The microbiological and metabolic outcomes of good cocoa fermentation practices can be standardized and influenced through the addition of starter culture mixtures composed of yeast and bacterial strains. The present study performed two spontaneous and 10 starter culture-initiated (SCI) cocoa fermentation processes (CFPs) in Costa Rica with local Trinitario cocoa. The yeast strains Saccharomyces cerevisiae IMDO 050523, Hanseniaspora opuntiae IMDO 020003, and Pichia kudriavzevii IMDO 060005 were used to compose starter culture mixtures in combination with the lactic acid bacterium strain Limosilactobacillus fermentum IMDO 0611222 and the acetic acid bacterium strain Acetobacter pasteurianus IMDO 0506386. The microbial community and metabolite dynamics of the cocoa pulp-bean mass fermentation, the metabolite dynamics of the drying cocoa beans, and the volatile organic compound (VOC) profiles of the chocolate production were assessed. An amplicon sequence variant approach based on full-length 16S rRNA gene sequencing instead of targeting the V4 region led to a highly accurate monitoring of the starter culture strains added, in particular the Liml. fermentum IMDO 0611222 strain. The latter strain always prevailed over the background lactic acid bacteria. A similar approach, based on the internal transcribed spacer (ITS1) region of the fungal rRNA transcribed unit, was used for yeast strain monitoring. The SCI CFPs evolved faster when compared to the spontaneous ones. Moreover, the yeast strains applied did have an impact. The presence of S. cerevisiae IMDO 050523 was necessary for successful fermentation of the cocoa pulp-bean mass, which was characterized by the production of higher alcohols and esters. In contrast, the inoculation of H. opuntiae IMDO 020003 as the sole yeast strain led to underfermentation and a poor VOC profile, mainly due to its low competitiveness. The P. kudriavzevii IMDO 060005 strain tested in the present study did not contribute to a richer VOC profile. Although differences in VOCs could be revealed in the cocoa liquors, no significant effect on the final chocolates could be obtained, mainly due to a great impact of cocoa liquor processing during chocolate-making. Hence, optimization of the starter culture mixture and cocoa liquor processing seem to be of pivotal importance.

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Van de Voorde D, Díaz-Muñoz C, Hernandez CE, Weckx S, De Vuyst L

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