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. 2021 Nov 9;11(1):21904.
doi: 10.1038/s41598-021-01427-8.

Dissecting fine-flavor cocoa bean fermentation through metabolomics analysis to break down the current metabolic paradigm

Fabio Herrera-Rocha #  1 , Mónica P Cala #  2 , Jenny Lorena Aguirre Mejía  3 , Claudia M Rodríguez-López  3 , María José Chica  3 , Héctor Hugo Olarte  3 , Miguel Fernández-Niño  4   5 , Andrés Fernando Gonzalez Barrios  6
Affiliations

Dissecting fine-flavor cocoa bean fermentation through metabolomics analysis to break down the current metabolic paradigm

Fabio Herrera-Rocha et al. Sci Rep. .

Abstract

Cocoa fermentation plays a crucial role in producing flavor and bioactive compounds of high demand for food and nutraceutical industries. Such fermentations are frequently described as a succession of three main groups of microorganisms (i.e., yeast, lactic acid, and acetic acid bacteria), each producing a relevant metabolite (i.e., ethanol, lactic acid, and acetic acid). Nevertheless, this view of fermentation overlooks two critical observations: the role of minor groups of microorganisms to produce valuable compounds and the influence of environmental factors (other than oxygen availability) on their biosynthesis. Dissecting the metabolome during spontaneous cocoa fermentation is a current challenge for the rational design of controlled fermentations. This study evaluates variations in the metabolic fingerprint during spontaneous fermentation of fine flavor cocoa through a multiplatform metabolomics approach. Our data suggested the presence of two phases of differential metabolic activity that correlate with the observed variations on temperature over fermentations: an exothermic and an isothermic phase. We observed a continuous increase in temperature from day 0 to day 4 of fermentation and a significant variation in flavonoids and peptides between phases. While the second phase, from day four on, was characterized for lower metabolic activity, concomitant with small upward and downward fluctuations in temperature. Our work is the first to reveal two phases of metabolic activity concomitant with two temperature phases during spontaneous cocoa fermentation. Here, we proposed a new paradigm of cocoa fermentation that considers the changes in the global metabolic activity over fermentation, thus changing the current paradigm based only on three main groups of microorganism and their primary metabolic products.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Comparison of total ion chromatogram of metabolic profiles for day 0 (D0) and day 8 (D8) during fermentation of fine-flavor cocoa. (A) Metabolic fingerprinting (MF) by LC–MS( +); (B) MF by LC–MS(-); (C) MF by GC–MS.
Figure 2
Figure 2
PCA score plots for all fermentation samples. (A) MF by LC–MS( +): R2(cum): 0.875, Q2 (cum): 0.826; (B) MF by LC–MS(-): R2(cum): 0.876, Q2 (cum): 0.77; (C) MF by GC–MS: R2(cum): 0.724, Q2 (cum): 0.229.
Figure 3
Figure 3
PLS-DA models for all cocoa fermentation samples and the proposed metabolic phases. ALL DAYS: (A) MF by LC–MS( +): R2(cum): 0.838, Q2 (cum): 0.137, cv-anova: 0.995; (B) MF by LC–MS(-): R2(cum): 0.696, Q2 (cum): 0.083, cv-anova: 0.993; (C) MF by GC–MS: R2(cum): 0.442, Q2 (cum): 0.084, cv-anova: 0.951. PHASES: (D) MF by LC–MS( +): R2(cum): 0.907, Q2 (cum): 0.899, cv-anova: 6.296e−07; (E) MF by LC–MS(-): R2(cum): 0.903, Q2 (cum): 0.804, cv-anova: 0.00053; (F) MF by GC–MS: R2(cum): 0.415, Q2 (cum): 0.45, cv-anova: 0.0188.
Figure 4
Figure 4
Heat map analysis of metabolite features detected in all analytical platforms for all fermentation days (MetaboAnalyst 5.0). The color spectrum ranging from red to green indicates the range of high to low signal intensities for each metabolite.
Figure 5
Figure 5
Temperature dynamic during the fermentation of fine-flavor cocoa. The dotted line divides cocoa fermentation temperature dynamics into two phases.
Figure 6
Figure 6
OPLS-DA models for two phases. (A) MF by LC–MS( +): R2(cum): 0.804, Q2 (cum): 0.962, cv-anova: 3.58e−13; (B) MF by LC–MS(-): R2(cum): 0.739, Q2 (cum): 0.96, cv-anova: 5.67e−07; (C) MF by GC–MS: R2(cum): 0.702, Q2 (cum): 0.864, cv-anova: 5.86e−12.
Figure 7
Figure 7
Box plots of metabolites linked with fine-flavor cocoa sensorial notes significantly differ throughout fermentation days (one-way ANOVA correcting for false discovery rate, FDR). Linalool xylosyl-glucoside (fruity, floral), irone (floral), coumaric acid (floral), cedrol (floral, woody,sweet), acetophenone (floral), maltol (caramel, fruity), methylcoumarin (fruity,floral), vanillin (vanilla, chocolate), and vanillin isobutyrate (caramel, chocolate, fruity).
Figure 8
Figure 8
Box plots of metabolites linked with undesirable cocoa sensorial notes with significant differences throughout fermentation days (one-way ANOVA correcting for false discovery rate, FDR). Salicylic acid (phenolic, faint), phenol (phenolic), benzoic acid (urine, faint), coumarin (green, bitter).
Figure 9
Figure 9
Box plots of bioactive metabolites with significant differences throughout fermentation days (one-way ANOVA correcting for false discovery rate, FDR).
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