Evaluation of the Effects of Enzyme Pretreatment Extraction on Lipid Recovery with Normal Phase HPLC

With an increasing interest in the commercialization of microalgal fermentation-based products, sustainable and efficient oil extraction methods have been developed for manufacturing and for analytical testing – one of the methods of interest being enzymatic treatment. Due to the variance and resilience of micro-algae cells walls, traditional methods like Folch et al and Bligh and Dyer are not always efficient. 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. Because of concern that the treatment with proteases might also degrade lipids, particularly phospho- and sphingolipids, a normal phase HPLC coupled with Evaporative Light Scattering Detector (ELSD) method was developed for analyzing lipid classes before and after the enzymatic treatment. Lipids classes including tri, di- and monoacylglycerols, free fatty acids, sterols, sterol esters, mono- and di-galactosyldiacylglycerols, phosphotidyl -inositol (PI), -cholilne (PC), and -ethanolamine (PE), sphingomyelin and lyso-PC were separated and quantified successfully on a polyvinyl alcohol column with hexane/tert-butyl methyl ether/ethyl acetate/aqueous mobile phase system. For analyzing neutral lipids and polar lipids simultaneously, the ELSD was optimized; the instrumental reproducibility of all lipid classes was less than 5% relative standard deviation. Absolute detections limits were 10, 200 and 800 ng for neutral lipids, phospholipids and sphingomyelin, respectively, with polynomial calibration curves. To determine the recoveries of all lipid classes due to enzymatic treatment, samples made up from two combinations of lipid standards, were treated with 1% of a commercially available protease product and extracted by a modified Folch method, followed by the HPLC-ELSD analysis. Compared to traditional Folch method, although gravimetric results showed no significant weight change of the lipid extracted by enzyme method, the recovery of cholesteryl ester, cholesterol, free fatty acids, monoacylglycerol and DHA in triacylglycerol form was significant lower than Folch method.