Separation and Identification of Anthocyanins Extracted from Blueberry Wine Lees and Pigment Binding Properties toward β-Glucosidase

Anthocyanins were isolated from blueberry wine lees using Sephadex LH-20 column chromatography and semipreparative high-performance liquid chromatography (semipreparative HPLC) and then identified by HPLC-DAD-ESI-MS/MS. Our results show that malvidin-3-hexose (Mv-3-hex) and malvidin-3-(6'acetyl)-hexose (Mv-3-ace-hex) are the major components in the anthocyanin extracts of blueberry wine lees (>90%). The binding characteristics of My-3-hex and Mv-3-acehex with beta-glucosidase were investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking. Spectroscopic analysis revealed that beta-glucosidase fluorescence quenched by My-3-hex and My-3-ace-hex follows a static mode. Binding of My-3-hex and My-3-ace-hex to beta-glucosidase mainly depends on electrostatic force. The result from CD spectra shows that adaptive structure rearrangement and increase of fi-sheet structure occur only in the presence of My-3-ace-hex. A molecular docking study suggests that My-3-ace-hex has stronger binding with beta-glucosidase than My-3-hex.