Abalone-derived immunomodulatory peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food biology, and discloses an abalone-derived immunomodulatory peptide as well as a preparation method and application thereof. The abalone source immunomodulatory peptide is obtained by combining double-enzyme enzymolysis and molecular docking screening, and comprises at least one of peptides with amino acid sequences shown as SEQ.ID.NO.1-6, and the docking energy of the peptides and a receptor TLR4 is-7.4 kcal / mol,-6.9 kcal / mol,-6.8 kcal / mol,-6.8 kcal / mol,-6.8 kcal / mol and-6.6 kcal / mol in sequence respectively. Experiments prove that the abalone peptide has high immunocompetence, can provide a basis for application of the abalone-derived immunomodulatory peptide in preparation of functional food from natural food sources, and has important significance for development of novel immunomodulatory peptide products.

Description

Technical Field

[0001] The present invention belongs to the field of food biotechnology, and particularly relates to an abalone-derived immunomodulatory peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Immunomodulatory peptides are a class of polypeptide molecules that can regulate the function of the immune system and participate in the processes of the body's defense, inflammation regulation, prevention and treatment of autoimmune diseases, etc. by activating or inhibiting immune responses. Immunomodulatory peptides have been widely used in the food field due to their natural, safe, non-toxic, high nutritional value and other characteristics. Developing immunomodulatory peptides with nutritional and safety properties using low-cost biological resources has always been a research hotspot in the polypeptide field.

[0003] At present, scientific researchers have extracted a variety of natural immunologically active peptides from various animals, plants, microorganisms and their by-products, and these peptides have been widely used in animal experiments and clinical medicine. The methods for preparing immunomodulatory peptides using natural proteins include acid hydrolysis, alkali hydrolysis, chemical hydrolysis, microbial hydrolysis, enzymatic hydrolysis, etc.

[0004] Toll-like receptors (TLRs) are key pattern recognition receptors in the immune system. TLR4 is an important member of TLRs, which is expressed on the cell surface and mainly recognizes microbial membrane components such as lipids, lipoproteins and proteins. In recent years, homology modeling and molecular docking have been widely used to reveal the binding sites between immunomodulatory peptides and their receptor TLR4, providing an effective way for the screening and discovery of immunomodulatory peptides. Therefore, exploring the interaction between abalone-derived immunomodulatory peptides and the TLR4 receptor and finding its immunomodulatory mechanism can provide a basis for the application of abalone immunomodulatory peptides in the preparation of functional foods from natural food sources. Summary of the Invention

[0005] The present invention provides an abalone-derived immunomodulatory peptide, a preparation method thereof, and an application thereof. The immunomodulatory peptide of the present invention is prepared and screened from the muscle tissue of Haliotis discus hannai and has significant immunomodulatory activity.

[0006] The specific technical solutions are as follows: One of the purposes of the present invention is to provide an abalone-derived immunomodulatory peptide, which comprises at least one of the peptides shown in the amino acid sequences SEQ.ID.NO.1~6.

[0007] Among them, SEQ.ID.NO.1 is FGDDSNNNPFYK, and its docking energy with the receptor TLR4 is -7.4 kcal / mol.

[0008] Among them, SEQ.ID.NO.2 is KPLQPFGLSSDVNPDIITR, and its docking energy with the receptor TLR4 is -6.9 kcal / mol.

[0009] Among them, SEQ.ID.NO.3 is DFNHNPFTK, and its docking energy with the receptor TLR4 is -6.8 kcal / mol.

[0010] Among them, SEQ.ID.NO.4 is AQSIEQLPMKPF, and its docking energy with the receptor TLR4 is -6.8 kcal / mol.

[0011] Among them, SEQ.ID.NO.5 is PLGPVGR, and its docking energy with the receptor TLR4 is -6.8 kcal / mol.

[0012] Among them, SEQ.ID.NO.6 is FSKPEDTFDYKK, and its docking energy with the receptor TLR4 is -6.6 kcal / mol.

[0013] Among them, the abalone is Haliotis discus hannai Haliotis discus hannai ), and the above-mentioned abalone-derived immunomodulatory peptides are prepared and screened from the muscle of Haliotis discus hannai.

[0014] The second object of the present invention is to provide a preparation method of the above-mentioned abalone-derived immunomodulatory peptides, comprising the following steps: S1. Obtain abalone peptides; S2. Identify the sequences of abalone peptides; S3. Perform molecular docking of abalone peptides with the receptor protein TLR4 to screen immunomodulatory peptides.

[0015] Furthermore, in step S1: abalone peptides are obtained by enzymatically hydrolyzing abalone raw materials.

[0016] Specifically, in step S1, the working conditions of enzymatic hydrolysis preferably include: adding pepsin to the abalone raw materials for enzymatic hydrolysis first, and then adding trypsin for enzymatic hydrolysis.

[0017] More specifically, in step S1, the working conditions of enzymatic hydrolysis preferably include: adding pepsin to the abalone raw materials, adjusting the pH to 1.5 - 3.5, enzymatically hydrolyzing at 36 - 38 °C for 2 - 5 h; then adding trypsin, adjusting the pH to 6.0 - 8.0, enzymatically hydrolyzing at 36 - 38 °C for 1 - 3 h, and inactivating the enzyme.

[0018] Among them, the dosage of pepsin is preferably 1000 - 3000 U / g based on the abalone raw materials.

[0019] Among them, the dosage of trypsin is preferably 1000 - 2000 U / g based on the abalone raw materials.

[0020] Specifically, in step S1: Before enzymatic hydrolysis of abalone raw materials, it is preferably pretreated. The pretreatment includes: After homogenizing the abalone raw materials, heat them in a boiling water bath for 10 - 30 min.

[0021] Further, in step S1: After enzymatic hydrolysis, the enzymatic hydrolysate is separated and purified.

[0022] 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, it is preferably to obtain a fraction with a molecular weight of 200 - 3000 Da by subjecting the enzymatic hydrolysate to nanofiltration and ultrafiltration.

[0023] Further, in step S2: Use LC - MS / MS for polypeptide sequence analysis, and conduct alignment analysis through a database to obtain all peptide sequences. The mass spectrometry database retrieval software is MaxQuant 2.4.14.0, and the sample use database is the uniprot protein database.

[0024] Specifically, in step S2: It is preferably to desalt the product obtained in step S1 first and then conduct polypeptide sequence analysis. Among them, it is preferably to use a C18 StageTip chromatographic column for desalting.

[0025] Further, in step S3: It is preferably to use vina - 2.0 inside the pyrx software for molecular docking to screen for immunomodulatory peptides. The Affinity value represents the binding ability between the two. The lower the docking energy, the more stable the ligand binds to the receptor.

[0026] The third object of the present invention is to provide the application of the above - mentioned abalone - derived immunomodulatory peptides in food, especially in functional food.

[0027] The fourth object of the present invention is to provide the application of the above - mentioned abalone - derived immunomodulatory peptides in the preparation of immunomodulatory functional food or immunomodulatory drugs.

[0028] The beneficial effects of the present invention are as follows: The present invention obtained six abalone - derived immunomodulatory peptides from abalone through double - enzyme hydrolysis combined with molecular docking screening. The docking energies of the immunomodulatory peptides shown in SEQ.ID.NO.1 - 6 with the receptor TLR4 are - 7.4 kcal / mol, - 6.9 kcal / mol, - 6.8 kcal / mol, - 6.8 kcal / mol, - 6.8 kcal / mol, and - 6.6 kcal / mol respectively. It has been experimentally proven that the above - mentioned abalone peptides have high immunological activity, which can provide a basis for the application of abalone - derived immunomodulatory peptides in the preparation of functional food from natural food sources and is of great significance for the development of new immunomodulatory peptide products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a molecular docking diagram of abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.1 and the receptor TLR4; Figure 2 It is a molecular docking diagram of abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.2 and the receptor TLR4; Figure 3 It is a molecular docking diagram of abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.3 and the receptor TLR4; Figure 4 It is a molecular docking diagram of abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.4 and the receptor TLR4; Figure 5 It is a molecular docking diagram of abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.5 and the receptor TLR4; Figure 6 It is a molecular docking diagram of abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.6 and the receptor TLR4; Figure 7 It is the effect of six synthetic peptides on the relative proliferation rate of RAW264.7 cells in the test; Figure 8 It is the effect of six synthetic peptides on the phagocytic ability of RAW264.7 cells in the test; Figure 9 It is the effect of six synthetic peptides on the NO secretion of RAW264.7 cells in the test. DETAILED DESCRIPTION OF THE INVENTION

[0030] 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.

[0031] In the detailed description: Pepsin used was purchased from Sangon Biotech; Trypsin used was purchased from Bomei Biotech. EXAMPLE

[0032] To prepare abalone-derived immunomodulatory peptide, the steps are as follows: S1. Preparation of abalone peptide: (1) Raw material pretreatment: After homogenizing the muscle tissue of Haliotis discus hannai, heat it in a boiling water bath for 15 min to obtain a homogenate, and let it cool for later use.

[0033] (2)Enzymatic hydrolysis: Add pepsin at 2000 U / g based on abalone muscle to the raw materials obtained in step (1), adjust the pH to 2.5, and hydrolyze at 37 °C for 4 h; then add trypsin at 1200 U / g based on abalone muscle, adjust the pH to 7.0, and hydrolyze at 37 °C for 2 h, and then inactivate the enzyme by boiling at 100 °C; then centrifuge at 7000 r / min for 10 min, take the supernatant to obtain the enzymatic hydrolysate.

[0034] (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 fraction of 200 - 3000 Da to obtain peptide powder, and store it at -20 °C for later use.

[0035] S2. Sequence identification of abalone peptides: Desalt the peptide powder obtained in step S1 using a C18 StageTip chromatographic column, and perform polypeptide sequence analysis using LC-MS / MS. Compare and analyze through a database to obtain all peptide sequences; the mass spectrometry database retrieval software is MaxQuant 2.4.14.0, and the sample uses the uniprot protein database.

[0036] S3. Molecular docking of abalone peptides with the receptor protein TLR4: Obtain the SDF format file of the main active ingredient of the core drug through the Pubchem database, collect the key target protein structure 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.

[0037] 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 to bind. The lower the docking energy, 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.

[0038] Six immunomodulatory peptides and their docking energies are screened. The amino acid sequences of the six immunomodulatory peptides are shown in SEQ.ID.NO.1 - 6 in sequence. The amino acid sequences of the six immunomodulatory peptides and their corresponding docking energies are shown in Table 1.

[0039] Table 1 Amino Acid Sequences and Docking Energies of Abalone-Derived Immunomodulatory Peptides Immunomodulatory peptide Peptide sequence TLR4 docking energy (kcal / mol) SEQ.ID.NO.1 FGDDSNNNPFYK -7.4 SEQ.ID.NO.2 KPLQPFGLSSDVNPDIITR -6.9 SEQ.ID.NO.3 DFNHNPFTK -6.8 SEQ.ID.NO.4 AQSIEQLPMKPF -6.8 SEQ.ID.NO.5 PLGPVGR -6.8 SEQ.ID.NO.6 FSKPEDTFDYKK -6.6 The molecular docking results of abalone-derived immunomodulatory peptides with the receptor TLR4, whose amino acid sequences are shown in SEQ.ID.NO.1-6, are shown in Figures 1 - 6 . From the binding modes and binding sites of the six immunomodulatory peptides and the receptor, it can be seen that the binding to TLR4 is mainly through interactions such as hydrogen bonds, carbon-hydrogen bonds, alkyl groups, and π-alkyl groups generated between the receptor amino acid residues.

[0040] As Figure 1 shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.1 to TLR4 are ASN-155, SER-182, ARG-233, ARG-337, LYS-360, LYS-263, GLY-315, SER-210, ARG-380, ASP-208, VAL-133, ALA-157, LEU-211, MET-358; as Figure 2 shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.2 to TLR4 are SER-182, SER-85, THR-109, ASN-155, ASP-83, ASP-208, ASN-105, TRP-81, ASN-57, LYS-153, HIS-178, THR-231, VAL-177, GLU-229, HIS-228, ASP-59, ASP-180, TYR-183, LEU-211, ALA-157, PHE-262, ARG-86; as Figure 3 shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.3 to TLR4 are ARG-380, ASP-377, THR-355, ASP-403, TYR-375, SER-334, GLU-286, SER-332, SER-353, GLN-423, THR-357, ILE-336, LYS-352; as Figure 4 shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.4 to TLR4 are SER-353, SER-309, ASP-403, SER-379, ARG-380, GLN-428, HIS-424, SER-332, ASP-377, TYR-375, LYS-352, ARG-400, ALA-310, ARG-288, MET-358, TYR-449; as Figure 5As shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.5 to TLR4 are TYR-375, THR-355, SER-334, SER-312, SER-353, SER-332, ASP-377, GLU-286, ALA-310; as Figure 6 shown, the binding sites of the peptide with the amino acid sequence shown in SEQ.ID.NO.6 to TLR4 are THR-473, ARG-380, SER-379, THR-357, ASN-472, HIS-424, HIS-401, LEU-448, PHE-498, MET-358. Test

[0041] According to the mass spectrometry sequencing results, the above six peptide sequences were chemically synthesized and tested, and their effects on the relative proliferation rate of RAW264.7 cells, the phagocytosis ability of RAW264.7 cells, and the NO secretion of RAW264.7 cells were tested.

[0042] 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, and after mixing the cells, cell counting was performed. The cell density was adjusted to 6×10 5 cells / mL. 100 μL of 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. The cells were cultured at 37°C in a 5% CO2 incubator. After 24 h, when the cells were completely adherent, the supernatant was removed. 100 μL of complete medium was added to the blank group and the control group, and 100 μL of solutions of each peptide at different concentrations (50, 100, 200, and 400 μg / mL) prepared with complete medium were added to each well of the experimental group. To prevent evaporation of the cell supernatant and affect the experimental results, 200 μL of PBS was added 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, 100 μL of CCK-8 culture solution containing 10% was added to each well, and the cells were incubated in a 37°C incubator for 1 h. The absorbance value (OD) at 450 nm was measured with an enzyme-linked immunosorbent assay reader. The experiment was set up with 5 replicates, and the relative proliferation rate of RAW264.7 cells was calculated to screen the experimental action concentration of the samples. The relative proliferation rate of the cells was calculated according to the following formula: Relative proliferation rate of cells (%) = (ODexperimental group - ODblank group) / (ODcontrol group - ODblank group) × 100%.

[0043] The effects of the six synthetic peptides on the relative proliferation rate of RAW264.7 cells are shown in Figure 7Synthetic peptides with concentrations ranging from 50 to 400 μg / mL all had a proliferative effect on RAW264.7 cells, and the proliferative effect increased with the increase in peptide concentration. Among them, PLGPVGR (SEQ.ID.NO.5) had the best effect on promoting cell proliferation. At a concentration of 400 μg / mL, the relative cell proliferation rate reached 150.22 ± 11.50%. Thus, it can be seen that the six synthetic peptides had no toxic effect on cells within the concentration range of 50 to 400 μg / mL, and the concentration range of 100 to 400 μg / mL was selected for subsequent experiments.

[0044] 2. Determination of the phagocytic ability of RAW264.7 cells When the RAW264.7 cells in the culture flask reached 80%, they were diluted with complete medium to a cell density of 2×10 5 cells / well. 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. The cells were cultured at 37 °C in a 5% CO2 incubator. After 24 h, the supernatant was discarded. 100 μL of complete medium was added to the blank group and the control group. Different concentrations of peptides (100, 200, and 400 μg / mL) prepared with 100 μL of complete medium were added to each well of the experimental group, and an equal volume of LPS (lipopolysaccharide O55:B5) solution with a final concentration of 1 μg / mL was added to the LPS group. After culturing for 24 h, the cell culture supernatant of each well was discarded. 100 μL of neutral red solution was added to each well and continued to be cultured in a 37 °C constant temperature incubator for 2 h and then taken out. After washing 3 times with PBS to remove the floating color, 100 μL of cell lysate was added to each well and lysed on a shaker at room temperature for 30 min. The absorbance value was measured at a wavelength of 540 nm. The experiment was set with 5 replicates, and the relative cell phagocytosis rate was calculated according to the following formula: Relative cell phagocytosis rate (%) = (ODexperimental group - ODblank group) / (ODcontrol group - ODblank group) × 100%.

[0045] The effects of the six synthetic peptides on the phagocytic ability of RAW264.7 cells are as Figure 8 shown. Compared with the control group, the six synthetic peptides could all improve the phagocytic ability of RAW264.7 cells, and the phagocytic ability of the cells increased significantly with the increase in concentration. PLGPVGR (SEQ.ID.NO.5) was the peptide with the strongest ability to promote cell phagocytosis among the 6 synthetic peptides. When the concentration was 400 μg / mL, its relative cell phagocytosis rate was up to 164 ± 3.92% at most, which was 0.64 times that of the control group, showing a strong immune stimulation effect.

[0046] 3. Determination of the NO secretion amount of RAW264.7 cells The experimental grouping and operation steps were the same as those for the determination of the phagocytic ability of RAW 264.7 cells. After culturing for 24 h, the cell culture medium was collected. According to the instructions of the NO kit, 50 μL of the centrifuged supernatant of each group of cells was added to each well of a 96-well plate. Then, 50 μL of Griess Reagent I and 50 μL of Griess Reagent II were added successively. The absorbance was measured at 540 nm. The standard curve of nitrite was measured and plotted. According to the standard curve equation Y = 0.0051x + 0.0006, R 2 = 0.9999, the sodium nitrite content in the cell culture medium was calculated to reflect the level of NO secreted by the cells.

[0047] The effects of different bioactive peptides on immune regulation were evaluated by detecting the content of NO in RAW264.7 macrophages. The effects of six synthetic peptides on the NO secretion of RAW264.7 cells are shown in Figure 9 . As Figure 9 shown, compared with the control group, the NO secretion in the LPS group was significantly increased ( P <0.05), and after intervention with each immunomodulatory peptide, the NO secretion was also significantly increased ( P <0.05), indicating that these six peptides can effectively stimulate the cells to release NO. NO can maintain the balance between host defense and self-protection by dynamically regulating the immune activation and immunosuppression of the body. An appropriate level of NO in the body can effectively enhance the immune capacity of the body. Based on the above test results, the six immunomodulatory peptides can be applied to the preparation of immune-regulating functional foods or immune-regulating drugs.

[0048] 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. An abalone-derived immunomodulatory peptide, characterized in that, At least one of the peptides comprising the amino acid sequences shown in SEQ.ID.NO.1 to 6.

2. The abalone-source immunomodulatory peptide according to claim 1, wherein The abalone is Haliotis discus hannai.

3. A method for preparing the abalone-derived immunomodulatory peptide as described in claim 1 or 2, characterized in that, Comprising the following steps: S1. Obtain abalone peptides; S2. Conduct sequence identification on the abalone peptides; S3. Perform molecular docking of the abalone peptides with the receptor protein TLR4 to screen for immunomodulatory peptides.

4. The preparation method according to claim 3, characterized in that, In step S1: The abalone peptides are obtained by enzymolysis of abalone raw materials.

5. The preparation method according to claim 4, characterized in that, In step S1, pepsin is first added to the raw material to be processed for enzymolysis, and then trypsin is added for enzymolysis.

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 fractionating the enzymolysis solution using ultrafiltration and nanofiltration.

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

8. Use of an abalone-derived immunomodulatory peptide as claimed in claim 1 or 2 in food.

9. The application according to claim 8, wherein The food is a functional food.

10. Use of an abalone-derived immunomodulatory peptide as claimed in claim 1 or 2 in the preparation of an immunomodulatory functional food or an immunomodulatory drug.

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