Food Sci. Technol. Res.

, 19 (5), 781 793, 2013

Influence of Temperatures and Fermentation Behaviour of Mixed Cultures of

Williopsis saturnus var. saturnus and Saccharomyces cerevisiae Associated with
Winemaking
Hasan Tanguler*
Nigde University, Department of Food Engineering, 51245 Nigde Turkey

Received September 12, 2012; Accepted June 25, 2013

Up to now, this has been the first study in which the influences of fermentation temperature on the
yeast growth and the production of yeast-derived volatile compounds during the fermentation of Emir
grape must at various temperatures were examined. The results demonstrate that the fermentation temperature plays an important role compared to all tested variables. Fermentations were completed in 6, 8
and 14 days at 12, 18 and 24, respectively. Increase in temperature resulted in an increase in glycerol,
total acidity, acetic and tartaric acid, 2-methyl butanol, propan-1-ol, isobutanol, acetaldehyde and acetone, but in a decrease in ethanol, malic and citric acid, isoamyl acetate, ethyl acetate, isobutyl acetate,
ethyl butyrate and ethyl hexanoate. Moreover, these mixed culture fermentations formed higher amounts
of isoamyl acetate in comparison with pure culture of S. cerevisiae. According to chemical composition
and volatile compounds the differences between obtained wines were found generally significant.
Keywords: white wine, temperature, Williopsis saturnus var. saturnus, Saccharomyces cerevisiae, flavour compounds,
isoamyl acetate

Introduction
Wine production is a combination of complex interactions involving grape variety, microbiota and winemaking
technology (Torija et al., 2003). Among the most important
factors affecting the quality of the wine are the clarification
and composition of the grape juice, the sulfur dioxide level
added, the interaction with other indigenous microorganisms, the supplementation with nutrients, strain and amount
of inoculated yeasts, and the fermentation temperature. The
fermentation temperature is one of the most important parameters for the production of wine since it can affect the
biochemical reactions and metabolism of the yeasts especially non-Saccharomyces and, as a result, the formation of
secondary metabolites such as glycerol, acetic acid and succinic acid, thus determining the chemical and organoleptic
qualities of the wine (Fleet and Heard, 1993; Torija et al.,
2003). White wines are often fermented in the range of 10
20. Nevertheless, some European wineries still prefer
*To whom correspondence should be addressed.
E-mail: htanguler@nigde.edu.tr; tangulehasanr@gmail.com

fermentation temperatures between 20 25 (Sener et al.,

2007). Recently, low temperature (10 15) fermentations
are becoming more frequent due to the winemakers tendency to enhance the production of some volatile compounds
and improve the wine aromatic profile (Beltran et al., 2008;
Andor et al., 2010).
Another important parameter for the production of wine
is yeast. It can strongly affect the quality and flavour of the
final product. Among several yeasts, Saccharomyces (S.)
cerevisiae is the most important specie existing during the
fermentation process (Blanco et al., 2008). It has been used
in wine fermentation due to its ability to induce reliable and
rapid fermentation, convenience and ease of control and
consistency of fermentations (Heard and Fleet, 1985; Fleet,
2003). Inoculation of grape juice with S. cerevisiae is also
common practice in winemaking. The recommended dosage
at the beginning of fermentation is approximately 106 107
cells/mL (Degre, 1993; Boulton et al., 1996; Erten, 2002).
Lately, a trend of using non-Saccharomyces yeasts is
emerging in winemaking to take advantage of their positive
role in imparting the organoleptic characteristics back to

782

wine (Fleet, 2003; Viana et al., 2011). But, previous studies

have shown that small-scale fermentations carried out with
single strains of Kloeckera (K.) apiculata, Candida (C.) stellata, C. pulcherrima and C. colliculosa were not able to complete the fermentation. High residual sugar levels remained
at the end of fermentation, and these wines significantly
differed from those produced by an industrial wine yeast
strain (Jolly et al., 2003). This has led to the current trend
toward using so-called mixed starter cultures, containing
one or more non-Saccharomyces yeasts as well as a selected
industrial S. cerevisiae wine yeast (Styger et al., 2011; Viana
et al., 2011). This combined use of different species has been
evaluated to be able to impart better organoleptic characteristics of wine than single fermentation by pure Saccharomyces
or non-Saccharomyces yeasts in several studies (Soden et al.,
2000; Toro and Vazquez, 2002; Ciani et al., 2006; Moreira et
al., 2008; Viana et al., 2011; Clemente-Jimenez et al., 2005;
Trinh et al., 2011).
Some authors have reported the effect of temperature on
yeast growth and the production of volatile compounds by
using S. cerevisiae (Torija et al., 2003; Molina et al., 2007;
Beltran et al., 2008; Reddy and Reddy, 2011), but there are
limited studies on the effect of temperature on wine production by using non-Saccharomyces yeasts such as K. apiculata
(Erten, 2002), K. corcitis, Hanseniaspora osmophila (Granchi et al., 2002), Hanseniaspora uvarum, Torulaspora delbrueckii, Kluyveromyces thermotolerans (Ciani et al., 2006).
However, more information is still necessary to understand
the behaviour of non-Saccharomyces yeasts during wine
fermentation. Williopsis (W.) saturnus formerly known as
Hansenula saturnus synthesizes important levels of volatile
esters, especially isoamyl acetate and ethyl acetate (Yilmaztekin et al., 2009; Erten and Tanguler, 2010; Trinh et al.,
2011). Generally, it is not found in the natural environment
grape surfaces and winery equipments, but with the production of desirable flavour, W. saturnus can potentially enhance
the fruity flavour in wines obtained from neutral cultivar
characteristics (Erten and Tanguler, 2010).
Although there are various studies on the effects of temperature of S. cerevisiae on wine and beer fermentation, there
is no study on the effect of temperature by using W. saturnus
on wine fermentation. The present work describes the influence of temperatures from 12 to 24 at 6 intervals on
yeast growth, wine composition and volatile compounds in
the mixed cultures of W. saturnus var. saturnus and S. cerevisiae at two different inoculation ratios (W. saturnus var.
saturnus : S. cerevisiae = 1:1 and 10:1 cfu mL1). Meanwhile,
in this research, Emir which is a native grape variety of Vitis
vinifera L. was used due to its potential to produce the best
white wines of Turkey (Erten et al., 2006) and W. saturnus

H. Tanguler

var. saturnus was chosen because of its ability in producing

high quantity of esters such as isoamyl acetate (Yilmaztekin
et al., 2009; Lee et al., 2010).
Materials and Method
Yeast cultures Commercial wine yeast S. cerevisiae
(Actiflore PM) was obtained from Laffort Company (Bordeaux, France). W. saturnus var. saturnus HUT 7087 was
obtained from HUT Culture Collection (Higashi-Hiroshima,
Japan). Yeasts were maintained on Malt Extract Agar (Merck
105398.0500), MEA, slants and re-cultured monthly.
Fermentation conditions White wine grapes of cv. Emir
were obtained from a vineyard in the Nevsehir-Urgup province of Cappadocia region. They were transported to the pilot
winery of the Department of Food Engineering, University
of Cukurova, Adana. They were de-stemmed and crushed
and the must was mixed with 50 mg/kg of sulphur dioxide,
and kept at 15 for 12 h. Then the must was pressed in a
horizontal press and grape juice was sterilised by autoclaving at 115 for 10 min. On the other hand, all equipments
in contact with the grape juices were autoclaved at 121
for 15 min. Emir must had a 5.26 g/L of titratable acidity
(TA) (as tartaric acid), 3.32 of pH, and 18,80 of initial brix.
All fermentations were conducted in duplicate in 1 L sterile
erlenmeyer flasks containing 800 mL of sterile grape juice.
The flasks fitted with foam bungs were incubated at three
different temperatures, 12, 18 and 24 ( 0.5). Fermentations were monitored daily by measuring the density. Upon
completion of alcoholic fermentation, samples were stored at
4 for two days to sedimentation before general wine analysis. For GC and HPLC analysis, the samples were cleared
by centrifugation at 8000 rpm, 0, 5 min, and then stored at
18 until analysed.
Yeast propagation and enumeration of yeasts S. cerevisiae was suspended in sterile warm water at 35 for 30 min
according to the producers instructions. The yeast cells were
centrifuged at 4000 rev/min for 10 min at 4, and washed
with cold sterile water. On the other side, a loopful of stock
cultures of W. saturnus was plated onto MEA and incubated
for 48 h at 25. Yeast cultures (Pellet of S. cerevisiae and
a single colony of W. saturnus) were propagated aerobically
in 100 mL of sterile grape juice in a 250 mL sterile conical
flask fitted with foam bung and incubated for 48 h at 25
with orbital shaking at 160 rev/min. The yeast cells were
centrifuged at 4000 rev/min for 10 min at 4, and washed
with cold sterile water. Pellets were re-suspended in 5 mL
of sterile grape juice. After counting by haemacytometer, W.
saturnus var. saturnus and S. cerevisiae yeasts were added
into all fermentation medium as mono- and mixed culture
(Erten and Tanguler, 2010). Mixed culture fermentations

Effect of Temperature and W. saturnus on Wine

were conducted with the addition of 5 106 cells/mL of S.

cerevisiae + 5 106 cells/mL of W. saturnus var. saturnus
(1:1) and 5 106 cells/mL of S. cerevisiae + 5 107 cells/mL
of W. saturnus var. saturnus (1:10) for each temperature (12,
18 and 24). Fermentation trials from 12 to 24 were designated from W1 to W6, respectively. On the other hand, the
addition of pure culture of 5 106 cells/mL S. cerevisiae (Sc)
was used as a control.
Samples were taken aseptically to count the yeasts during alcoholic fermentations every day. They were diluted in
0.25% saline as necessary, and 0.1 mL of diluted sample was
spread onto plates of MEA and Lysine agar (Sigma-Aldrich
L5910). Lysine agar was used to count non-Saccharomyces
yeasts because it is a synthetic medium with glucose, vitamins, inorganic salts and L-lysine as the sole nitrogen
source, and Saccharomyces spp. are unable to grow on it.
Total yeast and W. saturnus var. saturnus yeast were enumerated on MEA and Lysine agar, respectively. The plates were
incubated at 25 for 3 to 5 days and then examined for the
yeast counts. The total Saccharomyces spp. count was calculated from the total yeast and W. saturnus var. saturnus yeast
counts (Fleet and Heard, 1993; Erten et al., 2006).
Analytic determinations Density and pH were determined using a density meter (Densito 30PX Mettler Toledo
Portable LabTM), and a pH meter (Inolab WTW, Germany),
respectively. TA was measured by titrating sample and expressed as grams of tartaric acid/L (OIV, 1990).
Determination of organic acid, sugar, ethanol content
and volatile compounds Ethanol, glycerol, glucose, fructose and acetic, succinic, tartaric, malic and citric acids were
analysed by a HPLC (Shimadzu LC-20AD, Kyoto, Japan)
using an Aminex HPX-87H column (Bio-Rad, Richmont,
CA, 300 7.8 mm, USA) at 50. The eluent was 5 mmol/L
H2SO4 in high-purity water at a flow rate of 0,6 mL/min.
Concentrations of ethanol, glycerol, glucose and fructose
were calculated from RI detector and amounts of organic
acids from UV detector (Shimadzu) (Erten and Tanguler,
2010). Standards (Merck, Darmstadt, Germany) were used
to determine the concentration of organic acids, sugars, glycerol and ethanol.
Higher alcohols, esters and carbonyl compounds were
measured by a Gas Chromatograph (HP 5890; HewlettPackard, Stockport, UK). Cell-free samples were diluted to
4% (vv) ethanol and then 5 mL of diluted sample, 2 g of
NaCl and 50 L of internal standard (200 mg/L of 3-heptanone) were sealed and a 1 mL sample was injected into a 60
m 0,25 mm i.d. 0,4-m-thick Chrompac CP-Wax-57-CB
column (Middleburgh, the Netherlands), temperature-programmed from 40 to 80. The stream from the column was
split 1:1 to flame ionization detector (Erten et al., 2006). All

783

analytical determinations were carried out in duplicate. The

samples were stored at 18 until GC and HPLC analyses.
Statistical analysis Data of chemical composition and
volatile compunds were analysed for statistical significance
by one-way analysis of variance (ANOVA). Means were
compared by Duncan test statistical analysis using the software SPSS 10.0 for Windows (SPSS Inc. Chicago, IL, USA).
These programmes were run on a PC.
Results and Discussion
This is the first study in which fermentation of grape
must by adding W. saturnus var. saturnus together with S.
cerevisiae at various temperatures was examined. Fermentations were carried out at 12, 18 and 24 using cv.
Emir grape must, in order to estimate how temperature and
inoculation ratios affect fermentation, yeast growth, wine
composition and the production of volatile compounds.
Effect of temperature and inoculation ratios on fermentation and growth of yeast In spite of the different temperatures and inoculation ratios used in experiments, all of the
mixed and pure fermentation trials were finished (final sugar
concentration < 2 g/L). Fermentation was followed by measuring the density. The results of density determinations during alcoholic fermentation are presented in Figure 1.
Fermentation of Emir grape juice was completed on 8
days at 18 by using pure S. cerevisiae. The fermentation
characteristic of the mixed culture (W3) at 18 was similar
to that of the pure S. cerevisiae in term of density.
As expected, fermentations performed at 12 (14 days)
were longer than those performed at 18 (8 days) and 24
(6 days), respectively. It is well known that fermentation rate
is mainly dependent on temperature. The rate increases with
an increase in fermentation temperature (Fleet and Heard,
1993). Similar observations were also obtained with sugar
consumption (data not shown). Several authors (Torija et al.,
2003; Masneuf-Pomarde et al., 2006; Sener et al., 2007;
Beltran et al., 2008; Cortes et al., 2009) have also stated that
increasing temperature results in a faster fermentation rate.
Furthermore, increasing inoculation ratio accelerated the fermentation but, fermentation time did not changed. Similarly,
other studies have showed that increasing the inoculum ratio
leads to faster fermentations (Fleet and Heard, 1993; Mateo
et al., 2001; Erten et al., 2006).
Temperature affected not only the fermentation time, but
also the yeast growth. Changes in S. cerevisiae and W. saturnus var. saturnus viability in mixed cultures at 12, 18
and 24 during alcoholic fermentation is given in Figures 2,
3 and 4, respectively.
Figure 3 also shows the changes in pure S. cerevisiae
viability at 18. As can be seen from figures, the growth

H. Tanguler

784
Sc

1,08

W1

W2

W3

W4

W5

W6

1,07
1,06
Density

1,05
1,04
1,03
1,02
1,01
1
0,99
0

10

12

14

Fermentation time (days)

Fig. 1. The decrease in density during fermentations.

Sc (5 106 cells/mL pure S. cerevisiae, +), W1 (5 106 cells/mL S. cerevisiae + 5 106 cells/mL of W. saturnus var. saturnus, ), W2 (5
106 cells/mL S. cerevisiae + 5 107 cells/mL W. saturnus var. saturnus, ), W3 (5 106 cells/mL S. cerevisiae + 5 106 cells/mL of W.
saturnus var. saturnus, ), W4 (5 106 cells/mL S. cerevisiae + 5 107 cells/mL W. saturnus var. saturnus, ), W5 (5 106 cells/mL S.
cerevisiae + 5 106 cells/mL of W. saturnus var. saturnus, ), W6 (5 106 cells/mL S. cerevisiae + 5 107 cells/mL W. saturnus var. saturnus, ), The values are averages of duplicate determinations.

Numbers of viable yeast cells

(Log cfu/mL)

8
7
6
5
4
3
2
1
0
0

Fermentation time (days)

10

12

14

Fig. 2. Growth of S. cerevisiae and W. saturnus var. saturnus in mixed cultures at 12 during alcoholic fermentation.
(S. cerevisiae in W1), (W. saturnus var. saturnus in W1), (S. cerevisiae in W2), (W. saturnus var. saturnus in W2). Vertical bars
indicate standard deviations.

in yeast varied according to temperature. A low fermentation temperature affects the growth of the yeast and leads to
a slow growth rate. For this reason, fermentations at 12
began more slowly, as we can see by their longer lag phase
(Figure 2). This caused a delay in reaching the maximal
population. The lag phase is an important technological aspect in wine-making because it determines the adaptation
of the yeast cells after their inoculation in the grape must.
One common feature among low temperature fermentations
is very long lag phases (Torija et al., 2003). In contrast, in
present study, the lag phase was not observed at 18 and 24

(Figures 3 and 4). These results correlated with the previous

reports that the lag phase was not seen and fermentation was
completed more rapidly at higher temperature than 18
(Erten, 2002; Sener et al., 2007; Cortes et al., 2009).
Figure 3 shows the growth of pure and mixed cultures
at 18. The main wine yeast, S. cerevisiae, in pure culture
used as control increased rapidly after fermentation started,
and the highest amounts achieved 7.90 log cfu/mL within
the first 4 days of the fermentation. After maximum growth,
it exhibited a very long stationary phase until the end of
fermentation and its population was found as 7.35 log cfu/

Effect of Temperature and W. saturnus on Wine

785

Numbers of viable yeast cells (Log cfu/mL)

8
7
6
5
4
3
2
1
0
0

Fermentation time (days)

Fig. 3. Growth of S. cerevisiae and W. saturnus var. saturnus in pure and mixed cultures at 18 during alcoholic fermentation.
+ (pure culture of S. cerevisiae), (S. cerevisiae in W3), (W. saturnus var. saturnus in W3), (S. cerevisiae in W4), (W. saturnus var.
saturnus in W4). Vertical bars indicate standard deviations.

Numbers of viable yeast cells (Log cfu/mL)

9
8
7
6
5
4
3
2
1
0
0

Fermentation time (days)

Fig. 4. Growth of S. cerevisiae and W. saturnus var. saturnus in mixed cultures at 24 during alcoholic fermentation.
(S. cerevisiae in W5), (W. saturnus var. saturnus in W5), (S. cerevisiae in W6), (W. saturnus var. saturnus in W6). Vertical bars
indicate standard deviations.

mL. Furthermore, S. cerevisiae in the grape juice fermented

with mixed cultures also increased rapidly after fermentation
started at all temperatures studied, and dominated the alcoholic fermentation at two inoculation ratios (S. cerevisiae :
W. saturnus var. saturnus, Sc:Ws= 1:1 and 1:10) by the 5th
and 6th days at 12, by the 1st and 4th days at 18 and by
the 1st and 3rd days at 24, respectively. It can obviously
be seen that S. cerevisiae dominated the fermentation earlier with the increasing temperature. After proliferation and
domination the fermentation of S. cerevisiae, the counts of W.
saturnus var. saturnus yeasts declined or disappeared (Figures
2, 3 and 4).
On the other hand, at different temperatures, the maxi-

mum S. cerevisiae cell population was obtained in different

days. It was obtained within 3 (inoculation ratio of 1:1) and
3 days at 24 (inoculation ratio of 1:10) compared to 4 and
3 days at 18, and 7 and 7 days at 12, respectively. After
maximum growth at different temperatures and inoculation
ratios, S. cerevisiae exhibited a very long stationary phase
until the end of alcoholic fermentation. At the end, S. cerevisiae values were obtained between 6.92 and 7.53 log cfu/
mL (Figures 2, 3 and 4). In our previous study on different
species of W. saturnus yeast, we determined that S. cerevisiae was the dominant yeast in mixed culture fermentations,
exhibited a stationary phase and had the numbers of 7 7.64
log cfu/mL at 18 (Erten and Tanguler, 2010). It is observed

786

that the starting of fermentation, dominating to the alcoholic

fermentation, reaching of maximal S. cerevisiae population
and decline of population at 12 are slow compared to fermentations at 18 and 24. The results obtained by this
study confirmed the previous investigations on the S. cerevisiae growth in mixed cultures during alcoholic fermentation (Erten, 2002; Zohre and Erten, 2002; Reddy and Reddy,
2011).
At 12, after fermentation started at both inoculation
raios of 1:1 and 1:10, W. saturnus var. saturnus dominated
over S. cerevisiae during the first 4 and 5 days, respectively.
The maximum W. saturnus var. saturnus amounts were
7.20 and 8.06 log cfu/mL by day 4. However, the maximum
amounts at 18 and 24 were obtained as 7.0 and 8.06
log cfu/mL at two inoculation ratios by day 2 and 3, and
6.74 and 7.84 log cfu/mL by day 1 and 2, respectively. After
maximum growth, W. saturnus var. saturnus did not exhibited a stationary phase in all trials and, the decline phase occured during fermentation. In our previous study (Erten and
Tanguler, 2010) on different species of W. saturnus yeast, we
observed similar results. At the same time, Zohre and Erten
(2002) and Erten et al. (2006) stated that after maximum
growth, non-Saccharomyces yeasts didnt show the stationary phase and a decline phase was observed. In contrast,
(Trinh, 2011) described that W. saturnus var. mrakii at inoculation ratio of 1:1000 with S. cerevisiae var. bayanus showed
stationary phase in mixed culture fermentation. Additionally,
it was observed that as the inoculation ratios increased, the
reduction in the number of W. saturnus var. saturnus slowed.
Moreover, while W. saturnus var. saturnus disappeared from
the 11th day in trial W1 (inoculation ratio, 1:1), it survived
14 day in trial W2 (inoculation ratio, 1:10). Similarly, at
24, it disappeared from the 5th day in trial W5, but it survived 6 day in trial W6. At 18, they died off on 6th and 8th
day of fermentation at two inoculation ratios, respectively.
In previous studies, the researchers reported that W. saturnus
died off by 4 5 days at inoculation ratio of 1:1 (Erten and
Tanguler, 2010), by 10 days at inoculation ratio of 1:1000
(Lee et al., 2012b) and by 14 days at inoculation rate of 1:100
(Trinh, 2011). In contrast, Lee et al. (2010) and Trinh (2011)
stated that W. saturnus survived until the end of fermentation
that was terminated by 21 days at inoculation ratio of 1:1000.
In present study, W. saturnus var. saturnus multiplied at
higher populations and survived longer at lower temperatures
than higher temperatures. Similar conclusions were also
reported by Erten (2002) and Sener et al. (2007) for nonSaccharomyces yeasts. It is generally accepted that non-Saccharomyces yeasts grow and then begin to die off during the
early stages of fermentations due to their inability to tolerate
the increasing ethanol concentrations present in the must me-

H. Tanguler

dium. Contrary to general assumptions, recent studies have

demonstrated that some non-Saccharomyces yeasts such as
Hanseniaspora guilliermondii, K. apiculata and C. pulcherrima are able to stand much higher ethanol concentrations
than previously thought (Bilbao et al., 1997; Zohre and
Erten, 2002; Perez-Nevado et al., 2006). Explanations for the
early death of non-Saccharomyces yeasts in mixed cultures
with Saccharomyces spp., could rely upon other factors such
as competition for sugar uptake, oxygen availability, nutrient
limitation, presence of toxic compounds, cell-cell interaction and quorum sensing (Fleet, 2003; Perez-Nevado et al.,
2006; Lee et al., 2012b). Therefore, this study indicates that
the fermentation temperature and different inoculation ratios
are important in order to determine the inhibition of the nonSaccharomyces yeast. They exist not only in the early wine
fermentation stage but also even for longer periods.
Effect of temperature and inoculation ratios on general
wine composition Temperature and inoculation ratio effected not only the fermentation rate and length but also the
yeast metabolism, which determined the general composition
of the wine (Torija et al., 2003; Sener et al., 2007; Reddy and
Reddy, 2011). The general composition of wines in present
study is illustrated in Table 1. As can be seen, all parameter
values were dependent on the fermentation temperature. Increasing temperature from 12 to 24 resulted in a decrease
in ethanol, malic and citric acid (except W6) concentrations,
but in an increase in glycerol, total acidity, acetic and tartaric
acid concentrations. The highest ethanol values were found
in W1 (9.56 %v/v) produced at 12 and the lowest values
were in W6 (8.01 %v/v) produced at 24. However, it
determined as 9.25%v/v in control produced at 18. Temperature and inoculation ratio significantly affected ethanol
concentration (P < 0.001).
Glycerol is quantitatively a very important wine constituent. During yeast fermentation, it is the major end product
other than ethanol and carbon dioxide. Its concentrations
in wine varying between 1.0 27.6 g/liter (Ciani and Ferraro, 1996; Cortes et al., 2009; Styger et al., 2011) and the
maximum level acceptable in wines is 25.8 g/L (Toro and
Vazquez, 2002; Mendoza and Faras, 2010). The amount
of glycerol formed is influenced by several factors, such as
grape variety, degree of ripeness, fermentation temperature
and yeast strain (Ciani and Ferraro, 1996; Suarez-Lepe and
Morata, 2012). In present study, glycerol were changed between 4.42 5.99 g/L (P < 0.001) and it was determined as
5.42 g/L in control. Wines inoculated with equal ratios of S.
cerevisiae and W. saturnus var. saturnus have higher amounts
of glycerol when compared with pure S. cerevisiae fermented
wine used as a control. These results for glycerol were found
in acceptable levels and correlated with the previous reports

Effect of Temperature and W. saturnus on Wine

787
Table 1. The general composition of wines.

Temperature

18

12

18

24

Sc

W1

W2

W3

W4

W5

W6

Density (20)
Ethanol (v v)
Glycerol (g/L)
pH
Total acidity as
tartaric acid (g/L)

0.9944ab
9.25ab 0.08
5.42b 0.07
3.165bc

0.99445ab
9.56a 0.18
5.45b 0.07
3.17abc

0.9937ab
8.63cd 0.17
4.42d 0.1
3.21a

0.9938ab
9.42a 0.01
5.56b 0.06
3.175abc

0.9924c
8.2de 0.16
4.46cd 0.06
3.195ab

0.9948a
8.94bc 0.12
5.99a 0.13
3.145c

0.9934bc
8.01e 0.12
4.76c 0.16
3.2ab

*
***
***
*

6.045ab 0.04

5.93abc 0.2

5.54c 0.12

5.99ab 0.03

5.66bc 0.15

6.16a 0.06

5.66bc 0.1

Organic acids (g/L)

Acetic acid
Tartaric acid
Malic acid
Succinic acid
Citric acid (mg/L)

0.40c 0.02
2.07 0.02
1.72a 0.03
0.67c 0.02
35abc 4

0.53bc 0.04
1.785 0.1
1.73a 0.03
0.73bc 0.06
43.18a 1.9

0.70ab 0.05
1.655 0.07
1.48bc 0.04
1.02a 0.06
28.53bc 1.4

0.70ab 0.02
2.04 0.03
1.585ab 0.06
0.7bc 0.05
38ab 4

0.8a 0.01
2.105 0.2
1.47bc 0.08
0.89ab 0.07
25.5c 3.5

0.79a 0.04
2.09 0.07
1.275cd 0.09
0.7bc 0.08
30.62bc 0.7

0.86a 0.12
2.18 0.12
1.16d 0.1
1.06a 0.07
31.06bc 1.1

**
ns
**
**
*

Sugars (g/L)
Glucose
Fructose
Total sugar

1.1abc 0.02
0.78a 0.03
1.88a 0.05

1abc 0.18
0.57abc 0.03
1.57ab 0.21

0.675d 0.07
0.385c 0.08
1.06b 0.14

1.12ab 0.02
0.705ab 0.02
1.825a 0.04

0.79cd 0.07
0.49bc 0.04
1.28b 0.11

1.14a 0.06
0.67ab 0.09
1.81a 0.15

0.81bcd 0.1
0.48bc 0.11
1.29b 0.22

*
*
*

Sc (5 106 cells/mL pure S. cerevisiae), W1 (5 106 cells/mL S. cerevisiae + 5 106 cells/mL of W. saturnus var. saturnus), W2 (5
106 cells/mL S. cerevisiae + 5 107 cells/mL W. saturnus var. saturnus), W3 (5 106 cells/mL S. cerevisiae + 5 106 cells/mL of W.
saturnus var. saturnus), W4 (5 106 cells/mL S. cerevisiae + 5 107 cells/mL W. saturnus var. saturnus), W5 (5 106 cells/mL S. cerevisiae + 5 106 cells/mL of W. saturnus var. saturnus), W6 (5 106 cells/mL S. cerevisiae + 5 107 cells/mL W. saturnus var. saturnus),
P : Significance. ***, ** and * display the significance at 0.1%, 1% and 5% by LSD, respectively. a-f Values not sharing the same superscript letter within the horizontal line are different according to Duncan test. ns: not significant.

(Soden et al., 2000; Toro and Vazquez, 2002; Comitini et al.,

2011).
On the other hand, Balli et al. (2003) stated that glycerol
and ethanol concentrations were inversely related. Moreover,
in present study it is observed that as ethanol concentration
decreased, glycerol increased. Similarly, Torija et al. (2003),
Balli et al. (2003), Molina et al. (2007), Cortes et al. (2009)
and Reddy and Reddy (2011) also reported that ethanol
concentration decreases, but glycerol increases as the temperature increases. In contrast, some researchers also stated
that ethanol value increases (Sener et al., 2007; Erten, 2002),
and/or glycerol decreases by increasing temperature (Bilbao
et al., 1997). In addition, the inoculum ratio also influences
the production of glycerol (Suarez-Lepe and Morata, 2012).
In present study, it is determined that glycerol and ethanol
decreased by increasing inoculum ratios of W. saturnus var.
saturnus studied at all temperatures. On the contrary, Viana
et al. (2011) reported that ethanol and glycerol levels were
not affected by the type of inoculation.
Fermentation temperature is also known to affect yeast
metabolism, and as a result, the formation of other secondary
metabolites (acetic acid, succinic acid, etc.) besides glycerol
(Torija et al., 2003). Acetic acid, a by-product of yeast metabolism, leads to wine objectionable near its flavour threshold of 0.7 1.1 g/L of volatile acidiy as acetic acid (Henschke
and Jiranek, 1993). Succinic acid is one of the major acids

found in wine. It contributes a pleasant acidic taste (Patel

and Shibamoto, 2003). Acetic acid and succinic acid values
in wine produced by pure S. cerevisiae were 0.40 g/L and 0.67
g/L, respectively. Their value in mixed cultures were higher
than pure S. cerevisiae and determined between 0.53 0.86
g/L and 0.70 1.06 g/L, respectively (P < 0.01). This high
values are probably a result of the W. saturnus var. saturnus,
which is produced high amounts acetic and succinic acid
(Lee et al., 2010; Lee et al., 2012a). As acetic acid increased
by temperature and inoculation, succinic acid decreased by
temperature (except for W6), but increased by inoculation.
Similarly, Torija et al. (2003) and Cortes et al. (2009) also
stated that acetic acid concentration increased with increasing temperature. On the other hand, in present study, acetic
acid concentrations obtained in some wines are in disagreement with the results found by Soden et al. (2000), Cortes et
al. (2009) and Mendoza et al. (2011) who found the lower
amounts of acetic acid (0.3 0.70 g/L) in mixed cultures. In
this study, it was produced higher than the lower threshold
value throughout the fermentations which leads to negative
effect on wine quality (except for W1). Glucose, fructose, total sugar, total acidity and tartaric acid values in wines were
increased with increasing temperature, except for fructose in
W5 and W6, but decreased by increasing inoculation ratio
(except for tartaric acid in W6). These results are in good accord with the data given by Cortes et al. (2009), by contrast

788

H. Tanguler

with Masneuf-Pomarde et al. (2006) and Ciani et al. (2006).

The results also showed that fermentation temperature and
inoculation ratio had a significant effect on glucose, fructose,
sucrose, total sugar and total acidity values (P < 0.05), but
did not affect tartaric acid concentration (P > 0.05).
Effect of temperature and inoculation ratios on volatile
compounds of wines There are many factors that may have
an important impact on the formation of volatile compounds
in wine, and thus on its quality, including yeast type, the
population of individual yeasts in must, the composition of
the must, the fermentation conditions and temperature, the
winemaking technique followed, and the yeast strain used
and inoculum ratio (Molina et al., 2007; Trinh et al., 2010;
Suarez-Lepe and Morata, 2012). In this study, effect of temperature and also inoculation ratio on the volatile compositions of wine fermented by mixed cultures of yeasts were
studied. Yeast is the main producer of flavour compounds
such as esters, higher alcohols and carbonyl compounds
during alcoholic fermentation (Sarris et al., 2009). For this
reason, in this study, yeasts belonging to the Williopsis genus

was chosen. It is reported that this yeast is able to consume

sugar oxidatively for cell growth as well as produce desirable
fruity flavours. Particularly, W. saturnus strains are known
to convert higher alcohols into the corresponding acetate
esters. An example is the conversion of isoamyl alcohol into
isoamyl acetate (Yilmaztekin et al., 2009). Isoamyl acetate
(3-methylbutyl acetate) is important contributors to the pleasant fruity note of wine (Gil et al., 1996). In present study,
mixed cultures of S. cerevisiae and W. saturnus var. saturnus
formed relatively higher amounts of isoamyl acetate, between 3.18 5.12 mg/L, in comparison with control (2.49
mg/L) (Table 2). It could be said that W. saturnus var. saturnus was significantly able to enhance the production isoamyl
acetate (banana and fruity like aroma). Zohre and Erten
(2002) produced white wine with mixed cultures of Kloeckera apiculata, Candida pulcherrima and S. cerevisiae, and
they found the concentrations of isoamyl acetate in the range
of 0.995 1.905 mg/L. Findings in present work for isoamyl
acetate are slightly higher than study reported by Zohre and
Erten (2002), however slightly lower than previous study of

Table 2. The effect of temperature on flavour compounds of wines.

Temperature

18

12

18

24

Sc

W1

W2

W3

W4

W5

W6

40.97b 2.86
0.115 0.025
0.167 0.015
2.49c 0.253
0.475 0.103
0.26 0.034
44.477b 2.43

56.35b 5.09
0.137 0.004
0.203 0.005
3.98ab 0.038
0.488 0.014
0.256 0.008
61.41b 5.03

103.76a 5.88
0.143 0.005
0.125 0.004
4.13ab 0.026
0.334 0.006
0.21 0.016
108.70a 5.86

49.08b 1.17
0.129 0.013
0.193 0.003
3.90ab 0.319
0.427 0.115
0.272 0.069
54.001b 1.02

93.09a 5.03
0.112 0.012
0.107 0.007
5.12a 0.83
0.324 0.102
0.206 0.022
98.959a 4.3

48.18b 3.86
0.125 0.006
0.114 0.006
3.18bc 0.041
0.29 0.014
0.26 0.008
52.15b 3.8

99.76a 7.6
0.112 0.004
0.083 0.004
3.31bc 0.036
0.226 0.01
0.178 0.004
104.0a 8.01

***
ns
ns
*
ns
ns
***

23.79 0.95
116.16a 2.56
24.593e 0.82

22.07 1.03
95.79b 0.9
50.093c 0.8

20.91 0.98
68.09d 0.98
45.51d 1.05

24.22 2.14
123.22a 3.1
70.47b 0.92

25.79 2.93
84.01c 3.08
52.496c 1.6

26.83 0.95
115.32a 1.62
72.16ab 0.94

28.73 0.41
76.71c 2.61
74.4a 1.16

ns
***
***

29.55e 0.91
194.09d 0.12

22.30f 0.07
190.25d 1

32.79d 0.57
167.30e 0.33

31.11de 0.15
249.02a 0.19

39.74b 0.89
202.03c 0.86

35.58c 0.96
249.89a 2.57

49.55a 0.91
229.39b 2.45

***
***

20.78c 1.73

21.47c 1

30.24b 1.45

21.58c 2.06

31.28b 1.12

30.42b 1.56

56.47a 0.87

***

0.774d 0.07

0.975c 0.01

1.077bc 0.02

1.113bc 0.09

1.103bc 0.1

1.573a 0.02

1.264b 0.01

***

0.073c 0.01
0.026bc 0.003
21.653c 1.67

0.142b 0.01
0.064a 0.001
22.65c 0.99

0.2a 0.01
0.014d 0.001
31.531b 1.41

0.054c 0.01
0.077c 0.02
0.028bc 0.008
0.035b
c
22.775 1.97 32.495b 1.23

0.046c 0.003 0.08c 0.003

0.015d 0.001 0.021cd 0.002
32.054b 1.54 57.835a 0.86

***
***
***

260.22d 4.21

274.31d 5.02

307.531c 7.6

325.796bc 3.18 333.49b 6.38

334.093b 7.9

***

ESTERS (mg/L)
Ethyl acetate
Isobutyl acetate
Ethyl butyrate
Isoamyl acetate
Ethyl hexanoate
Ethyl octanoate
Total

HIGHER ALCOHOLS (mg/L)

2-Methyl butanol
3-Methyl butanol
Propan-1-ol
Isobutanol
(isobutyl alcohol)
Total

CARBONYL COMPOUNDS (mg/L)

Acetaldehyde
Acetone
(dimethyl ketone)
Butanedione
(diacetyl)
Pentanedione
Total
MAIN TOTAL

391.249a 9.6

Sc (5 106 cells/mL pure S. cerevisiae), W1 (5 106 cells/mL S. cerevisiae + 5 106 cells/mL of W. saturnus var. saturnus), W2 (5
106 cells/mL S. cerevisiae + 5 107 cells/mL W. saturnus var. saturnus), W3 (5 106 cells/mL S. cerevisiae + 5 106 cells/mL of W.
saturnus var. saturnus), W4 (5 106 cells/mL S. cerevisiae + 5 107 cells/mL W. saturnus var. saturnus), W5 (5 106 cells/mL S. cerevisiae + 5 106 cells/mL of W. saturnus var. saturnus), W6 (5 106 cells/mL S. cerevisiae + 5 107 cells/mL W. saturnus var. saturnus),
P : Significance. *** and * display the significance at 0.1% and 5% by LSD, respectively. a-f Values not sharing the same superscript letter
within the horizontal line are different according to Duncan test. ns: not significant.

Effect of Temperature and W. saturnus on Wine

Viana et al. (2011) who found between 4.09 and 5.83 mg/L
in wine fermented with Hanseniaspora vineae and S. cerevisiae. But, they used red grape must with an initial sugar
content of 257 g/L and supplemented with 1 g/L of complex
yeast nutrient in their study. Additionally, the production of
isoamyl acetate dependent of fermentation temperature and
inoculation rate (P < 0.05).
Increasing temperature led to a marked decrease in the
concentration of isoamyl acetate (except for W4), but its concentration increased when the inoculation ratio was raised.
Simpson (1979) and Etievant (1991) stated that isoamyl acetate has 1 mg/L flavour threshold and it is the most abundant
contributor to wine aroma. The results obtained by this study
confirmed the previous investigations on the effects of temperature (Beltran et al., 2008), inoculation ratios (Erten et al.,
2006; Trinh et al., 2011) and W. saturnus species (Erten and
Tanguler, 2010). On the contrary, Erten (2002) stated that the
production of isoamyl acetate was independent of fermentation temperature. Moreover, Lee et al. (2010) reported that
the production of isoamyl acetate with pure S. cerevisiae is
higher than produced with mixed cultures (the ratio of 1:100).
Ethyl acetate is the most abundant ester existing in wine
and its existence gives the positive effect on the fruity flavour
of wines, because the levels are higher than flavour threshold (Reddy et al., 2008). Non-Saccharomyces yeasts such
as Candida, Hansenula, Pichia and Williopsis species have
a greater capacity to produce ethyl acetate than wine strains
of S. cerevisiae (Lee et al., 2010; Manzanares et al., 2011).
Similar to isoamyl acetate, it was observed that the lowest
ethyl acetate (40.97 mg/L) and total ester (44.48 mg/L) were
also determined in wine used as control (Table 2). Moreover,
their concentrations in wines inoculated with equal ratios of
S. cerevisiae and W. saturnus var. saturnus decreased when
fermentation temperature increased to 24 with the exception for W6, but increased with increasing inoculation ratio.
This is probably a result of longer growth and survival of
W. saturnus var. saturnus at lower temperatures and and
higher amount of W. saturnus var. saturnus than equal ratio.
The concentrations of ethyl acetate and total esters in mixed
cultures were ranging from 48.18 to 103.76 mg/L and from
52.15 to 108.70 mg/L, respectively. In addition, fermentation
temperature and inoculation ratio were found significantly
important with regard to the amount of ethyl acetate and total esters (P < 0.001). Etivant (1991) and Lee et al. (2010)
state that concentrations of ethyl acetate below 50 mg/L do
not contribute to wine flavour, while amounts higher than
150 200 mg/L results in defects with solvent-like flavour
in wine quality. In this study, the amounts of ethyl acetate
were generally found between 50 150 mg/L and contribute
positively to the wine flavour. The results obtained for ethyl

789

acetate in this study are in agreement with the findings of

Kourkoutas et al. (2001), Erten (2002), Reddy et al. (2008)
and Reddy and Reddy (2011) that there was an increase
in ethyl acetate formation in wines at lower temperatures.
Moreover, Trinh et al. (2011) have reported similar result
that the concentration of ethyl acetate rose at different inoculation rates of W. saturnus. Erten and Tanguler (2010)
reported that ethyl acetate concentrations were below 50 mg/
L in wines produced with equal ratios of S. cerevisiae and W.
saturnus. However, some authors reported that ethyl acetate
level decreased with decreasing temperature (Ikonomopoulou
et al., 2003; Mallouchos et al., 2003).
Isobutyl acetate (2-methylpropyl acetate), ethyl butyrate
(ethyl butanoate) and ethyl hexanoate (ethyl caproate) are
synthesized greater at low fermentation temperatures (Table
2). Their values were found as 0.11 0.14 mg/L, 0.08 0.20
mg/L and 0.23 0.49 mg/L, respectively. These results are
consistent with previous reports (Erten, 2002; Molina et al.,
2007; Beltran et al., 2008), but are not in agreement with
data reported by Mallouchos et al. (2003). On the other hand,
fermentation temperature and inoculation ratio did not affect
isobutyl acetate, ethyl butyrate, ethyl hexanoate and ethyl
octanoate (P > 0.05).
Higher alcohols are quantitatively the largest group of
volatile compounds. They are mainly formed from Ehrlich
pathway in the presence of amino acids, and/or from sugars
via biosynthesis by yeasts when amino acids are absent in
the medium. Higher alcohols, characterized by their strong
and pungent smell and taste, can significantly influence wine
taste and character (Erten, 2002; Manzanares et al., 2011;
Suarez-Lepe and Morata, 2012). The most important ones
are 2-methyl butanol (active amyl alcohol), 3-methyl butanol
(iso amyl alcohol) and propan-1-ol (n-propyl alcohol) (Kandylis et al., 2010; Suarez-Lepe and Morata, 2012).
Wines produced at 18 fermented with W. saturnus var.
saturnus at inoculation ratios of 1:1 and 1:10 have higher
amounts of relative and total higher alcohol when compared
with pure S. cerevisiae fermented wine used as a control (except for 3-methyl butanol in W4). However, it was observed
that 2-methyl butanol (except W2) and isobutanol amounts
increased, propan-1-ol (except W6), 3-methyl butanol and
total higher alcohol decreased with the inoculation. These
results correlated with the previous report given by Erten
and Tanguler (2010) who studied with W. saturnus at equal
inoculation ratio (1:1). In contrast, Trinh et al. (2011) studied
also W. saturnus and they showed that 3-methyl butanol increased with two inoculation ratios of 1:100 and 1:1000. On
the other hand, Lee et al. (2012b) reported that all relative
higher alcohols increased at higher W. saturnus inoculation
ratio (1:1000). However, Lee et al. (2010) stated that relative

790

higher alcohols decreased at higher W. saturnus inoculation

ratio.
On the other hand, Increasing at temperature led to an
increase in concentration of relative (except for 3-methyl butanol) and total higher alcohol. 3-methyl butanol amount was
increased by increasing temperature from 12 to 18 and
the highest concentration was determined as 123.22 mg/L in
W3, but later on decreased by increasing temperature from
18 to 24. In addition, relative and total higher alcohol values formed were found statistically important (P < 0.001),
but 2-methyl butanol concentrations were trivial. Several
authors (Bardi et al., 1997; Erten, 2002; Reddy and Reddy,
2011) have also stated that higher alcohols increased by increasing temperaure. The concentrations of 2-methyl butanol
(20.91 28.73 mg/L), propan-1-ol (24.59 74.4 mg/L) and
isobutanol (22.3 49.55 mg/L) were much below than their
threshold values (300 750 mg/L, 75 500 mg/L and 300
330 mg/L, respectively) given by Etivant (1991) but within
the range of previous studies reported previously. Moreover,
3-methyl butanol concentrations in wines produced by mixed
cultures were determined between 68.09 123.22 mg/L and
their values were lower than control sample except for W3.
Their concentrations in all wines were higher than its threshold value (14.5 mg/L) given in the literature (Etivant, 1991).
Higher alcohols generally exist in wine at the concentrations
< 300 mg/L and they contribute to the aromatic complexity
of the product. When their concentrations exceed 400 mg/
L, they are considered to have a negative effect on flavour
(Manzanares et al., 2011). In present study, higher alcohol
concentrations were determined below the 300 mg/L. For
this reason, it could be said that higher alcohol had positive
effect on flavour of wines produced at different temperatures
with W. saturnus var. saturnus.
Due to their low perception threshold and the characteristics that they confer on the wine, volatile aldehydes are
among the most interesting carbonyl compounds. Acetaldehyde is the principal carbonyl compound in wine derived
from pyruvate during alcoholic fermentation (Berry, 1995)
and it constitutes more than 90% of the total aldehyde content of wines (Manzanares et al., 2011). In addition, it plays
an important role in the flavour and bouquet of wine but
not always desirable (Lee et al., 2010). Because, at higher
concentrations this turns into a pungent irritating odor reminiscent of green grass or apples (Styger et al., 2011). In
present study, the lowest concentration of acetaldehyde were
found in control sample as 20.78 mg/L. However, in mixed
cultures, its concentration increased with increasing temperature and inoculation ratio. Smogrovicova and Domeny
(1999) reported that acetaldehyde concentration increased
as the temperature increased in beers. Controversial results

H. Tanguler

were reported with the effect of temperature on acetaldehyde

production in wines by Erten (2002), Torija et al. (2003) and
Reddy and Reddy (2011). On the other hand, in this study, its
value ranged from 21.47 to 56.47 mg/L in wines produced
with mixed cultures and it was found that fermentation
temperature and inoculation ratio significantly affected to
acetaldehyde concentration (P < 0.001). The acetaldehyde
concentration in wines usually ranges from 13 to 40 mg/l,
but may reach 75 mg/L (Reddy et al., 2008; Kourkoutas et
al., 2001). The results obtained in this study are in agreement
with the results given by Kourkoutas et al. (2001) and Reddy
et al. (2008). The lowest acetone value was determined in
control sample as 0.77 mg/L. Acetone concentration were
increased by increasing temperature and inoculum ratio in
mixed cultures and its value ranged from 0.98 to 1.57 mg/L,
whereas, butanedione and pentanedione amounts were decreased (except W6 for butanedione and except W1 for pentanedione). The effect of temperature and inoculation ratio
on the amounts of these compounds were found important (P
< 0.001).
Conclusions
This is the first study that effect of fermentation temperature at 12, 18 and 24 on wine fermentation by adding
W. saturnus var. saturnus together with S. cerevisiae was
evaluated based on fermentation rate, duration, yeast growth,
wine composition and volatile compounds.
Fermentations performed at lower temperature were
found longer; and lag phase which is a desired trait of wine
yeast was seen only at 12. The increase in temperature
from 12 to 24 resulted in a decrease in ethanol, malic
and citric acid concentrations, but an increase in glycerol,
total acidity, acetic and tartaric acid concentrations at both
inoculation ratios. Fermentation temperature affected also
to volatile compounds. Higher amounts of esters, especially
isoamyl acetate, ethyl acetate, isobutyl acetate, ethyl butyrate
and ethyl hexanoate, and lower amounts of 2-methyl butanol, propan-1-ol, isobutanol, acetaldehyde and acetone were
formed at low temperature.
The results given in this study show the importance of
temperature and inoculation ratio. Fermentations at 12
at two inoculation ratios appear to be more suitable options
than fermentation conducted 18 and 24. On the other
hand, it could be said that W. saturnus var. saturnus can be
used in mixed starter cultures with S. cerevisiae. Regarding
on wine production with W. saturnus var. saturnus, research
is lacking with respect to: (1) application of multistarter fermentations with the other non-Saccharomyces yeasts; (2) sequential inoculation of W. saturnus; (3) determination of bioactive compounds; (4) comparison of the glucophylic or/and

Effect of Temperature and W. saturnus on Wine

fructophylic nature of W. saturnus; (5) determination of the

effect of oxygen, lipid, and complex yeast nutrient supplementation on the volatile composition and (6) evaluation of
organoleptic quality of wines. Therefore, more research is
needed for concerning these subjects.

791
Comitini, F., Gobbi, M., Domizio, P., Romani, C., Lencioni, L.,
Mannazzu, I. and Ciani, M. (2011). Selected non-Saccharomyces
wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiol., 28, 873-882.
Cortes, S., Salgado, J.M., Rivas, B., Torrado, A.M. and Dominguez,
J.M. (2010). Fermentation kinetics and chemical characterisa-

Acknowledgements

The author is grateful to Prof. Huseyin

ERTEN and Niyazi CETINKAYA for critical reading and Dr. Adnan BOZDOGAN for his assistance in statistical analysis.

tion of Vino tostado, A traditional sweet wine from galicia (NW

Spain). J. Sci. Food Agric., 90, 121-131.
Degre, R. (1993). Selection and commercial cultivation of wine
yeast and bacteria, In Wine Microbiology and Biotechnology,

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