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Extraction, identification and semi-quantification of oligopeptides in cocoabeans
Angela Marseglia, Stefano Sforza, Andrea Faccini, Mariangela Ben-civenni, Gerardo Palla, Augusta Caligiani
PII: S0963-9969(14)00215-4DOI: doi: 10.1016/j.foodres.2014.03.046Reference: FRIN 5162
To appear in: Food Research International
Received date: 13 December 2013Revised date: 20 March 2014Accepted date: 29 March 2014
Please cite this article as: Marseglia, A., Sforza, S., Faccini, A., Bencivenni, M., Palla,G. & Caligiani, A., Extraction, identification and semi-quantification of oligopeptides incocoa beans, Food Research International (2014), doi: 10.1016/j.foodres.2014.03.046
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EXTRACTION, IDENTIFICATION AND SEMI-QUANTIFICATION OF
OLIGOPEPTIDES IN COCOA BEANS
Angela Marseglia 1, Stefano Sforza
1, Andrea Faccini
2, Mariangela Bencivenni
1, Gerardo Palla
1,
Augusta Caligiani 1*
1Department of Food Science, University of Parma, Parma, Italy.
2 Centro Interdipartimentale Misure, University of Parma, Parma, Italy.
E-mail: [email protected]
*Corresponding author:
Augusta Caligiani
Department of Food Science
Università degli Studi di Parma
Parco Area delle Scienze 17A
43124-Parma, Italy
tel: +39 0521 905407; fax: +39 0521 905472; E-mail: [email protected]
Short Title: oligopeptides in fermented cocoa beans and related products
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ABSTRACT
Peptides and amino acids generated during cocoa bean fermentation are known to be the most
important precursors for the development of cocoa aroma. Although cocoa fermentation and aroma
development have been extensively studied, the cocoa oligopeptide fraction is under-investigated.
In particular, the identification of specific peptide sequences and the quantification of cocoa
peptides are scarce in the literature. The aim of this study was to investigate the presence of
oligopeptides in fermented cocoa beans. Peptides were analysed by reversed phase LC/ESI-MS and
LC/ESI-MS/MS, and the molecular masses of 44 different peptides were obtained by analysing the
mass spectra associated with the most intense chromatographic peaks. Peptides were identified
based on the exact molecular masses, mass fragmentation patterns and by comparison with vicilin
and 21 kDa cocoa seed protein sequences. Semi-quantitative data on peptide presence in fermented
cocoa samples of different geographic origin, different fermentation levels and on roasted products
were also provided.
Keywords
Cocoa beans, oligopeptides, vicilin, 21kDa cocoa seed protein
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1. Introduction
Cocoa beans, from the fruit of the cocoa tree (Theobroma cacao L.), are transformed into chocolate
and other cocoa products by a complex process involving fermentation, drying and roasting.
Fermentation is carried out by different methods in the countries of origin, and represents a crucial
stage in the development of the aromatic precursors and bioactive compounds characteristic of
chocolate and cocoa products. Proteins are the cocoa fraction that undergoes the most intensive
modification during fermentation, where microbiological and enzymatic reactions lead to extensive
breakdown of cocoa seed proteins, yielding peptides and amino acids which are the important
flavour precursors (Zak & Keeney, 1976a).
Proteins make up 10-15% of the dry weight of cocoa seeds, the second most abundant
constituent after cocoa fat. Zak and Keeney (1976b) suggest that cocoa beans contain four main
proteins - albumin, globulin, prolamin and glutelin, while Voigt, Biehl, Kamaruddin, & Wazir
(1993) report total cocoa seed protein content is 52% albumin and 43% globulin. Lerceteau et al.,
employing two-dimensional SDS-PAGE and silver staining, determined that globulin and the
albumin storage proteins represent 23 and 14% of the total seed protein, respectively, in addition to
other abundant unknown polypeptides (Lerceteau, Rogers, Petiard, & Crouzillat, 1999). Globulins
are vicilin-like storage proteins consisting of three subunits with molecular masses of 47 kDa, 31
kDa, and 15 kDa which are derived from a common 66-kDa precursor (Spencer & Hodge, 1992).
The albumin fraction was identified as a 21 kDa cocoa seed protein and its primary structure,
together with its trypsin inhibitory properties, were reported by Kochhar, Gartenmann, and Juillerat
(2000).
During fermentation, peptide–nitrogen and free amino acids increase and total protein
concentration decreases (Adeyeye, Akinyeye, Ogunlade, Olaofe, & Boluwade, 2010; Hashim,
Selamat, Muhammad, & Ali, 1998), and the globulin protein fraction is the most degraded during
fermentation (Amin, Jinap, & Jamilah, 1997; Voigt, Biehl, Kamaruddin, & Wazir, 1993).
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Cocoa proteins are cleaved to hydrophilic and hydrophobic peptides as well as amino acids
through autolysis by two endogenous enzymes, aspartic endoprotease and carboxypeptidase (Amin,
Jinap, Jamilah, Harikrisna, & Biehl, 2002; Voigt, Biehl, Heinrichs, Kamaruddin, Marsoner, & Hugi,
1994a). Fermentation of cocoa beans is fundamental for the activation of these two enzymes by
microbial metabolites (such as acetic acid), though there is no evidence that microbial enzymes
penetrate the beans and create flavour compounds (Schwan & Wheals, 2004). Changes in the
protein composition of cocoa beans have been noted not only during fermentation but also as a
consequence of roasting. A decrease in total protein, free amino acids and albumin (35.65% to
18.10%) are seen during roasting (de Brito, García, Gallão, Cortelazzo, Fevereiro, & Braga, 2000;
Abecia-Soria, Pezoa-Garcia, & Amaya-Farfan, 2005).
Although the degradation of cocoa proteins into oligopeptides and amino acids is well
documented, the role of peptides in the formation/development of cocoa flavour, specifically
peptides formed during cocoa fermentation and autolysis which are responsible for generating
cocoa flavour during roasting, remain uncharacterized. Buyukpamukcu, Goodall, Hansen, Keely,
Kochhar, & Wille (2001) report the identification of a hexapeptide and a nonapeptide with sequence
homologies in cocoa, demonstrating their formation from vicilin during fermentation, where the
hexapeptide is formed (in part) from the nonapeptide as fermentation progresses. However, other
authors report more complex peptide patterns of uncharacterized peptides (Rashidah, Jinap,
Nazamid, & Jamilah, 2007).
Besides generating the characteristic chocolate aroma, the potential biological activities of
oligopeptides found in the cocoa samples are of interest, though they are under-investigated in
cocoa literature. Two recent papers report the physiological effects (antioxidant, ACE inhibitory
and hypoglycaemic activities) of cocoa autolysates (containing peptides and amino acids), though
the identification of peptides responsible for the activities is lacking (Sarmadi, Amin, & Muhajir,
2011; Sarmadi, Farhana, Muhajir, Nazamid, Azizah, & Amin, 2012).
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The aim of this work was to provide a full identification and semi-quantitative assessment of
the main peptides found in cocoa beans from different geographical origins, fermentation levels and
roasted products by HPLC-ESI-MS-MS, providing new insights into the formation and evolution of
the oligopeptide fraction of cocoa.
2. Materials and Methods
2.1 Cocoa samples. Well fermented cocoa beans of Forastero variety of the following geographical
origin were kindly provided by Barry Callebaut Belgium nv: Mexico, Grenada, Trinidad, Perù,
Ghana, Ivory Coast, Sulawesi Makassar. A sample of Criollo variety from Mexico was also
analysed. A series of cocoa beans from Ivory Cost with different fermentation levels (fresh cocoa
beans, under-fermented and dried cocoa beans and well fermented cocoa beans) and a roasted cocoa
series from Ghana (well fermented cocoa beans, roasted nibs, cocoa liquor) were also considered.
2.2 Chemicals. All solvents and reagents were of HPLC grade and used as commercially available
without any further purification; deionized water was obtained by Millipore Alpha Q system
(Millipore Corporation, Billerica, MA); formic acid 99% was purchased from ACROS Organics
(Fair Lawn, NJ).
2.3 Sample Preparation. Peptides were extracted according to a method previously described
(Sforza, Ferroni, Galaverna, Dossena& Marchelli, 2003). A total of 10 g of finely grinded cocoa
sample was suspended in 45 mL of 0.1 N HCl. (L,L)-phenylalanylphenylalanine (Phe-Phe) was
added as an internal standard (2.25 mL of a 1mM solution) The suspension was homogeneized for
1.5 min by Ultra Turrax T50 at 4000rpm (Janke and Hunkel Italabortechnik) and then centrifuged at
4000 rpm for 30 min at 4 °C by an ALC 4237R centrifuge. The solution was filtered through paper
filters (pore dimensions 15-20 µm) and then extracted four times with 50 mL of ethyl ether. The
solution was filtered again with a Millipore 47 mm Steril Aseptic system through 0.45 µm HVLP
Millipore filters. A total of 1.5 mL of the resulting solution were mixed with 0.5 mL of a formic
acid solution (0.1%). The solution was diafiltered through Sartorius Vivaspin 2 filters (nominal
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molecular cut-off 10 000 Da) by using an Amicon Micropartition system MPS-1. The filtrate was
dried under nitrogen, redissolved in H2O (0.1% HCOOH), and analyzed by HPLC.
2.4 UPLC/ESI MS (WATERS, Milford, MA) analysis conditions: eluent A: H2O (0.2% CH3CN
and 0.1% HCOOH); eluent B CH3CN (0.1% HCOOH); gradient elution was performed according
to the following steps: 0–7 min isocratic 100% A, 7–50 min linear gradient from 100% A to 50% A,
50–52 min isocratic 50% A, 53– 58 min from 50% A to 0% A and reconditioning. Column:
AQUITY UPLC Beh C18 (1.7 mm, 2.1x150 mm). Flow rate: 0.2 ml/min. MS conditions: ESI,
positive ions, single quadrupole analyzer. Capillary voltage: 3.2 kV, cone voltage: 30V, source
temperature: 150 °C, desolvation temperature: 300 °C, cone gas flow (N2): 100 L/h, desolvation gas
(N2): 650 L/h, acquisition: 100:2000 m/z. All data were acquired and processed by the software
MassLynx 4.0 (Waters, Milford, MA).
2.5 HPLC/ESI/MS/MS conditions. In order to confirm the identity of the peptides, some samples
were analysed by Thermo LTQ ORBITRAP XL mass spectrometer (Thermo scientific, Waltham,
MA, USA) coupled to a DIONEX Ultimate3000 HPLC (Thermo scientific, Waltham, MA, USA) in
the following conditions. Eluent A: H2O (0.2% HCOOH); eluent B CH3CN (0.2% HCOOH);
gradient elution was performed according to the following steps: 0–5 min isocratic 98% A, 5–65
min linear gradient from 98% A to 60% A, 65– 70 min from 60% A to 5% A and reconditioning.
Column: Aeris Peptide 3.6u XB-C18 (2.1x150 mm). Column temperature 30 °C. Flow rate: 0.2
ml/min. Source parameter: ionization type: ESI+, Capillary temperature: 275°C, source voltage:
3.5kV, capillary voltage: 13V, tube lens: 100V, sheath gas flow (N2): 40, auxiliary gas flow (N2):
20, sweep gas flow (N2): 20. Intact peptides spectra acquisition: data type: profile, scan range:
250:2000 m/z, resolution 30000. Fragments spectra acquisition: data type: centroid, activation type:
CID, normalized collision energy: 35. For LC-MS/MS analysis, four CID spectra were acquired
following each full scan. All data were acquired processed by the software Xcalibur 2.0.7 SP1
(Thermo scientific, Waltham, MA, USA). Peptide and Protein Identification: Proteom Discoverer
1.0 (Thermo scientific, Waltham, MA, USA), protein database: Theobroma cacao (UniProt),
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peptide mass tolerance: 10ppm, fragment mass tolerance: 0.8Da, dynamic modification: oxidation
(M), decoy database search: FDR=0.01 e FDR=0.05.
3. Results and Discussion
A typical MS chromatogram (full scan acquisition) of a cocoa bean sample is shown in
Figure 1 and characteristic mass peaks for every peptide identified are reported in Table 1.
Although many peptides were present in the cocoa samples (complex chromatograms), this study
concentrated on those giving the most intense signals. Two main peaks eluting in the same peptide
region were identified by their respective MS spectra as theobromine and caffeine
(methylxanthines) with retention times (Rt) of 12.96 and 17.16 min, respectively.
3.1 Peptide identification
44 oligopeptides (Table 1) were characterised on the basis of their MW and in-source
fragmentation detectable with the single-quadrupole mass analyser. The identities were confirmed
on the basis of the exact molecular masses, mass fragmentation patterns and by the analyses of
vicilin and 21kDa cocoa seed protein sequences. Vicilin precursor (McHenry & Fritz), a 566 amino
acid globulin-type protein, has an apparent mass of 66 kDa (Figure 2) and the 21 kDa cocoa seed
protein, an albumin type protein, was 221 amino acids in length (Figure 3). Among the peptides
identified in the LC-MS chromatograms, 25 originated from vicilin and 14 from the 21 kDa cocoa
seed protein.
Analysing the distribution of peptides identified in the sequence of the two main cocoa
proteins indicated that peptide sequences were localized to limited zones in both proteins and not
spread throughout the sequences. This suggested cleavage at specific sites which generated peptides
that were further degraded, and possibly the cleavage of these peptides induced denaturation of the
remaining polypeptide chains and resistance to further proteolytic enzyme activity.
The peptides from vicilin were localized at amino acids sequences 131-168, 209-229, 276-
292 and 434-484, with the first and the last being richest in peptides. Region 131-168 of vicilin
yielded four different peptides - RSEEEEGQQ, NNPYYFPK, TRFR and DEEGNFKIL.
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DEEGNFKIL was further cleaved into lower Mw peptides all having a common N-terminal and
different C-terminal amino acids, a clear indication of carboxypeptidase enzyme activity. Amino
acid 131 was the starting point of the 31 kDa (aas 131-425) subunit recently identified in vicilin,
together with the 47 kDa (aas 139-566), 15.0 kDa (aas 314-434) and 14.5 kDa (aas 416-545)
subunits (Kratzer, Frank, Kalbacher, Biehl, Wöstemeyer, &Voigt, 2009). The other sequence rich in
peptides was aas 434-484, which contained four main peptides that were further cleaved to lower
Mw peptides.
It is interesting to note amino acid 434 represents the end point of the 15 kDa subunit
identified by Kratzer et al, 2009, but the sequence rich in peptides was 14.5 kDa. This region
contains a previously-reported peptide (APLSPGDVF) with a protonated molecular mass (MH+) at
m/z 902.8 (Buyukpamukcu, Goodall, Hansen, Keely, Kochhar, & Wille, 2001) which was cleaved
further to SPGDVF (MH+ m/z 621.5) and DVF (MH
+ m/z 380.2, not previously reported) present in
some cocoa bean samples. Protonated molecular masses at m/z 534.2 and 437.3, corresponding to
the PGDVF and GDVF fragments, were also found at retention times of 19.29 and 21.51 min,
respectively, likely indicating the action of an amino peptidase. Another peptide family was
generated from aas 477-484 (ASKDQPLN) of vicilin. The chromatograms of cocoa bean samples
contained ASKDQPL (MH+ m/z 758.4) and KDQPL (MH
+ m/z 600.3), which suggested the 31 kDa
and 14.5 kDa subunits were (most extensively) cleaved during cocoa fermentation, and may be the
precursors to cocoa aroma.
With respect to peptides generated from the 21 kDa cocoa seed protein, 14 peptides were
found whose sequences localized to two main regions, aas 48-59 and 75-108, while a tetrapeptide
from the N-terminal region of the protein was also found (aas 27-31). The 75-108 sequence was the
richest in peptides, containing five, some of which further fragmented to low Mw peptides. Voigt,
Heinrichs, Voigt, & Biehl, 1994b report the peptide pattern of cocoa is not due to the 21 kDa cocoa
albumin and that the protein is not involved in the generation of cocoa aroma. This was contradicted
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by the data in this study, which showed a significant number of oligopeptides were derived from
cocoa albumin, though further investigation is required to determine their aroma potentials.
Preferred cleavage sites in the N-terminal and C-terminal portions of peptides were
identified, where aspartic acid was the most frequent N-terminal amino acid, which suggests
endogenous aspartic endoprotease activity present in ungerminated cocoa seeds (Biehl & Passern,
1982). Asparagine and arginine were also common N-terminal aas identified in the peptide pattern.
In addition to the aspartic endoprotease, a carboxypeptidase is reported to generate typical aroma
precursors present in fermented cocoa seeds (Voigt, Biehl, Heinrichs, Kamaruddin, Gaimarsoner, &
Hugi, 1994a). Phenylalanine is the C-terminal aa most frequently found in peptides, and present in
ten different peptides. Bytof, Biehl, Heinrichs, and Voigt (1995) report that a carboxypeptidase
preferentially liberates hydrophobic amino acids, whereas acidic amino acids are released very
slowly, suggesting the majority of peptides liberated in cocoa beans should not have acidic C-
terminal amino acids. However, glutamic acid was observed as the C-terminal aa in three peptides
derived from vicilin and aspartic acid in three peptides derived from the 21 kDa cocoa albumin.
Peptides with C-terminal arginine, lysine or proline residues were resistant to degradation by the
cocoa seed carboxypeptidase. Bytof et al. (1995) reported the rate of hydrolysis is determined by
the C-terminal aa and the neighbouring amino acid residue, where the release of the C-terminal
amino acid is favoured by the hydrophobic side chain of the adjacent amino acid residue. The
findings of this study were not in agreement with this observation, as many peptides showed a C-
terminal valine-phenylalanine sequence. These data indicated other proteases (endogenous or of
microbial origin) could be involved in the generation of the cocoa bean peptide pattern, even though
the penetration of microbial enzymes into beans has not been reported (Schwan & Wheals, 2004).
Other peptides present in the chromatograms, but not present in vicilin and 21kDa cocoa
seed protein sequences (not shown in Table 1), were not identified, suggesting they originated from
other cocoa seeds proteins. This supports the hypothesis of Lerceteau, Rogers, Petaird, and
Crouzillat (1999) who show total cocoa protein electrophoretic patterns with a number of highly-
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expressed proteins (other than albumin and globulin) and hypothesized these abundant seed proteins
may be the source of cocoa flavour precursor peptides.
3.2 Peptide semiquantification.
Peptides identified were semi-quantified by comparison to the internal Phe-Phe standard.
For the correct integration of peaks, as the total ion chromatograms (TIC) were crowded, the
eXtract Ion Chromatogram (XIC) technique was applied. The oligopeptides were semi-quantified
by measuring the ratio between the XIC peptide area and the relative XIC area of Phe-Phe, as
previously described (Sforza, Galaverna, Schivazappa, Marchelli, Dossena, & Virgili, 2006). The
results for 44 cocoa bean peptides present in cocoa beans from different geographic origins, varied
fermentations and roasting levels, as well as fragments extracted for the quantification are reported
in Tables 2-4.
The total content of peptides (Table 1, Figure 4a) in fermented Forastero cocoa beans from
different geographical origins ranged from 152.06 mg/kg in Sulawesi beans to 293.10 mg/kg for
Mexican beans (Forastero). The Criollo variety, considered a fine cocoa, had a significantly higher
content (353.57 mg/kg) of peptides with respect to the Forastero variety from the same geographic
origin.
The total amount of peptides generated from vicilin was higher compared to peptides
generated from the 21 kDa cocoa albumin for all samples, which was in agreement with literature
indicating vicilin as the main precursor of cocoa aroma. The ratio of peptide amounts generated
from vicilin and 21kDa cocoa seed protein was constant among well-fermented cocoa beans from
different origins, varying from 1.54 (Criollo from Mexico) to 2.85 (Forastero Ivory Coast) (Figure
4b). An exception was Sulawesi beans that showed significantly higher amounts of peptides from
vicilin (vicilin/21kDa ratio of 12.93). Sulawesi beans had a lower total peptide content and a
peculiar peptide distribution, with reduced amounts for the majority of peptides compared to other
samples, except for peptides with m/z values of 579.2, 360.3, 546.1, 867.4, 799.2, 1099.0, 789.6
and 1245.5 (corresponding to TRFR, VLE, RSEEEEGQQ, KESYNVQ, PAGHAVTF,
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YYGAFSYEV, TVWRLD, RQDRREQEE) which were significantly higher. Specific peptide
amounts differed significantly between various cocoas from different origins. As an example, the
m/z 902.8 peptide (APLSPGDVF) and its fragments (such as SPGDVF, m/z 621.5) were abundant
in cocoa beans from the Ivory Coast (Buyukpamukcu, Goodall, Hansen, Keely, Kochhar, & Wille,
2001), but were nearly absent in Criollo beans (Figure 5).
Fermentation strongly influenced the formation of peptides in cocoa beans. The total peptide
content (Table 3, Figure 6a) was very low in fresh beans (10.19 mg/Kg) and under-fermented dried
beans compared to the corresponding well-fermented beans (74.5 mg/Kg vs 246.13 mg/Kg).
Considering the distribution of peptides from vicilin and 21 kDa, the peptide amounts derived from
the 21kDa protein were slightly higher in fresh beans than those from vicilin, while in under-
fermented beans, no peptides were formed from the 21kDa protein. The vicilin/21kDa ratio (Figure
6b) and the distribution of single peptides in under-fermented beans was similar to that of Sulawesi
beans, suggesting the Sulawesi bean sample was not well-fermented, which is supported by a
previously-published study indicating fermentation of cocoa beans is often omitted in Sulawesi
regions (Rohsius, Matissek, & Lieberei, 2006).
Data on roasted products (roasted nibs and liquor, Table 4) showed that thermal treatments
reduced the total content of peptides by ~30%. The effect of thermal treatment was the same for
both cocoa proteins, and the vicilin/21 kDa ratio did not change significantly in roasted products
compared to fermented unroasted beans.
4. Conclusions
44 peptides mainly generated during cocoa fermentation were identified and semi quantified in
cocoa beans of different geographic origin and in other cocoa products. To the best of our
knowledge this is the first exhaustive investigation on cocoa peptides pattern.
The knowledge about peptide structures is of primary importance to better understand the biological
activity of cocoa, to identify the aroma relevant peptides and to provide additional analytical
methods useful for the determination of cocoa beans quality.
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Results showed that peptide patterns of cocoa beans of different origin are variable, suggesting an
effect of different fermentation processes. The ratio of peptides generated from vicilin respect to
those generated from 21 kDa cocoa albumin seems to be promising as indicator of the fermentation
level.
Further work is in progress on a higher number of cocoa samples from different origin and
fermentation levels, with the aims to define a specific correlation among cocoa origin / peptide
fingerprint and to identify the specific cocoa aroma development from different peptide fractions.
Legends to Figures
Figure 1: Typical MS chromatogram of a cocoa bean sample (Criollo Mexico).
Figure 2: Sequence of cocoa vicilin precursor (McHenry & Fritz, 1992). Oligopeptide sequences
identified in cocoa beans are underlined; dipeptides are not evidenced. Amino acids 1-24 represent
the signal peptide.
Figure 3: Sequence of 21 kDa cocoa seed protein (P32765,UniProtKB). Oligopeptide sequences
identified in cocoa beans are underlined; dipeptides are not evidenced. Amino acids 1-26 represent
the signal peptide.
Figure 4: a) Semiquantitative amount (mg/Kg) of total peptides, total peptides from vicilin and total
peptides from 21 kDa cocoa seed protein and b) ratio of peptides amount from vicilin and 21kDa
cocoa seed protein in well fermented cocoa beans of different geographic origin
Figure 5: EXtracted Ion Chromatograms (XIC) for peptide of MH+ m/z 902.8 in a) Criollo Mexico
beans and b) Ivory Coast beans; c) corresponding mass spectrum
Figure 6: a) Semiquantitative amount (mg/Kg) of total peptides, total peptides from vicilin and total
peptides from 21 kDa cocoa seed protein and b) ratio of peptides amount from vicilin and 21kDa
cocoa seed protein in fresh, under-fermented and well fermented cocoa beans from Ivory Coast
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Sforza, S., Ferroni, L., Galaverna, G., Dossena, A., & Marchelli, R. (2003). Extraction, Semi-
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Zak, D. L., & Keeney, P. G. (1976a). Changes in cocoa proteins during ripening of fruit,
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24, 483−486.
Zak, D. L., & Keeney, P. G. (1976b). Extraction and fractionation of cocoa proteins as applied to
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Fig1
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Fig3
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Fig6
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Table 1: Identification of the molecular masses of the most significant peptides detected in Cocoa
beans by single quadrupole and confirmed by Orbitrap analyser. Mass fragments (y- and b-
fragments) detected from in-source collision or MS-MS fragmentation were reported. Peptide
retention
time
(min)
MH + (single
quadrupole)
PEPTIDE IDENTIFICATION (fragment
ions identified in the mass spectrum)
PROTEIN
11,50 203,2 AI/AL (y1*)
11,98 761,4 RRSDLD (y5*,y3*,y2*,b6,b5,b4,b2,y5) 21 kDa Cocoa Seed Protein
12,61 403,4 RLD (b1,b2,b3*) 21 kDa Cocoa Seed Protein
13,6 231,2 VI/VL (b2*,y1*)
14,52 1205,5 SNADSKDDVVR
(b10,b8,b7,b2,y9,y8,y7,y6,y4,y3,y2,y1)
21 kDa Cocoa Seed Protein
14,74 634,3 VSTDVN (b4,b5*,b6) 21 kDa Cocoa Seed Protein
15,13 487,3 ANSPV (b5,b3,y3,y2) 21 kDa Cocoa Seed Protein
15,4 838,4 DEEGNFK (b6, y6,y5,y4,y2) Vicilin
15,69 488,3 GAGGGGL (b7,b6) 21 kDa Cocoa Seed Protein
16,49 758,4 ASKDQPL (b7,b6,b5,b4,b3,y6,y5) Vicilin
16,80 265,3 MD/FV
17,52 276,1 AW (b2,y1*) 21 kDa Cocoa Seed Protein
17,58 393,3 FLN/SSIS Vicilin/21 kDa Cocoa Seed Protein
17,91 710,4 DEEGNF (b6,b5,b4,b3,b2,y4,y3,y1*) Vicilin
18,26 265,2 VF (b2,y1)
18,45 579,2 TRFR (b4) Vicilin
18,85 360,3 VLE(b3,b2) Vicilin
19,10 690,3 NGKGTIT (b7,b6,b5,b4,b3,y6,y5) Vicilin
19,29 534,2 PGDVF (b5,b4,b3) Vicilin
19,34 563,2 DEEGN Vicilin
19,55 1091,5 RSEEEEGQQ Vicilin
20,00 933,6 DSKDDVVR (b8,b7,y6) 21 kDa Cocoa Seed Protein
20,11 564,1 RRSF Vicilin
20,41 867,4 KESYNVQ (b8) Vicilin
20,51 360,3 EVL (b2,b3)
20,76 862,4 SSISGAGGGGL
(b11,b10,b9,b8,b7,b6,y8,y7,y6,y5)
21 kDa Cocoa Seed Protein
21,42 600,4 KDQPL (b5,b4,y1*) Vicilin
21,61 437,3 GDVF (b4,b3,y3*,y2,y1) Vicilin
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21,67 380,2 DVF (b3,b2,y1) Vicilin
22,22 601,3 NNKPE (b5) Vicilin
22,30 799,2 PAGHAVTF (b7,b6*,b5,b4,y7,y2,y1) Vicilin
22,40 279,3 GM/FI/FL/PT
23,02 1099,0 YYGAFSYEV (b9) Vicilin
23,11 1042,5 NNPYYFPK Vicilin
23,31 621,5 SPGDVF (b6,b5,b4,y5) Vicilin
23,44 579,2 unidentified
24,27 408,3 IEF (b3,b2,y2) 21 kDa Cocoa Seed Protein
24,76 789,6 TVWRLD (b6,b5,b4,y5,y4,y3) 21 kDa Cocoa Seed Protein
27,48 1064,5 DEEGNFKIL (b8,b7,b6,b1*,y4,y3,y2,y1*) Vicilin
27,96 475,4 ISQQ/IFPV/PVIF/FVPI Vicilin/21 kDa Cocoa Seed Protein
28,47 747,5 NGTPVIF (b7,b6,b5,y4,y2,y1*) 21 kDa Cocoa Seed Protein
28,62 1245,5 RQDRREQEE (b9) Vicilin
28,8 902,8 APLSPGDVF (b9,b8*,b7,b4,y8,y6,y5,y1*) Vicilin
29,37 820,5 DNEWAW (b6,b5,b4,b3,y3) 21 kDa Cocoa Seed Protein
* ions from in source fragmentation
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Table 2: Semiquantitative amount (mg/Kg) of peptides identified in fermented cocoa beans of
different geographical origins.
Peptide
Retention
time (min)
m/z for
quantitation
Mexico
(Criollo) Mexico
Ivory
Coast Trinidad Grenada Sulawesi Perù Ghana
11,50 203,2 66,66 63,49 24,12 30,99 17,04 3,69 44,11 38,86
11,98 761,4 2,12 0,31 0,11 0,88 0,63 0,00 0,11 0,36
12,61 403,4 11,19 3,85 1,39 7,04 3,25 0,31 1,71 2,71
13,60 231,2 30,39 24,50 10,59 14,20 8,33 0,00 12,65 14,15
14,52 603,3 3,47 0,68 0,67 2,68 2,25 0,20 1,69 1,71
14,74 634,3 3,15 1,54 1,26 2,70 2,90 0,47 2,85 1,77
15,13 487,3 8,83 4,73 4,82 8,32 5,66 0,58 7,36 7,44
15,40 838,4 0,84 0,31 0,28 1,21 0,89 0,08 0,67 0,73
15,69 488,3 21,03 8,78 1,80 10,16 4,90 0,49 3,21 4,03
16,49 379,9 1,42 1,47 5,87 2,67 2,00 1,61 8,90 6,17
16,80 265,3 14,22 15,82 9,59 14,79 10,12 4,27 14,99 13,31
17,52 276,1 17,68 17,06 7,18 11,96 6,24 1,32 7,44 10,86
17,58 393,3 6,34 6,78 4,35 7,07 7,60 5,39 10,45 7,25
17,91 710,4 5,16 2,31 1,50 5,28 4,00 0,25 2,24 3,04
18,26 265,2 24,68 31,99 20,97 23,74 20,14 3,79 15,42 30,93
18,45 579,2 28,07 7,89 7,70 16,74 13,36 41,00 16,49 9,34
18,85 360,3 1,62 1,74 1,66 1,17 2,37 7,92 1,90 2,07
19,10 690,3 2,96 1,06 0,33 1,30 1,78 0,02 0,46 0,78
19,29 534,2 3,85 2,84 1,96 5,06 5,51 0,37 2,14 2,16
19,34 563,2 12,71 7,52 3,64 11,51 8,68 0,84 5,21 8,12
19,55 546,1 0,00 0,26 0,41 0,18 0,19 15,00 1,06 0,29
20,00 933,6 1,29 0,56 0,44 0,83 0,97 0,27 1,35 0,31
20,11 564,3 2,80 1,62 0,38 0,75 0,84 0,04 0,37 0,77
20,41 867,4 5,71 1,91 2,37 5,16 3,90 11,46 4,24 2,81
20,51 360,3 0,97 0,75 0,63 1,76 1,51 1,80 1,95 0,61
20,76 862,4 2,41 1,32 1,01 2,21 2,22 0,23 2,20 1,13
21,42 600,4 11,90 5,96 3,95 5,83 10,87 0,79 4,33 3,82
21,61 437,3 3,36 4,33 1,08 1,70 1,97 0,42 0,93 1,35
21,67 380,2 16,56 26,49 7,58 9,90 11,23 1,22 13,48 17,23
22,22 601,3 2,37 0,85 0,13 0,58 0,29 0,00 0,01 0,22
22,30 799,2 0,01 0,01 0,95 0,54 0,38 17,70 0,27 0,36
22,40 279,3 8,84 12,98 9,14 13,86 10,70 3,68 21,27 12,95
23,02 1099 0,03 0,03 0,25 0,26 0,28 2,57 0,42 0,23
23,11 522 1,34 0,49 0,30 0,85 0,71 0,00 0,75 0,81
23,31 621,5 2,70 2,78 6,40 12,55 15,29 2,38 6,31 10,61
23,44 579,2 3,86 1,69 1,95 4,26 3,38 9,20 3,59 2,20
24,37 408,3 13,94 17,50 4,80 11,67 14,83 3,47 13,50 12,01
24,76 789,6 0,13 0,70 2,79 0,29 0,39 1,53 0,55 1,26
27,48 533,1 1,31 1,35 0,83 0,85 3,19 0,54 3,93 1,49
27,96 475,4 2,54 3,50 1,56 3,86 3,76 0,23 1,39 2,66
28,47 747,5 4,07 2,73 1,22 3,44 2,81 0,33 1,81 1,74
28,62 623,5 0,02 0,01 0,74 0,19 0,29 5,19 1,40 0,82
28,80 902,8 0,07 0,04 0,56 0,48 0,62 1,36 1,21 0,31
29,37 820,5 0,97 0,60 0,14 0,70 0,88 0,08 0,45 0,48
Total
peptides 353,57 293,10 159,40 262,19 219,13 152,06 246,72 242,24
Peptides
from Vicilin 139,29 116,96 79,60 124,12 120,97 120,01 108,30 118,01
Peptides
from 21kDa 90,28 60,35 27,63 62,88 47,92 9,28 41,57 45,80
Ratio
vicilin/21kDa 1,54 1,94 2,88 1,97 2,52 12,93 2,61 2,58
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Table 3: Semiquantitative amount of peptides identified in cocoa beans from Ivory Coast at
different fermentation level
Peptide
Retention
time (min)
m/z for
quantitation
Fresh unfermented
cocoa beans Under-fermented dried cocoa beans
Well fermented
dried cocoa beans
11,50 203,2 0,56 3,81 29,10
11,98 761,4 0,00 0,00 0,21
12,61 403,4 0,01 0,05 2,51
13,60 231,2 0,67 0,37 10,65
14,52 603,3 0,03 0,21 1,41
14,74 634,3 0,00 0,01 1,99
15,13 487,3 0,75 0,04 5,41
15,40 838,4 0,01 0,01 0,73
15,69 488,3 0,02 0,03 4,42
16,49 379,9 0,49 0,40 7,58
16,80 265,3 0,83 0,40 14,95
17,52 276,1 0,01 0,10 10,12
17,58 393,3 0,73 0,57 11,20
17,91 710,4 0,00 0,02 2,97
18,26 265,2 0,31 0,31 24,12
18,45 579,2 0,01 27,58 12,20
18,85 360,3 0,38 4,14 3,68
19,10 690,3 0,00 0,23 0,69
19,29 534,2 0,10 0,07 2,33
19,34 563,2 0,42 0,62 6,75
19,55 546,1 0,23 1,84 1,19
20,00 933,6 0,00 0,02 0,57
20,11 564,3 0,02 0,25 0,29
20,41 867,4 0,01 9,26 4,61
20,51 360,3 0,19 2,06 0,70
20,76 862,4 0,00 0,01 1,27
21,42 600,4 0,03 0,04 4,60
21,61 437,3 0,02 0,06 1,88
21,67 380,2 0,02 0,10 14,93
22,22 601,3 0,00 0,01 0,08
22,30 799,2 0,25 4,45 1,27
22,40 279,3 0,09 0,11 17,86
23,02 1099 0,02 0,28 0,40
23,11 522 0,01 0,01 1,15
23,31 621,5 0,01 0,09 14,11
23,44 579,2 0,00 8,13 3,97
24,37 408,3 0,30 0,53 13,82
24,76 789,6 3,15 2,78 1,37
27,48 533,1 0,07 1,56 2,55
27,96 475,4 0,04 0,02 1,62
28,47 747,5 0,00 0,01 1,77
28,62 623,5 0,40 3,90 1,66
28,80 902,8 0,00 0,05 0,95
29,37 820,5 0,01 0,00 0,55
Total sum 10,20 74,50 246,14
Peptides
from Vicilin 3,08 57,428785 129,24
Peptides
from 21kDa 4,29 3,78467 45,41
Ratio
vicilin/21kDa 0,72 15,174053 2,85
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Table 4: Semiquantitative amount of peptides identified in cocoa beans from Ghana and
corresponding roasted products
Peptide
Retention
time (min)
m/z for
quantitation
Fermented
beans Roasted nibs Liquor
11,50 203,2 38,86 29,70 31,93
11,98 761,4 0,36 0,16 0,15
12,61 403,4 2,71 2,36 2,28
13,60 231,2 14,15 9,67 10,94
14,52 603,3 1,71 0,57 0,44
14,74 634,3 1,77 0,65 0,60
15,13 487,3 7,44 4,93 5,11
15,40 838,4 0,73 0,51 0,36
15,69 488,3 4,03 2,09 2,06
16,49 379,9 6,17 4,80 5,22
16,80 265,3 13,31 7,87 9,04
17,52 276,1 10,86 7,31 7,83
17,58 393,3 7,25 4,70 4,66
17,91 710,4 3,04 1,97 1,99
18,26 265,2 30,93 19,09 20,31
18,45 579,2 9,34 6,74 6,86
18,85 360,3 2,07 0,29 0,28
19,10 690,3 0,78 0,48 0,42
19,29 534,2 2,16 2,28 2,65
19,34 563,2 8,12 3,19 4,21
19,55 546,1 0,29 2,19 0,11
20,00 933,6 0,31 0,18 0,22
20,11 564,3 0,77 0,90 1,05
20,41 867,4 2,81 1,28 1,24
20,51 360,3 0,61 0,54 0,46
20,76 862,4 1,13 0,54 0,59
21,42 600,4 3,82 2,47 2,66
21,61 437,3 1,35 3,11 3,49
21,67 380,2 17,23 14,37 14,90
22,22 601,3 0,22 0,19 0,27
22,30 799,2 0,36 0,01 0,04
22,40 279,3 12,95 5,12 5,06
23,02 1099 0,23 0,12 0,11
23,11 522 0,81 0,77 0,60
23,31 621,5 10,61 3,95 3,96
23,44 579,2 2,20 1,19 1,09
24,37 408,3 12,01 9,04 9,71
24,76 789,6 1,26 1,01 1,02
27,48 533,1 1,49 0,77 0,88
27,96 475,4 2,66 2,47 2,56
28,47 747,5 1,74 0,53 0,44
28,62 623,5 0,82 0,27 0,24
28,80 902,8 0,31 0,24 0,30
29,37 820,5 0,48 0,41 0,47
Total sum 242,24 160,99 168,81
Peptides from Vicilin 118,01 75,63 77,67
Peptides from 21kDa 45,8 29,75 30,91
Ratio vicilin/21kDa 2.58 2.54 2.51
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Manuscript: Extraction, identification and semi-quantification of oligopeptides in cocoa beans
Authors: Angela Marseglia, Stefano Sforza, Andrea Faccini, Mariangela Bencivenni, Gerardo Palla,
Augusta Caligiani
Highlights
- Sequences of 44 peptides of cocoa beans were provided for the first time
- Peptide pattern of fermented cocoa beans of different geographic origin was shown
- Ratio of peptides from cocoa globulins/albumin was proposed as fermentation indicator
