Molecular identification of yeasts involved in the alcoholic fermentation of Tchoukoutou and Atan in Benin using sequencing.

In Benin, the consumption of local beverages is increasing, particularly "Tchoukoutou" and "Atan". However, they face numerous challenges that hinder their recognition as fermented drinks with potential medical and cultural benefits. This study aims to determine the biodiversity of yeasts responsible for the fermentation of "Tchoukoutou" and "Atan" produced in Benin to promote their production and commercialization. To achieve this, beverage samples were randomly collected from six (06) municipalities in Benin and subjected to physicochemical and microbiological analysis. Yeast identification was performed through sequencing of the D1/D2 region of the 26S ribosomal RNA (26S rRNA) gene. Physicochemical analysis revealed that the pH values ranged between 3.65 and 3.92 for "Tchoukoutou" and between 3.71 and 4.04 for "Atan." Regarding sugar content (Brix degree), "Tchoukoutou" values ranged from 1 to 12° Brix, while those of "Atan" ranged from 1° to 14° Brix. Microbiological analysis showed that the yeast count in "Tchoukoutou" and "Atan" was 1.57 × 10³ and 5.29 × 10³ CFU/mL, respectively. A total of five (05) different yeast species were identified in "Tchoukoutou" and six (06) in "Atan". Schizosaccharomyces pombe (44.44%) was the predominant yeast species in the fermentation of "Tchoukoutou", followed by Saccharomyces cerevisiae (22.22%). In contrast, for "Atan", Saccharomyces cerevisiae (53.33%) was the most dominant species, followed by Saccharomyces boulardii (13.33%), and Candida parapsilosis (13.33%). Other yeast species present in lower percentages included Candida parapsilosis (11.11%), Pichia sp. Feni (11.11%) and Pichia manshurica (11.11%) in "Tchoukoutou" and Pichia kudriavzevii (6.66%), Pichia ethanolica (6.66%) and Schizosaccharomyces osmophilus (6.66%) in "Atan". These results indicate that Beninese "Tchoukoutou" and "Atan" fermentation is primarily driven by Schizosaccharomyces pombe, a yeast not commonly found in traditional Benin beverages, and Saccharomyces cerevisiae, respectively. This information is valuable for developing genetic engineering strategies to control their alcoholic fermentation, improve packaging, and extend shelf life.