International Journal of Food Microbiology 130 (2009) 179–187

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

Molecular identification and osmotolerant profile of wine yeasts that ferment a high
sugar grape must
Rosanna Tofalo a, Clemencia Chaves-López a, Federico Di Fabio a, Maria Schirone a, Giovanna E. Felis b,
Sandra Torriani b, Antonello Paparella a, Giovanna Suzzi a,⁎
a
Dipartimento di Scienze degli Alimenti, Università degli Studi di Teramo, Via C.R. Lerici 1, 64023 Mosciano Sant'Angelo, Teramo, Italy
b
Dipartimento di Scienze, Tecnologie e Mercati della Vite e del Vino, Università degli Studi di Verona, Via delle Pieve 70, 37029 San Floriano, Verona, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to examine the Saccharomyces and non-Saccharomyces yeast populations involved
Received 17 October 2008 in a spontaneous fermentation of a traditional high sugar must (Vino cotto) produced in central Italy. Molecular
Received in revised form 20 January 2009 identification of a total of 78 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and
Accepted 21 January 2009
sequencing of the D1/D2 domain of the 26S rRNA gene. In addition, the isolates were differentiated by RAPD-PCR.
Only a restricted number of osmotolerant yeast species, i.e. Candida apicola, Candida zemplinina and Zygosac-
Keywords:
Osmotolerance
charomyces bailii, were found throughout all the fermentation process, while Saccharomyces cerevisiae prevailed
Yeasts after 15 days of fermentation. A physiological characterization of isolates was performed in relation to the
Ethanol tolerance resistance to osmotic stress and ethanol concentration. The osmotolerant features of C. apicola, C. zemplinina and
Vino cotto Z. bailii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different
glucose concentrations (2%, 20%, 40% and 60% w/v). The ability of some C. apicola and C. zemplinina strains to
grow at 14% v/v ethanol is noteworthy. The finding that some yeast biotypes with higher multiple stress tolerance
can persist in the entire winemaking process suggests possible future candidates as starter for Vino cotto
production.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction Traditional Balsamic Vinegar, another Italian traditional product made

“Vino cotto” is a wine produced in the Marche and Abruzzo regions
(central Italy) according to traditional procedures that involve a
prolonged fermentation of cooked grape must. The flow-chart for the
traditional manufacture of Vino cotto is showed in Fig. 1. Cooked must
derives from white grapes of the Trebbiano, Passerina or Moscato
cultivars and is obtained by direct heating of the must in copper
boilers until its volume is reduced by 10–70%, according to the specific
process. During cooking, the must becomes dark and dense, and
reaching a very high sugar concentration (up to 55% w/v) (Di Mattia
et al., 2007; Piva et al., 2008). Cooking is carried out below boiling
temperatures (80–95 °C), and requires long processing times (up to
48 h). To start fermentation, fresh must is added to sterile cooked
must, at percentages that depend on the different manufacturing
practices. The indigenous yeasts of the added fresh must conduct the
alcoholic fermentation that proceeds slowly for about 40 days at room
temperature. Stuck or sluggish fermentations often occur during the
production of Vino cotto, as observed during the fermentation of
⁎ Corresponding author. Dipartimento di Scienze degli Alimenti, via C.R. Lerici 1,
64023 Mosciano S. Angelo (TE), Italy. Tel.: +39 0861 266938; fax: +39 0861 266915.
E-mail address: gsuzzi@unite.it (G. Suzzi).
with cooked grape must (Solieri and Giudici, 2005, 2008; Solieri et al.,
2006). These problems derive from the exposure of yeast cells to high
osmotic conditions of cooked must (hyperosmotic shock) that
determine a rapid loss of intracellular water (Hohmann, 2002),
followed by cytoskeleton collapse (Chowdhury et al., 1992), intracel-
lular damage and subsequent arrest of growth. The majority of yeasts
requires a minimum water activity (aw) of 0.85 for their growth, and
requires only xerophilic yeasts can grow, at slow rate, at aw values
between 0.61 and 0.75 (Martorell et al., 2005; Solieri and Giudici,
2008). In general, osmotolerant yeasts are able to retain the ability to
synthesize glycerol as a compatible solute or osmoregulator, and some
yeasts even have active glycerol uptake pumps (Hohmann, 2002).
There are only few studies dealing on the yeast population involved
in the fermentation of cooked grape musts. Recently, Solieri et al. (2006)
found a complex yeast population in Traditional Balsamic Vinegar, that
includes S. cerevisiae, several Zygosaccharomyces species, two species
belonging to the Hanseniaspora genus (Hanseniaspora osmophila and
Hanseniaspora valbyensis), and two Candida species (Candida stellata
and Candida lactis-condensi). Also in high sugar grape musts, such as
special wines from dried, botrytized grapes or late-harvest grapes,
osmotolerant non-Saccharomyces yeasts are commonly found (Mills
et al., 2002). Candida zemplinina has been recently identified as a
new osmotolerant and psychrotolerant yeast, fermenting sweet and

0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2009.01.024
180 R. Tofalo et al. / International Journal of Food Microbiology 130 (2009) 179–187
Fig. 1. Vino cotto flow chart.
botrytized musts (Sipiczki, 2003). Various authors have mentioned that
indigenous yeast species, such as Kloeckera apiculata, C. stellata and
Torulaspora delbrueckii, may have better ability than S. cerevisiae to grow
during fermentations conducted at high sugar concentrations (e.g.,
N200 g/L) (Benda, 1982; Lafon-Lafourcade,1983). Malacrinò et al. (2005)
reported that strains of osmosensitive species, such as S. cerevisiae, may
possess appreciable capability to overcome osmotic stress and to yield
ethanol by fermentation of grape musts with high sugar concentration
in winemaking conditions. These authors suggested to use these strains
as starter for winemaking of partially dried grapes.
Very little information is available about the yeast microbiota of Vino
cotto, as previous researches on this peculiar wine have been focused
more on the technological aspects of the winemaking process. There-
fore, the aims of this study were to monitor the Saccharomyces and non-
Saccharomyces yeast populations during Vino cotto manufacturing and
to characterize the yeast isolates for their tolerance to osmotic stress and
ethanol. Initially, yeast isolates were presumptively identified using
morphological identification on Wallerstein Laboratory Nutrient (WLN)
agar and speciation of the new isolates was carried out by applying two
well-recognized yeast identification procedures based on ribosomal
RNA (rRNA) gene analysis, i.e. restriction fragment length polymorphism
(RFLP) analysis of the ITS1–ITS2 region (Esteve-Zarzoso et al., 1999) and
sequencing of the genes encoding the D1/D2 domain of the large (26S)
subunit of rRNA (Kurtzman and Robnett, 1998). A combination of the
two procedures was required, since the fast and not expensive ITS-RFLP
approach is not as discriminatory as 26S rRNA gene sequence (Arias
et al., 2002). In addition, differentiation of the isolates at the subspecies
level was acquired by applying the random amplification of polymorphic
DNA (RAPD)-PCR technique. This whole genome PCR sampling method
works by priming at arbitrary sites and results in strain-specific
fingerprints from which differentiation of strains belonging to different
Table 1
Chemical composition of the musts and Vino cotto
Sample Reducing sugars Tartaric acid
(g/L)a (g/L)
Fresh Trebbiano mustc 172.5 ± 21.4 4.81 ± 0.08
Cooked must 554.5 ± 28.5 13.32 ± 0.35
Vino cotto mustd 304.5 ± 18.7 7.35 ± 0.21
Vino cottoe 64.5 ± 2.21 3.61 ± 0.11
a
Data are the means of 3 repetitions with the relative standard deviations.
b
GAE: galic acid equivalent.
c
Trebbiano d'Abruzzo must.
d
Cooked must added with fresh must.
e
Vino cotto must after 40 days of fermentation.
yeast species was possible (Quesada and Cenis, 1995; Torriani et al.,
1999; Bujdoso et al., 2001; Urso et al., 2008). From the results obtained in
the present study, it is anticipated that the traditional and region-
dependent handling makes Vino cotto a wide genetic source of diverse
yeast species and strains suitable for following starter selection
programmes.
2. Materials and methods
2.1. Wine production
Cooked must was prepared according to Piva et al. (2008). White
grapes of Trebbiano d'Abruzzo were removed from stalks and pressed.
The fresh Trebbiano must (FTM) (100 L) was concentrated in copper
boilers of 120 L capacity to about 70%. The masses were rapidly heated
to boiling point, then the temperature was set at 95 °C for the whole
process (up to 18 h).
2.2. Analytical determinations
Chemical analyses were conducted using the official EU Commu-
nity methods for the analyses of wines (EEC, 1990). The total
polyphenols were determined by the colorimetric reaction with the
Folin Ciocalteau reagent as reported by Di Mattia et al. (2007).
2.3. Fermentation characteristics and yeast population dynamics
Vino cotto must (VCM), obtained by mixing 70% cooked must (CM)
added with 30% FTM, was spontaneously fermented by indigenous yeasts
in tanks (120 L) at room temperature (approximately 18 °C) for 45 days.
Samples were taken from FTM, VCM and during the fermentation
process. For all samples, decimal dilutions in sterile physiological solution
(NaCl 8.5 g/L) were made and 0.1 mL of each dilution was spread on three
different media: Wallerstein Laboratory Nutrient agar (WLN; Oxoid,
Milan, Italy), Lysine-medium (LM; Oxoid) and Yeast Peptone Dextrose
agar (YPD; 1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose and 2%
w/v agar). All media were supplemented with chloramphenicol
(150 ppm). Plates were incubated at 25 °C for 3–5 days. According to
Pallman et al. (2001), the WLN medium plating was used to monitor
yeast population diversity during fermentations. The colony morpho-
logies are characteristic enough to identify the corresponding yeast's
genus and species. After counting, a total of 20 colonies of yeasts with
different colour and morphology was isolated from WLN plates. The
isolates were purified by repetitive streaking on YPD and then stored
at −20 °C in YPD broth supplemented with glycerol (25% final
concentration).
2.4. Molecular identification of the isolates
Yeast cells were grown aerobically in YPD at 28 °C. DNA was
isolated according to Querol et al. (1992). The 5.8 internal transcribed
Total acidity Volatile acidity Ethanol (%) pH Total phenolics
(g/L of tartaric acid) (g/L of acetic acid) (ppm GAEb)
7.41 ± 0.28 – – 3.08 ± 0.03 348 ± 12.34
17.64 ± 0.15 – – 2.75 ± 0.02 1754 ± 56.72
9.42 ± 0.26 – – 2.94 ± 0.02 1200 ± 31.21
8.59 ± 0.37 0.76 ± 0.05 15.35 ± 0.27 3.03 ± 0.02 569 ± 16.28
 R. Tofalo et al. / International Journal of Food Microbiology 130 (2009) 179–187 181

Fig. 2. Fermentation kinetics of Vino cotto must. The results are reported as mean of 3 repetitions; the coefficient of variability was lower than 10%.

spacer (ITS) rRNA region was amplified in a Bio-Rad thermocycler
(MyCycler, Bio-Rad Laboratories, Milan, Italy) using ITS1 and ITS4
primers as described previously (Esteve-Zarzoso et al., 1999). The
amplified DNA was digested with the restriction endonucleases HinfI,
Cfo I, HaeIII and MboI (Roche Diagnostics, Mannheim, Germany),
according to the supplier's instructions. The PCR products and their
corresponding restriction fragments were separated in 1.5% and 2%
agarose gels, respectively, in 1× TAE (40 mM Tris–acetate, 1 mM EDTA,
pH 8.2) buffer. After electrophoresis, gels were stained with ethidium
bromide, and documented by the Gel Doc 2000 (Bio-Rad, Milan, Italy).
2.5. Sequencing, sequence comparison and phylogenetic analysis
Amplification of the D1/D2 domain of the 26S rRNA gene was carried
out using NL1 and NL4 primers as described by Kurtzman and Robnett
(1998). PCR products were gel-purified with GFX™ PCR DNA and Gel
Band Purification Kit (Amersham Biosciences AB, Uppsala, Sweden),
according to the manufacturer's instructions and delivered to Centro
Ricerca Interdipartimentale Biotecnologie Innovative (Padua University,
Padua, Italy) for sequencing. The sequences obtained in FASTA format
were compared with those deposited in GenBank DNA database (http://
www.ncbi.nlm.nih.gov/) using the basic Blast search tools (Altschult
et al., 1997). The D1/D2 sequences analysed in this study were deposited
in the EMBL Nucleotide sequence Database under the accession
numbers C. apicola (FM201316); C. zemplinina (FM201317),
(FM201318); S. cerevisiae (FM201319); Z. bailii (FM201320).
Alignment of sequences obtained for the isolates and reference
sequences retrieved from the database was performed with CLUSTALX
(Thompson et al., 1997). Webcutter 2.0 programme, available at http://
rna.lundberg.gu.se/cutter2/, was used to predict restriction sites in ITS
molecular sequences.
coefficient, and correlation coefficients were calculated by the
unweighted pair group method with arithmetic averages (UPGMA).
2.7. Frequency percentage analysis
Colonies randomly collected from plates at the highest dilution give a
high probability to pick up strains belonging to the dominant species
(Pulvirenti et al., 2004). The method proposed by Solieri et al. (2006) was
applied to study the species distribution in the analysed samples. It was
considered how many times each species was detected in the samples,
without considering the strain number belonging to the species. In this
way it was obtained the number of positive samples to each species and
the corresponding frequency, defined as the number of samples positive
to a species divided by the total number of samples expressed in
percentage.
2.8. Sugar and ethanol tolerance
Overnight cultures of yeasts were centrifuged (3000 ×g) at 4 °C for
10 min and cells were washed twice with potassium phosphate buffer
(pH 7.4). Then, cells were suspended in the same buffer to give a
concentration of about 106 CFU/mL. An aliquot of 20 μL was inoculated
into Bioscreen C (M-Medical S.r.l., Italy) plates containing 180 μL of YPD
broth with 150 ppm of chloramphenicol and supplemented with 0
(control), 8, 14, or 20% v/v ethanol or 2, 20, 40, 60% w/v glucose and
incubated at 25 °C for 100 h. The increase in cell number was determined
by measuring the optical density (O.D.) of cultures at 600 nm.

2.6. RAPD-PCR analysis

Table 2
Viable counts during Vino cotto production
The M13 primer (5′-GAG GGT GGC GGT TCT-3′) was used in RAPD-
Counts (log CFU/mL) ona
PCR amplifications. Reactions were carried out in 25 μL reaction mix
YPD WLN LM
containing 2.5 μL of 10× PCR buffer (Invitrogen), 1.5 mM MgCl2,
Fresh Trebbiano must 6.11 ± 0.12 6.25 ± 0.35 6.30 ± 0.20
200 μM of each dNTP, 20 pmol of primer, 1 U of Taq polymerase and
Vino cotto must (VCM) 6.30 ± 0.24 6.55 ± 0.20 6.51 ± 0.45
20 ng of extracted DNA. RAPD-PCR products were visualized on a 1.5% VCM during fermentation 8.20 ± 0.15 8.20 ± 0.25 8.31 ± 0.20
agarose gel after staining with ethidium bromide and acquired using Vino cotto 5.15 ± 0.11 5.19 ± 0.17 5.26 ± 0.25
the Gel Doc 2000. Conversion, normalization and analysis of RAPD- YPD: yeast peptone dextrose agar. WLN: Wallerstein Laboratory Nutrient agar; LM,
PCR profiles were carried out using Fingerprinting II Informatix™ Lysine medium.
Software (Bio-Rad). Similarities of bands were analysed using Pearson a
The results are the means of 3 repetitions with the respective standard deviations.
182 R. Tofalo et al. / International Journal of Food Microbiology 130 (2009) 179–187

Table 3
Sizes of the 5.8S ITS rRNA gene amplicons and the restriction fragments of the yeast isolates

Profile No. of PCR product Restriction fragments (bp) with: Species

isolates (bp) Cfo I Hae III Hinf I MboI
I 31 834 345 + 365 + 100 318 + 231 + 171 + 130 367 + 128 S. cerevisiae
II 21 509 207 + 187 + 77 400 + 100 210 + 103 C. apicola
III 15 473 208 + 110 + 77 448 215 + 215 290 + 90 C. zemplinina
IV 11a 796 342 + 232 + 143 700 + 90 329 + 220 Z. bailii

Identification of these isolates was based on sequence analysis of the D1/D2 domain that revealed 100% identity with the species Z. bailii.
a
The RFLP pattern of these isolates did not match the data reported in literature (Solieri et al., 2006; Esteve-Zarzoso et al., 1999).

In order to evaluate yeast growth, the values were analysed over time subjected to Student's t test to identify significant differences between
according to the Gompertz equation modified by Zwietering et al. yeast species using STATISTICA package (StatSoft Inc., Tulsa, OK, USA).
(1990):
3. Results
n h
μ d e io
max
y = Ad exp − exp d ðλ − t Þ + 1
A 3.1. Chemical determinations

where y is O.D. value at time t (h), A represents the maximum O.D.
(when t → ∞), μmax is the maximum specific growth rate (as h− 1), and
λ is the lag time (h) for O.D. increase. For the modelling with
Gompertz equation, means of three replicates and two repetitions
were used. In all the cases, the variability coefficient of raw data (cell
load as O.D.) was b5%. The data relative to the growth kinetics were
Vino cotto represents a very peculiar environment, as can be
observed from the chemical parameters of the samples analysed
(Table 1). The fermentation of Vino cotto must started after 6 days
from the fresh Trebbiano must addition to cooked must, and was
carried out slowly for 45 days (Fig. 2), due to the high content of sugars
(304.5 g/L) and polyphenols (1200 ppm). At the end of the

Fig. 3. Banding patterns were clustered using the UPGMA, with correlation levels expressed as percentage values of Pearson correlation coefficients. Yeasts isolated at different times
are indicated as follows: normal character, Fresh Trebbiano must; italics, Vino cotto must; underlined, Vino cotto must after 15 days; bold, Vino cotto must after 45 days of
fermentation. Species validation of representative strains (⁎) of each cluster included D1/D2 domain sequence analysis.
 R. Tofalo et al. / International Journal of Food Microbiology 130 (2009) 179–187 183

sequence database. All the sequences obtained displayed similarity

values ranging from 99 to 100% to reference sequences, confirming the
identity of isolates with profiles I, II, and III as S. cerevisiae, C. apicola
and C. zemplinina, respectively, and allowing the inclusion of the
isolates with profile IV into the species Zygosaccharomyces bailii (data
not shown).
Isolates were further characterized genomically with RAPD-PCR, to
obtain information on the genomic relatedness of isolates and to
determine the probable number of the different yeast strains present
in the samples. Indeed, RAPD-PCR has proved to be an informative
method suitable for the study of a large number of strains in a short
time and has been used successfully for identification and intraspecific
differentiation of Saccharomyces and non-Saccharomyces yeast species
(Quesada and Cenis, 1995; Torriani et al., 1999; Bujdoso et al., 2001;
Urso et al., 2008).
Fig. 4. Evolution of yeasts during Vino cotto production. The dendrogram based on the numerical analyses of generated
digitized M13 PCR fingerprints is shown in Fig. 3. Four main clusters
were obtained, which corresponded to the species previously identified.
fermentation, the average of alcoholic content was 153.5 g/L and the Considering 95% similarity as the arbitrary threshold to define biotypes,
reducing sugars 64.50 g/L, confirming previous observations (Di it can be easily observed that 6 biotypes of C. apicola, 13 of S. cerevisiae, 6
Mattia et al., 2007). of C. zemplinina, and 4 of Z. bailii were distinguishable. Moreover, as the
isolates were collected at different stages of fermentation, the RAPD-PCR
3.2. Enumeration of yeast populations analysis allowed the observation of microbiota evolution during
winemaking.
To study the succession of yeast populations during the Vino cotto As regards S. cerevisiae, an ample genetic polymorphism was
manufacturing, yeasts were detected from FTM, VCM, during and at observed: three groups can be easily discerned with relatively low
the end of the fermentation process. WLN agar proved extremely genomic relatedness (less then 75%). In particular, subcluster I contained
useful in the isolation and initial presumptive identification of almost all of the strains isolated from fresh Trebbiano must at the
different yeast species from the samples (Table 2). At the beginning beginning of the fermentation, while subclusters II and III contained
of the spontaneous fermentation, Vino cotto must exhibited a total almost all the isolates from Vino cotto must after 15 days of
viable count on YPD medium of 6.30 log CFU/mL, and a maximum fermentation. Interestingly, F25, F66, and F288 isolates, constituting a
number of 8.2 log CFU/mL during fermentation, while at the late single biotype in cluster I, were isolated in FTM (F25) and VCM after
stages of the process the viable cells decreased to 5.15 log CFU/mL. 45 days of fermentation, thus indicating that this strain was able to
Counts on WLN and on LM showed a similar trend to that detected on persist during all the winemaking process. Further, different strains
YPD medium. were present at the same stage of fermentation, indicating a multistrain
process.
3.3. Identification and molecular characterization of yeasts strains from Considering the other three species which were found, a remark-
Vino cotto able biodiversity was observed for C. zemplinina, C. apicola, and Z.
bailii strains. C. apicola strains were present at all stages of
A total of 78 yeasts were isolated from the samples collected during fermentation, but apparently there was no persistence of a single
Vino cotto production, after boiling and addition of FTM, in order to
focus on the most probably osmotolerant species. Molecular character-
ization was carried out using a combination of different techniques to Table 4
identify and gain information on genomic relatedness among isolates. Growth parameters of the yeast isolates from Vino cotto at different glucose concentrations
Identification was performed by amplifying the 5.8-ITS rRNA, and
Glucose No. of Species Growth parameters°
subsequent restriction analysis was carried out with Cfo I, Hae III and (% w/v) isolates Amax (O.D) μmax (h− 1) λ (h)
Hinf I endonucleases (Esteve-Zarzoso et al., 1999). The results of PCR-
2 6⁎/6⁎⁎ C. apicola 1.53 ± 0.19a 0.05 ± 0.01a 2.66 ± 0.77a
RFLP analysis of the 5.8-ITS rRNA are reported in Table 3. Four different
6/6 C. zemplinina 1.44 ± 0.06b 0.06 ± 0.01a 1.21 ± 0.41bc
profiles, named from I to IV, were generated, and were compared to the 13/13 S. cerevisiae 1.65 ± 0.17c 0.04 ± 0.02ab 6.76 ± 13.54ab
data set previously described (Esteve-Zarzoso et al., 1999; Villa-Carvajal 4/4 Z. bailii 1.67 ± 0.05c 0.05 ± 0.01a 4.30 ± 0.98ac
et al., 2006; de Llanos Frutos et al., 2004). Thirty-three isolates showed 20 6/6 C. apicola 1.35 ± 0.08a 0.09 ± 0.01a 1.16 ± 0.11a
the restriction profile named I, coresponding to the profile of S. cerevisiae 6/6 C. zemplinina 1.47 ± 0.05b 0.12 ± 0.01b 1.63 ± 0.15b
CBS 1171T; therefore, these isolates were assigned to this species. The 13/13 S. cerevisiae 1.45 ± 0.11b 0.12 ± 0.02b 5.59 ± 13.25abc
4/4 Z. bailii 1.35 ± 0.05a 0.10 ± 0.01a 2.57 ± 0.50c
isolates with profile II showed a pattern similar to the one reported in
40 6/6 C. apicola 1.11 ± 0.01a 0.02 ± 0.01a 6.24 ± 0.84a
literature for Candida apicola (de Llanos Frutos et al., 2004). Profile III
6/6 C. zemplinina 1.15 ± 0.08a 0.03 ± 0.01b 6.72 ± 0.75a
corresponded to the pattern described for the species C. zemplinina and 13/13 S. cerevisiae 1.37 ± 0.06b 0.03 ± 0.01b 9.34 ± 3.50b
C. stellata by Sipiczki (2003); after the additional restriction with MboI, 4/4 Z. bailii 1.33 ± 0.07 b 0.03 ± 0.01b 8.26 ± 0.72b
the isolates generated two fragments of 290 and 90 bp that are typical of 60 2/6 C. apicola 0.60 ± 0.12a 0.008 ± 0.002a 15.13 ± 2.33a
C. zemplinina. Sequence analysis of ITS1-5.8S rDNA-ITS2 region of 1/6 C. zemplinina 0.44b 0.018b 9.35b
selected isolates confirmed the presence and positions of predicted 0/13 S. cerevisiae – – –
0/4 Z. bailii – – –
restriction sites. Isolates with profile IV showed a particular RFLP pattern
°
that did not match the data reported in literature (Solieri et al., 2006; Results are the means of 3 replicates for 2 repetitions; standard deviations are also
Esteve-Zarzoso et al., 1999). indicated; Amax maximum abs; μmax: maximum growth rate; λ: length of lag. –: no
growth.
To achieve a certain identification of the yeasts, the D1/D2 domain Means within a column in the same block with different letters are significantly
of the 26S rRNA gene from two isolates of each 5.8S-ITS profile was different (P b 0.05).
sequenced and compared with those available in the EMBL nucleotide ⁎: positive isolates. ⁎⁎: total isolates.
184 R. Tofalo et al. / International Journal of Food Microbiology 130 (2009) 179–187
genotype. On the contrary, and similarly to what previously observed
for S. cerevisiae, a biotype of C. zemplinina (isolates F36, F365, and
F479) was detected in all phases of production.
Interestingly, the predominant species during fermentation were
very different from those observed in FTM, before its addition to CM:
according to the counts obtained in WLN medium, in FTM the
S. cerevisiae yeast population was only 32%, of total population while
68% was formed by non-Saccharomyces wine yeasts belonging to the
genera Hanseniaspora, Kloeckera, Metschnikowia and Candida (data not
shown). The evolution of the species S. cerevisiae, C. apicola and Z. bailii
during Vino cotto production was determined also by plate count
(Fig. 4): in Vino cotto must, before the start of fermentation, only the
osmotolerant species C. apicola, and Z. bailii were isolated, whereas
yeasts belonging to the species S. cerevisiae and other non-Saccharo-
myces wine yeasts were not found. During fermentation S. cerevisiae was
the prevalent yeast, whereas at the end only few strains, belonging to
this species, were isolated. C. apicola was the dominant species at the
Fig. 5. Patterns of S. cerevisiae growth in responses to different glucose and ethanol concentrations. Glucose (w/v): (♦) 2%; (■) 20%; (□) 40%; (x) 60%. Ethanol (v/v): (–□–) 0%; (x)
8%; (- - -) 14%; (▬) 20%.
start and at the end of fermentation (Fig. 4). Solieri et al. (2006)
suggested that yeasts present in cooked must depend on the stochastic
contamination events that occur after cooking. In Vino cotto manufac-
turing the addition of fresh must contributes to provide the yeast
inoculum. Although S. cerevisiae was not isolated after Vino cotto must
blending, it performed the fermentation. Vino cotto appeared a stressful
environment for the survival of the majority of the primary contaminant
yeasts, so few species can grow and at the end C. apicola was the
prevailing species, followed by Z. bailii, C. zemplinina and S. cerevisiae.
3.4. Phenotypes of isolates
All the isolates obtained in the present study, belonging to the
species C. zemplinina, C. apicola, Z. bailii and S. cerevisiae, were tested
for growth at various concentrations of glucose (2, 20, 40, 60% w/v)
and ethanol (0, 8, 14, 20% v/v). These analyses were performed on all
the isolates, in order to verify if the 95% similarity level could be
 R. Tofalo et al. / International Journal of Food Microbiology 130 (2009) 179–187 185

effectively chosen as threshold for strain delineation. In other words, if and C. zemplinina, growing at high glucose concentrations, showed to
isolates presumptively constituting a single biotype display the same possess more chances that S. cerevisiae and Z. bailii to grow in VCM.
characteristics, then they can be considered a single strain, otherwise Considering ethanol resistance, as expected, all the S. cerevisiae
they have to be considered different strains even if genomic similarity strains grew at 8% ethanol, and several isolates also at 14% ethanol, as
is high, and the 95% chosen as threshold must be critically re- shown in Fig. 5. C. apicola strains developed at 8% ethanol, with strain-
evaluated and increased. specific growth curves; only three isolates grew slowly at 14%. A
Data relative to O.D. of three replications for each isolate were similar behaviour was observed for C. zemplinina, and three strains
analysed according to the modified Gompertz equation. The predicted grew at 14% ethanol. As a general trend, ethanol affected yeast growth
curves fitted well with the experimental points and their regression by increasing the lag phase, decreasing the growth rate and biomass
coefficients ranged between 0.95 and 0.98. A perfect correlation yield (Amax) (Table 5). All isolates had a similar µmax decrease starting
between genomic relatedness and phenotypic traits was observed, at 8% ethanol, while a relevant decrease of Amax was observed at 14%
therefore we validated the 95% similarity of RAPD-PCR profile as ethanol for all isolates except S. cerevisiae.
threshold to delineate biotypes or strains.
All the strains belonging to the three osmotolerant species 4. Discussion
C. zemplinina, C. apicola, and Z. bailii grew in the media supplemented
with 2, 20, 40% w/v glucose, and most of them grew faster in the During Vino cotto production, must cooking determines the
medium with 20% glucose than in that with 2% (Table 4). Glucose at concentration of naturally occurring chemicals in must among which
60% inhibited many isolates and only two strains of C. apicola and one are sugars, acids, polyphenols, metal ions, and the formation of Maillard
of C. zemplinina showed some growth. reaction products (Piva et al., 2008). The main objective of this study was
As shown in Fig. 5, S. cerevisiae strains had three patterns of growth to isolate, identify and characterize the predominant indigenous yeast
in response to different glucose concentrations (2%, 20%, 40% w/v), species and strains during Vino cotto production. Only four species were
that we called growth types A, B and C. Type A strains grew better in identified: S. cerevisiae, C. apicola, C. zemplinina and Z. bailii.
the medium added with 2% glucose, with a decreased growth in media As expected, C. apicola, C. zemplinina and Z. bailii showed osmoto-
with higher sugar concentrations. Preferred condition for growth was lerant characteristics, but also some strains of the species S. cerevisiae,
medium with 20% glucose for type B and type C strains, but they could that is not generally considered an osmotolerant yeast, showed this
be differentiated on the basis of the second preferred condition: characteristic. In fact, among the strains of S. cerevisiae isolated from
medium with 2% added glucose for type B strains and medium with Vino cotto, the sugar consumption was different with three different
40% glucose for type C strains. growth kinetics (Fig. 5) in the presence of 2, 20, 40, and 60% glucose.
C. apicola, C. zemplinina, and Z. bailii strains displayed growth Among the studied species, a general decline in O.D. was observed as the
curves similar to S. cerevisiae type B strains, i.e. best growth in glucose increased from 200 to 600 g/L; such a trend might be attributed
medium with 20% glucose, and 2% sugar concentration as second to a slower proliferation of yeast cells due to the osmotic effect generated
preferred condition. Table 4 shows reports the average of the lag by high glucose concentrations (Thomas and Ingledew, 1990; Zhao and
phase, the growth rate and maximum cell biomass of the strains, Lin, 2003). Strains of the species Z. bailii are known to possess unusual
growing at different glucose concentrations. As glucose concentration physiological characteristics, such as resistance to weak-acid preserva-
increased, a general decrease in Amax was observed for all the isolates. tives and extreme osmotolerance up to 4 M glucose (72% w/v) (Martorell
C. apicola and C. zemplinina, characterized by a minor Amax overall at et al., 2007). In this study, the Z. bailii strains from Vino cotto exhibited
2% and 40% glucose, had a shorter lag phase than Z. bailii and S. lower growth rates at high sugar concentrations than C. apicola and C.
cerevisiae, and few isolates showed some growth even in the presence zemplinina, and had growth kinetics like those of S. cerevisiae type B
of 60% glucose. On the contrary, S. cerevisiae and Z. bailii had always strains, that grew faster at 20% glucose than at 2%. As regards C.
the major lag phase extension and very low O.D. values at 60% glucose zemplinina, Sipiczki (2003) reported that isolates from sweet botrytized
that could not be modelled with the Gompertz equation. C. apicola wines were able to grow at 50% glucose and to have some growth even in
the presence of 60% glucose. Similar physiological characteristics were
detected in some of our strains of C. zemplinina, showing also the same
Table 5 osmotolerant growth kinetics of C. apicola and S. cerevisiae type B.
Growth parameters of yeast isolates from Vino cotto at different ethanol concentrations Among the non-Saccharomyces wine yeasts, K. apiculata and C. stellata
Ethanol No. of Species Growth parameters°
have been reported to possess a better ability than S. cerevisiae to grow
(% v/v) isolates during fermentations conducted in a chemically defined grape juice
Amax (O.D) μmax(h− 1) λ (h)
medium with high sugar concentrations of more than 200 g/L
0 6⁎/6⁎⁎ C. apicola 1.38 ± 0.09a 0.10 ± 0.03a 1.49 ± 0.50a
6/6 C. zemplinina 1.32 ± 0.10a 0.14 ± 0.01a 3.07 ± 0.25b (Charoenchai et al., 1998). Our data, obtained in YPD medium, showed
13/13 S. cerevisiae 1.33 ± 0.09a 0.17 ± 0.01b 3.36 ± 0.18b that C. apicola and C. zemplinina can grow like or better than S. cerevisiae
4/4 Z. bailii 1.56 ± 0.05b 0.11 ± 0.02a 7.62 ± 1.42c in media with high sugar contents (Table 4), but they were not able to
8 6/6 C. apicola 0.93 ± 0.08a 0.05 ± 0.01a 3.44 ± 0.32a dominate fermentation (Fig. 4). In fact, S. cerevisiae had the higher A
6/6 C. zemplinina 0.98 ± 0.05a 0.06 ± 0.01a 4.15 ± 0.51b values up to 40 days. This finding might be attributed to elevated and
13/13 S. cerevisiae 1.01 ± 0.07a 0.08 ± 0.01b 3.76 ± 0.20ab
combined osmotic effects, due to the presence of high glucose
4/4 Z. bailii 0.93 ± 0.05a 0.06 ± 0.01a 4.58 ± 0.41b
concentration and the accumulation of ethanol during fermentation of
14 2/6 C. apicola 0.50 ± 0.09a 0.07 ± 0.01a 3.70 ± 0.17a
2/6 C. zemplinina 0.54 ± 0.08a 0.07 ± 0.01a 8.74 ± 0.21b
Vino cotto up to 15–16% (v/v).
8/13 S. cerevisiae 0.91 ± 0.11b 0.07 ± 0.02a 10.33 ± 1.34c As expected, S. cerevisiae was the most ethanol-tolerant species, with
0/11 Z. bailii – – – some differences depending on the strain; in fact, only eight strains were
20 0/6 C. apicola – – – able to grow at 14% of ethanol with different but high cell yield. On the
0/6 C. zemplinina – – – other hand, Z. bailii was the most sensitive species. It is well known (Fleet
0/13 S. cerevisiae – – –
and Heard, 1993; Boulton et al., 1996; Cocolin et al., 2000) that non-
0/4 Z. bailii – – –
Saccharomyces wine yeasts are less tolerant to ethanol than Saccharo-
°
Results are the means of 3 replicates for 2 repetitions; standard deviations are also myces. The major target of ethanol is the plasma membrane, altering the
indicated; Amax maximum abs; μmax: maximum growth rate; λ: length of lag. –: no growth.
Means within a column in the same block with different letters are significantly
membrane organization and permeability (Alexandre et al., 1994;
different (P b 0.05). D'Amore et al., 1990), and consequently inhibiting glucose transport
⁎: positive isolates. ⁎⁎: total isolates. and fermentation rate under enological conditions (Ansanay-Galeote
186 R. Tofalo et al. / International Journal of Food Microbiology 130 (2009) 179–187
et al., 2001). Ethanol stress represents toxicity through intracellular ROS
generation in addition to deleterious damage to cell membrane and
functional proteins (Costa et al., 1997). Many ethanol protectants such as
trehalose and various plasma membrane components, including ergos-
terol, phosphatidylinositol, stearic acid, palmitic acid, as well as
phospholipid palmitoleic acid, in addition to inositol, proline and
tryptophan, might result in an even higher resistance to ethanol stress
(Mansure et al., 1997; Inoue et al., 2000; Furukawa et al., 2004; Takagi
et al., 2005, 2007; Lei et al., 2007; Hirasawa et al., 2007).
As regards the composition and dynamics of yeast populations
during the production of Vino cotto the 5.8S-ITS restriction profile of
all Z. bailii isolates was not in agreement with the data reported in
literature. Nevertheless, they were clearly identified by D1/D2 domain
sequencing. An unusual polymorphism for 5.8S-ITS DNA has already
been reported for other species. Fernandez-Espinar et al. (2000) and
Suzzi et al. (2006) described an atypical polymorphism for 5.8S-ITS
DNA for S. cerevisiae strains. Sequence divergence which can be
observed in ITS regions is usually higher than in ribosomal RNA
encoding genes; in fact, Solieri et al. (2006, 2008) reported similar
results for Z. rouxii and found a new putative species highly related to
Z. rouxii on the basis of ITS-region. Therefore, the sequence hetero-
geneity displayed by the Z. bailii isolates from Vino cotto may indicate
a taxonomic peculiarity, whose investigation was out of the aim of this
paper and will be deepened in a future study.
RAPD-PCR fingerprints of the isolates showed a different genetic
homogeneity within the four species (29 profiles for 78 isolates) but,
most importantly, they allowed to follow the dynamics of the
population, showing that only few biotypes were present during all
the process; in particular, only one S. cerevisiae strain was shown to be
present either in fresh Trebbiano must and at the end of fermentation,
with an intermediate osmotolerant behaviour. The relatively reduced
biodiversity in terms of yeast species and strains could be explained if we
consider that the selective pressure exerted by the environmental
conditions encountered by microbial cells during Vino cotto production,
such as high osmotic pressure and high concentration of some must
compounds, account for the consolidated dominance of selected species
of yeasts and strains.
In conclusion, the application of a multiphasic approach has lead
towards a better understanding of the microbial ecology of this
particular fermentation process. Indeed, the combination of micro-
biological, physiological and genetic analyses allowed to profile
population dynamics and to characterize single strains associated with
the fermentation of cooked musts. Nevertheless, further analysis are
required in future studies to discover the species and strains that give the
major contribution to the production and final quality of Vino cotto.
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