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 gFOS/ginitial 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® 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.