Food Chemistry 221 (2017) 1874–1882

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

Proteomic analysis of coffee grains exposed to different drying process

Kalynka Gabriella do Livramento a, Flávio Meira Borém a, Anderson Cleiton José a, Agenor Valadares Santos b,
Darlan Einstein do Livramento c, José Donizeti Alves a, Luciano Vilela Paiva a,⇑
a
Federal University of Lavras, CEP 37200-000 Lavras, MG, Brazil
b
Federal University of Para, Belém, PA, Brazil
c
State University of Minas Gerais, Passos, MG, Brazil

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

Article history: Many biochemical events occur inside grains during post-harvest processes. Several methods have been
Received 29 June 2015 developed to relate the chemical composition of the coffee grain to the beverage quality, including iden-
Received in revised form 13 October 2016 tification of possible molecular markers for flavor characterizing. This study was aimed at evaluating the
Accepted 16 October 2016
changes in the proteomic profile of pulped and natural C. arabica grains dried in a yard or dryer at 60 °C. It
Available online 18 October 2016
was observed that fruits dried in a dryer at 60 °C showed an altered proteomic profile, with a reduction in
the most abundant proteins compared to those yard-dried grains. Among the identified proteins, those
Chemical compounds studied in this article:
involved in the metabolism of sugars and stress response were highlighted. Results have shown that
Dithiothreitol (Pubmed CID: 19001)
Iodoacetamide (Pubmed CID: 3727)
post-harvest processes that impact coffee quality are related to changes in protein abundance, indicating
Coomassie blue G250 (Pubmed CID: that proteomic analysis may be effective in the identification of biochemical changes in coffee grains sub-
6333920) jected to different post-harvest processes.
Ammonium sulfate (Pubmed CID: 6097028) Ó 2016 Elsevier Ltd. All rights reserved.
Acetonitrile (Pubmed CID: 6342)
Ammonium bicarbonate (Pubmed CID:
14013)
a-Cyano-4-hydroxycinnamic acid (Pubmed
CID: 5328791)
Urea (Pubmed CID: 1176)
Thiourea (Pubmed CID: 2723790)

Keywords:
Coffea arabica
Two-dimensional electrophoresis
Beverage quality
Proteome

1. Introduction The beverage quality of coffee can be attributed to the species,

In 2013, Brazilian coffee production reached 43.24 million 60 kg
bags of processed product, and C. arabica production represents
75% of the total (CONAB, 2016). To improve production further,
Brazil has adopted new technologies for coffee production and pro-
cessing as well as investing in research to improve the quality of
the national product by generating special beverages with a unique
flavor and aroma (BSCA, 2014).
⇑ Corresponding author.
E-mail addresses: kalynkagabriella@yahoo.com.br (K.G. Livramento), flavioborem@
deg.ufla.br (F.M. Borém), acjose@dcf.ufla.br (A.C. José), agenorvaladares@yahoo.
com.br (A.V. Santos), delivramento@yahoo.com.br (D.E. Livramento), jdalves@dbi.
ufla.br (J.D. Alves), luciano@dqi.ufla.br (L.V. Paiva).
the cultivar, the environment and pre- or post-harvest factors.
Among the post-harvest processes, drying is the second most
important step because when optimally performed, it produces
coffee with superior aroma and flavor. Both mechanical (on drier)
and yard drying can effect the sensory quality of coffee, provided
that management techniques are monitored to minimize undesir-
able characteristics (Borém, 2014) and to avoid damage to the
enzymes and proteins that are essential for the preservation of fla-
vor and aroma (Taveira, Rosa, Borém, Giomo, & Saath, 2012).
Considering the variety and complexity of physiological mech-
anisms involved with the beverage quality of coffee, research is
needed to identify and characterize metabolic routes and/or mole-
cules that contribute to these biochemical changes, thus allowing

http://dx.doi.org/10.1016/j.foodchem.2016.10.069
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
 K.G.d. Livramento et al. / Food Chemistry 221 (2017) 1874–1882
the development of further studies that corroborate the signifi-
cance of such mechanisms.
A promising study that might elucidate the reason for differ-
ences in coffee beverage quality is the analysis of the total protein
profile expressed by the organism under determined physiological
conditions, also called the proteome.
The objective of the present work was to analyze the proteomic
profile and identify differentially abundant proteins in Coffea ara-
bica L. grains subjected to two different drying conditions.
2. Materials and methods
2.1. Plant material
The samples of coffee grains (Coffea arabica L.) cv. Topazio MG
1190 were provided by the Bom Jardim farm, located in the munic-
ipality of Bom Sucesso (21°060 5000 S, 44°490 22,3500 W), state of Minas
Gerais, from a plot at 1100 m altitude. Fruits were collected at the
end of maturation (bright yellow exocarp and fleshy mesocarp).
According to Köppen’s classification map, the climate is Cwa,
rainy (mesothermal) with dry winter and rainy summer, the hot-
test month reaches temperature higher than 22 °C, average annual
rainfall around 1530 mm.
2.2. Berry harvest and processing
Samples were processed using dry and wet process and dried at
11% (w.b.) according to methods established by Borém, 2014.
For each repetition, 800 L of coffee fruits were harvested. All the
raw material was standardized through hydraulic separation and
manual selection, where only denser density fruits were chosen.
Finally, the immature and floating fruits were manually removed.
Then, about 150 L of cherry coffee were taken directly to the
yard, thus constituting the natural coffee (dry process).
The pulped coffee samples (350 L) were obtained through the
wet method: the pulp was eliminated by a pulper and about
150 L of coffee with parchment were placed in a fermentation tank,
where they remained for a period of 20 h. Then, coffee was washed
(six times) until pulp was completely removed. Finally, the pulped
coffee grains were set on screens to be dried in the sun. Drying
started immediately after the processing (Borém, 2014). The coffee
grains were taken to the suspended drying beds. The suspended
beds were wooden frames (1 m  1 m) with net made of polyethy-
lene yarn on the bottom.
Natural and pulped coffee were divided into different plots in
the yard. Natural coffee was kept for two days and pulped coffee
for one day in the yard, in order to enable fruits undergo mechan-
ical drying at the same temperature and relative humidity condi-
tions. The other portions remained in the yard for total drying
under the sun (Borém, 2014).
During the time that the coffee remained in the yard, it was
mixed every half hour. At this time temperature and relative
humidity of grain mass were evaluated using a thermo-
hygrometer (Borém, 2014).
Mechanical drying was conducted in two fixed bed dryers. Both
natural and pulped coffee were placed in dryer adjusted to 60 °C.
During hot air drying, coffee mass temperature as well as environ-
mental air’s relative humidity were periodically monitored. Coffee
mass temperature was measured every 30 min using temperature
sensor Type J Thermocouple placed in the mass’s center in each divi-
sion of the drying chamber. To minimize a possible temperature
difference between the four divisions, due to the position of resis-
tance in the plenum, samples were rotated every hour (Borém,
2014).
1875
After drying, seeds were immediately frozen in liquid nitrogen
and kept under -80 °C. The flowchart below shows the coffee pro-
cessing and drying (Fig. 1).
2.3. Protein extraction
Total protein extracts were prepared from dried coffee grains
following the method described by Gallardo et al., 2002. After
grinding of grains using mortar and pestle in liquid nitrogen,
approximately 200–300 mg powder were used for extraction at
4 °C in 1.2 mL of lysis buffer (Harder et al., 1999), containing 7 M
urea and 2 M thiourea, 4% (w/v) CHAPS. This extraction buffer also
contained 18 mM Tris-HCl, 14 mM Trizma base, protease inhibitor
cocktail (GE Healthcare), 53 units m L1 DNase I (Sigma),
4.9 Kunitz units m L1 RNase A (Sigma), and 0.2% (v/v) Triton X-
100. After 10 min at 4 °C, 14 mM dithiothreitol (DTT) was added
and the protein extracts were stirred for 20 min at 4 °C and were
then centrifuged (35,000g for 10 min) at 4 °C. The supernatant
was submitted to a second clarifying centrifugation as above. The
final supernatant corresponded to the total protein extract.
(Gallardo et al., 2002). Protein concentrations in the various
extracts were measured according to Bradford (1976), by spec-
trophotometry, using the GeneQuant unit (Amersham HealthCare)
and bovine serum albumin as standard.
2.4. Two-dimensional gel electrophoresis (2-DE)
The Multiphor II (GE Healthcare) system was used for two-
dimensional electrophoresis following manufacturer’s instructions
(GE Healthcare Handbook, 2005). Two hundred and fifty
microgram of proteins were separated using gel strips forming
an immobilized linear pH gradient from 3 to 10 (Immobiline
DryStrip, pH 3–10, 18 cm; GE Healthcare). Strips were rehydrated
for 14 h at 22 °C with the thiourea/urea lysis buffer containing
2% (v/v) Triton X-100, 20 mm DTT and the protein extracts (rehy-
dration loading) (GE Healthcare Handbook, 2005).
Isoelectrofocusing was performed at 22 °C in the Multiphor II
(GE Healthcare) under the following conditions: a total of
55,000 V h at 15 °C; Gradient 150 V for 1 min; Gradient 150 V for
3 h; Gradient 500 V for 3 h; Gradient 3500 V for 5 h; Gradient
3500 V for 12 h; maximum current setting of 50 lA per strip. After
IEF, IPG strip were equilibrated for 20 min on a buffer (100 mM
Tris-HCl pH 8.8,6 M urea, 2% SDS, 30% glycerol, and 0.2 mg/mL bro-
mophenolblue) containing 5 mg/mL DTT (to reduce proteins), fol-
lowed by another 20 min equilibration in the same buffer, but
containing 25 mg/mL iodoacetamide (to alkylate proteins) instead
of DTT. The second dimension SDS-PAGE was performed at 15 °C
with 12% resolving gels (GE Healthcare) using the Multiphor II
apparatus (GE Healthcare), under the following conditions:
Fig. 1. Flowchart of different drying and processing methods for coffee grains.
1876 K.G.d. Livramento et al. / Food Chemistry 221 (2017) 1874–1882
Step-n-hold 600 V for 30 min; 600 V for 2 h (program as ‘‘step and
hold”), or until the bromophenol blue dye front had run off the gel.
The high molecular weight for SDS electrophoresis (LMW-SDS
Marker Kit – GE Healthcare) was used for molecular mass determi-
nations (GE Healthcare Handbook, 2005).
After electrophoresis, gels were stained with Coomassie blue G-
250 solution for 48 h and stored in ammonium sulfate 20% (p/v)
solution, according to Neuhoff, Arold, Taube, & Ehrhardt, 1988.
Stained two-dimensional gels were digitalized at high resolu-
tion using a scanner (ImageScanner, GE-Healthcare) and processed
inthe software Ulmax MagicScan 4.6. Image analysis was performed
using the software ImageMaster 2D Platinum 5.0 (GE-Healthcare). In
order to evaluate the differences in protein abundance across dis-
tinct 2-DE gels, it was used as parameter the normalized protein
spot volumes (spot area multiplied by its intensity, measured by
optical density). The relative spot volume (%Vol) represents the
ratio of a given spot volume (Vols.)o the sum of all spot volumes
 
detected in the gel with ‘‘n” spots %Vol ¼ Pn Vol  100 . Pro-
ðVols Þ
S¼1
tein spots with significant and reproducible changes were consid-
ered to be differentially expressed proteins. The normalized
volumes of the spots from replicate gels were subjected to stu-
dent’s ANOVA test (P < 0.05) and only statistically significant data,
with a difference of at least 1.7 between treatments were
considered.
2.5. Protein identification by MALDI-TOF MS/MS
The protein points with differential abundance were excised,
destained with 50% acetonitrile and 25 mM ammonium bicarbon-
ate, pH 8.0, dehydrated with 100% acetonitrile, dried in a SpeedVac
and finally treated with the enzyme trypsin (PromegaÒ) at 37 °C for
16–24 h. The peptides resulting from sample trypsinization were
desalted by micro-purification with ZiTipsÒ C18 (Millipore),
according to the manufacturer’s instructions, and were analyzed
by mass spectrometry.
The spectrometric analyses were performed by matrix-assisted
laser desorption-time of flight-tandem mass spectrometry
(MALDI-TOF-TOF MS) (Autoflex III Smartbeam, Bruker Daltonics,
Germany). The analysis was performed by applying 0.5 lL of sam-
ple and 0.5 lL of matrix a-cyano-4-hydroxycinnamic acid (CHCA)
in a Maldi Target Plate (MTP) AnchorChip 800/384.
The spectra generated by mass spectrometry (MS) and tandem
mass spectrometry (MS/MS) were obtained in the positive mode.
The operation mode for acquisition was linear, with a laser repeti-
tion rate of 50 Hz, ion source voltage 1 at 20 kV, ion source voltage
2 at 18.3 kV, ion source lens voltage 6.75 kV and number of shots of
200. The collision-induced dissociation (CID) was used to fragment
trypsinized peptides in the peptide mass fingerprinting (PMF).
The mass spectrometry results were obtained as the reason
mass/charge for each sample using the software FlexControl 3.0.
The analyses of the acquired datasets were performed with the
aid of FlexAnalysis 3.0 and Biotools 3.0 software. The apparatus
was calibrated with external standards (Protein Calibration Stan-
dard I and IV – Bruker Daltonics).
The molecular mass profiles of the samples obtained by MS and
MS/MS were subjected to comparative analysis using the software
MASCOT to compare the masses of the observed peptides with a
database and consequently identify the most probable proteins
in the sample. The databases used in the analysis were from Brazil-
ian Coffee Genome database (Vieira, Andrade, et al., 2006), NCBI
(http://www.ncbi.nlm.nih.gov/) and Coffee Genome Hub
(Dereeper et al., 2014).
3. Results and discussion
Extracting the proteins with urea/thiourea buffer was sufficient
to extract a final mean concentration of 15.3 mg/mL of total
protein.
The two-dimensional profile corresponding to pulped coffee
dried in the yard (Fig. 2B) had higher protein content (average of
360 spots), followed by natural coffee dried in the yard (Fig. 2D),
with an average of 252 spots. The other samples consisted of
pulped (Fig. 2A) and natural coffee (Fig. 2C) that were dried at
60 °C in a dryer, exhibited the lowest proteins content (spots), with
average of 190 and 210, respectively.
The first comparative proteomic evaluation corresponding to
pulped grains dried in a dryer compared to pulped grains dried
in the yard detected 18 proteins of different abundance ranging
from 1.7 to 14-fold difference, all of which were selected based
on the t test (p < 0.05). Most of the proteins were more highly
abundant in pulped grains dried in the yard. After sequencing the
18 proteins and comparing them to the database, 10 were identi-
fied and characterized as the storage proteins 11S Globulin,
glyceraldehyde-3-phosphate dehydrogenase and proteins present
in late embryogenesis – LEA (Table 1).
The second analysis was performed to compare the grains of
natural coffee dried in a dryer to those dried in yards. Twenty-
two proteins were determined as being significantly differently
abundant. Twenty-one of them were more abundant when the
drying occurred in the yard, whereas only 1 protein was more
abundant when natural grains were dried in a dryer. All 22 pro-
teins were sequenced, and 17 of them were identified by homology
with proteins in the database, such as 11S globulin,
glyceraldehyde-3-phosphate dehydrogenase, dehydrin and others
(Table 1).
In both analyses performed in the present work, the decreased
abundance of differentially abundant proteins in the extracts of
grains dried in the dryer compared to those dried in yards is prob-
ably due to the contrasting temperatures of drying, which clearly
caused a difference in the observed protein profiles (Fig. 2). A pos-
sible explanation is that grains dried in dryers at 60 °C have more
strongly affected structures, and the high rate of protein/enzyme
degradation or even a reduction of expression during this drying
process causes these differences in protein abundance.
It is known that the control of drying temperature is very
important to avoid internal damage to the grain, particularly once
high temperatures degrade the internal membranes, exposing oils
and other cellular components to oxygen (Borém, Reinato, Silva, &
Faria, 2006). A reduction in cell volume and the agglomeration of
cytoplasm components was also observed, increasing the molecu-
lar interactions that might cause the denaturation of proteins and
membrane fusion (Hoekstra, Golovina, & Buitink, 2001). Thus, it is
possible to infer that during drying, both the presence of different
proteins and the variation in the total amount of proteins might
generate variable qualities for the beverage because they tend to
contain different amino groups, peptides or free amino acids.
The sensorial quality of the beverage is likely related to the
amount of free amino acids (Selmar, Bytof, & Knopp, 2008) because
proteins and free amino acids together with reducer sugars present
in the grains engage in the Maillard reaction, which is responsible
for the aroma and flavor of the beverage after the process of coffee
roasting (Reineccius, 1995).
After the spectrometric analysis, different isoforms of 11S Glob-
ulin were identified in pulped and natural grains (Table 1); 11S
Globulin is one of the storage proteins most frequently found in
mature grains, eventually constituting 50% of the total proteins
(Marraccini, Deshayes, Petiard, & Rogers, 1999).
 K.G.d. Livramento et al. / Food Chemistry 221 (2017) 1874–1882
Fig. 2. Proteome maps of pulped grains dried at 60 °C in a dryer (A) or dried in a yard (C) and natural grains dried at 60 °C in a dryer (B) or dried in a yard (D). Proteins were
detected with Coomassie Blue G-250 protein gel stain.
Under reducing conditions, the mature precursor of 11S Globu-
lin form generates one high weight subunit (the a-component,
32 kDa) and a low molecular weight subunit (the b-component,
22 kDa) (Rogers et al., 1999). Each of these subunit generates dif-
ferent isoforms, which explains the occurrence of many 11S pro-
teins identified in this study.
Such proteins are deposited in the embryo during seed develop-
ment, and their functions include regulating the nitrogen supply
for seedling development and reuptake of nitrogen during embryo
development in the form of free amino acids (Silveira, Balbuena, &
Santa-Catarina, 2004).
During the procedure of post harvest treatments, many process
occurred within coffee seeds. Seed germination is initiated during
processing and, especially whilst drying (Selmar & Bytof, 2006).
This suggests that 11S protein decline during drying process at
60 °C caused reserve mobilization where both germination and
degradation can be triggered, once high temperatures disintegrate
the internal membranes and expose the cell contents to oxidative
processes.
A recent study revealed a relationship between the expression
of storage proteins and some proteinases (cysteine and aspartic
protease), which may result in the release of free amino acids, thus
influencing the quality of the coffee (Andrade et al., 2011).
In this way, it is supposed that the high concentration of Glob-
ulin 11S in the coffee grains (natural and pulped) dried in yards
might generate more free amino acids due to the action of specific
enzymes that may contribute to beverage quality.
Another abundant enzyme found in the grains of natural coffee
dried in yards was Uridyltransferase UPT-glucose-phosphate, also
known as UDP-glucose pyrophosphorylase (UGPase or UDPGP).
Previous research has shown a positive correlation between the
level of saccharose and the activity of UGPase in potato tubers at
low temperatures (Spychalla, Scheffl, Sowokinos, & Bevan, 1994).
Corroborating this result, a reduction was observed in the saccha-
rose content when the activity of UGPase was reduced approxi-
mately 30% in the tubers of transgenic potatoes (Borovkov,
Mcclean, Sowokinos, Ruud, & Secor, 1996).
It is important to highlight the existence of a relationship
between the accumulation of sugars and beverage quality because
together with amino acids and proteins, they are correlated with
several volatile components in roasted coffee (Bertrand et al.,
2006; Reineccius, 1995). Thus, the presence of UGPase might
increase the sugar content of the grains, contributing to the gener-
ation of flavor and aroma in the coffee.
1877
However, more studies are needed in order to unravel the rela-
tionship of this enzyme, sugars levels, post-harvest processes and
beverage quality.
Isoforms of the protein glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) were also identified, and they were found at
higher levels in grains dried in the yards in both analyses (Table 1).
The induction of GAPDH genes coincides with the morphologi-
cal alteration of mitochondria, as studied in mammals, barley and
soy root cells. The expression of this glycolytic gene, one of the key
enzymes in the glycolytic pathway, is necessary for the survival of
several organisms when mitochondrial functions are affected by
hydric stress, flooding or even reduced oxygen levels (Kosová, Ví
támvása, Prásil, & Renaut, 2011).
In the present work, most of the GAPDH presence can likely be
attributed to protecting the cells from the environmental thermal
stress during the drying of grains in the yard. In addition to GAPDH,
we identified two other proteins that might help in the process of
desiccation tolerance: the proteins LEA and dehydrin DH1a (pro-
tein LEA type II) were more abundant in the grains of natural coffee
dried in yards (Table 1).
The LEA proteins are highly hydrophilic molecules and act as
osmoprotectors of high molecular weight. They are present in
plants and microorganisms, and increased expression is related
to thermal stress, hydric deficit and salinity, thus providing a rela-
tive tolerance to cell dehydration. Furthermore, they are strongly
induced in conditions in which the organism requires the mini-
mization of the negative effects of an oxidative process (Mowla
et al., 2006; Kosová, Vítámvás, Prásil & Renaut, 2011).
Dehydrins are thought to avoid the aggregation of proteins sen-
sitive to dehydration and to stabilize large-scale hydrophobic
interactions such as those found in membranes and in hydrophobic
proteins (Tunnacliffe & Wise, 2007). However, the presence of LEA
proteins is not sufficient to guarantee tolerance to desiccation.
These proteins usually act synergistically with saccharose to form
a viscous cytoplasmic matrix, thus ensuring cellular stability in
an organism tolerant to desiccation (Berjak & Pammenter, 2007).
Previous studies showed that there is a higher content of redu-
cer sugars in the grains of coffee dried at low temperatures com-
pared to those dried in dryers at high temperatures (Afonso,
Corrêa, Goneli, & da Silva, 2004; Borém et al., 2006). Considering
this information, it is possible that the presence of LEA proteins,
together with larger amounts of saccharose in grains dried in
yards, might minimize the damage caused by the rupture of cell
membranes during the drying process, avoiding the interference
 1878
Table 1
Differentially expressed proteins identified by mass spectrometry analysis of Coffea arabica L. grains after drying in either a yard or a dryer at 60 °C.

Spot Matched Organism Name/function % Trend of regulation – (Up-regulated in) – Accession Sequence Exp/Tha Mr
number Identity affected by s.d. code
Analysis 01 – Proteomic changes in Coffea arabica L. pulped grains after drying either a yard or a dryer at 60 °C
50 C. canephora 11S Globulin 89.32 gi|2979526| K.IPILSSLQLSAER.G 1425.9062/1425.8191
K.LSENIGLPQEADVFNPR.A 1897.9798/1897.9534

57 C. canephora 11S Globulin 89.32 gi|2979526| K.LSENIGLPQEADVFNPR.A 1897.8501/1897.9534

85 C. canephora 11S Globulin 89.32 gi|2979526| K.IPILSSLQLSAER.G 1425.7947/1425.8191

K.LSENIGLPQEADVFNPR.A 1897.9030/1897.9534
R.GFLYSNAIFAPHWNINAHSALYVIR.G 2873.3892 /2873.4765

K.G.d. Livramento et al. / Food Chemistry 221 (2017) 1874–1882

R.FPSEAGLTEFWDSNNPEFGCAGVEFER.N 3091.2821/3091.3294
154 C. canephora Glyceraldehyde 3-phosphate dehydrogenase 100.0 gi|120666| K.YDTVHGQWK.H 1132.4/1132.5

158 C. canephora Trichome birefringence-like 63.1 gi|661898767| K.GMDALVFNTWHWWLHR.G 2067.9819/2067.9890

161 C. arabica Alpha galactosidase precursor – gi|158934007| K.VFFQGISPSHYDGK.E 1580.5294/1580.7624

267 C. canephora Dehydrin DH1a 100.0 gi|84314116| K.SQYDEYGNPVR.Q 1326.8196/1326.5840

331 C. canephora Heat Shock cognate 70 kDa 91.42 Cc_g12670 K.SSVHEIVLVGGSTR.I 1439.8099/1439.7733

357 C. canephora Major allergen Pru ar1 100.0 gi|661886094| K.AVEAYLHANPTAYN.- 1532.7078/1532.7259
M.APVTSSYEVTCSIPPAR.L 1833.8510/1833.8931
Table 1 (continued)

Spot Matched Organism Name/function % Trend of regulation – (Up-regulated in) – Accession Sequence Exp/Tha Mr
number Identity affected by s.d. code
361 C. canephora Heat Shock cognate 70 kDa 91.42 Cc_g12670 K.SSVHEIVLVGGSTR.I 1439.7682/1439.7733

Analysis 02 – Proteomic changes in Coffea arabica L. natural grains after drying either a yard or a dryer at 60 °C
1260 C. canephora 11S Globulin 89.32 gi|2979526| K.IPILSSLQLSAER.G 1425.9062/1425.8191
K.LSENIGLPQEADVFNPR.A 1897.9798/1897.9534

1303 C. canephora UTP-glucose-1-phosphate uridylyltransferase 88.39 gi|661894692| K.SNIEIHTFNQSQYPR.V 1832.9932/1832.8805

K.G.d. Livramento et al. / Food Chemistry 221 (2017) 1874–1882

1312 C. canephora 11S Globulin 89.32 gi|2979526| K.IPILSSLQLSAER.G 1425.7947/1425.8191
K.LSENIGLPQEADVFNPR.A 1897.9030/1897.9534
R.GFLYSNAIFAPHWNINAHSALYVIR.G 2873.3892 /2873.4765
R.FPSEAGLTEFWDSNNPEFGCAGVEFER.N 3091.2821/3091.3294

1314 C. canephora 11S Globulin 89.32 gi|2979526| K.LSENIGLPQEADVFNPR.A 1897.9119/1897.9534

1405 C. canephora Glyceraldehyde 3-phosphate dehydrogenase 85.93 gi|661883092 R.VPTVDVSVVDLTVR.L 1497.9519 /1497.8403
K.LVAWYDNEVGYSTR.V 1671.8935 /1671.7893
K.GVLGYTEEDVVSSDFIGDSR.S 2144.0737 /2143.9910

1417 Coffea arabica Alpha galactosidase precursor – gi|158934007 K.VFFQGISPSHYDGK.E 1580.5294/1580.7624

1419 C. canephora Glyceraldehyde 3 phosphate dehydrogenase 85.93 gi|661883092 K.LVAWYDNEVGYSTR.V 1671.8264/1671.7893

K.GVLGYTEEDVVSSDFIGDSR.S 2143.9737/2143.9910

(continued on next page)

1879
 1880
Table 1 (continued)

Spot Matched Organism Name/function % Trend of regulation – (Up-regulated in) – Accession Sequence Exp/Tha Mr
number Identity affected by s.d. code
1422 C. canephora Glyceraldehyde 3-phosphate dehydrogenase 100.0 gi|120666| K.YDTVHGQWK.H 1132.4/1132.5

1424 C. arabica Alpha galactosidase precursor – gi|158934007 K.YGRPDKQYLK.Y K.YGRPDKQYLK.Y

K.YAWKPDSCNLPR.F K.YAWKPDSCNLPR.F

1456 C. canephora Putative RmlC-like cupins superfamily protein 100.00 gi|661894374 R.IYAIFGNAGEDLR.E 1437.5839/1437.7252
K.VPEEVIEEITSGR.K 1456.6104/1456.7409
R.QPIISTGFGEVSGVR.V 1545.6629/1545.8151
R.LGSGSVFFIQSDVGLER.Q 1809.7581/1809.9261

K.G.d. Livramento et al. / Food Chemistry 221 (2017) 1874–1882

1482 C. canephora Late embryogenesis abundant 100.0 gi|661884049 R.VIAEAVGGQVVGGYATR.G 1645.7513/1645.8788

1540 C. canephora Dehydrin DH1a 100.0 gi|84314116 K.SQYDEYGNPVR.Q 1326.8196/1326.5840

1551 C. canephora Dehydrin DH1a 100.0 gi|84314116 R.QTDEYGNPAR.H 1149.6509/1149.505

K.SQYDEYGNPVR.Q 1327.7559/1326.7486

1565 C. canephora 11S Globulin 100.00 gi|661897526 K.LSENIGLPQEADVFNPR.A 1898.0508/1897.9534

1603 C. canephora Late embryogenesis abundant protein 100.00 gi|661899519| T.IGGLQQTGEQV.K 1358.78/1358.8
F.DVIEGN.E 2210.89/2210.9
 K.G.d. Livramento et al. / Food Chemistry 221 (2017) 1874–1882 1881

of hydrolytic and oxidative enzymes with previously compartmen-

1439.7682/1439.7733
1834.0683/1833.8931 talized biochemical components, thus affecting the color, flavor
and aroma of the beverage.
In the grains of coffee dried in the yard (pulped and natural), the
Exp/Tha Mr

precursor protein of alpha-galactosidase was also more abundant,

which is one of the three main enzymes involved in the modifica-
tion and degradation of galactomannans in the cell wall.
Analysis of two-dimensional electrophoresis of coffees showed
the accumulation of alpha-galactosidase during the maturation of
grains (Marraccini et al., 2005). More recent work has also demon-
strated increased expression of this enzyme during the ripening of
papaya, suggesting that it may be responsible for changes in the
texture of the fruit due to modifications on the cell wall
M.APVTSSYEVTCSIPPAR.L

(Nogueira et al., 2012).

K.SSVHEIVLVGGSTR.I

For grains of natural coffee dried in the yard, we found a protein

homologous to the Putative rMLC-like Cupin superfamily protein.
Theses proteins are found in a wide range of cell types and have
several biochemical functions including various enzymatic activi-
Sequence

ties related to cell wall synthesis, particularly reactions involving

sugar modifications. In higher plants this may be associated with
desiccation processes tolerance (Bäumlein, Braun, Kakhovskaya,
gi|661886094

& Shutov, 1995; Dunwell, 1998).

Cc_g12670
Accession

In our study, the presence of this protein can apparently be

associated with the response to stress caused by yard drying.
code

And like LEA proteins, dehydrin and alpha-galactosidase can con-

tribute to the integrity of cell membranes and subsequently to
Trend of regulation – (Up-regulated in) –

the conservation of metabolites responsible for coffee beverage

quality.
Theoretical (Th) and experimental (Exp) molecular weight (Mr) of identified proteins.PC Pulped grains and NC natural grains.

Another protein involved in the modification of the cell wall is

Trichome birefringence like, which was found more abundant in
the grains of pulped coffee dried in the yard. In Arabidopsis this
family includes 46 members (TBR, TBL1-45) and is proposed to
encode wall polysaccharide specific O-acetyltransferases (Bischoff
et al., 2010). Members of the TBL protein family had been shown
affected by s.d.

to impact on pathogen resistance, freezing tolerance, and cellulose

biosynthesis. The members of this family are predicted to have
Acyl esterase activity and known to modify cell-surface biopoly-
mers such as glycans and glycoproteins (Gille & Pauly, 2012).
The Heat Shock Cognate 70 kDa proteins were found in greater
Identity

quantities in natural and pulped grains both dryied in the yard.

91.42
100.0

Recent studies have reported the presence of HSPs in protein

%

profiles of different species under different stress. For example,

two-dimensional gels revealed five distinct sub-families of HSPs
(classified according to their molecular weight: HSP70, HSP60,
Hsp90, Hsp100 and small HSPs) in larger quantities for citrus fruits
stored under low temperature when as compared those at room
temperature (Yun et al., 2012).
We believe that all the synergistically efforts towards prevent-
Heat Shock cognate 70 kDa

ing cell damage and restore cellular homeostasis may contribute to

Major allergen Pru ar1

preservation of cell integrity and metabolites present within the

coffee grains.
Name/function

4. Conclusions

In the coffee post-harvest, drying has been considered as one of

the factors that cause impact on the final beverage quality hence
Matched Organism

determining distinct qualities for dry coffee in the yard or dryer.

This study is one of the first to provide data that will elucidate
C. canephora

C. canephora

the biochemical processes which occur within the grain after the
drying process. The significant differences in the abundance of
Table 1 (continued)

proteins between dry grain in the yard and dryer at 60 °C show

the potential of some proteins to be used as flavor markers and
number

beverage aroma and post-harvest processes quality control.

1617

1632
Spot

Among the abundant differentially, the proteins Globulin 11S,

a

glyceraldehyde-3-phosphate dehydrogenase, LEA Dehydrin,

1882 K.G.d. Livramento et al. / Food Chemistry 221 (2017) 1874–1882
UTP-glucose-1-phosphate uridyltransferase, Heat Shock Protein,
Trichome birefringence like protein, alpha-galactosidase and a
homologous protein to putative rMLC Termites like superfamily
protein were identified. Sensory studies should be carried out to
confirm the association of these protein changes to quality bever-
age, having in mind that other factors such as species, cultural
practices and environmental conditions could also affect the bever-
age quality.
Acknowledgments
We thank the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado
de Minas Gerais (FAPEMIG), the professor Dr. Adriano Pimenta
(Núcleo de Estrutura e Função de Biomoléculas/ICB/UFMG) and FINEP
for their financial support.
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