tap chi khoa hoc 2007 2

vNU journal of science, Natural

L.

sciences and rechnologv 23, No. 1s (2002) 1g1-1g6

Microorganisms in yeast cakes controlling the quality and
taste of traditional fermentation products

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::rl"

\

Tran Thi Le Quyent, Bui Thi Viet Hal,*, Dao Thi Luong2,
Dinh Th.ry Hang2, Duong Van Hop2

;r

tFaculty
2lnstitute

of Biotogt, college of science, vNU, 334 Nguyen Trai, Hanoi, I/ietlant
of Microbiolog,t and Biotechnologt, WU, 144 Xuan Thuy, Honoi, viefirum
Received 15 August 2007

Abstract. Alcohol fermentation was carried out with sticky rice using 15 most popular
sorts of

yeast cakes in Vietnam to produce different kinds of alcoholic beverages.

The products were
obtained by traditional distillation method and compared with each other for flavour
and taste
according to the standard qualification scale for alcoholic products. It revealed
that Ruou Can
yeast cake which has been widely used for hundreds years by local commirnities
yielded the best
product and therefore was selected for further studies. From this yeast cake, two yeast
shains were
first time isolated and identifie d as Saccharomyces cerevisiae and, Sqccharomycopsis
fibuliger
based on morphology and 263 rDNA Dl/D2 sequencing analyses. A sort yeast
cake remade by
using these two yeast strains yielded alcoholic product as good as the product
obtained from using
Ruou Can yeast cake.

hec
--x-

PCR/DGGE analysis of 165 rDNA (for the Prokaryotes) and l8S rDNA (for
the Eukaryotes)
showed differences in structures of microbial communities in the studied yeast
cakes. However, in
all cases, Saccharomyces sp., Saccharomycopsis sp., and Lactobacillus sp. accounted
for the most
abundant populations. Interestingly, the both best sorts ofyeast cakes, i.e
the Ruou Can yeast cake
and the remade yeast cake, contain mainly these groups of microorganisms.

l\E

Keywords : yeast cake, traditional fermentation, alcohol fermentation.

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1.

Introduction

on the sorts of "yeast cake", water source and
the nature of starch source [1]. Among the
above mentioned factors, yeast cake with high
stability is the most important for the success of
fermentation process and the taste of product, In
this study we investigate the relationship
between the microbial community in yeast
cakes and the quality of alcoholic drinks. The
obtained results would be applied in
improvement of the production process of a
good sort of yeast cake with stable microbial

hsp
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Yeast cake is a traditional microbial product

which has been used widely in Vietnam as
starter in fermentation to produce alcoholic
drinks. It is rich in microorganisms, especially
those involve in the transformation of starch
into fermentable sugars, then into ethyl alcohol.
Quality of alcoholic drinks pnmarily depends

to.r.rp*ang

author. Tel. : 84-4-85
E-mail:

88

856

181


182

T.T.L. Quyen et al. /

content

Wll

lournal of Science, Natural

for long term production of highly

qualifi ed alcoholic drink.

2.

Materials and methods
Yeast cakes. 15 yeast cake samples were

collected from different provinces which are
well known for traditional alcoholic dnnk
products in Northern Vietnam (data not shown).
Fermentation. '\east cakes" were ground
to dust, mixed with cooked sticlry rice and
incubated for 5-8 days at room temperature.
Alcoholic products were collected by
distillation and examined for quality according
to the national standard TCW3217-79.

Sequencing ond phylogenetic analysis.
Cells rvere gro\rm in the logarithmic $owth
phase. Isolation and purification of nuclear
DNA were done according to Takashima and
Nakase (2000)121. The sequence of the DllDz
regron of 265 rDNA, were determined after
ampliffing the DNA using PCR. The strains
were sequenced directly t3]. Generated
sequences were aligned with related species by
using the CLUSTAL W ver. 1.83 sorftware

program [4]. Reference sequences used for the
phylogenetic study were obtained from the

Sciences and Technology 23, No. 15

(2007) 181-1.g6

the primers EuklA (5'- CTG GTT GAT CCT
GCC AG-3') and Euk5l6r (5'- ACC AGA CTT
GCC CTC C-3'). In order to stabilize the
melting behavior of the amplified fragments in
the gradient denaturing gel, a GC-clamp (CGC
CCG CCG CGC GCG GCG GGC GGG GCG
GGG GCA CGG GGGG) was added at 5'-end
of the primers GM5F (for prokaryote) and
Euk516r (for eukaryotes). PCR were performed
in 50 r,1 volume, containing 5 pl of template
DNA, 5 pl buffer x 10, 5 rr1 BSA (3mg/m1), lpl
dNTPs 25mM, lpl primer (20p1), 0.6y1 Taq
DNA polymerase (2.5u/pl). The PCR program
for eukaryotic primer set included an initial
denaturation at 94 oC for 130 s, followed by 35
cycles of denaturation at 94 oC for 30 s,
annealing at 56 oC for 45s, and extensionatT2
oC for
130 s. The PCR program for prokaryotic
primer set included an initial denaturation at 94
oC for I min and 20 touchdown
cycles of
denaturation at 94 "C for 1 min, annealing at 65

'C (with the temperature decreasing 0.5 oC
each cycle) for 1 min, and extension at 72 oC
for 3 min, followed by 20 cycles of 94 oC for 1
min, 55 oC for 1 min, and 72 oC for 3 min.
During the last cycle of both programs, the
length of the extension step was increased to l0
min.

database. The phylogenetic kee was constructed

DGGE was run in DCode system (Bio-Rad)

from the evolutionary distance data according
to Kimura using the neighbor-joining method
ls l.
Denaturing gradient gel electrophoresis
(DGGE). Total DNA from "yeast cakes" was

as described by Muyzer et al l3l. A 6%
polyacrylamide gel with a gradient of DNAdenaturant agent was cast by mixing solution of
Uoh and 80% denaturant agent (100% :7 M
urea and 40o/o deionized formamide). Linear
gradients of denaturants (20-70% for

extracted by using method described by Zhou et
al. [6] with some modifications. Fragments of

the 163 rDNA (550 bp) for DGGE

analysis

were obtained with the bacterial-specific primer
pair GM5F (5'- CCT ACG GGA GGC AGC
AG-3) and 907R (5'- CCG TCA ATT CCT
TTR AGT TT-3'). Fragments of the 18S rDNA
(560 bp) for DGGE analysis were obtained with

prokaryotic DNA, 30-60% for eukaryotic
DNA) rvere used. PCR products were loaded on
the gels rvith the volume of 50 pl for each
sample and the electrophoresis was run at 200
V for 3.5 h at 60 oC in IxTAE

buffer.
Aftenvard, the gels were stained with ethidium
bromide (5 mg/ml) for 30 min, rinsed with

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84.1


T.T.L. Quyen et al.

;

/ WLI lournal of Science, Natural

5 min

and visualized under IfV.
Prominent bands were excised from the gels,
mounted in sterilized Mili-Q water overnight at
4'C. The DNA was reamplified (using the same
primer sets without GC-clamp) and purified
using QIAgen kit. Sequencing reactions u'ere
carried out with the primers GM5F for
prokaryotic DGGE bands, EuklA for
eukaryotic DGGE bands and sequencing rvas
performed in an automatic ABI PRISN{ 3100
sequencer. Sequences were submitted to the
BLAST Search of Gene Bank to determine the
phylogenetic affiliation [7].
water for

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Sciences and Technology 23, No. 15 (2007)

181-186

183

Saccharomyces cerevisiae (l00Yo homology)

and

Saccharomycopsis

fibuliger

(100%

homology), respectively (Fig. 1) [9].

In order to verify the role of the isolated
yeast strains in the "yeast cake" used for
alcoholic fermentation, we used them as the
only source of microorganisms for making a
new sort of yeast cake, called remade yeast
cake (BM16). The alcoholic drink obtained
with the remade "yeast cake" showed high
quality and pleasant taste, similarly to the drink
produced with Ruou Can "yeast cake". The
obtained results indicated that the isolated yeast
strains represented the key organisms,
responsible for the fermentation process.

3. Results and discussion

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It is known that "yeast cakes" contain highly

Fermentation. 15 most popular sorts of
"yeast cakes" presently used for the production
of alcoholic drinks were collected from
provinces in Northem Vietnam. By using these
"yeast cakes" for fermentation, we obtained 15
alcoholic products which differed from each
other in quality, alcohol content and taste.
According to the national standard TCW
j217-79, the drink produced by Ruou Can
"yeast cake" showed best quality and taste, the

"yeast cake" was therefore chosen for
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microbiological studies.

Two yeast strains, B4.1 and B4.2,

u'ere

isolated from the Ruou Can "yeast cake". Strain
B4.1 has round, smooth, white-cream clonies.

whose cells are spheroidal

or oval

and

reproduce by budding. On the other hand, strain
P.4.2 has round, rough, white colonies, whose

cells are of branched form and reproduce by
budding. Thus, morphologically these strains
resemble the representatives of genera

Saccharomyces and Saccharomycopcis,
respectively [8]. Phylogenetic analyses of 265
rDNA DllDz sequencing indicated that strains
B4.1 and B4.2 could be affiliated to species

diverse communities of microorganisms,
including yeasts, molds and bacteria that
directly or indirectly involve in the fermentation
process and determine the quality of alcoholic
products [10]. To investigate the communities
of microorganisms existing in different sorts of
"yeast cakes" and look inside into the role of
microorganisms in
fermentation we carried out analyses of PCR

major groups of

amplified fragments of 165 rDNA and 18S

rDNA from the total DNA pool of "yeast cakes'
using DGGE method. PCR fragments of
prokaryotic 165 rDNA (550 bp) and eukarl'otic
18S rDNA (560 bp) arnplifred from DNA pool
of eight different "1'east cakes" x-ere separated
on denaturing -eradient pol-varylamide gels
(Fig. 2 and 3). -{s references, PCR fragments
arrplified *-ith ttrre same primer set from the
1'east isotrates B\I4.1 and BM4.2 were also
included"

"

1'east cake" (BM4), and the
remade ")'east cake" @M16) showed similar
DCrGE pattern in eukaryotic rDNA analyses
(Fig. 2, lanes 3 and land 9) and consisted two
DCrGE bands (Fig. 2, E3 and E4) that were
identical to the bands produced by pure cultures

Rou Can


l.84

T.T.L. Quyen et al. /

WU lournal of Science, Natural

Saccharomyces cerevisiae BM4.1 and

Saccharomycopsis filuliger 8M4.2 respectively
(Fig.l, lane 10, 11). On the other hand, other
"yeast cake" samples contained besides these
two bands some other bands (Fig.1,lane 1,2,48), representing yeast populations other than
Saccharomyceis and Saccharomycopsis such as

Lepidoglyphus (Fig.1,

band E5).

Sciences and Technology 23, No. 15 (2007) 787-186

have influences on the flavour and taste of the
final products [4]. Further investigation on the

role of bacteria such as Lactobacillus sp. 1:
production of specific flavour or taste o:
alcoholic drinks are in process.

Srdrm]66i86

Since

Sacdrffi.r6ceDuisiae

Saccharomyces and Saccharomycopsis yeasts
are responsible for two steps in fermentation
process, the starch hydrolysis and alcoholic
fermentation on sugar, the presence of these
populations in Ruoucan and the remade "yeast

cakes" could explain for the high quality of the
products. At the same time, it seemed that the

existence

of

yeast populations other

,
C[ Sacdr@]@shtuiilzwi

SecdrwlesDaloc
Sacdrffi)espasbrin 6

Saodr@I,}osnagmiCli

Seodrmlsshmdiffi
Srcdrmr€stsinds
Secdrardrrlcas
Saodarurucop.ds

SacdrarnlopdsmabrgE
Strdlamt@pdsltDd&Dr

Weissella and Lactobacillus

groups,

of bacteria are

known for the production of acids or
respectively. These groups

antimrcrobial substances and therefore might
inhibit souring microorganisms, and at the same
time support growth of yeast populations. In
addition, these groups of bacteria could also

J.(

@r&_wi
Dobaympswrib
Cardida&ttu

it is presented for the first time that
"yeast cakes" with less diverse yeast

Although not being involved directly in the
process,
prokaryotic
fermentation
microorganisms such as many kinds of bacteria
in "yeast cakes" could be responsible for the
taste of the drink products, Different "yeast
cake" samples showed different DGGE pattern
in prokaryotic rDNA analysis (Fig.3), even the
two similar samples Ruou Can and the remade
"yeast cakes". The most prominent bands P1, P2
and P3 showed affiliation to Acetobacter,

crebogdrsb

Saodrmlopsisviri

this results,

Saccharomyces and Saccharomycopsis groups,
would be the key for a successful fermentation
and produce alcoholic drinks of high quality.

L

B]

Secdlrylopsislamit

than

only

Jakdrrrafl ds

F

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Seodrmlopsis&manbrs

Saccharomyces and Saccharomycopsis was the
reason for souring problems in products

obtained with other sorts of "yeast cakes". With

communities, ideally contain

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Sacdrm]@sporEam

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Pichbhntui

Phylogenetic tree based on 265 rDNA DliDl
sequences showing the affiliation of yeasts isolates
from Ruou Can yeast cake with other relative

Fig.

1.

cho

ofi

pler

specles.

Ruc

Sac
Sac,

mor
"Ye.

pro(
Ruo

Fig. 2. DGGE pattem of PCR fragments amplified
with 18S rDNA eukaryotic primers. Lane 1-8M1,
Lane 2-8M2, Lane 3-BM4, Lane 4-BM7,Late 5BM9, Lane 6-8M10, Lane 7-BM1l, Lane 8-BM15.
Lane 9-BM16, Lane 10-B4.1, Lane ll-P.4.2.

rDN

Sacc

Lact

popL
qual.


T.T.L. Quyen et al. I

WU lournal of Science, Natural

Sciences and Technology 23, No. 75 (2007)

1.81-1.86 185

References

f the
1 the

[]

p. in

eof

Nguyen Thi Hien et al., Technologt for
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[2] M. Takashima, T. Nakase, Four new spesies of

q

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[3] C. P. Kurtzman, C.J. Robnett, Identification of
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the large- subunit (265) ribosomal DNA gene.
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with l6s rDNA prokaryotic primers. Lane l-BMl,
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Lane

9-BMl.

[4]

4. Conclusions

l) Among
D1,D2
,olates

i'r'e

15 most popular "yeast cakes" in

Northern Vietnam, Ruoucan "yeast cake" was
choosen as the best "yeast cake" for production
of alcoholic drink with high quality and soft,

[5] N. Sa:tou, M. Nei, The neighbor-joining method:
a nel method for reconstructing phylogenetic
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[7] Olga

as

cerevisioe and
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produced product as good as the original

plified
-8M1,
ane 5-BM15.
A.2.

Biol. Evol.4 (1987) 406.

[6] Zhoul et al., Isolation of genomic DNA from

pleasant taste.

2) Two yeast strains were isolated

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oil

extraction

Mitobiologt

[8]

and handling,

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3) DGGE analyses of 165 rDNA and 18S
rDNA of different 8 yeast cakes showed that

Vu

Saccharomyces, Saccharomycopsis and
Lactobacillus were the major microbial
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quality and taste of the final drink products.

Czechoslovakia, 1990.

tel

Nguyen Thanh, Yeat Biodiversity and

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ll0l Muyezer et ?1., Denaturing Gradient Gel
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