Food Research International 90 (2016) 313–319

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Food Research International

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Antioxidant capacity of cocoa beans and chocolate assessed by FTIR

Nádia Nara Batista b, Dayana Pereira de Andrade a, Cíntia Lacerda Ramos a,
Disney Ribeiro Dias b, Rosane Freitas Schwan a,⁎
a
Department of Biology, Federal University of Lavras, 37.200-000 Lavras, MG, Brazil
b
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 a b s t r a c t

Article history: The total antioxidant capacity (TAC) and total phenolic compounds (TPC) of cocoa beans and chocolate produced
Received 2 August 2016 from spontaneous and inoculated fermentations of different cocoa varieties were evaluated. Fourier transform in-
Received in revised form 16 October 2016 frared spectroscopy (FTIR), as well as conventional methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-
Accepted 16 October 2016
azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), was used to determine TAC and TPC. Chocolate
Available online 17 October 2016
showed higher (p b 0.05) TPC (47.17–57.16 mg GAE/g) and TAC (1.66–2.33 mM TE/g and 8.86–11.35 mM TE/g
Keywords:
as measured by DPPH and ABTS, respectively) than cocoa beans (6.30–26.05 mg GAE/g, 0.24–1.17 mM TE/g
Antioxidant activity and 1.29–4.83 mM TE/g for TPC, DPPH and ABTS, respectively). Partial least square (PLS) model for infrared
Total phenolic compounds data showed a good calibration coefficient (R2cal N 0.94), indicating that the FTIR technique represents a fast
DPPH and reliable tool to evaluate TPC and TAC in cocoa beans and chocolate.
ABTS © 2016 Elsevier Ltd. All rights reserved.
PLS analysis
Chocolate

1. Introduction
Cocoa and its derivatives (cocoa powder, cocoa liquor and chocolate)
are sources of methylxanthines, phenolic compounds such as epicate-
chin, polyphenols, and anthocyanins, and a great variety of volatile com-
pounds (e.g. 4-methyl-2-phenyl-2-pentenal and 5-methyl-2-phenyl-2-
hexenal) which are responsible for the astringent and bitter taste of the
cocoa beans (Bonvehi & Coil, 1997; Drewnowski & Gomez-Carneros,
2000). Several physiological functions, including antioxidant and
antimutagenic activities, have been attributed to polyphenols
(Bomser, Singletary, Wallig, & Smith, 1999). Methylxanthines (e.g.
theobromine and caffeine) and the main groups of polyphenols (e.g.
(+) epicatechin, (+) catechins, anthocyanins and proanthocyanidines
dimers) are found in cocoa and cocoa products and have been described
to play important role in preserving human health, however their bio-
availability may vary among polyphenols and, for some of compounds,
among dietary sources, depending on the forms they contain. Isoflavone
is the polyphenol most well absorbed, followed by catechins and others
(e.g. flavanones, and quercetin glucosides) (Katz, Doughty, & Ali, 2011;
Manach, Williamson, Morand, Scalbert, & Rémésy, 2005).
Post-harvest processing, such as fermentation and roasting, is
known to affect the polyphenol and methylxanthine concentrations
⁎ Corresponding author at: Federal University of Lavras, Department of Biology, Campus
Universitário, 3037, 37.200-000 Lavras, MG, Brazil.
E-mail addresses: nadia.nb@hotmail.com (N.N. Batista), dayanaded@hotmail.com
(D.P. de Andrade), cintialramos@yahoo.com.br (C.L. Ramos), diasdr@dca.ufla.br
(D.R. Dias), rschwan@dbi.ufla.br (R.F. Schwan).
and the antioxidant activity of cocoa beans, thereby influencing the
quality of the final product (Aculey et al., 2010). During the fermenta-
tion process the combination of endogenous and exogenous enzyme ac-
tivities, along with diffusion of metabolites into the cotyledons, allows
for the polymerization of polyphenols, which decreases their solubility
thus reducing the bitterness and astringency of the beans and assisting
with the release of methylxanthines from the beans (Bonvehi & Coil,
1997; Hansen, Olmo, & Burri, 1998). During the roasting process, condi-
tions such as time and temperature affect the phenolic stability as well
as the characteristics of resulting flavor (Oracz, Nebesny, & Zyzelewicz,
2014).
Some researchers have proposed the use of starter culture, microor-
ganisms selected to initiate and dominate the fermentation, to better
control the fermentation process and to improve the quality of the
fermented cocoa (Batista, Ramos, Ribeiro, Pinheiro, & Schwan, 2015;
Schwan, 1998). However, their effects on the concentration of polyphe-
nols and methylxanthines, and on the antioxidant activity of fermented
cocoa beans and chocolate have not been studied.
The Fourier transform infrared spectroscopy (FTIR) technique is
based on the vibration of functional groups present in the sample
when exposed to infrared radiation. This technique have been used to
evaluate adulteration of cocoa butter and chocolate, and to determine
the biochemical quality of cocoa (Che Man, Syahariza, Mirghani, Jinap,
& Bakar, 2005; Moros, Iñón, Garrigues, & de la Guardia, 2007). Further,
FTIR has been used to determine the total antioxidant capacity (TAC)
and total polyphenolic content (TPC) of chocolate samples (Hu et al.,
2016). Compared to the conventional methods such as 2,2-diphenyl-
1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-

http://dx.doi.org/10.1016/j.foodres.2016.10.028
0963-9969/© 2016 Elsevier Ltd. All rights reserved.
314 N.N. Batista et al. / Food Research International 90 (2016) 313–319
sulfonic acid) (ABTS), β-carotene, ferric reducing antioxidant power
(FRAP) and oxygen radical absorbance capacity (ORAC) used to detect
the antioxidant activity in foods, FTIR shows increased versatility and ef-
fectiveness and a reduced analysis time (Tarantilis, Troianou, Pappas,
Kotseridis, & Polissiou, 2008).
The aim of this study was to evaluate the TAC and TPC of three differ-
ent varieties of cocoa beans (PH15, PS1319, and CEPEC 2004) before and
after fermentation, and the resulting chocolate produced from these
beans with or without inoculation of S. cerevisiae. The FTIR technique
was employed, along with conventional methods, to evaluate the TAC
and TPC. The methylxanthine (theobromine and caffeine) concentra-
tions were also determined by HPLC.
2. Material and methods
2.1. Fermentation
The fermentations were conducted at the Vale do Juliana cocoa farm
in Igrapiúna, Bahia, Brazil according to described by Menezes et al.
(2016) with minor modifications. The ripe cocoa pods from PH15
(Porto Híbrido/São José da Vitória), PS1319 (Porto Seguro/Uruçuca),
and CEPEC 2004 (llhéus/Bahia) varieties were harvested in November
2013 and used for the experiments. A total of 100 kg of cocoa seeds
with pulp were used for fermentations which were performed in 0.06
m3 wooden boxes covered with banana leaves. After 48 h, the cocoa
seeds were transferred to another box and then were turning every
24 h until end (168 h) of fermentation. Fermentations were performed
spontaneously or with inoculation of Saccharomyces cerevisiae CA11
(LNF-CA11, LNF Latino America, Bento Gonçalves, Rio Grande do Sul,
Brazil) at the beginning of the process. Samples (around 100 g) were ob-
tained at the initial and final times for analysis. The initial time was con-
sidered to be 2 h after the starter culture inoculation, which began the
fermentation process. The fermented beans were dried and sent to
Sartori and Pedroso Alimentos Ltda. (São Roque, SP, Brazil) for chocolate
productions. The chocolate contained 70% cocoa and 30% icing sugar.
Beans were roasted in a roaster cylinder at 120 °C until they reached a
moisture content of 1%. After roasting, cocoa liquor was obtained and
transferred to conching step at 70 °C for 20 h. The molded chocolates
(20 g) were wrapped and stored at 4 °C for analysis.
2.2. Methylxanthine determination
The methylxanthines, caffeine and theobromine, were extracted
from samples of cocoa beans and chocolates according to the methodol-
ogy described by Risner (2008), with minor modifications. For methyl-
xanthine extraction, 0.02 g of cocoa beans were homogenized with 5 mL
of MilliQ water for 5 min by vortexing. Two milliliters of the supernatant
was filtered through a 0.22 μm membrane (Millipore) for HPLC analysis.
The analysis was performed using a liquid chromatography system
(Shimadzu model LC - 10AI, Shimadzu Corp., Japan) equipped with a
UV–Vis detector (SPD 10Ai) at 273 nm. The Shimadzu C18 column
was used and operated at 30 °C. Acetonitrile and water (80:20 v/v)
were used as the eluent at a flow rate of 0.6 mL/min. Calibration curves
were constructed by injecting different concentrations (0.0625 to
0.0039 g/L for theobromine, and 0.0156 to 0.0009 g/L for caffeine) of
the standards (theobromine and caffeine) under the same conditions
as the sample analyses and the areas obtained were plotted on a linear
curve whose equation was used to estimate the concentration of the
compounds in the sample. The analysis was performed in duplicate.
2.3. Preparation of samples and polyphenol extraction
The samples of cocoa beans and chocolate were defatted in accor-
dance with the methodology described by Ioannone et al. (2015), with
minor modifications. Samples (4 g) were extracted three times with
20 mL of n-hexane in order to eliminate lipids from the samples. The
fat-free solid was air-dried for 24 h to evaporate residues of organic sol-
vent. The polyphenols were extract from 1 g of defatted samples by
adding 5 mL of the solvent extraction solution (acetone/distilled
water/acetic acid; 70:29.5:0.5 v/v/v) and stirred by vortexing for
1 min followed by sonication for 10 min in an ultrasonic bath (Treviglio,
Italy). The mixture was centrifuged at 2086g for 10 min, filtered with
cellulose paper and used for chemical analysis (Adamson et al., 1999).
The analysis was performed in triplicate.
2.4. Determination of total polyphenol content (TPC)
TPC of chocolate and cocoa extracts was determined spectrophoto-
metrically according to the Follin–Ciocalteau method (Singleton &
Rossi, 1965). Briefly, 500 μL of samples, 2.5 mL of Folin–Ciocalteau re-
agent (10%) and 2.0 mL of Na2CO3 (4% w/v) were homogenized and
stored in the dark for 2 h. The absorbance of the samples was measured
at 750 nm. The TPC concentrations were obtained from a standard curve
of gallic acid (ranging from 10 to 100 μg/mL) and the results were
expressed as mg of gallic acid/100 g of chocolate/cocoa. The analyses
were performed in triplicate.
2.5. TAC determined by ABTS radical scavenging assay
The ABTS assay was performed according to Re et al. (1999) with
minor modifications. Stock solutions of ABTS (7 mM) and potassium
persulfate (140 mM) were homogenized and kept at room temperature
for 16 h in the dark. The ABTS radical solution was diluted with ethanol
to obtain an absorbance of 0.70 (±0.05) at 734 nm. Aliquots of 30 μL of
samples were added to 3.0 mL of the ABTS radical solution, and after
6 min the absorbance readings were taken. A calibration curve
(y = − 0.0003x + 0.7305) was developed using a range of 0.1–1.0
mmol Trolox/mL and showed good linearity (R2 = 0.9991). The results
were expressed as mmol Trolox Equivalents (TE). The analyses were
performed in triplicate.
2.6. TAC determined by DPPH radical scavenging assay
The DPPH radical scavenging assay was performed as described by
Brand-Williams, Cuvelier, and Berset (1995) with minor modifications.
Briefly, 100 μL of sample was added to 3.9 mL of the DPPH radical solu-
tion (0.06 mM) and stored in the dark for 2 h at room temperature. The
free radical scavenging capacity was then evaluated by measuring the
absorbance at 515 nm. A calibration curve (y = −0.0006x + 0.6427),
in the range of 0.1–1.0 mmol Trolox/mL was used for the quantification
of antioxidant activity, and showed good linearity (R2 = 0.9912). The
results were expressed as mmol Trolox Equivalents (TE). The analyses
were performed in triplicate.
2.7. FTIR spectroscopy
For FTIR analysis, 2 g of cocoa beans without the testa or 2 g of choc-
olate previously crushed using liquid Nitrogen were frozen for 24 h at −
20 °C. Then, the frozen samples were subjected to lyophilizing step for
30 h. FTIR spectra of chocolate and cocoa beans were recorded on a
Digilab Excalibur, series FTS 3000 (United States), coupled to an attenu-
ated total reflectance (ATR) accessory equipped with a ZnSe reflection
crystal. The spectra were acquired at room temperature with 32
scans/sample in the range of 4400 to 600 cm− 1 at a resolution of 4
cm−1, using Origin 8.0 software.
2.8. Statistical analysis and chemometrics
Analysis of variance and the Scott-Knott test (p b 0.05) for antioxi-
dant activity, total phenolic content and methylxanthines concentra-
tions were performed by SISVAR software version 5.3. (Ferreira,
 N.N. Batista et al. / Food Research International 90 (2016) 313–319 315

2008). The Pearson correlation coefficient was used to calculate the cor-
relations between the data, using SAS.
Chemometric model development was performed, including pre-
liminary checking for outliers, setting up a calibration model for TPC
and TAC by spectrophotometer assay, and cross-validation of the
models. For the calibration step, partial-least squares (PLS) regression
was chosen from the most commonly used multivariate calibration
methods for the evaluation of FTIR spectra. PLS models were evaluated
in terms of latent variables, correlation coefficient (R2 value), standard
error and outlier diagnostics. The PLSR model was created, followed
by cross validation (leave–one–out) and the model was statistically
evaluated using RMSECV and R2cv. The calibration performance was
evaluated using the root mean square error of calibration (RMSEc)
and the squared correlation coefficient of calibration (R2cal) (Kiralj &
Ferreira, 2009). A Y-randomization test was carried out to attest to the
model robustness (statistically evaluated using RMSEy-rand and R2y-rand).
The validated calibration model could be performed to do prediction
of samples outside. The root mean square error of prediction (RMSEp)
and squared correlation coefficient of prediction (R2pred) were used as
statistical parameters to judge the model performance on prediction.
Among the 18 samples, 10 were methodologically selected by Kennard
Stone algorithm (Kennard & Stone, 1969) as test samples.
The wavenumbers (between 600 and 4400 cm−1) that character-
ized phenolic compounds were selected for the establishment of all che-
mometric models (PCA and PLSR) in the present study. The second
derivative and mean-center were applied to FTIR spectra to reduce
baseline variation and enhance spectral features. All calculations were
carried out in the Chemoface version 1.4 (Nunes, Freitas, Pinheiro, &
Bastos, 2012).
3. Results and discussion
The concentration of methylxanthines, TPC and TCA were evaluated
in cocoa beans and in the resulting chocolate (70% cocoa) of three differ-
ent cocoa varieties (PH15, PS1319, and CEPEC 2004) after spontaneous
and inoculated fermentation.
Flavonols and methylxanthines are important bioactive components
present in cocoa beans. Methylxanthines (caffeine and theobromine)
are considered to be beneficial for maintenance of health (Franco,
Oñatibia-Astibia, & Martínez-Pinilla, 2013). Caffeine and theobromine
contents were determined in the cocoa beans from the three cocoa va-
rieties (PH15, PS1319, and CEPEC 2004) after spontaneous and inoculat-
ed fermentation and their resulting chocolate and the results are shown
in Table 1. The highest level of theobromine and caffeine was found in
all samples of cocoa at beginning of fermentation (N 12.0 g/kg).
Methylxanthine concentrations decreased during fermentation and
chocolate production. The technological process for chocolate produc-
tion generally result in a decrease of polyphenols concentration
(Bordiga, Locatelli, Travaglia, Co, & Mazza, 2015). Langer, Marshall,
Day, and Morgan (2011) found higher methylxanthine concentration
in chocolate containing 100% of cocoa (nonfat cocoa solids). The theo-
bromine contents in cocoa beans and chocolate did not differ signifi-
cantly (p N 0.05) among the cocoa varieties or between samples that
underwent spontaneous or inoculated fermentation (Table 1). Howev-
er, the caffeine content differed among the cocoa varieties and types
of fermentation (spontaneous and inoculated with S. cerevisiae). Ac-
cording to Pereira-Caro et al. (2013) the accumulation of theobromine
and caffeine in T. cacao (var. Trinitario) occurs in the late period of de-
velopment of the seed and the cocoa genotypes revealed considerable
variations in the purine alkaloid content of the seed. In the present
study, only caffeine showed variation related to the genotype. Inocula-
tion with S. cerevisiae did not affect (p b 0.05) the theobromine content,
but did affect the caffeine levels (Table 1). Caffeine content was, on av-
erage, 6-fold lower compared to theobromine in all samples, consistent
with the general trend in T. cacao beans (Álvarez et al., 2012). The pre-
dominance of theobromine in cocoa is probably a consequence of the N-
methyltransferase-catalyzed metabolism of theobromine to caffeine
being a rate-limiting conversion in the four-step caffeine biosynthesis
pathway (Ashihara, Sano, & Crozier, 2008).
A standardized method for the determination of antioxidant proper-
ties of certain foods and beverages has not yet been established; there-
fore, it is highly advisable to use more than one method for evaluating
antioxidant capacity. It is recommended that at least two or more
methods be combined to provide comprehensive information on the
total antioxidant capacity of a foodstuff. ABTS and DPPH measure a
sample's free radical scavenging capacity. From a mechanistic stand-
point, ABTS and DPPH methods involve an electron transfer reaction
from phenoxide anions to ABT or DPPH, respectively (Foti, Daquino, &
Geraci, 2004; Huang, Ou, & Prior, 2005; Prior, Wu, & Schaich, 2005). In
this study, DPPH and ABTS were applied for the evaluation of TAC in
chocolate and cocoa beans. TPC was also determined and the results
are shown in Table 2.
Table 2
The total phenolic compounds (TPC) and total antioxidant capacity (TAC), evaluated using
the DPPH and ABTS methods, of cocoa beans (before and after fermentation) and choco-
late produced by inoculated (I) and spontaneous (W/I) fermentation of CEPEC 2004,
PH15, PS1319 cocoa varieties.
Samples TPC (mg GAE/g)
Initial Final Chocolate

Table 1 CEPEC 2004 I 8.17 ± 0.02c,D 19.66 ± 0.55b,B 65.24 ± 0.56a,A

Methylxanthine content of cocoa beans (before and after fermentation) and chocolate CEPEC 2004 W/I 12.27 ± 0.34c,A 18.48 ± 0.32b,C 53.60 ± 0.40a,D
produced by inoculated (I) and spontaneous (W/I) fermentation of CEPEC 2004, PH15, PH15 I 10.62 ± 0.10b,C 11.46 ± 0.12b,E 49.25 ± 0.59a,E
PS1319 cocoa varieties. PH15 W/I 9.74 ± 0.14c,C 26.05 ± 0.22b,A 47.17 ± 0.81a,F
PS1319 I 11.27 ± 0.05b,B 10.52 ± 0.11b,F 57.16 ± 1.06a,B
Samples Theobromine (g/kg) PS1319 W/I 6.30 ± 0.06c,E 13.37 ± 0.44b,D 55.43 ± 0.06a,C
DPPH (mM TE/g)
Initial Final Chocolate
Initial Final Chocolate
CEPEC 2004 I 17.41 ± 0.14a,B 8.48 ± 0.14b,A 6.05 ± 0.03c,A CEPEC 2004 I 0.35 ± 0.00c,A 0.79 ± 0.03b,B 2.20 ± 0.08a,A
CEPEC 2004 W/I 16.80 ± 0.19a,B 10.26 ± 1.35b,A 6.60 ± 0.53c,A CEPEC 2004 W/I 0.48 ± 0.01b,A 0.72 ± 0.02b,B 1.94 ± 0.06a,B
PH15 I 17.94 ± 0.36a,B 8.57 ± 1.88b,A 7.70 ± 0.37b,A PH15 I 0.49 ± 0.00b,A 0.49 ± 0.00b,C 2.28 ± 0.14a,A
PH15 W/I 26.10 ± 0.27a,A 10.46 ± 1.31b,A 7.02 ± 0.05c,A PH15 W/I 0.37 ± 0.00c,A 1.17 ± 0.00b,A 1.66 ± 0.10a,C
PS1319 I 12.87 ± 0.29a,B 12.00 ± 0.07a,A 6.44 ± 0.14b,A PS1319 I 0.39 ± 0.00b,A 0.26 ± 0.00b,C 1.99 ± 0.00a,B
PS1319 W/I 14.71 ± 0.22a,B 9.94 ± 0.28b,A 7.72 ± 0.26c,A PS1319 W/I 0.24 ± 0.00c,A 1.06 ± 0.08b,A 2.33 ± 0.46a,A
Caffeine (g/kg) ABTS (mM TE/g)
Initial Final Chocolate Initial Final Chocolate
CEPEC 2004 I 2.55 ± 0.03a,B 1.54 ± 0.03b,C 1.04 ± 0.06b,D CEPEC 2004 I 1.72 ± 0.07c,B 3.87 ± 0.07b,B 10.62 ± 0.11a,B
CEPEC 2004 W/I 6.41 ± 0.16a,A 1.41 ± 0.17b,B 1.36 ± 0.24b,C CEPEC 2004 W/I 2.61 ± 0.03c,A 3.73 ± 0.04b,B 10.27 ± 0.12a,B
PH15 I 0.58 ± 0.03a,C 0.55 ± 0.33a,D 0.72 ± 0.03a,E PH15 I 2.01 ± 0.20b,A 2.16 ± 0.13b,C 10.21 ± 0.61a,B
PH15 W/I 3.18 ± 0.02a,B 1.53 ± 0.14b,B 0.50 ± 0.08c,E PH15 W/I 1.98 ± 0.08c,A 4.83 ± 0.76b,A 8.85 ± 0.05a,C
PS1319 I 3.12 ± 0.03a,B 2.13 ± 0.12b,A 1.81 ± 0.06c,B PS1319 I 2.29 ± 0.10b,A 1.94 ± 0.21b,C 11.07 ± 0.04a,A
PS1319 W/I 2.29 ± 0.03a,B 1.81 ± 0.02b,B 2.10 ± 0.06b,A PS1319 W/I 1.29 ± 0.32c,B 2.54 ± 0.08b,C 11.35 ± 0.20a,A

Means followed by the same capital letter in the column do not differ. Means followed by Means followed by the same capital letter in the column do not differ. Means followed by
the same lower case letter in the line do not differ by Scott-Knott test (p b 0.05). the same lower case letter in the line do not differ by Scott-Knott test (p b 0.05).
316 N.N. Batista et al. / Food Research International 90 (2016) 313–319

Correlations among the results obtained for TAC (DPPH and ABTS)
and TPC were highly positive (r ~ 0.99, p b 0.05) which indicates that
chocolate and cocoa beans have comparable activities in all assays.
The TPC and TCA were significantly (p b 0.05) different when related
to the interaction between cocoa variety and processing time (initial
and final time of fermentation and chocolate) (data not shown). Choco-
lates showed higher (p b 0.05) TPC (47.17–57.16 mg GAE/g) and TAC
(1.66–2.33 mM TE/g and 8.86–11.35 mM TE/g for DPPH and ABTS, re-
spectively) than cocoa beans from both before and after the fermenta-
tion process (6.30–65.24 mg GAE/g, 0.24–1.17 mM TE/g and 1.29–4.83
mM TE/g of phenolic compounds, DPPH and ABTS, respectively). The
high TPC and TAC observed in chocolate may be due to the oxidation
and polymerization of procyanidins, related to the high temperature
and the residual activity of some oxidative enzymes (Summa et al.,
2006). Further, during the bean processing, the Maillard reaction con-
tributes to the formation of reducing substances (e.g. melanoidins)
whose reducing power is responsible for their free-radical scavenging
activity increasing the antioxidant effect of the bean (Summa et al.,
2006; Yamaguchi, Koyama, & Fujimaki, 1981). Todorovic et al. (2015),
while studying dark chocolates (65% to 75% cocoa) from Serbia, report-
ed lower TPC and TAC (around 11.99 mg GAE/g, 0.075 mM TE/g, and
0.077 mM TE/g, for phenolic compounds, DPPH and ABTS, respectively)
than those found in the present study. Variations in bioactive com-
pounds in cocoa beans depend on the genotypic characteristics, geo-
graphical area and post-harvest conditions and are directly related to
the quality of the final product (Wollgast & Anklam, 2000).
It is well known that fermentation affects the concentration of poly-
phenols and the antioxidant activity of the cocoa beans interfering in
the quality of the final product (Aculey et al., 2010). In general, there
was an increase (p b 0.05) in TAC and TPC during spontaneous fermen-
tation of all cocoa varieties. It seems that the starter culture may have af-
fected the microbiota during fermentation, as observed by Batista et al.
(2015), and consequently the fermentation process, TAC and TPC.
Among the cocoa varieties, the chocolate produced by spontaneous fer-
mentation of PH15 showed the lowest TPC and TAC. These facts may in-
dicate that the cocoa variety and the fermentation process may affect
the polyphenol content and antioxidant activity of chocolate. The
main polyphenol groups found in cocoa beans are catechins,

Fig. 1. FTIR-ATR spectra from cocoa beans and chocolate produced by spontaneous (W/I) and inoculated (I) fermentation of CEPEC 2004 (a and b, respectively), PH15 (c and d,
respectively), PS1319 (e and f, respectively) cocoa varieties. T0 represents the beginning of fermentation and TF the end of fermentation.
 N.N. Batista et al. / Food Research International 90 (2016) 313–319 317

epicatechins, anthocyanins and procyanidins, the presence of which are

directly related to the antioxidant activity (Wan et al., 2001).
The spectral features of cocoa beans and chocolate were obtained by
FTIR analysis and are shown in Fig. 1. Bands referring to the phenol
group were observed in the regions 3562–3322 cm− 1 (attributed to
the stretch of O\\H) and 1244–1064 cm−1 (attributed to C\\H stretch).
The bands related to the aromatic ring were observed in the regions
2925–2854 cm− 1 (attributed to the stretch of the C\\H of aromatic
ring), 992–680 cm−1 (attributed to angular deformation of C\\H of ar-
omatic ring) and 1645–1544 cm−1 (attributed to C\\C stretch of aro-
matic ring) (Barbosa, 2007; Silverstein, Webster, & Kiemle, 2006).
From this information, a principal component analysis (PCA) was
performed (Fig. 2). The PCs explained 92.6% of the total variance
among the samples. The first component (PC1) explained 70.06% and
the second component (PC2) explained 22.54% of the variance. The
evaluated chocolates showed a strong similarity being grouped at the
negative side of PC1 and PC2 differentiated by the following spectra:
987.5 cm−1, which was attributed to the angular deformation of the ar-
omatic ring C\\H and 2806.4 cm− 1, 2848.9 cm−1 and 2916.4 cm− 1,
which were attributed to the aromatic ring C\\H stretch (Fig. 3). In ad-
dition, chocolates differed from cocoa beans due to the C\\C stretch of
the aromatic ring represented by spectra 1643.4 cm−1 and 1550.8
cm−1 (Fig. 3). All selected spectra evaluated belong to phenolic com-
pounds group. Fig. 3. First two principal components (a) PC1 and (b) PC2 of selected spectra for
The inoculation of S. cerevisiae yeast seemed did not affect the profile antioxidant compounds obtained by FTIR.

of phenolic compounds during the fermentation process as observed by

the similarity between the initial and final inoculated samples for all va-
rieties (Fig. 2). According to PCA, the cocoa beans from PS1319 and
PH15 before and after fermentation with S. cerevisiae as well as
PS1319 and CEPEC 2004 before fermentation without inoculation
showed similar phenolic compound profiles as grouped on the positive
side of PC2. On the negative side of PC2 and positive side of PC1, the
samples of PH15 before and after spontaneous fermentation, CEPEC
2004 before and after inoculated fermentation and PS1319 and CEPEC
2004 after fermentation without inoculation were grouped according
to their phenolic compound profiles (Fig. 2). This result shows that
spontaneous fermentation probably had a more pronounced microbial
modification during fermentation than the process inoculated with S.
cerevisiae which affected the phenolic compound profiles. This result
agrees with those obtained for TPC and TAC by conventional methods,
where the spontaneous fermentation showed an increase in the TPC,
DPPH and ABTS values from beginning to final of fermentation. It has
been found that the inoculation of yeast including S. cerevisiae influ-
enced the microbial profile, which likely affected the volatile com-
pounds that affect sensory characteristics, resulting in chocolate with
different flavor. Furthermore, cocoa variety has direct influence on the
fermentation since the substrates (carbohydrates, citric acid) are differ-
ent between the varieties (Batista, Ramos, Ribeiro, Pinheiro, & Schwan,
2016; Menezes et al., 2016; Ramos, Dias, Miguel, & Schwan, 2016).
Thus, the S. cerevisiae inoculation as well as cocoa variety may also affect
phenolic compounds during fermentation and in the resulting
chocolate.
For quantitative analysis of TPC and TAC by FTIR, the obtained spec-
tra (600–4400 cm−1) were evaluated using the PLS model. The number
of latent variables (LV) used in each model was established according to
a small value of root square error of cross validation (RMSEcv). Table 3
shows the results of the PLS model to predict the TAC and TPC of choc-
olate and cocoa samples. The PLS models for content of TPC and TAC
(DPPH and ABTS) showed good correlation coefficients of calibration
(R2cal N 0.94) and squared correlation of cross validation showed accept-
able values (R2cv N 0.60) ranging from 0.80–0.90 (Kiralj & Ferreira,
2009).

Table 3
PLSR models for TAC and TPC in cocoa beans using FTIR for ABTS, DPPH and Folin–
Ciocalteu assays (TPC).a

TPC DPPH ABTS

LV 4 4 4
RMSEc 4.5802 0.1608 0.7177
R2cal 0.9492 0.9550 0.9643
RMSEcv 6.6096 0.2390 1.1613
R2cv 0.8949 0.9010 0.9074
RMSEp 6.7379 0.2524 1.0735
R2pred 0.917 0.9536 0.9416
R2m (TEST) 0.8388 0.8215 0.8998
RMSEy-rand 14.0083 0.4700 2.8143
R2y-rand 0.5179 0.6100 0.4452
Fig. 2. Principal component analysis (PCA) of chocolate (CHOC) and cocoa beans from
cR2p(y-rand) 0.6398 0.5739 0.7076
inoculated (I) and spontaneous (W/I) fermentation of CEPEC 2004, PH15, PS1319 cocoa
a
varieties. T0 represents the cocoa beans obtained at the beginning of fermentation and For ABTS and DPPH, the unit is l mol Trolox/g FW; for Folin–Ciocalteu, the unit is mg
TF from the end of fermentation process. gallic acid/g FW.
318 N.N. Batista et al. / Food Research International 90 (2016) 313–319
The robustness of PLS the model was checked by randomizing the y
values which was also used for validation of PLS. Low R2y-rand values and
high RMSEy-rand values were obtained, suggesting that the correlation
was not random. The external calibration suggests that the PLS models
showed good predictive ability (R2pred N 0.91) for TPC and TAC (DPPH
and ABTS). The R2m values higher than 0.5 confirmed good correlations
between the measured data and the predicted PLS index (Roy, Paul,
Mitra, & Roy, 2009). Therefore, FTIR can be used to predict the antioxi-
dant capacity of cocoa beans and chocolate with a precision similar to
that obtained by conventional chemical assays.
4. Conclusion
The content of the bioactive compound theobromine in cocoa beans
and chocolate did not differ significantly (p N 0.05) among the cocoa va-
rieties after spontaneous and inoculated fermentation. On the other
hand, the caffeine content differed among the cocoa varieties and varied
based on the type of fermentation (spontaneous and inoculated with S.
cerevisiae). The cocoa beans at beginning of fermentation showed
higher TPC and TAC than chocolate. Among the cocoa varieties, the
chocolate produced by spontaneous fermentation of PH15 showed the
lowest TPC and TAC indicating that the cocoa variety and the fermenta-
tion process probably affect the polyphenol content and antioxidant ac-
tivity of chocolate. Further, the results obtained in this study show that
the phenolic compound contend increased (p b 0.05) from the begin-
ning to final for the most of the spontaneous fermentations. This was
not the case for the fermentations inoculated with S. cerevisiae. It may
indicate that the spontaneous fermentation produces a more pro-
nounced microbial modification than the process with S. cerevisiae inoc-
ulation, and this microbial modification may affect the phenolic
compound profile, as observed by conventional and FTIR methods. The
FTIR technique was successfully used to predict the TPC and TAC of
cocoa beans and chocolate allowing a fast and reliable evaluation of
these parameters.
Conflict of interest
None.
Acknowledgements
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 financial support, and Fazenda Reunidas Vale do Juliana
(Igrapiúna, Bahia, Brazil) where the fermentations were performed.
The authors gratefully acknowledge the anonymous referees for their
comments and constructive suggestions for improving the quality of
this manuscript.
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