A COLORIMETRIC DETERMINATION OF FATTY-ACIDS AS A NEW ASSAY OF LIPASES IN REVERSE MICELLES

A new simple spectrophotometric method is described for the determination of the free fatty acid content in triglyceride oils or other lipophilic samples. The method utilizes phenol red as fatty acid indicator, which is solubilized phenol red as fatty acid indicator, which is solubilized in reverse micelles formed by AOT [sodium bis(2-ethylhexyl) sulfosuccinate] in isooctane. Fatty acid determinations in vegetable oils can be carried out rapidly with oil samples of less than 100 mg. The acid value of three different oils tested agreed quite well with the acid value obtained for the same samples with another colorimetric determination using cupric acetate. The method can be extended to a continuous determination of fatty acids which are released during the initial stage of a lipase catalyzed hydrolysis of triglyceride substrates in reverse micelles. This new sensitive lipase assay has been applied for a lipase of Pseudomonas bacteria with lipase concentrations as low as 0.1 μg/ml. Using trioctanoylglycerol as substrate in 50 mM AOT/isooctane with wo=[H2O]/[AOT]=11.1 (pH 9.0), the apparent overall Michaelis-Menten constant (Km′app,ov) is 27 mM and the turnover number (kcat) 44 sec-1. © 1990 AOCS Press.