Bacterial Succession Studies during Fermentation of African Locust Bean (Parkia biglobosa) to Iru Using Molecular Methods

Aims: The identity, diversity and dynamics of the bacterial community involved in the fermentation of African Locust Bean (Parkia biglobosa) to “Iru”, a protein-rich condiment in Western Nigeria; was studied using the 16S rRNA gene sequence analysis.
Study Design: 16S rRNA gene was used to study bacterial succession and diversity in this solid-state fermentation with a view to develop a framework for improving quality control of this important and nutritious solid-state fermentation product and possibly develop starter cultures for commercializing the product.
Place and Duration of Study: Biotechnology Centre of University of Agriculture, Abeokuta, Ogun State, Nigeria and Biological Sciences Department, Florida Atlantic University, Davie Campus, Florida U.S.A., between July 2008 and October 2009.
Methodology: Raw seeds were prepared in the traditional African way by boiling them for 6hr to soften the seed coat; and for another 1hr to soften the cotyledon. The boiled seeds were immediately transferred into a jute-bag and wrapped tightly to prevent heat loss. They were left at ambient temperature to ferment for 72hr. Total Bacterial Community of the seed was obtained by vigorously rinsing seeds in phosphate buffered saline, before boiling and immediately after boiling (0hr), and subsequently at intervals of 24hrs for three days. To compare cultivable phyllotypes and possible non-cultivable bacteria, subsamples of the extracted bacteria were cultured on Tryptic Soy Agar. Total community small subunit (SSU) rRNA was amplified from extracted genomic DNA by Plate wash polymerase chain reaction (for cultured bacteria) and classic PCR directly from seed-buffer extract (uncultured bacteria). Genomic DNA was extracted employing a modified protocol of the freeze-thaw and Qiagen DNA extraction methods. Extracted genomic DNA was run on 1% agarose gel to rule out shearing before PCR amplification of the 16S rRNA gene with the 27F and 1492R primer pair. The amplified samples were cloned using TOPO cloning vector and transformed samples were sequenced. Identity of samples were done by aligning samples in Ribosomal database Project and close relatives was identified.
Results: The process was found to be a classic alkaline fermentation (pH 6 – 8.39). Cultivable bacterial populations changed from 120CFU/g at start of fermentation to 1630000000 CFU/g on day 3. The most abundant organism present in the raw African Locust Beans isolates (Clone 1A) had 97% match to Acinetobacter sp. Cooked Locust beans isolates (Clone 2A) shared 100% identity to Bacillus subtilis. Organisms present at 0 hr, 24 hr and 48 hr of fermentation (Clones 3A, 4A and 5A) proved to have 100% match to Bacillus anthracis relatives; Bacillus cereus; and Bacillus sp. respectively. Enterobacter sp. (99% similarity to Clone 6A) was only detected after 72 hrs; amidst the bacilli. Even less abundant clones were identified as various Bacillus phyllotypes. Cultured and non-cultured bacterial phyllotypes in this system clustered similarly and appear to be the same; confirming that Bacillus species were primarily responsible for the fermentation products of iru. While the bacterial identity and low diversity index reported here, is not surprising given the resilience of bacterial endospores to boiling; it provides convincing evidence to explore the use of endospores from these cultivable non-pathogenic bacillus strains as starter cultures for the solid state fermentation.
Conclusion: It is concluded from this study that the use of molecular method in the identification of organisms present during African locust beans fermentation to Iru, revealed varieties of organisms that had not been previously reported by culture- dependent methods. The application of these techniques in the field of microbiology allows a better understanding of the ecology of food fermentation.