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TRNG I HC CNG NGHIP TP.H CH MINH VIN CNG NGH SINH HC V THC PHM
B mn V sinh an ton thc phm

Tiu lun:

BACILLUS CEREUS

Nhm thc hin: 1 Lp hc phn:2105016 GVHD: C Trn Th Mai Anh

Thnh ph H Ch Minh 10 / 2011

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- Mc lc. - Nhn xt ca gio vin hng dn v danh sch nhm.

PHN 1: LI M U
.PHN

2: NI DUNG

I.Gii thiu chung v BACILLUS CEREUS. 1.c im cu to. 2.c im nui cy. 3.Tnh cht sinh ha. 4.Ngun ly nhim. 5.Tnh cht gy bnh. 5.1.c t. 5.2.Triu chng ng c. 6.Cch phng nga. II.Tc hi khi nhim BACILLUS CEREUS. 1.C ch sn sinh c t v cc loi c t. 2.C ch gy bnh. 2.1.Triu chng nn ma. 2.2.Triu chng tiu chy. III.Cc phng php pht hin BACILLUS CEREUS trong thc phm. 1.c im v nguyn tc. 2.Mi trng v ha cht. 3.Quy trnh phn tch. nh lng BACILLUS CEREUS bng phng php m lc khun.
2

Nhm:1 3.1.Pht hin bng mi trng chn lc. 3.2.Cc phn ng khng nh.

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3.3.Cc th nghim phn bit cc loi trong BACILLUS CEREUS nhm 1. 3.4.Cch tnh kt qu. 4.Gii thiu chung cc phng php phn tch nhanh. 4.1 .Phng php min dch. 4.2. K thut latex agglutination (LA) . 4.3. K thut lai phn t( DNA- hybridization). 4.4. K thut Microarray, Macroarray. 4.5 .Phng php pht hin vi sinh vt bng k thut Real Time PCR.

PHN 3:KT LUN PH LC. NHN XT CA GIO VIN HNG DN.

Chn thnh nhn li gp ca gio vin hng dn : ..

IM CHO TNG SINH VIN

STT 1. 2. 3. 4. 5. 6. MSSV 10264641 10259831 10067621 10217411 10283711 10078591 H TN Trn ng Phng ng Trn Th Trc Tin Nguyn Nhn Tn Nguyn Th Kim Ngn NHIM V Phn 1 Phn 2/I/1,2,3,4 Phn 2/I/5,6 Phn 2/II IM GHI CH

Phm Th Nguyt Nhi Phn 2/II Nguyn Tun Huy Phn 2/III/1,2

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7. 8. 9. 10. 10205111 10274011 10249121 L Xun Thanh Loan Trn Minh Thun Trn Th Yn

GVHD:C Trn Th Mai Anh

Phn 2/III/3+ Powerpoint+presentation Phn 2/III/4 Phn 3+Ph lc Powerpoint+presentation

CAC BAN TIM HIEU VE NHUNG VAN DE SAU DE TRA LOI CAU HOI CUA LOP 1.VK KHONG TAO GIAP MO 2.VK DE MOC 3.MOI TRUONG NA, TSA, BA, MYP, MOSSEL, NB, TSB. 4.PHAN UNG VP (+) 5.PHOSPHOLIPID LA GI 6.AP XE 7.TRYPSIN, PEPSIN 8.PHUONG PHAP MIEN DICH, KI THUAT LATEX AGGLUTINATION KI THUAT LAI PHAN TU KI THUAT MICROARRAY KI THUAT REAL TIME PCR BCET - RPLA REAL TIME - PCR 9.PHUONG PHAP DEM KHUAN LAC PHUONG PHAP MNP 10.PEPTONE DEM 11.KHANG NGUYEN KHANG THE CHAY DIEN DI

PHN 1:M U
Ngay c khi c Php lnh v Thc phm, hng nm nc ta vn ghi nhn c hng vn ca mc bnh truyn qua ng thc phm. l mt vn cp thit ca x hi cn c quan tm nhiu hn na.Nguyn nhn chnh ca vic ng c thc phm l do thcca con ngi cn qu km v thc phm c s dng khng an ton.Vi khun l ngun gy bnh chnh qua con ng thc phm (cc vi sinh vt gy bnh) m c quan thuc v thc phm M khuyn co dn chng v tc hi, cch pht hin, c ch ly nhim v cch phng. Mt trong s nhng loi vi khun gy ng c thc phm l Bacillus cereus. L mt vi khun gram dng, hnhque, k kh, sinh bo t. Bacillus cereus c th lm ngi b ng c i ma tiu chy v trng hp nng hn c th gy t vong.Bt c ai cng c nguy c ly nhim Bacillus cereus. Thc phm nhim Bacillus cereus l rt ph bin. Ngi n phi thc phm ny thng mc hai bnh in hnh l tiu chy v nn ma.Dng ng c gy tiu chy

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b gy ra bi loi vi khun Bacillus cereus c cu trc phn t protein ln. Loi vi khun ny rt d dnh ln cc nhm thc phm nh tht, sa, rau v c. Triu chng chnh caloi ng c ny l i tin ra nc v c bng b chut rt khong sau 615 ting k t lc n phi. Triu chng ny ko di khong 24ting. Nn ma cng c th i km nhng thng him gp i vi loi ng c gy tiu chy bi Bacillus cereus trng lng phn t ln. Cc bc s ghi nhn c cc biu hin lm sng ng ch khc vi nhng ngi b ng c bi loi vi khun Bacillus cereus mt khi khng c cha tr kp thi v trit . l biu hin vim v, chng vim nhim nng, hoi t, vim mng no, nhimtrng, vim m t bo, vim ton mt, p xe phi, vim mng trong tim. i vi tr s sinh, thm ch c nguy c gy t vong. Cch phng c bn khi nhim loi vi khun Bacillus cereus l cn thn trng khu ch bin thc phm v nu nng. V kh nng kim tra vi sinh vt Bacillus cereus Vit Nam, theo Cc An ton thc phm (B Y t), c nc ch c 38 trung tm y t d phng tnh, chim 60% tnh thnh, c nng lc kim nghim. Vin 4 vng min u nng lc xt nghim loi vi khun ny. T 2001-2006, c nc ghi nhn c hn 5.600.000 ca tiu chy do nhim trng qua thc phm, trong c 84 ca t vong. Ring 9 thng u nm ngoi ghi nhn c hn 750.000 ca tiu chy trong c 12 ca t vong. Thct, theo cc chuyn gia, phi gp t nht 10 ln con s c cng b. V vy, vic nghin cu cc c im, cu trc ca Bacillus cereus mang ngha c bit quan trng trong vic phng chng v cha tr nhng trng hp b ng c do nhim Bacillus cereus. Vi phm vi l mt ti tiu lun, nhm chng em ch cp n Bacillus cereus trong Bacillus nhm 1.

PHN 2:NI DUNG

I.Gii thiu chung v BACILLUS CEREUS. Bacillius cereus l vi khun Gram dng khi cha trng thnh nhng c th thnh Gram m khi chng gi, hnh que, sinh bo t, k kh, c khp ni trong t nhin. Mt s chun vi khun Bacillus cereus gy ng c thc phm, trong khi mt s chun li c li cho h vi sinh vt ng rut ca ng vt, theo phn loi quc t thuc gii

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Bacteria, ngnh (phylum) firmicutes, lp (Class) Bacilli, b (Order) Bacillales, h (Family) Bacillaceaem,chi (Genius) Bacillus, loi (Species) Cereus. Trong chi Bacillus ny ngoi loi Cereus cn c mt s loi nh: Bacillus subtilis Bacillus coagulans

Bacillus thuringiensis

Bacillus natto

Paenibacillus larvae

Bacillus cereus l loi vi khun hiu kh, bo t dng hnh ovan, c kh nng sinh nha bo, c pht hin u tin trong mt ca nhim c thc phm vo nm 1955. T nhng nm1972 n 1986 c ti 52 trng hp trng c thc phm do Bacillus cereus c pht hin v bo co chim khong 2% s ca bnh thc phm, trn thc t con s ny ln hn rt nhiu. 1. c im cu to BACILLUS CEREUS. Trc khun, gram dng, to ni bo t. Kch thc 0,51,5 x 2-4 . Vi khun khng to gip m, khng c kh nng di ng. Hnh 1: Bacillus cereus trn knh hin vi

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Hnh 2: Khun lc Bacillus cereus trn mi trng BA

2. c im nui cy. L loi vi khun d mc Hiu kh v k kh ty nghi. Nhit 5-50oC, ti u 35-40oC. pH 4,5-9,3, thch hp 7-7,2 Trn mi trng NA hay TSA sau 24 gi to khm ln, nhn nheo, x x. Trn mi trng BA to dung huyt rng. Trn mi trng MYP (Mannitol Egg Yolk Polymixin): khm hng chung quanh c vng sng. Trn mi trng Mossel (thch cereus selective agar): khm to hng chung quanh c vng sng. Trn mi trng canh NB, TSB: c to vng, sau cn ln cn

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Khun lc B.cereus trn mi trng Mossel

3. Tnh cht sinh ha.

Khun lc B.cereus trn mi trng MYP

Trn mi trng ng: ln men glucose trong iu kin hiu kh v k kh, khng ln men mannitol. Kh nitrat thnh nitrit. Phn ng VP (+) . Phn gii Tyroxin . Catalase (+), Citrate (+) . Mc trn NB + 0,001% lyzozym .

4.Ngun ly nhim. Bo t ca B. cereus c th c tm thy rng ri trong t nhin, bao gm cc mu bi, bn, cy ng cc, nc, vv, v vy n l mt cht gy nhim ph bin cc mt hng nguyn liu nng nghip.Mc nhim bnh thng l <100/g.3 Cc loi thc phm giu tinh bt, chng hn nh go hoc khoai ty, thng i km vi nn B. cereus (nn) bng pht c t. Do qu trnh chun b, mt trong nhng chic xe thc phm ph bin nht truyn bnh nn B. cereus l cm chin, v c nhiu t bng pht bo co. Cc bo t ca B. cereus c kch hot

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trong vic chun b ban u ca la go, m nu bo qun nhit lm dng (khong 59 n 104 F hoc 15 n 40 C) cho mt thi gian di, s pht trin nhanh v sn xut mt loic t l nhit n nh v s khng c bt hot trong qu trnh nu n tip theo. Cc chng gy tiu chy c tm thy trong mt la chn thc phm hn. Cc ngun thng thng bao gm cc mt hng tht v rau, sp v cc sn phm sa. Khng ging nh cc cht c nn, cht c tiu chy b ph hy trong nu n. Nu cc bo t kinh nghim iu kin cho php tng trng, h c th pht trin n mc cht c c sn xut. 5.Tnh cht gy bnh. Vi khun Bacillus cereus phn b nhiu trong t nhin, nhim vo cc loi thc n qua m hay tr lnh lu, thng gy ng c thc phm. 5.1. c t. c t: vi khun sn sinh 2 loi c t c t gy tiu chy (Type 1): Diarrhoed toxin. Vi khun sn sinh c t trn tht, rau qu, gia v. Bn cht l mt loi protein gy hy hoi biu b v nim mc rut, gy tiu chy c th nguy him n tnh mng. c t gy nn ma (Type 2): Emetic toxin. Vi khun nhim trong go, cm ngui, u cc loi. Bn cht c t l phospholipid c tnh n nh cao khng b phn hy nhit cao v dch d dy. Ngoi ra vi khun cn c enzyme hemolyzin l mt protein gy c mnh c th gy cht ngi. c t ny c th trung ha bi cholesterol trong huyt thanh nhng n gp phn cho s pht trin ca vi khun. Bacillus cereus c th gy ra s nhim trng v nhim c khc nhau nh: nhim trng mu, vim mng no v nhim trng mt. 5.2. Triu chng trng c. Thc n cha mt vi khun 105 vi khun/g thc phm gy ng c. Biu hin: au bng, bun nn v nn sau 1-5 gi n phi thc phm nhim vi khun. Bnh c th ko di 24 gi. Nu ng c do c t vi khun, du hiu ng c r hn: nn, mt mi, nhc u, mch nhanhnu khng iu tr kp thi bnh s tin trin nng. C 2 triu chng lm sng ng c do Bacillus cereus gy ra: Trng hp nhim type 1 c triu chng au bng tiu chy nhng khng st. Bt u sau 4-16 gi sau khi n thc phm nhim khun v ko di 12-24 gi. Tiu chy c th l mt khi lng nh hoc di do v chy nc. Loi ny c gi l " bnh di" hay hnh thc ca bnh tiu chy, v n tng t nh ng c thc phm gy ra bi Clostridium perfringens Trng hp nhim type 2: Loi ny c c trng bi bun nn v nn ma v au bng v c mt khong thi gian bnh l t 1 n 6 gi, thi gian ko di bnh l 8 n 10 gi v trung bnh l 9 gi. N ging nh Staphylococcus aureus (t cu khun) gy ng c thc phm trong cc biu hin triu chng ca n v thi gian

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bnh. y l "hnh thc bnh ngn" hoc hnh thc nn ca bnh.

c tnh Bn vi nhit pH Tnh nhy

Bng so snh c t type 1 v type 2 Type 1 Type 2 0 0 45 /30 pht 120 /90 pht n nh pH 4-11 n nh pH 2-11 Nhy vi enzyme Khng pepsin v trypsin protease v trypsin

6. Cch phng nga. Thc phm c ngun gc gy bnh l sn phm ng cc, nc st, hoa qu, xi, cm da. M cm chin c xem l mt trong nhng nguyn nhn hng u ca nhim vi khun Bacillus cereus. Chnh v th m c ngi cn gi l hi chng cm chin. Nhng loi thc phm d nhim ny bo t Bacillus cereus thng c mt v c th sng st nu un nu khng k. Trc khun cn sng st pht trin s sinh c t. Sau khi nu i vi thc phm t, nu p lnh khng cng lm vi khun tng nhanh. Cn thc hin cc bin php kim sot phng bnh nh m bo v sinh khi ch bin, nu n thch hp v lu tr thc phm, c bit l go nu chn s dng sau ny, bo qun lnh nhit thch hp vi thc phm cha n ngay v khng nn ch bin thc phm qu lu trc khi n. II.Tc hi khi nhim BACILLUS CEREUS. 1.C ch sn sinh c t v cc loi c t. V ng c thc phm do Bacillus cereus khng phi l loi bnh c ni n nhiu, s tht l phm vi nh hng ca loi bnh ny t c bit n, c bo co l nguyn nhn gy ng c thc phm v chim khong 33% trong tng s cc nguyn nhn gy ng c thc phm (a s cc nguyn nhn l do vi rt) Norway (19881993), 47% Iceland (1985-1992). T l thp hn nhiu c bo co cc quc gia khc bao gm U.S (1.3%) v Canada (2.2%). Anh v x Wales, c n 468 trng hp t 1990 n 1995. Bacillus cereus l mt thc vt hoi sinh trong t rt ph bin. N c phn lp t nhiu ngun thc phm a dng, c bit l thc phm c ngun gc t thc vt, t tht, cc v cc sn phm t tht c cng vy. Pht hin u tin cc v cc mm bnh gy ng c thc phm l t nm 1949 khi Hauge phn lp mu t xt vani sau khi c mt ca ng c thc phm gy tiu chy ti bnh vin Oslo, Norway. Xt vani c nu trc khi tiu th v bo qun nhit phng cho n khi s dng. khng nh Bacillus cerus l nguyn nhn gy ng c, Hauge pht trin mu phn lp n nng khong 4x106ml-1 v ung 200ml cocktail. Sau 13h, ng cm thy au bng v i tiu ra nhiu nc, triu chng ny dai dng khong 8h. Hn 20 nm sau, mt triu chng gy ng c thc phm khc do chng Bacillus cereus gy ra li xut hin cc cng nhn ngi Anh. Triu chng ny nng hn triu chng nn ma v ko di mt thi gian ngn (cha n 5h), iu ny cho thy rng y l mt s nhim c. Ngy nay, bnh ny lin quan n triu chng gy nn ma do Bacillus cereus. Bacillus cereus c th gy ra s nhim trng v nhim c khc nhau, thm vo , nhng ng c khc do Bacillus cereus gy ra l s nhim trng mu, vim mng
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no, v nhim trng mt. Hai loi ng c thc phm l nguyn nhn bi rt nhiu nhn t gy c hi khc nhau.
2. C ch gy bnh. 2.1 Triu chng nn ma.

c im ca bnh nn ma v tnh cht ca c t gy nn ma c th hin bng 1-1. S gii thch v cu trc ca c t emetic a n nhng hiu bit su sc hn v triu chng ny. c t emetic c tn l cereulide v l mt chui polypeptide ba ln lp li ca bn amino v/hoc oxy-acid (bng 1-1)

Cereulide (polypeptide) l tn ca mt c t quan trng gy ra triu chng nn ma do Bacillus cereus sn sinh ra. Bi bo ny gii quyt c nghin cu v qu trnh sinh tng hp c t ny da trn s bt thng ca c t depsipeptide t Cacbon s 13 (13C) lit k ra trn 3 loi tin L- amino acid (Valin, Alanin, Leuzin) trn mi trng tng hp trung gian. S phn tch ny c thc hin da vo mc cu to phn t ca amino hay oxy acid qua NMR v ESI MS ca phng php knh quang ph trn cereulide v sn phm thy phn l cc dipeptide ca n. S hp nht ca nguyn t cacbon s 13 (13C) l chim n 95% trong O-Val, O-Leu v L-Val, trong khi ch c 40%13C l kt hp trong D-Ala ca Cereulide. Bacillus cereus c bit l nguyn nhn gy ra hai loi ng c thc phm, l triu chng nn ma v triu chng tiu chy. Phn ln nhng ng c do Bacillus cereus gy ra ch mi pht hin sau ny. Cereulide c bit n vi cu trc bc 1, vi 1 dodecadepsipeptide tun hon, vi 12 gc lp th trung tm. Ha hc lp th
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c ch ra t sn phm thy phn l cc dipeptide kim tnh, D-O-Leu, D-Ala, L-OVal v L-Val. l 1 ion Kali mnh, n lin kt yu vi cc ion Li+, ion Na+, ion Cs+, nhng vi ion Rb+ n li lin kt mnh nht, hn bt k ion kim loi kim no. Cereulide to cu trc bc 2 t NMR v s tnh ton c hc cc phn t c biu th hnh 1. c t ny l nguyn nhn dn n s hnh thnh cc ty lp th c cha ATP v cc enzyme lien quan n hot ng chuyn ha t bo trong cc m khc nhau. Chng ta bt u quan tm n con ng sinh tng hp ca c t ny cho cc chng trnh phng chng ng c thc phm trong tng lai. Nghin cu qu trnh sinh tng hp tng t nh dodecadepsipeptide, valinomycin. Agata mt trong nhiu tc gi nghin cu qu trnh pht trin v sinh c t ca Bacillus cereus trong qu trnh tng hp trung gian mt cch y t CADM (hn hp cc amino acid) v ng sucrose (ng ma). Ngi ta nhn thy rng, Bacillus cereus cho php (chp nhn) vic sn sinh ra cereulide cu trc bc 1 v 3 amino acid Val, Leu v Thr l cn thit. Nhng nghin cu khc v qu trnh sinh tng hp cereulide c l l cn thit thc y vic nghin cu tm ra phng php ngn chn nhng ng c t thc phm. Xt on t cu trc ca 1, tin thn ca D-Ala, L-O-Val v D-O-Leu s lien quan n cc amino acid nh L-Ala, L-Val, v L-Leu.

Trong th nghim ny ch trng n sinh tng hp trung gian ca 3 amino acid l LVal, L-Leu, v L-Ala (0.1g/L c n 99% nguyn t cacbon dng13C trong cacboxylic, tt c 15 amino acid cn li (0.1g/L), K2HPO4 (5g/L) v MgSO4.7H2O (0.05g/L), c tin hnh nhit 300C v thi gian l 24h, gi tc lc l 200 rpm thu c 2mg cereulide c cng c tnh iu kin ny c nh du phc v cho phn tch cc nghin cu tip theo.1H NMR ca mu ny l ng nht vi quang ph ng tin cy ngoi tr cht ng v ca cacbon13C. ESI mass (interpreting Electrospray Mass Spectra) ca n xut hin 7 nh trung tm vi m/z l 1201.48 kh hn mc bnh thng M + K vi m/z l 1191.55 (th hin hnh 2), v vy c trung bnh 10 n v mass th cao hn bi v s kt hp cao ca13Cs. Chnh v s lin hp cao ca13Cs vo phn t cereulide thc y chng ta trong vic o lng mc quang ph NMR13C ca ion K+ trong phc hp CDCl3 t c mc quang ph nh hnh 3, vi s liu ng k 171.4 (L-O-Val), 172.2 (D-O-Leu), 175.7
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(L-Val) v 176.2 (D-Ala) c chuyn sang mi tng quan gia C-H ca n. Tng ng vi cng ca 3 cacbon (C6, C9, C12) ca L-O-Val, D-O-Leu v LVal l nhiu hn hai ln C6 D-Ala.

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Tng t vi vic lm ring cc th nghim t c vi bc x proton ca 4 hp phn amino hay oxy acid. Loi b cc kt qu t c vi proton ca O-Val 4.61 ppm vi bc x -H ca n 2.30 ppm thay i t b ba sang b kp (J=3.8 Hz cp vi13C ca O- Val). Bc x -H ca L-Val 2.24 ppm lm thay i proton ca Val 3.82 ppm t b nm thnh b ba (J=5 Hz). C hai kt qu trn u ch ra c s kt hp cao cacbonyl cacbon ca13C tin thn ca cc amino acid i vi L-O-Val v c L-Val. Cha c kt qu chnh xc no v s kt hp ca13C, tuy nhin trng hp ny li ng i vi Ala v O- Leu. S chiu x gc metyl ca Ala 1.47 ppm gy ra -H ca n (4.27 ppm) ging nh s pha trn ca b ba v b kp biu kin vi J=4 v 5 Hz, theo th t nh sn (tch bit ra) c ngha l t l st nhp li to thnh cacbonyl cacbon l khng qu cao (khng t n 100%). S chiu x ca -Hs ca O-Leu trong khong 1.84 ppm l khng t kt qu bi v di sng t nhin ca n qu rng. Tuy nhin, quang ph NMR ca13C v nhng th nghim ring l l nhng minh chng gn nh 100% ca s kt hp13C tin amino acid vi 3 hp phn (O-Leu, Val v O-Val) ca cereulide.

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Figure 5.Decoupling experiments of a protons of the four components by irradiating b protons.

Mt phn trm s kt hp ca13C l c xc nh bi php o ph t cc sn phm thy phn cereulide, v vy hai dipeptide thu c t s thy phn bng kim cereulide (1) vi 0.1N KOH (hoc 1N NH4OH) rt trong 30 pht. Cc sn phm thy phn dipeptide l D-O-Leu-D- Ala v L-O-Val-L-Val, c phn tch bng phng tin ESI (electrospray ionization)- thc MS/MS trn Q-TOF ca php o ph (Mcro Mass Co.,Ltd, Manchester, UK). lm c iu ny cc cht ng v tha v13C kt hp li vi nhau thnh mu, pha long mu n ha tan 10-100 pmol/L trong 99,8% methanol: 0.2% acid formic trc khi b electrospray 5L/pht. Ngy nay, L-Leu v L-Ala c chng minh xc nh r l c c t qu trnh sinh tng hp cereulide, v hai acid amin ny l cn thit cho B. cereus. Mt trong nhng kh nng c th c nghin cu l c ba L-amino acid s t nht mt ln c bin i thnh cc - keto acid v sau bin i thnh D-O-Leu v L-O-Val hoc l s chuyn ha amin to thnh D-Ala. Trong trng hp ny ch c acid pyruvic s b pha long bi v s gc cao v L- Ala l khng cn thit cho B. cereus. Nghin cu ny c th gii thch n 95% s kt hp to thnh D-O-Leu, L-O-Val, v L-Val khong (40%) s kt hp to thnh D-Ala. 2.1. Triu chng tiu chy. Triu chng tiu chy do t nht hai loi c t ng rut sn sinh ra trong sut qu trnh sinh trng ca Bacillus cereus trong rut non. S hnh thnh c t ng rut y trong thc phm dn n ng c thc phm v l thuyt m ni l c th, nhng i vi thc phm phc v cho con ngi th y l iu khng th chp nhn. iu ny xy ra khi, s lng Bacillus cereus tn ti trong thc phm thp nht l 106/g hoc /ml v lng ln ca c t ng rut phi c hnh thnh chng chu c vi pH ca d dy v enzyme proteolytic ca t trng. Nhng nhn t ny
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s lm gim mt cch nhanh chng hot ng ca c t ng rut thp n 1% s vi mc ban u. Thi gian tng i di gia vic n vo nhng sinh vt ny vi vic xut hin nhng triu chng ca bnh. c im ca triu chng tiu chy c th hin trong bng 1-2. Mc bin i ca liu ly nhim c l do kh nng sn sinh ra cc c t ng rut khc nhau v do tnh nhy cm ca mi c nhn l khc nhau.

Na-Uy, hai ln bc pht xy ra vi rt nhiu ngi b nh hng sau khi n tht hm vi khoai ty v rau. Liu lng gy bnh xp x 104 105. Ln u tin bng pht (1992), 17 24 ngi b ng c, 3 trong s cc bnh nhn phi nhp vin t 1 3 tun, triu chng bt u nng 3 bnh nhn ny kh mun (>24h). Ln th hai, bnh bng pht vo thng 2 nm 1995 khi m 152 trong s 252 ngi Na-uy b nh hng trong sut thi gian tham gia gii v ch v trt tuyt. Cc i th tr tui (16 19 tui) b nhim triu chng ny sau hn 24 gi bnh v h b au t 1 n vi ngy. Bo t ca Bacillus cereus t m t u tin trn (phn gii thiu chung) l c phn bit cho thy rng chng c kh nng bm vo cc t bo Caco-2 (trn cc t bo biu m ca ngi). Sau khi bm vo, cc bo t ny ny mm mt cch nhanh chng (trong vng 1h), hnh thnh t bo Bacillus cereus sinh dng trn nh ca cc t bo biu m, tip l sn sinh ra c t, nu c t ny xut hin trong ng

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GVHD:C Trn Th Mai Anh

rut, c t ng rut s tp trung khoanh vng vng ngoi bin ca ng rut s tng cao hn trong lumen v v vy gy nn mi nguy ln hn v gy bnh mt cch trm trng. Mt iu c th xy ra i vi c ch ny l thi gian bnh s lu hn nh quan st. S lng v loi c t lin quan n triu chng tiu chy do ng c thc phm t Bacillus cereus l ti tranh ci t nhiu nm qua v cho n nay vn cn ang tranh lun. C dng n v dng phc ca c t ng rut c xc nh l nguy nhn gy ra bnh tiu chy. C hai s khc bit trong ba hp phn ca c t ng rut c sn sinh bi cc thc phm nhim Bacillus cereus, mt s nhm cng c m t c t ng rut mt hp phn vi cc phn t trng lng khong t 40 100 kDa Nhng nghin cu gn y v c t ng rut khng nh rng c c t ch c mt hp phn v c t nhiu hp phn u c lin quan. Nhng nghin cu v qu trnh sinh tng hp gip thit lp nn cc gii php ngn nga cc c t sinh ra t Bacillus cereus trong tng lai gn. Hoc c th a ra cc phng php gip pht hin ra c t cereulide trong t nhin bt c lc no. III.Cc phng php pht hin BACILLUS CEREUS trong thc phm. 1.c im v nguyn tc. Nhm cc phng php ny da trn c im pht trin ca vi sinh vt trn cc mi trng c trng v c im sinh l, sinh ho ca cc chng, cc loi vi sinh vt khc nhau. u im ca phng php ny l thao tc n gin, d lm, khng phi u t dng c, thit b t tin. Tuy nhin vi nhm phng php ny cn mt s hn ch nh nhy khng cao, tn nhiu nhn cng v thi gian phn tch thng ko di do hn ch trong cng tc phng nga. Bacillus cereus phn bit vi cc loi khc trong Bacillus nhm 1 nh B.anthracis gy bnh than cho ngi, B.thuringiensis to c t kt tinh gy bnh cho cn trng, B.mycoides, B.megaterium da vo cc c tnh sinh ha Cc khun lc c khng nh da trn cc th nghim sinh ha vi cc c im nh ln men glucose, sinh acid trong iu kin k kh, kh nitrate thnh nitrite, th nghim VP (+), thy phn Ltyrosine, tng trng c trong 0.001% lysozyme. 3.Qui trnh phn tch. 25g mu + 225ml mi trng pepton m (BPW) -> ng nht bng Stomacher/ 1pht c pha long 10.1 -> pha long thnh dy thp phn c cc pha long thch hp. nh lng Bacillus Cereus bng phng php m khun lc. 3.1.Pht hin bng mi trng chn lc: Tri 0.1ml mi pha long ln cc mi trng thch ri 24h 30oC.
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MYP: do Bacillus cereus khng ln men mannitol, to lecithinase v khng polymicin nn khun lc B.cereus c mu hng eosin, c bao quanh bi vng c ta, chng t lecithinase c to thnh. MOSSE: khun lc to, mu hng, xung quanh c vng sng. Chn t 5 khun lc (+) cy sang thch nghin chun b cho cc phn ng khng nh Bacillus cereus. 3.2.Cc phn ng khng nh: Nhum Gram: Cy ria cc khun lc c chn t mi trng MYP/MOSSEL sang ng thch dinh dng -> 30oC/ 24h -> nhum Gram -> quan st di knh hin vi t bo nhum bng vt knh 100X nhng trong du. Cc bc nhum Gram: Chn phin knh sch -> v ln knh 1 vng trn, ng knh 2cm -> v tr tng n vi vng trn, mt cn li nh vi git nc ct thnh 1 git ln -> chuyn mt t sinh khi khun lc vo git nc trn knh bng que cy vng -> khuy nh bng u que cy -> dung dch huyn ph ng nht -> bi u trong khu vc ca vng trn -> yn cho vt bi kh -> a phin knh v tr cnh vt bi qua li trn ngn la n cn/n Bunsen c nh vt bi (trnh vt bi tip xc trc tip vi ngn la) Tng t cho hai chng VSV i chng trong cng mt phin knh khc: E.coli lm chng i chng cho Gram (-) v Staphylococcus aureus lm chng i chng cho Gram (+). C hai phng php nhum Gram: Phng php Jensen: nh vi git dd methy violet ln vt bi -> gi yn 20giy -> dng bnh xt nc ln vt bi -> ra sch phm nhum -> nh vi git KI/I2 ln vt bi/ yn 1pht -> dng bnh xt cn 95% ln vt bi -> ra phm nhum n khi mt mu -> yn vi giy -> ra bng nc -> nhum bng dd safranin/30giy -> ra sch bng nc, thm nc d bng giy lc. Phng php Hucker: tng t nh phng php Jensen, nhum vt bi bng dd crystal violet/ 1 pht -> ra bng nc -> nhum bng dd KI/I2 / 1 pht -> kh mu bng cch xt cn 95% -> ra li bng nc -> nhum mu bng dd safranin/2pht. Kt thc qui trnh nhum, quan st di knh hin vi cho ta thy t bo nhum Gram (+) c mu xanh ta (S.aureus), t bo nhum Gram (-) c mu hng (E.Coli). Bacillus cereus l trc khun ln, Gram (+), thng kt hp vi nhau thnh dng chui; bo t hnh bu dc, khng c dng bo t nang. Dng que cy vng chuyn mt lng sinh khi chng th nghim trong ng thch dinh dng vo 0.5ml BPW v trng. To huyn ph ha dch cho cc phn ng sinh ha pha sau. Th nghim ln men glucose: Cy vi khun vo 3ml canh Phenol Red Glucose Broth -> 35oC/ 24h, k kh -> lc mnh ng nghim -> quan st c ( s pht trin); s chuyn mu mi trng t sang vng ( chng t c s sinh acid glucose trong iu kin k kh).

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GVHD:C Trn Th Mai Anh

Hnh 1:Th nghim kh nng kh nitrate. Hnh 2:Th nghim ln men glucose.

Th nghim kh nng kh nitrate: cy vi khun vo 5ml canh trng Nitrate Broth -> 35oC/24h -> b sung vi git dd ca thuc th nitrate -> c mu cam xut hin trong 10pht (chng t nitrate b kh thnh nitrit) ng A cy vi khun trn canh trng Nitrate Broth cha b sung thuc th. ng B v C c b sung thuc th alpha-naphthylamine v sulfanilic acid. ng B cho kt qu dng tnh (nitrate b kh thnh nitrite ), ng C cho kt qu m tnh vi nitrite. Th nghim VP: vi sinh vt, bin dng nng lng bng phng thc ln men t glucose qua con ng ng phn s to ra cht trung gian ch yu l pyruvic acid. khi phc d tr NAD+ trong t bo phc v cho con ng ng phn cc loi vi sinh vt, sau pyruvic acid tip tc c chuyn ha khc nhau to ra cc sn phm ln men cui cng khc nhau. H Enterobacteriacceace c c tnh chung l ln men sinh tng hp acid nh acid formic, acid acetic, acid succinic, ethanol, H2 v CO2. H ny c th c chia thnh hai nhm l nhm khng sinh 2,3-butanediol (v d : E.coli) v nhm sinh 2,3-butanediol (v d: Enterobacter). Phn t 2,3-butanediol c th c chuyn ha qua li thnh acetoin: trong iu kin c oxi v mi trng c tnh kim nh xc tc ca enzyme 2,3-butanediol dehydrogenase. Ngc li acetoin c th b kh thnh 2,3-butanediol do hot tnh ca enzyme diacetyl reductase; ngoi ra acetoin cn b oxi ha thnh diacetyl, cht ny tham gia vo phn ng to mu trong th nghim VP. Nh vy, th nghim VP c th gip phn bit cc loi trong Enterobacteriaceace da trn s oxi ha acetoin (acetylmethylcarbinol, AMC) t 2,3-butanediol thnh diacetyl. S oxi ha acetoin thnh diacetyl c tng cng nh xc tc ca -naphthol. Diacetyl kt hp vi nhn guanidine c trong peptone kt t thnh phc diacetylguanidine c mu . Trong thuc th Koblentz v OMeara c cha creatine c tc dng b sung ngun nhn guanidine. Cc bc tin hnh:
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Mi trng c s dng cho th nghim VP l mi trng lng Clark-Lubs (mi trng MR-VP), c pH 6.9. Dng que cy vng cy vo cc ng mi trng MR-VP mt t sinh khi (trng hp dng thuc th Koblenntz th cy nhu sinh khi) t khun lc ca chng thun 18-24h trn mi trng KIA hoc TSI. yn cc ng mi trng ny 37oC / 24-48h hoc n 10 ngy. Sau thi gian , b sung thuc th trc tip vo ng mi trng. C 3 loi thuc th VP l: Thuc th Barritt: gm dung dch A l 5% -naphthol trong cn tuyt i, dung dch B l 40% KOH hoc NaOH. Thuc th Koblentz: gm dung dch A l 5% -naphthol trong cn 95%, dung dch B l 0.3% creatine, 40% KOH hoc NaOH. Thuc th OMeara: dung dch 0.3% creatine, 40% KOH hoc NaOH Khi s dng cc loi thuc th 2 thnh phn, trc tin nh 6 git dung dch A, sau nh 2 git dung dch B, b sung 1ml thuc th vo ng mi trng. Lc nh ng 1 pht oxi ha acetoin. c kt qu sau 20 pht hoc chm nht 4h. Trc khi s dng nn kim tra thuc th bng cc chng i chng nh E.Coli (VP ) b mt mi trng khng i mu v Enterobacter cloacae (VP +) c mu trn b mt mi trng.

Th nghim kh nng thy phn tyrosine: cy vi khun vo thch nghing tyrosine -> 35oC/48h -> xut hin khun lc ( chng t tyrosine b phn hy) Th nghim vi canh Lysozyme Broth: cy vi khun vo 2.5ml mi trng Nutrient Broth cha 0.001% lysozyme, thc hin tng t vi mi trng khng cha lysozyme -> 35oC/24h -> kim tra s tng trng trong mi trng cha v khng cha lysozyme -> nhng ng c kt qu m tnh thm 24h -> kt lun vi khun c khng lysozme hay khng. Da vo bng khng nh dng chn l B.cereus hay khng. Loi c tnh B.cereus B.thuringiensis B.mycoides B.anthracis B.megaterium

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Nhm:1 Gram Catalase Di ng Kh nitrate Phn hy tyrosine Khng lysozyme Phn ng vi lng trng Ln men glucose Phn ng VP Sinh acid t manitol Tan mu (cu) +(a) + +/-(b) + + + +/+ + + -(c) +

GVHD:C Trn Th Mai Anh + + + + + +/-(d)

+ +

+ +

+/+

-(d) +

+/-

+ + +

+ + +

+ + +

+ + +

-(d)

3.3.Cc th nghim phn bit cc loi trong Bacillus nhm I: Th nghim tnh di ng: Phng php 1: Dng que cy vng cy thng dch 24h nui vo gia mi trng kim tra di ng -> 30oC/ 18-24h -> kim tra di nh n kiu mc, dc theo ng cy. K qu: loi di ng mc khuch tn vo mi trng theo hng xa ng cy, loi khng di ng mc trong v dc theo ng cy. Phng php 2: b sung 0.2ml nc ct v trng vo b mt mi trng thch nghing Nutrient Agar -> cy huyn ph vi khun vo -> thch nghing 30oC/68h -> nh nc v trng ln knh hin vi, t sinh khi vi khun vo -> quan st di knh hin vi kim tra s di ng. Hu ht cc chng B.cereus, B.thuringiensis l di ng; B.anthracis v B.mycoides khng di ng S hnh thnh r gi: chm nh que cy vng mang huyn ph 24 gi ln gia a Nutrient agar -> 30oC/48-72h -> kim tra s pht trin ca r gi (B.cereus khng

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to cu trc r gi, thng to nhm khun lc x x khc vi cu trc r gi c trng ca B.mycoides (cu trc ging nh r hoc tc m rng vi centimet t v tr cy).

B.Cereus khng to cu trc r gi

B.mycoides to cu trc r gi

Th nghim lm tan mu: cy chng ln mi trng thch mu Trypticase Soy -> 35oC/24h -> B.cereus lm tan mu mnh, to vng tan mu han tan () 2-4 mm xung quanh vng pht trin. B.thuringiensis v B.mycoides cng tan mu . B.anthracis thng khng lm tan mu sau 24h. S to c t protein dng tinh th: cy huyn ph t bo 24h ln ng thch nghing nutrient agar -> 30oC/ 24h -> yn nhit phng /2-3ngy -> nhum bng phm mu fuchsin -> quan st di knh hin vi. Tinh th c t ca B.thuringiensis xut hin sau 3 n 4 ngy nui cy, pht hin c bng k thut nhum khi bo t nang v ra (tinh th c t hnh t gic dng kim cng c nhum mu ti, nh hn bo t). Do nu khng quan st c bo t t do cn thm vi ngy ri kim tra li. Bacillus cereus v cc Bacillus khc cng nhm khng tinh th c. 3.4.Cch tnh kt qu. S t bo Bacillus cereus/1g mu da vo s khun lc mc mi pha lang v hiu chnh bng t l khng nh (phn trm khun lc c xc nhn l Bacillus cereus). V d: s khun lc m c pha lang 10-4 l 65, c 4 trong 5 khun lc c chn xc nhn l Bacillus cereus (c kim tra bng cc phn ng sinh ha). S t bo Bacillus cereus/1g thc phm = 65 x 4/5 x 10000 x 10 = 5200000 (nhn 10 v c 0.1 ml mu c tri da) 4.Cc phng php phn tch nhanh. Theo nguyn l ca cc phng php phn tch vi sinh vt c th chia phng php ny thnh hai nhm nh: nhm da trn nguyn tc ca s kt hp c hiu gia khng nguyn- khng th (phng php min dch) v nhm da trn s bt cp b sung cc nucleotit

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1 .Phng php min dch. Nhm cc phng php ny da trn phn ng ca khng th c hiu vi khng nguyn b mt ca t bo vi sinh vt. Da trn nguyn tc ny c rt nhiu k thut pht trin thnh phng php pht hin nhanh vi sinh vt 2. K thut latex agglutination (LA) . S dng cc ht cao su c mu c nh khng th c hiu nh tnh nhanh cc chng vi khun c phn lp. Khi c mt khng nguyn tng ng, qu trnh kt t (agglutination) s din ra v c th quan st trc tip bng mt thng (to ra ht hoc di c mu). K thut ny c th s dng xc nh nhanh vi sinh vt nhim tp trong mi trng thun sau khi phn lp t thc phm.

B Kit BCAT-RPLA (Oxoid test kid) 3. K thut lai phn t( DNA- hybridization). K thut ny c th s dng u d vi on rARN ch do vy c tnh chnh xc v nhy cao. y l k thut cho php pht hin s c mt ca nhiu loi vi sinh vt mt lc vi thi gian nhanh. Tuy nhin k thut cng phc tp v phi tn cng thit k cc mu d v s dng vt liu phng x. 4. K thut Microarray. K thut ny thc cht l da trn k thut lai phn t, trn cc phin knh hoc cc mng cellulose c gn hng chc nghn mu d ADN theo mt trnh t xc nh. Nh vo phn ng lai, bt cp ca ADN c trnh t tng ng m s c mt mt on gen ca mt vi sinh vt ch no c th c pht hin v s copy c th xc nh

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chnh xc. Vi k thut ny c th xc nh c nhiu gen ch ( vi sinh vt) trong cng mt thi im vi mc chnh xc cao v thi gian nhanh v chnh xc 5 .Phng php pht hin vi sinh vt bng k thut Real Time PCR. Nguyn tc : K thut Real Time PCR hay cn gi l PCR ng v c bn da trn nguyn tc ca k thut PCR. Tuy nhin n cho php hin th v theo di trc tip qa trnh nhn bn DNA ang din ra theo tng chu trnh nhit qua s dng k thut pht hunh quang. Da vo pht hunh quang ta c th nh lng cc on DNA hnh thnh Do mi c tnh pht quang cho nn c th nh du chng vi cc cht nhum khc nhau, nh th c th nhn cc on khc nhau trong cng mt phn ng PCR. Tuy nhin phng php ny c hn ch l phi tng hp cc mu d khc nhau cho cc trnh t nhn bit khc nhau.

Cc phng php nh lng bng Real time PCR Phng php s dng cht nhum mu SYBR Green: Cht nhum mu ny c gn vi rnh nh ca chui DNA kp, khi c gn vi si
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DNA kp th cng pht hunh quang tng ln, khi bn sao to ra cng nhiu th tn hiu pht ra ca cht mu s tng ln theo t l thun. u im ca phng php ny l SYBR s nh vo bt c chui DNA kp no c mt. Phng php u d thu phn TaqMan: K thut ny s dng cc u d c th pht hunh quang, cc mu d l mt oligonucleotit trong mt u c gn vi cht nhum mu c pht hunh quang, u kia c gn vi cht nhum c vai tr lm tt s pht hunh quang. Khi c mt on DNA cn nhn, mu d s gn vo DNA khun vng di ca mi theo hng 5. trng thi ny khng c hin tng pht hunh quang. Khi bt u tin hnh quy trnh PCR vi s hot ng ca enzyme taq-polymerase mi c ko di cho ti khi gp mu d th mu d s b phn gii bi hot tnh 5 nuclease ca taq-DNA polymerase.S phn gii mu d s lm gii phng cht nhum mu c kh nng pht hunh quang khi nh hng km hm ca cht nhum c vai tr lm tt s pht hunh quang. S pht hunh quang ny c th nhn bit c. Trong k thut ny mt u d c gn vi mt cht cho hunh quang u 3 v mt u d th hai gn vi mt cht nhum hunh quang. Khi hai u d ny tin li gn nhau, cch nhau khong 1-5 nucleotide, cht pht quang pht ra t cht cho hunh quang s kch thch cht nhn hunh quang v kt qu l pht ra tn hiu hunh quang.

Hnh : S mt s phng php ca k thut Real Time PCR u im : K thut ny khc phc c nhng nhc im ca PCR nh sau: Khng cn chy in di kim tra kt qu PCR C th nh lng chnh xc sn phm PCR, trong khi s dng PCR thng thng vic phn bit da trn ln ca vch thu c sau in di
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GVHD:C Trn Th Mai Anh

c hiu v nhy cao hn, thi gian tin hnh phn ng ngn C th tin hnh vi nhiu on DNA cng lc trong cng mt ng nghim nn gim kh nng nhim mu .

PHN 3: KT LUN
Cuc sng ngy cng pht trin, nhu cu v vt cht v tinh thn ca con ngi ngycng cao. V vy m vic sn xut ra cc loi thc phm sch, r, an ton.lun l nhng vn ht sc cp thit cho cc nh khoa hc. Tuy nhin i km vi thc phm cn c nhng yu t gy ng c thc phm gy nh hng xu n sc kho conngi. iu ny ang gy tm l hoang mang, lo s cho ngi dn trn th gii. c bit l nhng nc ng dn, chm pht trin nh Vit Nam. Bacillus cereus l loi vi khun gy bnh cho con ngi. Bacillus cereus pht trin 35-40 C, cc thc phm sau khi nu chin ngui d b nhim Bacillus cereus. Chng ta cn bo qun thc phm trn 60 C, hm nng thcn trc khi n. Hiu c cu to,tnh cht gy bnh, c t..ca Bacillus cereus , chng ta s bit cch phng nga v c nhng bin php cha tr kp thi nu khng may mc phi. Khi s dng mt loi thc phm, chng ta nn quan tm ngun gc xut x, cht lngcng nh an ton v sinh thc phm. Tnh hnh ng c thc phm s c ci thin gip bo v sc kho bn thn, ngi tiu dng v gip tit kim ngn sch quc gia.Nh nc nn c nhng bin php x l kp thi ,ng n i vi nhng c s sn xut, ch bin thc phm tri php gy nh hng xu n sc kho v tnh mng ca ngi dn.ng thi tng cng cng tc tuyn truyn v an ton v sinh thc phm cho ngi dn, khuyn khch ngi dn sn xut v s dng nhng sn phm sch,an ton, thc hin n chn ung si, v sinh sch s mi trng sng ca mnh hn ch vi khun cng nh nhng cht c hi i vi c th.

TI LIU THAM KHO.

1.T Minh Chu (2005), Gio trnh vi sinh c s 2. Nguyn Th Hin, Phan Th Kim, Trng Th Ho, L Th Lan Chi (2003), Vi sinh vt nhim tp trong cng ngh thc phm, NXB Nng Nghip H Ni. 3. Nguyn th Kim Hoa (2004), Pht trin k thut PCR trong phn tch listeria monocytogenes gy bnh thc phm, Lun vn thc s khoa hc, H Ni. 4. Lm Xun Thanh (2004), Gio trnh cng ngh cc sn phm sa, NXB KHKT, H
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Ni. 5. Trn Linh Thc, Cc phng php kim tra vi sinh vt trong nc v thc phm, NXB gio dc v o to. 6. Phng Th Thy (2006) Gp phn kim sot s nhim tp vi sinh vt trong qu trnh sn xut sa tit trng vi s h tr ca k thut PCR, Lun vn thc s khoa hc

PH LC TIU CHUN VI SINH CHO PHP TRONG THC PHM B Y T 4/1998

Gi hn cho php (CFU/g hoc CFU/ml thc phm) Nhm thc phm Nhm tht Tht ti, tht ng, tht xay nh, tht nghin, tht ch bin Sn phm ch bin t tht: tht hun khi, pat, xc xch Sn phm ch bin t ng cc, khoai c, u : Cn x l nhit trc khi dng: bt, min, m si Dng trc tip khng x l nhit: bnh bt Nhm thc n kh v cha dinh dng cho tr em, thc n thay th c bit Phi x l nhit trc khi s dng Dng trc tip, khng qua x l nhit 10 10 10 10 102 B.cereus 102

27