Abstract
Cocoa fermentation plays a key role in defining chocolate's flavor, with yeasts being central to this process. This study aimed to explore intraspecific genetic diversity of major indigenous yeasts (i.e., Saccharomyces cerevisiae and Pichia kudriavzevii), and their potential interaction in the cocoa pulp environment. Their metabolic intraspecific diversity was characterized in synthetic cocoa pulp medium. Then, Saccharomyces cerevisiae, Pichia kudriavzevii, and other strains were introduced to each other to evaluate their potential negative interaction. Interesting strain associations were selected to further explore their interaction in synthetic cocoa pulp medium as well as real fresh cocoa pulp. From a fermentation campaign in Ivory Coast, a set of Saccharomyces (S.) cerevisiae and Pichia (P.) kudriavzevii strains were isolated from batches classified according to their chocolate quality (i.e., standard, intermediate, or premium chocolate). Less abundant species (i.e., Torulaspora franciscae, Kluyveromyces marxianus) were also isolated and tested for their potential negative interactions with S. cerevisiae and P. kudriavzevii. A set of strains were selected and cultured in single and in co-culture in a minimal cocoa pulp synthetic medium and in fresh cocoa pulp to highlight potential positive and/or negative interactions regarding fermentative aroma profile (i.e., higher alcohols, acetate esters, medium-chain fatty acids, and ethyl esters). The results highlighted the dominance of S. cerevisiae in fermentation kinetics and medium- to long-chain ester production, contrasted with P. kudriavzevii's efficiency in short-chain ester synthesis. Intraspecific aroma profile variations can be pointed out. The co-cultures of P. kudriavzevii and S. cerevisiae strains isolated from the premium chocolate batch had a positive impact on the fermented pulp aroma profile. Negative interactions were observed with Torulaspora franciscae, which eliminated P. kudriavzevii's aroma expression. Finally, the comparison of the data obtained for the minimal cocoa pulp synthetic medium compared to the cocoa pulp allowed us to draw conclusions about the use of synthetic media for studying cocoa fermentation. These findings emphasize the complex microbial interactions in cocoa fermentation that could shape future cocoa bean aroma.