SINH TỔNG HỢP ENZYME AMYLASE TỪ NẤM Aspergillus niger VÀ Mucor

B GIO DC V O TO
TRNG I HC NNG LM TP. H CH MINH

KHOA CNG NGH SINH HC
******************
KHA LUN TT NGHIP
NGHIN CU CC YU T NH HNG N KH NNG

SINH TNG HP ENZYME AMYLASE T NM
Aspergillus niger V Mucor TRN MI
TRNG LN MEN BN RN
Ngnh hc: CNG NGH SINH HC

Sinh vin thc hin: NGUYN B PHNG THO
Nin kha: 2005 2009
Thng 08 nm 2009
B GIO DC V O TO
TRNG I HC NNG LM THNH PH H CH MINH
KHOA CNG NGH SINH HC
******************
KHA LUN TT NGHIP

NGHIN CU CC YU T NH HNG N KH NNG
SINH TNG HP ENZYME AMYLASE T NM
Aspergillus niger V Mucor TRN MI
TRNG LN MEN BN RN
Hng dn khoa hc
Sinh vin thc hin
TS. V VN
NGUYN B PHNG THO
CN. TH TUYN
Thng 08 nm 2009
LI CM N
Trc tin em xin by t lng bit n su sc n thy V Vn v c
Th Tuyn ht lng hng dn, gip v to mi iu kin thun li cho em trong
sut qu trnh thc hin ti.
Xin by t lng bit n chn thnh i vi qu thy c trong Khoa Cng Ngh
Sinh Hc trng i hc Nng Lm thnh ph H Ch Minh cng nh qu thy c l
gio vin thnh ging ti cc khoa, trng, vin nghin cu tn tnh ging dy em
trong thi gian hc tp ti trng.
Xin chn thnh cm n n ban lnh o Vin sinh hc Nhit i, qu c, ch,
anh, ch v cc bn sinh vin ang cng tc v lm vic ti vin cng ngh sinh hc
nhit i, v i bo v gip em trong qu trnh thc hin ti.
Xin gi li cm n n cc bn lp DH05SH, i hc chnh quy kha 2005
-2009 quan tm v ng vin ti trong thi gian hc tp v thc hin ti.
Cui cng, con xin by t lng bit n su sc n ba, m v nhng ngi thn
trong gia nh ng vin, gip v chia s cng vi con trong thi gian qua.
Nguyn B Phng Tho
iii
TM TT
Enzyme amylase l mt enzyme c th tm thy rt nhiu sinh vt. Hin nay,
ezyme ny c ng dng rng ri trong nhiu lnh vc nh nng nghip, cng nghip
v y hc. Mc tiu ti l nghin cu kh nng to enzyme amylase ca nm
Aspergillus niger v Mucor v nhng yu t tc ng n kh nng to amylase ca
chng. ti thu c mt s kt qu v s tc ng ca cc yu t mi trng n
kh nng sinh tng hp enzyme amylase t nm Aspergillus niger v Mucor.
Aspergillus niger khi sinh trng v tng hp enzyme amylase trn mi trng
bn rn vi c cht l cm go b sung thm tru v b u nnh chu tc ng ca rt
nhiu yu t mi trng nh t l c cht, pH mi trng, thi gian nui cy, nhit ,
m, nng dinh dng v t l ging. ti nghin cu s tc ng ca cc
yu t mi trng nu trn u tin l mc ring l tng yu t, sau l s
tng tc qua li ca nhng yu t c nh hng mnh n s tng hp enzyme. Kt
qu thu c cho thy vi nhng mi trng c t l Cm go: tru: b u nnh
(2:1:1), pH 5, thi gian nui cy l 28 gi, nhit 350C, m 55%, 2 ln nng
dinh dng, t l ging 0,8*108 bt/g l ti u cho Aspergillus niger sinh tng hp
amylase.
Kh nng tng hp enzyme amylase t Mucor khi sinh trng trn mi trng
bn rn vi c cht l cm go b sung thm tru v b u nnh chu tc ng ca rt
nhiu yu t mi trng nh t l c cht, pH mi trng, thi gian nui cy, nhit ,
m, nng dinh dng v t l ging. ti nghin cu s tc ng ca cc
yu t mi trng nu trn u tin l mc ring l tng yu t, sau l s
tng tc qua li ca nhng yu t c nh hng mnh n s tng hp enzyme. Kt
qu thu c cho thy vi nhng mi trng c t l Cm go: tru: b u nnh
(2:1:1), pH 5, thi gian nui cy l 32 gi, nhit 300C, m 55%, 3 ln nng
dinh dng, t l ging 1,6*108 bt/g l ti u cho Mucor sinh tng hp amylase.
iv
SUMMARY
Enzyme amylase can be found in many living organisms. Nowadays, this is
applied in many domains, such as agriculture, industry and medicine. The purpose of
the survey is to find out Aspergillus niger and Mucors ability to produce enzyme
amylase, and some factors can affect this ability. Finally, the survey has some
following results
The first, for Aspergillus niger, they are born and grow up on semi-solid
environments including rice bran, rice rind, and soya residuum. Growing up and
biosynthesized enzyme ability of A. niger depend on environment factors, such as ratio
of rice bran to rice rind and to soya residuum, time, humidity, nutritious concentration,
and ratio of spore. The survey finds out affect of environment factors in individual of
each and compose of some factors, which have stronger affect. Results showed with
environments have ratio of rice bran to rice rind and to soya residuum is 2:1:1; after 28
hours at 350C with 55% humidity, double nutritious concentration and ratio of 0,8*108
spore to one g environment is the most suitable for Aspergillus niger to biosynthesize
enzyme amylase on semi-solid fermatable environment.
The second, for Mucor, they are born and grow up on semi-solid environments
including rice bran, rice rind, and soya residuum. Growing up and biosynthesized
enzyme ability of A. niger depend on environment factors, such as ratio of rice bran to
rice rind and to soya residuum, time, humidity, nutritious concentration, and ratio of
spore. The survey finds out affect of environment factors in individual of each and
compose of some factors, which have stronger affect. Results showed with
environments have ratio of rice bran to rice rind and to soya residuum is 2:1:1, after 32
hours at 300C with 55% humidity, treble nutritious concentration and ratio of 1,6*108
spore to one g environment is the most suitable for Mucor to biosynthesize enzyme
amylase on semi-solid fermatable environments.
MC LC
Trang
LI CM N..............................................................................................................iii
TM TT....................................................................................................................iv
MC LC....................................................................................................................vi
DANH SCH CC CH VIT TT.........................................................................xii
Hnh 2.1 Cu trc phn t ca amylose Error: Reference source not found...............xiii
th 4.14 Hot tnh v hm lng theo cc nng ging ca Mucor Error:
Reference source not found........................................................................................xiv
DANH SCH CC BNG.........................................................................................xv
Chng 1.......................................................................................................................1
M U.......................................................................................................................1
1.1. t vn ...............................................................................................................1
Chng 2.......................................................................................................................3
TNG QUAN TI LIU..............................................................................................3
2.1. Gii thiu s lc v enzyme.................................................................................3
2.1.1. Khi nim chung v enzyme................................................................................3
2.1.2. Tnh cht u vit ca enzyme..............................................................................3
2.2. Tng quan v enzyme amylase...............................................................................4
2.2.1. Amylase...............................................................................................................4
2.2.2. Phn loi..............................................................................................................4
2.2.3. c tnh v c ch tc dng ca 3 loi amylase thng dng................................4
2.2.3.1. -amylase..........................................................................................................4
2.2.3.2. -amylase..........................................................................................................5
2.2.3.3. -amylase (hay gluoamylase hoc -1,4 glucan-glucanhydrolase)....................6
2.2.4.1. Thc vt............................................................................................................6
2.2.4.2. Vi sinh vt.........................................................................................................7
2.2.5. ng dng.............................................................................................................7
2.2.5.1. ng dng trong cng ngh thc phm..............................................................7
2.2.5.2. Trong cng nghip dt, giy..............................................................................8
vi
2.2.5.3. Trong y hc.......................................................................................................8

2.2.5.4. Trong nng nghip............................................................................................9
2.2.5.5. Trong cng ngh ty ra...................................................................................9
2.2.6. C cht ca enzyme amylase...............................................................................9
2.2.6.1. Tinh bt.............................................................................................................9
Tinh bt l mt cht bt v nh hnh, mu trng, khng tan trong nc lnh nhng khi
un nng s c kh nng tan trong nc. Tinh bt l carbonhydrate thc vt ch yu
trong cc loi c nh khoai m, khoai ty, khoai lang.., v l cht d tr nng lng
quan trng......................................................................................................................9
Hnh 2.1 Cu trc phn t ca amylose. (Wikipedia.com)...........................................10
2.2.6.2. Glycogen.........................................................................................................11
Hnh 2.3 Cu trc phn t glycogen. (Wikipedia.com)........................11
2.3. Gii thiu v nm mc Aspergillus niger v Mucor.............................................12
2.3.1. Nm mc Aspergillus niger...............................................................................12
2.3.2. Nm mc Mucor................................................................................................13
2.4. Nui cy vi sinh vt tng hp amylase.................................................................14
2.4.1. Sinh tng hp enzyme cm ng.........................................................................14
2.4.2. nh hng ca cc yu t dinh dng i vi qu trnh sinh tng hp enzyme
amylase........................................................................................................................14
2.4.2.1. Ngun carbon..................................................................................................14
2.4.2.2. Ngun Nit.....................................................................................................14
2.4.2.3. Cc nguyn t khong.....................................................................................15
2.4.2.4. Nhit nui cy............................................................................................16
2.4.2.5. pH ban u......................................................................................................16
2.4.2.6. m..............................................................................................................16
2.4.2.7. Mi trng khng kh.....................................................................................16
2.5. Mi trng ln men bn rn..................................................................................17
2.5.1. Cm go.............................................................................................................17
2.6. Phng php nui cy vi sinh vt sn xut enzyme..............................................18
2.6.1. Phng php nui cy b mt............................................................................18
2.6.2. Phng php nui cy chm...............................................................................19
2.7.2. Cc yu t nh hng n qu trnh li trch.......................................................20
vii
Chng 3.....................................................................................................................21
VT LIU V PHNG PHP NGHIN CU......................................................21
3.1. Thi gian v a im th nghim.........................................................................21
3.2. Vt liu th nghim................................................................................................21
3.2.1. i tng th nghim.........................................................................................21
3.2.2. C cht...............................................................................................................21
3.3. Phng php nghin cu.......................................................................................21
3.3.1. Phng php cy truyn.....................................................................................21
3.3.2. Phng php nhn ging cp 2 (gi ging trn mi trng la)........................22
3.3.3. Phng php m t hnh thi A. niger v Mucor...............................................23
3.3.3.1. Quan st hnh thi i th ...............................................................................23
3.3.4. Xc nh trc tip s lng bo t nm si bng bung m hng cu.............23
3.3.6. Phng php ln men bn rn thu nhn enzyme amylase.............................25
3.3.7. Phng php xc nh hm lng amylase........................................................26
Phng php ny da trn s thay i bc sng hp th cc i ca thuc th
Coomassie Brilliant Blue khi to phc vi protein. Trong dung dch mang tnh acid,
khi cha kt hp vi protein th thuc nhum c bc sng hp th cc i 465 nm,
khi kt hp vi protein th thuc nhum hp th cc i bc sng 595 nm. hp
th bc sng 595 nm c lin h mt cch trc tip vi nng protein. c bit
nu c mt cc acid amin nhn thm s bt mu mnh hn. Phng php ny c
nhy cao cho php pht hin ti vi g protein/ml, d thc hin v tit kim thi gian.
.....................................................................................................................................26
Bng 3.1 Cc bc tin hnh to dung dch albumin chun........................................27
3.3.8. Phng php xc nh hot tnh amylase...........................................................28
Enzyme amylase l enzyme thy phn tinh bt. N phn ct amylose v amylopectin
ca tinh bt thnh cc loi ng maltose, glucose,Hot tnh ca enzyme amylase
c xc nh da trn s thay i mu sc ca phc hp tinh bt iod trc v sau
khi thy phn. Mt quang ca phc hp tinh bt iod s c o bc sng 595
nm trn my o quang ph...........................................................................................28
Ha cht cn thit cho th nghim ny bao gm dung dch Lugol, dung dch m Naacetate 50 mM pH 5, dung dch h tinh bt 1%. Dung dch ny c pha bng cch
viii
cn 1 g tinh bt tan cho vo berche ng 100 ml dung dch m, un cch thy cho si
trong 5 pht, khuy u cho tan...................................................................................28
3.3.9. Phng php quy hoch thc nghim...............................................................29
3.3.10. Phng php b tr v x l s liu th nghim................................................29
Chng 4.....................................................................................................................30
KT QU V THO LUN.....................................................................................30
4.1. Kh nng sinh tng hp amylase ca nm mc Aspergillus niger trn mi trng
ln men bn rn
.....................................................................................30
4.1.1. Nm mc A .niger ............................................................................................30

Hnh 4.1 Hnh nh i th v vi th ca A. niger. (a)hnh nh i th, (b) hnh nh vi
th................................................................................................................................30
4.1.2. nh hng ca t l cm go: b u nnh: tru ..............................................30
th 4.1 Biu din hot tnh amylase v hm lng protein t canh trng nui cy
A. niger vi cc t l c cht khc nhau......................................................................31
4.1.3. nh hng ca pH ............................................................................................32
th 4.2 Biu din hot tnh amylase v hm lng protein t mi trng nui cy
A. niger vi cc pH mi trng ban u khc nhau. ................................................33
4.1.4. nh hng ca thi gian nui cy ....................................................................34
th 4.3 Biu din hot tnh enzyme v hm lng protein t canh trng nui cy
A. niger vi cc mc thi gian thu nhn khc nhau. ...................................................34
4.1.5. nh hng ca nhit ....................................................................................35
th 4.4 Biu din hot tnh amylase v hm lng protein t canh trng nui
cy A. niger vi cc nhit nui cy khc nhau.................................................36
4.1.6. nh hng ca m ban u ..........................................................................37

A. niger vi cc m mi trng ban u khc nhau. .............................................37
4.1.7. nh hng ca nng dinh dng .................................................................38
4.1.8. nh hng ca t l ging ................................................................................39
4.2. Kh nng sinh tng hp amylase ca nm mc Mucor trn mi trng ln men
bn rn.........................................................................................................................41
4.2.1. Nm mc Mucor ...............................................................................................41
Hnh 4.2 Hnh nh i th ca Mucor. (a)hnh nh i th, (b) hnh nh vi th. .........42
ix
Hnh nh vi th quan st c cho thy Mucor c cung bo t phn nhnh v mc ln

bt k ch no ca si nm, bo t nh nm trong bc bo t. Bc bo t dng trn
hoc hnh qu l, cha nhiu bo t pha trong.
..................................................42
4.2.2. nh hng ca t l cm go: b u nnh: tru ..............................................42

4.2.3. nh hng ca pH ............................................................................................43
Mucor vi cc pH mi trng khc nhau. ..................................................................44
4.2.5. nh hng ca nhit ....................................................................................47
Mucor vi cc nhit nui cy khc nhau.................................................................47
4.2.6. nh hng ca m ban u ..........................................................................48
4.2.7. nh hng ca nng dinh dng .................................................................49
th 4.13 Biu din hot tnh amylse v hm lng protein t canh trng nui cy
Mucor vi cc nng dinh dng trong mi trng khc nhau................................50
Nng dinh dng X3 l thch hp nht cho s tng hp enzyme amylase. nng
dinh dng ny, hm lng protein v hot tnh amylase thu c u cao nht
(43,391 mg v 488,279 UI/g CT). Sau th hm lng v hot tnh u gim dn, c
th l do nng thm thu ca mi trng nh hng ln Mucor.............................50
4.2.8. nh hng ca t l ging ................................................................................51
4.3. Qui hoch thc nghim.........................................................................................53
4.3.1. Xc nh gi tr ti u ca mt s yu t nh hng n kh nng sinh tng hp
amylase ca A. niger v Mucor....................................................................................53
4.3.1.2. Xc nh gi tr ti u ca mt s yu t nh hng n kh nng sinh tng
hp amylase ca Mucor...............................................................................................58
Bng 4.4 M ha cc bin s cc yu t nh hng n kh nng sinh tng hp
amylase ca Mucor......................................................................................................58
Bng 4.5 Ma trn k hoch ha i vi TY-24.........................................................58
Bng 4.6 Hot lc amylase ca Mucor theo thc nghim v theo phng trnh hi qui
.....................................................................................................................................59
V t13, t124 v t1234 u nh hn 4,3 (t(0,05;2)) nn b13, b124 v b1234 b loi ra
khi phng trnh hi qui do khng c ngha...........................................................61
Chng 5.....................................................................................................................63
x
KT LUN V NGH..........................................................................................63
5.1. Kt lun.................................................................................................................63
5.2. ngh..................................................................................................................63
TI LIU THAM KHO............................................................................................64
xi
DANH SCH CC CH VIT TT

A. niger
Aspergillus niger
B u nnh
bt
bo t
Cm go
CT
Canh trng
UI
Unit International, n v quc t
One-Way ANOVA
Phn tch phng sai mt yu t
PGA
Potato glucose agar
Pt
phng trnh
Tru
TY
K hoch thc nghim y cc yu t
UV
Ultraviolet
xii
DANH SCH CC HNH

Trang
Hnh 2.1 Cu trc phn t ca amylose...........Error: Reference source not found

Hnh 2.2 Cu trc phn t ca amylopectin
Hnh 2.3 Cu trc phn t glycogen
Error: Reference source not found
Hnh 4.1 Hnh nh i th v vi th ca A. niger.
Error: Reference source not
found
Hnh 4.2 Hnh nh i th v vi th ca Mucor. Error: Reference source not found
th 4.1 Hot tnh v hm lng theo t l c cht ca A. niger.
Error: Reference
source not found

th 4.2 Hot tnh v hm lng theo pH ca A. niger.
Error: Reference source
not found
th 4.3 Hot tnh v hm lng theo thi gian ca A. niger Error: Reference source
not found
th 4.4 Hot tnh v hm lng theo nhit ca A. nigerError: Reference source
not found
th 4.5 Hot tnh v hm lng theo m ca A. niger.
not found
th 4.6 Hot tnh v hm lng theo nng dinh dng ca A. niger. Error:
Reference source not found
th 4.7 Hot tnh v hm lng theo t l ging ca A. niger.
Error: Reference
source not found

th 4.8 Hot tnh v hm lng thteo t l c cht ca Mucor.
Error: Reference
source not found

th 4.9 Hot tnh v hm lng theo pH ca Mucor. Error: Reference source not
found
th 4.10 Hot tnh v hm lng theo thi gian ca Mucor Error: Reference source
not found
th 4.11 Hot tnh v hm lng thteo nhit ca Mucor.Error: Reference source
not found
th 4.12 Hot tnh v hm lng theo m cu Mucor.
not found
xiii
th 4.13 Hot tnh v hm lng theo cc nng dinh dng ca Mucor.

th 4.14 Hot tnh v hm lng theo cc nng ging ca Mucor. . .Error:
xiv
DANH SCH CC BNG

Trang
Bng 2.1 Thnh phn dinh dng ca cm go Error: Reference source not found
Bng 3.1 Cc bc tin hnh to dung dch albumin chun
not found
Bng 4.1 M ha cc bin s cc yu t nh hng n A. niger
Error: Reference
source not found

Bng 4.2 Ma trn k hoch ha i vi TY-24 ca A. niger Error: Reference source
not found
Bng 4.3 Hot lc amylase ca A. niger theo thc nghim v pt hi qui
Error:

Bng 4.4 M ha cc bin s cc yu t nh hng n Mucor
Error: Reference
source not found

Bng 4.5 Ma trn k hoch ha i vi TY-24 ca Mucor
not found
Bng 4.6 Hot lc amylase ca Mucor theo thc nghim v pt hi qui
xv
Error:
Chng 1
M U
1.1. t vn
Enzyme l mt loi protein c bit c sinh vt tng hp nn v tham gia cc
phn ng sinh hc, l thnh phn khng th thiu trong mi t bo sinh vt. Chng
ng vai tr quyt nh mi quan h gia c th sng v mi trng sng.
Enzyme amylase c vai tr rt quan trng trong nhiu ngnh cng nghip v
cha tr bnh, nh cng nghip thc phm, dt, ty ra, cha bnh km tiu ha
ngi v trong ngnh ch bin thc n gia sc
Cui th k 19 v sut th k 20, ngnh cng nghip sn xut enzyme rt pht
trin, ngy cng thin v xu hng sn xut enzyme da vo hot ng sng ca vi
sinh vt v nhiu li ch thit thc. Cng ngh sn xut enzyme v ang mang li
nhng ngun li ln cho nhiu quc gia trn th gii.
Hin nay, doanh thu t vic sn xut enzyme ln n hn 1,7 t USD, trong
gp phn khng nh l li nhun thu c t enzyme amylase. Th trng enzyme
cng ngh s cn tip tc gia tng trong tng lai n. nc ta, nhu cu s dng cc
ch phm enzyme trong c amylase trong ch bin thc phm, thc n gia sc v
trong cc ngnh cng nghip khc ngy cng nhiu. V vy, hng nm, nc ta phi
nhp khu lng ln nhng ngun enzyme ny.
Ngy nay, vic tn dng ph liu trong cng, nng nghip lm ngun carbon
sn xut amylase bi vi sinh vt bng phng php ln men chm v ln men bn rn
rt c quan tm nc ta v nhiu nc trn th gii. Vit Nam l mt nc nhit
i c nn nng nghip lu i v a dng, ang trn pht trin. V vy lng ph
phm nng nghip thi ra ngy cng gia tng, nh cm go, b u nnh l ngun
cung cp carbon thch hp cm ng nm mc sinh tng hp amylase bng phng
php ln men bm rn. Mc khc, y l c cht kh r tin v n nh c tim nng
s dng sn xut amylase qui m ln.
Hin nay, vin cng ngh sinh hc nhit i c mt s chng nm mc c kh
nng sinh tng hp enzyme amylase kh cao, trong c chng Aspergillus niger v
Mucor. Tuy nhin, thu c enzyme amylase c hiu sut cao ch cn chng vi sinh
vt c kh nng to ezyme amylase cao l cha . Cc vi sinh vt cn phi c
nui trn mi trng c cht thch hp c kh nng cm ng kch thch chng sinh
amylase. Mi trng nui cy cn c b sung thm nhng nng dinh dng v
cc vi sinh vt phi c nui nhit thch hp. Cui cng, thi gian thu nhn dch
chit enzyme t canh trng nui cy cng s nh hng rt ln n hm lng v
hot tnh amylase thu c.
Trn c s , chng ti chn ti Kho st mt s yu t nh hng n
kh nng sinh tng hp enzyme amylase t nm mc Aspergillus niger v Mucor trn
mi trng ln men bn rn nhm xy dng iu kin mi trng ti u cho hai
chng nghin cu to enzyme nhiu v c hot tnh cao cng nh iu kin thu
nhn enzyme tt nht. ti c thc hin ti phng cc cht c hot tnh sinh
hc - vin Sinh Hc Nhit i, thnh ph H Ch Minh.
1.2. Yu cu
- Xc nh cc gi tr ti u ca cc yu t mi trng nh hng n
kh nng sinh tng hp enzyme amylase ca hai chng th nghim.
- Xc nh mi tng quan gia cc yu t mi trng c tc ng mnh
n kh nng tng hp amylase ca hai chng th nghim.
1.3. Ni dung
- Kho st kh nng tng hp amylase ca hai chng th nghim.
- Kho st s nh hng ca cc yu t nh t l c cht, m, nhit
nui cy, thi gian nui cy, pH mi trng ban u, nng dinh dng,
v t l ging n kh nng sinh tng hp amylase ca hai chng th nghim.
- Ti u ha cc yu t trn bng phng php quy hoch thc nghim
nhm tm ra iu kin nui cy v thnh phn mi trng ti u cho hai chng
trn sinh ra lng amylase cao.
Chng 2
TNG QUAN TI LIU
2.1. Gii thiu s lc v enzyme
2.1.1. Khi nim chung v enzyme
Enzyme l mt cht xc tc sinh hc ch c to ra trong t bo sinh vt, v
ng vai tr quan trng khng th thiu c trong qu trnh trao i cht ca c th
sinh vt. Tt c cc phn ng chuyn ha cc cht trong c th sinh vt u c s tham
gia xc tc ca enzyme. Enzyme khng ch tham gia xc tc cc phn ng trong t bo
m ngay c ngoi t bo ca vi sinh vt. iu ny c ngha quan trng trong vic
ng dng enzyme vo nhiu lnh vc khc nhau ca cng nghip v i sng.
Enzyme c bn cht l protein c kh nng xc tc c hiu cho cc phn ng
ha hc v lm cht xc tc sinh hc. Di tc dng ca enzyme cc phn ng ha
sinh trong c th sinh vt xy ra rt nhanh, khng cn nhng iu kin v nhit , p
sut, nng cc cht mt cch qu khc nghit. Tn enzyme c bt ngun t ch
Hy Lp c ngha l cht trong con men. Tt c qu trnh bin i ha sinh u xy ra
di tc dng ca h men.
Nu c th thiu enzyme th cc qu trnh chuyn ha ha sinh s b nh ch,
sinh vt khng th sng, sinh trng, pht trin v sinh sn bnh thng. S sng ca
sinh vt s khng tn ti.
Ngy nay, enzyme cn ng vai tr quan trng trong cc hot ng sn xut
cng nghip v ngy cng c nhiu nhng nghin cu v ng dng enzyme vo trong
cc lnh vc khoa hc, cng nghip, kinh t quc dn, vn vn.
2.1.2. Tnh cht u vit ca enzyme
Th nht, enzyme c th thu nhn c t nhiu ngun khc nhau nh ng
vt, thc vt, v vi sinh vt, v hu nh cc enzyme khng c tnh cht c hi.
Th hai, chng c th hot ng xc tc phn ng trong iu kin nhit bnh
thng, p sut thng, pH acid yu, kim yu hay trung tnh.
Th ba, tnh c hiu ca enzyme cao hn hn cc xc tc khc, v ch c tc
dng ln nhng c cht nht nh v theo mt kiu phn ng nht nh.
Th t, vn tc phn ng do enzyme xc tc c th d dng iu chnh thm ch

ngng phn ng bng cch tc ng vo cc yu t nh: nhit , pH, cc cht kim
hm, cht hot ha, nng c cht, nng enzyme, vn vn (L Ngc T, 2002).
Cui cng, cng lc xc tc ca enzyme cao hn hn cc cht xc tc thng
thng, nn ch cn mt lng nh c th chuyn ha mt lng ln c cht trong thi
gian ngn. y l iu cc cht xc tc ha hc kh c th lm c.
2.2. Tng quan v enzyme amylase
2.2.1. Amylase
Amylase l enzyme c dng rt thng dng trong cc ngnh sn xut, ch
bin, c bit l trong ngnh cng nghip thc phm. Enzyme ny cng rt ph bin
trong th gii sinh vt. Cc enzyme amylase u thuc nhm enzyme thy phn, xc
tc phn gii cc lin kt ni phn t trong polysaccharide vi s tham gia ca nc.
R.R + H-OH RH + ROH
Amylase thy phn tinh bt, glycogen, v dextrin thnh glucose, maltose v
dextrin gii hn. H enzyme ny c trong nc bt, dch tiu ha ca ngi, ng vt,
trong ht ny mm, nm mc, nm men v vi khun. Amylase bao gm nhiu enzyme
khc nhau v tnh c hiu tc dng ln tinh bt (ct tinh bt ti nhng v tr khc nhau
trn mch tinh bt).
2.2.2. Phn loi
Hin nay, amylase c phn thnh 6 loi, gm c -amylase, -amylase, amylase hay glucoamylase (thy phn cc lin kt -1,4-glucoside ca tinh bt v cc
polysaccharide ng loi), dextrin-6-glucanhydrolase, amylopectin-6-glucanhydrolase
v oligodextrin-6-glucanhydrolase (hay dextrinnase thy phn cc lin kt oligodextrin
-1,6-glucoside trong polysaccharide v cc dextrin cui).
2.2.3. c tnh v c ch tc dng ca 3 loi amylase thng dng
2.2.3.1. -amylase
-amylase d tan trong nc, trong cc dung dch mui v ru long. Protein ca amylase c tnh cht acid yu v tnh cht ca globuline. im ng in nm trong
vng pH 4,2 5,7. -amylase l mt metaloenzym, cha t 1 n 30 nguyn t gam
Ca/mol. Khi tch hon ton Ca ra khi enzyme th -amylase mt ht kh nng thy
phn c cht. -amylase bn vi nhit hn so vi cc amylase khc. bn vi tc
dng acid th -amylase ca nm mc bn vng hn so vi -amylase ca malt v vi

khun. Tt c -amylase u b km hm bi kim loi nng (Cu2+, Ag+, Hg2+, vn vn).
C ch tc dng ca -amylase l phn ct cc lin kt -1,4 glucoside bt k
v tr no trong phn t tinh bt, glycogen v cc polysaccharide. Di tc dng ca
enzyme -amylase tinh bt c th chuyn ha thnh maltose, glucose v cc dextrin c
phn t lng thp, nhng thng thng th -amylase ch thy phn tinh bt ch yu
thnh dextrin c phn t lng thp, maltose v mt t glucose (Nguyn c Lng,
2004). Do vy -amylase c tc dng lm gim nht ca dung dch h tinh bt rt
mnh (dch ha).
S thy phn tinh bt bng -amylase:
-amylase
Tinh bt (hoc glycogen) --------------> -dextrin + maltose + glucose + H2O
2.2.3.2. -amylase
-amylase khc vi -amylase, n hu nh khng thy phn ht tinh bt
nguyn vn m thy phn mnh m h tinh bt. -amylase khng bn khi c Ca2+, amylase b kim hm bi Cu2+, Hg+, urea, iodoacetamide, iodine, ozon chu nhit
km hn - amylase nhng bn vi acid hn. -amylase b v hot nhit 700C,
hot ng ti u 550C, pH t 5,1 n 5,5.
Enzyme ny xc tc s thy phn cc lin kt -1,4 glucan trong tinh bt,
glycogen v polysaccharide ng loi, phn ct tun t tng gc maltose mt t u
khng kh ca mch. -amylase phn gii 100% amylose thnh maltose v phn gii
54 - 58% amylopectin thnh maltose. Qu trnh thy phn c tin hnh t u
khng kh ca cc nhnh ngoi cng. Mi nhnh ngoi cng c t 20 26 gc
glucose nn to c t 10 12 phn t maltose. Khi gp lin kt -1,4 glucoside
ng k cn lin kt -1,6 glucoside th -amylase ngng tc dng. Phn
polysaccharide cn li l dextrin phn t ln c cha rt nhiu lin kt -1,6 glucoside
cho mu tm vi iodine. nht ca dung dch gim chm.
Tc dng ca -amylase ln tinh bt c th c din t theo s sau:
-amylase
Tinh bt (glycogen) ------------> maltose (54 58%) + -dextrin (42- 46%)
Nu cho ng thi c -amylase v -amylase cng tc dng ln tinh bt th
tinh bt b thy phn ti 95%. (Nguyn c Lng, 2004).
5
2.2.3.3. -amylase (hay gluoamylase hoc -1,4 glucan-glucanhydrolase)

-amylase c kh nng thy phn hon ton tinh bt, glycogen, amylopectin,
dextrin, panose, isomaltose v maltose thnh glucose m khng cn c s tham gia ca
cc enzyme amylase khc. -amylase l enzyme ngoi bo, c nhiu tn gi khc
nhau: glucoamylase, amyloglucozidase, taka-amylase B, maltulasea s -amylase
u thuc loi enzyme acid. -amylase c hot lc cao nht vng pH t 3,5 5,5
v nhit 500C. -amylase bn vi acid hn -amylase, nhng li km bn hn di
tc dng ca ru etylic, aceton khng c bo v bng Ca 2+. Glucoamylase xc tc
phn ng thy phn lin kt -1,4 ln -1,6 glucoside, glucoamylase cn c kh nng
thy phn cc lin kt kt -1,2 v lin kt -1,3 glucoside. im ng in ca amylase t cc ngun khc nhau cng khng ng nht. a s glucoamylase bit
u thuc loi enzyme acid, th hin hot lc ti a vng pH t 3,5 n 5,5. So vi
-amylase, glucoamylase bn i vi acid hn, nhng li km bn di tc dng ca
ru v aceton do khng c bo v bi Ca2+.
Dy l enzyme thy phn lin kt -1,4 ln -1,6 glucoside. Khi thy phn lin
kt -1,4 glucan trong chui polysaccharide, -amylase tch ln lt tng phn t
glucose ra khi u khng kh ca mch to ra glucose. S thy phn tinh bt c
thc hin theo c ch a mch m khng phi theo c ch n mch.
Fukumoto ngh chia -amylase ca vi sinh vt lm hai loi theo kh nng
thy phn tinh bt ca chng. l enzyme kiu Rhizopus delemar v enzyme kiu
Aspergillus niger (Nguyn c Lng, 2004).
S thy phn glucid bi glucoamylase:
-amylase kiu
Rh.delemar
Tinh bt hay oligosaccharide ------------->
(c cc lin kt -1,4 v -1,6)
100% glucose.
-amylase kiu
Asp.niger
Tinh bt hay oligosaccharide -------------> 80 85% glucose + oligosaccharide.
2.2.4. Ngun thu nhn
2.2.4.1. Thc vt
T lu con ngi bit cch s dng amylase t ht ny mm s dng
trong ngnh ch bin thc phm (mch nha), nc gii kht (bia), hoc sn sut ch
phm hn hp bt enzyme b sung vo ch phm bt dinh dng dnh cho tr em,

ngi gi v nhng ngi b suy tiu ha. thc vt, ngun amylase c thu nhn t
nhng ht ny mm, c bit l ht ng cc ny mm.
2.2.4.2. Vi sinh vt
Ngy nay, do u th v nhiu mt, vi sinh vt tr thnh ngun thu enzyme
ch o. Hin nay, c rt nhiu loi vi sinh vt c kh nng tng hp c enzyme
amylase c nghin cu v a vo sn xut. Nhng chng vi sinh vt ny thng
c phn lp t t nhin. Nhng chng to amylase nhiu hn c l nm si, nm
men, vi khun v x khun.
thu nhn enzyme amylase ngi ta thng dng cc ging nm si
Aspergillus, Rhizopus. Nhiu loi thuc ging ny (A. orizae, A. niger, A. usamii) v
mt s loi ca Neurospora v Mucor sinh tng hp rt mnh m loi enzyme ny.
Nm men v gi nm men thuc cc ging Candida, Saccharomysec.
Endomycopis, Endomyces cng to nn nhiu enzyme amylase.
Nhiu vi khun cng c kh nng to lng ln amylase nh Bac. Polymyxa,
Phytomonas dptructans, Bact. casavanum, Cc vi khun a nhit c kh nng sinh
trng nhanh gp 4 - 6 ln vi khun a m v sinh trng tt nht t 65 0 - 700C, v to
amylase mnh nht 500C. V vy m loi vi khun ny thng c nui cy 700C
lm ging v nui thu enzyme 500C.
Trong x khun him gp loi no c kh nng sinh tng hp amylase mnh
m, tuy nhin cng c mt s t x khun c kh nng sinh tng hp enzyme ny nh
Micromonospora vulgaris.
2.2.5. ng dng
Ch phm amylase dng th hay tinh khit c ng dng kh rng ri trong
cng nghip, c bit l trong cng nghip thc phm (sn xut ru bia, bnh m,
bnh ngt, ng mt tinh bt, vn vn).
2.2.5.1. ng dng trong cng ngh thc phm
Trong cng ngh ru bia vic s dng amylase t vi sinh vt trong cng
nghip sn xut ru bia thay cho malt tit kim c hng vn tn nguyn liu
(i mch) loi tt, gip gim gi thnh v rt ngn quy trnh sn xut, tit kim cng
sc, tin bc. Bin php thay enzyme ch phm cho malt c ngha c bit quan
trng. Vic ny gip gim gi thnh m cht lng sn phm vn khng b thay i.
7
c bit l i vi ngnh cng nghip bia ca nc ta do hin nay vic gieo trng i
mch trong nc cha mang li nhiu kt qu, nn nc ta vn phi nhp malt t nc
ngoi vi gi thnh cao. V vy nu s dng enzyme amylase t vi sinh vt s gp
phn lm gim gi nguyn liu v gim s ph thuc vo vic nhp khu malt.
Trong sn xut bnh m s dng enzyme amylase c tc dng rt ngn qu
trnh ln men, tng nhanh th tch bnh m, lm tng mi thm ca bnh, mu sc ca
bnh p hn v tng xp ca bnh. Trong sn xut bnh ngt, nu thm ch phm
amylase vo bt s xy ra qu trnh thy phn bt thnh ng, do gim lng
nguyn liu tiu tn cho sn xut.
Trong sn xut mt, tinh bt, maltose, glucose nhu cu s dng enzyme
amylase cng ngy cng cao. Ban u cc sn phm mt, tinh bt thng c sn
xut theo phng php thy phn tinh bt bng acid hoc malt. Ngy ny, qui trnh
ny c thc hin nh ch phm amylase. Ch phm enzyme amylase ng dng rt
hiu qu trong sn xut tinh bt, glucose, v cc loi mt tinh bt. Khi thy phn tinh
bt bng acid khng cho php sn xut cc loi ng mt theo mun, bi v khi
phn t tinh bt b thy phn v trt t v khng th iu chnh c t lng ca cc
cu t ca dch thy phn. Phm cht ca ng mt, tnh cht ha l ca chng li
ph thuc vo t lng ca cc cu t chnh ny. Khi thy phn tinh bt bng ch
phm enzyme s khng ging nh thy phn bng acid, khng xy ra s thy phn cc
hp cht ln trong tinh bt nn lm dch thy phn tt hn, d dng tinh ch hn.
2.2.5.2. Trong cng nghip dt, giy
Trong cng nghip dt, amylase c s dng r h vi, ty lp h bt trn
vi lm vi mn, mm, d ty trng v d bt mu khi nhum. X l vi bng phng
php ezyme tt hn rt nhiu so vi vic x l bng cc cht ha hc. X l vi bng
enzyme khng ch nhanh m cn gi c bn, dai, khng hi vi, mao dn
tt v m bo v sinh. Do vy tng c nng sut lao ng.
2.2.5.3. Trong y hc
Amylase c dng phi hp vi cc coenzyme A, cytocrom C, carbonxydase
ch thuc iu tr cc bnh tim mch, bnh thn kinh, phi hp vi enzyme thy
phn cha bnh thiu enzyme tiu ha. Ngoi ra ch phm enzyme amylase dng
iu ch mi trng nui vi sinh vt phc v cho cng tc nghin cu khoa hc
cng nh chn tr bnh.
8
2.2.5.4. Trong nng nghip

Ch phm enzyme amylase c s dng ring l hay phi hp vi cc enzyme
khc nh protease, hemicellulase, cellulase, vn vn sn xut cc loi thc n d
tiu ha, hp thu trong chn nui gia sc, gia cm, c bit l gia sc non.
Trong ch bin thc n gia sc, thnh phn ng cc chim mt khi lng rt
ln. Trong khi lng ny, thnh phn tinh bt rt cao. tng hiu sut s dng nng
lng t ngun tinh bt, ngi ta thng cho thm enzyme amylase vo. Enzyme
amylase s tham gia phn gii tinh bt to thnh ng, gip cho qu trnh chuyn ha
tinh bt tt hn. S dng thc n c b sung ch phm enzyme s lm tng kh nng
hp thu, tiu ha, do lm gim lng thc n, tng trng nhanh, tng kh nng sinh
sn, chng bnh tt, vn vn.
C th s dng cc loi ch phm enzyme amylase chuyn ha cc ph liu,
c bit l cc ph phm nng nghip, ci to t phc v nng nghip. y cng l
hng mi trong nghin cu v ng dng ti nhiu quc gia.
2.2.5.5. Trong cng ngh ty ra
Enzyme -amylase ca vi khun l nhng enzyme khng bn nhit v pH kim
trong thi gian lu nn chng thng c bao li ri trn chung vi cc thnh phn
ca cht ty ra bo qun lu v tng kh nng hot ng ca chng. Cc enzyme
vi khun s lm cho cc vt bn carbonhydrate trong qun o d phn gii hn.
Thng nhng enzyme ny c ly t vi khun do chng c kh nng chu c
nhit cao v pH kim lu hn.
2.2.6. C cht ca enzyme amylase
2.2.6.1. Tinh bt
Tinh bt l mt cht bt v nh hnh, mu trng, khng tan
trong nc lnh nhng khi un nng s c kh nng tan trong nc.
Tinh bt l carbonhydrate thc vt ch yu trong cc loi c nh
khoai m, khoai ty, khoai lang.., v l cht d tr nng lng quan
trng.
Mc d tinh bt ly t cc ngun khc nhau khng hon ton ging nhau, song
t nm 1940, Meyer bit rng tinh bt mi ngun u c cu to t amylose v
amylopectin.
Di tc dng ca enzyme amylase, tinh bt b thy phn do cc lin kt

glucoside b phn ct. Khi tc dng vi iod tinh bt cho mu tm xanh c trng.
Cu to ca tinh bt l polysaccharide bc hai, ng th (homopolysaccharide
hay homoglucan). Cng thc tng qut l (C6H10O5)n, cu to t nhng n v c s l
-D-glucose. Lin kt vi nhau bng lin kt 1,4 glucoside v lin kt 1,6 glucoside.
Tinh bt tn ti hai cu t: amylose v amylopectin c cu to v tnh cht khc nhau.
Tuy nhin a phn tinh bt u c khong 20 - 30% amylose v phn cn li l
amylopectin.
Amylose
Hnh 2.1 Cu trc phn t ca amylose. (Wikipedia.com).

C trng lng phn t khong 50.000 n 160.000 dalton, c cu to t
khong 200 - 1000 phn t D-glucose ni vi nhau bi lin kt -1,4 glucoside
(carbon v tr th nht ca glucose s lin kt vi carbon v tr th 4 ca glucose k
cn), to thnh 1 chui mch thng. V vy amylase cn c gi l tinh bt mch
thng. Do cu to nn cu hnh khng gian ca amylose s cun li thnh vng xon
c, mi xon c s gm c 6 n v glucose, khi cho dung dch iod vo trong tinh bt
th iod s chim v tr trung tm ca xon c to thnh phc cht c mu xanh, y l
phn ng c trng ca amylose. Khi un nng th mch amylose nhn thm mt nng
lng nn dui thng ra khng cn xon c nn phc cht tinh bt iod khng cn na
v mu xanh s bin mt.
Amylopectin
10
H
Hnh 2.2 Cu trc phn t ca amylopectin. (Wikipedia.com.).
C trng lng phn t khong vi triu dalton, c cu to t khong 600
n 6000 phn t D-glucose, ni vi nhau bi lin kt -1,4 glucoside v lin kt -1,6
glucoside to thnh mch c nhiu nhnh.
Amylopectin cho mu tm vi iod, ha tan trong nc nng cho dung dch
c nht cao. Thy phn tinh bt nh nhng vi amylase, hay acid long thi gian
ngn s to thnh dextrin, mt t maltose, glucose. Thy phn thi gian lu hn vi
acid hoc enzyme vi nhit cao hn s to nhiu maltose v glucose hn.
2.2.6.2. Glycogen
Hnh 2.3 Cu trc phn t glycogen. (Wikipedia.com).
11
Glycogen l mt loi carbonhydrate d tr c th ng vt v c c th

ng vt chuyn ha t t s dng. Amylase c vai tr quan trng trong chuyn
ha glucid t bo ng vt, vi sinh vt. Glycogen c cu to tng t nh
amylopectin nhng c mch nhnh nhiu hn, phn t lng khong 2 triu n 3
triu dalton. Glycogen d tan trong nc. ng vt v ngi, glycogen tp trung ch
yu gan v bp tht.
2.3. Gii thiu v nm mc Aspergillus niger v Mucor

2.3.1. Nm mc Aspergillus niger
Aspergillus niger cu to dng si, sinh bo t (bo t h / bo t ngoi sinh)
v khng c dip lc, s dng ngun hu c c sn sinh sng. Aspergillus niger c
trong t, xc b thc vt v hoa qu vng nhit i. Van Tieghem l ngi u tin
pht hin v phn lp chng nm mc Aspergillus niger t qu, ht, c bit l ht u
phng, b o, da( Lng c Phm, 2004).
V tr phn loi ca A. niger
Lp : Deuteromyces.
B
: Moniliales.
: Monilaceace.
Ging: Aspergillus.
Nhm: Aspergillus niger.
A. niger thuc loi nm si a bo. u si nm sinh sn phnh to ra to thnh
t bo hnh chai v cc chui nh bo t. Cung ca nh bo t n bo, t bo hnh
chai v chui nh bo t to u trn u si nm sinh sn nh nhng tia nc i t
xa vo vi ti. Cung sinh bo t c sinh ra t khun ty kh sinh, c chiu di t 1
n 3 mm, c vch t bo trn bng v trong sut. nh cung phnh ra c dng hnh
cu c gi l bng, c ng knh t 50 75 m, sinh ra hai lp th bnh. Lp th
nht hnh tam gic cn ngc, lp th hai hnh chai. Bo t hnh cu, dng bo t nh
c ng knh 4 5 m, c mu nu en, vch bo t nh c dng x x.
12
Loi nm si ny sinh trng nhit ti thiu l 6 - 8oC, ti a l 45 - 470C,

ti u l 30 - 370C, sinh trng trong mi trng c m ti thiu l 23%. m
mi trng thch hp ln men bn rn l 50 - 65%, ch sinh trng v pht trin
trong s c mt ca O2 v pH ti u t 4 - 6,5. A. niger l nm si chu kh, bo t
nh c th ny mm trong iu kin mi trng c th nc 0,77 35 0C. A. niger c
th sinh trng pH 2 trong iu kin c th nc cao.
A. niger c xem l nm si khng sinh c t v c s dng rng ri trong
ch bin thc phm. Cc enzyme thu phn thu nhn t chng ny khng gy t bin
vi khun v cc m ca chut. Tuy nhin, theo thng tin gn y, c 2 trong s 19
chng A. nger c phn lp sinh ra ochratoxin A (Abraca, 1994).
Loi nm ny chim u th vng kh hu nhit i. Bo t nh mu en nn
khng chu nh hng t mt tri v tia UV. A. niger thng c phn lp t cc
thc phm phi nng nh c, cy gia v
2.3.2. Nm mc Mucor
Nm mc Mucor l nm n bo cu to dng si, sinh bo t. Bo t c to
thnh trong cc ti (bo t nang) v thuc loi bo t ni sinh. Cung bo t nang c
phn nhnh v mc ln t bt k ch no ca si nm (Lng c Phm, 2004).
V tr phn loi ca nm Mucor
Lp : Zygomycetes
B
: Mucorales
: Mucoraceace
Ging: Mucor
Mucor l ging ln nht trong b Mucorales. Chng bao gm cc loi: M.
mucedo, M. cremois, M. rouxii, M. hiemalis, M. silvaticus, M. subtilis, Mucor l
mt loi nm pht trin rng ri, thng tm thy trong t, phn bn, tri cy, bnh
m, v nhng thc phm giu tinh bt khc. Chng pht trin tt trong iu kin hiu
kh. iu kin ym kh Mucor. sppr phn tch thnh nhng si nm nh hnh trn
hay hnh bu dc v ny chi, v th ngi ta hay gi loi ny l men ca nm mc.
Chng c kh nng s dng mt cch hiu kh nhiu ngun carbon, ln men
carbonhydrate, c th s dng ammoniac hay nit hu c cho qu trnh sinh trng, v
cn c th pht trin trn mt thang nhit rng. V th chng c mt trn nhiu loi
mi trng, bao gm nhiu loi thc n khc nhau.
13
Nm Mucor c th c phn bit vi cc loi nm khc qua cc im c bit

ca khun ty (khng c vch ngn), nang bo t (hnh thnh trn tt c cc b phn kh
sinh ca nm), nang tr hnh nn, tr, hoc hnh qu l, bo t u, bng long, khng
c thn b v r gi.
Mucor. spp c vai tr quan trng trong mt s quy trnh sn xut cng ngh
thc phm, chng thc y qu trnh chn ca phomai hoc mt s thc phm ng
nh chao, tng, ng ho cc giai on u ca qu trnh ln men bia, ru.
2.4. Nui cy vi sinh vt tng hp amylase
2.4.1. Sinh tng hp enzyme cm ng
Mt qu trnh sinh tng hp enzyme c gi l cm ng nu n ch xy ra
mc ng k khi trong mi trng c c cht c hiu ca enzyme ny hoc cc
cht c cu trc tng t c cht. Cc c cht ny c gi l cc cht cm ng, v
enzyme ch c to ra khi c cht cm ng gi l enzyme cm ng. Amylase cng l
mt enzyme thuc h enzyme cm ng. Enzym amylase ch c sinh ra khi nm mc
sinh trng trn mi trng c cha tinh bt.
2.4.2. nh hng ca cc yu t dinh dng i vi qu trnh sinh tng hp
enzyme amylase
2.4.2.1. Ngun carbon
vi sinh vt tng hp enzyme amylase, trong mi trng nht thit phi c
cht cm ng l tinh bt. Tinh bt trong mi trng c th l cm go, cm m,
Nhng loi sinh vt khc nhau s c nhng ngun cm ng khc nhau. T nhng cht
cm ng vi sinh vt s to ra enzyme c hot tnh khc nhau.
Cc ngun carbon khc nh Glucose, Cellobiose, Acetat, Citrat, Oxalate,
Succinat v nhng sn phm trung gian ca chu trnh Krebs, nu trong mi trng c
nng rt t s kch thch vi sinh vt pht trin v to enzyme. Nhng nu nng
cao s c tc dng ngc li, km hm s to thnh amylase.
2.4.2.2. Ngun Nit
Hot tnh ca enzyme s thay i rt nhiu nu ta thay i cc thnh phn chnh
ca mi trng, c bit l ngun nit. Nitrat l ngun nit v c thch hp nht i
vi cc vi sinh vt. NaNO3 l ngun nit dinh dng nui nhiu loi nm si to
amylase, ngoi ra cn c cc mui khc nh NH4NO3, Mg(NO3)2, (NH4)2SO4.... Cho
ngun nit nht nh vo mi trng c th kch thch tng hp amylase ny v c ch
14
tng hp amylase khc. Theo mc tiu th mui, mi trng s chuyn dch mnh
v pha tng hp glucoamylase v c ch tng hp -amylase.
S to amylase cc i thng thy cc nng nit cao (0,25 0,4 %). Khi
lm mi trng nui cy nm mc to amylase thng dng NaNO 3 l tt nht. Cng
c th b sung nit hu c (gelatin, casein, nc chit ng) nm sinh trng tt,
nhng li khng c tc dng kch thch to amylase. Thm nc chit mm mch hay
nc chit ng cng nh cc amino acid ring r s lm tng sinh tng hp -amylase
ngoi bo, song hiu qu khng bng NaNO3 (Nguyn c Lng, 2004).
Tnh cn bng ca mi trng dinh dng v carbon v nit c ngha ln i
vi sinh tng hp sinh khi vi sinh vt v s to thnh amylase. Ch khi no trong mi
trng c lng carbon v nit cn thit th vi sinh vt mi c kh nng tch ly
amylase cc ln. S thiu ht cu t ny khng th b p bng s tha cu t kia.
Thng thng, t l carbon gp t 1 n 40 ln hm lng nit trong mi trng l
thch hp nht. Trong mi trng Zapeck th t l ny l 18 : 1.
2.4.2.3. Cc nguyn t khong
Cc nguyn t khong a lng v vi lng c nh hng ln ti sinh trng
v tng hp cc enzyme amylase ca vi sinh vt. V cc nguyn t khong ny cn
c b sung vo mi trng theo t l thch hp kch thch s sinh tng hp
amylase. Nu mi trng thiu hoc tha khong u c nh hng n s to enzyme
ca cc vi sinh vt.
Mg2+ c nh hng n bn nhit. Thiu MgSO 4 s c nh hng xu n s
tng hp enzyme amylase ca nm si. Khi s tng hp -amylase s b c ch
hon ton, cn lng -amylase s b gim hng chc ln ( Fenikxova, Muxaeva,
1967). Nng ti u khong 0,05%. Tuy nhin, mui ny khng nh hng n s
to oligo-1,6-glucosidase (Silova, 1967).
Phospho cn tng hp cc hp phn quan trng ca sinh cht v nhiu
coenzyme, ng thi phosphoril ha glucide trong qu trnh oxy ha sinh hc.
Phospho nh hng trc tip ti sinh sn ca nm si cng nh cc vi sinh vt khc, v
vy cng nh hng n s sinh tng hp enzyme ca chng. Nhiu nh nghin cu
cho bit nu thm phospho hu c (nh dng fitin) vo mi trng s lm tng s tng
hp amylase ln 2 3 ln.
15
Canxi cn cho s tng hp v n nh -amylase hot ng v n l cu t

khng th thiu ca enzyme ny. Canxi cn c tc dng bo v amylase khoi tc
ng ca proteinase (Hsiu, 1964). Lng Canxi cn thit chim khong 0,01 0,05 %.
Nhiu nguyn t vi lng cng c nh hng n s sinh tng hp enzyme
amylase nh coban, mangan, ng, thy ngn, vn vn. Chng c th c kh nng kch
thch to amylase nh coban, Ca, nit, hoc cng c th c tc dng km hm s tng
hp enzyme ny nh thy ngn.
2.4.2.4. Nhit nui cy
Nhit l yu t c nh hng mnh m n s pht trin v tng hp enzyme
ca vi sinh vt. Hot ng ca cc loi vi sinh vt da trn s chuyn ho ca hng
lot cc phn ng da theo nhng trnh t xc nh. Khi nhit tng s nh hng
n qu trnh sinh trng v pht trin ca vi sinh vt v th n c nh hng n s
sinh tng hp enzyme ca chng.
2.4.2.5. pH ban u
pH ca mi trng cng c nh hng rt ln n s pht trin v kh nng
sinh tng hp amylase t vi sinh vt. pH ca mi trng c nhng nh hng rt khc
nhau i vi cc chng vi sinh vt khc nhau. Nhiu loi nm pht trin v phn gii
tinh bt mnh pH 4 - 6. S sinh trng ca vi sinh vt c th lm thay i ng k
pH trong c cht. S thay i ny ph thuc ch yu vo s trao i cht ca vi sinh
vt v kh nng m ca c cht rn. Trong mi trng ln men bn rn rt kh
iu chnh v kim sot pH. Tuy nhin nu nui cy bng phng php ln men bn
rn th pH mi trng s c t nh hng hn so vi phng php nui cy chm do
mi trng ca phng php nui cy b mt s c dung lng m cao v hm m
thp nn pH khng thay i nhiu trong qu trnh nui.
2.4.2.6. m
m khng ch nh hng n s sinh trng v trao i cht ca vi sinh vt
m cn nh hng ng k n c tnh l ho ca c cht. Cc c cht khc nhau c
kh nng gi nc khc nhau. Do , qu trnh ln men bn rn c thc hin vi
hm lng nc trong c cht khc nhau t 30 - 80%.
2.4.2.7. Mi trng khng kh
Vi sinh vt hiu kh th mi trng nui cy phi m bo cung cp oxi vi
sinh vt sinh trng v pht trin. Khi nui cy trn mi trng lng thng trn
16
my lc, mi trng bn rn th c cht phi c xp to s thng thong gia cc

tiu phn c cht. Ngc li vi sinh vt k kh ch pht trin trn mi trng khng c
s hin din ca oxi. Ngoi ra cng c vi khun sng c c hai iu kin trn.
2.5. Mi trng ln men bn rn
2.5.1. Cm go
Cm go l sn phm ph ca qu trnh xay xt v ch bin go. Cm go c
gi tr dinh dng kh cao. Cm c thu hi di 2 dng l cm th v cm t.
Chng thng c dng bt, mu hi vng, mm v mn, chim khong 10-12% khi
lng la cha xay xt. Trong 100 g cm go c khong 28 g tinh bt, v vy y l
ngun c cht kh l tng dng trong ln men bn rn thu nhn amylase t cc vi
sinh vt do ngu cung n nh, gi thnh r v c lng tinh bt kh co
Bng 2.1 Thnh phn dinh dng ca cm go
Thnh phn
Calori
Tng s lipit
Cht bo bo ha
Cht x tiu ha c
Carbohydrat
ng
Protein
Vitamin E
Vitamin B6
Canxi
Kali
Khi lng/100g
316 KJ
21g
4g
21g
28 g
0,9 g
13,3 g
4,9 mg
4,1 mg
57 mg
20 mg
(http://www.nutritiondata.com/).
Theo bng trn th cm c lng dinh dng rt cao vi lng cht bo cha
bo ho cao, vitamin nhm E, nhm B, phylate, km, can xi, kali u rt cao, ngoi ra
trong cm cn c chc cht bo omega 3 kh cao. Theo nhiu nghiu cu cho thy
65% cht dinh dng ca go tp trung cm, thnh phn ca cm c nhiu loi
vitamin v cht bo tt, li cn i v c nhiu x d tiu nn c th xem l ngun
lng thc rt tt cho c con ngi.
Tuy nhin cm thng c cho cc loi gia sc v thy sn n ch khng cho
ngi. Nguyn nhn l do cm c mt s enzyme (cht men) ni ti hot ng rt
mnh s oxy ha cc nhm bo cha no ca cm rt nhanh ch vi gi sau khi ch bin
to mi hi kh chu. Thm vo do cng ngh xay xt go cha cao, li t c u
17
t theo hng thu cm sch nn cm thng ln rt nhiu loi tp cht (v tru, sn

vn vn). V l do m hu ht lng cm thu c thng c b con nng dn
dng nui cc loi gia sc, gia cm. Hng nm trn th gii c khong 40 45 triu
tn cm c sn sut v 90% l nm chu (http://www.bannhanong.com/, x l
cm go thay th ng cc).
2.5.2. B u nnh
B u nnh c hm lng x cao, bao gm cht x ho tan v cht x khng
ho tan. Cht x khng ho tan c tc ng chng to bn, ngn nga ung th rut kt,
ung th i trng, gim nguy c tr, chng bo ph v bnh tiu ng.
Cht x ho tan c kh nng tan trong nc thnh dung dch keo. Khi i qua
rut s to ra th ng lm chm qu trnh hp th mt s cht c hi nh cholesterol
vo mu. Ngoi ra, trong b u nnh cn c cht isoflavones c kh nng phng
chng nhiu loi bnh ung th v bnh tim mch. Theo t chc Y t th gii (WHO),
lng cht x s dng mi ngy nn m bo 27 40 g x tng, 16 27 g x khng
ho tan.
Tuy nhin, trong thc t b u nnh kh xay mn v c cu trc nhm, ri rc
nn khng th dng trong ch bin thc phm. V vy, cc nh nghin cu dng
phng php vi sinh x l b u nnh. H dng hai loi enzyme (men) c tn l
Pectinase v Cellulase vi t l thch hp thy phn b u nnh. Nh , gip
b u nnh qua x l tr nn mn v lm thay i hm lng cc hp cht x trong b
u nnh theo cch c li nht (http:// www.Vietnamnet.com).
2.6. Phng php nui cy vi sinh vt sn xut enzyme
Hin nay trn th gii ch yu p dng hai phng php sn xut enzyme l
nui cy b mt (ln men bn rn) v nui cy chm.
2.6.1. Phng php nui cy b mt
L phng php nui cy m vi sinh vt s mc trn b mt mi trng. Trong
nui cy b mt vi sinh vt s c nui cy trn mi trng lng hoc mi trng c
(cn gi l mi trng len men bn rn), nhng ch yu vn l nui cy trn mi
trng c. Phn ln cc nh my sn xut enzyme thng s dng mi trng c
nui cy thu nhn enzyme t vi sinh vt.
18
Mi trng ln men bn rn thng dng nguyn liu t nhin nh cm go,

cm m, b u nnh, ng mnh, Ty theo i tng vi sinh vt v mc ch nui
cy khc nhau m b sung thm cc cht khc nhau v mt s thnh phn khc cho
thch hp.
Khi nui cy theo phng php ln men bn rn, vi sinh vt pht trin trn b
mt mi trng, nhn cht dinh dng t ht mi trng, ri sinh tng hp ra enzyme
ngoi bo v ni bo. Cc enzyme ngoi bo s thm thu vo trong cc ht mi
trng, cn enzyme ni bo s nm trong sinh khi vi sinh vt.
Phng php ny c u im l hm lng v hot tnh enzyme cao, ch phm
d sy kh, t b gim hot tnh, d vn chuyn v s dng, khng cn s dng nhng
thit b c bit, tiu th nng lng t nn c th p dng nhiu a phng. Tuy
nhin nhc im ca n l tn din tch mt bng v i hi lng nhn cng ln,
kh c gi ha trong sn xut.
u im khc ca phng php ny l d thc hin v khi b nhim bi cc vi
sinh vt khc, thng ch xy ra hin tng nhim cc b nn d x l. Tuy nhin,
phng php ny c nhc im rt ln l tn nhiu din tch v kh c gii ha, t
ng ha.
2.6.2. Phng php nui cy chm
Cn gi l phng php nui cy b su. Phng php ny vi sinh vt c
nui trong mi trng lng v c nui trong cc thng ln men. Cc thit b ln men
c lp t h thng iu khin cnh khuy, h thng cung cp i, h thng iu
chnh pH v nng cc cht dinh dng. Trong h thng iu ha khng kh v
h thng khuy trn c ngha rt ln (Nguyn c Lng, 2004). Ty theo chng
loi vi sinh vt m thnh phn ca mi trng lng s c thay i cho thch hp.
Phng php ny c dng ph bin trong ln men qui m cng nghip. So
vi phng php nui cy b mt, phng php nui cy chm t chong din tch b
mt, d c gii ha v t ng ha trong qu trnh theo di. Tuy nhin phng php
ny i hi u t nhiu chi ph cho trang thit b.
Phng php ny c u im l d dng c gii ha, t ng ha, t tn din
tch, song chng i hi s u t ln, kh c thc hin nhiu ni, v s rt kh
khn trong vic x l s xm nhp ca cc vi sinh vt l.
19
2.7. Thu nhn enzyme t mi trng nui cy b mt

2.7.1. Trch ly enzyme t mi trng nui cy b mt
Enzyme ngoi bo s tn ti trong t bo vi sinh vt vi hm lng rt thp so
vi cc thnh phn khc. Khi chit rt enzyme t canh trng ta thu c khng ch
enzyme ha tan trong nc m cn thu c rt nhiu sn phm thy phn t c cht
mt trng v cc cht do nm mc to ra trong qu trnh sinh trng v pht trin.
Cc enzyme thng l nhng cht hu c khng bn d b bin tnh khi b tc
ng bi cc yu t bn ngoi. Do , vic tch v thu nhn enzyme l iu khng d.
chit rt enzyme t mi trng bn rn, ta thng dng nc, cc dung dch
mui trung tnh hay cc dung dch m thch hp. Trong , nc thng c dng
nhiu nht v s thun tin v cho kt qu cao. Enzyme t mi trng khuch tn vo
nc do s chnh lch nng . Nc c th chit tch 90 95% enzyme trong canh
trng. Nc thng dng chit tch c nhit 25 - 280C.
Trong qu trnh nm mc sinh trng trn mi trng bn rn, cc vt cht kh
trong mi trng s b ha tan thy phn khong 33 - 35%, tinh bt s b thy phn
hon ton v trong mi trng s cn li phn ng m sinh vt cha s dng ht.
2.7.2. Cc yu t nh hng n qu trnh li trch
T l nc c nh hng rt ln n s thu nhn ezyme t canh trng nui cy
vi sinh vt. Canh trng kh cn nhiu nc trch li enzyme hn canh trng m.
Vi cc t l nc: canh trng nui cy khc nhau ta s thu c nhng lng
enzyme khc nhau. Thng lng nc dng li trch gp 3 3,5 ln so vi lng canh
trng nui cy nm mc.
Tip n l s nh hng ca thi gian li trch. Enzyme thy phn d dng ha
tan vo nc, thi gian trch li cng lu cng c kh nng trch li c nhiu enzyme
hn nhng lm tng hm lng tp cht trong dch chit v gim hot tnh enzyme.
Khi nhit trch li cng tng th vn tc trch li cng tng theo nhng cng
lm tng lng tp cht trong dch chit lm gim hiu sut trch enzyme v gim hot
tnh ca enzyme. Nhit trch li ti thch l t 28 30 0C. Trong cng nghip nc
my vi pH 7 thng c dng li trch enzyme.
20
Chng 3
VT LIU V PHNG PHP NGHIN CU
3.1. Thi gian v a im th nghim
Th nghim c tin hnh t thng 2 n thng 7 nm 2009, ti phong cac cht
co hoat tinh sinh hc Vin sinh hoc nhit i.
3.2. Vt liu th nghim
3.2.1. i tng th nghim
Nm mc Aspergillus niger va Mucor do Vin Sinh Hc Nhit i cung cp
c nui cy trong ng nghim thch nghing trn mi trng PGA nhit
thng l 300C trong thi gian 2 - 3 ngy, sau o em bo qun nhit 40C.
3.2.2. C cht
C cht s dng trong ti bao gm lua, cm go, tru v b u nnh. Lua,
cm go, tru c mua ch Th c, cm phi tng i xp d thng kh, khng
c mi i. B u nnh c cung cp bi Vin Sinh Hc Nhit i. Cc c cht s
c phi trn vi nhau trong mi trng nui cy.
3.2.3. Hoa cht va thit bi
3.2.3.1. Hoa cht
Ha cht pha thuc th Lugol, ha cht pha mi trng Czapeck, mi trng
Zapeck, ha cht pha mi trng PGA, mi trng dinh dng Mandel, ha cht dng
xc nh hm lng protein, bao gm dung dch Abumin chun v thuc th
Bradford, ha cht dng chnh pH cho mi trng dinh dng (HCl 0,1M, NaOH
0,1M), cn 700, 900, tinh bt tan v dung dch m Na-acetate 50 mM pH 5.
3.2.3.2. Thit bi
Cc thit b gm c cn phn tch, b n nhit, phng m hng cu, knh hin
vi quang hc Olympus, ni hp Tommy-SS-325, my xay sinh t, my khuy t, t
lnh, my o pH, my o quang ph UV-Vis cng cc dng c phng th nghim nh
que cy, pipet, pipetman, ng nghim, ng ong, becher, erlen, vn vn.
3.3. Phng php nghin cu
3.3.1. Phng php cy truyn
Do c hai loi nm trn u sinh sn bng bo t nn ta s dng phng php
gi ging trn mi trng thch nghing, bng cch cy theo ng dch dc. Khi
21
khun lc mc u s cho vo t lnh gi nhit 5 - 90C trnh nm mc b gi

lm nh hng n cc th nghim sau.
u tin, cn 50g khoai ty, 5 g agar, 5 g glucose. Khoai ty ra sch gt v,
ct lt mng em cn, cho vo ni un si vi 250 ml nc ct trong khong 30 pht.
Tin hnh lc qua vi lc, ly phn nc trong, cho thm nc ct va 250 ml, cho
agar v glucose vo un si n khi tan ht.
Sau tin hnh to mi trng thch nghing. Phn phi mi trng vo cc
ng nghim, khong 1/4 chiu cao ng nghim. Lm nt bng v bao gi hp kh
trng 1210C v 1 atm, trong 15 pht. Sau khi hp kh trng xong, thch trong ng
nghim cn lng ta ng thch v tr nghing thch hp cho n khi thch ng c
hon ton. V tr nghing phi u trn ca mt nghing khng vt qua 2/3 chiu
di ng nghim, khng c thch chm vo nt bng, mt thch bng phng,
khng b t, chiu di va phi khng qu ngn hoc qu di.
Tip theo s thc hin vic cy gi ging trn mi trng thch nghing. Dng
que cy mc kh trng cy truyn ging t ng ging sang cc ng nghim
chun b trn. ng tc ny c thc hin trong t cy v trng. Sau bo t
pht trin nhit phng khong 2 ngy, bo t mc u, ri chuyn vo t lnh tr
nhit 5 - 90C dng cho cc th nghim sau.
3.3.2. Phng php nhn ging cp 2 (gi ging trn mi trng la)
Chun b cc dng c th nghim v pha mi trng dinh dng, cn v cc
ng nghim ging A. niger v Mucor. Dng c th nghim cn thit cho cc th nghim
ny bao gm becher, ng ong, ng nghim, a khuy, que cy v pipet kh trng,
erlen. T cc dung dch stock tin hnh pha dung dch mi trng dinh dng gm c
mi trng Mandel v Czapeck, chnh v pH 6. Thm nc ct vi t l thch hp
mi trng nhn ging t m 55%.
Tin hnh cn 20g la cho vo 4 erlen, b sung thnh phn khong chun b
trn. Dng a thy tinh nh ti mi trng. Sau em cc erlen hp kh trng
1210C, 1atm trong 15 pht. ngui. Thc hin cy ging trong t v trng. Dng
pipetman ht 1 ml nc ct c v trng cho vo ng nghim cha ging, dng
que cy vng ha bo t nm mc trong nc. Sau chuyn vo trong erlen. Tin
hnh tng t cho c 4 erlen. Khuy u, nh ti bo t ca mc ging va cy vo
mi trng bng a thy tinh v trng. em nhit phng. Sau 3 ngy quan st
22
thy lng bo t mc nhiu, em cc erlen cho vo tr trong t lnh nhit 5 90C dng cho cc th nghim sau.
3.3.3. Phng php m t hnh thi A. niger v Mucor
3.3.3.1. Quan st hnh thi i th
Dng que cy mc (v trng) gt nh bo t nh ca nm si, chuyn sang a
petri cha mi trng PGA v trng v ngui, cm u mc xung mt thch
thnh 3 im. Cc ng tc trn c thc hin trong t cy. Sau a petri nhit
phng trong khong thi gian 2 ngy. Quan st khun lc ca nm si (hnh dng
v mu sc).
3.3.3.2. Quan st hnh thi vi th
Lm phng m quan st hnh thi vi th. Lt giy thm y petri (ng
knh 12 cm) ri t vo ming lame, gi giy v em kh trng. Nu chy mi trng
PGA (v trng) rt chy dc theo na tm lame v ch ng li, lm m giy thm
bng nc ct v trng (va t t giy). Cy mt t bo t thnh mt hoc hai
im trn phn thch ca ming lame trong phng m, nui nhit phng. Sau 2
n 3 ngy ly ming lame ra khi hp petri v quan st. Quan st di knh hin vi
( phng i 40 v 100 ln) hnh dng vi th ca hai chng th nghim.
u tin dng que cy hnh mc cu mt t h si nm dn mng trn phin
knh. Tip theo cho vi git cn 95% mt lc, dng giy thm lau kh sau cho
vi git NaOH 10% y l knh ln, em soi. Hoc cho vi git cn 600 c vt
amoniac, thm kh ri cho thm vi git glycerin long ln tm lame, y l knh ln,
em soi. Mc ch ca cc tc dng trn l tng cng kh nng thm nc ca si
nm, ui bt kh trong si nm, lm si nm n to ra d quan st.
3.3.4. Xc nh trc tip s lng bo t nm si bng bung m hng cu
Bung m hng cu dng m vi sinh vt c kch thc ln (nm men, bo
t nm mc). Ngi ta thng dng 2 loi bung m hng cu; bung m Thomas
v bung m Goriep. Nguyn tc cu to ca 2 loi bung m ny u ging nhau.
Bung m u l 1 phin knh hnh ch nht, chia thnh 3 khong ngang. Khong
gia chia thnh 2 khong nh. Trn mi khong nh ny c k mt li m, gm rt
nhiu vung. Mi vung c din tch l 1/25 mm 2 li c chia thnh cc nh
(thng l 16 ), mi nh c din tch l 1/400 mm2 v chiu cao l 1/10.
Nh vy V nh = 1/400 mm2 x 0,1 mm = 1/4000 mm3 hay 1/4000000 ml.
23
Tin hnh cn 1 g canh trng ly t canh trng nhn ging cp 2 thc

hin trong mc 3.3.2 em i pha long 10-1, 10-2, 10-3, 10-4. Sau tin hnh m
trn phng m.
Chun b mt s ng nghim cha 9 ml dung dch sinh l cha Tween 80 0,1%
v trng v mt s ng ht 1 ml v trng tin hnh vic pha long mu. Cn 1 g
bo t nm si cho vo ng nghim s 1 cha 9 ml dung dch mui sinh l cha Tween
80 0,1% v trng, huyn ph u. Ta c pha long 1/10 hay 10-1. Sau li ht 1
ml ng nghim 1 cho vo ng s 2, trn u, ta c pha long 1/1000 hay10-2.
Tip tc nh vy t ng 2 sang ng 3, ng 3 sang ng 4. Ta s c cc pha long
tng ng 10-3, 10-4.
m s lng bo t bng bung m hng cu. Lc u ng nghim pha
long. y l knh ln li m. Dng ng ht v trng ly mu, cho vi git vo mp
l knh, do sc mao dn, dch mu trn vo mt trn li m. Ch khng hnh
thnh bt kh trong li m hoc trn dch mu xung rnh. t bung m ln bn
knh hin vi v yn trong 3 - 5 pht, sau tin hnh m bo t trong nm ln
cho nhau, ch m cc bo t nm trong lng nh v nhng bo t nm trn 2 cnh
lin tip cng chiu. m ln lt t con 1 n con 16.
Ch nng dch huyn ph pha long sao cho mt trong mi nh
khng qu 10 bo t. S bo t m c trong 5 ln phi ln hn 200 v phi m
bo c chnh xc ca phng php. Sau khi s dng xong bung m phi c
ra ngay v lau tht kh bo qun.
Kt qu m c tnh theo cng thc nh bn di.
Cng thc :
N=
a*4000*103*n
b
N : s lng bo t trong 1 ml dch huyn ph.

a : s lng bo t trong 5 ln (80 con).
b : s con trong nm ln (16 x 5 = 80 ).
103 : s i mm3 thnh ml (1000 mm3 = 1 ml).
n : pha long ca mu.
24
3.3.5. Phng php xc nh m

Cng thc tnh m
F = A*(B% - C%) / 100%.
Cch tnh m % :
V d: Ta c 100 g c cht c m 10% nhng cn m 50%.
Theo cng thc trn ta c:
100*(50 10)
F=
= 40 (ml)
100
Nh vy: trong 10g c cht ta b sung thm 40ml nc ct s c m 50%.
Vi :
A
: khi lng c cht c trong mi trng nui cy (g).
: m mun b sung vo mi trng (%).
: m trong c cht (%) ( vi c cht l la, cm go, b u nnh, tru th
m thng thng ca chng l 10%). (Trn Anh Tun, 2005).

100 : m 100%.
F : lng nc cn b sung vo mi trng t m thch hp (ml).
3.3.6. Phng php ln men bn rn thu nhn enzyme amylase
Phi trn cm: tru: b u nnh theo t l (ph thuc vo tng giai on ca
ti), cn 20g hn hp cho vo mi erlen. T nhng dung dch stock tin hnh pha mi
trng dinh dng gm c mi trng Mandel v Zapeck. Tin hnh iu chnh theo
m, pH, nng mi trng theo tng th nghim. Cho dung dch dinh dng vo
cc erlen chun b trn, nh ti. Kh trng mi trng 121 0C trong 15 pht,sau
lm ngui mi trn.
Cn 1 g canh trng la t cc erlen trong mc 3.3.2, cho thm 3 ml nc ct
vo, khuy bng a khuy v trng. Tin hnh m bo t, pha long t c
lng bo t mi loi l 0,8*108 bo t/g canh trng.
Chuyn huyn ph bo t ca nm mc trn vo cc bnh tam gic c
chun b nh trn, nui nhit phng trong thi gian thch hp ty theo tng th
nghim. xc nh thnh phn mi trng v cc iu kin ti u cho s sinh trng
v sinh tng hp enzyme, nm si c nui cy trn mi trng c t l b u nnh:
cm m: cm go khc nhau (1:2:1, 2:1:1, 1:1:2), m mi trng (t 40 - 75%), pH
(t 3 - 8), nng dinh dng (x1 - x8 hay t 1 - 8ln), t l ging (t 0,8*108 n
25
3,2*108 bo t/g mi trng), thi gian nui cy (t 8 n 44 gi) v yu t cui cng

l nhit nui cy (t 30 - 550C).
Sau qu trnh nui cy, tin hnh li trch enzyme t canh trng vi dung mi
l nc ct. B sung nc ct vi t l 1 g canh trng: 3 g nc ct, khuy trong 40
pht, lc thu dch chit enzyme.
3.3.7. Phng php xc nh hm lng amylase
Phng php ny da trn s thay i bc sng hp th cc i ca thuc th
Coomassie Brilliant Blue khi to phc vi protein. Trong dung dch mang tnh acid,
khi cha kt hp vi protein th thuc nhum c bc sng hp th cc i 465 nm,
khi kt hp vi protein th thuc nhum hp th cc i bc sng 595 nm. hp
th bc sng 595 nm c lin h mt cch trc tip vi nng protein. c bit
nu c mt cc acid amin nhn thm s bt mu mnh hn. Phng php ny c
nhy cao cho php pht hin ti vi g protein/ml, d thc hin v tit kim thi gian.
Ho cht bao gm dung dch mu protein (c th trong bi ny l dch chit
enzyme amylase thu t cc canh trng nui cy), dung dch albumin 0,1 mg/ml v
dung dch thuc th Bradford. Pha dung dch albumin bng cch cn chnh xc 10 mg
albumin pha trong 10 ml nc ct. Lc u cho tan. Gi nhit lnh 200C, khi
dng pha long 100 ln, c dung dch albumin c nng 0,1 mg/ml. Sau tin
hnh pha dung dch thuc th Bradford. pha thuc th Bradford, u tin cn 0,001
g Coomassie Brilliant Blue, 4,7 g dung dch ethanol 990 v 8,5 g dung dch acid
phosphoric 85%. Phm mu Coomassie Brilliant Blue c lm tan trong dung dch
ethanol 990 trong bnh nh mc c th tch l 100ml, thm vo bnh 60 ml nc ct,
ri thm t t acid phosphoric 85% v chnh n 100ml bng nc ct.
xc nh hm lng protein trong mu, u tin ta xy dng mt ng
chun vi dung dch protein chun bit trc nng . Dung dch protein chun
thng l Bovine plasma-globulin hoc Bovine serum albumin. Sau khi cho dung dch
protein vo dung dch thuc nhum, mu s xut hin trong 2 pht v bn ti 1 gi.
Tin hnh o dung dch bng my quang ph k ta c ODx hp th s t l vi
lng protein trong mu.
Thc hin mt mu i chng vi nc ct (OD0), ly gi tr ODx:
ODx = ODx OD0
26
Lng protein mu trong dung dch o c xc nh bng cch chm trn

ng chun theo ODx, ly gi tr ca im trn trc honh chnh l lng
protein trong mu dung dch o.
Xy dng ng chun (ch ra sch cuvette bng cn, trng li bng nc
ct trc v sau khi i mu o). c dung dch albumin chun c nng t 0-50
g/ml ta thc hin cc bc nh bng 3.1.
Bng 3.1 Cc bc tin hnh to dung dch albumin chun
ng nghim s
Nng albumin (g/ml)
Dung dch albumin (mg/ml)
Nc ct (ml)
Thuc th Coomassie (ml)
1
0
0,0
1,0
2
2
10
0,1
0,9
2
3
20
0,2
0,8
2
4
30
0,3
0,7
2
5
40
0,4
0,6
2
6
50
0,5
0,5
2
Dng ng h bm giy, canh thi gian 0 pht, cho 5 ml thuc th Bradford

vo ng nghim 1. Lc u, yn. Thc hin vic b sung 5 ml dch thuc th tun
t vo cc ng t 2 n 6 cc thi im cch nhau 1 pht. Lc u, yn. Tnh thi
gian ng 1 c 20 pht, tin hnh o hp thu ca dung dch bc sng 595nm
(OD595 nm). Tun t tin hnh o OD595 ca cc ng cn li cc thi im cch nhau 1
pht. Ly OD595 ca ng th tht (c protein) tr OD595 ca ng th khng (khng c
protein) c OD595. V ng tuyn tnh gia nng protein (g/ml) vi mt
quang OD595. o mu thu c, tnh hm lng protein. Vic o mu cng tin hnh
tng t vi thi gian cho phn ng l 20 pht. Mu c o pha long sao cho gi tr
OD595 nm trong ng chun. T lng protein tnh c trong dung dch em o,
suy ra tng lng protein c trong nguyn liu ban u.
Hm lng protein tnh theo cng thc:
HL =
b*m*V
w
(mg/g)
Vi:
b : nng protein trong mu suy ra t ng chun (g/ml).
m : h s pha long.
V: tng s ml dch chit thu c.
w: tng khi lng canh trng thu c.
27
3.3.8. Phng php xc nh hot tnh amylase

Enzyme amylase l enzyme thy phn tinh bt. N phn ct amylose v
amylopectin ca tinh bt thnh cc loi ng maltose, glucose,Hot tnh ca
enzyme amylase c xc nh da trn s thay i mu sc ca phc hp tinh bt
iod trc v sau khi thy phn. Mt quang ca phc hp tinh bt iod s c o
bc sng 595 nm trn my o quang ph.
Ha cht cn thit cho th nghim ny bao gm dung dch Lugol, dung dch
m Na-acetate 50 mM pH 5, dung dch h tinh bt 1%. Dung dch ny c pha
bng cch cn 1 g tinh bt tan cho vo berche ng 100 ml dung dch m, un cch
thy cho si trong 5 pht, khuy u cho tan.
i vi ng nghim cha enzyme mu (An) s c chun b bng cch ht 1
ml dung dch enzyme mu cho vo ng nghim. Cho thm 1 ml dung dch c cht
(dung dch h tinh bt 1%), cho thm 1 ml dung dch m pH 5 v 0,5 ml NaCl 3%
vo. Cho ng nghim vo b nhit 500C, 30 pht. Sau cho vo ng nghim 1 ml
HCl 1N, thm nc ct va 10 ml. Nh 1 git dung dch Lugol, i 20 pht ri
em so mu bc sng 595 nm trn my quang ph.
Mu chun c pha tng t mu enzyme nhng thay 1 ml dung dch enzyme
bng 1 ml dung dch nc ct.
Mu trng cng c pha tng t mu enzyme nhng dng 1 ml nc ct thay
cho 1 ml dung dch h tinh bt.
Mt n v hot tnh (UI) ca amylase l lng enzyme cn lm gim mu
tinh bt tan bi iodine trong 1 pht di cc iu kin phn ng nh trn.
Hot tnh amylase tnh theo gam canh trng:
HT =
(A0-An)*100*V*n
(UI/g CT)
A0*30*w
Nu tnh hot lc enzyme amylase trong 1ml dch enzyme:
HT =
(A0-An)*100*n
(UI/ml)
A0*30
Trong :
A0 : l mt quang ca mu i chng bc sng 595 nm.
An : l mt quang ca mu enzyme bc sng 595 nm.
n
: h s pha long
28
: th tch dch chit enzyme thu c sau khi lc (ml).
w : khi lng canh trng ban u em ly trch (g).

Hot tnh ring tnh theo cng thc:
HTR =
HT chung
(UI/mg protein)
HL protein
3.3.9. Phng php quy hoch thc nghim
Phng php quy hoch thc nghim khng ch cho php nghin cu tc ng
ng thi ca nhiu yu t ln qu trnh m cn cho php nh gi mt cch nh
lng mc nh hng ca tng yu t .
Sau khi thc hin cc th nghim kho st s nh hng ca tng yu t, ta rt
ra c thnh phn mi trng v cc iu kin nui cy c s c gi l mi trng
c s cho hai chng trn sinh trng v to thnh enzyme amylase.
T mi trng c s ta tin hnh ti u ho cc yu t trn bng cch tin hnh
cc th nghim theo k hoch thc hin y cc yu t (gi tt l TY). C s ca
vic qui hoch thc nghim theo yu t 2n l vic th hin tt c cc t hp c th c
gia cc yu t nghin cu. Mi yu t u c kim tra ng thi v khng ph
thuc ln nhau hai mc l trn (+) v di (-). Trung tm ca th nghim l mi
trng c s xc nh c (Nguyn Cnh, 1993).
3.3.10. Phng php b tr v x l s liu th nghim
Tt c cc th nghim c b tr theo kiu hon ton ngu nhin vi 3 ln lp
li. S liu thu c ca cc nghim thc s c phn tch bin lng n yu t
(One-way ANOVA) nh gi s khc bit ca chng. y l phng php phn
tch s liu th nghim da trn nhng bin ng gy ra cc sai s trong th nghim.
Mc ch ca ANOVA l xc nh mc sai khc gia cc trung bnh nghim thc
ca ch tiu nghin cu l do nghim thc hay ch mang yu t ngu nhin. Ngoi ra,
ANOVA cn cho php so snh nhiu s trung bnh ng thi vi nhau c kt lun
chung v s khc bit. Ni cch khc, nu qua ANOVA m xc nh c gia cc
trung bnh nghim thc khc nhau c ngha th chng t rng s sai khc l do
cc nghim thc th nghim gy ra.
29
Chng 4
KT QU V THO LUN
4.1. Kh nng sinh tng hp amylase ca nm mc Aspergillus niger trn mi
trng ln men bn rn
4.1.1. Nm mc A .niger
Bo t A. niger t ng nghim thch nghing c cy sang a Petri v ming
lame trong phng m cha mi trng PGA theo phng php m t mc 3.3.3,
nui nhit phng 2 - 3 ngy. Kt qu quan st i th v vi th c trnh by
hnh 4.1 v 4.2.
(b)
(a)
Hnh 4.1 Hnh nh i th v vi th ca A. niger. (a)hnh nh i th, (b)

hnh nh vi th.
Kt qu quan st hnh nh i th cho thy A. niger to khun lc trn, mc

nhanh trn mi trng PGA nhit phng, c dng nhung, phng v nhn, c
nhiu rnh. Khun lc cn non c mu trng, khi gi chuyn sang mu nu en hoc
en than do mu ca bo t nh. Mt tri c sc t vng, xp np.
Quan st hnh nh vi th ta thy khun ty phn nhnh, c vch ngn, bo t
nh khng nm trong bc bo t, bng hnh cu sinh ra nhiu th bnh k nhau v bao
ph ton b b mt bng, th bnh sinh ra bo t nh. Bo t nh c mu en, hnh
cu, xp thnh chui, cun nh bo t n bo.
4.1.2. nh hng ca t l cm go: b u nnh: tru
Trong th nghim ny tin hnh pha ch cc mi trng c t l c cht khc
nhau, lm m vi nc c b sung dung dch dinh dng c nng X1 n m
30
55%, pH 5. Thc hin cc bc nh mc 3.3.6. Cy huyn ph bo t A. niger vi mt

0,8*108 bo t/g vo cc mi trng chun b trn.
Sau 36 gi nui cy, thu dch chit enzyme t canh trng, em xc nh hot
tnh v hm lng enzyme amyase thu c. Kt qu phn tch phng sai ANOVA
cho thy s khc nhau gia cc nghim thc l c ngha.
A. niger vi cc t l c cht khc nhau.
Hot tnh amylase v hm lng protein cao nht (358,552 UI/g CT v 41,484
mg/g) mi trng c t l cm: tru: b u nnh l 2:1:1. Hm lng v hot tnh
amylase mi trng c t l cm: tru: b u nnh l 1:2:1 cng kh cao. Mi
trng c t l cm: tru: b u nnh l 1:1:2 cho hm lng v hot tnh amylase
thp. iu ny cho thy vic tng tinh bt (c trong cm go) trong trng hp ny l
c cht ca enzyme c tc ng n vic kch thch vi sinh vt sinh tng hp enzyme
tng ng. Kt qu th nghim cng ch ra rng vic tng cng lng b u nnh
lm gim ng k hm lng v hot tnh ca amylase. C th do trong b u nnh
cha nhiu lipid, l c cht cho vic tng hp enzyme lipase nn c ch s tng hp
enzyme amylase. Tuy nhin, vic b sung thm b u nnh vi t l thp vo mi
trng s cung cp thm ngun Nit cho vi sinh vt.
Nng carbon v cc ngun carbon khc nhau cng c nh hng ln n s
to thnh cc enzyme ring bit ca h enzyme amylase khi nui nm mc. Trong ,
m bo c hot lc -amylase cao cn c t nht 6% tinh bt, cn mun c hot
31
lc oligo-1,6-glucosidase cao ch cn 2% tinh bt. Bt u nnh s c ch vic to

thnh -amylase, nhng li kch thch mnh m oligo-1,6-glucosidase.
T lng gia carbon v nit cng c ngha rt ln i vi s sinh tng hp
enzyme amylase t vi sinh vt. Ch khi no trong mi trng c lng carbon v
nit cn thit mi tch ly c lng enzyme amylase cc ln. V vy, trong mi
trng c t l c cht l 2 cm: 1 tru: 1b u nnh c c t l gia carbon v nit
thch hp nht, v vy, lng enzyme v hot tnh amylase thu c canh trng ny
l cao nht.
Cht lng ca cm go cng c nh hng ln ti hot lc ca cc enzyme
amylase. Cm phi cha tinh bt trn 20%, nn s dng cm mi, cm tt, khng c
d v chua hay ng, khng hi mi mc. m ca cm khong 10%, tp cht c
khng qu 0,05%. Tuy l ph liu ca ngnh cng nghim xay xt nhng cm li c
thnh phn dng cht rt thch hp nui vi sinh vt.
Tuy cm go v b u nnh l ph liu ca cc ngnh cng nghip, nhng c
hai u tng i t tin. Hn na trong qu trnh nui vi sinh vt, ngun dinh dng
khng c s dng ht, v th c th thay mt phn cm v b bng cc cu t r tin
hn, nh tru chng hn. Cu t b xung ny s gp phn lm xp mi trng. C th
dng cn b ca canh trng rn sau khi li trch enzyme lm cu t chnh ca canh
trng nui cy nhng phi m bo ch tit trng.
4.1.3. nh hng ca pH
Trong th nghim ny, chn t l cm go: tru: b u nnh theo t l 2:1:1 v
m 55% kho st nh hng ca pH. Bng cch thm nc ct v mi trng c
nng dinh dng X1 v iu chnh pH ca chng c c mi trng nui cy
c m 55% v c pH 2 , pH 3, pH 4, pH 5, pH 6, pH 7, pH 8. Cc bc tip theo
thc hin nh mc 3.3.6. Cy dch huyn ph bo t A. niger vi mt 0,8*108 bo
t/g vo cc mi trng chun b trn.
32
th 4.2 Biu din hot tnh amylase v hm lng protein t mi trng nui cy
A. niger vi cc pH mi trng ban u khc nhau.
pH c nh hng rt mnh n sinh tng hp amylse ca A. niger. Khong pH
ti u cho loi nm mc ny sinh tng hp amylase l t 4 - 6, trong ti u nht l
ti pH 5. Ti pH ny, A. niger tng hp lng amylase nhiu nht v c hm lng
cao nht (354,333 UI/g CT v 41,961 mg/g). pH nm ngoi khong ti u ny, A.
niger tng hp rt t amylase v hot tnh enzyme cng rt thp.
Khi nui cy bng phng php b mt, pH mi trng t b thay i do mi
trng c dung lng m cao v hm m thp. V vy, pH ban u c ngha rt ln
n s pht trin ca nm mc v s to enzyme. Mi trng c pH trong khong t
4,5 5,0 s to iu kin chn lc cho nm mc pht trin do a s nm mc sinh
trng tt v to enzyme trong mi trng acid yu. V cng pH ny, nm mc to
h amylase tt nht. V th, canh trng nui cy c pH 5, lng enzyme v hot
tnh amylse thu c l cao nht. iu ny l ph hp vi cc nghin cu trc y.
pH cng c nh hng n vic to enzyme ring bit ca h enzyme amylase
ca nm si. Trong mi trng acid, qu trnh to enzyme s chuyn dch mnh v
pha tng hp tch cc glucoamylase v c ch s tng hp -amylase. pH thch hp
cho vic to glucoamylase namwg trong khong t 3 n 3,5, trong khi pH thch hp
cho vic to thnh -amylase nm trong khong pH 5.
33
4.1.4. nh hng ca thi gian nui cy

Trong th nghim ny, chn t l cm go: tru: b u nnh theo t l 2:1:1.
Bng cch thm nc ct v mi trng c nng dinh dng X1 v iu chnh pH
ca chng c c mi trng nui cy c m 55% v c pH 5. Cc bc tip
theo thc hin nh mc 3.3.6. Cy dch huyn ph bo t A. niger vi mt 0,8*108
bo t/g vo cc mi trng chun b trn.
Thu nhn enzyme t canh trng cc mc thi gian khc nhau: sau 8 gi, 12
gi, 16 gi, 20 gi, 24 gi, 28 gi, 32 gi, 36 gi, 40 gi v sau 44 gi nui cy, thu
dch chit enzyme t canh trng, em xc nh hot tnh v hm lng enzyme
amyase thu c. Kt qu phn tch phng sai ANOVA cho thy s khc nhau gia
cc nghim thc l c ngha.
th 4.3 Biu din hot tnh enzyme v hm lng protein t canh trng nui
cy A. niger vi cc mc thi gian thu nhn khc nhau.
Khong thi gian A. niger to enzyme amylase mnh m l sau 24 32 gi
nui cy. Hot tnh v hm lng enzyme cao nht t c sau 28 gi nui cy.
Trong khong thi gian t 8 n trc 24 gi nui cy, lng enzyme tng rt chm.
Hot tnh v hm lng enzyme tng mnh sau 24 n 28 gi nui cy. Sau 28 gi
nui cy, hot tnh enzyme gim nhanh nhng hm lng protein gim chm. Sau 36
gi nui cy, hot tnh ca amylase cn rt thp. C th sau 36 gi nui cy, A. niger
bt u to bo t, nn hm lng protein gim v hot tnh amylase gim nhanh.
34
Khi nui vi sinh vt thu enzyme amylase, c hai qu trnh lin quan mt thit
vi nhau, l qu trnh tng hp sinh khi vi sinh vt v qu trnh tch ly enzyme
trong mi trng. V trong thc t, hai qu trnh ny khng h khp vi nhau v mt
thi gian.
Theo nhiu kin, s to thnh amylase cc i thng xy ra sau khi qun th
t bo t nh sinh trng. Trong qu trnh sinh trng, vi sinh vt ch tng hp nn
mt lng amylase ngoi bo rt t, v ch sau khi kt thc pha sinh trng mi xy ra
s tng hp enzyme amylase cc ln. Ngi ta cho rng s tng hp enzyme amylase
bt u khi m vic to protein ca t bo kt thc hoc gn kt thc v cnh tranh
vi qu trnh ny.
Thi gian nui to lng amylase cc ln thng c xc nh bng thc
nghim. Ty thuc tnh cht sinh sinh l ca tng vi sinh vt v s ngng tng hp
enzyme m c th ngng sinh trng ca nm mc vo bt k lc no thy cn thit.
S to bo t l hin tng khng mong mun v thng lm gim hot lc ca
enzyme. i vi a s nm mc Aspergillus s to enzyme cc i thng kt thc
khi nm mc bt u sinh nh bo t.
4.1.5. nh hng ca nhit
m 55%. Bng cch thm nc ct v mi trng c nng dinh dng X1 v
iu chnh pH ca chng c c mi trng nui cy c m 55% v c pH 5.
Cc bc tip theo thc hin nh mc 3.3.6. Cy dch huyn ph bo t A. niger vi
mt 0,8*108 bo t/g vo cc mi trng chun b trn.
em nui cy trong cc t sy vi cc nhit nh sau: 30 0C, 350C, 400C,
450C, 500C v 550C. Thu dch chit enzyme t canh trng sau 28 gi nui cy, em
xc nh hot tnh v hm lng enzyme amyase thu c. Kt qu phn tch phng
sai ANOVA cho thy s khc nhau gia cc nghim thc l c ngha.
35
A. niger vi cc nhit nui cy khc nhau.
mi trng nui cy trong nhit 350C cho kt qu hm lng protein v
hot tnh amylase cao nht (631,443 UI/g CT v 55,386 mg/g), v nhit nui cy
300C canh trng cho hm lng protein v hot tnh amylase cng kh cao (580,875
UI/g CT v 52,449 mg/g). Vy nhit ti u cho s tng hp amylase A. niger l
t 30 350C. Khong nhit ny thch ng vi s tng trng v pht trin ca nm
mc do , nm mc tng hp enzyme tt nht v t gi tr cc i. Cn nhit t
40 550C do nm mc khng thch nghi c nn kh nng pht trin km dn n
kh nng tng hp enzyme thp. C th do nhit trn 400C l qu cao v chng nh
hng n qu trnh sinh trng v pht trin ca nm mc A. niger.
Ton b chu k sinh trng ca nm mc trn cm c th chia thnh 3 thi k.
u tin l thi k trng mm ca nh bo t. Trong thi k ny phi duy tr nhit
trong khong 30 - 320C. Tip n l thi k sinh trng nhanh ca h si. giai on
ny, nm mc h hp rt mnh v sinh ra mt lng nhit rt ln. Kt qu l trong lp
si nm ang mc nhit tng ln n 37 - 400C. V vy trong lc ny cn ch
gim nhit phng gip si nm mc u v p. Cui cng l thi k to amylase
mnh m. Trong thi k ny cc qu trnh trao i cht dn dn yu i, s ta nhit
gim mnh, cc enzyme amylase c tng hp mnh m. Trong giai on ny, nn
h nhit thp hn nhit ca giai on u t 3 - 40C. Nhn chung, nhit thch
hp nm si sinh trng v tng hp amylase trn mi trng rn nm trong
khong t 28 350C.
36
4.1.6. nh hng ca m ban u

m va nh hng n sinh trng va nh hng n trao i cht ca vi
sinh vt trong qu trnh ln men bn rn. Trong th nghim ny, tip tc chn t l cm
go: tru: b u nnh theo t l 2:1:1 kho st nh hng ca m. Bng cch
thm nc ct v mi trng c nng dinh dng X1 v pH 5 iu chnh m
cc mi trng n 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% v cc bc tip
theo thc hin nh mc 3.3.6. Cy dch huyn ph bo t A. niger vi mt 0,8*108
tnh v hm lng enzyme amyase thu c t canh trng nui cy. Kt qu phn
tch phng sai ANOVA cho thy s khc nhau gia cc nghim thc l c ngha.
A. niger vi cc m mi trng ban u khc nhau.
m mi trng thch hp cho s sinh trng v pht trin ca A. niger l
55%. mi trng c m ny hm lng protein v hot lc enzyme amylase thu
c l cao nht (583,331 UI/g CT v 55,375 mg/g). Trong khi mi trng c m
t 40% n 50% c hot lc amylase v hm lng protein thp hn nhiu. iu ny
c th do tng m thch hp c tc dng lm c cht phng ln, tng xp to
iu kin cho nm d dng tip xc vi c cht hn. Cn cc mi trng cn li,
m cng cao th c cht cng t nn kt dnh vo nhau, mi trng khng c xp
nn nh hng n kh nng tng hp enzyme ca nm mc. Hin tng ny c th
37
do c cht qu m, dn n xp ca mi trng gim, ngn cn s khuch tn O2 t

bn ngoi vo mi trng (theo Pandey, 1992).
Trong iu kin sn xut, m ban u ti thch ca mi trng ln men bn
rn cho nm si sinh trng v tng hp enzyme amylase l 55 60% v phi gi cho
vic n nh ca m trong sut qu trnh nui. m tng qu 70% s lm gim
thong kh, cn thp hn 50% s km hm s sinh trng v tng hp enzyme ca vi
sinh vt. Cn nu nui trong iu kin mi trng khng c v trng tuyt i cn
lu m mi trng sau khi cy ging khng c vt qu 60%, v cao hn na
s rt d b nhim cc vi sinh vt khc.
4.1.7. nh hng ca nng dinh dng
C cht m c kh nng cung cp tt c cc cht dinh dng cho vi sinh vt l
c cht l tng. Tuy nhin, nhiu cht dinh dng c th di mc ti u thm ch
khng c trong c cht, v vy cn b sung thm cc cht dinh dng t bn ngoi khi
pha ch mi trng ln men. Trong phn ny chn t l cm go: tru: b u nnh
theo t l 2:1:1 v m 55%, pH 5 v cc nng dinh dng X1, X2, X3, X4, X5,
X6, X7, X8. Cc bc tip theo thc hin nh mc 3.3.6. Cy dch huyn ph bo t
A. niger vi mt 0,8*108 bo t/g vo cc mi trng chun b trn.
A. niger vi cc nng dinh dng trong mi trng khc nhau.
38
Hot lc amylase cao nht thu c nng mi trng dinh dng X2.
Nhng hm lng protein thu c hi thp hn so vi mi trng c nng dinh
dng X1, nn mi trng X2, hot tnh ring ca amylase l cao nht so vi cc
mc mi trng cn li. iu ny c l l do A. niger khng chu c p sut thm
thu cao t mi trng nui cy nn nhng mi trng c nng dinh dng qu
s cao nh hng n kh nng sinh trng v pht trin ca nm A.niger, v th m
nh hng n kh nng to enzyme ca chng.
Yu cu c bn i vi thnh phn mi trng dinh dng nui nm si to
amylase cng ging nh yu cu i vi mi trng nui vi sinh vt to enzyme l
tnh hon thin. V vy, tng cng s sinh trng ca vi sinh vt cng nh tng
kh nng to enzyme mong mun, ngoi cc thnh phn dinh dng c trong c cht,
mi trng nui cy s c b sung thm cc cht khong vi lng v a lng vn
l nhng cu t khng th thiu cho sinh trng v pht trin ca vi sinh vt.
Nng khong b sung vo mi trng phi va , v nu thp s khng
cho nhu cu ca vi sinh vt, cn nu cao, vi sinh vt s sinh trng km do khng chu
c p sut thm thu.
4.1.8. nh hng ca t l ging
Mt ging i khi cng c nh hng ng k n s sinh trng ca vi sinh
vt trong ln men bn rn. Trong th nghim ny, chn t l cm go: tru : b u
nnh theo t l 2:1:1 v m 55% kho st nh hng ca pH. Bng cch thm
nc ct v mi trng c nng dinh dng X2 v iu chnh pH ca chng c
c mi trng nui cy c m 55% v c pH 5. Cc bc tip theo thc hin nh
mc 3.3.6. Cy dch huyn ph bo t A. niger vi cc mt 0,8*108; 1,6*108;
2,4*108; 3,2*108 bo t/g vo cc mi trng chun b trn.
Thu nhn enzyme t canh trng sau 28 gi nui cy, thu dch chit enzyme t
canh trng, em xc nh hot tnh v hm lng enzyme amyase thu c. Kt qu
phn tch phng sai ANOVA cho thy s khc nhau gia cc nghim thc l c
ngha v mt thng k hc.
39
A. niger vi cc t l ging khc nhau.
mi trng c t l ging l 0,8*10 8 c hot tnh v hm lng l cao nht
(619,68 IU/g CT v 51,833 mg/g). Mt bo t phi l 0,8*108 m bo cc tiu
phn c cht phi c bao ph bi h si nm nhm hn ch s tp nhim. Nu t l
ging qu cao dn n s cnh tranh v dinh dng gia cc c th nm dn n gim
s sinh trng v pht trin, nn hm lng protein v hot tnh amylase s gim theo.
Khi vi sinh vt c nui trong mi trng kn, khng c cung cp mi
trng dinh dng mi trong qu trnh , th s lng cht dinh dng s gim v
lng cht thi trong mi trng s tng ln. V vy, cng mt lng c cht ban u
nh nhau v thu hoch sau mt thi gian nui cy nh nhau, nu cc nng ging
cho vo l khc nhau s cho cc kt qu thu nhn enzyme khc nhau do kh nng sinh
trng ca qun th vi sinh vt cho vo.
Nu lng vi sinh vt cho vo thp, th thi gian thu hoch vi sinh vt cha tch
ly lng amylase trong canh trng do thi gian sinh trng t n phase cp
s qu di. Cn nu lng vi sinh vt cho vo l qu nhiu, nng dinh dng trong
qun th l khng , v vy chng s nhanh chng chuyn sang phase t vong do
thiu dinh dng v tch t c t trong mi trng.
40
V vy cn tnh ton lng vi sinh vt cho vo ph hp vi mi trng nui cy

m bo thu c lng enzyme cao nht khng ri vo trng hp tha cht
dinh dng hoc tha ging.
S sinh trng ca vi sinh vt tri qua 4 giai on. u tin l phase tim phc.
Khi vi sinh vt c a vo mt mi trng nui cy mi s khng c s gia tng
ngay lp tc s lng hay sinh khi t bo. y l lc nhng thnh phn mi ca t
bo c hnh thnh. Sau s chuyn sang phase cp s. Trong phase ny, vi sinh
vt s pht trin v phn chia vi tc cc i. Sau qun th vi sinh vt s bc
vo phase n nh. S tng trng v dn s ca vi sinh vt s dng li. Lc ny qun
th vi sinh vt t mt dn s ti a. Kch thc qun th ph thuc vo cht dinh
dng, cc nhn t khc v ty vo loi vi sinh vt. C nhiu l do khin dn s vi sinh
vt i vo phase n nh, d thy l s gii hn v cht dinh dng v s tch t cht
thi c hi. Cui cng l phase t vong, s lng t bo sng s b gim do mi
trng thiu dng cht v c t tch t. V vy cn phi cho mt lng ging thch
hp vo mi trng chng t mt tt nht ti phase n nh. iu ny s lm
tng kh nng thu enzyme t canh trng nui cy.
4.2. Kh nng sinh tng hp amylase ca nm mc Mucor trn mi trng ln
men bn rn
4.2.1. Nm mc Mucor
Bo t Mucor t ng nghim thch nghing c cy sang a Petri v ming
lame trong phng m cha mi trng PGA theo phng php m t mc 3.3.3,
nui nhit phng 2 - 3 ngy.
(b)
(a)
41
Hnh 4.2 Hnh nh i th ca Mucor. (a)hnh nh i th, (b) hnh nh vi th.

Hnh nh i th cho thy Mucor to khun lc hnh trn, mc nhanh trn mi
trng PGA nhit phng, c dng nhung, c t mn. Khun lc cn non c mu
trng c, khi gi chuyn sang mu trng sa, c t di.
Hnh nh vi th quan st c cho thy Mucor c cung bo t
phn nhnh v mc ln bt k ch no ca si nm, bo t nh nm
trong bc bo t. Bc bo t dng trn hoc hnh qu l, cha nhiu
bo t pha trong.
4.2.2. nh hng ca t l cm go: b u nnh: tru
Tin hnh pha ch cc mi trng c t l c cht cm go: tru: b u nnh
khc nhau, lm m vi nc c b sung dung dch dinh dng vi nng X1 t
m 55%, chnh v pH 5. Cc bc tip theo thc hin nh mc 3.3.6. Cy dch
huyn ph bo t Mucor vi mt 0,8*108 bo t/g vo cc mi trng chun b.
Mucor vi cc t l c cht khc nhau.
Hm lng protein thu c cao nht mi trng c t l cm: tru: b u
nnh l 1:1:2, nhng mi trng ny, hot tnh amylase thu c li l thp nht.
42
iu ny cho thy, vi t l b u nnh trong mi trng cao, nm Mucor s sinh

tng hp loi enzyme khc ph hp vi c cht cm ng l b u nnh. Trong mi
trng c t l cm: tru: b u nnh l 2:1:1, hm lng protein thu c cng
tng i cao, v hot tnh amylase thu c l cao nht (20,651 mg/g v 250,999
UI/g CT). iu ny chng t y l mi trng c t l c cht thch hp nht cho
vic cm ng Mucor sinh tng hp enzyme amylase. D b u nnh c th s kch
thch Mucor to enzyme khng mong mun, nhng vi s lng t trong mi trng
n c tc dng b sung thm ngun Nit cho mi trng nui cy.
Nng carbon v cc ngun carbon khc nhau c nh hng ln n s to
thnh cc enzyme ring bit ca h enzyme amylase khi nui nm mc. Trong ,
m bo c hot lc -amylase cao cn c t nht 6% tinh bt, cn mun c hot lc
oligo-1,6-glucosidase cao ch cn 2% tinh bt. Bt u nnh s c ch vic to thnh
-amylase, nhng li kch thch mnh m oligo-1,6-glucosidase.
Tuy cm go v b u nnh l ph liu ca cc ngnh cng nghip, nhng c
hia u tng i t tin. Hn na trong qu trnh nui vi sinh vt, ngun dinh dng
khng c s dng ht, v th c th thay mt phn cm v b bng cc cu t r tin
hn, nh tru chng hn. Cu t b xung ny s gp phn lm xp mi trng. C th
dng cn b ca canh trng rn sau khi li trch enzyme lm cu t chnh ca canh
trng nui cy nhng phi m bo ch tit trng. Cm v cc cht ph gia cha
nhiu bo t ca cc vi sinh vt khc nn cn thanh trng k bo m chng nui
pht trin bnh thng v canh trng sn xut khng cha vi sinh vt ngoi lai.
T lng gia carbon v nit cng c ngha rt ln i vi s sinh tng hp
enzyme amylase t vi sinh vt. Ch khi no trong mi trng c lng carbon v
nit cn thit mi tch ly c lng enzyme amylase cc ln. v vy, trong mi
trng c t l c cht l 2 cm: 1 tru: 1b u nnh c c t l gia carbon v nit
thch hp nht, v vy, lng enzyme v hot tnh amylase thu c canh trng ny
l cao nht.
4.2.3. nh hng ca pH
Chn t l cm go: tru: b u nnh theo t l 2:1:1, kho st nh hng ca
pH. Thm nc ct, mi trng dinh dng mi trng nui cy c nng dinh
dng X1, v m 55%. iu chnh pH c mi trng nui cy c pH t 2 n 8.
43
Cc bc tip theo thc hin nh mc 3.3.6. Cy dch huyn ph bo t Mucor vi

mt 0,8*108 bo t/g vo cc mi trng chun b trn.
Mucor vi cc pH mi trng khc nhau.
Hot tnh v hm lng thu c t mi trng pH5 l cao nht (23,046 mg/g
v 263,427 UI/g CT). cc mi trng c pH 4 v pH 6, hm lng protein v hot
tnh amylase cng tng i cao hn nhng mi trng cn li. Vy c l pH t 4 n
6 trong mi trng ban u l thch hp cho s sinh trng v pht trin ca Mucor.
Trong mi trng c pH 2, c gi tr hm lng v hot tnh thu c u thp, c th
do Mucor khng sinh trng c trong mi trng c tnh cht acid qua mnh.
mi trng pH 7 v 8, hot tnh amylase thu c l thp nhng hm lng protein
trong canh trng vn tng i cao do lng protein c sn trong mi trng. V t
biu trn ta cng thy c s nh hng ca pH n s ion ha ca c cht v
bn bn trong c cht.
pH c nh hng r rt ln s tng trng ca vi sinh vt. Mi loi ch pht
trin trong mt khong pH xc nh. a s nm mc pht trin trong mi trng hi
acid v chng l nhng vi sinh vt a acid thuc nhm acidophile.
44
Mc d chng sng trong pH acid yu, nhng chng li c pH ni bo gn vi

gi tr trung tnh. y c th l kt qu ca s thm thu ca cc proton qua mng t
bo cht. C th l cc ion OH- v H+ b loi ra bn ngoi duy tr mt pH ni bo
ph hp. Th nhng pH mi trng trong len men bn rn li t b thay i trong sut
qu trnh nui cy do chng c mt h m tt.
Nm mc tuy c kh nng sinh trng trong vng pH tng i rng, tuy nhin
vn c mc gii hn cho s chu ng ca chng. Nhng thay i mnh v pH ca
mi trng ngoi cng c th lm bi i s ion ha cc phn t dng cht v phn
ng kh ca chng s lm cho nm b thiu cht dinh dng.
4.2.4. nh hng ca thi gian nui cy
m 55%. Thm nc ct v mi trng c nng dinh dng X1 v iu chnh
pH ca chng c c mi trng nui cy c m 55% v c pH 5. Cc bc
tip theo thc hin nh mc 3.3.6. Cy dch huyn ph bo t Mucor vi mt
0,8*108 bo t/g vo cc mi trng chun b trn.
Thu nhn enzyme t canh trng cc mc thi gian khc nhau: sau 8 gi, 12
gi, 16 gi, 20 gi, 24 gi, 28 gi, 32 gi, 36 gi, 40 gi v sau 44 gi nui cy, thu
dch chit enzyme t canh trng, em xc nh hot tnh v hm lng enzyme
amyase thu c. Kt qu phn tch phng sai ANOVA cho thy s khc nhau gia
cc nghim thc l c ngha.
45
Mucor vi cc mc thi gian thu nhn khc nhau.
Thi gian thch hp nht thu amylase t mi trng nui cy Mucor l 32
gi sau khi cy (34,084 mg/g v 414,722 UI/g CT). Khong thi gian t sau 28 gi
cy, Mucor bt u sinh tng hp mnh m enzyme amylase. Trc , lng amylase
thu c khng nhiu v hot tnh cng khng cao. Nhng t sau 36 gi nui cy,
hot tnh amylase gim nhanh chng, song hm lng protein li gim chm hn, iu
ly c th do t sau 36 gi nui cy, Mucor chuyn sang dng bo t nn hm lng
amylase khng cn nhiu trong canh trng, dn dn hot tnh ca amylase gim. Vy
thi gian nui cy c ngha rt quan trng trong vic thu nhn amylase t mi trng
nui cy vi sinh vt.
Theo l thuyt hin i th gia vn tc sinh trng ring ca vi sinh vt v tc
sinh tng hp enzyme c mi tng quan ph thuc. Kiu ph thuc th nht l
vn tc sinh trng ca vi sinh vt hon ton ph hp chnh xc vi vn tc sinh tng
hp enzyme. Kiu ph thuc th hai l ngoi s tng hp enzyme trong pha sinh
trng, cn c s tng hp enzyme thm. S to thm ny khng lin quan t l thun
vi s sinh trng ca vi sinh vt. S to thm ny c thc hin bi cc t bo
chuyn sang qu trnh t phn v ph thuc vo bn vng ca cc ribonucleic acid
thng tin. Nh vy l kh nng khng trng khp ca cc giai on sinh trng vi
giai on sinh tng hp enzyme c xc inh bng bn ca ribonucleic acid thng
46
tin (ribonucleic acid khun). V vy m qua trnh tch ly enzyme cc i trong canh
trng ch xy ra sau qu trnh sinh sn ca nm.
Thi gian nui to lng amylase cc ln thng c xc nh bng thc
nghim. Ty thuc tnh cht sinh sinh l ca tng vi sinh vt v s ngng tng hp
enzyme m c th ngng sinh trng ca nm mc vo bt k lc no thy cn thit.
S to bo t l hin tng khng mong mun v t lm gim hot lc ca enzyme.
4.2.5. nh hng ca nhit
m 55%. Thm nc ct v mi trng c nng dinh dng X1 v iu chnh
pH ca chng c c mi trng nui cy c m 55% v c pH 5. Cc bc
tip theo thc hin nh mc 3.3.6. Cy dch huyn ph bo t Mucor vi mt 8*108
em nui cy trong cc t sy vi cc nhit nh sau: 30 0C, 350C, 400C,
450C, 500C v 550C, thu dch chit enzyme t canh trng sau 32 gi, em xc nh
hot tnh v hm lng enzyme amyase thu c. Kt qu phn tch phng sai
ANOVA cho thy s khc nhau gia cc nghim thc l c ngha.
Mucor vi cc nhit nui cy khc nhau.
Nhit ti u cho s sinh tng hp amylase l 300C, nhit ny hm
lng v hot tnh u cao. Chng t t l hm lng amylase trong canh trng khi
Mucor c nui 300C l tt nht. Hm lng v hot tnh amylase gim nhanh
47
chng nhng mi trng nui cy trn 400C, do nhit cao c ch kh nng sinh
trng ca Mucor. n 550C, lng protein cn rt thp, ko theo hot tnh ca
amylase trong mi trng cng rt thp.
Cng nh mi sinh vt sng, nm mc cng chu nh hng su sc ca nhit
mi trng. Chng c bit nhy cm do c cu to n bo v tnh bin nhit. V
th m c s bin thin rt mnh ca hot tnh enzyme khi c s thay i ca nhit
nui cy. Thn nhit ca c th nm phn nh trc tip nhit ca mi trng.
Mt trong nhng nhn t quan trng nht c lin quan n tc ng ca nhit
trn s tng trng, l nhit cm ng ca nhng phn ng thy phn c xc
tc bi enzyme. mc nhit thp, s tng nhit s t l thun vi s tng vn
tc tng trng. Khi hot ng bin dng s c gia tng v vi sinh vt cng s
pht trin nhanh hn. Nhng nu vt qu mt im no (im ti u), s tng
trng ca sinh vt s gim v nu tip tc tng nhit s gy cht vi sinh vt. Nhit
cao s lm cc vi sinh vt b tn thng do enzyme b bin tnh, ng thi cng nh
hng rt ln n cc h thng vn chuyn v cc protein khc. Nu cc enzyme chc
nng hot ng vi vn tc nhanh hn nhit cao vi sinh vt c th b tn thng
v s tng trng s b c ch.
S tng trng ca nm ni ring v ca vi sinh vt ni chung ph thuc c
bit vo nhit . C ba mc ca nhit sinh trng, gm nhit ti thiu, nhit ti
a v nhit ti thch. V d ng cong tng trng theo nhit c th bin i,
nhng nhit ti thch bao gi cng gn vi nhit ti a hn l nhit ti thiu.
Tuy nhin, nhit sinh trng cng cn ph thuc nhiu vo cc yu t khc ca mi
trng nui cy nh pH, cht dinh dng c sn.
4.2.6. nh hng ca m ban u
m va nh hng n sinh trng va nh hng n trao i cht ca vi
sinh vt trong qu trnh ln men bn rn. Trong th nghim ny, chn t l Cm go:
tru: b u nnh theo t l 2:1:1 kho st nh hng ca m. Thm nc ct v
mi trng c nng dinh dng X1 v pH 5 iu chnh m cc mi trng
n 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, cc bc tip theo thc hin nh
mc 3.3.6. Cy dch huyn ph bo t Mucor vi mt 0,8*108 bo t/g vo cc mi
trng chun b trn.
48
Sau 32h nui cy, thu dch chit enzyme t canh trng, em xc nh hot tnh
v hm lng enzyme amyase thu c. Kt qu phn tch phng sai ANOVA cho
thy s khc nhau gia cc nghim thc l c ngha.
Mucor vi cc m mi trng ban u khc nhau.
Hot tnh amylase v hm lng protein cao nht thu c t mi trng c
m ban u l 55%. Sau m gim mnh c th do mi trng qu m nn ngn
cn s khuch tn O2 vo mi trng. Cn trc , hm lng v hot tnh li c xu
hng tng dn v khi tng m trong phm vi thch hp ko theo tc sinh trng
v tiu th O2 tng theo, nh , amylase cng c sinh ra nhiu hn.
Trong iu kin sn xut, m ban u ti thch ca mi trng ln men bn
rn cho nm si sinh trng v tng hp enzyme amylase l 55 60% v phi gi cho
vic n nh ca m trong sut qu trnh nui. m tng qu 70% s lm gim
thong kh, cn thp hn 50% s km hm s sinh trng v tng hp enzyme ca vi
sinh vt. Cn nu nui trong iu kin mi trng khng c v trng tuyt i cn
lu m mi trng sau khi cy ging khng c vt qu 60%, v cao hn na
s rt d b nhim cc vi sinh vt khc.
4.2.7. nh hng ca nng dinh dng
Do nhiu cht dinh dng c th di mc ti u thm ch khng c trong c
cht ang s dng, nn cn b sung thm cc cht dinh dng t bn ngoi khi pha
ch mi trng ln men. Trong phn ny chn t l cm go: tru: b u nnh theo t
49
l 2:1:1 v m 55%, pH 5 v cc nng dinh dng X1, X2 , X3, X4, X5, X6,
X7, X8. Cc bc tip theo thc hin nh mc 3.3.6. Cy dch huyn ph bo t
Mucor vi mt 0,8*108 bo t/g vo cc mi trng chun b trn.
tnh v hm lng enzyme amyase thu c. Kt qu thu nhn th 4.13. Kt qu
ngha v mt thng k hc.
Nng dinh dng X3 l thch hp nht cho s tng hp enzyme amylase.
nng dinh dng ny, hm lng protein v hot tnh amylase thu c u cao
nht (43,391 mg/g v 488,279 UI/g CT). Sau th hm lng v hot tnh u gim
dn, c th l do nng thm thu ca mi trng nh hng ln Mucor.
Mucor vi cc nng dinh dng trong mi trng khc nhau.
Nng dinh dng X3 l thch hp nht cho s tng hp enzyme amylase.
nng dinh dng ny, hm lng protein v hot tnh amylase thu c u cao
nht (43,391 mg v 488,279 UI/g CT). Sau th hm lng v hot tnh u gim
dn, c th l do nng thm thu ca mi trng nh hng ln Mucor.
Yu cu c bn i vi thnh phn mi trng dinh dng nui nm si to
amylase cng ging nh yu cu i vi mi trng nui vi sinh vt to enzyme l
tnh hon thin. V vy, tng cng s sinh trng ca vi sinh vt cng nh tng
50
kh nng to enzyme mong mun, ngoi cc thnh phn dinh dng c trong c cht,
mi trng nui cy s c b sung thm cc cht khong vi lng v a lng vn
l nhng cu t khng th thiu cho sinh trng v pht trin ca vi sinh vt.
Vi sinh vt chu nh hng rt ln nu thay i nng thm thu ca mi
trng sng. iu ny l do n ch c phn cch vi mi trng bng mt mng
bn thm chn lc. Nu cho nng dinh dng vo mi trng qua niu hy qu t
s nh hng ln n p sut thm thu gy hi cho t bo sinh vt. Nu vi sinh vt
sng trong mi trng c nng dinh dng qu cao, nc s t c th sinh vt thot
ra noi mi trng. Khi , t bo v mng t bo s co li y l qu trnh co
nguyn sinh. V nh vy, t bo s b mt nc v mng t bo b tn thng. iu
ny lm t bo b bt hot v mt bin dng v s ngng pht trin.
Nc c sn ca vi sinh vt c th b gim, l do s tng tc vi cc phn t
cht tan hoc s hp thu ln b mt cc cht rn. Nng thm thu ca mi trng
sng c nh hng ln ln vi sinh vt v c dng din t mc nc c sn.
Cc nh vi sinh vt hc thng gi l hot ng ca nc. Cc vi sinh vt khc
nhau ng k v kh nng thch ng vi mi trng ngoi cnh c hot ngca
nc thp. Tuy nhin a s vi sinh vt ch sinh sn tt ti hot ng ca nc
khong 0,98 (xp x gi tr ca nc bin). V vy nu nng dinh dng qu cao s
l tng hot ng ca nc, nh hng n s sinh trng v tng hp enzyme.
4.2.8. nh hng ca t l ging
Mt ging i khi cng c nh hng ng k n s sinh trng ca vi sinh
vt trong ln men bn rn. Vi th nghim ny, chn t l Cm go: tru: b u nnh
theo t l 2:1:1 v m 55%.Thm nc ct v mi trng c nng dinh dng
X3, iu chnh pH ca chng c c mi trng nui cy c m 55% v c pH
5. Cc bc tip theo thc hin nh mc 3.3.6. Cy dch huyn ph bo t Mucor vi
cc mt 0,8*108; 1,6*108; 2,4*108; 3,2*108 bo t/g vo cc mi trng chun b.
Thu nhn enzyme t canh trng sau 32 gi nui cy, thu dch chit enzyme t
canh trng, em xc nh hot tnh v hm lng enzyme amyase thu c. Kt qu
ngha xt v mt thng k hc.
51
Mucor vi cc nng ging cho vo mi trng khc nhau.
mi trng c t l ging l 1,6*10 8 c hot tnh v hm lng l cao nht
(522,643 IU/g CT v 44,991 mg/g). Mt bo t 1,6*108 m bo cc tiu phn c
cht phi c bao ph bi h si nm nhm hn ch s tp nhim, v cng va
khng xy ra s cnh tranh v dinh dng gia cc c th nm nn khng b c ch s
sinh trng v pht trin ca chng.
S sinh trng ca vi sinh vt tri qua 4 giai on. u tin l phase tim phc.
Khi vi sinh vt c a vo mt mi trng nui cy mi s khng c s gia tng
ngay lp tc s lng hay sinh khi t bo. y l lc nhng thnh phn mi ca t
bo c hnh thnh. Sau s chuyn sang phase cp s. Trong phase ny, vi sinh
vt s pht trin v phn chia vi tc cc i. Sau qun th vi sinh vt s bc
vo phase n nh. S tng trng v dn s ca vi sinh vt s dng li. C nhiu l do
khin dn s vi sinh vt i vo phase n nh, d thy l s gii hn v cht dinh
dng v s tch t cht thi c hi. Cui cng l phase t vong, s lng t bo sng
s b gim do mi trng thiu dng cht v c t tch t. Nu lng vi sinh vt cho
vo thp, th thi gian thu hoch vi sinh vt cha tch ly lng amylase trong canh
trng do thi gian sinh trng t n phase cp s qu di. Cn nu lng vi sinh
vt cho vo l qu nhiu, nng dinh dng trong qun th khng , v vy chng
s chuyn sang phase t vong do thiu dinh dng v tch t c t trong mi trng.
52
4.3. Qui hoch thc nghim

4.3.1. Xc nh gi tr ti u ca mt s yu t nh hng n kh nng sinh tng
hp amylase ca A. niger v Mucor
Sau khi thc hin cc th nghim kho st nh hng ca cc yu t nh bn
trn, nhn thy rng cc yu t pH, m ban u, nng dinh dng v thi gian
nui cy c tc ng mnh. V vy ta b tr qui hoch thc nghim nghin cu kh
nng tc ng tng hp ca cc yu t ny.
53
4.3.1.1. Xc nh gi tr ti u ca mt s yu t nh hng n kh nng sinh

tng hp amylase ca A. niger
amylase ca A. niger
Cc bin s
Mc di
Mc c s
Mc trn
X1: pH
X2: Thi gian nui cy (h)
X3: m ban u (%)
X4: nng dinh dng
(-)
pH 4
20
45%
X1
Bng 4.2 Ma trn k hoch ha i vi TY-24

S th
Bin s m ha
nghim
X1
X2
X3
X4
1
2
+
3
+
4
+
+
5
+
6
+
+
7
+
+
8
+
+
+
9
+
10
+
+
11
+
+
12
+
+
+
13
+
+
14
+
+
+
15
+
+
+
16
+
+
+
+
17
0
0
0
0
(0)
pH 5
28
55%
X2
(+)
pH 6
36
65%
X3
K hiu hng (cc yu t v tc

dng tng h ca chng)
"1"
X1
X2
X1X2
X3
X1X3
X2X3
X1X2X3
X4
X1X4
X2X4
X1X2X4
X3X4
X1X3X4
X2X3X4
X1X2X3X4
T bng trn, tin hnh chun b 17 mi trng m trong c cc yu t

kho st trn hai mc. Cy dch huyn ph bo t A. niger vi mt 0,8*108 bo
t/g mi trng. Sau thi gian nui cy theo bng 4.16 tin hnh li trch enzyme
54
Bng 4.3 Hot lc amylase ca A. niger theo thc nghim v phng trnh hi qui
Stt th
Bin s m ha
nghim
X1
X2
X3
X4
Y
Y^
1
435.331
435.9468
2
+
395.221
395.8368
3
+
412.149
411.5328
4
+
+
384.872
384.2556
5
+
212.273
210.69645
6
+
+
210.185
208.60805
7
+
+
262.625
264.20195
8
+
+
+
259.129
260.70595
9
+
261.129
262.70595
10
+
+
356.950
358.52715
11
+
+
377.451
375.87405
12
+
+
+
352.539
350.96245
13
+
+
197.898
197.2816
14
+
+
+
188.092
187.4756
15
+
+
+
189.58
190.1964
16
+
+
+
+
157.949
158.5656
17
0
0
0
0
621.46
18
0
0
0
0
625.669
19
0
0
0
0
623.132
Phng trnh hi qui c dng:
Yî=bo+b1X1+b2X2+b3X3+b4X4+b12X1X2+b13X1X3+b14X1X4+b23X2X3
+b24X2X4+b34X3X4+b123X1X2X3+b124X1X2X4+b134X1X3X4+b234X2X3X4
+b1234X1X2X3X4
Cc h s trong phng trnh hi qui nh sau:
16
xjiyi
i=1
bj =
16
Cc hiu ng tng tc c xc nh nh nhng hiu ng tuyn tnh
55
16
(xj xi)iyi
i=1
bjl =
16
Theo cng thc trn ta tnh c cc h s ca phng trnh hi qui nh sau:
b1 =
-2.719
b24 =
0.480
b2 =
8.701
b34 =
4.301
b3 =
-81.12
b123 =
5.291
b4 =
-30.64
b124 =
-9.624
b12 =
-8.196
b134 =
-10.88
b13 =
-3.159
b234 =
-17.7
b14 =
6.403
b1234 =
7.072
b23 =
-1.096
b0
=
290.84
nh gi v mt thng k hc ngha ca cc h s hi qui theo tiu chun Student.
-
Gi tr trung bnh ca ba th nghim ti tm:

y1o + y2o + y3o
yo =
-
623.419
n1
Phng sai ti hin:
Sth = 2.12111268
-
Phng sai cc h s:
Sth
Sbj =
= 0.53027817 (vi N = 16)
nh gi mc ngha theo tiu chun Student:

bj
tbj =
> tlt bbj c ngha
Sbj
Vi mc tlt : mc p = 0,05, bc t do f = n-1 = 2)
tlt = t(0,05;2) = 4,3 (tra bng student)
56
Ta tnh c cc mc ngha nh sau:

t1
t2
t3
t4
t12
t13
t14
t23
=
=
=
=
=
=
=
=
5.127
16.408
152.975
57.776
15.456
5.957
12.074
2.067
> 4,3
> 4,3
> 4,3
> 4,3
> 4,3
> 4,3
> 4,3
<4,3
t24 =
t34 =
t123 =
t124 =
t134 =
t234 =
t1234 =
t0 =
0.906
8.110
9.979
18.149
20.526
33.378
13.336
548.459
<4,3
> 4,3
> 4,3
> 4,3
> 4,3
> 4,3
> 4,3
> 4,3
V t23 v t24 u nh hn 4,3 (t(0,05;2)) nn b23 v b24 b loi ra khi phng trnh
hi qui do khng c ngha.
Vy phng trnh hi qui nh sau:
Yî=290.84-2.719X1+8.701X2-81.12X3-30.64X4
-8.196X1X2-3.159X1X3+7.403X1X4+4.3015X3X4
+5.291X1X2X3-9.624X1X2X4-10.88X1X3X4
-17.7X2X3X4+7.072X1X2X3X4
(4.1)
nh gi mc tng thch ca phng trnh theo tiu chun Fisher.

-
Tnh cc gi tr y^: kt qu trnh by bng 4.17.

Phng sai tng thch:
16
(yi - yi^)2
i=1
S2tt =
= 9.4309
N-l
vi N : s th nghim
= 16
l: s h s c ngha = 14
S2tt
Ta c:
Ftn =
= 2.09617504
2
S th
Gi tr chun Fisher mc p = 0.05; f1 = N-l; f2 = n-1 l:
F lt (0,05;2;2 )= 19,0
=> Ftn < F lt
Vy phng trnh hi qui 4.1 tng thch vi thc nghim.
57
Trong bn yu t tc ng n kh nng sinh tng hp amylase ca A. niger,

yu t m ban u c tc ng ln nht (b3 = -81,12), tip theo l nng dinh
dng (b4 = -30,64), thi gian (b2 = 8,701), v cui cng l pH (b1 = -2,719).
Xt v s tng tc cc yu t, kt qu cho thy c 4 yu t u c s tng
tc vi nhau. Trong , s tng tc gia yu t thi gian, m ban u v nng
dinh dng l c tng tc ln nht (b234 = 14,539).
4.3.1.2. Xc nh gi tr ti u ca mt s yu t nh hng n kh nng sinh
tng hp amylase ca Mucor
amylase ca Mucor
Cc bin s
X1: pH
X2: Thi gian nui cy (h)
X3: m ban u (%)
X4: nng dinh dng
Mc di
(-)
pH 4
24
45%
X1
Mc c s
(0)
pH 5
32
55%
X3
Mc trn
(+)
pH 6
40
65%
X5
Bng 4.5 Ma trn k hoch ha i vi TY-24

S th
nghim
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
X1
+
+
+
+
+
+
+
+
0
Bin s m ha
X2
X3
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
0
X4
+
+
+
+
+
+
+
+
0
58
K hiu hng (cc yu t v tc

dng tng h ca chng)
"1"
X1
X2
X1 X2
X3
X1 X3
X2 X3
X1X2X3
X4
X1 X4
X2 X4
X1X2X4
X3 X4
X1X3X4
X2X3X4
X1X2X3X4
T bng trn, tin hnh chun b 17 mi trng m trong c cc yu t

kho st trn hai mc. Cy dch huyn ph bo t Mucor vi mt 1,6*108 bo
t/g mi trng. Sau thi gian nui cy theo bng 4.5 tin hnh li trch enzyme
amylase t canh trng.
Bng 4.6 Hot lc amylase ca Mucor theo thc nghim v theo phng trnh hi qui
Stt th
Bin s m ha
nghim
X1
X2
X3
1
2
+
3
+
4
+
+
5
+
6
+
+
7
+
+
8
+
+
+
9
10
+
11
+
12
+
+
13
+
14
+
+
15
+
+
16
+
+
+
17
0
0
0
18
0
0
0
19
0
0
0
Phng trnh hi qui c dng:
X4
+
+
+
+
+
+
+
+
0
0
0
Y
384.107
295.175
193.030
172.615
218.142
161.876
179.793
100.464
255.177
177.848
149.388
145.4104
158.206
147.338
110.523
75.544
480.137
485.279
479.215
Y^
384.1358
295.147
191.8501
173.7952
218.5874
161.4314
180.5004
99.75772
253.9973
179.0282
149.4171
145.3819
158.9126
146.6313
110.9676
75.09956
Yî=bo+b1X1+b2X2+b3X3+b4X4+b12X1X2+b13X1X3+b14X1X4+b23X2X3
+b24X2X4+b34X3X4+b123X1X2X3+b124X1X2X4+b134X1X3X4+b234X2X3X4
+b1234X1X2X3X4
Cc h s trong phng trnh hi qui nh sau:
16
xjiyi
i=1
bj =
16
59
Cc hiu ng tng tc c xc nh nh nhng hiu ng tuyn tnh

16
(xj xi)iyi
i=1
bjl =
16
Theo cng thc trn ta tnh c cc h s ca phng trnh hi qui nh sau:
b1 = -23.256
b24 = 9.731
b2 = -41.944
b34 = 9.277
b3 = -38.804
b123 =
-11.815
b4 = -30.361
b124 =
0.237
b12 = 5.9184
b134 =
3.857
b13 =
0.576
b234 = -12.195
b14 = 7.362
b1234 =
-0.367
b23 = 14.539
b0
= 182.79
nh gi v mt thng k hc ngha ca cc h s hi qui theo tiu chun Student.
-
Gi tr trung bnh ca ba th nghim ti tm:

y1o + y2o + y3o
yo =
-
481.54
n1
Phng sai ti hin:
3
(yoi - yo)2
i=1
S2th =
= 10.677 (vi n = 3)
n-1
Sth = 3.2676
Phng sai cc h s:
Sth
Sbj =
= 0.8169 (vi N = 16)
nh gi mc ngha theo tiu chun Student:

bj
tbj =
> tlt bbj c ngha
Sbj
Vi mc tlt : mc p = 0,05, bc t do f = n-1 = 2
tlt = t(0,05;2) = 4,3 (tra bng student)
Ta tnh c cc mc ngha nh sau:
60
t1 = 28.468
t2 = 51.345
t3 = 47.502
t4 = 37.166
t12 = 7.245
t13 = 0.705
t14 = 9.012
t23 = 17.798
V t13, t124 v t1234 u
> 4,3
t24 = 11.912
> 4,3
t34 = 11.357
> 4,3
t123 = 14.463
> 4,3
t124 = 0.289
> 4,3
t134 = 4.721
< 4,3
t234 = 14.929
> 4,3
t1234 =
0.449
>4,3
t0 = 223.762
nh hn 4,3 (t(0,05;2)) nn b13, b124 v
>4,3
> 4,3
> 4,3
< 4,3
> 4,3
> 4,3
< 4,3
> 4,3
b1234 b loi ra khi
phng trnh hi qui do khng c ngha.

Vy phng trnh hi qui nh sau:
Yî=182.79-23.256X1-41.944X2-38.804X3-30.361X4+5.918X1X2
+7.362X1X4+14.539X2X3+9.731X2X4+9.277X3X4
-11.815X1X2X3+3.857X1X3X4 -12.195X2X3X4
(4.2)
nh gi mc tng thch ca phng trnh theo tiu chun Fisher.

- Tnh cc gi tr y^: kt qu trnh by bng 4.20.
-
Phng sai tng thch:

16
(yi - yi^)2
i=1
S2tt =
= 2.8568
N-l
vi N : s th nghim = 1, l: s h s c ngha = 13.
S2tt
Ta c:
Ftn =
= 0.2676
2
th
S
Gi tr chun Fisher mc p = 0.05; f1 = N-l; f2 = n-1 l:
F lt (0,05;3;2 )= 19,2
=> Ftn < F lt
Vy phng trnh hi qui 4.2 tng thch vi thc nghim.
61
Trong bn yu t tc ng n kh nng sinh tng hp amylase ca

Mucor, yu t thi gian c tc ng ln nht (b2 = -41,944), tip theo l m
ban u (b3 = -38, 804), nng dinh dng (b4 = -30,361), v cui cng l pH
(b1 = -23,256).
Xt v s tng tc cc yu t, kt qu cho thy c 4 yu t u c s
tng tc vi nhau. Trong , s tng tc gia yu t thi gian v m ban
u l c tng tc ln nht (b23 = 14,539).
62
Chng 5
KT LUN V NGH
5.1. Kt lun
Gi tr cc yu t nh hng tng i thch hp A. niger sinh tng hp
enzyme amylase l t l C:T:B (2:1:1), pH 5, thi gian nui cy 28 gi, nhit nui
cy 300C, m ban u 55%, 2 ln nng dinh dng, t l ging 0,8*108 bt/g CT.
Trong cc yu t trn cc yu t c tc ng mnh n hm lng protein v hot tnh
enzyme amylase l pH, thi gian nui cy, m v nng dinh dng. Trong
yu t m l c tc ng mnh nht.
Gi tr cc yu t nh hng tng i thch hp Mucor sinh tng hp
enzyme amylase l t l C:T:B (2:1:1), pH 5, thi gian nui cy 32 gi, nhit nui
cy 350C, m ban u 55%, 3 ln nng dinh dng, t l ging 1,6*108 bt/g CT.
Trong cc yu t trn cc yu t c tc ng mnh n hm lng protein v hot tnh
enzyme amylase l pH, thi gian nui cy, m v nng dinh dng. Trong
yu t thi gian l c tc ng mnh nht.
A. niger sinh tng hp enzyme amylase mnh hn chng Mucor, hm lng
protein v hot tnh enzyme amylas do A. niger to ra u cao hn do Mucor to ra.
Hn na, nm mc Mucor c sc sinh trng km hn A. niger v rt d b xm
nhim bi cc loi vi sinh vt khc.
5.2. ngh
- Thc hin k hoch leo dc tm ra mi trng thch hp nht cho hai chng
nm mc nghin cu to enzyme amylase mnh m nht.
- Th nghim c tnh canh trng nui cy Asp. niger v Mucor trn chut
bch.
- Th nghim lm ch phm enzyme dng trong thc n chn nui.
- Thc hin tinh sch enzyme amylase t cc canh trng nui cy trn thu
nhn enzyme tinh khit hn.
- Th nghim c nh ch phm enzyme amylase t canh trng nui cy ti u
trn c cht l Chitosan v Natrialginate.
63
TI LIU THAM KHO

Trong nc
1. Nguyn Cnh. 1993. Qui hoch thc nghim. Trng i hc Bch Khoa tp H
Ch Minh.
2. Nguyn Ln Dng v cc tc gi. 1979. Mt s phng php nghin cu vi sinh
vt, tp 2. NXB Khoa hc v k thut H Ni.
3. Phm Th nh Hng.2004. K thut sinh ho. NXB i Hc Quc Gia
Tp.HCM.
4. Nguyn c Lng v cc tc gi.2004. Cng ngh enzyme. NXB i hc
Quc gia tp.HCM.

5. Lng c Phm.2004. Cng ngh vi sinh vt. NXB Nng nghip H Ni.
6. Trn Thnh Phong.2004. Kho st kh nng sinh tng hp enzyme cellulase t
Trichoderma reesei v Asperillus niger trn mi trng ln men bn rn. Lun vn

thc s khoa hc sinh hc, Trng i Hc Khoa Hc T Nhin Tp.HCM.
7. Hong Trng. 2002. X l d liu nghin cu vi SPSS for windows. NXB
Thng k, H Ni.
8. Trn Anh Tun. 2005. Kha st cc yu t nh hng n s sinh tng hp
enzyme lipase t Aspergillus niger trn mi trng ln men bn rn. Kha lun tt
nghip khoa sinh hc, Trng i Hc Khoa Hc T Nhin Tp.HCM
Nc ngoi
9. Amano Pharmaceutical Co.,Ltc. (APC), (2000), Assay method for amylase
activity. Amano Enzyme inc.

10. Helmut Uhlig, PH.D., Industial enzymes and their applications, Franslate and
updated by ELFRIEDE M. LINSMAIER-BED. NAR, PH.D,pp.180.

Trang web
11. http://www.bannhanong.com
12. http://www.wikipedia.com
64
PH LC
Hnh 1 ng chun protein.
Hnh 2 Asp. niger.
Hnh 3 Mucor.
Hnh 4 Mi trng ln men bn rn.
K hoch leo dc
Ti u ha thc nghim bng phng php leo dc trn mt mc tiu l giai
on th hai ca qu trnh ti u ha thc nghim. Do nghin cu i t m hnh tuyn
tnh m t cc b mt mc tiu khi tin dn n min cc tr th m t tng thch ny
s khng cn ph hp nn phi tin hnh xc nh im ti u bng phng php leo
dc. Mc ch ca k hoch leo dc l tm ra ta ca cc tr ca mt mc tiu bng
thc nghim.
Ni dung: k hoch leo dc mang ni dung ca phng php lp bao gm
nhng bc c bn sau:
1. Chn im xut pht: thng mi trng ti tm th nghim s c chn
lm im xut pht.
2. Chn hng di chuyn ca gradient: t im c s xc nh mc cc yu t
v tin hnh xy dng k hoch thc nghim trc giao cp 1.
3. Di chuyn theo hng gradient (cn tm khong cch di chuyn thch hp)
4. Tm iu kin dng (trong kha lun ny, iu kin dng th nghim leo
dc l khi tm thy gi tr yn <y n-1)

Nh vy mi ln di chuyn theo hng dc ng trn mt mc tiu m cha
tm c ti u cn phi tin hnh lp i lp li cc bc trn n khi tm c
ti u.
1. nh hng t l c cht n kh nng sinh tng hp enzyme amylase ca Asp.

niger
1.1. nh hng n hm lng protein
Bng 1.1 Bng s liu hm lng (mg/g)
Nghim thc
C cht
2C:1T:1B
1C:2T:1B
1C:1T:2B
1
40.417
36.075
Ln lp li
2
41.481
36.863
Trung bnh
3
42.494
37.974
41.484
36.971
40.372
41.17
42.271
41.271
Bng 1.2 Bng ANOVA cho hm lng da trn s liu bng 1.1
ANOVA Table for ham luong by ty le co chat
Source
P-Value
Between groups
Within groups
Total (Corr.)
Analysis of Variance
Sum of Squares Df
Mean Square
38.9025
5.6384
44.5409
8
2
6
19.4513
20.70
0.939734
F-Ratio
0.0020
The StatAdvisor
The ANOVA table decomposes the variance of ham luong into two components: a betweengroup component and a within-group component. The F-ratio, which in this case equals
20.6987, is a ratio of the between-group estimate to the within-group estimate. Since the Pvalue of the F-test is less than 0.05, there is a statistically significant difference between the
mean ham luong from one level of ty le co chat to another at the 95.0% confidence level.
1.2. nh hng n hot tnh amylase

Bng 1.3 Bng s liu hot tnh (UI/gCT)
Nghim thc
C cht
2C:1T:1B
1C:2T:1B
1C:1T:2B
1
357.45
307.27
199.48
Ln lp li
2
358.44
310.26
200.47
Trung bnh
3
359.76
308.59
201.8
358.552
308.373
200.584
Bng 1.4 Bng ANOVA cho hot tnh da trn s liu bng 1.3
ANOVA Table for hoat tinh by ty le co chat
Source
Value
Between groups
0.0000
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares Df
Mean Square
39090.3
19545.1
8.07446
39098.4
8
1.34574
F-Ratio
P-
14523.67
The ANOVA table decomposes the variance of hoat tinh into two components: a betweengroup component and a within-group component. The F-ratio, which in this case equals
mean hoat tinh from one level of ty le co chat to another at the 95.0% confidence level.
2. nh hng ca pH mi trng n kh nng sinh tng hp enzyme amylase

ca Asp. niger
Nghim thc
pH
2
3
4
5
6
7
8
1
19.934
22.709
29.705
40.861
27.531
24.465
22.224
Ln lp li
2
20.723
23.812
32.650
41.849
28.519
25.453
23.212
Trung bnh
3
21.811
24.621
30.404
43.173
29.843
26.777
24.536
20.822
23.714
30.586
41.961
28.631
25.565
23.324
ANOVA Table for ham luong by pH
Source
P-Value
Between groups
0.0000
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares Df
898.19
15.8364
914.026 20
6
14
Mean Square
149.698
F-Ratio
132.34
1.13117
mean ham luong from one level of pH to another at the 95.0% confidence level.

Nghim thc
pH
2
3
4
5
6
7
8
1
135.36
178.22
250.59
353.23
241.02
176.77
126.22
Ln lp li
2
136.35
179.2
251.57
354.22
242.01
177.76
127.21
Trung bnh
3
137.68
180.53
252.9
355.55
243.33
179.08
128.53
136.463
179.315
251.686
354.333
242.12
177.869
127.321
ANOVA Table for hoat tinh by pH
Source
Sum of Squares
Df
Mean Square
F-Ratio
P-Value
Between groups
113456.0
18909.4
18.8404
14
1.34574
14051.26
0.0000
Within groups
Total (Corr.)
113475.0
20
The StatAdvisor
mean hoat tinh from one level of pH to another at the 95.0% confidence level.
3. nh hng thi gian nui cy n kh nng sinh tng hp enzyme amylase ca

Asp. niger
Nghim thc
Ln lp li
Trung bnh
Thi gian (gi)

8
12
16
20
24
28
32
36
40
44
1
18.546
21.721
28.802
39.513
43.907
51.424
41.294
39.553
35.046
30.375
2
19.534
22.709
29.79
40.501
44.895
52.412
42.282
40.541
36.034
31.363
3
20.858
24.033
31.114
41.825
46.219
53.736
43.606
41.865
37.358
32.687
19.646
22.821
29.902
40.613
45.007
52.524
42.394
40.653
36.146
31.475
ANOVA Table for ham luong by thoi gian nuoi cay
Source
P-Value
Between groups
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares Df
Mean Square
2809.99
26.9149 20
2836.91
29
9
312.221
1.34574
232.01
F-Ratio
0.0000
mean ham luong from one level of thoi gian nuoi cay to another at the 95.0% confidence
level.

Nghim thc
Thi gian nui cy (h)
8
12
16
20
24
28
32
36
40
44
1
109.83
171.14
255.84
309.32
383.74
581.16
378.88
353.51
225.14
101.73
Ln lp li
2
110.82
172.13
256.82
310.31
384.73
582.15
379.87
354.5
226.13
102.72
Trung bnh
3
112.14
173.46
258.15
311.63
386.05
583.47
381.2
355.83
227.45
104.04
110.929
172.244
256.935
310.421
384.841
582.262
379.984
354.613
226.242
102.832
ANOVA Table for hoat tinh by thoi gian nuoi cay
Source
P-Value
Between groups
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares
Df
Mean Square
579567.0
9
26.9149
579594.0
29
64396.3 47851.86
20
1.34574
F-Ratio
0.0000
mean hoat tinh from one level of thoi gian nuoi cay to another at the 95.0% confidence level.
4. nh hng nhit nui cy n kh nng sinh tng hp enzyme amylase ca

Asp. niger
Nghim thc
nhit nui cy (0C)
30
35
40
45
50
55
1
51.349
54.286
43.549
36.336
23.671
15.572
Ln lp li
2
52.337
55.274
44.537
37.324
24.659
16.56
Trung bnh
3
53.661
56.598
45.861
38.648
25.983
17.884
52.449
55.386
44.649
37.436
24.771
16.67166
ANOVA Table for ham luong by nhiet do nuoi cay
Source
P-Value
Between groups
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares Df
Mean Square
3550.78
16.1489
3566.93 17
5
12
710.156
527.71
1.34574
F-Ratio
0.0000
mean ham luong from one level of nhiet do nuoi cay to another at the 95.0% confidence level.
4.2 nh hng n hot tnh amylase

Nghim thc
nhit nui cy (0C)
30
35
40
45
50
55
1
579.78
630.34
408.69
267.17
176.61
89.468
Ln lp li
2
580.76
631.33
409.68
268.16
177.6
90.456
Trung bnh
3
582.09
632.66
411
269.49
178.92
91.78
580.876
631.443
409.792
268.273
177.709
90.568
ANOVA Table for hoat tinh by nhiet do nuoi cay

Source
P-Value
Between groups
0.0000
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares
Df
717552.0
16.1489
717568.0
12
17
Mean Square
143510.0
F-Ratio
06640.15
1.34574
mean hoat tinh from one level of nhiet do nuoi cay to another at the 95.0% confidence level.
5. nh hng ca m n kh nng sinh tng hp enzyme amylase ca Asp.

niger
Nghim thc
m (%)
40
45
50
55
60
65
70
75
1
36.072
42.975
49.776
54.275
51.336
47.425
41.268
36.259
Ln lp li
2
37.06
43.963
50.764
55.263
52.324
48.413
42.256
37.247
Trung bnh
3
38.384
45.287
52.088
56.587
53.648
49.737
43.58
38.571
37.172
44.0752
50.876
55.375
52.436
48.525
42.368
37.3589
ANOVA Table for ham luong by do am
Source
P-Value
Between groups
Within groups
Total (Corr.)
Sum of Squares Df
Mean Square
986.874
21.5319
1008.41 23
7
16
140.982
104.76
1.34574
F-Ratio
0.0000
The StatAdvisor
mean ham luong from one level of do am to another at the 95.0% confidence level.

Nghim thc
m (%)
40
45
50
55
60
65
70
75
1
198.92
331.62
408.26
582.23
266.66
168.71
155.25
142.26
Ln lp li
2
199.91
332.6
409.24
583.22
267.65
169.7
156.24
143.25
Trung bnh
3
201.23
333.93
410.57
584.54
268.97
171.02
157.56
144.57
200.021
332.716
409.356
583.331
267.758
169.812
156.351
143.358
Source
Value
Between groups
0.0000
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares
Df
Mean Square
492316.0
21.5319
492337.0
16
23
70330.8
F-Ratio
P-
52261.66
1.34574
mean hoat tinh from one level of do am to another at the 95.0% confidence level.
6. nh hng ca nng dinh dng n kh nng sinh tng hp enzyme

amylase ca Asp. niger
Nghim thc
Nng dinh dng
X1
X2
X3
X4
X5
X6
X7
X8
Ln lp li
2
54.515
53.301
49.836
48.953
45.067
40.855
40.569
37.992
1
53.527
52.313
48.848
47.965
44.079
39.867
39.581
37.004
Trung bnh
3
55.839
54.625
51.16
50.277
46.391
42.179
41.893
39.316
54.627
53.413
49.948
49.065
45.179
40.967
40.681
38.104
Analysis of Variance for ham luong - Type III Sums of Squares
Source
Sum of Squares
Df
Mean Square
F-Ratio
P-Value
MAIN EFFECTS
A:nong do dinh duong
807.055
115.294
RESIDUAL
21.5319
16
TOTAL (CORRECTED)
828.587
23
85.67
0.0000
1.34574
All F-ratios are based on the residual mean square error.

The StatAdviso
The ANOVA table decomposes the variability of ham luong into contributions due to
various factors.
Since Type III sums of squares (the default) have been chosen, the
contribution of each factor is measured having removed the effects of all other factors. The Pvalues test the statistical significance of each of the factors. Since one P-value is less than
0.05, this factor has a statistically significant effect on ham luong at the 95.0% confidence
level.
6.2. nh hng n hot tnh amylase :

Nghim thc
Nng dinh dng
X1
X2
X3
X4
X5
X6
X7
X8
Ln lp li
2
576.71
624.86
547.18
461.53
416.78
378.52
373.77
338.01
1
575.72
623.87
546.19
460.54
415.79
377.53
372.79
337.02
Trung bnh
3
578.03
626.18
548.51
462.85
418.1
379.84
375.1
339.34
576.821
624.969
547.294
461.642
416.887
378.631
373.885
338.123
ANOVA Table for hoat tinh by nong do dinh duong
Source
Sum of Squares
Df
Mean Square
F-Ratio
P-Value
Between groups
237154.0
33879.2
Within groups
21.5319
16
1.34574
Total (Corr.)
237176.0
23
25175.05
0.0000
The StatAdvisor
mean hoat tinh from one level of nong do dinh duong to another at the 95.0% confidence
level.
7. nh hng ca t l ging n kh nng sinh tng hp enzyme amylase ca

Asp. niger
Nghim thc
T l ging (*108bt/g)
0.8
1.6
2.4
3.2
1
50.733
47.592
43.317
42.514
Ln lp li
2
51.721
48.48
44.805
43.502
Trung bnh
3
53.045
49.704
46.629
44.826
51.833
48.592
44.917
43.614
ANOVA Table for ham luong by ty le giong
Source
Value
Between groups
0.0001
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares
Df Mean Square
124.403
10.766
135.169 11
3
8
41.4678
F-Ratio
P-
30.81
1.34574
mean ham luong from one level of ty le giong to another at the 95.0% confidence level

Nghim thc
T l ging (*108bt/g)
0.8
1.6
2.4
3.2
1
618.58
546.96
434.47
404.75
Ln lp li
2
619.57
547.95
435.45
405.74
ANOVA Table for hoat tinh by ty le giong
Trung bnh
3
620.89
549.27
436.78
407.07
619.678
548.061
435.566
405.853
Source
P-Value
Between groups
0.0000
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares
Df
88881.3
Mean Square
29627.1
1.34574
10.766
88892.1 11
F-Ratio
22015.42
mean hoat tinh from one level of ty le giong to another at the 95.0% confidence level.
8. nh hng ca t l c cht n kh nng sinh tng hp enzyme amylase ca

Mucor
Nghim thc
T l c cht
2C:1T:1B
1C:2T:1B
1C:1T:2B
Ln lp li
2
21.181
18.564
24.995
1
19.351
16.734
23.165
Trunng bnh
3
21.421
18.804
25.235
20.651
18.034
24.465
ANOVA Table for ham luong by ty le co chat
Source
Value
Between groups
0.0013
Within groups
Total (Corr.)
The StatAdviso
Sum of Squares
Df
Mean Square
62.753
2
7.6914
70.4444
F-Ratio
31.3765
6
P-
24.48
1.2819
mean ham luong from one level of ty le co chat to another at the 95.0% confidence level.
8.2. nh hng n hot tnh enzyme amylase

Nghim thc
Ln lp li
Trunng bnh
T l c cht
2C:1T:1B
249.699
251.529
251.769
250.999
1C:2T:1B
224.825
226.655
226.895
226.125
1C:1T:2B
170.2
172.03
172.27
171.5
ANOVA Table for hoat tinh by ty le co chat
Sum of Squares Df Mean Square
Source
Value
Between groups
Within groups
Total (Corr.)
The StatAdvisor
9922.7
2
4961.35 3870.31
7.6914
6
1.2819
9930.39
8
F-Ratio
P-
0.0000
mean hoat tinh from one level of ty le co chat to another at the 95.0% confidence level. To
determine
9. nh hng ca pH n kh nng sinh tng hp enzyme amylase ca Mucor

Nghim thc
pH
2
3
4
5
6
7
8
1
6.883
11.83
17.938
21.746
17.271
13.742
13.023
Ln lp li
2
8.713
13.66
19.768
23.576
19.101
15.572
14.853
Trunng bnh
3
8.953
13.9
20.008
23.816
19.341
15.812
15.093
8.183
13.13
19.238
23.046
18.571
15.042
14.323
ANOVA Table for ham luong by pH
Source
Between groups
Sum of Squares
419.346
Df
Mean Square F-Ratio
6
69.8911
54.52
P-Value
0.0000
Within groups
Total (Corr.)
The StatAdvisor
17.9466
437.293
14
20
1.2819
mean ham luong from one level of pH to another at the 95.0% confidence level.
9.2. nh hng n hot tnh enzyme amylase:

Nghim thc
pH
2
3
4
5
6
7
8
Ln lp li
2
90.685
155.244
205.197
263.957
170.325
111.039
86.139
1
88.855
153.414
203.367
262.127
168.495
109.209
84.309
Trunng bnh
3
90.925
155.484
205.437
264.197
170.565
111.279
86.379
90.155
154.714
204.667
263.427
169.795
110.509
85.609
ANOVA Table for hoat tinh by pH
Analysis of Varianc
Source
Sum of Squares
Df
Mean Square
F-Ratio
Value
Between groups
76308.8
Within groups
17.9466
14
Total (Corr.)
76326.8
20
12718.1
1.2819
9921.32
0.0000
P-
The StatAdvisor
mean hoat tinh from one level of pH to another at the 95.0% confidence level. To determine
which means
10. nh hng ca thi gian nui cy n kh nng sinh tng hp enzyme

amylase ca Mucor
Nghim thc
Ln lp li
Trunng bnh
Thi gian nui cy (h)

8
12
16
20
1
12.857
15.159
17.063
20.619
2
14.686
16.983
19.992
22.449
3
14.925
17.226
20.365
22.689
14.155
16.456
19.163
21.919
24
28
32
36
40
44
22.472
25.295
32.784
22.009
19.013
16.365
24.302
27.125
34.614
23.839
24.743
18.195
24.542
27.365
34.854
24.079
20.183
18.435
23.772
26.595
34.084
23.309
21.313
17.665
ANOVA Table for ham luong by thoi gian nuoi cay
Source
Value
Between groups
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares Df
Mean Square
874.031
25.638
899.669
9
20
29
97.1146
1.2819
F-Ratio
75.76
P0.0000
mean ham luong from one level of thoi gian nuoi cay to another at the 95.0% confidence
level.

Bng 10.3 Bng s liu hot tnh (UI/g CT)
Trunng
Nghim thc
Thi gian nui cy (h)
8
12
16
20
24
28
32
36
40
44
Ln lp li
2
69.038
108.485
155.163
203.190
232.060
270.018
415.252
250.394
139.631
47.705
1
67.312
106.655
153.333
201.764
230.111
268.188
413.422
249.564
137.901
45.875
bnh
3
69.48
108.725
155.403
203.638
232.242
270.258
415.492
255.634
140.071
47.945
68.61
107.955
154.633
202.864
231.471
269.488
414.722
251.864
139.201
47.175
ANOVA Table for hoat tinh by thoi gian nuoi cay
Source
P-Value
Between groups
0.0000
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares
Df
Mean Square
324641.0
25.638
324666.0 29
36071.2
20
1.2819
F-Ratio
28138.86
mean hoat tinh from one level of thoi gian nuoi cay to another at the 95.0% confidence level.
11. nh hng ca nhit nui cy n kh nng sinh tng hp enzyme

amylase ca Mucor
Nghim thc
Ln lp li
Trunng
bnh
Nhit nui cy (0C)
40
45
50
55
60
65
70
75
1
23.860
27.958
33.121
38.656
33.986
30.633
23.667
22.187
2
25.691
29.793
34.951
40.486
35.816
32.463
25.407
24.017
3
25.932
29.926
35.191
40.726
36.056
32.703
25.827
24.257
25.161
29.159
34.421
39.956
35.286
31.933
24.967
23.487
ANOVA Table for ham luong by nhiet do
Source
Sum of Squares
Df
Mean Square
F-Ratio
P-
Value
Between groups
1176.66
Within groups
15.3828
12
Total (Corr.)
1192.04
17
235.332
183.58
0.0000
1.2819
The StatAdvisor
The ANOVA table decomposes the variance of ham luong into two components: a
between-group component and a within-group component. The F-ratio, which in this case
equals 183.581, is a ratio of the between-group estimate to the within-group estimate. Since
the P-value of the F-test is less than 0.05, there is a statistically significant difference between
the mean ham luong from one level of
nhiet do to another at the 95.0% confidence level.

Trunng
Nghim thc
Nhit nui cy (0C)
40
45
50
55
60
65
70
75
1
23.861
27.859
33.121
38.656
Ln lp li
2
25.691
29.089
34.951
39.486
bnh
3
25.931
30.829
35.191
41.726
25.161
29.159
34.421
39.956
33.985
30.633
23.667
22.186
35.817
32.460
25.497
24.015
36.056
32.706
25.737
24.258
35.286
31.933
24.967
23.487
Source
Between groups
Within groups
Total (Corr.)
Sum of Squares
Df
Mean Square F-Ratio
P-Value
105956.0
0.0000
15.3828
12
1.2819
529797.0
17
529782.0
82655.72
The StatAdvisor
mean hoat tinh from one level of nhiet do to another at the 95.0% confidence level.
12. nh hng ca m n kh nng sinh tng hp enzyme amylase ca

Mucor
Bng 12.1 Bng s liu hm lng (mg)
Nghim thc
m (%)
40
45
50
55
60
65
70
75
1
23.863
27.861
33.121
37.656
33.987
30.036
23.667
22.187
Ln lp li
2
25.691
29.690
34.951
40.986
35.815
32.460
25.497
24.017
Trunng bnh
3
25.928
29.932
35.191
41.226
36.056
33.303
25.737
24.257
25.161
29.159
34.421
39.956
35.286
31.933
24.967
23.487
Sum of Squares Df
Mean Square
Source
F-Ratio
PValue
Between groups
423.243
7
60.4633
47.17
0.0000
Within groups
20.5104 16
1.2819
Total (Corr.)
443.754 23
mean ham luong from one level of do am to another at the 95.0% confidence level.

Nghim thc
m (%)
40
45
50
55
60
65
70
75
1
164.261
264.405
284.545
409.743
165.133
142.998
116.833
115.695
Ln lp li
2
164.091
266.235
286.775
411.573
166.963
144.828
117.663
117.525
Trunng bnh
3
165.331
266.475
286.815
411.813
167.203
145.068
119.903
117.765
164.561
265.705
286.045
411.043
166.433
144.298
118.133
116.995
ANOVA Table for hoat tinh by do am

Source
Sum of Squares
Df
Mean Square
F-Ratio
P-Value
Between groups
69808.4
9972.63
Within groups
20.5104
16
1.2819
Total (Corr.)
69828.9
7779.57
0.0000
23
The StatAdvisor
The ANOVA table decomposes the variance of hoat tinh into two components: a
between-group component and a within-group component. The F-ratio, which in this case
equals 7779.57, is a ratio of the between-group estimate to the within-group estimate. Since
the P-value of the F-test is less than 0.05, there is a statistically significant difference between
the mean hoat tinh from one level of do am to another at the 95.0% confidence level.
13. nh hng ca nng dinh dng n kh nng sinh tng hp enzyme

amylase ca Mucor
Trunng
Nghim thc
Nng dinh dng
X1
X2
X3
X4
X5
X6
X7
X8
1
38.207
38.781
42.091
37.3871
35.591
32.634
30.117
29.316
Ln lp li
2
40.037
40.611
43.921
39.2171
37.421
34.464
31.947
30.146
bnh
3
40.577
40.851
44.161
39.4571
37.661
34.704
32.187
32.386
39.607
40.081
43.391
38.6871
36.891
33.934
31.417
30.616
ANOVA Table for ham luong by nong do dinh duong
Source
Sum of Squares
Df
Mean Square
F-Ratio
P-Value
Between groups
423.243
60.4633
Within groups
20.5104
16
1.2819
Total (Corr.)
443.754
23
47.17
0.0000
The StatAdvisor
mean ham luong from one level of nong do dinh duong to another at the 95.0% confidence
level.

Nghim thc
Nng dinh dng
X1
X2
X3
X4
X5
X6
X7
X8
1
402.621
417.348
486.979
417.529
410.567
389.136
334.487
295.962
Ln lp li
2
404.451
418.078
488.809
419.359
412.397
389.466
336.317
297.792
Trunng
bnh
3
404.691
419.918
489.049
419.599
412.637
391.206
336.557
298.032
403.921
418.548
488.279
418.829
411.867
389.936
335.787
297.262
ANOVA Table for hoat tinh by nong do dinh duong
Source
P-Value
Between groups
Within groups
Total (Corr.)
Sum of Squares
Df
Mean Square
69808.4
20.5104 16
69828.9 23
7
9972.63
1.2819
7779.57
F-Ratio
0.0000
mean hoat tinh from one level of nong do dinh duong to another at the 95.0% confidence
level.
14. nh hng ca t l ging n kh nng sinh tng hp enzyme amylase ca

Mucor
Nghim thc
T l ging (*108bt/gCT)
0.8
1.6
2.4
3.2
1
38.398
43.691
37.474
33.423
Trunng
bnh
Ln lp li
2
40.228
45.021
38.304
35.253
3
40.468
46.261
40.544
35.493
39.698
44.991
38.774
34.723
Source
P-Value
Between groups
Within groups
Total (Corr.)
Sum of Squares
Df
Mean Square
160.585
10.2552
8
170.841 11
3
53.5284
1.2819
41.76
F-Ratio
0.0000
The ANOVA table decomposes the variance of ham luong into two components: a between-group
component and a within-group component. The F-ratio, which in this case equals 41.7571, is a ratio of
the between-group estimate to the within-group estimate. Since the P-value of the F-test is less than
0.05, there is a statistically significant difference between the mean ham luong from one level of ty le
giong to another at the 95.0% confidence level.

Nghim thc
T l ging (*108bt/gCT)
0.8
1.6
2.4
3.2
1
485.805
520.343
423.038
380.165
Ln lp li
2
487.635
523.173
424.868
381.995
Trunng
bnh
3
487.875
524.413
425.108
382.235
487.105
522.643
424.338
381.465
Source
Value
Between groups
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares Df
Mean Square
35846.7
10.2552
35857.0 11
3
8
11948.9
1.2819
F-Ratio
9321.25
P0.0000
The ANOVA table decomposes the variance of hoat tinh into two components: a betweengroup The F-ratio, which in this case equals 9321.25, is a ratio of the between-group estimate
to the within-group estimate. Since the P-value of the F-test is less than 0.05, there is a
statistically significant difference between the mean hoat tinh from one level of ty le giong to
another at the 95.0% confidence level.
15. Xc nh gi tr ti u ca mt s yu t nh hng n kh nng sinh tng

hp amylase ca Asp. niger
STT th nghim
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1
388.9
298.97
217.32
175.51
222.94
167.27
183.59
104.36
268.97
166.45
155.18
151.12
160
151.13
107.32
87.337
483.14
Ln lp li
2
389.8
299.87
218.23
176.41
223.84
168.17
184.49
105.26
269.87
167.36
156.08
152.02
160.9
152.03
108.22
88.24
485.22
3
389.98
300.05
218.4
176.59
224.01
168.35
184.66
105.43
270.05
167.53
156.26
152.19
161.08
152.21
108.39
88.414
487.28
Trung bnh
389.56
299.63
217.98
176.17
223.6
167.93
184.25
105.02
269.63
167.11
155.84
151.78
160.66
151.79
107.98
87.997
485.21
ANOVA Table for hoat tinh by STT
Sum of Squares
Df
Mean Square
Source
P-Value
etween groups
Within groups
Total (Corr.)
The StatAdvisor
709835.0 16
44364.7 123691.54
12.1948 34
0.358672
709847.0 50
F-Ratio
0.0000
mean hoat tinh from one level of STT to another at the 95.0% confidence level.
16. Xc nh gi tr ti u ca mt s yu t nh hng n kh nng sinh tng

hp amylase ca Mucor
STT th
nghim
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Y1
434.67
384.56
411.49
384.21
211.61
209.52
261.97
258.47
260.47
356.29
376.79
351.88
197.24
187.43
188.92
157.29
623.46
Ln lp li
Y2
435.57
385.46
412.39
385.11
212.52
210.43
262.87
259.37
261.37
357.19
377.69
352.78
198.14
188.33
189.82
158.19
624.67
Y3
435.75
385.64
412.57
385.29
212.69
210.6
263.04
259.55
261.55
357.37
377.87
352.96
198.31
188.51
190
158.37
625.13
Trung bnh
435.33
385.22
412.15
384.87
212.27
210.18
262.63
259.13
261.13
356.95
377.45
352.54
197.9
188.09
189.58
157.95
624.42
ANOVA Table for hoat tinh by STT
Source
Value
Between groups
0.0000
Within groups
Total (Corr.)
The StatAdvisor
Sum of Squares
Df
Mean Square
520282.0
19.2747
520301.0
34
50
16
32517.6
F-Ratio
P-
57360.01
0.566904
mean hoat tinh from one level of STT to another at the 95.0% confidence level.

SINH TỔNG HỢP ENZYME AMYLASE TỪ NẤM Aspergillus niger VÀ Mucor

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SINH TỔNG HỢP ENZYME AMYLASE TỪ NẤM Aspergillus niger VÀ Mucor

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B GIO DC V O TO

TRNG I HC NNG LM TP. H CH MINH

KHA LUN TT NGHIP

NGHIN CU CC YU T NH HNG N KH NNG

Ngnh hc: CNG NGH SINH HC

KHA LUN TT NGHIP

Sinh vin thc hin

NGUYN B PHNG THO

Nguyn B Phng Tho

2.2.5.3. Trong y hc.......................................................................................................8

4.1.1. Nm mc A .niger ............................................................................................30

4.1.6. nh hng ca m ban u ..........................................................................37

Hnh nh vi th quan st c cho thy Mucor c cung bo t phn nhnh v mc ln

4.2.2. nh hng ca t l cm go: b u nnh: tru ..............................................42

DANH SCH CC CH VIT TT

Unit International, n v quc t

Phn tch phng sai mt yu t

Potato glucose agar

K hoch thc nghim y cc yu t

DANH SCH CC HNH

Hnh 2.1 Cu trc phn t ca amylose...........Error: Reference source not found

Error: Reference source not found

Error: Reference source not found

Hnh 4.1 Hnh nh i th v vi th ca A. niger.

Error: Reference source not

source not found

Error: Reference source

Error: Reference source

source not found

source not found

Error: Reference source

th 4.13 Hot tnh v hm lng theo cc nng dinh dng ca Mucor.

DANH SCH CC BNG

Error: Reference source

source not found

Reference source not found

source not found

Error: Reference source

Th t, vn tc phn ng do enzyme xc tc c th d dng iu chnh thm ch

dng acid th -amylase ca nm mc bn vng hn so vi -amylase ca malt v vi

2.2.3.3. -amylase (hay gluoamylase hoc -1,4 glucan-glucanhydrolase)

phm hn hp bt enzyme b sung vo ch phm bt dinh dng dnh cho tr em,

2.2.5.4. Trong nng nghip

Di tc dng ca enzyme amylase, tinh bt b thy phn do cc lin kt

Hnh 2.1 Cu trc phn t ca amylose. (Wikipedia.com).

Hnh 2.3 Cu trc phn t glycogen. (Wikipedia.com).

Glycogen l mt loi carbonhydrate d tr c th ng vt v c c th

2.3. Gii thiu v nm mc Aspergillus niger v Mucor

Loi nm si ny sinh trng nhit ti thiu l 6 - 8oC, ti a l 45 - 470C,

Nm Mucor c th c phn bit vi cc loi nm khc qua cc im c bit

Canxi cn cho s tng hp v n nh -amylase hot ng v n l cu t

my lc, mi trng bn rn th c cht phi c xp to s thng thong gia cc

t theo hng thu cm sch nn cm thng ln rt nhiu loi tp cht (v tru, sn

Mi trng ln men bn rn thng dng nguyn liu t nhin nh cm go,

2.7. Thu nhn enzyme t mi trng nui cy b mt

khun lc mc u s cho vo t lnh gi nhit 5 - 90C trnh nm mc b gi

Tin hnh cn 1 g canh trng ly t canh trng nhn ging cp 2 thc

N : s lng bo t trong 1 ml dch huyn ph.

3.3.5. Phng php xc nh m

: khi lng c cht c trong mi trng nui cy (g).

: m mun b sung vo mi trng (%).

: m trong c cht (%) ( vi c cht l la, cm go, b u nnh, tru th

m thng thng ca chng l 10%). (Trn Anh Tun, 2005).