The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis," was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).Cocoa beans are the principal raw material for chocolate production (39,43,73,81). These seeds are derived from the fruit pods of the cocoa tree (Theobroma cacao L.), which is cultivated in plantations in the equatorial zone, with the Ivory Coast, Brazil, and Ghana as the major producers (2). The cocoa beans are embedded in a mucilaginous pulp inside the pods. Raw cocoa beans have an astringent, unpleasant taste and flavor and have to be fermented, dried, and roasted to obtain the desired characteristic cocoa flavor and taste (26, 73). The final chocolate flavor is influenced by the origin and cultivar of the cocoa beans, the on-the-farm fermentation and drying process, and the roasting and further processing performed by the cocoa and chocolate manufacturer (4,10,32,33,57,75).After removal of the beans from the pods, the first step in cocoa processing is a spontaneous 3-to 10-day fermentation of beans and pulp in heaps, boxes, baskets, or trays, of which spontaneous heap fermentation is the most widely used method in Ghana ...

BACKGROUND: Spontaneous cocoa bean fermentation is characterised by a succession of microbial activities. Cocoa flavour precursors are developed during fermentation and drying of cocoa beans. Polyphenols and alkaloids contribute to astringency and bitterness of cocoa and chocolate.

The fermentation of the Theobroma cacao beans, involving yeasts, lactic acid bacteria, and acetic acid bacteria, has a major influence on the quality of the resulting cocoa. An assessment of the microbial community of cocoa bean heap fermentations in Ghana resulted in 91 yeast isolates. These were grouped by PCR-fingerprinting with the primer M13. Representative isolates were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences and partial actin gene sequences leading to the detection of 15 species. Properties of importance for cocoa bean fermentation, namely sucrose, glucose, and citrate assimilation capacity, pH-, ethanol-, and heat-tolerance, were examined for selected isolates. Pichia kudriavzevii (Issatchenkia orientalis), Saccharomyces cerevisiae, and Hanseniaspora opuntiae formed the major components of the yeast community. Hanseniaspora opuntiae was identified conclusively for the first time from cocoa fermentations. Among the less frequently encountered species, Candida carpophila, Candida orthopsilosis, Kodamaea ohmeri, Meyerozyma (Pichia) caribbica, Pichia manshurica, Saccharomycodes ludwigii, and Yamadazyma (Pichia) mexicana were not yet documented from this substrate. Hanseniaspora opuntiae was preferably growing during the earlier phase of fermentation, reflecting its tolerance to low pH and its citrate-negative phenotype, while no specific temporal distribution was recognized for P. kudriavzevii and S. cerevisiae.

The influence of turning and environmental contamination on six spontaneous cocoa bean heap fermentations performed in Ghana was studied through a multiphasic approach, encompassing both microbiological (culture-dependent and culture-independent techniques) and metabolite target analyses. A sensory analysis of chocolate made from the fermented, dried beans was performed as well. Only four clusters were found among the isolates of acetic acid bacteria (AAB) identified: Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter senegalensis, and a potential new Acetobacter lovaniensis-like species. Two main clusters were identified among the lactic acid bacteria (LAB) isolated, namely, Lactobacillus plantarum and Lactobacillus fermentum. No differences in biodiversity of LAB and AAB were seen for fermentations carried out at the farm and factory sites, indicating the cocoa pod surfaces and not the general environment as the main inoculum for spontaneous cocoa bean heap fermentation. Turning of the heaps enhanced aeration and increased the relative population size of AAB and the production of acetic acid. This in turn gave a more sour taste to chocolate made from these beans. Bitterness was reduced through losses of polyphenols and alkaloids upon fermentation and cocoa bean processing.Raw cocoa beans have an astringent, unpleasant flavor and have to be fermented, dried, and roasted to obtain the desired characteristic cocoa flavor (27,30,41,45). Fermentation is hence the first step in cocoa powder and chocolate production. It is carried out spontaneously in heaps, boxes, baskets, or trays in cocoa-producing countries in the equatorial zone, led by the Ivory Coast, Brazil, and Ghana (1, 27). During the last decade, knowledge about the spontaneous cocoa bean fermentation process has been increasing (2,5,26,33,34,37). The microbiota involved in natural cocoa bean fermentation reflects the environmental factors (temperature, pH, and oxygen tension) and the metabolism of substrates of the cocoa bean pulp. This results in production times of significant amounts of ethanol, lactic acid, and acetic acid, representing a succession of yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) during the cocoa bean fermentation course (2, 5, 36-38).In the last 5 years, the microbiology and biochemistry of Ghanaian cocoa bean heap fermentation processes have been studied in detail (5,18,26,33,34). During early and mid-time spontaneous fermentation of freshly harvested pulp and cocoa beans, piled into a heap, yeasts produce ethanol under anaerobic conditions and cause depectinization of the pulp, enabling the pulp to flow away and air ingress (37, 38). Concerning microaerophilic LAB, citrate-fermenting, acid-tolerant, and ethanol-tolerant Lactobacillus plantarum and Lactobacillus fermentum strains dominate the spontaneous cocoa bean fermentation process (5, 34). Citrate and sugars are converted into acetic acid, lactic acid, and mannitol, enabling a slight increase of the pH of the pulp (5). During the aerobic phase, ...

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