A walnut protein peptide having ADH and ALDH activating activity and a screening method thereof

Abstract

The application discloses a walnut protein peptide with ADH and ALDH activation activity and a screening method thereof, and belongs to the technical field of food-derived bioactive peptide development. The application creatively combines polypeptidomics, various screening conditions and molecular docking to construct a method for rapidly screening ADH and ALDH activating peptides, thereby avoiding the time-consuming and labor-consuming defects caused by step-by-step separation and purification. The application first identifies a high-activity ADH and ALDH activating polypeptide PGHFEAF from walnut protein, the polypeptide can resist gastrointestinal digestion, the docking and binding energy of the polypeptide with ADH and ALDH are-8.2 and-10.5 kcal / mol respectively, the ADH activation EC 50 value is 0.49+ / -0.09 mM, and the ALDH activation EC 50 value is 1.85+ / -0.28 mM.

Description

TECHNICAL FIELD

[0001] The present application relates to a walnut protein peptide with ADH and ALDH activation activity and a screening method thereof, and belongs to the technical field of food-derived bioactive peptide development. BACKGROUND

[0002] Drinking has become a common phenomenon worldwide, but excessive drinking seriously affects human health. The World Health Organization's Global Status Report on Alcohol and Health states that about 3 million people die from drinking alcohol every year, accounting for 5.3% of all deaths, and more than 60 diseases are related to long-term excessive drinking. Alcohol can cause varying degrees of damage to various organs of the human body, among which the brain and liver are the most affected, and the most representative are fatty liver, hepatitis and cirrhosis. In many countries, there is a linear correlation between alcohol intake and liver disease mortality. So far, people have not yet obtained satisfactory therapeutic drugs. Although some drugs are effective in promoting alcohol metabolism and improving liver condition, they can also cause varying degrees of side effects and physiological toxicity. Therefore, it is of great research significance and broad development prospects to find functional substances that are both effective and safe for sobering up and treating alcoholic liver damage.

[0003] Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) are the main enzyme systems in the liver that catalyze the oxidative metabolism of alcohol. 80-90% of alcohol is converted to acetaldehyde under the action of ADH, and acetaldehyde is then oxidized by ALDH to form non-toxic acetic acid. Acetic acid enters the blood circulation and participates in the tricarboxylic acid cycle in organs such as the kidney, and is ultimately metabolized to CO2 and H2O. ADH, as the first limiting factor in alcohol metabolism in the body, plays an important role in limiting the rate of alcohol metabolism; while ALDH, as the main substance that catalyzes the metabolism of toxic acetaldehyde, can alleviate the damage of acetaldehyde to the human body. When the alcohol intake is small, ADH is the main metabolic pathway of alcohol, and when excessive drinking occurs, the oxidative pathway involving ADH is inhibited, and the expression and secretion of CYP2E1 are highly induced. The CYP2E1 pathway produces a large amount of reactive oxygen species (ROS), which is one of the main causes of liver damage. Therefore, by activating ADH activity, inhibiting the expression of the CYP2E1 pathway, reducing the production of reactive oxygen species, increasing the activity of ALDH, accelerating the conversion of acetaldehyde to non-toxic acetic acid, and reducing the accumulation of acetaldehyde in the liver, alcoholic liver damage can be improved to some extent.

[0004] In recent years, the activity of bioactive peptides in the field of alcoholism prevention and liver protection has gradually become a research hotspot. However, the identification of peptide sequences with alcoholism prevention and liver protection activity and related research are relatively lacking, including the elucidation of the molecular mechanism of peptide activation of ADH and ALDH, therefore, it is urgent to develop a rapid, convenient and efficient screening method for alcoholism prevention and liver protection peptides. Walnut protein is a protein resource that is rich in nutrition, balanced in amino acid composition and high in bioavailability. So far, there has been no report on ADH and ALDH activating peptides from walnut protein. SUMMARY

[0005] The purpose of the present application is to provide a peptide segment with ADH and ALDH activation ability, which can be used for the application development of alcoholism prevention and liver protection products.

[0006] The present application provides a method for screening ADH and ALDH activating peptides from walnut protein, mainly comprising the following steps:

[0007] (1) Extracting walnut protein from walnut kernels by alkali dissolution and acid precipitation method

[0008] The walnut protein powder is mixed with water at a ratio of 1:20 (w:v), the pH is adjusted to 10.00-11.0, and then centrifuged (7000r / min, 15-20min) after continuous stirring at room temperature for 3-4h. The supernatant is collected and stored, and the supernatant is adjusted to pH 4.5 and centrifuged again (7000r / min, 15-20min). The precipitate is redissolved and the pH is adjusted to 7.0. After dialysis, freeze-drying is performed to obtain walnut protein powder.

[0009] (2) Hydrolyzing walnut protein to produce walnut peptides by using protease

[0010] The walnut protein powder obtained in step (1) is mixed with water at a certain ratio, and then protease is added to hydrolyze the walnut protein. After hydrolysis, the walnut protein hydrolysate is boiled in water to inactivate the enzyme. After cooling to room temperature, the pH is adjusted to 7.0, centrifuged, and the supernatant is freeze-dried to obtain walnut protein hydrolysate powder, which is stored at -20℃ for standby. The protease is: alkaline protease, neutral protease, complex protease and flavor protease.

[0011] (3) In vitro simulation of gastrointestinal digestion

[0012] The walnut peptides obtained in step (2) are mixed with pepsin hydrochloride solution (2000U / mL, pH 3.0) at a certain ratio, and incubated at constant temperature (37℃, 150rpm, 2h) to simulate gastric digestion (SGD). Then the pH of the digestion system is adjusted to 7.0, and the enzyme is inactivated and cooled quickly. At this time, it is recorded as 2h. 100U / mL trypsin is added to the remaining sample to simulate the intestinal digestion (SID) stage. Sample is taken at different time points, enzyme is inactivated and cooled quickly.

[0013] (4) Identification screening

[0014] The walnut protein enzymatic powder obtained in step (2) is identified by polypeptidomics to screen potential new ADH and ALDH activating peptides from walnut peptides under various screening conditions.

[0015] (5) Synthetic verification

[0016] The internal mechanism of activating ADH and ALDH is explored by molecular docking, and the target sequence is synthesized in vitro to detect the ADH and ALDH activation rate.

[0017] In one embodiment, in step (1), the walnut protein powder is a dried protein powder after removing fat.

[0018] In one embodiment, in step (2), when the protease is alkaline protease, the hydrolysis conditions are: temperature is 55℃, pH is 8.5, and hydrolysis time is 4h; when the protease is neutral protease, the hydrolysis conditions are: temperature is 45℃, pH is 7.0, and hydrolysis time is 4h; when the protease is complex protease, the hydrolysis conditions are: temperature is 55℃, pH is 8.0, and hydrolysis time is 3h; when the protease is flavor protease, the hydrolysis conditions are: temperature is 50℃, pH is 7.0, and hydrolysis time is 2.5h.

[0019] In one embodiment, in step (1), the following implementation conditions are used: walnut protein powder is mixed with water at a ratio of 1:20 (w / v), the pH is adjusted to 10.00-11.0, and after continuous stirring at room temperature for 3h, centrifugation (7000r / min, 15min) is performed, the supernatant is collected and stored, the supernatant pH is adjusted to 4.5 and centrifugation (7000r / min, 15min) is performed again, the precipitate is redissolved and the pH is adjusted to 7.0. After dialysis, freeze-drying is performed to obtain walnut protein powder. During hydrolysis, 1mol / L NaOH or HCl is used to maintain the pH of the solution constant.

[0020] In one embodiment, in step (2), the following implementation conditions are used: walnut protein powder is mixed with water at a ratio of 1:25 (w:v) to prepare a protein solution with a substrate concentration of 4%. After mixing, the pH and temperature are adjusted to the optimal pH and temperature conditions of the used enzyme, and protease is added at an addition amount of 10000U / g protein to perform hydrolysis, and stirring extraction is performed at room temperature for 2-4h. After hydrolysis to constant pH, enzyme is inactivated by boiling water bath for 10min, and after cooling to room temperature, the pH is adjusted to 7.0, and centrifugation is performed at 8000r / min for 20min. After collecting the supernatant, freeze-drying is performed after dialysis to obtain walnut protein enzymatic powder under different protease hydrolysis conditions.

[0021] In one embodiment, in step (3), the following implementation conditions are adopted: 1 g of walnut peptide protease hydrolysate is weighed into 10 mL of 2000 U / mL pepsin hydrochloride solution (pH 3.0), and simulated gastric digestion (SGD) is carried out by constant temperature oscillation culture (37°C, 150 rpm, 2 h). Subsequently, enteric digestion (SID) is carried out, the pH of the digestion system is adjusted to 7.0, and the volume is adjusted to 20 mL. 10 mL is sampled at this point, the enzyme is inactivated and cooled quickly, and this time is recorded as 2 h. 100 U / mL trypsin is added to the remaining sample, and samples are taken at different time points, the enzyme is inactivated and quickly cooled. The freeze-dried sample is used for determination of ADH and ALDH activation activity.

[0022] In one embodiment, step (4) can specifically adopt the following steps:

[0023] ① Preliminary screening conditions: the amino acid sequence obtained by sequencing is subjected to potential biological activity prediction, protein source prediction, toxicity and allergenicity prediction by Peptide Ranker software, protein database, Toxinpred software and AllerTOP software. From the sequenced peptide segments, the peptide segments belonging to protein sources, not modified by functional groups, with peak area > 1.00 x 106, peptide sequence containing 3-12 amino acids, and activity score ≥ 0.7 are selected.

[0024] ② Rapid screening conditions: select peptide sequences with amphiphilic property, steric hindrance ≤ 0.65, hydrophobic amino acid ratio ≥ 50%, and characteristic amino acid ≥ 50%. Peptides with such characteristics have potential ADH activation activity.

[0025] In one embodiment, in step (5), the following steps can be specifically adopted:

[0026] ① Obtain small molecule ligands: use Chem Draw (version ChemBio Draw 22.0.0) software and Chem3D (version ChemBio Draw 22.0.0) software to construct 2D and 3D structures of the peptide sequences that need to be docked.

[0027] ② Preparation of ADH and ALDH PDB files: retrieve the protein crystal structure of ADH and ALDH (5ENV and 1O04, respectively) from the protein database, and remove excess ligands and water molecules to facilitate subsequent docking with peptide segments.

[0028] ③Molecular docking: convert the format of ligand and receptor into PDBQT in AutoDock Tools 1.5.7, and use AutoDockVina software to perform molecular docking simulation experiment, and the result is output as predicted binding energy, and the lower the binding energy, the stronger the binding of the ligand with the protein pocket. The ADH and ALDH activating peptides of the prior art disclosed sequence are excluded, and the remaining polypeptides are determined as potential ADH and ALDH activating peptides, which are synthesized by solid phase synthesis in polypeptide chemical synthesis method and verified for activating activity.

[0029] ④ The peptide segments screened are synthesized by solid phase synthesis in polypeptide chemical synthesis method, and the ADH and ALDH activating activities are determined by substrate chemical method to determine the strength of ADH and ALDH activating activity to verify the screening results.

[0030] The application provides a polypeptide PGHFEAF, and the amino acid sequence of the polypeptide is

[0031] Pro-Gly-His-Phe-Glu-Ala-Phe.

[0032] The application further provides a product containing the polypeptide PGHFEAF.

[0033] In an embodiment, the product comprises food, medicine or health care product.

[0034] In an embodiment, the food comprises dairy product, fruit and vegetable product, meat product or bean product.

[0035] In an embodiment, the dosage form of the medicine comprises tablet, capsule, injection, liposome nanoparticle, sustained-release preparation, dispersible tablet or enteric granule.

[0036] In an embodiment, the product further contains a derivative of the polypeptide PGHFEAF; the derivative of the polypeptide refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, the amino terminal or the carbonyl terminal of the polypeptide.

[0037] The application further provides application of the polypeptide PGHFEAF in preparation of a medicine for sobering up and protecting liver.

[0038] In an embodiment, the medicine further contains a pharmaceutically acceptable carrier.

[0039] In an embodiment, the content of the polypeptide PGHFEAF in the medicine is at least 0.49 mM or 393.65 mg / g.

[0040] In an embodiment, the medicine has both alcohol dehydrogenase activating activity and acetaldehyde dehydrogenase activating activity.

[0041] The application also provides application of the polypeptide PGHFEAF in preparation of health products with auxiliary protection effect on chemical liver injury.

[0042] Beneficial effects:

[0043] The application first identifies the high-activity ADH and ALDH activating polypeptide PGHFEAF from walnut protein, and the docking binding energy of the polypeptide PGHFEAF with ADH and ALDH is-8.2 and-10.5 kcal / mol respectively, the ADH EC 50 value is 0.49±0.09 mM, and the ALDH EC 50 value is 1.85±0.28 mM; the polypeptide PGHFEAF can resist gastrointestinal digestion, and after in-vitro digestion for 4 h, the ADH and ALDH activation rates are retained by 58.43% and 41.58% respectively. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Effect of enzyme species on ADH and ALDH activation rates of walnut peptide;

[0045] Figure 2 ADH and ALDH activation rates of walnut protein hydrolysate produced by alkaline protease;

[0046] Figure 3 Effect of in-vitro simulation of gastrointestinal digestion on ADH and ALDH activation activities of walnut protein hydrolysate;

[0047] Figure 4 Molecular structural formula of PGHFEAF;

[0048] Figure 5 Molecular docking diagram of PGHFEAF and ADH;

[0049] Figure 6 Molecular docking diagram of PGHFEAF and ALDH;

[0050] Figure 7 EC 50 values of ADH and ALDH activation by PGHFEAF;

[0051] Figure 8 Effect of in-vitro simulation of gastrointestinal digestion on ADH and ALDH activation activities of PGHFEAF. DETAILED DESCRIPTION

[0052] The walnut kernels used in the following examples were purchased from Moore Farm Biotechnology Co., Ltd., and the commercial proteases used were purchased from Novozymes (China) Biotechnology Co., Ltd. The measured enzyme activity of neutral protease was 74898.8 U / mL, the measured enzyme activity of alkaline protease was 296816.9 U / mL, the measured enzyme activity of protease was 94187.9 U / mL, and the measured enzyme activity of flavor protease was 45268.1 U / mL.

[0053] The walnut protein powder used in the following examples is a dried protein powder after removing fat.

[0054] The following examples relate to a detection method for measuring the activation rate of ADH and ALDH by substrate chemistry:

[0055] (1) Detection method for measuring the activation rate of ADH

[0056] The detection principle is that ADH catalyzes the dehydrogenation of ethanol to produce acetaldehyde, and NAD + is reduced to NADH. NADH has a maximum absorption peak at 340 nm, while other components do not have color development. Therefore, the amount of NADH generated can be indirectly indicative of ADH activity.

[0057] In a 96-well enzyme plate, 50 μL of sample was mixed with 150 μL of working solution (reagent 1: reagent 2: reagent 3 = 0.65: 0.05: 0.70), incubated at 37°C for 5 min, then 50 μL of ADH solution (enzyme activity 0.2 U / mL) was added to initiate the reaction, and the reaction was carried out at 37°C for 10 min. The absorbance was measured at 340 nm using an enzyme marker, and distilled water was used instead of the sample as a blank control. The formula for calculating the activation rate of ADH is:

[0058]

[0059] (2) Detection method for measuring the activation rate of ALDH

[0060] The detection principle is that acetaldehyde is oxidized by ALDH to produce NADH, and then the colorless probe is reduced to a colored product, which has strong absorbance at 450 nm, so it can be indirectly indicative of ALDH activity.

[0061] In a 96-well enzyme plate, 50 μL of sample was mixed with 200 μL of working solution (a mixture composed of acetaldehyde, NAD + and commercial probe according to the requirements of the kit manufacturer), incubated at 37°C for 5 min, then 50 μL of ALDH solution (enzyme activity 0.5 U / mL) was added to initiate the reaction, and the reaction was carried out at 37°C for 10 min. Distilled water was used instead of the sample as a blank control. The formula for calculating the activation rate of ALDH is:

[0062]

[0063] In the following examples, the amino acid sequence of the PGHFEAF is Pro-Gly-His-Phe-Glu-Ala-Phe.

[0064] Example 1: Method for obtaining walnut protein hydrolysate from walnut protein

[0065] (1) Walnut protein was extracted from walnut kernels by alkali dissolution and acid precipitation

[0066] Defatted walnut powder was mixed with water at a ratio of 1:20 (w:v), the pH was adjusted to 10.00-11.0, and after continuous stirring at room temperature for 3 h, centrifugation was performed (7000 r / min, 15 min), the supernatant was collected and stored, the supernatant was adjusted to pH 4.5 and centrifuged again (7000 r / min, 15 min), the precipitate was redissolved and the pH was adjusted to 7.0. After dialysis, freeze-drying was performed to obtain walnut protein powder.

[0067] (2) Walnut peptides were produced by hydrolyzing walnut protein using proteases

[0068] Walnut protein powder was mixed with water at a ratio of 1:25 (w:v) to prepare a protein solution with a substrate concentration of 4%. After mixing, the pH and temperature were adjusted to the optimal pH and temperature conditions for the enzymes used, different proteases were added according to an enzyme-to-substrate ratio of 10000 U / g protein, and hydrolysis was performed at room temperature with continuous stirring until the pH was constant. The hydrolysis conditions are shown in Table 1.

[0069] Table 1 Hydrolysis conditions for different proteases

[0070]

[0071] During the hydrolysis process, 1 mol / L NaOH or HCl was used to maintain the pH of the solution constant.

[0072] After the hydrolysis was completed, the walnut protein hydrolysate was boiled for 10 min to inactivate the enzymes, and after cooling to room temperature, the pH was adjusted to 7.0, and centrifugation was performed at 8000 r / min for 20 min. The supernatant was collected, dialyzed, and freeze-dried to obtain walnut protein hydrolysate powder under different protease hydrolysis conditions.

[0073] (3) The ADH and ALDH activation activities of walnut protein hydrolysate powder obtained under different protease hydrolysis conditions were determined. The alkaline protease hydrolysate with high activation activity was selected Figure 1 ), and the results showed that the ADH and ALDH activation rates of the alkaline protease hydrolysate were as high as 73.21±2.68 and 83.71±2.18%, respectively, and the EC 50The values were 0.05546 ± 0.0047 mg / mL and 0.0606 ± 0.0096 mg / mL, respectively. The next step of sequencing was then performed.

[0074] Example 2: In vitro simulated gastrointestinal digestion of walnut protein enzymatic hydrolysate

[0075] The specific steps are as follows:

[0076] 1 g of the alkaline protease hydrolysate obtained in Example 1 was dissolved in 10 mL of a pepsin hydrochloride solution (pH 3.0) at 2000 U / mL, and constant temperature oscillation culture (37°C, 150 rpm, 2 h) was performed to simulate gastric digestion (SGD). Subsequently, the digestion system was adjusted to pH 7.0 and then diluted to 20 mL to enter the simulated intestinal digestion (SID) stage. 10 mL was sampled at this point, the enzyme was inactivated and cooled quickly, and this time was recorded as 2 h. 100 U / mL trypsin was added to the remaining sample, and samples were taken at different time points, the enzyme was inactivated and cooled quickly. The freeze-dried samples were used to determine the ADH and ALDH activation activities. The results showed that Figure 3 ), after 2 h of pepsin digestion, the ADH activation rate decreased from 73.61% to 68.25%, and the activity retention was 92.72% (the activity retention rate was the ratio of the activation rate after digestion to that before digestion); the ALDH activation rate decreased from 83.71% to 60.24%, and the activity retention was 61.04%. After the in vitro intestinal simulated digestion, the final ADH and ALDH activation rates were 53.14% and 40.80%, respectively, and a relatively high activation activity was still retained.

[0077] Example 3: Method for separating and identifying ADH and ALDH activation peptides from walnut protein enzymatic hydrolysate

[0078] The specific steps are as follows:

[0079] The specific implementation is the same as in Example 1, except that the protease is adjusted to be alkaline protease to obtain walnut protein enzymatic hydrolysate.

[0080] (1) Peptide sequence identification of walnut protein enzymatic hydrolysate by peptidomics

[0081] The walnut protein enzymolysis sample was first desalted by C18 desalination column, and then analyzed by LC-MS / MS equipped with online nanospray ion source. The whole system is a tandem EASY-nanoLC 1200 Orbitrap Exploris 480 mass spectrometer. A total of 2 μL sample (analysis column: Acclaim PepMap C18, 75 μm x 25 cm) was loaded, and the sample was separated with a 60 min gradient, the column flow was controlled at 350 nL / min, the column temperature was 40℃, the electrospray voltage was 2 kV, and the gradient was from 2.2% B phase, increased to 50% at 51 min, increased to 90% in 3.5 min, and maintained for 5.5 min.

[0082] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometer parameters are as follows: (1) MS: scan range (m / z): 350-1500; resolution: 60,000; Normalized AGC target: 300%; maximum injection time: 25 ms; (2) HCD-MS / MS: resolution: 15,000: Normalized AGC target: 50%; maximum injection time: 22 ms; collision energy: 30%; dynamic exclusion time: 30 s.

[0083] Tandem mass spectrum analysis was completed using PEAKS Studio version 10.6. The uniprot_Cicerarietinum (version 202112, 24812 entries) database was searched by PEAKSDB. The protein card value is: -10lgP≥0, containing at least 1 unique peptide; the peptide segment card value is: -10lgP≥20.

[0084] After polypeptide sequencing of walnut protein hydrolysate, 3554 polypeptides were identified.

[0085] (2) Using a variety of peptide screening conditions to screen high-activity ADH and ALDH activating peptides from the 3554 polypeptides identified in (1), mainly including the following steps:

[0086] ① Preliminary screening conditions: potential biological activity prediction, protein source prediction, toxicity and allergenicity prediction were performed by Peptide Ranker software, protein database, Toxinpred software and AllerTOP software respectively. From the sequenced peptides, select peptides belonging to the protein source, not modified by the functional group, peak area > 1.00 x 10 6 , peptide sequence contains 3-12 amino acids, and activity score ≥0.7.

[0087] (2) Rapid screening conditions: select peptides with amphiphilic, steric hindrance ≤ 0.65, hydrophobic amino acid ratio ≥ 50%, and characteristic amino acid ratio ≥ 50%. Peptides with such characteristics have potential ADH activation activity.

[0088] (3) Screening and verification of peptide segments obtained from (2) using molecular docking

[0089] The specific steps are as follows:

[0090] (1) Obtain small molecule ligands: use Chem Draw (version ChemBio Draw 22.0.0) software and Chem3D (version ChemBio Draw 22.0.0) software to construct the 2D and 3D structures of the peptide sequences that need to be docked.

[0091] (2) ADH and ALDH PDB file preparation: retrieve the protein crystal structure of ADH and ALDH (5ENV and 1O04, respectively) from the protein database, remove excess ligands and water molecules, and prepare for subsequent docking with peptide segments.

[0092] (3) Molecular docking: convert the format of the ligand and receptor to PDBQT in AutoDock Tools 1.5.7, use AutoDockVina software for molecular docking simulation, and the output is the predicted binding energy. The lower the binding energy, the stronger the binding of the ligand to the protein pocket. Exclude ADH and ALDH activating peptides disclosed in the prior art, and determine the remaining polypeptides as potential ADH and ALDH activating peptides, which are synthesized by in vitro solid phase synthesis and verified for their activating activity.

[0093] Specifically, a total of 5 polypeptides were screened, each with a binding energy to ADH and ALDH less than -7.5 kcal / mol (Table 2); the 5 polypeptides were synthesized by in vitro solid phase synthesis and their ADH and ALDH activation activities were determined.

[0094] Table 2 Peptide docking energy with ADH molecule

[0095]

[0096] The 5 screened peptide segments were synthesized by solid phase synthesis method in polypeptide chemical synthesis method, and the ADH and ALDH activation activity was determined by substrate chemistry method to determine the strength of ADH and ALDH activation activity to verify the screening results.

[0097] Table 3 ADH and ALDH activation activity of synthesized peptides

[0098]

[0099] In general, the binding energy of PGHFEAF with ADH and ALDH is low, and the binding degree is high. In addition, the in vitro activation ability is strong, and the EC 50 values of ADH and ALDH activation of PGHFEAF are 0.49±0.09 mM and 1.85±0.28 mM, respectively (Table 3), and the activation ability is strong. After conversion, the content of 0.49 mM polypeptide in the solid product is 393.65 mg / g. PGHFEAF is selected as the target polypeptide of this study. By searching the BIOPEP-UWM database, it is found that there is no literature reported on PGHFEAF. Therefore, PGHFEAF is a new bioactive peptide.

[0100] Example 4: ADH and ALDH activation peptide PGHFEAF

[0101] (1) Structure analysis of PGHFEAF

[0102] As shown in Figure 4 , the molecular structure formula of PGHFEAF is obtained by Chem Draw and Auto Dock.

[0103] (2) Molecular docking of PGHFEAF with ADH and ALDH

[0104] As shown in Figure 5 , PGHFEAF forms hydrogen bonds with eight amino acid residues HIS-171, ASN-166, THR-264, SER-289, ASN-262, ASN-107, and LYS-160 in the active pocket of ADH. As shown in Figure 6 , PGHFEAF forms hydrogen bonds with six amino acid residues GLY-449, ALA-448, VAL-451, VAL-453, and SER-443 in the active pocket of ALDH.

[0105] (3) EC 50 values of ADH and ALDH activation of PGHFEAFL

[0106] The polypeptide sample PGHFEAF is prepared into solutions with different concentrations to determine the ADH and ALDH activation rates and plot analysis, and the ADH and ALDH half-inhibitory concentrations (EC 50 ) are calculated, i.e. the polypeptide concentration required for the activation activity to reach 50%. As shown in Figure 7 , the ADH EC 50 value of the polypeptide PGHFEAF is 0.49±0.09 mM, and the ALDH EC 50 value is 1.85±0.28 mM.

[0107] (4) In vitro simulation of gastrointestinal digestion of PGHFEAFL

[0108] Reference to the method of Example 2, PGHFEAF was subjected to in vitro simulated gastrointestinal digestion, and the change of ADH and ALDH activation rate during the digestion was determined. The results are shown in Table 2. Figure 8 As shown in Table 2, after 2h of pepsin digestion, the ADH and ALDH activation rates of PGHFEAF were reduced to 70.20% and 52.45%, respectively, and the activities were retained at 87.44% and 84.82%, respectively; after in vitro intestinal simulated digestion, the final ADH and ALDH activation rates were 58.43% and 41.58%, respectively, which showed that PGHFEAF exhibited better digestion resistance and biological stability. The activity retention rate is the ratio of the activation rate after digestion to the activation rate before digestion.

[0109] Although the present application has been disclosed with reference to the preferred embodiments thereof, it is not intended to limit the application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A polypeptide PGHFEAF, characterized in that, The amino acid sequence of the polypeptide is Pro-Gly-His-Phe-Glu-Ala-Phe.

2. A product containing the polypeptide PGHFEAF of claim 1, characterized in that, The products include food, medicine, or health products.

3. The product as described in claim 2, characterized in that, The food products include dairy products, fruit and vegetable products, meat products, or soy products.

4. The product as described in claim 3, characterized in that, The dosage forms of the drug include tablets, capsules, injections, liposome nanoparticles, sustained-release formulations, or enteric-coated granules.

5. The use of the polypeptide PGHFEAF according to claim 1 in the preparation of a drug for sobering up and protecting the liver.

6. The application as described in claim 5, characterized in that, The drug has both alcohol dehydrogenase activating activity and acetaldehyde dehydrogenase activating activity.

Images