Bioactive peptide with antioxidant and hypoglycemic effects, preparation and application thereof

Abstract

The invention discloses a bioactive peptide with antioxidant and hypoglycemic effects, a preparation and application thereof, and belongs to the technical field of bioactive peptides. The bioactive peptide provided by the invention is as shown in at least one of SEQ ID NO: 1 to SEQ ID NO: 4. Degreased shiny-leaved yellowhorn meal is used as a raw material, after high-temperature heat treatment and ultrahigh-pressure pretreatment are performed, the bioactive peptide with antioxidant and hypoglycemic effects is separated through enzymolysis, ultrafiltration and a liquid chromatography separation technology in combination with a bioinformatics technology, and components and sequences of the bioactive peptide are preliminarily identified through a liquid chromatography-mass spectrometry technology. And carrying out chemical synthesis according to the identified peptide fragment sequence to obtain four bioactive peptides with antioxidant and hypoglycemic effects. Through detection, the peptide fragments and the compound peptide fragments thereof have good alpha-glucosidase and alpha-amylase inhibitory activity, and have good application prospects in preparation of products with antioxidant and hypoglycemic effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and in particular relates to bioactive peptides with antioxidant and hypoglycemic effects, preparations and applications thereof. Background Art

[0002] In recent years, with the increasing prevalence of diabetes worldwide, the research and development of hypoglycemic drugs has received increasing attention. In the current field of diabetes treatment, blood sugar management mainly relies on chemical synthetic drugs, such as glimepiride, metformin, acarbose, sitagliptin and repaglinide. These drugs exert their hypoglycemic effects by promoting insulin secretion, inhibiting hepatic glucose output, delaying carbohydrate absorption or enhancing the incretin effect. However, chemical synthetic drugs generally have potential adverse reactions and long-term safety risks, such as hypoglycemia, gastrointestinal discomfort and liver and kidney function burden. In addition, although metabolic intervention based on dietary regulation has a theoretical basis, it is limited by individual compliance differences and delayed effects, and its clinical application effect is often difficult to achieve expectations. Therefore, the development of new hypoglycemic preparations with both efficient biological activity, good safety profile and environmentally friendly characteristics has become an important direction for diabetes prevention and treatment research. Summary of the invention

[0003] In view of the problems existing in the prior art, the object of the present invention is to provide a bioactive peptide with antioxidant and hypoglycemic effects, a preparation and application thereof.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: A bioactive peptide with antioxidant and hypoglycemic effects, wherein the bioactive peptide with antioxidant and hypoglycemic effects is a peptide with an amino acid sequence shown in at least one of SEQ ID NO: 1 to SEQ ID NO: 4.

[0005] Application of the above bioactive peptides with antioxidant and hypoglycemic effects in the preparation of products with antioxidant and hypoglycemic effects.

[0006] Based on the above scheme, the product is food, medicine or health care product.

[0007] Based on the above solution, the product further comprises excipients acceptable to food, pharmacy or health care products.

[0008] A bioactive peptide preparation with antioxidant and hypoglycemic effects, wherein the active ingredient of the preparation is at least one bioactive peptide selected from FPPW, FPWP, WPVF and LPAWP.

[0009] Based on the above scheme, the active ingredients of the preparation are any two of the bioactive peptides shown in FPPW, FPWP, WPVF, and LPAWP.

[0010] On the basis of the above scheme, the active ingredients of the preparation are a combination of FPPW and FPWP, FPPW and WPVF, or LPAWP and WPVF.

[0011] Based on the above scheme, the mass ratio of the two peptide segments in the preparation is 2:1-1:2.

[0012] On the basis of the above scheme, the active ingredients of the preparation are the bioactive peptides shown as FPPW, FPWP, WPVF and LPAWP.

[0013] Based on the above scheme, the mass ratio of FPPW, FPWP, WPVF and LPAWP in the preparation is 1:1:1:1.

[0014] On the basis of the above scheme, the antioxidant activity is DPPH scavenging activity, hydroxyl ion free radical scavenging activity, and iron ion reducing ability; the hypoglycemic activity is the activity of inhibiting α-glucosidase and α-amylase.

[0015] Advantages of the technical solution of the present invention The present invention uses defatted Xanthoceras sorbifolia meal as raw material, and after high-temperature heat treatment and ultrahigh pressure pretreatment, separates bioactive peptides with antioxidant and hypoglycemic effects through enzymatic hydrolysis, ultrafiltration, liquid chromatography separation technology and combined with bioinformatics technology, and preliminarily identifies its components and sequences through liquid chromatography-mass spectrometry technology. And chemical synthesis is carried out according to the identified peptide sequences to obtain 4 bioactive peptides FPPW, FPWP, WPVF, and LPAWP with antioxidant and hypoglycemic effects. According to the test, these peptides and their composite peptides have good α-glucosidase and α-amylase inhibitory activities, and have good application prospects in the preparation of products with antioxidant and hypoglycemic effects, laying a foundation for the high added value utilization of defatted Xanthoceras sorbifolia meal and promoting the research and development and application of functional active peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the molecular docking diagram of FPPW and α-glucosidase; Figure 2 This is the molecular docking diagram of FPPW and α-amylase; Figure 3 This is the molecular docking diagram of FPWP and α-glucosidase; Figure 4 is the molecular docking diagram of FPWP and α-amylase; Figure 5It is the molecular docking diagram of LPAWP and α-glucosidase; Figure 6 This is the molecular docking diagram of LPAWP and α-amylase; Figure 7 This is the molecular docking diagram of WPVF and α-glucosidase; Figure 8 This is the molecular docking diagram of WPVF and α-amylase; Fig. 9 This is the primary mass spectrum of the chemically synthesized FPPW peptide; Fig.10 This is the primary mass spectrum of the chemically synthesized FPWP peptide; Fig.11 This is the primary mass spectrum of the chemically synthesized LPAWP peptide; Fig.12 This is the primary mass spectrum of the chemically synthesized WPVF peptide; Fig.13 is the inhibition kinetic curve of FPPW and α-glucosidase; Fig.14 is the Lineweaver-Burk double reciprocal curve of FPPW and α-glucosidase; Fig.15 is the inhibition kinetic curve of FPPW and α-amylase; Fig.16 is the Lineweaver-Burk double reciprocal curve of FPPW and α-amylase; Fig.17 Inhibitory activities of synthetic peptides against α-glucosidase and α-amylase (different letters indicate significant differences). DETAILED DESCRIPTION

[0017] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0018] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0019] 1. Determination method of hydrolysis degree: Use ninhydrin colorimetry to determine, specifically: Mix 2.0 mL of appropriately diluted protein hydrolyzate solution with 1.0 mL of ninhydrin solution in a centrifuge tube. Next, incubate the centrifuge tube in an 80°C water bath for 30 minutes and cool to room temperature. Then add 5 mL of distilled water, let stand for 10 minutes, and detect the absorbance of the solution at 570 nm. Use glycine standard solution (0, 8, 16, 24, 32, 40 μg / mL) instead of protein hydrolyzate solution as the working curve. Calculate the degree of hydrolysis DH according to the following formula.

[0020]

[0021] DH is defined as the percentage of cleaved peptide bonds, where ht is the total number of peptide bonds per protein equivalent and h is the number of hydrolyzed bonds.

[0022] 2. The method for determining the α-glucosidase inhibition rate is as follows: 200 μL of protein solution and 200 μL of 0.1U mL -1 α-glucosidase (0.2 M phosphate buffer, pH 6.8) was mixed at 37°C for 15 min, and then 200 μL of p-nitrophenyl-α-D-glucoside (2.5 mmol L -1 ), add PBS to 2 mL, and mix at 37 °C for 30 min. Add 0.2 mol L -1 The reaction was terminated by adding 2 mL of Na2CO3. The absorbance (OD C ) to calculate the α-glucosidase activity. A blank group (OD A ), control group (OD B ) and sample background group (OD D ). Calculate the α-glucosidase inhibition rate of the protein solution to be tested according to the following formula: .

[0023] 3. The method for determining the α-amylase inhibition rate is as follows: 100 μL of protein solution was mixed with 100 μL of α-amylase (0.5 U mL -1 Phosphate buffer, pH 6.8) was mixed at 37 °C for 10 min. Then 125 μL of 1% gelatinized starch (0.2 M phosphate buffer, pH 6.8) was added and incubated at 37 °C for 10 min. 150 μL of 3,5-dinitrosalicylic acid (DNS) reagent was added for 5 min to terminate the reaction. The absorbance OD was measured at 540 nm using a spectrophotometer. C , a blank group (OD A ), control group (ODB ) and sample background group (OD D ). Calculate α-amylase activity according to the following formula.

[0024]

[0025] 4. DPPH scavenging activity The DPPH scavenging activity of the peptide was determined, and glutathione (GSH) was used as a positive control. 2 mL of Xanthoceras sorbifolia peptide sample and 2.5 mL of DPPH-anhydrous ethanol solution (100 μM) were mixed evenly, kept in the dark for 30 min, and the absorbance was measured at 517 nm. A blank group (OD A ), control group (OD B ) and sample background group (OD D ). The scavenging rate of the sample to be tested on DPPH free radicals was calculated according to the following formula:

[0026] 5. Hydroxyl ion free radical scavenging rate The hydroxyl radical scavenging activity of the peptide was determined, and glutathione (GSH) was used as a positive control. 1 mL of FeSO4 solution (6 mM), 1 mL of salicylic acid solution (6 mM), 1 mL of Xanthoceras sorbifolia peptide (10 mg / mL), and 1 mL of H2O2 solution (6 mM) were added to a colorimetric test tube. They were shaken well and placed at 37°C for 30 minutes. Its absorbance OD was detected at 510 nm. C A blank group (OD A ), control group (OD B ) and sample background group (OD D ), the hydroxyl ion free radical scavenging capacity was calculated according to the following formula:

[0027] 6. Iron ion reducing ability The iron ion reducing activity of the peptide was determined, and glutathione (GSH) was used as a positive control. 1 mL of phosphate buffer solution (pH = 6.6, 0.2 mol / L), then 1 mL of K3[Fe(CN)6] (1%) and Xanthoceras sorbifolia peptide (10 mg / mL) were prepared and added to the colorimetric tube. After mixing evenly, the solution was reacted in a 50°C water bath for 20 min, and finally 1 mL of TCA (10%) was added to terminate the reaction. The mixture was centrifuged at 5000 × g for 10 minutes. After standing for 10 minutes, 2.5 mL of supernatant, 2.5 mL of distilled water and 1.2 mL of FeCl3 (0.1%) were taken and the absorbance value of the sample was measured at 700 nm (OD). C A blank group (OD A), control group (OD B ) and sample background group (OD D ), the iron ion reducing capacity was calculated according to the following formula:

[0028] 7. Determination of the inhibition kinetics and inhibition type of synthetic peptides on α-amylase The synthetic peptide was prepared into solutions with concentrations of 0, 1, and 2 mg / mL, and α-amylase (0, 0.5, 1, and 2 U / mL). The enzymatic reaction rate was calculated, and a graph was drawn showing the relationship between the enzymatic reaction rate and the α-amylase concentration.

[0029] Prepare purified peptide solutions with concentrations of 0, 1, and 2 mg / mL and 0.5, 1, 1.5, and 2% (W / V) starch solutions. Add 250 μL of peptide solutions of different concentrations and 250 μL of 10U / mL α-amylase solution to a centrifuge tube, place in a water bath at 37°C for 10 minutes, add 275 μL of starch solutions of different concentrations to each test tube, take out the corresponding test tubes of each concentration every 5 minutes, add 500 μL of DNS colorimetric agent, react in boiling water for 5 minutes, quickly cool to room temperature, and then make up to 10 mL. Use a UV-visible spectrophotometer to measure its absorbance at 540 mn. Calculate the enzymatic reaction rates of different substrate concentrations and different concentrations of peptides. Draw a Lineweaver-Burk double reciprocal curve to analyze and determine the type of inhibition of peptides on α-amylase.

[0030] 8. Determination of the inhibition kinetics and inhibition type of synthetic peptides on α-glucosidase The synthetic peptide was prepared into solutions with concentrations of 0, 1, and 2 mg / mL, and α-glucosidase (0, 1, 2, and 4 U / mL). The enzymatic reaction rate was calculated, and a graph showing the relationship between the enzymatic reaction rate and the α-amylase concentration was drawn.

[0031] Prepare purified peptide solutions with concentrations of 0, 1, and 2 mg / mL and 1, 2, 3, and 4 mM pNPG solutions. Add 1 mL of peptide solutions of different concentrations and 1 mL of 1U / mL α-glucosidase solution to a centrifuge tube, incubate at 37°C for 15 min, add 200 μL of pNPG solutions of different concentrations to each test tube, take out the corresponding test tubes of each concentration and 2 mL of Na2CO3 every 5 min, and dilute to 4 mL with PBS buffer. Measure the absorbance at 405 mn with a UV-visible spectrophotometer. Calculate the enzymatic reaction rates of different substrate concentrations and different concentrations of peptides. Draw a Lineweaver-Burk double reciprocal curve to analyze and determine the type of inhibition of the peptide on α-glucosidase.

[0032] Example 1

[0033] The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia meal is as follows: (1) Fresh defatted Xanthoceras sorbifolia meal was selected, and the meal was washed, dried, and crushed to obtain defatted Xanthoceras sorbifolia meal powder with uniform particle size. The defatted Xanthoceras sorbifolia meal powder was dissolved in deionized water at a mass ratio of 1:10, and stirred evenly to obtain a defatted Xanthoceras sorbifolia powder solution.

[0034] (2) Pretreatment: The defatted Xanthoceras sorbifolia powder solution was placed in a water bath and subjected to high temperature heat treatment at 80°C for 30 minutes, followed by ultrahigh pressure pretreatment at 200 MPa for 350 seconds. The pretreated solution exhibited a more uniform texture, which was conducive to the subsequent enzymatic hydrolysis process.

[0035] (3) Enzymatic hydrolysis: Add protease to the pretreated solution, mix well, and perform enzymatic hydrolysis at 50°C for 4 hours, with a pH of 7. The protease is a compound of alkaline protease, composite protease and flavor protease, and the amount of each protease is 4500U / g (4500U alkaline protease, 4500U composite protease and 4500U flavor protease are added per gram of substrate defatted Xanthoceras sorbifolia powder). During the enzymatic hydrolysis process, it is observed that the solution gradually becomes clear, indicating that the protein is effectively decomposed into peptides.

[0036] The solution after enzymatic hydrolysis was centrifuged at a speed of 8500rpm for 15 minutes. After centrifugation, the supernatant and the precipitate were separated. The supernatant was light yellow, highly transparent, and contained a large number of peptides. The supernatant was tested to have an inhibition rate of 77.35% for β-glucosidase and 81.58% for α-amylase.

[0037] (4) Ultrafiltration separation: Use ultrafiltration membranes with molecular weight cutoffs of 10KDa, 3kDa, and 500Da to separate the supernatant and remove large molecular impurities and small molecular substances. Components of different molecular weights are obtained: <10KDa (3kDa-10KDa), <3kDa (500Da-3kDa), and <500Da. The concentrated solution after ultrafiltration shows a higher peptide concentration and purity.

[0038] The peptide components of different molecular weights after ultrafiltration were lyophilized into dry powders, and then prepared into solutions with a concentration of 10 mg / mL, respectively. The inhibitory effects of the peptide components of different molecular weights after ultrafiltration on α-glucosidase and α-amylase and their antioxidant capacity were measured, and 10 mg / mL acarbose (Acarbose) and 10 mg / mL glutathione (GSH) were used as controls. The results are shown in Table 1.

[0039] Table 1 Inhibitory effects and antioxidant capacity of α-glucosidase and α-amylase of solutions with different molecular weights after ultrafiltration

[0040] Note: Different letters in the table indicate significant differences, p<0.05.

[0041] As shown in Table 1, the peptide component with a molecular weight of <3 kDa (specifically, the range is 500 Da-3 kDa) exhibited the best activity. Specifically, the inhibition rates of this component on α-glucosidase and α-amylase were as high as 66.12 ± 1.21% and 72.17 ± 0.76%, respectively; the antioxidant activity (DPPH and H2O2 scavenging ability, Fe 3+ The reducing capacity) was between 86.67±1.53% and 91.33±1.15%. In view of the excellent inhibitory effect, strong antioxidant activity and considerable yield of this component, based on the comprehensive consideration of the above factors, it was decided to select the component with a molecular weight <3KDa for subsequent experimental research.

[0042] (5) Separation and purification by high performance liquid chromatography-mass spectrometry: The ultrafiltration concentrate with a molecular weight of <3 kDa (specifically, in the range of 500 Da-3 kDa) was analyzed using LC MS / MS to obtain 4161 polypeptide sequences.

[0043] Liquid chromatography conditions: analytical column: 100 μm id × 180 mm, packing: Reprosil-Pur 120C18-AQ 3 μm; mobile phase A: 0.1% FA; mobile phase B: 0.1% FA, 80% ACN; flow rate: 400 nL / min; analysis time for each component: 60 min.

[0044] (6) For the 4161 peptide sequences obtained in step (5), Toxinpred (http: / / crdd.osdd.net / raghava / toxinpred / ) was used to predict whether the peptide was toxic; PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict the activity of the peptide; and Expasy ProtParamtool (https: / / web.expasy.org / protparam / ) was used to predict the hydrophobicity of the peptide.

[0045] 527 peptide sequences were screened based on protein score (>0.9); 324 were screened based on non-toxicity principle; 233 peptide sequences with active amino acids were screened; 149 peptide sequences were screened based on non-sensitization; 34 peptides were screened based on hydrophobicity (>90%). Finally, these sequences were docked.

[0046] (7) The peptide sequence obtained through online tool screening was drawn by ChemDraw and the stable structure was constructed by energy minimization in Chem3D software. AutoDock Tools 1.5.7 was used to select it as the receptor, remove water and add hydrogen, calculate the charge, add atom types, and save it in pdbgt format for molecular docking.

[0047] Use AutoDock Tools 1.5.7 to open the conformational files of the ligand small molecule and the receptor macromolecule, set the docking box and save it, open the saved file and run Autogrid, set the docking parameters and calculation method and run AutoDock. The docking results were analyzed and displayed using Protein Ligand Interaction Profiler and PyMOL software, and the docking information is shown in Table 2.

[0048] Table 2 Molecular docking information of Xanthoceras sorbifolia active peptides

[0049] Note: F means non-toxic and non-sensitizing; T means hydrophobic.

[0050] It is known that the binding energy of acarbose to α-glucosidase is -7.5 kcal / mol. Finally, four peptide sequences with lower binding energy (FPPW, FPWP, WPVF, LPAWP) were selected for chemical synthesis. The docking diagram of the four peptide molecules is shown in the figure below. Figure 1-Figure 8 The chemical synthesis mass spectra of the four peptide molecules are shown in Figure 9-12 shown.

[0051] Example 2

[0052] In vitro inhibitory activity analysis of four synthetic peptides against α-amylase and α-glucosidase.

[0053] The inhibition kinetics of the four peptides on α-amylase and α-glucosidase were studied by ultraviolet spectroscopy to explore the relationship between the concentration of the synthetic peptide, the enzyme concentration and the enzymatic hydrolysis rate, and to determine whether the inhibition is reversible. The results of the inhibition kinetic curves of the four peptides and the two enzymes are shown in Table 3. Among them, the results of the inhibition kinetic curves of FPPW and α-glucosidase and the inhibition kinetic curves of FPPW and α-amylase are shown in Table 3. Fig.13 and Fig.15 As shown, the straight lines basically pass through the origin. As the peptide concentration increases, the slope of the straight line gradually decreases, indicating that the inhibitory effect of the four peptides on α-amylase and α-glucosidase is reversible.

[0054] Table 3 Inhibition kinetic curves of four peptides on α-glucosidase and α-amylase

[0055] In order to determine the reversible inhibition type of the four peptides on α-amylase and α-glucosidase, the Lineweaver-Burk curve method was used to explore the relationship between peptide concentration, substrate concentration and enzymatic hydrolysis rate, and to determine the inhibition type and inhibition constant. The results of the inhibition kinetic curves of the four peptides and the two enzymes are shown in Table 4. Among them, the Lineweaver-Burk double reciprocal curves of FPPW and α-glucosidase and the Lineweaver-Burk double reciprocal curves of FPPW and α-amylase are shown in Table 4. Fig.14 and Fig.16 As shown. Fig.14 , Fig.16 As can be seen from Table 4, as the peptide concentration increases, the x-intercept decreases and the y-intercept increases. The straight lines of peptides with different concentrations intersect at one point in the third quadrant; the Michaelis constant K is monitored by the Lineweaver-Burk plot. m and the maximum enzymatic rate V max The change in value, the slope is K m and V max The ratio of the y-intercept is 1 / V max The slope of the straight line increases with the increase of peptide concentration. This feature is consistent with mixed inhibition, indicating that FPPW is a mixed inhibitor for both enzymes. In addition, the same conclusion was obtained for the other three peptides, that is, they are all mixed inhibitors.

[0056] Table 4 Lineweaver-Burk double reciprocal curve results of four peptides with α-glucosidase and α-amylase

[0057] As the peptide concentration increased, the activities of α-amylase and α-glucosidase were inhibited (Table 5), which resulted in V max and K m The value of changes. Therefore, only part of the structure of these four peptides binds to the active site of α-glucosidase (competitive inhibition mode), and at the same time, the four peptides can also bind to the binary complex of enzyme-substrate to form a ternary complex of peptide-enzyme-substrate (non-competitive inhibition mode), playing a non-competitive role in hindering the decomposition of substrate. Because pNPG occupies the active site of α-glucosidase first (or starch occupies the active site of α-amylase first), the polypeptide cannot enter the active site of α-glucosidase to exert inhibitory activity. Therefore, it can be inferred that the polypeptide has a clear inhibitory effect on these two enzymes. Michaelis constant (K m ) is an enzyme-catalyzed reaction kinetic parameter, and its value is inversely proportional to the affinity of the enzyme for the substrate. m Larger values ​​indicate weaker enzyme-substrate binding ability.

[0058] And by calculating the K of FPPW, FPWP, WPVF, LPAWP and α-glucosidase m The K values ​​of FPPW, FPWP, WPVF, LPAWP and α-amylase were 0.838 mg / mL, 0.502 mg / mL, 1.084 mg / mL and 0.878 mg / mL, respectively, indicating that FPPW and FPWP have the strongest binding ability with α-glucosidase. m They were 0.743 mg / mL, 0.442 mg / mL, 1.818 mg / mL, and 1.677 mg / mL, respectively. This indicated that FPPW and FPWP had the strongest binding ability with α-amylase.

[0059] Table 5 Km and Vmax values ​​of different concentrations of peptides for inhibition of α-amylase and α-glucosidase

[0060] Example 3

[0061] A biologically active peptide with antioxidant and blood sugar lowering effects, wherein the active peptide is a peptide having an amino acid sequence shown in at least one of SEQ ID NO: 1 to SEQ ID NO: 4: SEQ ID NO: 1: FPPW; SEQ ID NO:2: FPWP; SEQ ID NO:3: WPVF; SEQ ID NO:4: LPAWP.

[0062] Example 4

[0063] A bioactive peptide preparation with antioxidant and blood sugar lowering effects, wherein the active ingredient is a bioactive peptide with an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 4, wherein the concentration of the bioactive peptide is 1 mg / mL.

[0064] Example 5

[0065] A bioactive peptide preparation with antioxidant and hypoglycemic effects, wherein the active ingredients are bioactive peptides with amino acid sequences of FPPW and FPWP, wherein the mass ratio of FPPW to FPWP is 1:2; and the concentrations of FPPW and FPWP are 1 mg / mL and 2 mg / mL, respectively.

[0066] Example 6

[0067] A bioactive peptide preparation with antioxidant and hypoglycemic effects, wherein the active ingredients are bioactive peptides with amino acid sequences of FPPW and WPVF, wherein the mass ratio of FPPW to WPVF is 1:1.5; and the concentrations of FPPW and WPVF are 1 mg / mL and 1.5 mg / mL, respectively.

[0068] Example 7

[0069] A bioactive peptide preparation with antioxidant and hypoglycemic effects, wherein the active ingredients are bioactive peptides with amino acid sequences of LPAWP and WPVF, wherein the mass ratio of LPAWP to WPVF is 2:1; and the concentrations of LPAWP and WPVF are 2 mg / mL and 1 mg / mL, respectively.

[0070] Example 8

[0071] A bioactive peptide preparation with antioxidant and hypoglycemic effects, wherein the active ingredients are bioactive peptides with amino acid sequences of FPPW, FPWP, WPVF and LPAWP, wherein the mass ratio of FPPW, FPWP, WPVF and LPAWP is 1:1:1:1; and the concentrations of FPPW, FPWP, WPVF and LPAWP are all 1 mg / mL.

[0072] The synthesized peptides FPPW, FPWP, WPVF, and LPAWP were prepared into solutions with a concentration of 1 mg / mL using ultrapure water as single peptide solutions; The synthesized peptides FPPW and FPWP, FPPW and WPVF, LPAWP and WPVF were prepared into composite peptide solutions with ultrapure water at mass ratios of 1:2, 1:1.5, and 2:1, respectively. In the composite peptide solution of FPPW and FPWP, the concentrations of FPPW and FPWP were 1 mg / mL and 2 mg / mL, respectively; in the composite peptide solution of FPPW and WPVF, the concentrations of FPPW and WPVF were 1 mg / mL and 1.5 mg / mL, respectively; in the composite peptide solution of LPAWP and WPVF, the concentrations of LPAWP and WPVF were 2 mg / mL and 1 mg / mL, respectively.

[0073] The synthesized peptides FPPW, FPWP, WPVF and LPAWP were prepared into a composite peptide solution in a mass ratio of 1:1:1:1 using ultrapure water, wherein the concentration of each peptide was 1 mg / mL.

[0074] The inhibition rates of α-glucosidase and α-amylase of the above single peptide and compound peptide solutions were determined. The results are as follows: Fig.17As shown in the figure, the inhibitory activity of the four single peptide solutions on α-glucosidase ranged from 42.5% to 51.5%, which means that they can interact with the enzyme to varying degrees, thereby slowing down or preventing its catalytic activity. Similarly, for α-amylase, these synthetic peptides also showed an inhibitory activity range of 47.3% to 59.5%, indicating that they also have a regulatory effect on the enzyme. Among them, FPWP showed the best inhibitory activity, with inhibition rates of 51.5% and 59.5% on α-glucosidase and α-amylase, respectively. After the four peptides were combined in pairs or the four were compounded, the inhibition rates of α-glucosidase and α-amylase were significantly improved, and the inhibitory activity of the two-to-two combinations on α-glucosidase and α-amylase ranged from 55.3% to 66% and 59.3% to 72.8%. The four peptides were mixed in a mass ratio of 1:1:1:1 to measure their inhibitory activity against the two enzymes, and it was found that the inhibition rates against α-glucosidase and α-amylase reached 74.5% and 83.4% respectively.

[0075] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A bioactive peptide with antioxidant and hypoglycemic effects, characterized in that: The bioactive peptide with antioxidant and hypoglycemic effects is a peptide with an amino acid sequence shown in at least one of SEQ ID NO: 1 to SEQ ID NO:

4.

2. Use of the bioactive peptide with antioxidant and hypoglycemic effects as claimed in claim 1 in the preparation of products with antioxidant and hypoglycemic effects.

3. The use of the bioactive peptide with antioxidant and hypoglycemic effects according to claim 2 in the preparation of products with antioxidant and hypoglycemic effects, characterized in that: The product is food, medicine or health product.

4. The use of the bioactive peptide with antioxidant and hypoglycemic effects according to claim 3 in the preparation of products with antioxidant and hypoglycemic effects, characterized in that: The product further comprises excipients acceptable to food, medicine or health care products.

5. A bioactive peptide preparation with antioxidant and hypoglycemic effects, characterized in that: The active ingredient of the preparation is at least one of the bioactive peptides shown in FPPW, FPWP, WPVF and LPAWP.

6. The bioactive peptide preparation with antioxidant and hypoglycemic effects according to claim 5, characterized in that: The active ingredients of the preparation are any two of the bioactive peptides shown in FPPW, FPWP, WPVF and LPAWP.

7. The bioactive peptide preparation with antioxidant and hypoglycemic effects according to claim 6, characterized in that: The active ingredients of the preparation are a combination of FPPW and FPWP, FPPW and WPVF, or LPAWP and WPVF.

8. The bioactive peptide preparation with antioxidant and hypoglycemic effects according to claim 7, characterized in that: The mass ratio of the two peptide segments in the preparation is 2:1-1:

2.

9. The bioactive peptide preparation with antioxidant and hypoglycemic effects according to claim 5, characterized in that: The active ingredients of the preparation are the bioactive peptides shown by FPPW, FPWP, WPVF and LPAWP.

10. The bioactive peptide preparation with antioxidant and hypoglycemic effects according to claim 9, characterized in that: The mass ratio of FPPW, FPWP, WPVF and LPAWP in the preparation is 1:1:1:1.

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