Eur Food Res Technol (2009) 228:913–922
DOI 10.1007/s00217-008-1004-x
ORIGINAL PAPER
EVect of diVerent extraction methods on the recovery
of chlorogenic acids, caVeine and Maillard reaction products
in coVee beans
Graryna Budryn · Ewa Nebesny · Anna Podsddek ·
Dorota ôyrelewicz · Maigorzata Materska ·
Stefan Jankowski · Bogdan Janda
Received: 5 March 2008 / Revised: 17 December 2008 / Accepted: 27 December 2008 / Published online: 3 February 2009
© Springer-Verlag 2009
Abstract Water and ethanolic extracts were obtained
from green and roasted (3 diVerent roast degrees) Arabica
and Robusta coVee beans. Three types of water extracts
were prepared from the examined, Wnely ground material
through: (a) brewing with boiling water, (b) boiling in
water, and (c) boiling in water under elevated pressure. All
these extracts were lyophilized. Two types of ethanolic
extracts were derived from the examined material through
(a) extraction of the Wnely ground coVee beans and (b)
extraction of the solid residue that remained after boiling
the coVee beans in water under elevated pressure. These
ethanolic extracts were dried. Both water and ethanolic
G. Budryn (&) · E. Nebesny · D. ôyrelewicz
Faculty of Biotechnology and Food Sciences,
Institute of Chemical Technology of Food,
Technical University of Lodz, Stefanowskiego 4-10,
90-924 Lodz, Poland
e-mail: gbudryn@snack.p.lodz.pl
A. Podsddek
Faculty of Biotechnology and Food Sciences,
Institute of Technical Biochemistry, Technical University of Lodz,
Stefanowskiego 4-10, 90-924 Lodz, Poland
M. Materska
Department of Chemistry,
Faculty of Food Sciences and Biotechnology,
Agricultural Academy of Lublin, Akademicka 13,
20-950 Lublin, Poland
S. Jankowski
Faculty of Chemistry, Institute of Organic Chemistry,
Technical University of Lodz, ôeromskiego 116,
90-924 Lodz, Poland
B. Janda
Institute of Soil Science and Plant Cultivation,
State Research Institute, Czartoryskich 8,
24-100 Puiawy, Poland
extracts were analyzed for concentration of potential anti-
oxidants such as chlorogenic acids and caVeine (by HPLC)
and Maillard reaction products (measurements of absor-
bance at 420 nm). Concentration of chlorogenic acids in
Robusta extracts varied between 0.4 and 36.0 g £ 100 g¡1
dry extract weight (db.), while in Arabica extracts it ranged
from 0.1 to 22.4 g £ 100 g¡1 db. Extracts of dark roasted
Arabica contained more chlorogenic acids than those of
Robusta. Concentration of caVeine, which in green and
roasted coVee beans is maintained at the similar level,
tended to increase in Robusta extracts with the roast degree
and temperature of extraction with water, while in case of
Arabica extracts there was no noticeable tendency. CaVeine
concentrations varied between 0.12 and 8.41 g £ 100 g¡1
db. and between 0.03 and 6.53 g £ 100 g¡1 db. in Robusta
and Arabica extracts, respectively. Ethanolic extracts were
characterized by relatively higher caVeine concentrations
and lower contents of brown pigments and chlorogenic
acids as compared to water extracts. The richest in antioxi-
dants were extracts of green Robusta coVee beans derived
through boiling in water under elevated pressure.
Keywords CoVee · CoVee processing · Chlorogenic acids ·
CaVeine · Maillard reaction products · HPLC
Introduction
The main reasons of consumption of coVee brews are mild
stimulation in mood and palatable taste and aroma. Further-
more, these brews display an antioxidant activity and their
moderate consumption is believed to reduce the risk of devel-
opment of some types of cancer, like the cancer of large intes-
tine [1]. Antioxidant activity is ascribed to such components
of coVee brews as chlorogenic acids, caVeine and some of
123
914
Maillard reaction products [2–4]. A prerequisite of absorption
of chlorogenic acids from the gastrointestinal tract is their
hydrolysis to caVeic and quinic acids in the small intestine
where approximately one-third of consumed chlorogenic
acids is absorbed. This reaction is catalyzed by an intestinal
esterase. Chlorogenic acids are also hydrolyzed by microXora
of large intestine [5, 6]. Since the antioxidant capacity of
coVee beans is relatively high, they can be used as a source of
natural antioxidants [7–11]. Preparations of the latter can be
applied as supplements of diet or shelf-life increasing addi-
tives to foodstuVs which are susceptible to oxidation [12, 13].
CoVee beans are roasted prior to brewing; this process brings
about an appreciable degradation of chlorogenic acids and
thus lessening their antioxidant activity. But roasting gives
rise to some other antioxidants like Maillard reaction prod-
ucts, comprising melanoidins and heterocyclic volatile com-
pounds. Thus both green and roasted coVee beans can be used
to produce preparations of antioxidants. Since foods which are
particularly susceptible to oxidation usually contain fats that
are rich in unsaturated fatty acids, antioxidants should be
extracted from coVee beans not only with water but also with
ethanol to obtain preparations with higher aYnity to lipids.
Earlier investigations revealed the occurrence of chloro-
genic acids in green and roasted Arabica and Robusta
coVee beans and in de-caVeinated coVee beans [14–17].
Studies on their content in coVee extracts were limited to
typical brews of roasted coVee [18–21]. Some researchers
presented only their concentration in brews [22, 23]. The
similar investigations were carried out for caVeine and
brown pigments measurable at 420 nm [24, 25]. However,
an eYciency of extraction of particular chlorogenic acids
and other antioxidants from green coVee beans under diVer-
ent conditions with food-grade solvents has not been deter-
mined yet, although these beans are rich in such substances.
This study aimed at obtaining of dried preparations of
water and ethanolic extracts from Robusta and Arabica
coVee beans, estimation of concentration of selected poten-
tial antioxidants in these dried extracts and an assessment
whether the solid residue after extraction with water could
be used as a potential source of antioxidants. The antioxi-
dant activity of the examined extracts was determined as
described earlier [26].
Materials and methods
Materials
Green beans of two coVee varieties: Arabica (CoVea arab-
ica) from Columbia (prepared by wet method) and Robusta
(CoVea canephora) from Indonesia (prepared by dry
method) were harvested in 2005 and purchased from P.H.P.
Aspol Ltd. (Poland).
123
Eur Food Res Technol (2009) 228:913–922
5-O-CaVeoylquinic acid and caVeine were purchased
from Fluka Chemie (Helvetia). All other reagents were ana-
lytical grade.
Methods
Roasting
Robusta and Arabica coVee beans were roasted in a labora-
tory convective coVee roaster (Precission Heartware, USA)
with automatic cooling. Three diVerent roast degrees were
characterized by a decrease in weight shown in Table 1.
This decrease was calculated as a diVerence between the
weight of coVee beans before and after roasting and
expressed per 100 g beans. The temperature inside the
roasting chamber was measured by using NiCr–Al thermo-
couple (Poland). The humidity of Wnely ground coVee
beans was equivalent to the loss in weight after drying at
103 °C until constant weight. The humidity of medium and
dark roasted Arabica coVee beans was the same despite
longer duration of roasting and in consequence darker color
of the latter. It could stem from production of water during
Maillard condensation reactions and other reactions taking
place during roasting. Our Wnding is consistent with results
of Nunes et al. [27] who even observed a transitory increase
in humidity of Arabica coVee beans during roasting. Also
Pittia et al. [28] reported on maintaining of humidity of
Arabica coVee beans at the constant level in the Wnal phase
of roasting while the humidity of Robusta coVee beans
gradually decreased throughout this process. Green coVee
beans were ground in a laboratory mill WZ-1 ZBPP
(Poland) adapted for very hard beans grinding. Roasted
coVee beans were roasted in a mill Il Macinino F.A.C.E.M
Tre Spade (Italy). Diameters of ground particles were mea-
sured by using the micrometric screw NSK Digitrix Mark II
Electronic Micrometer, (Micrometer MFG Co. Ltd., Japan)
after dispersing in paraYn oil. Dimensions of these parti-
cles varied between 480 and 680
m. The color of ground
coVee beans was determined by CIE L*a*b* method with
an illuminant “C” by using the reXection spectrophotometer
Specord M-40 Carl-Zeiss Jena (Germany).
Preparation of coVee extracts
Samples of green and roasted (with 3 diVerent roast
degrees), ground coVee were extracted with water at the
coVee:solvent ratio of 1:5.75, ensuring approximately
5 g £ 100 g¡1 concentration of solid substance in extracts.
Water extracts were prepared by 3 methods such as (a)
brewing with boiling water followed by intermittent mixing
and Wltration after 5 min, (b) boiling in water for 10 min
followed by Wltration and (c) boiling in a pressure cooker
PS-5682 First (Austria) at 110 °C and 1.4 £ 105 Pa for
Eur Food Res Technol (2009) 228:913–922 915

Table 1 Physicochemical properties of green and roasted (3 diVerent roast degrees) Arabica and Robusta coVee beans
Type of coVee beans A decrease Time of Ultimate Dry basis Color
in weight during roasting temperature (g £ 100 g¡1)
roasting (g £ 100 g¡1) (min) of roasting (°C) L* a* b*

Green Arabica – – – 91.99 75.51 1.68 20.28b

Light roasted Arabica 10 5 200 96.58 47.93 9.29 26.26
Medium roasted Arabica 14 7 220 98.93a 43.15a 5.97 20.76a
a b
Dark roasted Arabica 17 9.5 235 98.92 41.84 4.68 19.47
Green Robusta – – – 91.61 70.33 2.83 21.57
Light roasted Robusta 10 5 210 98.10 54.89 10.03 30.34
Medium roasted Robusta 14 8 230 98.90a 45.87 7.50 23.98
Dark roasted Robusta 17 12 240 99.24 42.65a,b 5.34 20.66a,b
Values in each column bearing the same letters are not statistically diVerent (P > 0.05) from one another
L* lightness variable (0, black ¥ 100, white), chromatic coordinates: a* red (0, no pigment ¥ 50, maximum red), b* yellow (0, no pigment ¥ 50,
maximum yellow)

10 min followed by rapid cooling and Wltration. Each time,
Wltration was conducted under reduced pressure by using
vacuum pump KNF Neuberger N 035.3 AT.18 (Germany).
In the case of brewing, the time of contact of ground beans
with hot water was shortened to 5 min to minimize the
inXuence of high temperature and compare the results with
more drastic methods. These three diVerent extraction
methods were used in order to Wnd whether a rise in tem-
perature gives rise to more eYcient extraction of analyzed
compounds or, in contrary, it results in changes in their
structure or their oxidation (Wrst of all chlorogenic acids).
Two types of ethanolic extracts were derived by 2-h extrac-
tion in Soxhlet apparatus, i.e., either from ground coVee
beans or from solids remained after extraction with water
under elevated pressure. Prior to extraction with ethanol,
these solids were washed three times with water at 95 °C in
a proportion 1:20, drained oV and dried at 70 °C until the
moisture <1.5 g £ 100 g¡1. Water extracts were freeze-
dried in DELTA 1-24LSC Christ lyophilizer (Germany)
and ethanolic extracts were dried convectively at 80 °C in a
laboratory dryer. Dried extracts contained from 1.0 to
4.5 g £ 100 g¡1 of water. To compare contents of quanti-
Wed compounds in the extracts, dry extract weight was
determined as the loss in weight after drying at 103 °C until
constant weight.
Determination of chlorogenic acids and caVeine
Extracts were dissolved and puriWed on C18 Sep-Pak 51910
column (Millipore Waters, Milford, USA), which was ear-
lier equilibrated with 5 mL methanol and 3 mL distilled
water. The applied extract was eluted with 20 mL of metha-
nol:water (40:60 v/v). The eluate was diluted to 50 mL with
re-distilled water and an aliquot (20
L) was used for
HPLC analysis.
Table 2 Solvent mixing program during HPLC of phenolic acids and
caVeine
Time (min) A B
0 88 12
26 70 30
40 0 100
43 0 100
48 88 12
50 88 12
Solvents: A water:formic acid (90:10 v/v), B water:acetonitrile:formic
acid (40:50:10 v/v/v)
HPLC was conducted by using liquid chromatograph
KNAUER (Germany) equipped with 250 £ 4 mm LiChro-
spher 100 RP 18 (5
m) column and UV–VIS detector.
Detection of chlorogenic acids and caVeine was conducted
at 320 and 280 nm, respectively. Elution was carried out by
using the following solvents: A, water/formic acid (90:10
v/v), and B, water:acetonitrile:formic acid (40:50:10 v/v/v).
The samples were eluted according to the program: A:B
mixture (Table 2) for 50 min in a linear gradient and at a
Xow rate of 1 mL min¡1. Concentrations of chlorogenic
acids were calculated from the equation of regression (sur-
face area under the peak as a function of concentration of
5-caVeoylquinic acid or caVeine).
IdentiWcation of chlorogenic acids
(a) Isolation of chlorogenic acids Samples (500 g) of green
coVee beans were homogenized three times with 1-L etha-
nol:water solution (80:20 v/v) for 15 min (homogenizer
DIAX 900) and each homogenate was Wltered. The Wltrates
were pooled and evaporated at 45 °C to obtain an oil-like
concentrate (73.15 g) by using vacuum evaporator. This

123
916
fraction was dispersed in water acidiWed with H3PO4 to pH
of 2.6 and the obtained emulsion was Wltered through G3
sintered funnel containing approximately 2.5-cm layer of
silica gel C18 (60–40
m), which was earlier washed with
methanol and next with the acidiWed water. Chlorogenic
acids were eluted with methanol in the acidiWed water (pH
of 2.6) 1:1 (m/m) until the eluate was colorless. The eluate
was concentrated to almost dry matter (42.5 g).
(b) Separation of chlorogenic acids Separation was con-
ducted on glass (length of 50 cm, Ø 2.5 cm) column packed
with silica gel C18 (40–25
m), which was earlier washed
with methanol and acidiWed water (pH of 2.6). Phenolic
acids were dissolved in a small volume of the acidiWed
water and applied onto the column. The column was
washed with re-distilled water and 300-mL portions of
methanol solution in water (methanol concentration rose by
5 until 100 g £ 100 g¡1). Eluate fractions (10 mL) were
analyzed for phenolic acids by TLC on SiO2 F254 plates
resolved in a system: 3.45 mL formic acid, 1 mL toluene,
8 mL ethyl acetate and 0.05 mL water. After drying the
plates were observed under UV light at 254 nm. Fractions
which contained the same bands were pooled and analyzed
by HPLC.
(c) HPLC analysis of separated fractions of chlorogenic
acids HPLC was conducted by using WellChrom chro-
matograph (Knauer, Germany) equipped with UV detector
and Vertex Eurosil Bioselect 300 Å, endcapped column
(300 £ 4 mm, 5-
m mesh). Elution was conducted with
gradient: A, acetonitrile; B, re-distilled water acidiWed with
H3PO4 to pH of 2.6. The acids were detected at 320 nm.
Fractions with the highest purity were dried in a stream of
nitrogen.
(d) MS analysis Liquid secondary ion (LSI) mass spectra
were recorded on the Finnigan MAT 95 double focusing
(BE geometry) mass spectrometer (Finnigan MAT, Bre-
men, Germany). Samples were dissolved in methanol and
the solution was mixed with glycerol. For the ionization,
the beam of 13-keV cesium ions was used. Spectra were
recorded in positive or negative ion mode. At least ten
spectra for each mode were recorded and averaged.
(e) NMR analysis This analysis was carried out for 2
compounds with the highest purity. 1H, 13C, DEPT 135,
COSY DQF, HMQC and HMBC spectra were recorded at
Avance II Plus 700 MHz spectrometer (Bruker, Rheinstet-
ten, Germany) in methanol-D4 solutions. Chemical shifts
were measured from solvent methyl group at 3.31 ppm (1H)
and 49.15 ppm (13C).
5-O-CaVeoylquinic acid
FAB: 369.1[M + H], 195.0, 185.0, 177.0, 148.9, 132.8
1
H NMR (CD3OD),
: 2.07 (2H, m, C-2), 2.21 (2H, m,
C-6), 3.74 (1H, dd, J = 8.5, 3.0 Hz, C-4), 3.88 (3H, s,
123
Eur Food Res Technol (2009) 228:913–922
CH3O)4.18 (1H, m, C-4), 5.35 (1H, dt, J = 8.9, 4.4 Hz,
C-5), 6.34 (1H, d, J = 15.9 Hz, C-8⬘), 6.80 (1H, d,
J = 8.2 Hz, C-5⬘), 7.07 (1H, dd, J = 8.2, 1.7 Hz, C-6⬘), 7.17
(1H, d, J = 1.7 Hz, C-2⬘), 7.61 (1H, d, J = 15.9 Hz, C-7⬘).
13
C NMR (CD3OD),
: 38.3 (C-2), 38.9 (C-6), 56.6
(CH3O-C-3⬘)), 71.4 (C-3), 72.1 (C-5), 73.6 (C-4), 76.3
(C-1), 111.9 (C-2⬘), 115.8 (C-8⬘), 116.6 (C-5⬘), 124.2
(C-6⬘), 127.9 (C-1⬘), 147.1 (C-7⬘), 149.5 (C-3⬘), 150.7
(C-4⬘), 168.8 (C-9⬘), 177.3 (C-7).
3,4-di-O-CaVeoylquinic acid
ESI: 515.1[M-H], 1031.0 (a dimer)
1
H NMR (CD3OD),
: 2.15 (2H, m, C-2,6, axial pro-
tons), 2.26 (1H, m, C-6, equatorial proton), 2.37 (1H, m, C-2,
equatorial proton), 4.40 (1H, m, C-5), 5.02 (1H, dd, J = 9.1,
3.1 Hz, C-4), 5.65 (1H, m, C-3), 6.26 (1H, d, J = 15.7 Hz,
C-8⬘), 6.30 (1H, d, J = 16.1 Hz, C-8⬘), 6.74 (1H, d,
J = 8.1 Hz, C-5⬘), 6.78 (1H, d, J = 8.1 Hz, C-5⬘), 6.86 (1H,
dd, J = 8.1,1.7 Hz, C-6⬘), 6.92 (1H, dd, J = 8.1,1.5 Hz, C-6⬘),
7.03 (1H, d, J = 1.7 Hz, C-2⬘), 7.05 (1H, d, J = 1.5 Hz,C-2⬘),
7.55 (1H, d, J = 15.7 Hz, C-7⬘), 7.58 (1H, d, J = 16.1 Hz,
C-7⬘).
13
C NMR (CD3OD),
: .37.0 (C-2), 41.9 (C-6), 65.9
(C-5), 70.3 (C-3), 76.6 (C-4), 114.97 (C-8⬘), 115.13 (C-8⬘),
115.29 (C-2⬘), 116.57 (C-5⬘), 116.63 (C-5⬘), 123.25 (C-6⬘),
123.39 (C-6⬘), 127.77 (C-1⬘), 127.82 (C-1⬘), 146.72 (C-3⬘),
146.75 (C-7⬘), 147.46, 147.52 (C-4⬘), 149.57 (C-4⬘), 168.72
(C-8⬘), 168.79 (C-8⬘), 176.28 (C-7), 176.31 (C-7).
Chromatographic separation enabled determination of
the content of the most abundant chlorogenic acids and
caVeine in the extracts. On the basis of MS and NMR spec-
tra and their interpretation proposed by CliVord et al. [29]
the following compounds were identiWed:
1. CaVeine, the reference chromatogram with the stan-
dard;
2. 3-O-CaVeoylquinic acid, MS [29];
3. 4-O-CaVeoylquinic acid, MS [29];
4. 5-O-CaVeoylquinic acid, the reference chromatogram
with the standard, MS, NMR;
5. 3-O-Feruoylquinic acid, MS [29];
6. 4-p-Coumaric acid, MS [29];
7. 5-O-Feruoylquinic acid, MS [29];
8. 3,4-DicaVeoylquinic acid, MS, NMR;
9. 3,5-DicaVeoylquinic acid, MS [29];
10. 4,5-DicaVeoylquinic acid, MS [29];
11. 4,5-CaVeoylferuoylquinic acid, MS [29].
Measurements of absorbance at 420 nm
To determine concentration of brown pigments generated
during roasting, the extracts were dissolved in water or
Eur Food Res Technol (2009) 228:913–922
ethanol (in the same solvent as used for extraction) in
proportion 1:20 to obtain the same concentration as in fresh
extracts. The measurement of absorbance needed further
dilution of extracts in proportion 1:20 [30]. Absorbance of
this solution was measured in 1-cm glass cuvettes at
420 nm (vs. water or ethanol, respectively) by using Hitachi
UV/VIS 2800A spectrophotometer (Japan).
Statistical analysis
All analyses were carried out at least in triplicate and the
results were averaged. One-way ANOVA was carried out
to Wnd if the diVerences between the results were statisti-
cally signiWcant.
Results and discussion
Analysis of extractability of chlorogenic acids
Individual polyphenols and caVeine were separated by
HPLC and determined quantitatively. Ten chlorogenic
acids were detected and identiWed in all the analyzed
extracts (Fig. 1 a, b). The sum of identiWed and detected but
unidentiWed compounds contained in the fraction of pheno-
lic acids is shown in Table 3. Concentration of each identi-
Wed chlorogenic acid in the extracts is displayed in Table 4.
Fig. 1 Examples of chromato-
grams of water and ethanolic ex-
tracts of green Robusta coVee
A 1 2 3
beans extracted by: a brewing
with water, b ethanol; 1 3-O-
caVeoylquinic acid, 2 4-O-
caVeoylquinic acid, 3 5-O-
caVeoylquinic acid, 4 3-O-fer-
uoylquinic acid, 5 4-O-feruoyl-
quinic acid, 6 5-O-feruoylquinic
acid, 7 3,4- di-O- caVeoylquinic
acid, 8 3,5-di-O-caVeoylquinic
acid, 9 4,5-di-O-caVeoylquinic
acid, 10 4,5-O-caVeoylferuoyl-
quinic acid
B 1 2 3
917
These concentrations depended not only on the degree of
degradation during roasting, but also on losses of other sub-
stances that gave rise to the relative increase in phenolic
acids contents. Also the eYciency of extraction, which
depends on swelling of coVee beans during roasting and
changes in solubility of other components, e.g., increasing
solubility of polymeric substances, plays decisive role in
this respect.
5-O-CaVeoylquinic acid (5-CQA) dominated other
chlorogenic acids in all the examined fractions. It is to note
that its concentration in Arabica and Robusta extracts was
similar. More drastic extraction conditions than brewing,
such as boiling under normal and elevated pressure resulted
in gradual isomerisation of 5-CQA to 3- and 4-O-caVeoyl-
quinic acids (3- and 4-CQA) both in Arabica and Robusta
extracts. In Arabica extracts concentration of 3- and 4-CQA
was relatively lower in relation to that of 5-CQA as com-
pared to Robusta ones.
The most eYcient method of chlorogenic acids extraction
from green Robusta coVee was found to be boiling in water
under elevated pressure [36.0 g £ 100 g¡1 dry extract weight
(db.)], while for green Arabica coVee it was boiling under
normal pressure (22.4 g £ 100 g¡1 db.). Concentration of
chlorogenic acids in solid substance of green Robusta
extracts increased in the order from brewing, through boiling
in water under normal pressure to boiling under elevated
pressure. Longer extraction under more drastic conditions is
8
7
10
6 9
4 5
9
7
6
10
8
5
4
123
918 Eur Food Res Technol (2009) 228:913–922

Table 3 Concentration of chlorogenic acids in dried coVee extracts

Extraction conditions Roast degree Concentration of chlorogenic acids
(g £ 100 g¡1dry extract weight)

IdentiWed Total (comprising

unidentiWed)

Robusta brewed with water Green 24.36 24.51

Light roasted 23.42 25.49
Medium roasted 5.80 8.07
Dark roasted 2.80 3.54
Robusta boiled in water Green 27.52 30.57
Light roasted 21.88 23.51
Medium roasted 5.96 7.59
Dark roasted 1.32 2.42
Robusta boiled in water under elevated pressure Green 36.04 37.61
Light roasted 17.26 19.38
Medium roasted 10.18 12.13
Dark roasted 2.67 3.74
Robusta extracted with ethanol Green 11.67 12.94
Light roasted 7.60 9.46
Medium roasted 2.44 3.98
Dark roasted 1.09 1.91
Ethanolic extract from the solid residue remained Green 0.36 0.36
after extraction of Robusta with water under Light roasted 0.42 0.53
elevated pressure
Medium roasted 0.18 0.80
Dark roasted 0.04 1.31
Arabica brewed with water Green 16.56 16.87
Light roasted 13.93 15.97
Medium roasted 7.02 8.75
Dark roasted 2.62 3.77
Arabica boiled in water Green 22.40 22.98
Light roasted 12.63 17.36
Medium roasted 8.30 9.14
Dark roasted 5.40 8.82
Arabica boiled in water under elevated pressure Green 18.05 19.56
Light roasted 15.84 17.72
Medium roasted 6.59 8.73
Dark roasted 3.80 6.19
Arabica extracted with ethanol Green 8.89 10.92
Light roasted 5.70 8.04
Medium roasted 1.72 4.09
Dark roasted 1.54 3.76
Ethanolic extract from the solid residue remained Green 0.03 0.23
after extraction of Robusta with water Light roasted 0.30 1.15
under elevated pressure
Medium roasted 0.02 0.05
Dark roasted 0.01 0.70

thought to result in the cleavage of hydrogen bonds between genic acids in water extracts tended to increase with the
chlorogenic acids and caVeine, amino acids or proteins and intensity of extraction (time, temperature) and the solubility
this in turn increases their solubility. Our results indicate that of other components was rather comparable irrespective of
for green Robusta coVee the relative concentration of chloro- extraction method. By contrast, the highest concentrations of

123
Eur Food Res Technol (2009) 228:913–922 919

Table 4 Concentration of particular chlorogenic acids and caVeine in extracts of Arabica and Robusta coVee beans
Extraction conditions Roast degree Concentration of chlorogenic acids and caVeine (g £ 100 g¡1dry extract weight)

1 2 3 4 5 6 7 8 9 10 11

Robusta brewed Green 3.91d 3.00b 3.31c 11.16a 0.15a 0.15d 0.80c 1.84 a 1.61 1.67 0.67a
with water Light roasted 6.12 b
4.50 a
5.00 9.43 b
0.05 b
0.81 f
0.66 b
1.05 h
0.70 a
0.69 a
0.53a
b d d d d c g d f c
Medium roasted 6.06 1.41 1.37 2.20 0.01 0.19 0.11 0.16 0.12 0.12 0.11d
b f g e b f h c c c
Dark roasted 6.11 0.35 0.39 1.10 0.03 0.05 0.06 0.24 0.21 0.17 0.20c
Robusta boiled Green 5.04c 4.19a 4.53a 11.51a 0.14a 0.41b 1.28a 1.93a 1.46 1.29 0.78
in water Light roasted 5.20 c
4.61 a
4.43 a
9.35 b
0.02 d
0.50 a
0.66 b
0.77 b
0.47 b
0.62 a
0.45
Medium roasted 5.48c 1.55d 1.42d 2.19d 0.06b 0.18c 0.12 g 0.11e 0.07 g 0.04d 0.22c
b f g h d e i
Dark roasted 6.15 0.35 0.32 0.50 0.01 0.08 0.03 tr tr tr 0.03f
a b f a
Robusta boiled Green 6.72 8.16 8.42 10.11 0.57 0.77 1.22 2.61 1.74 1.44 1.00
in water under Light roasted 4.43 e
3.40 b
4.10 b
6.85 c
0.18 a
0.37 b
0.43 e
0.73 b
0.47 b
0.41 0.32b
elevated pressure b c e a a e c c e
Medium roasted 6.26 2.52 2.17 3.52 0.16 0.46 0.37 0.32 0.22 0.18 0.26c
c j e b d h f h
Dark roasted 5.33 0.73 0.60 0.93 0.04 0.11 0.08 0.07 0.04 tr 0.07e
c e d b c c h a b
Robusta extracted Green 5.78 1.05 1.36 4.81 0.11 0.23 0.85 0.98 0.74 0.93 0.61a
with ethanol Light roasted 6.67a 1.15e 1.43d 2.57d 0.02d 0.32b 0.49e 0.56 0.27c 0.48a 0.31b
a f b d g c g c
Medium roasted 6.64 0.41 0.48 0.73 0.06 0.15 0.12 0.21 0.08 0.09 0.11d
i b e i e h
Dark roasted 8.41 0.21 0.25 0.32 0.05 0.08 0.04 0.12 0.02 tr tr
Ethanolic extract from Green 0.12 h tr 0.12 0.12 tr tr tr 0.12e tr tr tr
the solid residue Light roasted 0.23 g 0.04 g tr 0.04f 0.05b 0.02 g 0.02j 0.05f 0.04 h 0.09 0.07e
remained after
Medium roasted 0.25 g 0.01 h 0.01 h 0.02f 0.03c 0.02 g 0.05 h 0.01 g 0.01i 0.01 0.01
extraction of Robusta
g h g g i
with water under Dark roasted 0.31 0.01 tr 0.01 tr 0.01 tr tr 0.01 tr tr
elevated pressure
Arabica brewed Green 2.54f 1.49d 1.86e 11.30a 0.03c 0.21c 0.24f 0.33c 0.79a 0.28e 0.03f
with water Light roasted 3.06 d
2.81 c
3.15 c
6.46 c
0.02 d
0.24 c
0.25 f
0.25 c
0.18 e
0.55 a
tr
Medium roasted 3.66d 1.50d 1.59d 3.01 0.04c 0.29c 0.11 g 0.10e 0.06 g 0.05d 0.27c
Dark roasted 2.38f 0.30f 0.71 1.27e 0.02d 0.14d 0.06 h 0.07f tr 0.05d tr
f c c b e e b b
Arabica boiled Green 2.91 2.48 3.31 13.38 0.06 0.09 0.36 0.83 1.03 0.80 0.06e
in water Light roasted 1.93 3.04 b
2.92 5.15 0.24 0.47 a
0.40 e
0.16 d
0.09 f
tr 0.16d
b d e d c e d e c
Medium roasted 6.03 1.62 1.82 3.98 0.01 0.23 0.15 0.19 0.15 0.15 tr
Dark roasted 6.53a 1.42d 1.28f 1.93d 0.12a 0.30b 0.21f 0.06f 0.03 h tr 0.05f
Arabica boiled Green 2.41f 3.81b 4.02b 7.71 0.11a 0.09e 0.18f 0.79b 0.66a 0.64a 0.04f
in water under Light roasted 3.35 d
3.60 b
3.48 c
7.13 c
0.06 b
0.26 c
0.23 f
0.42 0.31 c
0.29 e
0.06e
elevated pressure f d d d b d d d g c
Medium roasted 2.75 1.61 1.52 2.73 0.06 0.18 0.09 0.14 0.08 0.10 0.08e
d e i e b d d f h
Dark roasted 3.64 1.05 0.93 1.38 0.06 0.14 0.07 0.06 0.03 tr 0.08e
f j i c f e
Arabica extracted Green 2.65 0.60 0.84 6.06 tr 0.06 0.48 tr tr tr 0.85
with ethanol Light roasted 5.38c 1.05e 1.24f 2.28d 0.02d 0.28c 0.26f 0.18d 0.12f 0.17c 0.10d
Medium roasted 4.13e 0.34e 0.37 g 0.59 h 0.01d 0.15d 0.08d 0.09e 0.05 g tr 0.04f
d i g h b d d e g d
Dark roasted 3.43 0.27 0.30 0.49 0.06 0.14 0.07 0.09 0.06 0.06 tr
Ethanolic extract Green 0.03 tr 0.01 h tr 0.01d tr tr 0.01 g tr tr tr
from the solid Light roasted 0.22 g 0.04 g 0.05 0.09 tr tr 0.02j 0.03 g 0.03 h 0.04d tr
residue remained
Medium roasted 0.06 tr tr 0.01 g tr 0.01 g tr tr tr tr tr
after extraction
h
of Robusta with Dark roasted 0.15 tr tr tr tr 0.01 g tr tr tr tr tr
water under
elevated pressure
Values bearing the same letters for each compound (in the same column) are not statistically diVerent (P > 0.05) from one another
1 caVeine, 2 3-O-caVeoylquinic acid, 3 4-O-caVeoylquinic acid, 4 5-O-caVeoylquinic acid, 5 3-O-feruoylquinic acid, 6 4-O-feruoylquinic acid,
7 5-O-feruoylquinic acid, 8 3,4-dicaVeoylquinic acid, 9 3,5-dicaVeoylquinic acid, 10 4,5-dicaVeoylquinic acid, 11 4,5-caVeoylferuoylquinic acid,
tr traces

123
920
chlorogenic acids were observed in extracts from green
Arabica which were obtained through boiling under normal
pressure. Boiling under elevated pressure apparently
increased the extractability of compounds with higher molec-
ular mass. Presumably these conditions favored their solubi-
lization (as compared to Robusta) and this in turn decreased
relative concentration of chlorogenic acids. The diVerences
between Arabica and Robusta coVee beans could stem from
higher concentration of polysaccharides in the Wrst coVee
beans because the solubility of these polymers increases with
temperature of extraction [31].
Chlorogenic acids are degraded during roasting of
Arabica and Robusta coVee. In the Wrst step of this process
5-CQA is isomerized to 3- and 4-CQA that are observed in
brews of light roasted Arabica and Robusta coVee. They are
also detectable in brews of green coVee beans, but their
concentrations are smaller. Only extracts of green coVee
beans obtained by boiling under elevated pressure con-
tained more 3- and 4-CQA than extracts obtained from light
roasted coVee because of an advanced isomerization of
5-CQA under these conditions. In further steps of roasting
losses of chlorogenic acids are greater and their content in
dark roasted coVee beans was much lower than in medium
roasted coVee.
The diVerence in the total contents of chlorogenic acids
in extracts of green Arabica and Robusta (Table 3) reXects
the diVerence between phenolic acids concentrations in
these beans (Robusta is richer in these compounds) [32,
33]. Concentration of chlorogenic acids in dark roasted
Robusta was 14-fold lesser than in green beans whereas in
Arabica it was about Wvefold lesser. More intensive degra-
dation of chlorogenic acids during roasting of Robusta
coVee beans as compared to Arabica caused that the diVer-
ence in phenolic acids concentration gradually decreased
with a roast degree and extracts of dark roasted Arabica
contained more chlorogenic acids than their counterparts
obtained from Robusta. Also Clarke found that the losses of
chlorogenic acids were larger during roasting of Robusta
coVee beans (relative to Arabica). He postulated that this
diVerence was a result of diVerent humidity of these two
sorts of coVee during roasting, in particular in the Wrst
phases of roasting, and the latter discrepancy was caused by
their diVerent chemical composition.
Concentration of chlorogenic acids in ethanolic extracts
was approximately twice lower than in water extracts
although the overall extraction yields were comparable
(data not shown). It implies that ethanol is the worser than
water as a solvent of chlorogenic acids.
Ethanolic extracts from the solid residue remained after
boiling under elevated pressure contained 0.05–1.31 g
chlorogenic acids £ 100 g¡1 db. and the overall yield of
extraction was relatively low. Thus, the majority of pheno-
lic compounds were extracted during the Wrst process, with
123
Eur Food Res Technol (2009) 228:913–922
water. Because the ethanolic extracts, from material treated
earlier with water, both from green and roasted coVee
beans, contained a very small fraction of chlorogenic acids,
it was diYcult to Wnd any relationships between roasting
and extraction methods and barely detectable concentration
of these acids in ethanol since almost all chlorogenic acids
were removed by boiling in water.
CaVeine concentration
Roasting caused lesser losses of caVeine as compared to
those of chlorogenic acids and there was no correlation
between caVeine content in freeze-dried extracts and the
roast degree of coVee beans. The relative caVeine concentra-
tion was apparently aVected by caused by roasting changes
in concentration and solubility of other substances (Table 4).
The majority of Arabica extracts contained less caVeine than
Robusta extracts with an exception of extracts derived from
medium and dark roasted Arabica by boiling in water.
Brown pigment index
An absorbance at 420 nm was regarded to be equivalent to
concentration of brown pigments generated during roasting,
mainly by Maillard reactions. Compounds measurable at
420 nm were also detected in extracts of green coVee beans
(Fig. 2a, b). They can be generated during drying of beans
after removal of husks. A part of them could be also formed
during extraction and lyophilization of extracts because
coeYcients a* and b* were found to increase due to these
processes (results of CIE L*a*b* analysis of the extracts
were not shown because of the huge number of presented
results, they are available at the authors). Some of the prod-
ucts of Maillard reactions display antioxidant activity.
Their representatives are melanoidins with high molecular
weight and heterocyclic volatile compounds [34–37]. For
extracts obtained by boiling in water from light and dark
roasted Arabica coVee beans this absorbance was almost
the same (1.440 and 1.494, respectively). This implies that
the brown pigments are intensively formed already in the
Wrst step of roasting, which is equivalent to a drop in weight
of 10 g £ 100 g¡1. Brown pigments from light roasted
Arabica were better soluble in water than in ethanol, and
pigments from medium and dark roasted Arabica also were
better soluble in water. The most eYcient method of brown
pigments extraction from roasted Arabica coVee beans was
boiling in water under elevated pressure (absorbance of
1.638 for dark roasted Arabica).
Water extracts of Robusta coVee beans contained similar
amounts of brown pigments, but in contrast to the extracts
of Arabica coVee their concentration continually increased
with the roast degree. Extraction with ethanol was less
eYcient since only absorbance of extracts derived from
Eur Food Res Technol (2009) 228:913–922 921

Fig. 2 a, b Concentration of robusta

brown pigments in extracts of
Arabica and Robusta coVee A
beans: b brewed, l boiled,
b a 1.6
p boiled at elevated pressure, b
e ethanolic extract, r ethanolic b c
extract of the residue after water c
1.4
extraction. Values bearing the
same letters in a and b Wgures
1.2

Absorption at 420 nm
are not statistically diVerent
(P > 0.05) from one another
g 1
f
f e
0.8

0.6

c
0.4
h
dark 0.2
g
tin

middle
s

h
oa

0
r

light
of

r
e

e
re

green
g

p
de

l
b extraction method

arabica
B
1.8
a

b 1.6
b

Absorption at 420 nm
1.4
e
d 1.2
d g
1
g

0.8

h
0.6

0.4

dark
g

0.2
tin

middle
as o

light 0
fr o

r
ee

green e
p
gr

l
de

b extraction method

dark roasted Robusta was comparable to that of the afore-
said water extracts. Concentration of brown pigments in
extracts from Robusta was much stronger aVected by the
roast degree. Medium and dark roasted Robusta coVee
beans contained much more brown pigments than light
roasted ones. These diVerences between roasted beans of
Arabica and Robusta result from their diVerent chemical
composition. Usually green Arabica beans contain more
free amino acids and saccharides, which can combine with
each other (Maillard reactions of condensation). By con-
trast, generation of brown pigments during roasting of
Robusta coVee beans requires hydrolysis of proteins and

123
922
polysaccharides, and oxidation of chlorogenic acids to qui-
nones. These reactions take place during roasting and are
followed by Maillard processes of condensation.
Ethanolic extracts derived from the solid residue after
extraction with water under elevated pressure had the sur-
prisingly high absorbance as compared to ethanolic extracts
derived from ground coVee beans. Many brown substances
are soluble in water and ethanol (as evidenced by the rela-
tively high absorbance of ethanolic extracts) and the rela-
tively eYcient extraction of brown pigments from the solid
byproduct remained after extraction with water can be
explained by changes in cell wall structure caused by boil-
ing under elevated pressure, e.g., partial hydrolysis of poly-
meric components. This in turn, increased the eYciency of
extraction with ethanol.
Conclusion
Presented study provides evidence that application of an
appropriate extraction method brings about eYcient extrac-
tion of antioxidants like chlorogenic acids and caVeine
from green and roasted Arabica and Robusta coVee beans.
The highest concentration of chlorogenic acids was found
in Robusta extracts obtained through boiling under elevated
pressure, while for Arabica the richest in these compounds
were extracts obtained by boiling under atmospheric pres-
sure. Thus, 10-min extraction at elevated temperature did
not cause degradation of these acids. Concentration of
caVeine in extracts was to a lesser extent correlated with an
extraction method and a roast degree, in particular for
Arabica coVee beans since roasting of the latter did not give
rise to caVeine degradation. The most eYcient method of
manufacturing of rich in potential antioxidants coVee
extracts was found to be boiling of green Robusta coVee
beans under elevated pressure followed by lyophilization.
Sensory attributes of the latter preparations can be
improved through addition of extracts of roasted coVee
beans containing more Maillard reaction products. CoVee
extracts with high antioxidants content in small concentra-
tion can be applied as shelf-life increasing additives to
foodstuVs of diVerent sensory proWle.
Acknowledgments The studies were supported by grant number 2
P06 T 060 29 from the State Committee for ScientiWc Researches.
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