The Microbial Diversity and Characterization of Bacillus Species for the Enhanced Fermentation of Bambara Groundnut in the Production of African Food Condiments

Introduction: African food condiments are products from alkaline fermentation (pH 6 – 9) of several legumes native to Africa and they serve as a low-cost meat substitute. Typically, the fermentation is spontaneous without a starter culture and no standard process control; thus, food safety and product quality consistency is a concern. Production from bambara groundnut is limited, despite the legume’s high potential as substrate because it is high in carbohydrate and protein contents. Current identification methods of the microbial diversity of alkaline fermentation of African condiments have been less accurate phenotypic and biochemical tests with Bacillus spp. identified as the predominant genera. 

Purpose: This study endeavors to identify the microbial diversity of African food condiments using phenotypic and genotypic methods. Furthermore, determine the genetic diversity of Bacillus spp. involved in alkaline fermentation of bambara groundnuts.

Methods: Microbial enumeration of viable aerobic count, Enterobacteriaceae, fungi and aerobic endospore-forming bacteria (AEFB). Direct Bacterial Profiling by MALDI-TOF Mass Spectrometry of the Bacillus isolates. Further (GTG)₅-Rep-PCR fingerprinting and DNA sequencing of the 16S rRNA  and gyrA genes.

Results: The microbial diversity comprised mostly of aerobic endospore-forming bacteria (3.0 x 10⁹ CFU/g), Enterobacteriaceae (2.0 x 10³ CFU/g) and molds (4.0 x 10² CFU/g). B. subtilis groups dominated the alkaline fermentation; however, B. cereus was also identified. This was shown in the microbial clustering of AEFB based on MALDI-TOF MS and rep-PCR (GTG₅) fingerprinting. The phylogenetic inferences obtained from sequences of 16S rRNA and gyrA gene showed a strong correlation with B. subtilis group. Typed strains B. subtilis subsp. subtilis SFBA3, B. amyloliquefaciens SFBA2, B. pumilus P25 and B. licheniformis O16 were suggested as starter cultures for fermentation with bambara groundnut.  

Significance: Starter cultures from Bacillus strains identified by genotypic methods will enhance food safety and commercial production of alkaline fermented African condiments in developing countries.