Food Chemistry 194 (2016) 1089–1094

Contents lists available at ScienceDirect

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

Physicochemical properties and antioxidant capacity of raw, roasted and

puffed cacao beans
SuJung Hu, Byung-Yong Kim, Moo-Yeol Baik ⇑
Department of Food Science and Biotechnology, Institute of Life Science and Resources, Kyung Hee University, Seocheon 1, Yongin 446-701, Republic of Korea

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

Article history: The antioxidant capacity and attributable bioactive compounds of puffed cacao beans were investigated.
Received 28 February 2015 Roasting was carried out at 190 °C for 15 min and puffing was performed at 4–7 kgf/cm2. Cacao beans
Received in revised form 7 July 2015 puffed at 4 kgf/cm2 showed the highest total polyphenols (23.16 mg GAE/g sample) and total flavonoids
Accepted 28 August 2015
(10.65 mg CE/g sample) (p < 0.05). As the puffing pressure increased, the amount of total polyphenols and
Available online 29 August 2015
total flavonoids decreased. The antioxidant capacity of cacao beans reflected the total polyphenols and
flavonoids measured. The quantities of theobromine, catechin, epicatechin, and procyanidin B2 were
Keywords:
higher in cacao beans puffed at 4 kgf/cm2 than in roasted cacao beans. Puffed cacao beans received a good
Cacao beans
Puffing
sensory score in flavor, but sourness increased as puffing pressure increased. Thus, these results suggest
Roasting that, in cacao bean processing, puffing could be an alternative to roasting, which provide a rich taste and
Antioxidant capacity high antioxidant capacity.
HPLC Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction are present in cacao beans; theobromine and caffeine comprise

Cacao (Theobroma cacao L.) is a popular ingredient used in var-
ious foods such as chocolate, beverages and bakery products. A
number of studies have shown that the consumption of cacao-
containing foods shows positive health benefits. For example,
cacao may lower blood pressure, resulting in a lower risk of cardio-
vascular disease (Buijsse, Weikert, Drogan, Bergmann, & Boeing,
2010); reverse endothelial dysfunction (Heiss et al., 2005); inhibit
epinephrine-stimulated platelet activation (Pearson et al., 2002)
and ameliorate insulin sensitivity via improving b-cell function
(Grassi et al., 2008). These health benefits are associated with their
phytochemical contents (Tomás-Barbéran, Borges, & Crozier,
2011).
Cacao beans contain high concentrations of polyphenols
(approximately 12–18%, dry weight) in unfermented raw beans
(Kim & Keeney, 1984). The cacao polyphenols can be classified into
three groups: catechins or flavan-3-ols (ca. 37%), anthocyanins (ca.
4%) and proanthocyanidins (ca. 58%) (Wollgast & Anklam, 2000).
The main form of cacao flavanol monomers are (
)-epicatechin
and (+)-catechin (Tomás-Barbéran et al., 2011). Procyanidin is
comprised of oligomers and/or polymers of catechin and epicate-
chin, which are members of the proanthocyanidin family. Addi-
tionally, alkaloids such as theobromine, caffeine and theophylline
⇑ Corresponding author.
E-mail address: mooyeol@khu.ac.kr (M.-Y. Baik).
3.7% and 0.2% of dry weight in defatted beans, respectively (Pura
Naik, 2001).
The identities and amounts of these phytochemicals change
during manufacturing processes. For example, high levels of fla-
vanols in unfermented raw cacao beans, and the levels are dramat-
ically decreased during fermentation, roasting and alkalization
(Wollgast & Anklam, 2000). Fermentation is essential for the devel-
opment of desirable flavors and flavor precursors. However, more
than 80% of the catechin and epicatechin are lost in this process
(Payne, Hurst, Miller, Rank, & Stuart, 2010). During roasting, the
cacao flavor is further developed from the precursor compounds
that were formed during fermentation and drying of raw cacao
beans. Roasting time and temperature affect the stability of pheno-
lic compounds as well as the taste of the cacao-containing foods
(Mazor Jolić, Radojčić Redovniković, Marković, Ivanec Šipušić, &
Delonga, 2011). In particular, the roasting process leads to loss
and modification of flavanol, which leads to a loss of 14% of the
total phenolics content (Mazor Jolić et al., 2011), as well as epimer-
ization of (
)-epicatechin to (
)-catechin (Kothe, Zimmermann, &
Galensa, 2013).
The puffing process is another cooking method that uses heat
and pressure. Puffing is accomplished by either the atmospheric
pressure method or the pressure drop method (Hsieh, Huff, Peng,
& Marek, 1989). However, both methods are similar in that the
moisture within the matrix suddenly expands into vapor and the
materials are simultaneously cooked. The puffing process also

http://dx.doi.org/10.1016/j.foodchem.2015.08.126
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
1090 S. Hu et al. / Food Chemistry 194 (2016) 1089–1094
changes the physicochemical properties of food. Puffing creates an
aerated, porous structure and snack-like texture with the added
benefits of dehydration (Nath, Chattopadhyay, & Majumdar,
2007). The heat and pressure during puffing also cause a change
in the chemical composition of puffed materials. For example, it
has been reported that explosion-puffing significantly increased
in the total polyphenol content of jujubes, which led to a corre-
sponding increase in antioxidant properties (Du et al., 2013).
Therefore, puffing could serve as an alternative processing method
to roasting for cacao beans, thereby increasing yield, promoting a
rich taste and providing a high antioxidant capacity. The objective
of this study was to compare the physicochemical properties and
antioxidant capacity of puffed cacao beans and conventionally
roasted cacao beans.
2. Materials and methods
2.1. Chemicals and reagents
(+)-Catechin, (
)-epicatechin, theobromine, procyanidin B2,
ascorbic acid, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH),
Folin–Ciocalteu reagent, 2,20 -azobis (2-methylpropionamidine)
dihydrochloride (AAPH), (2,20 -azino-bis(3-ethylbenzthiazoline-6-s
ulfonic acid) diammonium salt (ABTS), and 2,4,6-tris(2-pyridyl)-
s-triazine (TPTZ) were purchased from Sigma Aldrich Co. (St. Louis,
MO, USA). Sulfuric acid, acetic acid, methanol, diethyl ether,
sodium carbonate, sodium nitrate, sodium hydroxide, and iron
(III) chloride hexahydrate were purchased from Duksan Co. (Ansan,
Korea). Aluminum chloride anhydrous was purchased from Kanto
Co. (Tokyo, Japan).
2.2. Sample preparation
Fermented and dried cacao beans (Forastero variety, Ghana)
were kindly donated from Sun Food Co. (Seongnam, Korea).
2.2.1. Preparation of roasted and puffed cacao beans
Roasting was performed at 190 °C for 15 min using an industrial
coffee roaster (Neotec Co., Reinbek, Germany) (sample R15). Alter-
natively, puffing was performed using a traditional gun puffing
machine. First, raw cacao beans (sample RB) were placed into the
chamber, and the chamber and beans were heated. The chamber
door was opened when the pressure reached 4 kgf/cm2,
5 kgf/cm2, 6 kgf/cm2 or 7 kgf/cm2 for samples P4, P5, P6 and P7,
respectively. After the roasting and puffing, all cacao beans were
cooled to room temperature and stored at
20 °C until use. The
cacao beans were manually deshelled and ground using a food
mixer (JNM-650, Shanghai Guosheng Industrial Co., Ltd, Shanghai,
China) for each experiment.
2.2.2. Fat extraction
A ground cacao bean sample (10 g) was defatted by the Soxhlet
method using diethyl ether for 17 h. After defatting, the diethyl
ether in the sample was pre-evaporated in the fume hood at room
temperature for 1 h, and then evaporated in 40 °C in a dry oven for
2 h. After drying, the sample was cooled in a desiccator and stored
at -20 °C until use.
2.3. Proximate analysis and pH
Moisture content, crude fat, crude protein and ash were
determined using the Korean Food Standards Codex.
2.4. Phenolic extraction
Phenolic extraction of the cacao sample was performed using a
previously described aqueous methanol with slight modification
(Othman, Ismail, Abdul Ghani, & Adenan, 2007). Briefly, 1 g of
ground cacao bean was mixed with 25 mL of 70% aqueous metha-
nol for 2 h in a 40 °C water bath. The mixture was filtered through
filter paper (Whatman No. 2) using a Buchner funnel.
2.5. Total polyphenols
Total phenolic content (TP) of cacao beans was measured using
Folin & Ciocalteu’s assay (Singleton & Rossi, 1965). Gallic acid was
employed as a calibration standard and the results are expressed as
gallic acid equivalents (GAE)/g sample.
2.6. Total flavonoids
Total flavonoid content (TF) was determined according to the
method of Jia, Tang, and Wu (1999). Catechin was employed as a
calibration standard and results are expressed as catechin equiva-
lents (CE)/g sample.
2.7. Antioxidant capacity
2.7.1. DPPH assay
The DPPH assay was performed according to Brand-Williams,
Cuvelier, and Berset (1995) with slight modification. DPPH solution
(0.1 mM) in 80% methanol was prepared. The absorbance of DPPH
solution was adjusted to 0.650 ± 0.020 using 80% methanol at
517 nm. The filtered sample (0.5 mL) was mixed with 2.95 mL of
distilled water, shaken vigorously and stored in the dark at 23 °C
for 30 min. Then, the final absorbance reading was measured at
517 nm. Ascorbic acid was employed as a calibration standard
and results are expressed as vitamin C equivalents (VCE)/g sample.
2.7.2. FRAP assay
A ferric reducing ability of plasma (FRAP) assay was performed
according to the method of Thaipong, Boonprakob, Crosby,
Cisneros-Zevallos, and Hawkins Byrne (2006). Ascorbic acid was
employed as a calibration standard and results are expressed as
vitamin C equivalents (VCE)/g sample.
2.7.3. ABTS assay
The ABTS assay was performed according to the method of van
den Berg, Haenen, van den Berg, and Bast (1999). Ascorbic acid was
employed as the calibration standard and results are expressed as
vitamin C equivalents (VCE)/g sample.
2.8. HPLC analysis
Reverse phase (RP) high performance liquid chromatography
(HPLC) was used to quantify catechin, epicatechin, theobromine
and procyanidin B2. Samples were diluted and filtered before
RP-HPLC (Shimadzu model LC-20AD, Kyoto, Japan) analysis. The
sample injection volume was 20 lL. The column was an Agilent
ZORBAX Eclipse Plus C18 (4.6  250 nm, 5 lm) (Santa Clara, CA,
USA). The mobile phase was a binary gradient with a flow rate of
0.5 mL/min and consisted of (A) ddH2O + 0.1% formic acid and (B)
acetonitrile + 0.1% formic acid. The starting mobile phase condition
was 8% (B), held isocratically for 0.1 min. Subsequently, solvent (B)
was increased to 60% (0.1–16 min) and then returned to 8% (16–
18 min), with a final isocratic run of 8% (B) from 18 to 22 min.
The peaks were detected on a photodiode array detector at 280 nm.
 S. Hu et al. / Food Chemistry 194 (2016) 1089–1094 1091

2.9. Sensory evaluation Table 2

Total polyphenols and total flavonoids of raw, roasted and puffed cacao beans.

Sensory evaluation of cacao beans was carried out by fifty-five Sample Total polyphenols Total flavonoids
experienced panelists (ages from 23 to 28, graduate students of (mg GAE/g sample) (mg CE/g sample)
Food Science and Biotechnology Department at Kyung Hee Univer- Cacao beans Defatted cacao Cacao beans Defatted cacao
sity). Scoring was carried out a 9-point hedonic scale, following the beans beans
scale: 1 = lowest and 9 = highest for bitterness, astringency, sour RB 27.74 ± 5.17a 55.58 ± 4.98a 18.97 ± 4.90a 38.07 ± 2.79a
taste and flavor; 1 = extremely dislike and 9 = extremely like for R15 19.14 ± 0.38c 36.21 ± 5.44c 7.51 ± 1.50c 15.55 ± 1.09c
overall acceptance. Each sample was assigned a random number P4 23.16 ± 1.58b 42.55 ± 4.08b 10.65 ± 1.52b 18.30 ± 1.01b
P5 19.47 ± 3.35c 29.50 ± 1.28d 8.94 ± 1.94bc 15.72 ± 1.14c
and presented to panel members. Panelists rinsed their mouths
P6 14.45 ± 0.42d 30.76 ± 2.69d 7.19 ± 0.78c 13.57 ± 2.94d
with water between samples. P7 14.41 ± 0.18d 29.80 ± 0.89d 7.31 ± 1.83c 15.78 ± 0.97c

2.10. Statistical analysis
All experiments were repeated three times. All data are
expressed as the mean ± standard deviation (SD). Experimental
data were analyzed using analysis of variance (ANOVA), and
expressed as the mean value ± SD. A Duncan’s multiple range test
was conducted to assess significant differences among experimen-
tal mean values (p < 0.05). All statistical computations and analy-
ses were conducted using SAS version 8.02 for Windows (SAS
Institute, Inc., Cary, NC, USA).
3. Results and discussion
3.1. Proximate composition and pH
Table 1 shows proximate composition and pH of raw, roasted
and puffed cacao beans. Roasted and puffed cacao beans had less
moisture than their raw counterparts. In line with our results,
others have observed an effect of roasting temperature and time
on the moisture content (Pittia, Dalla Rosa, & Lerici, 2001;
Özdemir & Onur Devres, 1999). Moisture contents of puffed cacao
beans were higher than that of roasted cacao bean. This might be
due to the difference between open and closed system. Roasting
is carried out in an open system, where moisture in the beans
can be easily and constantly dried. On the other hand, gun puffing
occurred in a closed system, where water within the beans was
vaporized during heating up to certain vapor pressure and vapor
is gone when the system is opened resulting in moisture loss in
cacao beans. Mariotti, Alamprese, Pagani, and Lucisano (2006)
reported that the moisture contents of rice and buckwheat
decreased from 9.1 to 8.1 g/100 g and from 14.1 to 7.1 g/100 g with
puff processing, respectively. On the other hand, steep decrease of
moisture from 11.45% to 2% has been reported in coffee bean after
20 min of roasting at 170 or 200 °C (Pittia et al., 2001).
The crude fat content was slightly less in puffed and roasted
cacao beans compared to raw cacao beans. However, the crude
fat contents of roasted and puffed cacao beans were similar
(Table 1). This result may be due to degradation of long chain fatty
acids into short chain and free fatty acids or conversion of lipids
into polyenes and acidic aroma product during puffing or roasting.
Values with the same superscript in the same column are not significantly different
at p < 0.05.
Values are presented as mean values ± standard deviation.
It has been reported that after convective roasting of cocoa bean
kernels, fat content was decreased because of transfer of fat from
cocoa kernel to cocoa husk and some fatty acids were transformed
into polyene acids (Krysiak, 2011). Moreover, roasted cacao butter
contains greater numbers and higher concentrations of certain
compounds such as pyrazines, thiazoles, oxazoles, and pyridines
due to thermal conversion of lipids and fatty acids (Carlin et al.,
1986).
The pH of raw and roasted cacao beans was higher than those of
puffed cacao beans indicating that acidic compounds might be pro-
duced during puffing. This acidification seemed to be a unique
characteristic of the puffing process, as roasted cacao beans dis-
played a similar pH as raw cacao beans.
3.2. Total phenolic content and total flavonoid content
Table 2 shows total polyphenol contents (TP) and total flavo-
noid contents (TF) of roasted and puffed cacao beans. The TP of
cacao beans decreased after roasting and puffing. Similarly, it has
been reported that the quantity of phenolic compounds decreased
32.63% in Arriba cacao powder (1.716 g CE/100 g sample) and
54.74% in Ghana cacao powder (1.423 g CE/100 g) after roasting
(Arlorio et al., 2008). Moreover, TP decreased with increasing puff-
ing pressure. Among the puffed cacao bean samples, P4 showed the
highest TP (22.86 mg GAE/g sample). P5 revealed a similar TP
(19.20 mg GAE/g sample) as R15 (19.06 mg GAE/g sample). Com-
pared to thermal processes, new food processing techniques (i.e.,
microwave, infra-red, high pressure processing) have been
reported to lessen the degradation of phenolic antioxidants in food
(Mohamed Ghoul, 2012). The loss of polyphenol contents in cacao
is a result of oxidation of phenolic compounds, followed by poly-
merization and formation of insoluble high-molecular weight com-
pounds (Mohamed Ghoul, 2012).
TF demonstrated a similar pattern as TP. P4 showed the highest
TF (10.65 mg CE/g sample) among puffed cacao beans. P5 demon-
strated slightly lower TF than P4 (8.94 ± 1.94 mg CE/g sample),

Table 1
Proximate composition (% DM) and pH of cacao beans.

Sample Moisture (%) Crude fat (%) Crude protein (%) Ash (%) pH
RB 5.01 ± 0.23a 45.63 ± 4.52a 14.34 ± 0.56a 2.83 ± 0.23b 5.46 ± 0.04a
R15 0.43 ± 0.32c 38.37 ± 5.55b 14.01 ± 0.10a 3.09 ± 0.07a 5.49 ± 0.02a
P4 1.29 ± 0.07b 32.75 ± 1.45b 14.66 ± 0.48a 2.79 ± 0.02b 5.06 ± 0.04c
P5 1.37 ± 0.15b 34.60 ± 1.64b 14.77 ± 0.45a 2.82 ± 0.03b 5.03 ± 0.02c
P6 1.43 ± 0.11b 33.20 ± 0.44b 14.48 ± 0.33a 2.82 ± 0.06b 5.05 ± 0.01c
P7 1.32 ± 0.06b 37.09 ± 2.91b 14.69 ± 0.28a 3.10 ± 0.07b 5.33 ± 0.04b

Raw cacao bean (RB), roasted cacao bean (R15) and puffed cacao beans according at pressure 4 kgf/cm2 (P4), 5 kgf/cm2 (P5), 6 kgf/cm2 (P6) and 7 kgf/cm2 (P7).
Values with the same superscript in the same column are not significantly different at p < 0.05.
Values are presented as mean values ± standard deviation.
1092 S. Hu et al. / Food Chemistry 194 (2016) 1089–1094
Fig. 1. Antioxidant capacity of raw, roasted and puffed cacao beans. Results are expressed as ascorbic acid equivalents. (A) Cacao beans. (B) Defatted cacao beans. Defatted
raw cacao bean (df RB), defatted roasted cacao bean (df R15) and defatted puffed cacao beans according to pressure 4 kgf/cm2 (df P4), 5 kgf/cm2 (df P5), 6 kgf/cm2 (df P6) and
7 kgf/cm2 (df P7).
and then P6 and P7 showed a similar TF (7.19–7.31 mg CE/g sam-
ple) as R15 (7.51 ± 1.50 mg CE/g sample). Thus, TP of R15 and P4
decreased 27% and 12% with processing compared to that of raw
cacao beans, respectively. On the other hand, TF of R15 and P4
decreased 58% and 42% with processing, respectively. These results
suggest that flavonoids are more sensitive to temperature and
pressure than polyphenols.
When defatting occurred after roasting or puffing, P4 showed
the highest TP (42.55 mg GAE/g sample) and TF (18.30 mg CE/
g sample) among all processed samples. As the puffing pressure
increased, TP and TF also decreased in the defatted cacao beans.
With defatting, TP and TF of the raw cacao bean more than dou-
bled, and those of roasted and puffed cacao beans increased, but
to a lesser extent. Defatted cacao beans showed higher TP and TF
than full-fat cacao beans after both puffing and roasting because
polyphenols and flavonoids are mainly located in non-fat cacao
solid. Indeed, a strong correlation between flavanols and amount
of non-fat cacao solid has been noted in cacao products (Miller
et al., 2009), and procyanidin contents have been strongly corre-
lated with the non-fat cacao solid content (Gu, House, Wu, Ou, &
Prior, 2006).
3.3. Antioxidant capacity
Fig. 1 shows the antioxidant capacity of raw, roasted and puffed
cacao beans. Because the mechanisms of the antioxidant capacity
experiments are different, they showed slightly different results.
There was a significant difference between P4 and R15 in DPPH,
FRAP and ABTS, respectively. As the puffing pressure increased,
the antioxidant capacity of puffed cacao beans decreased. This
may be possibly due to the loss of antioxidative polyphenols by
heat and pressure.
On the other hand, the antioxidant capacity of defatted cacao
beans showed relatively higher antioxidant capacity than full-fat
cacao beans after puffing and roasting. This is because antioxidant
compounds such as polyphenols and flavonoids are mainly located
in non-fat cacao solid. Also, results are reported per g of the full-fat
or non-fat powder, so when the antioxidant-poor lipid component
is removed, the antioxidants are essentially concentrated.
3.4. HPLC analysis
Table 3 shows the contents of bioactive compounds including
theobromine, catechin, epicatechin and procyanidin B2 in raw,
roasted and puffed cacao beans. Theobromine is a major compound
in the methylxanthine group of the plant T. cacao. It has pharmaco-
logical effects, including stimulating the nervous system and
inducing diuresis. Herein, theobromine contents were not signifi-
cantly different in puffed cacao beans compared to raw cacao
beans. Nazaruddin, Ayub, Mamot, and Heng (2001) reported a sim-
ilar result of theobromine contents in Ghana cacao beans
(26.64 mg/g sample). However, the theobromine contents of
roasted cacao beans were relatively low. Together, these data sug-
gest that theobromine has a relatively high resistance to pressure,
and a lower resistance to heat.
For raw cacao beans, defatted one displayed higher theo-
bromine contents than full-fat one. Also, a relatively slight loss of
theobromine was observed during defatting in roasted and puffed
cacao beans. This suggests that, like TP and TF, theobromine may
also be located in the non-fat cacao solid.
The main cacao flavanols are (
)-epicatechin and (+)-catechin
monomers (Tomás-Barbéran et al., 2011). Catechin contents
increased after roasting and puffing, and P4 showed the highest
catechin content (8.45 mg/g sample). However, with increasing
puffing pressure, the catechin content decreased. On the other
hand, epicatechin content decreased after roasting and puffing.
This phenomenon has two potential explanations: (1) large mole-
cules such as procyanidin degraded to small molecules such as
catechins by heat and pressure, and (2) (
)-epicatechin epimer-
ized to (
)-catechin by heat and pressure. Indeed, it has been
reported that approximately 42.4% (
)-epicatechin epimerized
to (
)-catechin in 100 °C purified water (Wang & Helliwell,
2000). The (
)-epicatechin and the (+)-catechin are present in
unfermented, dried and unroasted cacao beans (Kofink,
Papagiannopoulos, & Galensa, 2007). Roasted cacao beans and
cacao products contained the (
)-catechin (Kofink et al., 2007),
and higher temperatures in the roasting process accelerate more
epimerization and have a larger influence on the flavanol profile
(Kothe et al., 2013).
 S. Hu et al. / Food Chemistry 194 (2016) 1089–1094 1093

Table 3 of roasted and puffed cacao beans, the procyanidin B2 content of

Theobromine, catechin, epicatechin and procyanidin B2 contents of raw, roasted and
the defatted one tended to be less than that of the full-fat one.
puffed cacao beans.

Sample Theobromine Catechin Epicatechin Procyanidin B2

3.5. Correlation between antioxidant capacities and total polyphenols,
(mg/g sample) (mg/g sample) (mg/g sample) (mg/g sample)
total flavonoids, and bioactive components
RB 11.50 ± 0.88a 5.74 ± 0.57cd 4.74 ± 0.67a 2.31 ± 0.26a
R15 9.47 ± 0.39c 6.09 ± 0.05c 1.60 ± 0.06d 0.77 ± 0.15e
P4 11.09 ± 0.68ab 8.45 ± 0.96a 2.54 ± 0.11b 1.20 ± 0.14d
Table 4 shows the correlation coefficients between antioxidant
P5 10.72 ± 0.36b 6.97 ± 0.24b 1.96 ± 0.10c 1.03 ± 0.03de capacity and TP or TF. Both TP and TF were highly correlated with
P6 10.68 ± 0.77b 7.56 ± 0.53b 1.72 ± 0.19cd 1.56 ± 0.46c the antioxidant capacity. It has been reported that flavonoid con-
P7 10.69 ± 0.61b 5.10 ± 0.80d 1.55 ± 0.03d 1.91 ± 0.06b tent significantly contributes to reducing power and scavenging
df RB 24.74 ± 2.20a 11.24 ± 0.82a 6.43 ± 0.95a 3.38 ± 0.61a activity with r = 0.82 and 0.73, respectively, in cacao powder
df R15 18.28 ± 0.91cb 3.42 ± 0.42e 1.40 ± 0.22c 0.55 ± 0.02c (Maleyki & Ismail, 2010). Total phenolic contents and total pro-
df P4 18.85 ± 0.43b 6.89 ± 0.63cd 2.19 ± 0.35b 1.09 ± 0.27b
cyanidin content were also highly correlated with the antioxidant
df P5 16.92 ± 1.14c 8.17 ± 0.76b 1.74 ± 0.34bc 0.70 ± 0.22bc
df P6 17.28 ± 1.41bc 5.97 ± 1.17d 1.50 ± 0.17c 0.63 ± 0.15bc capacity (R2 = 0.9653 and 0.9510, respectively) (Mazor Jolić et al.,
df P7 19.09 ± 0.85bc 7.21 ± 0.85bc 1.77 ± 0.51bc 0.40 ± 0.05c 2011). In our study, TF was highly correlated with the antioxidant
capacity (R2 = 0.951–0.984) than TP (R2 = 0.808–0.911), in all meth-
Values with the same superscript in the same column are not significantly different
at p < 0.05. ods. This strong correlation suggests that total flavonoid content
Values are presented as mean values ± standard deviation. may contribute a major portion of the antioxidant capacity of cacao
beans.
When the antioxidant capacities (DPPH, FRAP and ABTS) were
plotted against bioactive compounds (theobromine, catechin, epi-
Table 4 catechin and procyanidin B2), theobromine showed the highest
Correlation coefficient between antioxidant capacity and total polyphenol contents,
correlation coefficient with antioxidant capacity (DPPH;
total flavonoid contents, theobromine, catechin, epicatechin and procyanidin B2
contents. R2 = 0.669, FRAP; R2 = 0.875, ABTS; R2 = 0.833) (Table 4). It has been
reported that theobromine has a quenching effect on the produc-
DPPH FRAP ABTS
tion of hydroxyl radicals, as well as on oxidative DNA breakage
Total polyphenols 0.808 0.925 0.911 by hydroxyl radicals (Azam, Hadi, Khan, & Hadi, 2003). Catechin
Total flavonoids 0.984 0.951 0.963 revealed a relatively low correlation with antioxidant capacity
Theobromine 0.669 0.875 0.833
Catechin 0.395 0.296 0.323
because it is easily changed by heat, pressure and defatting
Epicatechin 0.721 0.491 0.532 (Table 4). Epicatechin showed the highest correlation with antiox-
Procyanidin B2 0.441 0.203 0.244 idant capacity (DPPH; R2 = 0.721, FRAP; R2 = 0.491, ABTS;
R2 = 0.532) among the flavonoid compounds (Table 4). Similarly,
a positive and high correlation between epicatechin content and
The catechin content of the defatted raw cacao bean was antioxidant potential based on TEAC assay and FRAP assay has
increased compared to the raw cacao bean. However, the defatted been reported (Othman et al., 2010).
R15, defatted P4 and defatted P6 had lower catechin content com-
pared to full-fat raw cacao beans. The puffing pressure did not 3.6. Sensory evaluation
affect the catechin content. However, loss of catechin occurred
during defatting. On the other hand, epicatechin contents of defat- Sensory evaluation of roasted and puffed cacao beans was car-
ted cacao beans were not significantly different from full-fat cacao ried out. There were no significant differences in bitterness or
beans. astringency between the roasted and puffed cacao beans (Table 5).
Procyanidin is an oligomer and/or a polymer of catechin and On the other hand, a strong sour taste in puffed cacao beans was
epicatechin. It has been reported that there is limited evidence that acknowledged. The sourness might be related to the pH of puffed
procyanidin dimers were degraded or absorbed in human diet cacao beans, which was much lower than roasted and raw cacao
(Holt et al., 2002). For that reason, procyanidin B2, which is a typ- beans (Table 1). Acidic substances may have been generated during
ical procyanidin dimer in cacao beans was analyzed in this study. the puffing process, which gave the sour taste. Overall acceptance
Procyanidin B2 contents were decreased after roasting and puffing, was not significantly different between roasted and puffed cacao
but puffed cacao beans maintained higher procyanidin B2 contents beans (Table 5).
than roasted cacao beans. Also, with increasing puffing pressure,
procyanidin B2 contents increased, possibly due to decomposition 4. Conclusions
of large procyanidins to small procyanidins and catechins by heat
and pressure. The aim to this study was to investigate the possibility of puff-
There was more procyanidin B2 in the defatted raw cacao bean ing in cacao bean processing to reduce the loss of antioxidant com-
compared to the full-fat raw cacao bean. On the other hand, in case pounds compared to roasting. P4 showed higher TP and TF than

Table 5
Sensory evaluation of roasted cacao beans and puffed cacao beans.

Sample Bitterness Astringency Sourness Flavor Overall acceptance

bc abc b bc
R15 5.15 ± 2.11 5.07 ± 2.36 3.04 ± 2.09 4.98 ± 2.12 4.16 ± 2.25ab
P4 4.89 ± 2.13c 4.71 ± 2.35c 4.29 ± 2.27a 5.89 ± 2.00a 4.29 ± 2.00a
P5 5.25 ± 2.08abc 4.95 ± 2.13bc 4.04 ± 2.28a 5.45 ± 2.14ab 4.31 ± 2.42a
P7 5.60 ± 1.97ab 5.62 ± 2.09a 4.24 ± 2.57a 5.35 ± 1.64ab 4.04 ± 1.89ab
P7 5.85 ± 1.93a 5.56 ± 2.32ab 4.04 ± 2.43a 4.45 ± 2.27c 3.44 ± 2.03b

Values with the same superscript in the same column are not significantly different at p < 0.05.
Values are presented as mean values ± standard deviation.
1094 S. Hu et al. / Food Chemistry 194 (2016) 1089–1094
R15, although the TP and TF decreased with increasing puffing
pressure. The antioxidant capacity of cacao beans reflected TP
and TF contents. P4 showed a higher antioxidant capacity in DPPH,
FRAP and ABTS assay than R15. With assessment of the correlation
results between antioxidant capacity and TP or TF, TF contributes
more to the antioxidant activity of cacao beans than TP. The cate-
chin content of cacao beans was increased when roasting and puff-
ing, perhaps due to epimerization of epicatechin to catechin and
decomposition of procyanidin into small molecules by heat and
pressure. Procyanidin, which has health benefits and better bio-
absorption than high degree polymers, was found to be more
prevalent in puffed cacao beans than roasted cacao beans. Accord-
ing to the sensory evaluation results, a sour taste was introduced in
puffed cacao beans, but overall acceptance was not significantly
different between roasted and puffed cacao beans. Consequently,
loss of bioactive compounds and antioxidant capacity were able
to be reduced through puffing while maintaining good sensory
evaluation. Thus, puffing offers a good alternative to roasting for
cacao bean processing.
Acknowledgement
This research was supported by High Value-added Food Tech-
nology Development Program, Ministry of Agriculture, Food and
Rural Affairs, Republic of Korea.
References
Arlorio, M., Locatelli, M., Travaglia, F., Coïsson, J.-D., Grosso, E. D., Minassi, A., ...
Martelli, A. (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(3), 967–975.
Azam, S., Hadi, N., Khan, N. U., & Hadi, S. M. (2003). Antioxidant and prooxidant
properties of caffeine, theobromine and xanthine. Medical Science Monitor, 9(9),
BR325–BR330.
Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of free radical method
to evaluate antioxidant activity. Lebensmittel-Wissenschaft und-Technologie-Food
Science and Technology, 28, 25–30.
Buijsse, B., Weikert, C., Drogan, D., Bergmann, M., & Boeing, H. (2010). Chocolate
consumption in relation to blood pressure and risk of cardiovascular disease in
German adults. European Heart Journal, 31(13), 1616–1623.
Carlin, J., Lee, K., Hsieh, O. L., Hwang, L., Ho, C.-T., & Chang, S. (1986). Comparision of
acidic and basic volatile compounds of cocoa butters from roasted and
unroasted cocoa beans. Journal of the American Oil Chemists’ Society, 63(8),
1031–1036.
Du, L.-J., Gao, Q.-H., Ji, X.-L., Ma, Y.-J., Xu, F.-Y., & Wang, M. (2013). Comparison of
flavonoids, phenolic acids, and antioxidant activity of explosion-puffed and
sun-dried Jujubes (Ziziphus jujuba Mill.). Journal of Agricultural and Food
Chemistry, 61(48), 11840–11847.
Grassi, D., Desideri, G., Necozione, S., Lippi, C., Casale, R., Properzi, G., ... Ferri, C.
(2008). Blood pressure Is reduced and insulin sensitivity increased in glucose-
intolerant, hypertensive subjects after 15 days of consuming high-polyphenol
dark chocolate. Journal of Nutrition, 138(9), 1671–1676.
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(11), 4057–4061.
Heiss, C., Kleinbongard, P., Dejam, A., Perré, S., Schroeter, H., Sies, H., & Kelm, M.
(2005). Acute consumption of flavanol-rich cocoa and the reversal of
endothelial dysfunction in smokers. Journal of the American College of
Cardiology, 46(7), 1276–1283.
Holt, R. R., Lazarus, S. A., Sullards, M. C., Zhu, Q. Y., Schramm, D. D., Hammerstone, J.
F., ... Keen, C. L. (2002). Procyanidin dimer B2 [epicatechin-(4b-8)-epicatechin]
in human plasma after the consumption of a flavanol-rich cocoa. American
Journal of Clinical Nutrition, 76(4), 798–804.
Hsieh, F., Huff, H. E., Peng, I. C., & Marek, S. W. (1989). Puffing of rice cakes as
influenced by tempering and heating conditions. Journal of Food Science, 54(5),
1310–1312.
Jia, Z., Tang, M., & Wu, J. (1999). The determination of flavonoid contents in
mulberry and their scavenging effects on superoxide radicals. Food Chemistry,
64(4), 555–559.
Kim, H., & Keeney, P. G. (1984). (-)-Epicatechin content in fermented and
unfermented cocoa beans. Journal of Food Science, 49(4), 1090–1092.
Kofink, M., Papagiannopoulos, M., & Galensa, R. (2007). (-)-Catechin in cocoa and
chocolate: occurence and analysis of an atypical flavan-3-ol enantiomer.
Molecules, 12(7), 1274–1288.
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(4), 3656–3663.
Krysiak, W. (2011). Effects of convective and microwave roasting on the
physicochemical properties of cocoa beans and cocoa butter extracted from
this material. Grasas V Aceites, 62(4), 467–478.
Maleyki, M. J. A., & Ismail, A. (2010). Antioxidant properties of cocoa powder. Journal
of Food Biochemistry, 34(1), 111–128.
Mariotti, M., Alamprese, C., Pagani, M. A., & Lucisano, M. (2006). Effect of puffing on
ultrastructure and physical characteristics of cereal grains and flours. Journal of
Cereal Science, 43(1), 47–56.
Mazor Jolić, S., Radojčić Redovniković, I., Marković, K., Ivanec Šipušić, Ð., & Delonga,
K. (2011). Changes of phenolic compounds and antioxidant capacity in cocoa
beans processing. International Journal of Food Science & Technology, 46(9),
1793–1800.
Miller, K. B., Hurst, W. J., Flannigan, N., Ou, B., Lee, C. Y., Smith, N., & Stuart, D. A.
(2009). Survey of commercially available chocolate- and cocoa-containing
products in the united states. 2. comparison of flavan-3-ol content with nonfat
cocoa solids, total polyphenols, and percent cacao. Journal of Agricultural and
Food Chemistry, 57(19), 9169–9180.
Mohamed Ghoul, D. I. I. (2012). Biological activities and effects of food processing
on flavonoids as phenolic antioxidants. In P. M. Petre (Ed.), Advances in Applied
Biotechnology (pp. 288). France: InTech.
Nath, A., Chattopadhyay, P. K., & Majumdar, G. C. (2007). High temperature short
time air puffed ready-to-eat (RTE) potato snacks: Process parameter
optimization. Journal of Food Engineering, 80(3), 770–780.
Nazaruddin, R., Ayub, M., Mamot, S., & Heng, C. (2001). HPLC determination of
methylxanthines and polyphenols levels in cocoa and chocolate products.
Malaysian Journal of Analytical Sciences, 7, 377–386.
Othman, A., Ismail, A., Abdul Ghani, N., & Adenan, I. (2007). Antioxidant capacity
and phenolic content of cocoa beans. Food Chemistry, 100(4), 1523–1530.
Othman, A., Jalil, A. M. M., Weng, K. K., Ismail, A., Ghani, N. A., & Adenan, I. (2010).
Epicatechin content and antioxidant capacity of cocoa beans from four different
countries. African Journal of Biotechnology, 9(7), 1052–1059.
Özdemir, M., & Onur Devres, Y. (1999). The thin layer drying characteristics of
hazelnuts during roasting. Journal of Food Engineering, 42(4), 225–233.
Payne, M. J., Hurst, W. J., Miller, K. B., Rank, C., & Stuart, D. A. (2010). Impact of
fermentation, dying, roasting, and dutch processing on epicatechin and catechin
content of cacao beans and cocoa ingredients. Journal of Agricultural and Food
Chemistry, 58(19), 10518–10527.
Pearson, D. A., Paglieroni, T. G., Rein, D., Wun, T., Schramm, D. D., Wang, J. F., ... Keen,
C. L. (2002). The effects of flavanol-rich cocoa and aspirin on ex vivo platelet
function. Thrombosis Research, 106(4–5), 191–197.
Pittia, P., Dalla Rosa, M., & Lerici, C. R. (2001). Textural changes of coffee beans as
affected by roasting conditions. LWT – Food Science and Technology, 34(3),
168–175.
Pura Naik, J. (2001). Improved high-performance liquid chromatography method to
determine theobromine and caffeine in cocoa and cocoa products. Journal of
Agricultural and Food Chemistry, 49(8), 3579–3583.
Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology
and Viticulture, 16(3), 144–158.
Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., & Hawkins Byrne, D.
(2006). Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating
antioxidant activity from guava fruit extracts. Journal of Food Composition and
Analysis, 19(6–7), 669–675.
Tomás-Barbéran, F., Borges, G., & Crozier, A. (2011). Phytochemicals in cocoa and
flavan-3-ol bioavailability. In Teas, Cocoa and Coffee (pp. 193–217). Wiley-
Blackwell.
van den Berg, R., Haenen, G. R. M. M., van den Berg, H., & Bast, A. (1999).
Applicability of an improved Trolox equivalent antioxidant capacity (TEAC)
assay for evaluation of antioxidant capacity measurements of mixtures. Food
Chemistry, 66(4), 511–517.
Wang, H., & Helliwell, K. (2000). Epimerisation of catechins in green tea infusions.
Food Chemistry, 70(3), 337–344.
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(6),
423–447.