Ultrasound assisted interesterification for enhancing productivity of oxidative sensitive docosahexanoic acid ‐enriched medium‐and‐long chain triacylglycerol synthesis

Abstract The synthesis of docosahexaenoic acid (DHA)‐enriched medium‐ and long‐chain triacylglycerols (MLCT), particularly with DHA at the sn ‐2 position and medium chain fatty acids (MCFA) at the sn ‐1,3 positions, is a promising strategy to enhance DHA bioavailability. However, the synthesis of DHA‐enriched MLCTs is hindered by steric hindrance due to DHA's very long‐chain polyunsaturated fatty acids. This study investigates the enzymatic interesterification of microalgae oil and medium chain triacylglycerols (MCT) using regio‐selective lipases with different typo‐selectivities. We evaluated the impact of interesterification conditions (substrate ratio, type of lipase, lipase loading, temperature and time) on MLCT yield, DHA distribution at sn ‐2 and acyl migration. The optimal conditions, using 6% Rhizopus oryzae lipase at 50 °C for 8 h achieved a MLCT yield of 64.9% ± 0.4%, with 37.5% DHA at the sn ‐2 position. In comparison, Rhizomucor miehei lipase produced a lower yield (49.5% ± 1.0%) but a higher DHA distribution at sn ‐2 position (46.3% ± 0.2%). Given DHA's susceptibility to oxidation, we also integrated ultrasound (US) pre‐treatment with enzymatic interesterification to improve MLCT yield. Using 4% RO with US pre‐treatment (40% amplitude, 4 s on/6 s off duty cycle, 8 min) increased MLCT yield from 36.1% ± 0.4% to 42.0% ± 0.7% ( p < 0.05), with a lower reaction temperature (30 °C) and shorter duration (4 h), without compromising oil quality and lipase activity. These findings emphasize the importance of lipase selection, acyl migration, and US pre‐treatment for enhancing oxidative sensitive DHA‐enriched MLCT production, offering applications in nutritional and functional food formulations.