Biodiversity of aerobic endospore-forming bacterial species occurring in Yanyanku and Ikpiru, fermented seeds of Hibiscus sabdariffa used to produce food condiments in Benin

Yanyanku and Ikpiru made by the fermentation of Malcavene bean (Hibiscus sabdariffa) are used as functional additives for Parkia biglobosa seed fermentations in Benin. A total of 355 aerobic endospore-forming bacteria (AEFB) isolated from Yanyanku and Ikpiru produced in northern and southern Benin were identified using phenotypic and genotypic methods, including GTG5-PCR, M13-PCR, 16S rRNA, gyrA and gyrB gene sequencing. Generally, the same 5–6 species of the genus Bacillus predominated: Bacillus subtilis (17–41% of isolates), Bacillus cereus (8–39%), Bacillus amyloliquefaciens (9–22%), Bacillus licheniformis (3–26%), Bacillus safensis (8–19%) and Bacillus altitudinis (0–19%). Bacillus aryabhattai, Bacillus flexus, and Bacillus circulans (0–2%), and species of the genera Lysinibacillus (0–14%), Paenibacillus (0–13%), Brevibacillus (0–4%), and Aneurinibacillus (0–3%) occurred sporadically. The diarrheal toxin encoding genes cytK-1, cytK-2, hblA, hblC, and hblD were present in 0%, 91% 15%, 34% and 35% of B. cereus isolates, respectively. 9% of them harbored the emetic toxin genetic determinant, cesB. This study is the first to identify the AEFB of Yanyanku and Ikpiru to species level and perform a safety evaluation based on toxin gene detections. We further suggest, that the gyrA gene can be used for differentiating the closely related species Bacillus pumilus and B. safensis.

► Bacillus spp. were the dominant species identified in additives Yanyanku and Ikpiru.
► 54% of the isolates from Yanyanku and Ikpiru were members of the Bacillus subtilis group.
► Pathogenic Bacillus spp. were identified, emphasizing the need to produce starter cultures.
► The gyrA gene can be used for differentiating the closely related species B. pumilus and B. safensis.