Expression of Multidisciplinary Flavour Science
GRAPE ODOURLESS PRECURSORS OF SOME RELEVANT WINE
AROMAS
R. LOPEZ, N. Loscos, P. Hernández-Orte, J. Cacho, and V. Ferreira
Laboratory for Flavor Analysis and Enology, Department of Analytical Chemistry,
Faculty of Science, University of Zaragoza, C/ Pedro Cerbuna, 12. 50009 Zaragoza,
Spain
Abstract
The precursor extract obtained from different grape varieties (Chardonnay, Cabernet
Sauvignon, Verdejo, Merlot, Tempranillo and Grenache) was fractionated on a
reversed-phase HPLC system. A total of 15 fractions of 8 mL were collected. Each
fraction was submitted to the following hydrolytical procedures: harsh acid hydrolysis,
enzymatic hydrolysis and alcoholic fermentation. The released aroma compounds
were extracted and analysed by GC-MS. Some aroma compounds were mainly
found in two o more fractions, which indicates the presence of more than one
precursor. In addition, in some cases, the aromas were formed in different extent
depending on the treatment. The existence of a maximum of concentration of δoctalactone in one of the fractions for enzymatic hydrolysis suggests the existence of
a glycosidic precursor for this compound. However, δ-octalactone was formed in a
different fraction only after fermentation, which suggests the existence of a second
non-glycosidic precursor for this aroma. In the case of γ-nonalactone, the aroma was
formed only after fermentation or acid hydrolysis, which suggests that the main
precursor of this compound is not a glycosidic precursor.
Introduction
The chemistry of wine aroma precursors is extremely complex because there are
hundreds of potential aroma precursors in grapes (glycosides, polyalcohols and
some others) that only after many complex transformations form the aroma
molecules. While the precursors of some relevant aroma compounds, such as
linalool and β-damascenone, have been exhaustively studied, the precursors of
many other relevant wine aroma molecules, such as aliphatic lactones, are barely
known. The work presented here shows some preliminary results about the numbers
and type of precursors of some wine aromas.
Experimental
Precursors were extracted following the procedure described by Loscos et al. (1)
from different grape varieties (Chardonnay, Cabernet Sauvignon, Verdejo, Merlot,
Tempranillo and Grenache). A mixture of the extracts of each variety (corresponding
to 1.5Kg of grapes) was concentrated and then fractionated on a reversed-phase
HPLC system equipped with a semipreparative C18 column. A total of 15 fractions
were collected. Fractions (an aliquot of 800 μL per fraction) were submitted to the
following hydrolytical procedures: harsh acid hydrolysis, enzymatic hydrolysis and
alcoholic fermentation. Norisoprenoids compounds were mainly studied by harsh acid
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Expression of Multidisciplinary Flavour Science
hydrolysis and SPME extraction. Harsh acid hydrolysis was carried out in citric acid
buffer solution (0.2 M, 10% EtOH, pH 2.5, 100ºC, 1h). Enzymatic hydrolysis was
carried out in citrate/phosphate buffer (0.1 M / 0.2 M, pH 5) by incubating at 40ºC for
16 h after addition of 12 mg of enzyme preparation AR-2000. Alcoholic fermentation
was carried out in a synthetic media (30g/L of glucose, 0.9 g/L (NH4)2SO4, 0.9 g/l
(NH4)2HPO4) and sterile conditions using Fermicru LVCB yeast strain. Aroma
compounds released by enzymatic hydrolysis and alcoholic fermentation were
extracted by SPE following the procedure described by Ibarz et al. (2). Released
aroma compounds were determined by GC-MS under the conditions reported by
Loscos et al. (1). For each kind of hydrolysis, a control sample without added
precursors was analysed.
Results and discussion
According to the experimental procedure, the existence of a single precursor
molecule for a given aroma compound, will be evidenced by the presence of the
released aroma molecule in a single fraction. The hydrolysis more efficient at
revealing the compound gives some additional clues about the nature of the
precursor molecule.
Norisoprenoids - Acid hydrolysis
10
β-damascenone x 100
8
Area x 10
+6
Vitispirane x 10
Riesling acetal x 500
6
TPB x 1
4
TDN x 100
2
0
5
+1
14
3+
F1
2
F1
1
F1
0
F1
F9
F8
F7
F6
F5
F4
F3
2
+F
F1 l
t ro
on
C
Fractions
Figure 1. Area for the norisoprenoid compounds released by acid hydrolysis.
Some norisoprenoids are better revealed by acid hydrolysis (Figure 1). βdamascenone was found with maxima in fractions F5 and F7, indicating the presence
at least of two precursors, which is in accordance with data reported by Winterhalter
et al. (3). The presence of the β-damascenone in these two fractions was confirmed
after enzymatic hydrolysis and alcoholic fermentation. On the other hand, Riesling
acetal, vitispiranes, t-1-(2,36-trimethylphenyl)but-1,3-diene (TPB) and 1,1,6-trimethyl1,2-dihydronaphthalene (TDN) were found in several fractions with a single
maximum. Such broad chromatographic peak could be due to a deficient elution of a
single aroma precursor, but it could be also the likely result of the presence of many
similar precursor molecules. For example, TDN was clearly found in more than one
fraction (F6 and F7) after enzymatic hydrolysis of the fractions, which indicates the
presence of two precursors of different nature. Winterhalter et al. (3) described that at
least three classes of precursors could exist for TDN and vitispiranes.
Among terpene compounds, linalool and α-terpineol (Figures 2 and 3) were found
after alcoholic fermentation in almost all fractions in the same concentration, even in
the control (synthetic must fermented without aroma precursor fraction). This could
be due to the ability of yeast to synthesise de novo terpenes. However, after
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Expression of Multidisciplinary Flavour Science
enzymatic hydrolysis, linalool and α-terpineol were mainly found in one fraction,
which indicates the presence of a glycosidic precursor for these compounds. In the
case linalool, this glycosidic precursor seems to be slightly hydrolyzed during
alcoholic fermentation. Some other non-glycosidic precursors for this aroma molecule
should exist since the levels of linalool in fermentative hydrolysis show a relative
maximum.
100
Enzymatic hydrolysis
Alcoholic fermentation
Concentration (μg/L)
80
60
40
20
0
F13+F14+F15
F12
F11
F10
F9
F8
F7
F6
F5
F4
F3
F1+F2
Control
Fractions
Figure 2. Concentration (referred to 800 μL of fraction) of linalool after enzymatic
hydrolysis and alcoholic fermentation.
120
Enzymatic hydrolysis
Alcoholic fermentation
Concentration (μg/L)
100
80
60
40
20
0
F13+F14+F15
F12
F11
F10
F9
F8
F7
F6
F5
F4
F3
F1+F2
Control
Fractions
Figure 3. Concentration (referred to 800 μL of fraction) of α-terpineol after enzymatic
hydrolysis and alcoholic fermentation.
Among vanillin compounds, acetovanillone and ethyl vanillate (Figure 4) were
mainly found in only one fraction after the three hydrolytical procedures. In the case
of acetovanillone, the precursor, mainly present in fraction 5, was hydrolyzed more
easily by enzymatic hydrolysis. However, no significant differences between the three
hydrolytical procedures were observed for ethyl vanillate (mainly present in fraction
10).
The existence of a maximum of concentration of δ-octalactone in one of the
fractions for enzymatic hydrolysis (Figure 5) suggests the existence of a glycosidic
precursor for this compound. The structure of this compound could be similar to the
glycosidic precursor of whisky lactone found in wood (4). In addition, δ-octalactone
was formed in a different fraction after fermentation, which suggests the existence of
a second non-glycosidic precursor for this aroma.
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Expression of Multidisciplinary Flavour Science
7000
Acid hydrolysis
Enzymatic hydrolysis
Alcoholic fermentation
6000
Concentration (μg/L)
5000
4000
3000
2000
1000
0
Acetovanillone
Ethyl vanillate x 10
Figure 4. Concentration (referred to 800 μL of fraction) of acetovanillone and ethyl
vanillate (multiplied by a factor 10) for the three hydrolytical procedures.
8
Enzymatic hydrolysis
Alcoholic fermentation
Concentration (μg/L)
6
4
2
0
4+
F1
3+
F1
2
F1
1
F1
0
F1
F9
F8
F7
F6
F5
F4
F3
l
tro
on
2
+F
F1
C
5
F1
Fractions
Figure 5. Concentration (referred to 800 μL of fraction) of δ-octalactone after
enzymatic hydrolysis and alcoholic fermentation.
This work presents a preliminary study about the number of precursors of the main
wine aroma compounds. Although similar studies have been carried out, it is the first
time that the possible existence of two precursors of δ-octalactone (one of them, a
glycosidic compound) is reported. Nevertheless, further studies must be carried out
to clarify the structure of these aroma precursors.
References
1. Loscos N., Hernandez-Orte P., Cacho J., Ferreira V. (2007) J. Agric. Food
Chem. 55: 6674-6684.
2. Ibarz M.J., Ferreira V., Hernandez-Orte P., Loscos N., Cacho J. (2006) J. Chrom.
A 1116: 217-229.
3. Winterhalter P., Sefton M. A., Williams P. J. (1990) Am. J. Enol. Vitic. 41: 277283.
4. Raunkjaer M., Pedersen D.S., Elsey G.M., Sefton M.A.Skouroumounis G.K.
(2001) Tetrahedron Lett. 42: 8717-8719.
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