Curing of Cocoa Beans: Fine-Scale Monitoring of the Starter Cultures Applied and Metabolomics of the Fermentation and Drying Steps

Abstract

Starter culture-initiated cocoa fermentation processes can be applied to improve the quality of cured cocoa beans. However, an accurate monitoring of the microbial strains inoculated in fresh cocoa pulp-bean mass to assess their contribution to the cocoa bean curing process is still lacking. In the present study, eight different cocoa fermentation processes were carried out with Trinitario cocoa in vessels in Costa Rica to assess the contribution of two candidate yeast starter culture strains, namely Saccharomyces cerevisiae IMDO 050523 and Pichia kudriavzevii IMDO 020508, inoculated in combination with Limosilactobacillus fermentum IMDO 0611222 and Acetobacter pasteurianus IMDO 0506386. A multiphasic approach, consisting of culture-dependent selective plating and incubation, rRNA-PCR-DGGE community profiling of agar plate washes, and culture-independent high-throughput amplicon sequencing, combined with a metabolite target analysis of non-volatile and volatile organic compounds (VOCs), was performed on samples from the fermentation and/or drying steps. The different starter culture mixtures applied effectively steered the cocoa fermentation processes performed. Moreover, the use of an amplicon sequence variant (ASV) approach, aligning these ASVs to the whole-genome sequences of the inoculated strains, allowed the monitoring of these inoculated strains and their differentiation from very closely related variants naturally present in the background or spontaneous fermentation processes. Further, traits such as malolactic fermentation during the fermentation step and acetoin and tetramethylpyrazine formation during the drying step could be unraveled. Finally, the yeast strains inoculated influenced the substrate consumption and metabolite production during all starter culture-initiated fermentation processes. This had an impact on the VOC profiles of the cured cocoa beans. Whereas the P. kudriavzevii strain produced a wide range of VOCs in the cocoa pulp, the S. cerevisiae strain mostly influenced the VOC composition of the cured cocoa beans.

Cocoa By-Products: Characterization of Bioactive Compounds and Beneficial Health Effects

“…Of all the residues generated, the sweet and sour juice is drained as a result of the pulp liquefaction, due to the enzymatic action in pectin [ 100 , 101 ]. This translucent juice has chemical and sensory characteristics similar to the original pulp and is regionally called cocoa honey [ 100 ], also referred to in the literature as cocoa sweating and exudate [ 102 , 103 ].…”

Functional yeast starter cultures for cocoa fermentation

“…high acidity, high temperature, high ethanol concentration, high osmolarity), use of available carbohydrates and/or production of antifungal compounds determine their prevalence throughout the fermentation process. In this way, Hanseniaspora species are often found at the beginning of the fermentation process but disappear in favour of more ethanol-and temperature-tolerant yeast species, such as S. cerevisiae, P. kudriavzevii, P. manshurica or P. kluyveri (Daniel et al, 2009;De Vuyst & Leroy, 2020;De Vuyst & Weckx, 2016;Díaz-Muñoz et al, 2021;Ho et al, 2018;Koné et al, 2016;Lagunes Gálvez et al, 2007;Maura et al, 2016;Meersman et al, 2013;Nielsen et al, 2005;Papalexandratou et al, 2013).…”

“…However, sucrose can still be present upon cocoa pod opening and, therefore, yeast invertases (SUC genes in S. cerevisiae; Carlson & Botstein, 1983) are thought to play an important role in its conversion (De Vuyst & Weckx, 2016). Once the main fermentable monosaccharides become available, they are promptly used by highly fermentative yeasts to produce ethanol and carbon dioxide, sometimes leading to a slight temperature increase (Díaz-Muñoz et al, 2021;Ramos et al, 2014;Romanens et al, 2020). Additionally, the assimilation of glucose during anaerobic conditions leads to the production of glycerol to maintain the redox balance (Rodrigues et al, 2006).…”

“…Additionally, the assimilation of glucose during anaerobic conditions leads to the production of glycerol to maintain the redox balance (Rodrigues et al, 2006). This metabolite can migrate into the cocoa beans and may influence the taste and mouthfeel properties of chocolate (Díaz-Muñoz et al, 2021;Ho et al, 2014Ho et al, , 2018. Although there is a preference for glucose, most yeast species are capable of fermenting both glucose and fructose.…”

Investigating interspecific quorum sensing influence on cocoa fermentation quality through defined microbial cocktails

“…In other words, ASVs can separate sequencing errors from biological variations in sequences (Callahan et al, 2017) . In cocoa fermentation, ASVs were already employed by oligotyping methods (ASVs), which revealed a huge intraspecific variability of yeasts and bacteria (LAB and AAB) along fermentation (Díaz-Muñoz et al, 2021;Pacheco-Montealegre et al, 2020;Verce et al, 2021) . Therefore, to discriminate if the ASVs might represent intraspecific strains variability or if a given strain originated several ASVs, the comparison of gene markers (16S rRNA and ITS) against the ASVs provides a useful resolution (Verce et al, 2021;Díaz-Muñoz et al, 2021).…”