Bioactive peptides with antioxidant and blood sugar-lowering functions, compositions and applications

Abstract

The present invention discloses a bioactive peptide with antioxidant and hypoglycemic functions, a composition and an application, and belongs to the technical field of bioactive peptides. The amino acid sequence of the bioactive peptide with antioxidant and hypoglycemic functions of the present invention is shown in one of SEQ ID NO:1 to SEQ ID NO:4. The present invention combines a plurality of enzymes to hydrolyze defatted peanut meal, thereby improving the diversity of peptide segments, and obtaining highly active enzymatic hydrolysis products by optimizing conditions. The enzymatic hydrolysis products are separated and purified by ultrafiltration and high performance liquid chromatography-mass spectrometry, thereby ensuring the purity and structural clarity of the peptide segments. Screening is performed in combination with bioinformatics technology, thereby improving the activity and effect of the peptide sequence. In vitro and in vivo experiments have shown that the separated bioactive peptides have good antioxidant and hypoglycemic effects, and have broad application prospects in the preparation of antioxidant and hypoglycemic products.

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 functions, compositions and applications. Background Art

[0002] Diabetes Mellitus (DM) is a metabolic disease caused by multiple causes, mainly caused by insufficient insulin secretion or insulin resistance, and manifested as symptoms such as hyperglycemia and "three mores and one less". The acute and chronic complications of diabetes are extremely harmful and are the primary cause of diseases such as stroke, heart disease, blindness, renal failure and amputation. The pathogenesis of diabetes involves multiple aspects such as genetics, autoimmunity and environmental factors, which affect the synthesis of pancreatic cells, insulin secretion and the sensitivity of target cells to insulin. Therefore, the prevention and treatment of diabetes has become an important issue in the field of global public health.

[0003] In recent years, with the increasing prevalence of diabetes worldwide, the research and development of hypoglycemic drugs has received increasing attention. Among them, α-glucosidase inhibitors, as an important hypoglycemic drug, can inhibit α-glucosidase on the membrane of small intestinal epithelial cells, delay the digestion and absorption of carbohydrates, and thus reduce blood sugar levels.

[0004] At present, the main methods of treating diabetes include oral medication, insulin injection, etc., but due to the side effects of long-term medication and the inconvenience of insulin injection, plant-derived active peptides have attracted much attention due to their natural, safe and effective characteristics. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a bioactive peptide, composition and application having antioxidant and blood sugar lowering functions.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A bioactive peptide with antioxidant and blood sugar lowering functions, wherein the amino acid sequence of the bioactive peptide is shown in one of SEQ ID NO: 1 to SEQ ID NO: 4.

[0008] A bioactive peptide composition with antioxidant and blood sugar lowering functions, the composition comprising at least one bioactive peptide shown in SEQ ID NO: 1 to SEQ ID NO: 4.

[0009] On the basis of the above scheme, the composition is the bioactive peptides shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the mass ratio thereof is 1.5:1.

[0010] On the basis of the above scheme, the composition is the bioactive peptides shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the mass ratio thereof is 1:2.

[0011] Based on the above scheme, the composition is the bioactive peptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the mass ratio thereof is 1:2:1:1.

[0012] Based on the above scheme, the antioxidant activity includes DPPH scavenging activity, hydroxyl ion free radical scavenging activity and iron ion reducing ability.

[0013] On the basis of the above scheme, the blood sugar lowering agent has α-glucosidase inhibitory activity and α-amylase inhibitory activity.

[0014] Application of the above bioactive peptide with antioxidant and blood sugar lowering function or the above bioactive peptide composition with antioxidant and blood sugar lowering function in the preparation of drugs with antioxidant and blood sugar lowering function.

[0015] Based on the above scheme, the medicine further comprises pharmaceutically acceptable excipients.

[0016] The method for preparing bioactive peptides with antioxidant and hypoglycemic functions using defatted peanut meal as raw material comprises the following steps:

[0017] (1) Dissolving defatted peanut meal in water to prepare a defatted peanut powder solution;

[0018] (2) adding protease to the defatted peanut meal for enzymatic hydrolysis;

[0019] (3) After enzymatic hydrolysis, the enzyme was inactivated at 90°C for 10 min, and the enzymatic hydrolyzate was centrifuged to separate the supernatant and the precipitate;

[0020] (4) Separating the supernatant using an ultrafiltration membrane to remove macromolecular impurities and small molecular substances therein, obtaining component concentrates with molecular weights of 10-3K Da, 3K Da-500 Da, and <500 Da, and freeze-drying to obtain dry solid powder;

[0021] (5) Determine the inhibition rate of α-glucosidase and α-amylase and antioxidant activity of different molecular weight components in step (4); screen out the molecular weight range of peptides with high activity;

[0022] (6) subjecting the highly active molecular weight segments obtained by the membrane separation to high performance liquid chromatography separation; combining mass spectrometry technology to identify and quantify the separated peptide segments, thereby obtaining peptide segments with high purity and clear structure;

[0023] (7) Use bioinformatics technology to further analyze and screen the peptides isolated and purified to obtain peptides that are non-toxic, non-sensitizing, have a hydrophobicity of >50% and have good biological activity;

[0024] (8) The peptides obtained by screening were molecularly docked with α-glucosidase, and the binding energy of the peptides was compared with the binding energy of acarbose. The peptides with lower binding energy than acarbose were selected as the isolated bioactive peptides with antioxidant and hypoglycemic effects.

[0025] Based on the above scheme, the mass volume ratio of defatted peanut meal to water in step (1) is 1:10 (w / v);

[0026] On the basis of the above scheme, in step (2), the protease is at least one of alkaline protease, compound protease and flavor protease; preferably, the protease is alkaline protease, compound protease and flavor protease in a mass ratio of 1:1:1.

[0027] Based on the above scheme, the enzymatic hydrolysis conditions in step (2) are 40-65°C, 4-6 h, and pH 6.8; more preferably, the enzymatic hydrolysis conditions are 55°C, 5 h, and pH 6.8.

[0028] Advantages of the technical solution of the present invention:

[0029] The bioactive peptides with antioxidant and blood sugar lowering functions separated and purified from defatted peanut meal have good inhibitory effects on α-glucosidase and α-amylase, are derived from plants, have natural and safe ingredients, and have great application prospects in the preparation of antioxidant and blood sugar lowering products.

[0030] Compared with a single component, the bioactive peptides of the present invention have higher antioxidant and hypoglycemic activity when used in combination, and are less likely to develop drug resistance.

[0031] The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from peanuts provided by the present invention combines multiple enzymes to hydrolyze defatted peanut meal, thereby improving the diversity of peptide segments, and obtaining highly active hydrolyzed products by optimizing conditions. The hydrolyzed products are separated and purified by ultrafiltration and high performance liquid chromatography-mass spectrometry, thereby ensuring the purity and structural clarity of the peptide segments. The activity and effect of the peptide sequence are improved by screening in combination with bioinformatics technology. The antioxidant and hypoglycemic effects of the bioactive peptides are verified by in vitro and in vivo experiments, and thus the optimization of the preparation method provides strong technical support and reference basis for subsequent industrial production. At the same time, it also demonstrates the important role and broad prospects of biotechnology in food processing and drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Determination results of α-glucosidase and α-amylase inhibition rates of different molecular weight components;

[0033] Figure 2 Determination results of antioxidant activity of components with different molecular weights;

[0034] Figure 3 Details of the interaction between the peptide and α-glucosidase;

[0035] Figure 4 Determination of hydrolysis degree of defatted peanut meal at different temperatures;

[0036] Figure 5 Determination of hydrolysis degree of defatted peanut meal at different times;

[0037] Figure 6 Determination of hydrolysis degree of defatted peanut meal under the action of different proteases;

[0038] Figure 7 Determination of α-glucosidase inhibition rate of defatted peanut meal under the action of different proteases;

[0039] Figure 8 Determination of α-amylase inhibition rate of defatted peanut meal under the action of different proteases;

[0040] Fig. 9 The inhibition rates of α-glucosidase and α-amylase and the antioxidant activity test results of different bioactive peptide complexes (the left figure shows the inhibition rates of α-glucosidase and α-amylase, and the right figure shows the antioxidant activity test results);

[0041] Fig.10 The effects of changes in water intake in mice during dietary intervention;

[0042] Fig.11 Effects of dietary intervention on body weight changes in mice;

[0043] Fig.12 Effects of dietary intervention on blood glucose changes in mice;

[0044] Fig.13 Effects of dietary intervention on changes in glucose tolerance (OGTT) in mice;

[0045] Fig.14 The effect of peanut peptide on TG and TC levels in mice (TG on the left and TC on the right);

[0046] Fig.15 The effect of peanut peptide on the HDL-C and LDL-C levels in mice (the left picture is HDL-C, the right picture is LDL-C);

[0047] Fig.16The effect of peanut peptide on GSH and SOD in mouse liver (the left picture shows liver GSH, and the right picture shows liver SOD);

[0048] Fig.17 The effect of raw peptides on MDA and SOD in mouse kidneys (the left picture shows kidney MDA, and the right picture shows kidney SOD);

[0049] Fig.18 HE staining results of mouse liver tissue (magnification ×400);

[0050] Fig.19 HE staining results of mouse kidney tissue (magnification ×400);

[0051] Fig. 20 MASSON staining results of mouse kidney (magnification ×200, where the black arrow indicates the lesion area);

[0052] Fig.21 Statistics of MASSON staining area in mouse kidney;

[0053] Fig. 22 Oil red staining results of mouse liver (magnification ×200, where the black arrow indicates the lipid deposition area);

[0054] Fig.23 Area statistics of Oil red staining results of mouse liver. DETAILED DESCRIPTION

[0055] 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.

[0056] 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.

[0057] Determination method of hydrolysis degree: using ninhydrin method, the specific method is:

[0058] 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.

[0059] The degree of hydrolysis DH was calculated according to the following formula:

[0060]

[0061] 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. Peanut htot = 7.55 meqv / g. The number of hydrolyzed bonds was obtained according to the method in the prior art (Niu Jiale, Tian Qing, Yi Yanjie, et al. Preparation of peanut peptides by enzymatic hydrolysis and analysis of their antioxidant activity [J]. Journal of Henan University of Technology (Natural Science Edition), 2024, 45(04): 19-28. DOI: 10.16433 / j.1673-2383.202401180001.).

[0062] The method for determining the α-glucosidase inhibition rate is as follows:

[0063] 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 ), make up to 2 mL with PBS, and mix at 37 °C for 30 min. Add 2 mL of 0.2 mol L-1 Na2CO3 to terminate the reaction. Measure the absorbance (OD) 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 ).

[0064] The α-glucosidase inhibition rate of the protein solution to be tested was calculated according to the following formula:

[0065] .

[0066] The method for determining the α-amylase inhibition rate is as follows:

[0067] 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 (OD B ) and sample background group (OD D ).

[0068] The α-amylase activity was calculated according to the following formula:

[0069]

[0070] The method for determining the antioxidant index is as follows:

[0071] DPPH scavenging activity:

[0072] The DPPH scavenging activity of the peptide was determined, and glutathione (GSH) was used as a positive control. 2 mL of peanut 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 OD was measured at 517 nm. C A blank group (OD A ), control group (OD B ) and sample background group (OD D ).

[0073] The scavenging rate of the sample to be tested for DPPH free radicals was calculated according to the following formula:

[0074]

[0075] Hydroxyl ion free radical scavenging rate

[0076] 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 peanut 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 (ODD ).

[0077] The hydroxyl ion free radical scavenging capacity was calculated according to the following formula:

[0078]

[0079] Iron ion reducing capacity

[0080] 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 (0.2 mol / L at pH = 6.6), then 1 mL of K3[Fe(CN)6] (1%) and peanut 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 mLFeCl3 (0.1%) were taken and the absorbance value of the sample was measured at 700 nm. 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:

[0081]

[0082] In the following examples, the enzyme activity of alkaline protease is 300000 U / g, the enzyme activity of compound protease is 150000 U / g, and the enzyme activity of flavor protease is 200000 U / g. Flavor protease (Flavourzyme), compound protease (Protamex), and alkaline protease (Alcalase) were purchased from Novozymes Biotech, Denmark.

[0083] In the accompanying drawings of the present invention,

[0084] “#” indicates that there is a significant difference between the sample group and the control group, #p<0.05, ###p<0.01;

[0085] "*" indicates that there is a significant difference between the sample group and the model group, *p<0.05, ***p<0.01;

[0086] In the figures, data with different superscript letters in the same group represent significant differences among the groups, p < 0.05.

[0087] Example 1

[0088] A bioactive peptide with antioxidant and blood sugar lowering functions, wherein the amino acid sequence of the bioactive peptide is shown in one of SEQ ID NO: 1 to SEQ ID NO: 4.

[0089] SEQ ID NO: 1: AFPKFR;

[0090] SEQ ID NO:2: PFPIK;

[0091] SEQ ID NO:3: APPFDPNKPK;

[0092] SEQ ID NO:4: FATPVPLPK.

[0093] Example 2

[0094] The method for preparing bioactive peptides with antioxidant and hypoglycemic functions using defatted peanut meal as raw material comprises the following steps:

[0095] (1) Dissolve defatted peanut meal in water at a ratio of 1:10 (w / v) to prepare defatted peanut powder solution.

[0096] (2) Then, a composite enzyme was added in a mass ratio of 1:0.02 of defatted peanut meal to enzyme, wherein the composite enzyme was composed of alkaline protease, composite protease, and flavor protease in a mass ratio of 1:1:1. The enzymolysis was carried out at a temperature of 55°C and a pH of 6.8 for 5 h.

[0097] (3) After enzymatic hydrolysis of the above solution, the enzyme was inactivated at 90°C for 10 min. The enzymatic hydrolyzate was centrifuged at 8500 rpm for 15 min to separate the supernatant and the precipitate. The supernatant contained a large amount of peptides, which were the main source for the subsequent preparation of hypoglycemic peptides.

[0098] (4) Use an ultrafiltration membrane (molecular weight 10KDa, 3KDa, 500 Da, etc.) to separate the supernatant to remove large molecular impurities and small molecular substances, obtain component concentrates with molecular weights of 10-3KDa, 3KDa-500Da, and <500Da, and freeze-dry to obtain dry solid powder.

[0099] (5) Determine the α-glucosidase and α-amylase inhibition rates and antioxidant activities of the different molecular weight components in step (4); screen out the peptide molecular weight range with high activity.

[0100] Using acarbose as a control, the inhibition rates of α-glucosidase and α-amylase of different molecular weight components were determined. Figure 1 As shown, Alc+Pro+Fla refers to the inhibition of α-glucosidase and α-amylase in the supernatant before ultrafiltration.

[0101] Depend on Figure 1 It can be seen that, except for acarbose, the α-glucosidase inhibition rate is 49.5%-69.5%, and the α-amylase inhibition rate is 52.5%-75%; the components with a molecular weight cutoff <3KDa (3KDa-500Da) have the highest α-glucosidase and α-amylase inhibition activity.

[0102] Antioxidant activity of components with different molecular weights Figure 2 shown.

[0103] Depend on Figure 2 It can be seen that, except for the glutathione (GSH) group, the antioxidant activities of the different molecular weight components are: DDPH scavenging rate 67%-85%; hydroxyl free radical scavenging rate 77.7%-90.3%; Fe 3+ The reducing capacity is 80.3%-88.1%. The antioxidant activity of the components with a molecular weight cutoff <3KDa (3KDa-500Da) is equivalent to that of the components with a molecular weight cutoff <500Da. Based on the results of the inhibition rate of α-glucosidase and α-amylase, the components with a molecular weight cutoff <3KDa (3KDa-500Da) are selected for further separation and purification.

[0104] (6) The highly active molecular weight segment (3KDa-500Da) obtained by the above membrane separation is separated by high performance liquid chromatography; combined with mass spectrometry technology, the separated peptides are identified and quantified to obtain peptides with high purity and clear structure.

[0105] The liquid chromatography parameters were as follows: analytical column: 75 μm id×25 cm, NanoViper C18 1.9 μm, 100A; mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% ACN; flow rate: 600 nL / min; analysis time for each component: 60 min.

[0106] The mass spectrometry conditions were:

[0107] Primary mass spectrometry parameters: Resolution: 60,000; AGCtarget: Custom; MaximumIT: Custom; Scanrange: 350to1500m / z.

[0108] Secondary mass spectrometry parameters: Resolution: 15,000; AGCtarget: Custom; MaximumIT: Custom; NCE / steppedNCE: 27.

[0109] The peptide sequences with a molecular weight cutoff of <3 KDa (3 KDa-500 Da) were identified by LC-MS / MS, and a total of 3036 peptide sequences were identified.

[0110] (7) Bioinformatics technology was used to further analyze and screen the peptides isolated and purified to obtain peptides that were non-toxic, non-sensitizing, had a hydrophobicity of >50% and had good biological activity.

[0111] The specific steps are as follows:

[0112] The peptides obtained in step (6) were scored by PeptideRanker, and peptide sequences with a protein score > 0.9 were selected for subsequent screening, resulting in a total of 285 peptide sequences. Peptide Ranker (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / help / peptideranker / help.ph) was used to evaluate the toxicity and allergenicity of the peptide sequences ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred2 / index.html). ExPasy (SIB Swiss Institute of Bioinformatics | Expasy) was used to determine the isoelectric point, hydrophilicity, stability, etc. of the peptide sequences. The activity probability of the peptide sequences with higher credibility in the results was predicted, and peptides with no toxicity, no allergenicity, and a hydrophobicity > 50% were selected.

[0113] Most α-glucosidase inhibitory peptides with high biological activity are oligopeptides with less than 10 amino acid residues; arginine (Arg) and lysine (Lys) at the C-terminus of the peptide chain are necessary amino acid residues to ensure that the active peptides exert α-glucosidase inhibitory activity.

[0114] Based on the above conditions, a total of 7 peptides were selected for molecular docking.

[0115] The sequence information of α-glucosidase (PDBID: 2QMJ) was obtained from the Protein Data Bank (https: / / www.rcsb.org / structure). The 3D structure of the peptide was generated using Chem3D. Molecular docking was performed using the open source software AutoDock Vina 1.1.2 to evaluate the interaction between the peptide and protein. The molecular docking results were expressed in Vina fractions (kcal / mol). The interaction details between the ligand and receptor were visualized using PyMol 2.1.1.

[0116] The molecular weights and binding energies of the seven peptides are shown in Table 1. The binding energy of acarbose is -7.6 kcal / mol, among which the binding energies of AFPKFR, APPFDPNKPK, PFPIK, and FATPVPLPK are all lower than that of acarbose. Binding energy is an important criterion to consider in protein-ligand interactions, and low binding energy indicates higher stability.

[0117] The details of the interaction between the four peptides AFPKFR, APPFDPNKPK, PFPIK, and FATPVPLPK and α-glucosidase are as follows Figure 3 shown.

[0118] Table 1 Molecular weight and binding energy of 7 peptides

[0119]

[0120] (8) The binding energy of the peptides was compared with that of acarbose, and four peptides AFPKFR, PFPIK, APPFDPNKPK, and FATPVPLPK with binding energies lower than that of acarbose were selected for sequence synthesis.

[0121] Effect of temperature on enzymatic hydrolysis

[0122] Enzyme hydrolysis experiments were carried out at different temperatures (such as 45°C, 50°C, 55°C, and 60°C) to explore the effect of temperature on the enzymatic hydrolysis of defatted peanut meal. Specifically, defatted peanut meal was dissolved in water at a ratio of 1:10 (w / v) to obtain a defatted peanut powder solution; then a composite enzyme was added at a mass ratio of 1:0.02 between defatted peanut meal and enzyme, wherein the composite enzyme was composed of alkaline protease, composite protease, and flavor protease at a mass ratio of 1:1:1. Under different temperature conditions, the pH was 6.8 and the supernatant was centrifuged after the enzyme was inactivated to determine the degree of hydrolysis of different protein hydrolysates.

[0123] The results are as follows Figure 4 As shown in the figure, when enzymatic hydrolysis was performed at different temperatures, the degree of protein hydrolysis was 30.1% (40℃)-57.1% (60℃). The highest degree of hydrolysis reached 57.1% at 60℃. Considering the effect of temperature on the activity of protease, 55℃ was the optimal enzymatic hydrolysis temperature.

[0124] Effect of time on enzymatic hydrolysis

[0125] Comparative experiments were conducted at different times (such as 4h, 4.5h, 5.0h, 5.5h, and 6h) to explore the effect of enzymatic hydrolysis time on the enzymatic hydrolysis effect of defatted peanut meal. Specifically, defatted peanut meal was dissolved in water at a ratio of 1:10 (w / v) to obtain a defatted peanut powder solution; then a composite enzyme was added at a mass ratio of 1:0.02 between defatted peanut meal and enzyme, wherein the composite enzyme was composed of alkaline protease, composite protease, and flavor protease at a mass ratio of 1:1:1. Under the conditions of 55°C and pH 6.8, the enzymatic hydrolysis was performed for different times, and the supernatant was taken after the enzyme was inactivated by centrifugation to determine the degree of hydrolysis of different protein hydrolysates.

[0126] The results are as follows Figure 5 As shown in the figure, the degree of protein hydrolysis was 47.7% (4h)-58.1% (5h) when the enzymatic hydrolysis was performed at different time. When the enzymatic hydrolysis time was 5h, the highest degree of hydrolysis reached 58.1%, so 5h was the best enzymatic hydrolysis time.

[0127] Effects of different proteases on enzymatic hydrolysis

[0128] The effects of flavor protease, compound protease, alkaline protease, flavor protease + alkaline protease (mass ratio of 1:1), alkaline protease + compound protease (mass ratio of 1:1), flavor protease + compound protease (mass ratio of 1:1), and flavor protease + alkaline protease + compound protease (mass ratio of 1:1:1) on the enzymatic hydrolysis of defatted peanut meal were determined. Specifically, defatted peanut meal was dissolved in water at a ratio of 1:10 (w / v) to prepare defatted peanut powder solution; then protease or compound enzyme was added at a mass ratio of defatted peanut meal to enzyme of 1:0.02. The enzymatic hydrolysis was carried out at 55℃ and pH 6.8 for 5 h, and the hydrolysis degree of different protein hydrolysates was determined after the enzyme was inactivated.

[0129] The hydrolysis degree of defatted peanut meal by different proteases is shown in the following table. Figure 6 As shown: the hydrolysis degree of protein after multi-enzyme complex hydrolysis was significantly higher than that of single protease hydrolysis. The hydrolysis degrees of defatted peanut meal after enzymatic hydrolysis by flavor protease + compound protease (mass ratio of 1:1) and flavor protease + alkaline protease + compound protease (mass ratio of 1:1:1) were 58% and 55%, respectively, which were significantly higher than those of other groups.

[0130] Effects of different proteases on the α-glucosidase inhibition rate of defatted peanut meal enzymatic hydrolysates Figure 7As shown in the data, the α-glucosidase inhibition rate after protein hydrolysis by a combination of multiple enzymes was significantly higher than that by a single protease, and the α-glucosidase inhibition rates of different protease hydrolysis products were 26%-45%; among them, the α-glucosidase inhibition rate of alkaline protease + flavor protease (mass ratio of 1:1) was the lowest, at 26%; the α-glucosidase inhibition rate of the hydrolysis product of flavor protease + alkaline protease + composite protease (mass ratio of 1:1:1) was the highest, at 45%, which was significantly higher than that of other groups.

[0131] Effects of different proteases on the α-amylase inhibition rate of defatted peanut meal enzymatic hydrolysates Figure 8 As shown in the figure, the α-amylase inhibition rate after protein hydrolysis by a combination of multiple proteases was significantly higher than that by a single protease. Among them, the α-amylase inhibition rate of the hydrolysis product of flavor protease + alkaline protease + composite protease (mass ratio of 1:1:1) reached 55%, which was significantly higher than that of other groups.

[0132] Example 3

[0133] A bioactive peptide composition with antioxidant and hypoglycemic effects, the composition comprising the peptide segments described in SEQ ID NO:1-SEQ ID NO:4, wherein the mass ratio of the peptide segments AFPKFR, PFPIK, APPFDPNKPK, and FATPVPLPK is 1:2:1:1.

[0134] The inhibition rate of different bioactive peptides and their combinations on α-glucosidase and α-amylase and the antioxidant activity were determined. Fig. 9 shown.

[0135] The inhibition rates of APPFDPNKPK on α-glucosidase and α-amylase were 34.7% and 45.8%, respectively; the inhibition rates of FATPVPLPK on α-glucosidase and α-amylase were 45.5% and 52.5%, which were significantly higher than 17.7% and 38.1% of AFPKFR, and 13.9% and 34.4% of PFPIK. Experiments have shown that the four synthesized peptide sequences all have hypoglycemic activity, and the activity of FATPVPLPK is relatively the best. The four peptide segments were compounded in a ratio of 1:2:1:1 to measure the inhibitory activity of the two enzymes. It was found that the inhibition rates of α-glucosidase and α-amylase were as high as 74.5% and 84%, respectively, which significantly improved the hypoglycemic activity. In addition, the inhibition rates of the four peptides on DPPH, H2O2 and Fe after compounding were significantly increased. 3+ The reducing power exceeds 92%.

[0136] Example 4

[0137] A bioactive peptide composition with antioxidant and hypoglycemic effects, the composition is compounded by the peptide segments AFPKFR and PFPIK at a mass ratio of 1.5:1. The inhibition rate of the composition on α-glucosidase and α-amylase was determined. The results showed that the inhibition rate of the composition on α-glucosidase was 40.5%, and the inhibition rate on α-amylase was 52.3%; the inhibition rate on DPPH, H2O2 and Fe 3+ The reducing powers were 90.1%, 89% and 93.2% respectively.

[0138] Example 5

[0139] A bioactive peptide composition with antioxidant and hypoglycemic effects, the composition is prepared by compounding the peptide segments APPFDPNKPK and FATPVPLPK in a mass ratio of 1:2. The inhibition rate of the composition on α-glucosidase and α-amylase was determined. The results showed that the inhibition rate of the composition on α-glucosidase was 59.4%, and the inhibition rate on α-amylase was 71.2%; the inhibition rate on DPPH, H2O2 and Fe 3+ The reducing powers were 88.6%, 86.7% and 88.3% respectively.

[0140] Example 6

[0141] Antioxidant and hypoglycemic effects of bioactive peptide components on diabetic mice

[0142] The diabetic model was established by feeding C57BL / 6J mice with a high-fat diet and injecting streptozotocin. After the model was successfully established, the bioactive peptide components (molecular weight 500-3000Da) with antioxidant and hypoglycemic effects were administered for treatment; the mice were then killed and a series of biochemical and pathological tests were performed to study the effects of bioactive peptides on biochemical indicators in mice and their histological changes in diabetes-related target organs (such as kidneys, liver, etc.).

[0143] The experiment was divided into 6 groups in total, CON (control group), MOD (model group), MET (yang medicine group, metformin 100 mg / kg), PEP-H (high-dose group of bioactive peptides with antioxidant and hypoglycemic effects 600 mg / kg), PEP-M (medium-dose group of bioactive peptides with antioxidant and hypoglycemic effects 400 mg / kg), PEP-L (low-dose group of bioactive peptides with antioxidant and hypoglycemic effects 200 mg / kg); the drugs were administered by gavage once a day, and the intervention treatment lasted for 6 weeks.

[0144] During the administration period, the water intake, body weight, blood sugar and OGTT changes of mice were detected. Figure 10-13 shown.

[0145] The intervention period was 6 weeks, and the weight, blood sugar, and water intake of each group of mice were recorded every week. Fig.10 During the treatment, the water intake of mice in the model group (MOD) was significantly higher than that of the normal group (CON), indicating that after becoming ill, the mice drank more water, which was consistent with the symptoms of type 2 diabetes. Starting from the third week of intragastric administration, the water intake of mice in the bioactive peptide group (PEP) and metformin group (MET) with antioxidant and hypoglycemic effects was significantly less than that of the MOD group. By the sixth week, the water intake was reduced by about 40%-55%, indicating that peanut peptide can significantly alleviate the polydipsia of mice.

[0146] The weight changes of mice Fig.11 As shown, in the first two weeks, there was no significant difference in body weight between the mice in the treatment groups (PEP group, MET group) and the mice in the model group (MOD). As the treatment time prolonged, the body weight of the mice in the treatment groups gradually increased, showing a significant difference compared with the mice in the model group.

[0147] The fasting blood glucose of each group of mice during the treatment period was measured. Fig.12 As shown in the figure, after the establishment of the type 2 diabetes model, the fasting blood glucose values ​​of mice in each group increased significantly. Compared with the model group (MOD), the fasting blood glucose of mice in each treatment group did not decrease significantly in the early stage of gavage, indicating that short-term gavage cannot significantly improve the fasting blood glucose of mice. After three weeks of gavage, especially in the sixth week, the blood glucose of the model group decreased by about 50% compared with the treatment group, indicating that the bioactive peptides with antioxidant and hypoglycemic effects significantly reduced the fasting blood glucose of model mice.

[0148] After 6 weeks of gavage, the mice were fasted for 12 hours and then subjected to an oral glucose tolerance test (OGTT). The mice were given glucose solution orally and the blood glucose levels were measured at 0, 30, 60, 90 and 120 min. The area under the curve (AUC) was calculated according to the following formula:

[0149]

[0150] Where: C0, C1, C2, C3, C4 are the blood glucose values ​​at 0, 30, 60, 90 and 120 min respectively.

[0151] Fig.13 To determine the oral glucose tolerance (OGTT) of mice to evaluate the ability of pancreatic β cells to regulate glucose levels, after the diabetic model was established, the oral glucose tolerance of mice in each group was significantly increased, indicating that the establishment of the diabetic model inhibited the ability of mice to regulate glucose levels. The OGTT values ​​of mice in the treatment group were all reduced, with the bioactive peptide group with antioxidant and hypoglycemic effects reducing it by 15% and the metformin group reducing it by 26%, indicating that the glucose regulation ability of diseased mice can be significantly improved after treatment.

[0152] Mice were anesthetized with isoflurane, and blood was collected from the eye sockets and then killed by spinal dislocation. A portion of the blood was subjected to routine biochemical examinations. The blood was centrifuged (10,000 rpm, 15 minutes at 4°C). The supernatant was immediately taken and used with Elisa kits to measure TG (triglycerides), TC (total cholesterol), HDL-C (high-density lipoprotein), LDL-C (low-density lipoprotein), SOD (superoxide dismutase), GSH (glutathione reductase), MDA (malondialdehyde) and other indicators. The test results were determined according to the instructions provided by Nanjing Jiancheng Bioengineering Research Institute.

[0153] The blood lipid levels (TG, TC, HDL-C, LDL-C) and organ oxidative stress indicators (liver GSH, SOD and kidney MDA, SOD) of mice were measured. Figure 14-17 After treatment with bioactive peptides with antioxidant and hypoglycemic effects, the blood lipid indexes of mice were improved (e.g. Fig.14 and Fig.15 ), compared with the model group mice, the TG, TC, and LDL-C levels of the bioactive peptide group with antioxidant and hypoglycemic effects and the metformin treatment group were significantly reduced, with each index reduced by 35%-43%, respectively, and the HDL-C level was also significantly increased by about 30%, further indicating that the bioactive peptides of the present invention can alleviate lipid metabolism disorders in the liver. After treatment with bioactive peptides with antioxidant and hypoglycemic effects, the GSH and SOD concentrations in the liver of the model group (MOD) mice were significantly reduced compared with the control group (CON); compared with the MOD group, the livers of the bioactive peptide group with antioxidant and hypoglycemic effects and the metformin treatment group mice showed a significant increase in GSH and SOD concentrations after intervention, among which the contents of the two enzymes in the bioactive peptide group mice increased by 49% and 46%, respectively (such as Fig.16 as shown).

[0154] Fig.17 Effect of MDA and SOD antioxidant enzyme activity levels in the kidneys. Compared with the control group (CON), the MDA level in the kidneys of the model group (MOD) increased and the SOD enzyme activity decreased, indicating that the enzymatic antioxidant capacity of diabetic mice decreased and the degree of cellular oxidative damage increased; after intervention treatment, the MDA content in the kidneys of the bioactive peptide group mice increased by 29%, and the SOD content increased by 41%. The above experimental results show that after the intervention of bioactive peptides, the blood lipid indexes of the PEP group mice were improved, and the oxidative stress levels of the liver and kidneys were alleviated, further indicating that the bioactive peptides of the present invention have good hypoglycemic and antioxidant activities.

[0155] The liver and kidney tissues of mice were taken for HE staining and observation. The results were as follows: Fig.18 and Fig.19As shown. HE staining showed that the hepatocytes of mice in the CON group had normal morphology and uniform size, and the liver tissue structure was intact. The hepatocytes of mice in the MOD group were swollen, with disordered structures, unclear nuclei, and a large number of vacuoles of varying sizes around them. The vacuoles around the nuclei of mice in the PEP and MET groups were significantly reduced, and the hepatocyte structure tended to be normal ( Fig.18 Moreover, compared with the CON group, the glomerular volume of the mice in the MOD group was significantly enlarged, with granular degeneration, edema, protein deposition and inflammatory cell infiltration. After treatment, the above morphological abnormalities of the mice in the PEP group were significantly improved, indicating that bioactive peptides have a significant protective effect on the kidney damage of diabetic mice ( Fig.19 ).

[0156] MASSON staining was used to determine the renal fibrosis of mice. The results of MASSON staining of mouse kidneys are shown in Fig. 20 As shown, the MASSON area is statistically Fig.21 Compared with the CON group, the renal fibrosis area of ​​the mice in the MOD group increased significantly. After intervention treatment, the renal fibrosis area of ​​the mice in the PEP group and the MET group gradually decreased from 27% to 20% and 15%, respectively.

[0157] Oil red staining results of mouse liver Fig. 22 As shown, the oil red area is statistically Fig.23 As shown. Fig. 22 The middle red color represents fat in the tissue. There was almost no red fat in the slices of mice in the CON group, while there was a significant increase in red in the MOD group, indicating that diabetes can cause metabolic disorders in the liver of mice, resulting in fat accumulation. After six weeks of treatment, the oil-red area of ​​the mouse liver was significantly reduced, and the oil-red area of ​​the mice in the PEP group was reduced by at least 25%.

[0158] In summary, diabetic metabolic disorders characterized by elevated blood sugar may affect the body's organs, causing organ damage or loss of function. It can also be clearly seen that the organ lesions of diabetic mice were effectively improved after treatment with bioactive peptides.

[0159] 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 blood sugar lowering function, characterized in that: The amino acid sequence of the bioactive peptide is shown in one of SEQ ID NO: 1 to SEQ ID NO:

4.

2. Use of the bioactive peptide with antioxidant and blood sugar lowering function according to claim 1 in the preparation of a drug with antioxidant and blood sugar lowering function.

3. A bioactive peptide composition with antioxidant and hypoglycemic functions, characterized in that: The composition comprises at least one bioactive peptide shown in SEQ ID NO: 1 to SEQ ID NO:

4.

4. The bioactive peptide composition with antioxidant and hypoglycemic functions according to claim 3, characterized in that: The composition is the bioactive peptides shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the mass ratio thereof is 1.5:

1.

5. The bioactive peptide composition with antioxidant and hypoglycemic functions according to claim 3, characterized in that: The composition is the bioactive peptides shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the mass ratio thereof is 1:

2.

6. The bioactive peptide composition with antioxidant and hypoglycemic functions according to claim 3, characterized in that: The composition is the bioactive peptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the mass ratio thereof is 1:2:1:

1.

7. The bioactive peptide composition with antioxidant and hypoglycemic functions according to claim 3, characterized in that: The antioxidant activity includes DPPH scavenging activity, hydroxyl ion free radical scavenging activity and iron ion reducing ability.

8. The bioactive peptide composition with antioxidant and hypoglycemic functions according to claim 3, characterized in that: The blood sugar lowering activity comprises α-glucosidase inhibitory activity and α-amylase inhibitory activity.

9. Use of the bioactive peptide composition with antioxidant and hypoglycemic function according to any one of claims 3 to 8 in the preparation of a drug with antioxidant and hypoglycemic function.

10. Use of the bioactive peptide composition with antioxidant and hypoglycemic function according to claim 9 in the preparation of a drug with antioxidant and hypoglycemic function, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

Images