Esther Meersmana, Jan Steenselsa, Tinneke Paulusa, Nore Struyfa, Veerle Saelsa, Melissa Mathawanc, Leen Allegaertc, Gino Vranckenc and Kevin J. Verstrepen

Accepted manuscript posted online 6 July 2015, doi: 10.1128/AEM.00133-15

Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at the cocoa farms is allowed to ferment the pulp surrounding the cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is a growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, we therefore aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produces high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot-scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate shows that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.