Photometric assay for polyphenol oxidase activity in olives, olive pastes, and virgin olive oils

A photometric method is proposed that allows the determination of phenolase activity in olive fruits, olive pastes, and virgin olive oil. The method can also be used to quantify partially purified phenolase from olives, and is based on the coupling between 4-methyl-o-benzoquinone, the reaction product of phenolase toward its substrate 4-methylcatechol, and the aromatic amine 4-amino-N,N-diethylaniline. The deep-blue adduct arising from this reaction has been characterized by means of nuclear magnetic resonance and mass spectrometric techniques and identified as 4-(4′-diethylamino-phenylimino)-2-hydroxy-5-methyl-cyclohexa-2,5-dienone. This compound shows an absorption band, centered (in dichloromethane) at 617 nm, with an ε of 11.080 M-1 cm-1. The main advantage of the proposed method resides in the high absorption coefficient of the adduct and its ultraviolet/visible absorption pattern, with a λmax in a spectral region void of significant interferences by the pigments that ultimately will probably be present in the extracts to be tested by this proposed method. The method has proven to be sensitive, specific, and reliable.