SALMON ET ORTIZ-JULIEN, IMPROVEMENT OF ALCOHOLIC FERMENTATION IN EXTREME CONDITIONS, P. 1
IMPROVEMENT OF ALCOHOLIC FERMENTATION IN EXTREME CONDITIONS
SALMON Jean-Michel 1, ORTIZ-JULIEN Anne 2
1)
2)
INRA, UMR 1083, 2 pl. Viala, 34060 MONTPELLIER cedex1, France, [email protected]
Lallemand SAS, 19 Rue des Briquetiers, B.P. 59, 31703 Blagnac cedex, France.
INTRODUCTION
During alcoholic fermentation, yeast cells are progressively exposed to a very stressful environment
due to the strong decrease of external pH and to the rising accumulation of ethanol in the external
medium (Salmon, 1996). Such environment induces progressively a slow decrease of cell viability,
related to alterations of the cellular plasma membrane, which in very stressful conditions (nutrient
depletion, low and high fermentation temperatures, for example) may lead to a sudden fall of cellular
viability towards zero before alcoholic fermentation was complete (Fornairon-Bonnefond et al.,
2002). Such sluggish or stuck fermentations represent a great risk for winemakers since residual
sugars in finished wines represent always certain microbiological and sensory instabilities.
Since now about 25 years, the use of Active Dry Yeasts (ADY) as starters for winemaking allowed
more reproducible alcoholic fermentations. However, their use did not overcome the risks of cellular
viability losses when stressful environmental conditions are encountered. Addition of several
nutrients (nitrogen, vitamins or oxygen) during fermentation may only partially protect yeast cells
from such stressful conditions.
Today, recent research results obtained on the physiology of yeasts during ADY rehydration give a
challenging opportunity to design new products for the improvement of alcoholic fermentation in
extreme conditions. The aim of the present conference is to give an overview of such results with a
peculiar emphasis on their potential impact for winemakers.
Mean cellular volume (µm3)
NEW INSIGHTS ON YEAST PHYSIOLOGY DURING REHYDRATION
Active Dry Yeasts (ADY) were industrially obtained by drying and granulation of yeast paste until a
water contents of 6 to 8% is reached (Villetaz, 1992). This severe drying step induces a shrinkage of
the internal cellular volume (Beker et al., 1984). In these conditions, ADY conserve a high level of
viability after drying. Before utilization, ADY need therefore to be rehydrated in water at 37°C for
about 30 minutes. This peculiar and important step is necessary to restore a normal level of water
inside the cells (towards 99%). The important water flux into cells during ADY rehydration is
responsible for an important swelling of the cell as depicted on Figure 1. This swelling is
accompanied by the rapid restoration of the cell viability (Figure 2), which is a prerequisite for a good
acclimatization of yeast starters to must conditions, and therefore to the restoration of fermentative
metabolism capacity. Recent work (Soubeyrand et al., 2005) demonstrate, by using lipidic
fluorescent probes targeted to the plasma membrane, that in the same time a great mobilization of
lipids occurs during rehydration, specially in the initial 15 first minutes (Figure 3). During rehydration,
a fast mobilization of lipid storage within the
cells was indeed previously observed by
110
electron microscopy (Saulite et al., 1986). This
fast lipid mobilization allows yeast cells to
100
quickly recover functional cellular membranes.
90
Figure 1: Evolution of the mean cellular diameter
during rehydration of EC 1118 ADY (1 g) in 10 ml
of water containing 0.5 g glucose at 37°C. Mean
cellular diameter was obtained by observation and
photomicrography under light microscopy of at
least 100 to 200 cells, and automatic calculation
using the UTHSCSA Image Tool software (v 2.0 for
Windows)(Salmon, 2005, unpublished data).
80
70
60
50
0
5
10
15
20
25
30
Time (minutes)
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SALMON ET ORTIZ-JULIEN, IMPROVEMENT OF ALCOHOLIC FERMENTATION IN EXTREME CONDITIONS, P. 2
Figure 2: Evolution of mean cellular viability
during rehydration of EC 1118 ADY (1 g) in 10
ml of water containing 0.5 g glucose at 37°C.
Mean cellular viability was determined by
counting viable cells under epifluorescence
microscopy after staining dead cells with 1anilino-8-naphthalene sulfonic acid (King et al,
1981) (Salmon, 2005, unpublished data).
Mean cellular viability (%)
60
40
20
0
5
10
15
20
25
30
Time (minutes)
Figure 3: Total fluorescence kinetics of TMADPH probe during rehydration of EC 1118 ADY
(1 g) in 10 ml of water containing 0.5 g glucose
at 37°C. The black arrow indicates the addition
of TMA-DPH (Soubeyrand et al., 2005).
TMA-DPH total fluorescence
0
60
40
20
0
0
5
10
15
20
25
30
Time (minutes)
STEROLS AS KEY FACTORS IN THE CELLULAR PLASMA MEMBRANE
Cytoplasmic (or plasma) membrane is the unique barrier existing between intracellular cytoplasm
and the external medium. This subcellular part of the cell plays a very important role of interface
helping the cell fighting to the detrimental effects of ethanol and must acidity (Figure 4). This
membrane is non permeable by itself to various macromolecules and ions, at the exception of
protons (H+ ions) (Figures 4 and 6-1).
Ext.
Int.
Metabolism
ATP
H+
Must
acidity
A
H+
ADP + Pi
Amino-acids
H+
B
Amino-acids
H+
C
NH 4+
H+
NH 4+
D
Figure 4 : Scheme showing the
important role of the ATPase-proton
pump activity in the maintenance of pH
homeostasis within the yeast cell. A:
Plasma membrane ATPase-proton
pump activity, B: Amino-acids transport
systems, C: Ammonium ion transport
system, D: Free diffusion of protons
+
(H ) across plasma membrane
(Salmon, 1998).
H+
cytoplasmic
membrane
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SALMON ET ORTIZ-JULIEN, IMPROVEMENT OF ALCOHOLIC FERMENTATION IN EXTREME CONDITIONS, P. 3
Plasma membrane is mainly constituted by lipidic bilayer formed by a fluid arrangement of different
classes of phospholipids, creating a very non polar environment surrounding the yeast cell (Figure
5). This very fluid architecture is stabilized by smaller rigid molecules – sterols – in charge of the
stabilization of the membrane mainly in the membrane regions where important integral proteins
(transporters) are located.
Internal face of the plasma membrane
Figure 5 : Scheme showing
the global composition of the
lipidic bilayer constituting the
plasma membrane and the
role of sterols in its
stabilisation. A :
Phospholipids with saturated
fatty acids, B : Phospholipids
with unsaturated fatty acids,
C : sterol, D : Integral protein
within the plasma
membrane.
D
B
A
C
External face of the plasma membrane
Sterols play therefore major roles in the building and maintenance of yeast membranes: they mainly
regulate membrane fluidity and permeability, and ATPase-proton pump activity. They also regulate
the cell cycle, gene expression and the uptake of exogenous sterols (for a review, see Daum et al.
1998). Ethanol accumulation in the fermentation medium during alcoholic fermentation strongly
interact with the functioning of plasma membrane as an interface between this medium and the
internal part of the cell. Its main effects are to render the membrane leaky to protons, and to partially
inhibit the activity of the ATPase-proton pump.
1
Ext.
pH ≈ 3,0
2
Int.
H+
A
H+
B
ATP
pH ≈ 7,0
Ext.
pH ≈ 3,0
Int.
A
ATP
H+
= Cell DEATH
ADP + Pi
H+
Plasm a m em brane
pH << 7,0
ADP + Pi
Ethanol
B
H+
H+
Plasm a mem brane
Figure 6 : Scheme showing the deleterious effect of ethanol along fermentation. A: ATPase-proton pump
+
activity, B: Free diffusion of protons (H ions) (Salmon, 1998).
Yeast cells were therefore in the obligation to consume more energy (in the form of ATP) to
maintain pH homeostasis, until the equilibrium between passive entrance of protons and their
expulsion by the ATPase-proton pump activity cannot be sustained, leading to cell death (Figure 62). This toxic effect of ethanol is peculiarly enhanced by low (below 8-10 °C) and intermediate
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SALMON ET ORTIZ-JULIEN, IMPROVEMENT OF ALCOHOLIC FERMENTATION IN EXTREME CONDITIONS, P. 4
temperatures (in the range 20 to 30 °C), depending on the ethanol concentration (Sa-Correia and
Van Uden, 1986 ; Salmon, 1998). Membrane sterols play an important role in stabilizing plasma
membrane and strengthening membrane against the toxic effect of ethanol (Fornairon-Bonnefond et
al., 2002).
ORIGIN OF PLASMA MEMBRANE STEROLS DURING ALCOHOLIC FERMENTATION
In enological conditions, yeast cells are only able to synthesize sterols in the presence of trace
amounts of oxygen (Rosenfeld et al., 2003). In this condition, the main sterol of yeast plasma
membrane is ergosterol. In complete anaerobiosis, yeasts cannot synthesize ergosterol but can
incorporate a wide variety of exogenous sterols. For example, during wine fermentation, yeast
growth occurs by assimilating grape phytosterols, which are mainly localized in the cuticular wax
(Higgins and Peng, 1976) and berry skins (Le Fur et al., 1994) of grapes. These phytosterols are
generally extracted during the maceration phases (Valero et al., 1998). However, recent work has
revealed that, in enological conditions in the absence of oxygen, although phytosterol assimilation
can sustain yeast growth, the corresponding yeast cells cannot achieve complete alcoholic
fermentations by only assimilating grape phytosterols (Luparia et al., 2004). This effect may be likely
related to the differences in the chemical structures of ergosterol and phytosterols.
Rodriguez et al. (1985) demonstrated that ergosterol plays different roles according to its
concentration in yeast membranes. Trace amounts of ergosterol play a “sparking role”, initiating cell
growth. At higher concentrations, it fulfils a “domain role”, which represents the minimal amount of
sterol required for active growth and for the synthesis of intact membranes. Beyond this
concentration, sterols are incorporated into membranes as free sterols, acting as architectural
components of membranes. This continues until the “bulk role” concentration is reached, i.e. the
maximal amount of sterols that can be incorporated into membranes. A wide variety of sterols,
including phytosterols, may apparently play this “bulk” role in yeast membranes. In enological
conditions, the minimal ergosterol requirement for the maximal anaerobic growth of S. cerevisiae
was found to be about 0.1 µmol/109 cells (Rosenfeld et al., 2003).
However, many prefermentative treatments, and in particular clarification (settling) usually practiced
in white and rosé winemaking, involve flocculation of particle and pectic aggregates on which
phytosterols, which are very hydrophobic compounds, remain adsorbed (Cocito and Delfini, 1997).
Therefore, after such prefermentative treatments, grape musts could be strongly depleted in
assimilable sterols at the beginning of alcoholic fermentation.
Incorporated sterols
(µg/30 mg ADY) (
)
Figure 7: Kinetics of sterols
incorporation measured using [414
C] cholesterol during the
rehydration of EC 1118 ADY (1 g)
in 10 ml of water containing 0.5 g
glucose in the presence of a
preparation of solubilized sterols
(25 mg) at 37°C (Soubeyrand et
al., 2005).
100
6
80
4
60
40
2
20
0
0
0
5
10
15
20
Rehydration time (min)
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25
30
Sterols in the rehydration medium
(µg) (
)
NATURAL STEROLS SUPPLEMENTATION DURING REHYDRATION
Under these conditions, it is thus imperative to help yeast cells to recover an optimal conformation of
their cell membranes to support an efficient alcoholic fermentation. We therefore use the property of
yeast cells to mobilize their lipidic reserves during ADY rehydration to quickly recover functional
cellular membranes (see Figure 3). In a previous work we show that, during the ADY rehydration
process, yeast cells can easily
incorporate
exogenously
200
provided specific natural yeast
12
180
sterols during the first part of
160
rehydration (Figure 7), by
10
assimilating sterols under the
140
form of micelles (Soubeyrand
8
120
et al., 2005).
SALMON ET ORTIZ-JULIEN, IMPROVEMENT OF ALCOHOLIC FERMENTATION IN EXTREME CONDITIONS, P. 5
This early complementation of yeast membranes with yeast sterols during ADY rehydration allows
the maintenance of cell viability even very later in the fermentation process, when ethanol level is
maximum (Figure 8).
Cellular viability (%)
100
Figure 8: Effect of solubilized specific yeast
sterols addition during EC 1118 ADY
rehydration phase on the cellular viability at
the end of alcoholic fermentation.
Rehydration without addition (filled symbols),
or with addition of solubilized specific yeast
sterols (25 mg, open symbols), mean and
standard deviation of two replicates.
90
80
70
95
100
105
110
115
120
Fermentation time (hours)
This application of sterol supplementation during the ADY rehydration phase was now sustained by
an European patent between INRA and Lallemand SAS (PCT 04-12309). The corresponding
technique of rehydration has now proved its efficiency since two vintages in a wide variety of
extreme conditions such as high alcoholic potential (thermovinified musts), difficult temperatures
(cold skin maceration followed by a long stabulation at low temperature), altered musts (bothrytised
must which cannot support micro-oxygenation during alcoholic fermentation), or simply strongly lowturbidity deaerated musts.
CONCLUSIONS
Plasma membrane sterols play an important role in the maintenance of the membrane as a
permeability barrier, especially at the end of alcoholic fermentation, when the environmental
conditions became detrimental for the cell. ADY rehydration is a very important technological step
during starter elaboration, since rebuilding of cell membranes and therefore of metabolic capacity
occurred during this short phase. The enrichment of Active Dry Yeasts with natural sterols during
their rehydration is very efficient to complete fermentation when musts are limited in available
phytosterols or when micro-oxygenation is not desirable during fermentation. This represents a very
interesting alternative for providing sterols to fermentative yeasts in order to sustain a high cellular
viability throughout alcoholic fermentation, especially in extreme conditions, when fermentations are
suspected to become sluggish or stuck.
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loss from grape must related to clarification technique. J. Wine Res. 8: 187-197.
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