Antioxidant peptide from cantharellus cibarius sporocarp, antioxidant peptide detection reagent and application

Abstract

The invention belongs to the technical field of food biology, and particularly relates to an antioxidant peptide derived from cantharellus cibarius sporocarp, an antioxidant peptide detection reagent and application, and the amino acid sequence of the antioxidant peptide is at least one of FPYPY, YPPYF, YYPF and MPWY. The antioxidant peptide provided by the invention is derived from chanterelle, is an enzymolysis product of chanterelle protein, and is derived from edible and medicinal fungus chanterelle, so that the antioxidant peptide is small in toxic and side effects and high in safety. Meanwhile, the antioxidant peptide disclosed by the invention has good biological activity and a relatively strong antioxidant effect, can remarkably scavenge free radicals, and can be applied to repair damage caused by oxidative stress.

Description

Technical Field

[0001] The invention belongs to the field of food biotechnology, and particularly relates to an antioxidant peptide derived from chanterelle fruiting bodies, an antioxidant peptide detection reagent and applications. Background Art

[0002] Oxidative stress, also known as OS, is a negative effect caused by free radicals in the body and is considered to be an important factor leading to aging and disease. Exogenous hazards include cigarettes, exhaust gas from cars and industries, alcohol and various heavy metal pollution, and endogenous hazards such as inflammation, stress and anxiety also need to be taken seriously. These exogenous and endogenous hazards can cause the levels of reactive oxygen and reactive nitrogen in the body to exceed normal values, thus leading to the generation of oxidative stress. Continuous oxidative stress can lead to the occurrence of a variety of mental illnesses including depression, as well as neurodegenerative diseases including diabetes, cancer and Alzheimer's disease. In order to reduce or inhibit the accumulation of free radicals, antioxidants are widely used. In addition, since antioxidants can directly react with various free radicals such as superoxide anion radicals, hydroxyl radicals, hydrogen peroxide, etc., and remove them by providing electrons or hydrogen atoms, thereby reducing the oxidative damage of free radicals to skin cells, antioxidants are also used in the field of cosmetics.

[0003] Antioxidants are divided into synthetic antioxidants and natural antioxidants. Common synthetic antioxidants include tert-butylhydroquinone, butylated hydroxytoluene and butylated hydroxyanisole, but they have side effects such as affecting body metabolism, causing allergic reactions and potential carcinogenicity during use. Therefore, there is an urgent need to provide a natural antioxidant that can repair oxidative damage and is safe. Summary of the invention

[0004] In order to repair the damage caused by oxidative stress, the present invention provides an antioxidant peptide derived from chanterelle fruiting bodies.

[0005] The technical solution adopted by the present invention is: The invention provides an antioxidant peptide derived from chanterelle fruiting bodies, wherein the amino acid sequence of the antioxidant peptide is at least one of FPYPY, YPPYF, YYPF and MPWY.

[0006] The present invention provides an application of the antioxidant peptide, wherein the antioxidant peptide is used for removing free radicals.

[0007] Preferably, the concentration of the antioxidant peptide used to scavenge the free radicals is 62.5 μg / mL to 1000 μg / mL.

[0008] Preferably, the concentration of the antioxidant peptide used to scavenge the free radicals is 1000 μg / mL.

[0009] The present invention provides an application of the antioxidant peptide, and the antioxidant peptide is used for preparing an antioxidant.

[0010] Preferably, the antioxidant further comprises a pharmaceutically acceptable excipient.

[0011] Preferably, the pharmaceutically acceptable excipients include at least one of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

[0012] Preferably, the diluent includes any one of starch, lactose, sucrose and mannitol.

[0013] Preferably, the disintegrant includes at least one of starch, microcrystalline cellulose and low-substituted hydroxypropyl cellulose.

[0014] Preferably, the precipitation inhibitor includes any one of sodium lauryl sulfate, Tween-80, polyvinyl pyrrolidone and hydroxypropyl methylcellulose.

[0015] Preferably, the glidant includes any one of cationic polyacrylamide, polydiallyldimethylammonium chloride and cationic starch.

[0016] Preferably, the binder comprises any one of starch slurry, hydroxypropyl methylcellulose and povidone.

[0017] Preferably, the dispersant includes any one of sodium lauryl sulfate, polyvinyl pyrrolidone and sodium carboxymethyl cellulose.

[0018] Preferably, the suspending agent comprises any one of gum arabic, gum tragacanth, sodium carboxymethylcellulose and hydroxypropyl methylcellulose.

[0019] Preferably, the isotonic agent includes any one of sodium chloride, glucose and mannitol.

[0020] Preferably, the thickener includes any one of gum arabic, xanthan gum and sodium carboxymethyl cellulose.

[0021] Preferably, the emulsifier includes any one of sodium lauryl sulfate, benzalkonium chloride and sorbitan fatty acid.

[0022] Preferably, the preservative includes any one of benzoic acid, sorbic acid, methyl paraben and benzalkonium bromide.

[0023] Preferably, the stabilizer comprises any one of sodium sulfite, sodium bisulfite, tocopherol and disodium edetate.

[0024] The present invention also provides a reagent for detecting the activity of the antioxidant peptide, wherein the reagent comprises any one of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and 1,1-diphenyl-2-trinitrophenylhydrazine.

[0025] Preferably, the antioxidant is used to prepare a medicament for treating or preventing mental illness.

[0026] Preferably, the mental illness includes any one of depression, obsessive-compulsive disorder, anxiety disorder and schizophrenia.

[0027] Preferably, the antioxidant is used to prepare a medicament for treating or preventing diabetes.

[0028] Preferably, the antioxidant is used to prepare a medicament for treating or preventing neurodegenerative diseases.

[0029] Preferably, the neurodegenerative disease includes any one of Alzheimer's disease, Parkinson's disease and Huntington's disease.

[0030] Preferably, the antioxidant is used in the preparation of cosmetics.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The invention provides an antioxidant peptide derived from a chanterelle fruiting body, wherein the amino acid sequence of the antioxidant peptide is at least one of FPYPY, YPPYF, YYPF and MPWY. The antioxidant peptide of the present invention is derived from chanterelles, and is an enzymolysis product of chanterelles protein. Since it is derived from the edible and medicinal mushroom chanterelles, it has little toxic and side effects and is highly safe. Meanwhile, the antioxidant peptide of the present invention has good biological activity and strong antioxidant effect, can significantly remove free radicals, and can be applied to repair damage caused by oxidative stress.

[0032] 2,2'-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid can be oxidized to generate a stable blue-green cationic free radical ABTS+. When an antioxidant peptide is present, the antioxidant peptide will react with ABTS+ to make the solution lighter in color. By measuring the change in absorbance, the ability of antioxidants to scavenge ABTS+ free radicals can be evaluated, thereby measuring their antioxidant activity. It is widely used in the evaluation of antioxidant properties of antioxidant ingredients or products in the fields of food, health products, and cosmetics. 1,1-Diphenyl-2-trinitrophenylhydrazine is a stable free radical whose lone pair of electrons has strong absorption at around 517nm, making the solution dark purple. When an antioxidant peptide is present, the hydrogen atoms or electrons provided by the antioxidant peptide will combine with the 1,1-diphenyl-2-trinitrophenylhydrazine free radical, making the solution lighter in color and reducing the absorbance. By measuring the change in absorbance, the scavenging rate of the antioxidant peptide on the 1,1-diphenyl-2-trinitrophenylhydrazine free radical can be calculated to evaluate the antioxidant capacity of the antioxidant peptide. The present invention utilizes 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and 1,1-diphenyl-2-trinitrophenylhydrazine to determine the antioxidant capacity of the antioxidant peptide, and ultimately determines that the concentration range of the antioxidant peptide of the present invention to exert the antioxidant capacity is 62.5 μg / mL to 1000 μg / mL, and the optimal concentration is 1000 μg / mL.

[0033] In addition, the antioxidant peptides of the present invention are obtained by enzymatic hydrolysis, and the antioxidant peptides prepared by this method can enhance functional properties without affecting their nutritional value.

[0034] Chanterelles Cantharellus cibarius Fr) belongs to the ecto-rhizophorous fungus, belonging to the kingdom of Fungi, phylum Basidiomycetes, subphylum Agaricaceae, class Agaricaceae, order Cantharellales, family Cantharellaceae, genus Cantharellaceae, also known as yellow silk fungus or apricot fungus. The annual production of wild chanterelles in the world is about 150,000 to 200,000 tons. The sugars in chanterelles account for about 64%, the crude protein accounts for about 21%, and the fat accounts for about 6%. Its total amino acids are higher than those of general edible fungi. Chanterelles have biological activities such as antioxidant immunity enhancement, antibacterial and neuroprotective, and have high nutritional value and medicinal value. In addition to direct consumption or rough processing, there is relatively little in-depth development of chanterelle resources. The antioxidant peptides described in the present invention provide a new way to develop and study active peptide products using chanterelles, which promotes the development of the chanterelle industry. The antioxidant peptides of the present invention have the advantages of high safety, strong antioxidant activity and easy absorption, and can be used as raw materials and auxiliary materials for food, medicine and health products, so that chanterelle resources are fully developed and utilized, providing effective guarantee for the development of the chanterelle industry, and having long-term scientific theoretical guiding significance and high economic value significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the degree of hydrolysis of enzymatic chanterelle protein.

[0036] Figure 2 ABTS free radical scavenging ability of chanterelle protein hydrolysate.

[0037] Figure 3 DPPH free radical scavenging ability of chanterelle protein hydrolysate.

[0038] Figure 4 It is the reducing capacity of chanterelle protein hydrolysate.

[0039] Figure 5 It is the degree of hydrolysis of the double enzymatic hydrolysate of chanterelle protein.

[0040] Figure 6 It is the ABTS free radical scavenging ability of the double enzymatic hydrolysate of chanterelle protein.

[0041] Figure 7 It is the DPPH free radical scavenging ability of the double enzymatic hydrolysate of chanterelle protein.

[0042] Figure 8 It is the reducing capacity of the double enzymatic hydrolysate of chanterelle protein.

[0043] Fig. 9 ABTS free radical scavenging capacity of the ultrafiltration fraction of FA-CCPH.

[0044] Fig.10 The DPPH radical scavenging capacity of the ultrafiltration fraction of FA-CCPH.

[0045] Fig.11 is the reducing capacity of the ultrafiltration fraction of FA-CCPH.

[0046] Fig.12 It is the component obtained by eluting the F2 ultrafiltration component through a gel chromatography column.

[0047] Fig.13 It is the ABTS free radical scavenging ability of the gel chromatography elution fraction.

[0048] Fig.14 It is the DPPH free radical scavenging ability of the gel chromatography elution component.

[0049] Fig.15 It is the reducing ability of the eluted components of gel chromatography.

[0050] Fig.16 This is the amino acid length distribution diagram of the 2099 peptide sequences that were initially screened.

[0051] Fig.17 Activity scores were given to 636 sequences predicted to have antioxidant activity.

[0052] Fig.183D docking diagram of VDPWHPWP peptide and the active site of Keap 1.

[0053] Fig.19 3D docking diagram of FPYPY peptide and the active site of Keap 1.

[0054] Fig. 20 3D docking diagram of YPPYF peptide and the active site of Keap 1.

[0055] Fig.21 3D docking diagram of WYW peptide and the active site of Keap 1.

[0056] Fig. 22 3D docking diagram of YYPF peptide and the active site of Keap 1.

[0057] Fig.23 3D docking diagram of MPWY peptide and Keap 1 active site.

[0058] Fig.24 3D docking diagram of GYGPF peptide and the active site of Keap 1.

[0059] Fig.25 The ABTS free radical scavenging ability of chanterelle antioxidant peptides.

[0060] Fig.26 The DPPH free radical scavenging ability of chanterelle antioxidant peptides.

[0061] Fig. 27 The reducing capacity of chanterelle antioxidant peptides. DETAILED DESCRIPTION

[0062] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0063] The inventive concept of the present invention is as follows: In order to cope with the damage caused by oxidative stress, antioxidants are usually used for repair. Antioxidants are divided into synthetic antioxidants and natural antioxidants. Common synthetic antioxidants include tert-butylhydroquinone, dibutylhydroxytoluene and butylated hydroxyanisole, but they have side effects such as affecting body metabolism, causing allergic reactions and potential carcinogenicity during use. Therefore, there is an urgent need to provide a natural antioxidant that can repair oxidative damage and is safe.

[0064] Based on this, the present invention provides an antioxidant peptide derived from chanterelle fruiting bodies, wherein the amino acid sequence of the antioxidant peptide is at least one of FPYPY, YPPYF, YYPF and MPWY.

[0065] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are all commercially available, and the methods used are all conventional techniques in the art.

[0066] The abbreviation comparison table of the present invention is shown in Table 1.

[0067] Table 1 Abbreviations

[0068] Example 1 An antioxidant peptide derived from the fruiting body of chanterelles, specifically as follows: Experiment 1: Extraction of CCP, as follows: The degreased chanterelle powder was mixed with ultrapure water at a solid-liquid ratio of 1:50, and the solution pH was adjusted to 13.0 with 0.1M NaOH. The solution was extracted at 50℃ for 1h with 800W ultrasonic assistance. After filtration, the solution pH was adjusted to 3.4 with 0.1M HCl. After standing overnight, the solution was centrifuged and the precipitate and supernatant were collected at the same time. Ammonium sulfate powder was added to the supernatant so that the amount of ammonium sulfate dissolved in the supernatant reached 90% of the maximum amount of ammonium sulfate dissolved. After standing overnight, the precipitate was collected by centrifugation. The two precipitates were mixed to obtain CCP, and the pH was adjusted to 7.0, dialyzed, concentrated and freeze-dried. The protein content was determined by the BCA method. Three parallel tests were performed, and the average protein extraction rate was calculated to be 49.00%, with a relative standard deviation of 0.20%. 0.603g of ammonium sulfate powder was added to each 1mL of supernatant.

[0069] Experiment 2: Enzymatic hydrolysis of CCP, as follows: First, prepare a 2% CCP solution, adjust the pH to 7.0, and place in a 95°C water bath for 20 minutes to completely denature the protein. After that, continue with the following experiments: (1) Add 4000 U / g alkaline protease to the completely denatured CCP solution, perform enzymatic hydrolysis at 45°C and pH 10.5 for 4 h, inactivate the enzyme in a 95°C water bath for 20 min, and obtain the chanterelle hydrolysate A-CCPH. Add 1 g alkaline protease to every 50 mL CCP solution. (2) Add 4000U / g neutral protease to the completely denatured CCP solution, perform enzymatic hydrolysis at 40℃ and pH 7.0 for 4h, inactivate the enzyme in a 95℃ water bath for 20min, and obtain the chanterelle hydrolysate N-CCPH. Add 1g neutral protease to every 50mL CCP solution. (3) Add 4000U / g flavor protease to the completely denatured CCP solution, and perform enzymatic hydrolysis at 55℃ and pH 7.0 for 4h. After enzymatic hydrolysis, inactivate the enzyme in a 95℃ water bath for 20min to obtain the chanterelle hydrolysate F-CCPH. Add 1g flavor protease to every 50mL CCP solution. (4) Add 4000 U / g bromelain to the completely denatured CCP solution, and perform enzymatic hydrolysis at 55°C and pH 7.0 for 4 h. After enzymatic hydrolysis, inactivate the enzyme in a 95°C water bath for 20 min to obtain the chanterelle hydrolysate B-CCPH. Add 1 g bromelain to every 50 mL CCP solution. (5) First, add 4000U / g alkaline protease to the completely denatured CCP solution, and perform enzymatic hydrolysis at 45℃ and pH 10.5 for 4h; inactivate the enzyme in a 95℃ water bath for 20min, then add 4000U / g flavor protease, and perform enzymatic hydrolysis at 55℃ and pH 7.0 for 4h. After enzymatic hydrolysis, inactivate the enzyme in a 95℃ water bath for 20min to obtain the chanterelle hydrolysate AF-CCPH. Add 1g alkaline protease and 1g flavor protease to every 50mL CCP solution; (6) First, add 4000U / g flavor protease to the completely denatured CCP solution, and perform enzymatic hydrolysis at 55℃ and pH 7.0 for 4h. After inactivating the enzyme in a 95℃ water bath for 20min, add 4000U / g alkaline protease, and perform enzymatic hydrolysis at 45℃ and pH 10.5 for 4h. After the enzymatic hydrolysis, inactivate the enzyme in a 95℃ water bath for 20min to obtain the Chanterelle hydrolysate FA-CCPH. Add 1g flavor protease and 1g alkaline protease to every 50mL CCP solution.

[0070] Alkaline protease and neutral protease were purchased from Beijing Solebow Technology Co., Ltd., the alkaline protease product number is B8360, and the neutral protease product number is Z8031; flavor enzyme protease and bromelain were purchased from Shanghai Yuanye Biotechnology Co., Ltd., the flavor enzyme protease product number is S10153-25g, and the bromelain product number is S10009-25g.

[0071] Experiment 3: Determination of the degree of hydrolysis, as follows: Experiment 3 is to study the hydrolysis degree of CCP enzymatic hydrolysis reaction by different types of proteases. The chanterelle hydrolysates A-CCPH, N-CCPH, F-CCPH and B-CCPH were centrifuged at 8000rpm / s for 10min at 4°C, and the protein content of the supernatant was determined by the BCA method, which was calculated as C. The hydrolysis degree of the supernatant was determined by the o-phthalaldehyde method. 20μL of the supernatant of A-CCPH, N-CCPH, F-CCPH and B-CCPH were mixed with 150μL of OPA reagent for precise reaction for 2min, and the absorbance value at 340nm was measured and recorded as ODs. At the same time, 0.9516meqv / L standard serine and ultrapure water were used instead of the sample as the standard control and blank control, respectively, and recorded as ODs. st and OD b The degree of hydrolysis DH is calculated according to the following formula: ; Among them, serine NH2 The calculation formula is: ; In the formula, DH is the degree of hydrolysis, and the unit is %; is the concentration of the supernatant, in meqv / L; α is 0.970; β is 0.342; OD s OD is the absorbance value at 340 nm after 20 μL supernatant and 150 μL OPA reagent are mixed and reacted; b is the absorbance value of blank control at 340nm; OD st is the absorbance value of standard serine at 340nm; C is the protein concentration of the supernatant, in mg / mL; h hot is the total number of peptide bonds in the protein, and the chanterelle protein h hot is 8.

[0072] Results Figure 1 The hydrolysis degree of A-CCPH was 37.13%, with a relative standard deviation of 0.40%; the hydrolysis degree of N-CCPH was 33.07%, with a relative standard deviation of 0.36%; the hydrolysis degree of F-CCPH was 39.03%, with a relative standard deviation of 0.26%; the hydrolysis degree of B-CCPH was 21.45%, with a relative standard deviation of 0.25%. Among them, the hydrolysis degree of flavor protease was significantly higher than that of the other three proteases, followed by alkaline protease.

[0073] Experiment 4: Determination of antioxidant activity, as follows: The antioxidant activity of chanterelle hydrolysate CCPHs was evaluated by measuring the ABTS free radical scavenging ability, DPPH free radical scavenging ability and reducing ability, and 0.1 mg / mL ascorbic acid VC was used as a positive control.

[0074] (1) ABTS free radical scavenging activity: evaluated by absorbance at 734 nm. Dissolve 0.3841 g ABTS and 0.0662 g potassium persulfate in water and dilute to 100 mL to prepare ABTS base solution, which is stored at room temperature and protected from light overnight; dilute the base solution to an OD value of approximately 0.75 at 734 nm and a relative standard deviation of 0.02 to prepare the ABTS working solution.

[0075] Method: Add 200µL ABTS working solution to 50µL chanterelle hydrolysate, react for 10 minutes in the dark, and measure the OD value at 734nm three times. The ABTS free radical scavenging rate is calculated as follows: ; Wherein, the absorbance of the enzymatic hydrolysate of chanterelles after adding ABTS is A 1 The absorbance of the chanterelle hydrolysate after adding water is A 2 The absorbance of ABTS after adding water is A 3 .

[0076] (2) DPPH free radical scavenging activity: Take 200 µL of chanterelle hydrolysate, add 100 µL of DPPH solution, place in dark place for 10 min, and measure OD 517 The value was repeated 3 times and the average value was taken. The calculation formula of DPPH free radical scavenging rate is as follows: ; A 4 A is the absorbance value of the sample after adding DPPH and 99.9% ethanol solution to the enzymatic hydrolysate of chanterelles; 5 A is the absorbance value of the sample control group after adding 99.9% ethanol solution to the chanterelle hydrolysate; 6 A is the absorbance value of the blank group after adding DPPH solution to distilled water; 7 It is the absorbance value of the blank control group after adding 99.9% ethanol solution by volume to distilled water.

[0077] (3) Determination of reducing ability: Prepare different concentrations of chanterelle hydrolysate with distilled water, take 50 µL of sample, add 50 µL of pH 6.6 phosphate buffer and 50 µL of 1% potassium ferrocyanide chloride solution, react in a 50°C water bath for 20 min, then add 50 µL of 10% trichloroacetic acid and 50 µL of 0.1% ferric chloride. After standing for 10 min, measure the absorbance at 700 nm. Use ultrapure water instead of potassium ferrocyanide solution as the control group. Repeat 3 times, record the data and take the average value.

[0078] Reducing capacity = OD of the enzymatic hydrolysate of chanterelles 700 -Control group OD700 .

[0079] Experiment 5: Double enzymatic hydrolysis of CCP, as follows: The antioxidant activities of the four single enzymatic hydrolysates were determined by the method in Experiment 4. Figure 2~Figure 4 , and the corresponding IC50 values ​​were calculated. Among them, F-CCPH and A-CCPH showed better ABTS free radical scavenging ability, with IC50 values ​​of 0.3078mg / mL and 0.3165mg / mL respectively; B-CCPH showed better DPPH free radical scavenging ability with an IC50 value of 1.620mg / mL; and the reducing ability of A-CCPH was significantly higher than that of other enzymatic hydrolysates. Therefore, considering the hydrolysis degree and antioxidant activity, flavor protease and alkaline protease were selected for double enzymatic hydrolysis to obtain AF-CCPH and FA-CCPH for subsequent analysis. Double enzymatic hydrolysis showed a higher hydrolysis degree, see Figure 5 The hydrolysis degree of AF-CCPH was 60.12%, and the relative standard deviation was 0.80%; the hydrolysis degree of FA-CCPH was 63.39%, and the relative standard deviation was 0.71%. Figure 6~Figure 8 The ABTS free radical scavenging ability of the double enzymatic hydrolysis product was similar to that of the single enzymatic hydrolysis product, while the DPPH free radical scavenging ability and reducing ability were significantly improved compared with the single enzymatic hydrolysis product. The IC50 value of FA-CCPH's DPPH free radical scavenging ability was 1.324 mg / mL; the reducing ability was also significantly higher than that of the single enzymatic hydrolysis product and AF-CCPH. At 5 mg / mL, the reducing ability of FA-CCPH was 1.19, and the relative standard deviation was 0.01. After comprehensive consideration, FA-CCPH was selected for subsequent ultrafiltration separation.

[0080] Experiment 6: Ultrafiltration of FA-CCPH, as follows: To prepare a 50 mg / mL FA-CCPH solution, first use an ultrafiltration tube with a molecular weight cutoff of 10 kDa, ultrafiltration conditions of 4500 rpm / s, and centrifugation at 4°C for 40 minutes. After the ultrafiltration is completed, collect the components less than 10 kDa. Then use an ultrafiltration tube with a molecular weight cutoff of 3 kDa and ultrafiltration under the above conditions to separate F1 and F2. The size of F1 is 3 kDa~10 kDa, and the size of F2 is less than 3 kDa. The yields of the two components after freeze-drying are calculated to be 6.38% and 26.54%, respectively. The antioxidant activity was determined, see Figure 9~Figure 11 Both F1 and F2 have good antioxidant activity, and the IC50 value of F2's ABTS free radical scavenging ability is 0.2903 mg / mL. After comprehensive consideration, F2 component was selected for gel column chromatography separation. 0.1 mg / mL ascorbic acid, i.e., vitamin C, was used as a positive control.

[0081] Experiment 7: The F2 fraction was separated by dextran gel column chromatography as follows: The F2 component was separated using an AKTA protein purifier equipped with a 16 mm 80 cm Sephadex-25 gel column. The flow rate was 0.5 mL / min, and the absorbance at 280 nm, 254 nm, and 230 nm was monitored. After elution with ultrapure water until the baseline was stable, the sample volume was 2 mL and the sample concentration was 50 mg / mL. Four components were separated, recorded as S1, S2, S3, and S4. S4 was not studied because it was difficult to collect due to its small molecular weight. Fig.12 The antioxidant activity of the S1, S2 and S3 fractions was determined in Figure 13~Figure 15 Among them, the IC50 value of ABTS free radical scavenging ability of S2 component was significantly reduced to 74μg / mL. Therefore, S2 component was selected for identification and sequencing.

[0082] Experiment 8: Identification and database screening of S2 components, as follows: The S2 component was identified by LC-MS / MS to contain 15,570 peptide sequences, of which 2,099 met the following conditions: removal of repeated sequences, 3 to 10 amino acids, average peptide confidence ALC of more than 95%, and removal of peptides with an abundance Area of ​​0. The amino acid length distribution of the 2,099 peptide sequences is shown in Fig.16 .

[0083] First, 2099 peptide sequences were predicted to have potential bioactive peptides through the PeptideRanker database, with a threshold of 9.0, and a total of 637 peptides were predicted; then 637 peptide sequences were used to exclude a potential toxic peptide through the ToxinPred database. Finally, 636 peptide sequences were predicted to have potential antioxidant activity through the AnOxPePred-1.0 database, and the score distribution is shown in Fig.17 Among them, there are 7 peptide sequences with scores greater than 0.6, see Table 2.

[0084] PeptideRanker database: http: / / distilldeep.ucd.ie / PeptideRanker / , accessed on June 25, 2024.

[0085] ToxinPred Database: https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submitfreq_S.php?ran=20020 / , accessed on June 25, 2024.

[0086] ToxinPred Database: https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submitfreq_S.php?ran=20020 / , accessed on June 25, 2024.

[0087] Table 2 Seven antioxidant peptides from chanterelles predicted by the database

[0088] Experiment 9: Virtual molecular docking and synthesis verification, as follows: The docked receptor protein Keap 1 was downloaded from the database https: / / www.rcsb.org / . Keap 1 is 2FLU in PDB. The ligands and water molecules were removed and hydrogen bonds were added. The 7 peptides were constructed using ChemDraw22.0.0 and Chem3D, and the geometry was optimized to minimize the energy. Molecular docking was performed using AutoDockTools-1.5.6, with the docking running coordinates of X: 5, Y: 9 and Z: 2. The docking box size was set to 100×100×100 and 0.375 spacing, and docking was performed 50 times. PyMOL software was used for visualization analysis, see Figure 18 to Figure 24 The binding energies of FPYPY, YPPYF, YYPF and MPWY were all lower than -5 kcal / mol. These four sequences were synthesized and their antioxidant activities were verified. Figure 25~Figure 27 and Table 3, Figure 25~Figure 27 P1~P4 are FPYPY, YPPYF, YYPF and MPWY respectively.

[0089] Figure 1 to Figure 11 , Figure 13~Figure 15 and Figure 25~Figure 27 Different capital letters on the middle column represent significant differences within the group, P < 0.05; different lowercase letters represent significant differences between the groups, P < 0.05.

[0090] Table 3 Antioxidant activity of synthetic chanterelle peptides

[0091] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0092] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An antioxidant peptide derived from chanterelle fruiting bodies, characterized in that: The amino acid sequence of the antioxidant peptide is at least one of FPYPY, YPPYF, YYPF and MPWY.

2. The use of the antioxidant peptide according to claim 1, characterized in that: The antioxidant peptides are used to scavenge free radicals.

3. The use according to claim 2, characterized in that: The concentration of the antioxidant peptides used to scavenge the free radicals is 62.5 μg / mL to 1000 μg / mL.

4. The use according to claim 3, characterized in that: The concentration of the antioxidant peptide used to scavenge the free radicals was 1000 μg / mL.

5. The use of the antioxidant peptide according to claim 1, characterized in that: The antioxidant peptide is used for preparing antioxidant.

6. The use according to claim 5, characterized in that: The antioxidant also includes pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable excipients include at least one of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

8. A reagent for detecting the activity of the antioxidant peptide according to claim 1, characterized in that: The reagent includes any one of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and 1,1-diphenyl-2-trinitrophenylhydrazine.

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