No abstract

Forastero hybrid cocoa bean fermentations have been carried out in a box (B) and in a heap (H), with or without the inoculation of and 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). ,, ,, and 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 ( < 0.05), as well as a statistically significant difference between the initially inoculated starter cultures ( < 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 ( < 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. 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.

Microbial communities are responsible for the unique functional properties of chocolate. During microbial growth, several antimicrobial and antioxidant metabolites are produced and can influence human wellbeing. In the last decades, the use of starter cultures in cocoa fermentation has been pushed to improve nutritional value, quality, and the overall product safety. However, it must be noted that unpredictable changes in cocoa flavor have been reported between the different strains from the same species used as a starter, causing a loss of desirable notes and flavors. Thus, the importance of an accurate selection of the starter cultures based on the biogenic effect to complement and optimize chocolate quality has become a major interest for the chocolate industry. This paper aimed to review the microbial communities identified from spontaneous cocoa fermentations and focused on the yeast starter strains used in cocoa beans and their sensorial and flavor profile. The potential compounds that could have health-promoting benefits like limonene, benzaldehyde, 2-phenylethanol, 2-methylbutanal, phenylacetaldehyde, and 2-phenylethyl acetate were also evaluated as their presence remained constant after roasting. Further research is needed to highlight the future perspectives of microbial volatile compounds as biomarkers to warrant food quality and safety.

Next-generation sequencing has been used to strengthen knowledge about taxonomic diversity and ecology of fungi within food ecosystems. However, primer amplification and identification bias could edge our understanding into the fungal ecology. The aim of this study is to compare the performance of two primer pairs over two nuclear ribosomal RNA (rRNA) regions of the fungal kingdom, namely the ITS2 and 26S regions. Fermented cocoa beans were employed as biological material and the fungal ecology during fermentation was studied using amplicon-based sequencing tools, making use of a manually curated 26S database constructed in this study, and validated with SILVA database. To explore potential biases introduced by PCR amplification of fungal communities, a mock community of known composition was prepared and tested. The relative abundances observed for ITS2 suggest that species with longer amplification fragments are underestimated and concurrently species that render shorter amplification fragments are overestimated. However, this correlation between amplicon length and estimation is not valid for all the species analysed. Variability in the amplification lengths contributed to the preferential amplification phenomenon. DNA extracted from twenty fermented cocoa bean samples were used to assess the performance of the two target regions. Overall, the metataxonomic data set recovered similar taxonomic composition and provided consistent results in OTU richness among biological samples. However, 26S region provided higher alpha diversity index and greater fungal rRNA taxonomic depth and robustness results compared with ITS2. Based on the results of this study we suggest the use of the 26S region for targeting fungi. Furthermore, this study showed the efficacy of the manually curated reference database optimized for annotation of mycobiota by using the 26S as a gene target.

No abstract