5 μM of each primer, 2 μl of LightCycler-FastStart DNA Master SYBR Green I (Roche), and either 2 μl of template or water (no-template control). The thermal cycling conditions were as follows: an initial denaturation step at 95°C for 10 min followed by 40 cycles

of denaturation at 95°C for 15 s; primer annealing at 60°C (Bifidobacterium), 65°C Dactolisib (Lactobacillus and B. longum) and 63°C (L. helveticus) for 25 s; extension at 72°C for 25 s (Bifidobacterium), 20 s (Lactobacillus), 45 s (B. longum) and 10 s (L. helveticus) and a fluorescence acquisition step at 90°C (Bifidobacterium and B. longum) or 85°C (Lactobacillus and L. helveticus) for 5 s. For each step the temperature Entospletinib transition rate was 20°C/s. Quantification of rrn operons of Bifidobacterium, Lactobacillus and B. longum was done by using standard curves made from known concentrations of genomic DNA from the sequenced strains B. longum NCC2705 [30] and L. acidophilus NCFM [57]. For L. helveticus www.selleckchem.com/products/chir-98014.html species the probiotic strain included in the synbiotic was used as standard and the number of rrn operons in the genome was deduced from the sequenced genome of L. helveticus DPC 4571 [58]. Chromosomal DNA of the strains used as standards was extracted by using DNeasy Tissue Kit (Qiagen)

and serially diluted from 105 to 101 molecules/μl. Results obtained by PCR were converted to the average estimate of total rrn operons from each group present in 1 μg of total DNA, and standard deviations (SD) were calculated. GC-MS/SPME A carboxen-polydimethylsiloxane coated fiber (85 μm) and a manual SPME holder (Supelco, Bellefonte, PA) were used in this study after preconditioning according to the manufacturer’s instruction manual. Before each head space sampling, the fiber was exposed to the GC inlet for 5 min for thermal desorption at 250°C

in a blank sample. Five ml of fecal slurries (20%) were placed in 10 ml glass vials, added with 4-methyl-2-pentanol (4 mg/l) as internal standard. The samples were then equilibrated for 10 min at 45°C. The SPME fiber was exposed to each sample for 40 min and then was inserted into the injection port of Osimertinib the GC for a 5 min sample desorption. GC-MS analyses were performed on an Agilent 7890A gaschromatograph (Agilent Technologies, Palo Alto, CA) coupled to an Agilent 5975C mass selective detector operating in electron impact mode (ionization voltage 70 eV). A Supelcowax 10 capillary column (60 m length, 0.32 mm ID) was used (Supelco). The temperature program was: 50°C for 1 min, then programmed at 4.5°C/min to 65°C and finally at 10°C/min to 230°C which was maintained for 25 min. Injector, interface and ion source temperatures were 250, 250 and 230°C, respectively. The mass-to-charge ratio interval was 30-350 Da at 2.9 scans per second. Injections were performed in splitless mode and helium (1 ml/min) was used as carrier gas.