acidilactici KSW b [14] N8, N9, N10       Ped pentosaceus KSW b

acidilactici KSW b [14] N8, N9, N10       Ped. pentosaceus KSW b [14] P4, P5, S4       W. confusa KSW b [14] P2, P3, SK9-2, SK9-5,   SK9-7, FK10-9       Genotypic characterization Genomic DNA preparation for PCR and selleck chemicals llc this website Sequencing reactions Overnight-culture of each strain was streak-plated on MRS agar (Oxoid Ltd., CM0361, pH 6.2 ± 0.2, Basingstoke, Hempshire, England) and incubated at 37°C under anaerobic conditions (AnaeroGen, Oxoid) for 48 hrs. Genomic DNA was extracted from a single colony of each strain using the InstaGene Matrix DNA extraction kit (Bio-Rad,

Hecules, CA, USA) and following the manufacturer’s instructions. DNA was stored at −20°C and used for all PCR reactions mentioned in this study. Rep-PCR Genomic DNA was analysed with the rep-PCR fingerprinting method using the GTG5 (5’-GTG GTG GTG GTG GTG-3’) primer (DNA Technology A/S, Denmark) with the protocol of Nielsen et al. [21]. Electrophoresis conditions and image analysis with the Bionumerics software package (Applied Maths, Sint-Martens-Latem, Belgium) were performed as previously [8]. 16S rRNA gene sequencing PCR amplification of 16S rRNA gene of all the isolates was performed with the primers 7f (5′-AGA GTT TGA TYM

TGG CTC AG-3′) and 1510r (5′-ACG GYT ACC TTG TTA CGA CTT-3′) [36] (DNA Technology A/S, Denmark). The reaction mixture consisted; 5.0 μl of 10X PCR reaction buffer (Fermentas, Germany), 0.2 mM dNTP-mix (Fermentas, Germany), 1.5 mM MgCl2, 0.1 pmol/μl primers 7f and 1510r, 0.5 μl formamide (Merck), 0.50 μl of 1 mg/ml bovine serum albumin (New England Biolabs), 0.25 μl DreamTaq™ DNA polymerase (5 u/μl) (Fermentas, Germany) and 1.5 μl of the extracted genomic DNA. The volume of the PCR mixture was adjusted to 50 μl with sterile MilliQ water. PCR amplification was performed in DNA thermocycler (Gene Amp PCR System 2400, Perkin-Elmer) at the following thermocycling conditions; 5 min of initial denaturation at 94°C, followed by 30 cycles of 94°C for 90 seconds, 52°C for 30 seconds, 72°C for 90 seconds and a final elongation step of 72°C for 7 minutes. To check for successful PCR amplification, 10 μl of the PCR product was electrophoresed in a 2% agarose gel in 1X TBE (1 hr, 100 V).

PCR products were purified of DNA amplification reagents using NucleoSpin® DNA purification kit by following the selleck chemical manufacturer’s instructions. Sequencing was performed in both directions with the universal primers 27f (5’-AGA GTT TGA TCM TGG CTC AG-3’) and 1492r (5’-TAC GGY TAC CTT GTT ACG ACT T-3’) by a commercial sequencing facility (Macrogen Inc., Korea). The sequences were corrected using Chromas version 2.33 (Technelysium Pty Ltd). Corrected sequences were aligned to 16S rRNA gene sequences in the GenBank data base using the BLAST algorithm [37]. Differentiation of Lactobacillus plantarum, Lb. paraplantarum and Lb. pentosus by multiplex PCR using recA gene-based primers A multiplex PCR assay for differentiation of Lb. plantarum, Lb. paraplantarum and Lb.

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