Abstract
Two completely different analytical methods for lactose determination are presented. Three-enzyme biosensor involving β-galactosidase, glucose oxidase, and horseradish peroxidase was constructed with successive steps of optimization. Primary experiments were carried out with horseradish peroxidase biosensor in solution of hydrogen peroxide. Addition of poly(ethylene glycol) diglycidyl ether was examined and the best constant potential for chronoamperometric experiments was selected at −0.05 V using cyclic voltammetry. Ferrocene was chosen as the best from three tested mediators and optimum concentration of ferrocene was found out at 2 g dm−3. Two electrode materials were compared. Spectroscopic graphite electrode proved better electrochemical response for its twice larger electro-active surface than screen-printed carbon electrode. Phosphate buffer solution of pH 7.5, at which the three-enzyme system provided the highest current signal, was used for lactose determination in milk, milk powder, curd cheese, and yogurt. Gas chromatography with tandem mass spectrometric detection was used for determination of lactose, glucose, and galactose in the real samples with internal standard salicine using single ion monitoring mode. Determined content of lactose in the milk products was compared with declared values. Gas chromatography with tandem mass spectrometric detection offers higher sensitivity and orthogonal selectivity in comparison to enzyme biosensor.
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