Fig. 3.

Schematic representation of the ex vivo detection assay based on BONCAT. Stool samples stored frozen were thawed, filtered, and washed in PBS and then incubated in the presence of AX and the cellular activity marker L-azidohomoalanine (AHA) to detect AX-stimulated bacterial cells. A no-amendment control, containing only AHA, was incubated to detect possible basal activity in the absence of AX. Microscopic inspection showed no BONCAT signal for all controls; thus, no basal activity was detected. AX-incubated samples were then fixed in ethanol and active cells were stained using a Cu(I)-catalyzed click reaction using a Cy5 dye solution. A and B A representative picture of fecal microbiota incubated for 6 h A with AX and B without AX (BONCAT control). Stimulated cells, shown in pink as a Cy5-positive BONCAT signal, were sorted by FACS, with all microbial cells shown in blue (DAPI stained). DNA was extracted from both sorted cells and samples at 0-h and 6-h anaerobic incubations. The 16S rRNA gene was amplified by PCR and amplicons were sequenced using the Illumina Miseq platform. AX, arabinoxylan; BONCAT, bioorthogonal non-canonical amino acid tagging; FACS, fluorescence-activated cell sorting