Enzymatic glycerolysis in natural deep eutectic solvents for the preparation of punicic acid-enriched diacylglycerols

To enhance the efficiency of producing diacylglycerols (DAG) from pomegranate seed oil and overcome the mass-transfer limitations caused by poor miscibility between oil and glycerol in conventional solvent-free enzymatic glycerolysis, this study introduced natural deep eutectic solvents (NADESs) as a green reaction medium to establish an efficient and environmentally friendly DAG production process. Using pomegranate seed oil as the substrate, various NADES systems and lipases were systematically screened, and key reaction parameters-including temperature, substrate molar ratio, enzyme loading, and reaction time-were optimized. The compositions of the reaction products were analyzed by high-performance liquid chromatography, and thermodynamic properties were evaluated using the Arrhenius equation to elucidate the accumulation pattern and formation selectivity of glycerolysis products. The results showed that Lipozyme 435 exhibited the best catalytic performance in the choline chloride-glycerol (ChCl∶2Gly) system. Under the optimal conditions (70 ℃, substrate molar ratio 1∶2, enzyme loading 6%, and reaction time 8 h), the triglyceride conversion reached 47.11%, with a DAG yield of 43.09%. The obtained DAGs contained more than 84% punicic acid. Compared with the solvent-free system under the same conditions, the ChCl∶2Gly system significantly increased DAG yield (43.09% vs 38.02%) and markedly reduced the formation of the by-product monoacylglycerol (MAG) (3.90% vs 8.69%). Thermodynamic analysis further revealed that the activation energy for DAG formation (52.96 kJ/mol) was lower than that for MAG formation (56.70 kJ/mol), indicating a clear selectivity toward DAG generation in the NADES-based system. In conclusion, this study successfully established a green and efficient NADES-mediated process for producing punicic-acid-rich DAGs, providing theoretical support and technical guidance for the development of functional lipids derived from pomegranate seed oil.