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Molecules 2017, 22(5), 766; https://doi.org/10.3390/molecules22050766

Article
Impact of a Microbial Cocktail Used as a Starter Culture on Cocoa Fermentation and Chocolate Flavor
1
Post-Graduate Program in Food Science, Federal University of Lavras, Lavras 37.200-000, Minas Gerais, Brazil
2
Post-Graduate Program in Agricultural Microbiology, Federal University of Lavras, Lavras 37.200-000, Minas Gerais, Brazil
3
Department of Chemical Sciences and Natural Resources, Centro de Excelencia en Investigación Biotecnológica Aplicada al Medio Ambiente (CIBAMA), Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco 4811-230, Chile
4
CEB-Centre of Biological Engineering, Micoteca da Universidade do Minho, University of Minho, 4710-057 Braga, Portugal
*
Author to whom correspondence should be addressed.
Academic Editor: Luca Forti
Received: 29 January 2017 / Accepted: 2 May 2017 / Published: 9 May 2017

Abstract

:
Chocolate production suffered a vast impact with the emergence of the “witches’ broom” disease in cocoa plants. To recover cocoa production, many disease-resistant hybrid plants have been developed. However, some different cocoa hybrids produce cocoa beans that generate chocolate with variable quality. Fermentation of cocoa beans is a microbiological process that can be applied for the production of chocolate flavor precursors, leading to overcoming the problem of variable chocolate quality. The aim of this work was to use a cocktail of microorganisms as a starter culture on the fermentation of the ripe cocoa pods from PH15 cocoa hybrid, and evaluate its influence on the microbial communities present on the fermentative process on the compounds involved during the fermentation, and to perform the chocolate sensorial characterization. According to the results obtained, different volatile compounds were identified in fermented beans and in the chocolate produced. Bitterness was the dominant taste found in non-inoculated chocolate, while chocolate made with inoculated beans showed bitter, sweet, and cocoa tastes. 2,3-Butanediol and 2,3-dimethylpyrazine were considered as volatile compounds making the difference on the flavor of both chocolates. Saccharomyces cerevisiae UFLA CCMA 0200, Lactobacillus plantarum CCMA 0238, and Acetobacter pasteurianus CCMA 0241 are proposed as starter cultures for cocoa fermentation.
Keywords:
chocolate quality; GC-MS; sensory analysis; culture-independent analysis; starter culture

1. Introduction

The cocoa (Theobroma cacao L.) supply chain for the production of chocolate is complex. It involves several post-harvest steps, which can determine the quality of the final product. In Brazil, cocoa production suffered a vast impact with the emergence of “witches’ broom” disease [1,2]. In order to recover the cocoa production, many disease-resistant hybrid plants, such as PH9, PH15, PH16, PS1030, PS1319, CCN51, CEPEC2002, CEPEC2004, and FA13, have been developed [3,4].
As a matter of consequence, different cocoa hybrids generate cocoa beans that produce chocolate with variable quality [5,6,7]. In this context, PH15 hybrid has great relevance due to high-productivity, adaptation, and resistance to some diseases, such as “witches’ broom” and ceratocystis wilt [8,9,10].
The fermentation of cocoa beans is a microbiological process with enzymatic activity and the development of chocolate flavor precursors [11,12,13]. This traditional process is spontaneous and uncontrolled. After opening of the cocoa pods, the beans are transferred to the area of fermentation and placed in heap or fermentation boxes. These methods are the most commonly used among the cocoa producer countries [14,15].
Yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) are the main microbial communities involved during cocoa fermentation. Yeast species are reported as the primary colonizers of cocoa fermentation. Saccharomyces, Hanseniaspora (anamorph Kloeckera), and Pichia are the prevalent genera found in cocoa fermentation in different countries. Saccharomyces cerevisiae is particularly the most reported species in many fermentations [16,17,18,19,20].
Simultaneously with the yeast growth, LAB colonize the cocoa mass and degrade the pulp’s glucose into lactic acid and assimilate the citric acid also present in the pulp. Several studies concerning the microbial fermentation reported two LAB species as the most prevalent in this process: Lactobacillus plantarum and Lactobacillus fermentum [19,20,21].
Yeast populations, which are responsible for the ethanol production, decline together with the LAB populations. AAB dominates the process and are responsible to the exothermic reaction of ethanol conversion into acetic acid. Acetobacter pasteurianus is the most frequent species of AAB found in cocoa fermentation, but other species, such as Acetobacter aceti, Acetobacter ghanensis, Acetobacter fabarum, Gluconobacter oxydans, and Gluconobacter xylinus, have also been reported in the literature [16,17,20,22].
Species of Bacillus (e.g., Bacillus subtilis, Bacillus megaterium, and Bacillus flexus) may also grow during fermentation and can affect bean quality and cocoa flavor [16,17,23].
Different compounds, such as alcohols (e.g., 2-methyl-1-propanol, 2-phenylethanol, methanol), aldehydes (e.g., acetaldehyde, benzaldehyde), ketones (e.g., 2-pentanone, phenylmethyl ketone), esters (e.g., ethyl acetate, 2-phenylethyl acetate), and carboxylic acids (e.g., butanoic acid, nonanoic acid), are produced during fermentation, affecting the final flavor character in chocolate [13,17,24,25,26].
The aim of this work was to use a cocktail of microorganisms as a starter culture on the fermentation of the ripe cocoa pods from PH15 cocoa hybrid, and evaluate its influence on the microbial communities present on the fermentative process, on both the volatile and non-volatile compounds produced during the fermentation, and to perform the chocolate sensorial characterization.

2. Results

2.1. Culture-Independent Analysis with PCR–DGGE

Analyses of the microbial communities on both inoculated and non-inoculated samples of the PH15 cocoa hybrid were performed by Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis (PCR–DGGE) for prokaryote (Figure 1A) and eukaryote (Figure 1B) microorganisms. Identification of DGGE bands are shown in Table 1.
The bacterial and yeast communities changed according to different fermentation processes (inoculated or non-inoculated). The bacterial species Gluconobacter oxydans (bands 22–24), Lactobacillus plantarum (bands 3, 4, 8, and 9), uncultured bacterium (bands 11–15), Acetobacter pasteurianus (bands 15–20), and Fructobacillus pseudoficulneus (bands 1, 5, and 6) were detected for both inoculated and non-inoculated fermentations.
The species Leuconostoc sp. (band 2), Zymomonas mobilis (Band 10), Acetobacter sp. (Bands 21), Bacillus sp. (Bands 25 and 26) were detected in the non-inoculated fermentation. The Lactobacillus helveticus (Band 7) was only detected in inoculated fermentation. The yeast species Saccharomyces cerevisiae (Bands 3 and 5–10) and Pichia kluyveri (Bands 15–18 and 20) were detected for both fermentations. Saccharomyces sp. (Band 4), Hanseniaspora uvarum (Bands 1 and 2), and Theobroma cacao (Band 11) were only detected in the non-inoculated fermentation: this was possible because universal eukaryote primers were used. In the inoculated fermentation Rhodotorula mucilaginosa (Bands 12 and 13), Trichosporon asahii (Band 19), and Lentinula edodes (Band 14) were detected.

2.2. Chemical Changes During Fermentation

Temperature and pH values were measured during 120 h of fermentation. In the fermentation of PH15 NI the temperature varied from 30.7 °C at 0 h (with the maximum of 49.1 °C at 96 h) to 47.9 °C at 120 h. On the other hand, in the fermentation of PH15 I, the temperature varied from 26.62 °C at 0 h (with the maximum of 50.06 °C at 72 h) to 49.88 °C at 120 h. The pH value outside the bean (pulp) varied during fermentation. The pH value of PH15 NI ranged from 3.27 to 4.45, and the fermentation PH15 I pH value ranged from 3.27 to 4.81.

2.2.1. Sugar Consumption and Metabolite Production

During the six days of fermentation the concentrations of glucose, fructose, and citric acid were evaluated in the pulp, and the results are shown in Figure 2. Citric acid was fully metabolized at 24 h of fermentation in both assays (Figure 2A,B). In both fermentations, glucose and fructose were completely consumed at 72 h. After 24 h of inoculated fermentation the PH15 sample showed greater sugar consumption compared to the non-inoculated one, but in the following hours, both inoculated and non-inoculated fermentations presented the same profile of sugar consumption (Figure 2A,B).
Ethanol, lactic acid, and acetic acid were evaluated in the pulp and inside the beans, and are shown in Figure 3. The inoculated fermentation of PH15 showed the maximum value of ethanol in the pulp (8.44 g/kg at 48 h), and this compound inside the beans also reached higher values (6.65 g/kg at 48 h) when compared to the control fermentation (PH15 NI) (Figure 3A). However, at the end of the fermentation, the ethanol concentration was higher (4.34 g/kg) inside the beans in the non-inoculated fermentation.
The microbial inoculation accelerated the sugar consumption in the first 24 h of cocoa fermentation, whereas the ethanol production was accelerated in the first 48 h of fermentation (Figure 2B and Figure 3A). The pulp of PH15 I showed the highest acetic acid concentration between 48 h and 120 h of cocoa fermentation, and the lowest concentration at the end of the fermentation (144 h, Figure 3C). In contrast, PH15 NI showed the highest acetic acid concentration in the residual pulp at the end of the fermentation (144 h, Figure 3C). Overall, the concentration of acetic acid was higher in the fermentation of PH15 I (Figure 3C).
After 144 h, the pulp of PH15 I showed greater concentration of lactic acid than the pulp of PH15 NI (Figure 3B). In both PH15 I and PH15 NI samples there was no lactic acid penetration in the cotyledon. Acetic acid was higher inside the beans in PH15 I at later stages, at 120 h (2.70 g/kg), and at 144 h (2.53 g/kg).

2.2.2. Volatile Compounds

A total of 37 volatile compounds were detected by Gas Chromatography Mass Spectrometry (GC–MS) at the beginning (0 h), and a total of 38 volatile compounds at the end (144 h), of the non-inoculated fermentation. While in the inoculated fermentation, a total of 37 volatile compounds were detected at the beginning (0 h) and a total of 34 volatile compounds at the end (144 h), as presented in the Table 2.
Both non-inoculated and inoculated hybrid PH15 (0 h and 144 h) showed the following identified compounds: aldehydes and ketones, acids, alcohols, esters, terpenoids, and furans. Aldehydes and ketones occurred at the beginning (0 h) and esters occurred at the end (144 h). In both fermentations, the most important groups of volatile compounds were detected (Figure 4).
Chocolate samples of PH15 I and PH15 NI presented 58 and 54 volatiles compounds, respectively (Table 2 and Figure 4). The compounds identified were aldehydes and ketones, acids, alcohols, esters, pyrazines, pyrroles, and furans, and the most important groups detected, in both chocolate samples, were aldehydes and ketones, alcohols, and pyrazines (Figure 4).

2.3. Sensorial Analyses of Chocolate

The chocolate analyses by the Temporal Dominance of Sensations (TDS) technique are shown in Figure 5. The judges noted difference between the two samples of chocolate during the tasting time. The bitterness was the dominant taste in the final time (25–35 s) of PH15 NI Ch (no-inoculated chocolate). However, the fruity and cocoa flavors were significant at 17 and 22 s, respectively (Figure 5A).
The sample PH15 I Ch showed a mixture of sensations, alternating between bitterness, cocoa taste and sweetness (Figure 5B). The bitterness is the more dominant taste in the initial (5 to 10 s) and final time (25 to 35 s), while the cocoa taste was dominant in the intermediate (15 to 20 s) and final time (25 to 35 s). The sweetness taste showed significant levels in the final time (25 to 35 s) (Figure 5B).

3. Discussion

In order to evaluate their influence on the fermentation of cocoa beans and on the final sensorial characteristics of produced chocolate, S. cerevisiae UFLA CCMA 0200, Lactobacillus plantarum CCMA 0238, and Acetobacter pasteurianus CCMA 0241 were used as starter cultures for the cocoa PH15 fermentation. The organic compounds and microbial communities involved during the fermentation of non-inoculated and inoculated cocoa were analyzed. Furthermore, the sensorial characterization of chocolate (PH15 I Ch and PH15 NI Ch) produced from the fermented beans was evaluated.
The PCR–DGGE analyses showed that the bacterial and yeast communities were different according to the fermentation process. This may be explained by the use of starter cultures that may have generated changes in the natural microbiota, as shown in Figure 1A,B. Species of LAB and AAB were identified in both fermentations.
The L. plantarum and Fructobacillus pseudoficulneus species were the bacteria present in both fermentations. However, in PH15 I these species were only identified until in the middle of the fermentation period (72 h), but after this time, they seem not to be present. It is reported that the increase of ethanol concentration during cocoa fermentation inhibited L. fermentum growth [15,25]. This could explain the low population rate of L. plantarum in PH15 NI.
Gluconobacter oxydans and A. pasteurianus were AAB species identified in both fermentation processes. In addition, A. pasteurianus (Figure 1A—bands 15, 16, 17, 18, 19, and 20) was present at all fermentation times of PH15 I, and this did not happen in the fermentation without inoculum. These species have been described in cocoa bean fermentation in Brazil, Ghana, and Indonesia [3,16,19,20].
The LAB species Leuconostoc sp., Lactobacillus helveticus were detected in non-inoculated and inoculated fermentations, respectively. The Zymomonas mobilis, an ethanol strain producer, Acetobacter sp., an AAB species, and Bacillus sp. were only detected in non-inoculated fermentation (Figure 1A). The Bacillus sp. present in PH15 I fermentation can be explained as the fermentation was not in aseptic conditions. Bacillus sp. and filamentous fungi may participate in the spontaneous cocoa bean fermentation process after four or five days of fermentation [14,16,17,20,25].
Fingerprinting based on PCR–DGGE showed that the most common yeast Saccharomyces cerevisiae was present during the fermentation in both fermentations. This fact indicated that this yeast may be a promising starter culture used for the cocoa fermentation process. Some works using S. cerevisiae as a starter culture have been reported [4,7], and concluded that yeast inoculation accelerated the fermentation process.
The microbial activity and metabolites produced during the cocoa beans’ fermentation leads to an increase of temperature and pH value [19]. Therefore, this may explain the temperature and pH increase in both fermentations, but in PH15 I there was a greater increase.
According to the chemical results, carbohydrates were consumed faster in the inoculated assay (Figure 2B). This is likely due to the higher microorganism population in the inoculated assay than in the control. Further, higher ethanol concentrations (almost two times the concentration detected in the control) were observed in this assay (Figure 3A). However, this was not the case for acetic acid, similar to results previously described elsewhere [4]. However, in inoculated fermentation, acetic acid was detected in the cotyledon at the end of fermentation (Figure 3C). Sucrose was not detected in the fermentation probably because it was hydrolyzed into glucose and fructose when the pods were broken open, as previously described [4].
In addition, to produce primary metabolites, such as ethanol, and lactic and acetic acids during cocoa fermentation, starter cultures also produce a vast array of volatile secondary metabolites, such as higher alcohols, acids, esters, aldehydes, ketones [24,27], and others that could influence cocoa flavor [25].
The most important volatile compound groups detected were esters and alcohols, in both fermentations (Table 2 and Figure 4). These compounds are already described as important in cocoa products [24]. The esters are correlated to fruity notes [28] and the alcohols with flowery and candy notes [29,30], e.g., 2,3-butanediol, 2-heptanol, guaiacol, benzyl alcohol, and phenylethyl alcohol found in this study, being that the latter two compounds were found in all chocolate process stages of both fermentations (PH15 I and PH15 NI) (Table 2) [4].
Acids are generally related to unpleasant odors present in cocoa products [24,30,31]. A total of five compounds were identified, some being related to rancid, sour, or fatty odors. However, some acids detected here may present pleasant odors, e.g., 4-hydroxybutanoic acid and hexanoic acid, with sweetish odors, as shown in Table 2 [4].
In order to investigate the influence of a starter culture on the final product, two chocolates were produced and their sensory analyses were evaluated. The judges noted differences between the two chocolate samples (PH15 I Ch and PH15 NI Ch) during the tasting time. Significant differences were observed as described in Figure 5. Bitter was the dominant taste in PH15 NI Ch (Figure 5A) and, in PH15 I Ch, bitter, sweet, and cocoa tastes were dominant (Figure 5B). These results can be corroborated by the analysis of the volatile compounds in the chocolate samples. The 2,3-butanediol, which gives flavor to cocoa butter (sweet chocolate), and 2,3-dimethylpyrazine, which gives caramel and cocoa flavors, were detected only in PH15 I Ch (Table 2). Therefore, that dominant flavor detected in PH15 I Ch could be related to these compounds, indicating the inoculation influence in the final product.

4. Materials and Methods

4.1. Fermentation Experiments, Inoculation, and Sampling

The fermentation experiments were conducted at the Vale do Juliana cocoa farm in Igrapiúna, Bahia, Brazil. The ripe cocoa pods from PH15 were harvested during the main crop of 2015 (November).
The cocoa pods were manually opened with a machete, and the beans were immediately transferred to the fermentation house. The fermentation started approximately 4 h after the breaking of the pods and was performed in 0.06 m3 wooden boxes [17]. The fermentations were conducted with 100 kg of PH15 cocoa beans. The fermentations were performed using a cocktail of microorganisms (PH15 I) as starter culture containing S. cerevisiae UFLA CCMA 0200 (LNF-CA11, LNF Latino America, Bento Gonçalves, Rio Grande do Sul, Brazil), Lactobacillus plantarum CCMA 0238 and Acetobacter pasteurianus CCMA 0241 at the beginning of the process and without inoculation (PH15 NI-control). These microorganisms were reported in previous studies on cocoa fermentation around the world, mainly in Brazil [3,16,18,20,21]. The pH value and temperature were evaluated during the fermentations.
All of the microbial strains used in the study are preserved at the Culture Collection of Agricultural Microbiology of the Federal University of Lavras (CCMA, Lavras, Minas Gerais, Brazil, WDCM 1083). The S. cerevisiae UFLA CCMA 0200, which is commercialized by LNF (CA11), was weighed (as recommended by the manufacturer’s instructions) and mixed in the solution to reach a population of approximately 107 cells/g of cocoa.
The Lactobacillus plantarum and Acetobacter pasteurianus species were grown in MRS broth (De Man, Rogosa and Sharpe, Merck, Darmstadt, Germany) and YPD broth (10 g/L yeast extract (Merck); 20 g/L peptone (Himedia); 20 g/L dextrose (Merck)), respectively, at 30 °C and 150 rpm, and replicated every 24 h. The cells were recovered by centrifugation (7000 rpm, 10 min) and re-suspended in 1 L of sterile peptone water (1 g/L peptone (Himedia, Mumbai, India)). This solution was spread over the cocoa beans, reaching a concentration of approximately 105 cells/g of cocoa.
The samples were taken every 24 h during 144 h of fermentation and placed in sterile plastic pots. The samples were stored at −20 °C. The fermentations were performed in triplicate [32].

4.2. Culture-Independent Microbiological Analysis

4.2.1. DNA Extraction and Polymerase Chain Reaction

The total DNA extraction and PCR reaction from the cocoa pulp were conducted as previously described [3]. Cocoa pulp DNA total was extracted with a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions and stored at −20 °C.
The bacterial DNA was amplified with the primers 338fgc (5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG-3′) (the GC clamp is underlined) and 518r (5′-ATT ACCGCG GCTGCT GG-3′). The DNA from the eukaryotic community was amplified with the primers NL1GC (5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGGGCA TAT CAA TAA GCG GAG GAA AAG-3′) (the GC clamp is underlined) and LS2 (5′-ATT CCC AAA CAA CTC GAC TC-3′). All reactions were performed in 25 μL containing 0.625 U Taq DNA polymerase (Promega, Madison, WI, USA), 2.5 μL 10 X buffer, 0.1 mMdNTP, 0.2 mM of each primer, 1.5 mM MgCl2, and 1 μL of extracted DNA. The amplification was performed as previously described [4]. The amplified products (2 μL) were analyzed by electrophoresis on 1% agarose gels before the DGGE analysis.

4.2.2. PCR–DGGE Analysis

To conduct the DGGE analyses, the PCR products were analyzed using a Bio-Rad DCode universal mutation detection system (Bio-Rad, Richmond, CA, USA). The PCR products were purified, sequenced, and available according to the procedures previously described [3]. Denaturant solutions containing 35–70% (100% denaturant contains 7 M urea and 40% (v/v) formamide) were used for bacteria, and containing 30–60% for yeast. The electrophoresis was run at 60 °C for 6 h at a constant voltage of 120 V.

4.3. Chromatographic Analysis

4.3.1. Sugars, Alcohols, and Organic Acid Extraction and HPLC Analyses

The carbohydrates, alcohols, and organic acids were extracted (from pulp and from the content inside the beans) and analyzed as described in previous work [3]. The analyses were determined by HPLC (Shimadzu, model LC-10Ai, Shimadzu Corp., Kyoto, Japan) equipped with a dual detection system consisting of a Ultraviolet-Visible (UV–Vis) detector (SPD 10Ai) and a refractive index detector (RID-10Ai). The HPLC was operated at 50 °C for acids and detected via UV absorbance (210 nm), while the alcohols and carbohydrates were examined at 30 °C and detected via Refractive Index Detector (RID). The column used for separation was a Shimadzu ion exclusion column (Shim-pack SCR-101H, 7.9 mm × 30 cm, Shimadzu, Kyoto, Japan) with a mobile phase of Perchloric acid (100 mM) at a flow rate of 0.6 mL/min. All samples were analyzed in triplicate.
The chemical compounds used as standards (purity N 99.8%), glucose, fructose, and citric acid, were purchased from Sigma-Aldrich (Saint Louis, MO, USA); acetic acid and ethanol were purchased from Merck (Darmstadt, Germany); and lactic acid was purchased from Fluka Analyticals (Seelze, Germany).

4.3.2. Characterization of Volatile Compounds by Headspace-Solid Phase Microextraction Gas Chromatography-Mass Spectrometry

The volatile compounds from cocoa samples were extracted using the Headspace-Solid Phase Microextraction (HS–SPME) technique, as described in previous research [24], with modifications. Briefly, cocoa samples (2.0 g) from the beginning and end of fermentation (0 h and 144 h) and chocolate samples (2.0 g) were macerated using liquid nitrogen for headspace analysis. A divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 mm SPME fiber (Supelco Co., Bellefonte, PA, USA.) was used to extract volatile constituents from the cocoa and chocolate headspace. The fiber was equilibrated for 15 min at 60 °C and then exposed to the samples (cocoa and chocolate) for 30 min at the same temperature.
The compounds were analyzed using a Shimadzu GC model QP2010 equipped with a mass spectrometry and a capillary column of silica DB-FFAP (25 m × 0.25 mm i.d. × 0.25 mm). The temperature program began with 5 min at 50 °C, followed by a gradient of 50 °C to 190 °C at 3 °C/min; the temperature was then maintained at 190 °C for 10 min. The injector and detector temperatures were maintained at 230 °C.
The carrier gas (He) was used at a flow rate of 1.2 mL/min. Injections were performed by fiber exposition for 5 min. 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) and the retention time with literature data using the n-Alkane index. All samples were examined in duplicate.

4.4. Sensory Analysis

After fermentation, the beans were dried in the sun inside drying greenhouses. Thereafter, the dried beans were sent for chocolate production at Sartori and Pedroso Alimentos Ltda. (São Roque, São Paulo, Brazil). Dark chocolate (100 g chocolate bar) was produced (62% liquor, 30% icing sugar, 8% cocoa butter). The molded chocolate was rapped and stored at 4 °C for four weeks before sensory analysis.
For sensory analysis, the chocolate was kept at room temperature (±20 °C) for two hours before the tests. The attributes involved in the TDS analysis were established by the Kelly grid method (“Kelly’s repertory grid method”) [33]. The TDS analysis was performed with 31 selected and trained judges. The judges evaluated differences between the two chocolate samples (PH15 I Ch (from inoculated fermentation) and PH15 NI Ch (from non-inoculated fermentation)) during the tasting time (the analysis time was 35 s, with an addition of delay time 2 s) and the attributes selected were acid, bitterness, nutty, sweetness, astringent, coffee, fruity, and cocoa.
The judges were asked to choose the dominant flavor over the analysis time. The dominant flavor is that perceived with greater clarity and intensity among the other ones [34]. The samples (approximately 2.5 g of chocolate) were presented in plastic cups, coded with a three-digit bar. Crackers and water were provided for palate cleansing. The analysis was performed in triplicate.
In order to calculate the TDS curves for all analyses, the software SensoMaker, version 1.8 was used [35]. Two lines were drawn on graphics: the “chance level” and the “significance level”. The “chance level” is the dominance rate that an attribute can obtain by chance. The “significance level” is the minimum value of this ratio to be considered significant.

4.5. Statistical Analyses

Analyses of the variance and the Scott–Knott test were performed with SISVAR 5.1 software (Federal University of Lavras, Department of Statistic, Lavras, MG, Brazil). Differences in values were considered significant when the p value was less than 0.05 (p < 0.05).

5. Conclusions

The inoculation of microorganisms as a starter culture accelerated the fermentation process. The bacterial and yeast communities were different according to each process (PH15 I and PH15 NI), but the bacteria (Gluconobacter oxydans, Lactobacillus plantarum, Acetobacter pasteurianus, Fructobacillus pseudoficulneus) and yeast (Saccharomyces cerevisiae and Pichia kluyveri) species were found in both processes. Glucose and fructose were consumed faster in the inoculated assay in the first 24 h of fermentation. Different volatile compounds were identified in fermented beans and chocolate produced in the present study. According to the sensory analysis of PH15 I Ch and PH15 NI Ch significant differences on the dominant tastes were observed. The inoculation leads to a chocolate with higher bitter, sweet, and cocoa notes than the chocolate produced by spontaneous fermentation. Bitter was the dominant taste in PH15 NI Ch, whereas bitter, sweet, and cocoa tastes were dominant tastes in PH15 I Ch. These results were corroborated by the analysis of volatile compounds in both chocolate samples. The 2,3-butanediol, which gives flavor to cocoa butter (sweet chocolate), and 2,3-dimethylpyrazine, which gives caramel and cocoa flavors, were detected only in PH15 I Ch. Therefore, that dominant flavor detected in PH15 I Ch was related with these compounds, indicating the inoculation influence in the final product. In this context, the inoculation influenced the fermentation process and the final product. In order to generate a standardized fermentative process and improve the chocolate quality, Saccharomyces cerevisiae UFLA CCMA 0200, L. Plantarum CCMA 0238, and A. pasteurianus CCMA 0241 are, herein, proposed to be used as a cocktail of microorganisms for application in cocoa fermentation.

Acknowledgments

Cledir Santos thanks the Universidad de La Frontera (Temuco, Chile) for the partial funding from Project DIUFRO DI16-0135. The authors acknowledge the financial support from the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the samples obtained from the Fazendas Reunidas Vale do Juliana (Igrapiúna, Bahia, Brazil).

Author Contributions

Rosane Freitas Schwan, Nelson Lima, and Cledir Santos conceived and designed the experiments and made the final revision of the paper; Igor Magalhães da Veiga Moreira and Leonardo de Figueiredo Vilela performed the experiments and wrote the paper; Maria Gabriela da Cruz Pedroso Miguel, Igor Magalhães da Veiga Moreira, and Leonardo de Figueiredo Vilela analyzed the data.

Conflicts of Interest

Authors declare no conflicts of interest.

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Sample Availability: Samples of the compounds are not available from the authors.
Figure 1. Changes in prokaryote (A) and eukaryote (B) communities during fermentation of the PH15 cocoa hybrid without (PH15 NI) and with (PH15 I) inoculation, and fingerprints of the starter cultures (CCMA 0238, CCMA 0241, and CCMA 0200). The identities of the bands are presented in Table 1. Bands marked with numbers were excised, re-amplified, and sequenced.
Figure 1. Changes in prokaryote (A) and eukaryote (B) communities during fermentation of the PH15 cocoa hybrid without (PH15 NI) and with (PH15 I) inoculation, and fingerprints of the starter cultures (CCMA 0238, CCMA 0241, and CCMA 0200). The identities of the bands are presented in Table 1. Bands marked with numbers were excised, re-amplified, and sequenced.
Molecules 22 00766 g001
Figure 2. Course of glucose ( Molecules 22 00766 i001), fructose ( Molecules 22 00766 i002), and citric acid ( Molecules 22 00766 i003) during fermentation of PH15 NI (A) and PH15 I (B).
Figure 2. Course of glucose ( Molecules 22 00766 i001), fructose ( Molecules 22 00766 i002), and citric acid ( Molecules 22 00766 i003) during fermentation of PH15 NI (A) and PH15 I (B).
Molecules 22 00766 g002
Figure 3. Detection by HPLC of ethanol (A); lactic acid (B); and acetic acid (C) during 144 h of fermentation of PH15 NI ( Molecules 22 00766 i004) and PH15 I ( Molecules 22 00766 i005); full symbols correspond to metabolites detected in the pulp while open symbols to those detected inside the beans.
Figure 3. Detection by HPLC of ethanol (A); lactic acid (B); and acetic acid (C) during 144 h of fermentation of PH15 NI ( Molecules 22 00766 i004) and PH15 I ( Molecules 22 00766 i005); full symbols correspond to metabolites detected in the pulp while open symbols to those detected inside the beans.
Molecules 22 00766 g003
Figure 4. Profile of volatile compounds identified by HS–SPME GC–MS during non–inoculated fermentation ( Molecules 22 00766 i006 PH15 NI), inoculated fermentation ( Molecules 22 00766 i007 PH15 I), and in the chocolate samples. Fermentation times: 0 h (A) and 144 h (B). Chocolate samples (C). Total amount of compounds: PH15 SI 0 h (37), PH15 SI 144 h (38), PH15 SI Ch (58), PH15 I 0 h (37), PH15 I 144 h (34), and PH15 I Ch (54).
Figure 4. Profile of volatile compounds identified by HS–SPME GC–MS during non–inoculated fermentation ( Molecules 22 00766 i006 PH15 NI), inoculated fermentation ( Molecules 22 00766 i007 PH15 I), and in the chocolate samples. Fermentation times: 0 h (A) and 144 h (B). Chocolate samples (C). Total amount of compounds: PH15 SI 0 h (37), PH15 SI 144 h (38), PH15 SI Ch (58), PH15 I 0 h (37), PH15 I 144 h (34), and PH15 I Ch (54).
Molecules 22 00766 g004
Figure 5. Temporal Dominance Sensory of chocolate produced from cocoa fermented beans PH15 NI (A) and PH15 I (B). Sensorial attributes: Acid ( Molecules 22 00766 i008), Bitterness ( Molecules 22 00766 i009), Nutty ( Molecules 22 00766 i010), Sweetness ( Molecules 22 00766 i011), Astringent ( Molecules 22 00766 i012), Coffee ( Molecules 22 00766 i013), Fruity ( Molecules 22 00766 i014), and Cocoa ( Molecules 22 00766 i015).
Figure 5. Temporal Dominance Sensory of chocolate produced from cocoa fermented beans PH15 NI (A) and PH15 I (B). Sensorial attributes: Acid ( Molecules 22 00766 i008), Bitterness ( Molecules 22 00766 i009), Nutty ( Molecules 22 00766 i010), Sweetness ( Molecules 22 00766 i011), Astringent ( Molecules 22 00766 i012), Coffee ( Molecules 22 00766 i013), Fruity ( Molecules 22 00766 i014), and Cocoa ( Molecules 22 00766 i015).
Molecules 22 00766 g005
Table 1. Identification of the bands based on Basic Local Alignment Search Tool (BLAST) in comparison with those in GenBank as obtained by PCR–DGGE using universal primers for yeasts and bacteria.
Table 1. Identification of the bands based on Basic Local Alignment Search Tool (BLAST) in comparison with those in GenBank as obtained by PCR–DGGE using universal primers for yeasts and bacteria.
IdentificationBands aSimilarity (%)Accession NumberSample Found
Prokaryotes
Fructobacillus pseudoficulneus1, 5, 698AB498052.1PH15NI, PH15I
Leuconostoc sp.297DQ523491.1PH15NI
Lactobacillus plantarum3, 4, 8, 999KT327866.1PH15NI, PH15I
Lactobacillus helveticus799KP764179.1PH15I
Zymomonas mobilis10100CP003715.1PH15NI
Uncultured bacterium11, 12, 13, 1499LN875309.1PH15NI, PH15I
Acetobacter pasteurianus15, 16, 17, 18, 19, 20100KM983001.1PH15NI, PH15I
Acetobacter sp.2198KC796695.1PH15NI
Gluconobacter oxydans22, 23, 2499CP003926PH15NI, PH15I
Bacillus sp.25, 2699JF309224PH15NI
Eukaryotes
Hanseniaspora uvarum1, 299KC544511PH15NI
Saccharomyces cerevisiae3, 5, 6, 7, 8, 9, 1099KT229544.1PH15NI, PH15I
Saccharomyces sp.498KU350335.1PH15NI
Theobroma cacao1197JQ228377.1PH15NI
Rhodotorula mucilaginosa12, 13100HM588765PH15I
Lentinula edodes1498KM015456.1PH15I
Pichia kluyveri15, 16, 17, 18, 2099FM864201PH15NI, PH15I
Trichosporon asahii1997JQ425402PH15I
a Bands are numbered as indicated on the DGGE gel. PH15NI: PH15 non-inoculated; PH15I: PH15 inoculated.
Table 2. Volatile compounds identified by Headspace—Solid Phase Microextraction—Gas Chromatography Mass Spectrometry (HS–SPME GC–MS) during fermentation times (0 h and 144 h) and in chocolate samples, and the reference odor of each compound.
Table 2. Volatile compounds identified by Headspace—Solid Phase Microextraction—Gas Chromatography Mass Spectrometry (HS–SPME GC–MS) during fermentation times (0 h and 144 h) and in chocolate samples, and the reference odor of each compound.
CompoundsOdor Description aSample Found
Acids
4-Hydroxybutanoic acid PH15I Ch
4-Hydroxybutyric acid PH15NI Ch
Acetic acidSour, astringentPH15NI 144 h
Benzeneacetic acid PH15NI Ch, PH15I Ch
Butanoic acidRancid, butter, cheesePH15NI Ch, PH15I Ch
Hexanoic acidSweat, pungentAll
Isovaleric acidSweat, rancidAll
Octanoic acidSweaty, fattyPH15NI (0 h and 144 h), PH15I 144 h, PH15NI Ch
Pentanoic acid PH15NI Ch, PH15I Ch
Valeric acidSweat, acid, rancidPH15NI Ch, PH15I Ch
Alcohols
2-Ethyl-1-hexanol PH15NI Ch
1-HexanolFruity, greenPH15NI (0 h and 144 h), PH15I 0 h
1-Methoxy-2-butanol PH15NI Ch, PH15I Ch
1-Nonanol PH15NI Ch, PH15I Ch
1-OctanolFatty, waxyAll
1-Penten-3-ol PH15NI 0 h
2,3-ButanediolCocoa butterPH15NI (0 h and 144 h), PH15I 144 h, PH15I Ch
2,4-Pentanediol PH15NI 0 h, PH15I 0 h, PH15NI Ch, PH15I Ch
2-Furanmethanol PH15NI Ch
2-HeptanolSweet, citrusyPH15NI (0 h and 144 h), PH15I (0 h and 144 h)
2-HexanolFruity, greenPH15NI 0 h, PH15I 0 h
2-PentanolGreen, mild greenPH15NI Ch, PH15I Ch
3-Methyl-1-butanolMalty, chocolatePH15NI (0 h and 144 h), PH15I (0 h and 144 h)
Benzyl alcoholSweet, flowerAll
Furfuryl alcohol PH15I Ch
GuaiacolSmoke, sweetPH15NI 144 h, PH15I 144 h
Phenylethyl AlcoholHoney, rose, caramelAll
Aldehydes and Ketones
3-methylpentanal PH15NI 0 h, PH15I 0 h, PH15NI Ch, PH15I Ch
(E)-2-Heptenal PH15NI 0 h, PH15I 0 h
(E)-2-NonenalTallowy greenPH15NI 0 h, PH15I 0 h
(E)-2-OctanalFatty, waxyPH15NI 0 h, PH15I 0 h
(E)-2-Undecenal PH15NI Ch, PH15I Ch
(E,E)-2,4-heptadienal PH15NI 0 h, PH15I 0 h, PH15NI Ch, PH15I Ch
1-(2-hydroxyphenyl)ethanone PH15NI 0 h, PH15I 0 h
2(5H)-FuranoneCaramel-likePH15NI Ch, PH15I Ch
2-Heptanone PH15NI (0 h and 144 h), PH15I (0 h and 144 h)
2-Hydroxyphenyl methyl ketone PH15NI 144 h
2-Nonanone PH15NI 144 h, PH15I (0 h and 144 h)
2-Phenyl-2-butenalSweet, roastedPH15NI 144 h, PH15I 144 h, PH15NI Ch, PH15I Ch
2-Undecenal PH15NI (0 h and 144 h), PH15I (0 h and 144 h)
3-Methyl-1,2-cyclopentanedione PH15NI Ch, PH15I Ch
3-Penten-2-one PH15NI Ch
4-hydroxy-3-methylbutanal PH15NI 0 h, PH15I 0 h
4-Methylhexanal PH15NI Ch, PH15I Ch
5-Methyl-2-furaldehyde PH15NI Ch, PH15I Ch
5-Methyl-2-phenyl-2-hexenal PH15NI Ch, PH15I Ch
3-methylpentanal PH15NI 0 h, PH15I 0 h, PH15NI Ch, PH15I Ch
AcetophenoneFloralPH15NI (0 h and 144 h), PH15I (0 h and 144 h)
BenzaldehydeBitterAll
Benzeneacetaldehyde PH15NI Ch, PH15I Ch
Nonanal PH15NI 0 h, PH15I 0 h, PH15NI Ch, PH15I Ch
Octanal PH15NI 0 h, PH15I 0 h, PH15NI Ch, PH15I Ch
Pyranone PH15NI Ch, PH15I Ch
AcetoinButter, creamPH15NI 144 h, PH15I 144 h, PH15NI Ch, PH15I Ch
Esters
1-methylbutyl benzoate PH15NI 144 h, PH15I 144 h
1-Methylhexyl acetate PH15NI 144 h
2-Ethyl-1-hexyl acetate PH15I Ch
2-Pentanyl benzoate PH15NI 0 h, PH15I 0 h, PH15NI Ch, PH15I Ch
2-Phenethyl acetateFruityPH15NI 144 h, PH15I 144 h
3-methylbutyl formate PH15NI 0 h
Amyl acetateFruity, bananaPH15NI 144 h, PH15I 144 h
Dibutyl phthalate PH15NI 144 h, PH15I (0 h and 144 h)
Diisobutyl phthalate PH15NI Ch, PH15I Ch
Ethyl 2-hydroxypropanoate PH15NI 144 h, PH15I 144 h
Ethyl benzeneacetate PH15NI 144 h
Ethyl capratePear, grapePH15NI 144 h, PH15I 144 h
Ethyl caprylateFruity, floweryPH15I (0 h and 144 h), PH15I Ch
Ethyl laurateFruity, floralPH15NI 144 h, PH15I 144 h, PH15NI Ch, PH15I Ch
Ethyl myristateWaxy, soapyPH15NI 144 h, PH15I 144 h
Ethyl palmitateWaxy, greenPH15NI (0 h and 144 h), PH15I (0 h and 144 h)
Ethyl phenylacetate PH15NI 0 h
Hexyl acetate PH15NI 144 h
Isoamylformate PH15I 0 h
Isobutyl phthalate PH15NI (0 h and 144 h), PH15I (0 h and 144 h)
Isopropyl palmitate PH15I 0 h
Methyl Palmitate PH15NI 144 h, PH15I (0 h and 144 h)
Phenylethyl acetatefruity, sweetPH15NI Ch, PH15I Ch
Pyrazines
2,3,5,6-TetramethylpyrazineChocolate, coffeePH15NI Ch, PH15I Ch
2,3,5-Trimethyl-6-isopentylpyrazine PH15NI Ch, PH15I Ch
2,3,5-TrimethylpyrazineCocoa, rusted nutsPH15NI Ch, PH15I Ch
2,3-DimethylpyrazineCaramel, cocoaPH15I Ch
2,5-Dimethyl-3-isoamylpyrazine PH15NI Ch, PH15I Ch
2,5-DimethylpyrazineCocoa, rusted nutsPH15NI Ch, PH15I Ch
2,6-DimethylpyrazineNutty, coffee, greenPH15I Ch
2-Acetyl-3,5-dimethylpyrazine PH15NI Ch, PH15I Ch
2-Ethyl-3,6-dimethylpyrazineRoasted, smokyPH15NI Ch, PH15I Ch
2-Ethyl-6-methylpyrazine PH15NI Ch, PH15I Ch
2-EthylpyrazinePeanut butter, nuttyPH15NI Ch
2-Methyl-3,5-diethylpyrazine PH15NI Ch
2-Methyl-6-vinylpyrazine PH15NI Ch
2-MethylpyrazineChocolate, cocoa, nutsPH15NI Ch, PH15I Ch
Pyrroles
1,3-Dimethyl-5-pyrazolinone PH15NI Ch, PH15I Ch
2-AcetylpyrroleChocolate, hazelnutPH15NI Ch, PH15I Ch
2-Pyrrolidinone PH15NI Ch, PH15I Ch
Pyrrole-2-carboxaldehyde PH15NI Ch, PH15I Ch
Terpenoids
(E)-Linalool oxideFloral, greenPH15NI (0 h and 144 h), PH15I (0 h and 144 h)
(Z)-Linalool oxideFloralPH15NI 0 h, PH15I 0 h
LinaloolFlower, lavenderPH15NI (0 h and 144 h), PH15I (0 h and 144 h)
Others b
1-methoxy-2-methylpropane PH15NI (0 h and 144 h), PH15I 144 h
2-Butyltetrahydrofuran PH15NI Ch, PH15I Ch
2-Pentylfuran All
7-methyl pentadecane PH15I (0 h and 144 h)
Hexadecane PH15I (0 h and 144 h)
a Obtained from literature; b Includes: furans and alkanes; Abbreviations: Ch: chocolate.

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