Bioactive peptides with antioxidant and hypoglycemic effects, preparations and applications thereof

Abstract

The present invention discloses bioactive peptides, preparations and applications thereof with antioxidant and hypoglycemic effects, and belongs to the technical field of bioactive peptides. The bioactive peptide of the present invention is shown as at least one of SEQ ID NO: 1 to SEQ ID NO: 4. The present invention uses defatted Xanthoceras sorbifolia meal as raw material, and after high-temperature heat treatment and ultra-high pressure pretreatment, separates bioactive peptides with antioxidant and hypoglycemic effects by enzymatic hydrolysis, ultrafiltration, liquid chromatography separation technology and combined with bioinformatics technology, and preliminarily identifies its components and sequences by liquid chromatography-mass spectrometry technology. And chemical synthesis is carried out according to the identified peptide sequence to obtain 4 bioactive peptides with antioxidant and hypoglycemic effects. After testing, these peptides and their composite peptides all have good α-glucosidase and α-amylase inhibitory activity, and have good application prospects in the 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 blood sugar-lowering 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 chemically synthesized 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, chemically synthesized 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, its clinical application effect is often difficult to achieve the expected results due to individual differences in compliance and delayed effects. Therefore, the development of new hypoglycemic agents with high biological activity, a good safety profile, and environmentally friendly properties has become an important direction in 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 its application.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] 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.

[0006] Application of the above bioactive peptides with antioxidant and hypoglycemic effects in the preparation of products with antioxidant and hypoglycemic effects.

[0007] Based on the above solution, the product is food, medicine or health product.

[0008] Based on the above solution, the product further comprises excipients acceptable to food, medicine or health products.

[0009] A bioactive peptide preparation with antioxidant and blood sugar-lowering effects, wherein the active ingredient of the preparation is at least one bioactive peptide selected from FPPW, FPWP, WPVF, and LPAWP.

[0010] Based on the above scheme, the active ingredients of the preparation are any two of the bioactive peptides shown among FPPW, FPWP, WPVF, and LPAWP.

[0011] 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.

[0012] Based on the above scheme, the mass ratio of the two peptide segments in the preparation is 2:1-1:2.

[0013] Based on the above scheme, the active ingredients of the preparation are the bioactive peptides shown as FPPW, FPWP, WPVF and LPAWP.

[0014] Based on the above scheme, the mass ratio of FPPW, FPWP, WPVF and LPAWP in the preparation is 1:1:1:1.

[0015] 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.

[0016] Advantages of the technical solution of the present invention

[0017] 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. Chemical synthesis is carried out based on 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 compound 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-value-added utilization of defatted Xanthoceras sorbifolia meal and promoting the research and development and application of functional active peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the molecular docking diagram of FPPW and α-glucosidase;

[0019] Figure 2 This is the molecular docking diagram of FPPW and α-amylase;

[0020] Figure 3 is the molecular docking diagram of FPWP and α-glucosidase;

[0021] Figure 4is the molecular docking diagram of FPWP and α-amylase;

[0022] Figure 5 is the molecular docking diagram of LPAWP and α-glucosidase;

[0023] Figure 6 This is the molecular docking diagram of LPAWP and α-amylase;

[0024] Figure 7 This is the molecular docking diagram of WPVF and α-glucosidase;

[0025] Figure 8 This is the molecular docking diagram of WPVF and α-amylase;

[0026] Figure 9 This is the primary mass spectrum of the chemically synthesized FPPW peptide;

[0027] Figure 10 This is the primary mass spectrum of the chemically synthesized FPWP peptide;

[0028] Figure 11 This is the primary mass spectrum of the chemically synthesized LPAWP peptide;

[0029] Figure 12 This is the primary mass spectrum of the chemically synthesized WPVF peptide;

[0030] Figure 13 is the inhibition kinetic curve of FPPW and α-glucosidase;

[0031] Figure 14 is the Lineweaver-Burk double reciprocal curve of FPPW and α-glucosidase;

[0032] Figure 15 is the inhibition kinetic curve of FPPW and α-amylase;

[0033] Figure 16 is the Lineweaver-Burk double reciprocal curve of FPPW and α-amylase;

[0034] Figure 17 Inhibitory activities of synthetic peptides against α-glucosidase and α-amylase (different letters indicate significant differences). DETAILED DESCRIPTION

[0035] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. Below, in conjunction with specific examples, the present invention will be further described in detail with reference to data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0036] 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 this field or according to the product instructions. The experimental materials, reagents, and drugs used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0037] 1. Determination of hydrolysis degree: Use ninhydrin colorimetry to determine the degree of hydrolysis. Specifically:

[0038] Mix 2.0 mL of appropriately diluted protein hydrolysate solution with 1.0 mL of ninhydrin solution in a centrifuge tube. Next, incubate the tube in an 80°C water bath for 30 minutes and cool to room temperature. Add 5 mL of distilled water, let the solution stand for 10 minutes, and measure the absorbance at 570 nm. Replace the protein hydrolysate with glycine standard solutions (0, 8, 16, 24, 32, and 40 μg / mL) for the working curve. Calculate the degree of hydrolysis (DH) according to the following formula.

[0039]

[0040] 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.

[0041] 2. The method for determining the α-glucosidase inhibition rate is as follows:

[0042] 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) was measured at 405 nm using a spectrophotometer. 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:

[0043] .

[0044] 3. The method for determining the α-amylase inhibition rate is as follows:

[0045] 100 μL of protein solution was mixed with 100 μL of α-amylase (0.5 U mL -1 The mixture 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. The reaction was terminated by adding 150 μL of 3,5-dinitrosalicylic acid (DNS) reagent for 5 min. The absorbance (OD) was measured at 540 nm using a spectrophotometer. C , a blank group (OD A ), control group (OD B ) and sample background group (OD D ). α-amylase activity was calculated according to the following formula.

[0046]

[0047] 4. DPPH scavenging activity

[0048] 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 100 μM DPPH-anhydrous ethanol solution 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:

[0049]

[0050] 5. Hydroxyl ion free radical scavenging rate

[0051] The hydroxyl radical scavenging activity of the peptide was measured, with glutathione (GSH) 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. The mixture was shaken thoroughly and incubated at 37°C for 30 minutes. The absorbance (OD) was measured at 510 nm. C The experiment also set up a blank group (OD A ), control group (OD B ) and sample background group (OD D ), the hydroxyl radical scavenging capacity was calculated according to the following formula:

[0052]

[0053] 6. Iron ion reducing ability

[0054] 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 a 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. C The experiment also set up 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:

[0055]

[0056] 7. Determination of the inhibition kinetics and inhibition type of synthetic peptides on α-amylase

[0057] Prepare solutions of synthetic peptides at concentrations of 0, 1, and 2 mg / mL, and α-amylase at 0, 0.5, 1, and 2 U / mL. Calculate the enzymatic reaction rate and plot the relationship between the enzymatic reaction rate and α-amylase concentration.

[0058] Purified peptide solutions at concentrations of 0, 1, and 2 mg / mL and 0.5, 1, 1.5, and 2% (w / v) starch solutions were prepared. 250 μL of each peptide solution and 250 μL of a 10 U / mL α-amylase solution were added to centrifuge tubes. The solution was incubated at 37°C in a water bath for 10 minutes. 275 μL of the starch solution of varying concentrations was then added to each tube. Every 5 minutes, 500 μL of DNS colorimetric reagent was added to the corresponding tubes. The reaction was incubated in boiling water for 5 minutes. The tubes were rapidly cooled to room temperature and then diluted to 10 mL. The absorbance was measured at 540 nm using a UV-visible spectrophotometer. The enzymatic reaction rates were calculated for different substrate concentrations and peptide concentrations. Lineweaver-Burk double reciprocal plots were plotted to determine the type of peptide inhibition of α-amylase.

[0059] 8. Determination of the inhibition kinetics and inhibition type of synthetic peptides on α-glucosidase

[0060] 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 the relationship between the enzymatic reaction rate and the α-amylase concentration was plotted.

[0061] Purified peptide solutions at concentrations of 0, 1, and 2 mg / mL and pNPG solutions at 1, 2, 3, and 4 mM were prepared. 1 mL of each peptide solution and 1 mL of a 1 U / mL α-glucosidase solution were added to centrifuge tubes. The tubes were incubated at 37°C for 15 minutes. 200 μL of the pNPG solution of varying concentrations was added to each tube. Every 5 minutes, the corresponding tubes were removed along with 2 mL of Na₂CO₃ and the volume was adjusted to 4 mL with PBS buffer. The absorbance was measured at 405 nm using a UV-visible spectrophotometer. The enzymatic reaction rates were calculated for different substrate concentrations and peptide concentrations. Lineweaver-Burk double reciprocal plots were plotted to determine the type of peptide inhibition on α-glucosidase.

[0062] Example 1

[0063] The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia meal comprises the following steps:

[0064] (1) Fresh defatted Xanthoceras sorbifolia meal was selected, 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.

[0065] (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 beneficial for the subsequent enzymatic hydrolysis process.

[0066] (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, with the dosage of each protease being 4500U / g (4500U alkaline protease, 4500U composite protease, and 4500U flavor protease per gram of defatted Xanthoceras sorbifolia powder). During the enzymatic hydrolysis process, the solution gradually became clear, indicating that the protein was effectively decomposed into peptides.

[0067] The enzymatically hydrolyzed solution was centrifuged at 8500 rpm for 15 minutes. After centrifugation, the supernatant and precipitate were separated. The supernatant was light yellow, highly transparent, and contained a large number of peptides. Testing revealed that the supernatant had a 77.35% inhibition rate for β-glucosidase and an 81.58% inhibition rate for α-amylase.

[0068] (4) Ultrafiltration separation: Ultrafiltration membranes with molecular weight cutoffs of 10KDa, 3kDa, and 500Da were used to separate the supernatant to remove large molecular impurities and small molecular substances. Components of different molecular weights were obtained: <10KDa (3kDa-10KDa), <3kDa (500Da-3kDa), and <500Da. The concentrated solution after ultrafiltration showed higher peptide concentration and purity.

[0069] The ultrafiltered peptide fractions of different molecular weights were lyophilized into dry powders, and then prepared into solutions with a concentration of 10 mg / mL. The inhibitory effects of the ultrafiltered peptide fractions of different molecular weights on α-glucosidase and α-amylase and their antioxidant capacity were measured. 10 mg / mL acarbose and 10 mg / mL glutathione (GSH) were used as controls. The results are shown in Table 1.

[0070] Table 1 Inhibitory effects of α-glucosidase and α-amylase and antioxidant capacity of solutions with different molecular weights after ultrafiltration

[0071]

[0072] Note: Different letters in the table indicate significant differences, p<0.05.

[0073] As shown in Table 1, the peptide fraction with a molecular weight of <3kDa (specifically, the range of 500Da-3kDa) exhibited the best activity. Specifically, the inhibition rates of this fraction against 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 capacity, Fe 3+ Reducing capacity) ranged from 86.67±1.53% to 91.33±1.15%. Given the excellent inhibitory effect, strong antioxidant activity, and impressive yield exhibited by this fraction, based on a comprehensive consideration of the aforementioned factors, it was decided to select fractions with a molecular weight <3 kDa for subsequent experimental studies.

[0074] (5) Separation and purification by high performance liquid chromatography-mass spectrometry: The ultrafiltration concentrate with a molecular weight of <3 kDa (specifically, 500 Da-3 kDa) was analyzed using LC-MS / MS, and 4161 polypeptide sequences were obtained.

[0075] 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.

[0076] (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.

[0077] 527 peptide sequences were screened based on protein scores (>0.9); 324 peptide sequences were screened based on nontoxicity; 233 peptide sequences were screened based on active amino acids; 149 peptide sequences were screened based on non-allergenicity; and 34 peptide sequences were screened based on hydrophobicity (>90%). These sequences were then subjected to molecular docking.

[0078] (7) The peptide sequence obtained through online tool screening was drawn using ChemDraw and energy minimized in Chem3D software to construct a stable structure. AutoDock Tools 1.5.7 was used to select it as the receptor, remove water and hydrogen, calculate the charge, add atom types, and save it in pdbgt format for molecular docking.

[0079] Using AutoDock Tools 1.5.7, I opened the conformational files of the ligand small molecule and the receptor macromolecule, set up the docking box, and saved it. Then, I opened the saved file and ran Autogrid, setting the docking parameters and calculation method. The docking results were analyzed and displayed using Protein Ligand Interaction Profiler and PyMOL software. The docking information is shown in Table 2.

[0080] Table 2 Molecular docking information of Xanthoceras sorbifolia active peptides

[0081]

[0082] Note: F stands for non-toxic and non-sensitizing; T stands for hydrophobic.

[0083] It is known that the binding energy of acarbose to α-glucosidase is -7.5kcal / 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. Figures 1-8 The chemical synthesis mass spectra of the four peptide molecules are shown in Figures 9-12 shown.

[0084] Example 2

[0085] In vitro inhibitory activity analysis of four synthetic peptides against α-amylase and α-glucosidase.

[0086] 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, enzyme concentration and 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 FPPW and α-amylase are shown in Table 3. Figure 13 and Figure 15 As shown, the straight lines all pass through the origin. As the peptide concentration increases, the slope of the straight line gradually decreases, indicating that the inhibitory effects of the four peptides on α-amylase and α-glucosidase are reversible.

[0087] Table 3 Inhibition kinetics curves of four peptides against α-glucosidase and α-amylase

[0088]

[0089] 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. Figure 14 and Figure 16 As shown. Figure 14 、 Figure 16 As can be seen from Table 4, as the peptide concentration increases, the x-axis intercept decreases and the y-axis 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 hydrolysis rate V max The change in value, the slope is K m and V max The ratio of the y-axis intercept is 1 / V maxThe slope of the line increases with increasing peptide concentration. This characteristic is consistent with mixed-type inhibition, indicating that FPPW is a mixed inhibitor for both enzymes. The same conclusion was reached for the other three peptides: all are mixed inhibitors.

[0090] Table 4 Lineweaver-Burk double reciprocal curve results of four peptides with α-glucosidase and α-amylase

[0091]

[0092] As the concentration of peptide increased, the activities of α-amylase and α-glucosidase were inhibited (Table 5), which led to the 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). 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 reaction kinetic parameter, and its value is inversely proportional to the affinity of the enzyme to the substrate. m A larger value indicates a weaker enzyme-substrate binding ability.

[0093] And by calculating the K of FPPW, FPWP, WPVF, LPAWP and α-glucosidase m The K values ​​of FPPW, FPWP, WPVF, and LPAWP for 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 affinity with α-glucosidase. m The concentrations of FPPW and FPWP were 0.743 mg / mL, 0.442 mg / mL, 1.818 mg / mL, and 1.677 mg / mL, respectively. This indicates that FPPW and FPWP have the strongest binding affinity with α-amylase.

[0094] Table 5 Km and Vmax values ​​of different concentrations of peptides for inhibition of α-amylase and α-glucosidase

[0095]

[0096] Example 3

[0097] A bioactive 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:

[0098] SEQ ID NO: 1: FPPW;

[0099] SEQ ID NO: 2: FPWP;

[0100] SEQ ID NO: 3: WPVF;

[0101] SEQ ID NO: 4: LPAWP.

[0102] Example 4

[0103] 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.

[0104] Example 5

[0105] A bioactive peptide preparation with antioxidant and hypoglycemic effects, wherein the active ingredients are bioactive peptides with the amino acid sequences 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.

[0106] Example 6

[0107] A bioactive peptide preparation with antioxidant and hypoglycemic effects, wherein the active ingredients are bioactive peptides with the 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.

[0108] Example 7

[0109] A bioactive peptide preparation with antioxidant and hypoglycemic effects, wherein the active ingredients are bioactive peptides with the 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.

[0110] Example 8

[0111] A bioactive peptide preparation with antioxidant and hypoglycemic effects, wherein the active ingredients are bioactive peptides with the 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.

[0112] 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;

[0113] The synthesized peptides FPPW and FPWP, FPPW and WPVF, and LPAWP and WPVF were prepared into composite peptide solutions using 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.

[0114] 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.

[0115] 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: Figure 17 As shown, the inhibitory activity of the four single peptide solutions against α-glucosidase ranged from 42.5% to 51.5%, indicating that they all interacted with the enzyme to varying degrees, slowing or preventing its catalytic activity. Similarly, against α-amylase, these synthetic peptides exhibited inhibitory activity ranging from 47.3% to 59.5%, indicating that they also had a regulatory effect on the enzyme. FPWP exhibited the best inhibitory activity, with inhibition rates of 51.5% and 59.5% against α-glucosidase and α-amylase, respectively. Combining the four peptides in pairs or in combination significantly increased the inhibition rates of α-glucosidase and α-amylase, with the inhibitory activity of the two-peptide combinations ranging from 55.3% to 66% and 59.3% to 72.8% against α-glucosidase and α-amylase, respectively. The four peptides were mixed in a mass ratio of 1:1:1:1 to measure their inhibitory activity against the two enzymes. It was found that the inhibition rates against α-glucosidase and α-amylase reached 74.5% and 83.4% respectively.

[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A bioactive peptide with antioxidant and hypoglycemic effects, characterized in that: The bioactive peptide with antioxidant and blood sugar-lowering effects is a peptide with an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

2. Use of a bioactive peptide having antioxidant and hypoglycemic effects in the preparation of a product having antioxidant and hypoglycemic effects, characterized in that: The bioactive peptide is a peptide having an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO:

4.

3. 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. 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 any one of the bioactive peptides selected from WPVF and LPAWP.

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

7. The bioactive peptide preparation with antioxidant and hypoglycemic effects according to claim 6, characterized in that: The mass ratio of the two peptide segments in the preparation is 2:1-1:

2.

8. A bioactive peptide preparation with antioxidant and hypoglycemic effects, characterized in that: The active ingredients of the preparation are bioactive peptides represented by FPPW, FPWP, WPVF and LPAWP.

9. The bioactive peptide preparation with antioxidant and hypoglycemic effects according to claim 8, characterized in that: The mass ratio of FPPW, FPWP, WPVF and LPAWP in the preparation is 1:1:1:1.

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