Yak milk casein peptide with uric acid-lowering activity and application thereof

Abstract

The application discloses a yak milk casein peptide with uric acid reducing activity and application thereof, and belongs to the technical field of protein peptide development. The yak milk casein peptide with clear uric acid reducing activity is prepared by taking yak milk casein as raw material, and the yak milk casein peptide can inhibit xanthine oxidase activity, reduce uric acid generation, and has good anti-inflammatory activity. The yak milk casein peptide is rich in small molecule active peptides, can be directly absorbed and utilized by the body, has excellent bioavailability, and has good application potential in improving hyperuricemia and related inflammatory damage. The application provides a new raw material source for developing hyperuricemia conditioning functional food and auxiliary intervention.

Description

Technical Field

[0001] This invention relates to the field of protein peptide development technology, and in particular to a yak milk casein peptide with uric acid-lowering activity and its applications. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disease caused by purine metabolism disorder or uric acid excretion impairment. The clinical diagnostic criteria are serum uric acid (SUA) >7.0 mg / dL (approximately 420 μmol / L) in men and >6.0 mg / dL (approximately 360 μmol / L) in women. HUA is not only a direct cause of gouty arthritis, uric acid kidney stones, and chronic kidney disease, but also an independent risk factor for hypertension, cardiovascular disease, and other conditions, making it a global public health issue. Long-term high uric acid levels increase the risk of gout and various metabolic diseases, harming human health. The core pathogenesis of HUA is an imbalance between uric acid production and excretion: uric acid is the end product of purine metabolism, and its production depends on the catalysis of xanthine oxidase (XOD). Genetic factors, high-purine diets, alcohol consumption, and high fructose intake can enhance XOD activity, leading to excessive uric acid production. Approximately 90% of cases originate from renal excretion disorders (abnormal function of transport proteins such as URAT1 and GLUT9), and abnormal intestinal excretion also contributes to its pathogenesis.

[0003] Currently, clinical treatment for hyperuricemia (HUA) primarily relies on medication, mainly including xanthine oxidase inhibitors (allopurinol, febuxostat) and uricosuric agents (probenecid). Allopurinol can inhibit XOD activity and reduce uric acid synthesis, but it can cause gastrointestinal discomfort, rashes, and even severe allergic reactions. Febuxostat has a high uric acid-lowering efficiency, but it may increase the risk of cardiovascular events, and long-term use of chemical drugs can increase the burden on liver and kidney function, limiting its application in specific populations. Given the limitations of chemical drugs, the development of highly effective and low-toxicity naturally derived uric acid-lowering functional factors has become a research hotspot. Bioactive peptides, due to their small molecular weight, easy absorption, strong targeting, and safety, have broad application prospects in metabolic regulation. Studies have confirmed that bioactive peptides prepared from dietary proteins such as tuna, walnuts, and milk have clear anti-HUA activity, exerting their effects by inhibiting XOD activity, regulating uric acid transport proteins, and improving gut microbiota. Their efficacy is close to that of chemical drugs with fewer adverse reactions, providing theoretical and technical support for the development of functional uric acid-lowering products. Summary of the Invention

[0004] The purpose of this invention is to provide a yak milk casein peptide with uric acid-lowering activity and its application, thereby addressing the problems existing in the prior art. This invention prepares a yak milk casein protein hydrolysate with definite uric acid-lowering activity using yak milk casein as raw material. It can inhibit xanthine oxidase activity, reduce uric acid production, and also possesses good anti-inflammatory activity. The yak milk casein protein hydrolysate is rich in small molecule active peptides, which can be directly absorbed and utilized by the body, exhibiting excellent bioavailability and showing good application potential in improving hyperuricemia and related inflammatory damage.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a yak milk casein hydrolysate with uric acid-lowering activity, wherein the yak milk casein hydrolysate contains at least one of the following peptides with the amino acid sequence shown: NQFLPYPY, GPVRGPFPII, HPHPHLSFM, GPVRGPFPIIV, LNENLLRFF, FFVAPFPEVF, PFPKYPVEPF, FYPELFRQF, ARHPHPHLSFM, LPLPLLQSWMH, GPVRGPFPIIVSL, QPHQPLPPTVMFPPQ, QEPVLGPVRGPFPIIV, LPLPL, LPLPLL, YPFPGPI, YPFPGPIP, and GPVRGPFPI.

[0006] The present invention also provides a method for preparing the above-mentioned yak milk casein protein hydrolysate, comprising the steps of pretreating yak milk casein and adding a complex enzyme for enzymatic hydrolysis; The complex enzyme is composed of flavor protease and alkaline protease in a mass ratio of (0.5-2):(0.5-2); The enzymatic hydrolysis was performed at a temperature of 55-65℃ for 4-6 hours, with a pH of 6.8-7.2.

[0007] Optionally, the pretreatment involves preheating at 55-65°C for 0.5-2 hours.

[0008] Optionally, the amount of the compound enzyme added is 4%-8%.

[0009] This invention also provides a yak milk casein peptide with uric acid-lowering activity, wherein the yak milk casein peptide comprises at least one of the following amino acid sequences: NQFLPYPY, GPVRGPFPII, HPHPHLSFM, GPVRGPFPIIV, LNENLLRFF, FFVAPFPEVF, PFPKYPVEPF, FYPELFRQF, ARHPHPHLSFM, LPLPLLQSWMH, GPVRGPFPIIVSL, QPHQPLPPTVMFPPQ, QEPVLGPVRGPFPIIV, LPLPL, LPLPLL, YPFPGPI, YPFPGPIP, and GPVRGPFPI.

[0010] The present invention also provides the use of the above-mentioned yak milk casein hydrolysate or the above-mentioned yak milk casein peptide in the preparation of products with uric acid-lowering activity.

[0011] The present invention also provides the use of the above-mentioned yak milk casein hydrolysate or the above-mentioned yak milk casein peptide in the preparation of products that help maintain healthy blood lipid levels.

[0012] The present invention also provides the use of the above-mentioned yak milk casein hydrolysate or the above-mentioned yak milk casein peptide in the preparation of anti-inflammatory products.

[0013] The present invention also provides the use of the above-mentioned yak milk casein hydrolysate or the above-mentioned yak milk casein peptide in the preparation of products that inhibit xanthine oxidase.

[0014] The present invention also provides a product having uric acid-lowering activity, wherein the product contains the above-mentioned yak milk casein hydrolysate or the above-mentioned yak milk casein peptide.

[0015] The present invention discloses the following technical effects: This invention prepares yak milk casein protein hydrolysate and yak milk casein peptides with definite uric acid-lowering activity using yak milk casein as raw material. The core mechanism of action is the inhibition of xanthine oxidase (XOD) activity, reducing uric acid production. The yak milk casein protein hydrolysate is rich in bioactive peptides, with molecular weights mainly below 1500 Da, exhibiting typical characteristics of small peptides. These peptides can be directly absorbed and utilized by the body, with significantly higher bioavailability than natural yak milk casein. These bioactive peptides not only lower uric acid by regulating purine metabolism disorders but also possess good anti-inflammatory activity. They can synergistically improve the pathological symptoms of hyperuricemia through multiple targets and pathways, giving the yak milk casein protein hydrolysate both uric acid-lowering and anti-inflammatory biological functions, demonstrating good application potential in improving hyperuricemia and related inflammatory damage.

[0016] This invention relates to yak milk casein protein hydrolysate, which uses yak milk casein as a raw material. This facilitates the high-value utilization of specialty livestock by-products, increases the added value of dairy product processing, and aligns with the requirements of efficient resource utilization and green, low-carbon development. The yak milk casein protein hydrolysate is a natural bioactive component, free of chemical drug components, and lacks the toxic side effects of traditional uric acid-lowering drugs. It exhibits good safety and can be used as a functional ingredient in the development of functional foods, dietary supplements, and adjuvant improvement formulations, providing a safe and long-lasting health intervention solution for people with hyperuricemia and those in a sub-healthy state. This invention not only provides a novel, safe, and convenient intervention strategy for patients with hyperuricemia but also contributes to the high-value utilization of agricultural by-products, aligning with the concept of green and sustainable development. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The results show the characteristic analysis of different protease hydrolysates; where A is peptide content; B is peptide yield; C is XOD inhibition rate; and D is the IC50 of XOD. 50 ; Figure 2 The results show the activity and stability evaluation of YMCH; where A represents thermal stability; B represents pH stability; C represents gastrointestinal digestibility stability; and D represents ionic stability. Figure 3 The identification results of peptide sequences of YMCH are shown below; where A represents the amino acid sequence of the enzymatic hydrolysate identified by LC-MS / MS technology; B represents the statistical results of peptide content with different amino acid counts; C represents the statistical results of peptide counts with different molecular weights; and D represents the predicted results of peptides with a bioactivity score >0.8. Figure 4 This describes the docking of peptide YPFPGPI with xanthine oxidase. Figure 5 This describes the docking of peptide YPFPGPIP with xanthine oxidase. Figure 6 This describes the docking of peptide ARHPHPHLSFM with xanthine oxidase. Figure 7 This describes the docking of peptide GPVRGPFPIIVSL with xanthine oxidase. Figure 8 This describes the docking of peptide NQFLPYPY with xanthine oxidase. Figure 9The results are from molecular dynamics simulations; where A represents the RMSD analysis results of ligands in different complexes; B represents the RMSD analysis results of different complexes; C represents the RMSF analysis results of proteins in different complexes; D represents the Rg analysis results of different complexes; E represents the hydrogen bond number analysis results of different complexes; F represents the SASA analysis results of different complexes; G represents the residue energy decomposition results of the XOD / GPVRGPFPI system; H represents the residue energy decomposition results of the XOD / NQFLPYPY system; I represents the residue energy decomposition results of the XOD / YPFPGPI system; J represents the 2D and 3D morphological diagrams of the Gibbs free energy of the XOD / GPVRGPFPI system; K represents the 2D and 3D morphological diagrams of the Gibbs free energy of the XOD / NQFLPYPY system; and L represents the 2D and 3D morphological diagrams of the Gibbs free energy of the XOD / YPFPGPI system. Figure 10 Here is the UV absorption chromatogram of YMCH; Figure 11 The molecular weight distribution of YMCH; Figure 12 The effects of YMCH on the physiological and biochemical properties of mice in each group were shown; where A represents the liver index; B represents the kidney index; C represents liver XOD activity; D represents kidney XOD activity; E represents serum creatinine level; F represents serum blood urea nitrogen level; and G represents serum UA level. Figure 13 The effects of YMCH on the levels of Casp-3, IL-1β, and NLRP3 in the kidneys and livers of mice in each group were investigated. A represents the liver Casp-3 level; B represents the kidney Casp-3 level; C represents the liver IL-1β level; D represents the kidney IL-1β level; E represents the liver NLRP3 level; and F represents the kidney NLRP3 level. Figure 14 The effect of YMCH on lipid levels in each group of mice; where A represents TG content; B represents TC content; C represents HDL content; and D represents LDL content. Figure 15 H&E slice images of the kidneys of mice in each group; Figure 16 Images of H&E sections of the livers of mice in each group. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1 I. Experimental Methods 1. Preparation of yak milk casein protein hydrolysate Yak milk casein was dispersed in deionized water at a material-to-liquid ratio of 1:15 (g / mL) and preheated at 60℃ for 1 h. The pH of the system was adjusted to 8.0 using 0.1 mol / L NaOH and HCl solutions. Enzyme was added at a ratio of 5% (w / w) for enzymatic hydrolysis; specific enzymes and hydrolysis conditions are shown in Table 1. During hydrolysis, the pH of the system was maintained by adding 0.1 mol / L NaOH or HCl solutions dropwise. After hydrolysis, the reaction solution was heated in a 95℃ water bath for 10 min to inactivate the enzyme, cooled to room temperature, and the pH was adjusted to neutral. Subsequently, the solution was centrifuged at 4000 r / min for 15 min, and the supernatant was collected and stored at -20℃ for later use.

[0025]

[0026] 2. Peptide content determination The peptide content in the enzymatic hydrolysate was determined using the o-phthalaldehyde (OPA) method: the enzymatic hydrolysate and OPA reagent were thoroughly mixed at a volume ratio of 1:40 (v / v), and incubated at 25°C in the dark for 8 min. The absorbance of the system was then measured at 340 nm using a microplate reader. A standard curve for peptide content was established using glutathione (GSH) as a standard, and the regression equation obtained was: y = 0.553x + 0.0049 (R²). 2 =0.999), calculate the peptide content in the sample based on the standard curve. And calculate the degree of hydrolysis of the sample using the following formula: .

[0027] 3. Determination of xanthine oxidase inhibition rate Xanthine solutions with concentrations of 0.06 mg / mL and 0.05 U / mL xanthine oxidase solutions were prepared using 50 mmol / L, pH 7.4 phosphate buffer. 50 μL of sample solution (the blank group was replaced with an equal volume of buffer) was mixed thoroughly with 50 μL of xanthine oxidase solution and incubated at 37℃ for 30 min. Then, 150 μL of 0.06 mg / mL xanthine solution was added to initiate the enzymatic reaction. Immediately after the reaction mixture was added, the absorbance was measured at 292 nm using a multi-functional microplate reader. The experiment included a sample group, a sample blank group, a control group, and a blank group. The sample addition details for each group are shown in Table 2. The xanthine oxidase inhibition rate was calculated using the following formula: .

[0028]

[0029] 4. Stability Evaluation The lyophilized enzymatic hydrolysate was reconstituted with distilled water to a concentration of 1 mg / mL, and its efficacy was investigated at different pH values ​​(3, 5, 7, 9, 11), temperatures (room temperature, 37, 60, 80, 100℃), and ionic strengths (Ca). 2+ K + Na + Xanthine oxidase inhibitory activity was measured under conditions of 0, 20, 40, 60, 80, and 100 mmol / L. The simulated gastrointestinal digestion method was based on existing literature and slightly modified, as follows: Pepsin digestion: The pH of the peptide solution was adjusted to 2.0 with 1 mol / L hydrochloric acid, 0.2 mg of pepsin was added, and the mixture was incubated at 37 °C and 200 r / min for 2 h. The reaction was then terminated by adding 0.2 mg of pepsin inhibitor.

[0030] Trypsin digestion: The pH of the peptide solution was adjusted to 7.5 with 1 mol / L sodium hydroxide, 0.2 mg of trypsin was added, and the mixture was incubated at 37 °C and 200 r / min for 2 h. The reaction was terminated by adding 0.2 mg of trypsin inhibitor.

[0031] Simulated gastrointestinal sequential digestion: First, adjust the pH of the peptide solution to 2.0 with 1 mol / L hydrochloric acid, add 0.2 mg of pepsin, and incubate at 37℃ and 200 r / min for 1 h. Then, add a pepsin inhibitor to terminate the reaction. Next, adjust the pH of the system to 7.5 with 1 mol / L sodium hydroxide, add 0.2 mg of trypsin, and continue incubating at 37℃ and 200 r / min for 1 h. Finally, add a trypsin inhibitor to terminate the reaction.

[0032] After each group was treated, the xanthine oxidase inhibitory activity was measured according to the formula for calculating the xanthine oxidase inhibition rate, and the effect of the simulated digestion process on the stability of the inhibitory peptide was evaluated.

[0033] II. Test Results 1. Activity determination of yak milk casein protein hydrolysate Yak milk casein hydrolysate was obtained by enzymatic hydrolysis using flavor protease, alkaline protease, pepsin, trypsin, and a combination of flavor and alkaline protease. Peptide yield, peptide content, and xanthine oxidase inhibition rate were then determined.

[0034] Depend on Figure 1 As shown in A, the peptide content of the five enzymatic hydrolysates was determined by the OPA method, and the flavor and alkaline compound protease had the highest peptide content, at 74.1 ± 1.9%. Figure 1 As can be seen from B, the protease with both flavor and alkalinity exhibits the highest peptide yield, at 91.03%. Figure 1 As shown in C and D, the flavor and alkaline complex protease exhibits the best inhibition rate against xanthine oxidase (XOD), with an IC50 value of [missing value]. 50 The concentration was 1.06 ± 0.1 mg / mL. Considering the peptide content, yield, and activity, the product prepared from the flavor- and alkaline protease was ultimately determined to be the optimal enzymatic hydrolysate, and named YMCH.

[0035] 2. Stability Evaluation Thermal stability studies showed that heating from room temperature to 80°C had no significant effect on the inhibitory activity of the xanthine oxidase inhibitory peptide, which remained stable at high temperatures, but its activity decreased at 100°C. Figure 2 (A). pH is a key factor affecting peptide activity. In a strongly acidic environment (pH 3), the inhibition rate of xanthine oxidase drops to 46.48%, and in a strongly alkaline environment (pH 11), the inhibition rate of xanthine oxidase also decreases accordingly. This may be due to conformational changes in the peptide structure. Figure 2(B). Gastrointestinal digestive stability experiments showed that after 2 hours of pepsin treatment, the inhibition rate decreased to 44.13%, possibly due to the exposure of hydrophobic groups by enzymatic hydrolysis, thus weakening the activity; after 2 hours of trypsin treatment, the inhibition rate decreased to 44.44%, most likely due to the degradation of key bioactive peptide fragments. However, after multiple gastrointestinal digestion treatments, the inhibition rate remained at 40.95%. Figure 2 The presence of C in the hydrolysate indicates that the urate-lowering peptides in this enzymatic hydrolysate may possess good stability in vivo. Conversely, Ca... 2+ K + Na + Increased concentration leads to a decrease in inhibition rate, which may be attributed to changes in peptide structure. Figure 2 (D). Overall, the peptides in the enzymatic hydrolysate exhibited good stability during processing and digestion. However, to maintain optimal xanthine oxidase inhibitory activity, exposure to highly acidic or high-salt environments should be avoided as much as possible.

[0036] Example 2: Peptide sequence identification and screening of yak milk casein hydrolysate I. Experimental Methods 1. Identification of polypeptide sequences Bioactive peptides in yak milk caseinate hydrolysates were separated and identified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). 38 mg of sample was accurately weighed and dissolved in 0.25 mL of 0.1% (m / m) trifluoroacetic acid solution. After thorough vortexing, the solution was centrifuged at 4 °C and 14000 × g for 5 min. The supernatant was transferred to an ultrafiltration tube and centrifuged for another 15 min at the same temperature and centrifugal force. The filtrate was collected and concentrated by freeze-drying. The freeze-dried sample was reconstituted with 30 μL of 0.1% trifluoroacetic acid solution, vortexed for 1 min, and centrifuged. The supernatant was used for LC-MS / MS analysis. Liquid chromatography conditions: A chromatographic column with an inner diameter of 0.15 mm, a length of 150 mm, and a packing particle size of 3 μm was used; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid-acetonitrile solution; the gradient elution program was as follows: 0-50 min, phase B linearly increased from 4% to 50%; 50-54 min, phase B increased to 100%; 54-60 min, phase B maintained constant elution at 100%. Mass spectrometry was performed using electrospray ionization in positive ion mode. Based on the first-stage full scan, the top 10 ions by intensity were selected to collect secondary fragment mass spectra, and peptide sequence analysis was performed using mass-to-charge ratio information.

[0037] 2. Activity prediction and molecular docking Activity was predicted using the Peptides Bioactive database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ); the activity of the screened peptides was scored using the Peptide Activity Database (http: / / distilldeep.ucd.ie / PeptideRanker / ); peptide sequence toxicity was predicted using the Virtual database (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php); and peptide sequence solubility was calculated using the peptide parameter calculator (https: / / www.allpeptide.com / canshu.html).

[0038] 3. Molecular dynamics simulation Using the peptide-protein complex obtained through molecular docking as the initial structure, all-atom molecular dynamics simulations were performed using AMBER 24 software. The preprocessing steps before simulation were as follows: both peptides and proteins were described using the ff14SB protein force field; hydrogen atoms were added to each system using the LEaP module of the software, and the distance from the system was... Add a truncated octahedral TIP3P solvent box to the system, and simultaneously add Na to the system. + / Cl - After balancing the system charge and completing the preprocessing, the topology and parameter files are output for subsequent molecular dynamics simulations.

[0039] II. Test Results 1. Identification and screening of target peptide sequences To analyze the active components in yak milk casein protein hydrolysates, LC-MS / MS was performed for identification. Bioinformatics methods were used to analyze 778 peptides identified in the yak milk casein protein hydrolysates, with 416 peptides having a score >100 and 20 peptides having a PeptideRanker score >0.8. Detailed statistical analysis was conducted based on molecular weight distribution, types of active peptides, peptide bioactivity scores, and peptide lengths to gain a deeper understanding of the functional peptides in yak milk casein. The protein sources were identified as mainly β-casein, α-S1-casein, α-S2-casein, and κ-casein, accounting for 36.8%, 26.7%, 22%, and 14.5%, respectively. Figure 3 A). For example Figure 3 As shown in B, it is mainly composed of 6-10 amino acids, with a total of 445 peptides in this range, accounting for 57.2% of the total peptides; Figure 3From C, we can see that there is 1 peptide with a molecular weight <0.5kDa, accounting for 0.1% of the total peptides; 328 peptides with a molecular weight of 0.5-1.0kDa, accounting for 42.16% of the total peptides; 281 peptides with a molecular weight of 1.0-1.5kDa, accounting for 24.51% of the total peptides; 128 peptides with a molecular weight of 1.5-2kDa, accounting for 16.45% of the total peptides; 30 peptides with a molecular weight of 2-2.5kDa, accounting for 3.9% of the total peptides; and 10 peptides with a molecular weight >2.5kDa, accounting for 1.3% of the total peptides. Figure 3 As indicated by D, the bioactivity of peptides was scored using the Peptide Ranker database, with 18 peptides having a score >0.8. These peptides with bioactivity scores >0.8 were mainly concentrated in the 1.0-1.5 kDa range. Furthermore, based on the characteristics of urate-lowering peptides and comparison with unreported bioactive peptides in the BIOPEP-UWM database, 18 peptides with potential xanthine oxidase activity were selected (see Table 3).

[0040]

[0041] 2. Investigating the interaction between yak milk casein peptides and xanthine oxidase based on molecular docking. Molecular docking technology can accurately identify potential ligand binding sites on the surface of receptor proteins, deeply analyze the binding affinity and interaction mechanism between ligands and receptor molecules, and provide solid theoretical and technical support for the rapid screening, activity evaluation, and mechanism of action research of active substances by simulating the binding process at the molecular level. Molecular docking results showed that all five peptides could form numerous hydrogen bonds and hydrophobic interactions with xanthine oxidase (XOD). Hydrogen bonds are key to their specific recognition, while hydrophobic interactions stabilize the binding of the peptide to the XOD active region. Together, they provide the molecular basis for the peptide to inhibit XOD activity and reduce uric acid production. Generally, the lower the receptor-ligand binding score, the stronger the binding ability. Based on the binding energy, the five peptides with the most XOD activity (YPFPGPI, YPFPGPIP, ARHPHPHLSFM, GPVRGPFPIIVSL, NQFLPYPY) were selected, with docking scores of -7.7, -7.1, -6.624, -6.91, and -7.623 kcal / mol, respectively. Figure 4As shown in Table 4, YPFPGPI forms hydrogen bonds with amino acid residue Ser876; hydrophobic interactions with Ala1079, Pro1012, His875, Leu1014, Phe1013, Phe649, and Leu648; a salt bridge with Glu802; a π-σ interaction with Val1011; and π-π stacking interactions with Phe914 and Phe1009, filling the hydrophobic region of the XOD active pocket, reducing the hydrophobic interface between the ligand and the enzyme, thereby enhancing activity. Figure 5 It is known that YPFPGPIP forms two hydrogen bonds with ligand target proteins Ser876 and Phe1142, three hydrogen bonds with ligand target proteins Phe649, Leu648, and Pro1012, and a π-σ interaction with Leu1014. From Figure 6 It is known that ARHPHPHLSFM forms hydrogen bonds with amino acids Ser876, Phe1013, Glu879, and Thr1010, hydrophobic interactions with Pro1012, Phe649, Leu1014, and Leu712, and van der Waals forces with Ser710, thus enhancing the ligand-receptor binding affinity. Figure 7 It is known that GPVRGPFPIIVSL forms hydrogen bonds with ligand target proteins Asn650, Lys771, Gly647, Gln1016, Phe1142, and Arg871, hydrophobic interactions with Phe1013, Phe883, Leu648, Tyr1140, Leu712, and Pro1012, and π-σ interactions with Val1011 and Phe649, enhancing the ligand's localization accuracy in the pocket and helping to maintain conformation. Figure 8 It is known that NQFLPYPY has hydrogen bonding with ligand's target proteins His875, Ser710, Lys713, and Glu715, and forms hydrophobic interactions with Phe1142 and Val1011.

[0042]

[0043] 3. Molecular dynamics simulation To verify the reliability of the molecular docking results and effectively eliminate false positives, this invention performed a 100 ns molecular dynamics simulation on the screened peptide-enzyme complex and systematically evaluated the overall conformational stability of the complex through root mean square deviation (RMSD) analysis. Figure 9The Ligand RMSD (A) results show that the three peptides rapidly increased in the initial stage of the simulation and reached a relatively stable plateau after about 10-20 ns. The XOD / NQFLPYPY system had the highest average RMSD (0.457±0.095 nm) and maintained it in the 0.45-0.55 nm range after 20 ns, indicating that its conformational fluctuation within the binding pocket was relatively large. The XOD / GPVRGPFPI system had a RMSD of 0.334±0.078 nm, with moderate fluctuations. The XOD / YPFPGPI system had the lowest RMSD (0.304±0.068 nm) and was the most stable overall. None of the three systems showed persistent drift, indicating that the binding did not dissociate, but the stability ranking was YPFPGPI > GPVRGPFPI > NQFLPYPY.

[0044] Figure 9 The B (Complex RMSD) reflects the overall structural stability of the complex. After rapidly reaching equilibrium within the first 10 ns, the three systems maintained a range of 0.19–0.26 nm. The XOD / GPVRGPFPI system had the lowest average value (0.203 ± 0.014 nm) and the smallest fluctuation, indicating the weakest perturbation to the protein backbone; XOD / YPFPGPI had a value of 0.209 ± 0.023 nm; and XOD / NQFLPYPY had the highest value (0.240 ± 0.023 nm), suggesting a slightly larger impact on the overall structure. Overall, all three complexes maintained good conformational stability.

[0045] Figure 9 The C-squared (RMSF) values ​​reveal the flexible distribution of protein residues. The average RMSF values ​​for the three systems were 0.081 ± 0.031 nm (GPVRGPFPI), 0.082 ± 0.034 nm (NQFLPYPY), and 0.084 ± 0.032 nm (YPFPGPI), respectively. Overall fluctuations were concentrated between 0.05 and 0.12 nm, with only a few loop or terminal regions showing higher peaks. The residue fluctuation patterns of the different peptide systems were highly similar, indicating that peptide binding did not induce significant local conformational destabilization.

[0046] Figure 9 The complex Rg (D) reflects the overall compactness of the complex. The three systems initially showed a slight increase in Rg before stabilizing. The XOD / NQFLPYPY system had the lowest average Rg (3.161±0.008 nm), indicating a slightly more compact structure; XOD / YPFPGPI was 3.164±0.016 nm; and XOD / GPVRGPFPI was 3.167±0.008 nm. The overall differences were small and the fluctuations limited, with no observed sustained expansion or collapse trend, indicating that the overall folding state of the complex was stable.

[0047] Figure 9The E (Number of H-bonds) indicates the change in the number of hydrogen bonds between peptides and proteins over time. The XOD / NQFLPYPY system had the highest average number of hydrogen bonds (2.48±1.42), slightly higher than GPVRGPFPI (2.18±1.35) and YPFPGPI (1.89±1.25). However, the overall number of hydrogen bonds in all three systems was low and exhibited dynamic fluctuations, suggesting that hydrogen bonds play a supporting role in stability rather than a dominant one.

[0048] Figure 9 The F (Complex SASA) value shows the trend of the solvent-accessible surface area of ​​the complexes. The three systems initially showed a rapid increase before reaching a stable plateau. XOD / GPVRGPFPI and XOD / YPFPGPI had higher average SASA values ​​(431.80 ± 8.99 nm, respectively). 2 and 431.28±10.56nm 2 XOD / NQFLPYPY is slightly lower (427.05±6.87nm). 2 This suggests that the latter has a slightly more compact overall structure. Overall, all three systems maintained a stable equilibrium in the solvent environment, without exhibiting significant structural loosening.

[0049] As shown in Table 5, the binding energies of XOD_GPVRGPFPI, XOD_NQFLPYPY, and XOD_YPFPGPI are -27.35±6.40, -11.14±6.04, and -7.11±2.79 kcal / mol, respectively. Negative values ​​indicate that these molecules have a binding affinity to the target protein; lower values ​​indicate stronger binding. This result shows that XOD_GPVRGPFPI, XOD_NQFLPYPY, and XOD_YPFPGPI have very strong binding affinity. Energy decomposition reveals that the main contributing factor to the binding of XOD_GPVRGPFPI, XOD_NQFLPYPY, and XOD_YPFPGPI is electrostatic energy, followed by van der Waals energy, and then nonpolar solvation free energy.

[0050] Figure 9 G represents the residue energy decomposition results of the XOD / GPVRGPFPI system. Among them, LEU547 and VAL1217 made the most significant contributions (approximately -4.7 and -4.5 kcal / mol), which are the main hydrophobic energy hotspots, indicating that hydrophobic interactions dominate in this system. PHE1221, PRO1216, and PHE548 also provided stable contributions of -2 to -3 kcal / mol, indicating that aromatic stacking and hydrophobic synergy jointly stabilize the binding interface, and the overall system exhibits a multi-residue dispersed contribution characteristic.

[0051] Figure 9The H in the XOD / NQFLPYPY system shows that GLN1216 contributes the most (approximately -3.3 kcal / mol), indicating that polarity or hydrogen bonding is more important in this system. LEU1218 and TYR1220 also provide moderate negative energy, while the contributions of the remaining residues are relatively weak and evenly distributed. Overall, the contribution of a single residue is lower than that of the GPVRGPFPI system, suggesting that its binding depends on multi-site synergy rather than strong hot spots.

[0052] Figure 9 The I-type is the XOD / YPFPGPI system, with PRO1216 being the most contributing residue (approximately -4.6 kcal / mol). LEU772 and PHE912 also have significant negative energy contributions, indicating that hydrophobic and aromatic interactions play a central role in this system. The contributions of the remaining residues gradually decrease, and the energy distribution is relatively concentrated but the overall amplitude is moderate, indicating that the peptide maintains binding stability through a local key hydrophobic core.

[0053] Figure 9 The free energy landscape (FEL) of the three systems in the JL model exhibits a single, deep energy well, indicating that the systems are mainly distributed in a stable low-energy conformation region during the simulation. The RMSD and RoG values ​​corresponding to the lowest energy region have a narrow range of variation, indicating that the conformational fluctuations of the system are small during the equilibrium phase. After the acceptor and ligand bind, the system maintains a compact and stable conformational state without obvious energy transitions or multiple metastable states. These results demonstrate that the three complexes possess high conformational stability in the simulated system.

[0054]

[0055] 4. Molecular weight distribution Depend on Figure 10 and Figure 11 It was found that the molecular weight distribution of yak milk casein enzymatic hydrolysates can be divided into seven segments, including >10kDa, 5-10kDa, 3-5kDa, 2-3kDa, 1-2kDa, 0.5-1kDa, and ≤0.5kDa. The molecular weight is mainly distributed in the ≤0.5kDa range, followed by 5-10kDa, 3-5kDa, and 1-2kDa, with >10kDa components being less common. This trend indicates that during enzymatic hydrolysis, larger molecular weight proteins in yak milk are gradually degraded into long-chain peptides, short-chain peptides, and even free amino acids. Simultaneously, the already generated long-chain peptides can be further cleaved by proteases, generating more small-molecule peptides.

[0056] 5. Amino acid distribution Table 6 reflects the amino acid composition of the enzymatic hydrolysate. The analysis results show that glutamic acid (Glu) has the highest content, accounting for 18.73%; followed by proline (Pro) 10.48%, leucine (Leu) 9.07%, valine (Val) 6.85%, and tyrosine (Tyr) 6.13%, which is closely related to the original amino acid composition of yak milk casein.

[0057]

[0058] Example 3 Animal Experiment 1. Establishment of a mouse model of hyperuricemia Seventy-two 5-week-old ICR mice (weighing 18-22g) were purchased from Yunnan Besttech Biotechnology Co., Ltd. The ambient temperature was controlled at 24±2℃, and the relative humidity at 50%-60%. All mice underwent acclimatization for one week before the experiment. During the experiment, mice had free access to food and water, and were kept in a 12-hour light / 12-hour dark cycle. After the acclimatization period, the mice were randomly divided into six groups of 12 mice each: normal control group (NC), model control group (Model), positive control group (PO), low-dose yak milk casein hydrolysate group (ML, 50mg / kg·d), medium-dose yak milk casein hydrolysate group (MM, 150mg / kg·d), and high-dose yak milk casein hydrolysate group (MH, 450mg / kg·d). All groups were administered the drugs via gavage at a uniform volume of 10mL / kg. The dosages of the modeling drugs and yak milk casein hydrolysate were determined based on relevant literature reports and previous preliminary experimental results. Every day at 9:00 AM, except for the normal control group, all other groups of mice were administered a modeling drug solution (containing 200 mg / kg·d hypoxanthine and 200 mg / kg·d potassium oxonate, prepared with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution) by gavage for 21 consecutive days. One hour after gavage administration of the modeling drug solution, the positive control group was administered 10 mg / kg·d allopurinol by gavage, the ML, MM, and MH groups were administered the corresponding doses of yak milk casein hydrolysate by gavage, and the normal control group was administered an equal volume of physiological saline by gavage. During the experiment, the mice's mental state, fur color, and other general physical characteristics were observed and recorded daily, and their body weight was measured and recorded regularly.

[0059] 2. HE staining Dewaxing paraffin sections to water: Immerse sections sequentially in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then rinse with tap water. Restoration and fixation of frozen sections: Remove frozen sections from the -20℃ freezer and allow them to reach room temperature. Fix with tissue fixative for 15 min, then rinse with running water. Pretreatment: Immerse sections in high-resolution constant staining pretreatment solution for 1 min.

[0060] Hematoxylin staining: Immerse sections in hematoxylin solution for 3-5 min, rinse with tap water, differentiate with differentiation solution, rinse with tap water, apply blue solution for blue reversion, and rinse with running water. Eosin staining: Dehydrate sections in 95% alcohol for 1 min, then stain with eosin solution for 15 s. Dehydration and mounting: Sequentially immerse sections in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, and xylene. Finally, examine under a microscope and acquire and analyze the images.

[0061] 3. The regulatory effect of YMCH on hyperuricemia To investigate the effects of yak milk casein peptides on hyperuricemia (HUA), a mouse model of HUA induced by hypoxanthine and potassium oxonate was established, and mice were treated with yak milk casein hydrolysate from Example 1 at doses of 50 mg / kg, 150 mg / kg, and 450 mg / kg, respectively. Xanthine oxidase (XOD), a key rate-limiting enzyme in the purine metabolism pathway, catalyzes the oxidation of hypoxanthine and xanthine to produce uric acid. Inhibiting its activity can reduce uric acid production at its source.

[0062] Depend on Figure 12 As shown by the G-test, compared with the NC group (85.14±9.15 μmol / L), the serum UA level in the Model group (204.02±20.85 μmol / L) was significantly higher (P<0.01), proving that the HUA was successfully established. Figure 12 As shown in A, except for the positive drug group, there were no significant differences in liver index among the other groups of mice (p>0.05), indicating that hypoxanthine and potassium oxonate did not cause significant liver damage in the model group, and MH, MM, and ML showed no hepatotoxicity. Figure 12 As shown in Figure B, the kidney index of the model group rats was slightly higher than that of the normal control group (p<0.05). After MH administration by gavage, kidney damage in mice was significantly improved. In conclusion, MH, MM, and ML have no adverse effects on the liver.

[0063] Given that uric acid is primarily synthesized in the liver and excreted through the kidneys, this invention simultaneously detected the activity levels of XOD in rat liver and kidney tissues. Figure 12The CD results showed that, compared with the normal control group (NC), the XOD activity in the model group (MC) rats was significantly increased (P < 0.05); while all doses of YMCH and the positive control group (PO) significantly downregulated XOD activity. Different doses of YMCH showed significant inhibitory effects on XOD, and the inhibitory effect was similar to that of the positive control group; simultaneously, the XOD activity in the YMCH-treated groups was significantly lower than that in the normal control group. These results indicate that YMCH can effectively inhibit XOD activity in vivo, thereby reducing serum uric acid concentration in hyperuricemia model rats by reducing uric acid biosynthesis. Serum creatinine and blood urea nitrogen are important indicators for assessing renal function; elevated levels usually indicate impaired renal function, and the degree of elevation is closely related to the degree of renal damage. Figure 12 The EF (effects) analysis showed that serum creatinine and blood urea nitrogen levels in the model group mice were significantly higher than those in the normal control group, indicating that the combination of hypoxanthine and potassium oxonate successfully induced kidney damage in mice. Compared with the model group, the low, medium, and high dose groups, as well as the positive control group, all significantly reduced creatinine and blood urea nitrogen levels; among them, the high-dose YMCH group showed a similar downregulation effect on creatinine and blood urea nitrogen as the positive control group, and some indicators were even better than those of the positive control group, suggesting that this peptide has a good renal protective effect.

[0064] Compared with the NC group (85.14±9.15 μmol / L), the serum UA level in the Model group (204.02±20.85 μmol / L) was significantly increased (P<0.01), confirming the successful establishment of HUA. The serum UA level in the PO group (88.87±11.59 μmol / L) was significantly lower than that in the Model group (P<0.05). After oral administration of 50 mg / kg, 150 mg / kg, and 450 mg / kg, the uric acid levels in mice were 100.47±4.11 μmol / L, 96.38±1.27 μmol / L, and 90.93±6.83 μmol / L, respectively, and the UA levels decreased by 50.98%, 52.94%, and 55.88%, respectively, showing a dose-dependent relationship. Figure 12 (G).

[0065] 4. The regulatory effect of YMCH on inflammatory factors Hyperuricemia is often accompanied by renal inflammation. Elevated uric acid levels can activate caspase-associated inflammasomes, causing cell membrane damage and releasing large amounts of inflammatory factors, ultimately leading to chronic renal inflammation. The caspase family plays a central role in the regulation of apoptosis, especially caspase-3, which, upon activation, can further initiate downstream apoptosis-related proteases. The NLRP3 inflammasome is a key component of the body's innate immune system, which can be activated by various danger signals and participates in the body's immune response and the development of various diseases. IL-1β, as an important pro-inflammatory cytokine, not only participates in autoimmune inflammatory responses and cell proliferation, differentiation, and apoptosis, but also plays a role in neuroprotection and tissue remodeling and repair; its abnormally high expression is closely related to the pathophysiological processes of various diseases, playing an important role in acute and chronic inflammation and autoimmune diseases.

[0066] like Figure 13 As shown in Figures AB, the levels of Caspase-3 in the kidney and liver tissues of the model group mice were significantly increased. After YMCH intervention, the high-dose group (MH) had lower Caspase-3 levels than the model group, while the low-dose (ML) and medium-dose (MM) groups showed only slight improvement trends. The positive control group (PO) also showed a significant inhibitory effect. Impaired cell membrane integrity can trigger the release of a large number of inflammatory factors. Figure 13 As observed in CD, the IL-1β level in the model group was significantly higher than that in the normal control group; after YMCH intervention, all dose groups could reduce IL-1β levels, with the high-dose group (MH) showing a significantly better inhibitory effect than the low- and medium-dose groups. Figure 13 As shown in the EF, the inflammasome NLRP3 level in the model group was significantly higher than that in the normal control group. After drug treatment, the expression of NLRP3 was downregulated in the medium- and high-dose YMCH groups and the positive control group, with the high-dose group (MH) showing the most significant effect.

[0067] The combined results of the above three inflammatory factor detections show that the expression of Caspase-3, IL-1β, and NLRP3 in hyperuricemia model mice were significantly upregulated, indicating that the model mice had undergone significant renal inflammation. The inhibitory effect of different doses of YMCH on inflammatory factors was dose-dependent: only high-dose YMCH significantly inhibited NLRP3 expression; while low, medium, and high doses of YMCH all significantly inhibited Caspase-3 and IL-1β, with the highest dose showing the best overall effect. Although the commonly used clinical drug PO has certain therapeutic effects, it suffers from limited anti-inflammatory effects and accompanying toxic side effects. Therefore, developing safe and non-toxic peptides with both uric acid-lowering and anti-inflammatory activities has significant research value and application prospects. In conclusion, YMCH exhibits excellent uric acid-lowering and anti-inflammatory potential, possessing good development value and application prospects. These results can provide experimental evidence and theoretical support for the subsequent development and utilization of this active peptide.

[0068] 5. Effects of YMCH on lipids in HUA mice The lipid metabolism disorder induced by hyperuricemia can be directly reflected through serum lipid indicators. For example... Figure 14 As shown, compared with the normal control group, the serum levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in the model group mice were significantly increased. After allopurinol treatment, the synthesis of TG, TC, HDL, and LDL in hyperuricemic mice decreased, while their excretion increased, resulting in a significant improvement in blood lipid levels. Compared with the model group, low, medium, and high doses of YMCH all downregulated the levels of TG, TC, HDL, and LDL, showing a clear dose-dependent improvement trend; the high-dose YMCH group showed the most significant regulatory effect, with all blood lipid indicators approaching the levels of the normal control group. These results indicate that YMCH can effectively regulate blood lipid levels in hyperuricemic mice and significantly improve lipid metabolism disorders induced by hyperuricemia.

[0069] 6. Effects of YMCH on the kidneys and liver Hematoxylin and eosin (H&E) staining is a classic and widely used method in histopathological studies, allowing for clear observation of cell morphology, tissue structure, and the degree of pathological damage. Hyperuricemia can lead to various kidney diseases, mainly including asymptomatic uric acid nephropathy, urate stones, acute and chronic uric acid nephropathy, and in severe cases, it can progress to chronic renal failure. In renal tissue pathological sections, the typical manifestation is inflammatory cell infiltration; as the disease progresses, a series of pathological changes may also occur, such as glomerular basement membrane thickening, glomerular fibrosis, renal tubular degeneration and atrophy, and renal interstitial vascular dilation. Figure 15As can be seen, the renal tissue in the NC group was clear and intact, with cells arranged in a tight and orderly manner. Compared with the NC group, the MC group showed further aggravation of renal function damage due to tubular dilation, vacuolar degeneration, and inflammatory infiltration. This section indicates that the hyperuricemia model successfully induced severe renal pathological damage. Compared with the MC group, the tubular dilation was basically restored to normal, vacuolar degeneration was significantly reduced, cell nucleus morphology was normal, and inflammatory cells were reduced, showing a significant difference from the model group, verifying the effectiveness of the model and providing a reliable positive reference for the renal protection of YMCH. The positive drug successfully reversed the severe renal damage induced by hyperuricemia, but it also damaged the renal tissue to some extent. After drug intervention, different doses of YMCH could improve the renal tissue pathological damage to varying degrees. Among them, ML showed glomerular atrophy, inflammatory cell infiltration, and tubular dilation, which was chronic tubulointerstitial nephritis with glomerular damage, and the degree of inflammation and tubular dilation was obvious; MM showed mild renal damage with tubular dilation as the main feature, and the inflammation and glomerular damage were relatively mild; MH showed no obvious abnormalities. In summary, YMCH can effectively improve kidney damage associated with hyperuricemia and is a functional factor with potential for development in treating hyperuricemia.

[0070] In addition, elevated uric acid (UA) levels in the body can also induce oxidative stress damage to the liver. Figure 16 In the NC group, the livers of mice were normal, with neatly arranged cells and abundant cytoplasm. In the Model group, hepatocyte edema and degeneration were observed, along with disordered liver structure, dilation and compression / occlusion of sinusoids in some areas, liver inflammation, inflammatory cell infiltration (lower left corner), inflammatory cell aggregation, hepatocyte necrosis, and impaired metabolic function. The positive control group (PO) showed mild inflammatory infiltration and sinusoidal dilation, but the degree was significantly reduced. No abnormalities were observed in the medium-dose MM and high-dose MH, indicating that medium and high doses of YMCH can improve liver damage in hyperuricemic mice.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A yak milk casein protein hydrolysate with uric acid-lowering activity, characterized in that, The yak milk casein hydrolysate contains at least one of the following peptides with the amino acid sequence shown: NQFLPYPY, GPVRGPFPII, HPHPHLSFM, GPVRGPFPIIV, LNENLLRFF, FFVAPFPEVF, PFPKYPVEPF, FYPELFRQF, ARHPHPHLSFM, LLPLLQSWMH, GPVRGPFPIIVSL, QPHQPLPPTVMFPPQ, QEPVLGPVRGPFPIIV, LPLPL, LLPLL, YPFPGPI, YPFPGPIP, and GPVRGPFPI.

2. A method for preparing the yak milk casein hydrolysate according to claim 1, characterized in that, This includes the steps of pretreating yak milk casein and then adding a complex enzyme for enzymatic hydrolysis; The complex enzyme is composed of flavor protease and alkaline protease in a mass ratio of (0.5-2):(0.5-2); The enzymatic hydrolysis was performed at a temperature of 55-65℃ for 4-6 hours, with a pH of 6.8-7.

2.

3. The preparation method according to claim 2, characterized in that, The pretreatment involves preheating at 55-65℃ for 0.5-2 hours.

4. The preparation method according to claim 2, characterized in that, The amount of the compound enzyme added is 4%-8%.

5. A yak milk casein peptide with uric acid-lowering activity, characterized in that, The yak milk casein peptides include at least one of the following amino acid sequences: NQFLPYPY, GPVRGPFPII, HPHPHLSFM, GPVRGPFPIIV, LNENLLRFF, FFVAPFPEVF, PFPKYPVEPF, FYPELFRQF, ARHPHPHLSFM, LPLPLLQSWMH, GPVRGPFPIIVSL, QPHQPLPPTVMFPPQ, QEPVLGPVRGPFPIIV, LPLPL, LPLPLL, YPFPGPI, YPFPGPIP, and GPVRGPFPI.

6. The use of the yak milk casein hydrolysate of claim 1 or the yak milk casein peptide of claim 5 in the preparation of products with uric acid-lowering activity.

7. The use of the yak milk casein hydrolysate of claim 1 or the yak milk casein peptide of claim 5 in the preparation of products that help maintain healthy blood lipid levels.

8. The use of the yak milk casein hydrolysate of claim 1 or the yak milk casein peptide of claim 5 in the preparation of anti-inflammatory products.

9. The use of the yak milk casein protein hydrolysate of claim 1 or the yak milk casein peptide of claim 5 in the preparation of a product that inhibits xanthine oxidase.

10. A product with uric acid-lowering activity, characterized in that, The product contains either the yak milk casein hydrolysate of claim 1 or the yak milk casein peptide of claim 5.

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