PHÂN LẬP, TUYỂN CHỌN CHỦNG VI KHUẨN ACETOBACTER XYLINUM CÓ KHẢ NĂNG TẠO MÀNG BACTERIAL CELLULOSE CÓ ĐẶC TÍNH DAI MỎNG

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PHN LP, TUYN CHN CHNG VI KHUN ACETOBACTER XYLINUM C KH NNG TO MNG BACTERIAL CELLULOSE C C TNH DAI MNG

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<ul><li><p>TRNG I HC S PHM H NI 2 KHOA SINH KTNN </p><p>--------***-------- </p><p> NGUYN TH LAN </p><p>PHN LP, TUYN CHN CHNG VI KHUN ACETOBACTER XYLINUM C KH NNG TO MNG BACTERIAL </p><p>CELLULOSE C C TNH DAI MNG </p><p>KHA LUN TT NGHIP I HC Chuyn ngnh: Vi sinh vt hc </p><p>H Ni 2011 </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan ii </p><p>MC LC </p><p> Trang </p><p>PHN M U </p><p>1. L do chn ti 1 </p><p>2. Mc tiu ca ti 2 </p><p>3. Ni dung nghin cu 2 </p><p>4. ngha ca ti 2 </p><p>PHN NI DUNG </p><p>CHNG 1. TNG QUAN TI LIU </p><p>1.1. V tr v c im phn loi ca Acetobacter xylinum </p><p>trong sinh gii: 3 </p><p>1.1.1. V tr phn loi ca Acetobacter xylinum 3 </p><p>1.1.2. c im phn loi ca Acetobacter xylinum 3 </p><p>1.2. c im v c ch hnh thnh mng bacterial cellulose 7 </p><p>1.2.1. c im cu trc ca mng bacterial cellulose 7 </p><p>1.2.2 C ch hnh thnh mng bacterial cellulose 8 </p><p>1.3. Tnh hnh nghin cu Acetobacter xylinum trn th gii 9 </p><p>v Vit Nam </p><p>1.3.1 Tnh hnh nghin cu Acetobacter xylinum trn th gii. 9 </p><p>1.3.2 Tnh hnh nghin cu Acetobacter xylinum Vit Nam 11 </p><p>CHNG 2. VT LIU V PHNG PHP NGHIN CU </p><p>2.1. Vt liu v thit b 12 </p><p>2.1.1. Vi sinh vt 12 </p><p>2.1.2. Thit b v ho cht 12 </p><p>2.1.3. Mi trng 13 </p><p>2.2. Phng php nghin cu 13 </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan iii </p><p>2.2.1. Phng php phn lp vi khun Acetobacter 13 </p><p>2.2.2. Phng php nghin cu c im hnh thi nui </p><p>cy ca cc chng A.xylinum 14 </p><p>2.2.2.1. Phng php quan st hnh thi t bo trn </p><p>knh hin vi 14 </p><p>2.2.2.2. Phng php quan st hnh thi khun lc [1] 14 </p><p>2.2.3. Phng php nghin cu c im sinh l - sinh </p><p>ho ca cc chng Acetobacter xylinum 15 </p><p>2.2.3.1. Phng php kim tra hot tnh catalase [11] 15 </p><p>2.2.3.2. Phng php kim tra kh nng oxy ho </p><p>ru ethanol thnh acid acetic [11] 15 </p><p>2.2.3.3. Xc nh kh nng oxy ho acid acetic [11]. 15 </p><p>2.2.3.4. Phng php xc nh kh nng tng hp cellulose 16 </p><p>2.2.4. Phng php xc nh trng lng kh ca mng BC 19 </p><p>2.2.5. Phng php xc nh kh nng thm ht nc </p><p>ca mng BC (g/100cm2) 16 </p><p>CHNG 3. KT QU V THO LUN </p><p>3.1. Phn lp chng Acetobacter xylinum 17 </p><p>3.1.1. Lm giu mu nguyn liu 18 </p><p>3.1.2. Phn lp vi khun Acetobacter 19 </p><p>3.1.3. Tuyn chn cc chng A.xylinum to mng </p><p>Bacterial Celullose (mng BC) 21 </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan iv </p><p>3. 2. Tuyn chn cc chng c kh nng to </p><p>mng BC dai, mng 24 </p><p>3.3. Nghin cu mt s c tnh sinh hc ca cc chng </p><p>Acetobacter xylinum tuyn chn 27 </p><p>3.3.1 Nghin cu c im hnh thi t bo hc ca cc </p><p>chng Acetobacter xylinum 27 </p><p>3.3.2. c im nui cy ca cc chng Acetobacter xylinum 28 </p><p>3.3.3. c im sinh l - sinh ho ca cc chng </p><p>Acetobacter xylinum 29 </p><p>3.3.3.1. Kh nng ng ho ngun cacbon ca cc chng </p><p>Acetobacter xylinum 29 </p><p>3.3.3.2. Kh nng ng ho ngun nit ca cc chng </p><p>Acetobacter xylinum 30 </p><p>3.3.4. nh hng ca nhit n kh nng sinh trng </p><p> ca 2 chng R1 v B4 31 </p><p>PHN KT LUN 33 </p><p>TI LIU THAM KHO 34 </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan v </p><p>DANH MC HNH BNG BIU </p><p>HNH Trang </p><p>Hnh 1.1. T bo vi khun A.xylinum 4 </p><p>Hnh 1.2. Si cellulose ca mng BC v si cellulose ca thc vt 7 </p><p>Hnh 1.3. S hnh thnh nhng di ribbon trong mng BC ca A.xylinum 9 </p><p>Hnh 3.1. Qui trnh phn lp vi khun A.xylinum 17 </p><p>Hnh 3.2. Mt s mu mng thu c sau khi lm giu nguyn liu 19 </p><p>Hnh 3.3. Chuyn ha ethanol thnh acid acetic ca vi khun 20 </p><p>Hnh 3.5. Mt s loi mng do cc chng vi khun hnh thnh trn </p><p> b mt mi trng dch th 23 </p><p>Hnh 3.6. Kh nng to mng cellulose ca cc chng A.xylinum 23 </p><p>Hnh 3.7. Cc loi mng bacterial cellulose (BC) 26 </p><p>Hnh 3.8. Hnh dng t bo ca hai chng vi khun A.xylinum 27 </p><p>Hnh 3.9. Khun lc A.xylinum 28 </p><p>BNG </p><p>Bng 1.1. c im sinh ha ca chng vi khun A.xylinum 5 </p><p>Bng 1.2. Cc c im phn bit Acetobacter v Gluconobacter 6 </p><p>( so snh vi Pseudomonas ) </p><p>Bng 3.1. Mt s c tnh ca mng BC 25 </p><p>Bng 3.2. c tnh khun lc vi khun Acetobacter xylinum 28 </p><p>Bng 3.3. Kh nng ng ha ngun cacbon ca chng R1 v B4 30 </p><p>Bng 3.4. Kh nng ng ha ngun nit ca 2 chng R1 v B4 31 </p><p>Bng 3.5. nh hng ca nhit n kh nng sinh trng </p><p> ca chng R1 v B4 32 </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan vi </p><p>DANH MC T VIT TT </p><p>T vit tt T y </p><p>A. xylinum </p><p>BC </p><p>R1 </p><p>B4 </p><p>BHN2 </p><p>cs </p><p>Cty </p><p>Acetobacter xylinum </p><p>Bacterial cellulose </p><p>Acetobacter xylinum R1 </p><p>Acetobacter xylinum B4 </p><p>Acetobacter xylinum BHN2 </p><p>Cng s </p><p>Cng ty </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan 1 </p><p>M U </p><p>1. L do chn ti </p><p>Bng l mt tai nn thng gp trong lao ng v sinh hot hng ngy. </p><p>Ngoi tn thng da, trng hp bng nng cn li di chng n kh nng </p><p>vn ng, thm m v sc khe ca ngi bnh. Vit Nam, ring vin bng </p><p>quc gia H Ni mi nm tip nhn khong 3000 bnh nhn bng do cc </p><p>nguyn nhn v mc khc nhau. Hn na s bnh nhn bng l tr em v </p><p> s bnh nhn nhp vin b bng su. TS. Nguyn Vit Lng trng phng </p><p>hnh chnh tng hp ng thi l ngi iu hnh cc ti nghin cu ca </p><p>Vin Bng Quc gia cho bit: Trc y nhng ca bng su rt d t vong </p><p>do hi thiu nhng loi thuc c hiu. Thuc nhp khu rt him v t. </p><p>Nu bnh nhn b bng nh, din tch vt bng nh cc t bo xung </p><p>quanh t pht trin dn ph kn vt thng. Nhng nu l vt bng su th </p><p>buc phi cy ghp da. Song song vi vic cy ghp da l vic tm kim cc </p><p>loi da t thn, da d loi v cc loi mng sinh hc thay th da tm thi trong </p><p>iu tr. Mc d da t thn l vt liu l tng nhng trong nhiu trng hp </p><p>khng p ng hoc th trng bnh nhn khng cho php ly Vic tm </p><p>kim cc loi da d loi v mng sinh hc thay th cho da tm thi tr </p><p>thnh phng php hiu qu hn c. Tuy nhin cc loi da d loi nh da ch </p><p>ti, ng kh; da ln ti, ng lnh li c nhng nhc im nht nh nh </p><p>vic s dng th b ng, bo qun v x l phc tp, kh c th s dng </p><p>rng ri trn lm sng. Chnh v vy, vt liu che ph tm thi t cc loi </p><p>mng sinh hc l la chn s mt, c vai tr rt quan trng trong iu tr </p><p>bng. Mt trong cc loi mng sinh hc v ang c quan tm ngy nay </p><p>l mng cellulose vi khun. </p><p>Cellulose vi khun c cu trc ho hc ng nht vi cellulose thc vt </p><p>nhng li c mt s tnh cht ho l c bit nh bn c hc, kh nng </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan 2 </p><p>thm ht nc cao, ng knh si nh, tinh khit cao, polymer ln... </p><p>rt thch hp lm mng tr bng. Th gii c nhiu nghin cu v mng tr </p><p>bng t cellulose vi khun. Vit Nam cc nghin cu v vn ny cn rt </p><p>t, mi ch c nghin cu ca nhm tc gi Nguyn Vn Thanh (2008) v </p><p>nhm tc gi inh Th Kim Nhung (2009) c cng b. Cc loi mng tr </p><p>bng ca chng ta hin nay phn ln l nhp ngoi vi gi rt t . </p><p>T nhng l do ni trn, chng ti quyt nh chn ti nghin cu: </p><p>Phn lp, tuyn chn chng vi khun Acetobacter xylinum c kh nng </p><p>to mng Bacterial Cellulose ( mng BC ) c c tnh dai, mng </p><p> 2. Mc tiu ca ti </p><p>- Phn lp cc chng vi khun Acetobacter xylinum (A.xylinum) t mt </p><p>s ngun nguyn liu Vit Nam. </p><p>- Tuyn chn cc chng A.xylinum phn lp c kh nng to mng </p><p>Bacterial Cellulose dai, mng. </p><p>- Nghin cu mt s c tnh sinh hc ca cc chng tuyn chn. </p><p>3. Ni dung nghin cu </p><p>- Phn lp cc chng vi khun Acetobacter xylinum. </p><p>- Tuyn chn cc chng A.xylinum c kh nng to mng Bacterial </p><p>Cellulose (BC) dai, mng. </p><p>- Nghin cu mt s c tnh sinh hc ca cc chng A.xylinum. </p><p>4. ngha ca ti </p><p>- ti nghin cu la chn cc chng A.xylinum c kh nng to </p><p>mng BC dai mng. C trin vng ng dng trong ch to mng tr bng. </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan 3 </p><p>NI DUNG </p><p>CHNG 1. TNG QUAN TI LIU </p><p>1.1. V tr v c im phn loi ca Acetobacter xylinum trong sinh gii: </p><p>1.1.1. V tr phn loi ca Acetobacter xylinum </p><p>Tn gi: Acetobacter xylinum l mt tn gi chnh thc theo h thng </p><p>danh php quc t 1990. </p><p> Nm 1957, theo Bergeys manual of determinative bacteriology </p><p>A.xylinum c xp vo chi Acetobacter, thuc h Pseudomonadaceae, b </p><p>Pseudomonadales, lp Schizomycetes [13]. </p><p> n nm 1974, theo Bergeys manual of determinative bacteriology, </p><p>A.xylinum li c coi nh l mt loi ph ca Acetobacter aceti, thuc chi </p><p>Acetobacter v c nhm vo nhng chi khng r ngun gc [14]. </p><p> Nm 1984 Theo Bergey [4], Acetobacter xylinum c xp vo chi </p><p>Acetobacter thuc h Acetobacteraceae. H vi khun ny gm 2 chi l </p><p>Acetobacter v Gluconobacter. </p><p>Ngy nay, tn ti nhiu quan im khc nhau trong vic phn loi vi </p><p>khun acetic ni chung v A.xylinum ni ring. Vn ny vn ang gy </p><p>nhiu tranh ci, i hi cn c nhiu hn nhng nghin cu tip theo. </p><p>1.1.2. c im phn loi ca Acetobacter xylinum </p><p> * c im hnh thi - t bo hc </p><p>A.xylinum c dng hnh que, thng hay hi cong, kch thc khong </p><p>2m, t bo ng ring l hoc xp thnh tng chui, khng c kh nng di </p><p>ng, khng sinh bo t. Cc t bo c bao bc bi cht nhy to vng </p><p>nhn c cha hemicellulose. </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan 4 </p><p> Vi khun A.xylinum thuc nhm vi khun Gram m, hiu kh bt buc, </p><p>ho d dng. T bo ca chng thng tm thy trong gim, dch ru, nc </p><p>p hoa qu, trong t. </p><p>* c im nui cy </p><p> Trn mi trng thch a, vi khun A.xylinum hnh thnh khun lc </p><p>nhn hoc x x, ra mp khun lc bng phng hay gn sng, mu trng hoc </p><p>trong sut, khun lc bng phng hoc li ln d tch khi mi trng. Vi </p><p>khun A.xylinum khi nui cy trong mi trng lng iu kin tnh, chng </p><p>s hnh thnh trn b mt mi trng mt lp mng BC [18]. Ngc li trong </p><p>iu kin nui lc, cellulose hnh thnh dng ht nh vi kch thc khng </p><p>u nhau v phn tn trong mi trng dinh dng to ra nhng c tnh hnh </p><p>thi khc hn cellulose trong iu kin nui cy tnh [13]. </p><p>*c im sinh l sinh ho </p><p>+ c im sinh l </p><p> Vi khun A.xylinum pht trin nhit 25 - 350C, pH = 4 - 6. Nhit </p><p>v pH ti u tu thuc vo ging. 370C, t bo s suy thoi hon ton ngay </p><p>c trong mi trng ti u. Tuy nhin vn cn nhng kin khc nhau v </p><p>nhiu tranh ci v iu kin nhit , pH ti thch cho chng vi khun </p><p>A.xylinum sinh trng v to mng. </p><p>Hnh 1.1. T bo vi khun A.xylinum [36] </p><p>1m </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan 5 </p><p> A.xylinum c kh nng chu c pH thp, v th thng b sung thm </p><p>acid acetic vo mi trng nui cy hn ch s nhim khun l. </p><p> + c im sinh ho : </p><p>Nm 1950, Prateur chnh thc a ra mt kha phn loi mi cn c </p><p>vo cc tiu chun nh: kh nng oxy ha acid acetic thnh CO2 v H2O; hot </p><p>tnh catalase; kh nng sinh trng trn mi trng Hoyer [23] Theo quan </p><p>im ny, A.xylinum l chng thuc chi Acetobacter, h Pseudomonas, b </p><p>Pseudomonadales, lp Schizomycetes. c im phn bit vi cc chng khc </p><p>cng mt chi c trnh by bng 1.1 di y: </p><p>Bng1.1.c im sinh ha ca chng vi khun A.xylinum </p><p>STT c im Hin tng Kt qu </p><p>1 Oxy ho ethanol thnh acid acetic </p><p>Chuyn ho mi trng cha Bromphenol Blue 0,04% t mu xanh sang mu vng </p><p>+ </p><p>2 Hot tnh catalase Hin tng si bt kh + </p><p>3 Sinh trng trn mi trng Hoyer </p><p>Sinh khi khng pht trin _ </p><p>4 Chuyn ho glycerol thnh dihydroxyaceton </p><p>To kt ta gch trong dch sau ln men </p><p>+ </p><p>5 Chuyn ho glucose thnh acid </p><p>Vng sng xut hin xung quanh khun lc trn mi trng cha CaCO3 </p><p>+ </p><p>6 Kim tra kh nng sinh sc t nu </p><p>Khng hnh thnh sc t nu _ </p><p>7 Kim tra kh nng tng hp cellulose </p><p>Vng vi khun xut hin mu lam + </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan 6 </p><p>Theo Bergey (1984) [4], Acetobacter xylinum c xp vo chi </p><p>Acetobacter thuc h Acetobacteraceae. H vi khun ny gm 2 chi l </p><p>Acetobacter v Gluconobacter. c im phn bit gia 2 chi c trnh by </p><p>trong bng 1.2 </p><p>Bng 1.2. Cc c im phn bit Acetobacter v Gluconobacter </p><p>( so snh vi Pseudomonas ) </p><p>c im Gluconobacter Acetobacter Pseudomonas </p><p>1. V tr tin mao + +/- Tin mao cc </p><p>2. Pht trin pH = </p><p>4,5 + + - </p><p>3. Oxy ha aceton </p><p>pH=4,5 + + - </p><p>4. Oxy ha axid </p><p>acetic - + + </p><p>5. Oxy ha lactat - + + </p><p>6. Glucose </p><p>Gluconat + +/- +/- </p><p>7. S dng tinh bt, </p><p>lactose - - +/- </p><p>8. S dng gelatin - - +/- </p><p>9. Sc t hunh </p><p>quang - - +/- </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan 7 </p><p>1.2. c im v c ch hnh thnh mng bacterial cellulose </p><p>1.2.1. c im cu trc ca mng bacterial cellulose </p><p>Cellulose cu trc mng BC c thnh phn ha hc tng t cellulose </p><p>thc vt nhng cu trc v c tnh li khc xa nhau. </p><p>Cellulose c cu to bi chui polyme -1,4 glucopyranose mch </p><p>thng, c ng knh si nh hn rt nhiu so vi si cellulose ca thc vt, </p><p>ng thi kh nng kt tinh cao (khong 60 %), polimer ha ln nn mng </p><p>BC c bn c hc cao, kh nng thm ht nc ln [18]. y l c tnh </p><p>u vit hn hn so vi cellulose thc vt. Khi quan st trn knh hin vi in </p><p>t thy ng knh ca si cellulose vi khun ch bng 1/100 ca si cellulose </p><p>thc vt.[35] </p><p>Do c nhng c im u vit nh trn m mng BC c ng dng </p><p>rt nhiu trong cc lnh vc cng ngh khc nhau. Mt trong cc lnh vc </p><p>ng lu l lnh vc y hc. Trong lnh vc ny, mng BC c ng dng </p><p> sn xut mng tr bng [22], </p><p>Si cellulose ca mng BC Si cellulose ca thc vt </p><p>Hnh 1.2. Si cellulose ca mng BC v si cellulose </p><p>ca thc vt [35] </p></li><li><p> Kha lun tt nghip K33 Khoa Sinh KTNN </p><p>GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan 8 </p><p>1.2.2 C ch hnh thnh mng bacterial cellulose </p><p>*V tr tng hp </p><p>V tr tng hp cellulose ca t bo A.xylinum nm gia lp mng </p><p>lipopolysaccharide v lp mng sinh cht. [34]. Quan st di knh hin vi </p><p>in t khi nhum m bn v tiu bn cho thy: mi t bo c 46 im tng </p><p>hp cellulose. Khong cch gia mi im l 12 15nm, kch thc mi </p><p>im l 3,5 nm. Khi t bo phn chia, cc v tr tng hp c phn b u </p><p>vo hai t bo con [29] </p><p>*C ch tng hp </p><p>Qu trnh sinh tng hp BC l mt tin trnh bao gm nhiu bc c </p><p>iu ho mt cch chuyn bit v chnh xc bng mt h thng cha nhiu </p><p>loi enzyme, phc hp xc tc v cc protein iu ho [11], [12]. </p><p>Qu trnh tng hp c din ra nh sau: </p><p>Ti cc im tng hp cellulose, nh c h thng cc enzyme...</p></li></ul>