An antioxidant peptide derived from the fruiting body of Cantharellus cibarius, an antioxidant peptide detection reagent and applications thereof

Abstract

The present invention belongs to the field of food biotechnology, and specifically relates to an antioxidant peptide derived from the fruiting body of Cantharellus cibarius, an antioxidant peptide detection reagent and applications thereof. 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 Cantharellus cibarius, and is an enzymatic hydrolysis product of Cantharellus cibarius protein. Because it is derived from the edible and medicinal mushroom Cantharellus cibarius, it has low toxicity and side effects and high safety. At the same time, the antioxidant peptide of the present invention has good biological activity and strong antioxidant effects, can significantly scavenge free radicals, and can be applied to repair damage caused by oxidative stress.

Description

Technical Field

[0001] The present invention belongs to the field of food biotechnology, and specifically relates to an antioxidant peptide derived from the fruiting body of Cantharellus cibarius, an antioxidant peptide detection reagent, and applications thereof. Background Art

[0002] Oxidative stress, with the English name Oxidative Stress and abbreviated as OS, is a negative effect produced by free radicals in the body and is considered an important factor leading to aging and diseases. Exogenous hazards such as cigarettes, exhaust gases emitted by automobiles and industries, alcohol, and various heavy metal pollutions, and equally worthy of attention are endogenous hazards such as inflammation, stress, and anxiety. These exogenous and endogenous hazards will cause the levels of reactive oxygen species and reactive nitrogen species in the body to exceed the normal value, thereby leading to the generation of oxidative stress. Persistent oxidative stress will lead to the occurrence of various diseases such as multiple mental diseases including depression and 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, and hydrogen peroxide, and scavenge them by providing electrons or hydrogen atoms, etc., thereby reducing the oxidative damage of free radicals to skin cells, antioxidants are also applied in the cosmetic field.

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

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

[0005] The technical solution adopted by the present invention is as follows:

[0006] The present invention provides an antioxidant peptide derived from the fruiting body of Cantharellus cibarius, and the amino acid sequence of the antioxidant peptide is at least one of FPYPY, YPPYF, YYPF, and MPWY.

[0007] The present invention provides an application of the antioxidant peptide, and the antioxidant peptide is used for scavenging free radicals.

[0008] Preferably, the concentration of the antioxidant peptide for scavenging the free radicals is 62.5 μg / mL to 1000 μg / mL.

[0009] Preferably, the concentration of the antioxidant peptide for scavenging the free radicals is 1000 μg / mL.

[0010] The present invention provides an application of the antioxidant peptide, and the antioxidant peptide is used for preparing an antioxidant.

[0011] Preferably, the antioxidant further comprises a pharmaceutically acceptable excipient.

[0012] Preferably, the pharmaceutically acceptable excipient comprises at least one of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickening agent, an emulsifier, a preservative, and a stabilizer.

[0013] Preferably, the diluent comprises any one of starch, lactose, sucrose, and mannitol.

[0014] Preferably, the disintegrant comprises any one of starch, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose.

[0015] Preferably, the precipitation inhibitor comprises any one of sodium dodecyl sulfate, Tween-80, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.

[0016] Preferably, the glidant comprises any one of cationic polyacrylamide, poly(diallyldimethylammonium chloride), and cationic starch.

[0017] Preferably, the binder comprises any one of starch paste, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.

[0018] Preferably, the dispersant comprises any one of sodium dodecyl sulfate, polyvinylpyrrolidone, and sodium carboxymethylcellulose.

[0019] Preferably, the suspending agent comprises any one of gum arabic, tragacanth, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose.

[0020] Preferably, the isotonic agent comprises any one of sodium chloride, glucose, and mannitol.

[0021] Preferably, the thickening agent comprises any one of gum arabic, xanthan gum, and sodium carboxymethylcellulose.

[0022] Preferably, the emulsifier comprises any one of sodium dodecyl sulfate, benzalkonium chloride, and sorbitan fatty acid esters.

[0023] Preferably, the preservative comprises any one of benzoic acid, sorbic acid, methyl p-hydroxybenzoate, and benzalkonium bromide.

[0024] Preferably, the stabilizer is any one of sodium sulfite, sodium bisulfite, tocopherol and disodium ethylenediaminetetraacetate.

[0025] The present invention also provides a reagent for detecting the activity of the antioxidant peptide, and the reagent includes any one of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl.

[0026] Preferably, the antioxidant is used for preparing a drug for treating or preventing mental diseases.

[0027] Preferably, the mental disease includes any one of depression, obsessive-compulsive disorder, anxiety disorder and schizophrenia.

[0028] Preferably, the antioxidant is used for preparing a drug for treating or preventing diabetes.

[0029] Preferably, the antioxidant is used for preparing a drug for treating or preventing neurodegenerative diseases.

[0030] Preferably, the neurodegenerative disease includes any one of Alzheimer's disease, Parkinson's disease and Huntington's disease.

[0031] Preferably, the antioxidant is used for preparing cosmetics.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] The present invention provides an antioxidant peptide derived from the fruiting body of Cantharellus cibarius, and 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 Cantharellus cibarius and is an enzymatic hydrolysis product of Cantharellus cibarius protein. Because it is derived from the edible and medicinal mushroom Cantharellus cibarius, it has small side effects and high safety. At the same time, the antioxidant peptide of the present invention has good biological activity and strong antioxidant effect, can significantly scavenge free radicals, and can be applied to repair the damage caused by oxidative stress.

[0034] 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) can be oxidized to form a stable blue-green cation radical ABTS⁺. When antioxidant peptides are present, the antioxidant peptides will react with ABTS⁺, causing the color of the solution to fade. By measuring the change in absorbance, the ability of antioxidants to scavenge ABTS⁺ radicals can be evaluated, thereby measuring their antioxidant activity. It is widely used in the evaluation of the antioxidant properties of antioxidant components or products in the fields of food, health products, cosmetics, etc. 1,1-Diphenyl-2-picrylhydrazyl is a stable free radical, and its lone pair of electrons has a strong absorption at about 517 nm, making the solution dark purple. When antioxidant peptides are present, the hydrogen atoms or electrons provided by the antioxidant peptides will combine with the 1,1-diphenyl-2-picrylhydrazyl radical, causing the color of its solution to fade and the absorbance to decrease. By measuring the change in absorbance, the scavenging rate of antioxidant peptides for 1,1-diphenyl-2-picrylhydrazyl radicals can be calculated, and the antioxidant ability of antioxidant peptides can be evaluated accordingly. The present invention uses 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl to determine the antioxidant ability of antioxidant peptides. Finally, it is determined that the concentration range for the antioxidant peptides of the present invention to exert antioxidant ability is 62.5 μg / mL to 1000 μg / mL, and the optimal concentration is 1000 μg / mL.

[0035] In addition, the antioxidant peptides of the present invention are obtained by enzymatic hydrolysis. The antioxidant peptides prepared by this method can enhance functional properties without affecting their nutritional value.

[0036] Cantharellus cibarius ( Cantharellus cibarius Fr) belongs to ectomycorrhizal fungi, belonging to the fungal kingdom, Basidiomycota, Agaricomycotina, Agaricomycetes, Cantharellales, Cantharellaceae, Cantharellus genus, and is also known as yellow filamentous mushroom or apricot mushroom. The global annual output of wild Cantharellus cibarius is about 150,000 to 200,000 tons. Carbohydrates in Cantharellus cibarius account for about 64%, crude protein accounts for about 21%, and fat accounts for about 6%. Its total amino acids are higher than those of general edible fungi. Cantharellus cibarius has biological activities such as antioxidant, immune enhancement, antibacterial, and neuroprotection, and has high nutritional and medicinal values. In addition to direct consumption or rough processing, the deep development of Cantharellus cibarius resources is relatively less. Through the antioxidant peptides of the present invention, a new way is provided for the development and research of active peptide products using Cantharellus cibarius, which promotes the development of the Cantharellus cibarius 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 and auxiliary materials for food, medicine, and health products, enabling the full development and utilization of Cantharellus cibarius resources, providing an effective guarantee for the development of the Cantharellus cibarius industry, and having relatively long-term scientific theoretical guiding significance and high economic value significance. Brief Description of the Drawings

[0037] Figure 1 is the degree of hydrolysis of enzymatically hydrolyzed Cantharellus cibarius protein.

[0038] Figure 2 is the ABTS radical scavenging ability of the protease hydrolysate of Cantharellus cibarius.

[0039] Figure 3 is the DPPH radical scavenging ability of the protease hydrolysate of Cantharellus cibarius.

[0040] Figure 4 is the reducing ability of the protease hydrolysate of Cantharellus cibarius.

[0041] Figure 5 is the degree of hydrolysis of the double-enzyme hydrolysate of Cantharellus cibarius protein.

[0042] Figure 6 is the ABTS radical scavenging ability of the double-enzyme hydrolysate of Cantharellus cibarius protein.

[0043] Figure 7 is the DPPH radical scavenging ability of the double-enzyme hydrolysate of Cantharellus cibarius protein.

[0044] Figure 8 is the reducing ability of the double-enzyme hydrolysate of Cantharellus cibarius protein.

[0045] Figure 9 is the ABTS radical scavenging ability of the ultrafiltration fraction of FA-CCPH.

[0046] Figure 10 is the DPPH radical scavenging ability of the ultrafiltration fraction of FA-CCPH.

[0047] Figure 11 is the reducing ability of the ultrafiltration fraction of FA-CCPH.

[0048] Figure 12 is the fraction obtained by eluting the F2 ultrafiltration fraction through a gel chromatography column.

[0049] Figure 13 is the ABTS radical scavenging ability of the gel chromatography elution fraction.

[0050] Figure 14 is the DPPH radical scavenging ability of the gel chromatography elution fraction.

[0051] Figure 15 is the reducing ability of the gel chromatography elution fraction.

[0052] Figure 16 is the amino acid length distribution map of 2099 peptide sequences preliminarily screened.

[0053] Figure 17 is the activity score of 636 sequences predicted to have antioxidant activity.

[0054] Figure 18It is the 3D docking map of VDPWHPWP peptide and the active site of Keap 1.

[0055] Figure 19 It is the 3D docking map of FPYPY peptide and the active site of Keap 1.

[0056] Figure 20 It is the 3D docking map of YPPYF peptide and the active site of Keap 1.

[0057] Figure 21 It is the 3D docking map of WYW peptide and the active site of Keap 1.

[0058] Figure 22 It is the 3D docking map of YYPF peptide and the active site of Keap 1.

[0059] Figure 23 It is the 3D docking map of MPWY peptide and the active site of Keap 1.

[0060] Figure 24 It is the 3D docking map of GYGPF peptide and the active site of Keap 1.

[0061] Figure 25 It is the ABTS radical scavenging ability of Cantharellus cibarius antioxidant peptide.

[0062] Figure 26 It is the DPPH radical scavenging ability of Cantharellus cibarius antioxidant peptide.

[0063] Figure 27 It is the reducing ability of Cantharellus cibarius antioxidant peptide. Specific implementation mode

[0064] The present invention is further illustrated by the following specific examples, but does not limit the scope of the present invention. Without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications or substitutions all fall within the protection scope of the present invention.

[0065] The inventive concept of the present invention is as follows:

[0066] 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, butylhydroxyanisole, etc., but they have side effects such as affecting body metabolism, causing allergic reactions and potential carcinogenicity during application. Therefore, there is an urgent need to provide a natural antioxidant that can repair oxidative damage and has safety.

[0067] Based on this, the present invention provides an antioxidant peptide derived from the fruiting body of Cantharellus cibarius, and the amino acid sequence of the antioxidant peptide is at least one of FPYPY, YPPYF, YYPF, and MPWY.

[0068] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0069] The abbreviation list of the present invention is shown in Table 1.

[0070] Table 1 Abbreviation list

[0071]

[0072] Example 1

[0073] An antioxidant peptide derived from the fruiting body of Cantharellus cibarius is as follows:

[0074] Experiment 1: Extraction of CCP is as follows:

[0075] Mix the defatted Cantharellus cibarius powder with ultrapure water at a solid-liquid ratio of 1:50, adjust the solution pH to 13.0 with 0.1M NaOH, and extract with 800W ultrasonic wave at 50°C for 1h; after filtration, adjust the solution pH to 3.4 with 0.1M HCl, let it stand overnight and then centrifuge, collecting both the precipitate and the supernatant; add ammonium sulfate powder to the supernatant to make the dissolution amount of ammonium sulfate in the supernatant reach 90% of the maximum dissolution amount, let it stand overnight and then centrifuge to collect the precipitate; mix the two precipitates to obtain CCP, adjust the pH to 7.0, then dialyze and concentrate and freeze-dry. The protein content is determined by the BCA method, and three parallel experiments are carried out. The average value of the protein extraction rate is calculated to be 49.00%, and the relative standard deviation is 0.20%. 0.603 g of ammonium sulfate powder is added to each 1 mL of the supernatant.

[0076] Experiment 2: Enzymatic hydrolysis of CCP is as follows:

[0077] First, prepare a 2% CCP solution, adjust the pH value to 7.0, and then heat it in a water bath at 95°C for 20 min to completely denature the protein. After that, the following experiments are continued respectively:

[0078] (1) Add 4000 U / g of alkaline protease to the completely denatured CCP solution, and carry out enzymatic hydrolysis at 45°C and pH value of 10.5 for 4h. After enzymatic hydrolysis, inactivate the enzyme in a water bath at 95°C for 20 min to obtain Cantharellus cibarius enzymolysis product A-CCPH. 1 g of alkaline protease is added to every 50 mL of CCP solution;

[0079] (2) Add 4000 U / g of neutral protease to the completely denatured CCP solution, enzymatically hydrolyze it at 40 °C and pH 7.0 for 4 h, inactivate the enzyme in a water bath at 95 °C for 20 min after enzymatic hydrolysis to obtain the enzymatically hydrolyzed product N-CCPH of Cantharellus cibarius, add 1 g of neutral protease to every 50 mL of CCP solution;

[0080] (3) Add 4000 U / g of flavor protease to the completely denatured CCP solution, enzymatically hydrolyze it at 55 °C and pH 7.0 for 4 h, inactivate the enzyme in a water bath at 95 °C for 20 min after enzymatic hydrolysis to obtain the enzymatically hydrolyzed product F-CCPH of Cantharellus cibarius, add 1 g of flavor protease to every 50 mL of CCP solution;

[0081] (4) Add 4000 U / g of bromelain to the completely denatured CCP solution, enzymatically hydrolyze it at 55 °C and pH 7.0 for 4 h, inactivate the enzyme in a water bath at 95 °C for 20 min after enzymatic hydrolysis to obtain the enzymatically hydrolyzed product B-CCPH of Cantharellus cibarius, add 1 g of bromelain to every 50 mL of CCP solution;

[0082] (5) First add 4000 U / g of alkaline protease to the completely denatured CCP solution, enzymatically hydrolyze it at 45 °C and pH 10.5 for 4 h; after inactivating the enzyme in a water bath at 95 °C for 20 min, then add 4000 U / g of flavor protease, enzymatically hydrolyze it at 55 °C and pH 7.0 for 4 h, inactivate the enzyme in a water bath at 95 °C for 20 min after enzymatic hydrolysis to obtain the enzymatically hydrolyzed product AF-CCPH of Cantharellus cibarius, add 1 g of alkaline protease and 1 g of flavor protease to every 50 mL of CCP solution;

[0083] (6) First add 4000 U / g of flavor protease to the completely denatured CCP solution, enzymatically hydrolyze it at 55 °C and pH 7.0 for 4 h; after inactivating the enzyme in a water bath at 95 °C for 20 min, then add 4000 U / g of alkaline protease, enzymatically hydrolyze it at 45 °C and pH 10.5 for 4 h, inactivate the enzyme in a water bath at 95 °C for 20 min after enzymatic hydrolysis to obtain the enzymatically hydrolyzed product FA-CCPH of Cantharellus cibarius, add 1 g of flavor protease and 1 g of alkaline protease to every 50 mL of CCP solution.

[0084] The alkaline protease and neutral protease were purchased from Beijing Solarbio Science & Technology Co., Ltd. The product number of the alkaline protease is B8360, and the product number of the neutral protease is Z8031; the flavor protease and bromelain were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The product number of the flavor protease is S10153-25g, and the product number of the bromelain is S10009-25g.

[0085] Experiment 3: Determination of degree of hydrolysis, as follows:

[0086] Experiment 3 was to study the degree of hydrolysis of different kinds of proteases on the enzymatic hydrolysis reaction of CCP. The enzymatic hydrolysates of Cantharellus cibarius A-CCPH, N-CCPH, F-CCPH and B-CCPH were centrifuged at 8000 rpm / s for 10 min at 4 °C respectively. The protein content of the supernatant was determined by the BCA method and recorded as C. The degree of hydrolysis of the supernatant was determined by the o-phthalaldehyde method. 20 μL of the supernatant of A-CCPH, N-CCPH, F-CCPH and B-CCPH were taken and mixed with 150 μL of OPA reagent respectively for an accurate reaction for 2 min, and the absorbance value at 340 nm was measured and recorded as ODs. At the same time, 0.9516 meqv / L standard serine and ultrapure water were used to replace the samples as the standard control and the blank control, and were recorded as OD st and OD b . The degree of hydrolysis DH was calculated according to the following formula, and the calculation formula was as follows:

[0087] ;

[0088] where serine NH2 The calculation formula of is:

[0089] ;

[0090] In the formula, DH is the degree of hydrolysis, and the unit is %; is the concentration of the supernatant, and the unit is meqv / L; α is 0.970; β is 0.342; OD s is the absorbance value at 340 nm after mixing 20 μL of the supernatant with 150 μL of OPA reagent for reaction; OD b is the absorbance value at 340 nm of the blank control; OD st is the absorbance value at 340 nm of the standard serine; C is the protein concentration of the supernatant, and the unit is mg / mL; h hot is the total number of peptide bonds of the protein, and the Cantharellus cibarius protein h hot is 8.

[0091] The results are shown in Figure 1 , the degree of hydrolysis of A-CCPH was 37.13% and the relative standard deviation was 0.40%; the degree of hydrolysis of N-CCPH was 33.07% and the relative standard deviation was 0.36%; the degree of hydrolysis of F-CCPH was 39.03% and the relative standard deviation was 0.26%; the degree of hydrolysis of B-CCPH was 21.45% and the relative standard deviation was 0.25%. Among them, the degree of hydrolysis of flavor protease was significantly higher than that of the other three proteases, followed by alkaline protease.

[0092] Experiment 4: Determination of antioxidant activity, as follows:

[0093] The antioxidant activity of Cantharellus cibarius enzymolysate CCPHs was evaluated by measuring the ABTS radical scavenging ability, DPPH radical scavenging ability and reducing power. At the same time, 0.1 mg / mL ascorbic acid VC was used as a positive control.

[0094] (1) ABTS radical scavenging activity: It was evaluated by the absorbance at 734 nm. 0.3841 g of ABTS and 0.0662 g of potassium persulfate were dissolved in water and made up to 100 mL to prepare the ABTS stock solution, which was stored in the dark at room temperature overnight; the stock solution was diluted to an OD value of about 0.75 at 734 nm with a relative standard deviation of 0.02 as the ABTS working solution.

[0095] Method: Add 200 μL of the ABTS working solution to 50 μL of the Cantharellus cibarius enzymolysate, react in the dark for 10 min, and measure the OD value 3 times at 734 nm. The calculation formula for the ABTS radical scavenging rate is as follows:

[0096] ;

[0097] In the formula, the absorbance after adding ABTS to the Cantharellus cibarius enzymolysate is A 1 , the absorbance after adding water to the Cantharellus cibarius enzymolysate is A 2 , and the absorbance after adding water to ABTS is A 3 .

[0098] (2) DPPH radical scavenging activity: Take 200 μL of the Cantharellus cibarius enzymolysate, add 100 μL of the DPPH solution, place it in the dark for 10 min, measure the OD 517 value, repeat 3 times, and take the average value. The calculation formula for the DPPH radical scavenging rate is as follows:

[0099] ;

[0100] A 4 is the absorbance value of the sample after adding DPPH and 99.9% ethanol solution by volume to the Cantharellus cibarius enzymolysate; A 5 is the absorbance value of the sample control group after adding 99.9% ethanol solution by volume to the Cantharellus cibarius enzymolysate; A 6 is the absorbance value of the blank group after adding DPPH solution to distilled water; A 7 is the absorbance value of the blank control group after adding 99.9% ethanol solution by volume to distilled water.

[0101] (3)Determination of reducing ability: Enzymatic hydrolysates of Cantharellus cibarius were prepared with distilled water at different concentrations. Take 50 μL of the sample, add 50 μL of phosphate buffer at pH 6.6 and 50 μL of 1% potassium ferricyanide chloride solution, react in a water bath at 50 °C 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 to replace the potassium ferricyanide solution as the control group, repeat 3 times, record the data, and take the average value.

[0102] Reducing ability = OD of Cantharellus cibarius enzymatic hydrolysate group 700 - OD of the control group 700 .

[0103] Experiment 5: Double enzymatic hydrolysis of CCP, as follows:

[0104] The antioxidant activities of 4 single enzymatic hydrolysis products were determined by the method in Experiment 4, as shown in Figures 2 - 4 , and the corresponding IC50 values were calculated. Among them, F-CCPH and A-CCPH showed better ABTS radical scavenging ability, and the IC50 values were 0.3078 mg / mL and 0.3165 mg / mL respectively; B-CCPH showed better DPPH radical scavenging ability, and the IC50 value was 1.620 mg / mL; while the reducing ability of A-CCPH was significantly higher than that of other enzymatic hydrolysates. Therefore, considering the hydrolysis degree and antioxidant activity comprehensively, flavor protease and alkaline protease were selected for double enzymatic hydrolysis to obtain AF-CCPH and FA-CCPH for subsequent analysis. The double enzymatic hydrolysis showed a higher hydrolysis degree, as shown in 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%. In terms of antioxidant activity, as shown in Figures 6 - 8 , the ABTS radical scavenging ability of the double enzymatic hydrolysis product was similar to that of the single enzymatic hydrolysis product, while the DPPH radical scavenging ability and reducing ability were significantly improved compared with the single enzymatic hydrolysis product. The IC50 value of the DPPH radical scavenging ability of FA-CCPH 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. Considering comprehensively, FA-CCPH was selected for subsequent ultrafiltration separation.

[0105] Experiment 6: Ultrafiltration of FA-CCPH, as follows:

[0106] Prepare a 50 mg / mL FA-CCPH solution. First, use an ultrafiltration tube with a molecular weight cut-off of 10 kDa. The ultrafiltration conditions are 4500 rpm / s and centrifugation at 4 °C for 40 min. After ultrafiltration, collect the fraction less than 10 kDa. Then, use an ultrafiltration tube with a molecular weight cut-off of 3 kDa and perform 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. Calculate the yields of the two fractions after lyophilization to be 6.38% and 26.54% respectively. Measure their antioxidant activities, see Figures 9 - 11 , both F1 and F2 have good antioxidant activities. The IC50 value of the ABTS radical scavenging ability of F2 is 0.2903 mg / mL. Considering comprehensively, select the F2 fraction for gel column chromatography separation. Use 0.1 mg / mL ascorbic acid, that is, vitamin C, as the positive control.

[0107] Experiment 7: Separation of the F2 fraction by Sephadex gel column chromatography is as follows:

[0108] Use an AKTA protein purifier equipped with a 16 mm × 80 cm Sephadex-25 gel column to separate the F2 fraction. The flow rate is 0.5 mL / min, and monitor the absorbance at 280 nm, 254 nm, and 230 nm. After washing with ultrapure water until the baseline is stable, the sample loading volume is 2 mL, and the sample loading concentration is 50 mg / mL. Four fractions are separated, denoted as S1, S2, S3, and S4. Since S4 has too small a molecular weight and is difficult to collect, it is not studied, see Figure 12 . Measure the antioxidant activities of the S1, S2, and S3 fractions, see Figures 13 - 15 , among which the IC50 value of the ABTS radical scavenging ability of the S2 fraction is significantly reduced to 74 μg / mL. Therefore, select the S2 fraction for identification and sequencing.

[0109] Experiment 8: Identification and database screening of the S2 fraction are as follows:

[0110] Through LC-MS / MS, it is identified that the S2 fraction contains 15570 peptide sequences, of which 2099 meet the following conditions: screening out repeated sequences, the number of amino acids is 3 - 10, the average confidence level of the peptide segment ALC is above 95%, and removing peptide segments with an abundance Area of 0. The amino acid length distribution of the 2099 peptide sequences is shown in Figure 16 .

[0111] First, 2099 peptide sequences are predicted for potentially bioactive peptides through the PeptideRanker database, with the threshold set to 9.0, and there are 637 in total; then, one potentially toxic peptide is excluded from the 637 peptide sequences through the ToxinPred database. Finally, 636 peptide sequences are predicted for potential antioxidant activities through the AnOxPePred-1.0 database, and the score distribution is shown inFigure 17 Among them, there are 7 peptide sequences with scores greater than 0.6, as shown in Table 2.

[0112] PeptideRanker database: http: / / distilldeep.ucd.ie / PeptideRanker / , accessed on June 25, 2024.

[0113] ToxinPred database:

[0114] https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submitfreq_S.php?ran=20020 / , accessed on June 25, 2024.

[0115] ToxinPred database:

[0116] https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submitfreq_S.php?ran=20020 / , accessed on June 25, 2024.

[0117] Table 2 Seven Cantharellus cibarius antioxidant activity peptides predicted by the database

[0118]

[0119] Experiment 9: Virtual molecular docking and synthesis verification are as follows:

[0120] Download the docking receptor protein Keap 1 from the database https: / / www.rcsb.org / . Keap 1 is 2FLU in the PDB. Remove the ligand and water molecules and add hydrogen bonds. The 7 peptides are constructed using ChemDraw 22.0.0 and Chem3D, and the geometric shape is optimized to minimize the energy; AutoDockTools - 1.5.6 is used for molecular docking. The docking running coordinates are X: 5, Y: 9, and Z: 2. At the same time, the docking box size is set to 100×100×100 and the spacing is 0.375, and the docking is performed 50 times. PyMOL software is used for visual analysis, as shown in Figures 18 - 24 Among them, the binding energies of FPYPY, YPPYF, YYPF, and MPWY are all lower than -5 kcal / mol. Synthesize these four sequences and verify their antioxidant activities, as shown in Figures 25 - 27 and Table 3, Figures 25 - 27 P1 - P4 in it are FPYPY, YPPYF, YYPF, and MPWY in sequence.

[0121] Figures 1 - 11 、 Figures 13 - 15and Figures 25 - 27 Different capital letters on the columns represent significant differences within the group, P < 0.05; different lowercase letters represent significant differences between groups, P < 0.05.

[0122] Table 3 Antioxidant activity of synthetic cantharellus cibarius peptide

[0123]

[0124] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0125] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

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

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

3. The use according to claim 2, characterized in that: The antioxidant is used for preparing cosmetics for scavenging free radicals.

4. The use according to claim 2, characterized in that: The antioxidant also includes pharmaceutically acceptable excipients.

5. The use according to claim 4, 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.

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