Enhancing Extraction Of Astaxanthin From Haematococcus Pluvialis Using Aqueous Biphasic Systems Composed Of Ionic Liquids And Deep Eutectic Solvents

Astaxanthin shows a variety of biological activities and physiological functions.Astaxanthin extracted from natural sources,such as Haematococcus pluvialis,has extraordinary value and potential application.Aqueous biphasic systems（ABS）constructed by ionic liquid（IL）with good biocompatibility,high extraction rate and strong designability has been applied to the extraction process of biomolecules.However,the kosmotropic component in the IL-based ABSs were inorganic salts or organic salts,which was adverse to maintain the stability and biological activity of the biomolecules in a strong alkaline solution.In this study,deep eutectic solvents（DESs）were used as alternative kosmotropic components to form novel ABSs with ILs（chaotropic components）.The law of phase behavior at different temperature were investigated to screen out thermoreversible systems showing high critical solution temperature（UCST）and low critical solution temperature（LCST）.Moreover,the physicochemical properties,such as the viscosity,density,conductivity,and p H of the IL-DES-H₂O ternary systems as function of temperature were explored.Subsequently,the effects of type of solvents,concentration of IL and DES,solid-liquid ratio,water bath constant temperature oscillation speed,extraction temperature,extraction time,and condense time on the astaxanthin distribution were investigated.Results:（1）The aqueous biphasic system was constructed successfully by IL and DES synthesized with choline chloride（ChCl）as the hydrogen bond acceptor and polysaccharides,polyols,and polyacids as hydrogen bond donors.3 UCST-type and19 LCST-type IL-DES-based ABS were screened out.The p H range of the ternary system was 2～8,and the viscosity,density,and conductivity gradually decreased with increasing temperature.The standard molar volume（V_m）and standard entropy（S°）increased with increasing temperature,while the lattice energy(U_POT)gradually decreased with increasing temperature.Increasing temperature would be speed up the thermal movement of molecules,weaken association,increase distance,and make the particles in the system chaotic.（3）The LCST-type IL-DES ABS was more effective in extracting astaxanthin,in which astaxanthin ester accounts for about 99%,and free astaxanthin was only 1%.Astaxanthin was preferentially distributed in the IL-rich,and the partition coefficients of astaxanthin ester and free astaxanthin were in the ranges of 12.34～101.68 and 4.62～21.03,respectively.[P₄₄₄₈]Cl-[ChCl][Fru]and[N₄₄₄₄]CF₃COO-[ChCl][Suc]ABS were suitable for the selective separation of astaxanthin esters and free astaxanthin respectively.Among them,[P₄₄₄₈]Cl-[ChCl][Fru]was similar to the organic solvent extraction results,and the highest astaxanthin extraction amount obtained was 12.72±0.06 mg×g^-1.[P₄₄₄₈]Cl-[ChCl][Fru]ABS had the characteristics of high stability,low cost,recyclable and green.The research aimed to provide basic data and theoretical reference for the design of temperature-responsive ABS separation technology to extract natural astaxanthin biomolecules with different structures from biological resources.