Screening of lactic acid bacteria for bacteriocins by microbiological and PCR methods

Abstract

We describe a practical laboratory designed for third-year undergraduate students of Biotechnology as part of a Microbial Physiology and Genetics course. It comprises a five-session laboratory module to screen foods for lactic acid bacteria (LAB) 1 and to test isolated LAB for the presence of bacteriocins. Traditional Thai fermented foods are first screened for bacteriocin-producing LAB using microbiological methods. This is followed by a simple and rapid DNA extraction and by a multiplex polymerase chain reaction (PCR) using three pairs of specific primers to test for the presence or absence of various bacteriocin genes in the isolated LAB. PCR amplicons of 332, 412, and 608 bp indicate the presence of pediocin, enterocin, and nisin genes, respectively, whereas no amplicon band indicates the absence of these bacteriocins. The laboratory provides the students with experience in the use of microbiological and multiplex PCR methods and shows how the molecular biology techniques can be related to their daily lives. The module could easily be adapted to the study of fermented foods from other countries.

Partial Characterization of Bacteriocin Produced by Halotolerant Pediococcus acidilactici Strain QC38 Isolated from Traditional Cotija Cheese

“…Plasmid DNA isolation was performed by the modified alkaline lysis method (Anderson and McKay, 1983) and DNA was used as a template for the amplification of bacteriocin gene. PCR was performed using 20 µl reaction mixture consisting of 1X PCR buffer, 1 U of Taq DNA polymerase, 3 mM of MgCl 2 , 0.4 mM of dNTPs and 0.2 µM of each primer (PedF and PedR) (Suwanjinda et al, 2007). The PCR conditions were an initial denaturation step at 94°C during 5 min, followed by 30 cycles of denaturation at 94°C during 1 min, annealing (51°C, 40 s) and extension (72°C, 3 min).…”

Spoilage volatiles and sensory properties of a grilled stick meat product inoculated with Pediococcus acidilactici FLE07 as starter culture

“…Full identity of the isolate was obtained in the present study using 16S rDNA nucleotide sequencing after successful amplification by PCR. Deoxyribonucleic acid (DNA) was extracted using a boiling method [9]. PCR amplification was performed using the following set of primers [10]: Forward, 5′-CCTACGGGAGGCAGCAG-3′ and Reverse, 5′-ATTACCGCGGCTGCTGG-3′, targeting approximately 200 bp of 16S rDNA gene (V3 region).…”

Bacteriocinogenic Bacteria Isolated from Raw Goat Milk and Goat Cheese Produced in the Center of México

“…DNA from the twelve bacteria was used for PCR amplification of nisin, enterocin A and pediocin genes using Taq polymerase (Invitrogen, Carlsbad CA, USA) and gene specific primers; for nisin, nisRF (5 0 -CTATGAAGTTGC GACGCATCA-3 0 ), nisRR (5 0 -CATGCCACTGATACCC AAGT-3 0 ); for enterocin A, entAF (5 0 -GGGTACCACT CATAGTGGAA-3 0 ); enterocin, entAR (5 0 -CCAGCAGTT CTTCCAATTTCA-3 0 ); and pediocin, pedF 5 0 -GGTAAG GCTACCACTTGCAT-3 0 ), pedR (5 0 -CTACTAACGCT TGGCTGGCA-3 0 ) [15]. Amplification was performed in a C1000 Touch Thermal Cycler (Bio-Rad) using the following conditions: 5 min at 95°C; 30 cycles of 30 s at 95°C, 30 s at 58°C and 90 s at 72°C, with a final extension of 5 min at 72°C.…”