Acetic acid bacteria are involved in many food and beverage fermentation processes. They play an important role in cocoa bean fermentation through their acetic acid production. They initiate the development of some of the flavor precursors that are necessary for the organoleptic quality of cocoa, and for the beans’ color. The development of starter cultures with local strains would enable the preservation of the microbial biodiversity of each country in cocoa-producing areas, and would also control the fermentation. This approach could avoid the standardization of cocoa bean fermentation in the producing countries. One hundred and thirty acetic acid bacteria were isolated from three different cocoa-producing countries, and were identified based on their 16S rRNA gene sequence. The predominate strains were grown in a cocoa pulp simulation medium (CPSM-AAB) in order to compare their physiological traits regarding their specific growth rate, ethanol and lactic acid consumption, acetic acid production, and relative preferences of carbon sources. Finally, the intraspecific diversity of the strains was then assessed through the analysis of their genomic polymorphism by (GTG)₅-PCR fingerprinting. Our results showed that Acetobacter pasteurianus was the most recovered species in all of the origins, with 86 isolates out of 130 cultures. A great similarity was observed between the strains according to their physiological characterization and genomic polymorphisms. However, the multi-parametric clustering results in the different groups highlighted some differences in their basic metabolism, such as their efficiency in converting carbon substrates to acetate, and their relative affinity to lactic acid and ethanol. The A. pasteurianus strains showed different behaviors regarding their ability to oxidize ethanol and lactic acid into acetic acid, and in their relative preference for each substrate. The impact of these behaviors on the cocoa quality should be investigated, and should be considered as a criterion for the selection of acetic acid bacteria starters.

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.