Selective enrichment of n−3 polyunsaturated fatty acids with C18–C20 acyl chain length from sardine oil using Pseudomonas fluorescens MTCC 2421 lipase

Abstract

An extracellular lipase purified from Pseudomonas fluorescens MTCC 2421 was used to enrich sardine oil triglycerides with eicosapentaenoic acid (20:5 n−3) and linolenic acid (18:3 n−3) to 35.28% and 8.25%, respectively, after 6 h of hydrolysis. The corresponding n−6 fatty acids (18:2 n−6 and 20:4 n−6) exhibit a reduction (54.93% and 50%, respectively). Structure–bioactivity relationship analyses revealed that the lower hydrophobic (log P values) constants of 18:3 n−3 and 20:5 n−3 (5.65 and 5.85, respectively) result in their higher hydrolytic resistance towards lipase, leading to their enrichment in the triglyceride fraction after lipase-catalysed hydrolysis. Lipase-catalysed hydrolysis of sardine oil for 6 h followed by urea fractionation at 4 °C with methanol provided free fatty acids containing 42.50% 20:5 n−3 and 10.31% 18:3 n−3, respectively. Argentation neutral alumina column chromatography, using n-hexane/ethylacetate (2:1, v/v) resulted in 20:5 n−3 of high purity (83.62%), while 18:3 n−3 was found to be eluted with n-hexane/dichloromethane (4:1, v/v) as eluting solvent with a final purity of 75.31%.