Purification And Investigation On The Mechanism Of Eptide From Brewer’s Spent Grain With α-glucosidase Inhibitory Activity

Brewer’s spent grain (BSG) is the most abundant co-products of the brewing industry.For a long time, BSG has been mainly used as low value cattle feed. Since the direct disposalof such products causes a waste of resources and environmental problem, there is a growinginterest in diversifying the utilization of BSG to raise economic and environmental benefits.Since BSG is rich in protein, it can be used as a raw material to produce proteins andbioactive peptides which can significantly improve its utilization. The purpose of this studywas to isolate bioactive peptide with α-glucosidase inhibitory activity made from BSG and thestability, molecular structure and interaction mechanism with α-glucosidase of this fractionwere also studied. The main contents and results are shown as following:(1) The BSG peptides were passed through ultrafiltration membranes with the range ofmolecular weight cut off (MWCO) of10kDa and5kDa. The fraction of molecular weightless than5kDa possess a highest α-glucosidase inhibitory activity. This fraction wasseparated with a Sephadex LH-20gel filtration chromatography column and a Mono anionexchange column. The fractions collected were then desalted and fractionated on a SephadexG-10gel filtration column. Results indicated that the fraction Da-1after separation had asingle sharp peak on an analytical HPLC column and exhibited an effective α-glucosidaseinhibitory activity of47.5%when the peptide concentration was325μg/mL.(2) The results of stability test showed that the BSG peptide with α-glucosidaseinhibitory activity had a good thermal stability at37℃, but its stability decreased with thetemperature increasing. The retention of peptide and inhibitory activity were88.7%and84.8%after3h in a37°C water bath. The BSG peptide was stable when pH was between5.0and9.0, but its inhibitory activity was significantly reduced at extreme pH conditions. Theretention of inhibitory activity was81.3%and54.9%when the pH was2.0and12.0. Theα-glucosidase inhibitory activity of BSG peptide decreased slightly with increasing theextension of UV irradiation time and the retention of inhibitory activity was84.6%after UVirradiation for4h. The stability of BSG peptide in artificial digestive juice was also studied.The retention of peptide and inhibitory activity were84.0%and81.5%after3h incubation inartificial gastric juice. The BSG peptide was more sensitive to trypsin because the retention of peptide and inhibitory activity were only73.1%and65.7%after3h incubation in artificialintestinal juice.(3) The molecular mass of Da-1was determined to be560.2Da by LC-MS/MS and thesequence was determined to be Ser-Pro-Asp-Arg-Ser by Edman degradation method.(4) The results of fluorescence analysis showed that the peptide caused the flurescencequenching of α-glucosidase through a static quenching procedure. The decomposing constantof this peptide binding with α-glucosidase kept at the magnitude of10-4in differenttemperature which indicated that they had a good combination. The peptide and α-glucosidaseformed one binding site and the number of binding sites was increased slightly with theincreasing of the temperature.(5) Molecular docking was used to investigate the interaction of the peptide andα-glucosidase. The docking results suggested that the peptide could enter the binding pocketand interact with the catalytic center. The peptide occupied the catalytic site by combiningwith the calcium ion and other key amino acids around the catalytic center which resulted inthe α-glucosidase inhibitory activity.