Basic Areas | Article
Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
Characterisation of AC1: a naturally
Luciana Benjamim Benatti (
); Maria Bernadete Silvarolla (
); Paulo Mazzafera (
) UNICAMP, Instituto de Biologia, Departamento de Biologia Vegetal, Caixa Postal 6109, 13083-970 Campinas (SP), Brasil.
) Instituto Agronômico (IAC), Centro Análise e Pesquisa Tecnológica do Agronegócio do Café “Alcides Carvalho”, Av. Barão de
Itapura, 1481, 13012-970 Campinas (SP), Brasil.
(*) Corresponding author: email@example.com
Received: Mar. 3, 2012; Accepted: May 23, 2012
We compared the biochemical characteristics of the beans of a naturally decaeinated Arabica coee (AC1) discovered in
2004 with those of the widely grown Brazilian Arabica cultivar “Mundo Novo” (MN). Although we observed dierences during
fruit development, the contents of amino acids, organic acids, chlorogenic acids, soluble sugars and trigonelline were similar
in the ripe fruits of AC1 and MN. AC1 beans accumulated theobromine, and caeine was almost entirely absent. Tests on the
supply of [2-
C] adenine and enzymatic analysis of theobromine synthase and caeine synthase in the endosperm of AC1
conrmed that, as in the leaves, caeine synthesis is blocked during the methylation of theobromine to caeine. The quality
of the nal coee beverage obtained from AC1 was similar to that of MN.
Key words: Coea arabica, caeine, decaeination, beverage quality.
Caracterização de AC1: um café naturalmente descafeinado
Foram comparadas as características bioquímicas das sementes de um cafeeiro Arabica naturalmente descafeinado (AC1),
descoberto em 2004, com aquelas da cultivar Mundo Novo (MN), amplamente cultivada no Brasil. Apesar de terem sido observa-
das diferenças durante o desenvolvimento das sementes, os conteúdos de aminoácidos, ácidos orgânicos, ácidos clorogênicos,
açúcares solúveis e trigonelina foram similares nas sementes de frutos maduros de AC1 e MN. Sementes de AC1 acumularam
teobromina, e a cafeína estava praticamente ausente. Experimentos com o fornecimento de [2-
C] adenina e análises enzimáti-
cas de teobromina sintase e cafeína sintase nas sementes de AC1 conrmaram que, assim como em folhas, a síntese de cafeína
é bloqueada na metilação de teobromina a cafeína. A qualidade nal da bebida de AC1 foi similar a de MN.
Palavras-chave: Coea arabica, cafeína, descafeinado, qualidade de bebida.
Coea arabica was originated in Ethiopia, and the prod-
uct made from the infusion of its roasted beans spread
worldwide as a refreshing drink due mostly to the al-
kaloid caeine. Two Coea species dominate the world
market, C. arabica, which is known as Arabica coee
and represents approximately 70–75% of the world
market, and C. canephora, or Robusta, representing
nearly 25–30% of the market (D etal., 2007; see
also Statistics of Coee Trade at http://www.ico.org/
For people sensitive to caeine, drinking coee
may cause some unwanted eects, including palpita-
tions, gastrointestinal disturbances, anxiety, tremors,
increased blood pressure and insomnia (C etal.,
2012). Due in large part to these symptoms, the de-
caeinated coee market has grown signicantly since
the establishment of the rst decaeination patents
(M et al., 2009). Currently, the only com-
mercially available decaeinated beans are those that
have been articially treated using chemical processes.
e drawback of chemical decaeination methods is
that, along with the removal of caeine, there are also
losses of or changes to important chemical compounds
that contribute to the avour and aroma of the bever-
age (F etal., 2006a,b; T etal., 2006; A
etal., 2008). In an attempt to meet the demands of
customers sensitive to caeine while maintaining the
original quality of a coee product, studies have fo-
cused on the selection and breeding of coee trees
that produce beans with a low caeine content, which
would therefore not require chemical processing meth-
ods (M etal., 2009).
In 1987, the Agronomic Institute of Campinas
established a breeding program to reduce the
144144 Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
L.B. Benatti et al.
caeine content in Arabica coee beans (M and
C, 1992; M et al., 1997; S
etal., 1999; 2000). As part of this program, analysis was
performed on the alkaloids from C. arabica accessions
from Ethiopia and maintained in the germplasm collec-
tion of the IAC (S etal., 2000; 2004). Among
more than 3,000 trees representing 300 accessions, three
plants were discovered whose beans had very low concen-
trations of caeine, and these plants were named AC1,
AC2 and AC3 (S etal., 2004). e AC1 plant
was the most suitable candidate for the genetic transfer-
ral of the “no caeine” trait to cultivars with high pro-
ductivity (M.B. Silvarolla, unpublished data). e mea-
sured caeine content of AC1 beans was 0.76 mg g
eobromine, the immediate precursor of caeine but
which is also involved in caeine catabolism, was found
to accumulate in the leaves of this plant (S etal.,
2004; M etal., 2009). When AC1 leaves were in-
cubated with [2-
C] caeine or [2-
C] adenine (the latter
also a precursor of caeine), caeine was degraded normally,
but the biosynthesis of caeine (1,3,7-trimethylxanthine)
from the methylation of theobromine (3,7-dimethylxan-
thine) was blocked. e activity of the methyltransferase
responsible for this conversion, caeine synthase, was
found to be reduced in AC1 leaves. Unfortunately, the
three naturally decaeinated plants had low productivity,
a typical characteristic of wild plants that hinders large-
scale planting and commercialisation.
e AC1 plant has been used in breeding programs to
transfer the “no caeine” trait to commercial cultivars of C.
arabica, which have high productivity.However, the avail-
able biochemical characterisations of AC1 are preliminary
because they were limited to the biosynthesis of caeine
in the leaves (S etal., 2004). More recently, the
caeine synthase gene was analysed in AC1 fruits (M
etal., 2009). is analysis revealed some complexities of
the regulation of caeine biosynthesis, suggesting that this
pathway is subject to the transcriptional control of caeine
synthase. us, the present study aimed to characterise the
biochemistry of AC1 beans, a knowledge that is missing for
this naturally decaeinated plant.
2. MATERIAL AND METHODS
Plant material and sampling
We compared the AC1 (S etal., 2004) with the
“Mundo Novo” cultivar of Coea Arabica (MN), which is
commercially cultivated in Brazil and has a caeine con-
tent between 1% and 1.2% in the beans. Both cultivars
were grown in the same experimental plot at the Fazenda
Santa Elisa of the Agronomic Institute of Campinas, lo-
cated in Campinas, São Paulo, Brazil. e samples used in
the assays were collected during 2008–2009.
For the analysis of methylxanthines in dierent organs
of the AC1 and MN plants, we collected root fragments,
the rst three leaf pairs (the rst up to 1 cm, the second up
to 5 cm and the third up to 8 cm) and internode samples
(the rst, third and fth internodes). e roots were col-
lected by digging around the plants and harvesting the sec-
ondary and tertiary roots at a depth of 30 cm. Prior to their
analysis, the roots were washed with running water.
Flowers and fruits were also collected for analyses.
Fruits were collected at eight dierent phenological stages,
from fruit at a very young stage (“pinhead” stage) to fruit
that were fully developed and mature (“cherry” stage). At
each stage, the fruits were cut in half; the pericarp, peri-
sperm and endosperm were separated using a scalpel; and
the fresh and dried weights of the respective tissues were
determined. During this procedure, the tissues were kept
on ice and further lyophilised in liquid nitrogen. e dry
weight was determined using the lyophilised material. e
contents of methylxanthine alkaloids, trigonelline, soluble
sugars, amino acids, organic acids, chlorogenic acids and
free phenols were determined from extracts obtained from
the endosperm. e qualitative analysis of amino acid con-
tent was only performed for the last fruit samples collected.
Green fruits (with a liquid endosperm occupying the
entire locule) were collected from AC1 and MN plants
for activity analysis of theobromine synthase (the meth-
ylation of 7-methylxanthine to 3,7-dimethylxanthine,
E.C. 220.127.116.11) and caeine synthase (the methylation
of 3,7-dimethylxanthine to 1,3,7-trimethylxanthine,
E.C.18.104.22.168). Immediately after fruit collection, the
endosperms were separated and placed in liquid nitrogen
and maintained at -80 ºC until further analysis. Green
fruits at the same stage of development were used in the
radioactive tracer experiments with [2-
H] S-adenosyl-methionine (specic activity=14.9 Ci
) was obtained from Perkin-Elmer, Inc., USA, and
C] adenine (283 mCi mmol
) and [2-
(51.2 mCi mmol
) were obtained from GE Healthcare, UK.
e lyophilised tissues were macerated with a mortar and
pestle, and the extractions were performed using 100 mg
of tissue in 5 mL of methanol (70%). e extraction mix-
ture was maintained in a water bath at 50 ºC for 1 h with
occasional stirring. After centrifugation, the extracts were
recovered and stored at -40 ºC. ese extracts were used
for all of the biochemical analyses, except for the analysis of
organic acids, which utilised 100 mg of tissue in 5mLof 4
containing 5mM dithiothreitol. e samples
145Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
Characterisation of decaeinated coee
were stirred on ice for 1 h, and the extracts were centrifuged
at 14,000rpm for 10 min and stored in a freezer at -40 ºC
until they were used for HPLC analysis.
e analysis of caeine, theobromine, trigonelline and
chlorogenic acids was performed using a Shimadzu HPLC
system operating with a diode detector and a C18 reverse
phase column (4.6 mm x 250 mm, 5 µm particles, ACE).
e mobile phases used were 0.5% acetic acid (A) and meth-
anol (B), and the gradient used was as follows: 0–5min at
0 to 5% B, 5–30 min at 50 to 70% B, 30–32min at 70 to
100% B, 32–34 min at 100 to 0% B and 34–44 min main-
tained at 0% B. e ow was 0.8mLmin
. Caeine, theo-
bromine and trigonelline were detected at 272 nm, and chlo-
rogenic acids were detected at 313nm. e injection volume
was 10 µL of sample extract. e concentrations were calcu-
lated using calibration curves obtained from pure standards
(Sigma). Chlorogenic acids were identied from absorption
spectra obtained from the diode detector between 190 nm
and 350 nm, and the concentration was calculated relative to
a calibration curve obtained from the 5-caeoylquinic acid
isomer (Sigma), which is the most abundant chlorogenic
acid isomer in coee (F and D, 2006).
Glucose, fructose and sucrose were analysed using the
Shimadzu HPLC system (with peak tubing) and operat-
ing with electrochemical detector at 400 mV, a Dionex
CarboPac PA1 (4 mm x 50 mm) pre-column and a
Dionex CarboPac PA1 column (4 mm x 250 mm). e
mobile phase was 40 mM NaOH and a 15 min run was
used for each sample. e ow rate was 1.2 mL min
Sugar concentrations were calculated using calibration
curves derived from pure standards (Sigma).
e qualitative analysis of amino acids was performed
using the Shimadzu HPLC system equipped with a man-
ual Rheodyne injector and a uorescence detector operat-
ing at 250 nm (excitation) and 480 nm (emission). e
amino acids were separated in a C18 reverse phase column
(4mmx250mm, 5 µm, Supelco LC-18). e mobile phas-
es used were as follows: (A) 50 mM sodium acetate, 50 mM
, pH 7.25 adjusted with HCl, containing 0.2%
tetrahydrofuran and 0.2% methanol and (B) a mixture con-
taining 65% methanol and 35% water (Jarret etal., 1986).
e following gradient was used: 0–21 min at 25 to 46%
B, 21–26 min at 46 to 48% B, 26–35 min at 48 to 60% B,
35–45 min at 60 to 70% B, 45–49 min at 70 to 100% B,
49–64 min maintained at 100% B and 64–65min at 100
to 25% B. e ow rate was 0.8 mL min
. Derivatization of
the samples was carried out with o-phthalaldehyde (J
etal., 1986). e amino acid concentrations were calculated
using a mixture of 18 amino acids (AAS-18, Sigma) plus
glutamine and asparagine (Sigma) as the reference standard.
e analysis of organic acids was performed using
the Shimadzu HPLC system equipped with a Rheodyne
injector and a diode detector operating at 210 nm. e
substances were separated in an Aminex HPX-87H
column (Bio-Rad). Fifteen microlitres of sample was
applied at a ow rate of 0.6 mL min
. e mobile phase
was 4 mM H
, and the isocratic run lasted 30 min.
Concentrations were calculated using pure reference stan-
dards of oxalic, malic, citric and succinic acids (Sigma).
From the ethanolic extracts, the total free amino acid
content of these extracts was determined using the ninhy-
drin reagent (C and Y, 1954).
Activity of caeine synthase and
e endosperms stored at – 80 °C were macerated in liq-
uid nitrogen using a mortar and pestle, and 1 g of tissue
was combined with 5 mL of 200 mM Na
5 mM EDTA, 10 mM 2ß-mercaptoethanol, 1.5% ascor-
bic acid and 4% polyvinylpolypyrrolidone (K et al.,
1996). After homogenisation, the extract was maintained
on icefor 15 min with occasional agitation and then cen-
trifuged for15min at 30,000 g (4 ºC). e supernatant
was recovered, saturated to 80% with (NH
centrifuged for 15 min at 30,000 g (4 ºC). e recovered
precipitate was dissolved in 200 mM Na
desalted in a PD10-Sephadex G25 column (Amersham),
using the 50 mM Na
buer for protein elution.
e protein concentrations in the desalted extracts were
determined using a “ready-to-use” reagent from Bio-Rad
(B, 1976). e substrates used were 7-methylx-
anthine for the determination of theobromine synthase
activity and paraxanthine for caeine synthase activity. In
a 1.5 mL Eppendorf tube, the following were combined:
0.11 µCi of [
H]-S-adenosyl methionine, 100 µg of protein
and 10µL of substrate at 3.5 mM. e nal volume was
adjusted to 200 µL with 50 mM Na
buer. e re-
action was incubated at 28 ºC for 30 min and stopped with
100µL of 6 N HCl. One millilitre of chloroform was added
to the reactions containing the paraxanthine substrate, and
1mL of a chloroform:isopropyl alcohol (17:3, v/v) mixture
was added to the reactions containing the 7-methylxan-
thine substrate (M etal., 1994b). e Eppendorf
tubes were vortexed for 30 s, and the organic fraction was
collected and transferred to scintillation vials, and then
dried using owing air in a fume hood. Scintillation liquid
(5 mL) was added to the dried contents, and the amount
of radioactivity incorporated into the theobromine or caf-
feine was determined in a scintillation counter for the
isotope for 2 min.
Metabolism of [2-
C] adenine and
e addition of [2-
C] adenine and [2-
C] caeine was
performed in fruits as previously described (M
etal., 1994a). Briey, 0.2 µCi of [2-
C] adenine or 0.2µCi
146146 Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
L.B. Benatti et al.
C] caeine was applied onto a small incision made
at the fruit peduncle. e fruits were placed in a plastic box,
with the stalk positioned upward. Once the [2-
nine or [2-
C] caeine solutions were absorbed, 5x10µL
of 100 mM Na
buer, pH 6, was applied at the
incision area. A cotton ball soaked in water was placed at
the centre of the box to prevent the fruit from drying out.
e box was covered and maintained under white uo-
rescent light (150 µmol photons m
). e fruits were
removed after 24 h ([2-
C] adenine) or 48h ([2-
feine), and the endosperms were separated with a scalpel
and lyophilised. For the extraction procedure, the lyophi-
lised material was ground in a mortar, transferred to a screw
cap tube with 70% methanol (20 mg mL
) and incubated
in a 50ºC water bath for 1 h. e extracts were centri-
fuged for 10 min at 12,000 g, and 500 µL was dried in a
Speed-Vac (Savant). e dried samples were dissolved in
30 µL of water, constantly stirred for 1 h and subjected to
thin layer chromatography on silica sheets GF
A 10 µL volume of an aqueous solution containing 5 µg
of caeine, 5µg of theobromine and 5 µg of theophylline
was applied on to the sample spots. e chromatography
was developed with a chloroform:methanol mixture (9:1,
v/v), and after drying, the spots were visualised under UV
light (254 nm) and circled with a pencil. e reference val-
ues (Rf values) for caeine, theobromine and theophylline
were 0.51, 0.41 and 0.37, respectively. Spots visualized un-
der UV light were scraped from the plate with a spatula and
transferred to scintillation tubes, and 1 mL of methanol
and 5 mL of scintillation liquid were added to each tube.
Radioactivity was determined with a scintillation counter
C isotope for 2 min.
ree replicates were performed for all biochemical mea-
surements. e activity of theobromine synthase and caf-
feine synthase was estimated from ve replicates. Analysis
of variance and post-hoc comparison of means (Tukey’s
test, p≤0.05) were performed using the statistical analysis
program SISVAR (F, 2000). For the analysis of
the metabolism of [2-
C] adenine and [2-
extracts from ve replicates were used.
Although some dierences were observed, the fruits of
AC1 and MN showed similar patterns of development
(Figure 1). However, the mass of AC1 fruit was always
lower than those of MN, and the smaller size of these
fruits was also observed visually.
Methylxanthines in dierent plant organs
An analysis of the caeine content during AC1 and MN
endosperm development showed that AC1 had a higher
total (caeine + theobromine) alkaloid content (Figure2).
However, the AC1 theobromine levels were always similar
to the caeine levels found in MN. e nal concentra-
tion of caeine in MN was 8.59±0.17mg g
. AC1 theo-
bromine contents varied between 6.75 and 13.42 mg g
with the largest amounts present in the immature endo-
sperm. e theobromine content in the AC1 mature en-
dosperm was 6.48±0.48mg g
, and the caeine content
in the mature AC1 endosperm was 0.40±0.02 mg g
Methylxanthines were not detected in the roots of
either plant. In MN owers, the caeine content was
1.06±0.11 mg g
, and in AC1 owers, the caeine
content was 1.04±0.03 mg g
. In both MN and AC1,
the methylxanthine levels decreased with leaf maturity
(Figure 2). Caeine and theobromine were detected in
MN leaves, but in AC1 leaves, only theobromine was
In MN, theobromine was detected only in the rst
internode; in contrast, this methylxanthine was present in
the rst, third and fth internodes of AC1 plants. Caeine
was present in the three internodes analysed in MN, but
it was not detected in any of the AC1 internodes. It was
also observed that the caeine and theobromine contents
decreased sharply from the rst to the third internodes in
In the young endosperm, which did not fully occupy the
locule and had a milky appearance, there were higher
levels of reducing sugars (glucose and fructose) com-
pared with sucrose (Figure 3a,b). As the fruits ripened,
the sucrose content increased, and the reducing sugar
levels decreased. In ripe fruits, the sucrose content was
37.07±5.06 mg g
and 66.2±7.4 mg g
for AC1 and
Oxalic, citric, malic and succinic acids were observed in
MN and AC1 endosperms at dierent stages of fruit de-
velopment (Figure 3c,d). In MN, citric acid was present
at higher levels than the other acids at all development
stages; this was not the case in AC1. Nevertheless, citric
acid was the organic acid with the highest concentration
in mature AC1 endosperms. Quantitative dierences in
the contents of oxalic, succinic and malic acid were ob-
served in MN, and these acids were present at similar
levels in AC1.
147Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
Characterisation of decaeinated coee
Figure 1. Fresh (a,b) and dry (c,d) mass of fruit and fruit tissues of MN (a,c) and AC1 (b,d) coees. Each symbol is the mean value of
three replications. Campinas (SP), Brazil.
Figure 2. Methylxanthine concentrations in the endosperms of MN (a) and AC1 (b) coees, leaves (c) and internodes (d) of AC1 and MN
plants. Each symbol/bar is the mean value of three replications. Campinas (SP), Brazil.
148148 Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
L.B. Benatti et al.
MN had a greater total free amino acid content in the ma-
ture endosperm (34.98±3.28 nmol mg
) compared with
AC1 (25.2±1.47 nmol mg
). e prole of amino acids ob-
tained from the endosperms of mature fruits showed that
asparagine, glutamate, aspartate and alanine were the most
abundant amino acids in both MN and AC1 (Table 1).
In this order, MN and AC1, asparagine was present at
28.69mol% and 33.61 mol%; glutamate at 27.38 mol%
and 22.45mol%; alanine at 14.76 mol% and 7.28 mol%;
and aspartate at 9.50 mol% and 12.90mol%. Glycine and
threonine were detected at levels less than 0.1% in AC1,
which was almost 10 times less than the levels in MN.
In the endosperms 5-caeoylquinic acid (5CQA) was the
predominant chlorogenic acid (CGA) in both MN and
AC1 (Figure 3e), and the levels varied throughout fruit
Table 1. Amino acid prole of the endosperms of AC1 and MN coees
Asn 28.69±0.76 33.61±2.85
Glu 27.58±0.52 22.45±0.57
Ala 14.76±0.40 7.49±0.05
Asp 9.5±0.00 12.9±0.07
Ser 5.55±0.28 4.28±0.68
Gln 3.85±0.01 4.06±0.93
Phe 2.63±0.02 4.02±0.99
Met 2.4±0.06 1.94±0.35
Arg 1.31±0.09 1.40±0.62
Ile 0.71±0.05 2.71±1.48
Lys 0.68±0.02 0.46±0.02
Tyr 0.67±0.04 0.54±0.04
Gaba 0.51±0.17 0.92±0.14
His 0.50±0.03 0.47±0.16
Leu 0.42±0.08 0.77±0.01
Val 0.20±0.08 0.26±0.02
Gly <0.1 1.23±0.78
Thr <0.1 0.98±0.00
Figure 3. Concentrations of soluble sugars (a,b), organic acids (c,d), chlorogenic acids (e) and trigonelline (f) in the endosperms of AC1
and MN coees. Each symbol is the mean value of three replications. Campinas (SP), Brazil.
149Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
Characterisation of decaeinated coee
Table 2. Chlorogenic acid contents of the endosperms from AC1
and MN coees. 5CQA=5-caeoylquinic acid; and numbers 1 to
7 are seven other chlorogenic acids identied based on the UV
absorption spectra obtained in the HPLC detector diode and the
5CQA 53.90±1.59 51.52±1.23
1 4.83±0.16 7.20±0.02
2 7.49±0.51 9.52±0.03
3 1.03±0.14 0.95±0.10
4 5.91±0.10 6.21±0.66
5 1.80±0.06 4.53±0.12
6 4.46±0.07 8.57±0.06
7 1.50±0.40 4.57±0.25
Total 80.92 93.07
development. AC1 always had greater amounts of 5CQA,
with the greatest dierence observed approximately 162
days after owering. When the beans were fully devel-
oped, the levels of 5CQA were similar between AC1 and
MN. Seven other CGAs were detected based on the UV
absorption spectra obtained in the HPLC detector di-
ode, and the chromatographic prole of the CGAs (not
shown) was very similar to that reported by Cliord etal.
(2008). It was not possible to identify these acids due to a
lack of standards, but based on quantications made rela-
tive to 5CQA (Table2), these other CGAs were present
at higher levels in MN.
e amount of trigonelline found in the endosperm of
immature AC1 fruits was greater than that observed in
MN (Figure 3f). However, this dierence decreased
as the fruits ripened. When the fruits were fully devel-
oped,thequantities found in the two plants were similar,
approximately 11 mg g
in the endosperm.
Activity of theobromine and caeine synthase
e activities of both theobromine synthase and caeine
synthase were higher in the endosperm of MN than in
AC1 (Table 3). eobromine synthase activity in AC1
was approximately half of that observed in MN, while the
Table 4. Radioactivity incorporated in caeine, theobromine and theophylline in the endosperms of fruits of AC1 and MN fed with
C] Adenine or [2-
Radioactivity (KBq g
Caeine Theobromine Theophylline
C] Adenine 0.015 0.904 0.035
MN 0.292 0.085 0.054
C] Caeine 0.592 0.140 0.267
MN 0.585 0.186 0.135
Table 3. eobromine synthase and caeine synthase activities in
the endosperms of AC1 and MN coees
[fkat (g protein)
MN Theobromine synthase 100.0±4.2
Caeine synthase 63.2±9.5
AC1 Theobromine synthase 47.7±14.7
Caeine synthase 4.2±2.0
activity of caeine synthase was 15 times lower in decaf-
feinated plants than in MN.
Metabolism of [
C] adenine and [2-
caeine in fruits
C] adenine to AC1 fruits revealed that theo-
bromine was the main methylxanthine labelled with ra-
dioactivity (Table 4); after 24 h of incubation, we detect-
ed 60 times more radioactivity in theobromine than in
caeine. Contrary to what was observed in the decaein-
ated plants, radioactivity accumulated mainly in caeine
in the MN endosperm fed with [2-
C] adenine, although
there was also a small amount of radioactivity present in
theobromine. When [2-
C] caeine was administered,
radioactivity was detected in theophylline, which appears
only in the catabolism of caeine. Radioactivity was also
detected in theobromine, which is both a precursor and a
degradation product of caeine.
Although mature AC1 fruits were smaller than mature
MN fruits, the pattern of AC1 fruit development was
similar to that of MN fruits. During coee fruit devel-
opment, the perisperm is substituted by the endosperm
(M, 1941; DC and M, 2006),
which pattern was similar in both MN and AC1. However,
closer inspection of our data suggests that the AC1 fruits
developed earlier than the MN fruits. We observed that
the replacement of the perisperm by the endosperm oc-
curred more rapidly in AC1. At 100 days after ower-
ing (DAF), the fresh and dry weights of AC1 endosperms
were considerably greater than those of MNendosperms.
On the other hand, the ripe fruits of MN were larger,
with greater values for both the fresh and dry weights.
150150 Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
L.B. Benatti et al.
is dierence may be considered a disadvantage for
AC1 because fruit size is one of the desired physical at-
tributes in quality coees. However, necessarily this is not
related with the beverage quality. For example, the variet-
ies Mokka (C etal., 1991) and Laurina (K
etal., 1954), which have small beans, produce coee of
excellent quality, and the small beans are accepted as in-
trinsic characteristics. Analysis of the beverage produced
from the beans of AC1 indicated that the quality is good,
having obtained the classication of “exotic” in the sen-
sory characterisation (G.S. Giomo, IAC, personal com-
munication). Because the size of the beans, AC1 demands
special care for certain aspects of post-harvest processing,
as well as roasting time and grinding when particle size
and appearance of the nal product is dened.
e analysis in the present study showed that no
methylxanthines were detected in the roots of MN or
AC1 what conrms ndings previously reported for C.
arabica seedlings (Z and A, 2004).
In both AC1 and MN, we observed a reduction in the
leaf caeine content with increased leaf age, which was
previously observed in adult C. arabica plants (H
and W, 1964). We also found that younger inter-
nodes had greater amounts of methylxanthines in both
MN and AC1, with a greater amount of caeine in MN
than in AC1. e endosperm of young MN fruits had
higher caeine content than the endosperm of ripe fruits;
the same pattern was observed for theobromine in AC1
fruits. Both methylxanthines, caeine and theobromine,
exhibited the same distribution patterns when compar-
ing young and old tissues or mature and immature endo-
sperms in AC1 and MN. ese patterns were previously
observed for the caeine content in leaves (A
etal., 1996a,b), fruit (K etal., 2006) and branches
(Z and A, 2004).
Interestingly, we observed that MN and AC1 owers
had similar quantities of caeine. In C. Arabica owers
during the anthesis stage, caeine is the most abundant
purine alkaloid, with 0.58 mg g
in the petals and gynoe-
cium and 1.36 mg g
in the stamens (B, 2006).
e predominance of theobromine over caeine was
detected in all of the AC1 tissues analysed. Additionally,
AC1 leaves incubated with [2-
C] adenine accumulated
radioactivity in theobromine, not caeine, similar to pre-
vious observations in leaves (S et al., 2004).
ese ndings provide evidence that the blockade of caf-
feine biosynthesis occurs at the step when theobromine is
methylated to form caeine.
AC1 beans have signicantly lower amounts of caf-
feine than any wild or cultivated C. arabica tissues investi-
gated thus far (M etal., 2009). New hybrids de-
veloped in Madagascar, from crosses between C. arabica,
C. canephora and C. eugenioides, had 0.37% caeine and
undetectable levels of theobromine; however, insucient
data regarding production were presented to support the
commercial viability of these hybrids (N etal., 2008).
Quantitative PCR analyses showed that the expres-
sion of genes coding for theobromine synthase (CTS2) and
caeine synthase (CCS1) were signicantly reduced in the
decaeinated cultivar AC1 in comparison with a caein-
ated coee variety (M etal., 2009). ese results are
not consistent with our results for the enzymatic activities,
where caeine synthase activity was almost absent and there
was a partial reduction of theobromine synthase activity. A
possible explanation for the reduced enzymatic conversion
of 7-methylxanthine to theobromine is that caeine syn-
thase is a bi-functional enzyme, also mediating the biosyn-
thesis of theobromine from 7-methylxanthine (M
etal., 2003). en, the reduction of theobromine synthase
activity was in part because the lack of expression of caf-
feine synthase gene. e gene expression results are not sup-
ported also by the results of [2-
C] adenine feeding assays
carried out with fruits (this work) and with leaves of AC1
(S et al., 2004), which showed that the block-
ade in the caeine synthesis occurs between theobromine
and caeine. e signicant reduction of theobromine
synthase expression in AC1 (M etal., 2009) might be
explained by the fact that the three methyltransferases of
caeine biosynthesis pathway in coee share high sequence
similarity (M etal., 2003; K and M, 2004;
Y etal., 2006; A etal., 2008), what may
have resulted in a lack of specicity in the primers used in
the expression analysis in AC1 (M etal., 2009).
e pattern of accumulation of soluble sugars in AC1
and MN was similar to that observed in C. arabica cv. Caturra
(R etal., 1999). We observed high glucose and fruc-
tose contents in the endosperm of young fruits, which de-
creased with ripening, while the reverse was observed for the
sucrose content. Although the accumulation patterns were
the same in AC1 and MN, quantitative dierences were ob-
served primarily for the sucrose content. In MN, the sucrose
content in the mature endosperm reached 61 mgg
in AC1, the levels were much lower (37 mgg
sucrose is an important compound that aects the qual-
ity of the nal beverage product (G et al., 2006;
2008), quantitative dierences in the levels of this sugar
are frequently observed between dierent C. arabica culti-
vars. Previous studies have shown that the sucrose content
varies from 46 to 150 mg g
in mature beans (C,
1985a; R etal., 1999; K etal., 2001; C etal.,
2004; F etal., 2006a; M and D, 2006).
In addition to the intrinsic characteristics of each plant
and/or cultivar, one possible explanation for the variation in
sucrose levels in these studies is the exact stage of ripeness
when the beans were collected. Although the red colour of
the fruit may be indicative of maturation, varying intensi-
ties of red colouration may reect biochemical dierences,
such as those observed in fruits collected from shaded plants
(G etal., 2008).
151Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
Characterisation of decaeinated coee
The main criticism of decaffeination methods is
the removal of other substances that are important
for the development of the product and the quality
of the final beverage. T etal. (2006) reported that
whole C.arabica beans had a sucrose concentration
of 96.5mg g
and that the concentration dropped to
38.5 mg g
after decaffeination with dichlorometh-
ane. Thus, the concentration of sucrose found in AC1
coffee beans requires further investigation. Although
the sucrose concentration observed in the present
study is within the concentration range found in dif-
ferent reports on coffee beans, it will be important to
evaluate, over a period of several years, the sucrose
content in AC1 plants grown in different coffee re-
gions and subject to different environmental and cul-
e alkaloid trigonelline, which is largely degraded
during the process of roasting coee, gives rise to com-
pounds that contribute to the aroma and avour of the
coee beverage (C, 1962; A, 2006).
Our results showed that in both studied genotypes,
green fruits that had already developed the endosperm
showed an accumulation of trigonelline similar to the
levels found in mature beans. e levels of trigonelline
observed in AC1 and MN are similar to the levels pre-
viously reported for dierent C. arabica cultivars (K
etal., 2001; C etal., 2004; F etal., 2006a;
K etal., 2006).
In both AC1 and MN plants, citric acid was the
organic acid present at the highest concentration in
the endosperm of ripe fruit, with the highest content
observed in MN. Although the concentration of citric
acid in MN remained relatively constant during devel-
opment, the concentration of citric acid increased in
maturing AC1 plants. e same pattern of increased cit-
ric acid content was also observed in two varieties of C.
arabica (Caturra commercial and Caturra 2308); at full
maturity, the citric acid content in the beans of these
varieties was approximately 15 mg g
(R et al.,
1999), a value similar to that observed in AC1. A
etal. (A etal., 2003) also reported that citric acid
was the most abundant organic acid in a variety of C.
arabica varieties. In the present study, the accumulation
of malic acid was also found to vary during the develop-
ment of the endosperm in MN and AC1. In the Caturra
varieties studied by R etal. (1999), malic acid was
found to be the second most abundant organic acid in
beans, with a content between 4 and 5 mg g
A etal. (2003) reported a malic acid content of
4.14 mg/g. In the present study, the malic acid content
in AC1 was similar to these previously reported values,
and malic acid was the second most abundant acid in this
tissue. In MN, we observed lower malic acid levels than
the levels observed in other varieties of C. arabica. e
endosperm of the Caturra variety exhibited intermediate
oxalate levels compared to the levels observed in AC1
and MN (R etal., 1999).
In both MN and in AC1, the three most abundant
amino acids were asparagine, aspartate and glutamate, a
nding that is consistent with previous work (C,
1985a). Despite this qualitative similarity, MN had sig-
nicantly greater total free amino acid content than AC1.
ese three amino acids have also been observed at high
concentrations in other C. arabica plants. In unroasted
beans of Brazilian C. arabica cv. Typica, the most abun-
dant amino acids were glutamate, aspartate and GABA
(C et al., 2005). e observed dierences in the
levels of amino acids may be due to post-harvest pro-
cessing (A and L, 1996; DC and
Although the levels of 5CQA were similar in the tis-
sues studied, MN had a higher total amount of CGAs
than AC1. Cliord (C, 1985b) listed the con-
tents of CGAs in Arabica coee reported by several au-
thors, which varied between 40.7 and 84.0 mg g
concluded that in part the variation was a consequence of
the analytical method used. Samples of C. arabica from
dierent sources had 5CQA as the major isomer, with the
concentrations ranging from 3.44 to 56.1 mg g
an average concentration of 47.9 mg g
. e total con-
centration of several other CGA isomers was 65.7 mg g
with values ranging from 55.2 to 75.5 mg g
CGA concentrations dier greatly among dierent coee
varieties, and the results available in the literature depend
on the analysis method used (C, 1985b). In the
present study, however, the dierences between MN and
AC1 were not quantitatively large, and the same CGAs
were detected in both genotypes.
Assessments of CGA content during C. arabica fruit
development indicated that the overall content increases
with maturity, with a peak occurring four weeks before
full maturity (C, 1985b). Our analysis did not
match this level of detail, but approximately 30 days be-
fore harvest, CGA levels were close to the maximum and
e maintenance of similar levels of CGAs in AC1
and MN is important because these compounds con-
tribute to the quality of the nal beverage (F and
D, 2006). Larger quantities of caeoylquinic
and feruloylquinic acid isomers are associated with re-
duced coee quality, while larger quantities of dicaf-
feoylquinic acid are related to improved beverage qual-
ity (F et al., 2006b). e articial decaeination
processes, including water decaeination (F et al.,
2006b) and dichloromethane decaeination (F
etal., 2006b; T etal., 2006), aect the amount and
proportion of CGAs in Arabica coee. In this regard,
the AC1 cultivar has the advantage of maintaining these
compounds at levels similar to those found in commonly
consumed and appreciated coees.
152152 Bragantia, Campinas, v. 71, n. 2, p.143-154, 2012
L.B. Benatti et al.
In light of consumer interest in a caeine-free product
that maintains the characteristics of a good quality cof-
fee, AC1 has great potential to satisfy this consumer de-
mand. Despite some dierences described in the present
study, the beans of AC1 and MN have a similar chemi-
cal composition, and the latter is a commercially ex-
ploited variety that is widely consumed as a good quality
coee. Some of the observed dierences may result not
only from the genetic backgrounds of the plants but also
from environmental and cultivation factors, as previous-
ly demonstrated by reports on the chemical composition
of coee beans (C, 1985a). Another advantage
of AC1 coee pertains to the fact that decaeination
with dichloromethane can leave solvent residues in de-
caeinated products (C et al., 1980; P and
C, 1984). Although the levels of these sol-
vent residues are not considered harmful (M
and C, 1991), the consumer may prefer not to
consume such a product.
is work was supported by Financiadora de Estudos e
Projetos (FINEP-Brazil), Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP) and Consórcio
Brasileiro de Pesquisa e Desenvolvimento do Café. L.B.B.
is supported by a doctoral fellowship from CAPES, and
P.M. is supported by a research fellowship from CNPq.
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