A bird's nest peptide for inhibiting xanthine oxidase and application thereof

Abstract

The application discloses a bird's nest peptide for inhibiting xanthine oxidase and application thereof, and belongs to the technical field of functional polypeptides. The amino acid sequence of the bird's nest peptide for inhibiting xanthine oxidase is WGLL, can chelate an active center of xanthine oxidase (XOD), and is helpful for inhibiting the activity of XOD in catalyzing conversion of xanthine or hypoxanthine. The application further provides a bird's nest peptide powder containing the bird's nest peptide for inhibiting xanthine oxidase. The bird's nest peptide powder can significantly reduce the rate of conversion of xanthine or hypoxanthine into uric acid catalyzed by XOD, has good uric acid reducing effect, can be applied to development of uric acid reducing products or various uric acid reducing products, and has high market value.

Description

Technical Field

[0001] This invention specifically relates to a bird's nest peptide that inhibits xanthine oxidase and its application, belonging to the field of functional polypeptide technology. Background Technology

[0002] In modern society, hyperuricemia has become the fourth leading metabolic disease, attracting widespread attention. Bird's nest, a precious food ingredient, is rich in water-soluble protein, carbohydrates, trace elements, and various important amino acids such as sialic acid. It has been found to have certain uric acid-lowering effects, making it highly sought after. However, although bird's nest is highly nutritious and has significant medicinal value, its deep processing research is still in its early stages. Processing methods mainly involve simple steaming and boiling, which makes the large protein molecules in bird's nest difficult for the intestines to digest and absorb, resulting in the limited effectiveness of existing bird's nest products in lowering uric acid.

[0003] Xanthine oxidase (XOD) catalyzes the conversion of xanthine and hypoxanthine into uric acid, and is a major cause of elevated uric acid levels. Current treatments for hyperuricemia commonly use probenecid, benzbromarone, preclinique, labribase, and allopurinol, but these drugs have serious side effects and may cause potential liver and kidney damage. Therefore, isolating novel xanthine oxidase-inhibiting bird's nest peptides with bioactivity and bioavailability that can inhibit XOD activity is of great significance for promoting the development of uric acid-lowering products and facilitating the market application of bird's nest. Summary of the Invention

[0004] This invention provides a xanthine oxidase-inhibiting bird's nest peptide and its application. This xanthine oxidase-inhibiting bird's nest peptide can chelate the active site of xanthine oxidase (XOD), helping to inhibit the activity of XOD in catalyzing the conversion of xanthine or hypoxanthine. In addition, this invention also provides a bird's nest peptide powder containing the above-mentioned xanthine oxidase-inhibiting bird's nest peptide. This bird's nest peptide powder can significantly reduce the rate at which XOD catalyzes the conversion of xanthine or hypoxanthine into uric acid, thereby achieving a good uric acid-lowering effect. It can be applied to the development of uric acid-lowering products or various uric acid-lowering products and has high market value.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides a xanthine oxidase inhibitory bird's nest peptide, wherein the amino acid sequence of the xanthine oxidase inhibitory bird's nest peptide is WGLL (SEQ ID NO:1).

[0007] This invention utilizes enzymatic hydrolysis, ultrafiltration, gel chromatography purification, amino acid sequencing, and computer-aided virtual screening techniques to screen dried bird's nest powder, thereby obtaining a novel xanthine oxidase inhibitor bird's nest peptide (SEQ ID NO:1). This xanthine oxidase inhibitor bird's nest peptide has a molecular weight of 487.2794 Da and exhibits a high affinity for xanthine oxidase (XOD), chelating the active site of XOD and inhibiting its catalytic conversion of xanthine or hypoxanthine.

[0008] The bird's nest peptide that inhibits xanthine oxidase provided by this invention can also be used in the preparation of various uric acid-lowering products.

[0009] The present invention also provides a bird's nest peptide powder that lowers uric acid, wherein the bird's nest peptide powder contains the above-mentioned bird's nest peptide that inhibits xanthine oxidase.

[0010] This invention also provides a method for preparing the above-mentioned uric acid-lowering bird's nest peptide powder, which is obtained by ultrafiltration chromatography from the enzymatic hydrolysate of dried bird's nest powder, including the following specific steps:

[0011] S1. Weigh out the dried bird's nest powder and place it in water for ultrasonic crushing and even dispersion to obtain bird's nest liquid;

[0012] S2. Adjust the pH of the bird's nest liquid to 7.5-8.5, add 1-2% of the mass of dried bird's nest powder with trypsin and enzymatically hydrolyze at 40-50℃ for 4 hours, then inactivate the enzyme with boiling water to obtain trypsin hydrolysate.

[0013] S3. Adjust the pH of the trypsin hydrolysate to 1.5-2.5, add 1-2% of the weight of the dried bird's nest powder with pepsin, and perform enzymatic hydrolysis at 35-40℃ for 4 hours. Then, inactivate the enzyme with boiling water to obtain the pepsin hydrolysate.

[0014] S4. Centrifuge the pepsin hydrolysate obtained in step S3, ultrafilter out the components with a molecular weight <3 kDa from its supernatant, and use gel chromatography to separate the polypeptide with the highest xanthine oxidase inhibition rate. Freeze-dry to obtain the bird's nest peptide powder.

[0015] Furthermore, in step S1, the ratio of dried bird's nest powder to water in the bird's nest liquid is 1:30; the ultrasonic crushing power is 25 kW, and the crushing time is 25 s.

[0016] Furthermore, in step S2, the enzyme activity of trypsin is ≥2500 U / mg.

[0017] Furthermore, in step S3, the pepsin enzyme activity is ≥3000 U / mg.

[0018] Further, the separation method of the polypeptide with the highest xanthine oxidase inhibition rate in step S4 includes the following steps: using an AKTA system equipped with a Superdex™ peptide 10 / 300 GL gel chromatography column to perform column chromatography on the ultrafiltered components with a molecular weight <3kDa, a total of 4 elution peaks are obtained, the eluents corresponding to the 4 elution peaks are collected, and the eluents are freeze-dried to obtain 4 polypeptides; then, using the xanthine oxidase inhibition rate as the evaluation index, the polypeptide with the highest xanthine oxidase inhibition rate is screened from the 4 polypeptides through an in vitro xanthine oxidase inhibition experiment;

[0019] The column chromatography conditions were as follows: sample concentration 20 mg / mL, sample volume 1 mL, flow rate 300 μL / min, one tube collected every 200 μL, and absorbance curves were plotted at 220 nm.

[0020] Furthermore, the in vitro xanthine oxidase inhibition experiment described in step S4 includes the following steps:

[0021] The polypeptide component was mixed with water to prepare a polypeptide solution, which was then mixed with phosphate buffer (PBS) and xanthine oxidase. Xanthine and nitrotetrazole blue chloride (NBT) were then added sequentially, and the mixture was shaken and incubated at 37°C in the dark for 30 min. The ultraviolet absorbance at a wavelength of 560 nm was then measured.

[0022] Unlike existing technologies, this invention has the following advantages:

[0023] 1. This invention provides a bird's nest peptide that can inhibit xanthine oxidase activity. The amino acid sequence of the bird's nest peptide is WGLL and the molecular weight is 487.2794 Da. The bird's nest peptide can chelate the active site of xanthine oxidase, thereby inhibiting the activity of xanthine oxidase in catalyzing the conversion of xanthine or hypoxanthine. It can be applied to the development of uric acid-lowering products.

[0024] 2. The present invention also provides a bird's nest peptide powder containing the above-mentioned bird's nest peptide that inhibits xanthine oxidase. This bird's nest peptide powder can effectively inhibit the rate at which xanthine or hypoxanthine is converted into uric acid by xanthine oxidase, and has a good uric acid-lowering effect. It is safe and has no toxic side effects, and has high market economic value.

[0025] 3. The bird's nest peptide powder provided by this invention is extracted from dried bird's nest fragments. The raw materials are natural and clean. This invention decomposes and separates the polypeptide components with a molecular weight of less than 3 kDa through secondary enzymatic hydrolysis and analyzes and screens out the polypeptide components with the best xanthine oxidase inhibition effect. This maximizes the utilization of the nutritional components of bird's nest, broadens the development path of uric acid-lowering products, and improves the economic value and market position of bird's nest. Attached Figure Description

[0026] Figure 1 The absorbance curves are for the gel chromatography column precipitates with molecular weight <3 kDa obtained by ultrafiltration in Examples 1 and 2.

[0027] Figure 2 Xanthine oxidase inhibition rates of the four polypeptides labeled 1-1, 1-2, 1-3, and 1-4 separated by chromatography in Examples 1 and 2.

[0028] Figure 3 This is a secondary mass spectrum of the WGLL bird's nest peptide amino acid sequence.

[0029] Figure 4 The conformation of the WGLL bird's nest peptide in the XOD pocket, its binding mode in the XOD active site, and its interaction plane diagram are shown.

[0030] Figure 5 The xanthine oxidase inhibition rate of EBNP-1 in Example 2, EBNP-2 and EBNP-3 in Comparative Examples 1-2, and ad indicates significant differences between groups. P <0.05).

[0031] Figure 6 Example 2: EBNP-1 bird's nest peptide powder's cytotoxicity against LO2 cells. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0035] This invention provides a xanthine oxidase inhibitory bird's nest peptide, wherein the amino acid sequence of the xanthine oxidase inhibitory bird's nest peptide is WGLL (SEQ ID NO:1).

[0036] This invention utilizes enzymatic hydrolysis, ultrafiltration, gel chromatography purification, amino acid sequencing, and computer-aided virtual screening techniques to screen dried bird's nest powder, thereby obtaining a novel xanthine oxidase inhibitor bird's nest peptide (SEQ ID NO:1). This xanthine oxidase inhibitor bird's nest peptide has a molecular weight of 487.2794 Da and exhibits a high affinity for xanthine oxidase (XOD), chelating the active site of XOD and inhibiting its catalytic conversion of xanthine or hypoxanthine.

[0037] The bird's nest peptide that inhibits xanthine oxidase provided by this invention can also be used in the preparation of various uric acid-lowering products.

[0038] The following specific embodiments further illustrate the xanthine oxidase-inhibiting bird's nest peptide of the present invention and its applications:

[0039] The experimental indicators and methods involved in the following embodiments are as follows:

[0040] (1) Gel column chromatography

[0041] The gel chromatography column used was a Superdex™ peptide 10 / 300 GL gel chromatography column. The chromatography method was as follows: the sample concentration was 20 mg / mL, the sample volume was 1 mL, the flow rate was 300 μL / min, and one tube was collected every 200 μL. The absorbance curves of each chromatographic component were detected under a 220 nm wavelength UV detector and plotted.

[0042] (2) In vitro xanthine oxidase inhibition experiment

[0043] The experimental parameters and methods for the in vitro xanthine oxidase inhibition experiment involved in the following examples are as follows:

[0044] 100 μL of 0.1 M neutralized phosphate-buffered saline (PBS), 25 μL of peptide solution, and 25 μL of 5 U / mL xanthine oxidase (XOD) were added sequentially to a 96-well plate. The mixture was vortexed and incubated at 37°C for 10 min. Then, 50 μL of 1 mM xanthine and 25 μL of 1 mM nitrotetrazole blue chloride (NBT) were added sequentially. The mixture was vortexed and incubated at 37°C in the dark for 30 min. The absorbance at 560 nm was then measured. Allopurinol was used as the positive control. PBS was used instead of peptide solution and xanthine in the zeroing group; PBS was used instead of the sample in the complete reaction group; and PBS was used instead of xanthine oxidase in the sample control group. The inhibition rate of the peptide against XOD was calculated using the following formula:

[0045] XOD inhibition rate (%) = [1 - (C3 - C4) / (C2 – C1)] × 100%

[0046] In the formula: C1 is the zeroing group; C2 is the complete reaction group; C3 is the sample reaction group; C4 is the sample control group.

[0047] Example 1

[0048] This embodiment provides a method for obtaining bird's nest peptides that inhibit xanthine oxidase, comprising the following steps:

[0049] 1) Preparation of bird's nest peptide powder

[0050] This embodiment obtains bird's nest peptide powder by enzymatic hydrolysis, ultrafiltration separation, and gel chromatography purification of dried bird's nest powder, including the following steps:

[0051] S1. Weigh out the dried bird's nest powder and place it in water for ultrasonic crushing and even dispersion. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid.

[0052] S2. Adjust the pH of the bird's nest liquid to 8.5, add 1% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 50℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate.

[0053] S3. Adjust the pH of the trypsin hydrolysate to 2.0, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 37℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate.

[0054] S4. Centrifuge the pepsin hydrolysate obtained in step S3, and use an ultrafiltration tube with a molecular weight cutoff of 3 kDa to ultrafilter out the components with a molecular weight <3 kDa from its supernatant.

[0055] S5. The components with a molecular weight <3 kDa obtained in step S4 were separated by Superdex™ peptide 10 / 300 GL gel chromatography column chromatography. The peptide with the highest xanthine oxidase inhibition rate was screened by in vitro xanthine oxidase inhibition experiment, and then freeze-dried to obtain the bird's nest peptide powder.

[0056] The absorbance curves of the components obtained by gel chromatography in this embodiment under a 220 nm ultraviolet wavelength detector are shown in the figure below. Figure 1 As shown, the fraction with a molecular weight <3 kDa was separated into four polypeptides labeled 1-1, 1-2, 1-3, and 1-4 by gel chromatography; Figure 2 As shown, the four obtained polypeptides were verified by in vitro xanthine oxidase inhibition experiments, and polypeptides 1-4 were the polypeptides with the highest xanthine oxidase inhibition rate.

[0057] 2) Amino acid sequence identification

[0058] In this embodiment, liquid chromatography-mass spectrometry (LC-MS / MS) and PEAKS Studio 11 mass spectrometry software were used to identify the amino acid sequence of the bird's nest peptide powder obtained above. The library search conditions for LC-MS / MS and PEAKS Studio 11 mass spectrometry software are as follows:

[0059] Liquid chromatography (LC) method: The chromatographic column was a NanoViper C18 1.9 μm, 100A, with phase A being 0.1% FA and phase B being 0.1% FA and 80% ACN. The flow rate was 50 nL / min, with a 70-min chromatographic gradient. The specific elution gradient was as follows: 0–1 min, phase B increased uniformly from 8% to 12%; 1–9 min, phase B increased uniformly from 12% to 15%; 9–31 min, phase B increased uniformly from 15% to 25%; 31–38 min, phase B increased uniformly from 25% to 36%; 38–45 min, phase B increased uniformly from 36% to 60%; 45–53 min, phase B increased uniformly from 60% to 95%; 53–60 min, the mobile phase remained unchanged; 60–70 min, phase B decreased uniformly from 95% to 8%.

[0060] Mass spectrometry (MS) method: Primary mass spectrometry parameters: Resolution: 120000; AGC target: Custorm; Maximum IT: Custorm; Scan range: 100 to 1000 m / z. Secondary mass spectrometry parameters: Resolution: 15000; AGC target: Custorm; Maximum IT: Custorm; Cycle time: 3 s; NCE / stepped NCE: 30.

[0061] Search criteria: The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: Fixed modifications: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (Peptide N-term); Enzyme: Non-specific; Database: Apodiformes; Peptide Mass Tolerance (PMT): 20 ppm; Fragment Mass Tolerance (FMT): 0.02 Da.

[0062] 3) Molecular docking screening

[0063] This embodiment uses computer virtual screening technology to screen potential xanthine oxidase inhibitory peptides from the amino acid sequence obtained in step 2), and includes the following screening conditions and steps:

[0064] i) For the peptide amino acid sequences obtained by LC-MS / MS and PEAKS Studio 11 mass spectrometry analysis software, firstly, the activity of the peptides was assessed using the PeptideRanker tool, where a score greater than 0.9 was set (indicating that these peptides are likely to have biological activity); the potential toxicity of the peptides was predicted using the ToxinPred tool to exclude peptides with potential application risks; peptides with a score greater than 0.5 were screened using the CPPpred tool (indicating that the peptides have good cell penetration); and novel peptides in the obtained peptide amino acid sequences were identified using the Biopep database.

[0065] ii) The novel peptides were screened for bioactivity using the molecular docking software Autodock (version 1.5.7) to obtain peptide sequences with strong protein binding ability; the spatial structure of the peptides was drawn using ChemDraw 22 software and energy minimization was performed; the protonation state and hydrogen orientation were optimized in Autodock and saved as a pdbqt file as a ligand file;

[0066] iii) Obtain the 3D structure of XOD (PDB ID: 3BDJ) from the PDB database, remove repetitive sequences and water molecules, and add polar hydrogen atoms to impart a Ko11man combined diatomic local charge to the XOD. Set the gridbox within the predicted XOD active pocket (containing a MoS hydrophobic cavity), with the active center sites set as: x: 94.925, y: 44.105, z: 117.513, and a grid size of 22.11 Å × 27.63 Å × 28.74 Å.

[0067] In this embodiment, 16 new polypeptide sequences were identified from bird's nest peptide powder. Among them, the bird's nest peptide with the strongest interaction with xanthine oxidase (XOD) and the most potential uric acid-lowering effect is the bird's nest peptide with the amino acid sequence WGLL (SEQ ID NO:1). The MS spectrum of this bird's nest peptide is shown below. Figure 3 As shown. The interaction between the xanthine oxidase-inhibiting bird's nest peptide and XOD is as follows. Figure 4 As shown, the binding energy of WGLL to XOD can reach -11.2 kcal / mol. This xanthine oxidase inhibitory peptide can enter the active site of XOD, occupy the hydrophobic pocket, and form a bond with the surrounding amino acid residues. The binding is relatively tight, which can effectively prevent the entry of purine substrates and achieve the activity inhibition effect of xanthine oxidase.

[0068] Example 2

[0069] This embodiment provides a bird's nest peptide powder that lowers uric acid, which contains the bird's nest peptide that inhibits xanthine oxidase obtained in Example 1.

[0070] The preparation of the uric acid-lowering bird's nest peptide powder includes the following steps:

[0071] S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid.

[0072] S2. Adjust the pH of the bird's nest liquid to 8.5, add 1% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 50℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate.

[0073] S3. Adjust the pH of the trypsin hydrolysate to 2.0, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 37℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate.

[0074] S4. Centrifuge the pepsin hydrolysate obtained in step S3, and use an ultrafiltration tube with a molecular weight cutoff of 3 kDa to ultrafilter out the components with a molecular weight <3 kDa from its supernatant.

[0075] S5. Using Superdex™ peptide 10 / 300 GL gel chromatography column chromatography, the components with a molecular weight <3 kDa obtained in step S4 are screened by in vitro xanthine oxidase inhibition experiment to identify the peptide with the highest xanthine oxidase inhibition rate, and then freeze-dried to obtain the bird's nest peptide powder.

[0076] The absorbance curves of the components obtained by gel chromatography in this embodiment under a 220 nm ultraviolet wavelength detector are shown in the figure below. Figure 1 As shown, the fraction with a molecular weight <3 kDa was separated into four polypeptides labeled 1-1, 1-2, 1-3, and 1-4 by gel chromatography; Figure 2 As shown, the four obtained polypeptides were verified by in vitro xanthine oxidase inhibition experiments, and polypeptides 1-4 were the polypeptides with the highest xanthine oxidase inhibition rate.

[0077] Example 3

[0078] This embodiment provides a bird's nest peptide powder that lowers uric acid, which contains the bird's nest peptide that inhibits xanthine oxidase obtained in Example 1.

[0079] The preparation of the uric acid-lowering bird's nest peptide powder includes the following steps:

[0080] S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid.

[0081] S2. Adjust the pH of the bird's nest liquid to 7.5, add 2% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 40℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate.

[0082] S3. Adjust the pH of the trypsin hydrolysate to 1.5, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 40℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate.

[0083] S4. Centrifuge the pepsin hydrolysate obtained in step S3, and use an ultrafiltration tube with a molecular weight cutoff of 3 kDa to ultrafilter out the components with a molecular weight <3 kDa from its supernatant.

[0084] S5. The components with a molecular weight <3kDa obtained in step S4 were chromatographically analyzed using a Superdex™ peptide 10 / 300 GL gel chromatography column. The peptides with the highest xanthine oxidase inhibition rate were screened by in vitro xanthine oxidase inhibition experiment, and then freeze-dried to obtain the bird's nest peptide powder.

[0085] The absorbance of the components obtained by gel chromatography under a 220 nm ultraviolet wavelength detector and the results of the in vitro xanthine oxidase inhibition experiment in this embodiment are similar to those in Example 2.

[0086] Example 4

[0087] This embodiment provides a bird's nest peptide powder that lowers uric acid, which contains the bird's nest peptide that inhibits xanthine oxidase obtained in Example 1.

[0088] The preparation of the uric acid-lowering bird's nest peptide powder includes the following steps:

[0089] S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid.

[0090] S2. Adjust the pH of the bird's nest liquid to 7.0, add 1.5% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 45℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate.

[0091] S3. Adjust the pH of the trypsin hydrolysate to 2.5, add 1.5% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 35℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate.

[0092] S4. Centrifuge the pepsin hydrolysate obtained in step S3, and use an ultrafiltration tube with a molecular weight cutoff of 3 kDa to ultrafilter out the components with a molecular weight <3 kDa from its supernatant.

[0093] S5. The components with a molecular weight <3kDa obtained in step S4 were chromatographically analyzed using a Superdex™ peptide 10 / 300 GL gel chromatography column. The peptides with the highest xanthine oxidase inhibition rate were screened by in vitro xanthine oxidase inhibition experiment, and then freeze-dried to obtain the bird's nest peptide powder.

[0094] The absorbance of the components obtained by gel chromatography under a 220 nm ultraviolet wavelength detector and the results of the in vitro xanthine oxidase inhibition experiment in this embodiment are similar to those in Example 2.

[0095] Comparative Example 1

[0096] This comparative example provides a method for preparing bird's nest peptide powder, including the following steps:

[0097] S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid.

[0098] S2. Adjust the pH of the bird's nest liquid to 8.5, add 1% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 50℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate.

[0099] S3. Adjust the pH of the trypsin hydrolysate to 2.0, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 37℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate.

[0100] S4. Centrifuge the pepsin hydrolysate obtained in step S3, and use an ultrafiltration tube with a molecular weight cutoff of 3 kD and an ultrafiltration tube with a molecular weight cutoff of 10 kD to ultrafilter out the components with a molecular weight between 3 kD and 10 kD from its supernatant.

[0101] S5. Freeze-dry the components with molecular weights between 3 kD and 10 kD obtained in step S4 to obtain the bird's nest peptide powder.

[0102] Comparative Example 2

[0103] This comparative example provides a method for preparing bird's nest peptide powder, including the following steps:

[0104] S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid.

[0105] S2. Adjust the pH of the bird's nest liquid to 8.5, add 1% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 50℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate.

[0106] S3. Adjust the pH of the trypsin hydrolysate to 2.0, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 37℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate.

[0107] S4. Centrifuge the pepsin hydrolysate obtained in step S3, and use an ultrafiltration tube with a molecular weight cutoff of 10 kD to ultrafilter out the components with a molecular weight >10 kD from its supernatant.

[0108] S5. Freeze-dry the components with a molecular weight >10 kD obtained in step S4 to obtain the bird's nest peptide powder.

[0109] Performance testing

[0110] 1) In vitro xanthine oxidase inhibition experiment

[0111] The bird's nest peptide powder that promotes alcohol metabolism described in Example 2 was selected for an in vitro xanthine oxidase inhibition experiment and compared with the bird's nest peptide powder of Comparative Examples 1-2. The samples of Example 2 and Comparative Examples 1-2 were labeled as EBNP-1, EBNP-2, and EBNP-3, respectively.

[0112] Results of in vitro xanthine oxidase inhibition experiments are as follows Figure 5 As shown, different peptides all have good inhibitory effects on xanthine oxidase. Compared with the samples of Comparative Examples 1 and 2, the bird's nest peptide powder of Example 2 with a molecular weight of less than 3 kDa has the most prominent inhibitory effect on xanthine oxidase. This is due to the full enzymatic hydrolysis of the peptides, which allows the nutrients of bird's nest to be released and utilized to the maximum extent.

[0113] 2) Cytotoxicity test

[0114] The uric acid-lowering bird's nest peptide powder described in Example 2 was used for cytotoxicity experiments.

[0115] LO2 cells in logarithmic growth phase were prepared into a homogeneous single-cell suspension, counted, and diluted with culture medium to 10,000 cells / 100 μL. 100 μL of each cell was seeded into 96-well plates and cultured for 12–16 h. The old culture medium was discarded, and different concentrations of peptide-containing culture medium (culture medium:peptide solution = 99:1) were added, and the cells were cultured for another 24 h. Finally, under light-protected conditions, 100 μL of fresh culture medium and 15 μL of 5 mg / mL thiazolyl blue solution were added to each well, and the cells were incubated for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well, with shaking for 10 min in the dark. The absorbance was measured at 490 nm. Six parallel wells were set up for each sample group, with a PBS control group included. The cell viability was calculated using the following formula:

[0116] Cell viability (%) = [(A1–A0) / (A2–A0)] × 100%

[0117] In the formula: A0 is the blank group; A1 is the sample group; A2 is the control group.

[0118] The results are as follows Figure 6 As shown, the peptide promoted the growth of LO2 cells at concentrations of 0.1–10 μg / mL, and was non-toxic to cells at a concentration of 100 μg / mL, with cell viability ranging from 95% to 110%.

[0119] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A bird's nest peptide that inhibits xanthine oxidase, characterized in that, The amino acid sequence of the xanthine oxidase-inhibiting bird's nest peptide is SEQ ID NO:

1.

2. Applying the xanthine oxidase-inhibiting bird's nest peptide as described in claim 1 to the preparation of uric acid-lowering products.

3. A bird's nest peptide powder for lowering uric acid, characterized in that, The bird's nest peptide powder contains the bird's nest peptide that inhibits xanthine oxidase as described in claim 1.

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