Expression of Multidisciplinary Flavour Science
SENSORIAL
ASPECTS
OF
“BRETT
CHARACTER”:
REEVALUATION OF THE OLFACTORY PERCEPTION THRESHOLD OF
VOLATILE PHENOLS IN RED WINE
A. ROMANO, M.C. Perello, A. Lonvaud-Funel, G. de Revel
UMR 1219 Œnologie, Université de Bordeaux, INRA, ISVV; Faculté d’Œnologie, 351
Cours de la Libération, 33405 Talence, France
Abstract
Wine spoilage caused by the yeast Brettanomyces bruxellensis (sometimes referred
to as “Brett character”) results in the production of several volatile compounds and a
large spectrum of flavours and aromas. Ethylphenols are the best-known markers of
this defect but ethylphenol contents of wines do not always match tasting notes.
Sensory analysis was employed to demonstrate the complexity of “Brett character”. A
masking effect of isobutyric acid and isovaleric acid on the detection of ethylphenols
in wine was proven. This partly explained the poor correspondence between
ethylphenol concentrations and presence of “Bretty” descriptors.
Introduction
Worldwide wine production has been significantly distressed by Brettanomyces
bruxellensis spoilage [1]. The development of B. bruxellensis in wine entails the
synthesis of ethylphenols (namely 4-ethylphenol and 4-ethylguaiacol). These
compounds have a commonly used aggregate limit threshold of 400 μg l-1 [2]. This
limit carries significant economic importance as winemakers have made it the basis
for treating wines thought to be at risk of B. bruxellensis spoilage. In a previous study
[3] we demonstrated that volatile phenol production in wine by B. bruxellensis is
accompanied by the synthesis of organic acids (from C3 to C10) and short chain acid
ethyl-esters (from C2C6 to C2C10). This work was aimed at ascertaining the influence
of this metabolic activity on the development of Brett character and in particular on
the perception of “Bretty” notes.
Experimental
All reagents and standards were of the highest available purity grade. A 2006
Bordeaux red wine was used for detection threshold calculation (pH 3.5, ethanol 12.2
% (v/v), total reducing sugars 2.0 g/l, volatile acidity 0.55 g/l expressed as acetic
acid, total phenolics 49 expressed as absorbance at 280 nm, 4-ethylphenol + 4ethylguaiacol 13 μg/l). All the other red wines (pH 3.4, ethanol 12.5-13.7 % (v/v), total
reducing sugars 1.2-2.4 g/l, volatile acidity 0.51-0.84 g/l, 58-98 total phenolics) were
commercial samples that originated from wineries throughout the Bordeaux region
and belonged to the 2005 vintage. We chose the measurement of absorbance at 280
nm as a rapid method to estimate wine phenolic complexity [4].
Fifty-one red Bordeaux wines of the 2005 vintage were tasted with the aim of
wine profiling for commercial purposes. Four professionals of the wine sector
evaluated samples both orthonasally and retronasally and a list of freely perceived
245
Expression of Multidisciplinary Flavour Science
descriptors was obtained for each wine. Samples were presented to the tasters
employing tulip shaped tasting glasses.
Volatile phenols were determined by GC-MS analysis coupled to solid-phase
micro-extraction (SPME) on polyacrylate fibres [3]. For carboxylic acid and ethyl ester
determination 10-ml aliquots of wine were supplemented with 40 μl of an internal
standard solution (3-octanol at a concentration of 400 mg/l in water:ethanol 1:1 v/v).
Samples were then extracted twice with diethylether:isohexane (1:1 v/v) and wine
extracts were submitted to GC-FID analysis [5]. All analytical determinations were
carried out in duplicate. Correlation coefficients among different variables were
calculated by the general linear model. Calculations were performed employing
Minitab statistical software (Minitab Inc., State College, PA).
Detection thresholds were calculated following ISO guidelines [6]. Six sets of
three-alternative forced-choice (3-AFC) tests were performed. Each series contained
one positive sample supplemented with ascending (17 - 34 - 68 - 137 - 275 - 550
μg/l) concentrations of ethylphenols in a 10:1 concentration ratio of 4-ethylphenol and
4-ethylguaiacol.
Experimental data are interpolated on the basis of the equation (1). This
expresses the probability of orthonasal detection (p) as a function of detection
threshold (t). The parameter b is related to the slope of the curve. Detection threshold
corresponds to a 0.5 probability of correct detection after correction for chance
guessing (0.33 probability). Calculations were performed employing Excel software
(Microsoft, Redmond, WA).
2
(1)
p=
3
1+e
b (1-x)
+
1
3
The panel consisted of 10 expert judges belonging to the laboratory staff. The
judges were already trained in the perception of the sensory notes of 4-ethylphenol
and 4-ethylguaiacol in wine but they were not informed about the purpose of the test.
Samples were evaluated orthonasally and consisted in 4 ml wine aliquots presented
within 20 ml screw capped bottles with 2 cm neck diameter. The wine samples were
introduced into the bottles and these were capped at least one hour before the
experiment in order to allow for equilibration. All tests were performed in a room
equipped with individual tasting booths.
Results
“Bretty” notes and ethylphenol concentrations in commercial wines. Fifty-one
Bordeaux red wines were profiled for commercial purposes. On the basis of the
tasting notes samples were subdivided into three classes. “Heavily tainted” wines
presented “Bretty” descriptors for all judges, in “mildly tainted” wines such descriptors
were found by at least one judge and “non tainted” wines were virtually faultless.
Wines were submitted to GC-MS analysis for volatile phenols. 4-ethylphenol and 4ethylguaiacol were present in all samples with a 10:1 average concentration ratio,
typical of Bordeaux wines. Vinylphenols were instead not detectable.
The correlation between sensory data and ethylphenol contents appeared to be
very poor (Table 1). No significant differences were found in the average ethylphenol
content of each class.
246
Expression of Multidisciplinary Flavour Science
Table 1. Descriptive analysis
commercial wines.
Sensory profile
and
corresponding
ethylphenol
contents
of
4-Ethylphenol +
4-ethylguaiacol (μg/l)
Min-max
Averagea
196-746
375 (223)
8-563
272 (201)
5-1370
403 (389)
Number of wines
Heavily tainted
5
Mildly tainted
10
Non tainted
36
a
standard deviation between brackets
Wine analysis for carboxylic acids and ethyl-esters. Twenty-six commercial wines
(5 heavily tainted, 6 mildly tainted and 15 non tainted) were selected among the
previously mentioned 51 and submitted to GC-FID analysis for carboxylic acids and
ethyl-esters. A statistically significant correlation (1-α= 0.95) could be evidenced
between ethylphenols and isobutyric (iC4) and isovaleric (iC5) acids. All other
carboxylic acids (from C3 to C10) and ethyl-esters (from C6 to C10) showed instead
very poor correlations (Table 2). B. bruxellensis is presumably the only known
species that produces ethylphenols in winemaking conditions. Our results indicate
therefore that isobutyric acid and isovaleric acid are to be considered as important
markers of Brett character.
Table 2. Concentration of carboxylic acids and ethyl-esters of some commercial
wines (mg/l). Correlation coefficients (r) with ethylphenols are reported.
C3+C
4
Min
Max
<0.1
a
iC5
C6
C8
C10
C2C6
C2C8
C2C10
1.34
1.87
0.63
0.51
0.21
0.07
0.10
0.18
4.67
6.73
1.92
2.22
1.36
0.48
0.50
0.67
2.62
3.49
1.02
0.82
0.45
0.31
0.27
0.42
(0.79) (1.12) (0.35) (0.42) (0.22) (0.11) (0.10) (0.12)
Averagea
R
iC4
n.a.
0.66b
0.78b
0.05
0.14
0.07
0.19
0.13
0.29
standard deviation between brackets; b significant at 99% confidence level
Calculation of ethylphenol detection thresholds. An aggregate detection threshold
was measured in wine for the mixture 4-ethylphenol : 4-ethylguaiacol in a 10 : 1
concentration ratio. In a second experiment the detection threshold was calculated in
a wine supplemented with 1 mg/l isobutyric acid and 1 mg/l isovaleric acid. In a
preliminary trial we verified that “rancid” and “sweaty” olfactory notes typical of these
compounds were not detectable at these concentrations.
The comparison between the two detection thresholds of the ethylphenols
allowed us to ascertain that isobutyric acid and isovaleric acid do possess a masking
effect with respect to these ethylphenols: in fact the detection threshold value was
three times higher when wine was supplemented with the carboxylic acids (Figure 1).
The calculated detection threshold for ethylphenols in wine was about four times
lower than that reported in literature (Chatonnet et al., 1992), but it must be reminded
247
Expression of Multidisciplinary Flavour Science
prob. of correct detection
this is easily influenced by wine complexity. The wine employed for detection
threshold calculation, unlike the ones submitted to sensory profiling, was not barrel
aged and its phenolic complexity was significantly below average, as indicated by the
values of absorbance at 280 nm (experimental).
The results presented in this work demonstrate that some secondary metabolites
other than volatile phenols have a sensory impact on the spoiled wine. This partly
explains the poor correlation between sensory data and ethylphenol contents. Some
incoherencies between sensory and analytical data could also be explained on the
basis of the presence of other ethylphenols (e.g. 4-ethylcatechol) whose role in
relation to “Brett character” has not been fully disclosed yet [7].
wine
wine+iC4/iC5
0,9
92 μg/l
290 μg/l
0,7
0,5
0,3
3
4
5
6
ln concentration (4-ethylphenol + 4-ethylguaiacol)
Figure 1. Orthonasal detection thresholds of 4-ethylphenol and 4-ethylguaiacol in a
10 : 1 concentration ratio. Threshold is calculated in a red Bordeaux wine
and in the same wine supplemented with 1 mg/l isobutyric acid and 1 mg/l
isovaleric acid. Error bars represent 95% confidence limits.
References
1.
2.
3.
4.
5.
6.
7.
Loureiro V., Malfeito-Ferreira M. (2006) In Food Spoilage Microorganisms (de
Blackburn C., ed.), Woodhead Publishing, Cambridge, pp 354-398.
Chatonnet P., Dubourdieu D., Boidron J.N., Pons M. (1992) J. Sci. Food Agric.
60:1 65-178.
Romano A., Perello M.C., de Revel G., Lonvaud-Funel A. (2008) J. Appl.
Microbiol. 104: 1577-1585.
Somers T.C., Evans M.E. (1977) J. Sci. Food Agric. 28: 279-287.
Silva M.L., Malcata F.X., de Revel G. (1996) J. Food Compos. Anal. 9 : 72-80.
ISO 13301:2002 General guidance for measuring odour, flavour and taste
detection thresholds by a three-alternative forced-choice (3-AFC) procedure.
(http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnum
ber=36791)
Hesford F., Schneider K., Porret N.A., Gafner J. (2004) Am. J. Enol. Viticult. 55:
295A.
248