Bile salt induced aggregation and nanostructure formation of β-lactoglobulin in gastrointestinal environments

beta-Lactoglobulin (BLG) is an important food protein with great nutritional value. It exists as monomeric and dimeric forms at pH 3.0 (gastric conditions) and 6.2 (small intestine conditions), respectively. The structural stability of BLG at acidic pH prevents its denaturation in the stomach. Bile salts present in the intestine may influence BLG proteins, which are partially hydrolysed after reaching the small intestine. However, very little is known about the effect of bile salt on structure of BLG, which is crucial for evaluating the protein's bioavailability, absorption, and degradation during its passage through the gastrointestinal tract. The present study delves into the interactions between a bile salt, named sodium taurocholate (NaTC), and BLG protein, elucidating structural alterations with implications for protein digestion. We have utilized spectroscopy to understand structural changes, microscopic techniques to explore morphology changes and molecular modelling tools to unveil specific molecular interactions. A significant structural change in protein were noticed in the presence of bile aggregates. Particularly in gastric pH conditions, the protein undergoes denaturation followed by aggregation. On the other hand, minimal structural changes were observed at pH 6.2. Noticeable changes in critical aggregation concentration of bile salts indicate a strong hydrophobic interaction with BLG. Time-resolved fluorescence measurements reveal significant changes in average lifetime values at pH 3.0 (from 1.8 ns to 2.5 ns) compared to pH 6.2 (from 1.4 ns to 1.6 ns), providing evidence for structural changes induced by protein-bile complex. Electron microscopy and fluorescence microscopy data revealed unique large spherical structures of NaTC embedded with protein aggregates. Molecular modelling identifies specific binding sites for bile salt on BLG. The present study on hydrophobicity-driven unfolding (denaturation/aggregation) of proteins by biological surfactants, may find potential applications in formulating effective enzyme-based detergents and providing insights into protein digestion dynamics in diverse pH environments of gastric and small intestine systems.