An abalone-derived immunomodulatory peptide, its preparation method and application

Abstract

This invention belongs to the field of food biotechnology and discloses an abalone-derived immunomodulatory peptide, its preparation method, and its application. The abalone-derived immunomodulatory peptide of this invention is obtained through a combination of dual-enzyme digestion and molecular docking screening. It includes at least one peptide with amino acid sequences as shown in SEQ ID NO. 1-6, and the docking energies 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. Experiments have demonstrated that the above-mentioned abalone peptide possesses high immunomodulatory activity, providing a foundation for its application in the preparation of functional foods from natural food sources, and is of great significance for the development of novel immunomodulatory peptide products.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology, specifically relating to an abalone-derived immunomodulatory peptide, its preparation method, and its application. Background Technology

[0002] Immunomodulatory peptides are a class of polypeptide molecules that can regulate the function of the immune system, participating in processes such as defense, inflammation regulation, and prevention and treatment of autoimmune diseases by activating or inhibiting immune responses. Due to their natural, safe, non-toxic, and highly nutritious characteristics, immunomodulatory peptides are widely used in the food industry. Developing nutritious and safe immunomodulatory peptides using low-cost biological resources has always been a research hotspot in the field of peptides.

[0003] Currently, researchers have extracted a variety of natural immunomodulatory peptides from various plants, animals, microorganisms, and their byproducts. These peptides are widely used in animal experiments and clinical medicine. Methods for preparing immunomodulatory peptides from natural proteins include acid hydrolysis, alkaline hydrolysis, chemical hydrolysis, microbial hydrolysis, and enzymatic hydrolysis.

[0004] Toll-like receptors (TLRs) are key pattern recognition receptors in the immune system. TLR4 is an important member of the TLR family, expressed on the cell surface, and primarily 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 approach for the screening and discovery of immunomodulatory peptides. Therefore, exploring the interaction between abalone-derived immunomodulatory peptides and the TLR4 receptor, and seeking their immunomodulatory mechanisms, can provide a foundation for the application of abalone immunomodulatory peptides in the preparation of functional foods from natural food sources. Summary of the Invention

[0005] This invention provides an abalone-derived immunomodulatory peptide, its preparation method, and its application. The immunomodulatory peptide of this invention is prepared and screened from the muscle tissue of *Abalone fasciatus* and exhibits significant immunomodulatory activity.

[0006] The specific technical solution is as follows:

[0007] One objective of this invention is to provide an abalone-derived immunomodulatory peptide, comprising at least one of the peptides with amino acid sequences as shown in SEQ ID NO. 1 to 6.

[0008] Among them, SEQ.ID.NO.1 is FGDDSNNNPFYK, and its docking energy with the receptor TLR4 is -7.4 kcal / mol.

[0009] Among them, SEQ.ID.NO.2 is KPLQPFGLSSDVNPDIITR, and its docking energy with the receptor TLR4 is -6.9 kcal / mol.

[0010] Among them, SEQ.ID.NO.3 is DFNHNPFTK, and its docking energy with the receptor TLR4 is -6.8 kcal / mol.

[0011] Among them, SEQ.ID.NO.4 is AQSIEQLPMKPF, and its docking energy with the receptor TLR4 is -6.8 kcal / mol.

[0012] Among them, SEQ.ID.NO.5 is PLGPVGR, whose docking energy with the receptor TLR4 is -6.8 kcal / mol.

[0013] Among them, SEQ.ID.NO.6 is FSKPEDTFDYKK, and its docking energy with the receptor TLR4 is -6.6 kcal / mol.

[0014] The abalone mentioned is the wrinkled abalone (Abalone fasciatus). Haliotis discus hannai The above-mentioned abalone-derived immunomodulatory peptides were prepared and screened from the muscle of the wrinkled abalone.

[0015] A second objective of this invention is to provide a method for preparing the aforementioned abalone-derived immunomodulatory peptides, comprising the following steps:

[0016] S1. Obtain abalone peptides;

[0017] S2. Sequence identification of abalone peptides;

[0018] S3. Molecular docking of abalone peptides with receptor protein TLR4 was performed to screen for immunomodulatory peptides.

[0019] Furthermore, in step S1: abalone peptides are obtained by enzymatic hydrolysis of abalone raw materials.

[0020] Specifically, in step S1, the preferred working conditions for enzymatic hydrolysis include: first adding pepsin to the abalone raw material for enzymatic hydrolysis, and then adding trypsin for enzymatic hydrolysis.

[0021] More specifically, in step S1, the preferred working conditions for enzymatic hydrolysis include: adding pepsin to the abalone raw material, adjusting the pH to 1.5~3.5, and hydrolyzing at 36~38℃ for 2~5 h; then adding trypsin, adjusting the pH to 6.0~8.0, and hydrolyzing at 36~38℃ for 1~3 h to inactivate the enzyme.

[0022] The preferred dosage of pepsin is 1000~3000 U / g based on the abalone raw material.

[0023] The preferred dosage of trypsin is 1000-2000 U / g based on the abalone raw material.

[0024] Specifically, in step S1: before enzymatically hydrolyzing the abalone raw material, it is preferable to pre-treat it. The pre-treatment includes: homogenizing the abalone raw material and heating it in a boiling water bath for 10-30 minutes.

[0025] Furthermore, in step S1: after enzymatic hydrolysis, the hydrolysate is separated and purified.

[0026] Furthermore, in step S1: the separation and purification includes fractionating the enzymatic hydrolysate using nanofiltration and ultrafiltration. Nanofiltration removes salts and free amino acids, while ultrafiltration removes macromolecules. Specifically, it is preferable to obtain components with a molecular weight of 200-3000 Da by performing nanofiltration and ultrafiltration on the enzymatic hydrolysate.

[0027] Further, in step S2: peptide sequence analysis was performed using LC-MS / MS, and the sequences were compared and analyzed against a database to obtain the complete peptide sequences. The mass spectrometry database search software used was MaxQuant 2.4.14.0, and the sample database used was the uniprot protein database.

[0028] Specifically, in step S2: it is preferable to desalt the product obtained in step S1 before performing peptide sequence analysis. Preferably, a C18 StageTip column is used for desalting.

[0029] Furthermore, in step S3: it is preferable to use Vina-2.0 within 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 binding between the ligand and the receptor.

[0030] The beneficial effects of this invention are as follows:

[0031] This invention obtained six abalone-derived immunomodulatory peptides from abalone through a combination of dual-enzyme digestion and 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. Experiments have demonstrated that these abalone peptides possess high immunomodulatory activity, which is of great significance for the development of novel immunomodulatory peptide products. Attached Figure Description

[0032] Figure 1 The diagram shows the molecular docking of the abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.1 with the receptor TLR4.

[0033] Figure 2The diagram shows the molecular docking of the abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.2 with the receptor TLR4.

[0034] Figure 3 The diagram shows the molecular docking of the abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.3 with the receptor TLR4.

[0035] Figure 4 The diagram shows the molecular docking of the abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.4 with the receptor TLR4.

[0036] Figure 5 The diagram shows the molecular docking of the abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.5 with the receptor TLR4.

[0037] Figure 6 The diagram shows the molecular docking of the abalone-derived immunomodulatory peptide with the amino acid sequence shown in SEQ.ID.NO.6 with the receptor TLR4.

[0038] Figure 7 To test the effect of six synthetic peptides on the relative proliferation rate of RAW264.7 cells;

[0039] Figure 8 To test the effect of six synthetic peptides on the phagocytic capacity of RAW264.7 cells;

[0040] Figure 9 To test the effect of six synthetic peptides on NO secretion in RAW264.7 cells. Detailed Implementation

[0041] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0042] In the specific implementation: the pepsin used was purchased from Sangon Biotech; the trypsin used was purchased from Bomei Biotechnology. Example

[0043] The steps for preparing abalone-derived immunomodulatory peptides are as follows:

[0044] S1. Preparation of abalone peptides:

[0045] (1) Raw material pretreatment: After homogenizing the muscle tissue of the wrinkled abalone, heat it in a boiling water bath for 15 minutes to obtain a homogenate, and let it cool before use.

[0046] (2) Enzymatic hydrolysis: Add 2000 U / g of pepsin (based on abalone muscle) to the raw material obtained in step (1), adjust the pH to 2.5, and hydrolyze at 37℃ for 4 h; then add 1200 U / g of trypsin (based on abalone muscle), adjust the pH to 7.0, and hydrolyze at 37℃ for 2 h, then boil at 100℃ to inactivate the enzyme; then centrifuge at 7000 r / min for 10 min, take the supernatant, and obtain the enzymatic hydrolysate.

[0047] (3) Purification: The enzymatic hydrolysate obtained in step (2) is subjected to nanofiltration and ultrafiltration fractionation. First, a 200 Da nanofiltration membrane is used to remove salt and free amino acids, and then a 3000 Da spiral wound membrane is selected for ultrafiltration. The 200~3000 Da fraction is freeze-dried to obtain peptide powder, which is stored at -20℃ for later use.

[0048] S2. Sequence identification of abalone peptides:

[0049] The peptide powder obtained in step S1 was desalted using a C18 StageTip column and subjected to peptide sequence analysis using LC-MS / MS. The complete peptide sequence was obtained by comparison analysis with a database. The mass spectrometry database search software was MaxQuant 2.4.14.0, and the sample database used was the uniprot protein database.

[0050] S3. Molecular docking of abalone peptide with receptor protein TLR4:

[0051] The SDF format files of the main active ingredients of the core drug were obtained from the Pubchem database. The protein structures of key targets were collected from the PDB database. Pymol-2.1.0 software was used to optimize the targets by removing water molecules and small molecule ligands. The targets were then hydrogenated and charged using AutoDock Tools-1.5.6 and saved as pdbqt format.

[0052] Using the key target as the acceptor and its corresponding active ingredient as the ligand, molecular docking was performed using VIA-2.0 within the PyRX software. Binding energies were calculated, and the results were output. Finally, PyMol software was used for result visualization. The affinity (kcal / mol) value represents the binding strength between the two; the lower the docking energy, the more stable the binding between the ligand and acceptor. PyMol was used for visualization analysis, and 2D plots were visualized using Discovery Studio 2020 Client.

[0053] Six immunomodulatory peptides and their docking energies were obtained through screening. The amino acid sequences of the six immunomodulatory peptides are shown in SEQ.ID.NO.1~6. The amino acid sequences of the six immunomodulatory peptides and their corresponding docking energies are shown in Table 1.

[0054] Table 1. Amino acid sequence and docking energy of abalone-derived immunomodulatory peptides

[0055] Immunomodulatory peptides 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

[0056] The molecular docking results of the abalone-derived immunomodulatory peptides with amino acid sequences as shown in SEQ.ID.NO.1~6 with the receptor TLR4 are as follows: Figures 1-6 The binding modes and binding sites of the six immunomodulatory peptides and their receptors reveal that the binding to TLR4 is primarily through interactions with receptor amino acid residues, such as hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups.

[0057] like Figure 1 As shown, the binding sites of the peptide with the amino acid sequence 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, and MET-358; Figure 2 As shown, the binding sites of the peptide with the amino acid sequence 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, and ARG-86; Figure 3 As shown, the amino acid sequences of the peptide represented by SEQ.ID.NO.3 have the following binding sites to TLR4: ARG-380, ASP-377, THR-355, ASP-403, TYR-375, SER-334, GLU-286, SER-332, SER-353, GLN-423, THR-357, ILE-336, and LYS-352; Figure 4 As shown, the binding sites of the peptide with the amino acid sequence 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, and TYR-449; Figure 5As shown, the amino acid sequences of the peptide represented by SEQ.ID.NO.5 have the following binding sites to TLR4: TYR-375, THR-355, SER-334, SER-312, SER-353, SER-332, ASP-377, GLU-286, and ALA-310; Figure 6 As shown, the binding sites of the peptide with the amino acid sequence 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, and MET-358.

[0058] test

[0059] Based on the mass spectrometry sequencing results, the above six peptide sequences were chemically synthesized and their effects on the relative proliferation rate, phagocytic capacity, and NO secretion of RAW264.7 cells were tested.

[0060] 1. Determination of relative proliferation rate of RAW264.7 cells

[0061] When the RAW264.7 cell density reached 80%, the cells were resuspended in complete culture medium, mixed thoroughly, and then counted to adjust the cell density to 6 × 10⁻⁶ cells / year. 5 Cells were cultured at a density of 100 μL / well in a 96-well plate. 100 μL of complete culture medium was added to the control group, and the plates were incubated at 37°C in a 5% CO2 incubator. After 24 h of complete cell attachment, the supernatant was removed. 100 μL of complete culture medium was added to the control and blank groups, while 100 μL of different concentrations of peptide solutions (50, 100, 200, and 400 µg / mL) were added to each well in the experimental groups. To prevent evaporation of the cell supernatant and its impact on the results, 200 μL of PBS was added to the outermost well of each 96-well plate. After 24 h of further incubation, 100 μL of 10% CCK-8 culture medium was added to each well according to the CCK-8 kit instructions. The plates were incubated at 37°C for 1 h, and the absorbance (OD) at 450 nm was measured using a microplate reader. Five replicates were used to calculate the relative proliferation rate of RAW264.7 cells and to screen the effective concentrations of the samples. The relative cell proliferation rate is calculated using the following formula:

[0062] Relative cell proliferation rate (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%.

[0063] The effects of six synthetic peptides on the relative proliferation rate of RAW264.7 cells are shown in [the table below]. Figure 7Synthetic peptides at concentrations ranging from 50 to 400 µg / mL all exhibited proliferative effects on RAW264.7 cells, with the proliferative effect increasing with increasing peptide concentration. Among them, PLGPVGR (SEQ.ID.NO.5) showed the best effect in promoting cell proliferation, achieving a relative cell proliferation rate of 150.22 ± 11.50% at a concentration of 400 µg / mL. Therefore, the six synthetic peptides showed no cytotoxicity within the concentration range of 50–400 µg / mL, and subsequent experiments were conducted using a concentration range of 100–400 µg / mL.

[0064] 2. Assay of phagocytic capacity of RAW264.7 cells

[0065] When the RAW264.7 cells in the culture flask reached 80%, they were diluted with complete culture medium to a cell density of 2 × 10⁻⁶. 5 Cells were added to each well of a 96-well cell culture plate with 100 μL of cell suspension per well. A control group was added with 100 μL of complete culture medium. The plates were incubated at 37°C in a 5% CO2 incubator. The supernatant was discarded after 24 h. 100 μL of complete culture medium was added to each well of the control and control groups. Different concentrations of peptide solutions (100, 200, and 400 µg / mL) prepared with complete culture medium were added to each well of the experimental groups. An equal volume of LPS (lipopolysaccharide O55:B5) solution with a final concentration of 1 µg / mL was added to each LPS group. After 24 h of incubation, the cell culture supernatant was discarded. 100 µL of neutral red solution was added to each well, and the plates were incubated at 37°C for another 2 h. The plates were then washed three times with PBS to remove excess color. 100 μL of cell lysis buffer was added to each well, and the cells were lysed on a shaker at room temperature for 30 min. The absorbance was measured at 540 nm. The experiment was set up with 5 replicates, and the relative phagocytic rate of cells was calculated using the following formula:

[0066] Relative phagocytosis rate of cells (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%.

[0067] The effects of six synthetic peptides on the phagocytic capacity of RAW264.7 cells are as follows: Figure 8 As shown, compared with the control group, all six synthetic peptides enhanced the phagocytic capacity of RAW264.7 cells, with a significant increase in phagocytic capacity as the concentration increased. PLGPVGR (SEQ.ID.NO.5) was the peptide with the strongest phagocytic promotion among the six synthetic peptides. At a concentration of 400 μg / mL, its relative phagocytic rate reached a maximum of 164±3.92%, which was 0.64 times that of the control group, demonstrating a strong immunostimulatory effect.

[0068] 3. Measurement of NO secretion in RAW264.7 cells

[0069] The experimental grouping and operation procedures were the same as those for the RAW 264.7 cell phagocytic capacity assay. After culturing for 24 h, the cell culture medium was collected. Following the NO kit instructions, 50 μL of the supernatant from each group of centrifuged cells was added to each well of a 96-well plate. Then, 50 μL of Griess Reagent I and Griess Reagent II were added sequentially, and the absorbance was measured at 540 nm. A standard curve for nitrite was determined and plotted according to the standard curve equation Y = 0.0051x + 0.0006, R... 2 =0.9999 is used to calculate the sodium nitrite content in the cell culture medium, thus reflecting the level of NO secreted by the cells.

[0070] The effects of different bioactive peptides on immunomodulation were assessed by detecting NO levels in RAW264.7 macrophages. The influence of six synthetic peptides on NO secretion in RAW264.7 cells is shown in [reference needed]. Figure 9 .like Figure 9 As shown, compared with the control group, the NO secretion in the LPS group was significantly increased ( P <0.05), and after intervention with various immunomodulatory peptides, NO secretion was significantly increased in all cases. P The concentration of NO (<0.05) indicates that these six peptides can effectively stimulate cells to release NO. NO can maintain the balance between host defense and self-protection by dynamically regulating the body's immune activation and immunosuppression. Appropriate levels of NO in the body can effectively enhance the body's immunity.

[0071] 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 within the protection scope of the present invention.

Claims

1. An abalone-derived immunomodulatory peptide, characterized in that, It is selected from at least one peptide whose amino acid sequence is shown in SEQ ID NO. 1 to 6.

2. The abalone-derived immunomodulatory peptide according to claim 1, characterized in that, The abalone mentioned is the wrinkled abalone.

3. A method for preparing abalone-derived immunomodulatory peptides as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Obtain abalone peptides; add pepsin to the abalone raw material and hydrolyze for 2-5 hours; then add trypsin and hydrolyze for 1-3 hours; S2. Sequence identification of abalone peptides; S3. Molecular docking of abalone peptide with receptor protein TLR4 was performed to screen for immunomodulatory peptides. Using key targets as receptors and their corresponding active ingredients as ligands, molecular docking was performed using the Vina-2.0 tool within the PyRX software. The binding energy was calculated and the results were output. Finally, the results were visualized using PyMol software. PyMol was used for visualization analysis, and 2D plots were visualized using Discovery Studio 2020 Client.

4. The preparation method according to claim 3, characterized in that, In step S1: After enzymatic hydrolysis, the enzymatic hydrolysate is separated and purified; the separation and purification includes fractionating the enzymatic hydrolysate using ultrafiltration and nanofiltration.

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

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