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The goal of this research was to determine the effect of different doses of galactooligosaccharide (GOS) on the fecal microbiota of healthy adults, with a focus on bifidobacteria. The study was designed as a single-blinded study, with eighteen subjects consuming GOS-containing chocolate chews at four increasing dosage levels; 0, 2.5, 5.0, and 10.0 g. Subjects consumed each dose for 3 weeks, with a two-week baseline period preceding the study and a two-week washout period at the end. Cultural methods were used for bifidobacteria, Bacteroides, enterobacteria, enterococci, lactobacilli, and total anaerobes; culture-independent methods included denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qRT-PCR) using Bifidobacterium-specific primers. All three methods revealed an increase in bifidobacteria populations, as the GOS dosage increased to 5 or 10 g. Enumeration of bifidobacteria by qRT-PCR showed a high inter-subject variation in bifidogenic effect and indicated a subset of 9 GOS responders among the eighteen subjects. There were no differences, however, in the initial levels of bifidobacteria between the responding individuals and the non-responding individuals. In order to gain a community wide perspective of the impact of GOS on the fecal microbiota of the subjects, we then performed high throughput multiplex community sequencing of 16S rRNA tags. Multiplex sequencing of the 16s rRNA tags revealed that GOS induced significant compositional alterations in the fecal microbial populations by increasing the phyla Actinobacteria. The population shifts caused by consumption of 10 g of GOS were numerically substantial, leading for example, to a ten-fold increase in bifidobacteria in four subjects, enriching them to 18-33% off the fecal microbial community, and a five-fold increase in seven additional subjects. This increase in bifidobacteria abundance was generally at the expense of only one group of bacteria, namely the genus Bacteroides. Collectively, this study showed that a high purity GOS, administered in a confection product at doses of 5 g or higher, was bifidogenic, while a dose of 2.5 gram showed no significant effect. Our results also demonstrated that GOS is remarkable for its ability to enrich specifically for bifidobacteria in human fecal samples.