Oyster high-F-value oligopeptide for protecting alcoholic liver injury as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food biology, and discloses oyster high-F-value oligopeptide for protecting alcoholic liver injury as well as a preparation method and application of the oyster high-F-value oligopeptide. Four high-F-value oligopeptides for protecting the alcoholic liver injury are obtained from pacific oyster meat through two-step enzymolysis, and comprise at least one of peptides with amino acid sequences as shown in SEQ.ID.NO.1-4. The docking energies of the four oligopeptides with high F values and the ALDH receptor are respectively-7.1 kcal / mol,-6.5 kcal / mol,-8.0 kcal / mol and-7.2 kcal / mol. The peptide has a relatively high ALDH activation rate. The oyster-derived high-F-value oligopeptide for protecting alcoholic liver injury can be used for research and development of functional foods, and has a wide market prospect.

Description

Technical Field

[0001] The present invention belongs to the field of food biotechnology, and particularly relates to an oyster high-F value oligopeptide for protecting alcoholic liver injury, a preparation method thereof, and an application thereof. Background Art

[0002] Alcoholic liver disease (ALD) is one of the important diseases causing liver injury globally. With the expansion of the drinking population and the increase in alcoholism, its incidence rate shows an upward trend year by year. After alcohol enters the human body, it is mainly metabolized by the liver. Ethanol is converted into acetaldehyde under the action of alcohol dehydrogenase (ADH), and the toxicity of acetaldehyde is much higher than that of ethanol, which can directly damage hepatocytes, induce oxidative stress, inflammatory responses, and apoptosis. As a key metabolic enzyme, aldehyde dehydrogenase (ALDH) can further oxidize acetaldehyde into acetic acid, thereby reducing the toxicity of acetaldehyde. Research shows that about 540 million people in Asia carry the ALDH2 mutant gene. If the ALDH2 gene mutates or is deleted, the activity of the corresponding aldehyde dehydrogenase will be greatly reduced, resulting in the long-term accumulation of acetaldehyde in the body that cannot be metabolized, which will then cause capillary dilation, skin flushing and inflammation, and further induce vascular endothelial dysfunction, increasing the risk of cardiovascular and cerebrovascular diseases. High-F value oligopeptides are a class of functional short peptides. The F value refers to the molar ratio of the content of branched-chain amino acids (BCAAs) to aromatic amino acids (AAAs). Due to their special amino acid composition and structure, they show significant potential in aspects such as antioxidant, anti-inflammatory, and regulation of liver metabolism, and are regarded as new functional substances for treating alcoholic liver injury. After high-F value oligopeptides interact with ALDH, by regulating the conformation of ALDH, enhancing the activity of ALDH, and accelerating the metabolism of acetaldehyde, the concentration of acetaldehyde in the liver is reduced, thereby reducing the direct toxic effect of acetaldehyde on hepatocytes and protecting the structural and functional integrity of hepatocytes.

[0003] Bioinformatics tools are used to assist in screening active peptides. Based on the known amino acid sequences in active peptides, database search and software analysis are used to predict the possible biological activities, safety, and bioaccessibility of peptide segments. On this basis, target peptide segments are selected for chemical synthesis and their related properties are verified. Using bioinformatics tools can save a large amount of time and experimental costs. Therefore, using bioinformatics tools to assist in screening high-F value oligopeptides with the function of protecting alcoholic liver injury and exploring their interaction with the ALDH receptor can provide a basis for the development of functional foods for protecting alcoholic liver injury. Summary of the Invention

[0004] The present invention provides an oyster high-F value oligopeptide for protecting alcoholic liver injury, a preparation method thereof, and an application thereof. The high-F value oligopeptide of the present invention is an oyster-derived high-F value oligopeptide, which can effectively bind to the ALDH receptor and can accelerate ethanol metabolism and protect hepatocytes from ethanol-induced injury.

[0005] The specific technical solution is as follows: One of the objectives of the present invention is to provide an oyster high-F value oligopeptide for protecting alcoholic liver injury, which comprises at least one of the peptides shown in SEQ.ID.NO.1-4 in terms of amino acid sequence.

[0006] Among them, SEQ.ID.NO.1 is KVCT, and the docking energy of this peptide with the ALDH receptor is -7.1 kcal / mol.

[0007] Among them, SEQ.ID.NO.2 is IEKPMG, and the docking energy of this peptide with the ALDH receptor is -6.5 kcal / mol.

[0008] Among them, SEQ.ID.NO.3 is VEMPPHL, and the docking energy of this peptide with the ALDH receptor is -8.0 kcal / mol.

[0009] Among them, SEQ.ID.NO.4 is AEKL, and the docking energy of this peptide with the ALDH receptor is -7.2 kcal / mol.

[0010] The above four peptides are all high-F value oligopeptides. The F value refers to the molar ratio of branched-chain amino acids (BCAA: Val, Ile, Leu) to aromatic amino acids (AAA: Trp, Tyr, Phe). The F value of high-F value oligopeptides should be greater than 20. The high-F value oligopeptides in the present invention refer to peptides with an F value greater than 20 and composed of 2-10 amino acids. The above four peptides do not contain aromatic amino acids.

[0011] Specifically, the oyster is Crassostrea gigas ( Crassostrea gigas ), and the above high-F value oligopeptide for protecting alcoholic liver injury is isolated and extracted from Crassostrea gigas meat.

[0012] Another objective of the present invention is to provide a preparation method of the above oyster high-F value oligopeptide, which comprises the following steps: S1. Obtaining oyster peptides by enzymatically hydrolyzing oysters; S2. Identifying the sequences of oyster peptides; S3. Screening oyster high-F value oligopeptides that can effectively bind to the receptor ALDH.

[0013] Furthermore, in step S1: The exopeptidase pepsin and the endopeptidase papain are sequentially used to enzymatically hydrolyze oyster meat.

[0014] Specifically, in step S1, the working conditions of enzymatic hydrolysis preferably include: Add pepsin to the raw material to be processed, adjust the pH value to 2 - 4, enzymatically hydrolyze at 30 - 37 °C for 3 - 6 h, and inactivate the enzyme; then add papain, adjust the pH value to 6 - 8, enzymatically hydrolyze at 55 - 65 °C for 2 - 4 h, and inactivate the enzyme.

[0015] Among them, the addition amount of pepsin is preferably 800 - 1200 U / g based on the raw material to be processed (oyster meat).

[0016] Among them, the addition amount of papain is preferably 1800 - 2200 U / g based on the raw material to be processed (oyster meat).

[0017] Specifically, in step S1: Before enzymatically hydrolyzing the oyster meat, it is preferably pretreated. The pretreatment includes: adding water to the oyster meat, then homogenizing, and after heat treatment for 10 - 15 min, homogenizing again for standby.

[0018] Among them, the dosage ratio of oyster meat to water is preferably 1 kg:(3 - 8) L.

[0019] Among them, it is preferably to use a boiling water bath for heat treatment.

[0020] Among them, the water added to the oyster is preferably ultrapure water.

[0021] Furthermore, in step S1: After enzymatic hydrolysis, it is preferably to add activated carbon to the enzymatic hydrolysate to remove aromatic amino acids. Specifically, it is preferably to add 5wt% - 15wt% of activated carbon to the enzymatic hydrolysate and stir at 30 - 40 °C for 1 - 3 h to remove aromatic amino acids.

[0022] Furthermore, in step S1: After enzymatic hydrolysis, ultrafiltration and nanofiltration are used to separate and purify the enzymatic hydrolysate. Nanofiltration is used to remove salts and free amino acids in the enzymatic hydrolysate, and ultrafiltration is used to 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] Furthermore, in step S2: LC - MS / MS is used for polypeptide sequence analysis. By comparing and analyzing with the Pacific oyster protein database, the sequences of all peptides are obtained.

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

[0025] Furthermore, in step S3: It is preferably to use vina inside the pyrx software to screen for oyster high - F - value oligopeptides that can effectively bind to the ALDH receptor.

[0026] The third object of the present invention is to provide the application of the above - mentioned oyster high - F - value oligopeptides in food.

[0027] A fourth object of the present invention is to provide the application of the above-mentioned oyster high-F value oligopeptides in the preparation of functional foods for protecting against alcoholic liver injury or in the preparation of drugs for protecting against alcoholic liver injury.

[0028] The beneficial effects of the present invention are as follows: The present invention obtained four high-F value oligopeptides for protecting against alcoholic liver injury from Pacific oyster meat through two-step enzymatic hydrolysis. The docking energies with the ALDH receptor are -7.1 kcal / mol, -6.5 kcal / mol, -8.0 kcal / mol, and -7.2 kcal / mol respectively. Through experimental verification, the above-mentioned peptides have a relatively high ALDH activation rate. The oyster-derived high-F value oligopeptides for protecting against alcoholic liver injury of the present invention can be used in the research and development of functional foods and have broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a molecular docking result diagram of a high-F value oligopeptide with the amino acid sequence shown in SEQ.ID.NO.1 and the ALDH receptor; Figure 2 It is a molecular docking result diagram of a high-F value oligopeptide with the amino acid sequence shown in SEQ.ID.NO.2 and the ALDH receptor; Figure 3 It is a molecular docking result diagram of a high-F value oligopeptide with the amino acid sequence shown in SEQ.ID.NO.3 and the ALDH receptor; Figure 4 It is a molecular docking result diagram of a high-F value oligopeptide with the amino acid sequence shown in SEQ.ID.NO.4 and the ALDH receptor. 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. 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 all be obtained from commercial channels unless otherwise specified.

[0031] In the examples, pepsin and papain were purchased from Sangon Biotech Co., Ltd. Example

[0032] To prepare oyster high-F value oligopeptides for protecting against alcoholic liver injury, the steps are as follows: S1. Preparation of oyster peptides: (1) Raw material pretreatment: Weigh 1 kg of Pacific oyster meat, add 5 L of ultrapure water and homogenize, heat in a boiling water bath for 15 min, and homogenize again to obtain a homogenate for use.

[0033] (2)Enzymatic hydrolysis: Add pepsin at 1000 U / g based on the weight of oyster meat to the homogenate obtained in step (1), adjust the pH to 3, and hydrolyze at 35 °C for 6 h. Then, inactivate the enzyme by boiling at 100 °C. After cooling, add papain at 2000 U / g based on the weight of oyster meat, adjust the pH to 6.5, and hydrolyze at 55 °C for 4 h. Then, inactivate the enzyme by boiling at 100 °C. Subsequently, centrifuge at 5000 r / min for 15 min, take the supernatant to obtain the enzymatic hydrolysate.

[0034] (3)Activated carbon adsorption: Add 10 wt% activated carbon to the enzymatic hydrolysate obtained in step (2), stir at 35 °C for 2 h to remove aromatic amino acids, and obtain the de-aromatic enzymatic hydrolysate.

[0035] (4)Separation and purification: Perform nanofiltration and ultrafiltration fractionation on the de-aromatic enzymatic hydrolysate obtained in step (3). 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 to obtain a fraction of 200 - 3000 Da. Freeze-dry and store at -20 °C for standby.

[0036] S2. Sequence identification of oyster peptides: Desalt the component peptides obtained in step S1 using a C18 StageTip chromatographic column, and perform polypeptide sequence analysis using LC-MS / MS. By comparing and analyzing with the Pacific oyster protein database, all peptide sequences are obtained.

[0037] S3. Molecular docking of oyster peptides with ALDH receptor: 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 Pymol software to optimize the target by removing water molecules and small molecule ligands, etc., and perform hydrogenation and charge processing using AutoDock Tools and save it as the pdbqt format. Using the key target ALDH as the receptor and its corresponding active ingredient (oyster peptide) as the ligand, perform molecular docking using vina inside the pyrx software, calculate the binding energy and output the result file. Finally, use PyMol software for result visualization. The Affinity (kcal / mol) value represents the binding ability of the two. The lower the binding energy, the more stable the binding of the ligand to the receptor. Use Pymol (https: / / pymol.org / 2 / ) for its visualization analysis to obtain the docking energies of the four polypeptide sequences.

[0038] Using bioinformatics tools to assist in screening bioactive peptides: The biological activities of peptides were predicted by the Peptide Ranker tool. Sequences with scores exceeding 0.5 were considered to have potential activities. The novelty of peptides was queried through the BIOPEP database. The digestion resistance characteristics and cell membrane permeability of peptides were predicted by the Peptide Cutter tool and the CPP pred tool. Sequences with scores exceeding 0.5 were considered to have the potential for complete transmembrane absorption. The potential allergenicity of peptides was predicted by the Aller TOP v.2.0 tool, and the potential toxicity and physicochemical properties of peptides were predicted by the Toxin Pred tool. The four polypeptide sequences obtained above were verified, and their biological activity scores, digestion resistance scores, and cell membrane permeability scores were all qualified, and there was no potential toxicity or allergenicity. The amino acid sequences of the four polypeptides are shown as SEQ.ID.NO.1-4 in sequence, and their amino acid sequences and corresponding docking energies are shown in Table 1.

[0039] Table 1 Amino acid sequences and docking energies of oyster high-F value oligopeptides for protecting alcoholic liver injury High F-value oligopeptides from oyster Sequence ALDH docking energy (kcal / mol) SEQ.ID.NO.1 KVCT -7.1 SEQ.ID.NO.2 IEKPMG -6.5 SEQ.ID.NO.3 VEMPPHL -8.0 SEQ.ID.NO.4 AEKL -7.2 The molecular docking results of the peptides with amino acid sequences shown as SEQ.ID.NO.1-4 with the ALDH receptor are shown successively in Figures 1-4 . From Figures 1-4 the binding modes and binding sites of the peptides and the receptor protein ALDH, it can be seen that the binding of the peptides to the ALDH subunit is mainly through hydrogen bond interactions with the receptor amino acid residues.

[0040] As Figure 1 shown, the main binding sites of the peptide with amino acid sequence shown as SEQ.ID.NO.1 to the ALDH subunit are TRP-185, GLN-213, SER-263, GLN-366, TYR-442, GLU-212; as Figure 2 shown, the main binding sites of the peptide with amino acid sequence shown as SEQ.ID.NO.2 to the ALDH subunit are ARG-101, GLY-158, ARG-172, HIS-173, THR-202, GLU-174, THR-503; as Figure 3 shown, the main binding sites of the peptide with amino acid sequence shown as SEQ.ID.NO.3 to the ALDH subunit are GLN-213, GLN-366, ASN-186; as Figure 4 shown, the main binding sites of the peptide with amino acid sequence shown as SEQ.ID.NO.4 to the ALDH subunit are TRP-185, LYS-369, GLY-262, LEU-286, ASP-363. Test

[0041] Peptides with amino acid sequences as shown in SEQ.ID.NO.1 - 4 were synthesized and their efficacy was verified. The ALDH activation rate experiment was carried out using the method of Jianglai Biotech Aldehyde Dehydrogenase Kit. The results of the ALDH activation rate at a concentration of 0.5 mM are shown in Table 2.

[0042] Table 2 ALDH activation rate of oyster high-F value oligopeptides for protecting alcoholic liver injury High F-value oligopeptides from oyster Sequence ALDH activation rate / % SEQ.ID.NO.1 KVCT 28.53±3.65 SEQ.ID.NO.2 IEKPMG 23.18±4.39 SEQ.ID.NO.3 VEMPPHL 42.43±5.53 SEQ.ID.NO.4 AEKL 34.91±5.27 The results in Table 2 confirmed that the oyster high-F value oligopeptides of the present invention have a high ALDH activation rate, have the efficacy of protecting alcoholic liver injury, and can be applied to the preparation of functional foods for protecting alcoholic liver injury or the preparation of drugs for protecting alcoholic liver injury.

[0043] 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. Oyster high-F value oligopeptides for protecting alcoholic liver injury, characterized in that, At least one of the peptides comprising the amino acid sequences shown in SEQ.ID.NO.1 to 4.

2. The oyster high-F value oligopeptide according to claim 1, wherein The oyster is Crassostrea gigas.

3. The preparation method of oyster high-F value oligopeptide according to claim 1 or 2, characterized in that, Comprising the following steps: S1. Obtaining oyster peptides by enzymolysis of oysters; S2. Conducting sequence identification of the oyster peptides; S3. Screening for oyster high-F value oligopeptides that can effectively bind to the receptor ALDH.

4. The preparation method according to claim 3, characterized in that, In step S1: Pepsin and papain are successively used for enzymolysis of oyster meat.

5. The preparation method according to claim 3, characterized in that, In step S1: After enzymolysis, activated carbon is added to the enzymolysis solution to remove aromatic amino acids.

6. The preparation method according to claim 3, wherein In step S1: After enzymolysis, the enzymolysis solution is separated and purified by ultrafiltration and nanofiltration.

7. The preparation method according to claim 6, characterized in that, In step S1: By subjecting the enzymolysis solution to nanofiltration and ultrafiltration, a fraction with a molecular weight of 200 - 3000 Da is obtained.

8. The preparation method according to claim 3, characterized in that, In step S2: LC-MS / MS is used for polypeptide sequence analysis.

9. Use of the oyster high-F value oligopeptide according to claim 1 or 2 in food.

10. Use of the oyster high-F value oligopeptide according to claim 1 or 2 in the preparation of a functional food for protecting against alcoholic liver injury or in the preparation of a drug for protecting against alcoholic liver injury.

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