Alcalase immobilized on novel porous hydrogel beads via CO2 bubble strategy to reduce cow's milk allergenicity

• CO 2 bubbles can improve the activity and reusability of alcalase immobilized on hydrogel beads. • Immobilized alcalase shows superior pH, temperature, and storage stabilities to free alcalase. • Immobilized alcalase enables the controlled hydrolysis of cow's milk proteins to reduce their allergenicity. • Gas strategy is an efficient method for preparing safety and recyclable hydrogel-based biocatalyst. Cow's milk allergy poses major global health risks that limit the consumption of milk-based products. Meanwhile, recycling enzymes in the enzymatic methods for decreasing cow's milk antigenicity has been challenging. Here, we developed a green and simple approach using carbon dioxide (CO 2 ), generated by the reaction between sodium bicarbonate and acetic acid, as the gas porogen to fabricate novel porous carboxymethyl chitosan/alginate hydrogel beads to immobilize alcalase, aiming at reducing cow's milk antigenicity. This CO 2 bubble strategy notably enhanced the porosity of the hydrogel beads. The specific enzyme activity (SEA) of alcalase immobilized on the porous hydrogel beads prepared with 0.075 mM NaHCO 3 (Alc/GHB-G7.5) increased by 42.2% compared with those on supports without gas treatment. Additionally, after four cycles of operation at 40°C and pH 7.0, Alc/GHB-G7.5 maintained 84.0% of its initial SEA. Compared with free alcalase, the immobilized form exhibited superior pH and temperature tolerance, along with improved storage stability. The allergenicity of cow's milk decreased by 65.6% after one hour of enzymatic hydrolysis using Alc/GHB-G7.5. Importantly, all materials involved in the study were food-grade, fully ensuring food safety. Therefore, the proposed biocatalyst has great potential for producing hypoallergenic cow's milk-related foods.