Vi sinh thc phm

Nhm thc hin:Bi Thu LoanVn Th ThuTrn Minh Tun

SeminarOXYRASE

Tng quan v oxyrasec tnh v c ch hot ng ca oxyrase Phn on mng c chc nng tng t oxyrase Mt s sn phm oxyrase hin nayCc trin vng trong tng lai

1) Tng quan v oxyrase1.1 nh ngha1.2 Ngun gc1.3 Li ch

nh nghaOxyrase l tn thng mi c t cho enzyme c kh nng kh oxy (hay enzyme mang chui chuyn e- ng vai tr quan trng cho qu trnh h hp m bn cht l cytochrome)Theo nh ngha ca cng ty OXYRASE Inc. Oxyrase l hn hp thng mi ca 2 loi enzym monooxygenase v dioxygenase c kh nng loi b oxy mt cch nhanh chng v hon ton ra khi mi trng lng v bn rn (OXYRASE Inc., 1991).Mt n v hot tnh Oxyrase c nh ngha l lng enzyme kh 1,0% oxy ha tan trong 1 giy trong 1 ml.

Ngun gcPhn on mng E.coli : Adler pht hin oxyrase t phn on t bo c cha enzyme h hp c kh nng hnh thnh mi trng k kh. Schnaitman (1970): E.ColiPhn on vchPhn on mngCytochrome v cc enzyme kh kh O2 ho tan thnh H2O 1/3 protein mng bao1/2 phospholipidNh vy phn on mng t bo c cha Oxyrase, c th l bo quan Mesosome

Ngun gc

Li ch ca oxyraseNhng kh khn khi thao tc vi i tng l vi sinh vt k kh:Nhn v mt l thuyt: Tp hp vi sinh vt k kh ln, a dng Mt s c kh nng gy bnh cho ngiTrong thc nghim: VSV k kh rt kh nui cy v tng sinh. Nguyn nhn ct li chnh l phi to c mi trng c iu kin k kh m bo cho VSV k kh c th sng v sau l tng sinh. Ti sao vn ny gp kh khn???

Trong thc t: cho cht kh vo to mi trng k kh chnh cht kh to thnh cc peroxyde hu c c ch s sinh trng ca vi sinh vtS dng h thng rt khng kh, bm chn khng, to hn hp kh nhm loi oxy h thng ny li kh cng knh v phc tp.Nu to c mi trng k kh thao tc kh khnOxyrase gii quyt vn trn

OxyrasePhc hi v tng sinh nhanh chng cc VSVKKCc a nui cy VSV k kh c th chung vi cc a nui cy VSV hiu kh trong cng t m. Thao tc n gin, tit kim khng gian Kh oxy H2O, nhanh hiu qu

2) c tnh v c ch hot ng ca Oxyrase2.1 Cc c tnh ca Oxyrase2.2 C ch hot ng ca Oxyrase2.3 Tc ng ca Oxyrase ln s tng trng vi sinh vt k kh

Cc c tnh ca oxyraseKh O2 thnh H2O khi c mt ca cht cho in t. Phm vi hot ng ca oxyrase:pH: 3.5-7.0Nhit : nhit phng - 500CMi tng quan gia nng cht cho in t v nng O2 ho tan. Nng cht cho in t phi tng ng vi nng O2 ho tan (1-10mM;

C ch hot ng ca Oxyrase V mt bn cht: mono- v di-oxygenase. Xc tc kh O2 thnh nc. (ly trc tip nguyn t oxy t khng kh thc hin phn ng oxi ha c cht.) L nhng enzyme quan trng trong cc qu trnh bin dng nng lng v trao i cht.

c tnh ca oxyrase V mt lu tr: n nh trong 1 nm hoc lu hn nu Oxyrase c gi ng -100C hoc lnh hn. Qu trnh ng v gii ng khng nh hng nhiu n hot tnh ca n.Vn hot ng tt trong 30 ngy nu gi nhit 2-80C.Gi nhit phng rt mau mt hot tnh.

C ch hot ng ca oxyrase vi khun: Oxyrase nm trn mng t bo v thc hin chc nng h hp. Oxyrase s dng hai loi c cht khc nhau: phn t oxy v cht cho in t. Cc cht cho in t bao gm formate, lactate, succinate, -glycerophosphate.Kh nng hot ng ca oxyrase tng hay gim ph thuc vo bn cht ca cht cho in t

C ch hot ng ca oxyraseVic kh O2 ca enzyme mng ph thuc vo kh nng ca h cytochrome, trong , Ubiquinone ng vai tr ch o ngha l thiu Ubiquinone, h mng khng thc hin c chc nng kh O2 Cht cho in t sau khi b oxi ha thnh cht khc khng nh hng n s sinh trng ca VSV k kh nh: Lactate pyruvate, formate CO2 + H2O.* H thng Oxyrase khng lm thay i ng k pH ca mi trng nh h thng CO2 (Retsema et al,1991)

Tc ng ca oxyrase ln s tng trng ca vi sinh vt k khKhi dng Oxyrase cn ch : To c mi trng k kh hay khng? C tc dng kch thch sinh trng ca VSV mc tiu hay khng? Cc yu t nh hng hot tnh v tc kh O2 ca enzyme Oxyrase v cc phn on mng c tnh nng tng t khc. Tc ng ca Oxyrase ln s tng trng ca Salmonella, Listeria,Campylobacter, E.coli O157

Tc ng ca oxyrase ln s tng trng ca Salmonella v ListeriaTheo kt qu thc nghim ca Fahimeh NirooMand v Daniel Y.C. Fung trong nghin cu Tc ng ca Oxyrase ln s tng trng ca Salmonella spp. v Listeria monocytogenes trong mi trng tin tng sinh ph qut (1992)Tin tng sinh: mi trng ny c tc dng phc hi VSV mc tiu c trong mu trc khi thc hin tng sinh chng trong mi trng chuyn bit.

Tc ng ca oxyrase ln s tng trng ca CampylobacterMt s Campylobacter nui cy thnh cng khi c s hin din ca Oxyrase trong mi trng nui cy.Nui cy lc C. jejuni v C. coli 42oC trong dch nui cy brucella c s hin din ca Oxyrase. Khi gia tng nng Oxyrase t 0-0,8 n v/ml th s tng trng ca Campylobacter cng tng theo. Trong dy nng ny, s tng trng nhanh nht v thi gian th h ngn nht ca Campylobacter t c khi mi trng nui cy c s hin din ca Oxyrase nng 0,6 n v/ml. nh hng ca oxyrase ln Campylobacter ch yu trong pha log hn l pha n nh.

Tc ng ca oxyrase ln s tng trng ca E.coli O157OxyraseTM lm gia tng s tng trng ca E. Coli O157 O157 trong cc mu thc phm v mi trng(Phebus et al. 1993; Thippareddi et al. 1995; Jiang et al.1998)Jiang et al. (1998): mi trng c OxyraseTM gip hi phc lng nh E. Coli O157 trong mu. c bit l trong trng hp c cc vi sinh vt cnh tranh khc.

3. Phn on mng c chc nng tng t oxyrase3.1 Phn on mng c ngun gc vi khun3.2 Phn on mng c ngun gc phi vi khun (non- bacterial source)

3. Phn on mng c chc nng tng t oxyrase3.1 Phn on mng c ngun gc vi khun3.2 Phn on mng c ngun gc phi vi khun (non- bacterial source)

Phn on mng c ngun gc vi khun Chng th nghimCc bc thc hinTng sinhThu nhn cc phn on mngThanh trng cc phn on mng

Chng th nghim

Chng s dng: t American Type Culture Collection (ATCC), Rock ville, MD gm:Escherichia coli (E8) ATCC 11775Acetobacter xylinum ATCC 12878Gluconobacter oxydans subsp. suboxydans ATTC33448Ngoi ra cn s dng cc chng: Pseudomonas aeruginosa, Acetobacter vinlandii, Mycobacterium phlei v c Salnonella typhimurium

Phn on mng c ngun gc vi khun Chng th nghimCc bc thc hinTng sinhThu nhn cc phn on mngThanh trng cc phn on mng

Phn on mng c ngun gc vi khun Chng th nghimCc bc thc hinTng sinhThu nhn cc phn on mngThanh trng cc phn on mng

Escherichia coli (E8) ATCC 11775Tng sinhQui m nh: E.coli trong 250ml BHI, lc 250v/ph trong 24hQuy m ln: nui t bo E.coli trong cc thit b ln men 2l , vi mc thng kh l 0.5-1.0 th tch/din tch cht lng/pht 37oC trong 24hBo qun trn thch nghing BHI v cy chuyn hng thng

Acetobacter xylinum ATCC 12878Gluconobacter oxydans subsp. suboxydans ATTC33448

Khi phc t bo trn mi trng manitol c pH 6,0 0,2, 30oC trong 48h Tng sinhQui m nh: chng khi ng (1 2%) trong 250 ml BHI mannitol trong cc bnh 500 ml, sau t trn my lc vi tc 200 vng/pht Qui m ln: A. xylinum v G. oxydans c nui cy trong cc thit b ln men 2 lt vi mc thng kh l 0,5 1,0 dung tch kh / dung tch cht lng 300C trong thi gian 36h v 24h . Bo qun 40C cho ti khi cn s dng, cy chuyn hng thng duy tr trng thi sng.

Phn on mng c ngun gc vi khun Chng th nghimCc bc thc hinTng sinhThu nhn cc phn on mngThanh trng cc phn on mng

Thu nhn cc phn on mng

Phn on mng c ngun gc vi khun Chng th nghimCc bc thc hinTng sinhThu nhn cc phn on mngThanh trng cc phn on mng

Thanh trng cc phn on mngS dng phng php lc v trngPhn on mng ca E. coli (E8) v Gluconobacter c lc 2 ln bng lc c ng knh l l 0,45m.Cc phn on mng ca Acetobacter c lc bng lc c ng knh l l 1,2m, sau qua l 0,8m v cui cng qua l 0,45m 2 ln, bi v chng c lp polysaccharide.

3. Phn on mng c chc nng tng t oxyrase3.1 Phn on mng c ngun gc vi khun3.2 Phn on mng c ngun gc phi vi khun (non- bacterial source)

Phn on mng c ngun gc phi vi khunTi th ca cc loi nm, to, thc vt, c kh nng kh O2 thnh H2O nu c s hin ca cht cho in t.Qu trnh to phn on mng theo cc bc:

Siu ly tm siu lc

4. Mt s sn phm oxyrase hin nayOxyrase for BrothHn hp cha Oxyrase v cc cht c bn c tc dng cc i ho hot ng ca Oxyrase phm vi rng cc iu kin trong mi trng broth Cch s dng: nh Oxyrase for Broth vo mi trng vi t l 0,1ml:5,0ml (1 git/1 ml).

4. Mt s sn phm oxyrase hin nayOxyrase for Agar cn s dng kt hp Oxyrase for Agar vi OxyDishti u ho hot ng ca oxyraseH thng ny thng c s dng m vi khun k kh. S dng: s dng t l Oxyrase: mi trng = 1:30 ch thm vo mi trng lc trng thi tan chy v ch thm vo mi trng hp kh trng.

4. Mt s sn phm oxyrase hin nayOxyDishOxyDish l lng cha cc a mi trng nui cy sau khi thm Oxyrase for Agar nhm to iu kin thun li vi khun trong iu kin hiu kh.

4) Mt s sn phm oxyrase hin nayOxyplates Oxyplate thch hp cho cc phng th nghim nghin cu VSV gy bnh

5) Mt s sn phm oxyrase hin nay OxyPRAS Plates L loi mi trng lm sn, s dng tt nht so vi bung , bnh ym kh hoc ti ym kh to mi trng ym kh hon ton, cng l mi trng tt nht phn lp v tng sinh hu ht cc vi khun k kh. Pre-Reduced Anaerobically Sterilized

5. Cc trin vng trong tng laiTo mi trng k kh cho cc nghin cu lin quan Loi b oxy khi mi trng lng dung mi nc thng n nh mi v, mu sc thc phm, gii khtLoi b oxy khi mi trng du ngn i thiu sn phm du, thay i mu sc b, kem...

OxyPRAS plates differ from standard plates in important ways:They are made for Anaerobes.They come reduced - ready to use.They are specially packaged in oxygen barrier bags.They stay reduced in air for hours.They can be stored refrigerated, for very long shelf life.They can be stored at room temperature.They meet Best Practice criteria for working with clinical Anaerobes.

***30 u/ml, 50 ml, 1 bottle $210.00**Mesosome - infolding of plasma membrane to aid in compartmentalization*****hot ng tt nhit phng, vn cn hot ng 45-500C trong nhiu gi, bt hot nu nhit cao hn na.Nu ch mun to mi trng vi hiu kh, s dng nng c bit thp.***It can be kept at 40C for hours without substantial loss of activity. It is heat inactivated above 55CTo maintain stability, EC-Oxyrase must be stored at constant - 20C or lower. It may be thawed and refrozen several times without loss of activity. *The oxygen-reducing activity of Oxyrase starts when Oxyrase comes in contact with oxygen and a hydrogen donor*Kh nng kch thch qu trnh sinh trng ca VSV k kh ca Oxyrase l do cc c tnh ca h mng hn l c tnh kh O2 ho tan t mi trng nui cy.**************Sau khi len men, nguoi ta tien hanh ly tam loai bo dich noi thu phan lang la sinh khoi vi khuan.

*********Pre-Reduced Anaerobically SterilizedOxyrase is used during the manufacture of the plate to prevent oxidation products from forming during preparation***