A new PCR-based method for monitoring inoculated

wine fermentations
Victoria Lopez
a
, M. Teresa Fernandez-Espinar
a
, Eladio Barrio
b
,
Daniel Ramon
a
, Amparo Querol
a,
*
a
Departamento de Biotecnolog a, Instituto de Agroqu mica y Tecnolog a de Alimentos (CSIC), PO Box 73, 46100 Burjassot, Vale`ncia, Spain
b
Unitat de Gene`tica Evolutiva, Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de Vale`ncia, Edificio de Institutos,
Campus de Paterna, PO Box 2085, 46071 Vale`ncia, Spain
Received 21 December 2001; received in revised form 12 April 2002; accepted 25 April 2002
Abstract
A new PCR-based method has been developed to monitor inoculated wine fermentations. The method is based on the
variation in the number and position of introns in the mitochondrial gene COX1. Oligonucleotide primers homologous to the
regions flanking the Saccharomyces cerevisiae COX1 introns have been designed and tested for S. cerevisiae wine yeast strain
differentiation. Four primers were selected for their subsequent use in a multiplex PCR reaction and have proved to be very
effective in uncovering polymorphism in natural and commercial yeast strains. An important point is that the speed and
simplicity of the technique, which does not require the isolation of DNA, allows early detection of the starter yeast strain
throughout the fermentation process. The main advantage for the wineries is that the must sample can be used directly for the
PCR reaction obtaining very fast results (in approximately 8 h). This allows the wine industries to intervene quickly if
necessary. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Identification; Characterization; Yeast; PCR; COX1
1. Introduction
The role of Saccharomyces cerevisiae in wine
production has been pointed out by several authors
(for a review see Fleet and Heard, 1993). These
studies demonstrate that this yeast, present on the
surface of grape skins and winery equipment, domi-
nates the fermentation process due to its efficient
fermentative metabolism. Modern wine fermentation
practices have included the use of S. cerevisiae
starters, in the form of active dry yeasts, to ensure
the reproducibility of fermentations and the final
product quality. This is of particular interest for the
wine industry since the sensory properties of the final
product vary considerably from one year to another
depending on the microbial flora present on the grapes
(Querol et al., 1990). The importance of using suitable
strains has stimulated the selection of new S. cerevi-
siae strains from the vineyards and wine fermenta-
tions. Therefore, many strains are commercialised at
present and their characterisation is necessary for
quality control in dry yeast production. It is generally
assumed that indigenous yeasts are suppressed by the
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0168- 1605( 02) 00194- 0
*
Corresponding author. Tel.: +34-96-390-0022; fax: +34-96-
363-6301.
E-mail address: aquerol@iata.csic.es (A. Querol).
www.elsevier.com/locate/ijfoodmicro
International Journal of Food Microbiology 81 (2003) 6371
starter, however, studies show that indigenous yeasts
can still participate in fermentation (Querol et al.,
1992a; Schutz and Gafner, 1993). Moreover, in some
cases only a ratio of 50% of the inoculated strain was
detected (Esteve-Zarzoso et al., 2000). For these
reasons, rapid and simple methods for the routine
verification of yeast strain characterisation in fermen-
tations would be useful to ensure that the fermentation
is conducted by the inoculated yeast. These methods
should clearly differentiate between the inoculated S.
cerevisiae strain and the wild S. cerevisiae strains
present in must.
The S. cerevisiae strains differ significantly in their
fermentation performance and their contribution to the
final bouquet and quality of wine, but cannot be readily
distinguished and identified using classical biochem-
ical methods. The introduction of molecular methods
provides new approaches to yeast strain differentiation.
These methods include: mitochondrial DNA (mtDNA)
restriction analysis (Lee and Knudsen, 1985; Vezinhet
et al., 1990; Querol et al., 1992b; Versavaud et al.,
1995), comparison of chromosomal DNA profiles
(Schutz and Gafner, 1993; Versavaud et al., 1995;
Guillamo n et al., 1996), and DNA fingerprinting
(Degre et al., 1989). Among these techniques, mtDNA
restriction analysis has proven useful for monitoring
inoculated wine fermentations due to the marked
mtDNA polymorphism of wine Saccharomyces strains
(Querol et al., 1992a). The new methods developed for
mtDNA restriction analysis are faster and easier than
other techniques but they do not allow the wineries to
carry out effective interventions or corrections since
the time required for the results varies from 25 to 60 h
(Querol et al., 1992b; Lopez et al., 2001).
The development of PCR techniques has opened up
new avenues for yeast strain identification and implies
a substantial reduction of time in relation to other
molecular techniques. To date, two PCR-based meth-
ods have been developed to discriminate between wine
yeast strains: one of them uses specific primers that
target the y elements, repeat sequences that flank the
TY1 retrotransposons (Ness et al., 1993), and the other
primers with conserved sequences complementary to
all intron splice sites, consensus sequences flanking
introns (de Barros Lopes et al., 1996). Both PCR
approaches are fast and simple, however, one of the
most important problems is the poor reproducibility of
band patterns observed due to variations in the quantity
of template DNA and the low annealing temperature
used as we have noted for the former method in a recent
work (Fernandez-Espinar et al., 2001).
In the present study, we propose a new PCR
method based on the variation in the number and
position of introns in the mitochondrial gene COX1.
This mosaic gene encodes the largest sub-unit of the
cytochrome c oxidase and it has been reported to be,
by far, the most intron-rich gene. In yeasts, a variable
number of introns have been reported between species
(Hardy and Clark-Walker, 1991; Sekito et al., 1995;
Foury et al., 1998). The COX1 intron variation has
been recorded also between strains within a species.
This is the case of Kluyveromyces lactis and S.
cerevisiae that possess strains with one, three or four
introns (Skelly et al., 1991) and strains with six or
seven introns (Foury et al., 1998), respectively.
We show how useful the developed PCR-based
method is in the differentiation of wine Saccharomy-
ces strains and we describe its use to control wine
fermentations conducted by active dry yeast strains.
2. Materials and methods
2.1. Yeast strains and growth conditions
The Saccharomyces wine strains used, the three
provided by the Spanish Culture Collection (CECT)
and 13 commercial dry yeasts are listed in Table 1.
Yeast cells were isolated and grown overnight in a
GPYA plate (0.5% w/v yeast extract (Pronadisa,
Madrid, Spain), 0.5% w/v peptone (Oxoid, Basing-
stoke, England), 4% w/v glucose (Panreac, Barcelona,
Spain), 2% w/v agar (Pronadisa)) and the isolated
colonies were grown in a 1.5-ml microfuge tube with
1 ml of GPY (0.5% w/v yeast extract (Pronadisa),
0.5% w/v peptone (Oxoid), 4% w/v glucose (Pan-
reac)) by shaking overnight at 28 jC.
2.2. Fermentation experiments
Wine musts from Bobal grapes were sterilised by
incubation with 1 g/l of dimethyl dicarbonate (Sigma-
Aldrich Chemie, Steincheim, Germany) for 6 h at 20
jC. Seven microvinifications were carried out in
duplicate using the wine S. cerevisiae strains T
73
,
CECT 1881, Fermol Complet Killer, and CECT
V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 6371 64
1484 combined simultaneously at different ratios: 1
(70%, 10%, 10%, 10%), 2 (60%, 20%, 10%, 10%), 3
(50%, 30%, 10%, 10%), 4 (40%, 40%, 10%, 10%), 5
(30%, 50%, 10%, 10%), 6 (20%, 60%, 10%, 10%), 7
(10%, 70%, 10%, 10%). All the strain stocks used
were prepared in a final concentration of 10
6
CFU/ml
and the appropriate volume to reach the desirable
percentage was added to 250 ml of must in each case.
All fermentations were conducted at 22 jC for 14
days. Samples were taken aseptically at different
stages of the fermentation (days 1, 2, 3, 6, 7, 8, 9
and 10) until the sugar level was less than 2.5 g/l
(measured by jBrix). The samples were centrifuged at
10,000 rpm for 1 min and washed twice with sterile
water. After this, they were kept at 20 jC for their
subsequent PCR amplification.
2.3. Wine fermentations
Two red wine-making processes were studied in
the same Spanish winery (Torre Oria located in Utiel-
Requena, Valencia) in 1998. Both wines were pro-
duced from Tempranillo (fermentor A) and Cencibel
grapes (fermentor B) and were inoculated with the dry
yeasts T
73
and Uvaferm L2056, respectively. Samples
were taken aseptically at different stages of the
fermentation: fermentor A (days 1, 2, 3 and 4) and
fermentor B (days 2, 3, 4 and 5). The samples were
centrifuged at 10,000 rpm for 1 min and washed twice
with sterile water. Then, they were resuspended in 100
Al of sterile water and kept at 20 jC for their
subsequent PCR amplification.
2.4. Preparation of DNA template for PCR
The template used for PCR was purified DNA or
must. In the first case, DNA from yeasts was obtained
according to the method of Querol et al. (1992b) and
then diluted 10 times with water. One microlitre of the
DNA dilution was added to the PCR reaction. When
the yeast DNA from the must was used as PCR
template, a 1.5-ml must sample was centrifuged in
an eppendorf tube and the precipitate obtained was
washed twice with sterile water. Then the precipitate
was resuspended in 100 Al of sterile water and an
adequate volume was added to the PCR reaction in a
final concentration of 10
6
cells/ml.
2.5. Primers
The COX1 introns were amplified by PCR using
the primers 1L (5V-ATCATTAGATTAGARTTAG-
CYGC-3V), 1R (5V-GAAAATGATTAATACNATG-3V),
2L (5V-GGTCATGCTGTATTAATRATTTT-3V), 2R
( 5V- ACCTCCAATTAAAGCNGGCAT- 3V) , 3L
(5V-GCTTTAATTGGWGGWTTTGG-3V), 3R (5V-
ATTGTCATACCATTTGTYCTYAT-3V), 4L (5V-
GAAGTAGCAGGWGGWGGWGA- 3V) , 4R
(5V-AATCCTACAATATAYATRTGRTG-3V), 5L (5V-
ATGTATATTGTAGGATTRGAYGC-3V), 5R (5V-
GTTAGCTAAGGCWACWCCWGT- 3V) , 67L
(5V-GCCTCATTAGATGTRGCATTYC-3V) and 67R
(5V-CTGCGAAAGCATCAGGRTARTC-3V). The
physical location of the primers is described in Fig. 1.
The primers were designed by comparing available
sequences of the COX1 gene from different yeasts and
ascomycetes using the CLUSTAL X program (a Win-
dows version of the Clustal W program; Thompson et
al., 1994). The accession numbers of these sequences in
the EMBL database are: Aspergillus nidulans
(X00790), H. wingei (D31785), K. lactis (X57546),
Neurospora crassa (X14669), Saccharomyces capen-
sis (U00801), S. cerevisiae (AJ011856), Saccharomy-
ces douglasii (M97514), and Schizosaccharomyces
pombe (X54421).
Table 1
List of strains used in the present work
Strain Species Source
Reference wine strains
CECT 1483 S. cerevisiae Must
CECT 1484 S. cerevisiae Bobal grape
CECT 1881 S. cerevisiae Jumilla wine
Commercial wine strains
Fermol Complet Killer S. cerevisiae AEB Iberica
Fermol Cryoaromae S. cerevisiae AEB Iberica
Fermol Clarifiant S. cerevisiae AEB Iberica
Fermol Primeur S. cerevisiae AEB Iberica
Uvaferm-CEG S. cerevisiae Danstar
Uvaferm-CM S. cerevisiae Danstar
Uvaferm-VRB S. cerevisiae Danstar
Uvaferm-PM S. cerevisiae Danstar
Uvaferm-L2056 S. cerevisiae Danstar
Fermivin Crio 7303 S. cerevisiae Gist Brocades
Levuline Tirage Agglo S. bayanus GLO Group Lab
Boeroferm W S. cerevisiae Lallemand
T
73
S. cerevisiae Lallemand
V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 6371 65
2.6. PCR conditions
DNA was amplified in a Progene thermocycler
(Techne, Cambridge, UK). Amplification reactions
contained 0.1 AM of each primer, 80 AM of each
deoxynucleotide, 1 buffer (10 mM TrisHCl, pH
8.8 at 25 jC, 1.5 mM MgCl
2
, 50 mM KCl and 0.1%
Triton X-100) and 2 U of DyNAzymek II DNA
Polymerase (Finnzymes OY, Espoo, Finland) in 100
Al of final volume. BSA was added in a final concen-
tration of 0.05 Ag/Al only when must sample is used
directly for the PCR reaction. The thermal cycling
parameters were an initial denaturation at 95 jC for 5
min; 35 cycles of denaturation at 95 jC for 1 min 30
sg, annealing at 52 jC for 2 min 30 sg and extension
at 72 jC for 3 min 30 sg, and a final extension at 72
jC for 10 min. When the method was applied to
microvinifications and industrial vinifications, ampli-
fication of the 5.8S ribosomal gene and the intergenic
regions (ITS1 and ITS2) was performed parallelly in
the same samples as a positive control. In this case,
PCR amplification was carried out as described pre-
viously (Esteve-Zarzoso et al., 1999). PCR products
(20 Al loaded) were separated on 2% (w/v) agarose
gels with 1 TAE buffer. After electrophoresis, gels
were stained with ethidium bromide and visualised
under UV light. A 100-bp DNA ladder marker (Gibco
BRL, Gaithersburg, MD) served as the size standard.
3. Results and discussion
3.1. Design of primers and their utility to differentiate
Saccharomyces wine strains
Twelve primers homologous to the regions flaking
the S. cerevisiae COX1 introns (see Fig. 1 and
Table 2
Amplification patterns displayed by the 12 primers designed in the
present work using the DNA of four S. cerevisiae strains as template
Primers PCR products (kbp)
T
73
CECT 1484 CECT 1483 CECT 1881
1L1R 1.4 + 1.2 +
0.8
1.4 + 1.2 +
0.8
1.4 + 1.2 +
0.8
1.4 + 1.2 +
0.8
2L2R 2.5 2.5 2.5 2.5
3L3R 0.32 2.0 2.0 0.9
4L4R
5L5R 0.3 0.3 0.3 0.3
67L67R 0.3 0.3 0.3 0.3
4L5R 0.42 0.42 1.4 2.0
5L67R 0.6 0.6 0.6 0.6
3L3R
4L5R
a
0.42 +0.32 2.0 2.0 + 1.4 2.0 + 0.9
No PCR amplification.
a
Restriction patterns shown in Fig. 2.
Fig. 1. Schematic representation based on the amino acid sequence of the S. cerevisiae COX1 gene according to Foury et al. (1998) and location
of the primers used to amplify the intronic sequences. The arrows indicate the direction of transcription. Exonic sequences are represented by
filled boxes, intronic sequences by open boxes. The sizes of the exonic and of the intronic sequences in nucleotides are indicated.
Fig. 2. PCR profiles of different S. cerevisiae wine strains using the
primers 3L, 3R, 4L and 5R. Lane m: size marker, 100-bp DNA
ladder (Gibco BRL).
V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 6371 66
Materials and methods) have been designed in the
present work. In a first step, PCR ability to give
polymorphic patterns was tested for the 12 primers
in different combinations. For this purpose, DNA was
used from four S. cerevisiae wine strains (T
73
, CECT
1483, CECT 1484 and CECT 1881), which had
previously been characterised as different by mtDNA
restriction analysis (Guillamon et al., 1994). The PCR
products displayed for each strain are shown in Table
2. When the primer pair 1L1R was used, the same
pattern (three bands: 1.4, 1.2 and 0.8 kbp) was
obtained for the four strains. Primer pairs 2L2R,
5L5R, 67L67R and 5L67R also gave monomor-
phic patterns for S. cerevisiae. In this case, each
primer pairs displayed a PCR pattern consisting of
single bands of 2.5, 0.3, 0.3 and 0.6 kbp, respectively.
No PCR amplification was detected using the primer
pair 4L4R but when 4L was combined with 5R, a
1.4- and a 2.0-kbp bands were obtained for strains
CECT 1483 and CECT 1881, respectively, although
the strains T
73
and CECT 1483 were indistinguishable
(a single 0.42-kbp band). However, it is possible to
distinguish both strains using the primer pair 3L3R
(single 0.3- and 2.0-kbp bands, respectively). The
PCR products obtained with 3L3R for the other
two strains were: CECT 1484 (2.0 kbp) and CECT
1881 (0.9 kbp). The higher polymorphism was
obtained by a multiplex PCR amplification reaction
with the four primers (3L, 3R, 4L and 5R) since
unique patterns were obtained for each strain as
shown in Fig. 2 (single 2.0-kbp band for strain CECT
1484, and two bands of 0.32 and 0.42 kbp, 1.4 and 2.0
kbp, 0.9 and 2.0 kbp for the strains T
73
, CECT 1483
and CECT 1881, respectively). For this reason, all the
subsequent PCR reactions were carried out with this
combination of primers in the same multiplex PCR
reaction.
Thirteen Saccharomyces dry yeast strains, com-
monly used in the wine industry as starter cultures
for must fermentation, have been used for the evalua-
tion of the present technique (Table 1). The PCR
profiles of the totality of the strains using the primers
Fig. 3. Application of the method for the differentiation of commercial Saccharomyces wine strains. Lane m: size marker, 100-bp DNA ladder
(Gibco BRL).
V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 6371 67
3L, 3R, 4L and 5R in a single PCR reaction are
presented in Fig. 3. Results show that all Saccharo-
myces strains can easily be differentiated from each
other and from the reference strains CECT 1483, 1484
and 1881, the PCR of which is presented in Fig. 2.
The differences given by the bands in the region
ranging from 0.3 to 0.4 kbp lead to the differentiation
of four groups of strains: Fermol Complet Killer,
Levuline Tirage Agglo, Fermol Cryoarome, Boero-
ferm, Uvaferm-L2056, Fermol Clarifiant, Fermol Pri-
meur and T
73
presenting a 0.32-kbp band; Uvaferm-
CEC and Uvaferm-CM presenting a 0.35-kbp band;
Uvaferm-VRB and Fermivin Crio presenting both
bands; and Uvaferm-PM in which both bands were
absent. Within the groups, the differences are given by
the presence or the absence of specific bands: the 1-
kbp band present in strain Uvaferm-CEC but absent in
Uvaferm-CM, the 1.1-kbp band present in Uvaferm-
VRB but absent in Fermivin Crio and the 2.0-, 1.45-,
1.1-, 1.05-, 1.0-, 0.6- and 0.45-kbp bands that serve to
differentiate between strains Fermol Complet Killer,
Levuline Tirage Agglo, Fermol Cryoaromae, Boero-
ferm, Uvaferm-L2056, Fermol Clarifiant, Fermol Pri-
meur and T
73
.
Fig. 4. PCR amplification profiles found when seven microvinifications were monitored. The microvinifications (1 to 7, see Materials and
methods section) differed from each other in the ratio in which the strains T
73
, CECT 1881, Fermol Complet Killer and CECT 1484 were
combined. The microvinifications 3 and 4 showed the same PCR-profile during the fermentation (panel A), panel (B) corresponds to
microvinification 5, panel (C) to the microvinification 1 and 2 and (D) to microvinification 7. Lane m: size marker, 100-bp DNA ladder (Gibco
BRL). Samples were taken daily from start of the fermentation.
V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 6371 68
3.2. Sensitivity of the PCR method
To study the utility of the technique for monitoring
wine fermentations, we first tested the sensitivity of
the detection method in microvinifications under
laboratory conditions. With this objective, we per-
formed seven small-scale fermentations combining
four strains at different ratios (see Materials and
methods section). In this way, we tried to simulate
the strain competition occurring in industrial pro-
cesses between the strains used as starters and the
indigenous strains. The four strains chosen were
clearly distinguishable by their COX1 profiles: T
73
(Figs. 2 and 3), CECT 1881 (Fig. 2), FCK (Fig. 3),
CECT 1484 (Fig. 2). The must samples, taken up at
different stages (days 1, 2, 3, 6, 7, 8, 9 and 10), were
used directly for PCR reactions. The results obtained
are present in Fig. 4. First, we established the sensi-
tivity of the method detection since the strains used up
to 20% (strain T
73
in microvinifications 6 and 7 and
strain CECT 1881 in microvinifications 1 and 2) were
not detected. Nevertheless, both strains were clearly
detected when they were used in percentages over
30%. Second, we showed the usefulness of the
method for fermentation process control since it was
possible to detect which strain was imposed under
laboratory conditions. Different situations were found
depending on the initial concentration of the strains
T
73
and CECT 1881. When they were combined at
proportions 1:1, 3:5 and 5:3, the COX1 profile of both
strains was simultaneously observed for the first 2 or 3
days. Then, one of the two strains became dominant
until the end of sampling as we can see in Fig. 4(A
and B). However, when one of the two strains was
used at 60% or 70%, only the PCR profile corre-
sponding to this strain was detected from the begin-
ning of the fermentation and remained as unique
throughout the whole process (Fig. 4C and D).
3.3. Control of inoculated wine fermentations
To assess whether this method is effective for
verifying the domination of an inoculated yeast strain
during an industrial vinification, two wine fermenta-
tions conducted in a Spanish winery (Torre Oria,
Utiel-Requena, Valencia) were studied. One fermen-
tation was inoculated with the dry yeast T
73
and the
other with Uvaferm L2056. The PCR patterns dis-
played by each strain are shown in Fig. 3. Wine
samples, taken at different stages of the fermentation
process, were directly subjected to PCR amplification
after centrifugation. As can be seen in Fig. 5, the PCR
profiles corresponding to the inoculated strains were
Fig. 5. Application of the method for monitoring two industrial
vinifications (A) and (B) inoculated with the commercial dry yeast
strains T
73
and Uvaferm-L2056, respectively. Lane m: 100-bp DNA
ladder (Gibco BRL). Samples were taken daily from start of the
fermentation.
V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 6371 69
detected in both tanks after a few hours of fermenta-
tion and became dominant during the whole process.
This result indicated that the method can be used to
detect prematurely if the strain used as a starter is
imposed.
Parallelly, amplification of the 5.8S-ITS region of
the ribosomal RNA unit was carried out for all the
samples in order to detect false-negative signals by
our developed PCR-based assay. At the same time,
since the length of the 5.8S-ITS PCR products is
species specific (Esteve-Zarzoso et al., 1999), this
control can be used to detect possible contaminants
in the must. In all cases, a single PCR band of 880 bp,
corresponding to the Saccharomyces sensu stricto
group, was detected (data not shown).
4. Discussion
Due to the increased introduction of new commer-
cial starters for enology, there is a great need to
develop rapid, simple and efficient methods to mon-
itor the course of fermentation. In the present work,
we propose a technique based on the variability in the
number and position of COX1 introns. An observation
to be made from the available data on COX1 sequen-
ces is that the number of introns and the position can
be quite variable between species. That means that
intron polymorphism can be used as a tool for fungus
differentiation. Within species, variations in the
mtDNA size are also mainly due to optional introns
(Clark-Walker, 1992). For example, strains of N.
crassa can have from none to four introns in COX1
(Burger et al., 1982; de Jonge and de Vries, 1983).
Among yeasts, the intraspecific COX1 intron variation
is specially well exemplified in K. lactis strains that
can be divided into three classes depending on the
structure of the COX1 gene (Skelly et al., 1991).
There is also evidence that strains of S. cerevisiae
are polymorphic for COX1: strain FY1679 possesses
seven introns (Fig. 1), while strain D273-10B pos-
sesses six due to the loss of intron ai5a (Foury et al.,
1998). In the present work, we have used primers
deduced from the exonic sequences of the S. cerevi-
siae strain FY1679 and the corresponding sequences
of other yeast and fungal species. Although 12 differ-
ent primers were tested, particularly good results were
obtained if a multiplex PCR amplification reaction is
performed with the four primers named 3L, 3R, 4L
and 5R.
The developed technique allows the differentiation
of commercial Saccharomyces wine yeast strains.
When the method was applied for monitoring vinifi-
cation processes, we realised that it is not useful to
examine the evolution of the different strains of S.
cerevisiae present during the course of the wine
fermentation. However, it is particularly interesting
to detect prematurely if the inoculated strain is
imposed in those wine-making processes that require
the use of dry yeast cultures. The main advantage for
the wineries is that the must can be used directly for
the PCR reaction. That means a considerable time-
frame reduction in comparison with mtDNA restric-
tion analysis, which requires the previous yeast DNA
isolation, or with other PCR-based methods, which
use purified DNA as template. The time needed to
obtain results with our proposed method is approx-
imately 8 h (5 min for sample washes, 5 h for the PCR
reaction and 3 h for the visualisation of the PCR
fragments), which allows the wine-making industries
to carry out effective interventions and corrections
(restarting of stuck fermentations) when the inocu-
lated strain is not imposed. From an industrial point of
view this is fundamental since important economic
losses are avoided. In addition, the equipment
required (a centrifuge, a PCR-thermocycler and an
electrophoresis equipment) is easily available in wine-
ries and highly qualified staff is not required. As the
variability of COX1 gene is general from all yeasts,
the method might be used for other yeast species.
Experiments testing all the designed primers to adapt
the method to other strains of S. cerevisiae and
Candida spp. are being conducted.
Acknowledgements
This work was supported by a Spanish Govern-
ment (C.I.C.Y.T.) grant to D.R. (95-0038-OP) and by
FEDER project and a (C.I.C.Y.T.) grant to A.Q.
(1FD97-0854-C03). M.T. F.-E. is the recipient of a
Postdoctoral Contract from the Ministerio de Educa-
cion y Ciencia.
Thanks are due to the Estacio` de Viticultura i
Enologia del Institut Catala` de la Vinya i el Vi
(Vilafranca del Penede`s, Spain) for supplying the
V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 6371 70
different commercial S. cerevisiae strains. Thanks are
also due to the Torre Oria winery for industrial
vinifications.
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