phân lập và tuyển chọn một số dòng aspergillus niger sinh pectin methylesterase hoạt tính cao








MSHV: 62030803




ASPERGILLUS NIGER SINH











3

 



MSHV: 62030803




ASPERGILLUS NIGER SINH



 

Mã ngành: 62 42 01 07


 





3
2008 - 2012 
Chuyên ngành Vi sinh  i 

  



u ca NCS Trn Thanh Trúc vi s

ng dn ca PGS. TS. Nguyi và TS. Nguy liu
và kt qu trình bày trong luc công b riêng l
bi tác gi khác trong bt k công trình nào .






PGS. TS. 




TS. 







2008 - 2012 
Chuyên ngành Vi sinh  ii 



PGS. TS. . 
, tôi xin PGS. TS.   
 
 

 

.   luôn 
 , cùng tôi 
.
.  Giám
, ã giúp  


           


, 

 và các
phòng ng; 

 p,
  

này.
GS. TS. . TS. 
 

Xin cám 
ý và 


2008 - 2012 
Chuyên ngành Vi sinh  iii 




d.


 03 3
 




2008 - 2012 
Chuyên ngành Vi sinh  iv 


Aspergillus niger sinh pectin methylesterase



liên qu9.2012.
                 
enzyme pectin methylesterase (P  
dòng Aspergillus niger (A. niger

dòng Aspergillus niger 

 các  khoai tây (Potato Dextrose
Agar, PDA), Czapek Dox Agar (CZ) n các
dòng 
  60 dòng 
dòng  
các dòng  dòng  
dòng có , 
T
1
, N
1
, So
2
và R
1
 .
 dòng R
1
và So
2


 gene dòng T

1
, N
1
,
So
2
và R
1,

 A. niger  ÷
100%.
 A. niger So
2

và R
1




                   
ly trích
 
gian ly trích 
khô i 
  Quá trình

% MgCl
2
và 0,15% CaCl

2


5
 (mL/g)
2008 - 2012 
Chuyên ngành Vi sinh  v 
ly trích 
 1 v/w (mL/g).
ly trích  
.
 

protein



protein,


               A. niger  

m
mg/mL
 A. niger  ÷  
 CaCl
2
  A. niger.







Từ khóa: Aspergillus niger, cải thiện cấu trúc, cơ chất giàu pectin, hoạt tính cao, lên
men bề mặt cơ chất rắn, pectin methylesterase, phân lập, tuyển chọn.

2008 - 2012 
Chuyên ngành Vi sinh  vi 
SUMMARY

Aspergillus niger biosynthesize high activity pectin
atory of Food Technology Department, College
of Agriculture and Applied Biology, Can Tho University and other laboratories, from
11.2008 to 9.2012.
The thesis was conducted to investigate the biosynthesis process and application of pectin
methylesterase (PME, EC 3.1.1.11) in solid state fermentation (SSF) of isolated and
selected Aspergillus niger from the practical conditions of Vietnam.
Sixty species which have morphologically similar Aspergillus niger were isolated from the
peels of rich pectin fruits which have been found to be widely in Mekong delta, such as
orange, pomelo, citrus, apple and fig peels on Potato Dextrose Agar (PDA), Czapek Dox
Agar (CZ) and Malt Extract Agar (MEA). To indicate the pectinase activity of the
organisms, diameters of clear zone around colonies on pectin - agar medium were
measured. The result showed that all of sixty isolates have the ability of pectinolytic
activities, in which 14 out of the 60 isolates were able to produce a higher pectinase
activity, based on the average clear zone diameters were larger than 20 mm.
Further screening of PME production of the 14 selected isolates, the maximum PME
production was obtained by 4 isolates (T
1
, N

1
, So
2
and R
1
) which were isolated from the
peels of Ziziphus mauritiana (  Citrus lemon   Citrus sinensis L.
 and Citrus maxima R
two isolates of R
1
and So
2
with the proportion of 1:1, was conducted to investigate the
suitable conditions for PME biosynthesis by solid state fermentation. In addition, the
identification by sequencing of 28S rRNA gene, revealed that 4 selected isolates T
1
, N
1
,
So
2
and R
1
had 99 ÷ 100% identity to Aspergillus niger.
The optimal conditions of pectin methylesterase biosynthesis from A. niger So
2
and
R
1
in solid-state fermentation were evaluated, based on the individual factors (substrate

characteristics, nutrients, buffer solutions and incubation time) which influenced to PME
production were investigated. After that, the Response surface methodology (RSM) based
on four-variable central composite design (CCD) can be used to model the correlation of
the fermentation conditions such as inducer concentration, initial pH, incubation
temperature and moisture content of medium to PME activity. Moreover, some extraction
parameters consist of type of solvent, pH, ratio of solvent and fermented solids were
studied. In addition, the best combination of extraction temperature, contact time was also
determined.
Especially, the use of rich pectin of agricultural material in Mekong Delta (mixture of
apple pomace and pomelo peels at the ratio of 1:1 w/w) as a fermentation medium was
taken into consideration for the PME production of A. niger in this investigation. With the
2008 - 2012 
Chuyên ngành Vi sinh  vii 
addition of 0.1% urea combined with 0.5% MgCl
2
and 0.15% CaCl
2
into media, the
highest PME obtained after 96 hours incubation at a temperature of 35.5°C, pH 4.0
(adjusted by citrate buffer), fermentation temperature and moisture content adjusted
to 57.4% and 16.5% v/w of inducer concentration (10
5
cfu/mL). In addition, citrate buffer
(pH 3.6) at 35°C and 50 min provided the best recovery in order to obtain highest value
of PME. The ratio 2:1 (v/w) of solvent and substrate was determined as an optimal
condition for concentrated enzyme extracts. Maximum production of PME (about 79.1 U/g
of substrate) was recorded under optimum conditions of SSF and extraction.
Ethanol was the appropriate precipitant for PME purification with the optimal ratio of cold
ethanol and crude enzyme 3: 1 (v/v). In this case, specific activity of PME was 10.02
U/mg

protein
, the purification factor achieved 7.98 fold and the PME recovery yield obtained
91.48%. Combined with the cross flow membrane step, the specific activity of PME
reached to 15.63 U/mg
protein
and the purification factor achieved 12.40 ± 0.89 fold in
comparison to the crude enzyme solution. The molecular weight of the enzyme was found
to be 43 kDa and K
m
was 0.6014 g/L. The activity of the enzyme was stimulated by NaCl
or CaCl
2
. In addition, the optimum temperature and pH of A. niger PME
were 38 ÷ 40°C and 5.0, respectively. Isothermal inactivation of partial purified
A. niger PME could be described by a fractional  conversion model and the inactivation
rate constants increase with increasing temperatures from 50 to 70°C.
The obtained technical PME enzyme also proved the positive effects to improving of the
texture of frozen pineapples and fermented cucumbers.
Key words: Aspergillus niger, isolation, high activity, pectin methylesterase, rich pectin
substrate, screening, solid state fermentation, texture improvement.



2008 - 2012 
Chuyên ngành Vi sinh  viii 

 i
 ii
 iv
SUMMARY vi

 viii
 xiii
DANH SÁCH HÌNH xv
 xix
 xx
 1
1.  1
2.  2
3.  3
4.  3
5.  4
6.  5
  6
1.1  6
1.1.1  6
1.1.2  6
1.1.3  7
1.1.4 PME 8
1.2  10
1.2.1  10
1.2.2 
 12
1.2.3 V
 13
1.3 Aspergillus niger  19
2008 - 2012 
Chuyên ngành Vi sinh  ix 
1.3.1 A. niger 19
1.3.2      p
enzyme PME 20

1.3.3 A. niger 
 21
1.4 Aspergillus niger 22
1.4.1  22
1.4.2 Aspergillus niger 23
1.4.3  23
1.4.4 Aspergillus niger 24
1.4.5 Aspergillus niger  24
1.4.6 Aspergillus  25
1.5 Aspergillus niger 
 26
1.5.1  26
1.5.2 Aspergillus niger 
 27
1.6  . 27
1.6.1  27
1.6.2  29
1.6.3  30
1.7 
PME 30
1.7.1  30
1.7.2  30
1.7.3  31
1.7.4  32
1.7.5  33
1.8  34
1.9  35
1.9.1  36
1.9.2  37
2008 - 2012 

Chuyên ngành Vi sinh  x 
1.9.3  38
1.10             
 38
1.11              
 PME 39
  45
2.1  45
2.1.1  45
2.1.2  45
2.2  47
2.2.1 Ch 47
2.2.2  48
2.2.3  50
2.3  52
2.3.1  52
2.3.2  A. niger 
 54
2.3.3 
 60
2.3.4 A. niger 71
2.3.5 A. niger 
 77
  81
3.1  A.
niger  81
3.1.1 Aspergillus  
giàu pectin 81
3.1.2  83
3.1.3 

 85
3.2  88
3.2.1  88
2008 - 2012 
Chuyên ngành Vi sinh  xi 
3.2.2  92
3.2.3  94
3.2.4  
2
và R
1

 96
3.3  A. niger 
 98
3.4 
2 dòng A. niger R
1
và So
2
100
3.4.1  100
3.4.2 
A. niger R
1
và So
2
102
3.4.3 A. niger 104
3.4.4 

lên men SSF dòng A. niger So
2
và R
1
107
3.5                
A. niger  109
3.5.1 
  109
3.5.2 
 111
3.5.3 
A. niger 113
3.6 
lên men SSF 120
3.6.1 
PME 120
3.6.2 
A. niger 122
3.6.3  124
3.6.4  126
3.7                
A. niger So
2
và R
1
129
2008 - 2012 
Chuyên ngành Vi sinh  xii 
3.7.1 

A. niger 129
3.7.2 A. niger  135
3.7.3  A. niger 137
3.8 A. niger So
2

và R
1
 146
3.8.1  146
3.8.2  151
KT LU NGH 155
1.  155
2.  156
TÀI LI 157
DANH M 177

PHÂN TÍCH xxi
 xli
 lxiii
  cxv
2008 - 2012 
Chuyên ngành Vi sinh  xiii 


 21

Aspergillus sp. 28
 49
 50

 
 66

 67
 82
Bng 3.2: Phân nhóm nm mc hiu vi enzyme pectinase 89
Bng 3.3: Kt qu thng kê s khác bing kính vòng phân gii pectin (D) ca các
dòng nm mc thuc hiu cao vi pectinase 90

 93

PME 95

1
và So
2

 97
 101

 102

A. niger 105

A. niger 107
 
A. niger 108

 112
 114

 
 122

4
)
2
SO
4
130
2008 - 2012 
Chuyên ngành Vi sinh  xiv 
 131
 132
 133
3.19khác nhau 134
A. niger  135

A. niger 141
A. niger 142

2
A. niger 143


2
 148

 150

 152

Aspergillus và Penicillium xxiv
 xxix
 xxix
-phosphate xxx
Bng PL1.5: Bng tra n mui (NH
4
)
2
SO
4
bão hòa b sung trong tin trình kt ta
enzyme và protein xxxi
 xxxiv
 xxxix
Aspergillus niger 
 xli
A. niger  xlv
A. niger  xlvii
Bng PL2.4: A. niger xlix
 lix



2008 - 2012 
Chuyên ngành Vi sinh  xv 
DANH SÁCH HÌNH
 6
  11
 14
 15

 15

 16
Hình 1.7: Hình thái Aspergillus sp. 23
Hìn 25
 36
Hình 2.1:  47
 53
 54
 
 59
- 
 62
-  63
A. niger 63
- 
 68

4
)
2
SO
4
/NaCl 73
 74

3
82

3

82
 83
 83

2
và G
1

84
Hình 3.6: Dòng A. niger  85

2
 85

2
 86

2
 86
2008 - 2012 
Chuyên ngành Vi sinh  xvi 
A. niger 86

2
87
 
2
87

2

91
 
2
91
 91
A. niger V 91
- 
 93
 
- agarose 95
 98
 100

2
và dòng A. niger NBL, BR 28s
ribosomal RNA gene hay An03c0110 100

A. niger 106

A. niger 110
A. niger 111

 115

116

 117

 117


 117

 117

 118
 
 118
2008 - 2012 
Chuyên ngành Vi sinh  xvii 
 121
 125

o
  127
 127

o
 127
-18°C) 127
A. niger 136
A. niger 137
 th b m th m biu th s ia
pH và nhi n hot tính PME t A. niger 139
A. niger 140
HA. niger 145


2
  146


o


2
C, 30 phút 153
A. niger xxv
A. awamori (trái) và A. foetidus  xxvi
A.
foetidus (h, CZ), A. niger (i, MEA) và A. awamori (j, CZ) xxvii
 lx

  lxii
 
 lxviii
 
 lxx
 
 lxxii
 
 lxxiv
 
1
và So
2

 lxxv
 
1
và So
2


 lxxvi
Hình PL4.1: Các thi cxv
2008 - 2012 
Chuyên ngành Vi sinh  xviii 
 cxvi
Hình PL4.3 cxvi

cxvii

2008 - 2012 
Chuyên ngành Vi sinh  xix 

A. Aspergillus
B. Botrytis
bp base pair
BSA Bovine serum albumin
CBB Coomassie Brilliant Blue
cbk 
CCD 
cfu 
CMSepharose Carboxymethyl Sepharose
CTAB Cetyltrimethylammonium bromide
CZ Czapek Dox Agar
DE Degree of esterification (m ester hóa)
DNA Deoxyribonucleic acid
E. Erwinia
EDTA Ethylenediaminetetraacetic acid
EGCG Epigallocatechin gallate
GalA Galacturonic acid

HG Homogalacturonan
HMP High Methoxyl Pectin  methoxyl hóa cao)
IAA Iodoacetamide
ITS Internal transcribed spacer
kDa Kilodalton
LMP  methoxyl hóa thp)
LSU Large subunit 
MEA Malt Extract Agar
MI Methoxyl Index (ch s methoxyl)
NCBI National Center for Biotechnology Information
NCKH 
PCR Polymerase Chain Reaction
PDA Potato Dextrose Agar
PG Polygalacturonase
pI Isoelectric point 
2008 - 2012 
Chuyên ngành Vi sinh  xx 
PL Pectate lyase
PME Pectin methylesterase
PMEI Pectin methylesterase inhibitor (cht c ch pectin methylesterase)
-  
RAPD Random Amplification of Polymorphic DNA
rpm rounds per minute 
rRNA Ribosomal ribonucleic acid
RSM Response Surface Methodology 
SDSPAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SmF 
SSF 
SSU Small subunit 
TSS Total soluble solid 

v/v volume/volume 
v/w 
w/w 
U/g Units per weight (gram) of substrate 
U/mL Units per volume (mL) of substrate 



Pectin methylesterase   Aspergillus niger: PME     
trình ly trích quá trình lên men SSF hay SmF dòng A. niger
 
PME cà chua: PME ly trích 
2008 - 2012 
Chuyên ngành Vi sinh  1 


1. 
ng dng enzyme trong ch bin thc phm và nhi
mt trong nhng v mang tính cp thic tin. Hin nay, các
nc phát tri   ng các ch phm enzyme trong sn xu  to ra
nhng sn phm ct và cht lng cao vi giá thành h (Saurel, 2003;
Suutarinenn và Autio, 2004; Ngô Tin Hin, 2001). Vit Nam là nc giàu tim
 nông sn, nhu cu các loi enzyme phc v trong ch bin thc phm là
rt ln. Tuy nhiên,  Vit Nam hoàn toà  ng sn xut ch phm
enzyme, hc ta vn phi nhp ngoi mt khi lng ln nhng loi
ng Th Thu và Nguyn Th Xuân Sâm, 2009b).
Pectinase là nhóm enzyme xúc tác cho s thy phân các polymer pectin - 
c ng dng rng rãi trong công nghip thc ph lý cht thi và
nhic khác.  Vit Nam, ch phc s dng khá ph
bic nhiu nhà khoa hc quan tâm nghiên cu (et al.,

1978; Phí Quyt Tin, 1999; Hunh Ngc Oanh và Trn Ngc Hùng, 2008). Tuy
i vc ci thic tính cu trúc t bào rau qu - mt hn ch ln
v cht ng ca sn phm sau ch bin, vic s dng hn hp ch phm
pectinase không mang li hiu qu tích cectin methylesterase
(PME, EC 3.1.1.11)  mt enzyme thu   t quan
trng (Van Buren, 1979).
PME  c quan tâm s dng và phát trin  nhiu quc gia (Saurel, 2003;
Suutarinenn và Autio, 2004) vi các mn ch s phá hy cu trúc
ca t bào thc vu sut ly trích c qu y nhanh quá trình
sy rau qu (Van Buren, 1979; Sajjaanantakul và Pitifer, 1991
PME
ng thi vn có s nhm ln trong hiu
bit v PME và pectinase. Chính vì th, vic nghiên cu có h thng s thu nhn
PME và kh ng dng thc tin ca enzyme này tu kin hin ti ca
Vit Nam là v cn thit, c và thc tin cao.
Bên cnh PME t thc vt, ngun giàu PME c s dng nhiu nht trong
các ch phm pectinase thng mi là các PME t vi sinh vt (Solis  Pereira et
al.
et al., 1997, trích dn bi Patil và Dayanand, 2006). Mc dù vy,
2008 - 2012 
Chuyên ngành Vi sinh  2 
không phi tt c nm mc u có kh  nhau và ngay c
nhng dòng cùng mt gió kh h tng hp enzyme vi
hot tính ng (Nguy
  tng hp các    dòng Aspergillus    
A. niger nói riêng 
  ng hp          
thu nhn PME

không nhng gây bt li 

u king có n kh ng và tng hp enzyme
(Bhalla và Chatanta, 2000) tuyn chn dòng A. niger có kh 
sinh tng hp PME hoi nhiu thi gian và công sc nghiên c
PME
   methoxyl hóa cao (Sajjaanantakul và Pitifer, 1991; Bhalla và
Chatanta, 2000 các ngun ph ph
d  cm ng cho quá trình lên men sinh tng hp PME 

 
, 2005; Pilnik và Voragen, 1993).
Vi nhng m mà PME mang li cho công nghip thc phm và vai trò quan
trng ca khâu tuyn chn dòng A. niger trong công nghip sn xut enzyme,
nghitng hp PME
và tuyn chn dòng A. niger c hiu vi PME n vic nghiên cu
u kin lên men sinh tng hp enzyme phù hp, ng dng hiu qu
trong c ci thin cu trúc rau qu.
2. 
i. 
Nghiên cu sinh tng hp và ng dng PME t quá trình lên men b mt
ng rn dòng A. niger c hiu c phân lp và tuyn chn t các
loi v qu giàu pectin  ng bng sông Cu Long.
ii. 
(1) dòng A. niger 
vs
PME.
(2) dòng A. niger 
PME 
2008 - 2012 
Chuyên ngành Vi sinh  3 
(3)               PME,

 này.
(4) 
iii. 
Nghiên cc thc hin vi 4 ni dung chính:
các dòng A. niger 
 PME .
rên môi
PME A. niger .
PME 
men A. niger .
PME 
p .
3. 
i. 
Các dòng A. niger PME 
Dòng dòng A. niger hoa
n Lomonosov -
MGU)  và (ii) dòng A. niger 
 ST

PME 
ii. 
Các dòng A. niger 
sPME.
P  

e PME 

4.  
A. niger, bao

 

 