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The ability of wine yeast to consume fructose
1 Lallemand Inc., 1620 Rue Préfontaine, Montréal, QC Canada H1W 2N8
Introduction Stuck
fermentations have been the subject of numerous studies, and several
have determined the factors responsible for this fermentation
problem. Research has shown how certain fermentation conditions, such
as nutritional deficiencies, high initial levels of sugar, and the
presence of inhibiting compounds, can lead to fermentation problems.
The results of this type of research are helping winemakers lower the
risk of stuck fermentations significantly. Under
oenological conditions, the main sugars fermentable by Saccharomyces
cerevisiae are
glucose and fructose. Both of these hexoses are generally present in
musts in equivalent quantities, but the proportions may vary in some
musts. S.
cerevisiae prefers
to consume glucose, which explains why, when fermentations become
stuck, the remaining sugar is mainly fructose. The frequency of stuck
fermentations showing residual fructose raises the question of the
ability of yeast to consume this hexose. The kinetics of sugar
utilization by S.
cerevisiae during
fermentation is largely driven by sugar transport, and glucose is
typically consumed at a faster rate than fructose. In sluggish
fermentations, the maximal rate of fermentation is reduced after most
of the glucose is consumed, and fermentation can become stuck with a
significant concentration of fructose remaining. According to the
literature, the level of residual glucose in stuck wines is 10 times
lower than the fructose concentration. According to Gafner and Schûtz
(1996), it is possible to predict stuck fermentation when the
glucose/fructose ratio (GFR) is under 0.1. During
alcoholic fermentation, sugars are consumed mainly during the
stationary phase. During this phase, the available nitrogen gradually
becomes less available, and since it is an essential nutrient
involved in the transport of sugars into the cell via protein
synthesis, this partially explains why both the yeast metabolism and
the fermentation activity (Salmon, 1996) slow down. The alcohol level
also gradually increases, becoming toxic to the yeast cell, and the
use of fructose is even more compromised. At
the molecular level, research has confirmed the genes coding for the
hexose transporters in yeast. Under oenological conditions, several
genes are involved in sugar transport, which is regulated by a large,
multi-gene family called HXT. There are 20 HXT genes. Hxt1 and Hxt7
are the main transporters. Hxt2, Hxt6 and Hxt7 are high-affinity
carriers, whereas Hxt1 and Hxt3 are low-affinity carriers. Several
other Hxt carriers have intermediate affinity. Both the high- and
low-affinity carriers have greater affinity for glucose than
fructose, which may affect the rate of utilization of those hexoses.
Hexose concentrations in the medium will influence the expression of
individual HXT genes (Perez et al., 2005; Guillaume et al., 2007). It
has been shown that Hxt3 has the highest capacity to support
fermentation (Luyten et al., 2002) and very recent studies have also
identified that this gene is indeed responsible for the capacity for
consuming fructose among certain yeasts (Guillaume et al., 2007).
They also showed that a mutation on an allele of the Hxt3 gene was
responsible for improving the performance of wine yeast by utilizing
fructose during fermentation and in cases of stuck fermentation. It
is now established that variations exist in the capacity of yeast to
consume fructose. The objective of this study was to evaluate the
fermentation performance of selected yeasts under oenological
conditions, paying particular attention to their capacity to consume
fructose. A method was developed to measure the “fructophilic
index,” which would help determine the ability of a particular
yeast to consume fructose. The
“fructophilic” character of yeasts In
our experiments, we assessed the yeasts’ capacity to utilize
fructose, based on measurable phenotypical criteria. The
different commercial yeasts were selected for their capacity to
ferment high-sugar musts and for their aptitude for restarting stuck
fermentations. The
impacts of several oenological parameters were studied: The
initial levels of sugars. The
glucose/fructose ratio (GFR). The
initial level of yeast-assimilable nitrogen (YAN). The
temperature of fermentation. The
criteria evaluated for each yeast were: Fermentation
activity –
Fermentation kinetics are represented by the speed of fermentation
in terms of time or of CO2
released. The
kinetics of glucose and fructose consumption –
In order to evaluate and differentiate the capacity of yeasts
vis-à-vis their fructose uptake, the glucose and fructose
contents were measured throughout fermentation to evaluate the
kinetics of sugar consumption. The
fructophilic index was based on the calculation of the area between
the glucose and fructose consumption curves for the CO2
released (Figure 1) by the same yeast, and is the criteria selected
to evaluate each yeast’s capacity to consume fructose and to
compare them with each other. We focused on the area located in the
last half of the fermentation since it is the critical area where the
sugars are mainly consumed. The smaller the area, the closer the
fructose consumption kinetics is to the glucose consumption kinetics.
We chose this value to represent each yeast and to categorize the
oenological yeast strains according to their capacity to utilize
fructose. The yeasts whose fructose consumption kinetics are similar
to that of glucose are the yeasts that present a fructophilic
character and can perform better in high fructose situations. Figure
1. Evolution
of the glucose and the fructose during alcoholic fermentation. Comparison
of 5 strains of Saccharomyces cerevisiae. Milieu
MS300 Glucose/Fructose (130 g/L of each sugar); 24°C.
To
validate our ranking system, we included in the study a highly
reputable control yeast described as having a strong fructophilic
character (Guillaume et al., 2007). Materials
and methods Oenological
yeasts. We
utilized several commercially available oenological yeasts and in
some cases, yeasts selected for their ability to restart stuck
fermentations, such as UVAFERM 43 (YSEO®).
Nineteen commercially available yeasts were initially trialled, and
four remained, based on their outstanding performance to restart
stuck fermentation, in addition to the UVAFERM 43 (YSEO®).
They were coded Ref. 1 to Ref. 4. During
microvinification fermentations, 1.1 L of medium in fermenters with a
1.2-litre capacity were inoculated with the yeasts. The inoculation
rate is 25 g/hL (corresponding to about 5 x 106
cells/mL). Fermentation
environments.
In
order to compare different, commercially available oenological
yeasts, we chose to work in a standard environment: a synthetic
medium that mimics the composition of a must (MS300) described by
Bely et al. (1991), with some modifications to the initial sugar
level (we systematically utilized fructose in a quantity equal to
that of the glucose, or in a higher quantity for the experiments
where the GFR was <1). Similarly, we varied the total nitrogen
concentrations from 100 mg/L to 400 mg/L according to the experiment. Fermentation.
The
fermentations were carried out with constant stirring, at 18°C,
24°C or 28°C, in fermenters with a 1.2-litre capacity. Rate
of fermentations CO2.
The
quantity of CO2
released was determined by the automatic measuring of the loss of
weight from each fermenter every 20 minutes. The validity of this
technique, developed by the INRA in Montpellier by Jean-Marie
Sablayrolles to estimate the sugar and alcohol levels, has been
described in numerous papers, including El Haloui et al. (1988) and
Sablayrolles et al. (1987). Rate
of CO2
production (dCO2/dt).
The
speed of CO2
production was calculated by the polynomial smoothing of the 11 last
values of CO2
released. The frequent acquisitions of the release of CO2
and the precision of the weighing (0.1 g to 0.01 g) allow us to
repeatedly calculate the fermentation speed with great precision
(Bely et al., 1990). Glucose
and fructose consumption Samples
were taken during fermentation. After centrifugation, the sugars in
the supernatant were dosed with the help of the ENZYTECTM
D-Glucose/D-Fructose kits (Scil Diagnostics GmbH, Germany). Different
oenological conditions were studied, including the different initial
levels of sugars, but only the following oenological conditions were
reported: Temperature
of fermentation: 24°C.
Synthetic
medium high
in YAN (MS300)
and high in sugars,
total
sugars: 260
g/L, GFR = 1 (glucose = 130 g/L and fructose = 130 g/L). Temperature
of fermentation: 24°C.
Synthetic
medium high
in YAN
(MS300),
total
sugars: 260
g/L, GFR = 0.33 (glucose = 65 g/L and fructose = 195 g/L). Temperature
of fermentation: 24°C.
Synthetic
medium deficient
in YAN
(MS70),
total
sugars: 260
g/L, GFR = 0.33 (glucose = 65 g/L and fructose = 195 g/L). Temperature
of fermentation: 18°C.
Synthetic
medium high
in YAN (MS300),
total
sugars: 260
g/L, GFR = 0.33 (glucose = 65 g/L and fructose = 195 g/L). Temperature
of fermentation: 28°C.
Synthetic
medium high
in YAN (MS300),
total
sugars: 260
g/L, GFR = 0.33 (glucose = 65 g/L and fructose = 195 g/L). Given
the number of conditions tested, not all the data on fermentation
kinetics and rate of sugar consumption have been reported in this
article. Results The
impact of glucose/fructose ratio The
single variable between oenological conditions 1 and 2 was the GFR:
the respective levels of the two hexoses were identical in condition
1 while in condition 2 there were three times more fructose than
glucose. Both sugars were monitored during fermentation, and the
uptake difference of both sugars was calculated to show the
fructophilic index. Figure 2 shows the results of the five yeasts
tested in conditions 1 and 2, and regardless of the GFR level (equal
to 1 or 0.33), UVAFERM 43 (YSEO®)
was the yeast that showed the best ability to consume the fructose.
The ranking of the yeasts in terms of their capacity to consume
fructose is maintained for both these different glucose/fructose
ratios. It also shows that when the GFR is lower than 1, the
fructophilic index is also lowered. However, we notice that some
yeast is less affected than others. For example, UVAFERM 43 (YSEO®)
and Ref. 4 appear to be less affected than the other three, as shown
by the level of reduction of the fructophilic index. Figure
2. Impact
of the GFR on the fructophilic index for different commercial yeasts.
The
impact of the nitrogen content When
we compared oenological conditions 2 and 3, where the only variable
was the initial level of YAN, with a GFR <1, we observed that the
UVAFERM 43 (YSEO®)
yeast still presents the best performance vis-à-vis fructose
consumption (Figure 3), and that the capacity of the yeasts to
utilize the fructose is almost maintained, no matter whether YAN was
available or there was a nitrogen deficiency (<150 mg/L). Figure 4
shows the impact of nitrogen deficiency on the fermentation activity
of yeasts. Fermentation times are about four times longer in the
MS70, and there is a notable effect on the maximum speed of
fermentation, as in the case of a nitrogen deficiency, the yeast
metabolism is slowed significantly. This concurs with the literature
(Salmon, 1989, Salmon et al., 1993). Working with a medium deficient
in nitrogen is an opportunity to better discern the behaviour of the
yeasts, and to demonstrate the variability in the need for nitrogen
among yeasts. These findings are completely coherent with a prior
study (Julien et al., 2001). These findings also show that the
initial levels of nitrogen have a very significant influence on the
fermentation activity of yeasts, but do not impact their variable
capacity to utilize fructose. In both conditions, UVAFERM 43 (YSEO®)
completes the fermentation the earliest with a steady fermentation
rate. Figure
3. Ranking
of selected yeasts based on the difference in sugar consumption in a
medium with the glucose/fructose ratio = 0.33 and with different
levels of nitrogen (media deficient in nitrogen or high in nitrogen).
Figure
4. Comparison
of the fermentation behaviour of yeasts in a medium with the
glucose/fructose ratio = 0.33 and with different levels of nitrogen
(media deficient in nitrogen or high in nitrogen).
The
impact of temperature We
studied the impact of the temperature on the yeasts’ capacity
to uptake fructose (Figure 5). Results indicated that this capacity
increased with the temperature whatever the yeast, except for one
specific yeast (Ref. 3). In this case, we see the fructophilic index
increased significantly when fermentation was carried at 18°C,
compared to fermentation at a higher temperature, but also compared
to the other yeasts. This yeast (Ref. 3) is well known for being well
adapted to fermenting at low temperature and this could explain its
behaviour. Except
for this particular situation, the ranking among the selected yeasts
remains the same, with the better fructophilic index for the UVAFERM
43 (YSEO®),
whatever the temperature. The
fact that the yeasts’ capacity to uptake the fructose is lower
at low temperature can be explained by the slower yeast metabolism
when the fermentation temperature decreases. Figure
5. Ranking
of selected yeasts based on the difference in sugar consumption in a
medium with the glucose/fructose ratio = 0.33 at different
temperatures.
Conclusion The
UVAFERM 43 (YSEO®) yeast consistently showed the smallest area
between the glucose and fructose consumption curves during the last
half of the fermentation, and therefore has the highest fructophilic
index, which means this yeast has the best fructose uptake capacity,
whatever the GFR, the nitrogen or temperature levels. This behaviour,
although reported only on five yeasts in this paper, was tested on 19
other selected yeasts with the same results. The
selected yeasts differed in their capacity to consume fructose, and
that is an indicator of performance in potentially problematic must,
where the GFR is lower and/or the must conditions are difficult. The
fructophilic index measured as the area difference between glucose
and fructose consumption can be a tool used to evaluate the
fructophilic capacity of wine yeasts, and to characterize this
phenotype and avoid stuck fermentations. The
study of the characterization of the UVAFERM 43 (YSEO®)
continues with an in-depth investigation on its ability to restart
stuck fermentations and to develop reliable protocols for such
situations. Need
more information? In
case you may need more information on the product(s) and details
discussed in the above-mentioned
article, please contact Piet Loubser, the area manager for Lallemand
in South Africa, tel (021) 913-7555, fax (021) 913-5550 or
ploubser@lallemand.com. References Bely,
M., J.M. Sablayrolles
& P. Barre. 1990. Description of alcoholic fermentation kinetics:
its variability and significance. Am
J Enol Vitic. 41:
319 - 324. Bely,
M., J.M. Sablayrolles & P. Barre. 1991. Automatic detection of
assimilable nitrogen deficiencies during alcoholic fermentation in
enological conditions. J
Ferm Bioeng.
70: 246 - 252. Bely,
M., J.M. Salmon & P. Barre. 1994. Assimilable nitrogen addition
and hexose transport activity during enological fermentations. J
Inst Brew.
100: 279 - 282. Bisson,
L.F. Glucose transport in Saccharomyces
cerevisiae and
the role of potassium in stuck fermentation. Proceedings of the 2000
Entretiens
Scientifiques Lallemand,
Krems, Austria. 27 - 33. McClellan,
C.J., A.L. Does & L.F. Bisson. 1989. Characterization of hexose
uptake in wine strains of Saccharomyces
cerevisiae and
Saccharomyces
bayanus.
Am
J Enol Vitic.
40: 9 - 15. El
Haloui, N., D. Picque & G. Corrieu. 1988. Alcoholic fermentation
in wine-making: on line measurement of density and carbon dioxide
evolution. J
Food Eng.
8: 17 - 30. Gafner,
J. & M. Schütz. 1996. Impact of glucose-fructose-ratio on
stuck fermentations: practical experience to restart stuck
fermentations. Vitic
Enol Scien.
51: 214 - 218. Guillaume,
C., P. Delobel, J.M. Sablayrolles & B. Blondin. 2007. Molecular
basis of fructose utilization by the wine yeast Saccharomyces
cerevisiae:
a mutated HXT3 allele enhances fructose fermentation. Appl
Environ
Microbiol.
73(8): 2432 - 2439. Perez,
M., K. Luyten, R. Michel, C. Riou & B. Blondin. 2005. Analysis of
Saccharomyces
cerevisiae hexose
carrier expression during wine fermentation: both low- and
high-affinity Hxt transporters are expressed. FEMS
Yeast
Res.
5: 351 - 361. Sablayrolles,
J.M., P. Barre & P. Grenier. 1987. Design of laboratory automatic
system for studying alcoholic fermentations in anisothermal
oenological conditions. Biotech
Tech.
1: 181 - 184. Salmon,
J.M. 1989. Effect of sugar transport inactivation in Saccharomyces
cerevisiae on
sluggish and stuck fermentation. Appl
Environ Microbiol.
55: 953 - 958. Salmon,
J.M., O. Vincent, J.C. Mauricio, M. Bely & P. Barre. 1993. Sugar
transport inhibition and apparent loss of activity in Saccharomyces
cerevisiae as
a major limiting factor of enological fermentation. Am
J Enol Vitic.
44: 56 - 64. Salmon,
J.M. 1996. Sluggish and stuck fermentations: Some actual trends on
their physiological basis. Vitic
Enol Scien. 51:
137 - 140.
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