Diet impacts major components dynamics of the intestinal microbiota
Twenty healthy adults (10 males, 10 females), aged between 19 and 25 years, with no history of gastrointestinal problems, volunteered for the study. The protocol was carefully explained to the volunteers, and their written informed consent was obtained. Inclusion criteria were the following: no intake of antibiotics, laxatives or other gastrointestinal medications 3 months prior to the beginning of the study, body mass index [BMI; body weight (kg)/height (m2)] between 18 and 25. The study had a double blinded randomized cross over design with a washout period of two weeks and was carried out into the clinical center of Investigation (University hospital center, Grenoble, France). In order to assess the effect of dietary fibers, two different diet formulas were compared. The food intakes were made with the same ingredients and differed only on the type and quantities of dietary fibers. For better reproducibility, the food intakes were lyophilized.  Subjects were randomly assigned to receive each diet during two periods of five days and were not allowed to eat or drink anything else, except water. Three controlled meals (see supporting information) were dispensed each day to the subjects. Meals were eaten at home during five days. Fecal samples were collected from subjects into individual containers before and after four and five days ofBacterial DNA samples with four time points (two diets periods) from the nutritional intervention were used to construct DNA libraries. The following universal 16S rRNA primers were used for the PCR reactions: V3F (TACGGRAGGCAGCAG, 343-357 E. coli position) and V4R (GGACTACCAGGGTATCTAAT, 787-806 E. coli position) to target V3-V4 region described as the most informative for taxonomic assignation (Wang et al, 2007). Barcode sequences (GsFLX key) TCAG and MIDGsFLX (10 nucleotides) were attached between the 454 GsFLX adaptator sequence and the forward primer V3F. The GsFLX key and the 454 GsFLX adaptator were attached to the reverse primer. The concentration and quality of the PCR products were assessed with Picogreen. Equal amounts of each of the samples were pooled and the 16S rRNA amplicons were sequenced on staining Roche GS FLX 454 sequencer. the diet intervention period, and retrieved to the clinical center of investigation. Samples were also collected one week before and after the nutritional intervention as control. After homogenization and sampling fecal samples were numbered and frozen at -80°C for analyzes. DNA was extracted from about 200 mg aliquots of feces as described previously (Furet et al, 2009). After the final precipitation, DNA was resuspended in 150 µL of TE buffer and stored to -20°C for further analysis.DNA was extracted from about 200 mg aliquots of feces as described previously (Furet et al, 2009). After the final precipitation, DNA was resuspended in 150 µL of TE buffer and stored to -20°C for further analysis.
Genoscreen (Lille, FRANCE) standard protocol