Production Of Diacylglycerol With High Purity By Holoenzymatic Process

Diacylglycerols (DAGs) has been recognized as functional oil with definite function ofprevention of obesity in recent years and can be widely used in fields of food, pharmaceuticaland cosmetic. Recently, DAG has been prepared through enzymatic method catalyzed bysn-1,3regioselective lipase. There was TAG synthesized in the reaction mixture which leadedto the low DAG content. The low DAG purity could not satisfy the demand for its biomedicaland dietetic properties research. The aim of the present study was to produce DAG with highpurity by holoenzymatic process with the catalysis of lipase G50and lipase CBD-MGLP, andthen have a further study of properties of the DAG product. The results are as follows:1. Lipase G50(Mono-and di-acylglycerol lipase, which is strictly specific for MAG andDAG but not TAG) was selected to catalyze the esterification of free fatty acid (FFA) andglycerol. The optimal reaction conditions were: temperature35°C, molar ratio of glycerol tofatty acid4:1, initial water content2%(by total weight of the subtrates).2. Scale-up esterification reaction was conducted under the above conditions and theesterification product was composed of49.9%DAG,31.6%MAG and18.5%FFA after24hof reaction, no TAG was synthesized during the reaction process. Lipase CBD-MGLPprepared in our lab with an activity of5000U/g was applied to specifically hydrolyze MAGin the esterification mixture, the optimal reaction conditions were: enzyme load375U/g (U/w,with respect to the mass of MAG in the mixture), temperature of45°C, mass ratio ofesterification mixture to Tris-HCl buffer (w/w)10:10, and pH of Tris-HCl buffer9.0, thehydrolysis mixture was composed of49.0%DAG,1.9%MAG and49.1%FFA after48hreaction under the conditions, then DAG in the hydrolysis mixture can be further purified justby molecular distillation at130°C and the DAG purity reached98.0%. However, formolecular distillation, evaporation temperature of170°C was required to get DAG with apurity of99.8%, and the recovery (93.2%) and yield (46.5%) of the DAG product were lowerthan that for the method of enzymatic hydrolysis combined with molecular distillation (98.4%and48.2%, respectively). Thus, enzymatic hydrolysis combined with molecular distillation is an economical and feasible method for purification of DAG product.3. The results of physical and chemical properties of DAG suggested that the DAG productmeet the requirements of GB; The DSC results indicated that the crystallization and meltingthermograms of DAG were different from that of TAG, which made DAG applied to productsof margarine, whipped cream and other products; Oxidative stability of the DAG product wasevaluated and TBHQ can effectively improve oxidative stability of the DAG product.In this study, a holoenzymatic process to produce DAG product with high purity wassuccessfully developed. DAG product with high purity was obtained under the conditions oflower operating temperature, lower energy consumption, higher reaction efficiency and lessenvironmental pollution. This process showed an efficient approach to produce DAGscontaining specific fatty acids with various functional properties but poor temperaturetolerance.