Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium strains grown on fructose, oligofructose, or inulin were performed. A principal component analysis of the data sets, expanded with the results of a genetic screen concerning the presence of a ␤-fructofuranosidase gene previously encountered in Bifidobacterium animalis subsp. lactis DSM 10140 T , revealed the existence of four clusters among the bifidobacteria tested. Strains belonging to a first cluster could not degrade oligofructose or inulin. Strains in a second cluster could degrade oligofructose, displaying a preferential breakdown mechanism, but did not grow on inulin. Fructose consumption was faster than oligofructose degradation. A third cluster was composed of strains that degraded all oligofructose fractions simultaneously and could partially break down inulin. Oligofructose degradation was substantially faster than fructose consumption. A fourth, smaller cluster consisted of strains that shared high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin. For all strains, a metabolic shift toward more acetate, formate, and ethanol production, at the expense of lactate production, was observed during growth on less readily fermentable energy sources. No correlation between breakdown patterns and the presence of the ␤-fructofuranosidase gene could be detected. These variations indicate niche-specific adaptation of bifidobacteria and could have in vivo implications on the strain specificity of the stimulatory effect of inulin-type fructans on bifidobacteria.

Galactooligosaccharides (GOS) constitute an important class of prebiotic compounds used by the food industry as active ingredients with potential health benefits. GOS are enzymatically produced from lactose using β-galactosidases through a reaction known as transgalactosylation. Many studies have been conducted in an attempt to increase GOS yields by controlling the reaction conditions using β-galactosidases from a range of microorganisms. In this study, we have used high-throughput protein engineering for two GOS producing β-galactosidases, BbgIII and BbgIV from Bifidobacterium bifidum in an effort to enhance transgalactosylation activity (over hydrolysis) thus favouring GOS synthesis. A total of 36 and 11 C- and N-terminus deletion mutants were designed for BbgIII and BbgIV, respectively. The mutant constructs ranged from highly active to completely inactive enzymes. Selected constructs were tested for their transgalactosylation activity. An increase ranging between 5 and 10% (of total carbohydrates) was obtained with the mutant enzymes. Additionally, up to 2-fold increase in the higher degree of polymerization of GOS products was observed for selected mutants compared to the native enzyme. Structure determination of two highly active constructs at 2.0 Å resolution indicated that truncations affected the oligomeric state of the enzymes, which may have implications for activity.