XC NH HM LNG C T AFLATOXIN B1 TRONG BP V U PHNG BNG PHNG PHP SC K BN MNG GVHD: Th.

S Lm Thanh Hin Thc hin: Hunh Minh Duy, Nguyn Vn Ph Khoa Cng Ngh Sau Thu Hoch

Sumary : The research of Post-harvest Technology Facultys students and teachers have successfully applied the method of Thin Layer Chromotography with two columns: immuno-affinity column and silicagel column to determine Aflatoxin B 1 (produced by Aspergillus flavus and Aspergillus paraciticus) on 30 samples (of peanut and corn) at its minimum concentration of 2ppb. This rapid, simple and economic method has served extension work for developing food safety control. I. t vn :

Thc n gia sc, thc phm v cc loi ht c du, tho dc,u c kh nng nhim c t nguy him aflatoxin B 1 do 2 loi vi nm in hnh l Aspergillus flavus (hnh 1) v Aspergillus paraciticus gy nn. Cc aflatoxin l nhng tinh th mu vng tan trong mt s dung mi hu c (clorofoc, methanol, acetone,) v c tnh cao, rt bn vng vi tc nhn ha l..., ch b hu nhit trn 120 0C trong mi trng kim. Do cu trng ha hc c vng dihydrofuran (hnh 2) nn aflatoxin B1 lin kt vi mt s enzym lm cn tr trao i cht dn n t vong. Ngoi ra, aflatoxin B1 cn tng tc ng ha tr vi vt cht di truyn (DNA, RNA) gy tn thng gan v gy ung th gan. Vi ph n mang thai, hp thu lng nht nh s dn n di tt thai nhi hoc qui thai, nng c th cht non.

Hnh 1: Aspergillus flavus Hnh 2: Cu trc aflatoxin B1 (C17H12O6) Do , vic kim tra thc phm c nguy c nhim aflatoxin B1 l rt cn thit. Theo tiu chun Vit Nam, thc phm dng cho ngi lng aflatoxin khng vt qu 10ppb. pht hin v nh lng aflatoxin, hin nay 2 nhm phng php chnh sau: Nhm phng php ha l: sc k lp mng, sc k lng cao p (HPLC) Nhm phng php min dch hc: min dch phng x (RIA), min dch enzym (ELISA) v ct sc k i lc min dch. i vi phng php sc k lng cao p, trc khi pht hin phi tri qua rt nhiu thao tc phc tp nh chit ly aflatoxin t vt liu, sau phi c c trc khi nh lng nn tn nhiu thi gian thao tc k thut, hn na thit b u t rt t tin.

Vi phng php ELISA, li c nhiu hn ch v aflatoxin thng khng bm c hiu vo cc ging polystirene, khong pht hin hp, v cn phi c aflatoxin nh du hoc khng th nh du, sai s ln mc d c nhy cao. V nhng l do , chng ti tin hnh ti: Xc nh hm lng c t aflatoxin B1 bng phng php sc k bn mng s dng ct i lc min dch v ct silicagel trong mu hay nhim nht l u phng v bp vi nhng u im sau: C c trn ct vi nng thp v loi b c tp cht nh hng n qui trnh nh lng. Thi gian tin hnh nhanh, nhy cao, thit b n gin v ha cht t. II. Vt liu v phng php 2.1. Nguyn liu -Bp ht v u phng ly t ch B Chiu, Ch Tn Sn Nht. -Bp ht v u phng ly ca mt s cng ty. 2.2. Thit b v ha cht -Thit b: + Bt thit b chy sc k TLC + Dng c lm kh gia nhit, bnh thi kh, n soi + Cc tm silicagel 10 x 10 cm mua t Merk + Microsylinge Halmilton 10 + Cc dng c thy tinh cn thit -Ha cht : acetonlintril, methanol, benzen (tinh khit) v nc ct + Dung dch trin khai:

Chloroform 18 = ; Acetone 2

dung

dch

ha

cn:

+ Aflatoxin B1 chun 0,5ppm trong dung dch ha cn. 2.3. Phng php nghin cu: 2.3.1. Ni tin hnh nghin cu: Phng th nghim phn tch ha sinh, cng ty c phn gim nh v kh trng FCC. 2.3.2. Tin trnh nghin cu: ct IAC kt qu IAC Bp ht + u phng sc k bn mng TLC ct silicagel ktqu silicagel 2.3.3. Qui trnh thc hin: 2.3.3.1. S phn tch hm lng Aflatoxin B1 bng ct IAC Mu phn tch (25gam) chit xut (5g NaCl, 37,5ml nc ct, methanol 87,5ml, thi gian 30 pht) lc thng (thu 15 ml v cho thm 15ml nc ct) lc giy thy tinh (thu 45ml dch lc) qua ct IAC (2 git/pht) lm kh dch chit (trong N2/40 500C) nh lng bng TLC 2.3.3.2. S phn tch hm lng Aflatoxin B1 bng ct Silicagel -Chun b ct Silicagel: Sc k ct cho 5gam Na2SO4 khan cho CHCl3 ti ng cho 10g silicagel ra thnh ng 20ml CHCl3 cho t t 15gam Na2SO4 khan cho CHCl3 chy ti nh Na2SO4 -Tin hnh: Mu phn tch (50gam) chit xut (25ml nc ct, 250ml CHCl 3, y kn v lc mnh trong 30 pht) lc thng (thu 50 ml) ct silicagel ( chun b) ra gii tp (ln 1: 150ml C 6H6, ln 2: 150ml (C2H5)2O) ra gii Aflatoxin B1 (150ml CH3OH : CHCl3 = 3:97) thu dch Aflatoxin B1 vo bnh cu

Benzen 98 = Aceto ln itril 2

 lm kh vi my c quay chn khng ha cn (2ml CHCl3) lm kh (trong N2/40 500C hoc chn khng/400C) nh lng Aflatoxin B1. III. Kt qu v bn lun 3.1. T l v mc nhim Aflatoxin B1 trn cc mu phn tch

Bng 3.1. Hm lng Aflatoxin B1 trong cc mu ca khch hnh Ct IAC Ct Khch STT Tn mu Silicagel hng Hm lng Aflatoxin B1 (ppb) 1 Bp Cty. Thi 2 khng pht Dng hin 2 Bp Phng khng pht khng pht Hng Hi hin hin 3 Bp Phng khng pht khng pht Hng Hi hin hin 4 Bp Cty. G 10 7 Sao 5 Bp FCC H 20 14 Ni 6 Bp Cty. G Sao 7 8 9 10 11 12 13 14 15 16 Bp Bp u phng u phng u phng u phng u phng u phng u phng u phng Phng hin trng khng pht hin khng pht hin FCC H Ni FCC H Ni

Nhn xt: theo bng 3.1, mc nhim cc mu u 20ppb v ct IAC pht hin c 3 mu v ct silicagel pht hin c 2 mu. T l nhim 3/16 mu (vi ct IAC) v 2/16 mu (vi ct silicagel) Bng 3.2. Hm lng Aflatoxin B 1 trong cc mu ch B Chiu v Tn Sn Nht Ct IAC Ct Silicagel STT Tn mu Hm lng Aflatoxin B1 (ppb) 1 Bp khng pht hin khng pht hin 2 Bp 7 5 3 Bp 2 khng pht hin 4 Bp 1 khng pht hin

khng pht hin 6 u phng 4 2,5 7 u phng 1 khng pht hin 8 u phng khng pht hin khng pht hin 9 u phng 30 25 10 u phng 2 khng pht hin Nhn xt: theo bng 3.2, mc nhim cc mu u 20ppb v ct IAC pht hin c 7 mu/10 mu v ct silicagel pht hin c 3 mu/10 mu. Nhn chung, mc nhim trn bp v u phng 10ppb. Nh vy, nhy ca ct IAC cao hn so vi ct silicagel. So vi cc mu khc hng, cc mu thu cc ch c nguy c nhim cao hn. 3.2. So snh t l pht hin ca ct IAC v ct Silicagel Bng 3.3. So snh t l pht hin Aflatoxin B1 ca ct IAC v Silicagel Tng s mu Ct IAC Ct Silicagel S mu nhim T l nhim S mu nhim T l nhim (%) (%) 3 18,75 2 12,50 7 70,0 3 30,0 4 100 4 100 14 46,67 9 30,0 bng 3.3, ct c IAC nhy pht hin tt hn ct

Bp

khng pht hin

Mu

Khch hng 16 Mua ch 10 m 4 Tng 30 Nhn xt: theo Silicagel.

IV. Kt lun v ngh 4.1. Kt lun Trong tng s 34 mu thu nhn v phn tch ti phng Sinh ha, Cng ty FCC c 30,77% mu nhim Aflatoxin B1 vi mc nhim 20ppb v c 10 mu nhim vi mc < 50ppb. nhy pht hin Aflatoxin B1 ca ct IAC cao hn so vi ct Silicagel. Trong 30 mu, ct IAC pht hin 14 mu (chim 46,67%) v ct silicagel pht hin c 9 mu (chim 30%). Ct silicagel khng pht hin c mu nhim Aflatoxin B1 di hm lng 2ppb. 4.2. ngh Vi phng php n gin, t tn km v ph pht hin nhy v rng nn phng php dng ct IAC pht hin Aflatoxin B1 trong cc sn phm v nguyn liu c du l phng php ti u hin nay. Hi vng phng php ny sm c p dng rng c bit ti cc ch, cc ngun thu mua nguyn liu. Phng php ny ch th nghim mt s mu gii hn, ti ny cn c nghiu cu thm cc ni dung sau: Kho st thm mt s mu thuc cc ngun nguyn liu khc nhau Kho st thm mt s khu vc khc nhau Kho st thm mt s mu trong cc thi im khc nhau