Removal of dissolved oxygen by immobilized ascorbate oxidase onto a packaging film

In order to remove dissolved oxygen from liquid food, ascorbate oxidase (AO) was covalently immobilized onto ethylene-vinyl alcohol copolymer (EVOH) film. Maximal activity was observed at pH 6.0, 30°C (1.14 mU/cm²). The immobilized AO still retained a higher residual activity than free AO under practical conditions ; at pH 4.0 corresponding to citrus juice, 10°C and 60°C, the residual activities were 47.8%, 91.3%, and 49.4% whereas those of free AO were 3.1%, 63.3% and 0%, respectively. When the AO immobilized EVOH film was directly in contact with 2.5 mM L-ascorbic acid in 10 mM citrate buffer solution (pH 4.0), more than 85% of dissolved oxygen was removed within 4h. In addition, flavors (d-limonene, linalool, and citral) in the solution were successfully preserved from the oxidative deteriorations ; for d-limonene, the residual ratio (71.6%) with AO immobilized EVOH film was 2.5-fold that with non-immobilized one (29.0%) after 28 days' storage.