Mussel peptide and application thereof in preparation of uric acid reducing product

Abstract

The invention relates to the field of preparation and application of biological peptides, in particular to a mussel peptide and application thereof in preparation of uric acid reducing products. The amino acid sequences of the gene are as shown in SEQ ID NO.1-4. The mussel polypeptide is prepared through directional enzymolysis of a compound enzyme and a membrane separation and purification technology, and four novel polypeptides with potential biological activity are screened by combining LC-MS / MS analysis and activity prediction. Molecular docking and cell experiments prove that the four polypeptides can specifically inhibit the GLUT9 protein activity and reduce the uric acid level; animal experiments further verify the effect of the mussel in the aspect of reducing uric acid, and a scientific basis is provided for high-value development of mussel resources and development of functional uric acid-reducing products.

Description

Technical Field

[0001] The present invention relates to the field of preparation and application of bioactive peptides, and particularly to a mussel peptide and its application in the preparation of products for reducing uric acid. Background Art

[0002] Hyperuricemia is a metabolic disease characterized by abnormally elevated serum uric acid concentration, and its background involves complex physiological mechanisms and multiple pathogenic factors. Although traditional drugs such as allopurinol and benzbromarone can reduce uric acid, long-term use has problems such as liver and kidney toxicity and gastrointestinal reactions. Although traditional Chinese medicine compounds have relatively high safety, it is difficult to standardize them due to their complex composition and unclear mechanisms. Therefore, there is an urgent need to develop safe and efficient natural food-derived ingredients to alleviate hyperuricemia.

[0003] As low-molecular-weight fragments produced by protein enzymatic hydrolysis, bioactive peptides have the advantages of high safety, multi-target synergy, and resource sustainability, and have attracted much attention in the development of functional foods. Mussel is the most important cultured variety of economic shellfish along the coast of China. Mussels are rich in protein and peptide components. Recent studies have shown that mussel active peptides have shown significant effects in the fields of antibacterial, antioxidant, bone repair, antithrombotic, etc. Chinese Patent CN118496307A discloses a mussel xanthine oxidoreductase inhibitory peptide for treating hyperuricemia, its preparation method and application. The active peptide YQMCGW prepared in this application can significantly reduce the activity of xanthine oxidoreductase and significantly reduce the serum uric acid level, and has the effect of treating hyperuricemia and gout; YQMCGW mainly binds to xanthine oxidoreductase through hydrogen bonds, electrostatic forces, and hydrophobic interactions to inhibit its activity. Therefore, YQMCGW can be developed as a therapeutic drug and adjuvant treatment product for hyperuricemia and gout. However, only one peptide segment cannot meet the huge market demand, and more mussel peptides need to be further developed to meet the diversification of raw materials, and open up a broader market space for the efficient development and utilization of marine shellfish resources. Summary of the Invention

[0004] The technical problem to be solved by the present invention is that the current application research on mussel peptides in reducing uric acid is relatively single, and more mussel peptides need to be further developed to meet the diversification of raw materials, and open up a broader market space for the efficient development and utilization of marine shellfish resources.

[0005] To solve the above problems, the present invention prepares mussel polypeptides through composite enzyme-directed enzymatic hydrolysis and membrane separation and purification technology, and screens out four novel polypeptides with potential biological activities by combining LC-MS / MS analysis and activity prediction. Confirmed by molecular docking and cell experiments, these four polypeptides can specifically inhibit the activity of GLUT9 protein and reduce the uric acid level; animal experiments further verify their efficacy in reducing uric acid, providing a scientific basis for the high-value development of mussel resources and the development of functional uric acid-reducing products.

[0006] To achieve the above object, the present invention is realized by the following technical means: An application of mussel peptides in the preparation of products for reducing uric acid, including polypeptides with amino acid sequences shown in SEQ ID NO.1-4: SEQ ID NO.1: WTFFGA.

[0007] SEQ ID NO.2: PDPFYK.

[0008] SEQ ID NO.3: SPFFKV.

[0009] SEQ ID NO.4: TSPFFK.

[0010] The preparation method of the above-mentioned mussel peptides includes the following steps: Take fresh mussel meat after shelling, add distilled water for homogenization, add 3% compound protease and stir evenly, and adjust the pH value to 7.5 with HCL and NaOH, and hydrolyze at 50 °C for 5 h in a water bath constant temperature oscillator; after enzymatic hydrolysis, inactivate the enzyme, cool, centrifuge at 4 °C and 8000 rpm for 15 min, and collect the supernatant; the supernatant is further ultrafiltered at 0.1 MPa and 25 °C, select an ultrafiltration tube with a cut-off molecular weight of 3 kDa, collect the filtrate, desalt and freeze-dry to obtain mussel peptide powder.

[0011] Furthermore, distilled water is added at a ratio of 1:5 of the material-liquid ratio.

[0012] Furthermore, the concentrations of the HCL solution and the NaOH solution are both 1 mol / L.

[0013] Furthermore, the compound protease (S10155) is purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0014] Furthermore, inactivate the enzyme in a boiling water bath for 10 min.

[0015] The mussel peptides with the above amino acid sequences shown in SEQ ID NO.1-4.

[0016] Furthermore, the mussel peptides with the amino acid sequences shown in SEQ ID NO.1-4 are artificially synthesized.

[0017] This active peptide segment can inhibit the uric acid transporter GLUT9, reduce the reabsorption of uric acid, reduce the uric acid content in the culture medium in a cell model, and it is confirmed in an animal model that it can effectively reduce the serum uric acid and urea nitrogen levels and inhibit the activity of xanthine oxidase, and has the activity of reducing uric acid.

[0018] The beneficial effects of the present invention are as follows: Mytilus polypeptides are prepared by a combination of directed enzymatic hydrolysis with composite enzymes and membrane separation and purification techniques. Four novel polypeptides with potential biological activities are screened out through LC-MS / MS analysis and activity prediction. Molecular docking and cell experiments confirm that these four polypeptides can specifically inhibit the activity of GLUT9 protein and reduce uric acid levels. Animal experiments further verify their efficacy in reducing uric acid, providing a scientific basis for the high-value development of Mytilus resources and the development of functional products for reducing uric acid. Description of the Drawings

[0019] Figure 1 : Molecular docking results of the peptide segment of SEQ ID NO.1 with the XOD receptor protein molecule. Among them, A is the three-dimensional structure diagram and B is the two-dimensional structure diagram.

[0020] Figure 2 : Molecular docking results of the peptide segment of SEQ ID NO.2 with the XOD receptor protein molecule. Among them, A is the three-dimensional structure diagram and B is the two-dimensional structure diagram.

[0021] Figure 3 : Molecular docking results of the peptide segment of SEQ ID NO.3 with the XOD receptor protein molecule. Among them, A is the three-dimensional structure diagram and B is the two-dimensional structure diagram.

[0022] Figure 4 : Molecular docking results of the peptide segment of SEQ ID NO.4 with the XOD receptor protein molecule. Among them, A is the three-dimensional structure diagram and B is the two-dimensional structure diagram.

[0023] Figure 5 : Effect of Mytilus active peptide segments on the uric acid level in the supernatant of the HK-2 cell model.

[0024] Figure 6 : Effect on the kidney index of hyperuricemia rats.

[0025] Figure 7 : Effect on the serum biochemical indexes of hyperuricemia rats; A is the uric acid level, B is the creatinine level, and C is the urea nitrogen level.

[0026] Figure 8 : Effect on the urine biochemical indexes and uric acid excretion of hyperuricemia rats; A is the uric acid level, B is the creatinine level, and C is the fractional excretion of uric acid. Detailed Embodiments

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0028] In addition, all materials used in the embodiments of the present invention are obtained by purchasing from the market unless otherwise specified.

[0029] Example 1: This example provides a screening and identification technology for uric acid-lowering functional polypeptides derived from mussels, specifically including the following steps: Preparation of mussel peptides: Take 100 g of fresh mussel meat after shelling, add distilled water for homogenization at a ratio of 1:5, add 3% compound protease and stir evenly, and adjust the pH value to 7.5 with 1 mol / L HCl and 1 mol / L NaOH. Hydrolyze at 50 °C in a water bath thermostatic oscillator for 5 h. After enzymatic hydrolysis, inactivate the enzyme in a boiling water bath for 10 min, cool, centrifuge at 4 °C and 8000 rpm for 15 min, and collect the supernatant. The supernatant is further ultrafiltered at 0.1 MPa and 25 °C, and an ultrafiltration tube with a molecular weight cut-off of 3 kDa is selected. Collect the filtrate, desalt and freeze-dry to obtain mussel peptide powder. The compound protease (1.2 million U / mg, product number: S10155) is purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0030] Identification of mussel active peptide segments: Connect a Q Exactive mass spectrometer (Thermo, Waltham, MA, USA) to an Ultimate 3000 UPLC system (Thermo, Waltham, MA, USA). Use Proteome Discoverer 2.4 to search the original data file against the mussel protein database (Uniport mussel) downloaded from NCBI, and use Peptide Ranker to predict the activity of the identified peptide segments and screen out peptide sequences with higher scores.

[0031] Molecular docking screening of bioactive peptide segments: Using uric acid transporter (GLUT9) as the target protein, 5 peptide segments with top-ranked activity predictions were selected for homology modeling, and molecular docking was performed with the main binding active sites of the above two proteins respectively, using its own ligand uric acid (UA) as a negative control. Peptide sequence homology modeling: The Chemdraw3D 18.1 software was used to input the peptide sequence for 3D modeling to generate the peptide segment structure. The energy of the peptide structure was minimized through the CHARMm program in the molecular simulation software Discover Studio 2019.

[0032] Pre-treatment of the receptor: The X-ray crystal structure of the GLUT9 protein (PDBID: 8Y65) was downloaded from the PDB database. After removing the existing ligand, chemical bond correction was performed and charges were added. Further, the active pocket was searched for and the pocket site was covered. Molecular docking of the receptor and the peptide segment was performed through the CDOCKER program. Based on the result with the highest CDOCKER Energy score, indicators such as binding free energy, hydrogen bonds, hydrophobic groups, and charged groups were analyzed, and the docking score was calculated.

[0033] The amino acid sequence of the mussel peptide was determined using Q-Exactive plus, and through analysis with the software ProteomeDiscover 2.4 and searching in the Uniport mussel peptides database, a total of 1315 different peptide segment sequences were matched. Further, 4 peptide segments with a residual local confidence greater than 80% and a Peptide Ranker score of the peptide segment sequence greater than 0.9 were identified and screened out, namely WTFFGA, PDPFYK, SPFFKV, and TSPFFK. By comparing with the bioactive peptides already discovered in the BIOPEP database, it was found that none of the above 4 peptide segments had been reported, and they were considered to be new bioactive peptide sequences. The results are shown in Table 1.

[0034] Table 1 Identification and bioactivity prediction of mussel bioactive peptide segments: 。

[0035] The above 4 peptide segments were selected for homology modeling and molecular docking with the GLUT9 protein to analyze the interaction between the peptide segment and the amino acid residues at the active center of GLUT9. The docking results are as shown in Figure 1 A-B to Figure 4As shown in A-B, the target protein GLUT9 used in this experiment has its own uric acid ligand, and the binding energy is -107.60. The following docking modes between the peptide segments and the uric acid transporter are the best poses after simulation. The docking scores of the four peptide segments with the XOD protein are: WTFFGA (-232.48), PDPFYK (-273.87), SPFFKV (-238.90), and TSPFFK (-234.40). The structures of the 4 peptide segments can be embedded into the active pocket of GLUT9. By interacting with key amino acid residues, conventional hydrogen bonds and hydrophobic interactions are formed to block the binding of GLUT9 to uric acid. The above results indicate that the above 4 mussel active peptide segments are good GLUT9 inhibitors at the molecular docking level and have the potential to be developed into active ingredients for multi-target uric acid reduction.

[0036] Example 2: This example provides an exploration of the effect of a uric acid-lowering functional polypeptide derived from mussels on an in vitro cell model, which specifically includes the following steps: Artificial synthesis of mussel active peptide segments: WTFFGA, PDPFYK, SPFFKV, and TSPFFK were synthesized by the Fmoc-solid phase peptide synthesis method and analyzed by HPLC and MS sequence analysis. The purity of the four peptide segments was greater than 98.89%.

[0037] Determination of the in vitro uric acid-lowering activity of the active peptide segments: HK-2 cells were seeded in a 96-well plate at a density of 3x10 4 cells / well and divided into four groups: control group (Control), model group (Model), each active peptide segment group (100 μg / mL), and allopurinol (100 μmol / L) positive control group (Positive). Each group had 6 replicates. After pre-incubation at 37°C and 5% CO2 for 24 h, the culture medium in the well plate was aspirated. After washing with PBS, 250 μL of 4 mmol / L adenosine serum-free medium was added to each well of the model group, positive control group, and each peptide segment group, and the blank group was incubated with serum-free medium for 24 h. 50 μL of 0.01 U / mL xanthine oxidase was added to each well and continued to be treated for 8 h. The operation was carried out strictly according to the instructions of the uric acid kit, and the uric acid content in the culture medium supernatant was detected.

[0038] As Figure 5 shown, the uric acid content in the culture medium supernatant of the model group cells was significantly higher than that of the blank group (P<0.01), proving that the model was successfully established. And the uric acid content in the allopurinol group was significantly lower than that in the model group, indicating that 100 μmol / L allopurinol can be used as a positive control. After the intervention of the mussel active peptide segments, the uric acid content was significantly lower than that in the model group. All five peptide segments showed uric acid-lowering activity, and WTFFGA had the best activity.

[0039] Example 3: This example provides an exploration of the effect of a mussel-derived uric acid-lowering functional polypeptide on hyperuricemic rats, which specifically includes the following steps: Animal grouping and treatment: After 36 male SD rats were adaptively fed for 1 week, they were randomly divided into a control group (control), a model group (model), low, medium, and high-dose (WA6, 80, 100, and 120 mg / kg BW) WTFFGA groups, and an allopurinol group (positive, 25 mg·kg-1), with six rats in each group. Except for the control group, each group was intragastrically administered a suspension of hypoxanthine + potassium oxonate diluted with 5% CMC-Na (adenine 0.1 g / kg and potassium oxonate 1.5 g / kg) every day. The control group was given an equal amount of 5% CMC-Na solution every day. The corresponding test substance was given 1 h after intragastric administration. The control group and the model group were given an equal amount of 5% CMC-Na solution, and intragastric administration was continued for 21 d. On the 20th day of the experiment, the rats were placed in a metabolic cage where they could freely eat and drink, and 24-hour urine was collected. After the experiment, the rats were fasted but allowed to drink water for 6 h. After blood was taken from the abdominal aorta, the rats were sacrificed by cervical dislocation. After sacrifice, the kidneys and livers were quickly separated on ice, rinsed with physiological saline, blotted dry with filter paper, weighed, and stored at -80 °C in a refrigerator for later use.

[0040] Determination of organ index: The whole kidneys of the experimental rats were weighed to determine the organ index of the rats. Organ index (mg / g) = organ (mg) / body weight (g); Detection of serum and urine biochemical indexes: The levels of serum uric acid (SUA), creatinine (SCr), and blood urea nitrogen (BUN) were strictly detected according to the kit instructions. The levels of urine uric acid (UUA) and creatinine (UCr) were detected, and the uric acid excretion fraction (EUA, %) = UUA × SCr / (SUA × UCr) × 100%; Detection of xanthine oxidase XOD activity in the liver: 0.1 g of liver tissue was taken, added with 900 μl of physiological saline (added with 1% PMSF and phosphatase inhibitor), homogenized at 4 °C, centrifuged to obtain the supernatant, and the XOD activity was strictly determined according to the kit instructions.

[0041] As Figure 6 shown, the kidney index of the model group was significantly higher than that of the control group (P<0.05). After intervention with the mussel active peptide, the kidney index was significantly increased and showed a dose dependence, but there was no statistical significance.

[0042] As Figure 7As shown, after 3 weeks of modeling, the concentrations of SUA, SCr, and BUN in the rats of the model group were significantly higher than those in the control group. The SUA levels in the low-, medium-, and high-dose groups of the mussel active peptide segment WA were significantly lower than those in the model group (P<0.01, P<0.001, P<0.001). The UCr levels in the medium- and high-dose groups of the mussel active peptide segment WA were significantly lower than those in the model group (P<0.05). The low-dose WA gavage also decreased the UCr level but without significant difference (P>0.05). The BUN levels in the low-, medium-, and high-dose groups of the mussel active peptide segment WA were significantly lower than those in the model group (P<0.001, P<0.05, P<0.001).

[0043] As Figure 8 shown, compared with the control group, the UUA, UCr levels, and FEUA scores of the rats in the model group were significantly decreased. The UUC and UCr concentrations in the low-, medium-, and high-dose groups of the mussel active peptide segment WA were increased compared with the model group but without statistical significance (P>0.05). The FEUA score was significantly increased compared with the model group, and the effect was equivalent to that of the positive control group. It is suggested that the test substance targeted and inhibited the uric acid reabsorption mediated by GLUT9.

[0044] Finally, it should be noted that although the above embodiments describe the specific implementation manners of the present invention, they do not limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. And all modifications or equivalent replacements should be included in the protection scope of the present invention.

Claims

1. Use of mussel peptides in the preparation of products for reducing uric acid, characterized in that: It includes polypeptides with amino acid sequences shown in SEQ ID NO. 1-4.

2. A method for preparing the mussel peptide according to claim 1, characterized in that It includes the following steps: Take fresh mussel meat after shelling, add distilled water for homogenization, add 3% compound protease and stir evenly, adjust the pH value to 7.5 with HCL solution and NaOH solution, and hydrolyze at 50 °C for 5 h in a water bath constant temperature oscillator; after enzymatic hydrolysis, inactivate the enzyme, cool, centrifuge at 4 °C and 8000 rpm for 15 min, and collect the supernatant; ultrafilter the supernatant at 0.1 MPa and 25 °C, select an ultrafiltration tube with a molecular weight cut-off of 3 kDa, collect the filtrate, desalt and freeze-dry to obtain mussel peptide powder.

3. The preparation method according to claim 2, wherein: Add distilled water at a ratio of 1:5 of the feed liquid ratio.

4. The preparation method according to claim 2, characterized in that: The concentrations of both the HCL solution and the NaOH solution are 1 mol / L.

5. The preparation method according to claim 2, characterized in that: Inactivate the enzyme in a boiling water bath for 10 min.

6. Mussel peptide powder prepared by any of the methods according to claims 2-5.

7. The mussel peptide according to claim 1, wherein: The amino acid sequences are shown in SEQ ID NO. 1-4.

8. The mussel peptide according to claim 7, wherein: The mussel peptide is artificially synthesized.

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