ABSTRACT
Aims: The microbial diversity, fermentation dynamic and the predominant microorganisms involved in the fermentation of African oil bean (Pentaciethra macrophylla Benth) seeds to “Ugba” traditional African food in Eastern Nigeria were investigated by analyzing the microbial community DNA of the food using sequences of their 16S rRNA genes fragment analysis. Study Design: Universal bacterial conserved 16S rRNA gene region was used to study bacterial dynamics as well as the diversity during fermentation stages. Predominant microorganisms were investigated with the view to establishing the best possible starter culture for the production of high flavoured “Ugba”. Place and Duration of Study: Biotechnology Centre of Federal University of Agriculture, Abeokuta, Ogun State, Nigeria, between January 2007 and May 2009. Methodology: Raw seeds were boiled for two hours for easy removal of the seed coats. Peeled seed cotyledons were sliced, cooked for 4hrs until softened. Sliced cotyledons were washed, wrapped in local leafs for fermentation for a period of 96hrs. Sampling for analysis was performed, at every 24 hours interval. Bacterial Community of freshly fermenting “Ugba” was obtained by washing seeds at room temperature in 0.40% NaCl salt solution for 15 minutes. The supernatant was used for streaking on both Nutrient agar and “Ugba” agar plates and for Community DNA extraction. DNA extraction was carried out from community DNA extracts and culture isolates grown in LB (Luria – Bertani) broth at 37°C for 24 hours using Promega DNA extraction kit. Partial 16S rRNA genes of isolates DNA and entire microbial community DNA were amplified using 16S rRNA primers. Amplified fragments were cloned using the PCRTRAP. The transformed clones were sequenced and aligned with reference sequences in the NCBI data base for identification. Results: This analysis indicated that from community DNA, seventeen clones were identified as Bacillus subtilis, Nine as Bacillus pumilus, four as Bacillus licheniformis, two as Bacillaceae bacterium, two as Bacillus sp Van 22, and two as Staphylococcus spp. Also, of the ten sequenced cloned isolates from the cultural technique, eight were identified as Bacillus subtilis, while two sequences were identified as Bacillus pumilus. The percentage abundance revealed that Bacillus subtilis had the highest abundance of 47.2% followed by Bacillus pumilus with 25%. Conclusion: Bacillus subtilis is the predominant species in Ugba fermentation as it had high percentage abundance throughout the fermentation period. This study indicated that molecular analysis of community DNA provides a more accurate picture of diversity and dynamics of microbial communities.
ABSTRACT
In this work, 100 samples each of 'ugba' and 'okpiye' were evaluated for the presence of bacteriocin producing lactic acid bacteria. Thirty strains showing antibacterial activity against at least one of the indicator organisms were selected from a total of 752 colonies isolated from the condiments. Out of the 30, only five strains retained activity after the pH of the broth supernatant was adjusted to 6.5. When evaluated by the agar-well diffusion assay, the spectra of inhibitory activity showed that Staphylococcus aureus was the most sensitive indicator organism tested, while Listeria monocytogenes was the most resistant. One strain (UG 2) was active against Escherichia coli. The assays using the cell-free supernatant of the cultures showed that the bacteriocins were completely inactivated by the proteolyses as well as by the chloroform treatment. In ethanol, the activity of the compounds was only partially modified. When incubated in a water bath at 80°C for 30 min, no significant activity loss was recorded. The antimicrobial activity of the bacteriocins produced by the lactic acid bacteria has potential for use in biopreservation of condiments against the spoilage and food - borne pathogens.