Preparation Of Diacylglycerol From Hydrolysis Of Soybean Oil Catalyzed By Phospholipase A1 And Its Immobilization

The purpose of this study was to prepare diacylglycerol (DAG) rich functional oil from soybean oil catalyzed with phospholipase A1 (PLA1), and concentrated and purified by two-step molecular distillation, and to immobilize the catalysis enzyme.The optimal pH value and temperature for hydrolysis of soybean by PLA1 was 6.8 and 40℃, respectively. PLA1 showed good stability at pH range of 4 to 8 and temperature below 45℃. Using 0.1% of calcium chloride solution as the reactant at the circulated reactions, the residual activity of the enzyme was improved by calcium ion. The mixture of recycled enzyme and 50% of fresh PLA1 showed high hydrolysis activity. After used for six batches, the residual enzyme activity reached 86.88%.Soybean DAG standard was prepared by combination of molecular distillation and column chromatography. The purity of the standard was verified by high performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC-ESI-MS) and thin layer chromatography (TLC). The calibration curve of soybean DAG built by reversed phase (RP)-HPLC at UV=210 nm showed a good linear relationship at the concentration range of 1.0-12.0 mg/mL. The results of recovery experiments showed that the recovery of sample was at range of 104-109% with RSD at the range of 0.69-5.19%. The contents of DAG in soybean oil and its related DAG samples produced by molecular distillation were analyzed by this method.PLA1 was immobilized by chemical cross-linking and physical adsorption method. Compared to the free enzyme, the activity of the enzyme immobilized with gelatin and glutaraldehyde reached 30% and the activity of the enzyme immobilized with diatomite was 33%. Based on the screening test for adsorption capacity with six types of macroporous adsorption resin, the type D113 was chosen as the carrier for immobilization of PLA1.