CHNG I

M U
1.1.

T VN
Trn th gii hin nay, vic nghin cu mc nhim nm mc v c t nm trn

lng thc, thc phm l vn quan trng nhm bo v sc kho con ngi v vt nui.
c t aflatoxin ch yu do loi vi nm Aspergillus flavus v Aspergillus parasiticus to
ra, l c t nguy him nht v thng nhim trn nng sn, gy c cho ngi v gia
sc.
nc ta, vi c im kh hu nhit i nng m, m trong khng kh thng
cao, thi v canh tc, thu hoch thng ri vo ma ma trong khi cc phng tin thu
hoch, phi sy nng sn km, kho cha khng m bo kh ro thong mt l iu kin
rt thun li cho nm mc pht trin gy nhim c t cho thc phm v thc n chn
nui. Mt s phng php vt l (phn hy bng khng kh nng, nhit, tia x) v ha
hc (s dng cht oxy ha kh, kim, kh NH 3) c p dng ngn nga s sn
sinh aflatoxin trong thc phm v thc n gia sc. Tuy nhin, hu ht cc phng php
khng c ph bin rng ri do chi ph cao hoc kh thc hin, lm mt gi tr dinh
dng sn phm, cht lng cm quan. Mt trong nhng xu hng hin nay l p dng
phng php vi sinh vt hc. Cc loi nm mc (Rhizopus stolonifer, Rhizopus arhizus),
vi khun (Bacillus subtilis, Bacillus pulimus), nm men (Saccharomyces cerevisiae), x
khun c th nghim v kh nng lm gim aflatoxin thu c kt qu cng rt kh
quan.
c s cho php ca Khoa Chn Nui Th y v di s hng dn ca PGS.TS.
Nguyn Ngc Hi, chng ti tin hnh ti Phn lp vi khun Bacillus subtils t phn
heo, kh st kh nng c ch sn sinh aflatoxin ca cc chng phn lp c.

Mc tiu: nh gi kh nng c ch aflatoxin ca Bacillus subtilis.

Yu cu
- Phn lp v nh danh vi khun Bacillus subtilis t phn heo.
- nh gi kh nng c ch aflatoxin ca Bacillus subtilis.

CHNG II
TNG QUAN TI LIU
2.1. KHI QUT V VI KHUN BACILLUS SUBTILIS
2.1.1. Lch s pht hin
Bacillus subtilis c pht hin u tin trong phn nga nm 1941 bi t
chc y hc Nazi ca c. Lc u c s dng ch yu l phng bnh l cho
cc binh s c chin u Bc Phi. Vic iu tr phi i n nhng nm
1949 - 1957, khi Henrry v cc cng s tch c chng thun khit ca Bacillus
subtilis. T subtilis therapy c ngha l "thuc subtilis" ra i tr cc chng
vim rut, vim i trng, chng tiu chy trong ri lon tiu ho. Ngy nay, vi
khun ny tr nn rt ph bin, c s dng rng ri trong y hc, chn nui,
thc phm (trch L Kim Hu, 2005).
2.1.2. c im phn loi v s phn b ca vi khun Bacillus subtilis
2.1.2.1. c im phn loi
Theo c im phn loi ca Bergey (1994), vi khun Bacillus subtilis thuc
B: Eubacterriales
H: Bacillaceae
Ging: Bacillus
Loi: Bacillus subtilis
2.1.2.2. c im phn b
Vi khun Bacillus subtilis thuc nhm vi sinh vt bt buc ca ng rut,
chng c phn b hu ht trong t nhin. Phn ln chng c tr trong t, thng
thng t trng trt cha khong 10 - 100 triu CFU/g. t ngho dinh dng
vng sa mc, vng t hoang th vi khun Bacillus subtilis rt him. Nc v bn

ca sng cng nh nc bin cng c mt bo t v t bo Bacillus subtilis (V

Th Th, 1996).
2.1.2. c im hnh thi
Vi khun Bacillus subtilus l trc khun Gram dng c hai u trn, kch
thc 0,5 0,8 m x 1,5 3 m, thng ng n l hoc to thnh chui ngn,
c kh nng di ng, c kh nng sinh bo t nh hn t bo sinh dng, kch
thc 0,9 - 0,6 m. V tr ca bo t trong t bo sinh dng khng theo mt
nguyn tc cht ch no, c th lch tm hoc gn tm nhng khng chnh tm
(trch dn bi Nguyn Ln Dng,1983).
2.1.3. c im nui cy
iu kin nui cy: hiu kh, nhit ti u 370 C.
Nhu cu O2: Bacillus subtillis l vi khun hiu kh nhng c kh nng pht
trin trong mi trng thiu oxy.
pH: Bacillus subtilis thch hp nht vi pH = 7 - 7,4.
Trn mi trng thch a TSA: khun lc dng trn, ra rng ca khng
u, c tm sm mu, pht trin chm, mu vng xm, ng knh 3 5 mm. Sau
1 - 4 ngy b mt nhn nheo, mu hi xm.
Trn mi trng thch nghing TSA: d mc, to thnh mu hi xm, ra
gn sng.
Mi trng canh TSB: Bacillus subtilis pht trin lm c mi trng, to
mng nhn trn b mt mi trng canh, lng cn kt li nh vn my y, kh
tan u khi lc ln.
Dinh dng: cn cc nguyn t C, H, O, N v cc nguyn t khc.
2.1.5. c im cu trc khng nguyn
4

Bacillus subtilis c khng nguyn H v O, cu trc khng nguyn dng D v L ca

acid glutamic.
Sn sinh khng sinh subtilin v bacitracin c tc dng c ch vi khun G+ v G-.
Bnh hc: a s chng Bacillus subtilis khng gy bnh.
2.1.6. Bo t vi khun Bacillus subtilis
Bo t vi khun l mt kt cu do s bin i ca t bo sinh dng trong mt giai
on no ca qu trnh sinh trng ca vi khun. Mi t bo ch to thnh mt bo t
(trch dn bi Nguyn Khc Tun, 1996).
Bo t vi khun khng phi l mt b phn lm chc nng sinh sn m ch l mt
th bin i ca t bo nhm bo tn, i mi v nng cao sc sng ca vi khun.
Bo t l mt hnh thc tim sinh ca vi khun, c s khng cao vi cc nhn
t bt li ca ngoi cnh.
Theo Nguyn Xun Thnh v ctv (2005) th s tn ti lu di ca bo t l do
chng c cc c tnh sau:
-

Nc trong bo t phn ln trng thi lin kt, do khng c kh nng lm

bin tnh protein khi tng nhit .

Do trong bo t c mt lng ln ion Ca 2+ v acid dipicolinic. Protein trong bo
t kt hp vi dipicolinat canxi to thnh mt phc cht c tnh n nh cao i

vi nhit .
Cc enzyme v cc hot cht sinh hc khc trong bo t u tn ti di dng
khng hot ng lm hn ch s trao i cht ca bo t vi mi trng bn

ngoi.
S c mt ca cc acid amin cha lu hunh, t bit l cysteine gip bo t

khng vi tia cc tm .
Vi cu trc c nhiu mng bao bc v tnh t thm thu ca cc lp mng, lm
cho cc cht ha hc v cc cht st trng kh c th tc ng n bo t .

Khi gp iu kin thun li, bo t s ny mm v pht trin thnh mt t bo mi c

sc sng mnh m hn (trch dn bi Nguyn Khc Tun, 1996).
2.1.7. C ch i khng ca Bacillus subtilis vi vi sinh vt gy bnh
Vi vi sinh vt gy bnh, mi loi sinh vt khc nhau s thch hp iu
kin mi trng, sinh khun lc khc nhau. Thay i mi trng hoc
cc yu t mi trng bt li lm thay i iu kin sng, hn ch hoc c
ch s pht trin ca vi sinh vt. Thc t khi mi trng nui cy nm bnh c s
hin din ca Bacillus subtilis vi mt s lng ln s xy ra s cnh tranh dinh
dng. Cnh tranh khng gian sinh sng gia vi khun v nm, do vi khun pht
trin nhanh hn (trong 24 gi) s s dng phn ln cc cht dinh dng trong mi
trng, ng thi to ra mt s loi khng sinh nn s sinh trng ca nm b c
ch (Nguyn Ln Dng v Hong c Thun, 1976).
Phng thc dit nm v tc nhn gy bnh nh sau:
u tin vi khun Bacillus subtilis to thnh khi xung quanh tc nhn gy bnh
ngn chn khng cho chng bm vo vt ch gy bnh.
Sau , Bacillus tit ra hn hp gm nhiu lipopeptid (c gi l serenade)
lm thng vch t bo v mng t bo cht ca tc nhn gy bnh do ngn chn s
pht trin ca chng. Serenade c th c bo qun, s dng nh mt loi thuc tr su
ha hc tng hp, c kim tra v chng minh trong phng th nghim l khng gy
c i vi c hi, ong mt, chim ct,v nhiu loi khc. Serenade gm 3 nhm
lipopeptid c tn l surfactin, agrastain v iturin. Ba nhm ny kt hp vi nhau lm
tng hot tnh dit cc mm bnh k c vi sinh vt gy hi, nm mc v c bo t gy
bnh ca chng.
Surfactin l nhm cht c hot tnh sinh hc, bn cht l lipopeptid. Bn thn
surfactin khng gy c cho nm nhng khi kt hp vi iturin th tr thnh hp cht dit

nm. Surfactin c kh nng lm thng vch t bo ca cc tc nhn gy bnh v bo t

ca chng, gip cho agrastain v iturin pht huy tc dng.
Hnh 2.1. Cu trc ha hc ca surfactin
(http://www.sciencedirect.com/science/article/pii/S0927776511005807)

Agrastatin

va mi c pht hin cng c kh nng dit nm ging nh

surfactin v iturin.

Hnh 2.2. Cu trc ho hc ca Argastin

Inturin l nhm cht chim vai tr quan trng nht trong qu trnh dit nm, bn
cht l lipoprotein c chit t mi trng nui cy vi khun Bacillus subtilis. Iturin
dit nm bng cch tc dng ln mng t bo cht, lm tan mng, to thnh nhng l
thng trn lm mt tnh thm thu chn lc ca mng.

Hnh 2.3. Cu trc ha hc ca iturin

Ngoi ra, Bacillus subtilis cng i khng vi vi khun gy bnh nh Escherichia coli.
Bacillus subtilis c kh nng tng hp mt s cht khng sinh nn gy c ch qu trnh
sinh trng v pht trin ca Escherichia coli, iu c th hin qua s lng
Escherichia coli trong bng ( Nguyn Qunh Nam, 2006).
Bng 2.1. Bacillus subtilis i khng vi Escherichia coli trong mi trng TSB
Mu

Thi gian (gi)

0
12
24
1
4,45.1010
29,80.108
15,75.107
2
4,45.1010
19,95.108
9,55.107
3
4,45.1010
17,26.108
8,30.107
4
4,45.1010
19,95.108
13,67.107
5
4,45.1010
23,40.108
2,60.107
10
8
i chng
4,45.10
22,15.10
61,76.107
Mu (1,2,3,4,5): mi trng TSB cha Bacillus subtilis v Escherichia coli

36
<106
<106
<106
<106
<106
23.1012

Mu i chng: mi trng TSB cha Escherichia coli

2.1.8. Nhng nghin cu v tc dng i khng Aspergillus ca Bacillus subtilis
Trong nc:

Nguyn nh o nm 1993 tin hnh kim tra kh nng i khng ca chng

Bacillus subtilis AO1 vi Aspergillus flavus v Aspergillus parasticus v kt qu l c s
c ch nm mc Aspergillus (trch dn bi Nguyn Th Ngc Trn, 1995).
Nguyn Hu Phc v cng s nm 1993 s dng vi khun Bacillus subtilis
c ch sinh trng v sn xut aflatoxin ca Aspergillus flavus, Aspergillus parasticus
trn u phng (trch dn bi Lm Thanh Thy Trn, 1995).
Nguyn Th Ngc Trn nm 1995 tin hnh kim tra kh nng i khng ca
chng Bacillus subtilis AO1 (do trung tm cng ngh sinh hc thuc lin hip sn xut
ha cht cung cp) vi Aspergillus flavus trn mi trng thch khoai ty-glucose v kt
qu rt kh quan.
Nm 2006, Nguyn Ngc Thanh Xun vi ti Kho st kh nng s dng vi
khun Bacillus subtilis c ngun gc t t trong x l nhim aflatoxin trn nguyn liu
bp cho thy vi khun Bacillus subtilis phn lp t t c kh nng lm gim aflatoxin
trn mi trng bp. T l nui cy bo t nm mc/bo t vi khun l 1/10 3, hm lng
aflatoxin gim 99,6 ln.
Nm 2007, Phm Hng Thi thc hin ti Phn lp vi khun Bacillus
subtilis phn lp t t, kho st kh nng c ch sn sinh aflatoxin ca cc chng phn
lp c. Kt qu cho thy vi khun Bacillus subtilis phn lp t t c kh nng lm
gim aflatoxin trn mi trng bp. T l nui cy t nm mc/bo t vi khun l 1/10 3,
hm lng aflatoxin gim 8,8 ln.
Ngoi nc:
Mt s th nghim v kh nng c ch sinh tng hp aflatoxin ca mt s chng vi
khun Bacillus subtilis c thc hin vi Aspergillus flavus v Aspergillus
parasticus.
Norio Kimura (1990) s dng vi khn Bacillus subtilis ngn chn s pht
trin v sinh c t ca chng Aspergillus flavus v Aspergillus parasticus trn u
9

phng v bp. Cc chng Bacillus subtilis NK330 v NK-C-3 c ch mnh m s pht

trin ca nm mc v tng hp aflatoxin.
2.2. KHI QUT V NM MC SINH AFLATOXIN
2.2.1. Khi nim nm mc:
Nm mc l vi sinh vt (vi nm) c cu to gn ging vi thc vt, sng k sinh
hay hoi sinh trn nhiu loi cht khc nhau, c bit l cc cht hu c n gin nht l
hydrocacbon.
Nm mc khng cha dip lc t, cu to dng si c vch ngn hoc khng c
vch ngn.Nhng si nm ny phn nhnh, hnh thnh tng m chng chit gi l h
thng si nm. C 2 loi si:
Si nm dinh dng: nm trong lp mi trng, lm nhim v hp thu cht
dinh dng cho ton b h nm.
Si nm kh sinh: thng nh ln trn mi trng, gi vai tr sinh sn (to
bo t).
2.2.2. c t nm mc
L sn phm ph ca qu trnh trao i t nhin ca nm mc v c th gy c
cho con ngi v gia sc. c t nm mc c tnh bn vi nhit cao v khng b tiu
dit trong qu trnh ch bin thc n thng thng. Ty tng loi m c t nm mc c
th gy nhim c cp tnh v mn tnh.
Hin nay pht hin trn 300 loi c t mycotoxin t hn 100 loi nm mc.
Trong c khong 20 loi c t gy nguy him n sc khe con ngi v vt nui. 5
loi c t nguy him nht hin nay l aflatoxin, deoxynivalenol, zearalenone, achratoxin
v fumonisin (Trn Cc Phng, 2008).
2.2.3. c t aflatoxin
2.2.3.1. Lch s pht hin v qu trnh hnh thnh aflatoxin
Aflatoxin l c t vi nm c pht hin vo nm 1960 khi gy cht trn 10.000
g ty con nc Anh vi tn thng gan rt nng n nh hoi t, chy mu trong gan,
tng sinh ng dn mt. Tip sau ngi ta pht hin thm loi mycotoxin ny cng gy
tn thng gan sc vt th nghim khc nh vt con, chut, th, kh, heo, b, cu. n
nm 1961, Sargeant v ctv cy v phn lp c c t ny v t tn l aflatoxin
(Dng Thanh Lim, 2009).
10

Aflatoxin c sn sinh ra bi vi nm Aspergillus flavus v Aspergillus

parasiticus. Ngoi ra cn mt s loi khc nh Aspergillus nomius, Aspergillus
pseudotamarii,

Aspergillus

bombycis,

Aspergillus

ochraceoroseus,

Emericella

venezuelensis (Yu v ctv, 2004). Con ng tng hp aflatoxin bao gm t nht 18 phn
ng chuyn i bt u bng cch tng hp polypeptide t acetate, tng t nh qu
trnh tng hp axit bo. Theo Prieto v Woloshuk (1997), qu trnh hnh thnh aflatoxin
B1 nh sau: NOR averantin averufanin averufin hydroxyversicolorone
versiconal hemiacetal acetate versicolorin A saerigmatocystin Omethylsterigmatocystin aflatoxin B1.
Mt s gen tng ng quy nh cc enzyme tham gia vo qu trnh sinh tng hp
aflatoxin bao gm pksA, pksL1, fas1A, nor-1, NorA, avf1, vbs, ver1, stcP, omtA, ord1,
avnA v gen aflR (Prieto v Woloshuk, 1997). 25 gen nm trong khu vc DNA 70kb
c xc nh lin quan n qu trnh sinh tng hp aflatoxin (trch dn Yu v ctv, 2004).
Cc yu t nh hng n s hnh thnh aflatoxin c th c chia thnh 3 loi:
vt l, dinh dng v cc yu t sinh hc (Gourama v Bullerman, 1995). Trong yu
t vt l ng vai tr quan trng. Yu t vt l bao gm nhit , m, nh sng, s lu
thng kh v mc ca cc kh trong kh quyn. Aflatoxin c sn xut nhit t
12 410C v nhit ti u l 250C n 320C (Lillehoj, 1983). S sn xut aflatoxin c
bit a chung bi nhng iu kin rt m t. Tng hp aflatoxin tng ln 27 0C,
m khng kh ln hn 62% v m trong thc n chn nui trn 14% (Royes v
Yanong, 2002). S hin din ca CO2 v O2 cng nh hng n s tng trng ca nm
mc v sn xut c t aflatoxin. Nhiu nghin cu bo co rng pH ban u
khng nh hng ng k n sn xut c t aflatoxin, trong khi cc nh iu tra khc
cho thy rng pH axit yu, nm mc pht trin tt hn v sn sinh nhiu aflatoxin hn
(Landers v ctv, 1967).

11

Bng 2.2: Mt s loi nm mc c kh nng sn sinh aflatoxin

Loi nm mc
B1
Aspergillus flavus
Aspergillus parasiticus
Aspergillus nomius
Aspergillus oryzae
Aspergillus niger
Aspergillus wentii
Aspergillus ruber
Aspergillus ostianus
Aspergillus ochraceus
Penicillium puberulum
Penicillium variabile
Penicillium

+
+
+
+
+
+
+
+
+
+
+
+

Aflatoxin
B2
G1
+
+
+

+
+

Tc gi
G2
+
+

+
+

Sargeant v ctv, 1961

Coduer v ctv, 1963
Kurzman v ctv, 1987
Basappa v ctv, 1967
Kulik v Holaday, 1967
Kulik v Holaday, 1967
Kulik v Holaday, 1967
Scot v ctv, 1968
Van Walbeck v ctv, 1968
Kulik v Holaday, 1967
Kulik v Holaday, 1967
Kulik v Holaday, 1967

frequentans
Penicilium citrinum
+
Kulik v Holaday, 1967
Rhizopus. sp
+
Van Walbeck v ctv, 1968
Cc loi nm mc c vai tr quan trng trong sn sinh Aflatoxin l Aspergillus flavus v
Aspergillus parasiticus, cc loi nm khc: Penicillium spp v Rhizopus. Spp thng t c
vai tr gy bnh trong thc t (L Anh Phng, 2001).
2.2.4. Cu trc v tnh cht vt l, ha hc
Aflatoxin l hp cht hu c c nhn a mch vng l dn xut difurocoumarin.
Khong 20 loi aflatoxin c xc nh.Trong c 4 loi c sm nht v
nhiu nht l B1, G1, B2, G2. Trong nh sng cc tm bc sng di (380-315 nm), B1
v B2 pht ra mu xanh nc bin, G1 v G2 pht ra mu xanh l cy (Devero, 1999).

12

Hnh 2.4. Cu trc ho hc ca aflatoxin B1, B2, G1, G2

Mc d aflatoxin B1, B2, G1, G2 c ph bin trong cng mt mu thc n,
nhng aflatoxin B1 l ch yu, chim 60 80% tng s aflatoxin. Ngoi ra, trong hu ht
cc trng hp, aflatoxin G1 c tm thy nng cao hn aflatoxin B2 v G2
(Weidenborner, 2001).
Cc aflatoxin pht quang mnh di nh sng cc tm sng di. iu ny cho
php pht hin cc hp cht ny nng cc k thp ( 0,5ng hay thp hn trn mt vt
sc k bn mng). N cung cp c bn v mt thc hnh cho tt c cc phng php
ha l v vic pht hin nh lng. Nng aflatoxin M1 0,02mg/l c th c pht
hin trong sa lng.
Aflatoxin tinh khit rt bn bng nhit cao ln n im nng chy, khi c
lm nng trong khng kh. Tuy nhin, n tng i bn khi di khng kh v di tia
cc tm phin sc k bn mng, v c bit khi ha tan cc dung mi c phn cc
cao. Cc aflatoxin trong cc dung mi clorofom v benzene bn vng trong nhiu nm
nu c gi trong ch ti v lnh. Cc aflatoxin t hoc khng b ph hy di iu kin

13

nu bnh thng v lm nng khi thanh trng. Tuy nhin, khi c m v nhit cao
vn c th tiu hy aflatoxin trong mt khong thi gian nht nh.
Cc aflatoxin c ha tan trong cc dung mi phn cc nh nh clorofom,
methanol v c bit dimethylsufoit (dung mi thng c s dng nh phng tin
trong vic p dng cc aflatoxin vo cc ng vt th nghim). Tnh tan ca aflatoxin
trong nc dao ng t 10-20mg/l.
S c mt ca vng lacton phn t aflatoxin lm chng nhy cm vi vic thy
phn trong mi trng kim, c tnh ny l quan trng trong bt k qu trnh ch bin
thc phm v qu trnh x l kim lm gim hm lng aflatoxin ca cc sn phm, mc
d s c mt ca protein, pH v thi gian x l c th thay i cc kt qu. Tuy nhin
nu x l kim l nh th vic axit ha s lm phn ng ngc tr li to aflatoxin ban
u.
nhit cao (khong 100oC) s m vng decarboxylation xy ra v phn ng c
th tin xa hn, dn n s mt mt cc nhm methoxy t vng thm.
Khi c cc acid v c v b sung nc, aflatoxin B1 v G1 chuyn ha thnh
aflatoxin B2A v G2A. Cc sn phm cng hp tng t ca aflatoxin B1 v G1 cng
c hnh thnh vi clorua acid formic thionyl, clorua acid acetic v acid thionyl
trifluoroacetic.
Nhiu tc nhn oxy ha, chng hn nh hypochloride ratri, thuc tm, chlorine,
hydrogen peroxide, ozone v peborat natty phn ng vi aflatoxin v thay i cc phn t
aflatoxin, mt s phn ng lm mt hunh quang.
S hydro ha aflatoxin B1 v B2 sinh ra aflatoxin G1, G2 tng ng. S kh
aflatoxin B1 bng 3 mol hydro sinh ra tetrahydroxyaflatoxin. Kh aflatoxin B1 v B2
bng natriborohydride to ra RB1 va RB2 tng ng. Hin tng l kt qu ca vic
m vng lacton bi s kh nhm acid v nhm xeton vng cyclopentene.
2.2.5. C ch gy c
Aflatoxin B1 l phn t i lc mnh vi thnh rut, c trng lng phn t thp
nn d dng c hp th hon ton sau khi n.
14

Khi n rut non, aflatoxin B1 s nhanh chng c hp thu vo tnh mch v rut
non, t trng.
T ng tiu ha, theo tnh mch ca, aflatoxin tp trung vo gan nhiu nht (chim
khong 17% lng aflatoxin ca c th) tip theo l thn, c, m m, ty, lch v 80%
b bi tit ra ngoi trong khong mt tun v ng ch l n cn bi tit qua tuyn sa
gy bnh cho thai nhi ang b sa m. Chu k bn r trong huyt tng l 36,5pht,
lng phn phi l 14% trng lng c th, gii phng khi c th l 1,25l/kg/h.
aflatoxin M1 ch yu bi tit trong vng 48h (Hendrickse, 1991).
Cho n nay, cc lun chng khoa hc cng nhn kh nng tc ng ln t bo gan
ca aflatoxin tri qua 5 giai on sau:
-

Tc ng qua li vi AND v c ch cc polumeraza chu trch nhin tng hp

AND v ARN.
- Ngng tng hp AND
- Gim tng hp AND v c ch tng hp ARN truyn tin.
- Bin i hnh thi nhn t bo.
- Gim tng hp protein.
Hu qu ca qu trnh tc ng sinh ho ln t bo gan ny l gy ung th biu m
t bo gan.
2.2.6. Nhng tc hi do aflatoxin gy ra
Theo Dng Thanh Lim (2002), aflatoxin gy ra nhng tc hi rt ln cho c th con
ngi v ng vt. Nhng tc hi nh sau:
- Gy tn thng t bo gan: tt c cc trng hp xc nhn s ng c
aflatoxin u c bnh tch ging nhau l gan ca ng vt b nhim u h hi
nng. Tu theo mc nhim t hay nhiu, lu hay mau m bnh tch trn gan
c khc nhau. Biu hin chung l: ban u gan bin thnh mu vng ti, mt
sng. Sau gan sng to ln, mt cng phng v bt u ni mt nh trn b
mt gan lm cho n g gh i khi c nhng nt hoi t trng. Sau cng do
-

nhim khun m gan tr nn b, d b.

Thn cng b sng to lm cho vic bi thi cht c ra khi c th tr nn kh
khn. T lm cho triu chng ng c tr nn trm trng.

15

Bo mn ng tiu ho nn lm gim kh nng tiu ho cc cht dinh dng

trong thc n. i khi cng thy tn thng ming, lm cho th kh ly thc

n.
Lm gim kh nng khng ca ng vt, c ch h thng sinh khng th.
Do khi nhim aflatoxin c th rt mn cm vi cc bnh thng thng, c

th gy t vong cho th.

Lm thay i hot ng sinh l bnh thng, gy ri lon sinh sn.
Lm gim tnh ngon ming i vi thc n do s pht trin ca nm mc lm

mt mi thc n.
Lm h hi cc cht dinh dng trong thc n nh glucid, protein, acidamin,
vitaminLm thc n b gim gi tr nghim trng, mt mi t nhin nn th
khng thch n.

c bit, aflatoxin c c khuynh hng gy ung th.

T c th thy aflatoxin lm gim thp s sinh trng, sc sn xut ca th.
Hu qu cui cng l c th gy cht th.
2.2.7. Cc phng php c ch v loi tr aflatoxin
Phng php vt l
-

Loi b phn b nhim nm mc: cc ht b nhim nm mc thng c s thay i

v mu sc v c th c la ra.
X l nhit: nhit cao th c kh nng tiu dit nm mc sinh c t (Dng
Thanh Lim v ctv, 2006). Cn aflatoxin l cht chu nhit, v vy cc bin php
vt l bng nhit ch lm thay i nh v mc ca chng (Tripathi v Mishra,

2010).
Phng php chiu x: tia X, tia , tia UV, tia hng ngoi, nh sng mt tri c

nghin cu rng ri nhm lm gim c t aflatoxin.

Dng cc cht hp ph v cht kt dnh c t: dng mt s cht c kh nng hp
ph hoc lin kt c c t nm mc trong ng tiu ha ca ng vt nn lm
gim s hp thu qua nim mc rut (L Anh Phng, 2002).

Phng php ha hc

16

Phng php s dng cc dung mi ha cht c th trch xut cc hp cht ny m

ch gy nh hng ti thiu v cht lng dinh dng. Tuy nhin, cng ngh ny vn cn
tn km, kh thc hin trong thc t, bn cnh cn gy mi v khng tt. Ozon ha l
phng php ha hc c nghin cu nhiu nht cho vic kh nhim aflatoxin trong
thc phm.
Phng php vi sinh vt hc
Hin nay, mt s nghin cu ch ra rng aflatoxin d b mt s vi sinh vt nh
nm, vi khun v nm men phn hy sinh hc. Mt s loi vi khun, chng hn nh
Lactobacillus, Bacillus, Pseudomonas, Ralstonia v Burkholderia spp cho thy kh
nng c ch s pht trin ca nm v s sn xut aflatoxin bi Aspergillus spp trong
phng th nghim (Reddy v ctv, 2010). Taylor v ctv (2010) nghin cu mt s
enzyme thuc nhm actinomicetales Mycobacterium smegmatis c kh nng tc ng
vo nhm este ca aflatoxin bng cch kch hot cc phn t cho qu trnh t thy phn
v kh nhim. Niu v ctv (2008) nghin cu mt s vi sinh vt s dng coumarin nh
l mt ngun cacbon, kt qu ch ra rng tc ng lm gim aflatoxin c thc hin
bi enzyme protease. Nghin cu ca Cacciamani v ctv (2007) nh gi s dng
A.oryzae v Rhizopus sp. c th lm gim n 80% aflatoxin B1. Cc loi nm hoi sinh
nh Candida krusei v Pichia anomala cng ha hn cho thy l tc nhn kim sot sinh
hc aflatoxin (Reddy v ctv, 2010)

17

CHNG III
NI DUNG V PHNG PHP TIN HNH
3.1.
-

THI GIAN V A IM
Thi gian: Thi gian thc hin tiu lun t thng 10/2012 n thng 6/2013
a im: Phng thc hnh vi sinh, khoa Chn Nui- Th Y trng i hc Nng
Lm TPHCM; phng chn on xt nghim th y Hn Vit; phn tch hm lng

aflatoxin ti trm chn on, xt nghim v iu tr chi cc th y TPHCM

3.2. I TNG KHO ST
- Chng vi khun Bacillus subtilis c phn lp t phn heo.
- Chng nm mc Aspergillus flavus sinh aflatoxin do phng thc hnh vi sinh cp.
3.3. THIT B V DNG C TH NGHIM
- Thit b: knh hin vi, t sy, t m, ni hp tit trng (autoclave), cn in t,
-

my lc (vortex), l vi sng, n cc tm, ni un cch thu,

Dng c: ng nghim, a petri, que cy, n cn, bnh tam gic, bacher,
micropipette, a khuy thu tinh, ng ng, que trang, bung m Neubauer, gi
ng nghim, my in di,Tt c cc dng c thu tinh dng trong th nghim
phi c sy tit trng 170oC/45 pht. Dng c nha phi c hp tit trng

3.4.
-

bng autoclave 121oC/15 pht.

MI TRNG NUI CY
Mi trng thch nc ct da: dng kim tra kh nng sinh aflatoxin ca
Aspergillus flavus. Nc ct da ng hp c pha long vi nc theo t l 1:1.

Sau thm agar theo t l thch hp. Hp tit trng 121oC/15 pht.
Mi trng bp: bp ht trc khi dng phi c nghin nh nh ht tm. Sau
hp tit trng, xc nh hm lng aflatoxin v o hm lng m. iu chnh hm
lng m n 30% bng nc c hp tit trng, cho vo chai nu, mi chai
50g bp. Hp tit trng 121oC/20 pht trc khi tin hnh nui cy
( Thnh phn cc mi trng c trnh by phn ph lc)

3.5.

Mi trng phn lp, gi ging v m s lng vi khun: mi trng NA

Mi trng tng sinh khi vi khun : NB
Mi trng gi ging vi khun: Glycerin 30%
Mi trng gi ging nm: mi trng PDA
PHNG PHP NGHIN CU
18

3.5.1. Phn lp v nh danh vi khun Bacillus subtilis t phn heo

- Ly mu: mu phn heo c ly cho vo ti nilon, bo qun trong thng xp
ng , vn chuyn v phng th nghim.
- Tng sinh v phn lp:
Ly 10g phn heo cho vo 90 ml nc mui sinh l v trng cha trong bnh tam
gic 100ml, lc u, c nng pha long 10-1 .
Chun b 4 ng nghim, mi ng cha 9ml nc mui sinh l v trng v c
nh s th t t 1 n 4. Dng micropipette ht 1ml dch mu t nng pha long 10 -1
cho vo ng nghim th 1 v lc u bng my vortex c nng pha long 10 -2, tip
tc nh vy c 10-3, 10-4, 10-5.
Ht 0,1ml t nng pha long 10 -3, 10-4, 10-5 cho vo a thch Nutrient Agar,
mi nng 2 a. Dng que trang v trng trang u ln b mt thch. Sau em
370C/24 gi.
Quan st cc khun lc mc trn b mt thch, chn ra nhng khun lc nghi ng
l Bacillus subtilis (khun lc dng R c ra rng ca nh, kh, mu trng hi c) tin
hnh nhum Gram v quan st di knh hin vi quang hc c phng i 1000 ln.
Chn nhng khun lc c tiu bn vi khun ph hp vi nhng c im ca vi khun
Bacillus subtilis (trc khun Gram dng, 2 u trn, kch thc nh, ring l, sinh bo
t nh hn t bo, bo t nm chnh tm hoc lch tm, khng lm phnh t bo) tin
hnh cy gi ging trn mi trng thch nghing NA lm cc bc tip theo.
- K thut PCR xc nh loi: Phng php PCR c tin hnh theo m t ca
Abushady v ctv (2005), s dng cp mi 5 AAGAGTTTGATCATGGCTCAG 3 v
5 AGGAGGTGATCCAACCGCA 3. Chng trnh PCR c thit lp bin tnh
940C trong 1 pht, gn mi 50 0C trong 1 pht v ko di 72 0C trong 1 pht cho tng
s 35 chu k.
Bng 3.1. Thnh phn phn ng (Bacillus subtilis)
Thnh phn
H2O

Th tch (l)
4,5
19

GoTaq Green Master Mix, 2X

12,5

Mi xui (10 M)

1,5

Mi ngc (10 M)

1,5
20

Mu DNA

Tng th tch

25

Bng 3.2. Chu trnh nhit

Giai on

Nhit

Thi gian

Tin bin tnh

95oC

5 pht

Bin tnh

95oC

1 pht

Bt cp

55 oC

1 pht

Ko di

72 oC

1 pht

Ko di cui

72 oC

10 pht

Gi sn phm

4 oC

Primer

Trnh t

ytcP-F

GCTTACGGGTTATCCCGC

ytcP-R

CCGACCCCATTTCAGACATATC

S chu k
1
30
1

Kch thc
SP
460 bp

Pht hin
B. subtilis

3.5.2. Xc nh nh tnh kh nng c ch aflatoxin ca nhng chng Bacillus

subtilis phn lp c
Dng que cy vng ly 1 t vi khun Bacillus subtilis phn lp c c gi
trong ng mi trng NA nghing vo a thch nc ct da v trong t m 37 oC
trong 2 ngy. Mi a t 3 n 4 chng vi khun Bacillus subtilis phn lp c. Sau 2
ngy, dng que cy mc ly 1 t si nm mc Aspergillus flavus trong ng ging vo gia
a thch nc ct da cy vi khun xung quanh, gi nhit phng 3-5 ngy.

20

Theo di quan st mu hunh quang xung quanh khun lc Aspergillus flavus

cc pha c cy vi khun Bacillus subtilis v khng cy vi khun bng cch em soi di
n UV (bc sng 365nm).
Mu hunh quang cng sng t a cy vi khun no th chng vi khun c
kh nng c ch sn sinh aflatoxin cng mnh.
Sau chn nhng chng vi khun c kh nng c ch sn sinh aflatoxin mnh
nht tin hnh th nghim tip theo.
3.5.3. nh gi kh nng c ch aflatoxin ca chng Bacillus subtilis phn lp
c trn mi trng bp
3.5.3.1. Phng php thu hoch v xc nh bo t nm mc Aspergillus
flavus
Cy chng nm mc c kim tra kh nng sinh c t ln ng mi trng
thch nghing PDA, nhit phng 4 ngy. Sau cho khong 9ml nc mui sinh
l v trng vo ng mi trng PDA cy nm mc, dng que cy vng khuy nh v
ht ly huyn dch bo t bng micropipette ri cho vo mt ng nghim v trng khc.
Tin hnh m s lng bo t nm mc trong huyn dch thu hoch bng phng php
dng bung m Neubauer di knh hin vi c phng i x40 ln.
C nh bung m, t lamell ln, dng micropipette ht 1 t (khong 100l)
huyn dch bo t nm mc thu c nh vo rnh bung m cho dn u khp bung
m. Cng s bo t c c cc va m v p dng cng thc xc nh s
lng bo t nm mc/ml (xem ph lc).
3.5.3.2. Phng php thu hoch v xc nh s lng bo t vi khun
Bacillus subtilis
Cy chuyn vi khun Bacillus subtilis t ng ging sang mi trng ng thch
nghing NA, trong t m 370C trong 24h.
Dng que cy vng ly 1 t khun lc ho tan vo trong ng cha 9ml nc mui
sinh l thu c ng huyn dch vi khun. Pha long huyn dch vi khun thu c bng
cch dng cc ng nghim cha 9ml nc mui v trng, nh s th t trn cc ng
nc mui. Dng micropipette htt 1ml huyn dch vi khun cho vo ng nghim tip
21

theo trn v lc trn my vortex, thu c nng pha long 10 -2, tip tc pha long
nh trn thu c cc nng pha long 10 -3, 10-4, 10-5, 10-6. Sau em un cch thu
cc ng huyn dch c pha long 70 0C trong 15 ph tiu dit cc t bo sinh
dng v hot ho bo t vi khun. un xong, ngui, dng micropipette ht 100l
huyn dch bo t vi khun cho ln a mi trng NA (mi nng 2 a), trang u
bng que trang v trng, a NA trong tur aams 37 0C trong 24h. Sau m s lng
khun lc vi khun hnh thnh trn a
3.5.3.3. B tr th nghim
-

Chun b nguyn liu

Nguyn liu bp c x l bng cch xay nh, kch thc khong 2 3 mm, sau
c sy kh 1100C trong 30 pht nhm ngn chn s pht trin ca nm mc.
Xc nh m ca bp bng phng php sy: tin hnh sy cho n khi khi
lng bp khng thay i, t s thay i khi lng ca bp c th tnh c m ca
bp. Bp c gi trong nhng chai sm mu (khong 50gr/chai) c hp tit trng sau
b sung lng nc m t khong 30% trc khi tin hnh th nghim.
-

Nui cy chung bo t nm mc v bo t vi khun trn mi trng nguyn

liu bp

Th nghim c b tr thnh 5 l:
L 1: nui cy chung bo t vi khun Bacillus subtilis chng th nht v bo t
nm Aspergillus flavus trn mi trng nguyn liu bp ( c b sung nc t m
30%) theo t l bo t nm mc v bo t vi khun tng ng l 104/106.
L 2: nui cy chung bo t vi khun Bacillus subtilis chng th hai v bo t
nm Aspergillus flavus trn mi trng nguyn liu bp ( c b sung nc t m
30%) theo t l bo t nm mc v bo t vi khun tng ng l 104/106.
L 3: nui cy chung bo t vi khun Bacillus subtilis chng th ba v bo t
nm Aspergillus flavus trn mi trng nguyn liu bp ( c b sung nc t m
30%) theo t l bo t nm mc v bo t vi khun tng ng l 104/106.
L 4: i chng dng, nui cy 104 bo t nm mc aflatoxin trn mi trng
nguyn liu bp ( c b sung nc t m 30%).
22

L 5: i chng m, ch b sung nc.

Sau khi cy bo t nm mc v bo t vi khun theo t l nghin cu vo cc chai,
t cc chai nhit phng (25 300C). Sau 7 ngy, tin hnh phn tch hm lng
aflatoxin bng phng php sc k lng cao p.
3.5.3.3. Ch tiu theo di
Xc nh hm lng aflatoxin trong tng l th nghim nh gi kt qu c ch
s sn sinh aflatoxin ca tng chng nghin cu.
3.5.4. Phng php phn tch mu
Phn tch hm lng aflatoxin bng phng php sc k lng cao p (HPLC)
3.5.5. Phng php x l s liu
S liu c phn tch thng k trn phn mm Minitab 16.0, cc s liu c x l
bng trc nghim F (Chi-Square Test), trc nghim Turkeys (Anova).

23

CHNG IV
KT QU V THO LUN
4.1. KT QU
4.1.1. Kt qu phn lp Bacillus subtilis t phn heo
Phn heo c pha long cc nng 10 -2, 10-3, 10-4 un cch thu 700C trong 15
pht, ht ly 100l dch pha long em trang trn mi trng NA a, trong t m 37 0C
trong 18 gi. Kt qu c th hin hnh 4.1, bt khun lc c trng l khun lc kh
ra nhn, nm st b mt thch.

Hnh 4.1. Khun lc vi khun Bacillus subtils trn mi trng NA

Cc khun lc c trng c nhm gram quan st di knh hin vi ( phng i
x1000 ln) kho st cc c im hnh thi. Chng ti nhn thy cc chng vi khun
nghi ng c hnh thi ging vi
Bacillus subtilis, l nhng trc khun
bt mu gram (+), ngn v ng, kch
thc 0,5-0,8 m, hai u trn, thng
ng ring l, i khi xp thnh chui
ngn t 3-5 t bo, c bo t.

Hnh 4.2. Hnh thi vi khun Bacillus subtilis phng i x1000

24

Sau khi nhum gram xem hnh thi, chng ti chn c 150 chng nghi ng l vi khun
Bacillus subtilis
Tin hnh th i khng 150 chng nghi ng l vi khun Bacillus subtilis vi nm mc
Aspergillus flavus trn mi trng thch nc ct da. Tip tc chn ra 34 chng trn
150 chng nghi ng c kh nng c ch s pht trin ca nm mc theo hnh 4.3.

(a)

(b)

(c)

Hnh 4.3. Kt qu th i khng di n UV

(a) a khun lc i chng di n UV
(b) cc chng vi khun c ch s pht trin ca nm mc
(c) cc chng vi khun khng c ch c nm mc, vng sng aflatoxin cn r
T 34 chng Bacillus subtilis c chn sau khi th i khng c tin hnh chy PCR.
Kt qu thu c 11 chng vi khun Bacillus subtilis dng tnh (hnh 4.4).
Hnh 4.4. Kt qu in di xc nh cc chng vi khun Bacillus subtilis dng tnh

25

4.1.2. Kim tra kh nng sinh aflatoxin ca Aspergillus flavus v nh gi s b kh

nng c ch sn sinh aflatoxin ca cc chng Bacillus subtilis
4.1.2.1. Kim tra kh nng sinh aflatoxin ca Aspergillus flavus
Sau 4-5 ngy cy trn mi trng thch nc ct da em soi di n UV (365nm)
c kt qu nh hnh 4.5

Hnh 4.5. Khun lc Aspergillus flavus di n UV sau 4 ngy nui cy trn mi trng
thch nc ct da
Vng sng pht ra xung quanh khun lc nm mc l vng aflatoxin c Aspergillus
flavus sn sinh. Vng pht ra cng rng, cng sng th nng c t sn sinh cng cao,
cng mnh. Dng chng nm mc sau khi kim tra c sinh c t aflatoxin tin hnh
cc th nghim tip theo.
4.1.2.2. Th nghim nh gi s b kh nng c ch sn sinh aflatoxin ca cc chng
Bacillus subtilis phn lp c
26

Sau khi cy nm mc Aspergillus flavus ln mi trng thch nc ct da 2 ngy bt

u cy cc chng vi khun Bacillus subtilis kim tra s i khng.
Ngy u tin sau khi cy khun lc vi khun Bacillus subtilis cha ln su vo cc
khun lc nm mc Aspergillus flavus, lc ny quan st bn ngoi vn thy khun lc
nm mc cha b khuyt cc ni tip xc vi khun lc vi khun v khi xem di nh
n tia UV bc sng 365nm th vn pht hin vng sng u xung quanh khun lc
nm mc ca c t aflatoxin pht ra (hnh 4.6).

Hnh 4.6. Khun lc Bacillus subtilis (1 ngy nui cy) tip xc vi khun lc Aspergillus
flavus (3 ngy nui cy) so vi a i chng trn mi trng thch nc ct da
n ngy nui cy th 2, quan st bn ngoi thy khun lc nm mc b khuyt nhng
pha tip xc vi cc khun lc vi khun, di n UV th vng sng xung quanh
khun lc nm mc khng cn nguyn vn nh hnh 4.7.

Hnh 4.7. Khun lc vi khun (2 ngy nui cy) ln vo khun lc Aspergillus flavus (4
ngy nui cy) so vi a i chng di n UV
Sang ngy th 3, khun lc vi khun pht trin mnh ngn cn khun lc nm mc pht
trin, di n UV th vng sng aflatoxin m r nh hnh 4.8.

27

Hnh 4.8. khun lc vi

khun v nm mc sau 5
ngy nui cy

So snh kh nng c ch s
sinh aflatoxin ca 11 chng
vi khun Bacillus subtils c th hin qua sng ca vng aflatoxin di dn UV

bc sng 365nm (ti vi tr khun lc nm mc tip xc vi khun lc vi khun), kt qu

c trnh by hnh 4.9.

(chng 85)
(chng 52)

(chng 34)

hnh 4.9. Vng sng aflatoxin khng u cc pha ca khun lc

Aspergillus flavus.
Kt qu chn 3 chng vi khun Bacillus subtilis c kh nng i khng
vi nm mc Aspergillus flavus mnh nht l cc chng s 34, 52 v
85, tin hnh th nghim tip theo.

28

4.1.3. Kho st kh nng lm gim aflatoxin sn sinh trn mi trng bp ca cc

chng Bacillus subtils phn lp c
m ca bp xc nh c bng phng php sy 12%, b sung 9ml nc ct tit
trng m t khong 30%.
Bo t nm mc Aspergillus flavus c thu hoch v xc nh s lng bng phng
php dng bung m Neubauer, kt qu thu c l
Bo t vi khun Bacillus subtilis c thu hoch v xc nh bng phng php trang
a, kt qu cho thy s lng bo t vi khun ln lt l
Sau khi xc nh c s lng bo t nm mc v bo t vi khun th tin hnh nui
cy trn mi trng bp theo t l bo t nm mc/bo t vi khun l 10 4/106. Trc khi
nui cy, mi trng phi c hp tit trng 1210C/15 pht.

Hnh 4.10. Nui cy bo t vi khun v bo t nm mc trn mi trng bp.

t cc chai mi trng bp c trn bo t Aspergillus flavus v bo t Bacillus
subtilis nhit phng (hnh 4.10).
Sau 5 ngy nui cy, quan st cc mu bp cc l th nghim s c s khc bit v s
sinh trng v pht trin ca nm mc gia cc mu th nghim v mu i chng, s
khc bit ny c trnh by hnh 4.11.

29

Mu i chng (-)

Mu i chng (+)

Mu th nghim

Hnh 4.11. So snh cc mu bp.

Mu i chng (-): mi trng bp ch b sung m.
Mu i chng (+): mi trng bp ch b sung bo t nm mc Aspergillus flavus.
Mu th nghim: mi trng bp b sung bo t nm mc v bo t vi khun.
Hnh 4.10 cho thy mu i chng (-) do ch b sung m nn mi trng bp khng
c s thay i, mu i chng (+) do khng b sung bo t vi khun nn nm mc pht
trin rt mnh, lm cho mi trng bp c mu xanh, cn mu th nghim th nm mc
pht trin t hn nn t xanh hn v vn gi c 1 phn mu vng ca bp. Qua c
th thy c vi khun Bacillus subtilis c ch qu trnh sinh trng ca nm mc
Aspergillus flavus. kim tra s thay i hm lng aflatoxin trong cc mu th
nghim, chng ti chn ngu nhin 2 mu bp trn mi l th nghim gi i phn tch
hm lng aflatoxin bng phng php sc k lng cao p (HPLC).
Muthng
him

i chng
(+)

i chng
(-)

Chng s 34

Chng s
85

Chng s
52

30

Ch tiu XN
Aflatoin B1

7.8

5.95

3.9

51.10

176.45

6.5

6.35

28.10

39.65

Aflatoxin B2

254.65

257.4

3.55

179.15

76.35

282.6

262.20

264.05

248.6

Aflatoxin G1

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KP
H
KP
H

KPH

Aflatoxin G2

KP
H
KP
H
KP
H

KPH

KPH

KPH

KPH

KPH

KPH

Bng 4.1. Kt qu phn tch HPLC

Hm lng aflatoxin ca cc mu th nghim khng c s khc bit nhiu so vi mu i
chng (+).

31

CHNG IV
KT LUN V NGH
5.1. Kt lun
Sau thi gian thc hin tiu lun chng ti c nhng kt lun sau:
C th phn lp Bacillus subtilis t phn heo
Vi khun Bacillus subtilis c phn lp t phn heo c kh nng c ch s sinh trng
v pht trin ca nm mc Aspergillus flavus.
5.2. ngh
Chng vi khun Bacillus subtilis m chng ti phn lp c c kh nng c ch sinh
trng ca nm mc nhng cha lm gim hm lng aflatoxin nhiu nn cn tip tc
tm kim cc chng khc t nc, t, c kh...
Th thay i t l nui cu gia bo t nm mc/bo t vi khun kim tra kh nng lm
gim hm lng aflatoxin trn mi trng bp.
Cn tm hiu thm v phng thc tc ng lm gim aflatoxin ca vi khun Bacillus
subtilis.
Kho st kh nng lm gim aflatoxin ca cc sinh vt khc nh Bacillus pulimus,
Bacillus megaterium, Sacchaomyces cerevisiae,...

32

TI LIU THAM KHO

Ti liu ting Vit
1. Bi Xun ng, 2004. Nguyn l phng chng nm mc v mycotoxin. NXB khoa
hc v k thut.
2. u Ngc Ho, L Th Ngc Dip, 2003. Nm mc v c t aflatoxin trong thc
n chn nui. NXB nng nghip.
3. Dng Thanh Lim, Bi Huy Nh Phc, Dng Duy ng, 2002. Thc n v
dinh dng ng vt. NXB nng nghip.
4. Nguyn Hunh Nam, 2006. Phn lp vi khun Bacillus subtilis trong phn heo v
th i khng vi Escherichia coli gy bnh tiu chy trn heo. Lun vn tt
nghip Khoa cng ngh sinh hc, i hc Nng Lm TP.H Ch Minh.
5. Phm Hong Thi, 2007. Phn lp vi khun Bacillus Subtilis t t, kho st kh
nng c ch sn sinh Aflatoxin ca cc chng phn lp c. Lun vn tt nghip
Khoa cng ngh sinh hc, i hc Nng Lm TP.H Ch Minh.
6. Dng Thanh Lim, 2002. Gio trnh c cht hc. T sch i hc Nng lm
TP.H Ch Minh.
7. L Anh Phng, 2001. Bnh nhim c aflatoxin v cc phng php pht hin
aflatoxin. Chuyn cp tin s i hc Nng Lm TP.H Ch Minh.
8. Nguyn Duy Khnh, 2000. Kho st iu kin nui cy v sinh bo t vi khun
Bacillus subtilis. Lun vn k s chuyn ngnh cng ngh sinh hc.
9. Nguyn Khc Tun, 1996. Vi sinh vt hc. NXB nng nghip.
10. L Anh Phng, 2001. Nm mc sinh c t v nh hng ca c t nm mc i
vi ng vt. Hng khc phc c t ca nm mc. Chuyn cp tin s i
hc Nng Lm TP.H Ch Minh.
Ti liu tham kho ting Anh
11. Aflatoxin B1 degradation by Bacillus subtilis UTBSP1 isolated from pistachio
nuts of Iran.

33

PH LC
I.

II.
-

PHNG PHP NHUM GRAM

Cc bc tin hnh
Chun b vt bi: cho ln lame knh mt git nc mui sinh l 9%, dng
que cy vng v trng ly mt t khun lc mc trn b mt mi trng ho
vo git nc mui v dn u.
C nh vt bi: h lame knh bng cch a qua li trn ngn la n cn.
Nhum tiu bn: t giy lc ln vt bi, nh thuc nhum crystal violet
ln ming giy lc sao cho thm xung vt bi, 1-2 pht ri ra nc.
Nh dung dch lugol ln vt bi va mi c nhum, 1 pht ri ra
nc. Ty mu nhanh bng cn 96 0C, khoangr 15-30 giy, ra nc. t
mnh giy lc khc ln vt bi v nh thuc nhum fucshin kim long ln
ming giy lc sao cho thm xung vt bi, khong 1 pht v ra
nc. Thm kh tiu bn v xem di knh hin vi.
CNG THC M KHUN LC TRN MI TRNG THCH
A
S khun lc trung bnh trong 1ml dch mu 3 pha long lin tip
Cng thc: Y =

Trong :
Y: l s khun lc trung bnh cc pha long
x1, x2, x3: l s khun lc trung bnh c trong 1g mu hay 2 ml dch
mu cc nng pha long.
S khun lc trong 1ml dch mu d pha long
Cng thc: X =

III.

x 1+ x 2+ x 3
3

A.

1
h

1
V

Trong :
X: s khun lc c trong 1ml dch mu
A: s khun lc trung bnh c trong a (trong tng s 2 a cng
nng pha long).
H: l pha long ( 10-5, 10-6, 10-7,...)
V: th tch dch mu cy 1 a.
CNG THC M VI KHUN BNG BUNG M
Cng thc: Avsv/ml = a.4000.1000.H
Trong :
a: l s vsv trung bnh c trong 1 nh =

a1
n

a1: l s vsv m c trong tng nh

n: s nh c m
34

H: l h s pha long =

1
pha lo ng

th tch 1 nh

1000: h s chuyn thnh ml (1ml= 1000 mm3)

1
4000: h s chuyn thnh 1 mm = th t ch 1 nh
3

IV.
V.
VI.

1
4000 mm 3

THNH PHN CC MI TRNG

HO CHT
THUC NHUM
VI.1. Crystal violet ( C25H30Cl.9H2O = 570,11)
a) Crystal violet
0.4g
o
Cn 96
10ml
b) Phenol
1g
Nc ct
100ml
Trn 2 dung dch a v b vi nhau, khuy cho ho tan u ri em
lc. Bo qun dung dch ny trong chai mu, trnh nh sng.
VI.2. Lugol
KI
2g
Iod tinh th
1g
Nc ct
300ml
Ho tan 2g Ki vo 5ml nc ct, sau thm 1g iod, ch cho iod tan
ht mi thm nc va 300ml.
VI.3. Fuchsine kim long ( C9H18N3Cl= 323,82)
a)
Fuchsine kim
0.3g
o
Cn 96
10ml
b)
Phenol
15g
Nc ct
35ml
Trn dung dch a v b vi nhau ri khuy cho tan u, em lc, bo
qun trong chai mu.

35