In this study the effect of glucose depletion using glucose oxidase and catalase, simultaneously to the synthesis of prebiotic galactooligosaccharides (GOS) by P-galactosidase was studied. Considering total GOS yield, a strong dependency on the source of D-galactosidase was found. Using an Aspergillus oryzae lactase, a small increase in GOS yield (from 50.0 +/- 1.3 g/L to 54.1 +/- 1.9 g/L) was noted. Due to the decreased rate of GOS disaccharide formation by the Kluyveromyces lactis enzyme, the total GOS yield was diminished (from 47.4 +/- 0.1 g/L to 30.5 +/- 1.7 g/L). However, for both enzymes, the synthesis of GOS tri- and higher oligosaccharides increased. Additionally, the total sugar content, and thus caloric value, of the final product was reduced, also resulting in a more purified GOS mixture.