Antimicrobial Properties of Purified Bacteriocins Produced from Lactobacillus casei and Lactobacillus fermentum against Selected Pathogenic Microorganisms.

Aims: (1) To isolate bacteriocin samples produced from Lactobacillus using natural fermented foods which include: palm-wine, milk, locust beans, fufu (white solid food made from cassava), ogi (known as pap) and dairy fermented product (Yogurt); (2) extraction and purification of these bacteriocin samples using centrifugation and ammonium sulphate respectively and removal of impurities using dialysis. (3) to confirm the production of bacteriocin by performing antimicrobial assay against some selected pathogenic microorganisms. (4) to examine the effects of pH, heat and storage stability as well as biopreservative efficiency of the bacteriocin samples in pap, kunu (made from millet) and fresh orange juice; (5) to investigate the effect of viable antibiotics on the growth of isolates. Study Design: Data were analyzed using the statistical software package SPSS version 16.0 and standard errors of mean (SEM) for all the graphs plotted were represented with error bars while their characterization was designed using reciprocal of the highest dilution (2n) that resulted in the inhibition of the indicator lawn. Thus, the arbitrary units (AU) of the bacteriocin activity per milliliter (AU/ml) were defined as 2n× 1000/ 10μl. Place and Duration of Study: Microbiology Laboratory, Sacred heart Hospital, Abeokuta and Department of Microbiology, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria, between March 2010 and November 2012. Methodology: Bacteriocins, otherwise known as the antimicrobial compounds were produced from the Lactobacillus strains and then isolated from Nigerian fermented foods which include: palm wine, milk, yoghurt, locust beans, ogi and fufu. These foods were isolated using de Mann Rogosa and Sharpe medium. The isolated microorganisms (L. fermentum and L.casei) were identified phenotypically after isolation. Bacteriocins were extracted and purified from the Lactobacillus strains by centrifugation, followed by ammonium sulphate precipitation and dialysis. The antimicrobial activities of the crude bacteriocins were tested against nine selected pathogenic clinical isolates collected from the University College Hospital, Ibadan. The tested isolates were Shigella dysenteriae, Shigella flexneri, Enteropathogenic Escherichia coli type 1, Enteropathogenic Escherichia coli type 2, Enterohaemorrhagic E. coli type 3, Salmonella typhi, Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae. Results: The bacteriocins of Lactobacillus fermentum and Lactobacillus casei showed a broad range of activities and had higher significant effect (P< 0.05) on the selected pathogenic microorganisms. The effects of pH on the bacteriocins were active in range of 2 to 6. Bacteriocins produced by Lactobacillus casei were stable at 800C for 15 minutes while bacteriocins produced by Lactobacillus fermentum were stable at 1000C for 15 minutes. It was observed that these bacteriocins can be stored between -200C and 40C and they had significant difference on the selected pathogenic microorganisms (P<0.05). The preservative activities of the bacteriocins tested on different foods showed that the bacteriocin of Lactobacillus fermentum had maximum reduction on bacterial population. Lactobacillus fermentum and Lactobacillus casei isolates were resistant to erythromycin of 70% and 100% for cotrimoxazole, ciprofloxacin, augmentin and amoxicillin. Conclusion: This study showed that bacteriocins from fermented foods could be used as an effective control for pathogenic microorganisms as they were able to exhibit antimicrobial activity against the test organisms when investigated for bacteriocin production and when characterized.

In vitro Studies on Antimicrobial Activities of Lactic Acid Bacteria Isolated from Fresh Vegetables for Biocontrol of Tomato Pathogens.

Aims: This study was focused on using Lactic Acid Bacteria (LAB) isolated from fresh vegetables which has been molecularly identified for in vitro control of some tomato pathogens. Study Design: The inhibitory potentials of supernatant obtained from previously characterized LAB isolates or vegetable origin were investigated against some tomato phytopathogens using agar-well method with the view to develop biological agents for some tomato disease causing organisms. Place and Duration of Study: Biotechnology Centre of Federal University of Agriculture, Abeokuta, Ogun State, Nigeria, between January 2011 and February 2012. Methodology: The antimicrobial activities of LAB against some tomato phytopathogenic bacteria which include (Xanthomonas campestries, Erwinia caratovora, and Pseudomonas syringae) were obtained by using the agar well diffusion method. Results: The result indicates that cell free culture of LAB from fresh vegetables origin (Weissella paramesenteroides, Lactobacillus pentosus, Weissella cibaria, Pediococcus pentosaceus, Weissella kimchi and Lactobacillus plantarum) can inhibits these bacteria by creating clear zones of inhibition around the wells containing cell free supernatants of the above mentioned strains of lactic acid bacteria. Pediococcus pentosaceus showed the highest zone of inhibition against Xanthomonas campestries at 15 mm radius, Weissella kimchi was the least effective against Pseudomonas syringae at 3.67 mm and Erwinia caratovora at 3.50 mm radius. Conclusion: Tomato disease causing organisms can be most likely biologically controlled by using extracts from LAB. This finding will reduce the potential hazard from the use of chemical herbicides on plant.

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

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.