Lipase Activity Test

Method description

Samples are homogenized in a cold phosphate buffered saline solution (PBS), pH 8.5 and then centrifuged. A protein determination by the Method of Bradford is performed on the enzyme extract (supernatant) to normalize subsequent enzyme results to the efficiency of the protein extraction. The enzyme extract is incubated at 37C with a saturating amount of triglyceride that contains sulfhydryl groups substituted for the normal carboxylic acid groups on the fatty acids. As the extracted lipase in the supernatant cleaves the modified fatty acids from the glycerol backbone, the sulfhydryl groups are made available for reaction with a color reagent (DTNB). The color reagant absorbs visible light at 412nm in direct proportion to the number of sulfhydryl groups exposed by the lipase. The appearance of absorbance with time of reaction is monitored kinetically. The initial velocity of the reaction is determined from the reaction rate.

Acceptable matrices

cereal grains, doughs, pastas, and vegetables

Limit of quantitation

2783 Units/g

Equipment

Waterbath, spectrophotometer, centrifuge

Method reference

BioAssay Systems Lipase Assay Kit (DLPS-100)

Reportable unit

Units per gram. One unit of enzyme catalyzes the cleavage of 1 µmole of substrate per minute under the assay conditions (pH 8.5).

Sample size requirements

20 g

Information required by submitter

Please supply estimates