BIOELECTROCHEMICAL DETERMINATION OF LACTIC AND MALIC-ACIDS IN WINE

Electroanalytical methods based on biosensor technology and differenti al PH measurement for the determination Pf L-lactic and L-malic acids in wine were developed. Lactic acid was determined according to two pr ocedures consisting of the use of two lactate electrochemical biosenso rs, one based on a Clark 02 probe and the other based on the H2O2 prob e. The two probes were assembled employing a polymeric membrane where the enzyme lactate oxidase was previously immobilized. Both sensors ex hibited high storage and operational stabilities, and good reproducibi lity when used in wine matrix. Measurement of lactate was carried out using two different flow-through cells assembled with the lactate prob es. The low detection limit of these probes and the relatively high co ncentration of lactate in wine samples required a serial dilution in t he range 1:100 to 1:200, thus eliminating all potential electrochemica l or enzyme interferences present in the sample. Malic acid was determ ined using a differential commercially available pH-meter and an enzym atic procedure. For this analysis, we employed the malic enzyme (EC 1. 1.1.40) and the NADP+ cofactor. The pH variation due to the action of this enzyme on malic acid was found to be proportional to the malic ac id present in the sample. Experimental parameters such as pH, temperat ure, and co-factor concentration were optimized, resulting in malate d etermination in less than 1 min with good reproducibility. Various sam ples of wine and wine musts were assayed for lactate with the two bios ensor procedures, and for malate with differential pH-metry. The resul ts when compared with those obtained with the commonly used spectropho tometric procedure correlated well.