1 B Y T TRNG I HC DC H NI TRN TH HNG GP PHN NGHIN CU KHNG SINHC SINH TNG HP TSTREPTOMYCES 183.219 KHA LUN TT NGHIP DC S H NI 2014 2 B Y T TRNG I HC DC H NI TRN TH HNG GP PHN NGHIN CU KHNG SINHC SINH TNG HP TSTREPTOMYCES 183.219 KHA LUN TT NGHIP DC S Ngi hng dn:PGS.TS. Cao Vn Thu Ni thc hin:B mn Vi sinh v Sinh hc Trng i hc Dc H Ni H NI 2014 3 Li cm n hon thnh kha lun tt nghip ny, ti xin gi li cm n su scn PGS.TS Cao Vn Thu ngi thy trc tip hng dn ti thc hinkha lun. Ti xin chn thnh cm n cc thy, c gio, cc cn b, k thut vin ang ging dyv lm vic ti bmn Vi sinh Sinh hc tn tnh gip ti trong sut thi gian lm kha lun.Nhn dp ny, ti cng xin gi li cm n ti Ban gim hiu, cng ton th cc thy, c gio trong trng i hc Dc H Ni to iu kin thun li cho ti trong qu trnh hc tp ti trng. Ti xin gi li cm n ti gia nh, bn b gip , ng h ti trong sut thi gian thc hin kha lun. V iu kin hn hp v thi gian, v phng tin nghin cu v trnh ca bn thn, chc chn kha lun cn c nhiu thiu st, hn ch. Ti rt mong nhn c s gp ca cc thy c, bn b kha lun c hon thin hn. Ti xin chn thnh cm n! H Ni, ngy 14 thng 5 nm 2014 Sinh vin Trn Th Hng

4 MC LC T VN ....................1 CHNG 1. TNG QUAN.........2 1.1i cng v khng sinh............2 1.1.1nh ngha khng sinh ............2 1.1.2Phn loi khng sinh............2 1.1.3C ch tc dng ca khng sinh...........2 1.1.4Cc ng dng ca khng sinh..................3 1.2.i cng v x khun..............4 1.2.1.c im hnh thi ca x khun.............4 1.2.2. c im hnh thi x khun chi Streptomyces...................5 1.3.Tuyn chn, ci to v bo qun ging x khun....................6 1.3.1. Mc ch...................6 1.3.2. Chn chng c HTKS cao bng sng lc ngu nhin.......................7 1.3.3. t bin ci to ging.......................7 1.3.4. Bo qun ging x khun..................................................................................8 1.4. Ln men sinh tng hp khng sinh...................8 1.4.1. Khi nim ln men............................8 1.4.2. Cc phng php ln men.............................8 1.4.3. Mt s yu t nh hng n qu trnh ln men.......................9 1.5. Chit tch v tinh ch khng sinh t dch ln men.........................9 1.5.1Vai tr ca chit tch v tinh ch khng sinh.. ...............9 1.5.2Cc phng php chit tch ......................10 1.6Bc u nghin cu cu trc khng sinh.......................10 1.6.1Ph t ngoi - kh kin .........................10 1.6.2Ph hng ngoi.......................11 1.6.3Phng php ph khi.............................11 1.7.SnxutactynomycinDnhStreptomycessindenensismicphn lp.............................................................................................................................12 5 1.8.nh hng ca cc mc oxy ha tan ln pellet sn xut Rapamycin t Streptomyceshygroscopicus.................................................................................12 CHNG 2: I TNG V PHNG PHP NGHIN CU...................14 2.1. Nguyn vt liu v thit b...............................14 2.1.1. Nguyn vt liu...........................14 2.1.2. My mc thit b.................................17 2.2 . Ni dung nghin cu........................................................................................18 2.2.1. Chn lc, ci to ging ..................................................................................18 2.2.2. Ln men, chit tch khng sinh ti u............................................................18 2.2.3. Phn loi x khun Streptomyces 183.219......................................................19 2.2.4. S b xc nh mt s tnh cht ca khng sinh thu c..............................19 2.3. Phng php thc nghim..............................................................................19 2.3.1. Nui cy v gi ging x khun.....................................................................19 2.3.2. Phn loi x khun theo ISP ..........................................................................19 2.3.3. nh gi hot tnh khng sinh bng phng php khuch tn.......................20 2.3.4. Xc nh mi trng nui cy thch hp........................................................21 2.3.5. Sng lc ngu nhin........................................................................................22 2.3.6. t bin bng nh sng UV............................................................................22 2.3.7. Ln men chm tng hp khng sinh................................................................23 2.3.8. Xc nh bn ca khng sinh trong dch ln men......................................24 2.3.9. Chit khng sinh t dch ln men bng dung mi hu c...............................24 2.3.10. Tch cc thnh phn trong khng sinh bng sc k lp mng......................24 2.2.11. Thu khng sinh th bng phng php ct quay..........................................25 2.3.12. Tinh ch khng sinh th bng sc k ct......................................................25 2.3.13. S b xc nh khng sinh tinh khit thu c.............................................26 CHNG III: KT QU THC NGHIM V NHN XT..........................27 3.1. Xc nh tn khoa hc ca Streptomyces 183.219..........................................27 6 3.2.PhtcdngcakhngsinhdoStreptomyces183.219sinhtng hp............................................................................................................................28 3.3. Kt qu chn mi trng nui cy thch hp................................................28 3.4. Kt qu sng lc ngu nhin...........................................................................29 3.5. Kt qu t bin ci to ging ln 1...............................................................30 3.6. Kt qu t bin ci to ging ln 2...............................................................31 3.7. Kt qu chn mi trng ln men chm.........................................................32 3.8. Kt qu chn chng ln men...........................................................................33 3.9. Kt qu th bn pH v bn nhit.........................................................33 3.10. Kt qu chn dung mi v pH chit.............................................................34 3.11. Kt qu sc k lp mng chn h dung mi................................................35 3.12. Kt qu sc k ct..........................................................................................36 3.13. Kt qu o nhit nng chy, o ph ca khng sinh tinh khit.......38 KT LUN V KIN NGH...........................................................................39,40 TI LIU THAM KHO PH LC

7 DANH MC CC K HIU, CC CH VIT TT ANDAcid 2- deoxyribonucleic ARNAcid ribonucleic B1t bin ln 1 B2t bin ln 2 G(+)Gram dng G(-)Gram m HTKSHot tnh khng sinh ISPInternational Streptomyces Project ( Chng trnh Streptomyces quc t ) KSKhng sinh MCMu chng MTMi trng MTdtMi trng dch th MT2dtMi trng 2 dch th S. flexneriShigella flexneri B. pumilusBacillus pumilus SLNNSng lc ngu nhin VSVVi sinh vt VVa

8 DANH MC CC BNG STT Tn bngTrang 1Bng 2.1: Cc vi khun kim nh14 2Bng 2.2: Cc MT nui cy x khun14 3Bng 2.3: Cc MT nui cy VSV kim nh15 4Bng 2.4: Cc dung mi s dng17 5Bng 3.1: Cc c im phn loi ISP ca Streptomyces 183.219 v Streptomyces gougeroti 27 6Bng 3.2: HTKS caStreptomyces 183.219 trn mt s VSVkim nh 28 7Bng 3.3: HTKS ca Streptomyces 183.219 trn MT1, MT2, MT528 8Bng 3.4: Kt qu th HTKS sng lc ngu nhin29 9Bng 3.5: Kt qu th HTKS t bin ln 130 10Bng 3.6: Kt qu th HTKS t bin ln 231 11Bng 3.7: Kt qu chn mi trng ln men chm33 12Bng 3.8: Kt qu chn chng ln men33 13Bng 3.9: Kt qu th bn pH34 14Bng 3.10: Kt qu th bn nhit34 15Bng 3.11: Kt qu chn dung mi v pH chit (trn S. flexneri)35 16Bng 3.12: Kt qu chn h dung mi chy sc k35 17Bng 3.13: Kt qu chy sc k ct36 18Bng 3.14: Kt qu sc k lp mng sau chy ct (trn S. flexneri)37 19Bng 3.15: Kt qu chy sc k ct37 20Bng3.16:Ktquscklpmngsauchyctln2(trn S. flexneri) 37 21Bng 3.17: Kt qu IR38 9 DANH MC CC HNH V STTTn hnhTrang 1Hnh 1.1: S c ch tc dng ca cc h khng sinh chnh3 2Hnh 1.2: Khun lc x khun4 3Hnh 1.3: S b phn loi x khun5 4Hnh 1.4: Cc khun ty x khun6 1 T VN T khi ra i cho n nay, khng sinh l mt v kh quan trng chng li cc vi sinh vt gybnh.Tuy nhin vi tnh hnh s dng khng sinh khng kim sotnhhinnaydntimtlotcchqumconngiangphivtv khc phc n. Cc h qu c th thy ngay l tnh trng khng khng sinh xut hin v ngy cng tr nn nghim trng. V vy, vic tm ra khng sinh mi l iu ht sc cn thit v lun c c th gii quan tm. Bn cnh , Vit Nam l nc ang pht trinnm trong vng nhit i gi ma, c t l khng khng sinh thuc hng cao nht th gii (WHO). Mt khc cng chnh kh hu l mt iu kin thun li cho s sinh si, pht trin ca h vi sinh vtvcngtomitrngttchokhnngsinhtng hpkhngsinhcachix khun Streptomyces. Chnh v vy, chng ti la chn ti: Gp phn nghin cu khng sinh c sinh tng hp t Streptomyces 183.219 lm kha lun tt nghip. Trong khun kh kha lun ny, ti mong mun t c cc mc tiu sau y: -Phn loi theo ISP xc nh tn khoa hc ca Streptomyces 183.219, -Chn lc, ci to ging nng cao hiu sut sinh tng hp khng sinh, -Xc nh iu kin ln men, chit tch khng sinh tt nht, -S b xc nh mt s tnh cht l, ha ca khng sinh thu c. 2 CHNG 1. TNG QUAN 1.1. i cng v khng sinh 1.1.1. nh ngha khng sinh [5,6,14,21] Khngsinhlnhngsnphmcbitnhnctvisinhvthaycc ngun t nhin khc c hot tnh sinh hc cao, c tc dng km hm hoc tiu dit mt cch chn lc ln mt nhm vi sinh vt xc nh (vi khun, nm, nguyn sinh ng vt, ) hay t bo ung th nng thp. 1.1.2.Phn loi khng sinh [5,12,14] C nhiu cch phn loi khng sinh: theo ngun gc, theo tnh nhy cm ca vikhunvikhngsinh,theocch tcdng,theocutrcha hcPhnloi khng sinh theo cu trc ha hc l khoa hc nht v n gip cho ngi nghin cu nhanh chng nh hng c cc c im ca cht khng sinh mi pht hin khi bit c cu trc ha hc ca n, trnh lng ph thi gian nghin cu v cc c im khc. Phn loi khng sinh theo cu trc ha hc thng chia ra cc nhm cht sau y: - Cc khng sinh c cu trc -lactam (penicillin, cephalosporin) - Cc khng sinh cha nhn thm (chloramphenicol) - Cc khng sinh c cu trc aminoglycosid (streptomycin, gentamicin) - Cc khng sinh c cu trc 4 vng (tetracyclin) - Cc khng sinh polypeptid (polymyxin, bacitracin) - Cc khng sinh macrolid (erythromycin, spiramycin) - Cc khng sinh polyen (nystatin, amphotericin B) - Cc khng sinh chng ung th nhm antracyclin (daunorubicin) - Cc khng sinh chng ung th nhm actinomycin (dactinomycin ). 1.1.3. C ch tc dng ca khng sinh [5,6] Cckhngsinhtcdngchyuquaviccchccphnngtnghp khcnhaucatbovisinhvtgybnhbngcchgnvoccvtrchnhxc 3 hayccphntchcatbovisinhvt,lmbiniccphnng.Mi nhm khng sinh tc dng ln cc ch khc nhau. C 6 kiu ch yu: - Tc dng ln vic tng hp thnh t bo - Tc dng ln mng nguyn sinh cht - Tc dng ln s tng hp ADN - Tc dng ln s tng hp protein - Tc dng ln s trao i h hp - Tc dng ln s trao i cht trung gian. Hnh1.1diygiithiuscchtcdngcacchkhngsinh chnh. Hnh 5.1: S c ch tc dng ca cc h khng sinh chnh 1.1.4.Cc ng dng ca khng sinh [14] - Tronglnh vc y hc: khng sinh c s dng iu tr cc bnh do vi khun,nmgyra.Ngoiramtskhngsinhcncdngtrongiutrung th. - Trong chn nui: Bc s th y dng khng sinh cha bnh cho ng vt: Griseoviridiniutrvimphicp,vimvchotrubKhngsinhcnc 4 s dng nh cht kch thch tng trng n gia sc, gia cm, gim chi ph thc n, tng sn lng trng g, vt. -Trongtrngtrt:Khngsinhcsdngditnm,vikhun,virus gy bnh cho cy trng: Validamycin dng dit nm Rhizoctonia solani gy bnh kh vn hi la rt hiu qu. -Trongcngnghipthcphm:Khngsinhcdngboqunthc phm nh tc dng tiu dit vi sinh vt c trong thc phm: khng sinh subtilin (do Bacillussubtilistora),nisin(doBacilluslicheniformistora),pimaricin, amphotericin B... 1.2. i cng v x khun [6,8,9,15] Xkhun(Actinomycetes)thucnhmvikhuntht(Eubacteria)phnb rng ri trong t nhin, l cc vi khun Gram dng, c t l G + C > 55%. Trong mi gam t ni chung thng c cha hng triu x khun. i a s cc x khun l cc vi sinh vt hiu kh, hoi sinh, c cu to si dng phn nhnh. Do c th sinh tng hp c nhiu sn phm trao i cht quan trng nn cc x khun c cc nh khoa hc quan tm nghin cu rt nhiu. Trongshn16000khngsinh hin cbittrnthgiithkhong 60%ldoxkhuntora.Xkhuncncsdngsnxutnhiuloi enzyme (amylase, celllulase, protease), cng nh cc hp cht khc. 1.2.1. c im hnh thi ca x khun: -Hsicaxkhunchiathnhkhuntycchtvkhuntykhsinh. Khunlcxkhunkhcbit:khngtrntnhvikhun,nmmenm thngcdngthrp,dngphn,khngtrongsut,cccnpgptaratheo hnh phng x. (Hnh 1.2) Hnh 1.6: Khun lc x khun 5 - ng knh khun ty x khun thay i trong khong 0,3-1,0 m n 2-3 m. a s khun ty x khun khng c vch ngn. Mu sc khun ty x khun ht sc phong ph: da cam, en , nu, trng, vng, xm - X khun c phn loi s b theo hnh 1.3. Hnh 1.7: S b phn loi x khun 1.2.2. c im hnh thi x khun chi Streptomyces : CcloixkhunthucchiStreptomycesckhnngtoranhiukhng sinhccutrcphctp.Trongtngscckhngsinhctmthydox khun tng hp th c ti 55% l t Streptomyces. - c im hnh thi: h si ca x khun gm c khun ty c cht v khun ty kh sinh:+ Khun ty c cht: mc su vo mi trng nui cy, khng phn ct trong sut qu trnh pht trin, b mt nhn hoc sn si, c th tit ra mi trng mt s loi sc t, c sc t tan trong nc, c sc t ch tan trong dung mi hu c. + Khun tykh sinh: do khun ty ccht pht trin di ra trong khng kh. Sau mt thi gian pht trin trn nh khun ty kh sinh s xut hin cc chui bo t. Actinomycetes VSV ging x khunActinomycetales ActinoplanaceaeStreptomycetaceaeActinomycetaceae StreptomycesStreptoverticulumThemostreptomyces 6 +Chuibot(sibot) c rt nhiu hnh dngkhcnhau:thng,sng,mc cu, xon Chui bo t phn ct to thnh cc bo t trn, l cquan sinh sn chyu ca x khun. Bmtbo t c th c dng trn nhn (sm), x x da cc (wa), c gai (sp) hoc c tc (sp). Hnh 1.4 di y th hin cc loi khun ty x khun. Hnh 1.8: Cc khun ty x khun -c imsinhl: Streptomyceslsinhvtd dng,ctnhoxihacao. phttrin,chngphngiicchydratcarbonlmnguncungcpvtchtv nnglng,ngthithyphncchpchtnhgelatin,casein,tinhbt,kh nitratthnhnitrit.Streptomyceslloixkhunhhphiukh.Nhittiu ca chng l 25-30C, pH ti u thng l 6,8-7,5. - Kh nng to sc t: Sc t to thnh t Streptomyces c chia lm 4 loi: scthatan,sctcakhuntyccht,sctcakhuntykhsinh,sct melanoid. 1.3.Tuyn chon, ci to v bo qun ging x khun1.3.1. Mc ch [14,19] CcVSVthunchng cphnlptcc ngunt nhin (bn, t,nc, m thc vt ) thng c HTKS khng cao, hiu sut sinh tng hp thp. Do , 7 thucccchngcHTKScaoavosnxutihiphicito,chn ging bng cc phng php khc nhau v nghin cu iu kin nui cy, bo qun thch hp. 1.3.2. Chn chng c HTKS cao bng sng lc ngu nhin [14,19] Cc vi sinh vt c s bin d tnhin theo tn skhc nhau trong ng ging thun khit, c c th c hot tnh khng sinh tng ln 20-30% so vi nhng c th khc. Cn phi chn ly c th c hot tnh cao nht trong ng ging nghin cu tip. Trongthct,vicchnlctnhincccthcHTKScaochnghin cu ban u, n khng c gi tr p dng vo sn xut. thu c nhng chng c khnngsiutnghpkhngsinh,ngitapdngccphngphptbin nhn to. 1.3.3. t bin ci to ging [13,14,16] Cc tc nhn gy t bin c th c chia lm 3 nhm : - Tc nhn ha hc : 5-bromouracil, ethylenimin, 2- aminopurin, dimethylsulfat, hydoxylamin - Tc nhn vt l : nh sng UV, tia X, tia alpha, tia Beta .Tc nhn hay cdng l nh sng t ngoi (UV). Kh nng t bin cn ph thuc vo khong cch chiu, thi gian chiu, cng bc x. - Tc nhn sinh hc : Tc nhn sinh hc cc Tn, bacteriophage Mu, Cc tc nhn vt l v ha hc vi liu lng v thi gian thch hp sgit cht hu ht vi sinh vt. Nhng c th no cn sng s c s t bin gen, lm thay i cc tnh trng dn n lm mt kh nng to khng sinh (t bin m) hoc lm tng hiu sut sinh tng hp khng sinh ln mnh (t bin dng). to ra cc chng c hiu sut sinh tng hp khng sinh cao phi tin hnh t bin bc thang, kt hp cc phng php di truyn phn t nh ti t hp gi hu tnh, k thut tch dng gen, k thut to v dung hp t bo trn. 8 1.3.4. Bo qun ging x khun [14,18,21] BoqunvgigingVSVlmtviclmcnthitdoccgingrtdb thoiha,nhmln,mtmtnukhngclugimtcchkhoahc.Mc ch c th l: gi ging VSV c t l sng st cao, cc c tnh di truyn khng b bin i v khng b tp nhim bi cc vi sinh vt l. Thngthcthsdng4phngphpsauboqunccchngVSV: bo qun lnh cy chuyn (bo qun lnh trong l hoc ng mi trng, nh k cy chuyn sang mi trng mi), lm kh (trn t bo VSV vi gi mang v lm kh nhit phng) , ng kh (phn tn t bo VSV trong MT cht bo qun ri ng lnh, lm khmu ng lnh) , ng lnh (huyn dch t bo trong cht bo qun c ng lnh nhanh v bo qun nhit thp). gi ging x khun trong phng th nghim, cch n gin nht l nui cy x khun trn mi trng thch nghing thch hp, ct ng thch nghing trong t lnh v nh k 3-6 thng cy chuyn li mt ln. 1.4. Ln men sinh tng hp khng sinh 1.4.1. Khi nim ln men [13,14,22] Lnmenlqutrnhtraoichtsinhhcctinhnhdohotngca VSV nh s xc tc ca cc enzym vi mc ch cung cp nng lng v cc hp cht trung gian cho chng. Mt trong nhng sn phm ca qu trnh ln men x khun l khng sinh. Khng sinh l sn phm trao i bc hai, c to thnh gn vo lc kt thc qu trnh sinh trng ca x khun, thng vo gn hoc vo chnh gia pha cn bng. 1.4.2. Cc phng php ln men [7,13,14] Phng php ln men b mt : MT dng trong phng php ny th rn hay th lng ty VSV. VSV hp thu dinh dng t MT v s dng oxi khng kh h hp trn b mt MT. Phng php ny c nhc im l tn mt bng, hiu sut s dng mi trng thp, kh t ng ha. Do , ngy nay ch s dng phng php ln men b mt trong chn ging v gi ging. 9 Phngphplnmenchm:VSVcnuitrongMTlng,phttrinc3 chiu nn khc phc c tt c cc nhc im k trn ca ln men b mt nhng i hi u t trang thit b ban u ln. C 4 kiu ln men chm sau: - Ln men m (ln men chu k): VSV c nui gii hn trong bnh ln men vi mt th tch MT xc nh. VSV pht trin theo giai on v to ra sn phm. Kt thc qu trnh, ngi ta thu ly sn phm. - Ln men c b sung: Trong qu trnh nui cy c b sung thm mi trng dinh dnglmmttbotngln.Do,nngcaohiuqusdngbnhln men. - Ln men lin tc: Thit b c cu to c bit sao cho khi VSVpht trin n mtgiai on thch hp th s ly i mt th tch dch ln men v ng thi b sung thm ng lngMT mi vo bnh. -Lnmenbnlintc:Trongqutrnhlnmen,vicbsungthmMTdinh dng v rt bt dch ln men khng c thc hin lin tc m nh k sau nhng khong thi gian nht nh. 1.4.3. Mt s yu t nh hng n qu trnh ln men [18] pH mi trng: nh hng n qu trnh ln men c th do tc dng trc tip ca cc ion H+ hay OH- n tnh cht keo ca t bo, hot lc ca enzym hoc l tc dng gin tip. hatanoxyvsthngkh:Thiuoxynhtthisphvtraoicht trong t bo. V th, phi cung cp oxy sao cho tc ha tan oxy bng tc s dngoxycaVSV.ivinhiuVSV,sthngkhslmtngtcsinh trng, rt ngn pha tim pht. Nh vy, trong sn xut, cn nghin cu c th nh hng ca ccyu t ti qutrnhlnmencthtiuhaqutrnhny,toiukinthunlinht cho sinh tng hp cht mong mun. 1.5. Chit tch v tinh ch khng sinh t dch ln men 1.5.1. Vai tr ca chit tch v tinh ch khng sinh [14] 10 y l giai on c vai tr rt quan trng bi v sn phm thu c sau qu trnhlnmenthngkhngbnvng,hmlngkhngsinhtrongdchlnmen thngthp.Do,cntchringkhngsinhkhicctpchtkhcvtinhch sn phm t c cc tiu chun cn thit theo quy nh dng iu tr. Cng on tinh ch s gp phn quyt nh ti gi thnh ca sn phm. 1.5.2. Cc phng php chit tch [1,2,14,17] -Lc:lqutrnhphnringhnhpkhngngnhtqualpgiylc (hoc bng), b c gi li trn lp lc, dung dch chui qua do chnh lch p sut trn v di lp lc. -Lytm:lphngphptchccphntckhilngringkhcnhau, thng l tch pha rn khi pha lng nh lc ly tm. -Chitbngdungmihuc:chuynkhngsinhcntch(anghatan trong dch lc) sang pha dung mi hu c. - Cc phng php sc k: +Scklpmng:Lphngphptchccchttronghnhpdatrn kh nng b hp ph (l ch yu) khc nhau ca chng trn cc b mt mt cht rn (cht hp ph). Cht hp ph y c trng u thnh mt lp mng trn gi . i lng c trng cho mc di chuyn ca cc cht phn tch l h s Rf. +Scklngtrnct:Lphngphptchxc nhcccht datrns phnbkhcnhaucaccchtgiahaipha:phatnhlchtlngbaobcto thnh tm phim mng mng trn b mt mt cht rn tr (gi l cht mang c c ht nh) c nhi vo ct, pha ng l dung mi thm qua ton b b mt pha tnh. 1.6. Bc u nghin cu cu trc khng sinh[1,3] 1.6.1. Ph t ngoi - kh kin CcbcxUV-VIScnnglngkhlnnnckhnnglmthayi mc nng lng ca cc electron t trng thi c bn ln trng thi kch thch. Gia cutrchahccaphntchtcnnghincuviquangphhpthcmi quan h cht ch (V d: Nhng phn t no cng c nhiu lin kt i th s hp th cng chuyn v bc sng di hn, c bit l cc h lin hp). Cc phn t c 11 cim:niilinhp,nhnthm,dtN,S,Ockhnnghpthnng lng nh sng UV-VIS.1.6.2. Ph hng ngoi Nng lng ca bc x hng ngoi khng ln lm thay i trng thi nnglngcaelectronmchthayitrngthidaongcaphnt. Trong phn t khi c nhm nguyn t no hp th nng lng v thay i trng thidaongthtonnmtdihpthtrnphIR.Cmitngquangia nhmnguyntvdihpthnnphIRcsdngphthinccnhm chccbittrongphnt.MibcsnghpthccitrnphIRsc trng cho mt nhm chc. y l c s cho vic phn tch cu trc phn t cc cht bng IR.1.6.3. Ph khi Ph khi l tp hp cc tn hiu tng ng vi cc ion c th hin di dng cc pic c cng khc nhau. Cc tn hiu ny thu c khi cc ion c to thnhtrongbungionha,cgiatcvtchringnhbphntchkhica mykhi ph. Ph khi cho phpxc nh chnh xc khi lng cc ion, phn t, xcnhcngthccutocachtnghincuhocdngtrongphntchnh lng. -Nguyn tc chung: + Phn t trng thi kh va chm vi mt chm electron c nng lng trung bnh (50e 80e) phn t bt ra 1 -2e to thnh cc ion +1 (ch yu) v +2. + Cc ion va chm tip vi chm e to thnh nhiu mnh ion,gcv cc phn t trung ha khc. + My o ph khi tch ring r theo s khi Z = m/e (vi m l khi lng ion, e l in tch). Cc s khi khc nhau c cng xut hin I khc nhau. -Ph khi biu din s phthuc s khi Z v cng I.Da vo ph khi lngvccphUV,IR,phcnghngthtnhncthbinlunccu trc phn t khng sinh. 12 1.7.Sn xut actinomycin D nh Streptomyces sindenensis mi c phn lp [23] Actinomycin l khng sinh chromopeptid lacton ca hn 30 x khun trong t nhinvnhiuccbinthtnghpbit.Ttcccactinomycintnhinc cha thnh phn tng t nh phenoxazone chromophore, ch khc nhau v thnh phn amino acid ca chui peptid bn ngoi. Cc actinomycin v actinomycin D c nghin cu rng ri trong vic s dng iu tr khi u c tnh. Nhng hiu lc sinh hc ca actinomycin D c cho l c th lm tng gp i qu trnh c ch s tho xon ADN v hot ng ca ARN polymerase v do s c ch s tng hp protein. Actinomycin cng c xut iu tr AIDS v kh nng c ch hoc lm gim cc vn chuyn trong virus HIV-1. phn lp c Streptomyces sindenensisn MTCC 8122 tng hp khng sinh chngungthactinomycin-D.HuhtactinomycinDcsnxutt Streptomycesparvulus.Snxutkhngsinhphthucvothngkhvtc khuy trn ca qu trnh ln men. Khi MT t n thng kh 1,5 vvm v tc khuy trn t 600 rpm, pH 7-8, sn lng khng sinh t c ln nht l120 g/l, cao hn khong 50% sn lng t c t c so vi khi ln men ban u. 1.8. nh hng ca cc mc oxy ha tan ln pellet sn xut Rapamycin t Streptomyces hygroscopicus. [22] Rapamycincbitlcccchcnngcamtloikhngsinhvcch min dch, gn y n cng c cng nhn l c kh nng lm chm qu trnh lo ha. Nhng nh hng ca oxy ha tan (DO) ln pellet sn xut rapamycin ca Streptomyces hygroscopicus c nghin cu trong nghin cu ny. Kt qu cho thymtphnmcDOcaolcnthittngcngsnxutrapamycin.Tuy nhin, tin c c s tp trung rapamycin cao bng cch s dng kim sot DOmginguynvncdngpelletcaS.hygroscopicus.NumcDOqucaolmgiatngkchng,cthphvcchnhthica dngpellettrongcc bnh ln men v kt qu l gim sn xut rapamycin. Cc nng rapamycin ti a t c trong cc nghin cu ny l khong 780 mg/l trong 5 lt dch ln men vi 13 kim sot DO hn 30%, rt hp vi vic b sung oxy tinh khit trongkh u vo, v tc khuy gii hn n di 200 rpm. PhnmcDOqucaov hnhthi dng pellet,chai ugybtlitrong vic t c lng rapamycin cao trong ln men sn xut ca S.hygroscopicus . 14 CHNG II: I TNG V PHNG PHP NGHIN CU 2.1. Nguyn vt liu v thit b 2.1.1.Nguyn vt liu Chng x khun: chng Streptomyces 183.219 do B mn Vi sinh Sinh hc trng i hc Dc H Ni phn lp t mu t ti Qun 9 Thnh ph H Ch Minh. Chng vi sinhvtkim nh: do Bmn Vi sinh Sinh hc cung cp, c trnh by bng 2.1. Bng 2.5: Cc vi khun kim nh Vi khun Gram(+)Vi khun Gram(-) Bacillus cereus ATCC 9946Escherichia coli ATCC 25922 Bacillus pumilus ATCC 6633Proteus mirabilis BV 108 Bacillus subtilis ATCC 6633Pseudomonas aeruginosa VM 201 Staphylococcus aureus ATCC 1228Salmonela typhi DT 220 Shighella flexneri Mi trng Ccmitrngnuicyxkhun:thnhphnccMTctrnhby bng 2.2. Cc MT lnmen (MTdt): C thnh phn tng ng MT nui cyx khun nhng loi b thch. Cc MT nui cy VSV kim nh: thnh phn cc MT c trnh by bng 2.3. Trongqu trnh lm ccMT nui cy x khun, MT ln men v MT nui cy VSV kim nh u c tit trng 118-120C trong 30 pht. Bng 2.6: Cc MT nui cy x khun Thnh phnn vMT1MT2MT3MT4MT5MT6MT7 Tinh btG222,42 LactoseG3 15 GlucoseG21 SaccaroseG3 Cao ngG0,50,5 Bt u tngG1,5 Cao thtG0,30,3 PeptonG0,30,5 KNO3G0,1 KClG0,05 NaNO3G0,2 NH4NO3G0,20,2 CaCO3G0,30,40,40,2 (NH4)2SO4G0,2 FeSO4.7H2O G0,01 K2HPO4G0,050,10,10,05 MgSO4.7H2OG0,050,050,250,15 NaClG0,050,10,5 Cao nm menG0,5 ThchG1,8222221,8 NcMl100100100100100100100 pH6,8-7,2 Bng 2.3: Cc MT nui cy VSV kim nh Thnh phn NaClCao thtPeptonThch pH %%%% MT canh thang0,50,30,50 7,0-7,4 MT thch thng0,50,30,51,8 Cc mi trng phn loi theo ISP (k hiu t ISP1 n ISP9): DungdchmuivilngcaISP:MnCl2.4H2O:0,1g;FeSO4.7H20:0,1g; ZnSO4.7H2O: 0,1g; nc ct v 100ml; pH=7,0 7,2. 16 ISP1: Tripton: 5,0g; cao nm men: 3,0g; nc ct v: 1000ml; pH=7,0 7,2 ISP2:Caonmmen:4,0g;dchchitmalt10,0g;glucose:4,0g;thch:20,0g; nc ct v 1000ml; pH=7,3. ISP3: Yn mch: 20,0g; dung dch mui vi lng: 1,0ml; thch: 18,0g; nc ct v 1000ml; pH=7,2. ISP4:Tinhbt:10,0g;K2HPO4:1,0g;MgSO4.7H2O:1,0g;NaCl:1,0g; (NH4)2SO4: 2,0g; CaCO3: 2,0g; dung dch mui vi lng 1,0ml; thch: 20,0g; nc ct v 1000ml; pH=7,0-7,4. ISP5:L-asparagine:1,0g;glycerin:10,0g;K2HPO4:1,0g;dungdchmuivi lng: 1,0ml; thch 20,0g; nc ct v 1000ml; pH=7,0-7,4. ISP6:Dung dch A: Pepton: 20,0g; acid citric: 0,384g; FeSO4.7H2O: 0,556g; NH4OH 25%: 0,264g; Na2S2O3: 10ml; K2HPO4: 1g; nc ct v 1000ml. ISP6: Dung dch A: 36,0g; cao nm nem: 1,0g; thch: 15g; nc ct v 100ml; pH=7,0-7,2 ISP7:Glycerin:15,0g;L-tyrosine:0,5g;L-asparagine:1,0g;K2HPO4:0,5g; MgSO4.7H2O:0,5g;NaCl:0,5g;FeSO4.7H2O:0,01g;dungdchmuivilng: 1,0ml; thch dy: 20,0g; nc ct v 1000ml; pH=7,2-7,4. ISP9: Cc ngun ng c tit trng bng phng php Tyndal(A) 1. Glucose4. D-Manitol 7. Raffinose 2. Inositiol5. D-Fructose8. Rhamnose 3. D -Xylose6. L-Arabinose9. Saccarose DungdchmuiPridhamvGottlieb(B):CuSO4.5H2O:0,64g;FeSO4.7H2O: 0,11g; MnCl2.4H2O: 0,79g; ZnSO4.7H2O: 0,15g; nc ct v 1000ml. ISP9: (NH4)2SO4: 2,64g; KH2PO4: 2,38g; K2HPO4.3H2O: 5,65g; MgSO4.7H2O: 1,0g; dng dch B: 1,0ml; thch: 15,0g; nc ct v 1000ml; pH=6,8-7,0g, D*ISP9: ISP9 sau khi tit trng ngui ti 60C th cho 1 trong cc ngun ng A vo tng mi trng ISP9 ring r sao cho nng ng trong mi mi trng l 1%. 17 Dung mi Bng 2.4 gii thiu cc dung mi s dng cc nghin cu cng cc c trng c bn ca chng. Bng 2.7: Cc dung mi s dng Dung miKhi lng ring (g/ml)Nhit si (C) Aceton0,7956 Acetonitril0,782-0,78380-82 n-Butanol0,81116-118 Butylacetat0,880-0,885117-118 Cloroform1,470-1,48060-62 Diclorometan1,3240 Dimethylformamid0,95153 Ethanol0,789-0,79178,3 Ethyl acetat0,889-0,90170,4 NH4OH 25%0,880 Methanol0,791-0,79364,5 Triethylamin0,726-0,72890 Vt liu chy sc k - Bn mng sc k: Silicagel 60 F254, Merck - Dung mi chy sc k: Cc dung mi trong bng 2.4 trn -Bnhchysck,buret,Silicagel60F254htcngknh0,01-0,063mm. 2.1.2My mc thit b - Tc nhn gy t bin: nh sng UV (=254nm), ngun pht Toshiba 60W, in th 220V. - My lc Taitec Bio Shaker BR 300 Lf, - T m Memmert, Binder, - T lnh, - T cy v trng Aura VF 48, 18 - T sy SL shellab, - L vi sng Whirlpool, - Ni hp v trng Hirayama Model HL3030, - Cn k thut Sartorius TE 210, - Cn phn tch Sartorius BP 121 S, my o UV- VIS, - My o nhit nng chy E2-Melt, - My ct quay Buchi Waterbath B480, - Knh hin vi in t, - Thc kp Palmer chnh xc 0,02mm, - Hp Petri ng knh 9cm, - Buret, bnh chit, patuyn, pipet cc loi, ng nghim,bnh nn, ng ong, cc c m cc loi, nt thy tinh, bng gc, giy lc, que cy, giy th pH, n cn, a thy tinh, kim mi mc, 2.2. Ni dung nghin cu tcccmctiubanucati,chngtitinhnhccni dung nghin cu c th nh sau: 2.2.1.Chn lc, ci to ging- La chn MT nui cy tt nht cho Streptomyces 183.219 -Chn2chngvikhun(1vikhunG(-),1vikhunG(+))lmVSV kim nh cho cc nghin cu v sau. - Tin hnh sng lc ngu nhin, la chn 3 dng chng c HTKS cao nht. - t bin bng nh sng UV t 1 n 2 ln nng cao kh nng sinh tng hp khng sinh ca chng x khun gc. 2.2.2.Ln men, chit tch khng sinh- T 3 MT ln men b mt tt nht, chn 1 MT ln men chm tt nht. -Thchinlnmentccdngchngvbinchngthuc,lachn bin chng (dng chng) c kh nng ln men to khng sinh mnh nht. - Tm dung mi hu c chit dch lc ca dch ln men v pH chit tt nht. - Tm h dung mi khai trin c kh nng tch hn hp khng sinh tt nht. 19 - Tch, tinh ch khng sinh t dch chit dung mi hu c. 2.2.3. Phn loi x khun Streptomyces 183.219 - Xc nh cc c im phn loi ISP ca Streptomyces 183.219 - So snh vi c im phn loi ISP ca mt s chng Streptomyces xc nh tn khoa hc ca Streptomyces 183.219. 2.2.4. S b xc nh mt s tnh cht ca khng sinh thu c - Xc nh nhit nng chy- o ph IR, ph UV. 2.3. Phng php thc nghim 2.3.1. Nui cy v gi ging x khun -CyzigzacxkhuntrnngthchnghingcchaMTnuicythch hp, cho pht trin 28-29C. -Sau6-10ngy,xkhunphttrintt,choccnggingvotlnh bo qun 2-4C, nh k 3-6 thng cy chuyn. 2.3.2. Phn loi x khun theo ISP- Chun b ng ging: Ging gc c nui cy trong ng thch nghing 6-10 ngy tui. - Xc nh c im hnh thi v sc t ha tan: + MT xc nh: ISP1, ISP2, ISP3, ISP4, ISP5 c hp tit trng, vo hpPetri,miMTlm4a.CyzigzacbotcaStreptomyces183.219 lnb mt thch. 28-29C, tin hnh quan st sau khi nui cy 7, 14, 21 ngy. + Mu ca khun ty kh sinh: Quan st trc tip trn b mt khun lc, c th c: mu (R), mu vng (Y), xanh l cy (G), xanh da tri (B), tm (V), xm (Gy), trng (W). Trng hp c cc mu xen k th ghi cc mu lin nhau. + Mu ca khun ty c cht: Quan st mt sau ca khun lc (mt di a Petri), thng c cc mu: vng nu, nng nu nh hoc da cam,vng nu nh xanh da tri hoc tm, vng nu ln xanh l cy. Nu thy cc mu trn k hiu l (1), nu khng thy k hiu l (0). 20 + Chui bo t v b mt bo t c quan st di knh hin vi in t c phng i cao. Dng chui bo t: thng (R), un cong (Rf), mc cu (RA), l xo (S). Nu chui bo t c nhiu c im th ghi y ccc im (v d: SRA: chui l xo mc cu). Bmtbotcthccc dngsau:phngnhn(sm),snsimncm (wa), c gai (sp), c tc (ha). +Scthatannmngaytrongmitrngcccmu:vng,xanh,, tm, Nu c k hiu l (1), nu khng c k hiu l (0). -Xcnhsctmelaninoid(muenhocnuen):Nuicyxkhun trn cc mi trng ISP6, ISP7 hp tit trng, quan st ngy th 2, 4. Nu c k hiu l (1), nu khng c k hiu l (0). - Kh nng tiu th ngun carbon: Nui cy bo t trn ccmi trng c cha cc ngun ng khc nhau (cc ISP9), nh gi ngy th 12, 14, 16. NubotphttrinmnhhnhocbngkhinuicytrnMTISPcha ccngglucose(chngdng)thkhiul(+),nukhngphttrinmnh bng khi nui cy trn MT ISP khng c ngun ng (chng m) th k hiu l (-), nu pht trin khng r rng, mnh hn chng m v km hn chng dng th k hiu l (). 2.3.3. nh gi hot tnh khng sinh bng phng php khuch tn - Nguyn tc: Mu th c t trn lp thch dinh dng cy VSV kim nh, khng sinh t mu th khuch tn vo MT thch s c ch s pht trin ca VSV kim nh to thnh vng v khun. - Tin hnh:+ To hn dch VSV: Ly mt vng que cy VSV kim nh cy vo 2,5ml MTcanhthang,37Ctrong18-24h(ivivikhun)tothnhhndch VSV c nng 107-108 t bo/ml. 21 + Cy hn dch VSV vo mi trng thch thng tit trng v ngui n45-50Cvitl2,5:100(105tbo/1ml),lcu,voccaPetriv trng vi th tch 20ml/a. + a mu th vo mi trng kim nh theo 1 trong 3 cch sau: Phng php khoanh giylc: Khoanh giylc (=6,0mm) c tm 3 ln dch chit dung mi hu c ca mu th, sy kh nhit di 50C , sau t ln b mt mi trng cy VSV kim nh. (Xem hnh P5 ph lc) Phngphpkhithch:tkhithch(=6,0mm)cchaVSV sinh khng sinh ln b mt MT cy VSV kim nh. (Xem hnh P3) Phngphpgingthch:cccgingthch (=6,0mm)trnMT cy VSV kim nh. Nh 0,05ml dch th vo mi ging. (Xem hnh P4 ) + Cc a Petri c mu th c trong t m 37C trong 18-24h (i vi vi khun). Mi mu c th song song trn 3 a vi cng 1 loi VSV kim nh. + nh gi kt qu: o ngknh vng v khun bng thc kp Palmer cchnhxc0,02mm.Ktqucnhgidatrnngknhvngv khun x l theo cng thc: 21 1( )1n ni ii iD D DD sn n Trong : D(mm): ng knh trung bnh vng v khun iD (mm): ng knh vng v khun th i s: lch thc nghim chun c hiu chnh n: S thc nghim tin hnh song song (Thng thng n=3) 2.3.4. Xc nh mi trng nui cy thch hp -Mcch:TmmitrngnuicyvboqunchngStreptomyces 183.219 n nh v cho hot tnh khng sinh cao nht. - Tin hnh: Streptomyces 183.219 c cy v trng trn 7 mi trng c MT1, MT2, MT3, MT4, MT5, MT6, MT7 trong hp Petri, 28C trong 6 ngy. 22 Th hot tnh khng sinh bng phng php khi thch. Mi MT lm 3mu song song. Chn MT c HTKS mnh nht. 2.3.5. Sng lc ngu nhin - Pha hn dch bo t: Ly mt vng que cy bo tStreptomyces 183.219, chovongnghimcha10mlncvtrng,lcchobotphntnu,thu c hn dch bo t pha long 10-1 (nh c). Sau , pha longn nng 10-6 bng nc v trng. - Cy bo t: Dng pipet v trng ht chnh xc 0,1ml hn dch bo t cc phalong10-4, 10-5,10-6,nhlnbmtthch ca MT2trongaPetri.Dng que trang v trng dn u hn dch bo t ln b mt thch. Tin hnh trn 3 a Petri cho mi nng pha long. Cho cc a vo t m 28C trong 6 ngy cho xut hin cc khun lc mc ring r. - Chn lc ngu nhin: Chn cc khun lc pht trin a dng, mc ring r sau6ngyrilyracyzigzacsangMT2dttronghppetrisau6ngyemth HTKS bng phng php khi thch. 2.3.6. t bin bng nh sng UV - Pha hn dch bo t: Ly mt vng que cy bo tStreptomyces 183.219, cho vo ng nghim cha 10ml nc ct v trng, lc cho bo t phn tn u, thu c hn dch bo t pha long 10-1 (nh c). Sau dng 1ml hn dch bo tnyphalongnnng10-6lmmuchng,9mlcnlicavoa Petri v trng. - tbin di tc dng ca nh sng UV: 9ml hn dch bo t nng 10-1 ngtrongaPetric chiunhsngUV(=254nm)vikhongcchv thi gian thch hp (khong cch 60cm, thi gian 5 pht), sau ly ra, ch ti 2h, ri dng pipet v trng pha long n nng 10-5 bng nc ct v trng. - Cy cc bo t sau t bin: Dng pipet v trng ht chnh xc 0,1ml mu chng10-6v0,1mlhndchtbinccnng10-4v10-5vocca PetricchaMT2,saudngquetrangvtrngdnugithndchbot trn b mt thch, mi pha long lm 3 a song song. Cc a Petri sau khi cy 23 c nhit 28C trong 6 ngyn khi xut hin khun lc. m cc khun lc, xc nh % sng st theo cng thc:% sng st = moNN x 10-6 + k x 100% Trong : Nm: S khun lc sau t bin nng 10-k ( 10-4, 10-5 ) No: S khun lc trong mu chng 10-6. - Tin hnh sng lc vi cc khun lc sau khi t bin tng t nh phng php chn lc ngu nhin. Lm song song mu chng. Phn trm bin i hot tnh c xc nh theo cng thc: % bin i hot tnh =100%ioDD Trong :i D : ng knh trung bnh vng v khun ca bin chng th i, o D : ng knh trung bnh vng v khun ca mu chng. -Saukhitinhnhtbin,davoktquthuclachnracc bin chng c % bin i hot tnh (+) cao nht dng cho cc nghin cu tip theo. 2.3.7. Ln men chm tng hp khng sinh - Nguyn tc: Sau khi c kt qu xc nh c cc mi trng nui cy b mttithchchovicsnxutkhngsinh,sdnggingcp1lnmengin on trong cc mi trng lng tng ng. - Tin hnh + To ging cp 1: Chng ging Streptomyces 183.219 sau khi nui cy trn thch nghing (MT2) 6 ngy tui c cy v trng sang bnh nn 500 ml cha 100 ml MT2dt bng 10 ml nc ct v trng, lc trn my lc trn nhit 28C 0,1C, tc quay 140 vng/pht trong 48 h. +Lnmen:Sau48hnhnging,tinhnhlnmentrnccMTdchth khc nhau chn MTdt ln men tt nht. Ging cp 1 c cy v trng sang bnh nn 500 ml cha cc MTdt vi t l Vging:Vmi trng = 1:10. Cc bnh ln men c lc nhit 28C 0,1C, tc quay 140 vng/pht trong 120 h. chn chng 24 ckhnnglnmensinhtnghpkhngsinhttnht:gingcp1trnMT2dt c ln men trn mi trng ti u cho vic sinh tng hp khng sinh, sau la chn chng cho dch ln men c HTKS tt nht. 2.3.8.Xc nh bn ca khng sinh trong dch ln men -Mcch:Xcnhkhnngchunhitcakhngsinhvmcnh hng ca cc pH khc nhau ln bn vng ca khng sinh.- Tin hnh: + Th bn nhit: Dch lc c phn vo 3 ng nghim, mi ng khong 7 ml. Mt ng c un si trc tip 10 pht, mt ng c un cch thy 30 pht, mtngnhitbnhthng.Saukhibangtrvnhitbnhthng, em th HTKS bng phng php ging thch. +ThbnpH:Dchlccchovo5ngnghim(lm3dysong song), mi ng khong 7 ml. iu chnh pH trong cc ng ln lt l 3, 5, 7, 9, 11, bo qun trong t lnh. Sau 1 ngy v 5 ngy, chnh pH mi ng v pH trung tnh, th HTKS bng phng php ging thch. Theo di s thay i HTKS. 2.3.9. Chit khng sinh t dch ln men bng dung mi hu c - Mc ch: Xc nh dung mi v pH chit ti u cho dch lc. - Tin hnh: Dng phng php chit 1 ln. + Dch lc c chnh v cc pH 3, 5, 7, 9, 11 bng dung dch NaOH 1M v HCl 1M. +ChitdchlcchnhpHvinhiudungmikhcnhau:Dchlcv dung mi cho vo bnh gn theo t l Vdungmi:Vdch lc = 1:5. Sau lc k trong 1 pht. yn 1 h cho phn lp. Tch ring ly lp dung mi v dch lc. +Saumilnchit,lylpdungmivlpdchlcthhottnhkhng sinh theo phng php khoanh giy. 2.3.10. Tch cc thnh phn trong khng sinh bng sc k lp mng - Mc ch: Xc nh cc thnh phn khng sinh trong dch lc hoc xc nh khng sinh tinh khit hay cha. - Tin hnh: 25 + Ct bn mng c kch thc thch hp, hot ha 110C/30 pht. + Pha hn hp dung mi theo ng t l, cho vo bnh sc k (lp dung mi khng qu 1cm), yn bnh khong 1h bo ha dung mi. +Dngngmaoqunngknh0,5mmchmdungdchcnsckln bn mng. Chm nhiu ln, kh t nhin hoc sy 50C. + t bn mng vo bnh sc k. Cho dung mi chy ln khong chiu di bn mng th ly ra, nh du ng dung mi chy. + Xc nh vt theo cc phng php sau: Soi n t ngoi: t bn mng sc k vo n t ngoi, vt cht th s hin mu. PhngphphinhnhVSV:tbnmngvoaPetritit trng,mtlpvaphiMTthchthngcyVSVkimnhsaochobn mng chm trong thch. vo t 37C, trong 18-24 h, vng v khun xut hin nu c khng sinh. (Xem hnh P6 ph lc) Phng php hin mu ha hc: Bn mng c phun thuc th hin mu, sy 70C trong 10 pht pht hin vt. Dng thuc th ninhydrin 1% trong cn pht hin cc khng sinh c cha nhm amin. Quan st bng mt thng. + Tnh h s Rf (Retardation factor) faRbTrong :a: Khong cch t vch xut pht n tm ca vt hot cht b: Khong cch t vch xut pht n vch dung mi. 2.2.11.Thu khng sinh th bng phng php ct quay Phadungmihucthucsaukhichitcctquaybngmyct chn khng. Cn thu c c ha tan bng mt lng nh methanol, vo bnh nn sch, sy 50C n kh. Co, thu ly bt khng sinh th. 2.3.12. Tinh ch khng sinh th bng sc k ct - Pha h dung mi chy theo ng t l, 30 pht cho bo ha. 26 -Chunbctsck:Ctcngknh1cm,di50cmcrasch, trng cn, sy kh. Cn khong 8 g ht Silicagel nhi ct, hot ha 110C trong 30 pht, em ha thnh hn dch vi dung mi chy v a ln ct. - a mu th ln ct: Bt khng sinh th ha tan trong mt lng ti thiu methanolritrnvikhong2gSilicagelhotha.Sy50Cchobayht methanol ri cho ln ct, n nh ct trong 30 pht. - Cho dung mi chy qua ct vi tc 0,5 ml/pht. Ly cc phn on vo ng nghim sch, khong 30 phn on, mi phn on 5 ml. - Pht hin v xc nh cc phn on cha khng sinh cn tch: Th HTKS caccphnonbngphngphpkhoanhgiylc.CcphnoncHTKS c tin hnh sc k lp mng vi h dung mi tch tt nht. Cc phn on chnh l cc phn on t khi xut hin vt n khi khng cn vt c Rf tng ng v vn c hot tnh khng sinh mnh.- Cc phn on chnh sau khi chy ct c thu gom, ct quay chn khng n cn. Co thu bt tinh th khng sinh vo ng nghim sch. Sau , bt tinh th khngsinhckttinhlibnghnhpdungmi,lc,sykh50Cthu khng sinh tinh khit. 2.3.13. S b xc nh khng sinh tinh khit thu c Khngsinhtinhkhitcoccthngs:nhitnngchy,phhng ngoi, t ngoi bc u xc nh thnh phn, phn t lng v d on nhm cu trc khng sinh thu c. 27 CHNG III: KT QU THC NGHIM V NHN XT 3.1. Xc nh tn khoa hc ca Streptomyces 183.219 ThchinccbcthcnghimphnloixkhunStreptomyces 183.219 theo ISP, i chiu vi cc c im ca mt s chng Streptomyces, nhn thyStreptomyces183.219cnhiucimtngngnhtviStreptomyces gougeroti. Kt qu c th c trnh by trong bng 3.1. Hnh nh chui bo t v b mt bo t x khun Streptomyces 183.219 (sau 10 ngy nui cy trn MT ISP2) c gii thiu hnh P1 v hnh P2 phn ph lc. Bng 3.1: Cc c im phn loi ISP ca Streptomyces 183.219 v Streptomyces gougeroti c imStreptomyces 183.219Strepmomyces gougeroti Mu khun ty kh sinhWY (trng vng)WY Mu khun ty c cht00 Sc t melanoid00 Sc t ha tan10 Chui bo tRF (cong)RF B mt bo tSm (Nhn, phng)Sm L-Arabinose D-Xylose+ Inositol D-Mannitol++ D-Fructose++ Rhamnose Saccarose- Raffinose-- Nhn xt: Nhn vo bng 3.1 c th thy Streptomyces 183.219 c 11/14 c im(78,57%)phnloigingviStreptomycesgougerotinhngchathkt lun c tn khoa hc ca Streptomyces 183.219. 28 3.2 Ph tc dng ca khng sinh do Streptomyces 183.219 sinh tng hp Ktquthhottnhkhngmtsvikhuncakhngsinhdochng Streptomyces 183.219 sinh tng hp trn MT2 c trnh by trong bng 3.2. Bng 3.2: HTKS ca Streptomyces 183.219 trn mt s VSV kim nh Vi khunGramD (mm) Staphylococcus aureus(+)25,14 Bacillus pumilus(+)26,76 Bacillus subtilis(+)26,1 Bacillus cereus(+)23,86 Shigella flexneri(-)26,64 Escherichia coli(-)25,58 Proteus mirabilis(-)25,59 Pseudomonas aeruginosa(-)24,9 Salmonella typhi(-)22,37 Nhn xt: Khng sinh doStreptomyces 183.219 to ra c hot tnh trn 9/9 vi khun th bao gm c vi khun Gram(+) v vi khun Gram(-).3.3 Kt qu chn mi trng nui cy thch hp Streptomyces183.219cnuicyringrtrn7MT.Sau6ngy,x khun ch pht trin tt trn MT1, MT2, MT5. Cc MT khc x khun khng mc hocmcrtkm.ThHTKStrn9 chngVSVkimnh.Ktqucthc trnh by bng 3.3. Bng 3.3: HTKS ca Streptomyces 183.219 trn MT1, MT2, MT5 VSV MT1MT2MT5 D (mm) s D (mm) s D (mm) s Staphylococcus aureus24,841,1125,140,8725,20,90 Bacillus pumilus25,40,7826,760,2826,060,49 Bacillus subtilis25,220,4126,10,1127,240,81 Bacillus cereus23,151,5925,161,4224,180,26 Shigella flexneri26,881,1226,950,5825,990,57 29 Escherichia coli26,000,4425,580,9025,521,32 Proteus mirabilis26,40,3126,021,7425,630,5 Pseudomonas aeruginosa23,521,3424,90,4425,120,34 Salmonella typhi22,480,9823,551,1322,091,37 Nhnxt:Streptomyces183.219phttrintttrnccMT1,MT2,MT5. Trong mnh nht l trn MT2. Do , chn MT2 l MT nui cy b mt v lu gi chng Streptomyces 183.219. Trong10chngVSVkimnhmangththtrnShigellaflexneriv Bacillus pumilus,Streptomyces 183.219th hinHTKSmnhv r rng nht.Do , chn Bacillus pumilus (Gram(+)) v Shigella flexneri (Gram(-)) l 2 chng VSV kim nh i din, dng cho cc th nghim tip theo. 3.4. Kt qu sng lc ngu nhin tin hnh sng lc ngu nhin theo 2.3.5 v kt qu th HTKS ca 32 dngchngthucsausnglcngu nhinbngphngphpkhithchc trnh by bng 3.4. Bng 3.4: Kt qu th HTKS sng lc ngu nhin Dng chng Hot tnh khng sinh Dng chng Hot tnh khng sinh S. flexneriB. pumilusS. flexneriB. pumilus D(mm)sD(mm)sD(mm)SD(mm)s 0125,261,8426,991,401725,341,2224,721,46 0225,440,5424,902,241825,701,1325,861,38 0325,161,0626,061,381925,481,0023,260,94 0424,580,7923,942,482026,402,0222,561,12 0526,302,0625,421,142125,260,1625,020,82 0626,301,1624,882,282222,180,5826,541,14 0726,861,0427,121,702325,500,2423,941,15 0826,581,1025,602,182426,040,3225,420,87 0925,080,9826,142,002523,260,2625,461,08 1026,970.8826,931,982623,981,2626,202,20 1123,963,2424,001,662724,920,9726,260,98 1225,641,4426,320,862824,741,1824,072,10 1325,580,7626,261,452926,050,3325,680,96 1426,031,4026,092,103025,660,6126,041.16 1525,300,9523,921,163126,200,6925,820,84 1625,202,0824,361,543225,860,9726,061,13 30 Nhnxt:Chn3dngchngs1,7,10(SLNN.1,SLNN.7,SLNN.10) em nhn ging, bo qun v s dng cho cc nghin cu v sau. 3.5 .Kt qu t bin ci to ging ln 1 em t bin chng SLNN.10 bng nh sng UV (=254 nm), khong cch chiu 60 cm, thi gian 4 pht 30 giy, t l sng st l 5,17 %. Th HTKS 30 bin chng sau t bin bng phngphp khi thch. Kt quchnh c trnh by bng 3.5. Bng 3.5: Kt qu th HTKS t bin ln 1 Khiu dng bin chng Hot tnh khng sinh S. flexneriB. pumilus D (mm) s % hot tnh so vi MC D (mm) s % hot tnh so vi MC B1.0126,270,59113,4925,160,62107,61 B1.0226,421,26114,1326,801,12114,63 B1.0326,721,08115,4224,730,99105,79 B1.0427,310,86117,9723,861,02102,05 B1.0525,500,57110,1525,640,45111,16 B1.0626,781,47115,6825,991,54106,89 B1.0725,301,70109,2927,350,98116,98 B1.0825,161,12108,6826,101,18111,63 B1.0925,711,14111,0427,090,98115,86 B1.1025,291,09109,2326,992,01115,44 B1.1124,851,31107,3325,551,56109,28 B1.1226,491,62114,4126,061,26111,46 B1.1327,841,52120,2627,541,09117,79 B1.1426,972,08116,5024,791,94106,03 B1.1525,691,09110,9924,191,04103,46 B1.1624,531,06105,9527,022,16115,57 B1.1724,310,82105,0326,810,70114,67 B1.1823,960,69103,524,570,95105,09 B1.1924,530,80105,9525,260,98108,04 B1.2028,050,79121,1726,381,07112,83 31 B1.2125,251,73109,0926,901,13115,05 B1.2225,972,49112,1725,821,98110,44 B1.2323,281,89100,5625,661,34109,75 B1.2427,991,36120,9126,881,18114,97 B1.2524,013,26103,7025,182,68107,7 B1.2629,511,15127,7526,930,89115,18 B1.2725,561,39110,4125,980,91111,12 B1.2828,951,99125,0427,181.26116,25 B1.2926,491,55114,4126,211,15112,21 B1.3027,410,77118,3926,070,88111,5 MC23,150,22100,0023,380,19100,00 Nhnxt:Chn 3binchngs13,26,28(B1.13,B1.26,B1.28) nhn ging, lu gi v s dng cho cc nghin cu v sau. 3.6. Kt qu t bin ci to ging ln 2 Tip tc em chng B1.12 t bin bng nh sng UV (=254nm), khong cch 60 cm, thi gian 4 pht 30 giy. T l sng st l 0,32%. Mang 35 bin chng thucthHTKSbngphngphpkhithch.Ktquchnhcthhin trong bng 3.6 ca 30 bin chng. Bng 3.6: Kt qu th HTKS t bin ln 2 Khiu dng bin chng Hot tnh khng sinh S. flexneriB. pumilus D (mm) s % hot tnh so vi MC D (mm) s % hot tnh so vi MC B2.0127,021,02100,2227,151,46101,76 B2.0229,380,53108,9828,170,41105,58 B2.0327,350,54101,4528,080,80105,25 B2.0426,860,4799,6327,990,51104,91 B2.0526,960,62100,0026,980,80104,87 B2.0627,270,68101,1527,950,47104,76 B2.0729,960,69111,1328,760,63107,35 B2.0826,941,0299,9328,61,42107,20 B2.0926,440,0098,0728,410,14106,48 32 B2.1027,890,20103,4527,510,16103,11 B2.1128,610,73106,1226,560,1899,55 B2.1226.860,5499,6327,680,56103,75 B2.1327,830,30103,2326,230,3598,31 B2.1426,980,57100,0729,000,60108,70 B2.1528,521,53105,7927,960,69104,80 B2.1628,760,52106,6828,130,76105,43 B2.1727,220,54100,9627,950,49104,76 B2.1828,010,62103,8928,660,15107,42 B2.1927,540,49102,1529,030,30108,81 B2.2027,980,60103,7828,530,63106,93 B2.2129,681,60110,0829,610,22110,98 B2.2228,631,61106,1927,920,53104,65 B2.2327,960,48103,7128,841,02108,10 B2.2430,060,65111,5029,060,65108,92 B2.2529,400,11109,0527,630,59103,56 B2.2628,841,16106,9728,970,47108,58 B2.2727,980,09103,7828,290,84106,03 B2.2829,481,49109,3527,910,97104,61 B2.2927,741,31102,8928,840,38108,10 B2.3029,421,29109,1227,710,56103,86 MC26,960,63100,0026,680,53100,00 Nhn xt: Chn 3 bin chng c HTKS tt nht l bin chng th 7, 21, 24 (B2.7, B2.21, B2.24) nhn ging, lu gi cho cc nghin cu v sau. 3.7. Kt qu chn mi trng ln men chm T kt qu chn MT nui cy b mt, chn c 3 MT tt nht l MT1, MT2vMT5.ThchinlnmenchmStreptomyces183.219trn3MTdttng nglMT1dt,MT2dtvMT5dt.Lcdchlnmenquagiylcbsinhkhi. ThHTKStheophngphpgingthch.Ktqucthctrnhbybng 3.7.

33 Bng 3.7: Kt qu chn mi trng ln men chm VSV kim nh MT1dtMT2dtMT5dt D(mm) s D(mm) s D(mm) s S. flexneri 25,090,4926,330,6619,781.64 B. pumilus 17,191,6726,290,8118,611,02 Nhn xt: Chn MT2dt l MT ln men chm hiu qu nht. 3.8 Kt qu chn chng ln men T cc th nghim sng lc ngu nhin, t bin ln 1, ln 2, chn la v lu gi c 9 dng chng v bin chng ca Streptomyces 183.219. Thc hin ln menchmringrccbinchngnytrongMT2dt.Dchlnmenclcqua giylcloisinhkhirithHTKSbngphngphpgingthch.Ktqu c trnh by bng 3.8. Bng 3.8: Kt qu chn chng ln men Dng chng, bin chng Hot tnh khng sinh S. flexneriB. pumilus D(mm) sD(mm)s SLNN.126,510,2926,680,19 SLNN.726,940,3327,010,25 SLNN.1027,060,3127,180,47 B1.1328,040,8428,080,44 B1.2627,990,4028,220,26 B1.2828,120,5828,330,45 B2.729,060,2729,210,61 B2.2130,380,9230,650,23 B2.2429,560,1829,880,69 Nhn xt: Chn chng B2.21 ln men to khng sinh. 3.9. Kt qu th bn pH v bn nhit Dch lc c th bn pH ( cc pH 3, 5, 7, 9, 11) v bn nhit (un si cch thy 30 pht, trc tip 10 pht). Kt qu c th hin bng 3.9 v 3.10. 34 Bng 3.9: Kt qu th bn pH Mu th HTKS sau 1 ngyHTKS sau 5 ngy S. flexneriS. flexneri D(mm)sD(mm)s pH 325,350,7226,260,79 pH 525,030,526,480,82 pH 726,120,7827,020,43 pH 923,820,6223,660,81 pH 1120,180,6720,920,72 Mu th HTKS sau 1 ngyHTKS sau 5 ngy B. pumilusB. pumilus D(mm)sD(mm)s pH 324,750,9724,511,02 pH 524,621,1624,611,15 pH 725,290,5625,020,83 pH 923,971,0122,980,82 pH 1119,030,2617,821,10 Nhn xt: Chng Streptomyces 183.219 kh bn vi pH t acid n trung tnh, km bn vi pH base cao. Do bo qun dch lc pH acid hoc trung tnh. Bng 3.10: Kt qu th bn nhit Mu th S. flexneri B. pumilus D(mm) sD(mm)s Nhit thng 26,28 0,5027,832,19 un si trc tip (10) 25,88 0,3827,321,40 un si cch thy (30) 25,79 0,9727,441,64 Nhn xt: Khng sinh ca Streptomyces 183.219 kh bn vi nhit , hot tnh khng sinh hu nh khng thay i.3.10 .Kt qu chn dung mi v pH chit Thc hin chit dch lc vi 4 dung mi: ethyl acetat, n-butanol, butylacetat, dichloromethan5pH3,5,7,9,11.ThHTKScaphancvphadungmi bng phng php khoanh giy lc. Kt qu c trnh by c th bng 3.11. 35 Bng 3.11: Kt qu chn dung mi v pH chit (trn S. flexneri) pHPha Ethyl acetatn-ButanolButylacetatDichloromethan D (mm) s D (mm) s D (mm) s D (mm) s 3 Dung mi25,10,6224,391,2621,040,6522,560,64 Nc7,220,1611,630,7015,710,4514,21,10 5 Dung mi24,681,1824,660,7319.690,5422,780,82 Nc9,030,2212,070,0813,070,6514,660,86 7 Dung mi25,680,9225,131,3320.680,5623,241,20 Nc7,030,1213,610,7711,930,6314,500,76 9 Dung mi24,671,0824,991,4319,471,0422,900,90 Nc9,881,0511,391,6212,310,8814,500,76 11 Dung mi19,071,8621,451,3120,241,2118,601,30 Nc13,771,2111,452,7113,591,5415,200,90 Nhnxt:Dungmiethylacetalvn-butanoluchitckhngsinh trong dch lc cc pH khc nhau, HTKS cng tng ng nhau nhng la chn ethyl acetat bi v khi chit bng n-butanol to nhiu nh tng hn, lm gim hiu sut chit ng k. Ngoi ra, n-butanol l dung mi c hn, c nhit si cao hn so vi ethyl acetat. HTKS khi chit pH 7 c gi tr cao nht. Vy chn dung mi ethyl acetat chit v chit pH 7. 3.11. Kt qu sc k lp mng chn h dung mi Dch chit dung mi hu c c dng chy sc k. Tin hnh sc k lp mng,thtrinkhaivi4hdungmi.PhthinvtbnghinhnhVSV(Vi khun s dng l S. flexneri). Thnh phn cc h dung mi v kt qu th c th hin bng 3.12. Bng 3.12: Kt qu chn h dung mi chy sc k Dung miThnh phnT lRf 1Chloroform: methanol: amoniac 25%2: 2: 10,76 2Butanol: ethanol: dimethylformamid3: 1: 10,81 3Butylacetat: ethanol: triethylamin1: 2: 10,79 4Ethyl acetat:propanol:dichloromethan2: 2: 10,85 36 Nhn xt: C 4 h u cho kt qu c vt khng sinh. H 4 c kh nng tch tthncvvthinhnhVSVcdnggitncngc,khngkoui,trong khi vi cc h 1, 2, 3 vt gn nh hnh trn. 3.12 Kt qu sc k ct Dchlccadchlnmengplickhong5lt,emchitbng ethylacetat pH 7 thu c 800 ml dch chit hu c. Lng dch chit ny mang ct quay bng my ct chn khng Buchi Waterbath, thu c 0,2346 g bt khng sinh th. Lng khng sinh th c chy qua ct Silicagel vi h dung mi 4 (Ethyl acetat:propanol:dichloromethan(2:2:1)).Ly25phnon,miphnon3ml vong nghim sch. Th HTKS tng phn on bng phngphp khoanh giy lc (trn S. flexneri). Kt qu c th c th hin trong bng 3.13. Bng 3.13: Kt qu chy sc k ct Phn onD(mm)sPhn onD(mm)s 10,000,001212,190,68 27,890,481311,610,78 325,370,121412,461,48 425,531,151511,771,02 524,490,71611,290,55 621,551,681710,561,09 719,870,911810,051,92 816,430,82199,650,14 915,731.20208,370,30 1013,270,97216,540,20 1114,530,1422 n 250,000,00 Nhn xt: C t nht 3 cht c HTKS trong bt khng sinh th thu c. C th phn on khng sinh chnh l cc phn on t 3 n 11. Tin hnh sc k lp mng vi cc phn on , h dung mi chy l h ethyl acetat : methanol (25: 1) . Kt qu c th hin bng 3.14.

37 Bng 3.14: Kt qu sc k lp mng sau chy ct (trn S . flexneri) Phn on345678910 Rf Vt1 0,36 Vt2 0,50 Vt1 0,40 Vt2 0,52 Vt1 0,41 Vt2 0,54 0,40 0,37 0,51 0,43 0,38 Nhn xt: - Phn on chnh l phn on t 3 n 5, c gp li,sau emcchnkhng,kttinhc0,1062gKS.Nhvyhiusuttinhcht: 45,27%. -emlngkhngsinhsaukhitinhchln1,chyctvih dungmiethylacetat:methanol(25:1)thuc20phnon.ThHTKScc phn on, kt qu c th hin bng 3.15. Bng 3.15: Kt qu chy sc k ct Phn onD(mm)sPhn onD(mm)s 10,000,001116,791,31 218,270,421215,591,78 321,490,531315,701,80 424,081,031415,271,02 524,270,791512,320,55 625,210,671610,581,02 724,060,841710,051,20 822,730,98189,650,14 923,460,61198,370,30 1019,071,04T 20-250,000,00 Nhn xt: - Phn on chnh l phn on t 2 n 11. Tin hnh SKLM cc phn on trn h dung mi ethyl acetat : methanol (25: 1). Kt qu thu c th hin trong bng 3.16 Bng3.16: Kt qu sc k lp mng sau chy ct ln 2 (trn S. flexneri)Phn on 234567891011 Rf0,420,480,380,390,390,380,390,380,440,46 Nhn xt: - Phn on chnh l phn on t 4 n 9, em gp li, c chn khng, kt tinh c 0,0868 g khng sinh tinh khit. Hiu sut tinh ch ln 2 l81,73 % v 38 tngqutrnhtinhchl37,00%.Khngsinhtinhkhitnydng ophIR, UV, nhit nng chy v khi ph. 3.13 Kt qu o nhit nng chy, o ph ca khng sinh tinh khit Nhit nng chy ca khng sinh: 218,4C. Ph t ngoi: cho cc nh hp th : 334nm, 445nm v 572 nm. T , d on cu trc KS c nhn thm, nii lin hp, d t O, N, halogen, hoc kt hp cc c im trn. Phhngngoi:chothyccbcsnghpthccicngccnhm chc d on tng ng c trnh by trong bng 3.17.

Bng 3.17: Kt qu IR (nm)Nhm chc c trng 484; 579Nhm R-X(vi X l Br, I) 734; 840Alkyl clorid ( R-Cl) 1099; 1193;1271ctrngchoccnhmchcalcol,ether,acidcarboxylic, ester. 1478Nhm imin (>C=N-) 1584Nitro ( -NO2) 1655Amid (R-C=O-NRR) 1742Ester (R-CO-OR) 2926; 2975c trng cho nhm alcol v phenol 3406c trng cho cu trc alken v vng thm Ph khi : Khi lng phn t d kin ca khng sinh thu c l 744,10 vC. 39 KT LUN V KIN NGH KT LUN Sau mt thi gian nghin cu, chng ti c bn hon thnh c mc tiu ban u ca kha lun tt nghip. Cc kt lun c th nh sau: - Cha th kt lun c chnh xc tn khoa hc caStreptomyces 183.219, m ch xc nh c Streptomyces 183.219 gn ging vi Streptomyces gougeroti. - Xc nh c cc iu kin ln men v chit tch khng sinh : + Tm c MT nui cy b mt tt nht l MT2 v MT ln men chm tt nht vi chng Streptomyces 183.219 l MT2dt.+ Chit khng sinh t dch ln men bng dung mi ethylacetat pH 7 cho hiu qu tt nht. -Qua2lntbinbngUVthhottnhkhngsinhcaStreptomyces 183.219 tng ln mnh m . -KhngsinhdoStreptomyces183.219sinhtnghpcmtscim nh sau: + L khng sinh c ph tc dng rng, trn c vi khun Gram(+) v vi khun Gram(-), + Tng i bn vi nhit , + Bn vi pH acid v trung tnh, khng bn vi pH base, + C t nht 3 thnh phn hot ng trong khng sinh th thu c, + Hiu sut tinh ch khng sinh t khong 37,00%, + Khng sinh tinh khit c nhit nng chy 218,4C, hp th nh sng t ngoi,hngngoi.Cutrcphntkhngsinhcdoncthchanhn thm,niilinhp,dt;ccnhmchcctrongkhngsinhl:ester,alcol, ether, imin, amin, nitro, amid, vng thm. 40 XUT Tnhngktquthuc,chngtixuttiptcphttrinti nghin cu su hn theo cc hng sau: -Tinhnhgiitrnhtgenxcnhchnhxctnkhoahcca Streptomyces 183.219. -TiptctbinchngStreptomyces183.219(bngUV,bnghacht, phng php t bin bc thang, ) to ra chng c kh nng siu sinh tng hp khng sinh. - Kho st tm iu kin ln men ti u. -Nghincuccphngphpchit,tchtorakhngsinhvitinh khit v hiu sut cao hn. Chit tch thu ly cc khng sinh ph v nghin cu su hn. - Tin hnh ph khi, cng hng t ht nhn, xc nh cc tnh cht l, ha, xc nh chnh xc cu trc ha hc ca khng sinh do Streptomyces 183.219 sinh tng hp, v nghin cu kh nng ng dng. 41 TI LIU THAM KHO Ting Vit 1.Trn T An ( 2007), Ha phn tch, NXB Y hc, tp 2. 2.Trn T An (2002), Phng php chit ng dng trong kim nghim v c cht, Trung tmThng tin- Th vin H Dc H Ni, tr. 40-41, 49-59. 3.Trn Th Hng Anh ( 1993), Quang ph hp th t ngoi- kh kin v ng dng trong nh lng khng sinh, NXB Khoa hc v k thut. 4.B Y t ( 2009) , Dc in Vit Nam IV , trang PL129-PL131. 5.BYt(2007),Dclhc,NhxutbnYhc,HNi,tp2, tr.130-142. 6.B Y t (2008), Vi sinh vt hc, NXB Y hc, H Ni. 7.NguynVnCch(2004),Cngnghlnmenccchtkhngsinh, Nh xut bn Khoa hc k thut, H Ni, tr.11-16. 8.LHuyChnh(2007),Visinhvthc,NXBYhc,HNi,tr.15-16, 50-56. 9.Nguyn Ln Dng (2001), Vi sinh vt hc, NXB Gio dc, tr.39-67. 10. Nguyn Ln Dng, Nguyn NKim Tho (2006),Cc nhm vi khun ch yu- Phn loi x khun. http://vietsciences.free.fr/khaocuu/nguyenlandung/phanloaixakhuan01.htm 11. Bi Th H (2008) , Nghin cu x khun thuc chi Streptomyces sinh cht khng sinh chng nm gy bnh trn cy ch Thi Nguyn, Lun vn thc s Sinh hc, trng i hc S Phm, Thi Nguyn, tr.3-16. 12. Trn c Hu (2006), Ha dc, NXB Y hc, tp 2.42 13. TMinhKong(2004),Cscngnghsinhhcvsnxutdc phm, Nh xut bn Y hc, H Ni, tr.42-54. 14. T Minh Kong (2006), K thut sn xut dc phm II, NXB Y hc, tp 2. 15. on Th Nguyn(2009), Vi sinhvt, NXBGio dc Vit Nam, tr.7-37. 16. Lng c Phm (1999), Cng ngh vi sinh vt, NXB Nng nghip. 17. HVitQu(2002),Chittch,phnchia,xcnhchtbngdung mi hu c, Nh xut bn Khoa hc K thut, H Ni, tp 1, tr.9-27. 18. Nguyn Vn Thch (2009), Cng ngh sinh hc dc, NXB Gio dc Vit Nam, tr.35-37. 19. Trn Th Thanh (2001), Cng ngh vi sinh, NXB Gio dc, tr.40-52. 20. NguynVnThanh(2009),Cngnghsinhhcdc,Nhxutbn Gio dc, H Ni, tr.14-57. 21. Cao Vn Thu (1998), Bi ging v khng sinh v vitamin. Ting anh 22.DepartmentofChemicalandMaterialsEngineeringEffectsof dissolvedoxygenlevelonrapamycinproductionbypellet-formof Streptomyces hygroscopicus Tunghai University, Taiwan, ROC. 23.VandanaPraveen,ChandraKantManiTripathi,VinodBihari,Suresh Chandra Srivasstava (2008), Production of actinomycin D by a new isolate, Streptomyces sindenensis, Annals of Microbiology, 58, pp 109- 114. 43 PH LC Khng sinhNgun gcPh tc dng ActinomycinS. antibioticusUng th Acid clavulanicS. clavuligerusVi khun Gr(+) AdriamycinS. peuceticusUng th AmphotericinS. nodosusNm BleomycinS. verticillusUng th CandicidinS. griseusNm CloramphenicolS. venezuelaeVi khun Gr(+) DaunomycinS. peuceticusUng th ErythromycinS. erythreusVi khun Gr(+) KanamycinS. kanamyceticusVi khun Gr(-) Kasugamycins. kasugaenis VikhunGr(+)vGr(-)v nm LincomycinS. lincolnensisVi khun Gr(+) NystatinS. nourseiNm OxytetracyclinS. rimosusVi khun Gr(+) v Gr(-) Pymaricins. natalnensisNm SpiramycinS. ambofaciensVi khun Gr(+) StreptomycinS. griseus VikhunGr(+)v Mycobacterium TetracyclinS. aureofaciensVi khun Gr(+) v Gr(-) VancomycinS. orientalisVi khun Gr(+) Bng 1: Mt s khng sinh do Streptomyces to ra 44 Hnh P1: Hnh nh chui bo t Streptomyces 183.219 ( Sau 10 ngy nui cy trn MT ISP 2 ) Hnh P2: Hnh nh chui bo t Streptomyces 183.219 45 (Sau 10 ngy nui cy trn MT ISP2) Hnh P3: Th HTKS bng phng php khi thch Hnh P4: Th HTKS bng phng phng php ging thch 46 Hnh P5: Th HTKS bng phng php khoanh giy lc Hnh P6: Pht hin vt sc k bng phng php hin hnh VSV 47 A : Ph t ngoi B: Bng pic Hnh P7 : Ph t ngoi ca khng sinh 48 Hnh P8: Tinh ch khng sinh bng sc k ct Hnh P9: Sng lc ngu nhin x khun. 49 Hnh P10 : Kt quo ph khi ca khng sinh do Streptomyces183.219 sinh tng hp 50 Hnh P11: Ph hng ngoi ( IR)