2.3 Enzyme Hydrolysis of FAD
Enzyme hydrolysis of FAD was conducted in a reactor. The reactor consisted of a Pyrex glass beaker with volume capacity of 600ml, diameter of 9cm and height of 12.5cm, a magnetic stirrer used for agitation was in conjunction with hot plate equipment. This reactor was designed to function as continuous stirred tank reactor (CSTR). The reaction mixture at the beginning of each run constituted 50g melted FAD, distilled water (% v/w) and enzyme lipase (Aspergillus niger) (% w/w). The total weight of the reaction mixture was 150g. The reactor with its content was heated by a temperature-controlled hot plate at 65 under controlled stirring of 200 rpm. A piece of aluminium foil was used as a reactor cover to prevent the evaporation of the fluid during the progress of the experiment. Based on design of experiment protocol setup for enzymatic hydrolysis for different reaction of times, the reactions were stopped by filtering out the immobilized lipase with a double-layer cheese cloth. Water contained in the product mixture was separated by centrifugation at 3000 \(\times\) g for about 2 min. The resulting mixture of lipid complex was contained and stored in a refrigerator for further analysis. The quantities of free fatty acid and vitamin E were determined, and the lipase was not recycled for further use. The quantity of FAD was kept constant.