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

Abstract

The present invention discloses an antioxidant peptide derived from sea cucumber gonad, its preparation method and application, belonging to the field of food biotechnology. The antioxidant peptide is obtained by enzymolysis of sea cucumber gonad and molecular docking screening, and includes at least one of the peptides shown in the amino acid sequences SEQ.ID.NO.1-4. Among them, the docking energy of SEQ.ID.NO.1 with the receptor SOD is -7.5 kcal / mol, the docking energy of SEQ.ID.NO.2 with the receptor SOD is -7.5 kcal / mol, the docking energy of SEQ.ID.NO.3 with the receptor SOD is -7.4 kcal / mol, and the docking energy of SEQ.ID.NO.4 with the receptor SOD is -7.2 kcal / mol. Through experimental verification, the above antioxidant peptide has high antioxidant activity and has broad application space in the field of functional foods.

Description

Technical Field

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

[0002] As a natural antioxidant, antioxidant peptides are easily absorbed, have good stability, no immunoreactivity, and have both nutritional properties and functions such as blood pressure lowering and immune enhancement. Due to the characteristics of "natural, safe, non-toxic, and high nutritional value", they are 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] There are many methods for evaluating the activity of antioxidant peptides. 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). SOD 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 excrete them smoothly, 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 SOD, providing an effective way for the screening and discovery of antioxidant peptides.

[0004] A large number of by-products are generated during sea cucumber processing, such as viscera, gonads, etc. Research has found that sea cucumber gonads have a high protein content and are also rich in active ingredients such as sea cucumber polysaccharides. The development and utilization degree of sea cucumber gonads is low. Currently, no commercial products developed from sea cucumber gonads as raw materials have been found in the domestic market. Exploring the interaction between sea cucumber-derived antioxidant peptides and SOD receptors and finding their antioxidant mechanisms can provide a basis for the preparation of functional foods from natural food sources of sea cucumber-derived antioxidant peptides. Summary of the Invention

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

[0006] The specific technical solutions are as follows:

[0007] One of the purposes of the present invention is to provide an antioxidant peptide derived from sea cucumber gonad, which includes at least one of the peptides shown in the amino acid sequences SEQ.ID.NO.1~4.

[0008] Among them, SEQ.ID.NO.1 is VPYPR, and its docking energy with the antioxidant receptor SOD is -7.5 kcal / mol.

[0009] Among them, SEQ.ID.NO.2 is ATGPQGPAGQRGPAGPTGPTGPAG, and its docking energy with the antioxidant receptor SOD is -7.5 kcal / mol.

[0010] Among them, SEQ.ID.NO.3 is PGHPF, and its docking energy with the antioxidant receptor SOD is -7.4 kcal / mol.

[0011] Among them, SEQ.ID.NO.4 is NPWGQ, and its docking energy with the antioxidant receptor SOD is -7.2 kcal / mol.

[0012] Specifically, the sea cucumber gonad is the mixed gonad of male and female Apostichopus japonicus.

[0013] The second object of the present invention is to provide a preparation method of the above-mentioned antioxidant peptide from sea cucumber gonad, comprising the following steps:

[0014] S1. Obtain sea cucumber gonad peptide;

[0015] S2. Identify the sequence of the sea cucumber gonad peptide;

[0016] S3. Perform molecular docking of the sea cucumber gonad peptide with the receptor SOD to screen for antioxidant peptides.

[0017] Further, in step S1: The sea cucumber gonad peptide is obtained by enzymolysis of the sea cucumber gonad.

[0018] Specifically, in step S1, the working conditions of enzymolysis preferably include: adding pepsin to the raw material to be processed for enzymolysis, and then adding flavor protease for enzymolysis.

[0019] More specifically, in step S1, the working conditions of enzymolysis preferably include: adding pepsin to the raw material to be processed, adjusting the pH to 1.5 - 3.5, enzymolyzing at 36 - 38 °C for 2 - 5 h; then adding flavor protease, adjusting the pH to 6.0 - 8.0, enzymolyzing at 45 - 55 °C for 1 - 3 h, and inactivating the enzyme.

[0020] Among them, the dosage of pepsin is preferably 1000 - 3000 U / g based on the raw material to be processed.

[0021] Among them, the dosage of flavor protease is preferably 1000 - 2000 U / g based on the raw material to be processed.

[0022] Specifically, in step S1: Before enzymolysis of the sea cucumber gonad, it is preferably pretreated. The pretreatment includes: homogenizing the sea cucumber gonad and heating it in a boiling water bath for 10 - 30 min.

[0023] Further, in step S1: after enzymatic hydrolysis, the enzymatic hydrolysate is separated and purified.

[0024] Furthermore, in step S1: the separation and purification includes fractionating the enzymatic hydrolysate using nanofiltration and ultrafiltration. Nanofiltration can remove salts and free amino acids, and ultrafiltration can remove macromolecular substances. Specifically, preferably by subjecting the enzymatic hydrolysate to nanofiltration and ultrafiltration, a fraction with a molecular weight of 200 - 3000 Da is obtained.

[0025] Further, in step S1: after obtaining sea cucumber gonad peptides, their in vitro SOD activity is measured.

[0026] Further, in step S2: LC-MS / MS is used for polypeptide sequence analysis, and by comparing and analyzing through a database, all peptide sequences are obtained. The mass spectrometry database retrieval software is MaxQuant 2.4.14.0, and the sample use database is the uniprot protein database.

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

[0028] Further, in step S3: preferably, vina - 2.0 inside the pyrx software is used for molecular docking to screen antioxidant peptides. Its Affinity value represents the binding ability between the two. The lower the binding ability, the more stable the ligand and the receptor bind.

[0029] The third object of the present invention is to provide the application of the above-mentioned sea cucumber gonad-derived antioxidant peptides in food. The food is preferably a functional food.

[0030] The fourth object of the present invention is to provide the application of the above-mentioned sea cucumber gonad-derived antioxidant peptides in the preparation of antioxidant functional foods or antioxidant drugs.

[0031] The beneficial effects of the present invention are as follows:

[0032] Through enzymatic hydrolysis and molecular docking screening of sea cucumber gonads, the present invention obtained four antioxidant peptides. Among them, the docking energy of SEQ.ID.NO.1 with the receptor SOD is -7.5 kcal / mol, the docking energy of SEQ.ID.NO.2 with the receptor SOD is -7.5 kcal / mol, the docking energy of SEQ.ID.NO.3 with the receptor SOD is -7.4 kcal / mol, and the docking energy of SEQ.ID.NO.4 with the receptor SOD is -7.2 kcal / mol. Through experimental verification, the above-mentioned antioxidant peptides have high antioxidant activity and have broad application space in the field of functional foods. Description of the Drawings

[0033] Figure 1 Molecular docking diagram of sea cucumber gonad-derived antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.1 and receptor SOD;

[0034] Figure 2 Molecular docking diagram of sea cucumber gonad-derived antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.2 and receptor SOD;

[0035] Figure 3 Molecular docking diagram of sea cucumber gonad-derived antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.3 and receptor SOD;

[0036] Figure 4 Molecular docking diagram of sea cucumber gonad-derived antioxidant peptide with the amino acid sequence shown in SEQ.ID.NO.4 and receptor SOD;

[0037] Figure 5 Activation ability of sea cucumber gonad peptide obtained in step S1 in the example on SOD;

[0038] Figure 6 Ability of four antioxidant peptides and control group GSH to scavenge DPPH free radicals;

[0039] Figure 7 Ability of four antioxidant peptides and control group GSH to scavenge ABTS free radicals. Detailed implementation manners

[0040] The principles and features of the present invention are described below in conjunction with examples. The examples given are only for explaining the present invention and are not used 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.

[0041] In the detailed implementation manners: The pepsin used was purchased from Sangon Biotech; the flavor protease used was purchased from Solarbio.

[0042] In the detailed implementation manners: The sea cucumber gonads used were the mixed gonads of male and female Apostichopus japonicus. Example

[0043] Prepare sea cucumber gonad-derived antioxidant peptide, the steps are as follows:

[0044] S1. Prepare sea cucumber gonad peptide:

[0045] (1) Raw material pretreatment: After homogenizing the sea cucumber gonad tissue, heat it in a boiling water bath for 15 min to obtain a homogenate, and let it cool for later use;

[0046] (2) Raw material enzymatic hydrolysis: Add pepsin at 2000 U / g based on the mass of the homogenate obtained in step (1), adjust the pH to 2.5, and carry out enzymatic hydrolysis at 37 °C for 4 h; then add flavor protease at 1200 U / g based on the mass of the homogenate, adjust the pH to 7.0, and carry out enzymatic hydrolysis at 50 °C for 2 h, and then boil to inactivate the enzyme at 100 °C; then centrifuge at 5000 r / min for 15 min, take the supernatant to obtain the enzymatic hydrolysate;

[0047] (3) Purification: Carry out nanofiltration and ultrafiltration fractionation on the enzymatic hydrolysate obtained in step (2). First, use a 200 Da nanofiltration membrane to remove salts and free amino acids, and then select a 3000 Da spiral wound membrane for ultrafiltration. Freeze-dry the obtained fraction of 200 - 3000 Da to obtain sea cucumber gonad peptide powder, and store it at -20 °C for standby;

[0048] (4) In vitro SOD activity determination of sea cucumber gonad peptide: Set the mass concentration gradients of the peptide powder obtained in step (3) to 1, 2, 3, 4, and 5 mg / mL respectively, and measure the autoxidation rate of pyrogallol. The autoxidation rate is controlled at 0.07 (±0.002) OD / min; when measuring the sample, mix Tris-HCl buffer, the sample to be measured, and SOD solution, place it in a 25 °C water bath and heat for 10 min, then add pyrogallol solution for thorough mixing and start timing, and detect at a wavelength of 325 nm. Read the data once every 30 s, and the continuous reading time is 3 min; the test results are shown in Figure 5 ; It can be seen from Figure 5 that within the dose range of 1 - 5 mg / mL, sea cucumber gonad peptide shows an activation effect on SOD, and shows a significant dose-effect relationship.

[0049] S2. Sequence identification of sea cucumber gonad peptide:

[0050] Desalt the peptide powder obtained in step S1 using a C18 StageTip chromatographic column, and perform polypeptide sequence analysis using LC-MS / MS. Carry out comparison and analysis through a database to obtain all peptide sequences; the mass spectrometry database retrieval software is MaxQuant2.4.14.0, and the sample uses the uniprot protein database.

[0051] S3. Molecular docking of sea cucumber gonad peptide with receptor SOD:

[0052] Obtain the SDF format file of the main active ingredients of the core drugs through the Pubchem database, collect the key target protein structures in the PDB database, use the Pymol-2.1.0 software to optimize the targets by removing water molecules and small molecule ligands, etc., and use AutoDock Tools-1.5.6 for hydrogenation and charge processing and save as pdbqt format. Using the key target as the receptor and its corresponding active ingredient as the ligand, use 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 binding ability, the more stable the ligand binds to the receptor. Use Pymol for its visualization analysis, and the 2D map is visualized using Discovery Studio 2020 Client. Four antioxidant peptides and their docking energies were screened. The amino acid sequences of the four antioxidant peptides are shown in SEQ.ID.NO.1~4 in turn. The amino acid sequences of the four antioxidant peptides and the corresponding docking energies are shown in Table 1.

[0053] Table 1 Amino acid sequences and docking energies of sea cucumber gonad-derived antioxidant peptides

[0054] Antioxidant peptide Sequence SOD docking energy (kcal / mol) SEQ.ID.NO.1 VPYPR -7.5 SEQ.ID.NO.2 ATGPQGPAGQRGPAGPTGPTGPAG -7.5 SEQ.ID.NO.3 PGHPF -7.4 SEQ.ID.NO.4 NPWGQ -7.2

[0055] The molecular docking results of the peptides with amino acid sequences shown in SEQ.ID.NO.1~4 and the receptor SOD are shown in Figures 1 - 4 . As Figures 1 - 4 can be seen, the binding of antioxidant peptides to the SOD subunit is mainly through hydrogen bond interactions and electrostatic interactions generated with the receptor amino acid residues.

[0056] As Figure 1 shown, the binding sites of the peptide with amino acid sequence shown in SEQ.ID.NO.1 and the SOD subunit are GLU(A:135), GLY(A:143), ALA(A:133), THR(A:38), SER(A:136), ALA(A:144), ALA(A:120), LYS(A:138); as Figure 2 shown, the binding sites of the peptide with amino acid sequence shown in SEQ.ID.NO.2 and the SOD subunit are ARG(A:145), ALA(A:144), GLN(A:122), GLN(A:48), ASN(A:64), ASP(A:123), ASP(A:82), LYS(A:68), ASN(A:85), ARG(A:78), HIS(A:119), HIS(A:47), HIS(A:79), ILE(A:57), PRO(A:61); asFigure 3 As shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.3 to the SOD subunit are VAL(A:80), ARG(A:78), GLY(A:84), HIS(A:45), ASP(A:82); the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.4 to the SOD subunit are HIS(A:62), ASN(A:85), GLN(A:122), ASP(A:123), ARG(A:145), ASP(A:124), HIS(A:79), HIS(A:119), ASP(A:82), LYS(A:68). The binding sites of these four antioxidant peptides to the SOD subunit are mainly ARG(A:145), GLN(A:122), ASP(A:123), ASN(A:85), ARG(A:78), ALA(A:144), HIS(A:119).

[0057] Test

[0058] According to the mass spectrometry sequencing results, Jiangsu GenScript Biotech Co., Ltd. was commissioned to chemically synthesize the above four peptide sequences, and then the DPPH method and ABTS method were respectively used to verify their antioxidant activities. Reduced glutathione (GSH) at the same concentration was used as a control, and the test concentration was 5 mg / mL. The abilities of the four polypeptides and the control group GSH to scavenge DPPH free radicals are as Figure 6 shown, and the abilities of the four polypeptides and the control group GSH to scavenge ABTS free radicals are as Figure 7 shown. Figure 6 , Figure 7 In, the abscissa is the corresponding peptide sequence, where AT-AG represents the peptide shown in SEQ.ID.NO.2.

[0059] The test results show that the polypeptides with the amino acid sequences shown in SEQ.ID.NO.1-4 all have certain abilities to scavenge DPPH free radicals and ABTS free radicals, and the abilities of the polypeptides shown in SEQ.ID.NO.1 (VPYPR) and SEQ.ID.NO.4 (NPWGQ) to scavenge ABTS free radicals are stronger than those of the control group GSH.

[0060] 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 gonad-derived antioxidant peptide, characterized in that, At least one selected from the peptides having the amino acid sequences shown in SEQ.ID.NO.2 to 4.

2. A preparation method of the antioxidant peptide derived from sea cucumber gonad as described in claim 1, characterized in that, Comprising the following steps: S1. Obtaining sea cucumber gonad peptides by enzymatically hydrolyzing sea cucumber gonads; the working conditions for enzymatic hydrolysis include: adding pepsin to the raw material to be treated for enzymatic hydrolysis, and then adding flavor protease for enzymatic hydrolysis; after enzymatic hydrolysis, separating and purifying the enzymatic hydrolysate; the said separation and purification includes fractionating the enzymatic hydrolysate using nanofiltration and ultrafiltration; by subjecting the enzymatic hydrolysate to nanofiltration and ultrafiltration, a fraction with a molecular weight of 200 to 3000 Da is obtained; S2. Identifying the sequence of the sea cucumber gonad peptides; S3. Performing molecular docking of the sea cucumber gonad peptides with receptor SOD to screen for antioxidant peptides.

3. The preparation method according to claim 2, characterized in that, In step S2: Polypeptide sequence analysis is performed using LC-MS / MS.

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

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