An antioxidant peptide derived from sea cucumber, its preparation method and application

Abstract

The present invention belongs to the field of food biotechnology, and particularly relates to a sea cucumber-derived antioxidant peptide, a preparation method thereof, and an application thereof. The sea cucumber-derived antioxidant peptide of the present invention is prepared and screened from Apostichopus japonicus, and includes at least one of the peptides shown in SEQ.ID.NO.1-4 in terms of amino acid sequence, and the docking energies with the protein receptor Keap1 are -8.6 kcal / mol, -9.4 kcal / mol, -9.1 kcal / mol, and -8.8 kcal / mol respectively. The obtained antioxidant peptide of the present invention can be applied to foods, cosmetics or pharmaceuticals, and is of great significance for the development of new antioxidant products.

Description

Technical Field

[0001] The present invention belongs to the field of food biotechnology, and particularly relates to a sea cucumber-derived antioxidant peptide, a preparation method thereof, and applications thereof. Background Art

[0002] As a natural antioxidant, antioxidant peptides are easy to absorb, have good stability, no immunoreactivity, and have both nutritional properties and functions such as lowering blood pressure and enhancing immunity. Due to their natural, safe, non-toxic, and high nutritional value characteristics, they have been widely used in the food field and have become one of the natural products with the highest usage rate in functional foods. Developing antioxidant peptides with nutritional and safety properties from low-cost biological resources has always been a research hotspot in the polypeptide field.

[0003] Bioinformatics tools are used to assist in screening bioactive peptides. Based on the known amino acid sequences in bioactive peptides, database search and software analysis are used to predict properties such as the possible biological activities, safety, and bioaccessibility of peptide segments. Target peptide segments are selected for chemical synthesis and their related properties are verified. Using bioinformatics tools can save a large amount of time and experimental costs, but there is currently no research on screening sea cucumber antioxidant peptides using bioinformatics tools.

[0004] There are many antioxidant peptide activity evaluation methods. Currently, the most widely used methods are the DPPH method (the first method in GB / T39100-2020) and the ABTS method (the second method in GB / T39100-2020). The Keap1 enzyme can effectively scavenge free radicals in the body that damage health and cause diseases, decompose them into oxygen molecules and water molecules harmless to the human body, and discharge them smoothly out of the body, playing an important role in protecting cells from the toxicity of oxygen free radicals. In recent years, homology modeling and molecular docking have been applied to reveal the binding sites between antioxidant peptides and their receptor Keap1, providing an effective way for the screening and discovery of antioxidant peptides. The Keap1-Nrf2 pathway plays a key defensive role in oxidative stress damage in the body. It can activate the expression of cellular antioxidant genes, induce the expression of genes encoding antioxidant, detoxification, and metabolic enzymes as well as transporters, and enhance the resistance of cells to oxidative and xenobiotic stresses. The Keap1 protein is an important component of the Keap1-Nrf2 antioxidant response signaling pathway. By inhibiting Keap1, the protein-protein interaction between Keap1 and Nrf2 can be affected, and then Nrf2 can be dissociated and the Nrf2 pathway can be activated, playing an antioxidant role in protecting cells. Therefore, exploring the interaction between sea cucumber-derived antioxidant peptides and the Keap1 receptor and finding their antioxidant mechanism can provide a basis for the application of sea cucumber-derived antioxidant peptides in the preparation of functional foods from natural food sources. Summary of the Invention

[0005] The present invention provides a sea cucumber-derived antioxidant peptide, a preparation method thereof, and an application thereof. The antioxidant peptide of the present invention is prepared and screened from Apostichopus japonicus and has significant antioxidant activity.

[0006] The specific technical solution is as follows:

[0007] One of the purposes of the present invention is to provide a sea cucumber-derived antioxidant peptide, which comprises at least one of the peptides shown in SEQ.ID.NO.1 to 4 in terms of amino acid sequence.

[0008] Among them, SEQ.ID.NO.1 is PGGR, and the docking energy of this sea cucumber-derived antioxidant peptide with the receptor protein Keap1 is -8.6 kcal / mol.

[0009] Among them, SEQ.ID.NO.2 is PGPAGR, and the docking energy of this sea cucumber-derived antioxidant peptide with the receptor protein Keap1 is -9.4 kcal / mol.

[0010] Among them, SEQ.ID.NO.3 is PGPTGPAGPR, and the docking energy of this sea cucumber-derived antioxidant peptide with the receptor protein Keap1 is -9.1 kcal / mol.

[0011] Among them, SEQ.ID.NO.4 is GGQQGPR, and the docking energy of this sea cucumber-derived antioxidant peptide with the receptor protein Keap1 is -8.8 kcal / mol.

[0012] Among them, the sea cucumber is Apostichopus japonicus ( Apostichopus japonicus ), and the above-mentioned sea cucumber-derived antioxidant peptide is prepared and screened from the body wall and gonad of Apostichopus japonicus.

[0013] Preferably, the sea cucumber-derived antioxidant peptide comprises the peptide PGPAGR shown in SEQ.ID.NO.2 in terms of amino acid sequence. The docking energy of this peptide segment is the lowest, it consists of 6 amino acids, and the peptide segment is relatively short, which is of great significance for the development of new antioxidant products.

[0014] Another purpose of the present invention is to provide a preparation method of the above-mentioned sea cucumber-derived antioxidant peptide, which comprises the following steps:

[0015] S1. Obtain sea cucumber peptides;

[0016] S2. Identify the sequences of the sea cucumber peptides;

[0017] S3. Perform molecular docking of the sea cucumber peptides with the receptor Keap1 protein to screen antioxidant peptides.

[0018] Furthermore, in step S1: the sea cucumber peptides are obtained by enzymatically hydrolyzing sea cucumber raw materials.

[0019] Specifically, in step S1: first, pepsin is used for enzymatic hydrolysis, and then trypsin and chymotrypsin are used for enzymatic hydrolysis. The present invention performs biomimetic enzymatic hydrolysis on sea cucumber raw materials.

[0020] More specifically, in step S1: the working conditions for enzymatic hydrolysis are preferably as follows: add pepsin to the raw material to be processed, adjust the pH to 2 - 4, perform enzymatic hydrolysis at 35 - 40 °C for 4 - 6 h, and then inactivate the enzyme; add trypsin and chymotrypsin, adjust the pH to 6 - 8, perform enzymatic hydrolysis at 35 - 40 °C for 2 - 4 h, and inactivate the enzyme.

[0021] Among them, the addition amount of pepsin is preferably 800 - 1200 U / g based on the sea cucumber raw material.

[0022] Among them, the addition amount of trypsin is preferably 1000 - 1400 U / g based on the sea cucumber raw material.

[0023] Among them, the addition amount of chymotrypsin is preferably 1200 - 1600 U / g based on the sea cucumber raw material.

[0024] Specifically, in step S1: before enzymatic hydrolysis of the sea cucumber raw material, it is preferably pretreated. The pretreatment includes: adding water to the sea cucumber raw material, then homogenizing, performing heat treatment for 10 - 15 min, and then homogenizing again for use.

[0025] Among them, the usage ratio of the sea cucumber raw material to water is preferably 1 kg : (3 - 8) L.

[0026] Among them, it is preferred to use a boiling water bath for heat treatment.

[0027] Among them, the water added to the sea cucumber raw material is preferably ultrapure water.

[0028] Furthermore, in step S1: after enzymatic hydrolysis, the enzymatic hydrolysate is separated and purified.

[0029] Among them, the separation and purification includes using ultrafiltration and nanofiltration to fractionate the enzymatic hydrolysate to obtain peptides within a certain molecular weight range. Nanofiltration can remove salts and free amino acids, and ultrafiltration can remove macromolecular substances.

[0030] Furthermore, in step S2: LC - MS / MS is used for polypeptide sequence analysis. Through database comparison and analysis, the sequences of all peptides are obtained. Specifically, the mass spectrometry database retrieval software is MaxQuant 2.0.1.0, and the sample use database is the uniprot protein database.

[0031] Specifically, in step S2: it is preferred to first desalt the product obtained in step S1 and then perform polypeptide sequence analysis. It is preferred to use a C18 StageTip chromatographic column for desalting.

[0032] Furthermore, in step S3: Before performing molecular docking of sea cucumber peptides with the receptor protein Keap1, it is preferred to predict their antioxidant activity through the AnOxPePred 1.0 tool and select the peptide sequences with higher scores.

[0033] Furthermore, in step S3: Before performing molecular docking of sea cucumber peptides with the receptor protein Keap1, it is preferred to predict the water solubility, potential allergenicity, toxicity, and physicochemical properties of the peptides, and sequences without potential allergenicity and toxicity are used for subsequent synthesis and verification. Specifically, the water solubility of the peptides can be predicted through the Peptide Property Calculator tool, and the potential toxicity and physicochemical properties of the peptides can be predicted through the Toxin Pred tool.

[0034] Furthermore, in step S3: It is preferred to use vina-2.0 inside the pyrx software for molecular docking, calculate the binding energy, and screen for antioxidant peptides. The lower the docking energy, the more stable the binding of the ligand to the receptor. Peptides with lower docking energy to Keap1 are screened.

[0035] The third object of the present invention is to provide the application of the above-mentioned sea cucumber-derived antioxidant peptides in food or cosmetics. Through molecular docking screening and activity verification, it is proved that the above-mentioned sea cucumber-derived antioxidant peptides have a protective effect on the oxidative damage of Hacat cells and can be applied to food or cosmetics, especially functional foods.

[0036] The fourth object of the present invention is the application of the above-mentioned sea cucumber-derived antioxidant peptides in the preparation of antioxidant functional foods or antioxidant drugs. Through molecular docking screening and activity verification, it is proved that the above-mentioned sea cucumber-derived antioxidant peptides have a protective effect on the oxidative damage of Hacat cells and can be applied to the preparation of functional foods or antioxidant drugs.

[0037] The beneficial effects of the present invention are as follows:

[0038] Through biomimetic enzymatic hydrolysis and molecular docking screening, the present invention prepared and screened four antioxidant peptides from Apostichopus japonicus, and their docking energies with the protein receptor Keap1 are -8.6 kcal / mol, -9.4 kcal / mol, -9.1 kcal / mol, and -8.8 kcal / mol respectively. Among them, the peptide PGPAGR with the lowest docking energy has better antioxidant potential. The antioxidant peptides obtained by the present invention can be applied to food, cosmetics, or drugs, which is of great significance for the development of new antioxidant products. Description of the Drawings

[0039] Figure 1 It is a molecular docking result diagram of a sea cucumber-derived antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.1 and the protein receptor Keap1;

[0040] Figure 2 The molecular docking result diagram of sea cucumber-derived antioxidant peptide with amino acid sequence shown in SEQ.ID.NO.2 and protein receptor Keap1;

[0041] Figure 3 The molecular docking result diagram of sea cucumber-derived antioxidant peptide with amino acid sequence shown in SEQ.ID.NO.3 and protein receptor Keap1;

[0042] Figure 4 The molecular docking result diagram of sea cucumber-derived antioxidant peptide with amino acid sequence shown in SEQ.ID.NO.4 and protein receptor Keap1;

[0043] Figure 5 The test results of the survival rate of Hacat cells induced by H2O2 for the synthetic peptides of SEQ.ID.NO.1-4 in the test, as well as the control group, model group, and positive group. Detailed implementation manners

[0044] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0045] In the detailed implementation manners: Pepsin and trypsin were purchased from Pangbo Biology; Chymotrypsin was purchased from Bomei Biology. Example

[0046] S1. Preparation of sea cucumber peptides:

[0047] (1) Remove the viscera of Apostichopus japonicus, and retain the body wall and gonad, which is the sea cucumber raw material; 1 kg of sea cucumber raw material is added with 5 L of ultrapure water, then homogenized, heated in a boiling water bath for 15 min, and homogenized again to obtain a homogenate for use;

[0048] (2) Bionic enzymatic hydrolysis: Add pepsin at 1000 U / g based on the sea cucumber raw material to the homogenate obtained in step (1), adjust the pH to 3, and enzymatically hydrolyze at 37 °C for 5 h, then inactivate the enzyme by boiling at 100 °C; after cooling, add trypsin at 1200 U / g and chymotrypsin at 1400 U / g based on the sea cucumber raw material, adjust the pH to 7, enzymatically hydrolyze at 37 °C for 3 h, and then inactivate the enzyme by boiling at 100 °C; then centrifuge at 5000 r / min for 15 min, take the supernatant to obtain the enzymatic hydrolysate;

[0049] (3)Purification: The enzymolysis solution was fractionated using ultrafiltration and nanofiltration; first, a 200 Da nanofiltration membrane was used to remove salts and free amino acids, and then a 3000 Da spiral wound membrane was selected for ultrafiltration. The fraction with a molecular weight of 200 - 3000 Da obtained was freeze-dried to obtain peptide powder, which was stored at -20 °C for later use.

[0050] S2. Sequence identification of sea cucumber peptides:

[0051] The peptide powder obtained in step S1 was desalted using a C18 StageTip chromatographic column, and LC-MS / MS was used for polypeptide sequence analysis. After the sample was injected into the Trap Column (100 µm × 20 mm), gradient separation was carried out using a DrMaisch-GmbH-C18 chromatographic analysis column (75 µm × 150 mm) at a flow rate of 300 nL / min, and the elution program was set; a Q Exactive mass spectrometer was used for structure identification; comparative analysis was carried out through a database. The mass spectrometry database retrieval software was MaxQuant 2.0.1.0, and the sample database used was the uniprot protein database to obtain all peptide sequences. A total of 78 sequences were obtained from the enzymolysis solution of Apostichopus japonicus through peptide spectrum identification, and 1 of them was an active sequence that had been reported.

[0052] S3. Screening of antioxidant peptides from Apostichopus japonicus assisted by bioinformatics tools:

[0053] (1)The potential biological activities of polypeptides were predicted using the Peptide Ranker tool (http: / / distilldeep.ucd.ie / Peptide Ranker), and sequences with a score exceeding 0.5 were screened. Then, the antioxidant properties of the polypeptides were predicted using the AnOxPePred 1.0 tool (http: / / Bioinformatic Services / AnOxPePred 1.0), and sequences with a score exceeding 0.4 were considered to have potential activity for subsequent screening;

[0054] (2)The novelty of peptides was queried through the BIOPEP database (https: / / biochemia.uwm.edu.pl / ); the water solubility of peptides was predicted using the Innovagen tool, the potential allergenicity of peptides was predicted using the AllercatPro (https: / / allercatpro.bii.a-star.edu.sg / ) tool, and the potential toxicity and physicochemical properties of peptides were predicted using the Toxin Pred (http: / / crdd.osdd.net / raghava / toxinpred / ) tool. Sequences without potential allergenicity and toxicity were subjected to subsequent molecular docking, synthesis, and verification;

[0055] (3) Collect the structures of key target proteins from the PDB database, use the Pymol software to optimize the targets by removing water molecules and small molecule ligands, etc., and perform hydrogenation and charge processing using AutoDock Tools and save them as pdbqt format; use the Keap1 protein as the receptor and its corresponding active ingredient as the ligand, and utilize vina-2.0 inside the pyrx software for molecular docking, calculate the binding energy and output the result file. Finally, use the PyMol software for result visualization. Its Affinity (kcal / mol) value represents the binding ability of the two. The lower the docking energy, the more stable the binding of the ligand to the receptor; use Pymol (https: / / pymol.org / 2 / ) to perform visual analysis on it (the lower the binding energy, the better the binding), and obtain four antioxidant polypeptides, and their amino acid sequences are shown as SEQ.ID.NO.1~4 respectively.

[0056] The amino acid sequences of each sea cucumber-derived antioxidant peptide and the corresponding docking energy are shown in Table 1. The predicted antioxidant activity scores, water solubility, potential allergenicity, potential toxicity and physicochemical properties of each antioxidant peptide are shown in Table 2.

[0057] Table 1 Amino acid sequences of antioxidant peptides and docking energy

[0058] Antioxidant peptide Sequence Docking energy (kcal / mol) SEQ.ID.NO.1 PGGR -8.6 SEQ.ID.NO.2 PGPAGR -9.4 SEQ.ID.NO.3 PGPTGPAGPR -9.1 SEQ.ID.NO.4 GGQQGPR -8.8

[0059] Table 2 Activity scores of antioxidant peptides and prediction of allergenicity and toxicity

[0060] Peptide sequence SEQ.ID.NO.1 SEQ.ID.NO.2 SEQ.ID.NO.3 SEQ.ID.NO.4 Bioactivity score 0.826707 0.753197 0.717293 0.559352 Antioxidant activity prediction score 0.454306 0.463967 0.500148 0.469607 Allergy prediction No allergy No allergy No allergy No allergy Toxicity prediction No toxicity No toxicity No toxicity No toxicity Water solubility Readily soluble Readily soluble Readily soluble Readily soluble

[0061] The molecular docking results of the antioxidant peptide with the amino acid sequence shown as SEQ.ID.NO.1 and the Keap1 receptor are shown in Figure 1 ; the molecular docking results of the antioxidant peptide with the amino acid sequence shown as SEQ.ID.NO.2 and the Keap1 receptor are shown in Figure 2 ; the molecular docking results of the antioxidant peptide with the amino acid sequence shown as SEQ.ID.NO.3 and the Keap1 receptor are shown in Figure 3 ; the molecular docking results of the antioxidant peptide with the amino acid sequence shown as SEQ.ID.NO.4 and the Keap1 receptor are shown in Figure 4 .

[0062] Such as Figures 1 - 4As shown, from the binding modes and binding sites of the four antioxidant peptides and the receptor protein Keap1, it can be seen that the binding of the antioxidant peptides to the Keap1 subunit is mainly through hydrogen bond interactions and electrostatic interactions generated with the receptor amino acid residues. The main binding sites of these four sea cucumber-derived antioxidant peptides to the Keap1 subunit are GLU-191, HIS-71, SER-3, GLN-147, GLU-187, LEU-81, PRO-8, ASN-185, ASN-188, ARG-192, SER-75, THR-79, LEU-146. Among them, the binding sites of the antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.1 to the Keap1 subunit are VAL-418, VAL-512, VAL-465, LEU-365, LEU-557, VAL-604; the binding sites of the antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.2 to the Keap1 subunit are SER-363, ARG-380, VAL-463, ALA-510, VAL-604, GLY-367, VAL-606; the binding sites of the antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.3 to the Keap1 subunit are ARG-380, ASN-414, ARG-415, LEU-365; the binding sites of the antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.4 to the Keap1 subunit are VAL-418, SER-555, LEU-557, VAL-512, GLY-367, ILE-559, ILE-416, VAL-463.

[0063] Test

[0064] The activities of the peptides with the amino acid sequences shown in SEQ.ID.NO.1 to 4 obtained by screening were verified respectively.

[0065] According to the mass spectrometry sequencing and decomposition docking results, the four peptide sequences SEQ.ID.NO.1 to 4 were entrusted to Anhui Guoping Pharmaceutical Co., Ltd. for chemical synthesis. HaCaT cells are an immortalized cell line of normal human skin keratinocytes derived from non-tumor sources and are widely used to study the antioxidant mechanism and protective function of skin cells. Therefore, an H2O2-induced oxidative damage model of Hacat cells was selected to verify its antioxidant activity and anti-aging effect. The Hacat cells were seeded at 5×10 4Inoculate at a density of cells / mL into a 96-well plate, 100 μL per well. After 24 h, divide the cells into a normal control group, a model group, a positive group, and a sample group. In the sample group, add the synthetic peptides of SEQ.ID.NO.1-4 at a concentration of 0.3 mg / mL in sequence. In the positive group, add 0.3 mg / mL N-acetylcysteine (NAC) as a positive control. After incubating for 24 h, except for the normal control group, add 150 μmol / L H2O2 to each group and incubate for 4 h. Use a Cell Counting Kit-8 to detect the cell viability.

[0066] The test results of the synthetic peptides of SEQ.ID.NO.1-4 and the control group, model group, and positive group are as Figure 5 shown; Figure 5 Among them, S1, S2, S3, and S4 correspond to the synthetic peptides of SEQ.ID.NO.1-4 in sequence, with a concentration of 0.3 mg / mL.

[0067] The test results show that after treatment with H2O2, the survival rate of the model group decreased significantly to 53.5% ( P ≤0.01), while adding the synthetic peptides of SEQ.ID.NO.1-4 can significantly repair the damage caused by H2O2 to Hacat cells and significantly reduce the cell damage and apoptosis caused by oxidative stress ( P ≤0.01). Among them, the antioxidant capacity of the synthetic peptide of SEQ.ID.NO.1 (72.56%) is similar to that of the control group NAC (73.02%). The experimental data prove that the sea cucumber-derived antioxidant peptide has a protective effect on the oxidative damage of Hacat cells and can be applied to food or cosmetics, and can be used in the preparation of functional foods or antioxidant drugs.

[0068] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sea cucumber-derived antioxidant peptide, characterized in that, A peptide selected from the amino acid sequences shown in SEQ.ID.NO.3 - 4.

2. A method for preparing the sea cucumber-derived antioxidant peptide as described in claim 1, characterized in that, Comprising the following steps: S1. Obtaining sea cucumber peptides by enzymolysis of sea cucumber raw materials; first using pepsin for enzymolysis, then using trypsin and chymotrypsin for enzymolysis; after enzymolysis, separating and purifying the enzymolysis solution; the said separation and purification includes fractionating the enzymolysis solution using ultrafiltration and nanofiltration; S2. Identifying the sequence of the sea cucumber peptides; S3. Performing molecular docking of the sea cucumber peptides with the receptor Keap1 protein to screen for antioxidant peptides.

3. The preparation method according to claim 2, characterized in that, In step S2: Using LC-MS / MS for polypeptide sequence analysis.

4. According to the preparation method described in claim 2, characterized in that, Before performing molecular docking of the sea cucumber peptides with the receptor protein Keap1, predicting its antioxidant activity through the AnOxPePred 1.0 tool; Before performing molecular docking of the sea cucumber peptides with the receptor protein Keap1, predicting the water solubility, potential allergenicity, toxicity and physicochemical properties of the peptides.

5. Use of a sea cucumber-derived antioxidant peptide as described in claim 1 in the preparation of foods or cosmetics.

6. Use of a sea cucumber-derived antioxidant peptide as described in claim 1 in the preparation of antioxidant functional foods.

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