công nghệ enzyme

8/7/2019 cng ngh enzyme

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CNG NGH ENZIM GVC: Ths. Trn Xun Ngch

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c lcChng 1: NGUYN LIU THU ENZIM V PHN B....................................................................3

1.1. Ngunng vt:........................................................................................................................................31.2. Ngun gc thc vt:..................................................................................................................................41.3. Ngun vi sinh vt:......................................................................................................................................4

Chng 2: N XUT CC CHPHM ENZIM TVI SINH VT............................................52.1. u ho qu trnh sinh tng hp enzim trong mi trng nui cy vi sinh vt..............................52.2. Tuyn chn v ci to ging vi sinh vt cho enzim c hot lc cao:...............................................11

2.3. Phng php bo qun ging vi sinh vt :..........................................................................................122.4. Mi trng nui cy vi sinh vt sinh tng hp enzim:......................................................................132.5. Cc phng php nui cy vi sinh vt:................................................................................................172.6. Tch v lm sch chphm enzym :....................................................................................................22

Chng 3: THUT SN XUT CHPHM THT CC NY MM (MALT).........243.1. Nguyn liui mch:............................................................................................................................243.2. Lm sch v phn loi ht:.....................................................................................................................253.3. a, st trng v ngm ht:....................................................................................................................263.4. y mm:.................................................................................................................................................283.5. y malt:....................................................................................................................................................34

3.6. Tch mm, r, bo qun malt:...............................................................................................................373.7. thut sn xut mt s loi maltc bit:.........................................................................................38Chng 4: N XUT ENZIM TTHC VT..................................................................................40

4.1. n xut ureaza tu ra:.....................................................................................................................404.2. Thu nhn bromelain tda:..................................................................................................................40

Chng 5: ENZIM CNH.......................................................................................................................445.1. Gii thiu chung:......................................................................................................................................445.2. t sphng php chyu ch to enzim cnh :......................................................................445.3. t s lin kt trong vic cnh enzim.............................................................................................455.4. nh hng ca scnhn hottnh ca enzim...........................................................................46

5.5. Cc reactor cha enzim cnh:...........................................................................................................485.6. . Sdng enzim cnh trong y hc v trong cng nghip:.............................................................50Chng 6: GII THIU MT S LOI ENZIM CH YU V KH NNG NG

NG 556.1. Amylaza....................................................................................................................................................556.2. Proteaza.....................................................................................................................................................586.3. Pectinaza....................................................................................................................................................606.4. Xenluloza:.................................................................................................................................................64


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6.5. Saccaraza v glucooxydaza...................................................................................................................66Chng 7: PHNG PHP XCNH HOTMT S LOI ENZIM............................68

7.1. n vo hot:...................................................................................................................................687.2. Cc phng php xcnh hotenzim:.........................................................................................697.3. Chun bdch chit enzimxcnh ho....................................................................................71


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Chng 1: NGUYN LIU THU ENZIM V PHN B

1.1. Ngun ng vt:

1.1.1. Tu tng: (Pan creas)y l ngun enzim sm nht, lu di nht, c cha nhiu loi enzim nht nh:

tripxin, kimotripxin, cacboxy pectidaza A v B, ribonucleaza, amilaza, lipaza.+ Tripxin y hc phi l loi tinh ch.+ ng dng u tin ca ch phm tripxin l lm mn da lt da, kh cc vt nt

trn da.+ Sn xut sn phm thu phn protein y hc (dch truyn y t) v mi trng nui cy

vsv.+ Ch phm dch tu y hc cha bnh v tu (ri lon chc nng, b ct b tu).

+ Sn xut ch phm enzim ty ra (vt bn, mu kh tan) nhit va phi, khngthch hp vi nhit cao v pH thay i.

1.1.2. Mng nhy d dy ln:L ngun enzim pepxin A, B, C, D, gastrisin. Cc enzim ny c tit ra ngoi t bo

cng vi dch v ( khi tiu ho thc n). i vi cc typ pepxin c pHopt=1.32.2.1.1.3. dy b:

Trong ngn th t ca d dy b c tn ti enzim thuc nhm Proteaza tn l renin.Enzim ny t lu c s dng ph bin trong cng ngh phomat. Renin lm bin icazein thnh paracazein c kh nng kt ta trong mi trng sa c nng Ca2+.y l qu trnh ng t sa rt n hnh, c nghin cu v ng dng y nht.Trong thc t nu ch phm renin b nhim pepxin (trong trng hp thu ch phm reninb qu th. Khi , d dy b pht trin y c kh nng tit ra pepxin) th khng ng t sa km i.

n y c nghin cu sn xut proteaza t vsv c c tnh renin nh cc loiEudothia Parasitica v Mucor Purillus.

1.1.4. Cc loi ni tng khc:Gan, l lch, thn, phi, chonh tim, d con, huyt. Cc loi ny u c cha enzim,

a s tn ti trong t bo. Ch c mt s loi c sn xut di dng ch phm nh: gan,tim ln tch aspartat-glutamat aminotransferaza, huyt tng (t huyt) tch ratrombia (Proenzim chng ng mu)

Nhn chung nguyn liu ng vt dng tch enzim phi ti tt (ly ngay sau khigit m) hoc gi-200C c thc 112 thng vn khng lm gim hot tnh enzim.


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1.2. Ngun gc thc vt:

1.2.1. Cy u ra (Canavalin ensifirmis):y l cy thuc hu Canavalia c nhiu chu Phi, Vit Nam c ni k trn.

Trong tt c cc ni u ra u rt giu enzim Ureaza, hm lng c thn 20% chtkh.

1.2.2. da (Bromalaceae):Bao gm tt c cc ni da trng ly qu, ly si (k c cc ni da di). Trong cc b

phn khc nhau ca cy da (v, li, chi, thn, l,) u c cha enzim bromelain.Trong nhiu nht l phn li u qu da. Hot tnh ca enzim bromelain ph thucnhiu vo trng thi v u kin bo qun nguyn liu. Cc nghin cu ch ra rngcc nguyn liu sy kh nhit 400C s gic hot tnh enzim tt hn so vinguyn liu c bo qun lnh nhit 40C.

1.2.3. Nha u (Carica Papaya. L):y l loi cy n qu ph bin cc nc nhit i. T qu ti hoc thn thu c

nha (latex) chnh l ch phm papain th t tinh ch thnh papain thng phm.

Hin nay ngi ta to ra c cc ging u c sn lng m v hot tnh papaincao khai thc c hiu qu ngun enzim ny (khng t vn ly qu).

1.2.4. t s loi nguyn liu thc vt khc:Khi tin hnh nghin cu khoa hc, y sinh hc, nhiu khi cn xem xt (nh tnh, nh

ng, cu trc phn t, hot ng enzim, ) ca mt s loi enzim c trong bn thnnguyn liu nh lng s dng. ng ch hn c l:

Ch phm enzim Polyphenoloxydaza (EPPO): n hnh nht l eppo ca l ch, cai nh ht ca cao ti, nc p qu nho. Ch phm loi ny ph bin hn c l loi btaxeton.

1.2.5. t cc v mt s loi c cha tinh bt:

Trong ht cc ny mm (malt) v mt s loi c ny mm (n hnh l khoai lang) ct h enzim rt phong ph c ngi ta s dng t rt lu trong cc lnh vc: mt tinht (mch nha), ru v bia (thm ch c mt phng php sn xut ru etylic mang tn lphng php maltaza hay phng php malt)

1.3. Ngun vi sinh vt:

y l ngun enzim phong ph nht, c hu ht cc loi vi sinh vt nh: nm mc,vi khun v mt s loi nm men. C th ni vi sinh vt l ngun nguyn liu thch hpnht sn xut enzim qui m ln dng trong cng ngh v i sng. Dng ngun visinh vt c nhng li ch chnh nh sau:

+ Chng v nguyn liu nui cy vi sinh vt v ging vi sinh vt.+ Chu k sinh trng ca vi sinh vt ngn: 16100 ginn c th thu hoch nhiu lnquanh nm.

+ C thu khin sinh tng hp enzim d dng theo hng c li (nh hng sng v tng hiu sut tng thu hi).

+ Gi thnh tng i thp v mt trng tng i r, n gin, d t chc sn xut.Tuy nhin trong mi trng hp cn lu kh nng sinh c t (gy c, gy bnh)

c bin php phng nga, x l thch hp.


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sn xut ch phm enzim, ngi ta c th phn lp cc ging vi sinh vt c trong tnhin hoc cc ging t bin c la chn theo hng c li nht, ch tng hp u tht loi enzim nht nh cn thit no .

Chng 2: SN XUT CC CH PHM ENZIM TVI SINH VT

2.1. u ho qu trnh sinh tng hp enzim trong mi trng nui cy visinh vt

i mc ch nui cy thu hi enzim vi hiu sut cao, cn phi nhn r qu trnh uho sinh tng hp enzim c cc nh hng tc ng thch hp trong cng ngh. Tbo vi sinh vt ch tng hp enzim khi cn thit vi s lng thch hp mong mun.

2.1.1. u ho theo hng ng m bi gen operator (gen u khin) _hinng trn p :

+ hin tng trn p (c ch) (repression): l lm gim qu trnh sinh tng hp do snphm cui cng ca qu trnh nui cy. Hin tng ny thng gp i vi cc enzymxc tc qu trnh sinh tng hp mt chiu nh: qu trnh sinh tng hp axit amin,nucleotit.

V d: khi thm mt axit amin no vo mi trng nui cy th t bo s khng cnng hp ny na. Do cng snh ch qu trnh sinh tng hp enzym, xc tc cho qutrnh tng hp nn chnh axit amin . Enzym ny chc tng hp tr li khi c nhuu ngha l khi lm gim nng axit amin tng ng. i vi h thng phn nhnhngha l qu trnh dn n vic to thnh nhiu sn phm cui cng khc nhau t mt ccht chung ban u th cch trn p c thc thc hin theo cc cch khc nhau.

V d: Phn ng u tin ca qu trnh sinh tng hp cc axit amin lizin, methionin,treonin u do enzym aspactokinaza xc tc. Enzym ny c 3 izoenzim.

K hiu: al, am, at. Qu trnh sinh tng hp al s b trn p bi nng lizin. am camethionin. Ring i vi at th treonin va l sn phm cui cng ca c qu trnh va lcht ban u sinh tng hp izolxin. Do qu trnh sinh tng hp axitt ch b trnp khi c treonin v izolexin t nng cao vt qu nhu cu ca t bo. C th minhho cch trn p ny theo s:


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al

am

at

A B C D Methionin

E

G

Lizin

Treonin Izoloxin

Ghi ch: A Ccht Aspartic ban u.B, C, D, G l cc sn phm trung gian c tc dng trn p.

Nh vy y s trn p ch xy ra khi c s hp ng tc dng ca c 2 sn phm.u trn p hp ng ny cng xy ra i vi qu trnh sinh tng hp enzym ging nhauxc tc cho cc phn ng song song to thnh 2 sn phm cui cng khc nhau. V d:qu trnh sinh tng hp valin v izolxin do 4 enzym ging nhau xc tc theo s sau:

Valin

Izoloxin

432

1

Hin nay ngi ta cho rng ARN mi l yu t trn p thc s cho qu trnh sinh tngp cc enzym xc tc tng hp cc axit amin tng ng.

+ Hin tng cm ng (induction): l hin tng ngc li vi hin tng trn p lmng lng enzym ca t bo(Ghi ch s trn: 1: -axeto.-oxyaxtintetaza

2: reductoizomeraza (axetolactat mutaza)3: hydrooxyaxit dehydrataza4: amino transpheraza

4 Phn ng

4 Phn ng6 Phn ng

-axetolactat

-axeto-Ox butirat

-Dioxy metylvalerat

-Xeto - metyl

-Xetoizovalerat

-Dioxyizovalerat

CH3CHOhot ng


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Ngha l khi trong mi trng nui cy c cht cm ng s kch thch cho vi sinh vtsinh tng hp nn nhiu enzym hn so vi bnh thng.

Cht cm ng c xem nh l mt cht nn (Cht cs, b khung ccbon) sinhng hp enzym. Hin nay, ngi ta ch ra rng c th cc sn phm trung gian ca qutrnh bin i ng vai tr l cht cm ng, thm ch nhiu ccht ca enzym cng c

th l cht cm ng. n hnh l cc gluxit (monosaccarit v polysaccarit).Trong s cc enzim do vi sinh vt tng hp, c nhng enzim bnh thng chc

ng hp rt t i nhng khi thm mt s cht nht nh vo mi trng nui cy th hmng ca chng c th tng ln rt nhiu ln. Monod v Cohn (1925) gi cc enzim nyl enzim cm ng, cht gy nn hiu qu ny l gi l cht cm ng. Cc enzim cm ngthng l nhng enzim xc tc cho qu trnh phn gii nh: Proteinaza, amylaza,pectinaza, penixilinaza, _galactosidaza t bo E. coli. Khi nui cy E. coli trong mitrng glucoza v glyxerin, vi khun ch tng hp khong 10 phn t_galactosidaza/tbo. Nu nui cy trn mi trng lactoza l ngun cc bon duy nht th hm lngenzim l 67% tng hp lng protein ca t bo. Trch ra t t bo cha n 6000 phn

enzim, ngha l tng ln gn 1000 ln so vi khi nui cy trong mi trng c. cm ng thng c tnh cht dy chuyn. Trong h thng gm nhiu phn ng, ccht u tin ca h thng c th cm ng qu trnh sinh tng hp tt c cc enzim xctc cho qu trnh chuyn ho ca n. u ny c thc hin theo cch sau: Trc htcht cm ng lm tng qu trnh sinh tng hp enzim tng ng, sau sn phm ny lim ng tng hp enzim ph hu n, tip theo sn phm th 2 ny li cm ng tngp nn enzim th 3,

V d: Histidin c tc dng cm ng hng lot cc enzim xc tt cho qu trnh chuynho n thnh axt glutamic (Chasin v Magasamil (1968)).

+ Cchu ha theo kiu trn p v cm ng:

Zocob v Monod ra m hnh gii thch cch ca 2 hin tng trn p v cmng trn csdi truyn. Theo m hnh ny, s trn p v cm ng sinh tng hp enzimc thc hin theo cng mt cch chung da trn csu ho hot ng ca ccgene di tc dng ca cc cht phn t thp. Nhng cn c chnh ca thuyt ny nhsau:

1) C s phn ho chc nng ca cc giai n khc nhau trong phn t AND trongnhim sc th, da vo chc phn ca chng trong qui trnh sinh tng hp Protein c thchia thnh cc loi gene sau:

- Gene cu trc (k hiu: S1,S2,S3) : m ho phn t protein enzim c tng hp, tcl th t cc axit amin trong phn t enzim c tng hp l tu thuc vo th t ccnucleotit ca n gene ny. Cc gene m ha cc enzim c p xp lin nhau thnht nhm trn nhim sc th. Chng l khun tng hp phn t ARNtt.

- Gene Operator (k hiu: O): cnh nhm gene cu trc, khng m ho proteinnhng m bo cho qu trnh sao chp m gene cu trc theo cch ng m tanh cng tc ca mt dy n. Qu trnh sao chp ch c th tin hnh khi gene operatortrng thi m (khng kt vi cht no c) v ngng li khi n b ng (kt hp vit cht c bit gi l cht trn p represson). Mt gene operator c th ph trch mtnhm gene cu trc cc gene cu trc ny cng vi gene operator ca chng hp thnh


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t n v sao chp scp gi l mt operon. S tng hp ARNttc bt u mtu ca operon v chuyn qua cc gene cu trc n u kia ca operon.

- Gene Promotor (gene hot ho k hiu P) ng trc gene operator l n And mARN-polimeraza s kt hp v bt u qu trnh sao chp cc gene cu trc.

- Gene u ho regulator (k hiu R): Gene ny m ho cho mt protein c bit gi

l cht trn p (repressor). Cht trn p c vai tr ng-m gene operator. Do geneu ho c th kim tra qu trnh sao chp gene cu trc thng qua cht trn p ny.

+ Khng c repressor (sn phm cui cng)

R P O S1 S2 S3 ADN

E3E2E1

A B C D

ARNtt

+ C repressor (sn phm cui cng):

Phin m


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R P O S1 S2 S3 ADN

: ARN-polymeraza

: Repressor

: coreressor

R: Gene u ho, P: Gene promotor, O: Gene Operator,

S1, S2, S3: Cc gene cu trc.2) Trong trng hp u ho sinh tng hp enzim theo cch trn p, repressor do

gene u ho tng hp cn dng khng hot ng (aporepessor) cha c kh nng ktp vi gene operator nn qu trnh sao chp cc gene cu trc tin hnh bnh thng.Cc enzim c tng hp xc tc cho cc phn ng to thnh cc sn phm cui cng,n phm cui cng ny li c kh nng kt hp vi aporepessor v hot ho n.Aporepessor c hot ho s kt hp vi operator ngn cn qu trnh sao chp ccgene cu trc, lm ngng vic tng hp ARNtt tng ng do nh ch qu trnh sinhng hp cc enzim tng ng. Trong trng hp ny cc sn phm mi c coi nh lcht trn p (repressor).

3) i vi trng hp cm ng:+ Khng c cht cm ng

R P O S1 S2 S3 ADN

+ C cht cm ng:


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ADNS3S2S1OPR

ARNtt

A B C DE1 E2 E3

minh ho cch cm ng sinh tng hp enzim

: Cht cm ngKhi khng c mt cht cm ng, cht trn p (repressor) c tng hp trng thi

hot ng, n kt hp vi gene u khin operator, qu trnh sao chp m ca gene cutrc b bao vy nn cc enzim tng ng khng c tng hp.

Khi c mt cht cm ng th cht trn p repressor b mt hot ng, tch khi geneu khin operator v qu trnh sao chp m bt u, kt qu lm tng lng enzim cng hp.

Nh vy ta thy hin tng trn p v cm ng sinh tng hp enzim l hai mt i lp

a mt qu trnh ho sinh thng nht c thc hin thng qua hot ng ng-mgene di tc dng ca cc cht phn t thp2.1.2. u ho tng tc gia ARN-polymeraza vi gene promotor:

Nhiu du hiu thc nghim cho thy cc gene bo m sinh tng hp mt s enzimm ng xc tc cho qu trnh phn gii khng ch chu s kim tra theo cch cm ngnh trnh by trn m cn chu s kim tra theo mt cch khc nh tc dng caAMP vng (AMPv) gi l trn p phn gii (cactabolic repressor) AMPv c tc dngkch thch ca AMPvi vi qu trnh sao chp m ca cc operon phn gii. Hin tngny c nghin cu nhiu i vi operon lactoza. Theo nhiu tc gi, tc dng kchthch ca AMPvi vi qu trnh sao chp m c thc hin nhmt protein c bitlm trung gian gi l protein nhn AMP

v, hay cn gi l protein hot ho gene phn gii

CAP (catabolite gene activator protein). Khi AMPv kt hp vi CAP to thnh phc hpc tc dng hot ho gene promotor lm cho ARN-polymeraza d dng kt hp vi n t u qu trnh sao chp m. Nh vy AMPv c tc dng lm tng cng qu trnh saochp. Cng c kin cho rng phc hp AMPv-CAP-ARN-polymeraza cho php bt uqu trnh sao chp m.


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CAPP O Zi

i ZOARN-Polymeraza

CAP

a)

b)

c)i ZO

CAP

AMPv

AMPv

AMPv

ARN

Pol

Pol

ARN

CAPO Zi

d)

ARNtt

M hnh bt u sao chp m ca operon lactozaa Phc hp CAP-AMPv chun b kt hp vo min c bit ca ADN.b Sau khi phc CAP-AMPv kt hp vo, n lm yu n ADN.c - ARN-polymeraza kt hp vo min c bit ca n.d - ARN-polymeraza trc dc theo n ADN nh mt ci bt n min bt u.

Ngi ta cng nhn thy glucza v mt s loi ng khc khi thm vo mi trngnui cy vi khun thng lm giu lng AMPv trong t bo, do lm gim qu trnhsinh tng hp nhiu enzim cm ng, ngay c khi n c cht cm ng trong mi trng.Hin tng ny cn gi l hiu ng glucoza c quan st thy E. coli v mt s vikhun. Tuy nhin cho n nay vn cha bit r cch lm giu AMPv do glucoza v ccng khc

2.2. Tuyn chn v ci to ging vi sinh vt cho enzim c hot lc cao:

chn ging vi sinh vt c kh nng sinh tng hp enzim cao, ngi ta c th phnp t mi trng t nhin hoc c th dng cc tc nhn gy t bin tc ng ln b

my di truyn hoc lm thay i c tnh di truyn to thnh cc bin chng c khng tng hp c bit hu hiu mt loi enzim no , cao hn hn chng gc ban u.2.2.1. Phng php gy t bin:

y l phng php hay c dng nht nhm :- To nhng t bin b gim kh nng sinh tng hp repressor hoc tng hp

repressor c i lc thp vi gene opertor.- To nhng t bin tng hp enzim c cu trc bc 1 thay i do c th gim

thay i vi kiu km hm theo cch lin h ngc.

u vo

Min bt u vo


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u s thay i cu trc bc 1 xy ra vng trung tm hot ng hoc gn th cth lm thay i r rt hot tnh ca enzim.

- Gy t bin n gene hot ho promotor lm tng p lc ca n i vi ARN-polymaraza do lm tng tc sao chp m.. Dng bin php ny c th lm tngng glucoza-6-phosphatdehydrogenaza ln 6 ln.

Hin tng t bin thng lin h vi s thay i mt gene, chng hn b li mtbazkhi ti to phn t ADN. V dmt v tr no trn gene c th t nucleotit lG-X, nu n b thay th bng A-T, T-A hoc X-G th phn t ARNttc tng hp trnn gene b li ny cng s khc vi ARNtt bnh thng v tr tng ng vi ch litrn gene. Do s tng hp nn phn t enzim khc vi bnh thng mt s gc axtamin.

to mt t bin gene c th dng tc nhn vt l (tia t ngoi, tia phng x) hayho hc (cc ho cht) tc dng ln t bo sinh vt.

2.2.2. Phng php bin np:L s bin i tnh trng di truyn ca mt ni vi sinh vt di nh hng ca ADN

trong dch chit nhn c t t bo ca vi sinh vt khc. y yu t bin np l ADN. chuyn vt liu di truyn (ADN) t t bo cho n t bo nhn c th xy ra trong ngnghim (invitro) khi cho t bo nhn tip xc vi dch chit t t bo cho m khng c stip xc gia cc t bo.

Cc t bo c th nhn bt k loi ADN no ch khng i hi phi l ADN t ccging h hng. Tuy nhin t bo ch c th nhn mt sn ADN nht nh, thngkhng qu 10 n. Cc n ADN c di truyn trong bin np c M=106-107 v phic cu trc xon kp. T bo khng tip nhn cc n ADN c kch thc nh hn hoccc n khng c cu trc xon kp. Hin tng bin np ph bin nhiu loi vi sinht nh: Diplococus, Staphylococus, Hemophilus, Agrobacterium, Rhizobium, Bacillus,

Xantomonas.2.2.3. Phng php tip hp gene:Khc vi bin np, y vt liu di truyn chc truyn t t bo cho n t bo

nhn khi hai t bo tip xc vi nhau. Do vy cc vi sinh vt c kh nng bin np th skhng c kh nng tham gia tip hp gene na. Hin nay qu trnh tip hp gene cnghin cu mt s loi vi khun nh E. coli, salmonella, Pseudomonas aeruginosa.

2.2.4. Phng php ti np:t liu di truyn (ADN) c chuyn t t bo cho sang t bo nhn nhvai tr trung

gian ca thc khun th (phage). Trong qu trnh ti np, cc n ADN c chuyn t bo cho n t bo tip hp vi ADN ca t bo nhn. Do lm bin i tnh cht ditruyn ca t bo nhn.

2.3. Phng php bo qun ging vi sinh vt :

Khi s dng vi sinh vt sn xut enzim cn chn ging thun chng, c kimtra y v cc c tnh ho sinh, vi sinh, nui cy v cn c bit lu n u kino qun ging. Thc t khi bo qun ging gc trong mt thi gian di c th to ra ccbin d ngu nhin khng mong mun do nh k phi cy chuyn v kim tra li ccc tnh ban u.


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2.3.1. Phng php cy chuyn:y l phng php ph bin nht d thc hin bng cch gi ging trn mi trng

thch (thch nghing, hp petri,) vi thnh phn mi trng nui cy v u kin nuiy thch hp cho ging vi sinh vt . Sau khi ging mc tt cn bo qun nhit nh 3-40C v sau mi tun phi cy chuyn li. Khi cy chuyn ch ly bo t hoc

khun lc m khng nn ly c mi trng dinh dng bo m khng chuyn cc snphm trao i cht vo mi trng mi (c th gy nn nhng bin i bt li khng thng ht c). Nu l x khun th khng nn bo qun ging trn mi trng thchm nn gi trong t kh trng.

ko di thi gian bo qun ging t hng thng n 1 nm, ngi ta ph mt lpparaphin lng tit trng trn b mt ging hn ch s pht trin ca n. Cn lu ch ph lp du sau khi cy vi sinh vt t n chn sinh l.

Phng php cy chuyn rt c hiu qu bo qun cc ging nm men, vi khun vt hu hiu, d dng trin khai ging ra sn xut ln, hn ch cc tai bin c th dn n hng ging gc.

2.3.2. Phng php lm kh:ng cch gi ging trn ct, t, silicagen trong u kin kh ro (tt cu ckh trng cn thn). Trong u kin nh vy s hn ch s pht trin tip tc ca gingkhi bo qun. Phng php ny rt hay c s dng bo qun nm mc, x khun,t vi loi nm men, vi khun thi gian gi ging c thc 1 nm.

Phng php lm kh cng thc hin n gin, khng cn dng ct tin. Tuy nhinging nh phng php cy chuyn thi gian bo qun tng i ngn.

2.3.3. Phng php ng kh:c l lm kh bng sy chn khng thng hoa (nu nguyn tc), cn gi l sy lnh

to nn sn phm ng kh (thc phm ng kh, cc vt phm sinh hc, y hc ng

kh). y l phng php bo qun lu di n 10 nm m khng lm cho ging bbin i c tnh nhng i hi cng ngh cao, thit bt tin, chi ph bo qun ln.n na mt s loi vi sinh vt nh nm mc khng c bo t v mt s loi vi rt t rakhng thch hp khi bo qun ng kh.

2.3.4. Phng php lm lnh ng trong nitlng:Kh nitho lng nhit rt thp -1650C n -1960C nn nu bo qun vi sinh vt

mi trng ny s rt tt v ging c gi bt bin trn 10 nm. Tuy nhin y l lnhc cng ngh cao (cn nitnguyn cht v lnh thm ) nn chi ph bo qun rt cao.

2.4. Mi trng nui cy vi sinh vt sinh tng hp enzim:

y l yu tu tin nh hng trc tip n hot ng sng cng nh kh nng sinh

ng hp enzim ca vi sinh vt. Mi trng chn cha y cc cht C, N, H, O. Cccht v c: Mn, Ca, P, S, Fe, K v cc cht vi lng khc.

2.4.1. Ngun ccbon:Thng l hp cht hu c trong ch yu l gluxit, tu thuc vo c tnh ca

enzim v ni vi sinh vt m ngi ta la chn cho thch hp.- i vi cc h vi sinh vt sinh enzim amylaza: y l enzim cm ng n hnh v

y mi trng nui cy phi c cc cht cm ng: tinh bt, dextrin, mantoza. Qua


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nghin cu ngi ta nhn thy ba loi gluxit l ngun ccbon tt nht sinh tng hpamylaza t hiu qu cao. Chng hn hiu sut sinh tng hp trn mi trng gluxit khcnhau vi mt s loi enzim amylaza nh sau:

+ i vi -amylaza:Tinh bt > dextrin > mantoza > glucoza > saccaroza > galactoza > manit > avabinoza.

+ i vi Oligo-1,6-glucoridaza (dextrinaza):Tinh bt > dextrin > mantoza > saccaroza > glucoza > lactoza > galactoza > orabinoza

> manit.+ i vi -1,4-amyloglucoridaza :Tinh bt > dextrin > mantoza > saccaroza, glucoza, lactoza, orabinoza > rabinoza >

lactoza > manit.Khi nui cy theo phng php b mt nu dng cm th khng cn b sung tinh bt,

ngun tinh bt rt ph bin, ngoi cm c th dng bt ng, bt m, bo bo.n ch trong a s trng hp, mt s loi ng, n hnh nht l ng glucoza

i kn hm sinh tng hp cc enzim thu phn ni chung (chng hn theo cch trn p

phn gii do lm giu lng AMPv trong t bo).i vi cc h vi sinh vt sinh enzim Proteaza:C mt s ngun gluxit khi dng nui cy nm mc c kh nng sinh tng hp enzim

Proteaza c hot lc cao, chng hn theo th t sau:+ i vi Asp. Flavus 74: fructoza > glucoza > saccaroza > ramnoza > mantoza >

galactoza > orabinoza > lactoza.+ i vi Asp. Awamori 200: fructoza > manit > saccaroza > orabinoza > galactoza >

lactoza.+ i vi Asp. Oryae 79: fructoza > saccaroza > mantoza > glucoza > manit >

orabinoza > galactoza > lactoza.

Tinh bt l ngun ccbon ca nhiu chng vi khun sinh tng hp enzim proteaza. V: Vi khun Bac. Subtilis c kh nng sinh tng hp proteaza mi trng tinh bt>8%, ging x khun a nhit Micromonospora vulgaricus sinh tng hp proteaza trongmi trng 0.15-0.25% tinh bt.

Ngoi ra mt s loi hydrocacbon cng c ngun ccbon cho 125 chng vi sinh vt.Chng hn, mt s ging vi khun Pseudomonas semginosa c kh nng sinh tng hpproteinaza hot lc cao trn mi trng n-paraphin vi 12, 14, 16 nguyn t C hocproplylenglycol, hydrocacbon thm.

- i vi cc h vi sinh vt sinh enzim Pectinaza:Qu trnh sinh tng hp enzim pectinaza c lin quan n cht cm ng. chnh l

pectin, ng nhin l ngun ccbon. Nu s dng hn hp gluxit trong c pectin, nui cy vi sinh vt th hot lc ca pectinaza ngoi bo c th tng 4-6 ln so vi khinui cy khng c pectin.

Ging Asp. Niger c nui cy trn mi trng c nhiu ngun ccbon nh: Pectin,tnh bt, isulin, lactoza, saccaroza, mantoza, galactoza nng 2, 4, 6% s cho pectinazac hiu sut cao. Tuy nhin nn nui cy trn mi trng ch c monosacarit v glyxerinth hon ton khng th sinh tng hp enzim ny. ng glucoza c tc dng kn hm


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(cht trn p) sinh tng hp enzim pectinaza trn mi trng nui cy l pectin v lactozai vi loi Asp. Niger, Asp. Awamori.

- i vi cc h vi sinh vt sinh enzim xenluloza.Enzim xenluloza l enzim cm ng v vy trong mi trng nui cy vi sinh vt sinh

enzim ny nht thit phi c xenluloza l cht cm ng v l ngun ccbon.

Ngun xenluloza rt phong ph: giy lc, bng, bt xenluloza, li ng, cm, mn ca,m r, than bn. Ngoi ra c th k thm chit xut xenlobiozo-octa axetat, cm m,lactoza, balixyl cng c ngun ccbon tt. i vi ging Stachybotris atra, ngun gluxitt nht sinh tng hp enzim xenluloza l tinh bt 1%.

Cc ngun ccbon khc ni chung (glucoza, xenlobioza, axetat, xitrat, oxalat,). Likm hm sinh tng hp xenluloza, glyxerin khng phi l cht cm ng cho enzim ny.

- Ngoi ngun gluxit l ch yu cn phi kn cc ngun ccbon khc nh:+ Cc axit bo phn t lng ln (oleic, stearic, miniotic). V d: axit oleic c tc dng

kch thch tng hp glucoamylaza ln 2.5-3.5 ln so vi nng thch hp 2-3%.+ Etanol v glyxerin trong nhiu trng hp nui cy c dng lm ccbon b sung.

+ Trong s cc axit hu c th axit lactic hay c vi sinh vt hp th tng hpenzim. Tuy nhin ngi ta thng khng b sung trc tip axit ny vo mi trng nuiy m ch b sung loi nguyn liu hay ch phm c cha n hoc s gy sinh ra ntrong qu trnh nui cy.

2.4.2. Ngun nit:- i vi h vi sinh vt sinh enzim amylaza: nhiu loi nm mc, ngun nit tt nht l NaNO3 v NH4NO3, nng nitdi

c 0.05% nm mc vn pht trin c nhng sinh tng hp amylaza rt km. l ti u gia tinh bt v NaNO3 trong mi trng Zapec nui cy nm mc sinh

ng hp amylaza t hiu qu cao nht l 18:1.

Cc mui amoni v c (NH4H2PO4, (NH4)2SO4, NH4Cl), mt s ngun nit hu c(gelatin, cazein, cao ng) cho hiu qu sinh tng hp amylaza thp.Trong thc t, ngi ta thng dng ngun nitl cc axit amin c ngun gc t dch

thu phn protein (dch t phn nm men, nc chm, cao ng, dch chit malt) y val ngun nitva l ngun ccbon v cht cm ng sinh enzim.

Cc axit amin c tc dng tt nht trong nhng trng hp ny l asparagin, axitglutamic; D,L serin, histamin, alanin. Trong khi casein thm ch l c ch th dch thuphn casein li cm ng sinh tng hp amylaza ln gp 2 ln so vi ban u.

- i vi h vsv sinh enzyme proteaza:Ngun nits dng rt phong ph, bao gm 2 nhm: v cv hu c.+ i vi mt s loi nm mc thuc h Asp. (oryzae, awamori, niger, flavas) nu mi

trng c ngun nit hu c th s sinh tng hp proteinaza axit tnh cao. Trn mitrng Czapek nu thay NaNO3 bng cazein th hot lc proteinaza c th tng ln 3,5n. Sinh tng hp enzyme proteaza c nng cao khi mi trng nui cy c c haingun nithu cv v c. Nu mi trng ch c ngun nitv cs dn n ngngsinh tng hp enzyme ny.

+ Trong qu trnh nui cy vi khun, trong s cc ngun nitv cth NH4, H2PO4 lt hn c. Cc mui amon v nitrat khc u lm gim hot lc enzyme.


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+ i vi x khun a nhit Actynomyces Vulgaris U2 th pepton l cht cm ng sinh tng hp enzyme proteaza l tt nht.

+ Cc axit amin c nh hng r rt nht n qu trnh sinh tng hp enzyme vsv nichung. Chng hn glyxin, alanin, metionin, lxin lm tng hot lc proteaza ca chngt bin Asp. Oryzae 251-90 ln 16% v chng nguyn thu Asp. Oryzae 132-63 ln 7 -

14%. Nhiu axit amin li c tc dng c ch sinh tng hp enzyme nh: valin, axitglutamic, izolxin, treonin. Ni chung c khong 10 axit amin nh vy. Axit amin c tcng kch thch sinh tng hp enzyme khi trong t bo vsv khng t tng lng lngaxit amin t do so vi mi trng nui cy.

+ Ngoi ra, cc baz purin nh A (adenin), G (guanin) v cc dn xut ca chng,ARN v cc sn phm thu phn cng lm tng ng k sinh tng hp proteinaza vsv.

- i vi h vsv sinh tng hp enzyme pectinaza:ng ging nhi vi h vsv sinh tng hp enzyme proteaza, nu dng kt hp nit

u c v v c s c tc dng tt n qu trnh sinh tng hp pectinaza. Tuy nhin,mui nitrat kim loi kim li kim hm enzyme ny. i vi Asp. Niger, ngun nittn

km nht sinh tng hp pectinaza la NH4H2PO4. i vi Asp. Awamori th li l(NH4)2SO4. Trong khi th N t pepton, cazein thu phn l hon ton c ch s tothnh enzyme.

- i vi nm mc Asp. Foetidus th (NH4)2SO4, nc chit cm, nc chit nm menc tc dng nng cao hot lc polygalacturonaza. Ni chung, t l thch hp nht i viC/N khi tng hp pectinaza trong khong 7/1- 13/1.

- i vi h vsv sinh enzyme xenlulaza.:Ngun nitthh hp nht i vi nhm vsv ny l ngun mui nitrat. Trong NaNO3

lm cho mi trng kim ho to u kin thun li cho s to thnh xenlulaza. Cao ngv cao nm men ( k c nc chit nm men) cng c tc ng khc nhau n kh nng

sinh tng hp xenlulaza tu thuc ging vsv. Cc mui amoni c tc dng thm chcch qu trnh sinh tng hp v chng lm cho mi trng b axit ho gy c ch qu trnhsinh tng hp thm ch lm mt hot tnh enzyme ngay sau khi to thnh trong mitrng.

2.4.3. Ngun cc nguyn t khong v cc yu t (cht) kch thch sinh trng:- Mui khong rt cn thit cho hot ng ca vsv, c bit l i vi cc qu trnh

sinh tng hp cc enzyme kim loi. sinh tng hp -amylaza v glucoamylaza, nng MnSO4 thch hp nht l 0,05%. Nu thiu mui ny v mui photphat kali th vsvkhng th sinh tng hp c dextrinaza. Hot lc -amylaza v dextrinaza c nngcao nng KH2PO4 1% v hot lc glucoamylaza nng KCl 0,05%, dextrinaza

ng thch hp nht l 0,15%.-Ion Mg 2+ c tc dng sinh tng hp v n nh cc enzyme c hot tnh nhit cao. c bit Ca2+c trong thnh phn ca -amylaza (trong 1 phn t gam -amylazaa Asp. Oryzae c 20g Ca, ca Bac. Subtilis c 4g Ca). Trong mi trng nui cy Ca2+nng cao kh nng tng hp -amylaza, bo v enzyme ny khi s nh hng caproteaza.

-Lu hunh S vi ngun ch yu l cc axit amin cha S nh metionin, cystein, sistin,v cc mui sunphat (CuSO4). Cc mui khong c Fe, Mn, Zn, B, Mo, Cu nh hng


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n kh nng sinh tng hp xenlulaza. Trong a s trng hp biotin (VTM H) v mt sVTM cng rt cn thit cho qu trnh sinh tng hp enzyme .

Khi la chn mi trng cn ch n c thnh phn nh tnh v nh lng sao choqu trnh sinh tng hp enzyme mong mun l cao nht. Mun vy ngi ta c th sng mt s phng php sau:

1) Phng php ti u ho quy hoch thc nghim ton phn ( yu t): i hinhiu thi gian v khng c chnh xc lm.

2) Phng php ton hc m hnh ho thc nghim: cho php xc nh nhanh chngv ng n t l cc thnh phn mi trng nui cy v cc yu t cng ngh bo mcho hot ng sng v sinh tng hp enzyme cao nht.

2.4.4. Cc loi mi trng nui cy vi sinh vt sinh tng hp enzyme :C th chia lm 2 loi: mi trng tng hp v mi trng t nhin (phc hp).- Mi trng tng hp: l mi trng bao gm cc cht vi liu lng xc nh (qua

tm hiu, nghin cu), chng hn ngun cacbon c th l tinh bt, xenlulolaza, ng,axit, ru, ngun Nit v c hoc hu c (axit amin, peptin...). Loi mi trng ny

c s dng cho mc ch nghin cu (c khi n mang tn nh nghin cu ra n:Czpk-Dobrovonxki, Hasen...).- Mi trng t nhin: thng dng cc lai ph liu, nguyn liu( a s trong l

thc phm) c cha cc ngun cacbon, nit, khong(a lng, vi lng), cc yu t sinhng hp trng. Mt khc, cc nguyn liu ny li c sn, r tin nn c s dng rtnhiu trong cng nghip sn xut cc ch phm enzyme vi sinh vt.

- Cc nguyn liu d chun b lm mi trng t nhin bao gm: cm v bt ht cc,c chit ng, dch p hoa qu, rau, kh du, b ru, rng, sn phm phn hu nmmen bia, tru, li ng( lm cht n, to xp). Khi la chn s dng mi trng cnch n cc cht c tc dng u ho sinh tng hp enzyme, c bit cc cht cm

ng. Bng thc nghim, ngi ta thy rng cht cm ng (tng cng sinh tng hpenzyme) thng l ccht ch yu, cc sn phm thu phn ca n hoc cht tng t ccht. trn ta bit l cht cm ng thng kt hp vi cht trn p repressor lm chon khng hot ng( mt kh nng kt hp vi gene u khin operator). Nh vy, chtm ng phi i vo bn trong t bo do khng th l nhng cht i phn t nhprotein, tinh bt, xenluloza, pectin. Theo mt s tc gi th cc ccht ny l cc ccht'tin cm ng', di tc dng ca enzyme gc chng b thu phn mt phn to thnhcht c phn t lng b hn ng vai tr l cht cm ng thc s. Chng hn, t nm1972 Iurikievits cho rng cht cm ng thc s ca -amylaza khng phi l tinh bt ml sn phm thu phn mt phn ca n: erytrodextrin. Tng t nh vy, cht cm nga enzyme proteinaza l cc polypeptin, protein c phn t lng nh.

- Khi la chn mi trng nui cy v c bit l cht cm ng cn xem xt cn thncc yu t chi ph, gi thnh sn xut ra sn phm.

2.5. Cc phng php nui cy vi sinh vt:

nguyn tc c 2 phng php nui cy vsv thu enzyme l: phng php nui cy mt (cn gi l phng php ni) v phng php b su (cn gi l phng php


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nui cy chm), trong phng php b su cn c th chia ra 2 phng php c thn: l nui cy chm 1 bc (1pha) v nui cy chm 2 bc (2 pha).

2.5.1. Phng php nui cy b mt:- Phng php ny rt thch hp nui cy cc loi nm mc (sinh tng hp cc h

enzyme amylaza, xenlulaza, pectinaza, proteaza) do kh nng pht trin nhanh, mnh,

nn t b tp nhim. Khi nui, nm mc pht trin bao ph b mt ht cht dinh dngn, cc khun ty cng pht trin m su vo lng mi trng c tit trng, lm m(khun ty ccht). i vi mt s mc ch c bit, ngi ta nui vsv trc tip trn bt ht go (sn xut tng), ht u tng (u tng ln men - mis) c nu chntrn ht cc cn sng (lm men thuc bc, men dn tc, lm tng).

- Ngi ta thng dng cm m, cm go, ng mnh, bt ng, mnh ht bo bo c chtph gia l tru. Cm, tru, c b mt tip xc ln, mng, to c xp nhiu, khngc nhng cht gy nh hng xu n s pht trin ca nm mc. T l cc cht ph gia(cht n) phi bo m so cho hm lng tinh bt trong khi nguyn liu khng cthp hn 20%, c th b sung thm ngun nitv c ((NH4)2SO4, (NH4)2CO), photpho

(P2O5, H3PO4 k thut), nithu cv cc cht kch thch sinh trng nh malt, ncchit ng, nc lc b ru.*Quy trnh cng ngh:

Nguyn liu

Trn

Lm m

Thanh trng bng nhit

Lm ngui, lm ti

Gieo ging vsv Nui cy ging

Chuyn vo dng c nui cy

Nui cy, theo di, x l

+ Lm m mi trng :C ngha quan trng, trong u kin sn xut ln, hm m ti u ca mi trng

cm l 58-60%. Khi c nui cy trong u kin tit trng nghim ngt th st hot enzyme cao nht khi hm m 65-68%. Tuy nhin nu mi trng qu m s b dnht (khi hp thanh trng, lm ti, khi nui cy), d b nhim vi sinh vt tp (b ln menu, ln men dm.....). lm m c th dng nc trn vi nguyn liu (nho) rithanh trng hoc lm m sb ri thanh trng sau dng nc v trng (nc ngng, nc un si ngui) u chnh li m ca khi nguyn liu. Cch sau c th


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rt ngn thi gian lm ngui, khng chc m chnh xc hn nhng i hi phithanh trng nhit v p sut sao hn.

+ Thanh trng bng hi nhit:Lm cho mi trng c tinh khit hn v phng din vsv v lm cho chn (bin

hnh) mi trng (tinh bt, protein). Thng thng ngi ta thanh trng bng hi nc

trc tip nhit 120- 1300C trong 2-3h.+ Lm ngui v lm ti mi trng gieo ging:

Khi mi trng va hp xong cn nng v dnh bt. V vy phi lm ngui v lmi thun tin cho vic gieo ging v phn phi vo cc dng c nui. Yu cu thigian ny phi ngn hn ch nhim khun t bn ngoi. Nhit yu cu t c gieo ging l 35-390C.

+ Nui cy nm mc ging:Nhm lng bo t ging cho ton b mi trng nui cy. Quy trnh cng ngh

thc hin tng t nh trong sn xut ln nhng phi thc hin cc u kin k thutc bit v khc khe hn nh: nguyn liu phi tt, giu cht dinh dng hn, u kin

nui cy khng ch nghim ngt hn, thi gian nui cy di hn (gn gp i) nmc hnh thnh nhiu bo t v u.+ Tin hnh qu trnh nui cy :

Sau khi gieo ging v phn phi vo cc dng c nui (mnh hay khay c l) richuyn vo phng nui c u chnh nhit v m tng i ca khng kh ()ng nh mc thng kh. Qu trnh nui cy nm mc ko di 33-48h/mc triqua 3 giai n:

*Giai n 1: T khi nui cy mc gng n ginui th 10-12. Xy ra s trng nbo t v xut hin cung nm. bo m s ny mm nhanh v hn ch nhim tp,n gim nguyn liu 55-60%, =96-100%, T=30-32oC.

*Giai n 2: ko di trong 10-18h. Nm mc pht trin mnh, lan khp b mt vtrong ton khi mi trng trng (khun ty n su vo ccht) dn n hin tng ktbnh. Qu trnhh hp v to nhit mnh lm mi trng trng b kh xp, tng hmng CO2, nhit phng nui tng ln n 38-40

oC. khng ch nhit thch hp28-30oC cn thng gi (qut) v bo ho m khng kh phng nui.

Giai n 3: ko di trong 10-20h v c trng nht v to ra enzyme nhiu nht.ng trao i cht gim i cht t, nhit to ra t hn nn tc bc hi nc cami trng nui cy cng gim theo. Qu trnh nui cy c chm dt khi nm mc t gi chn sinh l v bt u to thnh bo t.

2.5.2. Phng php nui cy chm:

- Vi sinh vt c nui cy trong mi trng lng vi c cht ch yu trong a strng hp l tinh bt. Ch c mt s t ging vsv dng ngun ccht cacbon l ngglucoza, saccharoza. Thc t, trong mt s trng hp ngi ta ng ho sb tinh bttrc khi thanh trng (bng ch phm enzyme amylaza). Khi ng maltoza c tothnh l cht cm ng tt, mi trng trng b gim nht nn d dng cho qu trnhkhuy trn v sc kh.

-Mt s loi mi trng dinh dng sn xut ch phm enzyme amylaza dng trongCNSX ru etylic nh sau:


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+Mi trng nui cy 40m3 ca Trung Quc:t khoai lang : 400kgt bnh m : 240kgCm : 160kg.NaNO3 : 1,2kg

c va hm lng cht kh:3,33%+ Mi trng sn xut thnh my ru H Ni:c b ru trong : 100 phnt ng mn :1,5-2 phnc ng ho t ng16% : 5-10 phn(NH4)2SO4 : 0,4-0,5 phnP2O5 : 0,4-0,5 phnMgO : 0,15-0,20 phnu chnh pH t 5-5,5- Phng php nui cy b su i hi phi c v trng tuyt i cc khu v sinh

ng hp, thanh trng thit b, thanh trng mi trng dinh dng, thao tc nui cy ,khng kh cung cp cho qu trnh nui cy .Cc giai n ca qu trnh nui cy chm 1 bc (1pha) gm: chun b mi trng

nui cy, nui cy nm mc ging, nui cy nm mc sn xut.+ Chun b mi trng nui cy :Sau khi phi trn ng t l cc thnh phn sc khuy trn k ri thanh trng

ng hi nhit (trc tip hay gin tip bng ni 2 v), nhit 118-125oC, thi gian 15-60pht, sau c lm ngui n nhit 30oC th tin hnh gieo cy nm mc gingvo.

+ Nui cy nm mc ging:

c tin hnh qua 2 cp (bc), phng th nghim v men ging trung gian. cpPTN c thc hin trong cc bnh cu, tit trng mi trng lm ngui, cy ging rinui trn my lc (150-200ln/pht). Nm mc s dng oxy khng kh qua nt bng vqu trnh lc, thi gian nui 46-50h. cp pht trin ging trung gian ngi ta chuync ging PTN vo thit b nui cha sn mi trng tit trng v lm ngui. Nuiy c sc kh v trng vi lu lng 15-20m3/m3h, thi gian 36-40h. Th tch dch menging bng 10% so vi dch men sn xut v sau.

+ Nui cy nm mc sn xut:Trong qu trnh nui cy cn phi sc kh v trng v khuy trn, tip du ph bt nu

c hin tng to bt tro ra khi ni ln men. Thi gian nui 1-4 ngy tu theo ging visinh vt. Vic khng ch pH, ch sc kh v bo m v trng l nhng yu t quantrng quyt nh hiu qu qu trnh. Chng hn nu mi trng c thm mui amnia NH4NO3 th khi NH4

+c vi sinh vt s dng s chuyn mi trng v axit. Nu axit ho thng ny c nh hng su n sinh tng hp enzym th cn phi b sungCaCO3 trung ho hoc duy tr tng pHopt cho sinh tng hp. Nu ngun NaNO3 thkhi vi sinh vt s dng NO3

- s cn li Na+ s kim ho mi trng, lc li phi dngaxit trung ho. Tr s pH ban u ca mi trng nui cy c nh hng nht nh n


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to thnh enzym. V d: i vi enzym -amylaza th pHopt ca ccloi vi khun l 7,a cc loi nm mc l 5.65.7.

2.5.3. Phng php nui cy chm 2 bc: (ln men 2 pha)Vi sinh vt c nui trong thit bu tin (giai n u, bc u tin, pha th

nht) pht trin n mc cn thit, sau c chuyn sang thit b ln men tip

theo (giai n sau, bc th hai, pha th hai) c thnh phn khc vi thit bu sinhng hp enzym.

Pha th nht c gi l pha sinh trng (trophophase), pha th hai c gi l phach to enzym (idiophase).

n hnh cho phng php ny xut pht t vic pht minh qu trnh ln men chtkhng sinh streptomixin bi x khun streptomyces griseus vo nm 1944 bi Schatz,Bugie v Waksman.

Ngun gluxit m ging x khun ny ng ho c sinh tng hp streptomixin l:glucoza, tinh bt, dextrin, mantoza, galactoza, mannoza. Ngun Nitc s dng lprotein ca bt u nnh, bt c, men kh, bt ht bng, gluten bt m (nhm x khun

sinh tng hp khng sinh streptomixin ni chung u c hot lc proteaza rt mnh thu phn cc protein ni trn thnh cc axit amin cn thit). Ngun Nitv cbao gmcc mui amoni, photpho ho tan.

n thn qu trnh ln men streptomixin l cc qu trnh ln men 2 pha n hnh. Phasinh trng mnh, bo t ny chi v mc thnh si sau 6-8h. Pha th 2, khun ty phttrin v bt u sinh tng hp khng sinh. Trong qu trnh ny (pha th 2) ng thio thnh mt phc ca mannoza vi streptomixin gi l manozilostreptomixin c hottnh khng sinh km hn 6 ln so vi streptomixin v c th coi y l tp cht khngmong mun trong qu trnh sinh tng hp. Tuy nhin phc ny di tc dng ca enzym- manozilostreptomixinaza c tnh D-manoza gii phng streptomixin vo nm 1969

Inamine v cc cng s nghin cu sn xut enzym - manozilostreptomixinaza theophng php ni cy chm 2 bc nh sau:Pha th nht: T bo streptomyces gricus c nui trong mi trng dinh dng c

khuy trn v sc kh trong 17hnhit 280C to nhiu bo t. Sau bo tca sch v chuyn sang thit b tip theo.

Pha th 2: Tip tc nui cy sinh tng hp enzym - manozilostreptomixinazatrong 18-24h. Lc ny tc pht trin ca vi khun chm li, nhng s chuyn ho phccht manozidosteptomixin nhanh chng din ra di tc dng ca enzym thnh khngsinh streptomixin.

2.5.4. So snh phng php nui cy vi sinh vt sinh tng hp enzym :- Phng php nui cy b mt c nhng u nhc m sau:+ Nng enzym to thnh cao hn nhiu ln so vi dch nui cy chm sau khi

tch t bo vi sinh vt. Trong cng nghip ru mun ng ho 100kg tinh bt ch cn5kg ch phm nm mc b mt nhng phi cn n 100lt nm mc chm lc b v tbo vi sinh vt.

+ Ch phm d dng sy kh m khng lm gim ng k hot tnh enzym, ch phmkh, d bo qun, vn chuyn, nghin nh hoc s dng trc tip nu khng cn khutch v lm sch enzym.


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+ Tn t nng lng (n, hi nc, cng nhn) thit b, dng c nui cy n gin,c th thc hin qui m gia nh, trang tri cng nhqui m ln n 20T/ngy.

+ Nui cy trong u kin khng cn v trung tuyt i v trong qu trnh nui cyu c nhim trng phn no, khu vc no th ch cn loi b canh trng phn .

+ Tuy nhin phng php b mt c nng sut thp, kh ckh ho, tng ho, cn

din tch nui ln, cht lng ch phm cc m khng ng u.- Phng php nui cy b su c nhng u nhc m sau:+ Phng php nui cy hin i (cng ngh cao) d ckh ho, tng ho, nng

sut cao, d t chc sn xut tit kim din tch sn xut.+ C th nui cy d dng cc chng vi sinh vt t bin c kh nng sinh tng hp

enzym cao v la chn ti u thnh phn mi trng, cc u kin nui cy, enzym thuc tinh khit hn, m bo u kin v sinh, v trng.

+ Tuy nhin do thu c canh trng c nng enzym thp nn khi tch thu hienzym s c gi thnh cao (c t trc). Tn n nng cho khuy trn, nu khng bom v trng s b nhim hng lot, ton b gy tn tht ln.

2.6. Tch v lm sch ch phm enzym :(xem s tng qut trang 200 ca gio trnh)- Mc ch yu cu: Cc ch phm enzym c s dng cc dng khc nhau theo

c tinh khit (hot ring). Trong mt s trng hp, canh trng nui cy vi sinht c cha enzym c s dng trc tip di dng th khng cn tch tp cht nuchng khng gy nh hng ng kn sn phm v quy trnh cng ngh sau ny (V: sn xut ru, nc chm thc vt, da). Cng c khi ngi ta cn s dng ch phmenzym tinh khit trong cng nghip dt, cng nghip mch nha, y hc, nghin cu khoac.

Enzym ni chung rt d b gim hot tnh di tc dng ca cc tc nhn bn ngoi do khi tch v tinh ch enzym trnh s bin hnh protein nh hng ln n hot tnhenzym cn tin hnh nhanh chng nhit thp, pH thch hp khng c mt cccht gy bin hinh enzym.

2.6.1. Thu dch enzym :- i vi trng hp enzym cn nm trong t bo (enzym ni bo nui bng phng

php b mt) th cn phi gii phng enzym bng cch ph vt bo thu nhiu cch nh:+ Nghin nh, nghin vi ct, nghin vi vn thu tinh, nghin bi.+ t bo t phn hu.+ Dng tc dng ca siu m hoc to p sut thm thu cao, trch ly (chin) bng

mui, dung dch mui trung tnh, dung mi hu c

+ Kt ta enzym bng cc cht n ly thch hp.- i vi trng hp enzym tit ra mi trng (enzym ngoi bo nui theo phng

php chm), ngi ta thng tch sinh khi v cn b khoi canh trng bng cchc li tm, lc p c s dng tc nhn trlc (diatomit, t hot tnh) hoc cc tcnhn trkt ta (V d: hn hp CaCl2+(NH4)2SO4 CaSO4: lng cn ko theosinh khi nn lc d hn)


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2.6.2. Thu nhn ch phm k thut:Ch phm k thut l ch phm enzym cha c tinh ch; c th cha mt vi loi

enzym ch yu, mt s loi protein khng phi enzym, cc cht n nh v cc tp chtkhc. Dch enzym thu c trn thng c nng cht kh thp 4-6g/l, bc ungi ta c c chn khng nhit 350C n nng 15-20g/l ri tip tc x l nh

sau:- Tip tc c c chn khng nhit 40-45 0C t nng cht kh 30-35g/l,

sung thm cht bo qun nh NaCl, glyxerin, sorbitol, benzoat ta s thu cch phm enzym k thut dng c th bo qun nhit thng c 1-2 nm.

- sung thm cc cht n nh t nng cht kh 30-40g/l ri sy phun nhit 120 0C (nhit kh thi 60 0C), ch phm k thut thu c dng bt .

- t ta enzym bng cc dung mi thch hp nh: dung mi hu c (etanol,izopropanol, axeton), dng mui trung tnh ph bin nht l (NH4)2SO4 dung dchbo ho. Sau khi li tm tch kt ta c th trn thm cc cht n nh ri sy khv nghin mn thu c ch phm dng bt.

2.6.3. Thu ch phm enzym tinh khit:Vic tinh ch enzym c th tin hnh bng nhiu phng php qua nhiu giai n:- Ho tan ch phm k thut vo nc hoc dung dch nui CaCl2nng thch

p hoc dung dch m, kt ta tr li bng etanol, axeton hay (NH4)2SO4. Qutrnh ny cn tin hnh nhanh chng nhit thp trnh s v hot enzym.Ngoi ra mui NaCl cng c hay c dng kt ta cc enzym ngun gcng vt. Kt ta pH gn m ng n ca enzym. Sau khi kt ta cc mui vc loi i bng phng php thm tch, thm thu ngc hoc lc gel.

- Tch enzym bng phng php hp ph chn lc:Cho dch enzym chy t t qua ct cht hp ph (thng l hydrat oxit-nhm,

silicagel) cc enzym khc nhau sc hp ph vi kh nng khc nhau, sau dng ccdung dch m thch hp chit rt enzym ra khi ct. Phng php dng lm mc enzym.

- Tch enzym bng phng php trao i ion:a vo s trao i ion gia enzym c n tch vi cc ion tri du ca cht nha khi

cho dung dch enzym chy t t qua ct cha cc cht nha trao i ion. Sau khi ct no (ht hiu lc) cho dung dch ra (dung dch cht n gii) c nng tng dn chyqua ct y ra khi nha cc enzym va lin kt vi chng. Khi enzym no c pc (lin kt) vi nha km nht s by ra khi ct nha trc. Nh vy cc enzymkhc nhau sc chit ra khi ct theo tng phn chit khc nhau trong ca phnchit cha enzym cn thu vi nng cao nht.

Cc nha trao i ion thng l cc cht nha tng hu c : Dowex, Amberlit,Wolfatit, Permuit, cc dn xut ca xenluloza.

Sau khi lm sch cn sy kh chn khng nhit thp hoc sy thng hoa. Enzymtinh khit c hot tnh cao hn nhiu so vi ch phm ban u. Nhng do qu trnh lmch rt khc khe v tn km nn loi ny chc dng trong y hc, trong nghin cukhoa hc xc nh khi lng phn t, cu trc enzym.


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Chng 3: K THUT SN XUT CH PHM THT CC NYM (MALT)

Malt l loi ht ho tho (ht cc) ny mm trong nhng u kin nhn to (nhit ,m, thi gian) xc nh gi tc l qu trnh malt. Mc ch chnh trong qu trnh malt l trch luc mt lng ln cc enzym (ch yu l enzym amylaza) trong ht,c s dng trong cc lnh vc sau:

- Trong cng nghip sn xut ru etylic (cn, ru etylic) t nguyn liu tinh bt.Malt l tc nhn ng ho tinh bt (phng php sn xut ru ny c tn lphng php maltaza hay phng php malt). C th dng cc loi ht nh: ich, la mch en, yn mch, k, ng sn xut malt loi ny.

- Trong cng nghip sn xut bia malt va l tc nhn ng ho tinh bt va l

nguyn liu chnh (cng vi hoa houblon v nc) v c th c nguyn liu thayth (Khng phi malt i mch). Malt bia ch yu c sn xut ti mch, ngoira ngi ta c th dng mt t l malt thay th nh thc mm.

- Trong cng nghip sn xut mt tinh bt (ng nha, mch nha): malt va l tcnhn ng ho tinh bt va l nguyn liu chnh. Malt loi ny c sn xut tla, la m, ng, i mch, k thm ch t c khoai lang ny mm. Mch nha snxut t malt vn l ngon nht, cho cht lng tt nht.

- Trong mt s ngnh sn xut thc n sinh dng, thc n kin (cho ngi bnh,ngi gi, tr em, gia xc, gia cm non). Malt c dng phi ch vo thc na thc n.

Qu trnh sn xut malt bao gm cc khu sau:Thu nhn, x l, lm sch, phn loi v bo qun ht,a, xt trng v ngm ht.m mm (ny mm) ta s thu c malt ti.y malt ti. l v bo qun malt kh.

3.1. Nguyn liu i mch:

i mch l cy ht cc cc nc n i, c khong 30 ging khc nhau nhng chc mt ging c ngha kinh t l i mch ma (Hordeum sativum) cn li u l ich di. Hin nay din tch trng v sn lng i mch trn th gii ng v tr th 4

sau la m, la, ng. Thuc ging i mch ma c 130 loi khc nhau v c chia lm3 nhm chnh: i mch nhiu hng (6 hng v 4 hng)-Hordium hexatichum; i mch 2hng (Hordium disstichum) v i mch trung gian (H. intermedium). Nhm c gi trtrong sn xut malt v bia l i mch nhiu hng.

i mch sau khi thu hoch c phi sy n m di 13% bo qun cngging nh cc ht ho tho khc, cu to ht i mch gm v tru, v qu, v ht,alrng, ni nh v phi. T l trung bnh trong cc phn nh sau:


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Tn b phn ht % khi lng ton ht tru qu htAlrng

Phii nh

123,5 42 2,512 142,5 364,5 6,8

Thnh phn ho hc trung bnh ca i mch theo % cht kh nh sau:

phn ht

Protein(N*5,7)

Chtbo tinht

Pentosan Xenluloza Tro

tPhi

13,47,128,6

2,02,17,6

54,08,246,0

9,020,020,0

5,722,61,1

3,010,010,0

Khi lng 1000 ht c trng cho my ca ht nm trong khong 15 60 gam vngi ta chia thnh cc loi:

i mch ht nh: Khi lng 1000 ht ti 30 gami mch tng i nh: Khi lng 1000 ht ti 31 40 gami mch nng: Khi lng 1000 ht ti > 51gamKch thc ht di 8 10 mm, rng 3 4 mm, dy 2 3 mm.Ch s cht lng quan trng ca i mch sn xut malt v bia l ny mm v

ng lc ny mm. Ngoi ra, i mch cn c c trng bi chit v hm lngprotein. i mch tt c chit t ti 82% v hm lng protein khng qu 12%. Nu

hm lng t qu 12 % th chit s thp, bia s bc; cn nu hm lng proteinqu thp s lm gim bt v v bia.

3.2. Lm sch v phn loi ht:

Khi ht c cha nhiu tp cht (bi, tp cht nh nh c, rm r; tp cht nng nhi , vn kim loi...) do cn phi c lm sch trc khi a vo sn xut. Mtkhc, khi ht phi m bo ng u qu trnh ngm v ny mm c thun li vng u.

Thng thng ngi ta lm sch v phn loi bng cc h thng sng thch hp:- Sng khng: dng tch tp cht v phn loi ht theo chiu dy, chiu rng ca

n theo cc tnh cht khi ng (dng qut).u ht c phn loi theo chiu dy ht sn xut malt bia, ngi ta c 3 nhm:+ Loi I : b dy ht > 2,5 mm Dng sn xut malt bia tt nht.+ Loi II : b dy ht > 2,2 2, 5 mm Dng sn xut malt bia tt nht.+ Loi III : b dy < 2,2 mm Dphi liu, c dng lm vic khc.- Sng ng (Trier): phn loi ht theo chiu di- Tch tp cht kim loi dng nam chm kin trng.


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- S cng ngh lm sch v phn loi ht

3.3. a, st trng v ngm ht:

3.3.1. a v st trng ht:t c ra sch trong qu trnh ngm, cc cht bn theo nc v tp cht nh s ni

ln c tho ra ngoi. Trong qu trnh bo qun ht s b nhim vi sinh vt, trng b,t cht v h hng khc. V vy, khi ngm ngi ta thng kt hp vi ra, st trng khi ht c sch, ng nht, kch thch s ny mm.

t s cc ho cht dng st trng ht khi ngm:- CaOCl2, Ca(OCl)2, HCHO 40 %: 700 gam/m

3 H2O- H2SO4, KmnO4: 10 15 gam/m

3 H2O- Ca(OH)2: 2 3 lit/100 gam htTrong , Ca(OCl)2 st trng mnh, kch thch s ny mm nn hay c dng. Liu

ng 40 g Ca(OCl)2 33% / 100 kg ht.Ca(OH)2 bo ho ch dng ra st trng ln th 2, khng c cho vo ni giai n

ngm v n hay bm ln v ht lm nh hng n cht lng ca malt v bia. C thdng H2O2 st trng v kch thch ny mm: liu lng 3 lit/m3 H2O. Ch phm

Giberelia kch thch thc vt c s dng ln u tin nht Bn vo nm 1940 vi liung 40 200 mg/1 tn i mch, rt ngn thi gian ny mm t 8 ngy xung cn 5ngy, gim gi thnh Malt 12 15 %. Anh, 90 % ch phm Giberelia c dng trongcng nghip malt.

3.3.2. Ngm ht:- Mc ch ht ht nc cn thit chun b ny mm, ny mm v pht trin

m.- Bin i ca ht khi ngm:+ Trc khi ngm, ht c m trung bnh 13 % duy tr kh nng sng.+ Khi ngm, nc ngm (thm thu qua v) vo ht, khi m ca ht qu 15 % th

trong ht xut hin nc t do, ht trng ndn (th tch ht tng trung bnh khong1,45 ln so vi ban u). Nc t do thc y cc qu trnh ho sinh c lin quan nhot ng sng, h hp v hot ho enzim.

+ Bin i ho hc khi ngm khng ng k, ht h hp vn rt yu nn tiu tngluxit rt t, mt lng nh cc cht ho tan vo nc ngm: ng, pentosan, chtkhong, tiu hao cht kh khong 1 %.

+ Ht ht nc th nng lc h hp tng, khi ngm ht tiu th 63 mg O2/kg ht.h vthi ra 86 mg CO2/kg ht. nu khng lng oxi ht s h hp ym kh to ra C2H5OH,CO2, cc axit hu c... a s l cc cht c vi t bo, kim hm qu trnh sng bnhthng, dn n ph hu cu trc t bo v hin tng t phn hu (gy h hng ht, thia, nhim vi sinh vt).

+ Tc ngm nc ph thuc vo nhit ngm nc, mc thay nc, thng kh,kch thc ca ht. Nhit ca nc ngy cng cao th nc thm vo t bo ngy cngnhanh. V khi nhit s lm tng s trng n cc cht keo (protein, tinh bt,xenluloza,...), tng vn tc khuch tn (do chuyn ng nhit), gim nht, tng h


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p ca ht ng thi tng kh nng nhim v pht trin ca vi sinh vt tp, nhu cu oxia khi ht cng tng ln nhiu.

a vo nhit ca nc ngm, ngi ta chia ra cc ch ngm nh sau:

Ch ngm Nhit nc ngm

Ngm lnh < 10 oC

Ngm thng 10 15 oC

Ngm m 20 40 oC

Ngm nng 50 55 oC

Trong ch ngm thng c s dng rng ri nht, ngm m c s dngtrong ma ng, ngm nng thng kt hp vi ngm thng hay ngm m x l ht(kiu ngm 3 si 2 lnh).

*Qu trnh ht nc ca ht i mch khi ngm din ra theo th:

th biu din s thay i m ca ht khi ngm

Khi m khi ht t khong 40 % th tc ht nc ca ht bt u gim. Sau96 h ngm m khi ht t cao nht khong 47%. y cng chnh l mc ngmc i cn thit.

+Cc thnh phn khong trong nc ngm nh hng n tc ngm ht. Nc

m ngm nhanh hn nc cng, tt nht s dng nc c cng 7mg ngng/lit. In Ca2

+ lin kt vi polyphenol trong v ht to mui t tan c cu trc th keoo thnh bong bng trn v ngn cn s thm nc, mui Na2CO3 lm tng v malt chobia. Cc mui st gy kt ta dng Fe(OH)3, tc dng vi polyphenol lm cho ht c mnnu. NaCl lm chm qu trnh ngm v ny mm, nng mui ny qu cao c th gitcht mm.

- Cc phng php v ch cng ngh ngm ht: c nhiu phng php ngm ht:

5040302010

10 20 30 40 50 60 70 80 90 100

m ht(%)

Thi gian ngm(h)


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+ Ngm trong nc-khng kh gin n: Ht lc c ngm trong nc, lc cngm trong khng kh, mi chu k ko di 3 6 h. Khng kh nn c thi cp cho cchu k, mi ln thi t 3 5 pht, c 30 pht li thi kh mt ln.

+ Ngm trong lung nc v khng kh lin tc: Dng nc bo ho khng khc phun lin tc vo b ngm ht (cn c thit b bo ho khng kh). Vi phng

php ny mc ngm ht c nhanh, rt ngn c qu trnh ny mm.+ Ngm ht trong lung nc phun: Khi ht c phun nc lin tc chy thm qua

p ht t trn xung di ri chy vo rnh thot nc. Nh vy nc ch lu li trongt mt thi gain nht nh no . Phng php ny cho php ht c thng kh tnhin lin tc, mm ht mau xut hin v pht trin.

+Ngm ht trong lung khng kh - nc phun: y l phng php kt hp giangm trong nc lin tc v gi ht tng thi hiu kh (i mi khng kh) bng cchht kh ca khi ht.

Sau khi ra xong, ht c phun ti nc trong 15 pht. Sau ht khng kh tpha di (bng qut hay bm ht) trong 15 pht ri yn trong khng kh mt gi, ri

i phun ti nc...c nh th cho n khi ht t m yu cu. sn xut matl bia, mc ngm i mch khong 42 48 %. sn xut matlng ho khi sn xut ru etylic th thi gian ngm thng ngn: i mch, la mch,n mch: 12 18 h; mch en: 12 14 h, ngm nc 15 200C cho n mc ngm3840 %. Ht k, la l ging a m nn ngm trong nc 25300C trong 2224hn khit c mc ngm 3537%.

Thng qu trnh ngm kt thc th thy r con xut hin.

3.4. y mm:

Thc t qu trnh ny mm c th bt u giai n ngm khi mc ngm ht t25 ( 30 %. Lc cc h enzim t trng thi ngh c nh thc tr li hot ng u kin nhit , m thch hp. Kt qu l cc polyme d tr khng tan (tinh bt,protein, hemixenluloza, xenluloza) b thu phn mt phn (bin hnh) thnh cc cht tanc kh nng nui sng mm v r.

Trong giai n u ca qu trnh ny mm, trong ht xy ra cc qu trnh sinh l, hosinh tng t nh khi ht ny mm t nhin (trong t khi gieo ht), trong dung dch dinhng (thu canh), di tc dng ca m, nhit , kh CO2 trong khng kh, phi htpht trin s to nn mm v r phi, cht dinh dng nui phi ht c ly t ni nha ht. i , di tc dng ca h enzim s chuyn ho cc cht v tr cao phn t vng cc cht ho tan n gin phi hp thc. Tuy nhin, khi phi ht cng phttrin th tiu hao cht kh trong ht (ch yu ni nh) cng ln. Bng cch khng ch,

tc ng cc u kin cng ngh phi ht (mm v r) pht trin mt cch hp l nht tiu hao hm lng cht kh l nh nht nhng cho php tch luc nhiu enzimnht (tr h enzim oxy ho). y l mc ch ch yu ca qu trnh sn xut matl, vng l m khc bit gia ny mm nhn to v ny mm t nhin.

Khi ny mm, trong ht i mch s xy ra cc qu trnh ch yu sau:- Qu trnh sinh l: S pht trin ca phi xy ra ng thi vi s h hp v tng hp

t s cht mi, m quan trng nht l mt s enzim cn thit.


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- Qu trnh sinh ho: Thu phn cc cht d tr trong ni nh.- Qu trnh ho hc: Tc dng tng h gia cc cht to thnh khi thu phn, hnh

thnh cc cht thm, cht c v matl.- Qu trnh vt l: Vn chuyn m v cc cht d tr ho tan t ni nhi nui phi v

ngc li.

3.4.1. hnh thnh v hot ng ca cc h enzim ng thi nhng qu trnhy ra trong qu trnh ny mm:

t t chn v kh (W13%) th cc enzim trong ht dng lin kt (khng hotng) nm trong t bo ca ht. Trong qu trnh ny mm chng c gii phng ra ng t do hot ng, ng thi ht cng c kh nng hnh thnh nn nhng enzim hotng mi.

- H enzim oxy ho - kh:Hot ng ca h enzim ny l bc chun b ban u cho qu trnh ho sinh tip theo

a s ny mm.C th kn cc enzim oxy ho hn: oxydaza, peroxydaza v catalaza sn phm

phn ng xc tc qu trnh h hp ca ht bao gm: H2O, CO2 v O2. Cng h hpph thuc rt nhiu vo nhit v lng O2 tham gia. Trong khong nhit 10300C,

nhit tng s lm tng cng h hp, ng thi hot tnh ca a s cc enzim cngng dn n lm tng s tn tht cht kh.

Qu trnh h hp km theo s thi nhit: 673 kcal/phn t gam ng.Trong ht ngm c 23 % ng (tnh theo hm lng cht kh), trong qu trnh ny

m do thu phn nn lng ng tng thm khong 20%. Khong 910% lngng tn hao do h hp, 35% tn hao do s pht trin ca mm v r, cn li i vothnh phn ca malt. Nh vy, ta thy qu trnh h hp tiu th mt lng ng kh lnnn lng nhit to ra nhiu.

t s axit hu cc sn v hnh thnh trong qu trnh ny mm nh: oxalic, xitric,malic, lactic, formic, axetic, propionic,... Nu oxi khng cung cp y (trng thi ymkh) c bit giai n u ca qu trnh ny mm s xy ra hin tng h hp ym kho nn ru etylic C2H5OH v CO2. Nu hm lng C2H5OH sinh ra ln n mc nht nh sc ch t bo sng v nh ch s pht trin ca mm. Tip , cc sn phmng h ca ru v axetic l este to ra trong malt ti c mi c trng.

c trng cho mc h hp ngi ta dng h s h hp k=CO2/O2. Chng hn soxi ho ng trong u kin hiu kh di tc dng ca enzim oxydaza:

C6H12O6 + 6O2 6 + 6H2O + 2322kJ

K=2

2

O

CO= 1

Khi oxi ho cht bo : K = 0,5 ; protein K = 0,8Khi h hp ym kh K > 1 khc nhau gia qu trnh ngm v ny nm l ch h s h hp k khi ngm

thng > 1 , khi ny mm K = 0,74-1,00. T ngi ta khng nh khng phi s haon cht kh v lng nhit thi ra do h hp trong qu trnh ny mm l hon ton tgluxit (tinh bt v ng)


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- Enzim Sitaza: y l tp hp enzim gm sitoplastaza v sitaza chng hot ng utin ngay khi ht va ny mm v c gi l qu trnh sitoliza. Nhng enzim ny phnt hemixenluloza v polisacarit khc dng gel hnh thnh cc sn phm c gc C5,C8,... S phn ct hemixenluloza nh enzim sitaza khi ny mm ng vai tr rt quantrng trong sn xut malt. Nh mng ngn cch gia ni nh v v tru b ph vto

u kin cho cc enzim amylaza v proteaza d dng hot ng trong lng ni nh.-Enzim amylaza: Nhm enzim ny trong i mch ny mm bao gm -amylaza, -

amylaza v amylofattaza tp trung ch yu trong ni nh, ch c mt s t trong mngngn gia ni nh v v tru.

+Hot tnh -amylaza tng sau khi ny mm 36 ngy v hot tnh ny t cc i ph thuc nhit ny mm:

: tnm= 12140C Hmax= 1112 ngy

tnm= 18200C Hmax= 67 ngy

tnm= 25280C Hmax= 45 ngy

+-amylaza c sn trong ht i mch dng lin kt v t do. Trong qu trnhy mm hot tnh -amylaza t do tng ln 34 ln.+Amylophotphataza t do khng c trong i mch m chc hnh thnh sau

ngy ny mm th 2, sau 8 ngy th t cc i nn hot tnh tng 150200 ln. tng hot tnh chung ca amylaza sau khi ny mm - k hiu l DC (hot lc

diastaza) c xc nh bng lng ng maltose to thnh, c th biu din theo n Vindich-Cotback ( 0Wd ) hay theo n v Lintner (0L ) vi quan h:

oL= 5,316+Wdo

Tc dng ca amylaza n cc qu trnh ho sinh nh ny mm v c bit trong qutrnh ng ho sau ny c ngha n sc quan trng trong qu trnh sn xut biau, mch nha.

c ch ca qu trnh ny mm ch yu tch t tht nhiu amylaza hot ng khi ng ho chng c th chuyn tinh bt (ca chnh malt v ca cc nguyn liu thayth malt) thnh cc sn phm trung gian (dextrin) v cc ng nh maltotriaza,maltotetroza, maltodextrin, glucoza, maltoza. Ngoi ra khi ny mm cn to thnh mtng ng kng saccaroza v mt t ng fructoza (tng hp trong phi). Tt ccc loi ng lm cho malt c v ngt, hm lng ca chng trong malt tng ln 34 lnso vi trong ht ban u

( trong ht ban u c 2,32,5% ng, trong malt ti c 8,59% ng ).Trong thi gian ny mm c khong 10% tinh bt b bin i, trong khong4,55% tinh bt b hao tn do h hp v to r, mm phi, lng tinh bt cn li b binhnh (thu phn c mc ) nh trn trnh by. Kt qu lm cho tnh cht tinh bta malt khc vi tinh bt ban u: kch thc ht tinh bt gim, rng (xp) gia cct tinh bt tng ln, t l Am/Ap cng c thay i,

-Enzin proteaza: C hot tnh trong ht ban u khng ng k nhng khi ny mm ng ln 48 ln, tng nhanh hn hot tnh amylaza v t cc i vo khong ngy ny


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m th 5. S thu phn protein bt u bng tc dng ca proteinaza to thnhalbumoza, polypeptit, pepton v sau di tc dng ca peptidaza to thnh cc axitamin. Tuy nhin s bin i ny thng khng hon ton (n cc sn phm cui cngl cc axit amin) v cc u kin ny mm (cc thng s ny mm) thng khng phil u kin ti thch cho hot ng ca h enzim proteaza ca ht.

Chng hn: topt peptidaza < 500C pHopt peptidaza = 7,68

topt proteinaza = 60oC pHopt peptidaza = 4,5 5

ty mm 600C pH t= 6,87

Tuy nhin, v nhit ny mm tng i thp v thi gian ny mm ko di s c sthu phn protein tng i su sc to thnh mt lng ng k cc polypeptit vaxit amin. Tc ng ny ch din ra vi ni nh, trong khi protein lp ngn cch ninh vi v v c phn v malt th khng b bin i c trong cc qu trnh ch bin v sau(sy malt ti, bo qun malt, nu v ng ho khi sn xut bia, mch nha). Chng s

n ti trong b malt khi lng, lc dch ng. thu phn protein bi enzim proteaza lm tng lng nit ho tan (albumiza,pepton, polypeptit, axit amin), mt phn cc cht ny d b kt ta khiun si dch ng(vi hoa hublon - tc qu trnh hublon ho trong sn xut bia). Phn khc vn tn ti ng ho tan, phn ny c bit c gi tr trong sn xut bia (l ngun nithu cchom nem, to bt, kt ta t lm trong, to v bia...) vi t l thch hp, nu qu cao thbia d bc v c trli khi tn tr bia.

bin i cc sn phm thu phn proteaza khi ny mm i mch c th hintrong bng 4 (trang 139-1)

Qua bng ny, ta thy trong khong na thi gian ca qu trnh ny mm (t ngy 1 (

4), sn phm thu phn protein tng lin tc, n na thi gian sau th dng li hoc gimn. C th gii thch u ny l do hot tnh ca enzim proteaza gim dn v sn nh,v mt phn sn phm thu phn c dng vo vic to mm v r phi (chim 1112%ng protein ca ht).

Ch s quan trng c trng cho qu trnh thu phn protein khi ny mm ni ring vc thu phn ca malt ni chung l lng nitformol (lng sn phm nitbc thpc to thnh c cha nhm axit cacboxylic _COOH t do nh axit amin, peptic...).Hot tnh enzim proteaza c xc nh theo ch s ny (phng php chun formol)ng hiu s ca lng nitformol tng cng (chung) khi ng ho (ti thi m cnxc nh hot enzim) v lng nitformol ban u.

- Enzim esteraza: Xc tc cho qu trnh este ho, trong i mch ch yu l nhmenzim photphotaza bao gm: fitaza, saccarofotfataza, glicero fotfataza, nucleotidaza vamylofotfataza.

+Bin i quan trng c trng l s thu phn phitin di tc dng ca enzimfitaza gii phng ru inozit, mui fotfat v axit fotforic t do.

+Saccaro fotfataza, glicero fotfataza, nucleotidaza thu phn mi lin kt este cacc cht hu ccha fotfat tng ng v gii phng ra axit fotforic t do.


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+Amylofotfataza ct gc axit fotforic ca phn t amylopectin. Do kt qu caqu trnh tch t v bin i enzim m lng cc cht ho tan (cht chit) tng ng k:ban u ch c 6,58%, khi ny mm tng ln 26% (tnh theo cht kh) nhng do tn haoh hp v to mm nn cn li trong malt l 1415%.

ng h hp v cc qu trnh bin i ho sinh khi m mm ph thuc vo mc

thng kh, tc l ph thuc vo t l O2 /CO2 trong khi ht ang m mm. Trongkhong na u thi k m, xy ra s tch lu enzim ln nht th s thng kh l cnthit (ngy 14). Sau nn dng thng kh gi lng CO2 li (nng khng qu20% trong hn hp kh) hn ch ti a s h hp ca phi (nhm hn ch s hao hti a do h hp). Khng kh thi cho khi ht cn c m W=98 100% v nhit phi b hn nhit m 230C.

3.4.2. c m m mm ca cc loi ht khc nhau:Nh trn ni, vi mc ch s dng malt khc nhau m ngi ta dng cc loi ht

khc nhau sn xut vi ch cng ngh ph hp.- Trong sn xut malt dng lm tc nhn ng ho trong cng ngh ru etylic th

cht lng malt c ng gi bng hot tnh tng qut ca h enzim amylaza. Nu mm nhit 14160C trong ttg trong thi gian di th s tch luc rt nhiu ccenzim _amylaza, dextrinaza. Chng hn: i mch 10 ngy, yn mch 1012 ngy,ch en 78 ngy, k 56 ngy.

-Trong sn xut bia, sn xut malt sng mu (malt vng) ngi ta m mm nhit khng qu 180C. sn xut malt en (malt caramen) ngi ta m mm nhit cao hn nhng khng vt qu 250C, thi gian m di hn qu trnh thu phn tinht v protein c su hn.

- Trong sn xut mch nha, malt cng l tc nhn ng ho (chnh thc hoc b sung- ng ho sb), nu yu cu thi gian m mm di hn, nhit cao hn la :12

ngy, k: 7 ngy, ng: 8 ngy, to=2832

0

C.3.4.3. Cc phng php ny mm:- Ny mm khng thng gi:y l phng php cn hin vn cn s dng nhiu trong cc cssn xut malt

bia cng nh malt mch nha, nng sut t loi rt nhn rt ln (vi trm tn ht /ngy). Sn m mm bng xi mng phng, nhn, hi nghing d thot nc. Trongphng m cn t qut y, qut ht lu thng khng kh v h thng u ho nhit nhn to (nu sn m chm mt na sc li v nhit u ho nhng kh thotc v vn hnh).

t sau khi ngm c thnh ng trn sn ri san u thnh lp dy ban ukhong 40 cm, sau gim dn cho n 1012 cm giai n cui. Thng xuyn otrn lp malt, thng gi i CO2 v cung cp khng kh mi. C th chia lm 3 thik ny mm nh sau:

+ Thi k 1: (2 ngy u) tng dn nhit n 13140C, o trn 23 ln / ngy.+ Thi k 2: (3 ngy tip theo):nhit khi ht tng nhanh do h hp mnh. Nhit

cao nht cho php i vi malt vng 1617 0C, malt en 19200C, o trn 34 ln /ngy.


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+ Thi k 3: (2 ngy cui) cng h hp v ny mm gim, o trn 23 ln /ngy. R mm bng 1/5 ln chiu di ht, mm di 3/4 ln chiu di ht.

- Ny mm thng gi trong ngn:Phng ny mm gm cc ngn hhnh ch nht: di 1015 m, rng 34 m, cao 1,82

m, y hi nghing d thot nc, cch y 60 cm t mt li sn c l vi din tch

1520 % din tch sn, mi ngn c th cha c 1012 tn ht kh.t ngm xong vo cc ngn ri san u theo chiu dy ca lp malt trong ngy

y mm, lng nc ngm tha trong ht theo l li sn xung y ri ra ngoi. Maltc o trn nhmy o trc vt xon thng ng. Thi khng kh kh vo lp ht(qua sn y) sau 1820h th thi khng kh lnh v m ri o trn. Ch lm vic cangn ny mm xem bng sau. (bng 5-trang 115).

Sau khi ny mm xong, malt ti c vn chuyn n l sy (nu sn xut bia haych nha), hoc my nghin sa malt (lm tc nhn ng ho trong sn xut ru, tinht).

- Ny mm thng gi trong thng quay:

Thit b l thng hnh tr bng thp, t nm ngang trn hai gi v quay c trnhai gi ny nhng c quay thng vi h thng truyn ng bnh rng. C hai loithng quay: kn v h. Trong , thng dng hn l loi thng quay kn, chiu di thng46 m, ng knh 2,33 m m bo ny mm c 1012 tn ht kh. (xem hnh 47trang 156).

t ngm xong c a vo qua cc ca pha trn thng ri phn bu trn lithp. Khng kh lnh v m thi vo thng qua cc lp malt ang quay theo thng ritheo cc ng gp ra ngoi thng (nhqut y - ht). Thi gian ny mm 7 ( 10 ngy, tc quay thng 2,53 h/vng.

Sau khi ny mm xong cho thng quay vo v tr ngc vi lc np ht ri m ca

chn tho ht ra qua chnh ca np ban u.- Ny mm theo phng php bn lin tc v lin tc: (xem ti liu tham kho)- nh gi cht lng malt ti:Cht lng malt ti c nh gi qua phng php phn tch cm quan, hot

enzim v thnh phn ho hc v thng c gn lin vi mc ch s dng malt (snxut ru, bia hay mch nha). Malt ti tt c mi da chut, nu malt c mi ch, vthiu c th do malt bn, b nhim VSV, khi ny mm khng ng ch cng ngh. Htmalt phi mm, nu ht cn cng hay nho nt th coi nh phm cht xu.

u t quan trng ng gi cht lng malt l t l ny mm v chiu di, lna mm, r. Malt tt c t l ny mm trn 85%, i vi malt vng chiu di mm2/33/4 chiu di ht, malt en c chiu di mm 1/21 chiu di ht, r di 1,22 cm.

Ch tiu quan trong ca malt l hot tnh enzim. Sau y l bng ch tiu hot tnhenzim i vi malt dng cho sn xut ru.

t Kh Trung bnhLoi malt AC

(g/g.h)DC

(mg/g.h)AC

(g/g.)hDC

(mg/g.h)AC

(g/g.h)DC

(mg/g.h)


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i mchn mchMalt en v la mK

>6>5>7>3

>35>45>35>100

4-63-54-72-3

25-3535-4525-3570-100

3-42-33-41-2

15-2525-3515-2550-70

Ghi ch:

AC - hot tnh amylaza: kh nng thu phn tinh bt thnh mantoza v cc dextrinkhng lm i mu dung dch it, n v =g tinh bt / g malt trong 1 h nhit 30 oC.

DC - Nhkh nng thu phn cc dextrin thnh maltoza, n v = g dextrin / g malt nhit 300C.

Cht lng malt dng sn xut bia trc ht l hot tnh amylaza (AC) c biuth bng lng maltoza to thnht tinh bt di tc dng ca enzim c trong 100 g maltvng ti tt c AC bng 300 - 400, malt en ti tt c AC = 400 - 500.

3.5. y malt:

Malt ti c m cao (42 - 45%) nn khng th bo qun c (tr malt ti dng)

trong sn xut ru sc em i nghin thnh sa malt - l ch phm enzim ngho. Mt khc, mi v v thnh phn ho hc, cu trc ca malt ti khng thch hp chovic sn xut bia (trong mch nha) nh: hu nh khng c cht mu, cht thm (thmch c cht thm phn tc dng vi bia), thnh phn protein c th lm gim bn hol ca bia (bia d bc, bt khng bn), khng c tnh cht nghin vthnh bt...V limalt vn l nng sn ch bin c th trng ton cu (thng mi th gii) nn nht thitphi sy malt.

Khi sy phi ch n gi li c lng enzim ( tch luc trong qu trnhy mm) cho sn xut bia (hay mch nha). Mun vy khi xy dng th sy malt phihiu r quan h gia nhit sy v m ca malt (c tnh n bn ca enzim donhit v m. m malt khi sy cng cao th enzim v hot cng nhanh).

V d: Khi sy malt vng: giai n ht c m 30% th nhit sy cho php l400C. khi sy nhit 500C th m malt phi gim cn12%. Nu nhit sy tngn 600C th m malt cn 8%.

Malt en c th sy nhit cao hn malt vng, tuy nhin nhit sy khng cqu 1040C khng ph hu nhiu enzim .

Khi malt c m cao, nu tng nhit sy cao s to nn malt dng keo kh v khong nhit 60 oC xy ra hin tng h ho tinh bt, nu tip tc nng nhit th tinht chuyn sang trng thi thoi ho - to thnh ht cng. Trong khi , mt s protein btc dng bi enzim proteaza s ho tan v thm vo ht tinh bt v chuyn sang dngkeeo khi b mt nc. Malt dng keo kh do tinh bt v protein b bien hnh nn gim ho tan, gim chit.

3.5.1. Nhng bin i ca malt trong qu trnh sy:Theo tng dn ca nhit khi sy m ta c th chia ra lm 3 thi k nh sau:- Thi k sinh l: nhit sy tng dn v t n 450C: malt v r vn tip tc pht

trin, m cn khong 30%, s bin i do thu phn, do enzim vn tip tc.-Thi k men (enzim ): Nhit sy tng dn t 45 - 700C: Cc qu trnh sng b

ngng li: khng h hp, mm r khng cn pht trin nhng s thu phn v nhng


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bin i khc trong ni nh vn xy ra rt mnh v nhit ti thch ca a s men nmtrong khong 45 - 600C. thi k ny, m ca malt en t 20 - 300C, ca malt vngcn 10 %.

-Thi k ho hc: Nhit sy tng t 70 -1050C: nhit > 750C th cc hot nga enzim b ngng tr v mt s enzim b v hot mt phn (do nhit cao), s co li

cc cht keo ca ht hp th v chuyn sang trng thi khng hot ng. V d: nhit > 400C, enzim peptidaza ngng hot ng, nhit 600C cc enzim sitaza vphytaza mt hot tnh v enzim amylaza gim hn hot tnh. Ring i vi enzimproteinaza do kh nng chu nhit rt ln nn hot tnh gim rt t khi sy malt vng.

Thi k ho hc c c trng bng s to thnh cc cht thm, s bin hnh protein, lm yu hay v hot tng phn cc enzim.

Qu trnh quan trng nht xy ra trong qu trnh sy l phn ng melanoidin do tcng tng h gia nhng cht cha nhm cacbonyl (-COO) vi nhng cht c nhmamin (-NH2). Kt qu malt sy trnn c mu ti (sm) hn, c mi vc trng camalt bia (hay malt mch nha). Theo Hydge, qu trnh to melanoit trong khi sy malt c

th tin trin theo 3 giai n:+ Giai n u: c c (nng cao nng ) cc cht ng v axit amin do mtc khi sy to thnh cc tinh th khng mu.

+ Giai n gia: xy ra s kh hydro, b gy phn tng, phn t axit amin o thnh cc cht khng mu hoc c mu vng nht, c tnh kh mnh, tiu biu l mt cht nh:CH(OH)C(OH)CHO, hydroquinon (khi b kh hydro chngtr v dng quinon ban u). Nhng cht kh ny cng vi nhng cht khc tothnh trong cc qu trnh nu bia (ng ho, un si dch ng vi hoa hublon) ngvai tr rt quan trng trong qu trnh oxy ho - kh ca cng ngh bia. Chng trc tipo nn mi v, bt cho bia, l cc cht bo v chng li s oxy ho, chng li s kt

a keo lm cho bia c+ Giai n cui: to thnh nhng cht c mu sm.c phn ng melanoit ph thuc vo nhit , pH, thi gian, thnh phn cc cht

tham gia. Trong s cc axit amin tham gia mnh nht l glixin v alanin, sn phmmelanoit ca chng cho mu rt m v c mi bia, cho mi thm mnh hn c l valinv loxin, phenylalanin v valin cho sn phm c mu ti, mi hoa hng nh, lxin chon phm nht mu v c mi bnh m r. Trong s cc ng tham gia phn ngmelanoit theo th t l: arabinoza > glucoza > galactoza > fructoza, trong cc pentozanh nht l ng cexitoza. Thnh phn nguyn t cu melanoit nh sau: C= 54 - 60%,O= 31,3 - 35,1%, H=4,9 - 5,2, N= 3,5 - 5,3%

Chng c cha cc nhm -CO, -COOH, -OH, M 1480. Ttrong ngnh bia, melanoitc ngha rt quan trng: gy mu v mi thm c bit, do c kh nng hot ng bt rt mnh nn l nhng cht to bt tt, ng vai tr bo v cc keo ngn cn st ta keo khng bn vng, c kh nng kh mnh lm tng bn, chng hin tngc bia do b oxy ho.

3.5.2. Cc phng php v ch cng ngh sy malt:- L sy nm ngang hai tng hot ng tun hon:


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y l loi l sy cn vi phng php sy i lu cng bc, hin nay vn c dng rt ph bin nhng nc c sn xut malt. y l mt to nh cao 20 - 25 mhnh vung hay ch nht c 5 tng vi cc chc nng: tng 1 t l t, tng 2 l bungnhit c t clorifer, tng 3 la bung trn khng kh sy, tng 4 l mt sng li vng 5 l mt mt sng sy trn cng c ng ht v qut ht thi m ra ngoi. Sng sy

lm bng li thp an dy vi din tch l chim 30 - 35% din tch sng.+ Tin hnh sy malt vng:Malt ti nhgu ti chuyn ln sng sy trn vi chiu dy lp malt 14 - 22 cm. tng

nhit sy u n sau 6 git 30 - 350C, m cn 2%. Sau 6 h tip tc t 500C, m xung cn 8 - 10 %. Nh vy phn ln m ca malt c tch ra ti y. u kinquan trng thu c malt vng cht lng tt l nhit sy khng c qu 400Ckhi m cha h xung di 12 - 14%.

Sau 12h sy sng trn, malt c chuyn xung tip tc sy kit sng di. Sau 11- 12h, nhit sy tng ln 55 - 800C, m cn 2,5 - 3,3%. Sau khi sy xong, malt cn ng li chuyn sang khu gia cng tip tc. Ch sy malt vng (xem bgn 6

- trang 170 ca gio trnh).+ Tin hnh sy malt en:Malt en sy lu hn v phc tp hn malt vng. u tin malt ti cng c vo

v tri u ln sng sy trn vi lp malt dy 22 -30 cm. Giai n u di 14h tngnhanh nhit n 40 0C, m xung 20%. Trong 3- 5h tip theo tng n nhit 60- 650C, tng cng o trn. Sau 24h th sy xong sng trn, malt sc chuynxung sy sng di. Tip tc thi k sy th nht trong 9 - 10h nhit 50 -550C, m xung cn 8 - 10%.Thi k th hai nhit tng ln: trong 4 - 6h nhit tip tc t750C, m xung cn 5%, trong 3 - 4h. Sau nhit tng dn ln 100 -1050C cho nkhi sy xong vi hm m cui cng t 1,5 - 2%.

V malt en sn xut lu hn, hao tn cht kh nhiu hn nn gi thnh malt en vbia en t hn malt vng v bia vng. phn no h gi thnh, ngi ta sn xut mt loi malt c bit nh malt caf, malt mecan thay th mt phn hay ton b malten khi sn xut bia en.

Ch sy malt en (xem bng 7 - trang 171 ca gio trnh).- L sy nm ngang mt tng:y l loi l sy tng i mi theo phng php sy i lu cng bc. Tc l tc

nhn sy l khng kh nng c thi cng bc qua lp malt c dy 8 - 10cm mkhng o trn. Tin hnh sy theo phng php ngc chiu tnh tit kim nhit vt m cui cng ca malt. Ngha l tc nhn sy ban u c em i sy cho lpmalt c sy kh bng sng th hai bc hi tc nhn sy mang m ca chnh sngu tin ny. Sau khi malt sy xong chuyn i gia cng tip th li chuyn malt ti n sy sb tip tc (i chiu tc nhn sy)

- L sy hot ng lin tc:Trn csnhng loi thit b sy ht kiu thp li dng tnh t chy ca khi ht,

Vin Hng Lm Nng Nghip latvia (mt nc Cng Ho thuc Lin X c) thit kv ng dng thit b sy malt hot ng lin tc kiu thp, gi tt l LCXA. Thp sy cao


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15 m c chia thnh 3 khu chnh: khu tip nhn v sy sb malt ti, khu sy v khup nhit - thu nhn sn phm. Cu to ca l sy xem hnh 51 - trang 173 ca gio trnh.

Thit b sy lm vic nh sau:Malt ti c gu ti chuyn ln bonke cha v ti y n c sy nng sb

nhit 30 0C, m ban u ca malt ti khong 44%. Nhcc van u chnh, malt

ri thnh ng lin tc xung thn thp. Ti y c chia thnh 4 vng nhit sy(khng kh nng qua calorifer c trn vi khng kh ngoi tri nhqut ri cp chocc vng sy), malt i qua cc vng sy ny v t c m sau y:

Vng sy Nhit khng khoC m ca malt %Ban ke sy sb (malt ti)Vng syIVng sy IIVng sy IIIVng sy IV

3050678185

44261263

Malt lin tc chy qua cc ca thu, vo ban ke cha y ri nh vt ti ra ngoi.ng thi gian vn chuyn nguyn liu, sn phm v sy trong khong 9,5 - 10h (thigian sy 8 h), c th tng ho hon ton qu trnh sy.

3.6. Tch mm, r, bo qun malt:

- Tch mm, r malt:m v r malt kh c tnh ht nc mnh, c vng kh chu, v vy phi tch b

m r hn ch s ht m, lm p mt hng malt, m bo cht lng malt, nubia khng gy nn vng kh chu. Tch mm v r malt phi tin hnh ngay sau khi

y, v nu lu r s ht m trnn dai rt kh tch khi malt. tch mm v r malt ngi ta dng thit b ct l mt li thp hnh tr quay chmv t hi nghing trong mt thng kn, l li c kch thc 25x15 mm. Bn trong ctrc quay cng chiu nhng nhanh hn, trn c gn cc qut c cc l thp nh mp hicong ct mm, r (xem hnh sch tham kho). Khi quay thng th mm, r s b ctcng vi ht ra ngoi li, ri nhqut ht bi mm, r ra ngoi.

- Bo qun malt:t malt v nht l v tru sau khi sy rt kh v dn, thnh phn cha n nh, nn

u em i sn xut bia th s khng tt lm (khi nghin d b nt v tru nn khi nukh lc, dch ng c th ln men chm, bia km bn). V vy ngi ta thng boqun malt sau khi sy xong t nht l 3 -4 tun, nhiu nht n 2 nm. Trong qu trnho qun nh vy, m malt tng ln 5 -6% nn v tru trnn dai hn, ht do hn,th tch ht tng ln cht t, malt sn nh v thnh phn ho hc v hot tnh enzim.t nht l malt c bo qun trong thng st ty, nh k thng gi, nhit bo qun200C.

i nc ta hin nay, ton b lng malt cho sn xut bia u phi nhp khu nnthng qua qu trnh bo qun, chuyn ch (bng tu thu) rt lu, bo qun trong


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c nhit i m cao nn m ca malt khi em sn xut bia thng cao n 8-9%,nn malt b gim cht lng nhiu.

- nh gi cht lng malt kh:

i mch ban u

Malt sau khi syMalt sau khi bo

qun

100lit 100kg m 15%

90lit 75kg m 2%100lit 78kg m 4%

+Cm quan:Mu sc: Malt vng : vng ti

Malt en : ti sm, v ng nhKch thc, hnh dng tng t nhi mchMi v: c trng cho tng loi malt

Malt vng : ngt duMalt en : v caf, ngt mnh

+ sch: Khng ln tp chtt vti a 0,5%t khng ny mm: 5%

+ Ch s chc:* Dung trng:

Malt rt nh: 480 - 500 g/lMalt nh: 500 - 530 g/lMalt trung bnh: 530 - 560Malt nng: >560 g/l

* Trng lng 1000 ht: 25 - 38 g

* Ch s ho sinh v ho hc: xem gio trnh (trang 181, 182)3.7. thut sn xut mt s loi malt c bit:

lm tng cht lng bia, sn xut nhng loi bia c bit, ngi ta thng dngnhng loi malt cht lng c bit sau y:

- Malt caramen: dng sn xut bia en lm cho bia c v caramen, mi thm mnh,mu cnh gin, to bt tt. Malt caramen c ch bin t malt ti hay malt kh nhqu trnh ng ho ph v sy nhit cao hn n 110 1700C.

Ngi ta dng malt ti hay malt kh c chit cao (hot lc enzim cao) ti nc10 15 lit/100g - yn 12h din ra qu trnh thu phn nhng cht ni ti trong malt,sau malt c sy trong l c bit c kh nng nng nhit sy ln n 1700C, thigian sy 2 3h.

100g malt kh thng s thu c 90 95 kg malt caramen, dung trng 400 450g/l, m 5 6 %.

- Malt c ph: ch bin t malt vng c qu trnh ng ho ph v sy nhit cao220 2250C. Malt ngm vo nc nhit 700C trong 12h to th phu phn chtmu. Sy nhit cao trong 2 2,5h, h nhanh nhit sau khi sy. Ht malt phi c


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mu ging mu ht c ph rang, mi thm c ph rang. T 100kg malt thng thu c85 90 kg malt c ph dung trng 400 450 g/l, m sau khi sy 2 3%.

- Malt melanoit: cn gi l malt meland c ch bin ti mch c hm lngproteincao (>12%). Ny mm nhit cao 200C trong giai n cui 24h, ng sau 1 2 ngy nhit tng ln 50 520C, tip tc trong 16 24h. Sau tin hnh sy

nh sy malt en.


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Chng 4: SN XUT ENZIM TTHC VT

Enzim t thc vt cng chim mt t l thch ng trong cng ngh sn xut v sng enzim ni chung. Mt s loi enzim c s dng nhiu trong y hc, thc phmv cng nghip. c bit trong nghin cu khoa hc ngi ta rt ch trng chit tch v

xc nh c ch tc dng ca cc enzim trong cc m thc vt nh nhm enzimglycoxydaza, nhm enzim oxy ho kh polyphenoloxydaza (EPPO) mang li nhnggi tr l thuyt v thc tin rt cao trong thi gian gn y.

4.1. n xut ureaza tu ra:

Chi hu Canavalia chu phi, Vit Nam c loi Canavalia ensifomis - c gil cy u ra, u tc; ht dng cha bnh lui hn, nc ct, yu thn. Trong ht u ra,hm lng ureaza c tht n 20% cht kh do ay l ngun nguyn liu quantrng thu nhn ureaza.

- V mt ng dng: ureaza c s dng trong y t xc nh hm lng ur trong

huyt, bng quan, c trong thnh phn ca thuc chng thn nhn to. Trong ch bint s loi c c mi khai (i, nhm, mp) th dng ureaza kh mi khai rt hiuqu.Ureaza c th xc tc thu phn ur c trong v ngoi t bo (c th).

+ Chit xut ly enzim:Nghin k bt u trong dung dch HCl 0,4% c thm EDTA (trilon B, complexon III)

5.10-3 M v L.cystein 5.10-3 M. Sau ly tm tch b ly dch chit.

+ X l nhit: Nng nhit nhanh n 60

0

C, gi trong 30 pht em ly tm tch b cnt ta.+ Siu lc: dch ly tm c lc qua mng siu lc loi b peptit v polypeptit c

trng lng phn t b (M>500)+ Kt ta bng axeton:ch lc c x l bng axeton lnh (t l 1:1), ly tm tch kt ta. Phn kt ta em

ho tan trong trisbuffer 0,1M, pH = 7 cha EDTA v L.cystein 5.10-3 M.+Sc k trao i ionch enzim c chy sc k trao i ion trn ct cha DEAE xenluloza vi gradien

ng NaCl t 0 1M. Phn n cha enzim c sy thng hoa (ng kh) vi chtsacaroza lm cht n nh vi t l 2,5mg/1mg enzim.

Ch phm thu c c hot tnh tng 25 ln so vi ban u v hiu sut thu hi 43%.Xem bng 8 ca gio trnh (trang 186). Mt s thng s ca qu trnh thu nhn ureza

u ra.

4.2. Thu nhn bromelain tda:

- Gii thiu qu da: tn latinh: Bromelia ananas-L: thuc chi da n qu. Ngoi racn c chi da di ( ly si ).

(NH2)2CO + H2O ureaza 2NH3 + CO2


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Bromelain c nhiu trong ph liu da nh v, li, chi, li u.Gii thiu c m ch phm Bromelain (Xem qu trnh trang 187-189).Nguyn tc chung thu nhn Bromelain.

Ghi ch: kt ta bng (NH4)2SO2 lnh hay axeton lnh t l 1:1.- Thu nhn Bromelain bng phng php nhanh s dng CMC.Thu nhn Bromelain bng phng php m t nh trn i hi thi gian lu, kh

c, lng dch ln, kh bo qun. Hin nay mt s nh nghin cu dng phng phpnhanh tch Bromelain bng CMC. Cho php thu c Bromelain bt trng c hot tnhcao, thi gian nhanh, n gin nh sau:

+ Cng ngh ch to CMC (xem gio trnh trang 191).Ch : c th thay vi mn (hay gt) bng bng nn ( Xenluloxa ~ 100%).+ Tch chit Bromelain t dch chit da bng CMC.

CMC c tm t bng dung dch m photphat 0,05 M, pH= 6,1. Cho dch chi da( chi da nghin p lc c dch) vo, thnh thong khuy trn. EmzimBromelain s hp th ln b mt ca CMC. Sau 2 gi ly ra vt nc loi b cn bnbm vo CMC, ra tri cc prtin khng phi enzim bng m photphat pH=6,5. Sau cho phn hp th ln 1. Dung dch phn hp th l m photphat pH=7,1, NaCl 0,5 Nkhuy trn. Sau 2 gily ra vt c dung dch m dc cha bromelain. Tip tc phnp th nh ln th hai ri gp chung dch chit ca c hai ln kt ta enzim bng axeton

hay cn lnh.i phng php ny hiu xut thu hi t 0,1% so vi chi da ti, ch phm chot tnh 24 n v (mg ch phm enzim. So vi phng php kt ta t ban u bng(NH4)2SO4 th sch cao hn hai ln, thi gian nhanh hn, tin hnh thun li hn.

- Mt sng dng ca ch phm bromelain:+ Thu phn gan b:Gan b c x l bng ch phm bromelain trong10 gi 1-55 0C, sau v

hot enzim 100 0C trong 3-4 pht. Dch oc lc v ng kh thnh dng bt l chphm hn hp axit amin y t (sn xut cc dch truyn m y t). Thnh phn axit amina ch phm thu phn gan b bi bromelain c trnh by bng 12 (trang 193 ca

gio trnh).+S dng bromelain lm ng sa: lm ng sa trong phng php truyn thng (trong CN-SX photphat) ngi ta s

ng renin thu c t ngn th 4 ca d dy b. Hin nay, lng renin cha p xng nhu cu ca cng ngh. Trong nhng nm gn y xu hng s dng proteaza thct thay th mt phn renin trong ch bin phomat ang c pht trin. Trong

Ph liu da lm dp chit lc ly tm kt ta lytm lc ch phm k thut Sc k trao i ion sy thnghoa sn phm tinh khit .


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ng k nht l 2 loi ch phm: ficin t cy c ficus v bromelain t da. Kt qungng xem bng 13 (trang 194 ca gio trnh)

+ S dng bromelain thu nhn cc cht c ch proteaza.Trong mt s loi cquan ng vt, thc vt tn ti cc cht c ch enzim, trong

c cht c ch proteaza, chng c bn cht protein nh enzim. n hnh nh trongcc ht hu: u tng, u xanh, u vn, ht mt, ni tng ca mc nang, a, tronga (tn ti song song vi bromelain). Cc cht c ch thng tc ng mnh ln mt senzim, trong c c enzim trong ng tiu ho (ca ngi v ng vt) nh: ficin,bromelain, tripxin. thu hi c cc cht c ch ny, ngi ta dng phng php hini, hin nay l dng enzim cnh. Bromelain sau khi thu nhn bng CMC nh trnc cnh trong gel sepharoza ri nhi vo to ra ct phn ng. Dch nghin, lc phliu da c cho chy qua ct ny: u tin pH photphat 4,6, sau pH = 7,6 7,8,tip xy ra qu trnh lin kt gia bromelain vi cc cht c ch. Thu hi cht c ch

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