Peptide extracted from small yellow croaker and having uric acid reducing effect

Abstract

The invention discloses a peptide which is extracted from small yellow croakers and has a uric acid reducing effect, and belongs to the technical field of biology. The peptide is obtained by analyzing and identifying a product of papain enzymolysis of small yellow croaker by LC-MS / MS, and the amino acid residue sequence of the peptide is WDDMEKIW. The invention proves that the WDDEMEKIW peptide can effectively inhibit the rise of uric acid in serum of mice by improving intestinal flora, and also alleviates the symptom of weight loss of mice caused by too high uric acid. In conclusion, the invention not only provides a new thought for treating hyperuricemia, but also widens the application prospect of the small yellow croaker.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a peptide extracted from small yellow croaker and having a uric acid-lowering effect. Technical Background

[0002] In recent years, due to unhealthy lifestyles, more and more people suffer from diseases such as hyperuricemia, chronic kidney disease, gout, cardiovascular disease, hypertension and cancer. Among them, hyperuricemia is a metabolic disease caused by purine metabolism disorder. Patients with this disease need to take benzbromarone tablets, indomethacin tablets, febuxostat tablets, allopurinol tablets, hydrocortisone tablets and other drugs for a long time, and these drugs have huge side effects. On the other hand, studies have shown that food-derived active peptides containing 2 to 20 amino acid residues play an important role in antibacterial, blood pressure lowering, uric acid lowering, and anti-inflammatory effects. Compared with chemically synthesized drugs, food-derived bioactive peptides have a wide range of sources, rich and diverse activities, and are safe and reliable, and have great research value.

[0003] At present, microbial fermentation technology and enzymatic hydrolysis are the most commonly used modern biotechnologies for preparing food-derived active peptides. The biological activity of peptides prepared and separated by these two methods is usually stronger than that of the parent protein. Microbial fermentation is a method in which microorganisms (such as bacteria, yeast or endophytic fungi) act on proteins to produce complex reactions to release bioactive peptides. The degree of hydrolysis depends on the microbial strain, raw materials, protein type, fermentation time and temperature; enzymatic hydrolysis is the use of proteases to hydrolyze various proteins from plants and animals. The proteases are divided into endopeptidases and exopeptidases. The former mainly cuts the peptide chain at specific internal sites of the protein to produce internal peptides; while the latter mainly cuts the peptide chain at the end of the protein to produce short peptides or free amino acids. After the protein is broken down into polypeptides by endopeptidases, it becomes a substrate for the action of exopeptidases, and after further hydrolysis, smaller polypeptides and free amino acids are produced. Due to the differences in the cutting and cleavage specificity of proteases, the selection of appropriate protease hydrolysis plays a vital role in food degradation. Several proteolytic enzymes from plants, animals and microorganisms are mostly used to prepare bioactive protein hydrolysates or peptides, such as alkaline protease, trypsin, papain, pepsin, flavor protease, thermolysin and chymotrypsin. In the hydrolysis process, conditions such as hydrolysis time, degree of hydrolysis, enzyme activity and pretreatment of raw protein should also be considered. Because these factors are closely related to the degree of hydrolysis and affect the release of amino acids and peptide content in the raw materials. Compared with microbial fermentation, enzymatic hydrolysis can better control the hydrolysis process to obtain peptides with specific molecular weight and amino acid residue sequence, and has the advantages of short reaction time and easy mass production.

[0004] Small yellow croaker (Latin name: Larimichthys polyactis) belongs to the order Perciformes, family Sciaenidae, genus Larimichthys. It is a warm-temperate near-bottom fish and an important fishery species unique to the East China Sea, the Yellow Sea and the Bohai Sea. Its main production areas are coastal provinces such as Jiangsu, Zhejiang, Fujian and Shandong. Small yellow croaker has a high protein content and rich amino acid species, making it a high-quality protein source. However, the overall added value of the product is low. Although studies have shown that bioactive peptides developed and prepared using small yellow croaker as raw materials have antibacterial effects, there have been no reports on the presence of uric acid-lowering active peptides in the enzymatic hydrolysate of small yellow croaker. Summary of the invention

[0005] In view of the above problems, the present invention discloses a peptide extracted from small yellow croaker and having the effect of lowering uric acid, which belongs to the field of biotechnology. The peptide is obtained by analyzing and identifying the product of papain hydrolysis of small yellow croaker by LC-MS / MS, and its amino acid residue sequence is WDDMEKIW. The present invention confirms that the WDDMEKIW peptide can effectively inhibit the increase of uric acid in mouse serum by improving intestinal flora, and at the same time, alleviates the symptoms of weight loss due to excessive uric acid. In summary, the present invention not only provides new ideas for the treatment of hyperuricemia, but also increases the application prospects of small yellow croaker.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In one aspect, the present invention provides a peptide having uric acid-lowering function, wherein the peptide comprises an amino acid residue sequence of WDDMEKIW or APPERKYSVW, preferably WDDMEKIW.

[0008] The applicant used common small yellow croaker meat and fish bones as raw materials, selected common enzymes such as papain, neutral protease, alkaline protease as hydrolases, hydrolyzed the small yellow croaker under respective suitable reaction conditions, and tested the enzymatic products. It was finally found that the prepared papain enzymatic product had the highest inhibition rate on xanthine oxidase (XO), so the papain enzymatic hydrolysis scheme was selected; subsequently, LC-MS / MS was used to analyze the peptides with a molecular weight lower than 3kDa in the above papain enzymatic products, and 2 peptides (WDDMEKIW peptide or APPERKYSVW peptide) were identified. Through layers of screening and experimental verification, it was confirmed that the WDDMEKIW peptide has better biological activity and uric acid lowering effect.

[0009] It should be understood that studies have found that peptides with a molecular weight of less than 3 kDa have a stronger inhibitory effect on XO than other molecular weights; and the smaller the component, the stronger the effect; at the same time, the applicant has also confirmed that a polypeptide mixture with a molecular weight of less than 3 kDa has the strongest inhibitory ability on XO, which may be because the components less than 3 kDa have structural characteristics recognized by XO, thereby acting as substrates of XO. Therefore, the present invention analyzes peptides with a molecular weight of less than 3 kDa.

[0010] On the other hand, the present invention provides a method for preparing a peptide with uric acid-lowering function, wherein the peptide comprises a WDDMEKIW or APPERKYSVW amino acid residue sequence, preferably WDDMEKIW; the method comprises enzymatically hydrolyzing small yellow croaker using papain.

[0011] Specifically, the method comprises the following steps: washing small yellow croaker meat and fish bones (raw materials) and homogenizing; adding water, with the solid-liquid ratio of small yellow croaker to pure water being 1:18 (g / mL); adding papain at 4,000 U / g, with the pH of the enzymolysis being 6.4, the enzymolysis temperature being 50°C, and the enzymolysis time being 7 hours; centrifuging to obtain the supernatant, and freeze-drying.

[0012] On the other hand, the present invention provides a drug for treating hyperuricemia, wherein the drug and the peptide comprise an amino acid residue sequence of WDDMEKIW or APPERKYSVW, preferably WDDMEKIW.

[0013] On the other hand, the present invention provides a use of a peptide in the preparation of a preparation for treating hyperuricemia, wherein the peptide comprises an amino acid residue sequence of WDDMEKIW or APPERKYSVW, preferably WDDMEKIW.

[0014] Furthermore, the peptide is used at a concentration of 10 mg / kg to 1 g / kg.

[0015] On the other hand, the present invention provides an application of a peptide in the preparation of food or health products for gout patients, wherein the peptide comprises an amino acid residue sequence of WDDMEKIW or APPERKYSVW, preferably WDDMEKIW.

[0016] It is understandable that various auxiliary materials and raw materials such as fillers, adhesives, antioxidants, solvents, cosolvents, coating agents, pH adjusters, flavoring agents, sweeteners, flour, plant extracts, etc. can be added when preparing medicines, foods and health products containing the above peptides.

[0017] On the other hand, the present invention provides a use of a peptide for treating hyperuricemia by affecting intestinal flora, wherein the peptide comprises an amino acid residue sequence of WDDMEKIW or APPERKYSVW, preferably WDDMEKIW.

[0018] Furthermore, the intestinal flora belongs to any one or more of the phylum Firmicutes, phylum Bacteroidetes and phylum Proteobacteria.

[0019] Specifically, the WDDMEKIW peptide reduces the abundance of Bacteroidetes and increases the abundance of Firmicutes.

[0020] Furthermore, the intestinal flora belongs to any one or more of the genera Bifidobacterium, Bacteroides and Firmicutes.

[0021] Specifically, the WDDMEKIW peptide increased the species of Bifidobacterium, inhibited the decrease in the ratio of Bacteroides to Firmicutes (species), and increased the number of lactic acid bacteria.

[0022] On the other hand, the present invention provides a use of a peptide for alleviating complications of hyperuricemia, wherein the peptide comprises an amino acid residue sequence of WDDMEKIW or APPERKYSVW, preferably WDDMEKIW; the complications refer to weight loss.

[0023] The beneficial effects of the present invention include:

[0024] 1. The present invention discovered that the enzymatic hydrolysate of small yellow croaker has uric acid-lowering activity, which expands the application of small yellow croaker in functional foods and provides a new idea for the prevention and treatment of hyperuricemia;

[0025] 2. A peptide with uric acid-lowering effect (WDDMEKIW peptide) was identified and screened from the enzymatic hydrolysate of small yellow croaker, and the physiological function of the peptide was confirmed by experiments. This not only helps to develop drugs, foods or health products that can alleviate the symptoms of high uric acid in specific populations, but also creates higher added value and commercial value for small yellow croaker;

[0026] 3. WDDMEKIW peptide has high biological activity and is non-toxic to the body. It has a mild effect and can treat hyperuricemia by affecting intestinal flora and effectively alleviate the problem of weight loss caused by high uric acid.

[0027] 4. The WDDMEKIW peptide provided by the present invention consists of only 8 amino acids, which not only has a simple synthesis step and low cost, but is also easily absorbed by the body; in addition, because the molecular weight of the peptide is relatively small, only 1 kDa, theoretically the presentation efficiency of the delivery vector encapsulating the peptide will also be very high. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0029] Figure 1 :The effect of different proteases on the enzymatic hydrolysis of small yellow croaker; the group with the highest value is marked as "a" or "A", the second highest is marked as "b" or "B", and the lowest is marked as "c" or "C". The values ​​of the groups marked with different letters mean that there are significant differences (p<0.05);

[0030] Figure 2 : Mass spectra of the two polypeptides identified in Example 3;

[0031] Figure 3 : Computer three-dimensional structure simulation diagram of the two polypeptides identified in Example 3;

[0032] Figure 4 :The effect of small yellow croaker peptide on serum uric acid content in hyperuricemia mice; The sample size in each experimental group was 8 mice;

[0033] Figure 5 :Effects of small yellow croaker peptide on body weight of mice with hyperuricemia; The sample size in each experimental group was 8 mice;

[0034] Figure 6 :Analysis of intestinal bacterial community structure based on phylum level in hyperuricemia mice; Figure (a): The proportion of Bacteroidetes in the intestinal flora of mice in different treatment groups; Figure (b): The proportion of Firmicutes in the intestinal flora of mice in different treatment groups; Figure (c): The ratio of Firmicutes / Bacteroidetes in the intestinal flora of mice in different treatment groups (F / B);

[0035] Figure 7 : Analysis of intestinal bacterial community structure at the genus level in hyperuricemia mice; WW: polypeptide group (n=8), AP: allopurine group (n=8), MC: hyperuricemia group (n=8), NC: blank control group (n=8); DETAILED DESCRIPTION

[0036] The present invention is further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments of the specification. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0038] Example 1: Preparation of marine fish oligopeptides

[0039] 1.1 Experimental materials: small yellow croaker meat and fish bones (both processed from commercially available small yellow croaker), papain (food grade, purchased from Angel Enzyme Co., Ltd.)

[0040] 1.2 Preparation process:

[0041] The fish meat and bones of small yellow croaker were thawed overnight at 4°C; the fish paste was freeze-dried and ground into fine powder, filtered through an 80-mesh sieve, vacuum-packed, and stored in a -80°C ultra-low temperature refrigerator.

[0042] Purified water was added to the fish paste at a mass ratio of 1 (fish paste): 18 (purified water) and mixed well; 4,000 U / g papain was added, the pH was adjusted to 6.4, and enzymolysis was performed at 50° C. for 7 hours; the supernatant was taken by centrifugation, and freeze-dried to obtain marine fish oligopeptides.

[0043] The raw material of the marine fish oligopeptide prepared in this embodiment is fish meat, and the enzyme used for enzymatic hydrolysis is a single food-grade protease. Therefore, the marine fish oligopeptide has high safety and can be used in food.

[0044] Example 2: Screening of hydrolase types

[0045] There are many types of proteolytic enzymes, and the type of protease used determines the type and efficacy of the marine fish oligopeptides obtained by hydrolyzing small yellow croaker. Therefore, this example screens the type of protease used to hydrolyze small yellow croaker in Example 1 to obtain the fish-derived oligopeptides with the greatest potential for lowering uric acid. This example uses common small yellow croaker meat and fish bones as raw materials, selects papain, neutral protease, and alkaline protease as hydrolases, adds hydrolases at an amount of 4,000 U / g, and hydrolyzes the small yellow croaker under the most suitable reaction conditions (see Table 1 for details), and the remaining experimental steps are the same as those described in Example 1.

[0046] Table 1 Types of hydrolases and their optimal reaction conditions

[0047]

[0048] Next, the inhibitory effects of the hydrolysis products of the above three hydrolases on xanthine oxidase (XO) were determined respectively. The specific steps are as follows: a 0.48 mmol / L xanthine solution (Shanghai Aladdin Biochemical Technology Co., Ltd.) was prepared using 0.2 mol / L potassium dihydrogen phosphate buffer (pH=7.4); 50 μL of the sample solution to be tested and 50 μL of 0.07 U / mL XO solution (Shanghai Aladdin Biochemical Technology Co., Ltd.) were mixed, incubated at 37°C for 5 min, and then 150 μL of xanthine solution was added for reaction; a microplate reader (Molecular Biology, USA) was used to analyze the xanthine solution. Devices Company) measured the absorbance at 290nm, and continuously recorded the change trend of the absorbance within 10min; the calculation formula of XO inhibition rate is as follows: XO inhibition rate / % = (dA / dt) blank - (dA / dt) sample / (dA / dt) sample × 100%, where (dA / dt) blank and (dA / dt sample) are the reaction rates of blank and added with enzymatic hydrolysis products, respectively. Each experimental group was set up with 3 replicates. The specific results are shown in Figure 1 .

[0049] Under the same conditions of substrate concentration, amount of hydrolyzate added, time and temperature for XO enzymatic hydrolysis, the test was conducted and it was finally found that compared with the hydrolyzates of the other two enzyme preparations, the xanthine oxidase (XO) inhibition rate and degree of hydrolysis of the papain hydrolyzate were significantly higher (p<0.05). 50 The lowest value was 33.40±0.26 mg / mL, which means that it is most likely to screen out the polypeptide with the best uric acid-lowering effect from the products of papain hydrolysis. Subsequent experiments also confirmed that only by using papain to hydrolyze small yellow croaker can the two polypeptides described in the present invention (i.e., WDDMEKIW peptide and APPERKYSVW peptide) be screened out; therefore, when using enzymatic hydrolysis to prepare peptides derived from small yellow croaker with uric acid-lowering effect, papain enzymatic hydrolysis is preferred.

[0050] Example 3: Identification and screening of uricosuric acid-lowering peptides

[0051] 3.1 In order to more efficiently screen out highly active small yellow croaker hydrolyzed peptides that can lower uric acid, this example groups the mixed peptides in the papain hydrolysate of small yellow croaker according to their molecular weight (UF-1 of >10 kDa, UF-2 of 3 kDa to 10 kDa, and UF-3 of <3 kDa), and evaluates the uric acid-lowering efficacy of the mixed peptides in different groups. The specific operation is as follows: The marine fish oligopeptide prepared in Example 1 is dissolved in 0.2 mol / L phosphate buffer at a volume ratio of 1:20, and the prepared small yellow croaker total enzyme is firstly filtered using a 10 kDa ultrafiltration centrifuge tube (Thermo Fisher). The hydrolyzate (UF-0) was subjected to ultrafiltration centrifugation, the retentate was a mixed hydrolyzed peptide with a molecular weight greater than 10 kDa (UF-1 group), and the filtrate was a mixed hydrolyzed peptide with a molecular weight less than 10 kDa; then the filtrate was ultrafiltered using a 3 kDa ultrafiltration centrifuge tube (Thermo Fisher), the retentate after this ultrafiltration was a mixed hydrolyzed peptide with a molecular weight of 3-10 kDa (UF-2 group), and the filtrate was a mixed hydrolyzed peptide with a molecular weight less than 3 kDa (UF-3 group), and the conditions for each ultrafiltration centrifugation were 3,500 r / min, 40 min; the above liquid was taken and freeze-dried using a Biofugestratos desktop high-speed refrigerated centrifuge from Thermo Scientific, USA; and the XO inhibition rate of the three components was determined (the specific operation was the same as described in Example 2).

[0052] Comparing the test results of the enzymatic hydrolysate UF-0 group and the three ultrafiltration fractions, it was found that the XO inhibition rate IC 50 The value was 32.74±0.12 mg / mL, 41.32±0.08 mg / mL for UF-1 group, 30.91±0.13 mg / mL for UF-2 group, and 25.87±0.16 mg / mL for UF-3 group, which means that UF-3 with a molecular weight less than 3 kDa has the strongest inhibitory ability against XO, which may be because the components less than 3 kDa have structural characteristics recognized by XO, thus acting as substrates of XO. Therefore, the present invention analyzes peptides with a molecular weight less than 3 kDa.

[0053] 3.2 Then, the oligopeptide solution with molecular weight less than 3 kDa was subjected to liquid chromatography-tandem triple quadrupole mass spectrometry (LC-Tandem Mass Spectrometry, LC-MS / MS) (Waters, USA) analysis, specifically using a C18 chromatographic column (SIGMA, USA) with a flow rate of 300 nL / min; the elution conditions are as follows: mobile phase A is 0.1% formic acid in water (Macklin), mobile phase B is 0.1% formic acid in acetonitrile (Macklin); the column temperature is 40°C; the liquid phase separation gradient is as follows: from 6% to 25% mobile phase B within 42 minutes, increasing to 45% mobile phase B within 11 minutes, increasing to 80% mobile phase B within 0.5 minutes, and then maintaining at 80% mobile phase B for 6.5 minutes, with a continuous flow rate of 300 nL / min; PEAKS Studio version 10.6 software was then used to analyze the tandem mass spectra to identify the amino acid residue sequence of the small yellow croaker polypeptide. The results showed that a total of 2 small molecule peptides ( Figure 2 ); the online platform ToxinPre (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html), the program Prot Param (http: / / web.expasy.org / protparam / ), the Innovagen website (http: / / www.innovagen.com / proteomics-tools), Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) and Chem Bio Draw Ultra 14.0 were used to predict the potential toxicity, isoelectric point, water solubility, potential biological activity and three-dimensional structure of the above peptides. The results of the analysis using bioinformatics software / website are shown in Table 2, and the predicted three-dimensional structure of the peptide is shown in Figure 3 .

[0054] Table 2 Physicochemical properties of small yellow croaker peptides

[0055]

[0056] As shown in Table 2, the two polypeptides identified from the papain hydrolysate are both non-toxic and have good water solubility; and the predicted value of biological activity of WDDMEKIW polypeptide is greater than that of APPERKYSVW polypeptide. Generally speaking, the predicted result of biological activity is between 0 and 1. The closer the calculated value is to 1, the higher the activity of the peptide. In other words, compared with APPERKYSVW peptide, WDDMEKIW polypeptide has higher activity, which means that the latter can more effectively bind to the target receptor in vivo and produce more significant physiological or biological effects, further enhance its specific efficacy, and more effectively regulate the immune function of organisms, such as activating antioxidant, antibacterial, anti-tumor and other physiological functions, which can make WDDMEKIW polypeptide have significant advantages in drug efficacy development and reducing drug side effects.

[0057] In addition, the inhibitory effects of the above two polypeptides on XO were also determined. The WDDMEKIW polypeptide and the APPERKYSVW polypeptide were commissioned to Shanghai Shenggong Company for solid phase synthesis (purity>95%). According to the instructions, the powders of the two polypeptides were dissolved in 0.2 mol / L phosphate buffer respectively, and prepared into 1% to 5% polypeptide solutions (preferably 5% in this example); then, the effects of the two polypeptides on XO enzyme activity were determined respectively according to the method described in Example 2. The specific results are shown in Table 3: XO inhibition rate IC of polypeptide WDDMEKIW 50 The value is 3.16±0.03mmol / L, and the XO inhibition rate IC of peptide APPERKYSVW is 50 The value was 5.86±0.02mmol / L, which means that compared with APPERKYSVW polypeptide, WDDMEKIW polypeptide has a better inhibitory effect on XO enzyme activity. It is preliminarily believed that WDDMEKIW polypeptide has a better uric acid-lowering effect.

[0058] Based on the comprehensive biological activity, three-dimensional structure and XO inhibition rate, WDDMEKIW peptide was selected for subsequent functional verification experiments.

[0059] Table 3 Comparison of XO inhibition rates of various components of small yellow croaker polypeptide

[0060]

[0061] Example 4: Uric acid-lowering effect of small yellow croaker peptide

[0062] 4.1 The small yellow croaker peptide described in this example is the WDDMEKIW polypeptide, which was solid-phase synthesized by Shanghai Bioengineering Corporation (purity>95%).

[0063] 4.2 Mouse culture and grouping

[0064] The male ICR mice used were about 8 weeks old, with a single mouse weighing about 35g. They were all housed in a specific pathogen-free SPF-grade sterile laminar flow environment (22-25°C, 50-60% relative humidity, 12h light / 12h dark cycle), and all mice had free access to water and food. After ten days of adaptive feeding, they were divided into 4 groups according to different drug administration, namely, blank control group (NC group, n=8), high uric acid group (MC group, n=8), allopurine group (AP group, n=8) and polypeptide group (WW group, n=8).

[0065] 4.3 Hyperuricemia Modeling

[0066] The blank control group was gavaged with 0.05% C-hyperuric acid-Na (Shanghai Aladdin Biochemical Technology Co., Ltd.), and the other groups (hyperuric acid group, allopurine group and polypeptide group) were gavaged with 250 mg / kg / d potassium oxonate (Shanghai Aladdin Biochemical Technology Co., Ltd.) and 150 mg / kg / d hypoxanthine (Shanghai Aladdin Biochemical Technology Co., Ltd.) for 14 days.

[0067] 4.4 Uric acid-lowering drug therapy

[0068] After completing the modeling experiment in step "4.3", a treatment experiment was conducted, that is, the allopurinol group was gavaged with 8 mg / kg / d of allopurinol (Shanghai Aladdin Biochemical Technology Co., Ltd.) as a positive control; the peptide group was gavaged with 20 mg / kg / d of WDDMEKIW peptide, and the blank control group and the hyperuric acid group were replaced with an equal amount of normal saline instead of the uric acid-lowering drug; the gavage treatment lasted for 14 days.

[0069] 4.5 Detection of uric acid content in mouse serum

[0070] After 10 days of administration, the animals were fasted and provided with drinking water normally. All feed and bedding of the animals were removed 24 hours in advance. Each group of mice was anesthetized with ether (Shanghai McLean Biochemical Technology Co., Ltd.), and then 50 μL of blood was collected from the venous plexus of the fundus of the mice. The blood was collected in a sterile centrifuge tube, and after standing at room temperature for 1 hour, the blood was centrifuged at 3,500 r / min at 4°C for 10 minutes to obtain mouse serum. The serum was divided into 1.5 mL centrifuge tubes to prevent repeated freezing and thawing, and stored in a -80°C refrigerator. The uric acid content in the mouse serum was detected using a fully automatic biochemical analyzer (Shenzhen Lanyun Medical Equipment). The uric acid concentration in the mouse serum was measured once every 5 days ( Figure 4 ).

[0071] 4.6 Measurement of body weight

[0072] The body weight of mice was recorded every 5 days. Figure 5) and observe the physiological state of the mice to determine whether the polypeptide has toxic side effects on the growth of mice. After continuous gavage for 14 days, the mice were euthanized, their eyeballs were removed to collect blood, and the intestinal tissues were dissected and separated for intestinal microbial detection.

[0073] 4.7 Results Analysis

[0074] Figure 4 The results showed that the uric acid concentration of the blank group mice changed little during the experiment, that is, the uric acid concentration (200μM) was maintained in a normal range (0.2-0.8mg / dL). The uric acid level (>500μM) of the mice fed with hypoxanthine and potassium oxonate but not treated (hyperuric acid group) remained high compared with the other groups of mice (p<0.05), and there was a trend of continuous increase, indicating that the hyperuricemia model was successfully established; compared with the model control group (i.e., the hyperuricemia group), the hyperuricemia mice treated with allopurinol (allopurinol) The serum uric acid content of mice in the peptide group continued to decrease significantly after the 15th day (p<0.01), and even after the 20th day, its uric acid level was lower than that of the blank group, which may weaken the body's antioxidant capacity, damage neuroprotection, affect immune function, increase the risk of metabolic diseases, and even affect kidney and vascular health; the change trend of uric acid content in mice in the peptide group was similar to that in the allopurine group, but its rate of decline was relatively mild, avoiding the induction of acute gout attacks and increasing the risk of kidney stones. In addition, it also avoided problems such as kidney damage, cardiovascular problems and metabolic disorders. In short, the results of uric acid content determination showed that WDDMEKIW peptide (20 mg / kg / day) can effectively inhibit the uric acid content of hyperuricemic mice, and the therapeutic effect is relatively mild.

[0075] like Figure 5 As shown in the figure, during the animal experiment, the weight of mice in the blank control group gradually increased in 24 days; while the weight of mice in the hyperuric acid group suddenly dropped after the tenth day, and the downward trend was maintained in the later period, which indicates that long-term high uric acid in the blood, especially when it is much higher than 2.0 mg / dL, can cause gout, renal damage, metabolic disorders or drug side effects, and then affect appetite, leading to weight loss in mice. Compared with the hyperuricemia model group (hyperuric acid group), the weight loss rate of mice in the allopurine group and the polypeptide group was controlled, which shows that the intake of polypeptides or allopurinol can alleviate the adverse effects of uric acid on the growth of mice; further, the weight change fluctuation of mice in the polypeptide group was smaller than that in the allopurine group, which may be beneficial to reduce the probability of complications.

[0076] In summary, this example demonstrates that the WDDMEKIW peptide derived from small yellow croaker has the effect of lowering uric acid, and the therapeutic effect is mild, and can be used in the treatment of hyperuricemia.

[0077] Example 5: Effect of small yellow croaker peptide on intestinal flora of mice with hyperuricemia

[0078] 5.1 The CTAB method was used to extract the total DNA of the bacterial flora from the intestinal tract of mice treated with oral gavage for 14 days in Example 4, and the DNA quality was detected by agarose gel electrophoresis, and the DNA concentration was determined by UV spectrophotometer.

[0079] 5.2 Take an appropriate amount of the above DNA sample in a centrifuge tube and dilute the sample concentration to 150ng / μL with sterile enzyme-free water. Use 1% agarose gel and 100V voltage to run gel electrophoresis for 40min to detect the purity and concentration of DNA; and perform PCR amplification experiments according to the operation of the kit (Shanghai ELISA Biotechnology Co., Ltd.), the primers used are 5'-CCTACGGGNGGCWGCAG-3' and 5'-GACTACHVGGGTATCTAATCC-3', which were commissioned to Nanjing GenScript Biotechnology Co., Ltd. for synthesis; the amplification reaction system is shown in Table 3.

[0080] 5.3 The obtained PCR products were purified by beads (Shanghai Sangon Biotechnology Co., Ltd.), sequenced on the Illumina Miseq PE250 platform, and the sequencing results were analyzed by bioinformatics.

[0081] Table 4 PCR amplification reaction system

[0082]

[0083]

[0084] 5.4 Results Analysis

[0085] The intestinal flora is mainly composed of Bacteroidetes and Firmicutes. Bacteroidetes is likened to the "all-rounder" of the intestinal microbial community, with functions such as enhancing immune response, degrading polysaccharides, and dissociating bile acids; Firmicutes plays a key role in nutrition and metabolism. In addition, the ratio of Firmicutes to Bacteroidetes (F / B) plays an important role in maintaining intestinal homeostasis. A decrease in this ratio is usually associated with the occurrence of diseases such as intestinal inflammation. For example, a decrease in the F / B ratio may enhance the host's intestinal uptake of monosaccharides and promote the synthesis of triglycerides in the liver, leading to insulin resistance.

[0086] from Figure 6 It can be seen that compared with the blank (NC) group, the abundance of Bacteroidetes bacteria (72.35%) in the intestinal flora of mice in the high uric acid group (MC) was significantly increased. On the contrary, the abundance of Firmicutes (23.88%) was significantly decreased, resulting in the F / B ratio of only about 0.3, which was much lower than 1.4 in the NC group.

[0087] The Bacteroidetes microbial community in the peptide (WW) group was reduced to 61.60%. Figure 6 (a) shows. Another dominant phylum, Firmicutes, is Figure 6 As shown in (b), the relative abundance of the high uric acid group was the lowest, and the Firmicutes microbial community in the yellow croaker peptide group recovered to 28.54%. The ratio of Firmicutes to Bacteroidetes (F / B) in each group was further analyzed. Figure 6 As shown in (c), compared with the blank group, the ratio of Firmicutes to Bacteroidetes in the high uric acid group was significantly reduced, while the treatment with small yellow croaker peptide could increase this ratio.

[0088] exist Figure 7 In the comparison of genus levels, the lactic acid bacteria in the high uric acid group was significantly reduced compared with the blank group. Studies have shown that lactic acid bacteria are effective in treating hyperuricemia. Allopurinol and small yellow croaker peptide (WDDMEKIW peptide) treatment of hyperuricemia mice will cause an increase in Bifidobacterium, which has biological barriers, vitamin synthesis, immunity enhancement, blood lipid reduction, and anti-aging functions. Small yellow croaker peptide and allopurinol are similar, and can effectively treat hyperuricemia by promoting the increase of intestinal flora species in hyperuricemia mice, inhibiting the decrease of the ratio of Bacteroidetes to Firmicutes, and increasing the number of lactic acid bacteria.

[0089] In summary, small yellow croaker peptide (WDDMEKIW peptide) treats hyperuricemia by affecting intestinal flora.

[0090] The basic principles and main features of the present invention and the advantages of the present invention are shown and described above. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any figure marks in the claims should not be regarded as limiting the claims involved.

Claims

1. A peptide having uric acid lowering function, characterized in that: The peptide comprises a WDDMEKIW or APPERKYSVW amino acid residue sequence.

2. The peptide according to claim 1, characterized in that The peptide comprises the amino acid residue sequence WDDMEKIW.

3. A method for preparing a peptide having uric acid lowering function, characterized in that: The peptide is as described in any one of claims 1 to 2; the method refers to enzymatic hydrolysis of small yellow croaker using papain.

4. A drug for treating hyperuricemia, characterized in that: The drug comprises the peptide according to any one of claims 1 to 2.

5. Use of a peptide in the preparation of a preparation for treating hyperuricemia, characterized in that: The peptide is as described in any one of claims 1 to 2.

6. Use of a peptide in the preparation of food or health products for gout patients, characterized in that: The peptide is as described in any one of claims 1 to 2.

7. A use of a peptide for treating hyperuricemia by affecting intestinal flora, characterized in that: The peptide is as described in any one of claims 1 to 2.

8. The use according to claim 7, characterized in that The intestinal flora belongs to any one or more of the phyla Firmicutes, Bacteroidetes and Proteobacteria.

9. The use according to claim 7, characterized in that The intestinal flora belongs to any one or more of the genera Bifidobacterium, Bacteroides and Firmicutes.

10. A use of a peptide for alleviating complications of hyperuricemia, characterized in that: The peptide is as described in any one of claims 1 to 2; the complication is weight loss.

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