Multi-enzyme biosensors with amperometric detection for determination of lactose in milk and dairy products

Abstract

In home-made sensors coimmobilizing enzymes in thin-layer plexi-cells on natural protein membranes, three enzyme cells: β-galactosidase and galactose oxidase (A), β-galactosidase and glucose oxidase (B) and β-galactosidase, galactose oxidase and glucose oxidase (C) were built into a flow-injection-analyzer system. The lactose was decomposed and oxidized by the immobilized enzymes and the hydrogen peroxide generated during the enzymatic reactions was determined by amperometric detection. The parameters for biochemical and electrochemical reactions (concentration of buffer, temperature, flow rate) were optimized in each enzyme cell. The pH optima of the lactose measurement was determined in the three enzyme cells mentioned above. The pH optimum of the cells A, B and C were 6.4, 4.5 and 4.8, respectively. The measuring ranges were 1–5 mM, 2–10 mM and 1–5 mM, while the detection limits were 0.5, 1.0 and 0.5 mM, respectively. More than 600, 1000 and 800 samples could be measured with these cells, respectively. Commercial milk and instant dessert powder products were analysed also. Our results showed that the cells B and C were more suitable for the determination of the lactose content of milk. For samples of dairy products containing added glucose, starch and other carbohydrates, enzyme cell A could be used for the efficient determination of lactose in one step.