Abstract

(Meta)genomics is a scientific field that enables determination of the genetic material of microorganisms, which has led to new research opportunities in microbiology. The present study focused on the (meta)genomics of the cocoa bean fermentation process as a case study.
The fermentation of raw cocoa beans is an important step in the production of chocolate, during which the mucilaginous pulp surrounding the cocoa beans is removed, important colour and flavour precursors in the beans are formed, and the seed embryo is killed. Insights into the fermentation ecosystem composition and the functional roles of the microorganisms involved is of great interest, as this will provide knowledge to better select functional starter culture strains to perform controlled cocoa bean fermentation processes.
First, a combination of several similarity-based and composition-based taxonomic profiling tools was applied on metagenomic sequence data of the microbial community DNA of a single cocoa bean fermentation process sample from Brazil. Overall, this approach revealed a wider bacterial and fungal diversity than previously reported and identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing microbial species. Secondly, a functional analysis of the metagenomic sequence data of this single sample was carried out to gain insight into the metabolic capacities of the different microorganisms involved. By reconstructing bacterial meta-pathways, their association with the microbial members of the cocoa bean fermentation ecosystem was revealed.
Whole-genome shotgun sequencing and in-depth functional analysis of the lactic acid bacteria strains Lactobacillus fermentum 222 and Lactobacillus plantarum 80 revealed functional roles of these strains during the cocoa bean fermentation process. To investigate the mechanisms that enable Acetobacter pasteurianus 386B to dominate the cocoa bean fermentation process, a (comparative) genome sequence analysis using whole-genome mate-pair sequencing was applied. This revealed important characteristics for this strain, including the presence of strain-specific genes such as a gene coding for an endopolygalacturonase and mechanisms involved in tolerance towards various stress conditions. The genome sequences of Acetobacter ghanensis LMG 23848T and Acetobacter senegalensis 108B, species that are often found in the beginning of cocoa bean fermentation processes, were determined and analysed using whole-genome mate-pair sequencing. This approach revealed unique functional properties that are of interest for the cocoa bean fermentation process.
Overall, this work extended the knowledge of the cocoa bean fermentation ecosystem by using a metagenomic sequencing approach, in particular towards the microorganisms involved and the underlying mechanisms of their metabolic features. In addition, genome-based analysis of five (candidate) functional starter culture strains of potential application in the cocoa bean fermentation process was performed and proved to be a powerful tool to facilitate the selection of appropriate starter culture strains for controlled cocoa bean fermentation processes.
Date of Award6 Mar 2015
Original languageEnglish

Keywords

  • cocoa beans
  • (meta)genomics

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