Microbiota dynamics and diversity at different stages of industrial processing of cocoa beans into cocoa powder.

We sampled a cocoa powder production line to investigate the impact of processing on the microbial community size and diversity at different stages. Classical microbiological methods were combined with 16S rRNA gene PCR-denaturing gradient gel electrophoresis, coupled with clone library construction, to analyze the samples. Aerobic thermoresistant spores (ThrS) (100°C; 10 min) were also isolated and characterized (identity, genetic diversity, and spore heat resistance), in view of their relevance to the quality of downstream heat-treated cocoa-flavored drinks. In the nibs (broken, shelled cocoa beans), average levels of total aerobic microorganisms (TAM) (4.4 to 5.6 log CFU/g) and aerobic total spores (TS) (80°C; 10 min; 4.3 to 5.5 log CFU/g) were significantly reduced (P < 0.05) as a result of alkalizing, while fungi (4.2 to 4.4 log CFU/g) and Enterobacteriaceae (1.7 to 2.8 log CFU/g) were inactivated to levels below the detection limit, remaining undetectable throughout processing. Roasting further decreased the levels of TAM and TS, but they increased slightly during subsequent processing. Molecular characterization of bacterial communities based on enriched cocoa samples revealed a predominance of members of the Bacillaceae, Pseudomonadaceae, and Enterococcaceae. Eleven species of ThrS were found, but Bacillus licheniformis and the Bacillus subtilis complex were prominent and revealed great genetic heterogeneity. We concluded that the microbiota of cocoa powder resulted from microorganisms that could have been initially present in the nibs, as well as microorganisms that originated during processing. B. subtilis complex members, particularly B. subtilis subsp. subtilis, formed the most heat-resistant spores. Their occurrence in cocoa powder needs to be considered to ensure the stability of derived products, such as ultrahigh-temperature-treated chocolate drinks.

Theobroma cacao L., "The food of the Gods": quality determinants of commercial cocoa beans, with particular reference to the impact of fermentation.

The quality of commercial cocoa beans, the principal raw material for chocolate production, relies on the combination of factors that include the type of planting material, the agricultural practices, and the post-harvest processing. Among these, the fermentation of the cocoa beans is still the most relevant since it is the process whereby the precursors of the cocoa flavor arise. The formation of these precursors depends on the activity of different microbial groups on the beans pulp. A comparison of fermentations in different countries showed that a well-defined microbial succession does not always take place and that the role of Bacillus spp. in this process remains unclear. Considering the overriding importance of the fermentation to achieve high quality commercial cocoa beans, we discuss the need of addressing the impact of the farming system, the ripeness state of the pods, and the role of microbial interactions on the fermentation in future research. In addition, the problem of high acidification cocoa beans, aspects dealing with the volatile fraction of the flavor, and the cocoa butter properties, all were identified as critical aspects that need further investigation. The standardization of the microbiological methods and the application of metagenomic approaches would magnify the knowledge in this domain.

Microbiota of cocoa powder with particular reference to aerobic thermoresistant spore-formers.

The microbiological criteria of commercial cocoa powder are defined in guidelines instituted by the cocoa industry. Twenty-five commercial samples were collected with the aim of assessing the compliance with the microbiological quality guidelines and investigating the occurrence and properties of aerobic Thermoresistant Spores (ThrS). Seventeen samples complied with the guidelines, but one was positive for Salmonella, five for Enterobacteriaceae and two had mould levels just exceeding the maximum admissible level. The treatment of the cocoa powder suspensions from 100 °C to 170 °C for 10 min, revealed the presence of ThrS in 36% of the samples. In total 61 ThrS strains were isolated, of which the majority belonged to the Bacillus subtilis complex (65.6%). Strains resporulation and spore crops inactivation at 110 °C for 5 min showed a wide diversity of heat-resistance capacities. Amplified fragment length polymorphism analysis revealed not only a large intraspecies diversity, but also different clusters of heat-resistant spore-forming strains. The heat-resistance of spores of six B. subtilis complex strains was further examined by determination of their D and z-values. We concluded that B. subtilis complex spores, in particular those from strain M112, were the most heat-resistant and these may survive subsequent preservation treatments, being potentially problematic in food products, such as chocolate milk.