Investigations of Bifidobacterial Ecology and Oligosaccharide Metabolism in a Three-Stage Compound Continuous Culture System

Abstract

Background: Several different types of in vitro fermentation systems are currently employed to investigate pro- and prebiotic activities in the human large intestinal microbiota, ranging from simple batch cultures, with or without stirring and pH control, to more complex models involving pH controlled single and multiple-component continuous culture systems. Methods: In this investigation, we used a three-stage continuous culture model to study the activities of colonic bacteria. This fermentation system reproduces several of the nutritional and environmental characteristics of the proximal large intestine (vessel 1) and the distal colon (vessels 2 and 3), and was validated using bacteriological, metabolic and chemical measurements made with intestinal material obtained from different regions of the large bowel. In this paper, we report studies on prospective probiotic effects of Bifidobacterium longum NCFB 2259 in relation to other bacterial populations, production of tyrosine and phenylalanine metabolites, and bacterial synthesis of enzymes involved in the formation of putatively genotoxic metabolites, including ß glucosidase (GS), arylsulphatase (AS), ß-glucuronidase (GN), nitroreductase (NR) and azoreductase (AR). Results: Bacterial activities at two different retention times were studied (31.1 and 68.4 h), which correspond to large intestinal transit times. At R = 31.4 h, significant probiotic effects were observed with respect to reductions in GS and GN, upon adding B. longum. However, despite the fact that this organism does not ferment aromatic amino acids or produce significant amounts of genotoxic enzymes, dysbiotic manifestations occurred in that both NR synthesis and dissimilatory tyrosine metabolism were stimulated. In contrast, at R = 68.4 h, GS formation increased between five and 20-fold, while GN and NR activities increased by a factor of two after adding the bifidobacterium. These data are reviewed in relation to potential health hazards that may be encountered with long-term probiotic administration. In the prebiotic experiments, the three-stage fermentation system was operated at R = 65 h. Oligofructose was added to VI to give an initial concentration of 30 grams per litre, when the system was in steady state, to study its effects on a number of experimental parameters including bifidogenicity, bacterial growth, fermentation product formation and mutagenicity. After addition of the oligosaccharide, a multiplicity of effects were observed in VI, where synthesis of NR and AR, bifidobacterial populations and overall fermentation processes were stimulated, although these influences progressively diminished in V2 and V3. Conclusions: These studies indicate that bacterial metabolism and putative beneficial consequences associated with the breakdown of readily fermentable prebiotics in the large intestine may in some circumstances be spatially and temporally limited to the proximal bowel.

Key words:

• Arylsulphatase
• azoreductase
• β-glucosidase
• β-glucuronidase
• Bifidobacterium longum
• fructooligosaccharides
• in vitro fermentations
• nitroreductase
• prebiotics
• probiotics