Immobilization of -galactosidase in polyvinyl alcohol-chitosan-glycidyl methacrylate hydrogels based on directional freezing-assisted salting-out strategy for hydrolysis of RFOs

Raffinose family oligosaccharides (RFOs) in food are the main factors causing flatulence in Irritable Bowel Syndrome (IBS) patients and the development of effective approaches for reducing food-derived RFOs is of paramount importance. In this study, polyvinyl alcohol (PVA)-chitosan (CS)-glycidyl methacrylate (GMA) immobilized a-galactosidase was prepared by the directional freezing-assisted salting-out technique, aimed to hydrolyze RFOs. SEM, FTIR, XPS, fluorescence and UV characterization results demonstrated that a-galactosidase was successfully cross-linked in the PVA-CS-GMA hydrogels, forming a distinct porous stable network through the covalent bond between the enzyme and the carrier. Mechanical performance and swelling capacity analysis illustrated that a-gal @ PVA-CS-GMA not only had suitable strength and toughness for longer durability, but also exhibited high water content and swelling capacity for better retention of catalytic activity. The enzymatic properties of a-gal @ PVA-CS-GMA showed an improved Km value, pH and temperature tolerance range, antienzymatic inhibitor (melibiose) activity compared to the free a-galactosidase and its reusability was at least 12 times with prolonged storage stability. Finally, it was successfully applied in the hydrolysis of RFOs in soybeans. These findings provide a new strategy for the development of a-galactosidase immobilization system to biological transform the RFOs components in the food for diet intervention of IBS.