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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.. Interestingly, the both best sorts of

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vNU journal of science, Natural sciences and rechnologv 23, No 1s (2002) 1g1-1g6

Microorganisms in yeast cakes controlling the quality and

Dinh Th.ry Hang2, Duong Van Hop2

tFaculty

of Biotogt, college of science, vNU, 334 Nguyen Trai, Hanoi, I/ietlant

2lnstitute

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.

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.

Keywords : yeast cake, traditional fermentation, alcohol fermentation.

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Yeast cake is a traditional microbial product

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 88 856

E-mail: habtv@mu.edu.vn

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

improvement of the production process of a

good sort of yeast cake with stable microbial

181

182 T.T.L Quyen et al / Wll lournal of Science, Natural Sciences and Technology 23, No 15 (2007) 181-1.g6

content for long term production of highly

qualifi ed alcoholic drink

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

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

sequences were aligned with related species by

program [4] Reference sequences used for the

phylogenetic study were obtained from the

database The phylogenetic kee was constructed

ls l.

(DGGE) Total DNA from "yeast cakes" was

extracted by using method described by Zhou et

al [6] with some modifications Fragments of

were obtained with the bacterial-specific primer

TTR AGT TT-3') Fragments of the 18S rDNA

(560 bp) for DGGE analysis were obtained with

the primers EuklA (5'- CTG GTT GAT CCT GCC AG-3') and Euk5l6r (5'- ACC AGA CTT

melting behavior of the amplified fragments in the gradient denaturing gel, a GC-clamp (CGC

GGG GCA CGG GGGG) was added at 5'-end

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

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

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

length of the extension step was increased to l0

min

DGGE was run in DCode system (Bio-Rad)

as described by Muyzer et al l3l A 6%

polyacrylamide gel with a gradient of

DNA-denaturant agent was cast by mixing solution of

Uoh and 80% denaturant agent (100% :7 M

prokaryotic DNA, 30-60% for eukaryotic

sample and the electrophoresis was run at 200

Aftenvard, the gels were stained with ethidium

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T.T.L Quyen et al / WLI lournal of Science, Natural Sciences and Technology 23, No 15 (2007) 181-186 183

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Prominent bands were excised from the gels,

mounted in sterilized Mili-Q water overnight at

using QIAgen kit Sequencing reactions u'ere

carried out with the primers GM5F for

eukaryotic DGGE bands and sequencing rvas

performed in an automatic ABI PRISN{ 3100

sequencer Sequences were submitted to the

phylogenetic affiliation [7]

3 Results and discussion

Fermentation 15 most popular sorts of

"yeast cakes" presently used for the production

provinces in Northem Vietnam By using these

"yeast cakes" for fermentation, we obtained 15

alcoholic products which differed from each

j217-79, the drink produced by Ruou Can

"yeast cake" showed best quality and taste, the

"yeast cake" was therefore chosen for

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

respectively [8] Phylogenetic analyses of 265

Saccharomyces cerevisiae (l00Yo homology)

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

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

responsible for the fermentation process.

It is known that "yeast cakes" contain highly

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

fermentation we carried out analyses of PCR

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

1'east isotrates B\I4.1 and BM4.2 were also included"

Rou Can " 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

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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

1,2,4-8), representing yeast populations other than

Saccharomyceis and Saccharomycopsis such as

Lepidoglyphus (Fig.1, band E5) Since

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 than

Saccharomyces and Saccharomycopsis was the

reason for souring problems in products

obtained with other sorts of "yeast cakes" With

this results, it is presented for the first time that

"yeast cakes" with less diverse yeast

communities, ideally contain only

Saccharomyces and Saccharomycopsis groups,

would be the key for a successful fermentation

and produce alcoholic drinks of high quality

Although not being involved directly in the

fermentation process, prokaryotic

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

Weissella and Lactobacillus groups,

respectively These groups of bacteria are

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

have influences on the flavour and taste of the

role of bacteria such as Lactobacillus sp 1:

alcoholic drinks are in process.

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Fig 1 Phylogenetic tree based on 265 rDNA DliDl

sequences showing the affiliation of yeasts isolates from Ruou Can yeast cake with other relative

specles.

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 5-BM9, Lane 6-8M10, Lane 7-BM1l, Lane 8-BM15.

Lane 9-BM16, Lane 10-B4.1, Lane ll-P.4.2

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T.T.L Quyen et al I WU lournal of Science, Natural Sciences and Technology 23, No 75 (2007) 1.81-1.86 185

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Fig 3 DGGE pattern of PCR fragments amplified

with l6s rDNA prokaryotic primers Lane l-BMl,

Lane2-8M2, Lane 3-BM4, Lane 4-BM7,Lane 5-BM9, Lane 6-BM10, Lane 7-BM11, Lane 8-BM15'

Lane 9-BMl

4 Conclusions

l) Among 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,

pleasant taste.

2) Two yeast strains were isolated from Ruoucan "yeast cake" and were identified as

Saccharomyces cerevisioe and

Saccharomycopsis filuliger base on

morphology and 265 rDNA Dl/Dz sequencing.

"Yeast cake" remaded with the isolated yeasts

produced product as good as the original

Ruoucan "yeast cake".

3) DGGE analyses of 165 rDNA and 18S

rDNA of different 8 yeast cakes showed that

Lactobacillus were the major microbial

populations in the "yeast cakes", controlling the

quality and taste of the final drink products

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