B GIO DC V O TO

TRNG I HC NNG LM TP. HCM

B MN CNG NGH SINH HC
**

KHA LU N T T NGHI P

NUI C Y BACILLUS SUBTILIS

THU NH N -AMYLASE V NG D NG
TRONG S N XU T DEXTRIN

Ngnh: CNG NGH SINH HC

Nin kha: 2003 - 2007
Sinh vin thc hin: NGUYN THANH THY

Thnh ph H Ch Minh
- 2007 -

B GIO DC V O TO
TRNG I HC NNG LM TP. HCM
B MN CNG NGH SINH HC
**

NUI C Y BACILLUS SUBTILIS

THU NH N -AMYLASE V NG D NG
TRONG S N XU T DEXTRIN

Gio vin hng dn:

Trng Phc Thin
Hong Nguyn Nh Nht

Thnh ph H Ch Minh
- 2007 -

Sinh vin thc hin:

Nguyn
Thy

Thanh

iii

L I C M N
Xin t lng bit n su sc n ba m, ngi lun trn tr, lo lng v ng

vin ti c c ngy hm nay.

Xin t lng bit n n tt c qu thy c truyn t kin thc cho ti

trong sut qu trnh hc ti trng.
Xin c gi li cm n chn thnh nht n ch Tr ng Ph c Thin Hong

v anh Nguyn Nh Nht tn tnh hng dn, ng vin ti trong thi gian thc
hin kha lun.
Cm n cc anh v cc bn ti phng sinh ha ng dng ca trng i hc
Khoa Hc T Nhin to iu kin thun li v gip ti trong thi gian thc tp tt
nghip.
Cm n Hng Vy v Phng Uyn, hai ngi bn thn nht ca ti chia x v

gip ti trong 4 nm hc ti trng.

iv

TM T T
Nguyn Thanh Thy, i hc Nng Lm TP. HCM
ti nghin cu Nui cy Bacillus subtilis thu nhn -amylase v ng dng
trong sn xut dextrin, thc hin ti phng sinh ha v ng dng ca trng i hc
Khoa Hc T Nhin TP.HCM t 19/03/2007 - 19/07/2007.
GVHD: ThS. Trng Phc Thin Hong
ThS. Nguyn Nh Nht

i tng nghin cu l -amylase ca Bacillus subtilis

Chng ti s dng phng php Lowry nh lng protein ho tan trong nc,

phng php Heinkel xc nh hot -amylase kho st thi gian nui cy, t l

ta -amylase bng cn 96% v (NH4)2SO4, kho st mt s yu t nh hng n

kh nng thy phn tinh bt to dextrin ca ch phm -amylase.
Kt qu thu c nh sau:
- Thi gian nui cy Bacillus subtilis thch hp thu -amylase l 48 gi.
- T l ta -amylase bng cn 96% tt nht l 1 th tch dch chit enzyme v 3
th tch cn. Thi gian ta l 15 pht.
- pH v nhit ti u cho phn ng thy phn ca ch phm -amylase l 6,0
v 55oC.
- Lng dextrin thu c nhiu nht t s thy phn bt nng 10%.

ABSTRACT
NGUYEN THANH THUY, Nong Lam University of HCM city
Graduating thesis topic:

-amylase production by Bacillus subtilis solid state

fermentation and dextrin production application. This thesis was carried out at
biochemistry and application room of Natural Science University HCMC from
03/2007 to 09/2007.
We used -amylase activity measurement method

(Heinkel) and protein

concentration determination method (Lowry) to investigate fermentation time, amylase precipitation by 96% ethanol and solid ammonium sulfate. After
recovering -amylase from fermented medium we examined starch hydrolysis
ability and dextrin production of -amylase product.
Result experiments showed that:
- -amylase activity was hightest at 48h incubation.
- Precipiating -amylase by 96% ethanol was better than solid ammonium
sulfate and suitable volume ratio of extracted enzyme and ethanol for
precipitation was 1:3.
-

Optimum temperature and pH for hydrolysis of purified -amylase were

respective 55 degree C and 6,0.

- -amylase had the most effective hydrolysis in 10% cassava starch fluid.

vi

M C L C
TRANG
Li cm n............................................................................................................... iii
Tm tt.....................................................................................................................iv
Abstract.....................................................................................................................v
Mc lc.....................................................................................................................vi
Danh sch cc ch vit tt.........................................................................................ix
Danh sch cc hnh.....................................................................................................x
Danh sch cc bng...................................................................................................xi
Danh sch cc s .................................................................................................xii
Danh sch cc th.................................................................................................xii
Danh sch cc biu ..............................................................................................xii
Ch ng 1. M U ..........................................................................1
1.1. T VN ................................................................................................1
1.2. MC CH....................................................................................................2
1.3. YU CU......................................................................................................2
Chng 2. TNG QUAN TI LIU.......................................................3
2.1. BACILLUS SUBTILI....................................................................................3
2.1.1. c im vi khun...................................................................................3
2.1.2. Phn loi..................................................................................................6
2.1.3. B gen.....................................................................................................6
2.1.4. ng dng.................................................................................................7
2.2. ALPHA-AMYLASE.......................................................................................8
2.2.1. Khi nim................................................................................................8
2.2.2. Phn loi, danh php................................................................................8
2.2.3. Cu trc phn t....................................................................................... 8
2.2.4. Tnh cht..................................................................................................9
2.2.5. C ch xc tc........................................................................................10
2.2.6. ng dng...............................................................................................11
2.3. CC PHNG PHP NUI CY VI SINH VT......................................12

vii

2.3.1. Nui cy b mt......................................................................................12

2.3.2. Nui cy chm........................................................................................12
2.4. TCH V LM SCH ENZYME..............................................................13
2.5. DEXTRIN.....................................................................................................13
2.5.1. Phn loi................................................................................................14
2.5.2. Tnh cht................................................................................................14
2.5.2.1. nht.......................................................................................15
2.5.2.2. pH.........................................................................................16
2.5.2.3. Tui ca dung dch dextrin..........................................................16
2.5.2.4. ha tan...................................................................................16
2.5.2.5. Mu sc.......................................................................................16
2.5.3. Quy trnh sn xut dextrin......................................................................17
2.5.4. ng dng...............................................................................................18
Chng 3. VT LIU V PHNG PHP..........................................19
3.1. THI GIAN V A IM........................................................................19
3.2. VT LIU NGHIN CU...........................................................................19
3.3. PHNG PHP TH NGHIM..................................................................20
3.3.1. Xc nh mt t bo...........................................................................20
3.3.2. Kho st thi gian nui cy thch hp.....................................................20
3.3.3. Kho st t l ta enzyme bng cn 96%................................................20
3.3.4. Kho st t l ta enzyme bng (NH4)2SO4..............................................................................21
3.3.5. Kho st thi gian ta enzyme bng cn 96%.........................................21
3.3.6. Kho st mt s yu t nh hng n kh nng thy phn ca
-amylase........................................................................................................22
3.3.6.1. Nhit ......................................................................................22
3.3.6.2. pH..............................................................................................22
3.3.7. Kho st t l cn thch hp thu dextrin..................................................22
3.3.8. Kho st kh nng thy phn ca ch phm -amylase vi cc
ngun tinh bt......................................................................................................22
3.3.8.1. Kho st kh nng thy phn vi cc ngun tinh bt...................22
3.3.8.2. Kho st nng tinh bt thch hp............................................23
3.4. PHNG PHP X L S LIU..............................................................23

viii

Chng 4. KT QU V THO LUN................................................24

4.1. KT QU KHO ST THI GIAN NUI CY........................................24
4.2. KT QU TINH SCH -AMYLASE BNG CN 96% V
(NH4)2SO4........................................................................................................................................................................................... 25
4.2.1. Kt qu t l ta enzyme bng cn 96%.................................................24
4.2.2. Kt qu t l ta enzyme bng (NH4)2SO4.................................................................................27
4.3. KT QU KHO ST THI GIAN TA ENZYME BNG CN 96% .. 28
4.4. KT QU KHO ST CC YU T NH HNG N KH
NNG THY PHN CA -AMYLASE..........................................................29
4.4.1. Nhit .................................................................................................29
4.4.2. pH..........................................................................................................30
4.5. KT QU KHO ST T L CN THCH HP THU DEXTRIN...........31
4.6. KT QU KHO ST KH NNG THY PHN CA CH PHM
-AMYLASE VI CC NGUN TINH BT....................................................32
4.7. KT QU KHO ST NNG BT NNG V THI GIAN
THY PHN H BT NNG BNG CH PHM -AMYLASE...................32
Chng 5. KT LUN V NGH...................................................37
TI LIU THAM KHO.......................................................................38
PH LC

ix

DANH SCH CC CH VI T T T
B. subtilis

Bacillus subtilis

GH

Glycosidic Hydrolase

EC

Enzyme Commission

DAP

Diamoni Phosphate

MW

Molecular Weight

Da

Dalton

DE

Dextrose equivalent

CT

Canh trng

dOD

Delta optical density

DCE

Dch chit enzyme

CPE

Ch phm enzyme ( sy kh)

CP ti

Ch phm enzyme ti

UI

Unit International

DANH SCH CC HNH

HNH

TRANG

Hnh 2.1. Hnh thi Bacillus subtilis........................................................3

Hnh 2.2. Bacillus subtilis ang trong giai on to bo t.....................................4
Hnh 2.3. Khun lc B. subtilis trn thch a..........................................................4
Hnh 2.4. Cu trc khng gian ca -amylase.............................................................9
Hnh 2.4. V tr ct ca -amylase trn phn t tinh bt............................................11
Hnh 2.5. Dextrin.....................................................................................................14
Hnh 4.1. Mi trng bn rn trc khi nui cy......................................................25
Hnh 4.2. Canh trng nui cy sau 48 gi...............................................................25
Hnh 4.3. nh hng ca pH ln hot ca -amylase...........................................30
Hnh 4.4. Ch phm -amylase.................................................................................36
Hnh 4.5. Ch phm dextrin......................................................................................36

xi

DANH SCH CC B NG
BNG

TRANG

Bng 2.1. Mt s phn ng sinh ho ca B. subtilis........................................5

Bng 2.2. phn loi ca Bacillus subtilis....................................................6
Bng 2.3. Mt s tnh cht ca dextrin......................................................................15
Bng 4.1. nh hng ca thi gian nui cy ln hot -amylase..........................24
Bng 4.2. nh hng ca t l cn 96% ln hot -amylase.................................26
Bng 4.3. nh hng ca (NH4)2SO4 ln hot -amylase.....................................27
Bng 4.4. nh hng ca thi gian ta cn 96% ln hot -amylase....................28
Bng 4.5. nh hng ca nhit ln hot ca -amylase...................................29
Bng 4.6. nh hng ca pH ln hot ca -amylase...........................................30
Bng 4.7. Lng dextrin thu c cc t l cn 96%.............................................31
Bng 4.8. Dextrin to thnh do -amylase thy phn tinh bt 10%............................32
Bng 4.9. Hiu sut thu dextrin.................................................................................33
Bng 4.10. Hiu sut thu dextrin khi thy phn bt nng 10%...................................33
Bng 4.11. Hiu sut thu dextrin khi thy phn bt nng 15%...................................34
Bng 4.12. Hiu sut thu dextrin khi thy phn bt nng 20%...................................35

xii

DANH SCH CC S
S

TRANG

S 2.1. Tm tt qui trnh iu ch dextrin t tinh bt...........................................17

DANH SCH CC TH
TH

TRANG

th 4.1. nh hng ca thi gian nui cy ln hot -amylase.........................25

th 4.2. nh hng ca t l cn 96% ln hot -amylase...............................26
th 4.3. nh hng ca (NH4)2SO4 ln hot -amylase....................................27
th 4.4. nh hng ca thi gian ta cn 96% ln hot -amylase...................28
th 4.5. nh hng ca nhit ln hot ca -amylase..................................29
th 4.6. nh hng ca pH ln hot ca -amylase.........................................31

DANH SCH CC BI U
BIU

TRANG

Biu 4.1. Lng dextrin thu c t cc t l cn 96%.........................................31

Biu 4.2. Khi lng dextrin thu c khi thy phn h tinh bt 10%...................32
Biu 4.3. Khi lng dextrin thu c khi thy phn h bt nng 10%..................34
Biu 4.4. Khi lng dextrin thu c khi thy phn h bt nng 15%..................34
Biu 4.5. Khi lng dextrin thu c khi thy phn h bt nng 20%..................35

Ch ng 1
M U
1.1. T VN
Hin nay, -amylase l mt trong nhng enzyme c s dng rng ri trong

nhiu lnh vc nh cng nghip thc phm, chn nui th y, chn on

bnh

Trong cng nghip thc phm, -amylase ng vai tr c bit quan trng. Thng qua
s thy phn tinh bt, -amylase to ra nhng sn phm c gi tr dinh dng nh
dextrin, maltose, glucose
Trc y, ngi ta thu nhn -amylase t malt l ch yu. Ngy nay, vi nn
cng nghip pht trin mnh ko theo nhu cu v -amylase tng cao nn vic p dng
nhng k thut tin b trong nui cy vi sinh vt thu d dng hn vi lng ln amylase l rt cn thit.
Dextrin l sn phm do s thy phn tinh bt ca -amylase. Ngoi gi tr dinh
dng, dextrin cn l mt trong nhng yu t ci thin tnh cht, cm quan ca thc
phm. V vy, dextrin ngy cng c s dng ph bin trong ch bin thc phm v
cng nghip bnh ko.
Sn xut dextrin t tinh bt c th dng acid hay enzyme. Tuy nhin, sn xut
dextrin bng acid c rt nhiu hn ch l kh nh hng sn phm do tc dng khng
c hiu ca acid ln nguyn liu, c hi nn i hi phi c trang thit b t tin,
lng acid d gy h hng thit b v lm nhim mi trng. Mc khc, dextrin thnh
phm c mu v v ng.
Vic s dng -amylase thay th cho acid trong cc qui trnh sn xut
dextrin s khc phc c nhng hn ch trn.

Dextrin thu c nh -amylase sau khi sy phun c mu trng, pH trung tnh,

t ngt, sch khun, khong v cht hu c nn rt thch hp s dng trong

nhiu

ngnh cng nghip v trong y dc.

T thc tin trn chng ti tin hnh ti Nui cy Bacillus subtilis thu
nhn -amylase v ng dng trong sn xut dextrin.
1.2. MC CH
Thu nhn -amylase, xc nh iu kin ti u cho phn ng thy phn ca amylase v tinh bt thu nhn dextrin.
1.3. YU CU
- Kho st thi gian nui cy Bacillus subtilis trn mi tr ng bn rn

iu kin nui cy theo ti nghin cu tr c.

- Thu nhn v tinh sch s b -amylase t canh trng nui cy B.subtilis.

- Kho st mt s yu t nh hng n kh nng thy phn tinh bt to

dextrin ca ch phm -amylase.

Ch ng 2
T NG QUAN TI LI U
2.1. BACILLUS SUBTILIS
2.1.1. c im vi khun
Bacillus subtilis l trc khun, Gram (+), c kh nng sinh catalase, hiu
kh hay k kh ty . Thng c tm thy trong t.

C kh nng di ng, sinh ni bo t. T bo sinh dng c dng hnh que, kch

thc chiu rng t 0,7 - 0,8 m, chiu di t 2,0 - 3,0 m. Khng kt thnh chui, bt
phm nhum ng u, khng to bao nang.

Hnh 2.1. Bacillus subtilis

(URL: http://www.ami-tech.co.kr/customer/img/img_08.jpg)
Bo t B. subilis c dng ellip n hnh cu, kch thc chiu rng 0,6 0,9 m, chiu di 1,0 - 1,5 m, nm gia hay trong khong trung tm n gn cui
t bo, phn ln c to thnh 48 gi. Mi c th ch to mt bo t, bo t c
kh nng chu nhit, tia bc x, cht st khun, cht ht m (Trn Quyn, 2004).

Hnh 2.2. B. subtilis ang trong giai on to bo t. Cu trc

hnh

oval

trung tm l bo t (URL: ec.europa.eu/research/success/images/0291b.jpg).

Khi nui cy trn mi trng thch a khun lc trn, khng u hay phn

tn. ng knh khun lc t 3 - 5 mm, mu vng xm, ra c hnh rng ca. Sau 1
- 4 ngy b mt khun lc nhn nheo, mu hi nu (Nguyn c Duy Anh, 2005).

Hnh 2.3. Khun lc B. subtilis trn thch a

Trong mi trng lng sinh khi to mng mng c lp bao ph. Do bo t

chiu nhit cao nn B. subtilis c th gy h hng mt s thc phm hp to mi v

kh chu.
Sinh acid t xylose, arabinose, glucose, sucrose v mannitol nhng khng to
kh (s dng ngun nit l mui amonium).
C kh nng phn gii nitrate, sinh nitrit t nitrate. Trong iu kin k kh,
khng sinh kh t mi trng lng cha nitrate.
Mt c im dng phn bit vi cc vi khun khc l lm tan chy
gelatine nhanh chng.

Bng 2.1. Mt s phn ng sinh ho ca B. subtilis

Phn ng sinh ho

Kt qu

Hot tnh Catalase

Sinh Indol

MR

VP

S dng Citrate

Kh Nitrate

Tan chy gelatin

Phn gii tinh bt

Arabinose

Xylose

Saccharose

Manitol

Glucose

Lactose

Maltose

(Theo Holt, 1992)

Nhit ti thch ca B. subtilis vo khong 36 - 50oC. Nhiu loi vn pht
trin c 60oC. Nng mui n lm ngng pht trin l 10 - 15% (Lng c
Phm, 2000).
Ngi ta chng minh B. subtilis c tp tnh n ln nhau (cannibalism).

Chng dng cch ny nh mt phng php n gin thot khi nhng trng hp
iu kin sng gii hn.
B. subtilis c kh nng sinh tng hp hn 20 loi khng sinh khc nhau
nh: subtilin, subtilosin A, sublancin, chlorotetain, mycobacillin, rhizocticins,
bacillanene, difficidin(theo ti liu tng hp ca Nguyn Qunh Nam, 2006).

Phn b trong t v cc cht hu c b phn hy v l mt i tng dng

trong nghin cu kh nng ly bnh trong phng th nghim (Trn Quyn,
2004).
B. subtilis khng c xem l mm bnh gy bnh cho

thng c trong thc phm nhng him khi gy ng c thc phm.

ngi. Chng

2.1.2. Phn loi

Bng 2.2. Bng phn loi ca Bacillus subtilis
Phn loi khoa hc
Gii:

Bacteria

Ngnh: Firmicutes
Lp:

Bacilli

B:

Bacillales

H:

Bacillaceae

Ging:

Bacillus

Loi:

subtilis
Tn kp

Bacillus subtilis
(Ehrenberg 1835
Cohn 1872)

2.1.3. B gen
Nm 1997, ng i ta hon tt vic nghin cu v trnh t gen ca B.
subtilis v ln u tin cng b trnh t gen ca vi khun.
B gen cha 4,2 mega-base, xp x 4100 gen. Trong s , ch c 192 gen
khng th thiu c, 79 gen c d on l thit yu. Phn ln gen thit yu u c
lin quan vi qu trnh trao i cht ca t bo.

2.1.4. ng dng ca Bacillus subtilis

Trong cng nghip sn xut amino acid, thc n gia sc, Bacillus subtilis l
mt trong nhng chng vi sinh vt tng hp lysine c hm lng kh ln (15-20%)
t tinh bt.
Trong y dc, Bacillus subtilis c ng thnh ng thuc Subtilis 10 ml tr

bnh tiu chy cho tr em do vi khun Coliform gy ra, bnh dng rut do l trc
trng, p cc vt thng l lot ngoi da (Trn Quyn, 2004).

Sn xut cc khng sinh thc vt, ng dng trong phng tr vi sinh vt gy

bnh nh nm Rhizoctonia solani, Fusarium sp, Pylicularia oryzae,... Ngi
ta thy rng s pht trin ca Bacillus subtilis trong cy lm tng kh nng tng hp
cc peptide khng nm ca vi khun nt r (Rhizobacterium). Kh nng ny c
ng dng trong kim sot sinh hc.
ng dng trong sn xut ch phm sinh hc (probiotic) b sung trong thc
n nhm ci thin tiu ha, sc tng trng; gim s ti pht bnh tiu chy trn gia
sc; b sung vo ao nui nhm duy tr cht lng nc ao, hn ch bnh cho thy
sn nui.
H enzyme ca B. subtilis c s dng nhiu trong sn xut cht ty ra.
Chng c th bin i cc dng cht thi c hi thnh nhng dng hp cht v hi ca
nitrogen, carbon dioxide, v nc.

Mt chng Bacillus subtilis c bit trc y l Bacillus natto c

dng trong sn xut thc phm thng mi ca Nht tng t nh thc phm
cheonggukjang ca Hn Quc.
B. subtilis ti t hp c s dng trong sn xut polyhydroxyalkanoates

(PHA) v chng c th s dng malt ph thi nh l ngun cacbon, nh vy chi ph sn

xut PHA gim.

2.2. ALPHA-AMYLASE

2.2.1. Khi nim

-amylase l enzyme xc tc s thy phn cc lin kt -1,4-glucoside nm bn
trong phn t tinh bt v glycogen.
L mt enzyme kim loi. Nu khng c s hin din ca ion Canxi trong phn
t enzyme s khng hot ng c.
-amylase phn ct cacbonhydrate chui di to maltotriose v maltose t
amylose hay to maltose, glucose v dextrin t amylopectin.
Do c th hot ng bt k v tr -1,4 no trn c cht nn hot ng
phn ct ca -amylase nhanh hn -amylase. ng vt, -amylase l enzyme tiu
ha chnh.
2.2.2. Phn loi, danh php -amylase
-amylase: (1,4--D-glucan glucanhydrolase) thuc nhm enzyme thy
phn (hydrolase), h 13 (GH 13), m s EC 3.2.1.1.
Xc tc phn ng ni thy phn lin kt 1,4--D-glucosidic trong chui
ng a c cha 3 hoc nhiu hn nhng n v D-glucose lin kt vi nhau v
tr -1,4.
Tn khc: glycogenase; endoamylase; Taka-amylase A. Tn
h thng: 1,4--D-glucan glucanhydrolase.
2.2.3. Cu trc phn t
Gm 3 tiu n v A, B, C. A l tiu n v li c cu trc c trng helix

(/)8 - barrel, c ni vi tiu n v C (C-terminal) c cu trc 8 on -sheet

song song. Tiu n v B gm 2 on -sheet c lng vo gia on -sheet th

ba v on -helix th hai ca tiu n v A. Tiu n v B quyt nh bn v
lin kt enzyme v c cht. Mt ion Canxi ni gia hai tiu n v A v B.
Ty thuc vo dng enzyme m c th c mt s tiu n v khc c gn vo
u C- hay u N- ca phn t protein.
Trung tm hot ng ca -amylase c cha nhm -COOH v -NH2 (Trn
Quyn, 2004).

Khi lng phn t -amylase khc nhau cc loi Bacillus.

-amylase vi khun cng nghip ch yu c sn xut t B. subtilis var.

amyloliquefaciens v var. amylosacchariticus c trng lng phn t (MW) l

49.000 dalton (Da) (theo Fisher v Stein, 1960) v 60.000 Da (Geranum, 1979). Khi c
mt Zn, enzyme s lin kt to dng dimer c MW 100.000 Da.
-amylase khng cha co-enzyme, tuy nhin n cng cn 1 nguyn t gam Ca2+
cho 1 mol enzyme n nh cu trc v ngn khng cho protease phn hy enzyme
(Nguyn Tin Thng, 2005).

Hnh 2.4. Cu trc khng gian ca -amylase.

(URL: plaza.snu.ac.kr/~sewonsuh/home/structures.html)
2.2.4. Tnh cht -amylase
Tnh chy c trng ca -amylase l kh nng dextrin ha cao nn lm cho
phn ng mu ca tinh bt vi iod b bin i nhanh chng. Kt qu tc ng ca amylase th ng lm gim nhanh nht ca h tinh bt do ng i ta gi amylase l amylase dextrin ha hay amylase dch ha.
ru long.

-amylase d tan trong nc, trong cc dung dch mui, dung dch m v
-amylase hot ng khng cn cht hot ha v cn Ca vi vai tr l

mt ng yu t xc tc s thy phn tinh bt. Canxi c tc dng n nh tt amylase trong qu trnh thy phn bng nhit hay kim, tng bn ca -amylase

10

trc cc tc nhn gy bin tnh v tc dng phn hy ca protease (Lng c

Phm, 1998).
pH ti u ca -amylase vi khun trong khong 6,0 - 7,0. -amylase b
bt hot nhanh pH thp 4,5 - 4,7 v khi c mt cc tc nhn to phc vi Canxi nh
EDTA, polyphosphate, oxalate, chlorine t do v cc cht oxy ha (Nguyn Tin
Thng, 2005).
-amylase ca vi khun thng bn nhit hn -amylase t cc loi vi
sinh vt khc v cy trng, chng vn c th hot ng nhit m -amylase ca
nm mc v ng cc b bt hot . Nhit ti u ca -amylase trong khong 54 63oC (Hopek, 2006). i vi -amylase ca Bacillus subtilis nhit ti u c th
t n 70 - 80oC.
-amylase t cc ngun khc nhau c thnh phn cc amino acid khc
nhau, mi loi -amylase c cu to t mt t hp amino acid c hiu ring. Tuy

nhin, hm lng tryptophan, tyrosine thng chim u th. Cc glutamic acid v

aspartic acid chim khong 1/4 tng lng amino acid cu thnh phn t enzyme (Trn
Quyn, 2004).
2.2.5. C ch xc tc
Phn ng thy phn tinh bt bi -amylase thng xy ra qua hai giai on
Giai on u l giai on dch ha: ch mt s lin kt trong phn t b
t v nht ca h tinh bt gim nhanh.

Giai on tip theo l s thy phn cc dextrin phn t ln va to

thnh. Nh tinh bt c th chuyn thnh maltose, glucose v cc dextrin phn t

thp. Tuy nhin, thng -amylase ch thy phn tinh bt ch yu thnh dextrin
phn t thp, khng cho mu vi Iod v mt t maltose (Trn Quyn, 2004).

Tinh bt

- amylase
H2O

-Dextrin + Maltose + Glucose

11

Hnh 2.4. V tr ct ca -amylase trn phn t tinh bt

2.2.6. ng dng ca -amylase
L mt trong nhng enzyme quan trng hin nay do ng dng ca n trong nhiu
lnh vc nh thc phm, dc phm, cng nghip dt, chn nui
Trong cng nghip thc phm:
- Sn xut ru, bia, nc tri cy.
- Sn xut bnh, bnh m: trong tinh bt t nhin c amylase ca ng cc
nhng hm lng ca chng khng nn vic b sung -amylase c th ci thin tnh
cht ca bnh nh lm tng n xp v mi v ca bnh.

- Ch bin tinh bt, sn xut ng maltose, syro glucose, syro

fructose. Dch giu glucose c s dng trong cng nghip sn xut bt ngt, cng
nghip ln men, sn xut nc tri cy, lm mt. Glucose tinh th c s dng

trong y t, trong khu phn dinh dng cho bnh nhn, sn xut dch truyn. Syro

fructose c dng trong sn xut ung cha t nng lng, bnh ko, nc qu
v sn phm sa
Lm mm vi trong cng nghip dt:
Trong sn xut vi c giai on h ha lm vi chc hn, ngi ta h ha bng

tinh bt. Sau khi dt ngi ta phi loi b cht tham gia h ha bng cch x l vi amylase.
Sn xut thc n gia sc gip ci thin tiu ha:
B sung hn hp -amylase, cellulase, -glucanase vo sinh khi c chua
nhm tng nng sut tiu ha ca vt nui.

12

B sung -amylase, protease vo khu phn thc n cho heo con d i 5 tun
tui nhm gia tng tc pht trin ca heo con, ngn pht sinh bnh tiu chy ph bin
heo con (Nguyn Tin Thng, 2005).
Trong y hc: chn on bnh lm sng cc bnh thng thng v cc bnh
thiu enzyme.
2.3. CC PHNG PHP NUI CY VI SINH VT SN XUT ENZYME

Cng nghip sn xut enzyme hin nay trn th gii p dng hai phng php:

nui cy b mt v nui cy chm.

Phng php nui cy vi sinh vt trn mi trng rn (b mt) c mt s u

vit hn so vi phng php chm. Do lng nc trong c cht thp nn enzyme

v nhng sn phm khc trng thi c c v vy d tinh sch. Nui cy b mt d

sy kh v t b tn hao hot tnh enzyme. Khng cn trang thit b phc tp, ch
yu nui trn khay v bung nui gi nhit , m thch hp (L ng
c
Phm, 1998).
2.3.1. Nui cy b mt
Phng php nui cy b mt l phng php to mi trng cho vi sinh vt

pht trin trn b mt mi trng. Mi trng s dng l mi trng lng hoc s dng
mi trng c (mi trng bn rn).

mi trng lng vi sinh vt s pht trin trn b mt mi trng to thnh

vng khun ngn cch pha lng v pha kh. Vi sinh vt s s dng cht dinh dng t
dung dch mi tr ng, oxy khng kh, tin hnh qu trnh tng hp enzyme.

(Nguyn

c Lng, 2004).

2.3.2. Nui cy chm

Phng php ny ngi ta s dng mi trng lng v c thc hin trong
nhng thng ln men c trang b h thng sc kh, khuy trn, iu chnh pH.
Phng php nui cy chm c u im chim t din tch nhng c nhc im
l d b nhim ton b v chi ph cho trang thit b kh cao.

13

2.4. TCH V LM SCH ENZYME

Phn ln cc enzyme thy phn d ha tan trong nc nn c th tch chit

enzyme t canh trng nui cy vi khun bng cch lc hay ly tm.

tinh sch enzyme ngi ta thng dng phng php kt ta phn on bng

dung mi hu c hay cc dung dch mui trung tnh (K.Clarkson v cng s, 2000).
Phng php tinh sch enzyme c s dng rng ri nht hin nay l

phng php ta enzyme bng dung mi hu c (ethanol, isopropanol v aceton).

T l dung mi v dch enzyme l mt ch tiu quan trng, nu thiu dung mi
s khng thu hi ht enzyme trong dung dch (Ng Xun Mnh v cng s , 2005).
Thng thng ngi ta thm 3 - 4 th tch dung mi vo 1 th tch nc chit enzyme.
trnh mt hot tnh ca enzyme, dung mi v enzyme phi c lm lnh xung 3 5oC. Phng php ny cho hiu sut cao, t nh hng n hot tnh enzyme, d tch
dung mi khi enzyme bng cch sy nh. C th thu hi dung mi bng chng ct.

Phng php ph bin th hai tch enzyme l dng mui trung tnh kt

ta. Mui trung tnh thng dng l (NH 4)2SO4 vi t l 50 - 60 % so vi dch chit
enzyme (Lng c Phm, 1998).

2.5. DEXTRIN (theo ti liu tng hp ca Nguyn Xun Thy, 2001)

Dextrin l sn phm c to thnh do s thy phn tinh bt m gi tr

tng ng dextrose (DE) t c nh hn 20. Dextrin c cng thc tng qut

ging vi cacbonhydrate nhng chiu di chui ngn hn v mc phc tp cng

thp hn.

14

Hnh 2.5. Dextrin

(http://www.infomed.co.il/Images/Glossary/g_1014.jpg)
2.5.1. Phn loi
Dextrin c th dng polymer mch thng, phn nhnh hay polymer mch
vng (cyclodextrin).
Da vo phng php x l ngi ta chia dextrin thnh 4 nhm chnh:
Dextrin thu nhn di tc ng ca enzyme.

Cc dextrin vng Sardinger thu nhn d i

tc ng ca vi khun
Bacillus macerans ln tinh bt.
trng nc.

Cc dextrin thu c bng cch dng acid thy phn tinh bt trong mi
Cc pirodextrin gm cc sn phm thu nhn c bng x l nhit (c hay

khng c acid) ln tinh bt.

Da vo tnh tan, ngi ta phn loi dextrin nh sau (L Hng Ph, 2000):
Amylosedextrin: tan trong ru 25%, khng tan trong ru 40%
Erythrodextrin: tan trong ru 70%, khng tan trong cn tuyt i.
Achrodextrin: tan trong cn tuyt i.
2.5.2. Tnh cht
c tnh ca dextrin ty thuc vo ch s DE t c, thng c nht cao v

cha mt lng ln cc polysaccharide trng lng ln.

Dextrin c bn cht keo, nhng mc phc tp v phn t th li thp hn.

15

Dextrin c x l enzyme to maltose, thy gii bng acid to glucose. Cho

nhiu mu sc khc nhau vi dung dch Iod. Dextrin c mc phc tp cao cho mu
xanh hoc ta. Cc dextrin c mc phn t trung bnh cho mu v cc dextrin
c mc phn t thp hn s khng to mu vi Iod.
Dextrin tan trong nc to dung dch trong nh nc tri cy, nhng khi c
glycogen dung dch dextrin s c mu trng c.
Bng 2.2. Mt s tnh cht ca dextrin
DE

pH

Tnh cht
nht cao, rt t ht m, t ngt, ha tan ti 15% cht kh, bt

4-7

4,0 - 5,0

mn.
Glucose 0,3% cht kh, maltose 0,9% cht kh
Rt t ht m, t ngt, t ha mu nu, ha tan ti 30% cht

9 -12

4,0 - 4,7

kh
Glucose 0,8% cht kh
Maltose 2,9% cht kh
t ht m, t bin mu nu, ngt nh, ha tan ti 60 - 70%

13 - 19,5 4,0 - 5,1

cht kh
Glucose 1,3 - 1,6% cht kh, maltose 4,8 - 5,8% cht kh.

2.5.2.1.
nht
nht l mt trong nhng yu t quan trng trong ng dng ca dextrin, li
dng nht ny m ngi ta sn xut huyt tng trong y hc.

nht dextrin c o bng Sangtipuaz bi cc pipet chuyn dng hoc cc

cng c chun khc.

nht ph thuc vo ngun tinh bt, nhit , nng acid, pH v tui
ca dung dch.

16

2.6.2.2. pH
pH nh hng n dextrin thnh phm, keo t v kt dnh.
Trong qu trnh thy gii, acid lm bin i tinh bt hay dextrin phn t
lng ln to dung dch long. D c trung ha tr li bng Na 2CO3 hoc NH3
trc khi ng gi, nhng acid vi mt lng d vn nh hng tnh keo trong qu
trnh tch ly.
2.6.2.3. Tui ca dung dch dextrin
S gia tng nht gy ra do s ng li hoc do s thoi bin ca dung dch
dextrin, c bit l nhng dextrin t ha tan.
Khi thm vo dung dch dextrin cht lm ng c (Tetraborate Natri 5 20%) th nht ca dextrin gia tng, thm cc cht nh urea, dicyandiamide,
salisilic acid, formaldehyde, cc mui guanadin s lm gim nht. Thm urea c th
thch hp dung dch dextrin trng thi lng.
2.6.2.4. ha tan
Giai on u ca s dextrin ha, khng c bin i ln v tan ca
dextrin trong nc lnh. nhit 130 - 140 oC, khi c acid th ha tan nhanh
chng t 100% nh ng h nng t sn phm ny khng bn v lc lm lnh th
khng to nn cc gel c.
2.6.2.5. Mu sc
Mu ca dextrin ph thuc acid v nhit dextrin ha.
Cc sn phm thu c nhit thp thng cho mu trng, cn nhng sn

phm thu c nhit cao c th mu nu. acid cao mt nhit nht nh

thng lm sm mu sn phm do qu trnh caramel ha.

C th ty trng dextrin vi cc peroxide hydrogen v bisulfite natri trong h

thng acid hay peborat natri trong dung dch kim.

17

2.5.3. Qui trnh sn xut dextrin (Nguyn Xun Thy,

2001)
Dung dch tinh bt
pH thch hp
H ha
to = 90 - 100oC
t = 2 - 3 pht
+ enzyme
Dch ha
to thch hp
t = 30 - 150 pht
Bt hot enzyme
Gia nhit
H pH
Ly tm
Dung dch dextrin c ln cc loi ng
Ta dextrin bng cn 96o
Ly tm

loi nc v ng ha tan

Dextrin

sy kh

S 2.1. Tm tt qui trnh iu ch dextrin t tinh bt

18

2.5.4. ng dng
Dextrin l sn phm trung gian gia tinh bt v ng n hay ng i.

Bn cht l ng nn chng tng i c c th d hp thu hn tinh bt.

Dextrin c s dng rng ri trong cng nghip v chng khng c c t v gi

thnh thp. c s dng nh dch keo c th ha tan trong nc nh h dn, dng

lm tc nhn ha dy trong ch bin thc phm v tc nhn kt dnh

trong dc

phm.
ng malto-dextrin ch yu dng lm thc n cho tr em, thc phm n king

vi lng nh calo pha long (Janusz vaf Anna, 2004), lm cht thm hay mu thc
phm, dng trong cng nghip dc phm.

Mt s dextrin c s dng thay th cho cht bo trong cc sn phm nh b

v m gy.
Trong cng nghip ru bia, dextrin c a vo n nh lng dextrin trong

sn phm, va l c cht cho qu trnh ln men va tng cht lng, to cm quan tt cho
sn phm.
Trong y hc, dextrin c pha ch vi glucose 5%, NaCl 9 hay sorbitol 5%

to dung dch huyt tng, dung dch ny dng cha tr cho bnh nhn mt mu
nhiu (L Hng Ph, 2000).

19

Ch ng 3

V T LI U V PH NG PHP TH NGHI M
3.1. THI GIAN V A IM THC HIN
Kha lun c thc hin ti phng th nghim Sinh ha trng i hc Khoa
Hc T Nhin thnh ph H Ch Minh.
Thi gian tin hnh: t 19/03/2007 n 19/07/2007.
3.2. VT LIU NGHIN CU
Ngun gc vi khun
Ging vi khun Bacillus subtilis do cng ty TNHH Gia Tng cung cp.
Nguyn liu
- Cm bp, b u nnh, bt nng Vnh Thun, bt np Vnh Thun, bt go
Vnh Thun, bt bp Ti K.
Thit b v dng c
- Cn in t

- Pipet

- T lnh

- Micropipet

- B iu nhit

- Bercher

- Ni hp

- Erlen

- T sy

- Bnh nh mc

- My ly tm

- ng ong

- My o mt quang

- Khay nha

Ha cht
- Cn 96%

- Iod

- NaCl

- HCl

- NaOH

- Na2HPO4

- (NH4)2SO4

- NaH2PO4

20

Cc loi mi trng

Mi trng gi ging (mi trng nc chit gi u ng pepton agar). Mi

trng nhn ging (mi trng nc chit gi u ng pepton). Mi trng
nui cy bn rn.
Thnh phn v t l cc loi mi trng c trnh by chi tit mc 1 phn
ph lc.
3.3. PHNG PHP TH NGHIM
3.3.1. Xc nh mt t bo Bacillus subtilis trong dch
tng sinh Cch tin hnh:
Dng que cy vng cy chuyn 1 vng vi khun B. subtilis t ng ging
vo erlen cha 20 ml mi trng tng sinh kh trng, lc u v t trn my lc (120
- 180 vng/pht).
Sau 24 gi kim tra s l ng t bo vi khun trong dch tng sinh bng

phng php m khun lc (c trnh by mc 2.1 v 2.2 ca phn ph lc).

3.3.2. Kho st thi gian nui cy ti u

Mi trng nui cy bn rn (35 g/erlen) c kh trng 1atm trong 45 pht

v ngui khong 40 - 50oC. Cy 5% dch vi khun tng sinh 24 gi vo mi

trng nui cy. nhit phng (30 - 32oC), trong thi gian 24, 36, 48, 60 v 72 gi.
Canh trng sau khi nui cy c sy kh 50oC.

Cn 1 g canh tr ng sy kh ha trong 100 ml n c ct, em ly tm 3000

vng/pht trong 10 pht ta c dch enzyme pha long 100 ln. T dch pha long ny
tip tc pha long n pha long thch hp xc nh hot tnh amylase theo phng php Heinkel (xem mc 2.5 phn ph lc).
3.3.3. Kho st t l ta enzyme bng cn 96%

Trong th nghim ny dng phng php ta enzyme bng cn 96% lnh (xem

mc 2.3 phn ph lc).

Cn 5 g canh trng sy kh v nghin nh ha trong 80 ml nc ct,
lc u trong 15 pht. Ly tm 3000 vng/pht trong 10 pht, thu dch chit enzyme.

21

Ly 10 ml dch chit enzyme trn em ta vi cn 96% lnh khong 4oC

theo cc t l 1:1, 1:2, 1:3, 1:4 v 1:5, lc u, lnh trong 15 pht ri ly ra em ly
tm 4000 vng/pht trong 10 pht. Loi b cn, thu cn lng v ha trong 5 ml m
acetate pH = 5,0. nh mc vi nc ct thnh 100 ml.

Ht 1 ml dch enzyme ny em nh l ng
protein theo ph ng
php

Lowry (xem mc 2.6 phn ph lc) v xc nh hot tnh theo ph ngphp

Heinkel.
Hiu sut thu hi hot amylase t 10 ml DCE c tnh theo cng thc:
H (%) =

Hot -amylase sau ta

Hot -amylase trc ta

x 100

Hiu sut thu hi protein t 10 ml DCE c tnh theo cng thc:

H (%) =

Lng protein sau ta

Lng protein sau ta

x 100

3.3.4. Kho st t l ta enzyme bng (NH4)2SO4

Tng t, chun b dch chit enzyme t canh trng ging nh phn ta bng

cn 96%.
Cn chnh xc lng mui (NH4)2SO4 (xem mc 2.6 phn ph lc). Cho t t
vo cc erlen cha 10 ml dch enzyme lnh, lc u trong 10 pht. Sau em
lnh. Sau 10 pht, em ly tm 4000 vng/pht trong 10 pht. Cc bc k tip tin
hnh ging nh phn ta bng cn 96%.

3.3.5. Kho st thi gian ta cn 96%

Cc bc tin hnh ging nh phn kho st t l cn, nhng th tch cn th

nghim ny khng thay i. T l cn 96% v enzyme l t l thch hp xc nh

c mc 3.3.3.

Kim tra hot tnh -amylase v nh lng protein vi thi gian ta l 15, 30,

45, 60 v 75 pht.

22

3.3.6. Kho st cc yu t nh hng n kh nng nng thy phn

tinh bt ca ch phm -amylase
3.3.6.1. Nhit phn ng
Cn 0,03 g ch phm -amylase tinh sch s b bng cn 96% sy kh ha
trong 50 ml n c ct, pha long thm 100 ln. o hot tnh -amylase theo ph ng
php Heinkel cc gi tr nhit : 30, 40, 45, 50, 55, 60 v 65 oC.
3.3.6.2. pH
Tin hnh kho st nh hng ca cc gi tr pH: 5,8; 6,0; 6,2; 6,4 v 6,6 ln
hot -amylase nhit thch hp.
Kim tra hot tnh -amylase mi gi tr pH theo phng php Heinkel.

3.3.7. Kho st t l cn 96% thch hp thu dextrin

Tinh bt sau khi h ha c ngui n nhit thch hp v cho ch

phm enzyme (CPE) pha long vo (xem mc 2.7 phn ph lc). Sau 30 pht em
ly tm 3000 vng/pht trong 10 pht loi b tinh bt tha.
Dch ly tm c cho vo cc erlen c cha cn theo cc t l 1:2, 1:3 v

1:4, ly tm 4000 vng/pht trong 10 pht, loi dch cn v cn lng dextrin thu
c.

3.3.8. Kho st kh nng thy phn tinh bt ca ch phm -amylase

3.3.8.1. Kho st kh nng thy phn vi cc ngun tinh bt

Thc hin phn ng thy phn vi cc ngun tinh bt khc nhau: bt go, bt
nng, bt np, bt bp nng 10%.
Tin hnh: 2 g tinh bt c h ha trong 18 ml dung dch m gi tr pH ti

u; lm ngui n nhit thch hp v cho vo 0,5 ml ch phm -amylase pha

long 200, 400, 600 ln, khuy u, lin tc.

Sau 10 pht, cho 5 ml HCl 5% dng phn ng. Ly tm loi b tinh bt tha,
dch ly tm c ta cn thu dextrin. Cn lng dextrin to thnh v so snh khi
lng dextrin c c gia cc ngun tinh bt.

23

Hiu sut thu dextrin tnh theo cng thc:

H (%) =

Dextrin thu c (g)

x 100

Tinh bt ban u (g)

3.3.8.2. Kho st nng tinh bt thch hp

Sau khi chn c ngun tinh bt thch hp, tin hnh kho st l ng

dextrin to thnh cc nng tinh bt l 10%, 15% v 20%.

Cho th tch v pha long CPE thch hp vo 20 g h tinh bt vi nng
tinh bt 10% v 15% v 20%. Thi gian kho st l 10, 20, 30 v 40 pht.
3.4. PHNG PHP X L S LIU

S liu c x l sai s v v th, biu bng phn mm Microsoft Excel

2003.

24

Ch ng 4
K T QU V TH O LU N
4.1. KT QU KHO ST THI GIAN NUI CY B. SUBTILIS
Tin hnh th nghim theo phn 3 mc 3.3.2, canh trng nui cy sau cc thi

im 24, 36, 48, 60, 72 gi c kim tra hot tnh bng phng php Heinkel. Thi
gian nui cy 48 gi -amylase c hot cao nht l 468865 UI/g canh
(CT). Kt qu th nghim c trnh by bng 4.1 v th 4.1.

trng

Bng 4.1. nh hng ca thi gian nui cy ln hot -amylase

Thi gian
(gi)

Tinh bt b
pha long

OD

thy phn
(mg/ml)

Hot
(UI/g CT)

24

10000

0,296 0,0006

5,227 0,011

52272 105

36

80000

0,261 0,0018

4,585 0,032

366817 3802

48

100000

0,267 0,0012

4,689 0,021

468865 2108

60

100000

0,252 0,0017

4,427 0,030

442700 3042

72

100000

0,231 0,0009

4,044 0,016

404365 1610

Trong khong thi gian nui cy t 24 - 36 gi hot ca amylase tng

nhanh l do Bacillus subtilis trong giai on pht trin v phn chia ti a.
Ti thi im 48 gi, mt B. subtilis n nh v lng -amylase sinh ra t
cc i nn hot -amylase lc ny cao nht. T 48 gi tr i, vi khun cht dn do
cn dinh dng v hu nh khng sinh -amylase na; trong khi , lng nc c trong
canh trng trong thi gian di lm hot -amylase gim dn.

Hot (UI/g CT)

25

468865

500000

442700

400000

404365

366817

300000
200000
100000

52272

0
24

36

48

60

72

Thi gian
(gi)

th 4.1. nh hng ca thi gian nui cy ln hot -amylase

Hnh 4.1. Mi tr ng
bn
rn trc khi

nui

Hnh

4.2.

Canh

tr ng
nui c y

sau 48

4.2. K T QU TINH S CH S B -AMYLASE B NG C N 96% V

(NH4)2SO4
4.2.1. Kt qu kho st t l ta enzyme bng cn 96%
Hot -amylase trc ta l 50205,64 (UI/10ml dch chit enzyme), hm

lng protein c trong 10 ml dch chit enzyme trc ta l 97,032 mg/10ml. Sau

khi ta, ly tm loi b cn v o hot tnh bmg phng php Heinkel kt hp nh

lng protein bng phng php Lowry theo phn 3 mc 3.3.3; chng ti ghi nhn
t l cn 96% v enzyme l 3:1, -amylase c hot cao nht l 45607,75

26

UI/100ml ch phm -amylase ti). Kt qu kho st c trnh by bng 4.2 v

th 4.2.
Bng 4.2. nh hng ca t l cn 96% ln kh nng ta -amylase
Enzyme/cn

Hiu sut

Protein

thu hi

(mg/100ml CP ti)

protein (%)

Hiu sut

Hot
(UI/100ml CP ti)

thu hi hot
(%)

10,462 0,232

10,78

4366,90 190,003

8,698

1:2

15,963 0,568

16,45

44056,08 80,497

87,751

1:3

21,756 0,232

22,42

45607,75 185,067

90,842

1:4

23,127 0,154

23,83

44284,27 109,698

88,206

1:5

23,696 0,154

24,42

43219,40 270,422

86,085

Ho t (UI/100 ml CP t i)

1:1

60000
50000

44056

40000

45608

44284
43219

30000
20000
10000
4367

0
1

Vcn/Venzyme

th 4.2. nh hng ca t l cn 96% ln kh nng ta -amylase

t l 1:1, nng cn cn thp ch kt ta c mt lng nh protein

(-amylase) nn hot -amylase thp. Nh ng

t l 4:1 v 5:1, tuy l ng

protein thu c cao hn, song hot -amylase li thp hn t l 3:1 l do hai
t l trn nng cn qu cao lm protein b bin tnh nn hot -amylase
gim.

27

4.2.2. Kt qu kho st t l ta -amylase bng (NH4)2SO4

Th nghim c tin hnh theo phn 3 mc 3.3.4. Kt qu ghi nhn bng
4.3 v th 4.3.
Bng 4.3. nh hng ca (NH4)2SO4 ln kh nng ta -amylase
bo

Hiu sut

Hm lng protein

Hiu sut

Hot
(UI/100 ml CP ti )

thu hi hot

15,02

31835,132 205,852

61,73

15,846 0,101

16,08

36916,077 73,132

71,58

60

16,293 0,195

16,53

37575,282 20,283

72,86

65

16,683 0,108

16,92

37281,175 123,378

72,29

70

19,037 0,166

19,31

36926,219 126,669

71,60

(mg/100 ml CPti )

50

14,805 0,118

55

Hot (UI/100 ml CP ti)

ha (%)

thu hi
protein (%)

(%)

50000
40000

36916

30000

37575

37281
3693

31835

20000
10000
50
55

60

65

70

(NH4)2SO4 (% bo ha)

th 4.3. nh hng ca (NH4)2SO4 ln kh nng ta -amylase

Ta bng (NH4)2SO4 60% bo ha, -amylase c hot cao nht.
70%, hot -amylase gim, do nng mui qu cao lm bin tnh protein.
Theo bng 4.2 v 4.3, ta bng cn 96% hot ca -amylase v lng

protein thu hi c cao hn khi ta bng (NH4)2SO4. V vy, chng ti chn cn 96%
l tc nhn thch hp dng ta -amylase.

28

4.3. KT QU KHO ST THI GIAN TA CN 96%

Tin hnh th nghim kho st thi gian ta theo phn 3 mc 3.3.5. Theo
kt qu ghi nhn bng 4.4, thi gian ta tt nht i vi -amylase l 15 pht.
Thi gian ta cng di lng protein thu c cng nhiu nhng hot ca -

amylase khng tng do lng -amylase b bin tnh tng theo thi gian tip xc vi
cn 96%.

Bng 4.4. nh hng ca thi gian ta cn 96% ln kh nng ta -amylase

Hm lng protein

Hiu sut

(UI/100ml CP ti)

thu hi hot

22,15

50369,23 139,424

93,24

22,164 0,127

22,84

48741,50 30,425

90,23

45

23,069 0,101

23,77

45942,42 91,274

85,05

60

23,842 0,228

24,57

45729,45 121,699

84,65

75

24,324 0,077

25,07

44497,24 290,234

82,37

48742

ta (pht)

(mg/100ml CP ti)

15

21,493 0,134

30

thu hi

Hiu sut

Hot

Hot (UI/100 ml CP ti)

Thi gian

potein (%)

(%)

60000
50369
50000

45942

45729

40000

44497

30000
15

30

45

60

75

Thi gian (pht)

th 4.4. nh hng ca thi gian ta cn 96% ln kh nng ta amylase

29

4.4. KT QU KHO ST CC YU T NH HNG N KH NNG

THY PHN TINH BT CA CH PHM -AMYLASE
Thc hin th nghim theo phn 3 mc 3.3.6. Sau khi o hot tnh bng phng php

Heinkel, chng ti thu c kt qu nh sau:

4.4.1. nh hng ca nhit

Bng 4.5. nh hng ca nhit ln hot ca -amylase

Tinh bt b thy

Hot

phn (mg)

(UI/g CPE)

0,285 0,0041

5,032 0,075

838742 12462

40

0,320 0,0018

5,662 0,032

943708 5366

45

0,326 0,0015

5,781 0,027

963484 4421

50

0,423 0,0010

7,536 0,018

1256069 3042

55

0,446 0,0009

7,962 0,016

1327060 2683

60

0,414 0,0018

7,375 0,032

1229194 5366

65

0,309 0,0038

5,464 0,069

910748 11519

OD

30

Hot (UI/g CPE)

Nhit (oC)

1500000

1327061
1256069

1300000
1100000

943708

1229194

963484

900000

910748

838742
700000
500000
30

35

40
45
50
Nhit (oC)

55

60

65

th 4.5. nh hng ca nhit ln hot ca -amylase

30

Theo s liu bng 4.5 cho thy nhit ti u ca -amylase l 55oC vi hot
cao nht l 1327061 (UI/g ch phm enzyme). 60oC, hot -amylase bt u
gim xung.
4.4.2. nh hng ca pH
C nh nhit ti thch l 55oC, thc hin phn ng thy phn vi gi
tr pH thay i theo phn 3 mc 3.3.6.2. Kt qu c ghi nhn bng 4.6 v th
4.6.
pH 5,8

6,0

6,2

6,4

6,6

Hnh 4.3. nh hng ca pH ln hot ca -amylase

Bng 4.6. nh hng ca pH ln hot ca -amylase
Tinh bt b thy

pH

OD

5,8

0,433 0,0033

7,734 0,060

1255562 9988

6,0

0,438 0,0035

7,810 0,064

1301707 10685

6,2

0,419 0,0013

7,469 0,024

1244914 4057

6,4

0,421 0,0009

7,515 0,016

1238829 2683

6,6

0,402 0,0003

7,156 0,006

1192684 1014

phn (mg/ml)

Hot (UI/g CPE)

T bng 4.6, chng ti thy 55oC, pH ti u ca -amylase l 6,0 vi hot

l 1301707 (UI/g ch phm enzyme).

Hot (UI/g CPE)

31

1400000
1301707
1300000
1200000

1244914

1238829
1192684

1255562

1100000
1000000
5,8

6,2

6,4

6,6

pH

th 4.6. nh hng ca pH ln hot ca -amylase

4.5. KT QU KHO ST T L CN 96% TA DEXTRIN

Bng 4.7. Lng dextrin thu c cc t l cn 96%

Khi lng dextrin (g)

1:1

0,31 0,021

2:1

1,29 0,009

3:1

1,18 0,019

4:1

1,17 0,015

Dextrin (g)

Cn 96% : dch dextrin

1,5

1,29

1,18

1,17

1,0
0,5

0,31

0,0
1

Vcn/Vdextrin

Biu 4.1. L ng dextrin thu c t cc t l c n 96%

32

T l cn 96% thu dextrin l 2:1, t l cn tng th khi lng dextrin

thu c gim do lng cn cng nhiu lm cho lng nc trong dextrin tch ra
cng nhiu.

4.6. KT QU KHO ST KH NNG THY PHN CA -AMYLASE VI

CC NGUN TINH BT
Thc hin th nghim theo phn 3 mc 3.3.8 vi bt bp, bt np, bt go, bt
nng nng 10%, 0,5 ml enzyme, thi gian thy phn l 15 pht. Kt qu c ghi
nhn bng 4.8.
Bng 4.8. Lng dextrin (g) to thnh do -amylase thy phn tinh bt 10%
Khi lng dextrin (g)

pha long
CPE

Bt go

Bt np

Bt bp

Bt nng

200

1,03 0,058

1,31 0,032

0,99 0,023

1,39 0,018

400

1,40 0,084

1,58 0,061

1,23 0,026

1,94 0,044

600

1,35 0,087

1,56 0,04

1,45 0,031

1,87 0,053

2,50
1,94

Dextrin (g)

2,00

1,58

1,50
1,00

1,87

1,31
1,03

1,39

0,99

1,40

1,23

1,56
1,35

1,45

Bt go
Bt np
Bt bp
Bt nng

0,50
0,00
200

400

600

pha long enzyme (ln)

Biu 4.2. Kh i l ng dextrin thu c khi thy phn h tinh b t

33

Bng 4.9. Hiu sut thu dextrin

pha long

Hiu sut thu dextrin (%)

CPE (ln)

Bt go

Bt np

Bt bp

Bt nng

200

51,67 2,89

65,50 1,61

49,33 1,17

69,67 0,08

400

69,83 4,21

79,00 3,04

61,33 1,30

96,83 2,19

600

67,67 4,34

78,00 2,02

72,50 1,53

93,50 2,65

T bng 4.8 v bng 4.9 cho thy lng dextrin thu c cao nht khi s dng

c cht l bt nng pha long CPE l 400 ln vi lng dextrin thu c l 1,94
gam, hiu sut 96,83%.
4.7. KT QU KHO ST NNG BT NNG V THI GIAN THY
PHN H BT NNG BNG CH PHM -AMYLASE
Theo bng 4.8 v 4.9, chng ti thy -amylase thy phn tt pha
long 400 ln. V vy chng ti tin hnh th nghim ny vi ch phm -amylase pha
long 400 ln.
Sau khi tin hnh th nghim kho st s b s thy phn tinh bt vi cc th
tch -amylase pha long 400 ln, chng ti chn ra nhng th tch thch hp thc
hin th nghim ny: th tch -amylase cho vo 20 g h tinh bt nng 10% v 15% l
0,5 ml enzyme; 20 g h tinh bt nng 20% l 1,5 ml enzyme. Kt qu ghi nhn bng
4.10, 4.11 v 4.12.
Bng 4.10. Hiu sut thu dextrin khi thy phn bt nng 10%
Thi gian thy phn (pht)

Khi lng dextrin (g)

Hiu sut (%)

10

1,93 0,032

96,5 1,61

20

1,86 0,015

93,17 2,73

30

1,72 0,055

85,83 0,77

40

1,62 0,032

81,17 1,59

34

Dextrin (g)

2,5
2,0

1,93

1,86

1,72

1,62

1,5
1,0
0,5
0,0
10
20
30
40
Thi gian thy phn (pht)

Biu 4.3. Khi lng dextrin thu c khi thy phn h bt nng 10%
Sau khi thy phn ht tinh bt, -amylase s chuyn sang thy phn
dextrin. V vy, nu ko di thi gian thy phn lng dextrin thu c s thp dn.
Bng 4.11. Hiu sut thu dextrin khi thy phn bt nng 15%
Thi gian thy phn (pht)

Khi lng dextrin (g)

Hiu sut (%)

10

2,01 0,071

67 2,36

20

2,37 0,065

78,89 2,16

30

2 0,081

66,67 2,69

40

1,93 0,069

64,22 2,31

Dextrin (g)

3,0
2,5

2,01

2,37

2,03

1,93

30

40

2,0
1,5
1,0
0,5
0,0
10

20

Thi gian thy phn (pht)

Biu 4.4. Khi lng dextrin thu c khi thy phn h bt nng 15%

35

Bng 4.12. Hiu sut thu dextrin khi thy phn bt nng 20%
Thi gian thy phn (pht)

Khi lng dextrin (g)

Hiu sut (%)

10

2,25 0,072

56,25 1,81

20

2,507 0,086

62,67 2,14

30

2,363 0,102

59,08 2,54

40

2,303 0,064

57,58 1,59

Dextrin (g)

4,0
3,0
2,25

2,51

2,36

2,30

2,0
1,0
0,0
10
20
30
40
Thi gian thy phn (pht)

Biu 4.5. Khi lng dextrin thu c khi thy phn h bt nng 20%
i vi h bt nng 15% v 20%, do nng tinh bt cao lm cho kh
nng thy phn tinh bt ca -amylase gim nn hiu sut thu dextrin khng cao.
30 v 40 pht mc d lng tinh bt vn cn nhiu nhng lng dextrin thu c vn
khng tng.
Khi lng dextrin thu c nhiu nht khi thy phn bt nng 10% l 1,93 g
t hiu sut l 96,5% v thi gian thy phn l 10 pht (theo bng 4.10).
i vi dung dch bt nng 15%, lng dextrin nhiu nht l 2,37 g, hiu sut
78,89% v thi gian thy phn l 20 pht (theo bng 4.11).

36

Dung dch bt nng 20% c l

ng dextrin cao nht l 2,5 g, hiu sut
62,67% v thi gian thy phn l 20 pht (theo bng 4.12).

Hnh 4.4. Ch phm -amylase

Hnh 4.5. Ch phm dextrin

37

Ch ng 5
K T LU N V NGH
5.1. KT LUN
- Thi gian nui cy tt nht thu -amylase t Bacillus subtilis l 48 gi.
- Tinh sch s b dch chit -amylase t canh trng nui cy bng cn 96%
tt hn (NH4)2SO4 vi t l cn 96% thch hp nht l 1:3 vi hiu sut thu enzyme
theo hot l 90,84% v gian ta l 15 pht.
- Nhit v pH ti u cho hot ng thy phn ca -amylase tng ng l
55oC v 6,0.
- Lng dextrin thu c cao nht (1,94 g) t hiu sut 96,83% khi thy phn
bt nng (khoai m) 10%.
5.2. NGH
- Ti u cc iu kin nui trn khay thu -amylase c hot cao hn.
- Ta enzyme nhiu nng cn khc nhau.
- Xc nh lng tinh bt c trong cc ngun tinh bt kho st.
- nh lng dextrin tht trong ch phm dextrin.

- Tch v kho st tnh cht cc phn on dextrin t sn phm dextrin thu

c; nghin cu ng dng ch phm dextrin vo trong ch bin thc phm v y
dc.

38

TI LI U THAM KH O
Ti liu ting Vit
1. Lm Th Kim Chu, Nguyn Nh Nht, 2006. Phng php kim tra
mt s enzyme dng trong chn nui. Gio trnh khoa Sinh Hc B
mn
Sinh Ha i hc Khoa Hc T Nhin, TP. H Ch Minh, tr. 16 - 17.
2.

Lm Th Kim Chu, Vn c Chn, Ng i Nghip, 2004. Thc tp ln

sinh ha. Nh xut bn i Hc Quc Gia, TP. H Ch Minh, tr. 36 - 46.

3.

L Hng Ph, 2000. Kho st qui trnh iu ch dextrin t mt s loi

tinh bt bng s thy gii bi acid HCl. Kha lun tt nghip Khoa
Sinh Hc, i hc Khoa Hc T Nhin, TP. H Ch Minh.

4.

Lng c Phm, 1998. Cng ngh vi sinh v t . Nh xut bn Nng

nghip H Ni, tr. 249 - 257.

5.

Nguyn c Duy Anh, 2005. Xc nh mi tr ng t i u thu sinh

khi v enzyme ca vi khun Bacillus subtilis, Lactobacillus
acidophilus v th nghim sn xut ch phm sinh hc. Kha
lun tt nghip ngnh Cng Ngh Sinh Hc, i hc Nng Lm, TP. H
Ch Minh.

6.

Nguyn c L ng, 2004. Cng ngh s n xu t m chnh v cc s n

phm ln men c truyn. Nh xut bn Khoa Hc K Thut.

7.

Nguyn Qunh Nam, 2006. Phn l p vi khu n Bacillus subtilis trong

phn heo v th i khng vi Echerichia coli gy bnh tiu
chy tr n heo. Lun vn tt nghip Khoa Cng Ngh Sinh Hc, i
hc Nng Lm, TP. H Ch Minh.

8.

Nguyn Tin Thng, 2002. Gio trnh Cng ngh enzyme . Vin Sinh
Hc Nhit i, TP. H Ch Minh, tr. 42- 73.

9.

Nguyn Th Nh Thy, 2005. Kho st cc c tnh ca enzyme amylase

thu nhn t hai chng nm mc Aspergillus niger v
Aspergillus oryzae. Kha lun c nhn khoa hc ngnh Sinh Ha, i
hc Khoa Hc T Nhin TP. H Ch Minh.

10. Nguyn Th Thu Thy, 2006. Kho st kh nng tng hp -amylase v

mt s iu kin k thut nh h ng ln qu trnh tng sinh ca
Bacillus

39

sp. Bo co thc tp tt nghip ngnh Sinh Ha, i hc Khoa Hc T

Nhin TP. H Ch Minh.
11. Nguyn Xun Thy, 2001. Kho st qui trnh iu ch dextrin t
mt s
loi tinh bt bng s thy gii bi enzyme. Kha lun c nhn
khoa hc
ngnh Sinh Hc, i hc Khoa Hc T Nhin, TP. H Ch Minh.
12. Phm Th nh Hng, 2003. K Thut Sinh Ha. Nh xut bn i hc
Quc Gia TP. H Ch Minh.
13. Trn Quyn, 2004. Thu nh n v kh o st ho t tnh c a ch
phm
alpha amylase t Bacillus subtilis . Bo co thc tp tt nghip
ngnh
Sinh Ha, i hc Khoa Hc T Nhin TP. HCM.
14. Trn Linh Thc, 2005. Phng php phn tch vi sinh vt trong
thc
phm v m phm. NXB Gio Dc, tr. 65 - 71.
Ti liu ting Anh
15. K. Clarkson, B. Jones, R. Bott, B. Bower, G. Chotani, T. Becker,
Enzymes: Screening, Expression, Design and Production, Enzymes in
Farm Animal Nutrition (Michael R. Bedford, Gary G. Partridge). CABI
publishing, OCAB International, 2001, p. 337 - 346.
Ti liu t internet
16. Ng Xun Mnh, V Nhn Hu, Nguyn Th T. Nghin cu cc iu
kin ti u cho vic thu nhn -amylase chu nhit t vi
khun Bacillus
licheniformis.
< http://www.hau1.edu.vn/tc_khktnn/upload%5CVoNhanHau_kt&cn52005.pdf>
17. Steven J. McWethy, Paul A. Hartman. Purification and
Some
Propeerties of an Extracellular Alpha-amylase from
Bactriodes
amylophilus. Journal of Bacteriology, March. 1977, p. 1537 - 1544.
(URL: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=235133)
18. Janusz Kapusniak, Anna Tyflewska. The utilisation of dextrins from
the
thermolysis of starch with biogenic amino acids by
Lactobacillus

rhamnosus bacteria. Food, Agriculture & Enviroment Vol 2 (1): 153 159. 2004, Science and Technology, WFL Publisher.
<URL: http://www.world-food.net/scientificjournal/2004/issue1/pdf/food/f27.pdf>

40

19. Hopek M., Ziobro R, Achremowicz B., 2006. Comparison of the

effects
of microbial a-amylase and scalded flour on bread quality.
Acta Sci. Pol,
Technol. Aliment .5(1), 97 - 106.
<URL: http://www.food.actapol.net/pud/8_1_2006.pdf>
20. O. S. AZEEZ, 2005. Production of Dextrins from Cassava
Starch .
Chemical Engineering Department, Federal University of Technology,
Minna, Nigeria, Lenardo Journal of Sciences ISSN 1583 - 0233.
<URL: http://ljs.academicdirect.org/A07/09_16.pdf>
21. Dextrin. The Colombia Encylopedia, Sixthedition. Copyright 2001 - 05
Colombia University Press.
<URL:http:// www.bartleby.com/65/de/dextrin.html>
22. Bacillus subtilis
<URL: http://en.wikipedia.org/wiki/Bacillus subtilis>
<URL: http://www.epa.gov/oppt/biotech/pubs/fra/fra009.htm>

41

PH L C
1. THNH PHN MT S LOI MI TRNG

1.1. Mi trng nc chit gi u ng pepton agar

Gi u

100g

D-glucose

50g

Pepton

10g

Agar

20g

Nc ct

1000 ml

pH = 7
un si 100g gi vi 1000 ml nc ct trong 30 pht, ngui. Lc nc gi
bng vi lc v nh mc li cho 1000 ml. Cho glucose, pepton, agar vo v un
si, khuy u. Phn vo tng ng nghim, mi ng khong 10 ml mi trng, hp
kh trng 1atm trong 30 pht. Cc ng nghim sau khi hp c nghing.
1.2. Mi trng nc chit gi u ng pepton
Gi u

100g

D-glucose

50g

Pepton

10g

Nc ct

1000 ml

pH = 7
Cho Glucose, pepton vo nc chit gi, khuy u v cho vo cc erlen. Hp kh
trng 1atm trong 30 pht.

42

1.3. Mi trng nui cy bn rn

B u nnh

50g

Cm bp

50g

NaCl

0.36g

CaCl2

0.6g

DAP

0.2g

MgSO4

0.5g

Nc ct

135 ml

pH = 7
2. CC PHNG PHP THC HIN

2.1. Phng php m khun lc

Khc vi ph ng php m trc tip d i knh hin vi, ph ng php m

khun lc cho php xc nh s lng t bo vi sinh vt cn sng hin din trong

mu.
u im ca phng php ny l nhy cao, cho php nh lng vi sinh vt
mt thp trong mu.
Cch thc hin
- Pha long mu theo dy thp phn:
Mu c pha long tun t thnh dy cc nng thp phn: ht 1 ml mu (hoc

dung dch c pha long trc ) vo ng nghim cha 9 ml nc ct kh trng,

lc u.
- Sau khi pha long thnh cc nng 10-3,10-4, 10-5, 10-6, ht 1 ml dch pha
long mi nng cho vo a petri. khong 15 - 20 ml mi trng kh
trng v ngui n 45 - 50oC vo a petri c mu.
- Xoay nh a petri cng chiu v ngc chiu kim ng h vi ln dung dch

ging c trn u trong mi trng cy.

- y np a petri, ng t nhin, lt ngc a.

- Sau 24 gi tin hnh m s lng khun lc mc trn a.

43

Tnh kt qu

Mt t bo vi khun trong mu ban u tnh t s liu ca pha long D i c

tnh theo cng thc:
Mi (CFU/ml) = Ai x Di/V
Trong : Ai l s khun lc trung bnh /a
Di l pha long
V l dung tch huyn ph t bo cho vo mi a (ml).
Mt t bo trung bnh MI trong mu ban u l trung bnh cng ca Mi cc
nng pha long khc nhau.
2.2. Phng php o
c
Nguyn tc
Khi mt pha lng c cha nhiu phn t khng tan th s hnh thnh mt h
huyn ph v c c bi cc phn t hin din trong mi trng lng cn nh
sng, lm phn tn chm nh sng ti.
T bo vi sinh vt cng l mt thc th nn khi hin din trong mi trng cng lm

mi trng tr nn c. c ca huyn ph t l vi mt t bo.

Tin hnh

- Xy dng ng tng quan tuyn tnh gia c v mt t bo:

Cc huyn ph ca vi sinh vt cn kim nghim c pha long v o OD610nm t

cc gi tr ln cn 0,1; 0,2; 0,3; 0,4 v 0,5.

Dng phng php m khun lc xc nh mt t bo (N/ml) ca cc huyn ph
ny.
Tnh gi tr log(N/ml) cho mi gi tr mt N/ml tng ng vi mi c. V

ng biu din ca log(N/ml) (trc tung) theo OD 610nm (trc honh). Xc nh khong
tuyn tnh gia log(N/ml) v OD610nm.
- Xc nh mt t bo theo c:
o c ca huyn ph t bo cn xc nh mt
T tr s OD610nm o c, da vo ng tng quan gia log(N/ml) v c
OD610nm suy ra tr s log(N/ml) v tr s mt N/ml (N/ml = 10a vi a = log(N/ml).

44

2.3. Phng php tinh sch enzyme

bng

cn

Nguyn tc
ha tan ca protein trong dung dch ph thuc vo nhiu yu t, mt trong s
l hng s in mi ca dung dch. Nhn chung, nhng phn t c hng s in
mi ln (nh n c, dimethylsulphoxid) c th n nh cc t ng tc gia chng vi
cc phn t protein v to iu kin thun li cho s ha tan ca protein trong dung
dch. Ng c li, cc dung mi vi hng s in mi nh (aceton,

ethanol) ngn

cn s phn tn ca cc phn t protein trong mi trng. Do , ha tan ca nhng

phn t protein gim v xy ra s kt ta do s lm gim hng s in mi hin hu
ca mi trng.

Cch tin hnh

Sau khi cho cn vo, enzyme s b kt ta. Ly tm loi cn, thu ta enzyme.
Ta enzyme c ha tan trong dung dch m acetate (pH=5) v o hot tnh

enzyme bng phng php Heinkel kt hp vi nh lng protein bng phng php
Lowry.
2.4. Phng php ta enzyme bng
(NH4)2SO4
Nguyn tc
nng mui cao th kh nng ha tan ca enzyme gim mnh v c xu
hng kt lng v nng mui cao lm gim hng s in mi ca dung dch, lm
protein b mt nc.

Cch tin hnh

Cn chnh xc lng mui (NH4)2SO4 ng vi cc nng bo ha trong 10 ml dch

chit enzyme (xem bng 2.1).

Bng 2.1. Khi lng tng ng cc bo ho
bo ho
(%)
(NH4)2SO4
(gram)

50

55

60

65

70

3,1

3,51

3,9

4,3

4,72

45

Cho 10 ml dch chit enzyme vo erlen v

a vo my khuy t, cho t t
mui vo. Sau 30 pht ly ra ly tm.
Phng

2.5.

php

Heinkel
Thc hin
Cho vo ng nghim 1 ml dung dch tinh bt 1%, 1 ml dung dch NaCl 0,1%, 2
ml dung dch m phosphate 0,05M, pH 6,0. Lc u, ng nghim trong t m
30oC, 15 - 20 pht dung dch t n 30oC. Thm 1 ml dung dch enzyme pha
long v t n 30 oC vo ng nghim. Lc u v tip tc gi 30 oC trong 30
pht. Ly ng nghim ra khi t m cho ngay vo 5 ml dung dch HCl 1N dng
phn ng.
Song song vi mu th nghim, lm mu kim tra i chng tng t nh trn

nhng cho dung dch HCl vo trc ri cho enzyme vo.

Ly 1 ml mu th nghim v i chng cho vo ng nghim mi, thm vo 9 ml dung

dch Iod pha long 450 ln. Lc u, so mu bc sng 560 nm. Ly hiu s c c

trn my gia mu i chng v th nghim i chiu vi ng cong tiu chun, tnh s

mg tinh bt b thy phn.
Cch tnh
n v hot ng ca enzyme l lng enzyme c kh nng phn gii 1 mg tinh
bt thnh cc sn phm khng to mu vi Iod sau 30 pht iu nhit th
nghim.
Hot A = S mg tinh bt b phn gii * pha long (UI).
2.6. Phng php Lowry nh lng protein ha tan trong
nc

Nguyn tc
Hu ht cc protein u cha Tyrosin v Tryptophan. Hm lng ca nhng
amino acid ny ty thuc vo loi protein. V vy, nhng protein cng mt loi vi nhau
c hm lng cc amino acid ny ging nhau.
Khi cho protein tc dng vi thuc th Folin s to thnh mt phc cht c
mu. C ng mu ca phc ny t l vi hm l ng Tyrosin v Tryptophan

46

(cng l lng protein). V th ta c th dng phng php so mu xc nh hm

lng protein.

Ha cht
Dung dch albumin 0,1%: cn chnh xc 0,1 g albumin pha vi nc ct thnh
100 ml.
Dung dch A: cn 2 g Na2CO3 ha trong NaOH 0,1N thnh 100 ml
Dung dch B: 0,5 g CuSO4.5H2O ha trong natri citrate 1% thnh 100 ml
Dung dch C: hn hp A v B theo t l 49:1. dung dch C pha trc khi dng 30
pht.
Thuc th Folin: pha long 3 ln t Folin m c.
Cch tin hnh
+ Lp th chun:
Chun b dung dch protein chun c cc nng : 0, 50, 100, 150, 200, 250
g/ml t dung dch albumin chun 0,1% pha long vi nc ct theo cc t l khc nhau
(xem bng 2.1)
Bng 2.2. Cch pha cc nng albumin
S ng nghim

Albumin 1% (ml)

0,5

1,5

2,5

Nc ct (ml)

10

9,5

8,5

7,5

Protein (g/ml)

50

100

150

200

250

Ht 0,4 ml dung dch albumin pha long theo bng 3 t cc ng 1 - 6 cho vo 6

ng nghim mi theo th t. Thm vo 2 ml dung dch C, lc u, yn nhit
phng trong 5 pht. Thm 0,2 ml Folin, lc u, yn 5-10 pht, thm nc ct 5
ml. So mu bc sng 750 nm.

Xc nh tr s mt quang ca cc ng, t xy dng th biu din s bin

thin ca mt quang (OD) theo nng protein chun (g/ml).

47

+ Xc nh hm lng protein trong mu:

Ht vo ng nghim 0,4 ml dung dch mu th nghim. Thm vo 2 ml dung dch
C, lc u, yn trong 5 pht.
Thm 0,2 ml thuc th Folin, lc u, yn 5 - 10 pht, thm nc ct cho

5 ml. em o mt quang bc sng 750 nm.

Cch tnh

T ng chun so snh mt quang ca ng nghim cha mu protein, t suy

ra hm lng protein ca nguyn liu l a (g/ml)

Lng protein (M) c trong 1 g nguyn liu c tnh theo cng thc:
-3
M= a x 10 x n mg protein/g
m

Vi a: hm lng protein (g/ml dung dch)

n: h s pha long mu
m: khi lng nguyn liu ly phn tch (g)

2.7. Phng php thu nhn dextrin

Cc bc tin hnh:

- Dch ha: trong thi gian h ha tinh bt, ta dung dch enzyme nhit
thch hp enzyme c hot ha. Tinh bt c h ha v lm ngui khong
50oC, sau b sung enzyme vo dung dch h tinh bt. T lc cho enzyme vo,
khuy lin tc, khuy k to s tip xc gia enzyme v c cht ng u. Thi
gian t lc h ha tinh bt n lc lm ngui v cho enzyme vo khong 5 pht.
- Khi h tinh bt bt u lng ra, tnh thi gian.
- Bt hot enzyme: sau mi thi gian kho st ta bt hot enzyme nhm m bo
chnh xc cc thng s o ti thi im kho st bng cch lm lnh v thm 5 ml dung
dch HCl 5%.
- Sau ly tm loi b tinh bt tha, ta cn thu dextrin, em sy kh 70 80oC.

48

2.8. th chun xc nh mt t bo
Bng 2.3. Gi tr OD610nm t ng ng mt t bo
OD610nm

0,12

0,221

0,306

0,41

0,53

15,333x106

34x106

52,333x106

100,333x106

141x106

7,186

7,531

7,719

8,001

8,149

Mt trung bnh
(N/ml)

Log (N/ml)

Log (N/ml)

8,4

y = 2,3588x + 6,9686
R2 = 0,9725

8,0
7,6
7,2
6,8
0,1

0,2

0,3

0,4

0,5

0,6

OD610nm

th 2.1. S tng quan gia gi tr OD61onm v mt t bo

2.9. th chu n xc nh l ng tinh b t b th y phn theo
phng php Heinkel

Bng 2.4. Gi tr OD650nm tng ng hm lng tinh bt

Tinh bt (mg/ml)

10

OD trung bnh

0,016667

0,139

0,2497

0,3673

0,467

0,56167

dOD560nm

0,1223

0,233

0,3507

0,450333

0,545

dOD

49

0,6

y = 0,0561x

0,5

R2 = 0,997

0,4
0,3
0,2
0,1
0
0

10

tinh bt (mg/ml)

th 2.2. S tng quan gia gi tr OD560nm v hm lng tinh bt (mg/ml)

2.10. ng chun xc nh hm lng protein theo phng php LOWRY

Bng 2.5. Gi tr OD750nm tng ng vi cc ng albumin
0

50

100

150

200

250

OD750nm trung bnh

0,043

0,1117

0,1683

0,2253

0,277

0,33133

dOD

0,0687

0,1253

0,1823

0,234

0,28833

dOD

Albumin (g/ml)

y = 0,0012x

0,35
0,3
0,25
0,2
0,15
0,1
0,05
0

R = 0,9961

50

100

150

200

250

albumin (g/ml)

th 2.3. S tng quan gia gi tr OD750nm v hm lng protein

50

2.11. Th i gian nui cy Bacillus subtilis

Bng 2.6. nh h ng ca thi gian nui cy ln hot -amylase
S ln

Thi gian (gi)

Phng sai

Sai s

bnh

0,297

0,295

0,296

0,296

1E-06

0,0006

5,245

5,209

5,227

5,227

0,0003332

0,0105

Hot (UI/g CT)

52454,27

52089,17

52271,72

52272,38

33324,117

105,395

OD

0,264

0.262

0,258

0,261

9,33E-06

0,0018

4,597

4,524

4,585

0,0031103

0,0322

361948,7

375092,2

366817,31

43365784

3802,008

0,267

0,265

0,269

0,267

4E-06

0,0012

4,689

4,652

4,725

4,689

0,001333

0,0211

Hot (UI/g CT)

468865,3

465214,3

472516,2

468865,26

13329647

2107,894

OD

0,256

0,251

0,251

0,252

8,33E-06

0,0017

4,488

4,397

4,397

4,427

0,002777

0,0304

Hot (UI/g CT)

448784,9

439657,4

439657,4

442699,56

27770097

3042,482

OD

0,233

0,233

0,230

0,231

2,33E-06

0,0009

4,068

4,050

4,013

4,044

0,0007776

0,0161

406798,6

404973,1

401322,2

404364,3

7775627,2

1609,93

OD
L ng tinh bt
(mg/ml)

24

36

Trung

L ng tinh bt (mg/ml) 4,634

Hot (UI/g CT) 367790,03
OD
L ng tinh bt
(mg/ml)

48

Lng tinh bt
(mg/ml)

60

Lng tinh bt
(mg/ml)

72

Hot (UI/g CT)

2.12. Kt qu ta enzyme bng cn 96% v (NH4)2SO4

Bng 2.7. nh hng ca t l cn 96% ln hot -amylase
Trc ta

Enzyme/cn

Hot (UI/ml dch chit enzyme)

5020,56
Hot (UI/100ml CPE ti)
Ln 1

1:1
1:2

Ln 2

Ln 3

Hot (UI/10ml DCE)

50205,64

Trung

Phng

bnh

sai

4001,80 4640,72 4458,17 4366,90 108303,38

44025,66 43934,38 44208,21 44056,08 19439,07

Sai s

Hiu
sut

(%)
190,003 8,698
80,497 87,751

1:3
1:4
1:5

45303,50 45577,32 45942,42 45607,75 102749,4

44345,12 44071,29 44436,39 44284,27 36101,13
43341,10 43614,92 42702,18 43219,40 219383,77

185,067 90,842
109,698 88,206
270,422 86,085

51

Bng 2.8. Hm lng protein tng ng cc t l cn 96% v -amylase

Protein (mg/ml DCE)
9,703
Protein (mg/100ml CP ti )

Trc ta
Enzyme/cn

1:1
1:2
1:3
1:4
1:5

Ln 1

Ln 2

Ln 3

TB

10,900
14,883
22,193
22,894
23,638

10,112
16,809
21,405
23,069
23,463

10,375
16,196
21,668
23,419
23,988

10,462
15,963
21,756
23,127
23,696

Protein (mg/10ml DCE)

97,032
Hiu
Phng sai
Sai s
sut
(%)
0,1609487
0,9682472
0,1609487
0,0715328
0,0715328

0,232
0,568
0,232
0,154
0,154

10,78
16,45
22,42
23,83
24,42

Bng 2.9. Hot -amylase sau khi ta bng (NH4)2SO4

Hot (UI/ml DCE)
5157,47

Trc ta

Hot (UI/10ml DCE)

51574,76

Hot (UI/100ml CP ti)

bo
ha (%)

Ln 1

50
55
60
65
70

31571,450
37017,493
37595,565
37078,343
36743,670

Phng
sai

Ln 2

Ln 3

TB

31693,149
36774,095
37595,565
37504,290
36865,369

32240,796
36956,644
37534,715
37260,892
37169,617

31835,132
36916,077
37575,282
37281,175
36926,219

Sai s

Hiu
sut
(%)

127125,3 205,852 61,73

16044,95 73,132 71,58
1234,227 20,283 72,86
45666,38 123,378 72,29
48134,84 126,669 71,60

Bng 2.10. Hm lng protein tng ng cc bo ho (NH4)2SO4 dng ta

-amylase
Protein (mg/ml DCE)
9,85

Trc ta

Protein (mg/100ml CP ti)

bo ha
(%)

Ln 1

Ln 2

Ln 3

TB

50
55
60
65
70

14,825
16,021
15,904
16,605
19,173

14,591
15,846
16,488
16,546
19,231

15,000
15,671
16,488
16,897
18,706

14,805
15,846
16,293
16,683
19,037

Protein (mg/10ml DCE)

98,57
Hiu
Phng sai
Sai s
sut
(%)
0,0420113
0,0306569
0,1135441
0,0351987
0,0828872

0,118
0,101
0,195
0,108
0,166

15,02
16,08
16,53
16,92
19,31

52

2.13. Kt qu kho st nh hng ca thi gian ta cn 96%

Bng 2.11. Hot -amylase sau cc thi gian ta cn 96%
Hot (UI/ml DCE)
5020,56

Trc ta

Hot (UI/100ml CP ti)

Pht

15
30
45
60
75

Ln 1

Ln 2

Ln 3

50277,96
48680,65
45759,87
45486,05
44253,84

50186,68
48771,93
46033,69
45851,15
44162,57

50643,06
48771,93
46033,69
45851,15
45075,31

Trung
bnh

Hot (UI/10ml DCE)

50205,64
Hiu
Phng sai
Sai s
sut
(%)

50369,23 58317,204
48741,50 2777,010
45942,42 24993,087
45729,45 44432,155
44497,24 252707,884

139,424
30,425
91,274
121,699
290,234

93,24
90,23
85,05
84,65
82,37

Bng 2.12. Hm lng protein tng ng cc thi gian ta cn 96%

Trc ta
Pht

15
30
45
60
75

Protein (mg/ml DCE)

9,703
Protein (mg/100ml CP ti)
Ln 1

Ln 2

Ln 3

TB

21,230
22,193
23,244
23,463
24,294

21,580
22,368
23,069
23,813
24,469

21,668
21,931
22,894
24,251
24,207

21,493
22,164
23,069
23,842
24,324

Protein (mg/10ml DCE)

97,032
Hiu
Phng sai
Sai s
sut
(%)
0,0536496
0,0485401
0,0306569
0,1558393
0,0178832

0,134
0,127
0,101
0,228
0,077

22,15
22,84
23,77
24,57
25,07

53

2.14. Kt qu kho st nh hng ca nhit ln hot ca CPE

Bng 2.13. nh hng ca nhit ln hot ca ch phm -amylase

1

S ln
2

bnh

0,279

0,284

0,293

4,917

5,008

Hot (UI/g CP)

819473

OD

0,317

Sai s

0,285

5,03333E-05

0,0041

5,172

5,032

0,0167731

0,075

834686

862068

838742

4,66E+08

12462

0,323

0,320

0,320

9,33333E-06

0,0018

5,711
951821

5,674
945736

5,662
943708

0,0031103
86395857,8

0,032
5366

0,326

0,324

0,329

0,326

6,33333E-06

0,0015

5,775

5,738

5,830

5,781

0,0021105

0,027

962469,8

956385

971597

963484

58625760,65

4420

0,425

0,422

0,422

0,423

3E-06

0,0010

7,573

7,518

7,518

7,536

0,0009997

0,018

Hot (UI/g CP)

1262154

1253027

1253027

1256069

27770097,15

3042

OD

0,445

0,446

0,448

0,446

2,33333E-06

0,0009

7,938

7,956

7,992

7,962

0,0007776

0,016

1323004

1326046

1332131

1327060

21598964,45

2683

0,411

0,417

0,413

0,414

9,33333E-06

0,0018

7,327

7,436

7,363

7,375

0,0031103

0,032

1221081

1239336

1227166

1229194

86395857,8

5366

0,310

0,302

0,315

0,309

4,3E-05

0,0038

5,483

5,337

5,574

5,464

0,0143294

0,069

913790

889450

929003

910748

398038059

11519

OD
30

40

45

Lng tinh bt
(mg/ml)

L ng tinh bt (mg/ml) 5,601

Hot (UI/g CP)
933566
OD
L ng tinh bt
(mg/ml)
Hot (UI/g CP)

50

55

OD
L ng tinh bt
(mg/ml)

Lng tinh bt
(mg/ml)
Hot (UI/g CP)

60

OD
L ng tinh bt
(mg/ml)
Hot (UI/g CP)

65

Trung

Phng sai

Nhit (oC)

OD
L ng tinh bt
(mg/ml)
Hot (UI/g CP)

54

2.15. Kt qu kho st nh hng ca pH ln hot ca CPE

Bng 2.14. nh hng ca pH ln hot ca ch phm -amylase
pH

5,8

OD
L ng tinh bt
(mg/ml)
Hot (UI/g CT)

6,0

OD
L ng tinh bt
(mg/ml)
Hot (UI/g
CT)

6,4

6,6

0,427

Trung
bnh

Phng sai

Sai s

0,424

0,416

0,422

3,23333E-05

0,0033

7,619

7,564

7,418

0,010774798

0,060

1269760,
5

1260633

1236293

1255562

299299936

9988

0,4345

0,4445

0,4335

0,438

3,7E-05

0,0035

0,012329923

0,064

7,755

7,938

7,737

7,533

7,810

1292579

1323004

1289537

1301707

342497865

10685

0,4215

0,4175

0,4175

0,419

5,33333E-06

0,0013

7,518

7,445

7,445

7,469

0,001777286

0,024

Hot (UI/g CT)

1253027

1240857

1240857

1244914

49369062

4057

OD

0,4185

0,4165

0,4155

0,417

2,33333E-06

0,0009

7,463

7,427

7,409

7,433

0,000777563

0,016

Hot (UI/g CT)

1243899

1237814

1234772

1238829

21598964

2683

OD

0,402

0,402

0,401

0,402

3,33333E-07

0,0003

7,162

7,162

7,144

7,156

0,00011108

0,006

1193698

1193698

1190656

1192684

3085566

1014

OD
6,2

S ln
2

Lng tinh bt
(mg/ml)

Lng tinh bt
(mg/ml)

Lng tinh bt
(mg/ml)
Hot (UI/g CT)