HI NGH KHOA HC TON QUC V SINH THI V TI NGUYN SINH VT LN TH 6

PHN LP V SNG LC MT S CHNG Aspergillus niger

SINH PECTINASE T MT S V C, QU
(CHUI, TO, XOI, THANH LONG V C RT) THNH PH HU

TRN QUC DUNG, NGUYN TH KIM C,

NGUYN TH SNG, NGUYN TH THU CHUNG
Trng i hc S phm, i hc Hu
PHAN TH THANH DIM, NGUYN HONG LAN ANH
Trng i hc Qung Nam

Pectinase l cc enzyme phn gii cc c cht pectin, c s dng rng ri trong cng
nghip ch bin thc phm, ln men cotton, tch ri cc si thc vt, x l nc thi, lc du
thc vt, ln men tr v c ph, ty trng giy, trong nc gii kht c cn. Cc enzyme ny
thng lm cho vic chit xut, lc v tinh lc nc qu v nc gii kht c d dng cng
nh lm tng sn lng ca chng trong sn xut. Mt c im ni bt ca pectinase l cc
enzyme ny c dng khng gii hn trong thc phm v khng nh hng n sc khe con
ngi. S sn xut pectinase quy m cng nghip c thc hin ch yu t Aspergillus niger
(Yogesh, 2009). V vy vic tm kim cc chng A. niger c kh nng tng hp pectinase cao l
rt c ngha. Nhiu nghin cu v phn lp v tuyn chn cc chng A. niger c kh nng
tng hp pectinase cao (Dung, 2013; Trc, 2011, 2010; Hng, 2006), thu nhn v tinh sch
pectinase t A. niger (Oanh, 2008; Tm, 2002), sn xut pectinase (Mrudula, 2011; Yogesh,
2009) c thc hin.
Bi bo ny trnh by mt s kt qu ban u v phn lp v sng lc mt s chng A. niger
c kh nng tng hp pectinase t v ca 5 loi c, qu thnh ph Hu.
I. VT LIU V PHNG PHP NGHIN CU
1. Vt liu
Cc mu v ca 5 loi c, qu c thu thp t mt s ch thnh ph Hu, bao gm:
chui, to, xoi, thanh long v c rt.
Mi trng PDA: Dch chit khoai ty 1000 ml; agar 2%, glucose 2%; pH 5,5.
Mi trng Czapeck-pectin: K2HPO4 0,1%; MgSO4.7H2O 0,05%; NaNO3 0,3%; KCl 0,05%;
FeSO4.7H2O 0,001%; pectin 1,5%; pH 5,5.
Mi trng tuyn chn chng A. niger: Pectin 0,5%; agar 2% g; pH 5,5.
2. Phng php nghin cu
Phng php phn lp A. niger: Cc v qu ln mc t nhin. Ly mu v (phn b hng
do nm mc) ct thnh mnh 1-1,5 mm, sau nghin nh, cho vo ng nghim cha 9 ml
dung dch agar 0,1% v trng. Lc ng nghim cha mu trn my vortex dch tan u. Ht
200 l hn dch cho vo mi a petri cha mi trng PDA. Nui cy mu 30oC trong t m.
Sau 72 gi, ly mu ra quan st i th v vi th v nh loi A. niger theo kha phn loi ca
Diba v cs.(2007).
Sng lc cc chng A. niger: Cc chng A. niger c nui cy trong mi trng lng
Czapeck-pectin, 30oC, sau 3 ngy ly tm loi sinh khi, thu enzyme ngoi bo. Ly 100 l
dch enzyme ngoi bo cho vo l c trn a thch pectin. Sau em 37oC. Sau 24
gi ly ra nh dung dch lugol 1% ln mt thch. Xc nh hot tnh pectinase bng cch o

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ng knh vng thy phn pectin. Kh nng sinh tng hp pectinase ca cc chng c nh
gi thng qua ln ca ng knh vng thy phn pectin.
Xc nh hot pectinase: Hot pectinase c xc nh bng cch o lng ng kh
c gii phng t hot ng ca pectinase trn c cht pectin bng thuc th 3, 5-
dinitrosalicylic axit, DNS). Ly 3 ml hn hp phn ng bao gm 0,8 ml dung dch pectin v
0,2 ml dch enzyme c pha long thch hp trong 2 ml dung dch sodium acetate (0,1 M; pH
5). Hn hp phn ng c 40C trong 10 pht. Sau thm vo 1 ml NaOH v 1 ml DNS,
lm dng phn ng bng cch un si trong 10 pht. Tnh lng ng kh gii phng bi s
thy phn enzyme. Mt n v hot ca enzyme c nh ngha l lng enzyme cn thit
gii phng 1 mol ca cc nhm kh trong mt pht vi glucose l tiu chun trong cc iu
kin th nghim (Mrudula, 2011).
X l thng k: Mi th nghim c lp li 3 ln tnh trung bnh mu v phn tch
Duncans test, p<0,05) bng chng trnh SAS (ver.6.12).
II. KT QU V THO LUN
1. Phn lp cc chng A. niger c kh nng sinh tng hp pectinase
T cc v qu chui, to, xoi, thanh long v c rt tin hnh phn lp cc chng A. niger
trn mi trng Czapeck. Trong qu trnh nui cy 30C, cc khun lc c quan st u
tin bng mt thng thy c mu trng v sau nhanh chng chuyn thnh mu nu m v
en vi cc bo t nh v mt di ca a Petri c mu vng nht v cc rnh phng x xut
hin trn a thch (Hnh 1A, 1B). Sau quan st cc chng phn lp c di knh hin vi
thy cc si nm mnh, phn nhnh v c vch ngn. Cc bo t hnh cu c mu nu en,
cung bo t nhn. Bng nh gi c hnh cu hoc gn cu, c hai hng th bnh (Hnh 1C, 1D,
1E, 1F). Cc c im hnh thi ny hon ton ph hp vi kha nh loi ca Diba v cs.
(2007). Nh vy 50 chng nm phn lp c u l A. niger: 10 chng c ngun gc t v
chui (c k hiu l CH1, CH2, CH3, CH4, CH5, CH6, CH7, CH8, CH9 v CH10); 10 chng
c ngun gc t v to (c k hiu l T1, T2, T3, T4, T5, T6, T7, T8, T9 v T10); 10 chng
c ngun gc t v xoi (c k hiu l X1, X2, X3, X4, X5, X6, X7, X8, X9 v X10); 10
chng c ngun gc t v thanh long (c k hiu l TL1, TL2, TL3, TL4, TL5, TL6, TL7,
TL8, TL9 v TL10) v 10 chng c ngun gc t v c rt (c k hiu l CR1, CR2, CR3,
CR4, CR5, CR6, CR7, CR8, CR9 v CR10). Kt qu phn lp cc chng A. niger c trnh
by Bng 1.

A B
E F
C D
Hnh 1: Cc c im hnh thi ca
Aspergillus niger.
A, B: Cc khun lc sau 7 ngy nui cy , A:
mt trn, B: mt di); C. Bo t; D. Bng
bo t; E. Bng nh gi; F. H si
(nh: Trn Quc Dung v cs, 2015)

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Bng 1
Ngun gc v c im ca cc chng A. niger phn lp t mt s v qu
S chng A. c im ca chng
S mu
STT Ngun phn lp niger phn Thi gian xut Mu ca khun
phn lp
lp c hin bo t/ngy lc
1 V chui 10 10 2 en
2 V to 10 10 1 en v nu en
3 V xoi 10 10 2 en
4 V thanh long 10 10 2 en
5 V c rt 10 10 2 en
2. Sng lc cc chng A. niger c kh nng sinh pectinase
Kh nng sinh pectinase ca cc chng A. niger sau khi phn lp c nh gi gin tip qua
ng knh vng thy phn pectin. Kt qu xc nh ng knh vng thy phn pectin ca 50
chng A. niger c trnh by Bng 2 v Hnh 2.
Bng 2
ng knh vng thy phn pectin ca 50 chng A. niger phn lp
ng knh vng thy phn pectin (cm)
STT Ngun phn lp S mu phn lp
Trung bnh Khong bin thin
1 V chui 10 1,640,06 1,2-1,9
2 V to 10 1,880,04 1,7-2,1
3 V xoi 10 I 1,650,08 1,4-2,2
4 V thanh long 10 II 1,890,06 1,5-2,1
5 V c rt 10 III 1,990,08 1,3-2,2
Kt qu trnh by Bng 2 cho thy ng knh vng thy phn pectin trung bnh ca cc
chng A. niger c ngun gc t v c rt l ln nht (1,99 0,08), bin thin trong khong t 1,3-
2,2 cm. ng knh vng thy phn pectin ln nht ca cc chng A. niger l 2,2 cm. Trong ,
15 chng c ng knh vng thy phn pectin ln hn 2,0 cm (2,08 0,05); 31 chng c
ng knh vng thy phn pectin l 1,5-2,0 cm (1,74 0,13) v 4 chng c ng knh vng
thy phn pectin nh hn 1,5 cm (1,32 0,07). Kt qu ny tng ng vi kt qu nghin
cu ca Tran Thanh Truc v cs. (2011).

Hnh 2: ng knh vng thy phn ca hai chng A. niger CR10 v TL8
(nh: Trn Quc Dung v cs, 2015)

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3. Hot pectinase ca cc chng A. niger phn lp c

Kt qu phn tch hot pectinase ca 50 chng A. niger phn lp c trnh by Bng 3
cho thy: 2 chng t v qu xoi c hot pectinase cao nht t t 10,544-10,700 U/ml. Tip
n l chng phn lp t v qu chui c hot pectinase 8,806 U/ml v chng phn lp t v
qu xoi c hot pectinase 8,802 U/ml. Chng phn lp t v qu to c hot pectinase
thp nht ch t 0,407 U/ml.
Bng 3
Hot pectinase ca cc chng A. niger phn lp c
Hot , Hot , Hot ,
STT Chng STT Chng STT Chng
U/ml) U/ml) U/ml)
1 CH1 3.897de 17 T7 2.583e 35 TL5 2.029c
2 CH2 7.685b 18 T8 5.714b 36 TL6 0.609f
3 CH3 5.567c 19 T9 5.646b 37 TL7 1.112de
4 CH4 8.806a 20 T10 4.063c 38 TL8 2.000c
5 CH5 4.176ed 21 X1 8.850b 39 TL9 0.770ef
6 CH6 4.693cd 22 X2 6.693d 40 TL10 2,176c
7 CH7 3.228e 23 X3 6.130e 41 CR1 0.908f
8 CH8 5.601c 24 X4 4.527f 42 CR2 2.563d
9 CH9 3.890ed 25 X5 10.544a 43 CR3 3.040c
10 CH10 4.470d 28 X8 1.550g 44 CR4 2.549d
11 T1 4.676a 27 X7 8.802b 45 CR5 1.639e
12 T2 1.656f 29 X9 10.700a 46 CR6 1.245ef
13 T3 3.488d 30 X10 6.407de 47 CR7 5.155b
14 T4 1.307g 31 TL1 6.818a 48 CR8 2.985cd
15 T5 0.583h 32 TL2 1.307d 49 CR9 6.982a
16 T6 0.470h 33 TL3 1.452d 50 CR10 5.309b
17 T7 2.583e 34 TL4 2.660b
Ghi ch: Cc ch ci khc nhau trn cng mt ct ch s sai khc c ngha thng k ca cc trung
bnh mu vi p < 0,05 (Duncans test).
III. KT LUN
T 50 chng A. niger phn lp c t v qu chui, to, xoi, thanh long v c rt sng
lc c hai chng c kh nng sinh tng hp pectinase trn 10 U/ml sau 3 ngy nui cy l X5
v X9.
Li cm n: Cng trnh c hon thnh vi s ti tr kinh ph ca i hc Hu qua ti
m s DHH2014-03-54.
TI LIU THAM KHO
1. V Kim Dung, ng Th Thu, Nguyn Th Tr My, L Th Huyn, Nguyn Th Xun
Sm, 2013. Phn lp, tuyn chn cc chng Aspergillus niger sinh endo polygalacturonase
cao cho mc tiu sn xut pectic oligosacharide (POS), Bo co khoa hc Hi ngh Khoa
hc Cng ngh sinh hc ton quc 2013, Nxb. KHTN & CN, H Ni, trang 313-317.

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2. L Th Thanh Hng, Nguyn Thu Chu, 2005. Phn lp tuyn chn mt s chng nm
Aspergillus niger sinh enzym pectinaza cao t mt s ph ph phm nng sn, TC Nng
nghip v pht trin nng thn, 15: 37-38.
3. Hunh Ngc Oanh, Trn Ngc Hng, 2008. Thu nhn enzyme pectinase t A. niger - tinh
sch bng phng php lc gel v lc mng, Tp ch Pht trin Khoa hc v Cng ngh, 8
(11): 46-50.
4. Nguyn Quang Tm, L Th Hng Nga, ng Th Thanh Thu, Nguyn Th Tuyt
Thanh, 2002. Tinh sch v c nh enzyme pectinase thu nhn t mt s chng nm mc,
Bo co Hi ngh Khoa hc Trng i hc Khoa hc t nhin, HQG Thnh ph H Ch
Minh: 187-193.
5. Diba K., Kordbacheh P., Mirhendi S.H., Rezaie S., Mahmoudi, 2007. Identification of
Aspergillus species using morphological characteristics, Pak J Med Sci, 23(6): 867-872.
6. Mrudula S., Anitharaj R., 2011. Pectinase Production in Solid State Fermentation by
Aspergillus niger Using Orange Peel as Substrate, Global Journal of Biotechnology &
Biochemistry 6 (2): 64-71.
7. Tran Thanh Truc, Le Vuong Hai Nguyet, Nguyen Van Muoi, 2011. Journal of Food
Science and Technology, 49 (1A): 453-458.
8. Tran Thanh Truc, Duong Do Thanh Lan, Nguyen Thi Kieu Linh, Nguyen Van Thanh,
Nguyen Van Muoi, 2010. Isolation and screening Aspergillus niger strains producing
pectin methyl esterase from some kinds of orange and pomelo in Mekong river delta,
Vietnam, Food Innovation Asia Conference on Ligneous Food Research and Development
to Global Market, June 17-18, 2010, BITEC, Bangkok, Thailand: 453-458.
9. Yogesh, K., K. K. Vamsi, B. Amol, G. Nikhil, T. Soham, P. Prasad, G. Girish, G.
Mayank, J. Amol, M. Adarsh, B. Joshi, D. Mishra, 2009. International Journal of
Microbiology Research, 1 (2): 13-17.

ISOLATION AND SCREENING OF Aspergillus niger STRAINS FOR

BIOSYNTHESIS OF PECTIANSE FROM PEELS OF SOME FRUITS ,
BANANA, APPLE, MANGO, DRAGON AND CARROT) IN HUE CITY

TRAN QUOC DUNG, NGUYEN THI KIM CO, NGUYEN THI SUONG
NGUYEN THI THU CHUNG, PHAN THI THANH DIEM, NGUYEN HOANG LAN ANH
SUMMARY

Pectinase originated from Aspergillus niger strains are important in the food and other
processing industries. This investigation is for isolation and screening of Aspergillus niger
strains for biosynthesis of pectianse production from peels of some fruits such as banana, apple,
mango, dragon and carrot in Hue city. A preliminary result was isolated and selected 50 strains
of A. niger. Among these 2 strains, viz X5 and X9 are able to produce highest pectinase with 10
U/ml.

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