TI

GVHD: Ks Phm Minh Nht

Nhm 13_07DSH02
L Tun T Nguyn Thanh Xun Nguyn Th Hng Nh Nguyn Th Xun Kiu

- 2010 -

MC LC
Phn I: Tng quan v Pseudomonas fluorescens
1.1.Lch s pht hin 1.2.Phn loi 1.2.1. Gii thiu chung v Pseudomonas fluorescens 1.2.2. Pseudomonas fluorescens 1.3.c im 1.3.1. c im chung 1.3.2. c im sinh ha 1.4.Cu trc t bo 1.5.C ch hot ng ca Pseudomonas fluorescens 1.5.1. i vi thc vt 1.5.1.1. 1.5.1.2. 1.5.2. i vi ng vt 1.5.2.1. 1.5.2.2. 1.5.3. i vi thc phm 1.5.3.1. Pseudomonas fluorescens gy h hng trn b mt tht 1.5.3.2. Pseudomonas fluorescens gy h hng trn nhiu loi thc phm 1.5.4. Pseudomonas nh hng n vi khun Listeria 1.5.5. Bnh hc 1.6.Bnh v cc triu chng 1.7.ng dng ca Pseudomonas fluorescens 1.7.1. Sn xt thuc bo v thc vt 1.7.2. Sn xut thuc khng sinh Du hiu bnh Phng tr Vi sinh vt i khng Gy hi cy trng

1.7.3. Sn xut cht chuyn ha th cp 2,4-DAPG 1.7.4. Kim sot mm bnh 1.7.5. Lm sch nc thi cng nghip

Phn II: Cc phng php xc nh Pseudomonas fluorescens

2.1. Phng php truyn thng 2.1.2. nh tnh Pseudomonas fluorescens 2.1.2.1. Tin hnh 2.1.2.2. Kt qu 2.1.2.3. Quy trnh th nghim sinh ha 2.1.3. Th nghim amn ha 2.2. Phng php hin i 2.2.1. Phng php PCR 2.2.2. Phng php HPLC

Phn III: Kt lun v ngh Ph lc Ti liu tham kho chnh

LI M U
Ng c thc phm hin l vn c th gii quan tm nhiu v n c th xt hin mi ni, mi lc trn din hp hay din rng. Cc chng vi khun, nm c kh nng gy nhim khun thc phm c nghin cu mt cch t m t hnh thi, c ch n triu chng gy bnh. iu d thy nht thc phm bt u qu trnh h hng l mu sc. Vy ci g gy nn nhng vt mu trn thc phm? N c nh hng nhiu n sc kho khng nu ta b nhim? V bn c thc mc rng ti sao n c th lm nn nhng mu sc khng v ngoi tr thc phm ra n c nh hng n nhng th khc c lin quan n cuc sng ca ta khng?

PHN I:
TNG QUAN V PSEUDOMONAS FLUORESCENS

1.1.

Pseudomonas sp

Cc chi Pseudomonas c nh thc vt hc ngi c t tn nm 1894 v ng m t cc loi ca Pseudomonas nm 1895. Pseudomonas l vi khun gram m, hiu kh, hnh que c tin mao. Pseudomonas t ti trong t, nc v cng c tm thy trn cc b mt thc vt v ng vt. Vi khun Pseudomonas c bit n nh l mt loi chuyn ha linh hot v c th pht trin di nhiu iu kin tng trng. Pseudomonas l tc nhn gy bnh cy trnng tuy nhin mt s loi c th gy bnh cho ngi Pseudomonas fluorescens c kh nng pht trin 4C gy h hng cc thc phm ng lnh. N him khi gy bnh cho ngi do khng pht trin nhit c th. Mt s chng Pseudomonas fluorescens c ch mm bnh nng nghip nh nhim trng nm 1.2. Phn lai 1.2.1. Gii thiu chung v Pseudomonas fluorescens Gii: Bacteria Ngnh: Proteobacteria Lp: Gamma Proteobacteria B: Pseudomonadales Familia: Pseudomonadaceae Chi: Pseudomonas Loi: Pseudomonas fluorescens Tn: Pseudomonas fluorescens 1.2.2. Pseudomonas fluorescens Hnh 1. Vi khun Pseudomonas fluorescens

1.3.

c im 1.3.1. c im chung

Pseudomonas fluorescens l trc khun hiu kh khng nha bo gram m 16S rRNA, hnh que sng trong t, thc vt v nc b mt. Di chuyn linh hot nh c nhiu roi (flagella), pht trin ti u nht 77F 86 (25-30C). Pseudomonas fluorescens l thnh vin ca nhm vi khun hunh quang pseudomonas v l mt nguyn nhn khng thng xuyn ca cc bnh v nhim trng mu.

Hnh 2. Vi khun Pseudomonas fluorescens s dng flagella ca n y bn thn thng qua cc lp t nc Mt s chng Pseudomonas ngn chn dch bnh bng cch bo v ht v r trnh nhim nm. Tuy nhin chng Pseudomonas fluorescens li l nguyn nhn gy h hng thc phm v gy bnh cc loi thy sn. 1.3.2. c im sinh ha Pseudomonas fluorescens sn xut sc t ha tan pyoverdine l sc t pht hunh quang mu xanh vng khi c kch thch bi bc sng thp hn 260nm. Sc t ny c to ra trong mi trng c nng st thp, d khuch tn v c chc nng trong c ch trao i st ca vi khun

H Hnh 3. Pseudomonas fluorescens agar: mi trng dinh dng

Pseudomonas l mt vi khun hiu kh bt buc nhng mt s chng c kh nng s dng nitrat thay v oxi nh l mt cht nhn in t cui cng trong h hp t bo. Pseudomonas fluorescens cng vi cc pseudomonas tng t khc cho lipase nhit n nh v proteases. To cc enzyme gy h hng sa, h hng casein, v lm km cht lng thc phm do to cht nht, ngoi ra cng gy hin tng ng mu. Pseudomonas fluorescens khng pht trin iu kin ym kh v l vi khun quang hp khng thi oxi nn c vai tr lm sch ngun nc. Loi ny tng trng c trn mi trng ngho dinh dng ch gm khong v carbon. Mt s c im sinh ha chnh ca Pseudomonas fluorescens l: khng ln men glucose, c kh nng thy gii gelatin, tinh bt, oxidase (+), kh nitrate (+). 1.4. Cu trc t bo B gen Pseudomonas fluorescens PF-5 cha 1 NST trn vi 7.1Mbp v 63.3% hm lng GC, n cha 87 RNAs v 6137 protein, 5.7% ca chui gen ny gp phn vo s trao i cht th cp (ln nht trong Pseudomonas). B gen ca Pseudomonas fluorescens PfO-1 l mt NST vi 6.43841 Mbp v 60.5% hm lng GC, chng gm 95 RNAs v 5736 protein. 1.5. C ch hat ng ca Pseudomonas fluorescens 1.5.1. i vi thc vt: 1.5.1.1. Vi sinh vt i khng Nhiu cng trnh nghin cu v cc tc nhn phng tr sinh hc cng b t u th k 20 cho rng vi khun l mt tc nhn phng tr sinh hc, nhm vi khun hoi sinh m ph bin nht l Pseudomonas spp. Nhm vi khun ny sn xut ra cht kch thch tng trng cy, cc cht c ch hoc lm suy yu cc tc nhn gy bnh hoc c hai (Baker1988). C ch ban u c ch tc nhn gy bnh l tit ra cc cht khng sinh (Fravell1988). Cc yu t nh vic tit ra cht siderophore, HCN, s cnh tranh dinh dng cng c vai tr trong vic c ch tc nhn gy bnh. Pseudomonas fluorescens c kh nng i khng vi 10 loi nm bnh lu tn trong t, kch thch s pht trin ca cy trng bng cch s l ht c hay khng c cht mang hot chng vo t cng vi gi th lm thc n nn lm gim mc trm trng ca bnh ng thi gia tng s pht trin v tng nng sut cho cy trng. Vi khun Pseudomonas fluorescens k sinh trong phn v r cy trng, gip cho cy pht trin nhanh v chng li vi sinh vt gy hi, dn n tng nng sut (Lazarovits-1997). Pseudomonas fluorescens sn sinh nhiu hp cht ha hc c cu trc t n gin n phc tp vi c tnh nh nhng cht khng sinh.

Vi khun Pseudomonas fluorescens P217 c kh nng c ch s pht trin ca vi khun Ralstonia solanacearum v Pseudomonas solanacearum gy bnh ho xanh cho mt s cy trng c gi tr kinh t cao nh c chua, khoai ty, c tm, tDng PF AIR2 phn lp c trn than bn km hm s pht trin ca nm Rhizotina solani gy bnh kh vn, l c r. Vi khun Pf 2-79 rt d pht trin v c th nh c mt vi lai thc phm. Do n c th sng trong nhit ca my lnh nn n c th l mt tc nhn kim sot hu hiu i vi cc mm bnh c tnh chu lnh nh mm bnh Listeria monocytogenes v Yersinia enterocolitica. 1.5.1.2. Gy hi cy trng: L vi sinh vt ph sinh, thnh vin ca h vi sinh vt ht-c-r. Trc tip ph hoi t bo k ch, ht nhng vt cht sng trong t bo k ch. Chng Pseudomonas fluorescens Pf-5 enzym hy hoi bc tng t bo thc vt v cc thnh phn nh xenlulaza, pectinase, hoc lyase pectin. Tuy nhin n c th loi b mt s ngun carbohydrate, axit bo, v cc loi du v hydrolyze protein trong thc vt c th gy h hng sa, tht v c. 1.5.2. i vi ng vt: gy bnh nhim khun (bnh m ) trn cc lai thy sn, c bit l cc loi c nui. Vi khun l mt trong nhng tc nhn gy bnh kh quan trng, l tr lc ch yu km hm s pht trin ca c. Hu ht vi khun gy bnh l mt phn ca h vi sinh vt bnh thng trong mi trng (nc bin, ao, h, sng rch) v ni chung cc vi khun ny c xem l tc nhn gy bnh th cp hoc tc nhn gy bnh c hi. Tuy nhin cng c mt s t cc loi vi khun l tc nhn khi pht, bnh xy ra thng l do bin ng cc yu t mi trng hoc do stress nhng cng c th gy cht cao. T l cht do nhim khun c th ln n 100%, bnh c th xy ra di dng mn tnh, bn cp tnh v cp tnh. 1.5.2.1. Du hiu bnh: Xut huyt tng m nh trn da, xung quanh ming v np mang, pha mt bng, b mt c th c th b chy mu, tut nht nhng khng xut huyt vy v hu mn. Pseudomonas fluorescens gy nhim khun huyt thng xm nhp vo c th c qua cc thng tn mang, da, vy do cc tc nhn c hc, nui vi mt cao, dinh dng km, hm lng oxi gim

Hnh 4. Xut huyt c

1.5.2.2. Phng tr Gim mt nui, cung cp ngun nc tt. Dng vaccine phng bnh. Tm KMnO4 vi lng l 0.4g/100ml khng quy nh thi gian. 1.5.3. i vi thc phm Vi khun l tc nhn ca cc hin tng bin thoi v h hi thc phm. Thc n, thc ung, sa ti, fromage, rau quu c th b chua nht, ng vn, hi thi, c bt, c gaz hoc c nhng mu khc thng. 1.5.3.1. Pseudomonas fluorescens gy h hng trn b mt tht - Tht b ha nhy: thng c b mt tht p lnh khi m khng kh cao hn 90% (giai an u ca qu trnh h hng). Trn b mt tht hnh thnh mt lp dy c cc vi khun gy hi thc phm khc nh Micrococcus aureus, Micrococcus candidus v nm men. Tht c bo qun nhit t 0 n 2C vi m tng i t 85% n 90% l tt nht (c th bo qun trong 2 tun l m tht vn khng ha nhy). - Chng Pseudomonas fluorescens hnh thnh vt mu xanh lc trn b mt tht: di tc ng ca vi khun h hp hiu kh trn b mt tht xut hin nhng vt mu. Khi m b i thiu v xut hin perocid th mu vng s bin thnh mu xm ti v sau tr nn xanh. Nu tht c mi bnh thng v khng pht hin thy c c t th sau khi ty sch cc vt mu vn c th s dng bnh thng. - Khun Pseudomonas phn gii m thc y qu trnh oxy ha m, m b oxy ha do tc ng ng thi ca nh sng v khng kh. Hnh 5. Gy vt mu xanh lc trn b mt tht

1.5.3.2. Pseudomonas fluorescens gy h hng trn nhiu loi thc phm - Trn tht gia cm khun Pseudomonas gy ra mi hi, gy mi kh chu trong lng trng trng. - Chng Pseudomonas fluorescens gy cho sa c mu xanh l cy, lm cho v trng c mu xanh trng v cng gy vt mu xanh l trn c p lnh.

- Khun Pseudomonas gy nn s bin tm do astaxanthin b tch ra v b oxi ha khi tm nhit 30-40C sau t 8-12h sau khi nh bt. - Hin tng thi nhng rau ti cng l do Pseudomonas fluorescens gy nn. 1.5.4. Listeria Vi khun Listeria phn b rng ri trn mi trng t, nc, phn gia sc nn d ly nhim qua thc phm ch bin, khng nhng gy ng c thc phm m lai vi khun ny cn gy nn mt s bnh nguy him nh sy thai, vim no, nhim trng huyt. Sau giai an bnh (c khi n 2 thng) du hiu khi pht l st, kh chu, au lng. Nu c t mnh s gy ra ng c thc phm cp tnh n 12h vi chu chng nn ma, tiu chy, au qung bng, nhc u.Gy ng c trong bnh nhn tht, c st c, tht ng lnh, sa, phmai, rau ci ti sngVi khun Listeria c t chc y t th gii xp vo nhm tc nhn sinh hc c nguy c cao trong lnh vc an tan thc phm. Pseudomonas fluorescens nh hng n vi khun

Hnh 6. Vi khun Listeria monocytogenes Vi khun Listeria dnh bm vo b mt my mc ri t xm nhp vo cc sn phm thc n. Tuy nhin Listeria khng gii bm ln cc b mt bng thp khng g khi cnh tranh vi cc vi khun khc. Nhng khi Pseudomonas fluorescens bm vo b mt th Listeria c kh nng bm vo b mt mt cch hiu qu hn. Vy theo nghin cu ny th Pseudomonas fluorescens c nh hng trc tip n c ch gy c ca Listeria ln thc phm mt cch gin tip. 1.5.4. Bnh hc Ngoi tr gy ng c thc phm nh hng n sc khe con ngi th Pseudomonas fluorescens cn l mt nguyn nhn gy bnh c hi ngi, v thng nh hng n cc bnh nhn c h min dch km (nh l bnh nhn iu tr ung th). Khong nm 2004-2006 c mt dch ca Pseudomonas fluorescens ti Hoa K, n nh hng n khong 80 bnh nhn trn 6 tiu bang.

Ngoi ra cn c th tm thy Pseudomonas fluorescens trong cc bnh v nhim khun ng tiu. Tuy nhin ta c th iu tr d dng bng cc dc lc. 1.6. Bnh v cc triu chng S nhim bnh bt u t khi c nhng bin i lm suy yu h bo v ca t bo ch. Pseudomonas fluorescens c th gy ra: nhim trng huyt, ng tit niu,tc ng ln h tiu ha. Triu chng bnh khi b nhim khun Pseudomonas fluorescens thng l n lnh, st v lnh trong vi gi. 1.7. ng dng ca Pseudomonas fluorescens 1.7.1. Sn xut thuc bo v thc vt Ch phm thuc bo v thc vt sinh hc c ng dng rng ri tng nng sut, bo v cy trng. Vi khun hunh quang Pseudomonas fluorescens c s dng to ch phm phng tr bnh hi r c ph, vi thiu, u phng. Ch phm BioGro dng s l mi trng nui tm, phn hy rc thi trong ao nui tm, gim c lng kh c trong ao, to mu nc dn n n nh mi trng nui tm. 1.7.2. Sn xut thuc khng sinh Cc dng thuc khng sinh bi ti ch trn da thng c dng khi cc nhim khun ngoi da va v nh, cc trng hp nhim khun nng hn thng phi dng khng sinh tan thn bng ng ung, tim kt hp thuc bi. Hin nay c nhiu loi khng sinh bi ti ch tuy nhin khi s dng cn ch n c ch tc dng, ph tc dng, tc dng khng mong mun ca thuc nng cao hiu qu iu tr, gim khng thuc v cc phn ng c hi khc. Mt khc nhiu loi thuc khng sinh cn c bo ch kt hp vi 1, thm ch 2 nhm thuc khng sinh hoc corticoid, khng nm, khng histaminecng lm cho vn la chn iu tr cn c ch . Pseudomonas fluorescens c s dng iu ch mt loi khng sinh Mupirocin c ch trong iu tr cho da, tai v cc ri lon mt. N c tc dng dit khun do c ch RNA vn chuyn. Thuc c tc dng tt vi cc nhim khun da do vi khun gram (+). T cu thng rt nhy cm vi khng sinh ny tuy nhin d xy ra hin tng khng thuc ca cc vi khun khc nu s dng trn lan. Thng ch dng trong khong 10 ngy ri ngng trnh khng thuc. Hnh 7. Khng sinh Mupirocin

1.7.3. Sn xut cht chuyn ha th cp 2,4-DAPG Vi khun Pseudomonas fluorescens k sinh trong phn v r cy trng, gip cho cy pht trin nhanh v chng li vi sinh vt gy hi, dn n tng nng sut (Lazarovits-1997). Pseudomonas fluorescens sn sinh nhiu hp cht ha hc c cu trc t n gin n phc tp vi c tnh nh nhng cht khng sinh, trong 2,4-DAPG l hp cht c xem c vai tr xc lp tnh i khng. Gen m ha 2,4-DAPG c gi l PhlD, c phn lp v tm hiu cu trc trnh t cng nh qu trnh sinh tng hp. Bng cch tc ng vo cu trc gen m ha, mt s dng vi khun t bin c kh nng gia tng lng khng sinh sinh hc vo mi trng tng kh nng c ch s pht trin ca nm, vi khun, tuyn trng gy hi nh c trong vng r cy trng. S dng phng php sc k lng cao p pht hin v nh lng hp cht khng sinh 2,4-DAPG v dng PCR vi cc primer chuyn bit pht hin gen Phl m ha 2,4DAPG trong vi khun Pseudomonas fluorescens, cng nh nh gi tnh i khng ca cc dng chn lc vi mt s nm gy hi cy trng. Gip loi b khong 40% dng vi khun hunh quang i khng nhng c kh nng gy c cho cy trng, v gip chn chnh xc nhng dng vi khun cha gen khng sinh hin din trong r cy. 1.7.4. Kim sat mm bnh C th dng Pseudomonas fluorescens nh l mt vi khun c li chng li nhng vi khun tim n c hi, km hm s pht trin ca cc mm bnh. Theo mt nghin cu ca S Nng Nghip M (ARS) khi ngm t king trong dung dch nc c cha vi khun i khng Pseudomonas fluorescens 279 khong 2 pht th vic sinh si mm bnh hu nh b c ch hon ton.

Hnh 8: t chung ti v cha t mm bnh Kt qu cho thy nhng qu t chung cha c x l con s mm bnh nhn ln 100.000 ln nhit 68F trong khong thi gian ct tr l 2 ngy. Nhng nhng qu t chung qua x l vi vi khun Pf2-79 li km hm c s pht trin ca mm bnh. Cch x l ny c th lm tim nng ngn chn mm bnh sinh si nhiu n s lng c th gy bnh cho con ngi. 1.7.5. Lm sch nc thi cng nghip Cng ngh lm sch nc thi cng nghip t hydrocacbon nh tng ha s dng cht phn hy vi khun Pseudomonas fluorescens bt hot trn cht mang sinh hc phc ca phn ban u. Qu trnh lm sch din ra trong thit b phn ng sinh hc mikos hoc

trong thit b lc sau khi x l c hc hoc sinh ha. Cng ngh kh thi v sinh thi v kinh t, ging vi cc qu trnh t nhin.

Hnh 9. Nc thi cng nghip

PHN II
PHNG PHP XC NH PSEUDOMONAS FLUORESCENS

2.1.

Cc phng php truyn thng 2.1.1. nh tnh Pseudomonas fluorescens 2.1.1.1. Tin hnh

- chun b v pha long mu trong dung dch m pepton - cn 25g mu hoc 25ml cho vo bnh tam gic c 225ml dung dch mui m pepton v lc u (dung dch c pha long l 10-1 - tip tc pha long n nng cn thit s lng khun lc mc trn a petri khng vt qu 300. - ly 0.1ml mu cc dch pha long cy trn a Petri c cha mi trng thch Pseudomonas. - nui cy nhit 42C trong thi gian 24-48h (mi pha long cy t nht 2 a Petri v chn 2 nng pha long lin tip nhau). - quan st v m nhng khun lc c sc t mu xanh, trn u, mt nhn. 2.1.1.2. Kt qu Sau 48h, m s khun lc trn a Petri . khng nh cc khun lc nghi ng chc chn l Pseudomonas fluorescens th cn ly cc khun lc nghi ng cho nui cy trong mi trng lng BHI (Brain Heart Infusion) 37C trong 24h thu c canh trng thun nht. T canh trng thun nht ny xc nh hnh th v cc tnh cht sinh ha. 2.1.1.3. Quy trnh th nghim sinh ha Dng que cy vng tin hnh ria mu ln mt trong cc mi trng chn lc nh EMB agar, Endo agar, Deoxycholate agar, MacConkey agar, Hektoen Enteric agar v XLD agar phn lp h Enterobacteriaceae v ging Pseudomonas, 37C trong vng 24h. Trn mi trng XLD Pseudomonas cho khun lc mu khng c tm en. Cc khun lc c hnh thi c trng cho Pseudomonas c dng nhum gram, trc tin c th nghim TSI, oxidase, sau s c th nghim cc c trng sinh ha khc: Nhum gram: cy ria cc khun lc sang ng thch dinh dng, 30C trong 24h.Tin hnh nhum gram, quan st di knh hin vi (c th nhum gram bng phng php Jensen hay Hucker): - to vt bi vi sinh vt trn phin knh,chn mt phin knh sch, dng bt ghi trn knh v 1 vng trn ng knh 2cm mt di ca phin knh. t ti v tr tng ng vi vng nh du mt trn ca phin knh vi git nc ct thnh mt git ln. - dng que cy vng chuyn mt t sinh khi khun lc vo git nc trn phin knh, khuy nh bng u que cy c huyn ph ng nht v bi u trong khu vc ca vng trn

- yn cho vt bi kh - a phin knh qua li vi ln trn ngn la n cn - thc hin tng t to mt vt bi chung 2 chng vi sinh vt i chng trong cng mt phin knh khc (dng E.coli lm chng i chng gram (-) v Staphylococcus aureus lm chng i chng cho gram (+)). - theo phng php Jensen: nh vi git dung dch Methyl violet ln vt bi, gi yn 20 giy. Dng bnh xt bm nc ln vt bi ra sch phm nhum. Nh vi git KI/I2 ln vt bi, yn 1 pht. Xt cn 95% ln vt bi ra phm nhum n va mt mu. yn vi giy ri ra li vi nc. Nhum bng dung dch safranin 30 giy, ra li vi nc, thm nc d bng giy lc. - quan st mu nhum ca t bo bng vt knh 100X. T bo nhum gram (-) c mu hng Th nghim oxidase: - ct 1 ming giy lc thnh nhng di nh di 10-40 mm - nhng vo ha cht (dung dch 1% tetramethyl-p-phenylenediamine dihydrochloride hoc oxalate) - thm bt dch ha cht tha bng giy thm - kh 35C v gi trong chai ti mu nhit phng - dng que cy vng bng bch kim chuyn sinh khi ln mt phn giy lc - sau 10 giy c kt qu (khng c vt qu thi gian ny) - pseudomonas spp c phn ng oxidase (+): khi giy lc c mu ta m sau 10 giy Th nghim nitratase: - cy ria khun lc th ngnhim trn b mt v m su xung thch Nitrate Motility Agar trong ng thch su. - 35C trong 24h - nh vi git sulfanilic acid v naphthylamine: mu hng m hay mu chng t c s bin i nitrate thnh nitrite. Nu mi trng khng i mu nhng c s to thnh bt kh hoc rn nt mi trng cng c xem l phn ng (+) - Pseudomonas fluorescens cho kt qu (+) do kh nng kh nitrate thnh nitrite gii phng nit Th nghim to sc t pyoverdine: - cy ria khun lc th nghim ln mi trng thch Pseudomonas Agar F - 25C t nht l 3 ngy

- soi a di tia UV (260nm) - Pseudomonas fluorescens tit sc t pyoverdine pht hynh quang khuch tn trong agar dc theo ng cy.

Hnh 10 A. P.fluorescens pht trin trn mi trng thch dinh dng, nh sng bao quanh. B. P.fluorescens pht trin trn mi trng thch dinh dng, nh sng cc tm. C. P.fluorescens pht trin trn mi trng Pseudomonas Agar F, nh sng bao quanh. D. P.fluorescens pht trin trn mi trng Pseudomonas Agar F, nh sng cc tm.

2.1.2. Th nghim amon ha protein Vi khun Pseudomonas fluorescens c kh nng amon ha protein (gy hin tng thi ra): to thnh NH3 t cc hp cht nit hu c Th nghim: - ly 3-5g tht hoc c ct nh cho vo bnh tam gic - trn ming bnh cc giy ch th xc nh sn phm phn hy (giy qu hng xc nh s c mt NH3, giy lc tm dung dch 10% ch axetat xc nh s c mt ca H2S, giy lc tm dung dch nng ca oxalic axit bo ha xc nh s c mt ca indon). - y nt bng nhit t 28-35C Sau 1 ngy qu trnh phn hy prtin xy ra to mi hi thi c trng. Sau 57 ngy cc ming tht nt vn, mi trng ln men i mu, sau tht dn b v c ha thnh dch lng. Kim tra kt qu: ly 1 git dch phn hy lm tiu bn nhum mu c nh quan st vi sinh vt xem c phi l chng Pseudomonas fluorescens khng.

2.2.

Cc phng php hin i 2.2.1. Phng php PCR

Phng php PCR (Polymerase Chain Reaction) l mt phng php in vitro tng hp DNA da trn khun l mt trnh t ch DNA ban u, khuch i, nhn s lng bn sao ca khun ny thnh hng triu bn sao nh hat ng ca enzyme polymerase v mt cp mi (primer) c hiu cho an DNA ny. Hn hp phn ng nhn bn gen c th tch 25l vi thnh phn: 16,7 l nc 2,5 l m Taq (10x) 2,5 l dung dch dNTP (10mM) 1 l dch mi Prf (10mM) 1 l dch mi Prr (10mM) 1 l dch DNA khun 0,3 l Taq Polymerase Phn ng c thc hin vi chu trnh nhit nh sau: Giai an u: ADN bin tnh 95C trong 1 pht 30 giy, tip tc bin tnh 94C trong 1 pht, k tip mi c gn nhit 52C v phn ng ko di chui 72C trong 1 pht 20 giy. Giai an chnh: c lp li 30 ln chu k nhit c bn ging giai an u, ch khc ch ADN c bin tnh mt ln ngay nhit 94C trong 1 pht, tip sau pha cui cng phn ng ko di chui c duy tr 72C trong 10 pht v sn phm c bo qun 4C Kt qu c kim tra bng in di agarose 0.8%, nhum EtBr v hin hnh trn thit b hin hnh UV.

745 bp Hnh 11. Kt qu PCR da vo DNA khun mu trch t cc dng Pseudomonas fluorescens vi cc primer chuyn bit trn cc gen m ha cc cht khng sinh khc nhau

Hnh 12: Kt qu chy gel DNA ca Pseudomonas fluorescens t PCR 2.2.2. Phng php HPLC (High Performance Liquid Chromatography) dng pht hin kh nng to 2,4-DAPG ca Pseudomonas fluorescens 2.2.2.1. Phng php HPLC Phng php ny cn c gi l phng php sc k lng cao p: kh nng phn tch v tc phn tch cao hn nhiu so vi phng php c in, ct HPLC c th d dng ti s dng khng cn phi nhi ct li hoc ti sinh ct, lp li ln hn v kim sat c cc thng s gy nh hng, my c t ng ha cao v HPLC d dng s dng mc ln hoc mc thu mu. - HPLC nh c kt qu phn tch rt cao nh s dng cc ht hp ph rt mn < 20 m, c din tch b mt rt ln - HPLC s dng t dung mi, nhng i hi dung mi phi rt sch v khng cha bt kh

- HPLC s dng ct sc k bng thp khng r hoc ng thy tinh ng kinh la 2,1;3,2 v 4,5 mm

-teflon vi

Hnh 13: H thng phng php HPLC 2.2.2.2. Tin hnh - Chun b mu: chn lc vi khun Pseudomonas fluorescens da vo kh nng pht hynh quang v tr mu -80C trong dung dch glycerol. - Cc dng Pseudomonas fluorescens c nhn sinh khi trong mi trng KB lng 28C trong 36h. pH = 2 Ly tm thu dung dch, tin hnh acid ha vi trifluoroacetic 10% cho ti khi Chit rt 2 ln dung dch c acid ha vi ethyl acetate

- Thu dung dch chit rt v lm kh bng h thng BUCHI rotavapor R-114, ha tan trong 1ml methanol. - Phn tch trn h sc k dng Columm 100-5 C18AB Ser. No. 8075636, my HEWLETT PACKARD vi lng mu phn tch mi ln l 20l trong dung mi 35%

- acetonitrile cha 0,1% H3PO4, tc dng dung mi l 0,5ml/pht, bc song 270nm, thi im ghi nhn l 11,6 pht. - S dng mu chun 2,4-diacetylphlorogluinol (TRC) thit lp phng php chun v thit lp ng tng quan dng quy i nng c trong dung dch phn tch.

Hnh 14: Sc k HPLC pht hin hp cht khng sinh sinh hc 2,4-DAPG trong dung dch lng ca cc dng vi khun i khng Pseudomonas fluorescens A. Mu chun vi thi gian pht hin ng vi 2,4-DAPG l 11,6p vi nng 0,2ppm. B. Dung dch chit xt t vi khun PBV1 phn lp t bng vi. C. Sc k ca mu chun cng vi mu chit sut PBV1. D. Sc k ca vi khun PMT51 phn lp t r cy mng ti c nng 2,4-DAPG l 26,1g/ml dung dch chit xut.

III. KT LUN V NGH

Vi khun Pseudomonas fluorescens l dng vi khun thng thng hin din trong thc phm, nng gy ng c thc phm ca n n con ngi l khng cao tuy nhin n l tc nhn gy bnh i vi cc loi thy sn nui trong ao h. Mt khc chng Pseudomonas fluorescens c li cho cy trng, sn xut ra cc lai thuc bo v thc vt, khng sinh nn n c ch trng nghin cu trong thc vt. Tuy vy kh nng gy c ca n khng phi l khng c, n vn c kh nng gy bnh trn din rng i vi con ngi nn chng ta cn phi nghin cu chuyn su hn v tc nhn, triu chng gy bnh ca n ging nh trng hp nhim xc Pseudomonas fluorescens M 2004-2006. Nhng ng dng ca Pseudomonas fluorescens trong nng nghip cn c nghin cu v pht trin hn na c th s dng dng vi khun ny mt cch hiu qu nht.

PH LC:
Pseudomonas fluorescens 1. Kings B

mt s mi trng v thuc th dng cho vic xc nh

Proteose peptone No.3 Glycerol K2HPO4 MgSO4 Agar Nc ct

20g 10ml 1,5g 1,5g 15g 1 lt

Cho vo tt c cc thnh phn tr MgSO4. Lm nng v lc ha tan agar. Chnh pH n 7,2 0,2. Thm MgSO4 t t v trn. Phn phi 4ml vo cc ng nghim 13 x 100mm. Hp 121C trong 15 pht, nghing ng nghim.

Hnh 15: A- Pseudomonas fluorescens trn Kings B B- Pseudomonas fluorescens trn Kings B c quan st di n UV 2. Pseudomonas Agar F Proteose peptone No.3 (Difco) Tryptone (Difco) Dipotassium phosphate Magnesium sulfate Glycerol (Difco) Agar Nc ct 20g 10g 1,5g 0,73g 10g 15g 1 lt

Cho cc thnh phn mi trng vo nc, trn. un si ha tan cc thnh phn. Hp 121C trong 15 pht, pH cui l 7. 3. Brain Heart Infusion (BHI) Broth v Agar Dch no d Dch tim b Proteose peptone NaCl Na2HPO4 Dextrose Nc ct 4. Thuc th nhum Gram Phng php Jensen dung dch 0,25% methyl violet trong nc ct 200g 250g 10g 5g 2,5g 2g 1 lt

Ha tan cc thnh phn ca mi trng trong nc ct bng cch lc c gia nhit nh.

- dung dch iodine potassium iodide: ha tan 2g KI trong 20ml nc ct. B sung 1g iodine nghin nhuyn, qua m. Thm nc ct cho 300ml. - dung dch safranin: 1% safranin (hoc 1% neutral red) trong nc ct. Phng php Hucker - dung dch crystal violet: ha tan 2g crystal violet vo 20ml ethanol 95%. Ha tan 0,8g ammonium oxalate trong 80ml nc ct. Trn chung hai hn hp ny vi nhau, yn 24h, lc. - dung dch iodine potassium iodide: ha tan 2g KI trong 20ml nc ct. B sung 1g iodine nghin nhuyn, qua m. Thm nc ct cho 300ml. - dung dch safranin: nghin 0,25g safranin trong ci s vi 10ml ethanol 95%. Chuyn vo ng ong, ra bng nc ct v thu gp nc ra vo ng ong. B sung nc ct cho 100m Hnh 16. Cc bc tin hnh nhum gram

TI LIU THAM KHO CHNH

1- PGS. TS. L Thanh Mai, S.TS. Nguyn Th Hin, PGS.TS. Phm Thu Thy, TS. Nguyn Thanh Hng Cc phng php phn tch ngnh cng ngh ln men Nh xut bn khoa hc v k thut H Ni 2- Trn Linh Thc Phng php phn tch Vi Sinh Vt trong nc, thc phm v m phm Nh xut bn gio dc 3- GS. Nguyn Thnh t Sinh hc Vi Sinh Vt Nh xut bn gio dc 4- L Lng T, V Triu Mn Bnh vi khun v virus hi cy trng Nh xut bn gio dc 5- Ks.Phm Minh Nht Phn tch nh gi cht lng thc phm 6- http://en.wikipedia.org/wiki/Pseudomonas_fluorescens 7- http://microbewiki.kenyon.edu/index.php/Pseudomonas_fluorescens 8- http://www.uniprot.org/ 9- http://parts.mit.edu/ 10- http://genome.jgi-psf.org/ 11- http://inst.bact.wisc.edu/ 12- http://www.ncbi.nlm.nih.gov/ 13- http://www.fda.gov/ 14- http://www.cda.gov/ 15- http://www.mgc.ac.cn

GII P THC MC
Cu 1. Mi lien quan gia vi khun Listeria v vi khun Pseudomonas fluorescens ? Vi khun Listeria dnh bm vo b mt my mc ri t xm nhp vo cc sn phm thc n. Tuy nhin Listeria khng gii bm ln cc b mt bng thp khng g khi cnh tranh vi cc vi khun khc. Nhng khi Pseudomonas fluorescens bm vo b mt th Listeria c kh nng bm vo b mt mt cch hiu qu hn. Cu 2. Phng php HPLC l xc nh yu t no ca Pseudomonas fluorescens? Phng php HPLC c p dng xc nh kh nng to hp cht th cp 2,4-DAPG ca khun Pseudomonas fluorescens