Crayfish head protein peptide with uric acid reducing effect and application

Abstract

The invention discloses a procambarus clarkii head protein peptide with a uric acid reducing effect and application, and belongs to the technical field of biology. The amino acid sequence of the procambarus clarkii head protein peptide is as shown in any one of SEQ ID NO.1-4. Protein hydrolysis is assisted by an ultrasonic microwave technology, the xanthine oxidase inhibitory peptide is prepared by taking the crayfish heads as raw materials, the material basis of the activity of the xanthine oxidase inhibitory peptide is researched, and the action mechanism of the activity of the xanthine oxidase inhibitory peptide is researched; and a theoretical basis is laid for high-value utilization of crayfish processing byproducts and research and development of food-borne xanthine oxidase inhibitors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a crayfish head protein peptide with uric acid-lowering effects and applications thereof. Background Art

[0002] Crayfish, scientifically known as Procambarus clarkii, is rich in protein, essential amino acids, umami amino acids, and polyunsaturated fatty acids, and has high nutritional value. However, during the processing of crayfish, the heads and shells must be removed, resulting in a large amount of by-products being directly discarded or used as feed, causing environmental pollution and waste of biological resources. Crayfish heads are the main by-product of the processing process. They are rich in amino acids, astaxanthin, protein, etc., of which essential amino acids account for 45.3% of the crude protein, making them a high-quality source of protein. Studies have shown that the proteins in crayfish by-products can be used to prepare bioactive peptides with antifreeze, antioxidant, antibacterial, blood sugar-lowering and blood lipid-lowering effects, and have high utilization value.

[0003] Hyperuricemia (HUA) is a metabolic disorder caused by abnormal purine metabolism or impaired uric acid excretion. It can not only induce gout but is also closely associated with a variety of chronic diseases, including cardiovascular disease, kidney disease, and metabolic syndrome. Xanthine oxidase (XOD), a key enzyme in regulating uric acid metabolism, catalyzes the conversion of hypoxanthine to xanthine and further to uric acid. Currently, commonly used XOD inhibitors (such as allopurinol and febuxostat) and uricosuric drugs (such as benzbromarone and probenecid) can effectively control uric acid levels. However, long-term use may cause various adverse reactions, such as allergic reactions, hypertension, and even cardiovascular disease and chronic kidney disease. Research has shown that food-derived bioactive peptides, with their safety and ease of digestion, have great potential in controlling hyperuricemia.

[0004] Ultrasound and microwave technology are green and safe physical processing techniques. Using ultrasound and microwaves to assist protein hydrolysis can enhance enzyme-substrate contact and accelerate the catalytic reaction, effectively improving protein hydrolysis efficiency and producing more active protein hydrolysates. However, no relevant research on xanthine oxidase inhibitory peptides derived from crayfish has been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide a crayfish head protein peptide with uric acid-lowering effect and its application to solve the problems existing in the above-mentioned prior art. The present invention adopts ultrasonic microwave technology to assist protein hydrolysis, and uses crayfish heads as raw materials to prepare xanthine oxidase inhibitory peptides, explores the material basis of its activity, and studies the mechanism of its activity, laying a theoretical foundation for the high-value utilization of crayfish processing by-products and the research and development of food-borne xanthine oxidase inhibitors.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a crayfish head protein peptide with uric acid-lowering effect. The amino acid sequence of the crayfish head protein peptide is shown in any one of SEQ ID NOs. 1-4.

[0008] The present invention also provides a crayfish head enzymatic hydrolysate with uric acid-lowering effect, wherein the crayfish head enzymatic hydrolysate contains protein peptides with amino acid sequences as shown in SEQ ID NO.1-4.

[0009] The present invention also provides a method for preparing the above-mentioned crayfish head enzymatic hydrolysate, comprising the following steps:

[0010] (1) Dissolving crayfish head powder in water, sequentially performing ultrasonic pretreatment and microwave pretreatment; and then performing enzymatic hydrolysis with alkaline protease to obtain an enzymatic hydrolyzate;

[0011] (2) The enzymatic hydrolysate is subjected to ultrafiltration separation to collect components with a molecular weight of less than 1 kDa to obtain the crayfish head enzymatic hydrolysate.

[0012] Optionally, the ultrasonic pretreatment has a power of 300-500 W, a frequency of 20-30 kHz, and a time of 30 min.

[0013] Optionally, the microwave pretreatment has a power of 300-500 W, a frequency of 2200-2500 MHz, and a time of 30 min.

[0014] Optionally, the usage amount of the alkaline protease is 3%; the temperature of the enzymatic hydrolysis is 50° C., and the time is 2-4 h.

[0015] The present invention also provides the use of the crayfish head protein peptide or the crayfish head enzymatic hydrolysate in the preparation of a xanthine oxidase inhibitor.

[0016] The present invention also provides the use of the above-mentioned crayfish head protein peptide or the above-mentioned crayfish head enzymatic hydrolysate in the preparation of uric acid-lowering drugs.

[0017] The present invention also provides a xanthine oxidase inhibitor, the active ingredient of which is the above-mentioned crayfish head protein peptide or the above-mentioned crayfish head enzymatic hydrolysate.

[0018] The present invention also provides a uric acid-lowering drug, comprising the above-mentioned crayfish head protein peptide or the above-mentioned crayfish head enzymatic hydrolysate, and a pharmaceutically acceptable excipient or carrier.

[0019] The present invention discloses the following technical effects:

[0020] The present invention uses crayfish heads as raw materials and adopts ultrasonic microwave technology to assist protein hydrolysis to prepare crayfish head hydrolysates with xanthine oxidase inhibitory activity. Four bioactive protein peptides, namely WSPDAPF, SGGPWRPL, WTPDAPF and DWSPPYPT, were screened and identified from the crayfish head hydrolysates. It was verified that these four protein peptides all showed strong xanthine oxidase inhibitory activity, with IC values ​​of 50 The values ​​are between 2.00-2.33mM; the binding mode of WSPDAPF, SGGPWRPL, DWSPPYPT to XOD is reversible mixed inhibition, and the binding mode of WTPDAPF is reversible non-competitive inhibition.

[0021] This invention explores the uric acid-lowering activity of protein peptides in crayfish heads and studies the mechanism of action of their activity, laying a theoretical foundation for the high-value utilization of crayfish processing by-products and the research and development of food-borne xanthine oxidase inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 is the hydrolysis degree of crayfish head enzymatic hydrolysate under different preparation conditions;

[0024] Figure 2 is the inhibitory activity of samples on xanthine oxidase before and after ultrafiltration separation;

[0025] Figure 3 The results of reversible inhibition test of xanthine oxidase by active peptides WSPDAPF (A), SGGPWRPL (B), WTPDAPF (C) and DWSPPYPT (D) are shown;

[0026] Figure 4 The results show the effects of active peptides WSPDAPF (A), SGGPWRPL (B), DWSPPYPT (C) and WTPDAPF (D) on the proliferation activity of RAW264.7 cells. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0033] Example 1

[0034] Crayfish heads were used as raw material. 100g of the powder was crushed and added to 300mL of deionized water, mixing thoroughly. Subsequently, different pretreatment methods were used: a. no pretreatment; b. ultrasonic pretreatment (power 400W, frequency 30kHz) for 30min; c. microwave pretreatment (power 400W, frequency 2450MHz) for 30min; d. ultrasonic pretreatment for 30min followed by microwave pretreatment for 30min. Then, 3% alkaline protease (based on the weight of the raw material) was added, the pH was adjusted to 8.0, the enzymatic hydrolysis temperature was 50°C, and the enzymatic hydrolysis time was set at 2, 3, and 4h, respectively. After enzymatic hydrolysis, the enzyme was inactivated in a boiling water bath for 10min. After cooling, the mixture was filtered and the pH was adjusted to 7.0. The mixture was centrifuged at 4°C and 10,000 rpm for 20min. The supernatant was collected and filtered to remove suspended matter and precipitate, yielding the enzymatic hydrolyzate.

[0035] The total nitrogen content of crayfish heads was determined by the Kjeldahl method, and the ammoniacal nitrogen content of the enzymatic hydrolysis solution was determined by formaldehyde automatic potentiometric titration. The degree of hydrolysis (DH) was calculated as follows:

[0036] DH / %=M2 / M1×100 (1);

[0037] Where: M1 represents the total nitrogen mass of crayfish head (g); M2 represents the ammoniacal nitrogen mass of enzymatic hydrolyzate (g).

[0038] like Figure 1 As shown in the figure, compared with normal enzymatic hydrolysis without pretreatment, as the enzymatic hydrolysis time is extended (2, 3, 4h), ultrasound, microwave and ultrasound-microwave combined pretreatment can promote the enzymatic hydrolysis process and improve the hydrolysis degree of the sample. Among them, the effect of ultrasound-microwave combined pretreatment is the most significant. When the enzymatic hydrolysis is 4h, the hydrolysis degree is increased by 24.8% compared with the normal enzymatic hydrolysis group, and a crayfish head enzymatic hydrolysate with a higher hydrolysis degree can be obtained. Therefore, the optimal preparation method of crayfish head protein peptide is determined to be:

[0039] Crayfish heads were used as raw material. 100g of the powder was crushed and added to 300mL of deionized water, mixing thoroughly. The sample was then pretreated using an ultrasonic-microwave synergistic reaction system: ultrasonic (power 400W, frequency 30kHz) for 30min, followed by microwave (power 400W, frequency 2450MHz) for 30min. 3% alkaline protease (based on the weight of the raw material) was then added, the pH adjusted to 8.0, and hydrolysis was performed at 50°C for 2-4h. After hydrolysis, the enzyme was inactivated in a boiling water bath for 10min. After cooling, the sample was filtered and the pH adjusted to 7.0. The supernatant was collected and filtered to remove suspended matter and precipitate. The hydrolyzate was freeze-dried and stored at -20°C.

[0040] Example 2

[0041] Ultrafiltration was used to separate the crayfish head hydrolysate by molecular weight. The sample was then separated using 3kDa and 1kDa ultrafiltration tubes, collecting three fractions (F1: >3kDa, F2: 1-3kDa, and F3: <1kDa). After separation, the sample was freeze-dried, and each fraction was evaluated for in vitro XOD inhibitory activity.

[0042] The xanthine oxidase (XOD) inhibition rate was determined as follows:

[0043] Add 50 μL of the test sample or 50 μL of phosphate buffer (0.05 mM, pH 7.4) and 50 μL of 0.1 U / mL XOD solution to each well of a 96-well plate. Incubate the mixture at 37°C for 15 minutes. Then, add 150 μL of 0.40 mM xanthine solution and incubate at 37°C for 30 minutes. Measure the absorbance at 290 nm. Perform quadruplicates for each sample, and use phosphate buffer as a blank.

[0044] Inhibition rate = [1-(A1-A2) / (A3-A4)] × 100% (2);

[0045] Where: 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; A4 represents the absorbance of the blank group without enzyme added.

[0046] like Figure 2 As shown in the figure, the inhibitory activity of the F3 fraction was significantly higher than that of the sample before ultrafiltration, indicating that ultrafiltration fractionation had an enrichment effect on the fractions with XOD inhibitory activity.

[0047] Example 3

[0048] After desalting the F3 fraction using a C18 desalting column, peptide sequences were analyzed using an EASY-nano LC-Q Exactive Plus MS tandem liquid chromatography-mass spectrometer. Specific conditions were as follows: C18 column, 20 cm × 75 μm, 1.9 μm; mobile phase: 0.1% TFA-acetonitrile (A): 0.1% TFA-water (B) = 80:20 (v / v); injection volume: 5 μL; column temperature: 40°C; flow rate: 300 nL / min; elution conditions: 4% B phase (0-0.1 min); 50% B phase (0.1-53 min); 95% B phase (53-53.1 min); and 95% B phase (53.1-60 min). Mass spectrometry conditions were as follows: m / z range: 100-1500; electrospray voltage: 2 kV. Mass spectrometry data were processed using PEAKS Studio version 12 (Bioinformatics Solutions Inc., Waterloo, Canada).

[0049] The bioactivity scores of the identified oligopeptides were analyzed using the PeptideRanker database (http: / / distilldeep.ucd.ie / PeptideRanker / ), and peptides with an activity score ≥ 0.8, a -10lgP ≥ 40, and an amino acid length ≤ 10 were selected. The potential toxicity and allergenicity of the active peptides were predicted using the ToxinPred database (https: / / webs.iiitd.edu.in / raghava / toxinpred / ) and the AllerTOP database (https: / / www.ddg-pharmfac.net / allertop_test / ). The isoelectric points of the resulting peptides were predicted using PepDraw (https: / / www.pepdraw.com / ).

[0050] The molecular docking of XOD (PDB: 1N5X) was performed using AutoDock vina software. First, the target protein was pre-processed using DiscoveryStudio 2025 Client to add hydrogen atoms and remove water molecules to prepare XOD without ligand. Then, the oligopeptide was drawn using ChemDraw 19.0 and its energy was minimized. Finally, the molecular docking was performed using AutoDock Vina, and the GridBox was controlled to The coordinates of the active center are 119.830, 52.472, 23.335 (x, y, z), and the grid spacing is After docking, the ligand object with the lowest binding energy is saved in PDB format.

[0051] The results of screening and identification of peptides with XOD inhibitory activity in the F3 fraction are shown in Table 1. It can be seen that the peptides WSPDAPF, SGGPWRPL, WTPDAPF and DWSPPYPT all showed strong XOD inhibitory activity, and their IC 50 The values ​​range from 2.00 to 2.33 mM.

[0052] Table 1 Screening and identification results of active peptides from F3 component

[0053]

[0054]

[0055] Example 4 Determination of the reversibility of inhibition of XOD by active peptides

[0056] Using phosphate buffer (pH 7.4, 50mM) as the buffer system, active peptide solutions of different concentrations (0, 1, 2, 3mg / mL, 50μL, artificially synthesized) were mixed with XOD solutions of different concentrations (0, 0.02, 0.04, 0.08, 0.16U / mL, 50μL) and incubated at 37°C for 5min. Subsequently, xanthine solution (40mM, 150μL) was added. After mixing evenly, the absorbance of the reaction system at 290nm was immediately detected every 30s using a microplate reader for a total of 5min. The reaction rate v (min) was expressed as the XOD concentration (U / mL) on the horizontal axis. -1 ) is the vertical axis. The reversibility of the inhibitory effect of the active peptide on XOD is determined by correlation.

[0057] like Figure 3 As shown, all the fitted straight lines pass through the origin and have a good linear relationship. With the increase of the active peptide concentration, the slope of the fitted straight line gradually decreases, indicating that the inhibitory effect of the active peptides WSPDAPF, SGGPWRPL, DWSPPYPT and WTPDAPF on XOD is reversible.

[0058] Example 5 Determination of the inhibitory type of active peptides on XOD

[0059] Using phosphate buffer (pH 7.4, 50mM) as the buffer system, active peptide solutions of different concentrations (5mg / mL, artificially synthesized, 50μL) and XOD solution (0.1U / mL, 50μL) were thoroughly mixed in a centrifuge tube and incubated in a 37°C constant temperature water bath for 30min. Then, different concentrations of xanthine solution (0.2, 0.3, 0.4, 0.5, 0.6, 0.7mM, 150μL) were added respectively. After mixing evenly, the absorbance of the reaction system at 290nm was immediately measured every 1min using a microplate reader for a total of 5min. The reciprocal of the xanthine concentration 1 / [S] was used as the horizontal axis and the reciprocal of the reaction rate 1 / v was used as the vertical axis. A relationship curve was drawn according to the Lineweaver-Burk double reciprocal equation to determine the type of inhibition of the active peptide on XOD.

[0060] The results are shown in Table 2. It can be seen that the inhibition kinetic analysis confirmed that the binding mode of WSPDAPF, SGGPWRPL, and DWSPPYPT to XOD was a reversible mixed-type inhibition, while the binding mode of WTPDAPF was a reversible non-competitive inhibition.

[0061] Table 2 Effects of each sample on Km and Vmax in XOD enzymatic reaction and their inhibition types

[0062]

[0063]

[0064] Example 6 Biosafety Determination of Active Peptides

[0065] The biosafety of the active peptides was tested by examining their effects on the proliferation of RAW264.7 cells. The specific process is as follows:

[0066] RAW264.7 cells were digested with EDTA trypsin and then diluted to 5×10 3 A cell suspension of 100 μL / mL was placed in a 96-well plate, with 100 μL per well. A blank control was set up in a well without cells. After cell attachment, the culture medium was removed and complete culture medium containing different concentrations (10, 25, 50, 100, 200, 400, and 600 μg / mL) of active peptide or lipopolysaccharide (LPS, 1 μg / mL) was added. After 24 hours of incubation, 25 μL of 5 mg / mL thiazolyl tetrazolium blue (MTT) solution was added to each well. Culture was continued for another 4 hours, the supernatant was removed from the 96-well plate, and 150 μL of DMSO solution was added to dissolve the crystals. After shaking for 15 minutes, the absorbance was measured at a single wavelength of 490 nm on a microplate reader to calculate cell viability.

[0067] See the results Figure 4 It can be seen that the active peptide WSPDAPF ( Figure 4A)、SGGPWRPL( Figure 4 B), DWSPPYPT( Figure 4 C) and WTPDAPF( Figure 4 D) has no negative effect on the proliferation activity of RAW264.7 cells. This shows that the active peptide component prepared by the present invention is natural and safe and can be used to prepare xanthine oxidase inhibitors or uric acid-lowering drugs.

[0068] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A crayfish head protein peptide with uric acid lowering effect, characterized in that: The amino acid sequence of the crayfish head protein peptide is shown in any one of SEQ ID NO.1-4.

2. A crayfish head enzymatic hydrolysate with uric acid-lowering effect, characterized in that: The crayfish head enzymatic hydrolysate contains protein peptides with amino acid sequences as shown in SEQ ID NO.1-4.

3. The method for preparing the crayfish head enzymatic hydrolysate according to claim 2, characterized in that: The steps include: (1) Dissolving crayfish head powder in water, sequentially performing ultrasonic pretreatment and microwave pretreatment; and then performing enzymatic hydrolysis with alkaline protease to obtain an enzymatic hydrolyzate; (2) The enzymatic hydrolysate is subjected to ultrafiltration separation to collect components with a molecular weight of less than 1 kDa to obtain the crayfish head enzymatic hydrolysate.

4. The preparation method according to claim 3, characterized in that The power of the ultrasonic pretreatment is 300-500W, the frequency is 20-30kHz, and the time is 30min.

5. The preparation method according to claim 3, characterized in that The microwave pretreatment has a power of 300-500W, a frequency of 2200-2500MHz, and a time of 30 minutes.

6. The preparation method according to claim 3, characterized in that The usage amount of the alkaline protease is 3%; the temperature of the enzymolysis is 50° C., and the time is 2-4 hours.

7. Use of the crayfish head protein peptide according to claim 1 or the crayfish head enzymatic hydrolysate according to claim 2 in the preparation of a xanthine oxidase inhibitor.

8. Use of the crayfish head protein peptide according to claim 1 or the crayfish head enzymatic hydrolysate according to claim 2 in the preparation of uric acid-lowering drugs.

9. A xanthine oxidase inhibitor, characterized in that The active ingredient is the crayfish head protein peptide according to claim 1 or the crayfish head enzymatic hydrolysate according to claim 2.

10. A uric acid-lowering drug, characterized in that: It comprises the crayfish head protein peptide according to claim 1 or the crayfish head enzymatic hydrolysate according to claim 2, and a pharmaceutically acceptable excipient or carrier.

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