Pandan Lipophilic Bioactives Bind Milk Proteins with Gastric-Intestinal Stage-Specific Antioxidant Activity

Pandan (Pandanus amaryllifolius), a tropical aromatic plant, is a rich source of lipophilic bioactive compounds, notably characterized by high levels of squalene, phytol, and β-sitosterol, which exhibit antioxidant and lipid-lowering effects. Bovine milk proteins (α-casein, β-casein, and β-lactoglobulin) are widely used as delivery matrices owing to their nutritional value and technological functionality. In this study, molecular docking, fluorescence spectroscopy, circular dichroism spectroscopy, Fourier-transform infrared spectroscopy, and an in vitro digestion model were employed to systematically investigate the interactions between the major lipophilic bioactive compounds from pandan and bovine milk proteins. The results demonstrated that these compounds bound to milk proteins through hydrophobic interactions and van der Waals forces, resulting in the formation of protein-ligand complexes. Such interactions induced distinct conformational changes in the proteins, with caseins adopting a more compact structure, whereas β-lactoglobulin underwent localized unfolding. Furthermore, in vitro digestion revealed that the protein-bioactive systems exhibited distinct digestion-stage-dependent DPPH• and ABTS•+ radical-scavenging capacities, with the complexes displaying phase-dependent radical-scavenging responses during both gastric and intestinal phases. These findings provide molecular-level insights into the interactions between pandan bioactives and milk proteins and provide a theoretical basis for developing functional dairy products enriched with pandan-derived ingredients.