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The composition of cocoa pulp simulation media (PSM) was optimized with species-specific strains of lactic acid bacteria (PSM-LAB) and acetic acid bacteria (PSM-AAB). Also, laboratory fermentations were carried out in PSM to investigate growth and metabolite production of strains of Lactobacillus plantarum and Lactobacillus fermentum and of Acetobacter pasteurianus isolated from Ghanaian cocoa bean heap fermentations, in view of the development of a defined starter culture. In a first step, a selection of strains was made out of a pool of strains of these LAB and AAB species, obtained from previous studies, based on their fermentation kinetics in PSM. Also, various concentrations of citric acid in the presence of glucose and/or fructose (PSM-LAB) and of lactic acid in the presence of ethanol (PSM-AAB) were tested. These data could explain the competitiveness of particular cocoa-specific strains, namely, L. plantarum 80 (homolactic and acid tolerant), L. fermentum 222 (heterolactic, citric acid fermenting, mannitol producing, and less acid tolerant), and A. pasteurianus 386B (ethanol and lactic acid oxidizing, acetic acid overoxidizing, acid tolerant, and moderately heat tolerant), during the natural cocoa bean fermentation process. For instance, it turned out that the capacity to use citric acid, which was exhibited by L. fermentum 222, is of the utmost importance. Also, the formation of mannitol was dependent not only on the LAB strain but also on environmental conditions. A mixture of L. plantarum 80, L. fermentum 222, and A. pasteurianus 386B can now be considered a mixed-strain starter culture for better controlled and more reliable cocoa bean fermentation processes.

Four cocoa-specific acetic acid bacterium (AAB) strains, namely, Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848 T , Acetobacter fabarum LMG 24244 T , and Acetobacter senegalensis 108B, were analyzed kinetically and metabolically during monoculture laboratory fermentations. A cocoa pulp simulation medium (CPSM) for AAB, containing ethanol, lactic acid, and mannitol, was used. All AAB strains differed in their ethanol and lactic acid oxidation kinetics, whereby only A. pasteurianus 386B performed a fast oxidation of ethanol and lactic acid into acetic acid and acetoin, respectively. Only A. pasteurianus 386B and A. ghanensis LMG 23848 T oxidized mannitol into fructose. Coculture fermentations with A. pasteurianus 386B or A. ghanensis LMG 23848T and Lactobacillus fermentum 222 in CPSM for lactic acid bacteria (LAB) containing glucose, fructose, and citric acid revealed oxidation of lactic acid produced by the LAB strain into acetic acid and acetoin that was faster in the case of A. pasteurianus 386B. A triculture fermentation with Saccharomyces cerevisiae H5S5K23, L. fermentum 222, and A. pasteurianus 386B, using CPSM for LAB, showed oxidation of ethanol and lactic acid produced by the yeast and LAB strain, respectively, into acetic acid and acetoin. Hence, acetic acid and acetoin are the major end metabolites of cocoa bean fermentation. All data highlight that A. pasteurianus 386B displayed beneficial functional roles to be used as a starter culture, namely, a fast oxidation of ethanol and lactic acid, and that these metabolites play a key role as substrates for A. pasteurianus in its indispensable crossfeeding interactions with yeast and LAB during cocoa bean fermentation.

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