An optimized in vitro digestion protocol for evaluating zero-valent iron nanoparticles in plant-based food matrices

Zero-valent iron nanoparticles (Fe(0) NPs) are increasingly used in agriculture and food systems, raising concerns about their stability and bioaccessibility upon ingestion. This study investigated Fe(0) NP transformations during simulated oral-gastric-intestinal digestion using STEM imaging, single-particle ICP-MS/MS, and total iron quantification. The INFOGEST 2.0 protocol was adapted and optimized for FeNP-containing plant samples. Salivary amylase had no effect on Fe(0) NPs or plant matrix integrity. Gastric digestion with pepsin led to nanoparticle dissolution and aggregation at concentrations above 70 g·L^-1. The intestinal phase, with pancreatin at neutral pH, partially reversed dissolution, producing a mixture of ∼45 nm particles, aggregates, and ionic iron. The protocol was validated using FeNP-enriched tomato tissues and CRM tomato leaf. Iron distribution varied by plant part, with roots showing the highest accumulation. The optimized enzyme concentrations-0.5 g·L^-1 amylase, 70 g·L^-1 pepsin, and 7 g·L^-1 pancreatin-are recommended for in vitro studies of FeNP transformation and bioaccessibility in food-relevant plant systems.