Osborne & Edwards Inhibition of MLF 69

Inhibition of malolactic fermentation by Saccharomyces

during alcoholic fermentation under low- and high-nitrogen
conditions: a study in synthetic media
JAMES P. OSBORNE1,2 and CHARLES G. EDWARDS1
1
Department of Food Science and Human Nutrition,Washington State University, Pullman,WA 99163, USA
2
Coresponding author: Dr James P. Osborne, email Jamesoz@gmx.net

Abstract
The ability of different wine yeast (Saccharomyces cerevisiae) to inhibit malolactic bacteria (Oenococcus oeni)
and the influence of nitrogen were studied using a synthetic grape juice. Malolactic fermentation was
induced in fermenting synthetic grape juice or synthetic wines inoculated with different commercial strains
of S. cerevisiae. O. oeni was generally inhibited in wines that contained higher concentrations of total SO2
although many yeast strains only inhibited the bacteria during fermentation under high nitrogen
conditions. Yeast produced higher amounts of SO2 during fermentation under high nitrogen conditions
suggesting that nitrogen affected the malolactic fermentation by influencing yeast SO2 production.
However, the production of SO2 by yeast did not always account for the inhibition of O. oeni, suggesting
the presence of other inhibitory mechanisms.

Abbreviations
MLF malolactic fermentation; MRS de Man, Rogosa and Sharpe; YAN yeast assimilable nitrogen

Keywords: Malolactic fermentation, alcoholic fermentation, bacterial inhibition, SO2, nitrogen

Introduction Alternatively, bacterial inhibition has been suggested to

The malolactic fermentation (MLF), an important sec-
ondary fermentation of wines performed by lactic acid
bacteria, can be problematic to induce (Lonvaud-Funel et
al. 1988, Wibowo et al. 1988, Henick-Kling and Park
1994, Henick-Kling 1995, Alexandre et al. 2004, Osborne
and Edwards 2005) despite the development of starter cul-
tures of pure strains of malolactic bacteria (Kunkee et al.
1964, Henick-Kling 1993, Krieger et al. 1993, Pilone 1995,
Nielsen et al. 1996). Inhibition of MLF is often attributed
to the effects of low pH (Davis et al. 1986, Wibowo et al.
1988, Britz and Tracey 1990, Vaillant et al. 1995), ethanol
(Britz and Tracey 1990, Vaillant et al. 1995), temperature
(Britz and Tracey 1990), and/or antagonistic interactions
between wine yeast (Saccharomyces) and malolactic bac-
teria (Oenococcus oeni) (Beelman et al. 1982, King and
Beelman 1986, Lemaresquier 1987, Lonvaud-Funel et al.
1988, Wibowo et al. 1988, Cannon and Pilone 1993,
Henick-Kling and Park 1994). Some researchers have pro-
posed that the inhibition of MLF is due to the removal of
nutrients by the faster growing Saccharomyces (Kunkee
1967, Amerine and Kunkee 1968, Fornachon 1968,
Beelman et al. 1982), nutrients that are required by the
nutritionally fastidious malolactic bacteria (Du Plessis
1963, Garvie 1967). However, recent studies have
demonstrated that the removal of nutrients by yeast does
not always explain the observed inhibition of O. oeni
(Patynowski et al. 2002, Larsen et al. 2003).
be due to toxic metabolites produced by yeast during alco-
holic fermentation (King and Beelman 1986, Dick et al.
1992, Henick-Kling and Park, 1994) as Saccharomyces is
known to produce compounds inhibitory to Oenococcus.
These include ethanol (Costello et al. 1983, Davis et al.
1986, Britz and Tracey 1990), SO2 (Eschenbruch 1974,
Dott et al. 1976, Eschenbruch and Bonish 1976, Suzzi et
al. 1985, Romano and Suzzi 1993, Henick-Kling and Park
1994, Carrete et al. 2002), medium chain fatty acids
(Edwards and Beelman 1987, Lonvaud-Funel et al. 1988,
Edwards et al. 1990, Capucho and San Ramao 1994), and
antibacterial proteins and/or peptides (Dick et al. 1992,
Comitini et al. 2005). Of these compounds, SO2 is most
commonly implicated in causing bacterial inhibition
(Fornachon 1963, Henick-Kling and Park 1994, Larsen et
al. 2003) as it is an effective antimicrobial against malo-
lactic bacteria (Carr et al. 1976, Amerine et al. 1980, Liu
and Gallander 1983, Ough and Crowell 1987, Britz and
Tracey 1990).
Production of SO2 by yeast, coupled with that added to
a must or wine, has been suggested by many researchers
to be the primary mechanism of bacterial inhibition during
alcoholic fermentation (Fornachon 1968, Liu and
Gallander 1982, Lonvaud-Funel et al. 1988, Henick-Kling
1993, Henick-Kling and Park 1994, Larsen et al. 2003).
However, other studies have reported that yeast-produced
SO2 is not always responsible for the inhibition of MLF
70 Inhibition of MLF
(Eglinton and Henschke 1996, Larsen et al. 2003).
Eglinton and Henschke (1996) argued that S. cerevisiae
strain AWRI 838 and EC1118 did not produce sufficient
amounts of SO2 to inhibit MLF while Larsen et al. (2003)
reported that the concentration of total SO2 produced by
Saccharomyces did not always correspond to the extent of
bacterial inhibition.
SO2 is produced by Saccharomyces as an intermediate of
the sulfate assimilatory pathway (Eschenbruch 1974,
Amerine et al. 1980, Suzzi et al. 1985, Rauhut 1993,
Romano and Suzzi 1993, Thomas and Surdin-Kerjan
1997, Donalies and Stahl 2002) and can be released by
yeast during fermentation. Sulfate is taken up by the yeast
cell via two membrane-bound permease enzymes,
reduced to sulfite via adenosine-5’-phosphosulfate and 3’-
phosphadenosine-5’-phosphosulfate before being reduced
to sulfide by sulfite reductase. Depending on needs and
conditions, yeast actively excrete sulfite via a membrane
bound sulfite pump (Avram and Bakalinsky 1997). Most
strains of S. cerevisiae produce concentrations of 10 to 30
mg/L SO2 during alcoholic fermentation, but some strains
can produce amounts exceeding 100 mg/L (Rankine and
Pocock 1969, Eschenbruch 1974). The formation of SO2 is
influenced by a complex interaction of genetic, metabolic
and physiochemical factors including concentrations of
sulfate (Rankine 1968, Eschenbruch 1974), cysteine and
methionine (Eschenbruch 1972, Duan et al. 2004), ex-
posure to oxygen (Pearlstein 1988), sulfite binding
compounds (Rankine 1968, Rankine and Pocock 1969),
insoluble solids (Liu and Gallander 1982), and the
presence, absence, or relative activity of genes encoding
for the membrane bound sulfite pump (Avram and
Bakalinsky 1997, Park and Bakalinsky 2000, Donalies and
Stahl 2002).
Besides the above-mentioned factors, the concentra-
tion of assimilable nitrogen in grape musts can also affect
the metabolic pathways involved in the assimilatory
reduction of sulfate to sulfite and sulfide (Henschke and
Jiranek 1991, Giudici and Kunkee 1994, Jiranek et al.
1995, 1996, Spiropoulos et al. 2000, Wang et al. 2003).
For example, nitrogen deficiency results in the increased
production of sulfide by Saccharomyces (Vos and Gray 1979,
Giudici and Kunkee 1994, Jiranek et al. 1995, Hallinan et
al. 1999, Spiropoulos et al. 2000). Despite the fact that sul-
fide and sulfite production are within the same biological
pathway (Vos and Gray 1979, Tamayo et al. 1999,
Spiropoulos et al. 2000), the effect of assimilable nitrogen
on the production of sulfite by wine yeast has not been
reported.
Therefore, the objective of this research was to inves-
tigate the influence of nitrogen on SO2 production by
Saccharomyces and the effect of this SO2 on the malolactic
fermentation.
Materials and methods
Microorganisms
Active-dry forms of Saccharomyces cerevisiae used were
strains RUBY.ferm (Chr. Hansen, Hørsholm, Denmark), V-
1116 and UCD 522 Montrachet (Lallemand, Montreal,
Australian Journal of Grape and Wine Research 12, 69–78, 2006
Canada), UCLM S325 and Saint Georges S101 (Lesaffre
Group, Maisons-Alfort, France) and S. bayanus EC1118
Prise de Mousse (Lallemand). Yeast strains were main-
tained on potato dextrose agar (Difco) slants stored at 4ºC.
The strain of Oenococcus oeni used in this study was the
freeze-dried form of DSM 7008, Viniflora oenos (VFO)
(Chr. Hansen).
Enumeration
Microbial viabilities were determined using diluents con-
taining 0.1% peptone and using appropriate media and an
Autoplate 4000 spiral plating device (Spiral Biotech,
Bethesda, MD, USA). Yeast were grown on wort agar
while bacteria were enumerated using 2% de Man,
Rogosa and Sharpe (MRS) agar (pH 4.5) comprised of liver
extract (1 g/L) (Sigma, Saint Louis, MO, USA), tryptone
(20 g/L), glucose (5 g/L), peptone (5 g/L), yeast extract (5
g/L), 5% Tween 80 (1 mL/L), and non-concentrated apple
juice (250 mL/L). Plates were incubated aerobically at
25ºC for two (yeast) or seven (bacteria) days prior to
counting.
Starter culture preparation
Yeast were transferred from a slant into 1 L of acidic grape
juice broth (pH 4.5) (Dicks et al. 1990) and incubated aer-
obically at 25ºC for 72 h. The cells were then harvested
using centrifugation (4000 × g for 30 min) and resuspend-
ed in 0.2 M phosphate buffer (pH 7.0) before inoculation
into fermentation media. Bacterial cultures were in direct
inoculum freeze-dried form.
Alcoholic fermentations
Fermentations were conducted using a synthetic grape
juice medium (pH 3.5) as described by Wang et al. (2003)
with the following additions; 5% Tween 80 (1 mL/L), ade-
nine sulfate (5 mg/L), guanine.HCL (5 mg/L), cytosine (5
mg/L), thymidine (5 mg/L), xanthine (5 mg/L), and uracil
(5 mg/L). Concentrations of 10 µg/L biotin and 250 µg/L
pantothenic acid were used for all fermentations. A 2 × 6
factorial experimental design was employed with con-
centrations of yeast assimilable nitrogen (YAN) (60 and
250 mg/L) and yeast strain (Saint Georges S101, UCD 522,
EC1118, RUBY.ferm, UCLM S325, V-1116) as variables.
YAN was calculated as the sum of the concentrations of
ammonia and the molar proportion of the α-amino nitro-
gen present in amino acids except proline. Once prepared,
3 L of each medium was sterile filtered through 0.22 µm
PLUS membrane bottle-top filters (Millipore Corp.
Bedford, MA, USA) into 5 L Celstir fermenters (Wheaton
Science Products, Millville, NJ, USA) equipped with stain-
less steel headpieces (B. Braun Biotech, Allentown, PA,
USA). Yeast starter cultures (10 mL) were inoculated into
the fermentation media to yield initial viable cell con-
centrations of 1 × 106 CFU/mL. All fermentations were
replicated in triplicate and conducted at 22ºC with stirring
(75 rpm for 5 min) just prior to sampling. Aseptic
sampling was accomplished using a syringe-type system
obtained from New Brunswick Scientific Co. (model
1230–6000, Edison, NJ, USA). Reducing sugars were esti-
mated by Clinitest.
Osborne & Edwards
Malolactic fermentations
During alcoholic fermentation, 150 mL samples were
aseptically removed and 100 mL was sterile filtered
through 0.45 µm disposable Nalgene PES membrane filter
units (Nalge Nuno International, Rochester, NY, USA) into
sterilised milk dilution bottles. The remaining sample
volume (c. 50 mL) was used to analyse yeast viable cell
populations, and free/total SO2 by titration with iodine
(Amerine and Ough 1980). All analyses were performed
in triplicate. Freeze-dried O. oeni (0.1 g) was rehydrated in
20 mL of 0.2M sterile phosphate buffer (pH 7.0) for 30
min before inoculation into the sterile filtered 100 mL
samples at initial populations of approximately 5 × 106
CFU/mL. Bottles were incubated at 25ºC with bacterial
viable cell populations and L-malic acid (enzymatic test
kit, R-Biopharm, Darmstadt, Germany) being measured
twice-weekly.
In a secondary experiment, samples were removed
after 16 days from fermentations being conducted by S.
cerevisiae strains RUBY.ferm and UCD 522 in high- and
low-YAN-containing synthetic grape juices. These samples
were sterile filtered as above and free/total SO2 concen-
trations were measured by titration with iodine. Prior to
measuring the samples, synthetic wine samples were
spiked with known amounts of SO2 and measured using
iodine titration to check the accuracy of this methodology.
An addition of SO2 (as K2S2O5) was made to the low-YAN
samples so that the total SO2 concentration was the same
as that measured in the high-YAN samples. Samples were
then left for 24 h at room temperature to allow binding of
free SO2 before O. oeni was added to give an initial popu-
lation of 5 × 106 CFU/mL. Samples were incubated at 25ºC
and viable cell populations and malic acid were measured
twice-weekly as outlined previously.
Statistics
Statistical analysis was accomplished using SAS version
8.1 (SAS Institute Inc., Cary, NC, USA) with Tukey’s HSD
test for mean separation.
Results
Alcoholic fermentations
After inoculation into synthetic grape juice, all six yeast
strains achieved populations of 107 CFU/mL or greater
within two days under both high- and low-YAN con-
ditions (data not shown) with no differences between
maximum yeast populations of high- and low-YAN fer-
mentations being observed. In addition, all fermentations
reached dryness (< 2 g/L reducing sugars) within 44 days.
Despite no observable differences in yeast growth,
yeast strains produced varying amounts of SO2 under both
high- and low-YAN conditions as shown in Figure 1. Peak
total SO2 concentrations coincided with maximal yeast
populations with the highest amount of total SO2 being
produced by V-1116 on day 16 (44.3 mg/L) under high-
YAN conditions. Saint Georges S101 produced the lowest
concentrations of SO2 under both high- and low-YAN con-
ditions. Concentrations of free SO2 as measured by titra-
tion were less than 5 mg/L for all yeast strains under both
high- and low-YAN conditions (data not shown). All yeast
Inhibition of MLF 71
Table 1. Maximum total SO2 produced by different strains
of Saccharomyces during alcoholic fermentation of synthetic
grape juice with high or low amounts of YAN.
Maximum total SO2 (mg/L)1
Yeast strain High-YAN Low-YAN
Saint Georges S101 9.60ab 6.30a
UCD 522 22.9e 9.60ab
EC1118 18.6 cd
10.1ab
RUBY.ferm 21.3ed 11.2b
UCLM S325 27.7f 17.1c
V-1116 44.3g 23.3e
1
Values are the means from triplicate analyses made on triplicate
fermentations.
a-g
Mean values with different superscript letters are significantly different at
P ≤ 0.05.
strains except Saint Georges S101 produced significantly
higher concentrations of total SO2 under high-YAN con-
ditions than under low-YAN conditions (Table 1). There
were also significant differences between yeast strains with
regard to total SO2 production as V-1116 and UCLM S325
produced significantly more total SO2 than all other strains
under high-YAN conditions. EC1118, RUBY.ferm, and
UCD 522 produced similar amounts of total SO2 while
Saint Georges S101 produced the least. These trends were
generally mirrored under low-YAN conditions with V-
1116 producing the highest concentrations of total SO2
while Saint Georges S101 produced the lowest.
Malolactic fermentations
Samples of the fermenting synthetic grape juice were peri-
odically removed, sterile filtered, and inoculated with O.
oeni to induce MLF. The bacterium consumed malate in
samples fermented under low nitrogen conditions by Saint
Georges S101 (Figure 2a) UCD 522 (Figure 3a), EC1118
(Figure 4a), RUBY.ferm (Figure 5a), and UCLM S325
(Figure 6a) at every sampling time. However, slower
malate utilisations were observed in samples that were fer-
mented for 2, 9, 16, 23 and 44 days by V-1116 under low-
YAN conditions (Figure 7a). O. oeni grew well and malate
was consumed in musts fermented under high-YAN con-
ditions by Saint Georges S101 (Figure 2b), and EC1118
(Figure 4b) at every sampling time but bacterial viability
rapidly decreased and slower malate utilisations were
observed in musts fermented for 9, 16, 23 and 44 days
under high-YAN conditions by UCD 522 (Figure 3b),
RUBY.ferm (Figure 5b), UCLM S325 (Figure 6b), and V-
1116 (Figure 7b).
The time taken by O. oeni to complete the MLF was
influenced by the production of SO2 by Saccharomyces as
shown in Figure 8. In samples containing the highest con-
centrations of SO2 the MLF was not completed in less than
28 days under low (group I) and high (group III and IV)
nitrogen conditions. In contrast, MLF was completed in
less than 21 days in samples which generally had lower
72 Inhibition of MLF Australian Journal of Grape and Wine Research 12, 69–78, 2006

50 50
a b
40 40
Total SO2 (mg/L)

Total SO2 (mg/L)

30 30

20 20

10 10

0 0
0 14 28 42 0 14 28 42
Time (days) Time (days)

50 50
c d
40 40
Total SO2 (mg/L)

30 Total SO2 (mg/L) 30

20 20

10 10

0 0
0 14 28 42 0 14 28 42
Time (days)
Time (days)

50 50
e f
40 40
Total SO2 (mg/L)
Total SO2 (mg/L)

30 30

20 20

10 10

0 0
0 14 28 42 0 14 28 42
Time (days) Time (days)

Figure 1. Production of total SO2 by Saccharomyces strain Saint Georges S101 (a), UCD 522 (b), EC1118 (c), RUBY.ferm (d), UCLM S325
(e), and V-1116 (f) during alcoholic fermentation of synthetic grape juice with high () or low (  ) amounts of YAN. Arrows indicate
completion of alcoholic fermentation. Values are the means from triplicate analyses made on triplicate fermentations.
Osborne & Edwards Inhibition of MLF 73

Day 0 4
4 Day 0
Day 2
a a
Day 9 Day 2

Day 16 Day 9
3 Day 16
Malic acid (g/L)

Day 23 3

Malic acid (g/L)

Day 44 Day 23
Day 44

2 2

1 1

0 0
0 7 14 21 0 7 14 21
Time (days) Time (days)
4 4
Day 0
b b
Day 2 Day 0
Day 9 Day 2
3 3
Day 16 Day 9

Malic acid (g/L)

Malic acid (g/L)

Day 23 Day 16
Day 44 Day 23
2 2 Day 44

1 1

0 0
0 7 14 21 0 7 14 21
Time (days) Time (days)

Figure 2. Malic acid degradation by O. oeni VFO in synthetic grape Figure 4. Malic acid degradation by O. oeni VFO in synthetic grape
juice undergoing alcoholic fermentation induced by S. cerevisiae juice undergoing alcoholic fermentation induced by S. cerevisiae var.
Saint Georges S101 with low (a) or high (b) amounts of YAN. Values bayanus EC1118 with low (a) or high (b) amounts of YAN. Values are
are the means from triplicate analyses made on triplicate the means from triplicate analyses made on triplicate fermentations.
fermentations.
4 4
Day 0 Day 0
a a
Day 2 Day 2
Day 9 Day 9
3 Day 16 Day 16
3
Malic acid (g/L)
Malic acid (g/L)

Day 23 Day 23
Day 44 Day 44

2 2

1 1

0 0
0 7 14 21 0 7 14 21
Time (days) Time (days)
4 4
Day 0
Day 0 b
b Day 2
Day 2
Day 9
Day 9
3 3 Day 16
Day 16
Malic acid (g/L)
Malic acid (g/L)

Day 23
Day 23
Day 44
Day 44
2 2

1 1

0
0
14 21 0 7 14 21
0 7
Time (days) Time (days)

Figure 3. Malic acid degradation by O. oeni VFO in synthetic grape Figure 5. Malic acid degradation by O. oeni VFO in synthetic grape
juice undergoing alcoholic fermentation induced by S. cerevisiae juice undergoing alcoholic fermentation induced by S. cerevisiae
UCD 522 with low (a) or high (b) amounts of YAN. Values are the RUBY.ferm with low (a) or high (b) amounts of YAN. Values are the
means from triplicate analyses made on triplicate fermentations. means from triplicate analyses made on triplicate fermentations.
74 Inhibition of MLF Australian Journal of Grape and Wine Research 12, 69–78, 2006

4 Day 0 4 Day 0
a Day 2 a Day 2
Day 9 Day 9
Day 16
3 Day 16
Day 23 3
Malic acid (g/L)

Malic acid (g/L)

Day 23
Day 44
Day 44

2 2

1 1

0 0
0 7 14 21 0 7 14 21
Time (days) Time (days)

Day 0 4 Day 0
4
b Day 2 b Day 2
Day 9 Day 9
Day 16 3 Day 16
3
Malic acid (g/L)

Malic acid (g/L)

Day 23 Day 23
Day 44 Day 44

2 2

1 1

0
0
0 7 14 21
0 7 14 21
Time (days) Time (days)

Figure 6. Malic acid degradation by O. oeni VFO in synthetic grape Figure 7. Malic acid degradation by O. oeni VFO in synthetic grape
juice undergoing alcoholic fermentation induced by S. cerevisiae juice undergoing alcoholic fermentation induced by S. cerevisiae V-
UCLM S325 with low (a) or high (b) amounts of YAN. Values are the 1116 with low (a) or high (b) amounts of YAN. Values are the means
means from triplicate analyses made on triplicate fermentations. from triplicate analyses made on triplicate fermentations.

total SO2 concentrations (group II, V, and VI). However, Discussion

within groups I and III, there were samples that contained
similar concentrations of total SO2 to those found in sam-
ples in group VI, all of which completed MLF in less than
14 days. For example, under high nitrogen conditions
MLF was not completed by O. oeni in less than 28 days in
samples fermented by RUBY.ferm (group III) but was
completed in 14 days in samples fermented by EC1118
(group VI). In addition, in samples fermented by V-1116
under low nitrogen conditions the MLF was not com-
pleted in less than 28 days while in samples in group II
and VI that contained similar amounts of SO2 the MLF
was completed in less than 21 days
Inhibition of O. oeni was observed in samples fer-
mented by RUBY.ferm and UCD 522 under both high-
and low-YAN conditions if SO2 concentrations were
equalised through the addition of SO2 to low-YAN samples
(Figure 9). Results showed that the bacteria grew well
(Figure 9a) and consumed malic acid (Figure 9b) in sam-
ples fermented by S. cerevisiae RUBY.ferm and UCD 522
under low-YAN conditions (10.1 and 9.6 mg/L total SO2).
In contrast, O. oeni populations decreased below detectable
levels and little or no malic acid was consumed in samples
fermented by S. cerevisiae RUBY.ferm and UCD 522 under
high-YAN conditions (20.3 and 21.3 mg/L total SO2) and
under low-YAN conditions where SO2 had been added to
give 20 mg/L total SO2.
Growth of S. cerevisiae during the alcoholic fermentation
inhibited the MLF with the inhibition dependent on the
yeast strain used. Here, strains UCD 522, RUBY.ferm,
UCLM S325, and V-1116 inhibited the MLF while strains
Saint Georges S101 and EC1118 did not. These findings
were consistent with previous reports where yeast strains
were shown to vary in their antagonism of malolactic bac-
teria (Lemaresquier 1987, Cannon and Pilone 1993,
Henick-Kling and Park 1994, Larsen et al. 2003). The vari-
able ability of S. cerevisiae strains to inhibit O. oeni is
thought to be due to the production of antibacterial com-
pounds (Fornachon 1968, Lonvaud-Funel et al. 1988,
Cannon and Pilone 1993, Henick-Kling and Park 1994,
Caridi and Corte 1997, Larsen et al. 2003) leading to the
description of wine yeast strains as ‘malolactic friendly’ or
‘malolactic unfriendly’.
Variability between yeast strains with regard to their
antagonism of O. oeni however could not always explain
the observed bacterial inhibition in the present study. For
instance, the inhibition of the MLF also appeared to be
influenced by the nitrogen status of the grape juice, as
some yeast strains inhibited O. oeni only during fermen-
tation in high-nitrogen grape juice. The MLF was inhibited
by S. cerevisiae strains UCD 522, RUBY.ferm and UCLM
S325 during fermentation under high-nitrogen conditions
while inhibition was greatly reduced or not observed
Osborne & Edwards Inhibition of MLF 75

a
I a
Malolactic fermentation (days)

> 28

Bacterial growth (CFU/mL)

21

II
14

0
0 10 20 30 40 50 < 103 < 103
Total SO2 (mg/L)
0 7 14 21
Time (days)
b
b
Malolactic fermentation (days)

> 28

III IV
21

Malic acid (g/L)

V

14

VI
7

0
0 10 20 30 40 50
Total SO2 (mg/L)

0 7 14 21
Figure 8. Days required by O. oeni to degrade malic acid below 0.25 Time (days)
g/L in wine containing various concentrations of SO2 produced by
yeast strains Saint Georges S101 (), UCD 522 ( ), EC1118 (), Figure 9. Growth (a) and malic acid degradation (b) by O. oeni in
RUBY.ferm (
), UCLM S325 (  ), and V1116 (), during synthetic grape juice fermented 16 days by S. cerevisiae RUBY.ferm
fermentation under low (a) or high (b) nitrogen conditions. Roman (, ,
) and UCD 522 ( , ,
) under high- (, ) and low- (,
numerals refer to groups of samples with comparable amounts of SO2 ) YAN conditions and under low-YAN conditions with the addition
requiring a similar time to complete MLF. Data extracted from of SO2 (
,
).Values are the means from triplicate analyses made on
information in Figures 1 to 7. triplicate fermentations.

under low-nitrogen conditions. Previous studies on the for NADPH may result in sulfite reduction becoming redox
influence of nitrogen on the MLF have suggested that a limited to such an extent that SO2 accumulates. However,
lack of nitrogen may cause inhibition of O. oeni due to the whether this mechanism operates in yeast during growth
need for nitrogen for bacterial growth (Kunkee 1967, under high nitrogen conditions is debatable, as Jiranek et
Amerine and Kunkee 1968, Fornachon 1968, Beelman et al. (1996) reported that sulfite reductase activity was not
al. 1982). However, in this study the high nitrogen con- repressed under high nitrogen conditions when S. cerevisiae
centration of a grape juice was shown to contribute to the was grown in a chemically defined grape juice medium.
inhibition of O. oeni. In the present study it is not known whether com-
The influence of high nitrogen concentrations on the petition for reduced coenzyme was responsible for the ele-
MLF may have been due to the increased production of vated production of SO2 under high nitrogen conditions as
SO2 by the yeast during fermentation under high-nitrogen sulfite reductase activity was not measured. It should also
conditions. Evidence for this is provided in the present be noted that (NH4)2HPO4 and NH4Cl were the primary
study where all yeast strains, with the exception of Saint sources of assimilable nitrogen in the studies by Giudici
Georges S101, produced higher concentrations of SO2 and Kunkee (1994), Jiranek et al. (1996), and Duan et al.
during fermentation in high nitrogen grape juice. In sup- (2004), while amino acids were the primary source of
port, Giudici and Kunkee (1994) reported that sulfite for- assimilable nitrogen in the present study. Individual amino
mation by wine yeast increased with increasing amounts acids such as methionine, cysteine, asparagine and argi-
of nitrogen availability while Duan et al. (2004) reported nine have been shown to influence sulfite formation
that high nitrogen levels increased the amount of SO2 pro- (Eschenbruch 1972, Giudici and Kunkee 1994, Duan et al.
duced by brewing yeast. Duan et al. (2004) argued that 2004) and therefore direct comparisons between these
under high nitrogen conditions an increased production of studies may not be valid.
O-acetylhomoserine would compete with the redox and The increased production of SO2 during fermentation
energy requirements for the reduction of sulfite to sulfide. under high-nitrogen conditions contributed to the in-
Three molecules of NADPH are needed for the reduction hibition of the MLF by the various yeast strains. Samples
of sulfite to sulfate by sulfite reductase while O-acetyl- containing the highest concentrations of total SO2 were
homoserine production involves acetyl CoA, a compound inhibitory towards the MLF with bacterial inhibition only
whose formation also requires NADPH. This competition being observed in samples containing greater than 20
76 Inhibition of MLF
mg/L total SO2. For instance, strain V-1116 produced
greater than 20 mg/L total SO2 under both high- and low-
nitrogen conditions and was highly antagonistic towards
the bacteria, while the low-sulfite producing strain, Saint
Georges S101, did not inhibit bacterial growth, a finding
supported by Larsen et al. (2003). Strains UCD 522,
RUBY.ferm, and UCLM S325 only produced greater than
20 mg/L of SO2 during fermentation under high-nitrogen
conditions, suggesting that the elevated production of SO2
by yeast strains under high-nitrogen conditions was
responsible for inhibition of the MLF. Furthermore, exper-
iments where low-nitrogen samples were supplemented
with SO2 suggest that bacterial inhibition in the high-
nitrogen samples was due to the higher SO2 concentra-
tions produced by the yeast and not by the increased pro-
duction of other metabolites. In agreement with Larsen et
al. (2003), very low concentrations of free SO2 were mea-
sured at all stages of the alcoholic fermentation, suggest-
ing that the bound form of SO2 was responsible for the
observed inhibition of O. oeni.
The ability of nitrogen to cause an increase in the pro-
duction of SO2 by yeast is often not accounted for in stud-
ies investigating the inhibition of MLF. For example,
Henick-Kling and Park (1994) reported that yeast-
produced SO2 was responsible for MLF inhibition, while
Eglinton and Henschke (1996), using the same yeast
strains, concluded that yeast produced insufficient
amounts of SO2 to cause bacterial antagonism. However,
neither study reported the nitrogen content of the grape
juices used, which may have influenced SO2 formation by
the yeast leading to the contrasting conclusions of the two
studies. Results from this present study may also help
explain the findings of King and Beelman (1986) where
less bacterial antagonism by yeast was observed if grape
juice was diluted from 20°Brix to 10°Brix. The authors
proposed that the dilution of the grape juice resulted in a
dilution of inhibitory precursors. An alternative explana-
tion is offered based on the results from the present study.
The dilution of the grape juice would decrease the nitro-
gen content of the juice, resulting in lower SO2 production
by the yeast and reduced inhibition of the bacteria.
Furthermore, S. cerevisiae strain UCD 522 was used in the
study by King and Beelman (1986), a yeast strain inves-
tigated in this current study and found to be inhibitory to
bacteria only during fermentation under high-nitrogen
conditions.
Although the production of SO2 by yeast and the influ-
ence of nitrogen on its production often accounted for the
observed bacterial inhibition, a correlation between SO2
production and inhibition of O. oeni did not always exist.
For instance, O. oeni completed MLF in wine fermented
under high nitrogen conditions by EC1118, but did not
complete MLF in samples fermented by RUBY.ferm,
despite there being no statistical difference in the maxi-
mum concentration of SO2 produced by each yeast strain.
These findings suggest that inhibitory mechanism(s) other
than SO2 were responsible for the antagonism caused by
RUBY.ferm, corroborating the contention that inhibitory
mechanisms other than SO2 must exist (King and
Beelman 1986, Eglinton and Henschke 1996, Caridi and
Australian Journal of Grape and Wine Research 12, 69–78, 2006
Corte 1997, Larsen et al. 2003). However, O. oeni was not
inhibited in samples fermented by RUBY.ferm under low-
nitrogen conditions that contained lower SO2 concentra-
tions (11.2 mg/L maximum SO2 produced). This indicates
that although SO2 may not have been the primary cause
of bacterial inhibition by RUBY.ferm, it may have con-
tributed, in conjunction with other inhibitory mecha-
nism(s), to the antagonism of O. oeni.
If MLF is to be encouraged during winemaking then
the yeast strain used to conduct the alcoholic fermentation
and the nitrogen content of the grape juice should be con-
sidered. Certain yeast strains can produce sufficient SO2 to
inhibit O. oeni, especially under high-nitrogen conditions.
Furthermore, some yeast strains may inhibit MLF through
the production of other inhibitory substances. Because the
concentration of nitrogen in grape juice varies greatly
(Amerine et al. 1980, Henschke and Jiranek 1993, Spayd
and Andersen-Bagge 1996), there may be much variabil-
ity regarding the antagonism of O. oeni by specific yeast
strains. For example, in this study certain yeast strains in-
hibited O. oeni only when fermenting under high-nitrogen
conditions. Therefore, when considering the selection of
yeast strains to conduct the alcoholic fermentation the
nitrogen content of the grape juice should also be
accounted for if MLF is desired.
Conclusions
This research demonstrates that yeast strain and nitrogen
content can influence production of SO2 by Saccharomyces
during alcoholic fermentation with increased production
of SO2 under high-nitrogen conditions. The production of
SO2 by Saccharomyces may be involved in the inhibition of
O. oeni, with bound SO2 possibly being more inhibitory
then previously believed. However, results also indicate
that some yeast strains may inhibit malolactic fermenta-
tion by mechanisms other than SO2. The nature of this
mechanism remains unknown and requires further
study. In addition, the means by which nitrogen concen-
tration influences the production of SO2 by yeast also
needs further investigation. Future studies should also be
conducted using grape juice rather than synthetic grape
juice media to confirm the findings in the present study.
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Manuscript received: 9 September 2005
Revised manuscript received: 21 February 2006