L-LACTATE AND D-LACTATE ASSAY IN REAL MILK SAMPLES WITH IMMOBILIZED ENZYME REACTORS AND GRAPHITE ELECTRODE

A sensitive flow system for the determination Of L- and D-lactate in m ilk samples is described. L- and D-Lactate dehydrogenase, LDH, were im mobilized on aminopropyl-controlled pore glass beads. L- and D-Lactate are oxidized to pyruvate in the presence of NAD+ and NADH is produced . The electrochemical determination of NADH allows the measurement of the substrate involved in the reaction. We used a graphite-based anode sensor without any mediator at +500 mV vs. Ag/AgCl. The analytes were measured, in standard solutions, in the concentration range from 1 x 10(-6) to 4 x 10(-4) M using 1 mM NAD+ concentration and 0.1M Tris buf fer pH 9. Experiments with real milk samples showed large values of cu rrents probably due to electroactive substances usually contained in m ilk. To eliminate interfering compounds a microdialysis probe coupled with a pre-oxidizing cell was used. This method of pre-treatment remov es the interfering substances, but leaves the analytes under study una ffected. The procedure allows the determination of L- and D-lactate in milk samples in the concentration range from 1 x 10(-5) to 5 X 10(-4) M. The assay was applied to monitor continuously the bacterial fermen tation of Staphylococcus aureus in UHT milk as an example of possible contamination detection in the manufacturing process.