Molecular Identification of Bacteriocin-Producing Strains

DNA was extracted from an overnight culture of each strain using the wizard genomic DNA purification kit (Promega, Madison, WI, United States) as described by Fernandez et al. (2015). The 16S ribosomal DNA was amplified by PCR (Eppendorf Mastercycler gradient, Hamburg, Germany) with 27f 5′-AGAGTTTGATCMTGGCTCAG-3′ (Gürtler and Stanisich, 1996) and 1390r 5′-GACGGGCGGTGTGTACAA-3′ (Zheng et al., 1996) primers (Invitrogen, Carlsbad, CA, United States). The reaction volume was 50 μL, composed of 1 × Taq buffer (New England Biolabs, Beverly, MA, United States), 0.25 U of Taq DNA polymerase (New England Biolabs), 200 nM of each primer, 200 μM of a dNTP mixture (A, T, C, and G, Invitrogen), and 20 ng of bacterial DNA. The thermal cycle program consisted of an initial cycle of 94°C for 5 min for denaturation and polymerase activation, 35 cycles of 94°C for 45 s, 54°C for 45 s, and 68°C for 60 s, and a final extension step of 5 min at 68°C. PCR products were then subjected to gel electrophoresis (100 V, 1 h) on a 1% agarose gel in Tris-acetate-EDTA 1 × buffer (Ambion, Life Technologies Inc., Burlington, ON, Canada) and visualized by gelRed staining (Biotium, Inc., Hayward, CA, United States). Finally, pure PCR products were sequenced using an ABI 3730XL DNA analyzer (Applied Biosystems, Streetsville, ON, Canada).