A novel method for preparation of human milk fat substitute enriched with 1-oleoyl-2-palmitoyl-3-linoleoylglycerol: stepwise enzymatic acidolysis strategy

1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) is the most abundant triacylglycerol in Chinese human milk fat, which plays a key role in regulating lipid metabolism, improving nutrient absorption, and modulating gut microbiota. In this study, a stepwise enzymatic acidolysis strategy was innovatively developed for the synthesis of a human milk fat substitute (HMFS) enriched with OPL. Initially, 1,3-dioleoyl-2-palmitoylglycerol (OPO) was synthesized and purified as an intermediate before incorporating linoleic acid (LA). The first-step acidolysis using fractionated tripalmitin (PPP) and oleic acid (OA) yielded an OPO content of 69.71%. Subsequently, the second-step acidolysis of purified OPO with LA successfully produced OPL. Under the optimal conditions, the OPL content in the final product reached 50.49%, with the relative percentage of palmitic acid at the sn -2 position as high as 72.37%. Notably, this strategy required only a molar ratio of PPP/OA/LA of 1:8:1.66, which greatly reduced LA consumption compared to existing methods. These results demonstrate that the stepwise enzymatic strategy offers an efficient and industrially viable route for producing HMFS enriched with OPL. • A novel method for synthesizing HMFS enriched with OPL was developed. • The stepwise enzymatic acidolysis strategy is conducive to improving OPL yield. • High levels of OPL and OPL/OPO/LPL could be simultaneously obtained. • The consumption of high-cost LA was significantly reduced.