A new isolated P. citreonigrum URM 4459 was selected to produce fructooligosaccharides (FOS) in an efficient, economical and fast one-step fermentation. Optimal culture conditions were stablished by experimental design. Experiments run in bioreactor resulted in a high yield, content, productivity and purity of FOS (0.65 +/- 0.06 g(FOS)/g(initial) (Sucrose), 126.3 +/- 0.1 g/L, 2.28 +/- 0.08 g/L. h and 61 +/- 0%). The FOS mixture was purified up to 92% (w/w) with an activated charcoal column. FOS produced were able to promote lactobacilli and bifidobacteria growth. Higher bacteria cell density was obtained for microbial-FOS mixtures than commercial Raftilose (R) P95. Some strains grew even faster in the FOS mixture produced than in all other carbon sources. FOS were resistant to the simulated gastrointestinal conditions. A high amount of a reducing trisaccharide was identified in the FOS produced mixture, possibly neokestose, which may explain the great prebiotic potential of the FOS.