Stool form will be assessed using the BSFS, a simple tool for estimating intestinal transit time [
8]. The BSFS classifies stools into seven categories, including type 1, separate hard lumps, like nuts; type 2, sausage-shaped, but lumpy; type 3, like a sausage but with cracks on the surface; type 4, like a sausage or snake, smooth and soft; type 5, soft blobs with clear-cut edges; type 6, fluffy pieces with ragged edges, a mushy stool; type 7, watery, no solid pieces [
8]. These types are categorized into slow transit (types 1 and 2), normal transit (types 3–5), and fast transit (types 6 and 7).
GI symptoms
GI symptoms will be assessed using the GSRS, a 7-point scale that evaluates symptoms of GI disorders, where 1 represents no discomfort at all and 7 represents very severe discomfort over the past week [
9,
10]. Symptoms of the GSRS are grouped into five GI syndromes, which are abdominal pain, reflux syndrome, diarrhea syndrome, indigestion syndrome, and constipation syndrome.
Microbiota analysis
Subjects will be asked to collect one stool each during the baseline, intervention 1, washout 1, intervention 2, and washout 2 periods. Subjects will be provided with Fisherbrand® commode collection kits for stool collection. Samples will be processed and appropriately stored in the lab within 6 h after defecation. Stool samples will be analyzed for changes in microbiota. Changes in the concentration of Lactobacilli and Bifidobacteria genera as well as Bifidobacterium animalis sp. lactis will be measured in fecal samples by real-time PCR (qPCR).
Total DNA will be extracted from homogenized fecal samples using the QIAamp® Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions, with minor modifications, i.e., two 0.05 M phosphate buffer washes prior to the addition of InhibitEX (Qiagen) and a 1 mm zirconia/silica bead beating step (~250–350 mg/tube, 4 m/s for 1 min × 3) before centrifugation of samples to pellet stool particles. Purified DNA will be further processed for qPCR and 16S rRNA gene amplicon sequencing.
For qPCR, template DNA for standard curves will be generated by spiking feces with a bacterial suspension consisting of lyophilized bacterial powder (Lallemand Health Solutions) in HyClone phosphate buffered saline. Total cell count of each bacterial suspension will be determined by flow cytometry. Feces will be spiked with a volume equivalent to 109 bacteria, and then subjected to DNA extraction as described above.
Quantification of Lactobacilli, Bifidobacteria, and Bifidobacterium animalis sp. lactis will be performed by qPCR using the ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific). Standard curves will be generated by serially diluting DNA from spiked feces. DNA samples to be quantified will be diluted in molecular biology grade water prior to qPCR.
The qPCR reaction mixture will consist of the appropriate primers, 1X SYBR® Select Master Mix (Thermo Fisher Scientific), and diluted DNA. Standard curve samples will be run in duplicate and unknown samples will be tested in triplicate. Cycling conditions will consist of initial incubations, followed by 40 cycles of denaturation, annealing, and extension. A dissociation curve analysis (60°C to 95°C) will also be performed to ensure amplification specificity of the primers.
Libraries for sequencing will be prepared according to Illumina’s 16S Metagenomic Sequencing Library Preparation guidelines (Part # 15044223 Rev. B), with the exception of using Qiagen HotStar MasterMix for the first PCR (‘amplicon PCR’) and halving reagent volumes for the second PCR (‘index PCR’). As per Illumina’s guidelines, template-specific primers will target the V3-V4 region of the 16S rRNA gene (PMCID: PMC3592464) [
11]. Resulting sequence reads will be analyzed through the National Research Council’s (Montreal, Canada) 16S rRNA gene amplicon analysis pipeline, as previously described [
12,
13]. Reads will be QCed, paired-end assembled, and clustered at 97% similarity. Taxonomic summaries and alpha (observed) and beta (weighted or unweighted UniFrac and Bray-Curtis distances) diversity metrics, statistical analysis, and taxonomic classifications will be computed using QIIME software [
14] and downstream analyses will be performed with in-house Perl and R scripts at the National Research Council.