Basil Us Lacto

PHN II
CNG NGH VI SINH

Phn II: CNG NGH VI SINH 199
XC NH NHANH Lactobacillus BNG PHNG PHP
PCR (Polymerase Chain Reaction)
Trn Hong Ngc i, Trn nh Nguyt, L Th Hng Tuyt, Hong Quc Khnh
Phng Vi sinh ng dng, Vin Sinh hc Nhit i
M U
Lactobacillus c vai tr quan tr ng trong cng nghip sa, mui chua rau qu, l m
yaourt,... cng nh trong nng nghip v dc phm. V mt c tnh khc ca
lactobacillus tr nn c xem trng l chng c kh nng to ra bacteriocin (cht
khng khun) nh lactacin, brevicin, lacticin, helveticin, sakacin, plantacin,... c tc
dng c ch mt s vi sinh vt gy bnh, ngn chn s pht trin ca cc ngun bnh
trong thc phm (Batt, 1999; Duber net et al., 2002). V cc chng lactobacillus thng c
tr trong ng rut ca ngi v ng vt vi mt lng sn c s gip kch thch ti u
ho thc n v tiu dit mt s vi khun gy bnh ng rut khc nn hin nay trn th
trng c nhiu thc phm, dc phm c b sung cc chng probiotic c n sng.
Lactobacillus bao gm trn 25 loi v mc u tin phn bit chng l da
trn thnh phn ca sn phm cui; mt s lactobacillus l n men ng hnh trong khi
mt s khc th ln men d hnh. Ngoi phng php phn bi t da trn cc c tnh
pht trin nhng nhit , pH v nng mui khc nhau ca lactobacillus, c n c
phng php khc phn bit chng l dng ln men carbohydrat, thy gii arginine,
thy gii peptidoglycan v dng tng ng DNA-DNA.
Tuy nhin, do ging vi nhiu vi khun lactic khc v cc y u cu pht trin v
dinh dng nn thng kh khi s dng phng php vi sinh vt truyn thng xc
nh chng ngay c mc ging. V vy, nghin cu ny tp trung vo ng dng k
thut sinh hc phn t da tr n phn tch d liu trnh t gen rDNA16S xc nh
nhanh lactobacillus. Vi c s dng mi c hiu cho gen m ho rRNA 16S bng PCR
c thc hin nh l mt phng php xc nh.
VT LIU V PHNG PHP
Cc chng vi khun v iu kin nui cy
Chng vi khun
Cc chng vi khun Lactobacillus c dng trong nghin cu ny l c thu
nhn t ARS Culture Collection (NRRL) bao gm: Lactobacillus acidophilus,
Lactobacillus bulgaricus, Lactobaci llus johnsonii, Lactobacillus reuteri, Lactobacillus
sakei. Ngoi ra cn c chng Streptococcus faecium, Bacillus subtilis.
Cc iu kin nui cy
Mi trng: Vi khun Lactobacillus v Streptococcus c nui cy trong mi
Hi ngh KHOA HC V CNG NGH 2007 200
trng MRS (de Man-Rogosa-Sharpe); cn B.subtilis c nui cy trong mi
trng LB (Luria-Bertani )
Nhit : Cc chng vi khun ny c nui 37
o
C
Tch chit DNA (Theo Sambrook J. et al, 1989)
T bo vi khun lactic c nui cy trong mi tr ng MRS lng, cn B. subtilis
c nui cy trong mi trng LB 37
o
C qua m
Thu sinh khi ca 5ml dch vi khun trong eppendorf bng cch ly tm 10 000
vng/ pht trong 2 pht
Thm 500 l dch huyn ph phn gii t bo, vortex mnh dch t bo ng nht
Cho vo mi eppendorf 10mg ct trng, vortex mnh, trong 30 giy
Thm 150 l dung dch Potassium acetat 3M, pH 4.8
Vortex u, ly tm 10 000 vng/ pht trong 10 pht, l y dch ni cho vo
eppendorf mi
Thm vo 600 l isopropanol lnh, o ngc eppendorf 1-2 ln ta DNA, ly
tm 13 000 vng/ pht trong 10 pht, b dch ni mt cch nh nhng
Thm vo 500 l ethanol 70% ra ta, ly tm 13 000 vng/ pht trong 10
pht, b dch ni mt cch nh nhng, lm kh trn gi y thm, kh t nhi n
trong khng kh khong 20 pht
Thm vo 50 l dung dch TE, lc nh
Thm vo 2 l RNAase 1mg/ml v 37
o
C trong 3 gi
Bo qun DNA 4
o
C hoc - 20
o
C
Sau xc nh hm lng v tinh sch ca DNA bng cch o OD bc
sng 260nm, 280nm v chy in di trn gel agarose 0,8%.
Khuch i gen bng PCR
S dng hai cp mi khuch i gen rDNA 16S ( c tng hp t hng Proligo)
vi trnh t nh sau:
- Cp mi 1: Mi xui: 5- GGA AAC AGA TGC TAA TAC CG-3
Mi ngc: 5- CAC CGC TAC ACA TGG AG-3
- Cp mi 2: Mi xui: 5- AGC AGT AGG GAA TCT TCC A-3
Mi ngc: 5- ATT CCA CCG CTA CAC ATG-3
Thnh phn phn ng
Khun DNA 200ng
Taq DNA polymerase 1.25 U
Nng mi 1 M
dNTP 200 M
Tris-HCl, pH 8.3 20mM
KCl 100mM
MgCl
2
3mM
iu kin phn ng
95
o
C, 5 pht; 30 chu k (95
o
C, 1 pht; 55
o
C, 1 pht; 72
o
C, 2 pht); 72
o
C, 7 pht.
Sn phm PCR c phn tch bng in di tr n gel agarose 1.5% v thu c kch
thc gen rDNA 16S (khi c khuch i vi cp mi 1) l 700 bp v kch thc gen
rDNA 16S (khi c khuch i vi cp mi 2) l 340 bp.
Khuch i gen bng PCR khun lc
S dng Kit microLYSIS
TM
theo ch dn ca nh sn xut Microzone Limited vi
cc bc nh sau:
T bo vi khun lactic c nui cy trong mi trng MRS lng, cn B. subtilis
c nui cy trong mi tr ng LB 37
o
C qua m
Trn 3 l dch vi khun trn vi 17 l dung dch microLYSIS
t vo my chu k nhit vi ch : 65
o
C, 5 pht; 96
o
C, 2 pht; 65
o
C, 4 pht;
96
o
C, 1 pht; 65
o
C, 1 pht; 96
o
C, 30 giy; gi 20
o
C
Sau khi chy vi chu k nhit trn, tt c hn hp microLYSIS/DNA c th c
dng trc tip trong PCR hoc c th c ct gi - 20
o
C.
Vi PCR khun lc, ch s dng cp mi 2 vi tr nh t nh trn khuch i gen
rDNA 16S v cho kch thc l 340 bp. Cn iu kin phn ng vn khng thay i.
Thnh phn phn ng:
Hn hp microLYSIS/DNA 5 L
Hn hp DNA polymerase 1.2 U
Nng mi 1 M
dNTP 200 M
Tris-HCl, pH 8.3 10 mM
KCl 50 mM
MgCl
2
1.5 mM
Trong , hn hp DNA polymerase, Taq DNA polymerase, dNTP v thang DNA
Marker VI c mua t hng Roche.
KT QU
Khuch i gen bng PCR
Hnh 1. Sn phm khuch i gen rDNA 16S ca phn ng PCR tr n gel agarose 1,5%. M: thang
DNA Marker VI (c 11 vch vi kch thc t trn xung: 2.20, 1.80, 1.20, 1.0, 0.65, 0.52, 0.45, 0.39,
0.30, 0.23, 0.22 kb) ; 1, 4, 6: Lactobacillus acidophilus c khuch i vi cp mi 1; 2, 5, 7:
Lactobacillus acidophilus c khuch i vi cp mi 2; 3: B. subtilis c khuch i vi cp
mi 1; 8: B. subtilis c khuch i vi cp mi 2
T kt qu trn cho thy: Khi khuch i gen rDNA 16S vi cp mi 1 v 2 ca L.
acidophilus th u cho vch DNA t ng ng l 0.7 kb v 0.34 kb nh d kin; trong
khi B. subtilis cho kt qu m tnh.
Khuch i gen bng PCR khun lc
Hnh 2. Sn phm khuch i gen rDNA 16S ca phn ng PCR khun lc tr n gel agarose 1,5%.
M: thang DNA Marker VI; 1: Lactobacillus acidophilus; 2: Lactobacillus johnsonii; 3: Lactobacillus
sakei; 4: Lactobacill us bulgaricus; 5: Lactobacillus reuteri; 6: Streptococcus faecium
Qua kt qu trn c th kt lun: Mc d khc nhau v loi nhng cng l gi ng
Lactobacillus nn khi khuch i gen rDNA 16S vi cp mi 2 th sn phm PCR u
cho vch DNA l 0.34 kb; cn Streptococcus faecium th cho kt qu m tnh.
Nh vy, c hai mi 1 v 2 u c hiu cho ging Lactobacillus v gip ta c th
pht hin chng trong sn phm c nhiu ging khc nhau ca cng mt nhm hoc
ngoi nhm vi khun lactic.
KT LUN
Vi cp mi c hiu cho gen rDNA 16S, chng ti xc nh c Lactobacillus
mc ging khi s dng phng php PCR (vi khun l DNA c tch chit) v
phng php PCR khun lc. c bit phng php PCR khun lc cho kt qu
nhanh hn. y l k thut sinh hc phn t c dng xc nh, phn loi cc vi
khun thc (Eubacteria) v dng pht hin cc vi khun gy bnh trong thc phm,
nc, m phm, Do , t i gp phn cho vic pht hin v nh danh n mc
loi ca cc vi khun lactic ni ri ng v vi khun thc ni chung.
TI LIU THAM KHO
1. Batt CA. (1999). Latobacillus. Introduction. Encyclopedia of Food Microbiology.
Academic Press.
2. Dubernet S, Desmasures N, Gueuguen M. (2002). A PCR-based method for identification
of lactobacilli at the genus level. FEMS Microbiology Letters 214: 271-275.
3. Heilig H, Zoetendal EG, Vaughan E, Marteau P, Akkermans A, and deVos. (2002).
Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the
human intestine as determined by specific amplification of 16S ribosomal DNA.
Appl Environ Microbiol68:114-123.
4. Reid G. (1999). The scientific basis for probiotic strains of Lactobacillus. Appl
Environ Microbiol 65: 3763-3766.
5. Sambrook J, Fritsch EF and Maniatis T. (1989). Molecular Cloning. A Laboratory
Manual, 2
nd
ed. Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY.
6. Tannock GW, Munro K, Harmsen HJM, Welling GM, Smart J, and Gobal PK. (2000).
Analyses of the fecal microflora of human subjects consuming a probiotic product
containing Lactobacillus rhamnosus DR20. Appl Environ Microbiol 66: 2578-2588.
7. Walter J, Hertel C, Tannock GW, Lis CM, Munro K, and Hammes WP . (2001).
Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in
Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel
Electrophoresis. Appl Environ Microbiol 67: 2578-2585.
SUMMARY
The PCR- based rapid identifying method for Lactobacillus
Tran Hoang Ngoc Ai, Tran Anh Nguyet, Le Thi Hong Tuyet, Hoang Quoc Khanh
Institute of Tropical Biology
The sequencing and analysis of the 16S and 23S rDNA genes are considered as one
of the cornerstones of modern microbial taxonomy. These sequences are used to define
genus-specific PCR primers for a rapid detection of lactic acid bacteria (LA B).
Moreover, in recent years, several rapid methods for the detection and identification of
lactobacilli have been developed. Traditional microbiological assays for the
identification of lactobacillus species are very often time - consuming and can yield
rather variable results. LAB strains including Lactobacillus acidophilus, L. johnsonii, L.
reuteri, L. bulgaricus, L. sakei were collected from freeze-dried samples of ARS
Culture Collection (NRRL). They were activated and incubated in MRS broth medium
at 37
o
C to get biomass; then they were extracted DNA as a template of standard PCR or
used directly in colony PCR. Both of this two PCR methods were used to amplify 16S
rDNA gene with lactobacillus genus -specific primer. In this work, we used two primers
for amplifying 16S rDNA gene and generated PCR product size as 0.7 kb (for primer 1)
and 0.34 kb (for primer 2) for five lactobacillus species. Electrophoresis did not reveal
any discrete bands when Bacillus subtilis, Streptococcus faecium DNA were used as
template. Identification of lactobacillus in colony PCR have a result faster and more
high yield than standard PCR because there was a DNA polymerase mixture including
Taq DNA polymerase and Tgo DNA polymerase in Expand High Fidelity System
designed from Roche Ltd. Thus, we showed that the genus -specific primers could be a
useful tool for identification of the members of lactobacilli.
BIN NP DI TRUYN GIN TIP NH Agrobacterium
tumefaciens VO NM BNH CY Phytophthora palmivora
Trn Hong Ngc i v Hong Quc Khnh
M U
Bin np di truyn da tr n kh nng t nhin ca Agrobacterium tumefaciens l
chuyn on DNA t Ti plasmid ca n, T-DNA, vo t bo thc vt v nm v ha
nhp ngu nhin vo nhim sc th trong nhn. Tin tr nh chuyn T-DNA ph thuc
vo biu hin ca gen vir (gen gy c) ca A.tumefaciens. S biu hin ca gen gy
c ny c cm ng bi s tit ra ca mt s cht t vt th ng thc vt nh
acetosyringone (AS). S c mt ca bin (border) tri v bin phi T-DNA l cn thit
trong tin trnh ny.
Trong t nhin c rt nhiu bnh thc vt do nm gy ra, dn n tnh trng nng
sut ma mng thp nhiu ni trn th gii. V vy nm gy bnh thc vt l i
tng cn c nghin cu khng ch v c tnh nui cy, sinh ha, bnh l m cn
mc di truyn phn t c th bin i di truyn cng nh kim sot bnh gy ra.
Trong bo co ny trnh by cc k t qu thu c v bin np di truyn nm
Phytophthora palmivora, nm gy bnh chnh tr n cy cao su v cy n tri nh s u
ring, ca cao, bi.
VT LIU V PHNG PHP
Chng vi sinh vt
Cc chng Escherichia coli DH5o v Agrobacterium tumefaciens LBA 4404 m
t trong bi bo (3).
Phytophtora palmivora thu nhn t Trung tm Kim dch thc vt Vng II.
Plasmid pPK2 l vector th cp c thit k cho bin np vi nm (2).
Mi trng
Mi trng LB dng nui cy E. coli v A. tumefaciens.
Mi trng V-8 dng nui cy nm P. palmivora.
Mi trng nui cy chung vi khun v nm theo (3).
Mi trng chn lc th bin np P. palmivora theo (3).
Mi trng nui cy tch chit DNA ca P. palmivora l mi trng lng malt-
cao nm men (MY).
Cc vt liu khc
Cc vt liu dng trong th nghim tng t trong (3).
Phng php
Bin np plasmid pPK2 vo E.coli bng phng php CaCl
2
lnh theo Sambrook
v cs (6).
Tch chit plasmid pPK2 t E. coli theo b kit UltraClean 6 Minute Mini Plasmid
Prep (hng MO BIO)
Bin np plasmid pPK2 bng xung i n vo A. tumefaciens theo (4).
Tch chit plasmid pPK2 t A. tumefaciens theo Sambrook v cs (6).
Bin np Phytophthora palmivora gin tip qua A. tumefaciens bng phng php
nui cy chung (3).
Tch chit DNA genome ca nm P. palmivora:
Cy nm vo 30 ml mi trng MY, trong ti trong trng thi tnh 3 ng y.
Ly tm thu sinh khi ca 5 ml dch nm trong eppendorf 6000 v/p trong 5 pht.
Cho vo 500 l dung dch phn gii t bo v thm 10mg ct trng. Vortex mnh
trong 1 pht, t trong 1 pht, lp li 2 ln.
Thm vo 150 l potassium acetate 3M. Ly tm 10.000 v/p trong 10 pht, thu d ch
ni vo ng eppendorf mi.
Cho vo 600 l isopropanol lnh, o ngc trn, t trong 10 pht, ly tm
13.000 v/p trong 10 pht, rt nh nhng dch ni, thu ta.
Ra ta vi 500 l ethanol 70% lnh, ly tm 13.000 v/p trong 10 pht, rt dch ni,
thu ta, phi kh t nhin trong khng kh.
Ha tan ta vi 20 l m TE. Thm vo 1 l RNAse 1mg/ml, 37
0
C khong 3
gi. Gi 4
0
C.
Nng v sch ca ADN kim tra bng quang ph b c sng 260 v 280
nm. Ngoi ra DNA cn c kim tra bng in di tr n gel agarose 0,8%.
Kim tra s hin din ca gen hph (gen khnh hygromycin B) tr ong DNA genome
ca P.palmivora bng phn ng PCR:
Phn ng PCR thc hin vi cp mi c hiu cho gen hph l hph-F v hph-R trong iu
kin: 94
0
C, 5 pht; 30 chu k (94
0
C, 1 pht, 60
0
C, 1 pht, 72
0
C, 2 pht); 72
0
C, 10 pht.
Sn phm PCR c in di trn gel agarose 1,5 % v cho kch thc gen hph d
kin 1 kb.
KT QU V THO LUN
Bin np Agrobacterium tumefaciens:
Sau khi tch chit plasmid pPK2 mang gen khng kanamycin t E. coli v bin np
vo A.tumefaciens bng phng php xung in kt qu cho thy nhng khun lc bin
np mc c trn mi trng LB c b sung 50 g/ml kanamycin, trong khi chng
hoang di th khng (Hnh 1).
Hnh 1. Bin np pPK2 vo t bo A.tumefaciens. Chng A.tumefaciens LBA 4404 bin
np vi plasmid pPK2 mc tr n mi trng LB c kanamycin (phi),
chng khng bin np (tri)
Quan st s gn kt A. tumefaciens ln t nm P. palmivora
A. tumefaciens v P. palmivora c nui trn mi trng nui cy chung. Quan
st trn knh hin vi cho thy trong giai on u ca qu tr nh nui cy chung din ra
s tip xc ca t bo A. tumefaciens ln b mt ca t nm (Hnh 2). iu ny c
ngha quan trng trong qu tr nh chuyn T-DNA t vi khun vo nm. Cc th nghim
tip theo s tip tc l m sng t qu trnh ny.
Hnh 2. Gn kt ca A.tumefaciens ln t nm P.palmivora (40 X)
Bin np Phytophthora palmivora
Chn nng hygromycin B thch hp chn lc P.palmivora bin np. Cy
P.palmivora hoang di vo mi trng V8-agar c b sung nng hygromycin B l 0, 50,
100, 150, 200, 250 g/ml. Kt qu cho thy nng 250 g/ml nm khng mc c
(s liu khng cng b), v vy nng 250 g/ml hygromycin B c dng chn
lc th nm bin np. Cc khun lc nm bin np xut hin tr n mi trng chn lc
sau 6 ngy nui cy, sau cy sang mi trng V8-agar c b sung 250 g/ml
hygromycin B. Cc kt qu chn lc th hin tr n Hnh 3.
Hnh 3. Bin np P.palmivora nh A.tumefaciens mang plasmid pPK2. Cc th bin np
P.palmivora mc trn mi trng c 250 g/ml hygromycin (B) , th hoang di (A)
Kim tra s hin din ca gen hph trong genome ca P.palmivora bng PCR.
Thc hin phn ng PCR vi cp mi c hiu v DNA mch khun l DNA
genome nm. Sn phm PCR in di tr n gel agarose cho band c kch thc tng
ng vi kch thc on gen hph c trong on T-DNA ca plasmid pPK2. iu
chng t T-DNA ca pPK2 ha nhp vo trong DNA genome ca nm P.palmivora.
2 1 3 4 5 6
1.0 kb
2 1 3 4 5 6 2 1 3 4 5 6
1.0 kb
Hnh 4. Kim chng dng P.palmivora bin np c mang gen hph bng PCR. 1, Thang DNA marker VI
(Roche ); 2, Sn phm PCR t khun plasmid pPK2; 3, sn phm PCR t khun DNA ca chng hoang
di; 4, 5, 6, sn phm PCR t khun DNA ca chng bin np
y l nhng kt qu u ti n v bin np di truyn nm gy bnh thc vt
P.palmivora gin tin nh A. tumefaciens. Ni chung bin np di truyn vo vi nm gp
nhiu kh khn cho nn s pht trin ca bin np di truyn nh Agrobacterium gip
ch rt nhiu cho vic a vo cc gen mong mun, hoc to ra cc t bin c nh
hng gip cho vic nghi n cu c ch gy bnh trn thc vt (2, 5). ng thi, qua cc
th nghim bin np nh Agrobacterium cho thy k thut n y tng i n gin v
tn t cng sc so vi cc k thut bin np khc.
TI LIU THAM KHO
1. Cassago A., et al. (2002). Cellophane based mini -prep method for DNA extraction
from the filamentous fungus Trichoderma reesei. BMC Microbiology (xem:
http://www.biomedcentral.com/1471-2180/2/14).
2. Covert S. F., et al. (2001). Agrobacterium tumefaciens -mediated transformation of
Fusarium circinatum. Mycological Re search 105: 259-264.
3. Hong Quc Khnh, Trn Hong Ngc i (2003). Chuyn gen khng hygromycin B
vo vi nm Trichoderma harzianum bng phng php gin tip nh Agrobacterium
tumefaciens. Bo co khoa hc, Hi ngh ton quc ln th 2. Nhng vn nghin
cu c bn trong khoa hc s sng: 930-934, Nxb Khoa hc v K thut H Ni.
4. McCormac A. C., et al., (1998). A simple method for the production of highly
competent cells of Agrobacterium for transformation via electroporation. Molecular
Biotechnology 9:155-159.
5. Mullins E. D. and Kang S., (2001). Transformation: A tool for studying fungal
pathogens of plants. Cell Mol. Life Sci. 58: 2043-2052.
6. Sambrook J., et al., (1989). Molecular Cloning: A Laboratory Manual, 2
nd
Ed. Cold
Spring Harbor Laboratory Press.
SUMMARY
Agrobacterium tumefaciens-mediated genetic
transformation of the phytopathogen Phytophthora palmivora
Tran Hoang Ngoc Ai and Hoang Quoc Khanh
Agrobacterium tumefaciens-mediated transformation has been successfully applied
to the phytopathogen Phytophthora palmivora. The T -DNA was integrated into the
genome and was stable through cell division. The presence of hph gene in P.palmivora
genomic DNA was checked by PCR. These findings should facilitate future analysis of
P.palmivora pathogenicity and stimulate wider use of this valuable transformation
method in fungal research.
BIN NP DI TRUYN NM RM Volvariella volvacea
BNG PHNG PHP BIN NP GIN TIP NH
Agrobacterium tumefaciens
Trn Hong Ngc i, Hong Quc Khnh
M U
Nm rm Volvariella volvacea l mt nm n c nui trng ph bin nhng vng
nhit i v cn nhit i. Mc d hin nay nm rm chim 1/5 lng sn xut hng nm
trn khp th gii nhng hiu qu sinh hc (pht trin trn c cht hnh thnh qu th) th
thp so vi cc loi nm nui trng ph bin khc nh Agaricus bisporus, Lentinula edodes
v Pleurotus sajor - caju (Chen et al., 2003; Ding et al., 2001).
Nm rm c nui trng trn nhiu dng cht thi c lignocellulose nh rm ca
cc cy ng cc, l chui, b ma ng, v qu c du (Cai et al., 1999). Ging vi
nhiu vi nm c kh nng phn gii cellulose, V. volvacea sn xut ra mt h thng
nhiu enzym gm |-glucosidase, endo-1,4-|-glucanase, cellobiohydrolase chuyn ho
cellulose thnh glucose (Cai et al., 1998). Ngoi ra, V. volvacea cn to ra laccase l
mt enzym phn gii lignin v oxy ho cc hp cht phenol.
Do tm quan trng ca nm r m v mt sinh hc, c gi tr kinh t v cng l mt
loi nm ln ph bin nc ta nn vic tm hiu s di truyn ca chng l cn thit.
Bc u, chng ti chuyn gen khng hygromycin B vo V. volvacea bng phng
php bin np gin tip nh Agrobacterium tumefaciens da trn c tnh t nhin ca
vi khun ny l chuyn T-DNA t Ti plasmid ca chng vo nhim sc th trong nhn
ca t bo ch nh thc vt hoc vi nm.
VT LIU V PHNG PHP
Chng vi sinh vt
Escherichia coli DH5o [F
-
endA1 hsdR17(r
k-
/m
k-
) supE44 thi
-
recA1 gyrA96
lac U 169(|80 lacZ M15)] mang plasmid pPK2.
Agrobacterium tumefaciens LBA 4404 c dng lm th nhn trong th nghim
bin np, nhn plasmid v chuyn T-DNA t plasmid vo Volvariella volvacea.
Volvariella volvacea l i tng cn chuyn gen.
Plasmid
Plasmid pPK2 mang gen hph (gen khng hygromycin B) c kch th c l 10,77 kb
v chiu di ca gen hph l 1 kb.
Mi trng
Mi trng LB (Luria - Bertani): nui cy vi khun E. coli v A. tumefaciens; PDA
(Potato Dextrose Agar): nui cy nm rm V. volvacea; IM: nui cy chung A.
tumefaciens v V. volvacea; M-100agar: chn lc th bin np V. volvacea; CYM-R:
mi trng CYM (glucose 20g; cao nm men 2g; peptone 2 g; MgSO
4
. 7 H
2
O 0,5g;
KH
2
PO
4
0,46g; K
2
HPO
4
1g trong 1 lt nc ct ) cha 0,6 M Sucrose dng chn lc
th bin np V. volvacea ln 2; MY ( Malt-Yeast extract): nui cy nm rm V.
volvacea tch chit DNA.
Cc vt liu khc
Giy lc (Prolabo)
Cc ho cht dng cho bin np vi khun v in di gel agarose ca hng Merck
Cc ho cht dng cho phn ng PCR ca hng Roche
Mi c hiu cho gen hph: Mi xui (hph-F): 5-AAGCCTGAACTCACCGCGAC-3
Mi ngc (hph-R): 5-CTATTCCTTTGCCCTCGGAC-3
Phng php
Tch chit plasmid pPK2 t E.coli theo hng dn ca b kit Ultra Clean
TM
6
Minute Mini Plasmid Prep Kit
TM
ca hng MO BIO.
Bin np plasmid pPK2 bng xung in v o A.tumefaciens
Tch chit plasmid pPK2 t A.tumefaciens theo Sambrook v cng s (Sambrook J
et al.,1989)
Bin np V. volvacea gin tip qua A. tumefaciens bng phng php nui cy
chung:
Dng 5ml nc ct thu dch bo t v t nm V. volvacea mc 1 tun trn
mi trng PDA.
Dng kp v trng chuyn cc ming giy lc (1,5 x 1,5cm) ln mi trng IM
agar c 200 M AS. Bm 20 l dch bo t v t nm V. volvacea trc tip vo
gia cc ming giy lc; 28
o
C trong 3 ngy.
Cy A.tumefaciens - pPK2 vo 10 ml mi trng LB lng c 50 g/ml kanamycin,
nui cy lc 200 vng/ pht qua m nhit phng.
o OD
600
ca dch vi khun. Pha long dch vi khun vi mi tr ng IM c cha
200 M AS c OD = 0,15.
Nui cy lc 250 vng/ pht khong 4 gi nhit phng c OD
600
= 0,6-0,8.
Bm 50 l dch vi khun c cm ng vi AS vo gia mi ming giy lc c
nm; 28
o
C trong 4 ngy.
Sau 4 ngy, chuyn cc ming giy lc sang mi tr ng M-100 agar c 500 g/ ml
cefotaxime v 70 g/ ml hygromycin B; 28
o
C, loi b cc ming giy lc sau 1
ngy v tip trong 10 ngy na.
Cc khun lc nm xut hin tr n mi trng M-100 agar c 70 g/ ml hygromycin
B c cy chuyn sang mi tr ng CYM-R cng c 70 g/ ml hygromycin B
chn lc th nm bin np.
Tch chit DNA b gen ca nm V. volvacea:
DNA b gen ca nm cha bin np v nm bin np c tch chit t si nm
c pht trin trong 30ml mi tr ng MY; nui trng thi tnh trong 7 ng y v tin
hnh tch chit DNA theo Sambrook v cng s (Sambrook J et al.,1989).
Nng v sch ca DNA plasmid v DNA b gen c kim tra bng cch o
OD
260 nm
v OD
280 nm
. Ngoi ra, DNA cn c kim tra bng in di trn gel agarose 1%.
Kim tra s hin din ca gen hph (gen khng hygromycin B) trong DNA b gen
ca V. volvacea bng phn ng PCR:
Phn ng PCR c thc hin vi cp mi c hiu cho gen hph l hph-F v hph-R
trong iu kin: 94
0
C, 5 pht; 30 chu k (94
0
C, 1 pht; 60
0
C, 1 pht; 72
0
C, 2 pht);
72
0
C, 10 pht.
Sn phm PCR c in di trn gel agarose 1,5% v cho kch thc gen hph d
kin l 1 kb.
KT QU
Bin np Agrobacterium tumefaciens
Plasmid pPK2 mang gen khng kanamycin c tch chit t E.coli v bin np
vo A. tumefaciens, cho kt qu: nhng khun lc ca chng vi khun c bin np
xut hin trn mi trng LB c b sung 50 g/ ml kanamycin, trong khi chng
hoang di (i chng) th khng mc trn mi trng c khng sinh.
Hnh 1. A. tumefaciens trn mi trng LB-Kanamycin (50 g/ ml).
Chng hoang di (tri); chng bin np ( phi)
Bin np Volvariella volvacea
Chn nng hygromycin B chn lc V. volvacea bin np: Cy V. volvacea
hoang di vo mi trng M-100 agar c b sung nng hygromycin B l 0, 20,
40, 50, 60, 70, 80 g/ ml. Kt qu cho thy nng 70 g/ ml hygromycin B v
cao hn , nm khng mc c; v vy chn 70 g/ ml hygromycin B lm nng
chn lc th bin np.
Cc khun lc nm bin np xut hin trn mi trng M-100 agar c 70 g/ ml
hygromycin B sau 10 ngy nui c y (hnh 2), c cy sang mi trng CYM-R
cng c b sung 70 g/ ml hygromycin B, kt qu th hin qua Hnh 3.
A B
Hnh 2. Volvariella volvacea trn mi trng M-100 agar - Hygromycin B (70 g/ ml)
A: Chng hoang di; B: Chng bin np
A B
Hnh 3. Volvariella volvacea trn mi tr ng CYM-R - Hygromycin B (70 g/ml)
A: Chng hoang di; B: Chng bin np
Kim tra s hin din ca gen hph (gen khng hygromycin B) trong DNA b gen ca V.
volvacea bng phn ng PCR
Thc hin phn ng PCR vi cp mi c hiu cho gen hph v DNA mch khun
l DNA b gen ca nm, sn phm PCR c in di trn gel agarose cho band 1 kb
tng ng vi kch thc on gen hph c trong T-DNA ca plasmid pPK2. Nh
vy, T-DNA ca plasmid pPK2 ho nhp vo trong DNA b gen ca nm
Volvariella volvacea.
1 kb
Hnh 4. Sn phm khuch i gen hph ca
phn ng PCR trn gel agarose 1,5%
M: thang DNA Marker VI (c 11 v ch vi
kch thc t trn xung: 2.20, 1.80, 1.20,
1.0, 0.65, 0.52, 0.45, 0.39, 0.30, 0.23, 0.22
kb); 1: plasmid pPK2; 2: DNA ca
Volvariella volvacea hoang di; 3,4: DNA
ca Volvariella volvacea bin np
KT LUN
Chng ti thu nhn c chng nm rm Volvariella volvacea chuyn gen
khng hygromycin B bng phng php bin np gin tip nh A. tumefaciens. Phng
php bin np ny cho hiu qu bin np cao, nhanh chng v t tn km. Nm rm l
mt nm n c gi tr kinh t nn nghin cu ny l s m u cho nhng nghi n cu
chuyn su v di truyn c th l m bin i hiu qu sinh hc ca nm n y cng nh
gp phn cho s nghin cu nhng c tnh di truyn ca cc nm ln khc.
TI LIU THAM KHO
1. Cai YJ, Chapman S, Buswell J and Chang S. (1999). Production and distribution of
endoglucanase, cellobiohydrolase and |- glucosisade components of cellulolytic
system of Volvariella volvacea, the edible straw mushroom. Appl Environ
Microbiol 65(2): 553-559.
2. Cai YJ, Buswell J and Chang S. (1998). |- glucosisade components of cellulolytic
system of the edible straw mushroom, Volvariella volvacea. Enzyme and Microbial
Technology 22: 122-129.
3. Chen S, Ma D, Ge W and Buswell J. (2003). Induction of laccase activity in the
edible straw mushroom, Volvariella volvacea. FEMS Microbiology Letters
218:143-148.
4. Covert SF, Kapoor P, Lee M, Briley A and Nairn CJ. (2001). Agrobacterium
tumefaciens - mediated transformation of Fusarium circinatum. Mycological
Research 105: 259-264.
5. De Groot MJA, Bundock P, Hooykaas PJJ and Beijersbergen AGM. (1998).
Agrobacterium tumefaciens - mediated transformation of filamentous fungi . Nature
Biotechnology 16: 839-842.
6. Ding S, Ge W and Buswell J. (2001). Endoglucanase I from the edible straw
mushroom, Volvariella volvacea - Purification, characterization, cloning and
expression. Eur. J. Biochem. 268: 5687-5695.
7. Hong Quc Khnh, Trn Hong Ngc i (2003). Chuyn gen khng hygromycin
B vo vi nm Trichoderma harzianum bng phng php gin ti p nh
Agrobacterium tumefaciens. Bo co khoa hc, Hi ngh ton quc ln th 2.
Nhng vn nghin cu c bn trong khoa hc s sng, 930-934, NXB Khoa hc
v K thut H Ni.
8. Jia JH, Buswell JA and Peberdy JF. (1998). Transformation of the edible fungi,
Pleurotus ostreatus and Volvariella volvacea. Mycological Research 102 (7): 876-880.
9. Mikosch TSP, Lavrijssen B, Sonnenberg ASM and van Griensven LJLD. (2001).
Transformation of the cultivated mushroom Agaricus bisporus (Lange) using T-
DNA from Agrobacterium tumefaciens. Current Genetics 39: 35-39.
10. Sambrook J, Fritsch EF and Maniatis T. (1989). Molecular Cloning. A Laboratory
Manual, 2
nd
ed. Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY.
SUMMARY
Agrobacterium tumefaciens -mediated genetic transformation
of the edible straw mushroom Volvariella volvacea
Tran Hoang Ngoc Ai, Hoang Quoc Khanh
Agrobacterium tumefaciens is known to transfer parts of its tumor -inducing
plasmid, the T-DNA, to plants, yeasts and filamentous fungi; therefore, we have used
this system to transform germinating basidiospores and vegetative mycelium of the
cultivated basidiomycete Volvariella volvacea. Strain LBA4404 of A. tumefaciens was
provided by Plant Genetic Engineering Laboratory (Institute of Tropical Biology).
Plasmid pPK2, containing hygromycin B phosphotransferase( hph) gene with the
Aspergillus nidulans Pgpd. (promoter glyceraldehyd - 3 - phosphate dehydrogenase ),
was provided by Dr. Sarah F.Covert, University of Georgia, USA. Hph gene which is 1
kb long was designed between LB and RB of T -DNA in this plasmid DNA. Out of T-
DNA is kanamycin resistant gene used to select transformed bacteria. Spores of V.
volvacea were collected on glass Petri dishes. The standard growth medium for V.
volvacea was PDA and incubation was performed at 28
o
C. pPK2 was transformed into
A. tumefaciens via electroporation. Bact erial cultivation was performed as describes in
De Groot et al. (1998). For induction of virulence and T -DNA transfer, A. tumefaciens
was grown on induction medium (IM) with 200 M AS and, for negative controls,
without AS. Selection for transformed mycel ial colonies was performed on M-100 agar
containing cefotaxime (500 g/ml) and hygromycin B (70 g/ml) and on CYM -R
containing hygromycinB (70 g/ml). Hph gene sequence of T-DNA which integrated
genomic DNA of V. volvacea was checked by PCR. Analysis of t ransformants shows
that the T-DNA integrates at random sites into the host genome and that the selective
marker is stable during mitosis and meiosis. The transformation technique decribed
herein provides a practical method for using transgenic technology i n the genetic
improvement of this commercially important mushroom and represents an important
tool for the molecular genetic analysis of biological processes in this species.
PHN LP V NH DANH H NM MEN TI VN
QUC GIA NAM CT TIN
Ngo Duc Duy, Pham Tran To Nhu, Hoang Quoc Khanh
Sasitorn Jindamorakot
National Center for Genetic Engineering and Biotechnolog, Thailand
M U
Nm men c rt nhiu ng dng, ngoi nhng sn phm ln men truyn thng, ngy
nay chng c ng dng trong cng nghip hin i nh l sn xut enzym, vitamin, acid
hu c v thuc sinh hc (Domain 1998), u ny cho thy nm men l mt trong nhng vi
sinh vt rt quan trng trong i sng, kinh t v sc khe.
Gn y nm men c ghi nhn nh l nm n bo, hu ht c xc nhn Nm
men thuc b nm Ascomycetous v Basidiomycetous trong t bo sinh dng sinh sn
bng cch ny chi v phn i, trong s hnh thnh gii tnh khng km theo giai on to
qu th (Boekhour v Kurtman 1996). Trc y, trong thi gian di cc c im kiu
hnh c s dng nh l nhng tiu chun quan trng trong nh danh nm men, tuy nhi n
cc c im ny thng khng n nh bi s thay i do iu kin mi tr ng v i hi
nhiu thi gian kho st. Do , cc c im ny thng tri vi kt qu trong nghi n cu
sinh hc phn t, khc phc nhng vn ny, k thut nh danh vi sinh vt ni chung
v nm men ni ring c dng trc tip phng php sinh hc phn t. c bit l
phn tch trnh t 26S ca Ribosomal DNA trong phm vi vng D1/D2 cho s phn lp loi
ca nm men x dng ph bin rng ri v d liu cho thy rng hu ht cc loi nm
men c th nh danh bng trnh t ny.
Nm men khng nhng ng gp rt nhiu cho nghi n cu khoa hc c bn bao gm
nh danh, sinh l, sinh ha v a dng sinh hc m c nhiu ng dng trong nng nghip,
cng nghip v s l mi trng, c bit t bo nm men l m hnh nghin cu ca t bo
Eukaryotes (Graeme M. Walker, 1998).
Rt t nhng bo co v tnh a dng ca nm men Vit Nam hin nay (Lng et al.,
2000), trong phm vi nghin cu, vi mc ch l ng dng cc phng php v k thut
sinh hc phn t trong nh danh v phn loi vi sinh vt cng vi qu trnh kho st s a
dng sinh hc ca nm men ti Vn Quc gia Nam Ct Tin.
VT LIU V PHNG PHP
Vi sinh vt; cc chng nm men c phn lp t l, phn con tr ng, hoa v tri
cy ti Vn Quc gia Nam Ct Tin tnh ng Nai.
Ly trch trnh t 26S rDNA trong phm vi v ng D1/D2; nm men nui trong
mi trng YM agar trong 48gi nhit 25
0
C. Thu nhn t bo v ha tan trong
200l ca dung lch m ly trch (100mM tris (pH 8.0), 30nM EDTA (pH 8.0,
0.5% SDS) un si trong 15 pht. Thm vo 200l c a 2.5M potasium acetate, pH
7.5, v t chng trong lnh khong 1 gi, sau khi li tm 13000prm trong 5 pht
v thu nhn dch ni pha tr n, tip tc thm vo mt lng th tch
chloroform:isoamyl alcohol (24:1) b ng th tch dch ly trch v lc nh, thu nhn
DNA sau khi thm mt th tch isopropanal v ly tm 14,000prm trong 16 pht.
Phn ng PCR c thit lp th tch 50l bao gm 1X Taq buffer, 2mM MgCl
2
,
200M dNTP, 0.3pM ca mi cp mi, 1.25U Taq polymerase v 5l DNA mu cho
chu trnh PCR l 94
0
C trong 1 pht, 52
o
C thi gian 1.30 pht v 72
o
C trong 2.30 pht
cho khong 30 chu trnh. Sn phm DNA c lm sch bng QIAquick (QIAGEN) v
gii trnh t bng automate ABI.
Hai cp mi c ng dng cho tr nh t ny nh sau; mi xui F63 (5- GCA
TAT CAA GCG GAG GAA AAG -3) v mi ngc LR3 (5- GGT CCG TGG
TTC AAG ACG -3).
Phn tch BLAST (BLAST analysis); trnh t thu nhn c s so snh vi cc
trnh t khc trong ngn hng gen (Genbank) v s dng cng c BLAST ca NCBI
(National Center for Biotechnology Information - USA).
Phn tch cy ph h (phylogenetic analysis); s dng cc tr nh t tng ng
vi cc loi lin quan bng cch s dng chng trnh ClustalX 1.8 (Thompson et
al., 1997). Cu trc cy ph h c cu to t khong d liu tin ha theo
Kimura. M (1980) bng cch s dng phng php neighbor -joining (Saitou v
Nei., 1987), phn tch bootstrap (Felsentien, 1985) c hnh thnh t 1000 s ti
ly mu ngu nhin.
KT QU V THO LUN
T nhng mu thu c chng ti phn lp c 85 chng nm men t l , hoa, v
phn con trng ti Vn Quc gia Nam Ct Tin tnh ng Nai.
Hnh 1. Cc t bo sinh dng nm men trong mi trng YM broth, 2 ngy nhit 25
0
C
(Scale 10m, 100X)
P5 H7
L60
L52 L10 L5
L29 P2 L53 H9 L40 H5
L24
L67
Da trn cc kho st v phn tch sinh hc phn t chng ti ghi nhn 14 chng
(P2, P5, H5, H7, H9, L10, L5, L24, L29, L40, L52, L53, L60 v L67) c kh nng l
loi mi v tip tc kho st hnh thi, sinh l, sinh ha ca cc chng ny, 56 loi
bit v 15 loi cha xc nh r rng.
Mi bn chng nm men nui cy trong mi tr ng YM borth nhit 25
0
C
trong 2 ngy, sau quan st di knh hin vi cho thy a s cc chng ny ti sinh
bng ny chi, chng c dng hnh cu, hnh ovan v cc t bo to khun ty gi hnh
thon di v.v. (hnh 1).
Bn cnh s sinh sn bng cch ny chi, nm men c n c kh nng hnh thnh mt
s khun ty gi ging nh s hnh thnh si nm (hnh 3).
Hnh 3. Khun ty gi to ra trn mi trng PDA sau 1 tun nhit 250C bng k
thut a Dalmau
T kt qu thu nhn c 14 chng khc nhau t tt c nhng loi bit bi 5
nucleotitdes hoc nhiu hn trong phm vi vng D1/D2 thuc trnh t 26S rDNA. Cc chng
ny c ngh l hin din loi mi theo nh tho lun ca Kuztman v Robnett (1998), v
14 chng ny c phn lp trong 9 loi, t nhng loi gn chng nht (bng 1).
Bng 1. Danh sch cc chng (H7, H5 L24) cng vi cc chng so snh trong ngn
hng d liu gen
Strain GenBank
Accession No.
Nearest Species with Accession No. of DNA
DataBank
Nucleotide Identity
(%)
No. of Nucleotide
Difference (gap)
Ascomycetes
H7 AM076407 Candida naeodendra partial 520/530 (98.1) 10(2)
L5 AM076407 Candida naeodendra partial 520/530 (98.1) 10 (0)
L10 AM076407 Candida naeodendra partial 519/530 (97.9) 11(3)
H5 AF530612 Candida sp. UWO(PS)00-147.3 552/582 (94.8) 30(6)
P5 U74598 Pichia hampshirensis 525/553 (94.9) 28 (0)
H9 AF017412 Pichia lachance 513/572 (89.7) 59(11)
L67 AJ508573 Pichia sydowiorum 570/580 (98.3) 10 (1)
Basidiomycetes
L40 DQ640492 Cryptococcus liquefaciens strain MZKI K-490 588/591 (99.5) 11 (0)
L29 AF444704 Cryptococcus sp. CBS 8368 594/603 (98.5) 9(2)
P2 AF387128 Sporidiobolus ruineniae strain IGC 5692 574/580 (98.9) 6 (0)
L24 AF453938 Ustilago maydis 601/608 (98.8) 7(2)
L24
L29
H2
H7 H9 P2
Qu trnh nh danh da trn trnh t ca phm vi vng D1/D2 ca trnh t 26S
rDNA theo nh s hng dn ca Kuztman v Robnett (1998), kt qu cho thy 14 loi
mi hon ton ring bit da trn s phn tch cy ph h cho s hnh thnh loi mi
trong nghin cu ny c to thnh trong phm vi vng D1/D2 thuc trnh t 26S
rDNA cng vi cc chng bit gn nht theo s t ng ng trong ngn hng d liu
gen (hnh 2).
Hnh 2. Cy pht sinh loi ca 14 chng v cc loi lin quan da trn trnh t 26S rDNA
trong phm vi vng D1/D1
Ngoi nhng d liu m sinh hc phn t cho chng ta xc nh s pht hin lo i
mi v loi bit trong ngn hng ging v ngn hng gen cho vi c nh danh vi sinh
vt ni chung v nm nem ni ring, th vic kho st cc d liu v hnh thi, sinh l,
v sinh ha cng nh thng tin cu trc sinh hc khc s ging chng ta xc nh v
thng tin y v loi mi.
KT LUN
Da trn trnh t 26S rDNA trong phm vi vng D1/D2, 85 chng nm men c
xc nh t cc mu l, hoa v phn con trng t i Vn Quc gia Nam Ct Tin tnh
ng Nai theo nhm nm men Ascomycetous v Basidiomycetous nh sau;
+ 56 chng l loi bit (21 chng thuc nhm Ascomycetous v 35 chng thuc
nhm Basidiomycetous)
Saccharomyces cerevis iae
Williopsis saturnus var. mrakii
Williopsis saturnus KCTC 1724
Pichia meyerae
Pichia lachancei
Candida sp. CBS 5303
Pichia hampshirensis
Pichia strasburgensis
H5
Candida quercuum
Candida sp.
Candida ulmi
Pichia myanmaensis
Pichia anomala strain VTT C-04565
Pichia lynferdii
Pichia subpelliculosa.
L53
Pichia sydowiorum
L52
H7
Candida naeodendra
Candida diddensiae
Pichia sp. ST-335
Ustilago maydis
Pseudozyma prolifica
Ustilago vetiveriae
Cintractia sorghi -vulgaris
L29
basidiomycete yeast sp. BG02-7-16-015A-1-1
Cryptococcus sp. CBS 8368
Cryptococcus liquefaciens strain MZKI K -493
Cryptococcus liquefaciens strain MZKI K -490
Cryptococcus liquefaciens strain UWFP -357
Sporidiobolus ruineniae var. ruineniae
Sporobolomyces sp. TY-228
Sporidiobolus ruineniae
L24
L40
P2
L5
L10
P5
L60
L67
0.05
+ 14 chng l loi mi (10 chng thuc nhm Ascomycetous v 4 chng thuc
nhm Basidiomycetous)
+ 15 chng cha c xc nh (8 chng thuc nhm Ascomycetous v 7 chng
thuc nhm Basidiomycetous)
TI LIU THAM KHO
1. A. L. Demain, H. J. Phaff and C. P. Kurtzman. (1998). The industrial and
agricultural significance of yeasts. In: Kurtzman C. P and Fell J. W (eds), The
Yeasts, a Taxonomy Study Eleevier, Amsterdam, p. 13.
2. Graeme M. Wallker. (1998). Yeast physiology and biotechnology, pp. 1- 8.
3. Cletus P. Kurtzman and Christie J. Robnett. (1998). Identification and phylogeny of
ascomycetous yeast from analysis of nuclear subunit (26S) ribosomal DNA partial
sequences. Antonie van Leeuwenhoek 73, 311 -371.
4. Jack W. Fell, Teun Boelkhout, Alvaro Fonseca, Gloria Scorzetti and Adele Statzell -
Tallman. (2000). Biodiversity and systematics of basidiomycetous y easts are
determined by large-subunit rDNA D1/D2 domain sequence analysis. International
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5. Dao, T. L., M. Takashima, Pham, V. T., Nguyen, L. D and Nakase, T. (2000). Four
new species of Kockovaella isolated from plant leaves collected in Vietnam. J. Gen.
Appl. Microbiol., 46, 297-310.
6. Nagatsuka, Y., Kawasaki, H., Limtong, S., Mikata, K. and Seki, T. (2002).
Citeromyces siamensis sp. nov., a novel halotolerant yeast isolated in Thailand. In t.
J. Syst. Evol. Microbiol., 52, 2315-2319.
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Komagata. K (1987). Identification of yeasts isolated from fermented foods and
related materials in Thailand. J. Gen. Appl. Microbiol ., 33, 205-220.
8. Suzuki, M., Nakase, T. and Komagata, K. ( 1994). Candida stellimalicola, a new
species of anamorphic yeast isolated from star apple in Thailand. J. Gen. Appl.
Microbiol., 40, 115-121.
9. Saito, K., Hasuo, T., Sugano, N., Kitamoto, K., Watanabe , S., Tadenuma, M.,
Nakamura, K., Sato, M., Akiyama, H., Vongsuvanlert, V., Karuwanna, P. and
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SUMARY
Isolation and identification of Saccharomyces sp.
collected from Cat Tien national park
Ngo Duc Duy, Pham Tran To Nhu, Hoang Quoc Khanh(1), Sasitorn Jindamorakot(2)
(1)Institute of Tropical Biology, (2)BIOTEC, Thailand
Eighty five strains of yeasts were isolated from insect frass (5 strains), leaves (67
strains) and flowers (13 strains) which were collected from Cat Tien National Pa rk in
the Southeast of Vietnam. Based on sequences of D1/ D2 domain of 26S ribosomal
DNA, 39 strains belong to ascomycetous yeasts and 46 strains belong to
basidiomycetous yeasts. In these study, 14 new species (16.5%) differed by 6 to 59
nucleotides, according to discussed by Kurtzman and Robnett (1998) that these strains
are suggested to represent new species, 15 strains (17.6%) differed by 2 to 4 nucleotides
are not yet identified and the remaining 56 strains (65.9%) were identified as known
species. The eighty five species were identified as 7 genera of basidiomycetous yeasts;
Cryptococcus, Sporidiobolus, Sporobolomyces, Rhotosporidium, Rhodotorula,
Ustilago, Pseudozyma and 4 genera of ascomycetous yeasts; Pichia, Candida,
Metschnikowia and Saccharomyces . Fourteen strains assigned to new species these
strains close to the genera Candida (H5, H7, L5 and L10), Pichia (H9, P5, L52, L53,
L60 and L67), Sporidiololus (P2), Cryptococcus (L29, L40) and Ustilago (L24). The
phylogenetic tree were constructed based on D1/D2 domain of 26S rDNA sequences of
14 new species found in this study and related species, the characteristics cell
morphylogy, the production of ascospore, pseudomycelium, true mycelium and
maximu growth temperature were examined.
SN XUT V THNG MI HO CC SN PHM
SINH HC DNG TRONG NUI TRNG THU SN
V Th Hnh, L Th Bch Phng, L Tn Hng,
Trng Th Hng Vn, Trn Thnh Phong
M U
Nm 2006, ngnh thy sn hng ti mc tiu xut khu 2,8 t USD. Din tch
nui trng thy sn thm canh v bn thm canh nc ta ang gia tng nhanh, s
nhim mi trng nc ao nui tm do l ng thc n tha, cht thi hu c cng ngy
cng nghim trng nh hng n nng sut v cht lng nui trng, tng kh nng
nhim bnh cho tm.
Khng sinh v ha cht c s dng nhiu trong phng tr bnh cho cc i
tng thy sn nui trng s gy ra hin t ng ln thuc v s tn d khng
sinh trong cc mt hng ch bin l ro cn cho vic xut khu cc sn phm n y.
Mt gii php c chp nhn rng ri trn th gii l s dng cc ch phm
probiotics cha cc vi sinh vt sng hu ch c kh nng lm sch mi trng nc ao
nui tm, iu chnh pH, gim s l ng vi khun gy bnh cho tm. Trn th trng
hin c nhiu ch phm probiotics ca n c ngoi dng x l ao nui tm c ng
bao ti Vit Nam c gi thnh cao.
T thc t trn, chng ti nghin cu sn xut v th nghim cc ch phm sinh
hc probiotics nh BIOII, VEM v PB trn cc ao nui tm s cc tnh min Nam.
T kt qu t c, chng ti mnh dn kt hp vi cc cng ty sn xut thuc th y
v nui trng thy sn a cc ch phm ny ra th trng vi tn thng mi ca
cc cng ty.
VT LIU V PHNG PHP
Ging vi sinh vt: Cc chng Bacillus spp., Lactobacillus spp., Saccharomyces
spp., Rhodopseudomonas, Rhodospirillum c phn lp v chn lc ti Vit Nam
Thit b: Ni ln men (fermenter) 100 lt, my ly tm siu t c 20,000 vng/pht,
my sy phun, my ng kh, T sy tnh, my xay, my trn siu tc KBC-ST-20,
t lnh m...
Mi trng (MT) sn xut ch phm BIOII v PB: MT dch chit ng cc dng sn
xut sinh khi vi khun Bacillus spp; MT MRS dng thu sinh khi vi khun
Lactobacillus spp; MT Malt - Cao nm men dng thu sinh khi Saccharomyces spp;
Mi trng Norbert Pfennig dng nui cy vi khun quang dng.
Sn xut -amylase v protease: t vi khun Bacillus subtilis bng phng php ln
men bn rn.
Sn xut ch phm VEM: Ci tin qui trnh sn xut EM (Effective Microorganisms)
ca gio s Teruo Higa, Nht Bn sn xut VEM c mt vi sinh vt hu ch :
Bacillus spp., Lactobacillus spp., Saccharomyces spp. V vi khun quang hp to ra
ch phm VEM.
Sn xut ch phm PB: Nui cy vi khun Rhodopseudomonas v Rhodospirillum
trong mi trng Norbert Pfennig di nh sng mt tri, sau 2 tun nui cy, em
ng chai v bo qun trong ti.
Xc nh cc ch ti u sinh ha: Mt vi sinh vt bng phng php m khun
lc; hot lc o-amylase bng phng php Smith v Roe; hot lc enzym protease
bng phng php Anson ci tin; nh l ng NH
3
-N, H
2
S, NO
2
-N bng phng
php so mu.
Th nghim hiu qu ch phm BIOII tr n ao nui tm: Trn ao tm s 45 ngy
tui ti cc ao nui tm bn thm canh tnh B Ra-Vng Tu vi nng 0,2-0,5 ppm. S
dng ch phm BRF2 ca M cho ao i chng vi nng 0,5 ppm. Theo d i cc ch
tiu nh mu nc, pH, NH
3
-N, H
2
S, NO
2
-N v mt vi khun Vibrio trong nc ao.
Th nghim cc tnh Kin Giang, Tin Giang vi liu l ng 100g/1000m
2
/tun,
Th nghim hiu qu ch phm VEM tr n ao nui tm: Ch phm VEM c
th nghim trn cc ao nui tm t i B Ra-Vng Tu; theo di tr ng lng trung
bnh, t l sng v h s FCR (food conversion ratio), i chng l sn phm
Pond-Clear.
KT QU V THO LUN
Ch phm probiotics BIOII
Bng 3.1: Thnh phn ch phm probiotics BIOII
Thnh phn nh lng
Lactobacillus spp.
Bacillus spp.
Sacchromyces spp.
o-amylase
Protease
10
9
CFU/g
10
10
CFU/g
10
7
CFU/g
2000 UI/g
20 UI/g
Kt qu th nghim ch phm BIOII trn ao nui tm
Bng 3.2. Th nghim ti B Ra - Vng Tu
Cc t x l
X l t 1
(BIOII
0,2ppm;
BRF20,5ppm)
X l t 2
(BIOII 0,5ppm;
BRF2 0,5ppm)
X l t 3
(BIOII 0,5ppm;
BRF2 0,5ppm)
X l t 4
(BIOII 0,5ppm;
BRF2 0,5ppm)
Thi gian (ngy)
0 3 6 9 12 15 18 21 24 27 30
BIOII 1.3 1.12 1.22 0.6 1.3 0.95 0.85 1.45 0.45 0.75 0.70
NH
3
-N
(mg/l)
BRF2 0.63 0.53 0.35 1.40 0.90 0.95 0.45 1.50 0.90 0.75 0.72
BIOII 0.074 0.126 0.098 0.008 0.045 0.000 0.000 0.000 0.000 0.005 0.002
NO
2
-N
(mg/l)
BRF2 0.001 0.002 0.005 0.000 0.001 0.000 0.000 0.0005 0.002 0.002 0.001
BIOII 0.023 0.013 0.013 0.051 0.016 0.049 0.023 0.011 0.013 0.011 0.007
H
2
S
(mg/l)
BRF2 0.031 0.023 0.020 0.067 0.005 0.002 0.001 0.002 0.002 0.002 0.002
BIOII 6083 560 340 1140 2000 700 450 270 300 300 200
Vibrio
(CFU/ml)
BRF2 430 295 300 1350 1300 1000 500 400 300 200 200
BIOII 7.3 - 8.5
C
c

c
h

t
i
u

l
a
pH
BRF2 7.6 - 8.2
Khi tng nng BIOII t 0,2 ppm l n 0,5 ppm cc t x l th 2, 3 v 4, nng
NH
3
-N, NO
2
-N, H
2
S sau 30 ngy gim xung mc cho php. Cc ch ti u pH,
COD n nh, thch hp cho s pht trin ca tm. Mt Vibrio gim cn 200 CFU/
ml bng vi ao i chng x l vi BRF2 sau 30 ngy.
Bng 3.3. Kt qu th nghim ch phm BIOII (100 g/1000 m
2
/tun)
trn ao tm ti Kin Giang, Tin Giang
Kin Giang
Tin Giang
Cc ch tiu
Th nghim i chng Th nghim i chng
Cht lng nc Tt, khng c mi hi Xu, c mi hi Mu nc, trong c ci thin Xu, c mi hi
pH 7,8 - 8,1 8,5 9,3 7,5 8,3 7,5 9,5
T l bnh Khng ng k 15- 20% Khng ng k 15%
Nng sut (tn/ha) 7,8 6,4 5,7 4,7
Nng sut tng (%) 121.9 100 119,1 100
Bng 3.3 cho thy: Nng sut tm bnh qun tng t 19,1%- 21,9%
Ch phm VEM
Bng 3.4. Thnh phn vi sinh vt trong ch phm VEM
Vi sinh vt Ch phm VEM
Vi khun lactic
Vi khun Bacillus spp
Vi khun quang dng
Nm men
1x10
9
CFU/ml
1x10
10
CFU/ml
1x10
5
CFU/ml
1x10
7
CFU/ml
Th nghim hiu qu ch phm VEM trn ao nui tm
Bng 3.5. Kt qu th nghim ch phm VEM tr n cc ao nui tm ti B Ra-Vng Tu
Ch tiu Pond-Clear VEM
T l sng (%)
Trng lng trung bnh (g/con)
Kch c tm (S con/kg)
Sn lng bnh qun (kg/ha)
Tng lng thc n (kg)
H s FCR
63,96
28,2
35,5
4.545
3.502
1,75
62,73
28,9
34,5
4.546
2.671
1,78
Bng 3.5 cho thy: trng l ng trung bnh v t l sng ca tm thu hoch hai ao
s dng ch phm VEM khng khc bit nhiu so vi ao s dng sn phm Pond-Clear.
Ch phm PB
Qua th nghim ch phm PB trn cc ao nui tm s, kt qu cho thy c tc dng
phn gii thc n d tha v cht thi tch t di y ao, gim BOD, COD, n nh
cht lng nc, gip cn bng sinh thi, gim t l bnh cho tm.
Thng mi ha cc ch phm dng trong nui trng thy sn
Chng ti kt hp vi cc cng ty thuc th y v nui trng thy sn thng
mi ha ch phm BIOII v VEM mang thng hi u ca cc cng ty.
Bng 3.6. Thng mi ha ch phm BIOI, BIOII v VEM
STT Tn sn phm Ngun gc i tng Cng ty sn xut
1 NAVET Biozym BIO II Tm, c Thuc th y Trung ng
2
3
Enzym subtilis
Enzymmin subtilis
Bacillus subtitis
Bacillus subtitis
Tm, c
Tm, c
Thuc th y Si Gn
4 Biolactizym BIO II Tm, c Thuc Th y Long An II
5 NP-Lactizym BIO II Tm, c Thuc th y NAPHA
6
7
L-AKL
B-AKL
BIO II
BIO II
Tm, c, (nc kim)
Tm, c, (nc phn)
CP NTTS Khnh Long
7
8
9
10
11
BACILLUS
MAZAR
BS@
BACTERI
LASA
Bacillus subtitis
VEM
VEM
Pb
VEM
Tm, c
Tm, c
Tm, c
Tm, c
Tm, c
at Viet Co.LTD
12
13
BIOLAS-2
BIOLAS- Pb
BIOII
VEM
Tm
Tm
i Trng Sn
KT LUN
Ch phm BIOII, PB v VEM c hiu qu trong vic lm sch mi trng nc ao
nui tm, cn bng pH, gim s lng vi khun gy bnh cho tm. Nng sut tm bnh
qun thu hoch tng 19,1 % - 21,9% so vi sn phm BRF2, h s FCR thp h n so vi
sn phm Pond-Clear, trong khi gi thnh ca ch phm BIOII thp hn gn 10 ln.
Chng ti kt hp vi cc cng ty thuc th y v nui trng thy sn thng
mi ha ch phm BIOII v VEM mang thng hi u ca cc cng ty.
TI LIU THAM KHO
1. Havenaar, R., Ten Brisk, B., Selection of strains for probiotic use . In: R. Fuller
(ed.), Probiotics the scientific basic, Chapman and Hall, Londo n. P. 209-224, 1992.
2. V Th Hnh v cs (2003). Nghin cu sn xut ch phm hn hp vi sinh vt sng
v enzyme tiu ha dng trong chn nui v nui tr ng thy sn. Bo co nghim
thu ti, S Khoa hc v Cng ngh TPHCM
SUMMARY
Production and commercialize the bio-products using for
aquaculture
Vo T Hanh, Le Bich Phuong, Tran Thanh Phong, Le Tan Hung, Truong T Hong Van
The bioproducts such as BIOII, VEM and PB have been studying and producing;
these bioproducts have been using for aquaculture effectively. The BIOII and VEM had
effect on cleaning water, pH stability, reducing pathogenic bateria on shrimp. The
average yield increased 19,1% - 21,9% compared with BRF2 products, FCR (food
conversion ratio) lowered compared with Pond-Clear product, meanwhile, price of
BIOII lower 10 times BRF2.
GIM THIU NHIM MI HI CHUNG TRI V SN
XUT PHN VI SINH T PHN CHUNG BNG CH PHM
SINH HC
V Th Hnh, L Th Bch Phng, Trn Thnh Phong,
L Tn Hng, Trng Th Hng Vn
M U
Ngnh chn nui Vit Nam ang chuyn dn t chn nui truyn thng, phn tn,
quy m nh sang chn nui trang tri (TT), tp trung, qui m ln v sn xut hng ho
cht lng cao.
Tuy nhin, trong qu trnh pht tri n, chn nui trang tri bc l mt s hn ch
nh: pht trin t pht, thiu quy hoch. Trong vn nhim mi trng cn phi
c quan tm.
cc TT chn nui heo, lng phn v nc thi thi ra vi khi lng rt ln
(khong 5 tn/ 15.000 u heo), phn ti c bn vi gi r (khong 5.000 ng/ 40
kg). Do khng c x l hoc x l khng ng cch gy nhim mi hi cho khu
vc ln cn.
Do , chng ti a ra gi i php s dng ch phm VEM-K lm gim mi hi;
ch phm BIO-F dng sn xut phn vi sinh t phn v nc thi. Cc ch phm sinh
hc trn cha tp on vi sinh vt hu ch c phn lp v chn lc, chu c iu
kin kh hu v th nhng Vit Nam, do vy khng phi ph thuc v o ngun ging vi
sinh ca nc ngoi.
Qua gii php trn, chng nhn c gii thng Ngy sng to Vit Nam vi
ch Hnh ng v mi trng do Ngn hng Th gii ti Vit Nam t chc nm 2005.
VT LIU V PHNG PHP
Ging vi sinh vt: Cc chng Bacillus spp., Lactobacillus spp., nm men
Saccharomyces spp., Rhodopseudomonas, Rhodospirillum, x khun Streptomyces sp.
v nm si Trichoderma sp c phn lp v chn lc ti Vit Nam
Sn xut ch phm VEM-K: Ci tin qui trnh sn xut EM (Effective
Microorganisms) ca gio s Teruo Higa, Nht Bn sn xut VEM-K c mt vi
sinh vt hu ch cao v khong a vi lng d tan.
Th nghim ch phm VEM-K trn heo, g v sn xut phn vi sinh theo s sau:
KT QU V THO LUN
Ch phm VEM-K
Bng 3.1. Thnh phn vi sinh vt trong ch phm VEM-K
Ch phm VEM-K c th nghim trn heo tht giai on t 20 kg n 50 kg, ti
tri heo Gia Kim tnh ng Nai . Kt qu theo di cc ch tiu sau 55 ngy cho ung
(1lt VEM-K/500 lt nc sch) ghi nhn c bng 3.2.
Bng 3.2. Kt qu th nghim ch phm VEM-K (1lt/500 lt nc sch) trn heo tht.
Ch tiu theo di L th nghim L i chng
Chnh lch so
vi i chng
Trng lng trung bnh u vo (Kg) 19,10 19,30 -0,20
Tng trng lng ca l u vo (Kg) 191,00 193,00 -2,00
Trng lng trung bnh u ra (Kg) 48,10 42,70 5,40
Tng trng lng ca l u ra (Kg) 481,00 427,00 54,00
Tng tng trng/l (Kg) 290,00 234,00 56,00
Tng trng bnh qun/con (Kg) 29,00 23,40 5,60
Tiu th thc n bnh qun/ngy (Kg) 0,99 0,90 0,09
Tng lng thc n ti u th/l (Kg) 546,50 493,00 53,50
FCR (Kg thc n/kg th trng) 1,88 2,11 -0,23
Tng trng tuyt i (g/con/ngy) 527,30 425,50 101,80
Thi gian nui (ngy) 55,00 55,00 0
Vi sinh vt Ch phm VEM-K
Lactobacillus spp.
Bacillus spp
Saccharomyces spp.
Vi khun quang dng
Khong d tiu
10
9
CFU/ml
10
10
CFU/ml
10
7
CFU/ml
10
5
CFU/ml
500 mg/ml
VEM pha long
Cho heo, g
ung hng ngy
Phn gim mi hi ca
X l bng ch
phm BIO-F
Phn hu c vi sinh
Kt qu bng 3.2 cho thy, ch ti u tng trng bnh qun tng 20% v ch tiu tiu
tn thc n l th nghim gim 11% so vi i chng, mi hi ca phn v rui nhng
l th nghim t hn v phn thi ca heo l th nghim rn hn so vi l i chng.
Ch phm VEM-K c th nghim trn g (ging HIGRO, trang tri Trn Quc
Tun, x M Xun, Tn Thnh, B Ra - Vng Tu) vi liu lng 1lt VEM-K/1000 lt
nc sch ri cho khong 4000 con g giai on t 21 n 39 ngy tui ung. Kt qu
theo di cc ch tiu tng trng bnh qun v t l tiu tn thc n sau thi gian cho
ung c ghi nhn trong bng 3.3.
Bng 3.3. Kt qu th nghim ch phm VEM-K (1lt/1000 lt nc sch) trn g.
STT Ch tiu th nghim L i chng L th nghim
1 S lng g th nghim bt u (con) 3522 (100%) 3518 (100%)
2 Trng lng bnh qun lc 21 ngy tui (g/con) 765 766
3 Tng trng bnh qun (g/con/ngy) 75,83 80,0
4 Trng lng cui k (g) 2130 2206
5 Tiu tn thc n (FCR = Kg thc n/kg th trng) 1,88 1,80
6 T l sng (%) 98,18 98,49
7 Thi gian th nghim (ngy) 18 18
Bng 3.3 cho thy, cc chng vi sinh gip tng trng bnh qun tng 5,5%, gim
lng thc n tiu tn, nhng con g c ung VEM-K hng ngy c t l tiu tn
thc n l 1,80 so vi nhng con khng c ung VEM-K (1,88).
Sn xut phn vi sinh (PVS)
Phn chung c x l m , sau vi ch p hm BIO-F. Sau ba ngy, cc vi
sinh vt hu ch ni trn bt u pht trin mnh, phn gii cc hp cht hu c v lm
mt mi hi. Nhit trong khi cng tng l n ti 60-70
0
C, tiu dit cc vi sinh vt
gy bnh v trng giun trong phn. Sau 7-10 ngy, sn phm thu c l phn bn vi
sinh cht lng cao.
Bng 3.4. Cc ch ti u cht lng ca sn phm phn bn HCVS
Ch tiu
Hm lng
Ch tiu
Hm lng
m (%)
Ntng s (%)
P2O5 (%)
K2O (%)
Humic acid (%)
Mn, cht hu c (%)
25
0,78
1,06
0,61
2,61
4,65
Trichoderma spp.
Bacillus spp.
Streptomyces spp.
107 CFU/g
107 CFU/g
108 CFU/g
Loi phn bn hu c vi sinh ni trn c gi thnh cha t i 1.000 ng/kg.
Th nghim PVS trn cy con da leo
Sn phm phn HCVS c B mn Bo v thc vt, Tr ng i hc Nng lm
Tp. HCM th nghim trn rung da leo. Kt qu c ghi nhn nh sau:
T l (%) bnh ho cy con da leo sau 25 - 30 ngy gieo ht l 0 - 1% thp hn so
vi i chng (3-3,5%). Loi nm gy bnh cht c xc nh l Fusarium sp.
Chiu cao (18,43cm) v s l (6,2/cy) ca cy da leo trn cc nghim thc th
nghim giai on 24 - 31 ngy sau gieo ht tng so vi i chng (chiu cao: 17,56cm, s
l: 5,44/cy)
Thng mi ha ch phm VEM-K v BIO-F
Chng ti kt hp vi cc cng ty thuc th y v cng ty sn xut phn hu c vi
sinh thng mi ha ch phm VEM-K v BIO-F mang thng hiu ca cc cng ty.
Bng 3.7. Thng mi ha ch phm VEM-K v BIO-F
STT Tn sn phm Ngun gc i tng Cng ty sn xut
1 BIOLAS-789V
VEM-K
Heo, g
Cng ty TNHH - TMSX i Trng Sn
2
3
4
MAZAR
BS@
LASA
VEM-K
VEM-K
VEM-K
Heo, g at Viet Co.LTD
5 Phn hu c vi sinh BIO-F Cy ma Nh My ng Ty Ninh
6
Phn vi sinh Tricho BIO-F Cy hoa mu Cng Ty TNHH TM & SX Mai Xun - Tp. HCM
7 Phn hu c vi sinh BIO-F Cy hoa mu CTy Ging cy trng ng Ty - Tp. HCM
8 Phn hu c vi sinh BIO-F Cy cng nghip Cng Ty Hong Thnh - aklak
KT LUN
T cc ph ph phm cng nng nghip v cc chng vi sinh vt chn lc c,
sn xut c ch phm VEM-K v ch phm BIO-F.
Ch phm VEM-K c th nghim trn heo tht (1 lt VEM-K/500 lt nc sch)
cho heo ung trong 30-60 ngy, kt qu heo tng trng trung bnh tng 20%, s tiu hao
thc n gim 11%; v trn g (1lt VEM-K/1000 lt nc) cho g ung, g tng trng
trung bnh tng 5,5%, s tiu hao thc n gim 4,4%; c trn heo v g, VEM - K c tc
dng gim t l bnh ng rut v gim mi hi ca phn.
Phn hu c vi sinh c sn xut t phn chung v BIO-F c tc dng gim t l
bnh ho cy con da leo do Fusarium sp., gip cy tng tr ng v chiu cao v s l
nhanh so vi i chng.
Vi quy trnh n gin, r tin c th p dng i tr cho cc tri chn nui cng
nghip vi n heo t 10.000 - 100.000 con. Ngoi ra, quy trnh cn c th p dng cho
cc h chn nui vi quy m gia nh t nh hn 2.000 con.
TI LIU THAM KHO
1. Fuller R. (1989). Probiotic in man and animal, J. Applied. Bacteriol. 66: 365 -378
2. Havenaar, R., Ten Brisk, B. (1992). Selection of strains for probiotic use . In: R.
Fuller (ed.), Probiotics the scientific basic, Chapman and Hall, London, pp209 -224
3. V Th Hnh v cs (2003). Nghin cu sn xut ch phm hn hp vi sinh vt sng
v enzyme tiu ha dng trong chn nui v nui tr ng thy sn. Bo co nghim
thu ti, S Khoa hc v Cng ngh THCM
4. Thomashow LS., (1996). Biological control of plant root pathogens. Curr. Opin.
Biotechnol. 7: 343-347
5. V Th Hnh, L Th Bch Phng, Trn Thnh Phong, L Tn Hng, Trng Th
Hng Vn (2004), Nghi n cu sn xut ch phm BIO-F dng phng tr nm bnh
hi cy trng v sn xut phn bn vi sinh, Gii III Hi thi Sng to Khoa hc K
thut tnh Bnh Dng
6. V Th Hnh, L Th Bch Phng, Trng Hng Vn, L Tn Hng v Trn
Thnh Phong, D n Gim thiu nhim mi hi v s dng phn chung sn
xut phn bn vi sinh cht l ng cao ti trang tri nui heo, Gii th ng Ngy
sng to Vit Nam" nm 2005 do Ngn hng Th gii t chc.
SUMMARY
Reducing odour pollution at farms and producing
biofertilizer from manure by bio -products
Vo Thi Hanh, Le Thi Bich Phuong, Tran Thanh Phong,
Le Tan Hung, Truong Thi Hong Van
The VEM-K and BIO-F bioproducts are produced from agri -industrial by-products
and selected useful microorganisms. The VEM-K product prevent livestock and poultry
from intestinal diseases and reduce bad odour pollution at farms compared with
controls. By using biofertilizer produced from manure and BIO -F, the result of test
showed that the disease ratio on young cucumber caused by Fusarium s p. and other
pathogenetic fungi decreased and the growth of cucumber better compared with control.
TUYN CHN CC CHNG Lactobacillus SINH TNG
HP polyhydroxybutyrate (PHB)
Nguyn Duy Long, Phm Trn T Nh, Nguyn Minh Nht, Hong Quc Khnh
M U
Polyhydroxybutyrate (PHB) l mt dng polymer thuc h Polyhydroxyalkanoate
(PHA). Cc polymer thuc h PHA u c kh nng phn hy bng con ng sinh
hc. Tuy nhin, vi c tnh u vit ca PHB c bn vt l cao, nhit nng chy
171-182
0
C, bn vi tia uv tt , cc c im ny gn nh tng ng vi cc loi
polymer thng dng nh PP (polypropylene), PE (polyethylene)
[4]
. PHB c tng hp
v tch ly trong vi sinh vt rt a dng, s tch l y ny xy ra trong iu kin gii hn
ca ngun dinh dng v s d tha ca ngun carbon. PHB c sn xut quy
m cng nghip trn Alcaligenes eutrophus bi cng ty ZENECA [
2
] c dng lm
bao b thc phm, m phm, l m v bc cho thuc dc, lm cht m v thay th
xng trong gii phu, Tuy nhin vic gim gi thnh cho sn xut quy m ln vn
cn l mt vn ang c quan tm v nghin cu. Cc nghin cu tp trung vo
thay i cng ngh, iu kin l n men, k thut di truyn v s a dng PHB trong th
gii vi sinh vt. Lactobacillus l mt vi khun c s dng trong cc quy tr nh ln men
sn xut acid lactic, l n men rau, c qu trong mt s kt qu nghi n cu cho thy s
tch ly PHB trong cc loi Lactobacillus trong t nhin c th t n hn gn 30%
[5].
Trong nghin cu ny, chng ti chn lc cc chng Lactobacillus t cc ngun l n
men thc phm, kho st cc iu kin dinh d ng v iu kin ly trch hiu qu cho
qu trnh thu hi PHB t vi sinh vt.
VT LIU V PHNG PHP
Cc chng vi khun lactic c phn lp t cc ngun Yaourt, u n nh ngm, cc
loi da mui chua, nem chua, sa l n men, mt s loi rau qu, ng cc ln men[1]
trn mi trng MRS agar v nui cy trn MRS Broth, mi trng Elliker (pepton 10g,
cao nm men 5g, dextrose 5g, saccharose 5g, NaCl 1,5g, acid ascorbic 0,5g, nc ct
1000ml) v mi trng dch chit c chua (dch chit c chua 20g/l, glucose 10g/l). Qu
trnh xc nh cc chng Lactobacillus c thc hin trn mi trng carbonat agar,
quan st hnh thi, xc nh kh nng ln men glucose (peptone 10g, NaCl 5g, cao tht
1g, glucose 10g, nc ct 1000ml, phenol red 0.018g), xc nh kh nng sinh acid
lactic bng thuc nhum Uphenmen, quan st h nh thi, nhum gram v thc hin phn
ng String test [1] xc nh gram vi khun.
Cc khun lc Lactobacillus tr n cc mi trng MRS agar, Elliker agar, dch chit
c chua agar c nhum vi Sudan Black B xc nh kh nng sinh tng hp PHB.
Kho st kh nng sinh tng hp PHB tr n ngun carbon glucose 1%. sucrose 2%,
lactose 2%, manitol 1%, hn hp ngun carbon (mi tr ng Elliker), dch chit c chua
v ngun nit peptone, cao nm men, (NH
4
)
2
SO
4
0,4 % v glycine 2% [6, 7, 9, 10].
PHB c li trch v xc nh hm lng theo phng php Law Slepecky [14],
qu trnh kho st cc iu kin li trch PHB c thc hin vi Sodiumhypochlorite
5% v SDS 3% di hm lng thay i ca t l sinh khi t 0.5% n 6% (w/v).
ng thi kho st cc i u kin nhit li trch t 40
0
C - 90
0
C, theo thi gian t 20
pht - 120 pht.
KT QU
1. Kt qu phn lp v xc nh PHB trong t bo vi khun lactic
T kt qu phn lp, 78 chng vi khun lactic c chn lc cho nghi n cu kh
nng tch ly PHB. a s cc chng vi khun c kh nng sinh acid lactic, gram dng,
hiu kh ty , catalase m tnh, c kh nng ln men glucose vi 57 chng
Lactobacillus c xc nh. Cc chng cn st hin din ca PHB trong t bo khi
kim tra vi thuc nhum Sudan Black B l 30 chng chim 38,5% trong tong so vi
khun lactic phn lp nn. [bng 1, hnh 1, 2].
Hnh 1. Kt qu nhum gram v hnh thi mt s chng vi khun lactic. H1a. i chng:
E.coli (gram m) bt mu thuc nhum b sung nn c mu hng, B. subtillis (gram
dng) gi c phc mu gia tm kt tinh v glugol nn c mu tm. H1b, c, d, e, f: l
kt qu nhum gram ca cc chng N8, CK1, B3, DG5, M1.
Hnh 2. Kt qu nh tnh PHB; 2a: DG5, 2b: DG5 + E.coli, 2c PHB from DG5
1a
1b 1c 1d 1e 1f
2b
2a 2c
Bng 1: Kt qu phn lp v xc nh s hin din PHB trong t bo vi khun.
[
*
]: kt qu dng tnh ca khun lc vi thuc nhum Sudan Black B
Ngun phn
lp
Lactobacillus Pediococcus Streptococcus Entorococcus Cha xc
nh
Da ci chua C1, C2,C3, C4, C5, C6, C7,
C8, C9, C10, C11, C12,
C13, C14*, C15*, C16*,
C17, C18, C19
C9, C11
Ya-ua Y3, Y4,Y5, Y7 Y2* Y1, Y6
u nnh N1*, N3, N4*, N5*, N6*,
N7*, N8*, N9*
N2
Da kiu CK1*, CK2*, CK3*, CK4*,
CK6*
CK5, CK7
Da gi DG1*, DG5*, DG6, DG7,
DG8
DG2, DG3*, DG4*
Nem chua NC1*, NC2*, N3C*, NC4*,
NC5*, NC6*, NC7, NC8
Da c F3, F6, F7*, F8* F1*, F2*, F4, F5
Ng sen chua S5 S3
Da hnh CH1*, CH2*
Phn tr s
sinh
B41, B3* BB8
M M1*, M2
2. Kt qu kho st ngun dinh dng nh hng n kh nng tch ly PHB
trong t bo
Cc chng vi khun cho kt qu dng tnh cao DG5, M1, N8, CK1, B3, N1, NC3,
NC4, DG4, N4, CH5, N9 v NC2 c chn xc nh hm lng PHB trong t bo.
Qua kt qu th nghim, chng DG5, N8 v M1 cho hm lng PHB cao hn cc chng
cn li tng ng 27.870 %, 26.230% v 26.600% theo trng lng kh t bo v c
chn lm i tng cho tt c cc th nghim tip theo.
Hu ht 3 chng u thch hp vi glucose cho s sinh tng hp PHB. Tuy nhi n,
i vi chng M1 t hm lng PHB 32,302% trong mi tr ng b sung lactose,
peptone l ngun nit thch hp nht cho s sinh tng hp PHB, chng DG5 t h m
lng PHB cao nht vi peptone 27,870% (h nh 3 v hnh 4)
Hnh 3: nh hng ca ngun carbon ti s sinh tng hp PHB: 3a; DG5, 3b; M1, 3c; N8
Hnh 4: nh hng ca ngun nito ti s sinh tng hp PHB: 4a; DG5, 4b; M1, 4c; N8
DG5
0.00
0.05
0.10
0.15
0.20
Peptone yeast extract NH42SO4 gl ycyne
P
H
B
(
%
)
0
5
10
15
20
25
30
S
i
n
h

k
h
o
i
(
g
)
si nh khoi
PHB
M1
0.00
0.05
0.10
0.15
0.20
0.25
P
H
B
(
%
)
0
5
10
15
20
25
30
S
i
n
h

k
h
o
i
(
g
)
si nh khoi
PHB
N8
0.00
0.05
0.10
0.15
0.20
0.25
P
H
B
(
%
)
0
5
10
15
20
25
30
S
i
n
h

k
h
o
i
(
g
)
si nh khoi
PHB
4a
4b
4c
3. Kt qu kho st iu kin li trch PHB
nh hng ca tc nhn li trch: Sodiumhypochlorite (SH) 5%, Sodiumdodecylsulfate
(SDS) 3% c s dng lm tc nhn ph v t bo theo hm lng sinh khi. i vi
tc nhn sodiumhypochlorite cho hi u sut ph v t bo cao nht hm lng 1% sinh
khi t bo t 85,635% v thu c 32,865 % PHB theo trng l ng kh t bo. i
vi tc nhn SDS 3% cho hiu sut ph v t bo cao nht hm lng 5% sinh khi t
bo t 85,124% v thu c 34,635 % PHB theo trng l ng kh t bo. Tc nhn
SDS 3% c kh nng ly trch PHB hm lng sinh khi cao hn tc nhn
Sodiumhypochlorite 5%.
nh hng ca nhit li trch: Th nghim c thc hin vi 2 tc nhn
sodiumhypochlorite v SDS theo dy nhi t t 40
0
C n 90
0
C. i vi tc nhn
sodiumhypochlorite t hm lng PHB cao nht (32,182 %) ti 60
0
C vi hiu sut ph
v t bo 85,462%. i vi tc nhn SDS t hm lng PHB cao nht (35,623%) ti
80
0
C vi hiu sut ph v t bo 85,253% [hnh 6]. Kt qu cho thy, i vi tc nhn
Sodiumhypochlorite 5% nhi t cng cao qu trnh ph v t bo cng gim i vi
cc chng lactobacllus. Ngc li, hiu sut ph v t bo cng mnh khi nhit tng
ln i vi tc nhn SDS 3%, tuy nhin li nh hng n s ph v cu trc ca PHB
v cho hm lng thp xung.
Hnh 5: Kho st s ph v t bo vi tc nhn sodiumhypochlorite 5% v SDS 3%
theo hm lng sinh khi t bo
Hnh 6: Kho st nh hng ca nhit n qu tr nh li trch PHB
vi tc nhn sodiumhypochlorite 5% v SDS 3%
Sodi umhypochl ori te - SDS
0
10
20
30
40
50
60
70
80
90
0. 50% 1% 2% 3% 4% 5% 6%
Si nh k hoi ( % )
P
H
B
)
%
)
0
10
20
30
40
50
60
70
80
90 H
i
e
u

s
u
a
t
PH B ( %) - SH 5 %
H i e u su at ( %) - SH 5 %
PH B ( %) - SD S 3 %
H i e u su at ( %) - SD S 3 %
0
10
20
30
40
50
60
70
80
90
40 50 60 70 80 90
Nhi et o(
o
C)
P
H
B
)
%
)
0
10
20
30
40
50
60
70
80
90
H
i
e
u

s
u
a
t
PH B ( %) - SH 5 %
H i e u su at ( %) - SH 5 %
PH B ( %) - SD S 3 %
H i e u su at ( %) - SD S 3 %
nh hng ca thi gian li trch: Thi gian trong qu tr nh ly trch PHB c thit
lp trong khong thi gian t 20 pht n 120 pht cho 2 tc nhn Sodiumhypochlorite
ti nhit 60
0
C v SDS ti 80
0
C. Kt qu kho st cho thy qu tr nh ly trch PHB vi
tc nhn Sodiumhypochlorite t hm lng PHB cao nht ti thi im 60 pht ly trch
vi 32,246% PHB, hiu sut ly trch l 85,642% v vi tc nhn SDS t hm lng
PHB cao nht ti thi im 80 pht ly trch vi 34,575% PHB, hiu sut ly trch l
95,563% (hnh 7).
Hnh 7: Kho st thi gian li trch nh hng n qu trnh li trch PHB vi tc nhn
sodiumhypochlorite 5% v SDS 3%
KT LUN
Qua kt qu nghin cu, chng ti c mt s nhn xt nh sau: Vi khun lactic c
kh nng sinh tng hp PHB, phn lp c 78 chng vi khun lactic vi h n 50
chng thuc nhm Lactobacillus . Cc chng c s hin din ca PHB trong t b o l
30 chng chim 38,5% trong tng s vi khun lactic phn lp c. Trong chng
DG5, M1, N8 c kh nng sinh tng hp PHB cao nht. Glucose v peptone l 2
ngun dinh dng thch hp cho c 3 chng DG5, M1 v N8 sinh t ng hp PHB. Ring
chng M1 ng lactose hoc ho hm lng cao hn. Kt qu nghin cu cho thy vi
mi trng dch chit c chua b sung 1% glucose cng thch hp cho qu tr nh sinh
tng hp PHB ca c 3 chng. t i nghin cu xy dng c qu trnh tch chit
PHB da trn 2 tc nhn Sodiumhypochlorite 5% v SDS 3 %. Qu trnh tch chi t vi
tc nhn Sodiumhypochlorite 5% thch h p hm lng sinh khi t bo l 1%, nhit
ly trch 60
0
C v thi gian ly trch l 60 pht. i vi qu trnh tch chit bng tc
nhn SDS 3% thch hp vi hm lng sinh khi l 5%, nhit ly trch l 80
0
C v thi
gian ly trch l 80 pht.
TI LIU THAM KHO
1. Dng Th Thu L (2003). Phn lp v chn ging vi khun lactic l m yaourt
u nnh. Lun vn Thc s sinh hc. i hc Khoa hc T nhi n - TPHCM.
0
10
20
30
40
50
60
70
80
90
100
20 40 60 80 100 120
phut
P
H
B
)
%
)
0
20
40
60
80
100
120
H
i
e
u

s
u
a
t
PHB(%)-SH 5%-
Hi eu suat (%)- SH 5%
PHB(%)-SDS 3%
Hi eu suat (%)- SDS 3%
2. Kolybaba M. A.. Tabil L. G.. Panigrahi S. A. (2004). Recent Developments in the
Biopolymer Industry. The society for Engineering in agricultural food and
biological systems. pp. MB04 - 301.
3. Law. John H. & RalpH A. Splepecky (1960). Assay of Poly-|-hydroxybutyric acid.
J. Bacterial. Vol. 82. pp. 33 - 36.
4. Lee S. Y. (1996). Bacterial Polyhydroxyalkanoate. Biotechnology and Bioengineering.
Vol. 49. pp. 1 - 14.
5. Liddell J. M. (1999). Process for the recover y of polyhydroxyalkanoic acid. United
states Patent. Patent Number 5894062.
6. Luengo J. M.. Garcia B.. Sandoval A.. Naharro G. and Olivera E. R. (2003).
Bioplastics from microorganisms. Current Opinion in Microbiology. Vol. 6. pp.
251-260.
7. Mahishi L. H.. Tripathi G.. Rawal S. K. (2003). Poly (3-|-hydroxybutyrate)
synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens
PHB biosynthesis genes: Effect of various carbon and nitrogen sources. Microbial
Research. Vol. 158. pp. 19 - 27.
8. Saledizadeh. Loosdrecht (2004). Production of polyhyd roxyalkanoates by mixed
culture: Recent trends and biotechnological importance. Biotechnology Advances.
Vol. 22. pp. 261 - 279.
9. Steinbuchel A. (1996). PHB and Other Polyhydroxyalkanoic Acids.
Biotechnology. Vol. 6. pp 403 - 464.
10. Ugur. Sahin. Beyati (2002). Accumulation of Poly -|- hydroxybutyrate in
Streptomyces Species: During Growth with Different Nitrogen Sources. Tur J Biol.
Vol. 26. pp. 171 - 174.
SUMMARY
Screening of lacyobacillus strains for biosynthe sis of
polyhydroxybutyrate (PHB)
Nguyen Duy Long, Phm Tran To Nhu, Nguyen Minh Nhut, Hoang Quoc Khanh
In this study, we found 3 Lactobacillus strains (DG5, M1, N8) with a h igher
concentration of PHB than 78 strains collected with 27.87%, 26.23%, 26.60%
corresponding. Glucose and peptone are investigated and determined as nutrient factor
for produce PHB. For the accumulation of PHB from DG5, M1, and N8, the tomato
extract solution was found as a cheeper nutrient resourse to produce PHB. The
processing of PHB extraction was followed by Sodiumhypochlorite (SH) 5% in cell
mass 1% at 600C during 60 minutes and Sodiumdodecylsulphate (SD
PHN LP V THU NHN enzym phytase T MT S
NM MC Aspergillus TRN MI TRNG
NN MEN B MT
Nguyn Duy Long, Nguyn Th M Hnh, Ho ng Quc Khnh
Phng Vi Sinh ng dng, Vin Sinh hc Nhit i
M U
Photpho l nguyn t thit yu cn thit cho s tng trng, pht trin v sinh sn ca
ng vt. Tuy nhin, c khong 60-75% photpho trong ng cc v ht du trong thc n
ca ng vt c tm thy dng phytat (inositol hexaphosphat) hoc axit phytic. ng
vt khng th hp th photpho trong phytat v chng khng c enzym phytase trong ng
rut. V th phn ln photpho c tm thy trong phn ca ng vt. Ngoi ra nhng cht
dinh dng khc nh protein, Fe
2+
, Ca
2+
, Mg
2+
, Zn
2+
cng s b lin kt vo cu trc ca
axit phytic. Do c th ng vt khng th hp th c cc ion ny. Photpho trong phn
ca ng vt c hp th vo t, chng cn thit cho s pht trin ca thc vt. Lng
photpho d tha khng c cy hp th s b cun tri ra ao, h, sng, sui kch thch s
pht trin ca phiu sinh thc vt (to) gy ra hin tng ph dng ho. Ngoi ra, s thiu
ht oxy s lm c cht v gim s a dng ca h sinh vt c li trong nc.
Phytase l mt trong s cc enzym hin ang rt c quan tm. Phytase thy phn
lin kt gia photpho v vng phytat s phng thch photpho v c. Vic b sung phytase
vo thc n gia sc gip tn dng ti a ngun photpho trong cc loi nguy n liu ch bin
v h vi sinh vt ng rut ca ng vt hu nh khng c kh nng sinh tng hp
phytase, v ng thi gip gim chi ph b sung l ng photpho v c cn thit vo thc n
gia sc. Hn th na, phytase cn gip gim thiu nhim photpho vo mi trng do cht
thi ng vt. Enzym phytase c nghin cu trn nhiu i tng khc nhau nh nm
men, nm mc v vi khun nhng nhiu nht vn l trn cc loi nm mc c bit l
Aspergillus sp. v kh sinh tng hp enzym phytase cao v c th chu c pH thp.
VT LIU V PHNG PHP
Cc chng Aspergillus c phn lp t cc loi men ru (men cm ru, men ru)
v mt s chng Aspergillus khc (A.aculeatus, A.awamori, A.carbonarius, A.ellipticus,
A.ficuum, A.flavus, A.niger NRRL-363, A.ochraceus A175, A.oryzae, A.phoenisis,
A.tubingensis) t b su tp ging ca phng Vi sinh ng dng - Vin Sinh hc Nhit i
trn mi trng PGA .
Qu trnh nui cy v tch chit enzyme phytase c thc hin theo quy trnh di
(Hnh 1). Enzym phytase c phn tch hm lng protein theo phng php Bradford.
Hot tnh enzym phytase c xc nh trn c cht sodium phytate (mt n v hot tnh
phytase (U) c nh ngha l lng phytase gii phng c 1 mol photphat v c trong
mt pht t sodium phytat 37
o
C, pH5,5). Qu trnh kho st nh hng ca nhit , pH
n hot tnh enzym phytase c thc hin trong di nhit t 30-95
0
C, trong m
Glycin: 2,5pH, trong Sodium acetat : 3,0; 3,5; 4,0; 4,5; 5,0; 5,5, trong m Imidazol-HCl:
6,0; 6,5; 7,0, trong m Tris-HCl: 7,5; 8,0; 8,5; 9,0. Enzym phytase c phn tch trn gel
polyacrylamide theo phng php SDS-PAGE.
KT QU
1. Phn lp nm Aspergillus t men ru
Trong s 14 loi men ru thu nhn t cc ni khc nhau cho thy s phn b cc
chng nm mc khng c s khc bit nhiu. trong chng ti xc nh c 4
chng nm mc khc bit nhau r rt v hnh thi l Aspergillus.MCR1 (hnh 2.1; 2.2;
2.3), Aspergillus.MCR2(hnh 3.1; 3.2; 3.3), Aspergillus. MR5 (hnh 4.1; 4.2; 4.3),
Aspergillus.MR2 (hnh 5.1; 5.2; 5.3),
ng PGA gi ging Aspergillus
+ 5ml nc ct v trng
Ht 1ml dung dch bo t vo cc bnh ln men b mt (Tru:Bt m:Khong)[T l Tru:Bt m 1:2, 60% Khang]
37
0
C, 2 ngy
B sung 200ml dung dch m Sodium Acetate pH 5.5
Lc 200rpm, 3h
Lc dch qua vi mng
Dch chit enzym th
Cy truyn sang cc ng nghim PGA
Hnh 1: Quy trnh ln men b mt aspergillus sinh tng
hp enzyme phytase
Hnh 2.2. Hnh thi v kch
thc
Conidiophores chng MCR1; X 6
Hnh 2.1. Hnh thi chng MCR1
mc trn mi trng PGA
Hnh 2.3. Hnh thi v kch
thc
bo t chng
MCR1 (conidia); X
100
Hnh 3.2. Hnh thi v kch thc
conidiophores chng MCR2; X 6
Hnh 3.1. Hnh thi chng
MCR2
mc trn mi
trng PGA
Hnh 3.3. Hnh thi v kch thc bo
t chng MCR2 (conidia); X 100
Hnh 4.1. Hnh thi chng MR5
mc trn mi trng PGA
Conidiophores chng MR5; X 6
bo t chng MR5 (conidia); X 100
Hnh 5.1. Hnh thi chng MR2
trn mi trng PGA
bo t chng MR2 (conidia); X 100
Conidiophores chng MR2; X 6
2. Xc nh hot tnh enzyme phytase ca cc chng Aspergillus
Trong 4 chng c phn lp t cc loi men thng mi, c 3 chng Aspergillus
c kh nng sinh phytase l A.MCR2, A.MR2, A.MR5 v 1 chng khng sinh phytase
l A.MCR1. Trong s 3 chng c hot tnh phytase th A.MR2 cho hot tnh cao nht.
Ngoi ra, 1 chng trong B su tp ging- Phng Vi sinh ng dng cng khng c kh
nng sinh phytase l A.ochraceus. Hai chng Aspergillus cho hot tnh phytase cao nht
l A.niger NRRL-363 v A.oryzae. A.niger NRRL-363 c hot tnh tng (Ut) cao hn
A.oryzae nhng A.oryzae li cho hot tnh ring cao hn. Enzyme t cc chng cn li
c hot tnh trung bnh hoc thp (hnh 6a, 6b). i vi A.flavus, mt loi nm mc sinh
c t anfatocin cng c kh nng sinh enzym phytase, do cn t hn trng trong vic
tuyn chn nm mc. T cc chng tr n, chn ra 3 chng A.niger NRRL-363, A.oryzae,
A.MR2 cho hot tnh phytase cao nht kho st ph nhit , pH.
3. Kho st nh hng ca nhit n hot tnh enzyme phytase
T th cho thy nhit ti u cho hot tnh phytase cao i vi c 3 chng
A.niger NRRL-363, A.oryzae, A.MR2 l 50-55
0
C so snh vi cc kt qu nghin
Hnh 6a: Hot tnh tng Ut
(U/g mu)
ca cc chng nm mc
Chung
H
o
a
t

t
n
h

t
o
n
g
U
t

(
U
/
g

m
a
u
)
A.M CR1 A.M CR2 A.M R5
A.M R2 A.acul eat us A.awamori
A.carbonari us A.el l i pt i cus A.f i cuum
A.f l avus A.ni ger NRRL -363 A.ochraceus A175
A.ory zae A.phoeni ci s A.t ubi ngensi s
oi chng
0
500
1000
1500
2000
2500
3000
Chung
H
o
a
t

t
n
h

r
i
e
n
g
U
s

(
U
/
m
g

m
a
u
)
A.M CR1 A.M CR2 A.M R5
A.M R2 A.acul eat us A.awamori
A.carbonari us A.el l i pt i cus A.f i cuum
A.f l av us A.ni ger NRRL -363 A.ochraceus A175
A.ory zae A.phoeni ci s A.t ubi ngensi s
oi chng
Hnh 6b. Hot tnh ring Us (U/mg mu)
ca cc chng nm mc
cu a s phytase c nhit ti u trong khong 44-60
0
C [6,10]. Ti khong
nhit ti u ny, A.niger NRRL-363 cho hot tnh enzym phytase tng Ut cao
nht nhng li c hot tnh ri ng Us thp nht trong 3 chng. A.niger NRRL-363
c hm lng protein cao, hot tnh tng cao, hot tnh ri ng thp chng t chng
ny cho t enzym phytase hn trong t ng s nhiu loi protein c sinh ra, hnh
(7a, 7b). Trong sn xut, hot tnh ri ng cao c quan tm nhiu hn. A.oryzae v
A.MR2 p ng c yu t ny, c 2 chng u c hot tnh enzym phytase tng
thp hn A.niger NRRL-363 nhng l i c hot tnh ri ng cao hn hn nht l
A.oryzae. T kt qu kho st nh h ng ca nhit n hot tnh phytase t
A.niger NRRL-363, A.oryzae, A.MR2, chng ti c kt lun v nhn xt nh sau:
Cc tnh cht ny ca phytase t 3 chng nm mc tr n rt thch hp cho cc ng
dng cng nghi p, c bit trong ch bin thc n gia sc.
4. Kho st nh hng ca pH n hot tnh enzyme phytase
T th cho thy, c 3 chng A.niger NRRL-363, A.oryzae, A.MR2 c pH ti u
cho hot tnh phytase cao l 2,5 v 5,0-5,5. Hu ht enzym phytase t vi sinh vt th
hin pH ti u gia 4,5 v 5,5, c bit i vi phytase t nm mc [10,11]. Tnh cht
ny rt cn thit trong cc ng dng cng nghip v ph hp vi iu kin trong d dy
(pH 2,0-4,0) v rut non (pH 4,0-6,0) ca ng vt [9].
0
50
100
150
200
250
300
350
400
450
500
0 10 20 30 40 50 60 70 80 90 100 110
Nhiet o(oC)
H
o
a
t

t
n
h

t
o
n
g

U
t
(
U
/
g

m
a
u
)
A.ni ger NRRL-363
A.oryzae
A.MR2
0
500
1000
1500
2000
2500
3000
3500
0 10 20 30 40 50 60 70 80 90 100 110
Nhiet o(oC)
H
o
a
t

t
n
h

r
i
e
n
g

U
s
(
U
/
m
g

m
a
u
)
A.ni ger NRRL-363
A.oryzae
A.MR2
Hnh 7a. nh hng ca nhit l n hot
tnh tng ca
enzym phytase
ca A.niger NRRL-363, A.oryzae, A.MR2
Hnh 7b. nh hng ca nhit l n hot
tnh tng ca
enzym phytase
ca A.niger NRRL-363, A.oryzae, A.MR2
Hnh 8a. nh hng ca pH ln hot tnh tng enzym
phytase chng A.niger NRRL-363, A.oryzae, A.MR2
0
500
1000
1500
2000
2500
3000
0,0 2,5 5,0 7,5 10,0
pH
H
o
a
t

t
n
h

r
i
e
n
g

U
s
(
U
/
m
g

m
a
u
)
A.ni ger NRRL-363
A.oryzae
A.MR2
0
50
100
150
200
250
300
0,0 2,0 4,0 6,0 8,0 10,0
pH
H
o
a
t

t
n
h

t
o
n
g

U
t
(
U
/
g

m
a
u
)
A.ni ger NRRL-363
A.oryzae
A.MR2
Hnh 8b. nh hng ca pH ln hot tnh tng enzym
phytase chng A.niger NRRL-363, A.oryzae, A.MR2
Hnh 9: Phn tch enzym phytase trn gel polyacrylamide b ng
phng php i n di SDS PAGE.
5. Phn tch enzyme phytase trn gel polyacrylamide
Mc ch cu th nghim nhm xc nh mt s th nh phn protein c trong dch
chit enzym phytase ca ba chng A.niger NRRL-363, A.oryzae, Aspergillus MR2. Kt
qu xc nh c trnh by hnh 9 di y:
Band 1: cha cc protein marker c trng l ng phn t l 200.000, 116.250,
97.400, 66.200, 45.000, 31.000, 21.500, 14.400, 6.500 Daltons. Band 2: m u i chng
(control). Band 3: dch chit enzym th ca A.or yzae cha cc protein c trong l ng
phn t l 97.400, 89.200 v 58.000 Daltons. Band 4: d ch chit enzym th ca A.MR2
cha cc protein c trong l ng phn t l 97.400, 89.200 v 58.000 Daltons. Band 5:
dch chit enzym th ca A.niger NRRL-363 cha cc protein c trong lng phn t l
116.250, 97.400, 89.200 v 58.000 Daltons. Phytase t cc chng A.niger c hai loi l
PhyA v PhyB. PhyA c tr ng lng phn t nm trong khong 66-128 kDa. PhyB c
trng lng phn t khong 58-65 kDa [2]. Phytase t cc chng A.oryzae cng cho hai
loi phytase l PhyA v PhyB. PhyA c tr ng lng phn t khong 65-120 kDa. PhyB
c trng lng phn t khong 58 kDa [19]. Qua kt qu phn tch enzym phytase sinh
tng hp t cc chng A.niger NRRL-363, A.oryzae, A.MR2 trn gel polyacrylamide
bng phng php SDS - PAGE, cc band protein xut hin tng i r trong vng
kch thc ca phytase so vi kt qu nghi n cu trc [2,19].
TI LIU THAM KHO
1. Trn Th Tuyt (2003). Thu nhn v kho st enzym phytase ca nm Asperg illus
niger NRRL-363 trong mi trng ln men bn rn. Kha lun Trng i hc
Khoa hc T nhin TP HCM, 5-27.
2. Mullaney E. J. and Ullah A. H. J. (2003). The term phytase comprises serveral
different classes of enzymes. Biochemical and Biophysical Researc h
Communications 312, 197-184.
3. Barbara, George Szakacs, Ashok Pandey, Sabu Abdulhameed, James C. Linden, v
Robert P. Tengerdy (2003). Production of phytase by mucor racemosus in solid -
state fermentation. Biotechnol Prog. 19 (2), 312 -319.
4. Wodzinski R. J. and Ullah A. H. J. (1996). Phytase. Advances in Applied
Microbiology, vol 42, 263-298.
5. Lei X. G. and Porres J. M. (2003). Phytase enzymology, applications, and
biotechnology. Biotechnology Letters, 25 (21), 1787 -1794.
6. Oh B. C., Choi W. -C., Park S., Kim Y.-O., Oh T.-K. (2004). Biochemical
properties and substrate specificities of alkaline and histidine acid phytases. Appl
Microbiol Biotechnology , 63 (4), 362 -372.
7. Zimmermann B., Lantzsch H.-J., Langbein U. and Drochner W. (2002).
Determination of Phytase activity in cereal grains by direct incubation, J Anim
Physiol Anim Nutr (Berl), 86 (9-10), 374-352.
8. Bogar B., Szakacs G., Linden J. C., Pandey A. and Tengerdy R. P. (2003).
Optimization of phytase production by solid substrate fermentation. J Ind Microbiol
Biotechnol., 30 (3), 183-189.
9. Casey A. and Walsh G. (2003). Purification and characterization of extracellular
phytase from Aspergillus niger ATCC 9142. Bioresour Technol, 86 (2), 183 -188.
10. Vohra A. and Satyanarayana T. (2003). Phytases: Microbial Sou rces, Production,
Purification, and Potential Biotechnological Applications. Critical Reviews in
Biotechnology, 23 (1), 29-60.
11. Kerovuo J. (2000). A Novel Phytase from Bacillus. Characterization and
Production of the Enzyme, 1-66.
12. McKee T. and McKee J. R. (1999). An Introduction to Chemical Biology.
Biochemistry, vol 2.
13. Daniel M. Bollag, Michael D. Rozycki v Stuart J. Edelstein (1996). Protein
Methods. Wiley, Chichester.
14. Nagashima T., Tange T., and Anazawa H. (1999). Dephosphorylation of Phytate by
Using the Aspergillus niger Phytase with a High Affinity for Phytate. Appl Environ
Microbiol, 65 (10), 4682-4684.
15. Budy J. Hidayat, Niels T. Eriksen and Marylin G. Wiebe (2006). Acid photphatase
production by Aspergillus niger N402A in continuous flow culture. Federation of
European Microbiologycal Societ8ies Microbioal Lett , vol 254, 324 -321.
16. Chantasartrasamee K, Duangnetre Israngkul Na Ayuthaya, Sunthum Intarareugsorn,
Saovanee Dharmsthiti (2004). Phytase activity from Aspergillus oryzae AK9 cultivated
on solid state soybean meal medium. Process Biochemistry, 1 -5.
17. Shieh T. R. and Ware J. H. (1968). Survey of microorganisms for production of
extracellular phytase. Applied Microbiology, 16 (9), 1348 -1351.
18. Howson S. J. and Davis R. P. (1983). Production of Phy tase hydrolysing enzyme
by some fungi. Enzyme Microbiol Technol., 5, 377 -382.
19. Fujita J., Seiko Shigeta, Yu-Ichi Yamane, hisashi Fukuda, Yasuzo Kizaki, Saburo
Wakabayashi, and Kazuhisa Ono (2003). Production of two phytase from
Aspergillus oryzae during industrial Koji making. Journal of Bioscience and
Bioengineering, 95 (5), 460-464.
20. Wyss M, Brugger R, Kronenberger A, Rmy R, Fimbel R, Oesterhelt G, Lehmann
M, and Van Loon A. P. G. M. (1999). Biochemical Characterization of fungal
phytases (myo-Inositol hexakisphophate phosphohydrolases): catalytic properties,
65 (2), 367-373.
SUMMARY
Isolation and investigation of phytase from A spergillus
in solid-state fermentation
Nguyen Duy Long, Nguyen Thi My Hanh, Hoang Quoc Khanh
Institute of Tropical Biol ogy
Phytases are acid phosphatase enzymes have been found as a supplement to
increase not only the growth rate of monogastric animals but also the efficiency of
phosphate utilization in feeds, which significantly reduces phosphorus excretion effect
to environmental pollutants. In this study, phytases are extracted from Aspergillus
species are isolated from original raw material of fermented glutinous rice. Three
species A.MCR2, A.MR2, A.MR5 are found with a high positive of phytase activity.
The effects of pH and temperature conditionals are researched and compared with
others species of Aspergillus genus. Phytases are also assayed on polyacrylamide gel by
SDS-PAGE method and the results appearing with 65 kDa and 58 kDa respective to
phyA and phyB from A.MR2.
TI U HO KH NNG SINH TNG HP
enzyme cellulase
CA Tricoderma reesei TRN MI TRNG BN RN
Trn Thnh Phong, Hong Quc Khnh, V Th Hnh, L Th Bch Phng,
Nguyn Duy Long, L Tn Hng, Trng Th Hng Vn
M U
Nhiu loi nm si c kh nng sinh ra mt l ng ln cellulase thuc ging Trichoderma,
Aspergillus, Pinicillium, Trong hai ging nm si l Trichoderma v Aspergillus c
nhiu nh khoa hc nghin cu sn xut cellulase (Bothast & Saha, 1997).
Cellulase l enzym a c u t gm: exoglucanase hay C
1
(EC 3.2.1.91),
endoglucanase hay C
x
(EC 3.2.1.4) v |-glucosidase (EC 3.2.1.21), hot ng phi hp
thy phn cellulose thnh glucose. Cellulase c ng dng b sung vo thc n
gia sc, gia cm; ch bin thc phm; trch ly cc cht t thc vt, t cy thuc; ng
ha cc ph liu giu cellulose sn xut ethanol.
Vit Nam c lng ph ph liu nng nghip thi ra rt di d o, trong lng b
ma thi ra t cc nh my ng chim khong 20% ma nguyn liu, trong b ma c
hm lng cellulose khong 50% v hemicellulose khong 25% nn c th s dng nh
ngun carbon cm ng nm si sinh tng hp cellulase.
VT LIU V PHNG PHP
Nm si:Chng T. reesei VTT-D-80133 nhn c t bo tng ging Roal Oy,
Phn Lan. C cht: B ma v cm m.
Xc nh hot tnh cc enzym: Carboxymethyl cellulase (CMCase), FPU (Filter
Paper Unit), xylanase, -amylase, protease.
Thy phn giy: cho dch enzym cellulase (5 FPU/ ml) vo giy xay nh (10%)
50
0
C, pH 5 trong 24 gi. Hiu sut (%) = lng ng kh (g)*0,9*(100/lng giy in (g))
ien di pr otein: Sodi um Dodecyl Sul f ate Pol yacryl ami de gel (SDS-PAGE)
c thc hi en tren gel ng cha 10% (w/v) pol yacryl ami de.
Ti u ha thnh phn mi trng: T l BM: CM, m, nng dinh dng
v thi gian nui cy l 4 yu t c nh hng r rt n kh nng sinh tng hp
cellulase ca T. reesei nn c ti u theo phng php quy hoch thc nghim.
KT QU V THO LUN
Kt qu ti u ha thnh phn mi trng v cc iu kin nui cy.
Cc kt qu th nghim trc y, chng ti xc nh c thnh phn mi
trng c s cho chng T. reesei VTT-D-80133 sinh ra cellulase: t l BM: CM (4:6),
m ban u 54%, 5 ln nng dinh dng, thi gian nui 7 ngy, t l ging
6x10
6
bo t/g mi trng, hot tnh cellulase t c l 251,43 IU/g.
Tuy nhin, thnh phn mi trng v cc iu kin nui cy mi ch c nghin
cu nh hng mc ring r. Trong nm yu t trn th bn yu t l t l BM:CM,
m ban u, nng dinh dng v thi gian nui cy c nh hng ng k n
kh nng sinh tng hp cellulase ca T. reesei VTT-D-80133 nn c chn nghin
cu ti u ha theo phng php qui ho ch thc nghim.
Qui hoch c thc hin vi ma trn y vi s th nghim N = 2
4
= 16
Bng 1. M ha cc bin s
Cc bin s Mc di (-) Mc trung bnh (0) Mc trn (+)
X1: T l BM:CM 2:8 4:6 6:4
X2: Nng dinh dng (ln) x2 x5 x8
X3: m ban u (%) 50 54 58
X4: Thi gian nui cy (gi) 3 7 11
Tin hnh nui cy T. reesei trong cc mi trng m c cc yu t kho st trn
hai mc (theo bng 1). Kt qu xc nh hot tnh cellulase c ghi nhn trong bng 2.
Cc h s trong phng trnh hi qui c xc nh nh sau:
- Tnh b
0
:
16
16
1
0
y
b
i
i
=
= , b
0
= 87,30
- Tnh b
i
:
16
16
1
y
x
b
i
i
ji
j
=
= , b
1
= 2,2; b
2
= 5,82,
b
3
= 29,1, b
4
= 49,8
- Tnh b
ij
:
,
16
16
1
=
=
i
i i
l j
jl
y
x x
b
, b
12
= 9,66,
b
23
= 2,08, b
13
= 31,32, b
14
= 9,71, b
24
= 16,17, b
34
= -0,24, b
123
= 13,58,b
124
= - 21,27,
b
134
= 32,03, b
234
= 6,17, b
1234
= -10,19.
H s c ngha phi tha mn iu kin:
t
b
t lt
j
j
Sbj
) = , Vi
t
lt
: p = 0,05, f = n 1 = 2
t
) 2 ; 05 , 0 (
= 4,3 (Tra bng tiu chun Student)
S
bj
(phng sai ca cc h s):
N
S
S
th
bj
= =
4
03 , 1
= 0,26 (N = 16, =
S
th
2
1
3
1
0
0
|
.
|

\
|
=
n
i
i
y y
= 1,07
(vi n = 3) =
S
th
1,03))
So snh cc h s t
j
vi t
lt
ta thy,
cc h s ca phng trnh u c
ngha, ngoi tr cc h s t
34
. Vy
hm mc tiu c dng:
^
y = 87,3 + 2,2x
1
+ 5,82x
2
+ 29,1x
3
+ 49,8x
4
+ 9,66x
1
x
2
+ 31,32x
1
x
3
+
9,71x
1
x
4
+ 2,08x
2
x
3
+16,17x
2
x
4
+
13,18x
1
x
2
x
3
21,57x
1
x
2
x
4
+
32,03x
1
x
3
x
4
+ 6,17x
2
x
3
x
4
10,19x
1
x
2
x
3
x
4
T phng trnh hi qui thu c,
ta thy rng thi gian nui , m
ban u v nng dinh dng c nh
hng ng k n hot tnh cellulase
hn t l b ma:cm m.
Kim tra s tng thch ca m hnh theo tiu chun Fisher.
iu kin m hnh tng thch l:
F F lt tn
( , F
lt
: gi tr chun Fisher mc p =
0,05; f
1
= N l; f
2
= n-1; trong N = 16, l: s h s c ngha = 15, n =
3),
F F lt
= = 5 , 18
) 2 ; 1 ; 05 , 0 (
(Tra bng tiu chun Fisher)
F
tn
: 87 , 1
07 , 1
00075 , 2
2
2
= = =
S
S
F
th
tt
tn
(
l N
i
i i
tt
y y
S
\
|
|
|
.
|
=

=
16
1
2
^
2
). Ta c:
F F lt tn
( (v 1,87 < 18,5)
Vy: Phng trnh hi qui thu c tng thch vi thc nghim
Kt qu ti u ha theo k hoch leo dc: mi trng c t l BM:CM (7:3), 8
ln nng dinh dng, m 60%, thi gian nui cy 7 ng y cho hot tnh cellulase
cao nht: CMCase 280,64 IU/g v FPU 5 UI/g, thp hn 3,2 v 37 ln so vi ch phm
Amano T. Canh trng cn cha -amylase 368,75 UI/g, protease 12,43 UI /g v
xylanase 10073,25 BXU/g
Kt qu ng ha giy in qua s dng
Dch chit enzym cellulase (5 FPU/ml) ng ha khong 20% giy in qua s
dng (10%) trong 24 gi thy phn 50
0
C, pH 5,0; dch ng ha cha 23,62 mg
ng kh/ml c th c s dng ln men ethanol hoc ln men sn xut cc sn
phm c gi tr.
T
TN
X
1
X
2
X
3
X
4
y y^
1 2:8 x2 50 3 28,67 28,92
2 6:4 x2 50 3 0,15 0,4
3 2:8 x8 50 3 1,24 1,48
4 6:4 x8 50 3 2,57 2,8
5 2:8 x2 58 3 144,48 143,72
6 6:4 x2 58 3 18,07 17,82
7 2:8 x8 58 3 5,62 5,38
8 6:4 x8 58 3 99,18 98,94
9 2:8 x2 50 11 130,64 130,4
10 6:4 x2 50 11 58,36 58,12
11 2:8 x8 50 11 188,73 188,48
12 6:4 x8 50 11 55,23 55
13 2:8 x2 58 11 51,94 52,72
14 6:4 x2 58 11 219,5 219,74
15 2:8 x8 58 11 129,45 129,7
16 6:4 x8 58 11 262,94 263,18
17 4:6 x5 54 7 154,03
18 4:6 x5 54 7 155,07
19 4:6 x5 54 7 156,1
Bng 2. Hot lc CMCase t T. reesei theo
thc nghim v theo phng trnh hi qui.
Vi y: Hot tnh CMCase (UI/g) theo thc nghim;
y^: hot tnh CMCase (UI/g) theo phng trnh hi qui
Kt qu phn tch h cellulase trn gel SDS-PAGE
KT LUN
Mi trng ti u cho T. reesei VTT-D-80133 sinh ra cellulase l: t l BM:CM
(7:3), 8 ln nng dinh dng, m ban u 60 %, thi gian nui cy 7 ng y. Hot
tnh CMCase v FPU tng ng l: 280,63 IU/g v 5 FPU/g; thp hn 3,2 v 37 ln so
vi Amano T. Ngoi cellulase, canh trng cn cha -amylase 368,75 UI/g, protease
12,43 UI/g v xylanase 10073,25 BXU/g. Qua phn tch trn gel polyacrylamide,
cellulase thu nhn c c cc vch protein vi trng l ng phn t bng vi cc vch
protein ca ch phm AmanoT. Dch enzym cellulase ca T. reesei VTT-D-80133 vi
hot lc 5 FPU/ml, c kh nng ng ha khong 20% giy in qua s dng; dch
ng ha cha 23,62mg ng kh/ml c th c s dng ln men ethanol hoc
ln men sn xut cc sn phm c gi tr.
TI LIU THAM KHO
1. Nguyn Cnh (1993). Qui hoch thc nghim, Trng HBK Tp. H Ch Minh.
2. Chahal D. S., (1985). Solid-state fermentation with Trichoderma reesei for cellulase
production. Applied and Environmental Microbiology, Vol. 49, No. 1, p. 205-210.
3. Jeffries T. W. (1987). Production and applications of cellulase laboratory
procedures. Forest Products Laboratory, Madison, Wisconsin.
4. Bigelow M and Wyman C. E. (2002). Cellulase production on bagasse pretreated
with hot water. Applied Biochemistry and Biotechnology, pp. 98-100.
5. Raimbault M. (1998). General and microbiological aspects o f solid substrate. EJB
Electronic Journal of Biotechnology Vol. 1, No. 3.
1 2
3
kDa
66
45
36
29
24
20
Kt qu in di trn gel SDS-PAGE ca cc
dch enzym cellulase ghi nhn c nh hnh bn.
Ging 1: Dch enzym t T. reesei VTT-D-80133.
Ging 2: Dch enzym t ch phm Amano T
Ging 3: Thang phn t lng nh
Kt qu cho thy, ging 1 c cc vch protein rt
ging vi ging 2. Kt qu ny c th c gii
thch nh sau: theo Barnett (1991), h cellulase t T.
reesei gm CBHI, CBHII, EGI, EGII, EGIII, EGV
v mt |-glucosidase; theo bo co gn y th T.
reesei sinh ra hai loi |-glucosidase l Bgl1 v Bgl2
(Alinda A. Hasper et al., 2001). Da vo cc d liu
trn, ta c th kt lun rng canh tr ng nui cy T.
reesei VTT-D-80133 c y cc tiu phn ca h
cellulase
6. Yun Hyun Shik et al. (1998). Production of cellulase by Trichoderma reesei Rut
C30 in wheat bran media. Journal of Microbiology and Biotechnology , 8(3).
SUMMARY
Optimization of cellulase production by Tricoderma reesei
on solid substrate media
Tran Thanh Phong, Hoang Quoc Khanh,
Vo Thi Hanh, Le Thi Bich Phuong, Nguyen Duy Long,
Le Tan Hung, Truong Thi Hong Van
Sugarcane baggase/wheat bran ratio (7/3), initial moisture content of 60%, 8 times
concentration of basal medium and cultivation time of 7 days were optimum for
cellulase production in solid state fermentation (SSF ) by Trichoderma reesei VTT-D-
80133 mutant. The activity and productivity of ce llullase obtained after 7 days of
fermentation were 280,64 IU/g and 5 FPU/g of fermented medium; These activities are
lower than 3,2 and 37 times compared with commercial cellulase (Amano T) of
AMANO Company (Japan). Besides cellulase, fermented substrate contain activities of
-amylase 268,75 UI/g, protease 12,43 UI/g and xylanase 10073,25 BXU/g. The
cellulase extract was shown to have some bands that their molecular weights are
equivalent to commercial cellulase (Amano T) by sodium dodecyl sulphate poly-
acrylamide gel electrophoresis (SDS-PAGE). At the filter paper activity of cellulase
was 5 FPU/ml, the yield of enzymatic hydrolysis of paper waste was 21%.
PHN LP, NH DANH V TUYN CHN CC CHNG
Enterococcus
C TIM NNG probiotic T PHN TR S SINH
H Vn Tho, Hong Quc Khnh
Phng Vi Sinh ng dng, Vin Sinh hc Nhit i
M U
Trong rut ngi c khong 10 t vi khun trong 1 gram phn, gm v i trm loi vi
khun to nn h vi sinh ng rut ht sc phong ph [14]. Enterococci v
streptococci nhm D to nn mt phn quan trng c bit trong h vi sinh vt bn a
trong rut ngi v mt s loi ng vt, m ph bin l E. faecalis v E. faecium. c
bit E. faecalis thng xuyn c tm thy trong rut gi ca ngi, thnh thong cng
c tm thy trong rut g, nhng t khi tm thy trong cc ng vt khc.
Theo Shleifer v Kilpper- Balz (1984, 1987) m t cc c im v c p
dng cho hu ht cc loi thuc ging Enterococcus. y l nhm vi sinh vt k kh ty
, dng cu n hay kt chui, gram dng, catalase m tnh. Xut hin khp ni trong
mi trng, nhng ph bin trong cc sn phm sa v nht l trong rut ngi v mt
s loi ng vt c v. Enterococci c th sng v pht trin c trong bin nhit kh
rng ln, t 10
0
C - 45
0
C, ti u 35
0
C - 37
0
C, sng st t nht 30 pht 60
0
C. Nh c
bm Natri-ATPase nm trn mng t bo cht nn c th chu c nng NaCl ln n
6,5%. pH hot ng t 4,6 - 9,6, ti u khong 6,5 - 7,5. Hu ht cc loi thuc
Enterococci c th thy phn c esculine vi s hin din ca mui mt 40%.
VT LIU V PHNG PHP
Phn lp v nh danh [10,11, 13]
Ngun ging vi khun: ging vi khun c phn lp t phn tr s sinh thu thp
ti bnh vin T D v Bnh vin Gia nh thuc Thnh ph H Ch Minh, Vit Nam.
Mu c pha long v tri trn mi trng BEA (Bile Esculin Azid), c 37
0
C,
[4], [5]. Sau 2 ngy chn nhng khun lc tch ri c ng knh t 0,5 - 1mm, mu
trng c hoc hi trong, ng thi to thnh qung en xung quanh khun lc, lm
thun v gi ging trn mi trng MRS broth (c b sung thm 20% glycerol [16].
Cc chng vi khun thu c s c tin hnh nhum gram, test catalase, kho st
c tnh ln men glucose, kh nng sng st v pht trin trong mi trng MRS broth
c nng NaCl 6,5% v mi trng c nng dch mt 40% [4]. Kh nng pht trin
c 10
0
C, 45
0
C trong khong thi gian t 2 - 7 ngy v sng st c nhit
60
0
C trong thi gian 30 pht. Sau , kt hp vi thc hin k thut PCR khuch i
trnh t vng trung gian rDNA 16S - 23S (Tyrrel et al., 1997) [1], c bo tn tt
Enterococcus vng ITS gip cho vic nh danh ging Enterocccus. S dng cp
mi pS1490, 5-TGC GGC TGG ATC CCC TCC TT-3 v pL132, 5-CCG GGT TTC
CCC ATT CGG-3 (Normand et al., 1996)
2.2. Kho st cc c im probiotic
2.2.1. nh hng ca Enzyme v pH thp
2.2.2. nh hng ca trypsin pH 8
2.2.3. Kh nng kt bm
2.2.4. Kho st kh nng khng vi khun gy bnh
2.2.5. Kho st kh nng khng khng sinh
2.2.6. Kho st c im sinh bacteriocin khng khun
KT QU
Kt qu phn lp
Qua qu trnh phn l p v lm thun, thu c tt c 34 chng vi khun, phn gii
c esculine [2], dng cu chui, gram d ng, catalase m tnh, phn gi i arginine.
Trong c 13 chng t mu phn tr sinh ti Bnh vin T D, k hiu t TD1 -
TD13, v 21 chng t mu phn tr sinh ti Bnh vin Gia nh k hiu GD1 - GD21.
Kt qu nh danh
c im sinh l, sinh ha
Bng 1: Kt qu kho st cc c im sinh l c a 34 chng thu c
Chng NaCl 6,5% Dch mt pH 9,6 100C 450C 600C/30 Ln men Phn gii
Chng NaCl
6,5%
Dch mt
40%
pH 9,6 100C 450C 600C/30 Ln men
glucose
sinh acid *
Phn gii
arginine
GD1 + + + + + + + +
GD2 + + + + + + + +
GD3 + + + + + + + +
GD4 + + + + + + + +
GD5 + + + + + + + +
GD6 + + + + + + + +
GD7 + + + + + + + +
GD8 + + + + +
GD9 + + + + + + + +
GD10 + + + + + + + +
GD11 + + + + + + + +
GD12 + + + + + + +
GD13 + + + + + +
GD14 + + + + + + + +
GD15 + + + + + +
GD16 + + + + + + +
GD17 + + + + + + +
GD18 + + + + + + +
GD19 + + + + + + +
GD20 + + + + + + + +
GD21 + + + + + + + +
40% Glucose,
sinh aicd
arginine
Ef + + + + + + + +
TD1 + + + + + + + +
TD2 + + + + + + + +
TD3 + + + + + + + +
TD4 + + + + + + + +
TD5 + + + + + + + +
TD6 + + + + + + + +
TD7 + + + + + + + +
TD8 + + + + + + + +
TD9 + + + + + + + +
TD10 + + + + + + + +
TD11 + + + + + + + +
TD12 + + + + + + + +
TD13 + + + + + + + +
(+): vi khun sng v pht trin c;
(-): vi khun khng pht trin c.
* : test ln men glucose sinh acid lactic, khng sinh hi
c im sinh l: sau hng lot cc test nhn thy rng: a s cc chng u c kh
nng pht trin c trong NaCl 6,5%, dch mt 40%, mi tr ng c pH 9,6, mi
trng c nhit 10
0
C v 45
0
C, v 60
0
C trong thi gian 30 pht. Ngoi tr c mt s
chng cho kt qu m tnh nh: GD8, GD16, GD17, GD18, GD19 khng pht tri n
c trong NaCl 6,5%; GD8, GD13,GD15 khng pht trin c trong mi trng c
nhit 10
0
C ln 45
0
C.
c im sinh ha: tt c 34 chng u ln men c glucose sinh acid lactic v
khng sinh hi, phn gi i c arginine.
Nh vy a s cc chng kho st u c c im sinh l ph hp vi cc c
im ca Enterococcus thng hin din ng vt. Tuy nhi n c mt s chng cn
cha c s khng nh nh GD8, GD13, GD15, GD16, GD17, GD18, GD19. Do ,
cn phi kt hp vi k thut PCR khuch i v ng ITS c kch thc t 280 620 bp
cho s khng nh chnh xc hn.
Kt qu kho st cc c im probiotic trong iu kin in vitro
Kh nng chu c pH thp
pH 3 tt c cc chng u chu c v khng b nh hng ti sc sng sau 180
pht. pH 2 cho thy kh nng chu ng khc nhau ca cc chng, nh 30 pht i
vi TD3, TD4, TD5, TD6, GD10, GD11, GD12, GD20., 60 pht vi TD2 , GD3, GD21,
90 pht vi TD1, GD4, GD7, GD8 v 180 pht th khng c chng no sng c
Cc chng chu c pH 1 sau cc khong thi gian l 30 pht i vi TD6, 60
pht vi TD5, GD4, GD7, GD21. thi im 90 n 180 pht khng c n chng no
sng st c.
Kt qu kh nng chu c enzyme trypsin pH kim
Vi iu kin mi trng c cha trypsin 0,01% pH 8 trong khong thi gian t 0
- 180 pht, nhn thy nng trypsin v pH ny th khng lm nh hng n sc
sng ca tt c 31 chng kho st. Nh vy cng vi kh nng chu ng c nng
mui mt 40% cho thy cc chng kho st c th sng tt trong iu kin mi tr ng
rut non.
c tnh kt bm ca 31 chng kho st
T hai kt qu kho st v c tnh kt bm ca 31 chng Enterococcus nhn t hy
rng cc chng cho c tnh kt bm tt, th hin c th nghim kt t trong mui
amonium sulfat v kt bm vi dung mi. Kt qu cho thy tnh t ng ng kh cao,
gip nhn thy c mt s chng c tnh kt bm kh tt: Ef, TD10, TD12, TD13,
GD1, GD5, GD6, GD9, GD10, GD17, GD19.
c tnh khng cc vi khun gy bnh
C 31 chng kho st u khng tt i vi E. coli, Salmonella v khng yu hn
i vi Staphilococcus.
Ni bt c 13 chng khng tt vi c 5 loi vi khun ch th: TD3, TD10, TD11,
GD5, GD6, GD7.
Kh nng khng khng sinh
i vi khng sinh vancomycin (Va): a s cc chng nhy khi tip xc vi loi
khng sinh ny, tr mt s chng c hot tnh khng thuc l TD11, GD4, GD5, GD6,
GD7; v mt s chng th hin hot tnh khng trung g ian TD2, TD12, GD1, GD14,
GD17. Nhng chng ny c th cha gene khng vancomycin ang hot ng c th c
tnh chuyn i kh cao nn rt nguy him v c cc t chc cng nh cc nghin
cu ca nhiu nh khoa hc cnh bo l khng c s dng.
Kt qu kho st kh nng sinh bacteriocin c ch cc vi khun ch th
a s cc chng kho st u th hin hot tnh bacteriocin kh tt l n E. coli
ATCC 25922 v St. aureus ATCC 25923; nhng c 4 ch ng khng c Lactobacillus
acidophillus NRRL B-2092: TD1, TD5, GD1, GD10.
KT LUN
phn lp, kho st cc c im sinh l, sinh ha v cc c im probiotic, cui
cng chn c 6 chng: TD3, TD8, TD10, TD13, GD3, GD19 trong s 31 chng
thuc ging Enterococcus c cc c tnh tt kh ng u nh kh nng chu ng
c pH thp trong ng rut, kh nng kt bm v nh c trn t bo biu m rut,
chu c s tc ng ca enzyme rut v pH kim, ng thi c kh nng khng c
mt s loi vi khun gy bnh, khng c mt s loi khng sinh mt liu l ng
nht nh, v to c bacteriocin khng khun.
TI LIU THAM KHO
1. Alves P. I., Martins M. P., Semedo T. M., Marques J. J. F., Tenreino R. and Crespo M.
T. B., (2004). Comparison of phenotypic and genotypic taxonomic methods for the
identification of dairy enterococci. Antonie van Leeuwenhoek, vol 85, pp 237 - 252.
2. Chuard C., and Reller L. B., (1998). Bile-Esculin test for presiumptive identification of
enterococci and streptococci: effect of bile concentration, inoculation technique, and
incubation time. J. Clin. Microbial, Vol 36, pp 1135-1136.
3. Collins C. H., Lyne P.M., Grange J. M. and Falkinham J. O. Microbiological
methods. 8
th
ed, pp 61-345.
4. Domig K. J., Mayer H. K., Kneifel W., (2003). Methods used for isolation,
enumeration, characterisation and identification of Enterococcus spp. 1. Media for
isolation and enumeration. International Journal of Food Microbiology, vol 88, pp
147-164.
5. Falklam R. and Moody M. D., (1970). Presumptive identification of group D
streptococci: the Bile-Esculin test. Applied Microbiology, vol 20, pp 245 -250.
6. FAO/WHO Working Group (2002). Guideline for the evaluation of probiotic in
food. Food and Agriculture Organizatin of the United Nations.
7. Gusils C., Bujazha M. and Gonzalez S., (2002). Preliminary studies to des ing a
probiotic for use swine feed. Comunicaciones reports comunication, vol 27, pp
409-413
8. Karimi O. and Pena A.S. (2003). Probiotic: isolated bacteria strain or mixture of
different strain?. Drug of Today, vol 39, pp 565 597.
9. Laukova A, Strompfova V. and Ouwehand A. (2004). Adhesion properties of
Enterococci to intestinal mucus of different hosts. Veterinary Research
Communication, vol 28, pp 647-655.
10. MacFaddin J. F., (2000). Biochemical Tests for Identification of Media Bacteria.
Laboratory Manuals, 3
rd
ed, vol 1, pp 8-439.
11. Manero A. and Blanch A. R., (1999). Identification of Enterococcus spp. with a
biochemical key. Applied and Environmental microbiology, vol 65, pp 4425 -4430.
12. Marcinakova M., Simonova M., Laukova A., (2004). Probiotic properties of
Enterococcus faecium EF9296 strain isolated from silage. Bull Vet Inst Pulawy, vol
73, pp 513-519.
13. Naidu A. S, Bidlack W. R, and Clemens R. A., (1999). Probiotic spectra of
lactic acid bacteria (LAB). Critical Reviews in Food Science and Nutrition, vol
38, pp 13-126.
14. Rastall R. A. (2004). Bacteria in the gut: friends and foes and how to alter balance.
American society for nutritional sciences, pp 2022s
15. Tagg J. R. and Mc Given A. R., (1971). Assay system for bacteriocin s. Appl
Microbiol, vol 21, pp 934-938.
SUMMARY
Isolation, identification and selection of Enterococcus strains
with high potential probiotic from infant faeces
Ho Van Thao and Hoang Quoc Khanh
Bacteria of the genus Enterococcus are found in a wide variety of habitats such as
soil, water, plants, fermented food, and in the gastrointestinal tracts of humans. From
the metabolic point of view, Enterococcus spp. produce lactic acid as the main product
of carbohydrate fermentation.Thus, they may be considered to be lactic bacteria
(Doming et al. 2003). With the purpose of researching and studying the capacity
applying of this group in the field of probiotic, our group carried out isolations and
selection the Enterococcus types having applicability to use for probioti c micro-
organism having the origin from strong infant intestine. We isolated and identified
Enterococcus spp. by combining physiology tests and biological chemistry tests with
sequenced amplification of the intermediate ITS - PCR. As a result, we selected 31
bacteria strains belong to Enterococcus spp. Then we continued to survey probiotic
properties such as durable capability and survival in the low pH condition, digestive
enzyme, adhering to intestinal epithelial cell in in-vitro condition. In addition, we still
survey the resistance to pathogenic bacteria in the intestine, the capability of resistance
to some types of antibiotic products and the produce bacteriocin initially. Through the
research, we affirm that the isolates have potential in producing and applying, through
the survival capability in inclement condition and may live well in intestinal condition.
Besides, they can resist and compete to some pathogenic bacteria strains. Finally, the
survey initially help us pick out 6 genera belong to Enterococcus that having the most
probiotic potential to apply for researching and applying probiotic bacteria
KHO ST KH NNG TNG CNG KCH THCH
MIN DCH TRONG TM S Penaeus monodon ca beta-
glucan I VI virus GY HI CHNG M TRNG
(WSSV)
V Long Thun, Nguyn Duy Long, Hong Quc Khnh
Phng Vi sinh ng dng
Vn Th Hnh
Phng Cng ngh sinh hc ng vt, Vin Sinh hc Nhit i
M U
Lnh vc nui tm ang pht trin mnh vi t l tng tr ng hng nm t
khong 16% trong hn thp k qua. Tuy nhin vi c pht trin ngh nui tm b
hn ch bi: i) Dch bnh ho nh hnh nhng cc bi n php ngn chn v kim
sot bnh vn cn hn ch, ii) Cht l ng tm b m v tm post-larvae khng
ng nht, iii) Ngun thc n c cht l ng khng ph hp, iv) Qun l cht
lng nc khng thch hp. Trong nghi n cu ny, chng ti i u ch beta-
glucan bc u quy m phng th nghi m, ng thi cng tin h nh th
nghim hot tnh ca ch phm n y i vi virus gy hi chng m trng
(WSSV) trn tm s Penaeus monodon.
VT LIU V PHNG PHP
Tm s penaeus monodon thu nh n t cc tri nui tm c x l v nui
trong h thng b knh, cho n v nui dng di thc n c iu ch theo
quy trnh hnh 1, virus gy h i chng m trng (wssv) c s dng cho mc
ch ly nhi m, lm tc nhn gy bnh. Virus c pht hin tr n tm bng SV-
kt, phng php PCR km theo phng php xc nh hnh thi huyt bo tm.
Hm lng protein c xc nh bng phng php Bradford. Ch phm beta-
glucan c tch chit t nm men Saccharomyces cerevisiae theo quy tr nh bn
di (hnh 2):
KT QU
1. iu ch beta-glucan t Saccharomyces cerevisiae
Ch phm Beta-glucan c iu ch t Saccharomyces cerevisiae c mu xm
trng, c mi, v c trng v dng bt mn. Hiu sut iu ch c trnh by bng 1.
Bng 1: Kt qu iu ch Beta-glucan t Saccharomyces cerevisiae
2. Quan st hnh thi t bo haemocyte
y l cng on nm trong phng php m tng huyt bo trong huyt tng
tm, l c s khoa hc cng nh lm tng chnh xc cho phng php. Qua y cng
nhm mc ch xc nh s b hnh thi ca huyt bo (t bo haemocyte) tm. Cc
huyt bo c rt nhiu hnh dng khc nhau: hnh trn, oval, mng nga, ng knh
trong khong 5-12 m. Mu sc: c mu xanh dng nht khi bt mu vi thuc nhum
giemsa. trng thi sinh l bnh thng (hnh 3), huyt bo c mu xanh ng lt.
S ln iu ch 1 3 2 4 5
Trng lng nm men ban u (g) 50 150 200 300 500
Trng lng glucan kh thu c (g) 3,50 10,67 14,80 21,07 35,51
Hiu sut tch chit (%) 7,00 7,11 7,40 7,02 7,10
Hiu sut tch chit trung bnh (%) 7,13
t th h c c n n
( ( x xa ay y n nh hu uy y n n) )
b be et ta a- -g gl lu uc ca an n
( (d d n ng g b b t t) )
c ch h t t k k t t d d n nh h ( (g ge el la at ti in n
+ + t ti in nh h b b t t) )
t tr r n n u u v v l l m m m m b b n ng g
n n c c m m ( (n n c c u un n s s i i
n ng gu u i i n nh hi i t t 4 40 0- -5 50 0
o o
c c) )
t t o o d d o o c c n n t th hi i t t v v p p v vi i n n
t th h c c n n c ch h a a b be et ta a- -g gl lu uc ca an n
H H n nh h 1 1: : Q Qu uy y t tr r n nh h t t o o t th h c c n n v vi i n n c ch h a a b be et ta a- -
g gl lu uc ca an n
H H n nh h 2 2: : Q Qu uy y t tr r n nh h t t c ch h c ch hi i t t b be et ta a- -g gl lu uc ca an n
Huyen phu nam men trong dung dch NaOH 1M (1 : 5(w/v))
Utrong 90
o
C, 1 gi
Khuay eu , enguoi nhi et ophong
Li tam 4000 vong/10 phut, thu can, ra vi nc 3 l an , trung hoa can na y vi
dung dch aci d aceti c 2M, l i ta m thu can
Ra can thu c bang mot l ng tha acetone ( 4 acetone : 1 nc (v/v )), ra
l ai 3 l an vi nc
Thu can, tr i thanh l p mong tren mot tam pl asti c.
Say kho70
0
C, 8 gi
Nghi en bang may nghi e n
Beta -gl ucan da ng bot
Hnh 3 . Tiu bn Haemocyte nhum giemsa di knh hin vi quang hc x100;
A: tiu bn, B: trong bum m.
3. Kt qu nhim nhn to trn tm
Sau khi nhim khong 3 - 4 ngy, tm bt u biu hin v cht do wssv. Biu hin
c ghi li nh sau: tm b nhim m trng c biu hin vng m trng ng knh
0,3 - 0,5mm vng v u ngc, quanh mang, n rt t, phn x km (bt rt d) (hnh 4)
Hnh 4. Mu tm cht do bnh m trng nhim nhn to
4. Kt qu theo di khi ly nhim virus v cho n thc n cha Beta-glucan
Kho st t l sng st
T l sng st t 1: l i chng m ((-) wssv (-) glucan): t l sng c duy tr
mc trn 90% trong sut thi gian th nghim. L i chng dng ((+) wssv (-)
glucan): t l sng gim mnh, bt u t ng y th 3 (cn 77,78%). t ngy th 4 n
th 7, t l sng ny gim rt mnh (t 72,22% xung c n 0%). L th nghim ((+) wssv
(+) glucan): t l sng cng bt u gim vo ngy th 3 (cn 81,82%) v cng gim
mnh vo cc ngy sau . n ngy th 7, t l sng ca l th nghim c n 21,21% v
vn cn duy tr n ngy th 8 l 12,12%. Mc d, c 2 l (l i chng dng v l th
nghim) c t l sng u gim, nh ng t l sng ca l th nghim lun duy tr mc
cao hn l i chng dng. c bit l ngy th 7, t l sng l i chng dng l
0%, trong khi l th nghi m vn duy tr mc 21,21% (hnh 5)
B A
Hnh 5: t l sng st ca tm t 1 Hnh 6 : t l sng st ca tm t 2
T l sng st t 2: L i chng m ((-) wssv (-) glucan): t l sng c duy tr
mc trn 88,89% trong sut thi gian th nghim. L i chng dng ((+) wssv (-)
glucan): sau 4 ngy nhi m, t l sng bt u gim xung c n 96,67% v gim mnh
trong 3 ngy tip theo. n ngy th 7, t l sng ny ch cn 6,67%, v tm cht hon
ton vo ngy th 8. L th nghim ((+) wssv (+) glucan): t l sng bt u gi m vo
ngy th 5 (cn 75%) v cng gim mnh vo cc ngy sau . n ngy th 8, t l
sng ca l th nghim c n 12,5%. Cc kt qu ny cng gn ging nh t th
nghim, mc d, c 2 l (l i chng dng v l th nghi m) c t l sng u gim,
nhng t l sng ca l th nghim lun duy tr mc cao hn l i chng dng. c
bit l ngy th 8, t l sng l i chng dng l 0%, trong khi l th nghi m vn
duy tr mc 12,5% (hnh 6)
Tng huyt bo
Tng huyt bo t 1: L i chng ((-) wssv (-) glucan): tng huyt bo c duy
tr mc 14,95 - 15,29 x 106 huyt bo/ml trong sut thi gian th nghim. L i
chng m ((+) wssv (-) glucan): tng huyt bo gim vo ngy th 3 (9,03 x 106 huyt
bo/ml), n ngy th 6 ch cn 1,3 x 106 huyt bo/ml). L th nghi m ((+) wssv (+)
glucan): tng huyt bo gim cng vo ngy th 3 (cn 14,45 x 106 huyt bo/ml),
gim mnh ngy th 4 (ch cn 2,85 x 106 huyt bo/ml). Tuy nhin, t ngy th 5
n ngy th 8, tng huyt bo vn duy tr c mc t 5,82 - 7,83 x 106 huyt
bo/ml (hnh 7).
Tng huyt bo t 2: L i chng m (( -) wssv (-) glucan): tng huyt bo c
duy tr mc 13,98 - 14,23 x 106 huyt bo/ml trong sut thi gian th nghim. L i
chng dng ((+) wssv (-) glucan): tng huyt bo bt u gim t ngy th 2 (cn
12,67 x 106 huyt bo/ml), gim mnh ngy th 4 (cn 4,92 x 106 huyt bo/ml ) v
ch cn 2,36 x 106 huyt bo/ml ngy th 6. L th nghim ((+) wssv (+) glucan):
tng huyt bo cng gim vo ngy th 2 (11,87 x 106 huyt bo/ml), tip tc gim
mnh n ngy th 4 (ch cn 2,58 x 106 huyt bo/ml). tuy nhin, t ngy th 4 n
ngy th 7, tng huyt bo vn duy tr c mc t 4,71 - 6,89 x 106 huyt bo/ml.
Nh vy, tng lng haemocyte trong huyt t ng tm, qua 2 t th nghim, ch ti u
ny nhm cho n ch phm beta-glucan ((+) wssv (+) glucan) mc d c chiu hng
TLESONG SOT t I
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8
Ngay th nghi em
T

l
e

s
o
n
g

s
o
t

(
%
)
(-) WSSV (-) GLUCAN
(+) WSSV (-) GLUCAN
(+) WSSV (+) GLUCAN
TLESONG SOT t I I
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8
Ngay t h nghi em
T

l
e

s
o
n
g

s
o
t

(
%
)
(-) WSSV (-) GLUCAN
(+) WSSV (-) GLUCAN
(+) WSSV (+) GLUCAN
gim, nhng vn n nh v c duy tr mc cao hn nhm khng n ch phm beta-
glucan ((+) wssv (-) glucan) (hnh 8).
Hnh 7: th bin thin haemocyte t 1 Hnh 8: th bin thin haemocyte t 2
Hm lng protein trong huyt t ng
Bin thin hm lng protein t 1: L i chng m (( -) wssv (-) glucan): tng
hm lng protein trong huyt t ng duy tr mc n nh trong sut thi gian th
nghim (t 21,03 26,95 mg/ml). L i chng dng ((+) wssv (-) glucan): tng hm
lng protein trong huyt t ng bt u gim vo ngy th 3 (cn 20,63 mg/ml) v n
ngy th 6 ch cn 7,67 mg/ml. L th nghi m ((+) wssv (+) glucan): tng hm lng
protein trong huyt tng cng bt u gim t ngy th 3 (cn 15,76 mg/ml) v ti p
tc gim nhng ngy sau , n ngy th 8 ch cn 6,78 mg/ml. Tng hm lng
protein trong huyt tng ca l th nghim gim t hn v lun duy tr mc cao hn
tng hm lng protein trong huyt t ng ca l i chng dng (hnh 9).
Hnh 9: Bin thin hm lng protein t 1 Hnh 10: Bin thin hm lng protein t 2
Bin thin hm lng protein trong huyt t ng t 2: L i chng m ((-) wssv
(-) glucan): tng hm lng protein trong huyt t ng lun duy tr mc n nh trong
sut thi gian th nghim (t 23,04 24,21 mg/ml). L i chng dng ((+) wssv (-)
glucan): tng hm lng protein trong huyt tng bt u gim vo ngy th 4 (cn 19,64
mg/ml), tip tc gim nhng ngy tip theo v n ngy th 6 ch cn 9,56 mg/ml. L th
nghim ((+) wssv (+) glucan): t ng hm lng protein trong huyt t ng cng bt u
OTHBI EN THI EN LNG HAEMOCYTE THEO
THI GI AN t I
0
2
4
6
8
10
12
14
16
18
1 2 3 4 5 6 7 8
Ngay th nghiem
L
n
g

h
a
e
m
o
c
y
t
e
(
x
1
0
6

t
e

b
a
o
/
m
l
)
(-) WSSV (-)
GLUCAN
(+) WSSV (-)
GLUCAN
(+) WSSV (+)
GLUCAN
OT HBI EN T HI EN L NG HAEM OCYT E
T HEO T HI GI AN t I I
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7
Ngay t h nghi em
L
n
g

h
a
e
m
o
c
y
t
e
(
x
1
0
6

t
e

b
a
o
/

m
l
)
(-) WSSV (-) GLUCAN
(+) WSSV (-) GLUCAN
(+) WSSV (+) GLUCAN
BI EN THI EN HAM LNG PROTEI N HUYET
TNG THEO THI GI AN t I
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9
Ngay th nghi em
H
a
m

l
n
g

p
r
o
t
e
i
n
h
u
y
e
t

t
n
g

(
m
g
/
m
l
)
(-) WSSV (-)
GLUCAN
(+) WSSV (-)
GLUCAN
(+) WSSV (+)
GLUCAN
BI EN THI EN HAM LNG PROTEI N HUYET
TNG THEO THI GI AN t I I
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9
Ngay th nghi em
H
a
m

l
n
g

p
r
o
t
e
i
n
h
u
y
e
t

t
n
g

(
m
g
/
m
l
)
(-) WSSV (-)
GLUCAN
(+) WSSV (-)
GLUCAN
(+) WSSV (+)
GLUCAN
gim t ngy th 2 (cn 18,18 mg/ml) v dao ng tht thng nhng ngy tip theo,
nhng c khuynh hng cao hn l i chng dng. Nh vy, tng hm lng protein
trong huyt tng tm: qua 2 t th nghim, ch ti u ny nhm cho n ch phm
beta-glucan ((+) wssv (+) glucan) mc d c chiu hng gim, c lc dao ng cao
thp tht thng nhng vn duy tr mc cao hn nhm khng n ch phm beta-
glucan ((+) wssv (-) glucan) (hnh 10).
5. Chun on v pht hin virus WSSV bng phng php PCR v trn SV-kit.
Chun on v pht hin virus WSSV bng phng php PCR
Hnh 11. Kt qu phng php pht hi n virus wssv trn tm bng PCR; ging pos: mu
i chng dng, ging neg: mu i chng m ging 1: ( -) wssv (-) glucan, ging 3: (+)
wssv (-) glucan, ging 4: (+) wssv (+) glucan
Kt qu cho thy, Beta-glucan lm tng cng tnh min dch cho h thng ph ng v
tm. Cn l i chng dng, s gia tng huyt bo v cc protein min dch khng
b p ni s tiu huyt (v t bo haemocyte) do virus ti n hnh t nhn bn da trn
c s vt cht v nguyn liu ca t bo ny qu nhanh, khng c s h tr ca Beta-
glucan t ban u.
Chun on v pht hin virus WSSV trn SV-kit.
Hnh 12: Kt qu pht hin virus wssv tr n tm bng sv - kit
Mu i chng m (-) wssv (-) glucan m tnh. Mu i chng dng ((+) wssv (-)
glucan) v mu th nghim ((+) wssv (+) glucan) u cho kt qu d ng tnh. Da vo
cng mu trn mng nitrocellulose (hnh 12), chng ti k t lun mc nhim
bnh ca 2 l i chng dng (+) wssv (-) glucan v mu th nghim (+) wssv (+)
glucan l nh nhau. iu ny l hon ton ph hp vi phng php cm nhim. Cng
nh phng php chn on PCR bn trn, kt qu ca phng php ny khng c gi tr
nh gi tnh tng cng h min dch tm ca beta-glucan khi cm nhim virus vi
Bng c
hiu wssv
364 bp
mt liu lng cp tnh, m ch c gi tr nh gi phng php cm nhim l thnh cng.
nh gi xc ng tnh tng cng h min dch tm ca beta -glucan mt liu
lng cp tnh, chng ti da vo cc ch tiu sinh l mu v t l sng st ca tm trong
mt khong thi gian nht nh nh phn kt qu v bin lun trnh by.
IV. KT LUN
Ch phm beta-glucan iu ch t nm men bnh m saccharomyces cerevisiae c
hiu qu i vi vic tng cng h min dch v duy tr thi gian sng ti mt khong
thi gian nht nh ca tm s penaeus monodon liu gy nhim cp tnh. Do , mc
tiu ca ti l kho st kh nng tng cng kch thch min dch tr n tm s penaeus
monodon ca ch phm beta-glucan i vi virus gy hi chng m trng (wssv). Mc
tiu ny c bn thnh cng quy m phng th nghim.
Tm s dng cc h thng khc nhau chng li nhng tc ng do mi tr ng bt
li (d nhim bnh). Do cc bnh d ly nhim v cht lng nc xung quanh phn
nh thng qua huyt tng ca tm, cho nn cc thng s huyt tng (nh tng lng
huyt bo lu thng trong h tun hon, tng hm lng protein trong huyt t ng tm
ti mt thi im nht nh, cc ch ti u ny c bit c lin quan n cc thng s v
sinh l, mi trng v cc yu t c th gy sc cho tm) l mt thng s nhy v c
ngha nht nh i vi vic nh gi mm bnh v cc yu t gy sc t mi trng.
TI LIU THAM KHO
1. Vn Th Hnh (2001). Nghin cu s pht trin ca mt s Baculovirus trong t
bo cn trng nui cy in vitro v kh nng ng dng trong sn xut thuc tr su
sinh hc v kim sot virus gy bnh tr n tm, Lun n Tin s sinh hc, Vin Sinh
hc Nhit i, Tp. H Ch Minh.
2. Phm Vn Tnh (1996). K thut nui tm s, NXB Nng Nghip, trang 5 - 9.
3. Tr Thanh Tho (2003). Chn on bnh m trng tr n tm s (penaeus monodon)
bng k thut non-stop single-tube semi-nested PCR (Kiatpathomchai, 2001), Kha
lun tt nghip, Trng i hc Khoa hc T nhin TPHCM.
4. Nguyn Th Phng (2005). Nghin cu v phn tch mt s thnh phn
carbohydrate v phng php ph h y vch t bo nm men, Kha lun tt nghip,
Trng i hc Khoa hc T nhin.
5. Walker GM (1998). Yeast physiology and Biotechnology, Wiley, Chichester.
6. Reimund S., (2003). A new non-degrading isolation process for 1,3-b-D-glucan of
high purity from bakers yeast Saccharomyces cerevisiae, Switzerland.
7. Suphantharika M, Khuntae P, Thanardkit P, Verduyn C, (2003). Preparation of
spent brewers yeast -glucans with a potential applicat ion as an immunostimulant
for black tiger shrimp, Penaeus monodon. Bioresourse Technology, p. 88, 55-60.
8. Thanardkit P, Khunrae P, Suphantharika M and Verduyn C, (2002). Glucan from
spent brewers yeast: preparation, analysis and use as a potential immunost imulant
in shrimp feed, World Journal of Microbiology & Biotechnology, p. 18, 527 -539.
9. Sugimoto, (1976). Process for Autolysis of Yeast , U.S Patent 3,961,080.
10. Kado (2000). Process for producing yeast extract , U.S Patent 6,051,212.
11. Raa, J., (2000). The use of immune-stimulants in fish and shellfish feeds ,
University of Tromso, Norway.
12. T.W.Flegel (1998). Advances in Shrimp Biotechnology. Proceedings to the Special
Session on Shrimp Biotechnology, 5
th
Asian Fisheries Forum, Chiengmai,
Thailand.
13. Chang CF et al., (1999). Effect of dietary beta-1,3-glucan on resistance to white
spot syndrome virus (WSSV) in postlarval and juvenile Penaeus monodon ,
FishAquat Organ; 36; 163-8.
14. Cheng FC, Su MS, Chen HY, (1999). A rapid method to quantify total haemocyte
count of Penaeus monodon using ATP analysis , Fish Pathol.
15. Karin Van De Braak, (2002). Haemocytic defence in black tiger shrimp (Penaeus
monodon), Wagening University, Netherland.
16. Hazel Wade, Vinh Viet Nguyen, Hue Thu Nguyen, (2002). Preliminary Overview
of Shrimp Aquaculture in Vietnam.
17. Hong Phuoc Le, (2001). Haemocyte reactions against microorganisms in Black
Tiger Shrimp (Penaeus monodon).
18. Vetvicka V., Sima P, (2004). -Glucan in invertebrate, ISJ 1: 60-65.
SUMMARY
Investigation of immunostimulation of beta -glucan
for tiger shrimp penaeus monodon against virus WSSV
Vo Long Thuan, Nguyen Duy Long, Hoang Quoc Khanh, Van Thi Ha nh
Beta-glucan was found in yeast cell as a natural immunostimulants to reduce stress
and mortalities, to mai ntain good health of cultured organisms and to stimulate the non -
specific defence mechanism, is becoming increasingly important in aquaculture. In this
study, we investigate effect of beta -glucan to reducing susceptibility disease and
infection of WSSV virus to tiger shrimp Penaeus monodon. A SV-kit and PCR analysis
system are proceeded to determine cell infected by WSSV virus and the blood cells are
also investigate and counting during the treatment with beta -glucan. The results show
on treatments and supplies by beta-glucan into experimental design tanks by feed
increased survival percentage at 7
th
-days treatment with highest level is 21.2%.
CHN GING NM MEN Rhodotorula C KH NNG
PHT TRIN CNG VI MC Monascus sp TRN MI
TRNG GO TM THEO PHNG PHP NUI CY
B MT
Nguyn Th Minh Nguyt,
H Cng nghip
ng Th Anh o,
H Bch khoa TPHCM
Nguyn Hu Phc
Vin Sinh hc Nhit i
M U
Nm men Rhodotorula cn c gi l vi sinh vt sinh sc t carotenoid
(carotengensis). Cc sc t carotenoid chnh trong nm men Rhodotorula l: |-carotene,
torularhodin v torulene [2]. c nhiu cng trnh nghin cu v kh nng sinh tng
hp sc t carotenoid tr n nhiu cc ngun c cht khc nhau nhng ch yu l theo
phng php nui cy dch th [1], [3], [4], [6], [7]
Mc Monascus cn gi mc go t rt lu c dng lm cht mu hay
ph gia thc phm. Hin ti c hn 50 patent c lin quan n vic s dng sc t
Monascus vo thc phm c cng nhn ti Nht Bn, M, Php v c. [5] Cc
sc t c tit ra t Monacus l hn hp ca mu , cam v vng thng c dng
chung m khng cn tch ring ra tng loi. Khi t bo trng thnh, vch t bo
chuyn sang mu nu lm nh hng n cht lng ca sc t thu c [8].
tng hiu qu s dng tinh bt ca nm men Rhodotorula khi nui trn mi
trng go tm ng thi tng hm lng sc t carotenoid tit ra canh tr ng nui cy
mt cch t nhin khng dng cc tc ng c hc ph v thnh t bo nm men, ln
u tin chng ti thnh cng tin hnh nui hn hp nm men Rhodotorula v nm
mc Monascus sp. .
VT LIU V PHNG PHP
Vt liu, ha cht v mi trng:
Ging vi sinh vt: cc chng nm men Rhodotorula do chng ti phn l p v nh
danh gm: Rh.graminis (MN5), Rh.glutinis HUI-1(MN10) Rh.glutinis HUI-2
(MN17) v 4 ging cha nh danh n loi l Rhodotorula sp1(MN1),
Rhodotorula sp.3 (MN12), Rhodotorula sp.4 (MN16). Ging mc Monascus
sp.(As6) nhn t Vin Sinh hc Nhit i - Vin Khoa hc min Nam.
Go tm: go tm th phm mua t c s cung cp hng nng sn thc phm s
28- Nguyn Th Nh - ch Bnh Ty.
Thnh phn mi trng nui b mt gm :
- Ngun nitrogen: bt u nnh.
- Ngun carbohydarte b sung : du cooking oil T ng An.
- Ngun phospho gm: KH
2
PO
4
, K
2
HPO
4
, Na
2
HPO
4
.
- Ngun khong v vitamin gm: MgSO
4
.7H
2
O, MnSO
4
.H
2
O,
CaCl
2
.2H
2
O, CaCO
3
, CuSO
4
.5H
2
O, ZnSO
4
.7H
2
O, FeSO
4
.H
2
O, cao nm men.
Gi cc ging men Rhodotorula trn mi trng thch malt, mc Monascus sp. trn
mi trng PGA, nhit 8
0
C v cy chuyn nh k sau hai tun.
Nui cy b mt: theo quy m phng th nghim trong iu kin v tr ng, c tin
hnh trn hai mc, hot ha ging trn my lc v nui b mt trong cc khay nha
PP chu nhit dung tch 700ml. Nguyn liu go tm sau khi ngm 24gi c h
ha cho vo cc khay, b sung dinh dng iu chnh v pH 5,3 ri mang i thanh
trng nhit 100
0
C trong 30 pht. Cy ging vo cc khay nha v t phng
c nhit trung bnh 28 1
0
C, m khng kh 38-40%. m mi trng cho
nui cy b mt l 60-65%.
Cc ho cht, my mc, thit b chuyn dng trong phn tch ha sinh, vi sinh thu c
phng th nghim Khoa Cng ngh Thc phm v Sinh hc - Trng i hc Cng
nghip Thnh ph H Ch Minh.
Phng php:
Kho st t l ging men theo phng php m khun lc CFU, t l mc theo
phng php m bo t trn bung m hng cu. Dng ng chun v o mt
quang xc nh t l ging s dng. i vi nm men chng ti s dng
trung bnh: 10
7
-10
8
CFU/g nguyn liu khi nui c lp men. Khi nui hn hp t l
men 5.10
6
- 5.10
7
CFU/g v bo t mc trung bnh l 5.10
6
-5.10
7
/g.
nh tnh hot amylase theo phng php khuch tn trn a thch. Dng mi
trng Czapeck agar trong thay glucose bng tinh bt tan 1%, kh trng mi trng
1 atm trong 30 pht, vo cc a Petri cy cc chng vi sinh vt nghi n cu.
nhit phng trong thi gian 3 ngy i vi mc v 10 ngy i vi cc chng nm
men, cho vo a 1ml dung dch lugol 1%. Quan st vng phn gii tinh bt.
Xc nh cc chng ging pht trin nhanh tr n mi trng go tm c b sung
dinh dng, o hm lng protein xc nh sau khi pht hin mu bng thuc th
Foling ca Lowry (O. H. Lowry et al., 1951).
Xc nh hot alpha-amylase bng cch pht hin mu vi Ido v so mu bc
sng 620nm theo phng php Smith&Roe.
Xc nh hot protease theo phng php ca Anson ci tin.
Xc nh hm lng tinh bt ca nguyn liu v tinh bt st theo phng php kt
ta bng cn 96
0
.
Xc nh hm lng m tng theo phng php Kjeldahl vi h thng chng ct
m t ng bng h chun H
3
BO
3
-H
2
SO
4
.
Xc nh m bng phng php sy kh nhit 105
0
C n khi lng
khng i.
KT QU V THO LUN
1. nh tnh hot tnh amylase ca cc chng nghi n cu:
Sau khi tin hnh th nghim nh trnh by phn phng php, chng ti thu
c kt qu nh hnh 1.
Men Rhodotorula cho phn ng dng tnh khi nh lugol vo thch a nhng hot
amylase rt yu ch hnh thnh mt vng phn gii tinh bt rt nh bao ly khun lc nm
men. Trong khi mc Monascus sp. c vng phn gii tinh bt rt ln ln n 29,3mm.
2. Kh nng pht trin trn mi trng go tm khi nui hn hp men v mc
Cy men v mc vo mi trng go tm b sung dinh dng, vo cng mt lc.
nh k sau 24 gi ly mu xc nh gin tip hm lng protein ha tan bng phng
php Lowry. Kt qu thu c nh bng 1.
Bng 1: Hm lng protein ha tan khi nui hn hp tng chng men vi mc.
Hm lng proetin ha tan (mg/g) Thi gian
(ngy)
MN1-As6 MN5-As6 MN10-As6 MN12-As6 MN16-As6 MN17-As6
1 2,705 2,163 3,918 4,115 2,324 2,027
2 2,661 4,105 6,172 3,902 3,902 2,262
3 9,485 15,299 7,997 5,703 5,703 3,283
4 11,874 17,612 11,183 9,826 7,826 3,749
5 8,973 10,271 13,386 11,735 6,735 4,591
6 5,792 7,485 8,706 12,643 5,049 5,370
7 3,952 6,013 6,313 5,477 4,996 3,717
8 2,826 4,967 5,083 5,049 4,502 3,046
Hnh 1a: Vng phn gi i tinh bt Hnh 1b: Vng phn gii tinh bt ca
menRhodotorula graminis. ca mc Monascus sp.
0.000
2.000
4.000
6.000
8.000
10.000
12.000
14.000
16.000
18.000
20.000
1 2 3 4 5 6 7 8
Thi gian nui (ngy)
H
m

l
n
g

p
r
o
t
e
i
n

h
a

t
a
n
(
m
g
/
g

n
g
u
y
n

l
i
u
)
MN1- As6
MN5- As6
MN10 - As6
MN12- As6
MN16- As6
MN17- As6
Hnh 2: So snh hm l ng protein ha tan hnh thnh trong thi gian nui hn hp
tng chng men Rhodotorula vi mc Monascus sp.
Hnh 2 cho thy MN5-As6 pht trin chung vi nhau tt nht hm lng protein
tng ln hn (t 17,61mg/g ) ch trong thi gian 4 ngy. Trong khi nui c lp cc
men a s cc chng u t hm lng protein cc i sau 6-7 ngy nui cy.
3. Xc nh hot amylase, protease trong canh tr ng khi nui c lp cc chng men
trn mi trng go tm.
xc nh hot amylase, protease trong thi gian nui cy, chng ti ch kho
st trn 4 chng men cho kt qu nui cy hn hp hiu qu, tc tin hnh kho st trn
cc mu th nghim gm: MN1-As6, MN5-As6, MN10-As6 v MN12-As6. Kt qu thu
c nh bng 2.
Bng 2: Hot amylase, protease khi nui phi hp men -mc theo thi gian.
MN1-As6 MN5-As6 MN10-As6 MN12-As6 Thi
gian
(ngy)
Amylase
(UI/g)
Protease
(UI/g)
Amylase
(UI/g)
Protease
(UI/g)
Amylase
(UI/g)
Protease
(UI/g)
Amylase
(UI/g)
Protease
(UI/g)
1 0,0005 0,1112 0,0004 0,0848 0,0001 0,0576 0,0002 0,1324
2 0,0030 0,3425 0,0007 0,6672 0,0058 0,1340 0,0042 0,5473
3 0,0052 0,6742 0,0091 1,3168 0,0068 0,6742 0,0055 0,9647
4 0,0067 0,9640 0,0072 1,8480 0,0043 1,2678 0,0059 1,1240
5 0,0042 1,0340 0,0057 1,0000 0,0042 0,8640 0,0037 0,8930
6 0,0027 0,7813 0,0036 0,7712 0,0023 0,5378 0,0033 0,7624
7 0,0010 0,6497 0,0012 0,5872 0,0013 0,5034 0,0025 0,6724
8 0,0007 0,5647 0,0009 0,5134 0,0005 0,4397 0,0018 0,4034
0
0.002
0.004
0.006
0.008
0.01
1 2 3 4 5 6 7 8
Thi gian (ngy)
H
o

a
m
y
l
a
s
e

(
U
I
/
g
)
M1-As6
M5-As6
M10-As6
M12-As6
th trn cho thy khi nui hn hp MN5 v As6 cng cho hot amylase v
protease cng cao nht nhng thi gian t gi tr cc i c khc nhau. Hot lc
amylase v protease trong canh trng ln lt t 0,0091 v 1,8480 (UI/g). Kt qu thu
c ph hp vi c s l thuyt v bn cht ca cc enzyme l protein nn khi enzyme
tit ra mi trng nhiu th hm lng protein ha tan xc nh c cng s cao theo.
4. Xc nh hm lng tinh bt ca nguyn liu trc khi cy ging vo v so snh hm
lng tinh bt st sau thi gian nui cy c lp men MN5 v nui hn hp MN5 -As6.
Bng 3: Kt qu phn tch hm lng tinh bt.
S TT Mu th nghim Hm lng tinh bt (%)
1 Go tm nguyn liu 62,76
2 Go tm sau khi ngm 24 gi 54,58
3 Go tm sau khi h ha v thanh trng 27,35
4 Go tm nui men MN5 sau 7 ngy 13,71
5 Go tm nui hn hp MN5- As6 sau 7 ngy 4,17
Kt qu bng 3 cho thy qua giai on ngm, h ha v thanh trng nhit go tm,
mt phn tinh bt b thy phn thnh cc hp cht n gin hn cung cp cho hot
ng trao i cht ca nm men, tuy nhi n lng tinh bt st khi nui men c lp kh
cao. Ngc li nui hn hp men MN5 vi mc Monascus sp. sau 7 ngy nui cy
lng tinh bt st ch cn 4,17%.
5. Kho st s thay i m v pH theo thi gian nui cy
Sau mi ngy nui cy men hn hp MN5-As6, chng ti tin hnh xc nh s
thay i m v pH v thu c kt qu nh bng 4.
Bng 4: S thay i pH v m theo thi gian khi nui hn hp MN5 -As6.
Ngy 1 2 3 4 5 6 7 8
pH 6,7 7,24 6,12 6,81 5,16 5,14 4,48 4,04
m 65 65,74 67,38 68,45 69,19 69,35 70,12 70,68
0.0000
0.5000
1.0000
1.5000
2.0000
1 2 3 4 5 6 7 8
Thi gi an (ngy)
H
o

p
r
o
t
e
a
s
e
(
U
I
/
g
)
MN1 -As6
MN 5-As6
MN 10-As6
MN12 -As6
pH ban u l 5,5 sau 2 ngy nui cy pH tng mnh nhng sau pH gim dn v
pha acid. Gii thch iu ny do trong thi gian nui Monascus thng lm mi trng
chuyn sang acid yu. Ngc li m ca mi tr ng tng dn theo thi gian l do
hm lng nc to thnh tch t trong khay ln men lm m tng ln.
6. Kt qu phn tch hm lng protein tng v lipid tng
Mu nui hn hp MN5-As6 sau thi gian 7 ngy chng ti tin hnh xc nh hm
lng protein tng, lipid tng, tinh bt, ng st ca mu nui men MN5 v mu nui
hn hp men-mc. Kt qu thu c nh bng 5.
Bng 5: Thnh phn mi trng sau 7 ngy nui cy ca MN5 v MN5-As6.
Vi sinh vt nui cy Protein th (%) Lipid tng (%) Tinh bt (%)
Ch nui men MN5 19,46 36,63 13,71
Hn hp MN5-As6 23,37 55,13 4,17
KT LUN
Tin hnh nui cy hn hp tng chng men Rhodotorula vi mc Monascus
sp. theo phng php b mt chng ti chn c nm men Rhodotorula graminis
(MN5) cho kt qu nui cy hn hp vi mc Monascus sp. l cao nht. Vi t l ging
1:1 sau 7 ngy nui cy hn hp men MN5 v mc Monascus sp. trn mi trng go
tm qua h ha c b sung dinh dng, chng ti thu c kt qu nh sau:
Protein tng : 23,37%.
Lipid tng : 55,13%.
Tinh bt : 4,17%.
Kt qu trn gp phn lm tng ng k gi tr dinh dng ca go tm dng trong
chn nui v c bit l tng cht mu carotenoid lm ngun thc n phc v cho
chn nui gia cm trng.
TI LIU THAM KHO
1. Phan T Anh - "Sinh tng hp sinh khi Rhodotorula gi u Protein-Carotenoid"-
Bo co tng kt nghim thu ti chng trnh vn m sng to KHKT tr- S
Khoa hc Cng ngh- Thnh on Tp. HCM- Nm 2003.
2. Bhosale P., Jogdant V.V, Grade R.V. - "Stability of |-carotene in spray dried
preparation of Rhodotorula glutinis mutant 32"- Journal of Applied Micr obiology,
September 2003, Vol.95 (3), pp.584.
3. Buzzini Pietro, Martini Alessandro "Production of carotenoids by strains of
Rhodotorula glutinis cultured in raw materials of agro -industrial origin"-
Bioresource technology 71 (1999) 41-44.
4. Buzzini Pietro -"Batch and fed batch carotenoid production by Rhodotorula glutinis
-Debaryomyces castellii co-cultures in corn syrup"-Journal of Applied
Microbiology 2001, 90, pp.843-847.
5. Dufoss L. -Microbiol production of food grade pigments - Food Technol.
Biotechnol. Vol. 44(3), 2006, pp.313-321.
6. Frengova Ginka, Emilina Simova, Dora Beshkova - "Use of Whey untrafiltrate as a
substrate for production of carotenoid by the yeast Rhodotorula rubra"- World
Journal of Microbiology and Biotechnology, Applied Biochemistry and
Biotechnology, March 2004, Vol. 112, Issue 3, pp.133 -142.
7. Frengova Ginka, Emilina Simova, Dora Beshkova - "Beta-carotene-rich carotenoid-
protein preparation and exopolysaccharide production by Rhodotorula rubra GED
8 grown a yogurt starter culture" - Journal of Industry Microbiology and
Biotechnology, Applied Biochemistry and Biotechnology, ISSN 1367-5435, 2006,
Vol. 2, pp. 572-577.
8. Iturriaga E. A., Papp T., Breum J., Arnau J., Eslava A.P. - Strain and culture
condition improvement for |-carotene production with Mucor. In: methods in
biotechnology: Microbial processes and products - J. L. Barredo (Ed), USA (2005),
Vol. 18, pp. 239-256.
SUMARY
High nutrition production by Rhodotorula yeast co -cultivated
with Monacus sp. Mold on the broken grain of ordinary rice
by solid state method
Nguyen Thi Minh Nguyet(1), ong Thi Anh o (2), Nguyen Huu Phuc(3)
(1)Univ. Industry (2)Univ Technology (3)Institute of Tropical Biology
Broken grain of ordinary rice, obtained from the poultry feed agent, were soaked
overnight at room temperature, acidified to pH 4.0 with chloric acid. The broken grain
of ordinary rice were cooked for 20 minutes, at 95
0
C in water also at pH 4.0. Cooked
rice were added mineral and nutrition. Mixing followed and devided into each of 700ml
P.P. box. Media were sterilized by autoclaving at 121
0
C for 20 min. The cooked rice
were inoculated with yeast suspension of Rhodotorula and spore suspension of
Monascus sp. The suspension was prepared by growing on glucose 4% in 10ml tubes at
30
0
C for 24 hours and contained approximately 5.10
6
-5.10
7
mould spores/g and 5.10
6
-
5.10
7
CFU/g. Incubated conditions were: pH media = 5.3, moisture 65%, temperature
281
0
C. Depending on the solute protein content were obtained as co -culturing every
Rhodotorula species and Monascus sp., we chose Rhodotorula graminis (MN5) has the
best grown with Monacus sp.(the maximum protein content was 17,612 mg/g media).
After 7 days co-culture, nutrient components of solid state media were increased
worthily (total protein 23.37% , t otal lipid 55.23% and starch 4.17%). The aim of this
research was to increase the nutrition and carotenoid pigments of broken grain of
ordinary rice, as they are produced by co -culturing Rhodotorula species and Monascus
sp. It is necessary for poultry feed in order to increase the pigment of egg.
NGHIN CU CC C IM SINH HC V KH
NNG PHT TRIN TRN MI TRNG BN RN
CA MT S GING NM MEN Rhodotorula PHN LP
TI VIT NAM
Nguyn Th Minh Nguyt,
H Cng nghip Tp.HCM
ng Th Anh o,
H Bch khoa Tp.HCM
Nguyn Hu Phc
Vin Sinh hc Nhit i.
M U
Nm men Rhodotorula l mt trong rt t cc chi nm men c kh nng tng hp
tch lu mt lng ln cc sc t carotenoid trong ch yu l -carotene, torulene,
torularhodin.[7], [14] Ti cc nc c nn sinh hc pht trin nm men Rhodotorula
c nghin cu nhiu v c s dng nh mt ngun cung cao cht mu thc
phm an ton, [9] sinh khi nm men c gi tr dinh dng cao dng trong chn
nui.[7], [8], [15], [16] Nhng kt qu nghin cu cho thy ti Vit Nam rt ph bin
chi nm men Rhodotorula. Mt s chng c nghin cu v kh nng to sinh khi,
tch lu carotenoid, cht bo c bit l cc acid bo khng no c gi tr sinh hc cao
trn cc ph ph phm ch bin nng sn thc phm. [1], [3]
Vi xu th cng hng vo cc loi thc n t nhin c ngun gc sinh hc thay th cc
cht b sung c ngun gc tng hp ha hc nn vic phn lp v tuyn chn cc ging nm
men t t nhin c kh nng thch nghi vi iu kin sn xut cng nghip thc n gia sc
trn cc ngun nguyn liu th l mt hng nghin cu ng quan tm.
Mc ch ca chng ti l tm ra cc chng nm men Rhodotorula c kh nng
pht trin theo phng php nui cy b mt trn tng loi ph ph phm hu c nht
nh, c bit l cc ph phm ca ngnh cng ngh thc phm nhm nng cao cht
lng thc n gia sc gp phn to nn mt nn nng nghip sch, bn vng.
Trong phm vi bi vit ny chng ti ch trnh by kt qu bc u ca cng tc
phn lp, tuyn chn nm men sinh sc t carotenoid thuc chi Rhodotorula c kh
nng nui cy theo phng php b mt thu sinh khi gi u sc t carotenoid.
NGUYN LIU V PHNG PHP
1. Nguyn liu, ha cht v mi trng
Cc vt liu dng trong phn lp v nui cy bn rn.
Ngun carbohydrate gm: glucose, saccharose, maltose, i mch, tinh bt, bt
ng, cm go, go tm, b sn, du thc vt.
Ngun nitrogen gm: bt c, bt u tng, bt lc, monosodium glutamate, ure,
(NH
4
)
2
SO
4
, pepton.
Ngun phospho gm : KH
2
PO
4
, K
2
HPO
4
, Na
2
HPO
4
.
Ngun khong v vitamin gm : MgSO
4
.7H
2
O, MnSO
4
.H
2
O, CaCl
2
.2H
2
O, CaCO
3
,
CuSO
4
.5H
2
O, ZnSO
4
.7H
2
O, FeSO
4
.H
2
O, cao nm men, cao tht.
Mu phn lp t t trng, b mt l cy, cc loi hoa, v tri cy ch yu ly t
Long An v mt s ti Thnh ph H Ch Minh, nc bin ti thnh ph Vng Tu.
Tng s mu phn lp l 64.
Mi trng phn lp:
* Mi trng A (YMPG: Yeast extract - Malt extract- Pepton - Glucose -agar).
* Mi trng B (YPG: Yeast extract - Pepton - Glucose-agar).
* Mi trng C (PGA: Potatoes - Glucose - agar).
Cc ho cht, my mc, thit b chuyn dng trong phn tch ha sinh, vi sinh thu c
phng th nghim Khoa Cng ngh Thc phm v Sinh hc, Trng i hc Cng
nghip Thnh ph H Ch Minh.
2. Phng php
- Phng php phn lp v gi ging: cc mu th nghim c phn lp bng
phng php Koch l n lt trn c ba mi trng A, B v C, nui cc nhit
cn thit v tip tc lm thun cng trn mi trng phn lp. Chn cc khun lc
to, trn c mu t vng, cam n , quan st di kinh hin vi c hnh thi ca
nm men (c nhn in hnh). Gi ging trn mi trng thch malt nhit 5
0
C.
Cy chuyn nh k sau 2 tun.
- Cc c im sinh l, sinh ha v nh loi nm men theo cc kho phn loi ca
Lodder et al. [4]
- Kho st kh nng s dng cc ngun carbohydrate, ngun nitrate v mt s cc
phn ng sinh ha: dng cc mu test ID 32 E ca hng BioMrieux
sa (Php) kt
hp vi cc th nghim kim chng tr n thch a v trong ng nghim.
- Nui cy b mt: theo quy m phng th nghim trong iu kin v tr ng, c tin
hnh trn hai mc, hot ha ging trn my lc v nui b mt trong cc khay nha
PP chu c nhit thanh trng.
- Khi chn nm men c kh nng pht trin tch ly sinh khi nhanh tr n mi trng
nghin cu, chng ti xc nh hm lng protein sau khi pht hin mu bng
thuc th Foling (O. H. Lowry et al., 1951) .
- Nguyn liu sau ln men qua lc th thu dch v ly tm vi tc 6.000 vng/ pht
trong 20 pht, tch sinh khi t bo nm men mang xc nh m quy v khi
lng sinh khi t bo kh thu c trong 1kg mi trng.
- Kho st t l ging theo phng php m khun lc CFU v o mt quang
OD. Ging c hot ho trn mi trng glucose 4% trn my lc ngang trong 24
gi. T l ging cy vo t 8.10
7
- 10.10
7
CFU/ml/ 100g mi trng.
- Xc nh hm lng protein tng theo phng php Micro-Kjeldalh.
- Xc nh hm lng cht bo theo phng php Soxlet.
- Cc th nghim c lp li 3 ln.
KT QU V THO LUN
1. c im sinh hc
T 64 mu phn lp, bc u chng ti tuyn chn c 17 chng nm men sinh
sc t t kem, hng cam n cam, trong c 8 chng c cc c im nh:
- Vt cy mu hng do sinh sc t carotenoid.
- Khng ln men cc loi ng.
- Khng ng ha Inositol.
- Sinh enzyme urease.
- Khng to thnh hp cht loi tinh bt.
Theo kha phn loi ca Lodder, chng ti chn c 8 chng thuc chi
Rhodotorula v kt qu m t hnh thi t bo, khun lc nh sau:
Bng1: M t c im t bo, khun lc ca 8 chng nm men thuc c hi Rhodotorula
c
im
Chng
Ngun
phn lp
Hnh
dng
t bo
M t
khun lc
Kch
thc
khun lc
(mm)
MN 1 t vn Hnh di Mu hng cam hay cam , b mt nhn, bng sng,
mp khng c rng ca, tm khun lc nh ln.
7 - 10
MN 5 V to Hnh trn Mu hng hay cam, to, trn b mt mn, nhn bng, mp
khng rng ca, dy .
10 - 20
MN 7 Nc
bin
Hnh trn
cu
Mu vng kem hay hng cam, trn, b mt kh mn, m
c, mp khng rng ca, khun lc dy tm.
5 15
MN 8 Ma Hnh trn Mu cam n cam, to trn, b mt gh gh, xp m,
mp khng c rng ca, tm khun lc dy c cc np
gp.
12 - 16
MN 10 L la Hnh di Mu hng cam hay , to, tr n nhn bng, nhiu nht,
mp khng c rng ca, khun lc tng i dy nh
nhau ti tm cng nh gn mp.
8 - 14
MN 12 L dm
bt
Hnh
trng
Mu cam hay hng cam, to trn, b mt kh nhn, mp
khng c rng ca, b dy trung bnh.
12 17
MN 16 V da
l
Hnh elip
ko di
Mu cam hay , to trn i khi gh gh, b mt hi kh,
xp nhiu np, mp khng c rng c a, khun lc mng .
8 - 13
MN 17 Hoa bt Hnh trn
cu
Mu cam, to hnh ovan, b mt kh, mn , mp khun lc
khng c rng ca, khun lc mng hi nh ln tm
7 -12
2. Kt qu phn ng sinh ha:
Bng 2: Kt qu mt s phn ng ha sinh thc hin trong ng nghim tr n thch a.
c
im
Chng
k hiu
L
n

m
e
n

c
c

l
o
n
g

I
n
o
s
i
t
o
l
H
n
h

t
h
n
h

a
c
i
d

a
c
e
t
i
c
P
h
n
g

D
B
B
D
-
S
a
c
c
h
a
r
o
s
e
D
-
M
a
l
t
o
s
e
D
-
G
l
u
c
o
s
e
D
-

G
a
l
a
c
t
o
s
e
L
-
A
r
a
b
i
n
o
s
e
M
e
l
e
b
i
o
s
e
R
a
f
i
n
o
s
e
T
i
n
h

b
t
T
h
u

p
h
n

u
r
e
N
i
t
r
a
t
MN 1
- - - + + + + + - - - - + +
MN 5
- - - + + + + + -/+ - + - + +
MN 7
- - - + + + + + - - + + + +
MN 8
- - - + - - + - - - - - + -
MN 10
- - - + + + + - - - -/+ - + +
MN 12
- - - + + + + - - - - - + -
MN 16
- - - + + - + - - - - - + -
MN 17
- - - + + + + +/- - - - - + +
Kt qu t b test ID 32 E v kt hp cc th nghim tin h nh trn a peptri (
bng 2) chng ti nh danh c n v xc nh c 4 loi l: (MN 5) Rh.
graminis, (MN7) Rh. ingeniosa , (MN 10) Rh. glutinis HUI-1, (MN 17) Rh. glutinis
HUI-2. Bn chng cha nh danh n loi chng ti k hiu l: Rhodotorula sp.1 (MN
1), Rhodotorula sp.2 (MN 8), Rhodotorula sp.3 (MN 12), Rhodotorula sp.4 (MN 16).
3. La chn chng c kh nng tch lu sinh khi nhanh trn mi trng go tm theo
phng php nui cy b mt.
T 8 chng phn lp c chng ti chn c 4 c kh nng tch lu sinh khi
nhanh trn go tm qua h ha l: Rhodotorula sp.1 (MN 1), (MN 5) Rh. graminis,
Rh. glutinis HUI-1 (MN 10), Rh. sp3 (MN 12). i chiu bng 2 cho thy a s u
c phn ng m tnh (-) trn mi trng tinh bt (tr MN 7), nhng khi nui cy b mt
trn go tm qua h ha bi mi trng acid cho thy nm men ny li pht trin
mnh. Kt qu ny c gii thch nh sau mt s vi sinh vt khng ng ha tinh bt
sng, nhng li pht trin tt trn tinh bt chn. Go tm khi h ha bng n c c pH
thp tinh bt go b thu phn mt phn thnh cc phn t n gin hn to thun li
cho hot ng trao i cht ca nm men. Da vo hnh 1 thy (MN 10) Rh. glutinis
HUI-1 c nng lc tch lu sinh khi tr n mi trng nghin cu vt cc chng khc ,
s cch bit so vi Rhodotorula sp.1 (MN 1), (MN 5) Rh. graminis, Rhodotorula sp.3
(MN 12) l khng nhiu nhng sinh khi t bo c sc t cam m. Chng ti chn
(MN 10) Rh. glutinis HUI-1 l chng dng cho cc nghin cu tip theo.
Hnh 1: So snh kh nng tch lu sinh khi ca 8 chng phn lp c trn mi
trng go tm qua h ho, 27 - 28
0
C v pH = 4,7
4. Xc nh khong pH thch hp ca chng (MN 10) Rh. glutinis HUI-1.
S dng chng (MN 10) Rh. glutinis HUI-1 c la chn, chng ti tin hnh chn
khong pH thch hp trn mi trng go tm qua h ha c b sung dinh dng.
Kt qu hnh 2 cho thy khi nui cy bn rn tr n go tm qua h ha c 4 chng
nm men nghin cu u pht trin mnh trong vng pH 4,5 - 5,5. Khi tng pH mi
trng ngay t thi im u nhn thy nm men pht trin chm, pha thch nghi ko
di, sinh khi t bo gim dn, ti pH 7,0 sinh khi t kh thu c ch t 6,5g/ 1kg
mi trng. Tin hnh lm th nghim ln dc trong vng pH t 4,5 - 5,5 chng ti nhn
c gi tr pH cc i l 5,3.
pH 5,3 nm men (MN 10) Rh. glutinis HUI-1 pht trin cc i, sinh khi c sc
t cam, kt qu ph hp vi nghin cu trc y ca nhiu tc gi. (Pca v
Votruba 1991, Prell et al. 1991, Sigler et al. 1983, Orndorff et al. US4677072).
Hnh 2: nh hng ca pH n kh nng tch lu sinh khi ca Rhodotorula sp.3
nui bn rn 28
0
C trn mi trng go tm h ha sau 7 ngy.
0
2
4
6
8
1
0
1
2
1
4
1
6
MN
1
MN
5
MN
7
MN
8
MN1
0
MN1
2
MN1
6
MN1
7
Chng
S
i
n
h

k
h
i

k
h

(
g
/
k
g
m
i

t
r
n
g
)
0
2
4
6
8
10
12
14
16
3.5 4.0 4.5 5 5.5 6.0 6.5 7.0
pH
S
i
n
h

k
h
i

k
h

(
g
/
k
g

m
i

t
r
n
g
)
0
2
4
6
8
10
12
14
16
0
50 100 150 200 250 300
Thi gian nui cy (gi)
S
K
K

g
/
k
g

m
i

t
r
n
g
Dry cell
Protein
5. Xc nh ng cong sinh trng (MN 10) Rh. glutinis HUI-1 pH = 5,3 ca chng
Rhodotorula sp.3 nui bn rn trn c cht l go tm qua h ha, nhit 27-28
0
C.
Tin hnh nui cy b mt (MN 10) Rh. glutinis HUI-1 27 - 28
0
C trn mi trng
go tm qua h ha c pH = 5.3 v khng iu chnh pH trong sut qu tr nh nui
cy. Sau 24 gi ly mu phn tch h m lng protein theo phng php Lowry ng
thi xc nh s thay i pH trong sut qu tr nh nui cy. Kt qu nh hnh 3:
Hnh 3:S hnh thnh sinh khi v protein ha tan ca Rhodotorula sp.3
28
0
C, pH = 5,3 trn mi trng tm go h ha sau 7 ngy.
Sinh khi thu c sy 60
0
C cho n khi lng khng i, xc nh hm lng
protein tng, v hm lng cht bo cho kt qu: Sau 7 ngy thu c 14,45g/kg mi
trng. Sinh khi kh cha 52,47g protein v 28,68 g/100g sinh khi kh.
KT LUN
Kt qu kho st iu kin v mi trng ln men bn rn l:
- Nhit nui: 27-28
0
C.
- m khng kh (%): 38 - 40.
- pH mi trng: 5,3
- m c cht: 65-67%
- T l ging: s dng lng cy ging trung bnh 8.10
7
- 10.10
7
CFU/ml/ 100g mi
trng cho sn lng sinh khi khng thua km g so vi khi s dng lng ging
nhiu hn (1.10
6
- 2.10
6
CFU/g mi trng).
- Thnh phn mi trng nui cy gm:
Go tm nu chn.
1% (khi lng) bt u nnh.
1% (ml/g) du n.
Khong cht (g/lit): NaNO
3
= 2,55, MgSO
4
.7H
2
O = 0,75, K
2
HPO
4
= 1,75,
FeSO
4
= 0,1, KCl = 1,5 cho thm nc ct vo 1 lt. S dng: 40 (% ml/g).
- Hm lng protein ha tan sau 7 ngy nui cy t cc i l: 2,53 g/kg. Sinh khi
kh t cc i l 14,45g/kg mi trng.
- Hm lng cht bo v protein tng sau 7 ngy nui cy ln lt l: 28,68% v
52,47 % cht kh.
- T kt qu thu c bc u chng ti nhn thy phng php nui cy bn rn
nm men Rhodotorula trn mi trng go tm qua h ha c th t hiu qu
cao. Cn nghin cu thm iu kin t phn nm men s dng ton b sn phm
thu c sau nui cy bn rn nhm l m tng hm lng beta-carotene gp phn
ci thin dinh dng cho gia sc, gia cm.
TI LIU THAM KHO
1. Phan T Anh - "Sinh tng hp sinh khi Rhodotorula gi u Protein-Carotenoid"-
Bo co tng kt nghim thu t i chng trnh vn m sng to KHKT tr - S
Khoa hc Cng ngh - Thnh on Tp.HCM - Nm 2003.
2. Nguyn Ln Dng v cng s -"Mt s phng php nghin cu vi sinh hc "-
NXB Khoa hc v K thut, H Ni 1986, - trang 324.
3. Nguyn c Lng, Nguyn Th Minh Nguyt - Ti u ha mi trng nui cy
Rhodotorula glutinis thu nhn sinh khi gi u protein- Tuyn tp cc cng trnh
khoa hc 45 nm trng i hc Bch Khoa H Ni 2001, trang 188 -194.
4. Barnett J. A., Payne R. W., Yarrow D. - "Yeast- characteristics and identification" -
Cambridge University Press, Cambridge - London - New York - Melbourne, 1990,
pp. 70-71.
5. Bhosale P., Jogdant V. V, Grade R. V.- "Stability of |-carotene in spray dried
preparation of Rhodotorula glutinis mutant 32"- Journal of Applied Microbiology,
September 2003, Vol.95 (3), pp.584.
6. Bong Kyum Kim, Pyoung Kyu Park, Hee Jeong Chae and Eui Yong Kim - Effect
of phenol on -carotene content in total carotenoids production in cultivation of
Rhodotorula glutinis- Korean J. Chem. Eng., 2004, 21(3), 689 -692.
7. Buzzini Pietro, Martini Alessandro "Production of carotenoids by strains of
Rhodotorula glutinis cultured in raw materials of agro -industrial origin"-
Bioresource technology 71 (1999) 41-44.
8. Buzzini Pietro -"Batch and fed batch carotenoid production by Rhodotorula glutinis
-Debaryomyces castellii co-cultures in corn syrup"-Journal of Applied
Microbiology 2001, 90, pp. 843-847.
9. Dufoss L.-Microbiol production of food grade pigments - Food Technol.
Biotechnol. Vol. 44(3), 2006, pp. 313-321.
10. Frengova G. I., Emilina S. D., Beshkova D. M.- "Formation of carotenoids by
Rhodotorula glutinis in whey ultrafitrate" - Journal of Applied Microbiology and
Biotechnology, Bulgarian Academic of Sciences, 1994.
11. Frengova G. I., Emilina S. D., Beshkova D. M.-"Carotenoid production by lactoso-
negative yeast co-cultivated with lactic acid bacteria in whey ultrafiltrate" - Journal
of Applied Microbiology, Institute of Microbiology, Bulgarian Academic of
Sciences, Bulgaria 2003, Vol. 58 (7-8), pp. 562-567.
12. Frengova Ginka, Emilina Simova, Dora Beshkova - "Use of Whey untrafiltrate as a
substrate for production of carotenoid by the yeast Rhodotorula rubra" - World
Journal of Microbiology and Biotechnology, Applied Biochemistry and
Biotechnology, March 2004, Vol. 112, Issue 3, pp.133 -142.
13. Frengova Ginka, Emilina Simova, Dora Beshkova - "Beta-carotene-rich carotenoid-
protein preparation and exopolysaccharide production by Rhodotorula rubra GED 8
grown a yogurt starter culture" - Journal of Industry Microbiology and
Biotechnology , Applied Biochemistry and Biotechnology, ISSN 1367-5435, 2006,
Vol.2, pp.572-577.
14. Hayman P. et al. - "Carotenoids bionsynthesisi in Rhodotorula glutinis" -Journal of
bacterionlogy, American Society for Mi crobiology. Printed in U.S.A 1974,
Vol.120, No.3, pp.1339-1343.
15. Iturriaga E. A., Papp T., Breum J., Arnau J., Eslava A.P. - Strain and culture
condition improvement for |-carotene production with Mucor. In: methods in
biotechnology:Microbial processes and products- J. L. Barredo (Ed), USA (2005),
Vol. 18, pp. 239-256.
16. Jacob Z.-"Enrichment of wheat bran by Rhodotorula gracilis through solid -state
fermentation"- Folia Microbiol (Praha), 1991, Vol.36 (1), pp. 86 -91.
17. Vani Saxena C. D., Sharma C. D., Bhagat S. D., Saini V. S. and Adhikari D. K.-
"Lipid and fatty acid biosynthesis by Rhodotorula" -JAOCS Abstracts, April 1998,
Vol.75(4), pp. 501-505.
SUMMARY
Studying som biological characteristics and the solid -state
fermentation ability of some strains of red yeast Rhodotorula
isolated from south Viet Nam
Nguyn Th Minh Nguyt (1), ng Th Anh o (2), Nguyn Hu Phc (3)
(1)Univ. Industry (2)Univ. Technology HCMCity (3)Institute of Tropical Biology
Chosen 4 species of genus Rhodotorula were isolated from leaves, flowers and soils
in South Viet Nam were grown in broken gr ain of ordinary rice (size 1- 2mm) which
were starched by solid state method fo r receiving red yeast biomass. The medium
component and culture condition medium of solid -state culture method were
determined. Red yeast biomass were coloured from orange to red. Nutrition rate of
enriched broken grain of ordinary rice product is higher than other researches. The
result can be applied for Microbiol Production of Food grade pigments as well as
supplement production for animal feed technology .

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