Isolation and Molecular Characterization of Lactic Acid Bacteria Isolated from Fresh Fruits and Vegetables Using Nested PCR Analysis.
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Date
2013-07
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Abstract
Aims: The study investigated the diversity and identities of Lactic Acid Bacteria (LAB)
isolated from different fresh fruits and vegetables using Molecular Nested PCR analysis
with the view of identifying LAB with anti-microbial potentials.
Study Design: Nested PCR approach was used in this study employing universal 16S
rRNA gene primers in the first round PCR and LAB specific Primers in the second round
PCR with the view of generating specific Nested PCR products for the LAB diversity
present in the samples.
Place and Duration of Study: Biotechnology Centre of Federal University of Agriculture,
Abeokuta, Ogun State, Nigeria, between January 2011 and February 2012.
Methodology: Forty Gram positive, catalase negative strains of LAB were isolated from
fresh fruits and vegetables on Man Rogosa and Sharpe agar (Lab M) using streaking
method. Standard molecular methods were used for DNA extraction (Norgen Biotek kit
method, Canada), Polymerase Chain Reaction (PCR) Amplification, Electrophoresis, Purification and Sequencing of generated Nested PCR products (Macrogen Inc., USA).
Results: The partial sequences obtained were deposited in the database of National
Centre for Biotechnology Information (NCBI). Isolates were identified based upon the
sequences as Weissella cibaria (5 isolates, 27.78%), Weissella kimchi (5, 27.78%),
Weissella paramensenteroides (3, 16.67%), Lactobacillus plantarum (2, 11.11%),
Pediococcus pentosaceus (2, 11.11%) and Lactobacillus pentosus (1, 5.56%) from fresh
vegetable; while Weissella cibaria (4, 18.18%), Weissella confusa (3, 13.64%),
Leuconostoc paramensenteroides (1, 4.55%), Lactobacillus plantarum (8, 36.36%),
Lactobacillus paraplantarum (1, 4.55%) and Lactobacillus pentosus (1, 4.55%) were
identified from fresh fruits.
Conclusion: This study shows that potentially LAB can be quickly and holistically
characterized by molecular methods to specie level by nested PCR analysis of the bacteria
isolate genomic DNA using universal 16S rRNA primers and LAB specific primer.
Description
Keywords
Nested PCR, Molecular characterization, 16S rRNA gene, Lactic acid bacteria
Citation
Emerenini E C, Afolabi O R, Okolie P I, Akintokun A K. Isolation and Molecular Characterization of Lactic Acid Bacteria Isolated from Fresh Fruits and Vegetables Using Nested PCR Analysis. British Microbiology Research Journal. 2013 July; 3(3): 368-377.