Animal blood-derived antioxidant peptide and its preparation method and application

Abstract

The present invention relates to the technical field of antioxidant peptides, in particular to an animal blood-derived antioxidant peptide and a preparation method and application thereof, wherein the amino acid sequence thereof is at least one of GLWGKV and LIVYPW, as specifically shown in SEQ ID NO: 1-2. The preparation method thereof comprises the following steps: 1) preparation of a crude hemoglobin peptide product; 2) ultrafiltration; 3) gel exclusion chromatography separation; 4) screening out two polypeptides with antioxidant activity, GLWGKV and LIVYPW, by nano-LC-MS / MS detection, computer virtual screening of bioactive peptides, molecular docking and molecular dynamics simulation. The animal blood-derived antioxidant peptide of the present invention can be applied in functional foods, and the method of the present invention can accurately, quickly, efficiently and greenly prepare antioxidant peptides by means of computer simulation technology combined with cell experimental verification, and the antioxidant activity mechanism and structure-activity relationship of polypeptides in hemoglobin are explored from the molecular and cellular levels.

Description

Technical Field

[0001] The present invention relates to the technical field of antioxidant peptides, and specifically relates to an animal blood-derived antioxidant peptide and a preparation method and application thereof. Background Art

[0002] Excessive accumulation of ROS, RNS and other free radical substances in cells can cause oxidative stress, which is the driving force for the onset and development of many diseases, including chronic diseases such as diabetes, obesity and Alzheimer's disease. In order to prevent free radicals from damaging the human body, scholars have begun to study the role and prevention mechanism of free radicals, explore the relationship between free radicals and antioxidant effects, and find appropriate antioxidants for protection. Studies have shown that antioxidant peptides derived from dietary foods have excellent antioxidant potential and are the most effective strategy to address the occurrence of the above chronic diseases.

[0003] Livestock and poultry blood is rich in a variety of essential amino acids, proteins and minerals, and is a raw material with high value potential. More than 60% of the protein in blood is present in blood cells, and it is rich in amino acid residues such as phenylalanine, leucine, glutamic acid, aspartic acid, etc. that have direct or indirect antioxidant activity, and is a good source of antioxidant peptides. However, basic research on the efficient separation and extraction, structure, activity mechanism and structure-activity relationship of antioxidant peptides in blood globulin is still relatively weak. The effect of single separation and extraction technology is poor, the traditional purification, identification and screening methods are backward, and the functional mechanism of active substances is unclear, which seriously restricts its development and utilization.

[0004] At present, most antioxidant peptides are separated and purified by a series of membrane and chromatographic techniques to separate and purify peptides, use mass spectrometry to identify peptide sequences, and finally synthesize the identified peptides for functional activity verification. However, in most cases, the peptides identified after separation and purification will increase the workload of peptide synthesis and functional activity verification. Therefore, it is necessary to explore new technologies to study food-derived peptides.

[0005] Common evaluation methods for antioxidant peptides include evaluation based on chemical systems, evaluation based on in vitro cell models, and evaluation based on in vivo animal experiments. Generally speaking, the purpose of these detection methods is to relatively quantify the hydrogen atom transfer, single electron transfer, or metal ion chelation ability of the target substrate, depending on the chemical reactions involved. In vitro chemical tests take into account the fact that some peptides exhibit strong antioxidant activity in vitro, but are unable to effectively resist oxidation reactions in vivo, so there is uncertainty in their in vivo performance. Animal clinical trials are costly and time-consuming.

[0006] Computer simulation, which has been developed in recent years, is a new technology based on computer-assisted analysis such as bioinformatics analysis, molecular docking, and dynamics simulation, which can be used to screen, predict and clarify the potential activity and mechanism of peptides. The combination of molecular docking and molecular dynamics simulation is conducive to the rapid screening of functional active peptides, reducing the workload of peptide synthesis and verification, and can explain the structure-activity relationship between proteins and bioactive peptides from the perspective of molecular interactions. Summary of the invention

[0007] The purpose of the present invention is to provide an animal blood-derived antioxidant peptide and a preparation method and application thereof.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] An animal blood-derived antioxidant peptide, whose amino acid sequence is at least one of GLWGKV and LIVYPW, as specifically shown in SEQ ID NO: 1-2.

[0010] The method for preparing the animal blood-derived antioxidant peptide of the present invention comprises the following steps:

[0011] (1) Preparation of crude hemoglobin peptide

[0012] Fresh anticoagulated chicken blood was taken, and the blood was separated after centrifugation, and the red blood cells in the lower layer were collected, and washed three times with physiological saline to obtain fresh red blood cells; the red blood cells obtained above were crushed by an ultrasonic cell crusher, the crushed liquid was centrifuged, the lower layer of cell fragments were discarded, and the supernatant was collected as a hemoglobin extract, and vacuum freeze-dried into hemoglobin powder for later use;

[0013] Take hemoglobin powder and perform enzymolysis according to the material-liquid ratio of 1:11, enzymolysis time of 4.75h, and alkaline protease activity of 6280U / g. After the enzymolysis is completed, the enzyme is inactivated and the supernatant is centrifuged; the initial pH of the supernatant is adjusted to 7.8, and 8% of Aspergillus oryzae inoculation amount is added, and fermentation is carried out for 32h. The supernatant is centrifuged and freeze-dried to obtain a crude hemoglobin peptide product, which greatly improves the antioxidant activity and has higher stability under the influence of factors such as pH, temperature, NaCl and metal ions;

[0014] (2) Ultrafiltration

[0015] The supernatant was placed in a Buchner funnel and further filtered through a double-layer filter paper to collect the filtrate; the filtrate was ultrafiltered through a tangential flow filtration system; the three fractions (M 1 <3kDa,3kDa<M 2 <10kDa,10kDa<M 3 ) The filtrate was freeze-dried and the antioxidant activity was measured;

[0016] (3) Gel Exclusion Chromatography Separation

[0017] The concentration of the polypeptide solution was adjusted to 5 mg / mL, and the solution was filtered through a 0.22 μm polyethersulfone filter membrane to remove impurities before injection. The injection volume was 100 μL, the buffer was 0.01 M HCl, and the flow rate was constant at 0.35 mL / min at room temperature. A UV detector was selected to collect signals at a wavelength of 214 nm. All fractions were collected using an automatic collector system and stored at -20°C.

[0018] (4) Through nano-LC-MS / MS detection, computer virtual screening of bioactive peptides, molecular docking and molecular dynamics simulation, two peptides with antioxidant activity, GLWGKV and LIVYPW, were screened out.

[0019] Among them, in the nano-LC-MS / MS detection method, mobile phase A is 0.1% formic acid-98% aqueous solution, of which acetonitrile is 2%; phase B is 0.1% formic acid-80% acetonitrile solution, of which water is 20%; the flow rate is 300nL / min, and the gradient elution is 60min;

[0020] Mobile phase B ratio: 2-5% for 2 min, 5-22% for 34 min, 22-45% for 20 min, 45-95% for 2 min, 95% for 2 min;

[0021] Mass spectrometry conditions: MS1, resolution: 120k (@200m / z), AGC: 1E6, Max IT: 50ms, scanning range: The 20 ions with the highest intensity in the primary scan were screened by quadrupole and then fragmented by HCD for fragment ion scanning; the quadrupole isolation window was 1.2m / z, the standardized collision energy was 30%, AGC: 1E5, Max IT: 100ms. The secondary scan resolution was 15k; according to the chromatographic peak width, the dynamic exclusion time was set to 30s; single-charged ions and ions with a valence greater than 6 were not scanned in the secondary scan.

[0022] The computer virtual screening of bioactive peptides is as follows: ToxinPred virtually predicts the toxicity of peptides, and the peptide ranker https: / / distilldeep.ucd.ie / PeptideRanker / predicts the potential biological activity of peptides. The prediction mainly relies on the specific amino acid residues in the peptide sequence; the score of the peptide ranges from 0 to 1. The higher the score, the higher the biological activity. Peptides with a score of ≥ 0.5 are selected; the CPPpred software is used to predict the potential bioaccessibility of peptides. The software ranks peptides according to the probability of cell penetration; the score of the peptide ranges from 0 to 1. The higher the score, the higher the biopenetration; BIOPEP-UWM TM The bioactive peptide database is used to search for peptides and predict their functional properties.

[0023] Molecular docking used Autodock to analyze the interaction between the selected antioxidant peptides and Keap1; before docking with the antioxidant peptide, pymol was first used to remove water molecules and the original ligand Nrf2 from the crystal, and then Autodock was used to add hydrogen atoms and charges; the ligand was drawn as a secondary structure, and then the 3D structure was drawn and the minimum energy structure was calculated; Autodock was used for docking, and the Lamarckian genetic algorithm was used to find the optimal binding state of the ligand and the receptor; the number of genetic algorithms was set to 50, and the binding energy was used to evaluate the binding ability of the ligand to the receptor. The lower the energy, the more stable the complex; the 2D and 3D structures of the interaction between the peptide and the target protein were obtained using BIOVIA Discovery Studio 2021Client visualization software.

[0024] Molecular dynamics simulation is to use Gromacs2020 software to perform 200ns molecular dynamics simulation on the protein-ligand complex obtained by molecular docking; Charmm 36 is selected as the protein force field, Gaff2 is selected as the ligand force field, and the TIP3P water model is selected to add solvent to the protein-ligand system, establish a water box with a periodic boundary of 1.0nm, and add sodium ions and chloride ions to balance the charge in the system to maximize the restoration of the real experimental environment;

[0025] First, the simulation system was prepared by constraining the heavy atoms of proteins and small molecules, and performing 10,000 steps of energy minimization on water molecules; then, we released the constraints and performed 10,000 steps of energy minimization on the entire system; after optimizing the energy, the system was gradually heated to 300K, and after 1000ps, the system reached the target temperature; then, a 1ns equilibrium operation was performed in the NVT and NPT systems to ensure the stability of the system; finally, a 200ns molecular dynamics simulation was performed; the root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg) and hydrogen bond changes of the complex were analyzed, and the g_MMGBSA method in the Gromacs 2020 program was used to calculate the binding free energy of the ligand and the protein.

[0026] The animal blood-derived antioxidant peptide of the present invention can be widely used in foods, especially functional foods.

[0027] The present invention further purifies and identifies the crude peptide obtained, predicts its physicochemical properties and activity by means of computer simulation, and combines molecular docking and molecular dynamics simulation to explain the structure-activity relationship between proteins and polypeptides from the perspective of molecular interaction; realizes rapid screening of polypeptides with high antioxidant activity, and clarifies the interaction and structure-activity relationship between polypeptides and Keap1 protein.

[0028] At present, cell experiments have become an important tool for studying cells' resistance to oxidative stress as a supplement to in vitro chemical experiments. Considering that some peptides show strong antioxidant activity in vitro but cannot effectively resist oxidative reactions in vivo, cell experiments provide a window for directly observing the assimilation and metabolic processes of substances in cells. Compared with animal experiments, cell experiments are not only less time-consuming, but also relatively low in cost. The present invention adopts H 2 O 2 The induced Caco-2 cell oxidative damage model was used to explore the preventive effect of peptides on oxidative damage at the cellular level; the effects of peptides on antioxidant enzyme activity and MDA content in cells damaged by oxidative stress were studied, and the expression of key proteins in oxidative stress-related pathways was verified by Western Blot, revealing the potential antioxidant pathway of antioxidant peptides in preventing cellular oxidative stress damage, and verifying the interaction mechanism between antioxidant peptides and Keap1 protein, in order to better explore the relationship between structure and activity.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The preparation method of the present invention utilizes probiotics synergistic enzymatic hydrolysis technology to prepare crude hemoglobin peptides. Compared with hemoglobin peptides prepared by single enzymatic hydrolysis technology, the hydroxyl radical scavenging rate, DPPH radical scavenging rate, metal chelating rate, and reducing power are increased by 60.86%, 102.97%, 78.22%, and 46.43%, respectively, which greatly improves the antioxidant activity, and has higher stability under the influence of factors such as pH, temperature, NaCl, and metal ions.

[0031] (2) The present invention discovered the antioxidant mechanism of the polypeptide GLWGKV with antioxidant activity.

[0032] (3) The present invention further purified and identified the polypeptides, and used computer methods to predict their physicochemical properties and activities, thus achieving rapid screening and activity prediction of antioxidant peptides; the structure-activity relationship between Keap1 protein and polypeptides GLWGKV and LIVYPW was explained from the perspective of molecular interaction by combining molecular docking and molecular dynamics simulation; the binding sites were identified, and GT occupied 6 binding sites where amino acid residues in the Keap1-Kelch region formed hydrogen bonds with Nrf2, and LT occupied 2 binding sites where amino acid residues in the Keap1-Kelch region formed hydrogen bonds with Nrf2. The molecular dynamics simulation results showed that the two complexes of Keap1-GLWGKV and Keap1-LIVYPW reached dynamic equilibrium at around 100ns, and neither of them left the protein pocket and was able to bind firmly to the protein. In addition, the number of hydrogen bonds between GLWGKV and Keap1 protein is significantly greater than that between LIVYPW and Keap1, and the binding free energy of GLWGKV and Keap1 protein is -86.37 kJ / mol, which is significantly lower than the binding energy of LIVYPW and Keap1 protein -28.403 kJ / mol, indicating that the complex Keap1-GLWGKV is more stable than Keap1-LIVYPW. The present invention uses computer simulation technology combined with cell experimental verification to avoid the complex process and expensive cost of traditional methods, accurately, quickly, efficiently and greenly prepares antioxidant peptides, and explores the antioxidant activity mechanism and structure-activity relationship of polypeptides in hemoglobin at the molecular and cellular levels. The method can also be extended to the rapid screening, prediction and verification of functional activities other than antioxidant.

[0033] (4) The present invention found that the polypeptide GLWGKV is stable in the simulated gastrointestinal digestion process and has an overall enhanced antioxidant activity. 2 O 2 The Caco-2 cell oxidative damage model was induced to explore the protective mechanism of GLWGKV on cell oxidative damage at the cellular level. It was found that GLWGKV increased H 2 O 2 The induced oxidative damage in Caco-2 cells increased the activities of SOD, CAT and GSH-Px, reduced the content of MDA, and enhanced the expression of Nrf2, HO-1 and NQO1 proteins. The potential antioxidant pathway of GLWGKV in preventing cellular oxidative stress damage was revealed to be Keap1-Nrf2-ARE.

[0034] (5) The animal blood-derived antioxidant peptides of the present invention can be widely used in foods, especially functional foods, which can help improve the comprehensive utilization rate and economic added value of animal blood. It can also help to gain a deeper understanding of the potential of blood cell-derived polypeptides in preventing and treating sub-health, and provide a theoretical and technical basis for the development of high-value functional foods from by-products, thereby promoting the development of the big health food industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Effects of pH (A), temperature (B), NaCl (C) and metal ions (D) on the antioxidant activity of hydrolysates.

[0036] Figure 2 Antioxidant activity of different molecular weights.

[0037] Figure 3 Gel exclusion chromatography separation diagram (A) and antioxidant activity of each component (B).

[0038] Figure 4 The peptide chain length distribution of bioactive peptides (A) and the source protein ratio (B).

[0039] Figure 5 Molecular docking of peptide GLWGKV to Keap1 (A, B) and molecular docking of peptide LIVYPW to Keap1 (C, D).

[0040] Figure 6 The root mean square deviation (A), root mean square fluctuation (B), radius of gyration (C) and hydrogen bond (D) during the complex simulation process.

[0041] Figure 7 To simulate the effect of gastrointestinal digestion on the antioxidant activity of GLWGKV (A) and LIVYPW (B).

[0042] Figure 8 The effects of GLWGKV (GT) and LIVYPW (LT) on the cytotoxicity of Caco-2 cells.

[0043] Fig. 9 For GT, LT and H 2 O 2 Effects on the viability of Caco-2 cells.

[0044] Fig.10 This is the effect of GT on the antioxidant enzyme activities and MDA content in Caco-2 cells.

[0045] Fig.11 This is the effect of GT on the expression of proteins related to the Nrf2-Keap1 pathway. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] 1. Preparation and identification of animal blood-derived antioxidant peptides

[0048] (1) Preparation of crude hemoglobin peptide

[0049] Fresh anticoagulated chicken blood is taken, and the blood is separated after centrifugation, and the red blood cells in the lower layer are collected, and the blood is washed three times with physiological saline to obtain fresh red blood cells; the red blood cells obtained above are crushed by an ultrasonic cell crusher, the crushed liquid is centrifuged to discard the lower layer of cell fragments, and the supernatant is collected as a hemoglobin extract, and the hemoglobin powder is vacuum freeze-dried for standby use; the hemoglobin powder is enzymolyzed according to a solid-liquid ratio of 1:11, an enzymolysis time of 4.75 hours, and an alkaline protease activity of 6280U / g, and the enzyme is inactivated and centrifuged to obtain a supernatant; the initial pH of the supernatant is adjusted to 7.8, an Aspergillus oryzae inoculum amount of 8% is added, and the fermentation is carried out for 32 hours, and the supernatant is centrifuged and freeze-dried to obtain a crude hemoglobin peptide product, and the soluble peptide content and the ferrous heme content are measured.

[0050] (2) Effects of pH, temperature, NaCl and metal ions on the antioxidant activity of hydrolysates

[0051] ① Effect of metal ions on the antioxidant activity of hydrolysates

[0052] To evaluate the effect of metal ions on the hydrolyzate, 1.0 mg / mL samples were added to 10 mM solutions of different metal ions, including K+ (KCl), Zn 2+ (ZnSO 4 ), Ca 2+ (CaCl 2 ),Cu 2+ (CuSO 4 ) and Mg 2+ (MgCl 2 The mixture was placed at room temperature for 1 h and then subjected to the DPPH free radical scavenging activity test.

[0053] ② Thermal stability

[0054] To determine the thermal stability of the hydrolyzate, 1.0 mg / mL of the sample was transferred into a screw-capped test tube. The test tube was heated to different temperatures (20, 40, 60, 80, 100°C) for 1 h and then cooled in ice water. The DPPH free radical scavenging activity was then tested.

[0055] ③ pH stability

[0056] To measure the effect of pH on the hydrolysis products, samples (1.0 mg / mL) were adjusted to different pH values ​​(3.0, 5.0, 7.0, 9.0, 11.0) using 1 M HCl and 1 M NaOH and incubated at room temperature for 1 h. The pH of the samples was then adjusted to 7.0 for DPPH free radical scavenging activity testing.

[0057] ④Stability against NaCl concentration

[0058] The hydrolyzate was mixed with different concentrations of NaCl (1%, 3%, 5%, 7%, 9%), left to stand at room temperature (25°C) for 1 h, and then tested for DPPH free radical scavenging activity.

[0059] The experimental results are as follows Figure 1 shown.

[0060] (3) Ultrafiltration

[0061] The supernatant was placed in a Buchner funnel and further filtered through a double layer of filter paper to collect the filtrate. The filtrate was ultrafiltered through a tangential flow filtration system (P / N: S02-E003-05-N, Media / Rating: mPES / 3kDa / 10kDa). The three fractions obtained by ultrafiltration (M 1 <3kDa、3kDa<M 2 <10kDa、10kDa<M 3 ) The filtrate was freeze-dried and the antioxidant activity was measured.

[0062] (4) Gel Exclusion Chromatography Separation

[0063] The concentration of the peptide solution was adjusted to 5 mg / mL, filtered through a 0.22 μm polyethersulfone filter to remove impurities, and then injected with an injection volume of 100 μL, a buffer of 0.01 M HCl, and a constant flow rate of 0.35 mL / min at room temperature. Select a UV detector to collect signals at a wavelength of 214 nm. All fractions were collected using an automatic collector system and stored at -20 °C until further analysis.

[0064] (5) Detection

[0065] Chromatographic column (1.9 μm, Dr. Maisch, 100 μm ID×15 cm); mobile phase A is 0.1% formic acid-98% aqueous solution (acetonitrile is 2%), phase B is 0.1% formic acid-80% acetonitrile solution (water is 20%); flow rate is 300 nL / min, gradient elution is 60 min. Mobile phase B ratio: 2-5% for 2 min, 5-22% for 34 min, 22-45% for 20 min, 45-95% for 2 min, 95% for 2 min. Mass spectrometry conditions: MS1, resolution: 120k (@200 m / z), AGC: 1E6, Max IT: 50 ms, scanning range: The top 20 ions with the highest intensity in the primary scan were screened by quadrupole and then fragmented by HCD. The quadrupole isolation window was 1.2 m / z, the normalized collision energy was 30%, AGC: 1E5, and Max IT: 100 ms. The secondary scan resolution was 15k. The dynamic exclusion time was set to 30 s according to the chromatographic peak width; single-charged ions and ions with a valence greater than 6 were not scanned in the secondary scan.

[0066] (6) Computer virtual screening of bioactive peptides

[0067] ToxinPred virtually predicts the toxicity of peptides (https: / / webs.iiitd.edu.in / raghava / toxinpred / ). Peptide Ranker (https: / / distilldeep.ucd.ie / PeptideRanker / ) predicts the potential biological activity of peptides, and the prediction mainly relies on specific amino acid residues in the peptide sequence. The score of the peptide ranges from 0 to 1. The higher the score, the higher the biological activity. Peptides with a score ≥ 0.5 are selected. The potential bioaccessibility of peptides is predicted using CPPpred software (https: / / distilldeep.ucd.ie / CPPpred / ), which ranks peptides according to the probability of cell penetration. The score of the peptide ranges from 0 to 1. The higher the score, the higher the biopenetration. Using BIOPEP-UWM TM The bioactive peptide database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) was used to search for peptides and predict their functional properties.

[0068] (7) Molecular docking

[0069] To screen for potential peptides that directly inhibit Keap1-Nrf2 interaction, Autodock was used to analyze the interaction between the selected antioxidant peptides and Keap1. The crystal structure of Keap1 protein was obtained from the Protein Data Bank (PDB ID: 2FLU, https: / / www.rcsb.org / structure / 2FLU). Before docking with the antioxidant peptide, pymol was first used to remove water molecules and the original ligand (Nrf2) from the crystal, and then Autodock was used to add hydrogen atoms and charges. The ligand was drawn as a secondary structure using Chem Draw18.0, and then the 3D structure was drawn and the minimum energy structure was calculated using Chem3D 18.0. Autodock was used for docking, and the Lamarckian genetic algorithm was used to find the best binding state of the ligand to the receptor. The number of genetic algorithms was set to 50, and the binding energy was used to evaluate the binding ability of the ligand to the receptor. The lower the energy, the more stable the complex. The 2D and 3D structures of the interaction between the peptide and the target protein were obtained using the BIOVIA Discovery Studio 2021Client (Dassault Syst`emes, Paris, France) visualization software.

[0070] (8) Molecular dynamics simulation

[0071] Gromacs2020 software was used to perform a 200ns molecular dynamics simulation of the protein-ligand (Keap1-GLWGKV and Keap1-LIVYPW) complex obtained by molecular docking. Charmm 36 was selected as the protein force field, Gaff2 was selected as the ligand force field, and the TIP3P water model was selected to add solvent to the protein-ligand system, establish a water box with a periodic boundary of 1.0nm, and add sodium ions and chloride ions to balance the charge in the system to maximize the restoration of the real experimental environment.

[0072] First, the simulation system was prepared by constraining the heavy atoms of the protein (and small molecules) and performing 10,000 steps of energy minimization (including 5,000 steps of steepest descent and 5,000 steps of conjugate gradient method) for water molecules. Then, the constraints were released and the whole system was minimized for 10,000 steps. After optimizing the energy, we gradually heated the system to 300 K, and the system reached the target temperature after 1000 ps. Subsequently, 1 ns of equilibrium operation was performed in the NVT and NPT systems to ensure the stability of the system. Finally, a molecular dynamics simulation was performed for 200 ns. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg) and hydrogen bond changes of the complex were analyzed, and the g_MMGBSA method in the Gromacs 2020 program was used to calculate the binding free energy of the ligand to the protein.

[0073] (9) Stability of peptides to simulated gastrointestinal digestion

[0074] Briefly, the active peptides were dissolved in distilled water, adjusted to pH 2.0 using 1 M HCl, and the mixture was added with 4% pepsin (w / w) and incubated at 37 °C for 2 h in a continuously shaking water bath. 3 The pH of the reaction mixture was raised to 5.3, and then further raised to 7.5 with 1.0 M NaOH. 4% pancreatin (w / w) was added, and the mixture was continuously shaken and incubated at 37°C (duodenal conditions) for 2 h. Finally, the pH of the reaction mixture was adjusted to 7.0 with 1 M HCl or NaOH. The mixture was placed in a 100°C water bath for 10 min to terminate the digestion. The samples after gastrointestinal digestion were centrifuged at 5000 g and 4°C for 10 min, and the supernatant was freeze-dried and the antioxidant activity was measured.

[0075] (10) Caco-2 cell culture

[0076] Caco-2 cells were cultured in a 37°C, 5% CO2 saturated humidity incubator using DMEM medium (containing 20% ​​fetal bovine serum, 1% penicillin, 1% streptomycin). The culture medium was replaced every 1-2 days. When the cell density reached 80%-90%, the cells were subcultured approximately every 3-4 days. The cells were treated with different concentrations of polypeptides (0-10mM, dissolved in DMEM) for 24 hours, the culture medium was discarded, the cells were washed 3 times with PBS, CCK-8 solution was added, and the absorbance was measured at 450nm after incubation for 1 hour to determine the appropriate concentration of the polypeptide.

[0077] (11)H 2 O 2 Experiment on inducing oxidative damage model of Caco-2 cells

[0078] Each well of a 96-well plate was inoculated with 1 × 10 4 Caco-2 cells. After 24 h of incubation, different concentrations of H 2 O 2 The cells were treated with 100-1500 μM flavonoids for 4 h, the culture medium was discarded, the cells were washed three times with PBS, CCK-8 solution was added, and the absorbance was measured at 450 nm after incubation for 1 h to determine the H 2 O 2 The half inhibitory concentration (IC 50 ) and used in subsequent experiments.

[0079] (12) Peptide to H 2 O 2 Induced protection against Caco-2 cell injury

[0080] Caco-2 cells were plated at 1 × 10 4 The cells were plated in a 96-well plate at a density of 100 cells / mL and cultured for 24 h to allow them to adhere to the plate. Then, the cells were pretreated with GSH, LT, and GT (200 μmol / L, dissolved in complete medium) and added to each well. After 24 h of incubation, the cells were incubated with 800 μmol / L H 2 O 2 The culture medium was replaced with fresh culture medium and incubated for another 4 h. Afterwards, cell viability was assessed using the CCK-8 assay. 2 O 2 deal with.

[0081] (13) Peptide to H 2 O 2 Effect of inducing antioxidant enzyme activities and MDA content in Caco-2 cells

[0082] Caco-2 cells were plated at 1 × 10 6 The cells were seeded in a six-well plate at a density of 10 cells / mL and cultured for 24 h to allow them to adhere to the plate. The cells were pretreated with peptides at different concentrations (0, 50, 100, and 200 μmol / L, dissolved in complete culture medium) for 24 h. 2 O 2 The culture medium was replaced and the mixture was incubated for another 4 h to induce oxidative damage. 2 O 2 The untreated cells were considered as the blank group, and H 2 O 2 The cells treated with GSH were the model group, those treated with GSH were the positive control group, and those treated with GT or LT were the sample group. To obtain total cellular protein, the cells were trypsinized, lysed with RIPA buffer, and centrifuged. The protein concentration was determined using a BCA protein concentration assay kit, and the samples were stored at -20°C until analysis. The activities of CAT, SOD, and GSH-Px and the content of MDA were measured for each sample according to the instructions of the kit.

[0083] (14) Determination of protein expression levels in Caco-2 cells

[0084] Western blot was used to analyze the protein expression of Nrf2, HO-1 and NQO1. Caco-2 cells were plated at 1×10 6 The cells were seeded in a six-well plate at a density of 10 cells / mL and cultured for 24 h to allow them to adhere to the plate. 2 O 2After treatment. RIPA lysis buffer (add each protease inhibitor within a few minutes before use) was added and lysed on ice for 30 minutes to ensure complete cell lysis. Then, the lysed cell suspension was centrifuged (12000rpm, 4℃) for 10 minutes, and the supernatant was collected and its total protein content was determined. The supernatant was added to 5× reduced protein loading buffer at a ratio of 4:1 and denatured in a boiling water bath for 15 minutes. The protein solution of each group was separated by loading SDS-PAGE (5%) electrophoresis. The protein was transferred to a PVDF membrane (ice water was used to cool the membrane during transfer) and blocked with 5% skim milk for 30 minutes at room temperature. The membrane was incubated with diluted primary antibodies Nrf2 (1:1000), HO-1 (1:1000), NQO1 (1:1000) and ACTIN (1:2000) at 4℃ overnight. After washing 5 times with TBST, the membrane was incubated with HRP-conjugated secondary antibody HRP-goat anti-mouse (1:5000) at room temperature for 30 min and washed 3 times in PBST. Subsequently, the protein was detected using an enhanced chemiluminescence (ECL) kit, and the chemiluminescence was saved and the images were saved. Data analysis was performed using AIWBwellTM analysis software.

[0085] 2. Results and Analysis

[0086] (1) Antioxidant activity of each component separated by ultrafiltration

[0087] The hydrolyzate was separated by 10kDa and 3kDa ultrafiltration membranes to obtain three components (M 1 <3kDa, 3kDa <M 2 <10 kDa and M 3 >10 kDa). 1 The DPPH free radical scavenging activity of the component was 37.75%, and the metal ion chelating capacity was 70.31%, which was significantly higher than that of M. 2 20.18%, 56.46% and M 3 15.84%, 50.42% of the components, M 1 and M 2 There was no significant difference in the hydroxyl radical scavenging rate and reducing power of the components, which was significantly higher than that of M 3 The results are as follows Figure 2 shown.

[0088] (2) Gel Exclusion Chromatography Separation of Antioxidant Activity of Each Component

[0089] In order to purify the antioxidant peptides, the enriched M1 fraction was further separated and purified by Sephadex-G 25 column and divided into 4 peaks. Among the 4 peaks, the DPPH free radical scavenging activity of the P1 fraction was 93.69%, the hydroxyl free radical scavenging activity was 54.51%, the metal chelating ability was 89.00%, and the reducing power was 0.489. The results are as follows Figure 3 As shown, it had higher antioxidant activity than other components and was therefore selected for further characterization.

[0090] (3) Identification of P1 component peptide sequence

[0091] like Figure 4 As shown in the figure, the peptide composition of P1 was identified by nano-LC-MS / MS, and the mass spectrometry and database search results showed that 149 peptides were identified from P1. The length of the peptides ranged from 6 to 25 amino acids, the molecular weight ranged from 559.34 to 2812.47.63Da, and more than 50% of the peptides were less than 10. These peptides were derived from 6 different proteins, of which 47 were from hemoglobin subunit α-A (P01994), 47 were from hemoglobin subunit α-D (P02001), 25 were from hemoglobin subunit β (P02112), 23 were from hemoglobin subunit ε (P02128), 3 were from hemoglobin subunit pi (P02007), and 4 were from hemoglobin subunit rho (P02127).

[0092] (4) Analysis of computer virtual screening results of bioactive peptides

[0093] The 149 identified peptide fragments were first used to predict the toxicity of the peptides, and none of them were toxic. The PeptideRanker program was used to screen potential bioactive substances based on their structural patterns (scores > 0.5), and 26 of the 149 peptides were identified with scores greater than 0.5, and 4 peptides (RFFASF, ASFGNL, ALARLLIVYPWTQRFF, LIVYPW) had scores greater than 0.7. The CPP-pred software predicted the potential bioaccessibility, and only three peptides with scores greater than 0.5 were KLRVDPVNFKLL, GLWGKV, and AAWQKLVRVVAHALARKYH. It is worth noting that the potential biological activity and bioaccessibility scores of the peptides KLRVDPVNFKLL and GLWGKV were both greater than 0.5, which may have potential antioxidant activity and can play an antioxidant role in cells. Using BIOPEP-UWM TMThe database performed functional prediction and molecular docking on peptides with scores greater than 0.5, such as RFFASF, ASFGNL, ALARLLIVYPWTQRFF, AAWQKLVRVVAHALARKYH, KLRVDPVNFKLL, LIVYPW, and GLWGKV, and screened out two peptides with antioxidant activity, LIVYPW and GLWGKV. According to the results of peptidomics identification, the peptide GLWGKV is derived from the hemoglobin β subunit, and the peptide LIVYPW exists in the hemoglobin subunit β, hemoglobin subunit ε, and hemoglobin subunit rho.

[0094] (5) Molecular docking-based study of the interaction between peptides and Keap1

[0095] Two antioxidant peptides with potential biological activity were screened out by computer simulation prediction. In order to further evaluate the interaction between the most active peptides GLWGKV (GT) and LIVYPW (LT) and Keap1-Nrf2, molecular docking analysis was performed on Keap1. Both GT and LT interact with key amino acid residues in the central cavity of the Kelch domain, showing a binding mode similar to that of the native ligand (e.g. Figure 5 ). Specifically, GT forms hydrogen bonds with Arg380, Arg415, Arg483, Asn414, Gln530, Ser555, Tyr334, Try572, Tyr525, Gly364 and Ala556 of the receptor protein Keap1, and can interact with some residues to form van der Waals forces, etc. The docking binding energy is -11.48 kcal / mol. GT occupies 6 binding sites (Arg380, Arg415, Arg483, Gln530, Ser555, Tyr334) where amino acid residues in the Keap1-Kelch region form hydrogen bonds with Nrf2. LT forms hydrogen bonds with Arg415, Arg483, Ala510, Ala556, Ala366, Gly603, Gly364, Tyr525 and Phe478 of the receptor protein Keap1, and interacts with some residues to form van der Waals forces, etc. The docking binding energy is -10.85 kcal / mol. LT occupies two binding sites (Arg415 and Arg483) where amino acid residues in the Keap1-Kelch region form hydrogen bonds with Nrf2. The lower the docking energy, the stronger the affinity between the peptide and the protein. Therefore, the affinity of the two peptides is good, and GT is relatively good. Based on this, it can be speculated that the antioxidant properties of the two antioxidant peptides may be due to the destruction of the Keap1-Nrf2 interaction, occupying the binding site of Nrf2 and Keap1, thereby activating the Nrf2 signaling pathway.

[0096] (6) Study on the interaction between peptide and Keap1 based on molecular dynamics simulation

[0097] In order to further study the interaction between small molecules and proteins, molecular dynamics simulations of the complex of protein and small molecules were performed for 200 ns. Figure 6 As shown. The average RMSD of the complexes is less than 0.15nm, and the complexes basically reach dynamic equilibrium around 100ns, which indicates that the small molecule matches the target protein well and can form a stable complex. The complexes did not leave the protein pocket and were able to bind firmly to the protein to reach dynamic equilibrium. The fluctuation of the RMSF curve of the complex is within 0.3nm in the entire range, indicating that the addition of GT and LT has little effect on the overall structural stability of the Keap1 protein. The root mean square fluctuation of Keap1-GT is slightly smaller than that of Keap-LT, and the binding is slightly better. The gyration radius of the two complexes is reduced and reaches equilibrium. The number of hydrogen bonds between GT and Keap1 protein is significantly more than that between LT and Keap1, and the binding is more stable. As shown in Table 1, the binding free energies of polypeptides GT and LT with Keap1 protein are -86.37kJ / mol and -28.403kJ / mol, respectively, indicating that GT binds more stably to Keap1 than LT.

[0098] Table 1 Binding energy of MM GBSA (kJ / mol)

[0099]

[0100]

[0101] Note: ΔE VDW :Van der Waals energy Van der Waals interaction energy;

[0102] ΔE ELE :Eletrostatic energyElectrostatic interaction energy;

[0103] ΔE GB :Polar solvation energyPolar solvation energy;

[0104] ΔE SA :Non-polar contribution to solvation Non-polar solvation energy;

[0105] ΔG bind :Binding free energyBinding free energy.

[0106] (7) Stability results of peptides in simulated gastrointestinal digestion

[0107] like Figure 7 After digestion, the hydroxyl radical scavenging rates of peptides LT and GT were 41.89% and 46.37%, respectively, and the metal ion chelating abilities were 84.61% and 85.04%, respectively, which were significantly higher than those of undigested peptides. The antioxidant activity of peptide GT was enhanced overall during simulated gastrointestinal digestion, which was higher than that of peptide LT.

[0108] (8) Results of the antioxidant regulation mechanism of peptides on Caco-2 cells

[0109] like Figure 8 , After Caco-2 cells were incubated with GT for 24 h, the cell viability of Caco-2 cells remained above 90%.

[0110] like Fig. 9 As shown, Caco-2 cells were exposed to H 2 O 2 After 4 h of treatment, cell viability decreased significantly, showing a concentration-dependent pattern.

[0111] H 2 O 2 At a concentration of 800 μmol / L, cell viability decreased to 51.47±1.43%. The viability of Caco-2 cells pretreated with 200 μmol / L GT increased significantly by 35.91%, recovering to 87.22% of the untreated control group; similarly, LT also showed a similar cell protection effect.

[0112] like Fig.10 As shown in the results, the enzyme activities of GSH-Px, CAT and SOD in the model group were significantly inhibited, and decreased to 21.68%, 57.44% and 53.00% of the normal cell level, respectively. After GT pretreatment, the enzyme activities of GSH-Px, CAT and SOD increased by 150.21%, 110.70% and 99.08% respectively compared with the model group; 200μmol / L GT treatment restored the activities of CAT and SOD to 104.84% and 105.58% of the normal cell level. It is worth noting that at the same concentration, GT pretreatment increased the GSH-Px enzyme activity of cells compared with the positive control group (GSH). GT pretreatment can significantly reduce the generation of intracellular MDA in a concentration-dependent manner.

[0113] like Fig.11As shown, the protein expression of Nrf2, HO-1 and NQO1 in the model group was significantly reduced, indicating that H2O2 caused oxidative damage. In contrast, the protein expression of Nrf2, HO-1 and NQO1 in cells pretreated with GT was significantly enhanced and showed a dose-dependent manner. GT promoted the expression of Nrf2 in the nucleus and activated the Nrf2 signaling pathway, thereby upregulating the expression of HO-1 and NQO1 proteins downstream of the Nrf2 pathway. The potential antioxidant pathway of GT in preventing cellular oxidative stress damage is revealed to be Keap1-Nrf2-ARE.

[0114] In summary, the present invention prepares hemoglobin peptides by probiotics synergistic enzymolysis technology, which can significantly improve the antioxidant activity and stability of the product; the polypeptide GLWGKV with antioxidant activity is discovered and its antioxidant mechanism is clarified. The M1 component is screened out by ultrafiltration and purified by gel chromatography column to obtain 4 peaks, and it is clear that the P1 component has the best antioxidant activity. 149 polypeptides from 6 proteins were identified by nano-LC-MS / MS, and the proportion of polypeptides with a peptide chain length of less than 10 accounted for 55.7%. With the help of computer simulation, two polypeptides GLWGKV and LIVYPW with antioxidant activity were predicted to be molecularly docked with Keap1 protein, and the main binding sites of GLWGKV, LIVYPW and Keap1 were clarified. It was found that GLWGKV occupied 6 binding sites for Keap1 to form hydrogen bonds with Nrf2, and LIVYPW occupied 2 binding sites. The results of molecular dynamics simulation showed that the Keap1-GLWGKV and Keap1-LIVYPW complexes both reached dynamic equilibrium in about 100 ns, and neither of them left the protein pocket and was able to bind firmly to the protein. In addition, the number of hydrogen bonds between GLWGKV and Keap1 protein was significantly greater than that between LIVYPW and Keap1, and the binding free energy of GLWGKV and Keap1 protein was -86.37 kJ / mol, which was significantly lower than the binding energy of LIVYPW and Keap1 protein -28.403 kJ / mol, indicating that the Keap1-GLWGKV complex was more stable than Keap1-LIVYPW.

[0115] The present invention found that the polypeptide GLWGKV is stable in the process of simulating gastrointestinal digestion, and the antioxidant activity is enhanced overall. A H2O2-induced Caco-2 cell oxidative damage model was constructed to explore the prevention mechanism of GLWGKV on cell oxidative damage at the cellular level. It was found that GLWGKV increased the activity of SOD, CAT and GSH-Px in H2O2-induced oxidative damage Caco-2 cells, reduced the MDA content, and enhanced the expression of proteins such as Nrf2, HO-1 and NQO1. It was revealed that the potential antioxidant pathway of GLWGKV in preventing cell oxidative stress damage is: Keap1-Nrf2-ARE.

[0116] The present invention uses computer simulation technology combined with cell experiment verification to avoid the complex process and expensive cost of traditional methods, accurately, quickly, efficiently and greenly prepare antioxidant peptides, and explore the antioxidant activity mechanism and structure-activity relationship of polypeptides in hemoglobin at the molecular and cellular levels. The method can also be extended to the rapid screening, prediction and verification of functional activities other than antioxidant.

[0117] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An animal blood-derived antioxidant peptide, characterized in that: Its amino acid sequence is GLWGKV, as shown in SEQ ID NO: 1 is shown.

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