MORA, 2017 analise de tres especies de cacau e composição quimica

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Accepted Manuscript
Substantial equivalence analysis in fruits from three Theobroma species through
chemical composition and protein profiling
Walter Pérez-Mora, Jesús V. Jorrin-Novo, Luz Marina Melgarejo
PII: S0308-8146(17)31281-5
DOI: http://dx.doi.org/10.1016/j.foodchem.2017.07.128
Reference: FOCH 21512
To appear in: Food Chemistry
Received Date: 13 April 2017
Revised Date: 19 July 2017
Accepted Date: 25 July 2017
Please cite this article as: Pérez-Mora, W., Jorrin-Novo, J.V., Melgarejo, L.M., Substantial equivalence analysis in
fruits from three Theobroma species through chemical composition and protein profiling, Food Chemistry (2017),
doi: http://dx.doi.org/10.1016/j.foodchem.2017.07.128
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Substantial equivalence analysis in fruits from three Theobroma species through chemical 
composition and protein profiling 
 
Walter Pérez-Moraa*, Jesús V. Jorrin-Novob*, Luz Marina Melgarejoc,* 
a
 Laboratorio de Fisiología y Bioquímica Vegetal, Departamento de Biología, Universidad 
Nacional de Colombia, Bogotá. 
 
b
 Agroforestry and Plant Biochemistry, Proteomics and Systems Biology Research Group, Dpt. 
of Biochemistry and Molecular University of Córdoba-CeiA3 Córdoba, Spain, 
 *Corresponding authors: lmmelgarejom@unal.edu.co, bf1jonoj@uco.es, 
whperezm@unal.edu.co 
 
 Abstract 
Substantial equivalence studies were performed in three Theobroma spp., cacao, bicolor and 
grandiflorum through chemical composition analysis and protein profiling of fruit (pulp juice and 
seeds). Principal component analysis of sugar, organic acid, and phenol content in pulp juice 
revealed equivalence among the three species, with differences in some of the compounds that 
may result in different organoleptic properties. 
Proteins were extracted from seeds and pulp juice, resolved by two dimensional electrophoresis 
and major spots subjected to mass spectrometry analysis and identification. The protein profile, 
as revealed by principal component analysis, was variable among the three species in both seed 
and pulp, with qualitative and quantitative differences in some of protein species. The functional 
grouping of the identified proteins correlated with the biological role of each organ. Some of the 
 
identified proteins are of interest, being minimally discussed, including vicilin, a protease 
inhibitor, and a flavonol synthase/flavanone 3-hydroxylase. 
 
Biological Significance: Theobroma grandiflorum and Theobroma bicolor are endemic 
Amazonian plants that are poorly traded at the local level. As close relatives of Theobroma 
cacao, they may provide a good alternative for human consumption and industrial purposes. In 
this regard, we performed equivalence studies by conducting a comparative biochemical and 
proteomics analysis of the fruit, pulp juice and seeds of these three species. The results indicated 
equivalent chemical compositions and variable protein profiles with some differences in the 
content of the specific compounds or protein species that may result in variable organoleptic 
properties between the species and can be exploited for traceability purposes. 
 
Keywords- Theobroma bicolor, Theobroma cacao, Theobroma grandiflorum, protein profiling, 
traceability 
1. Introduction 
The Theobroma genus is composed of 22 species, nine of which are native to the Amazon region. 
These species constitute a vital resource for the indigenous communities of Latin America and 
other tropical countries in Asia and Africa. They are part of the wild animal and human 
Amazonian diet and are of potential agroindustrial interest (Motamayor et al., 2013). Theobroma 
fruits have a pleasing smell and flavor and are used in making juices, gelatins, ice cream, liquor, 
marmalades and other products (Rogez, Buxant, Mignolet, Souza, Silva & Larondelle 2004; 
González, Moncada, Idárraga, Rosenberg & Cardona 2016). Although only cultivated at the 
regional level, some species such as T. bicolor (macambo or bacao) and T. grandiflorum 
(cupuaçu) are in the process of domestication and technification (Hernández & Hernández 2012). 
 
They may constitute an alternative to T. cacao, the most cultivated and commercialized species 
of this genus. T. cacao as well as T. bicolor and T. grandiflorum seeds are used worldwide in the 
production of chocolate (toasted seeds) and locally in the production of candy and cosmetics 
(Motamayor et al., 2013). 
 
In addition, as close relatives of cocoa, other Theobroma species may be used in breeding 
programs as a source of genes related to desired phenotypes, such as disease resistance, drought 
tolerance, high protein content, and other agronomic and economic traits. The recent sequencing 
of the T. cacao genome (Argout et al., 2011) will favor research in this direction. The efficient 
utilization of these species requires detailed knowledge of their biology, especially from a genetic 
and molecular point of view; however, to date, few studies have been published on this topic. A 
substantial equivalence analysis between commercial cocoa and its wild relatives is necessary as 
a preliminary step prior to the validation, production and commercialization of Amazonian 
Theobromas as a food alternative. This result will improve consumer confidence and acceptance, 
open new markets to those interested in exotic fruits, avoid illicit crop problems and increase the 
incomes of indigenous farmers (Rogez et al., 2004; Motamayor et al., 2013). 
In the present study, partial chemical composition (sugars, organic acids, and total 
phenolics) as well as protein content and profiling analyses were performed on the pulp juice and 
seeds of mature T. bicolor and T. grandiflorum fruits, and the results were compared to those 
obtained from T. cacao. Proteomics is a useful approach for translational purposes and is used in 
food and plant material traceability (Jorrín Novo & Valledor, 2013). This proteomics study will 
provide a basis for future traceability and functional studies on the differences among 
provenances and the effects of environmental conditions and agronomic practices on the 
chemical composition of the fruit. To date, there are no published proteomics studies on wild 
 
Theobroma spp. or on the pulp and seeds of T. cacao fruits. A few proteomics studies on T. 
cacao have examined changes that occur in the seeds during chocolate production (Lerceteau, 
Rogers, Pétiard & Crouzillat 1999), changes in the fruit pericarp (Awang, Karim & Mitsui 2010), 
differences between zygotic and somatic embryos (Noah et al., 2013), characteristics of the 
leaves and roots of soil-flooded plants (Bertolde, Almeida & Pirovani 2014) and responses to 
Moniliophthora perniciosa (Pirovani et al., 2008). 
 
2. Materials and methods 
2.1.Plant material 
Fruits were obtained from the municipalities of Albania, Caquetá - Colombia (T. grandiflorum); 
Belén de los Andaquíes, Caquetá (T. bicolor); and Florencia, Caquetá (T. cacao). The fruits were 
mature (approximately 140 days after fruit set), and their condition was optimal for consumption. 
The fruits were sampled using a completely randomized design. Each fruit represented a single 
sample unit or biological replicate. Four biological replicates were obtained for each species, and 
3 analyticalreplicates were conducted, yielding a total of 12 replicates per specie. 
 
The pulp was manually separated from the seeds and squeezed using a commercial blender to 
obtain the juice. The seeds were thoroughly washed with tap water and dried with filter paper. 
Both the seeds and the juice were lyophilized using a Labconco Freezone 4.5 device (Labconco 
Corporation; Kansas City, USA) ground into small particulates and, stored at -80 °C until 
analysis. The pulp fresh weight/volume of juice/lyophilized powder ratio was 1 g/0.87 mL/165 
mg on average. 
 
2.2.Chemical analysis of pulp juice 
 
The chemical composition of the pulp juice was determined as these parameters are related to the 
quality of the fruit. The pH, total titratable acidity and total soluble solids of the fresh juice were 
determined directly before lyophilizing. Sugars, organic acids and phenolics were analyzed in the 
lyophilized juice powder. 
The pH was measured using the potentiometric method in accordance with AOAC 981.12 
(AOAC 2005). The total titratable acidity (TTA) was determined according to AOAC 942.15 
(AOAC 2005). The total soluble solids (TSS) expressed in units of °Brix units was determined 
using a Hanna Instruments HI96802 refractometer (Hanna instruments, Woonsocket, USA.) 
according to AOAC 932.12 (AOAC 2005). 
The following biomolecules were analyzed in the lyophilized juice powder from the three 
Theobroma spp.: total phenols, reducing sugars, sucrose, glucose, fructose, oxalic acid, malic 
acid and citric acid. The data are presented as relative to dry weight DW. 
The extraction and quantitative analysis of the phenolics and reducing sugars were performed as 
reported by Zeng et al. (2017). The total phenols were quantified by the Folin-Ciocalteu method 
using tannic acid as the standard. The reducing sugar content was determined by the 3,5-
dinitrosalicilic method using glucose as the standard. The sucrose, glucose and fructose content 
was measured by HPLC chromatography (column Phenomenex Ca++ monosaccharide; mobile 
phase: water; and refractive index detector) as previously reported (Solarte, Melgarejo, Martínez, 
Hernández & Fernández-Trujillo 2014). The organic acid contents (oxalic, malic and citric acids) 
were measured by HPLC chromatography (column ROA acid organic H+; mobile phase: 5 mM 
H2SO4; and a photodiode array detector at 207 nm) (Solarte et al., 2014). The HPLC analysis was 
performed using a Waters instrument (Waters, Milford, Massachusetts, USA). 
 
2.3. Protein extraction and quantification in seeds and pulp juice 
 
Proteins were extracted from 0.2 and 0.1 g of lyophilized powder of the seeds and the pulp juice, 
respectively. The preliminary experiments did not yield 2-DE patterns from seeds of sufficient 
quality or resolution. For this reason, prior to protein extraction, the lyophilized powder was 
delipidated in n-hexane (1:5, p/v with constant shaking) for 5 minutes. The TCA-acetone 
protocol reported by Maldonado, Echevarría-Zomeño, Jean-Baptiste, Hernández & Jorrín-Novo 
(2008) was employed to extract proteins. The final pellet was solubilized in 2 mL of rehydration 
solution (7 M urea, 1.2% (p/v) CHAPS, 43 mM DTT, 30 mM Tris HCl pH 8.5). Insoluble 
material was removed by centrifugation (15500 g, 10 min, 4°C), and the protein content was 
quantified in the supernatant according to the Bradford method (Valero, Valledor, Navarro, Gil 
Pelegrín, & Jorrín-Novo 2011) using a Bio-Rad Protein Assay kit and bovine serum albumin 
pattern (BSA) as the standard. 
 
2.4.Two-dimensional electrophoresis 
Protein extracts were subjected to 2D electrophoresis as reported by Maldonado et al. (2008) and 
Valero et al., (2011). In seeds, 4-7 pH IPG strips 18 cm in length were used, and 3-10 pH strips 
were used for the pulp juice (Bio-Rad, Hercules, USA). The pH gradient was chosen on the bases 
of preliminary experiments and after visualizing the pH range distribution of the spots. Strips 
were actively rehydrated at 50 V for 20 hours with 500 µg of extracted protein in 400 µL of IEF 
buffer (7 M urea, 2% p/v CHAPS, 0.2% (v/v) ampholytes (Bio-Lyte ampholytes 3-10, BioRad, 
Hercules, USA), 20 mM DTT and 0.01% (p/v) bromophenol blue). IEF was carried out in a 
Protean IEF Cell system (BioRad, Hercules, USA) at 20 °C using a gradual voltage increase 
between 250 V-10000 V until reaching 55000 V/h. The strips were immediately reduced and 
alkylated after IEF finished. Second dimension SDS-PAGE was carried out in a Protein Dodeca 
Cell (BioRad, Hercules, USA) using a 12% gel at 40 V for 3 hours and 80 V until the dye 
 
reached the bottom of the gel. Seed gels were stained with G-250 colloidal Coomassie blue 
(Noah et al., 2013), while the pulp gels were silver stained (Blum, Beier, & Gross 1987) as a 
more sensitive staining protocol was required to visualize most of the spots. The gels were 
digitized (UMAX PowerLook 2100XL-USB scanner), and images were analyzed with PDQuest 
8.0.1 (BioRad, Hercules, USA) software using tenfold over the background as a minimum 
criterion for the presence or absence. The analysis was re-evaluated by visual inspection. For 
each spot, normalized volumes (individual spot intensity/normalization factor calculated for each 
gel based on the total quantity in valid spots) were used for statistical analysis. The spots were 
manually cut with an EXQuest spot-cutter system and subjected to MS analysis. 
 
2.5.Mass spectrometry analysis and identification of the proteins 
The spots were digested with trypsin, and the resulting peptides were subjected to MS analysis 
(4700 Proteomics Analyzer MALDI–TOF/TOF Mass Spectrometer, Applied Biosystems) 
(Maldonado et al., 2008). A range of 800-4000 m/z with an acceleration voltage of 20 kV in the 
reflectron mode was employed. The spectrometer was calibrated using the trypsin autolysis peaks 
of m/z = 842.51 and m/z = 2211.10 as the internal standard. The 3 most abundant peptide ions 
were then subjected to MS/MS analysis, providing information that can be used to determine the 
peptide sequence. A peptide mass fingerprinting search and a combined search (+ MS/MS) were 
conducted. The peptide search was carried out by GPS Explorer TM v 3.5 (Applied Biosystems) 
from the NCBI database using MASCOT v 1.9 (Matrix Science Ltd., London; 
http://www.matrixscience.com) with the search restricted to Viridiplantae. One missed cleavage, 
100 ppm mass tolerance in MS and 0.5 Da for the CID data were allowed, and cysteine 
carbamidomethylation was selected as a fixed modification and methionine oxidation was 
selected as a modifier variable. The confidence in the peptide mass fingerprinting matches 
 
(pb0.05) was based on the MOWSE score (higher than 65) and CI> 99.8% and was confirmed by 
accurate overlap of the matched peptides with the major peaks of the mass spectrum. 
 
2.6.Statistical analysis 
Normality tests were conducted on the data using the Ryan-Joiner method and homoscedasticity 
tests as required for the analysis of variance ANOVA (p < 0.05). In addition, multiple mean 
comparison Tukey tests and principal component analysis (PCA) were carried out with Minitab 
statistics software, 15 trial free version commercialized by Minitab Inc. 
https://www.minitab.com. 
 
3. Results and discussion 
The pH, total soluble solids, titratable acidity, organic acids (oxalic, malic, and citric), reducing 
sugars, sugars (sucrose, glucose, and fructose) and total phenolics were determined and 
quantified in the pulp juice. These chemical data are of interest for comparative purposes but are 
also important due to their relationship with the nutritional and organoleptic properties of the 
fruit, such as the acidityand sweetness (pH, organic acids, and sugars), and the antioxidant 
activity (phenolics). The data are presented in Table 1. 
Principal component analysis (Figure 1A) of the data including the protein content revealed that 
the first two principle components accounted for 98% of the total variation. PC1 (91.0%) was 
positively influenced by phenols, reducing sugars, fructose and citric acid content, but it did not 
separate the Theobromas species. PC2 (7.0%) separated the three groups; T. cacao. and T. 
bicolor were closer, as also shown by hierarchical clustering analysis (Figure 1B). T. 
grandiflorum is positively influenced by the acidity parameters (titratable acidity, malic and citric 
acid content), and glucose. T. cacao and T. bicolor were positively influenced by the pH, the TSS 
 
the fructose and reducing sugar content. From these data, we could conclude that the three 
species examined are 90% equivalent according to the chemical analysis, with a 10% difference 
between individual parameters. 
The pH of the juice differed significantly between species. The T. grandiflorum and T. cacao 
juices were acidic (3.48 and 3.94 pH, respectively), and the T. bicolor juice was nearly neutral 
(6.65 pH). These data were correlated with the titratable acidity such that the acidic fruits 
exhibited the highest values (60.7 mg citric acid/g DW and 18.0 mg citric acid/g DW, for T. 
grandiflorum and T. cacao, respectively) and neutral fruits (T. bicolor) the lowest values (7.8 mg 
citric acid/g DW); these differences were statistically significant (Table 1). These data agree with 
those reported previously (Hernández and Hernández, 2012) and are correlated with the taste of 
the juice (more or less sweet). 
Total soluble solids is a standard measurement in fruits and their derived juices and is 
related to quality, organoleptic properties and consumer preferences. Large differences in the 
total soluble solids among species were not found, although T. cacao showed slightly higher 
values (13.9 °Brix) (Table 1). 
The sugar content in foodstuffs is related to nutritional value, organoleptic properties, and 
consumer acceptance, among other things. In this work, the total reducing sugars, as well as 
sucrose, fructose and glucose, were determined (Table 1). The amounts of total reducing sugars 
differed significantly between species. The ratio of the maximum (T. cacao) and minimum (T. 
grandiflorum) amounts (172 and 89 mg/g DW, respectively) was approximately 2. The highest 
value measured in T. cacao was highly correlated with the glucose content. While the highest 
monosaccharide values were found in T. cacao (69 and 72 mg/g DW of glucose and fructose, 
respectively), the values for sucrose were much higher in the other two species, T. bicolor and T. 
grandiflorum (77and 108 mg/g DW, respectively; Table 1). Rogez et al. (2004) reported similar 
 
findings in T. grandiflorum. The inverse correlation between sucrose and its monosaccharide 
components may be related to the activity of invertases and other sucrose hydrolyzing enzymes. 
The organic acid content was variable among species and depended on the compound that 
was analyzed. Citric acid was the most abundant in T. grandiflorum, and oxalic acid only was 
detected in T. cacao. The total organic acid values were higher in T. grandiflorum (224 mg/g 
DW), and did not differ greatly between T. bicolor and T. cacao (104 and 110 mg/g DW, 
respectively; Table 1). The sugar (glucose + fructose + sucrose) to acid (oxalic + citric + malic) 
ratios were 1.2 (bicolor), 1.6 (cacao) and 1.33 (grandiflorum), and were used to rank the 
sweetness. 
 
Phenolic compounds are also important nutraceutical components of food due to their 
antioxidant capacity and other qualities (Belchior & Genovese, 2013). Because of these 
properties, the phenolic compounds were quantified in pulp juice. The values ranged from 169 to 
340 mg/g DW, the lower value corresponding to T. bicolor and the higher value corresponding to 
T. cacao (Table 1). 
 
Protein content and profiling analysis results are parameters that are relevant in food 
analysis, as proteins are key elements of the nutritional value, may be toxic or allergenic, and are 
a source of bioactive peptides. On the other hand, protein profiling may be used in food 
traceability (Jorrín & Valledor, 2013). There have been no similar reports on this issue, with the 
exception of Lerceteau et al. (1999), who described the changes that occurred in cocoa seeds 
during chocolate production. Additionally, Reisdorff, Rohsius, de Souza, Gasparotto & Lieberei 
(2004) examined protease activities and seed storage globulins in T. grandiflorum and T. bicolor, 
as both are important in the formation of aroma precursors in cocoa. Recently, Bertazzo et al. 
 
(2011) analyzed whole seed extracts by MALDI-TOF TOF mass spectrometry; unfortunately, 
they did not identify individual proteins. 
Proteins were TCA-acetone extracted from pulp juice and seeds, quantified (Bradford 
assay) and resolved by 2-DE (Table 2, Figure 2). This protocol has provided good results using 
leaf tissues (Maldonado et al., 2008) and has been previously employed using the fruits and seeds 
of other plants (Sarry et al., 2004; Wu et al., 2014; Yin, Yang, Han & Gu 2015). Consistent with 
previous studies (Sotelo & Álvarez, 1991; Pugliese, Tomas-Barberan, Truchado & Genovese 
2013; Ribeiro, 2014), the protein content was higher in seeds than in pulp juice. 
In seeds, the highest value corresponded to T. bicolor (31.3 mg/g DW) and the lowest to 
T. grandiflorun (20.6 mg/g DW), with intermediate values for T. cacao (25.1 mg/g DW). Higher 
values have been previously reported for these species (Table 2). The differences were due to the 
analytical method employed. While the Kjeldahl technique (references in Table 2) determined the 
total protein content, the Bradford assay (this work) quantified only solubilized proteins. 
 
There were no statistically significant differences between the pulp of T. bicolor and T. 
grandiflorum (5.1 and 4.5 mg/g DW). Lower values were found for T. cacao (2.6 mg/g DW), 
indicating lower protein solubilization and more recalcitrance, which is also shown by the low 
number of spots resolved (Table 2). These values were lower than those previously reported 
(Table 2), reflecting the different methodology employed (Kjeldahl versus Bradford), but they 
were similar to the values published for other fruits, using Bradford assay (Prinsi et al., 2011, 
Sarry et al., 2004). 
Proteomic analysis in fruits is difficult due to the low protein content of this organ and the 
presence of proteases and metabolites such as polysaccharides, pigments, starch, polyphenols, 
organic acids and others that interfere with protein extraction and gel image quality and cause 
 
protein denaturation and inactivation, thus contributing to the recalcitrance of the plant material 
(Maldonado et al., 2008; Chan, 2013; Amoako-Andoh,, Daniëls, Keulemans & Davey 2014). 
Thus, for example, the phenolic content of T. cacao seeds was much higher than that of the other 
two species analyzed and correlated with the low protein levels in the extract. Using the same 
method, Valero et al. (2011) reported protein contents that ranged from 3-6 mg/g DW in Holm 
oak (Qurcus ilex L.) acorns (also a recalcitrant material); these values are lower than those 
reported for Theobroma in the current study. In contrast, the number of resolved spots (160) was 
slightly higher in the Quercus than in the Theobroma system. 
 
Proteins were resolved by 2-DE (Figure 2). The number of resolved spots was higher in 
seeds than in pulp and higher in T. grandiflorum than in T. bicolor and T. cacao,reflecting the 
protein content levels in the extracts. The increased recalcitrance of T. cacao can be deduced 
from the gel images themselves, which had the lowest resolution and higher striking. In the 
protein profile, there were common and variable spots, as revealed by the PD Quest gel image 
analysis (Figure 2). The number of variable spots was greater than the number of common spots. 
Only 4 spots in the pulp and 10 in the seeds were common to the three Theobromas. 
The spot intensity values for seeds were statistically analyzed using clustering and principal 
component analysis (Figure 3). As expected, considering the high number of variables (spots) 
and the dynamic character of the proteins, the variability was high, as is commonly reported in 
plants (Jiang et al., 2014; Valero et al., 2011). 
In the seed proteome, cluster analysis separated the three species; T. cacao and T. bicolor 
grouped together, which is consistent with the results of the chemical analysis (Figure 3-1A). The 
results of the PCA analysis showed that the first two components accounted for 69.6% of the 
variability, reaching 80 and 90% of the variability with 4 and 7 components, respectively. PC1 
 
(35.60%) discriminated the three species and was positively influenced by T. gandriflorum-
specific spots. PC2 (30.3%) separated T. bicolor and T. grandiflorum from T. cacao and was 
positively influenced by common spots, including vicilin and protease inhibitors (Figure 3-1B). 
Similar results and conclusions were obtained from the cluster and PCA analyses of the pulp 2-
DE spot intensity (Figure 3-2A and 3-2B). The first six components accounted for 82.7% of the 
variability, with values of 23.1% and 19.9% for PC1 and PC2 respectively. Both PC1 and PC2 
were positively influenced by spots present in T. glandiforum, including the ATPase subunit 4; 
PC2 was negatively affected by Maturase K, which is present in T. cacao and T. grandiflorum. 
The consistent spots, present in all the biological replicates, with the highest measured 
intensities were subjected to MS analysis: 52 from seeds (24 T. bicolor, 16 T. grandiflorum, and 
12 T. cacao) and 32 from pulp (13 T. bicolor, 16 T. grandiflorum, and 3 T. cacao). In Table 3, the 
identified proteins are shown. The high number of matches and identifications has been favored 
by the genome sequencing of T. cacao (Argout et al., 2011) and the availability of DNA 
sequences for the other species, mainly T. grandiflorum. The seeds for the three species from the 
Theobroma genus were found to have reserve proteins, with the most abundant proteins related to 
energy metabolism, carbohydrate metabolism, stress and genetic information processing, in 
addition to various other proteins whose function could not be identified through the database 
search. On the other hand, the pulp juice of the fruit from the three species contained proteins that 
were mainly related to the carbohydrate metabolism, stress, energy metabolism and genetic 
information processing, as well as various proteins without identified functions. This functional 
grouping represents the specific characteristics of each tissue and is common to other fruits 
whose proteomes have been studied. 
Common proteins in the seeds of the three Theobroma included reserve vicilin and trypsin 
inhibitors, two types of proteins of interest. Vicilin is a reserve protein that is commonly found in 
 
the seeds of various species of plants, such as legumes, and was also found in T. cacao embryos 
(Niemenak, Kaiser, Maximova, Laremore & Guiltinan 2015). Vicilin is considered to be very 
important, as some studies have revealed that it is the precursor of the specific aroma of 
processed cacao (Kratzer, Frank, Kalbacher, Biehl, Wöstemeyer & Voigt 2009). Trypsin 
inhibitors have been proposed to have different roles, including seed germination, protease 
activity regulation and insect and fungal pathogen defense (Li et al., 2013; Yin et al., 2015). 
Some of the species-specific proteins could be of interest as markers, but this has to be confirmed 
experimentally using a larger number of populations. Some other proteins of interest included the 
Flavonol synthase/flavanone 3-hydroxylase present in the proteome of T. grandiflorum seeds, 
which forms part of the flavonoid biosynthetic pathway from dihydroflavonol (Kuhn et al., 2011) 
related to antioxidants. This family of compounds contributes to the nutritional value of plant-
derived foods. Other interesting groups included stress- and defense-related proteins, such as heat 
shock proteins in the T. cacao and T. grandiflorum seeds that has also been reported in 
descriptive proteomic studies on the pericarp of T. cacao fruits (Awang et al., 2010). Other 
identified proteins have been previously reported, including a malic enzyme found in the T. 
bicolor seeds, which was also reported in the pericarp of T. cacao (Awang et al., 2010). 
In pulp juice extracts, common proteins were not identified, but more proteins were specific or 
common to just two species. This result increases the value of this tissue in the traceability of the 
species and derived products. Some of the proteins identified deserve some attention and 
discussion. Chitinases, which are involved in the protection against pathogens and the growth and 
development of fruits, have been reported as the most abundant family of proteins in the pulp of 
immature bananas (Torres et al., 2012) and other fruits. Other reports indicated that class I 
chitinases are relevant chestnut and avocado allergens and are likely the panallergens responsible 
for the latex fruit syndrome (Blanco et al., 1999). The presence of these proteins is the principal 
 
difference between T. bicolor and the other two species and suggests that the consumption of T. 
bicolor pulp is not recommended for persons susceptible to allergens. In T. bicolor pulp juice, we 
found a peroxidase, an enzyme widely used in clinical biochemistry and enzyme immunoassays 
to eliminate peroxide in foodstuffs and in waste water (Hamid & Rehman, 2009). An isocitrate 
dehydrogenase identified in T. bicolor and an ATP synthase in T. grandiflorum have been 
previously reported in T. cacao embryos (Noah et al., 2013) and pericarp (Awang et al., 2010). 
 
4. Conclusions 
To study the substantial equivalence among fruits from three species of the Theobroma genus 
(T. cacao, T. grandiflorum and T. bicolor), chemical analysis and protein profiling were 
performed in pulp juice and seeds. The results of the chemical analysis, which included a 
comparison of pH, titratable acidity, total soluble solids, total phenols, reducing sugars, soluble 
sugars and organic acids, showed high similarities between the three species according to PCA 
and clustering analysis. However, some statistically significant differences were found in some of 
the sugars, organic acids and total phenols quantified that might result in differences in 
organoleptic properties, such as sweetness and acidity, and nutraceutical value. 
Theobroma fruit is a recalcitrant tissue whose chemical composition (polysaccharides and 
phenolics) complicates proteome analysis, and this is demonstrated by the low protein yield and 
low number of spots resolved when compared with results from other plant tissues. PCA revealed 
that the seed 2-DE profile was quite variable among the three species. Consistent with previous 
findings in other fruits, seeds and pulp juice showed different functional groupings of the 
identified proteins, and this distribution represented the biological characteristics of both the pulp 
and seeds. 
The seeds of the three species shared major proteins, particularly vicilin and protease inhibitors. 
 
The firstis of great interest, as it is involved in the formation of the aroma precursors of 
fermented T. cacao seed products. Other proteins identified were enzymes implicated in the 
flavonoid pathway. Species-specific proteins can serve as markers in traceability analyses. The 
protein yield of the pulp juice was lower than that of the seeds and resulted in a lower number of 
spots on the 2-DE. The presence of putative allergens such as chitinase I in pulp juice should be 
highlighted. 
 
Acknowledgements 
Financial support was provided by COLCIENCIAS, DIEB and Facultad de Ciencias of the 
Universidad Nacional de Colombia (Contract 0729/2012). The fruits were provided by Amazon 
scientific research center SINCHI. The mass spectrometry analysis and identification were 
provided by UCO-SCAI proteomic facility, a member of the Carlos III Networked proteomics 
platform, ProteoRed-ISCIII, Spain. 
 
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FIGURE LEGENDS 
 
Figure 1. Principal component analysis (A) and clustering (B) of the data corresponding to the 
fruit chemical composition of the three Theobroma species under study. 
 
Figure 2. Master gels of T. bicolor (A), T. grandiflorum (B) and T. cacao (C) seed (1) and pulp 
(2) extracts. The numbers in the gels indicate the proteins selected for MS analysis and the 
protein identification (Table 3). Venn diagram (3) showing the number of common and species-
specific spots among the three Theobroma species (A, Pulp; B, Seeds). 
Figure 3. Clustering (A) and principal component analysis (B) of the spot intensity data in gels 
from the seeds (1) and pulp (2) of the three Theobroma species. 
 
 
Table 1. pH and chemical composition of T. bicolor, T. cacao and T. grandiflorum pulp juice. 
Unless otherwise indicated, the units are mg/g dry weight. Values represent the mean of four 
biological and three analytical replicates. The SD is given in parentheses. The same letter within 
each file indicates that there were no statistically significant differences (Tukey’s test, P < 0.05). 
n.d. not detected. DW dry weight 
 
 
T. bicolor T. grandiflorum T. cacao 
pH 6.65 (0.17)a 3.48 (0.12)c 3.94 (0.16)b 
 
Titratable acidity 
(citric acid equivalent) 
7.8 (1.6)c 60.7 (2.9)a 18.0 (2.1)b 
 
Total soluble solids 
(°Brix) 
12.2 (0.35)b 12.4 (0.36)b 13.9 (1.02)a 
 
Phenols 
(µg tannic acid/g DW 
169 (23)c 226 (26)a 340 (52)b 
 
Oxalic acid (mg/g DW) 
 
n.d. n.d. 1.45 (0.05) 
Citric acid (mg/g DW) 
 
72 (1)c 176 (3)a 78 (3)b 
Malic acid (mg/g DW) 
 
32 (2)b 48 (2)a 31 (1)b 
Reducing sugars 
(Glucose equivalents) 
 
120 (3)b 89 (10)a 172 (12)c 
Sucrose (mg/g DW) 
 
77 (2)b 108 (5)a 35 (1)c 
Glucose (mg/g DW) 
 
26 (1)b 24 (3)b 69 (2)a 
Fructose (mg/g DW) 18 (2)c 36 (2)b 72 (2)a 
 
 
 
 
 
 
 
Table 2. Protein content (as determined by the Bradford assay) and number of 2-DE resolved 
spots in the seed and pulp juice extracts from the Theobroma spp. fruits. Values correspond to 
the mean of 4 biological replicates with 3 analytical replicates each. The SD is given in the 
parentheses. Different letters within each column indicate statistically significant differences 
(Tukey’s test, P < 0.05). Values found in the literature are also reported. 
Species Tissue 
Protein content Protein content references Number 
of spots (mg/g DW) (mg/g DW) 
T. bicolor Seed 31.3a (3.4) 114 Sotelo et al. (1991) 94a (17) 
T. grandiflorum Seed 20.6c (1.3) 37-50 Pugliese et al. (2013) 122a (20) 
T. cacao Seed 25.1b (2.9) 63-79 Sotelo et al. (1991) 62b,c (8) 
T. bicolor Pulp 5.1d (0.9) 12,5 Sotelo et al. (1991) 46c (13) 
T. grandiflorum Pulp 4.5d (1.2) 8-12 Pugliese et al. (2013) 70b (10) 
T. cacao Pulp 2.6e (0.6) 7-11 Ribeiro (2014) 17d (3) 
 
 
 
 
Table 3: Identification of the physiological function of the sequenced proteins in the Theobroma genus. 
aNCBI ID: Protein identification number in the National Center for Biotechnology Information database. 
bMW. Molecular weight (kDa) 
cIP: Isoelectric point. 
 
 
Protein number/Protein Species NCBI IDa 
% of 
covered 
sequence 
Matched 
peptides 
Experimental Theoretical Characte
ristics 
 MWb IPc MWb IPc 
 
T
.
 
b
i
c
o
l
o
r
 
S
e
e
d
s
 
Nutrient reserve proteins 
 
 
1 Vicilin Theobroma 
cacao 
sp|Q43358|VCL_T 18 5 67.3 4.1 61.4 6.6 
Common 
in the 
three 
species 
2 Vicilin (Fragment) Theobroma bicolor tr|Q9SQ43|Q9SQ4 47 5 33.7 4.1 27.2 8.8 
3 Vicilin Theobroma 
cacao 
sp|Q43358|VCL_T 13 5 28.4 5.3 27.2 6.6 
Processing of genetic 
information 
 
 
4 Trypsin Inhibitor 
(Fragment) 
Theobroma 
microcarpum tr|Q3BD85|Q3BD8 14 2 15.8 5 16.8 4.7 
Common 
and the 
most 
abundant 
in the 
three 
species 
5 21 kDa putative trypsin 
inhibitor 
Theobroma 
bicolor tr|Q3BD85|Q3BD8 25 7 15.8 5.8 24.4 8.7 
6 21 kDa putative trypsin 
inhibitor 
Theobroma 
bicolor tr|Q3BD85|Q3BD8 21 6 11.1 6.1 24.4 8.7 
7 21 kDa seed proteins Theobroma 
cacao 
sp|P32765|ASP_T 45 9 15.8 4.5 24.2 5.7 
8 21 kDa seed proteins Theobroma 
cacao 
sp|P32765|ASP_T 28 7 16.3 6.3 24.4 5.7 
 
 
 
13 AAA-type ATPase 
family protein 
Arabidopsis 
thaliana Q0WM93 20 16 18.7 4.4 12.4 6.2 
Only in T. 
bicolor 
22 Hydroxyproline-rich 
glycoprotein-like 
(BLASTP 91%) 
Oriza sativa Q655Y7 33 5 9.7 4.3 19.6 4.7 Only in T. bicolor 
Metabolism of 
carbohydrates 
10 malic enzyme Oryza brachyantha tr|J3N3W6|J3N3W 36 11 9.6 5 68.1 8.5 
Only in T. 
bicolor 
Response to stress 
 
 
11 Glutathione S-
transferase GST 27 Zea mays tr|Q9FQB2|Q9FQB 55 7 7.9 5.1 25.2 5.3 
Only in T. 
bicolor 
 
T
.
 
g
r
a
n
d
i
f
l
o
r
u
m
 
s
e
e
d
s
 
Nutrient reserve proteins 
 
 
25 Vicilin Theobroma 
cacao 
sp|Q43358|VCL_T 11 5 7.1 5.5 27.2 6.6 
Common 
in the 
three 
species 
26 Vicilin Theobroma 
cacao 
sp|Q43358|VCL_T 11 5 13 5.8 27.2 6.6 
27 Vicilin Theobroma 
cacao 
sp|Q43358|VCL_T 7 3 10 5.1 27.2 6.6 
28 Vicilin Theobroma 
cacao 
sp|Q43358|VCL_T 7 3 7.7 5.3 27.2 6.6 
Processing of genetic 
information 
 
 
29 Trypsin inhibitor 
(Fragment) 
Theobroma 
grandiflorum tr|Q8RVF3|Q8RVF 29 6 16.1 4.9 17.1 4.3 
Common 
and the 
most 
abundant 
30 Trypsin inhibitor 
(Fragment) 
Theobroma 
mammosum 
tr|Q8S4Z8|Q8S4Z 22 3 15 4.1 17.1 4.3 
 
31 Trypsininhibitor 
(Fragment) 
Theobroma 
subincanum tr|Q8RUR7|Q8RUR 23 4 8.1 4.9 17 4.3 
in the 
three 
species 32 Trypsin inhibitor 
(Fragment) 
Theobroma 
speciosum tr|Q8S502|Q8S50 29 6 16.1 5 16.7 4.5 
33 21 kDa Putative 
Trypsin inhibitor 
Theobroma 
grandiflorum tr|Q3BD86|Q3BD8 26 5 16 5.4 22.5 5.3 
Related to stress 
 
 
35 18.2 kDa class I heat 
shock protein 
Medicago 
sativa sp|P27880|HSP12 34 6 13.8 5.8 18.1 5.8 
Common 
with T. 
cacao 
38 26.7 kDa heat shock 
protein 
Oryza sativa 
subsp. Indica Q10P60 26 10 15.9 5.3 26.6 6.2 
40 Chaperone function 
Putative protein 
Selaginella 
moellendorffii tr|D8T7N5|D8T7N 20 15 21.4 4.9 94.1 6 
Antioxidant metabolism 
 
 
36 Flavonol 
synthase/flavanone 3-
hydroxylase 
Medicago 
truncatula tr|G7JMI8|G7JMI 34 9 7.7 5.4 41.1 5.5 
Only in T. 
grandiflor
um 
 
 
 
 
 
 
 
T
.
 
c
a
c
a
o
 
s
e
e
d
s
 
Nutrient reserve proteins 
 
 
41 Vicilin Theobroma 
cacao 
sp|Q43358|VCL_T 12 5 14.7 4.5 27.2 6.6 Common in the 
three 
species 42 Vicilin 
Theobroma 
cacao 
sp|Q43358|VCL_T 7 8 7.8 4.9 27.2 6.6 
Processing of genetic 
information 
43 Trypsin inhibitor 
(Fragment) 
Theobroma 
cacao 
tr|Q8S4Z5|Q8S4Z 38 7 15.2 4.7 16.9 4.5 Common 
and the 
most 44 Trypsin inhibitor Theobroma tr|Q8RV88|Q8RV8 26 5 15.2 4.9 16.7 4.6 
 
(Fragment) sylvestre abundant 
in the 
three 
species 
45 Trypsin inhibitor 
(Fragment) 
Theobroma 
cacao 
tr|Q8S4Z5|Q8S4Z 30 8 15.2 5.2 16.9 4.5 
46 Trypsin inhibitor 
(Fragment) 
Theobroma 
cacao 
tr|Q8S4Z5|Q8S4Z 33 6 15.3 4.5 16.9 4.5 
47 21 kDa seed protein Theobroma 
cacao 
sp|P32765|ASP_T 21 6 11.6 6.3 24.4 5.7 
52 U6 snRNA-associated-
like-Smprotein (99% 
Blastp) 
Medicago 
truncatula A0A072UD03 48 13 7.6 5.2 10.7 6.8 
Only in T. 
cacao 
50 Protein disulfide 
isomerase (94% Blastp) Zea mays Q5EUD6 40 10 8.5 5.3 17.2 9.1 
Only in T. 
cacao 
Related to stress 
 
 
48 17c heat shock protein 
(Fragment) Quercus suber Q93WR2 25 10 7.4 5 40.3 6.4 
Common 
with T. 
grandiflor
um 
 
T
.
 
b
i
c
o
l
o
r
 
P
u
l
p
 
Response to stress 
52 Peroxidase Medicago 
truncatula tr|G7KFK8|G7KFK 36 9 24.3 5.6 36.4 9.38 
Only in T. 
bicolor 
53 9-cis-epoxycarotenoid 
dioxygenase 2t (Fragment) 
Sorghum 
bicolor 
tr|D7NMW9|D7NM
W 43 9 34.2 4.3 26.6 8 
Only in T. 
bicolor 
Carbohydrates 
metabolism 
54 UDP-
glucuronosyltransferase, 
putative 
Ricinus 
communis tr|B9SV12|B9SV1 28 10 25.1 4.2 55.6 5.2 
Only in T. 
bicolor 
55 Isocitrate 
dehydrogenase [NADP] 
Populus 
trichocarpa tr|B9GHS2|B9GHS 28 12 25.2 3.5 45.3 5.4 
Only in T. 
bicolor 
 
56 Chitinase Citrus unshiu tr|B2DD07|B2DD0 13 2 76.2 4.5 31 8.3 
Allergen 
found 
only in T. 
bicolor 
57 Chitinase Petroselinum 
crispum tr|Q9SPU0|Q9SPU 15 4 75.4 4 29.4 6.3 
58 Class I Chitinase Festuca 
arundinacea tr|B8R3R6|B8R3R 12 3 19.5 7.2 34.1 8.2 
59 Class I Chitinase Festuca 
arundinacea tr|B8R3R6|B8R3R 11 2 34.2 5 34.1 8.2 
Processing of genetic 
information 
60 Pentatricopeptide repeat 
containing protein 
Arabidopsis 
lyrata subsp. 
Lyrata 
tr|D7KX87|D7KX8 31 12 80.7 3.1 46 9.5 
Common 
with T. 
grandiflor
um 
61 Seryl-tRNA synthetase 
(68% Blastp) 
Theobroma 
cacao 
A0A061E4V8 28 10 19.3 5.2 51 7.1 Only in T. bicolor 
63 T-complex protein 1 
subunit gamma (98% 
Blstp) 
Viti vinifera D7TB48 31 13 45.7 8.6 60.5 5.5 Only in T. bicolor 
64 ATP synthase subunit d, 
putative isoform 1 (89% 
Blastp) 
Theobroma 
cacao 
A0A061DJY4 45 9 17.9 8.5 29.2 9.7 
Common 
with T. 
cacao 
 
 
 
 
 
 
T
.
 
g
r
a
n
d
i
f
l
o
r
u
m
 
p
u
l
p
 
Biosynthesis of fatty acids 
 
 
65 Protein from the Acyl-
CoA thioesterase family 
Arabidopsis 
thaliana tr|B3H5Z2|B3H5Z 25 10 37.9 6 43.2 6.3 
Only in T. 
grandiflor
um 
Energy metabolism 
 
 
 
66 ATPase subunit 4 Vitis vinífera tr|B6VJV3|B6VJV 52 9 39.6 7.1 22.4 9.6 
Only in T. 
grandiflor
um 
Processing of genetic 
information 
67 Pentatricopeptide repeat 
containing protein 
Medicago 
truncatula tr|G7IP45|G7IP4 36 10 53.7 6.2 42.2 8.1 Common 
with T. 
bicolor 68 Pentatricopeptide repeat 
containing protein 
Medicago 
truncatula tr|G7K3R0|G7K3R 28 13 40.2 5.1 68.6 7.8 
69 Maturase K (Fragment) Chamelaucium 
megalopetalum tr|Q6X2G1|Q6X2G 50 7 40.2 5.6 22.2 9.7 
Common 
with T. 
cacao 
73 Tetratricopeptide 
repeat-containing family 
protein (100% Blastp) 
Populus 
trichocarpa B9HVN0 33 11 29.3 3.6 38.1 5.2 
Only in T. 
grandiflor
um 
 
 
 
 
T
.
 
c
a
c
a
o
 
P
u
l
p
 
T. Cacao pulp 
 
 
81 Maturase K (Fragment) 
(processing of the genetic 
information) 
Neomarica 
variegata tr|I1UDI6|I1UDI 31 8 38.7 4 31.8 9.8 
Common 
with T. 
grandiflor
um 
82 ATP synthase subunit 
alpha (Fragment) (energy 
metabolism) 
Galium aparine tr|F8S0A2|F8S0A 28 10 80.3 4.1 46.6 5.2 
Common 
with T. 
bicolor 
 
 
 
 
 
 
 
 
 
 
 
 
 
Highlights 
• A substantial equivalence study in seed and pulp from Theobroma spp. was performed. 
• The protein profiles were variable as revealed by principal component analysis. 
• Some of the compounds analyzed differed quantitatively between the species. 
• Differences in protein species abundance may be used for traceability purposes.