Diversity of Bacterial Community in Fermentation of African Oil Bean Seeds (Pentaciethra macrophylla Benth) by comparison of 16S rRNA Gene Fragments

Aims: The microbial diversity, fermentation dynamic and the predominant micro-organisms 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 PCR-TRAP. 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.