Gels were electrophoresed at 60°C at 75 V for 15 h. Sybr Green I stained gels were

photographed and acquired by the Bio-Rad Gel Doc 2000 documentation system (Bio-Rad Laboratories). To compensate for internal distortions occurring during the electrophoresis, binding patterns LY411575 were digitally aligned using the Bionumerics software version 4.5 (Applied Maths, Belgium) by comparison with an external reference pattern obtained by appropriately mixing DGGE marker II, III and V (Nippon gene, Tokyo), depending on the gradient used. This normalization enabled comparison among DGGE profiles from different gels, provided that these were run under comparable denaturing and electrophoretic conditions. Comparison and cluster of profiles were carried out using the unweigthed pair-group method with the arithmetic average (UPGMA) clustering algorithm based on the Pearson product-moment correlation coefficient (r) [25, 48] and resulted in a distance matrix. DGGE fragments from primers Lac1 and Lac2 were cut out using sterile scalpel.

The DNA of each band was eluted in 100 μl of sterile water overnight at 4°C. Two μl of the eluted DNA were reamplified as described above. PCR products were separated by electrophoresis on 1.5% (wt/vol) agarose gel (Gibco BRL, France) stained with ethidium bromide (0.5 μg/ml). The amplicons were eluted from gel and purified by the GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences, Milan, Italy). DNA sequencing reactions were performed by MWG Biotech ifenprodil AG (Ebersberg, Germany). Sequences were

compared to buy EPZ-6438 the GenBank database with the BLAST program. Enumeration of cultivable bacteria Diluted faecal samples (20 g) were mixed with 80 ml sterilized peptone water and homogenized. Counts of viable bacterial cell were carried out as described by Macfarlane et al. [45, 49] The following selective media were used: MRS agar (lactobacilli); Beerens agar (bifidobacteria); Baird-Parker (staphylococci and micrococci); Blood Azide agar (enterococci); Wilkins-Chalgren agar (total anaerobes); Wilkins-Chalgren agar plus GN selective supplements (Bacteroides, Porphyromonas and Prevotella); Reinforced Clostridial Medium supplemented with 8 mg/l novobiocin, 8 mg/l colistin (Clostridium), MacConkey agar No2 (enterobacteria); and nutrient agar (total anaerobes) [50]. Lactic acid bacteria isolation Fifteen to twenty colonies of presumptive lactic acid bacteria were isolated from the highest plate dilutions of MRS and Blood Azide agar media. Gram-positive, catalase-negative, non-motile rods and cocci isolates were cultivated in MRS or Blood Azide broth (Oxoid Ltd) at 30, 37 or 42°C for 24 h, and re-streaked into the same agar media. All isolates considered for further analyses learn more showed the capacity of acidifying the liquid culture medium. All cultures were stored at -80°C in 10% (vol/vol) glycerol.