Genipin-crosslinked whey protein isolate for enhanced probiotic viability: Balancing protein structure and cell interactions during spray-dried encapsulation, storage, and digestion

This study explored the use of genipin-crosslinked whey protein isolate (WPI) to enhance the viability of Limosilactobacillus reuteri KUB-AC5 during spray drying, storage, and simulated gastrointestinal digestion. The effects of different genipin concentrations (0, 2.5, 5, and 10 mM) on WPI’s protein structure, particle size, microcapsule morphology, cell interactions, and probiotic survival were systematically examined. SDS-PAGE revealed that genipin crosslinking promoted protein aggregation, leading to the formation of higher molecular weight complexes. The ninhydrin assay confirmed a concentration-dependent increase in crosslinking. SEM and 13 C-NMR analyses indicated that WPI-2.5G provided the best balance between crosslinking and protein flexibility, resulting in strong cell-protein interactions. Viability tests demonstrated that WPI-2.5G offered the highest protection during spray drying, showing the lowest levels of cell wall and membrane injury. Encapsulated cells exhibited significantly improved survival under simulated gastrointestinal conditions compared to free cells, with WPI-2.5G yielding the best results. During 12 wk of storage, genipin-crosslinked WPI, particularly WPI-2.5G, maintained superior probiotic viability at 4 °C, with higher cell counts than at 25 °C. These findings suggest that genipin-crosslinked WPI, especially at 2.5 mM, effectively protects probiotics by optimizing protein structure and cell interactions during spray drying, storage, and digestion. • Genipin-crosslinked WPI forms protective matrix for probiotic encapsulation. • 2.5 mM genipin enhances WPI crosslinking to protect probiotics during spray drying. • WPI-2.5G boosts probiotic survival through gastrointestinal conditions. • WPI-2.5G extends probiotic shelf life, especially at cooler storage temperatures. • Genipin-crosslinked WPI enhances probiotic stability for food and pharma uses.