Novel Environmentally-Friendly Process for Selective Extraction and Enrichment of DHA/EPA-Containing Phospholipids from Krill Oil via Differential Temperature-Controlled Crystallization

This study presents a novel environmentally-friendly process for the selective extraction and enrichment of DHA/EPA-containing phospholipids (PL-DHA/EPA) from krill oil. The methodology leverages differential crystallization behavior between phospholipids and triacylglycerols in ethanolic solutions, exploiting their distinct freezing point thresholds to achieve precise fractionation. Response surface methodology optimization identified optimal extraction parameters: liquid-to-material ratio of 6:1 (<i>v</i>/<i>w</i>), freezing temperature of -20 °C, freezing duration of 25 h, and rotary evaporation temperature of 45 °C, yielding a final product with 39.40% PL-DHA/EPA content. Principal component analysis revealed substantial overlap in confidence ellipses among extraction methodologies, indicating effective preservation of core phospholipid signatures from the parent krill oil while maintaining critical structural characteristics and molecular species distribution. Comprehensive analysis of phospholipid fractions and heatmap analysis revealed distinctive molecular profiles compared to conventional organic solvent extraction, with selective enrichment of EPA-containing phospholipids, particularly PC-EPA and PI-EPA species. The green extraction method demonstrated comparable oxidative stability to conventional approaches, with superior protection against secondary oxidation as evidenced by significantly lower anisidine values. This sustainable approach achieves effective phospholipid enrichment while substantially reducing environmental impact through elimination of halogenated solvents, addressing the critical need for environmentally conscious technologies in marine lipid processing with potential applications in nutraceutical and functional food industries.