A crayfish head protein peptide with uric acid-lowering effect and its application

Abstract

This invention discloses a crayfish head protein peptide with uric acid-lowering effect and its application, belonging to the field of biotechnology. The amino acid sequence of the crayfish head protein peptide is shown in any one of SEQ ID NO. 1-4. This invention uses ultrasonic and microwave technology to assist protein hydrolysis, prepares xanthine oxidase inhibitory peptides from crayfish heads, explores the material basis of its activity, and studies its mechanism of action, laying a theoretical foundation for the high-value utilization of crayfish processing by-products and the development of food-derived xanthine oxidase inhibitors.

Description

Technical Field

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

[0002] Crayfish, scientifically known as *Procambarus clarkii*, is rich in protein, essential amino acids, umami amino acids, and polyunsaturated fatty acids, making it highly nutritious. However, the processing of crayfish requires the removal of the head and shell, resulting in a large amount of byproducts being discarded directly or used as animal feed, causing environmental pollution and wasting biological resources. Crayfish heads are a major byproduct of processing, containing abundant amino acids, astaxanthin, and protein. The crude protein contains 45.3% essential amino acids, making it a high-quality protein source. Studies have shown that the protein in crayfish byproducts can be used to prepare bioactive peptides, which have antifreeze, antioxidant, antibacterial, hypoglycemic, and hypolipidemic effects, demonstrating high utilization value.

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

[0004] Ultrasound and microwave technology is a green and safe physical processing technique. Using ultrasound and microwave-assisted protein hydrolysis can enhance the contact between enzymes and substrates and accelerate the catalytic reaction, effectively improving protein hydrolysis efficiency and yielding protein hydrolysates with superior activity. However, no relevant studies have been reported domestically or internationally regarding xanthine oxidase inhibitory peptides from crayfish. Summary of the Invention

[0005] The purpose of this invention is to provide a crayfish head protein peptide with uric acid-lowering effects and its applications, thereby addressing the problems existing in the prior art. This invention employs ultrasound and microwave-assisted protein hydrolysis to prepare xanthine oxidase inhibitory peptides from crayfish heads, explores the material basis of its activity, and studies its mechanism of action. This lays a theoretical foundation for the high-value utilization of crayfish processing by-products and the development of food-derived xanthine oxidase inhibitors.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a crayfish head protein peptide with uric acid-lowering effect, wherein the amino acid sequence of the crayfish head protein peptide is shown in any one of SEQ ID NO.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 a protein peptide with an amino acid sequence as described in SEQ ID NO.1-4.

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

[0010] (1) After dissolving crayfish head powder in water, it was subjected to ultrasonic pretreatment and microwave pretreatment in sequence; then it was enzymatically hydrolyzed with alkaline protease to obtain hydrolysate;

[0011] (2) The enzymatic hydrolysate is separated by ultrafiltration, and the components with a molecular weight <1kDa are collected to obtain the crayfish head enzymatic hydrolysate.

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

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

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

[0015] The present invention also provides the application of the above-mentioned crayfish head protein peptide or the above-mentioned crayfish head enzymatic hydrolysate in the preparation of xanthine oxidase inhibitor.

[0016] The present invention also provides the application 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 pharmaceutically acceptable excipients or carriers.

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

[0020] This invention uses crayfish heads as raw material and employs ultrasonic and microwave-assisted protein hydrolysis to prepare crayfish head enzymatic hydrolysates with xanthine oxidase inhibitory activity. Four bioactive protein peptides were screened and identified from the crayfish head enzymatic hydrolysates: WSPDPPF, SGGPWRPL, WTPDPPF, and DWSPPYPT. Verification showed that all four protein peptides exhibited strong xanthine oxidase inhibitory activity, with IC50 values... 50 The values ​​ranged from 2.00 to 2.33 mM; the binding modes of WSPDPPF, SGGPWRPL, DWSPPYPT and XOD were reversible mixed-mode inhibition, while the binding mode of WSPDPPF was reversible non-competitive inhibition.

[0021] This invention explores the uric acid-lowering activity of protein peptides in crayfish heads and studies the mechanism of their activity, laying a theoretical foundation for the high-value utilization of crayfish processing by-products and the development of food-derived xanthine oxidase inhibitors. Attached Figure Description

[0022] 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.

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

[0024] Figure 2 The inhibitory activity of samples before and after ultrafiltration on xanthine oxidase was measured.

[0025] Figure 3 The results show the reversibility of inhibition of xanthine oxidase by the active peptides WSPDPPF(A), SGGPWRPL(B), WSPDPPF(C), and DWSPPYPT(D);

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

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

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

[0033] Example 1

[0034] Using crayfish heads as raw material, the crayfish heads were crushed, and 100g of powder was added to 300mL of deionized water and mixed evenly. Subsequently, different pretreatment methods were used: a. no pretreatment; b. ultrasonic pretreatment (400W power; 30kHz frequency) for 30min; c. microwave pretreatment (400W power; 2450MHz frequency) for 30min; d. ultrasonic pretreatment for 30min followed by microwave pretreatment for 30min. Then, 3% alkaline protease (by raw material mass) was added, the pH was adjusted to 8.0, the enzymatic hydrolysis temperature was 50℃, and the hydrolysis time was set to 2, 3, and 4 hours, respectively. After hydrolysis, the enzyme was inactivated by boiling water bath for 10min, cooled, filtered, and the pH was adjusted to 7.0. The mixture was then centrifuged at 4℃ and 10000r / min for 20min. The supernatant was collected, filtered to remove suspended solids and precipitates, and the hydrolysate was obtained.

[0035] The total nitrogen content of crayfish heads was determined using the Kjeldahl method, and the mass of ammonia nitrogen in the enzymatic hydrolysate was determined using an automated potentiometric titration method with formaldehyde. The formula for calculating the degree of hydrolysis (DH) is as follows:

[0036] DH / % = M2 / M1 × 100 (1);

[0037] In the formula: M1 represents the total nitrogen mass (g) of the crayfish head; M2 represents the ammonia nitrogen mass (g) of the enzymatic hydrolysate.

[0038] like Figure 1 As shown, compared with normal enzymatic hydrolysis without pretreatment, with increasing hydrolysis time (2, 3, 4 h), ultrasonic, microwave, and ultrasonic-microwave combined pretreatment all promoted the enzymatic hydrolysis process and increased the degree of hydrolysis. Among them, ultrasonic-microwave combined pretreatment had the most significant effect; after 4 h of enzymatic hydrolysis, its degree of hydrolysis was increased by 24.8% compared with the normal enzymatic hydrolysis group, yielding crayfish head hydrolysates with a high degree of hydrolysis. Therefore, the optimal preparation method for crayfish head protein peptides was determined to be:

[0039] Using crayfish heads as raw material, the crayfish heads were crushed, and 100g of powder was added to 300mL of deionized water and mixed evenly. Subsequently, the sample was pretreated using an ultrasonic-microwave synergistic reaction system: first, ultrasonic pretreatment (power 400W; frequency 30kHz) for 30min, followed by microwave pretreatment (power 400W; frequency 2450MHz) for 30min. Then, 3% alkaline protease (by raw material mass) was added, the pH was adjusted to 8.0, and hydrolysis was carried out at 50℃ for 2–4h. After hydrolysis, the enzyme was inactivated by boiling water bath for 10min, cooled, filtered, and the pH was adjusted to 7.0. The mixture was then centrifuged at 4℃ and 10000r / min for 20min. The supernatant was collected, filtered to remove suspended solids and precipitates, and freeze-dried to obtain the enzymatic hydrolysate, which was then stored at -20℃.

[0040] Example 2

[0041] Ultrafiltration was used to separate crayfish head enzymatic hydrolysates by different molecular weight cutoffs. The samples were separated using 3kDa and 1kDa ultrafiltration tubes, and three fractions were collected (F1: >3kDa, F2: 1-3kDa, F3: <1kDa). After separation, the samples were freeze-dried, and the in vitro XOD inhibitory activity of each fraction was evaluated.

[0042] The method for determining the inhibition rate of xanthine oxidase (XOD) is 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 min, then add 150 μL of 0.40 mM xanthine solution and incubate at 37 °C for 30 min. Measure the absorbance at 290 nm. Perform quadruplicate tests for each sample, using phosphate buffer as a blank.

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

[0045] 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 solution instead of sample solution; A4 represents the absorbance of the blank group without enzyme added.

[0046] like Figure 2 As shown, the inhibitory activity of the F3 component was significantly higher than that of the sample before ultrafiltration, indicating that ultrafiltration fractionation has an enrichment effect on components with XOD inhibitory activity.

[0047] Example 3

[0048] After desalting the F3 fraction using a C18 desalting column, the peptide sequence of the sample was analyzed using an EASY-nano LC-Q Exactive Plus MS tandem liquid chromatography-mass spectrometry system. 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℃; flow rate: 300 nL / min; elution conditions: 0–0.1 min, 4% B phase; 0.1–53 min, 50% B phase; 53–53.1 min, 95% B phase; 53.1–60 min, 95% B phase. Mass spectrometry conditions were set as follows: m / z: 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 oligopeptides with an activity score ≥0.8, -10lgP ≥40, and amino acid length ≤10 were screened. The potential toxicity and sensitization of the bioactive 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 point of the obtained peptides was predicted using PepDraw (https: / / www.pepdraw.com / ).

[0050] Molecular docking of XOD (PDB: 1N5X) was performed using AutoDock Vina software. First, the target protein was pretreated using Discovery Studio 2025 Client, adding hydrogen atoms and removing water molecules to prepare ligand-free XOD. Then, the oligopeptide was plotted using ChemDraw 19.0, and its energy was minimized. Finally, molecular docking was performed using AutoDock Vina, controlling the GridBox to... The active center coordinates are 119.830, 52.472, 23.335 (x, y, z), and the grid spacing is [missing information]. After docking is complete, the ligand with the lowest binding energy is saved as a PDB file.

[0051] The screening and identification results of peptides with XOD inhibitory activity in fraction F3 are shown in Table 1. It can be seen that peptides WSPDPPF, SGGPWRPL, WTPDAPF, and DWSPPYPT all exhibited strong XOD inhibitory activity, with IC50 values ​​of [missing information]. 50 The value ranges from 2.00 to 2.33 mM.

[0052] Table 1. Screening and identification results of bioactive peptides in F3 component.

[0053]

[0054]

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

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

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

[0058] Example 5: Determination of the type of XOD inhibition by the active peptide

[0059] Using phosphate-buffered saline (pH 7.4, 50 mM) as the buffer system, different concentrations of active peptide solutions (5 mg / mL, synthetic, 50 μL) were thoroughly mixed with XOD solution (0.1 U / mL, 50 μL) in centrifuge tubes and incubated in a 37℃ water bath for 30 min. Then, different concentrations of xanthine solutions (0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mM, 150 μL) were added and mixed thoroughly. Immediately after mixing, the absorbance of the reaction system at 290 nm was measured every 1 min using a microplate reader for a total of 5 min. The reciprocal of xanthine concentration (1 / [S]) was plotted on the x-axis, and the reciprocal of reaction rate (1 / v) was plotted on the y-axis. The relationship curve was plotted using the Lineweaver-Burk double reciprocal equation to determine the type of inhibition of XOD by the active peptide.

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

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

[0062]

[0063]

[0064] Example 6: Biosafety determination of bioactive peptides

[0065] The biosafety of the bioactive peptides was assessed by examining their effect on the proliferation activity of RAW264.7 cells. The specific procedure is as follows:

[0066] RAW264.7 cells were digested with EDTA trypsin and then formulated into 5×10⁻⁶ cells. 3 Cell suspension of 100 μL / mL was added to each well of a 96-well plate, with a cell-free well serving as a blank control. After cell adhesion, the culture medium was removed, and complete culture medium containing different concentrations (10, 25, 50, 100, 200, 400, 600 μg / mL) of active peptides or lipopolysaccharide (LPS, 1 μg / mL) was added to each well. After culturing for 24 h, 25 μL of thiazolyl blue tetrazolium blue (MTT) solution (5 mg / mL) was added to each well, and the plates were cultured for another 4 h. The supernatant in the 96-well plate was then removed, and 150 μL of DMSO solution was added to dissolve any crystals. After shaking for 15 min, the absorbance was measured at 490 nm using a microplate reader, and cell viability was calculated.

[0067] See results Figure 4 Therefore, the active peptide WSPDPPF (prepared in this invention) Figure 4A), SGGPWRPL ( Figure 4 B), DWSPPYPT ( Figure 4 C) and WTPDAPF Figure 4 D) had no negative impact on the proliferation activity of RAW264.7 cells. This indicates that the active peptide components prepared in this invention are natural and safe, and can be used to prepare xanthine oxidase inhibitors or uric acid-lowering drugs.

[0068] 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 crayfish head protein peptide having a uric acid-lowering effect, characterized by, 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 having a uric acid lowering effect, characterized in that, The crayfish head protein peptide with an amino acid sequence shown in any one of SEQ ID NO. 1-4 is contained in the crayfish head enzymatic hydrolysate.

3. The method of claim 2, wherein the red swamp crayfish head hydrolysate is prepared by the steps of: The method comprises the following steps: (1) dissolving crayfish head powder in water, and then sequentially performing ultrasonic pretreatment and microwave pretreatment; and then performing enzymatic hydrolysis by using alkaline protease to obtain an enzymatic hydrolysate; (2) performing ultrafiltration separation on the enzymatic hydrolysate, and collecting a component with a molecular weight <1 kDa to obtain the crayfish head enzymatic hydrolysate; The power of the ultrasonic pretreatment is 300-500 W, the frequency is 20-30 kHz, and the time is 30 min; The power of the microwave pretreatment is 300-500 W, the frequency is 2200-2500 MHz, and the time is 30 min; The use amount of the alkaline protease is 3%, the temperature of the enzymatic hydrolysis is 50°C, and the time is 2-4 h.

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

5. Use of the crayfish head protein peptide of claim 1 or the crayfish head enzymatic hydrolysate of claim 2 in the preparation of a drug for reducing uric acid.

6. A xanthine oxidase inhibitor, characterized by, The effective component is the crayfish head protein peptide of claim 1 or the crayfish head enzymatic hydrolysate of claim 2.

7. A drug for reducing uric acid, characterized by comprising the compound according to claim 1. The effective component is the crayfish head protein peptide of claim 1 or the crayfish head enzymatic hydrolysate of claim 2. The effective component is the crayfish head protein peptide of claim 1 or the crayfish head enzymatic hydrolysate of claim 2.

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