Two‐step enzymatic synthesis of docosahexaenoic acid‐rich symmetrically structured triacylglycerols via 2‐monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two‐step process: (i) ethanolysis of bonito oil TG to 2‐monoacylglycerols (2‐MG) and fatty acid ethyl esters, and (ii) reesterification of 2‐MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2‐MG with 43.5% DHA content in 2 h. The 2‐MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C22 residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3‐dicapryloyl‐2‐docosahexaenoylglycerol to 1,2(2,3)‐dicapryloyl‐3 (1)‐docosahexaenoylglycerol ratio (based on high‐performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2‐MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3‐regiospecificity of Novozym 435 and the reduced acyl migration in the system.