International Journal of Food Sciences and Nutrition

ISSN: 0963-7486 (Print) 1465-3478 (Online) Journal homepage: http://www.tandfonline.com/loi/iijf20

Nutritional composition, bioactive compounds

and volatile profile of cocoa beans from different
regions of Cameroon

Giovanni Caprioli, Dennis Fiorini, Filippo Maggi, Marcello Nicoletti, Massimo

Ricciutelli, Chiara Toniolo, Biapa Prosper, Sauro Vittori & Gianni Sagratini

To cite this article: Giovanni Caprioli, Dennis Fiorini, Filippo Maggi, Marcello Nicoletti,
Massimo Ricciutelli, Chiara Toniolo, Biapa Prosper, Sauro Vittori & Gianni Sagratini (2016):
Nutritional composition, bioactive compounds and volatile profile of cocoa beans from
different regions of Cameroon, International Journal of Food Sciences and Nutrition, DOI:
10.3109/09637486.2016.1170769

To link to this article: http://dx.doi.org/10.3109/09637486.2016.1170769

Published online: 07 Apr 2016.

Submit your article to this journal

Article views: 9

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=iijf20

Download by: [University of California, San Diego] Date: 15 April 2016, At: 20:52
 INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION, 2016
http://dx.doi.org/10.3109/09637486.2016.1170769

RESEARCH ARTICLE

Nutritional composition, bioactive compounds and volatile profile of cocoa

beans from different regions of Cameroon
Giovanni Capriolia, Dennis Fiorinia, Filippo Maggia, Marcello Nicolettib, Massimo Ricciutellia, Chiara Toniolob,
Biapa Prosperc, Sauro Vittoria and Gianni Sagratinia
a
School of Pharmacy, University of Camerino, Camerino, Italy; bDepartment of Environmental Biology, University of Rome ‘‘La Sapienza’’,
Rome, Italy; cLaboratory of Medicinal Plant Biochemistry, Department of Biochemistry, Faculty of Science, Food Science and Nutrition,
University of Dschang, Dschang, Cameroon

ABSTRACT ARTICLE HISTORY

Analysis of the complex composition of cocoa beans provides fundamental information for evalu- Received 13 November 2015
Downloaded by [University of California, San Diego] at 20:52 15 April 2016

ating the quality and nutritional aspects of cocoa-based food products, nutraceuticals and supple- Revised 13 March 2016
ments. Cameroon, the world’s fourth largest producer of cocoa, has been defined as ‘‘Africa in Accepted 21 March 2016
miniature’’ because of the variety it habitats. In order to evaluate the nutritional characteristics of Published online 8 April 2016
cocoa beans from five different regions of Cameroon, we studied their polyphenolic content, vola- KEYWORDS
tile compounds and fatty acids composition. The High Performance Thin Layer Chromatography Fatty acids; GC–MS; HPTLC;
(HPTLC) analysis showed that the Mbalmayo sample had the highest content of theobromine HS–SPME; polyphenols
(11.6 mg/g) and caffeic acid (2.1 mg/g), while the Sanchou sample had the highest level of ()-epi-
catechin (142.9 mg/g). Concerning fatty acids, the lowest level of stearic acid was found in the
Mbalmayo sample while the Bertoua sample showed the highest content of oleic acid. Thus, we
confirmed that geographical origin influences the quality and nutritional characteristics of cocoa
from these regions of Cameroon.

Introduction nutraceuticals and food supplements has been gaining

Analysis of the complex composition of cocoa beans
affords key information for evaluating a number of uses
for this commercially important raw material. The vari-
ous components of cocoa beans exert several activities
and have a number of physiological effects; information
about these components is important for successful use
of this resource. For example, their nutritional value is
mainly determined by their fatty constituents, while
their diuretic effect, excitation of the central nervous
system, and influence on cardiovascular activity are due
to their content of xanthine alkaloids (Cooper et al.
2008; Rusconi & Conti 2010). The polyphenol content
in cocoa beans has been the object of recent attention
because of antioxidant properties and effective impact
against reactive oxygen species (ROS), as reported in
recent reviews and single articles (Serafini et al. 2003;
Han et al. 2007; And ujar et al. 2012). The volatile con-
stituents of cocoa powder are very important to impart
flavor notes to end product (Li et al. 2012). Besides
these substances, several other constituents should be
considered, including simple organic acids, gamma-ami-
nobutyric acid (GABA) and others. Because of these
activities and effects, the use of cocoa beans in
ground, though chocolate remains the primary product
made with this raw material (Ackar et al. 2013).
In order to have an analytic approach for studying
cocoa beans that is as complete as possible, study of
the activities and effects exerted by the components of
cocoa beans should be complemented by consideration
of differences in varieties and cultivars (De Almeida &
Valle 2007) and epigenetic and environmental factors,
often conditioned by the region of provenance (Saltini
et al. 2000). For example, several works have reported
the differences in constituents for cocoa beans collected
in different parts of the world, including a recent one
comparing the fatty acid composition in beans from
Ecuador and Ghana (Torres-Moreno et al. 2015).
Other researchers have reported that the Near Infrared
(NIR) Spectroscopy technique coupled with multivari-
ate classification methods has proven to be a powerful
tool for discriminating cocoa bean samples according
to their geographical origin (Teye et al. 2013).
This study analyzed cocoa from five regions of
Cameroon, the fourth largest producer of cocoa in the
world, with around 200,000 tons exported annually
(International Cocoa Organization 2012). Cameroon’s
cocoa bean varieties are priced for their characteristic

CONTACT Dr Gianni Sagratini gianni.sagratini@unicam.it School of Pharmacy, University of Camerino, Via Sant’ Agostino 1, 62032 Camerino, Italy
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
 2 G. CAPRIOLI ET AL.

reddish colored cocoa powder sought after by the bak-

ing, biscuit and ice cream industries; their relatively fla-
vorless cocoa butter is also appreciated in the chocolate
industry. Cameroon has been referred to as ‘‘Africa in
miniature’’, because its mountains, oceanic coasts,
deserts, savannahs and tropical belt of rain forests
reflect the continent’s great variety of climates and
lands. In the tropical belt, the fertile volcanic soil of
Mount Cameroon, called ‘‘Mongo ma Ndemi’’, the
Mountain of Greatness (Sanchou area) is particularly Sanchou
Obala
favorable for cultivation of the red cocoa, while other
regions also produce fine cocoa beans of a deep red
color. Bertoua
Cocoa beans are particularly rich in polyphenols.
The quantity of catechins are considered very import-
Downloaded by [University of California, San Diego] at 20:52 15 April 2016

ant for healthy properties of cocoa and related prod-

ucts, including chocolate. Secondary products, and in
particular polyphenols, can be analyzed by the High
Performance Thin Layer Chromatography (HPTLC).
HPTLC is the last evolution of planar chromatography
and it enables identification of these natural products,
even in very low concentrations. The study of volatile
fraction of cocoa beans by Solid-Phase Microextraction
(SPME) coupled to Gas Chromatographic (GC) Mass
Spectrometry (MS) systems is very important for defin-
ing the quality and the flavor of the end product as
chocolate bars or cocoa-based beverages. Moreover, the
fatty acids composition of cocoa beans is of enormous
interest for processing and quality control during
manufacture of the product (Torres-Moreno et al.
2015), and also because the content of Saturated Fatty
Acids (SFA), MonoUnsaturated Fatty Acids (MUFA)
and PolyUnsaturated Fatty Acids (PUFA) is a key fac-
tor in the health-promoting properties of cocoa.
In this paper, we report for the first time the results
of analysis of cocoa beans collected in different parts
of Cameroon, highlighting the opportunities afforded
by using different analytical devices to obtain comple-
mentary and thus more complete information.
Materials and methods
Collection areas and identification
Cocoa beans were collected in the principal areas for
farming cocoa in Cameroon: (1) Bertoua in the east,
(2) Mbalmayo in the south, (3) Penja on the Atlantic
coast, (4) Sanchou in the west, and (5) Obala in central
Cameroon (Figure 1). Theobroma cacao was authenti-
cated at the National Herbarium of Cameroon in
Yaounde. The variety of all samples of Theobroma
cacao was Trinitario and the voucher specimen was
60071HNC. One kilogram of cocoa bean samples were
Penja
Mbalmayo
Figure 1. The five areas in Cameroon where cocoa beans were
collected.
collected between December 2012 and January 2013 by
producers of each region. After collection of samples,
the external pods of cocoa beans have been removed,
and then they have been placed on banana leaves for
fermentation process for 3–5 days. After this time,
cocoa beans were dried using ‘‘sun drying method’’
(from 1 to 2 weeks) and then packaged under vacuum
at room temperature until the time of analysis.
Proximate analysis
Cocoa beans samples were analyzed for chemical com-
position (moisture, protein, carbohydrates, fats) using
AOAC procedures (AOAC 1995) (Table 1). The mois-
ture content was calculated by oven drying the sample
to a constant weight (24 h, 133  C). The crude protein
content of the samples was estimated by the Kjeldahl
method; the crude fat was determined by extracting a
known weight of powdered sample with petroleum
ether, using a Soxhlet apparatus; the ash content was
determined by incineration at 600 ± 15  C. Total energy
was calculated according to the following equation
(Manzi et al. 2004):
Energy ðKcalÞ ¼ 4  ðg protein þ g carbohydrateÞ
þ 9  ðg lipidÞ
Chemicals and reagents
The SPME fiber assembly used (Supelco, Bellofonte,
PA) was Divinylbenzene/Carboxen/Polydimethylsiloxane

Table 1. Proximate composition of Cameroonian cocoa bean samples.
Cocoa bean samples Moisture Proteins
1 Bertoua 4.25 ± 0.18 11.7 ± 0.24
2 Sanchou 3.24 ± 0.14 12.07 ± 0.24
3 Mbalmayo 3.12 ± 0.13 11.87 ± 0.24
4 Penja 3.85 ± 0.16 11.80 ± 0.24
5 Obala 3.21 ± 0.13 13.35 ± 0.27
a
Values, referred to fresh matter, are means of three determinations. Composition reported in %, ± standard deviation.
(DVB/CAR/PDMS) 50/30 lm. Methanol (99.9%),
chloroform, ethyl acetone, toluene, formic acid and the
alkane mixture were purchased from Sigma-Aldrich
(Milano, Italy). Acetic acid 99  100% was bought
from J.T. Baker B.V. (Deventer, The Netherlands),
whereas n-hexane was supplied by Fluka-Riedel-
Downloaded by [University of California, San Diego] at 20:52 15 April 2016
deHa€en (Milano, Italy). Sodium sulfate anhydrous,
sodium chloride and potassium hydroxide were pur-
chased from Panreac Quimica SA (Barcelona, Spain).
Deionized water (>18 MX cm resistivity) was obtained
from a Milli-Q SP Reagent Water System (Millipore,
Bedford, MA). All solvents and solutions were filtered
through 0.45-lm PTFE filters purchased from
Phenomenex (Bologna, Italy). The reference standards
Theobromine and (-)-Epicatechin were purchased from
Sigma-Aldrich (Milano, Italy) and Caffeic Acid from
Extrasynthese (Genay, France).
HPTLC and densitometric analysis
The HPTLC system (CAMAG, Muttenz, Switzerland)
consisted of a Linomat 5 sample applicator using
100 ll syringes and connected to a nitrogen tank; an
automatic developing chamber ADC 2 containing twin
20  10 cm trough chambers; an immersion device III;
a TLC Plate Heater III; and a TLC visualizer linked to
winCATS software (Alfatech, Genova, Italy). Glass
plates 20 cm 10 cm (Merck, Darmstadt, Germany)
with glass-backed layers silica gel 60 (2 lm thickness)
were prewashed with methanol and dried for 3 min at
100  C. Filtered solutions of extract and standards were
applied with nitrogen flow. The operating conditions
were: syringe delivery speed, 100 nl s  1; injection vol-
ume, 4 ll; band length, 8 mm; distance from bottom,
8 mm. The HPTLC plates were developed in ethyl acet-
one, toluene, formic acid (9:9:2 v/v/v) using the auto-
matic and reproducible developing chamber ADC 2,
saturated with the same mobile phase for 20 min at
25  C. The developing solvents (i.e. type of solvents
and ratios) were carefully optimized before the analysis.
The length of the chromatogram run was 70 mm from
the point of application.
The developed layers were allowed to dry at 100  C
for 5 min and then derivatized with a selected
INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION 3
Composition %a
Carbohydrates Fats Energy (kcal) Energy (kJ)
32.14 ± 0.96 36.34 ± 1.55 502 ± 15 2100 ± 63
33.16 ± 0.99 43.12 ± 1.84 569 ± 17 2382 ± 71
39.63 ± 1.19 30.03 ± 1.28 476 ± 14 1993 ± 60
43.35 ± 1.30 24.63 ± 1.05 442 ± 13 1849 ± 55
24.38 ± 0.73 42.60 ± 1.82 534 ± 16 2233 ± 67
solution, including Natural Product Reagent (NPR)
(1 g diphenylborinic acid aminoethylester in 200 ml of
ethyl acetate); the plate was heated at 100  C for
2–3 min and then dipped into anisaldehyde-sulfuric
acid (1 ml p-anisaldehyde, 10 ml H2SO4, 20 ml AcOH
in 170 ml MeOH). Finally, the plates were dried for
5 min at 120  C before inspection. All treated plates
were then inspected under a UV light at 254 or
366 nm or under reflectance and transmission white
light (WRT), respectively, at a Camag TLC visualizer,
before and after derivatization. WinCATS software
1.4.4 was used for the documentation of derivatized
plates. Therefore, for each plate at least six different
visualizations were obtained (Figure 2).
For the densitometric analysis, the scanner was set
at 250 nm, after a previous trial of multi-wavelength
scanning between 190 and 800 nm in the absorption
mode. Minimum background compensation was per-
formed on the x-axis during the scanning. The sources
of radiation were deuterium and tungsten lamps. The
slit dimension was kept at 6.00  0.45 mm and the
scanning speed used was 100 mm s  1. Sample solu-
tions of the extracts were found to be stable at 4  C for
at least 1 month and for at least 3 days on the HPTLC
plates. Repeatability was determined by running a min-
imum of three analyses. Retention factor (RF) values
for the main compounds selected varied at ±0.02%.
The effects of small changes in the mobile phase com-
position, mobile phase volume, and duration of satur-
ation were minute and reduced by direct comparison.
On the contrary, the results were critically dependent
on prewashing of HPTLC plates with methanol.
Headspace-solid-phase microextraction
Powdered cocoa sample was used for headspace solid
phase microextraction (HS–SPME) coupled with Gas
Chromatography–Mass Spectrometry (GC–MS). The
sample (1 g powdered cocoa) was placed in a 20 ml
vial, which was tightly capped with a PTFE–silicon
septum. The sample was kept in water bath at 60  C
with stirring at 1300 rpm for 15 min for conditioning,
then SPME fiber coated with DVB/CAR/PDMS was
exposed to the headspace of the sample for another
 4 G. CAPRIOLI ET AL.
(A) (B)
1 2 3 4 5 1
Figure 2. HPTLC fingerprint analysis of T. cacao beans extracts from Cameroon. (A) Visualization, UV 254 nm, without derivatization.
(B) Visualization, UV 366 nm, derivatization with Natural Product reagent. (C) Visualization, white light RT, derivatization with Natural
Product reagent and anisaldehyde. Visualization. Tracks: 1, Bertoua - East; 2, Mbalmayo - South; 3, Penja – Atlantic coast; 4, Sanchou
- West; 5, Obala - Center.
Downloaded by [University of California, San Diego] at 20:52 15 April 2016
15 min for extraction. The fiber was then retracted and
inserted into the GC injection port. Desorption was
performed for 3 min, with the injector temperature at
260  C and in splitless mode. The desorption time was
sufficient to desorb most of the analytes, and then the
extract was directly transferred to the analytical col-
umn for analysis.
GC–MS analysis for volatile compounds
A gas chromatograph equipped with mass selective
detector (GC-MSD) (Agilent, Santa Clara, CA; Agilent
6890N with Agilent 5973N) was used. Separation was
performed on an HP-5 MS column (5% phenylmethyl-
polysiloxane, 30 m, 0.25 mm i.d., 0.1 lm film thickness)
(J&W Scientific, Folsom, CA). An Agilent Chem work-
station was used with the GC-MS system. The flow
rate (He) was 1 ml min  1 under splitless mode. The
injector temperature was 260  C. The column tempera-
ture program was: from 40  C (5 min) to 200  C at
10  C min  1, then 200  C for 10 min. Data were
acquired in the electron impact (EI) mode with an ion-
ization voltage of 70 eV, using the scan ion monitoring
(SCAN mode). A mixture of aliphatic hydrocarbons
(C5–C30) (Sigma, Milan, Italy) diluted in n-hexane, was
absorbed onto the SPME fiber and injected under the
temperature program described above to calculate the
linear retention indices of each extracted compound.
The peak assignment was based on computer matching
with the WILEY275, NIST 08, ADAMS, taking into
account the coherence of the linear retention indices of
the analyzed compounds with those reported by
Adams (2007) and the NIST 08 library (NIST 08 2008)
in accordance with the standard of the International
Organization of the Flavor Industry (IOFI, http://www.
iofi.org/) statement. The results from the HS-SPME-
GC-MS analysis of volatile components extracted by
(C)
2 3 4 5 1 2 3 4 5
the SPME fiber were expressed as percentages,
obtained by dividing the area of each component by
the total area of all isolated components under the
conditions described above. Data were analyzed by
using MSD Chem Station software (Agilent, Version
G1701DA D.01.00).
Multivariate analysis (PCA)
To reveal the relationship among cocoa samples in
terms of volatile components, and to identify the main
constituents influencing variability, the composition
data matrix of five samples (22 variables 5 samples
¼110 data) was analyzed using principal component
analysis (PCA) with STATISTICA 7.1 (Stat Soft Italia
srl, 2005, www.statsoft.it) (Sagratini et al. 2012).
Eigenvalues were calculated using a covariance matrix
among 22 chemical compounds as input, and the two-
dimensional PCA biplot, including both samples of dif-
ferent origin and compounds, was generated.
Lipid extraction
Finely ground cocoa sample (2.5 g) was hydrated with
10 ml of deionized water, the minimum amount of
water required to wet the flour, for 1 h under magnetic
stirring at room temperature (Caprioli et al. 2016).
Each extraction was performed in triplicate. The solv-
ent combination of chloroform/methanol (CHCl3/
MeOH, 2/1, 86 ml) was added to the sample and
extracted for 3 min using a homogenizer (Ultraturrax,
IKA, Staufen, Germany) (Folch et al. 1957). The fil-
tered solution was collected into a separating funnel;
the solid residue was washed with 22 ml of the extract-
ive solvent that had also been collected in the separat-
ing funnel where the organic phase was washed with
21.4 ml 0.73% w/v NaCl aqueous solution. Afterwards,

the organic phase was siphoned, dried over anhydrous
sodium sulfate, filtered, and dried under reduced pres-
sure with a rotary evaporator. The lipid extract was
weighed and subjected to transesterification.
Transesterification and gas chromatographic
analysis
Derivatization following Ichihara’s procedure (Ichihara
et al. 1996) was performed on each lipid extract to obtain
fatty acid methyl esters (FAMEs). Briefly, a quantity
between 10 and 15 mg of lipid extract, dissolved in 1 ml
hexane, was added to 0.1 ml of 2 M KOH in methanol
and vortexed for 2 min. After the addition of 1.5 ml 0.15
M of aqueous acetic acid, the solution was vortexed for
1 min and centrifuged at 5000 rpm for 10 min. The
Downloaded by [University of California, San Diego] at 20:52 15 April 2016
upper phase was transferred while 1 ml hexane was
added to the remaining one, which was then vigorously
shaken and centrifuged at 5000 rpm for 5 min. This
operation was repeated three times. Traces of water in
the total hexane phase were removed with sodium sul-
fate. Finally, the n-hexane solution containing fatty acid
methyl esters (FAMEs) was sealed and stored at 20  C.
Before the analysis, FAMEs solutions were evaporated to
dryness with a nitrogen stream, dissolved in 1 ml of n-
hexane and analyzed in duplicate. The gas chromato-
graph used was an Agilent Technologies 6850 equipped
with a flame ionization detector (FID). The capillary
chromatographic column used was a 50% cyanopropyl-
phenyl-dimethylpolysiloxane DB-225 Agilent
Technologies (length 30 m, ID, 0.32 mm; film thickness,
0.25 mm) and the injected volume was 1 ll. The split
ratio was 1:30 and the injector temperature was 260  C.
The carrier gas was H2 and its flow was 3.7 ml/min in
the column. The oven temperature was held at 60  C for
3 min, then programed to increase to 220  C at
20  C/min, and held at 220  C for 8 min. The FID
detector temperature was 250  C (air flow 400.0 ml/min,
H2 flow 40.0 ml/min). Each analysis took 19 min.
Identifications were done by analyzing the standard
‘‘Supelco 37 Component FAME Mix’’ purchased from
Supelco (Bellefonte, PA) and by GC-MS. Peak areas
obtained from FAMEs were corrected using theoretical
response factors (Ackman & Sipos 1964).
Results and discussion
Chemical composition of cocoa bean samples
The chemical composition and estimated energy value
results for the five cocoa bean samples are shown in
Table 1. The moisture content ranged from 3.12% of
sample n.3 from Mbalmayo to 4.25% of sample n.1
from Bertoua. The most abundant nutrients in cocoa
INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION 5
samples were fats (24.63%–43.12%) and carbohydrates
(24.38%–43.35%), while proteins were present in lower
amounts (11.70%–13.35%).
The highest content of fats was found in sample n.5
from Obala, the lowest in sample n.4 from Penja. The
highest level of carbohydrates was found in sample n.4
from Penja, while the lowest was in sample n. 5 from
Obala. The lowest value of protein was found in sam-
ple n.1 from Bertoua and the highest in sample n. 5
from Obala. On the basis of the proximate analysis, it
was calculated that 100 g of cocoa bean provided from
1849 (sample n. 4) to 2382 kJ (sample n. 2).
Our results are in agreement with those published by
Torres-Moreno et al. (2015) on cocoa from Ecuador
and Ghana. They found that the moisture content was
less than 6%, and that fats were the major nutrient of
the cocoa examined, followed by carbohydrates and
proteins. However, our results concerning sample n.3
from Penja on the Atlantic coast and sample n.4 from
Sanchou in the west were particular: these two samples
displayed higher percentages of carbohydrates than of
fats.
On the other hand, the level of fats obtained in our
samples were a bit lower than those found by Liendo
et al. (1997) for Venezuelan cocoa, as they reported lev-
els ranging from 46.08% to 55.93%. These differences
can be explained by considering the different geographic
origin, genetic variability and post-harvesting processing
of the cocoa samples, which can significantly affect their
composition (Torres-Moreno et al. 2015).
HPTLC analysis
The HPTLC can be used to perform metabolome stud-
ies, such as determination of most of the constituents of
an extract (Gallo et al. 2011). Here, HPTLC was selected
to investigate the differences in composition among the
samples of cocoa beans. In particular, HPTLC was
adapted for analysis of the polyphenol content, by
selecting the adequate mobile phase and revelation
agents. The main product of HPTLC analysis is the
chromatographic fingerprint, consisting in the individ-
ual track typical of the extract or the product in a plate.
The chromatographic fingerprint analytic approach has
received important official recognition (TLC Atlas of
Chinese Pharmacopeia 2009). Plates can be visualized
and derivatized in several ways, obtaining multiple
pieces of information, as well as converted into a series
of peaks by densitometric treatment. In this way, com-
parison between samples is reliable and facilitated by
visual inspection, and samples can be analyzed side-by-
side and exactly in the same conditions (Gallo et al.
2011; Toniolo et al. 2014).
 6 G. CAPRIOLI ET AL.

The HPTLC fingerprints of the analyzed samples of
cocoa beans are reported in Figure 2. The same sam-
ples are visualized at different wavelengths, namely (A)
UV 254 nm, without derivatization, (B) UV 366 nm,
derivatization with Natural Product Reagent, (C) white
light RT, derivatization with Natural Product Reagent
and anisaldehyde, to obtain the evidence of substance
in different chromatographic conditions.
Representative polyphenols of cocoa beans (caffeic
acid, (-)-epicatechin) and theobromine were quantified
Downloaded by [University of California, San Diego] at 20:52 15 April 2016
by HPTLC analysis. The results are reported in Figure 3.
Caffeic acid was present in a concentration range of
1.1–2.1 mg/g dried extract, theobromine in a concentra-
tion range of 4.7–11.6 mg/g dried extract and (-)-epica-
techin in a concentration range of 1.1–142.9 mg/g dried
extract. The most interesting samples seemed to be
those from Mbalmayo, which showed the highest level
of theobromine (11.6 mg/g) and caffeic acid (2.1 mg/g),
and from Sanchou, which presented the highest level of
(-)epicatechin (142.9 mg/g). The lowest quantity of
theobromine (4.7 mg/g) and (-)-epicatechin (1.1 mg/g)
was detected in the sample from Penja, while samples
from Bertoua and Obala showed intermediate values of
concentration of these compounds. The levels of theo-
bromine found are in according with those reported by

Brunetto and collaborators (until 12 mg/g) (Brunetto

et al. 2007) that used HPLC-UV system for analyzing
this analyte in cocoa beans.

HS-SPME-GC/MS analysis of volatile compounds

Figure 3. HPTLC polyphenols and theobromine content of The volatile components of cocoa samples, extracted
Cameroonian cocoa samples. Each sample was analyzed in tripli- by HS-SPME, are reported in Table 2. A total of 22
cate (n ¼ 3) and in all cases RSD % was lower than 2.6. components were detected by GC-MS. Among these
Table 2. Volatile components identified in cocoa samples and related percentages of area values obtained using the PDMS/DVB/
CAR fiber in the HS-SPME-GC-MS analysis.
Mbalmayo
No. Componenta Calc. RI Lit.RI Bertoua Sanchou Area values %b,c Penja Obala
1 ethanol – nd nd 14.92 nd nd
2 methyl acetate – 11.30 4.11 nd 6.51 6.28
3 acetic acid 642 642 31.21 18.20 13.97 14.61 43.07
4 3-methylbutanal 650 649 19.10 3.26 nd 8.25 nd
5 2-pentanone 695 695 6.53 2.71 16.72 2.07 6.48
6 2-pentanol 732 730 nd 15.76 19.08 9.16 18.08
7 3-methylbutanol 736 738 nd 16.45 nd 6.92 8.49
8 4-methylheptane 761 762 4.02 3.74 2.57 3.92 nd
9 1,3-butanediol 787 788 nd 3.14 nd 3.89 1.86
10 2,3 butanediol 791 793 nd 2.28 nd 6.04 2.33
11 2,4-dimethylheptane 819 818 nd 3.24 nd nd nd
12 2,4 dimethyl-1-heptene 839 836 9.38 3.81 9.57 4.37 1.41
13 ethyl 2-methylbutanoate 851 850 nd nd nd 0.60 nd
14 2-pentanol acetate 852 854 nd 2.66 nd nd 1.47
15 3-methylbutyl acetate 880 881 nd 5.31 nd 0.77 3.67
16 benzaldehyde 977 974 1.62 nd nd 1.09 0.12
17 nonanal 1105 1103 nd 0.52 nd nd nd
18 2-phenylethanol 1116 1116 nd 4.76 nd 4.20 nd
19 benzocyclohexane 1154 1154 nd nd nd 0.30 nd
20 ethyl octanoate 1198 1197 nd 0.09 nd nd nd
21 1,3-di-tert-butylbenzene 1256 1249 0.59 0.09 2.35 1.92 nd
22 2-phenylethylacetate 1264 1264 nd 0.33 nd 0.14 nd
Total identified [%] 83.74 90.46 79.17 74.76 93.26
Aldehydes 20.72 3.78 nd 9.34 0.12
Ketones 6.53 2.71 16.72 2.07 6.48
Alkanes/alkenes 13.40 10.78 12.14 8.29 1.41
Esters 11.30 12.50 nd 8.03 11.42
Alcohols nd 42.39 34.00 30.21 30.75
Aromatics (benzene derivatives) 0.59 0.09 2.35 2.22 nd
Carboxylic acids 31.21 18.20 13.97 14.61 43.07
a
Compounds are listed in order of their elution from a 5% phenylpolymethylsiloxane GC column (detection at GC/MS); percentage values are means of three
determinations.
b
Relative standard deviations % ranged from 0.37 to 8.81%.
c
nd: not detected (area value below 500).
 INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION 7

volatiles, there were six esters, six alcohols, three alka-
nes, three aldehydes, two benzene derivatives, one
ketone and one carboxylic acid (acetic acid).
Results are reported as area percentages. The chem-
ical classes with the highest area percentages were car-
boxylic acids, in particular acetic acid
(13.97%–43.07%), present in all samples, and alcohols
(30.2%–42.39%), found in four samples but instead not
detected in the Bertoua sample. Concerning alcohols,
those present with the highest area percentages were 2-
pentanol (9.16%–19.08%) and 3-methylbutanol
(6.92%–16.45%). The latter was not detected in the
Bertoua or Mbalmayo samples. On the other hand,
total alkanes/alkenes area percentages ranged from
1.41% in the Obala sample to 13.40% in the Bertoua
Downloaded by [University of California, San Diego] at 20:52 15 April 2016
‘‘sour-vinegar’’. On the contrary, samples from
Sanchou and from Penja displayed low area percen-
tages for acetic acid (18.20% and 14.61%, respectively)
and corresponding high values for 3-methylbutanol
(amyl alcohol) and phenylethyl alcohol.
Alcohols are responsible for producing desirable fla-
vor notes (Jinap et al. 1998; Ducki et al. 2008); of
interest are 3-methylbutanol, which is associated with a
malty-chocolate odor, and 1,3-n-butanediol and 2,3-n-
butanediol, which instead are associated with a sweet-
citrusy odor.
Moreover, it has been suggested that the esterifica-
tion of amyl alcohols to amyl acetates could be used as
a fermentation index (Oberparleiter & Ziegleder 1997;
Rodriguez-Campos et al. 2011).

sample, with highest values given by 2,4-dimethyl-1-

heptene. Total esters area percentage ranged from
8.03% in the Penja to 12.50% in the Sanchou sample.
They were not detected in the Mbalmayo sample. The
ester detected with highest area percentage was methyl
acetate (4.11%–11.30%); only in the Mbalmayo sample
was this ester not detected.
In general, various differences in volatile chemical
profiles were detected among the analyzed samples.
For example, the Mbalmayo sample gave high area
percentage of ethanol (14.92%) and the lowest values
of acetic acid (13.97%). It is known that acetic acid is
produced by the biochemical synthesis from the oxida-
tion of ethanol during the first stages of fermentation
(Schwan & Wheals 2004). Samples from Bertoua and
Obala showed the highest values for acetic acid,
31.21% and 43.07%, respectively, and thus they could
be considered ‘‘fermented’’ and of low quality, since
acetic acid is associated with the odor description of
Multivariate analysis (PCA)
Multivariate analysis revealed that the cocoa samples
were well separated from each other, according to their
volatile constituents. The 2D graphical representation
of the principal-component analysis is shown in Figure 4,
and represents 74.3% of the total variance in the data
set. The variability of data was generated mostly by the
content of ethanol (values of eigenvectors: 5.16;
0.16) and acetic acid (of eigenvectors: 10.40; 5.97) in
the first PC, and by 3-n-methylbutanal (4.60; 6.49)
and 2-n-pentanol (4.70; 6.38) in the second PC.
Cocoa samples showed a weak correlation with each
other, as they were well separated in the score plot.
The Mbalmayo sample was strongly correlated with a
high level of ethanol and low level of acetic acid, while
Obala sample showed low level of ethanol and high
level of acetic acid. The Bertoua sample was

(A) 30 (B) 14

25 12

5 10
20 Obala
8
15 2-pentano l acetic acid
6
10
2
PC 2 : 31,20%
PC 2: 31,20%

4 1-butanol,3-m eth yl
Sanchou
5
3 1-b utanol,3-m ethyl acetate
2
Mbalm ayo Var14
Var9
0 2-pen tanone Var11
Var10
Var17
Var20
Var22
Var19
Var13
ethanol Var18
Var21
0 Var16
Var8
acetic acid m ethyl este r
-5
P enja -2 2,4-d im ethyl-1-hepte ne

-10 4
-4
Bertoua
-15 3-m ethyl butana l
1 -6

-20 -8

-25 -10
-30 -25 -20 -15 -10 -5 0 5 10 15 20 25 -8 -6 -4 -2 0 2 4 6 8 10 12

PC 1: 43 ,14% PC 1 : 43,14%

Figure 4. (A) Score plot (PCA) for the main variations in the cocoa seeds, depending on the volatile components; (B) the PCA load-
ing plot for contents of the analyzed volatile constituents.
 8 G. CAPRIOLI ET AL.

Table 3. Percentagea fatty acid composition, SFA, MUFA, PUFA, and n-6/n-3 ratio, in the cocoa samples under investigation.
C 18:1 MUFAs
No. Cocoa samples C 16:0 C 18:0 % C 18:2, n-6 C 18:3, n-3 SFAs % PUFAs n-6/n-3
1 Bertoua 24.84 ± 0.51 39.08 ± 1.07 33.23 ± 1.51 2.74 ± 0.19 0.12 ± 0.009 63.92 33.23 2.86 22.83
2 Sanchou 27.5 ± 0.89 37.66 ± 0.76 32.00 ± 0.39 2.76 ± 0.21 0.08 ± 0.012 65.16 32.00 2.84 34.50
3 Mbalmayo 28.2 ± 1.27 37.60 ± 0.27 31.50 ± 0.91 2.52 ± 0.17 0.19 ± 0.067 65.80 31.50 2.71 13.26
4 Penja 27.31 ± 2.17 39.54 ± 1.11 30.40 ± 2.19 2.32 ± 0.21 0.44 ± 0.052 66.84 30.40 2.76 5.27
5 Obala 26.03 ± 0.61 38.41 ± 0.44 32.39 ± 0.34 2.69 ± 0.11 0.48 ± 0.243 64.44 32.39 3.17 5.60
a
Results are the mean of three independent experiments. Composition reported in %, ± standard deviation. SFAs: Saturated fatty acids; MUFAs: monounsatu-
rated fatty acids; PUFAs: polyunsaturated fatty acids.

characterized by 3-methylbutanol, while samples from Conclusions

Sanchou and Penja were also influenced by other
In this work, we report for the first time the nutritional
components, such as 2-pentanol and 2,4-dimethyl-1-
composition, theobromine and polyphenolic profile,
n-heptene, respectively. In particular, 3-methylbutanol
volatile fraction, and fatty acid composition of cocoa
is a desiderable compound for high-quality cocoa
beans from different parts of Cameroon. The proxim-
Downloaded by [University of California, San Diego] at 20:52 15 April 2016

products and, both with other compounds (alde-

ate analysis showed that the principal nutrients of these
hydes), is considered as an important index for evalu-
cocoa beans were lipids and carbohydrates; the samples
ating the fermentation degree (Rodriguez-Campos
from Mbalmayo and Penja had a higher content of
et al. 2011).
carbohydrates at 39.63% and 43.35%, respectively,
PCA confirmed that the geographic origin of the
compared to lipids at 30.03% and 24.63%. HPTLC
samples, together with the type of processing used sig-
analysis showed particularly interesting results for the
nificantly influenced the volatile composition of the
samples from Mbalmayo, which displayed the highest
cocoa beans.
content of theobromine (11.6 mg/g) and caffeic acid
(2.1 mg/g), and Sanchou, which showed the highest
Fatty acid profile level of (-)-epicatechin (142.9 mg/g). The HS-SPME-
GC/MS analysis of the composition of the volatile
The fatty acid profile was determined in the cocoa
fraction of the cocoa beans identified 22 molecules
samples from the five areas. The percentage compos-
belonging to the classes of esters, alcohols, alkanes,
ition of individual fatty acids, and the total percentage
aldehydes, benzene derivatives, ketones and carboxylic
of SFAs, MUFAs and PUFAs obtained are reported in
acids. The Mbalmayo sample showed the lowest level
Table 3. Among the fatty acids detected, stearic acid
of acetic acid (13.97%), a component that is considered
(C18:0) was the most abundant (37.60%–39.54%) fol-
an index of poor quality in cocoa. Analysis of the com-
lowed by oleic acid (C18:1, n-9) found in the range
position in fatty acids showed that SFAs were most
30.40%–33.23% and palmitic acid (C16:0), found in
abundant (63.92%–66.84%), followed by MUFAs
the range 24.84%–28.2%.
(30.40%–33.23%) and finally by PUFAs (2.71%–3.17%).
The highest level of stearic acid was found in sam-
The lowest level of stearic acid was found in the
ple n.4 from Penja, followed by sample n.1 from
Mbalmayo sample (37.60%), while the Bertoua sample
Bertoua. The latter also displayed the highest level of
showed the highest content of oleic acid (33.23%).
oleic acid. Concerning the two MUFAs, the highest
In conclusion, the cocoa bean samples investigated,
level of linoleic acid was found in sample n.2 from
coming from different geographic areas of Cameroon,
Sanchou (2.76%); sample n.5 from Obala, instead,
showed different nutritional characteristics and, for this
showed the highest level of linolenic acid (0.48%).
reason, industries that use this raw material for the
Regarding the distribution among SFAs, MUFAs and
production of cocoa foods should take into account
PUFAs, cocoa samples were thus mainly constituted by
this fact. Moreover a detailed quantification of healthy
SFAs (63.92%–66.84%), followed by MUFAs
substances as theobromine, caffeic acid and ()-epica-
(30.40%–33.23%), and finally by PUFAs
techin related to specific areas and provenience should
(2.71%–3.17%), with a range of 5.27–34.50 for the
be considered by industries that produce food supple-
value of the ratio PUFA n-6/n-3.
ments and nutraceuticals.
The fatty acid composition found is in agreement
with the results already reported by Liendo et al.
Acknowledgements
(1997) and Torres-Moreno et al. (2015). The low con-
tent of PUFAs causes these samples to be less prone to The authors would like to thank Sheila Beatty for her editing
lipid oxidation. of the English usage in the manuscript.

Disclosure statement
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
This article does not contain any studies with human or ani-
mal subjects.
References
Ackar D, Lendıc KV, Valek M, Subaric D, Milicevic B, Babic J,
Nedic I. 2013. Cocoa polyphenols: can we consider cocoa and
chocolate as potential functional foods? J Chem. 2013:1–7.
Ackman RG, Sipos JC. 1964. Application of specific response
factors in the gas chromatographic analysis of methyl
esters of fatty acids with flame ionization detectors. J Am
Oil Chem Soc. 41:377–378.
Adams RP. 2007. Identification of essential oil components
by gas chromatography/mass spectrometry. Carol Stream
Downloaded by [University of California, San Diego] at 20:52 15 April 2016
(IL): Allured Publishing Corporation.
And ujar MCR, Giner RM, Rıos JL. 2012. Cocoa polyphenols
and their potential benefits for human health. Oxid Med
Cell Longev. 2012:1–23.
AOAC. 1995. Official methods of analysis. 16th ed. Arlington
(VA): Association of Official Analytical Chemists.
Brunetto MDR, Gutièrrez L, Delgado Y, Gallignani M,
Zambrano A, G omez A, Ramos G, Romero C. 2007.
Determination of theobromine, theophylline and caffeine
in cocoa samples by a high-performance liquid chromato-
graphic method with on-line sample cleanup in a switch-
ing-column system. Food Chem. 100:459–467.
Caprioli G, Giusti F, Ballini R, Sagratini G, Vila-Donat P,
Vittori S, Fiorini D. 2016. Lipid nutritional value of
legumes: evaluation of different extraction methods and
determination of fatty acid composition. Food Chem.
192:965–971.
Cooper KA, Donovan JL, Waterhouse AI, Williamson G.
2008. Cocoa and health: a decade of research. Br J Nutr.
99:1–11.
De Almeida AA, Valle RR. 2007. Ecophysiology of the cacao
tree. Braz J Plant Physiol. 19:425–448.
Ducki S, Miralles-Garcia J, Zumbe A, Tornero A, Storey
DM. 2008. Evaluation of solid-phase micro-extraction
coupled to gas chromatography-mass spectrometry for the
headspace analysis of volatile compounds in cocoa prod-
ucts. Talanta. 74:1166–1174.
Folch J, Lees M, Stanley GHS. 1957. A simple method for
the isolation and purification of total lipids from animal
tissues. J Biol Chem. 226:497–509.
Gallo FR, Multari G, Federici E, Palazzino G, Giambenedetti
M, Petitto V, Poli F, Nicoletti M. 2011. Chemical finger-
printing of Equisetum arvense L. using HPTLC densitom-
etry and HPLC. Nat Prod Res. 25:1261–1270.
Han X, Shen T, Lou H. 2007. Dietary polyphenols and their
biological significance. Int J Mol Sci. 8:950–988.
Ichihara K, Shibahara A, Yamamoto K, Nakayama T. 1996.
An improved method for rapid analysis of the fatty acids
of glycerolipids. Lipids. 31:535–539.
International Cocoa Organization. 2012; [cited 2011 Sep 08].
Available from: http://www.icco.org/about-us/inter-
national-cocoa-agreements/cat_view/30-related-documents/
45-statistics-other-statistics.html.
INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION 9
Jinap S, Wan-Rosli WI, Russly AR, Nordin LM. 1998. Effect
of roasting time and temperature on volatile component
profiles during nib roasting of cocoa beans (Theobroma
cacao). J Sci Food Agr. 77:441–448.
Li Y, Feng Y, Zhu S, Luo C, Ma J, Zhong F. 2012. The effect
of alkalinization on the bioactive and flavor related com-
ponents in commercial cocoa powder. J Food Compos
Anal. 25:17–23.
Liendo R, Padilla FC, Quintanab A. 1997. Characterization
of cocoa butter extracted from Criollo cultivars of
Theobroma cacao L. Food Res Int. 30:727–731.
Manzi P, Marconi S, Aguzzi A, Pizzoferrato L. 2004.
Commercial mushrooms: nutritional quality and effect of
cooking. Food Chem. 84:201–206.
NIST 08. 2008. Mass spectral library (NIST/EPA/NIH).
Gaithersburg (MD): National Institute of Standards and
Technology.
Oberparleiter S, Ziegleder G. 1997. Amyl alcohols as com-
pounds indicative of raw cocoa bean quality. Z Lebensm
Unter Forsch. 204:156–160.
Rodriguez-Campos J, Escalona-Buendıa HB, Orozco-Avila I,
Lugo-Cervantes E, Jaramillo-Flores E. 2011. Dynamics of
volatile and non-volatile compounds in cocoa (Theobroma
cacao L.) during fermentation and drying processes using
principal components analysis. Food Res Int. 44:250–258.
Rusconi M, Conti A. 2010. Theobroma cacao L., the Food of
the Gods: a scientific approach beyond myths and claims.
Pharmacol Res. 61:5–13.
Sagratini G, Maggi F, Caprioli G, Cristalli G, Ricciutelli M,
Torregiani E, Vittori S. 2012. Comparative study of aroma
profile and phenolic content of Montepulciano monovarie-
tal red wines from the Marches and Abruzzo regions of
Italy using HS-SPME-GC-MS and HPLC-MS. Food
Chem. 132:1592–1599.
Saltini R, Akkerman R, Frosh S. 2000. Optimizing chocolate
production through traceability: a review of influence of
farming practices on cocoa beans quality. Food Res Int.
33:449–459.
Schwan RF, Wheals AE. 2004. The microbiology of cocoa
fermentation and its role in chocolate quality. Crit Rev
Food Sci Nutr. 44:205–221.
Serafini M, Bugianesi R, Maiani G, Valtuena S, De Santis S,
Crozier A. 2003. Plasma antioxidants from chocolate.
Nature. 424:1013.
Teye E, Huang X, Dai H, Chen Q. 2013. Rapid differenti-
ation of Ghana cocoa beans by FT-NIR spectroscopy
coupled with multivariate classification. Spectrochim Acta
a Mol Biomol Spectrosc. 114:183–189.
TLC Atlas of Chinese Crude Drugs in Pharmacopoeia of the
People’s Republic of China. 2009. Chinese Pharmacopoeia
Commission. Bejing, China: People’s Medical Publishing
House.
Toniolo C, Nicoletti M, Maggi F, Venditti A. 2014. HPTLC
determination of chemical composition variability in raw
materials used in botanicals. Nat Prod Res. 28:119–126.
Torres-Moreno M, Torrescasana E, Salas-Salvad o J, Blanch C.
2015. Nutritional composition and fatty acids profile in
cocoa beans and chocolates with different geographical ori-
gin and processing conditions. Food Chem. 166:125–132.