Tp ch Khoa hc 2012:21a 11-18

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SN XUT MEN RU T SACCHAROMYCES

CEREVISIAE V ENZYME AMYLASE TRONG MM LA
Ng Th Phng Dung1, Bi Duy Nhn2 v Hunh Xun Phong1

ABSTRACT
In this study, the total yeast and mould counts of five collected commercial alcoholic
fermentation starters were determined (yeasts: 7,49 - 10,23 log cfu/g and moulds: 5,93
7,61 log cfu/g), and 26 isolates of yeasts were obtained. Five yeast isolates performing
their good fermentative capacity were selected and compared with the control yeast
Saccharomyces cerevisiae. This research also examined the production of enzyme
amylase activity of rice malt during 7 days of germination incubation, in which the
highest enzyme activity was obtained after 4 days (9,0 U/g). In addition, in this study the
laboratory-scale process for mixed starter including Saccharomyces cerevisiae and rice
malt powder was prepared following the mixture ratio at 1:3, 1:4 v 1:5, respectively.
Keywords: enzyme amylase, alcoholic starter, yeast, Saccharomyces cerevisiae
Title: Preparation of defined alcoholic starter using Saccharomyces cerevisiae and
amylase enzyme of rice malt

TM TT
Trong nghin cu ny mt s nm men v nm mc ca nm loi men ru th trng
c xc nh (nm men: 7,49 - 10,23 log cfu/g v nm mc: 5,93 7,61 log cfu/g), v
tng cng c 26 dng nm men c phn lp thun chng. Nm dng phn lp c kh
nng ln men mnh c s tuyn v tin hnh so snh vi dng men Saccharomyces
cerevisiae. ti cng kho st c s bin i hm lng enzyme amylase trong
mm la qua 7 ngy , mm la c hot tnh enzyme cao nht sau 4 ngy (9,0 U/g).
ti bc u th nghim sn xut c bt men ru bng phng php phi trn bt
men thun Saccharomyces cerevisiae v bt mm la 3 t l khc nhau (1:3, 1:4
v 1:5).
T kha: enzyme amylase, men ru, nm men, Saccharomyces cerevisiae

1 GII THIU
Thng thc ru l mt nt vn ha mang m bn sc ring ca tng dn tc v
tng vng. ng bng sng Cu Long l mt trong nhng vng c ngh sn xut
ru lu i v gn lin vi tng a danh l nhng thng hiu ni ting t xa
nh ru Xun Thnh (Tr Vinh), Ph L (Bn Tre), ru G en (Long
An),... Nguyn liu nu ru thng l go, np, sn (khoai m). Mi loi
nguyn liu to thnh mt hng v c trng ca ru. Ru go l loi ru ph
bin nht Vit Nam. Mi sn phm ru u c mt cng thc c trng ring
mang m tnh th cng truyn thng.
ng ha v ln men ru l hai giai on ch yu trong qui trnh sn xut ru
ln men. Giai on ng ho l mt qu trnh bin i ha hc, chuyn tinh bt
1
2

Vin Nghin cu v Pht trin Cng ngh Sinh hc, Trng i hc Cn Th

Sinh vin lp Cng ngh Sinh hc kha 32, Vin NC & PT CNSH, Trng i hc Cn Th

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Tp ch Khoa hc 2012:21a 11-18

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thnh ng ln men nh hot ng ca cc enzyme thu phn tinh bt amylase

sn c trong nguyn liu hay sn sinh t vi sinh vt. Giai on ln men ru l qu
trnh ln men ym kh ca nm men, chuyn ha ng thnh ru etylic v CO2
(Nguyn nh Thng v Nguyn Thanh Hng, 2005). H enzyme amylase gm
c -amylase, -amylase v amylophosphatase. Phn ln chng tp trung phi
mm v mt t tp trung phn di ca ni nh hoc trong mng ngn gia v
tru v ni nh ca ht i mch dng tin cht. Chng s c hnh thnh sau
ngy th 2 ca qu trnh m mm v t cc i sau 8 ngy vi hot ha tng
ln khong 150 - 200 ln (Bi i, 2005). Cc enzyme ny c th c phi trn
vo thnh phn men ru thay th mt phn vai tr ca vi sinh vt trong bnh men,
rt ngn thi gian sn xut.
H vi sinh vt trong men ru gi vai tr quan trng trong qu trnh sn xut ru,
nh hng trc tip n nng sut v cht lng sn phm. Nm mc thc hin
qu trnh ng ho v nm men thc hin qu trnh ln men ru, l hai nhm vi
sinh vt ch yu c trong men lm ru. Mt s dng nm mc ph bin nh
Amylomyces rouxii, Rhizopus spp., Mucor spp, Aspergillus spp. Mt s nm men
chnh gm c Saccharomyces cerevisiae, Hansenula spp., Endomycopsis spp.
(Steinkraus, 1997; Nout & Aidoo, 2002). Vic ci tin cht lng v hot tnh ca
men lm ru gp phn quan trng trong s hon thin quy trnh cng ngh sn
xut ru v ci tin cht lng ru thnh phm. Kt qu nghin cu sn xut
men lm ru vi dng ht gm t hp nm mc Amylomyces rouxii v nm men
Saccharomyces cerevisiae c xc nh c hot tnh cao (Dung et al., 2005) v
bc u th nghim cho thy kh nng ng dng trong qu trnh ln men ru t
mt s loi nng sn (go, np) khc nhau (Dung & Phong, 2011). Ngoi ra, gn
y hng ng dng enzyme amylase t mm la thay th cho ngun enzyme
t nm mc cng l mt vn cn c kho st vi mc ch gp phn rt ngn
thi gian sn xut ru v vn m bo cht lng, an ton cho ngi tiu dng.
Mc tiu ca ti l kho st thnh phn ngun bnh men th trng, tuyn chn
dng nm men c kh nng ln men ethanol tt v bc u nghin cu sn xut
bt men lm ru gm nm men thun v enzyme amylase t mm la.
2 PHNG TIN V PHNG PHP NGHIN CU
2.1 Phng tin
- Nguyn liu: la, go, men ru th trng v dng nm men Saccharomyces
cerevisiae c phn lp, th nghim v tuyn chn trong nghin cu ca
Dung et al. (2006).
- Ha cht: dng trong phn tch hot tnh enzyme amylase v nui cy nm
men.
- Mi trng: Rose Bengal Chloramphenicol Agar (RBC Agar, Merck), PG
(khoai ty 20%, glucose 1%, lactose 1%, (NH4)2SO4 0,2%, MgSO4 0,05%,
CaSO4 0,02%, KH2PO4 0,1%, nc), PGA (mi trng PG b sung 2% agar)
v PGY (khoai ty 20%; glucose 2%; yeast extract 1%).

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2.2 Phng php nghin cu

2.2.1 Thu thp men ru th trng v xc nh mt s nm men v nm mc
Mu men ru th trng c thu thp gm c: men ru Hi Anh Quan (HAQ),
Quang Minh (QM), Hong Anh (HA), Dung-c Thnh (DT) v Linh Chi (LC).
Mt s nm men v nm mc trong mu men ru c thc hin v xc nh trn
mi trng RBC Agar sau 24 gi, 48 gi v 72 gi 30oC.
2.2.2 Phn lp v nh gi s b hnh thi cc dng nm men
Cc khun lc nm men trn a mi trng RBC Agar c cy chuyn nhiu ln
trn mi trng PGA, 30oC cho n khi thu c khun lc thun nht.
rng v hnh thi nm men c xc nh bng cch quan st di knh hin vi
quang hc. Cc dng thun c tr trn mi trng thch nghing PGA 4oC.
2.2.3 So snh v tuyn chn nm men c kh nng ln men ethanol mnh
Cc dng thun c kim tra kh nng ln men ng glucose da trn lng kh
CO2 sinh ra trong qu trnh ln men ru. Sau 24 gi pht trin sinh khi trong
mi trng PGY 30oC, 1ml dung dch nm men c chng vo chai Durham
cha 9ml dung dch glucose 2% ( c kh trng 115oC trong 10 pht), lc
u cho dch ng trn y vo ng thu tinh p ngc nm bn trong chai
Durham, 30oC. Chiu cao ct kh CO2 sinh ra trong ng thu tinh p ngc
c ghi nhn ti cc thi im 6, 8, 10, 12 v 14 gi. Chn 5 dng nm men tt
nht t 5 loi bnh men so snh kh nng ln men vi dng nm men S.
cerevisiae ca Dung et al. (2006). Dch sinh khi nm men (1ml) c chng vo
100ml dung dch glucose 20%, 30oC v tin hnh theo di lng bt kh sinh
ra ti cc thi im tng t nh trn v o cn sau 48 gi ln men. Th nghim
c lp li 3 ln.
2.2.4 Xc nh hot tnh enzyme amylase trong mm la v bnh men
Hot tnh enzyme amylase trong mm la c kho st ti cc thi im ln mm
khc nhau la chn thi gian mm cho lng enzyme nhiu nht. La ging
c ra sch, loi b tp cht, cho ln mm nhit phng v thu hoch sau
1, 2, 3, 4, 5, 6 v 7 ngy. Mm la c sy kh 40oC trong 24 gi. Xc nh
hot tnh enzyme trong mm la v bnh men bng phng php Nelson (Nelson,
1994). Nghin mn 5g mu vi 20ml dung dch m, ly tm 7.000 vng/pht
(Hettich-Zentrifligen, c) trong 15 pht. S dng dung dch thuc th Arsenomolybdate v o quang ph bc sng 520nm tnh hot tnh enzyme trong
1ml dch mu.
2.2.5 Sn xut men ru gm nm men v enzyme amylase
Men ru c phi trn t bt nm men thun (sn xut t dng nm men
tuyn chn) v bt la cha h enzyme amylase. Bt men thun c phi trn vi
bt mm la cc t l khc nhau to thnh sn phm bt men. Bt men c tr
trong ti nha hn kn ming v bo qun nhit phng.
Bt men thun c sn xut nh sau: chng 100ml dch sinh khi nm men ( 16
gi trong mi trng PG) vo 100g bt go (sy 100oC v ngui), to m n
khong 40% bng dung dch mui khong (MgSO4 0,05%, (NH4)2SO4 0,2%,
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CaSO4 0,02%, KH2PO4 0,1%) c b sung 1% glucose v 1% lactose. Hn hp

c cho vo bc, xom l, 30oC trong 24 gi, thu hoch v sy kh 42oC,
xay nh to thnh bt men thun. Bt la cha enzyme c sn xut t la ln
mm tch ly enzyme nhiu nht (th nghim 2.2.4). Mm la c sy kh
40 - 42oC trong 24 gi v nghin thnh bt.
2.2.6 Kho st kh nng ln men ca bt men thnh phm
Bt men thnh phm c dng lm men ging cho qu trnh ln men ru go.
Th nghim c b tr 2 nhn t v 3 ln lp li. Hai nhn t bao gm t l bt
men thun v bt mm la (b tr 3 mc khc nhau, tu theo kt qu so snh
hot tnh enzyme trong th nghim 2.2.4) v hnh thc ln men (b tr 2 mc
khc nhau: chan nc ngay sau khi trn men, ln men 7 ngy; v chan nc sau
khi trn men 1 ngy, ln men 6 ngy). Tng cng c 6 nghim thc v 18 n v
th nghim.
Qui trnh tin hnh nh sau: 50g go ngm trong nc hp cch thy 100oC
trong 1 gi ngui 40 - 45oC trn men chan nc ngay (nghim thc 1)
hoc chan nc sau 1 ngy 30oC (nghim thc 2) (lng nc chan bng 2 ln
lng c cht em ) ln men trong 7 ngy (nghim thc 1) hoc 6 ngy
(nghim thc 2) chng ct v thu ru thnh phm. Ch tiu nh gi gm c
lng kh CO2 c sinh ra trong qu trnh ln men, cn thu c sau chng
ct v mi v sn phm ru thnh phm.
2.2.7 Phng php phn tch thng k
Cc s liu c x l bng phn mm MicroSoft Excel 2003 v thng k bng
chng trnh StatGraphics version 3.0.
3 KT QU THO LUN
3.1 Xc nh mt s nm men, nm mc v phn lp nm men
Mt s nm men v nm mc hin din trong 5 loi men ru th trng c
trnh by trong bng 1. Kt qu cho thy mt s nm mc v nm men trong cc
loi men ru th trng kh cao, ln lt l 5,93 - 7,61 v 7,49 - 10,23 log cfu/g.
Bng 1: Mt s nm mc v nm men trong 5 loi men ru th trng

STT
1
2
3
4
5

Tn loi men
Quang Minh (QM)
Hong Anh (HA)
Hi Anh Quang (HAQ)
Dung c Thnh (DT)
Linh Chi (LC)

Mt s vi sinh vt (log cfu/g trng lng kh)

Nm mc
Nm men
6,41
8,89
6,56
10,23
7,61
7,49
7,05
8,40
5,93
9,32

i vi bnh men c b sung enzyme, nh sn xut cng b thnh phn bt men

ch gm nm men v enzyme, song kt qu phn tch cho thy trong nhng loi
men ny cn c bo t nm mc vi mt s tng i cao, chng t nm mc c
vai tr quan trng khng th thay th trong qu trnh ln men ru.

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Cc khun lc nm men c cy chuyn nhiu ln trn mi trng PGA cho n

khi t c thun nht. Kt qu phn lp c 26 dng nm men, trong
gm c: 8 dng t men Quang Minh, 6 dng t men Hong Anh, 5 dng t men
Linh Chi, 4 dng t men Hi Anh Quang, v 3 dng t men Dung - c Thnh.
Cc dng nm men c nhiu hnh dng khc nhau: hnh cu, elip v ovan; tn ti
ring l hoc kt chui v c hnh thc sinh sn ny chi c trng. Hnh dng tiu
biu ca mt dng nm men phn lp c trnh by trong hnh 1.

Hnh 1: Hnh dng t bo nm men c phn lp (vt knh E40)

3.2 So snh kh nng ln men v tuyn chn dng nm men c hot tnh cao
Kh nng ln men ethanol ca 26 dng nm men c kho st bng phng php
o chiu cao ct kh CO2 (cm) sinh ra trong chung Durham. Chiu cao ti a ca
ct kh trong chai Durham l 4,2cm, v trn nguyn tc l trong qu trnh ln men
ru ct kh sinh ra cng cao th kh nng ln men ethanol cng mnh. Kt qu
cho thy sau 6 gi ln men, c 10 dng phn lp th hin kh nng ln men sm
v mnh vi trung bnh chiu cao ct kh t 2,2 - 2,58cm. Hu ht cc dng nm
men u cho thy kt qu chiu cao ct kh trong chung Durham khng khc bit
gia mc thi gian 12 gi v 14 gi. Kt qu sau 14 gi kt thc qu trnh ln
men, cc dng nm men c ct kh cao nht mi loi bnh men c tuyn chn
cho th nghim k tip gm c: Y2LC (3,73cm), Y2DT (3,58cm), Y3HA
(3,25cm), Y1QM (3,23cm) v Y2HAQ (3,17cm). Nm dng nm men s tuyn
ny c s dng so snh kh nng ln men vi dng men S. cerevisiae.
Kt qu o chiu cao ct kh v m s lng bt kh sinh ra trong qu trnh ln
men cho thy su dng nm men th hin kh nng ln men kh mnh v cho thy
s khc bit khng c ngha gia cc dng men. Qu trnh ln men c du hiu
gim mnh thi gian sau 24 gi ln men. Tuy nhin, Bng 2 cho thy kt qu
phn tch cn sinh ra thu c ( 20oC) sau 48 gi ln men c khc bit ngha
tin cy 95% gia mt s dng men. Dng men Y3HA c cn sinh ra thp
nht ( cn trung bnh l 8,83%w/v) khc bit c ngha so vi dng S.
cerevisiae c cn cao nht ( cn trung bnh l 9,67%w/v). Bn dng men cn
li c cn trung bnh l 9,50%w/v (Y2HAQ) v 9,17%w/v (Y2LC, Y2DT v
Y1QM) khc bit khng ngha so vi dng S. cerevisiae. Dng men S. cerevisiae
c chn thc hin cho th nghim tip theo, s dng nh ngun ging chng cho
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nghin cu sn xut v kho st kh nng ln men ca bt men gm nm men v

enzyme amylase.
Bng 2: Kt qu ethanol thu c sau 48 gi ln men ca su dng men

Dng men

cn 20oC (%w/v)
Ln 2
Ln 3
Trung bnh
9,5
8,0
8,83b
9,5
9,5
9,50ab
9,0
9,0
9,17ab
9,5
9,5
9,17ab
10,0
9,5
9,67a
9,0
9,5
9,17ab

Ln 1
9,0
9,5
9,5
8,5
9,5
9,0

Y3 HA
Y2 HAQ
Y2 LC
Y1 QM
Saccharomyces cerevisiae
Y2 DT

Ghi ch: Cc gi tr trung bnh c cng mu t th khng khc bit mc ngha 5%.

3.3 Xc nh hot tnh enzyme amylase trong qu trnh ln mm la

Hot tnh enzyme amylase trong mm la c kho st qua cc ngy ln mm
nhm xc nh thi gian mm la t hot tnh enzyme cao nht. Kt qu hnh
2 cho thy hot tnh enzyme trong mm la tng dn trong 4 ngy u tin v t
gi tr cao nht 9,0 U/g vo ngy th 4, sau gim dn.
Nh vy, mm la 4 ngy c hot tnh enzyme cao nht, khc bit c ngha
tin cy 95%, nn c chn tin hnh th nghim sn xut bt mm la. Tuy
nhin, so vi hm lng enzyme c trong 5g c cht kh ca bt men th trng
(21,96 U/g) th lng enzyme trong mm la ngy cao nht cn thp hn 2,44
ln. V th, t l pha trn nm men v mm la trong th nghim sn xut bt men
c xc nh l 1:3, 1:4 v 1:5.
10

9.00 a

Hot tnh enzyme (U/g

9
8
7

6.06 b

6.31b
bc

5.23

bc

5.10

3.97 cd

4
3

2.54d

2
1
0
1

7 Ngy

Hnh 2: Hot tnh enzyme amylase trong qu trnh ln mm

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3.4 Kh nng ln men ca bt men ru gm nm men v enzyme amylase

Kh nng ln men ca bt men ru gm t hp nm men v enzyme amylase c
trong bt mm la c kho st 3 t l phi trn tng ng l 1:3; 1:4 v 1:5
vi 2 phng php ln men khc nhau. Cc mu th nghim nh gi lng bt kh
CO2 sinh ra sau mi 12 gi trong 3 ngy v o cn sau 7 ngy. Kt qu m bt
kh sinh ra trong qu trnh ln men ru c trnh by trong hnh 3.
30

A1: Bt men : mm la = 1:3

A2: Bt men : mm la = 1:4
A3: Bt men : mm la = 1:5
B1: Chan nc ngay sau khi trn men
B2: Chan nc sau khi trn men 1 ngy

25

S bt kh

20
15
10
5
0
12

24

B1 A1

36

B1 A2

48

B1 A3

72 Thi gian (gi)

60

B2 A1

B2 A2

B2 A3

Hnh 3: Lng bt kh CO2 sinh ra trong qu trnh ln men

Kt qu cho thy phng php ln men chan nc trc tip sau khi trn men
khng c s chnh lch v s lng bt kh sinh ra c ngha gia 3 t l phi
trn. Lng bt kh sinh ra nhiu 12 v 24 gi cho thy enzyme pht huy tc
dng tt v nm men cng thch nghi vi phng thc ny. Cc nghim thc s
dng phng php ln men chan nc sau khi trn v hiu kh 1 ngy cho thy
s khc bit c ngha v lng bt kh sinh ra gia cc t l bt men v mm la,
trong t l 1:4 cho kt qu bt kh tng i cao sau 12 gi chan nc. Qu
trnh kt thc ln men ca 2 nghim thc xy ra cng thi gian (sau 7 ngy ).
Bng 3 th hin kt qu phn tch cn sinh ra thu c ( 20oC) sau qu trnh
ln men ru.
Bng 3: Kt qu ethanol sau qu trnh ln men ca cc nghim thc

T l bt
men :
mm la
1:3
1:4
1:5

Phng thc
chan nc
B1
B2
B1
B2
B1
B2

ru 20oC sau ln men (%w/v)

Ln 1

Ln 2

Ln 3

Trung bnh

5
6
6
6
6
6

6
5
5
6
5
7

6
5
6
6
7
6

5,7a
5,3a
5,7a
6,0a
6,0a
6,3a

Ghi ch B1: chan nc trc tip sau khi trn men; B2: chan nc 1 ngy sau khi trn men v .

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Kt qu cho thy ru gia 2 phng thc ln men cc t l phi trn bt

men v mm la khng c khc bit ngha. Lng ru sinh ra tng i thp,
trung bnh t 5,3 - 6,3%w/v. nghim thc B1 sau 7 ngy ln men sn phm c
mi kh chu v chua, chng t xy ra qu trnh ln men lactic sau qu trnh ln
men ru, v vy c kh nng mt lng ru tht thot trong qu trnh ln men
chua ny. Lng dch ln men trong sn phm nhiu v lng c cht cng b
phn hy tng i. Sn phm nghim thc B2 sau 6 ngy chan nc c hng
cm ru c trng, nhng lng c cht cha c s dng ht v lng dch ln
men sinh ra khng nhiu. iu ny chng t nm men vn hot ng nhng lng
enzyme c th cha c th hin tc dng ti a trong qu trnh ng ha to
ra lng ng kh nhiu cung cp cho qu trnh ru ha di hot tnh ca
nm men.
4 KT LUN
Kh nng ln men ru ca 26 dng phn lp t 5 loi men th trng c xc
nh v so snh vi kh nng ln men ca nm men S. cersvisiae. Bt men ru
gm t hp nm men S. cersvisiae v enzyme amylase c sn xut bng cch
phi trn bt mm la v bt men thun 3 t l khc nhau (1:3, 1:4 v 1:5).
Phng thc ln men thch hp hn c th hin khi chan nc 1 ngy sau chng
ging v . Kt qu ti ny cho thy ngun enzyme amylase c kh nng c
s dng thay th ngun ging nm mc trong qu trnh ng ha. Tuy nhin, kh
nng ln men ru ca bt men 3 t l ny khng c khc bit ngha v nng
cn sinh ra cha cao, v th cn c tip tc nghin cu cc t l phi trn
khc nhau, cng nh nn th nghim so snh vi bt men ru gm t hp nm
mc v nm men trong qu trnh ln men ru.
TI LIU THAM KHO
Bi i, 2005. Cng ngh ln men ng dng trong Cng ngh thc phm, Nh xut bn i
hc Quc gia TP. H Ch Minh.
Dung N.T.P, F.M. Rombouts, and M.J.R. Nout, 2005. Development of defind mixed culture
fungal fermentation starter granulate for controlled production of rice wine. Innovative
Food and Emerging Technologies 6, pp. 429-441.
Dung N.T.P, F.M. Rombouts, and M.J.R. Nout, 2006. Functionality of selected strains of
moulds and yeasts from Vietnamese rice wine starters. Food Microbiology 23,
pp.331-340.
Dung, N.T.P. and Phong, H.X., 2011. Application prospects for the innovation of defined
fungal starter in rice wine fermentation. Journal of Life Sciences 5 (4), pp. 255-263.
Nelson Norton, 1994. A photometric adaption of the Somogyi method for the determination
of glucose. J Biol Chem, 153 (2), pp. 375-380.
Nguyn nh Thng v Nguyn Thanh Hng, 2005. Cng ngh sn xut v kim tra cn
etylic, Nh xut bn Khoa hc v K thut, H Ni.
Nout, M.J.R. and Aidoo, K.E., 2002. Asian fungal fermented food. In The Mycota. Vol.X
Industrial applications. ed. H.D. Osiewacz, pp. 23-47. Berlin-Heidelberg-New York:
Springer-Verlag.
Steinkraus, K.H, 1997. Classification of fermented foods: worldwide review of household
fermentation technquies. Food Control 8, pp. 311-317.

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