Composite double-protein hypoglycemic peptide as well as preparation method and application thereof

Abstract

The invention discloses a composite double-protein hypoglycemic peptide as well as a preparation method and application thereof, and belongs to the technical field of animal and plant source double-protein active peptides. The method comprises the following steps: performing computer simulation enzyme digestion and virtual screening by utilizing bioinformatics to determine a protein raw material and protease, performing enzymolysis on animal and plant double proteins (soybean protein and casein) serving as raw materials by adopting neutral protease, and performing separation, purification and structural identification, thereby obtaining the protein. The protein peptide fragment with high alpha-glucosidase inhibitory activity is obtained by virtually screening molecular docking, and the amino acid sequences of the protein peptide fragment are shown as SEQ ID No: 6, SEQ ID No: 9 and SEQ ID No: 24. The compound double-protein peptide sequence provided by the invention has an inhibiting effect on the activity of alpha-glucosidase, can be used for preventing and treating diabetes mellitus, and can be used for long-term health care or treatment of people with impaired glucose tolerance or diabetics as a functional food ingredient.

Description

Technical Field

[0001] The present invention relates to a composite dual-protein hypoglycemic peptide and a preparation method and application thereof, and in particular to a preparation method and application of a dual-protein hypoglycemic peptide composited with soy protein and casein, belonging to the technical field of animal and plant-derived dual-protein active peptides. Background Art

[0002] Diabetes is a metabolic disorder caused by insufficient insulin production or insulin resistance. It has become one of the most serious and common chronic diseases in modern times, and is often accompanied by multiple complications such as cardiovascular and cerebrovascular diseases and kidney disease. Severe cases can cause disability or even life-threatening. Since drugs for treating diabetes have certain side effects, dietary intervention starting from daily diet is becoming increasingly important for the prevention and treatment of diabetes. Many studies have shown that α-glucosidase, which is involved in the regulation of postprandial blood glucose levels, is considered a key enzyme in the prevention and treatment of diabetes, and inhibiting its activity is an important strategy for controlling postprandial blood glucose through dietary intervention. Therefore, the development of new food-derived α-glucosidase inhibitors is of great value for the dietary prevention of diabetes.

[0003] Diets are rich in a variety of functional ingredients, including polyphenols, flavonoids, and bioactive peptides. Compared to other active ingredients, food-derived bioactive peptides offer advantages such as ease of absorption and processing stability. Furthermore, as protein supplements, they have a wide range of daily intake requirements, making them the most promising functional food ingredients. Research both domestically and internationally has demonstrated that peptides with antioxidant, immune-modulating, antihypertensive, and hypoallergenic properties can be produced from plant and animal-derived dual proteins through enzymatic hydrolysis or microbial fermentation. Furthermore, dual-protein complex peptides offer a richer amino acid profile than single-protein peptides, ensuring nutritional comprehensiveness. Existing literature lacks in-depth research on how to obtain highly active dual-protein complex hypoglycemic peptides, as traditional methods for isolating bioactive peptides have numerous limitations. Therefore, bioinformatics techniques can be used to predict peptide bioactivity, compare peptide sequence differences, and simulate protein hydrolysis processes. This could lead to the development of a stable preparation process for highly active dual-protein peptides with potential for preventing and treating diabetes. These peptides could be added to the diet as functional ingredients to improve overall health. Summary of the Invention

[0004] The present invention aims to overcome the above-mentioned shortcomings by providing a composite dual-protein hypoglycemic peptide, its preparation method, and its application. The active peptide has high α-glucosidase inhibitory activity, helping to lower postprandial blood sugar levels. It is derived from natural plant and animal proteins, is highly safe, has a more comprehensive amino acid composition, and its preparation process is suitable for large-scale industrial production.

[0005] The technical solution of the present invention is a composite dual-protein hypoglycemic peptide, wherein the amino acid sequence of the composite dual-protein hypoglycemic peptide comprises one or more of the following amino acid fragments:

[0006] SEQ ID No: 6: Leu Gln Ser Trp;

[0007] SEQ ID No:9: Val Val Val Pro Pro Phe Leu;

[0008] SEQ ID No: 24: Ala Ser Tyr Asp Thr Lys Phe Glu Glu.

[0009] Another technical solution of the present invention is an α-glucosidase inhibitor comprising one or more of the composite dual-protein hypoglycemic peptides.

[0010] The third technical solution of the present invention is a hypoglycemic protein peptide ingredient product, which includes the composite dual-protein hypoglycemic peptide and / or the α-glucosidase inhibitor 2.

[0011] The preparation method of the aforementioned composite dual-protein hypoglycemic peptide is as follows:

[0012] a. Preparation by virtual enzyme digestion and screening; the specific steps are: first, bioinformatics is used to simulate enzyme digestion, and virtual screening is used to obtain the preparation conditions of protein peptides with high α-glucosidase inhibitory activity, and the selected protein raw materials and proteases are determined;

[0013] b. Subsequently, the composite protein peptides were prepared and separated and purified under the conditions of simulated enzyme cleavage to obtain several peptide segments with high α-glucosidase inhibitory activity;

[0014] c. Virtually screen the peptides obtained in step b and verify the highly active protein peptides; finally, artificially synthesize the protein peptides with high α-glucosidase inhibitory activity screened out above.

[0015] Furthermore, the specific steps are:

[0016] (1) Protein peptide screening: Download the complete protein sequences of soy protein and casein from the protein database website, and use protease to simulate enzymatic digestion of each protein sequence. Through virtual screening, the preparation conditions of protein peptides with high α-glucosidase inhibitory activity were obtained;

[0017] (2) Preparation of composite protein peptides: fully mix the aqueous solution of casein and soy protein, heat and adjust the pH value to the optimal reaction condition of protease, add neutral protease for enzymatic hydrolysis, monitor and adjust the pH and temperature of the resulting enzymatic hydrolysate, boil and inactivate the enzyme after the enzymatic hydrolysis is completed, and obtain a composite protein hydrolysate;

[0018] (3) separation and purification: centrifuging the enzymatic hydrolysate obtained in step (2), and taking the supernatant, separating and purifying it by ultrafiltration and anion exchange resin column chromatography, and freeze-drying the component with high α-glucosidase inhibitory activity to obtain the composite dual-protein hypoglycemic peptide;

[0019] (4) Structural identification: LC-MS / MS was used to determine the amino acid sequence of the composite dual-protein hypoglycemic peptide in the fraction obtained in step (3), and several peptide segments with α-glucosidase inhibitory activity were obtained;

[0020] (5) Virtual screening: The peptides obtained in step (4) were virtually screened based on their α-glucosidase inhibitory activity, and the interaction between the highly active protein peptides and α-glucosidase was verified by molecular docking;

[0021] (6) Synthetic peptides: artificially synthesize the protein peptides with high α-glucosidase inhibitory activity obtained in step (5) to finally obtain a composite dual-protein hypoglycemic peptide.

[0022] Furthermore, in step (2), the composite mass ratio of soy protein and casein is 1:1, the optimum pH value of the protease is 6-8, the temperature is 40-60°C, the amount of neutral protease added is 2-10U / mg of composite double protein, and the enzymatic reaction time is 1-3h; the enzyme inactivation temperature is 90-100°C, and the enzyme inactivation time is 10-20min.

[0023] Furthermore, in step (3), the centrifugation condition is 8000 rpm / min and the centrifugation time is 20 min; ultrafiltration collects the <5 kDa component and passes it through a DEAE-52 anion exchange resin chromatography column, first pre-equilibrated with Tris-HCl buffer at pH 8, and then eluted with 0.1-0.8 M NaCl solution, and the eluted components are collected according to the absorbance curve at a wavelength of 220 nm, and their inhibitory activity against α-glucosidase is determined, and the component with the highest inhibitory activity is enriched, concentrated, and freeze-dried.

[0024] Furthermore, in step (4), the liquid chromatography separation used in step A was 0.1% formic acid in water, and in step B was 0.1% formic acid in acetonitrile. The gradients were set as follows: from 0 to 50 min, the linear gradient of liquid B was from 4% to 50%; from 50 to 54 min, the linear gradient of liquid B was from 50% to 100%; and from 54 to 60 min, liquid B was maintained at 100%. After liquid chromatography separation, mass spectrometry analysis was performed using a Q Exactive HF-X mass spectrometer for 60 min using positive ion detection. The mass-to-charge ratios of the polypeptide and its fragments were determined by collecting 10 fragment spectra after each full scan.

[0025] Furthermore, there are 26 peptide segments with α-glucosidase inhibitory activity, and their amino acid sequences are SEQ ID No. 1 to SEQ ID No. 26, respectively.

[0026] SEQ ID No. 1 is: Leu Arg Phe Leu

[0027] SEQ ID No.2 is: Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile

[0028] SEQ ID No.3 is: Leu Ala Phe Pro Gly Ser Ala Lys Asp Ile Glu Asn Leu

[0029] SEQ ID No. 4 is: Met Lys His

[0030] SEQ ID No.5 is: Leu Asp Ala Tyr Pro Ser Gly Ala Trp

[0031] SEQ ID No.6 is: Leu Gln Ser Trp

[0032] SEQ ID No.7 is: Leu Gln Pro Glu Val Met Gly

[0033] SEQ ID No.8 is: Phe Leu Phe

[0034] SEQ ID No.9 is: Val Val Val Pro Pro Phe Leu

[0035] SEQ ID No.10 is: Leu Gly Pro Val Arg Gly Pro Phe Pro

[0036] SEQ ID No.11 is: Leu Asp Ala Tyr Pro Ser Gly Ala Trp Tyr Tyr Val Pro

[0037] SEQ ID No.12 is: Leu Ser Ala Glu Phe Gly Ser Leu

[0038] SEQ ID No. 13 is: Val Leu Asn Glu Asn Leu

[0039] SEQ ID No.14 is: Val Val Ala Glu Gln Gly Gly Glu Gln Gly Leu-Glu TyrVal Val Phe Lys

[0040] SEQ ID No. 15 is: Phe Tyr Pro Glu Leu

[0041] SEQ ID No.16 is: Val Phe Asp Gly Glu Leu Arg Arg Gly Gln Leu

[0042] SEQ ID No.17 is: Leu Gly Thr Gln Tyr

[0043] SEQ ID No.18 is: Leu Ser Glu Asp Asp Val Phe Val Ile Pro

[0044] SEQ ID No.19 is: Leu Ser Ser Val Asp Ile Asn Glu Gly Ala Leu

[0045] SEQ ID No.20 is: His Lys Glu Met Pro Phe Pro Lys Tyr Pro Val Glu ProPheSEQ ID No.21 is: Leu Ser Glu Gln Asp Ile Phe Val Ile Pro Ala

[0046] SEQ ID No.22 is: Met His Gln Pro His Gln Pro Leu Pro Pro Thr

[0047] SEQ ID No.23 is: Ile Leu Leu Pro His His Ala Asp Ala Asp Phe Leu

[0048] SEQ ID No.24 is: Ala Ser Tyr Asp Thr Lys Phe Glu Glu

[0049] SEQ ID No.25 is: Leu Tyr Gln Glu Pro Val Leu Gly Pro Val Arg Gly ProPhe Pro Ile

[0050] SEQ ID No. 26 is: Tyr Phe Phe.

[0051] Furthermore, the virtual screening method in step (5) is the same as that in step (1), and the crystal structure of α-glucosidase is downloaded from the protein database, and molecular docking is performed using software.

[0052] The above products are used in the preparation of blood sugar lowering products or blood sugar lowering ingredients, as well as functional foods or health products.

[0053] The beneficial effects of the present invention are as follows: the present invention combines existing bioinformatics technology, simulates protein hydrolysis and virtual screening, predicts the biological activity of peptides, and conducts preliminary screening of raw materials; for the first time, a composite dual-protein peptide with amino acid sequences of SEQ ID No: 6, SEQ ID No: 9 and SEQ ID No: 24 is isolated and identified from a composite protein composed of soy protein and casein.

[0054] The present invention provides a method for treating diabetes through dietary intervention, wherein the main effect of the dietary intervention is a food-derived bioactive protein peptide with high α-glucosidase inhibitory activity, which reduces postprandial blood sugar levels and delays the rise of postprandial blood sugar.

[0055] The food-derived bioactive peptides described in the present invention are derived from natural animal proteins and plant proteins, are highly safe, easily absorbed by the human body, and have a strong targeted effect in lowering blood sugar. The raw materials for their preparation are soy protein and casein, which have a comprehensive amino acid composition, high nutritional value, low price, and a simple preparation process that can realize large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the mass spectrometry detection diagram of the composite dual-protein peptide SEQ ID No: 6.

[0057] Figure 2 This is the mass spectrometry detection diagram of the complex dual-protein peptide SEQ ID No: 9.

[0058] Figure 3 This is the mass spectrometry detection diagram of the complex dual-protein peptide SEQ ID No: 24.

[0059] Figure 4 This is a diagram showing the molecular docking results of the composite dual-protein peptide SEQ ID No: 6 and α-glucosidase.

[0060] Figure 5 This is a diagram showing the molecular docking results of the composite dual-protein peptide SEQ ID No: 9 and α-glucosidase.

[0061] Figure 6 This is a diagram showing the molecular docking results of the composite dual-protein peptide SEQ ID No: 24 and α-glucosidase.

[0062] Figure 7 This is a graph showing the inhibitory activity of three composite dual-protein peptides against α-glucosidase.

[0063] Figure 8 The effect of three complex dual-protein peptides on lowering postprandial blood glucose in mice.

[0064] Figure 9 The area under the curve (AUC) of the three complex dual-protein peptides affects blood glucose changes. DETAILED DESCRIPTION

[0065] The present invention will be further described in detail below with reference to specific examples. The following examples are not to be construed as limiting the scope of protection of the present invention.

[0066] Unless otherwise specified, the following examples are based on conventional methods. The materials and reagents used are all commercially available.

[0067] If no specific techniques or conditions are specified in the examples, all the processes were carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0068] Example 1 Preparation of Hypoglycemic Protein Peptide

[0069] 1. Simulation of protein cleavage and virtual screening

[0070] Bioinformatics screening of protein peptides: Soybean protein sequence P0DO16GLCA1_SOYBN and casein protein sequence P02663 CASA2_BOVIN were downloaded from the protein database websites of NCBI (http: / / www.ncbi.gov) and UniprotKB (http: / / www.uniport.org / ), and the proteases Trypsin (No. 1), Pepsin (No. 10), Proteinase K (No. 4), Flavorzyme (No. 29) and Papain (No. 7) provided by the peptidecutter tool in EXPASY software were used to simulate the enzyme cleavage of each protein sequence. The processing and optimization of the virtual screening of peptide sequences were performed by Protein preparation was performed using the Glide module in Maestro software. Protein preparation was performed using the Protein Preparation Wizard module. Receptors were pre-processed, optimized, and minimized (using the OPLS3e force field for constrained minimization). All peptides were prepared using the default settings of the LigPre module. During screening in the Glide module, the prepared receptors were imported to specify appropriate positions in the receptor grid generation.

[0071] First, the original ligand was redocked to confirm the feasibility of the selected docking method; then the data set was screened through SP docking; finally, the XP docking template was used to screen out the former ligands with higher scores determined by the SP method; and finally, the preparation conditions of the protein peptide with high α-glucosidase inhibitory activity were screened.

[0072] 2. Preparation of composite double protein hydrolysate

[0073] The enzymatic hydrolysis method was used to obtain a composite dual-protein peptide. The specific steps are as follows: First, soy protein and casein were weighed at a material-liquid ratio of 4%-8% and a composite mass ratio of 1:1. The two protein raw materials were added to ultrapure water and stirred until dissolved. After adjusting the pH to 7, 2-10 U / mg of neutral protease was added. The temperature was maintained at 50°C and the pH was not less than 6.5. The enzymatic hydrolysis was maintained for 2 hours. After the hydrolysis was completed, the enzymatic hydrolyzate was boiled in boiling water for 15 minutes to inactivate the enzyme. After cooling, the hydrolyzate was centrifuged at 8000g for 20 minutes. The supernatant was the composite dual-protein hydrolyzate.

[0074] 3. Isolation and purification of the composite dual-protein hypoglycemic peptide

[0075] The composite double protein hydrolysate was separated at room temperature using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa and an ultrafiltration pressure of 0.1-0.3 MPa. The inhibition rates of the two molecular weight fractions >5 kDa and <5 kDa on α-glucosidase activity were determined according to the method in Example 2.

[0076] The component with the highest inhibition rate of α-glucosidase activity (<5 kDa) obtained by ultrafiltration fractionation was further purified using anion exchange resin. DEAE-52 anion exchange resin was used as the chromatography column filler. The chromatography column was pre-equilibrated with Tris-HCl buffer at pH = 8, and then eluted with 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl solutions. The polypeptide solution eluted from each gradient eluent was collected according to the absorbance curve at 220 nm, and its inhibitory activity against α-glucosidase was determined according to the method in Example 2. The component with the highest inhibition rate was screened, enriched, concentrated, and freeze-dried.

[0077] 4. Structural identification of the composite dual-protein hypoglycemic peptide

[0078] The lyophilized fractions were subjected to LC-MS / MS analysis using a liquid chromatography column (0.15 mm x 150 mm, RP-C18) equilibrated with 95% solution A. Solution A consisted of 0.1% formic acid in water and solution B consisted of 0.1% formic acid in acetonitrile (84% acetonitrile). The gradient settings were as follows: 0-50 min, linear gradient of solution B from 4% to 50%; 50-54 min, linear gradient of solution B from 50% to 100%; and 54-60 min, solution B maintained at 100%. Mass spectrometry analysis of the isolates was performed, and amino acid sequences were identified using MaxQuant 1.5.5.1 software and the UniProt database. The peptide sequences shown below were obtained.

[0079] There are 26 peptide segments with α-glucosidase inhibitory activity, and their amino acid sequences are SEQ ID No. 1 to SEQ ID No. 26 respectively.

[0080] SEQ ID No. 1 is: Leu Arg Phe Leu

[0081] SEQ ID No.2 is: Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile

[0082] SEQ ID No.3 is: Leu Ala Phe Pro Gly Ser Ala Lys Asp Ile Glu Asn Leu

[0083] SEQ ID No. 4 is: Met Lys His

[0084] SEQ ID No.5 is: Leu Asp Ala Tyr Pro Ser Gly Ala Trp

[0085] SEQ ID No.6 is: Leu Gln Ser Trp

[0086] SEQ ID No.7 is: Leu Gln Pro Glu Val Met Gly

[0087] SEQ ID No.8 is: Phe Leu Phe

[0088] SEQ ID No.9 is: Val Val Val Pro Pro Phe Leu

[0089] SEQ ID No.10 is: Leu Gly Pro Val Arg Gly Pro Phe Pro

[0090] SEQ ID No.11 is: Leu Asp Ala Tyr Pro Ser Gly Ala Trp Tyr Tyr Val Pro

[0091] SEQ ID No.12 is: Leu Ser Ala Glu Phe Gly Ser Leu

[0092] SEQ ID No. 13 is: Val Leu Asn Glu Asn Leu

[0093] SEQ ID No.14 is: Val Val Ala Glu Gln Gly Gly Glu Gln Gly Leu-Glu TyrVal Val Phe Lys

[0094] SEQ ID No. 15 is: Phe Tyr Pro Glu Leu

[0095] SEQ ID No.16 is: Val Phe Asp Gly Glu Leu Arg Arg Gly Gln Leu

[0096] SEQ ID No.17 is: Leu Gly Thr Gln Tyr

[0097] SEQ ID No.18 is: Leu Ser Glu Asp Asp Val Phe Val Ile Pro

[0098] SEQ ID No.19 is: Leu Ser Ser Val Asp Ile Asn Glu Gly Ala Leu

[0099] SEQ ID No.20 is: His Lys Glu Met Pro Phe Pro Lys Tyr Pro Val Glu ProPhe

[0100] SEQ ID No.21 is: Leu Ser Glu Gln Asp Ile Phe Val Ile Pro Ala

[0101] SEQ ID No.22 is: Met His Gln Pro His Gln Pro Leu Pro Pro Thr

[0102] SEQ ID No.23 is: Ile Leu Leu Pro His His Ala Asp Ala Asp Phe Leu

[0103] SEQ ID No.24 is: Ala Ser Tyr Asp Thr Lys Phe Glu Glu

[0104] SEQ ID No.25 is: Leu Tyr Gln Glu Pro Val Leu Gly Pro Val Arg Gly ProPhe Pro Ile

[0105] SEQ ID No. 26 is: Tyr Phe Phe.

[0106] Example 2 Virtual screening and molecular docking of α-glucosidase inhibitory protein peptides

[0107] The peptide sequences shown in Table 1 were screened using the virtual screening method in Example 1, and the possible interaction between the screened composite dual-protein peptides and α-glucosidase was verified using the molecular docking software Auto Dock Tools 1.5.7. The crystal structure of α-glucosidase (PDB ID: 3W37, Resolution: 1.70 ) was downloaded from the Protein Data Bank (https: / / www.rcsb.org / structure / 3W37). Before docking, the receptor preparation steps included removing excess small molecules, nonpolar water molecules, and adding hydrogen atoms.

[0108] The peptide amino acid sequences obtained by the specific screening are SEQ ID No: 6, SEQ ID No: 9 and SEQ ID No: 24. The selected peptides were subjected to mass spectrometry detection, and the detection results were as follows Figure 1-Figure 3 shown.

[0109] For the selected peptide, the three-dimensional structure of the peptide was generated by Alphafold 3 as a ligand, and molecular simulation was performed using Auto Dock Vina 1.2.5 in Auto DockTools. The interaction mode between the peptide molecule and α-glucosidase was analyzed using Discover studio to obtain the interaction with each target residue, such as hydrogen bond, π-π interaction, hydrophobic interaction, etc., and the three-dimensional structure was saved. The docking results are as follows Figure 4-Figure 6 shown.

[0110] After multiple dockings of the three peptides with the original ligand, the binding mode of the original ligand to the active site was determined. Active site residues such as ASP-666, GLU-301, THR-681, GLU-792, ARG-676, THR-299, and MET-302 play a key role in stabilizing the ligand. GLU-301, THR-681, and GLU-792 bind to the original ligand multiple times in the three peptides through various mechanisms, including hydrogen bonds, salt bridges, van der Waals forces, and electrostatic interactions, contributing significantly to the stabilization of the small molecule in the protein pocket. These diverse interactions collectively stabilize the binding of the ligand to the protein, confirming the inhibitory mechanism of the peptide sequences represented by SEQ ID No: 6, SEQ ID No: 9, and SEQ ID No: 24 against α-glucosidase.

[0111] Example 3 Determination of the Inhibition of α-glucosidase Activity by the Composite Dual-Protein Hypoglycemic Peptide

[0112] 100 μL of 1.5 mmol / L 4-nitrophenyl-α-D-pyranoglucoside (α-PNPG) and 50 μL of the complex dual-protein peptide solution were pipetted into a 96-well microtiter plate and mixed, and incubated at 37°C for 10 minutes. Then, 100 μL of 0.5 U / mL α-glucosidase solution was added to initiate the reaction, which was incubated at 37°C for 30 minutes. The reaction was terminated with 80 μL of 1 M Na2CO3, and the absorbance was measured at a wavelength of 405 nm. The experiment was repeated three times, and the average value was taken.

[0113] The α-glucosidase activity inhibition rate was calculated according to the following formula:

[0114] α-glucosidase activity inhibition rate (%) = [1-(OD1-OD2) / (OD3-OD4)] × 100; where OD1 is the absorbance value of the sample group; OD2 is the absorbance value when the enzyme solution is replaced by an equal amount of 0.1 M phosphate solution (pH 6.8); OD3 is the absorbance value when the sample is replaced by an equal amount of 0.1 M phosphate solution (pH 6.8); OD4 is the absorbance value when the sample and enzyme solution are replaced by an equal amount of 0.1 M phosphate solution (pH 6.8). The α-glucosidase activity inhibition curves of the three composite dual-protein peptides are shown in FIG. Figure 7 shown.

[0115] The results show:

[0116] The IC50 value of SEQ ID No: 6 against α-glucosidase inhibitory activity was 77.99±12.49 μmoL / L.

[0117] The IC50 value of SEQ ID No: 9's inhibitory activity against α-glucosidase was 66.73±12.64 μmoL / L.

[0118] The IC50 value of SEQ ID No: 24's inhibitory activity against α-glucosidase was 112.90±13.3 μmoL / L.

[0119] Example 4 Effect of the Composite Dual-Protein Hypoglycemic Peptide on Postprandial Blood Glucose Changes in Normal Mice

[0120] Male CD-1 mice were used as research subjects. After one week of acclimatization, their fasting blood glucose was measured. The mice were then randomly divided into five groups of 10 mice each based on their fasting blood glucose and body weight. Group 1 received 1.5 g / kg maltose by gavage; Group 2 received 1.5 g / kg maltose and 100 mg / kg acarbose as a positive control by gavage; Group 3 received 1.5 g / kg maltose and 100 mg / kg SEQ ID No: 6 by gavage; Group 4 received 1.5 g / kg maltose and 100 mg / kg SEQ ID No: 9 by gavage; and Group 5 received 1.5 g / kg maltose and 100 mg / kg SEQ ID No: 24 by gavage. The gavage volume was 10 mL / kg. Blood was collected from the tail vein of mice at 0, 5, 15, 30, 60, 90 and 120 min after gavage to detect blood glucose. The blood glucose change curves of each group of mice over time were drawn and the area under the blood glucose curve of each group was calculated.

[0121] The results are as follows Figure 8 The results showed that compared with the negative control group in group 1, the blood sugar increase trend of mice in group 2 that were gavaged with acarbose was significantly reduced, and the peak blood sugar increase of groups 3-5 that were gavaged with the compound dual-protein peptide was also significantly lower than that of group 1, showing a good effect in alleviating blood sugar increase. Figure 9 The area under the blood glucose change curve (AUC) shows that the areas under the postprandial blood glucose change curves of the three composite dual-protein hypoglycemic peptides are significantly different from those of the control group, playing a good regulatory role in inhibiting the increase in postprandial blood glucose and effectively slowing down the increase in postprandial blood glucose.

[0122] The above are only several preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several modifications and improvements can be made without departing from the principles of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A composite dual-protein hypoglycemic peptide, characterized by: The amino acid sequence of the composite dual-protein hypoglycemic peptide comprises one or more of the following amino acid fragments: SEQ ID No: 6: Leu Gln Ser Trp; SEQ ID No:9: Val Val Val Pro Pro Phe Leu; SEQ ID No: 24: Ala Ser Tyr Asp Thr Lys Phe Glu Glu.

2. An α-glucosidase inhibitor, characterized in that: It comprises one or more complex dual-protein hypoglycemic peptides according to claim 1.

3. A blood sugar-lowering protein peptide ingredient product, characterized by: It comprises the composite dual-protein hypoglycemic peptide according to claim 1 and / or the α-glucosidase inhibitor according to claim 2.

4. The method for preparing the composite dual-protein hypoglycemic peptide according to claim 1, characterized in that: a. Preparation by virtual enzyme digestion and screening; the specific steps are: first, bioinformatics is used to simulate enzyme digestion, and virtual screening is used to obtain the preparation conditions of protein peptides with high α-glucosidase inhibitory activity, and the selected protein raw materials and proteases are determined; b. Subsequently, the composite protein peptides were prepared and separated and purified under the conditions of simulated enzyme cleavage to obtain several peptide segments with high α-glucosidase inhibitory activity; c. Virtually screen the peptides obtained in step b and verify the highly active protein peptides; finally, artificially synthesize the protein peptides with high α-glucosidase inhibitory activity screened out above.

5. The method for preparing the composite dual-protein hypoglycemic peptide according to claim 4, Its characteristic is that the steps are: (1) Protein peptide screening: Download the complete protein sequences of soy protein and casein from the protein database website, and use protease to simulate enzymatic digestion of each protein sequence. Through virtual screening, the preparation conditions of protein peptides with high α-glucosidase inhibitory activity were obtained; (2) Preparation of composite protein peptides: fully mix the aqueous solution of casein and soy protein, heat and adjust the pH value to the optimal reaction condition of protease, add neutral protease for enzymatic hydrolysis, monitor and adjust the pH and temperature of the resulting enzymatic hydrolysate, boil and inactivate the enzyme after the enzymatic hydrolysis is completed, and obtain a composite protein hydrolysate; (3) separation and purification: centrifuging the enzymatic hydrolysate obtained in step (2), and taking the supernatant, separating and purifying it by ultrafiltration and anion exchange resin column chromatography, and freeze-drying the component with high α-glucosidase inhibitory activity to obtain the composite dual-protein hypoglycemic peptide; (4) Structural identification: LC-MS / MS was used to determine the amino acid sequence of the composite dual-protein hypoglycemic peptide in the fraction obtained in step (3), and several peptide segments with α-glucosidase inhibitory activity were obtained; (5) Virtual screening: The peptides obtained in step (4) were virtually screened based on their α-glucosidase inhibitory activity, and the interaction between the highly active protein peptides and α-glucosidase was verified by molecular docking; (6) Synthetic peptides: artificially synthesize the protein peptides with high α-glucosidase inhibitory activity obtained in step (5) to finally obtain a composite dual-protein hypoglycemic peptide.

6. The method for preparing the composite dual-protein hypoglycemic peptide according to claim 5, characterized in that: In step (2), the composite mass ratio of soy protein and casein is 1:1, the optimum pH value of the protease is 6-8, the temperature is 40-60°C, the amount of neutral protease added is 2-10U / mg of the composite double protein, and the enzymatic reaction time is 1-3h; the enzyme inactivation temperature is 90-100°C, and the enzyme inactivation time is 10-20min.

7. The method for preparing the composite dual-protein hypoglycemic peptide according to claim 5, characterized in that: In step (3), the centrifugation condition is 8000 rpm / min and the centrifugation time is 20 min; ultrafiltration collects the <5 kDa component and passes it through a DEAE-52 anion exchange resin chromatography column, which is first pre-balanced with Tris-HCl buffer at pH 8 and then eluted with 0.1-0.8 M NaCl solution. The eluted components are collected according to the absorbance curve at a wavelength of 220 nm, and their inhibitory activity against α-glucosidase is determined. The component with the highest inhibitory activity is enriched, concentrated, and freeze-dried.

8. The method for preparing the composite dual-protein hypoglycemic peptide according to claim 5, characterized in that: In step (4), the liquid chromatography separation was performed using a 0.1% formic acid aqueous solution and a 0.1% formic acid acetonitrile aqueous solution in a liquid A solution. The gradients were set to be from 0 to 50 min, with a linear gradient of 4% to 50% for liquid B; and from 50 to 54 min, with a linear gradient of 50% to 100% for liquid B. Liquid B was maintained at 100% for 54-60 minutes. After liquid chromatography separation, mass spectrometry analysis was performed on a QExactive HF-X mass spectrometer for 60 minutes using positive ion detection. The mass-to-charge ratios of the peptides and their fragments were determined by collecting 10 fragment spectra after each full scan.

9. The method for preparing the composite dual-protein hypoglycemic peptide according to claim 5, wherein: Step (5) The virtual screening method is the same as that described in step (1). The crystal structure of α-glucosidase is downloaded from the protein database, and molecular docking is completed using software.

10. Use of the product according to any one of claims 1 to 3, characterized in that: The invention can be used in the preparation of blood sugar lowering products or blood sugar lowering ingredients, and functional foods or health care products.

Images