Trn Ngc Hng Nghin cu thu nhn pectinase t aspergillus niger

NGHIN CU THU NHN PECTINASE T ASPERGILLUS

NIGER NUI CY TRN MI TRNG BN RN CHA
CI BI NNG CAO HIU QU BC V TIU
Trn Ngc Hng, Nguyn Anh Dng, Mai Th Ngc Lan Thanh,
Trn Th Ngc Nh
Trng i hc Th Du Mt

TM TT
Trong quy trnh sn xut tiu s, vic b sung enzyme pectinase c th gip nng cao
hiu qu bc v tiu. Trong s 9 chng Aspergillus niger nghin cu, chng A. niger B2
sinh tng hp enzyme pectinase tt nht trn mi trng cha 12% bt ci bi, lng
nc b sung vo mi trng 40%, thi gian thch hp thu nhn enzyme l 5 ngy, hot
pectinase t 4,82 UI/g. Ch phm pectinase gip rt ngn thi gian bc v tiu. Hiu
sut bc v t 93% sau 28 gi i vi tiu en v 76 gi i vi tiu xanh. Kt qu cho
thy tim nng s dng pectinase c sn xut t ph liu nng nghip nng cao hiu
qu sn xut tiu s.
T kha: nui cy, mi trng, bn rn, ci bi, thu nhn pectinase, v tiu

1. T VN
Pectinase l nhm enzyme xc tc phn ng thy phn pectin, gii phng cc n phn
l acid galacturonic[4]. Trong lnh vc cng ngh thc phm, enzyme pectinase c s
dng lm mm vch t bo, tng qu trnh ly trch cc loi nc p tri cy; h tr qu
trnh lc v lm trong cc loi nc p tri cy, ru vang. Nhng nm gn y, enzyme
pectinase cn c s dng nng cao kh nng bc v tiu; rt ngn thi gian hoai mc
ca v c ph trong qu trnh ; x l lp nht bm trn ht c ph[3,6,7]. Mc d enzyme
pectinase c mt nhiu thc vt v vi sinh vt, nhng trong sn sut cng nghip, vi
khun Bacillus v nm mc Aspergillus lun l la chn hng u[4]. Vi kh nng pht
trin nhanh trn nhiu loi c cht khc nhau, c bit l trn cc ph liu nng nghip giu
pectin, nm mc Aspergillus niger lun thu ht c nhiu s quan tm ca cc nh nghin
cu. Vit Nam, c th k n mt lot cc nghin cu lin quan n enzyme pectinase t
Aspergillus trong nhng nm gn y: nm 2008, Hunh Ngc Oanh nghin cu tinh sch
enzyme pectinase bng phng php lc gel v lc mng[5]; nm 2009, Trn Th Thanh
Thun s dng ch phm c cha pectinase x l nhanh v c ph trong qu trnh
hoai[3]; nm 2011, L Th Thu Trang nghin cu kh nng tng hp pectinesterase v
polygalaturonic ca Aspergillus niger [8], nm 2012, Dng Vn Tun s dng pectinase
x l lp nht ca v c ph sau khi bc v[7]; nm 2013, Trn Thanh Trc phn lp
v tuyn chn dng Aspergillus niger sinh pectin methylesterase cao[9].
82
Tp ch Khoa hc i hc Th Du Mt S 5(30)-2016

Trong khi , hng nm, ngnh cng ngh ch bin thc phm thi ra mt lng ln
cc loi ph ph liu giu pectin nh: ci v cc loi tri cy c mi, b to, v c rt, ci
mt tn dng ngun ph ph liu ny b sung vo mi trng nui cy bn rn s cm
ng qu trnh sinh tng hp pectinase v gim thiu lng xc b thc vt thi ra mi
trng. Ch phm pectinase sau s c th nghim kh nng bc v tiu nhm nng
cao hiu qu ca quy trnh sn xut tiu trng, mt sn phm c gi tr cao v c th
trng cc nc pht trin a chung. T nh hng trn, chng ti tin hnh nghin cu:
Nghin cu thu nhn pectinase t Aspergillus niger nui cy trn mi trng bn rn cha
ci bi nng cao hiu qu bc v tiu (Piper nigrum L.).

2. VT LIU V PHNG PHP NGHIN CU

2.1. Vt liu
Cc ph liu giu pectin nh ci mt, ci bi, v cam, v c rt c thu gom ti cc
ch, cc quy nc p tri cy trn a bn thnh ph Th Du Mt, tnh Bnh Dng. Ci
mt: gt b phn v cng v phn x, xay nhuyn v sy kh cho n khi m cn
khong 10%. V cam, ci bi: gt b phn v xanh cha tinh du pha ngoi. Xay nhuyn
v sy kh cho n khi m cn khong 10%. V c rt: xay nhuyn v sy kh cho n
khi m cn khong 10%.
Chn (9) chng Aspergillus niger c phn lp trn cc c cht giu pectin do phng
th nghim Sinh hc, Trng i hc Th Du Mt cung cp. Bao gm cc chng Asp.
niger B1, B2, B3 v B4 phn lp trn v bi; cc chng Asp. niger C1, C2 v C3 phn lp
trn v cam; chng Asp. niger C4 v C5 phn lp trn v chanh.
Tiu xanh: ging tiu trung c dung trng khong 570g/lt, do nh vn ti huyn Ph
Gio tnh Bnh Dng cung cp. Tiu en: tiu xanh c phi nng cho n khi m
cn khong 20%.
2.2. Phng php nghin cu
Phng php nui cy bn rn thu nhn enzyme pectinase[5]: Cc chng Aspergillus
niger t mi trng thch nghing c cy vo cc erlen cha 25 g mi trng bn rn c
thnh phn: cm go 30%; tru 16%; bt c rt 8%, (NH4)2SO4 1%, m 45%. Mt
ging ban u khong 107 bo t/g canh trng. nhit phng (30oC). Sau 5 ngy,
thu nhn v sy kh canh trng 45oC cho n khi m cn khong 10%. Xc nh hot
enzyme pectinase trong canh trng bng phng php nh lng ng kh.
Xc nh hot pectinase bng phng php nh lng ng kh[1]: Trn u 0,5
ml dung dch enzyme pectinase vi 0,5 ml dung dch pectin 0,5% pha trong m acetate pH
5.0, 40oC trong 60 pht. Thm 1 ml thuc th DNS, un si cch thy trong 5 pht v
so mu bc sng 540 nm. Mt n v hot (UI) enzyme pectinase l lng enzyme
ti thiu trong iu kin chun (40oC, pH 5) thy phn pectin trong 1 pht to ra 1 mol
D-galacturonic acid.
Phng php x l s liu: Mi nghim thc c tin hnh lp li 5 ln. nh gi sai
s chun bng chng trnh phn tch phng sai ca phn mm Statgraphic Centurion 15.

83
Trn Ngc Hng Nghin cu thu nhn pectinase t aspergillus niger

2.3. B tr th nghim
Chn lc chng Aspergillus niger c hot tnh sinh tng hp enzyme pectinase cao: 09
chng Aspergillus niger trn mi trng thch nghing c cy vo cc mi trng bn
rn. Sau 5 ngy nui cy, xc nh hot tnh pectinase trong canh trng nui cy v chn
chng sinh tng hp pectinase cao nht.
nh hng ca thnh phn c cht giu pectin n kh nng sinh tng hp enzyme
pectinase: Chng Aspergillus niger chn lc t th nghim trc c nui cy trn mi
trng bn rn c b sung 8% cc c cht giu pectin khc nhau (ci mt, v cam, ci bi
v v c rt). Sau 5 ngy nui cy, xc nh hot tnh pectinase trong canh trng nui cy
v chn thnh phn c cht thch hp sinh tng hp pectinase cao nht.
nh hng ca t l c cht giu pectin n kh nng sinh tng hp enzyme pectinase:
Chng Aspergillus niger chn lc t th nghim trc c nui cy trn mi trng bn
rn c b sung c cht giu pectin chn t th nghim trc. T l c cht trong cc
nghim thc thay i: 4; 8; 12; 16 v 20%. Sau 5 ngy nui cy, xc nh hot tnh
pectinase trong canh trng nui cy v chn t l c cht thch hp sinh tng hp
pectinase cao nht.
nh hng ca lng nc b sung n kh nng sinh tng hp enzyme pectinase:
Chng Aspergillus niger chn lc t th nghim trc c nui cy trn mi trng bn
rn c b sung thnh phn v t l c cht giu pectin chn t th nghim trc. Lng
nc b sung vo mi trng bn rn trong cc nghim thc thay i: 35; 40; 45; 50 v
55%. Sau 5 ngy nui cy, xc nh hot tnh pectinase trong canh trng nui cy v chn
lng nc b sung vo mi trng bn rn thch hp sinh tng hp pectinase cao nht.
Xc nh thi gian thu nhn enzyme pectinase thch hp nht: Chng Aspergillus niger
chn lc t th nghim trc c nui cy trn mi trng bn rn c b sung thnh phn
v t l c cht giu pectin, lng nc b sung chn t th nghim trc. Xc nh hot
tnh pectinase trong canh trng nui cy sau cc khong thi gian khc nhau: 3; 4; 5; 6 v
7 ngy. Chn thi gian thu nhn pectinase c hot cao nht.
nh gi kh nng bc v tiu ca ch phm enzyme pectinase: 50 g tiu xanh hoc
tiu en ( m 20%) c ngm vi 50 g nc, gi nhit phng (30oC). Sau 24 gi;
48 gi; 72 gi hoc 96 gi, b sung 10 ml dung dch enzyme pectinase c tng hot 4 UI
v gi 40oC. Sau 4 gi x l pectinase, tiu c ch xt tch v. Sy kh tiu n khi
m cn khong 10%. Cc l i chng c tin hnh vi ch phm pectinase bt
hot bng cch un si 15 pht.
Hiu sut bc v tnh theo cng thc: H (%) = Khi lng tiu s/khi lng (tiu s +
tiu cn dnh v).

3. KT QU V THO LUN
3.1. Chn lc chng Aspergillus niger sinh tng hp enzyme pectinase cao
Chn (9) chng Aspergillus niger trn mi trng thch nghing c cy vo cc mi
trng bn rn. Sau 5 ngy nui cy, hot tnh pectinase ca cc chng c xc nh v
th hin trong biu 1.

84
Tp ch Khoa hc i hc Th Du Mt S 5(30)-2016

Trn mi trng cha c cht cm

ng l bt v c rt, cc chng
Aspergillus niger th hin nhng kh
nng sinh tng hp pectinase rt khc
nhau. Chng Asp B2 c hot cao
nht, t 2,55 UI/g canh trng, khc
bit v mt thng k so vi cc chng
cn li. Chng Asp B2 c phn lp
trn ci tri bi, do c kh nng sinh
tng hp enzyme pectinase cao.
Biu 1. Hot pectinase ca cc chng
Aspergillus niger
3.2. nh hng ca thnh phn c cht giu pectin n kh nng sinh tng hp
enzyme pectinase ca chng Aspergillus niger B2
Chng Aspergillus niger B2 c
nui cy trn mi trng bn rn c b
sung cc c cht giu pectin khc nhau.
Sau 5 ngy nui cy, hot tnh pectinase
trong cc canh trng c xc nh v
th hin trong biu 2. Kt qu th
nghim cho thy ngun gc pectin c
nh hng nhiu n kh nng cm ng
sinh enzyme pectinase. V cam cha
hm lng pectin cao nht (3,5 5,5%)
nhng kh nng cm ng sinh pectinase Biu 2. nh hng ca c cht n kh
li thp hn so vi cc c cht khc. nng sinh tng hp pectinase
Ci bi cha 3,1% pectin thch hp nht cho vic cm ng sinh enzyme pectinase,
hot pectinase t 3,42 UI/g canh trng. Kt qu th nghim cho thy c th s dng
nm mc Aspergillus niger B2 sn xut ch phm giu pectinase trn c cht l ci bi.
3.3. nh hng ca t l c cht giu pectin n kh nng sinh tng hp enzyme
pectinase ca chng Aspergillus niger B2
Chng Aspergillus niger B2 c
nui cy trn mi trng bn rn c b
sung ci bi vi cc t l khc nhau.
Sau 5 ngy nui cy, hot tnh pectinase
trong cc canh trng c xc nh v
th hin trong biu 3. T l bt ci
bi trong cc nghim thc c nh
hng ng k ti kh nng sinh tng
hp pectinase ca chng Aspergillus
niger B2. Hm lng bt bi b sung
vo mi trng tt nht l 12%, hot Biu 3. nh hng ca t l c cht n
enzyme trong canh trng t 3 UI/g. kh nng sinh tng hp pectinase

85
Trn Ngc Hng Nghin cu thu nhn pectinase t aspergillus niger

Vic gia tng t l bt ci bi trong canh trng s kch thch qu trnh sinh pectinase, tuy
nhin, hm lng bt ci bi tng qu cao li lm gim kh nng sinh tng hp enzyme
pectinase. C th hm lng pectin trong mi trng cao s lm gia tng nht ca canh
trng, tc ng n kh nng sinh trng ca chng nm Aspergillus niger B2[1].
3.4. nh hng ca lng nc b sung n kh nng sinh tng hp enzyme
pectinase ca chng Aspergillus niger chn lc
Chng Aspergillus niger B2 c nui
cy trn mi trng bn rn c b sung ci
bi vi t l 12%. Lng nc b sung
vo mi trng thay i t 35 n 55%.
Sau 5 ngy nui cy, hot tnh pectinase
trong cc canh trng c xc nh v th
hin trong biu 4. Trong qu trnh nui
cy bn rn, m l yu t rt quan trng
cho s sinh trng ca nm mc. Vic thay
i lng nc b sung ban u s lm
thay i m ca canh trng. Trong
phm vi ca th nghim, lng nc b
sung ban u thay i 35 - 55% khng nh Biu 4. nh hng ca lng nc b sung
hng nhiu n kh nng sinh tng hp n kh nng sinh tng hp pectinase
enzyme pectinase.
Hot tnh pectinase cao nht t 4,15 UI/g khi lng nc b sung vo canh trng
chim 40%. Kt qu ny khng khc bit so vi cc nghim thc c b sung nc nhiu
hn. Tuy nhin, trong sn xut, vic gim lng nc trong canh trng s gip tit kim
chi ph sy sn phm. Do , chng ti chn lng nc b sung ban u l 40% tin
hnh th nghim tip theo.
3.5. Xc nh thi gian thu nhn enzyme pectinase thch hp nht
Chng Aspergillus niger B2 c nui
cy trn mi trng bn rn c b sung ci
bi vi t l 12%. Lng nc b sung
vo mi trng 40%. Sau khong thi gian
nui cy t 3 n 7 ngy, hot tnh
pectinase trong cc canh trng c xc
nh v th hin trong biu 5. Thi im
thu nhn enzyme pectinase thch hp l sau
5 ngy nui cy, hot pectinase trong
canh trng t 4,82 UI/g. Thi gian nui
cy cng di, hot enzyme pectinase
cng gim. Sau thi gian 5 ngy, kh nng
sinh trng ca nm Aspergillus niger B2
t cc i nn chng khng tit thm
pectinase vo mi trng. Biu 5. nh hng ca thi gian n hot
pectinase trong canh trng

86
Tp ch Khoa hc i hc Th Du Mt S 5(30)-2016

Trong khi , lng enzyme c tit ra trc mt dn hot tnh nn hot

pectinase trong canh trng gim dn. So vi kt qu nghin cu ca Hunh Ngc Oanh
(2008), ch phm ca chng ti c hot thp hn. Tuy nhin, nhng th nghim sng lc
trc cho thy enzyme pectinase thu c gip nng cao hiu qu bc v tiu, nn
chng ti tip tc th nghim hng ng dng ny.
3.6. nh gi kh nng bc v tiu ca ch phm enzyme pectinase
Tiu xanh v tiu en sau khi ngm trong nc c x l enzyme pectinase vi hot
4 UI. 40oC, sau 4 gi, hiu qu bc v c th hin trong biu 6 v biu 7.

Biu 6. nh hng ca pectinase n Biu 7. nh hng ca pectinase n

hiu qu bc v tiu xanh. hiu qu bc v tiu en.

Cng vi cellulose, pectin l thnh phn chnh cu trc nn v tiu. Cho nn khi b
sung enzyme pectinase, qu trnh bc v tiu din ra d dng hn. Tiu xanh c lp v
cha b h hi nn enzyme pectinase kh xm nhp vo bn trong, dn n hiu qu bc v
gia tng khng nhiu. Sau khi tiu ngm trong nc 72 gi, v tiu mm v thm nc tt,
vic b sung enzyme pectinase s em li hiu qu cao nht, hiu sut bc v t 93.3%
sau tng thi gian 76 gi x l. Khi ngm tiu trong nc lu hn, hiu qu bc v trong
trng hp c b sung pectinase khng c s gia tng ng k. Trong khi , i vi tiu
en, enzyme pectinase gip gia tng r rt hiu qu bc v. Sau khi ngm 24 gi, tiu en
tip tc c x l vi enzyme pectinase trong 4 gi, hiu sut bc v tiu en t 93,2%
sau tng thi gian 28 gi x l, cao hn 22,7% so vi i chng. C th vic tiu xanh
c phi nng lm cho lp v tiu h hi, to iu kin cho enzyme pectinase d dng
thm vo bn trong, thy phn thnh phn pectin v lm cho v tiu d bc tch ra khi
phn s. cc nghim x l tiu 52 v 76 gi, hiu sut bc v khng c s khc bit so
vi khi x l pectinase sau khi ngm 28 gi.
Kt qu ca th nghim cho thy trin vng ca vic s dng ch phm pectinase
nng cao hiu qu trong qu trnh sn xut tiu s. So vi kt qu nghin cu ca mt s
tc gi nh Bi Vn Min (2004)[3], thi gian ngm tiu 6 7 ngy, lng nc s dng
gp 30 ln, cho hiu sut bc v t 95% hay Nguyn Th M Hin (2010)[2], s dng ch
phm c cha nm Aspergillus sp. thc y qu trnh bc v ca tiu, hiu sut bc v
t 93% trong thi gian 3 ngy, kt qu ca chng ti rt ngn ng k v mt thi gian
v gim bt c lng nc s dng. Theo , thi gian ngm tiu ch ko di t 1 n 2
ngy v lng nc s dng gim c 30 ln.

87
Trn Ngc Hng Nghin cu thu nhn pectinase t aspergillus niger

4. KT LUN
T 9 chng Aspergillus niger phn lp trn cc ph liu giu pectin, chng ti chn lc
c chng Aspergillus niger B2 sinh tng hp enzyme pectinase tt nht trn mi trng
bn rn cha bt ci bi vi t l 12%, lng nc b sung vo mi trng 40%, thi
gian nui cy thch hp l 5 ngy, hot enzyme pectinase trong canh trng t 4,82
UI/g. X l enzyme pectinase 40oC trong thi gian 4 gi gip gia tng hiu qu bc v
tiu en. T l bc v tiu t 93,2% sau 28 gi ngm. Vic x l enzyme pectinase trn
tiu xanh khng em li nhiu hiu qu. Sau 76 gi x l, t l bc v ch cao hn 7,8% so
vi i chng.

RESEARCH ON PRODUCING PECTINASE FROM

ASPERGILLUS NIGER ON SOLID STATE MEDIUM CONTAINING GRAPE
FRUIT PULP FOR IMPROVING PEPPER PEELING
Tran Ngoc Hung, Nguyen Anh Dung, Tran Thi Ngoc Nhu, Mai Thi Ngoc Lan Thanh
ABTRACT
In white pepper processing, adding the pectinase is able to help improve pepper
peeling. Among 9 Aspergillus niger strains, A. niger B2 strain biosynthetise the pectinase
have a highest activity on the medium that contains the powder of pomelo with the ratio of
12 percent; a amount of water is 40 percent (w/w). The best suitable time to receive
pectinase is 5 days, with the activity pectinase reaches 4.82 UI/gr. The pectinase
preparation help shorten the time of remove the husk of pepper. The productivity of taking
off the husk gets 93 percent after 28 hours to the black pepper and after 76 hours to green
pepper. This result shows potential of applying pectinase that produce from agricultural
residues to improve effect of white pepper processing.

TI LIUU THAM KHO

[1] Nguyn Vn B, Cao Ngc ip, Nguyn Vn Thnh (2005), Gio trnh mn nm hc, Trng
i hc Cn Th.
[2] Lm th Kim Chu, Vn c Chn, Ng i Nghip (2004), Thc tp ln sinh ha, NXB i
hc Quc gia thnh ph H Ch Minh.
[3] Nguyn Th M Hin, Nguyn Vn Hong (2010), S dng ch phm sinh hc ln men sn
xut tiu trng, lun vn thc s, Trng i hc Lc Hng.
[4] Bi Vn Min, Nguyn nh Kim Ln (2004), Bc u nghin cu quy trnh cng ngh v
th nghim ch bin tiu en thnh tiu trng ph hp vi quy m va v nh Vit Nam, Tp
ch Khoa hc K thut, Trng i hc Nng Lm, s 1.
[5] Nguyn c Lng (2004), Cng ngh vi sinh, Tp 2: Vi sinh vt hc cng nghip, NXB i
hc Quc gia thnh ph H Ch Minh.
[6] Hunh Ngc Oanh, Trn Ngc Hng (2008), Thu nhn enzyme pectinase t Aspergillus niger bng
phng php lc gel v lc mng, Tp ch Pht trin Khoa hc v Cng ngh, tp 11, s 8.
[7] Trn Th Thanh Thun, Nguyn c Lng (2009), Nghin cu enzyme cellulase v pectinase
t chng Trichoderma viride v Aspergillus niger nhm x l nhanh v c ph, Tp ch Pht
trin Khoa hc v Cng ngh, tp 12, s 3.

88
Tp ch Khoa hc i hc Th Du Mt S 5(30)-2016

[8] Dng Vn Tun, L Th Hong Linh, Trn Th X (2012), Nghin cu s dng h enzyme
pectinase, cellulase ca vi khun Bacillus subtilis, Lactobacillus plantarum, nm mc
Aspergillus niger, Phanerochaete chrysosporium x l lp nht ca v c ph, Tuyn tp
bo co Hi ngh sinh vin nghin cu khoa hc ln th 8, i hc Nng.
[9] L Th Thu Trang (2011), Nghin cu kh nng tng hp pectinesterase v polygalacturonase
ca A. niger, lun vn thc s, i hc Nng.
[10] Trn Thanh Trc (2013), Phn lp v tuyn chn dng Aspergillus niger sinh pectin
methylesterase hot tnh cao, lun n tin s, Trng i hc Cn Th.

Ngy nhn bi: 11/9/2016

Chp nhn ng: 11/10/2016
Lin h: Trn Ngc Hng
Trng i hc Th Du Mt
Email: gnuh1423@yahoo.com

89