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DOI 10.1007/s00217-012-1812-x
ORIGINAL PAPER
Introduction
The flavour of cocoa beans (seeds of Theobroma cacao)
and products thereof is not only dependent on the genotype
but largely determined by chemical and microbial reactions
during processing and manufacturing. Fermentation and
drying of the raw cocoa beans are the first essential steps
for the development of the characteristic flavour. Reactions
occurring during fermentation depend on the water content
and are catalysed by enzymes of the living seed; they are
followed by non-enzymatic reactions in a dry lipid phase.
The resulting dried cocoa beans are further treated to
produce various cocoa products and chocolates. Roasting
of the raw cocoa beans is the major step during this manufacturing and essential for the aroma of the final product.
Comprehensive overviews on cocoa processing have been
published [1, 2].
Since the first report on the study of cocoa aroma in
1912, in which linalool was identified amongst the main
volatile constituents in an extract prepared from about 2
tons of roasted cocoa beans [3], hundreds of volatile
compounds have been identified in different types of cocoa
[4]. In recent years, studies focused on the investigation of
the aroma-relevant compounds in cocoa and chocolate
[57] and their changes during the roasting process [8].
Within the terpene fraction of the cocoa aroma, linalool
(3,7-dimethyl-1,6-octadien-3-ol) is an important component and contributes to the flowery and tea-like flavour of
some cocoa varieties, for example, the Criollo type [9, 10].
Such most priced Criollo cocoas are classified as flavourgrade because of their aromatic, mild, flowery, and tea-like flavour quality. They are mainly harvested in Venezuela,
Trinidad, and Ecuador (Arriba) and contain significant
amounts of linalool (0.52 mg kg-1) and other terpenoids,
which have been reported to contribute to their valuable
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Table 1 Analytical data for linalool and benzaldehyde in roasted and unroasted cocoa beans
Grade
Provenience
Roasting
(C)
Basic
Basic
Ghana
Ivory Coast
(min)
(S)-(?)-Linalool
(%)
Linalool
(mg kg-1)
Benzaldehyde
(mg kg-1)
Ratio lin./
benz.
Replicates
(n)
Raw
96.7 0.5
0.8 0.1
4.1 0.6
0.2 0.1
165
25
95.5 0.9
0.5 0.2
3.4 0.5
0.2 0.03
Raw
96.5 0.1
0.7 0.4
3.9 1.0
0.1 0.1
110
25
95.7 0.5
0.5 0.02
3.9 0.1
0.1 0.01
135
25
96.2 0.5
0.5 0.1
3.9 0.4
0.1 0.03
New
Guinea
Raw
96.3 0.6
0.2 0.05
2.5 0.5
0.1 0.0
110
25
97.1 0.2
0.3 0.1
2.7 0.2
0.1 0.02
135
25
97.1 0.2
0.3 0.1
3.2 0.1
0.1 0.1
Flavour
Amazonia
Raw
96.7 0.5
0.5 0.1
2.9 0.2
0.2 0.0
165
25
94.6 1.4
0.9 0.8
2.6 0.5
0.4 0.4
Flavour
Arriba (A)
Raw
94.0 1.4
3.6 0.5
2.2 0.4
1.7 0.4
165
25
92.1 0.6
3.0 1.2
1.9 0.2
1.5 0.6
Flavour
Arriba (A)a
140
30
92.0
n.d.b
n.d.
n.d.
Flavour
Arriba (B)
Raw
145
20
95.3 0.4
94.8 2.3
1.2 0.2
1.7 0.3
4.5 0.4
4.4 0.4
0.3 0.1
0.4 0.1
5
5
Flavour
Arriba (C)
Raw
95.3 0.4
2.9 0.9
3.8 0.4
0.7 0.2
5c (3)d
Basic
Flavour
Undefined
Undefined
Venezuela
Colombia
Jamaica
110
25
94.2 0.8
1.4 0.9
3.7 0.5
0.4 0.2
135
25
94.1 0.4
2.9 1.0
3.2 0.5
1.0 0.5
Raw
96.1 0.5
0.3 0.03
3.8 0.5
0.1 0.02
110
25
96.4 1.0
0.5 0.1
3.5 0.1
0.1 0.04
135
25
95.6 2.0
0.4 0.1
3.7 0.3
0.1 0.03
Raw
97.1 0.1
0.8 0.2
1.8 0.3
0.5 0.2
110
25
97.5 0.3
1.2 0.2
1.9 0.3
0.7 0.2
4
4
135
25
97.5 0.2
1.1 0.04
2.3 0.2
0.5 0.1
Raw
96.7
0.5
2.5
0.2
110
25
96.4 0.3
0.4 0.1
2.4 0.3
0.2 0.04
135
25
96.9 0.1
0.4 0.1
3.1 0.2
0.2 0.04
Not determined
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from Arriba were in agreement with this correlation. However, the low ratios (0.2 and 0.1) obtained for the flavourgrade cocoas from Amazonia and Venezuela demonstrate the
potential limitations of this approach. An unequivocal
assignment of cocoa bean materials to one of the quality
grades solely on the basis of the ratio of the concentrations of
linalool and benzaldehyde seems questionable.
Influence of sample preparation on enantiomeric
composition
Considering the known instability of linalool in acidic
medium, several control experiments were performed.
First, (R)-linalool of known enantiomeric purity was
refluxed at different pH values; subsequent determination
of the enantiomeric composition by MDGC demonstrated
that linalool undergoes racemization only under acidic
conditions (Table 2). The basic mechanism of this process
is the previously described proton-catalysed 1-alken-3-ol/
2-alken-1-ol rearrangement [32, 43, 44]. In order to verify
the suitability of the simultaneous steam distillation
extraction (SDE) method, linalool of known enantiomeric
purity was analysed after SDE under extreme pH conditions (from pH 1.7 to 11.5) and different extraction periods
(Table 2). Comparable to the first experiments, linalool
only tends to racemize under acidic conditions. However,
when comparing a reflux period of 1 h with SDE for 1 h at
the same pH (3.5), considerably less racemization was
found with the SDE method. Even a prolonged period of
24 h for SDE at this pH did not cause a significantly higher
degree of racemization (data not shown). This may be
explained, as the proton-catalysed racemization occurs in
the aqueous phase, and most of the linalool is extracted
within the first 30 min [36].
Liquidliquid extraction of one of the cocoa samples
(Arriba) yielded identical results as SDE at pH 7, thus
proving the suitability of this isolation technique in conjunction with the determination of the enantiomeric distribution of linalool. The repeatability of the SDEMDGC
analysis of the enantiomeric distribution of linalool was
determined on a roasted (Amazonia) cocoa beans sample
(n = 5), resulting in 95.2 0.2 % (S)-(?)-linalool.
Enantiomeric distribution of linalool in cocoa beans
and cocoa products
The analysis of the linalool fraction from an aroma extract
of roasted cocoa beans (from Venezuela) via multidimensional GC is presented in Fig. 1. The evaluation of the
enantiomeric distribution in cocoa beans revealed a consistently high preponderance of (S)-linalool (Table 1). In
all cocoa samples analysed, linalool was found well above
90 % in favour of the (S)-enantiomer. Furthermore, the
Time (min)
(R)-(-)-Linalool
(%)
(S)-(?)-Linalool
(%)
3.5
67.1
32.9
7.0
9.0
99.3
99.3
0.7
0.7
1.7
60
62.0
38.0
3.5
60
92.0
8.0
3.5
240
91.0
9.0
7.0
60
99.3
0.7
7.0
120
99.3
0.7
7.0
240
99.3
0.7
11.5
60
99.3
0.7
LEa
SDE
A*
(mV)
25.6
19.2
12.8
cut
6.4
24
26
48
(min)
42
(min)
0
0
12
24
Linalool
36
(S)
OH
OH
(R)
34
36
38
40
123
R (%)
\0.1
13
87
7
9
93
91
Product
S (%)
Cocoa powders
Brand A
Brand D
\0.1
92
Brand E
\0.1
10
90
Brand F
0.3
11
89
Brand G
0.2
10
90
0.2
95
0.1
93
Brand B, Zartbittera
0.3
94
Brand C, Halbbittera
0.2
94
Brand D, Mocha
\0.1
11
89
Brand E, Mochab
\0.1
11
89
\0.1
91
Chocolates
from Colombia. With respect to the enantiomeric composition of linalool, no difference was found between flavourand basic-grade cocoa beans.
The data for the enantiomeric distribution of roasted and
unroasted cocoa beans are almost identical; they demonstrate that there is no significant influence on the original
enantiomeric distribution of linalool during the roasting
process, even though the milling of the beans to a size of
2 mm before the thermal treatment can be considered as
worst-case scenario compared to industrial conditions.
The results obtained for commercial cocoa powders and
chocolates (Table 3) indicate that even technological procedures used in the production of cocoa products such as
alkalisation (cocoa powders) or conching (chocolate) do
not change the original distribution significantly. Except
for a small increase of the proportion of the (R)-enantiomer, observed in particular for the Brand A sample, the
high excess of (S)-linalool can still be found in such
products.
Interestingly, the absolute concentration of linalool does
not change significantly from raw to roasted cocoa beans.
This has also been described recently in a comprehensive
study following changes of key aroma compounds of
Criollo cocoa beans during roasting [8]. Linalool is known
to be often found not only in its free form, but also bound
to glycosidic precursors, as has been described for Vitis
vinifera varieties [45], but also for others. If this would be
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Conclusion
Linalool could be successfully enantiodifferentiated in
SDE extracts from cocoa beans or products thereof by
MDGC and a chiral stationary phase. SDE conditions with
a neutral-buffered solution, allowed enantiodifferentiation
without the risk of partial racemization, which may occur
under acidic conditions. It was found that cocoa beans of
both flavour- and basic-grade varieties, as well as from
different origins, have a high proportion of (S)-linalool of
usually well above 90 %. This still remains in commercial
products, indicating that technological processes such as
roasting or alkalisation have no detrimental effect on the
high enantiomeric excess of linalool. Interestingly, the
linalool content does not change significantly from raw to
roasted beans. If this can be explained by a balance
between generation from potential precursors and losses
due to the roasting process still remains an open question.
Considering the differences in odour properties and odour
thresholds reported for the enantiomers of linalool, future
studies should also focus on the sensory contribution of the
(S)-enantiomer of this monoterpene alcohol to the cocoa
flavour.
Acknowledgments The authors gratefully acknowledge Claudia
Preis, Carsten Stein, and Lilian Simoes dos Santos for their contribution and technical assistance in this work. We also thank the
Fraunhofer Institut fur Lebensmitteltechnologie und Verpackung
(Freising, Germany), the Bundesverband der Deutschen Suwarenindustrie e.V. (Koln, Germany), HACHEZ (Bremen, Germany),
Kraft Jacobs Suchard R&D, Inc. (Munchen, Germany), as well as the
Verein der am Rohkakaohandel beteiligten Firmen e.V. (Hamburg,
Germany) for donating defined cocoa samples.
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