Food Chemistry 174 (2015) 256–262

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Flavanols, proanthocyanidins and antioxidant activity changes during

cocoa (Theobroma cacao L.) roasting as affected by temperature and time
of processing
F. Ioannone a,b, C.D. Di Mattia b, M. De Gregorio b, M. Sergi b, M. Serafini a, G. Sacchetti b,⇑
a
Agricultural Research Centre, Functional Food and Metabolic Stress Prevention Laboratory, CRA-NUT, Via Ardeatina 546, 00178 Rome, Italy
b
Faculty of Biosciences and Technologies for Food, Agriculture and Environment, University of Teramo, Via C.R. Lerici 1, Mosciano Stazione, 64023 Teramo, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The effect of roasting on the content of flavanols and proanthocyanidins and on the antioxidant activity of
Received 11 April 2014 cocoa beans was investigated. Cocoa beans were roasted at three temperatures (125, 135 and 145 °C), for
Received in revised form 6 October 2014 different times, to reach moisture contents of about 2 g 100 g1. Flavanols and proanthocyanidins were
Accepted 3 November 2014
determined, and the antioxidant activity was tested by total phenolic index (TPI), ferric reducing antiox-
Available online 8 November 2014
idant power (FRAP) and total radical trapping antioxidant parameter (TRAP) methods.
The rates of flavanol and total proanthocyanidin loss increased with roasting temperatures. Moisture
Keywords:
content of the roasted beans being equal, high temperature-short time processes minimised proanthocy-
Cocoa
Roasting
anidins loss. Moisture content being equal, the average roasting temperature (135 °C) determined the
Flavanols highest TPI and FRAP values and the highest temperature (145 °C) determined the lowest TPI values.
Procyanidins Moisture content being equal, low temperature-long time roasting processes maximised the chain-
Antioxidant activity breaking activity, as determined by the TRAP method.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction
A number of studies have shown that the consumption of cocoa
and chocolate products has positive health effects for humans.
These health effects have been assumed to be associated with
the presence of polyphenols, among which are monomeric fla-
van-3-ols, (+)-catechin and ()-epicatechin, as well as oligomeric
and polymeric procyanidins or proanthocyanidin, which show high
antioxidant capacity (Adamson et al., 1999).
Proanthocyanidins found in cocoa are tannins, ranging in size
from monomers to long chain polymers (Ortega et al., 2010), and
their size is described by degree of polymerisation (Gu, House,
Wu, Ou, & Prior, 2006).
In vitro studies have confirmed that proanthocyanidins exhibit
several biological properties, associated with antioxidant activity
(Adamson et al., 1999; Counet & Collin, 2003; Lee, Kim, Lee, &
Lee, 2003; Othman, Ismail, Ghani, & Adenan, 2007), such as the
ability to scavenge superoxide radicals and hydroxyl radicals,
reduce lipid peroxyl radicals and inhibit lipid peroxidation
(Kanner, Frankel, & Granet, 1994; Salah et al., 1995; Vinson &
Hontz, 1995). Recently, these compounds have attracted increased
⇑ Corresponding author. Tel.: +39 0861 266913; fax: +39 0861 266915.
E-mail address: gsacchetti@unite.it (G. Sacchetti).
attention in the fields of nutrition and health since they are associ-
ated with anti-inflammatory and anti-atherosclerotic activity,
blood pressure and immune function modulation and platelet acti-
vation (Ramiro et al., 2005; Katz, Doughty, & Ali, 2011; Latif, 2013).
In order to obtain chocolate, the cacao seeds must be subjected
to a multi-step post-harvest process, by which the beans can be
converted to cocoa and, eventually, into final industrial products.
Cocoa processing to chocolate dramatically affects the polyphenols
quantity and quality of cocoa beans (Caligiani, Cirlini, Palla,
Ravaglia, & Arlorio, 2007; Di Mattia et al., 2013; Gu et al., 2006;
Payne, Hurst, Miller, Rank, & Stuart, 2010; Wollgast & Anklam,
2000).
Roasting is the most important technological operation in pro-
cessing of cocoa beans since it brings about formation of character-
istic brown colour, mild aroma and texture of roasted beans
(Krysiak, 2006). Roasting affects also the ability of polyphenols to
interact with protein, causing a decrease in astringency
(Misnawi, Jamilah, & Nazamid, 2005).
Literature reviews suggest that the impact of roasting on quality
indices should be studied by taking into account the general
impact of process parameters; in fact, the degree of cocoa roasting
is dependent on both roasting time and temperature, with temper-
atures ranging from 120 to 150 °C and roasting times varying from
5 to 120 min, depending on the temperature (Krysiak, 2006, 2011).

http://dx.doi.org/10.1016/j.foodchem.2014.11.019
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
 F. Ioannone et al. / Food Chemistry 174 (2015) 256–262 257

It has long been reported that, during roasting, monomeric flav-
anols (catechin and epicatechin compounds) reduce in concentra-
tion (Caligiani et al., 2007; Kothe, Zimmermann, & Galensa, 2013;
Payne et al., 2010). Payne et al. (2010) and Kothe et al. (2013) stud-
ied the evolution of monomeric, dimeric and trimeric proanthocy-
anidins as a function of roasting temperature without considering
the effect of roasting times. Other authors determined the antiox-
idant activity of methanolic extracts of cocoa as affected by roast-
ing (Arlorio et al., 2008; Suazo, Davidov-Pardo, & Arozarena, 2014)
but these studies gave conflicting results, even though the same
roasting temperature and antioxidant activity assay were used.
The aim of this work is to study the evolution of monomeric and
condensed flavanols (proanthocyanidins), as well as changes in
antioxidant activity during the cocoa roasting process, as a func-
tion of roasting temperature and time, in order to identify the
time–temperature combinations which maximise the polyphenol
content and antioxidant activity of roasted beans.
2. Materials and methods
2.1. Materials
Fermented and dried cocoa (Theobroma cacao L.) beans (cv. Cri-
ollo) were purchased from the Peruvian company ‘‘Cacaotera’’ in
Rome (Italy). Reagents of analytical grade were provided by
Sigma–Aldrich (Steinheim, Germany).
2.2. Roasting process
Cocoa beans were subjected to roasting at three selected tem-
peratures (125, 135 and 145 °C) for different times (see Table 1)
until they reached about 1.8 g 100 g1 moisture content.
The roasting process was carried out in a ventilated electric
oven, model Air-o-steam COMBI 6GN 1/1, (Electrolux, Stockholm,
Sweden) by keeping air-flow rate (1 m s1) and relative humidity
(0.4%) constant. Approximately 35 g of cocoa beans, with uniform
size, were placed in single layer on an aluminium plate and placed
in the pre-heated oven. Due to the heat capacity of the oven, the set
temperature decreased by only 4 °C upon product loading and was
re-stabilized after 40 s; thus the temperature inside the oven could
be considered constant during processing. Portions of roasted
cocoa were taken from the oven after determined time limits
(Table 1) and brought to ambient temperature (20 ± 2 °C) before
analysis. The roasting process was carried out in duplicate.
2.3. Weight loss and moisture
The weight loss of all the cocoa samples was determined using a
technical balance (±0.01 g).
Samples were ground in a coffee mill (Super Junior ‘‘S’’,
Moulinex, Paris, F) to obtain a mean particle size of about
500 lm prior to analysis.
Moisture content of cocoa samples was determined gravimetri-
cally by a moisture analyzer (Sartorius MA150, Goettingen,
Germany) according to Di Mattia et al. (2013).
2.4. Chemical analysis
2.4.1. Sample extraction
Ground samples (4 g) were defatted three times by extracting
with 25 ml of hexane and, each time, the supernatant was dis-
charged and the lipid-free solids were dried under nitrogen flux.
Sample extraction was carried out according to the procedure
described by Adamson et al. (1999). 1 g of dried and defatted mate-
rial was extracted with 5 ml of 70:29.5:0.5 acetone/water/acetic
acid (v/v/v) by mixing for 1 min in a vortex mixture and by sonica-
tion at 20 °C for 10 min in an ultrasonic bath. The mixture was
eventually centrifuged (2086g for 10 min) and finally filtered
through cellulose filters. The filtered cocoa extract (CE) was used
for all chemicals analysis.
2.4.2. Flavanols determination
Chromatographic analyses were performed on a 1200 Agilent
Series HPLC (Agilent Technologies, Milano, Italy) equipped with a
G1322 degasser, a G1311A quaternary pump, a G136A Column
thermostat, an autosampler injection system and a diode array
detector. The system was controlled with Agilent ChemStation
for Windows (Agilent Technologies). Phenolics determination
was carried out according to Zuo, Chen, and Deng (2002). The sam-
ple (20 ll) was injected onto a C18 reversed-phase column, Kine-
tex 5 lm C18 100A 250  4.6 mm (Phenomenex, Bologna, Italy);
the column was equipped with a C18 SecurityGuard Column

Table 1
Flavanol and proanthocyanidin (P1–P10) contents of control cocoa beans (CNT) and cocoa beans roasted at three temperatures for different times.

T (°C) Time (min) Epicatechin Catechin Epigallocatechin Gallocatechin P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

CNT 0 1.12 1.34 0.00 0.00 3.76 2.24 2.00 1.58 1.17 0.60 0.38 0.26 0.15 0.12
125 6 1.11 1.34 0.37 0.26 4.22 2.29 2.09 1.56 1.24 0.71 0.42 0.28 0.17 0.12
125 22 0.88 1.06 0.93 1.70 2.54 1.35 0.86 0.80 0.39 0.17 0.15 0.09 0.07 –
125 28 0.53 1.01 1.00 1.38 3.52 2.00 1.40 1.16 0.59 0.14 0.11 0.11 0.11 –
125 40 0.43 0.94 0.92 1.80 2.33 1.34 0.80 0.44 0.38 0.11 0.08 0.07 0.05 –
125 46a 0.42 0.88 0.70 1.80 2.27 1.22 0.43 0.62 0.25 0.23 0.11 0.09 0.08 –
125 50 0.40 0.85 0.80 1.24 1.93 1.17 0.23 0.50 0.26 0.19 0.07 0.07 0.06 –
125 62b 0.39 0.84 0.58 1.14 2.12 1.41 0.68 0.58 0.40 0.43 0.16 0.13 0.10 0.08
125 74 0.38 0.80 0.55 1.45 2.40 1.37 0.26 0.56 0.23 0.20 0.10 0.09 0.08 0.06
135 5 0.68 1.19 0.54 0.18 3.76 2.24 2.00 1.58 1.17 0.60 0.38 0.26 0.15 0.12
135 15 0.53 1.14 1.30 1.84 4.03 2.40 2.10 1.75 1.40 0.61 0.35 0.22 0.11 0.07
135 25a 0.34 0.75 0.79 1.67 3.31 1.80 1.10 0.83 0.82 0.20 0.14 0.10 0.09 –
135 30b 0.24 0.55 0.54 1.26 2.60 1.69 0.58 0.71 0.53 0.10 0.09 0.07 0.07 –
135 35 0.25 0.54 0.53 1.10 2.99 1.75 0.37 0.67 0.22 0.25 0.11 0.09 0.07 0.06
145 9 0.50 1.17 0.54 0.18 2.23 1.33 0.28 0.60 0.25 0.20 0.09 0.10 0.08 0.05
145 13a 0.43 1.00 1.17 2.25 3.76 2.24 2.00 1.58 1.17 0.60 0.38 0.26 0.15 0.12
145 18b 0.32 0.84 1.09 1.76 2.82 1.65 1.09 0.77 0.81 0.22 0.13 0.11 0.07 –
145 20 0.32 0.79 0.82 1.36 3.22 1.82 1.22 0.77 0.66 0.16 0.10 0.07 0.07 –
145 30 0.24 0.55 0.69 1.21 2.54 1.38 0.75 0.78 0.59 0.11 0.06 0.06 0.06 –
145 40 0.21 0.52 0.56 0.70 2.44 1.36 0.49 0.38 0.30 0.08 0.06 0.06 0.04 –

Data expressed as mg g1

dw. RSD < 5%.
a
2.3 g 100 g1 moisture content.
b
1.9 g 100 g1 moisture content.
258 F. Ioannone et al. / Food Chemistry 174 (2015) 256–262
4  3.0 mm (inside diameter). Separation of phenolic compounds
was carried out at a flow rate of 1 ml/min with a non-linear gradi-
ent from A (1% acetic acid solution) to B (methanol). Gradient elu-
tion was as follows: from 5% to 25% B from 0 to 5 min, from 25% to
63% B from 5 to 45 min, from 63% to 5% B from 45 to 50 min, iso-
cratic from 50 to 53 min. The DAD acquisition range was set from
200 to 400 nm. Calibration curves were made with epicatechin,
catechin, catechin gallate, gallocatechin and epigallocatechin and
the results were expressed as mg per gdw of cocoa solids.
2.4.3. Proanthocyanidins determination
Proanthocyanidin determination was carried out according to
Di Mattia et al. (2013). The sample (20 ll) was injected onto a Phe-
nomenex 5 lm normal-phase Luna Silica column, 100A,
250 mm  4.6 mm (inside diameter), at 25 °C; the column was
equipped with SecurityGuard Cartridges Silica 4  3.0 mm (inside
diameter). Separation of proanthocyanidins was carried out at a
flow rate of 1 ml/min with a linear gradient from A (dichlorometh-
ane) to B (methanol) and a constant 4% level of C (acid acetic and
water, 1:1 v/v) according to Counet and Collin (2003). Gradient
elution was as follows: from 14% to 28% B from 0 to 30 min, from
28% to 50% B from 30 to 60 min, from 50% to 86% B from 60 to
65 min, isocratic from 65 to 70 min. Separation of the compounds
was previously done according to retention times by HPLC-UV
analysis and then the compounds were collected according to
Counet and Collin (2003) and submitted to mass spectrometric
(MS) analysis for their identification.
The MS analyses of the HPLC fractions (P1–P10) were carried
out by means of a triple quadrupole mass spectrometer API 2000
from AB-Sciex (Toronto, ON, Canada) equipped with a TurboIon-
Spray source. The spectra were acquired by injecting each solution
at a flow of 10 ll min1 by a syringe pump; all the analytes were
detected in negative ionisation with a capillary voltage of
4500 V, nebulizer gas (air) at 0.21 N mm2, curtain gas (nitrogen)
at 0.21 N mm2. The declustering potential was set at 22 V for m/
z < 1000 amu; 80 V for m/z > 1000 amu. For the MS/MS experi-
ments, the collision gas was set at 3 (in a scale 0–6) and the colli-
sion energy was 20 eV. The spectra were acquired using AB-Sciex
Analyst Software 1.5.
The quantification of single proanthocyanidins was carried out
by HPLC analysis. Since proanthocyanidins show similar absorp-
tion coefficients (Counet & Collin, 2003), a calibration curve, made
with ()-epicatechin, was used for their quantification and the
results for each proanthocyanidin class were expressed as mg epi-
catechin equivalents per gdw of cocoa solids.
2.4.4. Total phenolics index
The amount of total soluble phenolics was determined accord-
ing to the method proposed by Singleton and Rossi (1965). 20 ll
of diluted sample were pipetted into a 96-well plate. 100 ll of
Folin–Ciocalteu reagent, diluted 1:10 with water, and 75 ll of
10% (w/v) sodium carbonate solution were added to each well,
and the plate was stood for 2 h at room temperature in the dark.
Colour development at 740 nm was then determined using a Nano-
Quant spectrophotometer (Tecan, Segrate, Italy). TPI values were
calculated by a calibration curve, obtained with increasing concen-
trations of gallic acid, and results were expressed as mg of gallic
acid equivalents (GAE) per g of dry weight.
2.4.5. Antioxidant activity assays
Total antioxidant capacity was assessed by ferric reducing
power antioxidant parameter (FRAP) and the total radical trapping
antioxidant parameter (TRAP) assays, respectively, for the mea-
surement of reducing and chain-breaking antioxidant activity.
The reducing power was monitored by the FRAP assay (Benzie &
Strain, 1996). The reduction of the Fe3+-TPTZ complex to the
ferrous form, at low pH, was monitored at 595 nm by a Sunrise
absorbance plate reader (Tecan). Briefly, 160 ll of FRAP reagent,
prepared daily, were mixed with 30 ll of water and 10 ll of diluted
sample; the absorbance at 595 nm was recorded after a 30 min
incubation at 37 °C by a Tecan NanoQuant spectrophotometer.
FRAP values were calculated, using a calibration curve obtained
with increasing concentrations of Fe2+. FRAP results were
expressed as lmol of Fe2+ per gdw.
The chain-breaking activity was determined by the TRAP
method, which is based on the protection provided by antioxidants
on the fluorescence decay of R-phycoerythrin (lag phase) during a
controlled peroxidation reaction (Ghiselli, Serafini, Maiani, Azzini,
& Ferro-Luzzi, 1995). In brief, 50 ll of diluted sample were added
to 75 ll of PBS (pH 7.4), 15 ll of R-PE (4.30  103 lg ll1), and
60 ll of ABAP (7.5 mM); the reaction kinetics at 38 °C were
recorded for 60 min (kex = 495 nm, kem = 570 nm) by a Tecan
GENios standard fluorescence plate reader spectrometer. The
length of the lag phase, automatically calculated, was used to
assess TRAP values. TRAP values were calculated by a calibration
curve obtained from increasing concentrations of trolox. TRAP
results were expressed as lmol of trolox equivalents (TE) per
gdw.
2.5. Statistical analysis
Data presented are the averages of at least three independent
measurements taken from two different repetitions of each sample
type and reported as mean and relative standard deviation RSD.
One way ANOVA was applied to experimental data to determine
the significance of effects (roasting temperature and roasting
time).
Principal components analysis (PCA) was applied to describe
the proanthocyanidin data set and to detect the most important
variables for determining the data structure (only variables with
a loading >0.70 on PCs were used).
Epicatechin/catechin and total proanthocyanidins changes over
time were modelled, using the Weibull probabilistic cumulative
distribution, which was rewritten as:
C0  Ct a
¼ 1  eðt=bÞ ð1Þ
C0  CE
where C0, CE, Ct are initial, equilibrium and time t epicatechin/cate-
chin or proanthocyanidin concentrations; a is the shape parameter
and b (s) is a rate parameter.
The effect of temperature on the rate of proanthocyanidins loss
was evaluated by means of the Arrhenius equation:
K ¼ K 0  eðEa=RÞ ð2Þ
where K is the reaction rate, K0 the pre-exponential factor, Ea is the
apparent activation energy and R the universal gas constant. Since
the inverse of the rate parameter of Eq. (1) (b1), having the dimen-
sion of s1, was used as reaction rate, Ea could be considered as the
activation energy rather than a thermal dependence factor.
Non-linear regression was performed to compute Eq. (1)
parameters by the ‘‘Rosenbrock pattern search’’ algorithm and to
compute Eq. (2) parameters using the ‘‘Levenberg-Marquadt’’ algo-
rithm. The goodness of fit was evaluated by the coefficient of deter-
mination (R2). Statistical analyses were carried out using the
STATISTICA software (StatSoft™, Tulsa, OK).
3. Results
Cocoa beans roasting at three temperatures, from 125 °C to
145 °C, was carried out for different times (Table 1) until reaching
a 1.8 g 100 g1 residual moisture content, around 2 g 100 g1 being
 F. Ioannone et al. / Food Chemistry 174 (2015) 256–262
the optimal moisture content for cocoa beans grinding and fat
extraction (Krysiak, Adamski, & Zyzelewicz, 2013).
Roasting times of 62 and 46 min at 125 °C determined final
moisture contents of 1.9 and 2.3 g 100 g1, respectively; these val-
ues were not different from those reached by roasting for 30 and
25 min at 135 °C, respectively, and for 18 and 13 min at 145 °C,
respectively.
The content of flavanols was determined on the control and on
samples roasted at the three temperatures for different times
(Table 1). The epicatechin content of the control sample is within
the range reported by Payne et al. (2010) for dried fermented beans
but catechin content was much higher than those reported by
these authors and others (Caligiani et al., 2007; Kothe et al.,
2013) whilst it is more similar to that reported by Gu et al.
(2006) for natural cocoa powder. Epicatechin content in cocoa is
largely affected by fermentation and drying processes, which could
determine a 6-fold variation of epicatechin in dried fermented
cocoa beans (Kim & Keeney, 1984; Wollgast & Anklam, 2000) but
scarce data are available about the content of catechin as affected
by fermentation and drying.
A general reduction of catechin and epicatechin upon roasting
was observed (Table 1), in accordance with the results reported
by Payne et al. (2010) and Kothe et al. (2013). On the other hand,
the contents of gallocatechin and epigallocatechin content
increased during the first stages of roasting, and eventually
decreased in the last stages with epigallocatechin content reaching
its maximum value more rapidly than gallocatechin. The epicate-
chin to catechin ratio, which has been proposed as a useful and
sensitive indicator for the processing history of cacao beans
(Payne et al., 2010), decreased during roasting time (Fig. 1), thus
suggesting that epicatechin degradation is faster than that of cate-
chin, possibly due to isomerization of () epicatechin to (+) cate-
chin (Caligiani et al., 2007). The evolution of epicatechin to
catechin ratio over roasting time at three different temperatures,
as shown in Fig. 1, was well described by the cumulative function
of the Weibull probabilistic model, with R2 of 0.998, 0.996 and
0.984 for 125, 135 and 145 °C, respectively.
Roasting temperature elevation increased the overall rate of
epicatechin/catechin decrease, as described by the decrease of
the rate parameter (b), and the shape parameter (a), (Table 2).
The shape parameter (a) is a behaviour index that depends on
the process mechanism and, the higher its value, the slower is
the process in the initial phase (Cunha, Oliveira, & Oliveira, 1998;
Fig. 1. Epicatechin to catechin ratio evolution in cocoa beans during roasting at
different temperatures and Arrhenius plot of epicatechin/catechin decrease rate.
The broken lines connect samples with equal moisture contents: light grey
2.3 g 100 g1, dark grey 1.9 g 100 g1.
259
Sacchetti, Pittia, & Pinnavaia, 2005). The estimated values of b1
and absolute roasting temperatures (K) were used to compute
the Arrhenius equation parameters (Table 2) and b1 resulted
strictly (R2 = 0.982) temperature-dependent, as shown in the
Arrhenius plot (Fig. 1 insert).
In processes aimed to mimic conventional roasting (Table 1),
moisture content being equal, the epicatechin/catechin ratio con-
tent was inversely related to roasting temperature (Fig. 1). This
result suggests that HTST processes induce a higher epicatechin
epimerization than do LTLT. When roasting times were equal, tem-
perature negatively affected the epicatechin/catechin ratio, thus
confirming the results of Kothe et al. (2013), who suggested a
higher ()-epicatechin to (+)-catechin epimerization at higher
roasting temperatures.
The content of monomeric and polymeric proanthocyanidins
was determined on the control and on samples roasted at three
temperatures for different times (Table 1). The P1 content of the
control sample, when expressed on gdw of non fat cocoa solids,
was equal to 6.88 mg g1 dw; this value is higher than those reported
by Di Mattia et al. (2013) who reported values of 2.5–6 mg g1 dw for
dried fermented beans.
PCA analysis was applied to describe the data set and to deter-
mine the most important variables that influence the data struc-
ture. The results showed that the first principal component
explains almost 85% of total variability and that a second compo-
nent could only explain 9% of variability. All the proanthocyani-
dins but P10 showed a loading higher than 0.7 on the first PC.
The scores of all samples showed an increase along the first PC,
where P1–P9 proanthocyanidins had a negative loading, during
processing time (data not shown). P10, which decreased in the
initial phases of roasting until it completely disappeared and fur-
ther increased at the end of processing (Table 1), did not show a
loading higher than 0.7 either on the first or on the second PC
(data not shown).
The relative abundance of each single proanthocyanidin frac-
tion, in samples roasted at the three temperatures, was calculated
as a function of roasting time from data shown in Table 2. Roasting
time governed increases of the relative abundance of P1 and P2
that, in previous studies, were less oxidizable than other proanth-
ocyanidins (Di Mattia et al., 2013). When only P1 and P2 concen-
trations were analysed, no changes of their relative abundance
were observed between unroasted beans and beans roasted for
30 min (data not shown). This is not in accordance with Kothe
et al. (2013) who observed a decrease of the relative abundance
of monomeric flavanols in favour of dimers.
The relative abundance of P3, P4 and P5 (calculated from data
reported in Table 2) decreased during roasting. The relative abun-
dance of high molecular weight proanthocyanidins (P6–P10)
decreased in the first stages of roasting but increased at prolonged
roasting time, as a result of the polymerisation of proanthocyani-
dins with lower molecular weight. Final moisture content being
equal, the high molecular weight proanthocyanidins (P6–P10) con-
tent is higher in low temperature long time (LTLT) processes than
in high temperature short time (HTST) ones.
The total proanthocyanidins content, which is a useful index to
evaluate the processing history of cocoa (Di Mattia et al., 2013),
was computed on the basis of data reported in Table 2. The total
proanthocyanidins content of the control sample, when expressed
as gdw of non fat cocoa solids, was equal to 22.4 mg g1dw; this value
is higher than that reported by Di Mattia et al. (2013) who reported
values 9–15 mg g1 .dw The total proanthocyanidins content unequivo-
cally decreased upon heat treatment (Fig. 2) in accordance with the
results of Di Mattia et al. (2013).
The evolution of total proanthocyanidins content over roasting
time at three different temperatures, shown in Fig. 2, was well
described by the cumulative function of the Weibull probabilistic
260 F. Ioannone et al. / Food Chemistry 174 (2015) 256–262

Table 2
Estimation of the parameters and determination coefficient (R2) of the Weibull model (Eq. (1)) applied to model epicatechin/catechin ratio and total proanthocyanidins decrease
kinetics over time and calculation of the thermal dependence factor (Ea) using the Arrhenius equation.

Temperature (°C) a b (min) CEa R2 k0 (min1) Ea (J mol1) R2

Epicatechin/catechin
125 21.9 ± 3.37 27.2 ± 0.09 0.47 ± 0.00 0.998 6.93E+16 1.33E+05 0.982
135 1.03 ± 0.43 4.22 ± 0.49 0.45 ± 0.01 0.996
145 0.68 ± 0.19 1.13 ± 0.49 0.40 ± 0.01 0.987
Total proanthocyanidins
125 4.80 ± 1.62 24.5 ± 1.10 5.35 ± 0.26 0.974 2.16E+06 5.90E+04 0.985
135 2.11 ± 0.56 18.8 ± 2.11 5.32 ± 0.53 0.965
145 0.87 ± 0.31 7.03 ± 1.77 4.92 ± 0.77 0.987

± Signifies standard error.

a
CE unit for epicatechin/catechin is mg mg1; CE unit for total proanthocyanidins is mg g1
dw.

different reactivities with the Folin–Ciocalteu reagent (Naczk &

Shahidi, 2004) and that the mechanism is based on a oxidation/
reduction reaction, TPI can also be considered an antioxidant
method (Prior, Wu, & Schaich, 2005).

Fig. 2. Kinetics of total proanthocyanidin loss in cocoa beans during roasting at

different temperatures and Arrhenius plot of proanthocyanidins loss rate. The
broken lines connect samples with equal moisture contents: light grey
2.3 g 100 g1, dark grey 1.9 g 100 g1.

model, with R2 of 0.993, 0.963 and 0.950 for 125, 135 and 145 °C,
respectively.
Roasting temperatures increased the overall rate of total pro-
anthocyanidins loss, as described by the decrease of the rate
parameter (b), and decreased the shape parameter (a) (Table 2).
The estimated values of b1 and absolute roasting temperatures
(K) were used to compute the Arrhenius equation parameters
(Table 2) and b1 resulted strictly (R2 = 0.982) temperature-depen-
dent, as shown in the Arrhenius plot (Fig. 2 insert).
In processes aimed to mimic conventional roasting (Table 1),
moisture content being equal, the total proanthocyanidins content
was inversely related to roasting temperatures (Fig. 2), similarly to
the epicatechin/catechin ratio. These results confirm that HTST
processes better preserve the polyphenols content than do LTLT
ones (Mrkić, Cocci, Dalla Rosa, & Sacchetti, 2006).
The antioxidant activity was determined on the same sample
extracts as used for polyphenols analyses by means of the reaction
with the Folin–Ciocalteu reagent (TPI method), the reduction of
Fe3+ (FRAP method) and the chain breaking activity, as determined
by the TRAP method. These methods differ in several aspects, such
as the mechanism of action (radical or redox reaction) and the
environmental conditions (solvent polarity and pH).
The TPI is based on the capacity of phenolic compounds to
Fig. 3. Antioxidant activity of the cocoa beans during roasting at different
reduce the Folin–Ciocalteu reagent under basic conditions and thus temperatures as measured by TPI (A), FRAP (B), and TRAP (C). The broken lines
has been extensively used as a method for the estimation of total connect samples with equal moisture contents: light grey 2.3 g 100 g1, dark grey
phenolics; nonetheless, taking into account that polyphenols show 1.9 g 100 g1.
 F. Ioannone et al. / Food Chemistry 174 (2015) 256–262
In Fig. 3A, the TPI of cocoa beans subjected to roasting at different
temperatures is shown as a function of process time. The initial value
of TPI, which corresponds to the control sample, was higher than
those reported by Di Mattia et al. (2013) and underwent to signifi-
cant changes (p < 0.05) upon roasting. At the end of roasting time,
samples showed lower TPI values than did raw beans and this result
is in accordance with Arlorio et al. (2008) and Suazo et al. (2014).
A non-linear evolution of TPI during roasting, with a general
decrease in the first phases of the roasting process, followed by a
slight increase in the last phases, was observed. The initial decrease
of TPI values could be explained by the reduction of flavanols and
proanthocyanidins. The slight increase of TPI observed in the last
phases of roasting could be explained by the formation of proanth-
ocyanidins with high molecular weight and higher reducing power
(Di Mattia et al., 2013), as well as to the formation of reductones as
a consequence of non-enzymatic browning reactions (among
which is the Maillard reaction), which occur during roasting (Di
Mattia et al., 2011; Sacchetti, Di Mattia, Pittia, & Mastrocola,
2009; Summa et al., 2006).
Moisture content being equal, the average roasting temperature
(135 °C) determined the highest TPI values and the highest tem-
perature determined the lowest TPI values. Other studies proved
that colour formation upon roasting is maximum at 135 °C
(Ioannone, 2014; Krysiak et al., 2013), thus confirming that the for-
mation of coloured compounds, as a consequence of Maillard reac-
tion (MR), could have contributed to increase of the TPI value.
The temperature dependence of TPI decrease was different from
that observed for proanthocyanidins loss (Fig. 1), thus confirming
that TPI could not be indiscriminately used to measure the total
polyphenols content of cocoa beans.
In Fig. 3B the FRAP of cocoa beans subjected to roasting at
different temperatures is reported as a function of process time.
The initial value of FRAP, which corresponds to the control sample,
was higher than those reported by Di Mattia et al. (2013) and
underwent significant changes (p < 0.05) upon roasting. The results
achieved by means of the FRAP assay (Fig. 3) were similar to those
obtained by the TPI, because both the assays measure the reducing
power of the cocoa extracts. Moisture content being equal, the
average roasting temperature (135 °C) determined the highest
FRAP values whilst the other temperatures showed different
effects on FRAP, depending on the duration of the roasting process.
The antiradical properties of cocoa beans subjected to roasting
were evaluated by the TRAP method, which works by a mechanism
based on a hydrogen transfer reaction (Prior et al., 2005). Results,
expressed as TE antioxidant capacity, are illustrated in Fig. 3C.
The results achieved by means of the TRAP assay were different
from those obtained by the other two methods, since the increase
of antioxidant activity observed during roasting was much more
evident when the TRAP assay was used. This fact could be
explained by taking into account that MR products formed during
roasting process show a high chain-breaking activity although they
did not have high reducing potential (Nicoli, Toniolo, & Anese,
2004). Moisture content being equal, lower roasting temperature
(125 °C) led to the highest TRAP values and the LTLT roasting pro-
cesses maximised the chain-breaking activity. However, when
moisture contents were equal, TRAP values observed at 125 °C
were not significantly higher than those at 135 °C and this temper-
ature is generally preferred to 125 °C since it maximises colour
formation and it causes a lower polyphenol loss (Fig. 2) and a lower
formation of MR-induced potentially toxic compounds, such as
furans, (Ioannone, 2014) than does the latter.
HTST process carried out at 145 °C maximise polyphenols
retention and this result is important since the bioavailability
and function of proanthocyanidin monomers and dimers in human
metabolism have been widely investigated and their bio-efficacy is
generally accepted.
261
On the other hand, the metabolism of food-borne advanced
MRPs with in vitro antioxidant activity has not been completely
elucidated and it is still an open question whether isolated mela-
noidin structures are bioavailable, undergo metabolic biotransfor-
mation, and, subsequently, cause physiological effects in vivo.
4. Conclusion
The different processing conditions applied during roasting
caused dramatic differences in flavanols, proanthocyanidin con-
tents and antioxidant activity. In particular, high temperature-
short time (HTST) roasting better preserves the polyphenol content
than does low temperature-long time (LTLT), whilst this tendency
was not observed for antioxidant activity which is generally max-
imised by LTLT processes.
Since polyphenol bioavailability and function in human of
metabolism has been widely investigated and their bio-efficacy is
generally accepted, HTST processes are recommendable for pre-
serving the functional properties of cocoa upon roasting; however,
other quality indices should also be taken into consideration in
order to meet the market requirement.
References
Adamson, G. E., Lazarus, S. A., Mitchell, A. E., Prior, R. L., Cao, G., Jacobs, P. H., et al.
(1999). HPLC method for the quantification of procyanidins in cocoa and
chocolate samples and correlation to total antioxidant capacity. Journal of
Agricultural and Food Chemistry, 47, 4184–4188.
Arlorio, M., Locatelli, M., Travaglia, F., Coïsson, J. D., Del Grosso, E., Minassi, A., et al.
(2008). Roasting impact on the contents of clovamide (N-caffeoyl-L-DOPA) and
the antioxidant activity of cocoa beans (Theobroma cacao, L.). Food Chemistry,
106, 967–975.
Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as
measure of antioxidant power: The FRAP assay. Analytical Biochemistry, 239,
70–76.
Caligiani, A., Cirlini, M., Palla, G., Ravaglia, R., & Arlorio, M. (2007). GC–MS detection
of chiral markers in cocoa beans of different quality and geographic origin.
Chirality, 19, 329–334.
Counet, C., & Collin, S. (2003). Effect of the number of flavanol units on the
antioxidant activity of procyanidin fractions isolated from chocolate. Journal of
Agricultural and Food Chemistry, 51, 6816–6822.
Cunha, L. M., Oliveira, F. A. R., & Oliveira, J. C. (1998). Optimal experimental design
for estimating the kinetic parameters of processes described by the Weibull
probability distribution function. Journal of Food Engineering, 37, 175–191.
Di Mattia, C. D., Sacchetti, G., Neri, L., Martuscelli, M., Mastrocola, D., & Pittia, P.
(2011). Technological parameters and antioxidant activity of cocoa powders.
Progress in Nutrition, 13, 39–47.
Di Mattia, C., Martuscelli, M., Sacchetti, G., Scheirlinck, I., Beheydt, B., Mastrocola, D.,
et al. (2013). Effect of fermentation and drying on procyanidins, antiradical
activity and reducing properties of cocoa beans. Food and Bioprocess Technology,
6, 3420–3432.
Ghiselli, A., Serafini, M., Maiani, G., Azzini, E., & Ferro-Luzzi, A. (1995). A
fluorescence based method for measuring total plasma antioxidant capacity.
Free Radical Biology and Medicine, 18, 29–36.
Gu, L., House, S. E., Wu, X., Ou, B., & Prior, R. L. (2006). Procyanidin and catechin
contents and antioxidant capacity of cocoa and chocolate products. Journal of
Agricultural and Food Chemistry, 54, 4057–4061.
Kanner, J., Frankel, E., & Granet, R. (1994). Natural antioxidants in grapes and wines.
Journal of Agricultural and Food Chemistry, 42, 64–69.
Katz, D. L., Doughty, K., & Ali, A. (2011). Cocoa and chocolate in human health and
disease. Antioxidants and Redox Signaling, 15, 2779–2811.
Kim, H., & Keeney, P. G. (1984). (–)Epicatechin content in fermented and
unfermented cocoa beans. Journal of Food Science, 49, 1090–1092.
Krysiak, W. (2006). Influence of roasting conditions on coloration of roasted cocoa
beans. Journal of Food Engineering, 77, 449–453.
Krysiak, W. (2011). Effects of convective and microwave roasting on the
physicochemical properties of cocoa beans and cocoa butter extracted from
this material. Grasas y Aceites, 62, 467–478.
Krysiak, W., Adamski, R., & Zyzelewicz, D. (2013). Factors affecting the color of
roasted cocoa bean. Journal of Food Quality, 36, 21–31.
Kothe, L., Zimmermann, B. F., & Galensa, R. (2013). Temperature influences
epimerization and composition of flavanol monomers, dimers and trimers
during cocoa bean roasting. Food Chemistry, 141, 3656–3663.
Ioannone, F. (2014). Dark chocolate with high antioxidant activity: Cocoa roasting
optimization and oxidative stress modulation in humans (PhD thesis). University
of Teramo.
Latif, R. (2013). Chocolate/cocoa and human health: A review. The Netherlands
Journal of Medicine, 71, 63–68.
262 F. Ioannone et al. / Food Chemistry 174 (2015) 256–262
Lee, K. W., Kim, Y. J., Lee, H. J., & Lee, C. Y. (2003). Cocoa has more phenolic
phytochemicals and a higher antioxidant capacity than teas and red wine.
Journal of Agricultural and Food Chemistry, 51, 7292–7295.
Misnawi, S. J., Jamilah, B., & Nazamid, S. (2005). Changes in polyphenol ability to
produce astringency during roasting of cocoa liquor. Journal of the Science of
Food and Agriculture, 85, 917–924.
Mrkić, V., Cocci, E., Dalla Rosa, M., & Sacchetti, G. (2006). Influence of drying
conditions on bioactive compounds and antioxidant activity of dried broccoli
(Brassica oleracea, L.). Journal of the Science of Food and Agriculture, 86,
1559–1566.
Naczk, M., & Shahidi, F. (2004). Extraction and analysis of phenolics in food. Journal
of Chromatography A, 1054, 95–111.
Nicoli, M. C., Toniolo, R., & Anese, M. (2004). Relationship between redox potential
and chain-breaking activity of model systems and foods. Food Chemistry, 88,
79–83.
Ortega, N., Romero, M., Macià, A., Reguant, J., Anglès, N., Morelló, J. R., et al. (2010).
Comparative study of UPLC–MS/MS and HPLC–MS/MS to determine
procyanidins and alkaloids in cocoa samples. Journal of Food Composition and
Analysis, 23, 298–305.
Othman, A., Ismail, A., Ghani, N. A., & Adenan, I. (2007). Antioxidant capacity and
phenolic content of cocoa beans. Food Chemistry, 100, 1523–1530.
Payne, M. J., Hurst, W. J., Miller, K. B., Rank, C., & Stuart, D. A. (2010). Impact of
fermentation, drying, roasting, and Dutch processing on epicatechin and
catechin content of cacao beans and cocoa ingredients. Journal of Agricultural
and Food Chemistry, 58, 10518–10527.
Prior, R. L., Wu, X., & Schaich, K. (2005). Standardized methods for the
determination of antioxidant capacity and phenolics in foods and dietary
supplements. Journal of Agricultural and Food Chemistry, 53, 3101–3113.
Ramiro, E., Franch, A., Castellote, C., Cano, F. P., Permanyer, J., Pulido, M. I., et al.
(2005). Flavonoids from Theobroma cacao down-regulate inflammatory
mediators. Journal of Agricultural and Food Chemistry, 53, 8506–8511.
Sacchetti, G., Pittia, P., & Pinnavaia, G. G. (2005). The effect of extrusion temperature
and drying-tempering on both the kinetics of hydration and the textural
changes in extruded ready-to-eat breakfast cereals during soaking in
semi-skimmed milk. International Journal of Food Science and Technology, 38,
135–143.
Sacchetti, G., Di Mattia, C., Pittia, P., & Mastrocola, D. (2009). Effect of roasting
degree, equivalent thermal effect and coffee type on the radical scavenging
activity of coffee brews and their phenolic fraction. Journal of Food Engineering,
90, 74–80 [Erratum. Journal of Food Engineering, 91, 372].
Salah, N., Miller, N. J., Paganga, G., Tijburg, L., Bolwell, P., & Rice-Evans, C. (1995).
Polyphenolic flavonoids as scavengers of aqueous phase radicals and as
chain breaking antioxidants. Archives in Biochemistry and Biophysics, 322,
339–346.
Singleton, V. L., & Rossi, J. A. Jr., (1965). Colorimetry of total phenolics with
phosphomolybdic–phosphotungstic acid reagents. American Journal of Enology
and Viticulture, 16, 144–158.
Suazo, Y., Davidov-Pardo, G., & Arozarena, I. (2014). Effect of fermentation and
roasting on the phenolic concentration and antioxidant activity of cocoa from
Nicaragua. Journal of Food Quality, 37, 50–56.
Summa, C., Cordeiro Raposo, F., McCourt, J., Lo Scalzo, R., Wagner, K. H., Elmdfa, I.,
et al. (2006). Effect of roasting on the radical scavenging activity of cocoa beans.
European Food Research and Technology, 222, 368–375.
Vinson, J. A., & Hontz, B. A. (1995). Phenol antioxidants index: Comparative
antioxidant effectiveness of red and white wines. Journal of Agricultural and Food
Chemistry, 43, 401–403.
Wollgast, J., & Anklam, E. (2000). Review on polyphenols in Theobroma cacao:
Changes in composition during the manufacture of chocolate and methodology
for identification and quantification. Food Research International, 33, 423–447.
Zuo, Y., Chen, H., & Deng, Y. (2002). Simultaneous determination of catechins,
caffeine and gallic acids in green, oolong, black and pu-erh teas using HPLC with
a photodiode array detector. Talanta, 57, 307–316.