Rogosa

TiO₂ nanoparticles (anatase) with purity of more than 99% and particle size of 10–25 nm were obtained from US Research Nanomaterials, Inc. Na‐alginate was obtained from Behin Azma Co. Nutrient broth, Violet Red Bile Agar (VRBA), Cetrimide fucidin cephaloridine agar (CFC agar), De Man, Rogosa, Sharpe agar (MRS), Listeria Chrom agar, and plate count agar (PCA) were purchased from Merck Chemical Co.

Cultural analysis of the intestinal microbiota along the entire GI tract was performed as described previously [20] (link). Luminal contents taken from stomach, duodenum, jejunum, ileum, caecum, and distal colon were resuspended in PBS, weighted, and 100 µl aliquots of serial dilutions plated onto solid media (Oxoid). Bacteria were grown at 37°C for 2 days under aerobic or for 4 days under anaerobic conditions, and total numbers determined by colony counting on Columbia blood agar. Bile esculin, McConkey, anaerobe 5% sheep blood agar supplemented with kanamycin and vancomycin, and Rogosa (Merck) media were used for quantitative identification of enterococci, enterobacteria (such as E. coli and Proteus spp.), Bacteroides/Prevotella spp., and lactic acid bacteria (mainly lactobacilli), respectively. Bacteria were subcultivated and further investigated by Gram-staining and by biochemical analysis with the API20E, API50 CH, and API Rapid ID 32A systems (Biomérieux) and confirmed by sequence analyses in cases of inconclusive identification. Results were expressed as colony forming units (CFU) per g of luminal content. The detection limit of viable pathogens was ≈25 CFU per g.