Impact of different cocoa hybrids (Theobroma cacao L.) and S. cerevisiae UFLA CA11 inoculation on microbial communities and volatile compounds of cocoa fermentation

Food Research International 64 (2014) 908–918

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Impact of different cocoa hybrids (Theobroma cacao L.) and S. cerevisiae UFLA CA11 inoculation on microbial communities and volatile compounds of cocoa fermentation Cíntia Lacerda Ramos a, Disney Ribeiro Dias b, Maria Gabriela da Cruz Pedrozo Miguel a, Rosane Freitas Schwan a,⁎ a b

Department of Biology, Federal University of Lavras, 37.200-000 Lavras, MG, Brazil Department of Food Science, Federal University of Lavras, 37.200-000 Lavras, MG, Brazil

a r t i c l e

i n f o

Article history: Received 4 July 2014 Accepted 26 August 2014 Available online 4 September 2014 Chemical compounds studied in this article: Isobutyric acid (PubChem CID: 6590) Butyric acid (PubChem CID: 264) Hexanoic acid (PubChem CID: 8892) Heptanoic acid (PubChem CID: 8094) Nonanoic acid (PubChem CID: 8158) Decanoic acid (PubChem CID: 2969) Methanol (PubChem CID: 887) 1-Propanol (PubChem CID: 1031) 2-Methyl-1-propanol (PubChem CID: 6560) 1-Butanol (PubChem CID: 163) 2-Methyl-1-butanol (PubChem CID: 8723) 2-Heptanol (PubChem CID: 10976) Trans-3-hexen-1-ol (PubChem CID: 5284503) 1,2-Propanediol (PubChem CID: 1030) Furfuryl alcohol (PubChem CID: 7361) 2-Phenylethanol (PubChem CID: 6054) Acetaldehyde (PubChem CID: 177) Hexanal (PubChem CID: 6184) Furfural (PubChem CID: 7362) Ethyl acetate (PubChem CID: 8857) Propyl acetate (PubChem CID: 7997) Isobutyl acetate (PubChem CID: 8038) Ethyl butyrate (PubChem CID: 7762) Isoamyl acetate (PubChem CID: 31276) Ethyl pyruvate (PubChem CID: 12041) Ethyl lactate (PubChem CID: 7344) Ethyl octanoate (PubChem CID: 7799) Furfuryl acetate (PubChem CID: 12170) Diethylsuccinate (PubChem CID: 21615604) Diethyl malate (PubChem CID: 24197) Mono-ethyl succinate (PubChem CID: 1711967) b-Citronellol (PubChem CID: 7793) Geraniol (PubChem CID: 637566)

a b s t r a c t The aim of this work was to study the microbial communities and volatile compounds profile of different fermentations: using four different cocoa hybrids and adding Saccharomyces cerevisiae UFLA CA11 as starter culture. Each hybrid showed particular characteristics: size, peel, seed and pulp. The temperature of the cocoa mass increased during fermentations (24 °C to 47 °C). The hybrid FA13 inoculated with S. cerevisiae showed the lowest temperatures (26 to 37 °C). The pulp's compositions were different between the hybrids, mainly regarding citric acid (0.5 to 3.2 g/kg). The carbohydrates were more rapidly (60 h) metabolized in inoculated fermentations than in spontaneous fermentations (84 h). Thirty-nine volatile compounds were identified by GC–FID for all fermentation processes. Esters (14 compounds) and alcohols (12) were the most important groups. Yeast communities were similar among the different processes while bacterial communities were dependent on the hybrid and process. The inoculation accelerated the fermentation and the hybrid characteristics influenced on the fermentation requiring particular management. © 2014 Published by Elsevier Ltd.

⁎ Corresponding author at: Federal University of Lavras, Department of Biology, Campus Universitário, 3037, 37.200-000 Lavras, MG, Brazil. Tel.: +55 38291614. E-mail address: [email protected]fla.br (R.F. Schwan).

http://dx.doi.org/10.1016/j.foodres.2014.08.033 0963-9969/© 2014 Published by Elsevier Ltd.

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Keywords: Cocoa hybrids Cocoa fermentation Volatile compounds PCR-DGGE

1. Introduction In Brazil, generally, the farmers cultivate different hybrids in the same field to prevent the total destruction of the cocoa trees by the fungus. Thus, the beans from different hybrids are collected and spontaneously fermented into the same box. This procedure probably affects the quality of the chocolate. Besides the genetically inherited flavor potential of different cocoa varieties, the fermentation process is regarded as another important factor influencing the flavor of the chocolate (Schwan & Wheals, 2004; Thompson, Miller, & Lopez, 2001). Unprocessed cocoa beans have a bitter, unpleasant taste, and flavor and by the application of adequate fermentation, drying, and roasting processes it is possible to obtain the desired characteristics of cocoa powder and chocolate (Fowler, 2009). Thus, a properly conducted fermentation process is a prerequisite for the production of high quality chocolates (Beckett, 2009; Owusu, Petersen, & Heimdal, 2011; Thompson et al., 2001). Generally, cocoa is fermented spontaneously by a consortium of naturally occurring yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB). The importance of yeast metabolism on the development of chocolate aroma has recently been elucidated by Ho, Zhao, and Fleet (2014). Using natamycin to inhibit yeast growth, it was shown that cocoa, fermented in the absence of yeasts, yielded an acidic chocolate lacking the characteristic chocolate aromas. Besides being responsible for the production of ethanol and the release of pulp degrading enzymes, yeasts are major producers of esters and higher alcohols which have been suggested to contribute to the complex mixture of volatile aroma compounds that characterizes chocolate aroma (Crafack et al., 2013; Owusu et al., 2011; Schwan & Wheals, 2004). It is known that the aroma and flavor of cocoa depend on the genotype of the cocoa tree that has produced the beans, the origin, and how the beans have been fermented (Bonvehi, 2005; Counet, Ouwerx, Rosoux, & Collin, 2004). In this sense, the aim of this work was to study the volatile compounds and microbial communities involved

during the fermentation of different cocoa hybrids (Theobroma cacao L.). Inoculation with Saccharomyces cerevisiae UFLA CA11 was also performed in order to observe its impact on fermentation process.

2. Material and methods 2.1. Fermentation and sampling The fermentation experiments were conducted at the Vale do Juliana cocoa farm in Igrapiúna, Bahia, Brazil. The ripe cocoa pods from four different hybrids PH 16, PS1030, FA13 and PS1319, (Table 1) were harvested during the crop of 2012 (October and 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. Fermentations of the hybrids PH16, PS1030, FA13 and PS1319 were performed with inoculation of S. cerevisiae UFLA CA11 at the beginning of the process. The inoculum was grown in YPD broth [10 g/L Yeast extract (Merck); 20 g/L Peptone (Himedia); 20 g/L dextrose (Merck)] at 30 °C at 150 rpm and replicated every 24 h until reach a population of approximately 108 cells/L. The cells were recovered by centrifugation (7000 rpm, 10 min) and resuspended in 1 L of sterile peptone water [1 g/L Peptone (Himedia)]. This solution was spread over the cocoa beans reaching a concentration of approximately 106 cells/kg of cocoa. They were also spontaneously fermented (without inoculation). All fermentation was evaluated for 156 h, and the amount used for each one of the hybrids was 60 kg. Samples of approximately 100 g each were withdrawn at 0, 12, 36, 60, 84, 108, 132, and 156 h of the fermentation process. The samples were taken approximately 40 cm from the surface of the center of the fermenting cocoa mass, placed in sterile plastic pots and transferred to the laboratory. The samples for chemical and culture-independent analyses were stored at −20 °C.

Table 1 General aspects of the different cocoa hybrids used in this study. Hybrida

Source

Diameter (cm)

Rind weight (g)

Seeds per fruit

Seeds weight (g)

PH16

Porto Híbrido/São José da Vitória

20.5 ± 0.35

9.60 ± 0.37

665.01 ± 45.5

40 ± 7

4.39 ± 0.49

PS1030

Porto Seguro/Uruçuca

16.4 ± 1.07

8.2 ± 0.58

637.4 ± 83.52

47 ± 7

4.12 ± 0.31

FA13

Angola/ Itajuípe

17.5 ± 0.24

9.6 ± 0.11

743.2 ± 35.12

49 ± 6

2.99 ± 0.25

PS1319

Porto Seguro/Uruçuca

18.65 ± 1.01

9.6 ± 0.42

727.7 ± 122.68

48 ± 4

4.82 ± 1.16

a

Length (cm)

Values are expressed as the mean of five fruits ± standard deviation.

Photo

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2.2. Physicochemical analysis The pH values and temperatures were evaluated during fermentations by an average of five different points into the fermentation boxes. A portable pHmeter Q400HM (Quimis, SP, Brazil) was used. The same points were used for pH and temperature evaluations. 2.3. Metabolites extraction and HPLC analyses The carbohydrates, alcohols and organic acids were extracted as described by Rodriguez-Campos, Escalona-buendía, Orozco-Avila, Lugo-Cervantes, and Jaramillo-Flores (2011) with minor modifications. Ten grams of fermented cocoa beans from each sample was extracted twice with 10 mL of Milli-Q water with vortexing for 5 min, and each 10 mL homogenate was transferred to another tube. The tubes containing 20 mL of solution were centrifuged (7000 rpm, 10 min, 4 °C), and the supernatant was separated from the precipitate. The precipitate was re-suspended in an additional 5 mL of Milli-Q water, vortexed, and centrifuged as described above. The final volume of 25 mL of diluted pulp was centrifuged, and 2 mL of supernatant was filtered through a 0.22 μm membrane (Millipore) for the HPLC analysis of sugars, alcohols and organic acids. After the extraction of the cocoa pulp, the testa was also separated from the cocoa beans and the beans were crushed using a pestle and mortar. Carbohydrates, alcohols and organic acids from the beans were, then, extracted as described above. The carbohydrates (glucose and fructose), organic acids (acetic, lactic, and citric acids) and alcohol (ethanol) analyses were carried out using a liquid chromatography system (Shimadzu, model LC-10Ai, Shimadzu Corp., Japan) equipped with a dual detection system consisting of a UV–Vis detector (SPD 10Ai) and a refractive index detector (RID-10Ai). A Shimadzu ion exclusion column (Shim-pack SCR101H, 7.9 mm × 30 cm) was operated at 30 °C for carbohydrates and alcohols, and 50 °C for acids. Perchloric acid (100 mM) was used as the eluent at a flow rate of 0.6 mL/min. The acids were detected via UV absorbance (210 nm), while the alcohols and carbohydrates were detected via RID. All samples were analyzed in triplicate, and individual compounds were identified based on the retention time of standards injected using the same conditions. The sample concentrations were determined using an external calibration method. Calibration curves were constructed by injecting different concentrations of the standards under the same conditions of the samples analyses and the areas obtained were plotted a linear curve whose equation was used to estimate the concentration of the compounds in the sample. The chemical compounds, used as standard (purity N 99.8%), glucose, fructose, and citric acid were purchased from Sigma-Aldrich (Saint Luis, EUA); acetic acid and ethanol, were purchased from Merck (Darmstadt, Germany); and lactic acid from Fluka Analyticals (Seelze, Germany). 2.4. Volatile compounds extraction and GC–FID The volatile compounds of the cocoa samples (2.0 g) were extracted using the solid phase microextraction technique in the headspace (SPME-HS) described by Rodriguez-Campos et al. (2011) with minor modifications. A 50/30 μm divinylbenzene/ carboxene/polydimethylsiloxane (DVB/CAR/PDMS) fiber provided by Supelco was used to extract volatile compounds. The fiber was balanced for 15 min at 60 °C and then exposed to the cocoa powder for 30 min at the same temperature. The compounds were analyzed using a Shimadzu GC Model 17A equipped with a flame ionization detector (FID) and a capillary column of silica DB Wax (30 m × 0.25 mm i.d. × 0.25 μm) (J & W Scientific, Folsom, CA, USA). 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 and 240 °C, respectively. The carrier gas (N2) 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 retention times of the compounds in the samples with the retention times of standard compounds injected under the same conditions. Quantitative data of the identified compounds were obtained by integrating the peak areas of all identified compounds. The relative percentages of individual compounds were calculated from the total contents of volatiles on the chromatograms. All samples were examined in duplicate.

2.5. DNA extraction and PCR reaction The total DNA from the cocoa pulp was extracted from samples with a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions for DNA purification from tissues. The DNA was stored at −20 °C until further use. The DNA from the bacterial community 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 ACC GCG GCTGCT GG-3′), which span the V3 region of the 16S rRNA gene (Ovreas,Forney, Daae & Torsvik, 1997). A fragment of the D1/D2 region of the 26S rRNA gene was amplified with the eukaryotic universal primers NL1GC (5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA TAT CAA TAA GCG GAG GAA AAG-3′) (the GC clamp is underlined) and LS2 (5′-ATT CCC AAA CAA CTC GAC TC-3′), which amplified a fragment of approximately 250 bp (Cocolin, Bisson, & Mills, 2000). All reactions were performed in 25 μL containing 0.625 U Taq DNA polymerase (Promega, Madison,USA), 2.5 μL 10× buffer, 0.1 mM dNTP, 0.2 mM each primer, 1.5 mM MgCl2 and 1 μL of extracted DNA. The amplification was performed as previously described (Ramos et al., 2010). The amplified products (2 μL) were analyzed by electrophoresis on 1% agarose gels before the DGGE analysis.

2.6. PCR-DGGE analysis The PCR products were separated on polyacrylamide gels [8% (w/v) acrylamide:bisacrylamide (37.5:1)] in 1 × TAE buffer with a DCode System apparatus (BioRad Universal DCode Mutation Detection System, Richmond, CA, USA), according to the procedures previously described (Ramos et al., 2010). Solutions containing 35–70% denaturant [100% denaturant contains 7 M urea and 40% (v/v) formamide] were used for bacteria, and solutions containing 30–60% denaturant were used for yeast. The gels were run at 60 °C for 6 h at a constant voltage of 120 V. After electrophoresis, the gels were stained with SYBR-Green I solution (Molecular Probes, Eugene, UK) (1:10,000 v/v) for 30 min, and the images were visualized and photographed with a transluminator LPix Image (LTB 20 × 20HE, LPix®, Brazil). The DGGE bands were excised from the acrylamide gels. The DNA fragments were purified with a QIAEX II gel extraction kit (Qiagen, Chatsworth, CA, USA) and reamplified with the primers 338fgc and 518r for bacteria, and NL1 and LS2 for yeast. The PCR products obtained from the bands DNA were purified and sequenced with an ABI3730 XL automatic DNA sequencer (Advanced Genetics Technologies Center — AGTC, Kentucky, USA), and the sequences available in the GenBank database were compared with those in the BLAST algorithm (National Center for Biotechnology Information, Maryland, USA).

2.7. Statistical analyses Analyses of the variance and the Scott–Knott test were performed with SISVAR 5.1 software. Principal component analyses (PCA) were performed using the XLSTAT 7.5.2 software (Addinsoft's, New York, NY, USA) using the total concentrations of the volatile compound groups evaluated at T0 and TF of the different fermentation processes.

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25.4 25.2 26.4 28.5 43.2 44.1 45.5 49.4 0.11 0.08 0.09 0.09 0.05 0.10 0.12 0.11 ± ± ± ± ± ± ± ± 0.4 0.8 0.9 0.4 0.6 0.4 0.9 1.0

3.60 3.76 3.93 4.00 3.87 4.32 4.34 4.49

PS1319 (CA11)

Different hybrids of cocoa fruits were used in this study. Each fruit showed a particular size, peel, seed, pulp and number of beans. These characteristics are described in Table 1.

pH

3.1. General aspects of cocoa fruit

T °C

± ± ± ± ± ± ± ±

0.9 0.7 0.5 0.1 0.3 0.3 0.5 0.2

3. Results

25.8 27.0 31.9 32.7 33.5 34.6 37.3 35.8 0.12 0.08 0.10 0.08 0.05 0.06 0.09 0.09 ± ± ± ± ± ± ± ± 3.96 4.15 4.04 3.95 3.89 4.11 4.24 4.26 Values are expressed as the mean ± standard deviation.

± ± ± ± ± ± ± ± 3.48 3.63 3.46 4.00 4.14 4.40 4.21 4.62 0.3 0.0 0.5 0.6 0.5 0.4 0.3 0.9 ± ± ± ± ± ± ± ± 23.6 22.8 23.6 26.3 35.6 41.5 44.8 44.8 0.11 0.09 0.09 0.12 0.15 0.14 0.09 0.10

T °C

± ± ± ± ± ± ± ± 3.27 3.51 3.43 3.85 3.76 4.17 4.33 4.51 0 12 36 60 84 108 132 156

a

0.08 0.06 0.10 0.10 0.08 0.12 0.11 0.15

24.1 23.5 24.7 28.5 32.0 42.3 43.9 47.9

± ± ± ± ± ± ± ±

0.0 0.6 0.4 0.2 0.9 0.6 0.8 1.0

3.50 3.53 3.42 3.50 3.70 4.28 4.47 4.84

± ± ± ± ± ± ± ±

0.05 0.09 0.11 0.13 0.10 0.12 0.12 0.10

24.1 22.7 24.7 29.6 36.9 41.8 47.8 45.6

± ± ± ± ± ± ± ±

0.5 0.6 0.4 0.7 0.2 0.9 0.8 0.9

3.46 3.60 3.62 3.73 4.20 4.40 4.60 4.45

± ± ± ± ± ± ± ±

0.09 0.11 0.05 0.15 0.14 0.09 0.10 0.08

25.5 24.9 26.8 27.0 35.0 42.4 45.3 48.2

± ± ± ± ± ± ± ±

0.1 0.0 0.5 0.3 0.9 1.2 1.0 1.1

3.27 3.43 3.17 4.34 4.49 4.97 4.67 4.65

± ± ± ± ± ± ± ±

0.09 0.08 0.05 0.05 0.08 0.10 0.09 0.08

23.6 24.9 31.0 37.3 45.8 46.0 46.6 46.8

± ± ± ± ± ± ± ±

0.2 0.8 0.5 0.4 0.5 0.6 0.4 0.8

3.37 3.45 3.42 3.56 3.99 4.03 4.26 4.40

± ± ± ± ± ± ± ±

0.13 0.08 0.07 0.10 0.07 0.08 0.05 0.08

24.8 25.1 28.1 32.6 35.0 41.5 42.7 43.3

± ± ± ± ± ± ± ±

0.6 0.8 0.7 0.7 0.5 1.0 0.9 1.1

pH T °C

PS1030 (CA11)

pH T °C

PH16 (CA11)

pH T °C

PS1319

pH T °C

FA13

pH T °C

PS1030

pH

PH16

pH

Time (h)

Fermentations parametersa

Table 2 Measurements of the pH of the pulp and temperature during the 156 h of fermentation for the different hybrids.

The pH and temperature were measured during the different fermentations process and are presented in Table 2. The pH value of the pulp ranged from around 3.27 to 4.97, starting around 3.5 at the beginning of the fermentation and reaching values around 4.5 at 156 h for all fermentations. The highest variation was observed during the fermentation of the PH16 hybrid inoculated with S. cerevisiae UFLA CA11, which reached the lowest value (3.27) at 36 h and the highest value (4.97) at 108 h. At the end of the fermentation process (156 h) the pH was 4.7. The highest value of pH was 4.84 observed for FA13 hybrid. As observed in Table 2, the pH varied according to the fermentation process (hybrid and inoculation). Regarding the temperatures, there was an increase according to the time for all fermentation processes, which ranged from around 24 °C at 0 h to 47 °C at 156 h. The lowest temperatures were observed for the hybrid FA13 inoculated with S. cerevisiae UFLA CA11 ranging from 26 to 37 °C. The temperatures increased faster in the fermentations of the hybrids PS1319 and PH16 inoculated with S. cerevisiae UFLA CA11, than the non-inoculated. It was due to the conversion of glucose and fructose to ethanol by the yeast liberating heat to the cocoa mass. Carbohydrates (glucose and fructose), ethanol and organic acids (citric, lactic and acetic acids) were evaluated in the pulp and the beans of the different hybrids of cocoa for 156 h of fermentation and are shown in Figs. 1 and 2. The pulp compositions of the different hybrids (0 h) are different, mainly regarding citric acid, which ranged from approximately 0.5 to 3.2 g/kg (Fig. 1). The carbohydrates were faster metabolized in the fermentation inoculated with the yeast S. cerevisiae UFLA CA11 than in the spontaneous fermentation. Further, there was higher ethanol production in the inoculated fermentations, with peaks of production between 36 and 108 h of the process. The highest production of ethanol was observed to the inoculated hybrid PS1030 at 60 h (approximately 100 g/kg). All hybrids spontaneously fermented showed ethanol concentrations lower than 40 g/kg. After 108 h there was a decrease in the ethanol concentration in the fermentation of the all different hybrids, which coincided with the increase of the acetic acid. The highest acetic acid production was detected in the fermentation of the PH16 spontaneously fermented (0.66 g/kg) and the inoculated PS1030 hybrids (0.74 g/kg) at 132 h. The lactic acid production was variable for the different assays. The highest concentrations were detected during the fermentations of the inoculated hybrids (mainly PS1319 and FA13). The inoculated hybrid PS1319 showed a peak of lactic acid at 60 h with 0.97 g/kg, while the FA13 hybrid showed a peak at 36 h with 0.74 g/kg of lactic acid. From 108 h the lactic acid concentration increased again in the FA13 fermentations, being constant until 156 h with approximately 0.85 g/kg. The ethanol content detected in the cotyledons showed variable values (Fig. 2). In general, the hybrids spontaneously fermented showed lower concentrations than those inoculated, except for the PH16 hybrid. The PH16 hybrid spontaneously fermented presented a peak of 4.5 g/kg at 84 h, higher than the inoculated hybrid (highest concentration of 3.8 g/kg at 36 h). The highest ethanol concentration detected in the cocoa mass of the inoculated hybrids was 3.8 g/kg for both PH16 (36 h) and PS1319 (60 h), faster than that for the PH16 hybrid spontaneously fermented. The microbial inoculation accelerated the ethanol production in the cocoa pulp, which penetrated into the cotyledon.

FA13 (CA11)

T °C

± ± ± ± ± ± ± ±

3.2. Physical and chemical changes during fermentations

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Fig. 1. Carbohydrates, acids and alcohols detected in the pulp during 156 h of fermentation of different cocoa hybrids spontaneously fermented: PH16 (●), PS1030 (◆), FA13 (■) and PS1319 (▲); and inoculated with UFLA CA11: PH16 (○), PS1030 (◊), FA13 (□) and PS1319 (Δ). Standard deviation ranging from: 0.00–2.27 glucose, 0.00–1.90 fructose, 0.00–0.95 citric acid, 0.00–14.51 ethanol, 0.00–0.08 lactic acid, and 0.00–0.09 acetic acid.

Lactic and acetic acids were also determined. The highest concentrations of lactic acid were observed in the cotyledons of the inoculated hybrids PH16 (1 g/kg at 156 h) and FA13 (0.9 g/kg at 108 h), and the hybrid PH16 spontaneously fermented (0.8 g/kg at 84 h). The acetic acid was mainly detected from 108 h of fermentation for all different processes, except for PH16 spontaneously fermented, which showed the fastest (from 84 h) and the highest concentration (1.7 g/kg at 108 h). Among the hybrids spontaneously fermented, the PH16 showed the highest concentrations of ethanol, carbohydrates and organic acids (lactic and acetic). The volatile compounds detected in the different hybrid fermentations are shown in Table 3. A total of 39 volatile compounds were detected and quantified by GC–FID at the beginning (T0) and at the end (TF) of the different fermentation processes. These compounds included 6 acids, 12 alcohols, 1 aldehyde and ketones, 14 esters, 1 lactone and 2 terpenoids. A total of 14 ester compounds were detected. The hybrid PS1030 (inoculated and not inoculated) showed the highest concentrations of total esters, 87.15 and 85.68%, respectively at the end (156 h) of the fermentation. The ethyl octanoate, correlated to fruity and flowery odor, was the main ester produced during these fermentations when 74.29 and 67.31% were detected for PS1030 inoculated and not inoculated, respectively at 156 h. Twelve alcohol compounds were detected. The higher concentrations of the total alcohols were detected for the hybrids FA13 (without inoculation) and PS1319 (inoculated and not), showing values of 86.59% at 0 h decreasing to 16.43% at 156 h for FA13; 72.69% at 0 h to 66.36% at 156 h for PS1319 inoculated with S. cerevisiae UFLA

CA11; and 54.37% at 0 h to 40.28 at 156 h for PS1319 spontaneously fermented. The total concentrations of the volatile compound groups evaluated at T0 and TF of the different fermentation processes were submitted to PCA (Fig. 3). The first (F1) and second (F2) principal components explain 72.60 and 20.73%, respectively, of the total variance (93.33%). On the positive side of F1 and F2, the hybrids naturally fermented FA13 (T0) and PS1319 (T0 and TF) and the inoculated hybrids FA13 (TF) and PS1319 (T0 and TF) were correlated with ethanol presence. On the positive side of F1 and the negative side of F2, all the others were correlated with ester presence. Although, terpenoids, lactones, aldehydes and ketones, and acids were detected they were grouped on the negative side of the F1 and F2 and were not correlated with none of the fermentations. 3.3. Microbial communities detected during the fermentations Analyses of the succession of the microbial communities were performed by PCR-DGGE for prokaryote and eukaryote microorganisms (Fig. 4). The bacterial communities changed according to different fermentations process (hybrids), while for eukaryotes the profiles were almost similar, where S. cerevisiae and Hanseniaspora uvarum were present in all samples except for the spontaneously fermented hybrid PH16 which did not show bands corresponding to H. uvarum. The bacterial species Lactobacillus fermentum, Lactobacillus rhamnosus, and Gluconobacter liquefaciens were detected during the process for all

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Fig. 2. Carbohydrates, acids and alcohols detected inside the beans during 156 h of fermentation of different cocoa hybrids spontaneously fermented: PH16 (●), PS1030 (◆), FA13 (■) and PS1319 (▲); and inoculated with UFLA CA11: PH16 (○), PS1030 (◊), FA13 (□) and PS1319 (Δ). Standard deviation ranging from: 0.00–0.13 glucose, 0.00–0.21 fructose, 0.00–0.23 citric acid, 0.00–0.36 ethanol, 0.00–0.59 lactic acid, and 0.00–0.08 acetic acid.

different fermentations. The species Streptomyces sp., Acetobacter sp., Lactobacillus casei, Leuconostoc lactis, Acetobacter pastorianus, Lactobacillus plantarum and uncultured bacterium were also detected depending on the cocoa hybrid fermentation. Regarding the yeast species, further S. cerevisiae and H. uvarum, common for all processes, the species Hanseniaspora guilliermondii was also detected by PCR-DGGE analysis during the fermentations of the hybrid PS1030 inoculated with UFLA CA11, and FA13 and PH16 during the both processes (spontaneously fermented and inoculated). 4. Discussion With the financial crises in the international cocoa market and the ‘witches' broom’ disease in 90th year, disease-resistant hybrids were developed and studied. However, some characteristics of cocoa pods and seeds, such as size, color, quantity and weight of the seeds, pulp amount, flavor and chemical composition have changed (Clapperton, Lockwood, Yow, & Lim, 1994; Moreira, Miguel, Duarte, Dias, & Schwan, 2013). It is known that the amount, nature, and distribution of the microorganisms present in the cocoa pulp will determine the speed and intensity of the fermentation as well as the quality of the fermented beans and the chocolate made thereof (Camu et al., 2008). In this sense, the different hybrids with distinct characteristics probably favor the growth of different communities of microorganisms. Thus, a better understanding of the microbial communities and the physicochemical changes during the spontaneous fermentation of cocoa

hybrids is a prerequisite for developing management procedures and for the production of high quality cocoa. The cocoa fermentation consists of well-defined microbial successions that are initially dominated by yeasts and subsequently surpassed by lactic acid bacteria (LAB) and acetic acid bacteria (AAB) (Ardhana & Fleet, 2003; Garcia-Armisen et al., 2010; Lima, Almeida, Rob Nout, & Zwietering, 2011; Schwan & Wheals, 2004). The yeast metabolism results in an ethanol production which will be converted to acetic acid by AAB increasing the temperature during the fermentation and a very strong vinegar-like aroma is perceptible. The microbial activities consume the citric acid present in the pulp causing an increase in pH. These temperature and pH changes were observed for all different fermentations performed in the present study, however some hybrids showed greater and/or faster changes than others. The yeast inoculation seemed to accelerate the temperature and pH increase for the hybrids PH16 and PS1319. The FA13 hybrid inoculated with S. cerevisiae did not reach a high temperature, keeping it below to 40 °C, which probably affected the quality of the chocolate. The microbial activity generates metabolites and conditions (temperature and pH) that kill the beans, thereby triggering an array of biochemical reactions and chemical changes within the bean itself, that are essential for the development of the complex flavor of chocolate (Pereira, Miguel, Ramos, & Schwan, 2012). As expected, carbohydrates (glucose and fructose) and citric acid consumption were observed at the initial times of fermentation (until around 60 h). Sucrose was not detected at the beginning of the

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Table 3 Volatile compounds detected at the beginning (T0) and at the end (TF) of the beans of different hybrid fermentations inoculated with S. cerevisiae UFLA CA11. Concentration (%)b Group

Odor descriptiona

PH16 T0

PH16 TF

PS1030 T0

PS1030 TF

FA13 T0

FA13 TF

PS1319 T0

PS1319 TF

Isobutyric acid Butyric acid

Acid Acid

ND 4.66 ± 0.23c

0.60 ± 0.01j ND

ND 0.02 ± 0.01j

ND 0.11 ± 0.01g

ND 7.36 ± 0.01 e

ND 13.10 ± 0.74c

ND ND

0.05 ± 0.01h ND

Hexanoic acid Heptanoic acid Nonanoic acid Decanoic acid Total

Acid Acid Acid Acid

Rancid, butter Unpleasant smell and acrid taste. With a sweetish aftertaste Sweet, pungent Rancid, sour Green, fatty Rancid, fatty

0.46 ± 0.01f ND ND ND 5.12

ND ND ND ND 0.60

0.72 ± 0.01i 9.79 ± 0.06c 10.28 ± 0.14b 1.32 ± 0.03h 22.13

0.03 ± 0.00g ND ND ND 0.14

2.71 ± 0.01g ND ND ND 10.07

0.20 ± 0.01l ND ND ND 13.30

0.16 ± 0.02g ND ND ND 0.16

0.50 ± 0.01h ND ND ND 0.55

Methanol 1-Propanol 2-Methyl-1-propanol 1-Butanol 2-Methyl-1-butanol 2-Heptanol 3-Mehtyl-1-pentanol Trans-3-hexen-1-ol 2,3-Butanediol 1,2-Propanediol Furfuryl alcohol 2-Phenylethanol Total

Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols

Grassy-green Natural odor of cocoa butter No odor Faint burning odor and a bitter taste Honey, flowery

15.74 ± 0.35b 0.11 ± 0.01f ND 3.22 ± 0.03d 0.35 ± 0.08f ND 0.63 ± 0.06f 0.80 ± 0.06e ND ND ND ND 20.85

ND 0.22 ± 0.03l 10.33 ± 0.33d 1.44 ± 0.06h 1.61 ± 0.01h ND ND ND ND 0.17 ± 0.03l 10.71 ± 0.40c 6.87 ± 0.18f 31.35

ND ND 0.22 ± 3.20 ± 0.02 ± ND 7.64 ± 0.13 ± 9.60 ± ND ND ND 20.81

1.24 ± 0.02 ± 6.33 ± 1.00 ± 0.32 ± 0.01 ± 0.23 ± 0.47 ± 0.90 ± 0.07 ± ND ND 10.59

24.41 ± 0.58a 1.02 ± 0.03i 5.02 ± 0.03f 0.91 ± 0.02i 2.75 ± 0.17g 0.26 ± 0.05j 3.08 ± 0.12g 0.32 ± 0.04j 0.50 ± 0.15j 0.35 ± 0.20j ND 1.27 ± 0.32j 39.89

0.66 ± 0.01f ND ND 33.26 ± 0.52b ND ND 0.96 ± 0.06f 37.65 ± 0.49ª 0.16 ± 0.05g ND ND ND 72.69

9.83 ± 0.02d 0.03 ± 0.01h ND 10.50 ± 0.42c ND 0.09 ± 0.01h ND 43.75 ± 1.07ª 2.07 ± 0.18f 0.09 ± 0.01h ND ND 66.36

Acetaldehyde 1,1-Dietoxyethane Hexanal Furfural Total

Aldehydes and ketones Aldehydes and ketones Aldehydes and ketones Aldehydes and ketones

Pungent Acid taste Fruity Almonds

0.78 4.82 1.05 0.24 6.89

0.16 ± 0.05l 1.34 ± 0.23h 6.55 ± 0.64f ND 8.05

0.02 ± 0.00j 0.29 ± 0.06j 0.03 ± 0.01j ND 0.34

ND 0.12 ± 0.03g 0.01 ± 0.00g 0.02 ± 0.00g 0.15

0.85 ± 0.21i 21.65 ± 0.93ª 3.65 ± 0.21f ND 26.15

0.18 ± 0.03l 5.83 ± 0.24e 0.12 ± 0.11j ND 6.13

0.06 ± 0.01g 0.06 ± 0.01g 0.02 ± 0.03g ND 0.14

ND 0.39 ± 0.01h 0.01 ± 0.00h 0.25 ± 0.06h 0.65

Phenylethyl acetate Ethyl acetate Propyl acetate Isobutyl acetate Ethyl butyrate Isoamyl acetate Ethyl pyruvate Ethyl lactate Ethyl octanoate Furfuryl acetate Phenyl acetate Diethylsuccinate Diethyl malate mono-Ethyl succinate Total

Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters

Honey, flowery Pineapple

ND 1.88 ± 0.16e ND 0.43 ± 0.10f 3.99 ± 0.01d 56.50 ± 0.71ª 0.22 ± 0.11f 0.22 ± 0.11f ND 1.59 ± 0.57e ND 1.62 ± 0.53e ND ND 66.45

0.89 ± 0.15i 22.82 ± 0.25ª ND 0.84 ± 0.23i 4.18 ± 0.03g 17.65 ± 0.49b 7.97 ± 0.04e 1.77 ± 0.32h 0.91 ± 0.12i 1.32 ± 0.11h ND 0.93 ± 0.09i 0.18 ± 0.02l 0.52 ± 0.03j 59.98

0.36 ± 0.19j ND ND 0.14 ± 0.01j 0.26 ± 0.05j 5.91 ± 0.12e 0.17 ± 0.09j 1.64 ± 0.51h ND 27.04 ± 0.51ª 0.37 ± 0.03j 0.75 ± 0.35i ND 1.98 ± 0.17g 38.62

0.25 ± 0.07g ND 0.47 ± 0.04f 0.06 ± 0.05g 0.06 ± 0.05g 1.88 ± 0.17d 8.79 ± 0.58b 0.97 ± 0.04e 74.29 ± 0.41ª 0.13 ± 0.09g ND ND 0.25 ± 0.21f ND 87.15

0.45 ± 0.06j 17.85 ± 0.20b ND 1.97 ± 0.04h 1.64 ± 0.08h ND ND 1.04 ± 0.06i ND 1.98 ± 0.12h 0.54 ± 0.08j 11.65 ± 0.92c ND ND 37.12

0.17 ± 0.03l 11.59 ± 0.58d 0.40 ± 0.13j 1.98 ± 0.03h 0.43 ± 0.09j ND 1.78 ± 0.31h 0.38 ± 0.17j 21.11 ± 0.15b 1.57 ± 0.11h 0.14 ± 0.08l ND 0.44 ± 0.08j ND 39.99

0.28 ± 0.03g 0.42 ± 0.11g 0.09 ± 0.01g 0.28 ± 0.03g 1.29 ± 0.29e 21.57 ± 0.61c 0.88 ± 0.16f ND ND 1.87 ± 0.18d ND 0.06 ± 0.05g ND ND 26.74

0.88 ± 0.17g 0.35 ± 0.20h 1.34 ± 0.23g 0.89 ± 0.01g 0.08 ± 0.02h 2.81 ± 0.26f 20.88 ± 0.88b 0.99 ± 0.01g ND 3.57 ± 0.60e ND ND 0.26 ± 0.06h ND 32.05

Decalactone Total

Lactones

Fruity

ND ND

ND ND

2.61 ± 0.55f 2.61

ND ND

7.92 ± 0.11e 7.92

0.20 ± 0.13l 0.20

ND ND

ND ND

b-Citronellol Geraniol Total

Terpenoids Terpenoids/alcohol

Used in perfumes and insect repellents Fruity, flowery

ND 0.31 ± 0.13f 0.31

ND ND ND

ND ND ND

0.09 ± 0.01g ND 0.09

ND ND ND

0.41 ± 0.12j ND 0.41

ND ND ND

0.19 ± 0.01h ND 0.19

Sweet, candy Wine Fruity, grape Sweet, citrusy

Fruity Pineapple Fruity, banana Fruity Fruity, flowery Fruity, banana Pleasant aroma

± ± ± ±

0.30e 0.25c 0.07e 0.01f

0.03j 0.07f 0.03j 0.51d 0.03j 0.56c

0.06e 0.03g 0.18c 0.01e 0.03f 0.00g 0.03f 0.02f 0.14e 0.04g

1.84 ± 1.01 ± 0.22 ± 8.62 ± 3.36 ± ND 2.17 ± ND ND ND ND ND 17.22

0.07h 0.01i 0.03l 0.45d 0.19f 0.25h

The values are the mean of three determinations ± standard deviation. Values with different letters (a–l) in the same row are significantly different (p b 0.05) as determined by Scott-Knott's test. ND = not detected. a Obtained from the literature. b Relative percentage from total area of the peaks.

C.L. Ramos et al. / Food Research International 64 (2014) 908–918

Compounds

Table 4 Volatile compounds detected at the beginning (T0) and at the end (TF) of the beans of different hybrid fermentations without inoculation. Concentration (%)b Group

Odor descriptiona

PH16 T0

PH16 TF

PS1030 T0

PS1030 TF

FA13 T0

FA13 TF

PS1319 T0

PS1319 TF

Isobutyric acid Butyric acid

Acid Acid

ND 3.46 ± 0.08f

0.69 ± 0.06e ND

ND 14.94 ± 0.08c

0.25 ± 0.06f ND

ND ND

0.17 ± 0.03g 0.65 ± 0.01f

ND 1.39 ± 0.01g

0.29 ± 0.01m 3.79 ± 0.29g

Hexanoic acid Heptanoic acid Nonanoic acid Decanoic acid Total

Acid Acid Acid Acid

Rancid, butter Unpleasant smell and acrid taste. With a sweetish aftertaste Sweet, pungent Rancid, sour Green, fatty Rancid, fatty

1.95 ± 0.08g ND ND ND 5.41

ND ND ND ND 0.69

0.09 ± 0.01f 0.08 ± 0.02f ND 3.46 ± 0.05d 18.48

ND ND ND ND 0.25

0.44 ± 0.08f ND ND ND 0.44

0.28 ± 0.02g ND 32.69 ± 0.44a ND 33.79

0.17 ± 0.03h ND ND ND 1.56

6.57 ± 0.60d ND ND ND 10.65

Methanol 1-Propanol 2-Methyl-1-propanol 1-Butanol 2-Methyl-1-butanol 2-Heptanol 3-Mehtyl-1-pentanol Trans-3-hexen-1-ol 2,3-Butanediol 1,2-Propanediol Furfuryl alcohol 2-Phenylethanol Total

Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols Alcohols

21.56 ± 0.61b 0.19 ± 0.01f 0.72 ± 0.03e 0.31 ± 0.01f 0.67 ± 0.11e ND ND ND 5.86 ± 0.20d ND ND ND 29.31

ND 0.25 ND 2.77 ND ND 0.05 ND ND ND ND 0.27 3.34

3.36 ± ND 4.63 ± 0.38 ± ND 0.17 ± ND ND 2.88 ± 0.17 ± ND ND 11.59

81.89 ± 0.85a 0.20 ± 0.07g 0.22 ± 0.04g 3.22 ± 0.18b 0.56 ± 0.05f ND 0.41 ± 0.06f 0.09 ± 0.01g ND ND ND ND 86.59

7.67 ± ND 3.76 ± 0.97 ± 1.29 ± 0.09 ± 0.08 ± 0.30 ± 1.97 ± 0.08 ± 0.22 ± ND 16.43

0.46c

ND ND ND 35.84 ± 0.23a ND ND 2.67 ± 0.47e 15.86 ± 0.19c ND ND ND ND 54.37

10.64 ± 0.50b 2.27 ± 0.32i ND 4.70 ± 0.42f ND 0.60 ± 0.13l 1.72 ± 0.11j 5.81 ± 0.26e 13.81 ± 0.54a 0.40 ± 0.14m ND 0.33 ± 0.09m 40.28

Acetaldehyde 1,1-Dietoxyethane Hexanal Furfural Total

Aldehydes and ketones Aldehydes and ketones Aldehydes and ketones Aldehydes and ketones

Pungent Acid taste Fruity Almonds

0.08 ± 0.02i 0.17 ± 0.04i 9.01 ± 0.01c ND 9.26

0.44 ± 0.08f 0.89 ± 0.15e 0.36 ± 0.05f ND 1.69

0.09 ± 0.01f ND 0.46 ± 0.12f 0.40 ± 0.07f 0.95

0.08 1.41 0.26 0.09 1.84

0.18 ± 0.03g ND ND 0.60 ± 0.13f 0.78

0.05 0.39 0.07 0.12 0.63

0.01g 0.15g 0.02g 0.03g

0.08 ± 0.02h ND 1.71 ± 0.12f 0.09 ± 0.01h 1.88

5.68 ± 0.45d 5.84 ± 0.22d ND 0.91 ± 0.12l 12.43

Phenylethyl acetate Ethyl acetate Propyl acetate Isobutyl acetate Ethyl butyrate Isoamyl acetate Ethyl pyruvate Ethyl lactate Ethyl octanoate Furfuryl acetate Phenyl acetate Diethylsuccinate Diethyl malate mono-Ethyl succinate Total

Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters Esters

Honey, flowery Pineapple

0.94 ± 0.08h ND 0.45 ± 0.06i 1.89 ± 0.15g 6.40 ± 0.57e 17.70 ± 0.99b ND 1.09 ± 0.15h 21.45 ± 0.77a 1.58 ± 0.40g ND 6.22 ± 0.31e ND ND 57.72

ND ND ND 0.28 ± 0.03f 1.88 ± 0.16e 6.30 ± 0.43c 56.74 ± 1.05a ND ND 0.95 ± 0.06e 0.58 ± 0.12e ND 0.60 ± 0.14e ND 67.33

ND 39.48 ± 0.73a ND 0.51 ± 0.13f 0.09 ± 0.01f 31.12 ± 1.24b 0.28 ± 0.03f 3.72 ± 0.11d ND 0.41 ± .012f ND 0.31 ± 0.12f 0.22 ± 0.10f ND 76.14

ND 0.04 ± 0.02f 0.17 ± 0.03f 1.78 ± 0.30e 0.77 ± 0.18f 1.74 ± 0.23e 10.68 ± 0.45b 0.25 ± 0.07f 67.31 ± 0.98a 2.39 ± 0.15d 0.18 ± 0.03f 0.09 ± 0.01f 0.28 ± 0.03f ND 85.68

1.12 ± ND 3.16 ± 0.59 ± ND ND 0.66 ± 1.83 ± ND 3.12 ± ND 1.28 ± ND ND 11.76

0.47 ± .018g ND 0.93 ± 0.10f 0.63 ± 0.09f 0.30 ± 0.06g 0.20 ± 0.06g 8.94 ± 0.78b 1.34 ± 0.22f 34.50 ± 0.70a 0.83 ± 0.08f 0.08 ± 0.03g ND 0.23 ± 0.02g ND 48.45

ND 5.74 ± 0.79d ND 0.24 ± 0.08h 1.08 ± 0.16g 32.08 ± 0.29b ND ND ND 1.91 ± 0.12f ND 0.06 ± 0.01h 0.44 ± 0.08h ND 41.55

ND ND 1.82 ± ND 0.80 ± 4.62 ± ND 0.91 ± ND 8.28 ± ND 7.70 ± 2.38 ± 4.94 ± 31.45

Decalactone Total

Lactones

Fruity

ND ND

ND ND

ND ND

0.09 ± 0.01f 0.09

ND ND

ND ND

0.40 ± 0.13h 0.40

0.98 ± 0.02l 0.98

b-Citronellol Geraniol Total

Terpenoids Terpenoids/alcohol

Used in perfumes and insect repellents Fruity, flowery

0.12 ± 0.04i 2.96 ± 0.05f 3.08

ND ND ND

0.17 ± 0.08f ND 0.17

0.40 ± 0.14f 0.08 ± 0.03f 0.48

0.29 ± 0.08g ND 0.29

0.20 ± 0.06g ND 0.20

ND ND ND

1.23 ± 0.10l 2.86 ± 0.20h 4.09

Sweet, candy Wine Fruity, grape Sweet, citrusy Grassy-green Natural odor of cocoa butter No odor Faint burning odor and a bitter taste Honey, flowery

Fruity Pineapple Fruity, banana Fruity Fruity, flowery Fruity, banana Pleasant aroma

2.07 ± 0.28 ± 7.46 ± 2.51 ± 6.84 ± ND ND 1.83 ± 0.45 ± 0.35 ± 0.41 ± ND 22.20

0.10g 0.03i 0.66d 0.12f 0.22e

0.24g 0.06i 0.07i 0.13i

± 0.06f ± 0.32e

± 0.01f

± 0.03f

± ± ± ±

0.20d 0.09c 0.19f 0.04f

0.17d 0.03f

0.03f .012e 0.08f 0.01f

0.11e 0.01c 0.01f

0.12f 0.08d 0.18c 0.05e

0.25j 0.27l 0.53f 0.06l 0.31c 0.29c 0.30i 0.36f

915

The values are the mean of three determinations ± standard deviation. Values with different letters (a–l) in the same row are significantly different (p b 0.05) as determined by Scott–Knott's test. ND = not detected. a Obtained from the literature. b Relative percentage from total area of the peaks.

± ± ± ±

.018d 0.03f 0.01f 0.01g 0.03g 0.13g 0.04e 0.02g 0.04g

C.L. Ramos et al. / Food Research International 64 (2014) 908–918

Compounds

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C.L. Ramos et al. / Food Research International 64 (2014) 908–918

Fig. 3. Principal component analysis (PCA) of the total concentration of volatile compound groups and the fermentation assays at the beginning (T0) and final (TF) times of fermentation.

fermentation probably because it was hydrolyzed into glucose and fructose, since the fruit harvest were performed 3 days before the fermentation. This procedure is common in Brazilian cocoa farms because the producers have observed that this procedure accelerates the fermentation process. Ethanol and lactic acid were produced at the middle of the fermentation process (between 36 and 132 h) and acetic acid was detected at the end of the process (after 86 h). These results are consistent with the results of other cocoa fermentations which are related to microbial communities present during the fermentation (Camu et al., 2007; Nielsen et al., 2007; Pereira et al., 2012). It is generally accepted that yeast conduct a strong alcoholic fermentation, thereby creating conditions (e.g. production of ethanol and its conversion to acetic acid) that contribute to the death of the beans. Bean death initiates an array of endogenous biochemical changes that are essential for the development of characteristic chocolate flavor (Hansen, Olmo, & Burns, 1998; Lehrian & Patterson, 1983; Lopez & Dimick, 1995). Other functions of yeasts include degradation of pulp through the production of pectolytic enzymes and decreasing pulp and bean acidity through the utilization of citric acid (Schwan & Wheals, 2004). The secondary products of yeast metabolism (e.g. organic acid, aldehydes, ketones, higher alcohols, esters) and glycosidase production are likely to be significant and should impact on bean and chocolate quality. S. cerevisiae and H. uvarum were the dominant yeast species during the different fermentations even in those spontaneously fermented. This result is in concurrence with other authors (Moreira et al., 2013; Pereira et al., 2012; Schwan & Wheals, 2004), who suggested that S. cerevisiae and Hanseniaspora spp. could be used in cocoa fermentation to enhance the aroma and flavor of chocolate. As expected, faster (starting from 12 h) and higher (higher than 40 g/kg) ethanol concentrations were observed for the hybrid

fermentations inoculated with S. cerevisiae UFLA CA11. However, it was not the case for acetic acid. The hybrids PH16 and PS1319, spontaneously fermented, showed higher acetic concentrations at 156 h, 0.44 and 0.54 g/kg, respectively, than those inoculated with S. cerevisiae UFLA CA11, 0.18 and 0.37 g/kg, respectively. As ethanol is converted into acetic acid by AAB, it was expected to detect higher acetic acid concentrations in the fermentations inoculated with S. cerevisiae UFLA CA11. Ethanol was probably partially eliminated by evaporation and it did not infer in the acetic acid concentration at the end of the process. These are interesting results, the yeast S. cerevisiae UFLA CA11 accelerated the fermentation with a faster (until 36 h) consumption of the carbohydrates and high ethanol production preventing undesirable microorganisms growth. Further, it seems that the yeast did not interfere in the acetic acid concentration at the end of the fermentation. The PCR-DGGE analyses showed that the yeast communities were similar between the different fermentations. On the other hand, the bacterial communities were different according to the fermentation process. It may be explained by the different chemical composition (carbohydrate and citric acid) of the pulp of cocoa hybrids. Representatives of LAB and AAB were identified in all different fermentations. The Lb. fermentum, Lb. rhamnosus and G. liquefaciens species were the dominant bacteria present in all fermentations. The LAB metabolize pulp sugars, producing significant amounts of lactic acid that impacts on the acidity and quality of the bean. Moreover, some species may metabolize pulp citric acid (Schwan & Wheals, 2004; Thompson et al., 2001). The characteristic vinegar-like aroma of cocoa bean fermentations leads early investigators to conclude and demonstrate that acetic acid bacteria were significant contributors to the process. Species belonging to the genera Acetobacter and Gluconobacter were already described in different cocoa fermentations (Ardhana & Fleet, 2003; Garcia-Armisen

C.L. Ramos et al. / Food Research International 64 (2014) 908–918

917

Fig. 4. DGGE analyses of bacteria (A) and yeasts (B) communities found during the 156 h of fermentation of the different cocoa hybrids spontaneously fermented and inoculated with S. cerevisiae UFLA CA11. The closest relatives of the sequenced fragments were determined via GenBank searches for sequences with over 97% similarity. The letters represent the identified species. (A) Bacterial species: a = Streptomuyces sp.; b = Acetobacter sp.; c = Lb. fermentum; d = Lb. casei; e = Leuconostoc lactis; f = Lb. rhamnosus; g = Acetobacter pastorianus; h = Gluconobacter liquefaciens; i = Lb. plantarum; and j = uncultured bacterium. (B) Yeast species: a = H. uvarum; b = S. cerevisiae; and c = H. guilliermondii.

et al., 2010; Lima et al., 2011; Moreira et al., 2013; Pereira et al., 2012; Schwan & Wheals, 2004). The species A. pastorianus, G. liquefaciens and Acetobacter sp. were identified. The AAB oxidized the ethanol, produced by yeasts, to acetic acid, which contributed to the acidity and death of the beans. Acetic acid bacteria can also metabolize sugars and organic acids to produce various aldehydic, ketogenic and other volatile products (Drysdale & Fleet, 1998) that could impact on the sensory quality of beans. Although 39 volatile compounds were detected in this study, their presence and relative percentage varied according to the fermentation

process. It is probably a consequence of the microbial metabolism which was different for the cocoa hybrid fermentations. Esters and alcohols were the most important groups of volatile compounds detected in the different fermentation processes. Esters are already described as important compounds found in cocoa fermentation (Rodriguez-Campos et al.,2011) and are correlated to fruity flavor notes (Serra-Bonvehí, 2005). Regarding the alcohols, they are generally desired to obtain cocoa products with flowery and candy notes (Aculey et al., 2010; Frauendorfer & Schieberle, 2008), e.g. 1-Propanol, 2-Methyl-1-butanol, 2-Heptanol, 2-Phenylethanol found in this study. Acids are generally

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related to unpleasant odor present in cocoa products (Frauendorfer & Schieberle, 2008; Rodriguez-Campos et al., 2011). A total of 6 compounds were identified being some related to rancid, sour, or fatty odor. However, some acids here detected may present pleasant odor e.g. butyric acid and hexanoic acid with sweetish odor. Other volatile compounds such as aldehydes and ketones, lactones and terpenoids were also identified, however in lower concentrations. 5. Conclusion The results showed that the inoculation of S. cerevisiae UFLA CA11 accelerated the fermentation process. However, the hybrid characteristics had direct influence on the fermentation. The substrates (carbohydrates, citric acid) were different between the hybrids that may influence the microbial profile and volatile compounds produced by them. The yeast communities were similar between the hybrid fermentation where S. cerevisiae and H. uvarum were the most important yeasts detected. However, the bacterial communities were dependent on the hybrid and process. The Lb. fermentum, Lb. rhamnosus and G. liquefaciens were the common bacteria species found in all different hybrid and process. The volatile compounds identified were different for all hybrid and process fermentations. In this sense, the different characteristics of the hybrids influence the fermentation process requiring particular management. New studies should be performed in order to elucidate the quality of the chocolate produced by the different process and hybrids. Acknowledgments The authors thank the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support, and Fazenda Reunidas Vale do Juliana (Igrapiúna, Bahia, Brazil) where the fermentations were performed. References Aculey, P., Snitkjaer, P., Owusu, M., Bassompiere, M., Takrama, J., Nørgaard, L., et al. (2010). Ghanaian cocoa bean fermentation characterized by spectroscopic and chromatographic methods and chemometrics. Journal of Food Science, 75, S300–S307. Ardhana, M., & Fleet, G. (2003). The microbial ecology of cocoa bean fermentations in Indonesia. International Journal of Food Microbiology, 86, 87–99. Beckett, S. T. (2009). Conching. In S. T. Beckett (Ed.), Industrial chocolate manufacture and use (pp. 192–222). Chichester: John Wiley & Sons Ltd. Bonvehi, J. S. (2005). Investigation of aromatic compounds in roasted cocoa powder. European food research and technology, 221, 19–29. Camu, N., De Winter, T., Addo, S. K., Takrama, J. S., Bernaert, H., & De Vuyst, L. (2008). Fermentation of cocoa beans: Influence of microbial activities and polyphenol concentrations on the flavor of chocolate. Journal of the Science of Food and Agriculture, 88, 2288–2297. Camu, N., De Winter, T., Verbrugghe, K., Cleenwerck, I., Vandamme, P., Takrama, J. S., et al. (2007). Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana. Applied and environmental microbiology, 73, 1809–1824.

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