Oyster high F value oligopeptide for protecting alcoholic liver damage and its preparation method and application

Abstract

The present invention belongs to the field of food biotechnology and discloses an oyster high F value oligopeptide for protecting against alcoholic liver damage, as well as its preparation method and application. The present invention obtains four high F value oligopeptides for protecting against alcoholic liver damage from Pacific oyster meat through two-step enzymatic hydrolysis, including at least one of the peptides with amino acid sequences shown in SEQ.ID.NO.1~4. The docking energies of the four high F value oligopeptides with the ALDH receptor are ‑7.1 kcal / mol, ‑6.5 kcal / mol, ‑8.0 kcal / mol and ‑7.2 kcal / mol, respectively. The above peptides have a high ALDH activation rate. The oyster-derived high F value oligopeptides for protecting against alcoholic liver damage of the present invention can be used for the research and development of functional foods and have broad market prospects.

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 damage, and a preparation method and application thereof. Background Art

[0002] Alcoholic liver disease (ALD) is a major cause of liver damage worldwide. With the expansion of the drinking population and the rise in alcohol abuse, its incidence is increasing annually. After entering the body, alcohol is primarily metabolized in the liver, where it is converted to acetaldehyde by the enzyme alcohol dehydrogenase (ADH). Acetaldehyde is far more toxic than ethanol and can directly damage liver cells, inducing oxidative stress, inflammation, and apoptosis. Aldehyde dehydrogenase (ALDH), a key metabolic enzyme, further oxidizes acetaldehyde to acetic acid, thereby reducing its toxicity. Studies have shown that approximately 540 million people in Asia carry a mutation in the ALDH2 gene. Mutations or deletions in the ALDH2 gene significantly reduce the activity of the corresponding aldehyde dehydrogenase, leading to the long-term accumulation of acetaldehyde in the body and its inability to be metabolized. This can cause capillary dilation, skin flushing, and inflammation, which in turn can induce endothelial dysfunction and increase the risk of cardiovascular and cerebrovascular diseases.

[0003] High-F-value oligopeptides are a class of functional short peptides. The F-value refers to the molar ratio of branched-chain amino acids (BCAAs) to aromatic amino acids (AAAs). Due to their unique amino acid composition and structure, these peptides have demonstrated significant potential for antioxidant, anti-inflammatory, and liver metabolism regulation, and are considered novel functional substances for the treatment of alcoholic liver injury. After interacting with ALDH, these peptides modulate ALDH conformation, enhance ALDH activity, and accelerate acetaldehyde metabolism, thereby reducing acetaldehyde concentration in the liver. This mitigates the direct toxic effects of acetaldehyde on hepatocytes and protects their structural and functional integrity.

[0004] Bioinformatics tools assist in the screening of active peptides. Based on the known amino acid sequences of active peptides, database searches and software analysis are used to predict the peptide's potential biological activity, safety, and bioaccessibility. Based on this, target peptides are selected for chemical synthesis and their relevant properties are verified. Bioinformatics tools can save significant time and experimental costs. Summary of the Invention

[0005] The present invention provides an oyster high-F value oligopeptide for protecting against alcoholic liver damage, as well as its preparation method and application. The high-F value oligopeptide of the present invention is derived from oysters and can effectively bind to the ALDH receptor, accelerate ethanol metabolism, and protect liver cells from ethanol-induced damage.

[0006] The specific technical solutions are as follows:

[0007] One of the purposes of the present invention is to provide an oyster high F value oligopeptide for protecting alcoholic liver damage, comprising at least one of the peptides with amino acid sequences shown in SEQ.ID.NO.1-4.

[0008] Among them, SEQ.ID.NO.1 is KVCT, and the docking energy of this peptide with the ALDH receptor is -7.1 kcal / mol.

[0009] Among them, SEQ.ID.NO.2 is IEKPMG, and the docking energy of this peptide with the ALDH receptor is -6.5 kcal / mol.

[0010] Among them, SEQ.ID.NO.3 is VEMPPHL, and the docking energy of this peptide with the ALDH receptor is -8.0 kcal / mol.

[0011] Among them, SEQ.ID.NO.4 is AEKL, and the docking energy of this peptide with the ALDH receptor is -7.2 kcal / mol.

[0012] All four peptides described above are high-F-value oligopeptides. The F-value refers to the molar ratio of branched-chain amino acids (BCAAs: Val, Ile, Leu) to aromatic amino acids (AAAs: Trp, Tyr, Phe). High-F-value oligopeptides should have an F-value greater than 20. High-F-value oligopeptides in the present invention are peptides with an F-value greater than 20 and composed of 2 to 10 amino acids. None of the four peptides described above contain aromatic amino acids.

[0013] Specifically, the oyster is the Pacific oyster ( Crassostrea gigas ), the above-mentioned high F value oligopeptides that protect against alcoholic liver damage are separated and extracted from Pacific oyster meat.

[0014] A second object of the present invention is to provide a method for preparing the above-mentioned oyster high F value oligopeptide, comprising the following steps:

[0015] S1. Obtaining oyster peptides by enzymatic hydrolysis of oysters;

[0016] S2. Sequence identification of oyster peptides;

[0017] S3. Screening of oyster oligopeptides with high F values that can effectively bind to the receptor ALDH.

[0018] Furthermore, in step S1: the oyster meat is enzymatically hydrolyzed using the exoenzyme pepsin and the endoenzyme papain in sequence.

[0019] Specifically, in step S1, the working conditions of enzymatic hydrolysis preferably include:

[0020] Add pepsin to the raw material to be processed, adjust the pH value to 2-4, and enzymolyze at 30-37℃ for 3-6 hours to inactivate the enzyme; then add papain, adjust the pH value to 6-8, and enzymolyze at 55-65℃ for 2-4 hours to inactivate the enzyme.

[0021] The amount of pepsin added is preferably 800 to 1200 U / g based on the raw material to be processed (oyster meat).

[0022] The amount of papain added is preferably 1800-2200 U / g based on the raw material to be processed (oyster meat).

[0023] Specifically, in step S1, the oyster meat is preferably pretreated before being enzymatically hydrolyzed. The pretreatment includes adding water to the oyster meat, homogenizing the oyster meat, heat-treating the oyster meat for 10 to 15 minutes, and homogenizing the oyster meat again for later use.

[0024] Among them, the preferred ratio of oyster meat to water is 1 kg: (3~8) L.

[0025] Among them, heat treatment using a boiling water bath is preferred.

[0026] The water added to the oysters is preferably ultrapure water.

[0027] Furthermore, in step S1: after enzymatic hydrolysis, preferably activated carbon is added to the enzymatic hydrolysis solution to remove aromatic amino acids. Specifically, preferably 5 wt% to 15 wt% activated carbon is added to the enzymatic hydrolysis solution and stirred at 30 to 40°C for 1 to 3 hours to remove aromatic amino acids.

[0028] Furthermore, in step S1: after enzymatic hydrolysis, the enzymatic hydrolyzate is separated and purified using ultrafiltration and nanofiltration. Nanofiltration is used to remove salts and free amino acids from the enzymatic hydrolyzate, and ultrafiltration is used to 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 hydrolyzate.

[0029] Furthermore, in step S2: peptide sequence analysis is performed using LC-MS / MS, and the sequences of all peptides are obtained by comparison with the Pacific oyster protein database.

[0030] Specifically, in step S2, the product obtained in step S1 is preferably desalted before performing polypeptide sequence analysis, preferably using a C18 StageTip column for desalting.

[0031] Furthermore, in step S3: preferably, vina within the pyrx software is used to screen oyster high F value oligopeptides that can effectively bind to the ALDH receptor.

[0032] A fourth object of the present invention is to provide the use of the above-mentioned oyster high F value oligopeptide in the preparation of a drug for protecting alcoholic liver damage.

[0033] The beneficial effects of the present invention are as follows:

[0034] This study, using a two-step enzymatic hydrolysis method, extracted four high-F-value oligopeptides from Pacific oyster meat that protect against alcoholic liver damage. Their docking energies with the ALDH receptor were -7.1 kcal / mol, -6.5 kcal / mol, -8.0 kcal / mol, and -7.2 kcal / mol, respectively. Experimental validation confirmed that these peptides possessed high ALDH activation rates. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a diagram showing the molecular docking results of the high F value oligopeptide with the amino acid sequence shown in SEQ.ID.NO.1 and the ALDH receptor;

[0036] Figure 2 This is a diagram showing the molecular docking results of the high F value oligopeptide with the amino acid sequence shown in SEQ.ID.NO.2 and the ALDH receptor;

[0037] Figure 3 This is a diagram showing the molecular docking results of the high F value oligopeptide with the amino acid sequence shown in SEQ.ID.NO.3 and the ALDH receptor;

[0038] Figure 4 This is a diagram showing the molecular docking results of the high F-value oligopeptide with the amino acid sequence shown in SEQ.ID.NO.4 and the ALDH receptor. DETAILED DESCRIPTION

[0039] The principles and features of the present invention are described below with reference to examples. The examples are intended only to illustrate the present invention and are not intended 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.

[0040] In the examples, pepsin and papain used were purchased from Shenggong Bioengineering Co., Ltd. Example

[0041] The steps for preparing oyster high F value oligopeptides for protecting against alcoholic liver damage are as follows:

[0042] S1. Preparation of oyster peptides:

[0043] (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 the homogenate for use.

[0044] (2) Enzymatic hydrolysis: add 1000 U / g pepsin based on oyster meat to the homogenate obtained in step (1), adjust the pH to 3, perform enzymatic hydrolysis at 35°C for 6 h, and then boil at 100°C to inactivate the enzyme; after cooling, add 2000 U / g papain based on oyster meat, adjust the pH to 6.5, perform enzymatic hydrolysis at 55°C for 4 h, and 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.

[0045] (3) Activated carbon adsorption: 10 wt% activated carbon was added to the hydrolyzate obtained in step (2), and the mixture was stirred at 35 °C for 2 h to remove aromatic amino acids and obtain dearomatized hydrolyzate.

[0046] (4) Separation and purification: The dearomatization hydrolysate obtained in step (3) was subjected to nanofiltration and ultrafiltration classification. A 200 Da nanofiltration membrane was first used to remove salts and free amino acids, and then a 3000 Da spiral membrane was selected for ultrafiltration to obtain 200-3000 Da components; the components were freeze-dried and stored at -20°C for future use.

[0047] S2. Sequence identification of oyster peptides:

[0048] The component peptides obtained in step S1 were desalted using a C18 StageTip column, and peptide sequence analysis was performed using LC-MS / MS. The complete peptide sequence was obtained by comparison with the Pacific oyster protein database.

[0049] S3. Molecular Docking of Oyster Peptide and ALDH Receptor:

[0050] The Pubchem database was used to obtain SDF files of the core drug's main active ingredient. Key target protein structures were collected from the PDB database. Pymol software was used to optimize the targets by removing water molecules and small molecule ligands, and AutoDock Tools were used for hydrogenation and charge manipulation. The structures were then saved in pdbqt format. Using the key target ALDH as the receptor and its corresponding active ingredient (oyster peptide) as the ligand, molecular docking was performed using VINA within PyRx software. Binding energies were calculated and output as result files. Finally, PyMol software was used to visualize the results. The affinity (kcal / mol) value represents the binding strength of the two peptides; the lower the binding energy, the more stable the ligand-receptor binding. Pymol (https: / / pymol.org / 2 / ) was used for visualization and analysis, yielding the docking energies for the four peptide sequences.

[0051] Bioinformatics tools were used to assist in the screening of active peptides: The Peptide Ranker tool was used to predict the bioactivity of the peptides, and sequences with a score exceeding 0.5 were considered to have potential activity. The novelty of the peptides was checked against the BIOPEP database. The Peptide Cutter tool and the CPP Pred tool were used to predict the digestibility and cell membrane permeability of the peptides. Sequences with a score exceeding 0.5 were considered to have the potential for complete transmembrane absorption. The Aller TOP v.2.0 tool was used to predict the potential allergenicity of the peptides, and the Toxin Pred tool was used to predict the potential toxicity and physicochemical properties of the peptides. The four peptide sequences obtained above were verified to have acceptable bioactivity scores, digestibility, and cell membrane permeability, and showed no potential for toxicity or allergenicity. The amino acid sequences of the four peptides are shown in SEQ ID NOs. 1 to 4, and their amino acid sequences and corresponding docking energies are shown in Table 1.

[0052] Table 1 Amino acid sequence and docking energy of oyster high F value oligopeptides that protect against alcoholic liver damage

[0053] Oyster high F value oligopeptide 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

[0054] The molecular docking results of the peptides with amino acid sequences shown in SEQ.ID.NO.1~4 and ALDH receptors are shown in the following order. Figures 1 to 4 .Depend on Figures 1 to 4 The binding pattern and binding site of the peptide and the receptor protein ALDH show that the binding of the peptide to the ALDH subunit is mainly through the hydrogen bond interaction between the peptide and the receptor amino acid residues.

[0055] like Figure 1 As shown, the binding sites of the peptide shown in SEQ.ID.NO.1 and ALDH subunit are mainly TRP-185, GLN-213, SER-263, GLN-366, TYR-442, GLU-212; Figure 2 As shown, the binding sites of the peptide shown in SEQ.ID.NO.2 and the ALDH subunit are mainly ARG-101, GLY-158, ARG-172, HIS-173, THR-202, GLU-174, and THR-503; Figure 3 As shown, the binding sites of the peptide shown in SEQ.ID.NO.3 and the ALDH subunit are mainly GLN-213, GLN-366, and ASN-186; Figure 4 As shown, the binding sites of the peptide shown in SEQ.ID.NO.4 with the ALDH subunit are mainly TRP-185, LYS-369, GLY-262, LEU-286, and ASP-363.

[0056] test

[0057] Peptides with amino acid sequences shown in SEQ ID NOs. 1-4 were synthesized and their efficacy was verified. ALDH activation was performed using the Jianglai Biotechnology Aldehyde Dehydrogenase Kit. The results of ALDH activation at a concentration of 0.5 mM are shown in Table 2.

[0058] Table 2 ALDH activation rate of oyster high F value oligopeptides that protect against alcoholic liver damage

[0059] Oyster high F value oligopeptide 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

[0060] The results in Table 2 confirm that the oyster high F value oligopeptide of the present invention has a high ALDH activation rate, has the efficacy of protecting alcoholic liver damage, and can be used to prepare drugs for protecting alcoholic liver damage.

[0061] 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 scope of protection of the present invention.

Claims

1. Oyster high F value oligopeptide for protecting alcoholic liver damage, characterized by: The amino acid sequence is shown in SEQ.ID.NO.

3.

2. The method for preparing the oyster high F value oligopeptide according to claim 1, wherein: The steps include: S1. Oyster peptides were obtained by enzymatic hydrolysis of oysters. Oyster meat was enzymatically hydrolyzed using pepsin and then papain. After enzymatic hydrolysis, activated carbon is added to the hydrolyzate to remove aromatic amino acids; After enzymatic hydrolysis, the enzymatic hydrolysate is separated and purified by ultrafiltration and nanofiltration; by nanofiltration and ultrafiltration of the enzymatic hydrolysate, a component with a molecular weight of 200-3000 Da is obtained; S2. Sequence identification of oyster peptides; S3. Screening of oyster oligopeptides with high F values that can effectively bind to the receptor ALDH.

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

4. Application of oyster high F value oligopeptide for protecting alcoholic liver damage in the preparation of a drug for protecting alcoholic liver damage, characterized in that: The oyster high F value oligopeptide is selected from peptides with amino acid sequences as shown in SEQ.ID.NO.1~4.

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