LWT - Food Science and Technology 101 (2019) 789–798

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LWT - Food Science and Technology

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Preservation of diversity and oenological properties of wine yeasts during T

long-term laboratory maintenance: A study of strains of a century-old Tokaj
wine yeast collection
Zoltán Kállaia,b, Walter P. Pflieglerb,1, Judit Mitercsákb, Gergő Szendeic, Matthias Sipiczkib,∗
a
Research Institute for Viticulture and Oenology, Tokaj, Hungary
b
Department of Genetics and Applied Microbiology, University of Debrecen, Hungary
c
Szendei Wine Ltd, Budapest, Hungary

A R T I C LE I N FO A B S T R A C T

Keywords: The effect of long-term laboratory maintenance on the biological diversity and certain oenolological properties
Yeast of wine yeasts was investigated by the analysis of 17 strains isolated from Tokaj (Hungary) wines more than a
Wine century ago and maintained since then in culture collections. The analysis of the D1/D2 domains of the large
Diversity subunit ribosomal RNA genes and the ITS sequences assigned them all to S. cerevisiae but divided them into two
Fermentation
groups. One group showed sequence identity with wine strains occurring mainly in countries of the region. The
Taxonomy
combined results of the interdelta, RAPD, microsatellite-primed PCR (MSP), mtDNA and karyotype analyses
found both groups diverse and defined each strain as unique, demonstrating that the long period of maintenance
under identical conditions has not resulted in convergent evolution in their genomes. High diversity was de-
tected also in certain phenotypic traits of oenological relevance (osmotolerance, killer phenotype, production of
glycerol, ethanol, H2S and volatile acids). In the microvinification experiments all strains performed better than
the laboratory strain S288c and were comparable to the industrial strain used as a control or even surpassed it in
certain parameters. Thus, the oenological abilities of wine yeasts can be preserved over long periods of main-
tenance under laboratory conditions.

1. Introduction
Fermenting grape must is a continuously changing environment that
exposes the yeast biota to a variety of stress conditions including high
sugar concentration, rapidly increasing ethanol and acid concentration,
anaerobiosis and depletion of nutrients (reviewed in Marsit & Dequin,
2015). The fermentation process is usually dominated by strains of S.
cerevisiae, S. uvarum (reviewed in Liu et al., 2017) and various Sac-
charomyces strains of mosaic genomes (reviewed in Sipiczki, 2011;
Marsit & Dequin, 2015; Albergaria & Arneborg, 2016), which can adapt
to the changes by modulating their physiology by processes like FAGE
(fast adaptive genome evolution) (Sipiczki, 2011).
It has been realized that fermentation can be reproducibly directed
and controlled by inoculation of the must with pure-culture yeasts, so-
called starter cultures selected on the basis of their favourable oeno-
logical characteristics. It was in 1889, when the results of fermentation
of grape must inoculated with pure yeast cultures were published for
the first time (Müller-Thurgau, 1889), but this practice became widely
used only since the 1970s (for reviews, see Hornsey, 2007; Gonzalez,
Munoz, & Carrascosa, 2011). In modern wine biotechnology the starters
are commercialized as active dry yeasts (ADY). The yeasts of these
preparations have been selected from the natural yeast biota of spon-
taneously fermenting wines in various wine-growing regions of the
Globe. Strains maintained in culture collections are rarely used as
starters because they represent only a small fraction of the real diversity
of the natural populations and usually perform poorly in wine fer-
mentation. Their lower efficiency is assumed to be due to the loss of
good fermentation capabilities as a result of prolonged culturing under
conditions not selective for better fermentation performance (Pizarro,
Vargas, & Agosin, 2007), but solid experimental proofs supporting this
hypothesis have not been presented yet.
It was just 12 years after Müller-Thurgau's publication (Müller &
Thurgau, 1889) that yeasts were isolated in the Royal Hungarian Re-
search Institute for Ampelology from wine samples collected in various

∗
Corresponding author.
E-mail address: lipovy@gmx.com (M. Sipiczki).
1
Present address: Department of Biotechnology and Microbiology, University of Debrecen, Debrecen, Hungary.

https://doi.org/10.1016/j.lwt.2018.12.002
Received 14 June 2018; Received in revised form 4 November 2018; Accepted 2 December 2018
Available online 03 December 2018
0023-6438/ © 2018 Elsevier Ltd. All rights reserved.
Z. Kállai et al. LWT - Food Science and Technology 101 (2019) 789–798

regions. In Tokaj, the wine-growing region having the longest docu-
mented tradition in botrytised wine production in the world (e.g.
Gregor, 1881), strains were isolated from old wines, wine sediments,
grape berries and leaves in the years 1901–1906 (Soós & Ásvány, 1950).
Some of these strains have been preserved under laboratory conditions
and are now available in the National Collection of Agricultural and
Industrial Microorganisms, Budapest, Hungary under the taxonomic
name S. cerevisiae, although they have not been subjected so far to an
expert taxonomic examination. Their phenotypic characterisation based
on laboratory tests was published in 1950 (Soós & Ásvány, 1950). Our
aims were to determine their taxonomic affiliation, explore their mo-
lecular diversity and compare their phenotypic properties with those
observed seven decades ago to find out whether they have changed
during the long period of laboratory maintenance. To assess their ap-
plicability as starter cultures, we also investigated their additional
characteristics of oenological relevance and tested them for fermenta-
tion performance in microvinification experiments.
on Nickerson agar (DIFCO) (Nickerson, 1953).
2.2. Molecular taxonomy
DNA was isolated from cultures grown aerobically in 50 ml of YPGL
at 24 °C for one day, and purified according to Hanna and Xiao (2006).
The ITS (ITS1-5.8S-ITS4) regions and the D1/D2 domains of the large-
subunit (LSU) rRNA genes of the rDNA repeats were amplified and
sequenced using the primer pairs ITS1-ITS4 (White, Burns, Lee, &
Taylor, 1990) and NL1-NL4 (O'Donell, 1993), respectively. The se-
quences obtained were deposited in the GenBank database (accession
numbers are listed in Table 1) and compared to the corresponding se-
quences of the type strains of S. cerevisiae, S. kudriavzevii and S. uvarum
using the blast2 tool available at http://ncbi.nlm.nih.gov/blast. To
obtain multiple alignments for identification of polymorphic sites, the
sequences were aligned with the Clustal W 1.7 (Thompson, Higgins, &
Gibson, 1994) algorithm.

2. Materials and methods 2.3. Analysis of molecular diversity

2.1. Yeast strains and media
The Tokaj strains used in this study were obtained from NCAIM
(National Collection of Agricultural and Industrial Microorganisms,
Budapest, Hungary) and are listed in Table 1. Killer phenotype was
tested with the S. cerevisiae strains NCYC 232 (K1 killer), NCYC 738 (K2
killer) and NCYC 1006 (killer sensitive) obtained from the National
Collection of Yeast Cultures (NCYC), Norwich, UK. The S. cerevisiae
strain S288c was used as chromosome size control in electrophoretic
karyotyping and as a laboratory control strain in the microvinification
experiments. The oenological properties of the Tokaj isolates were
compared also to those of the commercial strain Uvaferm 43.
Yeast strains were maintained on YPGA (20 g l−1 glucose, 20 g l−1
agar, 10 g l−1 yeast extract and 10 g l−1 peptone) plates or cultured in
YPGL (YPGA without agar). The media used to test the strains for
sporulation, acid production on solid medium, assimilation of carbon
sources were prepared as described by Van der Walt and Yarrow
(1984). Hydrogen sulphide production on solid medium was examined
RAPD analysis with primers RAPD24 (Baleiras Couto, Eijsma,
Hofstra, Huisin't Veld, & van der Vossen, 1996) and RF2 (Paffetti et al.,
1995) and microsatellite-primed PCR (MSP) with (GTG)5 (Lieckfeldt
et al., 1993) were performed with amplification programmes described
in Pfliegler, Horvath, Kallai, and Sipiczki (2014). For interdelta geno-
typing (δ-PCR) the primers delta1, delta2 and delta12 (Legras & Karst,
2003) were applied in combinations delta1-delta2 and delta12-delta2
with parameters described in Pfliegler and Sipiczki (2016). PCR reac-
tions were performed with the following programmes: 94 °C for 5 min,
30x(94 °C 50 s, Tm 50 s, 72 °C 50 s), 72 °C 5 min. Tm was set to 36 °C for
primer RF2, to 38 °C for primer RAPD24, to 50 °C for (GTG)5 and to
55 °C for δ-PCR. The amplified DNA was run in 14 g l−1 [24, RF2,
(GTG)5] or 20 g l−1 (δ-PCR), 10-cm-long agarose gels stained with
ethidium-bromide. The interdelta band patterns and the concatenated
RAPD/MSP band patterns were converted in two separate binary ma-
trices and analysed with the DendroUPGMA software (using Dice
coefficient) available at http://genomes.urv.es/UPGMA/ (Garcia-
Vallvé, Palau, & Romeu, 1999).

Table 1
List of the Tokaj strains and the accession numbers of the rDNA sequences.
Strain Isolation Accession numbers

ID number in the laboratory Original ID number in Source Location in the wine Date Isolated by D1/D2 ITS1-5.8S-ITS2
collection designationa NCAIMb region

10–1343 Tokaj1 00240 Young wine Mád 1901 Requinyi, G. KF560467 MF150087
10–1344 Tokaj2 00252 Furmint wine Tarcal 1901 Groh, G. KF560468 MF150088
sediment
10–1345 Tokaj4 00303 Young wine Tarcal 1905 Requinyi, G. KF560469 MF150089
10–1346 Tokaj5 00302 Young wine Mád 1905 Requinyi, G. MF062690
10–1347 Tokaj7 00203 Young wine Tarcal 1905 Requinyi, G. KF560470
10–1348 Tokaj8 00366 Wine sediment Mád 1903 Requinyi, G. KF560471 MF169657
10–1349 Tokaj9 00225 Wine sediment Mád 1901 Requinyi, G. KF560472 MF169658
10–1350 Tokaj10 00250 Young wine Tarcal 1901 Groh, G. KF560473
10–1351 Tokaj11 00233 Wine sediment Tarcal 1901 Groh, G. KF582940
10–1352 Tokaj12 00261 Young wine Mád 1902 Requinyi, G. KF582939
10–1353 Tokaj13 00254 Furmint wine Tarcal 1901 Groh, G. KF582938 MF169659
sediment
10–1354 Tokaj15 00248 Furmint wine Mád 1904 Requinyi, G. KF582937 MF276987
sediment
10–1355 Tokaj18 00368 Wine sediment Tolcsva 1901 Requinyi, G. KF582936 MF276988
10–1356 Tokaj19 00369 5-year old aszú wine Mád 1906 Requinyi, G. MF062691 MF276989
10–1357 Tokaj21 00236 Young Muscat Lunel Tarcal 1901 Requinyi, G. MF062692 MF375632
wine
10–1358 Tokaj22 00207 Young Furmint wine Olaszliszka 1903 Requinyi, G. MF078469 MF375633
10–1359 – 00171 5-year old aszú wine Mád unknown Requinyi, G. MF078470 MF375634

a
Soós & Ásvány, 1950.
b
National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary.

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Z. Kállai et al. LWT - Food Science and Technology 101 (2019) 789–798

Fig. 1. Clustal alignment of rDNA sequences. A.

Alignment of the D1/D2 domain segments con-
taining variable sites. For the accession numbers
of the sequences of the Tokaj isolates, see
Table 1. The accession numbers of the type
strains are AY497669 (S. cerevisiae CBS 1171NT),
AB040995 (S. kudriavzevii CBS 8840T),
AF005703 (S. paradoxus CBS 432NT) and
AJ279065 (S. uvarum CBS 395T). B. Alignment of
the segments of the internal transcribed se-
quences ITS1 and ITS2 containing variable sites.
For the accession numbers of the sequences of
the Tokaj isolates, see Table 1. The accession
numbers of the type strains are KY105073 (S.
cerevisiae CBS 1171NT), FJ196779 (S. kudriavzevii
CBS 8840T), AJ229059 (S. paradoxus CBS 432NT)
and AY130306 (S. uvarum CBS 395T).

For mtDNA RFLP, mitochondrial DNA was extracted from ex- fragments were separated by electrophoresis in 7 g l−1 agarose
ponential-phase cultures grown overnight in 50 ml YPGL at 24 °C ac- 0.5 × TBE gels, visualized and analysed as described above.
cording to the method of Defontaine, Lecocoq, and Hallet (1991) and Electrophoretic karyotyping was performed as described by
digested with 0.5 μl of EcoRV (Thermo Scientific). The restriction Karanyicz, Antunovics, Kallai, and Sipiczki (2017) with the Bio-Rad

791
Z. Kállai et al.
CHEF Mapper system.
2.4. Phenotypic characterisation
For the examination of colony morphology, small loopful amounts
of cells were inoculated into the centre of YPGA plates and incubated at
25 °C for 30 days. Cell morphology was examined in overnight YPGL
cultures with phase-contrast microscopy. Sporulation was detected
microscopically after incubation of the cultures on the potassium
acetate sporulation medium at 25 °C for 30 days. To test the strains for
hydrogen sulphide production on solid medium, 10 μl of overnight
cultures grown in YPL medium (106 cells ml−1) were dropped on plates
of Nickerson agar and incubated at 12 °C for 5 days. The hydrogen
sulphide produced by the yeast cells turned the colony brown. To detect
acid secretion on solid medium, 10 μl of overnight cultures grown in
YPL medium (106 cells ml−1) were dropped on plates of Custer's
medium. After 5 and 30 days of incubation at 25 °C, the clear zones
around the cultures were measured. The effect of osmotic conditions on
the growth of the yeast strains was examined by dropping 5 μl of
overnight YPGL cultures (106 cells ml−1) on YPGA plates supplemented
with 20, 300, 400, 500 or 600 g l−1 glucose and incubated at 25 °C for 5
days. For the examination of the ability of the strains to utilise ga-
lactose, maltose, mannitol, raffinose, melibiose and inulin, 5-μl samples
of suspensions of stationary-phase cells of YPGA cultures suspended in
sterile water were dropped onto media containing these compounds as
sole carbon sources.
Killer activity and sensitivity was tested with the killer-sensitive
tester strain NCYC 1006, the K1-type killer NCYC 232 and the K2-type
killer NCYC 738 on YPGA plates supplemented with methylene blue
(Woods & Bevan, 1968).
2.5. Microvinification
Fifty ml autoclaved Yellow Muscat must (204.3 g l−1 sugar,
6.38 g l−1 acid, pH 3.38) prepared from grapes harvested in Tarcal was
inoculated with cells of an overnight culture to obtain 5 × 106 cell
ml−1 concentration and was incubated at 12 °C without shaking for 30
days. The fermentations were performed in duplicate. Total sugar,
glucose, fructose and alcohol concentrations were measured on every 5
day. On the 30th day, the fermentation was terminated and the residual
sugar content, glycerol content, the final alcohol concentration, as well
as the total and volatile acid contents were measured with a Bruker
Alpha FTIR spectrometer (Bruker Optic GmbH, Germany). The results
were processed with the Bruker OPUS software. Fisher's test (MANOVA)
was applied to determine the reliability of the analytical measurements
(Svab, 1979). The reliability of the determination of quantitative
parameters of completed wines varied in range of p0.001 to p0.1, where
the levels of ethanol (E) and total sugars (TS) could be determined with
higher accuracy (FE = 18.2 > FTS = 16.3 > F0.001 = 15.38). With the
exception of pH (F0.05 = 4.41 > FpH = 4.31 > F0.1 = 3.01), all para-
meters were determined at p0,05 level (Table 4). The correlation coef-
ficient of amount/time lines was over 0.9675 (r2 = 0.9362) in all but
one case (strain 10_1353's was0,9053 (r2 = 0,8169), that means that
the fittness of regression can be accepted at p < 0.01 level. Hydrogen
sulphide production under fermentation conditions was evaluated in
the gas phase by using lead acetate test strips (Sigma-Aldrich 06728).
Foam production was monitored visually.
3. Results
3.1. Taxonomy
The LSU D1/D2 sequences of most Tokaj strains (Type I) were
identical with that of CBS 1171NT, the neotype strain of S. cerevisiae and
differed from the corresponding sequences of the type strains of S. ku-
driavzevii, S. paradoxus and S. uvarum at 10, 7 and 11 positions,
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LWT - Food Science and Technology 101 (2019) 789–798
respectively (Fig. 1A). Four strains (Type II) differed at two positions
from the S. cerevisiae neotype strain, and by 12, 9 and 13 positions from
the other three type strains.
The ITS1-5.8S-ITS2 sequences of the Type-I strains differed at 1
position from that of the S. cerevisiae neotype strain, and at 19, 11 and
25 sites from the sequences of the other type strains. The corresponding
differences were 5, 23, 15 and 29 in the case of Type-II (Fig. 1B). The
high sequence similarity of their rDNA regions to those of S. cerevisiae
CBS 1171NT implies that both groups of the Tokaj strains belong to the
species S. cerevisiae.
Consistent with this conclusion, all Tokaj strains grew at 35 °C,
utilised maltose, galactose and raffinose as carbon sources, but could
not assimilate and ferment mannitol, melibiose, and inulin, which are
traits routinely used for the differentiation of S. cerevisiae from the other
three related species (Antunovics, Irinyi, & Sipiczki, 2005; Vaughan-
Martini & Martini, 2011).
3.2. rDNA sequence diversity
The Type-II sequences differed from the Type-I strains and the S.
cerevisiae type strain by a T-to-C substitution in D1 and a T insertion in
D2 (Fig. 1A). A blast search in the GenBank database found only 16
sequences in which both markers were present (Table 2) indicating that
this genotype is rare in S. cerevisiae. Thirteen sequences were amplified
from wine strains isolated in Slovakia (5), Spain (5), Austria (1), France
(1), and Australia (1).
In the ITS1-5.8S-ITS2 segments, the groups differed at three loca-
tions. The Type-II sequences had a TT insertion and a C insertion in
ITS1 and a TT insertion in ITS2, which were missing both in the Type-I
strains and in S. cerevisiae CBS 1171NT (Fig. 1B). Each of these markers
occurred individually quite frequently in the database sequences but we
found only 11 sequences harbouring all three ones. All but one se-
quences were from strains that also had both characteristic Type-II
markers in the D1/D2 segments (Table 2). Three flor strains isolated in
Jura (France) and one strain isolated from wine in Liběchov (Czech
Republic) had ITS sequences containing the Type-II markers, but no
D1/D2 sequences were available for them.
3.3. Karyotype diversity
Ten chromosome patterns could be distinguished in the Tokaj
strains (Fig. 2). The strains 10-1343, 10-1344, 10-1345, 10-1346 and
10-1350 had unique patterns. The rest of the strains formed five groups.
The largest group consisted of four strains (10-1347, 10-1348, 10-1349
and 10-1353) isolated from three different substrates, in three different
locations and in three different years. Each of the remaining four groups
consisted of only two strains: 10-1351/10-1352, 10-1354/10-1355, 10-
1356/10-1359 and 10-1357/10-1358. The members of these strain
pairs also originated from different substrates, locations and even years.
Obviously, there was no correlation between the chromosomal patterns
and the origin of the strains.
3.4. Interdelta, RAPD and microsatellite-primed PCR (MSP) diversity
The interdelta patterns allowed much better differentiation of the
strains than the combined RAPD/MSP patterns. The alignment of the
UPGMA dendrograms is shown in Fig. 3. The Type-II rDNA strains form
compact groups on both dendograms. Except for the two pairs of strains
(10-1347/10-1349 and 10-1354/10-1355) indistinguishable by the
analysis of these markers, neither dendrogram is fully in line with the
relationships among the chromosomal patterns.
3.5. Diversity of mitochondrial genomes
The restriction analysis of the mitochondrial DNA of the 17 Tokaj
strains resulted in 13 different patterns (Fig. 4). The patterns of
Z. Kállai et al. LWT - Food Science and Technology 101 (2019) 789–798

Table 2
List of database sequences containing the characteristic markers of the Type-II Tokaj strains.
Strain/clone Accession number of Origin of strain

a a
D1/D2 sequence containing both markers ITS1-5.8S-ITS2 sequence containing all three markers Substrate Country

ATCC 834 KU729158 KU729072 Whey USA

CBS 2183 KY109350 KY109350 Wine France
CBS 2247 KY109262 KY105059 Unspecified juice South Africa
CBS 2789 KY109365 KY105114 Wine Slovakia-Hungary
CBS 2805 KY109366 KY105143 Wine Slovakia
CBS 2807 (YJM270) KY109360 KY109360 Wine Slovakia
CBS 2808 n.a. KY105138 Wine Czech Republic
CBS 2814 CBS sequence KY105135 Wine Slovakia
CBS 2963 KY109417 CBS sequence Distillery Denmark
CBS 4054 KY109258 KY105036 Wine Spain
CBS 4079 KY109340 l.d. Wine Spain
CBS 5835 KY109263 l.d. Wine Spain
CECT 11762 AJ544259 l.d. Sherry Spain
CECT 12664 AJ544261 n.d. Sherry Spain
HA1835 AM262822 AM262824 Wine Austria
K-53-15-44 KY816824 n.a. Ice wine Slovakia
K-53-15-45 KY816825 n.a. Ice wine Slovakia
YJM1574 (AWRI1775) CP006432 d. Sherry Australia
CAV21 n.a. FM177897 Wine, flor France
MAC51 n.a. FM177898 Wine, flor France
LRJura n.a. FM177899 Wine, flor France

d.: different.
n.a.: not available.
l.d.: very different due to large deletions.
CBS sequence: sequence is available in the CBS Strain Database (http://www.westerdijkinstitute.nl/Collections/Biolomics.aspx?Table=CBS%20strain%20database).
a
For the description of the markers see Figs. 1 and 2.

10–1351 and 10-1352 were indistinguishable. Remarkably, this pair
also had identical karyotypes and MSP patterns, formed sister branches
on the interdelta dendrogram and had Type-I rDNA patterns. The
strains 10-1347, 10-1349 and 10-1355 also had identical mtDNA pat-
terns but they differed in karyotypes, interdelta and MSP patterns and
one of them had Type-II rDNA.
3.6. Morphology
All but two strains formed colonies with smooth or slightly sectored
surface and with entire or slightly undulate edges after one month of
incubation on YPGA (Fig. 5A, C and D). 10-1344 formed colonies that
had wrinkled surface and sectors of rough, venose edges (Fig. 5B). 10-
1358 had smooth but heavily sectored colonies with highly undulate
edges (Fig. 5E). The microscopic examination of the cell morphology
distinguished three main shape categories: globular, ovoid and elon-
gated (Fig. 5F–J). Irrespective of their shape, the cells of all strains
proliferated by multilateral budding. When cultured in liquid medium,
the cells of 10–1344 and to lesser extent also those of 10–1343 ag-
gregated (Fig. 5K and L). The morphological properties recorded 68
years ago (Soós & Ásvány, 1950) were compared with those observed in
this study and found essentially identical (data not shown). The results
of the sporulation tests are shown in Table 3.
3.7. Physiology and killer phenotype
Table 3 also compares the physiological properties investigated 68
years ago and in this study. Differences were found in the utilisation of

Fig. 2. Electrophoretic karyotypes of strains. Chromosome numbering and size in base pairs of S. cerevisiae S288c shown on the left side is according to https://www.
yeastgenome.org/strain/S288C.

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Z. Kállai et al.
Fig. 3. Alignment of midpoint-rooted interdelta and MSP UPGMA dendro-
grams. Roman II: strains having Type-II rDNA patterns. Strains marked with
identical symbols have identical karyotypes. Numerals on the branches of the
interdelta dendrogram: levels of alcohol at the end of fermentation (Table 4)
numbered from the highest (1 = 129.5 g l−1) to the lowest (12 = 119.0 g l−1)
level.
inulin and raffinose in certain strains. The strains were tested also for
properties of oenological relevance which were not investigated 68
years ago. Considerable diversity was found in all traits. Two strains
produced less and 14 strains produced more H2S than the commercial
strain when tested on agar plates at 12 °C (Table 3). Most strains were
less acidogenic on agar plates than the commercial strain (data not
shown) and all but one strain were more osmotolerant than the com-
mercial strain (Table S1). Two strains showed killer activity, two strains
Fig. 4. Band patterns of mitochondrial DNA digested with EcoRV. M: size ladder.
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LWT - Food Science and Technology 101 (2019) 789–798
were neutral (neither killer nor sensitive), whereas the rest was sensi-
tive to the toxins of both the K1 and the K2 tester strains (Table 3).
3.8. Microvinification
High diversity was detected in the fermenting vigour manifested in
diverse sugar consumption and ethanol production during the first five
days of fermentation (Table 4 and Fig. 6), but the initial differences
gradually diminished in the later phases (Table 2S and Fig. S1). By the
end of the 30-day fermentation the sugar content fell from the initial
204 g l−1 to 2.05–6.25 g l−1 in the experiments with the wine strains
and to 11.70 g l−1 with the laboratory strain (Table 4). The final
ethanol content only slightly varied; the difference between the lowest
(S288c) and the highest (10-1349) value was 13 g l−1. The high ethanol
content and the low diversity indicate that all Tokaj strains have fairly
good alcohol tolerance and good sugar-to-alcohol conversion cap-
abilities.
All Tokaj strains proved to be glucophilic (Table 4) but to varying
degrees. The wines fermented by 10–1343 and 10-1348 contained no
glucose in the residual sugar and the wine fermented by 10–1351 had
25 times less glucose than fructose. Only limited variations were de-
tected in total acidity and in the final glycerol content, but the acetic-
acid levels showed considerable diversity. All strains showed low foam
production.
Soós and Ásvány (1950) described the strain 10-1345 as the best
alcohol producer, and proposed the strains 10-1347 and 10-1358 for
application to inoculated wine fermentation because of their ability to
efficiently ferment grape musts of high sugar contents and produce
pleasant aroma compounds. In our examinations two of these strains,
10-1345 and the 10-1347, proved to be very good alcohol producers,
and the third one, 10-1358, formed the highest amount of glycerol
(Table 4). The good performance of these strains in our experiments
demonstrates that they have not lost their oenological values during the
more than six decades of maintenance in culture collections.
4. Discussion
To determine the taxonomic affiliation of the old Tokaj isolates, we
subjected them to molecular examination and physiological tests used
in modern yeast taxonomy. The analysis of the sequences of D1/D2
domains and the ITS regions of the rDNA arrays as well as the phy-
siological tests assigned them to S. cerevisiae. Remarkably, no S. uvarum
was found among them. S. uvarum is quite common in the Tokaj region
Z. Kállai et al. LWT - Food Science and Technology 101 (2019) 789–798

Fig. 5. Examples of morphological types. Colony morphology (A to E), cell morphology (F to J) and flocculation (K to O) of 10–1343 (A, F and K), 10-1344 (B, G and
L), 10-1347 (C, H and M), 10-1349 (D, I and N) and 10-1358 (E, J and O).

(e.g. Antunovics et al., 2005; Magyar & Bene, 2006; Minarik & Laho,
1962; Sipiczki, Romano, Lipani, Miklos, & Antunovics, 2001).
Albeit the rDNA analysis unambiguously assigned all strains to S.
cerevisiae, their rDNA sequences were not identical and could be divided
in two groups (Type I and Type II). The Type-II strains differed from the
S. cerevisiae neotype strain at two positions in the D1/D2 domain and at
five positions in the two ITS segments. We found the same divergence
from the neotype strain in database sequences of numerous wine strains
isolated in Slovakia and Austria. The simultaneous presence of all these
differences can be a specific molecular marker of an indigenous (sub)
population of S. cerevisiae wine strains of this Central-European geo-
graphical region. These strains appear to have been components of the

Table 3
Morphological and physiological properties of Tokaj strains.
Strain Sporulationa H2S production Killer Utilisation of galactosee Utilisation of inuline Utilisation of raffinosee
phenotyped
68 years agod Now On Nickerson During 68 years agof Now 68 years Now 68 years Now
agarb fermentationc agof agof

10–1343 ++ 0 1 – N + + – – + w
10–1344 + + (9.8%) 3 – S + + – – + +
10–1345 0 0 3 +++ S + + – – + w
10–1346 +++ ++ 4 + K + + – – + +
(11.6%)
10–1347 ++ 0 3 +++ S + + – – + +
10–1348 + 0 2 +++ S + + w – + +
10–1349 +++ 0 3 ++ S + + – – – +
10–1350 ++++ ++ 3 – S + + – – + +
(20.3%)
10–1351 ++++ + (2.6%) 4 – S + + + – – +
10–1352 0 + (7.6%) 4 + S + + – – + +
10–1353 0 0 4 ++ S + + – – + w
10–1354 +++ 0 4 + K + + w – + +
10–1355 + 0 4 ++ S + + w – + +
10–1356 ++ + (2.1%) 4 – S + + – – + w
10–1357 + + (4.0% 4 – S + + – – + +
10–1358 0 0 1 – N + + – – + w
10–1359 n.d. + (0.8%) 4 – S n.d. + n.d. – n.d. w
Uvaferm 43 n.d. n.d. 2 – S n.d. + n.d. -. n.d. +

n.d.: not determined.

a
0: no sporulation; +: 1–10% sporulation; ++: 10–50% sporulation; +++: 50-80: ++++: 80–90% sporulation.
b
At 12 °C. Colour scale from weak browning (1) to dark browning (4).
c
-: no reaction on the strip; + weak browning on the strip; intermediate reaction on the strip; ++: intensive browning on the strip.
d
K: killer; S: sensitive to both K1 and K2 killer toxin; N: neutral (neither killer nor sensitive).
e
-: no growth; w: weak growth; +: growth.
f
Described in Soós & Ásvány, 1950.

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Z. Kállai et al. LWT - Food Science and Technology 101 (2019) 789–798

Table 4
Wine composition on the 30th day of fermentation.
Strains Component (g l−1) pH

Ethanol Total sugar Fructose Glucose Glycerol Total acid Acetic acid

a a b,c,d a a,b,c d
10–1343 126.0 3.30 3.10 0.00 7.05 7.70 0.45b 3.05
10–1344 126.0a 3.85a 2.95b,c,d 0.70a,b,c 6.50a,b,c 7.30b,c,d 0.55b,c 3.04
10–1345 128.5a 3.35a 2.35a,b,c 0.70a,b,c 7.35a,b,c 7.30b,c,d 0.50b 3.01
10–1346 127.5a 2.65a 2.20a,b 0.20a,b 6.90a,b,c 7.00a,b,c,d 0.45b 3.07
10–1347 128.5a 2.40a 1.60a 0.60a,b,c 6.25a,b,c 6.60a,b,c 0.40a,b 3.11
10–1348 125.5a 2.05a 1.90a 0.00a 6.65a,b,c 6.75a,b,c,d 0.45b 3.05
10–1349 129.5a 3.75a 2.80a,b,c,d 0.95b,c 6.15a,b 6.55a,b,c 0.25a 3.06
10–1350 128.5a 3.85a 3.15b,c,d 0.35a,b,c 6.85a,b,c 6.50a,b 0.45b 3.05
10–1351 122.0a 4.10a 3.85,d,e 0.15a,b 6.50a,b,c 7.15a,b,c,d 0.40a,b 2.96
10–1352 127.0a 3.20a 2.80a,b,c,d 0.35a,b,c 6.05a,b 7.10a,b,c,d 0.39a,b 2.94
10–1353 126.5a 3.70a 2.95b,c,d 0.70a,b,c 6.75a,b,c 7.25b,c,d 0.45b 2.98
10–1354 124.5a 2.90a 1.95a,b 0.85a,b,c 5.90a,b 6.00a 0.25a 3.13
10–1355 124.5a 5.70b 4.40e 1.20c,d,e 6.30a,b,c 6.20a 0.39a,b 3.04
10–1356 122.5a 5.00a,b 3.70d,e 1.15c,d 7.70c,d 6.90a,b,c,d 0.48b 3.02
10–1357 124.5a 4.10a 3.55c,d,e 0.45a,b,c 6.55a,b,c 7.50b,c,d 0.46b 2.97
10–1358 121.5a 6.25b 3.85d,e 2.00d 8.90d 6.75a,b,c,d 0.52b,c 3.10
10–1359 119.0a 2.65a 2.25a,b 0.35a,b,c 7.40a,b,c,d 7.00a,b,c,d 0.69b,c 3.06
uva43 122.5a 2.75a 2,10a,b 0.35a,b,c 7,05a,b,c 6,95a,b,c,d 0.59b 3.06
S288 116.5a 11.70c 9.70+ 2.05e 6.95a,b,c 7.25b,c,d 0.45b 2.99

F 18.2*** 16.3*** 7.62* 4.18* 4.60* 5.41* 6.48* 4.3+

F: Reliability of the analytical determinations: +F0.1 = 3.01, *F0.05 = 4.41, **F0.01 = 8.40, ***F0.001 = 15.38. The values labeled with the same letter do not differ
significantly at P = 5% probability level.
The LSD05 of pH was 0.08, thus no significant difference could be revealed at P = 5% probability level with the applied analytical method between batches.

Fig. 6. Dynamics of ethanol production during microvinification.

autochthonous yeast biota persisting over long periods of time, since
the years of the isolation of the Tokaj strains (1901-1906) and of two
Slovak strains (1954) up to the present (2017) when the two ice-wine
yeast clones were found in Slovakia. The more sporadic appearance of
this pattern in certain Spanish, one French and one Australian wine
region and in different substrates might be due to radiation from
Central-European wineries. The similarity of the ITS sequences of the
Type-II strains to the ITS sequences of flor yeasts isolated in Jura
(Charpentier, Colin, Alais, & Legras, 2009) suggests close relationship
between the two groups of strains. Further experiments are needed to
verify this possibility because no D1/D2 sequences are available for the
Jura strains and the Tokaj strains are able to utilise galactose as a
carbon source and are glucophilic.
The comparative analysis of less conserved molecular markers (in-
terdelta, MSP, mitochondrial DNA and karyotype) revealed much
higher diversity among the strains. When all markers are considered
together, the overwhelming majority of strains possess unique patterns,
suggesting that most strains have unique genome structures. Exceptions
are the Type-I pair 10-1354/10-1355 and the Type-II pair 10-1347/10-
1349 whose members were indistinguishable in all tests. Interestingly,
neither pair was isolated from the same location and in the same year.
Apart from these identities, both the Type-I and the Type-II strain
groups were diverse, but remarkably, formed well-separated clusters as
if they were biologically isolated.
The different origin of the molecularly indistinguishable strains in-
dicates that in the early 1900s there must have been S. cerevisiae strains

796
Z. Kállai et al.
in the Tokaj region that were present in multiple wineries and remained
abundant for years. The identity of the molecular patterns of the 10-
1354/10-1355 and the 10-1347/10-1349 strain pairs further suggests
that their genomes have not changed over the decades of laboratory
maintenance. In contrast, certain strains which were isolated in the
same location and year (e.g. five strains isolated in Tarcal in 1901) had
different molecular patterns. These strains might have been different
already at isolation but it is equally possible that they originated from
the same clone and have diverged afterwards, during propagation
under laboratory conditions. However, when comparing the molecular
patterns, one has to bear in mind that with fingerprinting methods,
exactly identical conditions have to be applied to all strains in all re-
actions in order to obtain high levels of reproducibility (e.g. Pfliegler
et al., 2014) and minor changes in band patterns are often hard to
evaluate. Furthermore, the higher diversity observed in interdelta
analysis may not precisely reflect the diversity at the time of strain
isolation because the specific stresses exerted on the strains by various
preservation methods might have mobilized certain Ty1 retro-
transposons (which the delta sequences are partially associated with).
For example, in a previous study (Stamenova et al., 2008) freezing and
thawing were found to increase Ty1 transposition frequency.
Somewhat unexpectedly, no correlation was found between the
karyotypes and the origin of the strains. We do not know what the
karyotypes might have looked like at the time of isolation of the strains,
but it is clear that the long-lasting laboratory maintenance have not
made them uniform. It is pertinent to mention here that in a previous
study, we found no correlation between the chromosomal patterns and
the location and wine type from which the Saccharomyces strains were
isolated (Csoma, Zakany, Capece, Romano, & Sipiczki, 2010). In a dif-
ferent study, no correlation was found between the ability to form
velum and the karyotypes of the strains, while clear correlation was
observed between velum formation and certain interdelta and micro-
satellite patterns (Charpentier et al., 2009; Legras, Erny, & Charpentier,
2014).
The high molecular diversity was accompanied with comparably
high diversity in certain phenotypic properties and traits such as colony
morphology, cell morphology, sporulation efficiency and H2S produc-
tion. The diversity was lower in the killer phenotype and flocculation.
When all examined properties are taken together, practically each
strain has a unique phenotype. This is in line with the results of the
molecular (genetic) examinations which also defined almost all strains
as unique. The high phenotypic diversity is not surprising because the
phenotype is basically determined by the genotype. For example recent
results (e.g. Granek & Magwene, 2010) demonstrated that the mor-
phology of S. cerevisiae colonies is under the control of complex net-
works of genetic determinants which are involved also in other cellular
processes such as the integration of nutrient signals and the response to
certain changes in the environment. Sporulation is another trait that is
controlled by complex genetic interactions. The major determinant of
sporulation is mating-type heterozygosity (cells of homothallic strains
are usually heterozygous) but the efficiency of the process depends on
the function of a large number of downstream genes (for a review, see
Herskowitz, 1988). Inactivation or biased activity of any of these genes
can prevent sporulation or at least reduce its efficiency. Another reason
of poor sporulation can be chromosomal heterogeneity which impairs
the accuracy of chromosome pairing in the meiotic prophase. Improper
chromosome pairing occurs mainly in interspecies hybrids that have
two (partially) different sets of chromosomes (e.g. Karanyicz et al.,
2017). As none of the Tokaj strains had supernumerary bands in their
karyotypes, it is unlikely that any of them is interspecies hybrid or has
an alloaneuploid genome. Numerous studies have demonstrated that
the adaptation of yeast cultures to changing conditions is usually as-
sociated with genomic changes (adaptive evolution), and in different
cultures exposed to the same stress condition for longer periods of time,
usually variants of similarly changed genomes (including similar
changes in karyotypes) become dominant (convergent adaptive
797
LWT - Food Science and Technology 101 (2019) 789–798
evolution) (e.g. Gresham et al., 2008; Mangado, Morales, Gonzalez, &
Tronchoni, 2018; Piotrowski et al., 2012). Interestingly, the long-term
laboratory maintenance does not appear to have elicited detectable
convergent adaptive evolution in the genome structure and physiolo-
gical properties of the Tokaj strains.
Concerning the phenotypic traits of oenological relevance, the di-
versity of the individual traits varied considerably. Whereas the strains
were highly diverse in H2S production (both on the solid medium and
under fermentation conditions) and the initial fermentation vigour,
moderately diverse in glycerol production, most of them only slightly
differed from each other in fermentation power, ethanol production and
the acidity at the end of fermentation. Interestingly, most of them were
superior to the industrial strain and much better than the laboratory
strain in the fermentation of the must prepared from locally harvested
grapes. Again, we do not know what wine-making capabilities these
strains had at the time of their isolation, but their good performance in
our lab-scale fermentation trials demonstrates that the long-lasting
maintenance in non-wine-related environment has not been detri-
mental to their vinification capacity. This finding raises doubts about
the widespread view that prolonged culturing under laboratory condi-
tions compromises fermentation capabilities (e.g. Pizarro et al., 2007).
Thus, certain members of the century-old collection can be good al-
ternatives for the region-alien starters widely used in the Tokaj region.
Further investigations are needed to select the best candidates for such
purposes.
Conflicts of interest
The authors declared no conflict of interest.
Acknowledgements
The authors would like to thank Dr. Gyula Oros for his helpful ad-
vice on statistical analysis. The expert technical assistance of Anita
Bordán-Kovács is greatly appreciated. This research was financed from
the grant K-124417 provided by the National Research, Development
and Innovation Office of Hungary.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.lwt.2018.12.002.
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