I HC NNG

TRNG I HC BCH KHOA

KHOA HO K THUT
B MN CNG NGH THC PHM

GIO TRNH
CNG NGH SINH HC THC PHM II
THI LNG 45 TIT

NNG 2007

M U
Cng ngh sinh hc l mt ngnh khoa hc ang c th gii quan tm. Gi
tr ca cng ngh sinh hc l ch l mt cng c c th p dng cho nhiu ngnh
kinh t nh sn xut lng thc thc phm, chn nui th y, cng nghip dc v
cng nghip ho hc, chuyn ho sinh khi thnh nng lng, x l ph liu v ph
liu cng nng nghip, phng chng nhim v v sinh mi trng
- Cng ngh sinh hc gip cho cc chng trnh ci thin nng nghip,
ngh vn v ngh rng tng nng sut cy trng, tng cht lng dinh dng ca
nng phm, chn ging chng chu vi su bnh v vi thi tit t ai khng thun
li v to ra cc ging thch nghi vi cc iu kin th nhng v kh hu nht nh.
ng thi n cng to iu kin duy tr mt s a dng di truyn rng gia cc
ging cy trng v gi gn cc ngun gen c to nn t cc h hng hoang di
ca chng. Tnh a dng di truyn c th hin thng qua mt s lng cc ln cc
kiu kt hp gen c trong mt s c th ca mt loi v thng qua s khc nhau v
cc tnh trng ca cc ging trong cng mt loi: kiu sinh trng, tnh khng su
bnh, tnh khng vi ngoi cnh (sng mui, hn, nng) v nng sut. Sau khi
nghin cu kt qu ca nhiu t hp lai mt cch cn thn v nghim tc, cc nh
chn ging da v tnh a dng di truyn chn ra cc dng c tnh trng mong
mun. thnh cng, nh chn ging phi c c trong tay vn di truyn cng ln
cng tt. Vn di truyn ny bao gm cc ging cy trng, cc ging chng chu, cc
ging a phng (cc ging ny thng b b qun v do nng sut thp nhng n rt
qu v c tnh chng su bnh v cc iu kin ngoi cnh khng thun li). Trong
vn di truyn cn phi k n cc cy hoang di c tc dng tng sc sng cho cc
cy ging trng.
Tnh a ng di truyn l nhn t bo m cy trng khng b cc tai bin kh
hu hoc su bnh tiu dit hon ton. Cy trng cng thun nht th cng d b hi
khi c tai ho.
- Cng ngh sinh hc cng c ng dng rng ri trong lnh vc thc phm
v dinh dng. Ngnh cng ngh ln men l mt b phn ca cng ngh sinh hc
sn xut ra nhiu sn phm rt th v cho ngnh thc phm. Hoc vic sn xut nm
men cng c mt ngha rt quan trng. Ngi ta c th s dng mt lng nh nm
men b sung protein, vitamin v cc cht khong cho thc phm. Ngoi ra, sinh
khi nm men l ngun thc n b sung trong chn nui rt c hiu qu.
- Cng ngh sinh hc gip cho chn nui v th y to ra c nhng ging
nui mong mun v sn xut ra cc loi vacxin phng chng bnh tt cho cc vt
nui. V d: vic cy chuyn hp t b to ra c ging b tt, c sc chu ng
cao. K thut cy chuyn c thc hin nh sau: gy s rng trng mt con b ci
c cc c im m ngnh chn nui cn n v em th tinh nhn to bng tinh
trng ca mt con b c c nhng c im nh ngi ta mong mun. Cc hp t
hay phi c thu nhn bng cch ra d con. Lm ng lnh phi trong nit lng

-179oC v c th vn chuyn trng thi ny: 1000 phi ng lnh khng nng qu
50kg. Phi c cy vo b ci cha h. B con pht trin ln t cc phi ny s
ra i trong mi trng sng sau ny ca chng v khng phi ng u vi nhng
s bt li ca mi trng m cc sc vt nhp hay gp phi. S bo v bng cc
khng th ca b m v nh b sa ca b m m sau khi ra i b s sinh c th
chu ng tt hn i vi cc loi bnh tt thng thng.
- Cng ngh sinh hc cn tch cc gip ngnh y t bo v sc kho ca
cng ng. Ngi ta ni rng y hc d phng (v lu di hn l y hc d bo da trn
hiu bit v c im di truyn mi c th) s mang li li ch nhiu hn cc phng
php iu tr. Mt lnh vc ca cng ngh sinh hc c th gp phn quan trng trong
gii quyt cc vn y t, trong khun kh mt chnh sch u tin cho y hc d
phng, l ci tin cc vacxin hin c v ch to ra cc vacxin mi. ng thi n
cn gip ch trong vic sn xut cc loi khng sinh, vitamin v cc thuc cha bnh
khc. Nhng nm gn y, nh vo k thut di truyn ngi ta tm cch tch chit
cc hot cht ca thc vt bc cao lm vt liu xut pht cho hng lot loi thuc.
- Cng ngh sinh hc cn gp phn trong vic sn xut ra nng lng nh: sn
xut cn bng con ng ln men, chng trnh biogasv chnh cc ngun nng
lng ny li i phc v cho cc qu trnh sn xut khc.
- Cng ngh sinh hc cn tham gia vo vic chuyn ho cc cht v ngn chn
s nhim mi trng. Sn phm ph v cc cht thi cha hydratcacbon c th
c chuyn ho bng cch ln men nh cc vi sinh vt thng thng hay bng cc
qui trnh cng ngh sinh hc. Hoc cc k thut ti t hp AND cng s gp phn
tch cc tch c cc ging vi khun thch hp nht cho vic ti u ho nhng s
chuyn ho . V d: chuyn gen m ho cc enzym xenluloza v hemixenluloza ca
Clostridium thermocellum thnh nhng loi Clostridium khc c th iu khin c
s chuyn ho xenluloza v hemixenluloza thnh etanol, axeton, butanol, axt axetic
v axt lactic. Dng mt vi ging a nhit Clostridium (toop= 65-75oC) c li l ct
gim c chi ph trong vic chng ct v s h c gi thnh sn phm.
ngn chn s nhim mi trng ngi ta c th dng cc loi vi sinh vt
khc nhau. Cc k thut ti t hp AND to ra nhng chng vi khun c th phn
hu v hp th mt s ln cc cht do cng nghip ho cht thi ra.
Nh vy cng ngh sinh hc c lin quan n nhiu lnh vc v bao gm cc
ngnh nh:
- Cng ngh di truyn
- Cng ngh nui cy m v t bo
- Cng ngh enzyme
- Cng ngh vi sinh vt
Trong hc phn ny s nghin cu v mt s phn ca cng ngh vi sinh vt.
Cc qu trnh vi sinh c s dng rng ri trong cc lnh vc khc nhau ca nn
kinh t. Nhng thnh tu khoa hc k thut v sinh ho cho php to ra nhng qu
trnh sn xut c nng sut cao da trn cc phng php cng ngh c iu
chnh c mt s sn phm thc phm, chn nui, thuc cha bnh v cc cht hu
c.

CHNG I: NHNG NGUYN TC HO SINH TRONG

CNG NGH VI SINH VT
to ra bt k mt sn phm ln men no u phi qua cc bc sau:
- Chun b mi trng
- Chun b ging
- Ln men
- Thu hi v tinh ch sn phm
to ra sn phm c cht lng tt v nng sut cao th cn hiu r cc
vn sau.
1.1 Phn loi sn phm ca cng ngh vi sinh vt
Cc sn phm ln men cng nghip c phn loi theo cc tiu chun
sinh l trao i cht. S phn loi ny da vo sn phm chnh ca qu trnh ln
men v cc qu trnh sn xut nh vi sinh vt lun lun to thnh nhiu sn
phm.
1.1.1 Vt ch t bo (sinh khi)
C cht
t bo
V d: + protein n bo (trng thi cht)
+ vi khun c nh m (sng): Rhizobium, Azotobacter, VK tr su
Bacillus thuringiensis
Vic tng hp sinh khi hay vt cht t bo ng nht vi sinh trng
ca vi sinh vt. Sinh trng v sinh sn gn lin vi nhau. Sinh trng l tng
khi lng, cn sinh sn l tng s lng.
1.1.2 Cc sn phm trao i cht
C cht
sn phm + t bo
1. Cc sn phm cui cng ca s trao i nng lng (cc sn phm ln
men)
V d: etanol, axt lactic, axeton-butanol
Ln men l qu trnh ym kh ca s thu nhn nng lng, trong
hydro tch ra c chuyn n cc cht nhn hu c (n khng ng ngha vi
s ln men trong ngn ng quc t - n c hiu l cc qu trnh sn xut
cng nghip nh vi sinh vt)
Mt s c th tin hnh ln men khi khng c oxy lm cht nhn hydro
cui cng (k kh tu tin), nhng nhm c th ln men bt buc th khng
cha cc enzym h hp. Cc hp cht hu c nhn hydro l nhng hp cht
c hinh thnh trong qu trnh trao i cht d ho. Sau khi nhn hydro, cc
hp cht ny thi ra ngoi t bo ging nh cc sn phm cui cng ca s h
hp. T ny ra vn l trong sn xut cn chn cc iu kin nui sao cho
cng nhiu c cht c chuyn thnh cc sn phm ln men cng tt.
V d: trong sn xut ru tng hm lng ru th cn tng hm
lng ng v gim cc yu t quan trng cho sinh trng.

2. Cc cht trao i bc 1
V d: axt amin, nucleotit, vitamin, axt xitric.
Cc cht trao i bc mt l nhng vin gch cu trc c trng lng
phn t thp ca cc cao phn t sinh hc ca t bo cht: axt amin, nucleotit,
nucleozit, ng, axt bo, vitamin Ngoi ra cc sn phm trung gian ca qu
trnh trao i cht (cc axt hu c trong chu trnh ATC) cng l cc cht trao
i bc 1. Cc c ch iu ho pht trin trong qu trnh tin ho bo m sao
cho cc cht trao i bc 1 ch c tng hp n mc cn thit.
3. Cc cht trao i bc 2
V d: khng sinh, alcaloit
Cc cht trao i bc 2 l nhng cht trao i c trng lng phn t
thp, khng gp mi c th, s phn b ca chng ch gii hn nhng n
v phn loi nht nh. Chng khng c chc nng chung trong trao i cht
ca t bo v t bo cng c th tn ti m khng cn n chng. Tuy nhin,
cc cht trao i bc 2 c th c ngha vi s sinh trng v cc c th sn
sinh ra chng. Chng hn mt s cht trao i bc 2 c vai tr trong s hp th
st khi thiu nguyn t ny. Thng cc cht bc 2 c to thnh khi s sinh
trng kt thc. Cc chng tn ti trong t nhin thng ch to thnh rt t
cht trao i bc 2, nhng cht ny c tch lu trong t bo hoc thi ra
ngoi.
4.Cc enzym
V d: enzym ngoi bo: proteaza, amylaza
enzym ni bo : asparaginaza, penixilinaza.
T bo vi sinh vt cha khong 1000 enzym khc nhau. Mt s enzyme
ch c mt vi s lng vi phn t nhng nhiu enzym c mt vi s lng
ln. Ch nhng enzym chu trch nhim phn hu cc c cht khng ho tan
nh tinh bt, xenlulo, proteinmi c tit t t bo vo mi trng. Vi sinh
vt c kh nng s dng cc c cht khc nhau cho sinh trng v thch ng
vi cc iu kin sinh trng rt khc nhau. Trong s gii hn ca th tch t
bo ch tng hp nhng enzym m n cn. Trong cng ngh sn xut enzym
cn phi iu khin trao i cht sao cho enzym m ta mong mun c tng
hp cng nhiu cng tt.
1.1.3 Cc sn phm ca s chuyn ho cht
V d: s oxy ho khng hon ton to thnh axit axetic soboza.
Trong qu trnh chuyn ho cc t bo hot ng nh nhng h thng
xc tc cho mt hoc nhiu bc chuyn ho cht. V mt l thuyt nhng phn
ng ny cng xy ra nh cc enzym c lp, tuy nhin con ng ny khng th
thc hin c hoc khng kinh t (v d vi cc phn ng enzym ph thuc
nng lng). Cc enzym chuyn ho cc cht theo cch rt c hiu. V d: s

chuyn ho hoc tch hydro xy ra nh phn t steroid khng c ngha i

vi t bo (cng c th l nhng phn ng kh c).
Trong s chuyn ho to axt axetic gn lin vi sinh trng, n gn
lin vi s thu nhn nng lng.
1.2 ng hc ca sinh trng v ca s to thnh sn phm
Sinh trng ca mt qun th vi sinh vt din ra qua cc giai on khc
nhau. S sinh sn ca t bo bt u sau mt giai on tim pht. Trong giai
on log tip theo xy ra s sinh sn theo hm s m. Sau mt thi gian sinh
trng ngng li v thiu cht dinh dng c bn v v tch lu cc cht c ch.
Cc t bo chuyn vo giai on cn bng. Trong giai on ny khng din ra
sinh trng na nhng t bo vn cn hot ng trao i cht.
Ton b qu trnh nui gn lin vi s thay i ko di ca cc iu kin
nui. Cht dinh dng gim i, s lng t bo tng ln. ng thi hot tnh
trao i cht cng thay i.
V phng din chc nng ca cc sn phm trao i cht i vi t bo
c th phn bit 2 nhm:
- Cc sn phm m s hnh thnh ca chng gn lin vi s sinh trng.
V d: cc sn phm ln men, cc cht trao i bc 1, cc enzym. S tng hp
nhng sn phm ny xy ra trong thi gian sinh trng v cn c th tip din
sau khi sinh trng kt thc.
- Cc sn phm m s hnh thnh ca chng xy ra sau khi sinh trng
kt thc; v d cc sn phm trao i cht bc 2.

ng thi

Thi gian

Khng ng thi

Thi gian

Hnh 1: ng hc ca qu trnh sinh trng v to thnh sn phm

Nhiu sn phm chim mt v tr trung gian. V d s tng hp axt amin
mc d din ra trong thi gian sinh trng nhng vn tip din sau khi sinh
trng kt thc, v qu trnh tng hp tip din trn c s ca mt sai hng

di truyn. S tng hp ca nhiu enzym xy ra khng song song vi sinh

trng m gn lin vi mt trng thi sinh l nht nh ca t bo cho nn trong
ln men cng nghip cn phi tm ra trng thi sinh l ca nng sut cao nht v
duy tr n trong thi gian di.
1.3 S tng hp tha
Vi sinh vt tn ti trong t nhin sinh ra cc sn phm trao i cht v
cc thnh phn t bo ch mc cn thit cho s sinh sn ti u v cho s
duy tr loi. S trao i cht nh vy c bo m nh cc c ch iu ho.
V d: cc c ch ny cn hot ng sao cho cc axit amin khng c
tng hp qu nhu cu ca s tng hp protein.
Nh vy, trong iu kin t nhin khng c s sn sinh d tha cc sn
phm trao i cht bc 1, bc 2 v cc enzym. Nu trong t nhin c ch iu
ho ny b ri lon, v d do kt qu t bin th cc th t bin sai hng trao
i cht thng c hi t vi chng ban u.
1.3.1 Nhng nguyn tc iu ho trao i cht
C 3 c ch chu trch nhim iu ho trao i cht:
1. iu ho hot tnh enzym nh s c ch bng sn phm cui cng
hay cn gi l s km hm theo lin kt ngc.
2. iu ho tng hp enzym nh s kim ch bng sn phm cui cng
v s gii kim ch.
3. iu ho tng hp enzym nh s kim ch d ho.
1

Cc enzym cu trc
4
Glucoza

Kim ch d ho
Kim ch bng sn phm
cui cng

E.1

E.2

E.3

E.4

S.phm cui cng

c ch bng sn phm
cui cng

S 1: Cc nguyn tc ca s iu ho enzyme
* Trong c ch 1 sn phm cui cng ca 1 qu trnh sinh tng hp gy
ra s c ch qu trnh tng hp ca chnh n. y, sn phm cui cng d
c hnh thnh trong t bo hay c thu nhn t mi trng dinh dng,
iu cng ngha. Trong c ch ny, sn phm cui cng ni chung nh
hng n enzyme u tin ca chui sinh tng hp. Enzym c tnh quyt nh
ny l 1 protein d lp th. N c c im l thay i cu hnh khng gian khi

c mt sn phm cui cng nhm gim bt hot tnh xc tc. S c ch ny xy

ra nhanh v rt c hiu qu.
* Trong c ch 2, sn phm cui cng c ch s tng hp enzym cn cho
s to thnh sn phm y, trong vic c thng tin di truyn cn cho s tng
hp enzym (s phin m) b phong to. nng cao ca sn phm cui cng
s tng hp ca cc enzym tham gia vo chui phn ng b ngng hoc b ko
di mt cch ng k. Nu nng ca sn phm cui cng gim xung di 1
mc no th xy ra s gii kim ch, ngha l cc enzym c to thnh vi
tc cao hn. S iu ho theo kiu ny xy ra t t v n gn lin vi s
tng hp enzym.

Hot ng

Cht nh hng
d lp th

Cht nh hng
d lp th

Th t bin

Khng hot ng
Dng hoang di

Hnh 2: M hnh ca s c ch bng sn phm cui cng

*S kim ch d ho iu ho qu trnh tng hp cc enzym d ho xc
tc s phn hu c cht. Cc enzym ny c tng hp nh s cm ng enzym.
C ch ny tng t c ch kim ch tc l cng xy ra mc phin m.
Trong s cm ng enzym, mt cht dinh dng ng vai tr cht cm ng kch
thch s tng hp enzym xc tc cho s phn hu chnh n, ngha l cht ny
cm ng s tng hp. Do , vic tng hp cc enzym cm ng ch xy ra khi
c mt c cht tng ng trong mi trng.

* S kim ch d ho iu ho qu trnh tng hp cc enzym d ho xc

tc s phn hu c cht. Cc enzym ny c tng hp nh s cm ng enzym.
C ch ny tng t c ch kim ch tc l cng xy ra mc phin m.
Trong s cm ng enzym, mt cht dinh dng ng vai tr cht cm ng kch
thch s tng hp enzym xc tc cho s phn hu chnh n, ngha l cht ny
cm ng s tng hp. Do , vic tng hp cc enzym cm ng ch xy ra khi
c mt c cht tng ng trong mi trng.

Gen
iu
khin

cht kim ch khng

hot ng v khng
c sn phm cui cng

khng phong to s
truyn thng tin
tng
hp enzym

Cc
gen
cu
trc

cht kim ch
hot ng

cht kim ch khng hot ng v

b sai hng (ngay c khi khng c
sn phm cui cng )

khng phong ta s truyn

thng tin
tng hp enzym

cht kim ch khng hot ng

Sn phm
cui cng

Cht kim ch
hot ng

Khng c s truyn thng

tin
khng tng hp enzym
Dng hoang di

khng phong ta s truyn

thng tin
tng hp enzym

Th t bin

Hnh 3: M hnh kim ch bng sn phm cui cng

Nu trong mi trng c mt nhiu c cht th trc ht xy ra s tng

hp ca enzym no xc tc phn hu c cht d s dng nht. S tng hp ca
cc enzym khc b c ch bi s kim ch d ho. Thng thng th glucoza l
c cht thch hp nht.
1.3.2 Nhng sai hng di truyn ca iu ho trao i cht
Cc c ch iu ho trao i cht c th b thay i do nhng t bin
dn ti s tng hp tha cc cht trao i cht.
Nhng enzyme d lp th ngoi v tr phn ng vi c cht, chng cn
mt v tr khc i vi sn phm cui cng (hnh 2). V tr th 2 ny gi l
trung tm d lp th. Hai v tr ny tch bit nhau v khng gian v khc nhau
v cu trc.
Mt t bin c th dn n kt qu lm protein enzyme d lp th b
thay i bng cch mt i kh nng phn ng vi cht hiu ng nhng vn cn
hot tnh xc tc. Mt protein b bin i nh vy vn cn hot ng ngay c
khi c mt sn phm cui cng
n dn n s tng hp tha ca sn phm
cui cng tng ng (hnh 2 pha bn phi).
Trong s kim ch tng hp enzym xy ra nhng phn ng quyt lit
trong phm vi thng tin di truyn, s phin m (hnh 3).
S iu ho tng hp enzym c th b ri lon do nhng t bin khc
nhau. Nhng t bin c th ng chm n gen kim ch dn ti mt sai hng
ca cht kim ch hoc lm bin mt n; hay ng chm n gen iu khin v
lm cho gen ny mt kh nng tc dng vi cht kim ch (bn phi hnh 3).
Ton b nhng sai hng tng ng cng c th biu hin s cm ng
enzyme. Nh nhng s sai hng y m cc enzyme cm ng tr thnh cc
enzyme cu trc, ngha l chng tn ti trong t bo khng ph thuc vo c
cht
s kim ch d ho b mt i.

CHNG II: NHNG VN K THUT V PHNG

PHP CHUNG TRONG CNG NGH VI SINH VT
Sn xut sinh khi v cc sn phm trao i cht trong qu trnh ln men
c nhiu im ging nhau v phng php v k thut. Vic p dng k thut
v phng php tu tng i tng vi sinh vt v mc tiu sn phm cui cng.
Trn c s c th p dng nhng mc tiu v phng php ring. Chnh v
th khng th c mt phng php chung cho tt c cc sn phm. Vic p
dng k thut v phng php ch c th trn c s nhng nguyn tc chung
ca cc k thut v phng php trnh by. V th, trong chng ny s gii
thiu nhng nguyn tc chung ca k thut v phng php c p dng rng
ri trong cc ngnh vi sinh cng nghip. Cc nguyn tc chung bao gm
vic tuyn chn ging vi sinh vt, gi ging vi sinh vt, cc qu trnh v thit b

Nu trong mi trng c mt nhiu c cht th trc ht xy ra s tng

hp ca enzym no xc tc phn hu c cht d s dng nht. S tng hp ca
cc enzym khc b c ch bi s kim ch d ho. Thng thng th glucoza l
c cht thch hp nht.
1.3.2 Nhng sai hng di truyn ca iu ho trao i cht
Cc c ch iu ho trao i cht c th b thay i do nhng t bin
dn ti s tng hp tha cc cht trao i cht.
Nhng enzyme d lp th ngoi v tr phn ng vi c cht, chng cn
mt v tr khc i vi sn phm cui cng (hnh 2). V tr th 2 ny gi l
trung tm d lp th. Hai v tr ny tch bit nhau v khng gian v khc nhau
v cu trc.
Mt t bin c th dn n kt qu lm protein enzyme d lp th b
thay i bng cch mt i kh nng phn ng vi cht hiu ng nhng vn cn
hot tnh xc tc. Mt protein b bin i nh vy vn cn hot ng ngay c
khi c mt sn phm cui cng
n dn n s tng hp tha ca sn phm
cui cng tng ng (hnh 2 pha bn phi).
Trong s kim ch tng hp enzym xy ra nhng phn ng quyt lit
trong phm vi thng tin di truyn, s phin m (hnh 3).
S iu ho tng hp enzym c th b ri lon do nhng t bin khc
nhau. Nhng t bin c th ng chm n gen kim ch dn ti mt sai hng
ca cht kim ch hoc lm bin mt n; hay ng chm n gen iu khin v
lm cho gen ny mt kh nng tc dng vi cht kim ch (bn phi hnh 3).
Ton b nhng sai hng tng ng cng c th biu hin s cm ng
enzyme. Nh nhng s sai hng y m cc enzyme cm ng tr thnh cc
enzyme cu trc, ngha l chng tn ti trong t bo khng ph thuc vo c
cht
s kim ch d ho b mt i.

CHNG II: NHNG VN K THUT V PHNG

PHP CHUNG TRONG CNG NGH VI SINH VT
Sn xut sinh khi v cc sn phm trao i cht trong qu trnh ln men
c nhiu im ging nhau v phng php v k thut. Vic p dng k thut
v phng php tu tng i tng vi sinh vt v mc tiu sn phm cui cng.
Trn c s c th p dng nhng mc tiu v phng php ring. Chnh v
th khng th c mt phng php chung cho tt c cc sn phm. Vic p
dng k thut v phng php ch c th trn c s nhng nguyn tc chung
ca cc k thut v phng php trnh by. V th, trong chng ny s gii
thiu nhng nguyn tc chung ca k thut v phng php c p dng rng
ri trong cc ngnh vi sinh cng nghip. Cc nguyn tc chung bao gm
vic tuyn chn ging vi sinh vt, gi ging vi sinh vt, cc qu trnh v thit b

ln men c bn, mt s k thut v phng php chnh thu nhn sn phm

ln men.
2.1 Ging vi sinh vt
2.1.1 Yu cu v ging vi sinh vt cng nghip
Cc chng vi sinh vt mun em vo sn xut ln phi bo m cc tiu
chun sau y:
- Phi cho sn phm mong mun vi nng sut cao, cht lng tt, c
ngha kinh t trong sn xut v t sn phm ph khng mong mun.
- S dng nguyn liu sn c, r tin
- Thi gian ln men ngn.
- D tch sinh khi hay sn phm sau ln men.
- Vi sinh vt phi thun chng, khng cha vi sinh vt l, c bit khng
cha bacteriophage k sinh.
- Chng vi sinh vt phi kho, pht trin d dng v nhanh chng, cho
nhiu t bo dinh dng hoc cc bo t hoc cc hnh thc ti sinh khc.
- C kh nng chng, chu cc iu kin bt li ca mi trng.
- D bo qun, tn ti v n nh cc c tnh sinh l, sinh ha sut trong
thi gian s dng.
- C kh nng thay i cc c tnh bng cc k thut t bin, k thut
gen khng ngng nng cao nng sut v cht lng sn phm.
2.1.2 Ngun ging vi sinh vt
2.1.2.1 Phn lp trong t nhin
Thin nhin l ngun cung cp ging v tn cho cng nghip vi sinh vt.
Cc vi sinh vt s dng trong cng nghip thc phm c th d dng phn lp
chng t cc loi thc n, nc ung sn xut theo phng php c truyn: t
bnh men thuc bc, mc tng, sa chua Vi sinh vt s dng trong cng
nghip sn xut khng sinh thng c phn lp trong t, nc nhng
vng khc nhau.
phn lp c mt chng vi sinh vt mong mun t mt qun th hn
hp vi sinh vt trong t nhin chng ta phi thc hin cc bc sau: thu mu,
ho long, cy ln mi trng, nui, thun khit, kim tra
Cc cng vic ny u ging nhau khi nghin cu trn cc i tng vi
rt, vi khun, nm, to, nguyn sinh ng vt, cc ng vt khng xng sng
loi nh.
ho long mu ngi ta thng s dng nc mui sinh l (0,85%
dung dch NaCl) hoc nc ct. i vi mt s loi d cht trong nc ct hoc
nc mui sinh l nh Pseudomonas th dng dung dch peptone 0,1% lm
long. Sau khi pha long th em cy ra trn mi trng thch ri em nui
trong t m 1-3 ngy. Tip theo tch cc khun lc in hnh v lm tinh khit
mt s ln trn mi trng thch tch ring tng khun lc.

i vi nhm vi sinh vt ym kh nu cy trn mi trng thch trong

hp Petri ngi ta phi cho thm cc cht kh hay gn kn bng parafin hoc
nui trong mi trng khng c oxy.
phn lp cc chng sn xut khng sinh hoc khng khng sinh ngi
ta thng dng phng php nhanh: ho long mu 10 hoc 100 ln, sau ly
1ml dung dch ho long cy ln mi trng thch c sn vi sinh vt cn tiu
dit hoc c cht cn phn gii. Sau khi nui 1-3 ngy trong t m th em xem
xt nhng khun lc no c c tnh m chng ta mong mun th tin hnh
phn lp v thun khit nhng chng c hot lc cao.
phn lp c chng vi sinh vt mong mun th phi tin hnh lm
giu s lng vi sinh vt , tc l phi s dng nhng mi trng v iu kin
nui cy chn lc cc chng mong mun v c ch cc chng khng mong
mun.
2.1.2.2 Thu nhn t cc trung tm gi ging
Ngy nay c nhiu trung tm gi ging ging trn th gii c th cung
cp cho chng ta cc ging vi sinh vt phc v cho cng tc nghin cu, ging
dy, sn xut. Mt s trung tm gi ging nh:
- M: ABBOTT, ATCC, NRRL
- Canada: CANAD-212
- Nht: FERM, HIR
- c: CC
- Trung quc: IMASP
2.1.3 Ci to ging vi sinh vt
Tr mt s ngnh ca cng ngh thc phm ngi ta c th c s dng
vi sinh vt kiu di cn hu ht cc ngnh cng nghip khc nh sn xut
khng sinh, vitamin, enzyme v.v. ngi ta u dng cc bin chng.
2.1.3.1 u nhc im khi s dng bin chng
* u im:
- Cho nng sut cao
- Thi gian ln men ngn v t to bt trong khi ln men
- t to sn phm ph trong qu trnh ln men
- To c nhiu sn phm qu m chng kiu di khng c c, v d
nh: insulin, khng nguyn Hbs (chng vim gan B) v.v.
- Kh nng chng, chu cc iu kin bt li ca mi trng cao.
* Nhc:
- i hi cng ngh cao
- Trong qu trnh sn xut c th xut hin hin tng hi bin
2.1.3.2 Phng php to ging mi
to ra ging vi sinh vt mi ngi ta c th s dng nhiu phng
php khc nhau:

- t bin nhn to v chn la: y l cng vic ca cc phng th

nghim hoc cc trung tm nghin cu nhm tm ra cc chng vi sinh vt c
nng sut cao hoc sn xut c nhng sn phm m mnh mong mun.
- Ti t hp gen: s dng cc kh nng bin np, tip hp, ti np, lai t
bo cht vi sinh vt v cc k thut gen thc hin vic to ra cc c th
mi vi nhng b gen theo mun.
- La chn thng xuyn: cng vic ny thng thc hin cc phng
th nghim ca cc nh my nhm gi c cc c th tt trong qun th vi sinh
vt. Khi kim tra loi b cc t bin ngu nhin khng cho nng sut cao hoc
c nhng biu hin bt li v gi nhng c th thch nghi tt trong iu kin
sn xut.
2.1.4 Gi ging vi sinh vt
2.1.4.1 ngha v nhim v ca cng tc gi ging
Gi ging vi sinh vt l mt cng vic c ngha v cng ln. N gip
cho chng ta gi c cc c tnh qu (khng b thoi ho) ca vi sinh vt v
bo m cung cp ging cho cc qu trnh sn xut.
Nhim v ca cng tc gi ging l s dng cc k thut cn thit gi
cho vi sinh vt c t l sng cao, cc c tnh di truyn khng b bin i v
khng b tp nhim bi vi sinh vt l.
2.1.4.2 Cc phng php gi ging
C rt nhiu phng php gi ging vi sinh vt. Tu theo trang thit b
v loi vi sinh vt cn gi m chng ta c th s dng mt trong cc phng
php sau:
- Gi ging trn mi trng thch nghing
- Gi ging di lp du khong
- Gi ging trong ct
- Gi ging trong t
-Gi ging trn silicagen
-Gi ging trn cc loi ht ng cc (la, ng, k..)
-Gi ging trn giy lc
-Gi ging trn cc ming gelatin
-Gi ging bng phng php lnh ng
-Gi ging bng phng php ng kh
2.2 Dinh dng v mi trng nui cy vi sinh vt
2.2.1 Qu trnh v nhu cu dinh dng t bo vi sinh vt
Trong qu trnh sng t bo vi sinh vt khng ngng trao i cht vi
mi trng xung quanh. T bo vi sinh vt tuy rt nh nhng v hp thu cc
cht dinh dng v thi cc sn phm trao i cht qua ton b b mt t bo
cho nn cng trao i cht ca chng l rt ln. Cc cht dinh dng qua
mng vo t bo v c chuyn ho to thnh nhng cht ring bit cn

thit cho vic xy dng t bo. Nh qu trnh ng ho cc t bo mi c th

sinh trng, pht trin tng sinh khi, ng thi sinh ra cc sn phm trao i
cht.
S bin i cc cht dinh dng bao gm nhiu phn ng ho sinh khc
nhau nh h enzym theo con ng trao i cht :
1. To ra nhng cht c trong thnh phn ca t bo
2. Sn ra nng lng sinh hc cn thit cho hot ng sng
Nhng cht dinh dng khi l nhng hp cht c phn t nh c th
i qua mng t bo vi sinh vt v tham gia vo 2 loai phn ng:
- Bin i d ho lm xut hin nhng sn phm c cu trc n gin
hn. Nhng bin i d ho ny cung cp cho vi sinh vt nng lng chuyn
ho dng ATP hoc nhng hp cht giu nng lng khc. Mt s nhng sn
phm d ho thi i, mt s khc lm vt liu hoc lm tin cht cho cc phn
ng ng ho.
- Bin i ng ho, m bo s tng hp ca thnh phn mi c cu
trc phc tp hn v phn t lng cao hn - gi l qu trnh sinh tng hp.
Cc cht dinh dng ca vi sinh vt ch yu ly mi trng xung
quanh. Cho nn thnh phn ca mi trng dinh dng bo m cung cp cc
nguyn t C, H, O, N, P, S, Ca, Fe v cc nguyn t vi lng.
2.2.2 Mi trng nui cy vi sinh vt
2.2.2.1 Phn loi mi trng
1. Phn loi theo thnh phn c trong mi trng:
+ Mi trng t nhin: c thnh phn khng xc nh, c ch to t
ng hay thc vt.
+ Mi trng tng hp: gm nhng cht ho hc tinh khit v c ly
vi nng cho trc.
+ Mi trng bn tng hp.
2. Phn loi theo trng thi vt l:
+ Mi trng lng: dng tng sinh, tch lu cc sn phm trao i
cht, pht hin cc c tnh sinh l, sinh ho, gi ging v bo qun nhiu
loi vi sinh vt khng pht trin c trn mi trng c.
+ Mi trng c: dng phn lp thu khun lc ri, nghin cu nh
danh, gi ging, m s lng vi sinh vt.
+ Mi trng xp: dng trong cng ngh vi sinh vt.
2.2.2.2 Nguyn liu dng chun b mi trng trong cng ngh vi sinh vt
1. Nguyn liu cung cp ngun cacbon
Cacbon chim mt phn rt ln trong t bo vi sinh vt, chnh v vy
nhng hp cht cha cacbon c ngha hng u trong s sng ca vi sinh vt.
Ngun thc n cacbon ch yu ca vi sinh vt l hydratcacbon. Cc hp
cht c phn t thp nh mt s ng th vi sinh vt c th ng ho trc tip.

Cn cc hp cht cao phn t (tinh bt, xenlulo) s c phn hu nh cc

enzyme do vi sinh vt tit ra.
Trong cc hydratcacbon thi glucose l mt ngun cacbon vn nng i
vi vi sinh vt. Qu trnh bin i glucose trong t bo vi sinh vt c th din ra
bng 3 con ng: chu trnh Embden-Meyerhof-Parnas (EMP); pentoza;
Entner-Dondoroff. Tt c u dn ti to thnh axt pyruvic ri t i vo cc
qu trnh khc nhau.
Cc enzym

CO2, ru, cc sn phm k kh khc

Tinh bt glucose
amylaza

Qt. ngoi bo

Cc enzym

CO2, H2O, cc axt hu c v cc sn phm trao

i hiu kh

Ngoi ra vi sinh vt c th ng ho mt s cc cacbuahydro:

CH3-(CH2)n-CH3
CH3-(CH2)n-CH2OH
CH3-(CH2)n-CHO
CH3-(CH2)n-COOH

Ankan
alkanol
alkanal
axt bo
Axetyl-CoA.

i vi cht bo:
Cht bo
triglycerit
axt bo
palmityl-CoA
axetyl-CoA
2. Nguyn liu cung cp ngun nit
Vi sinh vt cn nit xy dng t bo v tt c cc thnh phn quan
trng ca t bo u c cha nit (protein, axt nucleic, enzym).
Axt amin to ra protein. Cc axit amin trong t bo vi sinh vt c to
thnh do qu trnh trao i cacbon v nit.
Nit trong khng kh rt phong ph nhng n bn vng v mt ho hc
v ch c cc vi sinh vt c nh nit mi c kh nng ng ho chng. Cn cc
ngun nit dng trong ln men ca cng ngh vi sinh vt l cc hp cht nit
v c v hu c.
Cc axit amin thng khng c vi sinh vt s dng trc tip ngay m
phi qua 2 loi phn ng: hoc kh amin hoc kh cacboxil.
Kh amin:
.deza min aza
R-CH-COOH + O2 E
R-C-COOH + NH3

NH2

Nh vy, ch c nhng vi sinh vt no c kh nng kh amin mi c kh

nng s dng axit amin. Qu trnh ny thng xy ra nhng vng pH=6,5
7,5.
Khi pH vng axt th xy ra phn ng kh cacboxyl:

E.
R-CH-COOH
R-CH2-NH2 + CO2

NH2

(Sn phm decacboxyl ca mt s cc axit amin c c t, v d: t

lizin
cadaverin).
Ngun nit hu c thng l cc hp cht phc tp nn u tin vi sinh
vt phi tit vo mi trng enzym proteaza thu phn.
Cc ngun nit v c
- Ur:
(NH2)2CO + H2O ureaza
2NH3 + CO2
- HNO3:
4AH2 + HNO3
NH3 + 3H2O
AH2 - cht kh c trong mi trng.
Hoc : HNO3
HNO2
(HNO)2
NH2OH
NH3
Qu trnh ny nh h enzym nitratreductaza
Tt c cc loi vi sinh vt u ng ho c mui amn.
Nhng s dng c nit vi sinh vt u phi tch NH3.
3. Nguyn liu cung cp cc cht khong
* Cc hp cht photpho:
nh hng ln qu trnh trao i cht ca t bo vi sinh vt. Trong t
bo vi sinh vt thng gp cc hp cht polyphotphat, chng c chc nng
tham gia vn chuyn glucose qua mng t bo
dn n qu trnh photphoril
ho glucose.
Ngun photpho c th l v c hoc c trong cc loi nguyn liu hu
c. Vi sinh vt s dng nhanh nht l cc hp cht photpho v c ho tan, cn
photpho hu c s dng t v chm. Tuy nhin nhu cu v photpho ph thuc
vo chng vi sinh vt, d cng nh hng xu.
* Cc cht khong khc:
Mg, Na, Fe, K, Al, Li, Rb, Mnly t mi trng dinh dng.
Cc cht ny c ngha khc nhau i vi vi sinh vt: c mt s kim loi
tham gia vo cu to phn t hoc lm thay i hot lc enzym.
Tuy nhin nng cc cht ny cng nm trong mt gii hn.
4. Cc cht kch thch sinh trng:
- Cao ng
- Cao nm men
- Dch p tri cy
5. Cht bo trong cng ngh vi sinh vt
- Du bo (lc, u tng, ng, lanh): thng dng lm ct ph bt.
- Mt khc nu vi sinh vt c kh nng tit ra enzym lypaza s phn hu
cht bo n axetyl-CoA v ng ho tip.
Ch : khng dng qu nhiu du ph bt
c th nh hng xu n
qu trnh ln men: nht ln, to nh tng ca cc ht x phng.
6. Nc

Trong cng ngh vi sinh vt nc c s dng nhiu v vi nhiu mc

ch khc nhau. Cht lng ca nc c nh hng n tin trnh cng ngh v
cht lng ca sn phm. Do , tu vo mc ch s dng m phi bo m
yu cu v cc ch tiu cht lng ca nc nh: cng, oxy ho, vi
sinh
2.3 Cc phng php ln men
C nhiu cch phn loi cc phng php ln men. Da vo cch np
liu v thu hi bn thnh phm sau ln men ngi ta chia ra ln men gin on,
lin tc v bn lin tc.
Ln men gin on thc hin theo tng m nn thng c nng sut thp
v chu k sn xut b ko di. Ln men lin tc th ngc li, nguyn liu lin
tc vo v sn phm sau ln men lin tc i ra. Ln men bn lin tc l hnh
thc kt hp gia hai phng php trn.
Da vo thnh phn ng nht hay khng ng nht ca canh trng
ngi ta chia ra ln men b mt (ni), ln men b su (chm) v bn rn.
Trong ln men b mt, vi sinh vt pht trin trn b mt ca mi trng
nui cy v ly khng kh t mt thong ca mi trng. Phng php ny
thng s dng sn xut axit citric v mt s enzyme. Nhc im ln nht
ca phng php ny l tn km b mt. Tuy nhin, do u t t nn chng
mc no vn c s dng.
Ln men bn rn l phng php trung gian gia ln men b mt v b
su. Hm lng nc trong mi trng chim khong 70% cht kh. Mt s
enzyme hin nay c sn xut theo phng php ny. Ngi ta ci tin
phng php ny bng thit b thng quay nhm cung cp oxy cho qu trnh
ln men v thc hin lun khu sy kh sau ln men. iu kh khn l do s
truyn nhit km ca cc cht n nn kh thc hin tt khu thanh trng.
Ln men chm l phng php c s dng nhiu nht. N c th cho
php kim sot c ton b qu trnh ln men mt cch thun li, t tn km
mt bng. Do h thng khuy trn tt nn ton b mi trng nui cy l mt
h thng nht.
2.4 Thu hi sn phm
thu hi sn phm phi qua nhiu bc. S lng cc bc ph thuc
vo nguyn liu ban u, nng ban u, s n nh v mt sinh hc ca sn
phm v mc yu cu v tinh khit ca sn phm.
Bc u ca vic thu hi sn phm l tch t bo v cc sn phm
khng ho tan ra khi dch ln men bng cch lc hoc lng. trch li cc sn
phm bn trong t bo th phi ph v t bo bng cc phng php vt l (siu
m, p, lnh ng ri tan gi) hoc cc phng php ho hc (x l bng axit,
trch li bng axeton) hoc bng phng php sinh hc (dng enzyme). Nu
s dng axit th sn phm phi bn vng trong mi trng axit.

Cc sn phm ln men c trong mi trng t th phi lm m c. Cc

phng php thng dng tch sn phm nh: lc, ly tm, lng, trch li, sc
k, c c, kt ta, kt tinh, tuyn ni, sy kh, nghin.

CHNG III: SN XUT SINH KHI VI SINH VT

3.1 Sn xut protein n bo
3.1.1 u, nhc im ca sn xut protein n bo
1. u:
- t tn din tch
- Tc sinh trng cao: gp 100 1000 ln so vi i gia sc
V d: sn xut 1t protein cn:
+ Trng 4ha u trong 3 thng
+ Nui 4 con b trong 15 18 thng
+ Nui 300m3 vi sinh vt trong 24h
- Khng ph thuc vo kh hu
- Thnh phn v gi tr dinh dng ca sinh khi c th iu
chnh bng cch thay i thnh phn mi trng, iu kin
nui cy hoc to ging mi.
- S dng nguyn liu sn c, r tin.
2. Nhc:
- Trong sinh khi ca vi sinh vt cha nhiu axt nucleic
(10 20%) khng c li cho sc kho ca con ngi.
- Protein vi sinh vt c hng v cha cao.
3.1.2 Cc yu cu c bn ca vic sn xut protein n bo
t c hiu qu kinh t cao trong vic sn xut sinh khi vi sinh vt
cn phi bo m cc yu cu sau:
1. S dng nguyn liu r tin vi thu hoch cao. t c nng sut
cao cn lu n hiu sut chuyn ho nguyn liu ca vi sinh vt. Cc dng
nguyn liu c quan tm nhiu:
- Cacbuahydro: vi sinh vt c kh nng chuyn ho 100% to thnh
sinh khi.
- Hydratcacbon (r ng, dch kim sunfit, xenluloza, tinh bt, cn
sa): vi sinh vt c kh nng chuyn ho 50% vt cht kh ny sang sinh
khi.
2. Tc sinh trng cao: ni chung tc sinh trng ca vi sinh vt
rt ln, thi gian nhn i ca chng ngn.
Vi khun 0,3-2 gi
Nm men v to 2-6 gi
Nm si khong 10 gi

Cc sn phm ln men c trong mi trng t th phi lm m c. Cc

phng php thng dng tch sn phm nh: lc, ly tm, lng, trch li, sc
k, c c, kt ta, kt tinh, tuyn ni, sy kh, nghin.

CHNG III: SN XUT SINH KHI VI SINH VT

3.1 Sn xut protein n bo
3.1.1 u, nhc im ca sn xut protein n bo
1. u:
- t tn din tch
- Tc sinh trng cao: gp 100 1000 ln so vi i gia sc
V d: sn xut 1t protein cn:
+ Trng 4ha u trong 3 thng
+ Nui 4 con b trong 15 18 thng
+ Nui 300m3 vi sinh vt trong 24h
- Khng ph thuc vo kh hu
- Thnh phn v gi tr dinh dng ca sinh khi c th iu
chnh bng cch thay i thnh phn mi trng, iu kin
nui cy hoc to ging mi.
- S dng nguyn liu sn c, r tin.
2. Nhc:
- Trong sinh khi ca vi sinh vt cha nhiu axt nucleic
(10 20%) khng c li cho sc kho ca con ngi.
- Protein vi sinh vt c hng v cha cao.
3.1.2 Cc yu cu c bn ca vic sn xut protein n bo
t c hiu qu kinh t cao trong vic sn xut sinh khi vi sinh vt
cn phi bo m cc yu cu sau:
1. S dng nguyn liu r tin vi thu hoch cao. t c nng sut
cao cn lu n hiu sut chuyn ho nguyn liu ca vi sinh vt. Cc dng
nguyn liu c quan tm nhiu:
- Cacbuahydro: vi sinh vt c kh nng chuyn ho 100% to thnh
sinh khi.
- Hydratcacbon (r ng, dch kim sunfit, xenluloza, tinh bt, cn
sa): vi sinh vt c kh nng chuyn ho 50% vt cht kh ny sang sinh
khi.
2. Tc sinh trng cao: ni chung tc sinh trng ca vi sinh vt
rt ln, thi gian nhn i ca chng ngn.
Vi khun 0,3-2 gi
Nm men v to 2-6 gi
Nm si khong 10 gi

G mi 500 gi
Heo 1000 gi
Tru b 2000 gi
Nh vy trong cng mt thi gian nu nui cy vi sinh vt thu nhn
sinh khi s thu c mt khi lng cao hn rt nhiu so vi cc sinh vt
khc.
3. Hm lng protein cao:
Hm lng protein ph thuc vo chng v chu nh hng nhiu iu
kin nui cy. Ni chung hm lng protein vi sinh vt t 50-60%.
4. Cht lng protein cao:
nh gi cht lng protein ngi ta hay quan tm n hm lng axit
amin khng thay th. Tiu chun ny cng mang tnh cht loi. Thnh phn axit
amin ca protein vi sinh vt ging nh trong tht v sa. Protein vi sinh vt giu
lizin nhng cc axit amin cha lu hunh li thp.
5. Kh nng tiu ho ca protein:
Kh nng tiu ho protein vi sinh vt b hn ch bi cc thnh phn nit
phi protein (nh axt nucleic, peptit ca thnh t bo). Tuy nhin nu protein
c tch khi t bo th khi quan tm. Do bn cht thnh t bo l tiu
chun la chn vi sinh vt dng trong sn xut protein vi sinh vt.
6. S an ton v c t:
Cc vi sinh vt gy bnh hoc c cha cc thnh phn gy c nghi ng
th khng c dng sn xut protein. Yu cu hm lng axt nucleic phi
thp, hm lng ca n cng cao th cng lm gim gi tr ca protein. Khi tiu
ho cc axt nucleic s phn hu tip thnh cc nucleotit, sau li c phn
hu tip thnh ademin hoc guanin v cui cng thnh axt uric. Trong c th
ngi khng c enzym urinaza do uric khng chuyn ho tip. S tch t
axt uric s gy nn bnh thp khp, to ra si thn, si bng quang do ho
tan thp ca axt ny. Lng axt nucleic hp th qua dinh dng khng c
qua 2g/ngy.
ng vt vn ny khng quan trng v chng c kh nng ng ho
axt uric. Ta c th lm gim lng nucleic bng nhng bin php sau:
- Gim mnh tc sinh trng.
- Chit rt ARN=dung dch NaCl 10% nng.
- Thu phn ARN bng kim v tch protein ho tan trong bng kt
ta.
- Phn hu ARN bi enzym nucleaza a vo hoc ca bn thn t bo.
Dng phng php gy chong nhit theo 3 bc: u tin cc t bo c un
nng ln 68oC trong 5 giy. Sau 52,5oC trong 2gi v cui cng 5556oC trong 1h. Vic x l ny lm bin tnh riboxom v hot ho cc
ribonucleaza. Cc sn phm thu phn c tch khi t bo, hm lng

protein khng b nh hng, hm lng axt nucleic gim t 1-2% lng vt

cht kh ca t bo.
7. Nhng vn k thut:
Vi sinh vt phi d tch v d x l. Cc t bo ln nh nm men c
tch ra bng li tm tt hn t bo vi khun. Chn cc chng c kh nng chu
nhit s lm gim chi ph cho vic lm ngui. Tnh khng mn cm vi s tp
nhim l tin cho vic lm ngui sn xut protein khng v trng. Ngoi ra
ngi ta cn ch n kh nng ng ho ng thi nhiu ngun cacbon khc
nhau.
3.1.3 Vi sinh vt dng trong sn xut protein n bo
3.1.3.1 Nm men
Trong cc i tng vi sinh vt c s dng thu nhn protein th
nm men l loi c nghin cu sm nht v n nay c p dng rng
ri trn nhiu nc. Nm men giu protein, vitamin (nht l nhm B). Hm
lng protein dao ng 40-60% khi lng cht kh. Protein ca nm men gn
ging protein ngun gc ng vt, cha khong 20 loi aminoacid, trong
y cc axit amin khng thay th. Thnh phn cc aminoacid ca nm men
cn i hn la m, km cht t so vi sa, bt c v sn phm ng vt ni
chung.
S dng rng ri: Candida, Torulopsis, Saccharomyces v cacs loi ny
kh nng chuyn ho cc cht cao, a dng v qui trnh cng ngh n gin.
3.1.3.2 Nm si
Khi dng nguyn liu l tinh bt v xenlulo th khng th s dng nm
men c v bn than nm men khng cha enzyme amylaza v xenlulaza, do
d phi dng nm si. Nm si c nhc im l thi gian nhn i di v hm
lng protein thp (khong 30%). Nhng nm si li c u im l rt d tch
sinh khi v to hng v c bit.
dng trong thc phm ngi ta s dng cc loi Morchella. Loi ny
c v ngon, ph hp cho ch bin thc phm. Tuy nhin, kh khn nht l nui
cy Morchella rt tn km v d b nhim.
Hin nay nhiu ni s dng hn hp ging Trichoderma virid v nm
men Sacch. Cerevisiae sn xut protein. Ngoi ra, ngi ta cn s dng hn
hp Trichoderma virid vi Candida utilis hoc Endomycopsis fibuliger vi
Candida utilis.
3.1.3.3 Vi khun
Vi khun dng sn xut protein n bo thng c nui trn
cacbuahydro. Ngi ta thng s dng cc ging Pseudomonas,
Flavobacterium, Mycobacterium, Nocardia. Cc ging vi khun ny c kh
nng ng ho cc ankal, cacbuahydro bo v thm.

i vi nguyn liu l metan ngita s dng cc ging Methylomonas

methanica, Methylococens capsulatus.
3.1.3.4 Vi khun lam v vi to
Tt c mi loi to c kch thc nh b v c th thch hp vi vic s
dng cc phng php nui cy i vi vi sinh vt u gi l vi to. Cn vi
khun lam trc y gi l to lam. V qui trnh cng ngh sn xut thu sinh
khi ca 2 loi ny v c bn l ging nhau.
Hm lng protein chim 40-60% lng cht kh, thm ch i vi
Spirulina cn ln n 60-70%. Hm lng cc aminoacid ca 2 loi ny kh
cn i, gn vi protein tiu chun.
Ngoi ra trong sinh khi ca cc loi to cn cha nhiu loi vitamin: A,
B, K, pantothenic v dng ti cn cha vitamin C. Spirulina cha nhiu B12
nn dng lm thc phm, m phm, TAGS (g cho trng , da vng). Trong
sinh khi vi khun lam cn cha khng sinh nn bo qun tt.
3.1.4 Qui trnh cng ngh sn xut protein n bo
3.1.4.1 Sn xut protein n bo t vi sinh vt
1.Qui trnh cng ngh:
Nguyn liu
x l
chun b mi trng dinh dng
Vi sinh vt thun khit
nui
cy vi sinh vt
Sn phmhon thinx ltch sinh khiln men
2. Nguyn liu: Thng s dng cc loi nguyn liu nh: r ng, nc thi
ca nh my sa, dch kim sunfit, dch thy phn g, tinh bt, dextrin,
cacbuahydro
3. X l: bao gm nhiu cng on khc nhau ph thuc vo loi nguyn liu
v chng vi sinh vt s dng:
+ Lm sch: (tch tp cht, loi b cc phn khng cn thit, lm sch vi
sinh vt).
+ Lm nh nguyn liu
+ Thu phn nguyn liu (tinh bt, xenlulo)
4. Chun b mi trng dinh dng: Ngoi c cht chnh l cacbon th trong
mi trng cn phi b sung thm cc cht dinh dng ngun nit, photpho,
kali, magie, cc nguyn t vi lng v cc cht sinh trng nh cao ng, cao
nm menS dng cc ho cht iu chnh pH ca mi trng n gi tr
thch hp.
5. Chun b ging: Nui cy nhn ging u tin c thc hin trong phng
th nghim, sau c nui cy tip phn xng sn xut. T l tip ging
chuyn cp l 1:10. Thi gian nui mi cp khong 15h. Trong qu trnh

nhn ging dng nc amoniac gi pH v phi sc kh v trng lin tc.

Trong giai on ny ch bo m vic kh trng mi trng nui cy.
6. Ln men: Thng s dng phng php ln men chm v c th tin hnh
ln men gin on, lin tc hoc bn lin tc. Khi ln men ch iu chnh cc
yu t: to, pH, mi trng dinh dng, O2, ph bt.
7.Tch sinh khi: Tu vo iu kin c th m c th s dng cc phng
php khc nhau tch sinh khi nh lc, lng, li tm
8. X l sinh khi: Tu vo mc ch s dng m qu trnh x l sinh khi gm
cc cng on sau:
+ Ra
+ Chit rt protein (loi bt ARN bng cc phng php khc nhau)
+ Sy: tt nht l dng phng php sy phun
3.1.4.2 Sn xut sinh khi to:
c im t bo ca cc loi to l c dip lc t nn chng c kh nng
quang hp. Phn ng quang hp ca to: CO2 + 4H2O
CH2O + 3H2O + O2 .
S c nh v kh CO2 thnh hydratcacbon ca to theo chu trnh Kalvin.
Nui to ngoi vic cung cp protein cn c tc dng trnh c s lm
giu dinh dng i vi cc thu vc do nc thi c lm sch bng vi
khun gy nn (trnh tch t cc cht khong nh NH4+, NO3-, PO43-)
1. Yu cu v to ging:
- Tc sinh trng nhanh
- Nng sut quang hp cao
- C sc chng chu vi iu kin ngoi cnh
- Sinh khi c thnh phn ho hc thch hp
- T bo lun trng thi huyn ph, khng dnh bt vo thnh b hoc
lng xung y.
- D tch sinh khi (nh vt, lc)
2. Cc loi to thng dng:
- Vito: Chlorela, Scendemus, Dunaliella
- Vi khun lam: Spirulina
3. Cng ngh nui :
* Qui trnh cng ngh:
Chun b mi trng cy to ging
nui to
thu hoch
* Mi trng: chun bi mi trng nui to ngi ta c th s dng
nc my, nc bin, nc thi qua x l. Cn b sung thm cc cht
khong cn thit vo mi trng cho to pht trin.
* iu kin nui: to thch hp vi mi trng c pH = 8,510 v nhit
20400C. Trong qu trnh nui bo m cung cp nh sng v CO2.

* Thu hoch: c th thu hoch to bng cch lc, vt hoc co. tn

dng ht cc cht khong ngi ta c th s dng li mi trng sau khi
tch to.
3.2 Sn xut nm men bnh m:
Nm men bnh m l sinh khi ca chng Sacch.cerevisiae vn cn sng.
3.2.1 Yu cu i vi ging nm men bnh m
- Sinh sn nhanh
- Chu c mi trng r ng
- C lc lm n bt cao: c kh nng ln men ng sacarose, glucese,
maltose, c hot lc zimaza v maltaza cao.
- t b thay i khi bo qun.
- T bo ln d tch sinh khi.
Hot lc zimaza v maltaza l thi gian (pht) sinh ra 10ml kh CO2
khi ln men dung dch 5% ng glucoza hoc maltoza bng men p vi t l l
2,5% so vi lng dch ng.
3.2.2 Cng ngh sn xut:
1. Qui trnh cng ngh:
Chun b mi trng dinh dng
cy ging
nui
tch nm men

x l
thnh phm.
2. Chun b mi trng dinh dng:
Ngi ta thng s dng r ng lm mi trng nui cy nm men
bnh m. Trc khi pha mi trng th cn phi x l r ng. R ng ngho
cht dinh dng, c bit l cc cht cung cp ngun nit v photpho cho nn
phi b sung. Ngoi ra, tng sinh khi nhanh trong mi trng cn phi c
mt s vitamin nh: biotin, inositon, axit pentotenic.v.v.
3. Cy ging:
Ging phi bo m ng yu cu v c nui cy trc cho s
lng t bo. Qu trnh nui cy ging cn thc hin nghim ngt v ch v
trng v nhu cu dinh dng ca nm men.
4. Nui nm men:
Mc ch ca giai on ny l to c lng sinh khi ln v c hot
lc cao. nm men sinh trng v pht trin tt th trong qu trnh ln men
cn ch n cc yu t nh: nhit gi trong khong 27300C; pH ca mi
trng 45,5; cung cp y oxy; bo m nng v thnh phn cc cht
dinh dng theo yu cu; nng t bo nm men tch lu c phi t mc
thch hp.
5. Thu nhn sinh khi:
Sau khi ln men xong th tin hnh tch sinh khi. C th tch sinh khi
bng cch lc hoc li tm.

6. X l nm men:
Tu theo yu cu ca sn phm (t hoc kh) m qu trnh x l khc
nhau. thu nhn nm men t th tin hnh ra sinh khi thu c ri em
p. Nm men t kh bo qun v thi gian bo qun khng lu.
bo qun lu th cn phi sy kh nm men. Khi sy nm men ch
khng nhit sy vt qu 300C.
3.2.3 Sn xut men nc:
1. Nguyn liu: bt, malt hoc ch phm amylaza ca nm mc.
2. Mi trng nhn ging: bt
trn nc (t l 1:3)
nu chin
lm
ngui 48-50oC
b sung ch phm amylaza (3% malt hoc 0,8 1% ch phm
enzym nm mc nh Asp.awamori hoc Asp.oryzae v dch ging vi khun.
Gi 8-14 gi, cho n khi mi trng t ti 11 12o axt (1o=1ml NaOH
1N/100ml) th a vo cy nm men.
3. Dch ging vi khun: ly nc malt c nng 12oBx thm 1 t
CaCO3. Cy vi khun Lactobacterium Delbruckii v gi nhit 50 52oC
trong 12 ngy. Trc khi a vo s dng phi nhn ging tip.
4. Cy nm men: mi trng sau khi axt ho c lm ngui n 28o
30 C ri tip ging nm men nui cy trc vo (t l 510%), gi nhit
ny 1415 gi khng sc kh hoc sc kh gin on. nhit ny vi
khun lactic ngng tch t axt. Nm men s dng ng v axt tng trng
sinh khi.
Trong mi trng c axt nm men s pht trin tt v hn ch cc vi sinh
vt l.

CHNG IV: SN XUT AXIT AMIN

Axit amin thng b sung vo thc n cho ngi v gia sc. Vi mc
ch ny ngi ta cn n cc axit amin khng thay th. Trong , quan trng
nht l cc axit amin L-lyzin, L-triptophan, L-methionin, L-treonin. Phn ln
cc loi protein c khai thc t thc vt do hoc thiu axit amin ny hoc
axit amin khc. V th, vic b sung axit amin vo thc phm s lm tng gi tr
ca protein. Ngoi ra, cc axit amin cn c tnh cht lm tng mi v ca cc
sn phm thc phm.
4.1 Cc phng php sn xut axit amin:
4.1.1 Phng php tng hp ho hc:
Phng php ny dng sn xut mt s axit amin nh: glyxin, alanin,
metyonin, triptophan. Phng php tng hp ho hc thng cho mt hn hp
cc dng ng phn L- v D-axit amin. Trong 2 dng ny ch c dng L-l thch
hp cho dinh dng. Do vic tch 2 dng ny rt kh khn v tr nn tn
km.

6. X l nm men:
Tu theo yu cu ca sn phm (t hoc kh) m qu trnh x l khc
nhau. thu nhn nm men t th tin hnh ra sinh khi thu c ri em
p. Nm men t kh bo qun v thi gian bo qun khng lu.
bo qun lu th cn phi sy kh nm men. Khi sy nm men ch
khng nhit sy vt qu 300C.
3.2.3 Sn xut men nc:
1. Nguyn liu: bt, malt hoc ch phm amylaza ca nm mc.
2. Mi trng nhn ging: bt
trn nc (t l 1:3)
nu chin
lm
ngui 48-50oC
b sung ch phm amylaza (3% malt hoc 0,8 1% ch phm
enzym nm mc nh Asp.awamori hoc Asp.oryzae v dch ging vi khun.
Gi 8-14 gi, cho n khi mi trng t ti 11 12o axt (1o=1ml NaOH
1N/100ml) th a vo cy nm men.
3. Dch ging vi khun: ly nc malt c nng 12oBx thm 1 t
CaCO3. Cy vi khun Lactobacterium Delbruckii v gi nhit 50 52oC
trong 12 ngy. Trc khi a vo s dng phi nhn ging tip.
4. Cy nm men: mi trng sau khi axt ho c lm ngui n 28o
30 C ri tip ging nm men nui cy trc vo (t l 510%), gi nhit
ny 1415 gi khng sc kh hoc sc kh gin on. nhit ny vi
khun lactic ngng tch t axt. Nm men s dng ng v axt tng trng
sinh khi.
Trong mi trng c axt nm men s pht trin tt v hn ch cc vi sinh
vt l.

CHNG IV: SN XUT AXIT AMIN

Axit amin thng b sung vo thc n cho ngi v gia sc. Vi mc
ch ny ngi ta cn n cc axit amin khng thay th. Trong , quan trng
nht l cc axit amin L-lyzin, L-triptophan, L-methionin, L-treonin. Phn ln
cc loi protein c khai thc t thc vt do hoc thiu axit amin ny hoc
axit amin khc. V th, vic b sung axit amin vo thc phm s lm tng gi tr
ca protein. Ngoi ra, cc axit amin cn c tnh cht lm tng mi v ca cc
sn phm thc phm.
4.1 Cc phng php sn xut axit amin:
4.1.1 Phng php tng hp ho hc:
Phng php ny dng sn xut mt s axit amin nh: glyxin, alanin,
metyonin, triptophan. Phng php tng hp ho hc thng cho mt hn hp
cc dng ng phn L- v D-axit amin. Trong 2 dng ny ch c dng L-l thch
hp cho dinh dng. Do vic tch 2 dng ny rt kh khn v tr nn tn
km.

4.1.2 Phng php trch ly t dch thu phn:

Phng php ny thng dng thu nhn L-cystein, L-cystin, L-leuxyn,
L-asparagin, L-tyrozin. sn xut axit amin theo phng php ny, u tin
phi tin hnh thu phn nguyn liu giu protein v sau dng cc phng
php khc nhau trch ly axit amin cn sn xut ra khi dung dch thu phn.
4.1.3 Phng php tng hp nh vi sinh vt:
Phng php ny c 2 phng n:
1. Ln men trc tip: cc axit amin c to thnh t cc nguyn liu r
tin nh vi sinh vt.
2. Chuyn ho cc tin cht ca axit amin nh vi sinh vt.
S ln men trc tip c ngha ln hn v hin nay hon chnh cng
ngh sn xut hng lot cc axit amin.
4.2 Sn xut axit amin nh vi sinh vt:
4.2.1 S tng hp axit amin t bo vi sinh vt:
Hystidin

Glucoza

Ribozo-5- P

Glucozo-6- P

Sixtein

Eritrozo-4- P

3-P-glyxerat

Serin

Triptophan Tirozin Semilalanin

Pyruvat
CH3COCOOH

Glyxin

-xetoizovalerat
Alanin

Valin
Lxin

+CO2
Oxalatxetat
(HOOCCOCH2COOH)
Asparagin

Aspartat

Fumarat

Xitrat

-xetoglutarat
Lyzin Treonin Methionin
Izolxin

Glutamat
Glutanin Prolin Arginin

S 2: S tng hp axit amin t bo vi sinh vt

Cc axit amin trong t bo vi sinh vt c to thnh do qu trnh trao i
cacbon v nit. Vic tng hp cc axit amin tri qua hng lot nhng phn ng
phc tp vi s xc tc ca nhiu loi enzym khc nhau, nhng c th qui v 2
loi phn ng l amin ho v chuyn amin:

1. Phn ng amin ho: gm 2 giai on:

Giai on 1: to iminoaxit. Giai on ny khng c s tham gia ca
enzym.
NH
R-CO-COOH + NH3
R-C-COOH + H2O
Giai on 2: chuyn iminoaxit thnh aminoaxit nh enzym
dehydrogenaza
NH
NH2
R-C-COOH + H2O dehydrogen
naza
R-C-COOH
NADH2 NAD
NADH2: nicotinamitadenin dinucleotit dng kh
2. Phn ng chuyn amin: phn ng ny xy ra nh s xc tc ca enzym
aminotransferaza
NH2
R1
R2
R1-CO-COOH + R2-CH-COOH
CH-NH2 + CO
COOH

COOH

4.2.2 Qui trnh cng ngh:

Nguyn liu
x l
chun b mi trng dung dch
cy vi sinh vt
nui
vi sinh vt ging
sn phm x l sn phm ln men
1.Nguyn liu: bo m cung cp ngun cacbon, nit v cc nguyn t khc
vi sinh vt pht trin v tng hp c nhiu sn phm.
- Cung cp ngun cacbon: dng r ng, nguyn liu giu tinh
bt hoc mt s loi nguyn liu khc.
- Ngun nit: thng dng l ur, cng c th dng cc loi
mui amoni.
- Cc hp cht khong: KH2PO4, MgSO4, MnSO4
- Cc cht kch thch sinh trng: vitamin, axit amin.
2.X l:
- Lm sch
- Thu phn

3.Chun b mi trng dinh dng:

- C nng v thnh phn thch hp ph thuc vo chng vi
sinh vt v ging sn xut.
- iu chnh pH cho mi trng v thanh trng mi trng.
4.Chun b ging
a.Chng vi sinh vt:
sn xut axit amin ngi ta c th s dng nhiu chng vi sinh vt
khc nhau, tuy nhin sn xut ngi ta phi to cc bin chng hoc loi b
s c ch bng sn phm cui cng ca vi sinh vt.
b.Nui ging:
lng ging cho sn xut ngi ta phi tin hnh nui cy qua
nhiu bc. Cc chng vi sinh vt dng trong sn xut axit amin phn ln l
cc t bin cho nn nu cy chuyn nhiu ln s gp th hi bin v nh
hng n sc sn xut. Trong sn xut axit amin khng bao gi c dng
sinh khi ca m trc lm ging cho m sau.
5.Ln men:
Thng s dng phng php ln men chm. Trong qu trnh ln men cn
ch iu chnh cc thng s k thut: nhit ln men, pH mi trng, s
thng kh, b sung cc cht dinh dng, ph bt v mt s cc yu t khc.
6.Thu nhn sn phm:
Tu thuc vo loi sn phm v mc ch s dng m qu trnh thu nhn
sn phm khng ging nhau:
Dng ch bin thc n gia sc:
- Thu nhn di dng canh trng:
Hn hp sau khi ln men
c c
bt kh
- Tch sinh khi
c c
sn phm
Dng trong ch bin thc phm hoc y hc:
Phi tinh ch qua nhiu cng on thu nhn cc ch phm k thut
hoc tinh khit.
4.3 K thut sn xut mt s axit amin:
4.3.1 Sn xut L-lyzin:
1. Chng vi sinh vt:
Chng sn xut l mt th t bin cn homoxerin ca Corynebacterium
glutamicum (cn gi l Micrococus glutamicus). Di iu kin ln men thch
hp chng ny c th sn xut ti 50g lyzin/lit mi trng. Nguyn liu thng
dng l glucoza hay mt r vi nng 150g/lit.
2. Phng trnh ln men tng qut:
.raffe 22

35C6H14N2O2 + 16C8H13O4 + 262CO2

100C6H12O6+219O2+86NH3 Brevib
+ 380H2O + 1669KJ

3. C ch:
Glucoza
Pyruvat
Oxalaxetat
1

Aspactat

-Aspactyl-photphat
Aspactat--semialdehyt
3

Lyzin

Homoxerin

Treonin

Methionin

Izoleuxin
S 3: C ch tng hp lyzin ca Corynebacterium glutamicum
1 -Enzym aspactokinaza; 2 -Enzym homoxerindehydrogenaza;3 -Enzym
dihydropicolinat-syntetaza.
Lyzin l mt axit amin thuc h aspactat v c tng hp qua mt con
ng trao i cht phn nhnh m qua homoxerin, metionin, treonin,
izolxin cng c to thnh.
Nhng ng chm chm biu din s c ch bi sn phm cui cng.
chng hoang di lyzin v treonin cng gy ra mt s c ch phi hp (E) i
vi aspactokinaza(1). Do khuyt homoxerindehydrogenaza(2) m khng c s
to thnh treonin. Dihydropicolinat-Syntetaza(3) khng mn cm d lp th nn
s c ch bi sn phm cui cng b trit tiu v c s tng hp tha lyzin
(50g/l).
4. Ln men :
Ngun hydratcacbon thch hp cho tng hp lyzin l glucose, fructose,
maltose v saccarosse. Cc ng nh lactose, rafinose, pentose cc chng sinh
lyzin khng ng ho c. Cho nn ngi ta thng s dng cc loi nguyn
liu nh r ng, dch thu phn t tinh bt (ng, sn) lm mi trng sn
xut lyzin.
Nng ng trong mi trng ln men khong 10-12%. Trong qu
trnh ln men, nhm tng hiu sut thu hi lyzin c th b sung thm ng
nng cao nng ng ln ti 25% (nhng cng thn trng v c khi hiu

sut sinh lyzin khng tng m cn nh hng xu n vi sinh vt do lm tng

p sut thm thu ca mi trng).
Ngun nit hay dng l ur, NH3 hoc cc mui amon. T l gia C:N
ng vai tr quan trng, n nh hng n hiu sut tng hp lyzin. Thng b
sung cc mui amon vi hm lng khong 2%.
Ngoi ra cn phi b sung vo mi trng cc cht cung cp ngun
phtpho nh KH2PO4 v K2HPO4 (bo m lng phtpho t 0,0080,02mg/lt; nu nng ny ln n 1,6-2mg/lt th s to thnh lyzin b
ngng). Do khng nn dng photphat amon lm ngun cung cp nit v
photpho.
Phi b sung cc mui c cha cc nguyn t Mg, Fe, Cu, Mn (thng
b sung MgSO4-0,03%; cn Fe, Cu, Mn c trong r ng v cao ng).
Cc cht kch thch sinh trng:
+ Biotin: cn khong 8-15mg/lt, nu qu t (1-2mg/l) th sn phm ch
yu s l axit glutamic, (biotin v B1 c trong cao ng, c bit trong r ng
ma c nhiu biotin)
+ Thiamin(B1): nu khng c s to thnh alanin
+ Treonin: khng c, hoc t s km hm s pht trin ca vi sinh vt
nhng d s dn ti s c ch bng sn phm cui cng. Hm lng thch hp
trong khong 200-800mg/l.
+ Methionin: n nh hng ti sinh trng ca vi sinh vt, hm lng
thch hp: 150-250mg/l.
C th dng homoxerin thay cho treonin v methionin.
Bo m mi trng c pH=7-7,6 v phi thanh trng. Lng ging cho
vo ln men l 5-10%. Nhit ln men l 30-32oC, cung cp O2 2-4g/1l.h;
trong qu trnh ln men dng ur hoc nc NH3 iu chnh pH. Tng thi
gian ln men 50-72h.
5.Thu nhn sn phm:
Tu vo dng ch phm (dch nui cy, dch c c, bt hoc tinh th)
m qu trnh thu nhn sn phm c khc nhau.
- Dch nui cy l ch phm thu c sau ln men v c th dng trc
tip pha vo thc n gia sc.
- Dung dch c c: chng h hng th dch sau ln men c axt ho
bng HCl n pH=5 v b sung dung dch NaHSO3 25% (t l 0,4% so vi dch
ln men) ri em c chn khng cho n khi t nng cht kh 35-40%.
Ch phm ny dng trong chn nui.
- Bt: t dch c c c th b sung thm cc cht n (bt xng,
cm) ri sy kh v nghin nh. Hoc dch c c em sy phun thu ch
phm dng bt.

- thu nhn cc tinh th lyzin th phi s dng nhiu phng php v

tin hnh qua nhiu bc:
Hn hp sau ln men
lc (li tm)
sinh khi
dung dch
nh hp phtrao i ion
Dng dung dch NH4OH 2-3,5% nh hp ph. Dng HCl axit ho n
pH=5 ri c c n khi t nng 30-50%
lm lnh 10-12oC tr tinh.
c ch phm tinh khit th em tinh th kt tinh ln 1 kt tinh li nhiu ln
(ho tan vo cn).
4.3.2 Sn xut L-treonin:
Cho n nay treonin cha c sn xut qui m ln bng con ng vi
sinh vt v vic gy to cc th t bin tng hp tha L-treonin gp nhiu kh
khn so vi lyzin.
C th t c mt s sn xut tha treonin nh 3 bc t bin sau:
- Mt tnh mn cm d lp th ca homoxerindehydrogenaza mn
cm vi treonin.
- Tr dng izolxin.
- Tr dng metionin.
4.3.3 Sn xut L-triptophan:
Nh vo s tng hp ca vi sinh vt, L-triptophan c th thu nhn bng
hai con ng: chuyn tin cht ca triptophan thnh triptophan nh s gip
ca cc h enzym vi sinh vt; hoc thu nhn triptophan nh s t bin do thiu
tirozin v fenilalanin ca cc chng vi sinh vt.
4.3.3.1 Phng php chuyn tin cht:
1. Tin cht:
Nh vi sinh vt c th thu nhn c triptophan t cc tin cht khc
nhau. Hiu sut thu hi sn phm t cc tin cht khc nhau l khng ging
nhau. Mt s tin cht hay s dng:
Axit antranilic
hiu sut thu hi 98,8%
Indol
87,2%
Indol + Cerin (1:2)
92,5%
Indol + Cistein (1:2)
89,5%
Indol + Alanin (1:4)
87,0%
2. Chng vi sinh vt:
chuyn cc tin cht ngi ta c th s dng nhiu chng vi sinh vt
khc nhau. Ring i vi axit antranilic ngi ta hay dng nht l nm men
Candida utilis hoc Hansunella.

3. C ch:
Khi s dng axit antranilic lm tin cht th c ch ca qu trnh nh sau:
COOH
NH2
NH
indol

axit antranilic
COOH

CH2CHCOOH+H2O

NH2

piridoxalfotfat

HOCH2CHNH2+
serin
NH

NH

Triptophan
S 4: C ch chuyn axit antranilic thnh triptophan
4. K thut ln men:
Qu trnh sn xut chia lm hai giai on: thu nhn sinh khi v chuyn
ho tin cht nh s gip ca sinh khi thu nhn c.
Giai on u khng khc my so vi qu trnh nui cy cc loi vi sinh
vt ging khc. Tc l qu trnh cng i t ng ging gc
ng nghim
bnh
tam giclng sinh khi thu c ph thuc vo phng php nui cy
ging. H thy rng nu lng ging a vo ln men cng ln th mc
chuyn ho ca axt antranilic cng ln.
Ngun cung cp cacbon cho ging pht trin l sacaroza hoc mt r (vi
hm lng t 6,3-20%), ngun cung cp nit l ur vi lng t 0,5-1%. Ngoi
ra cn b sung thm (%): K2HPO4-0,01, MgSO4-0,005,CaCl2-0,01. Bo m
mi trng c pH=7,58 v v trng. Tin hnh nui ging nhit
28300C, thi gian nui ca mi cp khng qu 24 gi. Yu cu ging t 35
gam sinh khi kh/1lt mi trng.
Giai on hai c thc hin trong thit b ln men. u tin chun b
mi trng nui ging trong thit b ln men v chuyn men ging nui
giai on 1 vo. Tip tc nui ging trong 24 gi nhit 28300C v thng
kh khng t hn 7g O2/l.h. Nu trong qu trnh nui ging c xut hin bt th
phi dng du ph bt.
Sau 24 gi nui ging cho vo thit b ln men dung dch axt antranilic
5% trong ru v dung dch amoniac 50% ln men. Lc ny thng kh bo
m cung cp 34 gam O2/l.h.

Sau khi cho dung dch axt vo c 3-4 gi th b sung thm dung dch
r ng 25%. Tip theo, c sau 12 gi th b sung dung dch r ng, sau 6
gi th b sung dung dch ammoniac v axit antranilic mt ln. Tng thi gian
ca giai on 2 l 120140 gi.
Qu trnh ln men (giai on 2) thc hin pH ca mi trng 8,0 v
nhit ln men khong 300C.
3. Thu nhn sn phm:
Dung dch sau ln men c cha 7,812,5% cht kh, trong c
0,30,5% triptophan. Phn ln triptophan (8588%) nm trong pha lng. Do
thu nhn ch phm tinh khit th phi tinh ch dch lc. Cn nu dng cho
chn nui th s dng dng c c c c sinh khi nm men.
lm thc n gia sc th dch sau ln men em c c cn li 1/3 th
tch, sau em sy trong thit b sy phun (to=110120oC) thu nhn bt
dng b sung cho thc n gia sc. Trong thnh phn ca bt ny gm c:
- Cht kh 90%
- Protein
4854%
- Triptophan 13%
- Vitamin (mg/kg):
+ B1 : 1518,5
+ B2 : 24,532,5
+ PP : 620680
- Axit amin khc 6%. Trong c cha nhiu cc axit amin nh: lyzin,
histidin, treonin, xerin, glutamate, prolin, glyxin, valin, tirozin, phenilalanin,
acparagin, acginin
thu nhn ch phm triptophan tinh khit th phi qua cc bc sau:
- Tch sinh khi bng li tm
- Dng HCl axit ho n pH = 1 v tch cc kt ta
- Tin hnh trao i ion
- Nh hp ph nh dung dch ammoniac 5%
- C c dung dch nhit 60700C
- Lm lnh n 460C tr tinh
- Tch tinh th triptophan
- Ra cc tinh th bng ru etylic
- Sy cc tinh th nhit 600C
Cc ph liu ca qu trnh tinh ch ny c th dng cho chn nui.
4.3.3.2 Thu nhn nh cc chng t bin:
S thu nhn triptophan cng nh tirozin v phenilalanin c th thc hin
trn cc chng t bin ca E. coli hoc Bacillus subtilis. Qu trnh tng hp
tirozin, triptophan v phenilalanin i theo s sau:

Glucose
Axit fotfoenol pyruvic

Eritrozo-4- P

Axit skimic
Axit corismic

Axit antranilic
Triptophan

Axit prephenic
Axit phenylpyruvic

Axit hydroxyphenylpyruvic

Phenylalanine
Tirozin
S 5: C ch tng hp triptophan, phenilalanin, tirozin trong
t bo vi sinh vt
tng hp tha triptophan ngi ta dng chng e. coli tr dng tirozin
v phenylalanine.

CHNG V: SN XUT CH PHM ENZYM

5.1 Nhng tnh cht u vit ca enzyme
Enzym l mt cht xc tc sinh hc. N c y cc tnh cht ca cht
xc tc ho hc, ngoi ra cn c nhng u vit sau:
1. C tnh chuyn ho cao (tnh c hiu): mi enzyme ch tc dng ln
mt c cht nht nh no theo mun m khng gy nn mt bin i no
khc. V d: amylaza ch thu phn tinh bt, proteaza ch thu phn protein.v.v.
2. Cng lc xc tc ln: vi mt lng nh enzyme c th lm xc tc
chuyn ho mt lng c cht ln. V d: 1g pepxin c kh nng thu phn
50kg trng, 1g rennin c th lm ng t 72 tn sa, 1mol catalaza c th lm
chuyn ho 5x106mol H2O2 trong 1 giy.
3. Tc dng trong mt iu kin m du: phn ln cc enzyme hot ng
nhit khng cao, pH thng l trung tnh v p sut thng.
4. Enzym khng c hi: tt c cc loi enzyme u c ngun gc t sinh
vt nn khng c hi i vi sc khe con ngi.
Chnh v nhng u vit m trong nhng nm gn y ch phm
enzyme c s dng rng ri v ngnh cng nghip enzyme cng c pht
trin khng ngng.
5.2 Ngun nguyn liu thu nhn enzyme:
Enzyme c th thu nhn t nhiu ngun nguyn liu khc nhau nh: ng
vt, thc vt v vi sinh vt.

Glucose
Axit fotfoenol pyruvic

Eritrozo-4- P

Axit skimic
Axit corismic

Axit antranilic
Triptophan

Axit prephenic
Axit phenylpyruvic

Axit hydroxyphenylpyruvic

Phenylalanine
Tirozin
S 5: C ch tng hp triptophan, phenilalanin, tirozin trong
t bo vi sinh vt
tng hp tha triptophan ngi ta dng chng e. coli tr dng tirozin
v phenylalanine.

CHNG V: SN XUT CH PHM ENZYM

5.1 Nhng tnh cht u vit ca enzyme
Enzym l mt cht xc tc sinh hc. N c y cc tnh cht ca cht
xc tc ho hc, ngoi ra cn c nhng u vit sau:
1. C tnh chuyn ho cao (tnh c hiu): mi enzyme ch tc dng ln
mt c cht nht nh no theo mun m khng gy nn mt bin i no
khc. V d: amylaza ch thu phn tinh bt, proteaza ch thu phn protein.v.v.
2. Cng lc xc tc ln: vi mt lng nh enzyme c th lm xc tc
chuyn ho mt lng c cht ln. V d: 1g pepxin c kh nng thu phn
50kg trng, 1g rennin c th lm ng t 72 tn sa, 1mol catalaza c th lm
chuyn ho 5x106mol H2O2 trong 1 giy.
3. Tc dng trong mt iu kin m du: phn ln cc enzyme hot ng
nhit khng cao, pH thng l trung tnh v p sut thng.
4. Enzym khng c hi: tt c cc loi enzyme u c ngun gc t sinh
vt nn khng c hi i vi sc khe con ngi.
Chnh v nhng u vit m trong nhng nm gn y ch phm
enzyme c s dng rng ri v ngnh cng nghip enzyme cng c pht
trin khng ngng.
5.2 Ngun nguyn liu thu nhn enzyme:
Enzyme c th thu nhn t nhiu ngun nguyn liu khc nhau nh: ng
vt, thc vt v vi sinh vt.

5.2.1 ng vt:
Enzyme nm trong cc c quan ni tng ca ng vt:
- Tu tng: cha nhiu enzyme amylaza, maltaza, proteaza, esteraza,
lipaza.v.v.
- D dy cha nhiu enzyme pepxin, cc enzyme tiu ho (trong ngn th
t ca d dy b c 1 loi vi khun c kh nng sinh tng hp xenlulaza), trong
d dy ca b v ngh c cha enzyme rennin lm ng t sa.
- Rut: cha ch yu l enzyme enterokinaza c kh nng ph v cc lin
kt peptit.
- Gan: l c quan cha rt nhiu enzyme.
Khng th thu enzyme t ng vt trn qui m cng nghip c v thi
gian nui ng vt qu di nn khng kinh t.
5.2.2 Thc vt:
Ngi ta c th thu c mt s enzyme trong thc vt thng ng nh
thu papain t u , bromelain t da, amylaza t malt, plazmin (c papain) t
h ficus (sung, v).
Trong cc h u c enzyme ureaza. Trong thuc l c cha enzyme
amylaza, esteraza, pectinaza, proteaza. Trong ch c cha nhiu enzyme oxy
ho kh nh polyphenoloxydaza. Trong rau qu cng cha nhiu loi enzyme
khc nhau.
Thu enzyme t thc vt cng khng kinh t v mun c nhiu thc vt
th phi trng nn phi c t v chi ph tn km trong vic chm bn.
5.2.3 Vi sinh vt:
So vi ng vt v thc vt th vic thu nhn enzyme t vi sinh vt c
nhiu li th hn.
- T vi sinh vt c th thu nhn c nhiu enzyme khc nhau, trong
c nhng loi khng th thu nhn c t ng vt hoc thc vt.
- Bng cch thay i iu kin nui cy hoc dng tc nhn iu chnh
ngi ta c th iu khin qu trnh sinh tng hp enzyme ca vi sinh vt theo
mun.
- Vi sinh vt c kh nng sinh sn v pht trin nhanh, tng hp enzyme
vi tc cc k ln trong mt thi gian ngn. Chnh v th vic sn xut
enzyme t vi sinh vt t tn thi gian.
- Enzyme thu nhn t vi sinh vt c hot tnh rt mnh, vt xa cc
enzyme t cc ngun sinh vt khc.
- Vi sinh vt c kh nng sinh sn, pht trin v tng hp enzyme trn
cc mi trng dinh dng n gin, d kim v r tin (c th l ph liu ca
cc ngnh sn xut khc nhau). Cho nn vic sn xut enzyme t vi sinh vt
cng rt kinh t.

Chnh v cc li th trn nn ngun enzyme t vi sinh vt c th a vo

ngnh sn xut ch phm enzyme cng nghip.
Bng 1: Kh nng s dng cc loi enzyme t vi sinh vt
Loi enzyme
Vi sinh vt
S dng
Amylaza
Bacillus Subtilis, Bacillus
Bia v ru (phn hu tinh bt
Stearotherophilus.
thnh ng ln men, sn xut
nha)
Asp.niger
Sn xut bnh m, bnh ngt
-amylaza
Asp.oryzac
(thu phn mt phn tinh bt)
Glucoamylaza Asp.niger
Sn xut glucoza, bia (lm
Asp.oryzac
gim hm lng dextrin)
Endomycopsis bispora
Pectnaza
Asp.niger
Sn xut v lm trong dch qu,
Asp.oryzac
cc ch phm rau cho dinh
Sclerotinia libertina
dng tr s sinh.
Cc xenluloza Trichoderma viride
CN thc phm (lm tan thnh
t bo thc vt rau), ru
(thu nhn ng t xenluloza).
extranaza
Penicillium funiculorum
Sn xut dexran vi kch thc
phn t nht nh. Cht b
sung cho thuc nh rng.
Invectaza
Sacch.Cerevisiac
Dch ho ng lm keo,
sn xut mt ong nhn to.
Cc proteaza
Bacil.Subtilis, Bacil.Cereus B sung vo bt git loi
Bacil.Stearotheroopilus,
protein, lm mm tht, cht tr
Streptomyces griseus,
tiu ho, dng trong cng ngh
Asp.oryzac
lm phim
Enzym lm ng Mucor pusillus
Sn xut fomat
sa
Keratinaza
Streptomyces fradiac
Ngnh da (kh lng)
Bo qun thc phm (loi O2)
-galactozidaza Asp.niger, Pen.notatum
Xc nh glucoza (chn on
bnh i ng).
Streptokinaza
Streptococcus,
iu tr (tiu cc nghn)
Spec.chotridium
iu tr cc vt thng, vt
histolyticum
bng
L-asparaginaza Eschericha coli, Erivinia
iu tr bnh bch cu
aroidac
Penixilinazylaza Escherichia coli
SX cc penixilin bn tng hp

5.3 Sn xut enzym t vi sinh vt:

Qu trnh sn xut gm 3 giai on:
- Tuyn chn vi sinh vt v ci to ging
- Nui cy vi sinh vt
- Tch v tinh ch enzym
5.3.1 Tuyn chn vi sinh vt v ci to ging:
1. Tuyn chn:
Kh nng to enzym ca vi sinh vt rt a dng v phong ph. Mi loi
enzym c th thu nhn c t nhiu chng vi sinh vt khc nhau v ngc li
mt chng vi sinh vt cng c kh nng sinh nhiu loi enzym.
V d:

Aspergillus oryzae
Amylaza c th thu nhn nh Bacillus Subtilis
Bac.Stearothermophilus
-Amylaza
Asp.niger
Asp.oryzae
Asp.awamori
Glucoamylaza
Asp.oryzae, niger
Endomycopsis, awamori
Asp.niger
Pectinaza
Asp. oryzae
Asp.awamori
Asp.oryzae
Proteaza
Bac.Subtilis
Streptomyces griseus
Proteinaza
Asp.awamori, flavus, Candidus
Bac.mesentericus, Bac.Subtilis
Xenlulaza
Trichoderma viride
Asp.terreus
Kh nng sinh enzym ca cc chng vi sinh vt khng ging nhau v
khc nhau ngay c trong cng mt chng. V vy, khi tuyn chn vi sinh vt
phi tin hnh tm kim, phn lp, la chn hng chc, hng trm thm ch n
hng nghn ging vi sinh vt khc nhau mi c c nhng chng vi sinh vt
c hot lc cao trong vic to nhng enzym cn thit.
2. Ci to ging:
nng cao sc sn xut enzym ca vi sinh vt cng nh lm tng cng
hot tnh ca enzym ngi ta phi s dng cc phng php khc nhau ci
to ging, c bit l phng php gy t bin. Bng phng php t bin
nhiu nc thu c nhng chng vi sinh vt c nhng c tnh tt, ch tng

hp mt hoc vi ba enzym nhng u c hot tnh cao so vi chng nguyn

thu ban u.
5.3.2 Nui vi sinh vt:
5.3.2.1 Cc yu t nh hng ti s tng hp enzym:
1. nh hng ca thnh phn mi trng dinh dng:
L yu t quan trng c nh hng ln n s pht trin v tng hp
enzym ca vi sinh vt. Cng mt loi vi sinh vt nhng nu nui cy trn cc
mi trng c thnh phn dinh dng khc nhau th mc to thnh enzym
v thnh phn enzym c to thnh cng khc nhau.
V d: nui Asp.oryzae bng phng php b mt trn mi trng cm i
mch th c th to c nhiu enzym, trong c cc loi enzym c hot tnh
cao nh amylaza, proteaza, maltaza, pectinaza, invectaza, ribonucleaza. Nhng
nu nui trn mi trng Kzapek ci tin vi nitrat v tinh bt th ch to thnh
ch yu l -amylaza, cn cc enzym khc to thnh vi lng khng ng k.
Hin tng ny c gii thch bng l thuyt sinh tng hp cm ng
enzym. Hin tng cm ng ny ch thy vi sinh vt ch khng thy ng
v thc vt. Khi trong mi trng c nhng cht kh ng ho vi sinh vt phi
tit vo mi trng mt hoc nhng enzym tng ng phn hu n thnh
nhng cht c th ng ho c. Cho nn khi thm cc cht cm ng vo mi
trng dinh dng th kh nng sinh tng hp enzym ca vi sinh vt c th
tng ln gp nhiu ln so vi trng hp bnh thng.
nh ngha: Mt qu trnh sinh tng hp c gi l cm ng nu nh
n ch xy ra mc ng k khi trong mi trng c c cht c hiu ca
enzym ny hoc cc cht trao i c cu trc tng t c cht. Cc enzym
thuc loi ny gi l enzym cm ng, cc c cht kch thch qu trnh tng hp
ny gi l cht cm ng.
Mun tng hp c enzym cm ng cn c 4 iu kin:
- Th nht: C gen tng ng trong nhim sc th
- Th hai: C nguyn liu y xy dng cc phn t enzyme
(cc axit amin, cc hp phn coenzyme).
-Th ba: C nng lng cn thit cho vic tng hp enzyme.
- Th t: C cht cm ng.
Nu c 3 iu kin trn nhng thiu cht cm ng th enzyme cng
khng c to thnh.
Trong cng ngh sn xut enzyme cn phi la chn cht cm ng thch
hp v xc nh nng ti u ca n trong mi trng c hiu sut sinh
tng hp cao nht.
2. nh hng ca m:
Trong phng php nui cy b mt, m ca mi trng c v tr c
bit quan trng. Khi m thp vi sinh vt b ri vo tnh trng bt li cho hot

ng sng nn c khuynh hng to bo t v hn ch s tng hp enzyme.

Khi m qu cao th ti xp v thong kh ca canh trng b gim, do
s tng hp enzyme kh khn.
m cho php vi sinh vt tng hp enzyme tt thng t 5365%, ph
thuc vo c im sinh l ca vi sinh vt, vo cu trc c hc ca mi trng
v iu kin thot m ca mi trng nui cy.
3. pH ca mi trng:
So vi phng php nui chm th phng php b mt t chu nh hng
ca pH mi trng n qu trnh sinh tng hp enzyme v trong qu trnh nui
pH ca mi trng kh n nh. Vi phng php nui cy chm pH mi
trng bin i kh r rt trong qu trnh nui v gy nh hng ln ti s
tng hp enzyme. Do , ngi ta thng iu chnh pH thu c enzyme
vi hiu sut cao.
Mi loi enzyme thng c tng hp mnh trong mt vng pH xc
nh i vi tng loi vi sinh vt. V d: amylaza v pectinaza ca nm mc
c tng hp mnh trong mi trng c pH=4,55, cn proteaza trong mi
trng c pH=66,5.
4. Nhit :
Phn ln cc vi sinh vt c kh nng tng hp mnh cc enzyme u
thuc loi a nhit trung bnh, c nhit ti thch 22320C. Vi vi khun c
loi thch nhit cao hn (35550C) v chng c kh nng tng hp cc
enzyme c bn nhit cao. Khi nng nhit mi trng ti qu mc thch
hp th kh nng sinh tng hp enzyme b gim r rt.
5. Oxy:
vi sinh vt sinh trng v pht trin c th phi c oxy. Do ,
trong thi k tch lu sinh khi phi cung cp y oxy cho mi trng.
6. Thi gian nui:
Thi gian nui cy thch hp ca mi loi vi sinh vt c xc nh bng
thi gian cho php tch t enzyme ti a. Thi gian ph thuc vo chng vi
sinh vt v t ph thuc vo enzyme chng tng hp.
5.3.2.2 La chn mi trng nui cy:
Thnh phn mi trng dinh dng l mt trong nhng yu t c nh
hng quyt nh n hot ng sng ca vi sinh vt cng nh kh nng sinh
tng hp enzyme ca chng.
bo m nhng yu cu ti thiu cho vi sinh vt pht trin v tng hp
enzyme th trong mi trng cn c cc cht cha cacbon, nit, hydro, oxy v
cc cht khong nh Mg, Ca, P,S, Fe, K .v.v.
- Ngun cung cp cacbon thng dng l gluxit. Khi s dng ng cn
nh rng mt s ng c tc dng km hm s sinh tng hp mt s enzyme

thu phn. V d: ng glucose c th km hm sinh tng hp mt s enzyme

h amylaza.
Ngoi gluxit cng c th s dng ngun cacbon khc nh m, du, axit
hu c, ru. Trong s cc axit h c th axit lactic c vi sinh vt hp th
d dng hn c. Axit ny c nhiu trong nc chit ng. Cho nn cao ng c
dng va l ngun b sung m va l ngun cung cp cacbon cho vi sinh vt
pht trin.
- Ngun cung cp nit c th l cc hp cht hu c hoc v c. Cc hp
cht hu c l nhng hp cht giu m nh cao ng, bt u tng, kh u,
kh lc Ngun nit v c nh ur, NH3, cc mui amn.
Ngoi ra, mt s axit amin, baz purin (adenine, guanine), pyrimidin
cng c b sung vo mi trng nui cy.
Cc axit amin c tc dng khc nhau i vi vi sinh vt cng nh vic
sinh tng hp enzyme. Nhiu axit amin c kh nng tng cng sinh tng hp
enzyme, mt s khc li km hm hoc khng c nh hng r rt.
- Cc nguyn t khong cng c nh hng r rt n qu trnh sinh tng
hp enzyme. Cc nguyn t ny bao gm c nguyn t a lng v vi lng.
Vi lng l st, km, ng, coban. Cc nguyn t ny thng c trong nc
hoc trong cc thnh phn hu c khc ca mi trng (bt, cao ng) do
khng cn b sung hoc ch b sung dng vt. Nng ca cc nguyn t
ny tng s km hm mnh s pht trin ca vi sinh vt.
Cc nguyn t a lng c a vo di dng mui v c nhng cn
tnh c hm lng ca chng c sn trong cc thnh phn ca mi trng.
Trong cc nguyn t ny cn lu n P v S v 2 nguyn t ny l thnh phn
cu to ca nhiu cht quan trng, nht l nucleotit, lipit, vitamin
- Cc yu t sinh trng: cc vitamin ch yu dng kch thch sinh
trng ca vi sinh vt. Tuy nhin cn nh rng, trong nhiu trng hp mi
trng thch hp cho s sinh trng ca vi sinh vt cha hn l thch hp
sinh tng hp enzyme. C khi mi trng km hm s tch lu sinh khi t bo
nhng vn tng cng sing tng hp enzyme.
xc nh thnh phn mi trng dinh dng thch hp cho mi vi sinh
vt c th tin hnh la chn trn c s kinh nghim hoc dng cc phng
php ton hc qui hoch thc nghim.
Khi la chn nguyn liu chun b mi trng cn ch rng, trong
cc nguyn liu t nhin c cha nhiu cht khc nhau, k c cc nguyn t
khong a lng cng nh vi lng v c c cc yu t sinh trng. Do vy,
trong nhiu trng hp khng cn b sung thm cc loi mui v c. Mt khc,
cc nguyn liu ny r tin do vy n thng dng trong cng nghip sn
xut ch phm enzyme t vi sinh vt.

Cc nguyn liu t nhin thng dng chun b mi trng l cm

go, bt ng, bt u tng, kh lc hoc cc ph liu nh men bia, r ng
Trong cc nguyn liu ny th cm go v bt ng l ngun cacbon tt nht.
Cn nm men bia t phn, kh lc l ngun nit tt nht.
Khi chun b mi trng cn cn phi ch n cc cht c tc dng iu
ho sinh tng hp enzyme, c bit l cc cht cm ng. i vi mi loi
enzyme c th c nhiu cht cm ng cho nn phi la chn cht cm ng c
hiu qu nht. V d nh cht cm ng ca sinh tng hp amylaza l tinh bt,
dextrin; ca proteaza l cc protein c trng lng phn t nh.
S dng cc cht cm ng thng lm tng ng k lng enzyme c
tng hp. Tuy nhin, khi la chn cht cm ng cn tnh ton cn thn v gi
thnh ca n.
5.3.2.3 Cc phng php nui cy vi sinh vt:
C nhiu phng php khc nhau nui vi sinh vt tng hp enzyme.
1. Phng php b mt:
Phng php ny thch hp nui cc loi nm mc v kh nng pht
trin ca n nhanh, mnh nn t tp nhim.
Qui trinh cng ngh:
Mi trng
phi trn
lm m
hp
nh ti v lm ngui
gieo
mc ging
vo thit b nui
vo phng nui
nui mc
canh trng.
Cc cht dinh dng thng l cc nguyn liu t nhin nh cc cht thi
ca cng ngh thc phm hoc hn hp ca nhiu cht. Trong mi trng cn
thm cht cm ng cn thit cho s tng hp enzym hoc b sung thm cht
dinh dng ngun nit, photpho nh nc chit ng, khoai ty, dch t phn
nm men bo m xp ca mi trng cn c cht lm xp nh tru
(1020%). Trc khi gieo cy cn thanh trng mi trng m bo khng b
nhim vi sinh vt l.
Qu trnh nui cy ko di t 3348h. C cc hin tng:
- Thi nhit, cn gi to=2832oC
- Gim m, gi phng=96100%
- Cung cp O2: trong qu trnh mc pht trin ly O2 v thi CO2

cn cung cp khng kh v trng.
u nhc im ca phng php:
u:
- Cho nng enzym cao hn so vi phng php b mt
- C th sy kh nhanh chng mi trng (dng th) m khng
nh hng n hot lc enzym.
- Trnh s nhim trng ton b khi canh trng
- Tn t in nng.

Nhc:
- Kh c gii ho, t ng ho.
- Nng sut thp.
- Tn nhiu lao ng.
- Tn nhiu mt bng.
2. Phng php nui cy chm:
Vi phng php ny vi sinh vt pht trin trong mi trng lng c
sc kh v khuy o lin tc. Ngun cacbon s dng trong phng php ny l
tinh bt, cc cht thi c ng nh r ng v cc nguyn liu khc. Ngun
nit hay dng l cht hu c nh cao ng, dch men bia, dch thu phn, kh
lcNgoi ra trong thnh phn mi trng cng cn b sung thm cc cht
khong. Mi trng cn c thanh trng bng cch sc hi trc tip
to=121125oC trong thi gian 6090 pht i vi mi trng cha nguyn liu
bt, m th v 2030 pht vi cc loi mi trng c cha ng.
Trong qu trnh nui cy chm cn phi sc kh v trng v khuy o
lin tc. Nu c bt phi dng du ph bt. Thi gian t 14 ngy tu chng.
Phi ch iu chnh pH.
u, nhc:
+ u:
- Tit kim din tch
- D c gii ho, t ng ho.
- S dng hp l cht dinh dng, trnh lng ph.
- Thu enzym t tp cht
- m bo v sinh, v trng nn t nhim vi sinh vt l.
+ Nhc:
- Nng enzym thp, cn lm c canh trng trc khi tch
nn gi thcao.
- Tn in do sc kh.
- Nu khng m bo v trng s nhim hng lot.
3. Phng php nui chm 2 bc:
- Bc 1: vi sinh vt nui trong thit b pht trin ti a.
- Bc 2: chuyn sinh khi sang thit b khc sinh tng hp
enzym.
5.3.3 Tch v tinh ch enzym:
Trong thc t enzym c s dng nhiu dng khc nhau. Mt s lnh
vc c th s dng di dng canh trng kh m cht lng sn phm khng
thay i (nh sn xut ru, cng ngh thuc da). a s cc ngnh cng nghip
(ch yu l thc phm v cng nghip nh) i hi phi s dng cc ch phm
enzym sch (gi l ch phm k thut). Cn trong y hc phi s dng cc ch
phm enzym tinh khit. Tuy nhin, cho n nay cha tm c mt phng

php chung tch mt enzym no m phi phi hp nhiu phng php

khc nhau.
Qu trnh lm sch enzym gm 2 giai on l chit enzym v tch enzym.
5.3.3.1 Chit enzym:
Mc ch ca cng on ny l tch cc enzym ra khi canh trng sau
ln men. Qu trnh ny ph thuc vo loi enzym (ngoi bo hay ni bo) v
mi trng ln men (lng hoc rn) m tin hnh khc nhau.
i vi enzym ngoi bo nm trong canh trng lng th tin hnh tch
sinh khi thu dung dch c cha enzyme. Nu enzym ngoi bo nm trong
canh trng rn (xp) th dng nc v trng hoc cc dung dch m thch
hp chit enzym.
thu cc enzym ni bo th phi tin hnh ph v t bo bng mt trong
cc phng php sau:
- Phng php ng ho (nghin trong my ng ho)
- Nghin vi bt thu tinh, ct
- T phn
- Lm lnh ng v tan gi lp i lp li nhiu ln
- Nh tc ng ca siu m
- Dng p sut thm thu cao
- Phng php khuych tn
Sau khi ph v t bo th dng nc ct hoc dung dch mui long hoc
cc dung dch m thch hp chit enzym.
5.3.3.2 Tch enzym:
Mc ch ca giai on ny l lm sch v tch cc enzym ra khi hn
hp ph thuc vo mc tinh sch ca cc ch phm enzym m qu trnh tch
cng rt khc nhau.
1. Thu nhn ch phm k thut:
Ch phm enzym k thut l ch phm enzym c tinh ch s b c th
cha mt hoc vi enzym ch yu.
thu ch phm enzym k thut th u tin phi c c dung dch
enzym nhit 30oC cho n khi t nng 20g/lit. Sau tip tc c
nhit 40oC cho t nng 3050g/lit. Khi c c th cho thm cht bo
qun hoc cht n nh. Tip theo em sy phun vi nhit khng vt qu
70oC.
Ngoi ra thu ch phm enzym k thut ngi ta cng c th kt ta
bng dung mi hu c hoc mui trung tnh v sau cho cht n nh vo sy
kh ri nghin.
Dung mi hu c thng dng l ru etylic, izopropylic, axetonkt
ta bng dung mi hu c da trn nguyn tc khi thm dung mi vo dung
dch enzym th lc ht gia cc phn t protein-enzym tch in v cc phn t

nc bao quanh chng b gim. Khi nng dung mi thm vo t n mc

lm cho nng lng hydrat ho gia cc phn t nc v protein nh hn nng
lng lin hp gia cc phn t protein-enzym th lc cc phn t proteinenzym lin hp li to kt ta.
Tu theo tnh cht ca enzym v dung mi hu c m mi mt loi
enzym c kt ta mt nng dung mi khc nhau. V d proteaza kt ta
trong etanol 7678%, -amylaza kt ta trong etanol 70%, glucoamylaza kt
ta trong etanol 45% v trong axeton 70%.
Dng dung mi hu c c u im l ct v thu hi c. Nhng dung
mi d lm cho enzym b v hot. khc phc nhc im phi tin hnh
kt ta enzym nhit thp (5oC) v thi gian ngn.
Cc mui trung tnh thng dng kt ta enzym l: (NH4)2SO4,
NaCltu theo tnh cht tng loi enzym m ngi ta dng nng mui
khc nhau. V d amylaza thng kt ta trong (NH4)2SO4 nng 0,75%
bo ho; glucoamylaza nng 0,80,9% bo ho.
Phng php dng mui trung tnh kt ta cn gi l dim tch. Phng
php ny da trn nguyn tc cc ion mui c tc dng ph v cc lp hydrat
bao quanh phn t protein do s ht cc phn t tri du v pha n. Kt qu
lm phn t protein b mt nc v ng t.
Sau khi kt ta phi loi cc mui trung tnh ra khi enzym bng phng
php thm tch. thc hin ngi ta cho enzym vo cc mng bn thm
(xenlofan) ri cho vo cc dung dch m thch hp ngm trong 24 gi v sau
th li nu loi ht mui th a i x l tip.
2. Thu nhn ch phm tinh khit:
thu nhn ch phm tinh khit phi kt hp nhiu phng php v qua
nhiu giai on. u tin phi tin hnh loi b cc protein khng hot ng
bng phng php bin tnh chn lc. Cc protein bin tnh c th tch ra bng
cch lc hoc ly tm. Sau enzym c kt ta bng dung mi hu c hoc
mui trung tnh. Tip theo dng sc k (sc k trao i ion, hp ph, phn b,
gel phn t v.v...) thu phn on enzym c hot lc cao nht.
Sau khi tinh ch xong phi dng phng php in di kim tra mc
ng nht ca enzym.
5.4 nh gi cht lng ca enzym:
5.4.1 Phng php nh gi:
Cht lng ca enzym c nh gi bng mc hot ng ca n tc
l hot ca enzyme. Thc t ngi ta o hot ca enzym thng qua vic
xc nh lng c cht mt i hay lng sn phm c to thnh trong phn
ng. V nguyn tc c th chia ra lm 3 nhm phng php xc nh hot
enzym:

1. o lng c cht b mt i hay lng sn phm c to thnh trong

mt thi gian nht nh vi mt nng enzym xc nh.
2. o thi gian cn thit thu c mt lng bin thin nht nh ca
c cht hay sn phm ng vi mt nng enzym nht nh.
3. o nng enzym cn thit lm bin i mt lng c cht hay thu
nhn mt lng sn phm nht nh trong mt thi gian nht nh.
5.4.2 n v:
o hot ca enzym ngi ta dng n v hot .
- n v enzym quc t (k hiu UI): l lng enzym c kh nng xc tc
lm chuyn ha mt micromol c cht sau mt pht iu kin tiu chun (
pHopt, toopt i vi enzym cn xc nh)
- n v Katal (Kat): l lng enzym c kh nng xc tc lm chuyn ho
mt mol c cht trong mt giy iu kin tiu chun
1UI=

1
-6
10 Kat=16,67 nKat (nanoKat)
60

- Hot ring ca ch phm enzym: l s n v UI hoc s n v Kat

ng vi 1ml ch phm (nu l dung dch) hoc 1mg ch phm (nu l bt).
Nu enzym tinh sch th hot c biu th bng s UI hoc s Kat
trn 1mg enzym.
- Hot phn t: s phn t c cht c chuyn ho bi mt phn t
enzym trong mt n v thi gian.
5.4.3 Nhng ch khi nh gi cht lng enzym:
nh gi chnh xc hot ng ca enzym th khi xc nh hot
ca n cn ch :
- Nng c cht trong phn ng phi trong mt gii hn thch hp
tha bo ho enzym nhng khng qu cao n mc km hm enzym.
- Vi nhng enzym cn c cht hot ho hoc cht lm bn th phi cho
cht ny vo enzym trc khi cho c cht vo phn ng.
- Xc nh hot cn tin hnh pHopt v c nh. pH thng thay i
ph thuc vo c cht v thnh phn dung dch m.
- Nhit dng xc nh hot phi nh hn nhit ti u ca enzym
phng s km hm ca enzym nhit cao.
- Thi gian xc nh hot thng 530 pht. Nhng cng c trng
hp ko di n 24 gi nu hot ca enzym qu thp. Trong thi gian di
phi cho thm nhng cht dit khun trnh hin tng tp nhim.
5.5 Enzym khng tan:
Enzym khng tan hay cn gi l enzym c nh l enzym nm trong mt
khng gian b gii hn. S gii hn linh hot ca enzym t c bng cch
a n vo mt pha cch li, tch ri khi pha dung dch t do v n vn

c kh nng tip xc c vi phn t c cht. Pha cha enzym thng khng

tan trong nc nhng cng c th l polymer cao phn t a nc.
5.5.1 u nhc im ca enzym khng tan:
1. u im:
- Gim gi thnh v c th s dng lp li nhiu ln mt lng
enzym xc nh.
- Enzym khng ln trong cc sn phm do trnh c nh
hng khng tt ca n i vi sn phm.
- C th lm ngng nhanh chng cc phn ng khi cn thit.
- Enzym khng tan bn hn enzym tan do kt hp tn dng
cc yu t nhit , pH
- Dng enzym c nh c th d dng thay th cc qu trnh ln
men bng cc phn ng enzym ngoi t bo.
2. Nhc im:
Enzym khng tan c hot ring nh hn enzym tan
5.5.2 Phng php sn xut enzym khng tan:
Gm c 3 phng php:
5.5.2.1 Gn lin kt ng ho tr:
a s cc enzym khng tan c sn xut bng phng php ny. N
chia ra lm hai phng php l gn enzym vo cht khng ho tan hoc gn cc
phn t enzym vi nhau to thnh i phn t khng ho tan.
1. Gn phn t enzym vo cht mang khng ho tan:
Yu cu v cht mang:
- C ho tan thp v bn vng vi cc tc ng c, nhit v
ho hc.
- Cc cht mang khng gy tc ng km hm enzym.
- Khng hp ph phi chn lc i vi protein.
- Nn ho nc v cht mang k nc c th gy c ch vi
enzym c mang.
- Vic gn s c hiu qu hn nu cht mang v enzym c in
tch tri du.
Nhng cht mang thng dng:
+ Cht mang hu c: nh l polypeptit, polysaccarit, cc dn xut ca
xenlulo (DEAE-xenlulo: dietyl animoetyl xenlulo, CM-xenlulo: cacboxyl metyl
xenlulo), dn xut ca dextran (DEAE-sephadex, CM-sephadex) hoc agaroza
(aga khc vi agaroza, trong thnh phn ca agaroza c gc sunfat).
Cht mang thng dng hn c l cc detran c lin kt ngang gi l
sephadex v agaroza dng ht gi l sepharoza. Ni chung c 2 loi ny u
c cu trc l xp lm cho cc phn t ln c th xm nhp vo gel mt cch
d dng.

Ngoi ra cn c cc polymer tng hp khc nh polyacrylamit,

polysterol, polyamit.
+ Cht mang v c: so vi cht mang hu c th cht mang v c bn hn
v nhit, c hc, ho hc, vi sinh vt v nht l khng thay i cu hnh ca
khun khi thay i tnh cht ca mi trng xung quanh. Cc cht mang v c
nh: silicagen, pentonit, nhm hydroxyt
2. Gn cc enzyme vi nhau
Ngi ta c th gn nhng enzyme li vi nhau nh nhng tc nhn a
hay lng chc to thnh nhng i phn t enzyme khng ho tan. Vi
phng php ny thu c nhiu dn xut enzyme khng tan, v d thu dn
xut tripxin khng tan bng cch dng andehyt glutaric (OHC-(CH2)3-CHO)
1% lin kt cc phn t enzyme.
Phng php ny c u im:
- t tn tht enzyme do gii phng ra mi trng
- Tng kh nng chng chu ca enzyme i vi cc yu t bin
tnh nn a vo sn xut c.
Nhc im ca phng php ny l s enzyme c nh t.
5.5.2.2 Gienzyme trong khun gel
Phi tin hnh trng hp ho cc gel ng thi vi s c mt ca enzyme.
Sau enzyme c gi li trong gel v gel c enzyme c th lm nh bng
cch nghin trong my ng ho hoc p qua cc l ry ri em sy kh nhit
khng qu 400C.
C th gi enzyme bng cc phng php sau:
- Cc gel c hnh thnh trong s polymer ho tng hp
- Cc enzyme b nht trong cc l nh ca cc si tng hp
- Gi trong bao vi th
- Phng php tin polymer
5.5.2.3 Phng php hp ph vt l
Phng php hp ph vt l khng thng qua lin kt ho tr gn cc
enzyme trn cht mang. Qu trnh thc hin hp ph kh n gin: cht hp
ph v enzyme c trn ln vi nhau trong mt khong thi gian cho php s
hp ph xy ra nh tng tc ca cc lin kt ion, k nc, hydrogen v lc
Van der Wals.
Phng php ny c nhc im l qu trnh gii hp ph enzyme d
dng xy ra bi s thay i pH, nhit v thnh phn ion.
5.5.3 Mt s tnh cht ca enzyme khng tan:
- Enzyme khng tan c hot ring nh hn enzyme tan
- Enzyme khng tan cng tun theo nh lut Michaelis Menten (s nh
hng ca nng c cht n hot tnh enzyme). Tuy nhin do c hin tng
cnh tranh gia c cht v cht mang, do hin tng khuych tn nn cng c

nhng sai khc ng k v tc phn ng ca enzyme: khi cht mang v c

cht tch in cnh du th Khngm khng tan > Km tan, nu tch in tri du
th ngc li (Km - Hng s Michaielis)
- Enzyme khng tan c tnh bn nhit cao hn enzyme tan
- Enzyme khng tan c s dch chuyn pH ti u sang min kim hoc
axit hn so vi enzyme tan.
- Enzyme khng tan c thi gian bo qun lu hn v bn vi cc cht
km hm cng nh cc tc nhn gy bin tnh.

CHNG VI: SN XUT AXIT HU C

Axit hu c c s dng rng ri trong ch bin v bo qun thc phm
cng nh trong cng ngh vi sinh vt. Chnh v th m hin nay axit hu c
c nghin cu v s xut rng ri.
6.1 Cc phng php sn xut axt hu c:
sn xut axt hu c ngi ta c th dng nhiu phng php khc
nhau nh:
- Phng php tng hp ho hc
- Phng php chit tch t nguyn liu thc vt
- Phng php sinh tng hp nh vi sinh vt
Trong cc phng php ny th phng php sinh tng hp nh vi sinh
vt rt c ngha v qu trnh sn xut nhanh, s dng nguyn liu sn c r
tin v c th to c nhiu loi axt hu c khc nhau.
6.2 K thut sn xut mt s axt hu c nh vi sinh vt:
6.2.1 Sn xut axt axetic:
Axit axetic c th thu nhn bng cch ln men ng hoc oxy ho ru
etylic. Sau y xt qu trnh sn xut axit axetic t ru etylic.
1. Chng vi sinh vt:
oxy ho ru etylic thnh axt axetic ngi ta c th s dng nhiu
loi vi khun khc nhau. Nhng s dng nhiu hn c l cc chng vi khun:
Bacterium schuzenbachi, Bacterium curvum. Cc loi vi khun ny c kh nng
oxy ha rt ln.
2. Phng trnh ln men:
CH3CH2OH + O2
CH3COOH + H2O + Q
3. K thut ln men:
C th s dng phng php ln men lin tc hoc gin on. qu
trnh ln men tin trin c tt th cn ch cc im sau:
- Nguyn liu v hn hp dinh dng: c th s dng cn th hoc cn
tinh ch lm mi trng. Theo kinh nghim cho thy sn xut t cn th th

nhng sai khc ng k v tc phn ng ca enzyme: khi cht mang v c

cht tch in cnh du th Khngm khng tan > Km tan, nu tch in tri du
th ngc li (Km - Hng s Michaielis)
- Enzyme khng tan c tnh bn nhit cao hn enzyme tan
- Enzyme khng tan c s dch chuyn pH ti u sang min kim hoc
axit hn so vi enzyme tan.
- Enzyme khng tan c thi gian bo qun lu hn v bn vi cc cht
km hm cng nh cc tc nhn gy bin tnh.

CHNG VI: SN XUT AXIT HU C

Axit hu c c s dng rng ri trong ch bin v bo qun thc phm
cng nh trong cng ngh vi sinh vt. Chnh v th m hin nay axit hu c
c nghin cu v s xut rng ri.
6.1 Cc phng php sn xut axt hu c:
sn xut axt hu c ngi ta c th dng nhiu phng php khc
nhau nh:
- Phng php tng hp ho hc
- Phng php chit tch t nguyn liu thc vt
- Phng php sinh tng hp nh vi sinh vt
Trong cc phng php ny th phng php sinh tng hp nh vi sinh
vt rt c ngha v qu trnh sn xut nhanh, s dng nguyn liu sn c r
tin v c th to c nhiu loi axt hu c khc nhau.
6.2 K thut sn xut mt s axt hu c nh vi sinh vt:
6.2.1 Sn xut axt axetic:
Axit axetic c th thu nhn bng cch ln men ng hoc oxy ho ru
etylic. Sau y xt qu trnh sn xut axit axetic t ru etylic.
1. Chng vi sinh vt:
oxy ho ru etylic thnh axt axetic ngi ta c th s dng nhiu
loi vi khun khc nhau. Nhng s dng nhiu hn c l cc chng vi khun:
Bacterium schuzenbachi, Bacterium curvum. Cc loi vi khun ny c kh nng
oxy ha rt ln.
2. Phng trnh ln men:
CH3CH2OH + O2
CH3COOH + H2O + Q
3. K thut ln men:
C th s dng phng php ln men lin tc hoc gin on. qu
trnh ln men tin trin c tt th cn ch cc im sau:
- Nguyn liu v hn hp dinh dng: c th s dng cn th hoc cn
tinh ch lm mi trng. Theo kinh nghim cho thy sn xut t cn th th

axit c mi mnh hn, tuy nhin trong cn th c cha nhiu du fuzen nn nh

hng xu n qu trnh ln men.
cho vi khun hot ng tt th trong mi trng ngoi nc, ru, axit
axetic cn phi c mui khong, hydrat cacbon v cc hp cht cha nit d
hp th.
- Nng ru v axit axetic: nng ru thch hp ph thuc vo
dnh vi khun s dng. Ni chung gii hn cao nht ca nng ru l 15%.
Khi trong mi trng ln men ht ru th vi khun s s dng axit. Do ,
trnh hin tng ny trong thit b ln men lun li mt t ru (0,30,5%).
Mi trng c phn ng axit rt thch hp cho hot ng ca vi khun
axetic. Tuy nhin khi axit tich lu nhiu s nh hng n hot ng ca vi
khun. bo ton tnh thun khit ca vi khun v chng s pht trin ca vi
sinh vt l, ng thi bo m cho qu trnh ln men tin trin bnh thng th
nn tin hnh ln men nng axit axetic khong 10%.
- S thng kh: s oxy ho ru ch xy ra khi vi khun tip xc trc tip
vi oxy ca khng kh. Do , s c mt nhiu oxy s to iu kin thun li
cho qu trnh ln men. Theo l thuyt th ln men 1 kg ru khan th cn 2,3
m3 khng kh.
- Nhit : nhit c ngha ln i vi qu trnh ln men axetic. Nhit
6100C l mc ti thiu i vi vi khun axetic, nu nhit thp hn na
th cc hot ng sng ca vi khun s b ngng. nhit nh hn 12150C
cc vi khun sinh sn chm, cn trong khong 15340C chng s pht trin
bnh thng v sinh sn nhanh chng. Khi nhit thp th qu trnh ln men
s din ra chm chp, ngc li, khi nhit qu cao th ru v axit axetic s
bc hi nhiu v do s gy tn tht ln trong sn xut.
Nhit thch hp ca cc loi vi khun khc nhau th khng ging nhau.
i vi Bacterium schuzenbachi th thch hp nht nhit 280C. Cn
Bacterium curvum 35370C. nhit cao hn th cng sinh sn v s to
axit bt u gim.
4. Thu nhn sn phm:
Sau khi ln men xong dung dch axit phi c lm trong v lc. lm
trong thng s dng mt s cht nh gelatin, than hot tnh. Sau khi x l
bng cc cht trn th yn vi ba ngy cho lng cn v tip theo a hn hp
i lc trong.
Sau khi lc dung dch axit rt nng nn phi tang tr t nht 2 thng mi
em s dng. Mun lm dm th pha long n nng yu cu ri em lc
trong ln na. gi c cht lng ca dm th c th thanh trng hoc s
dng cht bo qun.
Ngoi ra, trong mt s trng hp nng cao nng ca axit ngi ta
c th tin hnh chng ct hn hp sau ln men thu hi axt axetic.

6.2.2 Sn xut axt lactic:

1. Chng vi sinh vt:
S dng vi khun lactic ln men in hnh v thch nghi nhit cao.
Thng hay s dng chng Lactobacillus Delbruckii.
2. Phng trnh ln men:
C6H12O6
2C3H6O3 + Q
E.

C6H12O6
2NAD+

2NADH2

2CH3COCOOH

2CH3CHOHCOOH
E.
S 6: C ch tng hp axit lactic t bo vi sinh vt
3. K thut ln men:
chun b mi trng ln men c th s dng r ng hoc cc loi
nguyn liu khc. vi khun pht trin c th cn b sung thm vo mi
trng mt s cc cht dinh dng khc.
C th tin hnh ln men bng phng php lin tc hoc gin on. Ln
men nhit 500C v phi n nh mt cch nghim ngt. Gim nhit ln
men s lm cho hot lc ln men yu v lm pht trin cc vi sinh vt l.
Ngc li, tng nhit s lm v hot enzyme v lm chm qu trnh ln men.
Qu trnh sinh tng hp axt lactic thch nghi pH axt yu (6,36,5).
Tuy nhin, khi ln men do tch lu axit nn lm cho nng axit ca mi
trng tng ln. Nu pH qu thp s km hm s to thnh axt lactic v qu
trnh ln men s ngng trc khi tt c ng chuyn thnh axit lactic. Do
trong qu trnh ln men phi lun lun gi pH mc thch hp. Ngi ta
thng dng cc mui canxi (CaCO3) trung ho v to ra canxilactat
(Ca(C3H5O3)2).
4. Thu nhn sn phm:
Sau khi ln men ngi ta tin hnh x l thu hi dung dch canxilactat
v em kt tinh. Tip theo lm nng chy cc tinh th canxilactat v dng
H2SO4 chuyn n thnh CaSO4 v C3H6O3 nhit 60700C:
Ca(C3H5O3)2 + H2SO4 = CaSO4 + 2C3H6O3
Tip theo tin hnh tch dung dch axit ra khi hn hp. Sau phi tch
st v lm trong dung dch bng than hot tnh ri li tm tch tp cht.

Mun nng cao hn nng axt th em dung dch lm sch sy chn

khng. Bng cch nh vy c th nng cao nng axit ln n 70%.
6.2.3 Sn xut axt citric:
Axit citric c nhiu trong mt s loi qu nh cam, chanh, du ty.v.v.
Ln u tin axit citric c sn xut bng cch t dch qu chanh. Hin nay
axit citric c sn xut bng phng php ln men nh tc dng ca nm
mc.
Axit citric c s dng rng ri trong cng ngh thc phm sn xut
nc gii kht, hp, hoc c s dng trong cng nghip dt, trong in
nh, trong y dc
1. Chng vi sinh vt:
Thng s dng Asperillus niger sinh tng hp axt citric.
2. C ch tng hp:
C6H12O6

CH3COCOOH
CH3CHO + CO2

-2H
HO
CH3COOH -2H CH2COOH -2H CHCOOH +
CH2COOH

CH2COOH
CHCOOH
CHOHCOOH
Axit xuxinic ax.fumaric
ax.malic
2

oxalacetat
CH2COOH

COCOOH

COCOOH
+
CH3COOH

COOH
HOOCCH2CCH2COOH
OH
Axit citric
S 7: C ch tng hp axit citric t bo vi sinh vt
Phng trnh ln men tng qut:
C6H12O6 + 3O2 = 2C6H8O7 + 4H2O
3. K thut ln men:
Nguyn liu dng chun b mi trng c th l r ng, dch thu
phn tinh bt hoc c th l cc loi nguyn liu khc. Trong mi trng ngoi
ng cn cn phi c cc cht cung cp nit, photpho, kali, km v mt s
nguyn t vi lng khc.

Qu trnh tch lu axit citric thch nghi mi trng c pH axit. Tuy

nhin khi tch lu nhiu axit th pH ca mi trng s gim nhiu. Do vy cn
c bin php gi pH mc thch hp.
S thng kh cho mi trng cng rt quan trng v tt c mixen ca nm
mc l loi hiu kh in hnh. Trong qu trnh pht trin ca mnh nm mc
i hi mt lng oxy rt ln.
Nhit thch hp cho nm mc Aspergillus niger 31370C. nhit
34370C th sinh khi nm mc pht trin mnh, cn 31320C th axit to
nhiu. Nu nhit thp hn th pht trin sinh khi b chm v s to thnh
axit citric cng b gim m s tch lu axit gluconic li tng. Cn nhit cao
hn th sinh khi pht trin nhanh nhng qu trnh to axit b km hm.
4. Thu nhn sn phm:
Dung dch sau ln men l mt hn hp gm nhiu axit, trong ch yu
l cc axit citric, gluconic v oxalic.
Qu trnh tch axit citric gm cc bc:
- Trung ho dung dch sau ln men:
C6H8O7 + 3Ca(OH)2 = Ca3(C6H5O7)2 + 6H2O
Axit citric
Canxi citrate
2C6H12O7 + Ca(OH)2 = Ca(C6H11O7)2 + 2H2O
Axit gluconic
Canxi gluconat
C2H2O4 + Ca(OH)2 = CaC2O4
+ 2H2O
Axit oxalic
Can xxi oxalate
- Tch v ra kt ta trn thit b lc chn khng
- Chuyn canxi citrate thnh axit citric:
Ca3(C6H5O7)2 + 3H2SO4 = 2C6H8O7 + 3CaSO4
Ch : phi s dng d axit sunfuric
- Tch dung dch axit trn thit b lc chn khng.
- Sy dung dch axit trn thit b sy chn khng.
- Kt tinh thu nhn cc tinh th axit
- Li tm tch tinh th. Sau khi li tm s thu nhn c cc tinh th axit
citric c m 23% s em i sy kh v nc ci trong c cha t axit th
c s dng vo mc ch khc.
- Sy tinh th: c th dng cc thit b sy thng quay hoc sy bng ti.
Sy nhit khng vt qu 350C. Nu sy nhit cao hn th cc tinh
th ca axit b ph hu. Trc khi a i ng gi cn phi cho axit qua sn
loi b nhng tp cht tnh c ri vo.