The Effects of Enzyme Pretreatment Extraction on Lipid Recovery With Normal Phase <scp>HPLC</scp>

ABSTRACT With the increasing interest in the commercialization of microalgal fermentation‐based products, sustainable and efficient oil extraction methods have been developed for manufacturing and analytical testing. Due to the variability and resilience of micro‐algae cell walls, traditional lipid extraction methods such as Folch and Bligh and Dyer are not always efficient, leading to the development of enzymatic treatments to assist in lipid recovery. In this study, we used enzymatic treatment prior to a modified Folch extraction to maximize the extraction efficiency for the purpose of lipid profile analysis. The literature provides little evidence on whether nonspecific proteases degrade amide linkages in lipid structures like sphingolipids; therefore a normal phase HPLC method coupled with an Evaporative Light Scattering Detector (ELSD) was developed to determine changes in lipid class concentrations due to enzymatic treatment. We used lipid standards to compare the effects of enzymatic treatment and to determine the recoveries of each lipid class. Our lipid extraction model suggested that tridocosahexaenoylglycerol (TG‐DHA) undergoes significant oxidation induced by heat treatment during the extraction process, rather than by enzymatic activity.