Dietary protein-derived antioxidant peptide HYHE and application thereof

Abstract

The invention relates to the technical field of antioxidant peptides, in particular to an antioxidant peptide HYHE derived from dietary protein and application of the antioxidant peptide HYHE. The amino acid sequence of the antioxidant peptide is HYHE and is as shown in SEQ ID NO: 1. The antioxidant peptide is obtained based on prediction of an antioxidant peptide prediction platform Pred5AOP; 27 dietary proteins are virtually hydrolyzed through trypsin and pepsin, and the antioxidant peptide HYHE is screened out; compared with chemical drugs, the antioxidant peptide HYHE serving as the biological peptide has the advantages of high stability, low immunogenicity, high purity and the like; meanwhile, the invention further proves that the dietary protein has rich nutritional value, provides a new thought for resource recycling, and has good social and economic benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of antioxidant peptides, in particular to an antioxidant peptide HYHE derived from dietary protein and application thereof. Background Art

[0002] Excessive free radicals and reactive oxygen species in cells can lead to oxidative stress, which has a negative impact on human health and is associated with a variety of diseases, including chronic kidney disease, hypertension and cancer. The Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2) / antioxidant response element (ARE) signaling pathway is a key pathway for cellular oxidative stress and has become a hot topic in antioxidant research in recent years. Keap1 mainly maintains intracellular redox balance by regulating antioxidant stress responses.

[0003] In recent years, the study of antioxidant peptides obtained from dietary proteins has become a hot topic in the field of functional peptide research. Bioactive peptides are usually composed of 2-20 amino acids, and these peptides are inactive in the parent protein. After the protein is absorbed and digested in the gastrointestinal tract, the bioactive peptides are released from the parent protein and exhibit biological functions. Compared with synthetic antioxidants, antioxidant peptides extracted from dietary proteins have the advantages of being easy to absorb and non-toxic. Hydrolyzates and peptides extracted from various low-priced and low-utilization dietary proteins can be used to produce antioxidant peptides.

[0004] The present invention adopts a strategy combining machine learning with experimental exploration to screen antioxidant peptides in order to speed up the screening process and reduce costs; screens potential antioxidant peptides in 27 dietary proteins, and evaluates the antioxidant activity and protective effect of the peptides on cells in vitro; therefore, the method combining theoretical calculation with experimental evaluation can be used as an effective method for screening antioxidant peptides. Summary of the invention

[0005] The purpose of the present invention is to provide an antioxidant peptide HYHE derived from dietary protein and its application to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions: An antioxidant peptide derived from dietary protein, whose amino acid sequence SEQ ID NO:1 is described as: HYHE, hereinafter referred to as antioxidant peptide HYHE.

[0007] Application of the antioxidant peptide HYHE in the preparation of health products, food additives and cosmetics.

[0008] Compared with the prior art, the beneficial effects of this patent are: Multilayer Perceptron (MLP) is a feedforward neural network consisting of an input layer, a hidden layer, and an output layer. The neurons in each layer are connected to the neurons in the adjacent layer through weights. Its core advantage is that it can learn complex patterns through nonlinear activation functions. The present invention uses the MLP algorithm to develop an antioxidant peptide prediction platform Pred5AOP; through the virtual hydrolysis of 27 dietary proteins by trypsin and pepsin, the antioxidant peptide HYHE was screened out; compared with chemical drugs, polypeptide drugs have the advantages of strong stability, low immunogenicity, and high purity; at the same time, the present invention further proves the rich nutritional value of dietary protein, and provides a new idea for the recycling of resources. Antioxidant peptides have good application prospects in the fields of health products, food additives and cosmetics, and have good social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 It is a data set diagram of the present invention; Figure 2 The scavenging activity of the antioxidant peptide HYHE at different concentrations on DPPH free radicals in the present invention (* p<0.05, **p<0.01, *** p<0.001, **** p<0.0001); Figure 3 is the graph of ABTS free radical scavenging activity of the antioxidant peptide HYHE at different concentrations in the present invention (* p<0.05, **p<0.01, *** p<0.001, **** p<0.0001); Figure 4 The relative cell viability diagram of HepG2 cells after the antioxidant peptide HYHE of different concentrations in the present invention acts on the cells (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001): Figure 5 is a graph showing the relative cell viability of HepG2 cells after the action of different concentrations of D-galactose in the present invention (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001); Figure 6 The relative cell viability diagram of HepG2 cells damaged by oxidation after being treated with different concentrations of the antioxidant peptide HYHE in the present invention (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001); Figure 7is a graph showing ROS content after different concentrations of the antioxidant peptide HYHE in the present invention acted on oxidatively damaged HepG2 cells (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001); Figure 8 It is a fluorescence image of the antioxidant peptide HYHE of different concentrations in the present invention after acting on oxidatively damaged HepG2; Fig. 9 The results of quantum chemical analysis and molecular docking of the antioxidant peptide HYHE at different concentrations in the present invention. DETAILED DESCRIPTION

[0010] 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.

[0011] See also Figures 1 to 9 , the present invention provides a technical solution: Example 1: Rational design of antioxidant peptide HYHE

[0012] The present invention systematically screens out peptides with antioxidant potential by constructing a dietary protein source database containing 76,343 peptides. The data comes from 27 dietary proteins (such as soybeans, dairy products and fish), and uses pepsin and trypsin to simulate enzymatic hydrolysis to generate short peptides, and fully records the molecular weight, lipophilicity, solubility and other physical and chemical properties of each peptide. The database further integrates the functional data of public databases such as UniProt, takes peptides with antioxidant properties as positive samples, and introduces other functional peptides such as antibacterial and anticancer as negative samples, constructs a comprehensive training set and validation set, and provides a high-quality data foundation for subsequent screening. The specific data sets are as follows: Figure 1 shown.

[0013] During the screening process, the present invention adopts the Pred5AOP model based on deep learning to predict the antioxidant potential of peptides in the database; the model uses the multi-layer perceptron (MLP) algorithm to extract information from the characteristics of the amino acid composition, amino acid arrangement order and three-dimensional spatial structure of the peptides, and uses Morgan fingerprints to generate feature vectors, and the parameters of the feature vectors are set to radius 2 and length 2048 bits; the training data set includes 1020 known antioxidant peptides, and through five-fold cross-validation, the prediction accuracy of the model reached 92%; by batch prediction of a large database containing 76,343 peptides, the model calculated the antioxidant score of each peptide, and finally selected the top 5% peptides with the highest antioxidant scores (a total of 3817 peptides), which were used as candidate peptides to enter the subsequent verification and experimental stages.

[0014] Through Pred5AOP, 201 short peptides with scores greater than 0.999 were screened out from the top 5% peptides with the highest antioxidant scores, and cluster analysis was performed using the distance method, from which six types of representative antioxidant peptide molecules were obtained; among them, the antioxidant peptide HYHE is represented by a glutamic acid residue at the end.

[0015] In order to further evaluate the potential of the screened antioxidant peptides, the present invention also carried out a molecular property evaluation; the screened antioxidant peptides were subjected to ADMET (absorption, distribution, metabolism, excretion and toxicity) predictive analysis using the SwissAdme tool, focusing on the evaluation of water solubility, lipophilicity, gastrointestinal absorption, blood-brain barrier (BBB) ​​permeability and bioavailability. The evaluation results showed that the screened peptides generally had good water solubility and hydrophilicity, which is crucial for their effective absorption in the body; in addition, the metabolic pathways of these peptides were predicted using the ADMETLAB 3.0 platform, and the results showed that they were not involved in other metabolic pathways and did not show obvious side effects.

[0016] Example 2: Determination of free radical scavenging activity of antioxidant peptide HYHE

[0017] The antioxidant peptide HYHE standard product involved in the present invention is synthesized and provided by Liaoning Newkey Biochemical Technology Co., Ltd. Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0018] DPPH free radicals are dark purple. When DPPH free radicals react with antioxidants, the free radicals are scavenged, the color of the system becomes lighter, and the absorbance decreases. DPPH is dissolved in anhydrous ethanol to prepare 0.1mM DPPH free radicals. The present invention prepares five different concentrations (0.1, 0.3, 0.5, 1.0 and 2.0 mg / mL) of antioxidant peptide HYHE (the antioxidant peptide HYHE is dissolved in deionized water). The experimental group (As) is 100μL antioxidant peptide and 100μL DPPH free radical; the control group (Ac) is 100μL distilled water and 100μL DPPH free radical; the blank group (A) is 100μL antioxidant peptide and 100μL anhydrous ethanol (0.1mM); after reacting in the dark at room temperature (25°C) for 30 minutes, the absorbance is measured at a wavelength of 517 nm; the calculation formula for DPPH free radical scavenging rate (%) is as follows: DPPH free radical scavenging rate (%) = [(As - A) / (Ac - A)] × 100; DPPH free radical scavenging activity of antioxidant peptide HYHE Figure 2 As shown in the figure, with the increase of the concentration of antioxidant peptide HYHE, the DPPH free radical scavenging activity gradually increased; when the concentration of HYHE reached 1.0 and 2.0 mg / mL, the DPPH free radical scavenging rate reached 49.26% and 50.27%.

[0019] 15 mg of ABTS was dissolved in 4 mL of deionized water, and 10 mg of potassium persulfate was dissolved in 35 mL of deionized water. 500 μL of each of the ABTS solution and the potassium persulfate solution were mixed and placed in the dark for 12 hours. The mixture was diluted 30 times with phosphate buffered saline (PBS) to prepare ABTS free radicals. ABTS free radicals are green, and the color becomes lighter under the action of antioxidants. Five concentrations (0.1, 0.3, 0.5, 1.0 and 2.0 mg / mL) of antioxidant peptide HYHE were prepared (the antioxidant peptide HYHE was dissolved in deionized water). The experimental group (As) was 50 μL of antioxidant peptide and 150 μL of ABTS free radicals. The control group was 50 μL of distilled water and 150 μL of ABTS free radicals. The mixture was incubated in the dark at room temperature (25°C) for 10 minutes, and the absorbance at a wavelength of 405 nm was measured. The calculation formula for the scavenging rate (%) of ABTS free radicals is as follows: ABTS free radical scavenging rate (%) = [(A - As) / A] × 100; The ABTS free radical scavenging activity of the antioxidant peptide HYHE is shown in Figure 3, with the increase of HYHE concentration, the ABTS free radical scavenging activity gradually increased; when the concentration of HYHE reached 1.0 and 2.0 mg / mL, the DPPH free radical scavenging rate reached 85.18% and 86.54%.

[0020] Example 3: Effect of the antioxidant peptide HYHE on cytotoxicity

[0021] The present invention uses HepG2 cells (human liver cancer cells) to explore the antioxidant effect of the antioxidant peptide HYHE at the cellular level; cell viability is an important indicator for evaluating the cytotoxicity of exogenous additives; the present invention cultured HepG2 cells in DMEM medium (10% fetal bovine serum, 1% penicillin-streptomycin (10000 units / ml penicillin, 10 mg / mL streptomycin)), the DMEM medium was selected from Gibco, the following DMEM medium specifications are the same, and the cells are placed in a constant temperature CO 2 Incubator (37°C, 5% CO 2 ) in 96-well plates at 5 × 10 3 HepG2 cells in the logarithmic growth phase were cultured at a density of 24 h; after the cells adhered, the culture medium was discarded, and different concentrations (0.1, 0.3, 0.5, 1.0 and 2.0 mg / mL) of antioxidant peptide HYHE (the antioxidant peptide HYHE was dissolved in DMEM culture medium according to the concentration) were added to the HepG2 cells and cultured for another 24 h; the cell viability of each group was determined by the CCK-8 method, and the absorbance at a wavelength of 450 nm was determined by an enzyme marker; the calculation formula for cell viability (%) is as follows: Relative cell viability (%) = [(As - A) / (Ac - A)] × 100 Among them, As, Ac, and A are the absorbances of the sample group, control group (without addition of antioxidant peptide HYHE), and blank group (without addition of antioxidant peptide HYHE and HepG2 cells), respectively; The cytotoxic effects of five concentrations (0.1, 0.3, 0.5, 1.0 and 2.0 mg / mL) of the antioxidant peptide HYHE on HepG2 cells, such as Figure 4 As shown; when the concentrations of the antioxidant peptide HYHE were 0.1, 0.3, 0.5, 1.0 and 2.0 mg / mL, there was no significant effect on HepG2 cells after 24 hours of treatment; the relative viability values ​​of HepG2 cells were 108.61%, 100.74%, 94.14%, 102.63% and 97.60%, respectively; the results showed that the antioxidant peptide HYHE had no toxic effect on HepG2 cells.

[0022] Example 4: Protective effect of antioxidant peptide HYHE on oxidatively damaged cells

[0023] D-galactose is metabolized in the body by galactose oxidase; when D-galactose accumulates in large quantities in cells, it reduces the level of antioxidant enzymes, damages the body's antioxidant defense system, and induces cellular oxidative stress; the present invention uses D-galactose to act on HepG2 cells at different concentrations and different incubation times to construct a HepG2 cell oxidative damage model.

[0024] HepG2 cells growing in logarithmic growth were plated at 5 × 10 3 The density was in a 96-well plate and divided into two groups: a sample group with different concentrations of D-galactose (100, 200, 300, 400 and 500 mM) added (D-galactose was dissolved in the culture medium according to the concentration) and an untreated control group; the absorbance was measured at a wavelength of 450 nm using the CCK-8 method to determine the optimal induction concentration and time.

[0025] The relationship between HepG2 cell viability and D-galactose induction concentration and induction time, such as Figure 5 As shown; with the increase of D-galactose concentration and incubation time, cell viability gradually decreased; the cell survival rate used to construct the injury model should be around 50%; the results showed that when the concentration of D-galactose was 300 mmol / L and the induction time was 24 hours, the viability of HepG2 cells reached 52.12%, which was within the appropriate range for establishing a cell oxidative stress model.

[0026] In order to explore the protective effect of antioxidant peptides on oxidatively damaged cells, logarithmically growing HepG2 cells were cultured at 5×10 3 The cells were cultured in 96-well plates at a density of 1.50 μg / mL for 24 h. Before D-galactose induction, different concentrations (0.1, 0.3, 0.5, 1.0 and 2.0 mg / mL) of antioxidant peptide HYHE were added to the protected group and cultured for 24 h. The relative cell viability (%) was calculated as follows: Relative cell viability (%) = [(As - A) / (Ac - A) ]×100; Among them, As, Ac and A are the absorbance of the sample group, control group (without addition of D-galactose and antioxidant peptide HYHE) and blank group (without addition of D-galactose, antioxidant peptide HYHE and HepG2 cells), respectively.

[0027] The protective effect of antioxidant peptide HYHE on D-galactose-induced cell oxidative damage is shown in Figure 6; The cell survival rate after D-galactose induction dropped to 52.08%, which is within the survival rate range of the damage model and can be regarded as a successful modeling; after 24 hours of pre-protection culture with the antioxidant peptide HYHE, the relative cell viability of different concentrations (0.1, 0.3, 0.5, 1.0 and 2.0 mg / mL) of the antioxidant peptide HYHE was 43.67%, 55.63%, 67.19%, 86.68% and 90.94%, respectively; the relative cell viability was positively correlated with the concentration of the antioxidant peptide HYHE; the experimental results showed that the pre-protection treatment with the antioxidant peptide HYHE increased the relative viability of HepG2 cells subjected to oxidative stress.

[0028] Example 5: Antioxidant peptide HYHE reduces ROS content in HepG2 cells

[0029] Reactive oxygen species (ROS) are involved in a variety of physiological processes in the human body; however, excessive ROS may produce toxic effects, oxidize biomolecules, and even promote the development of various diseases.

[0030] In this study, the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to detect the intracellular ROS level. The intensity of green fluorescence was proportional to the ROS level. Five different concentrations (0.1, 0.3, 0.5, 1.0, and 2.0 mg / mL) of the antioxidant peptide HYHE were prepared using DMEM medium. HepG2 cells in the logarithmic growth phase were taken and cultured at 2×10 4 The cells were seeded at a density of 100 μg / mL in 6-well plates and cultured for 24 hours. After the culture medium was discarded, the cells were pretreated with different concentrations of antioxidant peptides for 24 hours. Subsequently, the cells were cultured with DMEM medium containing 300 mM D-galactose (containing 1% penicillin-streptomycin) for 24 hours. After the culture medium was discarded, 1 mL of ROS probe solution was added to each well and incubated for 30 minutes. Subsequently, the cells were washed three times with PBS, and the ROS level was observed using a green fluorescence microscope. The intracellular ROS level was quantitatively analyzed using Image J software. The content of ROS in HepG2 cells damaged by oxidative damage was shown in the results of antioxidant peptides Figure 7 and Figure 8 ; Compared with the damaged group (D-galactose induced group), the ROS level in the oxidatively damaged cells treated with antioxidant peptides decreased, and with the increase of the concentration of antioxidant peptide HYHE, the intracellular ROS fluorescence intensity gradually weakened.

[0031] Example 6: Taste characteristics analysis of antioxidant peptide HYHE

[0032] In order to evaluate the taste characteristics of antioxidant peptides, the antioxidant peptide HYHE sample was dissolved in deionized water and prepared at a concentration of 0.1 mg / mL. Each test included a 30-second initial taste measurement phase, followed by a 3-second wash with a reference solution and a 30-second aftertaste evaluation phase. The electronic tongue sensor was tested four times in total, and the first round of data was discarded due to calibration adjustments. The final result was the average of the remaining three rounds to determine the final taste evaluation result. The taste characteristics of the antioxidant peptide HYHE are shown in Table 1 below: Table 1: Taste characteristics of antioxidant peptide HYHE

[0033] The antioxidant peptide HYHE showed significant bitterness and astringency, and had a certain sourness and saltiness, but lacked umami and richness; compared with the tasteless group, the antioxidant peptide HYHE had a greater difference in taste characteristics, mainly showing a bitter taste characteristic.

[0034] Example 7: Molecular mechanism of antioxidant peptide HYHE

[0035] Kelch-like ECH-associated protein 1 (Keap1) is a key protein in the anti-oxidative stress signaling pathway Keap1-Nrf2-ARE; under conditions of excessive oxidative stress, Keap1 will be downregulated, thereby reducing the ubiquitination degradation of Nrf2 and increasing the expression of a series of antioxidant genes.

[0036] The crystal structure and sequence information of Keap1 protein were obtained from the PDB database (PDB ID: 2FLU). Molecular docking of the antioxidant peptide HYHE and Keap1 protein was performed using AlphaFold3, and the docking results were analyzed and visualized using Pymol 2.6. Quantum chemical calculations of the antioxidant peptide HYHE were performed using Gaussian 09, and the LUMO and HUMO orbitals of the antioxidant peptide were visualized using Multiwfn software.

[0037] HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energies are key parameters; the energy difference between the HOMO and LUMO orbitals reflects the stability and reactivity of the antioxidant peptide; a smaller energy difference generally indicates a more reactive molecule.

[0038] The results of quantum chemical analysis and molecular docking of antioxidant peptides are shown in Fig. 9; The LUMO energy level is 0.21 eV. The lower LUMO energy level indicates that the molecule may be more likely to accept electrons and may have a stronger binding ability with the Keap1 protein; the HOMO energy level is 3.74 eV; the distribution of the HOMO energy level reflects the ability of the molecule to release electrons; the energy gap is 3.53 eV, indicating that the antioxidant peptide has a certain stability and also has a certain chemical reactivity, and can effectively interact with the active site of the Keap1 protein.

[0039] Antioxidant peptides interact with the R91, R159, Y10, Y248 and Q206 residues of the Keap1 protein. R159 works synergistically with R91 to stabilize the carboxyl groups of glutamic acid and aspartic acid in antioxidant peptides, but adjacent tyrosine residues (such as Y248) usually form conjugated interactions with the side chains of adjacent amino acids in antioxidant peptides. This interaction enables R91 and R159 to anchor acidic amino acids, while Y248 acts with adjacent non-acidic amino acids, thereby fixing the antioxidant peptide in a stable conformation.

[0040] 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 antioxidant peptide HYHE derived from dietary protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the antioxidant peptide HYHE as claimed in claim 1 in the preparation of health products, food additives and cosmetics.

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