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).

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Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana.

Cocoa seeds and pulp were fermented for 144 h, followed by natural drying. The tegument was removed and the cotyledons were broken into nibs which were roasted at 150 °C for 30 min. Non-fermented material, material fermented for 24, 48 and 72 h, material fermented for 144 h and then dried, and also the roasted nibs, were all prepared for chemical and microscopic analyses. Light microscopy revealed the presence of anionic and cationic residues and of neutral sugars. During fermentation there was a reduction in the cytoplasmic content of phenolic compounds and in the number of protein bodies. The cell wall showed a reduction in anionic residues and a loss of crystallinity. These alterations were maximum after 72 h. Drying and roasting increased the number of damaged cells and reduced the amount of cytoplasmic material. The chemical analyses generally confirmed the microscopy results. The concentration of amino-terminal groups and total free amino acids increased during fermentation (up to 72 h), but returned to the initial values after roasting. The principal chemical changes were related to reducing sugars, free amino acids, proteins and phenols, and PCA was suggested as a useful tool to compare different samples. Microscopic analysis revealed the degradation of protein and phenolic bodies and cellular damage during roasting.

© 2000 Society of Chemical Industry

Structural and chemical changes in cocoa (Theobroma cacao L) during fermentation, drying and roasting

https://backend.orbit.dtu.dk/ws/files/20803556/Optimizing_chocolate_production.pdf

Optimizing chocolate production through traceability: A review of the influence of farming practices on cocoa bean quality

In-vitro formation of cocoa-specific aroma precursors: aroma-related peptides generated from cocoa-seed protein by co-operation of an aspartic endoprotease and a carboxypeptidase

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.

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Influence of Turning and Environmental Contamination on the Dynamics of Populations of Lactic Acid and Acetic Acid Bacteria Involved in Spontaneous Cocoa Bean Heap Fermentation in Ghana

R. Lieberei

Papers

Kinetics of the formation of free amino acids in cocoa seeds during fermentation