Enzymatic assay for the determination of olive oil polyphenol content: Assay conditions and validation of the method

A new spectrophotometric assay for the determination of the polyphenolic co ntent of olive oil is presented. It is a substrate-recycling assay for phen olic compounds that employs tyrosinase in the presence of excess NADH. The reaction of various phenols with the enzyme produces an o-quinone, which is detected by recycling between reactions with the enzyme and NADH. The meth od offers some advantages over the classical methods employed to determine the polyphenolic content of olive oil, that is, ease and reproducibility of the analysis, highly increased sensitivity, and selectivity toward phenoli c compounds. The amount of total polyphenols was determined in virgin olive oils both with the Folin-Ciocalteu reagent and with the proposed enzymatic method. The results suggest a better estimation of the polyphenol content, as compared with the colorimetric method. This has to be attributed to the different reactivities of the two methods toward phenols and catechols. Fi nally, the enzymatic method demonstrates that there is a linear relationshi p between the olive oil phenolic content and the antioxidative capacity of oil extracts.