Optimation of Enzymatic Diacylglycerol Production from CPO with Continuous Systems

Abstract

Diacylglycerol (DAG ) is categorized as one of the healthy oil types that has been used in daily diet by the society, especially in Japan. In addition, the DAG has been used as an emulsifier and surfactant in the food and in pharmaceutical and medical fields (Anggirasti et al. 2008; Noureddini et al. 2004) . DAG can be produced from Crude Palm Oil (CPO) that is abundantly produced in Indonesia. However, DAG production in Indonesia is constrained by the high cost of lipase that is still imported from abroad. To overcome this problem, research of DAG production has been conducted using crude extract of lipase produced by indigenous species of fungi Rhyzopus oryzae. The R. oryzae is edible indicating that it is safe to be used in the production of food products such as DAG. This study was aimed to automate the process of enzymatic glycerolysis in DAG production carried out using continuous system; to establish optimum conditions for DAG production through continuous glycerolysis which includes flow rate of CPO, glycerolysis time and the form of lipase for catalyzing the glycerolysis process; and to test the performance of lipase from indigenous fungi R. oryzae in catalyzing glycerolysis using continuous process for the production of DAG. The process of continuous glycerolysis was carried out in a bioreactor by reacting glycerol with CPO substrate in a hexane solvent that flowed continuously with a peristaltic pump and catalyzed by lipase dissolved in Tris-HCl buffer. The lipase used was in the form of free lipase (dissolved in buffer) and immobilized lipase (adsorbed in the zeolite). The process of continuous glycerolysis was carried out with variations of substrate (CPO) flow rate, i.e 1 and 3 mL/min and variations of glycerolysis time, using time increment of 3 hours for 24 hours. The results showed that the designed automation system for glycerolysis process was successful. The process of glycerolysis could run automatically, making the process could be run more easily, quickly and efficiently. The optimum conditions for continuous production of DAG via glycerolysis was achieved at the CPO flow rate of 3 mL/min, glycerolysis time at the 9th hour with free lipase which was two times faster than that from previous research with batch systems, and at the 21th hour with immobilized lipase, possibly due to immobilized lipase demonstrated increased stability during glycerolysis than free lipase. Products conversion of DAG was 29 % with free lipase and 33 % with immobilized lipase. In the process of continuous glycerolysis, substrate replacement treatment was also carried out 3 times to observe the performance of lipase in catalyzing the process of glycerolysis, and the results showed that lipase from indigenous mold R. oryzae was able to catalyze the glycerolysis process in hydrolysis or esterification reactions to form DAG products. This CPO substrate replacement treatment for 3 times caused an increase in conversion of DAG products to 37 %, it is suspected because of the new supply/acyl donor derived from each CPO substrate that replaced 3 times in the process of glycerolysis. A decrease in the stability of the immobilized lipase after 3 times repeated use, can be caused by several factors such as the weak bond of lipase-zeolite which causes desorption of lipase from zeolite, the imbalance of hydrophobic/hydrophilic properties of zeolite, the use of organic solvents that can damage the lipase, and the lack of buffer content in the zeolite granules.