3.
^
DE
TAI
KHOA HOC TRONG DIEM
TIM
KIEM
CAC CHUNG VI
SINH
VAT CO HOAT
TINH
SINH HOC NHAM TAO CHE PHAM LAM SACH
NlTdC
NUOI
TOM
MA
SO:
QG - TD
05-05
Chu tri de tai:
Cdc
thanh
vien:
GS.TS.
Pham Van Ty
GS.
TS. Nguyen Lan Dung
PGS.
TS.
Nguyin
Thanh

Du^crng
TS.
Bui Viet Ha
ThS.
Dao Thj
Lu-ong
ThS.
Tr§n
Thj Thanh
Huyin
ThS.
Nguyin
buy
Thjnh
CN.
Pham
Dii-c
Ngoc
KS.
Nguyen Thj Thuy Hang
HA
NOI
- 2007
DAI HOC
OUOC GIA
HA NOI
rPUNG TAM
THONG TIN
THU
VIEN

0006000666^
Lcn NOI
DAU
La mot trong nhung nuoc co dieu kien thien nhien
im
dai, nganh nuoi trong
thuy san cua Viet Nam trong nhung nam qua da phat trien
vugt
bac, dat dien tich
tren 700 nghin ha nuoi trong va vuon len thuoc nhom cac quoc gia co san lugng
nuoi
tr6ng
thuy san dung dau the
gioi.
Nam 2006 tong kim ngach xuat khau thuy
san dat tren 3 ty USD, trong do mot nua la tu tom, chi dung sau Trung Quoc va
Thai Lan. Nganh nuoi trong
thiiy
san
thirc sir
da giai quyet van de viec lam cho
ngudi lao
dong, tang thu nhap, gop phan xoa doi giam ngheo va tang
trudng
kinh
t6
cho dat nuoc.
Ngay
11/1/2006 Thii
tuong Chinh phu da ky

Quy^
dinh
s6
10/2006/QD-
TTG phe duyet Quy hoach tong the phat trien nganh thuy san den nam
2010
va
djnh
huong den nam 2020. Theo do nganh thuy san phai phan dau
tro
thanh nganh san
xuat hang hoa Ion, san pham phai co chat luong tot, co sue canh tranh cao tren thi
truong the gioi de dua nganh nay tiep tuc phat trien mot each ben
vmig.
De
nghj
quyet
ciia
chinh
phii
tro thanh hien thirc la mot nhiem vu day kho
khan, do chiing ta dang phai doi mat voi nhung thach thuc Ion ve 6 nhiem moi
truong, lam cho djch benh ngay cang nhieu va ngay cang phuc tap, khien cho
riii
ro
ciia
nganh nuoi torn ngay cang Ion, nhieu noi co nguy co mat trang do tom chet co
khi den 100%, gay thiet hai nang ne cho
nguoi
dan.

Truoc
day de phong chong benh cho tom, nguoi dan thuong dung hoa chat
va chat khang sinh. Ngay nay cac bien phap nay khong con
dugc
ap dung nua do du
lugng hoa chat va chat khang sinh ton du trong san pham. EU da ra thong bao se
kiem tra 100% tom nhap khau vao cac nuoc EU
tir
Viet Nam. Nam 2006 Nhat Ban -
ban hang Ion
ciia
Viet Nam,
ciing
da canh bao ve du lugng khang sinh trong torn
Viet Nam, ma neu khong cai thien thi se co nguy co mat thi truong nay,
Moi truong va benh la hai khia canh
ciia
mot van de, chiing co tac dong qua
lai voi nhau. Phan
Ion
dich benh
6
torn xuat phat
tir
6 nhiem moi
trudng.
Do vay,
viec tao ra mot che pham sinh hoc de xu ly
6
nhiem moi

trudng
co y
nghla
song con
doi vdi nganh nuoi trong thuy san noi chung va nganh nuoi tom noi rieng. Hien tai
chiing ta khong kiem soat het dugc cac che pham sinh hoc dang luu hanh tren thi
trudng. Cac che pham cua nude ngoai khong
nhimg
dugc ban vdi gia qua dat ma
con chua nhieu loai vi sinh vat ma mot khi tung vao thien nhien chiing ta khong
kiem soat dugc.
Xuat phat tu nhung ly do neu tren, muc tieu
ciia
de tai nghien curu cua chiing
toi nham:
- Tuyen chgn bo vi sinh vat cd kha nang sinh chat cd hoat tinh sinh hgc cao
giiip giiip
phan giai nhanh cac chat
hiru
co du
thira
va diet cac vi sinh vat gay benh
CO
hoi trong ao nuoi.
- Tu cac chiing da
lira
chon, tao che pham vi sinh than thien moi trudng vdi
gia re dung xu ly 6 nhiem moi trudng thay the cho viec su dung khang sinh trong
nghe nuoi torn.
Trong ban bao cao nay, ten de tai cung da dugc nit ggn, mot so ngi dung

nghien curu da dugc chinh sua (bo phan che pham
thiic
an, thu ggn dien tich thi
nghiem) theo y kien cua "Hoi dong danh gia de cuong de tai trgng diem" ngay
9/4/2005. Nhan
sir
tham gia de tai cung thay doi do thuyen chuyen cong tac.
Cuoi Cling chiing toi muon nhan co hoi nay
giri Idi
cam on chan thanh tdi
Dai hoc Quoc gia Ha noi da cap kinh phi, Ban
lanh
dao trudng Dai hgc Khoa hgc
Tu nhien, Ban Khoa hgc cong
nghe.
Ban Ke hoach - Tai chinh, phdng Khoa hgc -
Cong
nghe.
Ban Chu nhiem Khoa Sinh hgc da tao dieu kien thuan
Igi
de chiing toi
trien khai de tai.
Trong qua trinh thuc hien de tai chiing toi cung nhan dugc
sir
giiip d5 cua
nhieu can bg khoa hgc va dong nghiep, nhan dip nay chiing toi xin chan thanh cam
on
nhiing
su giiip do quy bau do
Hd noi, thdng 4 nam 2007

Tap the can bo tham gia de tai
CHrCfNG
1.
TONG QUAN
VAI
TRO CUA CHE PHAM
SINH
HOC TRONG
NUOI
TRONG THUY SAN
1.
DAT VAN DE
Viet Nam la
nude
cd tiem nang
Idn
trong nudi trong thiiy san, vdi bd bien dai
3260 km, khi hau am ap quanh nam, Viet Nam co tren 30 tinh nudi trong thiiy san.
Tinh den nam
2003,
ca nude cd 530.000 ha nuoi tdm, nam 2005 tong dien tich nudi
trong da len den 750.000 ha. Tuy nhien,
sir
phat trien 6 at nghe nudi trong thuy san,
dac biet la nghe nudi tom d cac tinh ven bien dan den
sir biing
no dich benh tren
dien rgng, gay thiet hai nang ne cho ngudi dan.
Sir
pha san cua

nhimg
ngudi dan d
Kien Thuy - Thai Binh cung nhu d nhieu dia phuong khac la bai hoc dau xdt do chi
chay theo quy mo nudi trong ma khong quan tam tdi yeu to mdi trudng.
Sir
6 nhiem
xay ra rat nghiem trgng trong chinh ao nudi va moi trudng sinh thai xung quanh do
thai ra mot lugng Idn cac chat
hiju
co
tir thiic
an du thua,
tir
phan va cac chat thai
khac cung nhu
sir
ton dong
ciia
cac loai hoa chat, chat khang sinh dugc sir dung
trong qua trinh nuoi trong ma khong dugc
xir
ly.
Benh cua thuy san
thu&ng
bat ngudn tir chinh moi
tru&ng
ao nudi. Viec
hinh thanh
Idp biin
day do

sir
tich tu
lau
ngay
ciia
cac chat hiru co, chat can va la noi
sinh song cua cac vi sinh vat gay thoi, cac vi sinh vat sinh cac khi doc nhu amoniac,
nitrit,
hydrosulflia
2.
VI SINH VAT GAY BENH
Cac vi sinh vat gay benh cho tom rat da dang, cd the la virut, vi
khuAn,
n4m
sgi va nguyen sinh dong vat. Mot so nhiem tir tdm gidng, cdn
phAn
Idn cac tac nhan
nay cd san trong ao ho nudi torn,
Benh do virut co khoang 15-16 loai, thudng lay lan nhanh va lam cho ty le
tdm chet cao.
Cac benh pho bien la:
- Benh BMV (Monodon Baculovirus)
thugc
ho Baculoviridae
- Benh dom trang (WSSV - White spot syndrome virus) do virut thuoc ho
Nimaviridae gay nen
-
B?nh
dau vang (YHV - Yellow head virus) do virut tuong tu Coronaviridae
gay nen.

- Benh hoai
tir
dudi vd va co quan tao mau
(IHHNV
- Infectious hypodermal
and hematoietic necrosis virus)
- Benh teo gan tuy do virut HPV - Hepatopancreatic parvolike virus thuoc
hg Parvoviridae.
Bfnh
do vi khuan rat da dang bao gom cac benh nhu den vd, den mang,
phat sang, nau mang, rung dudi, ddm den, phan trang. Vi khuan gay benh cd the
thugc chi Vibrio, Aeromonas. Pseudomonas Tuy nhien phd bien nhat van la cac
loai thuoc chi Vibrio nhu: V. harveyi. V. alginolyticus, V. parahaemolyticus, V.
vulnificus, trong do
V.
harveyi la loai chu yeu gay benh phat sang. Hau het vi khuan
gay benh la bgn co hoi, chiing thudng gay nhiem trong cac trudng hgp tdm da hi
nhiem tac nhan khac, bi stress do moi trudng,
bj Id loet
tir trudc hoac khi thirc an
thieu can doi ve mat dinh dudng.
Benh do nam: Nam Muco bam vao mang, Fusarium lam den mang, mot so
nam khac nhu
Lagenidium,
Haliphtoros phat trien
luon
dudi Idp vd chitin. Nam cdn
gay ra benh dd than do tiet ra aflatoxin.
Benh do nguyen sinh dong vat bao gdm Gregarina ky sinh trong rugt lam
tom cham Idn, benh tom bong do Microsporea bam vao co van lam

due
co. Mot
so ky sinh phia ngoai nhu Zoothammium spp., Epistylis spp. gay can trd chirc nang
ho hap
Phan lan cdc vi sinh vat gay benh ke tren
Id
mot phan cua he vi sinh vat
binh thu&ng trong moi
tru&ng (nude
bien, ao hd, song rach). Chiing dugc xem la
tac nhan gay benh thu cap hoac gay benh ca hot Mot khi
sir
can bang cua he vi
sinh vat trong ao nudi bi pha vd, cac vi sinh vat cd hai se phat trien d at va sdm
cong hudng vdi cac yeu to cd hai khac de gay
benh.
Sir
d nhiem mdi trudng trong
thdi gian ngan se tac dong tdi he than kinh, trong thdi gian dai se lam tang stress
dan den giam kha nang tieu thu thirc an, giam tang trudng, lam tang man cam doi
vdi cac tac nhan gay benh dac biet la cac virut. Tat ca cac yeu to neu tren se lam cho
thuy san chet hang
loat
chi trong mot thdi gian ngan.
Sir
canh bao tir phia Nhat Ban cuoi nam 2006 khien chiing ta can phai xem
xet lai mot each nghiem tuc ve ky thuat nudi trong de tir do dua ra giai phap hgp ly,
dam bao ve sinh an toan thuc pham neu nhu khdng mudn sau Nhat Ban, theo phan
ung Domino se la cac nude khac. Va nhu the khd Idng day manh xuat khau va thirc
hien thanh cdng nghi quyet cua Chinh phu.

Neu khdng sir dung hoa chat vd chat khdng sinh thi
giai
phap him hieu de
giai quyet van de
5
nhiem moi
tru&ng
trong ao nuoi chinh
Id
viec su dung cdc
che pham sinh hgc. Bien phap nay dd vd dang su dung pho bien tai cdc
nude
nuoi trong thuy san
&
Dong vd Tay Ban cdu nhu My,
Braxin,
Australia, Ixrael,
Thdi
Lan,
Ddi Loan, Trung
Quoc,
Malaysia
3.
CHE PHAM SINH HQC PROBIOTIC
Probiotic (trg sinh) la thuat ngir cd nguon gdc tir tieng Hy lap, bao gdm hai
tir: Pro co nghla la vi va biosis co nghTa la
sir
song,
la thuat
ngir

timg xuat hien nam
1965
nhung dugc nhac tdi nhieu trong nhung nam gan day do tinh uu viet
ciia
chung. Probiotic dugc dinh nghla la
che
pham
chira
cdc vi sinh vat song da chgn
Igc
ma khi dua vao ca the qua duang mieng vai lugng du lan se dnh
hu&ng
co lai den
sire
khoe vat chu.
Ddi vdi ngudi va dong vat (gia
siic,
gia cam), cac vi khuan probiotic thudng
la vi khuan lactic, dugc tuyen chgn cong phu vi chung dac trung cho timg loai. Tinh
hieu qua cua probiotic da dugc chung minh bao gdm kha nang chdng nhiem
triing
do
vi khuan va virut (vi du virut rota gay tieu chay), kha nang chdng ung thu, kich thich
hoat dong
ciia
he mien dich, lam giam cholesterol, cai thien sir dung lactoza d nhung
ca the khong dung nap lactoza (khdng udng dugc sira), giam tac dung phu
ciia
chat
khang sinh Sd

dT
probiotic cd dugc hieu qua nhu vay la vi chiing tac ddng lam on
djnh khu he vi sinh vat dudng ruot, lam tang cac vi khuan cd ich (cac vi khuan sinh
vitamin, sinh chat khang khuan, vi khuan phan giai dudng, tinh bgt., ), lam giam cac
vi khuan co hai (cac vi khuan canh tranh thirc an, sinh chat doc ).
Trong nudi trong thdy san dinh nghia ve probiotic dugc hieu vdi nghla
rgng hon, lien quan den cd mdi
tru&ng
nuoi trong. Khdc vai dong vat tren can, a
ddng vat thiiy sinh, khu he vi sinh vat duang rugt khong ton tai nhu
Id
mot
quhn
the
dgc
lap dac
thii
cho timg loai, md cd su tuong tac thu&ng xuyen vdi mdi
tru&ng
va
v&i ho^t
dong cua vat chu. Vi sinh vat cd mat trong mdi trudng
nude
anh hudng
true
tiep den thanh phan khu he vi sinh vat dudng rugt va ngugc lai. Cac
vi sinh vat cd mat trong dudng ruot hau het co ngudn gdc tir moi trudng. Thay cho
muc dich chu yeu la tieu diet cac bao tu vi khuan, che pham sinh hgc dugc san xuat
va dua vao sir dung vdi muc dich chu yeu la kich thich
sir

gia tang cua cac vi sinh
vat
CO
Igi trong ao nudi.
Ngay nay, che pham sinh hoc dugc coi la cong cu
him
hieu, tao nen tang
vung chac cho phan
Idn
hoat ddng nudi trong thuy san tren the gioi. Che pham sinh
hgc da dugc chap nhan rgng rai de khong che djch benh, tang
sire
de khang. Khac
vdi bien phap hda hoc va khang sinh, che pham sinh hgc cung cap mot phuong thirc
an toan ben vung ddi vdi ngudi nuoi va tieu
diing.
7.
CHE PHAM SINH HOC
HOAT DONG
THEO PHUONG
THlTC
NAO?
Mac
dii
nhieu loai probiotic dugc dua vao sir dung trong nuoi trdng thuy san
trong vai thap nien qua nhung viec sir dung cac che pham nay chu yeu theo kinh
nghiem, mdi chi ap dung cac ket qua nghien cim ve probiotic tren ngudi va ddng
vat d can cho ddng vat thiiy sinh ma chua thuc su hieu mot each tudng tan co che
tac dong cua probiotic ddi vdi nhdm dong vat nay. Tuy nhien ngudi ta cho rang, bat
ky mot che pham sinh hoc nao cung phai dat dugc 3 qua trinh sau:

- Khdng che sinh hgc: nhimg ddng vi khuan cd ich trong che pham cd kha
nang sinh cac chat khang khuan, vi du bacteriocin de tieu diet cac vi khuan gay
benh trong ao.
- Tao
sire
song mdi: Cac vi khuan trong che pham khi dua vao ao se phat
trien manh me ca ve so lugng va hoat tinh, cd kha nang tdn tai trong mdi trudng va
trong dudng ruot, anh hudng co Igi doi vdi vat nudi.
- Xir ly sinh hgc: Kha nang phan giai cac chat hiru co trong
nude
giai
phong cac axit amin, glucoza, cung cap thirc an cho cac vi sinh vat cd ich, giam
thieu thanh phan nito vd co nhu: NH3, NO2,
NO3' ,
giam
miii
hoi thoi, cai thien
chat lugng
nude.
8. VI SINH VAT
Sir
DUNG TRONG PROBIOTIC
• •
Viec sir dung cac vi sinh vat huu ich nham canh tranh vdi cac vi khuan gay
benh da dugc img dung rgng rai trong chan nudi nhu la mot bien phap tdi uu thay
thS
cho viec sir dung khang sinh, va ngay nay dang dan dugc ddn nhan nhu mot giai
phap quan trgng kiem soat benh trong nuoi trdng thuy san.
Sir
canh tranh cua vi sinh

vat la mot trong cac qua trinh sinh thai cd the dan den
sir
thay ddi thanh phan loai
cua bat ky he sinh thai nao, cho
dii
la dat,
nude,
trong co the ddng vat hoac trong
mdi trudng khac. Nhung thay doi nhd co the anh hudng tdi toe do phat trien hoac tir
vong dan den
sir
thay doi ve loai uu the trong quan xa. Mac
dii
chung ta con chua
biet het tat ca cac nhan td kiem soat
toe
do tang trudng
ciia
mot loai nao dd nhung
ciing du de co the thay ddi thanh phan loai theo quy
luat ire
che canh tranh. Vi sinh
vat CO the tiet ra cac chat khang khuan, tuy khong diet toan bg loai canh tranh
nhung tieu diet
dii
de thiet lap lai the can bang, lam giam sd lugng loai canh tranh
xuong dudi ngudng gay hai kinh te.
Thanh phan
ciia
che pham probiotic thudng la mot tap hgp cac chiing vi sinh

vat song, dugc tuyen chon, tdi uu hoa, lam kho bang phun say, ddng khd hoac bgc
trong alginat. Moi nha san xuat co the chon cac loai khac nhau, tuy nhien pho bien
nhat van la cac loai sau day:
- Vi sinh vat sinh phirc he enzym amylaza, proteaza, xenlulaza, lipaza phan
giai chat hiru co khong mong muon cd trong ao nuoi nhu thuc an thira, v6 tdm, xac
tao va ddng vat thiiy sinh. Cac vi sinh vat nay bao gom: Bacillus subtilis, B.
licheniformis, B. megaterium, B.
polymyxa
- Cac vi sinh vat sinh chat khang khuan (bacteriocin, pediocin, ) gdm
Lactobacillus plantarum, L. acidophilus , Bifidobacterium sp., Pediococcus sp.
- Ngoai ra mot so che pham con
chiia
nam men Saccharomyces cerevisiae, S.
boulardii,
cac vi khuan nitrat hoa nhu
Nitrosomonas,
Nitrobacter.
Mot thanh phan khac ciing thudng dugc thay trong che pham probiotic do la
tap hgp cac enzym cd nguon goc vi sinh vat nhu amylaza, proteaza, lipaza,
xenlulaza, chitinaza, mot so vitamin thiet yeu hoac axit amin va chat khoang
nham kich thich hoat tinh ban dau
ciia
vi sinh vat trong che pham va
xiic
tac cho
sir
hoat
dgng
cua enzym trong mdi trudng. Cac vi sinh vat dugc
lira

chgn phai cd cac
d^c
dilm sau:
- Khdng sinh dgc td, khdng gay benh cho vat chu va khdng anh hudng
\ku
d6n
he sinh thai mdi trudng.
- Cd kha nang bam dinh len niem mac tieu hda va cac md khac ciia vat
chii,
canh tranh vi tri bam vdi cac vi sinh vat gay benh, khdng cho chiing tiep
xiic true
tiep vdi cac co quan cua co the.
- Co kha nang sinh cac
ch§t ire
ch6,
ngan can su sinh trudng manh me
ciia
cac vi sinh vat gay benh. Cac chat nay gdm nhieu loai cd the tac ddng don le hoac
phdi hgp vdi nhau, bao gom cac chat khang sinh, bacteriocin, siderophore, lysozym,
protease,
hydroperoxit
- Co kha nang sinh trudng nhanh, canh tranh thuc an, hda chat, nang lugng
vdi cac vi sinh vat cd hai. Vi du vi khuan probiotic sinh siderophore co kha nang
lien
k6t
vdi ion sat, lam cho vi sinh vat gay hai khong sinh trudng dugc vi thieu sat.
- Tang cudng kha nang mien dich, tang cudng dap img mien djch tu nhien d
tdm va kha nang tao thanh khang the d ca.
- Co kha nang cai thien chat lugng
nude

ao nuoi do
sir
hinh thanh hang loat
enzym phan giai cac chat hiru co, lam giam ham lugng BOD, giam cac khi dgc nhu
H2S,
NH3
Khong nhimg the, san pham trao ddi chat ciia vi sinh vat trong probiotic
cdn cung cap cac enzym, cac nguyen td da, vi lugng cho vat
chii,
giiip chiing sir
dung thirc an hieu qua hon va do do tang trudng tot hon,
9. KET LUAN
Ro rang mdi
lo lang
ve
sir
xuat hien cac vi khuan khang thuoc tir ao nudi
trong thiiy san do sir dung chat khang sinh, cd the truyen gen khang thudc cho cac
vi khuan gay benh d ngudi (ton tai ngay trong ao nudi tdm), lam cho khang sinh
khong cdn hieu nghiem de dieu tri benh cho ngudi nira se dugc giai tda neu thay the
bien phap khang sinh bang sir dung che pham sinh hgc. Trong thdi diem hien nay,
viec sir dung che pham sinh hoc la giai phap uu viet nhat de cd dugc nang suat, chat
lugng, hieu qua va
sir
phat trien ben virng
ciia
nganh nuoi trdng thiiy san ndi chung
va nganh nudi tom ndi rieng.
2.1.
NGUYEN LIEU

2.1.1.
Vi sinh
vat
- Cac chung vi khuan phan lap tir dat thugc mot sd tinh khu
virc
phia Bac,
nude
dua mudi, nude
biin.
- Bg vi khuan kiem dinh: Proteus mirabilis. Pseudomonas sp., Escherichia
coli.
Vibrio
parahaemolyticus, Aeromonas
hydrophyla do
Bao tang Gidng chuan Vi sinh
vat- Trung tam Cdng nghe Sinh hgc- Dai hgc Quoc gia Ha ndi cung cap.
2.1.2.
Hoa chat
Cac hda chat lam moi trudng: -Pepton, cazein, gelatin,
(Merck-Dire),
Tinh bgt
tan (Anh), CMC, Kitin
Cac hoa chat
diing
trong phan loai va nghien
ctm:
Tris-HCl;
EDTA; SDS;
isoamyl alcohol; agaroza; ethilium bromid (Sigma), sorensen, axeton (Trung Qudc)
2.1.3.

Thiet bj, dung cu
May lac on nhiet (Nhat), noi len men (Nhat), may do pH (My), may Ii tam, may
do OD, may GeneAmp PCR System, may dgc trinh tu
ABI
PRISM 3100 Avant
Genetic Analyzer (My); may ly tam lanh (Sigma,
Dire);
may soi gel (My); may do
quang phd UV3000; may chay dien di
2.1.4. Moi
tru*dng
(xem phu luc)
2.2.
PHUOTVG
PHAP
2.2.1.
Phirong
phap phan lap va tuyen chon vi sinh vat
- Phan
lap
vi sinh vat
Can 10 g mau dat cho bao binh ndn dung tich 250 ml chira 90 ml
nude
vd
triing, lac deu 30
phiit
tren may lac. Pha loang
mlu
vdi ndng do giam
dSn

theo co sd
10.
Hiit
0,05 ml djch da pha loang d tir
10^-10^
cSy
gat
d8u
tren moi trudng
Hutchinson-Clayton u d
30'^C.
Sau 48 gid,
cSy
vi
khu4n
da mgc vao mdi trudng thach
NA de
giir
giong. Doi vdi cac chung lactic, phan lap tren mdi trudng MRS,
ii
d
30V
trong 48 gid.
- Tuyen chon
Cac chiing vi khuan dugc so tuyen bang phuong phap cay vach tren moi trudng
Hutchinson-Clayton cd bd sung cac co chat: CMC, tinh bot, kitin va tren moi trudng
thach cazein/tween. De tuyen chgn cac chiing sinh vi
khu^n
sinh khang sinh, vi khuan
dugc nudi

cSy
tren mdi trudng thach NA. Sau 72 gid, xac djnh hoat tinh khang sinh so
rong tu ddi vdi cac chiing vi khuan lactic, kiem tra
sir
xuat hien cac khuan lac cd vdng phan giai
CaCOa
bao quanh.
-
Phirorng
phap xac djnh boat tinh enzym va hoat tinh khang sinh
*Phuang
phap dat thdi thach
* Phuong phap nhd dich.
Hoat tinh enzym/hoat tinh khang sinh (HTE/HTKS) xac dinh theo cdng thirc:
HTE/HTKS = D - d (mm).
D:
Dudng kinh vdng phan giai/vdng vd khuan
+
dudng kinh
Id
khoan/dudng
kinh thdi thach.
d: Dudng kinh
16
khoan/dudng kinh thdi thach,
- Xac djnh sinh khoi bang phuong phap do mat do quang hgc-OD d budc sdng 620nm
-
Phuong
phap dem so lu-ong te bao
Djch nudi cay vi khuan d cac dieu kien thich hgp dugc pha loang vdi mdi ndng

do ke
ti^p
giam 10 lan, Sau dd cay gat 0,05 ml djch pha loang tren
dla
thach mdi
trudng,
giir
trong
tii
am
30^C.
Sau 24 gid
d^m
sd lugng
khuin
lac mgc tren
dla
Petri.
Cdng thuc tinh sd lugng
t6
bao: So
lu'ong
te bao = ax
1/Kxl/V
a: sd khuan lac trung binh moc tren
dTa
thach.
K: do pha loang.
V: the tich dich pha loang dugc cay gat tren dTa thach.
-

Phu-ong
phap xac djnh vi
khuSn
G(+) hay G(-)
Cd dinh tieu ban vi khuan bang ngon
lua
rdi nhudm
b^ng
dung dich tim
kk
tinh
trong khoang mot phut. Rira bang
nude.
Nhudm
tidp bang
dung dich
lot
(dung djch
Lugol)
trong mot phut. Rira
b^ng
nude.
Phii
len \k boi dung dich etanol 95%: axeton
(1:1)
trong mot phut va lai rira bang
nude.
Sau dd nhudm
tilp
b^ng

thu6c nhudm mau
dd
Saft-anin
trong 30-60 giay.
Rua
qua
nude,
dh
kho rdi soi kinh. Nhom G(+) se khong
bi dung moi
hiru
co (etanol, axeton)
xky
phirc
ch^t
mau giira tim
kit
tinh va idt nen se
bat mau tim. Nhom G(-) bj dung mdi tay mau thu6c nhudm mau
diu
nen se
bit
mau
d6(Safranin)[15].
- Phu-ong phap
chuSn
do Therner
De xac djnh ham lugng axit
ciia
cac chung vi

khuAn
lactic, \iy 10 ml djch sau
nudi
cky
b6 sung 20 ml
nude ck
va
1-2
gipt
chi thj phenolphtalein 1%.
Chuin
do bang
NaOH 0,1N den khi xuat hien mau hong nhat
b^n
trong 30 giay thi dimg lai. Xac dinh
so ml NaOH da chuan do. Do axit dugc tinh theo cong thirc:
°T
=
VwaOH X 10
% Axit lactic
- ^T
x 0,009
"^rr^rxn AA ^T
1Q
Ar\
Thorn*
"• ' '^o
Therner tuong
duong
vdi 9 mg axit lactic.

10
bacterioxin
Ly tam djch nudi cky vi
khuSn
lactic bang may ly tam lanh vdi
toe
do 10.000
vdng trong 15 phut, loai bd kk
tiia,
thu
iky
phkn dich trong. Bo sung axetol lanh vao
djch nay theo ty le
thi
tich
1:1.
Sau khi
giir
lanh
4^C
qua dem, ly tam thu ket
tiia,
lam
khd tua d nhiet do phdng va hda tan tua bang dem Sorensen 0,02M
dSn
pH 7. Thir hoat
tinh khang sinh theo phuong phap
khuSch
tan tren thach [58].
-

Phirong
phap djnh
lirong
kitinaza
Kitin la mot polyme dugc cku thanh tir cac don phan la cac phan tu
N-acetyl-D-
glucosamin
(GlcNAc)
n6i vdi nhau qua lien kk (3-1-4. Ham lugng kitinaza dugc xac
dinh d budc song 585 nm thdng qua ham lugng
N-acetyl-D-glucosamin
(GlcNAc) giai
phdng ra dudi tac dung
ciia
enzym nay.
Phuangphdp
tien
hanh:
tron
nhirng th6
tich bang nhau (0,2 ml) djch enzym tho
vdi 0,5% keo kitin trong dem axetat 0,05M, pH 6. Phan img dugc thuc hien d
37^C
trong 30 phut. Ham lugng GlcNAc dugc giai phong ra dudi tac dung
ciia
enzym dugc
do d budc sdng 585 nm bang may quang ph6. GlcNAc (Sigma, My) dugc diing lam
tieu chuan, vdi he sd hap phu la 2,4 x 10 [52].
Dua vao dinh luat Lambert-Beer ta tinh dugc ndng do
ciia

GlcNAc giai phong
ra theo phuong trinh:
OD585
=
KCul
(*)
Trong do: K la he s6 hkp phu
(K=2,4xl03)
Cu
la nong do chat hap phu (GlcNAc)
I la chieu dai anh sang qua dung dich
(1=1
cm).
-
Phirotig
phap xac djnh anh hudng cua cac dieu kien nuoi cay den kha nang tong
hop enzym va khang sinh
a. Lua chgn moi
tru&ng
nuoi cay thich hgp
Vi khuan dugc nudi tren mdi trudng thich hgp d
28-30^C
hoac
40-45°C.
Sau 48
gid nudi cay xac djnh hoat tinh enzym, pH sau nuoi cay, va sinh khdi te bao. Sau 72
gid nudi cay, xac dinh hoat tinh khang sinh, pH, va sinh khoi te bao.
b. Anh huong cua thdi gian
Vi khuan dugc nuoi cay tren mdi trudng thich hgp d
28-30'^C

hoac
40-45V.
De
xac dinh thdi gian thich hgp cho
sir
tdng hgp enzym, chat khang sinh va ddng thdi xac
djnh sinh khdi. Nuoi cay trong vong 72 gid, sau mdi 12 gid xac djnh hoat tinh enzym,
sau mdi 24 gid xac djnh hoat tinh khang sinh va xac djnh sinh khoi bang phuong phap
do mat do quang.
c. Anh huong cua pH nuoi cay
11
mdi trudng thich hgp d
28-30°C
hoac
40-45X,
pH
tir 4
dfin
9. Sau dd xac djnh pH sau nuoi cky, sinh khdi, hoat tinh enzym (sau 48 gid)
va khang sinh (sau 72 gid).
r
d.
Anh
hu&ng
cua nhiet do nuoi cay
Vi
khukn
dugc nudi cky d cac nhiet do: 20; 25; 30; 35; 40; 45;
50^C,
Sau 48

gid xac djnh pH sau nudi cky, sinh khdi, hoat tinh enzym. Sau 72 gid xac djnh pH sau
nudi cky, sinh khoi, hoat tinh khang sinh,
e. Anh
hu&ng
cua nguon cacbon vd nito
Vi
khuan
dugc nudi cay tren moi trudng thich hgp cd bo sung 1% cac ngudn
cacbon hoac nito hiru co, v6 co nhu: tinh bgt, glucoza, saccaroza, CMC, kitin, cam,
hot dau tuong, cao men, pepton Sau 48-72 gid, xac djnh kha nang sinh trudng va kha
nang sinh enzym, khang sinh
ciia
cac chiing.
- Anh hudng cua mot so yeu to den boat
tinh
enzym
Cac chung vi khukn cd kha nang sinh enzym dugc nuoi cay lac 220
vong/phiit
d
diSu
kien toi uu. Sau 48 gid, ly tam 8000
vong/phiit
trong 15
phiit d6
thu djch enzym
tho.
a, Anh huong
ciia
nhiet
dp

den hoat tinh enzym
TiSn
hanh theo phuong phap nho djch. Sau dd dat cac dTa Petri vao thang nhiet
dp:
20^ 30^ 40^ 50^ 60^
Sau 24 gid, do vdng phan giai co
chkt
b,
Anh
hu&ng cuapH
den hoat tinh enzym
Tikn
hanh theo phuong phap nho djch, nhung tren co chat dugc pha trong dung
djch dem tao ra gia trj pH 3, 4, 5, 6, 7, 8, 9. Sau 24 gid do vong phan giai co chat.
c, Anh huong
ciia
thdi gian bao quan den hoat tinh enzym
Djch enzym tho dugc bao quan d nhiet do
-4^C
trong 5 tuan. Tien hanh thir hoat
tinh theo phuong phap nhd djch sau mdi tuan.
- Xac djnh kha nang chju man
Vi sinh vat dugc cay tren mdi trudng thich hgp cd ndng do mudi tang dan tir
0% den 12%. Sau 48-72 gid, pha loang djch nudi d nong do thich hgp xac djnh kha
nang sinh trudng, hinh dang va kich thudc khuan lac theo phuong phap dem sd lugng
te bao va xac djnh hoat tinh sinh hpc cua chiing,
- Xac djnh tinh doi khang
Cac chiing vi sinh vat dugc tien hanh thir tinh doi khang theo phuong phap nho
djch tuong tu nhu xac djnh hoat tinh khang sinh.
-

Phuong
phap tao che pham
a, Lua chgn chat mang cho len men xdp: Cay gidng cap hai vao cac moi trudng
cd chira cac loai chat
manp khac
nhau nhu: Bgt gao, bgt dau tuong, cam, bgt thoc mam
12
m.
Bd sung nude den dp am
45-50%.
U 48 gid d
30**C,
sau dd xac djnh sd lugng
ik
bao
ciia
timg loai vi khukn.
b.
Ty le gidng cay thich hgp: Gidng ckp hai dugc bd sung vao mdi trudng chira
chat
mang thich hgp ddi vdi timg loai vi
khuSn
theo ty le khac nhau:
0,1-0,5-1-5-10-
13-15-20
%. Bd sung nude
dk
do im thich hgp va u 2 ngay, sau dd
dim
sd lugng

ik
bao cua timg loai vi
khuSn dk
xac dinh kha nang sinh trudng cua cac chiing.
c.
Thai
gian
ii
thich hgp: Chat mang sau khi bd sung gidng va lam
am
dugc u d
nhiet do
30V.
Cir sau 24 gid
tiln
hanh
lay mlu
xac dinh sd lugng te bao cua timg loai
vi khuan.
d.
Thai gian bao quan cua chi phdm: Djch len men sau thdi gian thich hgp
dugc trdn vdi chat mang theo ty le gidng cky thich hgp, sau khi u dem sky. Trdn cac
thanh phan va nghien nhd, ddng vao cac gdi polyme hai
Idp
250g. Kiem tra bien dong
s6 lugng timg loai vi khuan va hoat tinh sinh hgc cua che pham moi thang.
- Thir
nghif
m nuoi tom (xem
chuong

3)
a, Nuoi
torn
sd
trong
phdng thi nghiem
Tdm su dugc nuoi trong be kinh, the tich mdi be 100
lit,
nude
lay tir ao lang
nude
nudi tom, vdi dp man
25%o,
mdi be tha 100 con tdm su vdi trgng
lirgng
ban dau
0,7- 0,8g va kich thudc 2,5-3cm. Be thi nghiem bo sung
0,5gA
che pham vi sinh, be ddi
chung khdng bo sung che pham. Hang ngay cho tdm an
thiic
an cong nghiep va
sue
khi
trong qua trinh nuoi. Cir sau 7 ngay thay 1/3
nude
va can do trgng lugng
ciia
tom.
h,

Khao nghiem
che
phdm VSV ngodi ao nuoi
torn
Thi
nghiem dugc tien hanh d dia diem Nuoi tom cong nghiep: Xi nghiep nuoi
trong thiiy san Dinh Vu, Hai Phdng: Dac diem ao nudi d vu
thii
3, dp sau
ciia nude
dat
tir
1,0-1,2m,
cac ao tom xay dung tren nen rimg ngap man, trudc dd la bai
cay sii
vet
day dac. So do bd tri thi nghiem nhu sau: Tdng 3 ao nudi tdm, 2 ao thi nghiem, ao sd 1
CO
dien
tich
10.000m-, ao sd 3 cd dien tich
5.000m^,
ao sd 2 ddi chung cd dien
tich
5.000ml
Ao
1
2
3
Cong thuc

Thi nghiem
Ddi chiing
Thi nghiem
Cbe pham
EMUNIV-S
Khdng sir dung
EMUNIV-S
Mat do
(con/m^)
20
20
20
Nen day
Dat Cling
Dat Cling
Dat cung
+ Tat ca cac ao deu dugc xir ly trudc khi thi nghiem theo dung quy trinh cua ao
nudi cdng nghiep (xir ly phai day, xir ly
nude
bang chlorin, diet tap bang saponin, khir
frnno Kano vAi
hAt^
13
lay
nude,
han che sir dung hda chat trong chu ky
nudi. Trong cac ao deu cd he thdng quat
nude,
cung cac che do cham sdc Theo ddi
cac

chi
s6 mdi trudng thuy ly, thuy hda dinh ky 10 ngay mot Ian.
+ Trong qua
trinh
nudi tat ca cac ao
deu
sir dung cung
thiic
an tdm cdng nghiep.
+ Ao sd
1
va sd 3 bo sung che pham vi sinh chu ky 10 ngay mot lan. Thang
thii
nhat
2,5kg/ha/lSn.
Thang
thii
hai
4kg/ha/lSn.
Thang
thii
3 trd len
6-7kg/ha/ian.
+ Phan tich
nude
theo tai lieu "Hudng
dSn
phan tich
mSu
cho ao nudi trong thiiy

san" cua Claude E. Boyd & Craig Stueker vdi may quang pho UV/vis - Hitachi,
Model:
U-2010.
+ Xac dinh cac chi sd thuy ly, thuy hda theo tai lieu [26].
-H
Thu
mau
sinh vat
ndi
bang
ludi
vdt
hinh
chdp ndn, dudng kinh mieng
ludi
25cm, chieu dai 90cm. Vai ludi vdt thuc vat ndi cd 75 (75
sgi/cm^),
vai ludi ddng vat
ndi
CO
49.
+ Mau sinh vat ndi dugc cd djnh trong dung dich formalin 5%.
+
Kiem tra tdc dp sinh trudng cua tdm dinh ky 15 ngay mot lan. Danh gia ty le
sdng va nang suat tdm tinh toan sau khi thu hoach.
+ Kiem tra sinh trudng cua tdm bang phuong phap do chieu dai tdm theo dinh
ky bang thudc chia
dp.
Can trgng lugng tom bang
can

dien tir cd dp chinh
xaclO'^g,
-h
Ty le tdm sdng dugc xac dinh bang phuong phap "sir dung sang an" trong
thang nuoi
thii"
nhat, phuong phap quang chai va xac dinh sd lugng ca
the/m^
tir thang
nudi thir hai den khi thu hoach, ket hgp vdi ket qua can dem sau khi thu hoach.
+ Xac djnh nang suat nuoi tdm sau khi thu hoach, trir cac chi phi thii:c an, con
gidng, hda chat va cac chi phi khac.
+ Thdi gian khao nghiem bat dau tir 28/03/2006 den 30/08/2006.
-
Phuong
phap xac djnh mot so dac diem sinh hoc cua vi khuan
Xac dinh khd ndng sinh bdo tu
Cay vi khuan tren moi trudng thach thudng. Sau 3 ngay tien hanh xir ly nhiet d
80^C.
Diing vi khuan da xir ly nhiet cay lai tren dTa thach thudng dat d
30^C.
Sau 24
gid neu xuat hien khuan lac thi ket luan chiing vi khuan cd kha nang sinh bao tir. Neu
khdng thi ket luan ngugc lai.
Xac dinh khd ndng hda long gelatin
Cay vi khuan theo phuong thang dimg, sau vao ong nghiem moi trudng gelatin
dung. Sau 24 gid
ii
d 30V, lay ong nghiem ra giir d
4V

trong 1 gid. Kiem tra ket qua
neu thay mdi trudng
van
long thi ket luan co kha nang hoa Idng gelatin. Ngugc lai la
khdng cd kha nang.
14
C02
cky vi
khuSn
vao dng nghiem chua 5 ml mdi trudng thach thudng djch the da
dat dng Duham. Dat d
30°C,
sau 24 gid
kiim
tra
nSu
thdy trong dng Duham cd khi noi
len, kk luan cd kha nang sinh khi. Ngugc lai ket luan khdng cd kha nang sinh khi
CO2.
Xac
^nh
hoat tinh catalaza
cky vi
khudn
vao dng nghiem
chiia
5 ml mdi trudng thach thudng djch the, dat
d
30V.
Sau 24 gid, nhd 2 gigt

H2O2
vao djch nudi cay.
NSu siii
bgt ta kk luan cd hoat
tinh catalaza. Ngugc lai khdng co hoat tinh.
- Phan loai theo sinh hoc phan tir
O
Phuangphdp
tdch ADN vi khuan
-
Lay
2 vong que cky vi khukn hoa vao 200
^1
TE trong dng
Eppendoft
- Them lyzozym vao, trdn deu, sau dd u d
37°C
trong 30 phiit
-
Them
100
|il SDS
10%,
u d
37^C
trong 30 phiit
- Them 300
^il
PCI (phenol: chloroform: isoamyl alcohol) vao, trdn deu trong
da lanh, sau dd ly

tam
vdi van
toe
15.000
vdng/phiit.
Sau ly tam, \ky djch tren.
(Budc nay dugc lap lai 2
lan)
- Diing etanol lanh vdi the tich gap 2 lan the tich mau de
tiia
ADN.
- Rira
tiia
bang etanol 70%.
- Lam khd ADN bang may co chan khdng.
- Them 30-50
^il
nude,
bao quan de diing dan.
ODien
di tren gel agaroza
Day la ky thuat quan trpng vi do la
each chii
yeu lam cho cac doan axit nucleic
hien thj true tiep. Phuong phap nay dua tren mot dac tinh cua axit nucleic la d pH
trung tinh mang dien tich am nhd cac nhdm photphat nam tren khung photphodieste
ciia
cac sgi axit nucleic. Dieu do cd nghTa la cac phan tir se chay ve
circ
duong khi dat

trong dien trudng. Ky thuat nay dugc tien hanh tren mot dem gel cd tac dung phan
tach cac axit nucleic theo kich thudc.
- Tien hanh: Dun tan 1% agaroza trong dung dich dem TAE
Ix
dd vao khuon, dgi
cho ngudi va dat tam gel vao trong may dien di, ngap trong 300ml dung djch
IX
TAE.
Trpn deu
2|il
dung djch loading buffer 6x vdi
5|il
mau, nho vao gieng. Chay
dien
di
bang dong dien mot chieu vdi dien the
lOOV,
cudng do ddng dien 80mA trong 30
phiit, bd ra ngam trong dung djch EtBr (nong dp 0,5
|il/ml)
20 phiit vdt ra. Quan sat
vach ADN tren may soi gel.
15
Thanh phan
10 X
buffer
dNTP
1,25
mM
Moi xuoi

Moi ngugc
Taq polymeraza
Mau
Nuoc
The tich (%)
10
16
1
(10pmol/^l)
1
(10pmol/|il)
1,2
2
Du 100
Mdi xudi:
5'-
AGAGTTTGATCCTGGCTCAG -3' tuong img vdi vj tri
nucleotit 27 den 47 cua E.coli
Moi ngugc: 5'- AAAGGAGGTGATCCAGCC -3' tuong ung vdi vj tri
nucleotit 1525 den
1507 cm
E.coli
- Chu trinh nhiet
cho
phan img PCR
Buoc
tien hanh
1
2
3

4
Nhiet do
(°C)
94
lap lai 30 lan chu ky sau
94
55
72
72
4
Thai gian
1 phiit
30 giay
45 giay
2 phiit 30 giay
7 phiit
-
Kiem tra cdc san phdm cua PCR bang dien di: Tien hanh tuong tu nhu doi vdi
dien di genom.
O
Phan ung khuech dgi ADN
cho
giai trinh tu
Sir dung bp kit Cycle sequencing vdi hon hgp phan ung nhu sau :
Terminator Ready Reaction Mix
Mau
8|il
l^il
Mdi
Nude

cat
In!
dii
den 20|il
Chu trinh nhiet
Bu-tfc
tien hanh
1
2
3
Nhiet do
("C)
96
lap lai 25 lan chu ky sau
96
50
60
4
Thoi gian
1 phiit
10 giay
5 giay
4 phiit
-
16
nucleic
Do trong thuc
tk
thudng phai sir dung nhimg lugng axit nucleic rat nho (thudng
la micro-, nano- hoac picogram) khi tien hanh cac thi nghiem tach ddng. Khdng the

xac dinh sd lugng nay mot each true tiep ma ndng do
ciia
dung djch axit nucleic dugc
xac dinh bang each do do hap thu tai budc sdng 260 nm
(A260)
trong may do quang
phd kk. Mot don vj (1,0) gia trj
hap
thu budc sdng 260 nm tuong duong vdi ndng do
50|ag/ml
cua ADN sgi kep, hoac tuong duong vdi ndng do 40|ig/ml cua ADN hoac
ARN mach don. Ti so
A260/A280
la chi sd cho thay do
nhilm
cac chat nhu phenol hoac
protein. Ty sd
A260/A280 la
1,8
ddi vdi
mau
ADN sach.
O
Doc
trinh
tu ADN
Trinh tu
ciia
rADN 16S cua cac chiing vi khuan dugc dgc
true

tiep tren may
doc trinh tu tu ddng ABI
3100
Avant. Sau dd ket qua trinh tu dugc so sanh vdi cac trat
tu cua cac loai da co trong ngan hang gen qudc te de xac dinh den ten loai.
DAI HOC OUOC GIA HA
NQl
TPUNG
TAM
IHONG
TIN
THli
VIEN
0006000006^
17
KET QUA
VA
THAO LUAN
Tir 47
mlu dat
thu dugc tir Hda Binh, Ha Tay, Hai Phdng chiing tdi da phan
lap dugc 130 chung vi khuan tren mdi trudng Hutchinson-Clayton d cac nhiet do 30 C
va
40*^C,
tir dd so bg
tuy^n
chgn dugc
11
chiing cd kha nang sinh enzym phan giai
protein, CMC, tinh bgt, kitin,

lipit
(trong dd cd 5 chiing ua
am:
VI1,
MIO,
III,
112,
V13 va 6 chung ua nhiet: V20,
CB51,
L45,
L50,
T9, K8) va 4 chiing cd kha nang sinh
khang sinh manh: V72, V75, K9, K13.
3.1.
VI KHUAN PHAN GIAI HOP CHAT
HLTU
CO
Thanh
phan
cac chat hiru co du thira trong ao nuoi
vk
da dang chira ham lugng
protein, tinh bgt, kitin, lipit kha cao, cd ngudn gdc tir thuc
an
thira, xac ddng thuc vat,
chat thai tir tdm la mot trong nhung nguyen nhan chinh lam d nhilm mdi trudng ao
nuoi. Do dd, can ihik phai bd sung cac vi sinh vat cd kha nang
phan
giai cac chat hiru
CO

manh nham han che tinh trang 6 nhiem hiru co trong ao, tir dd giam thieu benh tdm.
3.1.1.
Tuyen chon cac chung vi khuan co kha nang sinh enzym cao
Bang
1.
Hoat tinh enzym ciia cac chiing phan lap
Chiing vi
khuan
V20
L45
L50
CB53
T9
K8
Vll
V13
MIO
111
112
Hoat
ti'nh
enzym (D - d, mm)
Kitinaza
21
16
17
21
23
24
29

19
20
20
26
Amylaza
28
21
17
22
22
24
24
21
17
20
20
Proteaza
27
15
18
18
22
10
23
17
18
16
21
Lipaza
16

5
12
14
9
8
17
13
10
10
9
CMC-aza
24
21
20
15
22
25
28
18
17
17
24
Vi khuan dugc nuoi
cay
lac 220 vong/phiit tren mdi trudng Hutchinson-
Clayton, pH 7 d
30^C
vdi cac chiing ua am va
40^C
doi vdi cac chiing ua nhiet. Sau 48

gid xac djnh hoat tinh enzym bang phuong phap khuech tan tren thach. Ket qua ghi d
hana 1
nhr^
thaw hai ^hnr^rr voo va V11 CO
hoat
tinh
phan giai liptit manh nhat. Chiing
oteaza manh, trong khi chung
Vll
lai cd kha nang
sinh kitinaza va CMC-aza manh hon so vdi cac chung khac. Chiing tdi
lira
chgn ra hai
chiing V20 va
VI1 dS ti6p
tuc nghien cuu.
Do cac chung dugc
lira
chgn trong
ch6 phkm
phai cd nguon goc rd rang, nen
chiing phai dugc xac dinh cac dac
di6m
hinh thai, dac dilm nudi cay, sinh ly, sinh hda
vaphan
loai.
3.1.2. Phan loai cac chung vi khuan
duac
lua chon
5.7.2.7. Phan loai vi khuan den cdp dg chi theo phuong phap truyen thong

Xac djnh cac dac dilm hinh thai, nhudm Gram, nhudm bao tu, kha nang di
dong, kha nang chju mudi, kha nang ddng hda cac loai dudng cua hai chiing vi khuan.
Kit qua
cho thay:
Bang 2. Dac dilm phan loai cua 2 chiing vi khuan
Vll
va V20
3f
Dac diem
Giong non co hinh que
Dudng kinh > 2,5
jim
Tiem mao
77.
^
Tiem mao co hinh que hoac cong
Noi bao tir
Kha nang di dong
Thuoc Gram (+)
Hieu khi bat buoc
Nhu cau
NaCI
cho sinh trudng
2-10%
Ket qua
+
-
-
-
+

-1-
+
+
+
• ,
Dac diem
Hieu khi
tiiy
tien hoac vi hieu
Kj
khi bat buoc
San pham len men toan lactat
Khir sunfat thanh
sunfit'~
^ .,
Catalaza
Oxidaza
Sinh axit tir glucoza
Khir nitrat thanh nitrit
Hoa long gelatin
Ket qua
^
-
-
—
-
+
+
-
-

+
10
Hinh
1.
Hinh dang khuan lac chung V20
Hinh 2. Hinh dang khuan lac chung
VI1
Chung
¥20:
Khuan lac hmh trdn, cd mep
viln
xung quanh.
BI
mat khuan lac
khd, san, cd mau
trSng
nga anh vang. Te bao hinh que, di
ddng.
Gram duong va bao tu
hmh trdn.
Chiing
Vll:
Hinh thai khuan lac trdn, be mat hoi
xii
xi, cd mep vien, mau trang
dye.
Te
bao
hinh que, di
dgng.

Gram duong va bao tir hinh trdn.
Theo md ta
ciia
Bergey (1994),
chimg
V20 va
VI1
thugc chi Bacillus.
20
intu
Trong thirc
tl,
khi diing phuong phap phan loai truyen thdng cd the phan loai
chinh xac
din
ckp dp chi nhung
din
ckp dp loai thi dp tin cay ddi khi khdng cao. Vi
vay,
vi?c
phan loai bang phuong phap sinh hpc phan tir la rat
can
thiet. De djnh ten
din
loai cua 2 chung vi
khuan
nay, trinh tu ADNr 16S dugc phan tich theo cac budc
nhu d
hinh 3.
Kilm tra san pham dien di tren gel agarose 1% cho thay tren ban gel thu dugc

bang ADN
ting
sd duy nhat, trong dd cd ADNr 16S. ADN nay dugc dung lam khudn
cho phan ung PCR
dl
nhan gen len nhieu
lan
bang cac cap mdi da
thilt
ke dua tren
trinh tu
bao
thu cua gen ARNr 16S
ciia
E.coli. Nhu vay, theo ly thuyet thi san pham
PCR thu dugc phai co chieu dai xap xi
1500bp.
Kit
qua dien di d hinh 4 cho thay chi
xuat hien bang cd kich thudc
1500bp,
rd net, khdng bj dut gay, cd dp tinh sach cao, cd
thi
sir dung trong cac phan img tiep theo. San pham PCR sau khi tinh sach bang bd kit
Vivapure dugc kiem tra do tinh sach va ham lugng ADN. Sau dd dugc khuyech dai
tilp
vdi cac mdi va hda chat cua bd kit Cycle sequencing de chuan bi xac djnh trinh
tir
tren may dpc trinh tu tu ddng ABI
3100

Avant. Ket
qua
trinh tu
dugc
so sanh vdi trinh
tu cua cac loai da cd trong
ngan
hang gen qudc te de djnh ten den loai va xay dung
cay
phat sinh chiing loai.
Tach ADN vi khuan
i
Dien di tren gel argarose
Khuyech dai ADN bang phan img PCR
i
Kiem tra san pham phan img PCR bang dien di
i
Khuyech dai ADN cho giai trinh tu
i
Xac dinh ham luong ADN
i
Doc trinh tu ADN tren may ABI
3100Avant
^ '
««•
^MA
IMMtt **"*•'
^ISOObp
Hinh 3. Cac budc tien hanh xac djnh Hinh 4.
Kit

qua dien di san pham PCR
trinh tu ADN vi
khuan
1 - Marker 2,3 - V20 4,5 -
VI1
91
ig
V20:
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC
GGACAGATGGGAGCTTGCTCCCTGATGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGC
CTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTT
CAATTATAAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTG
AGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGAC
TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT
GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAA
CAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTG
CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAACGCGCGCA
GGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGA
ACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGG
AACACCAGTGGCGAANGCGACTCTCTGGTCTGTACTGACGCTGAAGCGCGAAAGCGTGGGGAGCG
AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCG
CCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACT
CAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAG
AACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGT
GACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG
CAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGG
AGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATG
GGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGA
TCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGT
GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCG

GTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAA
GGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTT
Trinh
tu
ADNr
16S cua
chung
Vll:
AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC
GAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTG
CCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGT
TCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTG
AGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGAC
TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT
GACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAA
CAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTG
CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGC
AGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGA
GACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATGGAGGA
ACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCA
ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGC
CCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTC
AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGA
ACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTG
ACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC
AACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGA
GGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGG
ACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATT
GTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGA
ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGT

GGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAG
iGATCACCTCCTTT.
99
50,
100
100
78
uih
vdi trinh tu nucleotit cua ADNr
16S
da cong
)d tren ngan hang gen qudc tl
(http://wv^^.ncbi.nlm.nih.gov)
bang chuong trinh
3LAST.
Kit
qua:
• Chiing V20 cd dp tuong ddng so vdi chung 99% (1538/1543bp) B.
licheniformis
• Chung
VI1
cd dp tuong ddng 99,8% (1507/1510 bp) vdi chung B. subtilis.
PHYLIP_1
Bacillus
anthracisAYI 38383
Bacillus
cereus An
38279
I
I Bacillus thuringiensisAF

15595
ji
Bacillus
herbersteiniiAJ78102
I
I ;
I
Bacillus flexus
Bacillus megaterium
- Bacillus simplex
Bacillus asahii
Bacillus muralis
' Bacillus
psychrosaccharolyticus
- Bacillus
indicus AJ583158
100
Bacillus cibi AY550276
Bacillus firmus
Bacillus pumilus AB048252
Bacillus
atrophaeus
"
Bacillus mojavensis
V20
Bacillus licheniformis
Bacillus
vallismortis
Bacillus nematotocita
Bacillus subtilis

Bacillus amyloliquefaciens
86
99
100
98
51
52
100
-53
100
:
:
981
!
90"
55
55
54
0.01
Hinh 5. Cay phat sinh chiing loai
ciia
chung V20 so vdi cac loai
gan
gui dua tren trinh tu
ADNr
16S
9^
Bacillus thuringiensis
Bacillus anthracis
977 Bacillus cereus

r-
Bacillus herberstelnii
602 1000,
Bacillus
flexus
'• Bacillus megaterium
I
868 532i Bacillus
simplex
Bacillus
asahii
!
978
932 j
- Bacillus muralis
1
Bacillus psvchrosaccharolvticus
p
Bacillus
rirmus
602
1000 Bacillus indicus
I,
Bacillus cibi
'
i
-
_ Bacillus pumilus
Bacillus atrophaeus
999

I
^QQQ.
g43-
- Bacillus licheniformis
1
' Bacillus mojavensis
j
8931 "
Bacillus vallismortis
I
i
Bacillus nematotocita
V11
^^^
Bacillus subtilis
Bacillus amyloliquefaciens
700
0.01
Hinh 6. Cay phat sinh chiing loai cua chung
VII
so vdi cac loai
gan
gui dua tren trinh tu
ADNr
16S
3.1.3.
Lua chon cac dieu kien nuoi cay thich hop cho sinh tong hop enzym
5.7.5.7.
Lua chgn moi truong nuoi cay thich hgp
Hai chung vi

khuan
V20 va
VII
dugc nuoi
cay
lac 220
vdng/phiit
d tren 6 loai
mdi trudng khac nhau ky hieu tir
MTl
den MT6, pH 7, d
30^C
ddi vdi chiing
VI1
va
40 C doi vdi chiing V20. Sau 48 gid xac djnh hoat tinh enzym, gia trj pH sau nuoi
c^y
va kha nang sinh trudng. Ket qua dugc trinh bay d bang 3.
Chiing
VI1 CO
hoat tinh kitinaza va CMC-aza cao nhat tren mdi trudng MT3
vdi kich thudc vong phan giai kitin la 3 cm, CMC la 2,8 cm. Cdn chung V20 co vdng
hoat tinh phan giai tinh bpt la 3,0 cm va protein la 2,9 cm. Kha nang phan giai lipit cua
ca hai
chiing
deu khong cao vdi vong phan giai
1,7
cm.
24