Magnetic nanoparticle-immobilized trypsin for whey protein hydrolysis: catalytic performance, reusability and antioxidant peptide generation

Enzyme immobilization on magnetic nanoparticles (MNPs) is a promising strategy for developing reusable, operationally stable biocatalytic systems. In this study, we covalently immobilized trypsin on 3-aminopropyltriethoxysilane (APTES)-functionalized MNPs using genipin as a green crosslinker and evaluated the resulting biocatalyst for hydrolysis of cheese whey proteins, a complex dairy matrix, focusing on catalytic performance, reusability, and antioxidant peptide production. The immobilized system exhibited a specific enzymatic activity of 126.2 U·g -1 , corresponding to a recovered activity of 146.6% relative to the free enzyme, attributed to the favorable microenvironment of the magnetic support and reduced autolysis from genipin crosslinking. After five consecutive reuse cycles, the biocatalyst retained 66.8% of its initial activity, demonstrating satisfactory operational stability. Enzymatic hydrolysis with the immobilized trypsin yielded a maximum degree of hydrolysis (DH) of 42.6% at 4 h, comparable to the free enzyme (38.8%), while enabling biocatalyst recovery. The resulting hydrolysates exhibited strong antioxidant capacity, as assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH, 91.6%), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, 90.3%), ferric reducing antioxidant power (FRAP, 265 μM Fe 2+ ), and β-carotene/linoleic acid (BCAL, 60.8%) assays, with the immobilized enzyme producing hydrolysates with antioxidant activity superior in DPPH, statistically equivalent in ABTS and FRAP, and slightly lower in BCAL, relative to the free enzyme. Chromatographic analysis confirmed peptide fractions with distinct hydrophobic profiles consistent with antioxidant activity. These findings demonstrate that genipin-crosslinked trypsin immobilized on MNPs is an efficient, sustainable biocatalytic platform for whey protein bioprocessing and antioxidant peptide production from dairy byproducts.