Three-phase systems for simultaneous extraction and separation of bioactives from Haematococcus pluvialis: Role of thermo-switchable phase behavior

Astaxanthin (Ast), saccharides (Sac), and proteins (Prot) are the primary bioactive components in Haematococcus pluvialis ( H. pluvialis ). While previous studies have predominatly focused on Ast extraction or employed multi-step processes to isolate these compounds, this study introduces a thermo-switchable binary systems composed of deep eutectic solvents (DES) and water for their simulaneous extraction and separation from H. pluvialis . The system forms a three-phase configuration including two aqueous phases and a liquid-liquid interface which enhanced the partition efficiency . The binary systems exhibited lower critical solution temperature (LCST)-type phase behavior, and its phase formation, physicochemical properties, extraction efficiency, and separation coefficients were systematically investigated as functions of temperature. With increasing temperature, the system's conductivity rised while density decreased, improving phase formation and extraction performance. Notably, Ast preferentially migrated to the DES-rich phase, Sac to the water-rich phase, and Prot to the interfacial layer. Optimal performance was achieved using the system composed of tributyloctyl phosphonium bromide ([P 4448 ]Br):lactic acid and water at 323.15 K, yielding extraction efficiency of 44.50 mg/g (Ast), 37.03 mg/g (Sac), and 1.98 mg/g (Prot), with a partition coefficient of 291.58 for Ast and a Sac/Ast selectivity coefficients of 10.67. The water-rich phase shows optimal antioxidant activity, the interfacial phase has the strongest tyrosinase inhibitory activity, and the DES-rich phase achieves broad-spectrum antibacterial effects. This system enables polarity gradient-directed enrichment of diverse bioactive components, providing a novel strategy for the synchronized extraction of multifunctional natural products. This approach represents a significant advancement in natural product extraction, offering higher efficiency, reduced solvent use, and shorter processing times compared to sequential methods.