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B GIO DC V O TO
TRNG I HC CN TH

TRN THANH TRC
MSHV: 62030803

PHN LP V TUYN CHN
MT S DNG ASPERGILLUS NIGER SINH
PECTIN METHYLESTERASE HOT TNH CAO

LUN N TIN S SINH HC

Cn Th, nm 2013

B GIO DC V O TO
TRNG I HC CN TH

TRN THANH TRC
MSHV: 62030803

PHN LP V TUYN CHN
MT S DNG ASPERGILLUS NIGER SINH
PECTIN METHYLESTERASE HOT TNH CAO

LUN N TIN S SINH HC
Chun ngnh: VI SINH VT HC
M ngnh: 62 42 01 07

Ngi hng dn khoa hc
PGS TS NGUYN VN MI
TS NGUYN VN THNH

Cn Th, nm 2013
Lun n tt nghip in s Kh 2008 - 2012 rng H Cn h
Chuyn ngnh Vi sinh vt h i in Nghin u v Pht trin Cng ngh inh h
CNG HA X HI CH NGHA VIT NAM
c lp T do Hnh phc

LI CAM OAN

Ti xin cam oan y l cng trnh nghin cu ca NCS Trn Thanh Trc vi s
hng dn ca PGS. TS. Nguyn Vn Mi v TS. Nguyn Vn Thnh. Cc s liu
v kt qu trnh by trong lun vn l trung thc, cha tng c cng b ring l
bi tc gi khc trong bt k cng trnh no trc y.

Ngi hng dn khoa hc

PGS. TS. NGUYN VN MI

TS. NGUYN VN THNH
Tc gi lun vn

TRN THANH TRC

Chuyn ngnh Vi sinh vt h ii in Nghin u v Pht trin Cng ngh inh h
LI CM N

Hn c s bit n, ti cm thy tht hnh phc v may mn khi c thc hin lun n tin
s di s hng dn ca PGS. TS. Nguyn Vn Mi v TS. Nguyn Vn Thnh.
Li cm n u tin, ti xin c gi n PGS. TS. Nguyn Vn Mi gio vin hng
dn chnh. Tht s vinh d v t ho khi ti c l hc tr ca Thy. Thy chnh l ngi
truyn cho ti lng nhit huyt v thi ln ngn la am m khoa hc, khi dy ti s n
lc, t tin, c gng khng ngng v khng nn lng trc nhng kh khn trong sut tin
trnh thc hin lun n tin s. Xin cm n Thy dnh nhiu thi gian, cng sc v lun
gip em c c nh hng ng n trong cng vic.
Ti cng xin gi li tri n n TS. Nguyn Vn Thnh Thy lun ng gp cho ti rt
nhiu kinh nghim qu bu, khng ngi thi gian cng ti nghin cu nhng vn mi
pht sinh c kt qu thu nhn tt nht.
Ti c bit bit n s gip v h tr v c ng vin ca TS. Trn Nhn Dng Gim
c Vin Nghin cu v Pht trin Cng ngh Sinh hc, gip ti c th hon tt tt ni
dung nghin cu.
Tin trnh hn 3 nm thc hin cc th nghim trong lun n, ti lun c s ng
hnh, h tr ca cc em hc vin cao hc ngnh Cng ngh sinh hc, Cng ngh thc
phm, sinh vin i hc t cc kha 28 n kha 35. Cc em khng qun ngi kh khn,
thi gian cng vi ti thc hin cc nghin cu v hn th na, cc em cn lun tip
sc v h tr ti trong nhiu lnh vc v trong cuc sng,... n tn thi im ny.
Xin chn thnh cm n B Gio dc v o to, Ban Gim hiu Trng i hc Cn Th,
Phng T chc Cn b, Phng Qun l Khoa hc, Phng o to, Khoa Sau i hc v cc
phng ban chc nng khc ca Trng; c bit l Khoa Nng nghip v Sinh hc ng
dng, Vin Nghin cu v Pht trin Cng ngh sinh hc to iu kin thun li cho
vic thc hin ti nghin cu. Xin cm n Thy C B mn Cng ngh Thc phm,
Trng i hc Cn Th ni ti ang cng tc h tr v to iu kin thun li cho
nghin cu ny.
Ti cng xin c gi li cm n n GS. TS. Lu Dun, PGS. TS. ng Th Anh o v
nhiu bn b gn xa lun ng vin v gip ti c thm n lc phn u, hon thnh
nghin cu.
Xin cm n qu Thy C trong Hi ng chm Lun n Tin s cp c s c nhiu gp
v a ra nhiu li khuyn b ch, gip ti hon thin lun n tt hn.
c bit, xin gi li cm n su sc n hai phn bin c lp vi nhng gp c th,
nhng gi b ch, gip ti hon tt tt hn na cc ni dung nghin cu ca lun n.
Chuyn ngnh Vi sinh vt h iii in Nghin u v Pht trin Cng ngh inh h
Cm n cc Thy C gio c khng ngng ng vin, khuyn khch, cc bn b gn xa
lun khch l ti trong sut tin trnh hc tp.
Cui cng, ti xin c gi li bit n n gia nh vi tt c tnh yu v s khuyn khch,
ng h dnh cho ti trong chng ng cam go hon thnh c lun n nghin cu
ny. c bit, xin dnh tng ba m cc thnh qu m hm nay con t c.
Chn thnh cm n.
Cn Th, ngy thng 03 nm 2013
Nghin cu sinh thc hin

Trn Thanh Trc
Chuyn ngnh Vi sinh vt h iv in Nghin u v Pht trin Cng ngh inh h
TM LC

ti Phn lp v tuyn chn mt s dng Aspergillus niger sinh pectin methylesterase
hot tnh cao c thc hin ti phng th nghim B mn Cng ngh Thc phm, Khoa
Nng nghip v Sinh hc ng dng, Trng i hc Cn Th v cc phng th nghim c
lin quan, t thng 11.2008 n thng 9.2012.
ti c thc hin vi mc tiu nghin cu qu trnh sinh tng hp v ng dng
enzyme pectin methylesterase (PME, EC 3.1.1.11) t s ln men b mt trn mi trng
rn, s dng dng Aspergillus niger (A. niger) c hiu c phn lp v tuyn chn trong
iu kin thc t ti Vit Nam.
Kt qu nghin cu phn lp c 60 dng c c tnh ging Aspergillus niger t v
cc loi qu giu pectin ph bin khu vc ng bng sng Cu Long nh v cam, bi,
chanh, hnh, to v sung, s dng cc mi trng nc trch khoai ty (Potato Dextrose
Agar, PDA), Czapek Dox Agar (CZ) v Malt Extract Agar (MEA). Vic tuyn chn cc
dng nm mc c kh nng sinh tng hp pectinase c thc hin bng cch o ng
knh vng phn gii pectin cho thy, tt c 60 dng u c kh nng phn gii pectin, trong
14 dng nm phn lp c ng knh vng phn gii ln hn 20 mm.
Kt qu sng lc cc dng nm c kh nng sinh tng hp PME t 14 dng c hiu vi
pectinase tuyn chn c 4 dng c hot tnh PME thu c cao v n nh, k hiu
T
1
, N
1
, So
2
v R
1
phn lp t v to ta, v qu chanh nm, v cam son v bi Nm Roi.
S kt hp ca 2 dng R
1
v So
2
vi t l 1:1 c la chn trong nghin cu xc nh
iu kin thch hp cho qu trnh ln men rn sinh tng hp PME c hiu qu cao nht. K
thut gii trnh t gene 28S rRNA cng c p dng nhn din bn (4) dng T
1
, N
1
,
So
2
v R
1,
kt qu cho thy, 4 dng ny u thuc A. niger vi mc ng hnh 99
100%.
iu kin ln men rn sinh tng hp PME t t hp A. niger So
2
v R
1
c xc nh
da trn vic kho st mt s yu t n l (thnh phn c cht, dinh dng, tc ng ca
dung dch m v thi gian ) kt hp vi vic ti u ha mt s yu t chnh (pH ban u,
m mi trng ln men, t l bo t nm mc s dng v nhit ln men) theo
phng php b mt p ng vi m hnh phc hp ti tm. iu kin ly trch, thu nhn
enzyme sau ln men (pH ca dung dch m, t l dung mi v c cht, nhit v thi
gian ly trch tng ng) cng c xc nh. Kt qu cho thy hiu qu ca vic tn
dng ngun ph phm nng nghip l b to ta kh v v bi Nm Roi ti lm c cht
ln men chnh (vi t l b to ta v v bi l 1:1 w/w) cho vic thu nhn PME. Qu trnh
ln men t hiu qu cao nht khi mi trng nui cy c iu chnh n m 57,4%
bng dung dch m citrate pH 4,0, c b sung 0,1% urea, 0,5% MgCl
2
v 0,15% CaCl
2
, t
l huyn ph bo t nm mt s 10
5
cfu/mL s dng l 16,5% v/w (mL/g). Sau 96 gi
Chuyn ngnh Vi sinh vt h v in Nghin u v Pht trin Cng ngh inh h
nhit 35,5C, PME th c ly trch nhit 35C trong thi gian 50 pht bng
dung dch m citrate pH 3,6, s dng t l dung dch m v c cht l 2:1 v/w (mL/g).
c tnh hot tnh PME t c iu kin ln men v ly trch tt nht l 79,1 U/g c
cht.
PME th thu c sau khi tinh sch s b bng kt ta vi ethanol t l 3:1 (v/v), th
hin hot tnh ring l 10,02 U/mg
protein
, hiu sut thu hi 91,48% v tinh sch tng
gp 7,98 ln so vi mu enzyme th. Sau khi kt ta vi ethanol, tin hnh lc mng
khong phn on 50 kDa thu c PME c hot tnh ring t 15,63 U/mg
protein,
tinh
sch tng 12,40 0,89 ln khi so snh vi mu enzyme th. PME t A. niger c khi
lng phn t khong 43 kDa, hng s K
m
ca PME t c l 0,6014 mg/mL. Hot tnh
ca PME thu nhn t A. niger th hin cao nht 38 40C v pH mi trng 5,0. C hai
mui CaCl
2
v NaCl u c tc dng hot ha, lm tng hot tnh ca PME t A. niger.
ng thi, s bt hot nhit ca PME tun theo phng trnh bc 1 chuyn i mt phn
vi h s gc k tng dn khi nhit tng t 50 n 70C.
Vic nghin cu th nghim trn mt vi sn phm thc phm c cu trc khng n nh
cng khng nh hiu qu tch cc ca ch phm PME k thut t nm mc trong vic
ci thin cng chc ca sn phm sau ch bin, in hnh l khm lnh ng v da leo
mui chua.
T kha: Aspergillus niger, ci thin cu trc, c cht giu pectin, hot tnh cao, ln
men b mt c cht rn, pectin methylesterase, phn lp, tuyn chn.

Chuyn ngnh Vi sinh vt h vi in Nghin u v Pht trin Cng ngh inh h
SUMMARY

The thesis Isolation and screening of Aspergillus niger biosynthesize high activity pectin
methylesterase was performed in the laboratory 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 (tao ta), Citrus lemon (chanh num), Citrus sinensis L.
(cam soan) and Citrus maxima (Nam Roi pomelo), respectively. The combination of
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
Chuyn ngnh Vi sinh vt h vii in Nghin u v Pht trin Cng ngh inh h
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 35.5C, 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 35C 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 40C 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 70C.
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.

Chuyn ngnh Vi sinh vt h viii in Nghin u v Pht trin Cng ngh inh h
MC LC
LI CAM OAN .................................................................................................................. i
LI CM N ....................................................................................................................... ii
TM LC ........................................................................................................................ iv
SUMMARY ......................................................................................................................... vi
MC LC ......................................................................................................................... viii
DANH SCH BNG ........................................................................................................ xiii
DANH SCH HNH .......................................................................................................... xv
T VIT TT ................................................................................................................... xix
THUT NG ..................................................................................................................... xx
M U ............................................................................................................................... 1
1. Tnh cp thit ca ti ......................................................................................... 1
2. Mc tiu v ni dung nghin cu .......................................................................... 2
3. i tng v phm vi nghin cu ......................................................................... 3
4. Nhng ng gp mi, ngha khoa hc v thc tin ca lun n ..................... 3
5. Cch tip cn v gi thut khoa hc .................................................................... 4
6. Kt cu ca lun n ................................................................................................ 5
Chng 1 TNG QUAN TI LIU .............................................................................. 6
1.1 Tng quan v enzme pectin methlesterase ...................................................... 6
1.1.1 Tng quan ............................................................................................................ 6
1.1.2 Tnh cht sinh l ca PME .................................................................................. 6
1.1.3 Kiu phn ng ca PME ...................................................................................... 7
1.1.4 Cc yu t nh hng n ng hc ca enzyme PME ...................................... 8
1.2 Tm quan trng ca vic s dng PME trong thc tin ................................. 10
1.2.1 Cc ng dng c bn ......................................................................................... 10
1.2.2 Tng quan v cng ngh ch bin rau qu v s bin i c tnh cu trc ca
rau qu trong qu trnh ch bin .......................................................................................... 12
1.2.3 Vai tr ca pectin v PME i vi s ci thin c tnh cu trc ca thc phm
ngun gc thc vt ............................................................................................................... 13
1.3 Vai tr ca vic thu nhn PME t nm mc Aspergillus niger c hiu ........ 19
Chuyn ngnh Vi sinh vt h ix in Nghin u v Pht trin Cng ngh inh h
1.3.1 Tm quan trng ca vic thu nhn PME t nm mc A. niger ......................... 19
1.3.2 Vai tr ca c cht pectin i vi s pht trin ca nm mc sinh tng hp
enzyme PME ........................................................................................................................ 20
1.3.3 Ngun nguyn liu cho qu trnh phn lp v tuyn chn A. niger sinh tng hp
PME hot tnh cao ................................................................................................................ 21
1.4 Tng quan v nm Aspergillus niger ................................................................. 22
1.4.1 Gii thiu chung ................................................................................................ 22
1.4.2 c im h si ca nm Aspergillus niger ...................................................... 23
1.4.3 Cu to c quan sinh sn ................................................................................... 23
1.4.4 Chuyn lun phn loi Aspergillus niger .......................................................... 24
1.4.5 nh danh Aspergillus niger theo phng php truyn thng .......................... 24
1.4.6 nh danh nhm loi Aspergillus bng phng php sinh hc phn t ............ 25
1.5 Phng php tun chn dng nm mc Aspergillus niger c kh nng sinh
tng hp PME .................................................................................................................... 26
1.5.1 L thuyt chung ................................................................................................. 26
1.5.2 Tuyn chn dng Aspergillus niger sinh tng hp PME theo phng php sng
lc t nhin .......................................................................................................................... 27
1.6 nh hng ca thnh phn mi trng n qu trnh sinh tng hp PME . 27
1.6.1 Vai tr ca c cht trong qu trnh ln men sinh tng hp PME ...................... 27
1.6.2 nh hng ca thnh phn a lng ................................................................. 29
1.6.3 nh hng ca ngun khong dinh dng ....................................................... 30
1.7 Tc ng ca phng thc v iu kin ln men n hiu qu sinh tng hp
PME ............................................................................................................................... 30
1.7.1 nh hng ca nhit nui cy ...................................................................... 30
1.7.2 m mi trng ............................................................................................. 30
1.7.3 iu kin pH ban u ca mi trng............................................................... 31
1.7.4 nh hng ca thi gian ................................................................................ 32
1.7.5 nh hng ca phng thc ln men n qu trnh tng hp PME ................. 33
1.8 Cc u t nh hng n hiu qu thu nhn PME t canh trng b mt 34
1.9 Cc bin php tinh sch PME k thut ............................................................ 35
1.9.1 Tm quan trng ca ch phm enzyme k thut trong cng ngh thc phm .. 36
1.9.2 Tinh sch s b PME bng phng php kt ta .............................................. 37
Chuyn ngnh Vi sinh vt h x in Nghin u v Pht trin Cng ngh inh h
1.9.3 ng dng k thut lc mng trong tinh sch enzyme k thut ......................... 38
1.10 Qu hoch thc nghim xc nh iu kin ln men thch hp bng
phng php b mt p ng (RSM) .............................................................................. 38
1.11 Tnh hnh nghin cu trong v ngoi nc v lnh vc phn lp, tun
chn v sinh tng hp PME .............................................................................................. 39
Chng 2 PHNG TIN & PHNG PHP NGHIN CU ............................. 45
2.1 Phng tin nghin cu ...................................................................................... 45
2.1.1 a im, thi gian ............................................................................................ 45
2.1.2 Thit b, ho cht ............................................................................................... 45
2.2 Phng php nghin cu .................................................................................... 47
2.2.1 Chun b mu ..................................................................................................... 47
2.2.2 Phng php phn tch v o c ...................................................................... 48
2.2.3 Phng php phn tch v x l s liu ............................................................ 50
2.3 Ni dung nghin cu ........................................................................................... 52
2.3.1 S nghin cu tng qut ............................................................................... 52
2.3.2 Ni dung 1: Phn lp v tuyn chn dng nm mc A. niger c hiu cho qu
trnh sinh tng hp PME ...................................................................................................... 54
2.3.3 Ni dung 2: Xc nh cc yu t nh hng n qu trnh ln men rn sinh tng
hp PME ........................................................................................................................... 60
2.3.4 Ni dung 3: Tinh sch s b v xc nh c im ca PME t A. niger ......... 71
2.3.5 Ni dung 4: ng dng ch phm PME t A. niger trong ci thin c tnh cu
trc rau qu .......................................................................................................................... 77
Chng 3 KT QU V THO LUN ..................................................................... 81
3.1 Phn lp v xc nh c im hnh thi cc dng nm c c tnh ging A.
niger t v cc loi qu giu pectin .................................................................................. 81
3.1.1 Kt qu phn lp cc dng nm en thuc nhm Aspergillus t v cc loi qu
giu pectin ........................................................................................................................... 81
3.1.2 nh loi cc dng va phn lp da trn c im i th ca khun lc ....... 83
3.1.3 c im ca bo t nh v b my mang bo t ca cc dng nm mc phn
lp ........................................................................................................................... 85
3.2 Tun chn dng nm mc c hiu i vi PME ........................................... 88
3.2.1 nh gi kh nng sinh pectinase ca cc dng nm c phn lp ........... 88
Chuyn ngnh Vi sinh vt h xi in Nghin u v Pht trin Cng ngh inh h
3.2.2 Tuyn chn dng nm mc c kh nng sinh tng hp PME hot tnh cao ..... 92
3.2.3 Kh nng kt hp cc dng nm mc c hiu n hiu qu thu nhn PME .... 94
3.2.4 nh gi kh nng kt hp hai dng So
2
v R
1
c hiu theo cc t l khc
nhau n hiu qu sinh tng hp PME hot tnh cao .......................................................... 96
3.3 Bc u nh danh mt s dng A. niger phn lp bng k thut sinh
hc phn t ......................................................................................................................... 98
3.4 nh hng ca mi trng dinh dng n qu trnh sinh tng hp PME t
2 dng A. niger R
1
v So
2
................................................................................................. 100
3.4.1 Xc nh thnh phn c bn ca c cht ln men ........................................... 100
3.4.2 nh gi tc ng ca thnh phn c cht n hot tnh PME thu nhn t qu
trnh ln men SSF ca hai dng nm mc A. niger R
1
v So
2
........................................... 102
3.4.3 Vai tr ca vic b sung khong n kh nng tng hp PME t A. niger .... 104
3.4.4 Tc ng ca ngun nitrogen b sung n hiu qu thu nhn PME t qu trnh
ln men SSF dng A. niger So
2
v R
1
................................................................................ 107
3.5 Tc ng ca mt s iu kin ln men n hiu qu thu nhn PME t
A. niger trong qu trnh nui c SSF ........................................................................... 109
3.5.1 nh hng ca loi dung dch m s dng trong iu chnh m v pH ban
u ca mi trng ln men n hiu qu sinh tng hp PME ......................................... 109
3.5.2 S thay i hot tnh PME v thnh phn c cht ca mi trng nui cy theo
thi gian ......................................................................................................................... 111
3.5.3 S tng tc gia pH, m ban u ca mi trng, t l huyn ph bo t
nm v nhit n qu trnh sinh tng hp PME t A. niger ...................................... 113
3.6 nh hng ca iu kin l trch n hiu qu thu nhn PME sau qu trnh
ln men SSF ...................................................................................................................... 120
3.6.1 nh hng ca vic thay i gi tr pH dung dch m n hiu qu ly trch
PME ......................................................................................................................... 120
3.6.2 nh hng ca t l dung dch ly trch v canh trng b mt sau khi ln men
n hiu qu ly trch PME t nm mc A. niger ............................................................... 122
3.6.3 nh hng ca nhit v thi gian n hiu qu ly trch PME ................... 124
3.6.4 nh hng ca nhit bo qun n n nh ca dch trch PME th .... 126
3.7 Tinh sch v xc nh c im ca PME c sinh tng hp t t hp
A. niger So
2
v R
1
............................................................................................................ 129
Chuyn ngnh Vi sinh vt h xii in Nghin u v Pht trin Cng ngh inh h
3.7.1 nh hng ca loi ha cht v t l s dng n kh nng kt ta PME t
A. niger ......................................................................................................................... 129
3.7.2 Tinh sch s b PME t A. niger bng phng php lc mng ...................... 135
3.7.3 Mt s c im c bn ca ch phm PME k thut thu nhn t A. niger ... 137
3.8 Kh nng ng dng ca ch phm PME k thut thu nhn t A. niger So
2

v R
1
n kh nng ci thin cu trc rau qu ............................................................. 146
3.8.1 i vi sn phm khm lnh ng .................................................................. 146
3.8.2 Tc ng ca PME n s ci thin cu trc da leo mui chua .................... 151
KT LUN V NGH .............................................................................................. 155
1. Kt lun ................................................................................................................ 155
2. ngh ................................................................................................................. 156
TI LIU THAM KHO ............................................................................................... 157
DANH MC CC CNG TRNH CNG B ...................................................... 177
PH LC 1 MI TRNG V PHNG PHP NHN DIN NM MC, CC
PHNG PHP PHN TCH ....................................................................................... xxi
PH LC 2 MT S KT QU KHO ST ............................................................. xli
PH LC 3 KT QU THNG K ............................................................................ lxiii
PH LC 4 MT S HNH NH ............................................................................ cxv
Chuyn ngnh Vi sinh vt h xiii in Nghin u v Pht trin Cng ngh inh h
DANH SCH BNG

Bng 1.1: Thnh phn pectin v mc ester ha ca pectin mt s loi tri cy .......... 21
Bng 1.2: Cc cht dinh dng a lng, vi lng, ngun gc v chc nng i vi t bo
nm mc Aspergillus sp. ...................................................................................................... 28
Bng 2.1: Cc ch tiu, phng php phn tch v o c ................................................... 49
Bng 2.2: Cng thc tnh ton, nh gi hiu qu tinh sch PME ...................................... 50
Bng 2.3: Gi tr m ha v gi tr thc ca cc nhn t tng tc n hiu qu sinh tng
hp PME (U/g) .................................................................................................................... 66
Bng 2.4: Cc nghim thc kho st theo phng php b mt p ng, p dng thit k
trung tm phc hp .............................................................................................................. 67
Bng 3.1: S dng nm mc c phn lp t v cc loi qu giu pectin ....................... 82
Bng 3.2: Phn nhm nm mc c tnh c hiu vi enzyme pectinase ............................. 89
Bng 3.3: Kt qu thng k s khc bit ng knh vng phn gii pectin (D) ca cc
dng nm mc thuc nhm c hiu cao vi pectinase....................................................... 90
Bng 3.4: S thay i ng knh vng phn gii pectin v hot tnh PME sinh tng hp t
cc dng nm mc c hiu vi pectinase ........................................................................... 93
Bng 3.5: nh gi kh nng kt hp hai dng nm mc n hiu qu thu nhn enzyme
PME ..................................................................................................................................... 95
Bng 3.6: nh hng ca t l kt hp hai dng R
1
v So
2
n hiu qu sinh tng hp
PME hot tnh cao ................................................................................................................ 97
Bng 3.7: Thnh phn ha hc c bn ca cc ngun c cht ln men ............................ 101
Bng 3.8: S thay i hot tnh PME cc t l kt hp khc nhau ca b to ta v v qu
cam/bi ............................................................................................................................ 102
Bng 3.9: nh hng ca nng v loi khong b sung vo mi trng ln men n s
thay i hot tnh ca PME t A. niger ............................................................................. 105
Bng 3.10: nh hng ca vic b sung kt hp cc loi khong vo mi trng nui cy
n kh nng sinh tng hp PME t A. niger .................................................................... 107
Bng 3.11: nh hng ca hm lng v ngun nitrogen b sung n hot tnh PME t
qu trnh sinh tng hp A. niger ........................................................................................ 108
Bng 3.12: S thay i hm lng pectin, DE, m ca canh trng sau khi ln men v
pH ca dch trch enzyme sau thi gian nui cy .............................................................. 112
Bng 3.13: nh hng ca cc nhn t m ha i vi phng trnh hi quy ................. 114
Bng 3.14: nh hng ca t l dung dch m citrate v canh trng sau ln men n
hiu qu thu nhn PME ...................................................................................................... 122
Bng 3.15: Hiu qu tinh sch PME bng phng php kt ta vi (NH
4
)
2
SO
4
.............. 130
Chuyn ngnh Vi sinh vt h xiv in Nghin u v Pht trin Cng ngh inh h
Bng 3.16: Hiu qu tinh sch PME bng phng php kt ta vi NaCl ....................... 131
Bng 3.17: Hiu qu tinh sch PME bng phng php kt ta vi ethanol .................... 132
Bng 3.18: Hiu qu tinh sch PME bng phng php kt ta vi aceton...................... 133
Bng 3.19: So snh hiu qu tinh sch PME bng cc tc nhn khc nhau ...................... 134
Bng 3.20: Hiu qu tinh sch PME t A. niger sau qu trnh lc mng .......................... 135
Bng 3.21: Cc thng s ng hc ca qu trnh bt hot nhit PME sinh tng hp t
A. niger ............................................................................................................................... 141
Bng 3.22: nh hng ca nng NaCl n hot tnh PME t A. niger ....................... 142
Bng 3.23: nh hng ca nng CaCl
2
n hot tnh PME t A. niger ...................... 143
Bng 3.24: cng tng i trung bnh ca khm do tc ng ca tin x l vi PME
nm mc v CaCl
2
theo cc phng thc khc nhau ........................................................ 148
Bng 3.25: nh hng ca hot tnh PME nm mc b sung n s thay i cng
tng i v DE ca khm ng lnh .......................................................................... 150
Bng 3.26: nh hng ca thi gian tin x l nhit ( 50C) n s thay i ester ha
pectin v c tnh cu trc da leo..................................................................................... 152
Bng PL1.1: Phn bit mt s ging Aspergillus v Penicillium ..................................... xxiv
Bng PL1.2: Cch pha dung dch m citrate .................................................................. xxix
Bng PL1.3: Cch pha dung dch m acetate ................................................................ xxix
Bng PL1.4: Cch pha dung dch m citrate-phosphate .................................................. xxx
Bng PL1.5: Bng tra nng mui (NH
4
)
2
SO
4
bo ha b sung trong tin trnh kt ta
enzyme v protein ............................................................................................................. xxxi
Bng PL1.6: Chun b dung dch m CTAB (CTAB buffer) ....................................... xxxiv
Bng PL1.7: Bng tra lng ng nghch chuyn ....................................................... xxxix
Bng PL2.1: Hnh thi khun lc ca 60 dng Aspergillus niger phn lp trn mi
trng MEA v mi trng CZ ........................................................................................... xli
Bng PL2.2: c tnh vi th ca cc dng A. niger phn lp trn mi trng CZ ............ xlv
Bng PL2.3: c tnh vi th ca cc dng A. niger phn lp trn mi trng MEA ....... xlvii
Bng PL2.4: ng knh vng phn gii pectin (D) ca cc dng A. niger ..................... xlix
Bng PL2.5: Hiu qu thu nhn PME theo khi lng c cht ly trch khc nhau ............. lix

Chuyn ngnh Vi sinh vt h xv in Nghin u v Pht trin Cng ngh inh h
DANH SCH HNH
Hnh 1.1: S thy phn pectin ca PME........................................................................... 6
Hnh 1.2: Tc dng ca PME trong qu trnh lm trong nc qu...................................... 11
Hnh 1.3: Cu trc phn t pectin ........................................................................................ 14
Hnh 1.4: S m t cc cp cu trc ca thc phm ngun gc thc vt .................. 15
Hnh 1.5: M hnh cu trc ca vch t bo thc vt bc cao ............................................. 15
Hnh 1.6: S biu din cc chuyn ha ca pectin di tc ng ca enzyme thy phn
nh hng n cu trc t bo ............................................................................................. 16
Hnh 1.7: Hnh thi Aspergillus sp. ...................................................................................... 23
Hnh 1.8: Cu trc ca trnh t m ho cho rRNA sinh vt nhn tht .............................. 25
Hnh 1.9: Cc phng php thu ch phm enzyme ............................................................. 36
Hnh 2.1: Nguyn liu khm v cch chun b mu th nghim .......................................... 47
Hnh 2.2: S nghin cu tng qut ................................................................................. 53
Hnh 2.3: S b tr th nghim phn lp v tuyn chn nm mc .................................. 54
Hnh 2.4: S b tr th nghim 5 Kh nng kt hp cc dng nm mc c hiu n
hiu qu thu nhn PME ........................................................................................................ 59
Hnh 2.5: S b tr th nghim 8 - nh hng ca vic kt hp b to v v qu cam/bi n
qu trnh sinh tng hp PME iu kin ln men SSF ............................................................ 62
Hnh 2.6: S b tr th nghim 9a - nh hng ca tng thnh phn khong ................ 63
b sung ring l n hot tnh ca PME c tng hp bi A. niger .................................. 63
Hnh 2.7: S b tr th nghim 13 - nh hng tng tc ca cc iu kin ln men n
hiu qu thu nhn PME ........................................................................................................ 68
Hnh 2.8: S kt ta PME bng dung dch mui (NH
4
)
2
SO
4
/NaCl ................................ 73
Hnh 2.9: S kt ta PME bng dung mi hu c .......................................................... 74
Hnh 3.1: Mt trn ca mu phn lp M
3
............................................................................ 82
Hnh 3.2: Mt di ca mu phn lp M
3
.......................................................................... 82
Hnh 3.3: Hnh dng mt trn ca khun lc ...................................................................... 83
Hnh 3.4: Hnh dng mt di ca khun lc ..................................................................... 83
Hnh 3.5: c im khun lc ca dng S
2
v G
1
khi c cy 3 im trn mi trng CZ
............................................................................................................................................. 84
Hnh 3.6: Dng A. niger i chng (Klich, 2002) ............................................................... 85
Hnh 3.7: Dng nm mc H
2
trn mi trng MEA ........................................................... 85
Hnh 3.8: Si nm c vch ngn ca dng So
2
vt knh 4X ............................................. 86
Hnh 3.9: Hnh nh dng So
2
vt knh 4X v 10X ........................................................... 86
Chuyn ngnh Vi sinh vt h xvi in Nghin u v Pht trin Cng ngh inh h
Hnh 3.10: Hnh nh bo t di knh hin vi ca dng A. niger ........................................ 86
Hnh 3.11: Th bnh cn non (pht trin 1 tng) dng So
2
.................................................. 87
Hnh 3.12: Hai vng th bnh v bo t ca dng So
2
......................................................... 87
Hnh 3.13: Mt trn vng phn gii pectin ca dng nm So
2
........................................... 91
Hnh 3.14: Mt di vng phn gii pectin ca dng nm So
2
........................................... 91
Hnh 3.15: Mu i chng nc .......................................................................................... 91
Hnh 3.16: Mu i chng A. niger V .................................................................................. 91
Hnh 3.17: Vng phn gii pectin trn mi trng pectin - agarose sau 24 gi 37C v
nhn din vi thuc th CTAB 1% ..................................................................................... 93
Hnh 3.18: nh hng ca s kt hp cc dng nm mc khc nhau n s thay i vng
phn gii pectin trn mi trng pectin - agarose ............................................................... 95
Hnh 3.19: Ph in di ca sn phm PCR ca cc mu nm c khuch i .................. 98
Hnh 3.20: Biu biu din trnh t ca mt on RNA gene ca 4 mu nm .............. 100
Hnh 3.21: T l tng ng v gene ca dng nm So
2
v dng A. niger NBL, BR 28s
ribosomal RNA gene hay An03c0110 ............................................................................... 100
Hnh 3.22: th so snh nh hng ca cc loi khong b sung ti u i vi hiu qu
gia tng hot tnh PME t A. niger .................................................................................... 106
Hnh 3.23: nh hng ca loi dung dch m s dng trong iu chnh m v pH mi
trng ln men n hot tnh PME t qu trnh sinh tng hp A. niger ........................... 110
Hnh 3.24: nh hng ca thi gian n hot tnh PME thu nhn t A. niger .............. 111
Hnh 3.25: th tng quan gia hot tnh PME xc nh thc nghim v tnh ton theo
phng trnh hi quy ......................................................................................................... 115
Hnh 3.26: th biu din s tng tc ca tng cp nhn t n hiu qu thu nhn PME
........................................................................................................................................... 116
Hnh 3.27: th ng ng im biu din tng tc ca t l huyn ph bo t nm v
pH mi trng ban u n hot tnh PME thu nhn ........................................................ 117
Hnh 3.28: th ng ng im biu din tng quan ca t l huyn ph bo t nm
b sung v nhit n hot tnh PME thu nhn .......................................................... 117
Hnh 3.29: th ng ng im biu din tng tc ca t l huyn ph bo t nm v
m mi trng ban u n hot tnh PME thu nhn ................................................... 117
Hnh 3.30: th ng ng im biu din tng tc ca nhit v pH ban u ca
mi trng n hot tnh PME thu nhn .......................................................................... 117
Hnh 3.31: th ng ng im biu din tng tc ca pH v m ban u ca mi
trng ln men n hot tnh PME thu nhn .................................................................... 118
Hnh 3.32: th ng ng im biu din tng tc gia nhit v m ban u
ca mi trng ln men n hot tnh PME thu nhn ...................................................... 118
Chuyn ngnh Vi sinh vt h xvii in Nghin u v Pht trin Cng ngh inh h
Hnh 3.33: nh hng ca pH dung dch ly trch n hot tnh PME .............................. 121
Hnh 3.34: nh hng ca nhit v thi gian ly trch n hot tnh PME ................... 125
Hnh 3.35: S thay i hot tnh PME th (A/A
o
) theo thi gian bo qun ...................... 127
nhit lnh (4C).......................................................................................................... 127
Hnh 3.36: S thay i hot tnh PME th (A/A
o
) theo thi gian bo qun ...................... 127
nhit lnh ng (-18C) ............................................................................................. 127
Hnh 3.37: Kt qu in di trn gel SDSPAGE cc mu PME t A. niger ..................... 136
Hnh 3.38: nh hng ca nng pectin n hot tnh PME t A. niger....................... 137
Hnh 3.39: th b mt p ng v th ng ng im biu th s tng quan gia
pH v nhit n hot tnh PME t A. niger .................................................................. 139
Hnh 3.40: Phng trnh biu th s v hot nhit ca PME t A. niger ........................... 140
Hnh 3.41: Quy trnh tng hp ch phm PME k thut t A. niger ................................. 145
Hnh 3.42: S thay i ester ha ca pectin trong khm do tc ng ca tin x l vi
PME nm mc v CaCl
2
theo cc phng thc khc nhau ............................................... 146
Hnh 3.43: th biu din s thay i cng tng i (H/H
o
) ca da leo mui chua
theo cc t l PME v CaCl
2
b sung trong dch chn 50C, 30 pht ............................ 153
Hnh PL1.1: Hnh thi i th v vi th A. niger ............................................................... xxv
Hnh PL1.2: Bo t ca A. awamori (tri) v A. foetidus (phi) ...................................... xxvi
Hnh PL1.3: Khun lc 7 ngy ca dng nm mc trn mi trng nh loi c trng: A.
foetidus (h, CZ), A. niger (i, MEA) v A. awamori (j, CZ) ............................................ xxvii
Hnh PL2.1: nh hng ca cc cht km hm n hot ng ca PME nm mc ............. lx
Hnh PL2.2: So snh hiu qu ci thin cng ca khm do tc ng ca qu trnh tin
x l vi PME ngoi bo c ngun gc khc nhau ............................................................ lxii
Hnh PL3.1: th Box Whisker biu din nh hng ca dng nm mc n kh nng
thu nhn PME thng qua ng knh vng phn gii pectin.......................................... lxviii
Hnh PL3.2: th Box Whisker biu din s thay i hot tnh PME ca cc dng nm
mc khc nhau .................................................................................................................... lxx
Hnh PL3.3: th Box Whisker biu din nh hng kt hp 4 dng nm mc c hiu
n kh nng thu nhn PME thng qua ng knh vng phn gii pectin ..................... lxxii
Hnh PL3.4: th Box Whisker biu din nh hng ca s kt hp 4 dng nm mc
c hiu n hot tnh PME ............................................................................................. lxxiv
Hnh PL3.5: th Box Whisker biu din nh hng ca t l kt hp R
1
v So
2
n
hiu qu thu nhn PME da trn ng knh vng phn gii pectin ................................ lxxv
Hnh PL3.6: th Box Whisker biu din nh hng ca t l kt hp R
1
v So
2
n
hot tnh PME .................................................................................................................. lxxvi
Hnh PL4.1: Cc thit b s dng trong nghin cu .......................................................... cxv
Chuyn ngnh Vi sinh vt h xviii in Nghin u v Pht trin Cng ngh inh h
Hnh PL4.2: Mt s hnh nh chun b c cht v ln men SSF sinh tng hp PME ...... cxvi
Hnh PL4.3: Cc mu da leo mui chua ......................................................................... cxvi
Hnh PL4.4: Mt s cng on tin x l v ci thin c tnh cu trc khm ng lnh
......................................................................................................................................... cxvii

Chuyn ngnh Vi sinh vt h xix in Nghin u v Pht trin Cng ngh inh h
T VIT TT
A. Aspergillus
B. Botrytis
bp base pair
BSA Bovine serum albumin
CBB Coomassie Brilliant Blue
cbk cn bn kh
CCD Central composite design (m hnh phc hp ti tm)
cfu colony forming unit (mt s bo t hnh thnh)
CMSepharose Carboxymethyl Sepharose
CTAB Cetyltrimethylammonium bromide
CZ Czapek Dox Agar
DE Degree of esterification (mc ester ha)
DNA Deoxyribonucleic acid
E. Erwinia
EDTA Ethylenediaminetetraacetic acid
EGCG Epigallocatechin gallate
GalA Galacturonic acid
HG Homogalacturonan
HMP High Methoxyl Pectin (pectin c methoxyl ha cao)
IAA Iodoacetamide
ITS Internal transcribed spacer
kDa Kilodalton
LMP Low Methoxyl Pectin (pectin c methoxyl ha thp)
LSU Large subunit (tiu n v c kch thc ln)
MEA Malt Extract Agar
MI Methoxyl Index (ch s methoxyl)
NCBI National Center for Biotechnology Information
NCKH Nghin cu khoa hc
PCR Polymerase Chain Reaction
PDA Potato Dextrose Agar
PG Polygalacturonase
pI Isoelectric point (im ng in)
Chuyn ngnh Vi sinh vt h xx in Nghin u v Pht trin Cng ngh inh h
PL Pectate lyase
PME Pectin methylesterase
PMEI Pectin methylesterase inhibitor (cht c ch pectin methylesterase)
Q-BGDT Quyt nh B Gio dc v o to
RAPD Random Amplification of Polymorphic DNA
rpm rounds per minute (s vng trn pht)
rRNA Ribosomal ribonucleic acid
RSM Response Surface Methodology (phng php b mt p ng)
SDSPAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SmF Submerged fermentation (ln men b mt trn mi trng lng)
SSF Solid state fermentation (ln men b mt trn mi trng rn)
SSU Small subunit (tiu n v kch thc nh)
TSS Total soluble solid (tng cht kh ha tan)
v/v volume/volume (t l th tch/th tch)
v/w volume/weight (t l th tch/khi lng)
w/w weight/weight (t l khi lng/khi lng)
U/g Units per weight (gram) of substrate (hot tnh enzyme trong 1 g c cht)
U/mL Units per volume (mL) of substrate (hot tnh enzyme trong 1 mL c cht)

THUT NG

Pectin methylesterase (PME) t Aspergillus niger: PME c thu nhn t qu
trnh ly trch sinh khi nm mc sau qu trnh ln men SSF hay SmF dng A. niger
cc iu kin thch hp.
PME c chua: PME c ly trch t c chua iu kin thch hp.
Chuyn ngnh Vi sinh vt h 1 in Nghin u v Pht trin Cng ngh inh h
M U

1. Tnh cp thit ca ti
ng dng enzyme trong ch bin thc phm v nhiu lnh vc khc v ang l
mt trong nhng vn mang tnh cp thit v c ngha thc tin. Hin nay, cc
nc pht trin ang p dng cc ch phm enzyme trong sn xut to ra
nhng sn phm c nng sut v cht lng cao vi gi thnh h (Saurel, 2003;
Suutarinenn v Autio, 2004; Ng Tin Hin, 2001). Vit Nam l nc giu tim
nng v nng sn, nhu cu cc loi enzyme phc v trong ch bin thc phm l
rt ln. Tuy nhin, Vit Nam hon ton khng c xng sn xut ch phm
enzyme, hng nm nc ta vn phi nhp ngoi mt khi lng ln nhng loi
enzyme ny (ng Th Thu v Nguyn Th Xun Sm, 2009b).
Pectinase l nhm enzyme xc tc cho s thy phn cc polymer pectin - ang
c ng dng rng ri trong cng nghip thc phm cng nh x l cht thi v
nhiu lnh vc khc. Vit Nam, ch phm pectinase c s dng kh ph
bin v c nhiu nh khoa hc quan tm nghin cu (L Vn Nhng et al.,
1978; Ph Quyt Tin, 1999; Hunh Ngc Oanh v Trn Ngc Hng, 2008). Tuy
nhin, i vi lnh vc ci thin c tnh cu trc t bo rau qu - mt hn ch ln
v cht lng ca sn phm sau ch bin, vic s dng hn hp ch phm
pectinase khng mang li hiu qu tch cc. Trong khi , pectin methylesterase
(PME, EC 3.1.1.11) mt enzyme thuc nhm pectinase ng vai tr rt quan
trng (Van Buren, 1979).
PME c quan tm s dng v pht trin nhiu quc gia (Saurel, 2003;
Suutarinenn v Autio, 2004) vi cc mc ch nh hn ch s ph hy cu trc
ca t bo thc vt, tng hiu sut ly trch nc qu v thc y nhanh qu trnh
sy rau qu (Van Buren, 1979; Sajjaanantakul v Pitifer, 1991). Tuy nhin, vic
ng dng PME trong cng nghip ch bin cc sn phm c ngun gc t thc vt
Vit Nam vn cha c quan tm, ng thi vn c s nhm ln trong hiu
bit v PME v pectinase. Chnh v th, vic nghin cu c h thng s thu nhn
PME v kh nng ng dng thc tin ca enzyme ny trong iu kin hin ti ca
Vit Nam l vn cn thit, mang ngha khoa hc v thc tin cao.
Bn cnh PME t thc vt, ngun giu PME v c s dng nhiu nht trong
cc ch phm pectinase thng mi l cc PME t vi sinh vt (Solis Pereira et
al.,1993). Nm mc c bit n nh ngun ph bin nht cho sn xut enzyme
ny (Taragano et al., 1997, trch dn bi Patil v Dayanand, 2006). Mc d vy,
khng phi tt c nm mc u c kh nng sinh enzyme nh nhau v ngay c
nhng dng cng mt ging cng khng c kh nng sinh tng hp enzyme vi
hot tnh tng ng (Nguyn c Lng, 2004; Parenicova, 2000). Vit Nam,
nghin cu tng hp cc ch phm t dng Aspergillus ni chung cng nh
A. niger ni ring mi dng li nghin cu thm d, ng thi ch quan tm n
kh nng tng hp pectinase (Ph Quyt Tin, 1999; L Th Thanh Hng v
Nguyn Thy Chu, 2005). Nghin cu thu nhn PME t dng nm mc c sn
cng gp nhiu kh khn do ngn hng ging Vit Nam cha pht trin, vic
nhp ngoi khng nhng gy bt li v mt kinh t m cn c cc kh khn v
iu kin mi trng c nh hng n kh nng sinh trng v tng hp enzyme
(Bhalla v Chatanta, 2000). Do , vn tuyn chn dng A. niger c kh nng
sinh tng hp PME hot tnh cao i hi nhiu thi gian v cng sc nghin cu.
Hot ng ca PME c bit n ch yu nh vo tc ng ca enzyme ny trn
pectin c methoxyl ha cao (Sajjaanantakul v Pitifer, 1991; Bhalla v
Chatanta, 2000). Chnh v th, cc ngun ph phm giu pectin khng ch c s
dng nh c cht cm ng cho qu trnh ln men sinh tng hp PME, m cc
chng vi sinh vt c phn lp t nguyn liu giu pectin cng cho thy tim
nng ca chng trong vic thu nhn enzyme ny (L Th Thanh Hng v Nguyn
Thy Chu, 2005; Pilnik v Voragen, 1993).
Vi nhng u im m PME mang li cho cng nghip thc phm v vai tr quan
trng ca khu tuyn chn dng A. niger trong cng nghip sn xut enzyme,
nghin cu tng hp PME cn c tin hnh mt cch c th ngay t bc u
phn lp v tuyn chn cc dng A. niger c hiu vi PME n vic nghin cu
xc nh iu kin ln men sinh tng hp enzyme ph hp, ng dng hiu qu
trong lnh vc ci thin cu trc rau qu.
2. Mc tiu v ni dung nghin cu
i. M tiu hnh
Nghin cu sinh tng hp v ng dng PME t qu trnh ln men b mt
trn mi trng rn dng A. niger c hiu c phn lp v tuyn chn t cc
loi v qu giu pectin ng bng sng Cu Long.
ii. M tiu th
(1) Phn lp, tch rng v xc nh c im cc dng A. niger bn a t cc
v qu giu pectin ph bin ti ng bng sng Cu Long, lm ngun nm
mc s dng cho qu trnh sinh tng hp PME.
(2) Tuyn chn cc dng A. niger c hiu, ph hp cho qu trnh ln men sinh
tng hp PME t hiu qu cao.
(3) Xc nh iu kin ln men ph hp cho qu trnh sinh tng hp PME,
ng thi xc nh c im ca enzyme ny.
(4) nh gi hiu qu s dng thc t ca PME iu kin phng th nghim.
iii. Ni dung nghin u
Nghin cu c thc hin vi 4 ni dung chnh:
Phn 1: Phn lp v tuyn chn cc dng A. niger t v qu giu pectin c kh
nng sinh tng hp PME (mc tiu 1 v 2).
Phn 2: Xc nh cc yu t nh hng n qu trnh ln men b mt trn mi
trng rn sinh tng hp PME t A. niger (mc tiu 3).
Phn 3: Tinh sch s b v xc nh c im ca PME thu nhn t qu trnh ln
men A. niger c hiu (mc tiu 3).
Phn 4: Nghin cu ng dng ch phm PME vo vic ci thin cu trc hai sn
phm in hnh l khm ng lnh v da leo mui chua (mc tiu 4).
3. i tng v phm vi nghin cu
i. i tng nghin u
Cc dng A. niger c kh nng sinh tng hp PME hot tnh cao;
Dng nm mc i chng gm (i) dng A. niger i chng ngun gc Nga (khoa
Sinh hc, Trng i hc Tng hp quc gia Matxcova mang tn Lomonosov -
MGU) (k hiu R92) v (ii) dng A. niger c phn lp t t (k hiu V) c
tr mu Vin Nghin cu v Pht trin Cng ngh Sinh hc, Trng i hc
Cn Th.
PME k thut c kh nng s dng trong thc t.
ii. Phm vi nghin u
Cc dng A. niger bn a c phn lp t cc ngun nguyn liu giu pectin sn
c ng bng sng Cu Long, c tnh c hiu i vi PME.
Phng thc ln men b mt trn mi trng rn c chn la da trn iu kin
th nghim n gin.
Ch phm enzyme PME k thut, ch qua tinh sch s b c kh nng ng dng
thc tin trong ci thin cu trc t bo rau c qu.
4. Nhng ng gp mi, ngha khoa hc v thc tin ca lun n
Lun n nghin cu c tnh h thng v pectin methylesterase ca A. niger, bao
gm cc khu phn lp, nh danh, nui cy vi sinh vt sinh tng hp enzyme,
thu nhn, tinh sch s b v th nghim ng dng ch phm enzyme trong ch
bin thc phm (lnh vc ci thin c tnh cu trc rau qu).
iii. Nhng kt qu ngh kho h
Nghin cu phn lp c 60 dng nm mc t v tri cy giu pectin v tuyn
chn c 02 dng A. niger c ngun gc t cam son (So
2
) v bi Nm Roi
(R
1
) c hiu i vi PME.
ng dng k thut sinh hc phn t (PCR 28S- rRNA gene) kt hp vi phng
php truyn thng trong nh danh 04 dng A. niger c hiu i vi vic sinh
tng hp PME.
Mt s tnh cht c bn ca ch phm PME c thu nhn t canh trng nui
cy t hp hai chng A. niger So
2
v A. niger R
1
c xc nh: nhit v pH
hot ng ti u cho hot ng ca enzyme, kh nng lm tng hot tnh PME ca
mt s ion kim loi nng thch hp nh Na
+
v Ca
2+
.
iv. Nhng kt qu ngh v mt ng dng
Xc nh c thnh phn mi trng v iu kin nui cy sinh tng hp
PME hot tnh cao vi ngun ging A. niger ch ng c phn lp v tuyn
chn ti ch (t hp hai chng A. niger So
2
v A. niger R
1
).
Tn dng c ngun ph phm nng nghip sn c ti a phng l b to ta v
v bi Nm Roi lm c cht ln men chnh.
chn c iu kin ly trch PME t canh trng b mt, chn c tc nhn
kt ta v nng thch hp tinh sch enzyme.
th nghim ng dng ch phm PME thu c x l nguyn liu, gip ci
thin hiu qu c tnh cu trc t bo rau qu, vi hai sn phm in hnh l
khm lnh ng v da leo mui chua.
5. Cch tip cn v gi thut khoa hc
Qu trnh tuyn chn cng nh ln men sinh tng hp PME t dng A. niger c
da trn cc quy trnh tham kho cho cc loi nm mc, c cht v enzyme c c
tnh tng t (Klich, 2002; Schmitz, 2002; Joshi et al., 2006; Hamdy, 2005).
Th nghim thm d so snh hiu qu sinh tng hp PME ca hai phng thc ln
men b mt trn mi trng rn (SSF) v lng (SmF) khng nh SSF gip gia
tng hot tnh PME 5 ln so vi SmF (Nguyen et al., 2010).
Hot tnh PME thu c khi s dng c cht l mi trng c bn (cm, bt m,
v tru) thp hn 5 ln khi so snh vi trng hp s dng b to ln men (Tran et
al., 2009). Do , nghin cu xut s dng cc ph phm nng nghip sn c
nh b to ta, v cam, v bi l thnh phn c cht ln men chnh cho nghin
cu sinh tng hp PME.
6. Kt cu ca lun n
Lun n bao gm 3 phn: Phn M u (5 trang vi 6 tiu mc); Phn ni dung
gm 3 chng chnh (1) Chng 1: Tng quan ti liu (37 trang vi 11 tiu mc),
(2) Chng 2: Phng tin Phng php nghin cu (37 trang, gm 3 tiu mc
v 27 th nghim theo 4 ni dung c bn, ngoi ra cn c 3 th nghim ph ph
lc 2), (3) Chng 3: Kt qu - Tho lun (72 trang, 8 tiu mc); Phn Kt lun
v ngh (2 trang, 2 tiu mc). S bng v hnh trong ni dung chnh gm 32
bng v 61 hnh. Bi vit s dng 233 ti liu tham kho, bao gm 207 ti liu
ting Anh, 26 ti liu ting Vit.

CHNG 1 TNG QUAN TI LIU
1.1 Tng quan v enzyme pectin methylesterase
1.1.1 ng qun
Pectin methylesterase (PME, EC 3.1.1.11) l enzyme thuc nhm pectinase xc
tc phn ng thy phn lin kt C-O ca nhm methyl ester C
6
ca mt gc
GalA trong homogalacturonan (HG) ca pectin (hnh 1.1). Hiu sut thy phn c
th t n 98% (Gonzalez v Rosso, 2011).

Hn 11: S ty pn pectin ca PME
Ngun: Jayani et al., (2005)
Cc loi PME c tm thy trong rt nhiu loi thc vt v cng c sn xut t
cc loi vi sinh vt gy bnh cho cy. Ni PME ca thc vt c lin quan n s
chuyn ha ca pectin trong sut qu trnh pht trin ca thc vt (Bordenave,
1996). Phi hp vi cc enzyme pectinase khc, PME c trch nhim trong vic
iu khin s thoi ha ca pectin v s mm i ca m thc vt trong qu trnh
chn. PME t nhng ngun khc nhau c nhng c trng khng ging nhau
(Bordenave v Goldberg, 1993).
1.1.2 nh ht sinh l PME
PME c kch thc trung bnh hay phn t khi vo khong 25 54 kDa (Benen
et al., 2003). PME thu nhn t nhng ngun khc nhau c im ng in (pI)
khng ging nhau. im ng in ca PME thay i t 3,1 i vi mt s
enzyme c sinh tng hp t nm mc n gi tr pI l 11 trong trng hp PME
ly trch t c chua. Hu ht PME chit xut t thc vt c pI trung tnh n kim,
ch mt vi trng hp PME c pI mang tnh acid (Bordenave, 1996). Tuy nhin,
iu ny c th lin quan n phng php ly trch c s dng, bao gm giai
on loi b cc thnh phn ha tan trong m t bo ra khi cc thnh phn khng
ha tan ca vch t bo khi ngm trong dung dch m ion cao ly trch PME
(Micheli, 2001).
Nhiu loi PME khc nhau v phn t khi, im ng in v hot tnh sinh ha
c tm thy cc thc vt hai l mm (Bordenave, 1996; Bordenave v
Goldberg, 1994; Giovane et al., 2004). Vic o lng hot tnh ch ra c s
khc bit gia cc loi PME thu nhn t cc b phn khc nhau hoc trong cc
giai on pht trin khc nhau ca thc vt (Bordenave v Goldberg, 1993) tuy
nhin khng c s khc bit ln v tnh cht PME trong cng mt ngun vi sinh
vt, ngoi tr PME t Erwinia chrysanthemi (E. chrysanthemi) (Benen et al.,
2003).
1.1.3 Kiu phn ng PME
PME thy phn ch yu trn nhm methylester ca D-galacturonan. Ty theo s
khc bit v cch sp xp trong cu to ca cc loi PME thc vt, nm mc v vi
khun m c ch phn ng ca mi loi s khc nhau (Markovic v Janecek,
2004).
Hu ht cc PME c ngun gc thc vt u thy phn lin kt ester ca pectin t
u kh hoc u khng kh, hoc gn vi nhm carboxyl t do v tin dc theo
phn t bng c ch chui n (Nguyn c Lng, 2004). Phn ng kh ester
bt u gn nhm carboxyl t do hay t u kh ca mch polygalacturonic (ch
tc dng ti mt v tr trn mch), kt qu to thnh cc gc galacturonic b kh
nhm ester (Rexova - Benkova v Markovic, 1976; Markovic v Kohn, 1984;
Willats et al., 2001). Do hot ng cn c nhm carboxyl t do trn mch
galacturonic nn PME c ly trch t thc vt t hot ng trn cc pectin c
ester ha cao hn l cc pectin c ester ha t 20 30% (Evans v Machale,
1978; Seymour et al., 1991). Ngc li, PME c sinh tng hp t nm mc
(c bit t cc dng Aspergillus) c th phn ct c cht mt cch ngu nhin
(Ishi et al., 1979; Limberg et al., 2000). Ngoi tr hai trng hp ca enzyme
PME c ngun gc t nm mc Trichoderma reesei - kh gc ester theo tng
nhm (Markovic v Kohn, 1984) v PME t vi khun E. chrysanthemi - thc hin
phn ng kh gc ester tng t nh PME chit xut t thc vt (Christensen et
al., 2001). iu ny chi phi ng k n cc phn ng tip theo ca qu trnh kh
ester.
Khi phn ng thy phn theo v tr c nh thng xy ra do tc ng ca PME
ngun gc thc vt, cc pectin b kh ester s to gel khi c s hin din ca
ion calcium (Micheli, 2001) gip thnh t bo tr nn cng chc. Trong trng
hp khng c s kt hp ca Ca
2+
vo mch pectin, enzyme polygalacturonase
(PG) v enzyme pectate lyase (PL) s hot ng v thy phn ngu nhin mch
polymer ca pectin (Duvetter, 2007).
1.1.4 C yu t nh hng n ng h enzyme PME
Ging nh cc enzyme khc, cc nghin cu i vi PME cng khng nh, bin
i ng hc ca enzyme ny chu nh hng ca cc yu t nh nng
enzyme, nng c cht, pH, nhit cng nh tc ng ca cc cht hot ha v
km hm (Ly Nguyen, 2004; Duvetter, 2007; Arotupin et al., 2008).
i. nh hng nng ht nn petin methoxyl h o n t
phn ng PME
Do PME thy phn ch yu trn nhm methylester ca D-galacturonan, chnh v
vy, cc pectin c ester ha cao nh pectin t v chanh (pectin from citrus
peel, P9135, DE = 89%, Sigma), pectin to (apple pectin, P8471, DE = 70 75%,
Sigma) c s dng nh cht nn trong nghin cu ng hc PME (Ly Nguyen,
2004; Duvetter, 2007; Arotupin et al., 2008).
Cc nghin cu i vi PME cng cho thy, phn ng ca PME thuc trng hp
n gin nht - ch c mt c cht, theo phng trnh 1.1:
1
2
1
k
k
k
E S ES E P
+ +
(1.1)
Trong : E: enzyme, S: c cht, P: sn phm; k
1
, k
-1
l hng s tc phn ng
to thnh phc cht ES v phn ly phc cht ES thnh E v S, k
2
l hng s tc
phn ng to thnh E v P (Nguyn c Lng, 2004).
Vi s bin i ng hc phn ng theo phng trnh mt c cht, hng s
Michealis - Menten cng c ng dng th hin tc ca phn ng v c
trng cho PME. Duvetter (2007) khng nh i lc ca PME t A. aculeatus
i vi c cht phn ng pectin to, th hin qua gi K
m
1,4 0,07 g/L khi nhit
phn ng duy tr 22C. Christgau et al. (1996) tnh ton gi tr K
m
cho
PME t A. aculeatus l 2,7 g/L khi s dng cht nn l pectin t v chanh. i
vi PME c ly trch t thc vt nh du ty, chui, c rt v to, gi tr K
m
ln
lt l 0,42 g/L, 0,15 g/L, 0,20 g/L v 0,10 g/L (Denes et al., 2000, Ly Nguyen et
al., 2002). iu ny chng t, hng s K
m
ty thuc vo ngun gc enzyme,
phng php xc nh hot tnh. So vi gi tr K
m
tm c t PME ngun gc
thc vt, hng s K
m
ca PME t nm mc u c gi tr cao hn. Ni cch khc,
i lc ca PME t nm mc i vi c cht thp hn hay nhy ca PME t
nm mc km hn PME t thc vt (Duvetter, 2007).
ii. nh hng pH n hot ng PME
PME t thc vt rt nhy cm i vi mi trng ion, hu ht cc PME ngun gc
thc vt c pH ti u khong 6,0 8,0. khong pH ny, c s tng tc
mnh m gia enzyme tch in dng v nhm carboxyl t do ca acid
galacturonic, c cht ca enzyme (Ly Nguyen, 2004). Ti mt gi tr pH trn im
ng in pI, PME trng thi trung ha hay mang in tch m, iu ny c
gii thch do PME c thot ra khi c cht nh lc y tnh in gia nhm
carboxyl t do v enzyme mang in tch m (Nari et al., 1991). Ngc li, PME
t vi sinh vt c pH ti u t 4,0 cho n 9,0 (Bordanave, 1996). Tuy nhin, i
vi cc PME t nm mc, pH hot ng ti u dao ng trong khong hp t 4,0
4,5 (Bhalla v Chatanta, 2000).
iii. nh hng nhit n hot ng PME
Ging nh cc phn ng ca enzyme khc, tc ca phn ng phn gii pectin
do PME s gia tng cng vi s gia tng nhit n mt mc ti u, sau hot
tnh ca PME gim. Nhit ti u cho hot ng ca PME dao ng trong
khong t 45 55C, ph thuc vo ngun enzyme. Mc d vy, trong iu kin
thay i p sut, nhit ti u cho hot ng ca enzyme cng thay i (Verlent
et al.,2004; Castro et al., 2006; Sila et al., 2007).
iv. nh hng tion kim loi n hot ng PME
Cc cation kim loi thng c s dng vi vai tr cht hot ha i vi hot
ng ca enzyme (Marangoni, 2003). Cc cation kim loi thng c vai tr rt
cn thit i vi hot ng ca PME t thc vt, t vic m rng vch t bo n
s n nh c tnh cu trc nguyn liu trong qu trnh ch bin thc phm
(Aehle, 2004). Tuy nhin, cation kim loi t nh hng trn hot ng ca PME c
ngun gc t vi sinh vt (Nari et al., 1991; Bordenave, 1996). Nhn chung, hot
ng ca PME gia tng cng vi s tng nng cation kim loi v t n gi tr
ti u, trn im ny hiu qu hot ng ca enzyme thng gim. Trong phn
ng kh ester vi xc tc l PME ngun gc thc vt, cation kim loi phn ng
vi nhm mang in tch m (nhm carboxyl) cho php enzyme phn ng trn
lin kt ester v gii phng n. nng cao, cation kim loi c ch hot ng
ca enzyme. iu ny c hiu da trn nhm carboxyl k cn lin kt ester
c gii phng cho php phn ng tip din. Nu nhng nhm ny c kha
bi cation kim loi, phn ng enzyme khng xy ra v y l nguyn nhn c ch
khi c nng cation kim loi cao (Nari et al., 1991). Nng NaCl b sung ti
u cho hot ng PME l 0,2 M (Hultin et al., 1966; Lee v MacMillan, 1968;
Brady, 1976; Lourenco v Catutani, 1984; Lin et al., 1989). Theo Leiting v
Wicker (1997) cng mc ion ha, cc cation ha tr II kch hot PME khc
nhau. Hiu ng ca cation ha tr II trn hot ng ca PME xut hin phc tp
hn so vi cation ha tr I. Trong s nhng cation ho tr II, Ca
2+
l cation quan
trng nht. Ti nng thp (5 25 nM), Ca
2+
tng kh nng hot ng ca PME
v ngc li - nng cao tc phn ng ca PME gim, c l do s hnh
thnh gel pectate calcium (Leiting v Wicker, 1997). i vi PME c ly trch
t u xanh (Vigna radiata), Mg
2+
l mt trong nhng cht hot ha mnh hn
Ca
2+
(Goldberg et al., 1992).
v. nh hng ht h enzyme (PMEI) n hot tnh PME
Hot ng ca PME b c ch ch yu do tc ng ca glycoprotein c ly trch
t kiwi (Balestrieri et al.,1990; Giovane et al., 2004, Ly Nguyen, 2004). Ngoi ra,
cc nghin cu gn y cn cho thy catechin c trong tr xanh (epigallocatechin
gallate - EGCG) cng c kh nng c ch hot ng ca cc PME t thc vt
(Lewis et al., 2008).
Cht c ch enzyme pectin methylesterase (PMEI) c th phn ng cnh tranh v
ngn cn hot ng ca PME t thc vt, tuy nhin hu nh khng c nh hng
n hot tnh ca PME t vi sinh vt (Ly Nguyen, 2004). Nghin cu ca Giovane
et al. (2004) cho thy PMEI khng c ch c hot ng ca PME c sinh
tng hp t E. chrysanthemi.
Nh kh nng c ch ca PMEI i vi PME t thc vt, vic tinh sch v c c
PME ngun gc thc vt c th c tin hnh theo phng php sc k i lc
(Castaldo et al.,1996; Fachin et al., 2002; Ly Nguyen et al., 2004). Trong khi ,
vic tinh sch PME t vi sinh vt cn phi s dng mt quy trnh hon ton khc
bit.
Nh vy, ty theo ngun gc ca PME, tnh n nh cng nh hiu qu hot ng,
phng thc phn ng ca enzyme cng thay i.
1.2 Tm quan trng ca vic s dng PME trong thc tin
1.2.1 C ng dng bn
Trong nhiu th k qua, enzyme c s dng trong nhiu loi thc phm cng
nh cc ngnh cng nghip khc (Kashyap et al., 2001; Aehle, 2004). Cng ngh
enzyme c bit n vi nhiu li th so vi cc qu trnh ha hc thng thng
nh phn ng da vo c cht c hiu, hot ng di iu kin pH v nhit
n ha, hiu sut chuyn i cao. Ngoi ra, vi c tnh ca protein sinh hc, vic
s dng enzyme trong quy trnh ch bin thc phm v ung gip sn phm
thn thin vi mi trng so vi vic s dng ph gia, ha cht. iu ny c
bit c quan tm trong iu kin thc t v tc hi ca ha cht n mi trng
v bin i kh hu ton cu (Panesar et al., 2010). Nhm enzyme pectinase t vi
sinh vt chim t trng t 13% (Kashyap et al., 2001) ln n 25% (Jayani et al.,
2005) trong s cc enzyme thc phm tiu th trn ton cu.
Cc ng dng cng nghip ln nht v lu i nht ca enzyme PME c bit
n trong lnh vc h tr ch bin, gia tng hiu qu trch ly v lm trong nc
qu (Alkorta et al., 1998; Jayani et al., 2005; Rombouts v Pilnik, 1986). Trong
trng hp ny, PME thng c s dng vi enzyme PG v PL. Khi chit tch
dch bo, mt lng ln pectin s i theo v l nguyn nhn gy c nc qu
cng nh lm tng nht sn phm nu khng c loi ra khi dch qu. Trong
trng hp ny, hn hp enzyme pectinase c b sung vo sn phm. u tin,
PME c tc dng phn ct lin kt ester ca phn t pectin, t to iu kin
cho enzyme PG v PL thy phn lin kt glycosyl ca chui pectin to thnh
nhng phn nh, ri rc dn n d lng cn v thu c dch qu trong (Van
Buren, 1979; Aehle, 2004). Ngoi ra, do phc h protein - pectin trong t bo bao
gm protein mang in dng v lp v pectin mang in m, vi lp v ngoi
tch in cng du nn chng y nhau v trng thi l lng, gy vn c. Khi
x l vi enzyme pectinase, lp v pectin s b phn ct lm cc protein l ra. Khi
, cc ht s ht nhau v lng cn, kt ta (Bali, 2003; hnh 1.2).

Hn 12: Tc dng ca PME trong qu trn lm trong nc qu
Ngun: Bli (2003)
Ishii et al. (1972, trch dn bi Ishii et al., 1979) th nghim v cho thy hiu
qu tng t khi ng dng lm trong nc to bng cch kt hp hai enzyme
PME v PG hoc s dng enzyme PL c lp. Tuy nhin, n nm 1973, Ishii v
cng s (trch dn bi Bali, 2003) li cho thy vic s dng enzyme PL c lp
khng hiu qu bng vic kt hp PME v PG.
Bn cnh , s kt hp ca PME v cc enzyme khc thuc nhm pectinase cng
c s dng trong ch bin cc sn phm c s hin din ca pectin, in hnh
nh sn xut ru vang, ru to hoc mt (Benen et al., 2003; Grassin v
Fauquembergue, 1996; Heldt-Hansen et al., 1996; Kashyap et al., 2001).
Mc d vy, ng dng quan trng nht ca PME trong lnh vc ch bin rau c
qu l gim thiu s mt cu trc ca m t bo do tc ng ca iu kin x l.
Thm vo , PME cn c ng dng quan trng trong lnh vc sn xut thng
mi cc pectin k thut. Chit xut pectin c s dng rng ri trong cc ngnh
cng nghip thc phm ch yu da trn c tnh gel ca pectin. Ty thuc vo
nhu cu s dng khc nhau, cc pectin c methoxyl khc nhau c chn la.
Chnh v th, s kh gc methoxyl cc pectin c methoxyl ha cao s hnh
thnh pectin c methoxyl ha ph hp vi yu cu thc t. Ngoi ra, PME
cng nh enzyme nhm pectinase cn c s dng trong mt s ngnh cng
nghip khc nhm x l ngun nguyn liu giu pectin, v d nh trong ngnh dt,
x l nc thi, ngnh cng nghip giy v bt giy, khai thc du (Jayani et al.,
2005; Kashyap et al., 2001).
1.2.2 ng qun v ng ngh h bin ru qu v s bin i tnh u tr
c ru qu trong qu trnh h bin
Cc loi rau qu nhit i v bn nhit i hin nay l nhng loi thc phm c
tm quan trng cc nc sn xut v c nhng nc khng c iu kin trng
trt. mt vi quc gia, rau qu l ngun thu ngoi t ln (Skrede, 1996). Tuy
nhin, trc khi tip cn vi th trng tiu th, cc loi qu sau thu hoch phi
mt thi gian di vn chuyn, s ch v x l sau thu hoch. So vi cc loi tri
cy vng n i, cc loi tri cy nhit i v bn nhit i thng gp tr ngi
ln trong vic tn tr v vn chuyn do tnh cht d h hng ca chng, do vy
iu kin s ch, x l v vn chuyn n cc nc nhp khu thng km an
ton. Trong cc n lc ci thin cht lng ca sn phm tip cn c vi th
trng, chui dy chuyn cung ng nguyn liu t khu sn xut cho n qu
trnh tiu th cui cng phi c kim tra thng xuyn (Nguyn Vn Mi,
2007).
Tri cy sau khi thu hoch vn cn hot ng sng, cc qu trnh h hp v mt
nc vn c din ra nh cn trn cy, im khc bit ch l hot ng ny xy
ra trong mi trng sau thu hoch (Skrede, 1996). Chnh v vy, nng cao gi tr
thng phm ca rau qu, bn cnh cc bin php s ch v tn tr ti rau qu
sau thu hoch, nghin cu tr ng rau qu v p dng cc qu trnh ch bin khc
nh ng hp, sy kh hay ch bin sn phm mui chua cng c p dng
(Fellow, 2002). Tuy nhin, mt trong nhng bin i khng mong mun ca cc
sn phm x l nhit hay ch bin c nh hng n s chp nhn ca ngi tiu
dng l s mt cu trc t bo - mt trong nhng thuc tnh cht lng quan trng
i vi thc phm c ngun gc thc vt (Van Buren, 1979).
Chnh v vy, nghin cu cc bin php x l thch hp nhm hn ch s mt cu
trc ca t bo rau qu trong qu trnh ch bin lun c cc nh khoa hc quan
tm t nhiu thp k qua (Bell et al., 1950; Brett v Waldron, 1996; Jarvis et al.,
2003; Brummell, 2006).
M t bo rau c l nguyn liu sng, c tnh ti ca nguyn liu ph thuc rt
ln vo s thay i cu trc v thnh phn ho hc ca thnh t bo v gian bo
c bit l ph thuc vo s hin din ca thnh phn pectin. X l nhit (nhit
cao v lnh ng) l nguyn nhn dn n s mt cng do ph v mng v lm
mt s trng phng (Waldron et al., 1997). Tuy nhin, rau qu mm i ng k
khi s phn chia t bo gia tng (Van Buren, 1979; Waldron et al., 2007) do phn
gii pectic polysaccharide.
Cc nghin cu trc ca Bell et al. (1950) cng ch ra rng cng ca rau
mui chua ng hp tr nn mm hn nng acid lactic cao hn. cng nng
, acid lactic c nh hng bt li hn acid acetic i vi cng. ng thi, s
di chuyn Ca
2+
khi c cht pectin lin kt vi m da leo bi acid lactic c
xem l nguyn nhn chnh dn n s mt cu trc m qu, da b mm.
Nhn chung, cc tc ng ln pectin trn vch t bo rau qu v cc yu t nh
hng n s hnh thnh pectate calcium c vai tr quan trng nht trong vic duy
tr c tnh cu trc t bo, ci thin cht lng thc phm c ngun gc thc vt.
1.2.3 i tr petin v PME i vi s i thin tnh u tr th
phm ngun g th vt
i. Gii thiu petin
Pectin l mt phc hp cc polysaccharide, tn ti ph bin trong vch t bo thc
vt, gi vai tr lin kt cc t bo vi nhau (Ridley et al., 2001; Vincken et al.,
2003; Willats et al., 2001; Willats et al., 2006). Pectin chim khong mt phn ba
(1/3) khi lng kh ca vch t bo thc vt bc cao. thc vt, pectin tn ti
ch yu hai dng l pectin ha tan v protopectin khng ha tan. Di tc dng
ca enzyme hay cc phn ng thy phn khng enzyme (acid, tc ng ca qu
trnh gia nhit), protopectin chuyn thnh pectin (Waldron et al., 1997).
Cu trc phn t pectin do cc n phn D-galacturonic acid lin kt vi nhau
bng lin kt o - 1,4 - glucoside (Mukhiddinov et al., 2000). Pectin cha t vi
trm cho n mt ngn n v glucose, khi lng phn t trung bnh t 50.000
150.000 Daltons (Sriamornsak, 2002). Cc nhm carboxyl ca acid galacturonic
ny hoc b ester ha t nhin bi methyl, hoc b x l ha hc vi nhm amin
to thnh carboxamides (hay amino carbonyls) (hnh 1.3).
Tnh cht ca pectin c c trng bi hai ch s quan trng l: MI - ch s
methoxyl ha, biu hin cho phn trm khi lng nhm methoxyl (-OCH
3
) c
trong phn t pectin v DE - ch s ester ha, th hin mc ester ha ca phn
t galacturonic acid trong phn t pectin (Waldron et al., 1997; Nguyn c
Lng, 2004).

Hnh 1.3: Cu trc pn t pectin
()Phn t pectin (b) g carboxyl, (c) g ester, (d) amin
Ngun: rimornsk (2002)
Pectin thng mi c chia thnh hai loi:
- Pectin c methoxyl ha cao (High Methoxyl Pectin - HMP): DE > 50% hay
MI > 7%. Cht ny c th lm tng nht cho sn phm. Tc dng to gel ca
pectin nhm ny ch xy ra pH = 3,1 3,4 v nng ng trn 60%.
- Pectin c methoxyl ha thp (Low Methoxyl Pectin - LMP): DE < 50% hay
MI < 7%. Pectin ny c to thnh bng cch tch nhm methoxyl trong phn t
pectin. Pectin c methoxyl ha thp ch to gel trong mi trng khng c
ng v thng ng dng ph bin lm mng bao sn phm (Nguyn c
Lng, 2004).
ii. Cu tr th phm ngun g th vt
Cu trc l mt trong nhng thuc tnh cht lng quan trng i vi thc phm
c ngun gc thc vt. C nhiu khi nim c dng miu t tnh cht cm
quan v cu trc ca rau v tri nh cng, mm, gin, dai, ging bt, kh, mng
nc (Waldron, 1997). Cc cp cu trc c m t trong hnh 1.4.

Hn 14: S m t cc cp cu trc ca tc pm ngun gc tc vt
Ngun: Wldron et al., (1997)
Cp thp nht, c bn nht trong cu trc ca thc vt chnh l cc polymer
tham gia vo to nn vch t bo, bao gm cellulose, hemicellulose v pectin.
Cch sp xp v lin kt vi nhau ca cc phn t polysaccharide trong mng t
bo hay cc vi si phc tp ca cellulose, c v l hc v ha hc, quyt nh ln
i vi c tnh bn c hc ca vch t bo (Waldron et al., 1997; hnh 1.5).

Hn 15: M n cu trc ca vc t bo tc vt bc cao
Ngun: Dvidson, (2005); URL: http://micro.magnet.fsu.edu/cells/plants/cellwall.html
Cc lin kt c th b nh hng bi dy ca vch t bo v cc c tnh cu
trc bn trong (Jarvis et al., 2003). cng ca mi t bo chu nh hng bi
n hi ca vch t bo, c lin quan n s thay i p sut thm thu. p sut
thm thu l kt qu ca s thm cc thnh phn t dung dch c nng cht tan
cao vo trong t bo bi tnh bn thm ca mng t bo. p sut thm thu l yu
t quan trng i vi s gin n ca t bo, nh l kt qu ca s trt ln nhau
ca cc polymer trong vch t bo (Duvetter, 2007).
Cp th hai ca cu trc khc nhau hnh dng, kch c v thnh phn ca t
bo, ty thuc vo chc nng ca chng. Cc t bo c th lin kt vi nhau qua
phin gia vi phm vi v mc gn cht khc nhau to nn cc m. Cc t bo
v cc khong gian bo (nc hoc khng kh) c sp xp vo trong cc m;
hnh dng, s sp xp ca cc m to nn cp k tip ca cu trc (Jarvis et al.,
2003).
Cu trc ca m t bo thc vt b sp khi p sut thm thu b mt. Mc nhy
cm vi s mt cu trc ph thuc vo cc yu t nh cng ca vch t bo,
lc lin kt gia cc phin gia trong t bo v thnh phn ca t bo (Hounsome
et al., 2008). duy tr hnh dng ban u ca rau qu sau khi ch bin, vic tin
x l nhm lm tng cng chc ca cu trc thng c p dng. Mt trong
nhng phng php thch hp l s dng calcium nhm cng c t bo rau qu
nh vo s thay i cu trc pectin (Poovaiah, 1986; Garcia et al., 1996). Thm
vo , PME c th c s dng xc tc cho s phn tch lin kt ester ca
pectin, hnh thnh nhm COO
-
, nh ion Ca
2+
c th hnh thnh cu ni to
pectate calcium (hnh 1.6).

Hn 16: S biu din cc cuyn a ca pectin di tc ng
ca enzyme ty pn n ng n cu trc t bo
Ngun: Duvetter, (2007)
Pectate calcium
thit lp c s hnh thnh phc hp pectate calcium, ngn cn s ph hy
cu trc ca t bo thc vt, cn phi c s hiu bit c th hn v thnh phn
pectin trong vch t bo thc vt cng nh nh hng ca tnh cht ca pectin n
kh nng hnh thnh gel (Duvetter, 2007).
iii. C huyn h petin nh hng n tnh u tr t bo ru qu
S thay i v cu trc t bo rau qu trong qu trnh x l nhit hoc qu trnh
chn c lin quan cht ch vi kh nng ha tan v trng thi tn ti ca cc pectic
polysaccharide trong phin gia ca vch t bo. Cc chuyn ha ca pectin nh
hng n c tnh cu trc c mi lin quan vi cc enzyme nh protopectinase,
PG, PME v cc enzyme lyase (Brett v Waldron, 1996; Brummell, 2006; Jarvis
et al., 2003). Da vo tc ng ca h enzyme, cc chuyn ha ca pectin c th
chia thnh hai hng c bn l s ha tan v s n phn ha (Brett v Waldron,
1996).
S chun ha pectin khng ha tan v pectin ha tan
Trong tri cy sng, pectin trng thi lin kt vi cc vi si cellulose trong vch
t bo, c gi l protopectin v khng tan c trong nc, lm cho vch t bo
cng chc. Tuy nhin trong qu trnh chn ca tri, protopectin c chuyn ha
thnh pectin ha tan nh s xc tc ca enzyme protopectinase. Enzyme ny c
th c hai kiu hot ng: mt l tc ng vo vng polygalacturonic acid ca
pectin, hai l tc ng vo cc lin kt gia pectin vi cellulose, t lm thay
i cu trc ca pectin, tng kh nng ha tan ca pectin trong nc v chnh v
vy lm cho m thc vt mm i. Khi rau qu b nu chn, protopectin cng b
thy phn thnh pectin ha tan, kt qu l lm mm rau qu. Trong trng hp
ny, vic thit lp phc hp pectate calcium do phn ng gia thnh phn pectin
ha tan vi ion Ca
2+
rt cn thit. y l dng mui khng ha tan trong nc v
khi phc hp ny c to thnh s lp y cc m rau qu lm cho cu trc sn
phm tr nn rn chc hn (Van Buren, 1979; Brummell, 2006).
S n phn ha phn t pectin
Enzyme PG l enzyme chu trch nhim quan trng nht trong s n phn ha v
lm tan cc pectic polysaccharide. Enzyme PG thy phn lin kt glycosidic gia
2 n v GalA cha ester ha. V th hot ng ca enzyme ny i hi phn t
pectin phi c kh gc methoxyl trc nh vo hot ng ca PME v enzyme
-galactosidase gii phng gc galactose cui cng ra khi mch bn galactan.
Vic c ch hot ng ca enzyme PG trc khi chn c th lm gim ng k s
mm i ca qu, nh vy vic loi b mch bn galactan ca pectic c ngha
quan trng trong qu trnh chn v to ra xp v to iu kin thun li cho cc
enzyme gim cp tip cn vi c cht (Brummell, 2006). Bn cnh vic lm gim
s kt dnh ca t bo, cu trc ca tri cy chn cn c th nh hng bi s thay
i mc trng phng do mng t bo b ph hy v b kh nc (Waldron et
al., 1997; Brummell, 2006).
Nh vy, c rt nhiu thay i xut hin rau v qu trong qu trnh chn v trong
cc cng on ch bin sau thu hoch. Cc thay i ny c th c nh hng t
nhiu ln c tnh cu trc ca sn phm. S phn gii ca pectin lin quan n s
xc tc ca ion hydroxyl ph v lin kt glycosidic di iu kin khng acid v
thng xy ra trong ch bin nhit (Bemiller v Kumari, 1972).
Mc phn gii ph thuc ch s ester ha (DE) ca pectin - DE cng cao th
mc phn gii cng cao (Sajjanantakul, 1993). Pectin c methoxyl ha thp
bn vng hn nhit cao so vi cc pectin c methoxyl ha cao. Khi pectic
acid thiu nhm methyl ester C
6
th n c t proton acid C
5
. Hn th na, in
tch ca nhm carboxyl ngn cn s tip cn ca ion hydroxyl, s phn gii pectin
chu nh hng ln ca pH v c xu hng tng khi mi trng kim v c th
c kch thch bi nhng ion khc (Krall v McFeeters, 1998).
Chnh v th, vic nghin cu phng thc iu khin pectin trong t bo ph hp
cho s hnh thnh mng pectate calcium, gip n nh cu trc sn phm sau ch
bin c lin quan mt thit n hot ng ca PME.
iv. ng dng PME ngoi bo trong i thin u tr ru qu
Trong cc sn phm ch bin t rau qu i hi tnh gin, cng chc nh trong
sn phm rau mui chua, to ct lt, rau qu ng hp,Qu trnh tin x l
nguyn liu nhit thp, thi gian di c b sung dung dch mui CaCl
2
, s
lm kch hot PME ni bo phn ct nhm ester ca pectin, to iu kin cho
pectin lin kt vi ion Ca
2+
to cu trc theo mong mun.
i vi mt s loi rau qu cha rt t PME ni bo, ng thi vic tin x l
nhit n ha (50 70C) c th lm hot ha mt s loi enzyme khng mong
mun, vic b sung PME ngoi bo nh PME ly trch t thc vt hay PME t vi
sinh vt v ion Ca
2+
iu kin nhit thch hp c thc hin (Baker v
Wicker, 1996). PME ly trch t c chua, PME thu nhn t A. aculeatus l hai
enzyme in hnh c ng dng trong vic ci thin c tnh cu trc t bo rau
qu (Duvetter, 2007). cng ca du ty sau x l nhit c ci thin ng k,
ch gim khong 30% so vi ban u, trong khi mu khng c tin x l th
cng gim n 80% sau khi gia nhit nh tin trnh tin x l qua hai bc (i)
ngm trong chn khng vi dung dch PME c tng hp t A. aculeatus v
calcium, sau (ii) tin x l nhit n 60 80C (Duvetter, 2007).
Vic b sung kt hp PME t nm mc v ion Ca
2+
cng mang li hiu qu ci
thin cng cho du ty ct i v lnh ng (Suutarinen et al., 2000;
Suutarinen et al., 2002), to (Degraeve et al., 2003), c tm ct lt (Banjongsinsiri
et al., 2004) v c bit l xoi lnh ng (Sirijariyawat et al., 2012). Vic duy tr
cu trc ca da leo mui chua cng l mt vn quan trng cho ngnh cng
nghip ln men thng mi. Nghin cu v vn ny c bt u t rt sm
(Bell et al., 1950) v ngy cng c quan tm pht trin. Tang v McFeeters
(1983) bc u nghin cu tc ng ca vic ci thin cu trc da leo mui
chua da trn s thc y hot ng ca PME, lm thay i ester ha ca
pectin v hnh thnh pectate calcium. Tuy nhin, da leo ch cha mt lng rt
nh PME ni bo (10
-2
3.10
-2
U/g) (Bell v Etchell, 1951). Do nhiu phng
php khc nhau c ngh b sung enzyme vo m thc vt nguyn vn
(Baker v Wicker, 1996). y cng chnh l c s cho vic nghin cu b sung
PME ngoi bo nhm ci thin cu trc da leo mui chua trong iu kin hin ti
Vit Nam. Hn th na, vi tnh cht ca mt PME acid, PME t nm mc hot
ng c hiu qu hn PME t thc vt cc sn phm rau qu (Duvetter, 2007;
Sirijariyawat et al., 2012). y cng l nn tng cho vic nghin cu sinh tng
hp PME t vi sinh vt.
1.3 Vai tr ca vic thu nhn PME t nm mc Aspergillus niger c hiu
1.3.1 m qun trng vi thu nhn PME t nm m A. niger
Trong hu ht cc loi cy n tri u c s hin din ca PME vi hm lng
khc nhau. Vic ly trch PME t thc vt cng c tin hnh trn nhiu loi qu
nh c chua (Giovane et al., 2004), c rt en (nal v Bellur, 2009) hay b
(Awad v Young, 1980). Mc d vy, vic s dng vi sinh vt lm ngun nguyn
liu sn xut enzyme ang rt c quan tm. Hin c hn 50% tng lng
enzyme cng nghip c sn xut t nm men v nm mc, mt phn ba c
tng hp t ngun vi khun v phn cn li t thc vt, ng vt (Bhalla v
Chatanta, 2000).
Cc vi sinh vt c kh nng tng hp pectinase ch yu l vi sinh vt hiu kh, tiu
biu l cc dng sau:
- Nm men: Saccharomyces fragilis (Prade et al., 1999)
- Nm mc: Aspergillus sp., Penicillium glaucum, Botrytis cinerea, Fusarium
moniliforme, Rhizoctonia solani, Rhizopus stolonifer, Trichoderma sp.,
Neurospora crassa, (Reignault et al., 1994 ; Joshi et al., 2006).
- Vi khun: Bacillus polymyxa, Bacillus felineus, Flavobacterium pectinovorum,
Klebsiella aerogenes, Erwinia carotovora,... (Duvetter, 2007).
S phn b cc kiu enzyme pectinase cc vi sinh vt khng ging nhau, c c
th ch to ra c mt enzyme, song a s c th khc li to ra c mt phc
h enzyme pectinase khc nhau. Saccharomyces fragilis l v d in hnh ca vi
sinh vt loi th nht; c th ny hu nh ch to c mt enzyme phn gii
pectin duy nht l endo polygalacturonase (Prade et al., 1999). Cc nm mc
A. niger, A. awamori, A. saitoi, A. fonseanes, B. cinerea thng to ra c mt
phc h cc enzyme pectinase khc nhau (Panesar et al., 2010).
Mc d c nhiu nghin cu cho thy hiu qu ca qu trnh sinh tng hp
enzyme khc nhau t A. niger nh -amylase (EC 3.2.1.1), -galactosidase (EC
3.2.1.23), glucooxidase (EC 1.1.3.4), PG (EC 3.2.1.15) (Bhalla v Chatanta,
2000), tuy nhin PME l enzyme c sinh tng hp ch yu trong nhm
pectinase t A. niger (Prade et al., 1999). Cc nghin cu v vai tr ca dng nm
mc n kh nng sinh enzyme cng xc nhn hot tnh ca PME thu c t
A. niger mnh hn hn A. oryzae cng nh cc dng nm mc khc (Linde et al.,
2007). Hn th na, A. niger c xc nhn l dng nm c c tnh an ton
(GRAS), d pht trin v phn lp (Degraeve et al., 2003).
Tuy nhin, cc nghin cu thu nhn enzyme ch tp trung ch yu vo vic kho
st iu kin mi trng nui cy, c cht c hiu v phng php ln men (Bali,
2003; Hamdy, 2005), vn tuyn chn chng da trn ngun gc phn lp ca
chng c tc ng n hiu qu sinh PME vn cha c quan tm ng mc.
Cc nghin cu gn y cho thy s gia tng hiu qu thu nhn enzyme
pectinase ni chung v PME ni ring t cc dng A. niger c ngun gc t
nguyn liu giu pectin nh cam (Rogaiza Al - Gasgari, 2002), xc qu c ph, v
qu thanh long v v qu chanh (L Th Thanh Hng v Nguyn Thy Chu,
2005). Kt qu ny chnh l nn tng cho nghin cu chuyn su v tuyn chn
dng nm mc A. niger c hiu cho qu trnh sinh tng hp PME t hiu qu
cao trong iu kin Vit Nam.
1.3.2 i tr ht petin i vi s pht trin nm m sinh tng hp
enzyme PME
Cc loi nm mc trong t nhin c kh nng thch ng cao vi iu kin sng.
Vi mi ngun c cht nht nh, chng s sinh tng hp enzyme tng thch, c
hiu vi c cht nhm chuyn c cht t dng phc tp kh hp thu thnh dng
c cht n gin d hp thu. Chng hn nh, mi trng giu c cht pectin l
ng lc cho vic tit enzyme ngoi bo tng thch l pectinase (Nguyn c
Lng, 2004). Ni cch khc, mun thu nhn enzyme no th phi c s hin din
ca c cht c hiu vi enzyme trong mi trng nui cy (Pilnik v
Rombouts, 1981). Pectin c methoxyl ha cao, polyglacturonic acid, c
bit n l cht nn quan trng cho hot ng ca PME (Pilnik v Rombouts,
1981). Tc ng ca c cht c hiu t hiu qu cao khi mt nng nht
nh. Nu vt qu nng ti a cho php th kh nng sinh tng hp s gim
(Pilnik v Rombouts, 1981). Do vy, pectin c mt ngha c bit quan trng
i vi s pht trin ca nm mc sinh tng hp PME.
1.3.3 Ngun nguyn liu ho qu trnh phn lp v tuyn hn A. niger sinh tng
hp PME hot tnh o
Vi mc tiu to ra cc dng A. niger c hiu cho qu trnh ln men sinh tng
hp PME tip theo t hiu qu, vic phn lp dng A. niger t cc nguyn liu
giu pectin c ester ha cao c quan tm. Aehle (2004) cng b kt qu
xc nh thnh phn pectin v mc ester ha ca pectin ca mt s loi tri cy
ph bin (bng 1.1).
Bng 11: Thnh phn pectin v mc ester ha ca pectin mt s loi tri c
Loi tri cy Hm lng pectin %
*
Mc ester ha (DE) %
To
Nho
V qu h citrus
L
Khm
Du ty
0,5 1,6
0,1 1,4
3,5 5,5
0,7 0,9
0,04 0,1
0,5 0,7
80 92
50 65
65
50 70
22 40
20 60
*: % theo khi lng ti hy n bn t
Ngun: Aehle, (2004)
Da vo kt qu xc nh thnh phn pectin v mc ester ha ca pectin tng
ng, v qu h citrus (cam, chanh, hnh,), to, nho l ngun l tng cho vic
s dng lm c cht c hiu i vi qu trnh phn lp v sng lc dng c hot
tnh PME cao (Joshi et al., 2006; Huang et al., 2011; Ali et al., 2011).
Bn cnh , v qu bi c bit n l thnh phn cha hm lng pectin cao,
trong hm lng pectin ha tan trong nc (c DE cao, khong 69,32
78,68 %) chim n 8,12 10,87% v pectin ha tan trong oxalate (c DE
thp, t 21,01 55,41%) chim khong 4,89 8,62% (Chaidedgumjorn et al.,
2009). Mt nghin cu trc ca Phuangsinou et al. (2008) cho thy, mt
ging bi lai to ti a phng (Thi Lan) c th c DE rt cao (91,4%).
Thm vo , b mt v qu bi cn l mi trng thch hp cho s pht trin
ca mt s loi nm mc, trong c A. niger - y cng l nm mc c tuyn
chn cho kh nng sinh enzyme pectinase hot tnh cao (L Th Thanh Hng v
Nguyn Thy Chu, 2005).
1.4 Tng quan v nm Aspergillus niger
1.4.1 Gii thiu hung
Aspergillus niger l loi ph bin nht trong chi Aspergillus, phn b rng ri trn
cc c cht t nhin, trong cc sn phm nng cng nghip v nhiu vng a l
khc nhau trn th gii. Hin nay, A. niger c s dng ch yu trong cng
nghip sn xut enzyme (in hnh nh - amylase, glucoamylase, pectinase,
protease, cellulase), trong cng nghip ch bin thc phm, cng nghip sn xut
mt s acid hu c nh acid citric, acid gluconic,(Pansear et al., 2010)
Nm mc l vi sinh vt c thay i ng k trong lch s phn loi. Cc nghin
cu khi u phn chia nm thuc ngnh Thallophyta thuc gii ph
Cryptogamae, gii Plantae (thc vt). Cc kho st v c tnh cu trc di knh
hin vi tip theo chng minh nm khng thuc thc vt ln ng vt (Sumbali,
2005). Samson v van Reenen-Hoekstra (1988) phn chia nm tht thnh 4 lp:
Phycomycetes, Ascomycetes, Basidiomycetes v Deuteromycetes. H thng phn
loi ny tn ti mt thi gian di v A. niger c phn loi thuc gii nm
(Fungi), ngnh ph nm bt ton Deuteromycotina, lp Deuteromycetes, b
Moniliales, h Moniliaceae, chi Aspergillus v loi Aspergillus niger (Samson v
van Reenen-Hoekstra, 1988).
Tuy nhin, vi s tin b ca khoa hc k thut, cc nghin cu thp nin 70
ca th k XX, bt u t nghin cu phn chia nhm ca Whittaker (1969, trch
dn bi Sumbali, 2005), n gii (Alexopolous v Mims, 1979; trch dn bi
Sumbali, 2005) ngh kha phn loi mi cho gii nm ni chung v
A. niger ni ring thuc:
Ngnh: Amastigomycota
Ngnh ph: Ascomycotina
Lp: Ascomycetes
Lp ph: Plectomycetidae
B: Eurotiales
H: Eurotiaceae
Ging: Aspergillus
Loi: Aspergillus niger
Ngun: umbali, (2005)
Vi c im chnh trong h Eurotiaceae l bo t nh dng th bnh (Cao
Ngc ip v Nguyn Vn Thnh, 2010).
1.4.2 im h si nm Aspergillus niger
Khun ty ca nm ch tng trng ngn v c vch ngn, gm hai loi:
- Khun ty dinh dng: khun ty pht trin trong c cht, c kch thc nh, mu
trng. Cc khun ty bn cht thnh mt khi rt dai, n su vo mi trng nui
cy ht dng cht. Khi gi h si ng sang mu vng.
- Khun ty sinh sn: khun ty pht trin trong khng kh, c kch thc ln hn
khun ty dinh dng rt nhiu, trong sut. Khun ty sinh sn hng vo khng kh
ly oxy, c kh nng to bo t khi gi.
Tm li, khi gi h si c nhiu bin i: mt s khun ty dinh dng to thnh
cc hch nm lm h si ng sang mu vng. Trong khi , khun ty sinh sn hnh
thnh cc bo t nh lm cho b mt khun lc c mu en.
(Ngun: Klich, 2002; Samson et al., 2007)
1.4.3 Cu to qun sinh sn
Aspergillus niger l loi nm khng c giai on sinh sn hu tnh, A. niger sinh
sn v tnh ch yu bng bo t nh (Gams, 1985) (hnh 1.7).

Hnh 1.7: Hnh thi Aspergillus sp.
() khi bo t nh, (b) bo t nh, () t bo g, (d) ung th bnh v th bnh
Ngun: Onion et al., (1981)
C quan sinh sn c dng nh hoa cc, gm cc phn: bo t nh pht trin t
mt t bo c ng knh ln hn, mng dy hn cc on ln cn ca h si nm
gi l t bo gc. T t bo gc to thnh si cung khng vch ngn, ko di vi
phn nh phng to to thnh ti hnh cu, khng mu cho n vng nht. Xung
quanh b mt ti l cc cung th bnh mu nu, di 10 15 m, l ni sinh ra cc
th bnh. Th bnh c mt tng hoc hai tng. Cc bo t nh c to thnh ni
tip nhau trong ming th bnh thnh chui hng gc, khng phn nhnh (bo t
ngay ming th bnh l bo t non nht, cng xa ming th bnh l bo t cng
gi). Bo t nh hnh cu, thng dt, ng knh 4 5 m, nhng thng nh
hn (Onions et al., 1981). Loi A. niger c phn bit vi cc loi khc trong chi
Aspergillus bi khi bo t nh mu en.
1.4.4 Chuyn lun phn loi Aspergillus niger
Tn Aspergillus do Micheli t ra ln u tin nm 1729, dng m t mt loi
nm mc c h bo t nh (Micheli, 1729, trch dn bi Samson, 1994). Nm
1926, chuyn lun phn loi ging Aspergillus u tin mi c hnh thnh. c
im dng phn loi Aspergillus gm: mu sc khi bo t; mu sc v b mt
si cung; hnh dng ca ti; s hin din ca cung th bnh. Da vo cc c
im ny, ging Aspergillus chia thnh 13 nhm. Tuy nhin, c im hnh thi v
sinh l ca A. niger thay i rt nhiu khi chu tc ng ca mi trng sng
(Samson, 1994). Do c nhiu chuyn lun phn loi A. niger ln lt ra i.
Thom v Raper (1945, trch dn bi Raper v Fennell, 1965) chia nhm A. niger
gm 17 loi: 8 loi thuc A. niger (c cung th bnh v th bnh), 4 loi thuc
A. carbonarius v 5 loi thuc A. luchuensis (ch c th bnh). Tuy nhin, 2 trong
s 5 loi ch c th bnh l A. luchuensis v A. subfuscus c Raper v Fennell
(1965) phn loi li thnh 2 loi A. awamori v A. foetidus var. Pallidus - c
cung th bnh v th bnh. Hai nh nm hc ny phn chia nhm ny thnh 12
loi, vi 4 loi mi l A. tubingensis, A. ficuum, A. ellipticus v
A. heteromorphus. Al-Musallam (1980) tip tc phn loi vi s lng l 7 loi.
Samson (1994) phn chia nhm ca A. niger thnh 6 loi da vo c im hnh
thi.
1.4.5 nh dnh Aspergillus niger theo phng php truyn thng
Phng php truyn thng nh danh vi sinh vt thng da vo cc ch tiu
phn loi nh: hnh thi, sinh l, c im bin dng (Bukov et al., 2007; Zhao
et al., 2009).
Vic phn lp c tin hnh bng cch s dng mi trng c trng cho dng
A. niger, thng qua hai bc (i) Phn lp s b cc nm mc en trn mi trng
thch khoai ty (Potato dextrose agar, PDA), k n (ii) S dng mi trng
Czapek Dox Agar (CZ) v Malt Extract Agar (MEA) tch rng A. niger da
trn kha phn loi ca Klich (2002) (xem ph lc 1, phn 2).
Cc quy tc chnh trong tin trnh phn lp v nh danh da trn c im i th,
vi th:
- Dng nm mc tuyt i thun khit (khng ln nm mc v cc vi sinh vt
khc).
- S dng mi trng nui cy, nhit v thi gian nui cy ng qui nh ca
cc chuyn lun phn loi i vi tng ging (hoc tng nhm loi ca ging).
- Quan st v ghi chp y cc c im phn loi ca dng nm mc cn phn
loi (c im khun lc trn a petri, c im vi hc).
- Dng chuyn lun phn loi bc cao (ngnh, ngnh ph, lp, b, h) nh loi
n ging, sau dng chuyn lun phn loi ca ging xc nh nh loi
n loi (v th nu c).
(Bi Xun ng v Nguyn Vn Huy, 2000; Klich, 2002; Samson et al., 2007;
Bukov et al., 2007)
C b th hin hnh trong tin trnh phn lp:
Bc 1: Tm nhm phn loi c trong cc kha phn loi bng mt s c im
quy nh trong cc kha phn loi . Tip theo dng kha phn loi trong cc
chuyn lun phn loi ca chi xc nh loi. Khi xc nh tm thi, tin
hnh so snh tt c cc c im kho st ca dng nm A. niger vi cc c
im tng ng ca loi trong chuyn lun. Trng hp khng ph hp, cn tin
hnh li t u n khi c kt qu.
Bc 2: So snh dng nm A. niger va xc nh v cc c im chnh ca
khun lc, cc c im vi hc chnh ca dng nm mu. (Bi Xun ng v
Nguyn Vn Huy, 2000; Klich, 2002; Samson et al., 2007; Bukov et al., 2007).
1.4.6 nh dnh nhm loi Aspergillus bng phng php sinh h phn t
Ngy nay, vi s pht trin ca cc k thut sinh hc phn t hin i vic phn
loi nm men v nm mc cng ci tin hn, s chuyn nh danh nm men v
nm mc t cc c im hnh thi, sinh l, sinh ha sang phng php nh danh
da trn vt liu di truyn.
S phn tch vng trnh t D1/D2 ca rRNA tiu n v ln (LSU-rRNA;
25/26/28S) v/hay vng trnh t ITS-5,8S rRNA-ITS c th nh danh nhng loi
c mi quan h gn gi vi nhau (hnh 1.8).

Hn 18: Cu trc ca trn t m o co rRNA sin vt nn tt
Ngun: ugit & Nishikw, 2003
Do , vic gii trnh t on gene c trng ny c p dng trong phn loi
nm ti mc loi (Mirhendi et al., 2007; Etienne et al., 2009; Sugita & Nishikawa,
2003).
Nhiu nghin cu xc nh trnh t on mi thch hp khuch i on DNA
mc tiu i vi Aspergillus sp. c cng b. Trong , on mi IST1/NIG,
IST1/IST2 (Accensi et al., 1999) v IST1/IST4 (Henry et al., 2000; Tarai et al.,
2006) c s dng ph bin khuch i on gene trn vng trnh t ITS -
5,8S rRNA-ITS ; UNIR - 26/UNIF-26 (Xia et al., 2009) v U
1
/U
2
khuch i on
gene 260 bp (Sandhu et al., 1995; Nganou et al., 2012) trn vng D1/D2 ca 28S
rRNA.
Phng php gii trnh t vng D1/D2 ca rRNA tiu n v ln (LSU-rRNA;
25/26/28S) v/hay vng trnh t ITS-5,8S rRNA-ITS (Henry et al., 2000;
Hinrikson et al., 2005; Mirhendi et al., 2007; Etienne et al., 2009; Xia et al.,
2009; Nganou et al., 2012) kt hp vi vic xy dng cy pht sinh chng loi
hin nay tr nn kh ph bin nh danh nhanh cc dng nm men v nm
mc, c xy dng da trn s kt hp gia sinh hc phn t vi phn tch trn
my tnh. Nhng trnh t mi ny s c so snh vi nhng trnh t trong ngn
hng d liu gene bng cch s dng cng c BLAST (Hinrikson et al., 2005;
Mirhendi et al., 2007).
1.5 Phng php tun chn dng nm mc Aspergillus niger c kh nng
sinh tng hp PME
1.5.1 L thuyt hung
Vic sng lc cc dng vi sinh vt c hot tnh sinh hc mong mun thng ph
thuc vo tng sn phm c th. i vi trng hp tuyn chn dng A. niger
sinh enzyme hot tnh cao c th tin hnh bng phng php thi thch, c l
trn mi trng thch c c cht cm ng. Sau khi trong t m vi nhit
thch hp, tin hnh o ng knh ca vng phn gii (L Th Thanh Hng v
Nguyn Thy Chu, 2005; Khairnar et al., 2009).
Trong trng hp cc dng c phn lp v tuyn chn t t nhin c hiu sut
sinh tng hp enzyme mong mun thp, phi nng cao nng sut ging bng cc
bin php di truyn. Phng php c hiu qu tt nht trong vic chn lc ging
nng sut cao l chn lc gy t bin di tc ng mnh ca cc tc nhn nh
tia UV, tia X, tia Gamma hoc s dng cc ha cht gy t bin nh ethylenimin,
acid nitric, nitrosoguanidin, (L Gia Hy v Khut Hu Thnh, 2010). Tuy
nhin, vic p dng phng php t bin gene chn ging vi sinh vt sinh
enzyme thng khng nhiu khi so snh vi vic tuyn chn cc dng vi sinh vt
c hot tnh khng sinh. Trng hp chn lc t nhin trn mi trng c hiu
hay t bin t nhin da trn s cnh tranh ca cc dng vi sinh vt sinh enzyme
thng c p dng (Zhong - Tao, 2009).
1.5.2 uyn hn dng Aspergillus niger sinh tng hp PME theo phng php
sng l t nhin
Qu trnh tuyn chn dng A. niger c kh nng sinh tng hp PME thng c
tin hnh theo ba bc:
(i) Tuyn chn s b dng A. niger sinh tng hp pectinase hot tnh cao bng
cch s dng mi trng pectin 0,5% v agar 2% (Khairnar et al., 2009)
o kch thc vng pectin bng thuc th Lugol (iodine 2%) sau thi gian 48
gi, 30 2C vi i chng l nc.
- Kch thc vng 20 mm: hot tnh pectinase cao (H);
- Kch thc vng 15 cn 20 mm: hot tnh trung bnh (M);
- Kch thc vng 8 15 mm: hot tnh pectinase yu (W)
(ii) Tuyn chn dng A. niger c hiu vi PME bng cch s dng mi trng
pectin 0,5% v agarose 0,5% c b sung khong (Khanh et al., 1991).
(iii) Ln men b mt hay ln men rn trn mi trng c c cht cm ng
Qu trnh tuyn chn thng c thc hin bng cch s dng mi trng
Czapek c b sung pectin c ester ha cao (1% pectin), tin hnh 30C vi
thi gian 7 ngy (Sharma et al., 2011).
Hiu qu ca qu trnh ln men sinh PME cng nh cc enzyme khc ph thuc
vo nhiu thng s nh vi sinh vt, thnh phn c cht, cc vi cht dinh dng v
iu kin ln men. Sau qu trnh tuyn chn vi sinh vt c hiu, thnh phn c
cht v iu kin ln men l nhng nhn t thit yu, c nh hng ng k n
hiu qu ca qu trnh sinh tng hp enzyme mong mun (Patil et al., 2006).
1.6 nh hng ca thnh phn mi trng n qu trnh sinh tng hp
PME
1.6.1 i tr ht trong qu trnh ln men sinh tng hp PME
Thnh phn mi trng l yu t c bn nht quyt nh kh nng sinh tng hp
PME cng nh cc enzyme khc t vi sinh vt. Trong thnh phn mi trng phi
c cc cht m bo c s sinh trng bnh thng ca vi sinh vt v tng
hp enzyme (L Gia Hy v Khut Hu Thnh, 2010; bng 1.2).
Bng 12: Cc cht dinh dng a lng, vi lng, ngun gc v chc nng i vi
t bo nm mc Aspergillus sp.
Nguyn t Ngun gc Chc nng
Thc n a lng
Carbon Hp cht hu c hoc CO
2
Xy dng thnh phn vt cht c bn ca
t bo
Oxygen H
2
O, hp cht hu c, CO
2
,
nc

Xy dng nn vt cht v nc trong t
bo, O
2
l cht nhn in t trong h hp
hiu kh
Nitrogen NH
3
, NO
3
-
,
hp cht hu c,
N
2

Xy dng nn acid amin, nucleotide ca
acid nucleic v coenzyme
Hydrogen H
2
O, hp cht hu c, H
2
Xy dng nn cc hp cht hu c.
Tham gia cc qu trnh sinh nng lng
nh cc proton
Phospho Phosphate v c Xy dng nn cc acid nucleic,
nucelotide, phospholipid, acid teichoic
Thc n vi lng
Lu hunh SO
4
2-
,
H
2
S, SO
2
, hp cht
hu c cha lu hunh
Xy dng nn cystein, methionine,
glutathione v nhiu coenzyme
Kali Mui kali Xy dng nn cc cation v c ca t
bo v l ng nhn t ca cc enzyme
Magie Mui magie Dng cc cation v c ca t bo v l
ng nhn t cho nhiu phn ng
enzyme
Calcium Mui calcium Cation v c l ng nhn t cho nhiu
enzyme v l cu phn ca ni bo t
St Mui st Cu phn ca cytochrome v cc protein
khc cng l ng nhn t cho mt s
phn ng enzyme
Ngun : L Gia Hy v Khut Hu hnh, (2010)
Trong , ngun carbon l thnh phn u tin, ng vai tr c bit trong s pht
trin ca vi sinh vt. i vi qu trnh sinh tng hp PME, cng nh cc enzyme
khc trong nhm pectinase, ngun carbon giu pectin c methoxyl ha cao v
n phn t nh acid pectic, D-galacturonate v mt s ngun carbohydrate khc
thng c s dng (Sajjaanantakul v Pitifer, 1991). Cc thnh phn ny khng
ch ng vai tr n thun l ngun carbon cho s pht trin ca vi sinh vt, m
cn l mt c cht cm ng, xc tc cho qu trnh sinh tng hp PME
(Sajjaanantakul v Pitifer, 1991; Nguyn c Lng, 2004; Joshi et al., 2006).
Tuy nhin, vic s dng c cht pectin t ngun ph phm nng nghip c a
chung nht (Patil v Dayanand, 2006). Hm lng pectin cao (15 25%) trong
cc ph phm nng nghip nh cm m (Taragano et al., 1997), b ma, b c ci
ng (Solis-Pereira et al., 1993, 1996), xc u c ve (Boccas et al., 1994), v
chanh (Larios et al., 1989) v b to (Hours et al., 1988) l nhng ngun c cht
thch hp cho vic sn xut enzyme pectinase ngun gc vi sinh. Ngoi ra,
methoxyl ha cao (65 75% DE) ca pectin trong ph phm nng nghip chnh l
iu kin thun li cho hot ng sinh tng hp PME (Jayani et al., 2005).
Mt u im khc ca vic s dng ph phm nng nghip lm c cht cho qu
trnh nui cy thu nhn PME l s hin din mt s thnh phn carbohydrate khc
ngoi pectin, nitrogen, cht x v khong cn thit cho qu trnh ln men (Martin
et al., 2004; Favelar - Torres et al., 2006).
i vi tng loi enzyme ring bit, t l ti u cho hot ng cm ng ca c
cht thay i khc nhau. T l pectin dao ng trong khong 2% thng c la
chn cho qu trnh sinh tng hp PME (Grebechova et al., 2007). Tuy nhin, vic
gia tng nng c cht pectin c hiu qu rt tch cc trong vic y nhanh tc
ln men v gia tng hot tnh PME (Maldonado v Callieri, 2005). cng mt
t l pectin s dng, ngun gc nguyn liu khc nhau cng c nh hng n
hot tnh PME thu nhn c (Grebechova et al., 2007). Chnh v th, vic kho
st t l pectin thch hp cho qu trnh ln men sinh PME cn phi c tin hnh
cho tng c cht chuyn bit v iu kin nui cy c th.
1.6.2 nh hng thnh phn lng
c im chung ca mi trng nui cy vi sinh vt thu enzyme pectinase cng
nh PME l s c mt cht cm ng (Nguyn c Lng, 2004). Tuy nhin, t l
gia carbon v nitrogen trong mi trng, tnh cn bng ca mi trng dinh
dng v carbon v nitrogen c ngha ln i vi sinh tng hp sinh khi ca vi
sinh vt v s to thnh enzyme (Sajjaanantakul v Pitifer, 1991). Nitrogen tham
gia vo qu trnh to protein, acid nucleic v nhiu cht c c tnh sinh hc khc
ca t bo sinh vt. Mi trng c lng carbon v nitrogen cn thit s tch
ly lng enzyme ln nht. S thiu ht cu t ny khng c b p bng s
d tha ca cu t kia (Sajjaanantakul v Pitifer, 1991; Nguyn c Lng,
2004). Nghin cu ca Hossam (2005) cho thy s c mt ca NH
4
NO
3
, NH
4
Cl
trong mi trng nui cy gip ci thin hot tnh pectate lyase thu nhn.
Ngoi ra, do acid amin l nhng cu t hp thnh phn t enzyme, acid amin c
nh hng tt n sinh l ca vi sinh vt cng nh qu trnh sinh tng hp
enzyme. Acid amin c th ng thi va l ngun carbon, ngun nitrogen v l
ngun nng lng (Nguyn c Lng, 2004).
1.6.3 nh hng ngun khong dinh dng
Cc nguyn t a vi lng c nh hng ln n s sinh trng v tng hp
enzyme ca vi sinh vt. Phospho cn tng hp cc hp phn quan trng ca
sinh cht v nhiu coenzyme, ng thi phosphoryl ha glucid trong qu trnh
oxy ha sinh hc. Phospho nh hng trc tip n s sinh sn ca nm si v cc
vi sinh vt khc. Cation Mg
2+
c nh hng n bn nhit ca enzyme tuy
nhin MgSO
4
s c nh hng xu n s tng hp enzyme bi nm si. Trong
khi , lu hunh - c mt trong cc acid amin quan trng nh methionine,
cysteine, c vai tr tch cc trong vic kch thch s hnh thnh enzyme. Ngoi ra,
calcium, mangan, corban, cng nh hng n s tng hp enzyme (Nguyn
c Lng, 2004).
Theo nhiu tc gi, cc ion K
+
, Na
+
, Ca
2+
c nh hng ti hiu qu tc dng ca
PME (Kretovits v Iarovenko, 1982; Hossam, 2005; Joshi et al., 2006; Favelar -
Torres et al., 2006). Tuy nhin, KCl, HCl, HgCl
2
, H
2
O
2
khng c tc dng n s
thay i hot tnh PME trong cam trong khi s hin din ca acid ascorbic,
NaHSO
3
ngay c nng thp c kh nng nng cao hot tnh ca enzyme.
Nghin cu ca Hossam (2005) xc nhn EDTA, PbCl
2
, HgCl
2
v
Iodoacetamide (IAA) l nhn t c ch kh nng ln men ca Rhizopus oryzae,
lm gim hot tnh PME.
1.7 Tc ng ca phng thc v iu kin ln men n hiu qu sinh tng
hp PME
1.7.1 nh hng nhit nui y
Nhit nui cy khng c nh hng ln n hot ng ca h enzyme
pectinase ni chung. Trong khong nhit nui cy 25 40C hu nh khng c
tc ng n s hnh thnh sinh khi ca pectinase. Nhit ti u cho qu trnh
sinh tng hp PME cng thay i ph thuc vo ngun nm mc s dng, c cht
nui cy v iu kin kh hu (Maldonadoo v Callieri, 2005; Joshi et al., 2006;
Mandhania et al., 2011).
1.7.2 m mi trng
Thnh phn nc chim t 70 90% khi lng c th vi sinh vt, tt c qu trnh
phn hy thc n v cc phn ng chuyn ha cc cht trong t bo u din ra
vi s c mt ca nc. m cao nh hng n thong kh, ngc li m
thp qu s km hm s sinh trng v pht trin ca si nm cng nh kh nng
to enzyme (L Gia Hy v Khut Hu Thnh, 2010). m ca mi trng lin
h n s kt dnh, nh hng n thong kh. T nh hng n s sinh
trng v pht trin ca nm si cng nh kh nng to enzyme.
Trong qu trnh ln men trn mi trng lng (SmF), m khng gi vai tr
quyt nh n hiu qu tng hp enzyme. Ngc li, iu kin ln men b mt
trn mi trng rn (SSF), vi sinh vt pht trin v sn sinh ra sn phm ti gn b
mt c cht rn c hm lng m thp. Do , vic cung cp mt lng nc
thch hp v kim tra hot ng ca nc (a
w
) ca c cht ln men l cn thit.
Hm lng m cao hn hoc thp hn u nh hng bt li cho hot ng ca vi
sinh vt. Hn na, nc c nh hng su sc n cc tnh cht ha l ca c cht
v do nh hng n hiu sut ca ton b tin trnh nui cy (Pandey et al.,
2000).
Trong iu kin sn xut, m ban u ti u ca mi trng l 58 60% v s
duy tr m ca mi trng n nh trong qu trnh nui ng vai tr quan trng.
m tng qu 70% s lm gim thong kh, cn m thp hn 50% lm
km hm s sinh trng v pht trin ca vi sinh vt cng nh gim hiu qu hnh
thnh enzyme (Pandey et al., 2000). Khi nui cy trong iu kin khng c v
trng tuyt i th m mi trng sau khi cy ging khng c vt qu 60%
trnh s nhim khun, vi sinh vt l (Nguyn c Lng, 2004).
1.7.3 iu kin pH bn u mi trng
Khi nui cy bng phng php b mt, do mi trng c dung dch m cao v
hm m thp nn gi tr pH ca dch trch sau ln men thng t thay i trong
sut thi gian nui cy. Tuy nhin, gi tr pH ban u ca mi trng c nh
hng khng nh n s pht trin ca nm mc v s to thnh enzyme. Khi pH
mi trng dch v pha acid hoc pha kim, s to thnh sinh khi khng b nh
hng nhng s to thnh enzyme pectinase b km hm. Gi tr pH ti u ca
enzyme c ngun gc vi sinh vt trong khong 4,5 5,5 (Trn Xun Ngch,
2007). Mc d vy, ty thuc vo c im tng enzyme, loi c cht v ngun
nm mc, gi tr pH ti u cho hot ng ca vi sinh vt ln men, to ra enzyme l
khc nhau. Thm vo , trong qu trnh sinh tng hp enzyme, pH thng gim
nh vo thi gian u do s phn hy cc cht, in hnh nh s chuyn ha cc
protein thnh cc acid amin mang tnh acid, sau pH thng tng dn do qu
trnh trao i cht to ra cc cht mang tnh kim nh ammonium, Tuy nhin,
qu trnh ny cn ph thuc vo tng vi sinh vt v sn phm to thnh (L Gia
Hy v Khut Hu Thnh, 2010).
i vi qu trnh sinh tng hp PME hay pectinase t cc dng Aspergillus, vic
iu chnh pH ban u ca mi trng gi vai tr rt quan trng. Cavalitto (1996)
v Hours (1988) nghin cu kh nng pht trin v sinh enzyme t A. foetidus
v A. awamori trong cc h thng ln men SSF trn cc mi trng c gi tr pH
khc nhau. Kt qu cho thy rng, pectinase thu c c hot tnh cao khi mi
trng nui cy c b sung dung dch HCl nng cao.
Cc nghin cu chuyn bit cho qu trnh ln men sinh tng hp PME nhn thy,
ty thuc vo tng loi c cht ln men v vic iu chnh pH bng dung dch
m khc nhau cng c nh hng n hiu qu thu nhn enzyme. Tuttobello v
Mill (1961) cng cho thy hot tnh ca pectic enzyme t gi tr cao nht v n
nh khi iu chnh gi tr pH bng 4,0 vi m citrate. Kt qu ny cng tng
ng vi kt qu nghin cu ca Joshi et al. (2006). iu kin pH ban u ca
mi trng ln men 4,0 (iu chnh bng HCl) gip thu c PME c hot tnh
cao nht so vi khi iu chnh v cc gi tr pH khc. iu ny ng c vi iu
kin ln men SSF v SmF khi s dng c cht l b to t l pha long hai
iu kin ln men l khc nhau (t l pha long c cht : nc l 1 : 3 w/v i vi
ln men SSF v 1 : 6 w/v i vi ln men SmF) (Joshi et al., 2006). Tuy nhin,
nghin cu gn y ca Sharma et al. (2011) li a ra khong pH cao hn cho
qu trnh sinh tng hp PME v pH mi trng ln men ph thuc vo ngun
A. niger phn lp. Vi dng A. niger phn lp t v cam, pH ti u ca mi
trng nui cy l 5,4 trong khi nm mc A. niger phn lp t bt me hay A. niger
i chng MRCC 281 u c pH ti u cho qu trnh ln men 5,0. iu ny cho
thy, cn c s kho st cn thn iu kin mi trng, c bit l pH khi thay i
dng nm mc s dng, iu kin ln men.
1.7.4 nh hng thi gin
Thi gian ln men nh hng ng k n vic sinh tng hp enzyme cng nh
hot tnh ca enzyme. Vi sinh vt cn thi gian pht trin v sn sinh enzyme.
Tuy nhin, khi thi gian qu di, mi trng cn dn cht dinh dng, vi sinh
vt pht trin km v do hot tnh enzyme s gim. Kho st thc nghim l
phng php ph bin xc nh c thi gian nui thch hp gip thu c
enzyme c hot tnh cao v hiu sut thu hi ln (Joshi et al., 2006). Vic to bo
t l hin tng khng mong mun v thng lm gim hot tnh enzyme. i vi
A. niger, qu trnh sinh tng hp enzyme nhiu nht thng kt thc khi nm bt
u sinh nh bo t (Nguyn c Lng, 2004). PME c tng hp t A. niger
thng c nui cy trong thi gian 96 gi (Joshi et al., 2006).
1.7.5 nh hng phng th ln men n qu trnh tng hp PME
Qu trnh nui cy A. niger thu nhn PME c th c thc hin bng hai
phng php ln men khc nhau: ln men b mt trn mi trng rn (SSF) v ln
men b mt trn mi trng lng (SmF) (Fogarty et al., 1983; Sakai et al.,1993).
Cc nghin cu trc cho thy enzyme thu c t phng php SSF c hot
tnh cao hn so vi phng php SmF khi nui cy trn cng loi vi sinh vt v c
cht ln men (Viniegra-Gonzlez et al., 2003). Tuy nhin, l do vi sinh vt sn
xut ra enzyme trn mi trng rn SSF c hot tnh cao hn trng hp s dng
mi trng lng SmF vn cha c gii thch c th. Nhn chung, s khc bit
quan trng gia qu trnh ln men SmF v SSF l m mi trng. Do hot
ng ca nc trong mi trng ln men SmF rt cao nn mc nhim vi sinh
vt khng mong mun gia tng (Patil et al., 2006). Hesseltine (1977) ch ra mt
s thun li v bt li ca SSF so vi SmF.
i. hun li
- Mi trng nui cy n gin;
- Mt s c cht c th c s dng trc tip lm mi trng rn hoc b sung
thm mt s cht dinh dng;
- Sn phm thu nhn c nng cao, d dng tinh sch;
- S dng kt hp cc c cht t nhin c ngun gc thc vt, bo t hoc t
bo;
- m thp v mt s nm mc ln nn hn ch c s ly nhim ca nhiu
loi vi sinh vt khc;
- Lng tp cht sinh ra thp hn trong SmF;
- Enzyme t nhy cm vi cc cht c ch d ha hoc cm ng;
- Trong iu kin thit b n gin, khng c thit b phn ng sinh hc, vic
ln men SSF l phng thc hiu qu, d thc hin, c tnh kh thi cao.
ii. Bt li
Cc vi sinh vt nui cy bi phng php SSF b hn ch bi ro cn v m
ca mi trng;
Vic xc nh cc thng s nh m, pH, oxy t do, CO
2
l mt vn kh
khn do thiu thit b kim tra.
Nghin cu ca Nguyen et al. (2010) cng cho thy, khi nghin cu sinh tng hp
PME t A. niger theo phng php SSF, s dng c cht b to ta v v cam snh,
hot tnh PME thu c cao hn gn 5 ln khi so snh vi mu enzyme nui cy
trn mi trng ln men SmF.
1.8 Cc u t nh hng n hiu qu thu nhn PME t canh trng b
mt
Vi c im ca qu trnh ln men b mt trong mi trng rn l lng nc t
do c trong canh trng sau khi ln men rt t (Palit v Banerjee, 2001), cc
enzyme tit ra thng trng thi lin kt cht ch vi cc thnh phn c cht ln
men, ngn cn vic chit tch v thu nhn enzyme (Rezaei et al., 2011). Chnh v
vy, vic b sung mt t l dung mi thch hp thc hin qu trnh ly trch lng
- rn nhm ci thin ti a hiu qu thu nhn enzyme t canh trng b mt
(Castilho et al., 1999).
Qu trnh ly trch enzyme t canh trng b mt, da trn s phn tch cu t
(enzyme) t cht rn (canh trng b mt) tun theo nh lut Fick (McCable et
al., 1993):

*
.
A
Az AB
dx
J D
dz
= (1.1)
Trong :
J
*
Az
: thng lng cht khuch tn A theo phng z, kgmolA/m
2
.giy
D
AB
: h s khuch tn ca cht A vo B (dung mi), m
2
/giy
x
A
: phn mol cht A trong h thng
z: khong cch khuch tn (m)
Du (-) v mt vt l ngha l qu trnh khuch tn xy ra theo chiu gim
nng ca cht khuch tn.
Ni cch khc, vn tc truyn ca mt qu trnh truyn t l thun vi ng lc
ca qu trnh v t l nghch vi tr lc ca qu trnh (McCabe et al., 1993). ng
lc ca qu trnh ly trch enzyme ph thuc vo s chnh lch nng cht tan
gia dung mi v c cht rn (Lonsane v Krishnaiah, 1992); chu s chi phi ca
mc xuyn thu ca dung mi vo c cht (canh trng b mt sau ln men)
hay khong cch khuch tn tr lc ca qu trnh ly trch (Castilho et al., 1999;
Heck et al., 2005; Diaz et al., 2007; Shata v Farid, 2012).
Da trn phng trnh (1.1), hiu qu ca qu trnh ly trch ph thuc vo mt s
yu t c bn:
- S chnh lch nng
- Din tch tip xc
- Kh nng ha tan ca cht tan (hay enzyme) vo dung mi
- nht ca dung mi
- Kh nng khuch tn ca dung mi vo bn trong c cht rn
- Nhit trch ly
- Tc khuy trn ci thin hiu qu khuch tn.
(Rao, 2010)
i qu trnh ly trch c cht rn t dung mi lng ni chung v enzyme ni ring,
qu trnh khuch tn cht tan vo dung mi cn chu nh hng ca c tnh lin
kt ca enzyme v c cht, in hnh nh tng tc k nc, lin kt hydrogen,
lc Val de Walls (Palit v Banerjee, 2001).
Cc iu kin ly trch nh loi dung dch m, pH ca dung dch s dng ly trch,
thi gian cng nh nhit ly trch l nhng yu t c nh hng ng k n
hiu qu thu hi enzyme sau qu trnh nui cy rn SSF (Jayani et al., 2005). Mc
d vy, cc nghin cu v iu kin ly trch thch hp i vi PME ngoi bo cn
cha nhiu. Joshi et al. (2006) ngh ly trch PME sau qu trnh ln men SSF
ca dng A. niger trn c cht b to bng dung dch NaCl 0,025 M, c kt hp
khuy lc gin on. i vi enzyme pectinase ni chung hay cc enzyme khc
thuc nhm pectinase (PL, PG), cc nghin cu ngh s dng nc ct (Suresh
et al., 2009), dung dch tris HCl (Tewari et al., 2005) hay cc dung dch m
(Castilho et al., 2000). Castilho et al. (2000) l ngi u tin nghin cu t m
cc yu t nh hng n hiu qu ly trch pectinase t nm mc nh loi dung
dch m, nhit v thi gian ly trch cng nh t l dch trch v c cht. y
chnh l c s cho vic nghin cu iu kin ly trch PME thch hp t canh
trng b mt i vi nghin cu thc t.
Enzyme sau khi ly trch c th duy tr phm cht n nh v ng dng hiu qu,
cn phi c tinh sch qua cc bc khc nhau, ty theo mc ch s dng.
Trong lnh vc cng ngh thc phm hay mt s lnh vc k thut khc, ch phm
enzyme k thut thng c s dng rng ri nht (Jayani et al., 2005; Joshi
et al., 2011).
1.9 Cc bin php tinh sch PME k thut
Da vo c tnh ca enzyme, mt s phng php tch, tinh ch thu ch phm
enzyme c th s dng theo s hnh 1.9.
Trong s cc phng php, ph bin nht l phng php kt ta, sc k lng (sc
k trao i ion, sc k i lc, sc k lc gel) (Ishii et al., 1979) hay c ch chn
lc pectin polymerase (sc nhit, thay i pH hay s dng cht ha hc) (Schmitz,
2002). Vic chn la mc cng nh bin php tinh sch ph thuc vo yu cu
ca enzyme s dng.

Hn 19: Cc png pp tu c pm enzyme
Ngun: ng h hu v Nguyn h Xun m, (2009b)
1.9.1 m qun trng h phm enzyme k thut trong ng ngh th phm
Trong thc t, sn xut cc ch phm enzyme c th c s dng di cc dng
khc nhau, c th chia thnh 3 nhm: ch phm th, ch phm k thut hoc ch
phm tinh khit ty theo mc ch v yu cu s dng (ng Th Thu v Nguyn
Th Xun Sm, 2009b). Khng k n ch phm th, ch phm k thut thng
cha hn hp mt vi enzyme ch yu, thu c sau qu trnh tinh sch s b,
trong mt s protein v enzyme tp c tch ra (ng Th Thu v Nguyn
Th Xun Sm, 2009b).
Ch phm tinh khit - ch cha mt enzyme duy nht sau cc bc tinh sch phc
tp, c ngha quan trng trong cc nghin cu khoa hc c bn, xc nh c
tnh ca enzyme cng nh ng dng trong y hc (ng Th Thu v Nguyn Th
Xun Sm, 2009b). Tuy nhin, enzyme tinh khit rt kh ng dng trong cng
nghip do hiu sut thu hi thp, gi thnh cao. Hn th na, cc iu kin hot
ng ti u ca enzyme tinh khit khng th p dng i vi ch phm enzyme k
thut (Aehle, 2004; Jayani et al., 2005; Joshi et al., 2011).
Tc ng ca
acid hoc kim
Tc ng nhit
Enzyme
Sc k i lc
V tr lin kt
c hiu
Tnh ha tan
Tnh n nh Phn ly th lng-lng
- Vi lc
- Siu ly tm
- Thm tch
- Lc bng gel
Sc k
ng in
Sc k trao
i ion
in di trn gel
polyacrylamid
- Kt ta phn on bng
mui trung tnh
- Kt ta bng dung mi
hu c
- Kt ta im ng in
Khi lng
phn t
in tch
1.9.2 inh sh s b PME bng phng php kt t
Phng php kt ta c s dng tng i rng ri thu nhn cc phn t sinh
hc, c bit l cc phn t c bn cht protein nh enzyme. Cu to phn t
enzyme rt phc tp, chng c c nhng nhm k nc v a nc nn tnh tan
ph thuc vo s phn b ca cc nhm ny trn b mt. Kt ta c xem nh l
bc u tin trong quy trnh tinh sch enzyme th (Harris, 1995). C nhiu cch
khc nhau kt ta enzyme nh s dng mui, cc dung mi hu c hoc thay
i pH ca dung dch c cha enzyme. Cc dung mi hoc cc ha cht a nc
c i lc vi nc mnh hn enzyme nn c th li ko nc ra khi phn t
enzyme, lm cho chng mt lp o nc v ta xung. Vic kt ta bng mui
hay dung mi hu c l bin php ph bin nht (Banerjee, 2006).
i. Kt t bng mui
trng thi bnh thng, cc nhm mang in tch trn b mt enzyme s lin kt
vi cc phn t nc bao quanh bng lin kt hydrogen to lp o nc quanh
phn t. Khi thm mui vo vi nng cao, cc phn t mui s phn ly thnh
cc ion, cc ion ny lin kt vi cc phn t nc lm cho enzyme mt lp o
nc, lin kt li vi nhau v to kt ta, lng xung (Banerjee, 2006).
Cc loi mui thng c dng l (NH
4
)
2
SO
4
, Na
2
SO
4
, MgSO
4
. Nhiu nghin
cu cho thy mui (NH
4
)
2
SO
4
l tt nht v c kh nng n nh hu ht cc loi
enzyme (Frster v Rasched, 1985; Joshi et al., 2011). Mui (NH
4
)
2
SO
4
dng rn
hoc dng dung dch bo ha u c th s dng kt ta enzyme. Tuy nhin,
vic p dng bin php kt ta v thu phn on enzyme trong hn hp da trn
c s s khc nhau v kh nng kt ta ca cc protein cng nng mui c
p dng ph bin nht nhm hn ch tc ng cc b gy mt hot tnh enzyme
(Jakoby, 1971). Enzyme c kt ta bng mui t b bin tnh nhng cn phi
tin hnh thm bc thm tch loi mui ra khi dung dch trc khi tin hnh
sc k.
Ti thm tch l mt mng bn thm, c nhng l nh cho php cc phn t nh i
qua trong khi cc phn t ln nh enzyme c gi li. Khi t trong mi trng
c nng mui thp hay khng c mui, mui t trong ti s khuch tn ra bn
ngoi cn bng nng , lp li nhiu ln mui s c loi khi enzyme. Thi
gian thm tch thng l 24 n 28 gi ( Qu Hai v Trn Thanh Phong, 2005).
ii. Kt t bng dung mi hu
Phng php ny c tin hnh da trn c s ha tan ca protein ph thuc
vo s tng tc ca cc nhm tch in trong phn t enzyme vi cc phn t
nc. Kh nng hydrate ha ca enzyme s b gim khi thm vo dung dch cc
dung mi hu c, cc phn t enzyme lin kt li vi nhau v to kt ta. Dung
mi hu c thng dng l ethanol, isopropanol, acetone hoc hn hp cc loi
ru.
phng php ny cn ch tin hnh nhit thp (di 5C) nhm trnh
gy bin tnh enzyme. Cc dung mi hu c c s dng tch phn on
enzyme gn 0C s c tc dng tt n n nh ca enzyme. Khi c kt
ta, phi tch nhanh kt ta ra khi dung mi bng my ly tm.
Phng php ny c u im l khng cn loi mui, cc dung mi hu c c
nhit bay hi thp nn d dng tch khi enzyme bng cch sy nh nhng
nhc im l hay c mu (Lng c Phm, 2004). Mt s nghin cu ca
Reignault et al. (1994) hay Banu et al. (2010) cng xc nhn hiu qu ca vic kt
ta bng ethanol hay acetone i vi PME v pectinase.
1.9.3 ng dng k thut l mng trong tinh sh enzyme k thut
Enzyme cng l mt protein cao phn t, do vy cc phng php phn tch
protein s dng mng lc phn t kh hu hiu (Mohnen et al., 1996). Cc k
thut phn tch protein hay enzyme theo kch thc phn t ca chng nh mt
mng bn thm hoc mng lc phn t. Dung dch enzyme khi c phn
thnh hai pha: pha (1) khng i qua mng lc cha cc cao phn t, trong c
protein hay enzyme mun thu nhn v pha (2) i qua mng lc ch cha cc phn
t c kch thc nh hn kch thc mng lc (peptide, glucid n gin, mui,)
vi nng cn bng vi chng trong pha (1). Trong mt s trng hp, qu trnh
lc c p dng vi mc ch thu nhn thnh phn enzyme trong pha (2) - pha
cha phn t c kch thc nh hn. Enzyme c th c tch ra khi cc
protein/enzyme khc nh vo kch thc phn t ca chng, theo k thut sng
phn t hay vi lc vi khong phn on ph hp (ng Th Thu v Nguyn
Th Xun Sm, 2009a).
Mc d phng php tinh sch bng lc mng khng th loi b hon ton cc
hp cht cao phn t c cng khong khi lng phn t vi enzyme mun tinh
sch, tuy nhin y vn l gii php hu hiu do tnh tin dng, n gin, kinh ph
thp (Hunh Ngc Oanh v Trn Ngc Hng, 2008).
1.10 Qu hoch thc nghim xc nh iu kin ln men thch hp bng
phng php b mt p ng (RSM)
Phng php b mt p ng vi m hnh phc hp ti tm (CCD) thng c
s dng m phng iu kin hot ng ti u ca mt qu trnh thng qua cc
phng php thc nghim. Cp khc nhau hoc cc gi tr ca iu kin hot
ng bao gm cc yu t trong mi th nghim. Cc yu t ny c th l yu t
phn loi (v d ngun nguyn liu s dng) hay nh lng (t l thnh phn
kho st, nhit ,). Tuy nhin, trong thc t, cc bin phn loi phi c x l
mt cch ring bit bng cch so snh cc iu kin hot ng tt nht i vi cc
bin nh lng qua s kt hp khc nhau (Lenth, 2011).
S dng phng php bnh phng nh nht v cc ni dung phn tch hi quy,
phn tch phng sai xc nh gi tr ca cc h s trong phng trnh hi quy
a thc. Ty theo loi thc nghim m phng trnh hi quy l tuyn tnh hay phi
tuyn (Giovanni, 1983). Cc dng phng trnh hi quy thng p dng i vi
lnh vc cng ngh ha hc v cng ngh sinh hc gm:
Phng trnh bc hai tuyn tnh:
1 2
1 , 1
( , ,..., ) ...
k k
k o j j ju j u
j j u
j u
y x x x b b x b x x
= =
=
= = + + +

(1.2)
Phng trnh bc hai phi tuyn
2
1 , 1 1
...
k k k
o j j ju j u jj j
j j u j
j u
y b b x b x x b x
= = =
=
= + + + +

(1.3)
Vi : b
o
l h s hi quy bc 0
x
j
l nhn t c m ha nh hng n y
b
j
l h s tuyn tnh hay h s hi quy bc 1 m t nh hng ca nhn t x
j

i vi y
b
ju
l h s hi quy tng tc m t nh hng ng thi hai nhn t x
j
v x
u

i vi y
b
jj
l h s hi quy bc 2 m t nh hng ca nhn t x
j
i vi y
k l s yu t kho st (x
j
... x
k
)
M hnh thng k thc nghim ch c th s dng sau khi tha mn cc tiu
chun thng k (Student v Fisher) (Cheynier et al., 1983).
1.11 Tnh hnh nghin cu trong v ngoi nc v lnh vc phn lp, tun
chn v sinh tng hp PME
Nghin cu phn lp v tuyn chn vi sinh vt c hiu cho tng loi enzyme
c thc hin nhiu ni trn th gii (de Vries et al., 1999a,b; Lockington et
al., 2002). i vi qu trnh sinh tng hp PME v pectinase, cc ngun phn lp
giu pectin thng c quan tm. Rogaiza Al-Gasgari (2002) tin hnh phn
lp cc loi nm mc c trong 26 loi nc qu Saudi Arabia v s dng cc
nm mc sau phn lp ln men sinh pectinase. Kt qu kho st cho thy, A. niger
c mt hu ht cc ngun phn lp v c mt s cao cc loi cam hay nc
qu hn hp, tuy nhin khng tm thy loi nm mc ny trn hai mu nc to v
mt mu nc xoi. ng thi khi so snh kh nng sinh pectinase ca A. niger v
cc nm mc khc, A. niger cng c xc nhn l nhm c kh nng sinh
enzyme pectinase hot tnh cao. So snh hot tnh ca PME thu c t vic ln
men t hai dng nm mc khc nhau A. niger v A. oryzae, kt qu nghin cu
ca Linde et al. (2007) cho thy, hiu qu sinh enzyme pectinase t A. niger
cao hn hn (tng hn 46,1%) khi so snh vi A. oryzae. Hannan et al. (2009)
nghin cu phn lp 52 dng nm A. niger t to v chanh (thn, r, qu), u
phng, bp, xc tri cy, t, nc, khng kh da trn cc c im i th v vi
th trn mi trng CZ, tuy nhin hu ht cc dng phn lp t khng kh, nc,
t v u phng u khng to vng phn gii pectin ln. Vit Nam, L Th
Thanh Hng v Nguyn Thy Chu (2005) cng bc u nghin cu phn
lp v tuyn chn cc dng A. niger c kh nng sinh enzyme pectinase cao. Vi
50 dng A. niger c phn lp t 80 mu nguyn liu, ch c 4 dng c kh nng
sinh enzyme pectinase hot tnh cao, ng thi cc dng ny u phn lp t v
chanh, bi. Dng A. niger c phn lp t v thanh long hay c ph u cho
hot tnh enzyme pectinase km.
Vic phn lp v nh danh cc dng Aspergillus niger u tin hnh thng qua
cc bc (i) nui cy trn mi trng PDA tuyn chn cc mu mc en, tch
rng v (ii) nui cy trn mi trng MEA hay CZ c b sung cht cm ng ph
hp (Rogaiza Al-Gasgari, 2002; L Th Thanh Hng v Nguyn Thy Chu,
2005; Gupta et al., 2012). Nhng c tnh i th v vi th l c s ban u cho
vic nhn din A. niger, in hnh nh s hnh thnh cc khun lc hnh trn, hoc
gn trn, mp hnh tia, c mu en lan ta t cc m en trn mi trng PDA
(Gupta et al., 2012). Khun lc pht trin trn mi trng CZ v MEA thng c
kch thc nh hn khun lc trn mi trng PDA (McClenny, 2005; Gupta et
al., 2012). c bit, cc nghin cu cn ch ra c im quan trng ca nm mc
A. niger l th bnh 2 tng u vi bng nh gi ln, trong th bnh tng 1
di (gp hai th bnh tng 2) v bo t g gh mt cch bt quy tc (Klich, 2002;
McClenny, 2005). Vic ng dng k thut sinh hc phn t trong nh danh A.
niger cng pht trin trong nhng thp nin gn y (Mirhendi et al., 2007;
Etienne et al., 2009; Sugita & Nishikawa, 2003). Mirhendi et al. (2009) ng
dng k thut RAPD-PCR (Random Amplification of Polymorphic DNA) nhn
din A. niger v A. fumigatus, s dng on mi R151 (5-GCTGTAGTCT-3) v
UBC90 (5- GGGGGTTAGG-3). Trong khi , Susca et al. (2007) ng dng
k thut PCR n nhn din A. niger da trn calmodulin gene, s dng on mi
NIG1/NIG2, vi on gene 245 bp ca vng calmodulin c khuch i. Trc
, Tarai et al. (2006) nhn din cc dng A. niger bng phng php gii
trnh t trn on ITS vi cp mi ITS1/ITS4, trong khi cc nghin cu ca
Sandhu et al. (1995) v kho st gn y ca Nganou et al. (2012) xc nhn
tnh kh thi ca vic ng dng k thut sinh hc phn t nhn din A. niger
bng on mi U
1
/U
2
khuch i on 260 bp trn vng D1/D2 ca 28S rRNA.
y chnh l c s cho vic la chn phng php nh danh dng nm mc c
tuyn chn trong nghin cu.
Bn cnh cc nghin cu tuyn chn vi sinh vt sinh tng hp PME thch hp, cc
yu t nh hng n qu trnh ln men sinh tng hp enzyme cng c kho
st. PME c thu nhn bng vic ln men cc dng Aspergillus (Polizeli,
1991; Sols-Pereira, 1993), c bit l A. niger c kho st kh chi tit vi c
hai phng thc ln men rn v ln men lng (Joshi et al., 2006) trn nhiu loi
c cht khc nhau (Schmitz, 2002; Joshi et al., 2006). Nghin cu ca Joshi et al.
(2006) cho thy, nhit 25C, pH ban u 4,0 v thi gian 96 gi l iu
kin ti u cho vic ln men sinh tng hp PME t A. niger, kt hp s dng b
to lm c cht. Ngoi ra, t l pha long ca c cht v nc nhm to m cho
qu trnh ln men sinh tng hp PME thch hp cng c kho st. Theo , t l
pha long 1 : 3 (w/v) l thng s ti u cho qu trnh ln men SSF v t l 1 : 6
(w/v) c p dng cho ln men SmF (Joshi et al., 2006). Tuy nhin, PME c
sn xut theo phng thc ln men SSF c hot tnh cao hn 2,3 ln so vi ln
men trn mi trng SmF. Daz (2009) cng nghin cu sn xut cc loi
enzyme thy phn t A. awamori s dng c cht ln men l hn hp b nho v
v cam vi t l 1 : 1 (w/w).
Nhn chung, c im thnh phn c cht giu pectin v iu kin mi trng l
yu t chi phi mnh n hiu qu ln men sinh PME t vi sinh vt. Vi thnh
phn pectin trong ht hng dng l 21,36% (theo cn bn kh), Patil v
Dayanand (2006) xc nhn tnh kh thi ca vic s dng ht hng dng
trong sn xut PME t A. niger cho c hai trng hp ln men SSF v SmF.
Jayani et al. (2005) cng gii thiu rt nhiu ph phm nng nghip c th c
s dng lm c cht cm ng ca PME nh c rt, v cam, to, u, khoai ty v
b c ci ng,
Schmitz (2002) cng b bn quyn nui cy, thu nhn PME t A. niger theo
phng php nui cy SSF vi mi trng bt m, cm m, peptone v mt s
khong cht, sau tinh sch bng phng php lc mng v bo qun dng bt
sy phun.
Tuy nhin, hu ht cc kho st u tin hnh nh gi tc ng ring r ca tng
yu t mi trng ln men, thnh phn c cht n hiu qu sinh tng hp PME
m cha quan tm nhiu n tng quan ca cc thnh phn chnh, c bit l s
thay i ca iu kin ln men n hiu qu thu nhn enzyme. Gn y, Suresh et
al. (2009) cng bc u nghin cu ti u ha qu trnh ln men rn sn xut
pectinase t A. awamori bng phng php b mt p ng. Nghin cu ny
tin hnh kho st theo tha s 4 nhn t, 5 mc vi im trung tm, bao gm
nng c cht (5 25%), pH ban u ca mi trng (3,0 7,0), nhit ln
men (25 37C) v t l mc s dng (2 10%). Kt qu cho thy, m hnh bc
hai m rng c thit lp xc nh nh hng tng tc ca cc iu kin
ln men n hiu sut thu nhn enzyme pectinase.
Arotupin et al. (2008) nghin cu tinh sch v xc nh tnh cht ca PME t
nm mc A. repens. Theo , tinh sch PME t A. repens gp 7,93 ln ban u,
hot tnh ring ca PME gia tng t 0,82 U/mg
protein
n 6,5 U/mg
protein
sau cc
bc kt ta bng (NH
4
)
2
SO
4
, kh b mui bng Sephadex G100 v p dng sc
k trao i ion vi SP Sephadex C50.
Duvetter (2007) cng khng nh hiu qu ca vic tinh sch PME t
A. aculeatus thng qua sc k lc gel vi on mi Alka (Alka prime, GE
Helthcare), s dng ct Superdex 75. Kt qu cho thy, hot tnh ring ca PME
t A. aculeatus gia tng t 120 U/mg
protein
PME th (Novozyme) n gi tr
180 U/mg
protein
sau khi tinh sch.
Bn cnh vic kt ta s b hn hp protein cha enzyme sau qu trnh ln men
bng (NH
4
)
2
SO
4
, dung mi acetone lnh cng c s dng lm tc nhn kt ta
enzyme trc khi tinh sch bng sc k. PME t B. cinerea c th c tinh sch
bng acetone lnh vi t l dung mi v dch trch enzyme th l
4 : 1 (v/v) nhit 3 4C. Kt ta cha PME c phn tch bng cch ly tm
tc 27000 g trong thi gian 10 pht, sau ha tan li trong dung dch m
sodium acetate pH 4,5. Mc tinh sch PME (so vi enzyme th) tng 1,6 ln
sau khi kt ta bng acetone nhng gia tng n 37,5 ln sau qu trnh tinh sch
bng sc k trao i ion CMSepharose. Hot tnh ring t c l 1,69 U/mg
(so vi gi tr 0,04 U/mg enzyme th) (Reignault et al., 1994).
Vic ng dng lc mng theo vi lc, lc cho nano c ng dng kh thnh
cng nhm thu c ch phm enzyme pectinase k thut (Hunh Ngc Oanh v
Trn Ngc Hng, 2008). Nghin cu ca Hunh Ngc Oanh v Trn Ngc Hng
(2008) cho thy, sau khi kt ta bng ethanol, mc tinh sch ca enzyme
pectinase tng 3,1 ln khi enzyme c a qua h thng lc mng c khong
phn on 50 kDa, tc bm mu 150 rpm v p sut lc nh hn 5 psi.
Khi lng phn t ca PME t vi sinh vt vn cha c quan tm xc nh.
Nghin cu ca Pitkanen et al. (1992) xc nh khi lng phn t PME t
E. chrysanthemi B374 khong 36 kDa, PME t A. aculeatus c khi lng phn
t khong 41 kDa (Duvetter, 2007). Nhn chung, khi lng phn t ca PME t
vi sinh vt nh hn 50 kDa (Copeland, 2000).
Nghin cu cc yu t nh hng n hot ng enzyme cho thy, PME thu nhn
t nm mc c nhit ti u l 30 45C v b v hot 55 62C (Nguyn
c Lng, 2004). Khong pH ti u cho hot ng ca PME c thu nhn t
nm mc A. species l 3,7 4,2 trong khi PME t A. sojae th hin hot tnh mnh
pH 5,5 (Nguyn c Lng, 2004). Ring vi PME t A. repens th c hot tnh
mnh nht pH 6,5 (Arotupin et al., 2008). Theo nghin cu ca Nari et al.
(1991), hot ng ca PME tng cng vi s tng nng cation kim loi v t
n cc i, trn gi tr ny hot tnh enzyme gim dn. Ion Na
+
v Ca
2+
c nh
hng ln n hot tnh ca PME, nng 0,2 M v 5 25 nM l gi tr ti u
cho hot ng ca PME (Leiting v Wicker, 1997). ng thi, EDTA, Hg
2+
, Pb
2+
v IAA cng c xc nh c kh nng c ch hot ng ca PME t A. repens
(Arotupin et al., 2008).
T cc nghin cu thc t trong v ngoi nc lnh vc phn lp, tuyn chn
dng nm mc ph hp cho hot ng ca enzyme c hiu (PME), n qu trnh
sinh tng hp PME v kh nng ng dng ca enzyme ny, mt s vn c bn
cn quan tm l:
- Hot tnh ca PME thu nhn, hiu sut tng hp ph thuc vo dng vi sinh
vt c hiu - chu s chi phi ca iu kin mi trng sinh trng. Tuy
nhin, khng c nghin cu tuyn chn dng vi sinh vt c hiu ni chung v
nm mc, c bit l A. niger i vi enzyme PME Vit Nam. Cc ngn
hng ging vi sinh vt Vit Nam cng khng c chng c hiu ring cho
pectinase ni chung v hon ton khng c chng vi sinh vt c kh nng sinh
tng hp PME cao c lu tr v nhn ging.
- Vic s dng ph phm nng nghip giu pectin c ester ha cao lm c
cht chnh cho qu trnh sinh tng hp PME l c tnh kh thi - y cng l
ngun ph phm ph bin ng bng Sng Cu Long.
- Cc thng s k thut ca qu trnh ln men, sinh tng hp PME ph thuc
vo tng dng vi sinh vt kho st - cn phi c xc nh c th theo thc
nghim.
- Khng phi tt c PME nm mc, ngay c PME t A. niger u c tnh cht
ging nhau. c tnh ca enzyme thu nhn ph thuc vo dng vi sinh vt,
iu kin nui cy.
Chnh v vy, vic nghin cu phn lp ti ch dng Aspergillus niger c kh nng
sinh tng hp PME c hot tnh cao cng nh xut cc iu kin ln men SSF
thch hp thu nhn enzyme ny, trn c s s dng cc ph phm giu pectin
c ester ha cao in hnh l to, sung v cc loi qu h citrus c thc hin.
CHNG 2 PHNG TIN & PHNG PHP NGHIN CU
2.1 Phng tin nghin cu
2.1.1 im, thi gin
i. hi gin nghin u
Quyt nh cng nhn Nghin cu sinh s 6919/Q-BGDT ngy15/10/2008 ca
B trng B Gio dc v o to.
Thi gian o to theo h khng tp trung l 4 nm (11/2008 11/2012), theo
Quyt nh giao ti v ngi hng dn Nghin cu sinh s 467/Q-HCT
ngy 13/11/2008 ca Hiu trng trng i hc Cn Th.
Thi gian nghin cu ca nghin cu sinh cho n khi hon thnh cc ni dung
nghin cu theo cng ca ti: 11/2008 9/2012.
ii. im nghin u
Phng th nghim B mn Cng ngh Thc phm, Khoa Nng nghip v Sinh hc
ng dng, Trng i hc Cn Th.
Phng th nghim Cng ngh Protein Enzyme, Phng th nghim Cng ngh
sinh hc Thc phm, Vin Nghin cu v Pht trin Cng ngh sinh hc, Trng
i hc Cn Th.
Th nghim nhn din Aspergillus niger bng k thut sinh hc phn t da theo
PCR 28S rRNA c thc hin ti Phng Th nghim Sinh ha v Sinh hc
phn t - Phng xt nghim Nam Khoa (Thnh ph H Ch Minh).
2.1.2 hit b, ho ht
(i) hit b, dng
- Thit b ly tm nhit thp (Rotana 46 R, c; Herlme Z323K)
- My o pH 2 s l, chnh xc 0,01 (HANA pH 212, Trung Quc)
- My khuy t Toshiba (Magnestir MG-10, Nht)
- T Sanyo (MIR-262, Nht)
- T cy Dalton (Nht)
- T sy mu (Memmert,c)
- H thng in di Miniprotein II (BioRad)
- H thng chun in th (Metrohm, Thy S)
- My o cu trc Texture Analyser (TA XT 2i, Anh)
- B iu nhit (Memmert, c, iu chnh n cn 100C, chnh xc 0,1C)
- Cn in t (Ohaus, USA, chnh xc 0,0001 g v 0,01 g)
- B h thng lc mng QuixStand Benchtop Systems (hng Amersham
Biosciences) vi b lc mng (membrane) c khong phn on 50 kDa
- Mt s dng c v thit b khc.
(ii) H ht
- Mi trng thch khoai ty (Potato dextrose agar - PDA), Malt Extract Agar
(MEA), Czapek Dox Agar (CZ) (Merck)
- Agarose (Merck), cetyltrimethyl ammonium bromide (CTAB, Merck)
- Urea (Merck), Pepton (n )
- NaH
2
PO
4
.2H
2
O (Merck)
- Acid citric (Merck)
- EDTA (n )
- NaCl, KCl, CaCl
2
.2H
2
O, MgCl
2
, ZnCl
2
, HgCl
2
, PbCl
2
(Merck)
- Mui ammonium: (NH
4
)
2
SO
4
, NH
4
H
2
PO
4
, NH
4
NO
3
(Merck)
- Acetone (Merck), Ethanol (Merck)
- D (+) galacturonic acid monohydrate (Fluka)
- Coomassie Brillant Blue (CBB) G 250 (Merck)
- Bovine serum albumin (Merck)
- B protein chun ca GenScript gm 6 protein ti t hp (t 15 kDa - 100 kDa)
- Pectin to DE = 75% (Sigma), NaOH 0,1 N (Merck)
- Ha cht phn tch cc ch tiu thng thng
- Ha cht s dng nhn din Aspergillus niger
- on mi U
1
/U
2
(Sandhu et al., 1995) c s dng khuch i mt on
260 bp ca khu vc D1/D2 on gene 28S rDNA:
U
1f
GC (5' - CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GTG
AAA TTG TTG AAA GGG AA - 3'; Sigma)
U
2r
(5' - GAC TCC TTG GTC CGT GTT - 3'; Sigma)
- Thang DNA chun vi khong phn on 100 bp (GeneRuler 100 bp DNA
Ladder, 100 1000 bp, Thermo Scientific)
2.2 Phng php nghin cu
2.2.1 Chun b mu
i. Nguyn liu s dng trong phn lp A. niger
- - To Red Delicious, to Gala (Malus domestica), to ta (Ziziphus
mumularia) c mua ti ch Xun Khnh, Qun Ninh Kiu, Thnh ph Cn
Th.
- - Qu sung (Ficus racemosa) c thu hi ti cc h gia nh Thnh ph
Cn Th.
- - Bi Nm Roi (Citrus maxima), bi da xanh (Citrus grandis), chanh giy,
chanh nm (Citrus medica), cam son (Citrus sinensis), cam snh (Citrus nobilis
var nobilis) v hnh (Citrus microcarpa) mua trong cc nh vn ti Huyn
Phong in - Cn Th.
ii. C ht ln men sinh tng hp PME
- - B to ta c tch loi nc bng c hc v c sy nhit 60 1C
n khi m t 4 1% (Joshi et al., 2006). B to kh c nghin thnh bt
v ng gi trong cc bao b PA, chn khng 90%.
- - V cam snh v v bi Nm Roi: ly phn v trng bn trong, ra sch v
ct nh khong 1 mm (tch bt m v cam/bi xung cn 50% trc khi phi
trn).
iii. Ru qu nghin u i thin u tr
- Khm ging Queen, tri hnh chp (Ananas comosus) t chn k thut, thu
mua t rung khm V Thanh, Hu Giang, c s dng lm mu th
nghim. Tri khm c ct b hai u, sau s dng dao nh hnh, mu
c ct thnh hnh tr ng knh 20 mm, di 15 mm, khng ly phn li (hnh
2.1).

Hn 21: Nguyn liu km v cc cun b mu t ngim
- Da leo (Cucumis sativus L.) c cung cp t Hp tc x rau sch hai huyn
Bnh Tn v Tam Bnh, Vnh Long. Chn da c khi lng 60 75 g/tri, yu
cu thi gian da c mang v t rung cho n khi ch bin khng qu 21 gi.
iv. Dng nm m i hng
S dng 2 dng Aspergillus niger i chng gm dng V (phn lp t t, cung
cp bi Vin Nghin cu v Pht trin Cng ngh Sinh hc, i hc Cn Th) v
dng A. niger nhp ngoi c ngun gc t Nga (khoa Sinh hc, Trng i hc
Tng hp quc gia Matxcova mang tn Lomonosov - MGU), k hiu R92.
2.2.2 Phng php phn th v o
i. Phn phn lp v tuyn hn hng A. niger hiu i vi PME
- - Phn lp A. niger: Nui cy trn mi trng PDA, sau 2 3 ngy, nhit
30 2C, chn khun lc c mu nu hoc en (L Th Thanh Hng v
Nguyn Thy Chu, 2005).
- - nh loi dng nm mc: nh loi hnh thi trn mi trng CZ v MEA,
sau 7 ngy, nhit 25C, chn cc dng mang c im hnh thi ca A. niger
theo kha phn loi ca Klich (2002). p dng k thut PCR gii trnh t gene 28
S rRNA v tra cu bng phn mm Blast N nh loi chnh xc A. niger.
- - Xc nh kh nng sinh pectinase: S dng mi trng pectin - agar o
kch thc vng phn gii pectin bng thuc th Lugol sau thi gian 48 gi,
30 2C (Khairnar et al., 2009).
- - Tuyn chn A. niger sinh tng hp PME hot tnh cao: Cy cc dng A.
niger c vng phn gii pectin 20 mm ln mi trng c hiu i vi pectin
methylesterase
- 1. S dng mi trng pectin - agarose. o ng knh vng phn gii c
nhn thy bng s khc mu ca thuc th CTAB 1% trong nc ct (Khanh et
al., 1991).
- 2. Cy cc dng A. niger c vng phn gii pectin > 20 mm ln mi trng
CZ c b sung pectin c ester ha cao (1% pectin), tin hnh 30C trong 7
ngy (Sharma et al., 2011).
- - Mt s nm mc (cfu/mL): m khun lc nm mc (cfu/mL) khi trn
mi trng Sabouround (L Th Thanh Mai et al., 2009)
ii. Phng php x nh hot tnh PME
Phng php chun pH (theo Crelier et al., 1995; trch dn bi Duvetter,
2007), s dng pectin to (DE 75%) trong NaCl 0,12 M lm c cht phn ng,
chun bng NaOH 0,01 N. Mt n v (U) hot tnh ca PME c nh ngha
l lng enzyme c kh nng xc tc thy phn ca 1 mol lin kt methyl ester
trong mt pht theo cc iu kin p dng (pH 4,5 v 30 1C).
n v o hot tnh enzyme ph thuc vo tng trng hp c th:
- Dch trch enzyme th hay ch phm: U/mL enzyme
- i vi ln men SmF: U/mL c cht
- i vi ln men SSF: U/g c cht
iii. Phng php phn th v o h tiu h l
Cc ch tiu, phng php phn tch v o c cc ch tiu ha, l s dng trong
nghin cu c tng hp bng 2.1.
Bng 21: Cc ch tiu, phng php phn tch v o c
Ch tiu Phng php
m (%) Sy 105C n khi lng khng i (AOAC 934.06)
Pectin, (% cht kh) Phng php kt ta Calcium pectate (Marcon et al., 2005)
ester ha ca pectin
( DE, %)
Phng php chun acid - base (Food Chemical Codex,
FCC, 1981; trch dn bi Singthong et al., 2005)
Hm lng protein
tng (mg/mL enzyme)
Phng php Bradford (1976), s dng bovine serum albumin
(BSA) lm ng chun, vi ch th mu Coomassie Brillant
Blue v o bc sng 595 nm.
Protein th (%) Phng php Kjeldahl (984.13 A-D)
ng kh, ng
tng s (%)
Dng phng php phn tch Lane - Eynon, da vo tnh cht
kh oxy ca nhm aldehyde hay cetone t do c trong phn t
glucid nh lng (Ranganna, 1986)
iu chnh pH mi
trng
iu chnh bng dung dch m cc mc pH kho st.
S dng pH k theo tiu chun ISO 2917 : 1999 (E)
cng (g lc) S dng thit b o cu trc TA XT 2i (Anh).
i vi mu da leo (Fleming et al., 1978)
Mu da leo c ct thnh cc khoanh c chiu dy 1 cm.
o phn tht qu bng lc xuyn thu vi cc thng s:
- Lc tc ng: 25 kg; Tc nn: 2 mm/s
- u o (probe): P2; Khong cch xuyn thu: 50%
i vi mu khm: Khm c ct thnh cc mu hnh tr c
kch thc 20 mm x 15 mm. o lc ct mu vi cc thng s:
- Lc tc ng: 25 kg; Tc nn: 2 mm/s
- u o (probe): dao ct u bng, k hiu HDP/BSK
- Khong cch ph v: 50% (Nguyen et al., 2009)

iv. Phng php tnh ton, nh gi hiu qu tinh sh s b h phm PME k
thut
Hiu qu tinh sch s b ch phm PME k thut c nh gi qua cc thng s
c bn nh hiu sut thu hi, hot tnh ring, tinh sch (bng 2.2).
Ngoi ra, in di trn gel SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel
electrophoresis) c p dng nhn din PME v kim tra tinh khit ca
protein (ng Th Thu v Nguyn Th Xun Sm, 2009a).
Bng 22: Cng thc tnh ton, nh gi hiu qu tinh sch PME
Thng s Cng thc tnh ton
Hiu sut thu hi
PME, Y%
(Ngo et al., 2008)
100 (%)
0
1
=
TA
TA
Y

TA
0
: tng hot tnh PME c trong mu th (U)
TA
1
: tng hot tnh enzyme PME c trong mu sau ly trch (U)
Hot tnh ring S
(U/mg)
(Ngo et al., 2008)
protein
A
S
C
=
vi A: hot tnh PME (U/mL enzyme th);
C
protein
: nng protein trong mu tng ng (mg/mL)
tinh sch P
(Ngo et al., 2008)
0
1
S
S
P =
vi: S
1
v S
0:
hot tnh ring ca PME sau tinh sch v trong mu
th (U/mg)
2.2.3 Phng php phn th v x l s liu
Cc th nghim c b tr ngu nhin, lp li t nht 3 ln. Thng s tng ng
vi kt qu kho st c la chn t th nghim trc c s dng lm nhn t
c nh cho th nghim k tip.
i. hit lp phng trnh hi quy ti u h iu kin ln men sinh tng hp
PME
Vic xc nh phng trnh ti u ha iu kin ln men sinh tng hp PME da
trn phng php b mt p ng (Response Surface Methods). Phng trnh
tng quan c chn la da trn tc ng ca cc yu t kho st (4 tha s)
n kt qu thu nhn (hot tnh PME) bng cc th nghim phng trnh hi quy
a thc theo phng php bnh phng nh nht.
th m phng ( th ng ng im) c thit lp da trn nh hng
tng tc ca hai tham s kho st n hot tnh enzyme thu c trong iu kin
c nh hai thng s cn li gi tr ti u. Trn c s , tng tc ca mt tham
s vi cc tham s khc c nghin cu (Suresh et al., 2009).
ii. Phn th ng h s bin i nhit PME
S v hot enzyme mt iu kin nhit nht nh c th c m t bi m
hnh phng trnh bc mt (Ludikhuyze et al., 2001; Eagerman v Rouse, 1976).
ln( / )
t o
A A kt = (2.1)
Trong : A
t
: hot tnh ca enzyme thi im t; A
o
: hot tnh ban u ca
enzyme thi im t = 0 ; k: hng s tc v hot (pht
1
) v t: thi gian x l
nhit (pht)
Phng trnh (2.1) c gi tr iu kin ng nhit v ng p - isobaric ; hng s
tc phn ng (k) c th c xc nh bng cch thit lp th tng quan
gia ln(A
t
/A
0
) theo thi gian gia nhit. Hng s tc phn ng (k) c th suy ra
trc tip t h s gc ca ng hi quy.
Phng trnh chuyn i mt phn l mt trng hp c bit ca phn ng bc
mt, c ng dng trong trng hp hm lng ca cht nghin cu khng gim
n gi tr khng v hm lng ca cht ny sau khi gim n mt mc no s
khng i khi ko di thi gian x l nhit (A
) v c th c biu din nh:

=
A A
A A
f
0
0
(2.2)
Vi f: h s chuyn i mt phn; A
: hm lng ca mt cht cn li sau qu

trnh x l nhit ko di; A
gn bng 0.
Phng trnh (2.2) c th c vit thnh:

0
0
A
A A
f

= (2.3)
th logarith ca (1f) theo thi gian l mt ng thng vi hng s tc
phn ng c biu th bng gi tr m ca h s gc.
( ) kt f
A
A
= =
|
|
.
|
\
|
1 ln ln
0
(2.4)
Nh vy phng trnh (2.4) ging phng trnh (2.1) khi A
gn bng 0.
tnh ton hot tnh cn li sau qu trnh x l nhit ko di, m hnh chuyn
i mt phn sau nn c s dng:
( ) kt
A A
A A
f =
|
|
.
|
\
|
0
ln 1 ln (2.5)
Sp xp li phng trnh (2.5), nhn c phng trnh (2.6):
( ) ( ) kt A A A A + =

exp
0
(2.6)
Da vo quan im ng hc (phng trnh ng p, ng tch ca phn ng ha
hc), s ph thuc ca hng s tc phn ng vo nhit c th biu din bng
phng trnh Arrhenius :

|
|
.
|
\
|
=
T T R
E
k k
ref
a
ref
1 1
ln ln (2.7)
Trong : T l nhit x l (K), k l hng s tc phn ng (pht
1
); k
ref
: hng
s tc phn ng nhit tham chiu T
ref
(pht
1
); R: hng s kh l tng (R
= 8,314 J/mol.K) v E
a
: nng lng hot ha (kJ/mol)
(Duvetter, 2007).
iii. Phn mm phn th v x l thng k s liu
Kt qu ca cc th nghim so snh, chn nghim thc ti u v ti u ha qu
trnh ln men c thng k v phn tch theo chng trnh Stagraphic Centurion
16.1.
S dng phn mm Blast N c trong ngn hng d liu NCBI so snh tng
ng di truyn gia cc dng nm mc.
S dng phn mm Prism 5.0 tnh ton, xc nh hng s K
m
v V
max
.
S dng phn mm SAS 9.0 xc nh m hnh ng hc s n nh nhit ca
PME.
2.3 Ni dung nghin cu
2.3.1 nghin u tng qut
S b tr th nghim tng qut c tng hp hnh 2.2, th hin 4 phn kho
st chnh:
Phn 1: Phn lp v tuyn chn dng nm mc A. niger c hiu cho qu trnh
sinh tng hp PME.
Phn 2: Xc nh cc yu t nh hng n qu trnh ln men b mt trn mi
trng rn SSF sinh tng hp PME.
Phn 3: Tinh sch s b v xc nh c im PME t A. niger c hiu (tuyn
chn t phn 1).
Phn 4: Trin vng ng dng ca ch phm PME k thut trong ci thin cu trc
t bo rau qu (khm lnh ng, da leo mui chua).
PHN LP V TUYN CHN DNG A. NIGER T V QU GIU PECTIN,
C KH NNG SINH TNG HP PME HOT TNH CAO
Phn lp dng nm mc ging A. niger t v qu giu pectin
(To, sung, bi, chanh, cam v hnh)
+
Tuyn chn dng nm mc sinh pectinase hot tnh cao
+
Tuyn chn dng nm mc sinh PME hot tnh cao
+
Kho st kh nng cnh tranh ca cc dng nm mc sinh PME hot tnh cao
+
nh danh cc dng A. niger c hiu vi PME bng k thut PCR
+
XC NH CC YU T NH HNG N QU TRNH LN MEN SSF
SINH TNG HP PME
Xc nh c cht ln men thch hp i vi nm mc A. niger tuyn chn
+
nh hng ca thnh phn dng cht b sung (nitrogen, khong)
n hiu qu sinh tng hp PME
+
nh hng ca loi dung dch m v thi gian n hot tnh PME thu nhn
+
Ti u ha qu trnh ln men ( m, gi tr pH ban u ca mi trng, t l nm mc
b sung, nhit ) n hiu qu sinh tng hp PME
+
Tc ng ca iu kin ly trch n hiu sut thu hi v hot tnh PME
+
TINH SCH S B V XC NH C IM PME
+
Tinh sch s b PME (kt ta, lc mng)
+
Xc nh tnh cht ca PME t A. niger
(iu kin pH mi trng, nhit , bn nhit, nng ion CaCl
2
v NaCl,
nng c cht pectin s dng)
+
NG DNG CH PHM PME T A. NIGER CI THIN CU TRC RAU QU
+ +
Khm lnh ng Da leo mui chua
(Phng thc b sung, t l PME b sung) (T l PME b sung)

Hnh 2.2: S ngin cu tng qut
2.3.2 Ni dung 1: Phn lp v tuyn hn dng nm m A. niger hiu ho
qu trnh sinh tng hp PME

Hn 23: S b tr t ngim pn lp v tuyn cn nm mc
o ng knh vng phn gii pectin
Th nghim 3
Ct nh v v phn b qu

a petri cha mi trng PDA
nhit 30 2C, 2 3 ngy
Quan st khun lc
Khun lc mu en, nu thm
Lm thun khun lc
To, sung, bi, chanh, cam v hnh

Chn dng nm c kh nng sinh pectinase hot tnh cao

Ln men rn trn mi trng CZ cha pectin 1%
Xc nh hot tnh PME tuyn chn dng nm n l ph hp

nh gi kh nng cnh tranh ca cc dng nm tuyn chn
n hot tnh enzyme PME thu nhn
Th nghim 4
Nhn din cc dng tuyn chn bng k thut sinh hc phn t
Th nghim 5, 6
Th nghim 7
K hiu chng vi nm c c im ging A. niger
nhit 25C, 7 ngy
Cy chm 3 im trn mi trng CZ, MEA
nh loi cc dng nm da trn hnh thi i th, vi th
48 gi, nhit 30 2C

To huyn ph bo t nm

vo l gia thch agar pectin
Th nghim 2
Th nghim 1
i. h nghim 1: Phn lp dng nm m tnh hnh thi ging nhm
nm Aspergillus phn b trn ht v qu giu petin
Mc ch: Thu nhn cc dng nm c c im hnh thi ging nhm nm en
Aspergillus t b mt ca cc loi v qu giu pectin thuc nhm cam, chanh,
hnh, bi, to v sung.
Phng php
Mi trng phn lp: mi trng thch khoai ty (PDA) b sung 1% pectin (c
cht cm ng)
Nguyn liu phn lp:
- Cam: cam snh, cam son, cam mt
- Bi: bi Nm Roi, bi hng da xanh
- Hnh
- Chanh: chanh nm, chanh giy
- To: to ta, to Red Delicious, to Gala
- Sung
Phn lp 5 mu/loi
Cch tin hnh: p dng phng php phn lp gm cc bc sau:
- Ct nh phn v qu, phn b u vo b mt mi trng thch PDA b
sung 1% pectin trong a petri. Tin hnh trong iu kin v trng;
- Nui cy trong t nhit 30 2C;
- Phn lp t ngy th hai n ngy th ba cc khun lc mu en hoc nu
thm vo cc a petri;
- Cy truyn nhiu ln to thun chng;
- Mi khun lc trong a petri c xem l mt dng vi nm v ghi k hiu
lu tr v s dng cho cc th nghim tip theo.
Kt qu thu nhn: Cc dng phn lp c c im bn ngoi ging vi nm mc
en thuc nhm Aspergillus.
ii. h nghim 2: nh loi dng nm v phn lp
Mc ch: nh loi hnh thi i th, vi th cc dng nm phn lp v la
chn cc dng c c tnh ging A. niger.
Phng php
Mi trng nh loi: CZ, MEA
Cch tin hnh: Thng qua hai bc c bn l nh loi hnh thi da trn c
im khun lc theo phng thc cy 3 im v nh loi vi th theo c im bo
t nh.
nh loi hnh thi: Cy truyn cc dng nm phn lp sang a petri cha mi
trng CZ, MEA. Cy ba im trn mt mi trng. Mi dng nm c cy sang
3 a petri, nhit 25C. Quan st v ghi nhn tt c 3 a petri ca mt dng
nm v xc nh cc c im sau khun lc sau thi gian nui cy 7 ngy:
+ ng knh trung bnh ca khun lc;
+ Dng mt ca khun lc (dng ht, dng si nm nm ngang hay thng ng);
+ Mu sc ca mt trn khun lc (mu sc ca cc khi bo t trn to thnh
trn mt khun lc);
+ Mu sc ca mt di khun lc.
X nh im bo t nh v b my mng bo t nh 7 ngy
- Cc c im cn xc nh:
- Si nm: c hay khng vch ngn, mu sc, nhn hoc c gai;
- B my mang bo t trn: hnh dng, kch thc, mu sc, b mt c gai hay
nhn ca t bo gc, gi bo t nh, ti, cung th bnh, th bnh;
- Bo t nh: hnh dng, kch thc, mu sc, mt nhn hay c gai, kiu pht sinh
bo t, dng tp hp ca bo t trn (chui song song, chui phn ly, khi cu, tia
to trn,).
Kt qu thu nhn
- Cc dng nm c c tnh ging A. niger trn c cht giu pectin kho st
theo c im kha phn loi ca Klich (2002).
- nh gi nh hng ca loi nguyn liu n vic pht trin v phn lp
nm mc A. niger.
iii. h nghim 3: Kho st kh nng sinh tng hp petinse dng nm
m phn lp
Mc ch: S b chn cc dng nm mc c kh nng to ra pectinase t cc mu
nm phn lp c.
Phng php: o ng knh vng phn gii pectin trn mi trng thch
pectin. Hot tnh pectinase c nh gi trn da trn s thy phn pectin thng
qua ng knh ca vng phn gii pectin, nhn bit bng cht ch th Lugol 2%.
Cch tin hnh
+ Mi trng thch pectin un tan c phn phi vo cc hp petri, dng khoan
nt c l trn a thch.
+ Ht vo mi l 0,1 mL huyn ph bo t nm (mt s 10
5
cfu/mL), nui nhit
30 2C.
+ Sau 48 gi , nh dung dch Lugol ln b mt thch, sau o vng phn gii
pectin.
Kt qu thu nhn: Cc dng nm mc c kh nng tng hp pectinase cao da
trn ng knh vng phn gii pectin.
iv. h nghim 4: uyn hn dng nm m kh nng sinh tng hp
PME hot tnh o
Mc ch: Chn la cc dng nm mc c kh nng sinh tng hp PME hot tnh
cao t cc dng c hiu vi pectinase, lm c s cho cc nghin cu ln men
sinh tng hp PME tip theo.
Phng php: Tuyn chn dng nm mc c hiu vi PME bng hai phng
php: (i) phng php a, xc nh ng knh vng phn gii pectin trn mi
trng pectin - agarose v (ii) ln men lng trn mi trng CZ, s dng pectin
to 2% lm ngun carbon duy nht.
- Cch tin hnh: Chun b huyn ph (10
5
cfu/mL) cc dng nm mc c kh
nng sinh pectinase cao (ng knh vng phn gii pectin t nht 20 mm sau 48
gi nm mc trong mi trng thch pectin v nhn din bng thuc th Lugol).
Xc nh s b bng phng php a trn mi trng pectin - agarose:
Mi trng pectin - agarose c chun b v khoan l tng t phng php
tuyn chn dng nm mc c hiu vi pectinase. Chng 0,1 mL huyn ph bo
t nm (10
5
cfu/mL) vo mi l (i chng l mu nc ct). qua m 37C
v xc nh ng knh vng phn gii c nhn thy bng s khc mu ca
thuc th CTAB 1% trong nc ct.
Ln men lng trn mi trng CZ b sung pectin: Qu trnh ln men trong bnh
tam gic 250 mL cha 30 mL mi trng CZ lng vi 2% pectin to c
thanh trng nhit 121C trong 20 pht. Chng vo 3 mL huyn ph bo t
nm (10
5
cfu/mL) v nhit 30 2C trong 4 ngy. Enzyme th thu c sau
qu trnh lc sinh khi bng giy lc Whatman s 1, s dng xc nh hot tnh
PME (U/mL) theo phng php chun in th.
Kt qu thu nhn: Xc nh cc dng nm mc c kh nng tng hp PME hot
tnh cao da trn kch thc ng knh vng phn gii pectin trn mi trng
pectin - agarose ln v hot tnh PME o c c gi tr cao.
v. h nghim 5: Kho st kh nng kt hp cc dng nm mc hiu n
hiu qu thu nhn PME
Mc ch: La chn phng n kt hp cc dng nm mc c hiu hot tnh
PME thu c l cao nht (d kin 4 dng nm, k hiu 1, 2, 3 v 4, kt hp cp
i theo t l huyn ph bo t 1:1).
B tr th nghim: Th nghim c tin hnh vi mt nhn t
Nhn t A: Phng thc kt hp cc dng nm mc c hiu
A
1
: Dng 1
A
2
: Dng 2
A
3
: Dng 3
A
4
: Dng 4
A
5
: Kt hp dng 1 v 2
A
6
: Kt hp dng 1 v 3
A
7
: Kt hp dng 1 v 4
A
8
: Kt hp dng 2 v 3
A
9
: Kt hp dng 2 v 4
A
10
:Kt hp dng 3 v 4
S nghim thc kho st: 10 nghim thc
Phng php: Th nghim c thc hin tng t th nghim 4. S b tr th
nghim c trnh by hnh 2.4.
cc nghim thc kt hp ca hai dng nm mc, pha long huyn ph n mt
s mong mun, phi trn trong iu kin v trng (phi trn trong t cy kh
trng) huyn ph ca hai mu nm mc tng ng vi t l 1:1.
Kt qu thu nhn: Hai dng nm mc kt hp thch hp c kh nng to ra PME
nhiu nht da theo ng knh vng phn gii v hot tnh PME thu c.


Hn 24: S b tr t ngim 5 K nng kt p cc dng
nm mc c iu n iu qu tu nn PME
vi. Th nghim 6: Kho st vic kt hp hai dng nm mc c hiu theo cc t
l khc nhau n kh nng sinh tng hp PME hot tnh cao
Mc ch: Xc nh t l kt hp thch hp ca hai dng nm mc c hiu gip
gia tng hiu qu thu nhn PME.
Cc phng thc kt hp

Huyn ph bo t nm mc (10
5
cfu/mL)

4 dng nm mc c hiu (A
1
, A
2
, A
3
, A
4
)

A
1
A
2
A
3 A
4

A
1
x A
2
A
5

A
1
x A
3
A
6
A
1
x A
4
A
7
A
2
x A
3
A
8
A
2
x A
4
A
9
A
3
x A
4
A
10
nh gi kh nng kt hp ca cc dng nm tuyn chn
n hot tnh enzyme PME thu nhn
a trn mi trng
agarose - pectin
0,1 mL
huyn ph
bo t nm

(37 C - qua m)
Xc nh ng knh
vng phn gii
Thuc th
CTAB 1%
Ln men lng trn mi
trng CZ b sung pectin
(30 mL mi trng CZ lng
v 2% pectin to)

(30 2 C - 4 ngy)
Lc sinh khi
3 mL huyn ph
bo t nm
Xc nh hot tnh PME
Xc nh cc dng nm mc c kh nng tng hp PME hot tnh
cao
B tr th nghim: Th nghim c tin hnh vi mt nhn t
Nhn t B: T l kt hp ca hai dng nm mc tuyn chn
B
1
: 1:1 B
2
: 2:1 B
3
: 3:1 B
4
: 4:1
B
5
: 1:2 B
6
: 1:3 B
7
: 1:4
Cch tin hnh: Th nghim c thc hin tng t th nghim 5. Tin hnh
phi trn trong iu kin v trng (phi trn trong t cy kh trng) huyn ph
ca hai dng nm mc theo cc t l kho st.
Kt qu thu nhn: T l kt hp thch hp ca hai dng nm mc cho hot tnh
PME cao nht.
vii. h nghim 7: B u nhn din cc dng nm m tuyn hn bng
phng php sinh h phn t
Mc ch: Kim tra, nhn din cc dng nm mc c hiu vi PME nh loi
theo c tnh hnh thi (i th, vi th).
Phng php: Gii trnh t gene m ha 28S rRNA v tra cu bng phn mm
BLAST N so snh trnh t on DNA ca dng nm mc vi trnh t DNA ca
cc loi nm mc c trong ngn hng d liu NCBI.
Mu nm mc c thc hin cc bc nhn din t ly trch DNA n gii trnh
t ti Phng Xt nghim Nam Khoa TP. H Ch Minh, Vit Nam theo cc bc:
- Ly trch DNA ca nm mc theo phng php CTAB.
- Thc hin phn ng PCR, khuch i on DNA mc tiu.
- in di sn phm PCR trn agarose gel, nhm so snh kch thc DNA ca sn
phm PCR vi thang DNA chun v kim tra tinh sch ca DNA.
- Nhn din nm mc bng phng php gii trnh t on DNA.
(Phng php xc nh xem ph lc 1).
2.3.3 Ni dung 2: X nh yu t nh hng n qu trnh ln men rn sinh
tng hp PME
i. X nh thnh phn h h ht ln men
Mc ch : Xc nh cc thnh phn ha hc ca b to kh v v qu citrus (cam
snh, bi Nm Roi) ti, lm c s cho nghin cu nui cy sinh tng hp PME.
Cch tin hnh: B to kh v v qu citrus ti sau khi sy c xc nh
m, phn tch ng ha tan, hm lng pectin v xc nh mc DE, lm c s
cho vic tnh ton t l phi trn v iu chnh m mi trng thch hp.
S ln phn tch cho mt ch tiu: 3 5 ln
ii. h nghim 8: nh hng thnh phn ht n kh nng thu nhn
PME t A. niger
Mc ch: Tm ra loi c cht v kh nng kt hp ph hp (nu c) cho qu trnh
sinh tng hp PME t A. niger t hiu qu cao.
B tr th nghim: Th nghim c tin hnh vi 1 nhn t
Nhn t C: T l kt hp gia b to v v qu citrus
C
1
: 10 : 0 (ch c b to) C
4
: 6 : 4
C
2
: 8 : 2 C
5
: 5 : 5
C
3
: 7 : 3 C
6
: 4 : 6
C
7
: 0 : 10 (ch v cam hay bi)
Nhn t c nh: m mi trng (60%), t l huyn ph bo t nm b sung l
20% v/w (mL/g, vi mt s nm mc 10
5
bo t/mL), nhit 37C, thi gian
96 gi.
S nghim thc th nghim: 13 nghim thc
Tin hnh th nghim: Th nghim c thc hin theo s b tr c trnh
by hnh 2.5.
Ln men: Cn 5 g hn hp b to v v qu citrus (bi hay cam) cc t l phi
trn khc nhau cho vo cc bnh tam gic 250 mL. th nghim ny, nc ct
c s dng iu chnh m mi trng n 60%. Thanh trng cc bnh tam
gic cha c cht 121C trong 15 pht. Lm mt cc bnh sau khi thanh trng,
cho huyn ph bo t nm (hai dng nm mc vi t l kt hp c la chn
t th nghim 6, phn 1) vi t l 20% v/w (mL/g) vo mi bnh cy v nhit
37C trong 96 gi (4 ngy).
Ly trch enzyme: Dch cha PME c ly trch bng cch s dng nc ct (pH
6,7 6,8) t l dch trch v canh trng b mt l 2 : 1 v/w, tin hnh khuy ly
trch enzyme trong thi gian 30 pht. Tin hnh ly tm 4C, tc 8000 g thu
c phn dch trong (sau khi loi b phn kt ta) l dch trch enzyme th. Xc
nh th tch enzyme th thu c.
Xc nh li DE (%), lng pectin (%) trong mu sau khi thanh trng. o hot
tnh PME trong enzyme th (U/mL dch trch), t xc nh hot tnh PME
tng ng vi nghim thc kho st (U/g c cht) theo cng thc:
Hot tnh PME thu c (U/g)
o
C xV
C
m
=
Vi: C
o
: Hot tnh PME trong 1 mL dch trch
V: Th tch dch trch (mL)
m: Khi lng c cht chnh trc khi ln men (g)

Hn 25: S b tr t ngim 8 - n ng ca vic kt p b to v v qu
cam/bi n qu trnh sin tng p PME iu kin ln men SSF
Kt qu thu nhn: Thnh phn c cht chnh (hm lng pectin, ng ha tan
s dng) thch hp cho qu trnh ln men cho hot tnh PME cao nht c chn
lm thng s c nh cho cc th nghim sau.
iii. h nghim 9: Kho st nh hng thnh phn khong vi lng b sung
n kh nng sinh PME t A. niger
Th nghim 9a: Kho st nh hng ca tng thnh phn khong b sung ring l
n hot tnh ca PME c tng hp bi A. niger.
Mc ch: Xc nh loi khong v t l b sung thch hp nht cho s tng hp
PME ca A. niger t hiu qu cao.
B tr th nghim: Th nghim tin hnh vi 3 nhn t ring l
Nhn t D
1
: Hm lng KCl s dng
D
1.1
: KCl 0,5% D
1.3
: KCl 1,5%
D
1.2
: KCl 1,0% D
1.4
: KCl 2,0%
B to : v bi

C
1

o c ch tiu
B to : v cam

C
2

C
3

C
4

C
5

C
6

C
7

C cht

Ly trch enzyme
(4 ngy, 37C)
C
1

C
2

C
3

C
4

C
5

C
6

C
7

Chun b mi trng ln men
Nhn t D
2
: Hm lng CaCl
2
s dng
D
2.1
: CaCl
2
0,5% D
2.3
: CaCl
2
1,5%
D
2.2
: CaCl
2
1,0% D
2.4
: CaCl
2
2,0%
Nhn t D
3
: Hm lng MgCl
2
s dng
D
3.1
: MgCl
2
0,25% D
3.3
: MgCl
2
0,75%
D
3.2
: MgCl
2
0,50% D
3.4
: MgCl
2
1,00%
S nghim thc: 4 + 4 + 4 = 12 + 1 mu i chng (khng b sung khong)
Tin hnh th nghim: Kho st c tin hnh da trn kt qu ti u t cc th
nghim trc. Trc khi thanh trng c cht, cc loi mui cha ngun khong
vi t l cn kho st c b sung vo mi trng. Sau khi b sung 20% v/w
(mL/g) huyn ph bo t nm, mi trng ln men c nhit 37C trong
thi gian 96 gi (hnh 2.6). Trch ly dch cha enzyme th (ging nh th nghim
8) v xc nh hot tnh.

Hn 26: S b tr t ngim 9a - n ng ca tng tn pn kong
b sung ring l n ot tn ca PME c tng p bi A. niger
Dd NaCl 0,025M
Thanh trng
Lm ngui
Cy vi sinh vt
Thu nhn enzyme
Aspergillus niger

o hot tnh PME
Pha long Dung dch m
KCl

B sung khong
Mi trng nui cy
Dung dch m

CaCl
2 MgCl
2
D
1.1
D
1.2
D
1.3
D
1.4 D
2.1
D
2.2
D
2.3
D
2.4 D
3.1
D
3.2
D
3.3
D
3.4
Kt qu thu nhn
- Hot tnh ca PME (U/g) tng ng vi tng nghim thc th nghim.
- Thnh phn v t l khong b sung cho hot tnh ca PME cao (chn 3 loi
khong ti u) c chn lm iu kin c nh tin hnh cc th nghim kho
st tip theo.

Th nghim 9b: Kho st nh hng ca thnh phn khong b sung kt hp n
kh nng tng hp PME t A. niger
Mc ch: Xc nh kh nng kt hp cc thnh phn khong hot tnh PME
thu c t A. niger l cao nht.
Nhn t E: Thnh phn khong b sung kt hp trong mi trng ln men sinh
tng hp PME
E
1
: i chng E
2
: Khong 1 Khong 2
E
3
: Khong 1 Khong 3 E
4
: Khong 2 Khong 3
E
5
: Khong 1 Khong 2 v 3
S nghim thc: 5 nghim thc
nghim trc. B sung kt hp cc loi mui cha ngun khong vi t l b sung
thch hp thu c t th nghim 9a trc khi thanh trng v tin hnh cc bc
tip theo. Sau 96 gi , trch ly dch cha enzyme th v xc nh hot tnh.
Kt qu thu nhn: Hot tnh ca PME (U/g) tng ng vi tng nghim thc th
nghim. T , xc nh thnh phn v t l khong b sung kt hp cho hot tnh
ca PME cao nht c chn lm iu kin c nh tin hnh cc th nghim
kho st tip theo.
iv. h nghim 10: Kho st nh hng ngun nitrogen b sung n kh
nng thu nhn PME t A. niger
Mc ch: Xc nh thnh phn dinh dng b sung thch hp nht cho s tng
hp PME ca A. niger.
B tr th nghim: Th nghim c kho st vi 2 nhn t
Nhn t F
1
: Ngun dinh dng (nitrogen) b sung, thay i vi 3 loi
F
1.1
: Urea
F
1.2
: Ammonium dihydrogen phosphate NH
4
H
2
PO
4

F
1.3
: Ammonium sulfate (NH
4
)
2
SO
4
)
Nhn t F
2
: T l cht dinh dng b sung (%, tnh trn tng khi lng mi
trng rn, iu chnh m), thay i 4 mc
F
2.1
: 0,05% F
2.2
: 0,1%
F
2.3
: 0,15% F
2.4
: 0,2%
S nghim thc: 3 x 4 = 12 nghim thc + 1 mu i chng
Cc thng s khc c tin hnh da trn kt qu ti u tng ng t cc th
nghim trn.
nghim trc. B sung cc ngun dinh dng khc nhau vi cc t l b sung
kho st. Thanh trng mi trng 121C trong 15 pht, lm ngui v chng mt
t l thch hp huyn ph bo t nm ln mi trng, nhit phng trong 96
gi. Sau khi thi gian, trch ly dch cha enzyme th v xc nh hot tnh.
Kt qu thu nhn: Hot tnh ca PME (U/g) tng ng vi tng nghim thc, t
chn la ngun nitrogen b sung thch hp v t l s dng cho hot tnh PME
cao nht.
v. h nghim 11: Kho st nh hng loi dung dh m s dng iu
hnh m n kh nng sinh tng hp PME t A. niger
Mc ch: Xc nh vai tr ca cc dung dch m khc nhau ( gi tr pH ban
u ca mi trng nui cy c nh 4,0) n hiu qu thu nhn PME.
Nhn t G: Loi dung dch m (pH 4,0) b sung
G
o
: i chng (Nc ct pH 6,7 6,8)
G
1
: Acetate
G
2
: Citrate
G
3
: Citrate phosphate
G
4
: Nc ct, iu chnh pH 4,0 bng NaOH
nghim trc. Thnh phn c cht v dinh dng thch hp cho hot ng ca
PME c chun b. iu chnh m c cht n 60% bng cc dung dch m
kho st. Qu trnh ly trch enzyme vn s dng nc ct pH 6,7 6,8.
Kt qu thu nhn: Loi dung dch m thch hp gip qu trnh ln men n nh
v thu nhn hot tnh PME (U/g) cao nht c chn lm thng s c nh cho cc
th nghim sau.
vi. h nghim 12: thy i thi gin n kh nng sinh tng hp PME t
nm m A. niger
Mc ch: Xc nh hiu qu sinh tng hp PME theo thi gian nui A. niger
trong iu kin ln men SSF.
Nhn t H: Thi gian , c 6 mc thi gian c kho st
H
o
: 0 gi (i chng) H
1
: 24 gi H
2
: 48 gi
H
3
: 72 gi H
4
: 96 gi H
5
: 120 gi
in hnh th nghim: Kho st c thc hin da trn kt qu ti u ca th
nghim trn. Sau thi gian tng ng 24 gi, 48 gi, 72 gi, 96 gi v 120 gi
(so vi mu i chng), trch ly dch cha enzyme v xc nh hot tnh.
Kt qu thu nhn
- Hot tnh ca PME (U/g), s thay i thnh phn c cht ( m, pH, hm lng
pectin, DE) tng ng vi tng nghim thc th nghim.
- Thi gian thch hp sao cho hot tnh PME cao nht v hm lng c cht s
dng ti a c chn lm nhn t c nh cho cc th nghim tip theo.
vii. h nghim 13: nh hng tng t pH, m mi trng, t l huyn
ph bo t nm v nhit n qu trnh sinh tng hp PME t dng
A. niger tuyn hn
Mc ch: Xy dng phng trnh hi quy ca cc yu t tng tc gm pH v
m ban u ca mi trng, t l huyn ph bo t nm b sung v nhit n
qu trnh sinh tng hp PME t A. niger theo phng php b mt p ng.
B tr th nghim: Th nghim c b tr theo phng php b mt p ng vi
4 nhn t thay i 5 mc (bng 2.3) vi 7 im trung tm vi tng s nghim
thc kho st c thit lp l 31 (bng 2.4).
Bng 23: Gi tr m ha v gi tr thc ca cc nhn t tng tc n hiu qu sinh
tng hp PME (U/g)
Nhn t K hiu
Gi tr m ha
-2 -1 0 +1 +2
T l huyn ph bo t nm
(10
5
cfu/mL) b sung
X
1
(%v/w;
mL/100g)
12 14 16 18 20
pH ban u ca mi trng X
2
3,0 3,5 4,0 4,5 5,0
Nhit X
3
(
o
C) 28 31 34 37 40
m ban u ca mi trng X
4
(%) 50 55 60 65 70
Bng 24: Cc nghim thc kho st theo phng php b mt p ng, p dng
thit k trung tm phc hp
TT T l huyn ph bo
t nm (% v/w)
pH ban u ca
mi trng
Nhit (C) m
mi trng (%)
1 1 1 1 -1
2 -1 -1 -1 -1
3 -1 -1 1 -1
4 1 -1 1 -1
5 1 -1 1 1
6 0 0 0 0
7 0 0 0 -2
8 -2 0 0 0
9 0 0 0 0
10 0 0 0 0
11 -1 1 -1 1
12 0 0 0 0
13 -1 1 1 1
14 0 0 0 0
15 -1 1 -1 -1
16 0 2 0 0
17 0 0 0 0
18 1 -1 -1 1
19 1 1 1 1
20 -1 -1 -1 1
21 -1 1 1 -1
22 0 0 -2 0
23 0 -2 0 0
24 2 0 0 0
25 1 1 -1 -1
26 0 0 0 2
27 0 0 0 0
28 1 -1 -1 -1
29 1 1 -1 1
30 -1 -1 1 1
31 0 0 2 0
Tin hnh th nghim: Th nghim c chun b da trn cc thng s ti u t
cc kho st trc (th nghim 8 12) vi s b tr th nghim c trnh by
hnh 2.7.
Tin hnh pha long c cht n m vi cc t l kho st (X
4
) ng vi cc gi
tr ca pH (X
2
) bng dung dch m c xc nh th nghim 11. Sau khi cy
nm mc vi cc t l huyn ph bo t nm thay i (X
1
), mu kho st c
tin hnh ln men cc t c nhit kho st (X
3
) tng ng vi thi gian ln
men c xc nh t th nghim 12. Thu dch cha enzyme tng ng vi tng
mc thi gian kho st o c hot tnh PME c trong dch trch.
Hn 27: S b tr t ngim 13 - n ng tng tc ca cc iu kin
ln men n iu qu tu nn PME
Kt qu thu nhn
- Hot tnh ca PME (U/g) tng ng vi cc mu kho st.
- S dng chng trnh Statgraphic Centurion 16.1 phn tch tng tc ca cc
nhn t, xy dng phng trnh biu th hot tnh PME thu c trong mi tng
quan ca cc thng s ln men.
Dng nm mc

theo cc khong nhit X
3
(C)
(trong khong thi gin ti u t th nghim 12)
Thu dch cha enzyme tng ng o c hot tnh
PME c trong dch trch

Huyn ph nm mc A. niger X
1

So
2
v R
1
(10
5
cfu/mL)
iu chnh m theo X
4
(%)
Dung dch m (x nh t
TN11, h thnh phn khong
kho st TN 8,9) cc
mc pH khc nhau (X
2
)

Thanh trng 121C trong 15 pht

Lm ngui

C cht ln men (chn lc t th nghim 8)

9d
- V th ng ng im biu din hot tnh PME thu c da trn tng tc
ca 2 nhn t v 2 nhn t cn li c gi im trung tm. Xc nh cc thng
s ti u ha ca qu trnh ln men.
viii. h nghim 14: nh hng vi iu hnh pH dung dh ly trch n kh
nng thu hi PME t A. niger sau qu trnh ln men SSF
Mc ch: Xc nh gi tr pH thch hp ca dung dch m tng ng (dung dch
m ti u c s dng iu chnh pH mi trng ln men, th nghim 11) n
hiu qu ly trch PME.
B tr th nghim: Th nghim c tin hnh ngu nhin vi 1 nhn t.
C nh cc thng s: nhit phng (30 1C), t l dch trch : canh trng b
mt l 2:1 v/w, thi gian ly trch l 30 pht.
Nhn t I: Gi tr pH ca dung dch m ly trch, c iu chnh 7 mc :
I
1
: pH = 3,2 I
2
: pH = 3,6 I
3
: pH = 4,0
I
4
: pH = 4,4 I
5
: pH = 4,8 I
6
: pH = 5,2 I
7
: pH = 5,6
S nghim thc = 7 + 1 mu i chng (ly trch bng nc) = 8 nghim thc
Tin hnh th nghim: Cc bc chun b mu c thc hin da trn vic xc
nh iu kin ln men ti u phn 2. Sau khi thi gian, trch ly dch cha
enzyme th bng dung dch m la chn cc gi tr pH khc nhau v xc nh
hot tnh.
Kt qu thu nhn: Hot tnh ca PME (U/g) trong dch trch tng ng vi tng
nghim thc th nghim.
ix. h nghim 15: nh hng t l dung dh ly trh v ht n hiu
qu ly trch PME t A. niger
Mc ch: Tm t l thch hp gia dung dch m s dng v c cht hiu qu
ly trch PME t nm mc A. niger sau qu trnh ln men SSF t cao nht.
B tr th nghim: Th nghim c tin hnh hon ton ngu nhin vi 1 nhn t
C nh cc thng s : nhit phng (30 1C), thi gian ly trch l 30 pht.
Chn pH ti u ca dung dch m kho st da trn kt qu th nghim 14.
Nhn t J: T l dung dch m v c cht, iu chnh 6 mc
J
1
: 1 : 1 J
2
: 1,5 : 1 J
3
: 2 : 1
J
4
: 2,5 : 1 J
5
: 3 : 1 J
6
: 3,5 : 1
S nghim thc kho st = 6 nghim thc
Tin hnh th nghim: Kho st c tin hnh vi dung dch m v pH tng
ng chn t kt qu th nghim 14. Tuy nhin, t l dung dch m v c cht
thay i theo cc nghim thc kho st.
Kt qu thu nhn: Tng hot tnh (U) trong dch trch tng ng vi tng
nghim thc th nghim.
x. h nghim 16: nh hng nhit v thi gin n hiu qu ly trch
PME sau qu trnh ln men SSF
Mc ch: Tm ra nhit v thi gian ly trch thch hp nht cho vic thu PME
c hot tnh cao.
B tr th nghim: Th nghim c tin hnh ngu nhin vi 2 nhn t
Nhn t K
1
: Nhit ly trch, c 6 mc c kho st
K
1.1
: 5C K
1.2
: 15C
K
1.3
: 25C K
1.4
: 35C
K
1.5
: 45C K
1.6
: 55C
Nhn t K
2
: Thi gian ly trch, c 5 mc c kho st
K
2.1
: 10 pht K
2.2
: 30 pht
K
2.3
: 50 pht K
2.4
: 70 pht
K
2.5
: 90 pht
Nhn t c nh: Gi tr pH ti u ca dung dch m s dng ly trch enzyme
t th nghim 14 v t l thch hp ca dung dch m v canh trng b mt chn
c th nghim 15.
S nghim thc = 6 x 5 = 30 nghim thc
Tin hnh th nghim: Kho st c thc hin da trn kt qu thu nhn t 2 th
nghim trc (th nghim 14 v 15), tuy nhin qu trnh ly trch enzyme c thc
hin iu kin nhit v thi gian khc nhau.
Kt qu thu nhn: Hot tnh ca PME (U/g) trong dch trch tng ng vi tng
nghim thc th nghim. Trn c s , chn la iu kin ly trch ph hp.
xi. Th nghim 17: nh gi kh nng bo qun PME th
Mc ch: Xc nh nhit bo qun thch hp gip n nh hot tnh PME th.
B tr th nghim: Th nghim c b tr vi mt nhn t
Nhn t M: Nhit bo qun PME (C)
M
1
: Bo qun lnh 4C
M
2
: Tr ng -18C
Tin hnh xc nh s thay i hot tnh ca PME theo thi gian bo qun vi
mu i chng l mu khng bo qun (mu va mi ly trch).
Cch tin hnh: Dung dch enzyme th c ly trch sau qu trnh ln men SSF
c cha trong cc ng cha mu v tin hnh bo qun 2 mc nhit kho
st. ng vi tng iu kin bo qun, phn tch hot tnh ca PME theo thi gian.
Chu k phn tch: tn sut 1ngy/ln i vi mu bo qun lnh v 2 ngy/ln i
vi mu tr ng.
Kt qu th nghim: Mc thay i hot tnh ca PME theo thi gian bo qun
khi so snh vi mu i chng. nh gi nh hng ca nhit n n nh
ca PME.

2.3.4 Ni dung 3: inh sh s b v x nh im PME t A. niger
i. Kho st kh nng tinh sh s b PME t A. niger bng phng php kt
t v l mng
Th nghim 18: Nghin cu nh hng ca loi ha cht v t l s dng n kh
nng kt ta PME t A. niger.
Mc ch: Tm ra loi ha cht v t l s dng thch hp nhm t hiu qu kt
ta PME t A. niger tt nht m khng lm bin tnh enzyme.
B tr th nghim: Th nghim c tin hnh vi 1 nhn t
Nhn t N: Nng hay t l ha cht s dng kt ta PME trong dch enzyme
th, bao gm 4 nhm nhn t c kho st ring l (N
1
, N
2
, N
3
v N
4
)
Nhn t N
1
: Nng ammonium sulfate trong dung dch PME th t A. niger,
thay i 7 mc :
N
1.1
: 0 35% (F0 35)
N
1.2
: 35 45% (F35 45)
N
1.3
: 45 55% (F45 55)
N
1.4
: 55 65% (F55 65)
N
1.5
: 65 75% (F65 75)
N
1.6
: 75 85% (F75 85)
N
1.7
: 85 95% (F85 95)
Nhn t N
2
: Nng sodium chloride trong dung dch PME t A. niger th (v/w),
thay i 6 mc
N
2.1
: 0 10% (F0 10)
N
2.2
: 10 15% (F10 15)
N
2.3
: 15 20% (F15 20)
N
2.4
: 20 25% (F20 25)
N
2.5
: 25 30% (F25 30)
N
2.6
: 30 35% (F30 35)
Nhn t N
3
: T l ethanol v PME t A. niger th (v/v), thay i 6 mc
N
3.1
: 1,5 : 1 N
3.4
: 3 : 1
N
3.2
: 2,0 : 1 N
3.5
: 3,5 : 1
N
3.3
: 2,5 : 1 N
3.6
: 4 : 1
Nhn t N
4
: T l acetone v PME t A. niger th (v/v), thay i 4 mc
N
4.1
: 1 : 1 N
4.2
: 1,5 : 1
N
4.3
: 2 : 1 N
4.4
: 2,5 : 1
S nghim thc th nghim: 7 + 6 + 6 + 4 + 1 (i chng) = 24 nghim thc
Tin hnh th nghim: Dung dch enzyme th c lm lnh n 4C trc khi
tin hnh th nghim.
- i vi trng hp kt ta bng mui
Dch enzyme th c tin hnh kt ta phn on bng mui ammonium sulfate
bo ha bt u t 45% n 95% hoc sodium chloride bo ha t 15% n 35%.
u tin, cho mt th tch xc nh ca dch enzyme th lm lnh vo trong
ng ly tm. T bng tra nng bo ha mui (NH
4
)
2
SO
4
, NaCl, tnh ton c
khi lng mui cn thit phi cho vo dung dch t c nng bo ha
tng ng (hnh 2.8).
Cho mui t t vo dung dch enzyme v lc u cho n khi mui tan hon ton.
Qu trnh kt ta c thc hin trong vng 2 gi 4C, tip theo kt ta c
tch ra bng my ly tm 10000 vng/pht (khong 11173 g) trong 20 pht 4C.
Sau qu trnh ly tm, kt ta c tch ra v phn dung dch cn li c em i
xc nh th tch v tin hnh tnh ton xc nh lng mui cn thit cho vo
t c nng bo ha k tip. Cc bc tip theo cng c thc hin nh lc
u v cho n nng bo ha cui cng.
Sau , kt ta thu c cc phn on mui bo ha khc nhau s c ha tan
tr li bng dung dch m citrate phosphate (pH 4,5) vi th tch va . Tip
theo, phn kt ta c ha tan s em i thm tch trong dung dch m citrate-
phosphate (pH 4,5) bng mng cellophane trong vng 12 gi. Sau cng, ng vi
tng phn on mui bo ha xc nh hot tnh, hm lng protein ca PME v
tnh ton hiu qu tinh sch enzyme.

Hn 28: S kt ta PME bng dung dc mui (NH
4
)
2
SO
4
/NaCl
PME th (mL), lm lnh 4
C
(NH
4
)
2
SO
4
/
NaCl
35% (NH
4
)
2
SO
4
bo ha / 10% NaCl bo ha
kt ta 2gi, 4
C
Ly tm (10000 g, 20ph, 4
C)
Kt ta
Kt ta
Dung dch (mL)
.....
Dung dch (mL)
Ly tm (10000 g, 20ph, 4
C)
Ly tm (10000 g, 20ph, 4
C)
Ha tan trong dd m C-P (pH 4,5)
Thm tch (12gi)
Kt ta
Xc nh hot tnh enzyme, protein
(NH
4
)
2
SO
4
/
NaCl
45% (NH
4
)
2
SO
4
kt ta 2 gi, 4
C
(NH
4
)
2
SO
4
/
NaCl
95% (NH
4
)
2
SO
4
kt ta 2 gi, 4
C
- i vi kt ta bng dung mi hu c
Tin hnh lm lnh dung mi hu c trc n nhit t 5 0,5C, cho chm
chm vo cc ng nghim cha enzyme th theo cc t l nh b tr, lc nh v
n nh 2 gi 4C. Sau khi kt ta, hn hp c ly tm 4C, 10000
vng/pht trong 15 pht thu c phn kt ta. Phn kt ta c ha tan bng
dung dch m citrate phosphate (pH 4,5). Xc nh hot tnh PME, o hm lng
protein v tnh ton hiu qu tinh sch enzyme (hnh 2.9).

Hn 29: S kt ta PME bng dung mi u c
Lm lnh 5 0,5
C
Ethanol v PME
Lc nh, n nh 2 gi 4 C
Ly tm (10000 g, 20ph, 4 C)
Ha tan trong dd m C-P (pH 4,5)
Kt ta
Xc nh hot tnh enzyme, protein
Acetone v PME
N
3.1
N
3.2
N
3.3
N
3.4 N
3.5
N
3.6
N
4.1 N
4.2
N
4.3
N
4.4
Enzyme th (5 0,5
C)

Dung mi hu c
Kt qu thu nhn: Chn ra loi ha cht v t l s dng ph hp cho qu trnh
kt ta PME t A. niger da trn hot tnh ring, tinh sch v hiu sut thu hi.
Kt qu ti u ca th nghim ny s lm c s thc hin cc th nghim tip theo.

Th nghim 19: nh gi kh nng tinh sch PME t A. niger bng phng php
lc mng
Mc ch: nh gi kh nng tch loi, thu nhn PME bng cch s dng h
thng mng c khong phn on 50 kDa.
Tin hnh th nghim: Da trn kt qu kt ta protein bng mui v dung mi
hu c khc nhau, chn mu protein sau kt ta c hot tnh ring v tinh sch
cao nht tin hnh kho st lc mng. Tin hnh bm 50 mL kt ta ha tan
trong dung dch m citrate/phosphate c pH 4,5 vi tc 150 rpm qua h thng
lc mng QuixStand Benchtop Systems vi b lc mng c khong phn on 50
kDa. iu chnh sao cho p sut trn b mt mng khng qu 5 psi (da trn kt
qu ti u ca Hunh Ngc Oanh v Trn Ngc Hng, 2008). Thu dch qua lc,
xc nh hiu sut thu hi enzyme v hot tng ng. Nhit trong qu trnh
lc duy tr trong khong 4C.
Kt qu thu nhn: Hiu sut thu hi (%), tinh sch v hot tnh ring ca
PME sau qu trnh lc.
ii. X nh tnh ht PME thu nhn t A. niger tinh sh s b
Th nghim 20: Nghin cu nh hng ca nng c cht pectin n hot tnh
PME t A. niger.
Mc ch: Xc nh hng s K
m
v V
max
ca PME t A. niger vi c cht pectin.
Tin hnh th nghim: Tin hnh chun , xc nh hot tnh PME sau tinh sch
s b bng dung dch c cht pectin cc nng thay i t 0 10 g/L trong
dung dch NaCl 0,12 M.
S dng phng trnh Michaelis-Menten V
max
= f(S), phn mm Prism 5.0 xc
nh K
m
(mg/mL pectin) v V
max
(mol.min
-1
.mg
protein
-1
) ca ch phm PME.
Kt qu thu nhn: Hng s K
m
v V
max
ca PME t A. niger da vo s thay i
hot tnh ca enzyme ny theo nng c cht pectin.

Th nghim 21: nh hng ca nhit v pH mi trng n hot tnh PME t
A. niger.
Mc ch: Xc nh tng quan ca nhit v pH ti u cho hot ng ca ch
phm PME k thut thu nhn t A. niger.
Tin hnh th nghim: PME thu nhn c t qu trnh sinh tng hp A. niger,
sau khi tinh sch s b s c ha tan tr li bng dch m citrate phosphate 8
mc pH thay i t 2,5 6. C cht pectin cng c iu chnh pH n cc mc
tng ng bng dung dch HCl 0,01 M v NaOH 0,01 M. Tin hnh x l nhit
cc mu enzyme iu chnh pH 5 mc nhit (30 50C) trong b iu
nhit vi thi gian lu mu c nh 1 gi. Sau khi x l, cc mu enzyme c
lm lnh nhanh trong nc v xc nh hot tnh cn li ca PME tng ng
vi nhit v pH kho st.
Kt qu thu nhn: Gi tr nhit v pH ti u cho hot ng xc tc ca PME
t A. niger.

Th nghim 22: ng hc s v hot nhit ca PME t A. niger
Mc ch: Xc nh tnh n nh ca PME do tc ng ca nhit.
Tin hnh th nghim: Tin hnh x l enzyme 3 mc nhit 50, 60 v 70C
theo thi gian t 0 50 pht. Sau khi x l, cc mu enzyme c lm ngui
nhanh v xc nh hot tnh cn li ca PME trong tng mu.
Kt qu thu nhn: Hng s tc v hot k (pht
-1
) ca PME 3 mc nhit
v phng trnh ng hc s bin i hot tnh ca PME theo thi gian gia nhit.

Th nghim 23: Nghin cu nh hng ca NaCl v CaCl
2
n s thay i hot
tnh PME t A. niger
Mc ch: Tm ra quy lut thay i hot tnh ca PME t A. niger do nh hng
ca NaCl v CaCl
2
cc nng khc nhau.
Tin hnh th nghim: Chun b dung dch pectin 3,5 g/L ha tan trong dung
dch mui tng ng, c th: (i) 6 dung dch pectin 3,5 g/L trong NaCl ( 6 mc
kho st t 0; 0,05 M; 0,1 M; 0,12 M; 0,15 M v 0,2 M) v (ii) 6 dung dch
pectin 3,5 g/L trong CaCl
2
(nng thay i t 0; 0,005 M; 0,0075 M; 0,01 M;
0,0125 M v 0,015M). Tin hnh chun xc nh hot tnh ca enzyme v c
cht trong cc dung dch tng ng.
Kt qu thu nhn: S thay i hot tnh xc tc ca PME t A. niger cc nng
NaCl v CaCl
2
khc nhau.
2.3.5 Ni dung 4: ng dng h phm PME t A. niger trong i thin tnh
u tr ru qu
i. Nghin u iu kin tin x l thh hp ho hot ng PME i thin
ng h khm lnh ng
Th nghim 24: So snh nh hng ca phng thc b sung PME t nm mc
v CaCl
2
n s thay i c tnh cu trc khm lnh ng.
Mc ch: Xc nh th t b sung PME v CaCl
2
hiu qu nht cho s tng cu
trc khm.
B tr th nghim: Th nghim c thc hin vi 1 nhn t.
Nhn t O: Phng thc b sung PME v CaCl
2
vo khm, gm 7 nghim thc:
O
0
: i chng (0)
O
1
: Tin x l khm 40C, 20 pht, sau ngm trong dung dch CaCl
2

0,5% vi thi gian 30 pht (0A)
O
2
: Tin x l khm trong dung dch CaCl
2
0,15% 40C trong 20 pht
(0B)
O
3
: Tin x l khm vi PME 1 U/mL ha tan trong nc 40C, 20 pht,
sau ngm trong dung dch CaCl
2
0,5% vi thi gian 30 pht (1A)
O
4
: Tin x l khm vi PME 1 U/mL ha tan trong dung dch m citrate
phosphate pH 4,5 40C trong 20 pht, sau ngm trong dung dch CaCl
2
0,5%
vi thi gian 30 pht (1B)
O
5
: Tin x l khm vi dung dch PME 1 U/mL v CaCl
2
0,15% ha tan
trong nc 40C trong 20 pht (2A)
O
6
: Tin x l khm vi PME 1 U/mL v CaCl
2
0,15% ha tan trong dung
dch m phosphate pH 4,5 40C trong 20 pht (2B)
Cch thc hin
Bc 1: Chun b cc dung dch tin x l
Dung dh CCl
2
0,5% (th tch 1 lt): Ha tan 6,62 g CaCl
2
.2H
2
O trong nc ct,
nh mc bng nc ct n th tch 1 lt
Dung dch m phosphate pH 4,5 (xem ph lc 1)
Dung dh PME 1 U/mL: Da vo hot tnh PME sau tinh sch s b (C
1
), tin
hnh pha long vi nc theo cng thc:
C
1
.V
1
= C
2
.V
2

vi C
1
l hot tnh PME trong ch phm enzyme k thut (U/mL)
C
2
l hot tnh PME trong dung dch chn (1 U/mL)
V
1
l th tch ch phm enzyme c hot tnh C
1
cn s dng (mL)
V
2
l th tch dung dch PME 1 U/mL cn pha (mL)
Vy th tch dung mi b sung l V
2
V
1
(mL): s dng nc ct hay dung
dch m citrate pH 4,0 ty trng hp kho st.
Dung dh PME 1 U/mL v CaCl
2
0,15%: u tin, chun b dung dch PME
1 U/mL, sau b sung lng CaCl
2
thch hp t nng 0,15%. Gi s cn
1 lt dung dch PME 1 U/mL v CaCl
2
0,15%, lng CaCl
2
.2H
2
O cn s dng l
1,986 g.
Bc 2: Tin hnh th nghim
Th nghim c thc hin vi 2 phn:
a) S tng cu trc khm sau khi b sung PME t nm mc v CaCl
2

Cc mu khm vi kch thc nh nhau (20 mm x 15 mm) c b sung PME v
CaCl
2
theo cc phng thc k trn (t l khm v dch ngm l 150 g/200 mL).
Dung dch ngm hay chn phi ngp ht cc mu. o cng (lc ct) v DE
ca cc mu kho st.
b) S thay i cu trc khm qua tin x l sau 4 tun tr ng (-18C)
Khm sau khi c tin x l vi PME nm mc v CaCl
2
theo cc phng thc
k trn (7 nghim thc) s c cho cc bao PA (10 mu/bao), ng gi chn
khng 85%, tin hnh lnh ng nhanh nhit mi trng cp ng -86C
n nhit tm -18C. Tin hnh tr ng nhit -18C n 4 tun. Tan gi
bng khng kh nhit phng n khi nhit tm sn phm t 4C, o c
c tnh cu trc (th hin qua lc ct v DE) ca cc mu khm.
Kt qu thu nhn: cng ca cc mu khm th hin gi tr lc ct (g lc) v
c chuyn i sang cng tng i - t l gia cng ca tng mu so vi
mu khm ti. Thng k kt qu v xc nh nghim thc tin x l ti u gip
duy tr phm cht khm sau tr ng tt nht.

Th nghim 25: Kho st nh hng ca hot tnh PME t nm mc n s thay
i c tnh cu trc ca khm
Mc ch: Xc nh hot tnh PME t A. niger b sung thch hp n s n nh
c tnh cu trc ca khm lnh ng.
B tr th nghim: Th nghim c b tr vi 1 nhn t.
Nhn t P: Hot tnh PME (U/mL ch phm PME k thut), thay i cc mc
P
0
: 0 P
1
: 0,5 P
2
: 1 P
3
: 1,5 P
4
: 2
Trong th t b sung PME v CaCl
2
c xc nh t th nghim 24.
Cch thc hin
Chun b dung dh tin x l nng PME khc nhau: Tng t nh cch
chun b dung dch PME 1 U/mL (th nghim 25), trng hp ny C
2
thay i t
0 n 2 U/mL.
in hnh th nghim: Thc hin tng t th nghim 24. Cc mu khm vi kch
thc nh nhau (20 mm x 15 mm) c b sung dung dch PME cc nng
kho st cng vi s b sung CaCl
2
theo th t thch hp chn th nghim 24.
Kt thc tin x l, cc mu khm cng c xp vo bao b PA, ng gi chn
khng v lnh ng, tr ng -18C. Tan gi sau 4 tun tn tr v o c
cng (th hin qua lc ct) ca cc mu khm tng ng.
Kt qu thu nhn: cng ca cc mu khm th hin gi tr lc ct (g lc) v
c chuyn i sang cng tng i - t l gia cng ca tng mu so vi
mu khm ti. Thng k kt qu v xc nh hot tnh PME b sung thch hp
gip duy tr phm cht khm sau tr ng tt nht.
ii. nh gi t ng PME nm m trong i thin tnh u tr d
leo mui hu
Th nghim 26: Tc ng ca vic kch hot PME ni bo kt hp b sung CaCl
2

n s thay i cu trc da leo mui chua
Mc ch: Xc nh thi gian tin x l thch hp kch hot PME ni bo
nhm ci thin cu trc da leo mui chua.
Nhn t R: Thi gian tin x l (pht) da leo, thay i vi 5 mc
R
1
: 10 R
2
: 20 R
3
: 30 R
4
: 40 R
5
: 50
R
0
: i chng (khng x l)
Nhn t c nh: nhit tin x l 50C, nng CaCl
2
trong dung dch 0,4%.
Tin hnh th nghim: Quy trnh ch bin da leo mui chua c tin hnh da
trn quy trnh tham kho ca FAO (Battcock v Azam-Ali, 2001).
Nguyn liu da leo sau khi mua v c ra sch, o cu trc nguyn liu ban
u, sau em x l ch kho st vi thi gian tin x l c nh nhit
50C v hm lng CaCl
2
trong nc chn l 0,4%, Fleming et al., 1987). Tin
hnh cn mu chun b dch ngm (t l 1:1 hay 200 g mu/200 mL dch
ngm). Xp da leo vo dng c ln men ( ra sch, tit trng). Chun b dung
dch nc mui vi nng 4% ( thanh trng v lm ngui). Lu dung dch
nc mui s dng phi ngp b mt da leo nguyn liu t 3 n 5 cm, t l da
leo v nc mui 1:1 w/v. Sau y kn keo, tin hnh ln men, theo di qu
trnh v khi sn phm t n acid cn thit hay pH ca dch ln men nm
trong khong 3,0 3,3 qu trnh ln men dng li. Tin hnh o cu trc (lc
xuyn thu) v DE ca pectin.
Kt qu thu nhn
- Gi tr DE (%) sau khi tin x l v sau qu trnh mui chua.
- cng ca cc mu da leo mui chua th hin gi tr lc xuyn thu (g lc)
v c chuyn i sang cng tng i - t l gia cng ca tng mu sn
phm so vi mu da leo ti.
Th nghim 27: nh gi hiu qu ci thin cu trc da leo mui chua do tc
ng ca vic b sung ch phm PME t A. niger
Mc ch: Xc nh vai tr ca PME b sung v nng CaCl
2
s dng ph hp
i vi s n nh cu trc da leo mui chua.
B tr th nghim: Th nghim c thc hin vi hai nhn t
Nhn t S: Hot tnh ca PME (U/mL ch phm PME k thut) b sung trong qu
trnh tin x l, thay i 5 mc
S
1
: 0,5 S
2
: 1 S
3
: 1,5 S
4
: 2
S
0
: mu i chng, khng b sung PME
Nhn t T: Nng CaCl
2
(%) trong dch ngm, thay i 3 mc
T
1
: 0,3 T
1
: 0,4 T
3
: 0,5
S nghim thc kho st: 5 x 3 = 15 nghim thc.
Tin hnh th nghim
Chun b dh ngm: Tng t nh tin trnh chun b cho dung dch PME v
CaCl
2
phn tin x l khm lnh ng (th nghim 24).
Ch th hin: Tng t th nghim 26, dch ngm cha ng thi CaCl
2
v ch
phm enzyme b sung cc mc hot tnh khc nhau c kho st. Sau khi qu
trnh ln men kt thc, cu trc v DE ca da leo ban u cng nh trong sn
phm c o c.
Kt qu thu nhn: Hot tnh PME b sung thch hp v nng CaCl
2
ti u cho
hiu qu ci thin cu trc da leo mui chua tt nht.
CHNG 3 KT QU V THO LUN
3.1 Phn lp v xc nh c im hnh thi cc dng nm c c tnh ging
A. niger t v cc loi qu giu pectin
3.1.1 Kt qu phn lp dng nm en thu nhm Aspergillus t v loi
qu giu petin
Vic nui cy v tch rng, thu nhn cc dng nm mc en pht trin trn mi
trng PDA c b sung c cht cm ng l 1% pectin to t v cc loi qu giu
pectin (cam, hnh, chanh, bi, to, sung) c thc hin, da theo m t ca Bi
Xun ng v Nguyn Vn Huy (2000) v Klich (2002).
Kt qu quan st hnh thi cho thy, c s pht trin nhanh cc khun lc vi mu
trng sau 24 gi , sau 48 n 72 gi chuyn nhanh thnh mu nu en v en.
iu ny ph hp vi m t c tnh hnh thi bn ngoi cc dng nm thuc
nhm nm en Aspergillus ca Samson (1994) v Klich (2002). Trong nghin cu
phn lp v nhn din dng A. niger c kh nng sinh tng hp protopectinase,
Xia et al. (2009) cng xc nhn, s pht trin ca dng nm mc A. niger trn
mi trng PDA th hin s hnh thnh khun lc mu trng trong 12 24 gi
ngay sau khi , sau pht trin nhanh chng theo dng ta trn 48 72 gi ty
thuc c im tng dng. Nghin cu gn y ca Gupta et al. (2012), s pht
trin ca nm mc A. niger bt u vi s hnh thnh chm mu kem trn b mt
mi trng t ngy th hai sau khi trn mi trng PDA 30C, sau tng
trng theo dng xon c trn trn b mt mt cch nhanh chng trong vng 6
gi. Khi kch thc khun lc khong 2,5 3 cm, c s hnh thnh mt m ging
nh bt en trn b mt mi trng v chuyn sang mu en hon ton sau 72 gi
nui cy. L Th Thanh Hng v Nguyn Thy Chu (2005) khng m t c th
c im hnh thi ca cc dng nm c phn lp t cc ph ph phm nng
sn, tuy nhin nghin cu ny xc nhn, dng nm c xc nh c tnh hnh
thi l A. niger tip theo (k hiu C8.7/10, ngun phn lp l v qu chanh) u
c la chn t khun lc mu en - da trn kha phn loi ca Rapper v
Fennell, sau 72 gi nhit 30C.
La chn nhng khun lc ring r c mu en hoc mu nu en, em cy
chuyn nhiu ln trn mi trng PDA loi b tp nhim, thu c 60 dng t
12 ngun phn lp, c k hiu theo kt qu theo bng 3.1. Cc mu phn lp
ny u c mu nu en n en mt trn v mu vng hoc trng mt di
a petri (xem hnh 3.1, 3.2, 3.3 v 3.4).
Bng 31: S dng nm mc c phn lp t v cc loi qu giu pectin
TT Ngun phn lp S dng nm mc K hiu mu
1 Cam snh 3 Sa
1
, Sa
2,
Sa
3

2 Cam son 4 So
1
,So
2
,So
3
,So
4

3 Cam mt 6 M
1
,M
2
,M
3
,M
4
,M
5
,M
6

4 Hnh 4 H
1
,H
2
,H
3
,H
4

5 Bi da xanh 5 X
1
, X
2
, X
3
, X
4
, X
5

6 Bi Nm Roi 3 R
1
,R
2
,R
3

7 Chanh giy 5 G
1
,G
2
,G
3
,G
4
,G
5

8 Chanh nm 3 N
1
,N
2
,N
3

9 To Red Delicious 8 RD
1
, RD
2
, RD
3
, RD
4
, RD
5
,
RD
6
, RD
7
, RD
8

10 To Gala 8 GA
1
,GA
2
,GA
3
, GA
4
,GA
5
,
GA
6
,GA
7
, GA
8

11 To ta 6 T
1
,T
2
,T
3
, T
4
, T
5
, T
6

12 Sung 5 S
1
,S
2
,S
3
, S
4
,S
5

Tng s 60

Hnh 31: Mt trn ca
mu pn lp M
3

Hnh 32: Mt di ca
mu pn lp M
3

Mu M
3

Mu M
3

Tuy nhin, xc nh chnh xc y l dng A. niger, cc mu nm va phn lp
cn phi c nh loi da trn cc c im hnh thi c quan st di knh
hin vi th knh ph hp, s dng mi trng chuyn bit cho hot ng ca
dng nm mc ny.
Theo Klich (2002), mi trng MEA, CZ hay Czapek bin th nh Czapek Yeast
Agar (CYA25, CYA37), Czapek Yeast Agar vi 20% sucrose (CY20S) c th
c s dng. Tuy nhin, cc ti liu m t v hnh nh so snh s pht trin ca
nm mc thng tp trung hai loi mi trng CZ v MEA (Klich, 2002;
McClenny, 2005; Elfita et al., 2012). Chnh v th, mi trng CZ v MEA c
s dng nghin cu c im i th v vi th ca khun lc cc mu nm mc
va phn lp.
3.1.2 nh loi dng v phn lp d trn im i th khun l
Khi quan st cc khun lc ca cc dng nm mc phn lp trn c hai mi
trng MEA v CZ, kt qu nhn thy rng khun lc trn mi trng CZ u c
dng trn n gn trn, dng mt c nhiu chm en nhng tha. Si nm 3, 5
ngy c mu trng n vng nht, i khi mu kem sp hoc vng sm, trong khi
7 ngy th si nm a s nhn khng r. B mt khun lc trn mi trng MEA
t trn n gn trn, dng ht mn dy, thnh thong c rnh, cao thp khc nhau,
mt sau thng mu trng n vng v khng c sc t ha tan (bng PL 2.1).
Bn cnh , ng knh khun lc ca cc dng nm mc ny cng c o c.
Kt qu thu c cho thy, ng knh khun lc ca cc dng nm mc bin
thin trong khong 58 70 m trn mi trng CZ (bng PL 2.2) v 40 52 m

Hnh 33: Hn dng mt trn
ca kun lc
Hnh 34: Hn dng mt di
ca kun lc
trn mi trng MEA (bng PL2.3), ngha l khun lc nm mc thu c trn
mi trng MEA bao gi cng cho kch thc nh hn trn CZ. Tuy kch thc
khun lc trn hai loi mi trng l khc nhau, nhng c im chung v hnh
dng, mu sc gn nh ging nhau. iu ny cho thy hm lng dinh dng c
trong mi trng nh hng n kch thc khun lc (kch thc bin thin trong
khong xc nh) nhng khng lm bin i c im hnh thi ca khun lc
(Samson v van Reenen-Hoekstra, 1988). L Th Thanh Hng v Nguyn Thy
Chu (2005) cng ghi nhn c im hnh thi ca dng nm A. niger C8.7/10 sau
7 ngy trn mi trng CZ c cc tnh cht tng t nh cc mu nm kho st,
in hnh nh khun lc c hnh trn, mu en, mp hnh tia, c hch nhn, tuy
nhin ng knh khun lc nh hn (t 2,5 3 cm). Khi so snh c im ca 60
dng nm mc phn lp c vi c im ca nm mc A. niger c m t
trong kha phn loi ca Klich, kt qu cho thy 60 dng nm mc ny u c c
im rt ging nhm loi A. niger (Klich, 2002). Klich cng xc nhn, khun
lc trn mi trng MEA thng c ng knh hi nh hn so vi s pht trin
ca n trn CZ, mt s khun lc c ng knh t 30 m. iu ny cng ph hp
vi kt qu thc t kho st.
Da vo s khc bit ca mu cy 3 im khun lc ca 60 dng va phn lp
xc nh c im v chn la cc dng c tnh cht ca A. niger.

Hn 35: c im kun lc ca dng S
2
v G
1
ki c cy 3 im
trn mi trng CZ
Dng S
2
Dng G
1


Hnh 3.5 th hin c im khun lc ca hai dng in hnh S
2
v G
1
khi c
cy 3 im trn mi trng CZ v hnh 3.7 th hin c im khun lc ca dng
H
2
c cy 3 im trn mi trng MEA, so snh vi dng A. niger chun
hnh 3.6 (Klich, 2002).
Theo kho st ca Samson et al. (2007), khun lc ca nm mc A. niger khi c
cy 3 im trn mi trng CZ v MEA (7 ngy, 25C) c mu nu nh b c
ph, pht trin nh hnh r qut v 3 pha vi hch nhn gia, ta trn, khng c
vin ngoi. Trong khi , khun lc ca A. niger trn mi trng MEA c kch
thc nh hn, vn cn ng vin trng bn ngoi.
Nhng c im i th v khun lc ca 60 dng phn lp va c m t trong
bng PL 2.1 chng t 60 dng ny u mang c im i th ca A. niger c
m t trong kha phn loi ca Klich (2002). Bn cnh , khi so snh 60 dng
ny vi mu i chng l dng A. niger R92 v dng V cng cho thy s tng
ng v c im i th ca khun lc. Tuy nhin, nh loi chnh xc
A. niger, c im bo t nh v b my mang bo t cn c quan st mt cch
chi tit di knh hin vi phng i 10 10, 40 10 v 100 10 ln.
3.1.3 im bo t nh v b my mng bo t dng nm m
phn lp
Cc c im vi th ca bo t nh v b my mang bo t ca 60 dng nm mc
phn lp c m t chi tit bng PL 2.2. c im vi th gp phn xc nh
60 dng ny l A. niger.
Hnh 3.6: Dng A. niger i cng
(Klich, 2002)
Hnh 3.7: Dng nm mc H
2

trn mi trng MEA
Dng H
2

Trn mi trng MEA v CZ a s si nm dng mnh, phn nhnh v c vch
ngn (hnh 3.8). Cung bo t c kch thc (550) 600 2500 (10) 12,5 17,5
(20) m, vi thnh dy, phng, trong sut khng c vch ngn, c bit c mu
vng hoc nu sng gn nh (hnh 3.9, 3.11 v 3.12).

Hnh 38: Si nm c vc ngn ca dng So
2
vt kn 4X
Hn 39: Hn n dng So
2
vt kn 4X v 10X

Hnh 310: Hn n bo t di kn in vi ca dng A. niger
Vch ngn
10X
4X

Hn 311: T bn cn non (pt trin 1 tng) dng So
2

Hn 312: Hai vng t bn v bo t ca dng So
2

1 vh trn th = 1,25 m
Bng nh gi hnh cu r (hnh 3.11), c ng knh trung bnh t 32 83 m
(MEA) v 35 80 m (CZ). c bit c s hin din ca th bnh 2 tng vi tng
1 di gp i tng 2 v thnh thong c vch ngn (hnh 3.12). Tuy nhin, sau 7
ngy bo t trn bao ph xung quanh th bnh rt nhiu nn rt kh phn bit tng
1 v 2, v th khng th o kch thc tng tng. Kch thc th bnh dao ng t
(12,5) 17,5 30 (37,5) m v thng c hnh elip. Bo t trn hnh cu, sn si v
khng u mp trng nh c gai (hnh 3.10, 3.12), c ng knh dao ng t 3
5 m. Kiu pht sinh bo t dng hnh tia ta trn (hnh 3.12). Trong , c
im 2 tng th bnh v bo t c gai l 2 c im quan trng nht trong qu
trnh nh loi. Tht vy, khi iu kin mi trng nui cy thay i (MEA hoc
CZ) cc c im v kch thc s bin thin trong khong xc nh, ring 2 c
im th bnh 2 tng v b mt bo t sn si vn c th hin (hnh 3.10 v
3.12). Nghin cu ca Samson et al. (2007) cng ghi nhn, bo t A. niger trn
mi trng CZ c ng knh khong 3,5 5 m v bng nh gi c kch thc
45 80 m. Larone (2002) cng a ra cc thng tin v c im vi th ca
A. niger khi quan st di knh hin vi nh: th bnh 2 tng, cung bo t nhn,
trong sut, di gn 3000 m, chuyn mu sm dn gn nh v kt thc bng
bng nh gi hnh cu, c ng knh khong 30 75 m. Theo Larone (2002),
bo t A. niger cng c dng hnh cu, mu nu en, tuy nhin khng thy kt qu
o c kch thc bo t. Tng t, L Th Thanh Hng v Nguyn Thy Chu
(2005) cng ghi nhn s hnh thnh bo t ca A. niger c mu nu en, dng
hnh cu, ng knh khong 4 5 m v cung bo t nhn, kch thc 1,5 3
cm, bng nh gi hnh gn cu, c hai hng th bnh, kch thc 45 75 m. c
bit, nghin cu ny cng ghi nhn c im ca khi bo t pht sinh kiu hnh
tia lng lo, rt trng khp vi hnh nh minh ha s pht trin ca bo t hnh
3.12 (hnh bn tri). ng thi, kt qu quan st c im vi th ca 60 dng nm
phn lp cng rt ph hp vi m t loi A. niger trong kha phn loi cc loi
nm mc Aspergillus ca Klich (2002): Khun lc 7 ngy hnh trn hoc gn trn,
c ng knh (30) 50 70 mm (MEA), 55 70 m (CZ). Khun lc c mu en,
nu en, khun ty mu trng n mu vng, thng khng thy r. Cung bo t
dy, phng trong sut, kch thc (300) 400 3000 (7) 12 17 (20) m. Bng
nh gi hnh cu c ng knh (20) 30 75 (85) m. Th bnh 2 tng, tng th
nht c kch thc 12 20 (40) 3 6 m, thnh thong c vch ngn, kch
thc tng th hai dao ng trong khong 7 10 3 4 m v khng c vch
ngn. Bo t trn hnh cu, sn si, mp khng u, g gh v c ng knh (3)
3,5 4,5 (5) m.
Da trn cc c im v vi th c th khng nh, 60 dng kho st c c im
ging A. niger, khng c s trng lp vi dng A. awamori v A. foetidus cch
lin kt ca bo t nh (xem hnh PL 1.2 v PL 1.3).
Vi mc tiu ch yu ca ti l tuyn chn c dng nm mc A. niger c
hiu cho qu trnh sinh tng hp PME enzyme thuc nhm pectinase, bc tip
theo sau qu trnh phn lp l la chn nhanh cc dng nm phn lp c kh
nng thy phn pectin trc khi tin hnh xc nh chnh xc kh nng sinh tng
hp PME ca cc dng thch hp nht.
3.2 Tun chn dng nm mc c hiu i vi PME
3.2.1 nh gi kh nng sinh petinse dng nm phn lp
Vic kho st kh nng sinh tng hp pectinase ca cc dng nm mc va phn
lp l bc u tin cho vic tuyn chn dng nm thch hp cho vic thu nhn
PME tip theo. Da trn ng knh vng phn gii pectin trn mi trng chn
lc thch pectin sau 48 gi 30C, phn nhm nm mc c tnh c hiu vi
pectinase khc nhau, kt qu tng hp bng 3.2 (s liu th c tng hp
bng PL2.4). Ngoi ra, kt qu so snh hiu qu sinh tng hp pectinase ca cc
dng nm mc thuc nhm c hiu cao (D > 20 mm) c tng hp bng 3.3.
Bng 3.2: Phn nhm nm mc c tnh c hiu vi enzyme pectinase
ng knh vng
phn gii D (mm)
S dng Tn dng/ngun gc
D = 11,67 15 11 G
3
, G
5
(chanh giy), X
4
, X
5
(bi da xanh), M
1
, M
2
,
M
5
(cam mt), T
6
(to ta), GA
3
(to Gala), RD
7
, RD
8

(to Red Delicious)
D = 15,25 cn 20 35 + i
chng V
phn lp t
t
S
1
, S
2
, S
3
, S
4
, S
5
(sung), G
1
(chanh giy), N
2
, N
3

(chanh nm), H
1
, H
2
, H
3
(hnh), M
3
, M
4
, M
6
(cam
mt), Sa
1
(cam snh), So
1
, So
3
, So
4
(cam son), R
2
, R
3

(bi Nm Roi), X
1
, X
2
(bi da xanh), T
3
, T
4
, T
5
(to
ta), GA
4
, GA
5
, GA
6
, GA
7
, GA
8
(to Gala), RD
1
, RD
2
,
RD
3
, RD
5
, RD
6
(to Red Delicious), V (i chng)
D 20 14 dng
+ i
chng R92
G
2
, G
4
(chanh giy), N
1
(chanh nm), H
4
(hnh), Sa
2
,
Sa
3
(cam snh), So
2
(cam son), R
1
(bi Nm Roi),
X
3
(bi da xanh), T
1
, T
2
(to ta), GA
1
, GA
2
(to
Gala), RD
4
(to Red Delicious), R92 (i chng, Nga)

T kt qu tng hp bng 3.2 cho thy, hot tnh pectinase thu c ca 62
dng nm mc, trong gm 60 dng nm phn lp v 2 dng gc i chng l
R92 v dng V, c th chia thnh 3 nhm ring bit thng qua ng knh trung
bnh ca vng phn gii pectin dao ng t 11,67 n 24,33 mm (bng PL2.4).
Hu ht cc ngun t chanh giy, cam mt v bi da xanh u xut hin dng
nm mc cho hot tnh pectinase thp (th hin ng knh trung bnh vng
phn gii pectin nh), ng thi trong cc dng tuyn chn (D 20 mm), khng
c dng no c phn lp t sung. Ngc li, d ch c 4 dng c phn lp t
cam snh nhng c n hai dng thuc nhm ny cho hot tnh pectinase cao l
Sa
2
v Sa
3
. Vi 6 dng c phn lp t to ta thu c 2 dng T
1
v T
2
c
hot tnh pectinase cao. iu ny gp phn khng nh tc ng ca ngun phn
lp n kh nng sinh tng hp mt loi enzyme c hiu, c th y l
pectinase.
Bng 3.3: Kt qu thng k s khc bit ng knh vng phn gii pectin (D) ca
cc dng nm mc thuc nhm c hiu cao vi pectinase
TT Tn dng nm mc ng knh vng phn gii (mm)
1 G
2
20,00
ab
0,50
2 H
4
20,00
ab
0,50
3 GA
1
20,25
ab
0,50
4 T
2
20,59
abc
0,47
5 Sa
2
21,17
abcd
0,76
6 G
4
21,50
abcd
0,87
7 RD
4
21,75
bcd
1,15
8 X
3
21,83
cd
1,26
9 GA
2
22,00
cde
0,58
10 R92 22,25
def
0,44
11 Sa
3
22,33
def
1,15
12 T
1
23,38
efg
1,15
13 N
1
23,67
fg
1,15
14 So
2
24,33
gh
0,58
15 R
1
25,58
h
0,68
T s F 7,16
Gi tr P 0,00
C gi tr mu t i km ging nhu ng mt t kh bit khng ngh v mt thng k tin
y 95% theo php th Dunn tin y 95%
T 60 dng nm mc c phn lp v 2 dng i chng tuyn chn c 14
dng cho hot tnh pectinase cao nht, th hin ng knh trung bnh vng
phn gii t 20 mm tr ln, xp theo th t tng dn gm G
2
v H
4
(20 mm), GA
1

(20,25 mm), T
2
(20,59 mm), Sa
2
(21,17 mm), G
4
(21,25 mm), RD
4
(21,75 mm),
X
3
(21,83 mm), GA
2
(22 mm), V92 (22,25 mm), Sa
3
(22,33 mm), T
1
(23,38 mm),
N
1
(23,67 mm), So
2
(24,33 mm) v R
1
(25,58 mm). Mu i chng R92 thuc
nhm cho hot tnh pectinase cao (ng knh vng phn gii pectin l 22,25
mm). Trong khi , dng A. niger phn lp t t thuc nhm sinh pectinase trung
bnh (16,67 1,53 mm). Hnh 3.13 n hnh 3.16 th hin s phn gii vng
pectin ca cc dng nm mc khc nhau, c so snh vi mu i chng (nc)
khng lm thy phn pectin (hnh 3.15).
Kt qu thu c c tng ng cao vi cc nghin cu ca cc tc gi trong
v ngoi nc trong cc kho st c lin quan. Kt qu kho st ca L Th Thanh
Hng v Nguyn Thy Chu (2005) cho thy vi 50 chng A. niger c phn
lp t 80 mu nguyn liu, ch c 4 chng c kh nng sinh pectinase hot tnh
cao c ngun gc t v chanh, bi. Nm mc A. niger c phn lp t v thanh
long hay v qu c ph u cho hot tnh pectinase km. Trong khi , nghin cu
gn y ca Sharma et al. (2011) khi tin hnh phn lp A. niger trn b to, v
cam, bt me, c rt, u, t, nc thi t nh my tinh bt v bnh m cho thy
hot tnh PME cao nht t A. niger theo th t ngun phn lp:
Cam > bt me > c rt > v qu l > u phng > b to > bnh m > nc thi
nh my tinh bt > t.

Hn 313: Mt trn vng pn gii pectin
ca dng nm So
2

Hn 314: Mt di vng pn gii
pectin ca dng nm So
2

Nghin cu ca Patil v Chaudhari (2010) cng cho thy, nm mc phn lp t
ngun t cha cht thi l rau qu giu pectin nh my ch bin nc cam u
c hiu qu sinh tng hp pectinase rt cao.

Hnh 315: Mu i cng nc Hnh 316: Mu i cng A. niger V
/C dng V /C nc
So
2
(mt di)
So
2
(mt trn)
V cam cng l mt trong nhng ngun phn lp v tuyn chn dng A. niger c
hiu cho kh nng sinh tng hp PME cao (vi 3/14 dng). iu ny c l chu s
chi phi ca thnh phn c cht pectin c hiu cho hot ng ca PME. V cam,
bi v to c bit n l nhng ngun c ester ha ca pectin mc 65
75%, l iu kin thch hp cho hot ng thy phn pectinase, c bit l PME
(Aehle, 2004; Hamdy et al., 2005; Marcon et al., 2005) v l ngun phn lp cc
dng nm mc khc nhau c hiu vi pectinase (Phuangsinou et al., 2008,
Chaidedgumjorn et al.,2009).
Nh vy, 14 dng nm mc c ng knh vng phn gii pectin t 20 mm (G
2
,
H
4
, GA
1
, T
2
, Sa
2
, G
4
, RD
4
, X
3
, GA
2
, Sa
3
, T
1
, N
1
, R
1
, So
2
) cng vi dng A. niger
i chng R92 c tuyn chn cho kho st chn la dng nm mc c hiu i
vi qu trnh sinh tng hp PME.
3.2.2 uyn hn dng nm m kh nng sinh tng hp PME hot tnh o
nh hng ca dng nm mc n hiu qu sinh tng hp PME c xc nh
da trn kt qu thng k s khc bit v ng knh vng phn gii pectin v
hot tnh enzyme sinh ra t qu trnh ln men lng, c so snh vi mu i chng
(R92). Kt qu c tng hp bng 3.4.
Tin hnh tuyn chn cc dng nm mc c hiu vi PME t cc dng sinh
pectinase hot tnh cao (c xp theo th t c ng knh vng phn gii tng
dn) cho thy, khng c s tng quan hon ton gia cc dng c kh nng sinh
tng hp pectinase cao v PME, mi dng nm mc c hiu vi mi nhm
enzyme khc nhau trong h pectinase nh PG, PME hay PL. C th, cc dng
phn lp t chanh giy (G
2
v G
4
) c ng knh vng phn gii pectin trn mi
trng pectin agarose cng nh hot tnh PME thu nhn t qu trnh ln men
lng trn mi trng chuyn bit thp. Mc d vy, 4 dng c hiu cao vi
pectinase u c kh nng sinh tng hp PME tt, c th l dng T
1
(phn lp t
to ta), N
1
(phn lp t v qu chanh nm), So
2
(phn lp t v qu cam son) v
R
1
(phn lp t v qu bi Nm Roi). Kt hp vi nghin cu v kh nng sinh
tng hp pectinase c kho st, c th nhn thy rng, vi cng mt ngun
phn lp, khng phi tt c cc dng nm mc cng loi u c kh nng sinh
tng hp mt loi enzyme nht nh ging nhau (Sharma et al., 2011).
Bng 34: S tha i ng knh vng phn gii pectin v hot tnh PME sinh tng
hp t cc dng nm mc c hiu vi pectinase
Dng nm mc ng knh vng phn gii (mm) Hot tnh PME (U/mL)
G
2
10,95
ab
0,23 5,43
ab
0,08
H
4
11,43
bcd
0,59 5,53
bc
0,20
GA
1
11,77
d
0,25 5,52
bc
0,26
T
2
11,43
bcd
0,45

5,48
abc
0,68
Sa
2
11,80
d
0,26 6,00
cd
0,47
G
4
10,46
a
0,47 4,94
a
0,23
RD
4
11,37
bcd
0,30 5,30
ab
0,20
X
3
11,87
d
0,32 6,03
cd
0,37
GA
2
11,60
cd
0,46 5,44
ab
0,21
R92 (Nga) 10,97
abc
0,42 5,30
ab
0,33
Sa
3
11,73
d
0,30 6,03
cd
0,17
T
1
13,17
e
0,25 6,59
ef
0,27
N
1
13,10
e
0,30 6,34
de
0,09
So
2
14,87
f
0,35 7,50
g
0,13
R
1
14,47
f
0,60 7,00
fg
0,21
T s F 32,18 16,74
Gi tr P 0,00 0,00
Hnh nh minh ha hiu qu thy phn pectin ca 2 dng A. niger in hnh So
2

(hnh 3.17 b) v G
2
(hnh 3.17 a) cho thy, ng knh vng phn gii pectin ca
dng G
2
kh nh khi so snh mu So
2
.

Hn 317: Vng pn gii pectin trn mi trng pectin - agarose sau 24 gi
37C v nn din vi tuc t CTAB 1%
(a) Dng G
2
ng knh vng phn gii petin nh so vi vng sinh trng nm m
(b) ng phn gii petin v vng sinh trng nm m gn nh trng nhu i vi dng So
2

Cc nghin cu khc ca Manhania et al. (2010) cng cho thy, t 20 dng
Aspergillus c phn lp t cng mt loi qu to, ch c 5 dng c kh nng
phn gii pectin trn mi trng c hiu. Sharma et al. (2011) cng ch tm thy
hai dng A. niger phn lp t v cam v bt me trong s 48 dng A. niger phn
lp c tnh c hiu cao vi PME. Kt qu nghin cu v sinh hc phn t ca
Parenicov (2000) ghi nhn s khc nhau v gene hnh thnh enzyme ca mi
dng nm mc, c to thnh thng qua qu trnh phin m v dch m v chu
s chi phi ca ngun carbohydrate s dng. Chnh v th, i vi qu trnh chn
lc t nhin, cc dng nm mc c phn lp t v tr ph hp, pht trin trong
iu kin mi trng thch ng c kh nng sinh tng hp cc enzyme c hiu
khc nhau.
Tnh cht ngun nm mc l mt trong nhng yu t quan trng nht c tc ng
n hiu qu ln men sinh tng hp enzyme (ng Th Thu v Nguyn Th Xun
Sm, 2009b). Chnh v th, vic nghin cu hot ha s hot ng ca dng nm
mc c chn lc cho tng enzyme cn c quan tm. Vic kho st s tng
tc ca hai dng nm mc cng loi nhm tng cng hiu qu hot ng sinh
enzyme l bc c bn, u tin c th c p dng (Zhong-Tao et al., 2009).
Trn c s ny, ngh xc nh kh nng kt hp 4 dng nm mc c kh nng
sinh tng hp PME t hiu qu cao nht (dng T
1
, N
1
, So
2
, R
1
).
3.2.3 Kh nng kt hp dng nm m hiu n hiu qu thu nhn PME
Vic kho st kh nng kt hp tng cp ca 4 dng nm mc c hiu i vi
hiu qu thu nhn PME (dng T
1
, N
1
, So
2
, R
1
) l c s cho vic chn ra t l kt
hp gia hai dng nm mc thch hp sinh tng hp PME tip theo.
Khi quan st s pht trin ca nm mc v s thay i ng knh vng phn gii
pectin trn mi trng pectin agarose cho thy, vic kt hp hai dng nm mc
c cng hot tnh thp (T
1
v N
1
) hay cng hot tnh cao (So
2
, R
1
) u hnh thnh
vng phn gii dng ta trn, ng u (hnh 3.18a minh ha s kt hp ca hai
dng So
2
v R
1
), trong khi s kt hp ca mt dng mnh v mt dng yu (T
1
v
R
1
; N
1
v R
1
; N
1
v So
2
, T
1
v So
2
) u to nn vng trn khng ng u (hnh
3.18b).

Hnh 3.18: n ng ca s kt p cc dng nm mc kc nau n s tay i
vng pn gii pectin trn mi trng pectin - agarose
Kt qu o c s thay i ng knh vng phn gii pectin cng nh hot tnh
PME thu nhn cc phng thc kt hp c tng hp bng 3.5.
Bng 35: nh gi kh nng kt hp hai dng nm mc n hiu qu thu nhn
enzyme PME
Dng nm mc ng knh vng phn gii (mm) Hot tnh PME (U/mL)
T
1
13,22
a
0,25 6,55
ab
0,24
N
1
13,20
a
0,24 6,24
a
0,12
So
2
14,76
b
0,31 7,61
d
0,13
R
1
14,18
b
0,55

6,92
bc
0,21
T
1
N
1
14,40
b
0,66 7,20
cd
0,32
T
1
So
2
13,13
a
0,45 6,57
ab
0,23
T
1
R
1
13,00
a
0,46 6,50
a
0,23
N
1
So
2
12,73
a
0,25 6,37
a
0,13
N
1
R
1
12,43
a
0,49 6,22
a
0,25
R
1
So
2
16,10
c
1,01 8,05
e
0,50
T s F 14,12 16,90
Gi tr P 0,00 0,00
Kt qu bng 3.5 cho thy, 4 dng nm mc tuyn chn, c tnh c hiu cao
vi pectinase u c kh nng sinh tng hp PME tt, c th l dng T
1
(phn lp
t to ta), N
1
(phn lp t v qu chanh nm), So
2
(phn lp t v qu cam son)
v R
1
(phn lp t v qu bi Nm Roi).
t l kt hp hai dng nm mc l 1 : 1, vic kt hp hai dng nm c hot tnh
thp hn l T
1
v N
1
vi hai dng mnh (T
1
So
2
, T
1
R
1
hay N
1
So
2
v N
1
R
1
) u
khng cho hiu qu trong vic ci thin hot tnh PME. ng knh vng phn
gii pectin cng nh hot tnh PME thu nhn c u mc tng ng vi
dng i chng T
1
v N
1
. Trong khi , s kt hp ca hai dng c hot tnh
tng ng nhau (T
1
N
1
v R
1
So
2
) u cho hiu qu ci thin tt hn mu ring l.
ng knh vng phn gii pectin ca t hp T
1
N
1
c gi tr l 14,40 0,66 mm
cao khc bit c ngha v mt thng k khi so snh vi hai dng T
1
v N
1
ring
l (13,22

0,25 mm v 13,20 0,24 mm), hot tnh PME cng gia tng t 6,55
0,24 U/mL v 6,24 0,12 U/mL n gi tr 7,20 0,32 U/mL. Ring trng hp
kt hp hai dng c kh nng tng hp PME vi hot tnh cao hn (R
1
v So
2
),
hot tnh enzyme gia tng t 7,61

0,13U/mL v 6,92 0,21 U/mL n gi tr
8,05 0,50 U/mL, tng ng vi ng knh vng phn gii tng t 14,76
0,31mm v 14,18 0,55 mm ln n gi tr 16,10 1,01 mm. S kt hp ca hai
dng R
1
v So
2
cho hiu qu thu nhn PME cao vt tri v khc bit c ngha
v mt thng k khi so snh vi cc nghim thc cn li. Zhong-Tao et al. (2009)
khng nghin cu s kt hp ca hai dng mnh vi nhau, tuy nhin kt qu cng
cho thy, s kt hp hai dng yu hn s gip gia tng ng k hiu qu sinh tng
hp ng thi enzyme pectinase, protease v cellulase. Hiu qu sinh tng hp
pectinase trong trng hp kt hp hai dng rt mnh v rt yu (th hin hot
tnh enzyme tng ng ca hai dng trc khi kt hp l 19265 505 U/g v
1521 42 U/g, gp khong 12 ln) vn thp hn trng hp ch s dng mt dng
mnh n l (17864 530 U/g). Nghin cu ca Colombatto et al. (2005)
chng minh s tng tc ca hai dng ng kh nng nhm gia tng hiu qu sinh
tng hp mt loi enzyme khc (cellulase). iu ny hon ton ph hp vi quy
lut pht trin ca vi sinh vt trong qu trnh sinh trng v pht trin (ng Th
Thu v Nguyn Th Xun Sm, 2009b).
Da trn kt qu kho st, hai dng nm mc c phn lp t v qu cam son
(So
2
) v v qu bi Nm Roi (R
1
) c la chn nghin cu t l kt hp ca
hai dng ny trong qu trnh sinh tng hp PME.
3.2.4 nh gi kh nng kt hp hi dng So
2
v R
1
hiu theo t l kh
nhu n hiu qu sinh tng hp PME hot tnh o
nh hng t l kt hp gia hai dng nm mc So
2
v R
1
n kh nng sinh
PME c tng hp bng 3.6.
Bng 36: nh hng ca t l kt hp hai dng R
1
v So
2
n hiu qu sinh tng
hp PME hot tnh cao
T l kt hp ca R
1

v So
2

ng knh vng phn gii
(mm)
Hot tnh PME
(U/mL)
1: 1 16,10
bc
1,01 8,05
b
0,50
1: 2 16,27
c
0,57 8,12
b
0,23
1: 3 15,13
ab
0,40 7,52
a
0,12
1: 4 14,90
a
0,40

7,42
a
0,13
4: 1 14,43
a
0,68 7,15
a
0,23
3: 1 14,77
a
0,70 7,35
a
0,30
2: 1 15,23
abc
0,42 7,42
a
0,15
T s F 6,90 5,61
Gi tr P 0,014 0,038
T kt qu thng k ng knh vng phn gii pectin trn mi trng agarose
pectin v hot tnh PME sau qu trnh ln men lng s dng mi trng CZ
pectin, s thay i t l kt hp ca hai dng nm mc khng c s khc bit ln
n hiu qu sinh tng hp PME. cc t l kt hp ca R
1
v So
2
l 1 : 3, 1 : 4
hay ngc li, ng knh vng phn gii pectin cng nh hot tnh PME thu
c khng c s sai khc v mt thng k. iu ny c l l do s khc bit v
kh nng thu nhn PME ca tng dng nm mc ring l khng ln (Zhong - Tao
et al., 2009). t l kt hp ca dng R
1
v So
2
l 2: 1 cng khng cho hiu qu
ci thin hot tnh enzyme. Ch t l s dng 1 : 1 hay 1 : 2 ca hai dng nm
mc ny, hiu qu thu nhn PME t cao nht. Nghin cu ca Zhong - Tao et al.
(2009) cng tm ra t l 2 : 1 ca s kt hp hai dng A. niger sinh tng hp
pectinase mnh v yu cng nh t l 1 : 1 ca hai dng A. niger ng kh nng
(hot tnh thp) l iu kin kt hp thch hp cho s ci thin ng k s sinh
tng hp ng thi pectinase, cellulase v protease.
T cc kt qu thu nhn cho thy, vic nghin cu tuyn chn v kt hp cnh
tranh hai dng A. niger trong qu trnh sinh tng hp PME l c tnh kh thi. Hai
dng nm mc c hiu i vi PME l A. niger So
2
v R
1
c phn lp t v
cam son v bi Nm Roi c th gia tng hn na hot tnh PME khi c kt
hp vi nhau t l R
1
: So
2
l 1 : 1 hay 1 : 2. Thng qua kt qu kho st, t l
kt hp ca R
1
: So
2
l 1 : 1 c s dng cho nghin cu ti u ha iu kin
sinh tng hp PME vi vic iu khin c cht v qu trnh ln men.
3.3 Bc u nh danh mt s dng A. niger phn lp bng k thut
sinh hc phn t
Chn 04 dng nm mc c kh nng sinh tng hp PME hot tnh cao v dng i
chng A. niger R92, khuch i on 260 bp ca vng 28S rDNA bng cp mi
c chng U
1
v U
2
. Ph in di sn phm PCR c nhn ln t DNA ca 4
dng nm mc N
1
, T
1
, R
1
v So
2
c bng tng ng v tr 260 bp so vi thang
chun v tng ng vi bng ca dng i chng A. niger R92 (hnh 3.19).

Hnh 319: P in di ca sn pm PCR ca cc mu nm c kuc i
M: thng hun 100 bp plus; 5 ging n li tng ng vi 5 mu nm R92 (i hng), T
1
, N
1
, So
2
v R
1

Sn phm PCR ca 4 dng nm trn cng vi dng i chng A. niger R92 c
s dng gii trnh t DNA v s dng phn mm Blast N, so snh vi trnh t
DNA ca cc dng nm mc c trong ngn hng d liu NCBI.
Trnh t gene 28S rRNA ca cc dng ny c xc nh gm:
- Trnh t gene 28S rRNA ca dng T
1

TGTGAAATTGTTGAAAGGGAAGCGCTTGCGACCAGACTCGCCCGCGGG
GTTCGCCGGCATTCGTGCCGGTGTACTTCCCCGTGGGCGGGCCAGCGT
CGGTTTGGGCGGCGGTCAAAGGCCCCTGGAATGTAGTGCCCTCCGGGG
CACCTTATATAGCCAGGGGTGAATGCGGCCAGCCTGGACCGAGGAAC
GCGCTTCGGCACGGACGGACTGGCATAATGGTCGTAAACGACCCGTCT
TGAAACACGGACCAAGGAGTC (Tng s nucleotid: 258)
- Trnh t gene 28S rRNA ca dng R
1

CCTGGCTATAAGGTGCCCCGGAGGGCACTACATTCCAGGGGCCTTTGA
CCGGCCGCCCAAACCGACGCTGGCCCGCCCACGGGGAAGTACACCGC
CACGAATGCCGGCTGAACCCCGCGGGCGAGTCTGGTCGCAAGCGCTTC
CCTTTCAACAATTTCACA (Tng s nucleotid: 166)
200 bp
300 bp
- Trnh t gene 28S rRNA ca dng N
1

CGGGCCAGCGTCGGTTTGGGCGGCCGGTCAAAGGCCCCTGGAATGTAG
TGCCCTCCGGGGCACCTTATAGCCAGGGGTGCAATGCGGCCAGCCTGG
ACCGAGGAACGCGCTTCGGCACGGACGCTGGCATAATGGTCGTAAAC
GACCCGTCTTGAAACACGGACCA (Tng s nucleotid: 166)
- Trnh t gene 28S rRNA ca dng So
2
:
AATTGTTGAAAGGGAAGCGCTTGCGACCAGACTCGCCCGCGGGGTTCA
GCCGGCATTCGTGCCGGTGTACTTCCCCGTGGGCGGGCCAGCGTCGGT
TTGGGCGGCCGGTCAAAGGCCCCTGGAATGTAGTGCCCTCCGGGGCAC
CTTATAGCCAGGGGTGCAATGCGGCCAGCCTGGACCGAGGAACGCGCT
TCGGCACGGACGCTGGCATAATGGTCGTAAACGACCCGTCTTGAAACA
CGGACCAAGGAGTCA (Tng s nucleotid: 254).
Biu biu din mt on trnh t DNA ca mt mu nm in hnh So
2
cng
nh t l tng ng v gene ca dng nm v dng A. niger NBL c t l ng
hnh cao nht c th hin hnh 3.20 v 3.21 (cc biu tng ng ca cc
dng nm cn li xem Ph lc 2).
Kt qu cho thy, cc dng nm ny c t l tng ng di truyn vi nm mc
A. niger, c th:
- Dng T
1
c t l ng hnh vi A. niger contiq An03c0110 l 99%
- Dng R
1
c t l ng hnh vi A. niger US4, NBL v BR l 99%
- Dng So
2
c t l ng hnh 99% vi A. niger NBL, BR v contiq An03c0110
- Dng N
1
c t l ng hnh vi A. niger NBL, BR, RA402 v An03c0110 cng
l 100%.
T kt qu nh danh bc u ca 4 dng nm c phn lp, gi tn tng ng
cho 4 dng ny l :
Dng T
1
: Aspergillus niger T
1

Dng N
1
: Aspergillus niger N
1
Dng R
1
: Aspergillus niger R
1
Dng So
2
: Aspergillus niger So
2
Trong , 2 dng A. niger So
2
v R
1
c s dng trong nghin cu tip theo, xc
nh cc iu kin ln men SSF sinh tng hp PME.


Hn 320: Biu biu din trn t ca mt on RNA gene ca 4 mu nm

>gb HQ825091.1, Aspergillus niger strain NBL 28S ribosomal RNA gene, partial sequence
Length = 901
Score = 470 bits (254), Expect = 2e-129
Identities = 254/254 (100%), Gaps = 0/254 (0%)
Strand = Plus/Plus
Hn 321: T l tng ng v gene ca dng nm So
2
v dng A. niger NBL, BR
28s ribosomal RNA gene hay An03c0110

3.4 nh hng ca mi trng dinh dng n qu trnh sinh tng hp
PME t 2 dng A. niger R
1
v So
2

3.4.1 X nh thnh phn bn ht ln men
PME c tnh c hiu cao i vi nhm methylester ca acid polygalacturonic
(Ishi et al., 1979; Limberg et al., 2000). Chnh v th, mi trng nui cy giu
pectin vi mc DE cao s tr gip hiu qu cho vic thc y qu trnh ln
men sinh tng hp PME t nm mc.
Thnh phn ha hc c bn, c bit l pectin v DE (%) ca ba ngun c cht
chnh t ph phm nng nghip sn c ti a phng l b to ta, v qu cam
snh, v qu bi Nm Roi c xc nh, to c s cho vic nghin cu qu
trnh sinh tng hp PME tip theo. Kt qu phn tch c tng hp bng 3.7.
Bng 37: Thnh phn ha hc c bn ca cc ngun c cht ln men
Nguyn liu
m
(%)
Pectin
(% cbk)
DE*
(%)
ng
(%)
Protein
(%)
B to kh 4,29 1,19 34,83 0,67 64,17 1,44 47,58 2,82 4,93 0,08
V qu cam snh 72,41 1,43 3,91 0,36 66,67 1,23 4,11 0,72 0,27 0,04
V qu bi Nm
Roi
75,61 2,20 2,17 0,16 84,77 6,06 3,73 0,83 0,15 0,04
* DE x nh trn mu kh m 4 1%
T kt qu thu c bng 3.7 c th nhn thy, hm lng pectin trong b to ta
(Ziziphus mauritiana) kh kh cao (chim khong 34,83 0,67% tnh theo cn
bn kh) so vi cc ging to khc (pectin ch chim khong 15 18% thnh phn
cht kh, Pilnik v Voragen, 1993; Girdharilal et al., 1998), y chnh l iu kin
thun li cho hot ng sinh tng hp PME t qu trnh nui cy nm mc
A. niger. Tuy nhin, mc ester ha ca to ta cn thp (64,17 1,44%) so vi
cc ging to khc c kho st trn th gii. Cng b ca Aehle (2004) cho
thy, DE ca to thng dao ng t l 80 92%; Rao v Maini, (1999) tm
ra gi tr DE ca to l 74,0%. Mc d vy, y cng l mt kt qu kh quan khi
so snh vi DE ca to xanh, tri nh (ngun gc Thi Lan) c gi tr 57,19
2,31% (Tran et al., 2010). Hm lng pectin thu c t v cam snh (3,91
0,36%) v v bi Nm Roi (2,17 0,16%) u kh thp so vi cc ging cam
khc (4,0 5,5%; Aehle, 2004) nhng DE ca pectin t v cam c gi tr
tng ng ( DE 66,67 1,23% thu c t pectin ca v cam c kho st)
khi so snh vi thng s tham kho 65% theo nghin cu ca Aehle (2004). im
c bit l pectin thu nhn t v qu bi Nm Roi c DE cao (84,77%), cho
thy trin vng ca vic s dng nguyn liu ny lm c cht cho qu trnh sinh
tng hp PME t nm mc A. niger. Mt vn cn ch l hm lng protein
trong v qu cam v bi kh thp (0,27 v 0,15%) nhng b to kh c hm
lng ng cao cng vi t l protein 4,93 0,08% l ngun c cht cn thit
cho hot ng ca nm mc. Kt qu cho php ngh n s kt hp ca b to v
v qu citrus nhm ci thin thnh phn mi trng ln men.
3.4.2 nh gi t ng thnh phn ht n hot tnh PME thu nhn t
qu trnh ln men F hi dng nm m A. niger R
1
v So
2

Th nghim thm d cho thy, vic s dng v bi kh hay v cam kh c cng
m vi b to khng to iu kin thun li cho hot ng ca nm mc. V
cc loi qu h citrus sy kh, nghin mn ht nc v trng n mnh, lm gim
xp ca mi trng ln men. Vic s dng thm cc thnh phn to thong
kh cho mi trng ln men nh v tru vn khng cho hiu qu ci thin cht
lng ng k (Tran et al., 2009). Trn c s , th nghim c tin hnh vi
mc ch xc nh t l phi trn thch hp ca b to kh v v bi/cam ti
n hiu qu thu nhn PME t t hp 2 dng A. niger R
1
v So
2
. nghin cu
ny, m ban u mi trng ln men c c nh 60% (Joshi et al., 2006), t
l huyn ph bo t nm b sung l 20% (vi mt s nm mc 10
5
cfu/mL), nhit
l 37C (Tran et al., 2009). S dng mu i chng l v bi/cam ti n
l.
Bng 38: S tha i hot tnh PME cc t l kt hp khc nhau ca b to ta v
v qu cam/bi
Ngun c cht T l kt hp Hot tnh PME (U/g)
B to ta 10:0 4,83
b
0,29
V qu cam snh 10:0 1,20
a
0,21
V qu bi Nm Roi 10:0 1,90
a
0,17
B to ta : v qu cam 8:2 6,63
c
0,63
7:3 9,80
g
0,57
6:4 8,74
f
0,37
5:5 7,77
de
0,62
4:6 7,13
cd
0,54
B to ta : v qu bi 8:2 8,85
f
0,44
7:3 10,34
gh
0,38
6:4 11,14
h
0,64
5:5 13,17
i
0,51
4:6 8,29
ef
0,51

T s F 155,12
Gi tr P 0,00
C gi tr mu t i km ging nhu trong ng mt t kh bit khng ngh v mt thng k theo
php th Dunn tin y 95%.
T kt qu tng hp bng 3.8 cho thy, b to ta l ngun c cht rt thch hp
cho qu trnh ln men sinh tng hp enzyme PME, th hin hot tnh thu c l
4,83

0,29 U/g, cao gn gp 4 ln khi so snh vi hot tnh PME thu nhn khi ch
s dng n l v bi hay v cam lm c cht ln men. S tng trng v pht
trin ca nm mc rt cn s hin din ca cc nguyn t a lng v vi lng,
trong carbohydrate, protein l hai thnh phn c nh hng ln nht (Benen et
al., 2003). y cng chnh l nguyn nhn dn n s pht trin nhanh chng ca
nm mc trn b mt mi trng ln men l b to, trong khi , c s tng
trng nm mc rt chm khi s dng v bi hay v cam lm c cht ln men
ring l. Leuchtenberger et al. (1989) khng nh kh nng thu nhn PME t
A. niger trong iu kin ln men SmF l cao hn khi so snh vi cc enzyme thy
phn khc nh cellulase, -amylase v thm ch l PG khi s dng b to hay tht
qu to lm c cht cm ng. Nghin cu ca Joshi et al. (2006) cng xc nhn
hiu qu thu nhn PME t nm mc A. niger vi c cht l b to. Garzn
Hours (1992) cng ng s dng v qu citrus lm c cht cm ng cho qu
trnh sinh tng hp pectinase t nm mc, kt hp vi vic b sung cc thnh
phn dng cht cn thit tng t nh mi trng CZ.
Tuy nhin, khi s dng kt hp hai loi c cht b to ta v v bi hay b to ta
v v cam, hot tnh PME thu c u cao hn khi so snh vi hai mu i
chng. Trong trng hp s dng kt hp b to ta v v cam, t l kt hp tt
nht l 7 : 3 trong khi t l phi trn gia b to ta v v bi Nm Roi l 5:5
th kh nng sinh tng hp PME t nm mc l cao nht. t l kt hp ny, hot
tnh PME thu c sau 4 ngy nui l 13,17 U/g, tng gp 2,7 ln khi so snh
vi mu ch s dng c cht ln men l b to v tng gn 11 ln so vi trng
hp ch s dng ngun c cht ch yu t v cam.
Mc d thnh phn pectin trong v qu cam snh cao hn v bi Nm Roi, tuy
nhin v qu bi c hiu qu cao hn i vi vic thu nhn PME. Vic s dng
kt hp v bi ti lm c cht cho qu trnh ln men gp phn ci thin xp
ca mi trng - ci thin tc trao i kh. Nm mc A. niger l loi hiu kh
nn s thong kh l mt trong nhng iu kin thun li cho s phn trin v sinh
tng hp enzyme c hiu (Bayoumi et al., 2008). Kt qu nghin cu ca Daz et
al. (2009) cng cho thy, hot tnh ca cc loi enzyme thy phn c ngun gc t
A. awamori t cao nht trong qu trnh ln men s dng c cht l hn hp b
nho v v cam vi t l 1 : 1.
Kt qu tng hp bng 3.8 cng gp phn khng nh tm quan trng ca thnh
phn c cht n qu trnh sinh tng hp PME ca A. niger. t l phi trn gia
b to ta kh v v bi ti l 5 : 5 (hay 1 : 1), hm lng pectin cao trong b to
ta v mc ester ha cao ca pectin trong v bi Nm Roi l ngun c cht
cm ng thch hp nht cho A. niger pht trin v tng hp PME. Vic gia tng
nng c cht pectin c hiu qu rt tch cc trong vic y nhanh tc ln
men v gia tng hot tnh PME (Maldonadooo v Callieri, 2005). Hn th na,
DE cao ca pectin cng l mt ch s rt quan trng nh hng mnh m n hot
ng sinh tng hp PME ca A. niger (Grebechova, 2007).
Da trn kt qu kho st, vic s dng kt hp b to ta kh v v bi Nm Roi
ti vi t l phi trn l 1: 1 c s dng lm c cht ln men ch yu cho qu
trnh sinh tng hp PME.
Bn cnh thnh phn c cht cm ng thch hp th cc thnh phn dng cht
khc nh khong, ngun nitrogen cng c tc ng tch cc n qu trnh tng
trng, pht trin ca nm mc cng nh nh hng n hiu qu sinh tng hp
enzyme ni chung v PME ni ring (Joshi et al., 2006; Mrudula v Anitharaj,
2011). Do , cc thnh phn ny cn c kho st vi tng trng hp c th.
3.4.3 i tr vi b sung khong n kh nng tng hp PME t A. niger
i. nh hng nng v loi khong b sung ring l n hot tnh
PME sinh tng hp bi A. niger R
1
v So
2
trn mi trng b to t v
v bi Nm Roi
Thnh phn ca cc cht khong trong mi trng nui cy rt c ngha v nh
hng rt ln n kh nng sinh trng cng nh sinh tng hp enzyme ca vi
sinh vt. Cc cht khong nh hng trc tip n s sinh sn ca si nm, kch
thch s to enzyme cng nh nh hng n bn nhit ca enzyme (Nguyn
c Lng, 2004). nh hng ca cc loi khong (MgCl
2
, CaCl
2
v KCl) vi
cc nng b sung khc nhau c kho st trong th nghim ny nhm tm ra
loi khong v nng thch hp cho vic nui cy A. niger v sinh tng hp
PME c hot tnh cao. Kt qu th nghim c trnh by bng 3.9.

Bng 39: nh hng ca nng v loi khong b sung vo mi trng ln men
n s tha i hot tnh ca PME t A. niger
Loi khong Nng (%) Hot tnh ring (U/g)
i chng 0% 12,87
c
0,49

KCl
0,5% 12,08
ab
0,54

1,0% 13,83
cd
0,82

1,5% 16,87
g
0,17

2,0% 2,87
a
0,73

MgCl
2
0,25% 4,05
b
1,09

0,5% 16,55
g
0,40

0,75% 15,47
f
0,50

1,0% 13,72
cd
0,63

CaCl
2
0,5% 14,06
de
0,50

1,0% 14,90
ef
0,14

1,5% 16,90
f
0,27

2,0% 15,23
f
0,31

T s F 182,8
Gi tr P 0,00
T kt qu thng k bng 3.9 cho thy hot tnh ca dch trch enzyme c ci
thin khi tin hnh b sung K
+
, Mg
2+
v Ca
2+
t cc mui tng ng. Loi v hm
lng khong b sung khc nhau trong qu trnh nui cy cng nh hng r rt
n hot tnh ca dch trch enzyme thu c. i vi cc mu c b sung KCl,
hot tnh ca PME t A. niger thu c tng dn khi hm lng KCl b sung tng
t 0,5% n 1,5% v gim khi b sung KCl vi hm lng 2,0%. Hot tnh cao
nht ca dch trch enzyme thu c khi mu c b sung 1,5% KCl trong qu
trnh nui cy. iu ny cho thy ion K
+
phn ly t KCl cng nh hng n hot
tnh PME sinh ra bi nm mc A. niger. i vi vic s dng ngun khong
CaCl
2
trong mi trng ln men, kt qu o c cho thy, mu c b sung 1,5%
Ca
2+
cho hot tnh PME cao hn so vi cc mu c b sung 0,5%, 1% v 2%.
C th hn, hot tnh PME thu c t mu c b sung 1,5% ion Ca
2+
l
16,90 U/g v th hin s khc bit r rt so vi mu i chng. Ion Ca
2+
c cho
l c lin quan mt thit n qu trnh to kt ta calcium pectate (Van Buren,
1979). V vy, mc d ion Ca
2+
c kh nng kch hot PME thu nhn t A. niger
nhng nng cao hn, ion ny s tham gia to kt ta (Acikel et al., 2011).
Bn cnh , vic b sung MgCl
2
trong c cht ln men cng cho thy hot tnh
ca dch trch PME c ci thin so vi mu i chng. Mu c b sung 0,5%
MgCl
2
c hot tnh PME thu c cao nht (16,55 0,4 U/g) v hot tnh c
ci thin gn 1,5 ln so vi mu khng b sung khong (12,87 0,49 U/g). Hot
tnh PME ca dch trch enzyme th thu c tng dn khi hm lng MgCl
2
b
sung trong c cht ln men tng t 0,25% n 0,5% v gim khi hm lng
MgCl
2
c b sung mc 0,75% v 1%. iu ny th hin s nh hng ca
ion Mg
2+
n kh nng tng hp PME t A. niger. Nghin cu ca Joshi et al.
(2006) cng xc nhn vai tr ca Mg
2+
n vic gia tng tc hnh thnh PME,
Martin et al. (2004) cng nh gi cao vai tr ca Mg
2+
i vi qu trnh ln men
sinh tng hp pectinase.
Nhn chung, nm mc A. niger c kh nng sinh tng hp PME c hot tnh cao
khi b sung KCl, CaCl
2
vi hm lng 1,5% hoc 0,5% MgCl
2
trong mi trng
nui cy. Tuy nhin, hot tnh PME thu nhn khng c s khc bit ngha v
mt thng k gia cc nghim thc khong b sung ring l (hnh 3.22).

Hnh 3.22: t so sn n ng ca cc loi kong b sung ti u
i vi iu qu gia tng ot tn PME t A. niger
Hn th na, mc ci thin hot tnh PME ca cc thnh phn khong b sung
ring r khng cao (khong 1,3 ln khi so snh vi mu i chng, khng b sung
khong). Chnh v th, vic nghin cu kt hp 3 loi khong c tin hnh vi
mong mun gia tng qu trnh sinh tng hp enzyme ny mc ti a.
ii. nh hng s kt hp loi khong nng ti u n hot tnh
PME sinh tng hp t A. niger
Th nghim c tin hnh trn c s kt hp cc loi khong c mc ci thin
hot tnh cao nht th nghim trc (CaCl
2
1,5%; KCl 1,5%; MgCl
2
0,5%). Kt
qu c tng hp bng 3.10.
Bng 310: nh hng ca vic b sung kt hp cc loi khong vo mi trng
nui c n kh nng sinh tng hp PME t A. niger
Cc phng thc kt hp ca khong Hot tnh PME (U/g) Mc ci thin hot
tnh, (ln)
i chng 12,78
a
0,16 1
KCl 1,5% + MgCl
2
0,5% 20,16
b
0,02 1,58
KCl 1,5% + CaCl
2
1,5% 19,44
b
0,15 1,52
MgCl
2
0,5% + CaCl
2
1,5% 21,55
c
0,19 1,69
KCl 1,5% + MgCl
2
0,5% + CaCl
2
1,5% 19,14
b
0,23 1,50
T s F
Gi tr P
71,98
0,00

Kt qu t bng 3.10 cho thy, hot tnh PME trong dch trch enzyme thu c t
qu trnh nui cy A. niger gia tng t 1,5 n 1,7 ln so vi mu i chng
(khng b sung khong). Hot tnh PME thu c cao nht khi mi trng ln
men c b sung 0,5% MgCl
2
v 1,5% CaCl
2
. Kt qu o c cho thy, hot tnh
PME c ci thin n 1,69 ln khi so snh vi mu i chng. Vic kt hp ba
loi khong vi nng ti u t th nghim trc (KCl 1,5% - MgCl
2
0,5% -
CaCl
2
1,5%) cng khng cho hiu qu cao i vi vic ci thin s sinh tng hp
PME khi so snh vi cc nghim thc kt hp cn li. iu ny c l l do khi
nng khong b sung trong c cht ln men cng ln th lng protein tp sinh
ra cng nhiu nn hot tnh ca dch trch enzyme thu c khng cao (Malvessi
v da Silveira, 2004). Trc , nghin cu ca Leuchtenberger et al. (1989) cng
cho thy hiu qu ci thin hot tnh ca PME khi b sung KNO
3
v NaNO
3
vo
mi trng ln men lng SmF, nui cy A. niger.
Tm li, hot tnh ca PME thu c t qu trnh sinh tng hp nm mc A. niger
cao nht khi c b sung 0,5% MgCl
2
v 1,5% CaCl
2
vo mi trng nui .
Bn cnh ngun carbon cng nh cc khong cht, nitrogen l mt trong cc
thnh phn dng cht thit yu cho qu trnh sinh tng hp ca vi sinh vt, c
bit l cc nm mc sinh tng hp enzyme (Silva et al., 2002, Banu et al., 2010).
3.4.4 ng ngun nitrogen b sung n hiu qu thu nhn PME t qu
trnh ln men SSF dng A. niger So
2
v R
1

Th nghim b sung nitrogen vo mi trng ln men nhm thu dch trch PME c
hot tnh cao c thc hin vi ba ngun cung cp nitrogen l urea, (NH
4
)
2
SO
4
,
NH
4
H
2
PO
4
cc t l b sung khc nhau. S liu c thu nhn, thng k v tng
hp bng 3.11.
Bng 311: nh hng ca hm lng v ngun nitrogen b sung n hot tnh
PME t qu trnh sinh tng hp A. niger
Ngun nitrogen b sung T l b sung Hot tnh PME (U/g)
i chng 0 21,55
a
0,25
Urea 0,05% 25,66
c
0,32
0,10% 27,87
d
0,16
0,15% 25,82
c
0,19
0,20% 23,46
b
0,23
(NH
4
)
2
SO
4
0,05% 23,16
b
0,11
0,10% 25,27
c
0,21
0,15% 26,14
c
0,50
0,20% 25,93
c
0,61
NH
4
H
2
PO
4
0,05% 21,65
a
0,24
0,10% 23,59
b
0,16
0,15% 25,79
c
0,40
0,20% 23,33
b
0,08
T s F 20,77
Gi tr P 0,00
Kt qu kho st cho thy, vic b sung nitrogen t cc ngun khc nhau vo mi
trng ln men l mt bin php tch cc trong vic gia tng hiu qu ca qu
trnh sinh tng hp PME t A. niger. Hot tnh PME thu c t cc mu nui cy
trong mi trng c b sung nitrogen gia tng v khc bit c ngha khi so snh
vi mu i chng. Trong tng ngun nitrogen b sung ring l, ng vi cc nng
s dng khc nhau, hot tnh PME thu c cng c s khc bit ln.
i vi trng hp s dng NH
4
H
2
PO
4
l ngun nitrogen b sung, hot tnh PME
trong dch trch enzyme u c ci thin khi so snh vi mu i chng v tng
dn khi nng NH
4
H
2
PO
4
b sung tng t 0,05% n 0,15% nhng gim khi
nng NH
4
H
2
PO
4
tng n mc 0,2%. Tuy nhin, hiu qu ci thin hot tnh
PME ca NH
4
H
2
PO
4
km hn trng hp s dng urea hay (NH
4
)
2
SO
4
vi cng
mt nng . Kt qu cho thy y l loi mui c kh nng cung cp nitrogen
c phn ly t gc amonium lm tng hot tnh ca dch trch enzyme thu c
nhng khng phi l ngun nitrogen thch hp cho A. niger sinh tng hp PME c
hot tnh cao.
Mui (NH
4
)
2
SO
4
cng l mui c kh nng cung cp nitrogen t gc amonium
tng t nh NH
4
H
2
PO
4
. i vi mu c b sung mui ny, hot tnh ca PME
c s khc bit vi mu i chng (mu khng b sung nitrogen). T bng kt
qu cho thy, ng vi hm lng 0,15% (NH
4
)
2
SO
4
b sung trong mi trng
nui cy cho hot tnh ca dch trch enzyme thu c kh cao (26,14 U/g). Tuy
nhin, hot tnh ca PME thu c t cc mu c b sung mui (NH
4
)
2
SO
4
vi
hm lng khc nhau khng c s khc bit ln, mc ci thin hot tnh cng
khng cao khi so snh mu c hot tnh cao nht vi cc mu c hm lng b
sung ln cn.
Urea th hin r rt nht nh hng ca nitrogen ln kh nng sinh tng hp PME
ca nm mc A. niger. Cc mc hm lng nitrogen b sung t urea vo mi
trng nui cy u th hin s khc bit ngha vi nhau v khc bit hn so vi
mu khng b sung urea v hot tnh ca PME thu c, c bit hm lng
urea b sung 0,1% (hot tnh PME c gi tr 27,87 U/g). Kt qu cng cho thy,
khi urea b sung vo mi trng nui cy tng t 0,05% n 0,1% th hot tnh
ca enzyme thu c tng v hot tnh ny gim khi tng hm lng b sung t
0,15% n 0,2%. Hin tng ny c gii thch l do nm mc A. niger cn mt
lng nitrogen thch hp trong qu trnh sinh trng v tng hp enzyme. Khi b
sung nitrogen vi hm lng cao s to iu kin thun li cho s sinh trng ca
nm mc v lm hn ch s sinh tng hp PME. Ngoi ra, vi sinh vt ch tch ly
mt lng enzyme ln khi mi trng c va lng carbon v nitrogen cn
thit (Pandey et al., 2000; Mukherjee v Banerjee, 2006).
Nh vy, urea c b sung vo mi trng ln men vi t l 0,1% l mi trng
thch hp cho nm mc tng hp PME c hot tnh cao.
3.5 Tc ng ca mt s iu kin ln men n hiu qu thu nhn PME t
A. niger trong qu trnh nui c SSF
3.5.1 nh hng loi dung dh m s dng trong iu hnh m v pH
bn u mi trng ln men n hiu qu sinh tng hp PME
m v pH ban u ca mi trng ln men c bit n nh hai yu t quan
trng nht, c nh hng n kh nng sinh trng v tng hp enzyme t vi sinh
vt (Tewari et al., 2005). Tuy nhin, tc nhn iu chnh m v pH ban u ca
mi trng ln men cng c nh hng ng k n hiu qu sinh tng hp
enzyme. Mc d khng c nhiu nghin cu c th v nh hng ca tc nhn
iu chnh m v pH mi trng, tuy nhin c nhiu loi dung dch m khc
nhau thng c s dng trong cc kho st v mi trng ln men sinh tng
hp enzyme t vi sinh vt (Barredo, 2005; Murthy v Naidu, 2010). i vi qu
trnh nui cy nm mc sinh tng hp cc enzyme thy phn ni chung v PME
ni ring, vic iu chnh m v pH mi trng ban u thng c tin hnh
vi nc ct, c hay khng c b sung NaOH 0,1 N v HCl 0,1 N iu chnh
pH (Joshi et al., 2006; Mrudula v Anitharaj, 2011), hoc s dng Na
2
CO
3
1 N
(Tewari et al., 2005), m phosphate (Suresh v Viruthagiri, 2010). c bit,
Khan v Husaini (2006) bc u nghin cu nh hng ca vic iu chnh
pH ban u ca qu trnh ln men sinh tng hp amylase v cellulase bng 4 loi
m acetate, citrate, phosphate v tris HCl.
Da trn c s ny v cc th nghim thm d, nh hng ca vic iu chnh pH
ban u ca mi trng ln men n pH 4,0 (Joshi et al., 2006) bng 3 loi m
khc nhau (m citrate, phosphate, acetate) c thc hin. Hot tnh PME thu
nhn tng ng vi cc nghim thc kho st c th hin hnh 3.23.
27,85a
28,25a
32,44b
34,10b
41,17c
0
5
10
15
20
25
30
35
40
45
m citrate m citrate
phosphate
m acetate Nc ct (iu
chnh n pH
4,0)
i chng
(nc ct pH
6,7 6,8)
Dung dch m s dng
H
o
t

t
n
h

P
M
E

(
U
/
g
)

Hn 323: n ng ca loi dung dc m s dng trong iu cn m v
pH mi trng ln men n ot tn PME t qu trn sin tng p A. niger
Kt qu kho st cho thy, vic s dng dung dch m iu chnh pH v
m c l l nguyn nhn hn ch s bin i gi tr pH ban u ca mi trng ln
men (Doelle et al., 1992), hot tnh PME thu c gia tng, khc bit c ngha
khi so snh vi trng hp s dng nc ct. Hot tnh PME thu c cao nht
trong trng hp s dng m citrate iu chnh m v pH ban u ca mi
trng ln men. Hot tnh thu c trong trng hp ny l 41,17 0,72 U/g, khi
so snh vi mu enzyme c iu chnh m bng nc ct (c v khng c iu
chnh pH n 4,0), hot tnh PME ch t c gi tr 27,85 U/g v 28,25 U/g.
iu ny c th c gii thch do tc ng ca cc ion khc nhau ca dung dch
m (Khan v Husaini, 2006). Theo kho st ca Khan v Husaini (2006), s
dng m citrate lm tc nhn iu chnh m v pH ban u ca mi trng ln
men Bacillus sp. sinh tng hp cellulase cng cho hiu qu tt nht khi so snh
vi m phosphate, acetate v tris HCl. Tuy nhin, nghin cu ca Priest v Sharp
(1989) li nh gi cao vai tr ca ion phosphate n hiu qu thu nhn enzyme t
qu trnh nui cy Bacillus sp. iu ny chng t, vic s dng tc nhn m
thch hp ph thuc vo mi loi enzyme, vi sinh vt cng nh cc thnh phn
dng cht trong mi trng (Khan v Husaini, 2006).
Nh vy, dung dch m citrate c s dng lm tc nhn iu chnh pH mi
trng ln men, sinh tng hp PME trong cc nghin cu tip theo.
3.5.2 thy i hot tnh PME v thnh phn ht mi trng nui y
theo thi gin
Qu trnh sinh tng hp enzyme ph thuc rt ln vo s pht trin ca t bo
nm mc nn thi gian nui cy hay thi im thu nhn ch phm enzyme l mt
trong nhng nhn t quan trng quyt nh hot tnh ca enzyme cao hay thp. Do
, th nghim c tin hnh kho st nh hng thi gian nui cy nm mc
A. niger (24 gi, 48 gi, 72 gi, 96 gi v 120 gi) nhm tm ra thi im nm
mc pht trin ti u cho hiu qu sinh tng hp PME c hot tnh cao. Kt qu
c o c, thng k v tng hp hnh 3.24.
15,21c
0,36a
0a
12,80c
22,88d
41,16e
0
5
10
15
20
25
30
35
40
45
50
0 24 48 72 96 120
Thi gian (gi)
H
o
t

t
n
h

P
M
E

(
U
/
g
)

Hnh 3.24: n ng ca ti gian n ot tn PME tu nn t A. niger
Khi nh gi nh hng ca thi gian n hiu qu sinh tng hp PME, kt qu
hnh 3.24 cho thy hot tnh ca enzyme thu c tng dn khi thi gian ko
di n 96 gi. Sau hot tnh bt u gim khi thi gian nui cy tng t 96 gi
n 120 gi.
Hot tnh enzyme thu c lin quan cht ch n s pht trin ca t bo nm
mc. Trong 24 gi sau khi cy, nm mc A. niger ang dn thch nghi vi iu
kin mi trng nn cha gia tng mt s ng k. V th, vic thu ch phm
enzyme thi im ny l qu sm khin cho enzyme cha c tng hp nhiu,
PME thu c c hot tnh kh thp (0,36 U/g). Sau 24 gi nui cy, nm mc bt
u gia tng mt s, thc y s tng hp PME, hot tnh ca enzyme gia tng
(Patil et al., 2006). Thi gian nm mc pht trin ti u, sinh tng hp PME c
hot tnh cao nht l 96 gi nui , tng ng vi hot tnh PME thu c l
41,16 U/g 4,18. Kt qu ny tng ng vi nghin cu ca Patil et al. (2006)
sn xut pectinase t hoa hng dng trn c hai iu kin ln men SSF v SmF.
Venugopal et al. (2007) cng tm thy thi gian ti u gip thu nhn endo-
pectinase khi tin hnh nui cy A. niger trn c cht tht ht c ph l 96 gi.
Sau thi gian ti u (trng hp kho st l 96 gi), qu trnh tng trng ca
nm mc gim dn, ko theo s sinh tng hp enzyme hu nh ngng li nn hot
tnh enzyme thu c thp (Grebechova et al., 2007). Nu tip tc ko di thi
gian nui cy th hot tnh PME gim xung. Thi gian nm mc thch hp nht
nhm thu c PME c hot tnh cao nhit 37C l 96 gi trong iu kin
nui cy trn b mt mi trng SSF.
Trong qu trnh nui , nm mc s dng c cht sinh tng hp PME nn vic
xc nh hm lng DE, pectin, m canh trng sau khi ln men v pH dch
trch c thc hin nhm kho st tc s dng c cht ca A. niger v s thay
i thnh phn mi trng trong sut qu trnh ln men. Kt qu c tng hp
bng 3.12.
Bng 3.12: S tha i hm lng pectin, DE, m ca canh trng sau khi ln
men v pH ca dch trch enzme sau thi gian nui c
Thi gian
(gi)
Pectin (%) DE (%) m (%) pH dch trch
0 17,69
e
0,40 74,82
e
0,11 60,07
a
0,12 4,16
d
0,12
24 12,26
d
0,81 74,38
de
0,38 64,29
b
0,60 3,86
c
0,06
48 5,61
c
0,20 73,58
d
0,65 65,15
b
0,95 3,28
b
0,14
72 3,78
bc
0,16 70,46
c
0,50 68,54
c
0,47 2,96
a
0,26
96 1,67
ab
0,10 57,63
b
0,55 69,30
c
1,35 2,93
a
0,14
120 0
a
55,33
a
0,58 69,51
c
0,50 2,86
a
0,13
T s F 74,38 956,05 69,48 39,92
Gi tr P 0,00 0,00 0,00 0,00
T kt qu bng 3.12 cho thy c s gia tng m mi trng v gim dn hm
lng pectin, DE theo thi gian nui . iu ny c th chng t A. niger s
dng c cht trn cho qu trnh sinh tng hp enzyme. Trong thi gian u, do
mi trng giu dinh dng thun li cho nm mc s dng gia tng sinh khi
nn lng pectin gim nhanh, sau dinh dng cn dn nn hm lng pectin
gim chm li. S thay i DE chng t s c mt ca PME gia tng, thc y s
phn ct mch pectin trong mi trng (Venugopal et al., 2007). Mc ester ha
pectin gim t 74,82

0,11% trong c cht ban u n gi tr 55,33 0,58% sau
120 gi hay 5 ngy nui . Sau 24 gi u tin, c s gim ng k hm lng
pectin (t 17,69 0,40% xung cn 12,26 0,81%), tuy nhin khng c s thay
i ng k ester ha DE ca pectin (mc gim pectin sau 24 gi khong
0,5%, khng c s khc bit ngha v mt thng k). iu ny cng ph hp vi
hot tnh PME sinh ra rt thp sau 24 gi .
Sau 5 ngy ln men (120 gi), hm lng pectin trong c cht gim n mc thp
nht, ni cch khc, thnh phn dng cht thch hp cho hot ng ca enzyme
khng cn cho qu trnh tng hp enzyme. Bn cnh , PME sinh ra s thy
phn lin kt ester to iu kin cho cc enzyme PG, PL hot ng phn ct mch
polygalacturonic thnh acid galacturonic (Ishi et al., 1979) do lm gim pH ca
dch trch enzyme sau ln men. Trong trng hp kho st, pH gim t 4,16 thi
im bt u ln men xung n gi tr pH 2,86 sau 5 ngy nui . Ngoi ra, trong
qu trnh ln men, s hnh thnh cc hp cht hu c - trong c acid citric, lm
tng nng ion H
+
trong mi trng ln men, tc ng mt phn n s gim pH
dch trch. Nghin cu tng hp enzyme thy phn pectin t A. niger trn mi
trng SmF ca Mojsov (2010) cng xc nhn s gim dn pH mi trng ln
men (cng chnh l pH dch trch) theo thi gian nui . Ngc li, m ca mi
trng ln men li thay i theo chiu hng tng. Theo thi gian nui , s sinh
trng pht trin s ko theo s gia tng hot ng h hp ca bo t nm mc,
dn n s phn gii glucose to CO
2
v nc, gp phn gia tng m mi
trng.
Da vo cc thng s v s thay i thnh phn mi trng theo thi gian ln
men, thi gian nui 4 ngy (96 gi) l mc gii hn cho vic sinh tng hp PME
t gi tr cao nht.
3.5.3 tng t gi pH, m bn u mi trng, t l huyn ph bo
t nm v nhit n qu trnh sinh tng hp PME t A. niger
Da trn cc nghin cu sinh tng hp PME t qu trnh nui A. niger trn cc
ngun c cht khc nh ch s dng ring l b to (Tran et al., 2009; Joshi et al.,
2006) hay qu trnh sinh tng hp pectinase t A. awamori (Suresh et al., 2009),
t l huyn ph bo t nm s dng, vic iu chnh pH, m ban u ca mi
trng ln men v nhit l cc thng s c tc ng ln v c mi lin h
tng quan. qu trnh ln men b mt trn c cht chnh l b to ta, gi tr pH
ban u thch hp cho qu trnh sinh tng hp PME l 4,0 (Tran et al., 2009)
nhng khi s dng c cht kt hp l b to v v cam (t l 7 : 3), pH ban u
c iu chnh n gi tr 3,5 gip PME thu nhn c hot tnh cao nht (Tran et
al., 2010). Chnh v th, nghin cu xc nh hi quy tng quan ca 4 nhn t,
bao gm t l huyn ph bo t nm s dng X
1
(12 20% v/w), pH ban u ca
mi trng X
2
(3,0 5,0), nhit X
3
(28 40C) v m ban u mi trng
X
4
(50 70%) n hot tnh PME thu nhn (Y, U/g) c thc hin. Ma trn thit
k th nghim theo phng php b mt p ng (RSM) vi m hnh phc hp ti
tm (CCD) c thit lp vi 5 mc kho st tng ng mi bin v 7 im
trung tm. Kt qu phn tch nh hng ca cc nhn t m ha i vi phng
trnh hi quy c trnh by bng 3.13.
Bng 3.13: nh hng ca cc nhn t m ha i vi phng trnh hi quy
Nhn t
Tng
bnh phng
Bc
t do
Phng sai T s F Gi tr P
X
1
37,2504 1 37,2504 5,68 0,0299
X
2
54,3004 1 54,3004 8,28 0,0109
X
3
1351,5000 1 1351,5000 206,19 0,0000
X
4
424,2000 1 424,2000 64,72 0,0000
X
1
X
1
1284,1800 1 1284,1800 195,92 0,0000
X
1
X
2
155,6260 1 155,6260 23,74 0,0020
X
1
X
3
73,5306 1 73,5306 11,22 0,0410
X
1
X
4
64,4006 1 64,4006 9,83 0,0064
X
2
2
4170,3500 1 4170,3500 636,25 0,0000
X
2
X
3
43,8906 1 43,8906 6,70 0,0198
X
2
X
4
172,2660 1 172,2660 26,28 0,0010
X
3
2
2467,3100 1 2467,3100 376,43 0,0000
X
3
X
4
117,1810 1 117,1810 17,88 0,0060
X
4
2
979,0660 1 979,0660 149,37 0,0000
Sai s 104,8730 16 6,5546
T bng tng hp 3.13, tt c gi tr P ca cc tha s u nh hn 0,05 chng
t cc nhn t kho st u nh hng n qu trnh ln men b mt trn mi
trng SSF sinh tng hp PME t A. niger. H s hi quy bc mt ca X
1
, X
2
, X
3

v X
4
cng nh h s tng tc ca X
1
X
2
, X
1
X
3
, X
1
X
4
, X
2
X
3
, X
2
X
4
v X
3
X
4
u
khc bit c ngha v mt thng k tin cy 95%, ng thi h s hi quy
bc hai ca X
1
2
, X
2
2
, X
3
2
v X
4
2
cng khc bit ngha v mt thng k. iu ny
gp phn khng nh mc nh hng ca tng bin c lp cng nh cc
tng tc c ngha n qu trnh ln men c kho st.
Da trn kt qu phn tch ANOVA, phng trnh hi quy th hin s tng quan
ca iu kin ln men n hot tnh PME thu nhn c thit lp v s dng d
on hiu qu sinh tng hp PME. Hot tnh PME m phng (Y l thuyt) c
xc nh bng cch thay cc bin vi gi tr thc vo phng trnh (3.1) sau:
Y = -3331,94 + 66,60 X
1
+ 323,06 X
2
+ 73,38 X
3
+ 31,34 X
4
- 1,68 X
1
2
-
3,12X
1
X
2
+ 0,36X
1
X
3
- 0,20X
1
X
4
- 48,31X
2
2
+ 1,10X
2
X
3
+ 1,31X
2
X
4
- 1,03X
3
2
-
0,18X
3
X
4
- 0,23X
4
2
(R
2
= 0,989) (3.1)

Hot tnh PME thu c t thc nghim v tnh ton theo phng trnh c
tng thch cao gi tr r
2
= 0,9891 (hnh 3.25). Nh vy c th kt lun rng
phng trnh hi quy m t ng cc kt qu thc nghim. H s tng quan
cho bit 98,9% s bin i hot tnh PME l do nh hng ca cc bin c lp
X1, X2, X3 v X4 v ch c 1,1 % s thay i l do cc yu t khng xc nh c
gy ra.

Hnh 3.25: t tng quan gia ot tn PME xc n tc ngim v tn
ton teo png trn i quy

Chui th biu din tng quan ca 2 nhn t kho st (c nh 2 nhn t cn
li im trung tm) n hot tnh PME thu nhn c th hin hnh 3.26 (
th tng qut), 3.27, 3.28, 3.29, 3.30, 3.31 v 3.32 ( th ng ng im tng
ng vi 6 cp nh hng ng thi hnh 3.26).

Hnh 3.26: t biu din s tng tc ca tng cp nn t
n iu qu tu nn PME
T th tng qut biu din s tng tc ca tng cp nhn t n hot tnh
PME thu nhn hnh 3.26 cho thy, hu ht cc nhn t u c s nh hng
ng thi n qu trnh tng hp enzyme. Tuy nhin, nh hng ng thi ca
vic iu chnh t l huyn ph bo t nm b sung v pH ban u ca mi trng
ln men khng mang li hiu qu sinh tng hp PME vt tri, th hin nh ti
u nm mc thp khi so snh vi cc cp tng tc khc.
nh hng ng thi ca vic iu chnh pH v cc nhn t khc nh nhit ,
m ban u ca mi trng ln men cng khng to nn hiu qu thu nhn
PME cao khi so snh vi cc cp tng tc ca nhit v m hay t l
huyn ph bo t nm s dng (hnh 3.26b, 3.26c, 3.26f).
t l huyn ph bo t nm mc b sung thp (12%), vic iu chnh pH ban
u ca mi trng ln men mc cao (pH 5,0) cho hiu sut cao hn hn so vi
mc pH thp (pH 3,0). Khi t l huyn ph bo t nm mc tng n khong 16
17% th hot tnh PME thu c ng vi pH ban u cao nht (5,0) hay thp nht
(3,0) bng nhau. Khi tng t l huyn ph nm mc ln n 20% th hot tnh
PME thu c c xu hng cao hn pH thp (hnh 3.26a v 3.27).
Tng t, khi c nh hai bin m v pH ban u ca mi trng mc (hnh
3.26b v 3.28), hot tnh PME thu c thp khi nhit mi trng mc
kho st thp nht (28C) - ngay c t l nm mc s dng cao v ch th hin
hot tnh iu kin nhit thp vi lng nm mc s dng va phi. Ngc
li, nhit cao nht, mc m mi trng thp cho hiu qu thu nhn
enzyme cao hn trng hp iu chnh m mc cao.

Hnh 3.27: t ng ng im
biu din tng tc ca t l uyn p
bo t nm v pH mi trng ban u
n ot tn PME tu nn
( nh nhit 34C v m bn u
mi trng 60%)
biu din tng quan ca t l uyn
p bo t nm b sung v nit
n ot tn PME tu nn
( nh m v pH bn u mi trng l
60% v 4,0)

biu din tng tc ca t l uyn p
bo t nm v m mi trng ban
u n ot tn PME tu nn
( nh nhit 34C v pH bn u mi
trng 4,0)
biu din tng tc ca nit v
pH ban u ca mi trng
n ot tn PME tu nn
( nh t l huyn ph bo t nm 16% v m
bn u mi trng 60%)

biu din tng tc ca pH v m
ban u ca mi trng ln men n
hot tn PME tu nn
( nh t l huyn ph bo t nm 16% v nhit
34C )
biu din tng tc gia nit v
m ban u ca mi trng ln men
n ot tn PME tu nn
( nh t l huyn ph bo t nm 16% v pH bn
u mi trng 4,0)
Ni cch khc, t l huyn ph bo t nm b sung thp, s thay i nhit
khng nh hng nhiu n hot tnh PME thu c. iu ny cng xy ra tng
t i vi s nh hng ng thi ca m v nm mc (hnh 3.26c v 3.29),
pH v nhit (hnh 3.26d v 3.30), nhit v m (hnh 3.26f v 3.32).
nh hng ng thi ca m v nm mc dn n s gia tng hot tnh PME
cao hn khi iu chnh t l huyn ph bo t nm b sung v mc cao. c bit,
m thp l iu kin thch hp hn cho s to thnh PME t l nm mc b
sung gia tng. Trong khi , nhit tng cao gip s tng trng ca nm mc
tt hn, nh gia tng kh nng tng hp enzyme. iu kin ln men sinh tng
hp PME ti u khong trung bnh ca pH kho st, tuy nhin nhit thch
hp li lch dn v mc nhit cao. Mc d s tc ng ca pH khng cho hiu
qu thu nhn PME cao, tuy nhin mc pH thp, s thay i nhit hu nh
khng c nh hng mnh m n hot ng ca nm mc v s tng hp PME.
Khi gi tr pH ban u ca mi trng ln men tng, hot tnh PME thu c gim
dn nhit thp. th ng ng im hnh 3.30 v th tng tc ca
hai yu t nhit v m (hnh 3.26f) mt ln na khng nh, nhit thch
hp lch dn v mc cao (vt hn mc tm 34C). nhit cao nht (40C),
m mi trng cao c l l nguyn nhn dn n s cng hng v nhit do
s bc hi nc dn n tr ngi v iu kin ph hp cho s tng hp enzyme,
ng thi m v nhit cao s thc y s tng trng nm mc nhiu hn s
tch ly PME, dn n hiu qu thu nhn PME thp hn khi m tng cao cng
vi s gia tng nhit (Suresh et al., 2009). Nghin cu ca Patil v Dayanand
(2006) cng tm ra nhit ti u cho qu trnh sinh tng hp cc enzyme thuc
nhm pectinase (PL, PG v PME) t A. niger c hai phng thc ln men SmF
v SSF l 34C. Mc d vy, nhit thch hp cho qu trnh sinh tng hp PME
cng thay i ty thuc vo dng vi sinh vt s dng, v ngay c iu kin kh
hu bn ngoi. Bayoumi et al. (2008) xut nhit 37C l iu kin thch
hp khi nui cy Bacillus firmus I 4071, sinh tng hp pectinase. Trong khi
, rt nhiu cc nghin cu sinh tng hp pectinase ni chung v PME ni ring
li tm ra mc nhit ti u l 30C v 31C (Boccas et al., 1994; Castilho et al.,
2000).
Cui cng, nh hng ng thi ca m v pH (hnh 3.26e v 3.31) li to s
bin thin ngc khi so snh vi cc cp tng tc khc. gi tr pH cao (5,0), s
thay i m mi trng nui hu nh khng c nh hng n hiu qu thu
nhn enzyme.
Nhn chung, cc yu t nhit , m, pH v t l nm mc khng ch th hin
nh hng bc 1 v bc 2 n hiu qu thu nhn enzyme m cn chu s nh
hng ng thi ca tng cp nhn t n kh nng tng hp PME.
Tm li, phng php thng thng (thay i tng bin s kho st) theo truyn
thng c s dng cho ti u ha cc thit k a h s th nghim gp hn ch
do (i) to ra s lng ln cc d liu thng rt kh gii thch; (ii) tn km chi
ph v thi gian; (iii) b qua nh hng ca tng tc gia cc yu t. khc
phc nhng vn ny, vic s dng phng php b mt p ng (RSM) vi m
hnh phc hp ti tm (CCD) c p dng xc nh mc ti u ca cc
bin c nh hng n qu trnh sinh tng hp PME vi s th nghim va phi
(31 n v th nghim kho st ny). Cc kt qu nghin cu ch ra rng phng
php b mt p ng khng ch gip xc nh v tr cc thng s ti u ca cc
iu kin ln men gia tng hiu qu sinh tng hp PME m cn chng minh s
thch hp ca m hnh ny nh gi nhng tc ng chnh v tng tc ca cc
thng s cho vic ln men sinh tng hp PME t ph phm nng nghip t hiu
qu cao.
T phng trnh (3.1) cho thy, iu kin ti u t c hot tnh PME cao
nht (98,9%) nh sau:
- T l huyn ph bo t nm A. niger (10
5
cfu/mL) s dng l 16,5%
- Nhit 35,5C
- pH mi trng 4,0
- m mi trng ln men 57,4%
Hot tnh PME c m phng theo iu kin ln men ti u l 66 U/g.
T kt qu ny, qu trnh ln men SSF sinh tng hp PME t A. niger So
2
v R
1

di iu kin ln men ti u c tin hnh vi hot tnh enzyme iu kin
thc nghim (3 ln lp li) l 65,6 3,14 U/g.
Hin nay, nhiu nghin cu ti u ha qu trnh ln men sinh tng hp enzyme
theo phng php b mt p ng c thc hin. Suresh et al. (2009) cng p
dng thnh cng phng php ny cho vic ti u ha iu kin ln men sinh tng
hp pectinase t A. awamori vi cc thng s nhit , pH, hm lng c cht v
t l nm mc s dng c kho st. Tuy nhin, cc iu kin ti u cho qu
trnh sn xut pectinase ny c khc bit khi so snh vi cc thng s ang kho
st (nhit 31C v pH mi trng ln men 5,4). iu ny c l l do s khc
bit v dng nm mc, mt s ban u, ngun c cht ln men cng nh thi gian
nui cy. Trong khi , nghin cu gn y ca Suresh v Viruthagiri (2011) li
tm thy nhit thch hp cho qu trnh ln men sinh tng hp pectinase cng
t A. awamori l 35C vi mi trng ln men ngoi cm go cn b sung b ma.
Patil v Dayanand (2006) cng xut nhit 34C i vi qu trnh tng hp
pectinase t A. niger s dng c cht l mm ht hng dng. Trong nghin cu
iu kin sinh tng hp pectinase v PME ring l, pH 4,0 v m mi trng
ln men t 55 60% l thng s ti u, cho hot tnh enzyme cao nht (Joshi et
al., 2006; Malvessi v da Silveira, 2004).
Bn cnh qu trnh ln men, vic ly trch enzyme cng c vai tr c bit n hot
tnh enzyme thu nhn. S dng dung dch thch hp c th lm gim mt mt hot
tnh ca enzyme (L Ngc T, 2002). Hiu qu ly trch ph thuc vo loi dung
dch m v tng quan ca dung dch m v pH, nhit cng nh thi gian ly
trch v t l dch trch (Castilho et al., 2000). Chnh v vy, nng cao hiu qu
thu nhn PME t A. niger sau qu trnh ln men, mt s yu t ca qu trnh ly
trch c nh hng n hot tnh PME c kho st.
3.6 nh hng ca iu kin ly trch n hiu qu thu nhn PME sau qu
trnh ln men SSF
3.6.1 nh hng vi thy i gi tr pH dung dh m n hiu qu ly trch
PME
Vic chn la dung mi thch hp c tm quan trng trong vic thu hi enzyme t
canh trng b mt sau ln men (Shata v Farid, 2012). Cc nghin cu bc u
v ly trch enzyme PME sau qu trnh ln men SSF trn c cht b to ta cho thy,
vic s dng dung dch m t ra hiu qu hn khi so snh vi s dng dung dch
c tnh in ly cao nh mui NaCl (Trn Thanh Trc et al., 2012). Bn cnh ,
do mi enzyme ch hot ng trong mt khong pH nht nh, chnh v vy, vic
iu chnh pH ban u ca dung dch m s dng n hiu qu thu nhn enzyme
t canh trng b mt c kho st.
Da trn nghin cu nh hng ca vic iu chnh pH ban u ca mi trng
ln men n qu trnh sinh tng hp enzyme, dung dch m citrate cc gi tr
pH khc nhau c s dng nghin cu ly trch PME sau qu trnh ln men
SSF. Mu dch trch enzyme c ly trch bng nc ct l mu i chng. Kt
qu th nghim c tng hp hnh 3.33.
30,57d
16,87ab
29,14bc
66,71f
42,86d
64,14e
30,43c
14,57a
0
10
20
30
40
50
60
70
80
3,2 3,6 4,0 4,4 4,8 5,2 5,6 Nc ct
(pH
6,76,8) Gi tr pH ca dung dch m l trch
H
o
t

t
n
h

P
M
E

(
U
/
g
)

Hn 333: n ng ca pH dung dc ly trc n ot tn PME
T kt qu thu c hnh 3.33, c th nhn thy, gi tr pH ca dung dch m
citrate s dng ly trch c nh hng ng k n kh nng thu nhn PME. S
iu chnh pH gip ci thin ng k hot tnh PME thu c khi so snh vi mu
i chng l nc ct (khng iu chnh pH).
gi tr pH ca dung dch ly trch l 3,6 v 4,0, PME thu c c hot tnh cao
nht (hnh 3.33). Hot tnh PME cao nht (66,71 1,14 U/g) trng hp s
dng m citrate vi gi tr pH l 3,6, cao hn 2 ln hot tnh PME thu c
mu i chng (ly trch bng nc). Nghin cu ca Yoo et al. (2009) cng
ngh s dng m citrate pH 5,0 ly trch PME t A. aculeatus. Trong khi ,
Ali et al. (2010) li ngh ly trch pectinase t A. niger c nui cy trn c
cht l v cam bng m citrate 4,5. iu ny c l l do mi enzyme ch hot
ng mnh nht mt vng pH xc nh gi l pH ti u. S thay i pH mi
trng c nh hng n mc ion ha c cht, c bit l bn ca enzyme
(Phutela et al., 2005).
Bn cnh nh hng ca pH, t l dung dch ly trch v c cht, nhit v thi
gian ly trch l nhng nhn t quan trng nng cao hiu sut ly trch v hot
tnh ca PME. Chnh v th, vic xc nh nh hng ca t l dung dch ly trch
s dng v c cht, nhit v thi gian ly trch n PME cn phi c quan
tm. ng thi, m citrate vi pH 3,6 c s dng lm dung dch ly trch
enzyme cho cc kho st tip theo.
3.6.2 nh hng t l dung dh ly trh v nh trng b mt sau khi ln
men n hiu qu ly trch PME t nm m A. niger
S chnh lch nng cht tan gia dung mi v canh trng b mt sau ln men
cng ln s thc y qu trnh khuch tn cht tan (trng hp ny l PME) ra
ngoi dung mi cng tng. Trong h thng ln men SSF, lng nc hay dung
mi t do c trong canh trng rt gii hn, vic b sung lng dung mi thch
hp gip PME c th lin kt v khuch tn ra ngoi dung mi mc ti a l rt
cn thit (Palit & Banerjee, 2001). Do , vic kho st t l ca dung dch m
s dng ly trch v canh trng chim vai tr quan trng trong vic tng hot
tnh PME thu c t nm mc trong qu trnh ly trch iu kin ln men rn.
Trn c s , th nghim tin hnh kho st nh hng ca t l gia dung dch
m citrate c pH 3,6 v canh trng b mt sau qu trnh ln men SSF nhm thu
nhn PME t A. niger c hot tnh cao nht. Kt qu th nghim c tng hp v
trnh by bng 3.14.
Bng 3.14: nh hng ca t l dung dch m citrate v canh trng sau ln men
n hiu qu thu nhn PME
T l dung dch m citrate v
canh trng (v/w)
Th tch dch trch
(mL)
Hot tnh PME
(U/mL)
Hot tnh PME tng
(U)
1 : 1 7,36 1,04 24,43 0,57 179,80
a
1,65
1,5 : 1 11,66 1,17 25,64 0,62

289,96
d
5,67
2 : 1 12,89 1,22 32,36 1,15 417,12
e
5,25
2,5 : 1 14,87 2,01 27,93 1,14 415,31
e
5,34
3 : 1 19,08 2,24 14,62 0,93 278,95
c
6,79
3,5 : 1 33,40 2,38 5,99 0,79 200,07
b
5,96
T s F 1083,34
Gi tr P 0,00
T kt qu ca bng 3.14 cho thy, s thay i t l dung dch ly trch v c cht
(canh trng b mt) c tc ng ln n hiu sut thu hi PME ca nm mc, th
hin hot tnh ca dch trch enzyme thu c gia cc nghim thc kho st
u c s khc bit c ngha v mt thng k.
Hot tnh ca PME thu c tng dn khi t l dung dch m s dng v c cht
gia tng t 1 : 1 ln n gi tr 2 : 1 v sau li gim dn t l 3:1 v 3,5 : 1.
T l dung dch m s dng ly trch enzyme PME trong canh trng sau ln
men thp, PME tng s c thu hi thp. iu ny c l l do lng dung mi s
dng t, khng xm nhp vo ton b canh trng (Lonsane v Krishnaiah,
1992), khng cho s khuch tn cht tan (enzyme) vo dung mi (dung dch
m citrate s dng trch ly) (Madhusudhan et al., 2011). Hn na, mt lng
dung mi s c gi li trong canh trng cng l nguyn nhn lm gim hiu
qu thu nhn PME (Ikasari v Mitchell, 1996; Castilho et al., 1999), hiu qu thu
nhn enzyme thp.
Khi gia tng t l dung mi s dng, s chnh lch nng cht tan v c cht
tng, lm tng tnh tan v tng s khuch tn, v th lm tng hot tnh enzyme
trong qu trnh ly trch (Castilho et al., 2000; Madhusudhan et al., 2011). Tuy
nhin, cc t l dung mi s dng cao (3: 1 v 3,5: 1), th hot tnh PME thu
c s gim tr li do mc pha long ca PME trong dch trch tng (Lonsane
v Krishnaiah, 1992). Vi t l dung dch m s dng cng cao th th tch dch
trch (V) thu c cng nhiu, trong khi lng enzyme (m) sinh ra t qu trnh ln
men rn l khng i. Kt qu l hot tnh PME (U/mL) thu c t l dung mi
v c cht rn l 3: 1 v 3,5: 1 gim dn. Ngoi ra, khi t l dung dch m s
dng v c cht gia tng cng thc y qu trnh ha tan enzyme vo dung mi
tng nhanh giai on u. Vi nhit v thi gian ly trch c gi c nh,
lng enzyme c ha tan vo dung mi c th t n mc cao nht thi gian
ngn hn khi so snh vi cc t l dung mi v c cht thp hn; vic duy tr qu
trnh ly trch vt qu thi gian thu nhn ti u ny, enzyme c kh nng b v
hot v gim hot tnh (Shata v Farid, 2012). Mc d vy, th tch dch trch
enzyme khng gia tng t l thun cng vi s gia tng t l dung mi v c cht.
iu kin kho st, cc mu c t l dung mi s dng cao (3:1 v 3,5: 1), phn
kt ta thu c sau ly tm c khi lng cao hn khi so snh vi trng hp t l
dung mi s dng thp hn, iu ny cng gp phn lm gim th tch dch trch
v gim hot tnh PME tng. Nghin cu ca Shata v Farid (2012) cng cho
thy, hiu qu thu nhn enzyme kreatinase t cao nht khi s dng t l dung mi
(glycerol) v c cht rn l 5: 1(mL/g), vt qu t l ny, c hot tnh ring v
hot tnh tng ca enzyme u gim. Tuy vy, hu ht cc qu trnh thu hi
enzyme khc, hiu qu thu nhn enzyme tng l khng i khi t l dung mi v
c cht rn tng vt mc ti u; in hnh nh chit tch -amylase t qu trnh
ln men SSF Bacillus circulans GRS313 vi c cht cm go, hot tnh enzyme
tng khng i cc t l dung mi v canh trng s dng l 3: 1, 4: 1 v 5:1
(Palit v Banerjee, 2001). iu ny cho thy, vic mt mt enzyme v gim hot
tnh enzyme khi gia tng t l dung mi v canh trng b mt c l ty thuc vo
c tnh ca canh trng, loi dung mi v tnh cht ca enzyme.
iu kin kho st, t l dung dch m s dng ly trch: c cht l 2: 1, hot
tnh ca PME t cc i khi so snh vi cc mc pha long cao hay thp hn
(gi tr hot tnh PME tng ng l 32,36 1,15 U/mL dch trch). Tuy nhin,
hot tnh PME tng khc bit khng ngha v mt thng k hai t l dung dch
m v c cht l 2: 1 (417,12

5,25 U) v 2,5: 1 (415,31

5,34 U). T l dung
dch m s dng ly trch v c cht l 2: 1 vn c u tin chn la do
nhng l do sau: (i) Hot tnh ring t l 2: 1 vn cao hn so vi t l 2,5: 1;
(ii) Khi trong trng hp hiu sut enzyme thu c khng c s khc bit th
vic ly trch enzyme t l 2: 1 c tnh kinh t hn so vi t l 2,5: 1 nh tit
kim c mt phn th tch dung dch m s dng trong qu trnh ly trch v
gim lng ha cht s dng trong qu trnh tinh sch enzyme (Castilho et al.,
2000). Kt qu ny hon ton ph hp vi nghin cu ca Joshi et al. (2006),
nhm tc gi cng xc nh t l ti u ca dung dch ly trch s dng thu
nhn PME t A. niger sau qu trnh ln men SSF l 2: 1.
Da trn kt qu kho st, t l dung dch m citrate s dng cho qu trnh ly
trch PME trong c cht sau ln men l 2: 1 (v/w, mL/g).
3.6.3 nh hng nhit v thi gin n hiu qu ly trch PME
Bn cnh nh hng ca vic iu chnh pH dung dch m s dng trong ly trch,
t l dung dch m s dng v c cht hay canh trng b mt sau ln men, nhit
v thi gian ly trch l nhng yu t c nh hng ng k n hiu qu thu
nhn enzyme sau qu trnh nui cy (Jayani et al., 2005). Vic kho st nh hng
tng tc ca nhit v thi gian ly trch n hiu qu ly trch PME t A. niger
c tin hnh nhm tm ra nhit ph hp v thi gian ti u trong qu trnh ly
trch thu nhn PME c hot tnh cao.
C 6 mc nhit ly trch c kho st (5, 15, 25, 35, 45 v 55C) tng ng
vi 5 khong thi gian khc nhau (10 pht, 30 pht, 50 pht, 70 pht v 90 pht)
kho st s thay i hot tnh PME t A. niger trong qu trnh ly trch. Kt qu
kho st c trnh by hnh 3.34.
18,53 1,96
36,24 2,34
59,14 4,22
75,62 4,56
48,22 3,28
20,32 2,68
0
10
20
30
40
50
60
70
80
0 10 20 30 40 50 60 70 80 90 100
H
o
t

t
n
h

P
M
E

(
U
/
g
)
Thi gian (pht)
5C
15C
25C
35C
45C
55C

Hn 334: n ng ca nit v ti gian ly trc n ot tn PME
th hnh 3.34 cho thy, vic iu chnh nhit v thi gian ly trch cng c
nh hng ng k n tc truyn khi, ni cch khc chi phi n hiu qu
thu nhn PME, ng thi c s tng tc ca nhit v thi gian ly trch n
hot tnh PME thu nhn. cng thi gian ly trch, hot tnh PME thu c tng
dn khi nhit ly trch tng t 5C n 35C v bt u gim khi nhit ly
trch tip tc tng hn 35C. Trong iu kin kho st, khi nhit ly trch qu
thp (5 25C) hay qu cao (45 55C) hot tnh PME thu c s thp hn khi
so snh vi nhit khong (30 40C). Hot tnh PME cao nht thu c khi ly
trch nhit 35C.
Vic gia tng nhit ly trch c tc ng tch cc trong vic gia tng tnh tan ca
dung mi, lm tng vn tc chuyn ng v thc y qu trnh thm thu dung
mi vo c cht v gia tng kh nng khuch tn, hay ly trch PME. Tuy nhin,
nhit ly trch cao c th l nguyn nhn dn n s km n nh v c ch hot
ng ca enzyme. iu ny cng xy ra vi trng hp thi gian ly trch di.
Kt qu nghin cu ny cng tng ng vi mt s nghin cu khc cng b.
Castilho et al. (2000) xc nh rng nhit thch hp ly trch
polygalacturonase l 35C. Khi tng nhit ly trch t 5 35C s lm tng kh
nng ha tan v s khuch tn, v vy enzyme s hot ng mnh. Nhit c nh
hng rt ln n phn ng enzyme, cng nh cc phn ng ho hc thng
thng, vn tc phn ng enzyme tng khi nhit tng. nhit thp 5C v
15C enzyme b hn ch hot ng rt mnh, nhng khi tng dn nhit ly trch,
hot tnh enzyme cng tng dn u n mc ti u (35C). Nhit ti u ca
enzyme ph thuc rt nhiu vo s c mt ca c cht, kim loi, pH,... Phn ln
enzyme hot ng mnh nht nhit 40 50C (Madhusudhan et al., 2011).
Nhit ti u ca nhng enzyme khc nhau th hon ton khc nhau ty thuc
vo ngun gc ca chng. PME t nm mc c nhit ti u l 30 45C
(Nguyn c Lng, 2004). Theo nghin cu ca Duvetter (2007), nhit thch
hp ca A. aculeatus PME l 30 55C. Trong khi hot tnh ca PME c ngun
gc t A. niger c Nikolic v Mojovic (2007) nghin cu nhit ti u l
45C.
T kt qu hnh 3.34 cho thy a s PME ly trch t t hp A. niger So
2
v R
1
b
mt hot tnh nhit 55C khi thi gian ly trch ko di. Xt nh hng ca
vic thay i thi gian ly trch n s hiu qu thu nhn PME t A. niger, th
hnh 3.31 cng cho thy, tt c cc mc nhit ly trch kho st (t 5 55C),
hot tnh PME gia tng khi thi gian ly trch PME tng t 10 pht n 50 pht v
gim dn khi thi gian ly trch tip tc tng t 70 pht n 90 pht. Hot tnh
PME thu c cao nht thi gian ly trch l 50 pht. Khi thi gian ly trch qu
ngn (10 pht) hay qu di (90 pht) u lm gim hot tnh PME. Canh trng
ln men gm hai thnh phn chnh l b to v v bi, c cu trc xp, PME
trong canh trng c khuch tn vo dung mi phi xuyn qua khong trng
bn trong vi ng i quanh co, kt qu l khong cch khuch tn di (McCabe
et al., 1993). cng t l dung mi s dng, thi gian ly trch ngn s khng
cho qu trnh ngm dung mi vo canh trng, gim hiu sut thu nhn enzyme
(Shata, 2005; Negi et al., 2011; Shata, 2012). Ngc li, nu thi gian ly trch qu
di, di tc dng ca lc c hc (khuy, trn) s lm gim hot tnh ca enzyme
(Ghildyal et al., 1991). Kt qu nghin cu ny cng tng ng vi mt s
nghin cu khc cng b, in hnh nh Castilho et al. (2000) xc nh nhit
ti u ly trch polygalacturonase l 35C trong thi gian 30 pht.
T kt qu kho st cho thy hot tnh PME thu c sau qu trnh ln men SSF
c lin quan cht ch n nhit v thi gian ly trch. iu kin kho st, hiu
qu thu nhn PME t tt nht iu kin nhit 35C v thi gian ly trch 50
pht.
3.6.4 nh hng nhit bo qun n n nh dh trh PME th
Nghin cu kh nng bo qun enzyme th ng vai tr rt quan trng trong vic
chun b cho qu trnh tinh sch v ng dng tip theo. Ging nh cc enzyme
khc, PME rt d b bin i hot tnh do tc ng ca cc iu kin t nhin
(Nguyn c Lng, 2004). nhit phng, s ph v cu trc t bo dn n
bin tnh enzyme c th xy ra, h qu l enzyme b mt hot tnh (Price v
Steven, 2002). Chnh v th, nghin cu s n nh hot tnh ca PME th iu
kin bo qun lnh (4C) v tr ng (-18C) c tin hnh.
0a
0,16b
1,00c
1,63d
0,29b
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 1 2 3 4
Thi gian bo qun (ng)
A/A0

Hnh 3.35: S tay i ot tn PME t (A/A
o
) teo ti gian bo qun
nit ln (4C)
1,31a
2,12b
2,53b
2,22b
1,39a
1,00a
3,03c
1,34a
0
0.5
1
1.5
2
2.5
3
3.5
0 1 3 5 7 9 11 13
Thi gian bo qun (ng)
A/A0

Hnh 3.36: S tay i ot tn PME t (A/A
o
) teo ti gian bo qun
nit ln ng (-18C)
Kt qu kho st s thay i hot tnh ca PME theo thi gian bo qun c th
hin hnh 3.35 cho thy cc mu enzyme c bo qun hai iu kin nhit
khc nhau c s thay i hot tnh khc nhau hon ton.
Trong trng hp tr lnh enzyme nhit 4C, s thay i hot tnh PME theo
xu hng gia tng sau mt ngy bo qun v gim dn t ngy th hai. iu ny
c th c l gii l do trong qu trnh ly trch ban u, enzyme chu cc tc
ng c hc nh p, tch, ly tm lm nh hng n n nh ca h enzyme
trong dung dch. n ngy bo qun th 4, enzyme hon ton mt hot tnh. Do
trong h dung dch enzyme th thu c ngoi PME cn c nhiu phn t tp gy
cn tr cho hot ng ca PME, thm ch c th gy bin tnh PME. Do PME c
bn cht l protein nn ch cn mt nh hng nh ca cc phn t trong h s c
th nh hng rt ln n hot tnh enzyme (Deutscher, 1990).
So vi bo qun lnh, tr ng gip n nh hot tnh ca PME n hn 10 ngy
nhit -18C. Theo kt qu o c, hot tnh ca PME khng c s thay i
ng k trong 3 ngy u bo qun. S gia tng hot tnh ca enzyme th hin r
ngy th 5. iu ny c gii thch da trn kh nng km hm cc hot ng
sinh ha, vi sinh ca hp cht nh lnh ng. Do trong qu trnh ly trch ban u,
enzyme chu cc tc ng c hc nh khuy trn, ly tm, lm gim n nh
ca h enzyme trong dung dch; h dung dch enzyme th thu c ngoi PME
cn c nhiu phn t tp gy cn tr cho hot ng ca PME (Deutscher, 1990;
Price v Steven, 2002). iu kin lnh ng -18C, nc kt tinh, lng nc t
do trong dung dch cha enzyme gim. H qu l hot ng ca nc trong
dung dch gim thp, hu ht cc phn ng ha hc, sinh ha s b km hm. Sau
r ng, cc tp cht dung dch c phn tch v lng xung, mc n nh
ca enzyme gia tng (Copeland, 2000). Vic gia tng hot tnh PME trong trng
hp ny thc t l s duy tr hot tnh ca enzyme khi so snh vi trng hp
n nh ca enzyme gim ngay sau khi ly trch.
Has-Schn et al. (2005) xut s gia tng lin tc hot tnh enzyme trong
nhng ngy tn tr u tin ca lu tr l kt qu ca nhng thay i trong mi
trng t nhin (dch trch enzyme th), sau khi enzyme chit xut t mi trng
sinh hc ca chng, v / hoc c th c s gii phng ca mt s hp cht c tim
nng kch hot.
T cc kt qu thu thp v phn tch cho php kt lun, qu trnh kt ta
hiu qu nn s dng dung dch enzyme sau khi n nh mt ngy trong iu
kin nhit lnh. S dng phng php lnh ng bo qun mu cha
enzyme c th gip duy tr hot tnh enzyme trong thi gian di tt hn so vi
phng php bo qun lnh. Cc nghin cu v s n nh ca enzyme cng cho
thy, lnh ng nhanh nhit nh hn -20C (tt nht l -70C hay lnh ng
bng Nitrogen lng) l ch ti u nhm duy tr hot tnh ca enzyme
(Copeland, 2000).

3.7 Tinh sch v xc nh c im ca PME c sinh tng hp t t hp
A. niger So
2
v R
1
Qu trnh nui cy A. niger sinh ra mt hn hp nhiu loi enzyme nh PME, PG,
PL, hemicellulase, endo - - glucanase, protease,... Mi loi enzyme u c tc
dng khc nhau, thm ch tri ngc nhau trong ch bin thc phm. iu ny gy
kh khn cho vic ng dng hn hp enzyme th sau nui cy (Sajjaanantakul v
Pitifer, 1991). Nu ch phm PME c s dng cho mc ch lm gim mc
ester ha ca pectin th s c mt ca PG trong ch phm l khng cn thit.
Ngc li, nu ch phm c s dng cho mc ch lm gim nht ca dung
dch c cha pectin th s c mt ca PG s lm tng hiu qu s dng. Ngoi ra
cc nh sn xut khng mong mun s c mt ca protease trong ch phm v s
nh hng khng tt n hot tnh PME trong qu trnh bo qun v s dng.
3.7.1 nh hng loi h ht v t l s dng n kh nng kt t PME t
A. niger
Qu trnh kt ta protein c th c tin hnh da vo tnh cht ha tan ca
chng trong cc ha cht khc nhau. Hai mui trung tnh (NH
4
)
2
SO
4
v NaCl
thng c s dng cho kho st kh nng tinh sch s b enzyme (Scopes,
1994). Trong khi , tc ng ca dung mi hu c n kh nng ta enzyme
cng c nh gi da trn vic s dng ethanol v aceton cc nng khc
nhau (ng Th Thu v Nguyn Th Xun Sm, 2009a,b).
i. nh gi kh nng kt t PME t A. niger bng mui (NH
4
)
2
SO
4

Trong th nghim ny, PME th c kt ta bng mui (NH
4
)
2
SO
4
cc t l
khc nhau. Qua kho st s b cho thy t l (NH
4
)
2
SO
4
v PME th nh hn 30%
(w/v) gy kt ta rt t protein. Do , cc bc thm tch v xc nh hot tnh
PME ch tin hnh i vi cc mu enzyme thu c t phn on F35 45% n
F85 95%. Hiu qu ca qu trnh kt ta c nh gi thng qua cc ch tiu
nh hot tnh ring, hiu sut thu hi v tinh sch. S liu c thu thp, x l
v tng hp bng 3.15.
Da vo bng s liu (bng 3.15) c th thy, s thay i nng (NH
4
)
2
SO
4

trong dung dch PME th c nh hng n lng protein kt ta, hot tnh, hiu
sut thu hi v tinh sch ca enzyme.
Bng 3.15: Hiu qu tinh sch PME bng phng php kt ta vi (NH
4
)
2
SO
4

(NH
4
)
2
SO
4
:
PME th
(w/v)
Protein
(mg/mL)
Hot tnh
PME (U/mL)
Hot tnh
ring
(U/mg
protein)
Hiu sut thu
hi (%)
tinh sch
(ln)
i chng 38,30 3,91 44,60 0,28 1,26
a
0,13 100
g
0 1,00
a
0
F35 45% 7,75 0,21 14,50 0,42 1,87
a
0,01 32,51
cd
0,94 1,49
a
0,15
F45 55% 5,65 0,35 16,75 1,48 2,98
b
0,44 37,56
e
3,31 2,39
b
0,60
F55 65% 4,05 0,09 27,75 1,34 6,87
d
0,48 62,22
f
2,99 5,45
d
0,17
F65 75% 3,04 0,02 15,50 0,42 5,11
c
0,17 34,75
de
0,94 4,06
c
0,28
F75 85% 2,73 0,06 12,50 0,70 4,58
c
0,35 28,03
b
1,57 3,63
c
0,09
F85 95% 2,32 0,02 8,50 0,70 3,66
b
0,28 19,06
a
1,57 2,91
b
0,07
T s F 75,71 397,49 57,54
Gi tr P 0,00 0,00 0,00
C gi tr mu t i km ging nhu trong ng mt t biu th s kh bit khng ngh v mt
thng k theo php th Dunn tin y 95%
cc phn on mui ammonium sulfate cng cao th lng protein kt ta cng
nhiu. iu ny c gii thch l do cc ion ca phn t (NH
4
)
2
SO
4
lin kt vi
cc phn t nc lm cho protein mt lp o nc, lin kt li vi nhau v hnh
thnh kt ta (Mukherjee v Banerjee, 2006). Tuy nhin, kt qu t bng 3.20 cho
thy hm lng protein ha tan tr li sau khi kt ta cng thp khi nng ha
cht kt ta tng. Kt qu ny cng ph hp vi nghin cu tinh sch PME t A.
niger, s dng c cht b to ca Joshi et al. (2011).
Hm lng protein ha tan sau kt ta cho bit hiu qu thu hi ca tt c cc
enzyme v protein trong hn hp enzyme th. Tuy nhin, hot tnh PME, hot tnh
ring, hiu sut thu hi cng nh tinh sch c ngha quan trng hn trong
vic nh gi hiu qu gia cc phng php p dng. Kt qu th nghim cho
thy, hot tnh PME cng nh hot tnh ring v tinh sch khng quan h tuyn
tnh vi hm lng protein ha tan sau kt ta. iu ny l do mi loi enzyme c
mc ion ha, hydrate ha v i lc vi nc khc nhau (Mukherjee v
Banerjee, 2006). Do vy, i vi mu kt ta phn on F35 45% (NH
4
)
2
SO
4
,
hot tnh PME cng nh tinh sch kh thp mc d hm lng protein cao
nht. Ngc li, mu kt ta phn on F55 65% (NH
4
)
2
SO
4
cho hiu qu tinh
sch tt nht (hot tnh ring, tinh sch v hiu sut thu hi cao). Khi nng
(NH
4
)
2
SO
4
c s dng cc phn on cao hn F55 65% th hiu qu tinh
sch gim dn, ph hp vi kt qu v hm lng protein thu hi. iu ny cho
thy, vic s dng nng (NH
4
)
2
SO
4
qu cao cng lm bin tnh khng thun
nghch PME. Theo nghin cu ca Jaffar et al. (1993), nng mui bo ha
c s dng cho qu trnh kt ta enzyme ty thuc vo loi enzyme, ngun gc
thu nhn enzyme v thng t 60 n 80%. Arotupin et al. (2008) cng tm thy
hiu qu ca vic ng dng (NH
4
)
2
SO
4
trong kt ta PME th t A. repens, tuy
nhin hiu sut thu hi ca tin trnh ny cng ch t 56,82% v tinh sch tng
2,5 ln. Nghin cu gn y ca Joshi et al. (2011) xc nhn hiu qu ca vic
s dng (NH
4
)
2
SO
4
trong tinh sch pectinase (giu PME) t A. niger, vi s gia
tng hot tnh PME t 8,25 U/g n 21,5 U/g tng ng vi s gia tng nng
mui (NH
4
)
2
SO
4
t F0-20 n F60-80%. Tuy nhin, khng c gi tr v hot tnh
ring ca PME t c sau qu trnh kt ta bng (NH
4
)
2
SO
4
c cng b.
Trong iu kin kho st, phn on s dng thch hp nht ca (NH
4
)
2
SO
4
cho
qu trnh kt ta PME l F55-65%, khi hot tnh ring ca PME t 6,87 U/mg
protein, tinh sch 5,45 ln v hiu sut thu hi l 62,23%. Nhn chung, hiu
sut thu hi PME t qu trnh kt ta vi (NH
4
)
2
SO
4
vn kh thp. Chnh v vy,
nghin cu s dng loi cht tan khc trong qu trnh kt ta protein cng c
ngh.
ii. nh gi kh nng kt t PME t A. niger bng NCl
Bn cnh (NH
4
)
2
SO
4
, mui NaCl cng c tc dng tng t trong kt ta protein.
Mui NaCl c kh nng to p sut thm thu cao cng nh tnh ht nc mnh
nn d lm kt ta enzyme. Chnh v vy, th nghim c b tr vi cc phn
on NaCl t 15% n 35% (w/v) (thp hn so vi t l (NH
4
)
2
SO
4
v PME th).
Hiu qu ca qu trnh kt ta cng c nh gi thng qua cc ch tiu nh hot
tnh ring, hiu sut thu hi v tinh sch. Kt qu thu nhn trnh by bng
3.16.
Bng 3.16: Hiu qu tinh sch PME bng phng php kt ta vi NaCl
NaCl : PME
th (w/v)
Protein
(mg/mL)
Hot tnh
PME (U/mL)
Hot tnh ring
(U/mg protein)
Hiu sut
thu hi (%)
tinh sch
(ln)
i chng 38,30 3,91 44,60 0,28 1,26
a
0,13 100
f
0 1,00
a
0
0 15% 7,56 0,05 15,50 1,83 2,05
b
0,25 34,75
a
4,1 1,62
a
0,03
15 20% 5,37 0,22 23,50 1,34 4,34
c
0,43 52,13
b
2,99 3,44
b
0,01
20 25% 5,88 0,14 34,95 0,91 5,95
d
0,30 78,36
c
2,05 4,76
c
0,73
25 30% 4,94 0,16 41,30 1,13 8,37
f
0,04 92,60
e
2,52 6,67
d
0,65
30 35% 4,99 0,04 37,90 0,42 7,60
e
0,14 84,98
d
0,94 6,06
d
0,74
T s F 257,24 202,68 37,98
Gi tr P 0,00 0,00 0,00
thng k theo php th Dunn tin y 95%i
Tng t nh tc dng kt ta ca mui (NH
4
)
2
SO
4
, cc protein khc khng phi
PME kt ta nhiu nng mui thp, tng ng vi hm lng protein thu
c nhiu. Hiu qu tinh sch PME ti u phn on NaCl s dng l
F2530%. nng mui NaCl cao hn cng lm bin tnh bt thun nghch
PME nn hot tnh v hiu sut thu hi cng nh tinh sch thp. Ngo et al.
(2008) cng tm thy t l NaCl ti u 35% cho vic kt ta PG vi tinh sch
tng 5,3 ln, tuy nhin hiu sut thu hi ch t 48,87%.
Mui (NH
4
)
2
SO
4
v NaCl l cc cht kt ta protein r tin, d kim v t lm bin
tnh protein. Tuy nhin, mui phi c loi ra khi enzyme sau qu trnh kt ta.
Tri li, ethanol v aceton l cc dung mi hu c t tin nhng cng on loi
dung mi sau kt ta c thc hin d dng hn bng cch cho dung mi bay hi
t nhin. Hiu qu ca vic kt ta enzyme ph thuc vo nhiu yu t, trong
quan trng nht l c tnh ca tng enzyme v ngun gc ca n. Do vy, vic
kho st nh hng ca nhiu loi ha cht khc nhau n hiu qu kt ta PME
t A. niger c thc hin nhm tm ra bin php kt ta thch hp nht.
iii. nh hng vi s dng dung mi hu n kh nng kt t PME t
A. niger
Kt qu kho st nh hng ca dung mi ethanol v acetone n hiu sut thu
hi, tinh sch v hot tnh ring ca PME sau kt ta c trnh by bng
3.17 v 3.18.
Bng 3.17: Hiu qu tinh sch PME bng phng php kt ta vi ethanol
Ethanol v
PME th (v/v)
Protein
(mg/mL)
Hot tnh
PME (U/mL)
Hot tnh ring
(U/mg protein)
Hiu sut
thu hi (%)
tinh sch
(ln)
i chng 38,30 3,91 44,60 0,28 1,26
a
0,13 100
f
0 1,00
a
0
1,5 8,83 0,31 14,70 0,70 1,67
a
0,02 32,96
a
1,57 1,33
ab
0,12
2,0 7,19 0,03 22,80 0,56 3,17
b
0,09 51,12
c
1,26 2,53
b
0,33
2,5 6,80 0,03 34,60 1,69 5,09
c
0,27 77,58
d
3,78 4,04
c
0,20
3,0 4,07 0,08 40,80 0,98 10,02
e
0,03 91,48
e
2,20 7,98
d
0,80
3,5 4,13 0,11 35,80 0,56 8,67
d
0,37 80,27
d
1,26 6,93
d
1,01
4,0 6,54 0,60 18,25 0,91 2,81
b
0,40 40,92
b
2,05 2,26
ab
0,55
T s F 407,36 322,65 44,61
Gi tr P 0,00 0,00 0,00
Bng 3.18: Hiu qu tinh sch PME bng phng php kt ta vi aceton
Aceton v PME
th (v/v)
Protein
(mg/mL)
Hot tnh
PME (U/mL)
Hot tnh ring
(U/mg protein)
Hiu sut
thu hi (%)
tinh sch
(ln)
i chng 38,30 3,91 44,60 0,28 1,26
a
0,13 100
d
0 1,00
a
0
1,0 5,22 0,91 6,66 0,43 1,28
a
0,14 14,84
a
0,97 1,01
a
0,01
1,5 3,45 0,44 14,96 1,08 4,40
c
0,88 33,54
c
2,43 3,47
d
0,34
2,0 3,33 0,26 8,45 0,53 2,55
b
0,36 18,94
b
1,18 2,02
c
0,07
2,5 3,14 0,02 6,02 0,13 1,92
ab
0,02 13,49
a
0,29 1,52
b
0,13
T s F 17,76 1585,47 81,60
Gi tr P 0,00 0,00 0,00
Vic s dng mt t l thch hp ca acetone v dung dch enzyme c kh nng h
thp hng s in mi ca mi trng, gip cc phn t nc o bn ngoi protein
c khuynh hng b y i, v th cc phn t protein c c hi lin kt vi nhau
v to kt ta. Theo Rosenberg (2005), thm dung mi hu c vo dung dch
enzyme lm gim im ng in ca chng, v vy tng kh nng lin kt ca cc
phn t protein, dn n tng hiu sut thu hi ca enzyme thu c. cc t l
tip theo, hot tnh enzyme v hiu sut thu hi u gim dn. iu ny c th
c gii thch l do PME th thu c t bn vng, ng thi bn cht enzyme l
protein nn khi gp dung mi hu c c tnh phn cc mnh nh acetone, enzyme
d dng b ph hy khi b sung lng acetone qu nhiu vo kt ta. Trn th
gii cha c nhiu kt qu cng b v hiu qu kt ta protein tinh sch PME
t A. niger bng acetone, tuy nhin s thnh cng ca vic s dng acetone i
vi PME t ngun vi sinh vt khc hay mt vi enzyme khc cng nh c ghi
nhn. Reignault et al. (1994) s dng acetone trong tinh sch s b PME t
Botrytis cinerea vi t l acteone lnh v enzyme th l 66% (v/v). Qu trnh tinh
sch enzyme endo-PG t A. awamori cng cho thy hiu qu kt ta protein t
l enzyme v dung mi l 40: 60 (v/v) (Ngo et al., 2008). Nghin cu ca Rasouli
v Kulkani (1995) cho thy, hiu sut thu hi t 68% v tinh sch tng 3 ln
khi kt ta -galactosidase t A. niger UV-5 bng acetone vi t l enzyme v
acetone s dng l 1: 1,5 (v.v).
Vic ng dng phng php kt ta bng ethanol cho thy hiu qu tinh sch
PME tt hn trng hp s dng acetone. ng vi cc t l enzyme: ethanol tng
dn t 1: 1,5 n 1: 3, hiu sut thu hi enzyme, tinh sch cng nh hot tnh
ring ca PME gia tng. Khi lng dung mi s dng tng vt qu 1: 3 thc y
s ph hy enzyme, dn n hiu qu tinh sch gim. t l enzyme v ethanol l
1: 3, hot tnh ring t cc i (10,02 U/mg protein), hiu sut thu hi cng kh
cao (91,48%) v tinh sch tng ng k so vi mu i chng (3,47 ln). Banu
et al. (2010) cng kt ta hn hp pectinase, thu PME t Penicillium
chrysogenum bng ethanol vi t l dung mi: enzyme th l 3: 1. Farinas et al.
(2011) cng khng nh s thnh cng ca vic s dng ethanol tinh sch s b
endoglucanase v xylanase sau qu trnh nui cy A. niger, vi nng ethanol
trong hn hp ly trch l 60%, hiu sut thu hi tng ng vi hai loi enzyme l
61% v 25%. Nghin cu gn y ca Kants (2013, ng trn trang in t vo
16.4.2012) cho thy, kt ta PG t A. niger MTCC 3323 bng ethanol 60% gip
tinh sch tng 1,68 ln. Cc kt qu nghin cu gp phn khng nh vic
s dng ethanol trong kt ta, tinh sch s b PME t qu trnh nui cy A. niger
So
2
v R
1
l c tnh kh thi.
Mi loi enzyme c kh nng kt ta khc nhau vi tng loi ha cht c th cng
nh t l s dng. Do vy, bn cnh vic tm ra t l s dng thch hp nht ca
tng loi ha cht trong qu trnh kt ta PME th hiu qu s dng ca cc loi
ha cht cn c so snh nhm tm ra bin php ti u nht cho s kt ta PME
to enzyme c tinh sch cao, t l thu hi ln cng nh gia tng kh nng ng
dng enzyme. Hiu qu kt ta PME ca cc loi ha cht khc nhau t l thch
hp nht ca tng loi ha cht c thng k v trnh by bng 3.19.
Bng 3.19: So snh hiu qu tinh sch PME bng cc tc nhn khc nhau
Tc nhn
kt ta
T l
ha cht v PME th
Hot tnh ring
(U/mg
protein
)
Hiu sut
thu hi (%)
tinh
sch (ln)
i chng

1,26
a
0,13 100
d
0 1,00
a
0
(NH
4
)
2
SO
4
F55 65% (w/v)

6,87
c
0,48

62,23
b
2,99 5,45
c
0,18
NaCl F25 30% (w/v)

8,37
d
0,05 92,60
c
2,53 6,67
d
0,66
Ethanol 3 : 1 (v/v)

10,02
e
0,03 91,48
c
2,21 7,98
e
0,80
Aceton 1,5 : 1 (v/v)

4,40
b
0,89 33,54
a
2,43 3,47
b
0,34
T s F 113,45 292,05 55,40
Gi tr P 0,00 0,00 0,02
Da vo kt qu tng hp bng 3.19 cho thy phng php kt ta ci thin
ng k hot tnh PME. Hot tnh ring ca PME ca cc mu kt ta u cao hn
v c s khc bit ngha so vi mu i chng. Tuy nhin, cc loi ha cht
khc nhau c hiu qu kt ta PME rt khc bit mc ngha thng k 5%.
Phng php kt ta bng aceton cho hiu qu kt ta nh nht tt c cc ch
tiu v hot tnh ring cng nh hiu sut thu hi v tinh sch. iu ny c th
do hng s in mi do aceton to ra khng thch hp hoc cha kt ta PME.
c bit, PME d b bin tnh khi thm aceton vo dung dch enzyme. So vi
aceton th mui (NH
4
)
2
SO
4
cho hiu qu kt ta tng i cao hn v c s khc
bit mc ngha thng k 5%. V mt l thuyt, hiu qu kt ta protein ca ion
SO
4
2-
cao hn Cl
-
cng nh tc ng ca NH
4
+
n qu trnh tinh sch protein ni
chung mnh hn Na
+
(Michael, 1939). Tuy nhin kho st ny, hiu sut thu hi
cng nh cc ch tiu v tinh sch v hot tnh ring vn cn thp hn so vi
phng php kt ta bng NaCl. iu ny cho thy, kh nng kt ta protein ca
cc tc nhn khc nhau ph thuc vo tng loi protein, enzyme kho st cng
nh iu kin thc t.
Vic s dng dung mi ethanol t ra hiu qu i vi vic kt ta s b enzyme,
th hin hot tnh ring gia tng khc bit vi khi so snh vi cc loi ha cht
kt ta khc s dng v hiu sut thu hi tng ng vi vic s dng NaCl
(92,60% v 91,48%). ng thi, vic s dng ethanol cn gip gim bt cng
on thm tch sau qu trnh kt ta vi mui.
Da trn cc kt qu kho st, ethanol c s dng lm dung mi kt ta PME
vi t l ethanol: enzyme th l 3: 1.
3.7.2 inh sh s b PME t A. niger bng phng php l mng
Trong th nghim ny, mu enzyme c kt ta trc bng ethanol (t l
3:1) c ha tan li trong dung dch m pH 4,5 v bm qua h thng mng
khong phn on 50 kDa v tc bm mu 150 rpm. Kt qu t bng 3.20 th
hin hiu qu tinh sch PME t A. niger bng phng php lc mng.
Bng 3.20: Hiu qu tinh sch PME t A. niger sau qu trnh lc mng
Thng s Gi tr
Hot tnh ring (U/mg
protein
) 15,63 1,12
tinh sch (ln) 12,40 0,89
Hiu sut (%) 31,67 2,01
Kt qu thu c bng 3.20 cho thy, hiu sut thu hi PME t c cho
trng hp ny l 31,67% trong khi hot tnh ring ca PME qua kt ta bng
ethanol trc khi lc mng l 15,63 U/mg
protein
. Hot tnh ring cao ca mu
enzyme sau bc kt ta bng ethanol c l l iu kin thun li cho vic thu
nhn PME c tinh sch cao hn.
Kt qu cng cho thy hiu qu ca vic s dng ethanol trong vic kt ta s b
enzyme, tng hiu qu tinh sch PME cc bc tip theo.
Tin hnh chy in di PME sau khi lc mng nhm khng nh hiu qu tinh
sch PME v xc nh khi lng phn t ca enzyme thu c.

Hnh 3.37: Kt qu in di trn gel SDSPAGE cc mu PME t A. niger
0: hng hun; 1: Mu enzyme th su ly trh, vi l 0,2 m; 2: PME su qu trnh kt t ethnol 1: 3
(v/v), l mng 50 kD
T kt qu in di trn gel SDSPAGE hnh 3.37 c th nhn thy, mu enzyme
th ngay sau ly trch (ging 2, mu 1) c s hin din cao ca protein nm trong
khong khi lng phn t t 37 n 50 kDa, ph hp vi cc kho st trc
cho PME. Duvetter (2007) xc nh c khi lng phn t ca PME ti t
hp t A. aculeatus l 41 kDa, Reignaut (1994) cng xc nh c khi lng
phn t ca PME t Botrytis cinerea trn SDS-PAGE l 42 kDa. Khanh et al.
(1991) xc nh tnh cht ca PME c nui cy t A. niger RH5344 v xc
nh y chnh l glycoprotein cha 314 acid amin, c khi lng phn t 45 kDa.
Enzyme th sau khi qua vi lc (ng knh l lc 0,2 m) nhm loi b vi khun
v cc tp cht cho kt qu in di vi 4 bng phn tch r nm khong 100
kDa, 65 kDa, 37 50 kDa v 32 kDa. Hm lng protein tp trung cao nht
khong khi lng phn t t 37 50 kDa vi di bng r v m nht. Trong
trng hp PME qua kt ta ethanol kt hp vi lc mng, kt qu in di ch
cho thy c s hin din ca hai bng vi mt bng chnh ln nht c khi lng
phn t l 43 kDa v mt bng nht hn nm khong khi lng phn t 32
kDa. iu ny cho php kt lun PME t A. niger c khi lng phn t khong
43 kDa, ph hp vi cc nghin cu trc cho PME t vi sinh vt.
Tnh cht ca ch phm PME thu c:
- pH ca ch phm PME: 4,0
- Hot tnh: khong 15 U/mg protein
- Cm quan: trong, c mu vng nht
- Khng c s hin din ca orchatoxin A
(bng kt qu kim tra ph lc 2)
Do mc tinh sch enzyme sau lc mng cn thp, chnh v th vic xc nh
im pI ca enzyme ny cha c tin hnh. Mc d vy, vic kho st cc yu
t nh hng n hot tnh PME t A. niger vn c xc nh, lm c s cho
vic s dng enzyme ny trong ci thin c tnh cu trc ca t bo rau qu.
3.7.3 Mt s im bn h phm PME k thut thu nhn t A. niger
i. nh hng nng petin n hot tnh PME thu nhn t A. niger
Bn cnh nhit v pH th hng s tc phn ng K
m
v tc phn ng cc
i V
max
l hai thng s c bit quan trng trong nghin cu v ng dng
enzyme. Gi tr V
max
c vai tr quan trng trong vic la chn nng enzyme v
c cht thch hp trong cc qu trnh ng dng. ng thi, gi tr K
m
xc nh i
lc ca enzyme v c cht, K
m
ln cho thy i lc gia enzyme v c cht thp v
ngc li (Banu et al., 2010).
Gi tr V
max
v K
m
ca PME t A. niger c xc nh bng cch b sung PME
vo 30 mL dung dch pectin cc nng khc nhau. Mi quan h gia nng
pectin v hot tnh PME c trnh by hnh 3.38.
0 2 4 6 8 10
0
2
4
6
8
10
12
14
16
18
V
max
= 17,69 mol.min
-1
.mg
-1
protei n
K
m
= 0,6014 mg/mL
pecti n
Pectin (mg/mL pectin)
P
M
E

(
m
o
l
.
m
i
n
-
1
.
m
g
-
1
p
r
o
t
e
i
n
)

Hnh 3.38: n ng ca nng pectin n ot tn PME t A. niger
T kt qu th nghim cho thy nng c cht c nh hng quan trng n hot
tnh PME t A. niger. Nhn chung, s tng nng c cht s thc y s gia tng
hot tnh enzyme. Hot tnh enzyme tng nhanh khi nng c cht pectin tng t
0 n 4 mg/mL. S tng hot tnh enzyme khi tng nng c cht c th l do
gia tng s tip xc v lin kt gia c cht v enzyme. Tuy nhin, cc nng
c cht pectin cao hn (4 10 mg/mL) th hot tnh PME t A. niger hu nh
khng c s thay i. Murray et al. (1990) chng minh rng s tng nng
c cht vt qua nng ti u s khng lm tng hot tnh enzyme.
Thng qua vic thng k cc s liu th nghim v phng trnh Michaelis-
Menten, hng s K
m
ca PME kho st l 0,6014 0,0379 mg/mL v gi tr V
max

17,69 0,67 mol/pht.mg
protein
. Kt qu ny cho thy PME t A. niger c kh
nng ng dng cao do nhy ca enzyme ln (K
m
nh) so vi PME t cc dng
nm mc khc.
Xt v gi tr V
max
, mc d khng c nhiu nghin cu i vi PME cng b kt
qu ny, tuy nhin cc thng tin cng b u cho thy, gi tr V
max
ca PME
ngun gc thc vt thng thp hn khi so snh vi nm mc. Karaku &
Pekyardmc (2009) a ra gi tr V
max
ca PME ly trch t artiso
(Prunusarmeniaca L.) l 0,64 mol/pht.mg
protein
. Nghin cu ca nal v Bellur
(2009) tm c gi tr Vmax ca PME c ly trch t carrot en l
0,64 mol/pht.ml
protein
. Trong khi , gi tr PME ngun gc t A. oryzae l
14,9 mol/pht.ml
protein
(Abdel-Naby et al., 1999).
Duvetter (2007) kt lun gi tr K
m
ca PME t A. aculeatus l 1,4 0,07
mg/mL theo cng phng php xc nh. Bn cnh , Christgau et al. (1996)
xc nh gi tr K
m
ca PME t A. aculeatus l 2,7 mg/mL khi s dng phng
php xc nh hot tnh enzyme nhit 30C v khng b sung NaCl vo c
cht. So vi gi tr K
m
ca PME t nm mc, gi tr K
m
ca PME t thc vt
thng c gi tr thp hn. Gi tr K
m
ca PME t thc vt nh du, chui, c rt
v to ln lt l 0,42 mg/mL, 0,15 mg/mL, 0,20 mg/mL v 0,10 mg/mL (Denes
et al., 2000, Ly Nguyen et al., 2002). iu ny chng t, hng s K
m
v Vmax l
thng s thay i ph thuc vo ngun gc enzyme v phng php xc nh hot
tnh.
ii. nh hng tng t nhit v pH n hot tnh PME kho st
Nhit l yu t rt quan trng nh hng ln n hot tnh xc tc enzyme.
Cng nh cc phn ng ho hc thng thng, vn tc phn ng enzyme tng khi
nhit tng. Tuy nhin, enzyme c bn cht l protein nn khng bn di tc
dng ca nhit cao hn 70C. Ti nhit ny, phn ln phn t enzyme b
bin tnh hon ton dn n mt hot tnh xc tc ca n (Nguyn c Lng,
2004). Chnh v th, kho st nh hng ca nhit mi trng n hot tnh
PME sinh tng hp t nm mc A. niger c thc hin trong khong dao ng
nhit t 30C n 60C. Bn cnh nhit , hot ng ca enzyme rt nhy vi
s thay i pH ca mi trng. Mi enzyme u c mt khong pH thch hp m
ti tc phn ng xy ra nhanh nht. Ngoi khong gi tr pH th tc
phn ng ca enzyme s gim dn (Nguyn c Lng, 2004). Tuy nhin, mi
loi enzyme c pH ti u khc nhau ph thuc vo ngun gc, nhit , c cht,...
(Benen et al., 2003). V th, vic nghin cu nh hng tng tc ca pH v nhit
mi trng n hot tnh enzyme cn phi c thc hin cho tng loi
enzyme c th. T , c nhng xut thch hp cho qu trnh ng dng enzyme
ny. th biu din tng tc ca nhit v pH n s thay i hot tnh ca
ch phm k thut PME t A. niger c th hin hnh 3.39.

Hnh 3.39: th b mt p ng v th ng ng im biu th s tng
quan gia pH v nhit n hot tnh PME t A. niger
T th hnh 3.39 cho thy, hot tnh PME gim thp nht gi tr pH v nhit
cao (pH 6,0 v nhit 50C). cng nhit cao (50C), hot tnh PME t
l nghch vi s gia tng pH.
Nhit mi trng ti u cho hot ng ca PME thu nhn t qu trnh ln men
rn A. niger vo khong 38 40C. ng vi iu kin nhit ny, hot tnh
PME th hin cao nht. Khi nhit thp hn hay cao hn so vi nhit ti u
s lm gim hot tnh enzyme (Mathewson, 1998). Xt v tc ng ca pH, PME
c hot tnh cao nht tng ng vi pH mi trng khong 5,0. Trong iu kin
kho st, khi pH mi trng tng t gi tr 3,5 n 5,0 hot tnh PME c xu hng
gim. S ph thuc ca hot tnh enzyme vo pH do s thay i mc ion ha
ca c cht v trung tm hot ng ca enzyme (Phm Th Trn Chu v Phan
Tun Ngha, 2007).
Kt qu ny cng trng hp vi cc nghin cu trn th gii cho cc PME khc.
Maldonadooo et al., (1994) cng tm ra pH ti u cho hot ng ca PME t
A. niger (phn lp t t) l 5,0. Nghin cu gn y nht ca Huang et al. (2011)
cng ngh pH 5,0 v nhit t 40 50C l iu kin ti u gip gia tng
hiu qu hot ng ca PME t A. niger. Trong khi , Nikolic v Mojovic (2007)
ch ra nhit ti u cho hot ng ca PME t nm mc l 45C pH 3,5.
Ngc li, theo nghin cu ca Christgau et al. (1996), gi tr pH ti u ca PME
t A. aculeatus l 4,5 4,6 nhit 30C. Cng vi i tng l PME t
A. aculeatus, Duvetter (2007) li tm thy gi tr nhit v pH ti u cho hot
ng ca enzyme ny l khong nhit 40 45C kt hp vi pH dao ng
trong khong 4,1 4,2. Trong trng hp ca PME t A. repens, nhit ti u
cho hot ng ca enzyme ny l 30C v pH tng ng 6,5 (Arotupin et al.,
2008).
T kt qu thu c cho php kt lun, hot ca enzyme ph thuc rt ln vo
nhit v pH mi trng. Ty thuc vo ngun gc, phng php x l, mi
enzyme c gi tr nhit v pH ti u khc nhau. PME trong iu kin kho st
t hot tnh cao nht khong pH 5,0 v nhit mi trng khong 40C.
iii. ng h s v hot nhit PME thu nhn t A. niger So
2
v R
1

Nhit cao thng c s dng trong cc qu trnh ch bin thc phm nhng
li l nguyn nhn gy bt hot enzyme, ngay c PME, v th nh hng n hiu
qu s dng enzyme ny. Trong mt s tin trnh ch bin, PME c th c kch
hot trong cng on ny nhng s hot ng ca n cc giai on tip theo li
to s bt li cho sn phm. Do vy, bn cnh vic xc nh nhit hot ng ti
u th hiu qu v hot nhit PME cng khng km phn quan trng trong vic
ng dng enzyme ny (Sajjaanantakul v Pitifer, 1991). Th nghim c thc
hin nhm a ra nhng xut thch hp trong cc qu trnh ng dng PME
trong ch bin thc phm.
PME sau khi tinh sch s b c x l nhit 50C, 60C v 70C trong cc
khong thi gian khc nhau t 0 n 50 pht. Hot tnh cn li ca enzyme ny
c xc nh bng phng php chun in th. Kt qu nghin cu cho thy,
s v hot nhit PME t A. niger tun theo m hnh ng hc bin i mt phn
(Fractional conversion model) (hnh 3.35).

Hnh 3.40: Png trn biu t s v ot nit ca PME t A. niger
Kt qu t th hnh 3.40 cho thy s v hot nhit PME t A. niger ph
thuc ln vo nhit v thi gian x l. Qu trnh x l nhit cng di, nhit
cng cao th enzyme b v hot cng nhiu. S v hot nhit ph thuc ln vo
nhit khi x l nhit trong thi gian ngn. Tc v hot nhit tng khi tng
nhit x l tng t 50 n 70C. Khi thi gian x l nhit tng t 0 n 10 pht
th hot tnh enzyme gim hn 60%. Phn khng bn nhit ca enzyme chim
khong 60%, phn ny s b bt hot nhit rt nhanh trong thi gian ngn. Phn
bn nhit tng i n nh vi nhit , do hot tnh enzyme gim chm hn
trong thi gian sau . Tri li, tc v hot nhit hu nh khng c s khc bit
ngha khi x l nhit trong thi gian di. T sau khong thi gian 30 pht, tc
v hot nhit khng c s khc bit gia cc mu x l cc nhit khc
nhau.
H s tc v hot (k) PME t A. niger 50C, 60C v 70C ln lt l 0,135,
0,187 v 0,286 pht
1
tng ng vi gi tr D l 17,06, 12,32 v 8,04 pht (bng
3.21). Kt qu ny cho thy PME t A. niger km bn nhit hn so vi PME t
cc ngun khc. Christgau et al. (1996) kt lun PME t A. aculeatus c h s tc
v hot k l 0,009 pht
1
57C. Bn cnh , h s k ca PME chui, c rt
v du ty ln lt l 0,009 (65C); 0,6814 (60C); 0,1271 pht
1
(60C) (Ly
Nguyen et al., 2002).
Bng 3.21: Cc thng s ng hc ca qu trnh bt hot nhit PME sinh tng hp
t A. niger
Nhit
(C)
k (1/pht) D (pht) A
(%) Phng trnh ng hc

50 0,135 0,002 17,06 0,24 2,29 0,13 A = 2,29 + 43,71.exp(0,135t)
60 0,187 0,005 12,32 0,32 1,79 0,19 A = 1,79 + 44,21.exp(0,187t)
70 0,286 0,005 8,04 0,15 0,89 0,10 A = 0,89 + 45,11.exp(0,286t)
E
a
= 34,57
3,29 kJ/mol
R
Ea
2
= 0,995

Da trn kt qu thu c bng 3.21 cho thy, phn n nh nhit ca PME thu
nhn t A. niger nhit 50
o
C chim t l khong 2,29% so vi tng hot tnh
v gim dn cc iu kin nhit cao hn (A

60 v 70
o
C ln lt l 1,79%
v 0,89%). So snh vi kho st trc ca Ly Nguyen et al. (2002), phn n
nh nhit ca PME t du ty chim 11,2% 1,4 nhit 60C. Nng lng
hot ha cho tc v hot PME t A. niger l 34,57 3,29 kJ/mol, thp hn hn
cc PME c ngun gc t thc vt (E
a
ca PME t du ty l 206,7 38,4 kJ/mol)
(Ly Nguyen et al., 2002). iu ny gp phn khng nh mt ln na tnh khng
bn nhit ca PME t A. niger.
iv. nh hng vi b sung NCl v CCl
2
n hot tnh PME kho st
Hot ng ca enzyme cn chu s nh hng ca cc cht hot ha cng nh
cht km hm enzyme. Cc hp cht ion c bit n nh cht hot ha c nh
hng ng k n c tnh n nh nhit ca nhiu enzyme theo nhiu phng
thc khc nhau (Plaza et al., 2008). Nhiu nghin cu cho thy ion Na
+
v ion
Ca
2+
l nhng ion hot ha hiu qu cho hot ng ca PME. Kh nng hot ha
enzyme ca cc ion ny ph thuc rt ln vo nng s dng, loi enzyme v
ngun gc thu nhn enzyme (Arotupin, 2008; Plaza et al., 2008). Nng ion Na
+

v ion Ca
2+
ph hp s gip hot ha enzyme, nhng nu nng qu cao s c
ch, lm cho enzyme gim hot tnh. Do , vic la chn nng NaCl v CaCl
2

thch hp gip tng cao hot tnh PME t A. niger l iu cn thit.
Th nghim c tin hnh kho st s nh hng ca NaCl v CaCl
2
cc
nng khc nhau n hot tnh PME t A. niger. Kt qu thu nhn th hin
bng 3.22 v 3.23.
Bng 3.22: nh hng ca nng NaCl n hot tnh PME t A. niger
Nng NaCl (M) Mc thay i hot tnh (A/A
0
)
0 1,00
a
0
0,05 3,00
c
0,28

0,10 3,25
d
0,35
0,12 3,38
d
0,18

0,15 3,63
e
0,53

0,20 2,50
b
0,71
T s F 10,81
Gi tr P 0,0058
T kt qu phn tch thng k, c th nhn thy nng NaCl c nh hng tch
cc n hot tnh ca PME t A. niger. Hot tnh ca PME khi c o c trong
iu kin c s tham gia ca NaCl u gia tng khc bit c ngha (t 2,5 n
3,63 ln) khi so snh vi mu i chng (khng c NaCl). nng NaCl 0,15
M (trong dung dch pectin 3,5 g/L), hot tnh ca PME tng cao nht (3,63 ln)
khi so snh vi trng hp khng c s tham gia ca NaCl.
Tuy nhin, tc ng ca NaCl i vi s hot ha enzyme c gi tr ti hn.
nng NaCl cao hn gi tr ti u (trng hp ny l 0,2 M), mc hot ha
enzyme gim dn, th hin gi tr hot tnh ca enzyme gim, tuy nhin vn cao
hn khi khng c s hin din ca ion Na
+
. Kt qu ny cng ph hp vi mt s
nghin cu khc, mc nng NaCl ph bin (n 0,2 M), hot tnh ca PME
tng cao t nht 2 ln khi so snh vi trng hp khng c s tham gia ca NaCl
(Christgau,1996; Duvetter, 2007; Arotupin, 2008).
Trong trng hp s dng CaCl
2
,

nng ion Ca
2+
b sung c thay i 6 gi
tr t 0 n 0,015 M. Kt qu th nghim c trnh by bng 3.23.
Bng 3.23: nh hng ca nng CaCl
2
n hot tnh PME t A. niger
Nng CaCl
2
(M) Mc thay i hot tnh (A/A
0
)
0 1,00
a
0
0,0050 2,23
c
0,58
0,0075 2,33
c
0,29
0,0100 2,60
d
0,29

0,0125 2,67
d
0,29

0,0150 2,00
b
0,13
T s F 12
Gi tr P 0,02
thng k theo php th Dunn tin y 95%.
Kt qu bng 3.23 cho thy, CaCl
2
cng c vai tr tch cc trong vic hot ha
PME, tuy nhin hiu qu km hn trng hp s dng NaCl. Khi c s hin din
ca CaCl
2
, hot tnh PME t A. niger tng t 2 n 2,67 ln khi so snh vi trng
hp khng c s hin din ca ion ny. Hot tnh PME t gi tr cao nht trong
khong nng CaCl
2
t 0,005 0,0125 M. Duvetter (2007), Christgau et al.
(1996) cng a ra nhng kt qu v nhn xt tng t. Kt qu kho st hiu qu
hot ha ca CaCl
2
v NaCl i vi PME t A. aculeatus (Duvetter, 2007) cho
thy, hot tnh PME tng t 2,2 U (trong trng hp khng c s hin din ca
ion Na
+
v Ca
2+
) n gi tr 5,5 U khi b sung NaCl n gi tr 0,1 0,175 M v
hot tnh PME l 5U khi c s hin din ca CaCl
2
khong 0,005 0,015M.
S hot ng ca cc enzyme ni chung thng c kch hot bi cation kim
loi, nh vo lin kt vi cc nhm tch in m ca cc dng enzyme khng hot
ng v do lm thay i cu hnh tr thnh dng hot ng hn. Tuy nhin,
trong trng hp ca PME, s hin din ca ion kim loi, c bit l Na
+
v Ca
2+

c tc dng ch yu trn mch pectin, gip hnh thnh phc hp cation - carboxyl,
ngn cn vic c ch s hot ng ca PME bi cc nhm carboxy t do sn c
trn phn t pectin cng nh hnh thnh thm trong sut phn ng. S hnh thnh
phc hp ny t c hiu qu cao vi pH ti u t 4,0 4,5 v gim dn pH
ln n 8,0 (Gizisevangel, 1964). Ngoi ra, nghin cu ca Corredig & Wicker
(2000) cn cho thy, vic b sung Ca
2+
lm thay i nng lng t do ca phc
hp enzyme/c cht, thc y s hot ng ca PME. Chnh v vy, vic b sung
ion Na
+
v Ca
2+
nng thch hp thc y mnh s hot ng ca PME t
nm mc ang c kho st.
Quy trnh tng qut tng hp ch phm PME k thut t A. niger So
2
v R
1
trn
mi trng b to ta v v bi ti c tng hp hnh
3.41.

Hnh 3.41: Quy trn tng p c pm PME k tut t A. niger

Kt ta cha ch phm PME
Kt ta bng ethanol (3:1) 4C, 2 gi

Dng nm mc

trong 96 gi, 35,5C
Ly trch bng dung dch m citrate (pH = 3,6)
t l dung dch m: c cht 2:1 v/w
Nhit ly trch: 35C, thi gian 50 pht

16,5% v/w huyn ph nm mc A.
niger So
2
v R
1
(t l 1:1)
(10
5
cfu/mL)
Ch phm PME th

iu chnh m n 57,4% Dung dch m citrate (pH = 4,0)
cha 0,1% urea, 0,5% MgCl
2,
0,15%
CaCl
2

Thanh trng 121C trong 15 pht

Lm ngui

B to ( m khong 6%) + v bi ti (tch m cn 50%)
Vi lc 0,2 m

Ly tm 4C, 10000 vng/pht
(khong 11173 g) trong 15 pht
Ha tan bng m citrate pH 4,0
Lc mng 50 kDa
Ch phm PME k thut

3.8 Kh nng ng dng ca ch phm PME k thut thu nhn t A. niger
So
2
v R
1
n kh nng ci thin cu trc rau qu
3.8.1 i vi sn phm khm lnh ng
i. nh hng th t b sung PME v CCl
2
n s thy i tnh u
tr khm
Mt s phng thc kt hp b sung ch phm PME t A. niger v mui CaCl
2

vo nguyn liu khm c a ra bao gm (i) Tin x l khm 40C, 20 pht,
sau ngm trong dung dch CaCl
2
0,5% vi thi gian 30 pht (0A); (ii) Tin x
l khm trong dung dch CaCl
2
0,15% 40C trong 20 pht (0B); (iii) Tin x l
khm vi PME 1 U/mL ha tan trong nc 40C, 20 pht, sau ngm trong
dung dch CaCl
2
0,5% vi thi gian 30 pht (1A); (iv) Tin x l khm vi PME
1 U/mL ha tan trong dung dch m citrate phosphate pH 4,5 40C trong 20
pht, sau ngm trong dung dch CaCl
2
0,5% vi thi gian 30 pht (1B); (v)
Tin x l khm vi dung dch PME nng 1 U/mL v CaCl
2
0,15% ha tan
trong nc 40C trong 20 pht (2A); (vi) Tin x l khm vi PME 1 U/mL v
CaCl
2
0,15% ha tan trong dung dch m citrate phosphate pH 4,5 40C trong
20 pht (2B) v mu i chng (khng x l).
S bin i gi tr DE ca khm sau qu trnh x l vi enzyme v sau lnh ng
kt hp tr ng 4 tun nhit -18C cng nh cng tng i (H/H
o
) ca
mu khm sau qu trnh x l so snh vi khm ti cng iu kin kho st
cng c so snh v nh gi chn ra phng thc b sung hay th t b sung
PME v CaCl
2
thch hp nht, lm c s cho cc th nghim sau. Kt qu c
tng hp bng 3.24 v hnh 3.42 (s liu thng k s bin i gi tr DE ca cc
mu kho st c tng hp ph lc 3).

Hnh 3.42: S tay i ester a ca pectin trong km do tc ng ca tin x
l vi PME nm mc v CaCl
2
teo cc png tc kc nau
Da trn th hnh 3.42, c th nhn thy hiu qu hot ng ca PME do tc
ng ca qu trnh tin x l khi so snh vi mu i chng (mu 0). S thay i
DE ca khm do tc ng ca tin x l c phn r thnh 3 nhm v c
khc bit ngha v mt thng k (xem ph lc):
(i) Kch hot PME ni bo bng tin trnh tin x l nhit nhit thch hp.
i vi cc mu kho st khng b sung PME nm mc (mu 0A v 0B), ester
ha ca pectin cng gim thp, khc bit c ngha v mt thng k khi so snh
vi mu i chng. DE ca pectin trong mu khm i chng (mu 0) l
50,21% gim xung cn 45,89% v 45,33% (mu 0A v 0B). iu ny cho thy,
nhit tin x l 40C trong thi gian 20 pht kch hot PME ni bo trong
khm. Nghin cu ca Trn Thanh Trc et al. (2006, 2008) cng khng nh
mc nhit tin x l t 40 60C trong khong thi gian t 10 30 pht gip
kch hot PME ni bo, gp phn ci thin c tnh cu trc ca khm.
(ii) Trong trng hp tin x l vi PME nm mc v CaCl
2
c chun b trong
mi trng nc c pH 6,8 0,1 (mu 1A v 2A): DE gim thp hn khi so
snh vi mu i chng cng nh mu tin x l khng b sung PME ngoi bo.
Gi tr DE tng ng vi 2 mu ny ln lt l 43,54% v 43,59%.
(iii) Tin x l vi PME nm mc v CaCl
2
c chun b trong mi trng m
citrate phosphate pH 4,5 (mu 1B v 2B): DE ca pectin trong khm gim
thp nht v khc bit c ngha so vi cc mu x l cn li (DE ca mu 1B l
39,07% v 2B l 39,35%).
Nghin cu ca Van Buggenhout et al. (2006) cho thy, vic tin x l gip
kch hot PME trong du ty v lm gim ester ha pectin xung t 5 n 15%.
Trong khi , nghin cu ca Duvetter (2007) cho thy, vic tin x l du ty
trong dung dch m c cha PME t A. aculeatus gip lm gim DE ca
nguyn liu t 67 4% cn 25 4%.
Tuy nhin, hu nh khng c s thay i ester ha ca pectin trong tin trnh
tr ng khm; ng biu din s thay i DE ca cc mu khm tng ng
2 thi im sau khi tin x l v sau khi tr ng gn nh nm ngang. in hnh
nh gi tr DE ca mu 2B l 39,35% sau khi tin x l v 39,22% sau tr ng.
Ni cch khc, nhit tr ng c th km hm hot ng ca PME (Van
Buggenhout et al., 2004).
Xt v s thay i gi tr cng tng i (H/H
o
) ca khm, kt qu bng 3.24
cho thy, c s bin i tng ng gia H/H
o
v DE ca pectin trong khm
sau qu trnh tin x l. Tuy nhin, s bin i khng th hin r rt nh trng
hp o c v thng k DE.
Vic tin x l khm vi dung dch PME c gi n nh trong m citrate
phosphate pH 4,5 c tc ng nhanh chng n cng ca nguyn liu. cng
tng i ca khm (so snh vi khm ti) H/H
o
ca mu 1B (Tin x l khm
vi PME 1 U/mL ha tan trong m citrate phosphate pH 4,5 40C, 20 pht +
ngm trong CaCl
2
0,5% vi thi gian 30 pht) v mu 2B (Tin x l khm vi
PME 1 U/mL v CaCl
2
0,15% ha tan trong dung dch m citrate phosphate pH
4,5 40C trong 20 pht) c gi tr ln hn 1 (1,012 v 1,317). Tuy nhin, vic s
dng nc ct (pH 6,8 0,1) chun b dung dch tin x l khm khng mang
li hiu qu ci thin cng ca nguyn liu.
Bng 3.24: cng tng i trung bnh ca khm do tc ng ca tin x l vi
PME nm mc v CaCl
2
theo cc phng thc khc nhau
K hiu
mu
iu kin tin x l cng tng i (H/H
o
)
Sau tin x l Sau 4 tun tr ng
0 i chng (khng tin x l) 1,000
c
0,091 0,270
a
0,045
0A Tin x l khm trong nc (40C,
20 pht) + ngm trong dung dch
CaCl
2
0,5% (30 pht)
0,702
a
0,080 0,324
ab
0,022
0B Tin x l khm trong dung dch
CaCl
2
0,15% 40C trong 20 pht
0,748
ab
0,053 0,330
ab
0,034
1A Tin x l khm vi PME 1 U/mL
ha tan trong nc 40C, 20 pht +
ngm trong CaCl
2
0,5% (30 pht)
0,817
ab
0,087 0,362
b
0,038
1B Tin x l khm vi PME 1 U/mL
ha tan trong m citrate phosphate
pH 4,5 40C, 20 pht + ngm trong
CaCl
2
0,5% (30 pht)
1,012
c
0,101 0,395
bc
0,047
2A Tin x l khm vi dung dch PME
1 U/mL v CaCl
2
0,15% ha tan
trong nc 40C trong 20 pht
0,890
bc
0,103 0,383
bc
0,029
2B Tin x l khm vi PME 1 U/mL v
CaCl
2
0,15% ha tan trong dung
dch m citrate phosphate pH 4,5
40C, 20 pht
1,317
d
0,087 0,450
c
0,043
T s F 20,19 7,09
Gi tr P 0,00 0,013

iu ny c l l do dung dch m citrate phosphate pH 4,5 gip n nh hot
ng ca enzyme (Bemiller et al., 1972), im pH ny cng gn gi tr pH ti u
cho hot ng ca PME t A. niger c kho st mc 4.7.3 (phn ii) - nh
thc y s tip xc ca enzyme vi c cht pectin trong vch t bo thy
phn ester to ra cc gc acid t do, ngay khi s c mt ca ion Ca
2+
sn trong
dung dch c v thun li cho phn ng to phc pectate calcium din ra nhanh
chng. Cc vch t bo lc ny to c nhanh v nhiu cu ni calcium pectate
nn m qu tr nn cng chc hn (Van Buren, 1979). Ngoi ra, s tc ng nhit
cng c kh nng kch hot cc enzyme lyase v PG hin din ng thi trong ch
phm PME hay c sn trong khm, lm cho mch pectin khng n nh, cn tr
phn no s hnh thnh pectate calcium. Hn th na, khi PME xc tc thy phn
pectin lm gim mc ester ha m khng c ion calcium, cc gc carboxyl d
b tn cng hn bi polygalacturonase, khi pectin c th b phn ct mt phn
nn s to thnh pectate calcium din ra lc sau cng kh khn hn (Van Buren,
1979; Waldronet al., 1997). iu ny cng l gii cho gi tr cng tng i
ca mu tin x l n l trong dung dch enzyme, sau ngm CaCl
2
0,5% trong
thi gian 30 pht thng thp hn mu x l trong dung dch cha ng thi PME
v CaCl
2
.
Xt v cng ca khm sau khi lnh ng v tr ng 4 tun tng ng vi cc
phng thc tin x l khc nhau, kt qu o c v thng k cho thy, nh vo
tc ng ca qu trnh tin x l, kch hot PME ni vo ngoi bo, s hnh thnh
pectate calcium gip gia tng cng chc ca mng t bo m nguyn liu,
cng ca khm tt c cc trng hp c tin x l u c ci thin. Mc
duy tr cu trc tt nht trong trng hp khm c tin x l trong dung dch
m citrate phosphate pH 4,5 cha ng thi PME nm mc v CaCl
2
( cng
tng i cn li 45% khi so snh vi mc duy tr cu trc so vi ban u l
27%).
Nh vy, phng thc tin x l khm vi dung dch m citrate phosphate 4,5
cha ng thi ch phm PME k thut nng 1 U/mL v CaCl
2
0,15%
40C trong thi gian 20 pht cho kt qu kch hot PME hiu qu nht (th hin
DE gim thp) v tng cng ca khm cao nht. Phng thc ny c th
c s dng lm bc tin x l cho cc th nghim k tip.
ii. nh hng hot tnh PME b sung thh hp n s thy i u tr
khm lnh ng
Cc nghin cu b sung PME thc vt trong ci thin cu trc khm (Nguyen et
al., 2009) hay b sung PME t A. aculeatus trong ci thin cng du ty lnh
ng (Duvetter, 2007) cho thy, lng PME c nh hng trc tip n mc
thy phn pectin hay s lng gc acid t do hnh thnh, do cng c nh
hng n kh nng ci thin cu trc t bo rau qu. Cc mc hot tnh PME 0,5;
1; 1,5 v 2 U/mL c ln lt p dng trong qu trnh tin x l khm 40C
trong 20 pht cng vi s c mt ca 0,15% CaCl
2
. Trong kho st ny, cng
ca khm sau tin x l v sau tr ng 4 gi cng c xc nh. Tuy nhin,
ester ha pectin ca khm ch c o c ngay sau qu trnh tin x l. Kt qu
xc nh s thay i cng ca khm sau tin x l v tr ng c tng hp
bng 3.25.
Bng 3.25: nh hng ca hot tnh PME nm mc b sung n s tha i
cng tng i v DE ca khm ng lnh
Hot tnh PME
trong dch chn
DE
(sau tin x l)
cng tng i
H/H
o
(sau tin x l)
cng tng i H
e
/H
o

(sau 4 tun tr ng)
i chng 50,21
c
1,11 1,000
c
0,095 0,270
a
0,091
0,5 U/mL 43,60
b
1,11 0,765
a
0,127 0,272
a
0,051
1 U/mL 39,35
a
0,72 1,226
d
0,133 0,450
b
0,034
1,5 U/mL 39,36
a
1,05 0,865
ab
0,093 0,387
b
0,034
2 U/mL 39,04
a
1,00 0,784
a
0,087 0,295
a
0,037
T s F 67,96 10,89 10,73
Gi tr P 0,00 0,012 0,012
T kt qu tng hp bng 3.25 cho thy, khng c s gim DE khi tng nng
PME trong dch chn vt qu 1 U/mL. Ni cch khc, khng c phn ng
bin i pectin c methoxyl ha cao thnh cc pectin c methoxyl ha thp
hay acid pectinic v methanol khi b sung PME mc hot tnh cao hn 1,5
U/mL dch chn.
Vi hot tnh PME b sung thp 0,5 U/mL, ester ha pectin gim khng nhiu
(DE = 43,60 1,11%) cng vi gi tr cng tng i cn li sau khi tin x l
cng nh sau tr ng thp. iu ny c th do mc hot tnh ny to ra cha
nhiu gc carboxyl t do, s lin kt phc hnh thnh t v km bn. Khi tng hot
tnh n 1 v 1,5 U/mL, tc gim cu trc khm nh hn ng k v t l cu
trc cn li cao hn nhiu, cng cn li sau 4 tun tr ng c duy tr mc
cao nht, khc bit so vi cc trng hp x l cn li (H
e
/H
o
= 0,450 0,034).
iu ny ph hp vi gi thit rng hot tnh enzyme cng cao, s thy phn cc
gc ester ca pectin xy ra cng nhanh v nhiu, mch pectin cng c nhiu kh
nng kt hp vi ion Ca
2+
(Verlinden v De Baerdemaeker, 1997). Mng li cc
lin kt phc cng dy c th s kt dnh gia cc t bo cng cht ch nn cu
trc ca khm c duy tr tt.
Tuy nhin, khi tng hot tnh enzyme hn na, trng hp kho st l 2 U/mL, t
l H/H
o
v c H
e
/H
o
u gim thp (0,784 0,087 v 0,295 0,037). C th ni do
t l pectin trong khm cng khng cao (0,04 0,1% theo Aehle, 2004) nn ch
cn s dng lng va PME c th cho s ci thin cu trc tt. Mt khc,
c th c s hin din ng thi ca cc enzyme pectinase khc PG, trong iu
kin c s hin din ca pectin c ct t gc methoxyl s hnh thnh nn
cc pectin ha tan, lm gim n nh ca mch pectin v lm mm cu trc m
t bo (hnh 2.6, chng 2). Hn th na, phng thc tin x l khm trong dung
dch enzyme cng l nguyn nhn nh hng n hiu qu tc ng ca PME ln
mch pectin ca t bo thc vt (Duvetter, 2007). iu ny cho thy trong tng
lai, vic p dng cc gii php h tr qu trnh thm ca PME vo t bo, in
hnh nh ngm p lc cn c quan tm.
Nh vy, vic tin x l vi hot tnh PME b sung thch hp gip ci thin c
tnh cu trc ca khm lnh ng. Trong kho st ny, nng PME b sung
trong dch chn 1 U/mL gip hiu qu ci thin cu trc tt v c tnh kinh t cao
nht.
3.8.2 T ng PME n s i thin u tr d leo mui hu
Da leo mui chua l mt sn phm c a thch v ph bin trn ton th gii.
Cng vi s pht trin v s dng rng ri ca da leo mui chua l rt nhiu
nghin cu ci tin k thut v cht lng sn phm. Theo , cc thng s k
thut nh nng mui, pH, nhit thch hp vi tng loi da leo, khu v
tng vng cng c kho st. Tuy nhin, vic duy tr cu trc ca da leo
trong mui chua l mt vn quan trng cho ngnh cng nghip ln men thng
mi. Chnh v th, nghin cu iu kin tin x l nhm kch hot PME ni bo v
kh nng b sung PME n s ci thin cng chc ca sn phm l vn cn
c thc hin.
i. i tr PME ni bo n s n nh u tr d leo mui hua
Vi tin trnh tin x l nhit kt hp vi b sung CaCl
2
trong nc chn nhm
hnh thnh nhanh pectate calcium v ngn cn hot ng ph hy ca
polygalacturonase - ngun enzyme di do trong da leo, s dng pectin c
methoxyl thp (Tang v McFeeters, 1983) cn mt thi gian nht nh cho phn
ng xy ra trit . Chnh v th, nhit 50C c s dng nhm kho st thi
gian tin x l thch hp kch hot PME ni bo c trong da leo. Kt qu c
nh gi thng qua cng tng i ca da leo mui chua thnh phm, s thay
i ester ha ca pectin ngay sau tin x l v sau thi gian ln men, kt qu
c tng hp bng 3.26.
Bng 3.26: nh hng ca thi gian tin x l nhit ( 50C) n s tha i
ester ha pectin v c tnh cu trc da leo
Thi gian tin x l
(pht)
DE (%)
sau khi tin x l
DE (%)
ca sn phm
cng tng i
H/H
o
ca sn phm
0 45,07
bc
0,93 42,37
b
1,15 0,431
a
0,031
10 43,60
b
1,20 40,80
b
1,39 0,657
b
0,022
20 36,50
a
0,17 34,30
a
1,57 0,661
b
0,034
30 35,27
a
0,65 32,67
a
1,11 0,727
c
0,048
40 36,15
a
0,64 35,45
a
0,35 0,662
b
0,037
50 35,73
a
0,85 35,35
a
0,35 0,642
b
0,032
T s F 89,35 37,14 25,40
Gi tr P 0,00 0,00 0,00
Kt qu o c v thng k bng 3.26 cho thy, tin x l nhit 50C thc s
l iu kin thch hp nhm kch hot PME trong da leo, th hin r nht sau 20
pht gia nhit. ester ha ca pectin trong da leo gim thp, t gi tr 45,07
0,93% nguyn liu ban u (khng qua tin x l) cn 36,50 0,17% sau thi
gian tin x l 20 pht nhit 50C. Khi gia tng thi gian gia nhit di hn 30
pht (trng hp kho st l 40 v 50 pht), mc ester ha ca pectin khng c
s thay i. iu ny cho thy, thi gian tin x l t 20 pht gip kch hot
PME hiu qu. Ko di thi gian tin x l c l to tc ng ngc, thc y
hot ng ca cc enzyme pectinolytic khc, in hnh nh PG cng c cng iu
kin hot ng vi PME, dn n s thy phn cu trc pectin (Van Buren, 1979;
Waldron et al., 1997). iu ny c th hin s gim khc bit c ngha
v mt thng k cng tng i ca da leo mui chua khi nguyn liu c
tin x l 50C trong thi gian 40 v 50 pht. Thi gian gia nhit t 40 pht
lm hn ch hot ng ca PME cn th hin gi tr DE ca da leo sau ln men
hu nh khng thay i so vi mu sau khi tin x l. Vi thi gian tin x l 20
pht cho hot ng ca PME ni bo nhng khng cho qu trnh tng tc
hnh thnh phc hp pectate calcium, s thay i ester ha pectin tng t
nhau gia mu c x l nhit 20 pht v 30 pht nhng t l cng tng i
ca da leo thi gian x l 30 pht cao khc bit c ngha so vi cc mu cn
li cng nh mu x l 20 pht.
Tm li, vic tin x l da leo nhit 50C trong dung dch cha CaCl
2
0,4%
vi thi gian 30 pht l iu kin thch hp nhm kch hot PME ni bo c trong
nguyn liu, gip hn ch s mt cu trc sn phm sut qu trnh ln men v bo
qun tip theo. Mc d vy, mc ci thin cng ca da leo mui chua cha
tht s hiu qu, t l cng H/H
o
trong trng hp ny l 0,727 0,048. iu
ny cho thy, cn c mt gii php tch cc hn na, chng hn nh b sung PME
t nm mc nhm tc ng hu hiu vo mch pectin ca da leo v ngn cn
hot ng ca PG ni bo nhm to s cng chc n nh cho thnh phm da leo
mui chua.
ii. nh gi hiu qu i thin u tr d leo mui hu do t ng
vi b sung PME t A. niger
Tin hnh b sung kt hp PME t A. niger v CaCl
2
cc nng khc nhau
trong qu trnh tin x l da leo iu kin nhit 50C v thi gian 30 pht,
kt qu biu din s thay i cng da leo mui chua cc iu kin tin x l
kho st c th hin hnh 3.43.

Hnh 3.43: t biu din s tay i cng tng i (H/H
o
) ca da leo mui
cua teo cc t l PME v CaCl
2
b sung trong dc cn 50C, 30 pht
T th hnh 3.43 cho thy, c tnh cu trc ca da leo mui chua c ci
thin khi da leo c tin x l trong dung dch cha PME t A. niger kt hp
vi CaCl
2
cc nng khc nhau. Vic b sung PME nm mc ny t ra c hiu
qu tch cc trong vic ci thin cu trc da leo, ng biu din cng cn li
(H/H
o
) ca da leo mui chua trong trng hp c b sung PME u nm trn
mu i chng (ch kch hot PME ni bo 50C, 30 pht trong dung dch CaCl
2

cc nng 0,3; 0,4 v 0,5%). Khi tng hot tnh PME b sung n 1,5 U/mL,
tc gim cu trc da leo gim ng k. iu ny ph hp vi gi thit rng
hot tnh enzyme cng cao, s thy phn cc gc ester ca pectin xy ra cng
nhanh v nhiu, mch pectin cng c nhiu kh nng kt hp vi ion Ca
2+
. Mng
li cc lin kt phc cng dy c th s kt dnh gia cc t bo cng cht ch
nn cu trc ca da leo c duy tr tt. Tuy nhin, khi hm lng PME b sung
vt qu gi tr ti hn, cng ca da leo mui chua gim dn. iu ny c l
l do tc chuyn i thnh pectin c methoxyl ha nhanh hn tc hnh
thnh phc hp pectate calcium thc y cho cc hot ng ca enzyme thy
phn pectin c DE thp nh PG. Kt qu phn tch DE ca da leo mui
chua (bng s liu tng th khng c trnh by) cho thy, cng nng CaCl
2

trong dch ngm l 0,5%, gi tr DE tng ng vi hai mc b sung PME 1,5
U/mL v 2 U/mL hu nh khng khc bit (31,23 1,45% v 32,83 2,41%).
Khi xt nh hng ca vic tin x l trong dung dch c cha CaCl
2
cc nng
khc nhau cho thy, nng CaCl
2
qu cao (trng hp ny l 0,5%) th
lng Ca
2+
d khng nhng lm bt hot PME, gim cng ca da leo m
lng tha ny cn c tc ng khng mong mun n tnh cht cm quan sn
phm, to v ng, cht v mn (Sam et al., 1993).
Tm li, kt qu kho st cho thy mi tng quan trc tip gia qu trnh
demethylation pectin v s thay i cu trc sn phm. Mc d da leo l nguyn
liu c ester ha ca pectin ban u thp (thp hn 50%), tuy nhin nghin cu
tin x l da leo trong dung dch c b sung ch phm PME k thut t l 1,5
U/mL cng cho thy kh nng lm gim ester ha pectin n gi tr khong
30% - mc thch hp nht cho s hnh thnh mng pectate calcium khi s dng
PME ngun gc nm mc vi c ch ct mch ngu nhin (Duvetter, 2007), nh
gip cng ca da leo mui chua c gi gi tr cao. Tuy nhin, vic p
dng cc ch tin x l mc nhit trung bnh trong dung dch CaCl
2
cng
c hiu qu tch cc trong vic kch hot PME ni bo trong da leo, ci thin
ng k cu trc ca da leo mui chua. S gim cng ca da leo mui chua
l thp nht v tc hot ng ca PME ni bo cao nht khi x l nhit 50C
vi thi gian 30 pht trong dung dch CaCl
2
0,4%.
KT LUN V NGH
1. Kt lun
Nghin cu phn lp c 60 dng nm mc en c c tnh ging Aspergillus
niger t v cc loi qu giu pectin v c trng ph bin khu vc ng bng
sng Cu Long.
C 14 dng nm c tnh c hiu vi pectinase, 4 dng nm c phn lp t
chanh nm (k hiu N
1
), to ta (T
1
), bi Nm Roi (R
1
) v cam son (So
2
) c kh
nng tng hp PME tt nht.
Vic kt hp 2 dng nm mc Aspergillus niger So
2
v R
1
vi t l l 1:1 v s
dng c cht bao gm b to ta kh v v bi ti gip gia tng hiu qu sinh
tng hp PME.
Mi tng quan gia cc iu kin ln men sinh tng hp PME nh t l nm
mc s dng (X
1
,

v/w), pH mi trng (X
2
), nhit (X
3
) v m c cht (X
4
)
n hot tnh PME sinh ra (Y, U/g) sau 96 gi ln men c x l bng
phng php b mt p ng v c th hin qua phng trnh:
Y = -3331,94 + 66,60 X
1
+ 323,06 X
2
+ 73,38 X
3
+ 31,34 X
4
- 1,68 X
1
2
- 3,12X
1
X
2

+ 0,36X
1
X
3
- 0,20X
1
X
4
- 48,31X
2
2
+ 1,10X
2
X
3
+ 1,31X
2
X
4
- 1,03X
3
2
- 0,18X
3
X
4
-
0,23X
4
2
(R
2
= 0,981)
iu kin ln men sinh tng hp PME t A. niger c xc nh vi thi gian l
96 gi nhit 35,5C, pH 4,0, m mi trng ln men c iu chnh n
57,4% v t l huyn ph bo t nm (10
5
U/mL) s dng l 16,5% v/w (mL/g).
iu kin ly trch thch hp thu PME th t canh trng b mt l nhit
35C trong thi gian 50 pht, s dng dung dch m citrate pH 3,6 vi t l
dung dch m v c cht (canh trng sau ln men) l 2:1 v/w.
iu kin tinh sch s b, thu nhn ch phm PME k thut l kt ta protein
bng ethanol nhit di 4C vi t l ethanol v enzyme th l 3:1 (v/v), lc
mng khong phn on 50 kDa.
Cc tnh cht c bn ca ch phm PME : khi lng phn t khong 43 kDa,
hng s K
m
l 0,6014 0,0379 mg/mL, pH ti u khong 5,0 tng ng vi nhit
thch hp cho hot ng PME ny l 38 40C.
Ch phm PME k thut gip ci thin hiu qu c tnh cu trc khm ng lnh.
S mt cu trc khm l t nht khi p dng iu kin tin x l 40C, 20 pht
trong dung dch gm ch phm k thut PME t A. niger (nng 1 U/mL) v
0,5% CaCl
2
.
i vi da leo mui chua, c tnh cu trc ca sn phm c ci thin 1,7 ln
so vi mu i chng khi nguyn liu c tin x l nhit 50C vi thi
gian 30 pht trong dung dch gm 1,5 U/mL PME t A. niger v 0,4% CaCl
2
.
2. ngh
ng k ln ngn hng d liu gene NCBI (GenBank) 4 dng A. niger tuyn
chn v nh danh.
Nghin cu kh nng sinh tng hp PME t A. niger quy m ln hn v ng
dng PME trong iu kin ch bin thc t.

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Aspergillus niger, Oral Proceedings of Food Innovation Asia Conference
2010: Indigenous Food Research and Development to Global Market, June
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ti Nghin cu khoa hc (1 ti cp B, 4 ti cp C s)
(1) Trn Thanh Trc, Nguyn Vn Mi, Nguyn Vn Thnh, L Th Ngc
Hiu, V Th Kin Ho (2010), nh gi hiu qu sinh pectin methylesterase
t Aspergillus niger iu kin ln men khc nhau, ti NCKH p s,
rng i h Cn h, TNCS 2009-07.
(2) Trn Thanh Trc, Nguyn Vn Mi, Nguyn Vn Thnh, Nguyn Ngc
Hunh Trn, Vng T Trinh (2010), Xc nh mi trng ln men ti u
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dng c cht b to ta v v cam snh, ti NCKH p s, rng i
h Cn h, TNCS 2010-03.
(3) Trn Thanh Trc, Nguyn Vn Mi, Nguyn Vn Thnh, Dng
Thnh Ln, Nguyn Th Kiu Linh, L Vng Hi Nguyt, L Th Ngc
Hiu, Nguyn Tn t, V Th Kin Ho, Nguyn Ngc Hunh Trn, Vng
T Trinh (2012), Nghin cu kh nng sinh pectin methylesterase t mt s
dng Aspergillus niger c phn lp v tuyn chn, ti NCKH cp B,
B Gio D v o to, B2010-16-161.
(4) Trn Thanh Trc, Nguyn Vn Mi, Nguyn Vn Thnh, Nguyn Ngc
Hunh Trn, Vng T Trinh (2011), Cc yu t nh hng n tnh cht
pectin methylesterase t Aspergillus niger, ti NCKH p s, rng
i h Cn h, TNCS 2011-05.
(5) Trn Thanh Trc, Nguyn Vn Mi, Nguyn Vn Thnh, Nguyn Th T
Hng, T Th Trc Linh, Nguyn Th Hng Luyn (2011), Mt s gii php
ci thin hot tnh enzyme pectin methylesterase (EC 3.1.1.11), ti
NCKH p s, rng i h Cn h, T2011-07.

Chuyn ngnh i sinh vt h xxi in Nghin u v Pht trin Cng ngh inh h
PH LC 1 MI TRNG V PHNG PHP NHN DIN NM MC,
CC PHNG PHP PHN TCH
Phn 1: Cc mi trng s dng trong phn lp nm mc
1 Mi trng Czapek Dox Agar (CZ) s dng Aspergillus v Penicillium
Sucrose 30 g
K
2
HPO
4
1 g
KCl 0,5 g
Agar 15 g
Czapek Concentrate 10 mL
Nc 1000 mL
Pha ch: un nh la, khuy u n khi si ho tan cc cht. thch ngui
n 45 1C ri chnh pH n 4,5 5,5. Rt vo bnh cu c dung tch 250 mL mi
bnh 150 mL mi trng. Thanh trng trong ni hp nhit 121C/15 pht.
ngui n 45 1C ri vo cc a petri, mi a 20 25 mL mi trng. a
thch ng t nhin, bo qun trong t lnh nhit t 0 n 5C khng qu 15
ngy (Ngun: Atlas, 2010)
2. Czapek Concentrate (Ngun: Atlas, 2010)
NaNO
3
30 g
KCl 5 g
MgSO
4
.7H
2
O 5 g
FeSO
4
.7H
2
O 0,1 g
ZnSO
4
.7H
2
O 0,1 g
CuSO
4
.5H
2
O 0,05 g
H
2
O 100 mL
3 Mi trng Malt Extract Agar (MEA)
Malt extract 20 g
Peptone 1 g
Glucose 20 g
Agar 20 g
Nc 1000 mL
(Ngun: Klich, 2002)
Chuyn ngnh i sinh vt h xxii in Nghin u v Pht trin Cng ngh inh h
4 Mi trng Potato Dextrose Agar (PDA)
Nc chit khoai ty 200 g
Dextrose 20 g
Agar 20 g
Nc ct 1000 mL
un si 200 g lt ct khoai ty khng gt v trong 1 lt nc ct trong 30 pht. Lc
qua vi mng. Trn trong cc thnh phn khc v un si ha tan. Hp
121C/15 pht. Phn phi 20 25 mL vo cc a petri v trng. Gi tr pH cui
ca mi trng PDA l 5,6 0,2 (Ngun: Samson v van Reenen-Hoekstra, 1988).

Phn 2: Phng php phn lp v nh danh Aspergillus niger
1. Phng php phn lp A. niger
Chun b mi trng: Mi trng s dng thng l PDA, CZ, MEA c cho vo
bnh cu, bnh nn, ng nghim v hp tit trng (110C/30 pht hoc 121C/15
pht).
Chun b mu: Mu l cc ngun nguyn liu giu pectin nh v qu to ty, to
ta, sung. Ct nh v qu hoc xay nhuyn.
Phng php tin hnh
Bc 1 : Nui cy mu
- Ghi k hiu mu v cc thng tin cn thit ln a petri
- un nng chy thch, ngui n 45 1C. Trong iu kin v trng chnh pH
ca thch ph hp.
- Rt vo tng a petri khong 20 25 mL mi trng cc a thch ng t
nhin trn mt phng ngang.
- Rc mu cho vo gia mi a. Mi mu phi c nui cy t nht 3 a v
trng v dng pipet 1 mL v trng ring.
- Sau cc a thch vo t m 28 1C hoc nhit phng th nghim
tng ng trong 5 ngy. Khng lt ngc cc a.
- Lu : Thi gian t khi bt u pha long mu th n khi rt thch vo hp lng
khng c qu 30 pht.
Bc 2 : S b nhn nh kt qu
- Chn cc a c khng qu 50 khm nm s b c kt qu.
Chuyn ngnh i sinh vt h xxiii in Nghin u v Pht trin Cng ngh inh h
- m v ghi li s khm nm nghi ng l A. niger (c mu en, hoc nu thm
lm tm nh b cafe). Sau dng que cy nhn u ly mt t bo t cy ba im
cch u nhau trn a thch Czapek, m 28 1C trong 5 ngy. Khng lt ngc
cc a.
- xc nh tn nm mc, t nhng khm nm trn mi trng CZ tin hnh
nhn xt i th, vi th.
- i th: Bng mt thng, hay dng knh lp cm tay, nhn xt v kch thc,
mu sc ca khm nm.
- Vi th: nhn xt nm mc v vi th phi lm tiu bn nm mc, ri quan
st hnh thi hc di knh hin vi vt knh 10, 40.
- Lm tiu bn nm mc: Ly mt lam knh sch, trong, sy kh. Ly mt
on giy bng knh c kch thc rng khong 1 cm, di khong 2 3 cm, ri
chm vo khm nm mc trn a thch sao cho ly c ton b hnh thi ca nm
(t t bo chn n ht nh). Sau tri di giy bng knh trn lam knh.
Bc 3: Cy rng
- Dng que cy vng thao tc v trng thu ging;
- Ria cc ng trn a petri cha mi trng thch hp (ria ch T hoc ria 4
gc). Sau mi ng ria, t kh trng u que cy v lm ngui trc khi thc
hin ng ria tip theo;
- Gi a petri, nhit v thi gian thch hp trong t m.
2. Phng php nh danh Aspergillus niger
Sau khi cy truyn mu vi nm v thi gian thch hp c khun lc thun
khit, cn tin hnh ln lt cc bc sau:
- Xc nh cc c im hnh thi ca khun lc
- Xc nh cc c im ca bo t trn v b my mang bo t trn
- nh loi dng Aspergillus niger da theo kha phn loi ca Klich (2002)
Chuyn ngnh i sinh vt h xxiv in Nghin u v Pht trin Cng ngh inh h
2 c im cc loi thuc ging Aspergillus v Penicillium
Bng PL11: Phn bit mt s ging Aspergillus v Penicillium
Loi Si nm Khun lc Khi bo t Cung
bo t
Bng nh gi
A. niger

Mu trng
ng,c vch
ngn
Mu en, i
khi mu
nu. ng
knh 4 5
cm
Khi bo t
trn, nh
bng, hnh
cu, sau hnh
tia ta trn,
ng knh
700 800 m
Cung bo
t trn, nhn
khng c
vch ngn
ngang
Hnh cu hoc
gn cu, c 2
hng th bnh.
ng knh
4575 m
A. oryzae
Si nm c
vch ngn
ngang
Mu lc,
vng lc, lc
nu sau
chuyn thnh
nu lc, nu.
ng knh
5 6 cm
Khi bo t
trn, nh
bng, hnh
cu, ng
knh 150
300 m, i
khi 400
500 m
Cung bo
t di
thng
khng u
nhau to
thnh khun
lc c phn
tm cao hn
phn ra
Gn cu,
ng knh 40
50 m, mang
cung th bnh
hoc th bnh
A. flavus
Si nm c
vch ngn
ngang
Mu xanh
lc vng.
ng knh
3,5 4 cm
Khi bo t
trn, nh
bng. Hnh
tia sau hnh
ct, ng
knh 500 m
Trn, nhn,
thng di
500 m
Hnh gn cu,
ng knh
2,5 5 m.
Mang th bnh
Penicillium
Si nm
ngn vch,
phn nhnh
Mu lc,
vng lc,
xanh lc,
xm i khi
c mu vng,
, tm.
ng knh
4 6 cm
To thnh
chui di
trn ming
th bnh
Ngn, s
lng th
bnh phn
nhnh khng
ng nht.

Ngun: Bi Xun ng v Nguyn Huy n, 2000
Chuyn ngnh i sinh vt h xxv in Nghin u v Pht trin Cng ngh inh h
3. Hnh nh minh ha bo t A. niger
Hn PL11: Hn ti i t v vi t A. niger
Ct bn tri (t trn xung), khun l su 7 ngy trn mi trng CYA25, MEA v CY20S
Ct bn phi (t trn xung, tri sng phi): khi bo t trn, bng nh gi v bo t, th bnh tng 1 v
tng 2, bo t ( x 8000).
Ngun: Klih, 2002

Chuyn ngnh i sinh vt h xxvi in Nghin u v Pht trin Cng ngh inh h
4. Hnh nh minh ha bo t ca A. awamori v A. foetidus

Hn PL12: Bo t ca A. awamori (tri) v A. foetidus (pi)
T trn xung, theo tng dng nm: khi bo t trn, bng nh gi v bo t, th bnh tng 1 v tng 2, bo
t ( x 8000).
Ngun: Klich, 2002
Chuyn ngnh i sinh vt h xxvii in Nghin u v Pht trin Cng ngh inh h
5. S pht trin ca khun lc 3 loi A. niger , A. awamori v A. foetidus trn
a petri theo phng php chm 3 im

Hn PL13: Kun lc 7 ngy ca dng nm mc trn mi trng n loi c
trng: A. foetidus (h, CZ), A. niger (i, MEA) v A. awamori (j, CZ)
6. Cc tiu chun nhn bit Aspergillus niger
(i) im i th: da trn mu s v kh thc ca khun lc
ng knh ca khun lc sau 7 ngy nhit 25C trn cc mi trng khc
nhau: CYA25: 55 70 mm; CY20S 68 70 mm; MEA (30) 50 70 mm; CZ 40
62 mm. Ring mi trng CYA37, nhit thch hp l 30C, khi ng
knh khun lc dao ng trong khong 50 70 mm.
Mu sc v cu trc ca khun lc: Khun lc l vng en n nu en v dy tng
mng trn CYA25; si nm thng khng r, mu trng n vng nht; mng cng,
khi th mu kem sp, vng nu, hay hng nht; khng c dch r; mt tri khng mu
ti mu vng; sc t ha tan khng quan st c, khun lc dng ht nh n mc
thnh khm, thnh thong c tia rnh. Trn MEA, khun lc l vng en, khun ty
(si nm) mu trng v m; mt tri khng mu; khun lc dng ht nh n mc
thnh khm. Khun lc trn CY20S ging nh CYA25, nhng mu sc mt tri
thng mnh hn. Khun lc trn CYA37 v CZ ging nh CYA25.
(ii) im vi th: u bo t gai; kch thc cung bo t: (300) 400 3000 x
(7) 12 17 (20) m, thnh dy, nhn, mt, t trong sut n vng nht hoc nu
sng c bit gn nh cung; bng nh gi rng (20) 3075 (85), hnh cu, gn
cu; th bnh 2 tng (rt him khi 1 tng); th bnh tng 1 hu nh bao ph ton b
b mt ca bng nh gi, vi kch thc 12 20 (40) x 3 6 m, thnh thong c
vch ngn; th bnh tng 2 c kch thc 7 10 x 3 4 m. Bo t hnh cu, (3)
3,5 4,5 (5) m, thng rt sn si vi mp khng u v g gh ngu nhin.
(iii) im phn bit: Nhng loi ny c c im ring ph bin l khun lc
mu en hoc nu en, th bnh 2 tng u vi bng nh gi ln, trong th
Chuyn ngnh i sinh vt h xxviii in Nghin u v Pht trin Cng ngh inh h
bnh tng 1 di (gp hai th bnh tng 2) v bo t g gh mt cch bt quy tc
(Klich, 2002).

Phn 3: Mt s loi ha cht, dung dch m s dng trong tuyn chn v nui
cy, sinh tng hp PME
1. Mi trng v thuc th chun b tuyn chn A. niger c hiu vi enzyme
pectinase
- Mi trng thh petin
Pectin 5 g
Agar 20 g
Nc 1000 mL
pH 4,5 5,5
- Dung dh Lugol 2%
Iod tinh th 2 g
KI 4 g
Nc 294 mL
2. Mi trng v thuc th chun b tuyn chn A. niger c hiu vi PME theo
phng php o ng knh vng phn gii pectin
- Mi trng pectin - agarose: 1% agarose/l% apple pectin/40 mM Na acetate pH
4,8
3. Cc bng tra s dng cho chun b dung dch m, kt ta phn on
- Cc dung dch m s dng trong iu chnh mi trng ln men v ly trch
enzyme (Phm Th Trn Chu, 1988)
Nguyn tc chung: Chun b cc mui hay acid theo nng yu cu bng cch kt
hpt l th tch ca hai dung dch (mL) v nh mc n 100 mL theo s liu cc
bng PL1.2, PL1.3 v PL1.4.
Chuyn ngnh i sinh vt h xxix in Nghin u v Pht trin Cng ngh inh h
Bng PL12: Cch pha dung dch m citrate
pH Citric acid
0,1 M (mL)
Sodium
citrate 0,1 M
(mL)
pH Citric acid
0,1 M (mL)
Sodium citrate
0,1 M (mL)
3,0 18,6 1,4 5,0 8,2 11,8
3,2 17,2 2,8 5,2 7,3 12,7
3,4 16,0 4,0 5,4 6,4 13,6
3,6 14,9 5,1 5,6 5,5 14,5
3,8 14,0 6,0 5,8 4,7 15,3
4,0 13,1 6,9 6,0 3,8 16,2
4,2 12,3 7,7 6,2 2,8 17,2
4,4 11,4 8,6 6,4 2,0 18,0
4,6 10,3 9,7 6,6 1,4 18,6
4,8 9,2 10,8

Bng PL13: Cch pha dung dch m acetate
pH
Acid acetic
0,2 M (mL)
Sodium acetate
0,2 M (mL)
pH
Acid acetic
0,2 M
(mL)
Sodium
acetate 0,2 M
(mL)
3,6 46,3 3,7 4,8 20,0 30,0
3,8 44,0 6,0 5,0 14,8 35,2
4,0 41,0 9,0 5,2 10,5 39,5
4,2 36,8 13,2 5,4 8,8 41,2
4,4 30,5 19,5 5,6 4,8 45,2
4,6 25,5 24,5

Trong :
- Dung dch acid acetic 0,2 M (a): 11,55 mL CH
3
COOH bng (m c) v nh
mc n 1000 mL.
- Dung dch natri acetate 0,2 M (b): 16,4 g CH
3
COONa hoc 27,2 g
CH
3
COONa.3H
2
O c ha tan v nh mc n 1000 mL.
Chuyn ngnh i sinh vt h xxx in Nghin u v Pht trin Cng ngh inh h
Bng PL14: Cch pha dung dch m citrate-phosphate
pH Na
2
HPO
4
0,2 M (mL) Acid citric 0,1 M (mL)
2,2 0,40 19,60
2,4 1,24 18,76
2,6 2,18 17,82
2,8 3,17 16,83
3,0 4,11 15,89
3,2 4,94 15,06
3,4 5,70 14,30
3,6 6,44 13,56
3,8 7,10 12,90
4,0 7,71 12,29
4,2 8,28 11,72
4,4 8,82 11,18
4,6 9,35 10,65
4,8 9,86 10,14
5,0 10,30 9,70
5,2 10,72 9,28
5,4 11,15 8,85
5,6 11,60 8,40
5,8 12,09 7,91
6,0 12,63 7,37
6,2 13,22 6,78
6,4 13,85 6,15
6,6 14,55 5,45
6,8 15,45 4,55
7,0 16,47 3,53
7,2 17,39 2,61
7,4 18,17 1,83
7,6 18,73 1,27
7,8 19,15 0,85
8,0 19,45 0,55

Chuyn ngnh i sinh vt h xxxi in Nghin u v Pht trin Cng ngh inh h
- Bng tra nng mui (NH
4
)
2
SO
4
bo ha
Bng PL1.5: Bng tra nng mui (NH
4
)
2
SO
4
bo ha b sung trong tin trnh kt
ta enzyme v protein
(Ngun: Scopes, 1994)

Phn 4: Cc phng php sinh hc phn t
1. Phng php in di SDS PAGE xc nh khi lng phn t v im
ng in
Mu SDSPAGE c thc hin theo phng php Laemmli (1970, trch dn bi
ng Th Thu v Nguyn Th Xun Sm, 2009), trn h thng in di mini protein
II (BioRad), vi nng gel tp trung l 4% v gel phn tch l 10%. in di c
tin hnh vi cng dng in 20 mA v in th 40V, trong 2 gi. Gel c
nhum trong dung dch Coomassie Blue G 250 v gii nhum trong dung dch
methanol: acid acetic: nc (25:10:75).
2. Kim tra, nhn din nm mc bng phng php sinh hc phn t
* Ly trh DNA nm m theo phng php CAB (He et al., 2007).
1. Bt b iu nhit, chnh nhit 65C
2. Thm 500 L dung dch m CTAB (CTAB buffer) vo eppendorf 1,5 mL
Chuyn ngnh i sinh vt h xxxii in Nghin u v Pht trin Cng ngh inh h
3. Cho cc vin bi thy tinh (425 600 L, Sigma G9772) vo eppendorf
4. T mi trng chn lc, thu hoch bo t nm bng cch ht 50 L Triton-
X100 ln xung nhiu ln ti 1 im trn a
5. Cho hn hp bo t/ Triton-X100 vo eppendorf 1,5 mL
6. Vortex bng my Disruptor Genie trong 2 pht
7. eppendorf 65C trong 15 pht
8. Thm 233 L isopropanol v 32 L dung dch NH
4
OAc 7,5 M vo 1
eppendorf mi
9. Vortex eppendorf cha bo t bng my Disruptor Genie trong 1 pht
10. eppendorf 65C trong 15 pht
11. Thm 500 mL dung dch Chloroform : Isoaminalcohol (24 : 1)
12. Lc trn u
13. Ly tm tc ti a, 4C trong 10 pht
14. Chuyn 400 L dch ni vo eppendorf mi cha isopropanol v NH
4
OAc
15. Lc trn u
16. Ly tm tc ti a
17. B dch ni
18. Thm 500 L dung dch Ethanol 70% lnh
19. Ly tm tc ti a, 4C trong 10 pht
20. B dch ni (cn thn khng lm trc ta DNA)
21. Lm kh nhit phng
22. Thm 20 L nc ct ha tan mu
23. Chy in di kim tra v xc nh nng DNA
24. Tr mu -20C
* h hin phn ng PCR, khuh i on DNA m tiu
Tin trnh khuch i PCR: DNA sau khi ly trch c s dng nh trnh t
khun khuch i vng ITS trong b gene ca nm thng qua cp mi U
1f
v U
2r
.
Thit lp 30 chu k nhit khuch i trnh t mi 94C, thi gian 45 giy.
Phn ng PCR gm nhiu chu k, mi chu k c 3 giai on:
- Giai on tho xon: nhit 94C, giai on ny ko di 3 pht.
- Giai on gn mi: khi h nhit xung 50C, giai on ny ko di 50 giy.
Chuyn ngnh i sinh vt h xxxiii in Nghin u v Pht trin Cng ngh inh h
- Giai on ko di chui: nhit 72C, giai on ny ko di 90 giy. Mu chy
PCR c gi 72C trong thi gian 5 pht (Nganou et al., 2012)
* in di sn phm PCR trn garose gel
Nhm so snh kch thc DNA ca sn phm PCR vi thang DNA chun v kim
tra tinh sch ca DNA.
Qui trnh thc hin:
+ Chun b agarose gel:
- Cn 0,48 g agarose cho vo bnh tam gic ng 45 mL dung dch TAE 2%.
- un nng hn hp trong 3 pht cho agarose tan hon ton.
- ngui t nhin khong 10 pht.
- B sung 800 L ethidium bromide v lc nh cho u.
- nh dung dch agarose vo khun nh hnh gel (khi gel phi dt
khot trnh bt kh lm nh hng n s in di sn phm PCR).
+ Chy in di:
- t agarose gel c nh hnh vo b in di c cha dung dch m TAE 2%.
- Nh 5 L mu sn phm PCR ca DNA c trn u vi 2 L loading buffer
vo ging trn agarose gel.
- Tin hnh chy in di trn gel hiu in th 95 volt trong 45 pht.
- Quan st cc bng (band) DNA trn gel v so snh kch thc DNA ca sn phm
PCR vi thang DNA chun.
* Nhn din nm m bng phng php gii trnh t on DNA
- Cho vo tup sn phm PCR 2,5 L EDTA + 30 L cn 96% ta DNA.
- nhit phng 15 pht.
- Ly tm vi tc 10.000 vng/pht trong 45 pht, loi b phn dung dch.
- Ra vi 30 L cn 70%, ly tm vi tc 10.000 vng/pht trong 20 pht, b
phn dung dch (lp li 2 ln) v sy chn khng 10 pht 30C.
- Cho vo tup 20 L Hidye formamide, sau t tup vo my PCR nhit
95C trong 5 pht.
- Cho vo my gii trnh t ABI 3130 v c kt qu.
Chuyn ngnh i sinh vt h xxxiv in Nghin u v Pht trin Cng ngh inh h
- Dng chng trnh BLAST so snh trnh t on DNA ca dng nm mc vi
trnh t DNA ca cc loi nm mc c trong ngn hng d liu NCBI.
Cc ha cht chun b cho phn ng PCR
1. Dung dch CTAB stock (1 L)
- 100 mL 1 M Tris HCl pH 8
- 280 mL NaCl 5 M
- 400 mL EDTA 0,5 M
- 20 g CTAB (cetyltrimethyl ammonium bromide)
2. CTAB buffer: Thm vo dung dch CTAB stock cc thnh phn trong bng di
trc khi s dng (pha xong dung trong mt tun).
Bng PL16: Chun b dung dch m CTAB (CTAB buffer)
CTAB stock SDS 10% b-mercaptoethanol
5 mL 250 L (un nng) 25 L
20 mL 1000 L 100 L

Phn 5: Cc phng php p dng trong xc nh tnh cht enzyme v pectin
1. Phng php xc nh hot tnh ca PME (theo Crelier et al., 1995; trch dn
bi Duvetter, 2007 v Fachin et al., 2002)
(i) Nguyn tc
Phng php da trn c s thy phn dung dch pectin bi PME. Hot tnh PME
c xc nh bng cch o lng lng acid galacturonic gii phng ra trn n v
thi gian nhit v pH khng i.
(ii) Ha cht
- Dung dch pectin 3,5 g/L (Pectin to DE 75%, Sigma) trong dung dch NaCl
0,12M.
Cn khi lng pectin v NaCl cn thit. Trn u NaCl v pectin ( trnh pectin
vn cc), rc t t hn hp vo cc nc ct c un nng sn ng thi khuy
u bng my khuy t. yn 2 gi nhit phng pectin trng n v ha
tan, sau khong 12 gi nhit 4 6C. Sau dung dch c lm nng
n 20C v chuyn vo bnh nh mc c dung tch thch hp v nh mc n th
tch xc nh. Lc u v lc qua hai lp vi mn c bng gia. Bo qun dung
dch nhn c trong t lnh trong thi gian 2 ngy.
- Dung dch NaOH 0,01 N.
Chuyn ngnh i sinh vt h xxxv in Nghin u v Pht trin Cng ngh inh h
(iii) Tin hnh
i vi xc nh hot tnh PME nm mc: Chun b 2 cc thy tinh nh. Ly 30mL
dung dch pectin pha sn cho vo mi cc, iu chnh pH dung dch pectin n
4,5 bng dung dch NaOH 0,01 N. Sau b sung 250 L dung dch enzyme vo 1
cc, cc cn li b sung 250 L dung dch enzyme b v hot (100C, 30 pht)
lm mu i chng. Trong sut qu trnh enzyme thy phn pectin, pH dung dch
uc gi khng i (pH 4,5 nhit 30 1C) bng cch b sung NaOH 0,01 N.
i vi hot tnh PME thc vt: Tin hnh tng t nh xc nh PME nm mc.
Tuy nhin dung dch pectin 3,5 g/L c iu chnh pH n 7,0 v c gi n
nh trong qu trnh thy phn do tc ng ca PME cng bng NaOH 0,01 N
(Fachin et al., 2002)
(iv) Kt qu
Hot tnh PME l s mili ng lng ca este c thy phn trong 1 pht bi 1g
hay 1 mL enzyme.
PME (U/mL dch enzyme th) =
( ) 1000
b
V V M
v t

Vi: V: Th tch NaOH 0,01 N s dng iu chnh pH ca pectin v 4,5 (hay
7,0) trong qu trnh thy phn (mL)
V
b
: Th tch NaOH 0,01 N s dng chun mu trng (mL)
M: nng mol ca dung dch NaOH s dng (0,01 M)
v: Th tch ca enzyme s dng (mL)
t: Thi gian (thi gian thc hin phn ng) (pht)

PME (U/g c cht) =
[PME(U/mL dch enzyme) x tng th tch dch trch]
Khi lng c cht chnh trong mi trng ln men
2. Xc nh protein theo phng php Bradford (Nguyn c Lng v Cao
Cng, 2003).
Nguyn tc: Hm lng protein c xc nh vi thuc th Coomassie Brillant
Blue (CBB) G 250 theo Bradford (1976). CBB G 250 kt hp vi protein trong iu
kin acid to nn mt phc mu tm hp thu nh sng bc sng 595 nm. hp
thu t l thun vi hm lng protein trong dung dch.
Cch tin hnh: Thit lp ng chun protein, Bovin serum Albumin (BSA) vi
cc nng tng dn t 20 300 g/mL, da vo ng chun ny suy ra nng
protein trong mu.
Chuyn ngnh i sinh vt h xxxvi in Nghin u v Pht trin Cng ngh inh h
Chun b dung dch Bradford: ha tan 100 mg CBB G 250 trong 50 mL ethanol
95% , thm 100 mL acid phosphoric 85% khuy u, thm nc ct dn 1 lt. Sau
lc bng giy lc Whatman N
o
1 v tr trong chai ti nhit phng.
3. Phng php phn tch pectin (theo Marcon et al., 2005)
Nguyn t: Trong mi trng kim long, pectin ha tan s gii phng ra nhm
methoxyl thnh ru methylic v acid pectic t do. Acid pectic t do trong mi
trng c mt acid acetic s kt hp vi CaCl
2
thnh dng mui kt ta calcium
pectate. T hm lng mui kt ta c th tnh c hm lng pectin c trong
mu phn tch.
H ht: Dung dch NaOH 0,1 N; CH
3
COOH 0,1 N; CaCl
2
1 N; AgNO
3
1%
in hnh: Ly 20 mL dch qu (hay 5 g mu nghin vn) cho vo bnh tam gic
250 mL, cho thm 100 mL NaOH 0,1 N, hn hp trong 7 gi cho pectin x
phng ha hon ton thnh acid pectic. Sau , thm vo 50 mL dung dch acid
acetic 0,1 N v yn 5 pht, thm 50 mL NaCl 1 N 1 gi. Sau un si hn
hp trong 5 pht ri lc qua giy lc c sy kh n khi lng khng i,
ra kt ta calcium pectate bng nc ct nng cho n khi khng cn ion Cl
-
na
(th nc ra vi dung dch AgNO
3
1%). Sau khi ra xong, t giy lc c kt ta
vo cc, cn v sy 105
o
C n khi lng khng i.
Kt qu: Hm lng pectin trong dch qu c tnh theo cng thc sau:
Pectin= 1000
92 , 0
1
V
m
, g/L hay Pectin = 100
92 , 0
1
m
m

Trong : m
1
: khi lng cn calcium pectate thu c (g)
0,92: h s chuyn t calcium pectate sang pectin
V : Th tch dch qu ly mang i phn tch (mL)
m: khi lng mu cn phn tch (g)
4 Phng php xc nh mc ester ha (DE) (theo Food Chemical Codex,
FCC, 1981; trch dn bi Singthong et al., 2005)
Cch tin hnh: Cn 500 mg mu kh cho vo bnh tam gic 250 mL, thm 2 mL
ethanol v pha long bng 100 mL nc ct. Sau khi mu c ha tan hon ton,
thm 5 git phenolphtalein, mu c chun vi dung dch NaOH 0,5 N cho n
khi dung dch c mu hng nht v ghi nhn kt qu v ghi nhn kt qu V
b
. Sau
thm vo 10 mL NaOH 0,5 N v lc tht mnh ri yn khong 15 pht. 10
mL HCl c thm tip vo v lc cho n khi mu hng bin mt. Thm vo 5
git phenolphtalein v chun vi NaOH 0,5 N cho n khi dung dch c mu
hng nht bn trong 30 giy. Ghi li kt qu V
s
.
Chuyn ngnh i sinh vt h xxxvii in Nghin u v Pht trin Cng ngh inh h
Tnh ton kt qu: % DE =
s b
s
V V
V
+

Phn 6: Cc phng php ha hc c bn
1 Phng php xc nh m (AOAC 934.06)
Cch tin hnh: Ly cc s em sy 105C cho n khi khi lng khng i.
ngui trong bnh ht m v cn khi lng chnh xc n 0,001 g.Sau cho
vo cc khong 1 g mu. Cn tt c cn phn tch vi chnh xc nh trn.
Cho tt c vo t sy 105C, sy cho n khi khi lng khng i, thng ti
thiu l 6 gi. Trong thi gian sy, c sau 1 gi li dng a thu tinh u bt
nghin nh cc phn vn cc, sau dn u v tip tc sy.
Sy xong, lm ngui trong bnh ht m (20 25 pht) v em cn cn phn tch
vi chnh xc nh trn.
Cho li vo t sy 105C trong 30 pht, ly ra lm ngui trong bnh ht m (20 25
pht) v em cn nh trn ti khi khi lng khng i. Kt qu gia hai ln cn
lin tip khng c cch nhau qu 0,1 mg cho mi gam mu th.
Tnh kt qu: m theo phn trm tnh theo cng thc
0 1
0
100(%)
m m m
X
m
+
= -

Trong m: Khi lng cc (g); m
0
: Khi lng mu (g).
m
1
: Khi lng cc v ca mu sau khi sy (g).
Sai lch gia hai ln xc nh song song khng c ln hn 0,5%. Kt qu cui
cng l trung bnh ca 2 ln lp li.
2. Phng php xc nh hm lng ng kh (phng php Lane Eynon,
AOAC 942.15)
Nguyn tc: Da vo phn ng ca ng nghch o kh ng trong dung dch
Fehling thnh oxit ng 1 (Cu
2
O) c mu gch.
H ht x nh
1. NaOH 30%, 10%, 1 N
2. Pb(CH
3
COO)
2
30%
3. Na
2
SO
4
bo ha 30%
4. Xanh methylen 1% trong nc
5. Fehling A: CuSO
4
tinh th 69,28 g
Nc ct n 1000 mL
6. Fehling B. Potassium sodium tartrate 346 g
Chuyn ngnh i sinh vt h xxxviii in Nghin u v Pht trin Cng ngh inh h
NaOH 100 g
Nc ct n 1000 mL
7. Phenolphtalein 1% trong cn
Cch tin hnh
- Chun b mu
+ Cn 5 g mu cn phn tch
+ 50 mL nc ct
+ 5 mL HCl m c
- Trung ha vi NaOH vi nng gim dn t 30%, 10%, 1 N (dng
phenolphtalein lm cht ch th mu). Sau cho dung dch ny vo bnh nh mc
100 mL.
- Kh tp cht bng 7 mL Pb(CH
3
COO)
2
. yn 5 pht n khi thy xut hin mt
lp cht lng trong sut bn trn lp cn th coi nh kh tp cht xong.
- Loi b Pb(CH
3
COO)
2
bng 18 20 mL Na
2
SO
4
hoc Na
2
HPO
4
. Lc u v kt
ta lng xung. Nu c kt ta do Na
2
HPO
4
tc dng vi ch acetate tha th yn
10 pht, cn vi sodim sulfate, nu lp nc bn trn b c th phi lu n 24
gi. Kim tra li xem kt ta ch acetate tha cha bng cch cho ht sc cn
thn vi git disodium phosphate hay sodium sulfate. Nu khng thy vn c, cc
cht lng tip xc vi nhau th coi nh ht ch acetate. Thm nc ct vo dung
dch ny cho 100 mL.
- Lc, pha long khi s dng. Ty hm lng ng trong thc phm s tin hnh
pha long khc nhau. V d: hm lng ng 60% cn 1 gam pha long 2 3 ln.
- Cho vo cc hn hp gm 5 mL Fehling A v 5 mL Fehling B, thm vo 15 mL
dch lc. em t trn bp v chun . Mi ln chun nh 1 mL dung dch ng
n mu gch khng cn nh xanh. Th li bng cch nh mt git xanh
methylen vo dung dch ang si thy mt mu xanh tr v mu gch. c kt
qu v tra bng tnh ra hm lng ng.

Chuyn ngnh i sinh vt h xxxix in Nghin u v Pht trin Cng ngh inh h
Bng PL17: Bng tra lng ng nghch chun
mL dung
dch
ng
s
dng
Lng
ng
nghch
chuyn
mg/100
mL
mL dung
dch
ng
s
dng
Lng
ng
nghch
chuyn
mg/100
mL
mL dung
dch
ng
s
dng
Lng
ng
nghch
chuyn
mg/100
mL
mL dung
dch
ng
s
dng
Lng
ng
nghch
chuyn
mg/100
mL
15
16
17
18
19
20
21
22
23
336
316
298
282
267
254,5
242,9
231,8
222,2
24
25
26
27
28
29
30
31
32
213,3
204,8
197,4
190,4
183,7
177,6
171,7
166,3
161,2
33
34
35
36
37
38
39
40
41
156,06
152,2
147,09
143,9
140,2
136,6
133,3
130,1
127,1
42
43
44
45
46
47
48
49
50
124,2
121,4
118,7
116,1
113,7
111,4
109,2
107,1
105,1
Cng th tnh ton

3 Xc nh hm lng protein theo phng php Kjeldahl (984.13 A-D)
3.1. Cch tin hnh
(i) V c ha mu
Cn chnh xc 1g mu cn phn tch nghin nhuyn cho vo bnh Kjeldahl c th
tch 200 mL, sau thm 5 mL H
2
SO
4
m c. rt ngn thi gian v c ha,
thm vo bnh mt lng cht xc tc cn thit l 0,5 g. Hn hp cht xc tc ny
gm K
2
SO
4
: CuSO
4
: Se (100:10:1).
un si v lun gi cho bnh si nh khong 2 3 gi cho n khi dung dch trong
bnh Kjeldahl trong sut (thng ngi ta v c ha trong t rt hi). ngui,
kim tra kt thc s v c ha bng cch cho vo bnh mt lng nh nc ct
(thng bng bnh tia) ri lc nh trng thnh bnh, thy khng cn nhng ht mi
en li ti.

Hm lng ng =
S tra bng* HSPL*100
Khi lng mu *1000

% (mg/100mL)
Chuyn ngnh i sinh vt h xl in Nghin u v Pht trin Cng ngh inh h
(ii) Li cun m
- Cho 50 mL nc ct vo bnh Kjeldahl c cha sn mu va c v c ha,
thm vo 50 mL dung dch NaOH 10%.
- Ht chnh xc 20 mL dung dch acid boric 2% vo bnh tam gic 250 mL (bnh
hng), thm vo 6 git thuc th (hn hp ca methyl v bromocresol green).
- t bnh cha mu v bnh hng vo h thng chng ct mu, tin hnh chng ct
khong 15 pht, sau dng giy qu tm kim tra s kt thc qu trnh li cun
m.
(iii) Chun : Dng dung dch H
2
SO
4
0,1 N chun dung dch trong bnh
hng cho n khi dung dch chuyn t mu xanh sang hng nht. Tin hnh phn
tch mu i chng ging nh phn tch mu tht, cho vo bnh Kjeldahl ban u
1mL nc ct thay v 1 g mu.
3.2. nh ton kt qu
0,0014 *V
H2SO4
*100 * HSPL
Hm lng Nit tng s = (%)
m
Trong : m: khi lng nguyn liu v c ha (g)
0,0014: s g N tng ng vi 1 mL H
2
SO
4
0,1 N.
X nh hm lng protein
Da vo t l nit tng i n nh trong thnh phn cu to ca protein xc
nh h s protein. Thng thng, hm lng Nit ton phn trong protein c
xem nh 16%, khi h s protein H:
25 , 6
16
100
= = H
H s protein = 6,25 cn gi l h s trung bnh th.
Hm lng protein (%) = % N x H
Chuyn ngnh i sinh vt h xli in Nghin u v Pht trin Cng ngh inh h
PH LC 2 MT S KT QU KHO ST
Bng PL21: Hnh thi khun lc ca 60 dng Aspergillus niger phn lp trn mi
trng MEA v mi trng CZ
K
hiu
dng
Mi
trng
Hnh dng Hnh dng b mt
Mu sc
mt trn
Mu sc
mt di
ng
knh,
mm
Sa
1

CZ Gn trn
Chm ht tha, nh
cao
en Vng 65 72
MEA Gn trn
Ht mn, c rnh, nh
cao
Xm en Trng 35 42
Sa
2

CZ Gn trn Ht rt tha, nh cao en nht Vng 70 75
MEA Gn trn Ht mn, t nh cao en Trng 45 50
Sa
3

CZ Trn Ht tha, nh cao en nht Trng 50 77
MEA Trn Ht mn, c rnh, cao en nht Trng 50 65
So
1

CZ Trn Ht tha, nh cao en nht Vng nht 60 65
MEA Trn
Ht mn, c rnh, t
nh cao
en nht Trng 40 50
So
2

CZ Gn trn Ht tha, nh cao en nht Vng 40 75
MEA Gn trn
Ht mn, c rnh, nh
cao
Xm nht Trng 40 62
So
3

CZ Trn Ht tha, nh cao en nht Trng 40 65
MEA Elip
Ht mn, c rnh, nh
cao
Ht en Trng 40 50
So
4

CZ Gn trn Ht tha, nh cao en Vng 75 80
MEA Gn trn Ht mn, c rnh, cao en nht Trng 35 55
M
1

CZ Trn Ht dy, t nh cao en nht Vng 55 60
MEA Trn
Ht mn, c rnh, t
nh cao
en Trng 40 45
M
2

CZ Gn trn Ht tha, nh cao en Vng 55 65
MEA Gn trn Ht mn, c rnh, cao en Trng 35 45
M
3

CZ Trn Ht tha, nh cao en Trng 55 67
MEA Elip Ht mn, c rnh, cao en Trng 35 55
M
4

CZ Gn trn Ht mn, t nh cao en nht Vng 75 80
MEA Gn trn Ht mn, t nh cao Ht en Trng 30 60
M
5

CZ Elip Ht mn, t nh cao en Vng 50 65
MEA Elip Ht mn, nh cao en Trng 40 50
M
6

CZ Trn Mn, tha, t nh cao en nht Vng 55 69
MEA Trn Ht mn, rnh, t nh en Trng 36 50
Chuyn ngnh i sinh vt h xlii in Nghin u v Pht trin Cng ngh inh h
H
1

CZ Gn trn Ht lm chm, cao en Vng 60 62
MEA Gn trn Ht mn, nh cao Xm, en Trng 60 73
H
2

CZ Gn trn Ht lm chm, c si Chm en Vng 55 69
MEA Gn trn Ht mn, c rnh, cao en nht Trng 45 52
H
3

CZ Trn Ht mn, nh cao en Vng 70 80
MEA Trn Ht mn, t nh cao en nht Trng 40 60
H
4

CZ Gn trn Ht mn, nh cao en nht Vng 55 60
MEA Gn trn Ht mn, t nh cao Xm, en Trng 35 50
X
1

CZ Gn trn Ht mn, dy, nh cao en nht Trng 50 61
MEA Trn
Ht dy, nh cao, c
rnh
en xm Trng 40 50
X
2

CZ Gn trn Ht dy, nh cao en Trng 45 50
MEA Gn trn Ht mn, dy, nh cao en Trng 37 43
X
3

CZ Gn trn Ht mn, dy, cao
en nht,
gn nu
Vng nht 50 56
MEA Gn trn
Ht mn, tha, t nh
cao
en nht Vng nht 50 55
X
4

MEA Gn trn Ht mn, dy, nh cao en Trng 38 45
X
5

CZ Gn trn Ht mn, dy, nh cao en xm Trng 35 40
MEA Gn trn Ht mn, dy, nh cao en xm Trng 35 40
R
1

CZ Gn trn Ht mn, dy, nh cao en Trng 55 60
MEA Elip Ht mn, dy, nh cao en Trng 45 50
R
2

CZ Trn Ht mn, dy, nh cao en Trng 45 50
MEA Trn Ht mn, dy, nh cao en Trng 40 45
R
3

CZ Trn Ht mn, dy, nh cao en Trng 50 55
MEA Gn trn Ht mn, dy, nh cao en, nu Trng 35 45
G
1

CZ Gn trn Ht mn, dy, nh cao en sm Trng 51 58
MEA Gn trn Ht mn, dy, nh cao en sm Trng 48 52
G
2

CZ Gn trn Ht mn, dy, nh cao en Trng 55 60
MEA Trn Ht mn, dy, nh cao en xm Trng 41 50
G
3

MEA Gn trn
Ht mn, dy, nh
cao, c rnh
en nht Trng 40 45
G
4

CZ Trn Ht mn, dy, nh cao en Vng 45 50
MEA Trn Ht dy, c rnh, cao en than Vng nht 46 52
G
5
CZ Gn trn Ht mn, dy, nh cao en nht
Trng,
vng
35 40
Chuyn ngnh i sinh vt h xliii in Nghin u v Pht trin Cng ngh inh h
MEA Gn trn Ht mn, dy, nh cao en nht
Trng,
vng
35 37
N
1

CZ Gn trn Mn, tha, t nh cao en nht Vng 47 58
MEA Trn Mn, c rnh, tha en nht Vng 47 53
N
2

CZ Gn trn Ht mn, tha en nht
Trng,
vng
35 42
MEA Gn trn Ht mn, dy en sm Vng 38 45
N
3

CZ Trn Ht mn, dy en xm Trng 55 60
MEA Trn
Ht mn, dy,
nh cao, c rnh
en xm Trng 40 50
R
1

CZ Gn trn Ht tha, t nh cao en nht Trng 60 70
MEA Gn trn Ht mn, tha Xm Trng 42 50
R
2

CZ Gn trn Ht mn, tha en nht Trng 60 72
MEA Trn Ht dy, nh cao en Trng 40 45
R
3

CZ Gn trn Ht tha, t nh cao en nht Trng 62 74
MEA Gn trn Tha, t nh cao Nu thm Trng 32 45
RD
1

CZ Gn trn Tha, t nh cao Xm Trng 42 50
MEA Gn trn Tha, t nh cao en nht Trng 60 70
RD
2

CZ Gn trn Tha, t nh cao en nht Trng 45 55
RD
3

CZ Gn trn Tha, t nh cao Nu thm Trng 32 45
RD
4

CZ Gn trn Tha, t nh cao Xm Trng 34 50
MEA Trn Dy, nh cao en Trng 65 75
RD
5

CZ Elip Tha, t nh cao Xm Trng 32 40
MEA Gn trn Tha, t nh cao en Trng 62 75
RD
6

CZ Gn trn Dy, t nh cao Xm Trng 34 48
MEA Trn Dy, nh cao en Trng 65 75
RD
7

CZ Gn trn Tha, t nh cao Nu thm Trng 32 40
RD
8

CZ Trn Dy, t nh cao en Trng 35 55
MEA Trn Rt dy, nh cao en Trng 42 50
GA
1

CZ Gn trn Dy, nh cao en Trng 70 77
MEA Gn trn Dy, nh cao en Trng 35 52
GA
2

CZ Gn trn Dy, nh cao en Vng 60 75
MEA Gn trn Dy, nh cao en Vng 40 48
GA
3

CZ Trn Tha, nh cao en nht Vng 58 70
MEA Trn Tha, nh cao en nht Vng 32 45
Chuyn ngnh i sinh vt h xliv in Nghin u v Pht trin Cng ngh inh h
GA
4

MEA Gn trn Dy, nh cao en Trng 35 55
GA
5

CZ Elip Tha, nh cao en Trng 60 72
MEA Elip Tha, nh cao en Trng 38 52
GA
6

GA
7

CZ Trn Tha, nh cao en nht Trng 58 70
MEA Trn Tha, nh cao en nht Trng 32 45
GA
8

MEA Gn trn Dy, t nh cao en Trng 35 55
T
1

CZ Gn trn Tha, t nh cao en Trng 60 68
MEA Gn trn Tha, t nh cao en Trng 40 45
T
2

CZ Trn Tha, t nh cao en nht Trng 60 70
MEA Trn Tha, t nh cao en nht Trng 32 45
T
3

CZ Gn trn Tha, nh cao en Trng 58 65
MEA Gn trn Tha, nh cao en Trng 38 45
T
4

CZ Gn trn Dy, nh cao en nht Trng 60 70
MEA Gn trn Dy, nh cao en nht Trng 40 45
T
5

CZ Trn Dy, t nh cao en Trng 60 72
MEA Trn Dy, t nh cao en Trng 35 55
T
6

CZ Elip Tha, t nh cao en Trng 60 68
MEA Elip Tha, t nh cao en Trng 40 45
S
1

CZ Gn trn Tha, nh cao en Vng 60 75
MEA Gn trn Tha, nh cao en Vng 35 50
S
2

CZ Oval Dy, t nh cao en Trng 70 78
MEA Oval Dy, t nh cao en Trng 45 55
S
3

S
4

CZ Gn trn Dy, t nh cao en Trng 60 72
MEA Gn trn Dy, t nh cao en Trng 35 55
S
5

CZ Oval Dy, nh cao en Xanh 70 85
MEA Oval Dy, nh cao en Xanh 45 55
Chuyn ngnh i sinh vt h xlv in Nghin u v Pht trin Cng ngh inh h
Bng PL 22: c tnh vi th ca cc dng A. niger phn lp trn mi trng CZ
Dng
nm
mc
Cung bo t Bng nh gi Th bnh Bo t trn
Chiu di
(m)
Chiu rng
(m)
Kch thc
(m)
Hnh
dng
Kch
thc
(m)
Kch
thc
(m)
Hnh
dng
Sa
1
1100 2200 12,5 17,5 57,5 80,0 gl 20,030,0 3,5 4,0 gl
Sa
2
1700 2300 12,5 15,0 75,0 80,0 gl 35,040,0 3,5 4,5 gl/fr
Sa
3
1700 1950 10,0 12,5 55,0 80,0 gl 35,040,0 3,0 4,0 gl
So
1
1700 2300 12,5 17,5 62,5 75,0 gl 30,040,0 3,0 4,0 gl/fr
So
2
1500 2000 15,0 17,5 47,5 55,0 gl 20,535,0 4,0 5,0 gl/fr
So
3
970 1800 12,5 17,5 50,0 70,0 gl 20,030,0 3,0 4,0 gl/fr
So
4
1600 1850 12,5 15,0 70,0 75,0 gl 30,035,0 3,0 4,0 gl/fr
M
1
900 1300 7,5 10,0 42,5 50,0 gl 25,530,0 3,5 4,0 gl
M
2
1500 2400 10,0 14,0 45,0 55,0 gl 19,030,0 3,0 4,0 gl
M
3
1700 2800 12,5 15,0 50,0 65,0 gl 20,035,0 3,0 4,0 gl/fr
M
4
1400 1700 10,0 12,5 45,0 55,0 gl 20,025,0 3,0 3,5 gl
M
5
700 800 7,5 10,0 45,0 50,0 gl 25,535,5 3,0 4,0 gl
M
6
800 1400 7,5 12,5 35,0 42,5 gl 19,037,0 3,0 4,0 gl
H
1
1200 1500 12,5 13,5 62,5 75,0 gl 29,040,0 4,0 5,0 gl/fr
H
2
1400 1600 15,0 17,5 50,0 75,5 gl 20,030,0 3,0 4,0 gl/fr
H
3
1300 1800 12,5 17,5 47,5 57,5 gl 27,040,0 3,5 4,0 gl/fr
H
4
1200 1500 12,5 15,0 62,5 75,0 gl 28,035,0 3,0 4,5 gl
R
1
2150 2500 15,0 17,5 52,5 70,0 gl 20,030,0 4,0 4,5 gl/fr
R
2
1250 1500 11,3 12,5 45,0 75,0 gl 22,525,0 3,0 4,0 gl
R
3
2300 2400 10,0 15,0 50,0 62,5 gl 17,542,5 3,0 4,0 gl/fr
X
1
2000 2900 12,5 20,0 75,0 87,5 gl 25,037,5 3,0 4,0 el/fr
X
2
2000 3000 12,5 15,0 50,0 70,0 gl 25,030,0 4,0 5,0 gl/fr
X
3
2050 2500 17,5 20,0 50,0 62,5 gl 37,545,0 4,0 5,0 el
X
4
1950 3100 11,3 13,8 55,0 75,0 gl 32,537,5 3,0 4,0 gl
X
5
1500 2000 12,5 20,0 47,5 62,5 gl 25,037,5 3,0 4,0 gl/fr
N
1
1250 1800 15,0 20,0 62,5 87,5 gl 25,037,5 4,0 5,0 gl/fr
N
2
1100 1400 10,0 15,0 52,5 62,5 gl 27,532,5 3,0 4,0 gl/fr
N
3
2000 2300 10,0 15,0 45,0 62,5 gl 22,532,5 3,0 4,0 gl
G
1
1800 2500 10,0 12,5 47,5 50,0 gl 15,030,0 3,0 3,5 gl/fr
G
2
2200 2800 12,5 13,8 62,5 75,0 gl 25,035,0 3,0 4,0 el/fr
G
3
1500 2000 13,8 20,0 50,0 57,5 gl 22,540,0 3,0 4,0 gl/fr
G
4
1900 2200 10,0 12,5 55,0 65,0 gl 20,035,0 3,0 5,0 gl
G
5
1700 2250 10,0 12,5 50,0 55,0 gl 20,030,0 3,0 3,5 gl/fr
RD
1
1200 2000 11,5 15,5 56,5 80,0 gl 22,030,0 3,5 4,5 gl
Chuyn ngnh i sinh vt h xlvi in Nghin u v Pht trin Cng ngh inh h
RD
2
1700 2300 12,5 15,0 75,0 80,0 gl 35,040,0 3,5 4,5 gl/fr
RD
3
1500 1950 10,0 12,5 55,0 80,0 gl 31,040,0 3,0 4,0 gl
RD
4
1700 2200 12,5 17,5 62,5 75,0 gl 30,040,0 3,0 4,0 gl/fr
RD
5
1500 2000 15,0 17,5 47,5 55,0 gl 20,535,0 4,0 5,0 gl/fr
RD
6
970 1800 12,5 17,5 50,0 70,0 gl 20,030,0 3,0 4,0 gl/fr
RD
7
1500 1850 12,5 15,0 68,0 75,0 gl 30,035,0 3,0 4,0 gl/fr
RD
8
1650 1950 12,5 15,0 60,0 75,0 gl 32,040,0 3,0 4,0 gl/fr
GA
1
800 1250 8,5 17,0 42,5 50,0 gl 25,538,0 3,5 4,0 gl
GA
2
1600 2500 10,0 15,5 45,0 55,0 gl 19,030,0 3,0 4,0 gl
GA
3
1650 2750 12,5 15,0 55,0 65,0 gl 20,035,0 3,0 4,0 gl/fr
GA
4
1450 1750 10,0 14,5 45,0 65,0 gl 20,025,0 3,0 3,5 gl
GA
5
850 1200 11,5 16,0 55,0 70,0 gl 25,535,5 3,0 4,0 gl
GA
6
900 1500 8,5 12,5 46,0 72,5 gl 19,037,0 3,0 4,0 gl
GA
7
850 1450 9,0 14,5 35,0 47,5 gl
29,0
36,0
3,0 4,0 gl
GA
8
800 1450 11,5 15,5 35,0 42,5 gl 22,035,0 3,0 4,0 gl
T
1
1250 1550 12,5 15,5 62,5 75,0 gl 29,040,0 4,0 5,0 gl/fr
T
2
1550 1700 11,0 17,5 50,0 75,5 gl 20,030,0 3,0 4,0 gl/fr
T
3
1250 1600 12,5 15,0 62,5 79,5 gl 28,035,0 3,0 4,5 gl
T
4
1300 1550 9,5 15,0 56,5 75,0 gl 24,037,0 3,0 4,5 gl
T
5
1100 1950 12,5 15,0 62,5 78,0 gl 21,040,0 3,0 4,5 gl
T
6
1200 2500 11,0 15,5 62,5 75,0 gl 28,035,0 3,0 4,5 gl
S
1
1300 2200 12,5 17,5 51,5 67,0 gl 27,040,0 3,5 4,0 gl/fr
S
2
1300 2150 9,5 14,5 49,5 67,5 gl 25,040,0 3,5 4,0 gl/fr
S
3
1400 2200 12,5 17,5 47,5 57,5 gl 27,034,0 3,5 4,0 gl/fr
S
4
1250 1950 12,5 15,0 62,5 75,0 gl 28,035,0 3,0 4,5 gl
S
5
1350 1950 12,5 14,5 62,5 75,0 gl 28,035,0 3,0 4,5 gl/fr
* h bnh 2 tng; gl(globose): hnh u ; fr(finely roughened): g gh
Chuyn ngnh i sinh vt h xlvii in Nghin u v Pht trin Cng ngh inh h
Bng PL23: c tnh vi th ca cc dng A. niger phn lp trn mi trng MEA
Dng
nm
mc
Cung bo t Bng nh gi Th bnh Bo t trn
Chiu di
(m)
Chiu rng
(m)
Kch thc
(m)
Hnh
dng
Kch thc
(m)
Kch
thc
(m)
Hnh
dng
Sa
1
1200 1450 12,5 17,5 75,0 80,5 gl 25,0 35,0 3,0 4,0 gl/fr
Sa
2
1200 1400 10,0 12,5 50,0 55,0 gl 30,0 35,0 3,5 4,0 gl
Sa
3
1000 1200 12,5 15,0 37,5 52,5 gl 25,5 30,5 3,0 4,0 gl
So
1
1300 1350 10,0 15,0 55,0 60,0 gl 25,0 30,0 4,0 4,5 gl/fr
So
2
1000 1300 12,5 17,5 32,5 42,5 gl 20,0 35,0 3,5 4,0 gl/fr
So
3
1000 1300 15,0 20,0 50,0 70,0 gl 20,0 30,0 3,0 4,0 gl
So
4
900 1200 12,5 15,0 45,0 50,0 gl 25,0 30,0 3,5 4,0 gl/fr
M
1
980 1100 12,5 16,5 50,0 60,0 gl 20,0 30,0 3,0 4,0 gl/fr
M
2
900 1100 12,5 19,0 45,0 55,0 gl 20,0 25,0 3,5 4,0 gl
M
3
1000 1400 12,5 15,0 67,5 82,5 gl 25,5 30,5 3,5 4,0 gl/fr
M
4
970 1100 10,0 12,5 40,0 47,5 gl 20,0 25,5 3,0 4,0 gl
M
5
930 1200 10,0 12,5 40,0 50,0 gl 20,0 30,0 3,5 4,0 gl/fr
M
6
1100 1300 15,0 17,0 45,0 50,0 gl 20,0 30,0 4,0 5,0 gl/fr
H
1
1100 1400 10,0 12,5 62,5 83,0 gl 25,0 30,0 3,0 4,0 gl/fr
H
2
1100 1300 12,5 15,0 70,0 80,0 gl 25,0 35,0 3,5 4,0 gl/fr
H
3
700 1050 10,0 12,5 57,5 62,5 gl 19,0 25,0 3,0 4,5 gl/fr
H
4
1000 1210 10,0 12,5 37,5 50,0 gl 30,0 35,0 3,5 4,5 gl
R
1
850 1100 13,8 15,0 57,5 62,5 gl 30,0 37,5 3,5 4,0 gl/fr
R
2
1450 2400 12,5 15,0 45,0 55,0 gl 17,5 32,5 3,0 4,0 gl/fr
R
3
1500 2300 12,5 15,0 57,5 65,0 gl 22,5 25,0 3,5 4,5 gl
X
1
1260 2000 10,0 12,5 37,5 55,0 gl 12,5 20,0 3,0 4,0 el/fr
X
2
600 750 10,0 13,8 60,0 75,0 gl 30,0 35,0 4,0 5,0 el
X
3
900 1200 12,5 15,0 50,0 62,5 gl 17,5 25,0 4,0 5,0 gl
X
4
1600 1860 12,5 15,0 50,0 57,5 gl 20,0 30,0 3,0 4,0 gl/fr
X
5
2000 2500 12,5 15,0 50,0 55,0 gl 20,0 25,0 3,0 4,0 gl/fr
N
1
1000 1300 16,3 20,0 70,0 85,0 gl 17,5 37,5 3,5 5,0 gl/fr
N
2
600 700 10,0 12,5 40,0 60,0 gl 20,0 25,0 3,0 4,0 gl
N
3
1300 1380 12,5 15,0 37,5 40,0 gl 12,5 17,5 3,0 4,0 gl
G
1
550 650 12,5 15,0 55,0 62,5 gl 25,0 30,0 3,0 4,0 gl/fr
G
2
1700 2400 10,0 15,0 50,0 75,0 gl 25,0 37,5 3,0 4,0 el/fr
G
3
1600 1800 15,0 17,5 47,5 62,5 gl 20,0 25,0 3,0 4,0 gl
G
4
1350 1800 15,0 17,5 50,0 62,5 gl 25,0 37,5 4,0 5,0 gl/fr
G
5
1400 1900 12,5
13,75
55,0 62,5 gl 25,0 30,0 3,0 3,5 el
Chuyn ngnh i sinh vt h xlviii in Nghin u v Pht trin Cng ngh inh h
RD
1
900 1500 11,5 15,5 56,5 80,0 gl 22,0 30,0 3,5 4,5 gl
RD
2
750 1250 12,5 15,0 65,0 82,0 gl 25,0 35,0 3,5 4,5 gl/fr
RD
3
950 1400 10,0 12,5 55,0 80,0 gl 27,0 33,0 3,0 4,0 gl
RD
4
800 1350 11,5 18,5 62,0 75,0 gl 23,0 34,0 3,0 4,0 gl/fr
RD
5
1000 1350 12,0 17,5 47,0 55,0 gl 20,5 35,0 4,0 5,0 gl/fr
RD
6
950 1150 12,5 17,5 50,0 75,0 gl 20,0 30,0 3,0 4,0 gl/fr
RD
7
750 1300 12,5 16,0 60,0 75,0 gl 30,0 35,0 3,0 4,0 gl/fr
RD
8
1050 1650 12,5 18,5 55,0 75,0 gl 20,0 32,0 3,0 4,0 gl/fr
GA
1
950 1350 10,5 19,0 42,5 50,0 gl 25,5 31,0 3,5 4,0 gl
GA
2
1100 1600 12,0 20,0 45,0 55,0 gl 23,0 30,0 3,0 4,0 gl
GA
3
850 1350 12,5 15,0 50,0 65,0 gl 20,0 35,0 3,0 4,0 gl/fr
GA
4
1500 2200 10,0 12,5 45,0 75,0 gl 20,0 25,0 3,0 3,5 gl
GA
5
950 1400 12,5 20,0 45,0 50,0 gl 25,5 34,5 3,0 4,0 gl
GA
6
850 1450 7,5 12,5 35,0 42,0 gl 21,0 34,0 3,0 4,0 gl
GA
7
900 1500 7,5 12,5 35,0 42,5 gl 21,0 32,0 3,0 4,0 gl
GA
8
850 1450 7,5 12,5 36,0 47,5 gl 20,0 30,0 3,0 4,0 gl
T
1
1300 1550 12,5 13,5 61,5 79,0 gl 29,0 32,0 4,0 5,0 gl/fr
T
2
1500 1700 15,0 17,5 50,0 75,5 gl 20,0 30,0 3,0 4,0 gl/fr
T
3
1250 1600 12,5 15,0 62,5 75,0 gl 28,0 35,0 3,0 4,5 gl
T
4
950 1550 12,5 15,0 58,5 72,0 gl 22,0 34,0 3,0 4,5 gl
T
5
1300 1500 12,5 15,0 62,0 78,0 gl 28,0 35,0 3,0 4,5 gl
T
6
1250 1700 12,5 15,0 62,5 75,0 gl 27,0 31,0 3,0 4,5 gl
S
1
1300 1650 12,5 16,5 47,5 67,5 gl 20,0 35,0 3,5 4,0 gl/fr
S
2
1100 2200 11,0 17,5 49,5 62,5 gl 26,0 35,0 3,5 4,0 gl/fr
S
3
1100 1550 12,5 17,5 47,5 57,5 gl 27,0 34,0 3,5 4,0 gl/fr
S
4
950 1400 10,5 16,0 62,5 75,0 gl 28,0 35,0 3,0 4,5 gl
S
5
1300 1500 11,5 16,5 52,5 62,5 gl 27,0 35,0 3,5 4,5 gl/fr
* h bnh 2 tng; gl(globose): hnh u ; fr(finely roughened): g gh
Chuyn ngnh i sinh vt h xlix in Nghin u v Pht trin Cng ngh inh h
Bng PL2.4: ng knh vng phn gii pectin (D) ca cc dng A. niger
STT Dng nm mc K vng phn gii (mm)
Nhm I. Nhm c hot tnh pectinase thp D = 11,67 15 mm (11 dng)
1 G
3
11,67 1,15
2 G
5
11,67 0,58
3 M
5
12,33 0,58
4 X
5
12,33 0,58
5 X
4
12,67 1,16
6 M
1
13,33 0,58
7 M
2
14,00 0,61
8 T
6
15,00 0,74
9 GA
3
15,00 0,69
10 RD
7
15,00 0,64
11 RD
8
15,00 0,58
Nhm II. Nhm c hot tnh pectinase trung bnh D = 15,25 cn 20 mm (35 dng + C V)
12 S
5
15,25 0,58
13 N
2
15,33 0,58
14 N
3
15,67 0,58
15 M
6
15,67 1,52
16 H
3
15,67 0,58
17 S
2
15,75 0,58
18 RD
2
16,00 1,15
19 GA
7
16,50 0,58
20 S
4
16,50 1,15
21 GA
8
16,50 1,15
22 Sa
1
16,67 0,58
23 V 16,67 1,53
24 GA
4
16,75 0,11
25 M
3
17,00 1,01
26 S
3
17,00 0,58
27 R
2
17,10 0,67
28 R
3
17,30 0,55
29 G
1
17,33 0,58
30 So
3
17,33 0,44
31 T
4
17,50 1,04
32 GA
6
17,50 0,58
33 X
2
17,67 0,44
34 M
4
17,67 0,58
Chuyn ngnh i sinh vt h l in Nghin u v Pht trin Cng ngh inh h
35 RD
3
17,86 1,15
36 H
1
18,00 0,25
37 T
3
18,00 0,89
38 SO
4
18,00 0,14
39 GA
5
18,00 1, 11
40 RD
6
18,00 1,15
41 T
5
18,25 1,52
42 SO
1
18,33 0,58
43 X
1
18,50 0,50
44 S
1
18,50 0,58
45 RD
5
18,50 0,58
46 H
2
18,67 0,58
47 RD
1
19,14 1,15
Nhm III, Nhm c hot tnh pectinase cao D 20 mm (14 dng + i chng R92)
48 G
2
20,00 0,50
49 H
4
20,00 0,50
50 GA
1
20,25 0,50
51 T
2
20,59 0,47
52 Sa
2
21,17 0,76
53 G
4
21,50 0,87
54 RD
4
21,75 1,15
55 X
3
21,83 1,26
56 GA
2
22,00 0,58
57 R92 22,25 0,44
58 Sa
3
22,33 1,15
59 T
1
23,38 1,15
60 N
1
23,67 1,15
61 So
2
24,33 0,58
62 R
1
25,58 0,68

Chuyn ngnh i sinh vt h li in Nghin u v Pht trin Cng ngh inh h
Kt qu gii trnh t PCR ca 4 dng nm mc

Chuyn ngnh i sinh vt h lii in Nghin u v Pht trin Cng ngh inh h

Chuyn ngnh i sinh vt h liii in Nghin u v Pht trin Cng ngh inh h

Chuyn ngnh i sinh vt h liv in Nghin u v Pht trin Cng ngh inh h

Chuyn ngnh i sinh vt h lv in Nghin u v Pht trin Cng ngh inh h

Chuyn ngnh i sinh vt h lvi in Nghin u v Pht trin Cng ngh inh h

Chuyn ngnh i sinh vt h lvii in Nghin u v Pht trin Cng ngh inh h

Chuyn ngnh i sinh vt h lviii in Nghin u v Pht trin Cng ngh inh h

Chuyn ngnh i sinh vt h lix in Nghin u v Pht trin Cng ngh inh h
Mt s th nghim b sung
Th nghim P1: nh gi hiu qu ly trch PME cc m trch c thay i khi
lng (10 g. 50 g v 100 g)
Kt qu thu nhn
Vi mc ch m rng quy m ca kho st, qu trnh ly trch c thc hin vi
cc thng s k thut c la chn: s dng dung dch m citrate pH 3,6; t
l dung dch m s dng v c cht l 2 : 1, nhit v thi gian ly trch tng
ng l 35C, 50 pht. Khi thay i khi lng m kho st t 10 g c cht ln n
50 g v 100 g c cht, hiu qu ly trch PME c nh gi da trn s thay i
hot tnh PME (U/g c cht), kt qu c tng hp bng PL2.5.
Bng PL25: Hiu qu thu nhn PME theo khi lng c cht l trch khc nhau
Khi lng c cht (g) Hot tnh PME (U/g)
10 79,10 2,65
50 75,33 4,16
100 74,67 5,03
T s F 1,04
Gi tr P 0,4107 (ns)
T kt qu tng hp bng PL2.5 cho thy, khi gia tng khi lng m ly trch ln
10 ln (t 10 g n 100 g), hot tnh PME thu c (tnh trn khi lng c cht)
khc bit khng c ngha v mt thng k. iu ny cho thy, qu trnh ly trch
vi cc thng s k thut c xc nh l kh n nh. Porto et al. (2004) cng
xc nhn, vic thay i khi lng m ln men c nh hng ng k n hiu
sut thu nhn ascorbic oxidoreductase nhng vic gia tng khi lng m ly trch
enzyme sau qu trnh ln men khng c tc ng ln n hot tnh enzyme thu
c. Theo kt qu thu c bng PL2.5, mc dao ng ca hot tnh PME
thu nhn trong c 3 trng hp u di 10%, iu ny chng minh trin vng
ca vic m hnh ha qu trnh ly trch PME sau qu trnh ln men SSF.
Th nghim P2: nh hng ca cc cht km hm n hot ng ca PME nm
mc
Th nghim c thc hin vi 3 cht km hm: PbCl
2
v HgCl
2
b sung cc mc
0, 1, 3, 5, 7 v 9 mM; trong khi EDTA c kho st cc mc nng cao hn
(0, 1, 5, 10, 15 v 20 mM) vo trong dung dch pectin s dng chun . Tin
hnh kho st tng t nh th nghim i vi CaCl
2
v NaCl.
Kt qu kho st c th hin th hnh PL2.1.
Chuyn ngnh i sinh vt h lx in Nghin u v Pht trin Cng ngh inh h
0,42
0,31
0,22
0,08
0
0,64
0,41
0,36
0,29
0,22
0,86
0,71
0,51
0,46
0,41
0
0,2
0,4
0,6
0,8
1
1,2
0 5 10 15 20 25
Nng (mM)
Gi tr A/Ao
HgCl2
PbCl2
EDTA

Hnh PL2.1: n ng ca cc ct km hm n ot ng ca PME nm mc
Kt qu kho st cho thy, c 3 loi ph gia u c tc ng c ch hot ng ca
ch phm PME thu nhn. Trong , HgCl
2
c tc ng mnh nht, mc gim
hot tnh hn 50% ngay khi c s hin din ca HgCl
2
nng rt thp (1mM) v
lm mt hon ton hot tnh ca PME nng Hg
2+
b sung l 9 mM. Trong khi
, PbCl
2
vn duy tr 64% hot tnh mc b sung Pb
2+
l 1mM v tc mt hot
tnh chm dn, nng Pb
2+
b sung cao nht l 9mM, hot tnh PME vn cn
duy tr khong 22%. Nghin cu ca Arotupin et al. (2008) i vi PME ngun gc
t A. repens cho thy, hot tnh PME b mt hon ton nng Hg
2+
b sung l
10 mM, tuy nhin n mc Pb
2+
b sung n 15 mM, s hot ng ca PME vn
cn duy tr.
Trong khi , hiu qu c ch ca EDTA n hot ng ca PME t hn, ch
ngng EDTA b sung t 10 mM tr ln, hot tnh ca PME gim di 50%.

Th nghim P3: So snh hiu qu ci thin cu trc khm ca PME c chua v
PME t A. niger
- Ch phm PME t A. niger (hot tnh s dng, dung dch pha long, phng thc
b sung c chn t kt qu ti u ca th nghim 25 v 26).
- Ch phm PME c chua vi t l b sung 5 U/mL kt hp vi CaCl
2
0,15% trong
nc 45C, 30 pht (Nguyen et al., 2009)
Cch thc hin
Chun b dung dh tin x l nng PME nm m ti u: tng t nh cch
chun b dung dch PME 1 U/mL (th nghim 25) trong nng s dng, cch
Chuyn ngnh i sinh vt h lxi in Nghin u v Pht trin Cng ngh inh h
chun b da trn kt qu th nghim 25 v 26.
Chun b PME hu: PME c chua c ly trch v tinh sch s b theo quy
trnh Wicker ci tin (Nguyn Vn Mi et al., 2009).
C chua chn khi pht (breaker) c ct nh, lnh ng. Sau t nht 24 gi
tr ng, mu c ng ha bng my xay sinh t. Puree thu c em i ly tm
thit b ly tm lng tc 9000 vng/pht trong 30 pht nhit 4C. Dch
nc trong sau ly tm c loi b. Phn m qu c ha vo dung dch m
phosphate (KH
2
PO
4
/K
2
HPO
4
1/15 M, xem cch pha ph lc I) c cha NaCl 1,25
M, pH = 8,0 vi t l 2,5 kg nguyn liu ti cho 1 lt dung dch m v bo qun
qua m t mt c nhit 4C thc hin qu trnh ly trch enzyme. Sau qu
trnh ly trch, hn hp m qu - dung dch c em ly tm tc 9000
vng/pht trong thi gian 30 pht 4C. Thu ly phn dch trong cha PME, loi
b phn m qu. Phn dch trong tip tc c kt ta phn on ln th nht vi
mui ammonium sulfate 30% 4C, khuy trn trong 30 pht nhm kt ta b cc
protein tp. Ly tm dch kt ta 9000 vng/pht trong 30 pht nhit 4C, thu
phn dch trong. Phn dch trong em kt ta vi amonium sulfate 80% 4C,
khuy trn trong 30 pht. Tip tc ly tm dch kt ta 9000 vng/pht, trong 30
pht 4C - thu kt ta v ha tan li kt ta cha PME trong m phosphate pH
7,5.
Kt qu thu nhn
S khc bit v pH ti u cho hot ng ca PME vi sinh vt (pH ti u iu kin
kho st l 5,0) v PME thc vt (pH 8,0, Fachin et al., 2002) cng nh phng
thc hot ng (Rexova - Benkova v Markovic, 1976; Markovic v Kohn, 1984;
Willats et al., 2001) c th l nguyn nhn dn n s khc bit v hiu qu hot
ng ca PME i vi tng loi sn phm v qua , tc ng n kh nng ci
thin c tnh cu trc ca t bo rau qu do s khc bit v ngun PME b sung.
Chnh v th, nghin cu tc ng ca 2 ngun enzyme b sung l PME vi sinh vt
(PME t A. niger) v PME thc vt (PME c chua) n s thay i cng ca
khm ng lnh c thc hin. Kt qu so snh c th hin hnh PL2.2.
Chuyn ngnh i sinh vt h lxii in Nghin u v Pht trin Cng ngh inh h
0,448
1,317
1,136
0,349
0,270
1,00
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Sau tin x l Sau 4 tun tr ng
cng tng i
(H/H0)
PME nm mc
PME c chua
i chng

Hnh PL2.2: So sn iu qu ci tin cng ca km do tc ng ca qu trn
tin x l vi PME ngoi bo c ngun gc kc nau
Vi kiu phn ng ca cc PME c ngun gc thc vt l thy phn lin kt ester
ca pectin theo c ch chui n (Nguyn c Lng, 2004), ch tc dng ti mt
v tr trn mch (Rexova - Benkova v Markovic, 1976; Markovic v Kohn, 1984;
Willats et al., 2001), hot ng cn c nhm carboxyl t do trn mch galacturonic
nn PME c ly trch t thc vt t hot ng trn cc pectin c ester ha cao
hn l cc pectin c ester ha thp (Evans v Machale, 1978; Seymour et al.,
1991). Vi DE ca khm ban u ch khong 50,21%, hot ng ca PME c
chua t ra khng c hiu qu cao khi so snh vi PME c sinh tng hp t nm
mc c kh nng phn ct c cht mt cch ngu nhin, c bit t cc dng
Aspergillus (Ishi et al., 1979; Limberg et al., 2000). y c l l nguyn nhn chnh
dn n hiu qu ci thin cng chc ca khm nh vo vic b sung PME nm
mc t ra hiu qu hn. Ngoi ra, do khm l mi trng c pH thp (khong 3,7
4,4, Bartolom et al., 1995), gn vi pH ti u cho hot ng ca PME nm mc
(5,0), trong khi pH thch hp cho hot ng ca PME c chua nm khong pH
kim (8,0) c l cng c nh hng n hiu qu hot ng ca PME n s phn
ct pectin.
Kt qu kho st chng t vic b sung PME ngoi bo ngun gc vi sinh vt
(c th l ch phm PME k thut t A. niger) vi nng 1 U/mL trong nc chn
(kt hp 0,5% CaCl
2
) nhit tin x l 40C trong thi gian 20 pht l phng
thc kh thi gip ci thin ng k cu trc ca khm ng lnh. Gi tr cng
cn li ca khm c duy tr 45% so vi khm ti, tng gp 1,5 ln khi so snh
vi mu khng tin x l.

Chuyn ngnh i sinh vt h lxiii in Nghin u v Pht trin Cng ngh inh h
PH LC 3. KT QU THNG K
Phn 1 Tun chn dng nm mc thuc loi A. niger c hiu i vi PME
- o snh tnh hiu i vi petinse d trn ng knh vng phn gii
petin dng nm m thu loi A. niger
One-Way ANOVA - DK vong phan giai pectin by Chung nam moc
Dependent variable: DK vong phan giai pectin (mm)
Factor: Chung nam moc
Number of observations: 70
Number of levels: 15

ANOVA Table for DK vong phan giai pectin by Chung nam moc
Source Sum of Squares Df Mean Square s F P-Value
Between groups 132,554 14 9,46818 7,16 0,0000
Within groups 72,7622 55 1,32295
Total (Corr.) 205,317 69

Multiple Range Tests for DK vong phan giai_mm by Chung nam moc
Method: 95,0 percent Duncan
Chung nam moc Count Mean Homogeneous Groups
H4 3 20,0 XX
G2 3 20,0 XX
GA1 8 20,25 XX
T2 8 20,5 XXX
Sa2 3 21,1667 XXXX
G4 3 21,5 XXXX
RD4 8 21,75 XXX
X3 3 21,8333 XX
GA2 8 22,0 XXX
R92 3 22,25 XXX
Sa3 3 22,3333 XXX
T1 8 22,375 XXX
N1 3 23,6667 XX
So2 3 24,3333 XX
R1 3 25,6667 X
Contrast Sig. Difference
G2 - G4 -1,5
G2 - GA1 -,25
G2 - GA2 * -2,0
G2 - H4
G2 - N1 * -3,66667
G2 - R1 * -5,66667
G2 - R92 * -2,25
G2 - RD4 * -1,75
G2 - Sa2 -1,16667
G2 - Sa3 * -2,33333
G2 - So2 * -4,33333
G2 - T1 * -2,375
G2 - T2 -,5
G2 - X3 -1,83333
G4 - GA1 1,25
G4 - GA2 -,5
G4 - H4 1,5
G4 - N1 * -2,16667
G4 - R1 * -4,16667
G4 - R92 -,75
G4 - RD4 -,25
Chuyn ngnh i sinh vt h lxiv in Nghin u v Pht trin Cng ngh inh h
G4 - Sa2 ,333333
G4 - Sa3 -,833333
G4 - So2 * -2,83333
G4 - T1 -,875
G4 - T2 1,0
G4 - X3 -,333333
GA1 - GA2 * -1,75
GA1 - H4 ,25
GA1 - N1 * -3,41667
GA1 - R1 * -5,41667
GA1 - R92 * -2,0
GA1 - RD4 * -1,5
GA1 - Sa2 -,916667
GA1 - Sa3 * -2,08333
GA1 - So2 * -4,08333
GA1 - T1 * -2,125
GA1 - T2 -,25
GA1 - X3 * -1,58333
GA2 - H4 * 2,0
GA2 - N1 * -1,66667
GA2 - R1 * -3,66667
GA2 - R92 -,25
GA2 - RD4 ,25
GA2 - Sa2 ,833333
GA2 - Sa3 -,333333
GA2 - So2 * -2,33333
GA2 - T1 -,375
GA2 - T2 * 1,5
GA2 - X3 ,166667
H4 - N1 * -3,66667
H4 - R1 * -5,66667
H4 - R92 * -2,25
H4 - RD4 * -1,75
H4 - Sa2 -1,16667
H4 - Sa3 * -2,33333
H4 - So2 * -4,33333
H4 - T1 * -2,375
H4 - T2 -,5
H4 - X3 -1,83333
N1 - R1 * -2,0
N1 - R92 1,41667
N1 - RD4 * 1,91667
N1 - Sa2 * 2,5
N1 - Sa3 1,33333
N1 - So2 -,666667
N1 - T1 1,29167
N1 - T2 * 3,16667
N1 - X3 1,83333
R1 - R92 * 3,41667
R1 - RD4 * 3,91667
R1 - Sa2 * 4,5
R1 - Sa3 * 3,33333
R1 - So2 1,33333
R1 - T1 * 3,29167
R1 - T2 * 5,16667
R1 - X3 * 3,83333
R92 - RD4 ,5
R92 - Sa2 1,08333
R92 - Sa3 -,0833333
R92 - So2 * -2,08333
Chuyn ngnh i sinh vt h lxv in Nghin u v Pht trin Cng ngh inh h
R92 - T1 -,125
R92 - T2 * 1,75
R92 - X3 ,416667
RD4 - Sa2 ,583333
RD4 - Sa3 -,583333
RD4 - So2 * -2,58333
RD4 - T1 -,625
RD4 - T2 * 1,25
RD4 - X3 -,0833333
Sa2 - Sa3 -1,16667
Sa2 - So2 * -3,16667
Sa2 - T1 -1,20833
Sa2 - T2 ,666667
Sa2 - X3 -,666667
Sa3 - So2 * -2,0
Sa3 - T1 -,0416667
Sa3 - T2 * 1,83333
Sa3 - X3 ,5
So2 - T1 * 1,95833
So2 - T2 * 3,83333
So2 - X3 * 2,5
T1 - T2 * 1,875
T1 - X3 ,541667
T2 - X3 -1,33333
* denotes a statistically significant difference.

- nh gi kh nng thu nhn PME dng A. niger hiu vi pectinase
+Da trn ng knh vng phn gii pectin vi ch th CTAB

One-Way ANOVA - DK vong phan giai_mm by Chung nam moc
Dependent variable: DK vong phan giai_mm

ANOVA Table for DK vong phan giai_mm by Chung nam moc
Between groups 68,1508 14 4,86791 32,18 0,0000
Within groups 4,53833 30 ,151278
Total (Corr.) 72,6891 44

Multiple Range Tests for DK vong phan giai_mm by Chung nam moc
G4 3 10,4667 X
G2 3 10,95 XX
R92 3 10,9667 XXX
RD4 3 11,3667 XXX
T2 3 11,4333 XXX
H4 3 11,4333 XXX
GA2 3 11,6 XX
Sa3 3 11,7333 X
GA1 3 11,7667 X
Sa2 3 11,8 X
X3 3 11,8667 X
N1 3 13,1 X
T1 3 13,1667 X
R1 3 14,4667 X
So2 3 14,8667 X
Chuyn ngnh i sinh vt h lxvi in Nghin u v Pht trin Cng ngh inh h
G2 - G4 ,483333
G2 - GA1 * -,816667
G2 - GA2 * -,65
G2 - H4 -,483333
G2 - N1 * -2,15
G2 - R1 * -3,51667
G2 - R92 -,0166667
G2 - RD4 -,416667
G2 - Sa2 * -,85
G2 - Sa3 * -,783333
G2 - So2 * -3,91667
G2 - T1 * -2,21667
G2 - T2 -,483333
G2 - X3 * -,916667
G4 - GA1 * -1,3
G4 - GA2 * -1,13333
G4 - H4 * -,966667
G4 - N1 * -2,63333
G4 - R1 * -4,0
G4 - R92 -,5
G4 - RD4 * -,9
G4 - Sa2 * -1,33333
G4 - Sa3 * -1,26667
G4 - So2 * -4,4
G4 - T1 * -2,7
G4 - T2 * -,966667
G4 - X3 * -1,4
GA1 - GA2 ,166667
GA1 - H4 ,333333
GA1 - N1 * -1,33333
GA1 - R1 * -2,7
GA1 - R92 * ,8
GA1 - RD4 ,4
GA1 - Sa2 -,0333333
GA1 - Sa3 ,0333333
GA1 - So2 * -3,1
GA1 - T1 * -1,4
GA1 - T2 ,333333
GA1 - X3 -,1
GA2 - H4 ,166667
GA2 - N1 * -1,5
GA2 - R1 * -2,86667
GA2 - R92 ,633333
GA2 - RD4 ,233333
GA2 - Sa2 -,2
GA2 - Sa3 -,133333
GA2 - So2 * -3,26667
GA2 - T1 * -1,56667
GA2 - T2 ,166667
GA2 - X3 -,266667
H4 - N1 * -1,66667
H4 - R1 * -3,03333
H4 - R92 ,466667
H4 - RD4 ,0666667
H4 - Sa2 -,366667
H4 - Sa3 -,3
H4 - So2 * -3,43333
H4 - T1 * -1,73333
H4 - T2
Chuyn ngnh i sinh vt h lxvii in Nghin u v Pht trin Cng ngh inh h
H4 - X3 -,433333
N1 - R1 * -1,36667
N1 - R92 * 2,13333
N1 - RD4 * 1,73333
N1 - Sa2 * 1,3
N1 - Sa3 * 1,36667
N1 - So2 * -1,76667
N1 - T1 -,0666667
N1 - T2 * 1,66667
N1 - X3 * 1,23333
R1 - R92 * 3,5
R1 - RD4 * 3,1
R1 - Sa2 * 2,66667
R1 - Sa3 * 2,73333
R1 - So2 -,4
R1 - T1 * 1,3
R1 - T2 * 3,03333
R1 - X3 * 2,6
R92 - RD4 -,4
R92 - Sa2 * -,833333
R92 - Sa3 * -,766667
R92 - So2 * -3,9
R92 - T1 * -2,2
R92 - T2 -,466667
R92 - X3 * -,9
RD4 - Sa2 -,433333
RD4 - Sa3 -,366667
RD4 - So2 * -3,5
RD4 - T1 * -1,8
RD4 - T2 -,0666667
RD4 - X3 -,5
Sa2 - Sa3 ,0666667
Sa2 - So2 * -3,06667
Sa2 - T1 * -1,36667
Sa2 - T2 ,366667
Sa2 - X3 -,0666667
Sa3 - So2 * -3,13333
Sa3 - T1 * -1,43333
Sa3 - T2 ,3
Sa3 - X3 -,133333
So2 - T1 * 1,7
So2 - T2 * 3,43333
So2 - X3 * 3,0
T1 - T2 * 1,73333
T1 - X3 * 1,3
T2 - X3 -,433333
* denotes a statistically significant difference./CV= 0,648569
Chuyn ngnh i sinh vt h lxviii in Nghin u v Pht trin Cng ngh inh h
G2
G4
GA1
GA2
H4
N1
R1
R92
RD4
Sa2
Sa3
So2
T1
T2
X3
Box-and-Whisker Plot
10 11 12 13 14 15 16
DK vong phan giai_mm
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Hn PL31: t Box Wisker biu din n ng ca dng nm mc n k
nng tu nn PME tng qua ng kn vng pn gii pectin
+Da trn hot tnh PME thu nhn t phng php ln men lng vi c cht pectin
One-Way ANOVA - PME activity by Chung nam moc
Dependent variable: PME activity

ANOVA Table for PME activity by Chung nam moc
Between groups 21,025 14 1,50178 16,74 0,0000
Within groups 2,69213 30 ,0897378
Total (Corr.) 23,7171 44

Multiple Range Tests for PME activity by Chung nam moc
G4 3 4,93667 X
RD4 3 5,3 XX
R92 3 5,30333 XX
G2 3 5,43 XX
GA2 3 5,44333 XX
T2 3 5,48 XXX
GA1 3 5,52 XX
H4 3 5,53 XX
Sa2 3 6,0 XX
X3 3 6,03 XX
Sa3 3 6,03 XX
N1 3 6,34 XX
T1 3 6,59333 XX
R1 3 7,0 XX
So2 3 7,49667 X
G2 - G4 ,493333
G2 - GA1 -,09
G2 - GA2 -,0133333
G2 - H4 -,1
G2 - N1 * -,91
G2 - R1 * -1,57
G2 - R92 ,126667
G2 - RD4 ,13
G2 - Sa2 * -,57
Chuyn ngnh i sinh vt h lxix in Nghin u v Pht trin Cng ngh inh h
G2 - Sa3 * -,6
G2 - So2 * -2,06667
G2 - T1 * -1,16333
G2 - T2 -,05
G2 - X3 * -,6
G4 - GA1 * -,583333
G4 - GA2 -,506667
G4 - H4 * -,593333
G4 - N1 * -1,40333
G4 - R1 * -2,06333
G4 - R92 -,366667
G4 - RD4 -,363333
G4 - Sa2 * -1,06333
G4 - Sa3 * -1,09333
G4 - So2 * -2,56
G4 - T1 * -1,65667
G4 - T2 -,543333
G4 - X3 * -1,09333
GA1 - GA2 ,0766667
GA1 - H4 -,01
GA1 - N1 * -,82
GA1 - R1 * -1,48
GA1 - R92 ,216667
GA1 - RD4 ,22
GA1 - Sa2 -,48
GA1 - Sa3 -,51
GA1 - So2 * -1,97667
GA1 - T1 * -1,07333
GA1 - T2 ,04
GA1 - X3 -,51
GA2 - H4 -,0866667
GA2 - N1 * -,896667
GA2 - R1 * -1,55667
GA2 - R92 ,14
GA2 - RD4 ,143333
GA2 - Sa2 * -,556667
GA2 - Sa3 * -,586667
GA2 - So2 * -2,05333
GA2 - T1 * -1,15
GA2 - T2 -,0366667
GA2 - X3 * -,586667
H4 - N1 * -,81
H4 - R1 * -1,47
H4 - R92 ,226667
H4 - RD4 ,23
H4 - Sa2 -,47
H4 - Sa3 -,5
H4 - So2 * -1,96667
H4 - T1 * -1,06333
H4 - T2 ,05
H4 - X3 -,5
N1 - R1 * -,66
N1 - R92 * 1,03667
N1 - RD4 * 1,04
N1 - Sa2 ,34
N1 - Sa3 ,31
N1 - So2 * -1,15667
N1 - T1 -,253333
N1 - T2 * ,86
N1 - X3 ,31
Chuyn ngnh i sinh vt h lxx in Nghin u v Pht trin Cng ngh inh h
R1 - R92 * 1,69667
R1 - RD4 * 1,7
R1 - Sa2 * 1,0
R1 - Sa3 * ,97
R1 - So2 -,496667
R1 - T1 ,406667
R1 - T2 * 1,52
R1 - X3 * ,97
R92 - RD4 ,00333333
R92 - Sa2 * -,696667
R92 - Sa3 * -,726667
R92 - So2 * -2,19333
R92 - T1 * -1,29
R92 - T2 -,176667
R92 - X3 * -,726667
RD4 - Sa2 * -,7
RD4 - Sa3 * -,73
RD4 - So2 * -2,19667
RD4 - T1 * -1,29333
RD4 - T2 -,18
RD4 - X3 * -,73
Sa2 - Sa3 -,03
Sa2 - So2 * -1,49667
Sa2 - T1 * -,593333
Sa2 - T2 ,52
Sa2 - X3 -,03
Sa3 - So2 * -1,46667
Sa3 - T1 * -,563333
Sa3 - T2 ,55
Sa3 - X3
So2 - T1 * ,903333
So2 - T2 * 2,01667
So2 - X3 * 1,46667
T1 - T2 * 1,11333
T1 - X3 * ,563333
T2 - X3 -,55
G2
G4
GA1
GA2
H4
N1
R1
R92
RD4
Sa2
Sa3
So2
T1
T2
X3
4,7 5,2 5,7 6,2 6,7 7,2 7,7
PME activity
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Hn PL32: t Box Wisker biu din s tay i ot tn PME ca cc dng
nm mc kc nau
Chuyn ngnh i sinh vt h lxxi in Nghin u v Pht trin Cng ngh inh h
- nh gi hiu qu kt hp 4 dng nm m tnh hiu o i vi PME
+ Thng qua ng knh vng phn gii pectin vi ch th CTAB
One-Way ANOVA - DK vong phan giai by Phuong thuc ket hop dong nam
Dependent variable: DK vong phan giai (mm)
Factor: Phuong thuc ket hop dong nam


ANOVA Table for DK vong phan giai by Phuong thuc ket hop dong nam
Source Sum of Squares Df Mean Square F-Ratio P-Value
Between groups 34,2955 9 3,81061 14,12 0,0000
Within groups 5,3974 20 ,26987
Total (Corr.) 39,6929 29

Multiple Range Tests for DK vong phan giai by Phuong thuc ket hop dong nam

Level Count Mean Homogeneous Groups
N1R1 3 12,4333 X
N1So2 3 12,7333 X
T1R1 3 13,0 X
T1So2 3 13,1333 X
N1 3 13,2 X
T1 3 13,22 X
R1 3 14,18 X
T1N1 3 14,4 X
So2 3 14,76 X
R1So2 3 16,1 X
N1 - N1R1 ,766667
N1 - N1So2 ,466667
N1 - R1 * -,98
N1 - R1So2 * -2,9
N1 - So2 * -1,56
N1 - T1 -,02
N1 - T1N1 * -1,2
N1 - T1R1 ,2
N1 - T1So2 ,0666667
N1R1 - N1So2 -,3
N1R1 - R1 * -1,74667
N1R1 - R1So2 * -3,66667
N1R1 - So2 * -2,32667
N1R1 - T1 -,786667
N1R1 - T1N1 * -1,96667
N1R1 - T1R1 -,566667
N1R1 - T1So2 -,7
N1So2 - R1 * -1,44667
N1So2 - R1So2 * -3,36667
N1So2 - So2 * -2,02667
N1So2 - T1 -,486667
N1So2 - T1N1 * -1,66667
N1So2 - T1R1 -,266667
N1So2 - T1So2 -,4
R1 - R1So2 * -1,92
R1 - So2 -,58
R1 - T1 * ,96
R1 - T1N1 -,22
R1 - T1R1 * 1,18
Chuyn ngnh i sinh vt h lxxii in Nghin u v Pht trin Cng ngh inh h
R1 - T1So2 * 1,04667
R1So2 - So2 * 1,34
R1So2 - T1 * 2,88
R1So2 - T1N1 * 1,7
R1So2 - T1R1 * 3,1
R1So2 - T1So2 * 2,96667
So2 - T1 * 1,54
So2 - T1N1 ,36
So2 - T1R1 * 1,76
So2 - T1So2 * 1,62667
T1 - T1N1 * -1,18
T1 - T1R1 ,22
T1 - T1So2 ,0866667
T1N1 - T1R1 * 1,4
T1N1 - T1So2 * 1,26667
T1R1 - T1So2 -,133333
* denotes a statistically significant difference
N1
N1R1
N1So2
R1
R1So2
So2
T1
T1N1
T1R1
T1So2
12 13 14 15 16 17 18
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Hn PL33: t Box Wisker biu din n ng kt p 4 dng nm mc c
iu n k nng tu nn PME tng qua ng kn vng pn gii pectin
+ Thng qua hot tnh PME theo phng php ln men lng
One-Way ANOVA - Hoat tinh PME by Phuong thuc ket hop dong nam
Dependent variable: Hoat tinh PME (U/mL)
Factor: Phuong thuc ket hop dong nam

ANOVA Table for Hoat tinh PME by Phuong thuc ket hop dong nam
Between groups 10,3103 9 1,14559 16,90 , 0
Total (Corr.) 11,6663 29

Multiple Range Tests for Hoat tinh PME by Phuong thuc ket hop dong nam
N1R1 3 6,21667 X
N1 3 6,24 X
N1So2 3 6,36667 X
T1R1 3 6,5 XX
T1 3 6,55 XX
T1So2 3 6,56667 XX
R1 3 6,92 XX
T1N1 3 7,2 XX
So2 3 7,61 XX
R1So2 3 8,05 X
Chuyn ngnh i sinh vt h lxxiii in Nghin u v Pht trin Cng ngh inh h
N1 - N1R1 ,0233333
N1 - N1So2 -,126667
N1 - R1 * -,68
N1 - R1So2 * -1,81
N1 - So2 * -1,37
N1 - T1 -,31
N1 - T1N1 * -,96
N1 - T1R1 -,26
N1 - T1So2 -,326667
N1R1 - N1So2 -,15
N1R1 - R1 * -,703333
N1R1 - R1So2 * -1,83333
N1R1 - So2 * -1,39333
N1R1 - T1 -,333333
N1R1 - T1N1 * -,983333
N1R1 - T1R1 -,283333
N1R1 - T1So2 -,35
N1So2 - R1 * -,553333
N1So2 - R1So2 * -1,68333
N1So2 - So2 * -1,24333
N1So2 - T1 -,183333
N1So2 - T1N1 * -,833333
N1So2 - T1R1 -,133333
N1So2 - T1So2 -,2
R1 - R1So2 * -1,13
R1 - So2 * -,69
R1 - T1 ,37
R1 - T1N1 -,28
R1 - T1R1 ,42
R1 - T1So2 ,353333
R1So2 - So2 ,44
R1So2 - T1 * 1,5
R1So2 - T1N1 * ,85
R1So2 - T1R1 * 1,55
R1So2 - T1So2 * 1,48333
So2 - T1 * 1,06
So2 - T1N1 ,41
So2 - T1R1 * 1,11
So2 - T1So2 * 1,04333
T1 - T1N1 * -,65
T1 - T1R1 ,05
T1 - T1So2 -,0166667
T1N1 - T1R1 * ,7
T1N1 - T1So2 * ,633333
T1R1 - T1So2 -,0666667
Chuyn ngnh i sinh vt h lxxiv in Nghin u v Pht trin Cng ngh inh h
N1
N1R1
N1So2
R1
R1So2
So2
T1
T1N1
T1R1
T1So2
6 6,5 7 7,5 8 8,5 9
PME activity
K
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Hn PL34: t Box Wisker biu din n ng ca s kt p
4 dng nm mc c iu n ot tn PME
- thy i t l kt hp 2 dng R
1
v So
2
n s gi tng hiu qu sinh tng
hp PME
+ Thng qua ng knh vng phn gii pectin vi ch th CTAB
One-Way ANOVA - DK vong phan giai_mm by Ty le ket hop R1&So2
Dependent variable: DK vong phan giai_mm
Factor: Ty le ket hop R1&So2
Number of levels: 7
ANOVA Table for DK vong phan giai_mm by Ty le ket hop R1&So2
Between groups 8,37619 6 1,39603 6,90 0,014
Within groups 2,83333 14 ,202381
Total (Corr.) 11,2095 20

Multiple Range Tests for DK vong phan giai_mm by Ty le ket hop R1&So2
4_1 3 14,4333 X
3_1 3 14,7667 XX
1_4 3 14,9 XX
1_3 3 15,1333 XX
2_1 3 15,2333 X
1_1 3 16,1 X
1_2 3 16,2667 X
Contrast Sig. Difference +/- Limits
1_1 - 1_2 -,166667 ,787815
1_1 - 1_3 * ,966667 ,787815
1_1 - 1_4 * 1,2 ,787815
1_1 - 2_1 * ,866667 ,787815
1_1 - 3_1 * 1,33333 ,787815
1_1 - 4_1 * 1,66667 ,787815
1_2 - 1_3 * 1,13333 ,787815
1_2 - 1_4 * 1,36667 ,787815
1_2 - 2_1 * 1,03333 ,787815
1_2 - 3_1 * 1,5 ,787815
1_2 - 4_1 * 1,83333 ,787815
1_3 - 1_4 ,233333 ,787815
1_3 - 2_1 -,1 ,787815
1_3 - 3_1 ,366667 ,787815
1_3 - 4_1 ,7 ,787815
1_4 - 2_1 -,333333 ,787815
Chuyn ngnh i sinh vt h lxxv in Nghin u v Pht trin Cng ngh inh h
1_4 - 3_1 ,133333 ,787815
1_4 - 4_1 ,466667 ,787815
2_1 - 3_1 ,466667 ,787815
2_1 - 4_1 * ,8 ,787815
3_1 - 4_1 ,333333 ,787815

1_1
1_2
1_3
1_4
2_1
3_1
4_1
13 14 15 16 17 18
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Hn PL35: t Box Wisker biu din n ng ca t l kt p R
1
v So
2

n iu qu tu nn PME da trn ng kn vng pn gii pectin

+ Thng qua hot tnh PME theo phng php ln men lng
One-Way ANOVA - PME activity by Ty le ket hop R1&So2
Factor: Ty le ket hop R1&So2

Number of levels: 7
ANOVA Table for PME activity by Ty le ket hop R1&So2
Between groups 2,41911 6 ,403186 5,61 0,038
Within groups 1,007 14 ,0719286
Total (Corr.) 3,42611 20

Multiple Range Tests for PME activity by Ty le ket hop R1&So2

4_1 3 7,15 X
3_1 3 7,35 X
1_4 3 7,41667 X
2_1 3 7,41667 X
1_3 3 7,52667 X
1_1 3 8,05 X
1_2 3 8,12 X

1_1 - 1_2 -,07
1_1 - 1_3 * ,523333
1_1 - 1_4 * ,633333
1_1 - 2_1 * ,633333
1_1 - 3_1 * ,7
1_1 - 4_1 * ,9
1_2 - 1_3 * ,593333
1_2 - 1_4 * ,703333
1_2 - 2_1 * ,703333
Chuyn ngnh i sinh vt h lxxvi in Nghin u v Pht trin Cng ngh inh h
1_2 - 3_1 * ,77
1_2 - 4_1 * ,97
1_3 - 1_4 ,11
1_3 - 2_1 ,11
1_3 - 3_1 ,176667
1_3 - 4_1 ,376667
1_4 - 2_1
1_4 - 3_1 ,0666667
1_4 - 4_1 ,266667
2_1 - 3_1 ,0666667
2_1 - 4_1 ,266667
3_1 - 4_1 ,2
CV=0,469667
1_1
1_2
1_3
1_4
2_1
3_1
4_1
6,9 7,2 7,5 7,8 8,1 8,4 8,7
PME activity
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Hn PL36: t Box Wisker biu din n ng ca t l kt p R
1
v So
2

n ot tn PME

Chuyn ngnh i sinh vt h lxxvii in Nghin u v Pht trin Cng ngh inh h
Phn 2 Xc nh iu kin ln men v ly trch enzme PME thch hp t s kt
hp 2 dng A. niger R
1
v So
2
c hiu

- thy i hot tnh PME t l kt hp kh nhu b to t v v
qu m/bi
One-Way ANOVA - Hoat tinh PME by Co chat ket hop
Dependent variable: Hoat tinh PME (U/g)
Factor: Co chat ket hop

ANOVA Table for Hoat tinh PME by Co chat ket hop
Between groups 424,461 12 35,3718 155,12 0,00000
Within groups 5,92873 26 ,228028
Total (Corr.) 430,39 38

Multiple Range Tests for Hoat tinh PME by Co chat ket hop

Co chat ket hop Count Mean Homogeneous Groups
Orange peel 3 1,19667 X
Pomelo peel 3 1,9 X
Apple pomace 3 4,83 X
A8_O2 3 6,63 X
A4_O6 3 7,13 XX
A5_O5 3 7,77 XX
A4_P6 3 8,29 XX
A6_O4 3 8,74 X
A8_P2 3 8,85 X
A7_O3 3 9,8 X
A7_P3 3 10,34 XX
A6_P4 3 11,14 X
A5_P5 3 13,1667 X
A4_O6 - A4_P6 * -1,16
A4_O6 - A5_O5 -,64
A4_O6 - A5_P5 * -6,03667
A4_O6 - A6_O4 * -1,61
A4_O6 - A6_P4 * -4,01
A4_O6 - A7_O3 * -2,67
A4_O6 - A7_P3 * -3,21
A4_O6 - A8_O2 ,5
A4_O6 - A8_P2 * -1,72
A4_O6 - Apple pomace * 2,3
A4_O6 - Orange peel * 5,93333
A4_O6 - Pomelo peel * 5,23
A4_P6 - A5_O5 ,52
A4_P6 - A5_P5 * -4,87667
A4_P6 - A6_O4 -,45
A4_P6 - A6_P4 * -2,85
A4_P6 - A7_O3 * -1,51
A4_P6 - A7_P3 * -2,05
A4_P6 - A8_O2 * 1,66
A4_P6 - A8_P2 -,56
A4_P6 - Apple pomace * 3,46
A4_P6 - Orange peel * 7,09333
A4_P6 - Pomelo peel * 6,39
Chuyn ngnh i sinh vt h lxxviii in Nghin u v Pht trin Cng ngh inh h
A5_O5 - A5_P5 * -5,39667
A5_O5 - A6_O4 * -,97
A5_O5 - A6_P4 * -3,37
A5_O5 - A7_O3 * -2,03
A5_O5 - A7_P3 * -2,57
A5_O5 - A8_O2 * 1,14
A5_O5 - A8_P2 * -1,08
A5_P5 - A6_O4 * 4,42667
A5_P5 - A6_P4 * 2,02667
A5_P5 - A7_O3 * 3,36667
A5_P5 - A7_P3 * 2,82667
A5_P5 - A8_O2 * 6,53667
A5_P5 - A8_P2 * 4,31667
A6_O4 - A6_P4 * -2,4
A6_O4 - A7_O3 * -1,06
A6_O4 - A7_P3 * -1,6
A6_O4 - A8_O2 * 2,11
A6_O4 - A8_P2 -,11
A6_P4 - A7_O3 * 1,34
A6_P4 - A7_P3 ,8
A6_P4 - A8_O2 * 4,51
A6_P4 - A8_P2 * 2,29
A7_O3 - A7_P3 -,54
A7_O3 - A8_O2 * 3,17
A7_O3 - A8_P2 * ,95
A7_P3 - A8_O2 * 3,71
A7_P3 - A8_P2 * 1,49
A8_O2 - A8_P2 * -2,22
Apple pomace - Orange peel * 3,63333
Apple pomace - Pomelo peel * 2,93
Orange peel - Pomelo peel -,703333

Chuyn ngnh i sinh vt h lxxix in Nghin u v Pht trin Cng ngh inh h
+nh hng nng v loi khong b sung n s thy i hot tnh
PME t A. niger R
1
v So
2
trn ht b to t v v bi Nm Roi
One-Way ANOVA - PME activity by Khoang bo sung
Factor: Khoang bo sung


ANOVA Table for PME activity by Khoang bo sung
Between groups 727,183 12 60,5986 182,80 0,00000
Within groups 8,61887 26 ,331495
Total (Corr.) 735,802 38
Multiple Range Tests for PME activity by Khoang bo sung

Khoang bo sung Count Mean Homogeneous Groups
KCl 2% 3 2,87 X
MgCl
2
0,25 3 4,05 X
Control 3 12,87 X
KCl 0,5% 3 13,23 XX
MgCl
2
1% 3 13,72 XX
KCl 1% 3 13,83 XX
CaCl
2
0,5% 3 14,06 XX
CaCl
2
1% 3 14,9 XX
CaCl
2
2% 3 15,2267 X
MgCl
2
0,75 3 15,47 X
MgCl
2
0,5 3 16,55 X
KCl 1,5% 3 16,87 X
CaCl
2
1,5% 3 16,9 X

CaCl
2
0,5% - CaCl
2
1% -,84
CaCl
2
0,5% - CaCl
2
1,5% * -2,84
CaCl
2
0,5% - CaCl
2
2% * -1,16667
CaCl
2
0,5% - Control * 1,19
CaCl
2
0,5% - KCl 0,5% ,83
CaCl
2
0,5% - KCl 1% ,23
CaCl
2
0,5% - KCl 1,5% * -2,81
CaCl
2
0,5% - KCl 2% * 11,19
CaCl
2
0,5% - MgCl
2
0,25 * 10,01
CaCl
2
0,5% - MgCl
2
0,5 * -2,49
CaCl
2
0,5% - MgCl
2
0,75 * -1,41
CaCl
2
0,5% - MgCl
2
1% ,34
CaCl
2
1% - CaCl
2
1,5% * -2,0
CaCl
2
1% - CaCl
2
2% -,326667
CaCl
2
1% - Control * 2,03
CaCl
2
1% - KCl 0,5% * 1,67
CaCl
2
1% - KCl 1% * 1,07
CaCl
2
1% - KCl 1,5% * -1,97
CaCl
2
1% - KCl 2% * 12,03
CaCl
2
1% - MgCl
2
0,25 * 10,85
CaCl
2
1% - MgCl
2
0,5 * -1,65
CaCl
2
1% - MgCl
2
0,75 -,57
CaCl
2
1% - MgCl
2
1% * 1,18
CaCl
2
1,5% - CaCl
2
2% * 1,67333
CaCl
2
1,5% - Control * 4,03
CaCl
2
1,5% - KCl 0,5% * 3,67
Chuyn ngnh i sinh vt h lxxx in Nghin u v Pht trin Cng ngh inh h
CaCl
2
1,5% - KCl 1% * 3,07
CaCl
2
1,5% - KCl 1,5% ,03
CaCl
2
1,5% - KCl 2% * 14,03
CaCl
2
1,5% - MgCl
2
0,25 * 12,85
CaCl
2
1,5% - MgCl
2
0,5 ,35
CaCl
2
1,5% - MgCl
2
0,75 * 1,43
CaCl
2
1,5% - MgCl
2
1% * 3,18
CaCl
2
2% - Control * 2,35667
CaCl
2
2% - KCl 0,5% * 1,99667
CaCl
2
2% - KCl 1% * 1,39667
CaCl
2
2% - KCl 1,5% * -1,64333
CaCl
2
2% - KCl 2% * 12,3567
CaCl
2
2% - MgCl
2
0,25 * 11,1767
CaCl
2
2% - MgCl
2
0,5 * -1,32333
CaCl
2
2% - MgCl
2
0,75 -,243333
CaCl
2
2% - MgCl
2
1% * 1,50667
Control - KCl 0,5% -,36
Control - KCl 1% -,96
Control - KCl 1,5% * -4,0
Control - KCl 2% * 10,0
Control MgCl
2
0,25 * 8,82
Control MgCl
2
0,5 * -3,68
Control MgCl
2
0,75 * -2,6
Control MgCl
2
1% -,85
KCl 0,5% - KCl 1% -,6
KCl 0,5% - KCl 1,5% * -3,64
KCl 0,5% - KCl 2% * 10,36
KCl 0,5% - MgCl
2
0,25 * 9,18
KCl 0,5% - MgCl
2
0,5 * -3,32
KCl 0,5% - MgCl
2
0,75 * -2,24
KCl 0,5% - MgCl
2
1% -,49
KCl 1% - KCl 1,5% * -3,04
KCl 1% - KCl 2% * 10,96
KCl 1% - MgCl
2
0,25 * 9,78
KCl 1% - MgCl
2
0,5 * -2,72
KCl 1% - MgCl
2
0,75 * -1,64
KCl 1% - MgCl
2
1% ,11
KCl 1,5% - KCl 2% * 14,0
KCl 1,5% - MgCl
2
0,25 * 12,82
KCl 1,5% - MgCl
2
0,5 ,32
KCl 1,5% - MgCl
2
0,75 * 1,4
KCl 1,5% - MgCl
2
1% * 3,15
KCl 2% - MgCl
2
0,25 * -1,18
KCl 2% - MgCl
2
0,5 * -13,68
KCl 2% - MgCl
2
0,75 * -12,6
KCl 2% - MgCl
2
1% * -10,85
MgCl
2
0,25 MgCl
2
0,5 * -12,5
MgCl
2
0,25 MgCl
2
0,75 * -11,42
MgCl
2
0,25 MgCl
2
1% * -9,67
MgCl
2
0,5 MgCl
2
0,75 * 1,08
MgCl
2
0,5 MgCl
2
1% * 2,83
MgCl
2
0,75 MgCl
2
1% * 1,75

Chuyn ngnh i sinh vt h lxxxi in Nghin u v Pht trin Cng ngh inh h
- nh hng vi b sung kt hp loi khong n s thy i hot tnh
PME t A. niger R
1
v So
2
trn ht b to t v v bi Nm Roi
One-Way ANOVA - PME activity by Khoang ket hop
Factor: Khoang ket hop

Number of levels: 5

ANOVA Table for PME activity by Khoang ket hop

Between groups 134,883 4 33,7208 71,98 0,00000
Total (Corr.) 139,568 14

Multiple Range Tests for PME activity by Khoang ket hop

Method: 95,0 percent LSD
Khoang ket hop Count Mean Homogeneous Groups
Control 3 12,87 X
K_Mg_Ca 3 19,14 X
K_Ca 3 19,44 X
K_Mg 3 20,16 X
Mg_Ca 3 21,55 X

Control - K_Ca * -6,57 1,24524
Control - K_Mg * -7,29 1,24524
Control - K_Mg_Ca * -6,27 1,24524
Control - Mg_Ca * -8,68 1,24524
K_Ca - K_Mg -,72 1,24524
K_Ca - K_Mg_Ca ,3 1,24524
K_Ca - Mg_Ca * -2,11 1,24524
K_Mg - K_Mg_Ca 1,02 1,24524
K_Mg - Mg_Ca * -1,39 1,24524
K_Mg_Ca - Mg_Ca * -2,41 1,24524

- nh hng hm lng v ngun nitrogen b sung n hot tnh PME t
qu trnh sinh tng hp A. niger o2 v R1 iu kin ln men rn

One-Way ANOVA - Hoat tinh PME by Nitrogen bo sung
Dependent variable: Hoat tinh PME
Factor: Nitrogen bo sung

ANOVA Table for Hoat tinh PME by Nitrogen bo sung
Between groups 129,821 12 10,8184 20,77 0,0000
Within groups 13,5457 26 ,520987
Total (Corr.) 143,367 38

Chuyn ngnh i sinh vt h lxxxii in Nghin u v Pht trin Cng ngh inh h
Multiple Range Tests for Hoat tinh PME by Nitrogen bo sung
Control 3 21,55 X
NH
4
H
2
PO
4
0,05 3 21,65 X
(NH
4
)
2
SO
4
0,05 3 23,16 X
NH
4
H
2
PO
4
0,2 3 23,33 X
Urea 0,2 3 23,46 X
NH
4
H
2
PO
4
0,1 3 23,59 X
(NH
4
)
2
SO
4
0,1 3 25,27 X
Urea 0,05 3 25,66 X
NH
4
H
2
PO
4
0,15 3 25,79 X
Urea 0,15 3 25,82 X
(NH
4
)
2
SO
4
0,2 3 25,93 X
(NH
4
)
2
SO
4
0,15 3 26,14 X
Urea 0,1 3 27,8667 X
(NH
4
)
2
SO
4
0,05 - (NH
4
)
2
SO
4
0,1 * -2,11
(NH
4
)
2
SO
4
0,05 - (NH
4
)
2
SO
4
0,15 * -2,98
(NH
4
)
2
SO
4
0,05 - (NH
4
)
2
SO
4
0,2 * -2,77
(NH
4
)
2
SO
4
0,05 Control * 1,61
(NH
4
)
2
SO
4
0,05 - NH
4
H
2
PO
4
0,05 * 1,51
(NH
4
)
2
SO
4
0,05 - NH
4
H
2
PO
4
0,1 -,43
(NH
4
)
2
SO
4
0,05 - NH
4
H
2
PO
4
0,15 * -2,63
(NH
4
)
2
SO
4
0,05 - NH
4
H
2
PO
4
0,2 -,17
(NH
4
)
2
SO
4
0,05 - Urea 0,05 * -2,5
(NH
4
)
2
SO
4
0,05 - Urea 0,1 * -4,70667
(NH
4
)
2
SO
4
0,05 - Urea 0,15 * -2,66
(NH
4
)
2
SO
4
0,05 - Urea 0,2 -,3
(NH
4
)
2
SO
4
0,1 - (NH
4
)
2
SO
4
0,15 -,87
(NH
4
)
2
SO
4
0,1 - (NH
4
)
2
SO
4
0,2 -,66
(NH
4
)
2
SO
4
0,1 Control * 3,72
(NH
4
)
2
SO
4
0,1 - NH
4
H
2
PO
4
0,05 * 3,62
(NH
4
)
2
SO
4
0,1 - NH
4
H
2
PO
4
0,1 * 1,68
(NH
4
)
2
SO
4
0,1 - NH
4
H
2
PO
4
0,15 -,52
(NH
4
)
2
SO
4
0,1 - NH
4
H
2
PO
4
0,2 * 1,94
(NH
4
)
2
SO
4
0,1 - Urea 0,05 -,39
(NH
4
)
2
SO
4
0,1 - Urea 0,1 * -2,59667
(NH
4
)
2
SO
4
0,1 - Urea 0,15 -,55
(NH
4
)
2
SO
4
0,1 - Urea 0,2 * 1,81
(NH
4
)
2
SO
4
0,15 - (NH
4
)
2
SO
4
0,2 ,21
(NH
4
)
2
SO
4
0,15 Control * 4,59
(NH
4
)
2
SO
4
0,15 - NH
4
H
2
PO
4
0,05 * 4,49
(NH
4
)
2
SO
4
0,15 - NH
4
H
2
PO
4
0,1 * 2,55
(NH
4
)
2
SO
4
0,15 - NH
4
H
2
PO
4
0,15 ,35
(NH
4
)
2
SO
4
0,15 - NH
4
H
2
PO
4
0,2 * 2,81
(NH
4
)
2
SO
4
0,15 - Urea 0,05 ,48
(NH
4
)
2
SO
4
0,15 - Urea 0,1 * -1,72667
(NH
4
)
2
SO
4
0,15 - Urea 0,15 ,32
(NH
4
)
2
SO
4
0,15 - Urea 0,2 * 2,68
(NH
4
)
2
SO
4
0,2 - Control * 4,38
(NH
4
)
2
SO
4
0,2 - NH
4
H
2
PO
4
0,05 * 4,28
(NH
4
)
2
SO
4
0,2 - NH
4
H
2
PO
4
0,1 * 2,34
(NH
4
)
2
SO
4
0,2 - NH
4
H
2
PO
4
0,15 ,14
(NH
4
)
2
SO
4
0,2 - NH
4
H
2
PO
4
0,2 * 2,6
(NH
4
)
2
SO
4
0,2 - Urea 0,05 ,27
(NH
4
)
2
SO
4
0,2 - Urea 0,1 * -1,93667
(NH
4
)
2
SO
4
0,2 - Urea 0,15 ,11
(NH
4
)
2
SO
4
0,2 - Urea 0,2 * 2,47
Control - NH
4
H
2
PO
4
0,05 -,1
Chuyn ngnh i sinh vt h lxxxiii in Nghin u v Pht trin Cng ngh inh h
Control - NH
4
H
2
PO
4
0,1 * -2,04
Control - NH
4
H
2
PO
4
0,15 * -4,24
Control - NH
4
H
2
PO
4
0,2 * -1,78
Control - Urea 0,05 * -4,11
Control - Urea 0,1 * -6,31667
NH
4
H
2
PO
4
0,05 - NH
4
H
2
PO
4
0,1 * -1,94
NH
4
H
2
PO
4
0,05 - NH
4
H
2
PO
4
0,15 * -4,14
NH
4
H
2
PO
4
0,05 - NH
4
H
2
PO
4
0,2 * -1,68
NH
4
H
2
PO
4
0,05 - Urea 0,05 * -4,01
NH
4
H
2
PO
4
0,05 - Urea 0,1 * -6,21667
NH
4
H
2
PO
4
0,05 - Urea 0,15 * -4,17
NH
4
H
2
PO
4
0,05 - Urea 0,2 * -1,81
NH
4
H
2
PO
4
0,1 - NH
4
H
2
PO
4
0,15 * -2,2
NH
4
H
2
PO
4
0,1 - NH
4
H
2
PO
4
0,2 ,26
NH
4
H
2
PO
4
0,1 - Urea 0,05 * -2,07
NH
4
H
2
PO
4
0,1 - Urea 0,1 * -4,27667
NH
4
H
2
PO
4
0,1 - Urea 0,15 * -2,23
NH
4
H
2
PO
4
0,1 - Urea 0,2 ,13
NH
4
H
2
PO
4
0,15 - NH
4
H
2
PO
4
0,2 * 2,46
NH
4
H
2
PO
4
0,15 - Urea 0,05 ,13
NH
4
H
2
PO
4
0,15 - Urea 0,1 * -2,07667
NH
4
H
2
PO
4
0,15 - Urea 0,15 -,03
NH
4
H
2
PO
4
0,15 - Urea 0,2 * 2,33
NH
4
H
2
PO
4
0,2 - Urea 0,05 * -2,33
NH
4
H
2
PO
4
0,2 - Urea 0,1 * -4,53667
NH
4
H
2
PO
4
0,2 - Urea 0,15 * -2,49
NH
4
H
2
PO
4
0,2 - Urea 0,2 -,13
Urea 0,05 - Urea 0,1 * -2,20667
Urea 0,05 - Urea 0,15 -,16
Urea 0,05 - Urea 0,2 * 2,2
Urea 0,1 - Urea 0,15 * 2,04667
Urea 0,1 - Urea 0,2 * 4,40667
Urea 0,15 - Urea 0,2 * 2,36

- nh hng loi dung dh m (pH 4) n hot tnh PME t qu trnh
sinh tng hp A. niger o2 v R1 iu kin ln men rn

One-Way ANOVA - Hoat tinh PME by pH buffer
Dependent variable: Hoat tinh PME
Factor: pH buffer
Number of levels: 4

ANOVA Table for Hoat tinh PME by pH buffer
Between groups 649,189 3 216,396 111,54 0,00000
Within groups 15,52 8 1,94
Total (Corr.) 664,709 11

Multiple Range Tests for Hoat tinh PME by pH buffer

pH buffer Count Mean Homogeneous Groups
acetate pH 4 3 22,4333 X
citrate phosphate pH 4 3 24,1 X
Nuoc DW pH6,7 3 27,8667 X
citrate pH 4 3 41,1667 X
Chuyn ngnh i sinh vt h lxxxiv in Nghin u v Pht trin Cng ngh inh h
Nuoc DW pH6,7 - acetate pH 4 * 5,43333 2,62251
Nuoc DW pH6,7 - citrate pH 4 * -13,3 2,62251
Nuoc DW pH6,7 - citrate phosphate pH 4 * 3,76667 2,62251
acetate pH 4 - citrate pH 4 * -18,7333 2,62251
acetate pH 4 - citrate phosphate pH 4 -1,66667 2,62251
citrate pH 4 - citrate phosphate pH 4 * 17,0667 2,62251

- thy i hot tnh PME v thnh phn mi trng theo thi gin
+ Hot tnh PME
One-Way ANOVA - PME activity by Time_h
Factor: Time_h

Number of levels: 6
ANOVA Table for PME activity by Time_h
Between groups 3783,31 5 756,661 257,66 0,0000
Within groups 35,2405 12 2,93671
Total (Corr.) 3818,55 17

Multiple Range Tests for PME activity by Time_h
Time_h Count Mean Homogeneous Groups
0 3 0 X
24 4 ,3575 X
120 3 12,8 X
48 2 15,21 X
72 3 22,88 X
96 3 41,16 X

0 - 24 -,3575 2,85174
0 - 48 * -15,21 3,40848
0 - 72 * -22,88 3,04864
0 - 96 * -41,16 3,04864
0 - 120 * -12,8 3,04864
24 - 48 * -14,8525 3,23357
24 - 72 * -22,5225 2,85174
24 - 96 * -40,8025 2,85174
24 - 120 * -12,4425 2,85174
48 - 72 * -7,67 3,40848
48 - 96 * -25,95 3,40848
48 - 120 2,41 3,40848
72 - 96 * -18,28 3,04864
72 - 120 * 10,08 3,04864
96 - 120 * 28,36 3,04864

+ Hm lng pectin (%)
One-Way ANOVA - Pectin % by Time_h
Dependent variable: Pectin %
Factor: Time_h
Number of levels: 6

Chuyn ngnh i sinh vt h lxxxv in Nghin u v Pht trin Cng ngh inh h
ANOVA Table for Pectin % by Time_h
Between groups 719,74 5 143,948 73,48 0,0000
Within groups 23,5083 12 1,95902
Total (Corr.) 743,249 17

Multiple Range Tests for Pectin % by Time_h
120 3 0 X
96 3 1,67 XX
72 3 3,78 XX
48 2 5,61 X
24 4 12,255 X
0 3 17,69 X

0 - 24 * 5,435
0 - 48 * 12,08
0 - 72 * 13,91
0 - 96 * 16,02
0 - 120 * 17,69
24 - 48 * 6,645
24 - 72 * 8,475
24 - 96 * 10,585
24 - 120 * 12,255
48 - 72 1,83
48 - 96 * 3,94
48 - 120 * 5,61
72 - 96 2,11
72 - 120 * 3,78
96 - 120 1,67

+ ester ha ca pectin, DE (%)
One-Way ANOVA - DE % by Time_h
Dependent variable: DE %
Factor: Time_h
Number of levels: 6

ANOVA Table for DE % by Time_h
Between groups 1185,54 5 237,108 956,05 0,0000
Within groups 2,9761 12 ,248008
Total (Corr.) 1188,52 17

Multiple Range Tests for DE % by Time_h
120 3 55,33 X
96 3 57,63 X
72 3 70,46 X
48 2 73,58 X
24 4 74,375 XX
0 3 74,82 X
0 - 24 ,445
0 - 48 * 1,24
0 - 72 * 4,36
Chuyn ngnh i sinh vt h lxxxvi in Nghin u v Pht trin Cng ngh inh h
0 - 96 * 17,19
0 - 120 * 19,49
24 - 48 ,795
24 - 72 * 3,915
24 - 96 * 16,745
24 - 120 * 19,045
48 - 72 * 3,12
48 - 96 * 15,95
48 - 120 * 18,25
72 - 96 * 12,83
72 - 120 * 15,13
96 - 120 * 2,3

+ S thay i pH
One-Way ANOVA - pH by Time_h
Dependent variable: pH
Factor: Time_h

Number of levels: 6
ANOVA Table for pH by Time_h
Between groups 4,71403 5 ,942806 39,92 0,0000
Within groups ,2834 12 ,0236167
Total (Corr.) 4,99743 17

Multiple Range Tests for pH by Time_h

120 3 2,86 X
96 3 2,93 X
72 3 2,96 X
48 2 3,28 X
24 4 3,86 X
0 3 4,16 X

0 - 24 * ,3
0 - 48 * ,88
0 - 72 * 1,2
0 - 96 * 1,23
0 - 120 * 1,3
24 - 48 * ,58
24 - 72 * ,9
24 - 96 * ,93
24 - 120 * 1,0
48 - 72 * ,32
48 - 96 * ,35
48 - 120 * ,42
72 - 96 ,03
72 - 120 ,1
96 - 120 ,07
Chuyn ngnh i sinh vt h lxxxvii in Nghin u v Pht trin Cng ngh inh h
+ m (%)
One-Way ANOVA - Moist % by Time_h
Dependent variable: Moist %
Factor: Time_h
Number of levels: 6

ANOVA Table for Moist % by Time_h
Between groups 207,421 5 41,4842 69,48 , 0
Within groups 7,16467 12 ,597056
Total (Corr.) 214,585 17

Multiple Range Tests for Moist % by Time_h

0 3 60,07 X
24 4 64,2925 X
48 2 65,15 X
72 3 68,54 X
96 3 69,3 X
120 3 69,51 X
0 - 24 * -4,2225
0 - 48 * -5,08
0 - 72 * -8,47
0 - 96 * -9,23
0 - 120 * -9,44
24 - 48 -,8575
24 - 72 * -4,2475
24 - 96 * -5,0075
24 - 120 * -5,2175
48 - 72 * -3,39
48 - 96 * -4,15
48 - 120 * -4,36
72 - 96 -,76
72 - 120 -,97
96 - 120 -,21

- i u h qu trnh ln men (pH, nhit , m mi trng, t l nm m)
bng phng php b mt p ng
+ Thit k th nghim
Response Surface Design Attributes
Design class: Response Surface
Design name: Central composite design: 2^4 + star
Design characteristic: Rotatable
File name: <Untitled>
Comment: Optimal fermentation parameters of A.niger PME

Base Design
Number of experimental factors: 4
Number of blocks: 1
Number of responses: 1
Number of runs: 31, including 7 centerpoints per block
Error degrees of freedom: 16
Randomized: Yes
Chuyn ngnh i sinh vt h lxxxviii in Nghin u v Pht trin Cng ngh inh h
Factors Low High Units Continuous
Mold 12 20 %v/w Yes
pH 3 5 Yes
Temp 28 40
o
C Yes
Moist 50 70 % Yes
Responses Units
PME U/g

Analyze Experiment - PME
File name: F:\Luan an\New Folder\Toi uu hoa DK len men.sfx
Comment: Optimal fermentation parameters of A.niger PME

Estimated effects for PME (U/mL)
Effect Estimate Confidence Int. V.I.F.
Average 62,8429 +/- 2,82633
A:Mold 4,98333 +/- 6,10557 1,0
B:pH 6,01667 +/- 6,10557 1,0
C:Temp 30,0167 +/- 6,10557 1,0
D:Moist -16,8167 +/- 6,10557 1,0
AA -53,6107 +/- 11,1869 1,0288
AB -24,95 +/- 14,9555 1,0
AC 17,15 +/- 14,9555 1,0
AD -16,05 +/- 14,9555 1,0
BB -96,6107 +/- 11,1869 1,0288
BC 13,25 +/- 14,9555 1,0
BD 26,25 +/- 14,9555 1,0
CC -74,3107 +/- 11,1869 1,0288
CD -21,65 +/- 14,9555 1,0
DD -46,8107 +/- 11,1869 1,0288
99,0 confidence intervals are based on total error with 16 d.f. (t = 2,92079)

Analysis of Variance for PME - Optimal fermentation parameters of A. niger PME
A:Mold 37,2504 1 37,2504 5,68 0, 299
B:pH 54,3004 1 54,3004 8,28 0, 109
C:Temp 1351,5 1 1351,5 206,19 0, 00
D:Moist 424,2 1 424,2 64,72 0, 00
AA 1284,18 1 1284,18 195,92 0, 00
AB 155,626 1 155,626 23,74 0, 02
AC 73,5306 1 73,5306 11,22 0, 0 41
AD 64,4006 1 64,4006 9,83 0, 0 064
BB 4170,35 1 4170,35 636,25 0, 00
BC 43,8906 1 43,8906 6,70 0, 0198
BD 172,266 1 172,266 26,28 0, 01
CC 2467,31 1 2467,31 376,43 0, 00
CD 117,181 1 117,181 17,88 0, 06
DD 979,066 1 979,066 149,37 0, 00
Total error 104,873 16 6,55456
Total (corr.) 9590,72 30

R-squared = 98,9065 percent
R-squared (adjusted for d.f.) = 97,9497 percent
Standard Error of Est. = 2,56019

Chuyn ngnh i sinh vt h lxxxix in Nghin u v Pht trin Cng ngh inh h
Regression coeffs. for PME - Optimal fermentation parameters of A. niger PME
Coefficient Estimate
constant -3331,94
A:Mold 66,5982
B:pH 323,06
C:Temp 73,3754
D:Moist 31,3398
AA -1,67533
AB -3,11875
AC ,357292
AD -,200625
BB -48,3054
BC 1,10417
BD 1,3125
CC -1,03209
CD -,180417
DD -,234054
The StatAdvisor
This pane displays the regression equation which has been fitted to the data. The equation of the fitted model is
PME = -3331,94 + 66,5982*Mold + 323,06*pH + 73,3754*Temp + 31,3398*Moist - 1,67533*Mold^2 -
3,11875*Mold*pH + ,357292*Mold*Temp - ,200625*Mold*Moist - 48,3054*pH^2 + 1,10417*pH*Temp +
1,3125*pH*Moist - 1,03209*Temp^2 - ,180417*Temp*Moist - ,234054*Moist^2
where the values of the variables are specified in their original units. To have STATGRAPHICS evaluate this function,
select Predictions from the list of Tabular Options.
Estimation Results for PME
Observed Fitted Lower 95,0% CL Upper 95,0% CL
Row Value Value for Mean for Mean
1 46,5 45,4958 41,3506 49,6411
2 24,5 22,125 17,9798 26,2702
3 31,4 34,9458 30,8006 39,0911
4 53,3 51,975 47,8298 56,1202
5 25,0 27,5792 23,4339 31,7244
6 62,5 62,8429 60,7915 64,8942
7 50,0 47,8458 43,7006 51,9911
8 33,8 33,5458 29,4006 37,6911
9 63,0 62,8429 60,7915 64,8942
10 63,5 62,8429 60,7915 64,8942
11 27,1 29,075 24,9298 33,2202
12 63,0 62,8429 60,7915 64,8942
13 39,5 37,6958 33,5506 41,8411
14 62,0 62,8429 60,7915 64,8942
15 22,5 21,4958 17,3506 25,6411
16 18,7 17,5458 13,4006 21,6911
17 62,7 62,8429 60,7915 64,8942
18 18,6 17,0083 12,8631 21,1536
19 31,2 34,225 30,0798 38,3702
20 14,0 16,5792 12,4339 20,7244
21 38,7 40,9417 36,7964 45,0869
22 11,6 10,6792 6,53395 14,8244
23 12,6 11,5292 7,38395 15,6744
24 40,5 38,5292 34,3839 42,6744
25 14,4 17,475 13,3298 21,6202
26 31,1 31,0292 26,8839 35,1744
27 63,2 62,8429 60,7915 64,8942
28 27,2 30,5792 26,4339 34,7244
29 19,0 17,0292 12,8839 21,1744
30 21,0 18,575 14,4298 22,7202
31 42,0 40,6958 36,5506 44,8411
The StatAdvisor
This table contains information about values of PME generated using the fitted model. The table includes:
(1) the observed value of PME (if any)
(2) the predicted value of PME using the fitted model
(3) 95,0% confidence limits for the mean response

Chuyn ngnh i sinh vt h xc in Nghin u v Pht trin Cng ngh inh h
Optimize Response
Goal: maximize PME
Optimum value = 66,0029

Factor Low High Optimum
Mold 12,0 20,0 16,5083
pH 3,0 5,0 3,9968
Temp 28,0 40,0 35,526
Moist 50,0 70,0 57,3878

- iu kin ly trch PME su qu trnh ln men rn
+ pH citrate buffer
One-Way ANOVA - PME by pH citrate buffer
Dependent variable: PME (U/g)
Factor: pH citrate buffer
Number of levels: 8

ANOVA Table for PME by pH citrate buffer
Between groups 8124,45 7 1160,64 1237,15 0,0000
Within groups 15,0104 16 ,93815
Total (Corr.) 8139,46 23

Multiple Range Tests for PME by pH citrate buffer

5,6 3 14,57 X
5,2 3 16,87 X
4,8 3 29,14 X
4,4 3 30,43 X
DW 6,7-6,8 3 30,57 X
3,2 3 42,86 X
4 3 64,14 X
3,6 3 66,71 X
3,2 - 3,6 * -23,85
3,2 - 4 * -21,28
3,2 - 4,4 * 12,43
3,2 - 4,8 * 13,72
3,2 - 5,2 * 25,99
3,2 - 5,6 * 28,29
3,2 - DW 6,7-6,8 * 12,29
3,6 - 4 * 2,57
3,6 - 4,4 * 36,28
3,6 - 4,8 * 37,57
3,6 - 5,2 * 49,84
3,6 - 5,6 * 52,14
3,6 - DW 6,7-6,8 * 36,14
4 - 4,4 * 33,71
4 - 4,8 * 35,0
4 - 5,2 * 47,27
4 - 5,6 * 49,57
4 - DW 6,7-6,8 * 33,57
4,4 - 4,8 1,29
4,4 - 5,2 * 13,56
4,4 - 5,6 * 15,86
4,4 - DW 6,7-6,8 -,14
4,8 - 5,2 * 12,27
Chuyn ngnh i sinh vt h xci in Nghin u v Pht trin Cng ngh inh h
4,8 - 5,6 * 14,57
4,8 - DW 6,7-6,8 -1,43
5,2 - 5,6 * 2,3
5,2 - DW 6,7-6,8 * -13,7
5,6 - DW 6,7-6,8 * -16,0
* denotes a statistically significant difference. CV=1,67652

+ T l ca dung dch m v c cht sau ln men
One-Way ANOVA - Tong PME by Ty le dd dem va co chat
Dependent variable: Tong PME (U)
Factor: Ty le dd dem va co chat
Number of levels: 6

ANOVA Table for Tong PME by Ty le dd dem va co chat

Between groups 155798. 5 31159.6 1083.34 0.0000
Within groups 345.15 12 28.7625
Total (Corr.) 156143. 17

Multiple Range Tests for Tong PME by Ty le dd dem va co chat
Method: 95.0 percent LSD
1 : 1 3 179.8 X
3,5 : 1 3 200.07 X
3: 1 3 278.95 X
1,5 : 1 3 289.96 X
2,5 : 1 3 415.31 X
2 : 1 3 417.12 X
3: 1 - 1 : 1 * 99.15 9.54089
3: 1 - 2 : 1 * -138.17 9.54089
3: 1 - 1,5 : 1 * -11.01 9.54089
3: 1 - 2,5 : 1 * -136.36 9.54089
3: 1 - 3,5 : 1 * 78.88 9.54089
1 : 1 - 2 : 1 * -237.32 9.54089
1 : 1 - 1,5 : 1 * -110.16 9.54089
1 : 1 - 2,5 : 1 * -235.51 9.54089
1 : 1 - 3,5 : 1 * -20.27 9.54089
2 : 1 - 1,5 : 1 * 127.16 9.54089
2 : 1 - 2,5 : 1 1.81 9.54089
2 : 1 - 3,5 : 1 * 217.05 9.54089
1,5 : 1 - 2,5 : 1 * -125.35 9.54089
1,5 : 1 - 3,5 : 1 * 89.89 9.54089
2,5 : 1 - 3,5 : 1 * 215.24 9.54089

+ Khi lng m trch
One-Way ANOVA - Hoat tinh PME by KL me co chat
Dependent variable: Hoat tinh PME (U/g co chat)
Factor: KL me co chat
Number of levels: 3

ANOVA Table for Hoat tinh PME by KL me co chat
Between groups 34.2734 2 17.1367 1.04 0.4107
Within groups 99.258 6 16.543
Total (Corr.) 133.531 8
Chuyn ngnh i sinh vt h xcii in Nghin u v Pht trin Cng ngh inh h
Multiple Range Tests for Hoat tinh PME by KL me co chat
KL me co chat Count Mean Homogeneous Groups
50 3 137,563 X
100 3 138,31 X
10 3 139,153 X
10 - 50 1,59 11,3285
10 - 100 ,843333 11,3285
50 - 100 -,746667 11,3285

- hng k s n nh hot tnh PME th su ly trch trong qu trnh bo qun lnh
v lnh ng
+ Bo qun lnh
One-Way ANOVA - Hoat tinh PME duy tri (A/Ao) by Thoi gian BQ lanh
Dependent variable: Hoat tinh PME duy tri (A/Ao)
Factor: Thoi gian BQ lanh (ngy)
Number of levels: 5

ANOVA Table for Hoat tinh PME duy tri (A/Ao) by Thoi gian BQ lanh
Between groups 3,74719 4 0,936798 321,10 0,0000
Within groups 0,0145872 5 0,00291744
Total (Corr.) 3,76178 9

Multiple Range Tests for A/Ao by Thoi gian BQ lanh
4 2 0 X
3 2 0,155103 X
2 2 0,289916 X
0 2 1,0 X
1 2 1,62987 X
0 - 1 * -,62987
0 - 2 * ,710084
0 - 3 * ,844897
0 - 4 * 1,0
1 - 2 * 1,33995
1 - 3 * 1,47477
1 - 4 * 1,62987
2 - 3 ,134813
2 - 4 * ,289916
3 - 4 * ,155103
+ Bo qun lnh ng
One-Way ANOVA - Hoat tinh PME duy tri by Thoi gian tru dong
Dependent variable: Hoat tinh PME duy tri
Factor: Thoi gian tru dong (Ngay)
Number of levels: 8
ANOVA Table for Hoat tinh PME duy tri (A/Ao) by Thoi gian tru dong
Between groups 10,6438 7 1,52055 19,01 0,0000
Within groups 1,2796 16 0,079975
Total (Corr.) 11,9234 23
Chuyn ngnh i sinh vt h xciii in Nghin u v Pht trin Cng ngh inh h
Multiple Range Tests for Hoat tinh PME duy tri (A/Ao) by Thoi gian tru dong
Method: 95.0 percent Duncan
0 3 1,0 X
13 3 1,31 X
3 3 1,34 X
1 3 1,39 X
11 3 2,12 X
7 3 2,22 X
9 3 2,53 X
5 3 3,03 X
Contrast Sig, Difference
0 - 1 -0,39
0 - 11 <* -1,12
0 - 13 -0,31
0 - 3 -0,34
0 - 5 <* -2,03
0 - 7 <* -1,22
0 - 9 <* -1,53
1 - 11 <* -0,73
1 - 13 0,08
1 - 3 0,05
1 - 5 <* -1,64
1 - 7 <* -0,83
1 - 9 <* -1,14
11 - 13 <* 0,81
11 - 3 <* 0,78
11 - 5 <* -0,91
11 - 7 -0,1
11 - 9 -0,41
13 - 3 -0,03
13 - 5 <* -1,72
13 - 7 <* -0,91
13 - 9 <* -1,22
3 - 5 <* -1,69
3 - 7 <* -0,88
3 - 9 <* -1,19
5 - 7 <* 0,81
5 - 9 <* 0,5
7 - 9 -0,31

Chuyn ngnh i sinh vt h xciv in Nghin u v Pht trin Cng ngh inh h
Phn 3: Tinh sch s b v xc nh c im PME
- Kt t bng mui (NH
4
)
2
SO
4

One-Way ANOVA - Hoat tinh rieng S by (NH
4
)
2
SO
4
fraction
Dependent variable: Hoat tinh rieng S (U/mg protein)
Factor: (NH
4
)
2
SO
4
fraction

Number of levels: 7

ANOVA Table for Hoat tinh rieng S by (NH
4
)
2
SO
4
fraction
Between groups 45,1257 6 7,52095 75,71 0,0000
Within groups ,695373 7 ,0993389
Total (Corr.) 45,8211 13

Multiple Range Tests for Hoat tinh rieng S by ((NH
4
)
2
SO
4
fraction
Culture filtrate 2 1,26295 X
35-45% 2 1,87092 X
45-55% 2 2,97866 X
85-95% 2 3,66293 X
75-85% 2 4,58242 X
65-75% 2 5,1077 X
55-65% 2 6,86587 X

35-45% - 45-55% * -1,10774
35-45% - 55-65% * -4,99495
35-45% - 65-75% * -3,23678
35-45% - 75-85% * -2,7115
35-45% - 85-95% * -1,79201
35-45% - Culture filtrate ,607966
45-55% - 55-65% * -3,88721
45-55% - 65-75% * -2,12904
45-55% - 75-85% * -1,60377
45-55% - 85-95% -,684276
45-55% - Culture filtrate * 1,7157
55-65% - 65-75% * 1,75817
55-65% - 75-85% * 2,28345
55-65% - 85-95% * 3,20294
65-75% - 75-85% ,525275
65-75% - 85-95% * 1,44477
75-85% - 85-95% * ,91949

One-Way ANOVA - Hieu suat H by (NH
4
)
2
SO
4
fraction
Dependent variable: Hieu suat H (%)
Factor: (NH
4
)
2
SO
4
fraction

Number of levels: 7
Chuyn ngnh i sinh vt h xcv in Nghin u v Pht trin Cng ngh inh h
ANOVA Table for Hieu suat H by (NH
4
)
2
SO
4
fraction
Between groups 9197,69 6 1532,95 397,49 0,0000
Within groups 26,9963 7 3,85662
Total (Corr.) 9224,68 13
Multiple Range Tests for Hieu suat H by (NH
4
)
2
SO
4
fraction

85-95% 2 19,0583 X
75-85% 2 28,0269 X
35-45% 2 32,5112 XX
65-75% 2 34,7534 XX
45-55% 2 37,5561 X
55-65% 2 62,2197 X

35-45% - 45-55% * -5,04484
35-45% - 55-65% * -29,7085
35-45% - 65-75% -2,24215
35-45% - 75-85% 4,4843
35-45% - 85-95% * 13,4529
35-45% - Culture filtrate * -67,4888
45-55% - 55-65% * -24,6637
45-55% - 65-75% 2,80269
45-55% - 75-85% * 9,52915
45-55% - 85-95% * 18,4978
55-65% - 65-75% * 27,4664
55-65% - 75-85% * 34,1928
55-65% - 85-95% * 43,1614
65-75% - 75-85% * 6,72646
65-75% - 85-95% * 15,6951
75-85% - 85-95% * 8,96861

One-Way ANOVA - Do tinh sach by (NH
4
)
2
SO
4
fraction
Dependent variable: Do tinh sach (lan)
Factor: (NH
4
)
2
SO
4
fraction

Number of levels: 7

ANOVA Table for Do tinh sach by (NH
4
)
2
SO
4
fraction
Between groups 28,3588 6 4,72647 57,54 0,0000
Within groups ,574958 7 ,0821369
Total (Corr.) 28,9338 13

Chuyn ngnh i sinh vt h xcvi in Nghin u v Pht trin Cng ngh inh h
Multiple Range Tests for Do tinh sach by (NH
4
)
2
SO
4
fraction
35-45% 2 1,49029 X
45-55% 2 2,39264 X
85-95% 2 2,90562 X
75-85% 2 3,63498 X
65-75% 2 4,06127 X
55-65% 2 5,44816 X
35-45% - 45-55% * -,902347
35-45% - 55-65% * -3,95788
35-45% - 65-75% * -2,57098
35-45% - 75-85% * -2,14469
35-45% - 85-95% * -1,41533
45-55% - 55-65% * -3,05553
45-55% - 65-75% * -1,66864
45-55% - 75-85% * -1,24235
45-55% - 85-95% -,512987
55-65% - 65-75% * 1,38689
55-65% - 75-85% * 1,81318
55-65% - 85-95% * 2,54254
65-75% - 75-85% ,426292
65-75% - 85-95% * 1,15565
75-85% - 85-95% * ,729359
One-Way ANOVA - Hoat tinh rieng S by NaCl fraction
Factor: NaCl fraction
Number of levels: 6

ANOVA Table for Hoat tinh rieng S by NaCl fraction
Between groups 84,135 5 16,827 257,24 0,0000
Within groups ,392477 6 ,0654129
Total (Corr.) 84,5274 11

Multiple Range Tests for Hoat tinh rieng S by NaCl fraction
NaCl fraction Count Mean Homogeneous Groups
10-15% 2 2,05123 X
15-20% 2 4,33873 X
20-25% 2 5,95281 X
30-35% 2 7,59583 X
25-30% 2 8,36956 X

10-15% - 15-20% * -2,2875
10-15% - 20-25% * -3,90158
10-15% - 25-30% * -6,31833
10-15% - 30-35% * -5,54459
Chuyn ngnh i sinh vt h xcvii in Nghin u v Pht trin Cng ngh inh h
10-15% - Culture filtrate * ,788278
15-20% - 20-25% * -1,61408
15-20% - 25-30% * -4,03083
15-20% - 30-35% * -3,25709
20-25% - 25-30% * -2,41675
20-25% - 30-35% * -1,64301
25-30% - 30-35% * ,773735

One-Way ANOVA - Hieu suat H by NaCl fraction
Number of levels: 6

ANOVA Table for Hieu suat H by NaCl fraction
Between groups 6359,8 5 1271,96 202,68 0,0000
Within groups 37,6541 6 6,27568
Total (Corr.) 6397,46 11

Multiple Range Tests for Hieu suat H by NaCl fraction
10-15% 2 34,7534 X
15-20% 2 52,13 X
20-25% 2 78,3632 X
30-35% 2 84,9776 X
25-30% 2 92,6009 X

10-15% - 15-20% * -17,3767
10-15% - 20-25% * -43,6099
10-15% - 25-30% * -57,8475
10-15% - 30-35% * -50,2242
15-20% - 20-25% * -26,2332
15-20% - 25-30% * -40,4709
15-20% - 30-35% * -32,8475
20-25% - 25-30% * -14,2377
20-25% - 30-35% * -6,61435
25-30% - 30-35% * 7,62332

One-Way ANOVA - Do tinh sach by NaCl fraction

Number of levels: 6
Chuyn ngnh i sinh vt h xcviii in Nghin u v Pht trin Cng ngh inh h
ANOVA Table for Do tinh sach by NaCl fraction
Between groups 53,6847 5 10,7369 37,98 0,02
Within groups 1,69641 6 ,282735
Total (Corr.) 55,3811 11

Multiple Range Tests for Do tinh sach by NaCl fraction
10-15% 2 1,62273 X
15-20% 2 3,43738 X
20-25% 2 4,75571 X
30-35% 2 6,05768 X
25-30% 2 6,66546 X
10-15% - 15-20% * -1,81464
10-15% - 20-25% * -3,13298
10-15% - 25-30% * -5,04272
10-15% - 30-35% * -4,43495
15-20% - 20-25% * -1,31833
15-20% - 25-30% * -3,22808
15-20% - 30-35% * -2,62031
20-25% - 25-30% * -1,90975
20-25% - 30-35% * -1,30197
25-30% - 30-35% ,607771
* denotes a statistically significant difference.Cv=1,3011

One-Way ANOVA - Hoat tinh rieng S by Ty le ethanol
Factor: Ty le ethanol
Number of levels: 7

ANOVA Table for Hoat tinh rieng S by Ty le ethanol
Between groups 142,423 6 23,7371 407,36 0,0000
Within groups ,407898 7 ,0582711
Total (Corr.) 142,831 13

Multiple Range Tests for Hoat tinh rieng S by Ty le ethanol
Ty le ethanol Count Mean Homogeneous Groups
E 1,5 2 1,66536 X
E 4 2 2,8092 X
E 2 2 3,17351 X
E 2,5 2 5,0927 X
E 3,5 2 8,67341 X
E 3 2 10,0242 X
Culture filtrate - E 1,5 -,402407
Culture filtrate - E 2 * -1,91056
Culture filtrate - E 2,5 * -3,82974
Chuyn ngnh i sinh vt h xcix in Nghin u v Pht trin Cng ngh inh h
E 1,5 - E 2 * -1,50815
E 1,5 - E 2,5 * -3,42734
E 1,5 - E 3 * -8,35885
E 1,5 - E 3,5 * -7,00805
E 1,5 - E 4 * -1,14384
E 2 - E 2,5 * -1,91919
E 2 - E 3 * -6,8507
E 2 - E 3,5 * -5,4999
E 2 - E 4 ,364311
E 2,5 - E 3 * -4,93151
E 2,5 - E 3,5 * -3,58071
E 2,5 - E 4 * 2,2835
E 3 - E 3,5 * 1,3508
E 3 - E 4 * 7,21501
E 3,5 - E 4 * 5,86421
One-Way ANOVA - Hieu suat H by Ty le ethanol
Number of levels: 7

ANOVA Table for Hieu suat H by Ty le ethanol
Between groups 8126,54 6 1354,42 322,65 0,0000
Within groups 29,3843 7 4,19775
Total (Corr.) 8155,92 13

Multiple Range Tests for Hieu suat H by Ty le ethanol
E 1,5 2 32,9596 X
E 4 2 40,9193 X
E 2 2 51,1211 X
E 2,5 2 77,5785 X
E 3,5 2 80,2691 X
E 3 2 91,4798 X
Culture filtrate - E 1,5 * 67,0404
Culture filtrate - E 2 * 48,8789
E 1,5 - E 2 * -18,1614
E 1,5 - E 2,5 * -44,6188
E 1,5 - E 3 * -58,5202
E 1,5 - E 3,5 * -47,3094
E 1,5 - E 4 * -7,95964
E 2 - E 2,5 * -26,4574
E 2 - E 3 * -40,3587
E 2 - E 3,5 * -29,148
E 2 - E 4 * 10,2018
E 2,5 - E 3 * -13,9013
E 2,5 - E 3,5 -2,69058
E 2,5 - E 4 * 36,6592
E 3 - E 3,5 * 11,2108
E 3 - E 4 * 50,5605
E 3,5 - E 4 * 39,3498
Chuyn ngnh i sinh vt h c in Nghin u v Pht trin Cng ngh inh h
One-Way ANOVA - Do tinh sach by Ty le ethanol

Number of levels: 7

ANOVA Table for Do tinh sach by Ty le ethanol
Between groups 90,5056 6 15,0843 44,61 0,0000
Within groups 2,36709 7 ,338156
Total (Corr.) 92,8726 13

Multiple Range Tests for Do tinh sach by Ty le ethanol
E 1,5 2 1,32581 XX
E 4 2 2,25556 XX
E 2 2 2,53228 X
E 2,5 2 4,0449 X
E 3,5 2 6,92601 X
E 3 2 7,98415 X

Culture filtrate - E 1,5 -,325806
Culture filtrate - E 4 -1,25556
E 1,5 - E 2 -1,20647
E 1,5 - E 2,5 * -2,71909
E 1,5 - E 3 * -6,65835
E 1,5 - E 3,5 * -5,6002
E 1,5 - E 4 -,92975
E 2 - E 2,5 * -1,51262
E 2 - E 3 * -5,45187
E 2 - E 3,5 * -4,39373
E 2 - E 4 ,276723
E 2,5 - E 3 * -3,93925
E 2,5 - E 3,5 * -2,88111
E 2,5 - E 4 * 1,78934
E 3 - E 3,5 1,05814
E 3 - E 4 * 5,7286
E 3,5 - E 4 * 4,67045

One-Way ANOVA - Hoat tinh rieng S by Ty le acetone
Factor: Ty le acetone

Number of levels: 5

ANOVA Table for Hoat tinh rieng S by Ty le acetone
Between groups 13,4646 4 3,36615 17,66 0,037
Within groups ,952887 5 ,190577
Total (Corr.) 14,4175 9
Chuyn ngnh i sinh vt h ci in Nghin u v Pht trin Cng ngh inh h
Multiple Range Tests for Hoat tinh rieng S by Ty le acetone

Ty le acetone Count Mean Homogeneous Groups
A 1 2 1,28167 X
A 2,5 2 1,91549 XX
A 2 2 2,55403 X
A 1,5 2 4,39975 X

A 1 - A 1,5 * -3,11808
A 1 - A 2 * -1,27236
A 1 - A 2,5 -,633821
A 1 - Culture filtrate ,0187153
A 1,5 - A 2 * 1,84572
A 1,5 - A 2,5 * 2,48426
A 1,5 - Culture filtrate * 3,13679
A 2 - A 2,5 ,638541
A 2 - Culture filtrate * 1,29108
A 2,5 - Culture filtrate ,652536

One-Way ANOVA - Hieu suat H by Ty le acetone
Number of levels: 5

ANOVA Table for Hieu suat H by Ty le acetone
Between groups 10695,2 4 2673,8 1585,47 0,0000
Within groups 8,4322 5 1,68644
Total (Corr.) 10703,6 9

Multiple Range Tests for Hieu suat H by Ty le acetone
A 2,5 2 13,4865 X
A 1 2 14,843 X
A 2 2 18,935 X
A 1,5 2 33,5426 X
A 1 - A 1,5 * -18,6996
A 1 - A 2 * -4,09193
A 1 - A 2,5 1,3565
A 1 - Culture filtrate * -85,157
A 1,5 - A 2 * 14,6076
A 1,5 - A 2,5 * 20,0561
A 1,5 - Culture filtrate * -66,4574
A 2 - A 2,5 * 5,44843
A 2 - Culture filtrate * -81,065
A 2,5 - Culture filtrate * -86,5135
* denotes a statistically significant difference.CV= 3,33824

One-Way ANOVA - Do tinh sach by Ty le acetone
Number of levels: 5
Chuyn ngnh i sinh vt h cii in Nghin u v Pht trin Cng ngh inh h
ANOVA Table for Do tinh sach by Ty le acetone
Between groups 8,3118 4 2,07795 81,60 0,01
Within groups ,127332 5 ,0254664
Total (Corr.) 8,43913 9

Multiple Range Tests for Do tinh sach by Ty le acetone

A 1 2 1,01487 X
A 2,5 2 1,52483 X
A 2 2 2,0188 X
A 1,5 2 3,46613 X
A 1 - A 1,5 * -2,45126
A 1 - A 2 * -1,00393
A 1 - A 2,5 * -,509957
A 1 - Culture filtrate ,0148713
A 1,5 - A 2 * 1,44733
A 1,5 - A 2,5 * 1,9413
A 1,5 - Culture filtrate * 2,46613
A 2 - A 2,5 * ,493976
A 2 - Culture filtrate * 1,0188
A 2,5 - Culture filtrate * ,524829

One-Way ANOVA - Hoat tinh rieng S by So sanh DK ket tua
Factor: So sanh DK ket tua
Number of levels: 5

ANOVA Table for Hoat tinh rieng S by So sanh DK ket tua
Between groups 94,7782 4 23,6946 113,45 0,0000
Within groups 1,04432 5 ,208864
Total (Corr.) 95,8225 9

Multiple Range Tests for Hoat tinh rieng S by So sanh DK ket tua

Acetone 1,5:1 2 4,39975 X
Amoni 55-65% 2 6,86587 X
NaCl 25-30% 2 8,36956 X
Ethanol 3:1 2 10,0242 X
Acetone 1,5:1 - Amoni 55-65% * -2,46612
Acetone 1,5:1 - Culture filtrate * 3,13679
Acetone 1,5:1 - Ethanol 3:1 * -5,62447
Acetone 1,5:1 - NaCl 25-30% * -3,96981
Amoni 55-65% - Culture filtrate * 5,60291
Amoni 55-65% - Ethanol 3:1 * -3,15834
Amoni 55-65% - NaCl 25-30% * -1,50369
Culture filtrate - Ethanol 3:1 * -8,76126
Culture filtrate - NaCl 25-30% * -7,10661
Ethanol 3:1 - NaCl 25-30% * 1,65465
* denotes a statistically significant difference. 1,1748
Chuyn ngnh i sinh vt h ciii in Nghin u v Pht trin Cng ngh inh h
One-Way ANOVA - Hieu suat H by So sanh DK ket tua
Number of levels: 5

ANOVA Table for Hieu suat H by So sanh DK ket tua

Between groups 6167,47 4 1541,87 292,05 0,0000
Within groups 26,3971 5 5,27941
Total (Corr.) 6193,87 9

Multiple Range Tests for Hieu suat H by So sanh DK ket tua

Acetone 1,5:1 2 33,5426 X
Amoni 55-65% 2 62,2197 X
Ethanol 3:1 2 91,4798 X
NaCl 25-30% 2 92,6009 X
Acetone 1,5:1 - Amoni 55-65% * -28,6771
Acetone 1,5:1 - Culture filtrate * -66,4574
Acetone 1,5:1 - Ethanol 3:1 * -57,9372
Acetone 1,5:1 - NaCl 25-30% * -59,0583
Amoni 55-65% - Culture filtrate * -37,7803
Amoni 55-65% - Ethanol 3:1 * -29,2601
Amoni 55-65% - NaCl 25-30% * -30,3812
Culture filtrate - Ethanol 3:1 * 8,52018
Culture filtrate - NaCl 25-30% * 7,3991
Ethanol 3:1 - NaCl 25-30% -1,12108
* denotes a statistically significant difference. 5,90644

One-Way ANOVA - Do tinh sach by So sanh DK ket tua
Number of levels: 5

ANOVA Table for Do tinh sach by So sanh DK ket tua
Between groups 60,3889 4 15,0972 55,40 0,02
Within groups 1,36254 5 ,272508
Total (Corr.) 61,7515 9

Multiple Range Tests for Do tinh sach by So sanh DK ket tua
Acetone 1,5:1 2 3,46613 X
Amoni 55-65% 2 5,44816 X
NaCl 25-30% 2 6,66546 XX
Ethanol 3:1 2 7,98415 X

Acetone 1,5:1 - Amoni 55-65% * -1,98203
Acetone 1,5:1 - Culture filtrate * 2,46613
Acetone 1,5:1 - Ethanol 3:1 * -4,51802
Acetone 1,5:1 - NaCl 25-30% * -3,19932
Chuyn ngnh i sinh vt h civ in Nghin u v Pht trin Cng ngh inh h
Amoni 55-65% - Culture filtrate * 4,44816
Amoni 55-65% - Ethanol 3:1 * -2,53599
Amoni 55-65% - NaCl 25-30% -1,21729
Culture filtrate - Ethanol 3:1 * -6,98415
Culture filtrate - NaCl 25-30% * -5,66546
Ethanol 3:1 - NaCl 25-30% 1,3187

Gi tr tnh ton K
m
v V
max
cho PME t A . niger
PME (mol.min
-1
.mg
protein
-1
)
Michaelis-Menten
Best-fit values
Vmax 17,69
Km 0,6014
Std. Error
Vmax 0,3003
Km 0,04881
95% Confidence Intervals
Vmax 16,99 to 18,38
Km 0,4888 to 0,7139
Goodness of Fit
Degrees of Freedom 8
R square 0,9945
Absolute Sum of Squares 1,628
Sy.x 0,4511
Constraints
Km Km > 0,0
Number of points
Analyzed 10
Outliers (excluded, Q=1.0%) 0

Tham s ng hc s v hot nhit PME t A. niger
50
o
C
Sum of Mean Approx
Source DF Squares Square F Value Pr > F
Model 2 1.4305 0.7153 17540.7 <.0001
Error 9 0.000367 0.000041
Uncorrected Total 11 1.4309

Approx
Parameter Estimate Std Error Approximate 95% Confidence Limits
Ffinal 2.2905 0.1316 1.9929 2.5882
k 0.1350 0.00193 0.1306 0.1394

Chuyn ngnh i sinh vt h cv in Nghin u v Pht trin Cng ngh inh h
60
o
C
Sum of Mean Approx
Model 2 1.2246 0.6123 5326.66 <.0001
Error 9 0.00103 0.000115

Approx
Ffinal 1.7881 0.1943 1.3486 2.2277
k 0.1869 0.00483 0.1760 0.1979
70
o
C
Sum of Mean Approx
Model 2 1.0719 0.5360 14002.6 <.0001
Error 9 0.000344 0.000038

Approx
Ffinal 0.8884 0.1022 0.6571 1.1197
k 0.2863 0.00519 0.2746 0.2981

One-Way ANOVA - A/Ao by NaCl
Dependent variable: A/Ao
Factor: NaCl (M)

Number of levels: 6

ANOVA Table for A/Ao by NaCl
Between groups 9,16667 5 1,83333 10,81 0,0058
Within groups 1,0175 12 ,169583
Total (Corr.) 10,1842 17

Multiple Range Tests for A/Ao by NaCl

NaCl Count Mean Homogeneous Groups
0 3 1,0 X
,2 3 2,5 X
,05 3 3,0 XX
,1 3 3,25 XX
,12 3 3,375 XX
,15 3 3,625 X

0 - ,05 * -2,0
0 - ,1 * -2,25
0 - ,12 * -2,375
0 - ,15 * -2,625
0 - ,2 * -1,5
,05 - ,1 -,25
,05 - ,12 -,375
,05 - ,15 -,625
Chuyn ngnh i sinh vt h cvi in Nghin u v Pht trin Cng ngh inh h
,05 - ,2 ,5
,1 - ,12 -,125
,1 - ,15 -,375
,1 - ,2 ,75
,12 - ,15 -,25
,12 - ,2 ,875
,15 - ,2 * 1,125

One-Way ANOVA - A/Ao by CaCl
2

Dependent variable: A/Ao
Factor: CaCl2 (M)

Number of levels: 6

ANOVA Table for A/Ao by CaCl
2

Between groups 5,83333 5 1,16667 12,00 0,02
Within groups 1,16667 12 ,0972222
Total (Corr.) 7,0 17

Multiple Range Tests for A/Ao by CaCl
2

CaCl2 Count Mean Homogeneous Groups
0 3 1,0 X
,015 3 2,0 X
,0075 3 2,33333 XX
,005 3 2,33333 XX
,0125 3 2,66667 X
,01 3 2,66667 X

0 - ,005 * -1,33333 ,5547
0 - ,0075 * -1,33333 ,5547
0 - ,01 * -1,66667 ,5547
0 - ,0125 * -1,66667 ,5547
0 - ,015 * -1,0 ,5547
,005 - ,0075 ,5547
,005 - ,01 -,333333 ,5547
,005 - ,0125 -,333333 ,5547
,005 - ,015 ,333333 ,5547
,0075 - ,01 -,333333 ,5547
,0075 - ,0125 -,333333 ,5547
,0075 - ,015 ,333333 ,5547
,01 - ,0125 ,5547
,01 - ,015 * ,666667 ,5547
,0125 - ,015 * ,666667 ,5547

Chuyn ngnh i sinh vt h cvii in Nghin u v Pht trin Cng ngh inh h
Phn 4 ng dng ca PME c sinh tng hp t nm mc A. niger n hiu
qu ci thin cu trc rau qu
- i vi khm lnh ng
(i) Chn la phng thc tin x l ph hp

One-Way ANOVA - H/Ho sau TXL by DK tien xu ly
Dependent variable: H/Ho sau TXL
Factor: DK tien xu ly

Number of levels: 7

ANOVA Table for H/Ho sau TXL by DK tien xu ly
Between groups ,782456 6 ,130409 20,19 0,0000
Within groups ,0904453 14 ,00646038
Total (Corr.) ,872901 20

Multiple Range Tests for H/Ho sau TXL by DK tien xu ly
DK tien xu ly Count Mean Homogeneous Groups
0A 3 ,701667 X
0B 3 ,748333 XX
1A 3 ,817 XX
2A 3 ,89 XX
0 3 1,0 X
1B 3 1,012 X
2B 3 1,317 X

0 - 0A * ,298333
0 - 0B * ,251667
0 - 1A * ,183
0 - 1B -,012
0 - 2A ,11
0 - 2B * -,317
0A - 0B -,0466667
0A - 1A -,115333
0A - 1B * -,310333
0A - 2A * -,188333
0A - 2B * -,615333
0B - 1A -,0686667
0B - 1B * -,263667
0B - 2A -,141667
0B - 2B * -,568667
1A - 1B * -,195
1A - 2A -,073
1A - 2B * -,5
1B - 2A ,122
1B - 2B * -,305
2A - 2B * -,427
* denotes a statistically significant difference. CV = 0,140757

Chuyn ngnh i sinh vt h cviii in Nghin u v Pht trin Cng ngh inh h
One-Way ANOVA - He/Ho sau 4 w LD by DK tien xu ly
Dependent variable: He/Ho sau 4 w LD

Number of levels: 7

ANOVA Table for He/Ho sau 4 w LD by DK tien xu ly
Between groups ,0604818 6 ,0100803 7,09 0,013
Within groups ,0198933 14 ,00142095
Total (Corr.) ,0803751 20

Multiple Range Tests for He/Ho sau 4 w LD by DK tien xu ly
0 3 ,270333 X
0A 3 ,324333 XX
0B 3 ,33 XX
1A 3 ,362333 X
2A 3 ,383667 XX
1B 3 ,395 XX
2B 3 ,450333 X
0 - 0A -,054
0 - 0B -,0596667
0 - 1A * -,092
0 - 1B * -,124667
0 - 2A * -,113333
0 - 2B * -,18
0A - 0B -,00566667
0A - 1A -,038
0A - 1B -,0706667
0A - 2A -,0593333
0A - 2B * -,126
0B - 1A -,0323333
0B - 1B -,065
0B - 2A -,0536667
0B - 2B * -,120333
1A - 1B -,0326667
1A - 2A -,0213333
1A - 2B * -,088
1B - 2A ,0113333
1B - 2B -,0553333
2A - 2B -,0666667
* denotes a statistically significant difference. CV = 0,066013

One-Way ANOVA - DE sau TXL by DK tien xu ly
Dependent variable: DE sau TXL (%)
Number of levels: 7

ANOVA Table for DE sau TXL by DK tien xu ly
Between groups 270,095 6 45,0158 58,39 0,0000
Within groups 10,7925 14 ,77089
Total (Corr.) 280,888 20

Chuyn ngnh i sinh vt h cix in Nghin u v Pht trin Cng ngh inh h
Multiple Range Tests for DE sau TXL by DK tien xu ly

1B 3 39,0733 X
2B 3 39,35 X
1A 3 43,54 X
2A 3 43,59 X
0B 3 45,33 X
0A 3 45,89 X
0 3 50,21 X
0 - 0A * 4,32
0 - 0B * 4,88
0 - 1A * 6,67
0 - 1B * 11,1367
0 - 2A * 6,62
0 - 2B * 10,86
0A - 0B ,56
0A - 1A * 2,35
0A - 1B * 6,81667
0A - 2A * 2,3
0A - 2B * 6,54
0B - 1A * 1,79
0B - 1B * 6,25667
0B - 2A * 1,74
0B - 2B * 5,98
1A - 1B * 4,46667
1A - 2A -,05
1A - 2B * 4,19
1B - 2A * -4,51667
1B - 2B -,276667
2A - 2B * 4,24
* denotes a statistically significant difference.(1,53757)

One-Way ANOVA - DEe sau 4 w LD by DK tien xu ly
Dependent variable: DEe sau 4 w LD (%)
Number of levels: 7

ANOVA Table for DEe sau 4 w LD by DK tien xu ly
Between groups 271,001 6 45,1668 30,0000 0,0000
Within groups 21,0777 14 1,50555
Total (Corr.) 292,078 20

Multiple Range Tests for DEe sau 4 w LD by DK tien xu ly

1B 3 39,1333 X
2B 3 39,22 X
1A 3 43,02 X
2A 3 43,34 X
0B 3 45,12 XX
0A 3 45,98 X
0 3 50,15 X
0 - 0A * 4,17
0 - 0B * 5,03
Chuyn ngnh i sinh vt h cx in Nghin u v Pht trin Cng ngh inh h
0 - 1A * 7,13
0 - 1B * 11,0167
0 - 2A * 6,81
0 - 2B * 10,93
0A - 0B ,86
0A - 1A * 2,96
0A - 1B * 6,84667
0A - 2A * 2,64
0A - 2B * 6,76
0B - 1A 2,1
0B - 1B * 5,98667
0B - 2A 1,78
0B - 2B * 5,9
1A - 1B * 3,88667
1A - 2A -,32
1A - 2B * 3,8
1B - 2A * -4,20667
1B - 2B -,0866667
2A - 2B * 4,12

(ii) Xc nh hot tnh PME ph hp
One-Way ANOVA - H/Ho by Hoat tinh PME bo sung
Dependent variable: H/Ho
Factor: Hoat tinh PME bo sung (U/mL dung dich)
Number of levels: 5

ANOVA Table for H/Ho by Hoat tinh PME bo sung
Between groups ,435786 4 ,108947 10,89 0,012
Within groups ,100072 10 ,0100072
Total (Corr.) ,535858 14

Multiple Range Tests for H/Ho by Hoat tinh PME bo sung

0,5 U/mL 3 ,765 X
2 U/mL 3 ,784 X
1,5 U/mL 3 ,865 XX
0 3 1,0 X
1 U/mL 3 1,226 X
0 - 0,5 U/mL * ,235
0 - 1 U/mL * -,226
0 - 1,5 U/mL ,135
0 - 2 U/mL * ,216
0,5 U/mL - 1 U/mL * -,461
0,5 U/mL - 1,5 U/mL -,1
0,5 U/mL - 2 U/mL -,019
1 U/mL - 1,5 U/mL * ,361
1 U/mL - 2 U/mL * ,442
1,5 U/mL - 2 U/mL ,081

Chuyn ngnh i sinh vt h cxi in Nghin u v Pht trin Cng ngh inh h
One-Way ANOVA - He/Ho by Hoat tinh PME bo sung
Dependent variable: He/Ho
Number of levels: 5

ANOVA Table for He/Ho by Hoat tinh PME bo sung
Between groups ,0772696 4 ,0193174 10,73 0,012
Within groups ,0180033 10 ,00180033
Total (Corr.) ,0952729 14

Multiple Range Tests for He/Ho by Hoat tinh PME bo sung

0 3 ,270333 X
0,5 U/mL 3 ,272 X
2 U/mL 3 ,295 X
1,5 U/mL 3 ,387 X
1 U/mL 3 ,450333 X
0 - 0,5 U/mL -,00166667
0 - 1 U/mL * -,18
0 - 1,5 U/mL * -,116667
0 - 2 U/mL -,0246667
0,5 U/mL - 1 U/mL * -,178333
0,5 U/mL - 1,5 U/mL * -,115
0,5 U/mL - 2 U/mL -,023
1 U/mL - 1,5 U/mL ,0633333
1 U/mL - 2 U/mL * ,155333
1,5 U/mL - 2 U/mL * ,092

One-Way ANOVA - DE by Hoat tinh PME bo sung
Dependent variable: DE
Number of levels: 5

ANOVA Table for DE by Hoat tinh PME bo sung
Between groups 276,687 4 69,1717 67,96 0,0000
Within groups 10,1787 10 1,01787
Total (Corr.) 286,866 14

Multiple Range Tests for DE by Hoat tinh PME bo sung

2 U/mL 3 39,0433 X
1 U/mL 3 39,35 X
1,5 U/mL 3 39,3567 X
0,5 U/mL 3 43,6 X
0 3 50,21 X
0 - 0,5 U/mL * 6,61
0 - 1 U/mL * 10,86
0 - 1,5 U/mL * 10,8533
Chuyn ngnh i sinh vt h cxii in Nghin u v Pht trin Cng ngh inh h
0 - 2 U/mL * 11,1667
0,5 U/mL - 1 U/mL * 4,25
0,5 U/mL - 1,5 U/mL * 4,24333
0,5 U/mL - 2 U/mL * 4,55667
1 U/mL - 1,5 U/mL -,00666667
1 U/mL - 2 U/mL ,306667
1,5 U/mL - 2 U/mL ,313333

- Ci thin u tr d leo mui hu
(i) nh hng ca thi gian tin x l da leo nhm kch hot PME ni bo i
vi s thay i DE ca nguyn liu da leo
One-Way ANOVA - DE sau TXL by Thoi gian TXL
Dependent variable: DE sau TXL ((%))
Factor: Thoi gian TXL ((min))
Number of levels: 6

ANOVA Table for DE sau TXL by Thoi gian TXL
Between groups 289,538 5 57,9077 89,35 0,0000
Within groups 7,7768 12 ,648067
Total (Corr.) 297,315 17

Multiple Range Tests for DE sau TXL by Thoi gian TXL

Thoi gian TXL Count Mean Homogeneous Groups
30 3 35,27 X
50 3 35,73 X
40 3 36,15 X
20 3 36,5 X
10 3 43,6 X
0 3 45,07 X
0 10 * 1,47
0 20 * 8,57
0 30 * 9,8
0 40 * 8,92
0 50 * 9,34
10 20 * 7,1
10 30 * 8,33
10 40 * 7,45
10 50 * 7,87
20 30 1,23
20 40 ,35
20 50 ,77
30 40 -,88
30 50 -,46
40 50 ,42

Chuyn ngnh i sinh vt h cxiii in Nghin u v Pht trin Cng ngh inh h
(ii) nh hng ca thi gian tin x l da leo nhm kch hot PME ni bo i
vi s thay i DE ca da leo mui chua
One-Way ANOVA - DE san pham by Thoi gian TXL
Dependent variable: DE san pham ((%))

Number of levels: 6

ANOVA Table for DE san pham by Thoi gian TXL

Between groups 222,761 5 44,5521 37,14 0,0000
Within groups 14,3932 12 1,19943
Total (Corr.) 237,154 17

Multiple Range Tests for DE san pham by Thoi gian TXL

30 3 32,67 X
20 3 34,3 XX
50 3 35,35 X
40 3 35,45 X
10 3 40,8 X
0 3 42,37 X
0 10 1,57
0 20 * 8,07
0 30 * 9,7
0 40 * 6,92
0 50 * 7,02
10 20 * 6,5
10 30 * 8,13
10 40 * 5,35
10 50 * 5,45
20 30 1,63
20 40 -1,15
20 50 -1,05
30 40 * -2,78
30 50 * -2,68
40 50 ,1
(iii) nh hng ca thi gian tin x l da leo nhm kch hot PME ni bo i
vi s thay i gi tr cng tng i cn li ca da leo mui chua
One-Way ANOVA - H/Ho san pham by Thoi gian TXL
Dependent variable: H/Ho san pham
Number of levels: 6

ANOVA Table for H/Ho san pham by Thoi gian TXL

Between groups ,17877 5 ,035754 25,40 0,0000
Within groups ,016892 12 ,00140767
Total (Corr.) ,195662 17
Chuyn ngnh i sinh vt h cxiv in Nghin u v Pht trin Cng ngh inh h

Multiple Range Tests for H/Ho san pham by Thoi gian TXL

0 3 ,431 X
50 3 ,642 X
10 3 ,657 XX
40 3 ,662 XX
20 3 ,727 X
30 3 ,727 X
0 10 * -,226
0 20 * -,296
0 30 * -,296
0 40 * -,231
0 50 * -,211
10 20 -,07
10 30 -,07
10 40 -,005
10 50 ,015
20 30
20 40 ,065
20 50 * ,085
30 40 ,065
30 50 * ,085
40 50 ,02
Chuyn ngnh i sinh vt h cxv in Nghin u v Pht trin Cng ngh inh h
PH LC 4. MT S HNH NH

(a) Thit b thanh trng dng c (b) T Sanyo

(c)T cy Dalton (d) Thit b chun t ng (e) Ni thanh trng

(f) Thit b ly tm lnh (g) My quang ph

(h) T ng su (i) Thit b ng gi chn khng
Hn PL41: Cc tit b s dng trong ngin cu
Chuyn ngnh i sinh vt h cxvi in Nghin u v Pht trin Cng ngh inh h

C cht l v qu citrus C cht l b to C cht b to v bi

Mu ang nhit 37C Enzyme th sau trch ly
Hn PL42: Mt s n n cun b c ct v ln men SSF sin tng p PME

Mu i chng Da leo thnh phm Mu da leo hai iu kin tin x l thch hp
Hn PL43: Cc mu da leo mui cua
Chuyn ngnh i sinh vt h cxvii in Nghin u v Pht trin Cng ngh inh h

Khm chn 2 Chun b mu khm th nghim Dao ct khm

Mu khm dng hnh tr Tin x l trong PME nm mc Ngm trong PME c chua

Mu chun b lnh ng o lc ct ca cc mu khm

Cc mu khm sau tr ng v tan gi
Hn PL44: Mt s cng on tin x l v ci tin c tn cu trc
km ng ln

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