Preparation,Seperation And Purification Of Armillaria Mellea Peptides And Their Antioxidant Activity

Oxidative damage caused by high level of free radicals in the body is one of the main causes of human cancer,diabetes,atherosclerosis,neurodegenerative diseases.Antioxidant peptides can prevent or treat diseases by scavenging free radicals.Therefore,the development of antioxidants has become the current research hotspot.The scientific name of Armillaria mellea is honeysuckle,is rich in protein and is one of the good sources of bioactive peptides.At present,it is often consumed mainly,and research on its functional components is mainly focused on polysaccharides,with little research on proteins and peptides reported.In this study,Armillaria mellea protein was produced from Armillaria mellea,and then hydrolyzed by ultrasound-assisted flavored protease to obtain Armillaria mellea protein peptides;after purification of Armillaria mellea protein peptides by ultrafiltration and Sephadex gel column chromatography,antioxidant activity of Armillaria mellea protein peptide and Armillaria mellea peptide fraction obtained by stepwise separation and purification were measured by antioxidant activity methods in vitro: DPPH radical scavenging,hydroxyl radical scavenging,ABTS radical scavenging,and total reducing power.The structure identification of highly active Armillaria mellea antioxidant peptide fractions by mass spectrometry and the solid-phase synthesis of peptides were used to screen the peptide sequences with high antioxidant activity;the protective effects of Armillaria mellea antioxidant peptides on oxidatively damaged human neuroblastoma cells(SH-SY5Y)were further studied.The main results are as follows:1.The Armillaria mellea protein was extracted by ultrasonication and alkaline extraction in steps: the results showed that this stepwise extraction method could substantially improve the protein extraction rate.The conditions of the alkaline extraction method after response surface optimization were: material-liquid ratio 1:47,alkaline extraction pH 10.0,alkaline extraction time 1.55 h,and temperature 80℃.After the supernatant was collected by centrifugation,the residue was then sonicated with the following conditions: material-liquid ratio 1:47,sonication power 200 W,sonication time 15 min,sonication pH 9.0,sonication temperature 30℃.The extraction rate of Armillaria mellea protein under these conditions was 76.59%.The precipitation of Armillaria mellea proteins was performed by isoelectric point precipitation(p I=3.7)followed by salting with 90% saturated ammonium sulfate.The dried Armillaria mellea protein powder was obtained after freeze-drying.2.Preparation of Armillaria mellea peptides by ultrasound-assisted flavourzyme hydrolysis:The results showed that ultrasound could improve the hydrolysis degree of flavored protease and the antioxidant activity of hydrolysis products.The optimal hydrolysis conditions were obtained after response surface optimization: under the condition that the substrate concentration was 4%,the ultrasonic power was 200 W,the ultrasonic treatment time was 15 min,and then the flavourzyme was added and the pH was adjusted to 7.0,the enzyme concentration was 3.5%,the hydrolysis temperature was 41℃,and the hydrolysis time was 3 h.At this time,the degree of protein hydrolysis reached 16.32%.The Armillaria mellea peptide powder was obtained after freeze-drying.3.Seperation and purification of Armillaria mellea peptides,structural identification and analysis of their antioxidant activity: The results showed that the antioxidant activity of the <3kD fraction obtained in ultrafiltration was higher compared to the total peptide,of which the DPPH scavenging rate,hydroxyl radical scavenging rate,ABTS scavenging rate and total reducing power reached 92.37%,96.72%,97.68% and 1.26,respectively.The Sephadex G-25 column chromatography of the <3 kD peptide fraction revealed that the fraction A2 had higher antioxidant activity,of which the DPPH scavenging rate,hydroxyl radical scavenging rate,ABTS scavenging rate and total reducing power reached 97.01%,100%,100% and 1.62,respectively.Mass spectrometry and de novo sequencing were used for the structural identification of fraction A2.Combined with the BIOPEP database,seven peptides were selected for solid-phase synthesis,and two peptides with high antioxidant activity were screened using antioxidant activity in vitro,which was peptide 3(FDHEW)and peptide 6(WDPVH).4.The antioxidant activity of Armillaria mellea peptide was examined in cellular assay: the results showed that five kinds of antioxidant peptides(total peptide,<3 kD,A2,synthetic peptide 3,synthetic peptide 6)showed good protective effect on SH-SY5 Y cells after oxidative damage.At the concentration of 2 mg/mL,A2 component had the highest cell viability.When the concentration was 200 μM,the cell viability of peptide 3 was higher than that of peptide 6.Compared with model group,five kinds of antioxidant peptides could increase the activities of SOD,CAT,GSH-Px and ATPase.When the concentration was 2 mg/mL,the activities of GSH-Px and CAT were the highest under A2 treatment;The activity of SOD was the highest under <3 kD treatment.The ATPase activity was the highest under the treatment of total peptide.At the concentration of 200 μM,the activities of SOD,CAT and GSH-Px in cells treated with peptide 3 were higher than those of peptide 6,and the activity of ATPase in cells treated with peptide 6 was higher than that of peptide 3.