Nhom: TRAN DUY TIEN MAI HOANG VU NGUYEN QUOC CUONG Mc lc: Phn I: Tng quan v enzym amylase

I.Amylase l g? II.c tnh v c ch tc dng ca enzym -amylase II.1.c tnh ca enzym -amylase II.2. C ch tc dng ca enzym -amylase III. Lch s pht hin eznym Phn II: Phn lp, bo qun ging VSV Chng nm mc Aspergillus oryzae I.Vai tr ca ging trong cng ngh enzym II.Yu cu ging VSV trong cng nghip enzym III.Gii thiu v chng nm mc Aspergillus oryzae IV.Qa trnh phn lp ging VSV IV.1. Phn lp ging trong iu kin t nhin IV.2. Phn lp ging trong iu kin sn xut IV.3. Phn lp ging trong mu ging h hng V.Gii thiu phng php phn lp nm mc Aspergillus oryzae VI. Phng php bo qun ging VSV VI.1. Cy truyn v bo qun lnh VI.2. Bo qun ging trong t hay trong ct VI.3. Bo qun ging trong ht ng cc

Phn III: Cng ngh ln men to enzym -amylase I.Gii thiu v phng php ln men b mt I.1. u v nhc im ca phng php nui cy b mt I.1.1. Mi trng lng I.1.2. Mi trng c I.2. Qui trnh sn xut nm mc ging I.2.1. Nguyn liu I.2.2.Chun b mc ging I.2.2.1.Nui cy trong ng thch nghing I.2.2.2.Nui cy ging trong bnh tam gic I.2.2.3.Nui cy mc trn khay I.2.3. Qui trnh sn xut II. Qui trnh ln men cng nghip to enzym -amylase II.1. Nguyn liu II.2. Qui trnh cng ngh II.3. Thu nhn enzym -amylase t nm mc Aspergillus Oryzae II.3.1. Sinh trng v sinh tng hp amylase t nm II.3.2. Cc yu t nh hng n cht lng enzym II.3.3. Cc yu t nh hng n qu trnh ln men Phn IV : ng dng v kt lun I.ng dng I.1.ng dng amylase trong sn xut bia I.2. ng dng amylase trong sn xut cn I.3.ng dng amylase trong ch bin thc phm gia sc

I.4.ng dng enzym amylase trong cng nghip dt II.Kt lun Phn V: Ph lc v ti liu tham kho

Li gii thiu: Tinh bt l sn phm t nhin quan trng nht c nhiu ng dng trong k thut v trong i sng con ngi. Nhiu nc trn th gii s dng ngun tinh bt t khoai ty, la m, ng (sn), cn ring nc ta th s dng go v khoai m l ngun tinh bt ch yu. Trong ch bin tinh bt v ng, cng on quan trng nht l thu phn tinh bt v cc ng n gin. Sau , ch yu trn c s ng n nh ln men, ngi ta s nhn c rt nhiu sn phm quan trng nh: ru cn, ru vang, bia, cc loi acid hu c, amino acid,. Qu trnh thu phn tinh bt gm hai cng on ch yu l giai on h ho v giai on ng ho. thc hin hai cng on cng ngh ni trn, trong thc t sn xut ngi ta p dng hai cch: Thu phn tinh bt bng acid v bng enzym. thu phn tinh bt t lu ngi ta s dng acid v c nh HCl v H2SO4. Nhng kt qu cho thy, thu phn bng acid rt kh kim sot v thng to nhiu sn phm khng mong mun v khng p ng tiu chun an ton thc phm. Do vy, vic thay th v ng dng enzym thu phn tinh bt l mt kt qu tt yu ca lch s pht trin. Enzym amylase c tm ra gp phn quan trng cho nhiu ngnh cng nghip ch bin thc phm. Enzym amylase c th tm thy nhiu ngun khc nhau nh amylase t thc vt, ng vt v VSV. Amylase cng ngy cng c thay th acid trong sn xut qui m cng nghip. Hin nay, cc nh sn xut c th s dng amylase c kh nng chu nhit cao m khng b mt hot tnh, chng hn amylase c tch chit t VSV, c th l cc chng vi khun chu nhit c phn lp t nhng sui nc nng. Ngoi ra, amylase cn c nhiu u im hn khi s dng acid thu phn tinh bt: Nng lng xc tc thp, khng yu cu cao v thit b s dng, gim chi ph cho qu trnh tinh sch dch ng.

Ngun amylase c th ly t mm thc, mm i mch ( malt), ht bp ny mm, hay t nm mc, Nguyn liu cho sn xut l go, bp, khoai m, y l nhng ngun nguyn liu d tm, r tin c th tm thy d dng nc ta. Do , y l mt li th v l hng pht trin mnh lm c s cho nhiu ngnh khc pht trin. V d: sn xut bnh ko, bia, cn, sir v lm mm vi,

Phn I: Tng quan v enzym amylase

I.Amylase l g?

Amylase l mt h enzyme rt ph bin trong th gii sinh vt . Cc enzyme ny thuc nhm enzyme thy phn, xc tc phn gii lin kt ni phn t trong nhm polysaccharide vi s tham gia ca nc:

R.R

H-OH

RH

ROH

C 6 loi enzyme c xp vo 2 nhm: Endoamylase ( enzyme ni bo ) v exoamylase ( enzyme ngoi bo ). Endoamylase gm c -amylase v nhm enzyme kh nhnh. Nhm enzyme kh nhnh ny c chia thnh 2 loi: Kh trc tip l Pullulanase ( hay -dextrin 6 glucosidase); kh gin tip l Transglucosylase (hay oligo1,6-glucosidase) v maylo-1,6-glucosidase. Cc enzyme ny thy phn cc lin kt bn trong ca chui polysaccharide. Exoamylase gm c -amylase v -amylase. y l nhng enzyme thy phn tinh bt t u khng kh ca chui polysaccharide. C cht tc dng ca amylase l tinh bt v glycogen: Tinh bt: l nhm Carbohydrate thc vt, c ch yu trong cc loi c nh khoai lang, khoai ty, khoai m , trong cc ht ng cc, cc loi ht

v c cng thc tng qut l (C6H12O6)n.Tinh bt t mi ngun khc nhau u c cu to t amylase v amylopectin ( Meyer, 1940 ). Cc loi tinh bt u c 20-30% amylase v 70-80% amylopectin. Trong thc vt, tinh bt c xem l cht d tr nng lng quan trng. -Amylase c trng lng phn t 50.000 160.000 Da, c cu to t 200-1000 phn t D-glucose ni vi nhau bi lin kt -1,4-glucoside to thnh mt mch xon di khng phn nhnh. -Amylopectin c trng lng phn t 400.000 n hng chc triu Da, c cu to t 600-6000 phn t D-glucose, ni vi nhau bi lin kt -1,4glucoside v -1,6-glucoside to thnh mch c nhiu nhnh. Tinh bt khng tan trong nc lnh nhng khi hn dch tinh bt b un nng ( 60-85 0C ) th tinh bt s b h ha v c gi l h tinh bt . Di tc dng ca enzyme amylase, tinh bt b thy phn do cc lin kt glucoside b phn ct. S thy phn tinh bt bi enzyme amylase xy ra theo 2 mc : Dch ha v ng ha. Kt qu ca s dch ha l to ra sn phm trung gian dextrin v khi dextrin tip tc b ng ha th sn phm l maltose v glucose. -Cabohydrate trong thc phm l ngun cung cp nng lng quan trng cho c th con ngi. Rau v qu cng l ngun cung cp tinh bt v tinh bt ny mt phn c chuyn ha thnh disaccharide v glucose. Carbohydrate c mt trong hu ht cc loi thc phm nhng ngun cung cp ch yu l ng v tinh bt. Glycogen: l mt loi Cabohydrate d tr. ng vt c d tr trong c th ng vt v c c th chuyn ha s dng t t. Amylase c vai tr quan trng trong s chuyn ha glucid t bo ng vt, VSV, glucogen c cu to t cc glucose lin kt vi nhau bi lin kt -1,4glycoside cc v tr phn nhnh, glucose ni vi nhau bng lin kt -1,6glycoside. Glycogen c s mch nhiu hn tinh bt. Phn t lng trong khong 2 triu-3 triu Da. Glycogen d tan trong nc, nu nh chng ta n qu nhiu Carbohydrate th c th chng ta s chuyn ha chng thnh cht bo d tr. ng vt v ngi, glycogen tp trung ch yu trong gan.

II.c tnh v c ch tc dng ca enzym -amylase II.1.c tnh ca enzym -amylase

-amylase t cc ngun khc nhau c thnh phn amino acid khc nhau, mi loi -amylase c mt t hp amino acid c hiu ring. -amylase l mt protein giu tyrosine, tryptophan, acid glutamic v aspartic. Cc glutamic acid v aspartic acid chim khong tng lng amino acid cu thnh nn phn t enzyme: - -amylase c t methionine v c khong 7-10 gc cysteine.
-

Trng lng phn t ca -amylase nm mc: 45.000-50.000 Da ( Knir 1956;Fisher,Stein, 1960 ).

- Amylase d tan trong nc, trong dung dch mui v ru long.

-

Protein ca cc -amylase c tnh acid yu v c tnh cht ca globuline.

-im ng in nm trong vng pH=4,2-5,7 ( Bernfeld P, 1951 ). --amylase l mt metaloenzyme. Mi phn t -amylase u c cha 1-30 nguyn t gam Ca/mol, nhng khng t hn 1-6 nguyn t gam/mol Ca tham gia vo s hnh thnh v n nh cu trc bc 3 ca enzyme, duy tr hot ng ca enzyme ( Modolova, 1965 ). Do , Ca cn c vai tr duy tr s tn ti ca enzyme khi b tc ng bi cc tc nhn gy bin tnh v tc ng ca cc enzyme phn gii protein. Nu phn t -amylase b loi b ht Ca th n s hon ton b mt ht kh nng thy phn c cht. -amylase bn vi nhit hn cc enzyme khc. c tnh ny c l lin quan n hm lng Ca trong phn t v nng Mg2+. Tt c cc amylase u b kim hm bi cc kim loi nng nh Cu2+, Ag+, Hg2+. Mt s kim loi nh :Li+, Na+, Cr3+, Mn2+, Zn2+, Co2+, Sn2+, Cr3+, khng c nh hng my n -amylase. Thnh phn amino acid ca -amylase nm mc Aspergillus nh sau ( g/100 g protein ): alamine=6,8 ; glycine= 6,6; valine= 6,9, leucine= 8,3; Isoleucine= 5,2; prolin= 4,2, phenylalanine= 4,2; tyrosine=9,5; trytophan= 4,0; xetin= 6,5; trionin= 10,7, cystein + cystine= 1,6; glutamic acid= 6,9; amide= 1,5 ( Akabori et amiloza, 1954 ). Khng ging cc -amylase khc, amylase ca Asp.oryzae c cha phn phi protein l polysaccharide. Polyose ny bao gm 8 mol maltose, 1 mol glucose, 2 mol hexozamin trn 1 mol enzyme ( Akabori et amiloza, 1965 ). Vai tr ca polyose ny vn cha r, song bit c rng n khng tham gia vo thnh phn ca trung tm hot ng v nm pha trong phn t enzyme.

-amylase ca nm mc hu nh ch tn cng nhng ht tinh bt b thng tn. Sn phm cui cng ca thy phn amylase l glucose v maltose. i vi nm si t l l 1:3,79 ( Hanrahan, Caldwell, 1953 ) . Fenikxova v Eromsina (1991) cho bit rng cc maltopentose v maltohexose b thy phn theo s sau:

G5 G4+G1; G6 G2 + G4 hay 2G3 ( chnh ) hoc G5 + G1 ( t )

-amylase ca nm si khng tn cng lin kt -1,6 glucoside ca amylopectin, nn khi thy phn n s to thnh cc dextrin ti hn phn nhnh. y l mt cu trc phn t tinh bt do enzym -amylase phn ct to thnh dextrin ti hn phn nhnh.

Sn phm thy phn cui cng ca tinh bt di tc dng ca amylase nm si ch yu l maltose, th n l maltotriose. Khi dng nng amylase VSV tng i ln c th chuyn ha 70-85% tinh bt thnh ng ln men. Cn cc -amylase ca nm mc th mc ng ha n glucose v maltose c th ln ti 84-87%.

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 si l 4,0-4,8 ( c th hot ng tt trong vng pH t 4,5-5,8 ). Theo s liu ca Liphis, pH ti thch cho hot ng dextrin ha v ng ha ca ch phm amylase t Asp.oryzae trong vng 5,6-6,2. Cn theo s liu ca Fenixova th pH ti thch cho hot ng dextrin ha ca n l 6,0-7,0. bn i vi tc dng ca acid cng khc khc nhau. -amylase ca Asp.oryzae bn vng i vi acid tt hn l -amylase ca malt v vi khun Bac.subtilis. pH= 3,6 v 0oC, -amylase ca malt b v hot hon ton sau 15-30 pht; -amylase vi khun b bt hot n 50%, trong khi hot lc ca -amylase ca nm si hnh nh khng gim bao nhiu ( Fenilxova, Rmoshinoi 1989 ). Trong dung dch -amylase nm si bo qun tt pH= 5,0-5,5; -amylase dextrin ha ca nm si en c th chu c pH t 2,52,8. 0oC v pH= 2,5, n ch b bt hot hon ton sau 1 gi. Nhit ti thch cho hot ng xc tc ca -amylase t cc ngun khc nhau cng khng ng nht, -amylase ca nm si rt nhy cm i vi tc ng nhit. . Nhit ti thch ca n l 50 0C v b v hot 70OC( Kozmina, 1991 ). Trong dung dch m pH= 4,7, -amylase ca Asp.oryzae rt nhy vi tc ng ca nhit cao, thm ch 400C trong 3 gi hot lc dextrin ha ca n ch cn 22-29%, hot lc ng ha cn 27-85%. 500C trong 2 gi, -amylase ca nm si ny b v hot hon ton ( Miller v cng s ).

II.2. C ch tc dng ca enzym -amylase

-amylase ( 1,4--glucan-glucanhydrolase ). -amylase t cc ngun khc nhau c nhiu im rt ging nhau. -amylase c kh nng phn cch cc lin kt -1,4-glucoside nm pha bn trong phn t c cht ( tinh bt hoc glycogen ) mt cch ngu nhin, khng theo mt trt t no c. amylase khng ch thy phn h tinh bt m n thy phn c ht tinh bt nguyn song vi tc t rt chm. Qu trnh thy phn tinh bt bi -amylase l qu trnh a giai on.

+ giai on u ( giai on dextrin ha ):Ch mt s phn t c cht b thy phn to thnh mt lng ln dextrin phn t thp (-dextrin ), nht ca h tinh bt gim nhanh ( cc amylose v amylopectin u b dch ha nhanh ). + Sang giai on 2 ( giai on ng ha ): Cc dextrin phn t thp 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 disaccharide v monosaccharide. Di tc dng ca -amylase, amylose b phn gii kh nhanh thnh oligosaccharide gm 6-7 gc glucose( v vy, ngi ta cho rng -amylase lun phn ct amylose thnh tng on 6-7 gc glucopiranose 1 ). + Sau , cc poliglucose ny b phn cch tip tc to 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 amylose cha 13% glucose v 87% maltose. Tc dng ca -amylase ln amylopectin cng xy ra tng t nhng v khng phn ct c lin kt 1,6-glycoside ch mch nhnh trong phn t amylopectin nn d c chu tc dng lu th sn phm cui cng, ngoi cc ng ni trn ( 72% maltose v 19% glucose ) cn c dextrin phn t thp v isomaltose 8%. 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. Kh nng dextrin ha cao ca -amylase l tnh cht c trng ca n. V vy, ngi ta thng gi loi amylase ny l amylase dextrin ha hay amylase dch ha. Cc giai on ca qu trnh thy phn tinh bt ca -amylase: + Giai on dextrin ha: -amylase Tinh bt dextrin phn t lng thp + Giai on ng ha: Dextrin Amylase Maltose tetra v trimaltose oligosacharide maltotriose di & monosaccharide poliglucose maltotetrose

III.Lch s pht hin eznym

Vo u th k XIX, cc nh nghin cu tch c cc cht gy ra qu trnh ln men. Nm 1814 Kirchoff, vin s Saint Petercburg pht hin nc chit ca mm i mch c kh nng chuyn ha tinh bt thnh ng nhit thng. y l cng trnh u tin thu c ch phm dng dung dch v lch s enzyme hc thc s c xem nh bt u t y. Mi chn nm sau (nm 1833), hai nh khoa hc ngi Php l Payen v Pessoz chng minh cht c hot ng phn gii tinh bt thnh ng c th tch c dng bt. Th nghim c tin hnh bng cch cho etanol vo dch chit ca la i mch ny mm th thy xut hin kt ta. Kt ta c hnh thnh ny c kh nng chuyn ha tinh bt v nu un kt ta ny s mt tc dng chuyn ha. Danh t diastase (t ch Latinh diastasis - phn ct) l do Payen v Persoz dng gi enzyme lc by gi.

Phn II: Phn lp, bo qun ging VSV Chng nm mc Aspergillus Oryzae

Qu trnh ln men l hot ng sng ca t bo VSV trong mi trng. Qu trnh ny xy ra iu kin t nhin v qu trnh sn xut cng nghip. Bn cht ca 2 qu trnh ny c th ni l nh nhau nhng v mt hnh thc v phng din th khc nhau hon ton. Qu trnh ln men trong iu kin t nhin l mt quy lut sng cn ca VSV. T tham gia tng hp nn cht sng t vt liu ln men c trong t nhin. Cc VSV v vt cht s dng trong qu trnh ln men t nhin rt phc

tp, khng ng u v chng loi v v s lng. Mt khc, qu trnh ln men ny khng c kim sot, bao gm nhiu pha v khng nh hng. Sn phm ln men l a dng, khng n nh; do d, cht lng sn phm km khng ng nht. Qu trnh ln men trong sn xut cng nghip, tt c cc khu v phn lp ging VSV, c cht, nhit , pH, m cc qu trnh phn ng sinh hc, qu trnh thu nhn, tinh sch sn phm u c kim sot hon ton ( Sn phm to ra mang tnh nh hng r rng ngay t lc u khu chn ging VSV cho n cui qu trnh thu nhn sn phm ) . Do cht lng sn phm c ci thin . I.Vai tr ca ging trong cng ngh enzyme

Trong cng ngh enzyme t VSV, ging ng vai tr quyt nh: - Ging VSV quyt nh n nng sut enzyme ca nh my - Ging VSV quyt nh n cht lng sn phm sinh hc ( hay l hot tnh enzyme) - Ging VSV quyt nh vn u t cho sn xut - V cui cng l ging VSV quyt nh n gi thnh sn phm Nh vy, ging VSV c ngha to ln trong pht trin cng ngh VSV II.Yu cu ging VSV trong cng nghip enzyme

Cng ngh sn xut enzyme thuc nhm cng ngh ln men hin i v c sn xut theo quy m cng nghip. Do , ging VSV ng dng trong cng ngh enzyme cn phi c nhng yu cu v nhng chun mc nht nh. l: Ging VSV phi cho ra sn phm m ta mong mun. Sn phm ny phi c s lng v cht lng cao hn cc sn phm ph khc. V trong qu trnh trao i cht, chuyn ha mt khi lng sinh cht khng l ln gp hng nghn ln c th mnh trong mt khong thi gian cc k ngn th c th VSV cn tng hp nhiu cht. Do , sn phm to ra s cha nhiu loi
-

khc. Chnh v th, ging VSV dng trong sn xut mt sn phm no , th sn phm ny phi tri hn cc sn phm khc c v s lng v cht lng.
-

Ging phi cho nng sut sinh hc cao.

Ging VSV phi c kh nng thch nghi nhanh v pht trin mnh trong iu kin sn xut cng nghip. Ging VSV phi c kh nng ng ha cc nguyn liu r tin v d kim ti a phng ni nh my ang hot ng. Ging s dng trong cc qu trnh sn xut hin i phi l nhng VSV thun khit, c tc sinh sn nhanh. Tc trao i cht mnh to nhanh sn phm mong mun; d dng tch sn phm ra khi cc tp cht mi trng v sinh khi VSV ging.
-

Ging phi n nh trong bo qun v d dng bo qun.

to thun li nht v chng ging VSV cung cp cho qu trnh ln

men cng nghip, ta cn tin hnh phn lp ging VSV thun khit. III. Gii thiu v chng nm mc Aspergillus oryzae

Condium of Aspergillus oryzae

Asp. oryzae l mt loi nm vi th thuc b Plectascales, lp Ascomycetes ( nang khun ). C th sinh trng ca n l mt h si bao gm nhng si rt mnh, chiu ngang 5-7 m, phn nhnh rt nhiu v c vch ngang , chia si thnh nhiu bao t bo ( nm a bo ). T nhng si nm ngang ny hnh thnh nhng si ng thng gi l cung nh bo t, c c quan sinh sn v tnh. Cung nh bo t ca Asp.oryzae thng di 1-2 mm nn c th nhn thy bng mt thng. Pha u cung nh bo t phng ln gi l bng. T bng ny phn chia thnh nhng t bo nh, thun, di, gi l nhng t bo hnh chai. u cc t bo hnh chai phn chia thnh nhng bo t nh vo nhau, nn gi l nh bo t. nh bo t ca Asp.oryzae c mu vng lc hay mu vng hoa cau c im ca ging Asp.oryzae giu cc enzyme thy phn ni bo v ngoi bo ( amylase, protease, pectinasa, ), ta rt hay gp chng cc kho nguyn liu, trong cc thng cha ng bt, go ht nhng khng c ra sch, cn b bia, b ru, li ng, b sn Chng mc v pht trin c khi thnh lp mc, c mu sc en ,vng Mu do cc bo t gi c mu sc. Cc bo t ny, d b gi cun bay xa v ri vo u khi gp iu kin thun li s mc thnh mc mi.

IV.Qa trnh phn lp ging VSV

VSV phn b rt rng trong t nhin t ni c a hnh bnh thng n ni c a th phc tp, u u cng c mt VSV. nhng ni giu cht hu c, nhng ni ngho cht hu c, trong khng kh, trn b mt cc vt, trong c th ngi, ng vt, ni c nhit rt thp v hin din c ni c nhit cao. VSV c kh nng thch nghi trong mi hon cnh mi trng. Chnh nh kh nng tuyt vi ny m VSV c kh nng tn ti ngay c trong hon cnh khc nghit nht. Thng thng phn lp mt ging chng VSV thu nhn enzyme th c 3 cch phn lp:

+ Phn lp ging trong iu kin t nhin + Phn lp ging trong iu kin sn xut + Phn lp ging trong mu ging h hng Ty thuc vo kh nng v nhng iu kin thc t m ta chn cch phn lp cho ph hp nht. Mi cch phn lp trn u cho thy nhng u im ring bit. Sau y l 1 s u im: IV.1.Phn lp ging trong iu kin t nhin

-Trong iu kin t nhin, VSV c th tn ti v thch nghi nhanh c th cn phi c kh nng sinh tng hp tht nhiu loi enzyme chuyn ha nhanh c cht c trong mi trng thnh vt cht cung cp cho t bo. iu ny th khng thch hp cho vic sinh tng hp enzyme ( quy m sn xut cng nghip ) vi mt loi enzyme tht s mnh. -Do pht trin trong iu kin t nhin, VSV phi cng lc i ph vi hng lot cc yu t ngoi cnh v phi phn gii rt nhiu loi c cht khc nn VSV bt buc phi tng hp nhiu loi enzyme vi mt n lc rt ln. - iu kin t nhin v trong iu kin sn xut cng nghip th c s khc bit ng k cc ging VSV c kh nng sinh tng hp enzyme trong iu kin t nhin, c gi chung l cc chng VSV hoang di. Chng qu quen thuc vi s thay i tht thng ca iu kin t nhin. Khi chng ta a chng vo iu kin sn xut cng nghip vi nhiu iu kin mi trng c nh, i hi cc loi VSV ging phi c mt thi gian thch nghi vi iu kin sn xut cng nghip. Hun luyn chng thch nghi vi iu kin sn xut cng nghip l iu rt cn thit. -Cc loi VSV c kh nng sinh tng hp mt loi enzyme no thng tp trung vng mi trng cha nhiu c cht tng ng. Da v c im ny chng ta c th d dng xc nh v tr cn phn lp loi VSV sinh tng hp enzyme m ta cn. V d: Nu ta mun phn lp VSV c kh nng sinh tng hp protease cao, ta phi tm ni c cha nhiu protein trong t nhin, cn nu mun phn lp VSV c kh nng sinh tng hp amylase ta cn phi tm ni c ch nhiu tinh bt trong t nhin.

-Trong qu trnh sinh sn v pht trin, cnh tranh gia cc loi VSV, VSV trong iu kin t nhin lun xy ra nhng thng bin v t bin. Nhng t bin thng cho ra hai hiu ng: Th nht gy cho c th cht; th hai l nhiu t bin to ra loi mi c kh nng sinh tng hp enzyme rt cao. Vic tm ra nhng t bin kiu ny th ht sc c ngha v rt cn tin hnh. V mt li im na l, nhng t bin c li kiu ny thng rt bn vng. Rt thch hp a vo sn xut qui m cng nghip. IV.2. Phn lp ging trong iu kin sn xut -Cc ging c phn lp trong iu kin sn xut thng thch nghi vi iu kin sn xut. Nh , sau khi phn lp, cc ging ny khng cn qua giai on sn xut th, th nghim. -Cc ging c phn lp trong iu kin sn xut thng l nhng ging c chn lc hoc qua qu trnh bin i gen v c nhng c im sinh ho hn hn cc ging vi sinh vt hoang di. -Mt t bo vi sinh vt trong iu kin sn xut (trong dch ln men, dch nc thi, cht thi ca qu trnh ln men) thng rt cao. Do , kh nng thu nhn c nhng chng c bn nng sinh tng hp cao thng rt cao. IV.3. Phn lp ging trong mu ging h hng

Cc ng ging c th b nhim do qu trnh bo qun. Do b nhim, c th rt nhiu t bo VSV ging b thoi ho, nhng cng cn nhiu t bo khng b thoi ho. Vic phn lp li t ngun ging ny nhiu khi li t c nhng kt qu tt.

V.Gii thiu phng php phn lp nm mc Aspergillus Oryzae

Trong t c nhiu loi vi sinh vt c kh nng sinh tng hp enzyme amylase. nm mc ngun c cht thch hp cho qu trnh sinh tng hp enzyme amylase ny l tinh bt. Chng ta c th phn lp t t, thc n hay c th mua trc tip t cung cp nm mc ging. u im ca vic ny l ging mua th thi gian bo qun v hiu sut cht lng ging c m bo chc chn. Tuy nhin, qu trnh phn lp ging ny c th cho nhng kt qu y th v v c ngha trong vic b sung mt chng ging mi ti phng th nghim. Qu trnh ly mu: C th ly mu t t m khong 100g hay khoai ty ct lt em chn xung t. Sau khong 8 ngy, o h ly nhng ming khoai ty ra, r sch ct t bm trn b mt ming khoai ty. Sau cho vo bao nyclon v mang v phng th nghim. Tin hnh th nghim phn lp: Nghin mu i vi nhng mu khoai ty Ly 10g mu t hoc mu khoai ty ( lm nhuyn) cho vo 90ml nc ct v trng sau o trn mu. Dng pipet ht 10ml t dung dch mu ban u chuyn sang 1 ng nghim khc c cha 90ml nc ct v trng. Tip tc pha long mu nng 10-3, 10-4 nhng ng nghim tip theo. Ht 0,1ml cho vo a petri c cha mi trng dinh dng chn lc cho nm mc pht trin,mi trng PDA (Potato Dextro Agar).

Dng que tri, tri u phn dinh dng trn b mt mi trng em nhit phng trong vng 3 ngy. Mc s s dng ngun tinh bt lm c cht, nn nhng ch ny s xut hin nhng qung sng xung quanh khun lc. Nhn bit bng cch b sung Iodine vo trong mi trng trc khi cy. C tc dng l cht ch th cho tinh bt. Cy truyn nhng mc c trng trong mi trng PDA trong ng thch nghing vi 1% tinh bt cho php mc pht trin trong 72 gi sau c th d tr trong my lm ng. -Nhn ging vi sinh vt bnh tam gic (qui m nh). 10ml H2O ct v trng vo ng thch nghing c cha bo t nm. Lc u cho bo t ho trn vo mi trng n khi to dung dch huyn ph. Ht 0,1ml dch huyn ph c cha bo t nm cho vo bnh tam gic c cha mi trng sinh trng ca nm. KH2PO4 NH4NO3 6,5 KCl 0,5 h tinh bt 20 1,4 10 MgSO4.7H2O FeSO 4.7H2O 0,1 0,01 PH =

Phn phi khong 30 40ml mi trng vo erlen 50ml em kh trng bi Autoclave 1210C trong 15 pht sau ngui n nhit phng. Sau em 25 300C trong vng 72 gi trn my lc 200 vng/pht. Sau khi nhn ging thnh cng c th s dng ngay hoc em i bo qun v d tr,

 c hiu qu cao cho nm mc ging ta cn c nhng phng php bo qun thch hp.

VI. Phng php bo qun ging vsv

Mc ch ca bo qun ging VSV dng trong sn xut enzym l m bo tnh n nh trong qu trnh tng hp enzym v tnh n nh ca hot tnh enzym. C cc phng php thc hin qu trnh ny nh sau: VI.1. Cy truyn v bo qun lnh Phng php da trn nguyn tt l VSV s hn ch qu trnh trao i cht trong iu kin lnh mt khong thi gian nht nh. Trong thi gian ny VSV c kh nng bo tn c kh nng sinh tng hp enzym. Cch thc hin: ng ging VSV c cy truyn vo 3-5 ng nghim c mi trng ti thiu. Trong mt ng dng kim tra, mt ng dng cho sn xut hoc nghin cu v mt ng dng bo qun.C th lm thm hai ng trnh sai st do thao tc i vi nhng ngi mi bt u lm cng tc bo qun ging. Sau khi cy truyn, ng ging cn c bo qun iu kin nhit lnh t 4-70C. Sau thi gian nh k, s phi cy truyn tr li, thao tc ny c thc hin lin tc. VI.2.Bo qun ging trong t hoc trong ct Phng php da trn nguyn tt: Trong mi trng ti thiu c m thp, vsv c bo t c th bo tn kh nng sinh tng hp enzym trong thi gian di. Phng php ny rt ph hp v c hiu qu i vi nm mc Asp. oryzae. Trc khi s dng , t, ct phi c lm sch v sy n m < 5%. Asp. oryzae c nui cho n khi to bo t. Ngi ta trn bo t vi t hoc ct c lm sch v sy kh. Sau hn hp ny cho vo bao, hn kn v bo qun nhit thng.

VI.3.Bo qun ging trong ht ng cc

Phng php da trn nguyn tt bo t nm mc c gi trong ht ng cc x l nhit c m < 8% v gi c kh nng sinh tng hp enzym trong mt thi gian di. Ngi ta thc hin bo qun ging trong ht ng cc nh sau: Ging ng nm mc c nui trong mi trng ht ng cc cho n khi to nhiu bo t. Bo t cng thnh phn mi trng c sy kh nhit < 50 oC cho n khi m < 8%, a vo bao, hn kn v bo qun nhit thng.

Phn III: Cng ngh ln men to enzym -amylase

I. Gii thiu v phng php ln men b mt

Thit k cng ngh: Cui th k 19 u th k 20, vic nui cy VSV thng c thc hin theo phng php b mt. Phng php ny c pht trin rt rng ri, khng ch thu nhn ch phm enzyme m trc tin l phng php thu nhn khng sinh v mt s qu trnh ln men truyn thng. I.1.u v nhc im ca phng php nui cy b mt

Phng php nui cy b mt l phng php to iu kin cho VSV pht trin trn b mt mi trng. Nhng u im ca phng php nui cy b mt l:
-

Nui cy b mt rt d thc hin. Quy trnh cng ngh thng khng phc tp.

Lng enzyme c to thnh t nui cy b mt thng cao hn rt nhiu so vi nui cy chm. y l c im u vit rt quan trng trong gii thch ti sao phng php nui cy b mt hin nay pht trin mnh tr li. Ch phm enzyme th ( bao gm thnh phn mi trng sinh khi VSV, enzyme v nc ). Sau khi thu nhn rt d sy kh v d bo qun. Nui cy b mt khng cn s dng nhiu thit b phc tp, do vic vn hnh cng ngh cng nh vic u t va n gin va khng tn km. Trong trng hp b nhim cc VSV l, ta rt d dng x l. Mi trng c l mi trng tnh, khng c s xo trn nn khu vc no b nhim ta ch cn loi b khu vc khi ton b khi nui cy. Nhng khu vc khc s hon ton c an ton.

Phng php nui cy b mt cng c nhng u im cn quan tm khc phc v hon thin dn phng php ny. Nhc im ln nht v d nhn thy nht l: Phng php ny tn kh ln din tch cho nui cy. Trong phng php ny VSV pht trin trn b mt mi trng ( mi trng lng hoc mi trng bn rn) nn rt cn nhiu din tch.

I.1.1.Mi trng lng mi trn lng, VSV s pht trin trn b mt mi trng, to thnh khun lc ngn cch pha lng ( mi trng ) v pha kh ( khng kh ). y, VSV s s dng cht dinh dng t dung dch mi trng, O2 t khng kh, tin hnh qu trnh tng hp enzyme. Enzyme ngoi bo s c tch ra t sinh khi v ha tan vo dung dch mi trng. Enzyme ni bo s nm trong sinh khi VSV.

Nui cy b mt trn mi trng lng trong cc khay:

Nui cy VSV thu nhn enzyme trn mi trng lng theo phng php cy b mt thng c tin hnh trong cc khay c chiu cao khong 12-15 cm, chiu rng v chiu di c thit k ty theo kch thc phng nui sao cho thun tin trong thao tc. y ngi ta quan tm nhiu n chiu cao mi trng lng. Nu chiu cao mi trng lng qu ln, VSV s khng c kh nng ng ha ht cc cht dinh dng pha y khay nui cy. Nu chiu cao mi trng nh, s thiu thnh phn cht dinh dng, hiu sut thu nhn enzyme s khng cao. Trong nghiu nh my, ngi ta thng to mi trng trong khy nui cy c chiu cao mi trng t 5-7 cm l hp l.

I.1.2.Mi trng c

Phn ln cc nh my sn xut enzyme, khi nui cy VSV thu nhn enzyme, ngi ta thng s dng mi trng c . tng kh nng xm nhp ca khng kh vo trong lng mi trng, ngi ta thng s dng cm, tru, ht ng cc lm mi trng. Trong trng hp ny, VSV pht trin trn b mt mi trng, nhn cht dinh dng t ht mi trng v sinh tng hp ra enzyme ni bo v ngoi bo. Cc enzyme ngoi bo s thm thu vo trong cc ht mi trng, cn cc enzyme ni bo nm trong sinh khi VSV. VSV khng ch ph trin trn b mt mi trng, ni ngn cch pha rn ( mi trng ) v pha kh ( khng kh ) m cn pht trin trn b mt ca cc ht mi trng nm hn trong lng mi trng. Mi trng nui cy va c xp cao v va phi c m thch hp. Nu m qu cao s lm bt mi trng li , khng kh khng th xm nhp vo trong lng mi trng, nu c m thp qu s khng thun li cho VSV pht trin. Thng thng ngi ta thng to m khong 55-65% W l hp l.

Nu s dng cm lm nguyn liu chnh nui cu VSV thu nhn enzyme, ngi ta phi cho thm 20-25% tru lm xp mi trng, to iu kin thun li khng kh d xm nhp vo lng mi trng. Phng php nui cy b mt bn rn(mi trng c ) ny rt thch hp cho len men nm mc Asp.oryzae.

I.2.Qui trnh sn xut nm mc ging I.2.1Nguyn liu cho sn xut mc ging

Go np:Nc 14% ,Glucid 79,4%, Lipid 1,5% v protein 8,2% trong Protein ca go np ch yu l glutein(oryzaine)v glubuline ngoi ra cn mt t ln Cozine v Prolamin. Glucid ca go np ch yu l tinh bt, ng, cellulose v hemicellulose.Trong tinh bt ch yu l amylopectin.Ngoi ra cn cha mt s vitamin nh B1,B2,B6,PP v E. Go t:Nc 13,84%; Glucid 77,55%; Protein 7,35%; Lipid 0,52%; cellulose 0,18% v mui khong :0,54%. Bt M:Nc 11,6%; Glucid 72%; Lipid 4,8%,Protein 9%; cellulose 1,5% v mui khong 1,2%. Bp mnh:Nc 11,4%; Glucid 78,9%; Lipid0,8%; Protein8,5%,cellulose 0,4% v mui khong 0,4%. I.2.2.Chun b mc ging

Nui cy ging bao gm: -Trong ng thch nghing hay gi ging trong ng nghim -Trong bnh tam gic (nhn ging nh) -Trn sng, khay (nhn ging ln)

I.2.2.1.Nui cy trong ng thch nghing

Sau khi phn lp thnh cng trn mi trng chn lc t a petri cy chuyn nhng mc si sang (hay ly nm mc t ng ging) ng thch khc.Yu cu ng ging phi tuyt i m bo thun khit, khng c ln ln bt k mt loi VSV no khc.Mi trng thch nghing phi m bo y dinh dng.

I.2.2.2.Nui cy ging trong bnh tam gic

Cch lm mi trng trong bnh tam gic: Mi trng go:go t loi tt, nu cm nh nu bnh thng, ht cm chn u khng qa nho v khng qa kh. m khong 45% ngui bp ri thnh tng ht cho vo bnh tam gic thnh lp dy khong 1cm. y nt bnh tam gic bng giy chng m. Hp thanh trng P = 1atm trong vng 30-45 pht. Mi trng ng mnh: Ng mnh c kch thc khong 0,2-0,5 mm cho nc vo theo t l 90% trng lng so vi ng, trn u trong khay 1-2 gi cho ngm nc u. Bp ti cho vo bnh tam gic khc thnh lp dy 1cm. y nt bnh bng giy chng m. Hp thanh trng ng thi lm chn P = 1atm, 1200C, trong thi gian 60 pht, ly ra ngui lc cho khi vn cc. Mi trng cm: Chn cm tt, mi nhng loi th khng cn mn ht, sau lm tng t nh i vi ng mnh. Thng s dng cc bnh tam gic dung tch 0,3-0,5 lt hay 1 lt c c rng. Sau khi chun b mi ttrng trong bnh thu tinh ta tin hnh nui cy nm mc. Trc tin cn phi chun b ly 5ml nc v trng cho vo cc ng nghim. Sau , nc v trng cho bo t ho vo trong nc ng

thi cy chuyn chng sang bnh tam gic. Trung bnh c mt ng nghim c th cy chuyn sang 2-3 bnh tam gic c dung tch 1 lt, lc cho ging phn b u trong mi trng v tin hnh nui cy chng trong iu kin thch ng. Thng nui khong 5-6 ngy l c. Yu cu c bn trong giai on ny l lm sao to c nhiu bo t mnh kho. Trng hp no thy bnh b nhim th phi loi b ngay. I.2.2.3.Nui cy mc trn khay

Chun b mi trng lm mc trn khay: Nguyn liu thng dng l ng mnh c kch thc 0,2-0,5mm. Trn vi nc theo t l 80-90% trng lng so vi ng nu hp di p lc cao. Trn xong khong 3-4 gi cho ng ngm nc u ri hp chn. Thi gian hp khong 3-4 gi, di hn so vi thi gian hp cm hay go. Nguyn liu sau khi hp phi chn u, khng c qu bt hoc qu kh. m cn li 45-50% l va. Cch gieo cy v nui mc trn khay: Nguyn liu d ra, lm ngui nhanh. Nu t c th bp bng tay cho ti ra, nu nhiu cho qua my nh ti v dng qut thi. Sau khi lm ngui n 26-280C th trn nc ging t bnh tam gic vo vi t l 0,5-1% hoc vi t l cao hn. Sau khi trn ging, mi trng vo khay thnh lung cao (0,3m). t nhit 30-320C, m 85-100%. Thi gian 6-8 gi, nhit khi mi trng ln ti 34-360C v bo t ny mm gn ht nhng cha thnh si di. Lc ny cn ri mi trng thnh lp mng 2-3cm cn gi cho nhit mi trng khng vt qu 360C. Lng nhit ta ra nhiu nht khong 1014 gi sau khi trn ging. Sau khi nui 34-36 gi, nhit khi mi trng bt u gim, cn phi iu chnh nhit ln 34-350C, duy tr s hnh thnh bo t ca nm mc.

Thi gian nui mc ging trn khay thng vo khong 60 gi. Nu thy hnh thnh bo t chm th c th ko di n 70-72 gi. Mc ging khi ly ra thng c m 32-35 % c th dng ngay lm ging cho cng on sn xut. Nu khng dng ngay th phi em sy kh n m 8%, gi dng dn hoc l cung cp ging cho cc ni sn xut. Nhit phng sy khng c qu 400C. Cc bao mc ging cn c bo qun ni thong, mt ( c th bo qun lnh 4-50C ) trnh nh nng. Thi gian bo qun ty iu kin c th, t 1-2 thng hoc lu hn.

I.2.3.Qui trnh sn xut

Chun b mi trng thch nghing

ng mc ging

Cy chuyn Chun b mi trng trong bnh tam gic Nui cy 30-32 oC trong 5-6 ngy Mc ging trong bnh tam gic Mc ging cho sn xut Ng mnh hp thanh trng Lm ti

Nui cy 30-32oC trong 5-6 ngy Nc v trng Ging trong ng thch nghing Trn u bo t

Trn ging 0,5 10%

Nui mc 60 h Sy kh Bao gi

II. Qui trnh ln men cng nghip to enzym -amylase II.1. Nguyn liu

Ngun tinh bt: nc ta ngun nguyn liu cha tinh bt th v cng a dng v phong ph.V d tinh bt t go ( go t, go np), ng (bp), sn, Nhng ph hp cho sn xut enzym amylase qui m cng

nghip th cn phi tnh n chi ph cho gi thnh sn phm. Do vy, ngun nguyn liu cn c quan tm v ch trng v mc r tin v d kim. y l mt li th rt quan trng cho nh sn xut v cho c ngi tiu dng c gi thnh thp v c sn xut i tr.Sau y, gii thiu mt s ngun nguyn liu d tm:

+Thnh phn dinh dng ca ht go: Thnh phn dinh dng Protein Tinh bt Lipid (tng s) Cellulse Nc Nng lng n v tnh g g g g g kcal Hm lng/100g 6 82 0,8 0,6 10,2 361

(Vin Nghin cu dinh dng thuc i hc Mahidol, Thi Lan,1999)

- Protein ca go chim khong 8-9%, ch yu l glutelin v glubuline. Ngoi ra cn cln t : cozine v prolamin. Lng protein thoi ho v bin tnh dn trong qu trnh bo qun. - Glucid th ch yu l tinh bt, ng, cellulose hemicellulose.Trong tinh bt ch yu l amylopectin, c cha mt t cht khong nh: P,K,Mg, Ngoi ra cn c mt s Vitamin:B1, B2, B6, PP, H, C, E v carotenoid.

+Thnh phn dinh dng ca ng: Thnh phn dinh dng /100g Protein Tinh bt Lipid(tng s) Cellulose Nc Bp np 9,1 72,8 2,2 1,8 11,2 Bp ngt 12,9 69,3 3,9 2,9 9,5

Khong

2,9

1,5

(Cortez v Wild-Altamirano,1972) Nguyn liu s dng trong nui cy b mt thng l nhng nguyn liu c ngun gc t nhin nh cm m, cm go, go, ng mnh, u nnh v cc loi ht ng cc khc.Trong cc loi nguyn liu trn, cm go, cm m c s dng nhiu hn c. Hai loi ny c y cc cht dinh dng cn thit cho VSV pht trin. Mt khc khi to mi trng, chng thng c tnh cht vt l rt thch hp va m bo khi kt dnh cn thit, va m bo lng khng kh lu chuyn trong khi nguyn liu. Trong nhiu trng hp, to kh nng thong kh tt hn, ngi ta thng cho thm tru vi t l thch hp cho tng loi enzym c to ra t VSV. Thc cht cho tru vo l lm tng xp ca mi trng, to nn nhng khong trng khng kh c th lu thng trong lng mi trng. Chnh v th ta thy rng nm mc Asp. oryzae khng ch pht trin trn b mt mi trng m cn pht trin rt mnh trn b mt ht mi trng. Hay ni cch khc, chng nm mc Asp.oryzae c kh nng pht trin gia hai pha rn v pha kh ca mi trng.Trong trng hp ny, n c kh nng pht trin hn trong lng mi trng nhng n vn hon ton mang ngha ca qu trnh ln men b mt.

+Thnh phn dinh dng ca sn(khoai m): Sn l loi c cha nhiu tinh bt, rt thch hp l ngun c cht cm ng cho qu trnh tng hp enzym amylase nm mc Asp.oryzae. C sn gm ba phn chnh: v, tht c v li. Ngoi ra cn c cung v r c. -V gm hai phn: V g bn ngoi, cu to ch yu l cellulose, thng chim khong 1,5-2% khi lng c, v ci cng cu to t cellulose nhng trong v ci cn c m sn l cc polyphenol, chim ti 85-90% polyphenol ca c sn . -Tht cha nhiu tinh bt, t protein v mt lng du, lng polyphenol y c khong 10-15%, nhng cc polyphenol gy tr ngi khi ch bin, t bit l sn chy m s lm cho bt sn bin mu, thay i

mi v kh n trc tip khi luc, kh thot nc khi sy hay phi kh sn lt hay sn bt. -Thnh phn ho hc ti ca sn: Tinh bt:20-34%; protein: 0,8-1,2%; Nguyn cellulose: 1-3,1%; cht tro: 0.54%; polyphenol: 0,1-0,3%; cht bo: 0,3-0,4%; liu v nc: 60-74,2% .
X l nguyn liu

II.2. Qui trnh cng ngh

Hp thanh trng

Ging VSV

Lm ngui Trn ging VSV

Nhn ging

Ging cho sn xut

Nui cy Thu nhn ph phm enzyme th Ch phm enzyme th em tinh ch Ch phm ezyme th em s dng

Nghin mn Trch ly

Lc

Dng trong chn nui

Kt ta enzyme Thu nhn kt ta

Cn hoc Sulfat amon

Sy k ta Tinh ch kt ta Thu nhn ch phm enzyme tinh khit

S dng ch phm ezyme tinh sch

Enzyme tinh khit

Thuyt minh qui trnh: Nguyn liu: Trong phng php ln men b mt, cu trc chnh l cm m, cm go. Hai loi ny l nguyn liu hon ho v c th l mt cu t duy nht ca mi trng nui nm khng cn b xung thm cht khc na. Tuy nhin do l ph liu ca cng ngh xay xt, nhng lm cng tng i t tin. Hn na trong qu trnh nui nm, cc cht dinh dng khng c s dng ht, v th c th thay th hoc pha trn thm mt s cu t r tin hn. Mt khc cu t b sung vo (tru, mt ca,) lm tng xp gip cho h si nm pht trin tt hn tn dng ti a ngun c cht sinh ra hot lc enzyme cao nht. c bit ch , cm khng c cha tinh bt di 20-30%. Nn dng cm tt, cm mi khng c v chua hay ng, khng hi mi mc. m ca cm khng qu 15%, tp cht c khng qu 0,05%. Hp thanh trng: Cm v cc cht ph gia cha nhiu bo t VSV khc nn cn phi thanh trng m bo chng nui pht trin bnh thng v canh trng sn xut khng cha VSV ngoi lai, cn thanh trng di p sut hi 1-1,5 atm trong thi gian 45-60 pht. thun li cho vic thanh trng c hiu qu, cng nh to iu kin cho nm si pht trin, trc khi thanh trng, ngi ta thng dng HCl hay H2SO4 iu chnh pH mi trng. Sau khi thanh trng mi trng c lm ngui ngi ta bt u nui cy thu nhn enzyme.

Trn ging vi sinh vt: Sau khi lm ngui, tin hnh cy ging hoc rc bo t vo mi trng thanh trng, thnh ng vi gi, ging mc c th nui cy ring phng th nghim hay ti cc phn xng nhn ging hoc l ging thng phm c bn ni cung cp ging. Cc loi ging ny thng cha nhiu bo t. Khi cy vo mi trng dinh dng, chng s pht trin thnh t bo nm mcv to ra cc loi enzyme m ta mong mun.T l ging a vo nui cy thng vo khong 0,5-20% so vi khi lng ca mi trng. K thut nui cy: Sau khi trn ging, mi trng c tri u ra cc khay vi chiu di 2-3cm, ri c a vo phng nui cy, t trn nhng gi . Cc gi ny c thit k sao cho lng khng kh c lu thng thng xuyn. Phng nui cy phi c h thng iu chnh nhit v m khng kh.Nhit thch hp cho nm si pht trin l 28-320C. Nhit thp qu hoc cao qu u nh hng khng tt cho nm si pht trin. Trong qu trnh nui cy, ta hon ton khng cn iu chnh pH. Mi trng bn rn l mi trng tnh nn s thay i pH mt vng no t khi nh hng n ton b khi mi trng. Thi gian nui nm si thu nhn enzyme vo khong 36-60 gi. iu ny cn ph thuc vo chng nm mc Asp.oryzae v iu kin mi trng cng nh ph thuc vo iu kin nui cy. Qu trnh pht trin ca nm mc trong mi trng bn rn khi nui bng phng php b mt ny tri qua cc giai on sau: Giai on 1: Giai on ny ko di 10-14 gi k t thi gian bt u nui cy. giai on ny c nhng thay i sau: -Nhit tng rt chm. -Si nm bt u hnh thnh v c mu trng hoc mu sa.

-Thnh phn dinh dng bt u c s thay i. -Khi mi trng cn ri rc. -Enzyme mi bt u c hnh thnh. Trong giai on ny phi c bit quan tm n ch nhit . Tuyt i khng c a nhit cao qu 300C v thi k u ny ging rt mn cm vi nhit . Giai on 2: Giai on ny ko di 14-18 gi. Trong giai on ny c nhng thay i c bn sau: Ton b bo t pht trin thnh si nm v si nm bt u pht trin rt mnh cc si nm ny to ra nhng mng si chng cht khp trong cc ht mi trng trong lng mi trng. -Trong giai on ny ta c th hon ton nhn r cc si nm c mu trng xm bng mt thng. -Mi trng c kt li kh cht - m mi trng gim dn -Nhit mi trng s tng nhanh c th ln ti 40-450C. -Cc cht dinh dng bt u gim nhanh do s ng ho mnh ca nm si. -Enzyme amylase c tng hp mnh. -Lng O2 trong khng kh gim v CO2 s tng dn, do trong giai on ny cn phi c thng kh mnh v nhit c gng duy tr trong khong 29-300C l tt nht. Giai on 3: Giai on ny ko di 10-20 gi. giai on ny c mt s thay i c bn nh sau: -Qu trnh trao i cht yu dn, do mc gim cht dinh dng s chm li.

-Nhit ca khi mi trng gim, do lm gim lng khng kh mi trng xung 20-25 th tch khng kh /th tch phng nui cy/ 1gi. Nhit d nui duy tr 300C,trong giai on ny, bo t c hnh thnh nhiu do lung enzym amylase to ra s gim xung . Chnh v th vic xc nh thi im cn thit thu nhn enzym rt cn thit.

Thu nhn sn phm: Kt thc qa trnh nui cy ta thu nhn c ch phm enzym amylase, ch phm ny c gi l ch phm enzym th (v ngoi thnh phn enzym ra, chng cn cha sinh khi VSV, thnh phn mi trng v nc trong mi trng). m bo cho ch phm enzym th -amylase khng b mt hot tnh nhanh ngi ta thng sy kh ch phm enzym n mt m thp ( thit b sy thng dng y l my sy chn khng- c th xem phn ph lc). m cn t c sau khi qa trnh sy kt thc l nh hn10% m. m bo hot tnh enzym khng thay i ngi ta thng sy nhit 38-400C. Enzym -amylase nm mc Asp.oryzae s b bt hot nu nhit ln n 60-70 C. Ty theo mc ch s dng ta c th dng ch phm th ny ngay khng cn phi qa trnh tinh sch. Trong nhng trng hp cn thit khc, ta phi tin hnh lm sch enzym. sn xut enzym tinh khit ngi ta ph tin hnh nh sau: -Ton b khi lng enzym th amylase c em i nghin nh.Mc ch ca qa trnh nghin l va ph v thnh t bo va lm nh cc thnh phn ca ch phm th.Khi thnh t bo c ph v ,cc enzym ni bo cha thot ra khi t bo s d dng thot khi t bo.Phn ln enzym amylase ngoi bo khi c tng hp v thot khi t bo ngay lp tc thm vo thnh phn mi trng.Khi ta nghin nh, enzym thot ra khi cc thnh phn ny d dng hn. -Trong khi nghin ngi ta thng s dng nhng cht tr nghin trong trng hp ny cdng l ct thch anh v bt thy tinh. Cc cht ny l nhng cht v c khng tham gia vo phn ng v kh nng tng mc

 ma st trc khi s dng ct thch anh v bt thy tinh phi c ra sch, sy kh nhit ln hn 100oC loi b nc v tiu dit VSV. Trch ly:Sau khi nghin mn, ngi ta cho nc vo trch ly enzym -amylase. Cc loi enzyme thy phn c kh nng tan trong nc nn ngi ta thng dng nc nh mt dung mi ha tan. C mt phn ch phm enzym th, ngi ta cho 4-5 phn nc, khuy nh v sau lc ly dch, phn b thu ring dng lm thc phm gia sc ( ch cn loi b ct thch anh v bt thy tinh ra khi hn hp b ri mi cho gia sc n). Dch thu nhn c vn dng ch phm enzym th v trong c cha nc, cc cht ha tan khc t khi mi trng nui cy. Vic tip theo l lm sao tch enzym ra khi vt cht ny. Qa trnh kt ta enzym -amylase: lm vic trn ngi ta tin hnh kt ta enzym nh nhng tc nhn gy ta.Trong cng ngh tinh ch enzym, ngi ta thng dng cn v sunfat amon. Hai tc nhn kt ta ny d tm kim v gi r so vi nhng tc nhn gy ta khc. Trong khi tin hnh kt ta, ngi ta phi lm lnh c dung dch enzym th v c nhng tc nhn kt ta trnh lm mt hot tnh enzym. Khi cht lm kt ta enzym vo dung dch enzym th phi ht sc t t trnh hin tng bin tnh. Trong qa trnh kt ta ngi ta dng cn hoc sulfat amon vi liu lng nh sau: C mt phn dung dch enzym th ngi ta cho 2 n 2,5 ln cn hoc sulfat amon. Khi cho cht kt ta vo dung dich enzym th, ngi ta tin hnh khuy nh, sau yn trong iu kin nhit lnh (thng t 4-7OC) theo thi gian, cc enzym s c to kt ta v lng xung y, ngi ta tin hnh gn v lc thu nhn kt ta dng paste ( m ln hn 70%W). trng thi ny enzym rt d b bin tnh v cn nhiu nc d bo qun ngi ta sy kt ta enzyn -amylase 40OC cho n khi m cui cng t 5-8% W ( thit b sy thng dng l my sy phun sng c th xem phn ph lc). Trong nhiu trng hp ch phm enzym -amylase dng kt ta vn hon ton cha sch v mt ha hc v trong cn cha 1 s enzym ngoi enzym amylase ta quan tm, chng hn enzym - amylase ny cn cha

hot tnh protase c tnh acid v cellulose do mun thu nhn c enzym c tinh khit cao hn ta phi tinh ch enzym -amylase kt ta bng cc qa trnh lc Gel hay s dng phng php c nh enzym ,

II.3. Thu nhn enzym -amylase t nm mc Aspergillus Oryzae II.3.1. Sinh trng v sinh tng hp amylase t nm Khi nui VSV to amylase c 2 qu trnh lin quan mt thit vi nhau. Qu trnh tng hp sinh khi VSV v qu trnh tch t enzyme trong t bo hoc ngoi mi trng. mt s VSV, qu trnh sinh tng hp amylase tin hnh song song vi qu trnh sinh trng, ngha l s tch t enzyme ph thuc tuyn tnh vo s tng khi. Trong trng hp ny, sinh tng hp enzyme amylase kt thc pha logarit cng ng thi vi s ngng sinh trng v s bt u pha pht trin n nh tip theo sau. Tuy nhin theo kin ca nhiu tc gi, s to thnh amylase cc i thng xy ra sau khi qun th t bo VSV t im sinh trng. Trong trng hp ny, sinh trng ca VSV hu nh khng km theo s tch ly enzyme amylase trong canh trng, ch sau khi kt thc pha sinh trng mi xy ra s tng hp enzyme cc ln.

II.3.2. Cc yu t nh hng n cht lng enzym II.3.2.1.nh hng cc ngun Nit dinh dng

Ngun dinh dng Nit ht sc quan trng. Khi chun b mi trng dinh dng nui nm mc Asp.oryzae to enzym -amylase, ngi ta dng mui v c Natri Nitrat l ngun Nit dinh dng nui nhiu loi nm si to -amylase. Hm lng thng s dng l 0,91%. Nari Nitrat v Amoni Nitrat c hiu qa hn so vi cc loi mui khc ( Kali Nitrat, Magie Nitrat, Amoni Sunfat.). Cho ngun Nit nht nh vo mi trng c th kch thch tng hp amylase ny v c ch tng hp amylase khc .V d:Kt qa th nghim

Fenikvova v cng tc vin v vic s dng cc ngun Nit t nhng mui khc nhau.

Ngun Nit NaNO3 NaNO3 (NH4)2SO2 NH4NO3 NH4NO3 NH4H2PO4

Liu lng mui, % theo N

0,30 0,15 0,15 0,15 0,30 0,30

Hot ng enzyme sau 6 ngy nui i vi 100ml -amylase Glucomylase 145 8000 26 3300 98 4100 45 4900 5,5 8000

(bng : nh hng ca ngun nit ti sinh tng hp cc enzyme amylase). ( Sch Cng ngh enzym ca Nguyn c Lng ch bin, 2004)

Ngoi ra cng cn phi xc nh t l gia ngun carbon v Nit trong mi trng ph hp cho mc Asp.oryzae pht trin. Tnh cn bng ca mi trng dinh dng v carbon v Nit c ngha ln i vi s sinh tng hp sinh khi ca nm Asp.oryzae v s to thnh amylase.

II.3.2.2.nh hng ca Amino acid

Amino acid l nhng cu t hp thnh phn t enzym, trong c mt s cc amino acid khng ng nht v gi tr dinh dng nn s dng hn hp amino acid s c gi tr ln hn cho nhng cht lng mi. Amino acid c nh hng tt ti sinh l ca nm mc cng nh vic sinh tng hp enzym amylase do nhng nguyn nhn sau:

-Amino acid c th ng thi va l ngun carbon , ngun Nit va l ngun nng lng. -Mt s amino acid ring l (Glutamic acid ,Aspatic acid.) ng vai tr v cng quan trng trong trao i amino acid c th l sinh tng hp nhiu amino acid khc trong qa trnh chuyn amine ha. Nh mt s amino acid trc ht l glutamic acid, aspatic acid m thc hin ng ha d dng CO2 (Araiet al,1968). -Nhiu amino acid tham gia vo thnh phn ca cc vitamin tan trong nc nh: Pantotenic acid, biotin, folic acid, cc vitamin ny c ngha sinh hc rt ln. Ngoi ra cn c nhng nhn xt cho rng s tng hp ARN trong t bo mt s VSV ph thuc vo s c mt ca amino acid trong mi trng nui ( Kurland, Marloe, 1962 ). -Amino acid c tnh c hiu ca s cm ng v kim ch sinh tng hp enzyme. Mt s amino acid kch thch v lm tng cng sinh tng hp cc enzyme amylase, mt s li c tc dng c ch ngc li v mt s khc th khng c nh hng g c. II.3.2.3.nh hng ca ngun khong dinh dng

Cc yu t a lng v vi lng c nh hng ln n sinh trng v tng hp cc enzym, -amylsae nm mc Asp.oryzae. -Mg2+ c nh hng ti bn nhit ca enzym. Thiu MgSO4 s c nh hng xu n s tng hp mi amylase bi nm si. Khi s tng hp -amylase b c ch hon ton (Fenikxova, Muxaeva, 1967). Nng ti u ca mui ny cho tng hp -amylase l 0,05%. -Phospho cn tng hp cc hp phn quan trng ca sinh cht (nucleic phospholipide acid) v nhiu coenzym, ng thi phosphoril ha glucide trong qu trnh oxy ha sinh hc. Phospho nh hng trc tip ti sinh sn ca nm si, do vy m tng cng tng hp enzym. Cc mui phosphate thng c dng vi nng cao ti 0.15M. Hot ca amylase tng cao khi c 0,1% KH2PO4. -Canxi(Ca) cn cho tng hp v n nh -amylase hot ng v n l cu t khng th thiu c ca enzym ny. Canxi cn c tc dng bo v

amylase khi tc ng ca protease ( Hsiu et amiloza, 1964). Mun tch ly nhiu -amylase cn c lng Ca trong mi trng l 0,01-0,05%. -Lu hunh(S) kch thch s to amylase ( S c trong thnh phn ca cc amino acid quan trng : methionine, cystein, cystine). Lu hunh vi hm lng 0.04g/ml mi trng l thch hp nht cho Asp.oryzae to amylase. Ngoi nhng nguyn t a lng ny cn ch thm vi nhng nguyn t vi lng. Chnng hn nh: Coban, km,v cc nguyn t gy c ch s tng hp enzym amylase nh: ng, thy ngn,

II.3.3. Cc yu t nh hng n qu trnh ln men

m(w) mi trng: Tt nht cho s hnh thnh enzym ca nm mc Asp.oryzae l 55-60%. m mi trng thch hp cho s hnh thnh bo t l khong 45% nn cn gi cho m mi trng khng b gim trong qu trnh pht trin. nh hng ca vic gi m trong qu trnh sinh trng ti s to -amylase ca Asp. oryzae nui bng phng php b mt:
20 gi m Hot % amylase i vi canh I trng kh 27,8 46,4 15,0 20,4 34 gi 42 gi m Hot m Hot % amylase % amylase i /v g canh vi canh I trng trng kh kh 23,8 18,0 22,0 20,5 42,4 32,9 42,4 36,7

Phng n th nghim Khay h Khay y np

BNG: nh hng ca vic gi m trong qu trnh sinh trng ti s to amylase ca Asp.oryzae nui bng phng php b mt

iu ny tht s cn thit phi gi m cho mi trng mc ti thch. Cn thng kh lin tc trong sut thi k sinh trng ca nm mc v cn ph thuc vo kch thc, chiu dy ca lp mi trng nui.

- m tng i ca khng kh: m cho php t 80% tr ln n mc bo ho tc l 100% W u thch hp cho nm mc pht trin. Trong phng nui cn gi cho m khng kh bo ho trnh cho mi trng khi b kh. -nh hng ca khng kh: Asp.oryzae l VSV hon ton hiu kh, ch pht trin bnh thng khi y oxy. p ng iu kin nui ny, mi trng phi xp, ri thnh lp dy khng qu 2,5-3cm, phng nui phi thong. Theo thc nghim, tho mn cho s h hp ca nm mc Asp.oryzae trong ton b chu k pht trin c cch 1gi, mi trng cn khong 1,7m3 khng kh. Asp. oryzae pht trin bnh thng khi nng CO2 trong khng kh t 8%. -nh hng ca nhit : Nhit nui cng l mt yu t ht sc quan trng i vi s sinh trng ca chng nm mc ny v s to thnh cc enzym amylase. Nhit thch hp cho s pht trin v hnh thnh enzym l 28-320C. Nhit do nm mc ta ra mi trng c th b nng ln 40 0C hoc hn. Do vy cn gi cho nhit mi trng khng xung di 270C v khng cao hn 360C. Ton b chu k sinh trng ca nm mc cm c th chia lm hai thi k: +Thi k trng v ny mm ca nh bo t trong 11 gi u. Trong thi k ny cn phi cung cp nhit cho phng nui khng hn 23-300C . m tng i ca khng kh l 96-100%. +Thi k sinh trng nhanh ca h si (c th t t 4-18 gi).

-Thi gian nui nm mc: Hu ht cc chng nm mc Asp. oryzae c hot ng cc i ca amylase khong gi th 30-38, ri sau l cc i ca protease gi th 36-42. Mt s chng cho hai cc i ca enzym, v d: cc i ca enzym amylase gi th 36 v gi th 60. Do , thi gian nui mc c th ko di t 60-72 gi. Thi gian nui c lng amylase cc ln thng c xc nh bng thc nghim. S to bo t l hin tng khng mong mun v thng lm gim hot lc ca enzym. i vi Asp. Oryzae, s to hot lc ti a thng kt thc khi nm mc bt u sinh bo t. -nh hng ca pH: pH thch hp cho Asp.oryzae l mi trng acid yu khong 5,5-6.5. Cc mi trng t nhin t cm, nh thng c sn pH khong ny nn khng cn iu chnh. i khi kh nng sinh bo t ca

nm mc b yu hoc mt hn. khi phc kh nng ny c th nui nm mc trong nh sng khuch tn trong mt vi th h.

Phn IV: ng dng v kt lun I.ng dng

Hin nay, vic sn xut ch phm enzyme cc loi a v ang pht trin mnh m trn qui m cng nghip. Thc t a c hng nghn ch phm enzyme bn trn th trng th gii, cc ch phm ny a c khai thc v tinh ch c mc tinh khit theo tiu chun cng nghip v ng dng. Ch phm enzyme khng ch c ng dng trong y hc m cn c ng dng trong nhiu lnh vc cng nghip khc nhau, trong nng nghip, trong ha hc " ngha ca vic s dng enzyme trong cc lnh vc thc t khng km so vi ngha ca vic s dng nng lng nguyn t". Theo thi gian, enzym cng nghip ngy cng c ng dng vo nhiu lnh vc khc nhau, trong nhng enzym ng dng nhiu nht l protease, cellulose, ligase, amylase, v mt s enzym c bit khc thu c rt nhiu li nhun t ngnh ny. Di y l mt vi ng dng thc t:

I.1.ng dng amylase trong sn xut bia Trong cng ngh sn xut bia truyn thng, cc nc phng Ty ch yu s dng enzym amylase ca malt thy phn tinh bt trong malt, sau n giai on ru ha bi nm men Saccharomyces sp. C s khoa hc ca vic s dng amylase ca malt ch, khi i mch chuyn t trng thi ht sang trng thi ny mm (malt), enzym amylase s c tng hp v khi enzym ny s thy phn tinh bt c trong ht to ra nng lng v vt cht cho s to thnh mm. Nh vy vic ng ha tinh bt trong ht nh enzym ca chnh n. Khi ht ch tng hp ra lng enzym amylase va phn hy lng tinh bt c trong ht. Nh th cn rt nhiu mm i mch sn xut bia qui m ln, dn n chi ph cao cho sn xut v sn phm. khc phc iu ny, trong qu trnh ln men to bia th nh sn xut khng s dng hon tan 100% nguyn liu l malt i mch m c s pha trn theo mt cng thc no thay th malt v cn b sung ngun tinh bt cho qu

trnh ln men. L do l mt phn to hng v cho bia, mu sc, cn ph hp cho ngi tiu dng v mt phn l lm gim gi thnh cho sn phm bia nhng vn gi c c trng cho bia. Chnh v iu ny, cc nh sn xut bia quan tm n vic s dng ch phm enzym amylase cung cp cho qu trnh thy phn tinh bt .Enzym ny c ngha rt ln trong vic lm bia, gip sn xut bia qui m cng nghip. Ngoi ra, trong sn xut bia, ngi ta cn s dng ch phm enzym cellulose c tc dng ph v thnh t bo, to iu kin cc thnh phn c trong t bo ht that ra pha ngoi nh cht lng bia c nng cao hn. Mt loi enzym khc cng c s dng kh rng ri l gluco amylase, enzym ny c s dng loi tr O2 c trong bia, gip qu trnh bo qun bia ko di hn rt nhiu.

I.2. ng dng amylase trong sn xut cn

sn xut cn t ngun nguyn liu tinh bt, mi nc s dng cc loi nguyn liu khc nhau.V d, M ngi ta s dng nguyn liu t bt ng sn xut cn, cn Brazin li s dng khoai m, cc nc khc s dng go hoc tm t go. Qa trnh sn xut cn tri qua hai giai an: giai an ng ha v giai an ru ha. Giai an ng ha, ngi ta bt buc phi s dng enzym amylase ( khng th s dng phng php thy phn tinh bt bng acid ). Ngi Nht bit s dng enzym ca nm mc trong qu trnh ng ha sn xut ru Sake t cch y hn 1700 nm. Ngi Trung Quc th s dng nhiu loi nm mc ng ha ru trong sn xut ru cch y 4000 nm. Cn ngi Vit Nam bit sn xut ru t go cch y hng ngn nm. Ring M, mi n th k XIX khi Takamine ngi Nht a nm mc Aspergillus sang mi bit s dng enzym ny thay amylase ca malt sn xut cn. Chnh v th mi c phng php Micomalt ( mm mc) trong sn xut cn v ru. Nh s du nhp k thut ny t Nht m ngi M tit kim c mt khi lng malt khng l trong sn xut ru.

Giai an ru ha, nh nm men Saccharomyces cerevisiae, cng c th xem y l mt qu trnh p dng enzym. Qa trnh ru ha l qu trnh ht sc phc tp, tri qua rt nhiu giai on chuyn ha t ng thnh cn nh s tham gia ca nhiu enzym khc nhau. im khc vi enzym amylase l ch cc enzym tham gia qu trnh ru ha nm trong t bo nm men. Vic iu khin cc qu trnh chuyn ha bi enzym trong t bo thc cht l qu trnh trao i cht ca nm men trong mi trng cha ng.

I.3.ng dng amylase trong ch bin th c phm gia sc

Trong ch bin thc n gia sc, thnh phn ng cc chim mt khi lng rt ln. Trong khi lng ny, thnh phn tinh bt rt cao. tng hiu sut s dng nng lng t ngun tinh bt, ng i ta thng cho thm enzym amylase vo. Enzym amylase s tham gia phn gii tinh bt to thnh ng, gip cho qu trnh chuyn ha tinh bt tt hn.

I.4.ng dng enzym amylase trong cng nghip dt

Trong cng nghip dt, ngi ta thng s dng enzym amylase ca vi khun ty tinh bt v lm cho vi mm.Trong vi th thng cha khang 5% tinh bt v cc tp cht khc. Do , khi s dng ch phm enzym amylase ca vi khun vi s tt hn. Ngi ta thng s dng l ng ch phm amylase khang 0,3-0,6 g/l dung dch v thi gian x l 5-15 pht nhit 90oC. Tuy nhin ngoi ch phm enzym amylase c ngun gc t vi khun, hin nay ngi ta quan tm n vic s dng amylase t nm si.Cho n nay c rt nhiu nc s dng enzym trong cng nghip dt tng kh nng cnh tranh cc hng vi, si. Cc nc s dng lng enzym amylase nhiu nht trong lnh vc ny l M, Nht, Php, an Mch.

Ngoi ra, enzyme amylase cng c nghin cu ng dng rng ri trong sn xut ng bt, sn xut dextrin, maltodextrin, nha glucose, siro, glucose fructose, sn xut tng v nc chm quy m cng nghip.

II.Kt lun

Tm li, c th ni rng, vic nghin cu ng dng cc ch phm enzyme ngy cng c ch trng cc lnh vc khc nhau. Trong 20 nm cui th k XX v cc nm u ca th k XXI cc enzyme khc nhau c ng dng. Vit Nam bc u c nhiu nghin cu ng dng cc enzyme trong ch bin nng sn, thc phm, nht l trong lnh vc sn xut bia, ru, ch bin tinh bt (Vin cng nghip thc phm, Vin cng ngh sinh hc cng ngh thc phm, i hc Bch khoa H Ni). Vic nghin cu cc enzyme phc v nng nghip, cng nghip cng c quan tm v c cc kt qu ng khch l. V d, ch phm enzyme mi ra i phc v nng nghip E2001 c tc dng tng ph nhiu t, tng nng sut cy trng. c cc nghin cu ng dng protease trong sn xut ru bia, rt ngn thi k ln men cng nh sn xut nc mm ngn ngy bng cng ngh enzyme protease.

Phn V: Ph lc v ti liu tham kho Ph lc

My sy phun sng

My sy chn khng

My nghin siu mn kh nn

My sy ng kh

Ti liu tham kho 1. Nguyn c Lng v mt s tc gi, Cng ngh enzym, NXB i hc quc gia Tp.HCM, 2004
2. Nguyn c Lng, Vi sinh vt cng nghip ( Cng ngh vi sinh Tp

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