I HC QUC GIA THNH PH H CH MINH TRNG I HC BCH KHOA KHOA K THUT HA HC B MN CNG NGH HA V THC PHM

NM MC v C T AFLATOXIN

GVHD: TS. NGUYN THY HNG HVCH: HUNH NGUYN QU ANH

-20091

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1 M u ..................................................................................................................... 1 1.1 c im sinh hc ca nm mc ............................................................................ 1 1.2 Hnh dng, kch thc, cu to ca nm mc ......................................................... 2 1.2.1 Hnh dng v kch thc ...................................................................................... 2 1.2.2 Cu to................................................................................................................. 2 1.3 Dinh dng v tng trng ca nm mc ............................................................... 3 1.4 Sinh sn ca nm mc ............................................................................................ 4 1.4.1 Sinh sn v tnh ................................................................................................... 4 1.4.2 Sinh sn hu tnh ................................................................................................. 5 1.5 V tr v vai tr ca nm mc ................................................................................. 6 2 c t ...................................................................................................................... 7 2.1 Loi c t in hnh i vi chng nm mc ........................................................ 7 2.2 C ch tc dng ca c t trong c th con ngi v sc vt ................................ 8 2.2.1 Trn sc vt th nghim: biu hin 4 nhm bnh chnh ...................................... 8 2.2.2 Trn ngi ........................................................................................................... 8 3 C ch sinh tng hp c t ................................................................................... 11 3.1 Norsolorinic acid-averatin .................................................................................... 12 3.2 Versicolorin A sterigmatocystin ........................................................................ 13 3.3 Qu trnh chuyn Sterigmatocystin thnh Aflatoxin: ............................................ 13 4 Phng php xc nh c t ................................................................................. 14 4.1 Phng php truyn thng ................................................................................... 14 4.1.1 Dng Kit xc nh Aflatoxin trong thc phm ............................................... 14 4.1.2 Dng VERATOX .............................................................................................. 14 4.1.3 Dng Reveal ...................................................................................................... 17 4.1.4 Dng th nghim sinh ha pht hin c t Aflatoxin ........................................ 19 4.2 Phng php hin i ........................................................................................... 22 4.2.1 Phng php o mt hunh quang ................................................................ 22 i

4.2.2 Phng php sc k lng hiu nng cao ............................................................. 28 5.2.2.5 Phng php tin hnh................................................................................... 31 5 Bin php phng nhim c t Aflatoxin ............................................................... 34 6 Ti liu tham kho .................................................................................................. 35

ii

NM MC V C T AFLATOXIN

1 M u
c t nm mc t khi gy ng c cp tnh, n gy hi c th t t do lm ngi ta khng . Nhng khi pht hin ra triu chng th nhng c quan b phn h hi nghim trng kh lng cha tr. Ty theo lai nm mc v c t ca chng m tc ng chnh ca mi loi c khc nhau. Nu tng hp chung li th c t nm mc tc ng hu ht cc c quan b phn trong c th nh: Tc ng vo gan v thn gy vim, nu ko di c th gy ung th (Aflatoxin, Ochratoxm ); Tc ng vo h tun hon gy ra xut huyt mn tnh, ngng kt hng cu, gim lng khng th (Aflatoxin..); Tc ng h thn khinh gy ra hn m mt tnh ngon ming(Deoxynivalenol, DON); Tc ng ln h h hp gy vim nm phi(Aspergillus fumigatus); Tc ng vo h tiu ha gy ra vim l let ming, da dy Khc vi bnh nhim trng l khng sinh khng iu tr c nhim c t nm mc. Cch tt nht l ngn nga khng cho c t nhim vo thc n. 1.1 c im sinh hc ca nm mc Nm mc (fungus, mushroom) l vi sinh vt chn hch, th tn (thalophyte), t bo khng c dip lc t, sng d dng (hoi sinh, k sinh, cng sinh), vch t bo cu to ch yu l chitin, c hay khng c celuloz v mt s thnh phn khc c hm lng thp. Nm hc (Mycology) c khai sinh bi nh thc vt hc ngi tn l Pier Antonio Micheli (1729) qua ti liu cng b ging cy l (Nova Plantarum Genera) nhng theo Gio s Ekriksson Gunnan (1978) th ngi c cng nghin cu su v nm mc li l Elias Fries (1794 - 1874). Theo Elizabeth Tootyll (1984) nm mc c khong 5.100 ging v 50.000 loi c m t, tuy nhin, c tnh c trn 100.000 n 250.000 loi nm hin din trn tri t. Nhiu loi nm mc c kh nng k sinh trn nhiu k ch nh ng vt, thc vt, c bit trn con ngi, cy trng, vt nui, sn phm sau thu hoch cha hoc qua ch bin, bo qun. Mt s l tc nhn gy bnh, lm h cc thit b thy tinh bo qun khng tt nhng cng c nhiu loi c ch nh tng hp ra acid hu c, thuc khng sinh, vitamin, kch thch t tng trng thc vt c a vo sn xut cng nghip v c mt s nm c dng lm i tng nghin cu v di truyn hc.

NM MC V C T AFLATOXIN 1.2 Hnh dng, kch thc, cu to ca nm mc 1.2.1 Hnh dng v kch thc Mt s t nm th n bo c hnh trng (yeast = nm men), a s c hnh si (filamentous fungi=nm si), si c ngn vch (a bo) hay khng c ngn vch (n bo). Si nm thng l mt ng hnh tr di c kch thc ln nh khc nhau ty loi. ng knh ca si nm thng t 3-5m, c khi n 10m, thm ch n 1mm. Chiu di ca si nm c th ti vi chc centimet. Cc si nm pht trin chiu di theo kiu tng trng ngn (Hnh 1.1). Cc si nm c th phn nhnh v cc nhnh c th li phn nhnh lin tip to thnh h si nm (mycelium) kh sinh x x nh bng. Trn mi trng c v trn mt s c cht trong t nhin, bo t nm, t bo nm hoc mt on si nm c th pht trin thnh mt h si nm c hnh dng nht nh gi l khun lc nm (Hnh 1.2).

Hnh 1.1: Si nm v cu to

Hnh 1.2: Mt s dng khun lc nm (theo Samson v ctv., 1995)

1.2.2 Cu to T bo nm c cu trc tng t nh nhng t bo vi sinh vt chn hch khc c m t v trnh by nh Hnh 1.3 2

NM MC V C T AFLATOXIN

Hnh 1.3: Cu to t bo nh si nm Fusarium (theo Howard R J & Heist J R., 1979) (Ch thch: MT: vi ng, M: ty th, SC: b Golgi, V: bng(ti) nh, P: mng sinh cht 4 lp)

Vch t bo nm cu to bi vi si chitin v c hoc khng c celluloz. Chitin l thnh phn chnh ca vch t bo hu ht cc loi nm tr nhm Oomycetina. Nhng vi si chitin c hnh thnh nh vo enzim chitin syntaz T bo cht ca t bo nm cha mng ni mc (endoplasmic reticulum), khng bo (vacuoles), ty th (mitochondria) v ht d tr (glycogen v lipid), c bit cu trc ty th t bo nm tng t nh cu trc ty th t bo thc vt. Ngoi ra, t bo nm cn c rib th (ribosomes) v nhng th khc cha r chc nng. T bo nm khng c dip lc t, mt vi loi nm c ri rc trong t bo mt loi sc t c trng m Matsueda v ctv. (1978) u tin ly trch c v gi l neocercosporin (C29H26O10) c mu tm nm Cercosporina kikuchi. T bo nm khng nht thit c mt nhn m thng c nhiu nhn. Nhn ca t bo nm c hnh cu hay bu dc vi mng i m, bn trong mng nhn cha ARN vphospholipid v protein dy 0,02 ADN. 1.3 Dinh dng v tng trng ca nm mc Hu ht cc loi nm mc khng cn nh sng trong qu trnh sinh trng. Tuy nhin, c mt s loi li cn nh sng trong qu trnh to bo t (Buller, 1950). Nhit ti thiu cn cho s pht trin l t 2oC n 5oC, ti ho t 22oC n 27oC v nhit ti a m chng c th chu ng c l 35oC n 40oC, c bit c mt s t loi c th sng st OoC v 60oC. Ni chung, nm mc c th pht trin tt mi trng acit (pH=6) nhng pH ti ho l 5 - 6,5, mt s loi pht trin tt pH < 3 v mt s t pht trin pH > 9 (Ingold, 1967). Oxi cng cn cho s pht trin ca nm mc v chng l nhm hiu kh bt buc v s pht trin s ngng khi khng c oxi v d nhin nc l yu t cn thit cho s pht trin. Theo Alexopoulos v Minns (1979) cho bit nm mc c th pht trin lin tc trong 400 nm hay hn nu cc iu kin mi trng u thch hp cho s pht trin ca chng. 3

NM MC V C T AFLATOXIN Nm mc khng c dip lc t nn chng cn c cung cp dinh dng t bn ngoi (nhm d dng), mt s sng st v pht trin nh kh nng k sinh (sng k sinh trong c th ng vt hay thc vt) hay hoi sinh (saprophytes) trn xc b hu c, cng c nhm nm r hay a y sng cng sinh vi nhm thc vt nht nh. Theo Alexopoulos v Mims (1979) cho bit ngun dng cht cn thit cho nm c xp theo th t sau: C, O, H, N P, K, Mg, S, B, Mn, Cu, Zn, Fe, Mo v Ca. Cc nguyn t ny hin din trong cc ngun thc n v c n gin nh glucoz, mui ammonium... s c nm hp thu d dng, nu t ngun thc n hu c phc tp nm s sn sinh v tit ra bn ngoi cc loi enzim thch hp ct cc i phn t ny thnh nhng phn t nh d hp thu vo trong t bo. 1.4 Sinh sn ca nm mc Ni chung, nm mc sinh sn di 2 hnh thc: v tnh v hu tnh. Trong sinh sn v tnh, nm hnh thnh bo t m khng qua vic gim phn, tri li trong sinh sn hu tnh nm hnh thnh 2 loi giao t c v ci. 1.4.1 Sinh sn v tnh The Alexopoulos v Mims (1979), nm mc sinh sn v tnh th hin qua 2 dng: sinh sn dinh dng bng on si nm pht trin di ra hoc phn nhnh v sinh sn bng cc loi bo t. Mt s loi nm c nhng bo t c trng nh sau: a. Bo t ti (bo t bc)(sporangiospores): cc bo t ng (zoospores) (Hnh 1.4 a, b, c) c nm Saprolegnia

Hnh 1.4: Bo t ng (theo Samson v ctv., 1995)

b. Bo t nh (conidium): cc bo t nh khng c ti bao bc ging nm Aspergillus, Penicillium, ... Hnh dng, kch thc, mu sc, trang tr v cch sp xp ca bo t nh thay i t ging ny sang ging khc v c dng lm tiu chun phn loi nm. Cung bo t nh dng bnh c th khng phn nhnh nh Aspergillus (Hnh 1.5) hay dng th phn nhnh nh Penicillium (Hnh 1.6). 4

NM MC V C T AFLATOXIN

Hnh 1.5: Cc kiu cung bo t nh ca Aspergillus. a. 1 lp, b. 2 lp, c. phin, d. tia, e. t (theo Samson v ctv., 1995)

Hnh 1.6: Bo t nh v cung bo t nh Penicillium chrysogenum (theo Samson v ctv. 1995)

1.4.2 Sinh sn hu tnh Sinh sn hu tnh xy ra khi c s kt hp gia hai giao t c v ci (gametes) c tri qua giai on gim phn. Qu trnh sinh sn hu tnh tri qua 3 giai on: Tip hp t bo cht (plasmogamy) vi s ha hp 2 t bo trn (protoplast) ca 2 giao t Tip hp nhn (karyogamy) vi s ha hp 2 nhn ca 2 t bo giao t to mt nhn nh bi (diploid) Gim phn (meiosis) giai on ny hnh thnh 4 bo t n bi (haploid) qua s gim phn t 2n NST (nh bi) thnh n NST (n bi). Theo Machlis (1966) tt c cc giai on trn k c giai on to c quan sinh dc c iu khin bi mt s kch thch t sinh dc (sexual hormones). C quan sinh dc ca nm mc c tn l ti giao t (gametangia) c 2 loi: c quan sinh dc c gi l ti c (antheridium) cha cc giao t c (antherozoids), cn c quan sinh dc ci gi ti non (oogonium) cha giao t ci hay non, khi c 5

NM MC V C T AFLATOXIN s kt hp gia giao t c v non s to thnh bo t, bo t di ng c gi l bo t ng (zoospores). Kiu hai si nm c gii tnh c v ci tip hp nhau sinh ra bo t c tn l tip hp t (myxospores), tip hp t l c trng ca nhm nm Myxomycetes Bo t sinh dc khi hnh thnh c dng ti gi l nang (ascus) v ti ny cha nhng bo t gi l bo t nang (ascospores). Nang v bo t nang l c trng ca nhm Ascomycetes Trong nhm Basidiomycetes, 4 bo t pht trin phn tn cng ca cu trc th qu gi l m (basidium) v bo t c gi l bo t m (basidiospores) Nhm Nm bt ton (Deuteromycetes=Deuteromycotina)) gm nhng nm cho n nay cha bit r kiu sinh sn hu tnh ca chng.

Hnh 1.7: Cc kiu bo t m. a. Astrea, b. Bovista, c. Agaricales,

d. Clavulina, e. Dacrymyces, f. Sistotrema, g. Repetobasidium, h. Xenasma, i-n. bo t m c vch, n. Puccinia. (theo Kreisel, 1995) 1.5 V tr v vai tr ca nm mc Nm mc c nh hng xu n cuc sng con ngi mt cch trc tip bng cch lm h hng, gim phm cht lng thc, thc phm trc v sau thu hoch, trong ch bin, bo qun. Nm mc cn gy h hi vt dng, qun o... hay gy bnh cho ngi, ng vt khc v cy trng. Tuy nhin, cc qui trnh ch bin thc phm c lin quan n ln men u cn n s c mt ca vi sinh vt trong c 6

NM MC V C T AFLATOXIN nm mc. Nm mc cng gip tng hp nhng loi khng sinh (penicillin, griseofulvin), acit hu c (acit oxalic, citric, gluconic...), vitamin (nhm B, riboflavin), kch thch t (gibberellin, auxin, cytokinin), mt s enzim v cc hot cht khc dng trong cng nghip thc phm v y, dc ... c s dng rng ri trn th gii. Ngoi ra, nm cn gi vai tr quan trng trong vic phn gii cht hu c tr li mu m cho t trng. Mt s loi thuc ging Rhizopus, Mucor, Candida gy bnh trn ngi, Microsporum gy bnh trn ch, Aspergillus fumigatus gy bnh trn chim; Saprolegnia v Achlya gy bnh nm k sinh trn c. Nhng loi nm gy bnh trn cy trng nh Phytophthora, Fusarium, Cercospora.... c bit nm Aspergilus flavus v Aspergillus fumigatus pht trin trn ng cc trong iu kin thun li sinh ra c t aflatoxin. Bn cnh tc ng gy hi, mt s loi nm mc rt hu ch trong sn xut v i sng nh nm n, nm dc phm (nm linh chi, Penicillium notatum tng hp nn penicillin, Penicillium griseofulvum tng hp nn griseofulvin...), nm Aspergillus niger tng hp cc acit hu c nh acit citric, acit gluconic, nm Gibberella fujikuroi tng hp kch thch t gibberellin v mt s loi nm thuc nhm Phycomycetina hay Deuteromycetina c th k sinh trn cn trng gy hi qua c th dng lm thin ch dit cn trng. Ngoi ra, nhng loi nm sng cng sinh vi thc vt nh Nm r (Mycorrhizae), gip cho r cy ht c nhiu hn lng phn v c kh tan v cung cp cho nhu cu pht trin ca cy trng.

2 c t
2.1 Loi c t in hnh i vi chng nm mc Aspergillus flavus v A. parasiticus l hai loi nm mc quan trng trong cc lai nm mc c kh nng to c t mycotoxin gy ng c thc phm. c t mycotoxin c th c to ra t nhiu chun ca hai loi ny c gi chung l aflatoxin c kh nng gy ung th. Cc nm mc ny c th pht trin nhit t 7 40oC v ti u 24 28oC, do vy c kh nng tng tng tt trn cc lai ng cc, nng sn( la, go, bp ...) khng c phi sy tt, c bit l cc nc nhit i vi iu kin m v nhit khng kh cao. T cc nguyn liu ny, nm mc v c t c th nhim vo thc phm gy ng c cho ngi. cc chng c kh nng dinh c t, sau khi tng tng 1 3 ngy c t to ra v tin vo c cht. Bn loi aflatoxin c to ra bi Aspergillus flavus v A. parasiticus l aflatoxin B1, B2 pht hunh quang mu xanh (blue) va G1, G2 pht hunh quang mu lc (green). Khi n c hoc thc n cha B1 hoc B2, b sa c th chuyn ha chng thnh cc c t aflatoxin M1, M2 hin din trong sa b. Do aflatoxin l cht gy ung th mnh nn c kim tra nghim ngt. Hu ht cc nc u c 7

NM MC V C T AFLATOXIN tiu chun quc gia v hm lng ti a cho php s hin din ca c t ny trong nng sn, thc phm, thng la 10ppb.

Hnh 2.1: Nm si Aspergillus flav

2.2 C ch tc dng ca c t trong c th con ngi v sc vt 2.2.1 Trn sc vt th nghim: biu hin 4 nhm bnh chnh Nhng ph hy c tnh cht cp tnh gan - th hin mt nhim c cp tnh. Thng l do Aflatoxin B1, B2, G1, G2 trong c t c c tnh mnh nht l B1, sau n G1, ri n B2, v sau cng l G2. Bn cnh gan, cc c quan khc nh: phi, thn, mc treo, ti mt... cng b tn thng t nhiu. - Hin tng x gan: sau mt nhim c cp tnh nh trn c hai kh nng c th din ra: Mt l cc t chc mi gan s c ti to dn dn v gan tr li hi phc hon ton. Hai l chuyn thnh x gan. - Ung th gan: liu gy ung th gan trn chut nht trng l 0,4ppm, tc l cho chut n hng ngy vi liu 0,4mg aflatoxin/kg thc n. Sau 2-3 tun c th gy ung th gan. Ring Aflatoxin B1 liu gy ung th gan c th l 10ppm tc l mi ngy cho chut n 10mg/kg thc n. - Hin tng gy vim sng nng n dn n hoi t cc t chc v ni tng . 2.2.2 Trn ngi Nm 1986, Payet v cng s quan st trn 2 tr em b suy dinh dng Kwashiorkor, c nui bng thc n b sung m di dng bt lc, khng may bt lc ny b nhim c t Aflatoxin. Tr n mi ngy 70-100g bt lc b nhim Aflatoxin vi hm lng 0,5-1ppm n ko di trong 10 thng, n khi tr 4
-

NM MC V C T AFLATOXIN tui th thy xut hin cc triu chng ri lon chc nng gan. Sinh thit gan thy c hin tng lot m gan c 2 tr. - Bnh bch cu khng tng bch cu l bnh khng do c t nm Anatoxin gy ra, ln u tin xut hin Xiberi (Lin X c) v mt s vng khc thuc Lin X. nhng vng ny thc n c bn l k, la m, la mch. Sau ny, cc cng trnh nghin cu xc nh tc nhn gy bnh l nm fusarium. V lm sng bnh thng tin trin theo 3 giai on: Giai on 1: Ko di 3-6 ngy, biu hin u tin l vim nim mc ming, hng... sau lan xung d dy, rut. Sang ngy th 3 c i ngoi nhiu ln, au bng, nn ma. Giai on 2: cn gi l giai on bt sn ca h bch huyt v c quan to mu, ko di 15-30 ngy. Xt nghim mu: Bch cu gim, tiu cu gim v thiu mu r rt. S chuyn ha aflatoxin trong c th iu u tin chng ta cn bit aflatoxin l tinh th trng, bn vi nhit, khng b phn hy khi un nu nhit thng thng ( 120oC phi un 30 pht mi mt tc dng c) do vy n c th tn ti trong thc phm khng cn s c mt ca nm mc tng ng; ng thi n rt bn vi cc men tiu ha. Tuy nhin n li khng bn di nh sng mt tri v tia t ngoi, nn vic kh c thc phm s c nhiu bin php hn. C 17 loi aflatoxin khc nhau, nhng thng gp v c nht l aflatoxin B1. Aflatoxin B1 l phn t i m, c trng lng phn t thp, d dng c hp thu sau khi n, s hp thu l hon ton. Khi n rut non, aflatoxin B1 s c nhanh chng hp thu vo mu tnh mch mc treo, s hp thu rut non v t trng l nhiu nht. Nim mc ng tiu ha c kh nng chuyn dng sinh hc aflatoxin B1 nh s gn kt vi protein y l con ng chnh gii c aflatoxin B1 cho gan. T ng tiu ha, theo tnh mch ca, aflatoxin c tp trung vo gan nhiu nht (chim khong 17% lng aflatoxin ca c th) tip theo l thn, c, m m, ty, lch... Trong vng 24 gi c khong 80% b o thi theo ng tiu ha qua mt, ng tit niu qua thn v ng ch n cn bi tit qua c sa. c tnh trn ng vt th nghim Nhim c cc aflatoxin gy mt lot cc triu chng cp tnh v mn tnh. Nhim c cp thng biu hin bng ci cht ca cc ng vt th nghim vi cc triu chng thng gp l hoi t nhu m gan, chy mu gan v vim cu thn cp. Nhim c mn tnh thng biu hin bng n km ngon, chm ln, gan t mu, chy mu v hoi t nhu m. Loi mn tnh tc ng ti yu t di truyn tng ng vi 3 kiu gy ung th, gy qui thai v gy t bin. 9

NM MC V C T AFLATOXIN c tnh ca aflatoxin trn ngi Bnh do nhim aflatoxin cp tnh c thng bo cc nc, vi cc biu hin ch yu l suy chc nng gan cp, x gan v hoi t nhu m gan. Bn cnh cc nghin cu v vai tr ca virut vim gan, k sinh trng, dinh dng, cc cht c... i vi ung th gan, cc nh khoa hc cng ang tp trung nghin cu v vai tr ca aflatoxin vi cn bnh ph bin ny. Trn ngi, mt lot cc nghin cu cho thy t l mc ung th gan nguyn pht tng nhng vng c t l phi nhim cao vi aflatoxin, nhng c ch tc ng ca aflatoxin gy ung th gan ngi nh th no vn cn nhiu tranh ci, tuy nhin tm thy s gn kt ca aflatoxin B1 vi AND ca t bo gan nhng bnh nhn b ung th gan nguyn pht, phc hp ny cn c tm thy trong mu ngoi vi, trong mu rau thai v mu dy rn ca cc sn ph c phi nhim vi aflatoxin B1. Bn cnh cn c s lin quan gia phi nhim aflatoxin B1 vi s t bin gen cc bnh nhn ny, m nhiu nht l s t bin gen p53 mt gen kim sot s cht t bo theo chng trnh, khi t bin gen ny s lm i sng t bo tng ln ko theo nguy c t bo s chuyn thnh c tnh. Cho n nay, ngi ta tm thi cng nhn kh nng tc ng ln t bo gan ca aflatoxin qua 5 giai on. - Tc ng qua li vi AND v c ch cc polymeraza chu trch nhim 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 ha ln t bo gan ny l gy ung th biu m t bo gan. Nh vy, aflatoxin c kh nng gy c tnh cp v mn cc loi ng vt v con ngi. c tnh nguy him nht l kh nng gy x gan v ung th gan nguyn pht. Cc nh khoa hc gy c ung th gan nguyn pht trn thc nghim bng cch cho cc con vt n thc n c aflatoxin. Mt lot cc nghin cu cng cho thy s phi nhim aflatoxin tng lin quan n s gia tng mc bnh ung th gan nguyn pht trn ngi. Do vy vn bo qun lng thc thc phm, an ton lng thc thc phm, khng s dng cc thc phm b hng, b nm mc l mt vn ht sc quan trng c ngha trong vic hn ch tn sut xut hin bnh ung th gan nguyn pht. 10

NM MC V C T AFLATOXIN

3 C ch sinh tng hp c t
Aflatoxin l sn phm ca qu trnh trao i cht th cp ca A. flavus v A.parasiticus. Cc gene lin quan n qu trnh sinh tng hp Aflatoxin c trong b n trong h gene ca nhng loi nm si. Nhng nghin cu trc tip i su v sinh hc phn t ca qu trnh sinh tng hp. Cp gen sinh tng hp acid bo (fatty acid synthase) c xc nh c lin quan n qu trnh sinh tng hp. 2 gene ny c trng cho dng cytochrome P450 monooxygenase. Cc c t ny thng c thy trc qu trnh thu hoch bp, cy bng, u phng. Aflatoxin nh hng ln n thc n v cng ngnh cng nghip ht ging v n c c cao v c kh nng gy ung th. Aflatoxin B1 lin quan n qu trnh chuyn G T codon 249 ca h gene Gen u tin c tm ra c lin quan n qu trnh sinh tng hp aflatoxin l vo nm 1992. K t ngnh sinh hc phn t tin hnh nghin cu v b gen tng hp aflatoxin t A. Flavus v A. parasiticus nhanh v chnh xc hn. B gen trong A. Flavus nm trong 4.9 mb chromosome. Tc gi Prieto et al ch ra rng vic b sung vo b gene loi b cc t bin ca A. flavus l cc gen c tng hp aflatoxin trong gn 90kb on gen v tnh DNA. Yu et al sp xp li tng b gen trong A. Flavus v A. parasiticus, v c lng kch thc ca mi b khong 75kb. Cc v tr lin quan trong b v cc nucleotide to thnh ca cc b gene tng i ging nhau gia 2 loi, mc d A.parasiticus c bn sao b sung ca mt vi gen xc nh c mt s t phn trong gene nhn i. Vic tm hiu v qu trnh sinh tng hp Aflatoxin da vo h gene trong A.nidulans c lin quan n qu trnh sinh tng hp sterigmatocystin. qua xc nh c t l ca cc gen sinh tng hp aflatoxin cng tp trung thnh b. Ton b nucleotide c to thnh ca A.nidulans cng nh 25 qu trnh chuyn i to thnh cung nh bo t c xc nh, Vic sp xp cc gen trong nhm ny khc vi vic sp xp cc gen trong b to Aflatoxin nhng cc acid amin b gim i l ging nhau. Sinh tng hp acid Norsolorinic :

Polyketide l vin gch cu trc c bn to AFB1 lin quan n qu trnh ko di 1 n v hexanoate bi enzyme polyketide synthase (PKS) m enzyme ny l do tp hp ca 7 n v acetyl t malonyl CoA m khng qua s kh Ketone to noranthrone. Vic tng hp acid norsolorinic l giai on trung gian bn vng, c to thnh sau qu trnh oxi ha noranthrone t enzyme oxidase. PKS v enzyme tng hp acid bo (FAS) c ly t b gen c cha pksA (pksL1) v fas1. Tc gi Chang cho thy pksA c xc nh thng qua qu trnh chuyn mch NA ca 11

NM MC V C T AFLATOXIN A. parasiticus vi phn DNA tham gia tng hp AFlatoxin. Gn 5% cht vn chuyn b mt kh nng sn xut NA khi cht vn chuyn b ph v trong gene pksA. Vic ph v gene ca pksL1 l do trong cht vn chuyn khng lin kt vi Aflatoxin hay NA c xc nh qua qu trnh sinh tng hp Aflatoxin. Gene FAS, fas, c xc nh bng vic thm vo A.parasiticus mch, khng sn sinh ra Aflatoxin hay sn phm trung gian. Th nghim loi b gene thm vo l lc u fas1 tham gia to NA v gene khng cn thit cho qu trnh trng thnh ca nm. Gene FAS th 2, fas2, nm st fas1 lm nhim v nh nhm ph to chc nng cho FAS. Vic phn tch to nucleotide xc nh motif thng thng bit PKS v FAS c trong sn phm gene, c tn l enzyme cetoacyl synthase, acyltransferase v protein cht mang acyl. Gene FAS cng cha enzyme ketoacyl reductase, enoyl reductase v anoyl hydrase. Thiu enzyme ketoreductase trong PKS bao gm thiu bc kh trong phn ng PKS sn xut noranthrone. 3.1 Norsolorinic acid-averatin Vic chuyn t NA thnh averatin (AVN) thng qua enzyme ketoreductase Tng ng l vic b sung vo l hng do t bin ca A.parasiticus qua vic tp hp NA v aflatoxin. Nor1 m ha 1 protein 29kDa c acid amin tng t nh enzyme dehydrogenase c lin kt NADPH. Tng t NA t bin ban u, cht vn chuyn ct t nor1 khng tam gia sinh tng hp aflatoxin, ngi ta d on l c NA-reductase khc. Thc t th enzyme reductase th 2 ny c kh nng chuyn NA thnh AVN khi nghin cu in vitro to dng ng nht v cho protein ph khong 40kb. S dng khng th n tnh khng li enzyme reductase ny. Cary et al xc nh cDNA n tnh tng ng vi gen norA nm trong b gene ca Aflatoxin c trong loi A. parasiticus cng nh loi A.flavus. Cc chui acid amin ca NOR1 v NORA c ng nht thp (22%) nhng c 2 loi u c motif xc nh enzyme loi dehydrogenase vi phn lin kt adenine nucleotide. loi b 2 bn sao ca norA trong A.parasiticus cha xc nh c. Tuy nhin, ngi ta cha xc nh chnh xc chc nng cc gene ny. Averatin-versicolorin A:

Gene lin quan n qu trnh chuyn AVN thnh versicolorin A (VER A) xc nh gene trong qu trnh sinh tng hp Aflatoxin. Qua vic s dng cc on t bin, cht c ch v cc cht trung gian phng x, th qu trnh trao i cht trong qu trnh chuyn i ny s quyt nh: AVN Averufanin (AVNN) Averufin (AVF) 1- hydrocyversicolorone (HVN) versiconal hemiacetal cetate (VHA) Versiconal (VHOH) 12

NM MC V C T AFLATOXIN versicolorin B (VERB) VER A (hnh 1). S dng cDNA v tnh, Yu et al loi b c gene tng ng trong b gene tng hp Aflatoxin ca A.parasiticus v thu c cht vn chuyn tch ly vi AVN. Gene avnA m ha 56.3 kDa cytochrome P450 monooxygenase, gene c lin quan n qu trnh chuyn AVN thnh AVNN. Prieto et al thay i gen t bin ca A. flavus thiu ton b b gen Aflatoxin thnh 2 phage cos (5E6 v 8B9) v ly li cht vn chuyn tham gia vi AVF. Thm vo phage cos th 3 s gi c qu trnh sinh tng hp aflatoxin trong gene t bin ny, ngi ta cho rng c 1 gene lin quan n qu trnh chuyn AVF thnh VHA nm phage cos 13B9. Gene avf1 nm trong phn 7kb trn phage cos 13B9. Kt qu phn tch cho thy phn DNA cha 2 loi gene, aflB v aflW, m ha protein vi cc acid amin tng ng vi stc B v stcW. Qu trnh chuyn VHOH thnh VER A rt quan trng trong qu trnh sinh tng hp Aflatoxin v h thng dihidrobisfuran s c to thnh trong bc ny. Dihyrobisfuran l cn thit cho qu trnh t bin t nhin ca Aflatoxin B1. Lin kt i gia v tr 2 v 3 trong phn difuran l do enzyme cytochrome P450 hay lipozygenase to ra nhm epoxide linh ng c trong thn v gan. Cc enzyme dehydrative cyclization, l qu trnh oxy ha VER B thnh VER A cng enzyme versicolorin B synthase (VBS), to dihydrobisfuran. Gene m ha VBS , vbs, tng ng vi mi PCR c to ra da vo phn peptide ca enzyme . Sn phm gene vbs cho thy kh nng ng nht c trng ca cc enzyme t do flavin oxydase v dehydrogenase. Cc nghin cu gn y cho thy cu trc ca Aflatoxin B2 ng hn AFB2 khi n thiu lin kt i trong cu trc ca bifuran. 3.2 Versicolorin A sterigmatocystin Sterigmatocystin c to thnh t VERA thng qua mt s phn ng enzyme gm: phn ng oxy ha, kh ceton, decarboxyl ha v methyl ha. Vic thm gene vo VER A ca A.parasiticus dn n vic c lp c ver1A m ha 1 NADPH ketoreductase. Gene th 2, ver1B, trong A.parasiticus c 95% tng ng vi ver1 A v khng nm trong b gene. 3.3 Qu trnh chuyn Sterigmatocystin thnh Aflatoxin: Sterigmatoxystin c chuyn thnh O-methylsterigmatocystin (OMST) bi enzyme O-methyltransferase 40 kDa. Huyt thanh min dch c a vo chng li protein c dng che chn cDNA v tch omt1. Cc acid amin t cDNA xc nh OMT1 c trong chui amino nh lin kt S-adenosylmethionine. C A. flavus v A.parasiticus u cha 1 bn sao ca omt1. on gene chuyn OMST thnh AFB1 ca A. flavus xa i b gene Aflatoxin. Cht vn chuyn cha phage cos khng sn sinh ra Aflatoxin hay bt k cht trung gian no nhng c chuyn OMST thnh AFB1. Gen ord1 chu trch nhim hot ha 13

NM MC V C T AFLATOXIN enzyme nm phn gene DNA 3.3kb trn phage cos 8B9. Sn phm ca ORD1 l cytochrome P450 mono oxygenase (CYP64)

4 Phng php xc nh c t
4.1 Phng php truyn thng 4.1.1 Dng Kit xc nh Aflatoxin trong thc phm Kit xc nh Aflatoxin - Agri-Screen xc inh Aflatoxin - Reveal xc nh Aflatoxin - Veratox xc nh Aflatoxin - Veratox xc nh Aflatoxin th nghim n (AST) - Veratox xc nh Aflatoxin nhy cao 4.1.2 Dng VERATOX

4.1.2.1 Mc ch s dng Veratox xc nh Aflatoxin AST (th nghim n Aflatoxin) s dng phn tch nh lng Aflatoxin trong cc mt hng nh ng cc, bt ng cc, hn hp protein trong ng cc, hn hp ng cc/ u phng, la m, go, sa, tng, ht bng, bt ht bng, u phng, b u phng v hn hp khc. 14

NM MC V C T AFLATOXIN 4.1.2.2 Nguyn tc Th nghim ny dng phng php cnh tranh trc tip Elisa c th xc nh chnh xc nng Aflatoxin ti c phn t (ppb). Aflatoxin c chit c chit t mu nn vi dung dch methanol bng cch nh u v lc. c t c chit ra trong lc l mu wocj trn vi c t gn enzyme (tip hp). Dung dch hn hp ny c chuyn ti nhng ging tm khng th, ti y c t t do v cht tip hp cnh tranh lin kt . Sau khi ra, cht nn c thm vo, hp th ca mu v mu so snh 20ppb c c so snh vi ng chun. Kt qu mu c tnh ton sau . 4.1.2.3 Phng php Thm 100 ml cht tip hp vo mi ging trn. Thm 100 ml mu so snh vo ging th nht. Thm 100 ml ca mu u tin vo ging th hai v th ba. Trn u. Chuyn 100 ml vo mi ging khng th. khong 5 pht. Trt dung dch t ging khng th Ra sch vi nc loi ion Thm kh bng giy thm. Chuyn 100 ml cht nn t thuyn thuc th vo mi ging khng th s dng pipet 12 knh. Chuyn 100 ml cht hm t thuyn thuc th vo cc ging khng th. Kt qu c bng cch s dng b c ging vi knh lc 650 nm. Thng s k thut ca sn phm Gii hn xc nh thp nht :5 ppb Khong mh lng: 5 ppb - 320 ppb Mu so snh: 20 ppb Thi gian th: 10 pht Khng th phn ng Tng Aflatoxin (B1, B2, G1, G2) S test/kit 16 Ph chun USDA/GIPSA # 94-10

15

NM MC V C T AFLATOXIN 4.1.2.4 Cch tin hnh: B1:X l mu:

B2: Tin trnh th nghim

B3:c kt qu

16

NM MC V C T AFLATOXIN

4.1.3 Dng Reveal

4.1.3.1 Mc ch s dng Reveal xc nh Aflatoxin vi mc ch sng lc Aflatoxin trong ng cc. 4.1.3.2 Nguyn tc Reveal xc nh Aflatoxin s dng phng php mu min dch dng Lateral bc n trn c s s cnh tranh dng min dch. Dch chit c thm qua vng thuc th, c cha khng th c bit vi Aflatoxin tip hp vi nhng tiu phm mu. Nu Aflatoxin c mt, n s bt cc phc khng th tiu phn. Cc phc tiu phn khng th-Aflatoxin s thm vo mng c cha trong phn Aflatoxin tip hp ti cht mang protein. Vng ny s bt tt c cc khng th Aflatoxin khng to phc, cho php cc tiu phn tp trung v hnh thnh ng mu. 4.1.3.3 Phng php Mu phi c chit trc khi th: 17

NM MC V C T AFLATOXIN 1.Chuyn s cc mu thch hp v t n vo trong cc ph mu. 2.S dng pipet v u pipet mi, thm 200 ml cht pha long vo cc mu. 3.S dng u pipet mi thm 200ml mu chit vo cc mu. 4.t thanh th aflatoxin mi v mu cui ca cc mu. 5. thanh th c th pht trin trong cc 3 pht. 6.Loi b thanh v c kt qu bng mt ngc li vi nn trng hay my c Accuscan Reveal ca Neogen. Thng s k thut ca sn phm: nhy: 20 ppb Thi gian th: 3 pht Khng th phn ng AflatoxinB1 Bo qun : nhit phng, 18-300C S test/kit: 25 4.1.3.4 Cc bc tin hnh B1: X l mu

B2:tin hnh kim nghim 18

NM MC V C T AFLATOXIN

B3:c kt qu

SO SNH GIA CC KIT Veratox 20.88ppb 6 pht Tt c mt hng 8 thng/2-8 oC 36-44 Agri-Screen Reveal <50, >20ppb >20ppb 4 pht 4 pht Tt c mt hng Cho bp 6 thng/2-8oC 1nm/nhit phng 12-20 25

nhy Thi gian th nghim ng dng Hn s dng S ln test ti a

4.1.4 Dng th nghim sinh ha pht hin c t Aflatoxin 4.1.4.1 Th nghim trn vt con 1 ngy tui Cc th nghim u tin chng minh tnh c ca cc Aflatoxin c tin hnh trn vt con 1 ngy tui (nng khong 50g). 19

NM MC V C T AFLATOXIN Ngi ta ch x l lc hoc cc sn phm c ngun gc t lc bng cloroform ri chit cc cht ha tan bng methanol. Sau khi tinh ch bng phn chia gia cc dung mi khng ha ln (methanol: nc: ether du ha 10:1:10), cn c ha thnh nh tng trong nc (sao cho nng cui cng ca 1ml tng ng vi 40g mu ban u) sau a vo c hng vt con bng ng cht do. Ngy u tin cho vt nut mt lng cht chit bng 10g mu ban u, ri tng dn liu lng ln trong 4 ngy con vt tip nhn mt lng tng ng 80g. Mu th c xem l rt c khi con vt cht sau 7 ngy k t ngy bt u th nghim, c va hoc t ty theo tn thng nhiu hay t gan ca nhng con vt cn sng, c em gii phu ngy th 7. Vt l mt loi c bit mn cm vi c t Aflatoxin nn nh vy ta c th pht hin bng pp sinh hc nhng lng Aflatoxin rt nh. Th nghim vt mt ngy tui c dng rt nhiu, n cho php pht hin cc Aflatoxin bng cch: * Bit liu gy cht ca con vt : - Aflatoxin B1 : 0.36mg/kg Aflatoxin B2 : 1.69 mg/kg - Aflatoxin G1 : 0.78mg/kg Aflatoxin G2 : 2.45 mg/kg * Bit c con vt tip nhn mt liu Aflatoxin t nht 0.04mg/kg th thy cc ng mt pht trin d thng Tuy nhin cch th cng c mt s bt tin: phi t chc nui vt v con vt c th nn liu thuc c m ta va cho ung. 4.1.4.2 Th nghim trn phi g Th nghim trn phi g l th nghim bng sinh vt c dng nhiu sau th nghim vt con. Tin hnh th nghim bng cch tim nc chit vo bun kh hoc vo lng trng g Leghorn trng th tinh v p trong 5 ngy (mt s tc gi tim trc khi p hoc sau khi p 9 ngy). Phi g rt mn cm vi Aflatoxin, cht trong 2 ngy sau khi nhim, nu mu th tim vo trn 0.3g Aflatoxin. Th nghim ny c mn cm gp 200 ln hn th nghim bng vt con. Liu gy cht DL50 khi tim aflatoxin vo bun kh nh sau: Aflatoxin B1 : 0.025 g/ trng Aflatoxin B2 : 0.125 g/ trng Aflatoxin G1 : 1.2g/ trng Aflatoxin G2 : 2.7g/ trng 20

NM MC V C T AFLATOXIN Aflatoxin M1 : 0.2g/ trng. 4.1.4.3 Th nghim trn u trng gip xc Loi Artemia salina: Th nghim tin hnh trong vng 24 gi. Khng cn thit b t tin v nui dng sinh vt. Trng tm c sng c qua nhiu nm nu c bo qun kh ro. Cc trng ny 27 oC v nc mn ( t trng 1.02) s n trong vng 25 gi. Ta chn lc u trng cng mt sc sng bng cch cho chng bi t mt im ti ti mt im sng. Cc Aflatoxin nghi ng c trong mu c chit bng cloroform, sau loi b cht ny bng un cch thy. Dng pipet ly khong 30 - 50 u trng v 0.5 ml nc cho vo mt knh ng h c nc chit cn th. Sau 24 gi v 37.5o C tnh phn trm s u trng cht. Bng cch ny ngi ta xc nh rng trong cc iu kin trn hm lng 0.5g/ml aflatoxin B1 git cht 60% s u trng. Nng gp i nh vy git cht 90% u trng. 4.1.4.4 Th nghim trn c vng C Brachydanio rerio c trng trong sut, cho php ta d dng theo di s tin trin ca n. t u trng vo nhit mi trng xung quanh 3-4 ngy: Chng c mt non hong phi ln, chng khng cn phi nui dng hoc chm sc c bit. i vi chng aceton nng thp khng c.. ngi ta xc nh rng hm lng Aflatoxin B1 0.1g/ml (pha ch vi 0.8% aceton) sau 30 pht gy ra u trng nhng hot ng bt thng, sau 5 gi a n tnh trng hp hi, sau 20 gi lng b thm mu v ui xon li, sau 24 35 gi th lng v v cht 4.1.4.5 Th nghim trn nng nc Nhi Rana temporaria khi n phi Aflatoxin ch b xung huyt gan, nhng nng nc ca chng cht nng Aflatoxin trn 2g/ml. Di nng ny khng th bin nhi tr thnh nhi trng thnh. Nng nc ca Bufo melanostriclus, Racophorus leucomyslax maculatus v Uperodon sp th b cht v ngi ta nhn thy gan v thn ca chng b h.

21

NM MC V C T AFLATOXIN 4.2 Phng php hin i 4.2.1 Phng php o mt hunh quang 4.2.1.1 Nguyn l ca phng php Aflatoxin c chit tch t mu th bng clorofoc. Dch chit c lc v mt phn dch lc c lm sch qua ct silicagen. Lm bay hi dung dch ra gii v ho cn vi clorofoc hoc hn hp benzen axetonitril. Sau chm mt phn xc nh mu th ln bn mng,chy sc k v so snh Rf, mu sc vt sc k ca mu phn tch vi vt aflatoxin chun di n t ngoi c bc sng 365nm. Lng aflatoxin c th xc nh bng mt hay bng my o mt hunh quang. i tng p dng ca phng php : Xc nh hm lng aflatoxin B1, B2, G1, G2 trong cc sn phm ng cc, u , ht c du. 4.2.1.2 Dng c, ho cht, thuc th 4.2.1.2.1 Dng c - My xay nghin mu - My lc hoc my khuy t - Cn phn tch - My ct quay chn khng. - Bp cch thu. - n t ngoi c bc sng 365nm. - Quang ph k UV-VIS. - My o mt hunh quang. - Cc trang b ca sc k lp mng. - Ct sc k thu tinh, ng knh trong 22mm, di 300mm, c kho nt mi thu tinh hoc teflon v bnh ng dung mi pha trn, dung lng 250ml. - Bnh nn nt mi 250ml, 500ml. - ng ong 50ml, 100ml. - Phu lc, ng knh 8 10 cm. - Micropipet Hamilton:20 m l, 50 m l - Bn mng kch thc 20 x 20 cm trng sn lp mng Silicagel 60-G

22

NM MC V C T AFLATOXIN 4.2.1.2.2 Ho cht, thuc th Tt c cc ho cht u phi l loi tinh khit phn tch (TKPT) nu khng c cc ch dn ring no khc. - Ax tn - Clorofoc - n Hexan - Ete tylic khan - Metanol - Axetonitril - Toluen - Natri sunfat khan - Celite 545, t iatomit - Axit sunfuric 50% (v/v) - Axit trifluoraxectic - Kh nit - Bng ht nc (c loi bo bng clorofoc) hoc bng thu tinh. - Cc h dung mi khai trin sc k. - Clorofoc/ axeton: 9/1 (v/v) ( theo t l th tch) - Ete tylic/ metanol/ nc: 94/4, 5/1,5/1 (v/v/v/v) - Clorofoc/ axeton/ isopropanol/ nc: 88/12/1, 5/1 (v/v/v/v) - Toluen/ tyl axetat/ axit focmic: 6/3/1 (v/v/v) - Silicagel G 60 dng cho sc k ct, c ht 0,063 0,2 mm. Hot ho bng cch sy kh 105 o C trong mt gi, sau trt vo bnh tam gic nt nhm, thm nc vi t l 1ml nc cho 100g silicagel, y nt, lc n khi trn hon ton u, bo qun 15 gi trong bnh kn. - Chun b dung dch aflatoxin chun (iu kin tin hnh: trong phng mt, trnh nh sng): - Dung dch aflatoxin chun gc (dung dch A): - Dng ng chun 1 mg, mi loi aflatoxin chun B1, B2, G1, G2 ln lt cho vo 4 bnh nh mc 100ml v nh mc n vch bng hn hp toluen axetonitril 98: 2 (v/v) (nng dung dch aflatoxin chun l 10 m g/ml). 23

NM MC V C T AFLATOXIN - Dung dch aflatoxin chun lm vic (dung dch B): Cho vo mt bnh nh mc 10ml ln lt 0,5 ml aflatoxin B1; 0,5 ml aflatoxin G1; 0,1 ml aflatoxin B2, 0,1 ml aflatoxin G2 (t dung dch A trn), nh mc n 10ml bng hn hp toluen axetonitril 98: 2 (v/v), nng dung dch l 0,5 m g/ml i vi B1, G1, v 0,1 m g/ ml i vi B2, G2. Cn kim tra li nng dung dch aflatoxin chun (dung dch A m g/ml) bng quang ph hp th phn t UV-VIS, bc sng 350nm, tnh kt qu theo cng thc:
C mg / ml 1000 m A e

Trong : m Khi lng phn t aflatoxin A Mt quang bc sng hp th. e - hp th phn t ca aflatoxin Aflatoxin m Dung mi benzen l max (nm) e benzen axetonitril l max (nm) axetonitril (v/v) (v/v) B1 312 98: 2 350 19800 B2 314 98: 2 350 20900 G1 328 98: 2 350 17100 G2 330 98: 2 350 18200 0 Bo qun: nhit thp hn 0 C, dung dch A c 6 thng v dung dch B c hai tun trc khi dng hot ho 1100C trong 1 gi. 4.2.1.3 Phng php tin hnh: (iu kin tin hnh: phng mt, trnh nh nng ) 4.2.1.3.1 Chun b mu: Nghin mu sao cho c th qua c ry c l c 1mm. Ch thch: Vi cc mu th c hm lng cht bo ln hn 5%, cn loi cht bo bng cch chit vi dung mi te du ho ( si 40 600C) sau khi hon thnh cng on nghin mu. 4.2.1.3.2 Tin hnh xt nghim Chit sut: Cn 50g mu nghin nh cho vo bnh nt mi 500ml. Cho tip 25ml nc, 25g celite 545, 250ml clorofoc, y nt cht. Lc trong 30 pht bng my lc quay trn hay my khuy t (hoc lc k bng tay) ri lc qua giy lc gp cnh, b 24

NM MC V C T AFLATOXIN 10ml dch lc u, sau ly 50ml dch lc tip theo (tng ng 10g mu th). Nu tc dch lc xung chm, chuyn sang phu Bucher c cha 1 lp celite 545 dy 5mm trn giy lc v lc ht chn khng. Lm sch mu bng ct sc k: Chun b ct sc k: Cho mt t bng vo y ct sc k, thm 5g natri sunfat khan. clorofoc n 2/3 ct, sau cho 10 g silicagel dng cho sc k ct (5.1.2.2.2.15), va cho va g nh thnh ct cho silicagel lng u, dng a thu tinh khuy nh trnh bt kh. M kho cho clorofoc chy xung t t. Khi tc lng ca silicagel chm li, rt ht clorofoc ch li mt lp 5cm pha trn lp silicagel. Thm t t 15g natri sunfat khan, sau rt bt clorofoc n gn st lp natri sunfat khan trn. Ct silicagel thu c phi mn, khng c bt kh v ch khng ct b kh k t lp natri sunfat khan trn. Trn 50ml dch lc trn vi 150ml n hexan, cn thn vo ct sc k chun b nh trn. Loi b dung dch chy ra. Cho tip 150ml te tylic khan, loi b dung dch chy ra. Ch khng ct b kh, lu lng dng chy khong 8 12ml /pht. Dung dch r gii aflatoxin trn bng 150ml hn hp metanol clorofoc (3 : 97). Ly ton b dch chy ra t khi bt u cho dung dch ra gii cho n ht. Lu lng dng chy nh trn. Lm bay hi dung dch ra bng my ct quay chn khng nhit thp hn 500 C cho n khi cn khong 2 3 ml. Chuyn sang ng nghim 10ml, trng ra bnh cu bng clorofoc v lm bay hi n kh trn ni cch thu nhit thp hn 500C, tt nht di lung kh nit nh cn cn li trong ng nghim l mu phn tch. y kn ng nghim c cn chy sc k lp mng. Sc k lp mng mt chiu: Chun b: Ho cn thu c trn bng 0,5ml hn hp benzen axetonitril (98: 2) v ly dch ny chm ln bn sc k. Vi bn mng trng sn trc khi chm sc k phi co sch lp silicagel hai cnh bn tm sc k vi chiu rng 0,5cm. Sau cch cnh y 2 cm, dng mao qun hay micropipet chm cc vt dung dch chun aflatoxin v dung dch mu th nh sau: - Ly 2; 5;10; 20 m l dung dch chun B (5.1.2.2.2.16.2). - Ly 10 m l dch chit vi 10 m l dung dch chun B (5.1.2.2.2.16.2). - Ly 10, 20 m l dch chit. 25

NM MC V C T AFLATOXIN - Vi tng th tch nh trn, 4 dung dch aflatoxin B1, B2, G1, G2 chun c th c chm chng ln nhau. Khai trin bn sc k ch ti vi h dung mi clorofoc axeton (9: 1). Sau khi tip tuyn dung mi chy ln khong 10cm (khong 30 pht), ly bn sc k ra bay hi dung mi nhit phng, trnh nh sng. Sau soi di n t ngoi vi bc sng 365 nm. Cc vt aflatoxin B1, B2 c hunh quang mu xanh da tri; G1, G2 c hunh quang mu xanh l cy. Nu mu th c aflatoxin s xut hin cc vt cng gi tr Rf vi cc vt aflatoxin chun. Rf ca cc vt aflatoxin trong h dung mi clorofc - axtn (9:1) c tr s trung bnh nh sau: Aflatoxin B 1 : 0,72 Aflatoxin B 2 : 0,67 Aflatoxin G 1 : 0,59 Aflatoxin G 2 : 0,54 4.2.1.3.3 Th nghim khng nh s c mt ca aflatoxin: -Phun dung dch axit sunfuric 50% (v/v) ln bn sc k, hunh quang ca cc vt aflatoxin s chuyn sang mu vng di tia t ngoi c bc sng 365nm. -Rt dung dch it 5 10% trong te ln mt tm thu tinh 20 x 20cm, dn u khu vc gia, cho te bay hi ht cn li mt lp it mng. t p tm thu tinh ny ln bn sc k trin khai, cch khong 1 cm trong thi gian 20 30 giy. Ly bn sc k ra quan st di n t ngoi, nu hunh quang ca cht chun v dung dch th khng mt i, chng t s c mt ca aflatoxin. -Chia bn mng silicagel 20 x 20 cm lm hai phn bng nhau (cch 10cm gia), chm cc vt dch chit mu thv dung dch aflatoxin chun nh sau: Phn 1: Bn tri, chm 4 vt nh sau. - 10 m l dung dch aflatoxin chun (5.1.2.2.2.16.2) - 10 m l dch chit - 10 m l dch chit vi10 m l dung dch aflatoxin chun (5.1.2.2.2.16.2) - 10 m l dung dch aflatoxin chun (5.1.2.2.2.16.2) Phn 2: Bn phi,chm 4 vt nh sau. - 10 m l dung dch aflatoxin chun (5.1.2.2.2.16.2) - 10 m l dch chit - 10 m l dch chit vi 10 m l dung dch aflatoxin chun (5.1.2.2.2.16.2) 26

NM MC V C T AFLATOXIN - 10 m l dung dch aflatoxin chun (5.1.2.2.2.16.2) Chm xong dng my sy nh dung mi bay ht. Nh ln 4 vt chm phn 1 bn tri, mi vt mt git axt trifluoraxetic. Che phn 1 (bn tri) bng mt tm thu tinh khc, phun ln phn 2 (bn phi) dung dch axitsunfuric 50% (v/v). Dng my sy nh lm kh cc vt chm ri mi cho vo bnh khai trin. Dung mi khai trin: Nc/metanol/ete etylic 1/3/96 (v/v). Khi dung mi ln khong 13 cm, ly bn sc k ra kh nhit phng, trnh nh sng, soi di n t ngoi bc sng 365 nm, cch n 10 cm. 4.2.1.3.4 nh gi kt qu - Na bn tri: Cc vt hunh quang aflatoxin ca dung dch aflatoxin chun v ca vt dch chit (nu c) s cng c tr s Rf ngn hn bnh thng, vn gi nguyn mu hunh quang xanh . - Na bn phi: Cc vt hunh quang aflatoxin ca dung dch aflatoxin chun v ca vt dch chit (nu c)s cng c tr s Rf bnh thng, v mu hunh quang xanh chuyn sang mu vng. 4.2.1.3.5 nh lng - Bng mt: Xc nh hm lng aflatoxin trong dch chit bng cch so snh mt hunh quang ca vt dch chit vi mt hunh quang ca vt dung dch chun. Vt hunh quang ca vt chm dung dch aflatoxin chun chng ln vt dch chit phi mnh hn hunh quang ca vt 10 m l dch chit v phi trng nhau. Nu mt hunh quang ca vt 10 m l dch chit m hn mt hunh quang ca vt 20 m l dung dch chun th phi pha long dch chit v tin hnh lm sc k lp mng li. - Tnh kt qu Hm lng aflatoxin (C) tnh bng microgam trong 1 kilogam mu c tnh theo cng thc:
C S Y V WX

Trong : S Nng aflatoxin ca dung dch chun mg/ml (Dung dch B ) V Th tch cui cng ca dch chit k c nhng ln pha long nu c, ml. 27

NM MC V C T AFLATOXIN W Khi lng ca mu th, (tng ng vi th tch dch chit khi tin hnh lm sch qua ct) g. X Th tch dch chit c mt hunh quang ging th tch Y ca dung dch chun chm trn bn mng, ml. Y Th tch dung dch chun c mt hunh quang ging th tch X ca dch chit chm trn bn mng, ml. - Bng my o mt hunh quang. Bc sng kch thch l 365nm v bc sng pht x l 443 nm. nh lng aflatoxin trn cc chm ca dch chit bng cch so snh mt hunh quang ca vt dch chit vi mt hunh quang ca vt dung dch chun. Tnh kt qu. Hm lng aflatoxin (C), tnh bng microgam trong 1 kilogam mu th tnh nh sau:
C Am S V Y As W X

Trong : Am - mt hunh quang ca vt mu. As - mt hunh quang ca vt chun. X Th tch ca dch chit chm ln bn mng ( m l ). Y Th tch ca dch chun chm ln bn mng ( m l ). S Hm lng aflatoxin ca dung dch chun,( ng). V Th tch cui cng ca dch chit k c nhng ln pha long nu c, ( m l). W Khi lng ca mu th, tng ng vi th tch dch chit khi tin hnh lm sch qua ct,( g). 4.2.1.4 nhy ca phng php : 5 m g / kg (5 ppm) - H s thu hi ca phng php : 90 5%. - Sai s trung bnh ca phng php : 10%. 4.2.2 Phng php sc k lng hiu nng cao 4.2.2.1 Phm vi p dng Tiu chun ny qui nh phng php xc nh hm lng c t aflatoxin (B1, B2, G1, G2) trong thy sn v sn phm thy sn bng sc k lng hiu nng cao (sau y gi tt l HPLC). Gii hn pht hin ca phng php l 1,5 mg/kg. 28

NM MC V C T AFLATOXIN 4.2.2.2 Phng php tham chiu Tiu chun ny c xy dng da theo phng php chun s 49.2.05 ca Hip hi cc nh ho hc phn tch (AOAC) cng b nm 1997. 4.2.2.3 Nguyn tc Aflatoxin c trong mu thy sn bao gm cc nhm B1, B2, G1 v G2 c chit tch ra bng clorofom. Dch chit c lm sch bng phng php chit pha rn (SPE) trn silicagel. Hm lng aflatoxin c trong dch chit c xc nh trn h thng HPLC vi u d hunh quang theo phng php ngoi chun. 4.2.2.4 Thit b, dng c, ha cht, cht chun v dung dch th 4.2.2.4.1 Thit b, dng c 5.2.2.4.1.1 H thng HPLC vi u d hunh quang. 5.2.2.4.1.2 Ct sc k pha o LC18 kch thc L x ID l 25 cm x 4,6 mm, ng knh ht t 5 n 10 [NAD1]mm. 5.2.2.4.1.3 Mng lc mao qun kch thc 0,4 mm. 5.2.2.4.1.4 My nghin ng th tc 10 000 vng/ pht. 5.2.2.4.1.5 Cn phn tch c chnh xc 0,0001 g. 5.2.2.4.1.6 My ly tm tc 5 000 vng/pht. 5.2.2.4.1.7 B siu m. 5.2.2.4.1.8 H thng c quay chn khng. 5.2.2.4.1.9 Ct thy tinh c kha teflon, kch thc L x ID l 500 x 20 mm v 500 x 8 mm. 5.2.2.4.1.10 ng ly tm thy tinh dung tch 250 ml. 5.2.2.4.1.11 Bnh cu thy tinh dung tch 100 ml v 250 ml. 5.2.2.4.1.12 Bnh nh mc dung tch 5 ml v 10 ml. 4.2.2.4.2 Ha cht 5.2.2.4.2.1 Nc ct loi dng cho HPLC. 5.2.2.4.2.2 Metanol loi dng cho HPLC. 29

NM MC V C T AFLATOXIN 5.2.2.4.2.3 Clorofom loi dng cho HPLC. 5.2.2.4.2.4 Axetonitril loi dng cho HPLC. 5.2.2.4.2.5 n-hexan tinh khit loi dng cho phn tch. 5.2.2.4.2.6 Ete etylic tinh khit. 5.2.2.4.2.7 Sulfat natri khan. 5.2.2.4.2.8 Silicagel c ht t 60 n 200 mesh. 5.2.2.4.2.9 Silicagel c hot ha: cn 50,0 g silicagel tinh khit (4.2.8) vo t sy trong 2 gi nhit 110oC. Sau ngui v nhit ca phng trong bnh ht m v ngm trong clorofom 15 pht trc khi nhi ct. 4.2.2.4.3 Dung dch chun v dung dch th 5.2.2.4.3.1. Dung dch chun gc hn hp gm: B1 (nng 100,0 mg/l), B2 (nng 20,0 mg/l), G1 (nng 100 mg/l) v G2 (nng 20 mg/l). 5.2.2.4.3.2 Chun b dung dch chun gc hn hp aflatoxin c nng (nh iu 5.2.2.4.3.1) trong metanol t cc ng chun. Ty theo nng cc aflatoxin c trong ng chun, dng bnh nh mc v lng metanol thch hp. 5.2.2.4.3.3 Dung dch chun trung gian c nng l B1 (10,0 mg/l), B2 (2,0 mg/l), G1 (10,0 mg/l) v G2 (2,0 mg/l): ht chnh xc 1,0 ml dung dch chun gc hn hp (5.2.2.4.3.1) vo bnh nh mc 10 ml (5.2.2.4.1.12) ri nh mc ti vch bng metanol (5.2.2.4.2.2). 5.2.2.4.3.4 Dung dch chun: ht chnh xc ln lt 0,0 ml, 1,0 ml, 2,0 ml, 4,0 ml, 8,0 ml v 10,0 ml dung dch chun trung gian (5.2.2.4.3.3) vo cc bnh nh mc 10 ml (5.2.2.4.1.12) ri nh mc ti vch bng metanol. Cc dung dch chun thu c c nng aflatoxin ln lt nh sau: a. Chun 1 : B1 (0,0 mg/l), B2 (0,0 mg/l), G1 (0,0 mg/l), G2 (0,0 mg/l). b. Chun 2 : B1 (1,0 mg/l), B2 (0,2 mg/l), G1 (1,0 mg/l), G2 (0,2 mg/l). c. Chun 3 : B1 (2,0 mg/l), B2 (0,4 mg/l), G1 (2,0 mg/l), G2 (0,4 mg/l). d. Chun 4 : B1 (4,0 mg/l), B2 (0,8 mg/l), G1 (4,0 mg/l), G2 (0,8 mg/l). . Chun 5 : B1 (8,0 mg/l), B2 (1,6 mg/l), G1 (8,0 mg/l), G2 (1,6 mg/l). e. Chun 6 : B1 (10,0 mg/l), B2 (2,0 mg/l), G1 (10,0 mg/l), G2 (2,0 mge) 5.2.2.4.3.5 Dung dch pha ng: pha hn hp cc dung mi axetonitril, metanol v nc ct loi dng cho HPLC theo t l v th tch l 2 : 2 : 6. 30

NM MC V C T AFLATOXIN 4.2.2.4.4 Cch tin hnh 5.2.2.5.1 Chun b mu th 5.2.2.5.1.1 Dng cn phn tch (5.2.2.4.1.5) cn chnh xc 50,0 g mu (m) c bm nhuyn cho vo ng ly tm thu tinh dung tch 250 ml (5.2.2.4.1.10). 5.2.2.5.1.2 Thm 100,0 ml clorofom (5.2.2.4.2.3) vo ng ri trn u trong khong 2 pht bng my nghin ng th (5.2.2.4.1.4). Sau , ly tm ng bng my ly tm (5.2.2.4.1.6) trong khong 5 pht tc 5000 vng/pht. Tin hnh lc dch chit v ra phn b bng clorofom ri cho tt c dch thu c vo bnh cu thu tinh dung tch 250 ml (5.2.2.4.1.11). 5.2.2.5.2 Chun b mu trng Mu trng c nh ngha l mu thy sn c xc nh khng c aflatoxin. Tin hnh chun b mu trng ging nh chun b vi mu th theo qui nh ti (iu 5.2.2.5.1) 5.2.2.5.3 Chun b mu xc nh thu hi Thm 2,0 ml dung dch chun 3 (5.2.2.4.3.4.c) vo 10,0 g mu trng. ng nht mu bng my nghin ng th (5.2.2.4.1.4). Tin hnh chun b mu ging nh chun b vi mu th theo qui nh ti (iu 5.2.2.5.1). Phi chun b mu xc nh thu hi ng thi vi chun b mu th v mu trng. 5.2.2.5.4 Lm sch dch chit 5.2.2.5.4.1 Chun b ct t 1 lp bng thy tinh vo y ct thy tinh c kha teflon (4.1.9). ng kha v cho clorofom ti khong 2/3 ct ri thm ln lt 5,0 g sulfat natri khan (4.2.7), 20,0g silicagel hot ha (4.2.8), 15 g sulfat natri khan (4.2.7). Ch thch: trnh kh ct lun gi mc clorofom cao hn lp sulfat natri khan trn cng khong 1,5 cm. 5.2.2.5.4.2 Lm sch dch chit 5.2.2.5.4.2.1 C dch chit thu c (5.2.2.5.1.2) trn h thng c quay chn khng (5.2.2.4.1.8) cn khong 5,0 ml nhit 400C. Dng pipet chuyn dung dch t 31

NM MC V C T AFLATOXIN bnh cu (5.2.2.5.1.2) vo ct lm sch chun b (5.2.2.5.4.1) v trng ra bnh cu bng clorofom. iu chnh kho tc chy ca dung dch ra khi ct khong t 1,0 n 1,5 ml/pht. Trong giai on ny, aflatoxin s hp ph ln cc ht silicagel. 5.2.2.5.4.2.2 Thm vo ct ln lt 50,0 ml n-hexan (5.2.2.4.2.5), 50,0 ml ete etylic (5.2.2.4.2.6). iu chnh tc dung mi chy qua ct 1,0 ml/pht. Sau loi b dch chy ra khi ct. 5.2.2.5.4.2.3 Gii hp cc aflatoxin khi ct lm sch bng 50,0 ml hn hp clorofom v metanol theo t l v th tch l 97: 3 vi tc d 1,0 ml/pht. Hng dung dch chy ra khi ct vo bnh cu dung tch 100 ml (5.2.2.4.1.11). C dch thu c trn h thng c quay chn khng (5.2.2.4.1.8) nhit 40o C cho n kh hon ton. Ho tan cn bng 5,0 ml (V) dung dch pha ng (5.2.2.4.3.5) trong bnh nh mc 5 ml (5.2.2.4.1.12). Tin hnh phn tch hm lng cc aflatoxin trn HPLC theo qui nh ti( iu 5.2.2.5.5) 5.2.2.5.4.2.4 Tin hnh lm sch mu trng (5.2.2.5.2) v mu xc nh thu hi (5.2.2.5.3) ging nh vi mu th (5.2.2.5.1) theo qui nh ti (iu 5.2.2.5.4.2.1, iu 5.2.2.5.4.2.2 v iu 5.2.2.5.4.2.3.) 5.2.2.5.5 Tin hnh phn tch trn HPLC 5.2.2.5.5.1 iu kin phn tch a. Ct sc k : RP-LC 18, kch thc L x ID l 25 cm x 4,6 mm, ng knh ht 5 -10 [NAD2]mm. b. Nhit ct : 35oC. c. Pha ng : Hn hp gm: axetonitril, metanol v nc ct (5.2.2.4.3.5) d. Tc dng: 1,0 ml/pht. . Bc sng ci t cho u d hunh quang l: (kch hot) E x 365 nm, (pht x) Em 455 nm. e. Th tch tim: 20 ml. 5.2.2.5.5.2 Tim cc dung dch chun (5.2.2.4.3.4) vo my HPLC theo th t nng t thp n cao. Mi dung dch tim 2 ln, tnh din tch pic trung bnh. Dng ng chun biu th mi quan h gia cc din tch pic thu c v nng tng loi aflatoxin theo quan h tuyn tnh bc 1 (phng trnh y = ax + b).

32

NM MC V C T AFLATOXIN 5.2.2.5.5.3 Tim dung dch mu th, dung dch mu trng v dung dch xc nh thu hi vo h thng HPLC. Mi dung dch mu tim 2 ln. Tnh gi tr trung bnh. 5.2.2.5.6 Yu cu v tin cy ca php phn tch 5.2.2.5.6.1 lp li ca 2 ln tim lch chun (CVs) tnh theo din tch pic sc k ca 2 ln tim cng mt dung dch chun phi nh hn 0,5 %. 5.2.2.5.6.2 thu hi (R) thu hi c xc nh bng cch s dng 10 mu trng cho vo mt lng dung dch aflatoxin chun bit hm lng chnh xc (5.2.2.5.3). thu hi tnh c phi nm trong khong t 85 % n 115 %, thu hi trung bnh phi ln hn 90 %. 5.2.2.5.6.3 ng chun phi c tuyn tnh tt, h s tng quan quy hi tuyn tnh (R2) phi ln hn hoc bng 0,99. 5.2.2.6 Tnh kt qu Hm lng cc aflatoxin c trong mu c tnh trn c s ng chun thu c (5.5.2). Vi ng chun dng y = ax +b, hm lng cc aflatoxin c trong mu c tnh theo cng thc sau:
C (mg / kg ) Y b F a

Trong : - C l nng aflatoxin c trong mu, tnh theo mg/kg - Y l hiu s gia din tch pic ca dch chit v din tch pic c trong mu trng tim vo HPLC, tnh theo n v din tch - a, b l cc thng s ca ng chun y = ax + b, c xc nh theo (iu 5.2.2.5.5.2.) - F l h s pha long mu v c gi tr bng t s gia th tch dch chit thu c sau khi lm sch V (5.2.2.5.4.2) v khi lng mu m (5.2.2.5.1.1) s dng. 4.2.2.5 Kt lun c t nm Aflatoxin c th lm cho ngi nhim b ung th gan, ht sc nguy him. Do , vic phng nga rt quan trng v vn cha c thuc cha tr c hiu dnh ring cho bnh ny...

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NM MC V C T AFLATOXIN

5 Bin php phng nhim c t Aflatoxin

Hin nay thuc cha bnh c hiu khng c, v vy bin php phng bnh l quan trng. Aflatoxin l mt c t kh bn vng vi nhit. V vy bin php un si thng thng khng c tc dng i vi c t. phng ng c, bin php p dng l vn bo qun tt cc loi lng thc thc phm, trong ch yu l thc phm thc vt. - Vi lng thc nh go, ng, m: Yu cu bo qun l gi kh, thong mt khng b nhim mc. - Vi nhng thc phm thc vt kh nh lc, vng, c ph... l nhng thc phm d ht m v d mc. Mun bo qun tt cn c phi kh, gi nguyn v, cha trong cc dng c sch, kn nu lu, thnh thong phi em phi kh li. Yu cu m ca ht l di 15%. - Vi nc chm nh x du, tng: Nhng thng bo kt qu u tin nc ta cho thy nhim Aflatoxin trong nc chm l ng lo ngi. V vy vic kim tra v sinh cc x nghip sn xut nc chm v cc ca hng mua bn l cn thit v phi c tin hnh thng xuyn. Ni dung kim tra cn lm: Kim tra v sinh mi trng (ch yu l khng kh). Kim tra v sinh nc chm. Ngoi cc ch tiu v sinh c qui nh cho mt mu nc chm v mt mu khng kh, cn phi ch pht hin s c mt ca cc chng nm sinh c t nh: Aspergillus flavus, parasiticus v fumigatus.

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NM MC V C T AFLATOXIN

6 Ti liu tham kho

[1]. Abdollahi, A., and R. L. Buchanan, Regulation of aflatoxin, 1981 [2]. ADAMS, M.R., Et Al, Food microbiology, RSC, UK, 2002. [3]. Bhatnagar, D., S. P. McCormick, L. S. Lee, and R. A. Hill. 1987, Identification of Omethylsterigmatocystin as an aflatoxin B1/Gl precursor in Aspergillus parasiticus, Appi. Environ. Microbiol. [4]. GS.TS Nguyn Th Hin, GS.TS Phan Th Kim, Ts. Trng Th Ha, Th.S L Th Lan Chi, Vi sinh vt nhim tp trong lng thc - thc phm, i hc Bch Khoa H Ni, 2009. [5]. L Ngc T, c t hc v an ton thc phm, NXB Khoa hc K thut H Ni, 2006. [6]. Maggon, K. K., S. K. Gupta, and T. A. Venkitasubramanian 1977, Biosynthesis of aflatoxins. Bacteriol. Rev. 41:822-855. [7]. M. Herzberg (ed.), Toxic micro-organisms, UJNR Joint Panels on Toxic Micro-organisms and U.S. Department of the Interior, Washington, D.C. [8]. Vandegraft, and G. Shannon, Production of various aflatoxins, 1968. [9]. http:// www.biochem.duke.edu.

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