Pediatric digestive characteristics and in vitro safety evaluation of dairy-derived amyloid fibrils

Amyloid fibrils have garnered significant attention in the food industry as functional materials due to their high specific surface area and excellent mechanical resilience. Although their safety has been demonstrated in adults, differences in protein origin and digestive physiology may lead to distinct degradation behaviors. Particularly in populations with immature or impaired digestive systems, such as children, the digestibility and biosafety of dietary amyloid fibrils remain largely unknown. In this study, we simulated pediatric gastrointestinal conditions to characterize molecular size and structural changes during the digestion of amyloid fibrils formed from three major dairy proteins-α-lactalbumin, β-lactoglobulin, and casein-and to evaluate the safety of their digestive products. The formation of amyloid fibrils was confirmed by Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM). Upon simulated digestion, all three types of fibrils were degraded into short peptides (<10 kDa); ThT fluorescence returned to baseline, β-sheet structures were lost, and particle sizes dropped below 50 nm. Further seeding assays revealed no detectable seeding activity, and cell viability assays demonstrated survival rates above 82% even at 1000 μg/mL, confirming their in vitro safety. This study establishes Ia safety evaluation framework based on pediatric digestive physiology, offering a valuable reference for the risk assessment of dietary amyloid proteins.