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

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.

www.nature.com/scientificreports/ accountable for cocoa fermentation is still unclear 3,8,9 . Three significant phases during spontaneous cocoa fermentations have been previously proposed [10][11][12][13] due to the interactions between three main groups of microorganisms (yeast, lactic acid bacteria, and acetic acid bacteria) and their metabolic products 9,14 . An initial anaerobic phase occurs at the beginning of the fermentation, in which yeasts produce ethanol using the fermentable sugars from the cocoa pulp 9 . Then, when the pulp begins to disappear and oxygen penetrates at a higher rate the fermentation mass, a second (aerobic) phase occurs. In this phase, Lactic Acid Bacteria (LAB) utilizes the remaining sugars (mainly fructose) to produce lactic acid that accumulates 9,15 . Finally, the last phase occurs when acetic acid bacteria (AAB) convert ethanol into acetic acid that strongly affects the microbial diversity of fermentation 9,15,16 . Based on this paradigm, different mathematical models of cocoa fermentation have been previously proposed to further study the dynamics among these phases [10][11][12][13] . Apart from the general metabolic activity, other specific biochemical processes in cocoa fermentation have been modeled. For instance, genome-scale reconstructions for selected strains of acetic acid and lactic acid bacteria isolated from cocoa fermentations have been proposed in various studies, even using advanced techniques as fluxomic to improve the models [17][18][19] . However, recent metagenomics studies highlighted the relevance of other dominant groups of microorganisms during cocoa fermentations 8,13,14,[20][21][22][23] . The results of these studies question the traditional view of cocoa fermentation. For instance, the dominance of other microorganisms such as Bacillus, Pseudomonas, Aspergillus, Malassezia, and Pestalotiopsis at several phases during cocoa fermentations have been recently demostrated 8,13,14,[20][21][22][23] . The role of these microorganisms and their potential to produce valuable flavor and bioactive compounds during spontaneous cocoa fermentation still have to be elucidated. Therefore, a current essential challenge in cocoa research is identifying metabolites associated with these microorganisms to better understand their relevance during cocoa fermentation.However , only a few studies have dissected the variation in the metabolic profile during spontaneous cocoa fermentations [24][25][26][27][28] . Most of these studies focus on identifying metabolites during the fermentation of bulk cocoa (i.e., cocoa varieties with an ordinary flavor profile). In contrast, these kinds of analyses are rare and still in their infancy for fine-flavor cocoa. In this regard, to understand the metabolic dynamic of fine-flavor cocoa fermentation is a necessary previous step to connect the metabolites and microorganisms involved in this process.
Compared to bulk cocoa, fine-flavor offers a higher diversity of flavor attributes of high demand by the elite chocolatiers. Therefore, identifying changes in the metabolic fingerprint during spontaneous fermentation of fine-flavor cocoa is highly relevant to standardize this process and produce a high-quality bar of chocolate. Consequently, the goal of this study was to analyze the changes in the metabolic fingerprint during spontaneous fermentation of fine-flavor cocoa. Our data revealed two main phases of differential metabolic activity during spontaneous fermentation of fine-flavor cocoa that correlated with the observed variations on temperature and highlighted the relevance of comprehensive metabolomics studies to break down the current cocoa fermentation paradigm.

Experimental section
Cocoa beans fermentation. Cocoa fermentations were performed at the Luker Farm (Caldas, Colombia (5°4' N 75°41' W)) owned by CasaLuker S.A. Cocoa beans, from a standard, pre-designed, and a frequently-used mixture of Theobroma cacao clones (i.e., LUKER40, FSV41, FSA13, and TSH565), were selected for wooden box fermentations. Additional information regarding the used clones is available at the International Cocoa Germplasm Database (ICGD) (http:// www. icgd. readi ng. ac. uk/). Two independent fermentation boxes containing 400 kg of cocoa pulp-bean mass each was arranged using a ladder system in a pre-designed fermentation room as previously described 13 . Briefly, this fermentation room has an area of 84 m 2 with a metallic ceiling and acrylic walls to prevent air current entering the fermentation zone. The average temperature and during day time are 33 °C while at night time is 25 °C. Humidity is around 60% with a maximum level of 94%. The fermentation mass was mixed from the fourth day on, every 48 h,to allow aeration . Standard cutting tests were used to evaluate the fermentation quality over time and determine its final point following the standard protocols 13 .
Fermentation mass sampling. Three biological replicates, each consisting of 10 seeds, were collected from different fermentation mass locations-two of them from one fermentation box and the third one from the other fermentation box, as previously described 13 . Sampling was made at the beginning and every 24 h until the end of fermentation. To avoid chemical degradation, samples were frozen immediately at − 80 °C after collection. Furthermore, to connect the medium conditions and metabolic activity, we recorded the fermentation cocoa mass temperature variation every 4 min for each fermentation box using precision sensors placed in the center of the fermentation boxes.
This sampling process followed the guidelines and legislation settled by the ministry of environment and sustainable development of Colombia. We obtained a permission to access genetic and derivate resources according to resolution No 284 of 2020.
Sample preparation. Fermentation samples were initially milled using a clean and precooled coffee grinder. Aliquots of 100 mg were further macerated in the presence of liquid nitrogen and subsequently defatted by the addition of 500 µL of n-hexane and vigorous vortex as previously reported 29 . Defatting was repeated three times for each sample to maximize the lipid removal. Metabolite extraction was performed by adding 1 mL of methanol-ultrapure water (70:30) to each sample. Then these extracts were placed into an ultrasonic bath at room temperature for 10 min following by vortex-mixed for 10 min. After that, the samples were centrifuged at 6000 g, 25  To determine the reproducibility and stability of the analytical platforms used, several QC runs were performed before analyzing all cocoa samples until system equilibration was achieved and every five randomized samples.

Data treatment.
All raw data were processed as previously reported by Cala et al 30

Statistical analysis.
To determine statistically significant differences between metabolomics profiles, multivariate (MVA) statistical analyses were performed using SIMCA 16.0 (Umetrics, Umea, Sweden). Principal component analysis (PCA) was applied to evaluate the acquired data quality, verifying that the QC samples were correctly clustered in these models to guarantee the stability of the analytical system. After that, PLS-DA and OPLS-DA models were built to maximize and inspect the differences between study groups and select responsible metabolites for separating the groups. Pareto scaling were used before the statistical analysis. For all platform data, the significant variables were selected by keeping only the variables that fulfilled:1) MVA criteria (variance significant in projection (VIP) > 1.5 with Jack-knife confident interval (JK) not including the zero value from orthogonal partial least-squares discriminant analysis (OPLS-DA) with CV-ANOVA < 0.05) and 2) Change percent > 30%. www.nature.com/scientificreports/ PCA models ( Supplementary Fig. S1). A clear QC grouping was observed in PCA analysis for all analytical platforms, assuring the quality of acquired data and supporting that separating groups is related to biological and not analytical variations. Overall, we observed a significant difference between the metabolic fingerprint of the beginning (day 0) and the end (day 8) of the fermentation (Fig. 1), generally characterized by an increase in the signals of fermentation day 8. For all platforms, the PCA analysis revealed a clear separation between the metabolic fingerprint of day 0 and day 8 of the fermentation and a change in metabolite throughout all fermentation days (Fig. 2). However, the analysis of the metabolic fingerprint for each day revealed only a slight separation between specific days such as day 0 and day 1, day 2 and day 3, and the last three days of fermentation (i.e., day 6, day 7, and day 8), suggesting that these days exhibit a similar metabolic profile. This same behavior has been previously reported in several studies 24,25,28 .
Two different phases of metabolic activity were detected during spontaneous fermentation of fine-flavor cocoa beans. PLS-DA models were built to explore and maximize the differences in metabolic www.nature.com/scientificreports/ fingerprint throughout the fermentation process ( Fig. 3A-C). The PLS-DA models showed little discrimination in the metabolic fingerprint of the fermentation process on each day in all analytical platforms; however, in the PLS-DA score plots, it is possible to observe three clusters of fermentation days corresponding to D0-D3, D4, and D5-D8. Using this approach, additional PLS-DA models were built to explore the differences between these three groups ( Fig. 3D-F). A clear separation was observed in the score plots for all PLS-DA models between these groups with high quality, proven by significant variance values explained (R 2 ), variance predicted (Q 2 ), and CV-ANOVA. These results suggest that the most prominent metabolites changes cluster in two phases (D0 to D3 and D5 to D8) during the fermentation process of cocoa beans. To further explore the trends in metabolites modifications along fermentation days, a heatmap was built for all metabolite features detected in all analytical platforms using MetaboAnalyst 5.0 (Fig. 4). The groups (days) in the diagram were allocated using a hierarchical clustering algorithm, joining them by similarity, as indicates the dendrogram on the top of the figure. This heatmap shows two similar metabolic fingerprint with differential alteration of a significant number of metabolites between them. The first metabolic fingerprint goes from day 0 to day 3 and the second one from day fifth on, supporting the presence of two major metabolic phases during the fermentation of fine-flavor cocoa. Interestingly, these two phases are concomitant with the temperature profile in the cocoa mass through the entire process (Fig. 5).
The temperature of the fermenting mass is a crucial parameter during spontaneous fermentation of fine-flavor cocoa. Our data revealed two different temperature phases during spontaneous cocoa fermentation (Fig. 5) concomitant to the two phases of differential metabolic activity previously described (Figs. 3 and 4). The first phase is essentially exothermic. During this phase, the temperature of the fermenting mass significantly increased from 26.8 ± 0.3 °C to 49.4 ± 2.5 °C. Contrary, the second phase (from the fifth day on) is primarily isothermic. The temperature during second phase remained between 45 and 50 °C until the fermentation ended, a variation 81% lower compared with the exothermic phase. This kind of temperature profile was reported in different cocoa fermentations worldwide, with different cocoa varieties, fermentation methods, and weather conditions 20,21,32,33 . Temperature rise, usually more than 25 °C over the initial temperature of cocoa mass, could be associated with exothermic reactions (e.g., ethanol and weak acids production) from the first days of fermentation 10,34,35 . A maximum temperature increase rate was observed on fermentation day 4, which corresponds with the first turning of the fermenting mass to allow oxygenation. Thus, oxygen availably could partially explain the increase in temperature as a substantial increase in oxygen concentration may lead to a dramatic rise in exothermic oxidation www.nature.com/scientificreports/ reactions 36,37 . The temperature over 45 °C is a critical control point to determine the quality of the process, independently of the fermentation method, the region, the cocoa type, or the required fermentation time 20,33,37-39 .
As mentioned before, from the fifth day on, the temperature experienced minor changes and remained between 45-50 °C until fermentation ends. During this final phase, most microbial populations, and eventually its associated biochemical activity, decline as previously observed 13,14,40 . The relative abundance of microbial groups as yeast and LAB significantly decreases the last days of cocoa fermentation 13,15,40 . This drop is caused by a considerable decrease of fermentable sugars in the medium (e.g., glucose, fructose) 10,11 . Also, temperature plays a relevant role in controlling microbial populations considering that LAB and yeast growth decreases dramatically over 40 °C 41,42 . However, the elevated presence of lactic acid and oxygen prompts the growth of AAB that can resist higher temperature, producing acetic acid (an exothermic process) and releasing heat to the medium at a considerably lower rate but sufficient to maintain cocoa mass temperature relatively high, with a maximum variation of 5 °C 35,43 .
The temperature rise has a critical impact on flavor development during cocoa fermentation, as it guarantees the formation of molecules responsible for a high-quality sensorial profile 44 . The production of these compounds evidence the influence of temperature in metabolic activity across cocoa fermentation and the quality of the www.nature.com/scientificreports/ cocoa 10,44 . A high temperature causes a significant diffusion of acetic acid, lactic acid, and ethanol into the beans promoting the degradation of flavonoids, reducing the bitterness and astringency of the cocoa 1,39 . Also, it changes more than 25 °C during the fermentation 10,37,44 , stimulating a wide range of biochemical reactions and the growth of specific microorganisms in each increasing stage. These microorganisms carried out most of the transformations occurring in cocoa fermentation 37,45 . To achieve a high-quality sensorial profile, a proper succession of some specific microorganism genera is crucial. The development of this microbial progression depends on substrates present in the media and temperature 39 . www.nature.com/scientificreports/ Additionally, temperature plays a crucial role in proteolysis. The temperature reaches more than 45 °C during cocoa fermentation. This high temperature causes the activation of some native enzymes, and the protein heat denaturation produces a significant amount of peptides and amino acids 25,46 . These molecules are flavor precursors associated with the development of fruits and nutty notes in cocoa, qualities quite appreciated in the international markets 10,47,48 . The variation in dominant groups of metabolites diverge between the two phases of differential metabolic activity. We analyzed the effect of the temperature dynamic in the metabolic profile of cocoa fermentation using a multiplatform metabolomics analysis. This approach allowed us to propose a new paradigm of the phases of cocoa fermentation based on the global metabolic activity instead of the variation in only a few metabolites. We propose two phases of cocoa fermentation based on the temperature dynamic: an exothermic phase from day 0 to day 3 and an isothermic phase from day 5 to day 8. Once these phases were established, univariate (p-value < 0.05 from hypothesis testing) and multivariate (OPLS-DA models) analyses were performed to select the differential metabolites between each phase. For all analytical platforms, the OPLS-DA analysis allowed modeling the differences between the two phases with statistically significant values for R 2 , Q 2 , CV-ANOVA (Fig. 6). As a result, 44, 65, 20 differential metabolites were identified in LC-QTOF-MS( +), LC-QTOF-MS(-), and GC-QTOF-MS( +), respectively. Table 1 shows metabolites altered between the exothermic and isothermic phases of cocoa fermentation. The biggest group of metabolites differentially expressed correspond to amino acids, dipeptides, and tripeptides. The concentration of these 23 molecules increased in the isothermic phase. The formation of amino acids and peptides during cocoa fermentation is due to protein hydrolysis and denaturation processes. Protein content (e.g., albumin, prolamin, globulin, and glutein) represents 10 -15% of dry unfermented cocoa beans 46,47 and is significantly lower for fermented cocoa beans 38,47,49 . The drop in protein content, usually above 60%, is caused by proteolysis during fermentation that involves two groups of native cocoa proteases: endoproteases and carboxypeptidases 38,47,49 . The optimal temperature for these enzymes is 45-50°C 50,51 , precisely the same temperature range of the fermentation the isothermic phase. Also, this temperature itself is accountable for protein denaturation, breaking them down into peptides or even amino acids 25,49 . This protein degradation linked to enzymatic hydrolysis and denaturation by heat in cocoa fermentation has been widely documented in several studies 25,46-49 . Peptides and amino acids are highly relevant for the nutraceutical and sensorial properties of fine-flavor cocoa. Several cocoa peptides have been associated with bioactive properties such as antioxidant, antihypertensive, and antimicrobial 52 , although further research is still required. On the other hand, peptides and amino acids are precursors of pyrazines produced during drying and roasting through Maillard reactions 5,49 . These pyrazines are responsible for fine-flavor cocoa sensorial notes as fruity, floral, and cocoa 5,53 . In this regard, proteolysis could explain why cocoa fermentation in which temperature is consistently below 45 °C, the obtained chocolate usually has a poor sensorial profile.
Flavonoids are the second largest group of molecules differentially expressed with 18 compounds. In the isothermic phase, we observed a rise of different flavonoids such as dimers and trimers of procyanidins, polyphenol glycosides, and rhamnose-containing polyphenols. Also, our data show a decrease in some arecatannin types and butein and pelargonidin derivates. Although the overall content of flavonoids is expected to decrease throughout fermentation, these degradation processes can cause the emergence of polyphenolic dimers and trimers as well as polyphenolic acids as caffeic acid, benzoic acid, and coumaric acid 54,55 . For instance, oxidation and polyphenol oxidase hydrolysis of complex flavonoids such as anthocyanins, procyanidins, epigallocatechin, and kaempferol results in the formation of dimers and trimers of procyanidins and rhamnose-containing polyphenols 54,55 . However, these derivates tend to have lower bioactivity, leading to a significant drop in the bioactive properties of cocoa throughout fermentation, as previously reported 8,38,56 . Polyphenol degradation and eventual derivates formation can be more pronounced at high temperatures 56,57 . www.nature.com/scientificreports/ Contrasting that, carbohydrates are the compound group with a higher number of decreasing concentration metabolites from the exothermic to isothermic phase. These metabolites are a primary substrate for many yeasts and bacteria, eventually generating a rise in different organic acids beyond traditional ones (e.g., lactic acid, acetic acid) like benzoic acid, caffeic acid, coumaric acid, oxoglutaric acid, and salicylic acid, as our data revealed. The carbohydrates transformation into organic acids is more intense during the exothermic than isothermic phase due to temperatures under 40 °C and high content of pulp rich in carbohydrates 11,21,39 . These conditions facilitate yeast and bacteria growth as Saccharomyces, Candida, Mallasezia, Hanseniospora, Lactobacillus, and Bacillus produce organic acids as part of their central and secondary metabolism 13,35,36,40,58 .
Other small metabolite groups as phenols, coumarins, terpenoids, fatty acyls, and lipids were also found to varybetween the two phases. These compounds are likely a byproduct of the degradation of complex molecules as polyphenols using the central and secondary metabolism of many microorganisms associated with cocoa fermentations 13,40,54 . For instance, the yeast of genus such as Mallasezia, previously reported in cocoa fermentation, is highly active in the metabolism of lipids and fatty acyls 13 . However, as the link between microorganismsmetabolites is still not elucidated, further research is required to dissect the connection between microbial populations and altered metabolites during cocoa fermentations.

Changes in the concentration of relevant metabolites (i.e., flavor precursors and bioactive compounds) were observed during spontaneous fermentation of fine-flavor cocoa. To bet-
ter understand the development of some sensorial notes associated with fine-flavor cocoa (i.e., fruity, floral, and chocolate notes), we performed a search in the database FlavorDB 53 using the list of altered and annotated compounds. We found a rise between the exothermic and isothermic phases of cedrol, irone, acetophenone, coumaric acid, maltol, vanillin, vanillin isobutyrate, and methylcoumarin (Fig. 7) associated with fine-flavor cocoa sensorial notes 5,53 . Nevertheless, we also observed a significant increase in phenol, coumarin, benzoic acid, and salicylic acid. These molecules produce undesirable flavors previously associated with green, astringent, and bitter attributes (Fig. 8) 5,53 .
Interestingly, we also observed an increase in some specific flavonoids with documented bioactive properties from the exothermic to isothermic phase. For example, catechin, epicatechin, Resveratrol 3-sulfate, Isoflavonoid O-glycoside, and a group of procyanidins dimers and trimers experienced a considerable increase over fermentation (Fig. 9). These compounds are widely known due to their antioxidant properties 8,57 . Similarly, other metabolites as dopamine, nicotinamide riboside, and aspartic acid were observed to increase throughout cocoa fermentation. An attractive potential emerges from these molecules because they can act as neurotransmitters 59 , cardiovascular regulator 60 , and hormone regulator 61 , respectively. These bioactive molecules could be produced A. B.

C.
Exothermic Phase Isothermic Phase    www.nature.com/scientificreports/ from complex polyphenol degradation processes that can involve the action of weak acids and the temperature changes into the beans, oxidation reactions, and hydrolysis by polyphenol oxidases 62,63 or in the secondary metabolism of some microorganisms 64 , but the biochemical mechanisms behind their production remain unclear. Several studies associate non-conventional microorganisms' groups with the production of flavor molecules [64][65][66][67] . For instance, unconventional strains of Saccharomyces, Candida, Pseudomonas, and Bacillus species, widely reported in cocoa fermentations worldwide, can also produce vanillin and derivates from feluric acid 65,66 . Different fungi species produce coumarin and derivates naturally 64,67 . However, to fully connect the metabolite production with the microbiome of cocoa fermentation, an integration of metabolomics and metagenomic data is required, and it will be the focus of future research.

Conclusions
Our work reveals a clear connection between cocoa mass temperature and the metabolic activity during the fermentation of fine-flavor cocoa. Using temperature dynamic as a relevant parameter during cocoa fermentation, we proposed a new cocoa fermentation metabolic paradigm that offers a complete insight into how temperature regulates biochemical reactions during cocoa fermentation, considering global metabolic activity. This shift is a crucial step to develop strategies based on temperature control to drive cocoa fermentations toward better quality chocolate. Nevertheless, further research is required to further dissect the link between microorganisms and metabolites. This is a crucial step in order to understand the impact in the sensorial profile of a significant number of cocoa compounds.
We also elucidated metabolic modifications throughout fermentation associated with proteolysis and secondary metabolism. Our results reveal a potential for bioprospection beyond chocolate production of some peptides and polyphenols with attractive bioactive properties that arise during cocoa fermentation-considering that a significant proportion of these molecules are lost in post-fermentation processes. However, the bioactive properties of most of these identified metabolites also still need to be entirely dissected.

Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.