CNG NGH VI SINH NG DNG

EXOPOLYSACCHARIDES

LI NI U
Ngy nay, vi s tin b ca khoa hc cng ngh, con ngi ngy cng tip cn gn hn, hiu bit nhiu hn n th gii sinh vt ni chung v h vi sinh vt ni ring. V, vi nhng bc tin ca mnh, cc nh khoa hc nghin cu thnh cng rt nhiu nhng sn phm c sn xut t vi sinh vt, gp phn quan trng trong nn cng nghip sn xut, trong y hc, trong x l mi trng,....V mt trong nhng sn phm quan trng l exopolysaccharides.

GVHD: Th.S H THIN HONG

Page 1

CNG NGH VI SINH NG DNG

A. TNG QUAN EXOPOLYSACCHARIDES ( EPS ) & MNG SINH HC EXOPOLYSACCHARIDES I. NH NGHA Exopolysaccharides l polyme c trng lng phn t cao bao gm d lng ng, c tit ra bi mt loi vi sinh vt vo mi trng xung quanh. Vi sinh vt tng hp nhiu loi cc polysaccharides a chc nng bao gm polysaccharides trong t bo, cc polysaccharides cu trc v polysaccharides ngoi bo hoc exopolysaccharides (EPS). Exopolysaccharides thng bao gm cc monosacarit v mt s nhm th khng carbohydrate (nh acetate, pyruvate, succinate, v phosphate). Do s a dng, phong ph trong thnh phn, exopolysaccharides c ng dng phong ph trong nhiu ngnh cng nghip thc phm v dc phm. EPS ca vi sinh vt cung cp c tnh gn ging nh cht gm hin ang s dng. Vi phng php tip cn sng to, nhng n lc ang c tin hnh thay th cc thit b truyn thng v phn ln gm bi h vi sinh vt ca mnh. Hn na, mt tin b ng k c thc hin trong vic pht hin v pht trin cc EPS vi sinh vt mi c ngha trong cng nghip. II. CHC NNG Nhng li ch cm quan ca exopolysaccharides t vi khun axit lactic cng c thnh lp v c bng chng cho cc thuc tnh v sc khe do exopolysaccharides t vi khun axit lactic. Exopolysaccharides Capsular c th bo v vi khun gy bnh v gp phn vo kh nng ly bnh nhn to ca chng. Phn nh km ca vi khun c nh nit r cy v cc phn t trong t, c ngha quan trng i vi vng xung quanh trong t ca vng r v ly nhim ca thc vt, c th c trung gian bi exopolysaccharides. Mt v d cho vic s dng cng nghip ca exopolysaccharides l ng dng dextran trong bnh m panettone v cc ngnh cng nghip bnh. Exopolysaccharides cng c mt vai tr quan trng trong cc bnh nhim trng ni nha. III. DANH SCH CC EXOPOLYSACCHARIDES acetan (Acetobacter xylinum) alginate (Azotobacter vinelandii) cellulose (Acetobacter xylinum) chitosan (Mucorales spp.) curdlan (Alcaligenes faecalis var. myxogenes) cyclosophorans (Agrobacterium spp., Rhizobium spp. and Xanthomonas spp.)

GVHD: Th.S H THIN HONG

Page 2

CNG NGH VI SINH NG DNG

dextran (Leuconostoc mesenteroides, Leuconostoc dextranicum and Lactobacillus hilgardii) emulsan (Acinetobacter calcoaceticus) galactoglucopolysaccharides (Achromobacter spp., Agrobacterium radiobacter, Pseudomonas marginalis, Rhizobium spp. and Zooglea' spp.) gellan (Aureomonas elodea and Sphingomonas paucimobilis) glucuronan (Sinorhizobium meliloti) N-acetyl-glucosamine (Staphylococcus epidermidis) N-acetyl-heparosan (Escherichia coli) hyaluronic acid (Streptococcus equi) indican (Beijerinckia indica) kefiran (Lactobacillus hilgardii) lentinan (Lentinus elodes) levan (Alcaligenes viscosus, Zymomonas mobilis, Bacillus subtilis) pullulan (Aureobasidium pullulans) scleroglucan (Sclerotium rolfsii, Sclerotium delfinii and Sclerotium glucanicum) schizophyllan (Schizophylum commune) stewartan (Pantoea stewartii subsp. stewartii) succinoglycan (Alcaligenes faecalis var myxogenes, Sinorhizobium meliloti) xanthan (Xanthomonas campestris) welan (Alcaligenes spp.)

Succinoglycan t Sinorhizobium meliloti

GVHD: Th.S H THIN HONG Page 3

CNG NGH VI SINH NG DNG

IV. MNG SINH HC EXOPOLYSACCHARIDES Nhiu nhn nh v vi khun c da trn nhng nghin cu trong phng th nghim ni vi khun tn ti, l lng trong mi trng giu dinh dng. Th nhng, vi khun trong th gii t nhin th ng x khc vi vi khun trong phng th nghim. L v, thin nhin, ni k th th nhiu m thc n li khng bao nhiu, l mi trng khc nghit hn nhiu so vi phng th nghim. tn ti, vi khun phi hc cch bm ln b mt, lin kt cht ch vi cc loi khc cng sinh v t bo v mnh. Ci th gii thu nh gn kt li vi nhau bng mt th "cht nhy" polysaccharides do vi khun sn sinh ra , thnh mt th gi l "Mng Sinh hc". Mng sinh hc l cu trc thng gp trong th gii t nhin. Ngi yu thy sinh vn khng l g vi cn my lc hoc vng trn mt nc; (cn hoc vng trn mt nc) l nhng v d in hnh ca mng sinh hc. Nhng trng hp mng sinh hc c nghin cu nhiu nht, l: 1. cao rng; 2. bnh nhn b x nang v vim phi mn tnh; 3. ng nc v thn tu b n mn; 4. s nhim bn cc th nh knh p trng, tim nhn to v cc thit b cy ghp y khoa. do khin vi khun gn vo v to nn mng sinh hc ln b mt l v b mt l ni cht dinh dng tch t li. Chnh do mi b mt u c in tch m, in tch m th s ht ion dng v cacbon hu c ho tan. Ri cc hp cht mang in tch dng tch t li bn nhau li s thu ht cc hp cht mang in tch m. V th, ngay c trong mi trng nc ngho cht dinh dng, thng cng c va cht hu c bm vo b mt gip vi khun pht trin. hi cc hp cht hu c t li trn mt nc, chng s thu ht cc vi khun, to v ng vt nguyn sinh thch n chng n, theo thi gian s pht trin thnh mt mng sinh hc, c gi l neuston (sinh vt sng trong mng mt nc vng b mt). Vi khun bm vo b mt theo nhiu cch khc nhau. Vi loi vi khun t bn thn c tnh kt dnh: c bn chng l cc keo bao ph bi cc mng dnh lipopolysaccharide hoc bi cc phn ph gc prtin. Cc vi khun khc ch tng hp cht kt dnh cn thit khi xut hin b mt cho chng bm vo. Ch ng hn nh, trong vng 15 pht khi vi khun gy vim ng h hp Psuedomonas aeruginosa gp mt mt knh, n s kch hot ngay mt gen cn tng hp polysaccharide. hi vi khun gn vo b mt, chng chia ra v lin tc sn xut tht nhiu polysaccharides to nn mng sinh hc hon ch nh.

GVHD: Th.S H THIN HONG

Page 4

CNG NGH VI SINH NG DNG

Mt Mng Sinh hc han ch nh c th dy t 600-900 m, tc l dy gp my trm ln mt con vi khun n l.(mt con vi khun di khong 1m). Mng sinh hc khng phi l mt cht v nh hnh, hay mt khi c st cc polysaccharides v vi khun; n c t chc v cu trc. Thm ch l khu vc dy nht ca mng sinh hc cng cho lung nc chy qua. Nc chy qua cc cu trc hnh nm ca nhng khi cu vi khun, qua , cung cp dinh dng cho chng v em cht thi i. R rng, cu trc bn trong ca mng sinh hc khng c cu thnh theo cch ngu nhin. Cc nh nghin cu cho bit c s trao i thng tin lin tc din ra gia cc vi khun m bo mng sinh hc c hnh thnh mt cch chnh xc. (Cc vi khun t bin khng th truyn thng vi nhau to nn cc mng sinh hc bt thng.) Cc mng sinh hc khng lun lun ging y chang nhau, theo kiu gm nhiu lp vi khun hiu kh bn trn v nhiu lp vi khun k kh pha di. Do cc lung nc chy qua khuy ng nn cc vi khun k kh v hiu kh song song tn ti trong cc hc nh khp trong mng sinh hc. V th, cc nh nghin cu thit ngc nhin khi thy qu trnh kh nito xy ra trong mt b lc xc kh vn dng x l nc thi. H thy lng vi khun hiu kh, vi khun to nit (nitrat ha), vi khun kh nit, v vi khun k kh, c y v trn cng, bng y nh nhau. V lm thm nhng th nghim khc, h thy c cc hot ng trao i cht qua li gia cc vi khun to nito (hiu kh) v vi khun kh nito (ky kh) y nh nhau c tng y cng nh tng trn cng. C th vi khun to nito v cc vi khun khc lp c mt mi quan h tng h hai bn cng c li v cht ch trong cc mng sinh hc ca cc b lc sinh hc. V cc vi sinh hiu kh bnh thng phng thch NH3 trong qu trnh phn hy cc hp cht hu c, cc vi khun to nito c th dng NH3 (c nh m) lm ngun nng lng cho mnh. Ri n lt cc vi khun kh nito chuyn tiu th acid, nn, c th bo v cho cc vi khun to nito - vn c bit nhy cm vi tnh acid. Vi khun trong mng sinh hc c nhiu thun li hn l nhng vi khun l lng t do trong nc. Trc ht, chng chia s thng tin di truyn v trao i cht cho nhau. V d nh, trong mng sinh hc cao rng, vi khun Veillonella s dng lactate do vi khun Streptococcus sinh ra. Th nh, vi khun trong mng sinh hc c bo v khi k th v cc ho cht c hi. Trong th gii di nc, mng sinh hc bo v vi khun khi b cc ng vt nguyn sinh, cc loi to c hi (dinoflagellates - to roi) v khun c (Myxobacteria - nim khun) lm hi. V bnh ca ngi, mng sinh hc gip vi khun khng b thuc khng sinh, ha cht, khng th, t bo min dch,....lm hi.

GVHD: Th.S H THIN HONG

Page 5

CNG NGH VI SINH NG DNG

V th, cc t bo l lng ca trc khun m xanh - Pseudomonas aeruginosa s b tiu dit bi 0,050 mg/ml tobramycin - khng sinh dng trong thuc nh mt trong khi nhiu hn th 20 ln thuc ny (0.1 mg/ml) m vn khng dit c trc khun m xanh khi n l ho cht c ch nito - nitrapyrin, s tng trng ca vi khun trong trng hp cy mng sinh hc khng b nh hng g c, cn s tng trng ca vi khun sng trong mi trng cy l lng b gim 82 %. Cc nh nghin cu dng kt qu th nghim ca h gii thch ti sao cht c ch nito (nitrapyrin) khng ngn chn c qu trnh sinh ra nito ngoi thc t cho nng dn, nh tin liu theo nghin cu trong phng th nghim. V th, tuy cht c ch ni-t (nitrapyrin) l cht c ch hiu qu i vi N.europaea sng l lng trong mi trng giu dinh dng trong phng th nghim, nhng li khng c hiu lc trong nhng iu kin khi vi khun c th bm vo cc ht t v tr ng bn trong mng bo v sinh hc. Mt vi ng dng ca mng sinh hc l dng Lc sinh hc v lm mng tr bng Lc sinh hc: Lc sinh hc (biofiltration) l mt cng ngh iu khin s nhim mi. N bao gm s loi b v xi ha nhng hp cht kh b nhim bn nh vi sinh vt. Lc sinh hc c thit lp rt tt trong cng ngh iu khin nhim c v H an v n cng thu ht c s quan tm Bc M. Lc sinh hc c th x l nhng phn t kh hu c- nhng hp cht hu c bay hi ( Volatile Organic Compound- VOC's) hoc cc hp cht cacbon, hay nhng cht kh c v c- amoniac hay H2S. Lc sinh hc s dng vi sinh vt phn hy nhng hp cht hu c ( hoc bin i nhng hp cht v c) thnh cacbonic, nc v mui. Khi h thng lc sinh hc c lp t, vi sinh vt c sn trong nguyn liu m n c s dng nh mt lp lc. Trong cc phng th nghim, vi mc ch tng cng tc phn hy, vi sinh vt c cn nhc n u tin l hiu qu ca chng trong vic phn hy ca nguyn liu c nghin cu. Nguyn liu lc thng l than bn, t, phn compt hay cy thch nam, tuy nhin bt cacbon c hot ha v polysterene cng c th c s dng. S la chn nguyn liu lc l v cng quan trng bi v n phi cung cp cho vi sinh vt dinh dng, s pht trin v mt sinh hc, v c dung tch hp th tt. Qu trnh sinh hc l mt s xi ha nh vi sinh vt, v c th c vit nh sau: Hp cht gy nhim + Oxi -> CO2+ H2O + nhit + sinh khi Vi sinh vt sng trong lp mng sinh hc m , mng, ni c bao bc xung quanh cc phn t ca nguyn liu lc. Kh bn c khuych tn trong h thng lc v c hp th bn trn mng sinh hc. Thc t y l v tr m qu trnh xi ha c thc hin. Cc cht bn khng c lun chuyn c nh n nguyn liu lc.

GVHD: Th.S H THIN HONG

Page 6

CNG NGH VI SINH NG DNG

Mng tr bng: Ngi ta dng mng sinh hc ngn nga bin chng nhim trng vt thng bng, to iu kin che ph sm vt thng. Qua , rt ngn thi gian iu tr v gim thiu so xu trn vng bng su. Ch phm ny ca nhm nghin cu c kh nng thm nc cao, kh nng kt dnh cht ch v tr v mt ha hc nn n c vai tr nh mng sinh hc, c th thay th da tm thi. Mng c kh nng dit 100% vi khun thng gy ra cc nhim trng vt thng h da nh vt bng, hay cc vt thng mt. Ch cn p st mng vo vt thng v khng cn s dng bt c th g khc, mng c kh nng cn khun, ng thi, lm vt thng mau lnh do thc y qu trnh ti to m ht. B. TI U HA SN XUT EXOPOLYSACCHARIDE BNG Lactobacillus delbrueckii subsp. bulgaricus RR TRONG MI TRNG BN TNG HP Stacy A. Kimmel, Robert F. Roberts, Gregory R. Ziegler. (B phn Khoa hc Thc Phm, i Hc Tiu Bang Pennsylvania, University Park, Pennsylvania 16802.) Nhit ln men ti u, pH, v nng nito cho qu trnh sn xut exopolysaccharide bi Lactobacillus delbrueckii subsp. bulgaricus RR trong mt mi trng bn tng hp c xc nh bng cch s dng phng php phn ng trn b mt. Vic thit k bao gm 20 th nghim, 15 kt hp c o, v 5 ng dng. Tt c cc qu trnh ln men c thc hin trong ni ln men 2,5l th tch lm vic v kt thc khi 90% glucose trong mi trng c s dng. Qun th L. delbrueckii subsp. bulgaricus RR v hm lng exopolysaccharide c xc nh vo cui mi qu trnh ln men. Nhit ti u, pH, v nng nito trong sn xut exopolysaccharide l 38 C, pH = 5, v 30 g / lt, vi sn lng d on l 295 mg exopolysaccharide/lit . Nng sut thc t theo nhng iu kin ny l 354 mg exopolysaccharide / lt, trong khong tin cy 95% (217-374 mg ca exopolysaccharide / lt). Mt th nghim b sung thc hin theo iu kin ti u cho thy sn xut exopolysaccharide thu c s tng trng kt hp, vi sn xut ti im cui thch hp thu 101.4 mg / g t bo kh. Cui cng, c c vt liu cho cc c tnh hn na, mt qu trnh ln men 100 lt c tin hnh trong iu kin ti u. 29g exopolysaccharide c phn lp t ly tm, lc nc ln men v kt ta bi ethanol. S quan tm v vi khun axit lactic sn xut exopolysaccharide (EPS) tng ln gn y bi v y l nhng loi sinh vt thc phm sn xut polymer quan trng trong vic xc nh cc c tnh lu bin ca cc sn phm t b sa v c th ng dng vo thc phm khng cha b sa . Khi thm vo sn phm thc phm, polysaccharides mang chc nng nh cht lm c, n nh, cht chuyn th sa, tc nhn lm ng , v tc nhn gi nc . nh gi cc thuc tnh chc nng m
GVHD: Th.S H THIN HONG Page 7

CNG NGH VI SINH NG DNG

EPSs mang n li ch trong thc phm yu cu c sn ngun nguyn liu vi y s lng. iu ny thng i hi vic th nghim t ln men quy m th im n quy m cng nghip mt khi iu kin ti u c xc nh. S ti u ha mi trng tng trng l iu quan trng thu c EPS ti a sn xut bi cc sinh vt nh Xanthomonas, Pseudomonas, v Rhizobium spp.Nhng kt qu c cng b ti u ha sn xut EPS bi vi khun axit lactic bao gm cc nghin cu nh gi tc ng ca iu kin mi trng nh nhit v pH . Mt s cc nghin cu kim tra tc ng ca nhit bng cch s dng cc chng Lactobacillus delbrueckii subsp sn xut EPS . Garcia-Garibay v Marshall nhn thy rng s sn xut polymer tng ng (tng ng 1 1 mg dextran / 1 n v khun lc ) ca chng NCFB 2772 pht trin sa gy nhit (48oC) cao hn so vi nhit ti u cho s tng trng (37-42 C). S sn xut polymer tng ng cao hn (1 mg/1 gram cc t bo [trng lng kh]) nhit cao hn (45 C) cng c tm thy bi Grobben et al, ngi kim tra s sn xut EPS bi cng mt chng trong mi trng xc nh . Mozzi et al nhn thy mt mi tng quan gia nhit tng trng ti u (37 n 42 C) v sn xut polymer t chng CRL 870. Ngc li, Schellhaass bo co sn lng polymer cao hn bi chng RR nhit di mc nhit ti u cho s tng trng. Van den Berg et al. tin hnh mt nghin cu nh gi tc ng ca nhit v pH trong sn xut EPS t chng L. shake. 0-1. H nhn thy, bng cch s dng php theo di mt bin ti mt thi gian (OVAT), sn xut EPS ti a xy ra 20 C v pH 5,8. Mt nghin cu khc ca Mozzi et al. nhn thy rng s tng hp polymer ti a (488 mg / lt) t chng L. casei CRL 87 xy ra 30 pH 6,0. Tuy nhin, iu kin sn xut ti u nht (EPS c sn xut mi gram theo trng lng kh ca t bo) v nng sut EPS (gam EPS x 100/grams ca ng tiu th) c tm thy pH 4.0. i vi mt s vi khun sn xut EPS , ch ng hn nh Xanthomonas, Pseudomonas, v Rhizobium spp, trong nhng iu kin ngun nit hn ch kt qu sn xut EPS s tng ln. Tc ng ca nng nit nh hng n sn xut EPS bi cc chng lactobacilli khng c kim tra. L. delbrueckii subsp.bulgaricus RR l mt chng sn xut EPS ph bin. Hiu qu ca vic s dng L. delbrueckii subsp. Bulgaricus RR trong qu trnh khi u nui cy trn cc c tnh lu bin ca thch sa (sa chua) c kim tra di nhiu iu kin khc nhau . Ngoi ra, thnh phn monosaccharide v s lp i lp li cu trc ca EPS c sn xut bi L. Delbrueckii subsp. bulgaricus RR c xc nh .Tuy nhin, c tnh tng tc gia cc protein sa v EPS c sn xut bi L. delbrueckii subsp. bulgaricus RR, cng nh nh gi nh hng chc nng ca EPS n h thng thc phm khng ln men, b cn tr bi v khng c s lng cc polymer c chc nng.
GVHD: Th.S H THIN HONG Page 8

CNG NGH VI SINH NG DNG

Mc tiu ca cng vic hin ti l xc nh iu kin ti u v nhit , pH, v nng nito cho sn xut EPS bi chng subsp L. delbrueckii. bulgaricus RR trong mi trng bn xc nh bng cch s dng thit k b mt phn ng th nghim v s dng cc iu kin ny sn xut EPS mang nhiu c tnh hn na. I. VT LIU & PHNG PHP 1. Chng vi khun v qu trnh nui cy L. delbrueckii subsp. bulgaricus RR ban u thu c t phng th nghim ca H.A Morris (i hc Minnesota, St Paul) v duy tr trong mi trng MRS (Difco). Nguyn liu ca qu trnh nui cy c chun b bng cch trn vi mi trng nui cy thun khit t 12 n 16h 42 C trong canh trng MRS, kt hp vi 10% glycerol v c bo qun -75oC. Mi trng nui cy chnh thc c chun b bng cch chuyn 0,5 ml mi trng nui cy c ng lnh trn vi 10 ml canh trng MRS v n t 12 n 18 h 40 C. Mi trng lm vic ca chng RR c chuyn giao (1% [vol vol) ln 20 ml canh trng MRS v t 12 n 18 h 40 C. Dung dch tin nui cy ny c ly tm 8000 g v 4 C trong 10 pht v ra sch hai ln vi 20 ml nc ct v trng. Dch treo t bo (trong nc ct v trng) c s dng cy khi lng ln (2,0 lt) ca mi trng bn tng hp (semidefined medium - SDM). 2. Mi trng nui cy Mi trng bn tng hp ( SDM ) s dng nh mi trng c s cho cc th nghim EPS c cc thnh phn sau y (g l): dextrose, 20, 80 Tween, 1, ammonium citrate, 2, sodium acetate, 5; MgSO4 7H2O, 0.1, MnSO4, 0.05; 2HPO4, 2, nm men nit c s m khng amoni v axit amino (Difco), 5; Bacto.-casitone (Difco), 10. pH ca tt c mi trng c iu ch nh n 6,5 0,2 trc khi kh trng bng cch lm nng trong 15 pht 121 C. Glucose c kh trng mt cch ring bit v thm vo mi trng. Qu trnh lm vic trong phng th nghim ca chng ti ch ra rng thi gian th h mi cho dng RR MRS v SDM khng khc bit ng k 42 C v cc phng tin can thip ti thiu vi cc xt nghim c s dng nh lng EPS. 3.Thit k th nghim xc nh cc iu kin ti u cho EPS sn xut t chng RR, mt phn ng b mt c thit k th nghim bng cch s dng mt m hnh bc hai c to ra bng cch s dng Echip (eChip, Inc, Hockessin, Del). Ba yu t kh bin c a vo m hnh: nhit (35-45 C), pH (4-6), v Bacto.-Casitone (nit) tp trung (10 - 30 g / lt) ca mi trng tng trng. Cc thit k c 20 th nghim, 15 s kt hp, v nm ln lp li (xem Bng 1).
GVHD: Th.S H THIN HONG Page 9

CNG NGH VI SINH NG DNG

4. S ln men Tt c cc qu trnh ln men ti u ha c thc hin trong mt khi lng lm vic 2,5 lt Bio-flow III (New Brunswick, Edison, NJ) ln men chm. Sau khi cy, pH gim t 6,5 n im thit lp sn xut axit lactic. pH sau c duy tr im thit lp bng cch thm 5 N NaOH. S khuy trn c duy tr 200 rpm sut qu trnh ln men. Ngay trc khi cy, mi trng c ra vi nit trong 20 pht vi tc 1 lt / pht. Mu (khong 30 ml) c loi b theo chu k u n v phn tch cho s tng trng, glucose, v EPS nh m t di y. Qu trnh ln men c chm dt khi 90% glucose ban u c s dng. 5. Php o tng trng. Mc tng trng ny c theo di bng k thut o quang (650 nm) v nui cy trn a vi pha long thch hp trn mi trng thch MRS, tip theo l k kh trong 48 gi 42 C. Vi th nghim s b, xc nh trng lng kh t bo c thc hin trn bn sao mu 10 ml canh trng ln men. Mi mu c ly tm (8000 g trong 10 pht) v ra sch hai ln vi nc ct. Cc ht nh c tch huyn ph trong 5 ml nc ct, sy kh nhit 97 C trong 5 h, v sau c cn li. 6. Phn tch glucose Glucose ca mi trng trong qu trnh ln men c theo di bng sc k lng hiu sut cao. Cc column v u d c duy tr mc 35 C. Dung mi 0,005 M sulfuric acid, c chuyn ti mt tc dng chy 0,6 ml / pht. D liu c thu thp bng cch s dng h thng thu thp d liu SSI Vision IV (khoa hc Systems Inc, State College, Pa). Glucose c nh lng bng cch lin h cc khu vc cao im c mt ng cong tiu chun. 7. nh lng EPS Cc bc c s dng cho nh lng EPS c da trn m t bi Gancel v Novel. Khong 10 g mi trng nui cy c cn chnh xc vo mt ng ly tm 50 ml v sau un nng trong mt cc nc si trong 10 pht bt hot cc enzym c kh nng lm suy thoi polime. Mu c lm lnh ti nhit phng, 100 ml 5% (wt / vol) pronase E (EC 3.4.24.31, Sigma Chemical Co, St Louis, Missouri), dng dung dch c b sung, v hn hp c trong 1 gi 37 C trong mt bn nc. Sau khi protein ha, 250 ml 80% (wt vol) axit tricloaxetic c b sung v mu c trn u v c bo qun 4 C ti thiu l 30 pht v sau ly tm (8000 g trong 20 pht) loi b cc t bo v protein. Cc cht ni trn mt c chit ra bnh vo ng thm tch (trng lng phn t ct, 6.000 n 8.000) v thm tch 48 gi so vi bn thay i ca nc ct. Tt c cc xt nghim c thc hin nhiu ln, v cc th tc tng t c thc hin vi mi trng cha cy ging.
GVHD: Th.S H THIN HONG Page 10

CNG NGH VI SINH NG DNG

Nng carbohydrate ca retentate c xc nh bng cch s dng phng php phenol-sulfuric acid. Kho nghim carbohydrate c iu ch nh bng cch s dng mt hn hp OFD-galactose, D-glucose, andL-rhamnose (05:01:01), v kt qu c bo co bng s mg carbohydrate mi lt. Cc gi tr hin th cho EPS c tnh ton bng cch loi b s lng tp nhim trong mi trng cha cy ging (khong 44 mg carbohydrate / lt) t s lng trong canh trng ln men. 8. Sn xut vi quy m ln v thu hi EPS Sn xut EPS quy m ln c thc hin trong mt mch ln men 100 lt (khi lng) (Bio-service, Allentown, Pa). S khuy trn c duy tr mc 100 rpm, v h thng ln men kn c ra bng nit trong qu trnh tit trng c c mt mi trng k kh. Nhit , s khuy trn, v pH c theo di v kim sot vi mt my tnh Macintosh (Apple Computer Inc., Cupertino, California) v mt chng trnh pht trin trong nh bng cch s dng h thng phng th nghim pht trin (National Instruments, Austin, Texas). Mt m hnh 2700 Select Biochemistry Analyzer (YSI Biochemical Products, Yellow Springs, Ohio) c s dng gim st vic s dng glucose trong sut qu trnh ln men v phc hi. Cc thit b u cui (90% glucose c s dng), s khuy trn c tng ln n 350 rpm v nhit ca b cha c nng ln n 100 C. Sau 15 pht 100 C, b cha c lm lnh n 37 C v 100 ml 1 5% (wt / vol) pronase E (EC 3.4.24.31; Sigma Chemical Co) c b sung. Sau 60 pht 37 C, 250 ml 80% (wt / vol) axit tricloaxetic c b sung v b cha c lm lnh xung 17 C. Sau khi c x l, canh trng ln men c bm vo mt my ly tm Sharples A316Y (Sharples Inc, Warminster, Pa) v ly tm 16.800 rpm loi b cc t bo v protein. EPS c c c bng cch siu lc canh trng ln men bng cch s dng mt mng cellulose ti sinh trong h thng siu lc 50 RL (Millipore Corp.). Cc cht tan c kch thc nh c loi b bng 1 h thng mng lc ch loi b cc cht c kch thc nh v gi li cc phn t ln. Cc b lc cha EPS c t trong mt bnh 20 lt, v EPS b kt ta bng cch thm 2 khi lng ethanol 95% lnh v sau c lu tr trong 2 ngy 4 C. Lp trn cng ca cht lng c chit ra bnh, v lp di cng, trong c EPS kt ta, c t trong chai v ly tm 8000 g v 4 C trong 20 pht. Cc ht nh c g b, tch huyn ph trong nc ct, v ng kh (-40 C). Hm lng protein ca vt liu kh c o theo phng php Bradford , nng carbohydrate tng s c xc nh theo phng php phenol-sulfuric acid , v m c o bng trng lng vt liu trc v sau khi ng kh (-40 C). II. KT QU Ti u ha sn xut EPS. Mt thit k th nghim phn ng b mt c s dng xc nh nhit ti u, pH, v nng Bacto-casitone cho sn xut EPS bng
GVHD: Th.S H THIN HONG Page 11

CNG NGH VI SINH NG DNG

chng RR. Kt qu thu c t tt c cc th nghim trong thit k c trnh by trong bng 1. Chy 18( th nghim 9) b lai tr t cc phn tch d liu bi v iu kin sinh trng qu nghim ngt v im cui (90% s dng Glucose) khng t c trong vng 168 gi. Nhit ti u, pH, v nng Bacto-casitone sn xut EPS c xc nh 38C, 5v 30g l, tng ng (hnh 1),vi iu kin nh trn d an sn xut c nhiu nht l 295mg EPS/l.

HNH 1: Biu ng ng mc ca EPS sn xut bi L. subsp.bulgaricus delbrueckii RR 30 g ca Bacto-casitone mi lt nh mt hm s ca nhit v pH. ng ng mc vi nhng con s ng k khc nhau (P <0,05). Cc ng cho l ranh gii thit k, tc l, iu kin th nghim bn di dng ny khng c trong thit k. Nng sut thc t theo nhng iu kin ny l 354mg EPS l, iu ny chnh xc 95% so vi d an ( l thuyt).Mt l ca cc kt qu ch ra rng c th thu c sn lng EPS cao hn nu nng Bacto-casitone ln hn 30g l (hnh 2). kho st, mt th nghim c tin hnh nhit ti u (38C) v pH (5.0) vi nng Bacto-casitone 40g/l.Sn xut EPS di nhng iu kin ny c tm thy l 324g/l, thp hn sn lng ca EPS c sn xut trong iu kin ti u (Bng 1).Khi kt qu ny c a trong phn tch, d an ti u v ngha mi quan h gia cc iu kin tng trng v phn ng bin i (bin d) khng c s thay i. Bng 2 cung cp cc h s ca cc m hnh thng k thu c t nhng phn ng b mt, cng nh ngha thng k ca mi k.
GVHD: Th.S H THIN HONG Page 12

CNG NGH VI SINH NG DNG

to thnh cc iu kin ti u cho sn xut EPS bng chng RR, mt th nghim c tin hnh 38C, pH = 5, 30g/l Bacto-casitone.Tc tng trng v sn xut EPS c theo di trong sut qu trnh ln men.S to thnh cho thy sn xut EPS c lin quan n s tng trng, v d nh, sn xut EPS km theo s pht trin ca sinh vt, vi mt sn phm c th im cui 101.4mg EPS/g t bo kh v mt s lng sn phm c th trong qu trnh tng trng ca 0.472mg EPS/g t bo/h (xem hnh 4). S phn lp v phc hi ca EPS. c c vt cht tip tc xc nh c im, mt qu trnh ln men 100 lt c thc hin bng cch s dng nhng iu kin ti u cho sn xut EPS (38C, pH =5, 30g/l Bacto-casitone).Sau khi lc EPS lng li v lai b vt liu c phn t lng thp. 5 lt EPS c cha retentate c thu.Sau khi dng ethanol kt ta retentate v ng kh, thu c 29.2g semipure polysaccharide (bng 3). BNG 1: Kt qu th nghim ln men c thc hin ti u ha sn xut EPS L. RR subsp.bulgaricus delbrueckii

a. mi th nghim vi s ln khc nhau cho thy mt tp hp cc iu kin th nghim b. chy biu th th t m trong cc th nghim c tin hnh

GVHD: Th.S H THIN HONG

Page 13

CNG NGH VI SINH NG DNG

c. thi gian m 90% ca glucose trong mi trng pht trin c s dng c pht hin bng sc k lng cao p d. c tnh bng cch tr i s lng can thip sau trong mi trng cha cy ging t s lng trong canh trng dng trong ln men. e. kt qu ca mt th nghim c tin hnh kim tra d on iu kin ti u f. kt qu ca mt th nghim c thc hin vi Bacto-casitone 40g/lit III. BN LUN S ti u ha ca sn xut EPS,th nghim c thit k nghin cu ba iu kin sinh trng( nhit ,PH,v nng Bacto casitone) c kh nng nh hng n s sn xut EPS.S nh hng ca ngun carbon trong sn xut EPS khng c nghin cu bi v cc th nghim khc c biu hin,ni chung sn lng glucose cung cp(t 10 20 g lt) thu c sn lng EPS cao.

HNH 2: th ba chiu ca EPS sn xut bi L. delbrueckii subsp. bulgaricus RR pH 5.0 l mt hm s ca nhit v nng Bacto casitone.

GVHD: Th.S H THIN HONG

Page 14

CNG NGH VI SINH NG DNG

BNG 2: H s xc nh m hnh phn ng a Y = 0 + 1(T 40) + 2(pH 5) + 3(casitone 20) + 4(T 40) (pH 5) + 5(T 40)(casitone 20) + 6(pH 5)(casitone 20) + 7(T 40)2 + 8(pH 5)2 + 9(casitone 20)2, trong T l nhit ln men ( C), pH l pH ca mi trng trong qu trnh ln men, v casitone l s gam Bacto-casitone mi lt trong mi trng pht trin. b. Thi gian , 90% ca glucose trong mi trng pht trin c s dng bi cc sinh vt. Thi gian thit b u cui c mt thiu ht ng k ph hp (P <0,05) vi m hnh b mt phn ng. c. Khc bit ng k mc P <0,05 d. Khc bit ng k mc P <0,001 e. NI, khng c bao gm. Bao gm cc h s ny trong m hnh khng ci thin ph hp f. Khc bit ng k mc P <0,01. g. H s hi quy ca m hnh d on bin phn ng c th. h. gi tr P cho cc m hnh d on bin phn ng c th. Nhit ti u sn xut EPS nm trong khong nhit tng trng ti u ca RR (37-450C). Th nghim cng b tc ng ca nhit trong sn xut EPS bi vi khun acid lactic b tri ngc li.Mt vi bi bo co tm thy sn lng EPS ln
GVHD: Th.S H THIN HONG Page 15

CNG NGH VI SINH NG DNG

c sn xut ti nhit nm trong khong nhit sinh trng ti u,trong khi mt s khc li xut rng lng EPS c sn xut nhiu hn ti nhit thp hn nhit ti u.S khc bit ca nhng ti liu do nhiu l do,bao gm ci cch xc nh EPS khc nhau, mi trng sinh trng khc nhau,iu kin v thi gian khc nhau,khng c s iu ch nh PH v cc iu kin khc nhau sn xut nhanh EPS ( mg EPS,mg EPS/lt, mg EPS/CFU ).

HNH 3: ng vin l sn xut (sn phm) vi 30 g ca Bacto-casitone mi lt nh mt hm s ca nhit v pH. ng ng mc vi nhng con s ng k khc nhau (P <0,05). Cc ng cho l ranh gii thit k, tc l, iu kin th nghim bn di dng ny khng c trong thit k. Mt s t th nghim c lm nghin cu tc ng ca pH trong sn xut EPS bi actobacilli, v khng c kim sot bi sinh vt s dng trong nghin cu. Mozzi o lng lng polysaccharide c tng hp ti a ( 488mg l) v s lng t bo cao nht ti im pH = 6.0 ca vi khun . casei CR 87. Thm mt th nghim khc , s lng EPS (mg/l) iu kin pH c kim sot cao gp 3.6 ln so vi iu kin pH khng c kim sot. Trong nghin cu kim sot pH vi L.sake 0-1, pH ti u sn xut EPS l 5.8, nhng s lng t bo cao l pH = 6.2. T kt qu , cc nh nghin cu kt lun rng s bin i ng to EPS t hiu qu hn ti pH = 5.8, nhng lng ng to sinh khi cao l pH = 6.2.

GVHD: Th.S H THIN HONG

Page 16

CNG NGH VI SINH NG DNG

HNH 4: Thng tin ca EPS sn xut, trng lng kh t bo, v glucose s dng bi L. delbrueckiisubsp. bulgaricusRR pht trin trong iu kin ti u cho sn xut EPS (38 C, pH 5,0, 30-g/liter Bacto-casitone). , EPS; , glucose, , trng lng t bo kh. i vi 1 s vi khun sn xut EPS nh Xanthomonas, Pseudomonas, Rhizobium spp, s gii hn nitrogene l kt qu trong qu trnh tng sn xut EPS nhng n khng c p dng dng RR. Sn xut EPS ti u xy ra khi mi trng cha gp 3 ln nng Bacto-casitone (30g l) p ng nhu cu sinh trng (10g/l). S tng lng Bacto-casitone trong SDS n 40g/l khng c kt qu trong s tng mc sn xut EPS.iu c th din ra c bi v nhng cht dinh dng khc c nh ra gii hn va trong m nui cy. H phng trnh bc 2 dng trong ci biu th hin s tc ng ln b mt ca th nghim c s dng trong ci nghin cu ny. oi phng trnh ny rt l logic bi v nhng tha s tc ng n s sinh trng ca vi khun thng cho thy c 1 im ti a. S phn tch thng k cung cp thng tin v s nh hng ca iu kin sinh trng da trn nhng thay i ca tc ng. Mi quan h ng k c tm thy gia nhit v sn lng EPS. Ni chung, mi quan h gia nhit v s sinh trng ca vi khun cho thy c1 im ti a, nhng khng i xng, cng nh mi quan h c kho st y. Mi quan h gia pH v s sinh trng ca vi khun cho thy l mt vng tr s cc i. Trong nghin cu ny, c v ph hp v pH khng c tc ng ng k n sn xut EPS bi v khong s dng nghin cu (4 n 6) c th hin l ng tng i ph ng trn ng cong ca th. Trn thc t, hnh 1 th hin rng nhit ti u v iu kin pH ca qu trnh sn xut EPS tn ti trong ci ng elip hnh thnh bi 36-39oC v pH t 4.5 n 5.5. Cui cng,
GVHD: Th.S H THIN HONG Page 17

CNG NGH VI SINH NG DNG

nghin cu ny c c mi quan h c ngha gia nng Bacto-casitone v sn lng EPS, n c biu hin trn hnh 2. Nhng iu kin sinh trng v mi quan h ca chng c lin h ng k vi s lng CFU ml v thi im kt thc. N c bit chung l nhng iu kin sinh trng nh nng th nghim c nghin cu ny c tc ng da trn nhng thay i thu c. BNG 3: Bng thu hi cho EPS sn xut bi L. RR subsp.bulgaricus delbrueckii Sampling period material tested and Vol of medium (liters) EPS concn EPS yield EPS recovery (g)a (%)b

During recovery

Fermentation broth Concentrated fermentation broth

97.00

367.84c

35.68

100.0

5.32

6,647.27c

35.36

99.1

After recovery

Freeze-dried precipitate

964.0022.0e 29.20f

81.84

a. Nng sut (grams) = EPS (gram mi lt) khi lng trung bnh (lt) b. Thu hi (%) = g EPS / gram EPS c trong canh trng ln men 100. c. Trong miligam trn mt lt. d. Sau khi siu lc v diafiltration e. Trong mg mi gram. f. EPS c mt hm lng protein 1,42% (0,43 0,11 g) v m 0,3% (0,0073 0,0005 g). Nng sut, nh ngha l s lng sn xut ti a trong 1 khong thi gian ti thiu, l bin s quan trng t cng ngh trin vng. xc nh iu kin sn xut ti u, cc s tc ng thay i nng sut c kt hp li, dng cho d kin nng sut, c tnh ton bng tng trng lng ca nng EPS di nhng tc ng ca mt iu kin cng vi thi im kt thc. Bng cch s dng chng trnh o s ti u ha
GVHD: Th.S H THIN HONG Page 18

CNG NGH VI SINH NG DNG

thng thng, nhng tc ng ca mt iu kin c v theo t l v vy chng c th c kt hp chnh ng v im cc i ca nhng iu in tc ng c phi hp li cng i thi l im tc ng ti u nht ca tng iu kin. Nhng tc ng ca tng iu kin c cn x t mt cch ch quan phn nh chnh xc mi quan h ca chng. Trong trng hp ny, nng EPS c xc nh ti ci thi im kt thc. Nng sut ti u c tm thy ti 39oC, pH 5.6 v 30g l nng Bactocasitone. Di nhng iu kin ny, phng trnh ra c 236mg EPS l c sn xut ra trong 12h45. Trong trng hp cc thnh phn ca mi trng kh mt, cc thao tc chun b kh khn, kt qu l t l thi gian chun b v thi gian ln men cao,hoc chi ph gia cng cn d li cao, n s thch hp sn xut lng EPS vt qu thi im kt thc, do lm gim c ci lng mi trng d( thi gian ln men di hn) Nhng bn bo co th nghim trc y v tc ng ca mt hay nhiu iu kin i vi sn xut EPS c s dng phng php OVAT. Phng php OVAT cho rng khng c mi quan h no gia nhng bin s c nghin cu. H phng php tc ng b mt cho ph p ng thi c lng nhiu iu kin sinh trng v v cng cho thy mi quan h ca nhng bin s. M t v nhng iu kin sinh trng ti u. M t qu trnh len men c kim sot bi nhng iu kin ti u trong sn xut EPS cho thy sn xut EPS c gia tng nh s phi hp ca cc iu kin. Tng t kt qu thu c bi Grobben , ng s dng dng NCFB 2772. iu ny cho thy rng nhng iu kin ti u gia tng sn lng EPS ca sinh vt sn xut EPS s l kt qu thu c sn lng EPS ti a. Nhng kt hp ca cc iu kin trong sn xut EPS tng sn lng khng xy ra i vi nhng dng sn xut EPS khc, nh anthomonas v lcaligenes spp.

GVHD: Th.S H THIN HONG

Page 19

CNG NGH VI SINH NG DNG

TI LIU THAM KHO Bradford M. M.(1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248254. Breedveld M. W., Zevenhuizen L. P. T. M., Canter Cremers H. C. J., Zehnder A. J. B.(1993) Influence of growth conditions on production of capsular and extracellular polysaccharides by Rhizobium leguminosarum.Antonie Leeuwenhoek Cerning J. (1990) Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiol. Cerning J., Bouillanne C., Desmazeaud M. J. (1988) Extracellular polysaccharide production by Streptococcus thermophilus. Biotechnol. Lett.10:255260. Cerning J., Bouillanne C., Landon M., Desmazeaud M. (1992)Isolation and characterization of exopolysaccharides from slime-forming mesophilic lactic acid bacteria. J. Dairy Sci. 75:692699. Cerning J., Renard C. M. G. C., Thibault J. F., Bouillanne C.,Landon M., Desmazeaud M., Topisirovic L. (1994) Carbo n source requirements for exopolysaccharide production by Lactobacillus casei CG11 and partial structure analysis of the polymer. Appl. Environ. Microbiol.60:39143919. Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A., Smith F.(1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350356. Gancel F., Novel G. (1994) Exopolysaccharide production byStreptococcus salivarius ssp. thermophilus cultures. 1. Conditions of production. J. Dairy Sci. 77:685688. Garcia-Garibay M., Marshall V. M. E. (1991) Polymer production byLactobacillus delbrueckii ssp. bulgaricus. J. Appl. Bacteriol. 70:325328. Grobben G. J., Sikkema J., Smith M. R., de Bont J. A. M. (1995)Production of extracellular polysaccharides by Lactobacillus delbrueckii ssp.bulgaricus NCFB 2772 grown in a chemically defined medium. J. Appl. Bacteriol. 79:103107. Gruter M., Leeflang B. R., Kuiper J., Kamerling J. P.,Vliegenthart F. G. (1993) Structural characterisation of the exopolysaccharide produced by Lactobacillus delbrueckii subspeciesbulgaricus rr grown in skimmed milk. Carbohydr. Res. 239:209226.
GVHD: Th.S H THIN HONG Page 20

CNG NGH VI SINH NG DNG

Hess S. J., Roberts R. F., Ziegler G. R. (1997) Rheological properties of nonfat yogurt stabilized using Lactobacillus delbrueckii ssp. bulgaricusproducing exopolysaccharide or using commercial stabilizer systems. J. Dairy Sci. 80:252265.

Kojic M., Vujcic M., Banina A., Cocconcelli P., Cerning J.,Topisirovic L. ( 1992) Analysis of exopolysaccharide production byLactobacillus casei CG11, isolated from cheese. Appl. Environ. Microbiol.58:40864088. Mozzi F., de Giori G. S., Oliver G., de Valdez G. F. (1996)Exopolysaccharide production by Lactobacillus casei under controlled pH.Biotechnol. Lett. 18:435439. Mozzi F., Oliver G., de Giori G. S., de Valdez G. F. (1995) Influence of temperature on the production of exopolysaccharides by thermophilic lactic acid bacteria. Milchwissenschaft 50:8082. Mozzi F., Savoy de Giori G., Oliver G., Font de Valdez G. (1995)Exopolysaccharide production by Lactobacillus casei. II. Influence of the carbon source. Milchwissenschaft 50:307309. Oda M., Hasegawa H., Komatsu S., Kambe M., Tsuchiya F. (1983)Antitumor polysaccharide from Lactobacillus sp. Agric. Biol. Chem.47:1623 1625. Sanford P. A. (1979) Exocellular microbial polysaccharides. Adv. Carbohydr. Chem. Biochem. 36:265313. Schellhaass S. M. (1983) Ph.D. thesis. (University of Minnesota, St. Paul). Shu C., Yang S. (1990) Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris.Biotechnol. Bioeng. 35:454468. Sinclair C. G. (1987) Microbial process kinetics. in Basic biotechnology.eds Bulock J., Kristiansen B. (Academic Press, London, England), pp 75131. Souw P., Demain A. L. (1979) Nutritional studies on xanthan production by Xanthomonas campestris NRRL B1459. Appl. Environ. Microbiol.37:1186 1192. Sutherland I. W. (1990) Food usage of polysaccharides. in Biotechnology of microbial polysaccharides. ed Sutherland I. W. (Cambridge University Press, Cambridge, England), pp 117125. Sutherland I. W. (1990) Physiology and industrial production. inBiotechnology of microbial exopolysaccharides. ed Sutherland I. W.(Cambridge University Press, Cambridge, England), pp 7088.
GVHD: Th.S H THIN HONG Page 21

CNG NGH VI SINH NG DNG

Tamime A. Y. (1977) The behavior of different starter cultures during the manufacture of yogurt from hydrolysed milk. Dairy Ind. Int. 42:711. Teggatz J. A., Morris H. A. (1990) Changes in the rheology and microstructure of ropy yogurt during shearing. Food Struct. 9:133138. van den Berg D. J. C., Robijn G. W., Janssen A. C.,Giuseppin M. L. F., Vreeker R., Kamerling J. P., Vliegenthart J. F. G.,Ledeboer A. M., Verrips C. T. (1995) Production of a novel extracellular polysaccharide by Lactobacillus sake 0-1 and characterization of the polysaccharide. Appl. Environ. Microbiol. 61:28402844. Wheeler B., Betsch R., Donnelly T. (1993) EChips users guide version 6.0 for Windows. (EChip, Inc. Hockessin, Del).

GVHD: Th.S H THIN HONG

Page 22