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

Abstract

The invention belongs to the technical field of food biology, and particularly relates to sea cucumber gonad-derived anti-inflammatory peptide as well as a preparation method and application thereof. The anti-inflammatory peptide is obtained by performing enzymolysis and molecular docking screening on sexual glands of sea cucumbers, and comprises at least one of peptides with amino acid sequences as shown in SEQ.ID.NO.1-3. Experiments prove that the anti-inflammatory peptide has high anti-inflammatory activity, provides a basis for preparing functional food from natural food sources, and has important significance for development of novel anti-inflammatory products.

Description

Technical Field

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

[0002] Inflammation is an important progressive process mainly characterized by a defensive response after the body is damaged by inflammatory factors. When the body has chronic inflammation, the body's immune tolerance is damaged, activating relevant immune cells, which release inflammatory mediators. Inflammatory mediators stimulate immune cells to secrete excessive pro-inflammatory cytokines by activating intracellular and extracellular inflammatory signaling pathways, and pro-inflammatory cytokines promote cellular inflammatory responses. Excessive immune responses may ultimately lead to tissue damage and cause great harm to the human body.

[0003] Anti-inflammatory peptides are a class of small peptides with anti-inflammatory activity, which can reduce inflammatory symptoms by regulating the inflammatory response. They have the characteristics of easy absorption, good stability, no immunoreactivity, etc., and at the same time have nutritional and various biological activity functions, such as regulating immunity and promoting tissue repair. Natural anti-inflammatory peptides have application prospects in the fields of medicine, cosmetics and food due to their natural, safe, non-toxic, high nutritional value and other characteristics, and have become one of the important natural products in the fields of functional foods and biomedicines. Developing anti-inflammatory peptides with high anti-inflammatory activity and safety from low-cost biological resources has always been a research hotspot in the field of polypeptides. In the prior art, natural anti-inflammatory peptides are usually isolated from plant foods such as soybeans, citrus fruits, and amaranth by means of chromatography and ultrafiltration, or anti-inflammatory active peptides are prepared by enzymatic methods or microbial fermentation methods.

[0004] A large number of by-products are generated during sea cucumber processing, such as viscera, gonads, etc. It has been found that the sea cucumber gonad has a high protein content and is also rich in active ingredients such as sea cucumber polysaccharides. The development and utilization degree of sea cucumber gonads is low. At present, no commercial products developed from sea cucumber gonads have been found in the domestic market. In recent years, homology modeling and molecular docking have been used for the screening of active peptides, providing an effective way for the discovery of active peptides. Therefore, exploring the interaction between anti-inflammatory peptides derived from sea cucumber gonads and anti-inflammatory receptors TLR2 and TLR4 and finding its mechanism can provide a new way for the high-value utilization of sea cucumber gonads. Summary of the Invention

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

[0006] The specific technical solutions are as follows: One of the objects of the present invention is to provide an anti-inflammatory peptide derived from sea cucumber gonad, and the anti-inflammatory peptide derived from sea cucumber gonad includes at least one of the peptides shown in the amino acid sequences SEQ.ID.NO.1 - 3.

[0007] Among them, SEQ.ID.NO.1 is PSNLGTGLR, whose docking energy with the anti-inflammatory receptor TLR2 is -7.3 kcal / mol, and whose docking energy with the anti-inflammatory receptor TLR4 is -7.6 kcal / mol.

[0008] Among them, SEQ.ID.NO.2 is GDRGF, whose docking energy with the anti-inflammatory receptor TLR2 is -8.0 kcal / mol, and whose docking energy with the anti-inflammatory receptor TLR4 is -7.0 kcal / mol.

[0009] Among them, SEQ.ID.NO.3 is FDGPEGPRGPPGSEGRQG, whose docking energy with the anti-inflammatory receptor TLR2 is -7.5 kcal / mol, and whose docking energy with the anti-inflammatory receptor TLR4 is -7.3 kcal / mol.

[0010] Specifically, the sea cucumber gonad is the mixed male and female gonad of Apostichopus japonicus ( Apostichopus japonicu )

[0011] The second object of the present invention is to provide a preparation method of the above anti-inflammatory peptide, which comprises the following steps: S1. Obtain sea cucumber gonad peptide; S2. Identify the sequence of the sea cucumber gonad peptide; S3. Perform molecular docking of the sea cucumber gonad peptide with the receptor TLR2 and / or TLR4 to screen the anti-inflammatory peptide.

[0012] Furthermore, in step S1: The sea cucumber gonad peptide is obtained by enzymolysis of the sea cucumber gonad.

[0013] Specifically, in step S1, the working conditions of enzymolysis preferably include: adding pepsin to the raw material to be treated for enzymolysis, and then adding flavor protease for enzymolysis.

[0014] More specifically, in step S1, the working conditions of enzymolysis preferably include: adding pepsin to the raw material to be treated, 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.

[0015] Among them, the dosage of pepsin is preferably 1000 - 3000 U / g based on the raw material to be treated.

[0016] Among them, the dosage of flavor protease is preferably 1000 - 2000 U / g based on the raw material to be treated.

[0017] Specifically, in step S1: Before enzymolysis of sea cucumber gonads, it is preferably pretreated. The pretreatment includes: after homogenizing the sea cucumber gonads, heating in a boiling water bath for 10 - 30 min.

[0018] Furthermore, in step S1: After enzymolysis, the enzymolysis solution is separated and purified.

[0019] Even further, in step S1: The separation and purification includes fractionating the enzymolysis solution using nanofiltration and ultrafiltration. Nanofiltration can remove salts and free amino acids, and ultrafiltration can remove macromolecular substances. Specifically, it is preferably to obtain a fraction with a molecular weight of 200 - 3000 Da by subjecting the enzymolysis solution to nanofiltration and ultrafiltration.

[0020] Furthermore, in step S2: LC-MS / MS is used for polypeptide sequence analysis, and all peptide sequences are obtained through comparison and analysis with a database. The mass spectrometry database search software is MaxQuant 2.4.14.0, and the sample use database is the uniprot protein database.

[0021] Specifically, in step S2: It is preferably to desalt the product obtained in step S1 first and then perform polypeptide sequence analysis. It is preferably to use a C18 StageTip chromatographic column for desalting.

[0022] Furthermore, in step S3: It is preferably to use vina - 2.0 inside the pyrx software for molecular docking to screen for antioxidant peptides. The Affinity value represents the binding ability between the two. The lower the docking energy, the more stable the binding between the ligand and the receptor.

[0023] The third object of the present invention is to provide the application of the above-mentioned sea cucumber gonad-derived anti-inflammatory peptide in food, cosmetics or drugs. The food is preferably a functional food.

[0024] The fourth object of the present invention is to provide the application of the above-mentioned sea cucumber gonad-derived anti-inflammatory peptide in the preparation of anti-inflammatory functional foods or anti-inflammatory drugs.

[0025] The beneficial effects of the present invention are as follows: Through enzymatic hydrolysis and molecular docking screening, three anti-inflammatory peptides were obtained from sea cucumber gonads. Among them, the docking energy of SEQ.ID.NO.1 with the anti-inflammatory receptor TLR2 is -7.3 kcal / mol, and the docking energy with the anti-inflammatory receptor TLR4 is -7.6 kcal / mol; the docking energy of SEQ.ID.NO.2 with the anti-inflammatory receptor TLR2 is -8.0 kcal / mol, and the docking energy with the anti-inflammatory receptor TLR4 is -7.0 kcal / mol; the docking energy of SEQ.ID.NO.3 with the anti-inflammatory receptor TLR2 is -7.5 kcal / mol, and the docking energy with the anti-inflammatory receptor TLR4 is -7.3 kcal / mol. Through experimental verification, the above anti-inflammatory peptides have high anti-inflammatory activity, providing a basis for the preparation of functional foods from natural food sources and having important significance for the development of new anti-inflammatory products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a molecular docking diagram of the anti-inflammatory peptide from sea cucumber gonads with the amino acid sequence shown in SEQ.ID.NO.1 and the receptor TLR2; Figure 2 It is a molecular docking diagram of the anti-inflammatory peptide from sea cucumber gonads with the amino acid sequence shown in SEQ.ID.NO.2 and the receptor TLR2; Figure 3 It is a molecular docking diagram of the anti-inflammatory peptide from sea cucumber gonads with the amino acid sequence shown in SEQ.ID.NO.3 and the receptor TLR2; Figure 4 It is a molecular docking diagram of the anti-inflammatory peptide from sea cucumber gonads with the amino acid sequence shown in SEQ.ID.NO.1 and the receptor TLR4; Figure 5 It is a molecular docking diagram of the anti-inflammatory peptide from sea cucumber gonads with the amino acid sequence shown in SEQ.ID.NO.2 and the receptor TLR4; Figure 6 It is a molecular docking diagram of the anti-inflammatory peptide from sea cucumber gonads with the amino acid sequence shown in SEQ.ID.NO.3 and the receptor TLR4; Figure 7 It is the effect of three peptides on the relative proliferation rate of RAW264.7 cells in the test; Figure 8 It is the effect of three peptides on the NO secretion of RAW264.7 cells in the test. DETAILED DESCRIPTION OF THE INVENTION

[0027] The principles and features of the present invention will be described below in conjunction with examples. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0028] In the detailed description: The pepsin used was purchased from Sangon Biotech; the flavor protease used was purchased from Solarbio.

[0029] In the specific implementation: The sea cucumber gonads used are the mixed male and female gonads of Apostichopus japonicus ( Apostichopus japonicu ). Example

[0030] To prepare anti-inflammatory peptides from sea cucumber gonads, the steps are as follows: S1. Prepare sea cucumber gonad peptides: (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; (2) Raw material enzymatic hydrolysis: Add pepsin at 2000 U / g based on its mass to the homogenate obtained in step (1), adjust the pH to 2.5, and enzymatically hydrolyze 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 enzymatically hydrolyze at 50°C for 2 h, and then boil at 100°C to inactivate the enzyme; then centrifuge at 5000 r / min for 15 min, take the supernatant to obtain the enzymatic hydrolysate; (3) Purification: Perform 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 200 - 3000 Da fraction to obtain sea cucumber gonad peptide powder, and store it at -20°C for later use.

[0031] S2. Identify the sequence of sea cucumber gonad peptides: Desalt the peptide powder obtained in step S1 using a C18 StageTip chromatographic column, and perform polypeptide sequence analysis using LC-MS / MS. Conduct comparative 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.

[0032] S3. Perform molecular docking of sea cucumber gonad peptides with anti-inflammatory receptors TLR2 and TLR4: Obtain the SDF format file of the main active ingredient of the core drug through the Pubchem database, collect the key target protein structures in the PDB database, use the Pymol-2.1.0 software to optimize the target by removing water molecules and small molecule ligands, etc., and perform hydrogenation and charge processing using AutoDock Tools-1.5.6 and save it as the pdbqt format.

[0033] Using the key target as the receptor and its corresponding active ingredient as the ligand, molecular docking was performed using vina-2.0 inside the pyrx software to calculate the binding energy and output the result file. Finally, the results were visualized using the PyMol software. The Affinity (kcal / mol) value represents the binding ability between the two. The lower the docking energy, the more stable the binding between the ligand and the receptor. Visual analysis was performed using PyMol, and the 2D graph was visualized using Discovery Studio 2020 Client.

[0034] Three anti-inflammatory peptides and their docking energies were screened. The amino acid sequences of the three anti-inflammatory peptides are shown in SEQ.ID.NO.1 - 3 in sequence. The amino acid sequences of the three anti-inflammatory peptides and their corresponding docking energies are shown in Table 1.

[0035] Table 1 Amino acid sequences and docking energies of anti-inflammatory peptides from sea cucumber gonads Anti-inflammatory peptide Sequence TLR2 docking energy (kcal / mol) TLR4 docking energy (kcal / mol) SEQ.ID.NO.1 PSNLGTGLR -7.3 -7.6 SEQ.ID.NO.2 GDRGF -8.0 -7.0 SEQ.ID.NO.3 FDGPEGPRGPPGSEGRQG -7.5 -7.3 The molecular docking results of the peptides with amino acid sequences shown in SEQ.ID.NO.1 - 3 and the receptor TLR2 are shown in Figures 1 - 3 sequentially. The molecular docking results of the peptides with amino acid sequences shown in SEQ.ID.NO.1 - 3 and the receptor TLR4 are shown in Figures 4 - 6 sequentially. Through Figures 1 - 6 the binding modes and binding sites of the anti-inflammatory peptides and anti-inflammatory receptors shown, it can be seen that the binding of the anti-inflammatory peptides to TLR2 and TLR4 is mainly through interactions such as hydrogen bonds, carbon-hydrogen bonds, alkyl groups, and π-alkyl groups generated with the amino acid residues of the receptor.

[0036] As Figure 1 shown, the binding sites of the peptide with amino acid sequence shown in SEQ.ID.NO.1 and TLR2 are SER-424, SER-445, ASN-467, ARG-486, TRP-535, THR-532, LYS-561, ARG-508, ARG-447, LYS-422. As Figure 2 shown, the binding sites of the peptide with amino acid sequence shown in SEQ.ID.NO.2 and TLR2 are TYR-326, PHE-325, PHE-349, SER-346, LYS-347, VAL-348, LEU-266, PHE-284, LEU-289, LEU-317. As Figure 3As shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.3 to TLR2 are LYS-561, ASN-533, THR-532, ASN-487, HIS-398, ASN-397, GLN-396, LYS-422, SER-445, ARG-447, SER-424, GLY-566, SER-563, ARG-508, ASN-370, ASN-466, LEU-371, ARG-486. As Figure 4 As shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.1 to TLR4 are ASP-354, HIS-305, ASN-330, SER-334, LEU-249, ASN-248, LEU-216, ILE-378, ALA-301. As Figure 5 As shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.2 to TLR4 are ASN-330, LEU-302, SER-303, HIS-305, TYR-275, GLU-217, THR-250, LEU-249, ALA-301. As Figure 6 As shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.3 to TLR4 are THR-332, ILE-278, SER-277, SER-219, HIS-193, SER-279, LYS-166, ASN-280, GLU-217, HIS-305, ASN-253, VAL-195, PHE-123, ASP-354, MET-381. Test

[0037] According to the mass spectrometry sequencing results, the above three peptide sequences were chemically synthesized and tested, and their effects on the relative proliferation rate of RAW264.7 cells and the NO secretion of RAW264.7 cells were tested.

[0038] 1. Determination of the relative proliferation rate of RAW264.7 cells When the density of RAW264.7 cells reached 80%, the cells were resuspended with complete medium, the cells were mixed and counted, and the cell density was adjusted to 6×10 5 cells / mL. 100 μL of the cell suspension was added to each well of a 96-well cell culture plate, and 100 μL of complete medium was added to the blank group. In 5% CO 2Cultivate at 37°C in an incubator. After 24 h, when the cells are completely adherent, remove the supernatant. Add 100 μL of complete medium to the blank group and the normal group. To each well in the experimental group, add 100 μL of solutions of different concentrations of PSNLGTGLR, GDRGF, and FDGPEGPRGPPGSEGRQG (25, 50, 100, 200, and 400 μg / mL) prepared with complete medium. To prevent evaporation of the cell supernatant and affect the experimental results, add 200 μL of PBS to each well in the outermost periphery of the 96-well plate. After continuing to culture in the incubator for 24 h, according to the operation instructions of the CCK-8 kit, add 100 μL of CCK-8 culture solution containing 10% to each well, incubate in a 37°C incubator for 1 h, measure the absorbance value (OD) at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Set 5 replicate wells for the experiment, calculate the relative proliferation rate of RAW264.7 cells, and screen the experimental concentration of the sample. The relative proliferation rate of cells is calculated according to the following formula: Relative cell proliferation rate (%) = (ODexperimental group - ODblank group) / (ODnormal group - ODblank group) × 100%.

[0039] The effects of the 3 synthetic peptides on the relative proliferation rate of RAW264.7 cells are shown in Figure 7 . Synthetic peptides at different concentrations (50 - 400 μg / mL) have a proliferative effect on RAW264.7 cells, and the proliferative effect increases with the increase in peptide concentration. Among them, GDRGF has the best effect on promoting cell proliferation. At a concentration of 400 μg / mL, the relative proliferation rate of cells reaches 151.45 ± 2.33%. Thus, the 3 synthetic peptides have no toxic effect on cells within the concentration range of 50 - 400 μg / mL, and the concentration range of 100 - 400 μg / mL is selected for subsequent experiments.

[0040] 2. Determination of NO secretion in RAW264.7 cells Use a NO kit for determination. The experimental grouping and operation steps are the same as those for the determination of the relative proliferation rate of RAW264.7 cells. After culturing for 24 h, collect the cell culture medium. According to the instructions of the NO kit, add 50 μL of the centrifuged cell supernatant of each group to each well in a 96-well plate, and sequentially add 50 μL of Griess Reagent I and Griess Reagent II, and measure the absorbance at 540 nm. Measure and plot the standard curve of nitrite. According to the standard curve equation Y = 0.0051X - 0.0067, R 2 = 0.9991 to calculate the content of sodium nitrite in the cell culture medium, thereby reflecting the level of NO secreted by cells.

[0041] The effects of different bioactive peptides on inflammation were evaluated by detecting the content of NO in LPS-induced RAW264.7 macrophages. The effects of three synthetic peptides on the NO secretion of RAW264.7 cells are shown in Figure 8 . As Figure 8 shown, LPS stimulation significantly increased the production of NO (28.36 ± 0.856 μmol / L), while after intervention with PSNLGTGLR, GDRGF, and FDGPEGPRGPPGSEGRQG, the NO levels were all significantly decreased (P < 0.05), indicating that these three peptides can effectively inhibit the LPS-induced inflammatory response. Based on the above test results, the three anti-inflammatory peptides can be applied to the preparation of anti-inflammatory functional foods or anti-inflammatory drugs.

[0042] 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 within the protection scope of the present invention.

Claims

1. A sea cucumber gonad-derived anti-inflammatory peptide, characterized in that: It includes at least one of the peptides whose amino acid sequences are shown in SEQ.ID.NO.1~3.

2. A method for preparing the sea cucumber gonad-derived anti-inflammatory 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. Perform molecular docking of sea cucumber gonadal peptides with receptors TLR2 and / or TLR4 to screen anti-inflammatory 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 2, characterized in that: In step S2: peptide sequence analysis is performed using LC-MS / MS.

8. Use of the sea cucumber gonad-derived anti-inflammatory peptide as claimed in claim 1 in food, cosmetics or medicine.

9. The use according to claim 8, characterized in that: The food is a functional food.

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

Images