1

M U
1. Tnh cp thit ca ti nghin cu
Tm s l mt hng ch bin xut khu ch lc ca ngnh ch bin thy
sn Vit nam.ng thi vi khi lng ln tm xut khu hng nm th ph
liu ca n l u v v tm cng chim lng rt ln. Trong u tm cha
mt lng ln protein, chitin, cht mu astaxanthin v nhiu hp cht sinh hc
khc, c bit l h enzyme trong u tm c hot kh cao.
ti Nghin cu tch chit v ng dng enzyme protease t tm s
Penaeus monodon vo ch bin thy sn c tin hnh vi mong mun
kim tm nhng hiu bit y v enzyme protease trong tm nhm p ng
cc nhu cu thng tin v mt hng nui trng v ch bin ch lc ca ngnh
thu sn t nc, gip chng ta hiu v l gii c cc bin i ca tm
sau khi thu hoch, trong qu trnh ch bin cng nh bo qun, t ra
nhng bin php hu hiu gn gi cht lng tm. ti cng hng ti thu
nhn protease t ngun ph liu di do ny ng dng trong thy phn mt
vi i tng ph liu ch bin thu sn nhm nng cao hiu qu tn dng ca
cc ph liu thi ra v gp phn nh bo v mi trng.
2. Mc ch nghin cu ca lun n
Mc ch chung ca ti l nghin cu tch chit protease t tm s
nui Penaeus monodon v tnh cht ca n, nghin cu ng dng enzyme ny
trong thu phn protein mt vi ph liu ch bin thu sn (mu v gan c
basa Pangasiadon hypophthanus, ph liu u v tm) thu nhn cc sn
phm c gi tr kinh t cao hn.
3. i tng v phm vi nghin cu
ti tp trung vo i tng nghin cu l tm s nui vng bin Cn
Gi, thnh ph H Ch Minh. Ph liu ch bin thu sn c nghin cu tn
dng gm hai ngun: hn hp mu v gan c basa Pangasiadon hypophthanus
nui Tin giang; hn hp ph liu u v v tm s thi ra t qui trnh sn
xut tm s ng lnh xut khu vi ngun tm c nui Cn gi.
2


4. Phng php nghin cu
Phng php nghin cu ca ti l nghin cu thc nghim v x l s
liu da trn h tr ca ton hc v phn mm STATGRAPHIC Plus ti u
ha qu trnh.
5. Nhng ng gp mi ca lun n
Kt qu nghin cu l dn liu u tin Vit nam v cc thng tin khoa
hc ca h protease trong u, gan ty tm s nui thng phm Vit nam.
tm chn c cc thng s ti u cho qu trnh tch chit v tinh
sch enzyme protease t u v gan ty tm s c tinh khit tng ln ln
lt l 18,03 v 16,27 ln.
Xc nh c protease tm s thuc nhm protease serine, thnh phn
bao gm t nht nm loi protease ( gan ty tm) v by loi ( u tm),
trong ba loi c hot tnh rt mnh quyt nh hu nh ton b hot tnh vi
phn t lng t 20.200 n 25.000Da.
xc nh cc tnh cht c bn v ng hc ca protease tch chit t
u v gan ty tm.
Kt qu nghin cu ca lun n cng cn l nhng dn liu u tin v
vic s dng enzyme ny cho vic thu phn thu ch phm c gi tr
carotenoprotein t u, v tm v thu phn hn hp mu v gan c basa,
thu dch thu phn phc v cho sn xut thc n chn nui. a ra cc
quy trnh cng ngh vi cc thng s c ti u ha thc hin thu phn
hai loi ph liu ca ngnh ch bin thu sn, t c nhng kt qu bc
u, c th hon thin p dng cho thc t.
6. Kt cu ca Lun n:
Lun n gm 163 trang ni dung, 50 trang ph lc, 16 trang ti liu tham
kho (172 ti liu). Ni dung lun n gm ba chng, trong c 70 hnh
nh v th, 17 bng v 13 s .

3

CHNG 1
TNG QUAN V PROTEASE TRONG TM V CC
LOI THY SN, CC NG DNG CA CHNG
1.1. Protease trong tm v cc loi thy sn
Protease ca tm cng nh ca cc loi ng vt thu sinh khc l cc
protease ni bo, n tp trung nhiu nht c quan tiu ho, gan ty v sau
n c tht. c bit tm do c im h tiu ho gan ty nm phn u
nn h enzyme s tp trung nhiu nht phn u sau n cc c quan
khc. Protease tm khng c dng pepsin, ch yu dng trypsin hoc
protease serin ta trypsin v c kh nng hot ng rt cao. Ngoi ra, cn c
enzyme chymotrypsin, astacine, collagenase
Cc u im vt tri ca enzyme t ng vt thy sn khi ng dng vo
sn xut thc phm l chng c th cho hiu qu tt hn so vi phng php
c hc hay ha hc thng thng. Vic s dng cc enzyme ny cng khng
i hi phi kim tra an ton v chng c tch chit t cc phn n c
ca nguyn liu. Enzyme t thy sn thng th hin hot tnh cao nhit
thp hoc khng cao lm, v vy nh sn xut c th thc hin phn ng
nhit thp, gip tit kim nng lng, phn ng ny cng d dng ngng
li khi nng nhit ln khng qu cao v nh gim c nguy c h hng
do vi sinh vt.
1.2. Nhng ng dng ca enzyme t thy sn vo mc ch thc phm
Do bn cht sinh ha khc nhau gia cc b phn hnh thi hc ca ng
vt thy sn, ngi ta c th s dng enzyme thc hin qu trnh phn gii
mt cch c nh hng vo nhng mc ch nh loi da c ui, c trch, cc
loi c da trn, c ng i dng, c hi, tch v hu, hn, tm, mc, nghu
Phng php cho hiu sut cao hn nhiu ln so vi thc hin th cng hay
ha hc. Cc protease cng t ra hu dng trong vic lm sch vy c hay sn
xut ct ngc trai, bc tch mng v c quan ni tng thy sn. Qui trnh thu
nhn chitin v protein t ph liu ch bin tm c s dng protease gip gim
thiu lng ha cht s dng, ng thi thu nhn chitin c tnh cht bn v t
4

b deacetyl hay thy phn mch polymer. Protease t c v mt s loi thy
sn khc cn c s dng thay th rennet trong sn xut phomai cho kt qu
rt tt, c bit khi ng dng trong sn xut dch c (nc mm) hay bt m
c thy phn, thi gian thc hin c rt ngn ng k. S dng enzyme
protease trong chit rt carotenoprotein t ph liu ca qu trnh ch bin cc
loi gip xc l mt hng nghin cu ang rt c ch hin nay v gip
nng cao li nhun sn xut nh to ra sn phm gi tr gia tng v gim thiu
nhim mi trng. Cc carotenoprotein, thng c s dng nh cht b
sung cho sn xut thc n chn nui hoc cht to mu trong cng ngh thc
phm, c tnh cht bn vng v d bo qun hn nhiu so vi carotenoid ring
l. Carotenoprotein t u, v tm hin ang rt c quan tm s dng
lm thc phm chc nng cho ngi v thnh phn carotenoid ch yu l
astaxanthin, mt hp cht chng oxy ha thin nhin vi cc li ch k diu
cho sc khe ang c pht hin v khng nh.
1.3. Cc nghin cu trong nc v protease t ng vt thy sn v ng
dng ca chng
Vit nam, cc nghin cu v h protease t ng vt thy sn c
thc hin ch yu trn i tng tm bin, cc loi c nh thu, ng v mc
ng. Cc ng dng ca protease tp trung vo vic tn dng enzyme protease
c sn trong nguyn liu ch bin nc mm, mm c, mm tm chua
Cc ng dng khc nh thu nhn enzyme v dng vo cng ngh ch bin mi
ch dng mt s rt t nghin cu vi qui m phng th nghim nh dng
protease tng nhanh qu trnh ch bin c, s dng cho qu trnh thy phn
protein tht c mi sn xut dch m c m c.
Nghin cu ny hng vo s dng ph liu u tm s tn thu ch
phm enzyme protease v ng dng n trong thy phn hai i tng cng l
ph liu ch bin thy sn l hn hp mu, gan c basa v u, v tm. Cc
sn phm ca nghin cu gm dch m thy phn t mu, gan c, v bt
carotenoprotein s khng ch ph hp dng lm thc n vt nui m cn c
th l thc phm chc nng hu dng cho con ngi.
5

CHNG 2
NGUYN LIU V PHNG PHP NGHIN CU
2.1. Nguyn liu nghin cu
Nguyn liu tch chit v tinh sch enzyme l u (bao gm gan ty)
v gan ty tm s Penaeus monodon c phn tch t tm s sng, loi 40-
50 con/kg c nui Cn Gi.
Mu u tm s thu nhn CPE v ng dng vo thy phn c phn
tch ti cng ty C Phn Thy Sn S 1, ng gi v bo qun ng lnh
nhit -20
o
C trc khi s dng. Ph liu u, v tm ng dng vo thy
phn cng c thu nhn v bo qun tng t.
Mu mu v gan c basa Pangasiadon hypophthanus c phn tch v
ng gi bo qun ng lnh ti cng ty c phn thy sn Hng Vng, TP
M Tho, Tnh Tin Giang. Tin trnh ly mu c c thc hin bng cch ct
tit c v th vo bn cha nc vi t l c: nc l 5:1(v/w).
2.2. Phng php nghin cu
2.2.1. Thu nhn protease tinh sch:
Ton b qu trnh tch chit enzyme c thc hin 0-4
o
C. Hot tnh
protease v hm lng protein ha tan ca dch chit DC v ch phm enzyme
CPE c xc nh la chn thng s cho qu trnh tch chit CPE. Dung
mi chit th nghim l nc ct, nc mui sinh l, m phosphat v Tris-
HCl pH7,5, tc nhn ta nghin cu l ethanol, acetone v (NH
4
)
2
SO
4.
Qu
trnh tinh sch sau c thc hin bng sc k lc gel trn h thng sc k
ct p sut thp Bio-Rad s dng Bio-Gel P-100, kch thc ct: 50cmx1,5
cm. Flow adaptor: 1,5 cm, tc dng 0,14 ml/pht, th tch mu em phn
tch : 1ml, dung dch m Tris-HCl pH 7,5. Hm lng protein trong mi phn
on enzyme sau tinh sch (2ml) c o t ng bc sng 280 nm v ghi
nhn trn sc k nh phn mm Data View. Hot protease ca cc phn
on c xc nh bng phng php Amano.
2.2.2. Nghin cu tnh cht ca protease tm s: Trng lng phn t
protease c xc nh bng in di c cht Zymogram v Substrate Gel trn
6

in di ng Bio-Rad vi gel gom 4%, gel phn tn 12% hiu in th
110V, cng dng in 30A trong hai gi. nh hng ca nhit , pH,
nng mui n n hot protease xc nh bng cch cho enzyme tc
dng vi c cht casein cc pH, nhit , nng mui n quan tm khi xc
nh hot . bn nhit ca protease: kho st bng cch enzyme nhit
quan tm 30 pht, sau o hot tnh cn li bng phng php Amano.
nh hng ca mt s ion kim loi n hot protease nghin cu bng
cch b sung cht nh th vi nng 0,001M vo phn ng xc nh hot
enzyme. nh hng ca mt s cht c ch c hiu n hot protease
xc nh bng cch cht nh th vi cng th tch enzyme sao cho m bo
ng nng thch hp cn thit, gi 25
o
C trong 15 pht, sau o hot
tnh protease cn li. Cc thng s ng hc ca protease xc nh nh
phng trnh ng hc Hill trn c s phn tch s liu thu c v nng
v vn tc phn ng.
2.2.3. Nghin cu ng dng ch phm enzyme CPE thu nhn t u tm s
vo thy phn protein t hn hp mu v gan c basa thu dch m:
Hn hp mu v gan c basa c b sung CPE thc hin thy phn
thu dch m. Qu trnh thy phn hn hp mu v gan c basa ti v gia
nhit c so snh rt ra kt lun loi no ph hp hn cho thy phn thu
dch m. Nghin cu sau xc nh nh hng ca nng CPE b sung,
nhit v thi gian n hm lng peptid mch ngn v acid amin to
thnh trong dch m thy phn nh gi qu trnh.
2.2.4. Ti u ha qu trnh thy phn hn hp mu v gan c basa:
Cc s liu v qu trnh thy phn phn trn c xem xt v phn tch
la chn khong bin thin thch hp s dng cho ti u ha, sau dng
phn mm STATGRAPHICS Plus tm phng trnh hi qui. Thng s ti u
ha ca qu trnh c suy ra t nhng phn tch b mt p ng thu c sao
cho hm lng peptid mch ngn v acid amin t c cao nht. Kt qu ti
u sau c kim tra li bng thc nghim nhm m bo s thng nht
7

gia l thuyt ti u v thc t trc khi rt ra kt lun cui cng v thng s
ti u ca qu trnh thu phn.
2.2.5. Nghin cu ng dng ch phm enzyme CPE thu nhn t u tm s
vo thy phn ph liu u v v tm thu nhn carotenoprotein
Hn hp u, v tm c thy phn bng CPE trong mi trng Na
2
-
EDTA sau lc qua nhiu lp vi th thu dch, kt ta ng in vi
chitosan tr lng ri ly tm thu bt nho, ng kh thu bt carotenoprotein
thnh phm. Nghin cu so snh qu trnh thy phn u, v tm ti v
gia nhit chn la chn phng php x l s b thch hp, sau thc
hin thy phn vi cc nng CPE b sung, nhit v thi gian khc nhau,
nh gi qu trnh bng hm lng protein ha tan v carotenoid trong sn
phm bt carotenoprotein.
2.2.6. Ti u ha qu trnh thy phn hn hp u, v tm:
Thc hin tng t phn 2.2.4 vi tr gip ca phn mm
STATGRAPHIC Plus trn hai hm mc tiu l hm lng protein v
carotenoid trong sn phm bt carotenoprotein, trong hm u tin l hm
lng carotenoid v y chnh l thnh phn to nn gi tr kinh t vt tri
ca sn phm.
2.3. Cc phng php phn tch p dng:
Hot protease xc nh theo phng php Amano dng casein t sa
lm c cht. Hm lng protein ha tan xc nh theo phng php Bradford
dng albumine huyt thanh b lm cht chun. Hm lng carotenoid xc
nh bng phng php Tolasa. Hm lng peptid mch ngn v acid amin
xc nh bng so mu theo Amano. Hm lng nit tng s xc nh bng
phng php Kjeldahl, nit amoniac bng chng ct, nit formon bng
phng php Sorensen. Xc nh hm lng cht bo bng phng php
Soxhlet, hm lng tro bng phng php nung 600
o
C, m bng cch sy
n khi lng khng i 105
o
C v hm lng chitin bng phng php
Chakrabati.
2.4. Phng php x l s liu:
8

0
5
10
15
20
25
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55
H
o

p
r
o
t
e
a
s
e

(
U
/
m
l
)
A
-
2
8
0

n
m
Phn on
A-280 nm
Hot protease
(U/ml)
0
5
10
15
20
25
30
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55
H
o

p
r
o
t
e
a
s
e

(
U
/
m
l
)
A
-
2
8
0

n
m
Phn on
A-280 nm
Hot protease
(U/ml)
S liu thc nghim c x l theo phng php thng k ton hc da
trn cc phn mm Excel v STATGRAPHIC Plus.
CHNG 3
KT QU NGHIN CU V THO LUN
3.1. Thu nhn protease tinh sch:
Ch phm enzyme CPE protease t u (hoc gan ty) tm s c th thu
nhn c qua cc bc c bn: chit rt enzyme t nguyn liu nghin
nh bng Tris-HCl 0,05M pH7,5 vi t l 1:3 (w/v), thi gian chit 40 pht
(i vi u tm) v 60 pht (i vi gan ty ). Dch chit DC thu c bng
cch cho qua ry c l vi kch thc 1mm*1mm loi lng ln v tm,
sau ly tm 6000 vng/ph, 15 pht, loi cn em lm thc n gia sc. Kt
ta DC bng ethanol vi nng 80%, 50 pht iu ch CPE t u tm
hoc bng (NH
4
)
2
SO
4
70%, 70 pht thu CPE t gan ty tm. Cng on ly
tm thu CPE cng c thc hin 6000 vng/ph trong 15 pht.










Hnh 1. Sc k lc gel ch phm protease ta bng ethanol t u tm








Hnh 2. Sc k lc gel ch phm protease ta bng (NH
4
)
2
SO
4
t gan ty tm
9

Ch phm enzyme CPE thu t qu trnh tch chit protease t u v gan
ty tm s c em tch trn sc k lc gel Bio-Gel P-100. sch ca
protease sau sc k tng ln 16,27 ln i vi mu t gan ty v 18,03 ln i
vi mu t u tm.
3.2. Tnh cht ca protease tm s
3.2.1. Trng lng phn t ca protease gan ty v u tm
Trng lng phn t ca protease tm s c xc nh bng phng
php in di Zymogram v Substrate-Gel Electrophoresis. Phn tch cc bn
kt qu in di cho bit, h protease gan ty v u tm s P. monodon u
bao gm ba protease ch yu A, B, C vi phn t lng ln lt l 20.200,
22.000, 25.000 Da v hai protease t hot tnh hn l D, E (phn t lng theo
th t l 35.300, 40.200 Da). Ring u tm cn c thm hai protease na vi
hot tnh rt b l F v G (vi trng lng phn t 49.200 v 76.000 Da).







Hnh 3. in di Zymogram h protease gan ty (a) v u tm s (b)








Hnh 4. in di Substrate-Gel h protease gan ty (a) v u tm s (b)
Ch thch hnh 3, 4: pha long protease sau tinh sch a vo ging 1, 11:5 ln, ging 2, 10: 25
ln, ging 3, 9: 50 ln, ging 4, 8: 100 ln, ging 5, 7: 500 ln, ging 6: thang protein chun
10

0
20
40
60
80
100
120
37 42 47 52 57 62 67 72 77 82 H
o

n
g

i

(
%
)
Nhit (
o
C)
Gan ty
u tm
0
20
40
60
80
100
120
2 7 1217222732374247525762677277828792
H
o

n
g

i

(
%
)
Nhit (
o
C)
Gan ty
u tm
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
H
o

n
g

i

(
%
)
Nng mui n (%)
Gan ty
u tm
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8 9 10 11 12 13
H
o

n
g

i

(
%
)
pH
Gan ty
u tm
3.2.2. nh hng ca nhit n hot protease tm s sau tinh sch v
bn nhit ca n
Hnh 5. nh hng ca nhit n
hot tng i ca protease gan ty
v u tm
Hnh 6. bn nhit ca protease gan
ty v u tm cc nhit khc nhau

Kt qu thc nghim cho thy protease trong gan ty v u tm hot
ng tt trong khong 52-67
o
C vi nhit ti u l 62
o
C. Chng cng c
bn nhit rt tt so vi cc loi thy sn khc c nghin cu. Chng gi
hot tnh tt khong nhit kh cao 37-57
o
C, thm ch khi tng ln thnh
62
o
C th enzyme ny vn cn hot ng tt. y tht s l li th ng k khi
p dng vo thy phn protein v ch bin thc phm ni chung iu khin
phn ng thun li nht, bi v hu ht cc vi sinh vt gy thi khng pht
trin tt khong nhit cao m protease tm s vn hot ng tt 37-
57
o
C, thm ch 62
o
C.
3.2.3. nh hng ca pH v nng mui n n hot protease tm s
Hnh 7. nh hng ca pH n hot
ng ca protease thu nhn t gan ty
v u tm s
Hnh 8. nh hng ca nng mui n
n hot ng ca protease t gan ty
v u tm
Kt qu nghin cu cho thy protease t gan ty v u tm u th hin
hot tnh rt yu vng axit pH t 1 n 5, tng nhanh pH 6 tr ln, t cc
11

i ti pH 7,5 ri gim nhanh khi pH tng trong vng kim. Hai h protease
ny t ra rt nhy cm vi pH trong mi trng gn trung tnh n kim pH 6-
9, vic tng hoc gim nh pH t gi tr ti u c tc dng lm hot tnh
protease gim ng k, iu ny s l lu cn ghi nh khi ng dng enzyme
ny vo qu trnh thy phn protein to ra iu kin thun li nht v t
kt qu mong mun nht.
Kt qu thc nghim cng cho thy, nng mui n nh hng ln n
hot tnh protease ca tm. Nng mui 0-1% cho kt qu hot tnh protease
tm t cc i. Hot tnh ny gim kh u khi nng mui tip tc tng
ln. Protease ca gan ty v u tm chu mui tt hn nhiu so vi cc
protease t thc vt v ng vt nh protease ni tng c v gan mc.
3.2.4 nh hng ca mt s kim loi v cht c ch n hot protease tm
s sau tinh sch
Hnh 9. nh hng ca cc ion kim
loi n hot protease tm s
Hnh 10 nh hng ca mt s cht km
hm n hot ca protese tm s
Cc ion kim loi Mo
2+
, Pb
2+
, Mg
2+
, Ba
2+
, Co
2+
hu nh khng gy nh
hng n hot protease. Hg
2+
th li c tc dng c ch protease ng k,
sau l Zn
2+
. Cc kim loi c tc dng hot ha protease l Cu
2+
, Mn
2+
v
Fe
2+
, trong ng k nht l Mn
2+
, k tip l Cu
2+
, sau na l Fe
2+
.
Hot tnh protease tm s b gim mnh (ch cn 13-15%) khi c mt
PMSF, cht c ch c hiu ca nhm protease serin, l bng chng cho bit
protease tm s l thnh vin ca nhm ny. y l iu c khng nh li
qua kt qu xc nh hot khi thm SBTI vo phn ng, hot cn li ln
0
50
100
150
200
H
o

n
g

i

(
%
)
Kim loi thm vo phn ng
Gan ty
u tm
0
20
40
60
80
100
120
H
o

n
g

i

(
%
)
Cht thm vo phn ng
Gan ty
u tm
12

lt l 32 v 33% i vi gan ty v u tm. SBTI l cht c ch cc
protease serin, n km hm trypsin, t nhy hn mt cht vi chymotrypsin.
Khi c mt TLCK (cht c ch trypsin), hot protease cn li ch t con s
39 v 35% gan ty v u tm, nh vy, cc protease ch yu trong tm s
thuc v loi enzyme ta trypsin. Ngoi ra, trong tm s cn cc enzyme ta
chymotrypsin v TPCK c tc dng gim nh hot tnh protease, hot tnh cn
li khong 80%. EDTA (cht km hm cc metalloprotease), 1, 10-
phenanthroline (c hiu cao i vi km trong trung tm hot ng ca
metalloprotease) v iodoacetic acid hu nh khng nh hng n hot tnh
protease, cho thy rng, trong gan ty v u tm s c l khng c
metalloprotease (nh collagenase chng hn) hoc tn ti nhng vi hot tnh
rt thp.
3.2.5. ng hc ca protease gan ty tm sau tinh sch
Hnh 11 th Hill
Phng trnh ng hc ca protease:
log[

v
mcx
-
=2,8174log[S] +5,0508 (3.1)
h s Hill =2,8174
K =8,9x10
-6

Gi tr h s Hill h v hng s K cho php suy ra, i lc gia protease
tm s vi c cht c th coi l rt tt, v hp tc tng h gia cc trung tm
hot ng ca enzyme ny l dng tch cc, s gn kt ca mt c cht vo
trung tm hot ng ny s thc y, to iu kin thun li cho c cht gn
vo trung tm hot ng khc.
3.3. Nghin cu qu trnh thy phn hn hp mu v gan c basa bng
ch phm enzyme protease tch chit t u tm s
Hn hp mu v gan c basa c thy phn bng ch phm enzyme
protease tm s vi cc thng s ban u nh sau:
y =2.817x +5.050
R =0.989
-1
0
1
2
3
4
5
6
-2.1 -1.9 -1.7 -1.5 -1.3 -1.1 -0.9 -0.7
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13

T l mu v gan c: 80% mu v 20% gan c
pH hn hp thy phn: 7, iu chnh lc bt u thy phn bng cch
dng acid axetic iu chnh
Lng mui b sung: 1% (w/v)
Dch thy phn c khuy trn u sau mi na gi
3.3.1 So snh qu trnh thy phn bng CPE protease u tm trn hn hp
mu v gan c basa ti v gia nhit

Hnh 12 Bin ng hm lng peptid v acid amin ca qu trnh thy phn hn
hp mu v gan c basa ti v gia nhit
Theo thi gian thu phn, hm lng peptid v acid amin cao hn v tng
nhanh hn mu hn hp gia nhit. Cm quan cho thy sau 6 gi, hn hp
mu v gan c ti bt u c mi hi nng khc vi mi tanh c trng ca
mu, thi gian cng tng mi hi cng nng. i vi hn hp mu v gan c
qua gia nhit th thi gian cng tng mi tanh cng gim. Ti thi im 12
gi, cm quan c mi tanh gim nhiu.
Nh vy, dng hn hp mu v gan c ti cho thy phn l khng ph
hp thu thnh phm, trong phn tip theo, ti s dng hn hp mu v
gan c qua gia nhit thy phn nhm hn ch c nh hng ca cc vi
sinh vt gy phn hy nguyn liu, thu c HL peptid mch ngn v acid
amin cao hn, c ch tiu cm quan tt hn.
3.3.2 nh hng ca nng CPE protease n qu trnh thy phn hn hp
mu v gan c basa gia nhit
Khi tng nng enzyme th hm lng peptid v acid amin to thnh
cng tng. Nng CPE s dng 0,5% dng nh hi thp nn sn phm to
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 2 4 6 8 10 12
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Thi gian thu phn (h)
Hn hp ti
Hn hp gia nhit
14

thnh khng nhiu, v khi nng ln trn 2,5% th lng sn phm tng, tuy
nhin nh hng ca nng n lng sn phm to thnh khng cn rt r
rt nh trc. Vic tng nng enzyme v nhit ng thi trong khong
kho st s c tc dng tng tt hm lng peptid v acid amin to thnh.
Hnh 13 nh hng ca nng CPE n
bin i hm lng peptid v acid amin
trong dch thy phn 40
o
C
Hnh 14 nh hng ca nng CPE
n bin i hm lng peptid v acid
amin trong dch thy phn 50
o
C

Hnh 15 nh hng ca nng CPE
n bin i hm lng peptid v acid
amin trong dch thy phn 60
o
C

Hnh 16 nh hng ca nhit n
bin i hm lng peptid v acid amin
trong dch thy phn khi nng CPE b
sung l 2%
3.3.3 nh hng ca nhit ti qu trnh thy phn hn hp mu v gan
c gia nhit
Nhit c nh hng n qu trnh thy phn kh r, khi nhit tng
t 40
o
C ln thnh 50
o
C th hm lng peptid v acid amin to thnh tng
mnh. Tuy nhin khi nhit tip tc tng t 50
o
C thnh 60
o
C th hm lng
peptid v acid amin li tng khng ng k v gim hn khi thy phn nhit
65
o
C. Phn tch cm quan mu thu phn 40
o
C cho thy, t 8 gi tr i,
dch thy phn c mu nu sm, mi hi nng hn so vi cc mu 50, 60,
65
o
C vo cng thi im ly mu v bt u xut hin mi hi kh chu sau 16
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 2 4 6 8 10 12 14 16 18 20
Thi gian thu phn (h)
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2
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3
0 2 4 6 8 10 12 14 16 18 20
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2
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3
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40oC
50oC
60oC
65oC
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 2 4 6 8 10 12 14 16 18 20
Thi gian thu phn (h)
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2
3.5
4.5
15

gi thy phn. i vi cc mu thy phn nhit 50, 60, 65
o
C, vo thi
im 18 gi, dch c mu nu vng, long hn, mi tanh gim nhiu.
Nh vy, khng nn p dng nhit thy phn di 40
o
C, v vic tng
nhit ln trn 65
o
C cng l iu khng cn thit.
3.3.4 nh hng ca thi gian n qu trnh thy phn hn hp mu v gan
c gia nhit
Hnh 17 Bin i hm lng peptid v
acid amin trong dch thy phn theo thi
gian nng CPE b sung 3,5%
Hnh 18 Bin i hm lng peptid v
acid amin trong dch thy phn theo thi
gian nng CPE b sung 4,5%
Thi gian thy phn cng di, lng peptid v acid amin to nn cng
ln, t cc i, sau gim dn. S gim hm lng peptid v acid amin khi
thy phn ko di c l do mt lng acid amin b s dng to thnh cc
sn phm cp thp nh NH
3
hay cc hp cht d bay hi, v cng v th m
giai on cui, sn phm thy phn bt u c mi nng, t hp dn hn.
3.4 Ti u ha qu trnh thy phn hn hp mu v gan c basa bng
CPE t u tm s
Da trn cc phn tch s liu ca qu trnh thy phn trn, vic ti u
ha qu trnh c xem xt trong khong bin thin: Nhit : 40-65
o
C, nng
CPE s dng: 2-4,5% v thi gian thy phn 9-20 gi. Hm mc tiu ca
qu trnh thy phn l HL (hm lng peptid mch ngn v acid amin): HL
max. Qu trnh thy phn c dng ngay khi c du hiu ca s h hng.
Phn mm STATGRAPHICS Plus c s dng x l s liu. Vi cc
bin c lp l T, tg, C, T*tg, T*C, tg*C, cc php phn tch hi qui bc mt
v hai c thc hin. Kt qu hi qui c nh gi qua gi tr R
2
, mc
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 5 10 15 20 25
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50oC
60oC
65oC
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
4.000
0 2 4 6 8 10 12 14 16 18 20
Thi gian thu phn (h)
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50oC
60oC
65oC
16

ng tin cy ca phng trnh hi qui v ca tng bin s th hin qua gi tr
p-value. Phng trnh c chp nhn c dng nh sau:
HL = -17,0127 + 0,487788 T + 0,81786 tg + 0,0390555 C
2
0,00403577T
2
-
0,0255576 tg
2
+ 0,0118836 Ctg - 0,00202781 Ttg (3.2)
Trong :
HL Hm lng peptid v acid amin to thnh ca qu trnh thy phn (g/l)
T Nhit qu trnh thu phn (
o
C)
tg Thi gian thu phn (gi)
C Nng CPE s dng (%)
Phn tch cc h s ca phng trnh hi qui v mt p ng cho cc
thng s ti u ca qu trnh thy phn: Nhit 57
o
C, thi gian 14,5 gi.
xc nh nng CPE cn s dng cho thy phn, nghin cu thc hin
thm mt s th nghim bng cch p dng cc nng enzyme khc nhau
cho thy phn nhit v thi gian va ti u. Xt c n li ch v mt
kinh t, nng CPE ti u c chn l 4%.


Hnh 19 B mt p ng phng trnh hi qui HL nng CPE 4%
(T- Nhit (
o
C); tg - Thi gian (gi)

Dch thy phn thu nhn c theo ch ti u ha v sau lc c mu
nu vng, trong, khng c bt kh, thong mi tanh nh v mi thm ca c, v
ngt nht, c hm lng nit tng l 9,8g/l, nit acid amin l 3,4g/l v
ammoniac 2,7g/l. Sn phm ph hp cho ng dng trong sn xut thc n gia
sc hay thc n cho tm, c hoc thay nc mui cho li qua b chp lm
nc mm.
40
45
50
55
60
65
T
9
11
13
15
17
19
21
tg
1.8
2.2
2.6
3
3.4
3.8
4.2
H
L
17

3.5. Nghin cu qu trnh thy phn thu nhn bt carotenoprotein t u
v v tm bng ch phm enzyme protease tch chit t u tm s
Hn hp u, v tm xay nh 2-5 mm c thy phn bng ch phm
enzyme protease tm s trong mi trng Na
2
- EDTA vi cc thng s ban
u: T l ph liu: dung dch Na
2
-EDTA 0,5M pH 7 l 1:3; lng mui n b
sung: 1% (w/v); dch thy phn c khuy trn u sau mi na gi.
Bng 3.10 Thnh phn c bn ca ph
liu u, v tm P. monodon
Ch tiu Hm lng
m
Protein*
Cht bo*
Tro*
Chitin*
Carotenoid*
76,96 3,42 %
34,28 1,34 %
4,88 0,92 %
22,37 0,77 %
17,11 0,28 %
133,56 5,36 g/g
*Hm lng tnh trn nguyn liu kh
Hnh 20. ha tan ca protein dch
trong sau kt ta thu carotenoprotein
Vic thu nhn carotenoprotein t dch thy phn ph liu u, v tm
c thc hin bng cch kt ta ng in dch lc pH 4,5 ( ha tan
protein ca dch thy phn nh nht (hnh 20) bng dung dch HCl 10% vi
cht tr lng chitosan 100ppm iu kin lnh 4
o
C trong 2 gi.
3.5.1. So snh qu trnh thy phn bng CPE protease u tm trn ph liu
u v v tm ti v gia nhit
Hnh 21. Bin i hm lng carotenoid
trong sn phm carotenoprotein thu nhn
t qu trnh thy phn ph liu tm ti
v gia nhit

Hnh 22. Bin i hm lng protein
ha tan trong sn phm carotenoprotein
thu nhn t qu trnh thy phn ph liu
tm ti v gia nhit
V tm gia nhit chn v ph liu tm ti c em thy phn
nhit 50
o
C, nng CPE b sung 3,5%. Vic thy phn ph liu tm gia
0
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0.8
0 2 4 6 8 10 12 14
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Ph liu tm gia nhit
Ph liu tm ti
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Ph liu tm gia nhit
Ph liu tm ti
0
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4
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8
10
3.5 4 4.5 5 5.5 6
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pH
18

nhit t ra thun li hn nhiu, hm lng carotenoid v protein ha tan u
cao gp i, thm ch hn gp i so vi mu ti em thy phn, bt
carotenoprotein thu nhn t thy phn u v tm chn cng c mu vng cam
sng p hn nhiu so vi mu nu en khi thy phn nguyn liu ti. V
vy, trong nghin cu tip theo, ti s dng CPE protease thy phn ph
liu u v tm chn nhm hn ch nh hng ca polyphenoloxydase, hn
ch hot ng ca cc vi sinh vt gy thi, thu c sn phm carotenoprotein
giu carotenoid, giu protein v c mu sc, mi tt hn.
3.5.2 Phn tch nh hng ca nhit , thi gian thy phn v nng CPE
s dng ti hm lng carotenoid v protein ha tan ca sn phm
carotenoprotein
Qu trnh thy phn ph liu u v v tm gia nhit chn thu bt
carotenoprotein c thc hin vi ba nng CPE l 2; 3,5 v 5% cc
nhit 40, 50, 60 v 65
o
C theo thi gian.
Khi xem xt s liu phn tch thng k v hm lng carotenoid, qua gi
tr ca trung bnh bnh phng (Mean square), ta thy nh hng ca thi gian
thy phn ln hn nh hng ca hm lng CPE s dng v nhit c vai
tr thp hn c. Khi xt v nh hng ca cc tng tc i th th t gy nh
hng theo trnh t: nng CPE-thi gian, nhit -thi gian v cui cng l
nng CPE-nhit . iu ny c hi khc so vi kt qu phn tch yu t
nh hng hm lng protein ha tan, theo thi gian thy phn gy nh
hng ln nht, sau n nhit , v cui cng l hm lng CPE s dng,
mc nh hng ca cc tng tc i n hm lng protein ha tan tun
theo th t: nhit -thi gian, nng CPE-thi gian v cui cng l nng
CPE-nhit . Tuy nhin tt c nh hng ny u c ngha thng k, v tr
s p rt thp (< 0,001). Multiple Range Test cng c thc hin so snh s
khc bit gia cc ch thy phn khi thc hin cc khong hm lng
CPE, nhit v thi gian khc nhau, kt qu phn tch cho thy, mi bin i
ca tng yu t C, T hay tg u nh hng c ngha v to nn s khc bit
19

ng k n hm lng carotenoid v protein ha tan thu nhn c trong qu
trnh thy phn.
nh hng ca nng CPE s dng ti hm lng carotenoid v
protein thu nhn t qu trnh thy phn:
tt c cc nhit thy phn, hm lng CPE s dng cng cao th
hm lng carotenoid thu nhn c cng tng tng ng. Nng CPE s
dng 2% cho kt qu sn phm to thnh khng nhiu, v khi nng ln 3,5%
th hm lng carotenoid v protein tng ln rt ng k, vic tng CPE s
dng cho thy phn sau thnh 5% cng c tc dng tng sn phm to
thnh nhng hiu qu t c khng cn r rt nh trc.

Hnh 23. nh hng ca nng CPE
n hm lng carotenoid thu nhn khi
thy phn ph liu tm 50
o
C

Hnh 24. nh hng ca nng CPE
n hm lng protein thu nhn khi thy
phn ph liu tm 50
o
C
nh hng ca nhit ti hm lng carotenoid v protein thu
nhn t qu trnh thy phn:

Hnh 25. nh hng ca nhit n
hm lng carotenoid thu nhn khi thy
phn ph liu tm vi nng CPE 3,5%

Hnh 26. nh hng ca nhit n
hm lng protein thu nhnkhi thy phn
ph liu tm vi nng CPE 3,5%
Khi nhit thy phn tng ln, hm lng carotenoid v protein thu
nhn cng tng r rt. D thy rng, gi tr hm lng carotenoid cc i
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0.8
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3.50%
5%
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5%
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40oC
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65oC
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40
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Thi gian thy phn (gi)
40oC
50oC
60oC
65oC
20

cng nng CPE s dng lun cao hn khi nhit tng t 40
o
ln thnh
50
o
C, sau gim nh xung khi tip tc tng thnh 60
o
v gim ng k
nhit thy phn 65
o
C.
nh hng ca thi gian thy phn ti vic thu nhn carotenoid
Thi gian thy phn cng di, lng carotenoid v protein thu c cng
ln, t cc i sau gim dn. Cc s liu thc nghim cho php d on
thi gian cn thit l khong 9-10 gi.
3.6 Ti u ha qu trnh thy phn ph liu u, v tm thu sn phm
bt carotenoprotein
Qu trnh thy phn nhm ti mc tiu l thu nhn ti a lng
carotenoid CPmax v protein ha tan APmax, trong , ch s c ch
trng nhiu hn l carotenoid-astasanthin v chnh cht mu ny em li hiu
qu kinh t cao cho sn phm carotenoprotein.
Qua phn phn tch cc yu t nh hng n qu trnh thy phn u, v
tm trn, suy ra khong kho st tm thng s ti u ca qu trnh thy
phn s l: nhit thy phn: 40-65
o
C, thi gian thy phn: 6-15 gi, nng
CPE s dng: 2-5%. Phn mm STATGRAPHIC Plus c s dng
phn tch s liu, a ra phng trnh hi qui cho hm mc tiu CP, AP.
3.6.1. Phng trnh hi qui ca hm lng carotenoid CP v thng s ti u
ca qu trnh thy phn thu nhn carotenoprotein giu carotenoid
Theo cc phn tch thng k, phng trnh hi qui ca hm lng
carotenoid CP c dng:
CP = -4,46088 + 0,290656 C + 0,144341 T + 0,130811 tg 0,0307762 C
2

- 0,00136915 T
2
0,00643203 tg
2
(3.3)
Trong :
CP Hm lng carotenoid trong sn phm carotenoprotein ca qu trnh thy
phn (mg/g)
T Nhit qu trnh thu phn (
o
C)
tg Thi gian thu phn (gi)
C Nng CPE s dng (%)
21

Phn tch b mt p ng ca hm CP cc nng CPE khc nhau 2;
3,5 v 5% u cho kt qu hm mc tiu CP t ti a khi nhit thy phn
l 53
o
C sau thi gian 10 gi, t suy ra nng CPE ti u l 4,5%






Hnh 27 B mt p ng hm carotenoid CP sau 10 gi thy phn
Carotenoid- Hm lng carotenoid (mg/g); T- Nhit (
o
C); C- Nng CPE (%)
3.6.2 Phng trnh hi qui ca hm lng protein AP v thng s ti u ca
qu trnh thy phn thu nhn carotenoprotein giu protein
Theo kt qu phn tch thng k, phng trnh hi qui AP chp nhn
c c dng:
AP = -250.373 + 9.81032*C + 9.15861*T + 4.60104*tg - 0.966014*C*C
-0.0867718*T*T - 0.236527*tg*tg (3.4)
Trong :
AP Hm lng protein trong sn phm ca qu trnh thy phn (mg/g)
T Nhit qu trnh thu phn (
o
C)
tg Thi gian thu phn (gi)
C Nng CPE s dng (%)







Hnh 28 B mt p ng hm protein AP sau khi thy phn 9,5 gi
AP- Hm lng protein (mg/g); T- Nhit (
o
C); C- Nng CPE b sung (%)
Cc b mt p ng th hin ph thuc ca hm mc tiu AP vo nhit
T v thi gian tg cc nng CPE khc nhau 2; 3,5 v 5% u cho kt
2
2.5
3
3.5
4
4.5
5
C
40
45
50
55
60
65
T
0.24
0.34
0.44
0.54
0.64
0.74
C
a
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e
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i
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2 2.5 3 3.5 4 4.5 5
C
40
45
50
55
60
65
T
Carotenoid
0.29
0.34
0.39
0.44
0.49
0.54
0.59
0.64
0.69
2
2.5
3
3.5
4
4.5
5
C
40
45
50
55
60
65
T
15
19
23
27
31
35
39
A
P
2 2.5 3 3.5 4 4.5 5
C
40
45
50
55
60
65
T
AP
17.4
19.8
22.2
24.6
27.0
29.4
31.8
34.2
36.6
22

qu AP t ti a khi nhit thy phn l 53
o
C sau thi gian 9,5 gi. Xt b
mt p ng AP sau 9,5 gi, c th d on khi tng C t gi tr 3,5 n 4%
th AP tng nhng khng ng k, v khong tng nng CPE t 4 ln 5% s
cn t c tc dng tng AP hn na. Nng ti u C=4,5-5% c chp
nhn mt cch gn ng.
3.6.3. Thng s ti u chung ca qu trnh thy phn thu nhn
carotenoprotein
Mc tiu cn t ca qu trnh thy phn l CPmax v APmax, trong
CP c u tin v chnh hm lng carotenoid l thnh phn quyt nh
gi tr kinh t ca bt carotenoprotein thnh phm, do , nghin cu ny chp
nhn cc thng s ti u t c CP ln nht, ngha l thy phn u v
tm nhit 53
o
C, thi gian 10 gi nng CPE 4,5%. Cc thng s ti
u ny c p dng vo thy phn kim tra li tnh tng thch vi
thc t. Sn phm carotenoprotein thu c c dng bt mu vng cam sng
p, thm mi tht tm nu chn, v ngt nh d chu, giu m v acid amin,
ph hp cho s dng lm thc n cho vt nui hoc thc phm chc nng cho
ngi.
Bng 3.1 Thnh phn ha hc c bn ca bt carotenoprotein
Thnh phn Hm lng
Protein th
Lipid
Tro
Chitin
Carotenoid
70,68 0,36 %
16,89 0,23 %
6,37 0,18 %
3,25 0,11 %
0,7056 0,0257 mg/g
Bng 3.2 Thnh phn acid amin ca bt carotenoprotein
Thnh phn Hm lng (%) Thnh phn Hm lng (%)
Alanin
Glycine
Valine
Leucine
Isoleucine
Threonine
Serine
Proline
0,95
2,12
1,26
0,72
1,71
0,39
0,5
1,13
Methionin
4-hydroxyproline
Glutamine
Phenylalanine
Lysine
Histidine
Tyrosine
Asparagine
1,04
0,77
3,51
2,66
0,86
2,79
3,38
1,40
23

KT LUN V XUT KIN
1. xc lp c qui trnh tch chit ch phm enzyme CPE protease t gan
ty v u tm s. Protease t gan ty v u tm c th tinh sch bng sc k
lc gel s dng Bio-Gel P-100. sch ca protease sau sc k tng ln 16,27
ln i vi gan ty v 18,03 ln i vi u tm.
2. xc nh c thnh phn v tnh cht ca protease tm s nh sau:
Protease t gan ty v u tm s gm t nht nm loi protease khc nhau
vi phn t lng t 20.200 n 40.200 Da, ring u tm cn c thm
hai protease vi phn t lng l 49.200 v 76.000 Da. Ba protease chim
vai tr hot ng ch o c phn t lng 20.200 n 25.000 Da.
Protease gan ty v u tm s bn nhit, c nhit ti thch l 62
o
C, pH
hot ng ti u l 7,5 vi nng mui n NaCl thch hp cho hot ng
l 1%. Hot ca protease gan ty v u tm gim khi c mt ca mt
s ion kim loi, c bit l Hg
2+
. Ion Mn
2+
lm tng hot tnh ca protease
tm s.
Protease gan ty v u tm thuc nhm protease serine, trong cc
enzyme ta trypsin ng vai tr ch o hot ng.
Protease tm s c i lc vi c cht tt, hp tc tng h gia cc trung
tm hot ng ca enzyme ny l dng tch cc, s gn kt mt c cht
vo trung tm hot ng s thc y, to iu kin thun li cho c cht
gn kt vo trung tm hot ng khc.
3. s dng ch phm enzyme CPE t u tm vo thy phn hn hp mu
v gan c basa thu dch m c hm lng nit tng l 9,8 g/l, nit acid amin
l 3,4 g/l . iu kin thy phn ti u nh sau : Nhit : 57
o
C, thi gian 14,5
gi, nng CPE b sung: 4% so vi hn hp, nng mui n b sung 1%,
pH hn hp lc bt u thy phn l 7
4. s dng CPE u tm thy phn u, v tm v thu nhn bt
carotenoprotein vi hm lng carotenoid cao 0,706 mg/g; hm lng protein
cao 70,68%, rt giu acid amin (26,99%) ph hp dng cho vt nui v thc
phm chc nng cho ngi. Thng s thy phn ti u nh sau: Nhit :
24

53
o
C, thi gian thy phn 10 gi, nng CPE b sung: 4,5% so vi nguyn
liu u, v tm; t l ph liu u, v tm so vi dung dch Na
2
-EDTA0,5M
pH7 b sung vo thy phn: 1:3 (w/v), nng mui n b sung 1%, pH
lc bt u thy phn: 7
xut cho cc nghin cu tip theo
1. Tip tc nghin cu ng dng sn xut CPE t u tm qui m ln hn
p dng vo sn xut nhm nng cao gi tr s dng ca u tm
2. S dng ph phm ca qu trnh sn xut CPE v bt carotenoprotein l v
tm loi phn ln protein lm i tng nghin cu xc nh qui trnh
sn xut chitin thch hp cho n, nhm c ch x l thch ng t hao ph
ha cht, mang li li ch kinh t v tc dng bo v mi trng.
3. Tip tc nghin cu cc ng dng sn phm dch thy phn t hn hp mu
v gan c p dng vo sn xut, v d, s dng lm thc n thay th sa cho
vt nui, b sung vo thc n gia sc hoc dng n thay cho nc mui li
qua b chp sau khi rt nc mm ct.
4. Th nghim s dng mt s cht phng thi vo thy phn dch hn hp
mu v gan c basa, hn hp u, v tm nhm to iu kin thun li thu
dch m thu phn vi hm lng acid amin c nng cao hn.
5. Th nghim thy phn ph liu u v tm trong cc mi trng m khc
nh m citrate-phosphat hoc ln men ph liu trc khi thy phn bng
CPE protease u tm nhm c ch s thi ra do vi sinh vt gy ra.
6. Th nghim loi cht bo trc khi tin hnh thy phn i vi c hai i
tng mu v gan c basa thu dch m thy phn, ph liu u, v tm s thu
nhn bt carotenoprotein.
7. Kho st tc dng tr lng ca chitosan khi kt ta thu bt nho
carotenoprotein vi cc nng chitosan s dng khc nhau. Th nghim thu
bt nho caroteinoprotein vi thi gian lng lnh khc nhau.
8. Th nghim s dng cc loi protease khc vo thy phn u, v tm, so
snh hiu qu vi enzyme protease thu nhn t tm s.