A Combined Metagenomics and Metatranscriptomics Approach to Unravel Costa Rican Cocoa Box Fermentation Processes Reveals Yet Unreported Microbial Species and Functionalities

Abstract

Cocoa fermentation is the first step in the post-harvest processing chain of cocoa and is important for the removal of the cocoa pulp surrounding the beans and the development of flavor and color precursors. In the present study, metagenomic and metatranscriptomic sequencing were applied to Costa Rican cocoa fermentation processes to unravel the microbial diversity and assess the function and transcription of their genes, thereby increasing the knowledge of this spontaneous fermentation process. Among 97 genera found in these fermentation processes, the major ones were Acetobacter, Komagataeibacter, Limosilactobacillus, Liquorilactobacillus, Lactiplantibacillus, Leuconostoc, Paucilactobacillus, Hanseniaspora, and Saccharomyces. The most prominent species were Limosilactobacillus fermentum, Liquorilactobacillus cacaonum, and Lactiplantibacillus plantarum among the LAB, Acetobacter pasteurianus and Acetobacter ghanensis among the AAB, and Hanseniaspora opuntiae and Saccharomyces cerevisiae among the yeasts. Consumption of glucose, fructose, and citric acid, and the production of ethanol, lactic acid, acetic acid, and mannitol were linked to the major species through metagenomic binning and the application of metatranscriptomic sequencing. By using this approach, it was also found that Lacp. plantarum consumed mannitol and oxidized lactic acid, that A. pasteurianus degraded oxalate, and that species such as Cellvibrio sp., Pectobacterium spp., and Paucilactobacillus vaccinostercus could contribute to pectin degradation. The data generated and results presented in this study could enhance the ability to select and develop appropriate starter cultures to steer the cocoa fermentation process toward a desired course.

Microbial Diversity and Contribution to the Formation of Volatile Compounds during Fine-Flavor Cacao Bean Fermentation

“…Numerous researchers have characterized the microbial diversity of cacao bean fermentation processes by culture-dependent and next-generation sequencing (NGS) methods, such as metabarcoding sequencing. NGS stands out because of its sensitivity in community analysis and throughput [ 13 ], and was able to reveal a defined succession of yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) [ 7 , 14 , 15 , 16 , 17 , 18 , 19 ] during the cacao fermentation process. The microbiota of cacao bean fermentation and their metabolic activities can influence the production of volatile compounds that are related to fruity and floral aromas, as well as the removal of undesirable characteristics, such as astringency and bitterness [ 3 , 20 , 21 ].…”

Investigating interspecific quorum sensing influence on cocoa fermentation quality through defined microbial cocktails

“…In other words, ASVs can separate sequencing errors from biological variations in sequences (Callahan et al, 2017) . In cocoa fermentation, ASVs were already employed by oligotyping methods (ASVs), which revealed a huge intraspecific variability of yeasts and bacteria (LAB and AAB) along fermentation (Díaz-Muñoz et al, 2021;Pacheco-Montealegre et al, 2020;Verce et al, 2021) . Therefore, to discriminate if the ASVs might represent intraspecific strains variability or if a given strain originated several ASVs, the comparison of gene markers (16S rRNA and ITS) against the ASVs provides a useful resolution (Verce et al, 2021;Díaz-Muñoz et al, 2021).…”

“…plantarum , Lm. fermentum and Acetobacter species as core members □(Lefeber et al, 2011; Meersman et al, 2013; Papalexandratou et al, 2011; Verce et al, 2021)□, with evidence to Lm. fermentum to the detriment of Lp.…”

“…Total RNA extraction was performed according to the protocol described by Verce et al (2021) , with modifications. Briefly, three cocoa beans were homogenised in 30 ml of RNAprotect (Qiagen, Venio, The Netherlands) in a 50 ml falcon and vortexed for 30 seconds to disrupt the mucilaginous pulp from the seeds.…”

Microbiomes Associated With the Surfaces of Northern Argentinian Fruits Show a Wide Species Diversity

“…Two DNA extraction protocols (tackling both live and intact dead cells) were applied, as samples were taken in three different years and hence processed with adapted protocols. For the white guava and Japanese medlar flowers, a method based on different steps to break the cells, with centrifugation between each step, was used ( Vermote et al, 2018 ), whereas for the passion fruit and papaya samples the same method with an additional chitinase treatment step and with centrifugation only at the end of these steps was applied ( De Bruyn et al, 2017 ; Verce et al, 2021 ). Briefly, several enzymatic lysis steps (with chitinase, mutanolysin, lysozyme, lyticase, Zymolyase, and proteinase K) were applied, followed by chemical treatment with sodium dodecyl sulphate, and mechanical disruption with glass beads.…”