Synthesis of lactulose under repeated-batch operation was done with cross-linked aggregates of Aspergillus oryzae beta-galactosidase (CLAGs). The effect of the crosslinking agent to enzyme mass ratio and cross-linking time were first evaluated. Best results were obtained at 5.5 g de glutaraldehyde/g enzyme at 5 h of cross-linking, obtaining a specific activity of 15,000 IU g(-1), with 30% immobilization yield. CLAG was more stable than the free enzyme under non-reactive conditions with a half-life of 123 h at 50 degrees C and when operated in repeated-batch mode, yield and productivity was 3.8 and 4.3 times higher. Maximum number of batches was determined considering biocatalyst replacement at 50% residual activity. 98 and 27 batches could be performed under such criterion at fructose/lactose molar ratio of 4 and 20 respectively, reflecting that enzyme stability is strongly affected by the sugars distribution in the reaction medium. (C) 2015 Elsevier Ltd. All rights reserved.