Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex.

Abstract

Along the fermentation process yeasts are affected by a succession of stress conditions that affect their viability and fermentation efficiency. Among the stress conditions the most relevant are high sugar concentration and low pH in musts, temperature and, as fermentation progresses, ethanol accumulation. Nowadays, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing search for particular wine yeast strains possessing a wide range of optimized, improved or novel enological characteristics. Traditionally, the species S. cerevisiae and S. bayanus within the Saccharomyces sensu stricto species are considered some of the most important yeast species involved in fermentation processes. However, in the last years, hybrid strains between the species S. cerevisiae, S. bayanus and S. kudriavzevii have been described as yeasts conducting the alcoholic fermentations and some of them are commercially available. Our results indicate that yeasts in the Saccharomyces sensu stricto complex were not affected by low pH or high glucose content in the media; however temperature and ethanol concentration variables appreciably affected their growth. The strains pertaining to S. cerevisiae were able to tolerate high temperature stress, whereas strains within S. bayanus and S. kudriavzevii were better adapted to growth at lower temperatures. Regarding to alcohol tolerance, S. cerevisiae is tolerating alcohol better than S. bayanus or S. kudriavzevii. Surprisingly, the natural hybrids between these species have adapted to growth under ethanol and temperature stress by inheriting competitive traits from one or another parental species. These results open new perspectives in the construction of new hybrid strains with biotechnological interest, as the characteristics of the parents may result in interesting combinations in the hybrids.

Full text

Page 1

Fermentative stress adaptation of hybrids within the Saccharomyces
sensu stricto complex
Carmela Bellocha, Sandi Orlica,b, Eladio Barrioc, Amparo Querola,⁎
aDepartamento de Biotecnología, Instituto de Agroquímica y Tecnología de los Alimentos, CSIC, P.O. Box 73. E-46100 Burjassot, Valencia, Spain
bDepartment of Microbiology, Faculty of Agriculture, University of Zagreb, Svetosimunska 25, 10000, Zagreb, Croatia
cGenètica Evolutiva, Institut “Cavanilles” de Biodiversitat i Biologia Evolutiva, Universitat de València, Edificio de Institutos, Campus de Paterna,
E-46100 Burjassot, Valencia, Spain
Received 11 May 2007; received in revised form 9 November 2007; accepted 27 November 2007
Abstract
Along the fermentation process yeasts are affected by a succession of stress conditions that affect their viability and fermentation efficiency.
Among the stress conditions the most relevant are high sugar concentration and low pH in musts, temperature and, as fermentation progresses,
ethanol accumulation.
Nowadays, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing search for
particular wine yeast strains possessing a wide range of optimized, improved or novel enological characteristics. Traditionally, the species S.
cerevisiae and S. bayanus within the Saccharomyces sensu stricto species are considered some of the most important yeast species involved in
fermentation processes. However, in the last years, hybrid strains between the species S. cerevisiae, S. bayanus and S. kudriavzevii have been
described as yeasts conducting the alcoholic fermentations and some of them are commercially available.
Our results indicate that yeasts in the Saccharomyces sensu stricto complex were not affected by low pH or high glucose content in the media;
however temperature and ethanol concentration variables appreciably affected their growth. The strains pertaining to S. cerevisiae were able to
tolerate high temperature stress, whereas strains within S. bayanus and S. kudriavzevii were better adapted to growth at lower temperatures.
Regarding to alcohol tolerance, S. cerevisiae is tolerating alcohol better than S. bayanus or S. kudriavzevii. Surprisingly, the natural hybrids between
these species have adapted to growth under ethanol and temperature stress by inheriting competitive traits from one or another parental species.
These results open new perspectives in the construction of new hybrid strains with biotechnological interest, as the characteristics of the parents
may result in interesting combinations in the hybrids.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Saccharomyces sensu stricto; Saccharomyces hybrids; Fermentation stress; Domestication
1. Introduction
Fermentation has been used for several thousands of years as
an effective and low-cost resource to preserve the quality and
safety of foods. Among the fermenting microorganisms, yeasts
are the most important group of microorganisms that have been
used in production of alcoholic beverages such as wine and beer
(Romano et al., 2006).
The Saccharomyces sensu stricto species complex contains
some of the most important species involved in the fermentation
processes. The species S. cerevisiae is considered the agent of
wine, bread, ale beer, and sake fermentations. The species S.
bayanus orS. bayanus var.bayanus (Naumov,2000) isinvolved
in lager beer fermentation, whereas S. uvarum (Nguyen and
Gaillardin, 2005; Pulvirenti et al., 2000) or S. bayanus var.
uvarum (Naumov, 2000) has been isolated from wine and cider
fermentations(Naumovetal.,2000a,2001;2002).Otherspecies
in the sensu stricto group, namely S. paradoxus, has been
described asthemainyeastinCroatian wines(Redzepovicetal.,
2002). The other three species in the sensu stricto complex, S.
cariocanus, S. mikatae and S. kudriavzevii have been found in
natural environments (Naumov et al., 2000b) and are never
present in fermentative environments.
Available online at www.sciencedirect.com
International Journal of Food Microbiology 122 (2008) 188–195
www.elsevier.com/locate/ijfoodmicro
⁎Corresponding author. Tel.: +34 96 3900022x2306; fax: +34 96 3636301.
E-mail address: aquerol@iata.csic.es (A. Querol).
0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2007.11.083

Page 2

Hybrid strains between the species in the Saccharomyces
sensu stricto complex have also been described as yeasts
involved in alcoholic fermentations. In 1998, Masneuf et al.,
described a hybrid yeast strain possessing nuclear genomes
from both a S. cerevisiae and a S. bayanus-like yeast and, since
then, several wine and beer yeasts have been described as
hybrids of these two species (Naumov et al., 2000a; Casagerola
et al., 2001; de Barros Lopez et al., 2002). Recently, Rainieri
et al. (2006) have identified up to three different hybrid types in
lager brewing yeasts, namely, S. cerevisiae×S. bayanus (S.
pastorianus), S. bayanus×S. uvarum and a triple hybrid by S.
cerevisiae×S. bayanus×S. uvarum. Moreover, Gonzalez et al.
(2006) have described wine yeasts as hybrids between S.
cerevisiae and S. kudriavzevii, the latter being a yeast species
which isolates have been found growing on decayed leaves in
Japan.
The Saccharomyces yeasts that are isolated for industrial
purposes are selected for their special characteristics. Selective
pressures always favour the yeasts with the most efficient
fermentative catabolism, particularly strains of S. cerevisiae and
strains of closely related species such as S. bayanus (Pretorius,
2000). Similarly, several hybrids in the Saccharomyces sensu
stricto complex have been selected and commercialised like the
yeasts conducting the alcoholic fermentation; therefore, the
hybrid strains must be well adapted to the stress conditions
occurring during the alcoholic fermentation.
At the beginning of fermentation, yeast cells are affected by
osmotic stress due to the high sugar concentration, as well as
low pH; and as fermentation progresses other stress conditions
as ethanol accumulation become relevant. Moreover, depend-
ing on the fermentation process, other factors such as
temperature can be considered as stress factors (Cardona
et al., 2007).
Several authors have reported on variation of yeast growth
with sugar concentration (Carrasco et al., 2001; Zuzuarregui
and del Olmo, 2004). The concentration of fermentable sugars
(glucose and fructose) in grape musts varies between 125 and
250 g/L (Fleet and Heard, 1993) thus, it is likely that the initial
concentration of sugar in grape must will selectively influence
the species and strains of yeast responsible for the fermentation.
Different studies (Lafon-Lafourcade, 1983; Monk and Cowley,
1984) indicated that growth rate and completeness of
fermentation by S. cerevisiae were decreased as the initial
concentration of sugar in grape must increases above 200 g/L.
Grape must acidity is also considered as important for the
survival and growth of yeasts. Fleet and Heard (1993) observed
that growth rate and must fermentation by S. cerevisiae were
decreased as the pH was decreased from 3.5 to 3.0. On the other
hand, a recent study of pH influence on growth of S. bayanus
var. uvarum showed that pH does not have a significant
influence on growth (Serra et al., 2005).
The temperature at which the alcoholic fermentation is
conducted affects the yeast growth and duration of fermentation
(Fleet and Heard, 1993). The rate of yeast growth and alcoholic
fermentation increases as the temperature increases, with
maximum rates occurring at temperatures between 20 and
25 °C (Amerine et al., 1980). On the other hand, Serra et al.
(2005) reported that below the optimal growth temperature
yeast growth rate increases as the temperature increases, but
after the optimal temperature yeast growth rate decreases very
fast due to damage of cellular membranes and enzymes
denaturation. In recent years, there has been a preference by
winemakers to ferment white wines at temperatures in than
range of 10 to 15 °C to minimize the loss of aromatic volatiles,
and red wine fermentations performed at higher temperatures
(18–30 °C) to enhance extraction of anthocyanin pigments.
Similarly, beer fermentation occurs at different temperatures
depending on the type of beer; ale beer fermentation occurs
between 16 and 23 °C and lager beer fermentation occurs
between 8 and 15 °C (Hammond, 2000).
Grape must is typically prepared from fully matured grapes,
making for high flavour intensity, but also a considerable
concentration of sugar. This much sugar invariably leads to the
production of wines with high levels of ethanol, sometimes
reaching concentrations above 15% (v/v) (de Barros Lopes
et al., 2003). In spontaneous fermentations there is a progressive
growth pattern of indigenous yeasts in the middle stages when
the ethanol rises to 3–4% (Fleet and Heard 1993). The latter
stages of natural wine fermentations are invariably dominated
by the alcohol-tolerant strains of Saccharomyces cerevisiae.
Similarly, in beer fermentation ethanol content ranges between
4 and 9% although high gravity beer contains at least 8% of
ethanol (Campbell, 2003). The analysis of resistance to ethanol
of 14 wine strains by Zuzuarregui and del Olmo (2004) showed
that ethanol affected growth of some strains severely.
The aim of this study was to investigate the tolerance to
stress conditions occurring during wine fermentation of
different wine and beer yeasts pertaining to the Saccharo-
myces sensu stricto complex and the hybrids between them.
Besides, we tried to resolve the question of which parental
species bequeath the hybrids with the abilities to deal with the
different stress tolerances.
2. Materials and methods
2.1. Strains
Table 1 lists the strains used in this study. Species name,
strain number in different culture collections and isolation
source are indicated.
2.2. Medium and inoculum's preparation
Yeast cells were grown in GPY (peptone 0.5%, yeast
extract 0.5%, glucose 2%). Medium was solidified by adding
1.5% agar. 48 h fresh yeast cultures on GPYagar were used to
inoculate 5 mL of fresh GPY and shaken overnight at 30 °C.
The next day the cultures were adjusted to an absorbance at
655 nm of 0.3 by dilution with fresh GPY. After shaking at
30 °C for another 4 h the cells were diluted in sterile water to
an A655 of 0.3 and 5 μL of a 5-fold dilution series in water
were spotted onto all stress media. All plates were incubated
aerobically and checked 1 to 5 consecutive days for colony
development.
189C. Belloch et al. / International Journal of Food Microbiology 122 (2008) 188–195

Page 3

2.3. Analysis of yeast tolerance to several stress conditions
Four stress factors considered as key features in wine fer-
mentation were studied: pH, glucose, temperature and ethanol.
For each stress factor, several conditions were tested: pH (2.8,
3.0 and 3.2), glucose (200 g/L, 250 g/L and 300 g/L glucose),
temperature (10 °C, 16 °C, 30 °C and 37 °C), ethanol (5%, 10%,
12% and 15% ethanol), and combined stress (250 g/L glucose
and pH 2.8). Stress media were prepared using GPYA (peptone
0.5%, yeast extract 0.5%, glucose 2%, agar 1.5%) supplemen-
ted with the adequate amounts of ethanol and glucose. pH stress
was checked on GPYA adjusted to the desired pH, and tem-
perature stress was performed on GPYA medium incubated at
the above mentioned temperatures. Except for temperature
stress, all plates were incubated at 30 °C. In all cases, a positive
growth control was carried out with yeast cells that were not
exposed to any stress condition and incubated at 30 °C until
colonies appeared in all dilutions (24 h). To detect variations in
our results due to experimental errors, the tests were repeated at
least three times. However, the experimental design and the
method studying growth do not permit proper statistical
evaluation or error calculation.
3. Results and discussion
The primary role of fermenting yeast in the production of
alcoholic beverages is to catalyze the rapid, complete and
efficient conversion of grape sugar to ethanol, carbon dioxide
and other minor metabolites without the development of off-
flavours (Pretorius, 2000). As in other industrial processes,
Table 1
List of strains used in this study
SpeciesStrainsOriginpHGlucose (g l−1)Temperature (°C)Ethanol (%)
2,83,0 3,2200 250300101630 375 10 1215
S. cerevisiae CECT 1942T= CBS 1171T
CECT 1384=CBS 1636
CECT 1387 = CBS 7372
CECT 11001 = NCYC 2340
CECT 11036 = CBS 381
Lalvin T73, Lallemand
Uvaferm CEG, Danstar
Fermiblanc Arom DSM-Gist Broc.
Fermicru Primeur DSM-Gist Broc.
UCLM S-377, Springer Oenologie
CECT 11035T= CBS 380T
CECT 1991 = DSM 70411
CECT 11185 = NCYC 114
CECT 12627
CECT 12629
CECT 12638
CECT 12669
CECT 12930
54
11157 = CBS 2908
120 M
CECT 1939T= CBS 432T
IFO 1802T
CBS 8841T
CBS 8839T
Ale beer, The Netherlands
Spoiled beer, Ireland
Spoiled draught beer, UK
Lager beer, Belgium
Spoiled beer, Japan
wine, Alicante, Spain
Wine, Eppernay, France
Wine, Cognac, France
Wine, Beaujolais, France
wine, Spain
Turbid beer
Turbid beer
Beer contaminant, UK
Wine, Valladolid, Spain
Must, Zaragoza, Spain
Must, Cadiz, Spain
Grapes, La Rioja, Spain
Wine, Spain
Grapes, Croatia
Soil, South Africa
Pulque, Mexico
Tree sap, Russia
Decayed leaf, Japan
Drosophila sp., Brazil
Soil, Japan
6
6
6
6
6
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
4
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
4
6
6
6
6
6
6
6
6
6
5
6
6
3
3
4
3
4
3
3
3
3
3
5
6
6
6
6
6
6
6
4
4
4
4
6
3
6
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
3
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0
6
6
0
6
6
6
6
6
6
0
0
0
0
0
0
0
0
6
6
6
6
0
6
6
4
6
6
3
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
4
6
6
0
6
6
0
6
6
6
6
6
6
6
6
4
6
6
6
6
6
6
6
6
6
0
6
6
0
6
6
0
6
6
6
5
6
6
2
5
0
2
2
4
3
5
6
6
6
6
0
2
0
0
5
6
0
5
6
5
2
5
2
0
3
0
0
0
0
0
5
0
0
5
0
0
0
0
S. bayanus
S. paradoxus
S. kudriavzevii
S. cariocanus
S. mikatae
Hybrid strains
S.c.×S.b.
S.c.×S.b.
S.c.×S.b.
S.c.×S.k.
S.c.×S.k.
S.c.×S.k.
S.c.×S.k.
S.c.×S.k.
S.c.×S.k.
S.c.×S.b.×S.k.
S.c.×S.b. ×S.k.
Lalvin S6U, Lallemand
CECT 11000 = BRAS 12
CECT 11037 = CBS 1513
Lalvin W 27, Lallemand
Lalvin W 46, Lallemand
SPG 16-91
SPG 441
CECT 1388=NCYC 447
CECT 1990=DSM 1848
CBS 2834
CID 1
Wine, Italy
Ale beer
Lager beer
Wine, Wädenswill, Switzerland
Wine, Wädenswill, Switzerland
Wine, Wädenswill, Switzerland
Wine, Wädenswill, Switzerland
Draught beer
Draught beer, UK
Wine, Wädenswill, Switzerland
Cider, France
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
3
3
5
5
5
5
4
3
3
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
6
6
6
2
6
6
6
6
6
6
6
6
4
6
2
0
1
0
0
5
4
1
2
Tolerance to the different fermentation stress factors is indicated by numbers from 0 (absence of growth) to 6 (colony development at the sixth dilution).
Growth at low pH was observed at 24 h of incubation at 30 °C.
Growth at high glucose concentration was observed at 24 h of incubation at 30 °C.
Growth at 10 °C was recorded after 6 days of incubation; growth at 16 °C was recorded after 3 days of incubation; growth at 30 °C and 37 °C was recorded at 24 h of
incubation.
Growth at 5%, 10%, 12% and 15% of ethanol was observed at 48 h of incubation at 30 °C.
190 C. Belloch et al. / International Journal of Food Microbiology 122 (2008) 188–195

Page 4

yeast cells never find a physiologically optimal environment
during their use in wine production. In fact, they are exposed to
a continuous mix of several stress conditions (Attfield, 1997;
Bauer and Pretorius, 2000; Cardona et al., 2007).
In the present study we have tested the tolerance to four
stress conditions, namely, pH, high glucose concentration,
temperature and ethanol, of strains in the species within the
Saccharomyces sensu stricto complex and hybrids between
them. Table 1 shows the results strain growth of the strains at
different stress conditions considered. Colony development at
the sixth dilution is indicated with 6, colony development at the
fifth dilution is indicated with 5 and so on for the rest of
dilutions. Zero means that colony growth was not observed.
3.1. Growth under pH stress conditions
The majority of yeasts can grow at pH as low as 3.5 (Yarrow,
1998), however, pH in grape musts ranges between 2.75 and 4.2
(Fleet and Heard, 1993), whereas beer fermentation starts at pH
5.0–5.2 and falls to a final pH of 3.8–4.0 (Campbell, 2003).
Tolerance to low pH was observed by colony growth on
plates at pH 2.8, 3.0 and 3.2 after 24 h of incubation at 30 °C.
In general, the majority of strains were able to develop
colonies in all dilutions at the three pH conditions. Our results
showed similar pH tolerance for strains within all species, with
independence of their isolation source, wine or beer, or natural
environments. Only the type strain of S. kudriavzevii and the
strain CECT 11185 within S. bayanus were slightly affected by
pH 2.8 and were able to growth up to the fifth dilution only. Our
results indicate that ability to growth at low pH could be
considered common to all species and hybrids within the group
of Saccharomyces sensu stricto. Consequently, grape must or
beer with low pH should not be considered a stress factor for
yeasts in alcoholic fermentations. Other authors have obtained
similar conclusions; Charoenchai et al. (1998) observed that
variation of pH did not affect the growth rate of natural grape
species or S. cerevisiae, and Serra et al. (2005) also reported that
pH does not have a significant influence on Saccharomyces
growth.
3.2. Growth under osmophilic stress conditions
The majority of yeasts culture media contain 20 g/L of sugar,
although many yeast species are able to growth in glucose
concentrations up to 40% (Yarrow, 1998). Sugar content in
grape musts for table wine varies from 140 to 260 g/l (Amerine
et al., 1980). In the case of beer, sugar content in an all-malt
infusion mash produces wort with approximate glucose and
fructose content of 9 g/L and maltose content about 40 g/L
(Nevoigt et al., 2002; Campbell, 2003).
Colony growth on plates at 200 g/L, 250 g/L and 300 g/L
was observed after 24 h of incubation at 30 °C.
In general, the majority of strains were able to develop
colonies in all dilutions at the three glucose concentrations after
24 h of incubation at 30 °C. Even the type strain of S.
paradoxus, isolated from tree sap in Russia, was able of
growing at 300 g/L of glucose, although tree saps have very low
glucose content (around 1 g/L) (Escher et al., 2004). Also, the
type strain of S. cariocanus, isolated from soil, showed growth
at all sugar concentrations. The type strain of S. kudriavzevii,
isolated from decayed leaves, was slightly affected by 300 g/L
of glucose in the culture media. The strain CECT 11185 within
S. bayanus, isolated from beer, was clearly affected by high
glucose content in the culture media. Our results indicate that, in
general, species within the group of Saccharomyces sensu
stricto are able to growth at sugar concentrations in grape must.
Additional reports on winemaking of must from dried grapes
showed that yeast growth is though severely affected at 40° Brix
(approximately 400 g/l glucose) (Caridi et al., 1999).
3.3. Growth under pH and glucose stress conditions
Yeast growth was not affected by high glucose concentration
and lower pH separately. However, these stress conditions occur
simultaneously in grape must at the beginning of the
fermentation; consequently, yeast tolerance to both stress
conditions was simultaneously tested on agar plates at pH 2.8
and 250 g/L of glucose, which are normal conditions in wine
musts. In general, the majority of yeast strains were able to
develop colonies at all dilutions after 24 h of incubation at 30 °C
(data now shown) and no differences were observed between
these results and the ones obtained at pH 2.8. Consequently, the
ability to growth at both low pH and high sugar concentration
could be considered common for the species within the group of
Saccharomyces sensu stricto, and therefore these parameters do
not have a significant influence on yeast growth, even when
both are simultaneously considered.
Other combined stress conditions happening in alcoholic
fermentations such as temperature and ethanol were not studied
because these parameters produced the highest variations in
growth and, therefore, the evaluation of the accurate influence
of each parameter in the combined results might be very
difficult without complementary studies involving transcription
analysis of genes involved in tolerance to one and both
conditions simultaneously.
3.4. Growth under temperature stress conditions
Colony growth on GPY plates incubated at 10 °C, 16 °C,
30 °C and 37 °C was used to test yeast tolerance to low and high
temperatures. Growth at 30 °C and 37 °C was recorded after
24 h of incubation. Growth at 16 °C was registered after 3 days
of incubation. At 10 °C growth was observed at 6 days of
incubation.
The majority of yeasts are incubated between 20 °C and
25 °C because most of them are mesophilic; however,
temperatures between 4 °C and 15 °C are essential for
psychrophilic taxa. Higher temperatures 30 °C and 37 °C are
often required for yeasts that are strictly associated with warm-
blooded sources (Yarrow, 1998). Most of the yeast strains of our
study were able to develop colonies at 16 °C and 30 °C in all
tested dilutions.
At 37 °C, the majority of strains within S. cerevisiae were
able to grow except for the type strain of S. cerevisiae,
191 C. Belloch et al. / International Journal of Food Microbiology 122 (2008) 188–195

Page 5

isolated from ale beer and the strain CECT 11001, isolated
from lager beer, which was growing poorly at 30 °C and was
not able to grow at 37 °C. On the contrary, two S. cerevisiae
strains isolated from spoiled beer were able to grow at 37 °C.
The remaining S. cerevisiae strains isolated from wine were
able to grow at 37 °C. All strains within S. paradoxus were
able to grow at 37 °C independently of their isolation source
or geographical origin.
None of the strains within the species S. bayanus was able to
grow at 37 °C. The species S. bayanus is currently divided into
two varieties: var. bayanus and var. uvarum. Serra et al. (2005)
reported that temperature was the factor which had the main
influence on the yeast growth, and found that a strain of S.
bayanus var. uvarum (S. uvarum) was less sensitive to
temperature falls than a strain of S. cerevisiae, which is in
agreement with the classification of S. uvarum as cryotolerant
yeast in winemaking (Kishimoto and Goto, 1995; Naumov,
1996). On the other hand, S. bayanus var. bayanus (S. bayanus)
is a hybrid from S. uvarum and from the same non-S. cerevisiae
parental strain as that of lager brewing strains (Casagerola et al.,
2001; Tamai et al., 1998). Modern beer production was initiated
in temperate countries of Central Europe (Germany, Czech
Republic and Denmark) where rather seldom fermentation
temperatures raise up to 37 °C. Our results suggest that low
fermentation temperatures for beer production might have
favoured the cryotolerant trait in beer strains, and as a
consequence S. bayanus beer strains and also S. cerevisiae ale
and S. pastorianus lager beer strains are not able to grow at
37 °C. On the other hand, beer contaminants are able to grow at
37 °C, therefore they can contaminate not only at low
fermenting temperatures but also at higher temperatures during
transport or storing of beer.
Finally, the type strains of S. cariocanus and S. mikatae were
both able to grow at 37 °C, however the type strain of S.
kudriavzevii was not able to grow at 37 °C.
All hybrids between S. cerevisiae×S. bayanus (S. pastor-
ianus) and S. cerevisiae×S. kudriavzevii were able to grow at
37 °C with the exception of CECT 11037 isolated from lager
beer. Even CID1, the triple hybrid between S. cerevisiae×S.
bayanus×S. kudriavzevii, showed colony growth in the first
dilution at 37 °C.
These results indicate that wine yeast hybrids between S.
cerevisiae and S. bayanus or S. kudriavzevii might have
inherited the capability to growth at 37 °C from a wine strain
of the S. cerevisiae parental because S. bayanus and S.
kudriavzevii are not able to grow at 37 °C.
Nowadays, wine fermentation is done at controlled tem-
peratures; however, grape must fermentation was traditionally
done at the end of summer, during the warm days of September
in the Mediterranean countries at average temperatures between
25 to 30 °C and, in central Europe countries between 20 to
25 °C. Additionally, anaerobic fermentation generates waste
energy in the form of heat and yeasts must be able to maintain
viability and ferment at temperatures approaching 38 °C
(Boulton et al., 1996). Probably, after natural hybridization
occurred, natural selection and winemakers have favoured
keeping the ability to grow at 37 °C in wine yeasts.
Similarly, the beer hybrids are also able to grow at 37 °C.
However, the S. cerevisiae strains conducting ale and S.
pastorianus conducting lager beer fermentations are not able to
grow at 37 °C, for that reason, our results suggest that a S.
cerevisiae strain of possible wine origin might have been the
parental strain of the beer hybrids.
Most of the yeast strains were able to develop colonies in
all dilutions at 10 °C and 16 °C. Colony growth at 16 °C was
detected after 3 incubation days. Colony development at 10 °C
was recorded after 6 incubation days (Fig. 1). We observed
that strains within S. bayanus and S. kudriavzevii developed
visible colonies in the sixth dilution after 6 incubation days.
However, S. cerevisiae and S. paradoxus developed visible
colonies in the sixth dilution after 10 incubation days (data not
shown). Cryotolerance of S. bayanus strains was already
observed by several authors (Kishimoto and Goto, 1995;
Naumov, 1996; Giudici et al., 1998), and our results show that
S. kudriavzevii is better adapted to grow at lower temperatures
than S. cerevisiae and therefore could also be considered a
cryotolerant strain.
All hybrids between S. cerevisiae×S. bayanus (S. pastor-
ianus) and S. cerevisiae×S. kudriavzevii were able to grow at
10 °C better than any S. cerevisiae strain but not so well like the
strains of S. bayanus or S. kudriavzevii (Fig. 1). These results
indicate that wine yeast hybrids between S. cerevisiae and S.
bayanus or S. kudriavzevii might have inherited the capability
to growth at lower temperatures from the non-S. cerevisiae
parental. Accordingly, wine hybrids might be also better
adapted to low-temperature fermentations as they have inherited
the cryotolerance from S. bayanus or S. kudriavzevii. Nowa-
days, technological advances in winemaking favour fermenta-
tions at controlled low temperatures to increase the fruity
aromas from the grapes. Therefore wine yeast hybrids able to
ferment in the lower range of temperatures could be of great
biotechnological interest.
Research in this line has already been done for improving the
fermentative capabilities of beer-fermenting yeasts (Sato et al.,
2002). Ale beer-fermenting yeasts (top-yeasts) generally exhibit
good fermentability at a temperature of about 20 °C, but do not
function well at low-temperature as bottom-fermenting yeasts.
Therefore, hybridization with S. bayanus is useful for improv-
ing the low-temperature fermentability of the top-fermenting
yeast S. cerevisiae (ale beer). Additionally, the cryophilic
Fig. 1. Colony development at 10 °C after 6 incubation days.
192C. Belloch et al. / International Journal of Food Microbiology 122 (2008) 188–195