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Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS; Waters Ltd. Herts, United Kingdom) and analysed according to Brown et al. [56]. 4.5. DNA Extraction During the control and treatment period of the in vitro SHIME ® experiment, samples for microbial community analysis were collected once per week from each colonic reactor. Total DNA was isolated as described by Boon et al. [57], with some minor modifications as previously reported by Duysburgh et al. [55]. For the in vivo study, metagenomic DNA was extracted from all faecal samples by Teagasc (Cork, Ireland) using a modified version of the QIAGEN FAST Stool extraction kit (QIAGEN, Manchester, UK). The modification included an additional bead-beating step at the start of the procedure. Briefly, faeces were added to the buffer as described by the manufacturer’s instructions, after which homogenisation with a bead beater device was performed, followed by the protocol for Gram-positive bacteria as described by manufacturer’s instructions. 4.6. Microbial Community Analysis through qPCR qPCR assays to quantify Lactobacillus spp. and Bifidobacterium spp. were completed using a QuantStudio 5 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Each sample was analysed in technical triplicate and outliers (more than 1 C T difference) were removed. The qPCR assay, including primer sequences and amplification program, for Lactobacillus spp. was conducted as reported by Furet et al. [58], while the qPCR for Bifidobacterium spp. was previously described by Rinttilä et al. [59]. 4.7. Microbial Community Analysis through 16S-Targeted Illumina Sequencing In order to compare effects on microbial community composition of treatment with OFO and POF at a dose of 1.4 g β -glucan per day, microbial community profiling of the luminal PC and DC compartment of the in vitro SHIME ® experiment was performed using 16S-targeted Illumina sequencing. Library preparation and sequencing on an Illumina MiSeq platform with v3 chemistry (2 × 300bp) were conducted by LGC Genomics GmbH (Berlin, Germany) using the primers as reported by Klindworth et al. [60], with modifi- cation of the reverse primer (785Rmod; 5’-GAC TAC HVG GGT ATC TAA KCC-3’) to increase coverage. 4.8. Flow Cytometric Determination Samples were also collected once per week from each colonic reactor for enumeration of bacterial cells via flow cytometry. A 10-fold dilution series was prepared initially in phosphate-buffered saline. Assessment of the viable, non-viable, and total population of the microbial community was done by staining the appropriate dilutions with SYTO 24 and propidium iodide. Samples were analysed on a BD FACSVerse (BD Biosciences, Erembodegem, Belgium). The samples were run using the high flow rate. Bacterial cells were separated from medium debris and signal noise by applying a threshold level of 200 on the SYTO channel. Proper parent and daughter gates were set to determine all populations. Results were obtained as log counts/mL. 4.9. Data and Statistical Analysis For the analysis of 16S-targeted Illumina sequence data, read assembly and clean-up was adopted from the MiSeq procedure [61,62]. Briefly, mothur (v. 1.40.5, University of Michigan, Ann Arbor, MI, USA) was applied to assemble reads into contigs, perform alignment-based quality filtering (alignment to the mothur-reconstructed SILVA SEED alignment, v. 123), remove chimeras, assign taxonomy using a naïve Bayesian classifier [63] and SILVA NR v132 and cluster contigs into operational taxonomic units (OTUs) at 97% sequence similarity. All sequences classified as Archaea, Chloroplasts, Eukaryota, and Mitochondria were omitted, in addition to sequences that could not be classified at all. For each OTU, representative sequences were picked as the most abundant sequence within
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