Optimization of Covalent Immobilization of Extracellular Ice Nucleators from Erwinia herbicola on Magnetic Fe3O4/Chitosan Nanoparticles for Potential Application in Freeze Concentration

A magnetically separable extracellular ice nucleators (ECINs) immobilized system was constructed, ECINs from Erwinia herbicola were covalently immobilized on magnetic Fe3O4/chitosan nanoparticles by coupling via glutaraldehyde (GA) or epichlorohydrin (ECH). The immobilization process parameters were optimized, and under optimized conditions, ice nucleation activity (INA) of GA and ECH immobilized ECINs reached at 1.56 × 106 ± 1.54 × 105 Units/mg and 1.62 × 106 ± 1.91 × 105 Units/mg, respectively. The stability, reusability and cytotoxicity of GA and ECH immobilized ECINs were characterized and compared; both GA and ECH immobilized ECINs demonstrated good reusability performance after 15 freeze–thaw cycles and low cytotoxicity, while GA immobilized ECINs were recovered more easily and more stable at different pH compared to ECH immobilized ECINs. Application of the immobilized ECINs in freeze concentration was investigated, and an increase of ice nucleation temperature for water, seawater and apple juice (6.83 ± 0.18 °C, 8.08 ± 0.50 °C and 9.38 ± 1.12 °C) was achieved by using GA immobilized ECINs. The results collected so far show that there are extensively promising applications for GA immobilized ECINs in freeze concentration industry fields.