Identification and safety evaluation of Bacillus species occurring in high numbers during spontaneous fermentations to produce Gergoush, a traditional Sudanese bread snack

Gergoush is a naturally fermented Sudanese Bread snack produced in three fermentation steps (primary starter, adapted starter and final dough), followed by three baking steps for a half to one hour at above 200 °C. This study examines the microbiota of two sets of fermentations performed at a traditional production site in Khartoum, Sudan in 2006 and 2009, respectively. In 2006 four different milk/legume based primary starters (faba bean, chick pea, lentil and white bean) were sampled in order to enumerate and identify the Bacillus at species or group level. In 2009 specific focus was on the enumeration and safety evaluation of the dominant Bacillus cereus group species occurring during various Gergoush productions (including the three fermentations steps and after baking). In 2006, the primary starters contained Bacillus spp. in the order of between 7.7 and 8.1 log10 CFU/g. Species identifications were performed by M13-PCR typing using the Escherichia coli phage M13 derived primer PM13 combined with internally transcribed 16–23S rRNA PCR, 16S rRNA gene and gyrA or gyrB gene sequencing, and selected phenotypic tests. Depending on the legume used, 40–68% of the isolates were identified as B. cereus sensu lato, 16–27% as Bacillus licheniformis, 8–32% as Bacillus subtilis and 4–20% as Bacillus sonorensis. During the second set of fermentation trials performed in 2009, the Bacillus spp. and B. cereus occurred in numbers of between 7.7–9.9 and 6.1–7.8 log10 CFU/g, respectively, while no bacteria were detected after baking. A total of 180 B. cereus sensu lato isolates from four different primary starters, adapted starters and final doughs were further identified as B. cereus sensu stricto (118 isolates) and Bacillus thuringiensis (62 isolates). The safety of Gergoush was evaluated based on the counts and toxin gene profiles of the dominant B. cereus species. Considering that no bacteria survived the baking process, and that the cereulide synthetase gene cesB involved in the production of the heat stable emetic toxin cereulide was not detected in any of the investigated B. cereus isolates, the results indicate, that Gergoush produced at the traditional production site is safe for human consumption. This study is the first to identify the Bacillus of Gergoush to species level, and it is the first to perform a safety evaluation of the product, based on the dominant B. cereus species.

Research Highlights
► Gergoush Bacillus spp. were B. cereus , B. subtilis, B. licheniformis, B. sonorensis.
► None of the B. cereus harbored cesB of the cereulide synthetase gene cluster.
► No bacteria including B. cereus survived the baking process of Gergoush.
► Gergoush produced at local producer level in Khartoum is safe for human consumption.
► M13-PCR typing differentiates Bacillus subtilis sensu lato at species level.