FOOD

MICROBIOLOGY
Food Microbiology 22 (2005) 169178
Effectiveness of dimethyldicarbonate to stop alcoholic fermentation
in wine
Benoit Divol
a,b
, Pierre Strehaiano
b
, Aline Lonvaud-Funel
a,
a
Faculte d!nologie, Laboratoire de Biotechnologie et de Microbiologie Appliquee, Universite Victor Segalen Bordeaux 2, UMR !nologie-ampelologie,
351 cours de la Liberation, 33405 Talence cedex, France
b
Laboratoire de Genie Chimique, UMR CNRS 5503, INP-ENSIACET, 5 Rue Paulin Talabot, B.P. 1301, 31100 Toulouse cedex 1, France
Received 1 April 2004; received in revised form 1 June 2004; accepted 1 July 2004
Abstract
The alcoholic fermentation of Botrytis-affected wines is stopped by the addition of high concentrations of sulfur dioxide (SO
2
).
The natural microbial unstability of these wines and the binding phenomena forces winemakers to periodically add sulfur dioxide
during maturation, leading to a high concentration of a maximum of 400 mg/l in the bottled wine. Dimethyldicarbonate (DMDC) is
now considered as a reliable fungicide which could be partially used instead of SO
2
, especially just before bottling. This study
investigated the use of DMDC to stop alcoholic fermentation. The experiment was carried out on pure cultures of three yeast species
present in this type of wine (Saccharomyces cerevisiae, Candida stellata and Zygosaccharomyces bailii). The results were very
promising and suggested that DMDC was more effective than SO
2
. The yeast cells died after the addition of DMDC whereas they
partially entered into a viable but non-culturable (VBNC) state with SO
2
. However, the same experiment carried out on botrytized
must, whose fermentation was carried out using indigenous microora, was less conclusive. It pointed out that DMDC, used in a
concentration of 200 mg/l, was more effective than SO
2
but leading to the same results: the entering of a part of the cells into a
VBNC state. DMDC could be used to stop alcoholic fermentation, but could not replace SO
2
. Nevertheless, the concentrations of
SO
2
added in this type of wine could be reduced in this way.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Wine yeast; Stabilisation; SO
2
; Dimethyldicarbonate
1. Introduction
Botrytis-affected wines are sulted during alcoholic
fermentation to stop the activity of the yeast and to kill
them. But we have already observed (Divol and
Lonvaud-Funel, 2004) that yeast remain in wine after
the addition of sulfur dioxide, entering into a VBNC
state. This resistance phenomenon may trigger addi-
tional fermentation start during barrel maturation or
bottle-ageing. Musts and wines contain carbonyl com-
pounds, which bind free-SO
2
. Barbe et al. (2000, 2002)
showed that acetic acid bacteria, which are present on
the grape during infection by Botrytis cinerea, produce
such compounds. Furthermore, as the total SO
2
dose
was legally restricted to a maximum of 400 mg/l,
winemakers have to add the lowest SO
2
concentration
possible to stop alcoholic fermentation. All these factors
lead to the search for other products to stabilize the wine
after alcoholic fermentation. Sorbic acid is known to
have a fungicide action; however, if lactic acid bacteria
are present, they degrade the sorbic acid in 2-ethoxy-3,5-
hexandiene which gives off a geranium-like off-odor
(Crowell and Guymon, 1975). Silva et al. (2002)
suggested the possibility of using yeasts included
in alginate beads to control alcoholic fermentation.
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doi:10.1016/j.fm.2004.07.003

Corresponding author. Tel.: +33-5-40-00-64-66; fax: +33-5-40-00-

64-68.
E-mail address: aline.lonvaud@oenologie.u-bordeaux2.fr
(A. Lonvaud-Funel).
Winemakers have simply to remove the beads, thereby
limiting the dose of SO
2
added to the wine. Esters of
pyrocarbonic acid (Diethyldicarbonate (DEDC), Di-
methyldicarbonate (DMDC)) are known since 1938
(Boehm and Mehta, 1938). The action of these two
fungicides is roughly the same (Genth, 1972). DMDC is
currently selected instead of DEDC because the hydro-
lysis of the latter produces ethyl carbamate, which is a
carcinogen (Schlatter and Lutz, 1990). The formation of
methanol during the hydrolysis of the DMDC in wine
has for a long time concerned scientists. In 1976,
Stafford and Ough (1976) noted that the concentration
of methanol formed from the addition of DMDC was
linear with the substrate concentration and concluded
that the addition of DMDC in wine did not produce
toxicologically signicant levels of methanol. In fact, the
addition of DMDC to wine formed methanol, little ethyl
carbonate, very little methyl carbamate and a few
carbomethoxy derivatives (Ough, 1975). The hydrolysis
of DMDC is rapid: 1 h at 30 1C and 5 h at 10 1C (Delni
et al., 2002). The death of the microorganisms occurs
prior to complete hydrolysis. The action of DMDC is
particularly active when the pH is low, and/or when the
concentration of DMDC and the temperature are high
(Ough and Ingraham, 1961). The concentration of SO
2
also increased the sterilizing effect of DMDC even
though the combination of SO
2
and other preservatives
(such as sorbic acid) reduced the effect of each
component (Terrel et al., 1993). Synergic effects between
both inhibitors were also found by Ough et al. (1988).
Genth (1980) reported that DMDC did not interact with
sugars. Daudt and Ough (1980) reported on the amount
of DMDC needed to kill certain wine yeasts. These
results were completed by Delni et al. (2002) in a study
where DMDC was added to must. In most cases 50 mg/l
DMDC were sufcient to sterilize wine (Daudt and
Ough, 1980). But Delni et al. (2002) gave higher doses
to inhibit several yeast types in must (up 250400 mg/l
for Z. bailii, a yeast which is found during maturation of
sweet wines). The latter authors reported on the
possibility of using DMDC to decontaminate wine of
indigenous yeast species before adding commercial
yeast. Barbe and Bertrand (2000, unpublished data)
tested DMDC on botrytized wine instead of the
addition of SO
2
to stop the alcoholic fermentation.
They checked the efcacity of DMDC on yeast in
natural conditions of growth in barrels during wine-
making. They tested different doses of DMDC, alone or
with SO
2
, added in a lower dose than used to stop
alcoholic fermentation with SO
2
alone, by plating
samples on YPG medium. They conrmed that the
action of DMDC is rapid but brief, unlike the action of
SO
2
, which is more progressive but enduring. They
concluded that the addition of DMDC and SO
2
simultaneously was the most effective method. Threlfall
and Morris (2002) tested the ability of SO
2
and DMDC
to prevent a refermentation after contamination of wine
with Saccharomyces bayanus. They concluded that
DMDC was very effective alone starting at 100 mg/l
together with SO
2
starting at 50 mg/l. We already
observed that after the addition of SO
2
the yeast
survived in a viable but non-culturable (VBNC) state.
This expression was dened by Oliver (1993) as
metabolically active cells but unable to realize cell
division. Thus, they are unable to form colonies on plate
media in response to an environmental stress. Difference
with dormant cells was done by the residual activity of
VBNC cells (Kell et al., 1998). This state was reversible
and it could cause an additional fermentation to start
during maturation or bottle-ageing. Therefore, we
conducted additional experiments by observing cell
death with the direct epiuorescence method (DEFT)
in comparison with plating. DEFT had already been
used by Millet and Lonvaud-Funel (2000) to observe the
VBNC microorganisms in red wines. Observation of the
uorescence allowed for an assessment of the vitality of
the yeast. All these methods were used in this study to
test the effectiveness of DMDC against yeasts to stop
alcoholic fermentation. Experiments were carried out
rstly on pure cultures and then on indigenous micro-
ora in botrytized wine.
2. Material and methods
2.1. Yeasts
The commercial Saccharomyces cerevisiae Zymaor-
eST from Laffort !nologie (Bordeaux, France), Candida
stellata CBS 2649 and Zygosaccharomyces bailii CBS
680 strains were used for the experiments on pure
cultures. For further experiments, the native yeasts
taken from must or from wine during the alcoholic
fermentation, just before the addition of SO
2
, from six
cha teaux situated in Sauternes region (France), were
used.
2.2. Microbiological analysis
For the experiments carried out on indigenous yeasts,
samples were taken as described previously for the
isolation and counting of yeast using two different
methods. Samples were plated on YPG medium (1%
w/v yeast extract, 2% w/v peptone, 2% w/v glucose, 30g/
l agar). The pH was adjusted to 5 with HCl 1/4. Diphenyl
(0.015% in ethanol) to prevent mould contamination and
chloramphenicol (0.01% in ethanol) to prevent bacterial
growth were added. Plates were counted after a 3-day
incubation period at 251C. In all the gures presented
colony counts are in log CFU/ml. The value 0 represented
in fact less than 10CFU/ml (undetectable cells threshold).
VBNC cells are able to resuscitate under certain
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B. Divol et al. / Food Microbiology 22 (2005) 169178 170
conditions (Oliver, 1993). Some samples of wine after
alcoholic fermentation were diluted ve times in white
grape juice and water (1:1) to resuscitate VBNC yeasts.
After10 days of dilution, cells were isolated in YPD solid
medium. Colonies were then selected for identication.
DEFT was performed using the Chemunex (Ivry-sur-
Seine, France) system. The substrate ChemChrome V6
containing uoresceine diacetate is hydrolyzed by the
enzymatic activity of living cells in a uorescent. Wine
sample of 1 ml (with the appropriate dilution) was
ltered on the Chemlter CB04. A prelter Millipore
membrane, called Labelling Pad, was placed in a Petri
dish. 550 ml buffer (ChemSol B16) mixed with 5.5 ml
substrate (ChemChrome V6) were poured on the
Labelling Pad. The Chemlter was placed on the
Labelling Pad and incubated for 30 min at 30 1C. Cells
absorbed the substrate by capillarity. The Chemlter
was placed between a slide and a cover glass and
observed in a Olympus BX51 microscope with 1000
times magnication under UV light generated by a Hg
lamp, with oil immersion through an interferential green
lter (Olympus, ref 467803). Pictures of a few elds were
taken and transferred to a computer using Olympus DP-
soft program. Picture analysis were conducted with this
program: counting, the measure of cells size and
intensity of uorescence (intensity of cells were sub-
tracted to the background noise). Viability of cells, and
notably VBNC cells, were assessed by measuring the
uorescence intensity. Cell uorescence is linked to
enzymatic activity proving a global activity of the
microorganisms, thus differentiating these cells from
dormant cells. Fluorescence intensity unit was given in
an arbitrary unit. Invisible cells under UV microscopy
(uorescence intensity null) were considered as dead
cells.
To more precisely differentiate dead cells from living
cells, the live/dead yeast viability kit (Molecular Probes,
Leiden, The Netherlands) was used. 1 ml of wine was
centrifuged at 5300g for 5 min. The pellet was resus-
pended in 1 ml of the HEPES-Glucose buffer (10 Mm
HEPES (Boehringer Mannheim, Germany), 2% w/v
Glucose pH 7.2) and centrifuged for 5 min at 10 000g.
The cells were resuspended in the same buffer and 1 ml
Fun1 reagent was added. An incubation in the dark for
30 min at 30 1C was then carried out. 10 ml were placed
between a slide and a cover plate and the cells were
observed under a Olympus BX51 microscope with 1000
times magnication under UV light generated by a Hg
lamp, with oil immersion through triple band lter
(Olympus, ref E0197412).
In short, culturable cells could be detected by their
ability to form colonies on plate media, a high
uorescence intensity under UV microscopy (Chemunex
system) and a green uorescence using live/dead yeast
viability kit. VBNC cells were characterized and
visualized by their inability to form colonies on plate
media, their uorescence emission under UV micro-
scopy (DEFT), a green uorescence using live/dead
yeast viability kit, and their ability to resuscitate.
Resuscitation was determined by growth of colonies
on solid culture medium and increase of uorescence
intensity. Dead cells could be distinguished from VBNC
state by absence of uorescence under DEFT (Chemu-
nex system) and a red uorescence using live/dead yeast
viability kit.
2.3. Laboratory-scale fermentations
2.3.1. Fermentations of standard sterilized grape juice
Fermentations were conducted to compare the action
modes of DMDC and SO
2
. The yeast strain Zyma-
oreST was cultivated in YPG medium for 24 h. Cells
were centrifuged for 5 min at 10 000g. The pellet was
then washed with distilled water, and centrifuged once
again. The pellet was resuspended and put into grape
juice, whose sugar concentration was adjusted to 300 g/l.
After 24 h, the alcoholic fermentation was stopped by
adding either 250 mg/l SO
2
(the common dose used in
the cellars for this operation) or different concentrations
of DMDC (Velcorin
s
, Bayer AG, Leverkusen,
Germany).
2.3.2. Fermentations of botrytized must
Alcoholic fermentations were carried out using
indigenous yeasts in botrytized must. Botrytis-affected
must samples were taken in one cha teau of the Sauternes
region (France) just after pressing and immediately put
in sterilized 1.5 l bottles. The alcoholic fermentations
took place in these bottles.
2.4. Yeasts species identication by PCR-RFLP
Identication of yeast species was made using PCR
focused on the Internal Transcribed Spacer (ITS)5.8S
rRNA region, according to Esteve-Zarzoso et al. (1999).
PCR was directly from yeast colonies. Some of the
colonies selected were diluted in 20 ml of water. These
samples were heated at 95 1C for 10 min to disrupt the
cells. The Custom Made PCR Master Mix (QBiogene)
12.5X (comprising 10 mmol/l Tris-HCl, pH 9; 50 mmol/l
KCl; 0.1% Triton X100; 0.2 mg/ml BSA; 3.12%
glycerol; 1.5 mmol/l MgCl
2
; 200 mmol/l each dNTP;
0.1 u/ml Taq DNA-polymerase) was then added onto
these samples. Selected primers (ITS1 and ITS4)
synthetized by Proligo France SAS, were used in a
concentration of 0.2 mM. The PCR program used
has been described by the authors. Products were
visualized by electrophoresis on a 1% agarose gel.
RFLP was then conducted with HinfI from New
England Biolabs (Saint-Quentin-en-Yvelines, France)
on 10 ml PCR amplicon, according to the manufacturers
instructions. The identication was performed by
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B. Divol et al. / Food Microbiology 22 (2005) 169178 171
comparing digestion fragment proles with the data
bank (Esteve-Zarzoso et al., 1999). Uncertain identica-
tions were resolved by RFLP using the additional
enzymes HaeIII and HhaI.
3. Results
3.1. Action of DMDC on a pure culture of S. cerevisiae
(laboratory experiment)
As the major yeast for the alcoholic fermentation is S.
cerevisiae, this study began by testing rstly the
effectiveness of DMDC using a pure culture of this
yeast species. First experiment was carried out on the
strain S. cerevisiae Zymaore ST which was selected for
its resistance to high concentration of ethanol. Fig. 1A
shows the effect on this strain of the different conditions
tested to stop the alcoholic fermentation. The effective-
ness of DMDC was very positive: no other cells were
able to grow on solid medium 2 h after the addition of
any concentration of DMDC. It was not the case with
the addition of SO
2
, where the number of colony
forming units decreased slowly before it stabilized at
about 10
4
UFC/ml. The DMDC seemed to rapidly
prevent the ability to grow on solid medium. At the
same time, the uorescent cells under UV microscopy
were observed. In Fig. 1B, the evolution of the ratio
between the number of cells which gave uorescence
intensity greater than 2 and the total number of living
cells is represented. The number 2 was arbitrarily
chosen to discriminate between the cells which probably
had a high activity and those cells whose uorescence
intensity level had reached the stable minimum under
which they were considered to be dead. After SO
2
addition, the decrease of uorescence intensity (ob-
served as the representation of the cell activity) was fast
and similar for all the cells. On the contrary, with the
addition of DMDC, the decrease of uorescence was
heterogeneous and progressive. The rapidity to reach the
same uorescence of all the cells depended on the
concentration of added DMDC. The lowest concentra-
tion of added DMDC did not completely switch off the
uorescence.
This experiment conrmed that the action of both
fungicides is different. DEFT and colony count gave
complementary results. Moreover, DEFT allowed us to
observe the difference between cells in a viable and
culturable state (cells which are able to form colonies),
and cells in a VBNC, and dead cells. With DMDC, after
several days, cells were almost undetectable by DEFT
and no colonies were counted. Colony count remained
high (about 4 log CFU/ml) for condition D, instead of a
low ratio of uorescence. However, even if slightly
positive this ratio, indicated that some yeasts kept a high
uorescence intensity. Cells which conserved a uores-
cence intensity value greater than 2 are supposed to be
those able to form colonies.
3.2. Action of DMDC on C. stellata and Z. bailii
A previous study showed that not only S. cerevisiae
was able to persist in wine during maturation, but also
C. stellata and Z. bailii. S. cerevisiae (and especially
some strains which are very sensitive to SO
2
) and Z.
bailii were able to cause a refermentation. In spite of its
persistence, C. stellata was unable to resuscitate from
the VBNC state trigged by an addition of SO
2
, even
after canceling this stress. The effectiveness of the
DMDC was then tested on these two other yeast species.
In the previously cited study, after SO
2
addition, the
uorescence intensity of C. stellata decreased very
quickly to reach a minimum level. The same result was
observed after the addition of DMDC as shown in
Figs. 2A and B. The behavior of Z. bailii was very
different because of its higher tolerance to hostile
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Fig. 1. A: Number of cells after addition of fungicides (A: 100 mg/l
DMDC, B: 200 mg/l DMDC, C: 300 mg/l DMDC, D: 200 mg/l SO
2
, E:
100 mg/l DMDC and 100 mg/l SO
2
). The value 0 represented an
undetectable number of cells (lesser than 10 CFU/ml). B: Evolution of
the ratio between high uorescent cell number and total cell number
after the addition of the same fungicides.
B. Divol et al. / Food Microbiology 22 (2005) 169178 172
medium than C. stellata. At the end of the alcoholic
fermentation controlled by an addition of SO
2
, Z. bailii
did not react signicantly (Figs. 2C and D): the average
uorescence intensity varied slightly. In fact the
standard deviation was very high. Each cell did not
react similarly to the addition of SO
2
. Nevertheless, on
average the uorescence intensity remained high and
most of the cells did not lose their capacity to grow on a
solid medium. After the addition of DMDC, the
uorescence intensity decreased to a much greater
degree than the increase in the concentration of DMDC.
After a maximum time of 24 h, the uorescence intensity
reached a minimum and stable level.
Then, cells were diluted 5 times in half-diluted grape
juice. After a few days, a sample was plated on solid
culture medium. For C. stellata, the cells were never able
to resuscitate. On the contrary, Z. bailii treated with low
concentration of DMDC (lesser than 200 mg/l), was able
to resuscitate, even in the case of the combined addition
of SO
2
and DMDC. At greater concentration of DMDC
(starting form 200 mg/l), cells were unable to resuscitate
and they were invisible under UV microscopy, suggest-
ing that they died.
3.3. Effectiveness of DMDC on indigenous microora
during alcoholic fermentation
3.3.1. Test on Botrytized wine after laboratory-scale
winemaking
The same ve conditions previously described were
tested to stop alcoholic fermentation, after wine stayed
24 h at 4 1C, as it is generally done in some cellars of the
Sauternes region.
We checked the decrease of cell numbers using plating
(Fig. 3A) and uorescence according to two methods:
the rst one using uoresceine diacetate (Chemunex
system) to track the decrease of cell vitality (Fig. 3B),
and the second one using Fun1
s
cell stain which
revealed membrane integrity to visualize more precisely
the proportion of dead cells (Table 1).
The colony count number rapidly reached a value
lesser than 10 CFU/ml only in two conditions: C and D,
i.e. the addition of 300 mg/l of DMDC and the addition
of SO
2
(Fig. 3A). At lower concentration of DMDC or
combined addition of SO
2
and DMDC, the effectiveness
depended on the DMDC concentration. But two
months after the end of the alcoholic fermentation, no
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Fig. 2. Number of the CFU/ml after the addition of DMDC and/or SO
2
(A: 100 mg/l DMDC, B: 200 mg/l DMDC, C: 300 mg/l DMDC, D: 200 mg/l
SO
2
, E: 100 mg/l DMDC and 100 mg/l SO
2
) of Z. bailii (A) and C. stellata (B). In parallel, evolution of the intensity of uorescence for the same yeast
species (respectively, C and D). The value 0 represented an undetectable number of cells (lesser than 10 CFU/ml).
B. Divol et al. / Food Microbiology 22 (2005) 169178 173
culturable cell remained in any sample. At 100 mg/l
(condition A), the action of DMDC was very progres-
sive and some cells were able to form colonies even after
uorescence intensity reached its minimum stable value.
However, only 10 CFU/ml could be counted. Those cells
were probably uorescent, but not seen under UV
microscopy because too rare.
The decrease of uorescence intensity rapidly reached a
minimum level: the ratio between cells uorescence
intensity and background uorescence intensity was
nevertheless greater than 1 (Fig. 3B). Only batch 1 which
received only 100 mg/l DMDC needed a few more days to
reach the same level of uorescence intensity. However,
in spite of this low uorescence intensity level, cells
remained alive, since we were still able to see them.
To have a more precise idea of the proportion of live
and dead cells, the live-dead yeast viability kit was used
(Table 1). One week after the end of the alcoholic
fermentation, only the concentration of 300 mg/l
DMDC could achieve more than 50% of dead cells.
But after three months, in all the batches, more than
50% of dead cells could be counted. The proportion of
dead cells was close for 100, 200 mg/l DMDC and for
200 mg/l SO
2
. The best result was obtained by addition
of 300 mg/l DMDC. The combined addition of SO
2
and
DMDC seemed to be very effective (nearly 65% dead
cells), in spite of the low level of each fungicide.
Identication of the survival yeasts was realized by
PCR-RFLP three weeks after the end of the alcoholic
fermentation to visualize the effectiveness of both the
fungicides used on mixed yeasts. Results can be assessed
in Fig. 4 and in Table 2. At low concentration, the
survival of S. cerevisiae was dominant. When the
concentration increased, three species were able to
survive: S. cerevisiae, Z. bailii and Zygosaccharomyces
rouxii. The presence of SO
2
in wine (with or without
DMDC) allowed exclusively Z. rouxii to survive.
Three months after the end of the alcoholic fermenta-
tion, no cell was able to form colonies in any of the
conditions. However, because of the number of uor-
escent cells, the presence of VBNC cells was considered.
For these samples, a ve-time dilution of wine into half-
diluted grape juice was carried out. Ten days after, cells
were plated onto yeast solid culture medium. In these
conditions, cells recovered their ability to form colonies
excepted in the wines treated with 200 and 300 mg/l
DMDC. This method was already used by Divol and
Lonvaud-Funel (2004) to resuscitate VBNC yeast. By
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Fig. 3. A: Number of CFU/ml after the addition of DMDC and/or
SO
2
(A: 100 mg/l DMDC, B: 200 mg/l DMDC, C: 300 mg/l DMDC,
D: 200 mg/l SO
2
, E: 100 mg/l DMDC and 100 mg/l SO
2
) of indigenous
yeasts (micro-winemaking). The value 0 represented an undetectable
number of cells (lesser than 10 CFU/ml). B: Evolution of the intensity
of uorescence in parallel on the same samples.
Table 1
Total number of cells and the percentage of dead cells visualized by uorescence using the livedead yeast viability kit depending on the condition to
stop the alcoholic fermentation (micro-winemaking)
After 1 week After 12 weeks
Number of cells (cells/ml) Percentage dead cells Number of cells (cells/ml) Percentage dead cells
A 3.8 10
7
32.8 1.0 10
7
58.5
B 4.7 10
7
39.3 9.5 10
6
58.3
C 6.6 10
7
52.6 1.4 10
7
66.0
D 3.9 10
7
34.2 6.8 10
6
57.3
E 3.4 10
7
34.6 6.7 10
6
63.8
A: 100 mg/l DMDC, B: 200 mg/l DMDC, C: 300 mg/l DMDC, D: 200 mg/l SO
2
, E: 100 mg/l DMDC and 100 mg/l SO
2
.
B. Divol et al. / Food Microbiology 22 (2005) 169178 174
PCR-RFLP, Z. bailii was identied for the condition
100 mg/l DMDC, and C. stellata for the two others
(200 mg/l SO
2
and 100 mg/l DMDC plus 100 mg/l SO
2
)
as resuscitated yeasts.
3.3.2. Action of DMDC on yeasts after alcoholic
fermentation in barrels
As results on pure strains culture were promising, the
effectiveness of DMDC was tested on indigenous
microora after alcoholic fermentation in barrels. In
this experiment, wine samples were added with concen-
trations of DMDC alone and with SO
2
. They were
compared to the standard addition of SO
2
. The
effectiveness of both fungicides was simply tested by
plating (Fig. 5). The action of SO
2
was slower than
DMDC, which was brief and fast. The more effective
action was achieved by the simultaneous addition of
both fungicides. No additional yeasts were able to grow
after a number of hours.
4. Discussion
For Botrytis-affected wine, the maximum level con-
centration of total SO
2
, which may be added during
winemaking, accepted by the OIV and European
regulations, is 400 mg/l. This concentration is very high,
but necessary. The natural instability of these wines is
partially due to the high pH, which causes the decrease
of the active SO
2
in spite of high free-SO
2
concentration,
and to the high concentration of sugars, which allows
microorganisms to survive during maturation. More-
over, binding phenomena force winemakers in certain
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Fig. 4. Enzymatic digestion with HinfI of PCR products from amplication of ITS-5.8S rRNA region of survival culturable yeasts three weeks after
the end of the alcoholic fermentation. A: lanes 1 and 13: 100 bp marker (Promega); lanes 212: condition A, lanes 1422: condition B. B: lanes 1, 4
and 17: 100 bp marker (Promega); lanes 2 and 3: condition C; lanes 416: condition D; lanes 1821: condition E. (See legend of Fig. 3 for denition of
conditions.)
Table 2
Identication of yeasts surviving in wine three weeks after the addition
of the following fungicides
S. cerevisiae Z. bailii Z. rouxii
A 100 0 0
B 9.1 27.3 63.6
C 0 50 50
D 0 0 100
E 0 0 100
A: 100 mg/l DMDC, B: 200 mg/l DMDC, C: 300 mg/l DMDC, D:
200 mg/l SO
2
, E: 100 mg/l DMDC and 100 mg/l SO
2
. Numbers are
given as a percentage of total yeasts.
B. Divol et al. / Food Microbiology 22 (2005) 169178 175
years to add high quantities of SO
2
to maintain
acceptable free-SO
2
concentration. This problem, linked
with the health of the consumer, has led many authors
to think about the use of another fungicide. DMDC was
thus studied for its activity on yeasts, especially before
bottling.
In this study, we attempted to use DMDC, instead of
SO
2
, to stop alcoholic fermentation in Botrytis-affected
wines when the sugar/alcohol equilibrium is reached.
Experiments began by testing the DMDC on pure yeast
cultures in fermenting must. These experiments led to
the conclusion that these cells were dead. This phenom-
enon has never been observed after SO
2
addition, even
when the added concentration was high (Divol and
Lonvaud-Funel, 2004). After sulting, the yeasts entered
into a VBNC state. Cells were able to grow again when
medium conditions became more favorable. On the
contrary, after DMDC addition, the ability to start a
new alcoholic fermentation should be low since cells are
died. Moreover, results suggested, that DMDC at a
minimum of 200 mg/l was very effective even against Z.
bailii, which is very resistant to most fungicides. Its
action was more effective than SO
2
because it resulted in
total death of all the cells, unlike the addition of SO
2
which caused the death of many cells but left many
others to enter into the VBNC state.
This very promising conclusion led to the following
experiments. Botrytized must was inoculated with the
whole complex of indigenous microora. When the
standard equilibrium was reached, the alcoholic fermen-
tation was stopped by the addition of different
concentrations of DMDC (the same as the one tested
previously). Results were very distant from those
observed for pure cultures. The effectiveness of DMDC
was conclusive as seen before. Indeed, even if the
uorescence intensity of the cells rapidly decreased in all
the cases, the number of cells was less than 10 CFU/ml
after 2 months, even with 200 mg/l DMDC. The cell
vitality rapidly reached this low level, but most of the
cells remained alive. The lower the concentration of
added DMDC, the higher the number of live cells. In
fact, as the DMDC is quickly hydrolysed by the ethanol,
its effectiveness depends on the added concentration.
For a concentration of 300 mg/l of DMDC, the number
of CFU/ml decreased very rapidly and no colonies were
counted after a few days. However, 300 mg/l is too high
a concentration to be used in wine because of the
amount of methanol formed. Those results were
conrmed by the cell uorescence based on the
membrane integrity where dead cells are red and live
cells green. One week after the addition of both
fungicides, the percentage of dead cells depended on
the added concentration of DMDC. For 200 mg/l
added, results were very close to those corresponding
to the addition of SO
2
, and after the combined addition
of SO
2
and DMDC. This result suggested that DMDC
was as effective as SO
2
at the same concentration. It did
not conrm the observations in pure cultures. The
complex microora, composed of many competitive
yeast species and the complex medium (botrytized wine)
were probably important factors affecting the behavior
of yeast. Their natural slow fermentation in a difcult
medium probably made yeasts more resistant to
fungicides, even DMDC.
Moreover, three months after the addition of both
fungicides, the percentage of dead cells increased in all
cases, and the percentage in the case of the combined
addition of SO
2
and DMDC, reached the level of those
obtained with the addition of 300 mg/l DMDC. The
addition of DMDC alone was always more effective
than the addition of SO
2
at the same added concentra-
tion. DMDC was no longer active a few hours after its
addition to the wine. The remaining yeasts survived for
a few hours and then died or went into a VBNC state,
which was proved by the absence of colonies on plates
and the presence of live cells, observed by uorescence.
The effectiveness of DMDC when it was at least 200 mg/
l was conrmed by the absence of resuscitated yeasts in a
VBNC state three weeks after the end of alcoholic
fermentation, unlike the results at the same concentra-
tion of SO
2
.
DMDC, like SO
2
, seemed to make a selection in
mixed yeasts. The higher the concentration of DMDC
was, the more the selection was oriented to stress-
resistant yeasts, and S. cerevisiae disappeared at the high
level of DMDC. However, the selection of yeasts
belonging to Zygosaccharomyces genus took place at
ARTICLE IN PRESS
0 20 40 60
0
1
2
3
4
5
6
7
A
B
C
D
E
F
time (hours)
l
o
g
(
U
F
C
/
m
L
)
Fig. 5. Number of CFU/ml of indigenous yeasts (alcoholic fermenta-
tion in barrels with indigenous yeast population). (A: 250 mg/l SO
2
, B:
125 mg/l SO
2
, C: 125 mg/l SO
2
+100 mg/l DMDC, D: 50 mg/l DMDC,
E: 75 mg/l DMDC, F: 100 mg/l DMDC). The value 0 represented an
undetectable number of cells (lesser than 10 CFU/ml).
B. Divol et al. / Food Microbiology 22 (2005) 169178 176
the initial phase, since the yeasts were not able to
recover their ability to grow after some months. The
addition of at least 200 mg/l yielded consistent results.
Nevertheless, at a lower concentration, or for an
addition of SO
2
or of a mixed addition of low
concentrations of SO
2
and DMDC, wines appeared to
be microbiologically unstable. Cells from these wines
placed in simple culture conditions, rapidly recovered
their ability to grow. Fermenting yeasts such as Z. bailii
or C. stellata were likely to resuscitate. The resuscitation
of C. stellata was surprising and never previously
observed in barrels or in laboratory experiments. The
time between the addition of fungicides and the
resuscitation may have allowed the cells to adapt to
the wine. This resuscitation was observed to be unlikely
in barrels.
Nevertheless, the addition of low concentrations of
DMDC to botrytized wines for stopping the alcoholic
fermentation appeared to have the same consequences
as the addition of SO
2
. Yeasts were able to enter into a
VBNC state and to resuscitate when conditions were
better. For higher concentrations of DMDC, the
proportion of dead cells signicantly increased, and
few cells entered into a VBNC state. The exit from this
state seemed to be more difcult than with low
concentrations of DMDC or with SO
2
.
All these results were conrmed by the last experiment
on wines taken from barrels: the effectiveness of DMDC
depended on the added concentration. The combined
addition of SO
2
and DMDC was the best solution to
rapidly decrease the number of cells to zero CFU/ml.
In any case, the addition of DMDC without SO
2
to
the wine was not possible. Not only did SO
2
act as a
fungicide but also as an anti-oxidant. In the case of the
addition of DMDC alone, the wines discolored, reveal-
ing a high level of oxidation. Even in the case of the
combined addition of DMDC with SO
2
, the samples
showed the same oxidation phenomenon, thus pointing
to the necessity of adding a higher concentration of SO
2
.
This appeared to depend on the vintage and of the
oxidative power of the wine.
Finally, DMDC might be effective to stop alcoholic
fermentation even at low concentrations (100 mg/l). For
durable stability, the most effective level of DMDC
seemed to be 200 mg/l. However to avoid oxidation of
the wine and to preserve it against contaminations or
resuscitation of certain VBNC yeasts, the addition of
SO
2
was essential. Nevertheless, the addition of DMDC
appears to provide a solution to reduce the high
concentrations of SO
2
added in Botrytis-affected wines.
Acknowledgements
This study was supported by the Syndicat des Crus
Classe s de Sauternes et Barsac and by cha teau dYquem.
The authors thank Ce cile Miot-Sertier for her technical
assistance.
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