Abstract

This study explored how N-glycosylation influences the resistance of human lactoferrin (HLF) and bovine lactoferrin (BLF) to digestion during simulated infant gastrointestinal conditions. Native and deglycosylated lactoferrins were subjected to in vitro simulated infant gastrointestinal digestion, followed by structural and biochemical analyses. The results showed that natural HLF retained approximately 24.5% intact protein at I10 min, whereas BLF was almost completely degraded (<5%). The degree of hydrolysis at I10 min was significantly lower for HLF (19.1%) than BLF (34.2%) ( P < 0.05 ). Deglycosylation accelerated proteolysis by 1.23-fold at I10 min for HLF and 1.20-fold at I5 min for BLF ( P < 0.05 ), without altering secondary structure, but associated with premature tertiary unfolding (fluorescence red-shift ∼6 nm) and increased surface electronegativity at I5 min (ζ-potential: −21.1 vs −16.8 mV, P < 0.05). Molecular docking suggested glycan shielding may reduce enzyme binding by 0.3–2.1 kcal/mol. Glycoproteomic analysis identified the intact glycopeptide harboring Asn138-glycan adjacent to a trypsin cleavage site. These results indicated that N-glycans may confer resistance by sterically shielding proteolytic cleavage sites and modulating surface charge. The complex sialylated glycans of HLF were associated with greater resistance compared to the simpler oligomannose structures of BLF. These findings suggest N-glycosylation as an important structural contributor of lactoferrin digestibility and offer insights for designing glycoengineered lactoferrin for infant nutrition.