, αs2-casein, and β-casein), two whey proteins (α-lactalbumin and β-lactoglobulin), serum albumin (BSA), and lactoferrin. The corresponding PDB IDs for each protein are as follows: κ-casein (P02669), αs1-casein (P04653), αs2-casein (P04654), β-casein (P11839), α-lactalbumin (P09462), β-lactoglobulin (P67976), serum albumin (P14639), and lactoferrin (R9QXS6). Computer-simulated enzymatic digestion of these proteins was performed using the BIOPEP-UWM database.
[0041] Nine commercially available proteases were selected, covering animal-derived proteases (chymotrypsin, trypsin, pepsin, elastase), microbial-derived proteases (proteinase K, Bacillus subtilis subtilisin), and plant-derived proteases (papain, bromelain, figase). To simulate the in vivo digestive environment, a total of 129 enzymatic hydrolysis schemes were designed, including single-enzyme, dual-enzyme, and triple-enzyme combinations.
[0042] Example 3: Construction of Machine Learning Model and Screening of XOD Inhibitory Peptides
[0043] 202 experimentally validated XOD-inhibiting peptides were collected as positive samples; simultaneously, an equal number of non-XOD-inhibiting peptides were selected from the BIOPEP-UWM database and relevant literature as negative samples. This formed a binary classification dataset for model training.
[0044] To characterize peptides, the VHSE descriptor was used to transform each peptide sequence into a multidimensional feature matrix suitable for machine learning modeling. CatBoost was selected for predictive modeling of peptide activity. The CatBoost tree depth was set to 6, the learning rate to 0.05, and the number of iterations to 1000. All models were trained using a 5-fold cross-validation strategy to ensure the reliability of the model evaluation results.
[0045] A pre-trained CatBoost classifier was used to predict the activity probability score (range 0-1) of each peptide from a peptide database based on Examples 1 and 2. This score reflects the peptide's xanthine oxidase (XOD) inhibitory potential. Peptides with scores higher than 0.8 were selected as 538 high-confidence candidate peptides for subsequent molecular docking experiments and in vitro activity verification.
[0046] Example 5: Molecular docking screening of XOD-inhibiting peptides
[0047] Using the PDBParser and StructureBuilder modules in BioPython, a three-dimensional structure of a peptide is constructed from its N-terminus to its C-terminus and saved as a PDB file.
[0048] Download the xanthine oxidase (XOD) target protein (PDB ID: 3NVW) from the RCSB PDB database and preprocess it in AutoDock Tools software: remove ligands and water molecules from the protein structure, add polar hydrogen atoms, and calculate Geistig charge.
[0049] The coordinates of the center of the molecular docking grid were set to x = 38.142, y = 21.868, z = 19.497, and the grid box size was set to 24.111 Å to fully cover the active site of the target protein.
[0050] Molecular docking was performed using AutoDock Vina software with the following parameters set: exhaustiveness = 8, number of output conformations (num_modes) = 5, and energy range (energy_range) = 4. Each candidate peptide was docked with the XOD target protein, and the binding affinity (unit: kcal / mol) and the corresponding optimal binding conformation were recorded.
[0051] AutoDock Vina molecular docking was performed on 538 high-probability peptides. Peptides with binding affinity ≤ -8.0 kcal / mol were ultimately selected, with the highest binding energy (-9.7 kcal / mol) identified as the high-affinity peptide (docking diagram shown). Figure 2 (As shown).
[0052] Example 6: Synthesis of FAWP peptide and verification of its XOD inhibitory activity.
[0053] Solid-phase synthesis was used to artificially synthesize xanthine oxidase (XOD) inhibitory peptides selected from screening. The purity of the synthesized peptides was ≥95% as determined by liquid chromatography-mass spectrometry. The XOD inhibitory activity of the peptides was measured using a xanthine oxidase activity assay kit containing WST-8 reagent.
[0054] The experiment achieved quantitative analysis of XOD activity by measuring the absorbance at a wavelength of 450 nm. The results are as follows: Figure 3 As shown, the peptide exhibited dose-dependent inhibitory activity in the concentration range of 2-10 mg / mL, and the calculated half-maximal inhibitory concentration (IC50) of FAWP was [data missing].50 It is 12.99 ± 0.33 mM.
[0055] Example 7: Circular dichroism chromatographic analysis of the effect of FAWP peptide on the secondary structure of XOD
[0056] The circular dichroism (CD) spectra of xanthine oxidase (XOD) and its XOD-peptide complex in the far ultraviolet region (190–260 nm) were measured using a JASCO J-1500 circular dichroism spectrometer with a 0.1 cm path length quartz cuvette. The experimental procedure is as follows:
[0057] A 0.10 mg / mL XOD solution and a 2 mg / mL peptide solution were mixed at a volume ratio of 9:1 and incubated at room temperature for 1 hour before spectral analysis. Pure XOD solution under the same conditions was used as a control group. The spectral acquisition parameters were set as follows: bandwidth 1.0 nm, scan rate 2.0 nm·s⁻¹, and response time 0.5 s.
[0058] Experimental results are as follows Figure 4 As shown, far-ultraviolet circular dichroism analysis revealed that the binding of FAWP to XOD significantly reduced its negative ellipticity at 222 nm and 208 nm, and caused spectral changes in the 200–205 nm region. This indicates that the α-helix structure of XOD decreased, the β-sheet content decreased, and the random coil ratio increased significantly, thereby inhibiting enzyme activity by inducing structural loosening and partial unfolding of the catalytic pocket.
[0059] Example 8: Molecular dynamics simulation of FAWP peptide and XOD
[0060] Molecular dynamics simulations of xanthine oxidase-peptide complexes (XOD–peptide complexes) were performed using GROMACS v2022.03 software. The components were simulated using an amber99sb-ildn force field, with the complex placed in a dodecahedral box and solvated using TIP3P water molecules to ensure a minimum distance of 15 Å between the complex and the box boundary. Sodium ions (Na⁺) and chloride ions (Cl⁻) were then added to the system to neutralize the system charge and achieve a salt concentration of 0.1 mol / L. A steepest descent integrator was used to minimize the energy of the system, with an upper limit of 50,000 steps to eliminate conflicts between molecular geometry and solvent steric hindrance. Equilibrium simulations were performed sequentially under canonical ensemble (NVT) and isothermal-isobaric ensemble (NPT) for 200 ps, gradually increasing the system temperature to 300 K. The relevant parameter settings for the molecular dynamics simulations are as follows: temperature control uses a modified Berendsen thermostat, and pressure control uses a Parrinello-Rahman pressure thermostat; short-range nonbonded interactions (including Coulomb and van der Waals interactions) are calculated using a 10 Å Verlet cutoff scheme, while long-range electrostatic interactions are handled using the particle mesh Ewald method (PME). Finally, a yield simulation with a duration of 200 ns and a time step of 2 fs is performed, using a Verlet leapfrog integrator to evaluate the interactions of the system. Figure 5 As shown, FAWP is anchored by four hydrogen bonds: Phe1-Glu1261 / Gln767, Ala2-Gln1040, and Pro4-Ser1082. It also forms multiple stable bonds by relying on the hydrophobic network composed of residues such as Phe914 / Phe798 and the aromatic stacking effect between Phe1 / Trp3-Phe914, thereby occupying the active pocket of XOD and inhibiting its catalytic function.
[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A xanthine oxidase inhibitory peptide, characterized in that, The amino acid sequence of the xanthine oxidase inhibitory peptide is shown in SEQ ID NO.
1.
2. The xanthine oxidase inhibitory peptide according to claim 1, characterized in that, The xanthine oxidase inhibitory peptide is derived from goat milk protein, is a water-soluble polypeptide, and has a molecular weight of 519.6 Da.
3. A composition, characterized in that, The active ingredient of the composition comprises the xanthine oxidase inhibitory peptide of claim 1.
4. The use of the xanthine oxidase inhibitory peptide of claim 1 or the composition of claim 3 in the preparation of a product that inhibits xanthine oxidase.
5. The application according to claim 4, characterized in that, The products include xanthine oxidase inhibitors, pharmaceuticals, food, and beverages.
6. The use of the xanthine oxidase inhibitory peptide of claim 1 or the composition of claim 3 in the preparation of a product for regulating uric acid levels in vivo.
7. The application according to claim 6, characterized in that, The products include pharmaceuticals, beverages, food, functional foods, nutritional supplements, and food additives.
8. The application according to claim 6, characterized in that, The xanthine oxidase inhibitory peptide or the composition may be used as a food additive or beverage additive for dietary regulation in people with high uric acid.
9. The application according to claim 4 or 6, characterized in that, When the product is a pharmaceutical product, its dosage form includes powder, tablet, capsule, soft capsule, granule, pill, gel candy, oral liquid or drops.
10. The application according to claim 4 or 6, characterized in that, When the product is food or beverage, it includes dairy products, dairy beverages, solid beverages, and protein beverages.