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

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.