Abstract
Cocoa bean fermentation is an important microbial process, where most metabolites that affect chocolate quality and aroma are generated. Production of reproducible high-quality beans is a major challenge because most fermentations occur in open containers with a lack of variable control. Here we present a study that aims to identify the effect of farm protocols, climate, and bean mass exposure, in the dynamics and composition of microbial communities. Using high-throughput sequencing of molecular markers for bacteria and yeasts, complemented with culture-based methods, we evaluated the microbial diversity and dynamics associated to spontaneous cocoa fermentation in two distinct agro-ecological zones in Colombia. The bacterial communities were classified at two levels of evolutionary relationship, at a coarse resolution (OTU-level) and at a finer resolution (oligotype-level). A total of six bacterial OTUs were present in both farms, following a microbial succession that starts with the Enterobacteraceae family (one OTU), transitioning to the Lactobacillaceae family (three OTUs), and finishing with Acetobacteraceae family (two OTUs). When undesirable practices were done, OTUs were observed at unexpected moments during the fermentation. At a finer taxonomic resolution, 48 oligotypes were identified, with 46 present in both farms. These oligotypes have different patterns of prevalence. In the case of Lactobacillaceae a high evenness was observed among oligotypes. In contrast, for Enterobacteraceae and Acetobacteraceae a high dominance of one or two oligotypes was observed, these oligotypes were the same for both farms, despite geographic location and season of sampling. When the overall fermentations were compared using correlations matrices of oligotypes abundance, they show a clear clustering by farm, suggesting that farm protocols generate a unique fingerprint in the dynamics and interactions of the microbial communities. The comparison between the upper and middle layers of the bean mass showed that environmental exposure affects the paces at which ecological successions occur, and therefore, is an important source of cocoa quality heterogeneity. In conclusion, the results presented here showed that the dynamics of microbial fermentation can be used to identify the sources of variability and evidence the need for better fermentation technologies that favor the production of reproducible high-quality cocoa beans.
Highlights
Cocoa bean fermentation is a spontaneous process where microorganisms transform physically and biochemically all bean structures
Cocoa plantations in Colombia are distributed throughout the country and each productive region is delimited in agroecological zones (AEZ) based on climatic conditions, topography, and soil composition
The microbial composition and succession associated to the cocoa fermentation is in agreement with the reports obtained with methodologies such as PCR-DGGE and RFLP (Cocolin et al, 2013; AranaSánchez et al, 2015), in particular, identifying bacterial Operational Taxonomic Units (OTUs) belonging to the Enterobacteriaceae family, Lactobacillaceae family, Lactobacillus sp., Fructobacillus sp., Acetobacteraceae family and Acetobacter sp., and yeast OTUs belonging to Hanseniaspora opuntiae, Picha sp., Pichia kudriavzevi, and Wickerhamomyces pijperi
Summary
Cocoa bean fermentation is a spontaneous process where microorganisms transform physically and biochemically all bean structures (husk, mucilage, cotyledon, and embryo). The fermentation process lasts 3–7 days depending on variables such as the genetic origin of the seed, the agro-ecological conditions and the protocol used These microbial communities are composed mainly by yeasts, Enterobacteriaceae related bacteria (ENT group), Lactic Acid Bacteria (LAB group), and Acid Acetic Bacteria (AAB group) (Camu et al, 2007; Okiyama et al, 2017). LAB degrades the carbohydrate-rich pulp producing mainly lactic acid, AAB oxidizes ethanol into acetic acid (Kongor et al, 2016; Ozturk and Young, 2017) In this last step, the production of acetic acid lowers pH and increases bean mass temperature (40–50◦C), these conditions affect the seed and cause its death, promoting the subsequent release of the flavor precursors associated with chocolate quality (Schwan et al, 2014; De Vuyst and Weckx, 2016; Illeghems et al, 2016). Despite the understanding of the fermentation succession and function, the production of reproducible highquality beans still a major challenge for farmers, because most fermentations occur in open containers with a lack of variability control and without local standardization of fermentation protocols
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