Lipase extraction and activities assay

The extraction of crude lipases and LOX, as well as the determination of lipase, phospholipase and LOX activities were all carried out by the method of Jin et al. (2010).

4-methylumbelliferyl oleate was used as the substrate to measure the aforementioned lipase activities. The substrate will liberate fluorescent 4-methylumbelliferone after lipase hydrolysis, which was determined by using an Infinite F200 Pro microplate reader (Tecan Group Ltd., Männedorf, Switzerland). For determination of different lipase activity, different reaction buffers were used (0.1 M disodium phosphate/0.05 M citric acid [pH 5.0, containing 150 mM sodium fluoride, 0.05% (w/v) Triton X-100 and 0.8 mg/ml bovine serum albumin (BSA)] for phospholipase; 0.22 M tris/HCl (pH 7.5, containing 0.05% (w/v) Triton X-100) for neutral lipase; 0.1 M disodium phosphate/0.05 M citric acid (pH 5.0, containing 0.05% (w/v) Triton X-100 and 0.8 mg/ml BSA) for acid lipase). One unit (U) of lipase or phospholipase activity was defined as the nmol of released 4-methylumbelliferone per minute at 37 °C.

Crude LOX was extracted and determined by using the method of Jin et al. (2010). The activities were determined spectrophotometrically at 234 nm with a linoleic acid substrate solution. The LOX catalyzes the formation of conjugated diene hydroperoxide from linoleic acid while the conjugated double bonds have characteristic absorption at 234 nm. One unit (U) of LOX activity was defined as an increase absorbance of 0.001 per minute at 234 nm.