In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by speciesspecific PCR to determine bifidobacterial species and percent-G؉C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P ؍ 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G؉C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P ؍ 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P ؍ 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P ؍ 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G؉C profiles.The contribution of the human colonic microbiota towards improved host health and disease is presently a topical area of research. The beneficial effects of probiotic lactic acid bacteria (such as bifidobacteria and lactobacilli) have been documented and have a long history of use in humans (8, 9, 28). Indeed, many clinical trails have been carried out (in both human and animal models) where direct health benefits from probiotic intake has been investigated (16,23,33). More recently, interest has turned to the potentially advantageous effects of dietary substrates that can be fermented by populations of indigenous probiotic gut bacteria. These so-called prebiotics selectively stimulate beneficial flora components while suppressing, or having no stimulatory effect upon, less desirable bacteria, such as proteolytic bacteroides and clostridia and gram-negative pathogens such as Escherichia coli, shigellas, and salmonellas (11). Through not relying on the addition of exogenous bacteria, there are no survivability or safety issues. A number of prebiotic substrates already have scientific credibility and widespread acceptance, such as inulin, oligofructose (both fructooligosaccharides [FOS]), galacto-oligosaccharides, and lactulose (29,30,35). These substrates have been tested in various human trials for their prebiotic effects.Here we invest...