Enzymatic preparation of EPA-enriched glycerides by multi-step addition of water during transesterification

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are n-3 polyunsaturated fatty acids with EPA receiving increasing attention due to its documented beneficial effects on cardiovascular and cerebrovascular health. In this study, an effective enzymatic transesterification employing multi-step addition of water was developed to produce EPA-enriched glycerides from glycerol and fatty acid ethyl esters. Lipozyme 435 showed the best performance in EPA-enriched glyceride synthesis. Furthermore, multi-step water addition significantly enhanced glyceride synthesis efficiency, and the underlying mechanism was investigated using molecular dynamics simulations. Under the optimized conditions (65 °C, 6 h, 2.0 mbar, an ethyl ester to glycerol molar ratio of 3.5:1, and a lipase dosage of 6%), water was added at 2 h and 4 h, with 5% (w/w) relative to the mass of ethyl esters. The triacylglycerol (TAG) content in the product reached 82.31%, while the EPA and DHA contents in the glyceride fraction were 60.50% and 20.57%, respectively. A novel finding of this study is that the catalytic performance of the lipase in the transesterification is significantly influenced by the multi-step addition of water under vacuum.