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Journal of Food Quality ISSN 1745-4557

EFFECT OF FERMENTATION AND ROASTING ON THE PHENOLIC


CONCENTRATION AND ANTIOXIDANT ACTIVITY OF COCOA
FROM NICARAGUA
YADER SUAZO, GABRIEL DAVIDOV-PARDO1 and IÑIGO AROZARENA
Food Technology Department, Public University of Navarre, Campus Arrosadia s/n, Pamplona, Navarre 31006, Spain

1
Corresponding author. ABSTRACT
TEL: (34) 948169310;
FAX: (34) 948169893; The aim of this work was to quantify the changes in the total phenolic content
EMAIL: gabriel.davidov@unavarra.es (TPC), antioxidant activity (AA) and color hue (CH) of cocoa (Theobroma cacao)
beans of group Trinitario from Nicaragua after fermentation and roasting. Cocoa
Received for Publication February 14, 2013
beans with three different grades of fermentation (nonfermented, poorly fer-
Accepted for Publication November 10, 2013
mented and fermented) were put under three different roasting temperatures
10.1111/jfq.12070 (110, 130 and 150C). The kinetics of the roasting at 130C for 60 min was also
studied. After each treatment, TPC, AA and CH were determined. For all the
quantified parameters, significant differences (P < 0.05) were found between fer-
mentations and roasting conditions. TPC and AA showed decrease with fermenta-
tions and CH showed increases. Roasting decreased TPC except for the 150C
treatment, while AA and CH increased. When studying the kinetics at 130C, TPC
showed greater reductions with longer treatments while AA and CH showed
increases.

PRACTICAL APPLICATIONS
Cocoa (T. cacao) is a rich source of polyphenols with benefits for human health
and is the main raw material for elaboration of chocolate. However, the processes
to create the desirable organoleptic characteristics of the cocoa beans to produce
chocolate affect the concentration of polyphenols and the AA of the beans. Cocoa
beans are produced in developing countries in America, Asia and Africa. Nicara-
gua has become an important producer of cocoa turning it into a popular cash
crop and there are not any articles that study the profile and changes in the cocoa
from Nicaragua. The results of this paper could be used among the producing
cooperatives of cacao in Nicaragua, or even nongovernmental organizations
involved in cocoa production, to improve the processing (fermentation and roast-
ing) of cocoa to obtain cocoa beans richer in antioxidants, and therefore increase
their nutritional value to create a more competitive product.

cals and prevention of damage to DNA, chelation of metals,


INTRODUCTION
vasoprotective effects, improvements in the endothelial
Polyphenols from different sources such as plants, fruits, function, anti-inflammatory effects, amelioration of insulin
seeds and beverages like wine or tea have attracted the resistance, anticarcinogenesis effects, among others (Katz
attention of many researchers due to the beneficial effects et al. 2011).
of these compounds to human health. Cocoa (Theobroma Nowadays, cocoa beans are known because they are the
cacao) is a rich source of polyphenols because they consti- main raw material for elaboration of chocolate, which in
tute approximately 10% of the dry weight of the bean fact after fruits and vegetables is one of the main sources
(Rusconi and Conti 2010). The beneficial effects of cocoa of antioxidants to the diet of the United States (Rusconi and
polyphenols to human health are: the scavenge of free radi- Conti 2010). Although cocoa products are consumed all

50 Journal of Food Quality 37 (2014) 50–56 © 2014 Wiley Periodicals, Inc.


Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA EFFECT OF PROCESS ON COCOA FROM NICARAGUA

over the world, the beans are produced in developing coun- TABLE 1. CUT TEST FOR NONFERMENTED COCOA (NFC), POORLY
tries in America, Asia and Africa. Since 1991, after the civil FERMENTED COCOA (PFC) AND FERMENTED COCA (FC) SAMPLES
war, Nicaragua has become an important producer of cocoa Nonfermented Poorly Fermented
turning it into a popular cash crop. In Nicaragua, the main Parameter (%) fermented (%) (%)
production of cocoa is in the municipality of Waslala in the Slaty 36.7 0 0.7
north of the country (Trognitz et al. 2011). Lightly violet 4.7 20.2 2.6
To develop desirable organoleptic characteristics to Completely violet 31.5 0 1.5
produce chocolate and other cocoa products, the beans have Internal mold 0.2 0.3 0.8
White spots 0 0 0.2
to be submitted to fermentation and roasting. These two
processes affect the concentration of polyphenols and the
antioxidant activity (AA) of the beans (Aculey et al. 2010).
Basically, the fermentation is a spontaneous microbio- reagent 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
logical process that causes deep biochemical changes. The acid (Trolox) were purchased from Sigma-Aldrich Quimica
beans are fermented by a successive presence of microor- SL. (Madrid, Spain).
ganisms (fungi, yeasts, lactic acid and acetic bacteria). These
microorganisms are activated by the temperature changes
Samples
during the fermentation process that can last a minimum of
5 days to a maximum of 7 days (Wollgast and Anklam 2000; NFC, PFC and FC cacao beans of the same group
Schwan and Wheals 2004). The impact of the fermentation (Trinitario) were obtained from a collection center of Ritter
on the phenolic content of cocoa beans was studied by Sport in Matagalpa, Waslala, Nicaragua. During fermenta-
Efraim et al. (2010). After the first 3 days of fermentation tion (in Nicaragua), the beans were turned once a day
there was a 35% decrease in the phenolic content, and after during 7 days to reach a temperature between 45 and 50C.
7 days of fermentation only 45% of the original phenolic Table 1 shows the results of the cut test for all samples.
content remained. After fermentation, the beans are sun- Because of the cut test results, the PFC beans were consid-
dried or artificially dried, depending on the weather of the ered poorly fermented. The reasons to explain the cut
growing areas. The beans are dried to reach a moisture results for PFC were that the turn regime was not done
content of approximately 5–7% to prevent the growth of on time and the mass of the beans did not reach the aimed
molds during transportation and storage (Wollgast and temperature, as stated by the processing facility in Nicara-
Anklam 2000). During the drying process, the loss of poly- gua. Because of the lower quality of PFC, these kinds of
phenols continues mainly when the beans are artificially batches are usually commercialized in the local market.
dried (Efraim et al. 2010). All samples were sun-dried after fermentation until they
After fermentation and drying, the beans are usually reached a humidity of 6%, then the samples were sent to the
roasted. This process determines the final color, aroma and Public University of Navarre for analyses and roasting.
flavor of the cocoa products. The changes in the organolep- It was also stated by the processing facility in Nicaragua
tic characteristics of the beans are the result of oxidation that PFC beans reached the 6% humidity faster than usual.
and polymerization of polyphenols, degradation of pro- All samples had phytosanitary certificates from the
teins and Maillard reactions (Ramli et al. 2006). Convective Agropecuary Ministry of Nicaragua.
roasting is the most commonly roasting process in the
cocoa industry; the raw beans are exposed to temperatures
Experimental Design
between 130 and 150C for 15–45 min (Krysiak 2006).
In the light of this background, the aim of this work was Table 2 shows the experimental design to analyze the
to quantify the changes in the phenolic composition and AA changes in the polyphenolic concentration, AA and color
of nonfermented cocoa (NFC), poorly fermented cocoa hue of the beans after fermentation and roasting. The roast-
(PFC) and fermented cocoa (FC) beans (T. cacao) of group ing temperatures were chosen based on the work by Krysiak
Trinitario from Nicaragua after roasting. (2006). The combination of the selected roasting tempera-
tures and times is within the common temperatures used in
the cocoa industry and was determined to achieve a water
MATERIALS AND METHODS
content of 2%, which is the optimal humidity in terms
of beans grinding and fat extraction. The kinetics of the
Reagents
changes in the polyphenolic concentration, AA and color
Methanol, n-hexane, sodium carbonate and gallic acid (GA) hue when roasting the NFC and FC beans at 130C for 20, 40
were purchased from Panreac (Barcelona, Spain). 2,2- and 60 min was also studied. A sample of 100 g of the whole
Diphenyl-1-picrylhydrazyl (DPPH) and Folin–Ciocalteau beans with the shells was roasted in an oven Digitronic

Journal of Food Quality 37 (2014) 50–56 © 2014 Wiley Periodicals, Inc. 51


EFFECT OF PROCESS ON COCOA FROM NICARAGUA Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA

TABLE 2. EXPERIMENTAL DESIGN TO COMPARE THE POLYPHENOLIC Australia) double beam spectrophotometer. The phenolic
CONCENTRATION AND ANTIOXIDANT ACTIVITY OF COCOA SAMPLES content was expressed in GA equivalents after the preparation
AFTER THE TYPE OF FERMENTATION AND ROASTING CONDITIONS
of a standard curve of GA from 0 to 600 mg/L.
Cocoa samples Roasting temperature (C) Time (min)
Nonfermented cocoa (NFC) Nonroasted 0 AA
110 90
130 60 The antiradical activity was evaluated based on the tech-
150 45 nique by Rivero-Pérez et al. (2007). A 60-μM methanolic
Poorly fermented cocoa (PFC) Nonroasted 0 solution of DPPH (2,940 μL) was mixed with 60 μL of the
110 90 extract in a polystyrene cuvette. The absorbance at 515 nm
130 60
was measured before adding the extract, and 60 min after
150 45
adding it, using a Cintra 20 (GBC Scientific, Braeside, Aus-
Fermented cocoa (FC) Nonroasted 0
110 90 tralia) double beam spectrophotometer. The AA is reported
130 60 as mmoles of Trolox equivalents per gram of dry cocoa
150 45 powder after the elaboration of a standard curve of Trolox
based on the reduction of the absorbance after 60 min.

(Selecta, Barcelona, Spain). After roasting, the beans were Color Hue
left to cool for 1 h at ambient temperature before the phe-
nolic extraction. The color hue of the samples was measured through the
brown pigments ratio (Krysiak 2006), the ratio of the absor-
bance at 460 nm and the absorbance at 520 (A460/A520).
Extraction of Phenolic Compounds All the measures were made on the cocoa extracts using
polystyrene cuvettes in a Cintra 20 (GBC, Braeside, Austra-
The extraction of phenolic compounds from the cocoa
lia) double beam spectrophotometer.
beans was based on the defatted methodology employed
by Summa et al. (2006) and the methanolic extraction
based on the methodology employed by Jonfia-Essien et al. Statistical Analysis
(2008). Briefly, the cocoa beans were winnowed by hand Statistical analyses were conducted using Statgraphics
and grounded for 30 s in a coffee blender. To remove the Centurion XVI (StatPoint Technologies Inc., Warrenton VA,
fat, 5 g of the grounded beans were mixed with 25 mL of USA). Differences among the treatments were determined
n-hexane and thoroughly mixed for 3 min. The mixture using an analysis of variance and a Tukey test with a confi-
was centrifuged at 3,000 rpm during 15 min at 4C using a dence level of 95%. For the nonroasted samples, the extrac-
centrifuge Medifriger BL-S (Selecta, Barcelona, Spain). tion was made four times and analyzed in duplicate giving
The process was repeated four times and the defatted cocoa an n = 8 per sample. The roasting of the cocoa beans was
powder was left overnight at ambient temperature to made in duplicate and analyzed in duplicate giving an n = 4
remove the solvent. To extract the phenolic compounds, 2 g per treatment and sample. A bilateral Pearson correlation
of the defatted sample was mixed with 50 mL of an 80% analysis was performed between the TPC and the AA after
methanol solution and shaken for 2 h at 50C using an the roasting and fermentation treatments.
orbital mini shaker (VWR, Barcelona, Spain). The extract
was then filtered through a Whatman No. 1 filter paper and
filled up to 50 mL in a volumetric flask to compensate the
RESULTS AND DISCUSSION
losses of solvent during the extraction and filtration.
Effect of Fermentation
Table 3 shows the effect of fermentation on the cocoa beans.
Total Phenolic Content (TPC)
The results obtained in this work are in agreement with the
The Folin–Ciocalteu method was employed (EEC 1990) to results obtained by Efraim et al. (2010) who studied the
obtain the TPC. In a 100-mL volumetric flask, 1 mL of the fermentation and drying of Forastero cocoa samples from
diluted extract, 50 mL of deionized water, 5 mL of the Folin– Brazil. It is important to remark that there are no studies on
Ciocalteu reagent and 20 mL of a sodium carbonate solution the phenolic concentration of cocoa beans from Nicaragua
20% (w/v) were added in that order. The volumetric flask was to compare our results. It can be seen that for all the mea-
filled to its volume with deionized water. After 30 min, the sured parameters, there are significant differences among
absorbance of the samples was measured at 750 nm using all treatments except for the A460/A520, in which case the
polystyrene cuvettes in a Cintra 20 (GBC Scientific, Braeside, differences between the two fermented samples are not

52 Journal of Food Quality 37 (2014) 50–56 © 2014 Wiley Periodicals, Inc.


Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA EFFECT OF PROCESS ON COCOA FROM NICARAGUA

TABLE 3. EFFECT OF FERMENTATION ON THE


Phenolic content Antioxidant activity Color
EVALUATED PARAMETERS OF COCOA BEANS
Cocoa samples (mg/g dw*) (μM Trolox) (Abs 460/520 nm)
Nonfermented cocoa (NFC) 115 ± 2c 709 ± 17c 1.06 ± 0.03a
Poorly fermented cocoa (PFC) 56 ± 2b 154 ± 8b 1.27 ± 0.09b
Fermented cocoa (FC) 43 ± 1a 124 ± 4a 1.28 ± 0.04b

Note: a–cDifferent letters within a column are significantly different (P < 0.05).
* Grams of dry weight. The results are represented as means ± standard deviation.

significant. As expected, the polyphenolic concentration is the beans could be explained by nonenzymatic and enzy-
lower in the fermented than in the nonfermented samples. matic processes. Among the nonenzymatic reactions are
It is known that the fermentation and drying processes hydrolysis of anthocyanins into anthocyanidins and the
cause a decrease in the polyphenolic content due to oxida- later polymerization with simple catechins to form com-
tion and condensation reactions of single polyphenols to plex tannins. Another nonenzymatic process that happens
insoluble complex tannins that interact with proteins. These during fermentation and browning is Maillard reactions,
reactions can be nonenzymatic or catalyzed by oxidase which involve reducing sugar and the amino groups of pro-
enzymes. The loss of polyphenols diffused in the water per- teins to produce brown polymeric compounds. Finally, the
meated from the beans during fermentation also contrib- enzymatic browning during fermentation and drying of
utes to the decrease in the phenolic content (Wollgast and cocoa beans involves the formation of quinones from
Anklam 2000; Kyi et al. 2005). The reductions in the AA are phenolic compounds and the later formation of brown to
a direct consequence of the decrease in the polyphenolic black polymers under the action of the polyphenol oxidase
content. (Wollgast and Anklam 2000; Kyi et al. 2005).
After fermentation and drying, an increase in the brown
pigments ratio A460/A520 was observed; nevertheless, the
Effect of Roasting
results of the PFC and FC were not significantly different.
It is known that for a cocoa to be properly fermented in Figure 1 shows the evolution of phenolic concentration,
terms of color, A460/A520 should be higher than 1 (Krysiak AA and color hue of cocoa samples after the roasting
2006). In this case, both fermented samples had A460/A520 conditions. It can be seen that the TPC of the samples
values higher than 1. When cocoa beans are fermented and decreased after the roasting treatments (Fig. 1A), except
dried, their colors change from “slaty” and “purple” to for the roasting at 150C during 45 min in which NFC pre-
“brown” (Aculey et al. 2010). The changes in the color of sented an increase in TPC. In general, more intense roasting

d b
c B
a A
b
c a a a
d bc a ab
a cc
aa bb ab

c
b c
C
a bb b
a b
a
a
FIG. 1. IMPACT OF THE DIFFERENT ROASTING a

CONDITIONS ON THE THREE COCOA


SAMPLES. (A) TOTAL PHENOLIC CONTENT. (B)
ANTIOXIDANT ACTIVITY. (C) COLOR HUE
A460/A520
Different letters (a–d) are significantly different
for each extract separately (P < 0.05).

Journal of Food Quality 37 (2014) 50–56 © 2014 Wiley Periodicals, Inc. 53


EFFECT OF PROCESS ON COCOA FROM NICARAGUA Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA

conditions result in a greater loss of polyphenols due to high P > 0.05). The fact that the changes in the AA did not have
redox activity of polyphenols at those conditions (Wollgast the same magnitude and tendency compared to the changes
and Anklam 2000). The higher reductions in TPC for NFC in the TPC could be explained by the findings of Summa
when compared to PFC and FC are in agreement with the et al. (2006). In their work, Summa et al. (2006) found that
findings made by Kealey et al. (2001). The fact that the poly- the concentration of reducing substances, such as polyphe-
phenols contained in the NFC sample did not diffuse out of nols, measured by the Folin–Ciocalteau technique, does not
their storage cells during fermentation could be the reason always have a close relation with the AA measured with the
for the increase in the phenolic content with the 150C during DPPH technique.
45-min treatment. Kim et al. (2006) proved that using similar The color hue increased with the thermal treatments
heating conditions the phenolic content of grape seeds (Fig. 1C). This increment is consistent with the findings of
increased, because with the heat polyphenols were liberated Krysiak (2006) in which higher temperatures represented
out of their storage cells. The longer time of the other two greater increases in the A460/A520 value. The increase of
treatments could cause liberation of the polyphenols at the brown pigments ratio induced by the heat could be
the beginning, but degradation afterward. Another possible explained by the continuation and acceleration of some
explanation for the increase in TPC on NFC when roasting at of the reactions started during fermentation and drying
150C during 45 min could be the degradation of anthocyan- mainly Maillard reactions (Beckett 2000). Another factor
ins into other polyphenol monomers (Misnawi et al. 2005). that contributes to the increase in color hue could be the
Anthocyanins are only present in NFC beans because it is aromatic acid reductones, which are formed as a conse-
known that during fermentation anthocyanins are lost quence of reactions with phenols such as catechol and hyd-
(Wollgast and Anklam 2000). roquinone (Lee et al. 2001).
With regard to the AA, the changes were less notorious To analyze the influence of roasting time, the NFC and
than the changes of the TPC. The significant increment of FC samples were roasted at 130C for 20, 40 and 60 min and
AA with the treatment 150C during 45 min is consistent the kinetics of the changes for all the measured parameters
with the increment in TPC. The bilateral Pearson correla- was studied. As expected, the NFC had higher TPC and AA
tion between the TPC and the AA after the roasting and values and lower color values during the whole assay
fermentation treatments was not significant (r = 0.467, (Fig. 2). It can be seen that while TPC was reducing with

a
c
A B
b a a a
b
a

d c
b
a c c
b
a

C
a

a a
a

a,b
b b

FIG. 2. IMPACT OF THE DIFFERENT ROASTING TIMES AT 130C ON THE NFC AND THE FERMENTED COCOA SAMPLES. (A) TOTAL PHENOLIC
CONTENT. (B) ANTIOXIDANT ACTIVITY. (C) COLOR HUE A460/A520
Different letters (a–d) are significantly different for each extract separately (P < 0.05).

54 Journal of Food Quality 37 (2014) 50–56 © 2014 Wiley Periodicals, Inc.


Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA EFFECT OF PROCESS ON COCOA FROM NICARAGUA

longer treatment times, the AA was growing (Fig. 2A,B). BECKETT, S.T. 2000. The Science of Chocolate, Royal Society of
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polyphenol content and sensory acceptance. Cienc. Tecnol.
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of polyphenol monomers balancing the AA (Vignoli et al.
HAGERMAN, A.E., RIEDL, K.M., JONES, G.A., SOVIK, K.N.,
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