The total protein amounts contained in 50 mL of control samples [MRSC, GM17 supplemented or not with 0.1% or 1% (v/v)], or 40 μg of extracellular protein extracts were resolved by SDS-PAGE using a final polyacrylamide concentration of 12.5% (w/v) (Laemmli, 1970). Proteins whose electrophoretic bands showed changes in intensity with the presence of cecum extract were submitted to MALDI-MS/MS analysis and identified at the Proteomics Core Facility of CNIC (Madrid, Spain) using standard protocols. Relative expression of the genes coding for Imp11 and Imp23 was determined
by quantitative PCR (qPCR). Ten milliliters of MRS containing selleck chemical 0% or 1.0% (v/v) cecum extract was left in the anaerobic chamber MG500 (Don Whitley Scientific, West Yorkshire, UK) under 10% (v/v) H2, 10% CO2, and 80% N2 at 37 °C overnight. These aliquots AZD4547 order were inoculated (1% v/v) with overnight bacterial cultures made in MRSC; samples were taken after 90 min (early exponential phase), 3 h (middle exponential phase), and 12 h (early stationary phase). Cells were collected by centrifugation (9300 g, 5 min), and the protocols for cell lysis, RNA isolation, and cDNA synthesis were performed as previously described (Gueimonde et al., 2007). The qPCR experiments were run in an ABI Prism 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA). Specific primers were designed for imp11 (SABLF, 5′-CGTACGTGTGATCAAGCCCGCA-3′; SABLR, 5′-GGAATAGGTGTCTGCCTGGGCA-3′) and
for imp23 psacid (PSACIDF, 5′-TCAGCAGCCACTAATAGCGACTCA-3′; triclocarban PSACIDR, 5′-CACCTGGTACACCTCCAGGAGCT-3′). Their specificity was verified before the quantitative analysis. At least three independent qPCR runs were performed for each cDNA. Relative expression of stated genes under the experimental conditions was estimated according to ΔΔCt method using an intergenic spacer region between
the 16s and 23s rRNAs as an endogenous control, employing previously described primers (Gueimonde et al., 2004; Haarman & Knol, 2006). Expression rate was related to that of the corresponding genes in the absence of cecum extract, which was given the arbitrary value 1. Research studies focusing on characterization of food and probiotic bacterial strains generally involve the use of synthetic, defined, or complex culture media that do not reproduce adequately the conditions of the GIT, which is the natural habitat or the site of action of most of these bacteria. As a consequence, expression of some cellular and extracellular proteins may change with respect to the in vivo situation. Key proteins that might be potentially involved in interactions with the human host could be found by trying to mimic the environmental conditions that those bacteria face in the human intestine. Once released from the bacterial cell to the surrounding media, extracellular proteins would be able to interact directly with mucosal cells including epithelial and immune cells (Sánchez et al.