tFructo-oligosaccharides (FOS) obtained by fermentation of sucrose may be purified at large-scale bycontinuous chromatography (Simulated Moving Bed: SMB). In order to improve the efficiency of thesubsequent SMB purification, the optimization of the fermentative broth composition in salts and sugarswas investigated. Fermentations conducted at reduced amount of salts, using Aureobasidium pullulanswhole cells, yielded 0.63 ± 0.03 g of FOS per gram of initial sucrose. Additionally, a microbial treatmentwas proposed to reduce the amount of small saccharides in the mixture. Two approaches were evaluated,namely a co-culture of A. pullulans with Saccharomyces cerevisiae; and a two-step fermentation in whichFOS were first synthesized by A. pullulans and then the small saccharides were metabolized by S. cerevisiae.Assays were performed in 100 mL shaken flasks and further scaled-up to a 3 L working volume bioreactor.Fermentations in two-step were found to be more efficient than the co-culture ones. FOS were obtainedwith a purity of 81.6 ± 0.8% (w/w), on a dry weight basis, after the second-step fermentation with S.cerevisiae. The sucrose amount was reduced from 13.5 to 5.4% in total sugars, which suggests that FOSfrom this culture broth will be more efficiently separated by SMB.