Optimizing Assay and Extraction of Lipoxygenase in Wheat Germ

Using three-level seven-factor response surface methodology, wheat germ lipoxygenase (LPO) assay conditions were standardized. The important parameters were concentration of the substrate (linoleic acid), and surfactant (Tween 20), pH and temperature. The standardized LPO assay conditions for the 1 mL reaction volume were : 450 muM linoleic acid, 129 muM Tween 20, 175 mM ethanol, 1 mM EDTA, pH 6.2 (phosphate buffer), ionic strength 100 mM and 40-degrees-C. LPO extraction conditions were standardized by sequential variation of parameters. Optimum conditions were a single extraction of defatted wheat germ flour at 2-5-degrees-C with magnetic stirring of an extractant acetate buffer pH 4.5, ionic strength 100 mM, at buffer-to-solid ratio 10:1.