Food Chemistry 124 (2011) 863–868

Contents lists available at ScienceDirect

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

Antioxidant activity, polyphenols, caffeine and melanoidins in soluble coffee:

The influence of processing conditions and raw material
J.A. Vignoli a,*, D.G. Bassoli a, M.T. Benassi b
a
Companhia Iguaçu de Café Solúvel S.A., Research and Development Department, BR-369, Km 88, C.Procópio, PR, Brazil
b
Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Km 6, Londrina, PR, Brazil

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

Article history: The production of soluble coffee starts with the selection of beans and is followed by roasting, grinding,
Received 1 December 2009 extraction and drying. Lyophilised soluble coffees extracted by various methods from light, medium and
Received in revised form 28 June 2010 dark-roasted arabica and robusta beans were evaluated for antioxidant activity (AA) using ABTS, Folin,
Accepted 2 July 2010
DPPH and FRAP techniques. Caffeine, chlorogenic acid (5-CQA) and melanoidin content was also quantified.
The data were analysed by principal component analysis. The AA values derived from the various methods
used were correlated. Roasting resulted in the degradation of 5-CQA and formation of melanoidins, while AA
Keywords:
was largely unaffected by roasting. The extraction of soluble coffee more prominently affected the AA of
Coffea arabica
Coffea Canephora
light-roasted coffee, mainly because it favoured the extraction of 5-CQA. The larger caffeine content in
Roasting degree robusta coffee resulted in greater AA. All of soluble coffee products studied possessed antioxidant potential,
Extraction which was conferred by their concentrations of phenolic compounds, caffeine and melanoidins.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Coffee is a major commodity in the world economy, second only
to petroleum (Borreli, Visconti, Mennella, Anese, & Fogliano, 2002).
The most widely cultivated species are Coffea arabica (arabica) and
Coffea canephora (robusta). Despite the poorer sensory quality of C.
canephora, it has the advantage of allowing extraction of large
amounts of soluble solids, which enables its use in blends and in
the soluble coffee industry. Beverages prepared from roasted beans
have pleasant flavour and aroma in addition to their physiological
effects. The preference for different kinds of coffee beverages is
strictly associated with social habits and country cultures. The
company GEA Niro (Parma, Italy), a traditional producer of process
lines for instant coffee and tea, reported that soluble coffee is
widely consumed (45% in Eastern Europe, 53% in Asia/Pacific, and
79% in Australia), standing out in countries where tea is a tradi-
tional beverage (GEA-Group Coffee, 2010). Soluble coffee exports
are a major source of revenue in Brazil due to the higher aggregate
value of the product (ABICS, 2008).
Recently, scientific studies have pointed out the positive effect of
coffee on human health (Coughlin, 2006). In general, recent studies
report little evidence of health risks and considerable evidence of
health benefits for healthy adults as a result of moderate coffee con-
sumption (Higdon & Frei, 2006). The beverage also stands out as a die-
tary source of potential antioxidants, such as caffeine (Devasagayam,
* Corresponding author. Tel.: +55 43 3401 1542; fax: +55 43 3524 2542.
E-mail address: jvignoli@iguacu.com.br (J.A. Vignoli).
Kamat, Monhan, & Kesavan, 1996; Shi & Dalal, 1991), chlorogenic
acids (Gómez-Ruiz, Lake, & Ames, 2007; Moreira, Monteiro, Ribeiro-
Alves, Donangelo, & Trugo, 2005), hydroxycinnamic acids (Gallardo,
Jiménez, & Garcia-Conesa, 2006), and Maillard reaction products,
such as melanoidins (Borreli et al., 2002; Delgado-Andrade, Rufián-
Henares, & Morales, 2005). Thus, the antioxidant capacity of coffee
is related to the presence of both natural constituents and compounds
formed during processing. The antioxidant potential of coffee has
been evaluated by several methods, such as ferric reducing antioxi-
dant power (FRAP), 2,20 -azinobis(3-ethylbenzothiazoline-6-sul-
phonic acid assay (ABTS), 2,2-diphenyl-1-picrylhydrazyl assay
(DPPH) and determination of total phenolics (Borreli et al., 2002; Sán-
chez-Gonzalez, Jiménez-Escrig, & Saura-Calixto, 2005).
The FRAP method measures the ferric reduction of 2,4,6-tripyr-
idyl-S-triazine (TPTZ). This reaction detects compounds with redox
potentials lower than 0.7 V (the redox potential of Fe3+-TPTZ). Assays
using the ABTS radical, including TEAC, are based on the ability of
antioxidants to scavenge the long-lived ABTS radical; using a similar
mechanism, the DPPH assay measures the reduction of the stable
radical 2,2-diphenyl-1-picrylhydrazyl by monitoring the decrease
in its absorbance at 515 nm. The Folin–Ciocalteau method has been
used for years to measure total phenolic compounds and is also use-
ful in evaluating antioxidant activity (Benzie, 1996; Prior, Xianli, &
Schaich, 2005).
As compared to other beverages, coffee stands out for its antiox-
idant potential. Some authors have reported greater AA for soluble
and espresso coffee than those of red wine and green tea (Pellegrini
et al., 2003). AA is also affected by the green bean composition and

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.07.008
864 J.A. Vignoli et al. / Food Chemistry 124 (2011) 863–868
processing methods. Daglia, Papetti, Gregotti, Bertè, and Gazzani
(2000) studied beverages made from different coffee beans and re-
ported a greater antioxidant capacity for C. canephora than for C.
arabica. Del Castillo, Ames, and Gordon (2002) observed a decrease
in AA that was correlated with the degree of roasting, associated
mainly with the degradation of chlorogenic acid. In contrast, the
roasting process can also contribute to the formation of melanoi-
dins, which have already been reported to be responsible for AA
in high molecular weight fractions isolated from roasted coffee
(Daglia et al., 2000).
Most studies in literature refer to the AA of roasted coffee. De-
spite the great economic importance of soluble coffee, little has
been reported about its antioxidant potential or the influence of
processing conditions. To become solubilised, coffee undergoes
an extraction process. The beans are roasted, ground and subjected
to successive percolation with water at temperatures ranging from
100 to 180 °C. Chemically, percolation results in the selective solu-
bilisation of coffee solids. Depolymerisation and degradation may
occur during high-temperature extraction (Leloup, 2006), and pro-
cess variations may affect the product’s characteristics.
Thus, the objective of this study was to evaluate the effect of solu-
ble coffee processing conditions (roasting and extraction method), as
well as the effect of raw materials (C. arabica andC. canephora species),
on the antioxidant capacity of coffee as measured by different tech-
niques. In addition, representative compounds of the main classes
of antioxidants in soluble coffees (5-CQA, caffeine and melanoidins)
were analysed allowing to explain the variations observed in AA.
2. Materials and methods
2.1. Materials and samples
Coffee samples were provided by Companhia Iguaçu de Café
Solúvel (Cornélio Procópio, Paraná State, Brazil) and were repre-
sentative of different degrees of roasting, extraction conditions,
and raw materials.
Arabica (A) and robusta (B) coffee beans were roasted in a pilot
80-kg/h Rayar roaster for 7–10 min at 214–225 °C to obtain light
(L), medium (M), and dark (D) roasted beans, corresponding to
lightness values (L*) of 33, 25, and 14, respectively. Lightness
was assessed in triplicate with a Byk Gardner GmbH colorimeter
(Germany) with 45/0 geometry and CIE illuminant D65. Coffee
beans were ground to obtain adequate particle size for later pro-
cessing as follows: 30% retention in a 4-mm sieve, 60% retention
in a 2-mm sieve and 10% retention in a 1-mm sieve.
The samples were extracted by two processes: conventional (1)
and double-extraction systems (2). Generally, a set of extraction
columns is filled in sequence and extracted with heated water. In
the conventional process (1), water at 180 °C is fed into the first
stage (column with older coffee) and subsequently percolated in
the following stages until it reaches newer coffee. In the last stage,
the extract reaches the newly added coffee and extracts its soluble
solids, preserving the aroma and flavour. During the process, the
amount of soluble solids in the extract increase, while the temper-
ature decreases. The fresh coffee is extracted in the last column at
approximately 100 °C to minimise thermal damage. The resulting
extract is then lyophilised. In the double-extraction system (2),
one batch is fed into the selected stages at mild temperature and
pressure, which results in a better quality extract. A second batch
is fed into the remaining stages at higher temperature and pressure
to increase solubilisation. A mixture of 50% of the extracts from
each stream was lyophilised.
Antioxidant activity was determined by directly dissolving suit-
able concentrations of samples in water. All samples were pre-
pared in duplicate and measurements were made in triplicate.
2.2. Chemical and equipments
5-O-Caffeoylquinic acid (5-O-CQA), caffeine and gallic acid (HPLC
grade) were purchased from Sigma–Aldrich (St. Louis, MO, USA).
ABTS (2,2-azinobis-3 ethyl benzothiazoline-6-sulphonic acid) and
DPPH (2,2-diphenyl-1-picrylhydrazyl) were obtained from Sigma
Chemical CO. (St. Louis, MO, USA). Trolox (6-hydroxy-2,5,7,8,-
tetramethylchromane-2-carboxylic acid) and TPTZ (2,4,6-tri(2-pyr-
idyl)-S-triazine) were obtained from Fluka/Sigma–Aldrich (Vallens-
baek Strand, Nordic, Denmark). Folin–Ciocalteau, acetic acid and
acetonitrile were purchased from Merck (Darmstadt, Hessen,
Germany).
Melanoidins were separated by dialysis using 12–14 kDa cutoff
membranes (Spectra/Por, Irving, TX, USA).
AA measurements were taken in a UV–Vis–UV mini-1240 spec-
trophotometer (Shimadzu, Kyoto, Japan) and bioactive compounds
were detected by HPLC. The HPLC apparatus consisted of a Dionex
LC (Idstein, Hesse, Germany) equipped with a P680 gradient pump,
TCC-100 column oven, automated sample injector (model ASI-100)
and photodiode array detector (model PDA-100). The system is
operated by computer using Chromeleon version 6.6 software.
2.3. Determination of antioxidant activity
2.3.1. ABTS methodology (TEAC)
Antioxidant capacity was estimated in terms of radical scaveng-
ing activity using the procedure described by Sánchez-Gonzalez
et al. (2005). Briefly, ABTS radical cations (ABTS+) were produced
by reacting 7 mM ABTS stock solution with 2.45 mM potassium
persulphate and allowing the mixture to stand in the dark at room
temperature for 12–16 h before use. The ABTS+ solution (stable for
2 days) was diluted with 5 mM phosphate buffered saline (pH 7.4)
to an absorbance of 0.70 ± 0.02 at 730 nm. After addition of 10 lL
of sample or trolox standard to 4 mL of diluted ABTS+ solution,
absorbance readings were measured after 6 min using a UV–Vis
1240 Shimadzu spectrophotometer. Ethanol solutions containing
known concentrations of trolox, a water-soluble analogue of vita-
min E, were used for calibration. Results were expressed g of trolox
per 100 g of coffee (dry matter).
2.3.2. FRAP methodology
In FRAP experiments, the reduction power of various coffees
was estimated according to the procedure described by Sánchez-
Gonzalez et al. (2005). The FRAP reagent was prepared by mixing
of 2.5 mL of a 10 mM TPTZ solution in 40 mM HCl and 2.5 mL of
20 mM FeCl36H2O with 25 mL 0.3 mM acetate buffer pH 3.6. This
solution was incubated at 37 °C for 30 min.
Approximately 900 lL of freshly prepared FRAP reagent,
warmed at 37 °C, was mixed with 90 lL distilled water and either
10 lL of test sample or standard or appropriate reagent blank to
measure AA. After 30 min at 37 °C, readings at the absorption max-
imum (595 nm) were taken. Ethanol solutions containing known
trolox concentrations were used for calibration. Results were ex-
pressed g of trolox per 100 g of coffee (dry matter).
2.3.3. DPPH methodology
The DPPH assay has been widely used to evaluate the ability of
several free radical scavenger molecules. DPPH assays were per-
formed according to the procedure described by Casagrande et al.
(2007). Briefly, solutions of coffee were prepared to obtain 2, 3, 4,
6, 10 and 15 mg/mL. One millilitre of 100 mM acetate buffer (pH
5.5), 1 mL of ethanol and 0.5 mL of 250 lM ethanolic DPPH solu-
tion were mixed and 10 lL of each sample of the above prepared
solutions were added. After 10 min, absorbance was measured at
517 nm. A positive control was prepared in the absence of coffee
solutions, which indicates the maximum amount of free DPPH
 J.A. Vignoli et al. / Food Chemistry 124 (2011) 863–868
(considered 100% of free radicals in the solution), to calculate the
hydrogen-donating ability (IA%) of coffee. The blank was prepared
from the reaction mixture without DPPH solution (Eq. (1)). The
IC50 (the concentration of substance that provides 50% reduction
of free radical concentration) was determined
IA ð%Þ ¼ 100  ðAbs sample=Abs controlÞ  100
2.3.4. Folin–Ciocalteau methodology
Total polyphenol content was evaluated using the Folin–Ciocal-
teau reagent following a method adapted from Singleton, Orthofer,
and Lamuela-Raventos (1999). The sample (0.1 mL) was diluted
with deionised water to a volume of 7.5 mL. Then, 300 lL of
0.9 mol/L. Folin–Ciocalteau (FC) reagent and 1 mL of 20% Na2CO3
solution were added and deionised water was added up to a final
volume of 10 mL. Solutions were maintained at room temperature
for 60 min and total polyphenol content was determined at
765 nm using a UV–Vis 1240 Shimadzu spectrophotometer. Gallic
acid standard solutions were used for calibration. The results were
expressed as g equivalents of gallic acid per 100 g of coffee (dry
matter).
2.4. Determination of the bioactive compounds of soluble coffee
2.4.1. Determination of 5-caffeoilquinic acid (5-CQA) and caffeine
High performance liquid chromatography (HPLC) was used to
determine 5-CQA and caffeine content (Alves, Dias, Scholz, & Ben-
assi, 2006) using a 4.6  250 mm, 5 lm particle Spherisorb ODS2
column (Waters, EUA). Compounds were eluted with gradients
containing 5% acetic acid (A) and acetonitrile (B) as follows: 0–
5 min: 4% B; 5–10 min: 10% B; 10–30 min: 10% B; 30–40 min: 0%
B; 40–50 min: 4% B at a flow rate of 0.7 mL/min. Caffeine was de-
tected at 272 nm while 5-CQA was detected at 320 nm. Quantifica-
tion was carried out by external standardisation using a 5-point
calibration curve with triplicate measurements: the concentration
of 5-CQA ranged from 5 to 30 lg/mL while that of caffeine ranged
from 10 to 50 lg/mL. The samples were dissolved in ultra pure
water and passed through 0.22-lm filters directly into the chro-
matographic system.
2.4.2. Determination of melanoidins
A dialysis membrane separation system with a size exclusion
limit of 12–14 kDa was used as described by Bekedam, Schols, Boe-
kel, and Smit (2006), with modifications. A soluble coffee solution
(20 mL) with a concentration of 45% was prepared. The solution
was transferred to the membrane and placed in a beaker with
400 mL distilled water under agitation. The water was changed
every 8 h until it was colourless. The material retained on the mem-
brane was lyophilised and used to estimate the mass of the material
with molecular weight over 12–14 kDa in relation to the initial mass.
This fraction, here considered to be melanoidin, was expressed as g
of melanoidins/100 g of sample.
2.5. Statistical analysis
Antioxidant activity and chemical composition results were
subjected to principal components analysis by the ‘‘multivariate
exploratory techniques” and ‘‘principal components and classifica-
tion analysis” procedures using the Statistica 7.0 software package
(STATSOFT, São Caetano do Sul, Brazil).
3. Results and discussion
Soluble coffee beverages were assayed for their antioxidant
activity by monitoring their reaction with ABTS cation, their reduc-
865
ing capacity by the FRAP method and their capacity to scavenge
DPPH free radicals. Total phenolic compound content (Folin–Cio-
calteau) was also determined (Table 1). Coffee compounds related
to antioxidant capacity, such as caffeine, 5-CQA and melanoidins,
were also quantified (Table 2).
Principal component analysis was used to evaluate the correla-
ð1Þ
tion between antioxidant activity and the chemical composition of
the various products. The first two components (CP1 and 2) ex-
plained 87% of the total variance.
CP1 was determined by the antioxidant activity of the beverage
and its caffeine content, which was stable during the thermal pro-
cess (Fig. 1A). It is important to note that all methods used gave
similar antioxidant activity results for each the samples, despite
their differing underlying mechanisms (Table 1).
The Folin method was highly correlated with the other methods
used (Fig. 1A). This assay determines the total polyphenol content
of a substance based on a redox reaction, thus it can be considered
an evaluation of antioxidant activity (Prior et al., 2005). In a study
of the influence of coffee preparation methods on antioxidant activ-
ity, Sánchez-Gonzalez et al. (2005) also observed a correlation be-
tween the polyphenol content determined by the Folin method
and FRAP values. In this work, the strongest correlation was found
between the Folin and the DPPH methods (r = 0.88), which suggests
scavenging of this radical by phenolic compounds. It is important to
note that DPPH values are represented as IC50; smaller values of IC50
correspond to higher AA values. Furthermore, the Folin results may
also be related to the reducing capacity of the coffee beverage, which
was evaluated by the FRAP method (r = 0.72) and ABTS radical
scavenging (r = 0.82).
CP2 correlated the 5-CQA and melanoidin contents, compounds
that are degraded or produced during the roasting process. A neg-
ative correlation was observed between these compounds; that is,
the concentration of high molecular weight compounds increased
concurrently with a decrease in 5-CQA content. No correlation
was observed between the concentrations of 5-CQA or melanoidins
and the AA results (CP1). The AA of the light and dark-roasted sam-
ples was similar (Tables 1 and 2, Fig. 1A).
Thus, PCA separated the samples by AA (CP1) and/or roasting
(CP2) (Fig. 1B). The lower two quadrants show a distinct group
formed by dark samples, independent of the raw material or the
extraction methods used. In the higher quadrants are the light
and medium roasting samples, with the robusta coffee on the left
(larger AA) and the arabica coffee on the right.
Considering the influence of the raw material on AA, in general,
C. canephora samples yielded greater AA than the C. arabica sam-
ples (Table 1, Fig. 1B). Similar results, commonly attributed to
the larger CGA content of robusta coffee, were reported in other
studies of roasted coffee (Daglia et al., 2004). Nevertheless, this
study shows that isomer 5-CQA was present in similar or higher
concentrations in soluble arabica coffee than in robusta coffee (Ta-
ble 2). According to Leloup (2006), although green robusta beans
have a higher CGA content, around 10% versus 8% in arabica coffee,
these compounds seem to be more sensitive to the roasting process
in a robusta coffee matrix. Clifford (1997) reported that for a sim-
ilar degree of roasting, robusta coffee undergoes a larger absolute
loss of CGA, producing volatile phenols and guaiacol. Trugo and
Macrae (1984) and Dias (2005) also described a similar behaviour
for arabica and robusta coffees with different degrees of roasting.
Thus, the greater antioxidant capacity of robusta coffee could be
attributed to its higher caffeine content (Table 2), since it corre-
lated positively with AA (Fig. 1). In a study of brewing procedure
influence, Lopez-Galilea, De Pena, and Cid (2007) also reported a
significant correlation between caffeine content and AA results ob-
tained by DPPH (r = 0.83) and redox potential (r = 0.84). Parras,
Martinez-Tomé, Jiménez, and Murcia (2007) evaluated the antiox-
idant activity of coffee from different origins in beverages prepared
866 J.A. Vignoli et al. / Food Chemistry 124 (2011) 863–868

Table 1
Antioxidant activity of soluble coffee obtained from different species and processing conditions evaluated by assorted methods.*

Assays Roasting degree Arabica Robusta

Extraction 1 Extraction 2 Extraction 1 Extraction 2
ABTS (g trolox/100 g) Light 18.77 ± 1.00 23.27 ± 0.00 27.28 ± 0.25 32.29 ± 3.25
Medium 21.03 ± 2.25 23.53 ± 0.75 36.05 ± 1.75 32.79 ± 0.50
Dark 23.78 ± 0.25 24.78 ± 0.00 33.93 ± 1.50 27.79 ± 3.75
FRAP (g trolox/100 g) Light 19.27 ± 2.25 25.03 ± 0.00 26.28 ± 1.50 29.78 ± 0.50
Medium 25.03 ± 0.50 30.79 ± 0.50 34.04 ± 0.75 34.54 ± 1.00
Dark 27.78 ± 0.25 26.03 ± 0.50 35.04 ± 0.75 29.78 ± 1.75
Folin (g gallic acid/100 g) Light 12.08 ± 0.51 14.97 ± 0.17 14.97 ± 0.00 18.54 ± 0.51
Medium 13.09 ± 0.17 15.14 ± 1.02 16.67 ± 0.34 17.35 ± 0.34
Dark 13.44 ± 0.00 13.10 ± 0.34 15.82 ± 0.17 13.44 ± 0.34
DPPH IC50 (lg/mL) Light 24.92 ± 0.55 16.11 ± 0.26 16.35 ± 0.34 14.81 ± 0.37
Medium 19.87 ± 1.38 18.80 ± 0.47 16.14 ± 0.11 14.70 ± 0.20
Dark 20.51 ± 0.18 20.23 ± 1.02 16.79 ± 1.04 19.47 ± 0.35
*
Average of six measurements (duplicate in three repetitions) ± standard deviation.

Table 2
Contents of 5-CQA, caffeine, and melanoidins of soluble coffee obtained from different species and processes.*

Roasting degree Arabica Robusta

Extraction 1 Extraction 2 Extraction 1 Extraction 2
5-CQA (g/100 g) Light 3.53 ± 0.02 4.11 ± 0.11 2.79 ± 0.06 4.24 ± 0.05
Medium 1.87 ± 0.13 2.55 ± 0.03 1.71 ± 0.04 2.26 ± 0.11
Dark 0.62 ± 0.03 0.40 ± 0.03 0.21 ± 0.03 0.25 ± 0.03
Caffeine (g/100 g) Light 2.84 ± 0.00 3.44 ± 0.05 3.98 ± 0.08 5.33 ± 0.02
Medium 3.07 ± 0.36 4.12 ± 0.35 5.82 ± 0.11 5.54 ± 0.14
Dark 3.64 ± 0.04 3.34 ± 0.15 4.75 ± 0.11 4.88 ± 0.64
Melanoidins (g/100 g) Light 23.80 ± 0.77 20.13 ± 0.59 27.30 ± 0.51 22.89 ± 0.38
Medium 18.07 ± 0.45 22.08 ± 0.04 19.66 ± 2.04 21.28 ± 1.65
Dark 29.64 ± 1.59 25.42 ± 0.01 30.44 ± 1.84 26.50 ± 0.19
*
Average of two measurements ± standard deviation.

1,0 5-CQA 4
(A) (B)
3

0,5
A E2 L
2 R E2 L
FOLIN
A E1 L
PC 2 : 26%
PC 2 : 26%

1 R E2 M A E2 M
R E1 M R E1 L A E1 M
0,0
CAFFEINE 0

ABTS -1
FRAP DPPH R E2 D A E2 D
-0,5 -2 R E1 D A E1 D

-3
MELANOIDINS
-1,0 -4
-1,0 -0,5 0,0 0,5 1,0 -4 -3 -2 -1 0 1 2 3 4
PC 1 : 61% PC 1 : 61%

Fig. 1. Principal component analysis of antioxidant activity and composition of soluble coffee: variable projection (A) and scatter plot for the samples (B). Species: (A) arabica
and (R) robusta. Extraction process: conventional (1) and double extraction (2). Roasting degree: light (L), medium (M), dark (D).

by different procedures (espresso, italian, and brewed) and found
higher TEAC values for caffeinated products than in decaffeinated
products.
Regarding the influence of roasting on AA, it is worth noting
that there is little agreement in the literature, even for roasted cof-
fee, which has been the most extensively studied. Del Castillo et al.
(2002) observed greater AA in medium-roasted coffee. Duarte,
Abreu, Menezes, Santos, and Gouvêa (2005) found greater AA for
beverages made from light-roasted coffee. In contrast, Nicoli,
Anese, Manzocco, and Lerici (1997) reported greater AA for
medium and dark-roasted coffee beverages. No reports are avail-
able on soluble coffee; however, it must be taken into account that
for soluble coffee, after roasting, there is an additional thermal
extraction treatment at high temperature.
The degree of roasting showed little influence on the AA of soluble
coffees. Compounds that were affected by roasting (5-CQA and
melanoidins) are not directly related to the antioxidant potential
of the beverages. This may initially seem contradictory, given the
 J.A. Vignoli et al. / Food Chemistry 124 (2011) 863–868
acknowledged antioxidant activity of these compounds. Moreira
et al. (2005) described a strong correlation between the chlorogenic
acid content and the FRAP method, especially for caffeoylquinic iso-
mers. Borreli et al. (2002) evaluated AA and the melanoidin content
of green and roasted coffee (light, medium and dark roast). The ABTS
method yielded AA values for all fractions: low (1.0–3.0 kDa), med-
ium, and high molecular weight (over 100 kDa); however, the high
weight fractions that contained melanoidins and the low weight
fractions rich in phenolic compounds were the most active. Daglia
et al. (2004) obtained similar results for roasted coffee, correlating
AA and hydroxyl radical scavenging capacity. The authors reported
that high molecular weight fractions of green coffee were also eval-
uated; however, they did not present AA, which demonstrates that
roasting is responsible for the formation of AA compounds.
Frequently, studies of coffee AA available in the literature are
either restricted to just one roasting degree or focused on a specific
class of compounds (CGAs, caffeine, or melanoidins). A broader
analysis of these products as a function of roasting conditions,
extraction processes, and composition profiles showed that the fi-
nal AA of coffee beverages results from the balance of degraded
and newly formed compounds during roasting. Increasing roasting
will degrade 5-CQA and will form high molecular weight polymers,
ensuring antioxidant activity remains unaffected by roasting con-
ditions. In conclusion, under the conditions presently studied,
these two compounds contribute equally to AA. Furthermore, the
reduction of one is balanced by the formation of the other, which
explains the similar AA values found for light and dark-roasted cof-
fees. Thus, we can conclude that AA depends more on coffee com-
position than on roasting degree.
In relation to the soluble coffee extraction process, the results of
the processes studied varied according to roasting degree (Table 2).
Both the arabica- and the robusta-derived products presented high-
er 5-CQA contents for light and medium-roasted coffee in the dou-
ble-extraction system (2). Interestingly, this process also favoured
the extraction of caffeine from the two light-roasted coffee species;
only arabica was favoured by medium roasting. The extraction pro-
cess of dark-roasted coffee did not influence the release of these
compounds. It is likely that, after the matrix is extensively ‘‘dam-
aged” by the roasting process, the extraction of compounds is easier
and occurs more or less independently of which process is used. In
the case of the robusta coffee matrix, which was more sensitive to
degradation, this behaviour was observed for some compounds even
in medium-roasted coffee.
Although the double-extraction process increased both the caf-
feine and 5-CQA content of the light-roasted final product, this in-
crease probably occurred in a different way for each compound.
The 5-CQA is a thermolabile compound and was probably preserved
in the first extraction stream at milder temperatures. As caffeine is
thermostable, the double-extraction process may have released a
larger amount of compounds from the light-roasted matrix. Con-
cerning the behaviour of melanoidins, in general, their extraction
was favoured by the single-extraction process. Both the contents
and the composition of the higher molecular weight fraction, consid-
ered to be melanoidins, are influenced by the roasting process and
extraction conditions. As the molecular weight increases with the
thermal treatment, severe conditions also result in the degradation
of these compounds. Leloup (2006) evaluated the influence of tem-
perature on the molecular weight profile of carbohydrates and ob-
served the formation of mono- and disaccharides with increasing
extraction temperature; however, under mild conditions, molecules
of over 10 kDa were formed. As two water streams are used in the
double-extraction process, one with a higher temperature, com-
pounds with higher molecular weights are probably degraded.
More 5-CQA and caffeine were extracted by the double-extrac-
tion process, increasing the AA of the light-roasted product. This
demonstrates the effect of 5-CQA concentration on AA. For less in-
867
tense roasting systems, a process that yields higher concentrations
of 5-CQA is more advantageous. As this compound degrades and
the content of melanoidin increases, the extraction process has less
of an effect on AA.
Soluble coffee beverage thus possesses excellent antioxidant
potential, since its fabrication traditionally uses large amounts of
robusta coffee and the extraction processes favour the enrichment
of components with antioxidant potential, such as 5-CQA and
caffeine.
4. Conclusion
All of the studied soluble coffee products possessed antioxidant
potential conferred by balanced concentrations of phenolic com-
pounds, caffeine and melanoidins. AA was unaffected by roasting
conditions, since the degradation of 5-CQA was balanced by the
formation of melanoidins. The extraction process had a greater
influence on AA in lighter-roasted coffees. The higher caffeine con-
tent of C. canephora resulted in products with greater AA, indicat-
ing that the antioxidant activity of the final product depends
mainly on the species used in blends.
References
ABICS Associação Brasileira da Indústria de Café Solúvel (2008). Papel do café solúvel
na abertura de mercados para o café brasileiro. <http://www.abics.com.br/
relatorios/dodirex3-cafe-soluvel-futuro.pdf>.
Alves, S. T., Dias, R. C. E., Scholz, M. B. S., & Benassi, M. T. (2006). Metodologia para
análise simultânea de ácido nicotínico, trigonelina, ácido clorogênico e cafeína
em café torrado por cromatografia líquida de alta eficiência. Química Nova, 29,
1164–1168.
Bekedam, E. K., Schols, H. A., Boekel, T. Van, & Smit, G. (2006). High molecular
weight melanoidins from coffee brew. Journal of Agricultural and Food Chemistry,
54, 7658–7666.
Benzie, I. F. F. (1996). An automated, specific, spectrophotometric method for
measuring ascorbic acid in plasma (EFTSA). Clinical Biochemistry, 29, 111–116.
Borreli, R. C., Visconti, A., Mennella, C., Anese, M., & Fogliano, V. (2002). Chemical
characterization and antioxidant properties of coffee melanoidins. Journal of
Agricultural and Food Chemistry, 50, 6527–6533.
Casagrande, R., Georgetti, S. R., Verri, W. A., Jr., Borin, M. F., Lopez, R. F. V., & Fonseca,
M. J. (2007). In vitro evaluation of quercetin cutaneous absorption from topical
formulations and its functional stability by antioxidant activity. International
Journal of Pharmaceutics, 328, 183–190.
Clifford, M. N. (1997). The nature of chlorogenic acids. Are they advantageous
compounds in coffee? In Proceedings of the 17th ASIC colloquium, Nairobi, pp. 79–
89.
Coughlin, J. R. (2006). Coffee and health: The holistic approach. In Proceedings of the
21st ASIC colloquium, Montpellier, France, pp. 29–35.
Daglia, M., Papetti, A., Gregotti, C., Bertè, F., & Gazzani, G. (2000). In vitro antioxidant
and ex vivo protective activities of green and roasted coffee. Journal of
Agricultural and Food Chemistry, 48, 1449–1454.
Daglia, M., Rachi, M., Papetti, A., Lanni, C., Govoni, S., & Gazzani, G. (2004). In vitro
and ex vivo antihydroxil radical activity of green and roasted coffee. Journal of
Agricultural and Food Chemistry, 52, 1700–1704.
Del Castillo, M. D., Ames, J. M., & Gordon, M. H. (2002). Effect of roasting on the
antioxidant activity of coffee brews. Journal of Agricultural and Food Chemistry,
50, 3698–3703.
Delgado-Andrade, C., Rufián-Henares, J. A., & Morales, F. J. (2005). Assessing the
antioxidant activity of melanoidins from coffee brews by different antioxidant
methods. Journal of Agricultural and Food Chemistry, 53, 7832–7836.
Devasagayam, T. P., Kamat, J. P., Monhan, H., & Kesavan, P. C. (1996). Caffeine as an
antioxidant: Inhibition of lipid peroxidation induced by reactive oxygen
species. Biochimica Biophysica Acta, 1282, 63–70.
Dias, R. C. E. (2005). Discriminação de espécies de café (Coffea arábica e Coffea
canephora) em diferentes graus de torra. Tese de Mestrado. Departamento de
Ciência e Tecnologia de Alimentos, Universidade Estadual de Londrina, 106p.
<http://bibliotecadigital.uel.br/document/?code=vtls000108937>.
Duarte, S. M. S., Abreu, C. M. P., Menezes, H. C., Santos, M. H., & Gouvêa, C. M. C. P.
(2005). Effect of processing and roasting on the antioxidant activity of coffee
brews. Ciência e Tecnologia de Alimentos, 25, 387–393.
Gallardo, C., Jiménez, L., & Garcia-Conesa, M. T. (2006). Hydroxycinnamic acid
composition and in vitro antioxidant activity of selected grain fractions. Food
Chemistry, 99, 455–463.
GEA-Group (2010). Coffee – The drink that changed the world. <http://
www.geagroup.com/en/loesungen/kaffee.html>.
Gómez-Ruiz, J. A., Lake, D. S., & Ames, J. M. (2007). In vitro antioxidant activity of
coffee compounds and their metabolites. Journal of Agricultural and Food
Chemistry, 55, 6962–6969.
868 J.A. Vignoli et al. / Food Chemistry 124 (2011) 863–868
Higdon, J. V., & Frei, B. (2006). Coffee and health: A review of recent human
research. Critical Reviews in Food Science and Nutrition, 46, 101–123.
Leloup, V. (2006). Evaluation of the nutritive value of soluble coffee. In Proceedings
of 21st colloquium ASIC, Montpellier, France, pp. 80–87.
Lopez-Galilea, I., De Pena, M. P., & Cid, C. (2007). Correlation of selected constituents
with the antioxidant capacity of coffee beverages: Influence of the brewing
procedure. Journal of Agricultural and Food Chemistry, 55, 6110–6117.
Moreira, D. P., Monteiro, M. C., Ribeiro-Alves, M., Donangelo, C. M., & Trugo, L. C.
(2005). Contribution of chlorogenic acids to the iron-reducing activity of coffee
beverages. Journal of Agricultural and Food Chemistry, 53, 1399–1402.
Nicoli, M. C., Anese, M., Manzocco, L., & Lerici, C. (1997). Antioxidants properties of
coffee brews in relation to the roasting degree. Lebensmittel-Wissenschaft und-
Technologie, 30, 292–297.
Parras, P., Martinez-Tomé, M., Jiménez, A. M., & Murcia, M. A. (2007). Antioxidant
capacity of coffees of several origins brewed following three different
procedures. Food Chemistry, 102, 582–592.
Pellegrini, N., Serafini, M., Colombi, B., Del Rio, D., Salvatore, M. B., & Brighenti, F.
(2003). Total antioxidant capacity of plant foods, beverages and oils consumed
in Italy by three different in vitro assays. Journal of Nutrition, 133, 2812–2819.
Prior, R. L., Xianli, W., & Schaich, K. (2005). Standardized methods for determination
of antioxidant capacity and phenolics in foods and dietary supplements. Journal
of Agricultural and Food Chemistry, 53, 4290–4302.
Sánchez-Gonzalez, I., Jiménez-Escrig, A., & Saura-Calixto, F. (2005). In vitro
antioxidant activity of coffees brewed using different procedures (italian,
espresso and filter). Food Chemistry, 90, 133–139.
Shi, X., & Dalal, N. S. (1991). Antioxidant behavior of caffeine: Efficient scavenging of
hydroxyl radicals. Food Chemistry Toxicology, 29, 1–6.
Singleton, V. L., Orthofer, R., & Lamuela-Raventos, R. M. (1999). Analysis of total
phenols and other oxidation substrates and antioxidants by means of Folin–
Ciocalteau reagent. Methods in Enzymology, 299, 152–178.
Trugo, L. C., & Macrae, R. (1984). Chlorogenic acid composition of instant coffees.
Analyst, 109, 263–266.