Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the
synthesis of lactulose with -galactosidases GOS are also produced, but the ratio of lactulose and GOS in
the product can be tuned at will, depending on the operation conditions, so to obtain an optimal product
distribution in terms of prebiotic potential. The selectivity of fermentation of each carbohydrate alone as
well as mixtures of both was determined using pH-controlled anaerobic batch cultures with faecal inoculum.
Within the experimental range considered, lactulose/GOS molar ratio of 4 resulted in the highest
selectivity for Bifidobacterium and Lactobacillus/Enterococcus, so this ratio was selected as the target for
the synthesis of lactulose from fructose and lactose with Aspergillus oryzae -galactosidase. Synthesis
was optimized using response surface methodology, considering temperature, initial concentrations of
acceptor sugars and fructose/lactose molar ratio as key variables, with the aim of maximizing lactulose
yield at the optimal product distribution in terms of prebiotic potential (lactulose/GOS molar ratio of
4). Under optimal conditions (50 ◦C, 50%w/w total initial concentrations of sugars and fructose/lactose
molar ratio of 6.44), lactulose yield of 0.26 g of lactulose produced per g of initial lactose was obtained at
the optimal product distribution.