Food Research International 46 (2012) 22–29

Contents lists available at SciVerse ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano

d'Abruzzo red wine quality
Giovanna Suzzi, Giuseppe Arfelli, Maria Schirone, Aldo Corsetti, Giorgia Perpetuini, Rosanna Tofalo ⁎
Dipartimento di Scienze degli Alimenti, Università degli Studi di Teramo, Via C.R. Lerici 1, 64023 Mosciano Sant'Angelo, Teramo, Italy

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

Article history: The growth of selected, indigenous Saccharomyces cerevisiae added as starters (SRS1, MS72 and RT73) was
Received 20 July 2011 monitored during Montepulciano d'Abruzzo wine production. In all the fermentations the addition of the
Accepted 16 October 2011 starter, caused a decrease of the non-Saccharomyces yeasts. When strains MS72 and RT73 were used as
starters they were detected in the first phases of fermentations, while strain SRS1 competed successfully
Keywords:
with native yeasts during all the process. Wines obtained by fermentation with the indigenous starters
Saccharomyces cerevisiae
Native starter
showed some different characteristics, according to the chemical and sensory analyses. This study highlighted
PCR-DGGE that among selected starters with high fermentative capacity, some are able to dominate better than other natural
Wine flavor wine yeast biota, whereas some strains can interact and survive besides native yeast populations during the
fermentation. As a consequence, the dominance character can have a positive or negative effect on wine quality
and has to be considered in the frame of yeast selection in order to improve or characterize traditional wines.
Winemakers could choose among different degrees of yeast dominance to modulate the interaction among starter
and native wine yeast population.
© 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Traditionally, wine fermentation is carried out in a spontaneous
way by indigenous yeasts present on the grapes when harvested, or
introduced from the equipment and cellar during the vinification process
(Ciani, Mannazzu, Marinangeli, Clementi, & Martini, 2004; Mortimer &
Polsinelli, 1999). In the past years, strains of Saccharomyces cerevisiae
have been selected for their enological properties and have been
used as starters in winemaking to carry out alcoholic fermentation
with success. The native and starter strains of S. cerevisiae, involved in
fermentation, play an important role in the characteristics of wine
(King et al., 2008; Lambrechts & Pretorius, 2000; Thorhgate, 1998;
Vilanova & Sieiro, 2006). In fact the composition and the sensory quality
of the resulting wine are due to the diversity of S. cerevisiae strains and
to their dominance in fermentation (La Jeune, Enry, Demuyter, & Lollier,
2006; Torrens et al., 2008; Wondra & Boveric, 2001). Even if commercial
S. cerevisiae strains could be inoculated in grape must in order to establish
a high population and accomplish a well-controlled must fermentation,
in some cases, strains of S. cerevisiae inoculated as starter cultures were
not able to compete successfully with indigenous strains and, therefore,
do not dominate the fermentation as expected with important practical
consequences (Constantí, Poblet, Arola Mas, & Guillamón, 1997; Egli,
Edinger, Mitrakul, & Henick-Kling, 1998; Fleet, 2008; Ganga & Martinez,
2004; Gutierrez, Santamaria, Epifanio, Garijo, & Lopez, 1999; Querol,
⁎ Corresponding author. Tel.: + 39 0861 266911; fax: + 39 0861 266915.
E-mail address: rtofalo@unite.it (R. Tofalo).
Barrio, Huerta, & Ramon, 1992; Santamaria, Garijo, Lopez, Tenorio, &
Gutierrez, 2005). Many wine-researchers and winemakers prefer the
use of local, autochthonous, selected strains of S. cerevisiae as starters
(Martini & Vaughan-Martini, 1990). In fact active dried yeasts seem to
reduce the biodiversity of strains that perform natural fermentation,
and, as a consequence of this, to reduce the resulting wine complexity
(Frezier & Dubordieu, 1992). The native S. cerevisiae strains are better
acclimated to micro-area conditions of the wine production region
(Monk & Cowley, 1984) and therefore they can more easily dominate
on the natural biota (La Jeune et al., 2006).
Montepulciano d'Abruzzo is a native grape variety of Vitis vinifera
L., grown in central Italy and used for production of high quality red
wines. Limited studies have been carried out to improve its enological
characteristics through the use of indigenous strains of S. cerevisiae
(Suzzi et al., 2008). During 2006 a study on Montepulciano d'Abruzzo
“Colline Teramane DOCG” (Abruzzi Region, Italy) was started to select
autochthonous strains particularly useful to the specific fermentation
of musts produced in three defined homogenous winegrowing areas,
named A, B and C (Suzzi et al., 2008). The selection of these strains
was based on some desirable and traditional enological criteria,
such as high fermentation performance, low production of hydrogen
sulfide, resistance to ethanol and sulfur dioxide, among others. How-
ever, during must fermentation the presence of phenotypically dis-
tinct native yeast populations, perhaps reflecting an adaptation to
specific microenvironments, has very important implications for the
ecology and biotechnological use of autochthonous wild yeast strains,
as starters (Nadal, Colomer, & Pina, 1996). Although many producers
of red wine Montepulciano d'Abruzzo carry out spontaneous

0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.10.046
 G. Suzzi et al. / Food Research International 46 (2012) 22–29
fermentation of grape musts, commercial yeasts strains have been
used in the last decades as starters to reduce the risk of wine spoilage
and uniform wine quality. In particular, another aspect to be considered
is the determination of strain capacity to dominate natural Saccharomyces
populations, always present during must fermentation (Constantí et al.,
1997; Santamaria et al., 2005).
The aim of the present work was to monitor the succession of
yeast populations during Montepulciano d'Abruzzo fermentations in-
oculated with selected native S. cerevisiae strains from Teramo area
(Abruzzi Region, Italy). In the present study, the molecular detection
of yeasts was carried out directly from must or wine samples and, in
parallel, a culture-dependent approach was performed. In particular,
the ability of the inoculated strains to compete with native yeast
populations and conduct fermentation was evaluated by microsatel-
lite analyses. Furthermore, the impact of these strains competition
on chemical and aroma compounds was investigated.
2. Materials and methods
2.1. Yeasts and culture conditions
S. cerevisiae DBVPG 6171 T (Industrial Yeasts Collection of Peru-
gia, Italy) Saccharomyces bayanus DBVPG 6173 T, Saccharomyces
paradoxus DBVPG 6411 T, Saccharomyces pastorianus DBVPG 6047 T,
and Debaryomyces hansenii CBS 767 T, (Centraalbureau voor Schim-
melcultures, Baarn, The Netherlands) Hanseniaspora uvarum CBS
314 T were used. Three strains of S. cerevisiae (RT73, MS72 and
SRS1), belonging to the Department of Food Science (University of
Teramo), were used as starter cultures for Montepulciano d'Abruzzo
grape must fermentations (Suzzi et al., 2008). All yeast strains were
stored at -20 °C in yeast-peptone-dextrose broth (YPD; 1% w/v yeast ex-
tract, 2% w/v peptone, 2% w/v glucose) supplemented with glycerol
(25% v/v).
2.2. Small-scale Montepulciano d'Abruzzo vinifications
Fermentations were carried out in a local cellar in Teramo province
(Italy), during the vintage 2007. Three different Montepulciano d'Ab-
ruzzo grape musts, obtained by grapes harvested on three areas of Ter-
amo province, were used and identified as musts or wines A, B and C.
Each must was separated in four tanks of 50 L, after destemming and
crushing, and added with 80 mg/mL SO2.
Single strains of the autochthonous S. cerevisiae RT73, MS72, SRS1
were inoculated with 10 6 cells/mL from 24 h pre-cultures grown in
the same must. A commercial wine yeast starter of S. cerevisiae
(CS) was used as a control. Then, twelve fermentations were carried
out in the local winery according to the usual Montepulciano d'Ab-
ruzzo winemaking procedure. All fermentations were carried out
at room temperature (maximum temperature variation from 8 to
18 °C) and monitored by analyzing the sugar consumption at differ-
ent stages of fermentation (0, 24 h, 48 h, 7 days, 20 days). When
the fermentation was concluded, the yeast lees were allowed to settle
for 7 days and then wines were racked in 40 L tanks at room temper-
ature in the cellar for 3 months. Then the wines were placed
into glass bottles (750 mL), crown-sealed, and stored at 15–20 °C
for up to 6 months until chemical and sensorial analyses were
performed.
2.3. Analytical determination
Basic chemical analysis of wines, including pH, titratable and volatile
acidity, reducing sugars and dry extract was performed following the
official EU Community methods (EEC, 1990).
23
2.4. Enumeration and yeast isolation
Samples were taken in duplicate and collected at beginning, mid-
dle and end of fermentation. Viable yeast counts on must samples
were performed according to Tofalo et al. (2009). Counts of the
non-Saccharomyces and Saccharomyces yeasts were based on colony
morphology onto Wallerstein Laboratory Nutrient Agar (WLN,
Oxoid, Milan, Italy). Only the isolates that presumptively belonged
to the species S. cerevisiae were purified by repetitive streaking on
YPD. The isolates were maintained as culture stocks in 25% (v/v) glycerol
(Sigma-Aldrich, Milan, Italy) at -80 °C until further analyses.
2.5. Yeast identification
Yeast cells were grown aerobically in YPD at 28 °C. DNA was iso-
lated according to Querol et al. (1992) The 5.8 internal transcribed
spacer (ITS) rRNA region was amplified in a Bio-Rad thermocycler
(MyCycler, Bio-Rad Laboratories, Milan, Italy) using primers ITS1
and ITS4 as described previously (Esteve-Zarzoso, Belloch, Uruburu,
& Querol, 1999). The amplified DNA was digested with the restriction
endonucleases HinfI, CfoI and HaeIII (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% (w/v) 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).
2.6. Microsatellite PCR fingerprinting
After confirmation of S. cerevisiae species, DNA was used as a tem-
plate for microsatellite PCR fingerprinting, as described by Vaudano
and Garcia-Moruno (2008). A multiplex amplification of three micro-
satellite loci (SC8132X, YOR267C and SCPTSY7) was used. Each 25 μL
PCR reaction contained 1× PCR buffer, 3.1 mM MgCl2, 2 U Taq poly-
merase (Invitrogen, Milan, Italy), 10 pmol of each primer for locus
YOR267C, 15 pmol of primers for locus SC8132X and 40 pmol of
primers for locus SCPTSY7. PCR amplifications were performed in a
Bio-Rad thermocycler and amplification reactions were performed
with 4 min of initial denaturation at 94 °C, 28 cycles of 30 s at 94 °C,
45 s at 56 °C, 30 s at 72 °C and, finally, 10 min at 72 °C. The products
were run on a 2.5% (w/v) agarose gel 1 × TAE buffer at 100 V for
80 min. Gels were stained with ethidium bromide. Two lines of 1-kb
plus DNA ladder (Invitrogen) were used as molecular weight and nor-
malization gel standards for microsatellite profiles.
2.7. PCR-DGGE analysis
For the PCR-DGGE analyses, DNA was extracted directly from grapes
and musts using the PowerSoil DNA Isolation Kit (MoBio Laboratories).
For this purpose, a quantity of 10 mL of each sample, in duplicate, was
centrifuged to collected more cells. The DNA was then extracted accord-
ing to PowerSoil DNA Isolation Kit manufacturer's protocol. Quantification
of total DNA was achieved using a VersaFluor fluorimeter and a Fluores-
cent DNA Quantitation Kit (Bio-Rad). The D1–D2 region of 26S rRNA
gene was amplified by PCR using primer NL1-GC (5′-(GC)
CATATCAATAAGCGGAGGAAAAG-3′) and the reverse primer LS2 (5′-
ATTCCCAAACAACTCGACTC-3′). A GC-clamp which is needed to avoid du-
plex DNA artifacts was added to primer NL1 (Cocolin, Bisson, & Millis,
2000). PCR products were performed in a Bio-Rad thermocycler pro-
grammed as follows: initial denaturation at 95 °C for 5 min, followed by
30 cycles of 1 min at 94 °C for denaturing, 45 s at 52 °C for annealing,
1 min at 72 °C for synthesis, and a final extension step of 7 min at 72 °C.
PCR products were separated by DGGE by using the Dcode apparatus
(Bio-Rad). The PCR products were electrophoresed in a 0.8 mm polyacryl-
amide gel (8% w/v) acrylamide–bisacrylamide (37.5:1) and run with
1×TAE buffer using a denaturing gradient from 30% to 60% (v/v) urea
24 G. Suzzi et al. / Food Research International 46 (2012) 22–29
and formamide increasing in the direction of electrophoresis, as described
by Cocolin et al. (2000). The electrophoresis was performed at a constant
voltage of 120 V for 6 h with a constant temperature of 60 °C. The DGGE
running conditions were initially optimized on the reference strains to ob-
tain well-separated band profiles and to avoid co-migration (data not
shown). After electrophoresis the gels were stained with ethidium bro-
mide for 15 min, rinsed for 20 min in distilled water and photographed
under UV transillumination. Normalization of the gel was performed by
using band ladders of reference strains DNA in all gels.
2.8. Sequencing of DGGE bands
Bands of interest were excised from the gels with a sterile blade,
mixed with 500 μL of sterile water, and incubated overnight at 4 °C
to allow the DNA of the bands to diffuse out of the polyacrylamide
gel blocks. Two microliters of this solution was used to reamplify
the PCR product with NL1-LS2 (without GC-clamp) primer pair.
Then, PCR products were purified by use of the GFX TM PCR DNA and
Gel Band Purification Kit (Amersham Biosciences AB, Uppsala, Swe-
den) following the manufacturer's instructions, and delivered to
B.M.R. (Padua University, Italy) for sequencing. The sequences
obtained were compared with those deposited in the GenBank DNA
database (http://www.ncbi.nlm.nih.gov/) using the basic BLAST
search tools (Altschult et al., 1997).
2.9. Solid phase microextraction–gas chromatography (SPME–GC) analysis
of volatile compounds
Volatile compounds were determined by solid phase microextraction
coupled with gas chromatography (SPME–GC).
All reagents were purchased from Sigma-Aldrich (St. Louis, MO),
with a purity greater than 99%.
Five ml of wine samples was placed in 10 mL glass vials with 1 g
NaCl and 10 μL of 4-methyl-2-pentanol (final concentration 4 mg/L)
was added as internal standard. Both equilibration and adsorption
phases were carried out by stirring for 30 min at 40 °C. A carboxen–
polydimethylsiloxane-coated fiber (85 μm) was used (Sigma-Aldrich,
St. Louis, MO). Under the extraction conditions described above, the
recovery of the volatile compounds was between 88.9 and 103.5%.
For quantitative determination, a CP 380 capillary gas chromatograph
equipped with a 8200 autosampler SPME III (Varian, Italy) was used.
The fused silica capillary column was a CP-Wax 52 CB (50 m × 0.32 mm)
by Crompack (Netherlands), coated with polyethyleneglycol (film
thickness 1.2 μm), as stationary phase. The injector and FID temperature
was 250 °C. After extraction, the fiber was placed in the injector of the
GC for 15 min. The temperature program was the following: initial tem-
perature (50 °C) held for 2 min; first ramp, 1 °C min to 65 °C (0 min
hold); second ramp, 10 °C min to 150 °C (10 min hold); and third
ramp 10 °C min to 200 °C (1 min hold). The carrier gas (N2) flow rate
was 2.5 mL/min. The aroma compounds were identified by comparing
the retention time of standards and their identification was confirmed
by using GC-MS.
GC–MS analysis was performed using a GC-mass spectrometer
Finnigan Trace DSQ Quadrupole (Thermo Finnigan, San Jose, CA).
Mass spectrometer conditions were: Ion Source: Electron Ionization
(EI), Ion Polarity: POS, Ion Source Temperature: 250 °C, MS transfer
line: 220 °C, Turbomolecular Pump: 70 L/s, Acquisition: full Scan,
Mass range: 30–400 m/z, and Carrier gas: He. The data were processed
using Xcalibur Data System Software 1.4.1 SP3 (Thermo Finnigan, San
Jose, CA). The quantitative analysis of wine aroma compounds was car-
ried out on the basis of the relative peak area (Qi) calculated from head
space SPME (HS/SPME) gas chromatograms after addition of known
amounts of analyte standards, as well as the internal standard according
to De la Calle-Garcia et al. (1998). The chemical analyses were carried
out in the same period of the sensory analysis. Each determination
was carried out in duplicate. The presented data are the means of
three determinations.
2.10. Sensory analysis
Sensory tests were performed at room temperature (20 °C) and
sample position was randomized every time. In each session, 30 mL
samples were presented in coded, clear, 150 mL tulip glasses. Sensory
analyses were carried out in a sensory room designed according to
ISO 8589:2007 rules (ISO, 2007). Descriptive analysis was carried out
in only one session. Sensory profile was determined using 9 descriptors
(fruity, floral, grass, spicy, acidity, body, softness, persistence and inten-
sity) chosen by the panel in a preliminary session as stated in the ISO
11035: 1994 (ISO, 1994) rules and according to their importance in
Montepulciano d'Abruzzo wines. Skilled judges (n= 12) were trained
as stated in the ISO 8586-1: 1993 rules (ISO, 1993). Regarding each
topic, judges were asked to evaluate samples on a 0–10 point scale of in-
tensity. In that scale zero (0) indicated that the descriptor was not per-
ceived, while a score of ten (10) was equivalent to the highest
perception.
2.11. Statistical procedures
The mean and standard deviation were calculated for each experi-
mental parameter. An ANOVA and a Tukey test were used to highlight
the effects of yeast strains on sensorial descriptors. Discriminant analysis
was carried out to underline any differences due to yeast strain. Principal
component analysis (PCA) was performed using the statistical software
STATISTICA for windows (version 8.0, StatSoft Inc., Tulsa, OK, USA).
3. Results and discussion
3.1. Small scale vinifications of red wine Montepulciano d'Abruzzo
The alcoholic fermentations of twelve vinifications, four for each
grape musts belonging to the three homogenous winegrowing areas,
started quickly and proceeded to completion of must sugars between
6 and 19 days after grape crushing. Different kinetic responses were
found as a function of the yeast strain in the musts studied. The sugar
concentrations of the musts originating from the areas A and B were
24 and 25 °Brix, respectively, whereas must C was characterized by a
lower sugar content (19 °Brix), probably due to vineyard irrigation be-
fore the harvesting (data not shown).
The sugar concentration affected greatly the fermentation kinetics
of musts A and B, in which fermentation was slower and longer than
in must C. In this last must, sugars were completely fermented inde-
pendently by the starter used. Table 1 reports some parameters of
wines obtained by the inoculated fermentation with the starter
strains. The composition of the four wines from area C (Table 1), in
which fermentation was not affected by the initial sugar content,
was similar; strain SRS1 produced a wine with less acidity, while
the strain RT73 led to the lowest pH.
All the starters, growing in the musts from areas A and B, were able
to produce wines with a high alcoholic degree, even if some differences
in the residual sugars and total acidity were found.
3.2. Yeast identification
Identification was performed by amplifying the 5.8-ITS rRNA and
subsequent restriction analysis with the endonucleases CfoI, HaeIII
and HinfI (Esteve-Zarzoso et al., 1999), that resulted for all strains
tested in a single fragment with a molecular size of 850 bp. After com-
parison of these restriction products with those previously described
(Esteve-Zarzoso et al., 1999) all isolates belonged to S. cerevisiae spe-
cies, as expected. The addition of the starter produced a dilution of
 G. Suzzi et al. / Food Research International 46 (2012) 22–29 25

MS72

RT73
SRS1
Table 1
Chemical characteristics of wines obtained from must of three different areas (A, B and

M
C) inoculated with S. cerevisiae starter strains.

Characteristics Strains

CS MS72 SRS1 RT73

a
Alcohol (% v/v)
A 15.65 ± 0.07b 16.05 ± 0.25 16.05 ± 0.16 15.90 ± 0.18
B 16.20 ± 0.22 15.90 ± 0.21 16.00 ± 0.28 15.90 ± 0.14
C 12.25 ± 0.15 12.02 ± 0.22 12.53 ± 0.28 12.43 ± 0.29

Sugars (g/L)
A 2.65 ± 0.21 4.80 ± 0.14 4.45 ± 0.39 4.00 ± 0.28
B 6.65 ± 0.21 9.85 ± 0.49 4.45 ± 0.35 3.45 ± 0.49
C 2.50 ± 0.42 1.75 ± 0.07 2.20 ± 0.14 2.35 ± 0.35

pH
A 3.46 ± 0.01 3.52 ± 0.04 3.52 ± 0.35 3.56 ± 0.04
B 3.38 ± 0.02 3.47 ± 0.01 3.47 ± 0.03 3.36 ± 0.02
C 3.27 ± 0.02 3.16 ± 0.04 3.28 ± 0.03 3.08 ± 0.01

Volatile acidityc (g/L) Fig. 1. Electrophoretic patterns obtained for MS72, RT73 and SRS1 strains after micro-
A 0.52 ± 0.03 0.53 ± 0.02 0.48 ± 0.04 0.60 ± 0.05 satellite multiplex PCR (SC8132X, YOR267C and SCPTSY7) according to Vaudano and
B 0.60 ± 0.05 0.60 ± 0.06 0.60 ± 0.03 0.58 ± 0.07 Garcia-Moruno (2008). M: 1-kb plus DNA ladder (Invitrogen).
C 0.24 ± 0.03 0.20 ± 0.03 0.24 ± 0.05 0.17 ± 0.01

Titratable acidityd (g/L)

A 6.40 ± 0.14 6.30 ± 0.14 5.30 ± 0.42 7.05 ± 0.21 Paraggio, 2004). Recent studies suggest that the use of commercial start-
B 6.75 ± 0.21 7.20 ± 0.28 6.15 ± 0.35 7.05 ± 0.35
C 5.70 ± 0.20 6.50 ± 0.28 5.10 ± 0.28 6.30 ± 0.14
er has no significant effect on the development of indigenous micro-
biota. In some cases only a ratio of 50% of the inoculated strain was
Dry extract (g/L) detected (Esteve-Zarzoso, Gostincar, Bobet, Uruburu, & Querol, 2000;
A 31.80 ± 1.35 33.05 ± 1.22 32.50 ± 1.14 32.45 ± 1.44 Legras, Ruh, Didier, & Karst, 2005). Then, researchers and winemakers
B 34.60 ± 1.28 36.30 ± 1.11 33.10 ± 1.07 33.45 ± 1.19
are increasingly focusing on preserving the terroir characteristic of
C 26.00 ± 1.28 27.60 ± 0.98 26.10 ± 1.12 26.60 ± 1.18
each wine.
a
(mL of alcohol/100 mL of wine).
b
Data are expressed as average ± SD.
c
Expressed as acetic acid.
3.4. Yeast population fingerprinting by PCR-DGGE
d
Expressed as tartaric acid.
Information on yeast population dynamic during vinifications was
obtained by extracting DNA directly from musts and wines, followed
by the PCR-DGGE of amplicons obtained with the primers LS2/NL1-GC
the non-Saccharomyces biota (T0), and from first day of fermentation (Cocolin et al., 2000). Differences were not observed in the DGGE pro-
100% of the isolates corresponded to species S. cerevisiae. files when the triplicate samples were analyzed (data not shown). Fig. 2
shows the DGGE fingerprints obtained by the musts during fermenta-
3.3. Microsatellite analyses of S. cerevisiae populations tion. Some bands were present in all samples, while others were specific
only for some samples. Selected DGGE bands were excised and after
To verify the capacity of the inoculated strains to dominate the reamplification with primers LS2 and NL1, sent for sequencing. The re-
must fermentation, a total of 290 isolates were obtained from the sults of band sequencing are reported in Table 3. The fingerprints
12 vinifications. After identification by PCR–RFLP, S. cerevisiae isolates obtained by direct sample analysis consisted of 7 different bands
were subjected to microsatellite PCR fingerprinting as described by (Fig. 2). In general, S. cerevisiae (band 4) was the dominant species
Vaudano and Garcia-Moruno (2008). The starter strains were clearly throughout all fermentations in all the 12 vinifications. Also bands 1, 2
distinguishable by their microsatellite profiles. In fact, using only and 3 were visible throughout all fermentations and their closest rela-
three microsatellite markers, wine S. cerevisiae strains were differentiat- tives were Hanseniaspora spp., Cryptococcus magnus and Candida stellata,
ed unequivocally (Fig. 1). The comparison of these profiles with those respectively. These species were not detected on WLN plates. PCR meth-
obtained during fermentation of musts by the single starters enabled de- od can amplify also dead cells that can be detected as specific bands in
termination of implantation percentage at the beginning and middle of the DGGE gels, and evidence yeast populations not viable. The closest
fermentation. In this way, it was possible to evidence the competition relative corresponding to band 8 was Aspergillus sydowii and appeared
occurring in vinification process between the autochthonous starter at the start of fermentation only in the must inoculated by the strain
and the native wine yeasts. The microsatellite analyses indicated that MS72. However, not all the species present in the musts could be
strain SRS1 had a better capacity to dominate native yeast population detected as bands in the gel, due to the sensitivity of the PCR-DGGE pro-
than the other tested starters in all vinifications (Table 2). In fact its pres- tocol used that does not give a band in the gel underlining a population
ence was determined through all three must fermentations. Of interest less than 104 cfu/mL (Cocolin et al., 2000; Prakitchaiwattana, Fleet, &
was the presence of wild Saccharomyces during the must fermentations Heard, 2004). After 5 months, only the presence of S. cerevisiae was ob-
performed by the commercial strain and the autochthonous starter served (data not shown).
strains RT73, MS72, whereas starter strain SRS1 resulted to be alone.
Within each fermentation, there is an underlying growth of a succession 3.5. Aromatic profile of Montepulciano d'Abruzzo wines
of different strains. As many as 10 or more genetically distinct strains of
S. cerevisiae have been found to contribute to the one fermentation Flavor compounds give the wine its typical odor and taste. The
(Cocolin, Pepe, Comitini, Comi, & Ciani, 2004; Ganga & Martinez, 2004; main origin of these compounds is yeast metabolism during the fer-
Pramateftaki, Lanaridis, & Typas, 2000; Sabate, Cano, Querol, & mentation and over 60 compounds are known to have an impact on
Guillamon, 1998; Santamaria et al., 2005; Sipiczki, Romano, Capece, & wine flavor (e.g. higher alcohols, acids, esters, carbonyls, thiols). The
26 G. Suzzi et al. / Food Research International 46 (2012) 22–29

Table 2
Frequencies of the S. cerevisiae microsatellite patterns obtained in each sampling inoculated fermentation.

Fermentation A Fermentation B Fermentation C

Strain T0 T1 T2 T0 T1 T2 T0 T1 T2
a b
RT73 10 (90) 9 (89) 5 (100) 12 (98) 5(75) 5 (100) 9 (98%) 5 (100) 6 (100)
MS72 7 (72) 15 (93.3) 5 (100) 15 (80) 8 (100) 7(100) 14 (92.8) 5 (100) 5 (100)
SRS1 12 (100) 10 (100) 5 (100) 16 (100) 5 (100) 8 (100) 7 (100) 5 (100) 5 (100)
CS 10 (90) 10 (90) 5 (100) 14 (85.7) 5 (100) 6 (100) 10 (100) 5(100) 5 (100)
a
No. of isolates.
b
Frequency (%) of the microsatellite profiles of inoculated strain.

profile of these compounds varies with the yeast species and strains
T0 T1 T2 contributing to the fermentation (Antonelli, Castellari, Zambonelli, &
Carnacini, 1999; Regódon Mateos, Pérez-Nevado, & Ramírez
MS72

MS72

MS72

Fernández, 2006).
RT73

SRS1

RT73

SRS1

RT73

SRS1
CS

CS

CS

Table 4 shows the average concentrations of some volatile com-

pounds detected in the wines from the areas A, B, and C fermented by
the four different strains. The volatile composition of the wines showed
more quantitative than qualitative differences, according to other studies
5 1 (Callejon et al., 2010; Mateo, Jiménez, Pastor, & Huerta, 2001). It is well
Area A known that ester concentration is dependent on several main factors:
2
yeast strain, fermentation temperature, aeration degree and sugar con-
tent (Perestrelo, Fernandes, Albuquerque, Marques, & Camara, 2006). In
3
our experimental conditions, the applied winemaking technology was
4 the same for all the trials, and therefore, the different amounts of esters
found in the wines from the different trials could be related to yeast strain
mainly. Ethyl acetate was always below the level considered negative
T0 T1 T2 (>150 mg/L) (Mallouchos, Komaitis, Koutinas, & Kannelaki, 2002) for
all samples. The other esters, such as 3-methylbutyl acetate, butyric acid
MS72

MS72

MS72

ethyl ester, ethyl hexanoate and ethyl octanoate (Table 4) related to fruity
RT73

SRS1

RT73

SRS1

RT73

SRS1
CS

CS

CS

aroma are present in larger amounts to the odor threshold (Li, Tao, Wang,
& Zhang, 2008). The strains CS and RT73 lead to wine with the greatest
amount of these compounds. So, it is possible to affirm that yeast strain
has a great influence on ester compound formation during alcoholic
Area B
fermentation.
Ethyl esters of fatty acids are more abundant than higher alcohol ac-
etates; so according to Ferreira et al. (1995), the fruity character attribut-
6
ed to the aroma of Montepulciano wines is mainly related to fruity notes
7 (apple, pear, cherry, etc.). Higher alcohols are quantitatively the largest
8
group of the flavor compounds in wines, as normally in alcoholic bever-
9 ages (Nykänen, 1986), and they are important variables for differentiat-
ing the wine yeast strains (Romano, Fiore, Paraggio, Caruso, & Capece,
2003). Table 4 shows that only isoamyl alcohol and phenyl ethanol
were present in larger amounts to the odor thresholds which are
T0 T1 T2 30 mg/L and 14 mg/L, respectively (Li et al., 2008). Thus, in our condi-
tions, the production of higher alcohols in wines was substantially low
compared to Montepulciano d'Abruzzo wines on the market.
MS72

MS72

MS72
RT73

SRS1

RT73

SRS1

RT73

SRS1

The strain MS72 produced wines with the highest quantities of ethyl
CS

CS

CS

esters of fatty acids. 2-Phenyl ethanol (rose aroma) content ranged from
165.6 to 207.4 mg/L. Moderate concentrations of fusel alcohols, howev-
w er, contribute to the wine's aromatic complexity. In general the strain
Area C SRS1 was characterized by a low production of all the detected aromatic
compounds, the strain MS72 by the highest production of higher alco-
hols and strains CS and RT73 by a high ester formation. Due to the
11 large number of peaks and fragments that are found in the analysis of
one sample of wine, principal component analysis (PCA) has been
13 used to obtain biochemical fingerprints of wines and to elucidate differ-
ences in the volatile components of wine fermented by different strains
15 of Saccharomyces or in mixed cultures (Howell, Cozzolino, Bartowsky,
Fleet, & Henschke, 2006; Nurgel, Erten, Canbaş, Cabaroğlu, & Selli, 2002).
14 The volatile compounds produced by the four starter strains were
analyzed using PCA (Fig. 3). Firstly, the correlation matrix was comput-
Fig. 2. Results obtained by DGGE analysis of the DNA directly extracted from the fermen-
tation samples of the three areas (A, B and C). T0 (0 day), T1 (20 days) and T2 (20 days)
ed in order to discriminate the variables, thus selecting twenty four pa-
refer to days of fermentation. Purification and sequencing of faint bands (w) did not rameters (ethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl
prove to be successful. Species identification for band numbers is given in Table 3. heptanoate, ethyl dodecanoate, ethyl phenyl ester, methyl hexanoate,
 G. Suzzi et al. / Food Research International 46 (2012) 22–29 27

Table 3 3.6. Sensory profiles

Identification, based on BLAST comparison in GenBank, of the bands obtained by PCR-
DGGE using universal primers NL1/LS2.
Sensory analysis highlighted that some descriptors were statisti-
Band Closest relative % identity Closest relative accession number cally influenced by yeast starter (Fig. 4). Some significant differences
5 Eremothecium ashbyi 92% AB294409.1 related to fixed compounds can be easily explained as reported
1, 6, 10 Hanseniaspora spp. 92% AY953954 below. Wines fermented by strain RT73 were characterized by higher
2, 7, 11 Cryptococcus magnus 97% EF126360.1 acidity than the others, due to a lower pH than the other wines
3,8, 12 Candida stellata 99% AY188852
(Table 1), a parameter strictly correlated to this descriptor. The great-
4, 9, 13 S. cerevisiae 99% EU386754.1
14 Penicillium canescens 97% DQ658168.1 est softness of wines, obtained by using starter SRS1, was due to a
15 Aspergillus sydowii 98% AM883163.1 lower acidity, probably to a greater production of glycerol and/or a
greater release of mannoproteins as reported by Feuillat (2003).
The differences regarding fruity cannot be related to consider
wine flavor compound amount. As a matter of fact, wines fermented
octadecane, 1-butyl alcohol, isoamyl alcohol, ethanol, phenylethanol, 3- by the strains MS72 and SRS1 were characterized by a greater fruity
metil, 1-pentanol, propyl alcohol, diethyl succinate, 3-methylbutyl ace- than those fermented by strains CS and RT73; but these last starters
tate, butane-2,3 diol, acid acetic, butyric acid ethyl ester, dodecane, iso- formed a higher ester amount (Fig. 3). These compounds are normal-
butanol, isoheptanol, 1-hexanol and limonene). The PCA explained ly related to fruity descriptor, so it is possible to hypothesize that the
66.85% of the total variance. PC 1 accounted for 53.09% of the variance fruity descriptor in these wines was influenced by the lowest amount
and correlated with butyric acid ethyl ester, ethyl decanoate and of substances with a depressing or masking effect on fruity, as
3-methylbutyl acetate. PC 2 explained 13.76% of the variance; this reported by Campo, Ferreira, Escudero, and Cacho (2005). The spicy
dimension was mainly related positively to ethyl phenyl ester and was lower in wines fermented by strain RT73, probably because this
1-butyl alcohol and negatively with isobutanol. Then, in score plot strain produces lower amounts of some compounds able to influence
(Fig. 3a) it is possible to distinguish three different wines produced this descriptor than the other strains. The commercial strain CS, iso-
by four starters. The strain SRS1 is well differentiated from the lated in a different region, was able to produce good wines, but it
others for the highest concentration of isoheptanol, whereas strain doesn't preserve the typical characteristic. These results agree with
RT73 and CS were quite similar and mainly related to fermentative the findings of other authors (Antonelli et al., 1999). Sensory analysis
esters. The strain MS72 was grouped on the basis of the highest con- revealed that the best wines were those fermented by starter SRS1,
tent of 1-butyl alcohol and isoamyl alcohol. The discriminant analy- which had great olfactory and taste characteristics, typical of high
sis of SPME–GC profiles of the aromatic compounds present in all quality red wines. These results could be related to the general mod-
wines (Fig. 3b) showed the great influence of the single strains on erate production of several fruity esters that highlight the varietal
wine characteristics. For this it is very important to understand properties and typicity of Montepulciano d'Abruzzo red wine.
how particular species and strains of wine yeasts grow during fer-
mentations of musts inoculated by specific starters. If the starter
dominates on natural yeast population, the different yeasts growing 4. Conclusion
together in wine fermentation do not metabolically interact with
one another and the wine will display distinctive aroma and sensory High number of S. cerevisiae wine strains at the beginning of grape
profiles of the starter. If the starter grows but does not compete with must fermentation assured a right kinetic but their capacity to be
other natural yeasts, interactions can occur between yeasts giving a dominant can affect the natural S. cerevisiae populations in different
mixed culture wine with a composition that is different and variable patterns. However the matter of the dominance of some strains is
depending on the natural wine yeast population present. not well understand and is the object of many studies (Attfiled,

Table 4
Content of major volatile compounds produced by four S. cerevisiae strains in wine.

Volatile compounds Strains

(mg/L)
SRS1 MS72 CS RT73

Ethyl acetate 52.5 ± 4.95*b 74.7 ± 13.74a 78.7 ± 8.84a 77.3 ± 5.54a
3-Methylbutyl acetate 0.2 ± 0.05b 8.8 ± 5.81c 26.3 ± 5.39a 21.0 ± 1.24a
Butyric acid ethyl ester 0.9 ± 0.14b 0.4 ± 0.30a 1.5 ± 0.22d 1.2 ± 0.06c
Methyl hexanoate 54.0 ± 6.46a 52.4 ± 10.43a 130.9 ± 8.31b 117.1 ± 21.56b
Ethyl hexanoate 0.3 ± 0.08a 38.2 ± 20.54b 57.3 ± 21.42d 47.8 ± 2.22c
Ethyl heptanoate 0.8 ± 0.18a 0.7 ± 0.18a 1.2 ± 0.03b 1.2 ± 0.03b
Ethyl octanoate 52.9 ± 7.80a 97.8 ± 29.12b 162.9 ± 24.66d 126.4 ± 7.67c
Ethyl decanoate 25.6 ± 4.79b 16.3 ± 7.71a 60.5 ± 4.22d 51.3 ± 3.52c
Ethyl dodecanoate 3.0 ± 0.43a 4.1 ± 0.82a 7.8 ± 0.83b 6.7 ± 0.07b
Ethyl phenyl ester 5.8 ± 1.02a 7.1 ± 0.72a 3.3 ± 1.65b 5.7 ± 0.99a
Diethyl succinate 5.6 ± 2.33b 10.1 ± 3.76a 9.3 ± 3.75a 10.0 ± 2.07a
Propyl alcohol 1.0 ± 0.19b 3.0 ± 0.99a 3.1 ± 1.29a 2.4 ± 0.43ab
3-Methyl 1 pentanol 0.3 ± 0.09a 26.4 ± 22.15b 0.6 ± 19.23a 0.5 ± 10.63a
Isobutanol 17.1 ± 1.85a 4.9 ± 7.86b 9.1 ± 6.12c 14.5 ± 1.12a
1-Butyl alcohol 0.7 ± 0.49a 7.2 ± 5.47b 0.4 ± 4.53a 0.3 ± 2.58a
Isoamyl alcohol 128.5 ± 13.45a 160.8 ± 16.25b 124.6 ± 7.31a 114.0 ± 10.48a
1-Hexanol 4.4 ± 1.10b 6.9 ± 1.83a 6.9 ± 2.09a 7.5 ± 0.17a
Butane-2,3 diol 3.4 ± 1.31bc 5.7 ± 1.71c 0.7 ± 1.74a 1.2 ± 2.30ab
Isoheptanol 10.6 ± 1.94d 0.7 ± 6.31a 3.8 ± 7.02b 6.1 ± 0.33c
Phenyl ethanol 165.6 ± 6.01a 207.4 ± 30.25b 203.5 ± 28.16b 167.0 ± 14.21a
Dodecane 88.9 ± 31.40b 0.0 ± 0.0c 83.6 ± 71.15ab 44.1 ± 0.0a
Octadecane 0.0 ± 0.0a 0.0 ± 0.0a 0.5 ± 0.0b 1.1 ± 0.0c
Limonene 16.0 ± 12.51ab 0.1 ± 15.66a 11.0 ± 4.72a 32.9 ± 0.02b

*Data are expressed as average of the three production area (A, B and C) ± S.D. Different letters (a-d) in the same row correspond to statistically significant differences (P b 0.05).
28 G. Suzzi et al. / Food Research International 46 (2012) 22–29

CS MS72 SRS1 RT73

a4
Fruity*
3 10
Dimension 2: 13.76%

2 MS72 A Intensity Floral

RT73 C
MS72 C
1 MS72 B CS B

RT73 A
0 CS A
SRS1 B
SRS1 C RT73 B
-1 CS C Peristance Grass
0
-2 SRS1 A

-3

-4 Softness Spicy*
-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6
Dimension 1: 53.09%

b Body Acidity*
1.0
Fig. 4. Graphical representation of sensory scores of four wines obtained with S. cerevi-
Ethyl acetate
siae MS72, SRS1, RT73 and CS. In sensorial parameters indicated with an asterisk (*) a
difference among some trials is verified for p ≤ 0.01.
Ethyl hexanoate
0.5 Ethyl phenyl ester
1-Butyl alcohol 1-hexanol
Dimension 2 : 13.76%

Isoamyl alcohol Ethanol Ethyl octanoate

butane-2,3-diol
0.0
-0.5
-1.0
-1.0 -0.5
Dimension 1 : 53.09%
Fig. 3. Score plot (a) and loading plot (b) of the first and second principal components
(PC) after PC analysis encompassing volatile compounds (by name) for indigenous S.
cerevisiae strains (SRS1, MS72, RT73) and commercial strain (CS) inoculated in three
musts obtained from defined homogenous winegrowing zones (A, B and C).
1997; Barrajón, Arévalo-Villena, Rodríguez-Aragón, & Briones, 2009;
Kvitek, Will, & Gasch, 2008). The concentrations of most of the volatile
compounds were influenced by the autochthonous yeast strains that
were able to produce wines with different volatile profiles, some of
them rich in alcohols and others in fruity esters. In addition the domi-
nance or competitiveness of a starter strain could have an influence
on the sensorial quality of wine by imposing its aromatic profile or de-
leting the collaborative role of natural S. cerevisiae populations. The
wines produced by strain SRS1, that dominated natural S. cerevisiae
populations, had the aromatic fingerprinting of the strain, whereas
for the other strains, the aromatic role of the starters could have
been mediated by natural microbiota.
Acknowledgments
The authors wish to thank Tercas Foundation (project Agroes-
coampelos) for the financial support given. The authors are grateful
to “Azienda Di Giovanpietro” for kind cooperation.
3-metil 1-pentanol
Ethyl heptanoate References
Propyl alcohol Ethyl dodecanoate
Acetic acid Diethyl succinate
3-Methylbutyl aceta
acetate Altschult, S. F., Maddelen, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al.
Phenylethanol
(1997). Gapped BLAST and PSI-BLAST: A new generation of protein database
Limonene Ethyl decanoate search programs. Nucleic Acids Research, 25, 3389–3402.
Methyl hexanoate
Antonelli, A., Castellari, L., Zambonelli, C., & Carnacini, A. (1999). Yeast influence on volatile
Octadecane composition of wines. Journal of Agricultural and Food Chemistry, 47, 1139–1144.
Dodecane Attfiled, P. V. (1997). Stress tolerance: The key of effective strains of industrial baker's
Isoheptanol Butyric acid ethyl ester
yeast. Nature Biotechnology, 15, 1351–1357.
Barrajón, N., Arévalo-Villena, M., Rodríguez-Aragón, L. J., & Briones, A. (2009). Ecological
study of wine yeast in inoculated vats from La Mancha region. Food Control, 20,
Isobutanol
778–783.
Callejon, R. M., Clavijo, A., Ortigueira, P., Troncoso, A. M., Paneque, P., & Morales, M. L.
(2010). Volatile and sensory profile of organic red wines produced by different selected
autochthonous and commercial Saccharomyces cerevisiae strains. Analytica Chimica
0.0 0.5 1.0 Acta, 660, 68–75.
Campo, E., Ferreira, V., Escudero, A., & Cacho, J. (2005). Prediction of the wine sensory
properties related to grape variety from dynamic-headspace gas chromatography-
olfactometry data. Journal of Agricultural and Food Chemistry, 53, 2682–2690.
Ciani, M., Mannazzu, I., Marinangeli, P., Clementi, F., & Martini, A. (2004). Contribution
of winery-resident Saccharomyces cerevisiae strains to spontaneous grape must
fermentation. Antonie van Leeuwenhoek, 85, 159–164.
Cocolin, L., Bisson, L. F., & Millis, D. A. (2000). Direct profiling of the yeast dynamic in
wine fermentation. FEMS Microbiology Letters, 189, 81–87.
Cocolin, L., Pepe, V., Comitini, F., Comi, G., & Ciani, M. (2004). Enological and genetic
traits of Saccharomyces cerevisiae isolated from former and modern wineries.
FEMS Yeast Research, 5, 237–245.
Constantí, M., Poblet, M., Arola Mas, A., & Guillamón, J. M. (1997). Analysis of yeast
populations during alcoholic fermentation in a newly established winery. American
Journal of Enology and Viticulture, 48, 339–344.
De la Calle-Garcia, D., Reichenbacher, M., Dancer, K., Hurlbeck, C., Bartzsch, C., & Feller,
K. H. (1998). Analysis of wine bouquet components using headspace space solid-phase
microextraction-capillary gas chromatography. Journal High Resolution Chrome, 21,
373–377.
EEC (1990). Commission Regulation 2676/1990. Community methods for the analysis
of wines. Official Journal of the European Community, L272, 1–192.
Egli, C. M., Edinger, W. D., Mitrakul, C. M., & Henick-Kling, T. (1998). Dynamics of indigenous
and inoculated yeast populations and their effect on the sensory character of Riesling
and Chardonnay wines. Journal of Applied Microbiology, 85, 779–789.
Esteve-Zarzoso, B., Belloch, C., Uruburu, F., & Querol, A. (1999). Identification of yeasts
by RFLP analysis of the 5.8 rRNA gene and the two ribosomal internal transcribed
spacers. International Journal of Systematic Bacteriology, 49, 329–337.
Esteve-Zarzoso, B., Gostincar, A., Bobet, R., Uruburu, F., & Querol, A. (2000). Selection
and molecular characterization of wine yeasts isolated from the “El Penedes”
area (Spain). Food Microbiology, 17, 553–562.
Ferreira, B., Hary, C., Bard, M. H., Táisant, C., Olsson, A., & Lefur, Y. (1995). Effect of skin-contact
and setting on the level of the C18:2, C18:3 fatty acids and C6 compounds in Burgundy
Chardonnay musts and wines. Food Quality Preference, 6, 35–41.
Feuillat, M. (2003). Yeast macromolecules: Origin, composition, and enological interest.
American Journal of Enology and Viticulture, 54, 211–213.
 G. Suzzi et al. / Food Research International 46 (2012) 22–29
Fleet, G. H. (2008). Wine yeasts for the future. FEMS Yeast Reserach, 8, 979–995.
Frezier, V., & Dubordieu, D. (1992). Ecology of yeast strain Saccharomyces cerevisiae during
spontaneous fermentation in a Bordeaux winery. American Journal of Enology and
Viticulture, 43, 375–380.
Ganga, M. A., & Martinez, C. (2004). Effect of wine yeast monoculture practice on the bio-
diversity of non-Saccharomyces yeasts. Journal of Applied Microbiology, 96, 76–83.
Gutierrez, A. R., Santamaria, P., Epifanio, S., Garijo, P., & Lopez, R. (1999). Ecology of
spontaneous fermentation in one winery during 5 consecutive years. Letters in
Applied Microbiology, 29, 411–415.
Howell, K. S., Cozzolino, D., Bartowsky, E. J., Fleet, G. H., & Henschke, P. A. (2006). Metabolic
profiling as a tool for revealing Saccharomyces interactions during wine fermentation.
FEMS Yeast Research, 6, 91–101.
ISO (1993). Sensory analysis — General guidance for the selection, training, and monitoring
of assessors. Part 1: Selected assessors. International Organization for Standardization, ISO
8586-1:1993.
ISO (1994). Sensory analysis — Identification and selection of descriptors for establishing a
sensory profile by multidimensional approach. International Organization for
Standardization, ISO 11035:1994.
ISO (2007). Sensory analysis — General guidance for the design of test room. International
Organization for Standardization, ISO 8589:2007.
King, E. S., Swiegers, J. H., Travis, B., Francis, I. L., Bastian, S. E. P., & Petrorius, I. S. (2008).
Co-inoculated fermentations using Saccharomyces yeasts affect the volatile composition
and sensory properties of Vitis vinifera L. cv. Sauvignon Blanc wines. Journal of Agricultural
and Food Chemistry, 56, 10829–10837.
Kvitek, D. J., Will, J. L., & Gasch, A. P. (2008). Variations in stress sensitivity and genomic
expression in diverse Saccharomyces cerevisiae isolates. PLoS Genetics, 4, 2–11.
La Jeune, C., Enry, C., Demuyter, C., & Lollier, M. (2006). Evolution of the population of
Saccharomyces cerevisiae from grape to wine in a spontaneous fermentation. Food
Microbiology, 23, 709–716.
Lambrechts, M. G., & Pretorius, I. S. (2000). Yeast and its importance to wine aroma—A
review. South African Journal for Enolgy and Viticulture, 21, 97–129.
Legras, J. L., Ruh, O., Didier, M., & Karst, F. (2005). Selection of hypervariable microsatellite
loci for the characterization of Saccharomyces cerevisiae strains. International Journal of
Food Microbiology, 102, 73–83.
Li, H., Tao, Y. S., Wang, H., & Zhang, L. (2008). Impact odorants of Chardonnay dry white wine
from Ch'angli County (China). European Food Research and Technology, 227, 287–292.
Mallouchos, A., Komaitis, M., Koutinas, A., & Kannelaki, M. (2002). Investigation of
volatiles evolution during the alcoholic fermentation of grape must using free
and immobilized cells with the help of solid phase microextraction (SPME) headspace
sampling. Journal of Agricultural and Food Chemistry, 50, 3840–3848.
Martini, A., & Vaughan-Martini, A. (1990). Grape must fermentation: Past and pre-
sent. In J. F. T. Spencer, & D. Spencer (Eds.), Yeasts technology (pp. 105–123). Berlin:
SpringerVerlag.
Mateo, J. J., Jiménez, M., Pastor, A., & Huerta, T. (2001). Yeast starter cultures affecting
wine fermentation and volatiles. Food Research International, 34, 307–314.
Monk, P. R., & Cowley, P. J. (1984). Effect of nicotinic acid and sugar concentration of
grape juice and temperature on accumulation of acetic acid during yeast fermentation.
Journal of Fermentation Technology, 62, 515–521.
Mortimer, R., & Polsinelli, M. (1999). On the origins of wine yeast. Annual Review of
Microbiology, 150, 199–204.
Nadal, D., Colomer, B., & Pina, B. (1996). Molecular polymorphism distribution in
phenotypically distinct populations of wine yeast strains. Applied and Environmental
Microbiology, 62, 1944–1950.
Nurgel, C., Erten, H., Canbaş, A., Cabaroğlu, T., & Selli, S. (2002). Influence of Saccharomyces
cerevisiae strains on fermentation and flavor compounds of white wines made from
29
cv. Emir grown in Central Anatolia, Turkey. Journal of Industrial Microbiology
and Biotechnology, 29, 28–33.
Nykänen, L. (1986). Formation and occurrence of flavour compounds in wine and distilled
alcoholic beverages. American Journal of Enology and Viticulture, 37, 84–96.
Perestrelo, R., Fernandes, A., Albuquerque, F. F., Marques, J. C., & Camara, J. S. (2006).
Analytical characterization of the aroma of Tinta Negra Mole red wine: Identification
of the main odorants compounds. Analytica Chimica Acta, 563, 154–164.
Prakitchaiwattana, C. J., Fleet, G. H., & Heard, G. M. (2004). Application and evaluation
of denaturing gradient gel electrophoresis to analyze the yeast ecology of wine
grapes. FEMS Yeast Research, 4, 865–877.
Pramateftaki, P. V., Lanaridis, P., & Typas, M. A. (2000). Molecular identification of wine
yeasts at species or strain level: A case study with strains from two vine growing
areas of Greece. Journal of Applied Microbiology, 89, 236–248.
Querol, A., Barrio, E., Huerta, T., & Ramon, D. (1992). Molecular monitoring of wine
fermentations conducted by active dry yeast strains. Applied and Environmental
Microbiology, 58, 2948–2953.
Regódon Mateos, J. A., Pérez-Nevado, F., & Ramírez Fernández, M. (2006). Influence of
Saccharomyces cerevisiae yeast strain on the major volatile compounds of wine. Enzyme
and Microbial Technology, 40, 151–157.
Romano, P., Fiore, C., Paraggio, M., Caruso, M., & Capece, A. (2003). Function of yeast
species and strain in wine flavour. International Journal of Food Microbiology, 86,
169–180.
Sabate, J., Cano, J., Querol, A., & Guillamon, J. M. (1998). Diversity of Saccharomyces
strains in wine fermentation: Analysis for two consecutive years. Letters in Applied
Microbiology, 26, 453–455.
Santamaria, P., Garijo, P., Lopez, R., Tenorio, C., & Gutierrez, A. R. (2005). Analysis of
yeast population during spontaneous alcoholic fermentation: Effect of the age of
the cellar and the practice of inoculation. International Journal of Food Microbiology,
103, 49–56.
Sipiczki, M., Romano, P., Capece, A., & Paraggio, M. (2004). Genetic segregation of natural
Saccharomyces cerevisiae strains derived from spontaneous fermentation of Aglianico
wine. Journal of Applied Microbiology, 96, 1169–1175.
Suzzi, G., Tofalo, R., Chaves-Lopez, C., Ramazzotti, S., Stagnari, F., Di Fabio, F., et al.
(2008). Isolation and selection of Saccharomyces cerevisiae from Montepulciano
d'Abruzzo “Colline Teramane” areas with different microclimatic characteristics.
Proc OIV, 15 20 June, Verona, Italy.
Thorhgate, J. H. (1998). Yeast strain and wine flavor: Nature or nurture. In A. L. Waterhouse,
& E. E. Ebeler (Eds.), Chemistry of wine flavor (pp. 66–80). Washington, DC: Oxford
University Press.
Tofalo, R., Chaves-López, C., Di Fabio, F., Schirone, M., Felis, G. E., Torriani, S., et al.
(2009). Molecular identification and osmotolerant profile of wine yeasts that fer-
ment a high sugar grape must. International Journal of Food Microbiology, 130,
179–187.
Torrens, J., Urpi, P., Riu-Aumatell, M., Vichi, S., López-Tamames, E., & Buxaderas, S.
(2008). Different commercial yeast strains affecting the volatile and sensory profile
of cava base wine. International Journal of Food Microbiology, 124, 48–57.
Vaudano, E., & Garcia-Moruno, E. (2008). Discrimination of Saccharomyces cerevisiae
wine strains using microsatellite multiplex PCR and band pattern analysis. Food
Microbiology, 25, 56–64.
Vilanova, M., & Sieiro, C. (2006). Contribution by Saccharomyces cerevisiae yeast to
fermentative flavour compounds in wines from cv. Albari˜no. Journal of Industrial
Microbiology and Biotechnology, 33, 929–933.
Wondra, M., & Boveric, M. (2001). Analyses of aroma components of Chardonnay wine
fermented by different yeast strains. Food Technology Biotechnology, 39, 141–148.