Dry-cured ham skin source antioxidant peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antioxidant peptides, and particularly relates to a dry-cured ham skin source antioxidant peptide as well as a preparation method and application thereof. The antioxidative peptide comprises one or more of an antioxidative peptide I, an antioxidative peptide II, an antioxidative peptide III and an antioxidative peptide IV, wherein the amino acid sequences of the antioxidative peptide I, the antioxidative peptide II, the antioxidative peptide III and the antioxidative peptide IV are respectively shown as SEQ ID NO. 1 to SEQ ID NO. 4. The antioxidant peptide is extracted from dry-cured ham skin, antioxidant activity verification is carried out through a DPPH method, an ABTS method and a ferrous ion chelating ability method, it is found that the antioxidant peptide is good in antioxidant activity, the antioxidant peptide can be applied to food or drugs, and important raw materials and technical supports are provided for development of antioxidant products.

Description

Technical Field

[0001] The invention belongs to the technical field of antioxidant peptides, and in particular relates to a dry-cured ham skin-derived antioxidant peptide and a preparation method and application thereof. Background Art

[0002] Antioxidant peptides refer to peptide substances with antioxidant functions. They can inhibit the production of free radicals and protect cells from damage caused by oxidative stress. Some studies have shown that antioxidant peptides can significantly reduce the level of free radicals in the body, enhance the body's own antioxidant capacity, prevent diseases, and maintain the body's health. They are mostly used in the field of health care products and pharmaceutical preparation. At the same time, other studies have shown that because antioxidant peptides meet the characteristics of "natural, safe, non-toxic, and high nutritional value", they can delay the formation of lipid peroxides and help maintain the flavor and texture of food. A large number of antioxidant peptides are also widely used in the food field.

[0003] Antioxidant peptides have a wide range of sources, including soybean peptides, corn peptides, sea cucumber peptides, etc., and the development of nutritious and safe antioxidant peptides using low-cost or low-utilization food by-product resources has always been a research hotspot in the peptide field.

[0004] Dry-cured ham is a typical dry-cured meat product. It is a fermented meat product with special flavor, which is made by dry-curing, dehydration, maturation and post-ripening with salt as the main curing agent. It is welcomed by consumers because of its special flavor. For a long time, the people have always believed that ham has good nutritional functions and good nourishing effects. However, excessive sodium intake will bring health risks to consumers, and reducing the salt content of dry-cured ham is an important way to reduce the risk of consumption and ensure the health of consumers, which makes low-salt dry-cured ham more and more popular among dry-cured ham lovers. However, low-salt dry-cured ham skin is usually not eaten, and most of them are directly discarded, resulting in waste of resources. Therefore, if low-salt dry-cured ham skin is developed to prepare peptides with antioxidant activity, it can not only solve the current problem of resource waste, but also expand the application range of dry-cured ham, and realize high-value utilization, which is of great significance. However, there is no related research on the successful preparation of antioxidant peptides using dry-cured ham skin as raw material in existing research. Summary of the invention

[0005] The purpose of the present invention is to provide a dry-cured ham skin-derived antioxidant peptide and a preparation method and application thereof. The antioxidant peptide is extracted from the dry-cured ham skin and has high antioxidant activity.

[0006] The present invention provides an antioxidant peptide, including one or more of antioxidant peptide I, antioxidant peptide II, antioxidant peptide III and antioxidant peptide IV, wherein the amino acid sequences of the antioxidant peptide I, antioxidant peptide II, antioxidant peptide III and antioxidant peptide IV are shown in SEQ ID NO.1 to SEQ ID NO.4, respectively.

[0007] The present invention also provides a method for preparing the antioxidant peptides described in the above technical solution, comprising the following steps: enzymatically hydrolyzing dry-cured ham skin with an acidic protease, and performing solid-liquid separation on the obtained enzymatic hydrolysis mixture to obtain an enzymatic hydrolyzate;

[0008] Performing gel chromatography separation on the enzymatic hydrolysate, collecting the component with the highest DPPH free radical scavenging rate, and obtaining a gel chromatography chromatographic solution;

[0009] The gel chromatography liquid is subjected to solid phase extraction to obtain a solid phase extraction liquid, wherein the solid phase extraction liquid contains the antioxidant peptide.

[0010] Preferably, based on the dry product of the dry-cured ham skin, the dosage of the acid protease is 2500-10000 U / g of the dry product of the dry-cured ham skin.

[0011] Preferably, the enzymatic hydrolysis temperature is 45-55°C, the time is 5-8 hours, and the pH value is 2-5.

[0012] Preferably, the prepacked column for gel chromatography separation is Sephadex G-25.

[0013] Preferably, the mobile phase for gel chromatography separation is ultrapure water, and the flow rate is 1 to 4 mL / min.

[0014] Preferably, the solid phase extraction column is an Empore solid phase extraction column C18; the eluent of the solid phase extraction is a 0.1% by volume formic acid acetonitrile aqueous solution, the volume concentration of acetonitrile in the formic acid acetonitrile aqueous solution is 84%, and the flow rate is 250 μL / min.

[0015] Preferably, after the solid phase extraction, the method further comprises performing mass spectrometry sequencing and sequence comparison analysis on the solid phase extraction liquid to obtain the antioxidant peptide.

[0016] Preferably, before the enzymolysis, the dry-cured ham skin is sequentially cleaned, dried and ground to obtain dry-cured ham skin powder; the cleaning step comprises: mixing the dry-cured ham skin, water and anhydrous sodium carbonate and stirring for 24 hours to obtain a first mixture;

[0017] The first mixture, water and sodium chloride are mixed and stirred for a second time for 24 hours, and then clean dry-cured ham skin is obtained by separation.

[0018] The present invention also provides the use of the antioxidant peptide described in the above technical solution or the antioxidant peptide prepared by the preparation method described in the above technical solution in food and / or medicine.

[0019] Beneficial effects:

[0020] The present invention provides an antioxidant peptide, including one or more of antioxidant peptide I, antioxidant peptide II, antioxidant peptide III and antioxidant peptide IV, wherein the amino acid sequences of the antioxidant peptide I, antioxidant peptide II, antioxidant peptide III and antioxidant peptide IV are shown in SEQ ID NO.1 to SEQ ID NO.4, respectively. The antioxidant peptide of the present invention is extracted from dry-cured ham skin, and the antioxidant activity is verified by DPPH method, ABTS method and ferrous ion chelation ability method, and it is found that the antioxidant activity is good, and it can be applied to food or medicine, providing important raw materials and technical support for the development of antioxidant products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 This is a gel chromatogram of the peptides obtained after enzymatic hydrolysis of ham skin powder in step S1 of Example 1;

[0023] Figure 2 The antioxidant result of the best component F2 obtained by gel chromatography in step S1 in Example 1;

[0024] Figure 3 This is a diagram showing the molecular docking result of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.1 in Example 1 and the protein receptor Keap1;

[0025] Figure 4 This is a diagram showing the molecular docking result of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.2 in Example 1 and the protein receptor Keap1;

[0026] Figure 5 This is a diagram showing the molecular docking result of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.3 in Example 1 and the protein receptor Keap1;

[0027] Figure 6 This is a diagram showing the molecular docking result of the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.4 in Example 1 and the protein receptor Keap1;

[0028] Figure 7The results of the antioxidant peptides with amino acid sequences such as SEQ ID NO.1 to SEQ ID NO.4 and the synthetic glutathione (GSH) of the control group in Example 2 are shown in the figure below: DPPH free radical scavenging ability, ABTS free radical scavenging ability, and ferrous ion chelating ability of the antioxidant peptides;

[0029] Among them, Figures 3 to 6 In the figure, A is the cartoon interaction between the short peptide and Keap1 protein; B is the surface interaction between the short peptide and the protein; C is the 2D interaction details between the short peptide and the protein, where chain A is the short peptide, chain X is 2FLU, the gear shape is the hydrophobic amino acid, and the green dotted line represents the hydrogen bond. DETAILED DESCRIPTION

[0030] The present invention provides an antioxidant peptide, including one or more of antioxidant peptide I, antioxidant peptide II, antioxidant peptide III and antioxidant peptide IV, wherein the amino acid sequences of the antioxidant peptide I, antioxidant peptide II, antioxidant peptide III and antioxidant peptide IV are shown in SEQ ID NO.1 to SEQ ID NO.4, respectively.

[0031] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.4 of the present invention are as follows: SEQ ID NO.1: AAWGKVGGQAGAHGAEALER; SEQ ID NO.2: DQALKPTKPMRFLGDE; SEQ ID NO.3: GVNGFGRIGR; SEQ ID NO.4: SLTGEFKGKYYPL. The docking energies of the antioxidant peptide I, antioxidant peptide II, antioxidant peptide III and antioxidant peptide IV of the present invention with the receptor protein Keap1 are -6.835 kcal / mol, -6.429 kcal / mol, -8.367 kcal / mol and -6.451 kcal / mol respectively.

[0032] The present invention also provides a method for preparing the antioxidant peptides described in the above technical solution, comprising the following steps: enzymatically hydrolyzing dry-cured ham skin with an acidic protease, and performing solid-liquid separation on the obtained enzymatic hydrolysis mixture to obtain an enzymatic hydrolyzate;

[0033] Performing gel chromatography separation on the enzymatic hydrolysate, collecting the component with the highest DPPH free radical scavenging rate, and obtaining a gel chromatography chromatographic solution;

[0034] The gel chromatography liquid is subjected to solid phase extraction to obtain a solid phase extraction liquid, wherein the solid phase extraction liquid contains the antioxidant peptide.

[0035] The present invention preferably washes the dry-cured ham skin in sequence to obtain a clean dry-cured ham skin. The cleaning step of the present invention preferably includes: mixing the dry-cured ham skin, water and anhydrous sodium carbonate and stirring for 24 hours to obtain a first mixture; mixing the first mixture, water and sodium chloride and stirring for 24 hours, and separating to obtain a clean dry-cured ham skin. The dry-cured ham skin of the present invention is preferably low-salt dry-cured ham skin, and more preferably is derived from the low-salt dry-cured ham disclosed in the South African patent with patent number ZA202303077B (patent name METHOD FOR MAKING LOW-SODIUM FERMENTED FLAVORED MEATPRODUCT, public authorization time is May 31, 2023). During the first stirring process of the present invention, the mass of the dry-cured ham skin and the volume of water are preferably 1:4-8, more preferably 1g:4mL; the water is preferably ultrapure water; the mass ratio of the dry-cured ham skin to anhydrous sodium carbonate is preferably 4-6:1, more preferably 5:1. During the second stirring process of the present invention, the mass ratio of the volume of water to the dry-cured ham skin is preferably 4-8:1, more preferably 4mL:1g; the mass ratio of the dry-cured ham skin to sodium chloride is preferably 4-6:1, more preferably 5:1. The use of anhydrous sodium carbonate and sodium chloride to clean the dry-cured ham skin in the present invention is to use anhydrous sodium carbonate to remove residual grease on the surface of the ham skin, and sodium chloride to remove impurities on the surface of the ham skin. The temperatures of the first stirring and the second stirring of the present invention are preferably 4°C, and the methods of the first stirring and the second stirring are not particularly limited, and conventional stirring methods in the art can be used.

[0036] After obtaining the clean dry-cured ham skin, the present invention preferably dries the clean dry-cured ham skin to obtain dried dry-cured ham skin. The drying method of the present invention is preferably drying, the drying temperature is preferably 50-70°C, more preferably 55°C; the drying time is preferably 24-48h, more preferably 24h.

[0037] After obtaining the dried dry-cured ham skin, the present invention preferably grinds the dried dry-cured ham skin to obtain dry-cured ham skin powder. The present invention preferably uses a wall-breaking machine for the grinding, and the specific process is not particularly limited; the particle size of the dry-cured ham skin powder is preferably 60 to 80 meshes, more preferably 80 meshes.

[0038] After obtaining the dry-cured ham skin powder, the present invention preferably mixes and enzymolyzes the dry-cured ham skin powder, water and acidic protease to obtain an enzymolysis mixture. The mass ratio of the dry-cured ham skin powder and water in the present invention is preferably 1:5-20, more preferably 1:10. The dosage of the acidic protease in the present invention is preferably 2500-10000U / g dry-cured ham skin powder, more preferably 5000U / g dry-cured ham skin powder. The temperature of the enzymolysis in the present invention is preferably 45-55°C, more preferably 50-55°C; the time is preferably 5-8h, more preferably 6h; the pH value is preferably 2-5, more preferably 3.

[0039] After obtaining the enzymatic hydrolysis mixture, the present invention preferably performs an enzyme inactivation treatment on the enzymatic hydrolysis mixture, and the enzyme inactivation treatment is preferably performed by boiling the enzymatic hydrolysis mixture at 100° C. for 15 minutes.

[0040] After the enzyme inactivation treatment, the present invention preferably performs solid-liquid separation on the enzymatic hydrolysis mixture after the enzyme inactivation treatment, and takes the supernatant to obtain an enzymatic hydrolysis solution. The solid-liquid separation method of the present invention is preferably centrifugation, and the centrifugation speed is preferably 8000r / min, and the time is preferably 15min.

[0041] After obtaining the enzymatic hydrolysate, the present invention performs gel chromatography separation on the enzymatic hydrolysate, collects the component with the highest DPPH free radical scavenging rate, and obtains a gel chromatography chromatographic fluid. The pre-packed column for gel chromatography separation of the present invention is preferably Sephadex G-25, and the specification is preferably 16×80cm; the mobile phase for gel chromatography separation is preferably ultrapure water, and the flow rate is preferably 1 to 4mL / min, more preferably 2mL / min. The duration of collecting the gel chromatography chromatographic fluid each time (tube) of the present invention is preferably 4.5min, and the component with the highest DPPH free radical scavenging rate is preferably the gel chromatography chromatographic fluid of the second separation peak, that is, the gel chromatography chromatographic fluid composed of the 10th to 26th tubes.

[0042] After obtaining the gel chromatography liquid, the present invention preferably freeze-dries the gel chromatography liquid to obtain a freeze-dried powder of the gel chromatography liquid. The present invention has no special limitation on the freeze-drying process, and the conventional freeze-drying process in the art can be used.

[0043] After obtaining the freeze-dried powder of the gel chromatography liquid, the present invention performs solid phase extraction desalting on the freeze-dried powder of the gel chromatography liquid to obtain a solid phase extraction liquid. The extraction column of the solid phase extraction of the present invention is preferably an Empore solid phase extraction column C18; the eluent of the solid phase extraction is preferably a formic acid acetonitrile aqueous solution with a volume concentration of 0.1%, the volume concentration of acetonitrile in the formic acid acetonitrile aqueous solution is preferably 84%, and the flow rate is preferably 250 μL / min.

[0044] After obtaining the solid phase extract, the present invention preferably performs mass spectrometry sequencing and sequence alignment analysis on the solid phase extract to obtain the antioxidant peptide. The mass spectrometry sequencing method of the present invention is preferably LC-MS / MS, and the specific process is not particularly limited, and the conventional mass spectrometry sequencing process in the art can be used. The database for sequence alignment analysis of the present invention is preferably the Uniprot protein database.

[0045] The antioxidant peptides having the specific amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.4 prepared by the above preparation method have good antioxidant activity and can be used for the preparation of food and medicine.

[0046] Based on the above advantages, the present invention also provides the use of the antioxidant peptides described in the above technical solution or the antioxidant peptides prepared by the preparation method described in the above technical solution in food and / or medicine. The food described in the present invention preferably includes functional food.

[0047] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] In the following examples, acidic protease was purchased from Beijing Solebow Technology Co., Ltd. with the product number of G8410-250g.

[0049] The detection methods for antioxidant activity are as follows: the method for detecting the ability to scavenge DPPH free radicals refers to the national standard GB / T39100-2020 Determination of the antioxidant activity of polypeptides, the first method, the DPPH method; the method for detecting the ability to scavenge ABTS free radicals refers to the national standard GB / T 39100-2020 Determination of the antioxidant activity of polypeptides, the second method, the ABTS method.

[0050] Example 1

[0051] 1. A method for preparing low-salt dry-cured ham skin antioxidant peptides, comprising the following steps:

[0052] (1) Raw material pretreatment: weigh 200 g of low-salt dry-cured ham (the formula and preparation method of low-salt dry-cured ham are disclosed in South African patent No. ZA202303077B (patent name is METHOD FOR MAKING LOW-SODIUM FERMENTED FLAVORED MEAT PRODUCT, and the public authorization time is May 31, 2023)) peel, add 800 mL of ultrapure water and 40 g of anhydrous sodium carbonate, and place at 4°C for 24 h, stirring continuously;

[0053] Further, 800 mL of ultrapure water and 40 g of sodium chloride were added and placed at 4 °C for 24 h with constant stirring;

[0054] Furthermore, the cleaned ham skin is dried at 55° C. for 24 hours and crushed using a high-speed wall-breaking machine to obtain ham skin powder.

[0055] (2) Enzymatic hydrolysis: Ultrapure water and acidic protease were added to the ham skin powder obtained in step (1) at a solid-liquid ratio of 1 g:10 mL, and the amount of acidic protease added was 5000 U / g based on the ham skin powder. The pH value was adjusted to 3, and the enzymatic hydrolysis was performed at 50°C for 6 h, and then the enzyme was inactivated by boiling at 100°C for 15 min; after cooling, the mixture was centrifuged at 8000 r / min for 15 min, and the supernatant was collected. The supernatant was freeze-dried to obtain a peptide product.

[0056] (3) Purification: Use a 16×80 cm gel chromatography column (filled with Sephadex G-25) for separation; select ultrapure water as the mobile phase, the peptide sample concentration is 50 mg / mL, take 20 mL of sample at a time, set the elution flow rate to 2 mL / min, and set the automatic collection time to 4.5 min each time;

[0057] The separated components of each separated peak are collected and recorded as F1, F2 and F3 (such as Figure 1 As shown), F1 is the separated components of tubes 1 to 10, F2 is the separated components of tubes 10 to 26, and F3 is the separated components of tubes 27 to 60.

[0058] The antioxidant activity of the separated components F1, F2 and F3 was expressed by the ability to scavenge DPPH free radicals. The results are shown in Figure 2 As shown by Figure 2 It can be concluded that when the test sample concentration is 3 mg / mL, the DPPH free radical scavenging rates of the separated components F1, F2 and F3 are 22.75%, 44.33% and 27.02%, respectively. It can be seen that the separated component F2 has the strongest ability to scavenge DPPH free radicals, that is, its antioxidant activity is the strongest, and further screening shows that the component with the best antioxidant activity is the separated component F2. The separated component F2 is freeze-dried to obtain F2 peptide powder.

[0059] (4) The separated component F2 peptide powder obtained in step (3) was desalted using an Empore solid phase extraction column C18, and the eluent was a formic acid acetonitrile aqueous solution with a volume concentration of 0.1%, the volume concentration of acetonitrile in the formic acid acetonitrile aqueous solution was 84%, and the flow rate was 250 μL / min. LC-MS / MS was used for peptide sequence analysis, and the comparison analysis was performed through the database. The mass spectrometry database retrieval software was MaxQuant 1.5.5.1, and the sample database used was the uniprot protein database to obtain all peptide sequences.

[0060] The specific process is as follows: after desalting, the polypeptide components are freeze-dried in a vacuum, and finally re-dissolved using 40 μL 0.1% trifluoroacetic acid (TFA).

[0061] LC-MS / MS analysis was performed using the nano-flow HPLC system Easy nLC. The samples were loaded into Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) by an autosampler and then separated by a liquid chromatography column at a flow rate of 250 μL / min. The liquid phase gradient was set as follows: 0-50 min, liquid B linear gradient from 4% to 50%; 50-54 min, liquid B linear gradient from 50% to 100%; 54-60 min, liquid B maintained at 100%. The liquid A used in the liquid phase was a 0.1% formic acid aqueous solution, and the liquid B was a 0.1% formic acid acetonitrile aqueous solution (the volume concentration of acetonitrile was 84%). The liquid chromatography column (75 μm×150 mm, RP-C18, Column Technology Inc) was equilibrated with 95% liquid A. The samples were separated by HPLC and analyzed by mass spectrometry using Q Exactive mass spectrometer (Thermo Fisher). The analysis time was 60 min. Detection mode was positive ion. Scan range of parent ion was 300-1800 m / z. Primary mass spectrometry resolution was 70000 (at m / z 200). AGC target was 1e6. Primary maximum IT value was 10 ms. Number of scan ranges was 1. Dynamic exclusion was 20.0 s. The mass-to-charge ratio of the peptide and its fragments was collected according to the following method: 10 fragment spectra (MS2 scan) were collected after each full scan. MS2 Activation Type was HCD. Isolation window was 1.6 m / z. Secondary mass spectrometry resolution was 17500 (at m / z 200). Microscans was 1. Secondary maximum IT value was 60 ms. Normalized collision energy was 27 eV. Underfill ratio was 0.1%.

[0062] The raw data (raw file) of mass spectrometry test was searched with the corresponding database using the software MaxQuant 1.5.5.1, and finally the peptide identification and quantitative analysis results were obtained. The relevant parameters used for searching the database were as follows: Peptide mass tolerance: 20ppm; MS / MS tolerance: 0.1Da; Enzyme=None; Max Missed Cl eavages: 2; Variable modification: Oxidation (M); the database selected was uniprot_Sus_scrofa_46179_20230502 and combined with the reverse library; result filtering parameter: FDR<0.01.

[0063] 2. Bioinformatics tools assisted the screening of antioxidant peptides from low-salt dry-cured ham skin. The steps are as follows:

[0064] (1) The Services Peptide Ranker tool was used to predict the antioxidant activity of active peptides from the sequences obtained in step 1. Sequences with a score greater than 0.5 were considered to have potential activity and were subsequently screened.

[0065] (2) The isoelectric point (pI) and hydrophobicity of the peptides were analyzed using the Expasy-pI / Mw tool (https: / / web.expasy.org / computepi / ) and the PepDraw server (https: / / pepcalc.com).

[0066] Innovagen (https: / / www.innovagen.com / proteomics-tools) and ProtParam (https: / / web.expasy.org / protparam / ) tools were used to analyze water solubility and stability (instability index) online. FP software (v.1.0; https: / / www.ddg-pharmfac.net / AllergenFP) and ToxinPred (https: / / crdd.osdd.net / raghava / toxinpred / ) were used to analyze allergenicity and toxicity. Sequences without potential allergenicity and toxicity were subsequently subjected to molecular docking, synthesis, and verification;

[0067] (3) The structures of ABTS and DPPH were downloaded from the PubChem database. Molecular docking was performed using the .3D structure of the peptide sequence used for docking was obtained using Maestro (build 13.0). The crystal structure of the Keap1 protein used for docking was downloaded from the Protein Data Bank (PDB) database (PDB ID 2FLU). Before docking, the charge state and minimum energy conformation of ABTS and DPPH in physiological environment were calculated using the LigPrep module.

[0068] In addition, the structure of Keap1 was optimized using the ProteinPreparationWizard module of Maestro, hydrogen atoms were added, and intermolecular clashes were eliminated using the OPLS2005 force field. After the peptide sequence and target molecule were prepared, a mesh file defining the docking pocket was generated by the receptor mesh generation module of Maestro. The center of mass of the co-crystal ligand (NRF2) of the crystal structure was selected as the center of the active pocket.

[0069] Different peptide sequences were docked with Keap1 based on The size generates the calculation results of the lattice file.

[0070] Finally, the prepared files were molecularly docked using the SPPrecision algorithm in the Glide docking module. The docking results were visualized and analyzed using PyMOL 2.5.4 and LigPlus 2.2.5, and the binding energy was calculated using the MMGBSA method with the Prime module (the lower the binding energy, the better the binding), and four antioxidant peptides were obtained, whose amino acid sequences are shown in SEQ ID NO. 1 to 4, as shown in Table 1.

[0071] Table 1 Antioxidant peptide amino acid sequences and docking energy

[0072] Antioxidant Peptides sequence Docking energy (kcal / mol) SEQ ID NO.1 AAWGKVGGQAGAHGAEALER -6.835 SEQ ID NO.2 DQALKPTKPMRFLGDE -6.429 SEQ ID NO.3 GVNGFGRIGR -8.367 SEQ ID NO.4 SLTGEFKGKYYPL -6.451

[0073] The potential allergenicity, potential toxicity and physicochemical properties of the antioxidant peptides in Table 1 are shown in Table 2 .

[0074] Table 2 Prediction of antioxidant peptide properties

[0075]

[0076]

[0077] From Tables 1 and 2, it can be concluded that the four peptides are non-allergenic, non-toxic, soluble in water, and have a stability of less than 0, indicating that they are stable, indicating that they have good processing performance and high safety, and the binding energy is less than -5 kcal / mol, indicating that they have good binding ability with Keap1 protein.

[0078] The molecular docking results of the antioxidant peptide with amino acid sequence as shown in SEQ ID NO.1 and receptor protein Keap1 are shown in Figure 3 The molecular docking results of the antioxidant peptide with amino acid sequence as shown in SEQ ID NO.2 and receptor protein Keap1 are shown in Figure 4 The molecular docking results of the antioxidant peptide with amino acid sequence as shown in SEQ ID NO.3 and receptor protein Keap1 are shown in Figure 5 The molecular docking results of the receptor protein Keap1 with the amino acid sequence shown in SEQ ID NO.4 are shown in Figure 6 .

[0079] Combination Figures 3 to 6 It can be concluded that the binding modes and binding sites of the four antioxidant peptides and the receptor protein Keap1 show that the binding of the antioxidant peptides to the Keap1 subunit is mainly through hydrogen bonding and hydrophobic interactions with the receptor amino acid residues. Among them, the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.1 forms eight conventional hydrogen bonds and eight hydrophobic interactions with the receptor protein Keap1; the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.2 forms six hydrogen bonds and four hydrophobic interactions with the receptor protein Keap1; the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.3 forms six hydrogen bonds and two hydrophobic interactions with the receptor protein Keap1; the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.4 forms six hydrogen bonds and two hydrophobic interactions with the receptor protein Keap1.

[0080] It can be concluded that the amino acid residues in the short peptide can effectively interact with Keap1 protein, thus endowing it with strong DPPH· and ABTS·+ scavenging activity, thus showing beneficial antioxidant properties. Therefore, it can be concluded that the short peptide exhibits in vivo antioxidant activity by participating in cellular antioxidant activity in the Keap 1-Nrf2 signaling pathway.

[0081] Example 2

[0082] The antioxidant activity of the antioxidant peptides in Example 1 was determined in the following steps:

[0083] According to the amino acid sequence of the antioxidant peptide prepared in Table 1 of Example 1, Shanghai Hongpeptide Biotechnology Co., Ltd. was commissioned to carry out chemical synthesis.

[0084] The antioxidant activity of the synthesized antioxidant peptides was measured using the DPPH method, ABTS method and ferrous ion chelation ability method, and the same concentration of synthetic glutathione (GSH) was used as a control.

[0085] The determination steps of the ferrous ion chelating ability method are as follows:

[0086] Prepare a 3 mg / mL peptide solution by mixing 50 μL of the peptide solution with 5 μL of 2 mM FeSO 4 After mixing, add 140 μL of distilled water and place at 37°C for 15 minutes. Finally, add 17 μL of 5 mM ferrozine to activate the reaction and measure A1. Deionized water is used instead of peptide solution to mix with other reagents as blank group A0. After incubation at room temperature for 10 minutes, measure the absorbance at 562 nm, measure three times, and take the average value.

[0087] Ferrous ion chelating capacity = (A0-A1 / A0) x 100%.

[0088] The ability of antioxidant peptides and the control group GSH to scavenge DPPH free radicals, scavenge ABTS free radicals, and chelate with ferrous ions is shown in Figure 2. Figure 7 As shown. Figure 7 In the equation, AR20, DE16, GR10, and SL13 represent AAWGKVGGQAGAHGAEALER, DQALKPTKPMRFLGDE, GVNGFGRIGR, and SLTGEFKGKYYPL, respectively, and Figure 7 Different letters in the middle bar graph indicate that the same antioxidant capacity of different peptides is significantly different.

[0089] Depend on Figure 7 It can be concluded that the antioxidant peptides shown in amino acid sequences such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 have stronger abilities to scavenge DPPH free radicals and ABTS free radicals and to chelate with ferrous ions than the control group GSH.

[0090] It can be concluded from the above examples that the antioxidant peptides isolated from low-salt dry-cured ham skin of the present invention have good antioxidant activity, and their antioxidant activity is stronger than that of the positive control GSH.

[0091] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antioxidant peptide, characterized in that It comprises one or more of antioxidant peptide I, antioxidant peptide II, antioxidant peptide III and antioxidant peptide IV, and the amino acid sequences of the antioxidant peptide I, antioxidant peptide II, antioxidant peptide III and antioxidant peptide IV are shown in SEQ ID NO.1 to SEQ ID NO.4 respectively.

2. The method for preparing the antioxidant peptide according to claim 1, characterized in that: The method comprises the following steps: enzymolyzing dry-cured ham skin with acidic protease, and performing solid-liquid separation on the obtained enzymolysis mixture to obtain an enzymolysis solution; Performing gel chromatography separation on the enzymatic hydrolysate, collecting the component with the highest DPPH free radical scavenging rate, and obtaining a gel chromatography chromatographic solution; The gel chromatography liquid is subjected to solid phase extraction to obtain a solid phase extraction liquid, wherein the solid phase extraction liquid contains the antioxidant peptide.

3. The preparation method according to claim 2, characterized in that: Calculated on the basis of the dry product of the dry-cured ham skin, the dosage of the acidic protease is 2500-10000 U / g of the dry product of the dry-cured ham skin.

4. The preparation method according to claim 2 or 3, characterized in that: The enzymatic hydrolysis temperature is 45-55° C., the time is 5-8 hours, and the pH value is 2-5.

5. The preparation method according to claim 2, characterized in that: The pre-packed column for gel chromatography separation is Sephadex G-25.

6. The preparation method according to claim 2 or 5, characterized in that: The mobile phase of the gel chromatography separation is ultrapure water, and the flow rate is 1-4 mL / min.

7. The preparation method according to claim 2, characterized in that: The solid phase extraction column is Empore solid phase extraction column C18; the eluent of the solid phase extraction is a formic acid acetonitrile aqueous solution with a volume concentration of 0.1%, the volume concentration of acetonitrile in the formic acid acetonitrile aqueous solution is 84%, and the flow rate is 250 μL / min.

8. The preparation method according to claim 2, characterized in that: After the solid phase extraction, the method further includes performing mass spectrometry sequencing and sequence comparison analysis on the solid phase extraction liquid to obtain the antioxidant peptide.

9. The preparation method according to claim 2, characterized in that: Before the enzymolysis, the dry-cured ham skin is sequentially cleaned, dried and ground to obtain dry-cured ham skin powder; the cleaning step comprises: mixing the dry-cured ham skin, water and anhydrous sodium carbonate and stirring for 24 hours to obtain a first mixture; The first mixture, water and sodium chloride are mixed and stirred for a second time for 24 hours, and then clean dry-cured ham skin is obtained by separation.

10. Use of the antioxidant peptide according to claim 1 or the antioxidant peptide prepared by the preparation method according to any one of claims 2 to 9 in food and / or medicine.

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