Hierarchical magnetic core-shell nanoreactor (Fe3O4@SA/Trypsin@UiO-66) for efficient sensing of protease inhibitors in food and feed products.

Trypsin inhibitors (TIs) are common anti-nutritional factors in legumes and grains, necessitating reliable detection for food nutrition assessment and feed safety control. This study took advantage of immobilized enzymes and successfully constructed an efficient magnetic core-shell composite material, Fe3O4@SA/Trypsin@UiO-66. Within this structure, the sodium alginate (SA) hydrogel provides a mild microenvironment for trypsin immobilization, while the outer UiO-66 metal-organic framework (MOF) shell not only enhances the nanoreactor's stability but also improves substrate enrichment and catalytic efficiency through a nanoconfinement effect. The nanoreactor exhibits robust magnetic separability, well-retained catalytic activity, and long-term applicability. Systematic performance studies revealed that, compared to free enzyme, the nanoreactor exhibited enhanced stability under freeze-thaw cycles and across broad pH (2-11) and temperature (25-70 °C) ranges, maintaining over 70% of its initial activity after multiple uses. In practical sample detection, the established nanoreactor system, which uses the nanoreactor as the core material and benzoyl-l-arginine ethyl ester (BAEE) as the substrate, was successfully applied to analyze TI content in various grain flours and fish feeds. In addition, the results provide evidence supporting the role of thermal processing in reducing TI activity. This research provides a reliable approach for the efficient detection of anti-nutritional factors in complex matrices.