Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.

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.

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Among various lactic acid bacterial strains tested, cocoa-specific strains of Lactobacillus fermentum were best adapted to the cocoa pulp ecosystem. They fermented glucose to lactic acid and acetic acid, reduced fructose to mannitol, and converted citric acid into lactic acid and 2,3-butanediol.Fermented dry cocoa beans are the basic raw material for chocolate production. Cocoa beans are the seeds of the cocoa tree, Theobroma cacao L. The key microorganisms for successful cocoa bean fermentation processes are yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) (1, 3-5, 14, 18-21). Although the LAB species diversity involved in the onset of any spontaneous cocoa bean fermentation is wide, not much is known about how cocoa-specific LAB species, such as Lactobacillus fermentum, adapt physiologically and what their targeted functional roles are during fermentation (1,3,4,14,(19)(20)(21). The present study aimed at the kinetic investigation of carbohydrate fermentation and citric acid conversion by various LAB strains to unravel this.The LAB strains used throughout this study are listed in Table 1. Monoculture fermentations were performed in 1.5 liters of a cocoa pulp simulation medium (CPSM) for LAB (16) in Biostat B-DCU fermentors (Sartorius AG/B. Braun Biotech International, Melsungen, Germany) anaerobically for 48 h. Inoculum build-up, fermentor setup, online control of temperature (Table 2), pH profile, agitation, and sampling were as described previously (13, 16). All fermentations were performed in duplicate. The results and figures presented hereinafter are representative for both fermentations.During fermentation, bacterial growth (CFU per ml) was quantified through plating of 10-fold serial dilutions of the samples in saline (0.85% [wt/vol] NaCl solution) on CPSM agar (CPSM containing 1.5% [wt/vol] agar, pH 5.5) that was incubated at the appropriate fermentation temperature for 24 h. Metabolite concentrations were determined through high-performance anion-exchange chromatography using a standard addition protocol (glucose, fructose, mannitol, and citric acid) (15, 23) and high-performance liquid chromatography using external standards (lactic acid, acetic acid, and ethanol) (16). Cell count and metabolite (with external standards) measurements were performed on three independent samples. The errors on the measurements are represented as standard deviations. Gas chromatography (GC) was used for the qualitative determination of 2,3-butanediol and acetoin (15). Concentrations of carbon dioxide in the fermentor gas effluents were determined online through GC (13). The carbon recovery (CR, expressed as percentage) was calculated by dividing the total amount of carbon recovered in the metabolites by the total amount of carbon present in the carbon sources.All LAB strains tested were able to grow in CPSM ( Fig. 1 and T fermented fructose but not glucose and converted citric acid. Due to the initial low pH (3.5), Enterococcus casseliflavus M484 and Lactobacillus amylovorus DCE 471 were not able to...