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:Taxonomic and functional analysis of Costa Rican cocoa bean fermentation ecosystems through shotgun metagenomic and metatranscriptomics sequencing.
Project description:Cocoa is a crop of cultural, nutritional and social importance in Latin America. Cocoa production is mainly supported by smallholders and is central for the food security of these farmer families. Despite being part of their everyday diet and an important source of antioxidants and other healthy bioactive compounds, cocoa cropping is also a solid source of stable incomes supporting the livelihood of farmer families. Water deficit stress is one of the main limiting factors affecting crop yields. The ability of plants to tolerate or recover from the effects associated with this abiotic stress is of immense importance in terms of improvement in the context of climate change. Despite the emergence of functional genomics and phenotyping tools to approach these responses, many of these mechanisms are still little understood for many tropical food crops such as cocoa. For a transcriptomic analysis were selected 2 cocoa genotypes, from a hydric stress assay established in a greenhouse. 5-month-old plants of T. cacao of the genotypes EET 8 and TSH565 were tested for water deficit trial. A divided plot experimental design was applied: the hydric state of the 2 genotypes was evaluated with two levels: field capacity and water deficit by irrigation suspension during a period that generates severe stress (Leaf Water Potential of -3.0 Mpa). The irrigation suspension lasted 52 days.
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.
Project description:Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin fraction. Although both these classes of proteins have been fully characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nanoUHPLC-ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that a total of 906 proteins could be identified using a species-specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. However, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non-specific databases confirmed that using a species-specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools which can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins.