Isolation and Molecular Characterization of Lactic Acid Bacteria Isolated from Fresh Fruits and Vegetables Using Nested PCR Analysis

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.