Spontaneous cocoa bean fermentation carried out in a novel-design stainless steel tank: influence on the dynamics of microbial populations and physical-chemical properties.

Spontaneous cocoa bean fermentations carried out in a novel-design 40-kg-capacity stainless steel tank (SST) was studied in parallel to traditional Brazilian methods of fermentation in wooden boxes (40-kg-capacity wooden boxes (WB1) and 600-kg-capacity wooden boxes (WB2)) using a multiphasic approach that entailed culture-dependent and -independent microbiological analyses of fermenting cocoa bean pulp samples and target metabolite analyses of both cocoa pulp and cotyledons. Both microbiological approaches revealed that the dominant species of major physiological roles were the same for fermentations in SST, relative to boxes. These species consisted of Saccharomyces cerevisiae and Hanseniaspora sp. in the yeast group; Lactobacillus fermentum and L. plantarum in the lactic acid bacteria (LAB) group; Acetobacter tropicalis belonging to the acetic acid bacteria (AAB) group; and Bacillus subtilis in the Bacillaceae family. A greater diversity of bacteria and non-Saccharomyces yeasts was observed in box fermentations. Additionally, a potentially novel AAB belonging to the genus Asaia was isolated during fermentation in WB1. Cluster analysis of the rRNA genes-PCR-DGGE profiles revealed a more complex picture of the box samples, indicating that bacterial and yeast ecology were fermentation-specific processes (wooden boxes vs. SST). The profile of carbohydrate consumption and fermentation products in the pulp and beans showed similar trends during both fermentation processes. However, the yeast-AAB-mediated conversion of carbohydrates into ethanol, and subsequent conversion of ethanol into acetic acid, was achieved with greater efficiency in SST, while temperatures were generally higher during fermentation in wooden boxes. With further refinements, the SST model may be useful in designing novel bioreactors for the optimisation of cocoa fermentation with starter cultures.