Tp ch Khoa hc Trng i hc Cn Th

Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 37 (2015)(2): 39-46

NGHIN CU KH NNG THY PHN DCH PROTEIN

CA THT U TM S BNG ENZYME PROTEASE NI TI
Trn Thanh Trc1, Vi Nh Tun2, V Th Anh Minh3 v Nguyn Vn Mi1
1

Khoa Nng nghip & Sinh hc ng dng, Trng i hc Cn Th

Sinh vin lp Cng ngh Thc phm K36, Trng i hc Cn Th
3
Hc vin Cao hc Cng ngh Thc phm K19, Trng i hc Cn Th
2

Thng tin chung:

Ngy nhn: 22/07/2014
Ngy chp nhn: 27/04/2015

Title:
Study on enzymatic
hydrolysis of protein from
head meat of black tiger
shrimp using intracelllar
protease
T kha:
Tht u tm s, thi gian
thy phn, tin x l, pH mi
trng, protease
Keywords:
Black tiger shrimp head
meat, hydrolysis time,
pretreatment, pH, protease

ABSTRACT
A study on enzymatic hydrolysis of protein from head meat of Black Tiger
shrimp using intracelllar protease was carried out. Factors affecting the
hydrolysis of protein including time and pH were investigated. Thermal
pre-treatment conditions to activate the intracellular protease were
optimized using response surface method consisting of 24 experiments.
The experimental results showed that, the optimal hydrolysis time for the
best quality product was 8 hours. The optimal conditions for the best
hydrolysis included pH of 9.0 combined with the pre-treatment
temperature of 50.73C and the pre-treatment time of 4.43 minutes.
TM TT
Mc tiu ca nghin cu l kho st kh nng thy phn protein bng
enzyme protease ni bo c trong tht u tm s. Thng qua ni dung
nghin cu, mt s yu t nh hng n qu trnh thy phn protein nh
thi gian thy phn thch hp, nh hng ca pH mi trng thy phn
c quan tm. c bit, iu kin tin x l nhit nhm kch hot enzyme
protease ni bo c ti u ha theo phng php b mt p ng
vi 2 tha s bao gm 24 n v th nghim. T cc kt qu th nghim cho
thy, thi gian thy phn protein tt nht, dch thy phn khng c mi l
l 8 gi. ng thi, qu trnh thy phn protein t tt nht khi iu chnh
pH mi trng l 9, kt hp vi thng s ti u ca iu kin tin x l
nhit nguyn liu l 50,73C v thi gian 4,43 pht.
xut chitin v asthaxanthin (Shahidi & Synowiecki,
1991). i vi tm ch s dng 65% phn c th
n c, phn cn li c loi b nh cht thi
(u ngc v b xng ngoi). Trong nhiu nm
qua, cc k thut c pht trin trong vic khai
thc v thu hi nhng sn phm ph c gi tr sinh
hc cao (Ferrer et al., 1996) nh phn bn, thc n
gia sc, thc n thy sn. Ngoi ra, dch thy phn
protein c ng dng trong nhiu ngnh cng
nghip (Duarte de Holanda & Netto, 2006). Gn
y, thy phn protein t cc nguyn liu thy sn

1 T VN
Vit Nam lun l quc gia c th mnh v xut
khu thy hi sn, trong tm s l mt trong
nhng mt hng ch lc. Sn lng thy sn nm
2013 khong 5,9 triu tn, ring mt hng tm s
t 193.000 tn tng 6,4% so vi cng k nm
trc (http://www.seafood.vasep.com.vn/FisheryStatistics/123_8987/Vietnam-seafood-exports -in2013.htm). Cc ph phm t quy trnh ch bin
tm c xc nh l mt ngun protein ln,
ng thi cng l mt ngun quan trng sn
39

Tp ch Khoa hc Trng i hc Cn Th

Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 37 (2015)(2): 39-46

ni ti c tin hnh.

tr thnh ph bin trong ngnh cng nghip

thc phm do hm lng protein cao (CrdovaMurueta et al., 2007). Vic thy phn protein lm
gim kch thc cc peptide, dch thy phn l
ngun acid amin c sn cho sinh tng hp protein
(Gildberg & Stenberg, 2001). Mt s ha cht
c p dng thy phn ph phm tm, v d
HCl 1M (Kjartansson et al., 2006), sodium sulfite
(Mizani et al., 2005) NaOH v KOH (Duarte de
Holanda & Netto, 2006). Tuy nhin, vic s dng
acid mnh hoc baz mnh thy phn protein l
nguyn nhn to ra cc sn phm ph c tc hi vi
mi trng. V vy, thc hin thy phn protein t
tht u tm s theo phng php enzyme c nhiu
thun li trong vic kim sot cc qu trnh thy
phn v do gim thiu cc phn ng khng
mong mun, gim nhim mi trng. Tin trnh
ny c th thc hin bng cch to iu kin thun
li cho hot ng ca protease ni ti c trong tht
u tm. Trn c s , vic nghin cu tc ng
ca qu trnh tin x l n hiu qu thy phn
protein t tht u tm s bng enzyme protease

2 PHNG PHP NGHIN CU

2.1 Nguyn liu
Tht u tm s (Penaeus monodon) tch v,
c thu mua huyn Thi Bnh, tnh C Mau
(bo m mu c gi lnh nhit di 4C
v thi gian ti a 12 gi t cng on tch u
n khi thu nhn tht u tm). Nguyn liu c
cha trong cc bao PE (2 lp), ct cht ming bao
v bo qun trong thng xp nhit t 0 4C
bng nc , sau em v phng th nghim b
mn Cng ngh thc phm, Trng i hc Cn
Th. Thi gian vn chuyn ti a l 5 gi. Nguyn
liu sau khi ly v n phng th nghim tin hnh
x l s b, ng gi vi mi mu tin x l c
khi lng 200g v cp ng nhit 25C (m
bo tm nguyn liu t -18C).
2.2 Phng php b tr th nghim
2.2.1 S b tr th nghim

Nguyn liu tht u tm s

Tr ng

T l nguyn liu v
dung mi l 1:1 (w/v, g/mL)

Xay nhuyn

Tin x l
(iu chnh nhit , thi gian, pH)
Th nghim 2, 3
Thy phn (nhit phng, 300 rpm)

Th nghim 1

Hnh 1: S b tr th nghim tng qut

Lc, ly tm nhit 4 C

B (b)

Dch lc trong
Hnh 1: S b tr th nghim tng qut
2.2.2 Th nghim 1: Xc nh thi gian thy
phn thch hp gip dch thy phn khng c mi
l v hiu sut thy phn protein cao

thay i t 5 gi n 9 gi. Nguyn liu tht u

tm s sau khi x l s b c nghin nh vi t l
khi lng nguyn liu/dung mi l 1:1. Tin hnh
thy phn vi nhit thy phn c nh l nhit
phng (30C). Ch tiu theo di l hiu sut
thy phn protein do tc ng ca enzyme protease
ni bo c trong mu tht u tm sau khi x l v

Th nghim tin hnh vi mc ch tm ra thi

gian thy phn thch hp nht gip hiu sut thy
phn protein t tht u tm s cao v dch thy
phn khng pht sinh mi l. Thi gian thy phn
40

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Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 37 (2015)(2): 39-46

2.2.4 Th nghim 3: nh hng pH mi

trng n hiu sut thy phn protein bng
enzyme protease ni bo trong tht u tm

hm lng protein ha tan c trong dch thy phn.

2.2.3 Th nghim 2: Tc ng ca iu kin
tin x l gip kch hot protease ni bo n hiu
sut thy phn protein t tht u tm s

Th nghim tin hnh tng t nh th nghim

1, thay th nc ct bng cc dung mi c pH thay
i t 4 n 10. Sau qu trnh thy phn tin hnh
xc nh hiu sut thy phn protein v hm lng
protein ha tan c trong dch thy phn.

Mc ch th nghim xc nh c iu
kin tin x l thch hp nht gip ci thin hiu
sut thy phn protein t tht u tm. Th nghim
c b tr cc khong nhit tin x l tht u
tm thay i t 40C n 65C v thi gian t 1
n 7 pht.

2.3 Phng php phn tch v nh gi kt qu

Cc ch tiu c bn v phng php phn tch

c th hin Bng 1.

Th nghim c tin hnh tng t nh th

nghim 1. Tuy nhin, mu tht u tm sau khi
nghin vi t l dung mi (nc ct) c nh 1:1 s
c tin x l cc ch nhit v thi gian
khc nhau. Mu sau tin x l c chuyn sang
thu phn ngay vi thi gian thy phn c la
chn t th nghim 1 v nhit thy phn cng c
nh nhit phng (30C). Ch tiu theo di l
hiu sut thy phn protein v hm lng protein
ha tan c trong dch thy phn.

S ln lp li: 3 ln. ln ca mu th nghim

200 g/mu. S liu c thu thp v x l bng
phn mm thng k Statgraphics Centrution 16.1
v phn mm Excel. Phn tch phng sai
(ANOVA) v kim nh LSD so snh trung bnh
gia cc nghim thc. S dng quy hoch
thc nghim theo phng php a nhn t thit
lp phng trnh hi quy th hin s tng quan
gia nhit v thi gian tin x l tht u tm
thch hp.

Bng 1: Phng php v thit b s dng phn tch cc ch tiu

Thnh phn
m tng s
Hm lng protein ha tan
pH
Hiu sut thy phn

Phng php
Xc nh bng phng php Kjeldahl, TCVN 8125:2009.
Xc nh bng phng php Biuret, TCVN 8125:2009.
S dng pH k, theo ISO 2917:1999(E).
Xc nh bng phng php Adler Nissen, 1986.
thy phn l nguyn nhn lm cho nguyn liu
3 KT QU V THO LUN
chuyn sang hin tng t phn, to ra mi hi
3.1 Thi gian thy phn thch hp n c
thi, khng chp nhn c. Chnh v vy, vic xc
tnh cm quan dch thy phn v hiu sut thy
nh thi gian thu phn thch hp protein t tht
phn protein
u tm s l iu kin cn thit u tin cn c
quan tm. S thay i hiu sut thu phn protein
Thi gian l mt trong nhng yu t nh hng
t tht u tm s theo thi gian c th hin theo
ln n qu trnh thu phn. Thi gian qu ngn
Hnh 2.
khng enzyme protease ni bo hot ng,
hiu qu thy phn thp. Vic ko di thi gian

41

Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 37 (2015)(2): 39-46

8000

4,5

7200

6400

3,5

5600

4800

2,5

4000

3200

1,5

2400

1600

0,5

800

Hm lng protein ha tan (mg/100g cht kh)

Hiu sut thy phn (%DH)

Tp ch Khoa hc Trng i hc Cn Th

0
5h

6h

7h

8h

9h

Thi gian (gi)

%DH

Hm lng protein ho tan

Hnh 2: nh hng thi gian n hiu sut thu phn protein

bng protease ni bo trong tht u tm
tra l 18 gi. Tuy nhin, dch thy phn c s
dng trong ch bin thc n gia sc. Cc nghin
cu ng dng dch thy phn protein trong ch
bin cc sn phm thc phm, in hnh nh
nghin cu ca Nilsang et al. (2005) xut
thi gian thy phn ph phm c sardine (t qu
trnh sn xut c ng hp) l 6 gi to dch
protein c c c hay bt protein c (fish protein
hydrolase). Nghin cu ca L Th Minh Phng
(2013) ngh thi gian thy phn 8 gi l thch
hp sn xut dch thy phn t tht hu bin v
sau s dng trong ch bin thc phm. Chnh v
vy, thi gian thy phn tht u tm 8 gi c
la chn kho st bin php nng cao hiu qu
thy phn protein tip theo.
3.2 nh hng nhit v thi gian tin x
l n hiu sut thy phn protein bng enzyme
protease ni bo t u tm s

T kt qu th hin th Hnh 2 cho thy

hm lng protein ho tan tng tuyn tnh vi s
gia tng thi gian thy phn. Thi gian thu
phn 5 gi, hm lng protein ho tan ch c
3649,125 mg/100g cht kh nhng khi tng thi
gian ln 9 gi th hm lng protein ho tan
tng gn 2 ln. Bn cnh , hiu sut thu phn
cng tng khi tng thi gian thu phn. thi gian
5 gi, hiu sut thy phn ch c 0,86% nhng khi
thi gian 9 gi tng gp 5 ln. Qua cho thy
thi gian thy phn nh hng rt nhiu n hiu
sut thy phn ln hm lng protein ha tan. Khi
thi gian thy phn 5 6 gi th khng
enzyme tip xc v phn ct protein nn hiu sut
thy phn rt thp di 1%. Tng thi gian thy
phn 7 8 gi, enzyme thi gian tip xc v
phn ct protein nn hiu sut thy phn tng
mnh. thi im 9 gi hiu sut thu phn vn
tng nhng dch thu phn c mi hi v bt kh.
iu ny do cc vi sinh vt hiu kh pht trin sinh
ra protease phn gii protein v sn phm ca qu
trnh l hydro sulfua, indol, scatol, butyric, to
mi kh chu. l hin tng thi ra tht, khng
p ng mc ch s dng cho thc phm. V vy,
thi gian thu phn thch hp l 8 gi c hiu
sut thu phn tt v cha pht sinh mi l. Mc
d thi gian thy phn 8 gi l kh ngn so vi
mt s cc kho st khc, in hnh nh nghin
cu ca Randriamahatody et al. (2011) ngh
thi gian thy phn tht u tm s l 22 gi hay
nghin cu ca Trn Th Hng Nghi v ctv. (2009)
xut thi gian thy phn protein t ph phm c

Da trn cc kho st trc v tc ng

ring l ca yu t nhit , thi gian n kh nng
hot ng protease (Nguyn L H, 2011) cho
thy, hai nhn t ny u c s chi phi ng k
n hot ng ca protease iu ny c th l
nguyn nhn chnh nh hng n vic thay i
hiu qu thy phn protein t tht u tm s. V
vy, nghin cu xc nh hi quy tng quan ca 2
nhn t, bao gm nhit tin x l X1 (40
65C) v thi gian tin x l X2 (1 7 pht) n
hiu sut thy phn protein (Y, %) c thc
hin. Kt qu phn tch nh hng ca cc nhn t
m ha i vi phng trnh hi quy c trnh
by Bng 2.

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Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 37 (2015)(2): 39-46

Bng 2: nh hng cc nhn t n phng trnh hi quy

Nhn t
X1
X2
X1X1
X1X2
X2X2
S ln lp li
Sai s

Tng bnh phng

19,0142
13,4096
101,625
7,40544
77,5012
0,0448028
9,12282

Bc t do
Phng sai
T s F
Gi tr P
1
19,0142
133,39
0,0000
1
13,4096
94,07
0,0000
1
101,625
712,94
0,0000
1
7,40544
51,95
0,0000
1
77,5012
543,70
0,0000
2
0,0224014
0,16
0,8549
64
0,142544
tin
x
l
n
hiu sut thu phn c thit lp v
Bng tng hp 2 cho thy, kt qu kho st c
s
dng

d
on hiu qu vic thu phn
tin cy cao khi s ln lp li th nghim u c
protein.
Hiu
sut
thu phn m phng (Y l
s khc bit khng c ngha v mt thng k, th
thuyt) c xc nh bng cch thay cc bin vi
hin gi tr P = 0,8549 (gn bng 1, ln hn
gi tr thc vo phng trnh sau:
0,05). ng thi, tt c gi tr P ca cc tha s
u nh hn 0,05 chng t cc nhn t kho st
Y = 50,1566 + 2,00738X1 + 3,1498X2
u nh hng n qu trnh thu phn protein do
0,0190512X12 0,0167962X1X2 0,259375X22 (R2
tc ng ca vic kch hot protease ni bo t tht
= 0,9751)
u tm s. H s hi quy bc mt ca X1, X2 cng
Hiu sut thu phn thu c t thc nghim
nh h s tng tc ca X1X2 u khc bit ngha
v
tnh
ton theo phng trnh c tng thch
v mt thng k tin cy 95%, ng thi h s
cao gi tr R2 = 0,9751 (hnh 3). Nh vy, c th
hi quy bc hai ca X12, X22 cng khc bit ngha
kt lun rng phng trnh hi quy m t ng
v mt thng k. iu ny gp phn khng nh
cc kt qu thc nghim. H s tng quan cho
mc nh hng ca tng bin c lp cng nh
bit 97,51% s thay i hiu sut thu phn protein
cc tng tc c ngha n qu trnh thu phn
l do nh hng cc bin c lp X1, X2 v ch c
c kho st.
2,59% s thay i l do cc yu t khng xc nh
Da trn kt qu phn tch ANOVA, phng
gy ra.
trnh hi quy th hin s tng quan ca iu kin

Hnh 3: th tng quan gia hiu sut thu phn theo thc nghim
v tnh ton theo phng trnh hi quy
trong u tm th hin hiu sut thy phn cao
nht trong khong nhit 50 55C v thi gian
l 3 5 pht. ng thi, c s tng tc gia nhit
v thi gian tin x l n hiu sut thy phn
protein ca tht u tm s. Hiu sut thy phn c
gi tr thp nht iu kin nhit thp (40C) v

th b mt p ng v th ng ng
im biu din s tng tc ca nhit v thi
gian tin x l nguyn liu ln hiu sut thy phn
protein ca enzyme protease c biu din Hnh
4. V mt tng qut, h enzyme protease ni bo
43

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Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 37 (2015)(2): 39-46

thi gian ngn (1 pht). iu ny cho thy mc

thi gian thp l khng kch hot hot tnh
enzyme protease trong tht u tm. Hn th na,
nhit 40C cng khng nm trong khong nhit
ti u cho hot ng ca protease (55 60C,
Nguyn L H, 2011). Tuy nhin, khi iu kin
nhit cao (65C) v thi gian tin x l ko di
(7 pht) th hiu sut thy phn vn thp. iu ny
l do protease v ngay c protein ca tht u tm
b bin tnh nhit cao trong thi gian di.

Vi mi trng nhit trung bnh (50 55C) th

hiu sut thy phn tng nhanh trong khong thi
gian x l (3 5 pht).
Qua th Hnh 4, hiu sut thy phn t cc
i khi nhit 50,73C v thi gian x l l 4,43
pht. Kt qu ny kh ph hp vi nghin cu tch
chit v ng dng enzyme protese t u tm s
Penaeus monodon vo ch bin thy sn ca
Nguyn L H (2011).

8,00

16000

7,00

14000

6,00

12000

5,00

10000

4,00

8000

3,00

6000

2,00

4000

1,00

2000
0

0,00
i chng (khng tin x l)

%DH

Hm lng protein ha tan (mg/100g cht kh)

Hiu sut thy phn (%DH)

Hnh 4: th biu din s tng tc ca nhit v thi gian tin x l

nguyn liu ln hiu sut thy phn protein bng protease ni bo

Ti u (tin x l)

Hm lng protein hoa tan

Hnh 5: th so snh hiu sut thy phn v hm lng protein ha tan

gia hai mu i chng v ti u
3.3 nh hng pH n hiu sut thy phn
protein bng enzyme protease ni bo t tht
u tm s

Hnh 5 cho thy hiu sut thy phn ca mu

ti u qua tin x l nhit 50,73C, thi gian
4,43 pht l 7,7% cao gp 2 ln mu i chng
(khng tin x l). Bn cnh , hm lng protein
ha tan mu ti u cao hn mu i chng. iu
ny chng t l vic tin x l trc khi thy phn
l vic rt cn thit nhm nng cao hiu sut thy
phn. Bi v hu ht cc vi sinh vt gy thi khng
pht trin tt trong iu kin nhit cao m
enzyme protease t u tm s vn hot ng tt.

S thay i pH nh hng rt ln n hot tnh

enzyme do s tc ng n hiu sut thy phn.
Mi enzyme ch hot ng mnh nht mt pH
xc nh, gi l pH ti u. Khi pH qu cao hay qu
thp c th lm bin tnh enzyme. V vy, vic xc
nh pH thch hp cho qu trnh thy phn t hiu
sut thy phn cao nht l rt cn thit. Kt qu thu
nhn c th hin Hnh 6.
44

Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 37 (2015)(2): 39-46

12

18000

10

15000

12000

9000

6000

3000

0
4

10

Hm lng protein ha tan (mg/100g cht

kh)

Hiu sut thy phn (%DH)

Tp ch Khoa hc Trng i hc Cn Th

pH
%DH

Hm lng protein ha tan

Hnh 6: th biu din s thay i hiu sut thy phn protein theo Ph
th biu din Hnh 6 cho thy khi pH ban
u ca dung mi s dng trch ly protease
c iu chnh t 4 n 6, hiu sut thy phn
protein thu c l rt thp (<1%) v hm lng
protein ha tan cng mc nh hn 1000mg/100g
cht kh. Khi pH tng t 7 n 9 hiu sut thy
phn tng t 6,18% ln 9,88% v hm lng
protein ha tan cng tng t 10390,13 mg/100g
cht kh ln 16610,75 mg/100g cht kh. Tuy
nhin, khi tng pH ln 10 hiu sut thy phn gim
t 9,88% xung 6,18% v hm lng protein ha
tan gim t 16610,75 mg/100g cht kh xung
10390,13 mg /100g cht kh. Hiu sut thy phn
protein t cao nht iu kin pH ca dung mi
s dng l 9. So snh vi mu i chng (s dng
nc ct l dung mi cho qu trnh thy phn
protein), hiu sut thy phn protein ca mu s
dng nc ct v mu c pH bng 8 l khng c s
khc bit v mt thng k. iu ny cng ph hp
vi l thuyt v s hot ng ca protease theo pH
(pH ca nc ct s dng dao ng trong khong
7,2 7,5).

4 KT LUN
Nghin cu thy phn dch protein t tht u
tm s bng phng thc kch hot protease ni
bo cho thy tnh kh thi. Vic kch hot
protease ni bo nhit v thi gian ln lt
50,73C v 4,43 pht l iu kin tin x l thch
hp nht gip gia tng hiu qu trch ly protein.
Dch thy phn protein t tht u tm s bng
protease ni bo t hiu sut cao nht khi tin
hnh thy phn nhit phng (30C), thi gian
thy phn 8 gi v iu chnh pH ca dung mi s
dng thy phn n gi tr pH 9.
TI LIU THAM KHO
1. Adler Nissen J., 1986. Enzymic
Hydrolysis of Food Proteins. Elsevier
Science Ltd, 451 pp.
2. Crdova-Murueta J.H., M. de los ngeles
Navarrete del Toro, and F. Garca Carreo,
2007. Concentrates of fish protein from
bycatch species produced by various drying
processes. Food Chemistry 100: 705-711.
3. Duarte-De-Holanda H. and F.M.Netto,
2006. Recovery of components from shrimp
(Xiphopenaeus kroyeri) processing waste by
enzymatic hydrolysis. Journal of Food
Science 71: C298-C303.
4. Ferrer J., G. Paez, Z. Marmol, E. Ramones, H.
Garcia & C.F. Forster, 1996. Acid hydrolysis
of shrimp-shell wastes and the production of
single cell protein from the hydrolysate.
Bioresource Technology 57: 55-60.

Tm li, hiu qu thy phn protein t tht u

tm bng enzyme protease ni bo chu s chi
phi rt ln ca cc iu kin kch hot enzyme,
in hnh l nhit , thi gian v pH. iu kin
kho st, khi s dng dung mi hu c glycine
NaOH c pH 9 v t l nguyn liu v dung mi l
1 : 1, tin hnh kch hot enzyme nhit v thi
gian ti u c xc nh (50,43C v 4,43 pht)
gip hiu qu thy phn protein t tht u tm t
cao nht.

45

Tp ch Khoa hc Trng i hc Cn Th

Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 37 (2015)(2): 39-46

5. Gildberg A. and E. Stenberg, 2001. A new

process for advanced utilisation of shrimp
waste. Process Biochemistry 36: 809-812.
6. Kjartansson G., S. Zivanovic, K.
Kristbergsson, and J. Weiss, 2006.
Sonication-assisted extraction of chitin from
shells of fresh water prawns (Macrobrachium
rosenbergii). Journal of Agricultural and
Food Chemistry 54: 3317-3323.
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