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UMR 1219 oenologie, Universit Bordeaux 2, INRA, ISVV, 351 cours de la Libration, 33400 Talence, France
b
ENITA de Bordeaux, 1 cours du Gnral de Gaulle, CS 40201, 33175 Gradignan cedex, France
Received 10 May 2007; received in revised form 23 November 2007; accepted 30 December 2007
Abstract
Conventional wine yeasts produce high concentrations of volatile acidity, mainly acetic acid, during high-sugar fermentation. This alcoholic
fermentation by-product is highly detrimental to wine quality and, in some cases, levels may even exceed legal limits. In this study, a nonconventional species, Torulaspora delbrueckii, was used, in pure cultures and mixed with Saccharomyces cerevisiae yeast, to ferment botrytized
musts. Fermentation rate, biomass growth, and the formation of volatile acidity, acetaldehyde, and glycerol were considered. This study
demonstrated that T. delbrueckii, often described as a low acetic producer under standard conditions, retained this quality even in a high-sugar
medium. Unlike S. cerevisiae, this species did not respond to the hyper-osmotic medium by increasing acetic production as soon as it is inoculated
into the must. Nevertheless, this yeast produced low ethanol and biomass yields, and the fermentation was sluggish. As a result, T. delbrueckii
fermentations do not reach the required ethanol content (14%vol.), although this species can survive at this concentration. A mixed culture of T.
delbrueckii and S. cerevisiae was the best combination for improving the analytical profile of sweet wine, particularly volatile acidity and
acetaldehyde production. A mixed T. delbrueckii/S. cerevisiae culture at a 20:1 ratio produced 53% less in volatile acidity and 60% less
acetaldehyde than a pure culture of S. cerevisiae. Inoculating S. cerevisiae after 5 days' fermentation by T. delbrueckii had less effect on volatile
acidity and acetaldehyde production and resulted in stuck fermentation. These results contribute to a better understanding of the behaviour of nonSaccharomyces and their potential application in wine industry.
2008 Elsevier B.V. All rights reserved.
Keywords: Wine; High-sugar fermentation; Torulaspora delbrueckii; Mixed culture; Volatile acidity; Acetaldehyde
1. Introduction
Spontaneous alcoholic fermentation of grape must is a complex process, carried out by sequential action of several yeast
genera and species, found on grapes and in must. Saccharomyces cerevisiae is not the only species that grows during
natural wine fermentations. The early stages in fermentation are
dominated by the growth of non-Saccharomyces yeasts, characterized by low fermentative capacity. After the first few days
of fermentation, they die off, due to the increasing concentration
Corresponding author. Tel.: +33 5 4000 6489; fax: +33 5 4000 6468.
E-mail address: marina.bely@univ-pau.fr (M. Bely).
0168-1605/$ - see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2007.12.023
313
The aim of this work was to evaluate the ability of a nonconventional, non-Saccharomyces yeast species, T. delbrueckii,
often described as a low acid acetic producer, to ferment highsugar must. This paper reports the impact of pure, mixed, and
sequential cultures of T. delbrueckii and S. cerevisiae on fermentation behaviour, biomass growth, and analytical profile.
2. Materials and methods
2.1. Microorganisms and media
Two T. delbrueckii strains from the MUCL collection
(Mycothque de l'Universit Catholique de Louvain-la-Neuve),
T. delbrueckii 27828 and T. delbrueckii 31703, and one S.
cerevisiae strain, Zymaflore ST (Laffort Oenologie, France), were
used. The Zymaflore ST strain was selected from native microflora during spontaneous fermentation of a botrytized must for its
ability to produce small amounts of volatile acidity and SO2binding compounds (Barbe et al., 2000, Masneuf and Dubourdieu, 2000), and is commonly used in Sauternes.
Yeasts were grown at 30 C on complete YPD medium
(1% yeast extract, 1% peptone, 2% dextrose) solidified with 2%
agar.
2.2. Cell population count
Cell populations were determined by measuring absorbance
at 610 nm. One absorbance unit corresponded to 6 106 cfu/mL
for the two T. delbrueckii strains and to 7 106 cfu/mL for S.
cerevisiae ST. Cell population was measured every day until no
further increase in O.D was observed (O.D610 max.).
Yeast growth was also determined by plate counting in some
cases. Samples were withdrawn throughout fermentation and
diluted appropriately in dilution medium. Non-Saccharomyces
cells were counted using lysine agar (LA) (Per litre: 66 g lysine
medium (Oxoid), 10 mL 50% potassium lactate, and 1 mL 10%
lactic acid, pH 4.8). Total yeast cells were counted using YPDA
medium supplemented with chloramphenicol (100 mg/L). The
number of S. cerevisiae was given as the difference between the
total plate count using YPDA and the plate count using LA.
YPDA and LA plates were incubated at 25 C for 4 days before
counting.
2.3. Must
The musts were obtained from botrytized Semillon grapes,
harvested in vineyards in the Sauternes and Barsac appellations
(20032005 vintages). They were collected in the cellar after
settling and frozen. Initial sugar concentrations were 360 g/L.
Musts were supplemented with nitrogen by adding ammonium
sulphate (NH4)2 SO4 to a level of 190 mgN/L. According to
previous studies (Bely et al., 2003), this nitrogen concentration
is optimum for minimising volatile acidity production by this
strain. The indigenous yeast population was estimated by plate
counting.
Pasteurized musts were heated to 90 C for 15 min and the
effectiveness of this treatment was verified by plate counting.
314
of CO2 required (35 g/bottle, corresponding to an ethanol concentration of approximately 14%vol.), while both T. delbrueckii
27828 and T. delbrueckii 31703 stopped fermenting when only
18.7 and 23.9 g/bottle CO2, respectively, had been released.
The fermentation rate varied markedly depending on the yeast
used (Fig. 1). Alcoholic fermentation with S. cerevisiae ST
was completed 11 days after inoculation, whereas both T.
delbrueckii 27828 and T. delbrueckii 31703 strains had slower
fermentation rates and fermentations stopped after 20 and
26 days, respectively. It is worthwhile noting that, in both T.
delbrueckii, the fermentation rate was the same during the first
15 g/bottle CO2 released (corresponding to an ethanol content
of 6%vol.). S. cerevisiae ST had the highest cell densities (12.4)
after 3 days (not shown), while both T. delbrueckii 27828 and
T. delbrueckii 31703 stopped growing after 4 days' fermentation at cell densities of 4 and 8.4, respectively (Table 1). This
is consistent with previous results, which reported that T.
delbrueckii species had lower growth rates than S. cerevisiae
(Mauricio et al., 1991; Ciani and Picciotti, 1995; Ciani, 1997).
However, this reduced growth activity was not due to a deficit
of assimilable nitrogen compounds, as they did not consume all
that was available. Similar behaviour was reported by Ciani
et al. (2006).
The higher ethanol concentration in the S. cerevisiae ST
culture (15.1% vol.) was not the only difference. In comparison
with the S. cerevisiae ST strain, the two T. delbrueckii fermentations also had a lower ethanol/sugar yield, which differs
from a previous study using a low-sugar must (Ciani and Picciotti,
1995). Glycerol production by T. delbrueckii 27828 was lower
than that of the other two yeasts.
T. delbrueckii 27828 and T. delbrueckii 31703 produced 2.7and 1.9-fold less volatile acidity, respectively, than S. cerevisiae
ST, confirming the specific characteristic of low acetic acid
production of these yeasts species, as already reported in previous studies under standard winemaking conditions (Cabrera
et al., 1988; Herraiz et al., 1990; Martinez et al., 1990; Ciani and
Picciotti, 1995; Ciani, 1997; Ciani and Ferraro, 1998).
Max.cell population
(O.D610max)
Residual nitrogen (mgN/L)
Residual sugar (g/L)
Ethanol (% vol.)
Glycerol (g/L)
Volatile acidity
(g/L acetic acid)
Acetaldehyde (mg/L)
Yield ethanol/sugar (g/g)
T. delbrueckii T. delbrueckii
27828
31703
12.4
8.4
12.00 1.41c
106.33 1.15c
15.10 0.07a
17.24 0.34a
1.17 0.08a
77.00 5.66b
219.63 3.79a
7.40 0.21c
14.05 0.92b
0.43 0.01c
100.50 2.12a
187.00 10.82b
9.38 0.25b
15.95 0.07a
0.62 0.01b
80.7 16.31a
0.48
18.4 3.50b
0.43
27.00 3.00b
0.46
315
pointing out that the S. cerevisiae ST strain was selected for its
low volatile acidity and acetaldehyde production (Barbe et al.,
2000, Masneuf and Dubourdieu, 2000).
As expected, inoculating high-sugar musts with T. delbrueckii yeast resulted in stuck fermentations. Although this
yeast species had a low ethanol yield, it had the best secondary
metabolite profile. Low acetic acid and acetaldehyde production
is a positive feature for fermenting botrytized must, so a combination of T. delbrueckii species and S. cerevisiae was envisaged to improve wine quality, while ensuring that fermentation
would be completed. T. delbrueckii 31703 was chosen for the
following study as it had a higher ethanol production.
3.2. Fermentation behaviour of mixed culture
Pure and mixed fermentations were carried out using T.
delbrueckii 31703 and S. cerevisiae ST in pasteurized (PM) or
non-pasteurized must (NPM). Non-pasteurized musts were used
to mimic winemaking practices as closely as possible. Pure
cultures were inoculated with 2 106 cells/mL. Mixed fermentation trials were inoculated with 5.106 cells/mL of T. delbrueckii
31703 and 106 cells/mL of S. cerevisiae ST (ratio T. delbrueckii/
S. cerevisiae 5:1).
Yeast growth was monitored by plate counting. Non-Saccharomyces cells (corresponding to T. delbrueckii 31703 in
pasteurized must) were counted using lysine agar (LA). The
number of Saccharomyces cells was calculated as the difference
between the total plate count using YPDA and LA. In
pasteurized must, the estimated Saccharomyces count corresponded to the S. cerevisiae ST population. The Saccharomyces
and non-Saccharomyces populations in the non-pasteurized
must were estimated at 2 105 cfu/mL and 1.4 104 cfu/mL,
respectively.
The required amounts of ethanol (up to 14%vol.) were obtained in all fermentations except the pure T. delbrueckii 31703
culture in PM, which stopped fermenting at 9.7%vol. (not
shown). This result suggested that the indigenous yeasts
contributed sufficiently to fermentation behaviour to produce
the amount required of ethanol for the pure culture of T.
delbrueckii 31703 in NPM. Fermentation time varied, depending on the must treatment and yeast culture (Fig. 2). The fastest
fermentation occurred in the pure S. cerevisiae ST culture in
NPM, followed by S. cerevisiae ST in PM and mixed culture in
NPM, then mixed culture in PM. The pure T. delbrueckii 31703
cultures in NPM and PM took the longest to ferment (22 days).
As described above, the pure T. delbrueckii 31703 cultures
had the lowest maximum cell populations (O.D610 max.) and
the highest nitrogen residues (Table 2). However, nitrogen
consumption was higher in NPM, indicating competition between T. delbrueckii 31703 and the indigenous yeasts in the
NPM. On the contrary, in pure and mixed S. cerevisiae ST
cultures, irrespective of the must treatment, the nitrogen was
almost completely consumed and maximum cell populations
were higher.
Biomass evolutions during fermentation are shown in Fig. 3.
No significant differences in the Saccharomyces populations
were observed between the pasteurized and non-pasteurized
316
Fig. 2. Fermentation courses at 23 C. Pasteurized (open symbols) or nonpasteurized must (filled symbols). Pure S. cerevisiae ST culture (S). Pure T.
delbrueckii culture (T). Mixed T. delbrueckii 31703/S. cerevisiae ST (T/S).
Means of triplicate fermentations, max. SD 0.9.
treatments (Fig. 3a). The maximum concentration of Saccharomyces cells varied from 6.9 to 7.9 Log (cfu/mL), with the
highest population in pure S. cerevisiae ST cultures (in NPM or
PM) and the lowest in NPM inoculated with pure T. delbrueckii
31703. Intermediate populations were observed in mixed cultures,
suggesting possible competition between non-Saccharomyces
and Saccharomyces species, at critical nitrogen concentrations,
which restricted Saccharomyces cell growth.
The biomass evolutions of non-Saccharomyces species
(Fig. 3b) revealed similar maximum cell populations, 7.5 Log
(cfu/mL), except for the pure S. cerevisiae ST inoculum. In PM,
the pure T. delbrueckii 31703 culture slowly began to die off
after the level reached 6%vol. (corresponding to 12 g/bottle
CO2 released), while in NPM the non-Saccharomyces yeasts
population subsisted at a higher level, 6.6 Log(cfu/mL), until
fermentation was stopped. It is noteworthy that, in PM the pure
T. delbrueckii 31703 culture stopped fermenting when 20 g/
bottle CO2 had been released (at a cell concentration of 6.6 Log
cfu/mL), while, in mixed culture, it was still present in similar
cell concentrations at the end of fermentation. This suggests
that, although T. delbrueckii 31703 is a low ethanol producer, it
is able to survive at high ethanol concentrations, up to 14%vol.
In NPM, the non-Saccharomyces cells in the mixed culture were
present at maximum concentrations throughout fermentation.
Fig. 3. Evolutions of the biomass in function of CO2 released at 23 C. Saccharomyces populations (Fig. 3a) or non-Saccharomyces populations (Fig. 3b).
Pasteurized (open symbols) or non-pasteurized must (filled symbols). Pure S.
cerevisiae ST culture (S). Pure T. delbrueckii 31703 culture (T). Mixed T.
delbrueckii 31703/S. cerevisiae ST (T/S). Means of duplicate fermentations,
max. SD 0.15.
Table 2
Main oenological characters of pure and mixed fermentations at 23 C
Pasteurized must
Non-pasteurized must
Culture trial
Max.cell population
Residual nitrogen
Glycerol
Volatile acidity
O.D610 max.
(mgN/L)
(g/L)
13.8
8.4
14.2
14.8
9.6
15.0
16.67 0.58b
130.5 3.54a
11.00 1.00c
25. 33 0.58b
68.33 10.02a
21.00 0.00b
17.07 0.81a
14.70 0.53b
16.17 0.23a
17.05 0.35a
17.03 1.25a
16.40 0.99a
1.00 0.06a
0.56 0.10b
0.73 0.09b
0.94 0.06a
0.79 0.02b
0.75 0.11b
Initial sugar concentration: 360 g/L. Initial nitrogen concentration: 190mgN/L. Initial glycerol concentration: 6.6 g/L.Stuck fermentation.
Means of triplicate fermentations SD. Values followed by different superscript letters, within each column, are different according to the Newman-Keuls test
( = 5%).
317
Fig. 5. Fermentation courses at 23 C. Pasteurized (open symbols) or nonpasteurized must (filled symbols). Pure S. cerevisiae ST culture (S). Pure T.
delbrueckii culture (T). Means of triplicate fermentations, max. SD 0.6.
318
Table 3
Main oenological characters of pure, mixed and sequential fermentations at 23 C
Culture trial
Pasteurized must
Non-pasteurized must
Pure S. cerevisiae
Pure T. delbrueckii
Pure S. cerevisiae
Pure T. delbrueckii
Mixed T/S 5:1
Mixed T/S 10:1
Mixed T/S 20:1
Mixed T/S 50:1
Mixed T/S 100:1
Sequential T/S
Max.cell
population
Residual
nitrogen
Ethanol
Glycerol
Volatile acidity
Acetaldehyde
Yield
ethanol
A610max
(mgN/L)
(% vol.)
(g/L)
(mg/L)
(g/g)
11.8
6.2
11.2
7.2
11.1
10.8
11.0
9.4
9.4
nd
16.00 2.00
107.00 10.00
21.67 2.08d
79.00 9.54a
23.67 1.53d
28.33 0.58cd
35.00 2.65c
45.00 3.61b
48.67 4.93b
85.00 5.66a
14.23 0.11
6.92 0.33
14.10 0.35a
10.47 0.35c
14.27 0.35a
14.43 0.04a
13.92 0.08a
14.15 0.10a
14.08 0.08a
13.15 0.41b
15.80 0.36
10.93 1.02
15.60 1.06a
13.47 0.38b
16.00 0.10a
15.90 0.30a
15.07 0.91a
15.30 0.44a
15.90 1.51a
13.17 0.15b
1.05 0.02
0.27 0.10
0.89 0.16a
0.47 0.04cde
0.41 0.03e
0.40 0.03e
0.42 0.04de
0.50 0.01bcd
0.52 0.05bc
0.56 0.04b
38.50 5.33
25.20 5.00
45.63 5.86a
41.83 3.06a
30.90 4.00b
25.90 3.90b
18.20 1.47c
14.77 0.81c
16.47 0.12c
20.30 1.84c
0.47
0.44
0.48
0.45
0.46
0.47
0.47
0.47
0.47
0.46
Initial sugar concentration: 360 g/L. Initial nitrogen concentration: 190mgN/L. Initial glycerol concentration: 5.5 g/L.Stuck fermentation.
S. cerevisiae ST (S); T. delbrueckii 31703 (T); Mixed T. delbrueckii 31703/S. cerevisiae ST (T/S) with 107 T. delbrueckii cells/mL.
Means of triplicate fermentations SD. Values followed by different superscript letters, within each column, are different according to the Newman-Keuls test
( = 5%).
the most advantageous, irrespective of the S. cerevisiae concentration. Final volatile acidity and acetaldehyde concentrations were up to 55% and 68% lower, respectively. Considering
the reduction in both volatile acidity and acetaldehyde production, the best multistarter was T. delbrueckii 31703 mixed
with S. cerevisiae at a ratio of 20:1, with 107 T. delbrueckii
cells/mL.
4. Conclusion
This study of fermentation in high-sugar must shows that T.
delbrueckii is characterized by pure fermentation, with very
low volatile acidity and acetaldehyde production, confirming
previous studies under standard winemaking conditions. Even
if it is a low ethanol producer, T. delbrueckii still has useful
potential in sweet wine fermentation, as it does not seem to
respond to osmotic stress in the same way as S. cerevisiae.
Volatile acidity production remains constant throughout fermentation, in contrast to S. cerevisiae, where over 35% of the
total production occurs in the initial stage of fermentation.
Several studies have linked this overproduction by S. cerevisiae
to increased glycerol production, induced by osmotic stress.
One solution to the problem of excessive volatile acidity
formation in high-sugar fermentations was to use mixed cultures
of T. delbrueckii and S. cerevisiae, with a higher concentration of
T. delbrueckii to promote its growth. This mixed inoculum results
in combined, rather than successive, fermentations, producing
lower levels of acetic acid and acetaldehyde without affecting
the glycerol content. Reduction of the final volatile acidity and
acetaldehyde productions up to 55% and 68% were obtained,
respectively. This phenomenon depends on biochemical mechanisms involving both yeast metabolisms. We show that a high
population of T. delbrueckii in mixed culture reduces the acetate
production during the first stage of fermentation. Further investigations are required to clarify the role of mixed culture in
reducing volatile acidity production: could it be that the reduced
growth of the S. cerevisiae induced by a high concentration of
T. delbrueckii, may explain the acetate reduction or are there any
acetate exchanges between the two yeasts?
Acknowledgements
The authors thank Chteau Rayne Vigneau for its collaboration and acknowledge SARCO for technical assistance.
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