Self-assembly of hydrophobin and its co-assembly with hydrophobic nutraceuticals in aqueous solutions: Towards application as delivery systems

Hydrophobins (Hyd) are small cysteine-rich, amphipathic proteins with molecular masses of about 10kDa. They are present in large amounts in fungal cell walls, where they form part of the outermost layer ("rodlet layer"); some of them are secreted into the medium. Secreted Hyd have the ability to convert hydrophobic surfaces to hydrophilic ones and vice versa by self-assembly into an amphipathic protein membrane. Hyd are among the most surface-active molecules known. Due to their amphiphilic nature and self-assembly properties, hydrophobins have been suggested to serve as surfactants, emulsifiers and encapsulating agents for drugs, but their application in nanoencapsulation of nutraceuticals for food enrichment has apparently not yet been explored. Using pyrene fluorescence, we determined the critical micellization concentration (CMC) of a commercially available Hyd CMC=(0.041±0.006) mg/mL. Using intrinsic fluorescence quenching we showed that both vitamin D₃ (VD₃) and Nile Red (NR) bind to Hyd: NR-Hyd K_a=(8.2±0.5)×10⁵M^-1; VD₃-Hyd K_a=(0.65±0.04)×10⁵M^-1. Using dynamic light scattering we showed that while VD₃ particles had a mean diameter of 235nm, VD₃-Hyd complexes were much smaller (two subpopulations of particles were formed: 79% of the particles had a mean diameter of 30nm, and 21% had a mean diameter of 100nm). While VD₃ in phosphate buffer showed low stability, with ~70% loss in 21 days of storage, the VD₃-Hyd nano-particles system was colloidally and chemically very stable, without any loss of VD₃ during that time. Hyd were thus found to be promising nanovehicles of hydrophobic nutraceuticals for food and clear beverages enrichment.