Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Molecular identification and characterization of wine yeasts

isolated from Tenerife (Canary Island, Spain)
S.S. Gonzalez1, E. Barrio2 and A. Querol1
1 Instituto de Agroqumica y Tecnologa de Alimentos (CSIC), Valencia, Spain
2 Instituto Cavanilles de Biodiversidad y Biologa Evolutiva, Universidad de Valencia Edif Institutos del Campus de Paterna, Valencia, Spain

Keywords
58S-internal transcribed spacer region,
restriction fragment length polymorphism,
mitochondrial DNA, spontaneous
fermentation, wine yeast
Correspondence
Amparo Querol, Instituto de Agroqumica y
Tecnologa de Alimentos (CSIC), Apdo. 73,
46100 Burjassot, Valencia, Spain.
E-mail: aquerol@iata.csic.es

2006/0424: received 27 March 2006, revised

and accepted 21 July 2006
doi:10.1111/j.1365-2672.2006.03150.x

Abstract
Aims: The present study was aimed at the identification, differentiation and
characterization of indigenous yeasts isolated from Tenerife vineyards (viticulture region that has never been characterized before). Microbiota were studied
from 14 samples taken during fermentations carried out in the 2002 vintage,
from 11 wineries belonging to five wine regions on Tenerife Island.
Methods and Results: Yeasts strains were identified and characterized through
restriction analysis of the 58S-internal transcribed spacer region and the mitochondrial DNA. At the beginning of alcoholic fermentation, 26 yeast species
were found, where 14 species were present in significant frequencies in only
one sample. Likewise, the Saccharomyces cerevisiae strains isolated are very specific, as they were only present in one wine region.
Conclusions: There were isolated specific yeasts from each region on Tenerife
Island. The founded yeasts may be responsible for distinctive and interesting
properties of the studied wines.
Significance and Impact of the Study: This study forms part of an extensive
taxonomic survey within the ecological framework of vineyards in Tenerife.
This investigation is an essential step towards the preservation and exploitation
of the hidden oenological potential of the untapped wealth of yeast biodiversity
in the grape growing regions of this island. The results obtained demonstrate
the value of using molecular genetic methods in taxonomic and ecological surveys. The results also shed some light on the ecology and oenological potential
of S. cerevisiae strains isolated from this unique environment.

Introduction
Tenerife is one of the seven Canary Isles and, of them all,
it boasts the greatest wealth of microclimates, thanks to
its volcanic mountainous terrain, which rises to a maximum height of 3718 m in Spains highest peak, the Teide.
As well as enjoying an exceptional subtropical location,
Tenerife can also lay claim to the added advantage of the
trade winds that make the seasons less severe and bless
the island with an ideal climate for viticulture.
The origins of vineyards in the Canary Islands, and
particularly in Tenerife, date back to the time of the conquest by the Kingdom of Castile. The Canary wines in
1018

the past were very famous for their liquor-like characteristics (malvasia wine). The quality of the grape varieties
introduced by settlers, along with the fact that Tenerife
was unaffected by the philoxera plague that ravaged European vineyards, made it possible to create an excellent
wine-growing reserve in the islands.
Tenerife has five wine-growing areas with a Denominacion de Origen (DO), covering a total of 7814 ha of
vines on the island. Tacoronte-Acentejo DO is the largest and most densely populated wine-growing area in
the Canary Islands, with 2422 ha of vines between 50 and
850 m above sea level. This was the first area in the Canary Islands to win a DO. Valle de Orotava DO has

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S.S. Gonzalez et al.

Wine yeast from Tenerife

926 ha of vines between 275 and 675 m above sea level.

YcodenDauteIsora DO has an area of 1003 ha of vines
between 75 and 925 m above sea level. Abona DO has
1995 ha of vines between 210 and 1780 m above sea level.
Valle de Guimar DO has 1468 ha of vines between 175
and 1400 m above sea level.
Wine fermentation is a complex microbial process involving the transformation of must into wine by the action of
different species of yeasts and lactic acid bacteria originally
present in the grapes and winery equipment. The yeast
population is especially important during spontaneous fermentation in young wines to be consumed without ageing.
It is generally accepted that apiculate species are the most
frequent yeasts on ripe grapes and fresh must (Pretorius
2000). On the contrary, fermentative species of the genus
Saccharomyces, predominantly Saccharomyces cerevisiae,
occur in extremely low numbers on healthy undamaged
berries or in soils (Frezier and Dubourdieu 1992; Martini
et al. 1996). Other species from the genera Candida, Brettanomyces, Cryptococcus, Kluyveromyces, Metschnikowia,
Pichia, Torulaspora, Rhodotorula and Zygosaccharomyces,
whose characteristics are useful to preserve the oenological
characteristics of wine-producing regions, are often present
in grapes and early stages of fermentation (Pretorius 2000).
The low ethanol tolerance of these yeasts and their inability
to ferment all sugars present in musts are the reasons why
they are naturally replaced by S. cerevisiae.
While a relatively large amount of information about
spontaneous fermentation exists in other European areas
(e.g. Guillamon et al. 1998; Pramateftaki et al. 2000; van
Keulen et al. 2003), no studies have been published on
the indigenous yeast from Tenerife so far. Because some
characteristics of this island make it unique, there may be
differences in the indigenous yeast population and some
yeast might be useful to obtain a region-specific character

Table 1 Winery, town, wine region and type

of wine where samples were taken

for the wines. Wine quality is also a consequence of the

diversity and composition of micro-organisms and their
dynamics and the frequency with which they appear.
Therefore, it is very important to gain more knowledge
about the changes the entire microflora undergo during
the alcoholic fermentation process.
This investigation was aimed at the identification, differentiation and characterization of indigenous yeasts isolated from Tenerife vineyards (viticulture region that has
never been characterized before). In this article, we study
the yeast microbiota present in 14 samples taken throughout fermentations carried out during the 2002 vintage,
from 11 wineries belonging to the five wine regions of
Tenerife Island. Yeast strains were identified and characterized through restriction analysis of the 58S-internal
transcribed spacer (ITS) region and of the mitochondrial
DNA (mtDNA).
Materials and methods
Sampling of yeast strains
Spontaneous fermentations were made using red (Listan
Noir, Negramoll, Tintilla and Vijariego) and white (Listan
Blanc, Malvasa, Gual, Verdello, Marmajuelo and Moscatel) grape varieties in 11 wineries belonging to the five
wine regions of Tenerife Island (Table 1). Sampling was
carried out during the 2002 vintage. At the selected wineries, the winemaking process involved manual harvest and
the typical vinification practices followed in these areas. It
is worth mentioning that dry yeast was never used in any
of these wineries (except in the winery belonging to Valle
de Orotava DO); however, in some wineries, different
types of wine were studied, because of the grape variety,
the grape origin or the vinification practice.

Sample no. DO

Winery

Town

Wine

S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14

Monje
Monje
Monje
Fajanetas
Fajanetas
Jose Lugo
Agustn Daz
Cumbres de Abona
Leoncio
Casiano
Felipe
Basilio
Coralia
Valle Oro

Santa Ursula
El Sauzal
Santa Ursula-Victoria-Sauzal
Taganana
Taganana
Guimar
Guimar
Arico
El Palmar (Buenvista)
Icod de los Vinos
La Guancha
San Juan de la Rambla
Tierra del Trigo (Silos)
La Orotava

Red
Red
Red (CM)
White
Red
White
White
White
Red
Rose
White
White
Red
White

Tacoronte-Acentejo
Tacoronte-Acentejo
Tacoronte-Acentejo
Tacoronte-Acentejo
Tacoronte-Acentejo
Valle de Guimar
Valle de Guimar
Abona
Ycoden-Daute-Isora
Ycoden-Daute-Isora
Ycoden-Daute-Isora
Ycoden-Daute-Isora
Ycoden-Daute-Isora
Valle de Orotava

CM, carbonic maceration (special vinification practice).

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1019

S.S. Gonzalez et al.

Wine yeast from Tenerife

Samples (100 ml) were taken aseptically at the beginning, middle and end of alcoholic fermentation. The criteria to define the middle and the end of fermentation
were based on sugar consumption. Aliquots (01 ml) of
several dilutions in 1% saline solution were spread onto
Wallerstein laboratory (WL) nutrient agar (Oxoid CM
309; Oxoid, Basingstoke, UK), adding 5% of ethanol to
make it specific for growing species of the genus Saccharomyces, and lysine agar selective medium (Oxoid CM
191) on which only the non-Saccharomyces yeasts of the
musts can grow (Heard and Fleet 1986; Mora and Mulet
1991). WL nutrient agar was supplemented with 50 ppm
SO2 (Panreac, Barcelona, Spain), 14% (v/v) ethanol
(Merck, Darmstadt, Germany) and 10 ppm of streptomycin (Sigma, Steinheim, Germany) to inhibit bacterial
growth. Lysine medium was also supplemented with
100 ppm of streptomycin. Plates were incubated at 28C
for 26 days. Plates containing between 30 and 300 colonies were counted. Fifty colonies from each fermentation
and medium were randomly selected for isolation and
identification. This number is statistically significant in
accordance with Snedecor and Cochram (1956).
The colonies selected were maintained by spreading in
new medium as the genomic DNA was extracted. As a
result of the high number of yeasts in this study, the
genomic DNA was extracted in different stages and then
frozen until the identification and characterization of the
yeasts.
Yeast identification
Colonies isolated at each sampling point were identified
by PCR amplification of the region spanning ITS1 and
ITS2 and the 58S rRNA gene (58S-ITS region) and
subsequent restriction analysis according to the work by
Esteve-Zarzoso et al. (1999).
PCR mixture contained 10 ll Taq polymerase buffer
(BioTools, B&M Labs S.A., Madrid, Spain), 100 lmol l)1
deoxynucleotides, 1 lmol l)1 of each primer, 2 U of Taq
polymerase. A volume of 4 ll of DNA diluted to
150 ng ll)1 was pipetted and transferred to a PCR tube
before adding the reaction mixture, in 100 ll of final volume. PCR amplification was carried out in a Techgene
thermocycler (Techne, Cambridge, UK). Amplification
was performed as follows: denaturation at 95C for
5 min, then 40 PCR cycles at 94C for 1 min, annealing
at 555C for 2 min and extension at 72C for 2 min,
followed by final extension at 72C for 10 min. PCR
products were run on a 14% agarose (Pronadisa, Laboratorios Conda S.A., Madrid, Spain) gel in 05 TBE
(45 mmol l)1 Trisborate, 1 mmol l)1 EDTA) buffer. After
electrophoresis, gels were stained with ethidium bromide
(05 lg ml)1) (AppliChem, Darmstadt, Germany) and
1020

visualized under UV light. A 100-bp DNA ladder marker

(Roche Molecular Biochemicals, Mannheim, Germany)
served as the size standard.
PCR products were digested without further purification with the restriction endonucleases CfoI, HaeIII and
HinfI (Roche Molecular Biochemicals) to identify all yeasts isolated from sampled wines. The PCR products and
their restriction fragments were separated on 14% and
3% agarose gels, respectively. Fragment lengths were
estimated by comparison to a mix (100 bp and 50 bp)
ladder. Restriction patterns were compared using the
basic local search tool program online at the IATA web
page http://motor.edinfo.es/iata/.
Strain differentiation
mtDNA restriction analysis was used to differentiate
strains of S. cerevisiae. Total DNA extraction and mtDNA
restriction analysis were performed according to the work
by Querol et al. (1992a,b)). HinfI was used as the most
suitable restriction endonuclease to differentiate among
S. cerevisiae strains (Querol et al. 1994). Fragments were
separated in 1% agarose gels in 05 TBE buffer.
Results
Yeast population dynamics during alcoholic fermentation
A total of 2546 yeast colonies were isolated, 1239 from
lysine medium and 1307 from WL nutrient agar. The
ITS1 and ITS4 primers were used to amplify the region
of the rDNA repeat unit that includes the two noncoding
regions designated as ITS1 and ITS2 and the 58S rRNA
gene (White et al. 1990). The PCR products digested with
CfoI, HaeIII and HinfI enzymes were analysed for all the
isolates comparing the molecular mass of the restriction
products with those described previously (Esteve-Zarzoso
et al. 1999). Tables 24 show the identification results
and the percentage of colonies corresponding to the species found in each sample at the beginning, middle and
end of alcoholic fermentation, respectively. At the beginning of the process, the most common yeast species are
non-Saccharomyces; however, after 34 days of fermentation, and as a result of the increasing concentration of
ethanol produced by S. cerevisiae, this species begins to
predominate and is responsible for alcoholic fermentation.
In Valle de Orotava DO, there is extensive use of active
dried yeast strains, and we could not find any wineries
that did not use this practice. Samples from the winery
corresponding to this area were taken from an uninoculated fermentation tank, but it is known that yeasts
present in the cellar equipment spread throughout the

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Wine yeast from Tenerife

Table 2 Distribution of yeast (%) in all the musts studied

Species
Candida sp.
Candida amapae
Candida apicola
Candida diversa
Candida glaebosa
Candida maritima
Candida parapsilosis
Candida solani
Candida stellata
Candida vinnaria
Candida vanderwaltii
Cryptococcus bhutanensis or Cryptococcus kuetzingii
Cryptococcus laurentii
Kluyveromyces thermotolerans
Debaryomyces hansenii, Torulaspora globosa or
Debaryomyces pseudopolymorphus
Hanseniaspora uvarum
Metschnikowia pulcherrima
Pichia fermentans
Pichia galeiformis
Pichia guilliermondii
Pichia kluyveri
Pichia petersonii
Pichia populi or Pichia thermotolerans
Saccharomyces cerevisiae
Torulaspora delbrueckii
Zygosaccharomyces florentinus

S1

S2

46

S3

S4

S5

S6

14

40

10
30

S7

S8

S9

S10

S11

S12

S13

611

467

50

40
133

267
33

74

2
6
146
10
185
74
20

278
294

279

60

852

22

5
112

29
73
44

28

59
219

147
147

28

10
6

15
88
132

37

25
30

37
111

588

278
389
55

182
24
454

133

37

67

2
30
37

59
111
364

Table 3 Distribution of yeast (%) in the middle of alcoholic fermentation

Species

S1

Candida sp.
Candida amapae
Candida vanderwaltii
Hanseniaspora uvarum
Metschnikowia pulcherrima
Pichia kluyveri
Rhodotorula glutinis or Rhodotorula mucilaginosa
Saccharomyces cerevisiae
Zygosaccharomyces bailli
Zygosaccharomyces bisporus
Zygosaccharomyces cidri or Zygosaccharomyces fermentati

375

S2

S3

S4

S5

S6

S7

S8

289

S9

S10

20

S11

S12

12

S13

273
87
87

83
55

40

2
545
153

179
89
357

826

winery, and inoculated yeast strains compete with the

native strains, quickly becoming the predominant yeast
strains (Querol and Ramon 1996). Therefore, the active
dried yeast strain used in the winery was found in these
samples from the beginning in large numbers. Thus, these
samples were not considered in this study.
Twenty-six yeast strains were identified by PCRrestriction fragment length polymorphism from all the analysed
yeasts isolated from the musts. As can be seen, in most
cases, we could differentiate each yeast species with preci-

945

917

73
638

50

29

100

80

100

88

96

98

10

sion according to the size of its PCR product and the

restriction patterns obtained with CfoI, HaeIII and HinfI.
However, in the present study, we observed that a group
of Candida species were indistinguishable by comparing
their 58S-ITS restriction patterns. They have been called
Candida sp. Other authors have recorded the same in
other studies (de Llanos Frutos et al. 2003).
Nearly half of the species found in the musts (4673%)
belong to the Candida genus (1772% Candida sp.,
1539% Candida vanderwaltii, 368% Candida stellata,

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1021

S.S. Gonzalez et al.

Wine yeast from Tenerife

Table 4 Distribution of yeast (%) at the end of alcoholic fermentation

Species

S1

S2

S3

S4

S5

S6

S7

S8

S9

S10

S11

S12

S13

Hanseniaspora uvarum
Saccharomyces cerevisiae
Zygosaccharomyces bisporus

98
2

100

2
98

38
962

100

100

100

100

100

100

100

100

100

288% Candida amapae, 226% Candida vinnaria, 142%

Candida parapsilosis, 112% Candida glaebosa, 077% Candida maritima, 057% Candida solani, 046% Candida apicola, 046% Candida diversa). Candida vanderwaltii was
present in all the samples taken in Tacoronte-Acentejo
DO, and it is generally found as the most frequent yeast
(especially in the Monje winery). Furthermore, this species has not been isolated from any other DO. Likewise,
C. amapae was present in the two samples taken from
Valle de Guimar DO, but nowhere else. The indistinguishable Candida species, C. vanderwaltii, C. stellata,
C. amapae and C. vinnaria were isolated at frequencies of
over 2%. The other species were isolated at low frequencies and can be considered sporadic. In fact, C. glaebosa,
C. maritima and C. solani were present in only one sample and they have not been described as wine yeasts, so it
is possible that they were from soil or water contaminants. However, C. parapsilosis appeared at a low frequency compared with the total number of yeasts isolated
from all the musts, but it was only present in a large percentage (185%) in Agustn Daz winery.
Hanseniaspora uvarum (1749%) and Metschnikowia
pulcherrima (1373%) were isolated from nine of the 13
samples studied. They seem to be common species in all
the wine-growing areas from Tenerife, and in some cases
they appeared to be the most frequent yeast (M. pulcherrima in Cumbres de Abona winery, H. uvarum in Agustn
Daz winery and both in Leoncio winery). This result is
in accordance with those obtained by other authors
(Schutz and Gafner 1993; Fleet 2003).
The second most important genus in the studied musts
is the Pichia genus (1067%) with six species belonging to
it (530% Pichia galeiformis, 231% Pichia populi or Pichia
thermotolerans, 181% Pichia kluyveri, 068% Pichia guilliermondii, 042% Pichia fermentans, 015% Pichia petersonii). As well as in the Candida genus, the last three
species could be yeasts from soil or water contaminants,
because they were only present in one sample and in low
frequency. Pichia populi or P. thermotolerans were also
isolated from one of the 13 samples studied (Jose Lugo
winery), at a frequency representing 30% of the total
yeasts in this winery.
At this initial stage, 14 yeasts were present in only one
sample (C. diversa, C. glaebosa, C. maritima, C. parapsilosis, C. solani, C. vinnaria, Cryptococcus bhutanensis or
1022

Cryptococcus kuetzingii, Cryptococcus laurentii, P. fermentans, P. guilliermondii, P. petersonii, P. populi or P. thermotolerans, Torulaspora delbrueckii and Zygosaccharomyces
florentinus) and some of them in significant frequencies
(364% Z. florentinus in Casiano winery, 30% P. populi or
P. thermotolerans in Jose Lugo winery, 294% C. vinnaria
in Cumbres de Abona winery and 185% C. parapsilosis in
Agustn Daz winery).
In the middle of alcoholic fermentation, S. cerevisiae
was the most frequent yeast (7426%), as at this stage the
level of alcohol is deadly to most of the non-Saccharomyces yeasts. Species belonging to the Candida genus represented 1128% of the total yeasts isolated (787% Candida
sp., 210% C. amapae and 131% C. vanderwaltii). Hanseniaspora uvarum (432%), three Zygosaccharomyces species
(Zygosaccharomyces bailli, Zygosaccharomyces bisporus and
Zygosaccharomyces cidri or Zygosaccharomyces fermentati)
(420%), M. pulcherrima (419%), P. kluyveri (118%) and
Rhodotorula glutinis or Rhodotorula mucilaginosa (057%)
were the rest of species isolated during this phase.
Once alcoholic fermentation had finished, 997% of the
yeast isolated belonged to the species S. cerevisiae. Only
four colonies belonged to other genus: three colonies to
H. uvarum (two in white wine from Fajanetas winery and
one in the sample of carbonic maceration from Monje
winery) and the other one to Z. bisporus (the sample of
rsula from Monje winery).
Santa U
Characterization of Saccharomyces cerevisiae strains
A rapid and simple method to characterize Saccharomyces
yeast based on mtDNA restriction analysis was used to
study S. cerevisiae population dynamics during alcoholic
fermentation. This method has been described for monitoring wine fermentations (Querol et al. 1994), while HinfI
is shown to be the restriction endonuclease recovering the
highest mtDNA variability.
There were 47 different mtDNA restriction patterns of
the 1307 Saccharomyces colonies isolated in this study,
that is to say, 47 different Saccharomyces strains (Table 5).
The table represents the frequencies of all mtDNA patterns obtained in the different samples analysed. Only
two of them (pattern I and XX) were present in musts,
likewise these patterns were represented by only two and
three colonies, respectively. In the middle of alcoholic fer-

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S.S. Gonzalez et al.

Table 5 Frequencies (%) of the different

mtDNA patterns obtained in each sample

Wine yeast from Tenerife

Sample

Must

S1

S2

S3

Pattern I 100%

S4

S5

S6

S7
S8

S9

S10

S11

S12

S13

Pattern XX 100%

Middle
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern

Final
I 60%
II 10%
III 10%
IV 10%
V 10%
I 375%
XV 375%
II 125%
VIII 125%
I 80%
XVI 20%
VI 364%
VII 138%
VIII 91%
IX 91%
X 91%
XI 45%
XII 45%
XIII 45%
XIV 45%
XV 45%
VI 644%
VII 214%
VIII 71%
IX 71%
XVII 50%
XVIII 25%
XIX 25%
XX 100%
XXI 375%
XVII 25%
XXII 25%
XXIII 125%
XXIV 571%
XXV 286%
XXVI 143%
XXVII 334%
XXVIII 222%
XXIX 222%
XXX 111%
XXXI 111%
XXXII 363%
XXXIII 91%
XXXIV 91%
XXXV 91%
XXXVI 91%
XXXVII 91%
XXXVIII 91%
XXXIX 91%
XL 50%
XLI 334%
XLII 83%
XLIII 83%
XLIV 545%
XLV 273%
XLVI 91%
XLVII 91%

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Journal compilation 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 10181025

Pattern I 100%

Pattern XV 667%
Pattern I 333%

Pattern I 100%
Pattern VI 667%
Pattern VII 333%

Pattern VI 100%

Pattern XVII 100%

Pattern XX 100%
Pattern XXI 100%

Pattern XXIV 100%

Pattern XXVII 60%

Pattern XXVIII 40%

Pattern XXXII 100%

Pattern XL 100%

Pattern XLIV 100%

1023

S.S. Gonzalez et al.

Wine yeast from Tenerife

mentation, the 47 different patterns were isolated. The

number of patterns isolated in each sample varied from
one (Cumbres de Abona winery) to ten (the white wine
of Fajanetas winery). Samples taken from Cumbres de
Abona winery belong to ecological wine, therefore selected commercial yeast was not employed, but this winery
makes another type of wine for which they use dried
yeasts. Therefore, the only pattern isolated in this sample
may belong to a dried yeast strain habitually used in this
winery. Despite the diversity of strains observed in the
middle of fermentation, only one or two dominated the
process in the latter stages.
Discussion
Yeasts are the prominent organisms in wine production
and determine wine flavour and other qualities by a
range of mechanisms and activities (Fleet 2003). While
S. cerevisiae is the main species in wine production, other
species play important roles. More recently, wine makers
and wine researchers have come to realize that nonSaccharomyces yeasts also contribute to the flavour and
quality of wine to a greater extent than previously
thought (Romano et al. 2003). This has led to the studies
of the presence and changes these non-Saccharomyces
yeasts undergo on grapes and in must, in order to determine their potential effects on the organoleptic qualities
of the final product. The differences in the composition
of wines made from different yeast species appear to be
quantitative rather than qualitative (Romano 1997). The
products of fermentation are usually identical, but relative amounts of the various compounds differ between
different yeast species.
Numerous studies from Spain, Portugal, Greece, Italy
and USA have focused on the spontaneous fermentation
of wines. The present study has been carried out in a viticulture region that has never been characterized before.
In terms of yeast diversity, our results are similar to those
found in other areas of the world (Schutz and Gafner
1993; Guillamon et al. 1998; Pramateftaki et al. 2000; van
Keulen et al. 2003). In this manner, H. uvarum and
M. pulcherrima seem to be common species in all the
wine-growing areas from Tenerife, and in some cases they
appeared to be the most frequent yeast (M. pulcherrima
in Cumbres de Abona winery, H. uvarum in Agustn Daz
winery and both in Leoncio winery). This result is in
accordance with those obtained by other authors (Schutz
and Gafner 1993; Fleet 2003).
In any event, the fact that we have found different
yeasts associated to a wine region (C. vanderwaltii from
Tacoronte-Acentejo DO and C. amapae from Valle de
Guimar DO) or specific to a winery (Z. florentinus in
Casiano winery, P. populi or P. thermotolerans in Jose
1024

Lugo winery, C. vinnaria in Cumbres de Abona winery

and C. parapsilosis in Agustn Daz winery). These yeasts
may be responsible for distinctive and interesting wine
properties.
Grapes are a primary source of yeasts that enter the
winery environment. Consequently, the ecological interactions that occur on grapes will contribute to the species
diversity in the winery. In this study, no correlation was
established between grape variety or origin of the grapes,
and the number of yeasts. Differences in population density on grapes seemed to be related to differences in climatic conditions in the town where the grapes came
from. Most yeast species isolated from musts were apiculate yeasts (H. uvarum and M. pulcherrima). This study
was carried out during the 2002 vintage, which was a wet
and rainy year, therefore in some cases grape quality was
not adequate. As known, damaged grapes increase the
incidence of these species (Fleet 2003).
The principal wine yeast, S. cerevisiae, is not prevalent
on wine grapes. Some researchers have not been able to
isolate this species from healthy, mature grapes, and these
observations have raised speculations and controversies as
to its origins in wine production (Martini et al. 1996;
Mortimer and Polsinelli 1999). The increase in ethanol
concentrations during alcoholic fermentation could also
explain the sequential growth of strains within a species.
Strains of S. cerevisiae, as well as those of other species,
vary in their tolerance to ethanol stress (Fleet 1992; Bauer
and Pretorius 2000). Previous results from several studies
of S. cerevisiae populations during spontaneous wine fermentations showed a great diversity of strains, of which
only one or two represented more than 50% of the total
biomass (Querol et al. 1992a; Schutz and Gafner 1993).
Generally speaking, our results are similar to these. Moreover, the S. cerevisiae strains isolated are very specific as
they were only present in one wine region.
The use of commercial strains of S. cerevisiae is becoming a common practice in winemaking. This practice
ensures a reproducible product, reduces the risk of wine
spoilage and allows a more predictable control of fermentation and quality. However, the use of pure yeast culture
could also reduce the production of some desired metabolites. Selected strains can be used as inoculum in wine fermentation only if the major characteristics of wine flavour
remain essentially unchanged. For this purpose, it is seen
as more advantageous to use natural autochthonous
strains, selected on the basis of desirable technological
characteristics including a preferred metabolic profile
(Romano et al. 2003). This study could be considered as a
first step in the selection of an autochthonous yeast strain
to avoid the frequent problem of breaks in fermentation,
or over long fermentation periods, while simultaneously
maintaining the characteristics of the wines of the island.

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Journal compilation 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 10181025

S.S. Gonzalez et al.

Acknowledgements
This work was supported by CICYT grant (ref. BIO200303793-CO3-01 and 02) from the Spanish Ministerio de
Ciencia y Tecnologa to AQ and EB.
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