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

Abstract

The invention discloses sea cucumber gonad-derived antioxidant peptide as well as a preparation method and application thereof, and belongs to the technical field of food biology. The antioxidant peptide is obtained by carrying out enzymolysis and molecular docking screening on sea cucumber gonads, and comprises at least one of peptides with amino acid sequences as shown in SEQ.ID.NO.1-4. Wherein the docking energy of the SEQ.ID.NO.1 and the receptor SOD is-7.5 kcal / mol, the docking energy of the SEQ.ID.NO.2 and the receptor SOD is-7.5 kcal / mol, the docking energy of the SEQ.ID.NO.3 and the receptor SOD is-7.4 kcal / mol, and the docking energy of the SEQ.ID.NO.4 and the receptor SOD is-7.2 kcal / mol. Tests prove that the antioxidant peptide has high antioxidant activity and has wide application space in the field of functional food.

Description

Technical Field

[0001] The invention belongs to the field of food biotechnology, and specifically relates to a sea cucumber gonad-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 characteristics of "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] 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). SOD enzyme can effectively remove free radicals in the body that damage health and cause diseases, decompose them into oxygen molecules and water molecules that are harmless to the human body, and smoothly excrete them from the body. It plays 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 receptors SOD, providing an effective way for the screening and discovery of antioxidant peptides.

[0004] A large number of by-products are produced during the processing of sea cucumbers, such as viscera and gonads. Studies have found that sea cucumber gonads have a high protein content and are rich in active ingredients such as sea cucumber polysaccharides. The development and utilization of sea cucumber gonads is low. At present, no commercial products developed using 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 mechanism can provide a basis for the preparation of functional foods from sea cucumber-derived antioxidant peptides in natural food sources. Summary of the invention

[0005] The present invention provides a sea cucumber gonad-derived antioxidant peptide and a preparation method and 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: One of the purposes of the present invention is to provide sea cucumber gonad-derived antioxidant peptides, which include 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 VPYPR, and its docking energy with the antioxidant receptor SOD is -7.5 kcal / mol.

[0008] Among them, SEQ.ID.NO.2 is ATGPQGPAGQRGPAGPTGPTGPAG, and its docking energy with the antioxidant receptor SOD is -7.5 kcal / mol.

[0009] Among them, SEQ.ID.NO.3 is PGHPF, and its docking energy with the antioxidant receptor SOD is -7.4 kcal / mol.

[0010] Among them, SEQ.ID.NO.4 is NPWGQ, and its docking energy with the antioxidant receptor SOD is -7.2 kcal / mol.

[0011] Specifically, the sea cucumber gonads are mixed male and female gonads imitating those of Apostichopus japonicus.

[0012] The second object of the present invention is to provide a method for preparing the above-mentioned sea cucumber gonad-derived antioxidant peptide, comprising the following steps: S1. Obtaining sea cucumber gonadal peptides; S2. Sequence identification of sea cucumber gonadal peptides; S3. Molecular docking of sea cucumber gonadal peptide and receptor SOD was performed to screen antioxidant peptides.

[0013] Further, in step S1: sea cucumber gonad peptides are obtained by enzymatic hydrolysis of sea cucumber gonads.

[0014] Specifically, in step S1, the working conditions of enzymatic hydrolysis preferably include: adding pepsin to the raw material to be processed for enzymatic hydrolysis, and then adding flavor protease for enzymatic hydrolysis.

[0015] More specifically, in step S1, the working conditions of enzymatic hydrolysis preferably include: adding pepsin to the raw material to be processed, adjusting the pH to 1.5-3.5, and performing enzymatic hydrolysis at 36-38°C for 2-5 h; then adding flavor protease, adjusting the pH to 6.0-8.0, and performing enzymatic hydrolysis at 45-55°C for 1-3 h to inactivate the enzyme.

[0016] The dosage of pepsin is preferably 1000-3000 U / g based on the raw material to be processed.

[0017] The amount of flavor protease used is preferably 1000-2000 U / g based on the raw material to be processed.

[0018] Specifically, in step S1: before enzymatic hydrolysis of the sea cucumber gonad, it is preferably pretreated. The pretreatment comprises: homogenizing the sea cucumber gonad and heating it in a boiling water bath for 10 to 30 minutes.

[0019] Furthermore, in step S1: after enzymatic hydrolysis, the enzymatic hydrolyzate is separated and purified.

[0020] Furthermore, in step S1: the separation and purification includes classifying the enzymatic solution by nanofiltration and ultrafiltration. Nanofiltration can remove salts and free amino acids, and ultrafiltration can remove macromolecular substances. Specifically, it is preferred to obtain a component with a molecular weight of 200 to 3000 Da by nanofiltration and ultrafiltration of the enzymatic solution.

[0021] Further, in step S1: after obtaining the sea cucumber gonadal peptide, an in vitro SOD activity assay is performed on it.

[0022] Further, in step S2: peptide sequence analysis was performed using LC-MS / MS, and all peptide sequences were obtained by comparison analysis through the database. The mass spectrometry database retrieval software was MaxQuant 2.4.14.0, and the sample database used was the uniprot protein database.

[0023] 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.

[0024] Further, in step S3: preferably, VINA-2.0 in the Pyrx software is used for molecular docking to screen antioxidant peptides. The Affinity value represents the binding ability between the two. The lower the binding ability, the more stable the binding between the ligand and the receptor.

[0025] The third object of the present invention is to provide the use of the sea cucumber gonad-derived antioxidant peptide in food, which is preferably a functional food.

[0026] A fourth object of the present invention is to provide the use of the above-mentioned sea cucumber gonad-derived antioxidant peptides in the preparation of antioxidant functional foods or antioxidant drugs.

[0027] The beneficial effects of the present invention are as follows: The present invention obtains four antioxidant peptides by enzymatic hydrolysis and molecular docking screening of sea cucumber gonads. Among them, the docking energy of SEQ.ID.NO.1 and the receptor SOD is -7.5 kcal / mol, the docking energy of SEQ.ID.NO.2 and the receptor SOD is -7.5 kcal / mol, the docking energy of SEQ.ID.NO.3 and the receptor SOD is -7.4 kcal / mol, and the docking energy of SEQ.ID.NO.4 and the receptor SOD is -7.2 kcal / mol. It has been verified by experiments that the above antioxidant peptides have high antioxidant activity and have broad application space in the field of functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1It is a molecular docking diagram of the sea cucumber gonad-derived antioxidant peptide and the receptor SOD as shown in the amino acid sequence SEQ.ID.NO.1; Figure 2 It is a molecular docking diagram of the sea cucumber gonad-derived antioxidant peptide and the receptor SOD as shown in the amino acid sequence SEQ.ID.NO.2; Figure 3 It is a molecular docking diagram of the sea cucumber gonad-derived antioxidant peptide and the receptor SOD as shown in the amino acid sequence SEQ.ID.NO.3; Figure 4 It is a molecular docking diagram of the sea cucumber gonad-derived antioxidant peptide and the receptor SOD with the amino acid sequence shown in SEQ.ID.NO.4; Figure 5 The activation ability of the sea cucumber sex gland peptide obtained in step S1 of the embodiment on SOD; Figure 6 The ability of four antioxidant peptides and the control group GSH to scavenge DPPH free radicals; Figure 7 The ability of four antioxidant peptides and the control group GSH to scavenge ABTS free radicals. DETAILED DESCRIPTION

[0029] 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.

[0030] In the specific implementation manner: the pepsin used was purchased from Shenggong Biotechnology; the flavor protease used was purchased from Solebao Biotechnology.

[0031] In a specific embodiment, the sea cucumber gonads used are mixed male and female gonads of Apostichopus japonicus. Example

[0032] The steps for preparing sea cucumber gonad-derived antioxidant peptides are as follows: S1. Preparation of sea cucumber gonadal peptide: (1) Raw material pretreatment: After the sea cucumber gonad tissue is homogenized, it is heated in a boiling water bath for 15 min to obtain a homogenate, which is then cooled for later use; (2) Enzymatic hydrolysis of raw materials: add 2000 U / g of pepsin based on the mass of the homogenate obtained in step (1), adjust the pH to 2.5, and perform enzymatic hydrolysis at 37°C for 4 h; then add 1200 U / g of flavor protease based on the mass of the homogenate, adjust the pH to 7.0, and perform enzymatic hydrolysis at 50°C for 2 h, then boil at 100°C to inactivate the enzyme; then centrifuge at 5000 r / min for 15 min, collect the supernatant, and obtain the enzymatic hydrolyzate; (3) Purification: The enzymatic hydrolysate obtained in step (2) is subjected to nanofiltration and ultrafiltration classification. A 200 Da nanofiltration membrane is first used to remove salt and free amino acids, and then a 3000 Da spiral membrane is selected for ultrafiltration. The obtained 200-3000 Da components are freeze-dried to obtain sea cucumber sex gland peptide powder, which is stored at -20 °C for future use; (4) Determination of SOD activity of sea cucumber sex gland peptide in vitro: the mass concentration gradient of the peptide powder obtained in step (3) was set to 1, 2, 3, 4, and 5 mg / mL, and the self-oxidation rate of pyrogallol was measured. The self-oxidation rate was controlled at 0.07 (±0.002) OD / min. When measuring the sample, the Tris-HCl buffer, the sample to be tested and the SOD solution were mixed, placed in a 25°C water bath for 10 min, and then the pyrogallol solution was added for thorough mixing and timing. The detection was carried out at a wavelength of 325 nm, and the reading was taken once every 30 s. The continuous reading time was 3 min. The test results are shown in Figure 5 ;Depend on Figure 5 It can be seen that within the dosage range of 1~5 mg / mL, sea cucumber gonadal peptide showed an activating effect on SOD and showed a significant dose-effect relationship.

[0033] S2. Sequence identification of sea cucumber gonadal peptides: The peptide powder obtained in step S1 was desalted using a C18 StageTip column, and the peptide sequence was analyzed using LC-MS / MS. The entire peptide sequence was obtained by comparison analysis through the database; the mass spectrometry database retrieval software was MaxQuant2.4.14.0, and the sample database used was the uniprot protein database.

[0034] S3. Molecular docking of sea cucumber gonadal peptide and receptor SOD: The SDF format files of the main active ingredients of the core drugs were obtained through the Pubchem database, and the key target protein structures were collected in the PDB database. The targets were optimized by removing water molecules and small molecule ligands using Pymol-2.1.0 software, and hydrogenation and charge treatment were performed using AutoDock Tools-1.5.6 and saved as pdbqt format. The key targets were used as receptors, and the corresponding active ingredients were used as ligands. Molecular docking was performed using vina-2.0 within the pyrx software to calculate the binding energy and output the result file. Finally, the results were visualized using PyMol software. Its Affinity (kcal / mol) value represents the binding ability of the two. The lower the binding ability, the more stable the binding between the ligand and the receptor. Pymol was used for visualization analysis, and the 2D graph was visualized using Discovery Studio 2020 Client. Four antioxidant peptides and their docking energies were screened and obtained. The amino acid sequences of the four antioxidant peptides are shown in SEQ.ID.NO.1~4. The amino acid sequences of the four antioxidant peptides and the corresponding docking energies are shown in Table 1.

[0035] Table 1 Amino acid sequence and docking energy of sea cucumber gonad-derived antioxidant peptides Antioxidant Peptides 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 QUR -7.2 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 to 4 .like Figures 1 to 4 It can be seen that the binding of antioxidant peptides to SOD subunits is mainly through hydrogen bonding and electrostatic interactions with receptor amino acid residues.

[0036] like Figure 1 As shown, the binding sites of the peptide shown in SEQ.ID.NO.1 and 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); Figure 2 As shown, the binding sites of the peptide shown in SEQ.ID.NO.2 and SOD subunits 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), and PRO (A: 61); Figure 3As shown, the binding sites of the peptide shown in SEQ.ID.NO.3 and SOD subunits are VAL (A: 80), ARG (A: 78), GLY (A: 84), HIS (A: 45), ASP (A: 82); the binding sites of the peptide shown in SEQ.ID.NO.4 and SOD subunits 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 and SOD subunits are mainly ARG (A: 145), GLN (A: 122), ASP (A: 123), ASN (A: 85), ARG (A: 78), ALA (A: 144), HIS (A: 119). test

[0037] According to the results of mass spectrometry sequencing, Jiangsu GenScript Biotechnology Co., Ltd. was commissioned to chemically synthesize the above four peptide sequences, and then the DPPH method and ABTS method were used to verify their antioxidant activities, with the same concentration of reduced glutathione (GSH) as the control, and the test concentration was 5 mg / mL. Figure 6 As shown, the ability of the four peptides to scavenge ABTS free radicals compared with the control group GSH is as follows Figure 7 shown. Figure 6 , Figure 7 In the figure, the abscissa represents the corresponding peptide sequence, wherein AT-AG represents the peptide shown in SEQ.ID.NO.2.

[0038] The test results show that the polypeptides with amino acid sequences as shown in SEQ.ID.NO.1~4 all have a certain ability to scavenge DPPH free radicals and ABTS free radicals, and the ability of the polypeptide (VPYPR) shown in SEQ.ID.NO.1 and the polypeptide (NPWGQ) shown in SEQ.ID.NO.4 to scavenge ABTS free radicals is stronger than that of the control group GSH.

[0039] 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 gonad-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. A method for preparing the sea cucumber gonad-derived antioxidant peptide according to claim 1, characterized in that: The steps include: S1. Obtaining sea cucumber gonadal peptides; S2. Sequence identification of sea cucumber gonadal peptides; S3. Molecular docking of sea cucumber gonadal peptide and receptor SOD was performed to screen antioxidant peptides.

3. The preparation method according to claim 2, characterized in that: In step S1: sea cucumber gonad peptides are obtained by enzymatic hydrolysis of sea cucumber gonads.

4. The preparation method according to claim 3, characterized in that: In step S1, the working conditions of enzymatic hydrolysis include: adding pepsin to the raw material to be processed for enzymatic hydrolysis, and then adding flavor protease for enzymatic hydrolysis.

5. The preparation method according to claim 3, characterized in that: In step S1: after enzymolysis, the enzymolysis solution is separated and purified.

6. The preparation method according to claim 5, characterized in that: In step S1: the separation and purification includes classifying the enzymatic hydrolysate using nanofiltration and ultrafiltration.

7. The preparation method according to claim 6, characterized in that: In step S1: the enzymatic hydrolysate is subjected to nanofiltration and ultrafiltration to obtain a component with a molecular weight of 200-3000 Da.

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

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

10. Use of the sea cucumber gonad-derived antioxidant peptide as claimed in claim 1 in the preparation of antioxidant functional foods or antioxidant drugs.

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