Antarctic krill small molecule active peptide, preparation method and application thereof

Abstract

The application discloses a small molecule active peptide of Euphausia superba and a preparation method and application thereof. The application utilizes defatted Euphausia superba powder to prepare XOD inhibiting peptides, and the obtained polypeptides have high XOD inhibition rates. Through LC-MS / MS identification, five small molecule polypeptides with XOD inhibiting activity, i.e. LPPYSKE, EDVEGAVR, LDLPGWVK, LDDAFNHL and WDRPLVE, are screened from the enzymatic hydrolysate of the defatted Euphausia superba powder. The five polypeptides all have XOD inhibiting activity in different degrees. The Euphausia superba peptide prepared by the application has good XOD inhibiting capacity and can be applied to the field of deep processing of medicines and the like. The application utilizes aquatic product processing by-products to realize high-value utilization, and can effectively improve resource utilization rate and industrial economic value.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to small molecule bioactive peptides from Antarctic krill, their preparation methods, and applications. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disease caused by abnormal purine metabolism or insufficient uric acid excretion, mainly characterized by abnormally high serum uric acid levels (uric acid >7 mg / dL in men and >6 mg / dL in women). With socioeconomic development and changes in lifestyle and dietary structure, HUA has gradually gained public attention as a common disease. Related data shows that the prevalence of hyperuricemia among Chinese adults is as high as 13.3%, and it is showing a trend towards affecting younger people. Furthermore, HUA plays an important role in related metabolic diseases; high serum uric acid is often considered a prognostic indicator for chronic kidney disease, diabetes, cardiovascular disease, and inflammation. Currently used uric acid-lowering drugs such as allopurinol, febuxostat, and benzbromarone have significant therapeutic effects, but they have serious adverse reactions such as allergic reactions and liver and kidney damage, and are expensive. Therefore, developing safe, effective, and economical uric acid-lowering drugs has become a key research focus.

[0003] Bioactive peptides are polypeptides obtained through fermentation, enzymatic hydrolysis, chemical hydrolysis, and other methods, possessing special physiological functions such as anti-tumor, immunomodulatory, antibacterial, analgesic, and anti-inflammatory effects. In recent years, bioactive peptides have become a hot topic in the field of uric acid-lowering research due to their advantages such as easy absorption, low preparation cost, and high safety.

[0004] In the human metabolic process, xanthine oxidase (XOD, EC 1.17.3.2) is a key enzyme regulating uric acid production. XOD is mainly found in the liver and small intestine, where it continuously oxidizes hypoxanthine and xanthine to produce uric acid. It can also convert purines ingested from food into uric acid. Furthermore, during XOD-catalyzed purine metabolism, reactive oxygen species such as superoxide anion radicals and hydrogen peroxide are generated, participating in oxidative stress and further damaging health. Therefore, inhibiting XOD activity to lower uric acid levels is an important method for treating hyperuricemia.

[0005] Antarctic krill possesses enormous biomass, representing the largest animal protein resource on Earth. Defatted Antarctic krill meal, an economic byproduct of krill oil production, contains high-quality protein. However, these products are primarily used as food and feed additives, resulting in low resource utilization. Therefore, effectively utilizing the high-quality protein in defatted Antarctic krill meal has become a pressing issue. Summary of the Invention

[0006] The purpose of this invention is to provide small molecule bioactive peptides from Antarctic krill and their preparation methods, and another purpose is to provide their applications.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0008] A small molecule bioactive peptide from Antarctic krill, the amino acid sequence of which is: LPPYSKE, or EDVEGAVR, or LDLPGWVK, or LDDAFNHL, or WDRPLVE.

[0009] Specifically, LPPYSKE stands for: Leu-Pro-Pro-Tyr-Ser-Lys-Glu;

[0010] EDVEGAVR specifically stands for: Glu-Asp-Val-Glu-Gly-Ala-Val-Arg;

[0011] LDLPGWVK specifically stands for: Leu-Asp-Leu-Pro-Gly-Trp-Val-Lys;

[0012] LDDAFNHL specifically stands for: Leu-Asp-Asp-Ala-Phe-Asn-His-Leu;

[0013] WDRPLVE is specifically: Trp-Asp-Arg-Pro-Leu-Val-Glu.

[0014] The preparation method of the Antarctic krill small molecule bioactive peptides includes the following steps:

[0015] (1) Enzymatic hydrolysis of Antarctic krill powder was obtained by degreasing it with enzymes;

[0016] (2) High temperature inactivates enzymes;

[0017] (3) Centrifugation was used to obtain the enzymatic hydrolysis supernatant of Antarctic krill;

[0018] (4) The supernatant of Antarctic krill enzymatic hydrolysis was desalted and freeze-dried to obtain Antarctic krill enzymatic hydrolyzed freeze-dried powder, namely, Antarctic krill small molecule active polypeptide mixture.

[0019] Furthermore, the preparation method also includes further purification, using an affinity ultrafiltration method: the Antarctic krill enzyme lyophilized powder is dissolved and co-incubated with XOD enzyme. After incubation, ultrafiltration and centrifugation are performed to obtain the retentate, which is then eluted to denature the XOD enzyme and release it from the binding of small molecule peptides. The collected permeate is dried to obtain the purified Antarctic krill active polypeptide mixture.

[0020] Preferably, in step (5), the concentration of the lyophilized powder enzymatic hydrolysate is 50-250 mg / mL, the XOD enzyme concentration is 0.1-0.5 U / mL, the volume ratio of the hydrolysate to the XOD enzyme is 1:1-1:5, and the incubation time is 20-60 min. More preferably, the concentration of the hydrolysate is 200 mg / mL, the XOD enzyme concentration is 0.2 U / mL, the volume ratio of the hydrolysate to the XOD enzyme is 1:2, the incubation temperature is 37°C, and the incubation time is 30 min.

[0021] Furthermore, based on LC-MS / MS mass spectrometry, small molecule bioactive peptides of Antarctic krill were identified and then screened to finally obtain the aforementioned five small molecule bioactive peptides of Antarctic krill.

[0022] Preferably, in step (1), defatted Antarctic krill powder and water are mixed at a certain mass ratio (1:1-10), adjusted to the optimal pH value for enzymatic hydrolysis, and enzyme is added to start the enzymatic hydrolysis reaction. During the reaction, the pH value of the solution is kept constant. The protease is selected from alkaline protease, neutral protease, papain, and flavor protease.

[0023] Preferably, an alkaline protease is selected, with a hydrolysis pH of 9.5, a hydrolysis temperature of 55℃, and a protease activity of 2×10⁻⁶. 5 U / g; the mass fraction of alkaline protease used was 1.6%; the enzymatic hydrolysis time was 4.6 h.

[0024] Preferably, the enzyme inactivation temperature in step (2) is 100°C and the enzyme inactivation time is 10-20 min, preferably 15 min.

[0025] Preferably, the centrifugation speed in step (3) is 2000-5000 r / min and the centrifugation time is 20-60 min, with the preferred centrifugation speed being 4000 r / min and the centrifugation time being 30 min.

[0026] Preferably, the desalination method in step (4) is dialysis desalination, in which a 200 Da dialysis bag is desalted in pure water for 48 hours, the freezing temperature is -80℃, and the drying time is 24 hours.

[0027] The application of the Antarctic krill small molecule bioactive peptide in the preparation of drugs that inhibit xanthine oxidase (XOD).

[0028] Furthermore, the application of the Antarctic krill small molecule active peptide in the preparation of drugs to lower high uric acid.

[0029] Advantages and beneficial effects of the present invention:

[0030] This invention utilizes defatted Antarctic krill powder to prepare XOD-inhibiting peptides, and the obtained peptides exhibit high XOD inhibition rates. By LC-MS / MS identification, five small molecule peptides with XOD inhibitory activity were screened from the enzymatic hydrolysate of defatted Antarctic krill powder: LPPYSKE, EDVEGAVR, LDLPVGVK, LDDAFNHL, and WDRPLVE. All five peptides exhibit varying degrees of XOD inhibitory activity.

[0031] The Antarctic krill peptides prepared by this invention exhibit excellent XOD inhibition capabilities and can be applied in deep processing fields such as pharmaceuticals. This invention also enables the high-value utilization of aquatic product processing byproducts, effectively improving resource utilization and industrial economic value.

[0032] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0033] Figure 1 This is the basepeak mass spectrometry diagram of the sample in Example 6.

[0034] Figure 2 This is a secondary mass spectrum of a small molecule active polypeptide with the amino acid sequence LPPYSKE.

[0035] Figure 3 This is a secondary mass spectrum of a small molecule active polypeptide with the amino acid sequence EDVEGAVR.

[0036] Figure 4 This is a secondary mass spectrum of a small molecule active peptide with the amino acid sequence LDLPGWVK.

[0037] Figure 5 This is a secondary mass spectrum of a small molecule active peptide with the amino acid sequence LDDAFNHL.

[0038] Figure 6 This is a secondary mass spectrum of a small molecule active polypeptide with the amino acid sequence WDRPLVE.

[0039] Figure 7 This is a diagram showing the molecular docking results between the peptide LPPYSKE and XOD. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to the embodiments. The specific examples described herein are only for explaining the present invention and are not limited thereto.

[0041] In the following embodiments, the experimental methods for determining the XOD inhibition rate of each sample are as follows:

[0042] (1) Solution preparation:

[0043] Xanthine oxidase solution (0.05 U / mL): Take 77 μL of enzyme solution and dilute to 10 mL with 1×PBS buffer;

[0044] Xanthine solution (0.40 mmol / L): Weigh 6.08 mg of xanthine powder, dissolve it in 400 μL of 1 mol / L NaOH, and then dilute to 100 mL with buffer solution.

[0045] (2) Experimental methods:

[0046] Add 50 μL of the test sample (diluted to 9 mg / mL) and 50 μL of xanthine oxidase solution with a concentration of 0.05 U / mL to each well of a 96-well plate, shake for 30 s, incubate at 25 °C for 5 min, add 150 μL of 0.40 mM xanthine solution, shake for 30 s, incubate at 25 °C for 25 min, and measure the absorbance at 290 nm.

[0047] (3) Calculation formula:

[0048] The formula for calculating xanthine oxidase inhibitory activity is as follows:

[0049]

[0050] In the formula, A1 represents the absorbance of the sample solution with enzyme added; A2 represents the absorbance of the sample solution without enzyme added; A3 represents the absorbance of the blank group with buffer instead of sample solution; and A4 represents the absorbance of the blank group without enzyme added.

[0051] Example 1:

[0052] A method for preparing Antarctic krill bioactive peptides with XOD inhibitory activity, the method comprising the following steps:

[0053] (1) Enzymatic hydrolysis: Defatted Antarctic krill powder and water are mixed at a mass ratio of 1:5, dispersed evenly by a high-speed disperser, preheated to 50°C, pH adjusted to 9, alkaline protease is added to start the enzymatic hydrolysis reaction, and the solution is shaken in a water bath. During the reaction, the pH value of the solution is kept constant.

[0054] (2) High-temperature enzyme inactivation: Inactivate enzymes in a boiling water bath at 100℃ for 15 minutes;

[0055] (3) Centrifugation: Centrifuge at 4000 r / min for 30 min, and filter to obtain the enzymatic hydrolysate;

[0056] (4) Desalting and freeze-drying: The supernatant obtained in step (3) is desalted and freeze-dried to obtain Antarctic krill enzymatically hydrolyzed freeze-dried powder.

[0057] Example 2:

[0058] A method for preparing Antarctic krill peptides with XOD inhibitory activity, the method comprising the following steps:

[0059] (1) Enzymatic hydrolysis: Defatted Antarctic krill powder and water are mixed at a mass ratio of 1:5, dispersed evenly by a high-speed disperser, preheated to 50°C, pH adjusted to 7, neutral protease is added to start the enzymatic hydrolysis reaction, and the solution is shaken in a water bath. During the reaction, the pH value of the solution is kept constant.

[0060] (2) High-temperature enzyme inactivation: Inactivate enzymes in a boiling water bath at 100℃ for 15 minutes;

[0061] (3) Centrifugation: Centrifuge at 4000 r / min for 30 min, and filter to obtain the enzymatic hydrolysate;

[0062] (4) Desalting and freeze-drying: The supernatant obtained in step (3) is desalted and freeze-dried to obtain Antarctic krill enzymatically hydrolyzed freeze-dried powder.

[0063] Example 3:

[0064] A method for preparing Antarctic krill peptides with XOD inhibitory activity, the method comprising the following steps:

[0065] (1) Enzymatic hydrolysis: Defatted Antarctic krill powder and water were mixed at a mass ratio of 1:5 and dispersed evenly using a high-speed disperser. The mixture was preheated to 55°C, and the pH was adjusted to 6.5. Papain was added to start the enzymatic hydrolysis reaction. The mixture was shaken in a water bath, and the pH of the solution was kept constant during the reaction.

[0066] (2) High-temperature enzyme inactivation: Inactivate enzymes in a boiling water bath at 100℃ for 15 minutes;

[0067] (3) Centrifugation: Centrifuge at 4000 r / min for 30 min, and filter to obtain the enzymatic hydrolysate;

[0068] (4) Desalting and freeze-drying: The supernatant obtained in step (3) is desalted and freeze-dried to obtain Antarctic krill enzymatically hydrolyzed freeze-dried powder.

[0069] Example 4:

[0070] A method for preparing Antarctic krill peptides with XOD inhibitory activity, the method comprising the following steps:

[0071] (1) Enzymatic hydrolysis: Defatted Antarctic krill powder and water are mixed at a mass ratio of 1:5, dispersed evenly by a high-speed disperser, preheated to 50°C, pH adjusted to 7, flavor protease is added to start the enzymatic hydrolysis reaction, and the solution is shaken in a water bath. During the reaction, the pH value of the solution is kept constant.

[0072] (2) High-temperature enzyme inactivation: Inactivate enzymes in a boiling water bath at 100℃ for 15 minutes;

[0073] (3) Centrifugation: Centrifuge at 4000 r / min for 30 min, and filter to obtain the enzymatic hydrolysate;

[0074] (4) Desalting and freeze-drying: The supernatant obtained in step (3) is desalted and freeze-dried to obtain Antarctic krill enzymatically hydrolyzed freeze-dried powder.

[0075] Example 5:

[0076] This invention uses the UV method to determine the XOD inhibitory activity of enzyme extract.

[0077] XOD inhibition rate analysis was performed on the freeze-dried Antarctic krill peptide powders obtained in Examples 1-4. The XOD inhibition rates of the four freeze-dried peptide powders are shown in Table 1. The results showed that the product obtained by alkaline protease hydrolysis had significantly higher XOD inhibition activity than the products obtained under the other three conditions. Using alkaline protease to degrease Antarctic krill powder is more conducive to the preparation of XOD inhibiting peptides. Therefore, the XOD inhibiting peptides prepared by the method of this invention achieve efficient utilization of aquatic product processing by-products and can be used as pharmaceutical raw materials for the treatment and prevention of hypersensitivity to respiratory infections (HUA). This method effectively improves the utilization efficiency of defatted Antarctic krill powder, and the use of protease hydrolysis to obtain active peptide powder is inexpensive, economical, environmentally friendly, and safe, with promising application prospects.

[0078] Table 1. XOD inhibition rate of Antarctic krill peptides obtained from different proteases

[0079] Serial Number XOD inhibition rate (%) Example 1 82.16 Example 2 49.21 Example 3 62.98 Example 4 73.78

[0080] The Antarctic krill enzymatically hydrolyzed lyophilized powder prepared in Example 1 was further purified: the Antarctic krill enzymatically hydrolyzed lyophilized powder was dissolved and co-incubated with XOD enzyme. After incubation, ultrafiltration and centrifugation were performed to obtain the retentate, which was then eluted with 70% (v / v) methanol solution to denature the XOD enzyme and release it from the binding of small molecule peptides. The collected permeate was dried to obtain the purified Antarctic krill small molecule active peptide mixture. The concentration of the lyophilized powder enzymatic hydrolysate was 200 mg / mL, the XOD enzyme concentration was 0.2 U / mL, the volume ratio of enzymatic hydrolysate to XOD enzyme was 1:2, the incubation temperature was 37℃, and the incubation time was 30 min.

[0081] Example 6:

[0082] LC-MS / MS mass spectrometry identification of small molecule inhibitory peptides for XOD in Antarctic krill includes the following steps:

[0083] (1) Solution quantification: The sample obtained after purification in Example 1 was desalted using C18 StageTip and vacuum dried. The dried peptides were reconstituted with 0.1% FA, and the peptide concentration was determined by OD280 for LC-MS analysis.

[0084] (2) The XO inhibitory peptide sequence was determined using LC-MS / MS. Detection was performed based on the previously described method. Buffer A was a 0.1% formic acid (v / v) aqueous solution, and buffer B was a 0.1% formic acid and 80% acetonitrile (v / v) aqueous solution. The column was equilibrated with 100% buffer A. The sample was injected into a C18 capillary capture column (100 μm * 20 mm, 5 μm, Dr. Maisch GmbH) and then subjected to gradient separation using a C18 separation column (75 μm * 150 mm, 3 μm, Dr. Maisch GmbH) at a flow rate of 300 nl / min. The LC separation gradient is as follows:

[0085] 2-5% B phase lasts 0-2 min, 5-28% B phase lasts 2-44 min, 28-40% B phase lasts 44-51 min, 40-100% B phase lasts 51-53 min, and B phase is maintained at 100% for 53-60 min.

[0086] After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive mass spectrometer. The analysis time was 60 min, detection mode: positive ion; precursor ion scan range: 350-1800 m / z; primary mass resolution: 60000@m / z200; AGC target: 3e6; primary maximum IT: 50 ms. The primary mass spectrum obtained after sample analysis is shown below. Figure 1 As shown.

[0087] Peptide secondary mass spectrometry analysis was performed using the following method: Secondary mass spectra of the 20 highest-intensity precursor ions were acquired after each full scan (MS2 scan). Secondary mass spectrometry resolution: 15000 m / z 200; AGC target: 1e5; Secondary maximum IT: 50 ms; MS2 Activation Type: HCD; Isolation window: 1.6 m / z; Normalized collision energy: 28.

[0088] (3) The Uniprot protein database was analyzed using PEAKS Studio 10.6 software to obtain the identified peptides.

[0089] Table 2 PEAKS Studio Analysis Parameter Settings

[0090]

[0091] After searching the mass spectrometry data, PSM FDR ≤ 0.01 and Protein FDR ≤ 0.01 were used as screening criteria for peptide, site, and protein identification, respectively. The final sample identification yielded 943 peptides and 13 protein sequences.

[0092] Example 7:

[0093] Screening peptides via molecular docking includes the following steps:

[0094] (1) The polypeptide obtained in Example 6 was modeled in 3D using ChemDraw 21.0.0 software and used as a ligand for energy minimization.

[0095] (2) Download the XOD containing the ligand TEI (PDBID: 1N5X) from the protein database PDB (http: / / www.rcsb.org / ). Import the obtained XOD crystal structure into Discovery Studio 2019 Client (DS2019) and perform some preparatory operations such as dehydration and hydrogen atom addition before molecular docking.

[0096] (3) Using 1N5X as the receptor protein, the virtually enzymatically hydrolyzed polypeptide as the ligand, and (X: 96.574, Y: 55.310, Z: 39.332) as the docking center, with... Molecular docking was performed using the docking radius. The semi-flexible docking tool LibDock in Dock Ligands was used for molecular docking, with parameters set to rigid optimization and fast screening, and the rest left as default. The selected peptides were then proceeded to the next step based on their scores.

[0097] Results: From the 943 peptides in Example 6, 82 peptides were selected for DS2019 simulated docking with XOD. Based on the LibDock Score, 5 peptides were selected for synthesis; the secondary mass spectra of the 5 peptides are shown below. Figure 2-6 As shown in the diagram, the docking schematic is as follows: Figure 7 As shown.

[0098] Example 8:

[0099] The peptides screened in Example 7 were subjected to solid-phase synthesis and activity verification, including the following steps:

[0100] (1) Peptides LPPYSKE, EDVEGAVR, LDLPUGWVK, LDDAFNHL and WDRPLVE were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method.

[0101] (2) Prepare a 0.1-5 mg / mL solution of the polypeptide and determine the in vitro XO inhibition rate of the synthesized polypeptide by UV method.

[0102] Table 3. Peptide Information and XOD Inhibition Rate

[0103]

[0104] Note: (1) Due to the randomness of molecular docking results, the LibDock Score shown in the table is the average of the highest values ​​of the 5 docking results.

[0105] (2) The toxicity of the peptide was calculated using http: / / crdd.osdd.net / raghava / / toxinpred / .

[0106] As shown in Table 3, all five peptides exhibited high XOD inhibitory activity, with the peptide containing the amino acid sequence LPPYSKE showing the highest XOD inhibitory activity. Furthermore, the IC50 of the prepared Antarctic krill enzymatically hydrolyzed freeze-dried powder was [not specified in the original text]. 50 The value is 3.11 mg / mL, meaning that the XOD inhibition activity of the five small molecule active peptides prepared by this invention is up to 10 times higher than that of the present invention, and the five small molecule active peptides have extremely significant XOD inhibition activity.

[0107] The embodiments described above are merely preferred embodiments of the present invention and do not constitute a limitation on the scope of the claims. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be within the protection scope of the present invention.

Claims

1. A small molecule active peptide of Euphausia superba, characterized in that, The amino acid sequence of the active peptide is: LPPYSKE.

2. The preparation method of the small molecule active peptide of Euphausia superba of claim 1, characterized in that, The method comprises the following steps: (1) obtaining an enzymatic hydrolysate by enzymatic hydrolysis of defatted Euphausia superba powder, adjusting the pH to 9, and adding alkaline protease to start the enzymatic hydrolysis reaction; (2) high-temperature enzyme inactivation; (3) centrifugal separation to obtain an Euphausia superba enzymatic hydrolysate supernatant; (4) desalting and freeze-drying the Euphausia superba enzymatic hydrolysate supernatant to obtain Euphausia superba enzymatic hydrolysate freeze-dried powder, i.e., an Euphausia superba small-molecule active polypeptide mixture; (5) selecting an affinity ultrafiltration method: dissolving the Euphausia superba enzymatic hydrolysate freeze-dried powder and incubating it with XOD enzyme, centrifuging and ultrafiltering to obtain a retentate, eluting to denature the XOD enzyme and release the combination with small-molecule peptides, and drying the obtained permeate to obtain a purified Euphausia superba active polypeptide mixture; the Euphausia superba enzymatic hydrolysate freeze-dried powder has a concentration of 50-250 mg / ml, the XOD enzyme has a concentration of 0.1-0.5 U / ml, the volume ratio of the enzymatic hydrolysate to the XOD enzyme is 1:1-1:5, and the incubation time is 20-60 min; (6) identifying the Euphausia superba small-molecule active peptide based on LC-MS / MS mass spectrometry, and then screening to obtain an Euphausia superba small-molecule active peptide, which has an amino acid sequence of: LPPYSKE.

3. Use of the Euphausia superba small-molecule active peptide of claim 1 in the preparation of a drug for treating hyperuricemia.

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