The Origin of Ethylphenols in Wines
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J . Sci Food Agric 1992, 60, 165-178
The Origin of Ethylphenols in Wines
Pascal Chatonnet,* Denis Dubourdieu, Jean-Noel Boidron and Monique Pons
Institut d’CEnologie, UniversitC de Bordeaux 11, 351 Cours de la LibCration, 33405 Talence Cedex,
France
(Received 6 February 1992; revised version received 21 April 1992; accepted 3 June 1992)
Abstract: Ethylphenols are important aromatic compounds of red wines. These
compounds are formed in wines by some yeast species belonging to the genus
BrettanomyceslDekkera in the presence of hydroxycinnamic acids. These volatile
phenols are responsible for the ‘phenolic’, animal’ and ‘stable’ off-odours found
in certain red wines. The results presented show that the synthesis of the high
quantities of ethylphenols found in the ‘phenolic’ red wines can occur during the
ageing of wines having normally completed their alcoholic and malo-lactic
fermentations. This olfactory fault caused by BrettanomyceslDekkera is found
more frequently than the classical ‘mousy-taint’ attributed to this yeast genus. In
addition, the study of the mechanisms of biosynthesis of ethylphenols by
BrettanomyceslDekkera has shown the sequential activities of two enzymes. The
first, is a cinnamate decarboxylase (CD), which assures the transformation of
certain cinnamic acids into the correspondent vinylphenols; the second is a
vinylphenol reductase, which catalyses the reduction of vinylphenols into
ethylphenols. The CD activity of BretfanornyceslDekkerais not inhibited by the
polyphenolic compounds of red wines (procyanidins and catechins) while these
compounds do inhibit the C D activity of Saccharomyces cereuisiue. On the other
hand, the substrate specificities of the CD activities of BrettanomyceslDekkera
and Saccharomyces are different.
Key words: ethylphenols, off-odours, red wines, BrettanomyceslDekkera sp.
INTRODUCTION (hydroxystyrenes) in white wines and ethylphenols in red
wines (Boidron et af 1988).
Volatile phenols represent a large family of substances, The origin of vinylphenols is well known in beer
some of which possess a strong odorous activity which (Steinke and Paulson 1964; Dadic et af 1971; Albagnac
can influence the aroma of smoked food stuffs and 1975; Thurston and Tubb 1981). These compounds
numerous fermented beverages. Nykanen and Suoma- come from the enzymic decarboxylation of hydroxy
lainen (1983) and more recently Maarse and Visscher cinnamic acids of malt by certain yeast strains (Goodey
(1989) have presented interesting reviews on this subject. and Tubb 1981 ; Steward 1983) during the alcoholic
These substances are naturally part of the composition of fermentation.
wines (Chaudary et a1 1968; Dubois et af 1971; Del Re Although the quantities of hydroxycinnamic acids
et af 1977; Schreier et a1 1979; Schreier et al 1980; Present in grape musts are much less important than
Etievant 1981; Shimidzu and Watanabe 1982; Versini those of malts (Ribereau-Gayon 1964; Nykanen and
and Tomasi 1983; Versini 1985; Baumes e t a / 1986; Suomalanein 1983), they can be decarboxylated into
Chatonnet and Boidron 1988). Although grape musts amounts of vinylphenols SLlffiCient to actively participate
contain only trace amounts of such phenols (Baumes in the aroma of certain white wines (Boidron et a/ 1988).
e t a f 1988), the wines can have from 10 to several The precise origin of ethylphenols in red wines is not
hundred pg litre-1. These are principally vinylphenols known. Since the identification of these molecules (4-
ethylphenol, 4-ethylguaiacol) in beer (Steinke and
Paulson 1964) and in red wines (Dubois and Brule 1970),
* In charge of research for Seguin-Moreau Cooperage (16103 different theories have been proposed to explain the
Cognac, France) at the Institute of (Enology. origin of these substances. They rest, for the most part,
165
J Sci Food Agric 0022-5142/92/$05.00 0 1992 SCI. Printed in Great BritainI66 P Chatonnet, D Dubourdieu, J-N Boidron, M Pons
on the works of Whiting and Carr (1957, 1959), who (5 min, 400 rpm). The ether phase was decanted, dried
showed that certain bacteria (Lactobaccilus pastorianits over anhydrous sodium sulphate, then gently concen-
var quinicus)are capable of decarboxylating and reducing trated under nitrogen to 1 ml in a graduated test tube.
cinnamic acids into their correspondent ethylphenols. The extract was then analysed by GC and MS in
Some authors have therefore proposed that ethylphenols scanning or mass fragmentometry mode.
are produced during or immediately after the malo-lactic
fermentation (Dubois and Dekympe 1982; Dubois 1983; Identijication of the volatile phenols by coupled GCIMS
Baumes er a1 1986), by lactobacilli capable of trans- The volatile phenols were identified after injection in
forming the phenolic acids, and vinylphenols of wines, splitless mode (temperature, 230°C; injection volume,
into ethylphenols. This interpretation is not very satis- 3 p l ; purge time, 30 s; purge rate, 70) on a capillary
fying because Di Stefan0 (1985) has clearly showed that column of Carbowax 20 M (BP-20, SGE, Melbourne,
the accumulation of ethylphenols in red wines is Australia), 50 m x 0.22 mm. 0.25 pm, He N55. 18 psi
completely independent of whether or not the malo-lactic (124.2 kPa)) installed on an HP 5970-1 chromatograph.
fermentation occurs. Nevertheless, Etievant (1989) The temperature was set at 45 to 230°C at the rate of
retains the hypothesis of non-identified lactobacilli to 3°C min-' with a final isotherm of 30 min. The detection
explain the increase in levels of ethylphenols, during the was performed by an HP-5970-b mass spectrometer
ageing in bottle of wines produced by carbonic macer- working in electronic impact (ionisation energy, 70 ev;
ation. It has been shown (Chatonnet e t a / 1990) that source temperature, 250°C). The acquisition was made
storage in barrels, notably in used barrels, favours the in scanning mode (mass range, 30-300amu, 1.9
presence of high levels of ethylphenols in wines. spectrum s-'), or in fragmentometry mode on fragments
However, the oak wood is not directly responsible for the previously selected. The identification of molecules was
ethylphenols content because it is only able to give small realised by library research using the PBM algorithm
amounts of 4-ethylguaiacol (Chatonnet ei a1 1989a). (McLafferty et al 1974).
Significant ethylphenol concentrations found in nu-
merous red wines (Etievant ei al 1989; Chatonnet et al
1990) may be responsible for their disagreeable 'phe- Microbial analyses
nolic'. 'animal' and 'stable' odours.
In this paper, the microbial origin, the paths of their Cultures and isolations of microorganisnis
biosynthesis and the organoleptic impact of ethylphenols Liquid culture media.
in red wines are defined. -Liquid YPG: glucose 20 g litre-', yeast extract
(DIFCO) 20 g litre-', bacto-peptone (DIFCO)
20 g litre-'. The pH was adjusted to 4-5. Autoclaving
EXPERIMENTAL
10 min at 115°C. The cultures were run at 20°C on
an agitation table, aerobically, in beakers of 250 ml
Chromatographic analysis
filled with 100 ml of medium.
-Lanaridis (1984) medium: glucose 85 g litre-', fruc-
Voluiile phenols
tose 85 g litre-', L-tartaric acid 3 g litre-', citric
The volatile phenols of wines are measured according to
acid 0.3 g litre-', asparagine 2 g litre-', mono-pot-
the method previously described (Chatonnet and
assium phosphate 2 g litre-'. ammonium sulphate
Boidron 1988). After adjustment of the ionic strength of
2 g litre-', magnesium sulphate (7 H,O) 0.2 g litre-':
the medium and addition of an internal standard (3,4-
manganese sulphate ( H 2 0 ) 0.01 g litre-', meso-
dimethylphenol), a liquid-liquid extraction by dichloro-
inositol 0.3 g litre-l. The pH was adjusted to 3.2.
methane was performed. The extract obtained was
Autoclaving 10 min at 105°C.
washed several times at different pH values (pH 8.5 and
13) in order to isolate the phenolic aroma fraction. The The cultures were prepared at 20°C. anaerobically. At
final extract was concentrated under nitrogen and the moment of seeding, 10 ml litre-' of a sterile solution
analysed by capillary G C with FID detection. of growth factors was added (biotin, 4 mg litre-' ;
thiamine, 100 mg litre-'; pyridoxine, 100 mg litre-'; nic-
Stitdy of' ihc irunsforniarion products of phenolic acids otinic acid, 100 mg litre-l; pantothenic acid, 100 mg
in culture media litre-'; p-amino benzoic acid, 100 mg litre-'; sterile
The culture medium (10 ml), previously clarified by filtration 0.22 pm).
centrifugation (500 x g, 15 min), was extracted three
times by 5 ml of diethyl ether (magnetic stirring, 5 min, -Grape juice medium: juice from white grapes was
350 rpm) containing 4 mg litre-' of di-teri-butyl-p-cresol sterile filtered through 0.45 and 0.22 pm membranes.
as the internal standard. The organic phases were -Modified Carr medium: glucose, 5 g litre-'; yeast
separated by static decantation, pooled together, then extract (DIFCO), 4 g litre-'; casamino acids
washed by 10 ml of 5 % aqueous sodium bicarbonate (DIFCO). 5 g litre-'; potassium chloride, 0.45 gThe origin of phenols in wines 167
litre-'; mono-potassium phosphate, 0-6 g litre-'; AUX and API 50 CHL (API Systeme, La-Balmes-les-
calcium chloride (2 H,O), 0.13 g litre-'; magnesium Grottes, France) were used to identify the isolated
sulphate, 0.13 g litre-'; manganese sulphate (H,O), microorganisms. In case of doubtful identification, the
0.003 g litre-'; DL-malic acid, 10 g litre-'; tomato yeasts were sent to the mycology laboratory of Leuwen
juice (DIFCO), 10 ml litre-'. The pH was adjusted University (MUCL, Prof G Hennebert, B-1348 Louvain-
to 4.8. Autoclaving 15 min at 110°C. Culture in La-Neuve, Belgium), and the bacteria to the bacteria
closed tube (anaerobically) at 25°C. laboratory at the University of Ghent (LMG, Prof Dr K
-Wine medium : a red wine was de-alcoholised under Kerters, B-9000, Ghent, Belgium).
vacuum at 35°C and supplemented with the fol-
lowing: glucose, 80 g litre-'; fructose, 80 g litre-'; Decarboxylationlreduction test of p-coumaric acid
yeast extract, 0.5 g litre-'; growth factors solution, (coumarate media). To 10 ml of YPG liquid medium (for
2.5 ml litre-l. The pH was adjusted to 3.5. Sterile yeasts) or modified Carr medium (for bacteria),
filtration 0.22 pm. Culture anaerobically at 25°C. 100 mg litre-' of trans-4-hydroxycinnamic acid (p-
coumaric acid) were added. The cultures were run, in
Solid media. The cultures on solid medium in a Petri dish
both cases, at 25"C, semi-anaerobically for 72 h for the
of 100 mm in diameter were realised either by inclusion
yeasts and 120 h anaerobically for the bacteria. The
or by plating on membrane after sterile filtration (0.45 or
cultures were then centrifuged, 9 ml of the supernatant
0.22 pm) at 25°C.
were added to 1 ml of 3,4-dimethylphenol (20 mg litre-'
-Yeasts: YPG medium solidified by 20 g litre-' agar in ethanol). The mixture was extracted twice by 5 ml of
and added before the use of 100 pl penicillin G 0-1YO diethyl ether. The organic phases were separated by
(250000 UI in 0.22 pm filtered distilled water), of decantation, pooled together and washed by 10ml of
100 pl of gentamycin sodium sulphate 0.1 YO sodium hydrogenocarbonate (5 YO). The extract was then
(0-22pm filtered distilled water) and of 1OOpl of concentrated under nitrogen (1 ml) and analysed by
diphenyl at 0.75 YOin ethanol. GC/MS. The acquisition was realised in fragmentometry
-Acetic bacteria : YPG medium solidified by 20 g mode with the following fragments: mlz 107 for the 3.4-
litre-' agar, 200 p1 of pimaricin 0 2 Yo,200 pl of peni- dimethylphenol (internal standard), m/z 122 for the 4-
cillin G 0.1 YO,100 pI of diphenylO.75 Yo before use. ethylphenol, and m/z 120 for the 4-vinylphenol.
-Lactic bacteria (modified Dubois medium) : yeast
extract (DIFCO) 5 g litre-' ; neopeptone (DIFCO), Sensory analysis
5 g litre-'; DL-malic acid, 10 g litre-'; magnesium
sulphate (H,O), 0.05 g litre-'; manganese sulphate Determination of perception thresholds
(H,O), 0.05 g litre-'; tomato juice (DIFCO), The olfactory perception thresholds of volatile phenols
250 ml litre-'; bacto-agar (DIFCO), 20 g litre-'. were determined in different media (water, model
The pH was adjusted to 4.5. Autoclaving 10 min at aqueous alcoholic solution, red wines). The methodology
115°C. Upon seeding, 200 p1 of pimaricin 0.2 YOwas used was that described previously (Boidron et al 1988).
added; the incubation was performed in a hermetic The thresholds presented correspond to the minimum
jar equipped with a carbon dioxide generator. concentration under which 50% of the tasters in a 70
-Brettanomyces/Dekkera specific medium (DHSA) : person jury, statistically fail to taste the difference from
yeast extract (DIFCO), 5 g litre-'; bacto-peptone a control. PT designates the perception threshold
(DIFCO), 5 g litre-'; trehalose, 5 g litre-'; sacchar- determined in a hydro-alcoholic model medium of
ose, 45 g litre-'; mono-potassium phosphate, reference, having a composition close to that of wine and
0-55 g litre-'; potassium chloride, 0-125 g litre-l; RT, the recovery threshold, the perception threshold
magnesium sulphate (7 H,O), 0.125 g litre-'; ferric determined in a standard wine. The thresholds obtained
sulphate, 00025 g litre-' ; manganese sulphate in this latter case, have only an indicative value because
(H,O), 0.0025 g litre-' ; bacto-agar (DIFCO), of the great variability of composition from one wine to
20 g litre-'; green of bromocresol, 0.030 g litre-'; another. Nevertheless, this value is important because it
cycloheximide, 0.005 g litre-'; sorbic acid, indicates the concentration at which the odour of the
0-25 g litre-'. The pH was adjusted to 4.8; 10 ml of substance studied can be perceived over the overall
the medium was placed in a tube and autoclaved aroma of wine.
(10 min, 120°C); 100 pl of penicillin 0.1 %, 100 p1 of
gentamicin sodium sulphate and 100 p1 of diphenyl Determination of limit preference threshold (Chatonnet
at 0.75 O h in ethanol were added just before seeding. et a1 1990)
For each substance studied and its mixture, the per-
ception threshold was determined of each taster in a
Identification of the microorganisms standard red wine (individual recovery threshold RT) by
API system galleries and reference culture collections. The a triangular directional test ; the threshold represented
assimilation and fermentation tests API 20 C, API 20 the minimum concentration perceived by 50% of the168 P Chatonnet, D Dubourdieu, J-N Boidron, M Pons
tasters. For each concentration, a preference test is also
conducted. The control and supplemented samples were
submitted to the taster who indicated his preference. In
this way, the maximum quantity admissible. for a given
substance, in a standard red wine, beyond which more
than 50% of the tasters rejected the sample (limit
preference threshold LT) was determined. The responses
from each taster were only taken into account con-
sidering their individual perception threshold. The jury
was composed of 20 trained tasters.
A r onint ic inck.u
The contribution of a volatile compound to the aroma of (min)
the wine is estimated according to its aromatic index (I): I
h
* *Medicinal
I = C/PT *cc Spicy. phenolic
9 **
where C is the concentration of the substance in the wine,
determined by analysis, and PT its perception threshold. P
u)
Although I is a number without dimension, it is 6 Phenolic,animal, ,+
c stable *
**
expressed, by convention, in number of olfaction units ._
(NOU). 9
J
The recovery index (RI) was calculated in the same
-
manner, with the aid of the RT. RI measured the state of
domination of the wine aroms by the odour of the
considered substance :
RI = C/RT
GC/olfactory detection coupling
The extract of aroma was injected in the same chroma-
tographic conditions as previously described, but here,
the human nose functions as the detector. Sniffing of the having the odours already described. The analysis of
column effluent was conducted with the device ODO-1 these extracts by GC coupled with olfactory detection
(SGE, Melbourne, Australia). The method of quantifica- enabled the detection of different aromatic zones cor-
tion of sensorial detection used has already been respondent to these odours (Fig 1). The identification by
described (Chatonnet 1991). The chromatograms obtain- MS and by co-injection with the reference compounds,
ed in ionic detection and sniffing detection were showed that they are three volatile phenols: 4-
compared, which enabled the location of the peaks ethylguaiacol, 4-ethylphenol and 4-vinylphenol. Of these
corresponding to the odours in question. compounds, 4-ethylphenol is the most concentrated and
possesses an intense odour of ‘stable’. 2-Ethylphenol,
present at trace levels in certain red wines (Etievant
RESULTS 1981), was not found in this experiment.
The analysis of different wines showed that only
Levels of ethylphenols in wines-organoleptic impact certain wines can contain important quantities of
ethylphenols. Table 1 gives the perception (PT), the
Different red wines with ‘phenolic’, ‘animal’ and ‘stable’ recovery (RT) and the limit (LT) thresholds of the 4-
characteristics were analysed in order to determine which ethylphenol, the 4-ethylguaiacol and of the mixture 4-
volatile substances were responsible for the olfactory ethylguaiacol/4-ethylphenol (1 : lo), the rough propor-
faults. tion found in red wines. The 4-ethylguaiacol is more
In the wines, these odours were stable after acidi- easily perceived than the 4-ethylphenol (Table 1). The
fication by sulphuric acid (pH 2) and after alkalinisation recovery threshold of the mixture (4-ethylguaiacol/4-
by sodium hydroxide to pH 8.5. In the case of a stronger ethylphenol, 1 : 10) was obtained by proportional additive
alkalinisation (pH > 10.5), they seemed to decrease or effect. The LT of each phenol differs only slightly from its
disappear. These observations suggest that the sub- recovery threshold (1 c LT/RT < 1.3). However, in
stances concerned behave like weak acids. wines, one should always consider the sum of the two
The extraction of the weak acid fraction of the same ethylphenols because the presence of the 2-methoxylated
wines according to the method described by Chatonnet derivative lowers simultaneously the RT and the LT of
and Boidron (1988) obtained a concentrated extract these molecules (LT/RT = 1.5, LT,,,* = 426 pg litre-l).The origin of phenols in wines 169
TABLE 1
Olfactory perception thresholds of ethylphenols
Volatile phenols Perception threshold ( T ) Recovery Limit Ratio
threshold threshold LTIRT
Water Model for red wine for red wine
solution (RT) (LT)
4-Ethylphenol 130 440 605 620 1.02
CEthylguaiacol 25 47 110 140 1.27
4-Ethylphenol +4-ethylguaiacol (10: 1) - - 369 426 1.15
TABLE 2
Examples of the sensory impact of ethylphenols in different wines
Volatile phenols White wine Rose' wine Red wine
a b C d e
Concentration @g litre-l)
4-Ethylphenol 0 7 32 330 410 1241 2260
4-Ethylguaiacol 0 21 5 30 40 74 285
c Ethylphenols 0 28 37 360 450 1285 2505
Aromatic Index (NOU)
4-Ethylphenol 0I70 P Chatonnet, D Dubourdieu, J-N Boidron, M Pons
I70 P Chatonnet, D Dubourdieu, J-N Boidron, M Pons
TABLE 3 TABLE 4
TABLE
Concentration range 3
of ethylphenols in wines Ethylphenol content of redTABLE 4
wines from different wineries and
Concentration range of ethylphenols in wines Ethylphenol content ofdifferent
red wines
vintages different wineries and
from
different vintages
Volutile phenols White RosP Red
Volutile phenols White
wines RosP
wines Red
wines Vintage Elhylphetrols in diflerent wineries
wines wines wines
(n = 54)" (n = 12) (n = 83) Vintage Elhylphetrols in diflerent wineries
. -- (n = 54)" (n = 12) (n = 83) A B C
. --
4-Ethylphenol - A B C
4-Ethylphenol -
Minimum 0 0 1 1979 46 86 512"
Minimum
Maximum 0
28 0
75 60471 1979
1980 466 86
- 512"
266
283 75 1980 -
Maximum
Average 20 6047
440 198 I 306 253 266
395
Average of variation (%)
Coeflicient 2293 20
122 440
179 198 I
1982 30
276 253
I06 395
630"
Coeflicient of variation (%) 229 122 179 1982
1983 276
243 I06
54 630"
926"
4-Ethylguaiacol 1983 243 545 926"
4-Et hylguaiacol 1984 - 40 I
Minimum 0 0 0 1984 - 5
Minimum
Maximum 70 0
15 0
1561 1985 198 924" 40 46I
Maximum 7 15 1561 1985
1986 198
15 924"
975" 46
950"
Average 0.8 3 82 1986 15 975" 950"
Average of variation (%)
Coefficient 2250.8 3
159 82
230 1987 4 429 715"
1
Coefficient of variation (%)
1 225 159 230 1987
1988 43 429
654" 715"
275
" 11- Number of samples. 1988
1989 35 654"
I47 275
655"
" 11- Number of samples. 1989
1990 14 5 I47
3 655"
-
1990 14 3 -
Frequency 0 113 I /2
Frequency
of phenolic 0 113 I /2
of phenolic
taint
taint
Concentration over the limit threshold (phenolic taint).
Concentration over the limit threshold (phenolic taint).
Fig 3. Ethylphenol content of different red wines.
Fig 3. Ethylphenol content of different red wines.
4-ethylphenol/4-ethylguaiacol(8:1) is fairly regular. (Fig
4-ethylphenol/4-ethylguaiacol(8:1) is fairly regular. (Fig
4). It corresponds faithfully to the ratio of p-coumaric
4). It corresponds faithfully to the ratio of p-coumaric Fig 4. Relationship between the concentration of 4-
acid (4-hydroxycinnamic) and ferulic acid (4-hydroxy-3- Relationship and
Fig 4. ethylguaiacol between the concentration
4-ethylphenol in red wines. of 4-
acid (4-hydroxycinnamic) and ferulic acid (4-hydroxy-3- ethylguaiacol and 4-ethylphenol in red wines.
methoxy-cinnamic acid), which are respectively the
methoxy-cinnamic acid), which are respectively the
potential precursors of 4-ethylphenol and of 4-
potential precursors
ethylguaiacol (results not 4-ethylphenol and of 4-
of shown). TABLE 5
ethylguaiacol (results not shown). Incidence of the malo-lacticTABLE 5
fermentation (MLF) on the volatile
Incidence of the malo-lactic fermentation
phenol content of wines(MLF) on the volatile
Origin of ethylphenols in the red wines
Origin of ethylphenols in the red wines phenol content of wines
Influencr of the tnalo-lactic jertnentation Volatile phenols White wine Red wine
Influencr of the tnalo-lactic jertnentation Volatile phenols
@g litre-')
White wine Red wine
Most of the red and white wines analysed after the end @g litre-')
Most of the red and white wines analysed after the end Before Affer Bejore Ajkr
of the alcoholic a n d malo-lactic fermentations contained Before Affer Bejore A jkr
of the alcoholic a n d malo-lactic fermentations contained
very low quantities of ethylphenols. o r indeed none a t all
MLF M LF MLF MLF
MLF MLF MLF MLF
very low quantities of ethylphenols. o r indeed none a t all
(Table 5). 4-Vinylguaiacol I68 104 5 5
(Table 5). I68 104
4-Vinylguaiacol
4-vinyl phenol 84 89 355 305
Eroiution of elhylphenols during ageing in oak barrels 4-vinyl phenol 84 89 35 30
Eroiution of elhylphenols during ageing in oak barrels 4-Ethylguaiacol 0 3 0 Trace
The enrichment of the wines in ethylphenols usually 4-Ethylguaiacol
4-Ethylphenol 0
0 3
0 0
0 Trace
5
The enrichment of the wines in ethylphenols usually 4-Ethylphenol 0 0 0 5
takes place during ageing and in particular during the
takes place during ageing and in particular during theThe origin of phenols in wines 171
0
- I
8
I
0 100 2 00 300 1 2 3 4
Time ( d a y s ) Different tanks in the same cellar
Fig 5. Evolution of the ethylphenol content of a red wine during Fig 6. Ethylphenol content of different tanks of red wine in the
its ageing in barrels. ( 0 )-Ethylphenol, and ( 0 )4-ethylguaiacol. same cellar. (m) 4-Ethylguaiacol, and (0) 4-ethylphenol.
Study of the microjlora of a phenolic wine. In a second
TABLE 6 stage, several wines from the 1989 vintage and rich in
Incidence of the ageing conditions on the ethylphenol content ethylphenols, were sampled under sterile conditions in
of the same red wine order to isolate, in selected media, the different classes of
microorganisms; they were then tested in YPG-
Wine-ageing vessel 4-Ethylguaiacol 4-Ethylphenol
coumarate for the yeasts and Carr-coumarate medium
(pg litre-') @ g litre-')
for the bacteria. The isolates from these wines were acetic
Stainless-steel tank 5 32 bacteria, yeasts and some rare lactic bacteria. No culture
New oak barrels 41 455 of either lactic bacteria (Leuconostoc a?nos) or acetic
Old barrels 74 121 1 bacteria (Acetobacter sp frequently atypical) were
Old barrels after shaving 105 1125 capable of forming 4-ethylphenol (Table 9). Among the
Old barrels after shaving 70 48 1 yeasts isolated were, Saccharomyces cerevisiae (bayanus),
and disgorging Saccharomyces cerevisiae (cerevisiae) and more rarely
Pichia sp and Candida sp, which are also incapable of
forming the ethylphenol. The only yeasts which have
revealed themselves capable of synthesising the 4-
summer months (Fig 5). The excessive levels are ethylphenol from the p-coumaric acid are the
frequently found in the wines aged in barrels, particularly Brettanomycesf Dekkera, which were identified by the
in the case of used barrels (Table 6 ) , but one can equally classical biochemical tests. However, small populations
find high concentrations of phenols in tanks. In the same of Brettanomyces sp have equally been found in young
cellar, before blending, only certain lots might present non-phenolic red wines and in certain phenolic bottled
this type of alteration (Fig 6) wines (Table 10).
Microbial origin of ethylphenols in wines Production of ethylphenols in a red wine inoculated by
Ability of diflerent microorganisms to form ethyl- Brettanomyces intermedius. A red wine (500 ml), con-
phenols. Different microorganisms (yeasts, acetic bac- taining only small quantities of ethylphenols after
teria, lactic bacteria), isolated from different fermented alcoholic and malo-lactic fermentation, was sterile
media (wines, beers, pineaux, ciders, vinegars) were filtered through 0.45 pm membranes and divided into
inoculated at the laboratory in their specific model two samples of 250ml. One of the two samples was
medium containing the trans-p-coumaric acid as the inoculated with a culture in stationary phase of Bret-
substrate. Numerous microorganisms decarboxylate, at tanomyces intermedius in YPG liquid medium
a variable intensity, the p-coumaric acid into 4- (lo5 cells ml-l); the other was not inoculated and was
vinylphenol (Tables 7 and 8). A few of them form only used as the control. The two samples were placed at 25°C
trace amounts of 4-ethylphenol (Lactobacillus hilgardii, in semi-aerobic conditions. It was noted (Table 11) that
Pediococcus pentosaceus, Pediococcus damnosus). Only inoculation in the laboratory of a red wine, dry and
certain yeasts are capable of massive transformation biologically stable, by Brettanomyces intermedius pro-
(5&60%) of the substrate into 4-ethylphenol. These are voked, after 30days, the appearance ofsufficient quantities
the yeasts belonging to the genus Brettanomyces and of ethylphenols to depreciate the organoleptic charac-
Dekkera (sporogenous form of the Brettanomyces genus). teristics of the wine (C > LT).
12-2I72 P Chatoiinet, D Dubourdieu, J-N Boidron. M Pons
TABLE 7
Synthesis of volatile phenols from p-coumaric acid by different bacteria
~ ~~ ~~
Microorganisms Transforniation of p-coumaric acid"
4- Vinylphenol 4- Ethylphenol
-_
Leuconostoc oinos ICEB 8403 + -
Leirconosloc oinos ICEB 841 I +++ -
Leuconostoc oinos ICEB 84 13 + -
Lactobacillus hilgardii ICEB 85 10 +++ -
Lactobacillus hilgardii I E R 720 ++++ -
Lactobacillus hilgardii ICEB 8290 + -
Lactobacillus hilgardii ICEB R77 1 +- +/-
Lactobacillus brevis ICEB 8401 +/-
Lactobacillirs breois ICEB 8404 +++ -
Lactobacillus brevis ICEB 851 1 + -
Pediococcus sp ICEB Ja2 + -
Pediococcus pentosaceus ICEB 33316 +++ +I-
Pediococcus damnosus ATCC + +
Acetobacter aceti ICEB 15973 + -
Acetohacrer sp LMG 1662 ++ -
Acetobacter quasi aceti LMG 1657 ++ -
Acetobacter sp NCIB 8619 +++ -
quasi Acetobacter acefi NCIB 8956 ++ -
quasi Acetobacter aceti NCIB 8957 ++ -
" --Negative; +/----very weak 60°/0.
Relationship between the development of Brettanomyces Brettanomyces intermedius MUCL 8519 and Dekkera
sp in red wines and the level of ethylphenols interniedia MUCL 27704.
Contamination during ageing in a cellar Mechanism of uolatile phenols synthesis hj.
In the same ageing cellar of red wines in the region of BrettanomyceslDekkera sp
Bordeaux (Graves). the level of ethylphenols and the A YPG liquid medium supplemented with 100 mg litre-'
population of Brettanomyces sp was compared in wines ofp-coumaric acid, was inoculated with lo4 cells ml-' of
from different new barrels, after 9 months of ageing, Brettattomyces, then placed aerobically at 25°C with
which is the period corresponding to the appearance of stirring. Measurements were taken periodically of the
the phenolic character. The level of ethylphenols in the level of 4-vinylphenol and 4-ethylphenol as the yeast
wine was related to the population of Brettanomyces sp developed in the medium (Fig 8). From the latency phase
(Fig 7). to the end of the exponential phase (from 24 to 72 h of
culture). there was a strong synthesis of 4-ethylphenol
Coiitamitiation in bottled wines while the level of 4-vinylphenol decreased. The pre-
In the same batch of wine, bottled for 12 months (wine sumption was made that a first stage of phenolic acid
A) and 24 months (wine B), only certain bottles presented decarboxylation, by a cinnamate decarboxylase (CD),
an intense phenolic character due to the presence of was followed by a reductase (vinylphenol reductase,
important quantities of 4-ethylphenol. The micro- VPR) which induced the accumulation of 4-ethylphenol
biological analysis of these wines on DHSA medium in the medium. The synthesis of volatile phenol from a
revealed the presence of Brettanomyces sp only in the phenolic acid precursor by Brettanoniycesl Dekkera is
samples presenting the phenolic characteristic (Table 12). described in Fig 9.
Specificity of phenolic acid decarboxylation atid
Mechanisms of synthesis of volatile phenols by reduction by BrettanomycesIDekkera sp
BrettanomyceslDekkera sp The specificity of phenolic acid decarboxylationl
reduction by Brettanornycesl Dekkera intertziedius and
All the tests conducted at the laboratory on the Saccharomyces cerevisiae was studied in YPG media
metabolism of BrettanoniyceslDekkera s p were done on supplemented by different phenolic acids (100 mg litre-l).The origin of phenols in wines 173
TABLE 8
Synthesis of volatile phenols from p-coumaric acid by different yeasts
Microorganisms Transformation of p-coumaric
acih
4- Vinylphenol 4-Ethylphenol
Candida vini MUCL 21120 -
Candida freychussi MUCL 21114 ++++
Hanseniaspora uvarum MUCL 21110 +
Metchnikovia pulcherina MUCL 211816 -
Pichia menbranefaciens MUCL 21134 -
Hansenula anomala MUCL 21153 ++++
Saccaromyces cerevisiae (italicus) I E B +++
Saccharomyces cerevisiae (chevalieri) I E B +
Saccharomyces cerevisiae (uvarum) I E B ++
Saccharomyces cerevisiae (carlbergensis) I E B -
Saccharomyces cerevisiae (capensis) I E B +
Saccharomyces cerevisiae (cerevisiae) I E B +
Saccharomyces cerevisiae (cereoisiae) UTAD23 ++++
Saccharomyces cerevisiae (cerevisiae) EG8C +++
Saccharomyces cerevisiae (cerevisiae) I E B VLI +
Saccharomyces cerevisiae (bavanus) I E B ++
Kluyveromyces thermotolerans MUCL 28822 +
Torulaspora delbruchi MUCL 21816 -
Pichia carsenis I E B +
Pichia canadensis MUCL 21122 +
Saccharomycopsis fibuligera MUCL 1 1443 +
Zigosaccharomyces bailii I E B +
Brettanomyces intermedius CBS +
Dekkera intermedia MUCL 11989 +
Brettanomyces lambicus I E B SA +
~~ ~ ~ ~~~ ~ ~~~ ~ ~~
--Negative; +/--very weak < I YO of the substrate; +--1-20%;
+ + - 2 0 4 0 % ; + + + 4 & 6 0 % ; + + + +->6O%.
After 5 days of culture at 25"C, the products of total inhibition of Saccharomyces CD activity by the
transformation were studied with the aid of GC/MS as polyphenolic compounds of red wines, these same
described previously. compounds do not inhibit the C D activity of Brer-
Both Saccharomyces and Brettanomyces are incapable tanomyces. On the contrary, the procyanidins seem to
of transforming benzoic acids (Table 13). However, increase the activity of these microorganisms in that the
Brettanomyces is very active on the p-coumaric and duration of fermentation of the media enriched in
ferulic acids and equally capable of transforming the polyphenols was greatly reduced (18 compared to 45
sinapic acid (4-hydroxy-3,5-dimethoxy-cinnamicacid) days) and the synthesis of volatile phenols augmented.
into 2,6-dimethoxy-4-vinyI and ethylphenol (Cvinyl
and 4-ethylsyringol) while Saccharomyces is not.
DISCUSSION AND CONCLUSION
Study of an inhibition of Brettanomyces C D b y the
polyphenols of red wine In accordance with Di Stefan0 (1985), and contrary to
A Lanaridis model culture medium containing certain previous works (Dubois and Dekympe 1982;
5 mg litre-' of p-coumaric acid was supplemented with Dubois 1983), the present results confirm that the
procyanidins B (2 g litre-'), then inoculated with either presence of ethylphenols in wines is not at all linked to
Brettanornyces intermedius (I x lo6 cells ml-') or with the incidence of the malo-lactic fermentation. These
Saccharomyces cereuisiae EG8C (INRA, Colmar, molecules appear essentially during the ageing of the
France, 5 x lo5cells ml-'). The fermentation proceeded wines. Although certain bacteria may possess CD
anaerobically at 25°C. Upon conclusion of the alcoholic activity, none is capable of forming significant quantities
fermentation, the volatile phenols were measured. of ethylphenols in the wines. Only certain yeasts can
Table 14 clearly shows that, although there is almost form important concentrations of ethylphenols in the174 P Chatonnet, D Dubourdieu. J-N Boiciron, M Pons
TABLE 9
Isolation of different microorganisms from a ‘phenolic‘ red wine versus the ability of these microorganisms to produce volatile
phenols from p-coumaric acid
Nuniher Microorganism tyye Volatile phenols“ Ident$cation
st’ -
isolation 4- Vinylphenol 4-Ethylphenol
~ _ _ _ _ _ _ _____
I Gram-negative bacteria + - Acetobacter sp
?. Gram-negative bacteria + - Acetobacter sp
3 Gram-negative bacteria + - Acetobacter sp
4 Gram-negative bacteria + - Acetobacter sp
5 Gram-negative bacteria + - Acetobacter s p
6 Gram-positive bacteria + - Leuconostoc @nos
7 Gram-positive bacteria +/- - Leuconostoc m o s
8 Gram-positive bacteria + - Leuconostoc @nos
9 Yeast +++ - Saccharomy ces ceretiisiae (ceraisiaej
10 Yeast ++ - Saccharompces cerevisiae (cererisiae)
I1 Yeast +++ - Saccharomyes cererisiae (cerevisiae)
13 Yeast +++ - Saccharonijices cerecisiae (bajanus)
14 Yeast ++ - Sacchuromyces cereiiisiae (bajwius)
15 Yeast +++ - Saccharornvces cereclisiae (bavanus)
16 Yeast +++ - Saccharoni.vces cereuisiae (bayanus)
17 Yeast ++ +++ Brettwiomyces intermedius
18 Yeast + +++ Brettariomyces interinedius
19 Yeast + +++ Brettariomyces interrnedius
20 Yeast ++ +++ Brettanomyces intermedius
21 Yeast +++ ++ Brett an omyces int ermedius
I’
--Negative‘ + I - - -very weak < 1 YO of the substrate; +--1-20Y0; + +-2WO%; + + + A O - 6 O % .
TABLE 10
Isolation of Bretfanompces sp strains from different red wines
Reference Geographical Vintage Conditioning Ethylphenols ( p g litre-‘) Identification
of’ origin Oj’the wine of the wine ______ of yeasts
isolation 4- Ethylguaiacol 4- Ethylphenol
__ -____ -_____
(3.89.1 Haut-Medoc 1989 New barrels 177 1506 Br intermedius
Margaux + Br lambicus
JNB.89.3 Montagne- 1989 Cement tank 155 I504 Br intermedius
St Emilion
(3.89.3 Haut-Medoc 1989 Old barrels 1 30 Br intermedius
Margaux
CBX.89 Pessac-Leognan 1989 New barrels 71 53 1 Br intermedius
CM89.ES.91 St Emilion 1989 New barrels 200 1800 Br intermedius
HB87.91 Pessac- Leognan 1987 Bottle - - Br intermedius
(after 8 months)
N88.4.91 Montagne- 1988 Bottle I I3 71 1 Br intermedius
St Emilion (after 12 months)
P + 2-10.91 Madiran 1990 New barrels 253 1412 Br intermedius
P.5.91 Madiran 1990 New barrels 6 27 Br intermedius
+ Br lambicus
CV89-9 I . 1 Napa valley 1989 Barrels I83 1828 Br interniedius
presence of hydroxycinnamic acids. These are the species present results, one can conclude that the ethylphenols
belonging to the genus Brettanomj~ces and to its frequently identified in red wines come from a de-
sporogenous form Dekkera. In wines, the Brettanomjres velopment of yeasts of the genus Brettcniomjws/
species essentially found is intermedius. In light o f the Dekkera.The origin of phenols in wines 175
TABLE 11 ,-inn
Production of ethylphenols in a red wine inoculated by c
n
Brettanomyces intermedius
Conditions of storage" 4- Vinylphenol 4-Ethylphenol
(pg litre-') (pg litre-')
Control
t = 0 days 100 7
t = 30 days 95 6
Inoculatedb with 30 I 1230
Brettanomyces intermedius,
t = 30 days
Storage at 25OC, semi-aerobic. Time (h)
1 x lo5 cells rn1-l. Fig 8. Synthesis of volatile phenols from p-coumaric acid
during the culture of Brettartom)ices/DekXera sp. (El) 4-
Vinylphenol, ( 0 )4-ethylpheno1, and (*) population.
Brettanomycesl Dekkera is capable of synthesising the
4-ethylphenol, the 4-ethylguaiacol and the 4-
ethylsyringol from the phenolic acids present in the
grapes or in the oak wood. These results confirm the
work of Heresztyn (1986) who showed that these yeasts
were capable of forming ethylphenols during alcoholic
fermentation of white grape juice. It has been demon-
strated in this work, that the same microorganisms can
produce significantly high quantities of phenols during
the ageing of red wines after having normally completed
o* . I I their fermentations, which has, to the authors' knowl-
0 2 4 6 8 edge, never been previously reported.
Population of Brettanomyces (103cells ml-') Initially isolated from ciders (Osterwalder 1912), these
Fig 7. Relationship between the population of Brettanomyces yeasts have been described many times in musts and in
sp and the concentration of ethylphenols in a red wine aged in
a barrel. wines. Schanderl and Draczinsky (1952) found such
microorganisms in sparkling wines, Agostini (1 950) in a
'voile' of acetified wine, and Barret et a1 (1955) in a
'voile' of Jura wine. Several authors (Peynaud and
TABLE 12 Domercq 1956; Van Der Walt and Van Kerken 1958,
Relationship between the ethylphenol content of some red 1961; Larue eta1 1991) have reported on the different
wines in bottles and the presence of Brettanomyces sp
~~~~~~~~~~~
species present in the musts and the wines and on their
metabolism. Froudiere and Larue (1990) clarified the
Sample in 4-Ethylguaiacol 4-Ethylphenol Brettanomyces sp
bottle (pg litre-') (pg litre-') (cells ml-') conditions of survival of these microorganisms in the
musts and the wines. The development of these yeasts
Wine A has always been considered unfavourable to the quality
1 6 45 0 of the wines because it usually induces a profound
2 16 36 0 modification of the aroma. The grape musts inoculated
3 7 39 0 with Brettanornyces exhibited a marked butyric character
4 113 71 I 180 as well as a particular odour, described as 'mousy',
5 8 36 0 recalling acetamide (Ribereau-Gayon et a1 1975).
6 10 37 0
Australian works attribute this aroma to the presence of
Wine B acetyltetrahydropyridines but clarify that other micro-
1 40 236 1 organisms (Lactobacillus breois, Lactobacillirs hilgardii)
2 41 230 0
are equally capable of forming them (Craig and
3 42 24 I 1
Heresztyn 1984; Strauss and Heresztyn 1984; Heresztyn
4 34 232 2
5 30 239 1 1986). Curiously, there is no reference linking, in an
6 205 1769 306 obvious manner, the development of these yeasts to the
phenolic characteristic of red wines. This olfactory fault176 P Chatonnet, D Dubourdieu, J-N Boidron, M Pons
PH OH
I
1
CH
It CH,
1
CH
I CH3
COOH
Cinnamate Decarboxylase Vinylphenol Reductase
R = H : p-cournaric acid R = H : 4-vinylphenol R = H : 4-ethylphenol
R = OCH3 : ferulii acid R = OCki : 4-vinylguaiaco1 R = O C h : 4-ethylguaiacol
Fig 9. Biosynthesis of ethylphenols in wine by BrettanomyceslDekkera sp. .
TABLE 13
Synthesis of volatile phenols by different yeasts from some phenolic acids
~~ ~ ~~ ~
Phenolic acid Common name Saccharomyces Brettanomycesl Phenol MS
cerevisiae Dekkera product identification
m / z (YO)
cerevisiae bayanus intermedius lambicus
Benzoic series
3,4,5-Trihydroxy-benzoic Gallic
4-H yd rox y- benzoic p-H ydroxy-
benzoic
3,5-Dihydroxy-benzoic Protocatechnic
4-H ydroxy-3methoxy-benzoic Vanillic
4-Hydroxy-3,5-dimethoxy- Syringic
benzoic
Cinnamic series (trans isomers)
Cinnamic - - - -
4-Hydroxy-cinnamic p-Coumaric + + + + 4-Vinylphenol 120 ( IOO),
119 (22),
91 (34), 39 (28)
4-Ethylphenol 122 (26),
107 (IOO),
91 (41, 77 (7)
4,5-Dihydroxy-cinnamic Cafeic - -
4-Hydroxy-3-methoxy- Ferulic + + 4-Vinylguaiacol 150 (97),
cinnamic 135 (loo),
107 ( 5 3 , 77 (36)
4-Ethylguaiacol 152 (36),
137 (loo),
122 (14), 91 (12)
4-Hydroxy-3.5-dimethoxy- Sinapic + + 4-Vinylsyringol 180 (loo),
cinnamic 165 (40).
137 (26), 91 (13)
4-Ethylsyringol 182 (58),
167 (loo),
123 (7). 107 (8)The origin of phenols in wines 177
TABLE 14
Influence of procyanidins on the synthesis of volatile phenols by Saccharomyces
cereoisiae and Brettanomyces intermedius
Conditions 4- Vinyphenol 4-Ethyiphenol Duration of Inhibition
@g litre-’) (pg litre-‘) fermentation (YO)
(days)
Control 34 0 - -
Saccharomy ces 770 0 15 0
cereuisiae
(control)
Saccharomy ces 31 0 15 95
cerevisiae
+ procyanidins
(2 g litre-’)
Brettanomyces 42 1100 45 0
intermedius
(control)
Bret tanomyces 0 3080 18 0
intermedius
+ procyanidins
(2 g litre-’)
caused by Brettanomyces/Dekkera is found more fre- ACKNOWLEDGEMENTS
quently in wines than the ‘mousy taint’ attributed to this
yeast genus. The authors would like to thank Mrs Annick Joyeux, of
The study of the mechanisms of biosynthesis of the the Applied Microbiology Laboratory (Prof A Lonvaud)
ethylphenols by BrettanomyceslDekkera has shown the of the Institute of (Enology of Bordeaux, for supplying
sequential activities of two enzymes not previously the reference cultures of lactic and acetic bacteria and
described.The first is a CD which assures the decarboxyl- Miss Cristina Vazquez for her help in the translation of
ation of the cinnamic acids into the corresponding the text.
vinylphenols. This enzyme has a specificity radically
different from that of Saccharomyces cerevisiae. More-
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