TRNG I HC LC HNG KHOA CNG NGH SINH HC MI TRNG --------

BO CO NGHIN CU KHOA HC
TI:

NGHIN CU NG DNG NM MC T BNH MEN THU NHN ENZYME AMYLASE

CVKH:ThS. PHM TH LAN THANH SVTH : NGC PHM TH XUN NGC

BIN HA, THNG 12 NM 2010

LI CM N
Chng con xin chn thnh bit n su sc n cha m nui dy v cho chng con n hc nn ngi. Chng em xin chn thnh cm n Khoa Cng Ngh Sinh Hc Mi Trng Trng i hc Lc Hng to iu kin tt cho chng em thc hin ti tt nghip ny. Chng em xin chn thnh cm n c Phm Th Lan Thanh tn tnh hng dn, gip ch bo chng em trong thi gian thc hin ti, gip cho chng em tch ly c nhiu kinh nghim hu ch v qu bu. Chng em xin chn thnh cm n cc Thy C trong Khoa Cng Ngh Sinh Hc Mi Trng tn tnh ging dy, trang b cho chng em nhng kin thc thit thc lm hnh trang trong tng lai. Chng em xin chn thnh cm n cc Thy C qun l phng th nghim Khoa Cng ngh Sinh hc Mi trng gip chng em trong qu trnh thc hin ti. Cm n bn b quan tm, gip v ng vin chng ti trong sut thi gian thc hin ti.

MC LC
Trang ph ba Mc Lc Danh mc cc bng Danh mc cc hnh nh Trang LI M U ..................................................................................................1 CHNG 1 TNG QUAN TI LIU ......................................................2 1.1. Gii thiu v nm mc ...............................................................................2 1.1.1. Khi nim v nm mc .................................................................2 1.1.2. Hnh thi v cu trc ca nm mc ..............................................2 1.1.3. Sinh sn ca nm mc ..................................................................4 1.1.3.1. Sinh sn v tnh ...............................................................4 1.1.3.2. Sinh sn hu tnh .............................................................5 1.1.4. iu kin sinh trng ca nm mc.............................................6 1.1.4.1. Nhu cu dinh dng ........................................................6 1.1.4.2. m ..............................................................................9 1.1.4.3. Nhit .........................................................................10 1.1.4.4. pH ..................................................................................11 1.2. ng dng ca nm mc ...........................................................................11 1.3. Vi sinh vt trong bnh men cm ru .....................................................12

1.3.1. Nm mc.....................................................................................13 1.3.2. Nm men.....................................................................................13 1.3.3. Vi khun......................................................................................14 1.4. Enzyme amylase t vi sinh vt.................................................................14 1.4.1. Gii thiu v enzyme amylase....................................................14 1.4.2. c im ca vi sinh vt trong sn xut enzyme .......................19 1.4.3. Mt s k thut nui cy vi sinh vt thu nhn enzyme ..............20 1.4.4. Vi sinh vt sn xut enzyme amylase .........................................20 1.4.5. Thu nhn enzyme amylase t vi sinh vt....................................21 1.4.5.1. Sinh tng hp enzyme amylase vi sinh vt

....................................................................................................21 1.4.5.2. Cc yu t nh hng ca mi trng n tng hp enzyme amylase..........................................................................22 1.4.5.3. Cc phng php thu nhn enzyme amylase ............... 24 1.4.6. ng dng ca enzyme amylase ..................................................27 1.4.6.1. Amylase trong cng nghip ru bia ........................ 28 1.4.6.2. Trong sn xut bnh m ................................................ 29 1.4.6.3. Trong sn xut mt, tinh bt, malto, gluco .................. 29 1.4.6.4. Trong mt s ngnh cng nghip thc phm khc ...... 30 1.4.6.5. Trong cc ngnh cng nghip dt, giy........................ 30 1.4.6.6. Trong chn nui............................................................ 31

1.4.6.7. Trong y hc ....................................................................................... 31 CHNG 2 VT LIU V PHNG PHP ......................................32 2.1. Vt liu .....................................................................................................32 2.1.1. i tng nghin cu .................................................................32 2.1.2. Vt liu v ha cht ....................................................................32 2.1.2.1. Dng c v thit b ........................................................32 2.1.2.2. Ha cht.........................................................................33 2.1.2.3. Mi trng.....................................................................34 2.2. Phng php.............................................................................................34 2.2.1. a im v thi gian nghin cu...............................................34 2.2.2. Phng php nghin cu ............................................................35 2.2.2.1. Phng php phn lp ...................................................35 2.2.2.2. Phng php quan st hnh thi nm mc ....................35 2.2.2.3. Phng php xc nh kh nng phn gii tinh bt ......35 2.2.2.4. Phng php thu nhn enzyme amylase t nm mc ...36 2.2.2.5. Phng php xc nh hot ca enzyme amylase....38 CHNG 3 KT QU V BIN LUN ...............................................40 3.1. Kt qu phn lp cc chng nm mc c trong bnh men cm ru......40 3.2. Kt qu quan st hnh thi nm mc ........................................................42 3.3. Kt qu xc nh kh nng phn gii tinh bt ca cc chng nm mc ..43 3.4. Kt qu thu nhn enzyme amylase t nm mc ......................................46 3.5. Kt qu xc nh hot ca enzyme amylase .......................................49

CHNG 4 KT LUN V NGH..................................................50 4.1. Kt lun ....................................................................................................50 4.2. ngh ....................................................................................................51 TI LIU THAM KHO ........................................................................... 52

DANH MC CC BNG
Trang Bng 2.1: Cc mu bnh men cm ru ........................................................32 Bng 3.1: Cc chng nm mc phn lp t bnh men cm ru ..................40 Bng 3.2: Hnh thi khun lc ca cc chng nm mc phn lp t bnh men cm ru ................................................................................................42 Bng 3.3: Kh nng phn gii tinh bt ca cc chng nm mc phn lp t bnh men cm ru.........................................................................................44

DANH MC CC HNH NH
Trang Hnh 1.1: Nm mc Mucor sp..........................................................................3 Hnh 1.2: Pho mt xanh Stilton ......................................................................11 Hnh 1.3: Chao ...............................................................................................12 Hnh 2.1: Quy trnh thu nhn enzym amylase t nm mc ...........................37 Hnh 3.1: Khun lc ca cc chng M2, M4, M7, M10 ................................41 Hnh 3.2: Kh nng phn gii tinh bt ca cc chng M3, M4, M9, M11 ....45 Hnh 3.3: Kh nng phn gii tinh bt ca cc chng M6, M7 ................... 46 Hnh 3.4: Dch chit enzyme amylase sau ly tm ......................................... 47 Hnh 3.5: Ch phm enzyme amylase c ta bng cn 960 lnh............. 48 Hnh 3.6: Ch phm enzyme amylase bn tinh khit t nm mc ............... 48

LI M U

Amylase l mt trong nhng enzyme c ng dng rng ri trong cc ngnh cng nghip, c bit l ngnh cng nghip thc phm nh cng nghip sn xut ru bia, nc p tri cy, bnh ko, bnh m... Hin nay, enzyme amylase c sn xut ch yu t vi sinh vt, do c nhiu u im nh: tc sinh sn ca vi sinh vt nhanh, enzyme thu nhn t vi sinh vt c hot tnh cao, ngun nguyn liu dng sn xut enzyme r tinNhiu vi sinh vt c kh nng to ra enzyme amylase nh nm mc, vi khun, nm men, x khun ti nghin cu ny phn lp cc chng nm mc t cc mu bnh men cm ru, tuyn chn cc chng nm mc c kh nng phn gii tinh bt v ng dng chng thu nhn enzyme amylase, nhm gp phn to ra ngun enzyme c gi tr t vi sinh vt.

CHNG 1: TNG QUAN TI LIU

1.1.Gii thiu v nm mc: 1.1.1.Khi nim v nm mc: [2] Nm mc l tt c cc nm khng phi l nm men v cng khng sinh m nm nh cc nm ln. Nm mc cn c gi l nm si, tc l tt c cc mc mc trn thc phm, trn chiu, trn qun o, trn sch v Chng pht trin rt nhanh trn nhiu ngun c cht hu c khi gp kh hu nng m. Nhiu nm mc k sinh trn ngi, trn ng vt, thc vt v gy ra cc bnh nm kh nguy him. Nhiu nm mc sinh ra cc c t nm c th gy ra bnh ung th v nhiu bnh tt khc. Trong t nhin, nm mc phn b rt rng ri v tham gia tch cc vo cc vng tun hon vt cht, nht l qu trnh phn gii cht hu c v hnh thnh cht mn. 1.1.2.Hnh thi v cu trc ca nm mc:[2] Nm mc c thnh t bo, ri n mng t bo cht, bn trong l t bo cht vi nhn phn ha. Mng nhn c cu to hai lp v trn mng c nhiu l nh. Trong nhn c hch nhn. Bn trong t bo nm cn c khng bo, ti th, mng li ni cht nh si nm bao gm mt chp nn, khng tng trng v c tc dng che ch bo v cho phn ngn ca si nm. y l phn m cht nguyn sinh khng c nhn v t cha cc c quan t. cc loi nm khun ti khng c vch ngn gi l cc t bo a nhn v bn trong c nhiu nhn. cc loi nm c vch ngn th do kh nng di chuyn ca nhn m tng t bo c th cha t 1 nhn, 2 nhn, nhiu nhn hoc khng c nhn no.

Hnh 1.1. Nm mc Mucor sp. [9] H si nm c th bin ha thch nghi vi cc iu kin sng khc nhau thnh cc dng c bit sau y: R gi: Trng gn ging nh mt chm r phn nhnh, c tc dng gip bm cht vo c cht v hp th cht dinh dng t c cht. Si ht: Chng c mc ra t khun ti v phn nhnh ri m su vo t bo vt ch, chng c th bin thnh hnh cu, hnh nn tay hay hnh si. Chng s dng cc si ht ny ht cht dinh dng t c th ca vt ch. Si p: Gp cc nm k sinh thc vt. Phn si nm tip xc vi vt ch s phng to ra, tng din tip xc vi vt ch. Phn ny thng c hnh a, c nhiu nhn t bo, p cht vo vt ch. Si b hay thn b: l on si nm kh sinh khng phn nhnh, pht sinh t cc si nm c cht, c hnh thng hoc hnh cung. u mt ca cc si b chm vo c cht pht trin thnh cc r gi bm chc vo c cht.

Vng nm: Vng nm c th c dng bng dnh ra t nhng cung ngn xp thng gc vi si nm chnh. nh ca cc cung ny phnh to ra thnh bng hnh cu. Bng ny tit ra mt cht dnh trn khp b mt. 1.1.3. Sinh sn ca nm mc: [1] Nm mc sinh sn di 2 hnh thc: v tnh v hu tnh. Trong sinh sn v tnh, nm mc hnh thnh bo t m khng qua vic gim phn, tri li trong sinh sn hu tnh nm mc hnh thnh 2 loi giao t c v ci. 1.1.3.1. Sinh sn v tnh: Nm mc sinh sn v tnh th hin qua 2 dng: sinh sn dinh dng bng on si nm pht trin di ra hoc phn nhnh v sinh sn bng cc loi bo t. Mt s loi nm c nhng bo t c trng nh sau: Bo t ti (bo t bc): cc bo t ng c ging nm Saprolegnia v bo t ti ging nm Mucor, Rhizopus cha trong ti bo t ng v ti bo t c mang bi cung ti bo t. Bo t nh: cc bo t nh khng c ti bao bc ging nm Aspergillus, Penicillium ... Hnh dng, kch thc, mu sc, trang tr v cch sp xp ca bo t nh thay i t ging ny sang ging khc v c dng lm tiu chun phn loi nm. Cung bo t nh dng bnh c th khng phn nhnh nh Aspergillus hay dng th phn nhnh nh Penicillium. Bo t nh hnh thnh t nhng cm trn nhng cung bo t nh Trichoderma. Bo t tn: trong nhiu loi nm men v nm mc c hnh thc sinh sn c bit gi l bo t tn. Bo t tn c th c nhng loi sau:

+ Chi hnh thnh t t bo nm men: Cryptococcus v Candida l nhng loi bo t tn n gin nht, gi l bo t chi. + Ging Ustilago c nhng si nm c xut hin t bo c vch dy gi l bo t vch dy cn gi l bo t o. V tr ca bo t vch dy si nm c th khc nhau ty loi. + Ging Geotrichum v Oospora c si nm ko thng, vung hay ch nht v t bo vch dy gi l bo t t. 1.1.3.2. Sinh sn hu tnh: Sinh sn hu tnh xy ra khi c s kt hp gia hai giao t c v ci c tri qua giai on gim phn. C quan sinh dc ca nm mc c tn l ti giao t, c 2 loi: c quan sinh dc c gi l ti c cha cc giao t c, cn c quan sinh dc ci gi ti non cha giao t ci, khi c s kt hp gia giao t c v giao t ci s to thnh bo t, bo t di ng c gi l bo t ng. Kiu hai si nm c gii tnh c v ci tip hp nhau sinh ra bo t c tn l tip hp t, tip hp t l c trng ca nhm nm Myxomycetes. Bo t sinh dc khi hnh thnh c dng ti gi l nang v ti ny cha nhng bo t gi l bo t nang. Nang v bo t nang l c trng ca nhm Ascomycetes. Trong nhm Basidiomycetes, 4 bo t pht trin phn tn cng ca cu trc th qu gi l m v bo t c gi l bo t m. Nhm Nm bt ton gm nhng nm cho n nay cha bit r kiu sinh sn hu tnh ca chng.

1.1.4. iu kin sinh trng ca nm mc: [3] 1.1.4.1. Nhu cu dinh dng: Nm mc l sinh vt dinh dng ha nng hu c. Chng ch c kh nng thu nhn nng lng t mi trng bn ngoi nh qu trnh oxy ha hiu kh hoc qu trnh ln men k kh cc cht hu c ngoi bo. Kiu dinh dng carbon hu c ny c gi l kiu dinh dng carbon d dng. Nm mc l nhng loi nm c kiu carbon d dng thuc loi hoi sinh, s dng cc hp cht hu c lm cht dinh dng. a. Ngun carbon: Nm mc c th s dng ngun carbon rt khc nhau. Cc loi thc n carbon c th dng lm ngun nng lng v ngun vt liu xy dng t bo i vi nm mc c th k n cc loi hydradecarbon (monosaccharide, oligosaccharide, polysaccharide), cc dn xut ca hydradecarbon, cc loi ru, cc loi axit hu c, cc loi axit amin, protein, lipid....Trong nhiu trng hp nu c mt trong mi trng vi ngun carbon khc nhau, nm mc s pht trin mnh m hn so vi khi ch c ring bit tng loi mt. i vi cc ngun carbon phc tp (tinh bt, cenlulose,...) trc ht nm mc phi sinh ra cc enzyme phn hy cc hp cht ny thnh cc hp cht n phn t sau mi ng ha c chng. b. Ngun Nit: Cc loi nm mc khc nhau c th c nhu cu khc nhau i vi cc ngun nit. Nm mc thng c kh nng s dng c cc ngun nit hu c ln cc ngun nit v c. Nhiu loi nm mc c kh nng ng ha c mui amon ln nitrat. i khi c nhng loi nm mc khng pht trin c trn

cc mi trng cha ngun nit l mui amon nhng nguyn nhn khng phi bn thn gc NH4+ m chua sinh l do cc mui amon to ra. Sau khi nm mc ng ha gc NH4+ trong mi trng s tch ly cc anion v c ( SO42- , HPO42- , Cl- ...) v lm h pH ca mi trng xung. Ngc li vi cc mui amon, nitrat l nhng mui c tnh kim sinh l. Sau khi nm ng ha gc NO3- trong mi trng s tch ly li cc cation (Na+ , K+...) v lm tng r rt pH ca mi trng. Kh nng ng ha amon sunfat ca nm mc s tng cng r rt khi b sung vo mi trng mt t axit hu c ( acid lactic, acid malic...). Ngoi cc ngun nit v c, nhiu loi nm mc c th s dng ngun nit hu c (protein, peptid, acid amin, ...). c. Cc nguyn t khong: Nm mc cn cc nguyn t a lng nh: S, P, K, Ca, Mg v Fe. Cc nguyn t c nm mc i hi vi nhng s lng rt nh c gi l cc nguyn t vi lng nh: Mn, Zn, Cu, Co, Ni, ... Lng cha tng nguyn t khong trong t bo nm mc khng nhng thay i ty thuc vo tng loi nm mc m cn ph thuc c vo iu kin nui cy chng. Sau y l vai tr cc cht khong trong t bo nm mc: - Phospho: thng chim t l cao nht trong thnh phn khong ca nm mc. Phospho tham gia vo cu to ca nhiu cht hu c rt quan trng trong t bo nm mc nh: phosphoprotein, phosphatid... ). nucleoprotein, nucleotid,

- Lu hunh: tham gia vo thnh phn ca mt s acid amin: cystin, cystein, metionin. Lu hunh cng c mt trong nhiu coenzyme quan trng nh acid lipoic, biotin, coenzyme A ... - Kali: c lng kh ln trong cc t bo nm mc, chng tham gia vo qu trnh trao i glucid v c nh hng n nhiu qu trnh trao i cht khc ca cc t bo ny. - Magnesium: tham gia vo qu trnh hot ha khong 80 enzyme trong t bo. C nghin cu cho bit vic thiu Mg2+ dn n vic c ch qu trnh hnh thnh bo t mt s loi nm mc. - Canxi: khng tham gia cc thnh phn cc cht hu c trong t bo nhng chng rt cn thit v ng vai tr cu ni trung gian gia nhiu thnh phn quan trng trong t bo (gia cc nucleotid, gia protein v acid nucleic). Canxi cn c nh hng n s hnh thnh cc cu trc khng gian n nh ca ribosome, nhn ... - St: tham gia vo kt cu porphirin st ca cc h thng enzyme chuyn vn electron. St cn tham gia vo cu trc ca cc enzyme catalase, peroxidase. d. Cc cht sinh trng: Cng ging nh i vi cc loi vi sinh vt khc, nm mc c nhng quan h rt khc nhau i vi cc loi vitamin v cc cht sinh trng khc. Vic mt loi nm mc khng i hi mt loi cht sinh trng no y c th do hoc l chng t tng hp ra c cht sinh trng ny hoc l trong qu trnh trao i cht chng khng cn ti loi coenzyme c cha cht sinh trng ny.

Ngi ta chia nm mc thnh hai nhm cn c vo mi quan h ca chng i vi cc cht sinh trng : Nhm t dng cht sinh trng: c th t tng hp tt c cc cht sinh trng cn thit i vi hot ng sng ca chng. Nhm d dng cht sinh trng: cn phi c cung cp mt vi hoc nhiu cht sinh trng dng c sn.. Nhu cu v cht sinh trng ca mt loi nm mc c th thay i ty theo iu kin nui cy, ty theo tui ging. Phn ln cc vitamin nhm B c cc loi nm mc s dng to ra cc enzyme cn thit i vi hot ng sng ca chng. Nhiu loi vitamin tan trong lipid (A, D, E, K) c tm thy trong cc lp mng sinh hc, s tn ti v s lng ca cc loi vitamin ny trong mi trng c nh hng r rt i vi cu trc v i vi s hot ng ca cc lp mng ny. 1.1.4.2. m: Ngoi cc cht dinh dng nm mc cng nh tt c sinh vt khc cn c nhu cu v nc cho cc hot ng sinh l, sinh ha ca t bo. Lin quan n lng nc ny l m, bao gm : hm lng nc ca sn phm, m tng i ca khng kh. Gia hm lng nc ca sn phm v m ca khng kh mi trng ngoi c mi lin quan (trao i qua li). Chnh mi lin quan dinh dng ny khng nhng nh hng n cht lng ca sn phm, m cn nh hng n kh nng s dng nc ca t bo nm mc cho cc qu trnh sinh trng, pht trin ca chng.

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a. Hm lng nc ca sn phm: Nc trong cc sn phm nh: lng thc, thc phm, thuc men, dc liu gm phn nc lin kt v phn nc khng lin kt. Nm mc cng nh cc vi sinh vt khc ch s dng c phn nc t do c trong sn phm, v cng ch phn nc ny thc hin s trao i qua li vi nc c trong khng kh. b. m tng i ca khng kh: Mi loi nm mc thch ng vi mt khong gi tr m tng i. Vic gim m tng i s lm ngng hoc ko di thi gian ny chi ca bo t nm mc. Nm mc sng trong mi trng c m tng i thp, chng c kh nng thch nghi dn c th tip tc sinh trng, pht trin bnh thng hoc trng thi sng ngh. m tng i thp cng vi hm lng nc thp trong sn phm, t bo nm mc u tin mt nc t do, sau mt nc lin kt. Ty theo lng nc mt i, nm mc chuyn sang trng thi sng ngh hoc b cht. Phn ln nm mc khng sinh trng, pht trin trong iu kin m tng i ca khng kh mi trng di 60%. 1.1.4.3 Nhit : Mi loi nm mc cng c nhit ti u cho s pht trin, sinh trng. Phn ln nm mc sinh trng, pht trin nhit 15-300C v cng thng c nhit tng trng ti u trong khong 20-250C ty tng loi. Mt s rt nh cc loi nm mc c th tng trng cc kho lnh nhit -60C, thm ch -200C v mt s t loi khc c nhit tng trng n 500C. Cn c vo nhit tng trng, cc loi nm mc c chia thnh 5 nhm: nhm a nng, nhm chu nng, nhm a nhit trung bnh, nhm chu lnh, nhm a lnh.

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1.1.4.4. pH: Mt trong nhng yu t nh hng n s sinh trng, pht trin ca nm mc l pH. Nm mc ny mm v sau tng trng bnh thng pH 7. mi trng kim hoc axt, nm mc khng hoc tng trng rt yu. 1.2. ng dng ca nm mc: [11] Nm mc c ng dng rt rng ri, nhiu loi c s dng trong cng ngh thc phm, sn xut cht khng sinh v sn xut nhiu loi enzyme Nm mc c s dng ch bin v bo qun thc n rng ri v lu di. Mt s loi nm mc thng c s dng sn xut sinh khi protein. Mc Aspergillus oryzae c s dng rng ri nhiu nc nh sn xut tng . Nhiu loi thc phm khc cng c ch bin bi mc nh tng, chao, pho mt

Hnh 1.2: Pho mt xanh Stilton [11]

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Hnh 1.3: Chao [10] Trong s 12.000 loi khng sinh c bit nm 1995 c khong 22% c sn xut t nm mc. Trong s , khng sinh Penicillin, c Alexander Fleming tng hp t nm Penicillium chrysogenum vo nm 1928, c s dng rt rng ri trong cha tr y hc th k 20. Mt loi khng sinh -lactam ph bin khc l Cephalosporin cng c tng hp nm 1948 t nm Cephalosporium acremonium. Mt s enzyme c tng hp t nm mc nh: amylase, glucoamylase, protease, pectinaseNm mc gip tng hp acid hu c: acid oxalic, citric, gluconic..., vitamin nhm B, riboflavin; kch thch t: gibberellin, auxin, cytokinin. Mt s loi nm k sinh cn trng c s dng lm thuc tr su sinh hc. Nm mc cn l i tng nghin cu v di truyn hc dng tng hp AND. Ngoi ra, nm mc c ng dng sn xut ru, nm mc l thnh phn quan trng trong bnh men cm ru. 1.3. Vi sinh vt trong bnh men cm ru: [4] Bnh men thuc bc c cha nhiu ging vi sinh vt nh nm mc, nm men, vi khun.

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1.3.1. Nm mc: Trong bnh men thuc bc thy pht trin rt nhiu nm mc khc nhau thuc ging Aspergillus, Penicillium, Mucor v Rhizopus. Trong , ging Mucor v Rhizopus thy pht trin nhiu hn c c bit l Mucor rouxii c nhiu c tnh rt qu nh kh nng chu nhit cao (32 350C), chng va c kh nng ng ha v va c kh nng ru ha. 1.3.2. Nm men: Trong mi gam bnh men c t vi chc triu n vi trm triu t bo nm men. Chng gm hai chi khc nhau: Endomycopsis (ch yu l Endomycopsis fibuligenes) v Saccharomyces (ch yu l Saccharomyces cerevisiae). Endomycopsis: Endomycopsis fibuligenes l loi nm men rt giu enzyme amylase, glucoamylase. Do chng va c kh nng ng ha, va c kh nng ru ha. Saccharomyces: Saccharomyces cerevisiae c kh nng ln men rt nhiu loi ng khc nhau nh glucose, saccharose, maltose, fructose, rafinose, galactose. Chng c kh nng ln men c nhit cao khong 36 - 400C. Chng c kh nng chu c axit. c bit chng c kh nng chu c thuc st trng Na2SiF6 vi nng 0.02 0.025%. c im ny rt thun li cho ln men khi cn phi s dng cht st trng. c im quan trng hn ht l loi nm men ny c kh nng ln men cc loi nguyn liu rt khc nhau nh: go, ng, khoai, sn, vi lng ng trong dung dch t 12 14% c khi 16 18%. Nng ru trong dung dch ln men l 10 12%. Nhit ln men thch hp l 28 320C.

14

Ngoi hai chi nm men c bn trn, trong men thuc bc cn thy nhiu loi nm men di khc nhau. Chng va c kh nng thy phn tinh bt, va c kh nng chuyn ng thnh cn, tuy rng s chuyn ha ny cn thp. iu c bit l cc loi nm men di ny chu nhit rt cao, c khi ln ti 60 650C v chu c cht st trng nng 0.05 1%. 1.3.3. Vi khun: Trong bnh men thuc bc thy nhiu loi vi khun pht trin. Trong thy ch yu l cc loi vi khun lactic v vi khun axetic. Cc loi vi khun thng lm chua mi trng. Thi gian u ca qu trnh ln men, qu trnh ny xy ra c li v pH mi trng do chng to ra s thch hp cho nm men v nm mc pht trin. Tuy nhin pH xung qu thp li nh hng xu cho qu trnh ln men. Mt khc nu trong dch ln men c mt ca oxy th vi khun axetic s oxy ha ru thnh axit axetic. Qu trnh ny lm tn hao lng cn to thnh. 1.4. Enzyme amylase t vi sinh vt: [5], [8] 1.4.1. Gii thiu v enzyme amylase: Enzyme amylase l mt trong s cc enzyme c s dng rng ri nht trong cng nghip, y hc v nhiu lnh vc khc. Nhng nghin cu thc nghim u tin v enzyme ni chung v v enzyme amylase ni ring c bt u vo nhng nm 1811 1814. Nhng nghin cu ny gn lin vi tn tui ca nh bc hc ngi Nga,Vin s K.S Kirhof. ng nghin cu qu trnh phn gii tinh bt di tc dng ca dch chit i mch ny mm v nhn thy rng trong malt c cha cc cht phn gii tinh bt thnh ng. Nm 1833, hai nh khoa hc ngi Php l Payen v Persor tch c cht phn

15

gii tinh bt l t i mch ny mm. Cc tc gi dng ru kt ta n trong dung dch chit malt v thu c enzym dng bt, ng thi c tn l diaslase. Sau ny theo ngh ca Duclo, enzyme phn gii tinh bt gi l amylase. Cc enzym amylase c trong nm mc, x khun, nm men v vi khun, nc bt, dch tiu ha ca ngi v ng vt, trong ht ny mm. By gi, ngi ta thu chng ch yu t canh trng nm mc, vi khun v mt s loi nm men. Amylase c th thy phn ht tinh bt cha h ha cng nh ht tinh bt h ha. Theo tnh cht v c ch tc dng ln tinh bt ca amylase ngi ta phn bit amylase ra cc loi sau: -amylase, -amylase, glucoamylase Cc enzyme amylase t cc ngun khc nhau thng khc nhau v tnh cht, c ch tc dng cng nh sn phm cui cng ca s thy phn. -amylase c kh nng phn cch cc lin kt -1,4 glucoside nm pha bn trong phn t c cht: tinh bt, glycogen v polyose ng loi mt cch ngu nhin, khng theo mt trt t no c. V th ngi ta gi n l enzyme amylase ni phn. -amylase khng ch thy phn h tinh bt m n thy phn c ht tinh bt nguyn lnh, song vi tc rt chm. Qu trnh thy phn tinh bt bi -amylase l qu trnh a giai on. giai on u (giai on dextrin ha) ch 1 s phn t c cht b thy phn to thnh mt lng ln dextrin phn t thp nht ca h tinh bt gim nhanh sang giai on th hai (giai on ng ha) cc dextrin phn t thp va c to thnh b thy phn tip tc to ra cc tetra-trimaltose khng cho mu vi iodine. Cc cht ny b thy phn rt chm bi -amylase cho ti di v

16

monosaccharide. Di tc dng ca -amylase, amylase b phn gii kh nhanh thnh oligosaccharide gm 6-7 gc glucose. Sau cc polyglucose ny li b phn cch tip tc nn cc mch polyglucose colagen c ngn dn v b phn gii chm n maltotetrose v maltotriose, v maltose. Qua mt thi gian tc dng di, sn phm thy phn ca amylase cha 13% glucose v 87% maltose. Tm li, di tc dng ca -amylase, tinh bt c th chuyn thnh maltotetrose, maltose, glucose v dextrin phn t thp. Tuy nhin, thng thng -amylase ch thy phn tinh bt thnh ch yu l dextrin phn t thp khng cho mu vi iodine v mt t maltose. -amylase d tan trong nc, trong cc dung dch mui v ru long. Protein ca cc -amylase c tnh cht acid yu v tnh cht ca globulin. im ng in nm trong vng pH 4,2-5,7. Khi tch hon ton Ca ra khi enzyme th -amylase mt ht kh nng thy phn c cht. V Ca tham gia vo s hnh thnh v n nh cu trc bc Bacilus subtilis ca enzyme, duy tr cu hnh hot ng ca enzyme. Canxi cn c tc dng bo m cho amylase c bn cc ln i vi cc tc ng gy bin tnh v s phn hy bi cc enzym phn gii protein. -amylase bn nhit hn so vi cc amylase khc. -amylase b km hm bi kim loi nng. -amylase ca nm mc ch tn cng nhng ht tinh bt b thng tch, cn -amylase vi khun li c kh nng phn hy c cc ht tinh bt nguyn vn ln h tinh bt. giai on thy phn cui, tc dng ca -amylase t nm mc khc tc dng ca -amylase t vi khun. Dextrin do amylase vi khun to ra c mch di hn dextrin do -amylase ca malt v nm mc to ra mt s ln. iu kin hot ng ca -amylase t cc ngun khc nhau thng khng ging nhau. pH ti thch cho hot ng ca -amylase t nm mc l

17

4,5-4,8, ca i mch ny mm v thc mm l 5,3 v ca vi khun l 5,8-6,0. bn i vi tc dng ca axit cng khc nhau. -amylase ca nm mc Aspergillus oryzae bn vng i vi axit tt hn l -amylase ca malt v vi khun Bacilus subtilis. Nhit ti thch cho hot ng xa tc ca -amylase t cc ngun khc nhau cng khng ng nht, -amylase ca nm mc rt nhy i vi tc ng ca nhit. Nhit ti thch ca n l 500C. Amylase ca thc mm v ca malt bn nhit hn v hot ng ti thch 58-600C. Amylase ca vi khun c bn nhit cao hn c. Nhit ti thch ca n l 70-750C . Amylase ca nm mc b v hot 700C, trong khi amylase vi khun c th chu c nhit 920C. Nhng khc bit v nhit v pH ti thch, mc thy phn v c tnh thy phn ca -amylase t cc ngun khc nhau ang m ra nhiu kh nng to ln trong vic ng dng chng mt cch thch hp v y hiu qu cc giai on khc nhau ca qu trnh sn xut. -amylase xc tc s thy phn cc lin kt -1,4 glucan trong tinh bt, glucogen v polysaccharide ng loi, phn ct tun t tng gc maltose mt t u khng kh ca mch. Maltose to thnh c cu hnh , v th amylase ny c gi l -amylase. Theo c tnh tc dng ln tinh bt, -amylase khng thy phn ht tinh bt nguyn lnh m thy phn mnh m h tinh bt. -amylase phn gii 100% amylose thnh maltose v phn gii 54-58% amylopectin thnh maltose. Qu trnh thy phn amylopectin c tin hnh t u khng kh ca cc nhnh ngoi cng. Khi gp lin kt -1,4 glucoside ng k cn lin kt -1,6 glucoside th -amylase ngng tc dng. Phn saccharide cn li l dextrin phn t ln c cha rt nhiu lin kt -1,6 glucoside v c gi l

18

-dextrin, -dextrin cho mu tm ca iodine. nht ca dung dch gim chm. -amylase l mt albumin. -amylase l enzym ngoi phn c i lc vi cc lin kt -1,4 glucoside cch u khng kh ca mch mt lin kt 1,4. N rt bn khi khng c Ca2+, -amylase b km hm bi Cu2+, Hg2+, iodine, pH ti thch trong dung dch tinh bt thun khit ca -amylase l 4,6 cn trong dung dch nu l 5,6. Nhit ti thch trong dung dch tinh bt thun khit 40-500C, trong dch nu li l 60-650C, -amylase b v hot 700C. -amylase ch ph bin trong th gii thc vt, c bit c nhiu trong cc ht ny mm. Trong vi khun khng c -amylase. Glucoamylase thy phn lin kt -1,4 glucan trong polysaccharide, tch tun t tng gc glucose mt khi u khng kh ca mch. Glucoamylase c kh nng xc tc thy phn lin kt -1,4 ln -1,6 glucan. Glucoamylase l enzym ngoi phn, n thy phn polysaccharide t u khng kh to ra glucose. Khi thy phn tinh bt cng vi glucose cn c th to thnh cc -oligosaccharide. Ngoi cc lin kt -1,4 v -1,6 glucoside, glucoamylase cn c kh nng thy phn lin kt -1,2 v -1,3 glucoside. Glucoamylase c kh nng thy phn hon ton tinh bt, glucogen, amylopectin dextrin cui, panose, isomatose v maltose ti glucose. a s glucoamylase bit u thuc loi enzyme axit . Th hin hot lc ti a vng pH 3,5-5,5. So vi -amylase, glucoamylase bn i vi axit hn, nhng li km bn hn di tc dng ca ru etylic, aceton khng c bo v bng Ca2+.

19

im ng in ca glucoamylase t cc ngun khc nhau cng khng ng nht. a s cc ch phm glucoamylase ca nm mc, vi khun, nm men v ca m ng vt u c pH hot ng ti thch vng axit tr glucoamylase ca Saccharomyces italicus v mt s m ng vt c pH hot ng ti thch vng trung tnh. Glucoamylase axit c pH hot ng ti thch l 4,0-5,0, glucoamylase trung tnh pH ti thch l 6,0-7,5. Nhng nghin cu v amylase vi sinh vt t nn mng cho vic sn xut cc ch phm enzyme amylase s dng trong sn xut v i sng. Nhng nghin cu c bn v ng dng ca enzyme amylase ngy cng c pht trin mnh m hn v ang m ra nhng phng hng mi, trin vng mi to ln hn i vi vic ng dng enzyme amylase rng ri trong cc ngnh cng nghip. 1.4.2. c im ca vi sinh vt trong sn xut enzyme: Tc sinh sn ca vi sinh vt rt mnh. Trong mt khong thi gian ngn, khng nhng ta thu c lng sinh khi ln v enzyme ni bo m cn thu c lng enzyme ngoi bo va nhiu, va c hot tnh rt cao. Enzyme thu nhn t vi sinh vt c hot tnh rt cao. Vi sinh vt l gii sinh vt thch hp cho sn xut theo quy m cng nghip v qu trnh sinh trng, pht trin v sinh tng hp enzyme ca vi sinh vt khng ph thuc vo kh hu bn ngoi. Ngun nguyn liu dng sn xut enzyme theo quy m cng nghip r tin v d kim. V th, gi thnh sn phm s gim v c th sn xut mi ni trn th gii.

20

Vi sinh vt c th tng hp cng mt lc nhiu loi enzyme khc nhau. c im ny c lin quan n c th n bo. C th n bo phi thc hin tt c chc nng ca s sng, do chng phi to ra nhiu loi enzyme khc nhau m bo s sng pht trin tt. 1.4.3. Mt s k thut nui cy vi sinh vt thu nhn enzyme: K thut to ging La chn phng php nui cy Thit k thit b nui cy K thut ln men Tch, tinh ch thu nhn enzyme 1.4.4. Vi sinh vt sn xut enzyme amylase: Trong thin nhin, enzyme amylase c hu ht mi vi sinh vt, thc vt v ng vt. Song ch c mt s loi vi sinh vt v mt s ht thc vt mi l i tng dng lm ngun thu cc ch phm enzyme amylase, do chng c kh nng tch ly mt lng ln cc enzyme ny trong nhng iu kin xc nh. Ngy nay do c u th v nhiu mt, vi sinh vt tr thnh ngun thu enzyme ch o. Ngi ta bit nhiu loi vi sinh vt c kh nng tng hp cc enzyme amylase. Nhng chng vi sinh vt to nhiu enzym amylase thng c phn lp t cc ngun t nhin, bi cc loi khc nhau v cc chng vi sinh vt khc nhau cng thng sn sinh ra nhiu enzyme khc nhau. Vi sinh vt to enzyme amylase c dng nhin hn c l nm mc, gi nm men v vi khun, cn x khun th t hn.

21

- thu enzyme amylase ngi ta thng dng cc ging nm mc Aspergillus, Rhizopus v mt s loi ca ging Mucor sinh tng hp rt mnh m -amylase v glucoamylase. Nm men v gi nm men thuc cc ging Candida, Saccharomyces, Endomycopis, Endomyces cng to enzyme amylase. - Nhiu vi khun c kh nng to lng ln enzyme amylase nh Bacillus subtilis, Bacillus polymyxa, Clostridium acetobutylicum, Pseudomonas saccharophila, Bacillus circulans - Trong nhm x khun rt him gp loi to enzyme amylase rt mnh m, tuy nhin cng c mt s to enzyme nh x khun a nhit. 1.4.5. Thu nhn enzyme amylase t vi sinh vt: 1.4.5.1. Sinh tng hp enzyme amylase vi sinh vt: Khi nui vi sinh vt to enzyme amylase c hai qu trnh lin quan mt thit vi nhau: qu trnh tng hp sinh khi vi sinh vt v qu trnh to ra enzyme amylase trong t bo hay ngoi mi trng. mt s vi sinh vt, qu trnh sinh tng hp enzyme amylase xy ra song song vi qu trnh sinh trng, s to ra enzyme amylase ph thuc tuyn tnh vo s tng sinh khi. S to thnh enzyme amylase cc i xy ra sau khi qun th t bo vi sinh vt t im sinh trng.

22

1.4.5.2. Cc yu t nh hng ca mi trng n tng hp enzyme amylase: a. nh hng ca ngun nit: Ngun nit trong mi trng dinh dng ca phng php nui b su c th l mui v c hay cc hp cht hu c cha nit. NaNO3 l ngun nit dinh dng nui nhiu loi nm mc to enzyme amylase. Cho ngun nit nht nh vo mi trng c th kch thch tng hp enzyme amylase ny v c ch tng hp enzyme amylase khc. Theo mc tiu th mui, mi trng b axit ha, qu trnh chuyn dch mnh v pha tng hp nhiu glucoamylase v c ch tng hp -amylase. S to glucoamylase cng nh -amylase cc i thng thy cc nng nit cao (0,25 0,4%), khi lm mi trng nui vi khun to enzyme amylase thng dng (NH4)2HPO4. Ngun nit hu c nh: galatin, casein, nc chit ng m bo cho Aspergillus awamori sinh trng tt, nhng khng tng cng tng hp cc enzyme amylase. Thm nc chit mm mch hay nc chit ng cng nh cc amino axit u lm tng sinh tng hp -amylase ngoi bo, song hiu qu km hn NaNO3. Tnh cn bng ca mi trng dinh dng v carbon v nit c ngha ln i vi sinh tng hp sinh khi vi sinh vt v s to thnh enzyme amylase. Trong mi trng c lng carbon v nit cn thit mi to c enzyme cc ln. b. nh hng ca amino acid: Amino acid l nhng cu t hp thnh enzyme. Amino acid c nh hng tt ti sinh l ca vi sinh vt cng nh sinh tng hp enzyme amylase do cc nguyn nhn sau: Amino acid l ngun carbon, ngun nit, ngun nng lng.

23

Mt s amino acid ng vai tr v cng quan trng trong trao i amino acid c th l sinh tng hp nhiu amino acid khc v trong qu trnh chuyn amine ha. Nhiu amino acid tham gia vo thnh phn ca cc vitamin tan trong nc nh: biotin, folic acid Mt s amino acid kch thch v lm tng cng sinh tng hp cc enzyme amylase, mt s th c ch qu trnh, mt s khng c nh hng g. c. nh hng ca ngun dinh dng khong: Cc nguyn t a lng v vi lng c nh hng ln ti sinh trng v tng hp cc enzyme amylase ca vi sinh vt. Mg
2+

: c nh hng ti bn nhit ca enzyme amylase. Thiu

MgSO4 s nh hng n s tng hp mi enzyme amylase ca nm mc. Khi , s tng hp -amylase b c ch hon ton, cn lng glucoamylase gim xung. Nng ti u ca mui ny cho sinh tng hp -amylase v glucoamylase l 0.05%. Phospho: cn tng hp cc hp cht quan trng ca nucleic phospholipid acid v nhiu coenzyme, ng thi phosphoril ha glucide trong qu trnh oxy ha sinh hc. Phospho nh hng trc tip ti sinh sn ca nm mc v cc vi sinh vt khc, do vy m tng cng tng hp cc enzyme amylase. Calcium: cn cho tng hp v n nh -amylase hot ng v n l cu t khng th thiu ca enzyme ny, c tc dng bo v amylase khi tc ng ca proteinase.

24

Lu hunh: kch thch s to amylase v lu hunh c trong thnh phn cu cc amino axit quan trng nh: methionine, cystein. Ngun lu hunh tt nht to enzyme amylase l methionine , c th dng cc mui sulfate ngoi tr CuSO4. Nhiu nguyn t vi lng c nh hng ti s tng sinh khi v tng hp cc enzyme amylase nh Coban c tc dng kch thch tng hp enzyme amylase, trong sinh sn v hot ng sng ca vi sinh vt. Mn, Cu, Hg km hm sinh tng hp enzyme amylase vi sinh vt trong pH mi trng. d. nh hng ca pH nguyn liu: Nui cy bng phng php b mt th pH mi trng nh hng t, do mi trng c dung dch m cao v hm m thp nn pH khng thay i nhiu.Tuy nhin, pH ban u ca mi trng cng c nh hng khng nh ti s pht trin ca nm mc v s to enzyme amylase. Nu dng nc my lm m cm th pH mi trng l 5 6. Nu dng HCl, H2SO4 v axit lactic lm m th pH mi trng gim n 4,5 5,0 to iu kin chn lc cho nm mc pht trin. pH 4,5 5,0 nm mc to enzyme amylase tt nht. 1.4.5.3. Cc phng php thu nhn enzyme amylase: a. Nui vi sinh vt to enzyme amylase bng phng php b mt: Thnh phn chnh ca mi trng vi sinh vt to enzyme amylase bng phng php b mt l cm m, cm go. l nguyn liu hon ho v c th l thnh phn duy nht ca mi trng nui vi sinh vt m khng cn phi b sung thm cc cht khc na.

25

Cht lng ca cm go, cm m c nh hng ln ti hot lc ca cc enzyme amylase. Cm khng c cha tinh bt di 20 30%, nn dng cm tt, cm mi khng hi mi mc. m ca cm khng qu 15%, tp cht khng qu 0,05%... Thnh phn b sung c a vo mi trng c th l cht lm xp mi trng hoc lm giu thm cht dinh dng v cht sinh trng m cm khng c . Thnh phn ny thng l mm mch, tru, mn ca Cm v cc cht ph gia cha nhiu vi sinh vt khc cn phi thanh trng m bo mi trng khng c vi sinh vt ngoi lai, cn thanh trng di p sut hi 1 1,5 atm trong vng 4 - 6 gi. m ti thch ca mi trng: trong iu kin sn xut, m ban u ti thch ca mi trng i vi Aspergillus niger, Aspergillus awamori, Aspergillus oryzae l 58 60% v phi gi cho mi trng c m trong sut qu trnh nui. m m qu 55 70% s lm gim thong kh, cn thp hn 50 55% th km hm sinh trng v pht trin ca vi sinh vt cng nh to enzyme amylase. Ch thng kh c th lin tc, gin on ty thuc vo chiu dy ca lp mi trng nui, vo khong cch gia cc tng khay v dy khay. giai on sinh trng th nht phi thng kh vo phng nui khong 4 5 ln th tch khng kh trn mt th tch phng trong mt gi, giai on th hai l 30 60 th tch khng kh trn th tch phng nui trong 1 gi, giai on ba gim i ch cn 10 12 th tch khng kh. Nhit nui: Ton b chu k sinh trng ca nm mc trn cm chia lm ba thi k: Thi k trng v ny mm ca nh bo t (i vi nm mc 10 11 gi u tin, vi khun 3 4 gi). Trong thi k ny phi t nng

26

khng kh phng nui v gi cho nhit phng nui khng thp hn 23 300C i vi nm mc. m tng i ca khng kh l 96 100%. Thi k sinh trng nhanh ca h si ko di trong 4 18 gi. giai on ny nm mc h hp rt mnh v to ra mt lng nhit sinh l ln. V vy cn phi h nhit phng nui khong 28 290C. Thi k to enzyme amylase mnh m ko di t 10 20 gi. Trong thi k ny cc qu trnh trao i cht dn dn yu i, s ta nhit gim mnh. Cc enzyme amylase c tng hp mnh m. i vi a s vi sinh vt giai on ny nn h nhit xung 3 40C so vi giai on u. Nhit ti thch cho sinh trng ca a s nm mc trn mi trng rn l 28 300C. Thi gian nui c lng enzyme amylase cc ln: Ty thuc vo tnh cht sinh l ca chng vi sinh vt v s ngng tng hp enzyme amylase m c th ngng sinh trng ca nm mc vo bt k lc no thy cn thit. S to bo t l hin tng khng mong mun v thng lm gim hot lc enzyme. a s nm mc Aspergillus, s to enzym amylase cc i thng kt thc khi nm mc bt u sinh nh bo t. b. Nui vi sinh vt to enzyme amylase bng phng php b su: Mi trng dinh dng: mi trng nui vi sinh vt to enzyme amylase l c cht cm ng l tinh bt, dextrin hay maltose. to iu kin cho vi sinh vt pht trin tt v sinh nhiu enzyme amylase ngi ta cho thm vo mi trng cc loi nc chit nh nc chit mm mch, nc chit ng, nc chit u nnh

27

axit ca mi trng c xc nh bi thnh phn v tnh cht ca cc mui v c thm vo mi trng cng nh s tiu th cc mui ny bi vi sinh vt, pH ca mi trng ph thuc vo tnh cht ca ngun nit v c . Nu thm vo mi trng cc mui ammonium th khi vi sinh vt tiu th ion ammonium, cc anion c gii phng ra s axit ha mi trng. V th cn phi thm CaCO3 vo trung ha hoc duy tr gi tr pH thch hp cho vic tng hp enzyme amylase. Nhit nui: l mt yu t quan trng i vi sinh trng ca vi sinh vt v s to thnh enzyme amylase. Nhit nui ti thch i vi nm mc thuc ging Aspergillus l 30 320C, Bacillus subtilis l 370C. Sc kh v khuy trn. Phn ln vi sinh vt to enzyme amylase l nhng vi sinh vt hiu kh. V vy sinh trng ca chng ph thuc vo lng oxy phn t ha tan trong dch nui cy. Trong qu trnh sinh trng, vi sinh vt s dng oxy phn t cho hot ng sng nn lng oxy ha tan trong mi trng lng phi lun lun c b sung. V th vic sc kh v khuy o mi trng c tc dng tt ti sinh trng v tch ly sinh khi cng nh sinh tng hp enzyme amylase ca vi sinh vt. Ch sc kh i vi nm mc l 10 12 m3 khng kh v trng trn 1 m3 mi trng trong 1 gi vi thi gian nui khong 68 72 gi. 1.4.6. ng dng ca enzyme amylase: Ch phm enzyme amylase c ng dng rng ri trong cng nghip c bit l trong cng nghip thc phm, sau y l mt s ng dng quan trng ca enzyme amylase:

28

1.4.6.1. Amylase trong cng nghip ru bia: Ch phm enzyme amylase t nm mc Aspergillus oryzae, Aspergillus awamori, Aspergillus usamii, Aspergillus niger, Rhizopus delemar hoc t Endomycopsis bispora, t vi khun Bacillus subtilis, Bacillus diastaticus c thay th malt lm tc nhn ng ha tinh bt trong sn xut ru,bia t nguyn liu c bt. Vic s dng ch phm amylase vi sinh vt thay malt tit kim c hng vn tn i mch loi tt, gim gi thnh, rt ngn quy trnh sn xut. Trong giai on ng ha, glucoamylase chim v tr hng u v ng vai tr quyt nh trong vic chuyn ha tinh bt thnh ng ln men, do vy cn chn chng vi sinh vt c kh nng sn sinh nhiu glucoamylase. Trong sn xut ru ngi ta thng dng hn hp canh trng b mt hay b su ca cc nm mc. Vit Nam chng ta dng canh trng b mt ca nm mc Aspergillus oryzae, Aspergillus usamii, Aspergillus awamori v Rhizopus delemar ng ha. mt s nc, ngoi enzyme amylase nm mc ngi ta cn s dng enzyme amylase t vi khun, nm men cng cho kt qu ng ha kh cao. Ch phm enzyme amylase t vi khun Bacillus subtilis, Bacillus diastaticus chu nhit cao, dng trong giai on dch ha tinh bt lc u, trc khi ng ha rt tt. nhit 80 - 900C trong 10 - 15 pht amylase ca vi khun b v hot mt phn rt nh. Trong sn xut bia, thng dng Aspergillus oryzae v Bacillus

subtilis ng ha thay malt, l nguyn liu kh t tin v cn thit trong sn xut bia. Dng ch phm enzyme ny ngi ta c th thay t 50 - 100% malt bng i mch khng ny mm, thay th 50 - 60% malt th cht lng

29

bia tt hn. Khi dng ch phm enzyme vi sinh vt thay malt, cn b sung ngun nguyn liu c bt nh cc loi ng cc nh: bp, khi, sn, go, i mch khng ny mm, tiu mch. Bin php thay th malt bng ch phm enzyme nh trn c ngha c bit quan trng trong vic gim gi ng k gi thnh ca sn phm, m cht lng bia vn bo m. 1.4.6.2. Trong sn xut bnh m Trong sn xut bnh m ngi ta thng s dng ch phm enzyme amylase ca nm mc Aspergillus oryzae, Aspergillus awamori, hay ca vi khun, nhng ch phm t nm mc p dng c hiu qu v rng ri hn. Dng vi lng 0,002 - 0,05% ch phm enzyme amylase so vi bt nho s tng cng qu trnh ln men bt, rt ngn thi gian ln men xung 20 - 30%, tng xp 5 - 8%, tng th tch bnh ln men 15 - 25% mu sc v bnh p hn, rut bnh xp v sng mu hn tng hng v thm ngon ca bnh. Khi lm bnh m ngt, nu thm ch phm enzyme amylase vo bt, s xy ra qu trnh thy phn bt thnh ng, do gim lng ng tiu tn cho sn xut. 1.4.6.3. Trong sn xut mt, tinh bt, malto, gluco T lu cc sn phm mt tinh bt nh: mt gluco, mt malto c sn xut bng phng php thy phn tinh bt bng axit hay bng malt. Ngy nay ngi ta s dng rng ri ch phm enzyme amylase vi sinh vt trong sn xut mt tinh bt, gluco, malto t nguyn liu tinh bt. cc nc tin tin khc, phng php dng enzyme t vi sinh vt trong lnh vc ny cng c p dng c hiu qu v ph bin, hay phi hp phng php axit v enzyme. Trong sn xut mt tinh bt, gluco, hai enzyme ch yu l amylase v glucoamylase t nm mc v vi khun.

30

Enzyme -amylase dch ha tinh bt v to malto, cn glucoamylase dng ng ha to gluco, ch phm amylase cho sn xut gluco c sn xut t vi khun Bacillus sublitis, Bacillus mesentericus. Ch phm glucoamylase thng sn xut t nm mc: Aspergillus niger, Aspergillus awamori, Rhizopus delamar, Mucor hay t mt s nm men Saccharomyces, Endomycopsis 1.4.6.4. Trong mt s ngnh cng nghip thc phm khc Trong mt s ngnh cng nghip thc phm khc nh bnh ko, nc gii kht, hp, nc p tri cy, tng Ch phm enzyme amylase gip cho qu trnh thy phn tinh bt thnh ng, lm trong cc thnh phn dng lng, d c c, tng lng ng trong sn phm, gim lng ng tiu tn, tng lng ng n gin d hp thu cho ngi, c bit l thc n cho tr em, gim thi gian nu, ch bin Hin nay ngi ta dng hn hp cc enzyme: protease, amylase, pectinase trong sn xut cc sn phm trn, to sn phm d tan, d tiu ha mu sc p, d lc, d c c, rt ngn thi gian sn xut, gim gi thnh sn phm 1.4.6.5. Trong cc ngnh cng nghip dt, giy Trong cng nghip dt, ch phm enzyme amylase ca vi khun Bacillus subtilis, Bacillus mesentericus, Bacillus diastaticus, Bacillus amylosolvens c tnh u vit chu nhit cao, dng trong r h vi, to iu kin tt d dng khi nhum, ty vi sau ny. Ngoi ra ngi ta cn dng ch phm enzyme amylase trong sn xut keo tinh bt cho cng nghip giy.

31

1.4.6.6. Trong chn nui Ch phm enzyme amylase c s dng ring r hay phi hp vi cc enzyme khc nh: protease, cenlulase, pectinase sn xut cc loi thc n d tiu ha, hp thu trong chn nui gia sc, gia cm. S dng thc n c ch phm enzyme amylase s lm tng kh nng hp thu, tiu ha do gim lng thc n, tng trng nhanh, tng kh nng sinh sn, chng bnh tt 1.4.6.7. Trong y hc Enzyme amylase cng cc enzyme khc, c dng cho cha bnh do thiu enzyme, km kh nng chuyn ha cht, bnh v tiu ha, tim, thn kinh Ngoi ra ch phm enzyme amylase dng iu ch mi trng nui vi sinh vt phc v cho nghin cu khoa hc, chun tr bnh.

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CHNG 2: VT LIU V PHNG PHP

2.1. VT LIU: 2.1.1. i tng nghin cu: i tng nghin cu l cc chng nm mc phn lp t cc mu bnh men cm ru cc a im khc nhau theo bng 2.1: Bng 2.1: Cc mu bnh men cm ru STT 1 2 3 4 5 6 7 8 K hiu mu HA T Q9 Q5 LBT BH BL BD Ni ly mu Ch Ha An Bin Ha ng Nai Ch T c Qun Th c TP.HCM Ch Phc Long Qun 9 TP.HCM Ch Kim Bin Qun 5 TP.HCM Ch Long Bnh Tn Bin Ha ng Nai Ch Bin Ha Bin Ha ng Nai Ch Bu Long Bin Ha ng Nai Ch Tn Ba Tn Uyn Bnh Dng

2.1.2. Vt liu v ha cht: 2.1.2.1 Dng c v thit b: - Cc dng c th nghim ph bin trong phng th nghim vi sinh nh: ng nghim, a petri, que cy, erlen - T cy - T m

33

- Ni hp - My ly tm Centrifuge 5430 - My sy - My o OD Beckman Coulter - My lc iu nhit 2.1.2.2. Ha cht: a. Thuc th Lugol I2 : 1 g KI : 2 g Nc ct :300 ml b. Dung dch m axetat pH 4,7: Axetat : 82 g Axit CH3COOH : 32,3 ml Nc ct : 1000 ml c. Dung dch tinh bt 1%: Tinh bt : 1 g Dung dch m axetat : 10ml Nc ct : 100 ml

34

2.1.2.3. Mi trng: a. Mi trng PGA (Potato Glucose Agar) Khoai ty Glucose Agar Nc ct pH=6.5 b. Mi trng thch tinh bt KH2PO4 K2HPO4 MgSO4.7H2O (NH4)2SO4 Tinh bt tan Agar Nc ct 2.2. PHNG PHP: 2.2.1. a im v thi gian nghin cu: Th nghim c thc hin ti phng th nghim Khoa Cng Ngh Sinh Hc Mi Trng Trng i hc Lc Hng. Thi gian thc hin: t thng 03/2010 11/2010. 0.5g 0.5g 0.2g 0.2g 10g 20g 1000ml 200g 20g 20g 1000ml

35

2.2.2. Phng php nghin cu: 2.2.2.1. Phng php phn lp: Mi trng phn lp nm mc c s dng l mi trng PGA. Cc chng nm mc c phn lp t cc mu bnh men cm ru. Bnh men c b lm i, ly mt t bnh men gia ri u ln b mt a petri cha mi trng PGA, sau em nhit phng, trong 48 gi. Sau khi nm mc mc trn b mt mi trng ca a petri. Dng que cy chuyn nm mc qua a petri c cha mi trng PGA mi lm thun. Nm mc sau khi lm thun c cy vo ng nghim v c bo qun trong t lnh 40C dng cho cc th nghim tip theo. 2.2.2.2. Phng php quan st hnh thi nm mc: a. Quan st khun lc nm mc: Cy chng nm mc ln mi trng PGA, nhit phng trong 48 gi. Sau quan st v ghi nhn hnh thi khun lc nm mc trn a nh: mu sc, hnh dng si nm v o kch thc khun lc. b. Quan st t bo nm mc di knh hin vi: Lm tiu bn t bo nm mc trong xanh methylen, quan st hnh thi t bo di knh hin vi quang hc. 2.2.2.3. Phng php xc nh kh nng phn gii tinh bt: Mi trng s dng xc nh kh nng phn gii tinh bt ca cc chng nm mc l mi trng thch tinh bt. Cy chng nm mc ln mi trng thch - tinh bt, nhit phng, trong 48 gi. Sau , dung dch Lugol ln mi trng thch - tinh bt. Nu c vng phn gii xung

36

quanh khun lc th chng nm mc c kh nng phn gii tinh bt, nu khng c vng phn gii xung quanh khun lc th chng nm mc khng c kh nng phn gii tinh bt, dng thc o vng phn gii tinh bt v ghi nhn kt qu. 2.2.2.4 Phng php thu nhn enzyme amylase t nm mc [6] S dng mi trng PGA khng cho agar nui cy nm mc.

37

Thu nhn ch phm enzyme amylase theo phng php nui cy chm:
100 ml MT PGA lng Ging nm mc Lc 200v/p trong 48 gi t0 phng

Ly tm 6000v/p trong 5 pht

Thu dch lng, b cn

Ta bng cn 960 lnh

Gi lnh 40C trong 24 gi

Ly tm 6000v/p trong 20 pht

Thu cn, b dch

Sy di 400C

Enzyme bn tinh khit

Hnh 2.1: Quy trnh thu nhn enzym amylase t nm mc

38

Chun b 100ml mi trng trong erlen 250ml. em tit trng 1210C trong 30 pht v ngui. Cho 10ml nc ct v trng vo trong ng nghim ging nm mc, khuy nh cc bo t ha trong nc. ton b 10ml dch trong ng nghim ny vo trong erlen 250ml c 100ml mi trng. Tt c thao tc ny c thc hin trong t cy v trng. t cc erlen ny vo my lc c tc 200 v/p nui trong 48 gi nhit phng. Sau , em ly tm, thu ly dch. Dch nui cy ny c cha enzyme ha tan. Bo qun dch nui cy ny trong t lnh 40C. Dng 200ml cn 960 lnh t t vo 100ml dch lc enzyme lm lnh, khuy rt nh cn ha u vi dch enzyme. Sau , hn hp ny vo t lnh. Sau 24 gi, hn hp s phn thnh hai lp. em ly tm thu enzyme dng ta. Enzyme dng ta ny cn cha nhiu nc. Nc tn ti trong s lm gim hot tnh enzyme. Do , cn phi loi nc bng cch sy nhit <400C, ta c ch phm enzyme amylase bn tinh khit. 2.2.2.5. Phng php xc nh hot ca enzyme amylase: [5],[7] Hot enzyme amylase c trng cho kh nng thy phn tinh bt bi enzyme amylase ti dextrin cho mu vi iodine khc vi mu ca tinh bt ban u. n v hot enzyme amylase l lng enzyme xc tc thy phn 1g tinh bt tan nhit 300C trong 1 gi vi pH = 4,7. Cho vo 2 erlen 50ml, mi erlen 10ml dung dch tinh bt 1%. t vo my nhit 300C, gi 10 pht. Thm vo erlen 2 (erlen th nghim) 5 ml dch chit enzyme. Khuy u v gi 10 pht. Ly t mi erlen 0,5 ml hn

39

hp cho vo 2 erlen khc c cha 50 ml dung dch iodine, lc u. Erlen i chng c mu xanh, erlen th nghim c mu tm. o hp th bc sng 656nm. Mt quang ca erlen i chng (OD1) l lng tinh bt ban u. Mt quang ca erlen th nghim (OD2) l lng tinh bt cn li sau khi b -amylase thy phn. S khc nhau gia mt quang ca erlen i chng v erlen th nghim l lng tinh bt b enzyme amylase thy phn. - Lng tinh bt b thy phn:
C= OD1 OD2 0,1 (g) OD1

Trong : OD1 mt quang ca dung dch i chng, OD2 mt quang ca dung dch nghin cu, 0,1 lng tinh bt phn tch (g). - Hot enzyme amylase ca ch phm enzyme t nm mc:
HdAn = 7,264.C 0,03766 1000 (v/g) w

Trong : w lng ch phm enzyme amylase em th nghim (mg). C lng tinh bt b thy phn (g). 1000 h s chuyn miligam thnh gam. 7,264; 0,03766 l cc h s ca phng trnh tnh hot thu c bng phng php x l ton hc s liu thc nghim v s ph thuc ca lng tinh bt b thy phn v lng enzyme ly nghin cu. Trong cc h s ny c a vo tha s tnh chuyn thnh 1 gi tc dng ca enzyme.

40

CHNG 3: KT QU V BIN LUN

3.1. Kt qu phn lp cc chng nm mc c trong bnh men cm ru: - T 8 mu bnh men ly nhng a im khc nhau, chng ti phn lp c 11 chng nm mc theo bng 3.1: Bng 3.1: Cc chng nm mc phn lp t bnh men cm ru STT 1 2 3 4 5 6 7 8 9 10 11 Chng M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 Mu HA HA T T Q9 Q9 Q5 LBT BH BL BD

Qua bng trn thy rng t cc mu bnh men HA, T v Q9 mi mu chng ti phn lp c 2 chng nm mc, ring cc mu bnh men Q5, LBT, BH, BL, BD mi mu ch phn lp c 1 chng nm mc. Di y l mt s hnh nh khun lc nm mc m chng ti phn lp c:

41

a. Chng M2

b. Chng M4

c. Chng M7

d. Chng M10

Hnh 3.1: Khun lc ca cc chng nm mc M2, M4, M7, M10

42

3.2. Kt qu quan st hnh thi nm mc: Sau khi quan st khun lc ca cc chng nm mc phn lp c t cc mu bnh men, chng ti ghi nhn mt s c im hnh thi khun lc nh bng 3.2: Bng 3.2: Hnh thi khun lc ca cc chng nm mc phn lp t bnh men cm ru
STT Chng Kch thc (2 ngy) Tc pht trin Mu sc Hnh dng si nm

1 2 3 4 5 6 7 8 9 10 11

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11

7.7 cm 6 cm 8.8 cm 3.9 cm 7.5 cm 6 cm 9.5 cm 9.5 cm 9.5 cm 9.5 cm 9.5 cm

Nhanh Nhanh Nhanh Va phi Nhanh Nhanh Nhanh Nhanh Nhanh Nhanh Nhanh

Trng Mu xm, vin trng Trng Trng xm, nht Trng Mu xm, vin trng Trng Trng Trng Trng Trng

Dng bng Dng bng Dng bng Dng bng, dy c Dng bng Dng bng Dng bng Dng bng Dng bng Dng bng Dng bng

Qua bng trn cho thy rng, v c im hnh dng si nm: a s cc chng nm mc phn lp c u c dng bng, ring chng nm mc M4 c dng bng dy c.

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- V mu sc khun lc: cc chng nm mc M1, M3, M5, M7, M8, M9, M10, M11 phn lp t bnh men u c mu trng. Cc chng nm mc c mu xm, vin trng l M2 v M6. Ring chng nm mc M4 th c mu trng, xm nht. - V kch thc khun lc nm mc trong 2 ngy sau khi cy, c kch thc t 3,9 9, 5 cm. - Tc pht trin ca cc chng nm mc phn lp c u nhanh. Ring chng M4 c tc pht trin va phi. Sau khi quan st di knh hin vi, da vo kha phn loi ca TS. ng V Hng Min, chng ti phn loi s b c 3 chng n ging: - Chng M4 thuc ging Mucor. - Chng M2 v M6 thuc ging Rhizopus. Do iu kin hn ch v thi gian, thit b my mc cng nh ha cht, chng ti ch phn loi s b c 3 chng nm mc n ging. 3.3. Kt qu xc nh kh nng phn gii tinh bt ca cc chng nm mc : Kt qu th nghim xc nh kh nng phn gii tinh bt ca cc chng nm mc cho thy 9 chng: M1, M3, M4, M5, M7, M8, M9, M10, M11 c kh nng to vng phn gii tinh bt v 2 chng: M2, M6 khng c kh nng to vng phn gii tinh bt. Cc chng nm mc c kh nng phn gii tinh bt do chng c kh nng to ra enzyme amylase. Nm mc to ra enzyme amylase phn gii lin kt 1,4 glycoside gia chui to thnh dextrin phn t thp. Di tc dng

44

ca enzyme amylase, tinh bt trong mi trng nhanh chng b mt kh nng to mu vi dung dch iodine. Nh vy, 9 chng nm mc: M1, M3, M4, M5, M7, M8, M9, M10, M11 c kh nng to enzyme amylase v 2 chng nm mc: M2, M6 khng c kh nng to enzyme amylase. Kt qu o vng phn gii tinh bt ca cc chng nm mc theo bng 3.3: Bng 3.3: Kh nng phn gii tinh bt ca cc chng nm mc phn lp t bnh men cm ru STT 1 2 3 4 5 6 7 8 9 10 11 Chng M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 ng knh phn gii tinh bt (cm) 1,85 3,1 2,25 1,55 1,25 2,6 2,75 2,1 2,23

Qua kt qu th nghim kh nng phn gii tinh bt ca cc chng nm mc cho thy cc chng nm mc phn lp c t bnh men c ng knh phn gii t 1,25 3,1 cm. Trong c 2 chng nm mc khng c kh nng phn gii tinh bt l chng M2 v M6. Kh nng phn gii tinh bt ca chng M3 l ln nht c vng phn gii l 3, 1 cm, trong khi chng M7 c kh nng phn gii tinh bt nh nht, vng phn gii tinh bt l 1,25 cm.

45

a. Chng M3

b. Chng M4

c. Chng M9

d. Chng M11

Hnh 3.2: Kh nng phn gii tinh bt ca cc chng M3, M4, M9, M11

46

e. Chng M6

f. Chng M7

Hnh 3.3: Kh nng phn gii tinh bt ca cc chng M6, M7 3.4 Kt qu thu nhn enzyme amylase t nm mc: Do thi gian thc hin ti c hn, chng ti ch s dng chng nm mc M4 thu nhn enzyme amylase. Chng ti ch chn chng M4 thu nhn amylase v l do sau: Chng M4 l 1 trong 9 chng nm mc phn lp c t bnh men c kh nng phn gii tinh bt cho vng phn gii tng i ln l 2,25 cm. Chng M4 c nh danh s b l ging Mucor. Kt qu th nghim cho thy, t 1 lt mi trng nui cy chng M4, chng ti thu nhn c 1,23 g ch phm enzyme amylase bn tinh khit. Theo ti liu tham kho [8], thng thng bng phng php nui b su, t 1 lt mi trng nui cy thu c t 1 4 gam ch phm enzyme kh.

47

Di y l mt s hnh nh trong qu trnh thu nhn enzyme amylase bn tinh khit t chng nm mc M4.

Hnh 3.4: Dch chit enzyme amylase sau ly tm

48

Hnh 3.5: Ch phm enzyme amylase c ta bng cn 960 lnh

Hnh 3.6: Ch phm enzyme amylase bn tinh khit t nm mc

49

3.5. Kt qu xc nh hot ca enzyme amylase bn tinh khit: Mt quang ca dung dch i chng: OD1 = 0,023 Mt quang ca dung dch nghin cu: OD2 = 0,014 - Lng tinh bt b thy phn:
C= OD1 OD2 0,023 0,014 0,1 = 0,1 = 0,039 (g) OD1 0,023

- Hot amylase ca ch phm enzyme:

HdAn =

7,264.C 0,03766 7,264.0,039 0,03766 1000 = 1000 = 4912,72 (v/g) 0,05 w

Ch phm enzyme amylase bn tinh khit m chng ti thu nhn c t chng nm mc M4 c hot cao l 4912,72 v/g, iu ny c ngha quan trng gp phn to ra ngun enzyme amylase c gi tr t chng nm mc ny v ng dng rng ri trong nhiu lnh vc khng nhng trong ngnh cng nghip thc phm m cn trong cc ngnh khc nh: ngnh cng nghip dt, sn xut cht ty ra, y hc, chn nui

50

CHNG 4: KT LUN V KIN NGH

4.1. KT LUN: Sau mt thi gian tin hnh nghin cu, chng ti : Phn lp c 11 chng nm mc t bnh men cm ru 8 a im khc nhau. nh danh s b c 3 chng n ging: Chng M4: ging Mucor Chng M2 & M6: ging Rhizopus Trong c 9 chng nm mc u c kh nng phn gii tinh bt bao gm cc chng: M1, M3, M4, M5, M7, M8, M9, M10, M11 v 2 chng khng c kh nng phn gii tinh bt l chng M2, M6. Nh vy, 9 chng nm mc c kh nng to enzyme amylase, 2 chng nm mc khng c kh nng to enzyme amylase. Chng ti tin hnh nui cy chng M4 thu nhn c enzyme amylase bn tinh khit theo phng php nui cy chm. T 1 lt mi trng nui chng M4 thu c 1,23 g ch phm enzyme amylase bn tinh khit. Ch phm enzyme amylase bn tinh khit thu c c hot cao l 4912,72 v/g.

51

4.2. KIN NGH: Do thi gian c hn v iu kin my mc thit b cn hn ch, chng ti khng th tin hnh cc nghin cu su hn. Vi nhng kt qu t c, chng ti ngh: Tin hnh nh danh cc chng n loi. Nghin cu ti u qu trnh sn xut enzyme amylase thu c lng enzyme ti a, cht lng vi chi ph thp. Nghin cu sn xut enzyme amylase t cc chng khc c kh nng phn gii tinh bt ngoi chng M4.

52

TI LIU THAM KHO

[1] Nguyn Vn B Cao Ngc ip Nguyn Vn Thnh, Gio trnh mn Nm hc, Vin nghin cu v pht trin cng ngh sinh hc Trng H Cn Th, 2005. [2] Nguyn Ln Dng, Vi sinh hc hc, NXB Gio Dc, s 04 2009/CXB/301 2117/GD. [3] Bi Xun ng, Nguyn l phng chng nm mc v mycotoxin, NXB Khoa hc v K thut H Ni, 2003 [4] Nguyn Th Hin (Ch Bin), Cng ngh sn xut m chnh v cc sn phm ln men c truyn, NXB Khoa hc v K thut H Bch Khoa H Ni, 2006. [5] Nguyn c Lng (Ch bin), Cao Cng Nguyn nh Tuyt L Th Thy Tin T Thu Hng Hunh Ngc Oanh Nguyn Thy Hng Phan Th Huyn, Cng ngh enzyme, NXB i hc Quc gia TP.H Ch Minh Trng H Bch Khoa, 2004. [6] Nguyn c Lng (Ch bin) Phan Th Huyn Nguyn nh Tuyt, Th nghim cng ngh sinh hc, tp 2, Th nghim vi sinh vt hc, NXB H Quc gia TP. H Ch Minh, 2006. [7] Nguyn Vn Mi, Thc hnh Ha sinh hc, NXB Khoa hc v K thut, 2000. [8] ng Th Thanh Thu, Sinh ha ng dng (Trong cng nghip thc phm v mt s lnh vc khc), NXB i hc Quc gia TP.H Ch Minh Trng H Khoa hc T nhin, 2003.

53

TI LIU TRN MNG [9].http://commons.wikimedia.org/wiki/File:Mature_sporangium_of_a_Muco r_sp._fungus _PHIL_3960_lores.jpg [10].http://muivi.com/muivi/index.php?option=com_content&task=view&id= 3087&Itemid=431 [11].http://vi.wikipedia.org/wiki/N%E1%BA%A5m