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

Cristian Díaz-Muñoz,Dario Van De Voorde,Carlos Eduardo Hernandez,Andrea Comasio,Marko Verce,Stefan Weckx,Luc De Vuyst

doi:10.3389/fmicb.2020.616875

Cristian Díaz-Muñoz, Dario Van De Voorde + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2020.616875

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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.

Highlights

This study further aimed at confirming the prevalence of the inoculated yeast, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) strains at DNA level by comparing rRNA-polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) community profiling data, commonly used to follow up starter cultures, with data obtained through a high-throughput amplicon sequencingbased approach, in particular by refining the amplicon sequence variant (ASV) corresponding with species of the microbial genera involved and aligning them to the genomes of the inoculated strains
The pH of all cocoa fermentation processes increased from the start till 33–36 h of fermentation for the starter cultureinitiated ones (PC, adapted functional starter cultures (AFSCs) I, and AFSC II)
Concerning the Saccharomyces ASVs, a unique variant was present at high relative abundances only in those fermentation processes inoculated with S. cerevisiae IMDO 050523 (PC and AFSC II)

Summary

Introduction

Cocoa fermentation and drying are key steps in the curing of cocoa beans to make them ready for the production of chocolate (Schwan and Wheals, 2004; Saltini et al, 2013; De Vuyst and Weckx, 2016; Pereira et al, 2016; Ozturk and Young, 2017; Castro-Alayo et al, 2019; Figueroa-Hernández et al, 2019; De Vuyst and Leroy, 2020; Santander Muñoz et al, 2020). Cocoa fermentation processes are spontaneous, uncontrolled, on-farm processes (Schwan and Wheals, 2004; De Vuyst and Weckx, 2016; Pereira et al, 2016; Ozturk and Young, 2017; Figueroa-Hernández et al, 2019; De Vuyst and Leroy, 2020; Santander Muñoz et al, 2020) During the following aerobic phase, the microbial group of AAB oxidizes the ethanol produced by the yeasts into acetate These microbial activities cause an increase of the temperature of the fermenting cocoa pulp-bean mass, because of the exothermic effects of fermentation and, in particular, the ethanol oxidation and further overoxidation of acetate. The effect of the temperature and pH shift, together with the flow of ethanol and acetate from the pulp into the beans, is responsible for the killing of the seed embryo, the destruction of the seed structure, and the activation of endogenous enzymes that give the final flavor and color of the cured cocoa beans, in turn influencing the flavor characteristics of the chocolates produced from the concomitant roasted beans (Schwan and Wheals, 2004; Afoakwa et al, 2008; Saltini et al, 2013; Kongor et al, 2016; De Vuyst and Leroy, 2020; Santander Muñoz et al, 2020)

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