PH N IICNG NGHVI SINHPh n II: CNG NGHVI SINH 199XC NH NHANH Lactobacillus B NG PH NG PHPPCR (Polymerase Chain Reaction)Tr n Hong Ng c i, Tr n nh Nguy t, L Th H ng Tuy t, Hong Qu c KhnhPhng Vi sinh ng d ng, Vi n Sinh h c Nhi t iM ULactobacillus c vai tr quan tr ng trong cng nghi p s a, mu i chua rau qu , l myaourt,...c ngnh trongnngnghi pvd cph m. Vm t ctnhkhcc alactobacillustr nn cxemtr ngl chngckh nngt orabacteriocin(ch tkhngkhu n)nh lactacin,brevicin,lacticin,helveticin,sakacin,plantacin,...ctcd ng cch m ts visinhv tgyb nh,ngnch ns phttri nc accngu nb nhtrong th c ph m (Batt, 1999; Duber net et al., 2002). V cc ch ng lactobacillus th ng ctr trong ng ru t c a ng i v ng v t v i m t l ng s n c sgip kch thch ti uho th c n v tiu di t m t svi khu n gy b nh ng ru t khc nn hi n nay trn thtr ng c nhi u th c ph m, d c ph m c bsung cc ch ng probiotic c n s ng.Lactobacillusbaog mtrn25loivm c utin phnbi tchngl d atrnthnhph nc as nph mcu i;m ts lactobacillusl nmen nghnhtrongkhim ts khcthlnmend hnh.Ngoiph ngphpphnbi td atrncc ctnhphttri n nh ngnhi t ,pHvn ng mu ikhcnhauc alactobacillus,c ncph ng php khc phn bi t chng l d ng ln men carbohydrat, th y gi i arginine,th y gi i peptidoglycan v d ng t ng ng DNA-DNA.Tuynhin,dogi ngv inhi uvikhu nlactickhcv ccy uc uphttri nvdinhd ngnnth ngkhkhis d ngph ngphpvisinhv ttruy nth ng xcnhchngngayc m cgi ng.Vv y,nghinc unyt ptrungvo ngd ngkthu tsinhh cphnt d atr nphntchd li utrnht genrDNA16S xcnhnhanh lactobacillus. Vi c sd ng m i c hi uchogen m horRNA 16S b ng PCR c th c hi n nhl m t ph ng php xc nh.V T LI U V PH NG PHPCc ch ng vi khu n v i u ki n nui c yCh ng vi khu nCcch ngvikhu nLactobacillus cdngtrongnghinc unyl cthunh nt ARSCultureCollection(NRRL)baog m: Lactobacillusacidophilus,Lactobacillusbulgaricus,Lactobaci llusjohnsonii,Lactobacillusreuteri,Lactobacillussakei. Ngoi ra cn c ch ng Streptococcus faecium, Bacillus subtilis.Cc i u ki n nui c y Mitr ng:Vikhu nLactobacillusvStreptococcus cnuic ytrongmiH i ngh KHOA H C V CNG NGH2007 200tr ngMRS(deMan-Rogosa-Sharpe);cn B.subtilis cnuic ytrongmitr ng LB (Luria-Bertani ) Nhi t : Cc ch ng vi khu n ny c nui 37oCTch chi t DNA (Theo Sambrook J. et al, 1989) Tbo vi khu n lactic c nui c y trong mitr ng MRS l ng,cnB. subtilis c nui c y trong mi tr ng LB 37oC qua m Thusinhkh ic a5mldchvikhu ntrongeppendorfb ngcchlytm 10000vng/ pht trong 2 pht Thm 500 l dch huy n ph phn gi i tbo, vortex m nh dch tbo ng nh t Cho vo m i eppendorf 10mg ct tr ng, vortex m nh, trong 30 giy Thm 150 l dung dch Potassium acetat 3M, pH 4.8 Vortex u,lytm 10000vng/phttrong10pht,l y dchn ichovoeppendorf m i Thmvo600 lisopropanoll nh, ong ceppendorf1-2l n t a DNA,lytm 13 000 vng/ pht trong 10 pht, bdch n i m t cch nhnhng Thmvo500 lethanol70% r at a,lytm 13000vng/phttrong10pht, bdch n i m t cch nhnhng, lm kh trn gi y th m, kh tnhi ntrong khng kh kho ng 20 pht Thm vo 50 l dung dch TE, l c nh Thm vo 2 l RNAase 1mg/ml v 37oC trong 3 gi B o qu n DNA 4oC ho c - 20oC Sauxcnhhml ng v tinhs chc aDNA b ngcchoOD b csng 260nm, 280nm v ch y i n di trn gel agarose 0,8%.Khu ch i gen b ng PCRSd ng hai c p m i khu ch i gen rDNA 16S ( c t ng h p thng Proligo)v i trnh tnhsau:- C p m i 1: M i xui: 5- GGA AAC AGA TGC TAA TAC CG-3M i ng c: 5- CAC CGC TAC ACA TGG AG-3- C p m i 2: M i xui: 5- AGC AGT AGG GAA TCT TCC A-3M i ng c: 5- ATT CCA CCG CTA CAC ATG-3Thnh ph n ph n ngKhun DNA 200ngTaq DNA polymerase 1.25 UN ng m i 1 MdNTP 200 MTris-HCl, pH 8.3 20mMKCl 100mMMgCl23mMi u ki n ph n ng95oC, 5 pht; 30 chu k(95oC, 1 pht; 55oC, 1 pht; 72oC, 2 pht); 72oC, 7 pht.Ph n II: CNG NGHVI SINH 201S n ph m PCR c phn tch b ng i n di tr n gel agarose 1.5% v thu c kchth c gen rDNA 16S (khi c khu ch i v i c p m i 1) l 700 bp v kch th c genrDNA 16S (khi c khu ch i v i c p m i 2) l 340 bp.Khu ch i gen b ng PCR khu n l cSd ng Kit microLYSISTMtheo ch d n c a nh s n xu t Microzone Limited v icc b c nhsau: T bovikhu nlactic cnuic ytrongmitr ngMRSl ng,cn B.subtilis c nui c y trong mi tr ng LB 37oC qua m Tr n 3 l dch vi khu n trn v i 17 l dung dch microLYSIS tvomychuk nhi tv ich : 65oC,5pht; 96oC,2pht; 65oC,4pht;96oC, 1 pht; 65oC, 1 pht; 96oC, 30 giy; gi20oCSau khi ch y v i chu knhi t trn, t t ch n h p microLYSIS/DNAc th cdng tr c ti p trong PCR ho c c th c c t gi - 20oC.V i PCR khu n l c, ch sd ng c p m i 2 v i tr nh tnhtrn khu ch i genrDNA 16S v cho kch th c l 340 bp. Cn i u ki n ph n ng v n khng thay i.Thnh ph n ph n ng:H n h p microLYSIS/DNA 5 LH n h p DNA polymerase 1.2 UN ng m i 1 MdNTP 200 MTris-HCl, pH 8.3 10 mMKCl 50 mMMgCl21.5 mMTrong , h n h p DNA polymerase, Taq DNA polymerase, dNTP v thang DNAMarker VI c mua thng Roche.K T QUKhu ch i gen b ng PCRHnh 1. S n ph m khu ch i gen rDNA 16S c a ph n ng PCR tr n gel agarose 1,5%.M: thangDNA Marker VI (c 11 v ch v i kch th c ttrn xu ng: 2.20, 1.80, 1.20, 1.0, 0.65, 0.52, 0.45, 0.39,0.30, 0.23, 0.22 kb) ; 1, 4, 6: Lactobacillus acidophilus c khu ch i v i c p m i 1; 2, 5, 7:Lactobacillus acidophilus c khu ch i v i c p m i 2; 3: B. subtilis c khu ch i v i c pm i 1; 8: B. subtilis c khu ch i v i c p m i 2H i ngh KHOA H C V CNG NGH2007 202Tk t qutrn cho th y: Khi khu ch i gen rDNA 16S v i c p m i 1 v 2 c a L.acidophilusth uchov chDNAt ng ngl0.7kbv0.34kbnhd ki n;trongkhi B. subtilis cho k t qum tnh.Khu ch i gen b ng PCR khu n l cHnh 2. S n ph m khu ch i gen rDNA 16Sc a ph n ng PCR khu n l c tr n gel agarose 1,5%.M: thang DNA Marker VI; 1: Lactobacillus acidophilus; 2: Lactobacillus johnsonii; 3: Lactobacillussakei; 4: Lactobacill us bulgaricus; 5: Lactobacillus reuteri; 6: Streptococcus faeciumQuak tqu trncth k tlu n:M cdkhcnhauv loinh ngcnglgi ngLactobacillus nn khi khu ch i gen rDNA 16S v i c p m i 2 th s n ph m PCR ucho v ch DNA l 0.34 kb; cn Streptococcus faecium th cho k t qum tnh.Nhv y, chai m i 1 v 2 u c hi u cho gi ng Lactobacillus v gip ta c thphthi nchngtrongs nph m cnhi ugi ngkhcnhauc acngm tnhmho cngoi nhm vi khu n lactic.K T LU NV i c p m i c hi u cho gen rDNA 16S, chng ti xc nh c Lactobacillusm c gi ng khi sd ng ph ng php PCR (v i khun l DNA c tch chi t) vph ngphpPCRkhu nl c. cbi tph ngphpPCRkhu nl cchok tqunhanhh n.ylk thu tsinhh cphnt cdng xcnh,phnlo iccvikhu n th c (Eubacteria) v dng pht hi n cc vi khu n gy b nh trong th c ph m,n c, mph m, Do , t i gp ph n cho vi c pht hi n v nh danh n m c loi c a cc vi khu n lactic ni ri ng v vi khu n th c ni chung.TI LI U THAM KH O1. BattCA. (1999). Latobacillus.Introduction. EncyclopediaofFoodMicrobiology.Academic Press.2. Dubernet S, Desmasures N, Gueuguen M. (2002). A PCR-based method for identificationof lactobacilli at the genus level. FEMS Microbiology Letters 214: 271-275.3. Heilig H, Zoetendal EG, Vaughan E,Marteau P, Akkermans A, and deVos. (2002).Molecular diversityofLactobacillusspp. and other lactic acid bacteriainthehuman intestineas determinedby specific amplificationof16S ribosomalDNA.Appl Environ Microbiol68:114-123.Ph n II: CNG NGHVI SINH 2034. Reid G.(1999). ThescientificbasisforprobioticstrainsofLactobacillus. ApplEnviron Microbiol 65: 3763-3766.5. Sambrook J, Fritsch EF and Maniatis T. (1989). Molecular Cloning. A LaboratoryManual, 2nded. Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY.6. Tannock GW, Munro K, Harmsen HJM, Welling GM, Smart J, and Gobal PK. (2000).Analysesofthefecalmicrofloraofhumansubjectsconsumingaprobiotic productcontaining Lactobacillus rhamnosus DR20. Appl Environ Microbiol 66: 2578-2588.7. Walter J,Hertel C,Tannock GW, Lis CM,Munro K, and Hammes WP . (2001).DetectionofLactobacillus,Pediococcus,Leuconostoc,andWeissellaSpeciesinHuman Feces by Using Group-Specific PCR Primers and Denaturing Gradient GelElectrophoresis. Appl Environ Microbiol67: 2578-2585.SUMMARYThe PCR- based rapid identifying method forLactobacillusTran Hoang Ngoc Ai, Tran Anh Nguyet, Le Thi Hong Tuyet, Hoang Quoc KhanhInstitute of Tropical BiologyThe sequencing and analysis of the 16S and 23S rDNA genes are considered as oneof the cornerstones of modern microbial taxonomy. These sequences are used to definegenus-specificPCRprimersforarapiddetectionoflacticacidbacteria(LA B).Moreover, in recentyears, several rapid methods for the detection and identification oflactobacillihavebeendeveloped. Traditionalmicrobiologicalassaysfortheidentificationoflactobacillusspeciesareveryoftentime -consumingandcanyieldrather variable results. LAB strains including Lactobacillus acidophilus, L. johnsonii, L.reuteri, L.bulgaricus, L.sakeiwerecollectedfromfreeze-driedsamplesofARSCulture Collection (NRRL). Theywere activatedand incubated in MRS broth mediumat 37oC to get biomass; then they were extracted DNA as a template of standard PCR orused directly in colony PCR. Both of this two PCR methods were used to amplify 16SrDNA gene with lactobacillus genus -specific primer. In this work, we used two primersfor amplifying 16S rDNA gene and generated PCR product sizeas 0.7 kb (for primer 1)and 0.34 kb (for primer2) for five lactobacillus species. Electrophoresisdid not revealanydiscretebandswhen Bacillussubtilis, StreptococcusfaeciumDNAwereusedastemplate.IdentificationoflactobacillusincolonyPCRhavearesultfasterandmorehigh yield than standard PCR because there was a DNA polymerasemixture includingTaqDNApolymeraseandTgoDNApolymeraseinExpandHighFidelitySystemdesignedfrom RocheLtd. Thus, we showed that the genus -specific primers could be auseful tool for identification of the members of lactobacilli.H i ngh KHOA H C V CNG NGH2007 204BI N N P DI TRUY N GIN TI P NH Agrobacteriumtumefaciens VO N M B NH CY Phytophthora palmivoraTr n Hong Ng c i v Hong Qu c KhnhPhng Vi sinh ng d ng, Vi n Sinh h c Nhi t iM UBi nn pditruy nd atr nkh nngt nhinc a Agrobacteriumtumefacienslchuy no nDNAt Tiplasmidc an,T-DNA,vot both cv tvn mvhanh png unhinvonhi ms cth trongnhn.Ti ntr nhchuy nT-DNAph thu cvobi uhi nc agen vir(gengy c)c a A.tumefaciens.S bi uhi nc agen gy cny cc m ngb is ti trac am ts ch tt v tth ngth cv tnhacetosyringone (AS). Sc m t c a bin (border) tri v bin ph i T-DNA l c n thi ttrong ti n trnh ny.Trong tnhin c r t nhi u b nh th c v t do n m gy ra, d n n tnh tr ng nngsu tmamngth p nhi un itrnth gi i.Vv yn mgyb nh th cv tl it ng c n c nghin c u khng ch v c tnh nui c y, sinh ha, b nh l m cn m c di truy n phn tc thbi n i di truy n c ng nh ki m sot b nh gy ra.Trongboconytrnhbycck tqu thu cv bi nn pditruy nn mPhytophthorapalmivora,n mgyb nhchnhtr ncycaosuvcyntrinh s uring, ca cao, b i.V T LI U V PH NG PHPCh ng vi sinh v t Cc ch ng Escherichia coli DH5o v Agrobacterium tumefaciens LBA 4404 mttrong bi bo (3). Phytophtora palmivora thu nh n tTrung tm Ki m dch th c v t Vng II. Plasmid pPK2 l vector thc p c thi t kcho bi n n p vi n m (2).Mi tr ng Mi tr ng LB dng nui c y E. coli v A. tumefaciens. Mi tr ng V-8 dng nui c y n m P. palmivora. Mi tr ng nui c y chung vi khu n v n m theo (3). Mi tr ng ch n l c thbi n n p P. palmivora theo (3). Mi tr ng nui c y tch chi t DNA c a P. palmivora l mi tr ng l ng malt-cao n m men (MY).Cc v t li u khcCc v t li u dng trong th nghi m t ng ttrong (3).Ph n II: CNG NGHVI SINH 205Ph ng phpBi nn pplasmidpPK2vo E.colib ngph ngphpCaCl2l nhtheoSambrookv cs (6).Tch chi t plasmid pPK2 t E. coli theo bkit UltraClean 6 Minute Mini PlasmidPrep (hng MO BIO)Bi n n p plasmid pPK2 b ng xung i n vo A. tumefaciens theo (4).Tch chi t plasmid pPK2 t A. tumefaciens theo Sambrook v cs (6).Bi n n p Phytophthora palmivora gin ti p qua A. tumefaciens b ng ph ng phpnui c y chung (3).Tch chi t DNA genome c a n m P. palmivora: C y n m vo 30 ml mi tr ng MY, trong t i trong tr ng thi tnh 3 ng y. Ly tm thu sinh kh i c a 5 ml dch n m trong eppendorf 6000 v/p trong 5 pht. Cho vo 500 l dung dch phngi i tbo v thm 10mg ct tr ng.Vortex m nhtrong 1 pht, t trong 1 pht, l p l i 2 l n. Thm vo 150 l potassium acetate 3M. Ly tm 10.000 v/p trong 10 pht, thu d chn i vo ng eppendorf m i. Cho vo 600 l isopropanol l nh, o ng c tr n, t trong 10 pht, ly tm13.000 v/p trong 10 pht, rt nhnhng dch n i, thu t a. R a t a v i 500 l ethanol 70% l nh, ly tm 13.000 v/p trong 10 pht, rt dch n i,thu t a, ph i kh tnhin trong khng kh. Hatant av i20 l mTE.Thmvo1 lRNAse1mg/ml, 370Ckho ng3gi . Gi40C.N ng v s chc aADNki mtrab ngquangph b csng260v280nm. Ngoi ra DNA cn c ki m tra b ng i n di tr n gel agarose 0,8%.Ki m tra shi n di n c a gen hph (gen khnh hygromycin B) tr ong DNA genomec a P.palmivora b ng ph n ng PCR:Ph n ng PCR th c hi n v i c p m i c hi u cho gen hph l hph-F v hph-R trong i uki n: 940C, 5 pht; 30 chu k(940C, 1 pht, 600C, 1 pht, 720C, 2 pht); 720C, 10 pht.S n ph m PCR c i n di trn gel agarose 1,5 % v cho kch th c gen hph dki n 1 kb.K T QUV TH O LU NBi n n p Agrobacterium tumefaciens:Sau khi tch chi t plasmid pPK2 mang gen khng kanamycin t E. coli v bi n n pvo A.tumefaciens b ng ph ng php xung i n k t qucho th y nh ng khu n l c bi nn pm c ctrnmitr ngLBcb sung50 g/mlkanamycin,trongkhich nghoang d i th khng (Hnh 1).H i ngh KHOA H C V CNG NGH2007 206Hnh 1. Bi n n p pPK2 vo tbo A.tumefaciens. Ch ng A.tumefaciens LBA 4404 bi nn p v i plasmid pPK2 m c tr n mi tr ng LB c kanamycin (ph i),ch ng khng bi n n p (tri)Quan st sg n k t A. tumefaciens ln tn m P. palmivoraA. tumefaciensv P. palmivora cnuitrnmitr ngnuic ychung.Quanst trn knh hi n vi cho th y trong giai o n u c a qu tr nh nui c y chung di n ras ti pxcc at bo A. tumefacienslnb m tc at n m(Hnh2). i unycngha quan tr ng trong qu tr nh chuy n T-DNA tvi khu n vo n m. Cc th nghi mti p theo sti p t c l m sng tqu trnh ny.Hnh 2. G n k t c a A.tumefaciens ln tn m P.palmivora (40 X)Bi n n p Phytophthora palmivoraCh nn ng hygromycinBthchh p ch nl c P.palmivorabi nn p.C yP.palmivora hoang d i vo mi tr ng V8-agar c bsung n ng hygromycin B l 0, 50,100, 150, 200, 250 g/ml. K t qucho th y n ng 250 g/ml n m khng m c c(sli u khngcng b ), v v y n ng 250 g/ml hygromycin B cdng ch nl c thn m bi n n p. Cc khu n l c n m bi n n p xu t hi n tr n mi tr ng ch n l csau6ngynuic y,sauc ysangmitr ngV8-agarcb sung250 g/mlhygromycin B. Cc k t quch n l c thhi n tr n Hnh 3.Ph n II: CNG NGHVI SINH 207Hnh 3. Bi n n p P.palmivora nh A.tumefaciens mang plasmid pPK2. Cc thbi n n pP.palmivora m c trn mi tr ng c 250 g/ml hygromycin (B) , thhoang d i (A)Ki m tra shi n di n c a gen hph trong genome c a P.palmivora b ng PCR.Th chi nph n ngPCRv ic pm i chi uv DNAm chkhunlDNAgenomen m.S nph mPCRi nditr ngelagarosechobandckchth ct ng ng v i kch th c o n gen hph c trong o n T-DNA c a plasmid pPK2. i u ch ng tT-DNA c a pPK2 ha nh p vo trong DNA genome c a n m P.palmivora.2 1 3 4 5 61.0 kb2 1 3 4 5 6 2 1 3 4 5 61.0 kbHnh 4. Ki m ch ng dng P.palmivora bi n n p c mang gen hph b ng PCR. 1, Thang DNA marker VI(Roche ); 2, S n ph m PCR tkhun plasmid pPK2; 3, s n ph m PCR tkhun DNA c a ch ng hoangd i; 4, 5, 6, s n ph m PCR tkhun DNA c a ch ng bi n n pylnh ngk tqu uti nv bi nn pditruy nn mgyb nhth cv tP.palmivora gin ti n nh A. tumefaciens. Ni chung bi n n p di truy n vo vi n m g pnhi ukhkhnchonns phttri nc abi nn pditruy nnh Agrobacteriumgipchr tnhi uchovi c avoccgenmongmu n,ho ct oracc tbi ncnhh ng gip cho vi c nghi n c u cchgy b nh trn th c v t (2, 5). ng th i, qua ccthnghi mbi nn pnh Agrobacteriumchoth yk thu tn yt ng i ngi nvt n t cng s c so v i cc kthu t bi n n p khc.H i ngh KHOA H C V CNG NGH2007 208TI LI U THAM KH O1.CassagoA.,etal.(2002).Cellophanebasedmini -prepmethod forDNAextractionfromthefilamentousfungus Trichodermareesei.BMCMicrobiology(xem:http://www.biomedcentral.com/1471-2180/2/14).2.CovertS. F.,etal.(2001). Agrobacteriumtumefaciens -mediatedtransformationofFusarium circinatum. Mycological Re search 105: 259-264.3. Hong Qu c Khnh, Tr n Hong Ng c i (2003). Chuy n gen khng hygromycin Bvo vi n m Trichoderma harzianum b ng ph ng php gin ti p nh Agrobacteriumtumefaciens. Bo co khoa h c, H i ngh ton qu c l n th2. Nh ng v n nghinc u cb n trong khoa h c ss ng: 930-934, Nxb Khoa h c v Kthu t H N i.4.McCormacA. C.,etal.,(1998).Asimplemethodfortheproductionofhighlycompetent cells of Agrobacterium for transformation via electroporation.MolecularBiotechnology 9:155-159.5.MullinsE. D. andKangS.,(2001).Transformation:Atoolforstudyingfungalpathogens of plants.Cell Mol. Life Sci. 58: 2043-2052.6.SambrookJ.,etal.,(1989).MolecularCloning:ALaboratoryManual,2ndEd.ColdSpring Harbor Laboratory Press.SUMMARYAgrobacterium tumefaciens-mediated genetictransformation of the phytopathogenPhytophthora palmivoraTran Hoang Ngoc Aiand Hoang Quoc KhanhInstitute of Tropical BiologyAgrobacterium tumefaciens-mediated transformation has been successfully appliedtothephytopathogenPhytophthorapalmivora.TheT -DNAwasintegratedintothegenome and was stable through cell division. The presence of hph gene in P.palmivoragenomic DNA was checked by PCR. These findings should facilitate future analysis ofP.palmivorapathogenicityandstimulatewideruseofthisvaluabletransformationmethod in fungal research.Ph n II: CNG NGHVI SINH 209BI N N P DI TRUY N N M R M Volvariella volvaceaB NG PH NG PHP BI N N P GIN TI P NHAgrobacterium tumefaciensTr n Hong Ng c i, Hong Qu c KhnhPhng Vi sinh ng d ng, Vi n Sinh h c Nhi t iM UN m r m Volvariella volvacea l m t n m n c nui tr ng phbi n nh ng vngnhi t i v c n nhi t i. M c d hi n nay n m r m chi m 1/5 l ng s n xu t hng nmtrn kh p thgi i nh ng hi u qusinh h c (pht tri n trn cch t hnh thnh quth ) thth p so v i cc loi n m nui tr ng phbi n khc nh Agaricus bisporus, Lentinula edodesv Pleurotus sajor - caju (Chen et al., 2003; Ding et al., 2001).N m r m c nui tr ng trn nhi u d ng ch t th i c lignocellulose nh r m c acc cy ngc c, l chu i, b ma ng, vqucd u (Cai et al., 1999). Gi ng v inhi uvin mckh nngphngi icellulose, V.volvaceas nxu tram th th ngnhi uenzymg m |-glucosidase,endo-1,4-|-glucanase,cellobiohydrolasechuy nhocellulosethnhglucose(Caietal.,1998).Ngoira, V.volvacea cnt oralaccaselm t enzym phn gi i lignin v oxy ho cc h p ch t phenol.Do t m quan tr ng c a n m r m vm t sinh h c, c gi tr kinh tv c ng l m tloi n m l n phbi n n c ta nn vi c tm hi u sdi truy n c a chng l c n thi t.B c u, chng ti chuy n gen khng hygromycin B vo V. volvacea b ng ph ngphp bi n n p gin ti p nh Agrobacterium tumefaciens d a trn c tnh tnhin c avi khu n ny l chuy n T-DNA tTi plasmid c a chng vo nhi m s c thtrong nhnc a tbo chnhth c v t ho c vi n m.V T LI U V PH NG PHPCh ng vi sinh v tEscherichiacoli DH5o[F-endA1hsdR17(rk-/mk-)supE44thi-recA1gyrA96 lac U 169(|80 lacZ M15)] mang plasmid pPK2.AgrobacteriumtumefaciensLBA4404 cdnglmth nh ntrongthnghi mbi n n p, nhn plasmid v chuy n T-DNA tplasmid vo Volvariella volvacea.Volvariella volvacea l i t ngc n chuy n gen.PlasmidPlasmid pPK2 mang gen hph (gen khng hygromycin B) c kch th c l 10,77 kbv chi u di c a gen hph l 1 kb.H i ngh KHOA H C V CNG NGH2007 210Mi tr ngMi tr ng LB (Luria - Bertani): nui c y vi khu n E. coli v A. tumefaciens; PDA(PotatoDextroseAgar):nuic yn mr m V.volvacea;IM:nuic ychung A.tumefaciens v V.volvacea;M-100agar:ch nl cth bi nn p V.volvacea;CYM-R:mi tr ngCYM(glucose20g;caon mmen2g;peptone2 g;MgSO4.7H2O0,5g;KH2PO4 0,46g; K2HPO4 1g trong 1 lt n c c t ) ch a 0,6 M Sucrosedng ch n l cth bi nn p V.volvaceal n2;MY(Malt-Yeastextract):nuic yn mr m V.volvacea tch chi t DNA.Cc v t li u khcGi y l c (Prolabo)Cc ho ch t dng cho bi n n p vi khu n v i n di gel agarose c a hng MerckCc ho ch t dng cho ph n ng PCR c a hng RocheM i c hi u cho gen hph: M i xui (hph-F): 5-AAGCCTGAACTCACCGCGAC-3M i ng c (hph-R):5-CTATTCCTTTGCCCTCGGAC-3Ph ng phpTchchi tplasmidpPK2t E.colitheoh ngd nc ab kitUltraCleanTM6Minute Mini Plasmid Prep KitTM c a hng MO BIO.Bi n n p plasmid pPK2 b ng xung i n v o A.tumefaciensTch chi t plasmid pPK2 t A.tumefaciens theo Sambrook v c ng s(Sambrook Jet al.,1989)Bi nn p V.volvaceaginti pqua A. tumefaciensb ngph ngphpnuic ychung: Dng 5ml n c c t thu dch bo tv tn m V. volvacea m c1 tu n trnmi tr ng PDA. Dngk pvtrngchuy nccmi nggi yl c(1,5x1,5cm)lnmitr ngIMagarc200 MAS.B m20 ldchbot vt n m V.volvacea tr cti pvogi a cc mi ng gi y l c; 28oC trong 3 ngy. C y A.tumefaciens - pPK2 vo 10 ml mi tr ng LB l ng c 50 g/ml kanamycin,nui c y l c 200 vng/ pht qua m nhi t phng. o OD600c a dch vi khu n. Pha long dch vi khu n v i mi tr ngIM cch a200 M AS c OD = 0,15. Nui c y l c 250 vng/ phtkho ng 4 gi nhi t phng cOD600 = 0,6-0,8. B m 50 l dch vi khu n c c m ng v i AS vo gi a m i mi ng gi y l c cn m; 28oC trong 4 ngy. Sau 4 ngy, chuy n cc mi ng gi y l c sang mi tr ng M-100 agar c 500 g/ mlcefotaxime v 70 g/ ml hygromycin B; 28oC, lo i bcc mi ng gi y l c sau 1ngy v ti p trong 10 ngy n a. Cc khu n l c n m xu t hi n tr n mi tr ng M-100 agar c 70 g/ ml hygromycinPh n II: CNG NGHVI SINH 211B c c y chuy n sang mi tr ng CYM-R c ng c 70 g/ ml hygromycin B ch n l c thn m bi n n p.Tch chi t DNA bgen c a n m V. volvacea:DNA bgen c a n m ch a bi n n p v n m bi n n p c tch chi t ts i n m c pht tri n trong 30ml mi tr ng MY; nui tr ng thi tnh trong 7 ng y v ti nhnh tch chi t DNA theo Sambrook v c ng s(Sambrook J et al.,1989).N ng v s ch c a DNA plasmid v DNA bgen c ki m tra b ng cch oOD260 nm v OD280 nm. Ngoi ra, DNA cn c ki m tra b ng i n di trn gel agarose 1%.Ki mtras hi ndi nc agenhph(genkhnghygromycinB)trongDNAb genc a V. volvaceab ng ph n ng PCR:Ph n ng PCR c th c hi n v i c p m i c hi u cho gen hph l hph-F v hph-Rtrongi uki n:940C,5pht;30chuk (940C,1pht;600C,1pht;720C,2pht);720C, 10 pht.S nph mPCR ci nditrngelagarose1,5%vchokchth cgenhphdki n l 1 kb.K T QUBi n n p Agrobacterium tumefaciensPlasmidpPK2manggenkhngkanamycin ctchchi tt E.colivbi nn pvo A. tumefaciens, cho k t qu : nh ng khu n l c c a ch ng vi khu n c bi n n p xu thi ntrnmitr ngLBcb sung50 g/mlkanamycin,trongkhich nghoang d i ( i ch ng) th khng m c trn mi tr ng c khng sinh.Hnh 1. A. tumefaciens trn mi tr ng LB-Kanamycin (50 g/ ml).Ch ng hoang d i (tri); ch ng bi n n p ( ph i)Bi n n p Volvariella volvacea Ch nn ng hygromycinB ch nl c V.volvacea bi nn p:C y V.volvaceahoang d i vo mi tr ng M-100 agar c bsung n ng hygromycin B l 0, 20,40, 50, 60, 70, 80 g/ ml. K t qucho th y n ng 70 g/ ml hygromycin B vH i ngh KHOA H C V CNG NGH2007 212cao h n , n m khng m c c; v v y ch n 70 g/ ml hygromycin B lm n ng ch n l c thbi n n p. Cc khu n l c n m bi n n p xu t hi n trn mi tr ng M-100 agar c 70 g/ mlhygromycinBsau10ngynuic y(hnh2), cc ysangmitr ngCYM-Rc ng c bsung 70 g/ ml hygromycin B, k t quthhi n qua Hnh 3.A BHnh 2. Volvariella volvacea trn mi tr ng M-100 agar - Hygromycin B (70 g/ ml)A: Ch ng hoang d i; B: Ch ng bi n n pA BHnh 3. Volvariella volvacea trn mi tr ng CYM-R - Hygromycin B (70 g/ml)A: Ch ng hoang d i; B: Ch ng bi n n pKi m tra shi n di n c a gen hph (gen khng hygromycin B) trong DNA b gen c a V.volvaceab ng ph n ng PCRTh c hi n ph n ng PCR v i c p m i c hi u cho gen hph v DNA m ch khunlDNA b gen c a n m, s n ph m PCR c i n di trngel agarosecho band 1 kbt ng ngv ikchth co ngenhphctrongT-DNAc aplasmidpPK2.Nhv y,T-DNAc aplasmidpPK2honh pvotrongDNAb genc an mVolvariella volvacea.1 kbHnh 4. S n ph m khu ch i gen hphc aph n ng PCR trn gel agarose 1,5%M: thang DNA Marker VI (c 11 v ch v ikch th c ttrn xu ng: 2.20, 1.80, 1.20,1.0, 0.65, 0.52, 0.45, 0.39, 0.30, 0.23, 0.22kb); 1: plasmid pPK2; 2: DNA c aVolvariella volvacea hoang d i; 3,4: DNAc a Volvariella volvaceabi n n pPh n II: CNG NGHVI SINH 213K T LU NChngtithunh n cch ngn mr m Volvariellavolvaceachuy ngenkhng hygromycin B b ng ph ng php bi n n p gin ti p nh A. tumefaciens. Ph ngphp bi n n p ny cho hi u qubi n n p cao, nhanh chng v t t n km. N m r m lm t n m n c gi tr kinh tnn nghin c u ny l sm u cho nh ng nghi n c uchuyn su vdi truy n c thl m bi n i hi u qusinh h c c a n m n y c ng nhgp ph n cho snghin c u nh ng c tnh di truy n c a cc n m l n khc.TI LI U THAM KH O1. Cai YJ, Chapman S, Buswell J and Chang S. (1999). Production and distribution ofendoglucanase,cellobiohydrolaseand |-glucosisadecomponentsofcellulolyticsystem of Volvariellavolvacea,theediblestrawmushroom. ApplEnvironMicrobiol 65(2): 553-559.2. Cai YJ, Buswell J and Chang S. (1998). |- glucosisade components of cellulolyticsystem of the edible straw mushroom, Volvariella volvacea. Enzyme and MicrobialTechnology 22: 122-129.3. ChenS,MaD,GeWandBuswellJ.(2003).Inductionoflaccaseactivityintheediblestrawmushroom, Volvariellavolvacea. FEMSMicrobiologyLetters218:143-148.4. CovertSF,KapoorP,LeeM,BrileyAandNairnCJ.(2001). Agrobacteriumtumefaciens - mediatedtransformationof Fusariumcircinatum. MycologicalResearch 105: 259-264.5. DeGrootMJA,BundockP,HooykaasPJJandBeijersbergenAGM.(1998).Agrobacterium tumefaciens - mediated transformation of filamentous fungi . NatureBiotechnology 16: 839-842.6. DingS,GeWandBuswellJ.(2001).EndoglucanaseIfromtheediblestrawmushroom, Volvariellavolvacea - Purification,characterization,cloningandexpression. Eur. J. Biochem. 268: 5687-5695.7. Hong Qu c Khnh, Tr n Hong Ng c i (2003). Chuy n gen khng hygromycinBvovin m Trichodermaharzianum b ngph ngphpginti pnhAgrobacteriumtumefaciens.Bocokhoah c, H inghtonqu cl nth 2.Nh ng v n nghin c u cb n trong khoa h c ss ng, 930-934, NXB Khoa h cv Kthu t H N i.8. JiaJH,BuswellJAandPeberdyJF.(1998).Transformationoftheediblefungi,Pleurotus ostreatus and Volvariella volvacea. Mycological Research 102 (7): 876-880.9. MikoschTSP,LavrijssenB,SonnenbergASMandvanGriensvenLJLD.(2001).Transformationofthecultivatedmushroom Agaricusbisporus(Lange)usingT-DNA from Agrobacterium tumefaciens. Current Genetics 39: 35-39.H i ngh KHOA H C V CNG NGH2007 21410. Sambrook J, Fritsch EF and Maniatis T. (1989). Molecular Cloning. A LaboratoryManual, 2nded. Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY.SUMMARYAgrobacterium tumefaciens -mediated genetic transformationof the edible straw mushroom Volvariella volvaceaTran Hoang Ngoc Ai, Hoang Quoc KhanhInstitute of Tropical BiologyAgrobacteriumtumefaciensisknowntotransferpartsofitstumor -inducingplasmid,theT-DNA,toplants,yeastsandfilamentousfungi;therefore,wehaveusedthissystemtotransformgerminatingbasidiosporesandvegetativemyceliumofthecultivatedbasidiomycete Volvariellavolvacea.StrainLBA4404of A.tumefacienswasprovidedbyPlantGeneticEngineeringLaboratory(InstituteofTropicalBiology).PlasmidpPK2,containinghygromycinBphosphotransferase(hph)genewiththeAspergillusnidulansPgpd.(promoterglyceraldehyd -3 -phosphatedehydrogenase),was provided by Dr. Sarah F.Covert, University of Georgia, USA. Hph gene which is 1kb longwas designed betweenLB and RB of T -DNA in this plasmid DNA. Out ofT-DNAiskanamycinresistantgeneusedtoselecttransformedbacteria.Sporesof V.volvaceawerecollectedonglassPetridishes.Thestandardgrowthmediumfor V.volvacea was PDA and incubation was performed at 28oC. pPK2 was transformed intoA.tumefaciensviaelectroporation.Bact erialcultivationwasperformedasdescribesinDe Groot et al. (1998).For induction of virulence and T -DNA transfer, A. tumefacienswasgrownoninductionmedium(IM)with200 MASand,fornegativecontrols,without AS. Selection for transformed mycel ial colonies was performed on M-100 agarcontainingcefotaxime(500 g/ml)andhygromycinB(70 g/ml)andonCYM -RcontaininghygromycinB(70 g/ml).HphgenesequenceofT-DNAwhichintegratedgenomicDNAof V.volvaceawascheckedbyPCR.Analysisoft ransformantsshowsthattheT-DNAintegratesatrandomsitesintothehostgenomeandthattheselectivemarkerisstableduringmitosisandmeiosis.Thetransformationtechniquedecribedhereinprovidesapracticalmethodforusingtransgenictechnologyi nthegeneticimprovementofthiscommerciallyimportantmushroomandrepresentsanimportanttool for the molecular genetic analysis of biological processes in this species.Ph n II: CNG NGHVI SINH 215PHN L P V NH DANH HN M MEN T I V NQU C GIA NAM CT TINNgo Duc Duy, Pham Tran To Nhu, Hoang Quoc KhanhPhng Vi sinh ng d ng, Vi n Sinh h c Nhi t iSasitorn JindamorakotNational Center for Genetic Engineering and Biotechnolog, ThailandM UN m menc r t nhi u ng d ng, ngoi nh ng s n ph m ln men truy n th ng, ngynay chng c ng d ng trong cng nghi p hi n i nhl s n xu t enzym, vitamin, acidh u cv thu c sinh h c (Domain 1998), u ny cho th y n m men l m t trong nh ng visinh v t r t quan tr ng trong i s ng, kinh tv s c kh e.G n y n m men c ghi nh n nhl n m n bo, h u h t c xc nh n N mmen thu c bn m Ascomycetous v Basidiomycetous trong t bo sinh d ng sinh s nb ng cch n y ch i v phn i, trong shnh thnh gi i tnh khng km theo giai o n t oqu th (BoekhourvKurtman1996).Tr cy,trongth igiandicc ci mki uhnh c sd ng nhl nh ng tiu chu n quan tr ng trong nh danh n m men, tuy nhi ncc c i m ny th ng khng n nh b i sthay i do i u ki n mi tr ng v i h inhi u th i gian kh o st. Do , cc c i m ny th ng tri v i k t qutrong nghi n c usinh h c phn t , kh c ph c nh ng v n ny, kthu t nh danh vi sinh v t ni chungvn m men ni ring c dng tr c ti pph ngphpsinh h c phn t . c bi t lphn tch trnh t26S c a Ribosomal DNA trong ph m vi vng D1/D2 cho sphn l p loic a n m men xd ng phbi n r ng ri v dli u cho th y r ng h u h t cc loi n mmen c thnh danh b ng trnh tny.N m men khng nh ng ng gp r t nhi u cho nghi n c u khoa h c cb n bao g mnh danh, sinh l, sinh ha v a d ng sinh h c m c nhi u ng d ng trong nng nghi p,cng nghi p v sl mi tr ng, c bi t tbo n m men l m hnh nghin c u c a tboEukaryotes (Graeme M. Walker, 1998).R t t nh ng bo co v tnh a d ng c a n m men Vi t Nam hi n nay (L ng et al.,2000), trong ph m vi nghin c u, v i m c ch l ng d ng cc ph ng php v kthu tsinh h c phn ttrong nh danh v phn lo i vi sinh v t cng v i qu trnh kh o st sad ng sinh h c c a n m men t i V n Qu c gia Nam Ct Tin.V T LI U V PH NG PHPVisinhv t;ccch ngn mmen cphnl pt l,phncontr ng,hoa vtricy t i V n Qu c gia Nam Ct Tin tnh ng Nai.H i ngh KHOA H C V CNG NGH2007 216Lytrchtrnht 26SrDNAtrongph mviv ngD1/D2;n mmennuitrongmi tr ng YM agar trong 48ginhi t 250C. Thu nh n tbo v ha tan trong200lc adunglch mlytrch(100mMtris (pH8.0),30nMEDTA(pH8.0,0.5% SDS) un si trong 15 pht. Thm vo 200l c a 2.5M potasium acetate, pH7.5, v t chng trong l nh kho ng1 gi , sau khi li tm 13000prm trong 5 phtv thunh ndchn iphatr n,ti pt cthmvom tl ngth tchchloroform:isoamylalcohol(24:1)b ngth tchdchlytrchv l cnh ,thunh nDNA sau khi thm m t thtch isopropanal v ly tm 14,000prm trong 16 pht.Ph n ng PCR c thi t l p thtch 50l baog m 1X Taq buffer, 2mM MgCl2,200MdNTP,0.3pM c am ic pm i,1.25UTaqpolymerasev 5lDNAm uchochu trnh PCR l 940C trong 1 pht, 52oC th igian 1.30 pht v 72oC trong 2.30 phtcho kho ng 30 chu trnh. S n ph m DNA c lm s ch b ng QIAquick (QIAGEN) vgi i trnh tb ng automate ABI.Hai c p m i c ng d ng cho tr nh tny nhsau; m i xui F63 (5- GCATATCAAGCGGAGGAAAAG -3)vm ing cLR3(5-GGTCCGTGGTTC AAG ACG -3).PhntchBLAST(BLASTanalysis);trnht thunh n cs sosnhv icctrnht khctrongngnhnggen(Genbank)vs d ngcngc BLASTc aNCBI(National Center for Biotechnology Information - USA).Phn tch cy phh(phylogenetic analysis); sd ng cc tr nh tt ng ngv i cc loi lin quan b ng cch sd ng ch ng trnh ClustalX 1.8 (Thompson etal.,1997).C utrccyph h cc ut ot kho ngd li uti nhatheoKimura.M(1980)b ngcchs d ngph ngphpneighbor -joining(SaitouvNei., 1987), phn tch bootstrap(Felsentien,1985) chnh thnh t 1000stil y m u ng u nhin.K T QUV TH O LU NTnh ng m u thu c chng ti phn l p c 85 ch ng n m men tl , hoa, vphn con trngt i V n Qu c gia Nam Ct Tin tnh ng Nai.Hnh 1. Cc tbo sinh d ng n m men trong mi tr ng YM broth, 2 ngy nhi t 250C(Scale 10m, 100X)P5H7L60L52 L10 L5L29 P2 L53 H9 L40 H5L24L67Ph n II: CNG NGHVI SINH 217D atrncckh ostvphntchsinhh cphnt chngtighinh n14ch ng(P2,P5,H5,H7,H9,L10,L5,L24,L29,L40,L52,L53,L60vL67)ckh nnglloi m i v ti p t c kh o st hnh thi, sinh l, sinh ha c a cc ch ng ny, 56 loi bi t v 15 loi ch a xc nh r rng.M ib nch ngn mmennuic ytrongmitr ngYMborth nhi t 250Ctrong2ngy,sauquanstd iknhhi nvichoth yas ccch ngnytisinhb ng n y ch i, chng c d ng hnh c u, hnh ovan v cc tbo t o khu n tygihnhthon di v.v. (hnh 1).Bn c nh ssinh s n b ng cch n y ch i, n m men c n c khnng hnh thnh m tskhu n ty gigi ng nhshnh thnh s i n m (hnh 3).Hnh 3. Khu n ty git o ra trn mi tr ng PDA sau 1 tu n nhi t 250C b ng kthu t a DalmauT k tqu thunh n c14ch ngkhcnhaut t tc nh ngloibi tb i5nucleotitdes ho c nhi u h n trong ph m vi vng D1/D2 thu c trnh t26S rDNA. Cc ch ngny c ngh l hi n di n loi m i theo nhth o lu n c a Kuztman v Robnett (1998), v14 ch ng ny c phn l p trong 9 loi, tnh ng loi g n chng nh t (b ng 1).B ng 1. Danh sch cc ch ng (H7, H5 L24) cng v i cc ch ng so snh trong ngnhng dli u genStrain GenBankAccession No.Nearest Species with Accession No. of DNADataBankNucleotide Identity(%)No. of NucleotideDifference (gap)AscomycetesH7 AM076407 Candida naeodendra partial 520/530 (98.1) 10(2)L5 AM076407 Candida naeodendra partial 520/530 (98.1) 10 (0)L10 AM076407 Candida naeodendra partial 519/530 (97.9) 11(3)H5 AF530612 Candida sp. UWO(PS)00-147.3 552/582 (94.8) 30(6)P5 U74598 Pichia hampshirensis 525/553 (94.9) 28 (0)H9 AF017412 Pichia lachance 513/572 (89.7) 59(11)L67 AJ508573 Pichia sydowiorum 570/580 (98.3) 10 (1)L60 AJ508573 Pichia sydowiorum 562/575 (97.7) 13 (1)L52 AJ508573 Pichia sydowiorum 564/572 (98.6) 8 (0)L53 AJ508573 Pichia sydowiorum 563/572 (98.4) 9 (0)BasidiomycetesL40 DQ640492 Cryptococcus liquefaciens strain MZKI K-490 588/591 (99.5) 11 (0)L29 AF444704 Cryptococcus sp. CBS 8368 594/603 (98.5) 9(2)P2 AF387128 Sporidiobolus ruineniae strain IGC 5692 574/580 (98.9) 6 (0)L24 AF453938 Ustilago maydis 601/608 (98.8) 7(2)L24L29H2H7 H9 P2H i ngh KHOA H C V CNG NGH2007 218Qu trnh nh danh d a trn trnh tc a ph m vi vng D1/D2 c a trnh t26SrDNA theo nhsh ng d n c a Kuztman v Robnett (1998),k t qucho th y 14 loim ihontonringbi td atrns phntchcyph h chos hnhthnhloim itrong nghin c u ny c t o thnh trong ph m vi vng D1/D2 thu c trnh t26SrDNA cng v i cc ch ng bi t g n nh t theo st ng ng trong ngn hng dli ugen (hnh 2).Hnh 2. Cy pht sinh loi c a 14 ch ng v cc loi lin quan d a trn trnh t26S rDNAtrong ph m vi vng D1/D1Ngoi nh ng dli u m sinh h c phn tcho chng ta xc nh spht hi n lo im i v loi bi t trong ngn hng gi ng v ngn hng gen cho vi c nh danh vi sinhv t ni chung v n m nem ni ring, th vi c kh o st cc dli u vhnh thi, sinh l,vsinhhac ngnh thng tinc utrcsinhh ckhcs gi ngchngtaxcnhv thng tin y vloi m i.K T LU ND a trn trnh t26S rDNA trong ph m vi vng D1/D2, 85 ch ng n m men cxcnht ccm ul,hoavphncontrngt i V nQu c giaNamCtTintnh ng Nai theo nhm n m men Ascomycetous v Basidiomycetous nhsau;+ 56 ch ng l loi bi t (21 ch ng thu c nhm Ascomycetous v 35 ch ng thu cnhm Basidiomycetous)Saccharomyces cerevisiaeWilliopsis saturnus var. mrakiiWilliopsis saturnus KCTC 1724Pichia meyeraePichia lachanceiCandida sp. CBS 5303Pichia hampshirensisPichia strasburgensisH5Candida quercuumCandida sp.Candida ulmiPichia myanmaensisPichia anomala strain VTT C-04565Pichia lynferdiiPichia subpelliculosa.L53Pichia sydowiorumL52H7Candida naeodendraCandida diddensiaePichia sp. ST-335Ustilago maydisPseudozyma prolificaUstilago vetiveriaeCintractia sorghi -vulgarisL29basidiomycete yeast sp. BG02-7-16-015A-1-1Cryptococcus sp. CBS 8368Cryptococcus sp. CBS 8365Cryptococcus sp. CBS 8364Cryptococcus liquefaciens strain MZKI K-493Cryptococcus liquefaciens strain MZKI K-490Cryptococcus liquefaciens strain UWFP -357Sporidiobolus ruineniae var. ruineniaeSporobolomyces sp. TY-228Sporidiobolus ruineniaeL24L40P2L5L10P5L60L670.05Ph n II: CNG NGHVI SINH 219+ 14ch nglloim i(10ch ngthu cnhmAscomycetousv 4ch ngthu cnhm Basidiomycetous)+ 15ch ngch a cxcnh(8ch ngthu cnhmAscomycetousv 7ch ngthu c nhm Basidiomycetous)TI LI U THAM KH O1. A. L.Demain,H. J.PhaffandC. P.Kurtzman.(1998).Theindustrialandagriculturalsignificanceofyeasts.In:KurtzmanC. PandFellJ. W(eds),TheYeasts, a Taxonomy Study Eleevier, Amsterdam, p. 13.2. Graeme M. Wallker. (1998). Yeast physiology and biotechnology, pp. 1- 8.3. Cletus P. Kurtzman and Christie J. Robnett. (1998). Identification and phylogeny ofascomycetousyeast from analysis of nuclear subunit (26S) ribosomal DNA partialsequences. Antonie van Leeuwenhoek 73, 311 -371.4. Jack W. Fell, Teun Boelkhout, Alvaro Fonseca, Gloria Scorzetti and Adele Statzell -Tallman.(2000).Biodiversityandsystematicsofbasidiomycetousy eastsaredetermined by large-subunit rDNA D1/D2 domain sequence analysis. InternationalJournal of Systematic and Evolutionary Microbiology (50): 1351 -1371.5. Dao, T. L., M. Takashima, Pham, V. T., Nguyen, L. D and Nakase, T. (2000). Fournew species of Kockovaella isolated from plant leaves collected in Vietnam. J. Gen.Appl. Microbiol., 46, 297-310.6. Nagatsuka,Y.,Kawasaki,H.,Limtong,S.,Mikata,K.andSeki,T.(2002).Citeromyces siamensis sp. nov., a novel halotolerant yeast isolated in Thailand. In t.J. Syst. Evol. Microbiol., 52, 2315-2319.7. Suzuki, M., Nakase, T., Daengsubha, W., Chaowsangket, M., Suyanandana, P. andKomagata.K(1987).Identificationofyeastsisolatedfromfermentedfoodsandrelated materials in Thailand. J. Gen. Appl. Microbiol ., 33, 205-220.8. Suzuki,M.,Nakase,T.andKomagata,K.( 1994). Candidastellimalicola,anewspeciesofanamorphicyeastisolatedfromstarappleinThailand.J.Gen.Appl.Microbiol., 40, 115-121.9. Saito,K.,Hasuo,T.,Sugano,N.,Kitamoto,K.,Watanabe ,S.,Tadenuma,M.,Nakamura,K.,Sato,M.,Akiyama,H.,Vongsuvanlert,V.,Karuwanna,P.andKumnuanta, J. (1983) Microorganisms isolated in Thailand. Rept. Res. Inst. Brew.,155, 24-41.10. Jindamorakot,S.(2000).Identification,preservationandpolyolsp roductionofhalotolerant yeasts isolated in Thailand. MSc. Thesis, Kasetsart University. 225 pp.1-225.11. Jindamorakot, S. (2006). The species diversity of yeasts in some natural habitals ofThailand. Thesis of Graduate school, Kasetsart University 2006.H i ngh KHOA H C V CNG NGH2007 22012. Leda.C.M,Eli.A.T.G,Allen.N.H(1998).YeastcommunitiesassociatedwithsugarcaninCampos,RiodeJaneiro,Brazil.Internationalmicrobiology.Vol.11998, pp. 205-208.13. Kimura,M.,(1980).Asimplemethodforestimatingevolutionaryrateofbasesubstitutionsthroughcomparativestudiesofnucleotidesequences. JournalofMolecular Evolution 16: 111-120.SUMARYIsolation and identification of Saccharomyces sp.collected from Cat Tien national parkNgo Duc Duy, Pham Tran To Nhu, Hoang Quoc Khanh(1), Sasitorn Jindamorakot(2)(1)Institute of Tropical Biology, (2)BIOTEC, ThailandEightyfivestrainsofyeastswereisolatedfrominsectfrass(5strains),leaves(67strains)andflowers(13strains)whichwerecollectedfromCatTienNationalPa rkintheSoutheastofVietnam.BasedonsequencesofD1/D2domainof26SribosomalDNA,39strainsbelongtoascomycetousyeastsand46strainsbelongtobasidiomycetousyeasts.Inthesestudy,14newspecies(16.5%)differedby6to59nucleotides, according to discussed by Kurtzman and Robnett (1998) that these strainsare suggested to represent new species, 15 strains (17.6%) differed by 2 to 4 nucleotidesarenotyetidentifiedandtheremaining56strains(65.9%)wereidentifiedasknownspecies. The eighty five species were identified as 7 genera of basidiomycetous yeasts;Cryptococcus,Sporidiobolus,Sporobolomyces,Rhotosporidium,Rhodotorula,Ustilago,Pseudozymaand4generaofascomycetousyeasts;Pichia,Candida,MetschnikowiaandSaccharomyces .FourteenstrainsassignedtonewspeciesthesestrainsclosetothegeneraCandida(H5,H7,L5andL10),Pichia(H9,P5,L52,L53,L60andL67),Sporidiololus(P2),Cryptococcus(L29,L40)andUstilago(L24).Thephylogenetic tree were constructed based on D1/D2 domain of 26S rDNA sequences of14newspeciesfoundinthisstudyandrelatedspecies,thecharacteristicscellmorphylogy,theproductionofascospore,pseudomycelium,truemyceliumandmaximu growth temperature were examined.Ph n II: CNG NGHVI SINH 221S N XU T V TH NG M I HO CC S N PH MSINH H C DNG TRONG NUI TR NG THUS NV Th H nh, L Th Bch Ph ng, L T n H ng,Tr ng Th H ng Vn, Tr n Th nh PhongPhng Vi sinh ng d ng, Vi n Sinh h c Nhi t iM UNm 2006,ngnhth ys nh ngt i m ctiuxu tkh u2,8t USD. Di ntchnuitr ngth ys nthmcanhvbnthmcanh n ctaanggiatngnhanh,s nhi m mi tr ng n c ao nui tm do l ng th c n th a, ch t th i h u c cng ngycngnghimtr ng nhh ng nnngsu tvch tl ngnuitr ng, tngkh nngnhi m b nh cho tm.Khng sinh v ha ch t c sd ng nhi u trong phng tr b nh cho cc it ngth ys nnuitr ngs gyrahi nt ngl nthu cvs t nd khngsinh trong cc m t hng chbi n l ro c n cho vi c xu t kh u cc s n ph m n y.M tgi iphp cch pnh nr ngritrnth gi ils d ngccch ph mprobiotics ch a cc vi sinh v t s ng h u ch c khnng lm s ch mi tr ng n c aonuitm,i uchnhpH,gi ms l ngvikhu ngyb nh chotm. Trnthtr nghi n c nhi u chph m probiotics c a n c ngoi dng xl ao nui tm c ngbao t i Vi t Nam c gi thnh cao.Tth c ttrn, chng ti nghin c u s n xu t v thnghi m cc chph m sinhh c probiotics nh BIOII, VEM v PB trn cc ao nui tm s cc tnh mi n Nam.Tk t qu t c, chng ti m nh d n k t h p v i cc cng ty s n xu t thu c th yvnuitr ngth ys n accch ph mnyrathtr ngv itnth ngm ic acc cng ty.V T LI U V PH NG PHPGi ngvisinhv t: Ccch ngBacillusspp.,Lactobacillusspp.,Saccharomycesspp., Rhodopseudomonas, Rhodospirillum c phn l p v ch n l c t i Vi t NamThi tb: N ilnmen(fermenter)100lt, mylytmsiut c20,000vng/pht,my s y phun, my ng kh, Ts y tnh, my xay, my tr n siu t c KBC-ST-20,tl nh m...Mi tr ng (MT) s n xu t chph m BIOII v PB: MT dch chi t ngc c dng s nxu tsinhkh ivikhu n Bacillusspp; MTMRSdng thusinhkh ivikhu nH i ngh KHOA H C V CNG NGH2007 222Lactobacillus spp; MT Malt - Cao n m men dng thu sinh kh i Saccharomyces spp;Mi tr ng Norbert Pfennig dng nui c y vi khu n quang d ng.S n xu t -amylase vprotease: t vi khu n Bacillus subtilis b ng ph ng php lnmen bn r n.S n xu t chph m VEM: C i ti n qui trnh s n xu t EM (Effective Microorganisms)c agios TeruoHiga,Nh tB n s nxu tVEMcm t visinhv th uch :Bacillus spp., Lactobacillus spp., Saccharomyces spp. V vi khu n quang h p t o rachph m VEM.S nxu tch ph m PB: Nuic yvikhu n RhodopseudomonasvRhodospirillumtrongmitr ng NorbertPfennig d inhsngm ttr i,sau2tu nnuic y,emng chai v b o qu n trong t i.Xcnhccchti usinhha: M t visinhv tb ngph ngphp mkhu nl c; ho tl c o-amylaseb ngph ngphpSmithvRoe; ho tl cenzymproteaseb ngph ngphpAnsonc iti n;nhl ngNH3-N,H2S,NO2-Nb ngph ngphp so mu.Th nghi mhi uqu ch ph mBIOIItr naonuitm: Trnaotms45ngytu i t i cc ao nui tm bn thm canh tnh B Ra-V ng Tu v i n ng 0,2-0,5 ppm. Sd ng chph m BRF2 c a Mcho ao i ch ng v i n ng 0,5 ppm. Theo d i cc chtiu nh mu n c, pH, NH3-N, H2S, NO2-N v m t vi khu n Vibrio trong n c ao.Th nghi m cc tnh Kin Giang, Ti n Giang v i li u l ng 100g/1000m2/tu n,Thnghi m hi u quchph m VEM tr n ao nui tm: Chph m VEM cthnghi m trn cc ao nui tm t i B Ra-V ng Tu; theo di tr ng l ng trungbnh,tl s ngvh s FCR(foodconversionratio), ich ngl s nph mPond-Clear.K T QUV TH O LU NChph m probiotics BIOIIB ng 3.1: Thnh ph n chph m probiotics BIOIIThnh ph n nh l ngLactobacillus spp.Bacillus spp.Sacchromyces spp.o-amylaseProtease109CFU/g1010CFU/g107CFU/g2000 UI/g20 UI/gK t qu thnghi m chph m BIOII trn ao nui tmPh n II: CNG NGHVI SINH 223B ng 3.2. Thnghi m t i B Ra - V ng TuCc t xlX l t 1(BIOII0,2ppm;BRF20,5ppm)Xl t 2(BIOII 0,5ppm;BRF2 0,5ppm)Xl t 3(BIOII 0,5ppm;BRF2 0,5ppm)Xl t 4(BIOII 0,5ppm;BRF2 0,5ppm)Th i gian (ngy)0 3 6 9 12 15 18 21 24 27 30BIOII 1.3 1.12 1.22 0.6 1.3 0.95 0.85 1.45 0.45 0.75 0.70NH3-N(mg/l)BRF2 0.63 0.53 0.35 1.40 0.90 0.95 0.45 1.50 0.90 0.75 0.72BIOII 0.074 0.126 0.098 0.008 0.045 0.000 0.000 0.000 0.000 0.005 0.002NO2-N(mg/l)BRF2 0.001 0.002 0.005 0.000 0.001 0.000 0.000 0.0005 0.002 0.002 0.001BIOII 0.023 0.013 0.013 0.051 0.016 0.049 0.023 0.011 0.013 0.011 0.007H2S(mg/l)BRF2 0.031 0.023 0.020 0.067 0.005 0.002 0.001 0.002 0.002 0.002 0.002BIOII 6083 560 340 1140 2000 700 450 270 300 300 200Vibrio(CFU/ml)BRF2 430 295 300 1350 1300 1000 500 400 300 200 200BIOII 7.3 - 8.5Cc ch tiu l hapHBRF2 7.6 - 8.2Khi tng n ng BIOII t0,2 ppm l n 0,5 ppm cc t xl th2, 3 v 4, n ng NH3-N,NO2-N,H2Ssau30ngygi mxu ng m cchophp.Ccchti upH,COD n nh, thch h p cho spht tri n c a tm. M t Vibrio gi m cn 200 CFU/ml b ng v i ao i ch ng xl v i BRF2 sau 30 ngy.B ng 3.3. K t quthnghi m chph m BIOII (100 g/1000 m2/tu n)trn ao tm t i Kin Giang, Ti n GiangKin GiangTi n GiangCc ch tiuThnghi m i ch ng Thnghi m i ch ngCh t l ng n c T t, khng c mi hi X u, c mi hi Mu n c, trong c c i thi n X u, c mi hipH 7,8 - 8,1 8,5 9,3 7,5 8,3 7,5 9,5T lb nh Khng ng k 15- 20% Khng ng k 15%Nng su t (t n/ha) 7,8 6,4 5,7 4,7Nng su t tng (%) 121.9 100 119,1 100 B ng 3.3 cho th y: Nng su t tm bnh qun tng t19,1%- 21,9%Chph m VEMB ng 3.4. Thnh ph n vi sinh v t trong chph m VEMVi sinh v t Chph m VEMVi khu n lacticVi khu n Bacillus sppVi khu n quang d ngN m men1x109 CFU/ml1x1010 CFU/ml1x105CFU/ml1x107 CFU/mlH i ngh KHOA H C V CNG NGH2007 224Thnghi m hi u quchph m VEM trn ao nui tmB ng 3.5. K t quthnghi m chph m VEM tr n cc ao nui tm t i B Ra-V ng TuCh tiu Pond-Clear VEMT ls ng (%)Tr ng l ng trung bnh (g/con)Kch ctm (Scon/kg)S n l ng bnh qun (kg/ha)T ng l ng th c n (kg)HsFCR63,9628,235,54.5453.5021,7562,7328,934,54.5462.6711,78B ng 3.5 cho th y: tr ng l ng trung bnh v t ls ng c a tm thu ho ch hai aosd ng chph m VEM khng khc bi t nhi u so v i ao sd ng s n ph m Pond-Clear.Chph m PBQua thnghi m chph m PB trn cc ao nui tm s, k t qucho th y c tc d ngphngi ith cnd th avch tth itcht d iyao,gi mBOD,COD, nnhch t l ng n c, gip cn b ng sinh thi, gi m t lb nh cho tm.Th ng m i ha cc chph m dng trong nui tr ng th y s nChng ti k t h p v i cc cng ty thu c th y v nui tr ng th y s n th ngm i ha chph m BIOII v VEM mang th ng hi u c a cc cng ty.B ng 3.6. Th ng m i ha chph m BIOI, BIOII v VEMSTT Tn s n ph m Ngu n g c i t ng Cng ty s n xu t1 NAVET Biozym BIO II Tm, c Thu c th y Trung ng23Enzym subtilisEnzymmin subtilisBacillus subtitisBacillus subtitisTm, cTm, cThu c th y Si Gn4 Biolactizym BIO II Tm, c Thu c Th y Long An II5 NP-Lactizym BIO II Tm, c Thu c th y NAPHA67L-AKLB-AKLBIO IIBIO IITm, c, (n c ki m)Tm, c, (n c phn)CP NTTS Khnh Long7891011BACILLUSMAZARBS@BACTERILASABacillus subtitisVEMVEMPbVEMTm, cTm, cTm, cTm, cTm, cat Viet Co.LTD1213BIOLAS-2BIOLAS- PbBIOIIVEMTmTm i Tr ng S nK T LU NChph m BIOII, PB v VEM c hi u qutrong vi c lm s ch mi tr ng n c aonui tm, cn b ng pH, gi m sl ng vi khu n gy b nh cho tm. Nng su t tm bnhPh n II: CNG NGHVI SINH 225qun thu ho ch tng 19,1 % - 21,9% so v i s n ph m BRF2, hsFCR th p h n so v is n ph m Pond-Clear, trong khi gi thnh c a chph m BIOII th p h n g n 10 l n.Chng ti k t h p v i cc cng ty thu c th y v nui tr ng th y s n th ngm i ha chph m BIOII v VEM mang th ng hi u c a cc cng ty.TI LI U THAM KH O1. Havenaar,R.,TenBrisk,B., Selectionofstrainsforprobioticuse . In:R.Fuller(ed.), Probiotics the scientific basic, Chapman and Hall, Londo n. P. 209-224, 1992.2. V Th H nh v cs (2003). Nghin c u s n xu t chph m h n h p vi sinh v t s ngv enzyme tiu ha dng trong chn nui v nui tr ng th y s n. Bo co nghi mthu ti, SKhoa h c v Cng nghTPHCMSUMMARYProduction and commercialize the bio-products using foraquacultureVo T Hanh, Le Bich Phuong, Tran Thanh Phong, Le Tan Hung, Truong T Hong VanInstitute of Tropical BiologyThebioproductssuchasBIOII,VEMandPBhavebeenstudyingandproducing;these bioproducts have been using for aquaculture effectively. The BIOII and VEM hadeffectoncleaningwater,pHstability,reducingpathogenicbateriaonshrimp.Theaverageyieldincreased19,1% -21,9%comparedwithBRF2products, FCR (foodconversionratio)loweredcomparedwith Pond-Clearproduct, meanwhile, priceofBIOII lower 10 times BRF2.H i ngh KHOA H C V CNG NGH2007 226GI M THI U NHI M MI HI CHU NG TR I V S NXU T PHN VI SINH TPHN CHU NG B NG CHPH MSINH H CV Th H nh, L Th Bch Ph ng, Tr n Th nh Phong,L T n H ng, Tr ng Th H ng VnPhng Vi sinh ng d ng, Vi n Sinh h c Nhi t iM UNgnh chn nui Vi t Nam ang chuy n d n tchn nui truy n th ng, phn tn,quy m nh sang chn nui trang tr i (TT), t p trung, qui m l n v s n xu t hng hoch t l ng cao.Tuy nhin, trong qu trnh pht tri n, chn nui trang tr i b c lm t sh n chnh : pht tri n tpht, thi u quy ho ch. Trong v n nhi m mi tr ng c n ph i c quan tm. ccTTchnnuiheo, l ngphnvn cth ith irav ikh il ngr tl n(kho ng 5 t n/ 15.000 u heo), phn t i c bn v i gi r (kho ng 5.000 ng/ 40kg). Do khng c xl ho c xl khng ng cch gy nhi m mi hi cho khuv c ln c n.Do,chngti aragi iphps d ngch ph m VEM-Klmgi mmihi;chph m BIO-F dng s n xu t phn vi sinh tphn v n c th i. Cc chph m sinhh ctrnch at ponvisinhv th uch cphnl pvch nl c,chu ci uki n kh h u v thnh ng Vi t Nam, do v y khng ph i phthu c v o ngu n gi ng visinh c a n c ngoi.Quagi iphptrn,chngnh n cgi ith ngNgysngt oVi tNamv ichHnh ng v mi tr ng do Ngn hng Thgi i t i Vi t Nam tch c nm 2005.V T LI U V PH NG PHPGi ngvisinhv t: Ccch ng Bacillusspp., Lactobacillusspp.,n mmenSaccharomycesspp., Rhodopseudomonas,Rhodospirillum, x khu n Streptomycessp.v n m s i Trichoderma sp c phn l p v ch n l c t i Vi t NamS nxu tch ph mVEM-K: C iti nquitrnhs nxu tEM(EffectiveMicroorganisms)c agios TeruoHiga,Nh tB n s nxu t VEM-K cm t visinh v t h u ch cao v khong a vi l ng dtan.Ph n II: CNG NGHVI SINH 227Thnghi m chph m VEM-K trn heo, g v s n xu t phn vi sinh theo ssau:K T QUV TH O LU NChph m VEM-KB ng 3.1. Thnh ph n vi sinh v t trong chph m VEM-KChph m VEM-K c thnghi m trn heo tht giai o n t20 kg n 50 kg, t itr i heo Gia Ki m tnh ng Nai . K t qutheo di cc ch tiu sau 55 ngy cho u ng(1lt VEM-K/500 lt n c s ch) ghi nh n c b ng 3.2.B ng 3.2. K t quthnghi m chph m VEM-K (1lt/500 lt n c s ch) trn heo tht.Ch tiu theo di L th nghim L i ch ngChnh l ch sov i i ch ngTr ng l ng trung bnh u vo (Kg) 19,10 19,30 -0,20T ng tr ng l ng c a l u vo (Kg) 191,00 193,00 -2,00Tr ng l ng trung bnh u ra (Kg) 48,10 42,70 5,40T ng tr ng l ng c a l u ra (Kg) 481,00 427,00 54,00T ng tng tr ng/l (Kg) 290,00 234,00 56,00Tng tr ng bnh qun/con (Kg) 29,00 23,40 5,60Tiu thth c n bnh qun/ngy (Kg) 0,99 0,90 0,09T ng l ng th c n ti u th /l (Kg) 546,50 493,00 53,50FCR (Kg th c n/kg thtr ng) 1,88 2,11 -0,23Tng tr ng tuy t i (g/con/ngy) 527,30 425,50 101,80Th i gian nui (ngy) 55,00 55,00 0Vi sinh v t Chph m VEM-KLactobacillus spp.Bacillus sppSaccharomyces spp.Vi khu n quang d ngKhong dtiu109 CFU/ml1010 CFU/ml107 CFU/ml105 CFU/ml500 mg/mlVEM pha longCho heo, gu ng h ng ngyPhn gi m mi hi c aXl b ng chph m BIO-FPhn h u cvi sinhH i ngh KHOA H C V CNG NGH2007 228K t qub ng 3.2 cho th y, ch ti u tng tr ng bnh qun tng 20% v ch tiu tiut n th c n l th nghi m gi m 11% so v i i ch ng, mi hi c a phn v ru i nh ngl th nghi m t h n v phn th i c a heo l th nghi m r n h n so v i l i ch ng.Ch ph m VEM-K cth nghi mtrng (gi ngHIGRO,trangtr iTr nQu cTu n, x MXun, Tn Thnh, B Ra - V ng Tu) v i li u l ng 1lt VEM-K/1000 ltn c s ch r i cho kho ng 4000 con g giai o n t21 n 39 ngy tu i u ng. K t qutheodiccchtiu tngtr ngbnhqunvt l tiut nth cnsauth igianchou ng c ghi nh n trong b ng 3.3.B ng 3.3. K t quthnghi m chph m VEM-K (1lt/1000 lt n c s ch) trn g.STT Ch tiu th nghi m L i ch ng L th nghi m1 Sl ng g th nghi m b t u (con) 3522 (100%) 3518 (100%)2 Tr ng l ng bnh qun lc 21 ngy tu i (g/con) 765 7663 Tng tr ng bnh qun (g/con/ngy) 75,83 80,04 Tr ng l ng cu i k(g) 2130 22065 Tiu t n th c n (FCR = Kg th c n/kg thtr ng) 1,88 1,806 Tls ng (%) 98,18 98,497 Th i gian th nghi m (ngy) 18 18B ng3.3 choth y,ccch ngvisinhgip tngtr ngbnhquntng5,5%, gi ml ngth cntiut n,nh ngcong cu ng VEM-Khngngyct l tiut nth c n l 1,80 so v i nh ng con khng c u ng VEM-K (1,88).S n xu t phn vi sinh (PVS)Phn chu ng c xl m , sau v i chp h m BIO-F. Sau ba ngy, cc visinh v t h u ch ni trn b t u pht tri n m nh, phn gi i cc h p ch t h u c v lmm t mi hi. Nhi t trong kh i c ng tng l n t i 60-700C, tiu di t cc vi sinh v tgyb nhvtr nggiuntrongphn.Sau7-10ngy,s nph mthu clphnbnvisinh ch t l ng cao.B ng 3.4. Cc ch ti u ch t l ng c a s n ph m phn bn HCVSCh tiuHm l ngCh tiuHm l ng m (%)Nt ng s(%)P2O5 (%)K2O (%)Humic acid (%)Mn, ch t h u c (%)250,781,060,612,614,65Trichoderma spp.Bacillus spp.Streptomyces spp.107CFU/g107CFU/g108CFU/gLo i phn bn h u cvi sinh ni trn c gi thnh ch a t i 1.000 ng/kg.Thnghi m PVS trn cy con d a leoS nph mphnHCVS cB mnB ov th cv t,Tr ng ih c NnglmTp. HCM thnghi m trn ru ng d a leo. K t qu c ghi nh n nhsau:Ph n II: CNG NGHVI SINH 229T l(%) b nh ho cy con d a leo sau 25 - 30 ngy gieo h t l 0 - 1% th p h n sov i i ch ng (3-3,5%). Lo i n m gy b nh ch t c xc nh l Fusarium sp.Chi ucao (18,43cm)vs l(6,2/cy)c acyd aleotrnccnghi mth cthnghi m giai o n 24 - 31 ngy sau gieo h t tng so v i i ch ng (chi u cao: 17,56cm, sl: 5,44/cy)Th ng m i ha chph m VEM-K v BIO-FChng ti k t h p v i cc cng ty thu c th y v cng ty s n xu t phn h u cvisinh th ng m i ha chph m VEM-K v BIO-F mang th ng hi u c a cc cng ty.B ng 3.7. Th ng m i ha chph m VEM-K v BIO-FSTT Tn s n ph m Ngu n g c i t ng Cng ty s n xu t1 BIOLAS-789VVEM-KHeo, gCng ty TNHH - TMSX i Tr ng S n234MAZARBS@LASAVEM-KVEM-KVEM-KHeo, g at Viet Co.LTD5 Phn h u cvi sinh BIO-F Cy ma Nh My ng Ty Ninh6Phn vi sinh Tricho BIO-F Cy hoa mu Cng Ty TNHH TM & SX Mai Xun - Tp. HCM7 Phn h u cvi sinh BIO-F Cy hoa mu CTy Gi ng cy tr ng ng Ty - Tp. HCM8 Phn h u cvi sinh BIO-F Cy cng nghi p Cng Ty Hong Thnh - aklakK T LU NT ccph ph ph mcngnngnghi pv ccch ngvisinhv tch nl c c,s n xu t c chph m VEM-K v chph m BIO-F.Chph m VEM-K c thnghi m trn heo tht (1 lt VEM-K/500 lt n c s ch)cho heo u ng trong 30-60 ngy, k t quheo tng tr ng trung bnh tng 20%, stiu haoth cngi m11%;vtrng(1ltVEM-K/1000ltn c)chogu ng,gtngtr ngtrung bnh tng 5,5%, stiu hao th c n gi m 4,4%; ctrn heo v g, VEM - K c tcd ng gi m tlb nh ng ru t v gi m mi hi c a phn.Phn h u cvi sinh c s n xu t tphn chu ng v BIO-F c tc d ng gi m t lb nh ho cycon d a leo do Fusarium sp., gipcy tng tr ng v chi u cao v slnhanh so v i i ch ng.V iquytrnh ngi n,r ti ncth pd ng itr chocc tr ichnnuicngnghi p v i n heo t10.000 - 100.000 con. Ngoi ra, quy trnh cn c th p d ng chocc hchn nui v i quy m gia nh tnhh n 2.000 con.TI LI U THAM KH O1. Fuller R. (1989). Probiotic in man and animal, J. Applied. Bacteriol. 66: 365 -3782. Havenaar,R.,TenBrisk,B.(1992). Selectionofstrainsforprobioticuse . In:R.Fuller (ed.), Probiotics the scientific basic, Chapman and Hall, London, pp209 -224H i ngh KHOA H C V CNG NGH2007 2303. V Th H nh v cs (2003). Nghin c u s n xu t chph m h n h p vi sinh v t s ngv enzyme tiu ha dng trong chn nui v nui tr ng th y s n. Bo co nghi mthu ti, SKhoa h c v Cng nghTHCM4. ThomashowLS.,(1996).Biologicalcontrolofplantrootpathogens.Curr.Opin.Biotechnol. 7: 343-3475. V Th H nh, L Th Bch Ph ng, Tr n Th nh Phong, L T n H ng, Tr ng ThH ng Vn (2004), Nghi n c u s n xu t chph m BIO-F dng phng tr n m b nhh i cy tr ng v s n xu t phn bn vi sinh, Gi i III H i thi Sng t o Khoa h c Kthu t tnh Bnh D ng6. VThH nh,LThBchPh ng,Tr ngH ngVn,LT n H ngvTr nTh nhPhong,D nGi mthi unhi mmihivs d ngphnchu ngs nxu tphnbnvisinhch tl ngcaot itrangtr inuiheo,Gi ith ng Ngysng t o Vi t Nam" nm 2005 do Ngn hng Thgi i tch c.SUMMARYReducing odour pollution at farms and producingbiofertilizer from manure by bio -productsVo Thi Hanh, Le Thi Bich Phuong, Tran Thanh Phong,Le Tan Hung, Truong Thi Hong VanInstitute of Tropical BiologyThe VEM-K and BIO-F bioproducts are produced from agri -industrial by-productsand selected useful microorganisms. The VEM-K product prevent livestock and poultryfromintestinaldiseasesandreducebadodourpollutionatfarmscomparedwithcontrols.ByusingbiofertilizerproducedfrommanureandBIO -F,theresultoftestshowedthatthediseaseratioonyoungcucumbercausedbyFusariums p.andotherpathogenetic fungi decreased and the growth of cucumber better compared with control.Ph n II: CNG NGHVI SINH 231TUY N CH N CC CH NG Lactobacillus SINH T NGH P polyhydroxybutyrate (PHB)Nguy n Duy Long, Ph m Tr n TNh , Nguy n Minh Nh t, Hong Qu c KhnhPhng Vi sinh ng d ng, Vi n Sinh h c Nhi t iM UPolyhydroxybutyrate(PHB)lm td ngpolymerthu ch Polyhydroxyalkanoate(PHA).Ccpolymerthu ch PHA uckh nngphnh yb ngcon ngsinhh c. Tuy nhin, v i c tnh u vi t c a PHB c b n v t l cao, nhi t nng ch y171-1820C, b nv itiauvt t ,cc ci mnyg nnh t ng ngv icclo ipolymer thng d ng nhPP (polypropylene), PE (polyethylene)[4]. PHB c t ng h pv tch l y trong vi sinh v t r t a d ng, stch l y ny x y ra trong i u ki n gi i h nc angu ndinhd ngvs d th ac angu ncarbon.PHB cs nxu t quymcngnghi ptrn Alcaligeneseutrophus b icngtyZENECA[2] cdnglmbaobth cph m,m ph m,l mv b cchothu cd c,lm ch t mvthaythx ngtronggi iph u,Tuynhinvi cgi mgithnhchos nxu t quyml nv ncnlm tv n ang cquantmvnghinc u.Ccnghinc ut ptrungvothay i cng ngh , i u ki n l n men, kthu t di truy n v sa d ng PHB trong thgi i vi sinh v t. Lactobacillus l m t vi khu n c sd ng trong cc quy tr nh ln mens n xu t acid lactic, l n men rau, cqu trong m t sk t qunghi n c u cho th y stch l y PHB trong cc loi Lactobacillus trong t nhin c th t n h n g n 30%[5].Trongnghinc uny,chngtich nl cccch ngLactobacillust ccngu nl nmenth cph m,kh ostcci uki ndinhd ngvi uki nlytrchhi uqu choqu trnh thu h i PHB tvi sinh v t.V T LI U V PH NG PHPCc ch ng vi khu n lactic c phn l p tcc ngu n Yaourt, u n nh ngm, cclo i d a mu i chua, nem chua, s a l n men, m t slo i rau qu , ngc c ln men[1]trn mi tr ng MRS agar v nui c y trn MRS Broth, mi tr ng Elliker (pepton 10g,caon mmen5g, dextrose5g, saccharose5g,NaCl1,5g, acidascorbic0,5g,n cc t1000ml) v mi tr ng dch chi t c chua (dch chi t c chua 20g/l, glucose 10g/l). Qutrnhxc nhccch ngLactobacillus cth chi ntrnmitr ng carbonatagar,quan st hnh thi, xc nh khnng ln men glucose (peptone 10g, NaCl 5g, cao tht1g, glucose10g, n cc t1000ml, phenolred0.018g),xcnhkh nngsinhacidlactic b ng thu c nhu m Uphenmen, quan st h nh thi, nhu m gram v th c hi n ph n ng String test [1]xc nh gram vi khu n.H i ngh KHOA H C V CNG NGH2007 232Cc khu n l c Lactobacillus tr n cc mi tr ng MRS agar, Elliker agar, dch chi tc chua agar c nhu m v i Sudan Black B xc nh khnng sinh t ng h p PHB.Kh o st khnng sinh t ng h p PHB tr n ngu n carbon glucose 1%. sucrose 2%,lactose 2%, manitol 1%,h n h p ngu n carbon (mi tr ng Elliker), dch chi t c chuav ngu n nitpeptone, cao n m men, (NH4)2SO4 0,4 % v glycine 2% [6, 7, 9, 10].PHB clitrchvxcnhhml ngtheoph ngphpLawSlepecky[14],qutrnhkh ostcci uki nlitrchPHB cth chi nv iSodiumhypochlorite5%vSDS3%d ihml ngthay ic atl sinhkh it 0.5% n6%(w/v). ngth ikh ostcci uki nnhi tli trcht 400C -900C,theoth igiant 20pht - 120 pht.K T QU1. K t quphn l p v xc nh PHB trong tbo vi khu n lacticTk t quphn l p, 78 ch ng vi khu n lactic c ch n l c cho nghi n c u khnng tch l y PHB. a scc ch ng vi khu n c khnng sinh acid lactic, gram d ng,hi ukhty,catalasemtnh,ckh nnglnmenglucose v i 57 ch ngLactobacillus cxcnh. Ccch ng cnsthi ndi nc a PHBtrong t bo khiki mtra v i thu c nhu m Sudan Black B l 30 ch ng chi m 38,5% trong tong so vikhu n lactic phn l p nn. [b ng 1, hnh 1, 2].Hnh 1. K t qunhu m gram v hnh thi m t sch ng vi khu n lactic. H1a. i ch ng:E.coli (gram m) b t mu thu c nhu m bsung nn c mu h ng, B. subtillis (gramd ng) gi c ph c mu gi a tm k t tinh v glugol nn c mu tm. H1b, c, d, e, f: lk t qunhu m gram c a cc ch ng N8, CK1, B3, DG5, M1.Hnh 2. K t qunh tnh PHB; 2a: DG5, 2b: DG5 + E.coli, 2c PHB from DG51a1b 1c 1d 1e 1f2b2a 2cPh n II: CNG NGHVI SINH 233B ng 1: K t quphn l p v xc nh shi n di n PHB trong tbo vi khu n.[*]: k t qud ng tnh c a khu n l cv i thu c nhu m Sudan Black BNgu nphnl pLactobacillus Pediococcus Streptococcus Entorococcus Ch axcnhD a c i chua C1, C2,C3, C4, C5, C6, C7,C8,C9,C10,C11,C12,C13,C14*,C15*,C16*,C17, C18, C19C9, C11Ya-ua Y3, Y4,Y5, Y7 Y2* Y1, Y6 u nnh N1*,N3,N4*,N5*,N6*,N7*, N8*, N9*N2D a ki u CK1*,CK2*,CK3*,CK4*,CK6*CK5, CK7D a gi DG1*,DG5*,DG6,DG7,DG8DG2, DG3*, DG4*Nem chua NC1*,NC2*,N3C*,NC4*,NC5*, NC6*, NC7, NC8D a c F3,F6, F7*, F8* F1*, F2*, F4, F5Ng sen chuaS5 S3D a hnh CH1*, CH2*Phntr ssinhB41, B3* BB8M M1*, M22.K tqu kh ostngu ndinhd ng nhh ng nkh nngtchl yPHBtrong tboCc ch ng vi khu n cho k t qud ng tnh cao DG5, M1, N8, CK1, B3, N1, NC3,NC4, DG4, N4, CH5, N9 v NC2 c ch n xc nh hm l ng PHB trong tbo.Qua k t quth nghi m, ch ng DG5, N8 v M1 cho hm l ng PHB cao h n cc ch ngcn l i t ng ng 27.870 %, 26.230% v 26.600% theo tr ng l ng kh tbo v cch n lm i t ng cho t t ccc th nghi m ti p theo.H u h t 3 ch ng u thch h p v i glucose cho ssinh t ng h p PHB. Tuy nhi n, iv ich ngM1 thml ngPHB32,302%trongmitr ngb sunglactose,peptone l ngu n nitthch h p nh t cho ssinh t ng h p PHB, ch ng DG5 t h ml ng PHB cao nh t v i peptone 27,870% (h nh 3 v hnh 4)Hnh 3: nh h ng c a ngu n carbont i ssinh t ng h pPHB: 3a; DG5, 3b; M1, 3c; N8Hnh 4: nh h ng c a ngu n nitot i ssinh t ng h pPHB: 4a; DG5, 4b; M1, 4c; N8DG50.000.050.100.150.20Peptone yeast extract NH42SO4 gl ycynePHB(%)051015202530Sinh khoi(g)si nh khoiPHBM10.000.050.100.150.200.25Peptone yeast extract NH42SO4 gl ycynePHB(%)051015202530Sinh khoi(g)si nh khoiPHBN80.000.050.100.150.200.25Peptone yeast extract NH42SO4 gl ycynePHB(%)051015202530Sinh khoi(g)si nh khoiPHB4a4b4cH i ngh KHOA H C V CNG NGH2007 2343. K t qukh o st i u ki n li trch PHB nhh ngc atcnhnlitrch: Sodiumhypochlorite(SH)5%,Sodiumdodecylsulfate(SDS) 3% c sd ng lm tc nhn ph v tbo theo hm l ng sinh kh i. i v itc nhn sodiumhypochlorite cho hi u su t ph vtbo cao nh t hm l ng 1% sinhkh i tbo t 85,635% v thu c 32,865 % PHB theo tr ng l ng kh tbo. iv i tc nhn SDS 3% cho hi u su t ph vtbo cao nh t hm l ng 5% sinh kh i tbo t85,124%vthu c34,635%PHBtheotr ngl ngkht bo.TcnhnSDS3%ckh nnglytrchPHB hml ngsinhkh icaoh ntcnhnSodiumhypochlorite 5%. nhh ngc anhi t litrch:Thnghi m cth chi nv i2tcnhnsodiumhypochloritevSDStheodynhi t t 400C n900C. iv itcnhnsodiumhypochlorite t hm l ng PHB cao nh t (32,182 %) t i 600C v i hi u su t phvtbo 85,462%. i v i tc nhn SDS t hm l ng PHB cao nh t (35,623%) t i800C v i hi u su t ph vtbo 85,253% [hnh 6]. K t qucho th y, i v i tc nhnSodiumhypochlorite5%nhi t cngcaoqutrnhphv t bocnggi m iv icc ch ng lactobacllus. Ng c l i, hi u su t ph vtbo cng m nh khi nhi t tngln i v i tc nhn SDS 3%, tuy nhin l i nh h ng n sph vc u trc c a PHBv cho hm l ng th p xu ng.Hnh 5: Kh o st sph vtbo v i tc nhn sodiumhypochlorite 5% v SDS 3%theo hm l ng sinh kh i tboHnh 6: Kh o st nh h ng c a nhi t n qu tr nh li trch PHBv i tc nhn sodiumhypochlorite 5% v SDS 3%Sodi umhypochl ori te -SDS01020304050607080900. 50% 1% 2% 3% 4% 5% 6%Si nh k hoi ( % )PHB)%)0102030405060708090 Hieu suatPH B ( %) - SH 5 %H i e u su at ( %) - SH 5 %PH B ( %) - SD S3 %H i e u su at ( %) - SD S 3 %010203040506070809040 50 60 70 80 90Nhi eto(oC)PHB)%)0102030405060708090Hieu suatPH B ( %) - SH 5 %H i e u su at ( %) - SH 5 %PH B ( %) - SD S3 %H i e u su at ( %) - SD S 3 %Ph n II: CNG NGHVI SINH 235 nh h ng c a th i gian li trch: Th i gian trong qu tr nh ly trch PHB c thi tl p trong kho ng th i gian t20 pht n 120 pht cho 2 tc nhn Sodiumhypochloritet i nhi t 600C v SDS t i 800C. K t qukh o st cho th y qu tr nh ly trch PHB v itc nhn Sodiumhypochlorite t hm l ng PHB cao nh t t i th i i m 60pht ly trchv i32,246%PHB,hi usu tlytrchl85,642%vv itcnhnSDS thml ngPHBcaonh tt ith ii m80phtlytrchv i34,575%PHB,hi usu tlytrchl95,563% (hnh 7).Hnh 7: Kh o st th i gian li trch nh h ng n qu trnh li trch PHB v i tc nhnsodiumhypochlorite 5% v SDS 3%K T LU NQua k t qunghin c u, chng ti c m t snh n xt nhsau:Vi khu n lactic ckh nngsinht ngh pPHB,phnl p c78ch ngvikhu nlacticv ih n50ch ngthu cnhmLactobacillus. Ccch ngcs hi ndi nc aPHBtrongt b ol30ch ngchi m38,5%trongt ngs vikhu nlacticphnl p c.Trongch ngDG5,M1,N8ckh nngsinht ngh pPHBcaonh t.Glucosev peptonel2ngu n dinh d ng thch h p cho c3 ch ng DG5, M1 v N8 sinh t ng h p PHB. Ringch ng M1 ng lactose hoc ho hm l ng cao h n. K t qunghin c u cho th y v imitr ngdchchi tcchuab sung1%glucosec ngthchh pchoqutr nhsinht ng h p PHB c a c3 ch ng. t i nghin c u xy d ng c qu trnh tch chi tPHB d a trn 2 tc nhn Sodiumhypochlorite 5% v SDS 3 %. Qu trnh tch chi t v itc nhn Sodiumhypochlorite 5% thch h p hm l ng sinh kh i tbo l 1%, nhi t lytrch600Cvth igianlytrchl60pht. iv i qutrnhtchchi tb ngtcnhn SDS 3% thch h p v i hm l ng sinh kh i l 5%, nhi t ly trch l 800C v th igian ly trch l 80 pht.TI LI U THAM KH O1. D ngThThuL(2003).Phnl pvch ngi ngvikhu nlactic l myaourt u nnh. Lu n vn Th c ssinh h c. i h c Khoa h c Tnhi n - TPHCM.010203040506070809010020 40 60 80 100 120phutPHB)%)020406080100120Hieu suatPHB(%)-SH 5%-Hi eu suat (%)- SH 5%PHB(%)-SDS 3%Hi eu suat (%)- SDS 3%H i ngh KHOA H C V CNG NGH2007 2362. Kolybaba M. A.. Tabil L. G.. Panigrahi S. A. (2004). Recent Developments in theBiopolymerIndustry. ThesocietyforEngineeringinagriculturalfoodandbiological systems. pp. MB04 - 301.3. Law. John H. & RalpH A. Splepecky (1960). Assay of Poly-|-hydroxybutyric acid.J. Bacterial. Vol. 82. pp. 33 - 36.4. LeeS. Y. (1996).BacterialPolyhydroxyalkanoate. BiotechnologyandBioengineering.Vol. 49. pp. 1 - 14.5. Liddell J. M. (1999). Process for the recover y of polyhydroxyalkanoic acid. Unitedstates Patent. Patent Number 5894062.6. LuengoJ. M.. GarciaB..SandovalA..NaharroG.andOliveraE. R.(2003).Bioplasticsfrommicroorganisms. CurrentOpinioninMicrobiology.Vol.6.pp.251-260.7. MahishiL. H..TripathiG..RawalS. K.(2003).Poly (3-|-hydroxybutyrate)synthesisbyrecombinant Escherichiacoliharbouring StreptomycesaureofaciensPHBbiosynthesisgenes:Effectofvariouscarbonandnitrogensources. MicrobialResearch. Vol. 158. pp. 19 - 27.8. Saledizadeh.Loosdrecht(2004).Productionofpolyhyd roxyalkanoatesbymixedculture:Recent trends and biotechnological importance. Biotechnology Advances.Vol. 22. pp. 261 - 279.9. SteinbuchelA.(1996).PHBandOtherPolyhydroxyalkanoicAcids.Biotechnology. Vol. 6. pp 403 - 464.10. Ugur.Sahin.Beyati(2002).AccumulationofPoly -|-hydroxybutyrateinStreptomyces Species: During Growth with Different Nitrogen Sources. Tur J Biol.Vol. 26. pp. 171 - 174.SUMMARYScreening of lacyobacillus strains for biosynthe sis ofpolyhydroxybutyrate (PHB)Nguyen Duy Long, Ph m Tran To Nhu, Nguyen Minh Nhut, Hoang Quoc KhanhInstitute of Tropical BiologyInthisstudy,wefound3Lactobacillusstrains(DG5,M1,N8)withah igherconcentrationofPHBthan 78strainscollectedwith27.87%,26.23%,26.60%corresponding.GlucoseandpeptoneareinvestigatedanddeterminedasnutrientfactorforproducePHB.FortheaccumulationofPHBfromDG5,M1,andN8,thetomatoextractsolutionwasfoundasacheepernutrientresoursetoproducePHB.TheprocessingofPHB extractionwasfollowedbySodiumhypochlorite(SH)5%incellmass 1% at 600C during 60 minutes and Sodiumdodecylsulphate (SDPh n II: CNG NGHVI SINH 237PHN L P V THU NH N enzym phytase TM T SN M M C Aspergillus TRN MI TR NGNN MEN BM TNguy n Duy Long, Nguy n Th MH nh, Ho ng Qu c KhnhPhng Vi Sinh ng d ng, Vi n Sinh h c Nhi t iM UPhotpho l nguyn tthi t y u c n thi t cho stng tr ng, pht tri n v sinh s n c a ngv t.Tuynhin,ckho ng60-75%photphotrongng c cvh td utrongth cnc a ngv t c tm th y d ngphytat (inositol hexaphosphat) ho caxitphytic. ngv t khng thh p thphotpho trong phytat v chng khng c enzym phytase trong ngru t. V thph n l n photpho c tm th y trong phn c a ng v t. Ngoi ra nh ng ch tdinhd ngkhcnh protein,Fe2+,Ca2+,Mg2+,Zn2+c ngs blink tvoc utrcc aaxit phytic. Do cth ng v t khng thh p th c cc ion ny. Photpho trong phnc a ng v t c h p thvo t, chng c n thi t cho spht tri n c a th c v t. L ngphotpho dth a khng c cy h p thsb cu n tri ra ao, h , sng, su i kch thch spht tri n c a phiu sinh th c v t (t o) gy ra hi n t ng ph d ng ho. Ngoi ra, sthi uh t oxy slm c ch t v gi m sa d ng c a hsinh v t c l i trong n c.Phytase l m t trong sccenzymhi n angr t cquan tm. Phytaseth y phnlin k t gi a photpho v vng phytat sphng thch photpho v c . Vi c bsung phytasevo th c n gia sc gip t n d ng t i a ngu n photpho trong cc lo i nguy n li u chbi nvh visinhv t ngru tc a ngv th unh khngckh nngsinht ngh pphytase, v ng th i gip gi m chi ph bsung l ng photpho v cc n thi t vo th c ngia sc. H n thn a, phytase cn gip gi m thi u nhi m photpho vo mi tr ng do ch tth i ngv t.Enzymphytase cnghinc utrnnhi u it ngkhcnhaunh n mmen,n m m cvvikhu nnh ngnhi unh tv nltrncclo in mm c cbi tlAspergillus sp. v khsinh t ng h p enzym phytase cao v c thchu c pH th p.V T LI U V PH NG PHPCc ch ng Aspergillus c phn l p tcc lo i men r u (men c m r u, men r u)vm ts ch ngAspergilluskhc(A.aculeatus,A.awamori,A.carbonarius,A.ellipticus,A.ficuum,A.flavus,A.nigerNRRL-363,A.ochraceusA175,A.oryzae,A.phoenisis,A.tubingensis) tbs u t p gi ng c a phng Vi sinh ng d ng - Vi n Sinh h c Nhi t itrn mi tr ng PGA .Qutrnhnuic yvtchchi tenzymephytase cth chi ntheoquytrnhd i(Hnh1).Enzymphytase cphntchhml ngproteintheoph ngphpBradford.Ho t tnh enzym phytase c xc nh trn cch t sodium phytate (m t n v ho t tnhphytase (U) c nh ngha l l ng phytase gi i phng c 1 mol photphat v ctrongH i ngh KHOA H C V CNG NGH2007 238m t pht tsodium phytat 37oC, pH5,5). Qu trnh kh o st nh h ng c a nhi t , pH nho ttnhenzymphytase cth chi ntrongd inhi t t 30-950C,trong mGlycin:2,5pH,trongSodiumacetat :3,0;3,5;4,0;4,5;5,0;5,5,trong mImidazol-HCl:6,0; 6,5; 7,0, trong m Tris-HCl: 7,5; 8,0; 8,5; 9,0. Enzym phytase c phn tch trn gelpolyacrylamide theo ph ng php SDS-PAGE.K T QU1. Phn l p n m Aspergillus tmen r uTrong s14 lo i men r u thu nh n tcc n i khc nhau cho th y sphn bccch ngn mm ckhngcs khcbi tnhi u.trongchngti xc nh c4ch ng n m m c khc bi t nhau r r t vhnh thi l Aspergillus.MCR1 (hnh 2.1; 2.2;2.3), Aspergillus.MCR2(hnh3.1;3.2;3.3), Aspergillus.MR5 (hnh4.1;4.2;4.3),Aspergillus.MR2 (hnh 5.1; 5.2; 5.3), ng PGA gigi ng Aspergillus+ 5mln c c t v trngHt 1ml dung dch bo tvo cc bnh ln men bm t (Tr u:B t m:Khong)[T lTr u:B t m 1:2, 60% Khang]370C, 2 ngyBsung 200ml dung dch m Sodium Acetate pH 5.5L c 200rpm, 3hL c dch qua v i m ngDch chi t enzym thC y truy n sang cc ng nghi m PGAHnh 1: Quy trnh ln men bm t aspergillus sinh t ngh p enzyme phytaseHnh 2.2. Hnh thi v kchth cConidiophores ch ng MCR1; X 6Hnh 2.1. Hnh thi ch ng MCR1m c trn mi tr ng PGAHnh 2.3. Hnh thi v kchth cbo tch ngMCR1 (conidia); X100Hnh 3.2. Hnh thi v kch th cconidiophores ch ng MCR2; X 6Hnh 3.1. Hnh thi ch ngMCR2m c trn mitr ng PGAHnh 3.3. Hnh thi v kch th c botch ng MCR2 (conidia); X 100Ph n II: CNG NGHVI SINH 239Hnh 4.1. Hnh thi ch ng MR5m c trn mi tr ng PGAHnh 4.2. Hnh thi v kch th cConidiophores ch ng MR5; X 6Hnh 4.3. Hnh thi v kch th cbo tch ng MR5 (conidia); X 100Hnh 5.1. Hnh thi ch ng MR2trn mi tr ng PGAHnh 5.3. Hnh thi v kch th cbo tch ng MR2 (conidia); X 100Hnh 5.2. Hnh thi v kch th cConidiophores ch ng MR2; X 62. Xc nh ho t tnh enzyme phytase c a cc ch ng AspergillusTrong 4 ch ng c phn l p tcc lo i men th ng m i, c 3 ch ng Aspergillusc khnng sinh phytase l A.MCR2, A.MR2, A.MR5 v 1 ch ng khng sinh phytaselA.MCR1.Trongs 3ch ngcho ttnhphytaseth A.MR2choho ttnhcaonh t.Ngoi ra, 1 ch ng trong Bs u t p gi ng- Phng Vi sinh ng d ng c ng khng c khnng sinh phytase l A.ochraceus. Hai ch ng Aspergillus cho ho t tnh phytase cao nh tl A.nigerNRRL-363v A.oryzae. A.nigerNRRL-363cho ttnht ng(Ut)caoh nA.oryzae nh ng A.oryzae l i cho ho t tnh ring cao h n. Enzyme t cc ch ng cn l ic ho t tnh trung bnh ho c th p (hnh 6a, 6b). i v i A.flavus, m t lo i n m m c sinh c tanfatocin c ng c khnng sinh enzym phytase, do c n t h n tr ng trong vi ctuy n ch n n m m c. Tcc ch ng tr n, ch n ra 3 ch ng A.niger NRRL-363, A.oryzae,A.MR2 cho ho t tnh phytase cao nh t kh o st phnhi t , pH.3. Kh o st nh h ng c a nhi t n ho t tnh enzyme phytaseT th cho th y nhi t t i u cho ho t tnh phytase cao i v i c3 ch ngA.niger NRRL-363, A.oryzae, A.MR2 l 50-550C so snh v i cc k t qu nghinHnh 6a: Ho t tnh t ng Ut(U/g m u)c a cc ch ng n m m c050100150200250300ChungHoat tnh tongUt (U/g mau)A.M CR1 A.M CR2 A.M R5A.M R2 A.acul eat us A.awamoriA.carbonari us A.el l i pt i cus A.f i cuumA.f l avus A.ni ger NRRL -363 A.ochraceus A175A.ory zae A.phoeni ci s A.t ubi ngensi soichng050010001500200025003000ChungHoat tnh riengUs (U/mg mau)A.M CR1 A.M CR2 A.M R5A.M R2 A.acul eat us A.awamoriA.carbonari us A.el l i pt i cus A.f i cuumA.f l av us A.ni ger NRRL -363 A.ochraceus A175A.ory zae A.phoeni ci s A.t ubi ngensi soichngHnh 6b.Ho t tnh ring Us (U/mg m u)c a cc ch ng n m m cH i ngh KHOA H C V CNG NGH2007 240c uas phytasecnhi t t i utrongkho ng44-600C[6,10].T ikho ngnhi t t i uny, A.niger NRRL-363choho ttnhenzymphytaset ngUtcaonh tnh ngl icho ttnhri ngUsth pnh ttrong3ch ng. A.nigerNRRL-363c hm l ng protein cao, ho t tnh t ng cao, ho t tnh ri ng th p ch ng tch ngnychotenzymphytaseh ntrongt ngs nhi ulo iprotein csinhra,hnh(7a, 7b). Trong s n xu t, ho t tnh ri ng cao c quan tm nhi u h n. A.oryzae vA.MR2 p ng cy u t ny,c 2ch ng ucho t tnhenzymphytase t ngth ph nA.nigerNRRL-363nh ngl icho ttnhri ngcaoh nh nnh tlA.oryzae.T k tqu kh ost nhh ngc anhi t nho ttnhphytasetA.nigerNRRL-363, A.oryzae, A.MR2,chngtick tlu nvnh nxtnh sau:Cc tnhch t nyc aphytaset 3 ch ngn m m c tr nr t thchh p chocc ngd ng cng nghi p, c bi t trong chbi n th c n gia sc.4. Kh o st nh h ng c a pH n ho t tnh enzyme phytaseTth cho th y, c3 ch ng A.niger NRRL-363, A.oryzae, A.MR2 c pH t i uchoho ttnhphytasecaol 2,5v5,0-5,5.H uh tenzymphytaset visinhv tthhi n pH t i u gi a 4,5 v 5,5, c bi t i v i phytase tn m m c [10,11]. Tnh ch tny r t c n thi t trong cc ng d ng cng nghi p v ph h p v i i u ki n trong ddy(pH 2,0-4,0) v ru t non (pH 4,0-6,0) c a ng v t [9].0501001502002503003504004505000 10 20 30 40 50 60 70 80 90 100 110Nhieto(oC)Hoat tnh tong Ut(U/g mau)A.ni ger NRRL-363A.oryzaeA.MR205001000150020002500300035000 10 20 30 40 50 60 70 80 90 100 110Nhieto(oC)Hoat tnh rieng Us(U/mg mau)A.ni ger NRRL-363A.oryzaeA.MR2Hnh 7a. nh h ng c a nhi t l n ho ttnh t ng c aenzym phytase c aA.niger NRRL-363, A.oryzae, A.MR2Hnh 7b. nh h ng c a nhi t l n ho ttnh t ng c aenzym phytase c aA.niger NRRL-363, A.oryzae, A.MR2Hnh 8a. nh h ng c a pH ln ho t tnh t ng enzymphytase ch ng A.niger NRRL-363, A.oryzae, A.MR20500100015002000250030000,0 2,5 5,0 7,5 10,0pHHoat tnh rieng Us(U/mg mau)A.ni ger NRRL-363A.oryzaeA.MR20501001502002503000,0 2,0 4,0 6,0 8,0 10,0pHHoat tnh tong Ut(U/g mau)A.ni ger NRRL-363A.oryzaeA.MR2Hnh 8b. nh h ng c a pH ln ho t tnh t ng enzymphytase ch ng A.niger NRRL-363, A.oryzae, A.MR2Ph n II: CNG NGHVI SINH 241Hnh 9:Phn tch enzym phytase trn gel polyacrylamide b ngph ng php i n di SDS PAGE.5. Phn tch enzyme phytase trn gel polyacrylamideM cchcu thnghi mnh mxcnhm ts th nhph nproteinctrongdchchi t enzym phytase c a ba ch ng A.niger NRRL-363, A.oryzae, Aspergillus MR2. K tquxc nh c trnh by hnh 9 d i y:Band1:ch accproteinmarkerctr ngl ngphnt l200.000,116.250,97.400, 66.200, 45.000, 31.000, 21.500, 14.400, 6.500 Daltons. Band 2: m u i ch ng(control).Band3:dchchi tenzymthc aA.or yzaech accproteinctrongl ngphn tl 97.400, 89.200 v 58.000 Daltons. Band 4: d ch chi t enzym th c a A.MR2ch a cc protein c trong l ng phn tl 97.400, 89.200 v 58.000 Daltons. Band 5:dch chi t enzym th c a A.niger NRRL-363 ch a cc protein c trong l ng phn tl116.250, 97.400, 89.200 v 58.000 Daltons. Phytase t cc ch ng A.niger c hai lo i l PhyA v PhyB. PhyA c tr ng l ng phn tn m trong kho ng 66-128 kDa. PhyB ctr ng l ng phn tkho ng 58-65 kDa [2]. Phytase t cc ch ng A.oryzae c ng cho hailo i phytase l PhyA v PhyB. PhyA c tr ng l ng phn tkho ng 65-120 kDa. PhyBc tr ng l ng phn tkho ng 58 kDa [19]. Qua k t quphn tch enzym phytase sinht ngh pt ccch ngA.nigerNRRL-363,A.oryzae,A.MR2trngelpolyacrylamideb ngph ngphpSDS -PAGE,ccbandproteinxu thi nt ng irtrongvngkch th c c a phytase so v i k t qunghi n c u tr c [2,19].TI LI U THAM KH O1. Tr n Th Tuy t (2003).Thu nh n v kh o st enzym phytase c a n m Asperg illusnigerNRRL-363trongmitr nglnmenbnr n.Khalu nTr ng ih cKhoa h c T nhin TP HCM, 5-27.2. MullaneyE.J.andUllahA.H.J.(2003).Thetermphytasecomprisesserveraldifferentclassesofenzymes.BiochemicalandBiophysicalResearc hCommunications 312, 197-184.H i ngh KHOA H C V CNG NGH2007 2423. Barbara, George Szakacs, Ashok Pandey, Sabu Abdulhameed, James C. Linden, vRobertP.Tengerdy(2003).Productionofphytasebymucorracemosusinsolid -state fermentation. Biotechnol Prog. 19 (2), 312 -319.4. WodzinskiR.J.andUllahA.H.J.(1996).Phytase.AdvancesinAppliedMicrobiology, vol 42, 263-298.5. LeiX.G.andPorresJ.M.(2003). Phytaseenzymology,applications,andbiotechnology. Biotechnology Letters, 25 (21), 1787 -1794.6. OhB.C.,ChoiW. -C.,ParkS.,KimY.-O.,OhT.-K.(2004). Biochemicalpropertiesandsubstratespecificitiesofalkalineandhistidineacidphytases.ApplMicrobiol Biotechnology , 63 (4), 362 -372.7. ZimmermannB.,LantzschH.-J.,LangbeinU. andDrochnerW.(2002).DeterminationofPhytaseactivityincerealgrainsbydirectincubation,JAnimPhysiol Anim Nutr (Berl), 86 (9-10), 374-352.8. BogarB.,SzakacsG.,LindenJ. C.,PandeyA.andTengerdyR.P.(2003).Optimization of phytase production by solid substrate fermentation. J Ind MicrobiolBiotechnol., 30 (3), 183-189.9. CaseyA.andWalshG.(2003).Purificationandcharacterizationofextracellularphytase from Aspergillus niger ATCC 9142. Bioresour Technol, 86 (2), 183 -188.10. VohraA.andSatyanarayanaT.(2003).Phytases:MicrobialSou rces,Production,Purification,andPotentialBiotechnologicalApplications.CriticalReviewsinBiotechnology, 23 (1), 29-60.11. KerovuoJ.(2000).ANovelPhytasefrom Bacillus.CharacterizationandProduction of the Enzyme, 1-66.12. McKeeT.andMcKeeJ.R. (1999).AnIntroductiontoChemicalBiology.Biochemistry, vol 2.13. DanielM.Bollag,MichaelD.RozyckivStuartJ.Edelstein(1996).ProteinMethods. Wiley, Chichester.14. Nagashima T., Tange T., and Anazawa H. (1999). Dephosphorylation of Phytate byUsing the Aspergillus niger Phytase with a High Affinity for Phytate. Appl EnvironMicrobiol, 65 (10), 4682-4684.15. Budy J. Hidayat, Niels T. Eriksen and Marylin G. Wiebe (2006). Acid photphataseproductionby AspergillusnigerN402Aincontinuousflowculture. FederationofEuropean Microbiologycal Societ8ies Microbioal Lett , vol 254, 324 -321.16. ChantasartrasameeK,DuangnetreIsrangkulNaAyuthaya,SunthumIntarareugsorn,Saovanee Dharmsthiti (2004). Phytase activity from Aspergillus oryzae AK9 cultivatedon solid state soybean meal medium. Process Biochemistry, 1 -5.17. ShiehT.R.andWareJ.H.(1968).Surveyofmicroorganismsforproductionofextracellular phytase. Applied Microbiology, 16 (9), 1348 -1351.18. Howson S. J. and Davis R. P. (1983). Production of Phy tase hydrolysing enzymeby some fungi. Enzyme Microbiol Technol., 5, 377 -382.Ph n II: CNG NGHVI SINH 24319. FujitaJ.,SeikoShigeta,Yu-IchiYamane,hisashiFukuda,YasuzoKizaki,SaburoWakabayashi,andKazuhisaOno(2003).ProductionoftwophytasefromAspergillusoryzaeduring industrialKojimaking.JournalofBioscienceandBioengineering, 95 (5), 460-464.20. Wyss M, Brugger R, Kronenberger A, Rmy R, Fimbel R, Oesterhelt G, LehmannM,andVanLoonA.P. G. M.(1999).BiochemicalCharacterizationoffungalphytases(myo-Inositolhexakisphophatephosphohydrolases):catalyticproperties,65 (2), 367-373.SUMMARYIsolation and investigation of phytase from A spergillusin solid-state fermentationNguyen Duy Long, Nguyen Thi My Hanh, Hoang Quoc KhanhInstitute of Tropical Biol ogyPhytasesareacidphosphataseenzymeshavebeenfoundasasupplementtoincreasenotonlythegrowthrateofmonogastricanimalsbutalsotheefficiencyofphosphateutilizationinfeeds, whichsignificantlyreducesphosphorusexcretioneffecttoenvironmentalpollutants.Inthisstudy,phytasesareextractedfrom Aspergillusspeciesareisolatedfromoriginalrawmaterialoffermentedglutinousrice.Threespecies A.MCR2, A.MR2,A.MR5arefoundwithahighpositiveofphytaseactivity.TheeffectsofpHandtemperatureconditionalsareresearchedandcomparedwithothers species of Aspergillus genus. Phytases are also assayed on polyacrylamide gel bySDS-PAGEmethodandtheresultsappearingwith65kDaand58kDarespectivetophyA and phyB from A.MR2.H i ngh KHOA H C V CNG NGH2007 244T I U HO KHNNG SINH T NG H Penzyme cellulaseC A Tricoderma reesei TRN MI TR NG BN R NTr n Th nh Phong, Hong Qu c Khnh, V Th H nh, L Th Bch Ph ng,Nguy n Duy Long, L T n H ng, Tr ng Th H ng VnPhng Vi sinh ng d ng, Vi n Sinh h c Nhi t iM UNhi u loi n m s i c khnng sinh ra m t l ng l n cellulase thu c gi ng Trichoderma,Aspergillus, Pinicillium, Trong hai gi ng n m s i l Trichoderma v Aspergillus cnhi u nh khoa h c nghin c u s n xu t cellulase (Bothast & Saha, 1997).Cellulaselenzymac ut g m:exoglucanasehayC1(EC3.2.1.91),endoglucanase hay Cx (EC 3.2.1.4) v |-glucosidase (EC 3.2.1.21), ho t ng ph i h pth y phn cellulose thnh glucose. Cellulase c ng d ng bsung vo th c ngia sc, gia c m; chbi n th c ph m; trch ly cc ch t tth c v t, tcy thu c; ngha cc phli u giu cellulose s n xu t ethanol.Vi t Nam c l ng phphli u nng nghi p th i ra r t d i d o, trong l ng bma th i ra t cc nh my ng chi m kho ng 20% ma nguyn li u, trong b ma chm l ng cellulose kho ng 50% v hemicellulose kho ng 25% nn c thsd ng nhngu n carbon c m ng n m s i sinh t ng h p cellulase.V T LI U V PH NG PHPN ms i:Ch ng T.reeseiVTT-D-80133nh n ct b otnggi ngRoalOy,Ph n Lan. Cch t: B ma v cm m.Xcnhho ttnhccenzym: Carboxymethylcellulase(CMCase), FPU(FilterPaper Unit), xylanase, -amylase, protease.Th y phn gi y: cho dch enzymcellulase(5FPU/ ml) vogi y xaynh(10%)500C, pH 5 trong 24 gi . Hi u su t (%) = l ng ng kh(g)*0,9*(100/l ng gi y in (g))iendipr otein: Sodi umDodecyl Sul f atePol yacryl ami degel (SDS-PAGE)c thc hi en tren gelng cha 10% (w/v) pol yacryl ami de.T i u ha thnh ph n mi tr ng: TlBM: CM, m, n ng dinh d ngvth igiannuic yl 4y ut c nhh ngrr t nkh nngsinht ngh pcellulase c a T. reesei nn c t i u theo ph ng php quy ho ch th c nghi m.K T QUV TH O LU NK t qut i u ha thnh ph n mi tr ng v cc i u ki n nui c y.Ph n II: CNG NGHVI SINH 245Cck t qu thnghi mtr cy,chngtixc nh cthnhph nmitr ng cscho ch ng T. reesei VTT-D-80133 sinh ra cellulase: t lBM: CM (4:6), mban u54%,5l nn ng dinhd ng,th igiannui 7ngy,t l gi ng6x106 bo t /g mi tr ng, ho t tnh cellulase t c l 251,43 IU/g.Tuy nhin, thnh ph n mi tr ng v cc i uki n nui c y m i ch c nghinc u nh h ng m c ring r . Trong nm y u ttrn th b n y u tl tlBM:CM, mban u,n ng dinhd ngvth igiannuic yc nhh ngngk nkhnng sinh t ng h p cellulase c a T. reesei VTT-D-80133 nn c ch n nghinc u t i u ha theo ph ng php qui ho ch th c nghi m.Qui ho ch c th c hi n v i ma tr n y v i sth nghi m N = 24 = 16B ng 1. M ha cc bi n sCc bi n s M c d i (-) M c trung bnh (0) M c trn (+)X1: TlBM:CM 2:8 4:6 6:4X2: N ng dinh d ng (l n) x2 x5 x8X3: m ban u (%) 50 54 58X4: Th i gian nui c y (gi ) 3 7 11Ti n hnh nui c y T. reesei trong cc mi tr ng m c cc y u tkh o st trnhai m c (theo b ng 1). K t quxc nh ho t tnh cellulase c ghi nh n trong b ng 2.Cc hstrong ph ng trnh h i qui c xc nh nhsau:- Tnh b0:161610ybii== , b0 = 87,30- Tnh bi:16161yxbiijij== , b1 = 2,2; b2 = 5,82,b3 = 29,1, b4 = 49,8- Tnh bij: ,16161==ii il jjlyx xb, b12 = 9,66,b23 = 2,08, b13 = 31,32, b14 = 9,71, b24 = 16,17, b34 = -0,24, b123 = 13,58,b124 = - 21,27,b134 = 32,03, b234 = 6,17, b1234 = -10,19.Hsc ngha ph i th a mn i u ki n:tbt ltjjSbj) = , V itlt: p = 0,05, f = n 1 = 2t) 2 ; 05 , 0 ( = 4,3 (Tra b ng tiu chu n Student)Sbj (ph ng sai c a cc hs ):NSSthbj ==403 , 1= 0,26 (N = 16, =Sth213100|.| \|=niiy y = 1,07(v i n = 3) = Sth1,03))H i ngh KHOA H C V CNG NGH2007 246So snh cc hstj v i tlt ta th y,cch s c aph ngtrnh ucngha,ngo itr cch s t34.V yhm m c tiu c d ng:^y = 87,3 + 2,2x1 + 5,82x2 + 29,1x3+49,8x4+9,66x1x2+31,32x1x3+9,71x1x4+2,08x2x3+16,17x2x4+13,18x1x2x321,57x1x2x4+32,03x1x3x4+6,17x2x3x410,19x1x2x3x4Tph ng trnh h i qui thu c,tath yr ngth igiannui , mban u v n ng dinh d ng c nhh ngngk nho ttnhcellulaseh n tlb ma:cm m.Ki m tra st ng thch c a m hnh theo tiu chu n Fisher.i u ki n m hnh t ng thch l:F F lt tn( , Flt: gi tr chu n Fisher m c p =0,05; f1=N l; f2=n-1;trongN=16, l:s h s cngha=15,n=3),F F lt= = 5 , 18) 2 ; 1 ; 05 , 0 ( (Tra b ng tiu chu n Fisher)Ftn: 87 , 107 , 100075 , 222= = =SSFthtttn (l Nii itty yS

\|||.|= =1612^2). Ta c:F F lt tn((v 1,87 < 18,5)V y: Ph ng trnh h i qui thu c t ng thch v i th c nghi mK tqu t i uhatheok ho chleod c: mitr ngct l BM:CM(7:3),8l n n ng dinh d ng, m 60%, th i gian nui c y 7 ng y cho ho t tnh cellulasecao nh t: CMCase 280,64 IU/g v FPU 5 UI/g, th p h n 3,2 v 37 l n so v i chph mAmanoT. Canhtr ngcnch a -amylase368,75UI/g,protease12,43UI /gvxylanase 10073,25 BXU/gK t qu ng ha gi y in qua sd ngDchchi tenzymcellulase(5FPU/ml) nghakho ng20%gi yinquasd ng(10%)trong24gi th yphn 500C,pH5,0;dch nghach a 23,62mg ng kh /ml c th c sd ng ln men ethanol ho c ln men s n xu t cc s nph m c gi tr.TTNX1X2X3X4y y^1 2:8 x2 50 3 28,67 28,922 6:4 x2 50 3 0,15 0,43 2:8 x8 50 3 1,24 1,484 6:4 x8 50 3 2,57 2,85 2:8 x2 58 3 144,48 143,726 6:4 x2 58 3 18,07 17,827 2:8 x8 58 3 5,62 5,388 6:4 x8 58 3 99,18 98,949 2:8 x2 50 11 130,64 130,410 6:4 x2 50 11 58,36 58,1211 2:8 x8 50 11 188,73 188,4812 6:4 x8 50 11 55,23 5513 2:8 x2 58 11 51,94 52,7214 6:4 x2 58 11 219,5 219,7415 2:8 x8 58 11 129,45 129,716 6:4 x8 58 11 262,94 263,1817 4:6 x5 54 7 154,0318 4:6 x5 54 7 155,0719 4:6 x5 54 7 156,1B ng 2. Ho t l c CMCase tT. reesei theoth c nghi m v theo ph ng trnh h i qui.V i y: Ho t tnh CMCase (UI/g) theo th c nghi m;y^: ho t tnh CMCase (UI/g) theo ph ng trnh h i quiPh n II: CNG NGHVI SINH 247K t quphn tch hcellulase trn gel SDS-PAGEK T LU NMitr ngt i ucho T.reesei VTT-D-80133sinhracellulasel:t l BM:CM(7:3), 8 l n n ng dinh d ng, m ban u 60 %, th i gian nui c y 7 ng y. Ho ttnh CMCase v FPU t ng ng l: 280,63 IU/g v 5 FPU/g; th p h n 3,2 v 37 l n sov iAmanoT. Ngoi cellulase,canhtr ngcnch a -amylase368,75UI/g,protease12,43UI/gvxylanase10073,25BXU/g.Quaphntchtrngelpolyacrylamide,cellulase thu nh n c c cc v ch protein v i tr ng l ng phn tb ng v i cc v chproteinc ach ph mAmanoT. Dchenzymcellulasec a T.reesei VTT-D-80133 v iho tl c5FPU/ml,ckh nng nghakho ng20%gi yinquas d ng;dch ng ha ch a 23,62mg ng kh /ml c th c sd ng ln men ethanol ho cln men s n xu t cc s n ph m c gi tr.TI LI U THAM KH O1. Nguy n C nh (1993). Qui ho ch th c nghi m, Tr ng HBK Tp. HCh Minh.2. Chahal D. S., (1985). Solid-state fermentation with Trichoderma reesei for cellulaseproduction. Applied and Environmental Microbiology, Vol. 49, No. 1, p. 205-210.3. JeffriesT.W.(1987).Productionandapplicationsofcellulaselaboratoryprocedures. Forest Products Laboratory, Madison, Wisconsin.4. BigelowMandWymanC.E. (2002).Cellulaseproductiononbagassepretreatedwith hot water. Applied Biochemistry and Biotechnology, pp. 98-100.5. RaimbaultM.(1998).Generalandmicrobiologicalaspectso fsolidsubstrate. EJBElectronic Journal of Biotechnology Vol. 1, No. 3.1 23kDa664536292420K tqu i ndi trngelSDS-PAGEc accdch enzym cellulase ghi nh n c nhhnh bn.Gi ng 1: Dch enzym t T. reesei VTT-D-80133.Gi ng 2: Dch enzym tchph m Amano TGi ng 3: Thang phn tl ng nhK t qucho th y, gi ng 1 c cc v ch protein r tgi ngv igi ng2. K tqu nycth cgi ithch nhsau: theo Barnett (1991), hcellulase t T.reeseig mCBHI,CBHII,EGI,EGII,EGIII,EGVvm t |-glucosidase;theobocog n yth T.reesei sinh ra hai lo i |-glucosidase l Bgl1 v Bgl2(Alinda A. Hasper et al., 2001). D a vo cc dli utrn, ta c thk t lu n r ng canh tr ng nui c y T.reesei VTT-D-80133 c y cc ti u ph n c a hcellulaseH i ngh KHOA H C V CNG NGH2007 2486. YunHyunShiketal. (1998).Productionofcellulaseby TrichodermareeseiRutC30 in wheat bran media. Journal of Microbiology and Biotechnology , 8(3).SUMMARYOptimization of cellulase production by Tricoderma reeseion solid substrate mediaTran Thanh Phong, Hoang Quoc Khanh,Vo Thi Hanh, Le Thi Bich Phuong, Nguyen Duy Long,Le Tan Hung, Truong Thi Hong VanInstitute of Tropical BiologySugarcane baggase/wheat bran ratio (7/3), initial moisture content of 60%, 8 timesconcentrationofbasalmediumandcultivationtimeof 7dayswereoptimumforcellulaseproductioninsolidstatefermentation(SSF ) by Trichodermareesei VTT-D-80133mutant. Theactivityandproductivityofce llullaseobtainedafter 7days offermentation were 280,64 IU/gand 5 FPU/g of fermented medium; These activities arelowerthan3,2and37timescomparedwithcommercialcellulase (Amano T) ofAMANO Company (Japan). Besides cellulase, fermented substratecontain activities of -amylase268,75UI/g,protease12,43UI/gandxylanase10073,25BXU/g.Thecellulaseextract wasshowntohave somebandsthattheirmolecularweightsareequivalenttocommercial cellulase (Amano T) bysodiumdodecylsulphatepoly-acrylamidegelelectrophoresis(SDS-PAGE). Atthe filterpaperactivityof cellulasewas 5 FPU/ml, the yield of enzymatic hydrolysis of paper waste was 21%.Ph n II: CNG NGHVI SINH 249PHN L P, NH DANH V TUY N CH N CC CH NGEnterococcusC TI M NNG probiotic TPHN TRSSINHHVn Th o, Hong Qu c KhnhPhng Vi Sinh ng d ng, Vi n Sinh h c Nhi t iM UTrong ru t ng i c kho ng 10 t vi khu n trong 1 gram phn, g m v i trm loi vikhu nt onnh visinh ngru th ts cphongph[14].Enterococciv streptococci nhm D t o nn m t ph n quan tr ng c bi t trong hvi sinh v t b n atrong ru t ng i v m t sloi ng v t, m phbi n l E. faecalis v E. faecium. cbi t E. faecalis th ng xuyn c tm th y trong ru t gi c a ng i, thnh tho ng c ng c tm th y trong ru t g, nh ng t khi tm th y trong cc ng v t khc.TheoShleifervKilpper-Balz(1984,1987)mt cc ci mv cpd ng cho h u h t cc loi thu c gi ng Enterococcus. y l nhm vi sinh v t kkh ty, d ng c u n hay k t chu i, gram d ng, catalase m tnh. Xu t hi n kh p n i trongmi tr ng, nh ng phbi n trong cc s n ph m s a v nh t l trong ru t ng i v m tsloi ng v t c v. Enterococci c ths ng v pht tri n c trong bin nhi t khr ng l n, t100C - 450C, t i u 350C - 370C, s ng st t nh t 30 pht 600C. Nhcb m Natri-ATPase n m trn mng tbo ch t nn c thchu c n ng NaCl ln n6,5%.pHho t ngt 4,6 -9,6,t i u kho ng6,5 -7,5.H uh tccloithu cEnterococci c thth y phn c esculine v i shi n di n c a mu i m t 40%.V T LI U V PH NG PHPPhn l p v nh danh [10,11, 13]Ngu ngi ng vi khu n: gi ng vi khu n c phn l p tphn trssinh thu th pt i b nh vi n TDv B nh vi n Gia nh thu c Thnh phHCh Minh, Vi t Nam.M u c pha long vtr i trn mi tr ng BEA (Bile Esculin Azid), c 370C,[4],[5].Sau2ngych nnh ngkhu nl ctchr ic ngknht 0,5 -1mm,mutr ng cho ch itrong, ngth it othnhqu ngenxung quanhkhu nl c,lmthu n v gigi ng trn mi tr ng MRS broth (c bsung thm 20% glycerol [16].Cc ch ng vi khu n thu c s c ti n hnh nhu m gram, test catalase, kh o st c tnh ln men glucose, khnng s ng st v pht tri n trong mi tr ng MRS brothc n ng NaCl 6,5% v mi tr ng c n ng dch m t 40% [4]. Khnng pht tri n c 100C,450Ctrongkho n