Study On Preparation,Purification And Structure-function Of Dandelion Seeds

In this dissertation,dandelion seed protein was extracted from dandelion seed and then was hydrolyzed by suitable protease to obtain antioxidant peptides.Several isolation and purification methods were applied to purify peptides.Furthermore,the sequence and antioxidant activity in vitro of peptides were evaluated.Finally,the possible antioxidant mechanism of the peptides was explored.The process conditions for protein extraction were optimized by response surface methodology?RSM?.The result showed that the optimal extraction conditions were as follows:ration of solid to liquid was 1:20,alkali concentration was 0.1mol/L,temperature was 39.87 ? and time was 139.43 min.Under these conditions,the isoelectric point of protein was 4.0,the foaming and emulsifying capacities of dandelion protein was 145%and 1.089 m2/g respectively under a suitable pH,the water-binding capacity approximately ranged from 2.5 to 4.0 g/g,the oil-binding capacity approximately ranged from 1.0 to 3.0 g/g.A Box-Behnken design was used to optimize the hydrolysis technology and the optimal enzymolysis conditions were substrate concentration of 2%,temperature of 45 ?,time of 4.86h,pH of 8.45 and enzyme/substrate ratio of 7.76%.Under the optimal enzymolysis conditions,DPPH radical scavenging rate of the hydrolysates was 79.13%.Two separation steps,including gel filtration chromatography by Sephadex G-25 and RP-HPLC were adopted for the purification of dandelion seed protein hydrolysate.Finally,four peptides with higher antioxidant activity were obtained and the amino sequences were identified as Asn-Lys-Leu?NL-3?,Val-Phe?VF-2?,Trp-Thr-Leu-Asn?WN-4?and Thr-Pro-Thr-Leu-Asn?TN-5?.Especially,WN-4 could effectively quench free radicals and showed well capacity on inhibiting linoleic acid peroxide.The interaction between WN-4 and ABTS radical was also studied.The full wavelength scan experiment indicated that ABTS radical was reduced as ABTS.According to the calculation by Benesi-Hildebrand equation,the interaction between WN-4 and ABTS radical was a 1:1 interaction and the equilibrium constant was 2.58×104.The ABTS radical scavenging curve showed that WN-4 cleared ABTS radical completely in 400 seconds.To sum up,WN-4 might offer a proton to scavenge ABTS radical.Trp was replaced with Ala in the WN-4 sequence to get a new pepetide named as AN-4.Compared with WN-4,the antioxidant activity of AN-4 decreased significantly,which indicated that Trp made a great contribution to the antioxidant activity of WN-4.The mechanism of Cu²⁺-mediated inactivation of WN-4 was investigated by fluorescence spectra.Fluorescence quenching spectra demonstrated that Cu²⁺ could quench WN-4 endogenous fluorescence in both static and dynamic states.The dynamic quenching constant of interaction was 1.46×106 L/?mol·s?and quenching rate constant was 1.46×1014 L/?mol·s?.In the static quenching process,the binding constant between Cu²⁺ and WN-4 was 8.8×103 and the molecular coupling ratio was 1:1.