Cured cocoa beans are obtained through a post-harvest, batchwise process of fermentation and drying carried out on farms in the equatorial zone. Fermentation of cocoa pulp-bean mass is performed mainly in heaps or boxes. It is made possible by a succession of yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) activities. Yeasts ferment the glucose of the cocoa pulp into ethanol, perform pectinolysis and produce flavour compounds, such as (higher) alcohols, aldehydes, organic acids and esters. LAB ferment the glucose, fructose and citric acid of the cocoa pulp into lactic acid, acetic acid, mannitol and pyruvate, generate a microbiologically stable fermentation environment, provide lactate as carbon source for the indispensable growth of AAB, and contribute to the cocoa and chocolate flavours by the production of sugar alcohols, organic acids, (higher) alcohols and aldehydes. AAB oxidize the ethanol into acetic acid, which penetrates into the bean cotyledons to prevent seed germination. Destruction of the subcellular seed structure in turn initiates enzymatic and non-enzymatic conversions inside the cocoa beans, which provides the necessary colour and flavour precursor molecules (hydrophilic peptides, hydrophobic amino acids and reducing sugars) for later roasting of the cured cocoa beans, the first step of the chocolate-making.

Functional role of yeasts, lactic acid bacteria and acetic acid bacteria in cocoa fermentation processes.

Formation of aromatic compounds precursors during fermentation of Criollo and Forastero cocoa.

High-throughput DNA sequencing (HTS) was used to investigate the microbial community of cocoa bean spontaneous fermentation in the Amazon biome. The characterization was described in three localities from Para state (Amazon biome) and one locality from Bahia state, the top Brazilian state for cocoa production, for comparison of the microbial community structure. The microbial community profile was determined through a metagenomics approach using the Illumina HiSeq platform. Our data showed that the microbial community is composed of a consortium consisting of yeast (i.e., Pichia and Candida), filamentous fungi (i.e., Aspergillus, Penicillium and Fusarium), lactic acid bacteria (i.e., Leuconostoc, Lactococcus, and Lactobacillus), acetic acid bacteria (i.e., Acetobacter and Gluconobacter), enterobacteria and bacilli. Interestingly, Acetobacter pasteurianus was found in higher relative abundance than expected, and three acetic acid bacteria species were identified for the first time in cocoa fermentation (i.e., Komagataeibacter hansenii, Roseomonas sp. and Acidisphaera sp.). Some microorganisms were only detected in Amazonian cocoa and not detected in Bahia cocoa, including six yeast species (i.e., Starmerella bacillaris, Candida temnochilae, Zygosaccharomyces bailii, Candida glabrata, Saccharomycopsis crataegensis and Saccharomycopsis amapae) and seven lactic acid bacteria (i.e., Lactococcus lactis, Fructobacillus pseudoficulneus, Weissella confusa, Pediococcus pentosaceus, Lactobacillus reuteri, Lactobacillus murinus and Lactobacillus pontis). This study provides the first description of the microbiome in cocoa fermentation from the Amazon region, and these data open new perspectives for exploring these microorganisms in biotechnology applications, as well as their use as a starter culture to obtain high-quality cocoa beans.

Determination of the microbial community in Amazonian cocoa bean fermentation by Illumina-based metagenomic sequencing

Forastero hybrid cocoa bean fermentations have been carried out in a box (B) and in a heap (H), with or without the inoculation of Saccharomyces cerevisiae and Torulaspora delbrueckii as starter cultures. The bacteria, yeasts, and microbial metabolites (volatile and nonvolatile organic compounds) were monitored during fermentation to assess the connection between microbiota and the release of metabolites during this process. The presence of starter cultures was detected, by means of culture-dependent analysis, during the first 2 days of both fermentations. However, no statistical difference was observed in any of the physicochemical or microbiological analyses. Plate counts revealed the dominance of yeasts at the beginning of both fermentations, and these were followed by acetic acid bacteria (AAB) and lactic acid bacteria (LAB). Hanseniaspora opuntiae, S. cerevisiae, Pichia pijperi, Acetobacter pasteurianus, and Lactobacillus fermentum were the most abundant operational taxonomic units (OTUs) during both fermentation processes (B and H), although different relative abundances were observed. Only the diversity of the fungal species indicated a higher level of complexity in the B fermentations than in the H fermentations (P < 0.05), as well as a statistically significant difference between the initially inoculated starter cultures (P < 0.01). However, the microbial metabolite analysis indicated different distributions of the volatile and nonvolatile compounds between the two procedures, that is, B and H (P < 0.05), rather than between the inoculated and noninoculated fermentations. The box fermentations showed faster carbohydrate metabolism and greater production of organic acid compounds, which boosted the formation of alcohols and esters, than did the heap fermentations. Overall, the microbial dynamics and associations between the bacteria, yeasts, and metabolites were found to depend on the type of fermentation.IMPORTANCE In spite of the limited effectiveness of the considered inoculated starter strains, this study provides new information on the microbial development of box and heap cocoa fermentations, under inoculated and noninoculated conditions, as we coupled yeast/bacterial amplicon-based sequencing data with microbial metabolite detection. The information so far available suggests that microbial communities have played an important role in the evolution of aroma compounds. Understanding the pathways that microorganisms follow during the formation of aromas could be used to improve the fermentation processes and to enhance chocolate quality.

Yurelkys Fernandez Maura

Papers

The environmental and intrinsic yeast diversity of Cuban cocoa bean heap fermentations