Analysis of volatile compounds of five varieties of Maya cocoa during fermentation and drying processes by Venn diagram and PCA

Journal Pre-proofs Analysis of volatile compounds of five varieties of Maya cocoa during fermentation and drying processes by Venn diagram and PCA Marycarmen Utrilla-Vázquez, Jacobo Rodríguez-Campos, Carlos Hugo Avendaño-Arazate, Anne Gschaedler, Eugenia Lugo-Cervantes PII: DOI: Reference:

S0963-9969(19)30720-3 https://doi.org/10.1016/j.foodres.2019.108834 FRIN 108834

To appear in:

Food Research International

Received Date: Revised Date: Accepted Date:

3 July 2019 12 November 2019 18 November 2019

Please cite this article as: Utrilla-Vázquez, M., Rodríguez-Campos, J., Hugo Avendaño-Arazate, C., Gschaedler, A., Lugo-Cervantes, E., Analysis of volatile compounds of five varieties of Maya cocoa during fermentation and drying processes by Venn diagram and PCA, Food Research International (2019), doi: https://doi.org/10.1016/ j.foodres.2019.108834

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Analysis of volatile compounds of five varieties of Maya cocoa during fermentation and drying processes by Venn diagram and PCA

Marycarmen Utrilla-Vázqueza, Jacobo Rodríguez-Camposa, Carlos Hugo Avendaño-Arazateb, Anne Gschaedlera, Eugenia Lugo-Cervantesa*

aCentro

de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Unidad de Tecnología

Alimentaria. Camino Arenero 1227, El Bajío del Arenal. 45019. Guadalajara, Mexico. bInstituto

Nacional de Investigaciones Forestales, Agrícolas y Pecuarias-INIFAP. Campo experimental Rosario

Izapa, Carretera Tapachula-Cacahoatán Km. 18. 30780. Rosario Izapa, Chiapas, Mexico. * Corresponding author: [email protected]

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Abstract Fermented cocoa beans can be described as a complex matrix that integrates the chemical history of beans, their processing, and environmental factors. This study presents an analysis that aims to identify volatile compounds of five varieties of fine-aroma cocoa types. The cocoa types studied were Carmelo, Rojo Samuel, Lagarto, Arcoiris, Regalo de Dios, that grow in the Maya lands of Chiapas, Mexico. Profile of volatile compounds was obtained from each cacao type during fermentation and drying process. This profile of volatile compounds also was compared with beans unfermented, using a statistical analysis of Venn diagram and a multivariate Analysis of Principal Components (PCA). One hundred nine different compounds were identified by SPME-HS GC-MS, these compounds mainly related to desirable aromatic notes generated by esters, aldehydes, ketones, and alcohols. The differences in chemical composition of the volatile compounds were associated mainly with the process and not to cocoa varieties. Fermented dry cocoa beans showed a higher content of esters, aldehydes, pyrazines, alcohols, some acids, and furans where Lagarto (CL), Rojo Samuel (CR), and Regalo de Dios (TRD) cocoas type showed a more interesting aromatic profile. On the other hand, as expected dry unfermented cocoas presented a few numbers of aroma compounds, in the five cacao types, where alcohols, ketones and hydrocarbons predominated.

Keywords: cocoa beans; Criollo and Trinitario; fermented and unfermented; volatile profile, aromatic quality. Chemical compounds studied in this article: Isobutyl benzoate (PubChem CID: 61048); 2-Methylbutanal (PubChem CID: 7284); Benzeneacetaldehyde (PubChem CID: 998); 2-Nonanone (PubChem CID: 13187); Acetophenone (PubChem CID: 7410); 2-Heptanone (PubChem CID: 8051); Furfural (PubChem CID: 7362); β-Myrcene (PubChem CID: 31253); Benzonitrile (PubChem CID: 7505); Trimethylpyrazine (PubChem CID: 26808).

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1. Introduction Cocoa beans (Theobroma cacao L.) are the main ingredient for the manufacturing of chocolate. In particular, premium chocolate represents a rapidly growing industry of significant financial importance in many parts of the world (Le Gresley & Peron, 2019). Around 600 volatile compounds have been identified as being responsible for taste and aroma in chocolate and cocoa products (Engeseth & Pangan, 2018). These have been identified as pyrazines, amines, amides, carboxylic acids, esters, and hydrocarbons. The quantity and type of volatile compounds in beans are considered to be the most important aspects of quality. These define the commercial value of cocoa, and result in the unique and complex tastes and aromas of chocolate (Braga et al., 2018; Kongor et al., 2016; Lima, Almeida, Nout, & Zwietering, 2011; Magagna et al., 2017). Taste and aroma are among the main attributes in sensory profiles. They are a key factor in obtaining products and subproducts, which are adapted to high-quality demands by consumers (Engeseth & Pangan, 2018). The profile of volatile compounds can exhibit great variations, depending on the genotype and geographical origin of the cocoa beans (Menezes et al., 2016). Also, the profile may vary with agricultural practices and processing (fermenting, drying, and roasting), both of which determine its final aromatic quality (Ascrizzi, Flamini, Tessieri, & Pistelli, 2017; Cevallos-Cevallos, Gysel, Maridueña-Zavala, & Molina-Miranda, 2018). Fresh unprocessed cocoa beans are immersed within a sweet, mucilaginous pulp, which can also be acidic and aromatic. However, they do not contain sufficient aroma precursors for the development of volatile compounds for obtaining a final product with high aromatic quality (Aprotosoaie, Luca, & Miron, 2016; De Vuyst & Weckx, 2016; Kadow, Bohlmann, Phillips, & Lieberei, 2013; Koné et al., 2016). The fermentation of cocoa beans is a fundamental process whereby a series of biochemical, enzymatic, and microbiological reactions occur. This results in the development of key volatile fractions, which are alcohols and esters, and in the generation of flavor and aroma precursors, such as amino acids, free peptides, and reducing sugars (CastroAlayo, Idrogo-Vásquez, Siche, & Cardenas-Toro, 2019). Polyphenols and alkaloids (of bitter and astringent taste) are defunded out of cocoa bean (De Vuyst & Weckx, 2016). The development of flavor continues during drying, whereby the typical reddish color (of the bean) appears, and acidity and astringency levels decrease upon a reduction in the levels of volatile acids and total polyphenols (Afoakwa et al., 2008; Frauendorfer & Schieberle, 2008; Mirković et al., 2018; Schwan & Wheals, 2004). Based on volatile compounds, cocoa beans are classified in two groups: ordinary cocoa and fine or flavor cocoa (Aprotosoaie et al., 2016; Cevallos-Cevallos et al., 2018). Ordinary cocoa refers to the Forastero type, which represents approximately 95-97% of the cocoa beans produced worldwide (Cevallos-Cevallos et

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al., 2018; Figueroa-Hernández et al., 2019). These beans have a very strong acidic flavor, are dark red color in color, are rich in cocoa butter, have a high content of phenolic compounds, and are less aromatic. Fine or flavor cocoa refers to the Criollo or Trinitario cocoa types and are considered to be of high and medium aromatic quality, respectively. These represent about 5-6% of the total cocoa production worldwide and comprise the ideal raw material for high-quality or premium chocolates; thus, they are up to five times more expensive than Forastero cocoa (Lachenaud & Motamayor, 2017). Criollo cocoa beans are white, aromatic, with a sweet pulp, little bitterness, and a flavor reminiscent of caramel and nuts. However, Criollo cocoa trees have lower yields and are more susceptible to plagues and diseases. Trinitario cocoa trees are a hybrid species with the combined features of Criollo and Forastero types, which means they are less aromatic, but with higher yields and resistance to plague and diseases (Ascrizzi et al., 2017). The aromatic notes produced by the fine cocoa beans or aroma are fruit (fresh and ripe), flowers, herbs and woods, notes of nuts and candies, as well as monoterpenes such as linalool and notes of rich and balanced chocolate bases after fermenting and roasting the beans (Ziegleder, 1990; Beckett, 2009). In this context, cocoa (T. cacao L.) is one of the most important agricultural and cultural products in the Mexican tropics (Díaz-Jose, Díaz-Jose, Mora-Flores, Rendón-Medel, & Téllez-Delgado, 2014). Mexico possesses a great diversity of genetic materials, cocoa pods, and bean morphologies (Avendaño-Arrazate et al., 2011; Gutierrez-López et al., 2016). In 2017, Mexico held the 13th position worldwide in cocoa production, harvesting 27,287 tons from 60,000 hectares, located mostly in the states of Tabasco (65%), Chiapas (34%) and Guerrero (1%) (SIAP, 2018). The main types of cocoa grown in Mexico are Forastero, Criollo, and Trinitario. There are reports specifying that Criollo cocoa was domesticated and used as a food source in Central America in pre-Columbian times, about 3,800 years ago (Powis, Cyphers, Gaikwad, Grivetti, & Cheong, 2011; Lachenaud & Motamayor, 2017). In Chiapas, where Criollo and Trinitario cocoas have been located, cocoa processing is still carried out in a traditional and uncontrolled manner, from harvesting and fermentation to drying (Avendaño-Arrazate et al., 2011). In 2017, Chiapas has reported an increase in cocoa production and a better price for its harvest (SIAP, 2018). The main varieties of Criollo and Trinitario cocoa that have been identified in Chiapas include the following: Lacandón; Carmelo; Lagarto; Rojo Samuel; Arcoiris and Regalo de Dios, among others (Avendaño-Arrazate & Cueto-Moreno, 2018; Avendaño-Arrazate, GuillénDíaz, & Hernández-Gómez, 2018; Avendaño-Arrazate et al., 2011). Some of these varieties are grown mainly because of their better adaptation, yield, quality, and aroma (Avendaño-Arrazate et al., 2013) . To our knowledge, there are few studies in the literature regarding the volatile compounds of cocoa varieties from Mexico. These investigations have been carried out mainly in the Forastero cocoa type and in

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several cocoa hybrids, whereby the dynamics of the volatile compounds during fermentation and drying have been characterized (Ramos, Dias, Miguel, & Schwan, 2014; Rodriguez-Campos et al., 2012; RodriguezCampos, Escalona-Buendía, Orozco-Avila, Lugo-Cervantes, & Jaramillo-Flores, 2011). There are no reports concerning the dynamics of volatile compounds of Criollo and Trinitario cocoa types that appear during the fermentation and drying processes. These are the means to evaluate the quality of beans during fermentation and the manufacture of high-quality uniform products and to increase their commercial value. The main aims of this study will now be summarized briefly. The first objective was to identify the volatile compounds during the fermentation and drying processes of five varieties of cocoa beans (Carmelo, Rojo Samuel, Lagarto, Arcoiris, and Regalo de Dios) from Maya lands in Chiapas, Mexico, that are important in manufacturing gourmet chocolate. The second objective was to compare the similarities and differences between varieties and processes in terms of their volatile compounds profile using the Venn diagram and Principal Component Analysis (PCA). The outcome could improve post-harvest practices locally.

2. Materials and methods 2.1. Cocoa samples Five varieties of cocoa (Table 1) were used: three Criollo types (Carmelo (CC), Rojo Samuel (CR), and Lagarto (CL), and two Trinitario types, Arcoiris (TA) and Regalo de Dios (TRD). The cocoa pods were collected in the following three municipalities of the Soconusco region: Tapachula (N 14°54′13″, O 92°15′26″); Cacahoatán (N 15°3'41", O 92°9'31"), and Tuxtla Chico (N 14°55′27″, O 92°11′23″) in Maya lands in Chiapas, Mexico. The cocoa pods were donated by two producers and by the Cacao program (INIFAP Rosario Izapa Experimental Field, Tuxtla Chico, Chiapas). The traditional methods of harvesting (ripe cocoa pods were manually separated from the trees with the aid of a knife) were used in the main harvest season of 2017 (March-August). The cocoa pods that showed visible physical damage were discarded. The cocoa pods were collected in jute sacks and transported to the fermentation site. The experiment was conducted at the INIFAP Field. The cocoa pods were opened (broken) with a machete and the mass was collected.

2.2. Dried unfermented cocoa beans The traditional process of the producers of the Soconusco region in Chiapas, Mexico, was employed. This process consists of washing 200 g of beans of each type of cocoa with potable water to remove the mucilage. Then, the washed beans were extended (separately for each type of cocoa) on a wooden box for sun-drying until a humidity of 7% was reached in the beans. To improve drying, the mass of unfermented

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cocoa was spread out, forming a layer approximately 0.02-m thick. The cocoa beans were mixed manually each day to promote and enhance uniform drying. The drying process started at 9:00 and ended at 15:00 (6 h of sun per day). At the end of each day, the cocoa beans were picked and saved for the next day.

2.3. Fermentation and drying processes The fermentation of the cocoa beans occurred spontaneously at ambient temperature and relative humidity. Fermentation began approximately 4 h after all the pods had opened; this point was considered as F0 (0 h of fermentation). For the fermentation process, wooden boxes (0.012 m3) were utilized. These included perforations in the boxes’ lower parts for draining the exudate. The mass of cocoa beans was covered with banana leaves. Fermentation lasted 6 days (144 h). Every 24 h, the mass of cocoa beans was turned manually to obtain homogeneous fermentation and promote aeration of the grains. The drying process was carried out in covered solar dryers (greenhouse type). The mass of fermented cocoa was spread on tables set one meter above ground level. The beans were mixed manually each day to obtain uniform drying. The drying process started at 9:00 and ended at 15:00 (6 h of sun per day). At the end of each day, the beans were picked and saved for the next day. All varieties were dried to a final moisture content of 7%.

2.4. Sample collection Sampling was always carried out at the same time (every 24 h) and depth (10 cm from the upper surface). Samples were placed in plastic bags. All samples were frozen at −20°C and transported to the laboratory. Samples weighing 100 g were removed from the fermentation and drying processes daily, starting with the fermentation of raw cocoa beans at 0 (F0), 1 (F1), 2 (F2), 3 (F3), 4 (F4), 5 (F5), and 6 (F6) days. Afterward, dried fermented cocoa-bean samples were taken at the start (D1) and end (D3) of drying. Also, dried unfermented cocoa bean samples (DU) were taken at the end of drying.

2.5. Analysis of volatiles 2.5.1. Extraction of volatile compounds The volatile compounds from cocoa samples (2.0 g) were extracted using the Headspace-Solid Phase Microextraction (HS–SPME) Gas Chromatography-Mass Spectrometry (GC–MS) technique, as previously described (Rodríguez-Campos, Escalona-Buendía, Orozco-Ávila, Lugo-Cervantes, & Jaramillo-Flores, 2011). In

order

to

extract

the

volatile

constituents

from

the

cocoa

headspace,

a

DiVinylBenzene/CARboxen/PolyDiMethylSiloxane (DVB/CAR/PDMS) 50/30 mm SPME fiber (Supelco Co.,

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Bellefonte, PA, USA) was used. The fiber was equilibrated for 15 min at 60°C and then exposed to the samples of cocoa for 30 min at the same temperature.

2.5.2. Separation and identification of volatile compounds The fiber with volatile compounds was analyzed by Gas Chromatography–Mass Spectrometry (GC– MS) (Agilent Technologies, 6890N), equipped whit an Innowax capillary column (60 m by 0.25 mm inside diameter (id), 0.25-μm film thickness). The oven temperature was set at 40°C for 5 min, which was then increased up to 200°C at a rate of 10°C min−1 and finally maintained at 200°C for 30 min. The carrier gas was high-purity helium at 0.7 mL min−1. The splitless injection mode was at 240°C (5 min). The selective mass detector was a quadrupole (Agilent Technologies, 5975), with an electronic impact ionization system at 70 eV and at 260°C. Volatile compounds were identified by comparing the mass spectra of the compounds in the samples with the database of the National Institute of Standards and Technology (NIST Library, Gaithersburg, MD, USA) with a match of at least 85% and the retention time with literature data. All samples were analyzed in duplicate. A maximal acceptable coefficient of variation was 30% for a given compound (Cevallos-Cevallos et al., 2018). Aroma descriptors were obtained for each compound detected using the online databases Flavornet

(http://www.flavornet.org/flavornet.html),

The

Good

Scents

Company

(http://www.thegoodscentscompany.com/indeX.html), and the literature.

2.6. Statistical analysis The peak area of each volatile was obtained and aligned by using an in-house alignment protocol. Areas of volatile compounds were subjected to Venn Diagram, Principal Component Analysis (PCA) and a Cluster Analysis (CU). Scores obtained from each PCA were analyzed to a one-way ANalysis Of VAriance (ANOVA) to test for significant differences between samples. Tukey test was applied at 5% probability (p <0.05) with a 95% Confidence Level (95% CL) to analyze the differences among the means of the volatile compounds. All statistical analyses were performed using Statgraphics Centurion XVI Software Version 16.1.03 (Statgraphics, 2010).

3. Results and Discussion 3.1. Profile of volatile compounds produced during the fermentation, drying and dried unfermented beans of five cocoa varieties

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A total of 109 compounds was identified during fermentation (F0 and F6), drying (D3), and in dried unfermented (DU) cocoa beans. These were found in all five varieties of cocoa: Carmelo (CC); Rojo Samuel (CR); Lagarto (CL); Arcoiris (TA), and Regalo de Dios (TRD) (Table 2). The chemical groups present in greatest were esters (25), aldehydes (21), alcohols (19), ketones (15), and acids (9). At a smaller proportion, pyrazines (6), furans (4), hydrocarbons (4), and other compounds, including nitrile (1), pyridine (1), and pyrrole (1) were also found, as shown in Table 2. The odor descriptor of each compound is depicted in the same table. Some of these volatile compounds have been reported as being responsible for producing both desirable and undesirable flavor notes in cocoa beans during the fermentation, drying, and roasting processes (RodríguezCampos et al., 2011). Table S1 shows volatile compounds that were identified during fermentation days (F0F6), drying days (D1 and D3) and dried unfermented (DU) cocoa beans. At the beginning of fermentation (F0), it was observed that the highest number of volatile compounds (43) was detected in Arcoiris cocoa (TA). On the other hand, Lagarto cocoa (CL) was that with the smallest number, with 32. By the end of the fermentation (F6), 51 compounds had been identified in Arcoiris cocoa (TA), whereas in Carmelo (CC) and Rojo Samuel (CR) cocoa, 33 compounds were identified (in each one). At the end of the drying process (D3), Arcoiris (TA) and Regalo de Dios cocoa (TRD) presented 46 compounds (in each one), while in Carmelo cocoa (CC), 42 compounds were identified. In contrast, in dried unfermented cocoa beans (DU), the highest number of compounds identified in Rojo Samuel (CR) and Lagarto cocoa (CL), with 30 and 21 compounds identified, respectively (Fig. 1A). The differences between variety and processes allowed obtaining unique aromatic profiles, as well as the number of different compounds in the dried beans.

3.2. Volatile compounds produced during fermentation of five cocoa varieties The aldehydes formed the group with the most peak area (70-90%), both at the beginning (F0) and at the end of the fermentation (F6) for all of the varieties studied, except at the beginning of the fermentation (F0) for Rojo Samuel (CR) and Arcoiris (TA) cocoa, in which alcohols and ketones occupied a largest peak area (42 and 37%, respectively) (Figs. 1B and 1C). It is known that ketones and aldehydes favor the aromatic quality of cocoa (Aprotosoaie et al., 2016) and also tend to increase over time (Rodríguez-Campos et al., 2012). On the other hand, ethanol is produced mainly by yeasts during the anaerobic phase of fermentation (first 48 h) (Schwan & Wheals, 2004), while higher alcohols are produced from carbohydrates or can result from the thermal degradation of amino acids (Aprotosoaie et al., 2016). Alcohols are oxidized into acetic acid or esters; therefore, a high content of alcohols is desirable to obtain cocoa products with floral or sweet notes (Rodríguez-Campos et al., 2012).

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At the beginning of fermentation (F0), acids, esters, furans, hydrocarbons, and other compounds accounted for less than 8% of peak area. At the end of fermentation (F6), the area of acids, esters, and hydrocarbons increased two- to ten-fold, whereas furans increased only for Lagarto cocoa (CL). In the specific case of acids, the greater part comprises acetic acid, which is produced by acetic acid bacteria. The acids generate a series of biochemical changes necessary for the development of taste and for the death of the embryo (Cevallos-Cevallos et al., 2018; Qin et al., 2016)(Cevallos-Cevallos et al., 2018; Qin et al., 2016)(Cevallos-Cevallos et al., 2018; Qin et al., 2016)(Cevallos-Cevallos et al., 2018; Qin et al., 2016)(Qin et al., 2016). Some studies suggested that chocolates produced from Criollo beans have higher acidic tones, compared to Forastero and Trinitarios beans (Qin et al., 2016). Esters are the second most important class of volatile compounds after pyrazines, which provide a fruity flavor. Esters originate from the reaction of an organic acid with an alcohol during the anaerobic phase of fermentation (Aprotosoaie et al., 2016; Qin et al., 2016). However, pyrazines represented approximately 0.1% of peak area at the end of the fermentation (F6) in Rojo Samuel cocoa (CR) (Figs. 1B and 1C). The moderate temperatures that are reached during fermentation and drying can contribute to the final pyrazine levels. However, the formation of pyrazines is directly related to the roasting process of cocoa beans, wherein pyrazines are formed by condensation reactions between acetoin and 2,3-butanediol with amino acids (Crafack et al., 2014; Lima et al., 2011). Additionally, it has been reported that pyrazines are produced by Bacillus spp. or Bacillus subtilis or Bacillus megaterium, which are present at the end of cocoa fermentation (Hamdouche et al., 2019).

3.3. Volatile compounds produced during the drying process of the fermented and unfermented beans of five cocoa varieties In dry fermented cocoa beans (D3), acids and aldehydes were the groups with the largest peaks areas (50-85%) in all five varieties, followed by alcohols (9-32%), esters (3-11%), and ketones (2-6%). In the case of pyrazines, approximately 1% was detected for the varieties studied, except for Arcoiris cocoa (TA), where these were not detected. Furans decreased for all varieties of cocoa (Figs. 1B and 1C). In dried unfermented cocoa beans (DU), alcohols had a larger peak area in Carmelo cocoa (CC) (57%) and in Regalo de Dios cocoa (TRD) (38%). Contrary to dry fermented beans, there were no furans or pyrazines to be detected in dried unfermented beans (DU), whereas hydrocarbons were detected in greater abundance in this group (2-6%) (Figs. 1B and 1C.) Hydrocarbons have been reported as one of the main contributors to the aroma differentiation of fermented fine-flavors cocoa (Cevallos-Cevallos et al., 2018) .

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It has been reported that the content of alcohols, esters, and pyrazines increases during sun-drying. However, the content of acids, aldehydes, and ketones decreases. Pyrazines are generated in the drying process due to Maillard reactions initiated by a reduction in moisture content and temperatures between 30°C and 50°C. On the other hand, furanones and pyrans are generated during drying and roasting by the degradation of monosaccharides. Moderate temperatures and relatively high humidities favor the formation of these compounds (Aprotosoaie et al., 2016; Jinap, Wan Rosli, Russly, & Nordin, 1998; Rodriguez-Campos et al., 2012, 2011). 3.4. Key-aroma and technological marker Figure 2 shows the behavior of 16 volatile compounds that are considered key-aroma markers and technological markers, which were defined by Magagna et al. (2017). These aid in determining the aromatic properties and quality of cocoa beans. In this work, nine compounds were found in all varieties during the process and were classified as key-aroma markers. These key-aroma markers are as follows: 3-methylbutanal, 2-heptanol, 2-phenylethyl acetate, 2-phenethyl alcohol, isobutyric acid, isovaleric acid, acetic acid; benzeneacetaldehyde, and 2,3,5-trimethylpyrazine. In the case of technological markers, the following seven were

found:

methylpyrazine,

acetoin,

furfural,

2,3-dimethylpyrazine,

2,5-dimethylpyrazine,

2,6-

dimethylpyrazine, and tetramethylpyrazine. The markers were monitored at different steps of the process (fresh, fermented, and dried) and in the dried unfermented (DU) cocoa beans. In the case of esters, 2-phenylethyl acetate (honey-like aroma) presented a larger peak area in fermented dry beans. A high concentration of 2-phenylethyl acetate is desirable because it favors obtaining desirable aromatic notes (Ascrizzi et al., 2017). On the other hand, aldehydes such as 3-methylbutanal (malty and chocolate aroma) comprise a compound that contributes to the intensity of the chocolate aroma, which was found in all varieties during fermentation. However, in the dried cocoa a smaller area was observed, as it probably volatilized during the drying process (Castro-Alayo et al., 2019; RodriguezCampos et al., 2012). Benzeneacetaldehyde (honey-like aroma) is derived from L-phenylalanine (CevallosCevallos et al., 2018; Liu et al., 2015) and was detected from the beginning of fermentation until fermented dry beans were obtained, albeit in smaller area. Acetoin (buttery aroma) is desirable for flavor development in the fermentation process. In this study, the latter was found at the end of the fermentation process (D3) in all samples, with the exception of Rojo Samuel cocoa (CR). During drying, acetoin was not detected in Regalo de Dios cocoa (TRD) and it presented a smaller area in Lagarto (CC) and Arcoiris (TA) cocoa, while it showed a larger area in Carmelo (CC) and Rojo Samuel (CR). Acetoin can be produced from pyruvate and butanediol during alcoholic fermentation and can be a precursor of tetramethylpyrazine (Rodríguez-Campos et al., 2012).

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The 2-heptanol (citrus, fresh, lemon aroma) exhibited a greater area in Rojo Samuel (CR) at the beginning of the fermentation (F0) and it remained nearly constant until drying. Phenylethyl alcohol (floral, spicy, and honey aroma), a superior alcohol, increased its area in the course of fermentation and at the end of the drying of fermented and unfermented beans (Ho, Zhao, & Fleet, 2014; Magagna et al., 2017; RodriguezCampos et al., 2011) . Furfural (sweet, bread-like) was found in fresh beans (F0), but Lagarto cocoa (CL) was the variety that presented the largest peak area. The presence of furfural is associated with bacteria of the Bacillus type. In fermented dried beans (D3), the amount of trimethylpyrazine (cocoa, roasted nuts) and tetramethylpyrazine (earthy) was greater than methylpyrazine (buttery), 2-3 dimethylpyrazine (caramel, cocoa), 2-5 dimethylpyrazine (chocolate, nutty), and 2-6 dimethylpyrazine (nutty, herbal). The drying process converts flavor precursors into two main classes of active flavor components, pyrazines and aldehydes, although flavor development continues during the removal of volatile acids and moisture (Rodríguez-Campos et al., 2011). On the other hand, in the same figure (Fig. 2), acetic acid (sour, vinegary and produced during fermentation) was found in greater abundance than in the other acids at the end of drying, especially in Lagarto (CL) and Regalo de Dios (TRD). The presence of isobutyric acid (rancid) and isovaleric acid (rancid) was also observed at a smaller proportion, mainly at the end of fermentation and drying. Similar results were found during the fermentation of Criollo and Nacional cocoa in Ecuador (Cevallos-Cevallos et al., 2018). In the case of dried unfermented cocoa beans (DU), it is observed that there is no generation and development of the main key-aroma and technological marker. However, acetic acid and phenylethyl alcohol were identified, which are related to microorganisms in the first phase of fermentation associated with the pulp of the grain and the environment.

3.5. Comparison between varieties and processes according to the composition of volatiles analyzed by Venn diagram According to Fig. 3, the five varieties and two processes were analyzed using the Venn diagram. In the upper part (Fig. 3A), from left to right, the compounds present in fresh cocoa beans (F0) of all varieties were grouped. The next group corresponds to compounds obtained from the end of the fermentation process (F6). In the following group, we found the compounds obtained after the drying process (D3). Finally, the following group corresponds to the compounds obtained from dried unfermented cocoa (DU). In the lower section (Fig. 3B), from left to right, common and different compounds of fresh, fermented, and dry fermented

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cocoa of the five varieties are grouped. The next group (Fig. 3C) corresponds to similar and different compounds comparing dry fermented cocoa with dried unfermented cocoa. In the first analysis, we determined which compounds are common to all three steps, both fresh, fermented, and dry, as well as the compounds that are different and that will differentiate each variety by showing different aromatic profiles. In fresh cocoa beans, the different compounds of each variety are listed briefly. In Carmelo (CC), we find guaiacol, butyl butyrate, and 2-phenythyl acetate with fruity, banana, and spicy aromas, whereas Rojo Samuel (CR) had more volatiles, which were 2-nonanol, 2-octanone, hexadecanal, 2-undecanone, ethyl benzoate, methyl benzoate, and (E)-3-penten-2-one with green, fruity, floral, and sweet aromas. As for Lagarto (CL), no different compound was observed. In Arcoiris cocoa (TA), 1octanol, D-limonene and 2-phenylpropenal were found with sweet, citrus, and green aromas. Finally, 2-pentyl acetate (banana aroma) was found in Regalo de Dios (TRD). As is known, at the beginning of fermentation, alcohols and acids predominate and there is little presence of esters, as observed in this work. Regarding fermented cocoa (F6), the compounds detected will be described briefly. In Carmelo, cocoa beans (CC) with fruity, honey and cacao aromas, ethyl benzoate, ethyl palmitate, and 5-methyl-2phenyl-2-hexenal were found. As for Rojo Samuel (CR) with sweet, banana, chocolate, and banana aromas, ethanol, benzophenone, ethyl myristate, 2-acetophenol, isobutyl benzoate, and 2,5-dimethylpyrazine were found. In for Lagarto (CL) with creamy and cinnamon aromas. 2,3 Butanediol and benzenepropanol were detected. Pyridine, B-myrcene, 1-octanol, 2,4 pentadione, and 3-phenyl-2-propenal were found in Arcoiris (TA) with popcorn, nutty, creamy, and spicy aromas. Finally, in Regalo de Dios (TRD) with herbal, fruity, sweet, and cheesy aromas, methyl benzoate, 4-phenyl-3-buten-2-one, propanedioic acid, 1-pentanol, heptanoic acid, and isoamyl isovalerate were found. As can be observed esters and some pyrazines predominate in fermented cocoa. It has been reported that some pyrazines derive from the presence of the genus Bacillus during the fermentation process. In dried fermented cocoa beans (D3), the different compounds among the varieties of cocoa are mentioned in this paragraph. In Carmelo cocoa beans (CC) with pineapple and sweet aromas, ethyl acetate and 5-methyl-2-furancarboxialdehyde were detected, whereas in Rojo Samuel (CR) with nutty, butter, honey, herbal, and floral aromas, 1-octanol, 2-octanone, ethyl cinnamate, methyl benzoate, and ethyl 3phenylpropionate were to be found. In Lagarto (CL) with a fruity aroma, propanoic acid, ethyl benzoate, and (E)-cinnamaldehyde could be detected. In Arcoiris cocoa (TA) with sweet and fruity aromas, guaiacol, ethanol, 2-pentyl acetate, (E)-3-penten-2-one, isoamyl isovalerate, and 4-ethylbenzaldehyde were detected. Finally, in

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Regalo de Dios (RD) with fruity and chocolate aromas, hexanoic acid, 2-undecanone, 2-acetylpyrrole, and benzyl acetate were to be found. On the other hand, in dried unfermented cocoa beans (DU), the different compounds are enumerated briefly. Pyridine and 2,3-butanediol were found in Lagarto cocoa beans (CL) with popcorn and caramel aromas. Butyl butyrate (fruity aroma) was the only distinct compound found in Arcoiris cocoa (TA). In Carmel (CC), Rojo Samuel (CR), and Regalo de Dios (TRD), no different compounds were identified (Fig. 3A). In dried unfermented beans, there were lower numbers of compounds, as depicted in Table 2. In Figure 3B, differentiators per process step are presented from the common compounds, without considering those that were different; only 12 compounds overlapped. The compounds shared by stages during the fermentation and drying processes were mainly alcohols (isoamyl alcohol, phenylethyl alcohol, benzyl

alcohol,

2-heptanol),

aldehydes

(3-methylbutanal,

2-methylbutanal,

2-methylpropanal,

benzeneacetaldehyde, benzaldehyde), and a few ketones and acids (acetophenone, 2-nonanone and acetic acid), suggesting the important roles of these compounds in the flavor of dried fermented cocoa. At the beginning of fermentation (F0), the different compounds will be listed here. An alcohol (1-phenylethanol), two aldehydes (3-ethyl-benzaldehyde and 3-(methylthio)-propanal), an ester (isobutyl benzoate), and a furan (furfural) were detected. No different compounds were found at the end of the fermentation (F6). During the drying of fermented cocoa beans (D3), the different compounds included acids (isobutyric and isovaleric acid), an aldehyde (nonanal), and four esters (2-phenethyl acetate, ethyl octanoate, isobutyl acetate, and ethyl phenylacetate). In Fig. 3C, the differentiating compounds obtained from dry fermented and dried unfermented (DU) cocoa beans were compared, as well as those pertaining to common compounds. The compounds shared were eight compounds: two acids (acetic acid and benzoic acid), three alcohols (isobutanol, benzyl alcohol, and phenylethyl alcohol), an aldehyde (benzaldehyde), and two ketones (2-heptanone and acetophenone) were to be found in both processes. fermented dried cocoa beans were differentiated because they possessed the following compounds: two acids (isobutyric and isovaleric acid); three alcohols (2-pentanol, 2-heptanol and isoamyl alcohol); five aldehydes (nonanal, 3-methylbutanal, 2-methylbutanal, 2-methylbutanal and 2methylpropanal), a ketone (2-nonanone), and four esters (isoamyl acetate, ethyl octanoate, isobutyl acetate, ethyl phenylacetate, and 2-phenethylacetate), while in dried unfermented (DU) cocoa, the different compounds were an alcohol (1-pentanol), an aldehyde (4-ethyl-benzaldehyde), a ketone (butyrolactone), an ester (ethyl hexanoate), and a nitrile (benzonitrile).

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3.6. Principal Component Analysis (PCA) For further completing this study, a comparison of profiles of volatile compounds between the varieties and the processes was carried out with the aid of Principal Component Analysis (PCA). PCA can compress data based on their similarities and differences. The analysis was carried out with the most abundant volatile compounds of each variety. The Principal Components (PC) were chosen according to the greatest explanation of the variation of the data. For fresh cocoas at the beginning of fermentation (F0), PCA (Figs. 4a and 4b) explains 71.51% of the variation of the data in two components: PC1 (44.21%) and PC2 (27.30%). According to the statistical analysis of distribution of the scores (Fig. 4a) and loading plot (Fig. 4b), two of the Criollo cocoas, Carmelo (CC) and Lagarto (CL), were located on the positive axis of the PC1 component and were associated with a greater abundance of different aldehydes (benzeneacetaldehyde, 4-ethyl-benzaldehyde, 2-methylbutanal, 3methylbutanal, and (E)-2-methyl-2-butenal) and one alcohol, 2, 3-butanediol. Conversely, Rojo Samuel (CR) cocoa was located in the negative axis of the component (PC1) and exhibited a lower abundance of the previous compounds and a greater abundance of ketones (acetophenone, 2-heptanone, 2-nonanone, and 1(2-hydroxyphenyl)-ethanone), alcohols (2-pentanol, 2-methyl-3-buten-2-ol, 1-phenylethanol, 2-heptanol, and benzyl alcohol), and one hydrocarbon (beta-myrcene). On the other hand, Arcoiris (TA) and Regalo de Dios (TRD) cocoas were located on the positive axis of the PC2 component, with particular characteristics. Regalo de Dios (TRD) cocoa was correlated with a greater abundance of compounds such as 2-pentyl-furan, 1pentanol, acetic acid, and different aldehydes such as hexanal, octanal, 3-(methylthio)-propanal and (E,E)2,4-heptadienal (the latter in lower abundance in the Arcoiris cocoa (TA)), while 2-pentanone was more abundant in Arcoiris (TA). Finally, 2-methyl-3-buten-2-ol, ethyl alcohol, benzonitrile, and benzaldehyde demonstrated no significant differences in their abundance at the start of fermentation (F0). Finally, 2-methyl3-buten-2-ol, ethyl alcohol, benzonitrile, and benzaldehyde revealed no significant differences in abundance at the start of fermentation (F0). The Criollo cocoas showed a greater abundance of 2-methyl-1-propanol, phenylethyl alcohol, benzoic acid, and 2-methylpropanal than Trinitario cocoas. The predominant aromatic notes in the F0 stage for Criollo cocoa varieties were as follows: In Carmelo (CC) cocoa, sweet, malty, and fruity aromas predominated, but in Lagarto (CL) cocoa, there were aromatic notes to wine, in addition to the notes mentioned for CC. In the case of Rojo Samuel (CR) cocoa, the notes were caramelized, balsamic, fruity, floral, and almond. As for Trinitario cocoas, citrus and sweet notes were found in Arcoiris (TA), in addition to chocolate. almonds caramelized, and others in Regalo de Dios (TRD). The predominant aromatic notes in this stage (F0) for Criollo cocoa varieties were sweets, malty, and fruit trees for Carmelo (CC). Lagarto (CL) cocoa

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showed notes that ranged from aromatic to wine, in addition to the previous notes. In the case of Rojo Samuel (CR) cocoa, the notes were caramelized, balsamic, fruity, floral, and almond. As for Trinitario cocoas citrus and sweet notes were found in Arcoiris (TA) and chocolate, almond caramelized and other notes in Regalo de Dios (TRD). Figs. 4c and 4d present the distribution of the scores and the loading plot obtained from the Principal Components (PC) at the end of the fermentation (F6). Therein, the PC explain 72.86% of the variability of the major compounds in two components: PC1 (47.94%) and PC2 (24.92%). Regalo de Dios (TRD) and Lagarto (CL) cocoas were located in the positive axis of PC1 and were correlated with a greater abundance of aldehydes (including (E)-2-methyl-2-butenal, 2-methylbutanal, benzaldehyde, benzeneacetaldehyde, 3(methylthio)-propanal, E-cinnamaldehyde, 4-ethyl-benzaldehyde, and 3-ethyl-benzaldehyde), acids (isovaleric acid, isobutyric acid, acetic acid, and benzoic acid), alcohol (benzyl alcohol, guaiacol, propanoic acid biphenyl, and benzyl alcohol), esters (methyl 3-methylbut-2-enoate and hexyl butyrate), and 1-(3-methylphenyl)ethanone, a ketone. Conversely, Criollo Rojo Samuel (CR), and Carmelo (CC) cocoas were located in the negative axis of PC1, revealing a greater abundance of alcohols (2-methyl-3-buten-2-ol, 2-heptanol and 2methyl-1-propanol) and a ketone, 1-(4-ethylphenyl)-ethanone, and a lower abundance of the compounds was present in Regalo de Dios (TRD) and Lagarto (CL) cocoas. On the positive axis of PC2, Arcoiris (TA) cocoa was observed, which was correlated with a higher abundance of alcohols (1-phenylethanol, 2-pentanol, phenylethyl alcohol, and 3-methyl-1-butanol), ketones (acetophenone and (E)-3-penten-2-one 2-undecanone), esters (ethyl octanoate, 2-phenethyl acetate, ethyl decanoate, isobutyl benzoate, and ethyl pentanoate), acids (ocatanoic acid), and aldehydes (nonanal). Some compounds exhibited particular behaviors. For example, 2ethyl-5-methyl-furan, acetoin, and ethyl phenylacetate were present with equal abundance for Arcoiris (TA) and Lagarto (CL) cocoas. The 2-methyl-benzaldehyde demonstrated greater abundance for Arcoiris (TA) and Regalo de Dios (TRD) cocoas. Benzonitrile did not show a statistically significant difference among the varieties of cocoas. The volatile compounds present in this stage are related to the aromatic notes that are required in products a high aromatic quality in the market of gourmet chocolates. Finally, a comparison between the volatile compounds of the fermented dry grains (D3) and dried unfermented grains (DU) (Figs. 5a and 5b) is offered. PC explain 57.66% of the variability of the data in two components: PC1 (41.95%) and PC2 (15.77%). Lagarto (CL) and Regalo de Dios (TRD) cocoas were located at the positive axis of PC1. Lagarto (CL) cocoa showed a greater relationship with compounds with greatest abundance with two pyrazines (2,3-dimethylpyrazine and trimethylpyrazine) and an aldehyde (benzaldehyde), while in Regalo de Dios (TRD) cocoa, there was greater correlation with 5-methyl-2-phenyl-2-hexenal, benzyl

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alcohol, and benzoic acid. All dried unfermented cocoas (DU) were located on the negative axis of PC1, where the previous compounds were not observed. DU cocoas correlated well with various alcohols (isopropyl alcohol, 1-hexanol, 3-hexen-1-ol, and 2-methyl-3-buten-2-ol) and some ketones (butyrolactone), hydrocarbons (beta-myrcene, D-limonene, beta-pinene) and an ester (isoamyl lactate), which were more abundant in these cocoas. On the other hand, from the positive axis of PC2, dried fermented cocoas (D3), Rojo Samuel (CR) was found to correlate with the compounds in greater abundance, such as the pyrazines, 2,5-dimethylpyrazine and methyl pyrazine, an alcohol (2-heptanol), and an aldehyde (2-methyl-benzaldehyde), while a lower abundance of these compounds was shown in Carmelo (CC) and Arcoiris (TA) cocoas. Carmelo (CC) cocoa showed a strong correlation with two aldehydes, (E)-2-methyl-2-butenal and 2-methylbutanal) and one furan (2-ethyl-5-methyl-furan) which were more abundant for this cocoa. On the other hand, Arcoiris (TA) cocoa correlated highly with compounds in greater abundance, such as some aldehydes (3-ethyl-benzaldehyde and 4-ethyl-benzaldehyde), esters (ethyl acetate and ethyl hexanoate), alcohols (1-pentanol and 2-pentanol,) and a ketone (1- (4-ethylphenyl)-ethanone). The Cluster Analysis (CA) was carried out to show the affinity of the sample, according to the type of cocoa (Criollo or Trinitario) or the process (fresh (F0), fermented (F6), dried fermented (D3) or dried unfermented (DU)) (Figure S1). We can observe that the samples were grouped according to the process and not to the type of cocoa. These observations confirm the results of the PCA, whereby the process has a greater influence on the profile of volatile compounds and, to a lesser extent, on the type of cocoa. Furthermore, based on the Euclidean distance, it can be concluded that the volatile compound profile is different in most varieties of cocoa.

4. Conclusions This study highlights not only the importance of the process of fermentation of cocoas but also reveals the particular characteristics of the chemical composition of the volatile compounds of five varieties of fine-aroma cocoas (Carmelo, Rojo Samuel, Lagarto, Arcoiris, Regalo de Dios) from the Maya lands in Chiapas, Mexico. The statistical analyses allowed the differentiation and characterization of the samples, with a particular emphasis on the impact on the fermentation process. The results show that there is a large number of volatile compounds present in fermented dry beans and that these are not present in dried unfermented (DU) beans. These compounds, along with the key-aroma and the technological-markers, strongly suggest that the fermentation process comprises a primordial stage

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in the generation of the volatile compounds, which provide desirable aromatic notes in cocoa products, such as dark chocolate. In this regard, fermented dry cocoa beans were characterized as having a greater number of compounds such as esters, aldehydes, pyrazines, alcohols, some acids, and furans. DU cocoas beans presented a lower number of compounds and these were mainly alcohols, ketones, hydrocarbons, and certain other compounds such as nitrile, pyridine, ester, and lactone. Last, a comparison among cocoa varieties revealed a specific composition for each cacao, highlighting the importance of the initial composition and postharvest processes. These preliminary results show that the cocoa Lagarto (CL), Rojo Samuel (CR), and Regalo de Dios (TRD) are the varieties that generate the largest area of the main volatile compounds that give rise to desirable aromatic profiles.

Acknowledgments This research was supported by SAGARPA-CONACYT (Mexican National of Science and Technology) Project No. 2017-02-291417: “Desarrollo de innovaciones tecnológicas para el manejo integral sustentable del cultivo de cacao (Theobroma cacao L.) en Mexico. Utrilla-Vázquez thanks to PRODEP-SEP, México by PhD scholarship grant.

Conflict of interest The authors declare that they have no conflicts of interest.

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TABLE LEGENDS Table 1. General characteristics of the different cacao varieties used in this study. aAroma

descriptors were obtained for each compound detected using the online databases Flavornet

(http://www.flavornet.org/flavornet.html),

The

Good

Scents

Company

(http://www.thegoodscentscompany.com/indeX.html), and the literature.

Table 2. Volatile compounds identified by Headspace-Solid Phase Microextraction-Gas Chromatography Mass Spectrometry (HS-SPME GC-MS) during different fermentation times (F0 and F6) and drying (D3) and, dried unfermented (DU), of the different types of Criollo and Trinitario cocoa bean samples.

FIGURE LEGENDS Figure 1. Volatile compounds identified by HS-SPME GC–MS during the fermentation and drying of five varieties beans cacao. (A) Number of compounds in major classes of volatile in cacao samples. (B) Area units of major classes of volatile compounds. (C) Relative concentration of major classes of volatile compounds. CC: Criollo-Carmelo; CR: Criollo-Rojo Samuel; CL: Criollo Lagarto; TA: Trinitario Arcoiris; TRD: TrinitarioRegalo de Dios. Fermentation times: 0 (F0) and 6 (F6) days. Dried fermented beans (D3) and dried unfermented beans (DU).

Figure 2. Levels of significant the key-aroma and technological marker in Criollo-Carmelo (CC), Criollo-Rojo Samuel (CC), Criollo-Lagarto (CL), Trinitario-Arcoiris (TA), and Trinitario-Regalo de Dios (TRD) beans at different steps process. Levels of each metabolite are presented as area units.

22

Figure 3. Venn diagram showing the similarities and differences of volatile compounds. A) Similarities and differences of volatile compounds between varieties cacao. B) Similarities and differences of volatile compounds between processing steps. C) Similarities and differences of volatile compounds between dried fermented and unfermented cacao beans The Venn diagram was generated with the web tool provided by the Bioinformatics and Systems Biology of Gent (URL: http://bioinformatics.psb.ugent.be/webtools/Venn/). CC: Criollo-Carmelo; CR: Criollo-Rojo Samuel; CL: Criollo Lagarto; TA: Trinitario Arcoiris; TRD: Trinitario-Regalo de Dios. Fermentation times: 0 (F0) and 6 (F6) days. Dried fermented beans (D3) and dried unfermented beans (DU).

Figure 4. Principal component analysis. Score plot (a) and loading plot (b) of PC1 and PC2, from volatile compound in the samples at the beginning of fermentation at F0. Score plot (c) and loadings plot (d) of PC1 and PC2, from all volatile compounds in the samples at the end of fermentation (F6).

Figure 5. Principal component analysis. Score plot (a) and loadings plot (b) of PC1 and PC2, from all volatile compound in the samples of fermented and dried beans (D3), and dried unfermented beans (DU).

23

Figure 1. (A)

(B)

(C)

Fermentation and drying days

24

Figure 2.

25

Figure 3.

26

Figure 4.

27

Figure 5.

28

Table 1. Samle codes1

Type and variety cacao

Pod weight (g)

Pod length (cm)

Pod width (cm) 8.33+0.52

Beans number per pod 35+7

Beans weight per pod (g) 130.61+23.36

CC

Criollo Carmelo

544.48+105.7 7

18.20+1.47

CR

Criollo Rojo Samuel

533.18+127.2 2

19.03+2.39

7.72+0.84

32+6

105.71+36.17

CL

Criollo Lagarto

663.38+124.4 8

24.50+2.67

7.94+0.63

28+6

129.59+49.07

Photo

29

1

TA

Tinitario Arcoíris

508.67+112.7 2

19.63+1.12

8.24+0.44

35+6

100.09+25.92

TRD

Trinitario Regalo de Dios

400.09+93.30

18.76+1.40

7.45+0.57 a

32+9

79.02+19.61

Data are expressed as mean of fifty pods ± standard deviation.

30

Compound

CC

CR

CL

TA

TRD

CC CR CL TA TRD

F0 F6 D3 F0 F6 D3 F0 F6 D3 F0 F6 D3 F0 F6 D3

DU

Odor descriptora

Acids Acetic acid Propanoic acid Isobutyric acid Isovaleric acid Hexanoic acid Propanedioic acid Heptanoic acid Octanoic acid Benzoic acid Alcohols Isopropyl alcohol Ethanol 2-Methyl-3-buten-2-ol Isobutanol 2-Pentanol Isoamyl alcohol 1-Pentanol 2-Heptanol 3-Ethyl-2-pentanol 1-Hexanol 3-Hexen-1-ol 2-Nonanol

X

X

X

X X

X

X

X

X

X

X

X

X

X

X

X X

X X

X X

X

X X X X

X X X

X

X

X

X

X

X

X

X

X

X

X X X X X

X X X X X X X

X X X

X

X

X X X X

X

X

X

X X X X X

X

X

Phenylethyl alcohol

X

X

Guaiacol

X

X

X X X X

X

X

X

X

X

X

X X

X

X

X

X

X

Vinegar, peppers, green, sour

X

Pungent, rancid, soy Rancid, butter, cheese

X

X

X

Sweat, rancid, blue cheese,

X X

X X

X X X X X

X X X X X X X

X X

X X X X X

X X

X

X X X X X X X

X X

X X X X

X X X X X

X X X

X X

X X X X X

X

X

X

X

X X

X X

X

X X

X

X

X

X

X

X

X X

X X

X

X

X

X

X X

X X

X

X

X X

X

X

X

X

X

X

X

X

X

X

X

Green, fruity

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Sweet, flowery

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Honey, rose, flowery, caramel

X

X

X

X

X

Smoky, phenol, spicy

X X X X X X

X X X

X X X

X

X

X X

X

Benzaldehyde 5-Methyl-2furancarboxaldehyde

X

X

2-Methyl-Benzaldehyde Benzeneacetaldehyde Hexadecanal 3-Ethyl-benzaldehyde 4-Ethyl-benzaldehyde 2-Phenylpropenal 3-Phenyl-2-propenal (E)-Cinnamaldehyde

X X X X

X X X X X X

X X X

X X X

X X X

X X X

X

X

X

X

X X

X X

X

X

X

X

X

X X

X

X

Spicy, cinnamon, fruity, floral

X X X

X

X

X

Alcohol, musty, woody Ethanol-like, sweet Herbal Wine, solvent, bitter, green Light, seedy, sharp, green Burnt, malt, whiskey Sweet, pungen Sweet, citrusy Fruity, green Grassy, green Fat, green Nut, sweet, flowery Fruity, creamy, buttery

X X

Sweat, pungent Rancid, sour, cheesy, sweat Sweaty, fatty Urine

X

Benzenepropanol

Ketones 2-Pentanone

X X

X X X

2-phenylbut-2-enal 5-Methyl-2-phenyl-2hexenal

X

X

1-Phenylethanol Benzyl alcohol

3-(Methylthio)-propanal (E,E)-2,4-Heptadienal

X

X

1-Octanol 2,3-Butanediol

Aldehydes 2-Methylpropanal 2-Methylbutanal 3-Methylbutanal Hexanal (E)-2-Methyl-2-butenal Octanal Nonanal

X

X X X X X X X X X

X X X

X X X

X

X

X X

X

X

X

X

X X

X

X

X

X X X X

X

X X

X X X

X

X

X X X

X X X

X

X

X X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X X X X X X X X X

X

X

X X

X X

X X

X

X

X

X

X

X X

X X

X X

X X

X X X X X

X X

X X

X X

X X

X X

X

X

X

X X

X X

X

X

X

X X

X X X

X

X

X

X

Cherry Green Honey floral rose sweet Fruity Green spicy Cinnamon, paint Floral, honey, powdery, cocoa

X X

Candy, almond, burnt sugar Caramellic, bready, coffee-like

X X

X X

Malt and chocolate Malt and chocolate Malt, dark chocolate, cocoa Green, herbal, grassy, tallow Fruity, green Fatty, sharp Fatty, waxy, pungent Sulfurous, onion, vegetable Nut, fat

Cocoa X

X

X

X

Sweet, fruity, cheesy

31

(E)-3-Penten-2-one 2-Heptanone 2,4-Pentanedione 2-Octanone Acetoin 2-Nonanone 2-Undecanone Butyrolactone Acetophenone 3-Methylacetophenone 2-Acetophenol 4-Phenyl-3-buten-2-one 1-(4-Ethylphenyl)ethanone

X

X X

X X

X X

X X

X X

X

X

X

X

X

X X X

X X

X X

X

X X X

X

X

X X

X

X

X

X X X

X

X X

X X

X X

X X

X

X

X

X

X

X

X

X

X

X

X

X X

X

X X

X

X

X X

X

X

X

X X X

X X

X

X

X

X

X

X

X

X

X

X

X X

X X

X

X

X X

X

X X

X

X X

Benzophenone Esters Ethyl acetate Isobutyl acetate Methyl isovalerate Ethyl pentanoate 2-Pentyl acetate Isoamyl acetate Ethyl hexanoate Isoamyl isovalerate Ethyl heptanoate Isoamyl lactate Hexyl butyrate Ethyl octanoate Ethyl decanoate Methyl benzoate Ethyl benzoate Benzyl acetate Methyl phenylacetate Ethyl phenylacetate 2-Phenethyl acetate Isobutyl benzoate Butyl butyrate Ethyl 3phenylpropionate Ethyl myristate Ethyl cinnamate Ethyl palmitate Furans 2-ethyl-5-methyl-furan 2-pentyl-furan Furfural 2-ethenyl-benzofuran Hydrocarbons α-Pinene β-Myrcene D-Limonene Biphenyl Nitriles Benzonitrile Pyridines Pyridine Pyrroles 2-Acetylpyrrole Pyrazines Methylpyrazine 2,5-dimethylpyrazine 2,6-dimethylpyrazine 2,3-dimethylpyrazine Trimethylpyrazine

X

X

X

X

X

X

X

X

X X

Balsam, rose, geranium

X X X

X X

X

X X X

X

X

X X X

X X

X

X

X

X X

X X X

X X X X

X

X

X

X

X

X

X

X

X X

X

X

X X

X X

X X

X

X X

X X X

X

X X X

X X

X

X X X

X

X X

X X

X

X

X

X X

X

X X

X X X

X X X

X

X X X

X

X X

X X X X

X X X

X

X

Fresh, floral, jasmine

X X X X

Sweet, honey, jasmine Fruity, sweet, honey-like Fruity, sweet, floral Balsamic Fruity

X

X

Floral

X

Waxy, soapy Honey, cinnamon Waxy, green

X X

X X X

X

X

X X

X

X X X

X

X

X X X

X

X

X

X

X

X

X

X

X

Fresh gassy burnt Green bean, butter, fruity Bread, almond, sweet X

X X

X

X

X X

X

X

X

X

X

X X X

X X

X

X X X X

X X

X X X

X

X

X X

X X X

Pine, turpentine Balsamic, must, spice Citrus Pungent rose green geranium

X

X

Almond

X X

X

X

X

X

X

Pineapple Fruity, apple, banana Fruity Fruity, apple Banana Fruity, banana Apple peel, fruit Fruity Fruity Fruity creamy nutty Apple peel Fruity, floral, pineapple Pear, grape, brandy Ppune, lettuce, herb, sweet Camomile, flower, celery, fruity

X

X

X

Phenolic, sharp, benzaldehyde Sweet, spicy, cinnamon, fruity Sweet, anisic, vanilla-like

X X X X X

Fruity acetone phenolic fishy Pear, grape, brandy, fruity, floral Cream, butter, Blue cheese, Butter, cream Buttery, sour milk, caramel Fruity, musty Fruity Sweet, aromatic, creamy Flower, almond, pungent, sweet

X X

Pop corn

X

Chocolate, hazelnut

X X X X X

Nutty, chocolate, cocoa, roasted Chocolate, nutty Nutty, herbal Caramel, cocoa Cocoa, rusted nuts, peanut

32

Tetramethylpyrazine

X

X

X

Chocolate, cocoa, coffee

33

Graphical abstract

34

Highlights 

The volatile compounds of fermented and unfermented cocoa from five varieties of Mayan cocoa are different.



Fermented cocoa beans obtained showed different volatile compounds.



The quantity and quality of the profile of volatile compounds depend of the process and not of the cocoa variety.

35

Author contributions Maycarmen Utrilla carry out out the experiments and wrote the manuscript. Jacobo Rodriguez helped to carry out the GC analysis and helped with stadistical analysis. Carlos Hugo Avendaño helped to carry out the experiments in INIFAP, Chiapas. Anne Gschaedler helped with the project supervision. Eugenia Lugo designed the project and helped to supervise the research and draft the manuscript.

36

Zapopan, Jalisco, México, October 10th, 2019.

Conflict of Interest form

We have no conflicts of interest to disclose. The manuscript has been read and approved for submission by all the named authors.

37