Abstract
Glucose oxidase (GOX) and catalase (CAT) were co-immobilized in silica-calcium-alginate hydrogels to degrade must glucose. The effect of the enzyme dose (1.2-2.4 U/mL), the initial must pH (3.6-4.0), and the incubation temperature (10-20 °C) on the glucose consumption, gluconic acid concentration, pH, and color intensity of Verdejo must was studied by using a Box-Behnken experimental design and comparing free and co-immobilized enzymes. A reduction of up to 37.3 g/L of glucose was observed in co-immobilized enzyme-treated must, corresponding to a decrease in its potential alcohol strength of 2.0% vol. (v/v), while achieving a slight decrease in its pH (between 0.28 and 0.60). This slight acidification was due to a significant reduction in the estimated gluconic acid found in the must (up to 73.7%), likely due to its accumulation inside the capsules. Regarding the operational stability of immobilized enzymes, a gradual reduction in glucose consumption was observed over eight consecutive cycles. Finally, co-immobilized enzymes showed enhanced efficiency over a reaction period of 48 h, with an 87.1% higher ratio of glucose consumed per enzyme dose in the second 24 h period compared with free enzymes. These findings provide valuable insights into the performance of GOX-CAT co-immobilized to produce reduced-alcohol wines, mitigating excessive must acidification.