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


MSHV: 62030803

ASPERGILLUS NIGER SINH


 3

 

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. 




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



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





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

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

 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

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 

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 

 
 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

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

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

 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



 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

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.,

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,

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