b
a
z
i
l
i
a
n
j
o
u
n
a
l
o
m
i
c
o
b
i
o
l
o
g
y
4
7
(2
0
1
6)
181–190
h
p://www.bjmic obiol.com.b /
Food
Mic obiology
Isola ion,
iden ifica ion
and
cha ac e iza ion
o
egional
indigenous
Saccha omyces
ce e isiae
s ains
Hana ˇ
Su anská∗,
Dana
V áno á,
Jiˇ
ina
Omelko á
B no
Uni e si y
o
Technology,
Facul y
o
Chemis y,
Depa men
o
Food
Science
and
Bio echnology,
Pu kyˇ
no a
464/118,
612
00
B no,
Czech
Republic
a
i
c
l
e
i
n
o
A icle
his o y:
Recei ed
29
July
2014
Accep ed
19
May
2015
Associa e
Edi o :
Ma icê
Noguei a
de
Oli ei a
Keywo ds:
Saccha omyces
genus
Oenological
p ope ies
ITS-PCR-RFLP
In e del a
PCR
yping
Species-specific
p ime s
a
b
s
a
c
In
he
p esen
wo k
we
isola ed
and
iden ified
a ious
indigenous
Saccha omyces
ce e isiae
s ains
and
sc eened
hem
o
he
selec ed
oenological
p ope ies.
These
S.
ce e isiae
s ains
we e
isola ed
om
be ies
and
spon aneously
e men ed
mus s.
The
g ape
be ies
(Sau i-
gnon
blanc
and
Pino
noi )
we e
g own
unde
he
in eg a ed
and
o ganic
mode
o
a ming
in
he
Sou h
Mo a ia
(Czech
Republic)
wine
egion.
Mode n
geno yping
echniques
such
as
PCR-finge p in ing
and
in e del a
PCR
yping
we e
employed
o
di e en ia e
among
indige-
nous
S.
ce e isiae
s ains.
This
combina ion
o
he
me hods
p o ides
a
apid
and
ela i ely
simple
app oach
o
iden ifica ion
o
yeas
o
S.
ce e isiae
a
s ain
le el.
In
o al,
120
isola es
we e
iden ified
and
g ouped
by
molecula
app oaches
and
45
o
he
ep esen a i e
s ains
we e
es ed
o
selec ed
impo an
oenological
p ope ies
including
e hanol,
sul u
dioxide
and
osmo ic
s ess
ole ance,
in ensi y
o
floccula ion
and
desi able
enzyma ic
ac i i ies.
Thei
abili y
o
p oduce
and
u ilize
ace ic/malic
acid
was
examined
as
well;
in
addi ion,
H2S
p oduc ion
as
an
undesi able
p ope y
was
sc eened.
The
oenological
cha ac e is ics
o
indigenous
isola es
we e
compa ed
o
a
comme cially
a ailable
S.
ce e isiae
BS6
s ain,
which
is
commonly
used
as
he
s a e
cul u e.
Finally,
some
indigenous
s ains
coming
om
o ganically
ea ed
g ape
be ies
we e
chosen
o
hei
p omising
oenological
p ope -
ies
and
hese
s ains
will
be
used
as
he
s a e
cul u e,
because
applica ion
o
a
selec ed
indigenous
S.
ce e isiae
s ain
can
enhance
he
egional
cha ac e
o
he
wines.
©
2015
Sociedade
B asilei a
de
Mic obiologia.
Published
by
Else ie
Edi o a
L da.
This
is
an
open
access
a icle
unde
he
CC
BY-NC-ND
license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
In oduc ion
The
quali y
o
e men ed
oods
and
be e ages
is
pa ially
de e mined
by
he
mic oo ganisms
used
o
hei
p oduc ion.
∗Co esponding
au ho .
E-mail:
[email p o ec ed]
(H. ˇ
Su anská).
Fo
ins ance,
he
seconda y
cha ac e
o
wine
is
de e mined
by
senso y
cha ac e is ics
ha
a ise
om
he
di ec
ac ion
o
mic oo ganisms
on
he
subs a e.
The
e men a ion
o
g ape
mus
in o
wine
is
an
ecologically
complex
p ocess,
in
which
bac e ia
and
o he
mic oo ganisms,
especially
yeas s,
play
a
h p://dx.doi.o g/10.1016/j.bjm.2015.11.010
1517-8382/©
2015
Sociedade
B asilei a
de
Mic obiologia.
Published
by
Else ie
Edi o a
L da.
This
is
an
open
access
a icle
unde
he
CC
BY-NC-ND
license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
182
b
a
z
i
l
i
a
n
j
o
u
n
a
l
o
m
i
c
o
b
i
o
l
o
g
y
4
7
(2
0
1
6)
181–190
c ucial
ole.
The
s ains
o
Saccha omyces
ce e isiae
in ol ed
in
e men a ion
play
an
impo an
pa
in
he
cha ac e is ics
o
he
final
p oduc
and
he
di e si y
o
S.
ce e isiae
s ains
p esen
in
spon aneous
e men a ion
con ibu e
o
he
chem-
ical
composi ion
and
senso y
quali ies
o
he
esul ing
wine.1
The e o e,
one
o
he
mos
impo an
echnological
ad ances
in
wine-making
was
he
inocula ion
o
g ape
juice
wi h
selec ed
cul u es
o
S.
ce e isiae.2This
app oach
is
based
on
he
e idence
ha
mic obiological
con ol
o
he
e men-
a ion
p ocess
allows
be e
managemen
o
his
alcoholic
e men a ion.
I
is
known
ha
selec ed
s ains
o
S.
ce e isiae
supp ess
indigenous
non-Saccha omyces
species
and
domina e
he
e men a ion
p ocess.3–5
Nowadays,
no el
bio echnological
app oaches
in
wine-
making
a e
used
in
se e al
aspec s
o
he
e men a ion
indus y.
Apa
om
he
moni o ing
o
he
mic obial
popu-
la ions
and
he
con ol
o
he
spoilage
yeas s,
a en ion
is
ocused
also
on
he
selec ion
and
u iliza ion
o
he
s a e
cul-
u es
coming
om
one’s
own
ineya d,
which
can
enhance
he
egional
cha ac e
o
he
wine.6,7 The
me abolic
peculia i-
ies
and
he
physiological
p ope ies
o
S.
ce e isiae
yeas
may
lead
o
he
o ma ion
o
me aboli es
and
he
ans o ma ion
o
g ape
subs ances
ha
may
en ich
he
wine
fla o .7Ce ain
c i e ia
need
o
be
me
in
o de
o
gua an ee
he
desi able
ea-
u es
o
he
yeas
s ains
selec ed.
The
mos
impo an
ones
a e:
ole ance
o
e hanol;
g ow h
a
high
suga
concen a ions;
esis ance
o
sul u
dioxide;
low
p oduc ion
o
hyd ogen
sul-
fide;
p oduc ion
o
kille
oxins
o
some
enzyma ic
ac i i ies.8
Fu he mo e,
only
eliable
and
apid
iden ifica ion
o
he
yeas
species
du ing
he
p ocess
and
he
quali y
con ol
enables
enologis s
o
assess
he
ole
o
yeas s
as
a
main
p o agonis
o
alcoholic
e men a ion
o
as
a
con aminan .
The
u iliza ion
o
molecula
me hods
enabled
apid
and
p ecise
iden ifica ion
o
he
yeas s
a
he
species
o
s ain
le el.1Me cado
e
al.9 epo ed
ha
Saccha omyces
popula-
ions
a e
ep esen ed
by
mul iple
s ains,
e en
in
inocula ed
e men a ions.
The e o e,
i
is
impo an
o
ha e
simple
and
app op ia e
me hods
ha
allow
disc imina ion
a
he
s ain
le el.
This
s udy
is
ocused
on
indigenous
S.
ce e isiae
s ain
(i)
selec ion,
(ii)
iden ifica ion
and
(iii)
echnological
cha ac-
e iza ion.
Yeas s
we e
isola ed
om
g apes
and
mus s
du ing
he
p oduc ion
o
wines.
We
selec ed
wo
ypes
o
wine
a i-
e ies
–
Sau ignon
blanc
and
Pino
noi
coming
om
an
o ganic
and
in eg a ed
ea ed
ineya d
si ua ed
in
Sou h
Mo a ia,
Czech
Republic.
Ou
objec i e
was
also
ocused
on
he
selec-
ion
o
he
iden ifica ion
app oaches
ha
will
be
simple
and
sui able
o
apid
and
eliable
s ain
iden ifica ion.
The e-
o e,
isola es
o
S.
ce e isiae
we e
iden ified
and
g ouped
by
se e al
molecula
app oaches
such
as
ITS-PCR-RFLP,
PCR-
finge p in ing,
species-specific
p ime s
and
in e del a
PCR
yping.
The
combina ion
o
hese
echniques
enabled
apid
de ec ion,
iden ifica ion
and
yping
o
di e en
S.
ce e isiae
s ains.
Finally,
he
isola ed
s ains
we e
sc eened
o
selec ed
echnological
p ope ies
impo an
in
he
winemaking
p o-
cess
and
o
u he
applica ion
as
he
s a e
cul u es.
To
sum
up,
his
s udy
has
demons a ed
he
impo ance
o
selec ion
o
an
app op ia e
and
apid
iden ifica ion
ech-
nique
and
also
de e mina ion
o
some
impo an
oenological
p ope ies.
Me hods
Yeas
species
isola ion
and
cul i a ion
Au och honous
(indigenous)
s ains
belonging
o
Saccha-
omyces
genus
we e
isola ed
om
g ape
be ies
and
also
om
spon aneously
e men ed
mus s
in
di e en
s ages
o
he
e -
men a ion
p ocess.
G apes
we e
collec ed
a
he
I aˇ
n
ineya d
si ua ed
in
he
Sou h
Mo a ia
egion
and
belong
o
he
Mikulo
wine
egion,
Czech
Republic.
Red
wine
Pino
Noi
(Pn)
and
whi e
wine
Sau ignon
blanc
(Sg)
cul i a s
o
Vi is
ini e a
we e
chosen.
Bo h
a ie ies,
ypical
cul i a s
o
whi e
and,
espec i ely,
ed
Mo a ian
wine
p oduc ion,
we e
cul i a ed
using
he
o ganic
(O)
and
also
in eg a ed
(I)
a ming
p ocedu e.
G ape
be ies
(heal hy
and
undamaged)
we e
collec ed
be o e
ha es
(in
Sep embe
2009,
2010,
2011)
in o
s e ile
glasses.
Immedia ely
a e
anspo a ion
o
he
labo a o y,
15–20
g ape
be ies
we e
placed
in o
150
mL
o
Mal
ex ac
medium
MEM
(Himedia;
2%
mal
ex ac ,
0.1%
pep one,
2%
dex ose,
2%
aga )
and
cul i-
a ed
o
10
days
a
labo a o y
empe a u e
(app ox.
23◦C).
A e
ha ,
cul u e
media
(300
␮L)
inocula ed
on o
MEM
aga
pla es
supplemen ed
wi h
250
mg
L−1s ep omycin
sul a e
(Himedia,
India)
we e
incuba ed
a
26◦C
o
3–5
days.
The
single
colonies
we e
ob ained
by
Koch’s
dilu ion
me hod.
The
e men a ion
p ocess
was
pe o med
in
he
cella
in
I aˇ
n
(du ing
he
in age
2009,
2010,
2011),
which
was
sepa a ed
om
common
e men a ions
o
comme cial
wine
p oduc ion.
The
e men a ion
p ocess
ollowing
he
s anda d
p ocedu e
was
conduc ed
in
a
1000
L
ba el.
The
cella
em-
pe a u e
was
app ox.
10◦C
and
he
empe a u e
o
he
e men a ion
was
app ox.
18◦C.
The
mus
was
spon aneously
e men ed
and
he
samples
we e
collec ed
3
imes
pe
week
du ing
he
whole
e men a ion
p ocess.
Yeas
popula ions
om
mus
we e
isola ed
as
desc ibed
p e iously10 and
cul i a ed
on
mal
ex ac
medium
(MEM)
supplemen ed
wi h
250
mg
L−1s ep omycin
sul a e
(Hime-
dia,
India).
The
single
colonies
(pu e
cul u e)
we e
ob ained
by
Koch’s
dilu ion
me hod.
In
o al,
120
Saccha omyces
sp.
s ains
we e
isola ed
and
iden ified.
Pu e
cul u es
we e
p ese ed
on
MEM
aga
unde
pa a fin
oil.
The
mos
p omising
s ains
will
be
deposi ed
in
he
yeas
cul u e
collec ion
CCY
B a isla a
(s ain
S.
ce e isiae
1-09
has
al eady
been
deposi ed
he e).
DNA
isola ion
Genomic
DNA
was
isola ed
om
single
colonies
using
he
comme cial
ki
Ul aCleanTM Mic obial
DNA
Isola ion
Ki
(MoBio,
USA)
acco ding
o
he
manu ac u e ’s
p o ocol.
ITS-PCR-RFLP
To
dis inguish
Saccha omyces
sp.
om
o he
isola es,
ITS-PCR-
RFLP
was
employed.
The
in e nal
ansc ibed
space s
(ITS)
(ITS1
and
ITS2)
and
5.8S
DNA
gene
egions
we e
amplified
by
using
specific
p ime s
ITS1
(5-TCCGTAGGTGAACCTGCGG-3)
and
ITS4
(5-TCCTCCGCTTATTGATATGC-3).11 DNA
amplifica-
ion
was
ca ied
ou
in
he
final
olume
o
50
␮L
con aining
b
a
z
i
l
i
a
n
j
o
u
n
a
l
o
m
i
c
o
b
i
o
l
o
g
y
4
7
(2
0
1
6)
181–190
183
0.2
mM
o
dNTP,
0.5
␮L
o
each
p ime
100
pmol
␮L−1,
1×
PCR
eac ion
bu e
and
1
U
o
Taq
DNA
polyme ase
(Kapa
Biosys ems,
USA).
PCR
condi ions
we e
as
ollows:
ini ial
dena u a ion
cycle
a
94◦C
o
4
min
ollowed
by
25
cycles
o
amplifica ion,
dena u a ion
a
94◦C
o
1
min,
annealing
a
48◦C
o
30
s,
and
ex ension
a
72◦C
o
1
min;
final
ex ension
a
72◦C
o
10
min.
Fo
RFLP,
PCR
p oduc s
we e
pu ified
by
e hanol
p e-
cipi a ion
and
diges ed
by
es ic ion
endonucleases
HaeIII
(Fe men as,
USA)
ollowing
he
manu ac u e ’s
ins uc ions.
Species-specific
p ime s
Fo
S.
ce e isiae
species
iden ifica ion,
a
se
o
pai s
o
species-
specific
p ime s
Sce F2
(5-GCGCTTTACATTCAGATCCCGAG-
3)
and
Sce R2
(5-TAAGTTGGTTGTCAGCAAGATTG-3)
we e
used.12 DNA
amplifica ion
was
ca ied
ou
in
a
final
olume
o
25
␮L
con aining
0.2
mM
o
dNTP
(In i ek,
Ge many),
0.5
␮L
o
each
p ime
100
pmol
␮L−1(Gene iBio ech,
Czech
Republic),
1×
PCR
eac ion
bu e ,
1.5
mM
MgCl2and
1.25
U
Taq
DNA
poly-
me ase
(BioRad,
USA)
and
0.5
␮L
o
empla e
DNA.
PCR
cycling
condi ions
used
we e:
ini ial
dena u a ion
cycle
a
94◦C
o
4
min
ollowed
by
30
cycles
o
amplifica ion,
dena u a ion
a
94◦C
o
1
min,
annealing
a
55◦C
o
1
min,
and
ex ension
a
72◦C
o
1
min;
final
ex ension
a
72◦C
o
2
min.
PCR-finge p in ing
and
in e del a
PCR
yping
In
o de
o
dis inguish
di e en
S.
ce e isiae
s ains,
PCR-
finge p in ing
by
using
M13
p ime
and
in e del a
p ime s
we e
used.
DNA
amplifica ion
was
ca ied
ou
in
a
final
olume
o
25
␮L
con aining
0.2
mM
o
dNTP
(In i ek,
Ge -
many),
0.5
␮L
o
each
p ime
100
pmol
␮L−1(VBC
Bio ech,
Ge many),
1×
PCR
eac ion
bu e
and
1
U
Taq
DNA
polyme ase
(Kapa,
USA)
and
0.5
␮L
o
empla e
DNA.
PCR-assays
by
using
M13
p ime
(5-GAGGGTGGCGGTTCT-
3):13 ini ial
dena u a ion
cycle
a
95◦C
o
5
min,
ollowed
by
40
cycles
o
amplifica ion
ollowed
by
dena u a ion
a
93◦C
o
0.75
s,
annealing
a
50◦C
o
1
min,
and
ex ension
a
72◦C
o
1
min;
final
ex ension
a
72◦C
o
6
min.
The
second
PCR-assay
o
M13
p ime :
ini ial
dena u a ion
cycle
a
94◦C
o
4
min,
ollowed
by
35
cycles
o
amplifica ion
ollowed
by
dena u a ion
a
94◦C
o
30
s,
annealing
a
36◦C
o
45
s,
and
ex ension
a
72◦C
o
45
s;
final
ex ension
a
72◦C
o
7
min.
The
condi ions
o
in e del a
PCR
yping
by
␦1
(5-CAAAATTCACCTATATTCTCA-3)
and
␦2
(5-GTGGATTT-
TTATTCCAACA-3);
␦2
(5-GTGGATTTTTATTCCAACA-3)
and
␦12
(5-TCAACAATGGAATCCCAAC-3)14,15 we e
as
ollows:
ini-
ial
dena u a ion
cycle
a
94◦C
o
4
min,
ollowed
by
30
cycles
o
amplifica ion–dena u a ion
a
94◦C
o
30
s,
annealing
a
49◦C
o
1
min
and
ex ension
a
72◦C
o
2
min.
The
final
ex ension
was
a
72◦C
o
10
min.
De ec ion
o
PCR
p oduc s
PCR
p oduc s
and
es ic ion
agmen s
we e
sepa a ed
and
de ec ed
by
elec opho esis
on
2%
(w/ )
aga ose
gel
in
1×
TBE
bu e
a
5
V
cm−1 o
2
h.
DNA
amplified
by
single
epe i i e
p ime s
and
by
del a
p ime s
we e
sepa-
a ed
on
1.5%
aga ose
gel
o
3–4
h.
The
gels
we e
s ained
by
e hidium
b omide
(10
mg
L−1),
isualized
unde
UV
ligh
(Ul a
Lum.
Inc.,
USA)
and
documen ed
by
ScionImage
so wa e
(Scion,
India).
Finally,
elec opho eog ams
we e
p ocessed
by
he
BioNume ics
6.5
so wa e
employing
UPGMA
clus e
analysis.
Sc eening
o
oenological
p ope ies
Floccula ion
p ope ies
o
selec ed
s ains
we e
es ed
acco d-
ing
o
Bony
e
al.16 wi h
sligh
modifica ion.
Yeas s
we e
cul u ed
o
3
days
a
26◦C
in
ubes
con aining
10
mL
o
YPD
(2%
pep one,
1%
yeas
ex ac
and
2%
glucose)
medium
unde
pe manen
shaking
(150
pm).
Cells
we e
collec ed
by
cen-
i uga ion
and
washed
wi h
deionized
wa e .
A e
ha ,
cells
we e
suspended
in o
10
mL
o
50
mM
ace a e
bu e
(pH
4.5)
en iched
by
3
mM
CaSO4.
The
ubes
o
suspended
cells
we e
mixed
o
30
s
and
he
u bidi y
o
yeas
suspension
was
e al-
ua ed
by
he
naked
eye.
Floccula ion
deg ee
was
de e mined
using
a
subjec i e
scale
which
means
ha
he
sedimen a ion
was
obse ed
depending
on
he
ime.
The
ollowing
e alua-
ion
was
used:
+
he
cells
floccula e
and
sedimen
a e
15
min
(pa ially
clea
solu ion);
++
immedia e
floccula ion;
w
–
floc-
cula ion
a e
1
h.
E hanol
ole ance
was
es ed
in
5
mL
o
YPD
medium
sup-
plemen ed
by
12,
14,
15,
16
and
17%
( / )
e hanol
and
he
ubes
we e
inocula ed
by
100
␮L
o
cell
suspension
(cell
concen a-
ion
app ox.
5
log
CFU
mL−1).
Inocula ed
ubes
we e
cul i a ed
a
26◦C
o
10
days.
The
cul u e
densi y
was
measu ed
daily
by
DEN-1B
densi ome e
(Biosan,
La ia).
Specific
g ow h
a e

(h−1)
and
he
leng h
o
he
lag
phase
(h)
we e
es ima ed.
Osmo ole ance
o
selec ed
s ains
was
es ed
in
5
mL
o
YPD
medium
wi h
40%
(w/ )
and
50%
(w/ )
glucose.
The
cells
densi y
was
measu ed
as
desc ibed
abo e.
H2S
p oduc ion
by
selec ed
s ains
was
es ed
on
Biggy
aga
(Himedia,
India)
which
con ains
bismu h
as
an
indica o .
A e
incuba ion
a
26◦C
o
3–5
days,
he
zone
su ounding
he
colony
was
e alua ed
as
he
ollows:
−
no
p oduc ion;
+
whi e
colonies;
++
ligh
b own;
+++
b own;
++++
da k
b own/black.5
Malic
and
ace ic
acid
u iliza ion
by
selec ed
s ains
was
es ed
on
0.67%
yeas
ni ogen
base
(YNB,
Himedia)
aga
pla es
con aining
0.5%
(w/ )
malic
acid,
espec i ely,
0.25%
(w/ )
ace ic
acid.
The
g ow h
o
colonies
was
sc eened.
Ace ic
acid
p oduc ion
was
sc eened
on
CaCO3aga
pla es
(Cus e ’s
chalk
medium)
con aining
0.5%
yeas
ex ac ,
5%
glucose,
0.5%
CaCO3and
2%
aga .
The
ace ic
acid
p oduc ion
enabled
solu-
bili y
o
CaCO3which
esul ed
as
a
clea
zone
su ounding
he
colonies.5
The
isola ed
yeas s
we e
also
es ed
o
some
enzyma ic
ac i i ies
such
as
␤-glucosidase
and
glycosidase
ac i i ies.
The
ac i i ies
we e
de e mined
by
aga
pla ing.
The
pla es
we e
incuba ed
a
26◦C
o
3–5
days.17
␤-Glucosidase
ac i i y
was
sc eened
on o
selec i e
medium
con aining
0.67%
yeas
ni ogen
base
(YNB,
Hime-
dia),
0.5%
a bu in
and
2%
aga .
The
pH
o
he
medium
was
adjus ed
o
5
be o e
au ocla ing.
Two
millili e s
o
a
fil e
s e ilized
1%
e ic
ammonium
ci a e
solu ion
was
added
o
100
mL
media
be o e
pla es
pou ing.
Colonies
showing
ac i i y
we e
iden ified
by
a
da k
b own
halo
a ound
he
colonies.17
184
b
a
z
i
l
i
a
n
j
o
u
n
a
l
o
m
i
c
o
b
i
o
l
o
g
y
4
7
(2
0
1
6)
181–190
Table
1
–
Numbe
o
Saccha omyces
sp.
isola es.
G ape
a ie y
Vin age Numbe
o
Saccha omyces
sp.
isola es
In eg a ed
(I)
O ganic
(O)
G apes
(B)/mus
(M)
G apes
(B)/mus
(M)
Sau ignon
blanc
(Sg)
2009
1/14
1/11
Pino
noi
(Pn)
2010
0/14
0/17
Sau ignon
blanc
(Sg)
2011
0/20
0/17
Pino
noi
(Pn)
2011
1/14
0/10
To al
120
To al
o
yeas s
isola es
524
Glycosidase
ac i i y
was
de e mined
using
he
pla es
wi h
he
selec i e
medium
con aining
0.67%
yeas
ni ogen
base
(YNB,
Himedia),
0.2%
u in
and
2%
aga .
The
glycosidase
ac i -
i y
was
de ec ed
as
a
clea
zone
a ound
he
colonies.5,17
Resul s
and
discussion
Selec ion
and
iden ifica ion
o
S.
ce e isiae
s ains
In
o al,
we
isola ed
120
single
colonies
o
au och honous
Saccha omyces
sp.
s ains
om
524
o al
yeas s.
Yeas s
we e
isola ed
om
Sau ignon
blanc
(Sg)
and
Pino
noi
(Pn)
coming
om
o ganic
(O)
and
in eg a ed
(I)
a ming.
The
yeas
species
we e
isola ed
om
g ape
be ies
as
well
as
om
spon aneously
e men ed
mus
du ing
he
in age
2009
(09),
2010
(10)
and
2011
(11).
The
lis
o
he
Saccha omyces
isola es
and
he
sou ces
o
hei
isola ion
a e
shown
in
Table
1.
Yeas s
o
he
genus
Saccha omyces
we e
dis inguished
om
he
o he
isola es
( om
non-Saccha omyces
species)
by
ITS-
PCR-RFLP
( he
leng h
o
PCR
amplicon
was
880
bp).
Fu he ,
o
S.
ce e isiae
species
iden ifica ion
we
employed
species-
specific
p ime s
Sce F2
and
Sce R2.12 Species-specific
p ime s
enable
us
o
iden i y
and
dis inguish
S.
ce e isiae
species
om
o he
species
belonging
o
he
Saccha omyces
sensu
s ic o
com-
plex,
which
includes
he
species
which
can
also
be
ound
in
e men ed
mus
( o .
ex.
Saccha omyces
bayanus,
Saccha omyces
pas o ianus,
Saccha omyces
kud ia ze ii).
Based
on
ou
esul s,
all
he
isola es
belonging
o
he
Saccha omyces
genus
we e
iden ified
by
species-specific
p ime s
as
S.
ce e isiae
( he
leng h
o
he
PCR
p oduc s
was
150
bp)
(da a
no
shown).
Fu he ,
wo
di e en
PCR-assays
by
using
M13
p ime
we e
used.
These
assays
di e ed
in
annealing
empe a u e
(50◦C
s.
36◦C).
The
isola es
we e
di ided
in o
h ee
g oups
by
he
fi s
assay
wi h
annealing
empe a u e
50◦C
and
in o
ou
g oups
by
he
second
PCR-assay
(36◦C).
Da a
a e
p esen ed
as
pa
o
he
dend og am
(Fig.
1).
Two
isola es
(ma ked
as
U,
T)
exhibi ed
a
di e en
finge p in ing
p ofile
han
he
es
o
he
isola es;
hese
isola es
may
be
hyb ids
o
S.
ce e isiae
and
ano he
species
belonging
o
he
Saccha omyces
genus.
Hence,
PCR-finge p in ing
echniques
using
M13
p ime
a e
able
o
g oup
he
species
membe s
o
Saccha omyces
genus
bu
hey
a e
no
sui able
o
esol ing.
In
o de
o
dis inguish
a ious
S.
ce e isiae
s ains,
we
employed
␦1–␦2
and
␦12–␦2
p ime s
ampli ying
in e -del a
sequences.
These
del a
elemen s
a e
desc ibed
as
app o-
p ia e
gene ic
ma ke s
o
iden ifica ion
o
polymo phisms
because
he
numbe
and
loca ion
ha e
a
ce ain
in aspecific
a iabili y.18 The
p ime
pai
␦1–␦2
gene a ed
om
h ee
o
eigh
di e en
agmen s
pe
s ain
wi h
one
common
band
o
he
size
o
app oxima ely
1000
bp.
On
he
o he
hand,
p ime
pai
␦12–␦2
p o ided
significan ly
mo e
agmen s
pe
sample
and
some
o
hem
we e
o
low
in ensi y.
The
low
numbe
o
agmen s
pe
sample
is
asc ibed
o
he
weak
homology
exhibi ed
by
he
p ime
␦1–␦2
owa ds
he
whole
sequence
o
S.
ce e isiae
genom.19 Howe e ,
by
combina ion
o
hese
wo
se s
o
del a
p ime s,
we
iden ified
45
di e en
S.
ce e isiae
s ains.
The
majo i y
o
he
isola ed
and
iden ified
S.
ce e isiae
s ains
came
om
spon aneous
e men ed
mus s
and
belonged
o
g oup
A
(s ain
A).
Despi e
he
ac
ha
some
au ho s20,21 epo ed
ha
i
is
almos
impossible
o
isola e
Saccha omyces
sp.
popula ions
om
be ies
and
ini ial
mus
by
s anda d
di ec
aga
pla ing
p ocedu e
due
o
hei
low
coun s
(>10–100
CFU
cm−2)
we
isola ed
h ee
s ains
om
g ape
be ies
(see
Table
1).
The
elec opho e ic
pa e ns
and
final
dend og am
showing
he
gene ics
simila i y
o
a ious
iden ified
S.
ce e isiae
s ains
a e
shown
in
Fig.
1.
The
no el
combina ion
o
he
mode n
molecula
app oaches
used
in
his
s udy
o
s ain
iden ifica ion
and
yping
seems
o
be
sui able
o
apid,
eliable,
simple
and
ep oducible
iden ifica ion
o
S.
ce e isiae
s ains.
Schulle
e
al.,22 Maqueda
e
al.23 o
O iz
e
al.24 epo ed
applica ion
o
mi ochond ial
DNA
es ic ion
analysis
o
ka yo yping
in
o de
o
dis inguish
a ious
s ains
o
S.
ce e isiae.
Howe e ,
hese
me hods
a e
labo
in ensi e
and
he
esul s
a e
influ-
enced
by
complexi y
o
da a
in e p e a ion
due
o
he
high
numbe
o
gene a ed
agmen s
a e
m DNA
RFLP.
On
he
con a y,
we
employed
di e en
app oaches
o
he
g oup
and
dis inguish
Saccha omyces
a
s ain
le el
based
on
consequen
employmen
o
PCR-finge p in ing.
Fo
ins ance
Schulle
e
al.22 epo ed
ha
in e del a
PCR
yping
had
e y
simila
esol ing
powe
a
s ain
le el
as
m DNA
and
ka yo yping,
he e o e,
he
unique
combina ion
o
he
me hods
u ilized
in
his
wo k
can
be
conside ed
as
a
simple
and
apid
al e na i e
o
m DNA
o
ka yo yping.
Sc eening
o
selec ed
oenological
p ope ies
o
a ious
S.
ce e isiae
s ains
Because
no
all
yeas
s ains
a e
ele an
o
he
specific
con-
di ions
and
cha ac e is ics
o
wine,
a
numbe
o
c i e ia
ha e
been
p oposed
o
he
selec ion
o
new
yeas
s ains
o
use
in
he
winemaking
p ocess.
One
o
he
mos
impo an
c i-
e ions
o
high-quali y
wine
p oduc ion
is
use
o
S.
ce e isiae
s ain
wi h
sui able
echnological
p ope ies.
Thus,
in
o de
b
a
z
i
l
i
a
n
j
o
u
n
a
l
o
m
i
c
o
b
i
o
l
o
g
y
4
7
(2
0
1
6)
181–190
185
Composi e
40
60
80
100
M13-50°C M13-37
°C d1-d2 d12-d2
W1
Z
A3
A2
K1
X1
Y
BS6
25-10
E1
F1
A1
V
I
M
J
H
R
Q
O
N
K
D
F
G
E
C
13-10
27-10
P
A
B
L
1-09
2-09
13-09
5-09
A4
X
W
20-09
S
15-09
T
U
Fig.
1
–
Dend og am
based
on
he
simila i y
o
PCR-finge p in ing
pa e ns
o
S.
ce e isiae
isola es.
S.
ce e isiae
BS6
–
con ol
s ain.
The
desc ip ion
on
he
igh
side
o
he
dend og am
is
he
label
o
he
isola ed
S.
ce e isiae
s ains.

186
b
a
z
i
l
i
a
n
j
o
u
n
a
l
o
m
i
c
o
b
i
o
l
o
g
y
4
7
(2
0
1
6)
181–190
Table
2
–
Oenological
p ope ies
o
di e en
indigenous
Saccha omyces
ce e isiae
s ains
and
one
comme cial
S.
ce e isiae
s ain
BS6
as
a
con ol.
Pn
–
Pino
Noi ;
O,
I
–
o ganic,
in eg a ed;
M,
B
–
mus ,
g ape
be ies;
09–11
–
mean
yea
o
isola ion
(2009–2011).
The
g ay
labelled
fields
–
he
s ains
which
physiological
p ope ies
we e
be e
han
con ol
s ain
BS6.
Isola ed
s ain
Sou ce
o
isola ion
Desi able
echnological
p ope ies
E hanol
esis ance
(%) Osmo ole ance
12%
e hanol
14%
e hanol
E hanol
(%)
40%
glucose
50%
glucose

(h−1)blag
phase
(h)

(h−1)blag
phase
(h)
15
16
17

(h−1)blag
phase
(h)

(h−1)blag
phase
(h)
A
PnIB-11
0.061
22
0.019
58
+
−
−
0.037
19
0.029
24
A1
SgOM-11
0.054
22
−
−
−
−
−
0.044
19
0.029
24
A2
PnOM-11
0.058
24
0.025
59
+
−
−
0.049
19
0.021
24
A3
PnIM-11
0.055
24
−
−
−
−
−
0.037
28
0.013
24
A4
SgIM-11
0.092
24
0.017
39
−
−
−
0.052
19
0.022
24
B
PnOM-10
0.060
24
0.028
85
−
−
−
0.055
19
0.023
30
C
SgOM-09
0.060
24
0.033
58
−
−
−
0.057
19
0.020
24
D
PnOM-10
0.075
24
0.018
42
−
−
−
0.054
19
0.014
24
E
PnIM-10
0.080
22
0.013
42
−
−
−
0.051
19
0.020
43
E1
SgOM-11
0.057
24
0.033
80
−
−
−
0.051
19
0.020
24
F
SgOM-09
0.097
24
0.019
42
−
−
−
0.059
19
0.021
35
F1
SgIM-11
0.073
16
0.017
43
−
−
−
0.051
19
0.020
24
G
PnOM-10
0.058
24
0.018
45
−
−
−
0.065
19
0.021
28
H
SgIM-09
0.056
22
0.014
42
+
−
−
0.064
19
0.025
67
I
SgOM-09
0.090
22
0.063
24
−
−
−
0.052
19
0.019
51
J
PnOM-10
0.055
24
0.046
24
−
−
−
0.058
19
0.024
52
K
PnIM-10
0.061
14
0.034
68
−
−
−
0.060
19
0.016
43
K1
PnIM-11
0.065
24
0.032
68
−
−
−
0.060
24
0.022
45
L
SgIB-09
0.071
22
0.018
40
−
−
−
0.071
19
0.019
43
M
PnOM-10
0.061
14
0.017
40
+
w
−
0.051
19
0.023
51
N
PnIM-10
0.068
20
0.023
58
−
−
−
0.055
19
0.022
45
O
PnIM-10
0.055
20
0.005
150
−
−
−
0.061
19
0.028
67
P
PnOM-10
0.080
24
0.028
50
−
−
−
0.068
24
0.020
51
Q
SgOM-09
0.078
24
0.014
42
−
−
−
0.061
19
0.012
43
R
SgIM-09
0.060
24
0.013
24
−
−
−
0.062
19
0.018
43
S
SgIM-09
0.063
24
0.011
25
+
−
−
0.064
19
0.017
43
T
SgIM-09
0.070
24
0.010
24
+
w
−
0.065
19
0.017
43
U
SgIM-09
0.062
22
0.013
42
−
−
−
0.057
24
0.020
35
V
SgOM-11
0.049
4
0.010
38
−
−
−
0.064
19
0.022
51
W
SgIM-11
0.060
20
0.013
24
+
+
−
0.062
19
0.020
43
b
a
z
i
l
i
a
n
j
o
u
n
a
l
o
m
i
c
o
b
i
o
l
o
g
y
4
7
(2
0
1
6)
181–190
187
Table
2
–
(Con inued)
Isola ed
s ain
Sou ce
o
isola ion
Desi able
echnological
p ope ies
E hanol
esis ance
(%)
Osmo ole ance
12%
e hanol
14%
e hanol
E hanol
(%)
40%
glucose
50%
glucose

(h−1)blag
phase
(h)

(h−1)blag
phase
(h)
15
16
17

(h−1)blag
phase
(h)

(h−1)blag
phase
(h)
W1
PnIM-11
0.076
14
0.016
38
−
−
−
0.077
19
0.020
43
X
SgIM-11
0.064
24
0.018
42
−
−
−
0.072
19
0.022
43
X1
PnOM-11
0.075
25
0.016
25
+
+
−
0.064
19
0.018
51
Y
SgOM-11
0.073
22
0.006
24
−
−
−
0.065
19
0.022
67
Z
PnOM-11
0.064
16
0.007
24
−
−
−
0.058
19
0.018
43
1-09
SgOB-09
0.067
22
0.009
24
+
+
−
0.071
19
0.021
52
2-09
SgIM-09
0.058
24
0.011
40
+
+
−
0.064
19
0.016
55
5-09
SgIM-09
0.062
22
0.010
42
−
−
−
0.065
19
0.024
46
13-09
SgOM-09
0.058
16
0.008
24
−
−
−
0.063
19
0.021
51
15-09
SgOM-09
0.067
22
0.013
24
−
−
−
0.069
24
0.020
46
20-09
SgOM-09
0.059
22
0.006
26
+
w
−
0.062
19
0.019
46
13-10
PnOM-10
0.062
20
0.009
40
−
−
−
0.060
19
0.020
60
27-10
PnIM-10
0.065
24
0.003
80
−
−
−
0.065
19
0.021
44
25-10
PnIM-10
0.063
20
0.014
42
−
−
−
0.060
19
0.020
26
BS6
Con ol
0.064
20
0.012
24
−
−
−
0.070
24
0.018
26
Posi i e
(%) 100
−
96
−
24
9
−
−
−
100
−
Weak
(%)
−
−
4
−
−
7
−
−
−
−
−
Nega i e
(%) −
−
−
−
76
84
100
−
−
−
−
Isola ed
s ain
Sou ce
o
isola ion
Desi able
echnological
p ope ies Undesi able
p ope ies
Osmo ole ance H2S
p oduc ion
SO2 ole ance MA
u iliza ion AA
u iliza ion
AA
p oduc ion
Glycosidase
ac i i y
Floccula ion
0.5%
0.25%
A
PnIB-11
+
+
+
w
−
+
++
A1
SgOM-11
+
+
+
w
−
+
++
A2
PnOM-11
+
+
+
+
−
+
+++
A3
PnIM-11
+
+
+
+
−
+
++
A4
SgIM-11
+
+
+
+
−
+
++
B
PnOM-10
+
w
w
w
−
++++
−
C
SgOM-09
+
w
+
+
−
+
+++
D
PnOM-10
+
+
+
w
−
+
+++
E
PnIM-10
+
+
+
w
−
+
++++
E1
SgOM-11
+
+
+
+
−
+
++++
F
SgOM-09
+
+
+
+
−
+
+
F1
SgIM-11
+
+
+
w
−
+
++
188
b
a
z
i
l
i
a
n
j
o
u
n
a
l
o
m
i
c
o
b
i
o
l
o
g
y
4
7
(2
0
1
6)
181–190
Table
2
–
(Con inued)
Isola ed
s ain
Sou ce
o
isola ion
Desi able
echnological
p ope ies
Undesi able
p ope ies
Osmo ole ance H2S
p oduc ion
SO2 ole ance
MA
u iliza ion
AA
u iliza ion
AA
p oduc ion
Glycosidase
ac i i y
Floccula ion
0.5%
0.25%
G
PnOM-10
+
+
+
w
−
+
++++
H
SgIM-09
+
+
+
+
−
w
++++
I
SgOM-09
+
+
+
++
−
+
++
J
PnOM-10
+
+
+
++
−
+
++
K
PnIM-10
+
w
+
++
−
+
++
K1
PnIM-11
+
w
+
w
−
+
++
L
SgIB-09
+
+
+
w
−
+
++
M
PnOM-10
+
+
+
++
−
+
+
N
PnIM-10
+
+
+
w
−
+
++++
O
PnIM-10
+
+
+
w
−
+
++++
P
PnOM-10
+
+
+
w
−
+
+
Q
SgOM-09
+
+
+
−
−
+
+
R
SgIM-09
+
+
+
w
−
+
+
S
SgIM-09
+
+
+
+
−
+
+
T
SgIM-09
+
+
+
−
−
+
++
U
SgIM-09
+
+
+
−
−
+
++
V
SgOM-11
+
+
+
−
−
+
++
W
SgIM-11
+
+
+
−
−
+
+
W1
PnIM-11
+
+
+
−
−
+
++++
X
SgIM-11
+
+
+
−
−
+
+
X1
PnOM-11
+
+
+
w
−
+
+
Y
SgOM-11
+
+
+
w
−
+
+
Z
PnOM-11
+
+
+
+
−
+
+
1-09
SgOB-09
+
+
+
w
−
+
+
2-09
SgIM-09
+
+
+
w
−
+
+
5-09
SgIM-09
+
+
+
−
−
+
++++
13-09
SgOM-09
+
+
+
−
−
w
++
15-09
SgOM-09
+
+
+
−
−
+
++++
20-09
SgOM-09
+
+
+
−
−
+
+
13-10
PnOM-10
+
+
+
−
−
w
+++
27-10
PnIM-10
+
+
+
−
−
+
++
25-10
PnIM-10
+
+
+
−
−
+
++
BS6
Con ol
+
+
+
−
−
+
++
Posi i e
(%)
100
91
98
29
−
91/2
9a/20
Weak
(%)
−
9
2
38
−
7
31/38
Nega i e
(%)
−
−
0
33
100
−
2
No e:
no
glucosidase
and
glycosidase
ac i i ies
o
all
es ed
indigenous
s ains
and
also
o
comme cial
BS6
s ain.
aS ong
posi i e;
o
H2S
p oduc ion:
posi i e
(+++
and
++++),
weak
(+
and
++).
bSpecific
g ow h
a e;
he
s anda d
de ia ions
we e
no
highe
han
20%.
b
a
z
i
l
i
a
n
j
o
u
n
a
l
o
m
i
c
o
b
i
o
l
o
g
y
4
7
(2
0
1
6)
181–190
189
o
in es iga e
pheno ypic
di e ences
among
45
indigenous
S.
ce e isiae
s ains,
which
we e
iden ified
by
molecula
me hod
as
di e en
s ains
(see
abo e),
we
sc eened
hem
o
he
selec ed
echnological
p ope ies.
The
s ain
S.
ce e isiae
BS6,
which
is
comme cially
a ailable,
was
used
as
a
con ol
s ain.
The
comple e
lis
o
esul s,
summa izing
oenologi-
cal
p ope ies
ha
a e
impo an
o
s ain
selec ion
and
hei
applica ion
in
he
winemaking
p ocess,
is
p o ided
in
Table
2.
The
use
o
local,
au och honous,
selec ed
s ains
o
S.
ce e-
isiae
as
s a e s
is
a he
p e e able,
since
hese
yeas s
a e
be e
acclima ed
o
pa icula
condi ions
cha ac e is ics
o
he
specific
egion/a ea
o
wine
p oduc ion8and,
mo eo e ,
u iliza ion
o
he
local
isola e
o
S.
ce e isiae
is
likely
o
aise
he
egional
cha ac e
o
he
wine.
Ba ajón
e
al.25 epo ed
ha
high
e men a ion
powe
is
ob iously
ela ed
o
he
capaci y
o
he
s ain
o
o e come
he
s ess
associa ed
wi h
wine
e men a ion.
Fo
his
pu pose,
we
es ed
osmo ic
and
e hanol
s ess
ole ance.
The
majo
a ge
o
e hanol
is
he
memb ane,
al e ing
he
memb ane
o ganiza ion
and
pe meabili y
and
consequen ly
inhibi ing
glucose
anspo
and
e men a ion
a e
unde
enological
condi ions.26 As
expec ed,
he
physiological
di e -
ences
among
he
s ess
ole an
species
S.
ce e isiae
depended
on
he
s ain.
The
g ow h
pa ame e s
o
all
he
es ed
s ains
including
specific
g ow h
a e
and
leng h
o
he
lag
phase
a e
lis ed
in
Table
2.
Eigh een
isola ed
s ains
e ealed
be e
g ow h
cha ac e is ics
han
con ol
s ain
BS6
in
he
p es-
ence
o
12%
e hanol
and
29
showed
highe
g ow h
a e
when
exposed
o
14%
e hanol.
Fu he ,
ou
isola ed
s ains
(W,
X1,
2-09,
1-09)
we e
able
o
g ow
in
16%
e hanol
bu
none
o
he
es ed
s ains
was
able
o
g ow
in
he
p esence
o
17%
e hanol.
Highe
suga
con en
o
he
g ape
mus
can
esul
in
inhi-
bi ion
o
he
yeas
me abolism
and,
he e o e,
in
sluggish
e men a ion.24 The e o e,
ole ance
owa ds
osmo ic
p es-
su e
is
a
desi able
p ope y
o
he
yeas
s a e
cul u e.
All
he
isola ed
s ains
we e
capable
o
g owing
in
he
p esence
o
40%
and
50%
glucose.
Fou
s ains
(W1,
X,
L
and
1-09)
exhibi ed
highe
g ow h
a e
han
he
con ol
s ain
BS6
in
he
p es-
ence
o
40%
glucose.
In e es ingly,
despi e
he
ac
ha
To alo
e
al.26 did
no
no ice
any
g ow h
o
S.
ce e isiae
s ains
in
he
p esence
o
50%
glucose,
mos
o
ou
isola es
we e
capable
o
g owing
when
cul i a ed
in
he
p esence
o
50%
glucose.
Mo e-
o e ,
he
majo i y
o
he
sc eened
s ains
e ealed
a
highe
g ow h
a e
on
50%
glucose
han
he
con ol
s ain.
Resul s
o
floccula ion
es s
showed
ha ,
as
also
epo ed
in
o he
s udies,24,27 mos
o
he
s ains
(91%)
emained
in
suspension
a e
10
min
a
es .
This
is
an
impo an
ea u e
when
selec ing
ac i e
d ied
yeas s
o
inifica ion,
whe e
yeas
should
ideally
emain
in
suspension
du ing
e men a ion.24
Enzymes
play
a
defini i e
ole
in
he
p oduc ion
o
wine.
The
enzyma ic
ac i i ies
do
no
only
o igina e
om
he
g apes
i sel ,
bu
also
om
yeas s
and
o he
mic oo ganisms.17 Based
on
ou
esul s,
es ed
s ains
lacked
␤-glucosidase
ac i i y.
Unlike
he
o he
au ho s,5,28 we
did
no
de ec
any
glycosidase
p oduc ion
by
S.
ce e isiae
s ains
es ed.
To
p oduce
a
high-quali y
wine,
i
is
impo an
o
ob ain
a
fine
balance
be ween
he
a ious
chemical
cons i uen s,
espe-
cially
be ween
he
suga
and
acid
con en .29 As
epo ed
by
Suá ez-Lepe
and
Mo a a,7yeas s
migh
be
selec ed
o
hei
abili y
o
p oduce
and
deg ade
ace ic
and
malic
acid.
Ou
esul s
showed
ha
91%
(98%)
o
ou
isola es
we e
able
o
g ow
on
he
aga
medium
con aining
malic
o
ace ic
acid
as
sole
ca -
bon
sou ces.
The
capabili y
o
deg ada ion
o
malic
as
well
as
ace ic
acid
may
be
conside ed
as
a
desi able
p ope y
o
yeas
s ains
because
i
leads
o
he
deacidifica ion
o
wine.30 On
he
con a y,
ace ic
acid
p oduc ion
was
obse ed
o
29%
o
es ed
s ains.
Se e al
s udies
ha e
linked
he
p oduc ion
o
ace ic
acid
o
inc eased
glyce ol
p oduc ion
which
gi es
he
wine
a
desi able
p ope y.31,32
Hyd ogen
sulfi e
has
nega i e
o ganolep ic
impac
on
wine
due
o
o ma ion
o
o -fla o s.7Fi een
isola es
(29%)
syn-
hesized
H2S
and
emaining
s ains
exhibi ed
only
low
H2S
p oduc ion.
Only
one
s ain
(B)
did
no
p oduce
H2S,
howe e ,
his
s ain
is
no
sui able
o
applica ion
as
a
s a e
cul u e
due
o
s ong
floccula ion
p ope ies.
Yeas
selec ion
o e s
he
bes
way
o
ob ain
s ains
o
S.
ce e isiae
o
o he
oenological
species
wi h
p ope -
ies
ha
migh
imp o e
he
senso ial
p ofile,
echnolog-
ical
p ope ies
o
egional
cha ac e
o
he
wine.7The
assays
o
de e mine
yeas
p ope ies
ha
could
influ-
ence
e men a i e
capaci y
and
he
abili y
o
adap
o
s ess ul
condi ions
ela ed
o
he
wine-p oduc ion
p o-
cess
e ealed
di e ences
o
some
s ains.
Some
indigenous
s ains
exhibi ed
be e
adap ion
o
s ess ul
condi ions
han
he
con ol
s ain.
On
he
con a y,
some
o
hem
p oduced
a
high
amoun
o
hyd ogen
sulfi e,
causing
o -fla o .
Thus,
i
we
compa e
all
he
es ed
s ains,
only
15
(A,
A4,
F,
F1,
I,
J,
L,
M,
R,
S,
X1,
Z,
1-09,
2-09,
27-10)
o
hem
a e
sui able
o
u he
es ing
as
s a e
cul u es.
Based
on
ou
esul s,
hese
s ains
showed
p ope
echnological
p ope ies
ha
a e
e y
simila
o
he
con ol
comme cial
S.
ce e isiae
BS6
s ain.
These
s ains
can
be
chosen
o
u u e
la ge-scale
e men a ion
p ocesses
ins ead
o
comme cially
a ailable
s ains.
Conclusions
The
p esen
s udy
demons a ed
applica ion
o
app op i-
a e
mode n
molecula
echniques
ha
a e
sui able
o
apid
S.
ce e isiae
s ain
iden ifica ion
and
u he
es ing
o
a ious
s ains
o
hei
echnological
po en ial.
The
combina ion
o
used
mode n
molecula
echniques
including
species-specific
p ime s,
and
in e del a
PCR
yping
enabled
us
o
iden i y
S.
ce e isiae
a
s ain
le el.
Also
impo an
physiological
cha -
ac e is ics
o
he
yeas s
used
in
his
s udy
a e
sui able
o
apid
selec ion
o
he
di e en
S.
ce e isiae
s ains
ha
can
be
applied
in
he
winemaking
p ocess.
Applica ion
o
he
selec ed
s ains
wi h
sui able
echnological
p ope ies
in
he
wine
e -
men a ion
p ocess
should
inc ease
he
quali y
o
he
wine
and
enhance
he
egional
cha ac e
o
adi ional
Mo a ian
wines.
Hence,
some
isola ed
indigenous
s ains
will
be
es ed
in
a
la ge-scale
e men a ion
p ocess
by
a
small
Mo a ian
wine y.
Conflic s
o
in e es
The
au ho s
decla e
no
conflic s
o
in e es .