Lactulose synthesis was done in repeated-batch mode with Aspergillus oryzae beta-galactosidase immobilized in glyoxyl-agarose (GA-beta G), amino-glyoxyl-agarose (Am-GA-beta G) and chelate-glyoxyl-agarose (Che-GA-beta G), at fructose/lactose molar ratios of 4, 12 and 20. Highest yields of lactulose in batch were obtained with Che-GA-beta G (0.21, 0.29 and 0.32 g.g(-1)) for 4, 12 and 20 fructose/lactose molar ratios respectively; when operating in 10 repeated batches highest product to biocatalyst mass ratios were obtained with Am-GA-beta G (1.82, 2.52 and 2.7 g.mg(-1)), while the lowest were obtained with Che-GA-beta G (0.25, 0.33 and 0.39 g.mg(-1)). Operational stability of Am-GA-beta G was higher than GA-beta G and Che-GA-beta G and much higher than that of the free enzyme, at all fructose/lactose molar ratios evaluated. Efficiency of biocatalyst use for GA-beta G were 64.4, 35.5 and 18.4 kg(lactulose)/g(protein), for fructose/lactose molar ratios of 4, 12 and 20 respectively, while for Che-GA-beta G were 1.46, 1.05 and 0.96 k(glactulose)/g(protein). (C) 2017 Elsevier Ltd. All rights reserved.