Automated determination of lactulose in milk using an enzyme reactor and flow analysis with integrated dialysis

A highly simplified and rapid enzymatic procedure for the determination of lactulose was developed. Lactulose was hydrolysed to fructose and galactose by beta-galactosidase, and the amount of fructose was determined by using immobilized fructose dehydrogenase and K-3[Fe(CN)(6)] as mediator. The reduced mediator was re-oxidised at a screen-printed Pt-electrode at a potential of 385 mV vs. a screen-printed Pt-pseudo reference electrode. An automated flow analysis (FA) method was developed allowing in-line hydrolysis of lactulose by soluble beta-galactosidase from Aspergillus oryzae and separation of the analyte from the milk matrix by a dialysis unit. Optimised choice of reaction temperature (50 degrees C) and reaction time (210 s) led to the applicability of the device to ultra-heat treated (UHT) and pasteurized milk samples without sample pretreatment. Good correlation of data to the official photometric test was obtained when the standard addition method was used to calculate lactulose concentrations. The automated FA system allowed a frequency of analysis of 17 samples h(-1), compared to at least 12 h analysis time for the determination of the lactulose concentration in one milk sample with the German official method.