Detailed analyses of the bacterial populations in processed cocoa beans of different geographic origin, subject to varied fermentation conditions

• High-throughput sequencing (HTS) assessed bacterial diversity in four processed samples of cocoa beans from different geographic origins.
• Specific pattern of acetic acid bacteria (AAB) and lactic acid bacteria (LAB).
• HTS analyses of bacterial 16S DNA may represent a novel approach to define marker strains for geographic traceability.

The quality of chocolate is influenced by several parameters, one of which is bacterial diversity during fermentation and drying; a crucial factor for the generation of the optimal cocoa flavor precursors. Our understanding of the bacterial populations involved in chocolate fermentation can be improved by the use of high-throughput sequencing technologies (HTS), combined with PCR amplification of the 16S rRNA subunit. Here, we have conducted a high-throughput assessment of bacterial diversity in four processed samples of cocoa beans from different geographic origins. As part of this study, we also assessed whether different DNA extraction methods could affect the quality of our data. The dynamics of microbial populations were analyzed postharvest (fermentation and sun drying) and shipment, before entry to the industrial process. A total of 691,867 high quality sequences were obtained by Illumina MiSeq sequencing of the two bacterial 16S rRNA hypervariable regions, V3 and V4, following paired-read assembly of the raw reads. Manual curation of the 16S database allowed us to assign the correct taxonomic classifications, at species level, for 83.8% of those reads. This approach revealed a limited biodiversity and population dynamics for both the lactic acid bacteria (LAB) and acetic acid bacteria (AAB), both of which are key players during the acetification and lactic acid fermentation phases. Among the LAB, the most abundant species were Lactobacillus fermentum, Enterococcus casseliflavus, Weissella paramesenteroides, and Lactobacillus plantarum/paraplantarum. Among the AAB, Acetobacter syzygii, was most abundant, then Acetobacter senegalensis and Acetobacter pasteriuanus. Our results indicate that HTS approach has the ability to provide a comprehensive view of the cocoa bean microbiota at the species level.