Project description:Taxonomic and functional analysis of Costa Rican cocoa bean fermentation ecosystems through shotgun metagenomic and metatranscriptomics sequencing.
Project description:Fermentation is essential for cocoa flavour development, as during this process key flavour precursors are formed from the degradation of the major cocoa bean storage proteins. This work characterises the peptide and protein profiles of Theobroma cacao beans of the genotype IMC 67 at different fermentation stages, using the Styrofoam box fermentation method and employing UHPLC-ESI MS/MS for the analysis of peptides and proteins extracted from the beans. A total of 1058 endogenous peptides were identified and quantified over four fermentation time points. The majority of these peptides were formed during the early stage of fermentation and originated predominantly from the proteolysis of two storage proteins - vicilin and a 21 kDa albumin. The changes in the peptide profile over fermentation were subsequently evaluated, and potential markers for assessing the degree of fermentation were identified. In particular, changes of the relative abundance of the major cocoa proteins detected can be proposed as potential markers for the fermentation stage. Furthermore, PCA analysis of both the peptidomic and proteomic data has allowed differentiation of beans at different fermentation stages.
Project description:Cocoa bean fermentation relies on the sequential activation of several microbial populations, triggering a temporal pattern of biochemical transformations. Understanding this complex process is of tremendous importance as it is known to form the precursors of the resulting chocolate's flavour and taste. At the same time, cocoa bean fermentation is one of the least controlled processes in the food industry. Here, a quantitative model of cocoa bean fermentation is constructed based on available microbiological and biochemical knowledge. The model is formulated as a system of coupled ordinary differential equations with two distinct types of state variables: (i) metabolite concentrations of glucose, fructose, ethanol, lactic acid and acetic acid and (ii) population sizes of yeast, lactic acid bacteria and acetic acid bacteria. We demonstrate that the model can quantitatively describe existing fermentation time series and that the estimated parameters, obtained by a Bayesian framework, can be used to extract and interpret differences in environmental conditions. The proposed model is a valuable tool towards a mechanistic understanding of this complex biochemical process, and can serve as a starting point for hypothesis testing of new systemic adjustments. In addition to providing the first quantitative mathematical model of cocoa bean fermentation, the purpose of our investigation is to show how differences in estimated parameter values for two experiments allow us to deduce differences in experimental conditions.
Project description:Amongst other compounds, cocoa flavour is dependent on peptides that are formed during fermentation of cocoa beans from proteins such as albumin and vicilin. In this study the proteomic profiles of cocoa beans from four genotypes (ICS 1, ICS 39, IMC 67 and SCA 6) with different genetic background and flavour profiles have been analysed by employing a bottom-up label free LC-MS/MS approach. From a total of 430 identified proteins, 250 proteins were found significantly differentially expressed among the four cocoa genotypes analysed. Of these, 61 proteins with a fold change of 2 or more were further investigated, showing that the majority is involved in stress response. Furthermore, several of these 61 proteins could also be linked to oxidation-reduction processes. PCA analysis allowed a clear separation of the genotypes based on their proteomic profile, with an aminohydrolase and a sulphite oxidase greatly contributing to the separation. Aspartyl protease was more abundant in the genotypes ICS 1 and ICS compared to IMC 67, while a serine carboxypeptidase was significantly more expressed in the genotype ICS 39 in comparison with the other genotypes. Both these enzymes catalyse the degradation of storage proteins during fermentation. A Beta-amylase, an enzyme which catalyses the release of maltose was detected at a significantly higher level in the genotype SCA 6 compared to ICS 1 and IMC 67. Two Amine oxidases were significantly more abundant in the genotype SCA 6 compared to ICS 39, and in the genotype ICS 1 versus ICS 39, while two alcohol dehydrogenase were higher expressed in the genotype SCA 6 compared to IMC 67. These enzyme catalyse oxidation of amines and alcohols with release of aldehydes and ketones. The data shows that UHPLC-MS/MS can be employed to differentiate cocoa beans from various varieties, and thus in theory be linked to differences in their flavour profile.