Gastric pH-induced liquid-liquid phase separation between lactoferrin and osteopontin protects against proteolysis and modulates intestinal uptake.

Harnessing heteroprotein interactions to control the gastrointestinal fate of bioactive milk proteins represents a promising strategy for improving infant nutrition and developing food formulations. Nonetheless, the influence of protein-protein interactions on the stability and digestibility remains largely unexplored. Here, we investigate the interaction between lactoferrin (LF) and osteopontin (OPN) and its impact on gastric stability and intestinal uptake. We demonstrate that LF and OPN undergo electrostatically driven liquid-liquid phase separation under mildly acidic conditions (pH ∼ 4), forming dense, spherical coacervates, while yielding only soluble complexes at neutral (pH ∼ 7) or irregular precipitates at extreme acidic conditions. This coacervation is tunable by environmental factors: physiological temperature (37°C) promotes droplet formation, whereas high ionic strength (50-100 mM) disrupts assembly. Importantly, in vivo studies demonstrate that LF-OPN coacervates formed at gastric pH confer substantial protection against proteolysis, resulting in 2.2-fold increased OPN uptake and 1.52-fold increased LF uptake in the jejunum compared to single-protein administration. These findings establish LF-OPN coacervation as a tunable, food-grade delivery system that leverages intrinsic protein interactions to enhance the oral bioavailability of bioactive proteins, offering a bio-inspired strategy for optimizing infant formula and functional foods.