3.7. Oil Hydrolysis and Glycerol Esterification

The partially purified lipase (3 mL) was subjected to hydrolysis using 300 µL of oil (crude fish oil, coconut oil) or 0.3 g of butter. The mixtures were incubated at 30 °C, shaking at 140 rpm for 24 h in a solvent free system. Samples were taken for analysis. Free glycerol is quantified after derivatization [24]. Potassium periodate solution, 10 mM (1.2 mL) was added to a sample (2 mL) and was shaken for 30 s. Then, 0.2 M acetylacetone solution (1.2 mL) was added. The mixture was incubated at 70 °C for 1 min, with manual stirring. Subsequently, the mixture was cooled under tap water and the absorbance was read at 410 nm against an enzyme-free control. Esterification was carried out using glycerol and fatty acids were mixed according to their molar ratios in a solvent free system. The partially purified lipase (4%) was added. The mixture was incubated at 30 °C, shaking at 140 rpm for 5 days. Samples were taken daily and evaluated using thin layer chromatography (TLC) analysis (TLC silica gel 60 F254 Al plates, Merck, Kenilworth, NJ, USA). The plates were then visualized under iodine vapor treatments and UV 254 nm.