Professional Documents
Culture Documents
Basil Us Lacto
Basil Us Lacto
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
Hi ngh KHOA HC V CNG NGH 2007 224
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
Phn II: CNG NGH VI SINH 225
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
Institute of Tropical Biology
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.
Hi ngh KHOA HC V CNG NGH 2007 226
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
Phng Vi sinh ng dng, Vin Sinh hc Nhit i
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.
Phn II: CNG NGH VI SINH 227
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
Hi ngh KHOA HC V CNG NGH 2007 228
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:
Phn II: CNG NGH VI SINH 229
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
Hi ngh KHOA HC V CNG NGH 2007 230
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
Institute of Tropical Biology
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.
Phn II: CNG NGH VI SINH 231
TUYN CHN CC CHNG Lactobacillus SINH TNG
HP polyhydroxybutyrate (PHB)
Nguyn Duy Long, Phm Trn T Nh, Nguyn Minh Nht, Hong Quc Khnh
Phng Vi sinh ng dng, Vin Sinh hc Nhit i
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.
Hi ngh KHOA HC V CNG NGH 2007 232
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
Phn II: CNG NGH VI SINH 233
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
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
N8
0.00
0.05
0.10
0.15
0.20
0.25
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
4a
4b
4c
Hi ngh KHOA HC V CNG NGH 2007 234
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 %
Phn II: CNG NGH VI SINH 235
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%
Hi ngh KHOA HC V CNG NGH 2007 236
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
Institute of Tropical Biology
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 II: CNG NGH VI SINH 237
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
Hi ngh KHOA HC V CNG NGH 2007 238
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
Phn II: CNG NGH VI SINH 239
Hnh 4.1. Hnh thi chng MR5
mc trn mi trng PGA
Hnh 4.2. Hnh thi v kch thc
Conidiophores chng MR5; X 6
Hnh 4.3. Hnh thi v kch thc
bo t chng MR5 (conidia); X 100
Hnh 5.1. Hnh thi chng MR2
trn mi trng PGA
Hnh 5.3. Hnh thi v kch thc
bo t chng MR2 (conidia); X 100
Hnh 5.2. Hnh thi v kch thc
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
Hi ngh KHOA HC V CNG NGH 2007 240
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
Phn II: CNG NGH VI SINH 241
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.
Hi ngh KHOA HC V CNG NGH 2007 242
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.
Phn II: CNG NGH VI SINH 243
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.
Hi ngh KHOA HC V CNG NGH 2007 244
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
Phng Vi sinh ng dng, Vin Sinh hc Nhit i
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.
Phn II: CNG NGH VI SINH 245
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))
Hi ngh KHOA HC V CNG NGH 2007 246
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
Phn II: CNG NGH VI SINH 247
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
Hi ngh KHOA HC V CNG NGH 2007 248
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
Institute of Tropical Biology
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 II: CNG NGH VI SINH 249
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
Hi ngh KHOA HC V CNG NGH 2007 250
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 + + + + + + + +
Phn II: CNG NGH VI SINH 251
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
Hi ngh KHOA HC V CNG NGH 2007 252
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.
Phn II: CNG NGH VI SINH 253
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
Hi ngh KHOA HC V CNG NGH 2007 254
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
Institute of Tropical Biology
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
Phn II: CNG NGH VI SINH 255
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):
Hi ngh KHOA HC V CNG NGH 2007 256
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
Phn II: CNG NGH VI SINH 257
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
Hi ngh KHOA HC V CNG NGH 2007 258
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
Phn II: CNG NGH VI SINH 259
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
Hi ngh KHOA HC V CNG NGH 2007 260
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
Phn II: CNG NGH VI SINH 261
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.
Hi ngh KHOA HC V CNG NGH 2007 262
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
Institute of Tropical Biology
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%.
Phn II: CNG NGH VI SINH 263
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.
Hi ngh KHOA HC V CNG NGH 2007 264
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
.
Phn II: CNG NGH VI SINH 265
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.
Hi ngh KHOA HC V CNG NGH 2007 266
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
Phn II: CNG NGH VI SINH 267
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
Hi ngh KHOA HC V CNG NGH 2007 268
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.
Phn II: CNG NGH VI SINH 269
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.
Hi ngh KHOA HC V CNG NGH 2007 270
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.
Phn II: CNG NGH VI SINH 271
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.
Hi ngh KHOA HC V CNG NGH 2007 272
- 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
Phn II: CNG NGH VI SINH 273
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
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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.
Hi ngh KHOA HC V CNG NGH 2007 274
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
)
Phn II: CNG NGH VI SINH 275
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).
Hi ngh KHOA HC V CNG NGH 2007 276
- 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
Phn II: CNG NGH VI SINH 277
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 .