Phân lập, định danh và xác định các đặc tính có lợi của chủng bacillus SPP từ ao nuôi tôm ở tỉnh bến tre

Tap chi KHOA HOC DHSP TPHCM

Si; 64 flan? 2014

PHAN LAP, DINH DANH
VA XAC DINH CAC DAC TINH CO LQI
CUA CHUNG BACILLUS SPP. AO NUOI TOM O TiNH BEN THE
NGUYEN VAN PHOC., PHAN THI PHU'O'NG TRANG.TOM TAT

Bacillus la nhom vi khuan có 10 hien dien trong da so cac che ph am vi sinh dung
cho nuoi trong thily hai san dac biet la nuoi torn. nu& to cac che pham vi sinh clang
trong nuoi trong thily hai sem deu phai nh Op
' ngoai, gib thanh cao. Trong nghien cieu nay,
nham phcin lap cac cluing Bacillus tit cac ao nuoi tom o tinh Ben Tre va sang loc, dinh
danh, khao sat dac diem sinh trwang caa cac Chung mang cac dac tinh cc') l07 lam ca ser
cho viec (mg dung san xucit che pham vi sinh.
Tie kh6a: Bacillus, che pham vi sinh, enzyme ngoai bao, Vibrio parahaemolyticus,
ao nuoi torn, tinh Ben Tre.

ABSTRACT
Isolating, identifying and determining the beneficial properties of Bacillus spp. strains
from shrimp ponds in Ben Tre province

Bacillus is a group of bacteria that presents in the majority of biological products for
aquaculture, especially for shrimp. In our country, microbial products used in aquaculture
have to be imported and at a very high price. In this study, we isolated some Bacillus
strains from the shrimp ponds of Ben Tre province and screened the beneficial properties
for shrimp. Potential Bacillus strains were. classified and growth characteristics were
investigated to serve as a basis for manufacturing biological products.
Keywords: Bacillus, biological products, extracellular enzymes, Vibrio
parahaemolyticus, shrimp ponds, Ben Tre province.

Mir diu
Ben Tre là mOt trong cac tinh co dien tich nuoi tom khd 18n, th8ng ke len den
32.086 ha chiem 73% tong dien tich nuoi trong thily san, dat san lugng 47.397 tar
' vac)
nam 2013. Tuy nhien viec nuoi tom v8i dien tich Ian va lien tuc clan den mat can bang
sinh thai, 8 nhiem nguOn nuorc do lugng thirc an thira va chAt thai tir tom nuoi din den
tom nuoi de nhiem cac benh, do virus va vi khuAn gay ra, lam thiet hai nang
cho
nguiri nuoi tom. Hien nay che pham vi sinh chira cac vi sinh vat co lgi dugc dung nhu
mOt phuang tien de kiem soat dich benh bang each ire che, canh tranh vOi nhting sinh
vat c6 hal, hi!) trg he tieu h6a, can bang vi Ichuan d0mg rutt [12]. NgoAi ra the phatil
cling dugc dung trong xir li ao nuoi bAng each phan 114 cac chat thong tieu h6a dugc,
tilde an thira, dac biet IA rat than thien voi moi toting [7]. Bacillus IA nhom vi IchuAn co
1.

ThS, Trung tam Khoa hoc va Cong nghe Sinh hoc
TS, TrulYng Dai hoc Khoa hqc Tyr nhian, DHQG TPHCM
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Tap chi KHOA HOC DHSP TPHCM

Nguyin Van Phtic va tgk

mat chii yeu trong cac the pham vi sinh vi c6 nhiing dac tinh c6 lqi nhu: (i) lam sach
mai truang nha kha nang sinh cac loaf enzyme protease, amylase, cellulase, lipase phan
huy cac hop chat him ca, (ii) kie'm soat su phat trien qua mirc,ciia cac vi sinh vat gay
b8nh nhu Vibrio do co the canh tranh nguen dinh &rang (iii) tit cac chat khang khuan,
giir cho mei truang luon a tang thai can bang sinh h9c. [4]

Do dO chiing toi lien hanh phan lap Bacillus tir cac ao torn a Ben Tre nham tuyen
ch9n nhang chung co cac dac tinh co Iqi de sir dung trong cac the pham vi sinh cho
tom.
2. V4t ligu va phuong phap nghien cuv
2.1. V(it ligu
2.1.1. Doi ttrong nghien citu
Chung Bacillus phan lap tir mAu dat, mau nuerc va mL torn trong cac ao torn 6
tinh Ben Tre.
Chung vi sinh vat kiem dinh V. parahaemolyticus &roc phan lap tir ao torn 138nh
chet (duqc cung cap bai Trung tam Khoa hoc va C6ng ngh8 Sinh h9c, Truang Dai h9c
Khoa h9c Tu nhien TP He Chi Minh).
2.1.2. moi trtrong sit dung nghien caw
MOi truang LB: tryptone 10 g, cao nam men 5 g, NaC1 5 g, nuac cat vira di: 1 lit.
M6i truang kich thich tao bao tir DSM: KCI 1 g, MgSO4.7H20 0,12 g, NaOH
0,06, pH = 7,6, mrac cat vira du 1 lit. Hap khir trung a 121°C, 15 phut. Lam nguei den
50°C, be sung them cac dung dich: 1 ml Ca(NO3)2 (1 M), 1 ml MnC12 (0,01 M), 1 ml
FeSO4 (1 mM).
Mai truang TSA khao sat dei khang val. V. parahaemolyticus: tryticase peptone
15 g, phytone peptone 5 g, NaC115 g, nuac cat vira du 1 lit.
Mai twang thach: thanh phan nhu ten co b6 sung them 2% agar. Cac moi twang
ten dm:lc hap kha tiling a 121°C, 15 phiit truck khi sir dung.
2.2. Phtrang phdp nghien cult
2.2.1. Phtrang phap pilau 10p va lam thu0n
Loai b6 to bao sinh duerng bang cach den hanh gia nhi8t mAu trong be en nhi8t
80°C trong 10 phut. Pha loang mAu den 10-2, but 100 µl dich gec va dich da pha loang
trai ten moi twang LB - agar. U a 37°C trong 24 gia. ChQn khuan lac dac trung cho
Bacillus va tin hanh lam thuAn bang each cAy ria ten moi truang LB - agar, cho tai
khi quan sat thAy chi c6 met clang khan lac duy that tr8n moi tnaing. [11]
2.2.2. Phuong phap dinh danh Bacillus
De xac dinh cac chimg vi khuan thuc gi6ng Bacillus, mgt s6 thir nghi8m cho

sang 19c buck dau duqc thuc hien nhu: nhuOm Gram [2], quan sat hinh thai to bao,
nhuem bao tir [2], thir nghi8m catalase bang phuong phap sir dung dung dich H202 3%,
thir nghi8m oxidase ten dia gi'a'y c6 tam N-dimethyl-para phenylenediamine, thir
nghi8m kha nang di ding tren moi truang thach mem. [3]
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Tap chi KHOA HOC DHSP TPHCM

Si; 64 nit nt 2014

Sau buck sang lgc tin hanh dinh danh bang phuang phdp giai trinh to 16S
rRNA: Tach chiet be gen vi khuan bang b0 kit dm QIAgen, khuech dui trinh to 16S
rRNA bang phan img PCR voi cap moi c6 trinh to nhu sau:
27F (5'-AGAGITTGATCMTGGCTCAG-3).
1492R (5'-TACGGYTACCTFGTTACGACTT-3').

San phAm PCR dugc tinh the va giri giai trinh tu. Cdc trinh to nucleotide hoan
chinh dugc so sanh vbi ngan hang du lieu gen cua NCBI bang each sir dung cong cu
BLAST.
2.2.3. Burang phap nghien cfru ccic cla'c tinh có lai
!Chi nang sinh enzyme ngoui bio: Khao sat kha nang sinh enzyme ngoai bao
tren mei tnrarng LB — agar c6 bo sung ca chat thich hgp. Te bao cua chimg Bacillus
dugc nuoi cay trong mei truong LB long a 37°C trong 24 gib. ChAm dich Bacillus sau
24 gib nuoi cay len dia m6i truong LB — agar c6 be sung tirng loai ca chit: 0,5% CMC
(carboxy methyl cellulose), 1% tinh bet, 1% gelatin, 1% casein, 1% (v/v) ddu oliu
tuang img cho khao sat khd nang sinh cdc enzyme cellulase, amylase, protease va
lipase. U cdc dia khao sat b 37°C 12 gib va quan sat vOng phan giai teen dia. [3]
Khi nang
khfing viti Vibrio parahaemolyticus: Sir dung phucmg phdp cay

vach thang vuong goc (Cross-streak) [8], [9] va phuang phdp khuech tan qua to thach
[6], [10], de khan sat ac tinh doi khdng vOi vi khudn V. paraheamolyticus.
2.2.4. Phtrcmg pheip khao sat dijc tinh sinh trur'mg
Kha nang chit' pH: Cdy cdc chimg Bacillus tuyen ch9n dugc trong mei truong
LB dugc dieu chinh pH bang HC1 va NaOH theo cdc gid tri pH: 4, 5, 6, 7, 8, 9, 10.
Nuoi cay lac a 37°C, do OD600
xac dinh mat de vi khuAn sau 18 gib. [5]
Khi nang chin min: Cdy caz chimg Bacillus tuyen ch9n dugc trong mei tnrong
LB be sung them NaC1 vbi cdc neng a: 0, 1, 2, 3, 4, 5%. Nuoi cay lac 6 37°C, do
OD600. de xdc dinh mat de vi khudn sau 18 gig. [5]
Khi nang twang thich giira cfic ching tuyen ch9n: Sir dung phuang phdp cay
vach thing vuemg gOc.
3. Ket qui va thio luin
3.1. Phdn 1(ip vet sang 1pc so bp' chang Bacillus

Tir caz m'Au &at, mau Junk, mdu tom c6 ki hieu tucmg Ong la BD, BN, BT lua
ch9n dugc 70 chimg c6 cac dac di6'm hinh thai khuAn lac ging ved Bacillus nhu: IchuAn
lac tron, r1a rang cua khong au, mau yang xam (hinh 1). Cac chimg nay tiep tuc dugc
lam thudri tren mei truorng LB — agar va 'cep vao 9 clang khuan lac nhu hinh 2.

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Tap chi KHOA HOC DHSP TPHCM

Nguyin Van PI !tic va tgk

7

Hinh 1. Hinh dang cac khudn lac

ph& kip tren WO' Throng LB

Hinh 2. 9 clang khudn lac ddc trong
dwc lam thucin tren mot trufmg LB

Tir 70 chi:mg c6 hinh clang dd'c tnmg dm Bacillus tienhanh met so thin nghiem de
sang 19c so bO thong qua cac ddc diem can thiet dm Bacillus (te bao hinh que, c6 sinh
bao tir, Gram duong, catalase duong tinh, oxidase duong tinh va c6 kha nang di &Ong)
ch9n duqc 61 chimg.
3.2. Khdo sat cac d#c tinh a 10 caa chang Bacillus phan dwpr
3.2.1. Khci Wing sinh enzyme ngogi bao
Cac vi sinh vat trong cac the phdm thuong co kha nang tiet cac enzyme ngoai bao
de phan hOy cac chdt can bA, thirc an thira ten de'ng trong ao, han che kha nang gay
benh cua cac chi:mg Vibrio. Do vay, cac chung da ducc phan lap va sang 19c so b0 se
duqc tien hanh khao sat kha nang tiet enzyme ngoai bao nhu amylase, cellulase,
protease, lipase ten cac did LB — agar c6 bo sung co chat thich h9p. Sir dung cac .thuOc
nhuOm lugol, do congo, TCA tuong
von co chAt tinh bet, CMC, gelatin de hien
Ong phan giai tren dia thach. NhUng chimg co kha nang sinh enzyme ngoai bao khi
xuat hien yang phan giai xung quanh khudn lac (hinh 3).

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Tap chi KHOA HOC DHSP TPHCM

So 64 nain 2014

4111111k
Hinh 3: Jiang phan giai tinh b5t (1), cellulose (2), gelatin (3), casein (4), lipase

(5) clic: cac chUng Bacilhis phan lap dux
Da s6 cac chung Oran lap dugc deu c6 kha nang sinh enzyme amylase. Trong do
c6 3 chAng BT21.1, BD17, BD22.2 kha nang phan giai tinh Not manh nhAt voi ban kinh
vong phan giai Ian nhAt 6 mm. Tuang to nhu tray, 3 chAng BT21.1, BT22.1, BD23.1 c6
kha nang sinh enzyme cellulase math vOi ban kinh yang phan giai la 6,25 — 7 mm.
Tat ca cac chAng deu c6 kha nang tiet gelatinase va caseinase. Trong do co 5
chAng BT15.2, BT24.2, BT21.2, BT22.2, BD23.1 cho ban kinh yang phan giai gelatin
lOn nhAt la 7 - 7,75 mm. VA 6 chung BN12, BD1, BN3, BN5, BN8, BNIO c6 ban kinh
yang phan giai casein lan nhAt la 6,5 — 7 mm.
Rieng kha nang sinh enzyme lipase thAp, chi co 8 chAng co kha nang tiet enzyme
lipase nhtmg ban kinh yang phan giai chi 1 mm.
TA ket qua tr'en cho thAy cac chAng Bacillus phan lap dugc co kha nang tiet cac
loai enzyme ngoai bao b cac mAc dq khac nhau. Cac chAng tiep tic dugc khao sat kha
nang doi khang vii V. parahaemolyticus.
3.2.2. Kha nang den khang V. parahaemolyticus
V. parahaemolyticus la vi khuan gay benh eh& sant tren torn gay thiet hai lan cho
nganh nuoi tom trong ca nuck not chung va tinh Ben Tre not rieng. Do do tien hanh thi
nghiem khao sat de tuyen chon nhang chAng c6 kha nang khang V parahaemolyticus
nharn huong ten Cmg ding trong cac the pharn vi sinh cho tom. Khao sat kha nang Mi
khang ctia 61 chung tuyen chon bang phuong phap vach ding vuong goc va dpc16 thach.
Veri phucmg phap vach thing vuong goc chung CO doi khang khi duAng cay ctia
chiing V. parahaemolyticus moc cach xa dtrong cay chung Bacillus khao sat (Hinh 4A).
\Teri plurcmg 014 chic 16 thach chAng c6 d6i khang khi xung quanh 16' thach dugc
nho dich nuoi cay vi khuan Bacillus c6 yang vo khuan (Hinh 4B).

Hinh 4. Ket qua dii khang ctia chting BN1
A. Phumg phap vach thing vuong g6c;B. Phutmg phap duc to thach
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Tap chi KHOA HOC DHSP TPHCM

Nguyin Van Ph tic va tgk

Ke't qua khao sat trong 61 chang cho tha'y c6 5 .chang c6 d8i khang vai V.
parahaemolyticus la BN1, BT15.3, BT21.1, BT24.2, BD23.1.
Qua ket qua khao sat kha Pang sinh enzyme ngoai bao va &Si khang vai V.
parahaemolyticus ch9n doge 17 chang trong Bang 1. CO kha nang sinh it nhat 3
enzyme ngoai bac). Trong do 2 chang BN1 va BD23.1 ductc ch9n dal dien de dinh danh
den mac la bang phucmg phap giai trinh to 16S rRNA. Vi 2 chang nay c6 kha nang
Binh hau het cac loci enzyme ngoai bao khao sat va c6 kha nang doi khang vai V.
parahaemolyticus.
3.3. Kit qua dinh dank bdng phwang phcip giai trinh tot 16S rRNA
138 gen oh hai chang tuyen ch9n la BN1 va BD23.1 ducic tach chiet. Trinh tir
16S rRNA tren bi) gen dugc nhan ban bang phacmg phap PCR va chay dien di tren gel
agarose 1% kiem tra ket qua (hinh 5). Tren hinh 5 cho thy a gieng BN1 va BD23.1
da thu nhan ducfc doan trinh to DNA (— 1500 bp) ma h6a cho 16S rRNA caa 2 chang
BN1 va BD23.1. Wing trinh tir 16S rRNA dirge giai trinh ttr va so sanh (18 tuang d8ng
di truyen vai cac loci tren ngan hang gen NCBI bang cong cu BLAST. Ket qua &cm
trinh bay trong Bang 2.

a

M BN1 BD23.1

by

20000
7000
4000

2500
1500
1000
700
400
200

Hinh 5. Kit qua dien di tren gel Agarose 1% san phdin PCR rang 16S rRNA
cluing BN1 va BD23.1; M: thang DNA.

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Tpp chi KHOA HOC DHSP TPHCM

So 64 nam 2014

. Bang 1. Tang hop ket qua vong phcin giai ca chat cart enzyme ngoai bao
via dai khang v&i V. parahaemolyticus
Diii khang
Vach thing
Due to
yang goc
thach
(mm)
(mm)

Amylase
(mm)

Casein
(mm)

Gelatin
(mm)

Cellulase
(mm)

Lipase
(mm)

2

BN12
BDI

3
0,5

6,5
6,5

3,25
6

3,5
4,5

1

0

0
0

0
0

4
5
6
7
8
9
10
11
12
13
14
15
16

BN3
BN5
BN8
BNIO
BT15.2
BT15.3
BT21.1
BT21.2

BT22.1
BT22.2
BT24.2
BD17
BD22.2

4
3
3,5
2
0
2
6
0,5
3,5
2,25
0
4,5
4,5

7
6,5
6,5
6,5
3,75
3,5
5,5
0,25
5,5
2,25

1,5
2,75
3

4
5,5
4,25
6,25
7,25
5,5
3
7
1,5
7,5
7,75
4,5
4

3,25
2
3
5
0
4,5
7
0
6,25
0
0,75
0

0

1
1
0
0
0
1
0
0
1
0
1
0
0

0
0
1
0
0
1
4
0
1
0
2
0
0

0
0
0
0
0
0
1
0
0
0
3
0

Ki hi?u
chfing

STT
1

0

Bang 2. Tom tat Ic- t qua dinh danh hai chung BN1 va BD23.1
Max score

Query
Coverage

E-Value

Max

Identities

Idt loan

BN1

2494

100%

0,0

100%

B. subtilis

BD23.1

2508

100%

0,0

100%

B. subtilis

Chung

Dua tren ket qua phan tich trinh to vang 16S RNA xac dinh dugc 2 chang BN1
va B23.1 la chang B. subtilis. So sanh giaa 2 chang BN1 va BD32.1, chang BN1 la
chang doi khang v6i V. parahaemolyticus manh hcm. COn chang BD23.1 la chang tiet
enzyme ngoai bao manh hon. Nhu vay 2 chang nay nen dugc khao sat tier) theo de ph6i
trgn trong che pham vi sinh.
3.4. DOc tinh sinh truiing ceta chiing
Kha Jiang chit.' pH va chit' man: tir ket qua ten D6 thi 1 chO thay 2 chang BN1
va BD23.1 c6 kha nang chlu dugc pH tir 5 - 9, n6ng ci(5' mmSi to 0 - 5%. Va a pH 7,
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ATglOn Van Pinuc va tgk

Tap chi KHOA HOC DHSP TPHCM

n6ng d6 mu6i tir 0 — 2% la dieu kien sinh truang t6i uu. .
So sanh vai khoang pH trong,dieu kien ao nuoi torn nuac 1g a nu6c ta la 7,5 - 8,2
va nemg dO mue>i la 1,5 — 2,5 % [I], dieu nay cho they chung BN1 va BD23.1 deu co
kha nang chiu khoang pH va n6ng d'6 mu6i rOng phis hop vai dieu kien ao nuoi torn
nuac ta, do do, thich hop de lam the pham sinh hoc trong nuoi torn. Ket qua nay phir
hop vai du loan vi cac chimg vi sinh vat nay dugc phan lap chinh tir cac ao nuoi torn.
12

a pH4
• pH5

a pH6
pH7
• pH8
• pH9

BN1

A

BD23.1

Chimg

• pH10

BD23.1

BN 1

Chong

flo thi 1. OD 600 nrn cue hai chting BN1 va BD23. 1 a cac nang dO pH (A) va njng
dO muoi NaCI (B) khcic nhau sau 18 gia nuoi coy
Kha nang ttrcrng thich cue hai chfing tuytn chip: Cac the pham sinh hoc
thuomg dugc sir dung duai clang lion hop cac
chimg vi khuan nham irng..dong town b¢ cac tiem
nang cue tong chimg cfing nhu dam bao tinh thich
nghi va hieu qua 6 moi diet' kien moi tnr6ng. Do
do, viec tien hanh kiem tra tuong thich gifra cac
chimg tuyen chon nham c6 co sof phi trOn phis
hqp. Phuong phap dugc tien hanh nhu da mo ta
phan ten. Ket qua the hien 6 Hinh 6.
Duang cay hai chung BN1 va BD23.1 IncIC
sat nhau chimg to hai chimg CO kha nang tuong

thich vai nhau. Hai chung BN1 va BD23.1 co kha
nang Ichang vi khuan V. parahaemolyticus nhung
Ichong Ichang vai nhau, chimg co kha nang tuong
Su turmg thich gifia hai cluing
thich vai nhau nen chung hoan town CO the ph6i Hinh 6.
BN1 va BD23.1 sau 18 gia nuoi cay
trOn vai nhau trong the pham.
4. Ket Juan
Tir 70 chimg phan lap dugc trong cac mau &At, mAu ntrac, m'au torn thu dugc tir
31 ao nuoi torn a BenTre sang loc so b6 chon dugc 61 chung co cac dac diem cira
Bacillus. Trong dO tuyen chon dugc 17 chung c6 kha nang sinh enzyme ngoai bac va
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Tap chi KHOA HOC DHSP TPHCM

Si) 64 nam 2014

kha rang khang vai V. parahaemolyticus. Rieng hai chfing BN1 va BD23.1 có kha
nang khang vai V. parahaemolyticus va sinh enzyme mann nen dugc dinh danh va
phan tich ket qua djnh danh cho thy hai chimg nay thuOc B. subtilis. Hai chimg BN1
va BD23.1 có (lac diem sinh truemg phi" hgp vai dieu kien nuoi tom Cr nuac ta nhu
khoang chili pH (5-9) va chili man (0-5%) rOng , ngoai ra hai chfing nay tu.cmg thich
vai nhau va dac tinh có 19i dm timg chimg se bo sung cho nhau nen thich hgp cho img
dung trong san xuAt the pha'rn vi sinh. Tuy chi la buck dau nghien ciru trong phong thi
nghiem niumg la nghieri ciru ca ban có mang tinh img dung cu the cho dieu kien san
xudt the phdm vi sinh phyc vu trong nuoi tom a nu& ta.
TAI LIEU THAM KHAO
1. Nguyen Van Hao va ctv (2004), Huang dan quan li chat luting mac trong ao nuoi
tom sa,130 Thay san.

2. Trail Linh Thu& (2010), Phuung phap phan tich vi sinh vat trong mac, thyv pham
va mr pham, Nxb Giao dyc, TPHCM.
3. Pham Van Ty (2006), Cong nghe sinh hoc — cong nghe vi sinh va moi trtrang, Nxb
Giao dyc, TPHCM.
4. Altan, A. (2004), "Isolation and Molecular Characterization of Extracellular Lipase
and Pectinase Producing Bacteria from Olive Oil Mills", Izmir Institute of
Technology.
5. Arici, M., Bilgin, B., Sagdic, 0. and Ozdemir, C. (2004), "Some characteristics of
Lactobacillus isolates from, infant faeces", Food Microbiology, 21(1), pp.19-24.
6. Balcazar, J.L., de Blas, I., Ruiz-Zarzuela, I., Cunningham, D., Vendrell, D. and
Muzquiz, J.L. (2006), "The role of probiotics in aquaculture", Veterinary
microbiology, 114(3-4), pp.173-186.
7. BalcAznr, J.L. and Rojas-Luna, T. (2007), "Inhibitory Activity of Probiotic Bacillus subtilis
UTM 126 Against Vibrio Species Confers Protection Against Vibriosis in Juvenile Shrimp
(Litopenaeus vannamei)", Current Microbiology, 55(5), pp.409-412.
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V. and Prasad, D.N. (2005), "Application of in vitro methods for selection of
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Bacillus subtilis BT23", a possible probiotic treatment for black tiger shrimp
Penaeus monodon, Letters in applied microbiology, 36(2), pp.83-87.
(Ngay Tda soon nh$n ettioc bat 13-8-2014; ngdy phan bin danh gia: 18-8-2014;
ngay chip nhin ding: 21-11-2014)

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