Microbial ecology of the cocoa chain : quality aspects and insight into heat-resistant bacterial spores

Cocoa beans (<em>Theobroma cacao</em>L.) are the basis for chocolate and cocoa powder production. The first step in the production of these food products consists of a spontaneous fermentation of the beans in the tropical producing countries, in order to allow the formation of the essential precursor compounds of the cocoa flavour. Following this stage, cocoa beans are industrially transformed into a range of different products. In the first part of this thesis, the state of literature of cocoa bean fermentation was revisited with the aim of interlinking post-harvest processing practices and characteristics of the fermenting microbiota, with the quality of commercial cocoa beans obtained. This literature survey resulted in the identification of a number of research needs that can be used to design agricultural and operational measures, towards improving the quality of commercial cocoa beans. The second part of this thesis addressed the occurrence, levels and diversity of bacterial Thermoresistant Spores (ThrS) in commercial cocoa powder and in cocoa being transformed to cocoa powder in an industrial setting. Thermoresistant Spores were defined as spores which survive a heat-treatment of at least 100°C for 10 min, as opposed to the standard treatment at 80°C for 10 min for Total Spores (TS) determination. It was found that strains of <em>Bacillus licheniformis</em> and of the <em>B. subtilis</em> complex were the predominant species in the ThrS library and that strains of <em>B. subtilis</em> complex, including <em>B. subtilis</em> subsp. <em>subtilis </em>formed the most heat-resistant spores <em>in situ</em> and <em>in vitro</em>. Interestingly, a high heat-resistant spore phenotype was associated with diverse genotypes. In addition, a strategy was devised in order to identify biomarkers of spore heat-resistance. The comparison of physicochemical and ultrastructural properties among spores of different heat-resistance allowed the identification of manganese as a potential physiological biomarker for <em>B. subtilis</em> subsp. <em>subtilis </em>spores heat-resistance. On the other hand, a study of various genomic features conducted across strains of the phylum of <em>Firmicutes</em>, did not yield genomic biomarkers related to spore high heat-resistant phenotype, suggesting the primary role of spore physicochemical composition and ultrastructural characteristics as determinant factors of spore differential heat-resistance. In conclusion, this study presents suggestions to delve into the functionality of the microbiota involved in cocoa bean fermentation and to improve the quality of commercial cocoa beans. Furthermore, it contributes towards the understanding of the ecology of high heat-resistant aerobic bacterial spores in the cocoa chain and provides knowledge regarding their heat-resistance. The data gathered in this thesis are useful input for predictive modelling and to assist in the production of safe and high quality food.