Sea cucumber-derived antioxidant peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food biology, and particularly relates to a sea cucumber-derived antioxidant peptide as well as a preparation method and application thereof. The sea cucumber source antioxidant peptide is prepared and screened from apostichopus japonicus and comprises at least one of peptides with the amino acid sequence shown as SEQ ID NO.1-4, and the docking energy of the peptides and a protein receptor Keap1 is-8.6 kcal / mol,-9.4 kcal / mol,-9.1 kcal / mol and-8.8 kcal / mol respectively. The antioxidant peptide obtained by the invention can be applied to food, cosmetics or medicines, and has important significance on the development of novel antioxidant products.

Description

Technical Field

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

[0002] As a natural antioxidant, antioxidant peptides are easily absorbed, stable, and non-immunoreactive. They have nutritional properties and functions such as lowering blood pressure and improving immunity. They are widely used in the food field because of their natural, safe, non-toxic, and high nutritional value. They have become one of the most commonly used natural products in functional foods. The use of low-cost biological resources to develop antioxidant peptides with nutritional and safety has always been a research hotspot in the field of peptides.

[0003] Bioinformatics tools are used to assist in screening active peptides. Based on the known amino acid sequences in active peptides, database searches and software analysis are used to predict the possible biological activity, safety, and bioaccessibility of peptides, select target peptides for chemical synthesis, and verify their related properties. Bioinformatics tools can save a lot of time and experimental costs, but there is currently no research on using bioinformatics tools to screen sea cucumber antioxidant peptides.

[0004] There are many methods for evaluating the activity of antioxidant peptides. The most widely used methods are the DPPH method (GB / T39100-2020 Method 1) and the ABTS method (GB / T39100-2020 Method 2). Keap1 enzyme can effectively remove free radicals that damage health and cause diseases in the body, decompose them into oxygen molecules and water molecules that are harmless to the human body and excrete them smoothly from 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 used 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 and transporter-related genes, and enhance the resistance of cells to oxidative and heterologous stress. 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, thereby causing Nrf2 to dissociate and activate the Nrf2 pathway, thus playing an antioxidant role in protecting cells. Therefore, exploring the interaction between sea cucumber-derived antioxidant peptides and Keap1 receptors 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 and a preparation method and application thereof. The antioxidant peptide of the present invention is prepared and screened from sea cucumbers and has significant antioxidant activity.

[0006] The specific technical solutions are as follows: 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 with amino acid sequences as shown in SEQ.ID.NO.1-4.

[0007] 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.

[0008] Among them, SEQ.ID.NO.2 is PGPAGR, and the docking energy between this sea cucumber-derived antioxidant peptide and the receptor protein Keap1 is -9.4 kcal / mol.

[0009] 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.

[0010] Among them, SEQ.ID.NO.4 is GGQQGPR, and the docking energy between this sea cucumber-derived antioxidant peptide and the receptor protein Keap1 is -8.8 kcal / mol.

[0011] Wherein, the sea cucumber is an imitation sea cucumber ( Apostichopus japonicus ), the above-mentioned sea cucumber-derived antioxidant peptides were prepared and screened from the body wall and gonads of Apostichopus japonicus.

[0012] Preferably, the sea cucumber-derived antioxidant peptide includes the peptide PGPAGR with an amino acid sequence as shown in SEQ.ID.NO. 2. This peptide segment has the lowest docking energy, is composed of 6 amino acids, and is relatively short, which is of great significance for the development of new antioxidant products.

[0013] The second object of the present invention is to provide a method for preparing the above-mentioned sea cucumber-derived antioxidant peptide, comprising the following steps: S1. Obtaining sea cucumber peptides; S2. Sequence identification of sea cucumber peptides; S3. Molecular docking of sea cucumber peptides with receptor Keap1 protein was performed to screen antioxidant peptides.

[0014] Further, in step S1: sea cucumber peptides are obtained by enzymatically hydrolyzing the sea cucumber raw material.

[0015] Specifically, in step S1: firstly, pepsin is used for enzymolysis, and then trypsin and chymotrypsin are used for enzymolysis. The present invention performs bionic enzymolysis on the sea cucumber raw material.

[0016] More specifically, in step S1: the working conditions of enzymatic hydrolysis are preferably as follows: add pepsin to the raw material to be processed, adjust the pH to 2-4, enzymatic hydrolyze at 35-40°C for 4-6 hours, and then inactivate the enzymes; add trypsin and chymosin, adjust the pH to 6-8, enzymatic hydrolyze at 35-40°C for 2-4 hours, and then inactivate the enzymes.

[0017] Among them, the added amount of pepsin is preferably 800~1200 U / g based on the sea cucumber raw material.

[0018] Among them, the addition amount of trypsin is preferably 1000~1400 U / g based on the sea cucumber raw material.

[0019] Among them, the addition amount of chymosin is preferably 1200~1600 U / g based on the sea cucumber raw material.

[0020] 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, heat treating for 10 to 15 minutes, and then homogenizing again for standby use.

[0021] Among them, the usage ratio of sea cucumber raw material to water is preferably 1kg: (3~8)L.

[0022] Among them, heat treatment using a boiling water bath is preferred.

[0023] Among them, the water added to the sea cucumber raw material is preferably ultrapure water.

[0024] Furthermore, in step S1: after enzymatic hydrolysis, the enzymatic hydrolyzate is separated and purified.

[0025] The separation and purification includes classifying the enzymatic hydrolysate by ultrafiltration and nanofiltration to obtain peptides within a certain molecular weight range. Salts and free amino acids can be removed by nanofiltration, and macromolecular substances can be removed by ultrafiltration.

[0026] Further, in step S2: peptide sequence analysis is performed using LC-MS / MS. The sequences of all peptides are obtained by comparison analysis through the database. Specifically, the mass spectrometry database retrieval software is MaxQuant 2.0.1.0, and the database used for the sample is the uniprot protein database.

[0027] Specifically, in step S2: the product obtained in step S1 is preferably desalted first, and then the polypeptide sequence analysis is performed. Preferably, a C18 StageTip column is used for desalting.

[0028] Further, in step S3: before molecular docking of the sea cucumber peptide with the receptor protein Keap1, the antioxidant activity thereof is preferably predicted by the AnOxPePred 1.0 tool, and a peptide sequence with a higher score is selected.

[0029] Further, in step S3: before molecular docking the sea cucumber peptide with the receptor protein Keap1, the water solubility, potential allergic reaction, toxicity and physicochemical properties of the peptide are preferably predicted, and the sequence without potential allergic reaction and toxicity is subsequently synthesized and verified. Specifically, the water solubility of the peptide can be predicted by the Peptide Property Calculator tool, and the potential toxicity and physicochemical properties of the peptide can be predicted by the Toxin Pred tool.

[0030] Further, in step S3: preferably, VINA-2.0 in the pyrx software is used for molecular docking, binding energy is calculated, and antioxidant peptides are screened. The lower the docking energy, the more stable the binding of the ligand to the receptor. Peptides with lower Keap1 docking energy are screened.

[0031] The third object of the present invention is to provide the application of the above sea cucumber-derived antioxidant peptide in food or cosmetics. Molecular docking screening and activity verification have shown that the above sea cucumber-derived antioxidant peptide has a protective effect on oxidative damage of Hacat cells and can be applied to food or cosmetics, especially functional foods.

[0032] The fourth purpose of the present invention is to use the above-mentioned sea cucumber-derived antioxidant peptide in the preparation of antioxidant functional foods or antioxidant drugs. Molecular docking screening and activity verification have shown that the above-mentioned sea cucumber-derived antioxidant peptide has a protective effect on oxidative damage of Hacat cells and can be used in the preparation of functional foods or antioxidant drugs.

[0033] The beneficial effects of the present invention are as follows: The present invention obtains four antioxidant peptides from imitation sea cucumbers through bionic enzymatic hydrolysis and molecular docking screening, 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. 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 medicines, which is of great significance to the development of new antioxidant products. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a molecular docking result diagram of the sea cucumber-derived antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.1 and the protein receptor Keap1; Figure 2 The molecular docking result diagram of the sea cucumber-derived antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.2 and the protein receptor Keap1; Figure 3The molecular docking result diagram of the sea cucumber-derived antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.3 and the protein receptor Keap1; Figure 4 The molecular docking result diagram of the sea cucumber-derived antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.4 and the protein receptor Keap1; Figure 5 The synthetic peptides of SEQ.ID.NO.1~4 and the control group, model group and positive group were tested for H 2 O 2 Results of the Hacat cell survival test after induced injury. DETAILED DESCRIPTION

[0035] The principles and features of the present invention are described below in conjunction with examples, which are only used to explain the present invention and are not used to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0036] In a specific embodiment: pepsin and trypsin were purchased from Pangbo Biotechnology; chymotrypsin was purchased from Bomei Biotechnology. Example

[0037] S1. Preparation of sea cucumber peptide: (1) Remove the internal organs of the imitation sea cucumber and retain the body wall and gonads to obtain the raw material of the sea cucumber; add 5L of ultrapure water to 1kg of the raw material of the sea cucumber, then homogenize it, heat it in a boiling water bath for 15 minutes, and homogenize it again to obtain the homogenate for later use; (2) Bionic enzymatic hydrolysis: add 1000 U / g pepsin based on the raw material of sea cucumber to the homogenate obtained in step (1), adjust the pH to 3, perform enzymatic hydrolysis at 37°C for 5 h, and then boil at 100°C to inactivate the enzymes; after cooling, add 1200 U / g trypsin and 1400 U / g chymosin based on the raw material of sea cucumber, adjust the pH to 7, perform enzymatic hydrolysis at 37°C for 3 h, and then boil at 100°C to inactivate the enzymes; then centrifuge at 5000 r / min for 15 min, take the supernatant, and obtain the enzymatic hydrolyzate; (3) Purification: Use ultrafiltration and nanofiltration to grade the enzymatic hydrolysate; first use a 200 Da nanofiltration membrane to remove salts and free amino acids, then select a 3000 Da roll membrane for ultrafiltration, freeze-dry the obtained 200-3000 Da components to obtain peptide powder, and store it at -20°C for later use.

[0038] S2. Sequence identification of sea cucumber peptides: The peptide powder obtained in step S1 was desalted using a C18 StageTip column, and peptide sequence analysis was performed using LC-MS / MS. The sample was injected into a Trap Column (100 µm×20 mm) and then passed through a DrMaisch-GmbH-C18 column (75 µm×150 mm) for gradient separation. The flow rate was 300 nL / min and the elution program was set. The structure was identified using a Q Exactive mass spectrometer. The database was compared and analyzed using the database. The mass spectrometry database retrieval software was MaxQuant 2.0.1.0, and the database used for the sample was the uniprot protein database. All peptide sequences were obtained. A total of 78 sequences were obtained from the sea cucumber hydrolysate through peptide spectrum identification, of which 1 active sequence has been reported.

[0039] S3. Bioinformatics tools assisted the screening of sea cucumber-like antioxidant peptides: (1) The potential biological activity of peptides was predicted using the Peptide Ranker tool (http: / / distilldeep.ucd.ie / Peptide Ranker), and sequences with scores exceeding 0.5 were screened. The peptides were then predicted for antioxidant activity using the AnOxPePred 1.0 tool (http: / / Bioinformatic Services / AnOxPePred 1.0). Sequences with scores exceeding 0.4 were considered to have potential activity and were subsequently screened. (2) The novelty of the peptides was queried through the BIOPEP database (https: / / biochemia.uwm.edu.pl / ); the water solubility of the peptides was predicted through the Innovagen tool, the potential allergenicity of the peptides was predicted through the AllercatPro tool (https: / / allercatpro.bii.a-star.edu.sg / ), and the potential toxicity and physicochemical properties of the peptides were predicted through the Toxin Pred tool (http: / / crdd.osdd.net / raghava / toxinpred / ). Sequences without potential allergenicity and toxicity were subsequently docked, synthesized, and verified; (3) Collect key target protein structures in the PDB database, use Pymol software to optimize the targets by removing water molecules and small molecule ligands, and use AutoDock Tools for hydrogenation and charge processing and save as pdbqt format; use Keap1 protein as the receptor and its corresponding active ingredient as the ligand, use vina-2.0 within the pyrx software for molecular docking, calculate the binding energy and output the result file. Finally, use PyMol software to visualize the results. Its Affinity (kcal / mol) value represents the binding ability of the two. The lower the docking energy, the more stable the binding between the ligand and the receptor; use Pymol (https: / / pymol.org / 2 / ) to visualize and analyze it (the lower the binding energy, the better the binding), and obtain four antioxidant peptides, whose amino acid sequences are shown in SEQ.ID.NO.1~4.

[0040] The amino acid sequences and corresponding docking energies of each sea cucumber-derived antioxidant peptide are shown in Table 1. The antioxidant activity prediction scores, water solubility, potential allergenicity, potential toxicity, and physicochemical properties of each antioxidant peptide are shown in Table 2.

[0041] Table 1 Amino acid sequences and docking energies of antioxidant peptides Antioxidant Peptides 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

[0042] Table 2 Antioxidant peptide activity scores and predictions of allergic and toxicity 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 Non-allergenic Non-allergenic Non-allergenic Non-allergenic Toxicity prediction Non-toxic Non-toxic Non-toxic Non-toxic Water Solubility Soluble Soluble Soluble Soluble The molecular docking results of the antioxidant peptide with amino acid sequence as shown in SEQ.ID.NO.1 and Keap1 receptor are shown in Figure 1 The molecular docking results of the antioxidant peptide with amino acid sequence as shown in SEQ.ID.NO.2 and Keap1 receptor are shown in Figure 2 The molecular docking results of the antioxidant peptide with amino acid sequence as shown in SEQ.ID.NO.3 and Keap1 receptor are shown in Figure 3 The molecular docking results of the antioxidant peptide with amino acid sequence as shown in SEQ.ID.NO.4 and Keap1 receptor are shown in Figure 4 .

[0043] like Figures 1 to 4As shown in the figure, through the binding patterns and binding sites of the four antioxidant peptides and the receptor protein Keap1, it can be seen that the binding of antioxidant peptides to Keap1 subunits is mainly through hydrogen bonding and electrostatic interactions between the amino acid residues of the receptor. The binding sites of these four sea cucumber-derived antioxidant peptides to Keap1 subunits are mainly GLU-191, HIS-71, SER-3, GLN-147, GLU-187, LEU-81, PRO-8, ASN-185, ASN-188, ARG-192, SER-75, THR-79, and LEU-146. Among them, the binding sites of the antioxidant peptide shown in the amino acid sequence of SEQ.ID.NO.1 and the Keap1 subunit are VAL-418, VAL-512, VAL-465, LEU-365, LEU-557, and VAL-604; the binding sites of the antioxidant peptide shown in the amino acid sequence of SEQ.ID.NO.2 and the Keap1 subunit are SER-363, ARG-380, VAL-463, ALA-510, VAL-604, GLY-367, and VAL-604. L-606; the binding sites of the antioxidant peptide shown in the amino acid sequence of SEQ.ID.NO.3 and the Keap1 subunit are ARG-380, ASN-414, ARG-415, and LEU-365; the binding sites of the antioxidant peptide shown in the amino acid sequence of SEQ.ID.NO.4 and the Keap1 subunit are VAL-418, SER-555, LEU-557, VAL-512, GLY-367, ILE-559, ILE-416, and VAL-463. test

[0044] The activity of the peptides obtained by screening, whose amino acid sequences are shown in SEQ.ID.NO.1~4, was verified.

[0045] According to the results of mass spectrometry sequencing and decomposition docking, the four peptide sequences SEQ.ID.NO.1~4 were entrusted to Anhui Guoping Pharmaceutical Co., Ltd. for chemical synthesis. HaCaT cells are immortalized cell lines of normal human skin keratinocytes from non-tumor sources and are widely used to study the antioxidant mechanism and protective function of skin cells. Therefore, H 2 O 2 Hacat cells were induced to undergo oxidative damage and their antioxidant activity and anti-aging effects were verified. 4The cells were inoculated at a density of 100 μL / well in a 96-well plate. After 24 h, the cells were divided into a normal control group, a model group, a positive group, and a sample group. The sample group was added with 0.3 mg / mL of SEQ.ID.NO.1~4 synthetic peptides, and the positive group was added with 0.3 mg / mL N-acetylcysteine ​​(NAC) as a positive control. After incubation for 24 h, 150 μmol / L H 2 O 2 After incubation for 4 h, the cell survival rate was detected using a cell proliferation-toxicity detection kit (Cell Counting Kit-8).

[0046] The test results of SEQ.ID.NO.1~4 synthetic peptides and the control group, model group, and positive group are as follows Figure 5 As shown; Figure 5 In the assay, S1, S2, S3 and S4 correspond to SEQ.ID.NO.1~4 synthetic peptides, respectively, with a concentration of 0.3 mg / mL.

[0047] The test results show that H 2 O 2 After treatment, the survival rate of the model group dropped significantly to 53.5% ( P ≤0.01), and the addition of SEQ.ID.NO.1~4 synthetic peptides can significantly repair H 2 O 2 The damage to Hacat cells significantly reduced the cell damage and apoptosis caused by oxidative stress ( P ≤0.01). Among them, the antioxidant capacity of SEQ.ID.NO.1 synthetic peptide (72.56%) was similar to that of the control group NAC (73.02%). The experimental data proved that sea cucumber-derived antioxidant peptides have a protective effect on oxidative damage of Hacat cells and can be used in food or cosmetics, and can be used in the preparation of functional foods or antioxidant drugs.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A sea cucumber-derived antioxidant peptide, characterized in that: It includes at least one of the peptides whose amino acid sequences are shown in SEQ.ID.NO.1~4.

2. The sea cucumber-derived antioxidant peptide according to claim 1, characterized in that: The sea cucumber is an imitation sea cucumber.

3. A method for preparing the sea cucumber-derived antioxidant peptide according to claim 1 or 2, characterized in that: The steps include: S1. Obtaining sea cucumber peptides; S2. Sequence identification of sea cucumber peptides; S3. Molecular docking of sea cucumber peptides with receptor Keap1 protein was performed to screen antioxidant peptides.

4. The preparation method according to claim 3, characterized in that: In step S1: sea cucumber peptides are obtained by enzymatically hydrolyzing sea cucumber raw materials.

5. The preparation method according to claim 4, characterized in that: In step S1: firstly, enzymatic hydrolysis is performed using pepsin, and then enzymatic hydrolysis is performed using trypsin and chymotrypsin.

6. The preparation method according to claim 4 or 5, characterized in that: In step S1: after enzymolysis, the enzymolysis solution is separated and purified; the separation and purification includes classifying the enzymolysis solution by ultrafiltration and nanofiltration.

7. The preparation method according to any one of claims 3 to 5, characterized in that In step S2: peptide sequence analysis is performed using LC-MS / MS.

8. The preparation method according to any one of claims 3 to 5, characterized in that Before molecular docking of sea cucumber peptides with the receptor protein Keap1, the antioxidant activity of sea cucumber peptides was predicted using the AnOxPePred 1.0 tool; Before molecular docking of the sea cucumber peptide with the receptor protein Keap1, the water solubility, potential allergenicity, toxicity and physicochemical properties of the peptide were predicted.

9. Use of the sea cucumber-derived antioxidant peptide as claimed in claim 1 or 2 in food or cosmetics.

10. Use of the sea cucumber-derived antioxidant peptide according to claim 1 or 2 in the preparation of antioxidant functional foods or antioxidant drugs.

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