I. Eshkenazi - Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat-Aviv 69978, Israel
E. Maltz - Institute of Agricultural Engineering, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
B. Zion - Institute of Agricultural Engineering, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
J. Rishpon - Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat-Aviv 69978, Israel

The increasing demand for on-line measurement of milk composition directs science and industry to search for practical solutions, and biosensors may be a possibility. The specific objective of this work was to develop an electrochemical biosensor to determine lactose concentration in fresh raw milk. The sensor is based on serial reactions of three enzymes—β-galactosidase, glucose oxidase, and horseradish peroxidase—immobilized on a glassy carbon electrode. The sequential enzymatic reactions increase the selectivity and sensitivity of the sensor. The sensor requires dilution of the raw milk and the addition of 5-aminosalicylic acid. Lactose concentrations in raw milk measured by the sensor were in good agreement with those measured by a reference laboratory using infrared technology. The results were obtained in milk samples that varied in fat and protein composition. From the results, we conclude that an electrochemical biosensor for determination of lactose concentration in fresh raw milk can be developed, and that the biosensor presented in this study maintained the qualities required for further development into an online sensor in the milking parlor.