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,*

aDepartamento de Biotecnologıa, Instituto de Agroquımica y Tecnologıa de Alimentos (CSIC), PO Box 73, 46100 Burjassot, Valencia, SpainbUnitat de Genetica Evolutiva, Institut ‘‘Cavanilles’’ de Biodiversitat i Biologia Evolutiva, Universitat de Valencia, Edificio de Institutos,

Campus de Paterna, PO Box 2085, 46071 Valencia, 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) 63–71

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;

Guillamon 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 T73,

CECT 1881, Fermol Complet Killer, and CECT

V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 63–7164

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 106 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 T73 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 106 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

T73 S. cerevisiae Lallemand

V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 63–71 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 Tris–HCl, pH

8.8 at 25 jC, 1.5 mM MgCl2, 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)

T73 CECT 1484 CECT 1483 CECT 1881

1L–1R 1.4 + 1.2 +

0.8

1.4 + 1.2 +

0.8

1.4 + 1.2 +

0.8

1.4 + 1.2 +

0.8

2L–2R 2.5 2.5 2.5 2.5

3L–3R 0.32 2.0 2.0 0.9

4L–4R – – – –

5L–5R 0.3 0.3 0.3 0.3

67L–67R 0.3 0.3 0.3 0.3

4L–5R 0.42 0.42 1.4 2.0

5L–67R 0.6 0.6 0.6 0.6

3L–3R–

4L–5Ra

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) 63–7166

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, DNAwas

used from four S. cerevisiae wine strains (T73, 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 1L–1R was used, the same

pattern (three bands: 1.4, 1.2 and 0.8 kbp) was

obtained for the four strains. Primer pairs 2L–2R,

5L–5R, 67L–67R and 5L–67R 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 4L–4R 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 T73 and CECT 1483 were indistinguishable

(a single 0.42-kbp band). However, it is possible to

distinguish both strains using the primer pair 3L–3R

(single 0.3- and 2.0-kbp bands, respectively). The

PCR products obtained with 3L–3R 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 T73, 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) 63–71 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 T73 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 T73.

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 T73, 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) 63–7168

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: T73

(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 T73 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

T73 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 T73 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 T73 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) 63–71 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 Penedes, Spain) for supplying the

V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 63–7170

different commercial S. cerevisiae strains. Thanks are

also due to the Torre Oria winery for industrial

vinifications.

References

Burger, G., Scriven, C., Machleidt, W., Werner, S., 1982. Subunit 1

of cytochrome oxidase from Neurospora crassa: nucleotide se-

quence of the coding gene and partial amino acid sequence of

the protein. EMBO Journal 1, 1385–1391.

Clark-Walker, G.D., 1992. Evolution of mitochondrial genomes in

fungi. International Revisions in Cytology 14, 89–127.

de Barros Lopes, M., Soden, A., Henschke, P.A., Langridge, P.,

1996. PCR differentiation of commercial yeast strains using

intron splice site primers. Applied Environmental Microbiology

62, 4514–4520.

Degre, R., Thomas, D.Y., Ash, J., Mailhiot, K., Morin, A., Dubord,

C., 1989. Wine yeasts strains identification. American Journal of

Enology and Viticulture 40, 309–315.

de Jonge, J.C., de Vries, H., 1983. The structure of the gene for

subunit I of cytochrome c oxidase in Neurospora crassa mito-

chondria. Current Genetics 7, 21–28.

Esteve-Zarzoso, B., Belloch, C., Uruburu, F., Querol, A., 1999.

Identification of yeasts by RFLP analysis of the 5.8S rRNA

gene and the two ribosomal internal transcribed spacers. Interna-

tional Journal of Systematic Bacteriology 49, 329–337.

Esteve-Zarzoso, B., Gostıncar, A., Bobet, R., Uruburu, F., Querol,

A., 2000. Selection and molecular characterisation of wine

yeasts isolated from the ‘El Penedes’ area (Spain). Food Micro-

biology 17, 553–562.

Fernandez-Espinar, M.T., Lopez, V., Ramon, D., Bartra, E., Querol,

A., 2001. Study of the authenticity of commercial wine yeast

strains by molecular techniques. International Journal of Food

Microbiology 70, 1–10.

Fleet, H., Heard, G.M., 1993. Yeast-growth during fermentation. In:

Fleet, G.H. (Ed.), Wine. Microbiology and Biotechnology. Har-

wood Academic Publishers, Chur, pp. 27–54.

Foury, F., Roganti, T., Lecrenier, N., Purnelle, B., 1998. The com-

plete sequence of the mitochondrial genome of Saccharomyces

cerevisiae. FEBS Letters 440, 325–331.

Guillamon, J.M., Barrio, E., Huerta, T., Querol, A., 1994. Rapid

Characterization of four species of the Saccharomyces sensu

stricto complex according to mitochondrial DNA patterns. In-

ternational Journal of Systematic Bacteriology 44, 708–714.

Guillamon, J.M., Barrio, E., Querol, A., 1996. Characterization of

wine yeast strains of the Saccharomyces genus on the basis of

molecular markers: relationships between genetic distance and

geographic or ecological origin. Systematic Applied Microbiol-

ogy 19, 122–132.

Hardy, C.M., Clark-Walker, G.D., 1991. Nucleotide sequence of the

COX1 gene in Kluyveromyces lactis mitochondrial DNA: evi-

dence for recent horizontal transfer of a group II intron. Current

Genetics 20, 99–114.

Lee, S.Y., Knudsen, F.B., 1985. Differentiation of brewery yeast

strains by restriction endonuclease analysis of their mitochon-

drial DNA. Journal of International Brewing 91, 169–173.

Lopez, V., Querol, A., Ramon, D., Fernandez-Espinar, M.T., 2001.

A simplified procedure to analyse mtDNA from industrial

yeasts. International Journal of Food Microbiology 68, 75–81.

Ness, F., Lavallee, F., Dubordieu, D., Aigle, M., Dulau, L., 1993.

Identification of yeast strains using the polymerase chain reac-

tion. Journal of Science and Food Agriculture 62, 89–94.

Querol, A., Jimenez, M., Huerta, T., 1990. Microbiological and

enological parameters during fermentation of musts from poor

and normal grape-harvests in the region of Alicante (Spain).

Journal of Food Science 55, 1603–1606.

Querol, A., Barrio, D., Huerta, T., Ramon, D., 1992a. Molecular

monitoring of wine fermentations conducted by active dry yeast

strains. Applied Environmental Microbiology 58, 2948–2953.

Querol, A., Barrio, E., Ramon, D., 1992b. A comparative study of

different methods of yeast strain characterization. Systematic

Applied Microbiology 15, 439–446.

Schutz, M., Gafner, J., 1993. Analysis of yeast diversity during

spontaneous and induced alcoholic fermentations. Journal of

Applied Bacteriology 75, 551–558.

Sekito, T., Okamoto, K., Kitano, H., Yoshida, K., 1995. The com-

plete mitochondrial DNA sequence of Hansenula wingei reveals

new characteristics of yeast mitochondria. Current Genetics 28,

39–53.

Skelly, P.J., Hardy, C.M., Clark-Walker, G.D., 1991. A mobile

group II intron of a naturally occurring rearranged mitochondrial

genome in Kluyveromyces lactis. Current Genetics 20, 115–

120.

Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W:

improving the sensitivity of progressive multiple sequence

alignment through sequence weighting, positions-specific gap

penalties and weight matrix choice. Nucleic Acids Research

22, 4673–4680.

Versavaud, A., Courcoux, P., Roulland, C., Dulau, L., Hallet, J.-N.,

1995. Genetic diversity and geographical distribution of wild

Saccharomyces cerevisiae strains from the wine-producing area

of Charentes, France. Applied Environmental Microbiology 61,

3521–3529.

Vezinhet, F., Blondin, B., Hallet, J.-N., 1990. Chromosomal DNA

patterns and mitochondrial polymorphism as tools for identifi-

cation of enological strains of Saccharomyces cerevisiae. Ap-

plied Microbiology Biotechnology 32, 568–571.

V. Lopez et al. / International Journal of Food Microbiology 81 (2003) 63–71 71