L- and d-Lactate assay in real milk samples with immobilized enzyme reactors and graphite electrode

Abstract

A sensitive flow system for the determination of l- and d-lactate in milk samples is described. l- and d-Lactate dehydrogenase, LDH, were immobilized 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 × 10 −6 to 4 × 10 −4 M using 1 m M NAD + concentration and 0.1 M Tris buffer pH 9. Experiments with real milk samples showed large values of currents probably due to electroactive substances usually contained in milk. To eliminate interfering compounds a microdialysis probe coupled with a pre-oxidizing cell was used. This method of pre-treatment removes the interfering substances, but leaves the analytes under study unaffected. The procedure allows the determination of l- and d-lactate in milk samples in the concentration range from 1 × 10 −5 to 5 × 10 −4 M. The assay was applied to monitor continuously the bacterial fermentation of Staphylococcus aureus in UHT milk as an example of possible contamination detection in the manufacturing process.