B CNG THNGTRNG I HC CNG NGHIP TP. H CH MINHVIN CNG NGH SINH HC THC PHM

MN: VI SINH THC PHM

BACILLUS CEREUS V CC LOI BACILLUS KHC

Lp: DHTP7AGVHD: Ths. o Hng H

TP HCM, 26 thng 5 nm 2013.

Danh sch nhm

1Phm L Huyn Trang10200061

2on Anh T10270801

3Trn Thu Trinh10267711

4Trng Vn Trung10216951

5ng Trn Th Trc Tin10259831

6Nguyn Tn Vy10216171

7Nguyn Lm Thin Nguyt10262011

8Cao Th Vn Anh10237711

9Trn Th Hng Chu10303081

10L Quyn Hng10213431

Bng phn chia cng vic

Bacillus cereuson Anh TGii thiu hnh thi, c tnh + bnh do vsv gy ra

Phm L Huyn TrangPhn lp, nhn dng + quan h vi thc phm + tng kt

Bacillus subtilisTrng vn TrungGii thiu hnh thi, c tnh + bnh do vsv gy ra

ng Trn Th Trc TinPhn lp, nhn dng + quan h vi thc phm + tng kt

Bacillus thuringiensisNguyn Tn VyGii thiu hnh thi, c tnh + bnh do vsv gy ra

Trn Thu TrinhPhn lp, nhn dng + quan h vi thc phm + tng kt

Bacillus pumilisL Quyn HngGii thiu hnh thi, c tnh + bnh do vsv gy ra

Nguyn Lm Thin NguytPhn lp, nhn dng + quan h vi thc phm + tng kt

Bacillus mensentericusTrn Th Hng ChuGii thiu hnh thi, c tnh + bnh do vsv gy ra

Cao Th Vn AnhPhn lp, nhn dng + quan h vi thc phm + tng kt

Phn 1: BACILLUS CEREUS1. Gii thiu Mt bo co trc y v v ng c thc phm vi vi khun Bacillus spp. c thc hin vo nm 1906 khi Lubenau m t mt t bng pht trong mt vin iu dng ni 300 bnh nhn v nhn vin mc cc triu chng ca bnh tiu chy, au bng v nn ma. Xut hin bo t vi khun trong ba n sau khi tin hnh phn lp mn tht vin. Mc d Lubenau t tn l sinh vt ny l Bacillus peptonificans, nhng cc c tnh ng m t ging vi chng B. cereus. Sau , bo t hiu kh ny tip tc lin quan n mt s dch bnh chu u vo gia nm 1936 v 1943, chng b nghi ng gy ra 117 trong 367 trng hp iu tra ca y BanY t Stockholm Bacillus cereus vn khng c kt lun chn chn nh l mt nguyn nhn gy ng c thc phm cho n nm 1950, sau khi nguyn tc phn loi ca cc ging c lm r.Hauge m t bn dch bnh Na Uy lin quan n 600 ngi. Cc xe thc phm cha nc st vani c chun b trc mt ngy v bo qun nhit phng trc khi n. Mu ca nc st sau khi kim tra cha t 2,5* 10^7 n 11*10^8 B. cereus ml. Bo co ny v rt nhiu nhng ngi u tin chu u m t cn bnh ny, trong ni bt l triu chng tiu chy. By gi n c xc nh ,B. cereus l nguyn nhn gy ra hai loi bnh ca bnh thc phm: mt loi tng i khi pht mun, "hi chng tiu chy v mt khi pht nhanh, "hi chng nn c m t ln u tin vo nm 1971 Anh T nm 1975 mt s loi vi khun Bacillus khc c lin quan vi bnh t thc phm. Trong cc giai on ny, cc php kim tra khng tm thy c mm nhng thc phm tha v / hoc cc mu lm sng pht hin mt s lng ln vi khun Bacillus spp. t ph bin hn so vi s bng pht loi c trng B. cereus,chng thng lin quan rt cht ch n cc loi nh B. licheniformis v B. pumilis hoc B. subtilis. B. thuringiensis cng c bo co l nguyn nhn gy ra mt t bng pht Canada. Nhn chung, bo co phn ln cc trng hp bnh thc phm u do Bacillus spp. Anh th thp hn nhiu so vi vi khun Salmonella. K t nm 1992 c ti 10 dch bnh mi nm lin quan n tng s 67 trng hp. S liu thng k cho thy mc d c th nh gi thp hn mc thc s so vi vi khun Salmonella t cc d liu ch n t cc dch m khng c c tnh cc trng hp l t. Trong mt s khu vc cc nc Bc u cc sinh vt dng nh c tm quan trng ln hn nhiu.N chim 33% tng s v ng c thc phm do vi khun Na Uy t nm 1988 n nm 1993, 47% Iceland (1985-1992), 22% Phn Lan (1992) v 8,5% H Lan (1991). Ti an Mch, Anh v x Wales, Nht Bn, Hoa K v Canada con s ny dao ng trong khong 0,7 v 5,0%.

2. Hnh thi v c tnh Cc Bacillus l vi khun _Gram dng

Hnh 1: Hnh nh nhum gramvi khun Bacillus cereus _ Trc khun_Hiu kh_Bo t dng ovan v c kh nng sinh nha bo _C th di chuyn bng lng roi Mc d vy, c vi trng hp li l Gram m hoc c nhng bin i a dng . Phn loi chng kh phc tp v c sa i nhiu ln trong nhng nm gn y.Chng cha khong 80 loi, nhng loi gy ra ng c thc phm thng l vi khun Bacillus cereus, v mt s loi c lin quan kh cht ch vi B. cereus v Bacillus subtilis Bacillus cereus l loi k kh ty tin vi cc t bo thc vt ln,thng l chui t1,0 mm bng 3,0-5,0 mm. N pht trin nhit trong khong t 8-55 C, ti u l khong 28-35 C, v khng c kh nng chu ng r rt pH thp (ti thiu 5,0-6,0, ty thuc vo acidulant) hoc hot nc (ti thiu ~0.95). Bo t l trung tm, hnh dng elip v khng gy ra sng ti bo t.V l mt bo t, B. cereus c kh nng phn b rng ri trong cc mi trng v c th c phn lp t t, nc v thc vt. S ph bin ny ngha l n cng l mt thnh phn ph bin ca h sinh vt tm thi trong rut ngi. Cc bo t cho thy mt kh nng chu s bin i nhit; ghi nhn gi tr D ti 95 C trong khong b m phosphate t 1 pht n36 pht.Sc chu ng dng nh thay i theo chng huyt thanh. Ti Anh, mt gin cc kiu huyt thanh da trn khng nguyn(H) cc roi c a ra, da trn mt tp hp ca 29 dnh kt khng huyt thanh tng ln chng li cc chng bng pht v khng c dch bnh no c phn lp t thc phm. Trong khong 90% dch bnh c th type huyt thanh sinh vt gy bnh mc d mi trng phn lp c phn loi ch khong mt na ca mi trng. Chng khng c kh nng lin kt gia hai loi khc nhau nh B.cereus gy ng c thc phm v trong huyt thanh. Mt s c lin kt vi c hai triu chng, nhng trong mt nghin cu t 200 dch bnh th triu chng gy nn ma t khp ni trn th gii, huyt thanh loi 1 c kh nng chu nhit cao so vi cc loi huyt thanh khc, chng c phn lp t nhng thc phm lin quan, mu phn lp hoc t mu cht nn ra 63,5% trng hp.

Bo t ca B.cereusB. cereus hnh thnh bo t khi cc cht dinh dng ang khan him trong mi trng v khi c sn chng ny mm thnh cc t bo thc vt. Bo t c mt v ngoi bn chc c lm bi keratin.

3. Cc triu chng v bnh lm sn: 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.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 , rauqu, gia v. Bn cht l mt loi protein gy hy hoi biu b v nim mc rut gy tiuchy c th nguy him n tnh mng. c t gy nn ma (Type 2): emetic toxin. Vi khun nhim trong go, cm ngui, ucc loi. Bn cht c t l phospholipit 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 chtngi. c t ny c th trung ha bi cholesterol trong huyt thanh nhng n gp phn chos pht trin ca vi khun3.1 Triu chng nn ma:Tnh cht/ hot ngM t

Liu nhim c S lng t bo B.cereus:105-108 tb/g thc phm

Khi lng c t12-32g/kg

Nhit thch hp25-30oC

Thi gian bnh30 pht 5 ting

Khong thi gian mang bnh6-24h

Loi thc phm thng gpCm, m ng, ph, u

Tn c tCereulide

3.2 Triu chng tiu chy:Tnh cht/ hot ngM t

Liu nhim c105 tb/g hoc / ml

c t c sn sinh raTrong rut non

Loi c tAmin

Thi gian bnh8-16 ting

Khong thi gian mang bnh12-24h

Triu chng thng gpau bng di dng, thnh thong bun nn

4. Phn lp v nhn dng4.1 Truyn thng: Nhm cc phng php ny da trn c im pht trin ca vi sinh vt trn cc mitrng c trng v c im sinh l, sinh ho ca cc chng, cc loi vi sinh vt khc nhau.u im:Thao tc n gin, d lm, khng phi u t dng c, thit b t tin. Nhc im:- nhy khng cao.- Yu cu k nng thao tc cao.-Tn nhiu nhn cng.- Thi gian phn tch thng ko di. 4.1.1 nh lng B.cereus bng phng php m khun lc:

-Tri 0.1ml mi pha long ln cc mi trng thch ri 24h 300C-MYP: do B.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 nhB.cereus.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 nhumbng 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 ng 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 dungdch huyn ph ng nht bi utrong khu vc ca vng trn yn cho vt bikh 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). -Kt thc qui trnh nhum, quan st di knh hin vi cho ta thy t bo nhum Gram (+) c mu xanh ta, t bo nhum Gram (-) c mu hng. B.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.4.1.2 nh lng B.cereus bng phng php MPN

10g mu v trng ng nht vi 90ml dd m phosphat bng my dp mu

DD mu pha long 10-1

Pha long mu 10-2, 10-3, 10-4

Chn 2 nng pha long thch hp

Ht 0.1ml dch pha long cho vo mi a MYP ( mi nng hai a)

300C trong 24-48 gi

Chn v m cc a c t 15-150 khun lc c trng ca Bacillus areus (dt, ng knh 2-3 mm, b hnh rng ca, mu hng, xung quanh c vng c)

Chuyn 5 khun lc nghi ng ny ln mi trng phc hi TSA

300C qua m

Tin hnh th nghim sinh ha

Do c hnh thi c trng trn cc mi trng thch chn lc nh: MannitoEgg-Yolk-Polymycin(MYP),CereusSelectiveAgar(MOSSEL),PolymicinElgelbMannitolBromothymol Blue Agar (PEMBA), nn B.cereus cn c pht hin v nh lng bng mi trng ny.

Khun lc B.Cereus trn PEMBAKhun lc B.cereus trn MYPKhun lc B.cereus trn Bacara. B. cereus trn thch MYPPhn ng sinh ha: Th nghim ln men glucose:Cy vi khun vo 3ml canh Phenol Red Glucose Broth 350C/ 24h, k kh lc mnh ng nghim,quan st c (s pht trin). Nu mi trng t sang vng th chng t c s sinh acid glucose trong iu kin k kh.

Kt qu th nghim ln men glucose Th nghim kh nng kh nitrate:Cy vi khun vo 5ml canh trng Nitrate Broth 350C/24h, b sung vi git thuc th nitrate, c mu cam xut hin trong 10 pht th chng t nitrate b kh thnh nitrit.

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 tnhvi nitrite. Th nghim VP:Cy vi khun trong mi trng MR-VP 48h, nhit 370C b sung thuc th (-Napthol) vo mi trng, lc nh. C 2 loi thuc th VP thch hp trong trng hp ny l:Thuc th Barritt:gm dung dch A l 5%-naphthol trong cn tuyti, dung dch B l 40% KOH hoc NaOH.Thuc th Koblentz: gmdung dch A l5%-naphthol trong cn 95%, dung dchB l 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, chm nht l 4h To phc hng l (+).

Th nghim kh nng thy phn tyrosine:Cy vi khun vo thch nghing tyrosine, 350C/48h xut hin khong trng xung quanh khun lc (chng t tyrosine b phn hy). Th nghim khng Lysozyme:Cy vi khun vo mi trng Nutrient Broth cha 0.001% lysozyme, thc hin tng t vi mi trng khng cha lysozyme, 350C/24h kim tra s tng trng trong 2 mi trng. 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.Cc th nghim phn bit cc loi trong Bacillus nhm I: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 L-tyrosine, tng trng c trong 0.001% lysozyme, c trnh by trong bng sau: c tnhLoi

BacilluscereusBacillusthuringiensisBacillusmtcoidesBacillusanthracisBacillusmegaterium

Gram+(a)++++

Catalase+++++

Di ng+/-(b)+/--(c)-+/-

Kh nitrate-(d)

Phn hy tyrosine+++/--(d)+/-

Khng lysozyme++++-

Phn ng vi lng trng++++-

Ln men glucose++++-

Phn ng VP-

Sinh acid t manitol----+

Tan mu (cu)+++-(d)-

a +:90 - 100% cc chng dng tnh;b +/-: 50% cc chng dng tnh;c - : 90 100% cc chng m tnh;d - : Hu ht cc chng m tnh. Th nghim tnh di ng:Phng php 1: Dng que cy vng cy dch 24 gi nui cy thng vo gia mi trng kim tra di ng cho B.cereus. 300C,t 18-24h v kim tra di nh n kiu mc dc theo ng cy. Kt qu: loi di ng mc khuych 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 trong 6-8h 300C. Nh nc v trng ln phin knh v 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

Th nghim lm tan mu:Cy chun ln mi trng thch mu Tripticase Soy. 250C trong 24h. B.cereus lm tan mu mnh v to vng tan mu hon ton () 2-4mm xung quanh vng pht trin. B.thuringiensis v B.mycodes cng lm tan mu . B.anthracis thng khng tan trong mu sau 24h.

B.anthracis khng lm tan mu B.cereus lm tan mu Th nghim hnh thnh r gi:Chm nh que cy vng mang huyn ph 24 gi ln gia a Nutrient agar, 30oC/48-72h. Sau kim tra s pht trin ca r gi (B.cereus khng 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 c r gi

B.Mycoides c r gi S to c t protein dng tinh th:

Cy huyn ph t bo 24h ln ng thch nghingnutrient agar, 300C/ 24h yn nhit phng /2-3ngy. Sau nhum bng phm mu fuchsin, quan st di knh hin vi. Tinh th c t caB.thuringiensis xut hin sau 3n 4 ngy nui cy, phthin cbng 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. B.cereus v cc Bacillus khc cng nhm khng tinh th c.Cch tnh kt qu:S t bo B.cereus/1g mu da vo s khun lc mc mi pha lang v hiu chnhbng t l khng nh (phn trm khun lc c xc nhn l B.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 B.cereus (c kim tra bng cc phn ng sinh ha).S t bo B.cereus/1g thc phm = 65 x 4/5x 10000 x 10= 5200000 (nhn 10 v c 0.1ml mu c tri da)4.2 Hin i4.2.1 Phng php ELISANguyn tc: k thut ELISA l s dng khng th n dng ph ln nhng a ging. Nu c s c s hin din ca khng nguyn mc tiu trong mu, khng nguyn ny s gi li trn b mt ging. Cc khng nguyn ny s c pht hin bng cch s dng khng th th cp c gn vi enzyme nh horseradish peroxidase hay alkaline phosphatase. Khi b sung 1 c cht c hiu ca enzyme vo ging, xc tc phn ng thy phn c cht to ra cc sn phm c mu hay pht sang. Bng cnh theo di mu, c th pht hin s hin din v nh lng lng khng nguyn.4.2.1.1 Phng php Elisa trc tip (Direct Elisa)Trong trng hp ny, khng nguyn nm trn ln trong dung dch m s c cho vo y ging. Khng nguyn s lin kt c nh vo b mt ca y ging. mt thi gian ngn, sau tin hnh ra tri. Nhng khng nguyn no khng lin kt s c a ra ngoi.Thm dung dch cha khng th c gn enzyme vo ging, khng th s lin kt vi khng nguyn to phc hp gn cht vo ging. mt thi gian phc hp n nh, sau tin hnh ra tri. Nhng khng th no khng lin kt vi khng nguyn s c ra tri.Thm dung dch c cht c h m vo ging. Nu c s tn ti ca phc hp khng th_Enzym_khng nguyn th c cht di tc dung ca E s to mu cho dung dch. Da vo s xut hin mu v cng mu trong dung dch s nh tnh v nh lng c khng nguyn.

4.2.1.2 Phng php Elisa gin tip (Indirect Elisa)Khng nguyn s c cho vo y ging. Khng nguyn s lin kt c nh vo b mt ca y ging. mt thi gian ngn, sau tin hnh ra tri. Nhng khng nguyn no khng lin kt s c a ra ngoi.a khng th mc tiu c hiu vo ging, mt thi gian v tin hnh ra tri, ch nhng khng th c hiu mi c gi li cng khng nguyn trn b mt y ging.a tip dung dch m cha khng th th cp c gn emzyme vo, khng th ny s lin kt vi khng th mc tiu. To phc hp khng nguyn_khng th mc tiu_khng th th cp_E. Cng tin hnh v ra tri.Khi cho dung dch cha c cht vo ging, c cht di xc tc ca E s c phn ng to mu.Phng php ny ch khc vi dng trc tip l phi s dng khng th th cp gn E do khng th mc tiu khng th gn vi E.

4.2.1.3 Phng php Elisa Sandwich:Trc tipMu cn phn tch c cho v trong a ging c ph khng th chuyn bit. Khng nguyn trong mu s kt hp vi khng th trong a ging v tr nn bt ng. a ging ny tip tc c ra loi b nhng khng nguyn khng kt hp c vi khng th. Sau , phc hp khng th - enzym (gm khng th lin kt vi enzym qua lin kt cng ha tr) c thm v. Khng th ca phc hp ny s kt hp vi khng nguyn bt ng trong a ging cn li sau khi ra hnh thnh 1 bnh sandwich gm: khng th khng nguyn khng th/enzyme trong a ging.Sau khi loi b khng nguyn hoc khng th, cc cng hp t do, c cht v cht pht mu tng ng vi enzyme s c cho v v mu s xut hin trong cc ging th. Vic nh lng khng th hoc khng nguyn c thc hin bng cch o mt quang gia mu v cht chun nh my c ELISA.

Gin tip:

Xc nh Bacillus Cereus Bng Phng Php ElisaHai loi c t gy ra trng c thc phm do Bacillus cereus sinh ra l Emetic Toxin (c t gy nn) v Diarrhoeal Enterotoxin (c t gy tiu chy). Phng php xc nh Bacillus Diarrhoeal Enterotoxin (BDE) bng k thut phn tch Elisa c Terca International Pty a ra qua b Kit Tecra Bacillus Diarrhoeal Enterotoxin Visual Immuno Assay.4.2.2 Phng php PCRK thut PCR (Polymerase Chain Reaction) do Kary Mullis (Nobel 1993) pht minh, l mt k thut hin ph bin trong sinh hc phn t, da trn phn ng khuych i gene. Trong mt on DNA c nhn ln rt nhiu ln mt cch nhn to bi enzyme DNA Polymerase m khng cn s dng vi sinh vt sng.Trong t nhin, enzyme DNA Polymerase c trong ni bo sinh vt chuyn thc hin chc nng xc tc cho cc phn ng nhn i si DNA theo nguyn tc b sung. Phn ng ny xy ra trong giai on phn chia t bo.Tuy nhin qu trnh ny cng c kh nng thc hin trong iu kin ng nghim (Invitro). , si DNA cng c tch i thnh hai si n bi nhit cao (95oC). Vi xc tc Enzyme DNA Polymerase, mi si DNA n c b sung thm to si kp. Tuy nhin v nhit cao nh th ny th E rt d b phn hy v mt i hot tnh.Hin nay, ngi ta tm ra c nhng loi E chu nhit rt cao, c tch t loi vi khun chu nhit (Thermophilic) nh vi khun Thermus aquaticus, nhng E ny gi l Tag, Flu. Nhit 110oC Tag v Flu vn khng b bin tnh.V c bn, thc hin c k thut PCR cn mt s cc phn sau : on DNA mu (Template) cha on DNA cn khuch i on mi (Primer) xc nh im bt u v im kt thc Enzyme DNA Polymerase nhn i on DNA Nucleotide l nguyn liu cho phn ng nhn i DNA Dung dch mNgoi ra, do PCR thc hin trong chu trnh nhit (khong 20-30 ln) nn cn c my un nng v lm ngui mi trng phn ng trong cc ng nghim. Thit b in di cn thit xc nh kch thc DNA (thc t l trng lng on DNA c khuch i).

My PCRNguyn l ca s khuych i PCR c th tm tt nh sau :Mt chu trnh phn ng gm 3 bc, trong my PCR tng s chu trnh lp li l 30 ln vi thi gian rt nhanh. Bc 1 : Dui xon DNA nhit 94oC trong thi gian 1 pht. Chui DNA kp c tho xon to 2 si n. Bc 2 : Gn mi 54oC trong khong 45 giy. Primer s bt dnh trn on DNA n cn nhn i mt cch c hiu Bc 3 : Ko di 72oC trong 2 pht. Enzyme s xc tc phn ng ko di mch DNA b sung cho ti khi gp im kt thc.

Hn hp sau khi chy PCR s c nhum mu v phn tch bng in di trn bn gel Agarose. Lng DNA sau khi khuych i s c cng trong lng phn t v tp trung cng mt vi tr trn bn gel. Kt qu in di s em so snh vi thang DNA chun bit trong lng phn t ca on DNA khuch i, cng chnh l mnh DNA lc ban u.Xc nh B.cereus bng PCR:Nh trnh by phn m u, Bacillus cereus l loi vi khun gy ng c thc phm vi hai triu chng : Nn ma (Emetic) v tiu chy (Diarrheic). Trong c t gy mn ma (Emetic toxin) l loi bn nhit do mt loi gene c tn Cereulide Synthetic (CRS) ca vi khun tng hp.Kt qu sau khi in di th hin s c mt ca cc on gene nh sau: Cereulide synthetic enzyme (CRS) gene: 426 bp Lecithinase (LE) gene: 227bp Internal Control (I.C.): 106 bp

Kt qu in di Hnh nh in di A c ngha l c Bacillus cereus hoc cc loi bacillus khc trong mu th c s lng t hn gii hn di ca php th, hoc cng c th do nhim loi vi sinh vt khc loi bacillus. Hnh nh B c ngha l trong mu c cha Bacillus cereus gy c t Cereulide (CRS). Hnh nh C chng t mu th cha Bacillus cereus khng gy c t Cereulide (CRS), hoc cha cc loi cng chi bacillus khc (Bacillus thuringensis, bacillus antharcis).Mu phn ng m Nagative control reaction ( mu phn tch l nc tit trng) s cho kt qu in di trn hnh A.Nu mu phn ng m cho kt qu in di nh hnh B,C th xy ra qu trnh nhim khun l khc trong sut qu trnh thc hin php phn tch. Lc ny cn phi r sot li qu trnh m bo s v trng trong phn tch v tin hnh th li.Nu mu m khng cho kt qu in di th phn ng PCR chy khng ng k thut, hoc cng c th c cc yu t khc nh hng n nh: ha cht l, b kit b h hngDo mi c tnh pht quang cho nn c th nh du chng vi cc cht nhum khcnhau, nh th c th nhn cc on khc nhau trong cng mt phn ng PCR. Tuy nhinphng php ny chn ch lphi tng hp cc mu d khcnhau cho cc trnh t nhnbit khc nhau.[10] Cc phng php nh lng bng Real time PCRPhng php s dng cht nhum mu SYBR Green: Cht nhum mu ny c gn vi rnh nh ca chui DNA kp, khi c gn visi DNA kp th cng pht hunh quang tng ln, khi bn sao to ra cng nhiu th tnhiu pht ra ca cht mu s tng ln theo t l thun. u im ca phng php ny lSYBR 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, ukia 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 utin hnh quy trnh PCRvi s hotng ca enzyme taq-polymerasemi c ko di cho ti khi gp mu d th mu d s b phn gii bi hot tnh 5nuclease ca taq-DNA polymerase. S phn gii mu d s lm gii phng cht nhum muc kh nng phthunh quang khi nh hng km hm ca cht nhum cvai tr lm tts pht hunh quang. S pht hunh quang ny c th nhn bit c.Phng php u d lai:Trong k thut ny mt u d c gn vi mt cht cho hunh quang u 3 vmt 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.

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 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 nngim kh nng nhim mu

4.2.3 K thut latex agglutination (LA)S dng cc ht cao su c mu c nh khng th c hiu nh tnh nhanh cc chngvi 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 hocdi c mu). K thut ny c th s dng xc nh nhanh vi sinh vt nhim tp trongmi trng thun sau khi phn lp t thc phm.

4.2.4 K thutlaiphn 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 vtmt lc vi thi gian nhanh. Tuy nhin k thut cng phc tp v phi tn cng thitk cc mu d v s dng vt liu phng x.5. Quan h vi thc phmB.cereus gy ng c thc phm ti Hungary, ni m t nm 1960 n 1968 l ln th ba gy ng c thc phm ph bin nht, thng k khong 15,2% ngi b nh hng. S liu gn y cho thy tm quan trng ca n gim i phn no, nhng liu iu ny l do s thay i trong cch thc nu n, ci thin v sinh, gim s nhim bn ca cc loi gia v hoc cc vt phm.B.cereus c kh nng sn xut bo t khng cc yu t nh sy kh v nhit lm cho trc khun gy ng c thc phm c phn b rng trong thc phm. Tuy nhin hu ht cc trng hp chng ch l mt lng nh trong tng s v khng s lng gy bnh. Tt l B.cereus trong sa tit trng (thng l 35-38% mu dng tnh) cao hn so vi sa ti (~9% mu dng tnh). Trong hu ht cc trng hp s lng c pht hin thp hn 103 ml. Vi sa v kem bo qun nhit khng lnh, B.cereus c th pht trin gy hin tng h hng nh ng vn (sweet curdling) hay ni bt (bitty cream)Cc hi chng nn ma ch yu lin quan ti cc sn phm tinh bt nh cc mn n t go v m ng. Bo t, thng l cc huyt thanh khng nhit ny, tn ti trc lc nu n ny mm, pht trin v sn xut cc cht c trong thi gian lu tr. iu ny c th c ngn nga bng cch lm lnh xung di 80C, nhng t l lm mt v ngay c khi lu tr lnh cng ch c th lm chm s pht trin v sn xut c t ca cc bo t vi sinh vt. Hm nng cm trc khi n s khng lm bt hot nhng c t v khng lm cho thc phm c tnh an ton. Mt s lng ln thc phm lin quan n triu chng tiu chy bao gm cc sn phm t tht, sp, rau, bnh trng ming v nc st. Cc loi tho mc v gia v kh c s dng trong ch bin thc phm c th l mt ngun quan trng ca B.cereus.

TNG KTBo t v cc t bo sinh trng ca Bacillus cereus xut hin mt cch rng ri trong t nhin, c bit l trong t (trong t c th cha 105 106bo t/g), n c th c tm thy trong cc ngun thc phm dng ti hay qua ch bin ngoi tr cc thc phm c thanh trng bng nhit hay chiu x. Phn ln cc ging vi khun B.cereus c th pht trin trong cc thc phm c chua thp nhit di 150C v trn 550C (nhit ti thch l 30 350C), pht trin c trog khong pH 5.0 v 8.8.c t cereulide (gy nn ma) ca B.cereus rt bn nhit, c th tn ti 1260C trong 90 pht. Ngc li, c t diarrheal (gy tiu chy) rt nhy cm vi nhit v b v hot khi nu chn nhit 560C trong 5 pht.Chng loi, cu trc v thnh phn cu to c t ng rut ca B.cereus cn c nghin cu xa hn. Cn cc thao tc kim tra trn ng nghim i vi c t gy nn v kim tra c th hot ng v mt sinh hc i vi c t tiu chy.Tm li vi sinh vt tn ti tt c mi ni, trong khng kh, t, nc thm ch l trn c th ca mi chng ta. Bi th nn vic vi sinh vt xut hin trong thc phm khng phi l iu g to tt, tuy nhin vic n kt hp vi cc vi sinh vt khc v gy ra cc triu chng ng c m ngay c con ngi cng khng th xc nh c cch iu tr th y thc s l mt vn nan gii, cho d l cc vi sinh vt nh l Bacillus cereus, B.cereus, Salmonella, B. Subtilis, hay B.haemolysis. Nu chng ta khng bit cch bo v nhng loi thc n m mnh s dng th chnh n cng s gy ra v vn nhng bnh l m chnh chng ta cng khng bit. V vy, chng ta cn phi bit cch phn lp v nhn dng cc loi vi sinh vt khc nhau mt cch c bn nht nhm hn ch n mc thp nht qu trnh gy ng c thc phm nu chng xy ra.

Phn 2: Cc chng loi Bacillus khcA. Bacillus Subtilis

1. S lc v vi khun Bacillus subtilis

1.1 Lchsphthin

Bacillus subtilis cphthinutintrongphnnganm1941bit chcy hcNazi cac.Lcucsdngchyulphng bnhl cho ccbinhscchinuBcPhi.Viciutrphiinnhngnm1949-1957,khiHenrry vcccngstchcchngthunkhitcaBacillus subtilis.Tsubtilis therapy cnghal"thucsubtilis" raitrccchng vimrut,vimitrng,chngtiuchytrongrilontiuho.Ngy nay,vi khunny trnnrtphbin,csdngrngritrongyhc,chnnui, thcphm(trchLKimHu,2005).

1.2 cimphnloivsphnbcavikhunBacillussubtilis

cimphnloi:

TheophnloicaBergy(1994)Bacillussubtilisthuc: B:Eubacteriales

H:Bacillaceae

Ging:Bacillus

Loi:Bacillussubtilis

Hnh Vi khun Bacillussubtilis

cimphnb:

VikhunBacillus subtilis thucnhmvisinhvtbtbuc,chngc phnbhuhttrongtnhin.Phnlnchngctrtrongt,thngthng t trngtrt chakhong10-100 triuCFU/g.t nghodinhdng vngsamc, vngthoangthvikhunBacillus subtilisrthim.Ncvbncasngcng nhncbincngc mtbotv tboBacillussubtilis(VThTh,1996).1.3 cimhnhthiBacillussubtilisl trckhunnh,haiutrn, G+, kchthc

0,5-0,8mx1,53m,ngnlhocthnhchuingn.Vikhunckhnngding, c8-12lng,sinhbothnhbudcnhhntbovikhunvnmgiat bo,kchthct0,8-1,8m.Bo tpht trinbngcchnymmdosntca bot,khngkhngacid,ckhnngchunhit,chum,tiatngoi,tiaphng x(TMinhChu,2000).

1.4 cimnuicyiukinphttrin:hiukh,nhittiul370C

NhucuO2:Bacillussubtilislvikhunhiukhnhnglickhnng phttrinyutrongmitrngthiuoxy.pH:BacillussubtilisthchhpnhtvipH =7,0-7,4.

Mitrng

MitrngthchaTSA:khunlcdngtrn,rarngcakhngu, ctmsm mu,mu vngxm,ngknh35mm. Sau 1-4ngybmtnhn nheo,muhinu.a) MitrngthchnghingTSA:dmc,tothnhmuxm,ranhngnsng. b) Mitrnggelatin:phttrinvlmtanchygelatin.c) Thchkhoaity:phttrinu,muvnglmtmht.d) Mitrng canhTSB:Bacillussubtilisphttrinlmcmitrng,to mngnhn,lngcnktlinhvnmyy,lclnkhtanu.

1.5 cimsinhho

Ln men khngsinh hi cc loi ng:glucose, maltose,mannitol,saccharose,xylose,arabinose.

Indol(-),VP(+),Nitrat(+),H2S(-),NH3 (+),catalase(+),amylase(+),casein(+),citrat(+),ding(+),hiukh(+).

PhnngsinhhoKtqu

Hottnhcatalase+ Sinhindol- MR+VP+ Sdngcitrate+ Khnitrate+ Tanchygelatin+ Ding+Phngiitinhbt+ Arabinose+ Xylose+ Saccharose+ Mannitol+ Glucose+ Lactose-Maltose+

(TheoHolt,1992)(trchLKimHu,2005).

1.6 BotvkhnngtobotcavikhunBacillussubtilis

1.6.1 Cutobot

Ngoi cngcabotlmt lpmng, dilpmnglv.Vbotc nhiulp.y lnhnglpctcdngngnchnsthmthucancvcc chthotantrongnc. Di lpvllpmngtrongcabotvtrongcngl mtkhitbochtngnht.Trongccbottdokhngtntistraoi cht,vvycthgitrngthitimsinhtrongnhiunm(LMaiPhng,2004).Botkhctbodinhdngvcutrc,thnhphnhohcvtnhchtsinhl.

1.6.1.1 Khnngtobot

Mt trongnhngc imquantrngcaBacillussubtilislkh nngto bottrongnhngiukinnhtnh.Bacillussubtilisckhnnghnhthnh bottheochutrnhphttrintnhinhockhivikhungpiukinbtli (dinhdngtrongmitrngbkitqu)(TMinhChu,2000).Stobotdinragm nhiugiaion,tngcnggn8gihontt. Lculpnguyn sinhchttrongtbocsdng.Tbochtvnhntp trungtimtvtrnhtnhtrongtbo.Tbochttiptccclivto thnhtinbot(Prospore).Tinbotbtucbaobcdnbicclp mng.Tinbotphttrinvtrthnhbot.Khibottrng thnh,tbo dinhdngtphngiivbotcgiiphngkhitbom.Khigpiu kinthunli,botshtncvbtrng ra.Sau,vcachngbphhu vbotnymmphttrinthnhtbomi.Mitbodinhdngchtora mtbot(LMaiPhng,2004).

1.7 Tnhchtikhng

Vivisinhvtgybnh,miloisinhvtkhcnhausthchhpiu kinmitrngkhcnhau,sinhkhunlckhcnhau.Thayimitrnghoc ccyutmitrngbtlillmthay iiukinsng,lmhnchhocc chsphttrincavisinhvt. Thctkhimitrng nuicy nm bnhcs hindincaBacillussubtilisvimtslnglnsxyrascnhtranhdinh

dng. Cnhtranhkhnggiansinhsnggiavikhunvnm. Dovikhunpht trinnhanhhn(trong24gi)ssdngphnlnccchtdinhdng trongmi trng,ngthitoramtsloikhngsinhnnssinhtrngcanmbc ch(NguynLnDngvHongcThun,1976).2. Tinh an toan.Bacillus subtilisl loi vi khun c s dng Nht Bn trong cc mn n ctruyn t hng nghn nm trc v l thc phm an ton i vi con ngi. chu u v M, vi khun ny c ch nh l iu kin v an ton thc phm, ting Anh gi l QPS (Qualified Presumption of Safety) hay GRAS (Generally Regarded As Safe).Bacillus subtilis c cc nh khoa hc ca Trng i hc Hong gia Holloway Lun n, Anh Quc chng minh rt l an ton v khng h c tc dng ph vi liu ung ln n 1x1011CFU/ngy (Hong v ng s 2008). HU58cng c cc nh khoa hc caTrng i hc Hong gia Holloway Lun n, Anh Quc nghin cu tnh cht, s an ton v kh nng tn ti v pht trin rut (Hong v ng s, 2009). Theo hng dn ca Cng ng chu u v M, xut bi t chc Lng thc Quc t (FAO) v T chc Y t th gii (WHO) (FAO/WHO, 2002), C quan An ton Thc phm chu u EFSA (European Food Safety Authority, EU) v Cc An ton Thc phm v Thuc ca M FDA (Food & Drug Administration, USA), HU58 c nh danh tn chng bng cng ngh gen, v vy mang li thng tin y v an ton hn cho ngi s dng, khc hn vi nhiu sn phm khc trn th trng hin nay l vi khun s dng ch c xc nh tn loi bng cc phng php c in.3. Phn lp bacillus subtilis.Cn 10g muchovobnh tam giccha 90g ncmuisinhlvtrngvlcu, cnngphalong 10-1 .Chunb 4 ngnghimmingcha 9ml ncmuisinhlvtrng ,nhsthtt 1 n 4, dng micropipette ht 1ml dchmutbnh tam gicchamutphnlpcnngphalong 10-1chovongnghimthnhtvlcu, tiptcchovoccngnghimcphalong nngtiptheo.Chnccngnghim 10-3, 10-4, 10-5emuncchthy 70 trong 30 pht, saukhiunthdng micropipette ht 0,1ml tccnngphalongcholnmitrng TSA vtrangubngquetrangvtrng, a 37 / 24h trongtmsauquanstcckhunlchnhthnh , chnrahngkhunlcnghingl B. subtilisdngquecyvngbtvgiginglitrnmitrngngthch TSA nghing .3.1 Khostcimcavikhunphnlpc.Lymtngsinhkhivikhuntng TSA lmtiubnnhum Gram quanstdiknhhin vi, phngiln 1000 ln. saukhiquanstthytiubnnophhpvicimca bacillus subtilisnhkhun lac mclantrnmtthch, bmtth, muxmtrng, vinrngca, trckhun Gram dng, ngn, nh, haiutrnthngngringl, ikhixpthnhchuingn, cbotthtiptcthccphnngsinhhakhngnh.

a. Phnng catalaseChunb : dung dch H2O2 lametinhnh: dngquecyvngthaotcvtrnglymttsinhkhi vi khunphtln lam knhschvkhsaunhgit H2O2 30% lnvtpht vi khun. cktqusau 15 giy.phnng catalase (+): chintngsibtphnng catalase (-): khngsibt.b. PhnnglecithinaseChunb: MitrnglngtrngDngquecyvng, bngthaotcvtrnglyvcymttsinhkhivikhunlnmitrng long trng, 37 /24h.Phnng (+): cvngtrongxungquanhkhunlcPhnng (-): khngcvngtrongxungquanhkhunlcc. Phnng nitrate.Chunb: Mitrng nitrate Dung dchthucthgiessA,giessBDngquecythng, bngthaotcvtrnglyvcymttsinhkhivikhunvongnghimmitrng nitrate, 37 /24h. sau 24h nh 2-3 gitgiessA,saunhtip 2-3 gitgiess B.Phnng (+): mitrngcmu cam Phnng (-): mitrngcmuvng.d. Phnng MRChunb: Mitrngclarklubs Thucth: MRDngquecyvngbngthaotcvtrnglyvcymttsinhkhi vi khunvoonngsnghim, 37 / 24h, saunh 2-3 git MRPhnng (+): mitrngcmu camPhnng (-): mitrngcmuvnge. Phnng VPChunb: Mitrngclurklubs Thucth: NaOH 40%, -naphtol 10%Dngquecyvngbngthaotcvtrnglyvcymttsinhkhivomitrngclurklubs, 37 /24h.nh 3-5 gitNaOH 40% v 3-5 naphtolPhnng (+): mitrngcmuPhnng (-): mitrngcmuvngf. Phnng citrateChunb: mitrngsimmon citrateDngquecyvnglyvcymttsinhkhivikhunvongmitrng citrate, 37 /24hPhnng (+): mitrngchuyn sang muxanhdngPhnng (-): mitrngcmuxanhlmnong. Phnngln men ng maltoseChunbmitrng maltoseDngquecyvng, lyvcymttsinhkhi vi khunvomitrng maltose, 37 /24hPhnng (+): mitrngcmuvngPhnng (-): mitrngcmu

CcphnngsinhhaKtqu

Hottnhcatalase

Sinhindol

MR VPSdngcitrat

Khnitrate

Tanchygelatin

Ding

Phngiitinhbt

Arabinose Xylose SaccharoseManitol Glucose Lactose Maltose+

-

+

+

+

+

+

+

+

+

+

+

+

+

-

+

MR(-) VP(-) MR(+) VP(+)

Citrate(+) citrate (-)4. MtsnghincungdngcaBacillussubtilis

CcctnhcchcaBacillussubtilis

-Snsinhenzyme amylase, pectinase, protease, lipase, trypsin, urease, mannase,..- Snsinhccacidhuc:acidlactic,acidaceticlmgimpHngrut.

- SnsinhvitaminnhmB

- Cnhtranhvtrbmdnhvivisinhvtgybnh.

- Snxutcckhngsinhcchccvisinhvtgybnh

-Bacillussubtiliscncxemltcnhnkchthchmindchtrongiutrmtsbnh.Trongcngnghip

Bacillussubtiliscngdngrngritrongvicsnxutraenzyme vmtschthahcchocngnghipnhamylase, protease, inosine, ribosides, aminoacid,Trong,proteasecngdngrngritrongcngnghipchttyra.Trongyhc

VictraoigengianhngchngthucBacillussubtiliskhichngcngphttrintrongtcbitnkhlu.Kliervcngschngminh khnngtraoiplasmidgiaBacillussubtilisvBacillusthuringensis. MtsnghincuchrarngBacillus subtilischuyngenckhnngkchnghaylmcchkhnngbiuhincthay1sthnhphncttrongccvikhuntrongccbnh hog,bchhu,vimphi,iunycnghartlniviyhc.Tuynhin, cngcntnti1vnlBacillussubtilishontoncthnhnccgenquynhtnhctcc vikhunccquanhtngignvmtditruynvgyhi.

Trongnngnghip

BacillussubtilisngdngphngtrvisinhvtgybnhnhnmRhizoctoniasolani,Fusariumsp.,Pyliculariaoryzae,ngoiracnngdngnhiutrongcngtcbovnngsnsauthuhoch.Bacillus subtiliscngdngvosnxutccchphmnhmlmgimtiphtbnhtiuchygyratrnheosoviutrbngkhngsinh.

B. Bacillus thuringiensis1. .Gii thiuBacillus thuringiensis (vit tt Bt ) l vi khun Gram dng , l vi khun t in hnh,Bt c kh nng tng hp protein gy t lit u trng ca mt s loi cn trng gy hi nn thng c s dng nh mt loi thuc tr su sinh hc ; cc c t Cry c th c trch xut v s dng lm thuc tr su. 2. Hnh thiBacillus thuringiensis l thnh vin ca nhm I, chi Bacillus. y l loi vi khun sinh bo t, Gram ng, h hp hiu kh hoc hiu kh khng bt buc. T bo c kch thc 3x6um, c ph tim mao khng dy, c kh nng chuyn ng, t bo ng ring r hoc xp thnh chui.L mt loi thuc chi Bacilus nn khi gp iu kin khng thun li nh thiu dinh dng, nhit cao, kh hn, Bt c kh nng sinh ni bo t gip cho chng chng chu vi iu kin khc nghit . Bo t ca vi khun c dng hnh trng vi kch thc 1,5x2 um v c th ny mm thnh t bo sinh dng khi c iu kin thun li.S hnh thnh bo t din ra ng thi vi s to thnh protein tinh th c kh nng dit cn trng. Tinh th c nhiu hnh dng khc nhau ( hnh vung, ch nht, hnh thp, hnh ovan hoc v nh hnh). Khi t bo phn gii tinh th v bo t c gii phng ra ngoi. Tinh th c kch thc khong 0,6x0,2um v c th chim ti 30% trng lng kh ca t bo. Tinh th ny c gi l th vi. Tinh th v bo t c th quan st di knh hin vi di pha vt knh u. tinh th bt mu sm cn bo t khng bt mu nhng c mp sng. Hnh dng ca tinh th c quyt nh bi gen m ha cho tng hp protein ca tinh th nm trn plasmid.3. t tnhB. thuringiensissn xut hai loi c t.Cc loi chnh lCry(tinh th), nhim c, m ha bng cch khc nhaugen gc, v y l cch loi khc nhau ca Bt c phn loi.Loi th hai l nhngCyt(tiu t bo), nhim c, c th lm tng thm cc c t Cry, nng cao hiu qu ca kim sot cn trng.Hn 50 ca cc gen m ha c t Cry hin nay c gii trnh t v cho php cc c t c giao cho hn 15 nhm.Bng di y cho thy trng thi ca mt phn loi vo nm 1995:GenTinh th hnhKch thc protein (kDa)Hot ng cn trng

nhiu phn nhm:A (a), A (b), A (c), B, C, D, E, F, G]bipyramidal130-138u trng Lepidoptera

khc II [nhm A, B, C]cuboidal69-71Lepidoptera v Diptera

khc III [nhm A, B, C]phng / bt thng73-74Coleoptera

khc IV [nhm A, B, C, D]bipyramidal73-134Diptera

khc V-IXkhc nhau35-129khc nhau

4. Cc bnh v triu chng do vi sinh vt gy raNhng nghin cu t nn mng trong lch s pht trin Bt l s pht hin ngu nhin ra loi Bacillus gy hi u trng tm. Trong vng 100 nm qua, nhng nghin cu v Bt lin tc pht trin. Cc ch phm Bt c sn xut v s dng rng ri khp cc chu lc (chu u, , M, Phi). Nhiu nc trn th gii xy dng v nghin cu tp on Bt nhm tm hiu vai tr ca Bt trong mi trng cng nh s phn b gen Cry v thu nhn nhng chng Bt c hot lc dit cn trng cao a vo ng dng.Vit Nam nm trong khu vc nhit i gi ma, thin nhin phong ph v a dng. Cc vng trong lnh th Vit Nam c s khc bit kh m nt v iu kin kh hu, v tr a l, to nn s a dng v chng loi ng vt, thc vt v vi sinh vt.Ngi ta cho rng vng t no nhiu cn trng th cng xut hin nhiu Bt dit cn trng. iu kin kh hu ca nc ta thch hp cho nhiu loi cn trng sinh trng v pht trin v vy cng ha hn c mt tp on Bt phong ph v a dng v chng loi cng nh gen Cry. Khun Bt trng nh chic gy gc, di cha y 5/1000mm. Khi n ln n mt mc nht nh, mt u s hnh thnh mt nha bo hnh trng sinh sn, u kia sinh ra mt th kt tinh khi thoi hoc gn khi vung. V cng sinh ra ng thi vi nha bo, ngi ta gi l tinh th bn, rt c. Khi su hi ph hoi ma mng n phi khun Bt, tinh th bn s ph hng h thng tiu ha ca su, khin chng ngng n, nn ma, a chy; cn nha bo vo mu s gy bnh thiu mu cho su, cui cng su cht.Pht hin khun Bt m ra mt trin vng sng sa ca bin php tr su sinh hc. Hin nay ngi ta sn xut khun Bt quy m ln, ri ch bin thnh cc loi thuc nh Thuricide, Biotrol, Bathurin... c tc dng dit hng trm ging su hi.Ngy nay cc ch phm sinh hc an ton cho phng tr su hi cy trng v nng sn bo qun c khuyn khch, ng dng thay th dn cc thuc tr su ho hc, trong thuc tr su vi sinh Bacillus thuringiensis (gi tt l Bt) c dng rng ri nht. Trong nhng nm gn y cc nh khoa hc c rt nhiu c gng phn lp vi khun ny t mi trng ca nhiu nc trn th gii vi hy vng tm ra nhng chng B. thuringiensis c ph gy bnh mi v khong vt ch rng hoc nng cao hot tnh cc chng B.thuringiensis i vi cc nhm cn trng mi, hay tm kim ngun gen t nhng chng phn lp cho cc k thut di truyn tip theo. Vi s pht trin mnh m ca cng ngh sinh hc hin i, c bit l cng ngh gen m ra nhng tim nng ht sc to ln cho vic pht trin thuc tr su sinh hc B. thuringiensis. Vic lm giu thm b su tp cc chng B. thuringiensis phn lp Vit Nam v ngun gen qu t nhng chng ny nhm to ra cc chng ti t hp mi c ph dit su rng hn i vi mt s loi cn trng quan trng l rt cn thit. th nghim hot tnh dit su ca 10 chng B. thuringiensis var. kurstaki phn lp c tinh th hnh qu trm, trong 9/10 chng c hot tnh dit 90 - 100% i vi su t P. xylostella v su keo S. exiqua; 7/10 chng c hiu qu dit 80-90% i vi su khoang S. litura; 6/10 chng c hot tnh dit 50-60% i vi su bng H. armigera. 10/10 chng c hiu qu dit 100% i vi su ngi go C. cephalonica.

5. Phn lpv nhn dng Bacillus thurigiensisCho n nay cc phng php phn loi Bacillus thuringiensis kh ph bin l da trn cc c tnh huyt thanh hc. c tnh huyt thanh hc ca chng Bacillus thuringiensis c xc nh ch yu da trn khng nguyn tin mao H v c tin hnh bng phn ng ngng kt cc t bo sinh dng vi cc khng huyt thanh tng ng. Tuy nhin vic phn loi ch da trn huyt thanh vn cha phn nh c mi lin h gia chng ging v hot lc dit cn trng. Chnh v vy gn y cc nh khoa hc ngh a ra phng php phn loi mi da trn kt qu xc nh huyt thanh, hnh dng tinh th, hot lc dit cn trng v thnh phn protein tinh th. nhn dng Bacillus thuringiensis cn tin hnh cc th nghim sau:5.1 Cc phn ng c trng Mi trng Methyl red(+): Bacillus thuringiensis chuyn ha glucose thnh acid pyruvic, ri tip tc chuyn ha acid pyruvic thnh ethanol, acid lactic, acid succinic. Cc acid to ra lm pH mi trng gim mnh, pH= 4- 4,5 methyl red mu , ngc li pH cao hn th methyl red s chuyn sang mu vng. Tin hnh: cy khun lc nghi ng vo mi trng Clark- Lubs pH=7. 370C trong thi gian 48 gi. Sau , nh thm 5- 10 git methyl red, quan st v c kt qu Phn ng methyl red dng tnh: dung dch methyl red trong mi trng vn gi nguyn mu . Phn ng methyl red m tnh: dung dch methyl red trong mi trng chuyn sang vng. Phn ng VP(+): Bacillus thuringiensis chuyn ha glucose thnh acid pyruvic ri tip tc chuyn ha acid pyruvic thnh acetyl methyl carbinol( ACM). ACM tc dng vi - naphtol trong mi trng kim to thnh diacetyl, diacetyl phn ng vi nhn guanidine c trong pepton cho hp cht mu hng . Tin hnh: cy khun lc vo mi trng Clark- Lubs pH=7. 370C trong 24 gi. Sau thi gian ny, nh 10 git NaOH 40%, h nng nh ri nh tip 10-15 git - naphtol, lc u 5- 10 pht. Kt qu: phn ng VP dng tnh khi mi trng chuyn mu cam. m tnh khi mi trng c mu vng hoc mu nu t. Kh nitrat(+): Bacillus thuringiensis s dng nitrat lm ngun nhn hydro c cht trong qu trnh h hp k kh ng thi s dng nitrat lm ngun nito do c kh nng tng hp enzyme nitratreductase. Nitrat bi kh thnh nitrit v ri c th b kh tip thnh NH3 hoc N2. Tin hnh: cy khun lc vo mi trng nitrat, 370C trong 24 gi. Tip theo ly ra nh vo git thuc th Gress A( acid sulfanilic) v vi git Gress B( - naphthylamin) Kt qu: mi trng c mu hng l dng tnh: trong mi trng xut hin nhm NO2 do nhm NO3- chuyn ha. Mi trng khng chuyn mu l m tnh, trong mi trng khng c nhm NO2-. Nh vy c 2 kh nng:+ Trng hp 1(-) : NO3- khng chuyn thnh NO2-+ Trng hp 2( +): NO3- chuyn thnh NO2-, sau nitrit tp tc chuyn thnh NH3 hay N2 nn thuc th Gress A v Gress B khng pht hin c. Tip tc cho bt km vo nu xut hin mu th m tnh, nu khng i mu l dng tnh.5.2 Th nghim tnh di ng Mc ch ca mi trng th nghim tnh di ng l xc nh mt vi khun c th di ng c hay khng. Cc vi khun khng di ng l do khng c lng roi, cc vi khun di ng s dng lng roi di chuyn ra ngoi ng cy v xut hin m khi trong mi trng. Mi trng th nghim: Mi trng Nutrient agar: thch dinh dng c cha cc cht dinh dng ng vt v c bit l hm lng protein thp. Chng phn lp pht trin c trn mi trng ny th cng c ngha l chng rt d moc v khng i hi bt c thnh phn c bit no. Mi trng th tnh di ng: thng l mi trng bn rn vi hm lng agar 0,5% khng cng, khng lng pht hin tnh di ng tt nht Tin hnh: Dng que cy vng cy dch 24h nui cy vo gia mi trng th nghim. 300C t 18- 24h v kim tra di nh n kiu mc dc theo ng cy. Loi di ng moc khuch tn vo mi trng theo hng xa ng cy. Loi khng di ng ch mc trong v dc theo ng cy. 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 t 6-8h 300C Nh nc v trng ln phin knh v t sinh khi vo. Quan st ngay di knh hin vi kim tra s di ng. Chng Bacillus thuriniensis l di ng.5.3 Th nghim lm tan mu Mc ch:th nghim kh nng cng hng tan huyt gia cc vi sinh vt. C s: c 3 loi tan huyt+ Tan huyt hon ton ( ): vng tan huyt khng r+ Tan huyt khng hon ton () :xung quanh v di khun lc chuyn c m c mu khc.+ Khng tan huyt.CAMP+ lysin gy tan huyt mnh Mi trng th nghim: thch mu Tryticase Soy Polymycin Tin hnh: cy chng nghi ng l Bacillus thuringiensis ln mi trng thch mu Trypticase soy polymycin( TSP), 350C trong 24h. Bacillus thuringiensis lm tan mu () 2-4 mm xung quanh vng pht trin.5.4 S to c t protein dng tinh th Mi trng th nghim: Nutrient agar - thch dinh dng c cha cc cht dinh dng ng vt v c bit l hm lng protein thp. Chng phn lp pht trin c trn mi trng ny th cng c ngha l chng rt d moc v khng i hi bt c thnh phn c bit no. Tin hnh: Cy huyn ph t bo 24h ln ng thch nghing Nutrient agar, 24h 300C sau nhit phng 2-3 ngy. Thc hin nhum bng mu fuchsin v quan st di knh hin vi nhng tinh th c t hnh t gic( dng kim cng) c nhum mu ti nh hn bo t. Tinh th c t ca B. thuringiensis xut hin nhiu sau 3- 4 ngy nui cy nhng khng th pht hin c bng k thut nhum cho n khi bo t nang v ra. Do nu khng quan st c bng bo t t do th cn thm vi ngy nhit phng ri tin hnh kim tra li.6. Bacillus thuringiensis trong ngnh thc phm6.1 Vai tr- Bacillus thuringiensis to ra cht c t c th dit u trng ca mui v rui rt hiu nghim sau khi chng n phi vo c th. - Vi khun ny thch hp x l nc n v ti tiu, c tc dng dit cc loi u trng khng ha cht. N c tc dng phn tn nhanh trong mi trng v c ti s dng theo nh k.- Bacillus thuringiensisthng c xem l mt sn phm sinh hc, ch yu l vi khun cht, bo t sng v cc tinh th c trong cc bo t khng sinh sn, v vy sn phm ny c tc dng nh l mt cht dit cn trng sn xut bng phng php sinh hc.6.2 C ch tc ng ca Bacillus thuringiensis- Bacillus thuringiensisc tinh th protein c cao giai on bo t.- Khi b nhim ch phm Bt, c th su b t lit v cht sau 2-4 ngy6.3 Quy trnh sn xut ch phm Bacillus thuringiensis( Bt)Bt c sn xut ch yu theo 2 cch ln men thng( ln men b mt) v ln men chm c sc kh. Hin nay ngi ta thng s dng phng php ln men chm v mang li hiu qu cao hn.

Chng Bt thun khit

Nhn ging cp 1Nhn ging cp 2Thi gian ln men 48-72h, pH=7 300CLn menLc v ly tmThu sinh khiHon thin sn phm

Ln men chm tin hnh trong cc ni ln men, ngoi mi trng dinh dng cn ch ti mt s thng s khc: ch thi kh, ch nhit , ch lun chuyn ging hn ch cc thc khun th ph hy cc bo t v tinh ch c. Ch thi kh: l ch tiu quan trng trong qu trnh hnh thnh bo t v tinh ch c. ngng thi kh tt nht trong qu trnh ln men l 0,5- 0,6 m3 mi trng/ m3 khng kh. Ch thi kh thp bo t pht trin yu, mt tha. Ch thi kh cao bo t pht trin nhanh, thi gian ln men ngn, tinh th c nh hiu qu dit su khng cao. Nhit : nh hng n qu trnh hnh thnh bo t, nhit qu cao hoc qu thp s rt ngn hoc ko di qu trnh ln men, nhit ph hp l 300C. Ch lun chuyn ging: nu s dng ging lien tc s xy ra hin tng nhim thc khun th. Bnh thng ch ln men 10- 15 ln ging c th phi thay ging mi khc phc hin tng phn t, hin tng to ra t bo t v t tinh th c t. Chn chng ln men: ty theo vic phng tr loi su no m nh sn xut chn chng vi khun ph hp ln men. Chn mi trng ln men: trn c s mi trng c bn, ty thuc chng vi khun cn ln men m thm cc cht ph hpSau khi ln men ngi ta lc v ly tm dch ln men thu sinh khiBc hon thin sn phm: sn phm c ng gi v ch thnh cc dng ch phm khc nhau. Sng ch phm cng l yu t quan trng quyt nh hiu qu phng tr su bnh. Ch phm Bt c dng nc, bt, bt thm nc, dng keo.7. Kt lunNgi ta ng dng s to c t dng tinh th ca Bacillus thuringiensis vo vic tiu dit su bnh gy bnh trn cy trng ( c chua, khoai ty, cy n tri, hoa mu) m khng gy nhim mi trng, khng git cht thin ch su hi v sinh vt c ch. C th cn bng sinh thi, khng nh hng n cht lng nng sn.Bacillus thuringiensis khng gy c hi cho ngi v ng vt bc cao, khng c hiu lc tip xc v xng hi.C th s dng phng php truyn thng nh m khun lc, MNP nh lng Bacillus thuringiensis vi u im l khng phi u t dng c v thit b t tin

C. Bacillus pumilis

1. Gii thiuBacillus pumilus l mt loi vi khun c bo t hnh que, l vi khun Gram dng, hiu kh, thuc chi cc sinh vt i din rng ri nht trong t. Do kh nng d to thnh bo t vchu c mt lot cc iu kin mi trng thay i, vi khun Bacillus spp. thch nghi d dng vi a dngmi trng sng. Sinh l a dng ca vi khun Bacillus spp. yu cu xt nghim sinh ha phc tp nhn dng. Nhng tin b trong phn tch sc k ca ton b t bo bo methyl axiteste (FAME) h s thc hin k thut ny nhy cm v ng tin cy cho cc nhmBacillus cp loi (31). Hn na, acid nucleic k thut xy dng nh 16S rDNA (3,34)v gyrase B (gyrB) phn tch trnh t c chng minh l c gi tr to ln cho pht sinh loi phn tch vi khun . Da trn 16S rDNA phn tch trnh t, 5 nhm xc nh trong chi Bacillus, trong nhm 1 (B. subitilis nhm) bao gm B.amyloliquefaciens, B. subtilis v B. pumilus .cu trc b gen B. pumilus c mt nhim sc th trn v c chiu di khc nhau 3,7-3,8 Mbp. Chng SAFR-031 v ATCC 7061 l 3.704.465 v 3.833.998 cp base tng ng. Khong 41% s cp base DNA l GC. Nhim sc th trn c cha khong 4.000 gen v protein 3600-3900. Nhn rng ADN vng l phng php c s dng bi hu ht cc B. pumilus plasmid. ng ho ca plasmid l hu ch v c s dng trong cc qu trnh chuyn gen. Cu trc t bo, chuyn ha v vng i, kh nng di ng ca B. pumilus ging vi cc loi vi khun khc trong chi Bacillus bao gm B. subtilis, B. megaterium, v B. cereus. N l mt vi khun Gram dng, hnh que, bo t vi khun c tm thy trong t, nc, v nhiu mi trng sng khc. Ging nh hu ht cc vi khun Gram dng, axit teichoic v lipoteichoic bao gm lp ngoi cng ca cc peptidoglycan qua cc lin kt. N ng vai tr trong s bm dnh vo trong cc t bo v cc b mt khc c tm thy trong mi trng, cng nh l cc khng nguyn b mt ph hp. Cc axit c to thnh t pht pht polyglycosyl (tc l, glycerol-P hoc ribitol-P) vi mono v disaccharides trong cc n v lp i lp li. Cc pht pht trong chui axit teichoic cho b mt t bo mt in tch m tng th, cho php cho s hp thu hiu qu ca cc cation khc nhau nh Ca2 + v Mg2 + vo trong t bo. Kh nng bit ha thnh mt endospore lm cho B. pumilus cao kh nng sng trong mi trng ngho dinh dng, H2O2, ha cht kh trng, v cc iu kin khc nghit khc .Cng ging nh cc thnh phn t bo, B. pumilus vng i tng t nh trc khun khc. Gi nh bt k ca ba giai on sng: bc bo t, t bo thc vt, hoc bo t t do. Khi iu kin khng thun li cho s tng trng, B. pumilus s tri qua hnh thnh bo t v pht mt bo t vo mi trng. Giai on bc bo t l gia tnh trng thc vt v bo t t do, trong mt bo t cha trng thnh c lng bn trong t bo c th t hnh thnh bo t hn na nu cc iu kin ph hp. Trong s hnh thnh ca t bo thc vt t mt bo t t do, cc bo t ny mm s thng tri qua khi cc iu kin ph hp vi n. Mt thnh phn quan trng cho s ny mm v khi phc tc tng trng ph hp vi thi gian ny mm l s thay i nhit hoc lng cht dinh dng.Mt s xt nghim sinh ha c tm thy trong cc ch s phn tch h s c nhn (API) c s dng trong vic phn loi. B. pumilus l amylase, lipase, protease v protease dng. N c mt lot cc c ch kh nitrat, sn xut kh t t ng, v sn xut axit t nhiu ngun khc nhau carbon, c th l arabinose, mannitol, xylose, glucose v lactose. ng ch , B. pumilus c th to ra acetylbutanediol (ABD) t acetoin. ABD l mt phn ca mt lp hc ca diol rt quan trng trong qu trnh tng hp vi khun c nh hng ca polymer khc nhau v cng c kh nng s dng nh mt thay th cho tiu chun nguyn liu nhin liu ha thch.2. Sinh thi hoc ca Bacillus pumilusBacillus pumilus tham gia vo mt lot cc mi quan h cng sinh. B. pumilus c th hot ng nh mt cy pht trin thc y rhizobacteria trong vng r ca cy nng nghip quan trng nh mu t (Capsicum annuum L.) v la m (Triticum aestivum) . Trong la m, B. pumilus cng khin sc khng cy vi tt c (Gaeumannomyces graminis), bnh nm c th gy thit hi ng k cy la m . Ngoi ra, B. pumilus c cho l c chc nng nh mt thc y tng trng thc vt endophyte trong cy nho Vitis vinifera . Tm s, c th lu tr Bacillus pumilus trong rut, ni n c ch s nhim trng do vi khun Vibrio harveyi, V. alginolyticus, v V. parahaemolyticus, tt c u c bit n l vi khun ca tc nhn gy bnh nghim trng ca tm .B. pumilus l quan trng sinh ha h sinh thi v n c chc nng nh mt vi khun c nh nit c kh nng trao i cht nit phn t chuyn (N2) vo amoniac (NH3) .Nhim trng ngi do vi khun Bacillus pumilus l rt him, tuy nhin trong nm 2006 B. pumilus trong go c tm thy c trch nhim vi 3 trng hp ng c thc phm. S cng thng c lp sn xut mt phc tp ca lipopeptides gi pumilacidins, c tc dng c hi trn t bo biu m. Cc triu chng do nhim trng bao l chng mt, au u, n lnh, au lng, au bng, tiu chy . Hn na, trong nm 2007, mt bo co tng kt 3 nghin cu c cng b kt lun rng mt chng vi khun Bacillus pumilus c nh hng cho s pht trin ca cc tn thng da c triu chng tng t nh nhng ngi b gy ra bi vi khun Bacillus anthracis .Bacillus pumilus tn tai trong sinh phm trang thai bao t, nh vy khi ung vao da day no khng bi axit cung nh cac men tiu hoa dich vi pha huy . rut, bao t nay mm va phat trin thanh th hoat ng. Giai oan nay, B. pumilus tng hp nhiu cht co hoat tinh sinh hoc co li cho c th nh cac enzym thuy phn, cac vitamin, axit amin Mt s enzym nh protease, -amylase va mt s enzym khac hoat ng manh co li cho tiu hoa rut, chung co vai tro: Lam cho pH rut n inh, han ch s phat trin cua vi khun sinh hi va vi khun gy bnh Cung cp ngay cho c th mt s men cn thit, lam cho tiu hoa tr lai binh thng trong khi h vi khun rut cha lp lai trang thai cn bng. thanh bao t B. pumilus co enzyme ging nh lysozyme co kha nng dung giai trc tip mt s vi khun gy bnh nh: Proteus, Staphylococus, E.coli.Cac cht khang sinh do B. pumilus tit ra co tac dung c ch s phat trin va tiu dit mt s loai vi khun gy bnh tao iu kin cho cac vi khun chi binh thng rut phat trin tai lp lai trang thai cn bng. trong rut, cac cht sinh hoc nay khng chi c giai phong khi B. pumilus con sng ma ngay ca khi chung a cht, xac vi khun vn tip tuc giai phong ra cac enzym, khang sinh, vitamin co li cho c th.Co nhiu nghin cu cho thy rng khng nht thit la probiotic sng mi co li cho ai trang vi tac dung nay xay ra gian tip qua chui AND kich thich min dich khng methyl hoa. Vi khun probiotic co th c ch vim ai trang thng qua cac thanh phn cu truc c nhn din bng thu th c hiu cua min dich bm sinh nh tng t thu th Toll. B. pumilus khng nhn ln trong rut, chung chi chuyn t dang bao t sang dang hoat ng ri bi ao thai hoan toan. Nh vy kha nng canh tranh cua B. pumilus i vi vi khun gy bnh khng chi s lng t bao vi khun ma con nh vao cac hoat cht sinh hoc ma no tit ra. Trong nhng nm gn y, khoa vi sinh vt cua bnh vin Bach Mai a anh du hn dich B. pumilus bng ng vi phong xa trong iu tri bnh ng rut va kt lun: Hn dich B. pumilus sau khi vao c th chut lang, c tp trung rt nhiu trong rut gia, lu gi tai o mt lng ang k cho n ngay th ba va khng i vao mau. Nh vy iu tri cac bnh ng rut, khc phuc tinh trang ri loan tiu hoa sau khi dung khang sinh lu dai thi B. pumilus la mt vu khi li hai, re tin, d s dung, co th ap dung rng rai trong iu tri. Cac ch phm tr sinh t B. pumilus khi i vao c th co tac dung theo bn c ch chu yu: Trung hoa c t. Canh tranh vi mm gy bnh Thay i chuyn hoa cua vi sinh vt Kich thich min dich cua c th vt chu.Nh cc c ch tc dng ny B. pumilus mang cc c tnh tr liu v hin nay c s dung rng ri lam ch phm sinh hoc di dang men tiu hoa sng.

3. Phn lp v nhn dng.

Bacillus pumilus c phn lp thnh rt nhiu chng: B. pumilus (AMCTSA36b) B. pumilus (AMCAC12) B. pumilus (MAIIIM4a) B. pumilus 51-3c (AF526907) B. pumilus SA 175001 (AY289549) B. pumilus ARBG2 (AY496869S2) B. pumilus (DQ011675) B. pumilus (AB098578) B. pumilus (Ay112667) B. pumilusAY496869 B. pumilusAY167886 B. pumilus NM1C3 (EU221329) B. pumilus M1-9-1(AB048252) B. pumilus S9 (AY548955) B. pumilus B07032 (FM205013) B. pumilus SAFS-032 (CP000813) B. pumilusUW-02 B. pumilus dcs r1 B. pumilus:301B-1 B. pumilusQST 2808..............

Phn lp v nhn dng mt s chng B. pumilus:B. pumilus c th c phn lp t nhiu loi t, thc vt, b mt mi trng v thm ch c t bn trong sa mc Sonoran bazan. C s tng ng gia B. pumilus chng AMCTSA36b v AMCAC12. Bacillus pumilus (MAIIIM4a) AM c phn lp t cy sn. B. pumilus c phn lp t mi trng ven bin Cochin, n . Nhn dng bnh t chi do VK B. pumilus.

3.1 Phn lp B. pumilus MAIIIM4a t cy sn.

Phn lp B. pumilus MAIIIM4a hot ng c ch mnh m chng li cc loi nm Rhizoctonia solani, Pythium aphanidermatum v Sclerotium rolfsii. Cht chuyn ha trung gian ca dng ny c trch ly bng hexane, dichloromethane v ethyl acetate. Chit xut phi chu bioautography v phn tch LC/MS, cho php xc nh cc pumilacidin, mt hp cht khng nm sn xut bi B. pumilus MAIIIM4a.

3.2 Xc nh vi khun bng cch sp xp cc on gen 16S rRNA

Mt phn trnh t 16S rRNA c khuch i t DNA ca B. pumilus MAIIIM4a bng cch s dng mi F968 (5'-AACGCGAAGAACCTT AC-3 ') (Nbel et al., 1996) v R1387 (5'-CGGTGTGTACAAGGCCCGGGAACG-3') (Heuer et al., 1997). Khuch i c thc hin trong mt h thng Amp PCR gen 2400 (Applied Biosystems, Foster City, California, Hoa K) s dng 50 hn hp PCR ml cha 10 ng DNA, 0.2 M mi mi oligonucleotide, 200 M dNTP, 3,75 mM MgCl2, m 1 PCR v 5,0 U polymerase Taq DNA (Invitrogen, Carlsbad, California, Hoa K). Khuych i PCR sau c phn tch bng in s dng 1% gel agarose c nhum vi ethidium bromide. Gel c xem trn mt h thng Alpha DigiDoc h thng ti liu gel (Alpha Innotech, San Leandro, California, Hoa K) v c ghi li.3.3 Th nghim:

Cc cy sn non cha vi sinh vt axenic t do thu c bng cch nui cy m. Ba bnh cha 400 ml dung dch Murashige & Skoog (Murashige & Skoog, 1962) v ba bnh vi nc ct tit trng c chun b. Cy sn c chuyn qua cc bnh v c tim chng 108 CFU ml-1 ca B. pumilus MAIIIM4a. Mu cha cy sn khng tim c kim sot. Sau 5 ngy cc cy sn c tch thnh thn, r v l cy v mi m c ct thnh on khong 2 cm2. Mi phn m sn c c nh trong mt gii php Karnovksy ciu chnh (Kitajima & Leite, 1999) v cc mu c gi mc 8C qua m. Cc mu c ra 3 ln vi m cacodylate (0,05M) trong 10 pht v c ngm trong 1% OsO4 (trong 0,05 M m cacodylate, pH 7.2) trong 1 gi. Cc mnh sn c ra nm ln, 15 pht i nc ct mt ln. Cc mu b loi nc trong mt dy ethanol (30, 50, 70, 90 v 100%), c 15 pht thay i mt tng nng ethanol mt ln. Lp i lp li ba ln. Cui cng, nguyn liu sn t c im kh, my bn mc vng phun ln b mt v kim tra bng cch s dng mt trng pht x qut knh hin vi in t (SEM) (LEO Hp tc Zeiss / Leica, Oberkochem, c). Tt c cc th nghim c thc hin trong ba ln.

Theo sng lc khng nm, cho thy tnh khng nm mnh m ca vi khun Bacillus pumilus MAIIIM4a nn n c la chn nghin cu su hn, bao gm c cc c tnh ca cc cht chuyn ha ca n. Do dng ny c nui trong cht lng PD (khoai ty-dextrose) trung bnh v trong mt shaker (150 rpm) 28C trong 72 gi. Vo cui giai on ny, chng c ly tm 10.000g trong 5 pht. v 500 mL ni c s dng chit vi hexane (200 ml), diclometan (200 ml) v ethyl acetate (200 ml). Cc giai on hu c tng ng c thu thp v lm kh vi natri sunfat v bc hi, sau cc cht chit xut du th t c: 7 mg t s khai thc hexane, 8 mg t vic khai thc dichloromethane v 8 mg chit xut ethyl acetate.

Qut phn tch hin vi in t cho thy B. pumilus MAIIIM4a c li nhim cc m l, thn v r cy sn. Tuy nhin, s phn b ca loi ny l ty theo mi trng sng. Qun th B. pumilus MAIIIM4a trong thn cy v r sn c th hin trong hnh 3.

Phn ln cc t bo ca B. pumilus MAIIIM4a xm chim cc ni bo v gian bo ca cc m sn. Hot ng khng nm (t l phn trm ca s c ch chng li P. aphanidermatum) ca B. pumilus MAIIIM4a cao: 54%. Phn tch TLC s b ca B. pumilus MAIIIM4a ni tch ra thnh ethyl acetate 10 phn thu c, trong khi khai thc hexane ch c hai s thp phn (Bng 3). Cc bioautography c thc hin ch dnh cho cc chit xut ethyl acetate. Khu c ch r rng ti Rf l 0,62, 0,56 v 0,42 cho B. pumilus MAIIIM4a chit xut c quan st i vi P. aphanidermatum, cho thy rng cc hp cht chu trch nhim v hot tnh khng nm mnh m l apolar.

D liu o ph khi lng chng thc cho vic xc nh cc hp cht hot tnh sinh hc nh mt mt lipopeptide, pumilacidin, mt c tnh ca B. pumilus. y l ln u tin m mt endophytic B. pumilus c m t nh l nh sn xut pumilacidin.

3.4 Bacillus pumilus phn lp t mi trng ven bin Cochin, n 3.4.1 c tnh sinh ha v phn t ca vi khun Bacillus pumilus phn lp t mi trng ven bin Cochin, n .Trong nghin cu ny, cc chng vi khun Bacillus phn lp t mi trng ven bin Cochin, n c xc nh bng cc phng php ha sinh thng thng, phn tch acid bo methyl ester (FAME) v 16S rDNA mt phn trnh t. Phn tch d liu cho thy rng vi khun Bacillus pumilus l loi chim u th nht trong khu vc c nghin cu tip theo B. cereus v B. sphaericus. B. pumilus phn lp c c trng hn na bng cch c quyn mi nc PCR (AP-PCR), khng sinh bin hnh v mng PCR cho bit gen c t c lin kt vi vi khun Bacillus spp. Tt c phn lp B. pumilus c lm sinh ha ging nhau, protease v lipase hot ng cao v nhy thng nht vi khng sinh c th nghim trong nghin cu ny. Mt chng B. pumilus nui dng trong gen cereulide synthetase cesB ca B. cereus m khng th phn bit t phn tch ra ca cc chng sinh ha bi AP-PCR. Bo co nghin cu ny, ln u tin, s hin din ca gen c gy nn cesB trong B. pumilus.

Cc vi khun Gram dng, hiu kh, vi khun hnh que endospore ca vi khun ging Bacillus l nhng sinh vt i din rng ri nht trong t. Do kh nng ca chng to thnh bo t v chu c mt lot cc iu kin mi trng thay i, vi khun Bacillus spp thch nghi d dng vi mi trng sng khc nhau. Sinh l a dng ca vi khun Bacillus spp yu cu xt nghim sinh ha phc tp cho s nhn dng ca chng. Nhng li ch trong phn tch sc k ton b mt nghing t bo acid bo methyl ester (FAME) lm cho k thut ny nhy cm v ng tin cy cho cc nhm vi khun Bacillus cp loi. Hn na, k thut da trn axit nucleic nh 16S rDNA v gyrase B (gyrB) phn tch trnh t c chng minh l c gi tr to ln cho pht sinh loi phn tch vi khun. Da trn 16S rDNA phn tch trnh t, 5 nhm c xc nh trong loi Bacillus, trong nhm 1 (nhm B. subitilis) bao gm B. amyloliquefaciens, B. subtilis v B. pumilus.

Mt s loi vi khun Bacillus sng mi trng bin v ven bin, mc d rt kh phn loi chng ng nh vi nhng mi trng sng bn a. Cng vi B. cereus v B. subtilis, B. pumilus c coi l mt thnh phn chnh ca cng ng vi khun bin. Gn y, B. pumilus cng c bo co l loi vi khun Bacillus ch yu th hai trong tu v tr. Vi khun ny c bn cao vi iu kin mi trng khc nghit nh cn rt t hoc khng cn cht dinh dng, kh, chiu x, H2O2 v cc cht ty ha hc. Vai tr sinh thi ca B. pumilus c nhn mnh bi thc t l chng sn xut cc hp cht i khng vi cc mm bnh nm v vi khun. Do , B. pumilus l mi quan tm nghin cu ng k hiu s a dng sinh l ca n, quan h di truyn vi vi khun Bacillus spp khc. v s hin din ca cc yu t c th sinh c t. Trong bo co nghin cu ny, chng ti m t i) vi khun Bacillus spp b c lp v xc nh t cc mu mi trng bng cc phng php thng thng, FAME v trnh t rDNA 16S ii) tip tc kiu hnh v c tnh di truyn ca B. pumilus, nhm Bacillus ch yu ca vi khun vng ven bin ang c nghin cu.

3.4.2 S cch ly v c tnh sinh ha ca vi khun Bacillus.Nc bin, trm tch, thy sn c thu nhn t Cochin, b Ty bin ca n v x l phn lp cc loi vi khun Bacillus. Mu c, ng vt c v hoc trm tch c ng nht trong mui phosphate buffered (PBS 0,05 M, pH 7,2), pha long trong cng mt mi trng v ly lan cy trn mi trng thch dinh dng vi 50% nc bin. 100 vi lt mu nc bin c lan truyn trc tip m trn cng mt mi trng v 30oC trong 24-48 h. a thch c tinh sch v bo qun -80C trong mi trng dinh dng c cha 30% glycerol. nhn dng phn loi, phn lp phi chu mt lot cc xt nghim sinh ha, trong bao gm gim nitrat, tng trng k kh, sn xut kh t t ng, Voges-Proskauer (VP), tng trng nng NaCl khc nhau, nhit v phm vi pH, suy thoi tinh bt, casein, ur, gelatin, chitin, sn xut axit t arabinose, mannitol, xylose, glucose, lactose, s dng citrate v hot ng ca DNase. Sn xut cc enzym ngoi bo c th l caseinase, chitinase, protease, phosphatase kim, gelatinase v lipase c nghin cu theo Ngh nh th c m t bi Smibert v Krieg.Th nghim khng sinh nhy cm S c ch ca cc chng B. pumilus bng khng sinh khc nhau c th nghim theo tiu chun a khuch tn k thut. Cc vi khun cy c pht trin trong nc dinh dng qua m v m trn thch Muller Hinton (Hi-Media, Mumbai). Cc a khng sinh sau y vi nng ghi trong ngoc n c s dng: amoxicillin (25 mcg), penicillin (10 mg), ciprofloxacin (5 mcg), gentamycin (10 mg), cotrimaxazole (25 mcg), chloramphenicol (30 mcg), bacitracin (8 mg), tetracycline (30 mcg), kanamycin (30 mcg), erythromycin (15 mcg), vancomycin (30 mcg). c tnh ca B. pumilusMi su chng phn lp B. pumilus t cc ngun khc nhau (Bng 2) trng by cc thuc tnh kiu hnh thng nht (Bng 3). Th nghim sinh l cho thy c th nhn ra s sn xut ca protease v lipase nhng khng thy amylase, phosphatase, DNase, gelatinase v chitinase. Mt phn trnh t 16S rDNA ( 500 bp) ca B. pumilus xc nh trong nghin cu ny cho thy 99-100% tng ng vi B. pumilus trnh t 16S rDNA trong Ngn hng gen. Tnh nhy cm vi khng sinh ca B. pumilus l ng nht. Tt c 16 phn lp c u b c ch bi amoxicillin, ciprofloxacin, gentamycin, cotrimaxazole, chloramphenicol, bacitracin, tetracycline, kanamycin, erythromycin, vancomycin. Tt c cc chng biu hin tnh khng vi penicillin.

3.5 Kiu AP-PCR phn lpMi ngu nhin CRA 22, m lun mang li 4-10 di bng vi B. pumilus, c la chn g cc dng phn lp ca AP-PCR. Vic phn tch du vn tay AP-PCR cho thy s khng ng nht gia cc B. pumilus c lp vi 10 mu khc nhau (Hnh 1). Mc d tng th di truyn c s a dng ny, s gn ng nht thu c gia cc chng NIOB 485 v NIOB 525 (t cua), chng NIOB111 (c) v NIOB 426 (t trm tch), chng NIOB096 (t cua) v NIOB169 (t c), chng NIOB163 (t c) v NIOB431 (t sao bin).3.5.1 Pht hin gen synthetase cereulide trong B. pumilus bng phng php PCRMt c lp B. pumilus NIOB 133 phn lp t mt c ca sng mang li 635 bp amplicon vi cesB nhm mc tiu mi EM1-f v EM1-r (Hnh 2). Cc sn phm ca cesB PCR c gii trnh t v phn tch trnh t nucleotide ca sn phm PCR cho thy 96% tng ng vi trnh t tng ng ca cesB ca B. cereus (GenBank nhp khng DQ889676), trong khi cc trnh t amino acid cho thy 92% tng ng vi mt vi bt xng hp amino acid (Hnh 3). Dng ny l tiu cc bi CESA PCR s dng mi CER1 v EMT1.

Nhn dng bnh t chi do VK B. pumilus: Chi b bin dng, co cm, mu nu v cc phn on trn l cnh chm hoa u ngn v nh li, nhn xa dng cy chi. Cc hoa b bnh s km pht trin v kh nng u tri rt km. Cnh b bnh khng pht trin, thoi ha dn ri kh v cht. L ca cnh b bin dng khng m m xon nh li, bn ngoi l c lp lng t. Bnh nng c th gy cht c cy.

4. Quan h vi thc phm.Gn y, mt pumilacidin-sn phm ca B. pumilus lin quan n mt trng hp ng c thc phm. Mt nghin cu ca Brophy v Knoop bo co th nghim cm ng ca rut ln guinea, trong khi mt s hp cht c sn xut bi B. pumilus c bo co c hi i vi chut, cc t bo c nhn in hnh v con ngi.Ngoi ra, ngi ta cn pht hin rng B. pumilus l tc nhn gy ng c thc phm, vi khun ny c pht hin trong go v to ra cht c l pumilacidin (From et al., 2007). Cc triu chng do hu qu ca nhim trng bao gm chng mt, au u, n lnh, au lng, au bng, tiu chy.Hn na, trong nm 2007, mt bo co tng kt 3 nghin cu c cng b kt lun rng mt chngvi khun Bacillus pumiluslm pht trin cc tn thng da hnh thi tng t nh bnh dovi khun Bacillus anthracis.

5. Tng kt.

B. pumilusbo t-ngoi tr t bin ATCC 7061-ni chung cho thy kh nng chu p lc mi trng, bao gmtip xcnh sng cc tm, kh hn, v s hin din cacht oxy hanh hydrogen peroxide. Mt chng caB.pumilusphn lp t tm s c tm thy, c kh nng chu mn cao v c ch s pht trin ca tc nhn gy bnh hng hi, bao gm cvi khun Vibrio alginolyticus, khi lin kt vi nhau.Bacillus pumilus GB34 c s dng nh mt thnh phn hot cht trong thuc dit nm trong nng nghip.Tng trng ca vi khun trn r cy ngn cnRhizoctoniavFusariumt bo t ny

D. Bacillus mensentericus1. Gii thiu,c im hnh thi,c tnh ca Bacillus Mensentericus:Cc vi sinh vt phn hy cc cht hu c cha N thng c gi l vi sinh vt thi ra,trong s ny vi khun ng vai tr quan trng.Vi khun thi ra c th l hiu kh,k kh to thnh hoc khng to thnh bo t.Chng l cc c th a m,a nhit hoc a lnh.a s chng rt nhy cm vi axit v hm lng NaCl trong mi trng.Trong c mt loi trc khun thng gp nht l Bacillus mensentericus.Bacillus mensentericus: l nhng trc khun chuyn ng c tin mao xung quanh , bo t hnh bu dc,trc khun kt thnh tng chui di,ngn khc nhau to v t bo c th ng ring r.Chng c hot lc protease kh cao,bo t chu nhit kh ln.Trc khun ny gn ging Bacillus subtilus c dng mnh di hoc ngn(3-10)x (0,5-0,6)Mm.Khi phn gii protid cha S to thnh H2S.T bo Bacillus mensentericus khng b phnh to khi mang bo t.Khun lc thng bm cht vo mi trng thch c khi dnh vo mi trng.Loi Bacillus mensentericus cn tm thy trong tt c cc loi hp,loi ny thng khng c,hiu kh,c nha bo pht trin nhanh nhit 37 .Nha bo b ph hy 100 trong 1 gi.Phn loi:gm c 2 loi Bacillus mensentericus Fuscus v Bacillus Mensentericus Niger.Bacillus Mensentericus sinh trng v pht trin tt nhit ti u l36-45,ti a khong 50-55Khi pH t gi tr 4,5-5 th n s ngng pht trin.N c hot tnh amylase v protase hn hn Bacillus Subtilis nhng ln men ng li km hn.Loi trc khun ny thng ph bin trong t nhin,chng ly nhim v lm h hng thc phm.Ngoi ra n cn sinh ra 1 loi hp cht c hot tnh khng li mt s vi khun khc.Bacillusmensentericus(gy bn khoai ty),cc vi khun ny c kh nng ln men ng glucose v sinh bo t c th pht trin trong bt nu bt b m.Nu bt b nhim vi khun ny khi em ch bin s gy hin tng ko si gi l bnh si dnh.Bt v tinh bt l sn phm ca cc loi ng cc(la,la m,la mch),mt s loi c(khoai m) v mt s loi u.Bn cht ca bt l sn phm c hm lng m thp.V th bt v tinh bt nu c bo qun tt s c rt t kh nng vi sinh vt xm nhp v pht trin.Qu trnh nhim vi sinh vt ca bt v tinh bt ch xy ra khi m ca chng vt qu gii hn.Lc ny hot ng ca enzym c trong sn phm thy phn tinh bt thnh glucose gip cho vi sinh vt pht trin.S pht trin mnh m ca vi sinh vt nhanh chng lm h hng thc phm.V th,bt v tinh bt l nhng sn phm cn duy tr mt mc m ti thiu khng cho m xm nhp.Bao b c kh nng chng thm thu m l vt liu cn thit trong bo qun.Ngoi ra kho cha cng cn lu khng b ngp v m t.2. nh hng ca vi sinh vt trong mt s sn phm bt v tinh bt trong bo qunBt m:Nu bo qun khng tt bt m s b vn cc,mt s vi sinh vt c kh nng s dng ng glucose s pht trin.Bnh m:H vi sinh vt ca bnh m bt ngun t ngun bt m, t men bnh m v tp nhim.Khi nho bt mt s thnh phn trong h vi sinh vt pht trin,c bit l men bnh m v hot ng ln men to cn sinh CO2 lm n bt nho.Khi nh hnh v em nng nhiu vi sinh vt b cht di tc dng ca nhit cao.c lm t bt m ha vi nc v mui ln men bng men bnh m v nng nhit 180-200.Khi nng bnh,cc vi sinh vt ngoi v bnh cht v trong rut bnh s nng dn ln nhung khng qu 95-98 nn cc t bo sinh dng ca vi sinh vt b cht nhng bo t ca chng vn cn sng.Nhng bo t ca trc khun khoai ty pht trin thnh nhng t bo mi lm hng bnh m m ngi ta vn gi l bnh khoai ty.H hng bnh do vi sinh vt:Trong qu trnh bo qun,vn chuyn thng b nhim cc vi sinh vt t bn ngoi vo. chnh l nguyn nhn gy h hng bnh m.Bnh nht rut bnh m do trc khun khoai ty gy ra.Trong bt m c bit l bt m loi 2 hoc loi xu c ln bo t ny v khi nng bnh chng khng b cht sau s ny mm v pht trin gy hng bnh.Kt qu l ru bnh b nht dnh,thm mu v c mi kh chu.Bnh m bnh khoai ty khng c dng n,nu b nh c th lm thc n gia sc,b nng c th t hoc chn.Cc trc khun ny pht trin tt pH=6.5-7,cn pH=4.5-5 chng tm thi b ngng pht trin.V vy ta c th p dng cc bin php thch hp nh nng cao axit cho bt nho km hm cc trc khun ny.

3. Quan h vi thc phm

Thuc nhm vi khun gy c tht, h vsv tL vi khun hiu kh gy ln men pectinL vi sinh vt phn gii hiu kh cc cht pectin theo 2 giai on : thy phn ym kh pectin v cc sn phm thy phn b oxy ha to thnh CO2 v H2O. Thuc h vsv gy thi ra:Trc khun c tin mao xung quanh, cho bo t hnh bu dc, t bo khng ln lm v thng xp thnh chui, hnh thnh H2S khi phn gii protitTc nhn ca qu trnh ln men vi da chut, da gang mui chuaTrong c ng lnh: tn ti dng tim sinhC hp: trong c hp c cht bo v protein, cc phn t ca n c th vy quanh vi khun v bo v vi khun khi b cht khi gia nhitThuc h vsv tht,gy thi tht,hoat ng trn b mt tht. C hot tnh proteaza cao, tit ra enzyme proteaza lm phn gii protein ti cc sn phm c mi hi thi nh NH3, indola, scatola, mercaptan.C trong cc sn phm ln men, ban u cc acid hu c c ch vi khun gy thi ra pht trin, nhng mi trng acid, nm mc d pht trin v hot ng mnh to thnh NH3 v kim nitrua, nhng cht ny trung ha mi trng acid, to iu kin cho vi khun gy thi ra pht trinH vsv trong tht muiTht hp : trong qu trnh thanh trng, bo t khng b tiu dit. bo t cha qua gia nhit s ny mm sao 1-2 ngy. Nu qua gia nhit cao s ny mm 370C, sau 20-27 ngyC trong xc xchNng NaCl lm vi khun ngng pht trin 10-15%Vi khun to nhn trong nc ngC trong ng thVi khun xm nhp vo ng ct trng t siro qua cc khu ng gi, ty mu, bo qunNgay t khi cha c thu hoch, c khoai ty, c sn, bp ng, b nhim vsv t t. Vi khun gy h hng trong qu trnh bo qun v ch bin ng thnh bt v gluco tinh khitTrong giai on bo qun tinh bt khi sn xut gluco v mt tinh bt cng rt d b vsv ph hoi. Tinh bt t l nn cho vi khun ny pht trin, kt qu l tinh bt b chua v i mu.Vi khun xm nhp vo dy chuyn cng ngh t nguyn liu , nc ra. c kh nng pht trin cng on tinh bt t, c bit l i vi qu trnh knKhng gy ln men ng sa, l vi khun sinh bo t, c kh nng sinh enzyme phn gii protein sa to pepton v bin chng thnh cc acid t do, NH3. Pepton lm sa ng v NH3 lm sa c phn ng kim.vi khun chu nhit tt nn tuy hnh thnh sa vn c th pht trin gy h hng saMng nguyn sinh cht cha 55-75% ton b lipit c trong t bo Vi khun Bacillus Mesentericus thng c trong h vi sinh ca ng cc. Nu nng bo t vt qu 1000 t bo / g gi l bnh xy ra mc ca bnh m.Mc ca bnh xy ra cc bnh sau khi nng, mt s lng ng k cn li vi khun. Cn bnh ny l ph bin hn trong bt mu trng trong sut ma h v thc s th hin sau 24-48 gi sau khi nng, khi cc sn phm c trong tay ca ngi tiu dng. Mc d cc vi khun gy ra mc bnh b bt hot nhit cao, cc bo t khng nhit nu n thng thng vn bnh thng. Sau khi lm mt bo t c nhn vi mt tc rt cao. Enzyme sn xut bi cc vi khun phn hy cc dy chuyn t protein li bnh m, bin n thnh mt khi keo nu. ng thi, cc li c mi hi ngt ca qu thi, khi b bnh.Khi bnh c nng cao hn c quan st thy dy rt mng xy ra do vi khun c enzyme phn gii protein lm suy gim gluten v ko di n thnh si. Sau tinh bt thy gim hm lng tinh bt 64-16%.Cn bnh ny l ph bin hn trong ma h, bnh m mt m t 45oC n 25oC (s pht trin ti u ca cc vi khun. Hot ng ca cc vi khun trong thc n c thay i v khng th c tiu th, gy ra ri lon tiu ha.

4. Bacillus mesentericus (trc khun khoai ty)4.1 Ly mu1. S lng mu: 5 mu hin trng2. Thu thp mu: theo nguyn tc chung3. Chun b mu phn tch: ly 25g mu v thm 225ml nc pepton m, sau tin hnh quy trnh pha long

4.2 Kim tra trc khun khoai tyBt m b nhim trc khun khoai ty em lm bnh m th bnh s b bnh khoai ty, cn gi l bnh si dnh.Trc khun khoai ty c nha bo chu ng c nhit cao, nu nhit khng qu 100oC th vi khun vn cn sng, nht l trong rut bnh m. Khi gp nhit thch hp vi khun li pht trin trong bnh m, gy mi kh chu, b bnh m s thy trong rut c nhng si nh mng nhn, l bnh khoai ty.Nhit 40oC rt thch hp cho trc khun khoai ty pht trin, do bnh ny thng pht trin vo ma h. Tuy trc khun khoai ty khng phi l mt vi khun gy bnh cho ngi, nhng bnh m b bnh ny cng lm nh hng n cm quan v khng s dng c. Bi vy ngn nga bnh m b h hng th trc khi s dng bt m cn phi kim tra trc khun khoai ty.

4.3 Cch lm4.3.1 Phng php vi khunLy 1g mu, cho vo bnh c cha sn 30ml nc v khun, lc mnh ha tan. un cch thy nhit 80oC trong 30 pht dit vi khun khng c nha bo. Dng ng ht v khun ly dung dch trong bnh cy vo mi trng thch v mi trng canh thang ri m 24h.Trc khn khoai ty s mc thnh vng nhn nheo trn mt mi trng canh thang, cn dch th hu nh trong sut. mi trng thch th khun lc nhn nheo, mu xm sm, em pht knh, nhum v soi th thy trc khun Gram (+) c nha bo. Khi th phn ng sinh vt th thy phn gii glucose v saccharose, sinh acid, khng sinh hi,khng phn gii lactose.4.3.2 Phng php cng ngh

Ly 1200g bt m, sau khi ha vi nc mui v men vo nhit 25-30oC, bt s n ra.Chia khi bt ny thnh ba phn trng lng bng nhau, v thnh bnh, nng l.Sau khi bnh hi ngui, bc bnh bng mt loi giy khng thm khng kh m 37oC. Qua 24h, 48h, 72h ly ra b i tm du hiu bnh khoai ty.Trng hp kim tra ln u (24h) nu c du hin bnh khoai ty, chng t bt m b nhim nng.Nu kim tra ln th hai (48h) thy du hiu bnh khoai ty, bt m b nhim trung bnh.V nu kim tra ln th ba (72h) mi thy du hiu bnh khoai ty, th bt m coi nh bnh thng.Bt m c trc khun khoai ty th khng nn dng sn xut bnh m, m nn dng vo ch bin cc thc phm khc nh m si.Trng hp cn phi s dng bt m lm bnh m (v cha c loi bt m tt khc), th bnh m phi nn nh, trng lng mi chic bnh khng qu 200gram.Bnh m sau khi ly l ra phi ngui ngay v phi s dng trong 24h.Nu hp thu m do bo qun khng tt bt m s b vn cc, mt s vsv bo t c kh nng s dng ng glucose (Bacillus mesentericus) s pht trin. Bnh m c lm t bt m ha vs nc v mui ln men hoc men bnh m , nng nhit 220-250oC. Bacillus mesentericus lm bnh m ki b ra c nht ko thnh ch.

Hnh: Vi khun Bacillus mesentericus gy bnh khoai ty trong cc mi trng nui cy v nhn di knh hin vi.

5. Cc bin php c khuyn co:Lm mt nhanh chng bnh m v n chng ti lm gim nng di 200 bo t / g bt nhim trng c th kt hp ba n cht lng ttNhanh ln men c tng cng bng cch gim gi tr pH 5,0-5,2 vi khun v nm l lm chm li rt nhiu, tng ng vi pH bnh thng ca 5,4-5,7. Cng d dng c thc hin bng cch thm vo thc n gim 0,5-1,0% (9% chua) so vi ba n. Hiu ng tng t thu c bng cch s dng mt mui axit nh canxi hydrogen phosphate vi t l 227 g cho mi kg bt 45 (0,5% bt)Vic s dng cc cht c ch vi khun v nm, canxi propionate E 282 (cho cc sn phm lng vi men) v natri propionate (sn phm ha cht lng), bng cch s dng t l 142 g n 45 kg bt (0,3% trn bt).6. Kt lun

Bacillus mesentericus bnh thng c trn b mt ca cc ht ng ccBacillus Mesentericus hnh que, vi khun Gram dng, vi khun di ng hn v bo t tn ti trong t.N hin ang c nghin cu trong bnh nhim trng ng h hp v mt s ri lon tiu ha.

Ti liu tham kho1. Trung tm Quc gia v Thng tin Cng ngh sinh hc.(2008).Bacillus pumilus: Mt sinh vt t khp mi ni [tp tin d liu].Ly thttp://www.ncbi.nlm.nih.gov/genome/?term=bacillus% 20pumilus2.Hendrick C mt, Johnson LK, Tomes NJ, Smiley BK, Gi JP.Chn Tn916 vo Bacillus pumilus pMGD302 plasmid v bng chng chuyn plasmid bi s kt hp.Plasmid.Nm 1991; 26 (1) :1-9.C sn ti:http://www.ncbi.nlm.nih.gov/pubmed/1658832.3.Pan J, Q Huang, Zhang Y. gen nhn bn v biu hin ca mt serine protease c chc nng co lng t vi khun Bacillus pumilus.Hin vi sinh.Nm 2004; 49 (3) :165-9.C sn ti:http://www.ncbi.nlm.nih.gov/pubmed/15386098.4.Trung tm Quc gia v Thng tin Cng ngh sinh hc.(2008).Cc chi tit v b gen ca vi khun Bacillus pumilus [d liu tp tin].Ly thttp://www.ncbi.nlm.nih.gov/genome/genomes/440?subset =5.Zhang ZH, Tian W, Liu DY, et al.c tnh ca mt pPZZ84 plasmid kh hiu t vi khun Bacillus pumilus.Plasmid.Nm 2010; 64 (3) :200-3.C sn ti:http://www.ncbi.nlm.nih.gov/pubmed/206201626. Trn Linh Thc, Cc phng pho kim tra vi sinh vt trong nc v thc phm7. Martin R. Adam &Maurice O. Moss (2008), Food Microbiology 3rd edition, University of Surrey, Guidford, UK

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