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The effect of molecular sulphur dioxide on the growth, acetic and gluconic acid production of selected South African acetic acid bacterial strains
Wessel du Toit and Mia Pengilly, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch
INTRODUCTION
Micro-organisms
causing the spoilage of wine are a problem yet to be solved in the
wine industry. The metabolic activity of these spoilage
micro-organisms before, during or after fermentation can alter the
chemical composition of wine. This can adversely affect the sensory
properties (appearance, aroma and flavour) of the end-product (Du
Toit et al.,
2005). One of the micro-organisms frequently associated with the
spoilage of wine is acetic acid bacteria (Bartowsky et
al., 2003).
Although knowledge of their occurrence, metabolism, interactions with
other micro-organisms, and methods of inhibition in the winemaking
process are limited, the ability of acetic acid bacteria to affect
wine quality has been the subject of renewed interest in recent years
(Du Toit & Lambrechts, 2002; Du Toit et
al., 2005).
Acetic acid
bacteria are Gram-negative, catalase-positive rods that belong to the
family Acetobacteraceaea (Du
Toit & Pretorius, 2002). The best-known characteristic of acetic
acid bacteria is their ability to oxidize ethanol to acetic acid.
Acetic acid is the major volatile acid found in wine and is
considered to be detrimental to wine quality at concentrations
ranging from 0.7 to 1.2 g l-1
and higher, depending on the type of wine. Acetic acid bacteria are
also able to produce SO2-binding
compounds such as gluconic acid (Du Toit et
al., 2005).
To produce quality
wine, sound winemaking practices were considered to be sufficient to
inhibit the development of these aerobic micro-organisms. Some of
these practices include the maintenance of anaerobic conditions by
filling containers completely or blanketing the wine with an inert
gas, as well as the correct use of sulphur dioxide (SO2)
(Amerine & Kunkee, 1968). It has, however, become increasingly
evident that, in some cases, acetic acid bacteria can survive and
even multiply under the anaerobic or semi-anaerobic conditions found
during the winemaking process (Du Toit & Pretorius, 2002). Due to
the relatively anaerobic conditions in a bottle, it was believed that
the number of acetic acid bacteria normally decreases rapidly after
bottling. However, it has been shown that the excessive addition of
oxygen during the bottling process can lead to an increase in the
number of acetic acid bacteria (Millet & Lonvaud-Funel, 2000).
The risk of wine
spoilage by acetic acid bacteria is greatest during bulk storage of
wines in the cellar prior to bottling, but is managed by avoiding
exposure of wines to oxygen and by addition of sulphur dioxide to
wines (Bartowsky et al.,
2003). In wine, SO2
consists of the free and the bonded form of which the free form
consists of molecular SO2,
bisulphate and sulphite ions. The molecular form is the most active
anti-microbial form, although only 0.1-5% of the free SO2
occurs in the molecular form at normal wine pH. It has been found
that the levels of SO2
used in wine are not always adequate to inhibit these bacteria, since
it has been shown that acetic acid bacteria can grow in wine
containing 20 mg l-1
of free SO2
(Du Toit & Pretorius, 2002). Strain differences, however, could
exist and according to our knowledge, has not yet been investigated
in detail before. Over recent decades, the maximum level of sulphur
dioxide permitted in wine has progressively been decreased, and some
wine makers have significantly decreased the use of this
preservative. Whereas levels of approximately 300 mg l-1
were once common in French white wines, an average concentration of
74 mg l-1
of total sulphur dioxide is now found. Consequently, the risk of
unwanted microbial growth has greatly increased for some wines
(Bartowsky et al.,
2003). Acetic acid bacteria can be enumerated from must and wine with
traditional plating methods, as well as epifluorescent
microscopy (Millet & Lonvaud-Funel,
2000).
The purpose of this
study was thus to investigate the effect molecular SO2
has on the growth, survival, acetic acid and gluconic acid production
of certain South African acetic acid bacteria strains in an
artificial grape juice media, which has not been done before.
MATERIALS AND METHODS
Isolation, identification and
culturing conditions of bacterial strains
Three acetic acid bacterial strains,
designated F14, CI27 and CI40, were used during this study. Strains
CI27 and CI40 were isolated from two different South African
fermenting red wines and strain F14 from a rebate wine (collection of
IWBT, Stellenbosch University). These three acetic acid bacterial
isolates were identified as Acetobacter pasteurianus (Du Toit
& Lambrechts, 2002).
Freeze cultures of these three strains
were streaked on a culture medium which consisted of 57 g l-1
Man Rogosa Sharp (MRS) medium (pH 5 with HCl, 20 g l-1
agar) to which 2% v/v ethanol was added after autoclaving. Single
colonies from these plates were inoculated into 10 ml liquid MRS
medium containing 2% v/v ethanol. This pre-culture was then grown
aerobically at 30ºC for 2 to 3 days until an optical density
(O.D. at 600 nm) of 1 was obtained and used to inoculate the MS300
media. This procedure was followed for all experiments.
Procedure for SO2
experiment
Three molecular SO2
concentrations were chosen: A control with no SO2
addition, as well as 0.5 mg l-1 and 1.0 mg l-1
molecular SO2 additions. A synthetic grape media, MS300
(Bely et al., 1990), was used in these experiments, but the pH
was adjusted to 3.8 (KOH) in order to simulate that of red wine pH
levels. One hundred millilitres of sterile filtered MS300 was placed
in three sterile 120 ml glass bottles, one for each molecular SO2
concentration, and hermetically sealed with a rubber cap. The MS300
in each bottle was then vigorously sparged with sterile nitrogen to
remove excess oxygen. This was done by inserting a sterile syringe
through the rubber cap to the bottom of the glass bottle. This served
as an inflow for the nitrogen gas, while a second sterile syringe,
inserted through the rubber cap without making contact with the
media, served as an outflow for the oxygen and nitrogen. Sterile high
purity nitrogen gas (Afrox, SA, filtered through a 0.22 µm
filter) was used to vigorously sparge the medium in each glass bottle
for 20 min (at 20ºC). A 1% SO2 solution was used to
adjust the SO2 content of the artificial must (MS300). The
medium was left for one hour after the addition of SO2 and
the free SO2 concentration measured with Metrohm
titration unit (Metrohm Ltd., Switzerland). This was then used
to calculate the molecular SO2 concentration (Du Toit et
al., 2005).
Three test tubes containing 8 ml MS300
media of each molecular SO2 level were prepared for each
bacterial strain and inoculated at 80 µl from the
preculture of OD 1 (600 nm) and left to stand for 45 min. This was
done to ensure that SO2 uptake took place before aeration
and possible oxidation of the SO2 on the wheel could
occur. One set of dilution series was prepared for each bacterial
strain and plated out for colony counts. All test tubes were then
incubated on a spinning wheel at 30ºC. At regular intervals, a
dilution series of each sample was prepared and plated out. Each
dilution was plated out in duplicate and the plates incubated at 30ºC
for seven days before enumeration.
Samples were also taken at Day 6 and
Day 10 to determine the amount of acetic acid as well as gluconic
acid that was produced by each bacterial strain at different
molecular SO2 concentrations. This was done by filtering 2
ml of the media through a 0.22 µm filter and freezing it
at -20ºC before the analyses were conducted. Samples were
analysed using the acetic acid and D-gluconic acid assay kits for
enzymatic analysis of acetic acid D-gluconic acid respectively in
foodstuffs and other materials (Boehringer Mannheim/R-Biopharm,
Roche). Results given show the average of quadruplicates.
Percentage survival of acetic
bacteria using fluorescent microscopy
The percentage survival of the three
acetic acid bacterial strains with molecular SO2 additions
was determined using the fluorescent LIVE/DEAD BacLight bacterial
viability kit for microscopy (Molecular Probes, Inc.). The kit
consists of two dyes: SYTO 9® is a green fluorescent
nucleic acid stain, which stains the nucleic acids of both living and
dead bacteria, and the traditional red fluorescent nucleic acid stain
propium iodide, which only stains bacteria that have damaged and
leaky membranes. The live bacteria will thus fluoresce in green while
the dead bacteria will fluoresce in red. The number of live cells is
then determined by subtracting the red from the green number of
cells. Equal volumes (2 µl) of each stain were added to
1 ml Milli-Q dH2O and thoroughly mixed.
Samples were taken as previously
described for the plate counting method. To an undiluted 30 µl
bacterial sample was then added in equal volume the dye mixture and
incubated in the dark for 30 min at room temperature. After
incubation, 10 µl of the suspension was sampled and
trapped between a slide and a cover slip for observation under a
Nikon Eclipse E400 microscope. For each sample, 20 microscopic fields
(each field containing at least 10 cells)
were analysed after which the percentage live cells (green) versus
dead cells (red) were determined. All experiments were conducted in
triplicate and results given show the average of the triplicates.
In all experiments, cell counting was
performed simultaneously by both plate counting and epifluorescence
microscopy on the same samples.
RESULTS All
three acetic acid bacterial isolates were identified as Acetobacter
pasteurianus according to
the biochemical and physiological tests described in the Materials
and Methods. The
cell number and percentage survival as determined with plate counting
and epifluorescence microscopy of the three different acetic acid
strains after the addition of sulphur dioxide were monitored for 10
days. As can clearly be seen from Fig. 1, strain F14 was very
sensitive to the addition of molecular SO2,
with even a concentration of 0.5 mg l-1
killing it after 72 h. This was even faster where 1 mg l-1
molecular SO2
was added, with the control exhibiting good growth in the MS300
media. Strain CI27 however, exhibited much more resistance towards
SO2
as can be seen from Fig. 2. Strain CI27 also grew slower than strain
F14 as growth only occurred after 48 h, but after 240 h the initial
addition of 1 mg l-1
molecular SO2
lowered cell numbers as determined with plating. A different growth
pattern was observed with strain CI40 where the addition of molecular
SO2
inhibited growth, but only for 144 h in the case of the 0.5 mg l-1
addition, with cell numbers increasing (Fig. 3). In this strain, high
molecular SO2
additions thus seemed to prevent growth to a certain extent, but did
not lead to complete cell death as in strain F14. It is clear from
Fig. 4, that the percentage survival of strain F14 with molecular SO2
additions decreased very quickly over time. The same tendency was
also observed for strains CI27 and CI40 (results not shown).
From
Table 1 it can be seen that strain CI40 produced the highest amount
of acetic acid at all three molecular SO2
concentrations for both Day 6 and Day 10, except in the 0 mg l-1
molecular SO2
additions after ten days where strain F14 produced 4.51 g l-1.
The addition of 1 mg l-1
molecular SO2
led to almost no or very little acetic acid being produced in all
three strains. Strain CI27, however, produced the highest amount of
D-gluconic acid at all three molecular SO2
concentrations after both Day 6 and Day 10. Substantial differences
could be observed between the different strains within each molecular
SO2
concentration regarding gluconic acid production, but even 1 mg l-1
molecular SO2
could not prevent high gluconic acid concentration being formed by
strain CI27.
DISCUSSION
Only a few spoilage organisms are able
to survive the strong selective pressures in fermenting grape must
and in wine (Du Toit & Pretorius, 2000). It has been a long-term
goal of the wine industry to reduce the occurrence of microbial
spoiled wine, which alters the chemical composition of wine, thereby
affecting the sensory properties (volatile acidic and phenolic
aromas) of the end-product (Du Toit et al., 2005). Due to
their aerobic nature, acetic acid bacteria have for long been
considered to play no major role during winemaking operations, since
winemaking in general is a relative anaerobic process and the growth
and survival of these bacteria under this and other unfavourable
conditions found in wine seems unlikely (Du Toit & Lambrechts,
2002). However, some studies have shown that acetic acid bacteria can
survive during fermentation, producing high amounts of acetic acid as
well as contributing significantly to volatile acidity in must and
wine (Joyeux et al., 1984; Drysdale and Fleet, 1989).
Since SO2 addition is one
of the winemaker’s most efficient tools to eliminate or inhibit
the growth of acetic acid bacteria, the present study focused on the
effects of different molecular SO2 concentrations on the
growth, acetic and gluconic acid production of three different acetic
acid bacterial strains, namely F14, CI27 and CI40. Results showed
that although molecular SO2 addition seemed to inhibit the
growth of acetic acid bacterial strains to some extent it did not
lead to complete cell death in two of the three strains tested. Even
at high molecular SO2 levels (1 mg l-1), the
growth of strains CI27 and CI40 were not inhibited although the
growth of strain F14 was completely inhibited after 24 h, suggesting
that strain variability can also play a role. Watanabe
and Iino (1984) found that up to 100 mg l-1
of molecular SO2
was needed to inhibit the growth of an Acetobacter
specie in grape must. Du Toit et
al. (2005)
found an acetic acid bacterial strain isolated from French wine to be
viable under relative high concentrations of molecular SO2.
Strain F14 used in this study was the most sensitive towards
molecular SO2
addition. This strain was isolated from rebate wine, where the SO2
level has to be kept much lower than in other types of wine. This
could have resulted in this strain being less resistant to SO2
than strains CI27 and CI40, both which were isolated from red wines
with higher molecular SO2
levels. Gonzàlez et al.
(2005) indicated that yeast inoculation could also influence
diversity of acetic acid bacteria in wine. Acetic
acid bacteria are well-known to be able to oxidize ethanol in wine
first to acetaldehyde and then to acetic acid, which is catalysed by
an alcohol dehydrogenase and an aldehyde dehydrogenase (Saeki et
al.,
1997). Du Toit (2000) however, found certain acetic acid bacterial
strains being able to produce high amounts of acetic acid when grown
in grape juice. Barbe et al.
(2001), found certain acetic acid bacterial strains being able to
produce up to 51 g l-1
gluconic acid in grape juice. Gluconic acid can bind SO2
very effectively; thereby limiting its anti-microbial and
anti-oxidative capacities drastically. Our
results indicated that strain differences also exist regarding acetic
and gluconic acid production of South African acetic acid bacteria.
The two strains that exhibited the best growth in the MS300 media
without SO2,
F14 and CI40, also produced the highest concentration of acetic acid.
The 1 mg l-1
molecular SO2
addition prevented high concentrations of acetic acid being formed.
The production of gluconic acid was also inhibited by molecular SO2
additions in all the strains, but to a much lesser extent in strain
CI27, which also produced the most gluconic acid without molecular
SO2
addition.
The results
obtained during this study, suggests that wine producers can suppress
the unwanted growth of acetic acid bacteria in wine by maintaining a
relatively high concentration of molecular SO2
throughout the process of winemaking, although no complete inhibition
were obtained with some strains. Therefore it is advisable to always
use SO2 in conjunction with other best winemaking
procedures to limit the risk of inducing bacterial growth in wine.
These include the maintenance of lower pH levels, temperature control
and the exclusion of oxygen in wine to a large extent (Du Toit &
Pretorius, 2001). There is a worldwide trend to use less SO2
in wine, due to mounting consumer complaints, but this should not
prevent the winemaker from using this preservative in a responsible
manner at appropriate levels (Du Toit & Pretorius, 2002). The
information generated in this study will assist winemakers to
eliminate the problem of acetic acid bacteria causing spoilage in
wine, thus improving the quality of South African produced wines.
ACKNOWLEDGEMENTS
Winetech and Thrip for financial
support.
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