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ORIGINAL ARTICLE
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
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
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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
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
1019
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
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
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
945
917
73
638
50
29
100
80
100
88
96
98
10
1021
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
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-
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%
Pattern I 100%
Pattern XV 667%
Pattern I 333%
Pattern I 100%
Pattern VI 667%
Pattern VII 333%
Pattern VI 100%
Pattern XX 100%
Pattern XXI 100%
Pattern XL 100%
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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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