Heterocyclic acetals from glycerol and acetaldehyde in port wines: Evolution with aging

Article abstract of DOI:10.1021/jf011391j

In Port wine, isomers of glycerol and acetaldehyde acetals have been found at total contents ranging from 9.4 to 175.3 mg/L. During oxidative aging, the concentrations of the 5-hydroxy-2-methyl-1,3-ioxane and 4-hydroxymethyl-2-methyl-1,3-dioxolane isomers increased with time showing a linear correlation (r > 0.95). The flavor threshold for the mixture of the four isomers was evaluated in wine at 100 mg/L. Thus, it is expected that they contribute to old Port wine aroma in wines older than 30 years. Experiments with model solutions and wine clearly demonstrated that SO₂ combines with acetaldehyde and blocks the acetalization reaction.

Full text of DOI:10.1021/jf011391j

2560 J. Agric. Food Chem. 2002, 50, 2560−2564
Heterocyclic Acetals from Glycerol and Acetaldehyde in Port
Wines: Evolution with Aging
ANTONIO CESAR DA SILVA FERREIRA,*,†,‡ JEAN-CHRISTOPHE BARBE,†,§ AND
†
ALAIN BERTRAND
Faculte´ d’Œnologie, Unite´ Associe´e INRA/Universite´ Victor Segalen Bordeaux II,
351 Cours de la Libe´ration, F-33405 Talence Cedex, France; Escola Superior de Biotecnologia,
Universidade Catolica Portuguesa, Rua Dr. Antonio Bernardino de Almeida, PT-4700-072 Porto,
Portugal; and De´partement de Biologie Expe´rimentale, Ecole Nationale d’Inge´nieurs des Travaux
Agricoles de Bordeaux, 1, Cours du Ge´ne´ral de Gaulle, BP 201, F-33175 Gradignan Cedex, France
In Port wine, isomers of glycerol and acetaldehyde acetals have been found at total contents ranging
from 9.4 to 175.3 mg/L. During oxidative aging, the concentrations of the 5-hydroxy-2-methyl-1,3-
dioxane and 4-hydroxymethyl-2-methyl-1,3-dioxolane isomers increased with time showing a linear
correlation (r > 0.95). The flavor threshold for the mixture of the four isomers was evaluated in wine
at 100 mg/L. Thus, it is expected that they contribute to “old Port wine” aroma in wines older than 30
years. Experiments with model solutions and wine clearly demonstrated that SO2 combines with
acetaldehyde and blocks the acetalization reaction.
KEYWORDS: 1,3-Dioxanes; 1,3-dioxolanes; acetalization; oxidative wine-aging
INTRODUCTION
MATERIALS AND METHODS
Cyclic Acetal Synthesis. Synthesis was performed from glycerol
and acetaldehyde by a method previously described (5). Chemicals were
obtained from Aldrich (Lyon, France). Glycerol, (69.7 g), acetaldehyde
(43.03 g), toluene (150 mL), sodium sulfate (70 g), and p-toluene-
sulfonic acid (300 mg) were mixed in a round-bottomed flask fitted
with a reflux condenser cooled by water at 0 °C. The mixture was
heated at the boiling point for 10 h. It was then purified by distillation
and extraction of a 5-mL sample with ether (SDS, France). The organic
phase was evaporated with a Rotavapor, and the final residue was
injected into the GC. Acetals were identified by their retention index
and mass spectrum (Figure 1) as compared with those previously
described (6), and purity was calculated on the basis of flame ionization
detection peak area.
The condensation reaction between glycerol and acetaldehyde
under acid conditions (at wine pH) leads to the formation of
four isomers: cis- and trans-5-hydroxy-2-methyl-1,3-dioxane and
cis- and trans-4-hydroxymethyl-2-methyl-1,3-dioxolane. These
compounds were identified in sherry wine (1, 2). In a compara-
tive work of the volatile aroma composition of two blended
red fortified wines of 20 and 100 years old from Australia, the
cis-5-hydroxy-2-methyl-1,3-dioxane was reported in higher
concentrations in the older wine (3).
Oxidative reactions occurring during the aging process of Port
wine stored in barrels increased the contents of aldehydes and
methyl ketones (4). Among these substances, acetaldehyde was
the major aliphatic aldehyde in wine and the one that presented
the most significant increasing trend with time of barrel storage.
Glycerol is one of the major wine components, with concentra-
tions in Port wine ranging from 4 to 8 g/L, and, consequently,
acetal formation should be high. Thus, as expected, large
amounts of the four isomers produced by this reaction were
found in Port wine. Therefore, special attention was devoted to
the study of these compounds, to establish their formation
mechanism and their impact on Port wine aroma, and to
determine whether these substances can be used as indicators
of wine age.
Acetal Analysis. To 50 mL of wine were added 50 µL of octan-3-
ol in aqueous alcoholic solution (1/1, v/v) at 432.9 mg/L as internal
standard and 5 g of anhydrous sodium sulfate. The wine was extracted
twice with 5 mL of CH2Cl2 (SDS, France). The two extracts were
combined and dried over anhydrous sodium sulfate. A 2-mL portion
of this organic phase was concentrated 5-fold under a nitrogen stream
with a 1 L/min gas flow.
A 2-µL portion of extract was injected into the GC with an MS
detector. Chromatographic conditions were the following: Hewlett-
Packard HP 5890 gas chromatograph coupled with a mass spectrometer
(HP 5972), electronic impact 70 eV; detection mode was selected ion
monitoring (SIM) with ions of m/Z 83 (internal standard quantification),
m/Z 103 (acetal quantification), and m/Z 117 (acetal qualification);
column BP21 (SGE), 50 m × 0.25 mm i.d., 0.25 µm film; helium 5.6
Aga pressure: 55 kPa; injector temperature, 220 °C; detector temper-
ature, 280 °C; oven temperature, 40 °C for 1 min programmed at a
rate of 2 °C/min to 220 °C, the final step lasting 30 min; splitless time,
30 s; split flow, 30 mL/min.
* Corresponding author. Tel: +35 1 225 580 095. Fax: +35 1 225 580
088. E-mail: ferreira@morango.esb.ucp.pt.
† Unite´ Associe´e INRA/Universite´ Victor Segalen Bordeaux II.
‡ Universidade Catolica Portuguesa and Escola Superior de Biotecnologia.
§ Ecole Nationale d’Inge´nieurs des Travaux Agricoles de Bordeaux.
10.1021/jf011391j CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/21/2002

Evolution of Heterocyclic Acetals in Port Wines
J. Agric. Food Chem., Vol. 50, No. 9, 2002 2561
Figure 1. Mass spectra of the four acetaldehyde and glycerol acetal isomers.
Figure 2. Evolution of glycerol and acetaldehyde cyclic acetals with age.
Precursor Analysis. The determination of acetaldehyde levels was
performed by gas chromatography according to the method we
previously described (7).
and 12 g/L glycerol were divided into 10 aliquots, placed in stoppered
flasks, and kept at 40 °C. The levels of the four isomers were measured
periodically between 3 and 334 h after blending.
Glycerol was quantified by HPLC (reversed-phase chromatography)
with refractometric detection.
Port Wine Samples. Twenty-five samples of a single harvest ranging
from 1 to 60 years old and matured in oak barrels until analysis were
supplied by the Instituto do Vinho do Porto.
Synthetic Solutions and Supplemented Wine Preparation. To
study the acetal formation from glycerol and acetaldehyde, 600 mL of
a 20% aqueous alcoholic model solution (4 g/L tartaric acid adjusted
to pH 3.5 by 1 M NaOH) initially containing 100 mg/L acetaldehyde
To evaluate the influence of free SO2 level on the formation of these
acetals, two 2-L samples were prepared: the first was a 5-year-old
Port wine, and the second was a 20% aqueous alcoholic model solution
(4 g/L tartaric acid adjusted to pH 3.4 by 1 M NaOH). Wine was aerated
until free SO2 was not detected. Acetaldehyde and glycerol were also
added to samples in order to obtain approximately 100 mg/L and 15
g/L as initial concentrations of each. The volume of each of the two
samples was divided into two equal parts, one of each being
supplemented with a sufficient quantity of SO2 to obtain 50 mg/L of

2562 J. Agric. Food Chem., Vol. 50, No. 9, 2002
da Silva Ferreira et al.
Figure 3. Relative evolution of the 1,3-dioxane and 1,3-dioxolane forms with age.
Figure 4. Glycerol and acetaldehyde acetal formation in mild conditions.
free SO2. The wine and model solution of each set (with and without
free SO2) were placed in 150-mL screw-capped flasks and stored in
the dark at 20 °C. Samples were collected and analyzed after 0, 47,
67, 111, and 140 days of storage.
Sensory Evalution. The aroma evaluation of the four acetals was
performed by gas chromatography/olfactometry. Chromatographic
conditions were the same as those used for quantification with GC/
MS. The odors corresponding to each compound were qualified by 5
specialists. The perception threshold of the mixture of the four acetals
in ratios similar to those of old port wines was established by a
triangular test at five increasing concentrations in a 5-year-old Port
wine. The solutions were tasted by a 10-person jury.
always present at higher levels than the trans form for each
isomer. The ratio between cis and trans forms remained con-
stant independently of age; the calculated values were 2.5 (
0.19 for the cis/trans dioxane and 1.3 ( 0.06 for the cis/trans
dioxolanes.
Acetal Formation. At the first analysis (t ) 3 h), the
4-hydroxymethyl-2-methyl-1,3-dioxolane content was higher
than that of 5-hydroxy-2-methyl-1,3-dioxane (Figure 4). This
finding is in agreement with the mechanism proposed by Gelas
(8). Indeed, according to the reactional mechanism (Figure 5),
two carbocations led to the formation of acetals and the carbo-
cation [C2+], which leads exclusively (2.a and 2.b intramolecular
reactions) to the dioxolanes, was formed the most quickly;
moreover, the cyclization of the carbocation [C1+] according
to 1.b leads to the same compounds too. However, from the
fourth sampling (t ) 22 h), cis-5-hydroxy-2-methyl-1,3-dioxane
showed the highest level, and then the 5-hydroxy-2-methyl-
1,3-dioxane content was greater than that of 4-hydroxymethyl-
2-methyl-1,3-dioxolane. The four forms tended to an equilibrium
similar to that observed in wines after aging for several years.
This phenomenon may be explained by the mutual intercon-
version of these isomers (9) which leads to a thermodynamic
balance. The relative abundances of the cis and trans forms was
explained by Gelas and Rambaud (10). On one hand, an
RESULTS AND DISCUSSION
Wine Composition. The concentrations of the four iso-
mers increased with time, and their correlation coefficients with
age were high (r > 0.95) as shown in Figure 2; cis-5-hy-
droxy-2-methyl-1,3-dioxane was the isomer present in the
highest quantities. Dioxolane levels were higher than dioxane
levels in young wines up to 2 years old, but gradually an
inversion was observed with time between these two iso-
mers. In fact, after 10 years of barrel storage, 75% of the total
acetal contents were in the 1,3-dioxane form (Figure 3). This
proportion remained constant thereafter and could mean that
the thermodynamic equilibrium was attained. The cis form was

Evolution of Heterocyclic Acetals in Port Wines
J. Agric. Food Chem., Vol. 50, No. 9, 2002 2563
Figure 5. Formation mechanism of glycerol and of acetaldehyde acetals (10).
Figure 6. Acetaldehyde and cyclic acetal contents in synthetic media and supplemented wine with or without SO .
2
intramolecular hydrogen bond strongly stabilizes the cis-5-
hydroxy-2-methyl-1,3-dioxane form, whereas for trans-5-hy-
droxy-2-methyl-1,3-dioxane the bond is only partial. In contrast,
4-hydroxymethyl-2-methyl-1,3-dioxolane diastereoisomers present
an identical partial intramolecular hydrogen bonding for these
two forms.

2564 J. Agric. Food Chem., Vol. 50, No. 9, 2002
da Silva Ferreira et al.
LITERATURE CITED
Role of SO2. As shown in Figure 6, the total concentration
of the four acetals was higher in the samples without free SO2
both in wine and model solution, whereas no acetal appeared
when samples were supplemented with SO2. Moreover, when
free SO2 was absent, the total of the four acetals increased with
time of storage, unlike acetaldehyde. Therefore, SO2 clearly
combined with acetaldehyde and blocked the acetalization
reaction because acetaldehyde-bisulfite cannot react with glyc-
erol to give acetals. This underlines the importance of sulfur
dioxide in wine storage and the difference between oxidative
and reductive aging.
Aroma Impact. Odor evaluation of the four acetals by
sniffing in gas chromatography rated the trans-5-hydroxy-2-
methyl-1,3-dioxane as having the highest intensity aroma, the
odor being described as sweet and old port-like. The flavor
threshold in wine was estimated for the mixture of the four
isomers at a total concentration of 100 mg/L. Therefore, these
compounds might contribute to the old port wine aroma in wines
older than 30 years.
Moreover, high acetaldehyde quantities are consumed by the
acetalization reaction, with the corresponding heterocycles
accounting for about 30-40% of the “total” acetaldehyde. By
this reaction, glycerol protects the wine against an excessive
acetaldehyde content and thus indirectly plays an essential role
in port wine aroma.
Because of the constant increase in acetaldehyde content
and the absence of free SO2 during Port wine aging in bar-
rels, the concentrations of the acetals cis- and trans-5-hy-
droxy-2-methyl-1,3-dioxane and cis- and trans-4-(hydroxy-
methyl)-2-methyl-1,3-dioxolane increase regularly with age.
Their levels are well correlated with the time of storage, and it
seems that they can be used as effective age indicators of
port wine kept under oxidative conditions. Moreover, they
participate in the old port wine aroma detected in wines over
30 years old, and their formation is strictly depending on the
free SO2 content.
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Received for review October 19, 2001. Revised manuscript received
January 25, 2002. Accepted January 28, 2002.
JF011391J