Preparation method and application of rainbow trout fishbone polypeptide compound

Abstract

The invention relates to a preparation method and application of a rainbow trout fishbone polypeptide compound. The preparation method of the rainbow trout fishbone polypeptide compound comprises the following step: carrying out compound enzymolysis on rainbow trout fishbone by using alkaline protease-trypsin. The rainbow trout fishbone polypeptide is obtained through further ultrafiltration, liquid chromatography-mass spectrometry analysis, computer-aided screening technology and in-vitro verification, and the amino acid sequence of the rainbow trout fishbone polypeptide comprises any one or more of DRG, ARG, FDIIK, FVDI, IALEK and AAVK. The rainbow trout fishbone polypeptide compound and rainbow trout fishbone polypeptide provided by the invention have good uric acid reducing activity, can reduce the content of uric acid, and have the advantages of good water solubility, no toxicity and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological products, and particularly relates to a preparation method of a rainbow trout fish bone polypeptide complex and application thereof. BACKGROUND

[0002] Hyperuricemia (H∪A) is a metabolic syndrome caused by high levels of uric acid in the serum, with a threshold of 6.0 mg / dL or above for women, 7.0 mg / dL or above for men, and 5.5 mg / dL or above for adolescents, which is harmful to health. Possible causes of hyperuricemia include disorders of purine metabolism in the body and elevated uric acid concentration in the blood due to disorders of purine metabolism. Xanthine oxidase is a key enzyme in the process of uric acid formation, which can catalyze the conversion of xanthine and hypoxanthine to uric acid, so inhibiting the activity of xanthine oxidase (XOD) is one of the effective means to reduce uric acid.

[0003] Bioactive peptides are increasingly attracting attention due to their high efficiency and high stability. The high bioavailability of bioactive peptides makes them an ideal choice for developing functional foods and nutritional supplements, and the key is to choose which bioactive peptides. The number of amino acids and amino acid sequences of bioactive peptides obtained from different sample sources and different preparation methods are often different.

[0004] Traditional bioactive peptide preparation mainly obtains peptide mixtures through enzymatic hydrolysis technology, and single enzymatic hydrolysis technology is mainly used. Single enzymatic hydrolysis method for preparing bioactive peptides mainly involves enzymatic hydrolysis of the outer layer of amino acids of proteins, followed by enzymatic hydrolysis of the inside of proteins after the outer layer of amino acids is broken down. The enzymatic hydrolysis time is long, the efficiency is low, and the bioactivity is low. The presence of complex peptide components and non / low bioactive peptides in the mixture may affect the activity efficiency. Traditional single peptides are usually obtained by separation and purification technology, which is tedious and time-consuming. With the development of bioinformatics, computer-aided technology is gradually emerging in bioactive peptide screening. Its high-throughput analysis method can save peptide screening time and improve efficiency, but there may be a phenomenon that the screening results do not match the actual activity. The low degree of greenization of rainbow trout processing industry by-products is currently a bottleneck restricting the development of the industry, and the waste of a large amount of by-products has caused huge economic and resource waste. Therefore, it is urgent to provide a simple, efficient and rapid method for preparing rainbow trout fish bone polypeptides. At present, there is no report on the use of alkaline protease- trypsin complex for rainbow trout fish bones to prepare active peptides and their complexes with xanthine oxidase inhibition and uric acid content reduction. SUMMARY

[0005] In view of the low utilization rate and single activity of existing rainbow trout, the present application provides a preparation method and application of a rainbow trout fish bone polypeptide compound treated by a complex enzyme. On this basis, the present application uses the rainbow trout fish bone polypeptide compound as a raw material to realize the preparation of a biological activity peptide capable of reducing the content of uric acid and inhibiting the biological activity of xanthine oxidase by combining traditional separation and purification technology with computer-aided technology.

[0006] The technical solution of the present application to solve the above technical problems is as follows:

[0007] The present application provides a preparation method of a rainbow trout fish bone polypeptide compound, which comprises the following steps of using alkaline protease-tryptase complex to hydrolyze rainbow trout fish bones.

[0008] The beneficial effects of the above technical solution include:

[0009] In the research process, it is found that the preparation of biological activity peptide compound by single enzyme preparation to treat rainbow trout fish bones has the disadvantages of enzyme hydrolysis dead angle, long time consumption, uneven effect, low stability, poor reproducibility, etc. The present application prepares a rainbow trout fish bone polypeptide compound with xanthine oxidase inhibitory activity by using complex enzymes, which can promote the extension of the rainbow trout industry chain and improve the degree of deep processing of industrial by-products of rainbow trout. The preparation method has the advantages of high efficiency and good reproducibility, and the prepared product can reduce the content of uric acid, has high xanthine oxidase inhibitory activity, small peptide molecular weight, etc.

[0010] In the preparation process, the present application uses alkaline protease and trypsin to hydrolyze synchronously, uses trypsin to cut the peptide bond with basic amino acids on the carboxyl side, further exposes the internal hydrophobic amino acid and aromatic amino acid enzyme cutting site. At the same time, the alkaline protease accurately recognizes and cuts, realizes the "layer-by-layer" enzyme cutting of rainbow trout protein, increases the number of small molecules with hydrophobic amino acids and aromatic amino acids as the terminal in the complex, improves the xanthine oxidase enzyme inhibitory activity, and realizes the preparation of rainbow trout fish bone polypeptide compound.

[0011] Further, the rainbow trout fish bones can be defatted and decalcified rainbow trout fish bone powder.

[0012] The defatted and decalcified rainbow trout fish bone powder can be obtained by treating with hydrochloric acid and n-hexane.

[0013] Further, the enzyme hydrolysis is carried out at 40℃ for 4h, and then the enzyme is inactivated.

[0014] Further, the enzyme inactivation conditions include heating at 95℃ for 15min.

[0015] Further, the mass ratio of alkaline protease-trypsin to substrate is 2250U / g.

[0016] Further, the ratio of enzyme activity of alkaline protease to trypsin is 1:2.

[0017] Further, the pH of the enzymatic hydrolysis is 8.

[0018] Further, the method further comprises a step of ultrafiltrating the product after the enzymatic hydrolysis, and collecting components with a molecular weight > 5 kDa, components with a molecular weight 3 kDa-5 kDa and components with a molecular weight < 3 kDa, respectively.

[0019] The present application provides a rainbow trout fish bone polypeptide complex, which is prepared by the above method.

[0020] Further, the rainbow trout fish bone polypeptide complex comprises any one or any combination of DRG, ARG, FDIIK, FVDI, IALEK and AAVK.

[0021] The present application provides an application of the rainbow trout fish bone polypeptide complex in preparing a product for reducing uric acid content, a product for inhibiting xanthine oxidase activity or a product for resisting hyperuricemia; the rainbow trout fish bone polypeptide complex is prepared by the above method.

[0022] The product includes but is not limited to food, health care product, cosmetic, medicine and the like.

[0023] The present application provides a rainbow trout fish bone polypeptide, and the amino acid sequence of the rainbow trout fish bone polypeptide comprises any one or any combination of DRG, ARG, FDIIK, FVDI, IALEK and AAVK.

[0024] The beneficial effects of the above technical solutions include: the rainbow trout fish bone polypeptide provided by the present application can improve the added value of rainbow trout by-product, reduce resource waste and prolong the industrial chain of rainbow trout by-product. It has been verified by experiments that DRG, ARG, FDIIK, FVDI, IALEK and AAVK screened by the present application can effectively inhibit xanthine oxidase activity. The molecular weight of the rainbow trout fish bone polypeptide is less than 20 KDa, small molecule peptides are easily digested and absorbed by the human body, and act on the target. The rainbow trout fish bone polypeptide provided by the present application has good solubility and is non-toxic to cells.

[0025] Further, the amino acid sequence of the rainbow trout fish bone polypeptide is any one or any combination of DRG, ARG, FDIIK, FVDI, IALEK and AAVK.

[0026] The present application provides a preparation method of the above rainbow trout fish bone polypeptide, comprising the following steps: solid-phase synthesis of the above rainbow trout fish bone polypeptide.

[0027] The present application provides an application of the above rainbow trout fish bone polypeptide in preparing a product for reducing uric acid, a product for inhibiting xanthine oxidase activity or a product for resisting hyperuricemia.

[0028] The products include, but are not limited to, food, health products, cosmetics, pharmaceuticals, and the like.

[0029] The rainbow trout fish bone polypeptide provided by the application has excellent inhibitory activity on xanthine oxidase and can reduce the content of uric acid. 50 The IC50 is 0.14-0.85 mg / mL.

[0030] The application provides a screening method of the rainbow trout fish bone polypeptide, comprising the following steps:

[0031] (1) The rainbow trout fish bone is continuously stirred in 0.05 mol / L hydrochloric acid solution at 4℃ for 48h to remove calcium, and then continuously stirred in n-hexane at 4℃ for 24h to remove fat, and then freeze-dried to obtain the treated rainbow trout fish bone; then, the rainbow trout fish bone is crushed to obtain the rainbow trout fish bone powder;

[0032] (2) The rainbow trout fish bone powder is subjected to mixed enzymolysis by alkaline protease and trypsin to obtain a rainbow trout fish bone enzymolysis liquid;

[0033] (3) The rainbow trout fish bone enzymolysis liquid obtained in step (2) is subjected to ultrafiltration and freeze-drying to obtain an active peptide freeze-dried powder;

[0034] (4) The active peptide freeze-dried powder is analyzed by liquid chromatography-mass spectrometry to obtain a peptide amino acid sequence;

[0035] (5) The known amino acid sequence of the peptide obtained in step (4) is screened by computer-aided screening technology;

[0036] (6) The screening peptide is synthesized according to the screening result of step (5) and verified in vitro, for example, the xanthine oxidase inhibitory activity is detected, and the content of uric acid is detected.

[0037] The beneficial effects of the above technical solutions include that the method provided by the application is a high-efficiency, rapid and accurate peptide acquisition method, the traditional separation and purification technology is combined with computer-aided technology, the screening time of the active peptide is saved, the efficiency is improved, the component with inhibitory activity on xanthine oxidase is obtained, and then the accuracy of the peptide is improved. The obtained peptide is screened by computer-aided technology, and then the screening time, efficiency and accuracy are improved.

[0038] The alkaline protease-tryptase mixed enzyme hydrolysis can realize the cutting of the carboxyl side peptide bond of the alkaline amino acid and the hydrophobic amino acid, respectively. In the enzyme hydrolysis process, the tryptase first recognizes and acts on the surface amino acid of the rainbow trout fish bone polypeptide, so that the protein structure is loose. At the same time, the alkaline protease precisely cuts the hydrophobic and aromatic amino acids, thereby improving the xanthine oxidase inhibitory activity of the peptide.

[0039] Further, the specific operation process of step (1) can include: weighing 100g of rainbow trout fish bones, placing them in 500mL of 0.05mol / L hydrochloric acid solution, controlling the temperature to be 4℃, continuously stirring for 48h, replacing the hydrochloric acid solution every 12h, until the fish bones are white and soft in texture; then, placing the fish bones in 50mL of n-hexane, controlling the temperature to be 4℃, continuously stirring for 24h, obtaining the pretreated rainbow trout fish bones, and then freeze-drying, using a powder machine to break, running for 1-2min, until the fish bones are completely in powder form.

[0040] Further, the specific operation process of step (2) can include: weighing the rainbow trout fish bone powder, dissolving it in deionized water, adjusting the pH to 8, adding mixed enzymes, the enzyme activity of the mixed enzymes to the substrate mass ratio is 2250U / g, and the enzyme activity ratio of alkaline protease to trypsin in the mixed enzymes is 1:2; enzyme hydrolysis at 40℃ for 4h, heating at 95℃ for 15min to inactivate the enzyme, centrifuging at 10000r / min for 10min, and retaining the supernatant as the rainbow trout fish bone enzyme hydrolysate.

[0041] Further, the specific operation process of step (3) can include: sequentially grading the rainbow trout fish bone enzyme hydrolysate through 5kDa and 3kDa ultrafiltration membranes, and collecting each component.

[0042] Further, the specific operation process of step (5) can include: preliminarily screening the active peptide segments obtained by mass spectrometry according to the length of the peptide chain and the hydrophobic property; predicting the water solubility and toxicity of the peptide through online tools innovagen and ToxinPred, and retaining the active peptide sequence with good water solubility and no toxicity for molecular docking; using the CDOCK mode in Discovery Studio 2019Client to perform molecular docking with the XOD receptor, and screening the polypeptide according to the binding energy.

[0043] Further, the specific operation process of step (6) can include: synthesizing the screened peptide by solid phase synthesis method, and verifying its biological activity by xanthine oxidase inhibition rate. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1Figure 6 is a graph showing the effect of different concentrations of the rainbow trout bone polypeptide compound powder on the survival rate of HK-2 cells, wherein the abscissa represents the concentration of the enzymatic product and the ordinate represents the cell proliferation rate.

[0045] Figure 2 Figure 7 is a graph showing the effect of different concentrations of the rainbow trout bone polypeptide compound powder on the uric acid content of HK-2 cells in a hyperuricemia model, wherein the abscissa represents the concentration of the enzymatic product and the ordinate represents the uric acid concentration.

[0046] Figure 3 Figure 8 is a graph showing the results of the determination of the inhibition rate of xanthine oxidase by components of different molecular weights, wherein the abscissa represents the molecular weight range of the enzymatic product and the ordinate represents the inhibition rate of xanthine oxidase.

[0047] Figure 4 Figure 9 is a graph showing the results of the interaction of DRG with xanthine oxidase.

[0048] Figure 5 Figure 10 is a graph showing the results of the interaction of ARG with xanthine oxidase.

[0049] Figure 6 Figure 11 is a graph showing the results of the interaction of FDIIK with xanthine oxidase.

[0050] Figure 7 Figure 12 is a graph showing the results of the interaction of FVDI with xanthine oxidase.

[0051] Figure 8 Figure 13 is a graph showing the results of the interaction of IALEK with xanthine oxidase.

[0052] Figure 9 Figure 14 is a graph showing the results of the interaction of AAVK with xanthine oxidase.

[0053] Figure 10 Figure 15 is a graph showing the results of the determination of the inhibition rate of xanthine oxidase by synthetic peptides, wherein the abscissa represents different types of synthetic peptides and the ordinate represents the inhibition rate of xanthine oxidase.

[0054] Figure 11 Figure 16 is a graph showing the effect of different concentrations of various polypeptides on the survival rate of HK-2 cells, wherein the abscissa represents the concentration of the active peptide and the ordinate represents the cell proliferation rate.

[0055] Figure 12 Figure 17 is a graph showing the results of the determination of the inhibition rate of xanthine oxidase by the rainbow trout bone polypeptide compound, wherein 1 represents a sample of rainbow trout bone protein enzymatically digested by a complex of alkaline protease and trypsin, 2 represents a sample of rainbow trout bone protein enzymatically digested by alkaline protease, and 3 represents a sample of rainbow trout bone protein enzymatically digested by trypsin. DETAILED DESCRIPTION

[0056] The principles and features of the present application are described below in conjunction with the accompanying drawings, which are presented only for the purpose of illustrating the present application and are not intended to limit the scope of the present application.

[0057] The present application provides a preparation method of a rainbow trout fish bone polypeptide compound for inhibiting xanthine oxidase activity based on alkaline protease-trypsin treatment, which can comprise the following steps: obtaining a rainbow trout fish bone enzymatic hydrolysate by using alkaline protease-trypsin mixed enzymolysis on the rainbow trout fish bone. The obtained rainbow trout fish bone enzymatic hydrolysate or freeze-dried powder of the enzymatic hydrolysate can also be separated and purified by ultrafiltration.

[0058] Further, the above preparation method can comprise the following steps: taking defatted and decalcified rainbow trout fish bone powder as raw material, and simultaneously enzymolyzing by using alkaline protease and trypsin, and the enzymolysis process comprises: the ratio of enzyme activity of the mixed enzymes to the mass of the substrate is 2250 U / g, the mixed enzymes comprise alkaline protease and trypsin, the ratio of enzyme activity of the alkaline protease to the trypsin is 1:2, the enzymolysis time is 4 h, the enzymolysis temperature is 40℃, and pH is 8. After the enzymolysis is completed, enzyme inactivation treatment is performed by heating at 95℃ for 15 min, centrifugation is performed at 10000 r / min for 10 min, and the supernatant is taken to obtain the rainbow trout fish bone enzymatic hydrolysate.

[0059] Further, the above preparation method can comprise the following steps: after the rainbow trout fish bone enzymatic hydrolysate is freeze-dried, the freeze-dried product is prepared into a solution, and the solution is sequentially subjected to fractionation by using 5 kDa and 3 kDa ultrafiltration membranes, the xanthine oxidase inhibition rates of different components are determined, and the elution components with high xanthine oxidase inhibition capacity are collected and freeze-dried.

[0060] The present application provides a rainbow trout fish bone polypeptide compound, which can be prepared by using the above method.

[0061] The present application provides the application of the above rainbow trout fish bone polypeptide compound in the preparation of products for reducing uric acid and / or inhibiting xanthine oxidase activity. The products include but are not limited to food, health products, cosmetics, medicines, etc.

[0062] The present application provides a rainbow trout fish bone polypeptide with xanthine oxidase activity, a preparation method, a screening method and the application thereof.

[0063] The present application provides six kinds of rainbow trout fish bone polypeptides with xanthine oxidase activity, and the amino acid sequences of the polypeptides are DRG, ARG, FDIIK, FVDI, IALEK and AAVK, respectively. The above rainbow trout fish bone polypeptides can effectively inhibit xanthine oxidase activity.

[0064] The preparation method of the above rainbow trout fish bone polypeptide can comprise the following steps: solid-phase synthesis of the rainbow trout fish bone polypeptide with the above amino acid sequence.

[0065] The application provides application of the above-mentioned oncorhynchus nerka fish bone polypeptide in preparation of products for reducing uric acid and / or inhibiting xanthine oxidase activity. The products include but are not limited to food, health products, cosmetics, medicines and the like.

[0066] The application provides a screening method of the above-mentioned oncorhynchus nerka fish bone polypeptide, comprising the following steps:

[0067] (1) the obtained oncorhynchus nerka fish bone polypeptide complex is subjected to liquid chromatography-mass spectrometry to identify the peptide amino acid sequence.

[0068] (2) the known amino acid sequence of the peptide obtained in step (a) is subjected to computer-aided screening technology to obtain a peptide with potential high xanthine oxidase inhibitory activity.

[0069] (3) the peptide is synthesized by solid phase synthesis, and the xanthine oxidase inhibitory activity is verified in vitro.

[0070] Further, the identification of the oncorhynchus nerka fish bone polypeptide can comprise the following steps: the components separated by reverse phase high performance liquid chromatography are subjected to amino acid sequence identification by liquid chromatography-mass spectrometry (LC-MS / MS). The mobile phase A is 0.1% formic acid; the mobile phase B is 0.1% formic acid-80% acetonitrile solution (V / V); the gradient is started from 4% B phase, and increased to 55% in 23 minutes by non-linear gradient, and then increased to 95% in 2 minutes, and maintained for 5 minutes. The above percentages are all volume percentages.

[0071] Further, the screening of the oncorhynchus nerka fish bone polypeptide can comprise the following steps: the active peptide segments identified by LC-MS / MS are preliminarily screened according to the length of the peptide chain and the hydrophobic property. The water solubility and toxicity of the polypeptide are predicted by online tools Innovagen (http: / / www.innovagen.Com / proteomics-tools) and ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / design.php), and the active peptide sequence with good water solubility and no toxicity is reserved for molecular docking.

[0072] Further, the activity of the oncorhynchus nerka fish bone polypeptide can also be verified by experiment, comprising the following steps: the screened peptide is synthesized by solid phase synthesis method, and the biological activity is verified by xanthine oxidase inhibition rate experiment.

[0073] The application provides a method for isolating and purifying rainbow trout fish bone polypeptides based on alkaline protease-trypsin synchronous enzymolysis, computer screening and the like.

[0074] The experimental methods used in the following examples are conventional methods in the art unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels or prepared by conventional methods. The solutions involved in the application are water unless otherwise specified.

[0075] The rainbow trout was purchased from Jinzhou, Liaoning Province; the xanthine oxidase was purchased from Beijing Solabio Technology Co., Ltd.; the xanthine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the HK-2 cells were purchased from China Center for Type Culture Collection (CCTCC); the PBS was purchased from Biyun Tian Biotechnology Co., Ltd.; the CCK-8 kit was purchased from Biyun Tian Biotechnology Co., Ltd. (C0037); the uric acid kit was purchased from Nanjing Jiancheng Biological Engineering Institute (C012-2-1); the MEM medium was purchased from Wuhan Punsun Life Science and Technology Co., Ltd.; the fetal bovine serum was purchased from Wuhan Punsun Life Science and Technology Co., Ltd.; the 1% double-antibiotic MEM medium containing penicillin G sodium salt and streptomycin sulfate was purchased from Wuhan Punsun Life Science and Technology Co., Ltd., the content of penicillin G sodium salt was 1% (mass percent), and the content of streptomycin sulfate was 1% (mass percent). The alkaline protease was purchased from Beijing Solabio Technology Co., Ltd., the trypsin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., and the adenosine was purchased from Jiangsu Aidi Sheng Biological Technology Co., Ltd.

[0076] The following will be introduced through specific examples.

[0077] Example 1

[0078] 1.1 Preparation of rainbow trout fish bone polypeptide composite powder, comprising the following steps:

[0079] Take 100 g of rainbow trout fish bones and place them in 500 mL of 0.05 mol / L hydrochloric acid solution, control the temperature at 4°C, and continuously stir for 48 h, replace the hydrochloric acid solution every 12 h until the fish bones are white and soft in texture; then place the fish bones in 50 mL of n-hexane, control the temperature at 4°C, and continuously stir for 24 h to obtain pretreated rainbow trout fish bones; then freeze-dry and use a powder machine to break up for 1-2 min until the fish bones are completely powdered to obtain rainbow trout fish bone powder.

[0080] Take the rainbow trout fish bone powder, dissolve it in deionized water, the volume ratio of deionized water to rainbow trout fish bone powder is 30:1, adjust the pH to 8, add mixed enzymes, the enzyme activity of the mixed enzymes is 2250 U / g of the mass of the rainbow trout fish bone powder, the mixed enzymes include alkaline protease and trypsin, the enzyme activity ratio of alkaline protease to trypsin is 1:2 (for example, for every 1 g of substrate rainbow trout fish bone powder, the amount of alkaline protease added can be 750 U, and the amount of trypsin added can be 1500 U); 40°C for 4 h, 95°C for 15 min to inactivate the enzyme, 10000 r / min centrifugation for 10 min, reserve the supernatant as the rainbow trout fish bone enzymatic hydrolysate, freeze-dry to obtain the rainbow trout fish bone polypeptide complex powder.

[0081] 1.2 Detect the xanthine oxidase inhibitory activity of the rainbow trout fish bone polypeptide complex powder, the experimental method is based on the method in the following reference with slight modifications: Qi Xiaofen. Improvement of whey protein peptides on hyperuricemia in rats and its mechanism [D]. Harbin Institute of Technology, 2023. DOI: 10.27061 / d.cnki.ghgdu.2023.005390.

[0082] Set up four groups A, B, C, and D, prepare solutions for each group according to Table 1, and dissolve or dilute xanthine oxidase and the sample to be tested with ultrapure water.

[0083] Table 1 Reaction liquid grouping and dosage

[0084]

[0085]

[0086] Shake each group for 30 s, and incubate in a 37°C water bath for 5 min; then add 300 μL of 0.48 mM xanthine solution to each group, shake for 30 s, and incubate in a 37°C water bath for 30 min; measure the absorbance at 290 nm, and calculate the inhibition rate of different concentrations of active substances on xanthine oxidase according to the following formula.

[0087]

[0088] In the above formula, A AA is the absorbance of Group A at 290 nm; A B B is the absorbance of Group B at 290 nm; A c C is the absorbance of Group C at 290 nm; A D D is the absorbance of Group D at 290 nm.

[0089] The detection results show that the xanthine oxidase inhibition rate IC 50 is 6.46 ± 0.12 mg / mL.

[0090] 1.3 Detection of whether the rainbow trout fish bone polypeptide compound powder has toxicity

[0091] The rainbow trout fish bone polypeptide compound powder obtained by the method of 1.1 is subjected to cell proliferation rate determination, and the experimental method comprises the following steps: the biological safety of the peptide is evaluated by using the CCK-8 method, the HK-2 cells are cultured by using MEM, and the cells are cultured in a logarithmic growth phase. The HK-2 cells are inoculated in a 96-well plate at a density of 1×10 5 After 24 h of culture in an incubator, the culture medium is removed. The cells are divided into a control group and an experimental group. 100 μL of different concentrations of peptide samples (0.1875 mg / mL, 0.375 mg / mL, 0.75 mg / mL, 1.5 mg / mL, and 3.0 mg / mL, dissolved in MEM) are added to the experimental group, and 100 μL of MEM culture medium without peptide samples is added to the control group. The blank group is added with 100 μL of MEM culture medium without HK-2 cells. Then 10% CCK-8 solution is added to each well, and incubated at 37°C for 1 h. The absorbance is measured at 450 nm, and the cell survival rate (i.e. cell proliferation rate) is calculated according to the following formula.

[0092]

[0093] The experimental results are shown in Table 1. Figure 1 The rainbow trout fish bone polypeptide compound powder with a concentration of 3 mg / mL or less has no effect on the survival rate of HK-2 cells, indicating that the rainbow trout fish bone polypeptide compound prepared by the present application has no toxic side effects.

[0094] 1.4 Detection of the ability of the rainbow trout fish bone polypeptide compound powder to reduce the content of uric acid at the cellular level

[0095] The rainbow trout fish bone polypeptide compound powder obtained by the method of 1.1 is subjected to cell uric acid content determination, and the experimental method comprises the following steps:

[0096] (1) Cell culture: HK-2 human renal tubular epithelial clone cells were cultured in double-antibiotic MEM medium containing fetal bovine serum (FBS) with a volume percentage of 10%, and the double-antibiotic was penicillin G sodium salt and streptomycin sulfate, with a content of 1% (mass percentage) of penicillin G sodium salt and 1% (mass percentage) of streptomycin sulfate. The cells were cultured in a 37°C, 5% CO2 cell incubator to the logarithmic growth phase.

[0097] (2) The logarithmic growth phase HK-2 cells were taken and added to MEM medium at a density of 1×10 5 The HK-2 cells were plated in a 24-well plate at a density of 1×10

[0098] The cells were divided into a blank group, a model group, and an experimental group. To the experimental group, 1 mL of peptide samples of different concentrations (0.125, 0.25, 0.5, 1.0, 2.0 mg / mL, dissolved in MEM) was added, and to the blank group and the model group, 1 mL of MEM medium was added. Each group was incubated in a 37°C, 5% CO2 incubator for 24 h. Then, to the experimental group and the model group, 1 mL of 2.5 mmol / L adenosine solution (dissolved in MEM) was added, and to the blank group, 1 mL of MEM medium was added. Each group was incubated in a 37°C, 5% CO2 incubator for 24 h. Then, to the experimental group and the model group, 1 mL of 0.005 U / mL xanthine oxidase solution (dissolved in MEM) was added, and to the blank group, 1 mL of MEM medium was added. Each group was incubated in a 37°C, 5% CO2 incubator for 4 h.

[0099] The uric acid (UA) test kit was used to determine the uric acid content, with 5 μL of blank / standard / sample added to each well, and 250 μL of reagent one, incubated at 37°C for 10 min. The above reagents were provided by the UA kit of Nanjing Jiancheng Biological Engineering Institute. The absorbance was measured at a wavelength of 510 nm.

[0100]

[0101] In the above formula, A 测定 is the absorbance of the experimental group or the model group at 510 nm; A 空 is the absorbance of the blank group at 510 nm; A 标准 is the absorbance of the standard at 510 nm; C 校准品 is 400 μmol / L. The standard and the calibrator were provided with the UA test kit, and the operation was performed according to the kit instructions.

[0102] The experimental results are shown in Table 1. Figure 2As shown, the rainbow trout fish bone polypeptide composite powder can effectively reduce the content of uric acid in cells, and with the increase of the concentration of the rainbow trout fish bone polypeptide composite powder, the content of uric acid gradually decreases, indicating that the prepared rainbow trout fish bone polypeptide composite has the ability to reduce uric acid.

[0103] Example 2

[0104] The rainbow trout fish bone polypeptide composite powder obtained in 1.1 of Example 1 was configured into a solution with a concentration of 20 mg / mL with ultrapure water, and was sequentially subjected to ultrafiltration membranes with molecular weights of 5 kDa and 3 kDa, respectively, to collect components with molecular weights >5 kDa, components with molecular weights of 3 kDa-5 kDa, and components with molecular weights <3 kDa. The xanthine oxidase inhibition rate of each collected component was determined, and the detection method referred to 1.2 in Example 1. According to the screening results above, the component with higher clearance rate was selected and freeze-dried for later use.

[0105] The screening results are shown in Table 2 and Figure 3 As shown, it can be seen that the xanthine oxidase inhibition rate of the component with molecular weight <3 kDa is significantly better than that of other components, therefore, the component with molecular weight <3 kDa is selected for subsequent mass spectrometry identification.

[0106] Table 2 Xanthine oxidase inhibition rate of each ultrafiltration component

[0107]

[0108] Example 3 Identification of amino acid sequence of rainbow trout fish bone polypeptide

[0109] The amino acid sequence identification was performed by Beijing Baitai Pailake Biological Technology Co., Ltd., including the following steps: using liquid chromatography-mass spectrometry (LC-MS / MS) to identify the amino acid sequence of the components separated by reverse phase high performance liquid chromatography. The mobile phase A is 0.1% formic acid; the mobile phase B is 0.1% formic acid-80% acetonitrile solution (V / V); the gradient starts from 4% B phase, and increases to 55% in 23 min with a nonlinear gradient, and then increases to 95% in 2 min, and maintains for 5 min. The above percentages are all volume percentages.

[0110] By comparing with the protein database, 150 sequences were obtained, and Table 3 only shows part of the results of 10 peptide segments with higher scores identified from one sample.

[0111] Table 3 Identification of peptide sequences in SCP-2-4 by LC-MS / MS method

[0112]

[0113] Example 4 Screening and molecular docking of rainbow trout fish bone polypeptide

[0114] From the 150 sequences obtained from Example 3, the peptide sequences were preliminarily screened according to the molecular weight and the number of amino acids, and peptides with a molecular weight less than 1500 Da and an amino acid number ≤10 were selected for subsequent screening.

[0115] The water solubility and biological toxicity of the polypeptides were screened and predicted by online tools Innovagen (www.innovagen.Com / proteomicstools) and ToxinPred (http: / / crdd.osdd.net / raghava / / toxinpred), and the polypeptides with the software results showing “good solubility” and no toxicity were retained.

[0116] Molecular docking screening (LibDock) was performed using the CDOCK mode in Discovery Studio 2019 Client with the XOD (PBDID: 3NVV) receptor, and the LibDock score and docking score were recorded. Finally, 6 new peptide segments with potential good biological activity, good water solubility and no toxicity were screened (Table 4).

[0117] Table 4 Biological activity prediction, toxicity, solubility and other characteristics of rainbow trout fish bone polypeptides

[0118]

[0119]

[0120] Note: “-” in the table indicates that it cannot be docked at the same time, and no docking score is given. C(+57.021) in No. 18 represents Carbamidomethylation (C) alkylation modification, which occurs on cysteine, and the molecular weight changes by +57.021 Da.

[0121] The molecular docking results of DRG, ARG, FDIIK, FVDI, IALEK and AAVK are shown in Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Figure 9As shown, DRG binds most tightly to xanthine oxidase, indicating that DRG may have a potentially good inhibitory ability on xanthine oxidase. The bioactive peptide DRG is mainly linked to the SER928, ALA938, LEU998, GLU1292, and PHE1294 residues of XOD via hydrogen bonds; and is linked to GLU929, GLU939, ARG942, LYS981, and ASP1276 via electrostatic interactions. The bioactive peptide ARG is mainly linked to the LEU936, ARG1279, GLU1292, and PHE1294 residues of XOD via hydrogen bonds; and is linked to GLU929 and ARG942 via electrostatic interactions. The bioactive peptide FDIIK is mainly linked to the ALA938, TYR977, and PHE1272 residues of XOD via hydrogen bonds; and is linked to LYS981, ASP985, LYS1275, and ASP1276 via electrostatic interactions. The bioactive peptide FVDI is primarily linked to the SER928, ALA938, ASP1276, and ARG1279 residues of XOD via hydrogen bonds; and is also linked to LYS981, LYS1275, ASP1276, and ARG1279 via electrostatic interactions. The bioactive peptide IALEK is primarily linked to the ALA939, LEU998, PHE1272, and ASN1289 residues of XOD via hydrogen bonds; and is also linked to ASP1276, ARG1279, and GLU1292 via electrostatic interactions. The bioactive peptide AAVK is primarily linked to the SER928, LEU936, ASP1276, ARG1279, and GLU1292 residues of XOD via hydrogen bonds; and is also linked to GLU929 and ARG942 via electrostatic interactions.

[0122] Example 5

[0123] (1) Detection of the inhibition rate of active peptides on xanthine oxidase

[0124] The six selected bioactive peptides (DRG, ARG, FDIIK, FVDI, IALEK, and AAVK) were synthesized in a solid-phase manner by Sangon Biotech (Shanghai) Co., Ltd., with a purity exceeding 98%. The xanthine oxidase inhibition rate of the six bioactive peptides was verified, and the experimental method was as described in section 1.2 of Example 1.

[0125] Test results as follows Figure 10 As shown, DRG, ARG, FDIIK, FVDI, IALEK, and AAVK can all effectively inhibit xanthine oxidase. All six of these bioactive peptides can effectively inhibit xanthine oxidase activity, and their IC50 values ​​are [not specified]. 500.14±0.01mg / mL(0.4mmol / L), 0.26±0.02mg / mL(0.8mmol / L), 0.85±0.03mg / mL(1.3mmol / L), 0.77±0.03mg / mL(1.5mmol / L), 0.36±0.01mg / mL(0.6mmol / L), 0.44±0.01mg / mL(1.1mmol / L), respectively. It is proved that the polypeptide in the application has a great improvement on the inhibition rate of xanthine oxidase.

[0126] And the xanthine oxidase inhibition rates of the rainbow trout fish bone polypeptides DRG, ARG, FDIIK, FVDI, IALEK and AAVK are significantly higher than those of the rainbow trout fish bone polypeptide complex. Through further purification and identification of the rainbow trout fish bone polypeptide complex, a single peptide with high xanthine oxidase inhibition rate can be obtained.

[0127] (2) Biological safety evaluation of active peptides

[0128] The cytotoxicity of cells was detected, and the experimental method was the same as that in Example 1.3. 100μL of different concentrations of peptide samples (0.125mg / mL, 0.25mg / mL, 0.5mg / mL, 1.0mg / mL, 2.0mg / mL, dissolved in MEM) were added to the experimental group, and the rest were the same as in Example 1.3.

[0129] The experimental results are shown in Table 1. Figure 11 Different concentrations of DRG, ARG, FDIIK, FVDI, IALEK and AAVK have no effect on the survival rate of HK-2 cells, indicating that the polypeptides prepared in the application have no toxic side effects.

[0130] Comparative Example 1

[0131] On the basis of Example 1.1, alkaline protease was used to replace the mixed enzyme (alkaline protease-trypsin) in 1.1. The enzyme amount of alkaline protease was 2250U / g (i.e. the enzyme activity of alkaline protease to the mass ratio of rainbow trout fish bone powder was 2250U / g), and the enzyme was hydrolyzed at pH=8 and temperature 40℃ for 4h. The enzyme was inactivated by heating at 95℃ for 15min, and centrifuged at 10000r / min for 10min. The supernatant was reserved as the rainbow trout fish bone hydrolysate, and the rest was the same as in 1.1.

[0132] Comparative Example 2

[0133] On the basis of Example 1.1, trypsin is used to replace the mixed enzyme (alkaline protease-trypsin) in 1.1, the amount of trypsin added is 2250 U / g (i.e. the mass ratio of trypsin enzyme activity to rainbow trout fish bone powder is 2250 U / g), the enzyme is hydrolyzed at pH = 8 and a temperature of 40℃ for 4h. The enzyme is inactivated by heating at 95℃ for 15min, and centrifuged at 10000r / min for 10min. The supernatant is reserved as the rainbow trout fish bone hydrolysate, and the rest is the same as 1.1.

[0134] Effect Example 1 detects the biological activity of the rainbow trout fish bone hydrolysate

[0135] The rainbow trout fish bone hydrolysate prepared in Example 1, Comparative Example 1 and Comparative Example 2 is freeze-dried and used for xanthine oxidase inhibition activity detection, and the detection method is referred to Example 1.2.

[0136] The xanthine oxidase inhibition activity detection results are shown in Table 5 and Figure 12 As can be seen from Table 5, the xanthine oxidase inhibition activity of the rainbow trout fish bone polypeptide complex prepared by the method of Example 1 of the present application is better than that of the sample prepared by a single enzyme.

[0137] Table 5 Biological activity evaluation of peptide complex

[0138]

[0139] The six polypeptides (DRG, ARG, FDIIK, FVDI, IALEK and AAVK) prepared by the present application have much better xanthine oxidase inhibition ability than the prior art. Taking the literature (Qi Xiaofen. Improvement effect and mechanism of whey protein peptides on hyperuricemia in rats [D]. Harbin Institute of Technology, 2023. DOI: 10.27061 / d.cnki.ghgdu.2023.005390.) as an example, the xanthine oxidase inhibition activity of the active peptides IC 50 of the final screening in the literature are LLW (3.02 mmol / L) and PEW (3.46 mmol / L), respectively. The IC 50 of the polypeptides of the present application for xanthine oxidase inhibition is 1.4-1.5 mmol / L, which shows that the polypeptides of the present application have stronger xanthine oxidase inhibition effect and uric acid lowering effect.

[0140] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a polypeptide complex of rainbow trout fish bone, characterized by, The method comprises the following steps: The fish bone of rainbow trout is hydrolyzed by using alkaline protease-trypsin complex.

2. The method of claim 1, wherein the oncorhynchus mykiss bone polypeptide complex is prepared by the steps of: The enzyme is inactivated after hydrolysis for 4 hours at 40℃.

3. The method of claim 1 or 2, wherein the preparation of the polypeptide complex of rainbow trout fish bones is characterized by, The mass ratio of alkaline protease-trypsin to substrate is 2250 U / g.

4. The method of claim 1 or 2, wherein the rainbow trout bone polypeptide complex is prepared by the steps of: The ratio of alkaline protease to trypsin is 1:

2.

5. The method of claim 1 or 2, wherein the preparation of the polypeptide complex of rainbow trout fish bone is characterized by, The pH of enzyme hydrolysis is 8.

6. The method of claim 1 or 2, wherein the rainbow trout bone polypeptide complex is prepared by the steps of: The product after enzyme hydrolysis is further subjected to ultrafiltration, and components with molecular weight >5 kDa, molecular weight 3 kDa-5 kDa and molecular weight <3 kDa are collected respectively.

7. The use of polypeptide complex of fish bone of rainbow trout in the preparation of products for reducing uric acid content, products for inhibiting the activity of xanthine oxidase or products for resisting hyperuricemia; the polypeptide complex of fish bone of rainbow trout is prepared by the method according to any one of claims 1-6.

8. A rainbow trout bone polypeptide, characterized in that, The amino acid sequence of the polypeptide of fish bone of rainbow trout comprises any one or any combination of DRG, ARG, FDllK, FVDl, IALEK and AAVK.

9. The method of claim 8, wherein the oncorhynchus mykiss bone polypeptide is prepared by the steps of: The method comprises the following steps: The polypeptide of fish bone of rainbow trout is synthesized by solid phase synthesis.

10. The use of the polypeptide of fish bone of rainbow trout according to claim 8 in the preparation of products for reducing uric acid, products for inhibiting the activity of xanthine oxidase or products for resisting hyperuricemia.

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