GVHD: Th.

S Nguyn Th Thu Sang

B CNG THNG
TRNG I HC CNG NGHIP THC PHM
KHOA CNG NGH THC PHM

BO CO TIU LUN
MN: NG DNG CNG NGH SINH HC
TRONG CNG NGH THC PHM
ti: QUY TRNH SN XUT ENZYME PECTINASE

V NG DNG TRONG LM NC QU
GVHD: Th.S Nguyn Th Thu Sang
SVTH: Trn Th M Nhung

3004110234

Nguyn Th Ngc Thi

3005110299

Phm Th Kim Ti

3005110263

Phng Th Hng

3005110064

V Ngc Khim
V Th Tuyt Trang

3005110125
3005110357

Tp.HCM 06/2014

Nhm 20Trang 1

TPHCM. T04/2013

GVHD: Th.S Nguyn Th Thu Sang

MC LC
LI M U......................................................................................................................................2
CHNG 1: TNG QUAN V ENZYME PECTINASE...............................................................3
1.1.

nh ngha Enzyme pectinase ............................................................................................3

1.2.

Cu to..................................................................................................................................4

1.3.

Phn loi...............................................................................................................................4

1.4.

c im ca Enzyme pectinase:........................................................................................7

1.4.1.

Enzym pectinase t ngun thc vt:............................................................................7

1.4.2.

Enzym pectinase t vi sinh vt......................................................................................9

1.5.

Trung tm hot ng ca pectinase...................................................................................12

1.6.

C ch tc dng ca enzyme pectinase...............................................................................12

1.7.

Li ch khi s dng enzyme pectinase..............................................................................13

CHNG 2: CNG NGH SN XUT ENZYME PECTINEASE............................................16

2.1.

Ngun gc thu nhn...........................................................................................................16

2.2.

Phng php nui cy b mt............................................................................................17

2.3.

Tch v lm sch................................................................................................................20

2.3.1.

Phng php thu nhn...............................................................................................20

2.3.2.

T mi trng nui cy b mt..................................................................................21

2.3.3.

T mi trng nui cy b su...................................................................................23

2.4.

Phng php tinh sch......................................................................................................24

2.4.1.

Gii thiu chung.........................................................................................................24

2.4.2.

Phng php kt ta..................................................................................................26

2.4.3.

K thut siu lc.........................................................................................................30

2.4.4.

Phng php sc k...................................................................................................31

2.4.5.

Phng php thm tch.............................................................................................33

2.4.6.

Kt ta i lc..............................................................................................................34

2.5.

Thu ch phm t ASP.NIGER 72-32................................................................................34

2.5.1.

nh hng thi gian nui..........................................................................................34

2.5.2.

nh hng nhit nui...........................................................................................34

2.5.3.

nh hng m ban u ca mi trng nui cy..............................................35

2.6.

Xc nh hot tnh enzyme................................................................................................37

2.6.1.

Phng php nht k................................................................................................37

2.6.2.

Phng php ng chung(Cu-pectat).......................................................................37

2.6.3.

Phng php so mu:................................................................................................38

Nhm 20Trang 2

GVHD: Th.S Nguyn Th Thu Sang

CHNG 3: NG DNG ENZYME PECTINEASA TRONG LM NC QU...................39
3.1.

Gii thiu v ng dng enzyme pectinase........................................................................39

3.2.

ng dng enzyme pectinase sn xut nc qu..............................................................40

3.2.1.

Cc ch phm enzyme................................................................................................40

3.2.2.

ng dng ch phm pectinase trong sn xut nc qu.........................................41

TI LIU THAM KHO.................................................................................................................48

Nhm 20Trang 3

GVHD: Th.S Nguyn Th Thu Sang

LI M U

Hin nay nc ta ang trong thi k pht trin mnh, cng vi xu th hi nhp nn
kinh t quc t, th cc ngnh khoa hc k thut v dch v ngy cng pht trin, theo
ngnh cng ngh thc phm cng tng bc c ci tin cung cp cc sn
phm y cht dinh dng, p mt, tin li p ng nhng nhu cu ngy cng cao
ca con ngi.
S dng enzyme trong sn xut v i sng tr thnh ph bin trong ngnh sn
xut thc phaamt v mang li li ch kh ln.
Trc y, ngun enzyme ch yu thu nhn t thc vt v ng vt. Ngy nay,
ngi ta nghin cu tm ra ngun enzyme t vi sinh vt phong ph v a dng v r
tin. V c ng dng rng ri trong cc ngnh sn xut khc nhau.
hiu su hn v ngun enzyme ny nhm em thc hin ti tiu lun quy
trnh sn xut enzyme pectinase v ng dng trong sn xut nc qu.
V thi gian hon thnh bo co khng nhiu, cng nh kin thc cn hn ch nn
khng trnh khi nhng sai st, em knh mong nhn c s ng gp kin ca c,
cc bn bi bo co ca em c hon thin hn v gip em tin b hn trong
nhng bi lm sp ti.

Nhm 20Trang 4

GVHD: Th.S Nguyn Th Thu Sang

CHNG 1: TNG QUAN V ENZYME PECTINASE

1.1.

Enzyme l g?
Enzyme pectinase l mt nhm enzyme thy phn pectin , sn phm ca
qu trnh ny l acid galacturonic, galactose, arabinose, methanol y l mt
nhm ezyme c ng dng rng ri trong cng nghip ch ng sau amylase
v protease. Enzyme ny ban u c pht hin trong cc dch chit tri cy
nh c rt, c chua hay i mch. u tin phi k n l pht hin ca E.fremi
(1840) trn i tng c rt.

1.2.

Cu to
Pectin l polysaccharide d th, ch yu l mt mch chnh gm cc gc
acid D1,4 galacturonic, lin kt vi nhau bng lin kt 1,4O glucozic cn
gi l acid polygalacturonic hay acid pectic. Pectine ha tan trong t nhin l
ester metylic ca acid pectic.
Trong thc t khng phi tt c cc nhm COOH C6 ca ng galactose
cng b metyl ha (to ester metylic), m i khi mt s nhm COOH b
decacboxyl ha (kh CO2), mt s nhm COOH thay th -H bng kim loi,
cng c lc gi nguyn dng COO. Ngi ta cho rng protopectine l hp
cht gia pectine v araban, galactose hay tinh bt.
Trng lng phn t t 20.000 200.000 vC.

1.3.

Phn loi
Enzyme pectinase c th phn loi theo c ch tc dng ca chng:

Nhm 20Trang 5

GVHD: Th.S Nguyn Th Thu Sang

Bng 1.1: Phn loi enzyme pectinase

St

Enzym (tn gi theo Enzym(tn

h thng)

thng gi)

Pectin-pectinhydrolase

pectinesterase

Poly--1,4 galacturonidglycano hydrolase-PG

Endopoly
galacturonase
(endo-PG)

Poly--1,4-D-

Exopoly

galacturonidgalacturon-

galacturonase

hydrolase

(exo-PG)

Poly--1,4-D-

Endopolymetil-

galacturonidmetilester-

galacturonase

glycanohydrolase

(Endo-PMG)

Phn ng xc tc
Pectin + H2O = n metanol +
pectic acid
Thy phn lin kt -1,4-Dgalacturonid

trong

galacturonid khng theo mt

trt t no
Thy phn lin kt -1,4-Dgalacturonid

trong

pectat,

trong galacturonic vi s t
mch ca acid galacturonic
Thy phn lin kt -1,4-Dgalacturonid

trong

pectin

khng theo mt trt t nht

nh.
Thy phn lin kt -1,4-D-

Poly--1,4-D5

galacturonid
digalacturonoliase

Exo-pectatliase
(exo-PKTE)

galacturonid trong pectat vi

s

to

thnh

-4,5

aciddegalacturonic khng theo

mt trt t nht nh.
Thy phn lin kt -1,4-D-

Poly--1,4-D-

Endopectatliase

galacturonid glicanoliase

(PETE)

galacturonid

trong

trong galacturonic vi s to
thnh ni i khng theo mt
trt t nht nh

Nhm 20Trang 6

pectat,

GVHD: Th.S Nguyn Th Thu Sang

Thy phn lin kt -1,4-D-

Poly--1,4-D7

galacturonid

metylester

glicanoliase

Endopectinliase

galacturonid trong pectin vi

(Endo-PTE)

s to thnh ni i khng
theo mt trt t nht nh

Pectinesterase (PE): xc tc s thy phn ca cc nhm methyl ester.

Enzyme thng tn cng vo cc nhm ester methyl ca n v galaturonate
nm k n v khng b ester ho, phn ct cc nhm methoxy (COOCH 3)
ng cnh cc nhm COOH t do, to thnh acid pectinic hoc acid pectic v
methanol. Pectinesterase thu c t cc ngun khc nhau c gi tr pH ti u
khc nhau. Nu thu t ngun VSV th PH ti u t 4,5-5,5, cn nu t ngun
thc vt th c pH ti u t 5,0-8,5. Pectinesterase t nm mc c nhit ti
u l 30-40oC v b v hot 55-62 oC. Pectinesterase thng c hot ho
bi cc ion Ca2+ v Mg2+.
Polygalacturonase: cn c tn gi l poly -1,4-galacturoniglucanohydrolase,
xc tc s phn ct cc mi lin kt 1,4-glycosid. Cc exo-PG (exo-poly 1,4-D-galacturonide) galacturonohydrolase, phn ct t cc u khng kh, v
endo-PG (endo-poly1,4--D-galacturonide) glycanohydrolase, tn cng ngu
nhin vo gia mch c cht. Polygalacturonase t gp trong thc vt nhng c
ch yu mt s nm mc v vi khun. Polygalacturonase l mt phc h
enzyme gm c nhiu cu t v thng c tnh c hiu cao i vi c cht.
Trn c s tnh c hiu v c ch tc dng vi c cht, enzym
polygalacturonase c chia lm bn loi:
Polymethylgalacturonase

hay

cn

gi

-1,4-galacturonite-

methylesglucanohydrolase, tc dng trn polygalactorunic acid c

methoxyl ho (tc l pectin). Enzyme ny li c phn thnh hai nhm nh
ph thuc vo kh nng phn ct trong hay cui mch trong phn t pectin,
l endo-glucosidase-polymethyl galacturonase kiu I v exo-glucosidasepolymethylgalaturonase kiu II.

Nhm 20Trang 7

GVHD: Th.S Nguyn Th Thu Sang

Polygalacturonase, enzyme tc dng trn pectic acid hoc pectinic, cng c
chia thnh hai nhm nh l endo-glucosidase-polygalacturonase kiu II v exoglucosidase-polygalacturonase kiu IV. Enzyme endo-glucosidase-Polymethylgalacturonase kiu I l enzyme polymethylgalacturonase dch ho pectin c
mc methyl ho cng cao th b thy phn bi enzyme ny cng nhanh v
cng c hiu qu. Trong dung dch, khi c mt ca enzyme pectinesterase th
hot ca enzyme ny thng b gim. Enzyme ny rt ph bin trong cc
VSV, c bit l nm mc A. niger, A, awamori.
Pectate lyase (PEL): xc tc s phn ct cc n v galacturonate khng b
ester ho. C hai enzyme exo-PEL (exo-Poly(1,4--D-galac turonide) lyase) v
endo-PEL (endo-poly(1,4--D-galacturonic lyase) u tn ti. Pectate v pectin
c lng methoxyl thp l cc c cht thch hp hn c cho cc enzyme ny.
Ni chung, c hai enzyme ny u c khong pH ti a nm trong khong t
8,0-11, u cn ion Ca2+ hot ng. Pectate lyase khng c tm thy trong
cy xanh, nhng c vi khun v nm. Cc enzyme VSV ngoi bo ny ng
mt vai tr rt quan trng trong qu trnh gy bnh thc vt, gy ra s phn
hy m ca thnh t bo, lm mm v lm mc m thc vt.
Ngoi ra cn c:
Pectin-transeliminase hay cn c gi l poly

1,4-galaturonite

methylesteglucanoliase, l enyme tc dng trn pectin v pectinic acid.

Polygalactorunate-transeliminase cn c gi l poly -1,4D-galaturonite
glucanoliase, l enzyme tc dng trn pectic acid v pectinic acid.
Pectin lyase (PNL): xc tc s phn ct cc n v galacturonate b ester
ho. Tt c cc PNL u l endo-enzyme.
1.4.

c im ca Enzyme pectinase:

1.4.1. Enzym pectinase t ngun thc vt:

Pectinesterase:
Trong thc vt, hu ht cc loi cy cho tri u cha enyme
PE.Enzyme ny thng tn ti di nhiu hnh thc khc nhau, nm trong
Nhm 20Trang 8

GVHD: Th.S Nguyn Th Thu Sang

phn v t bo. PE thc vt ni chung c hot ti u trong khong pH hi
kim. Cc cation kim loi nng thp, nh Ca 2+ chng hn, c khuynh
hng lm tng nng hot ng ca enzyme.
-

C chua cha t nht hai loi PE. C PE1 v PE2 u tng trong giai
on u ca qu trnh chn. Khi bc vo giai on chn, nng enzyme
PE1 gim xung, nhng PE2 tch lu dn cho n khi tri cy c mu c trng
ca tri chn. PE2 c khi lng phn t 23kD, pH ti u 7,6. Enzyme ny b
bt hot 50% sau 5 pht un 67 oC. Cc ion Ca2+ v Na+ lm tng hot ca
enzyme ln ti a cc nng 0,005M v 0,05M, theo th t.

PE ca u nnh l protein c khi lng phn t 33kD, hot ng ti

u ti pH gn 8. Polygalacturonic acid, l sn phm c hnh thnh do qu
trnh methyl ho cc phn t galacturonic acid.

Trong tht qu chui c hai isoenzyme PE. C hai c cng khi lng
phn t l 30kD, nhng c im ng in khc nhau: 8,8 v 9,3. Cc enzyme
ny hot ng pH ti a l 7,5. Hot enzyme tng ln khi thm vo dung
dch NaCl nng 0,2M, v a pH ca dung dch v 6,0. Cc enzyme ny
b c ch bi nhiu loi.

Polyol c khi lng phn t thp, nh glycerol, sucrose, glucose,

maltose v galactose.
PE trong qu cam c hai loi: l hai isoenzyme PE1 v PE2 c khi lng
phn t 36,kD, nhng c im ng in khc nhau l

10,05 v 11,0, theo th t. pH ti u ca PE1 l 7,6, cn ca PE2 l 8,0.

Trong thnh phn ca nhiu tht qu khc cng cha hai isoenzyem,
mt trong hai enzyme ny c tnh bn nhit hn, cn enzyme kia th t mn cm
hn khi b tc ng ca protease. n nh ca enzyme c th lin quan n
mc glycosyl ho ca cc phn t enzyme. Enzyme bn nhit hn v
enzyme cn li c khi lng l 51kD v 36kD, theo th t.

Nhm 20Trang 9

GVHD: Th.S Nguyn Th Thu Sang

Hnh 2.1: Cu trc ca pectinesterase t carrot

-

C to v kiwi cng cha hai loi isoenzyme. Cc isoenzyme ca kiwi c cng

khi lng phn t l 57kD v cng im ng in l 7,3. Tuy nhin, chng
khc nhau v mc bn nhit.

Polygalacturonase:
Hu ht cc nghin cu v PG u trn c s cc ngun VSV. PG
thng c tm thy trong cc phn tit ngoi bo ca cc loi nm v vi
khun gy bnh, chng hn nh Sacchromyces gragilis, Aspergillus niger,
Lactobacillus plantarum, Cochiliobolus carbonum, Neurospora crassa, cc
loi Ascomycete, Phizopus arrchizus, v Fusarium osyporum. Tuy nhin, trong
thc t, PG ca thc vt bc cao c nghin cu rt nhiu c chua chn.
Cc enzyme PG trong c chua chn tn ti di hai dng, v c hai u
l endo-enzyme.PG1 c khi lng phn t 84kD v c khong 50% b bt
hot nhit 78oC.PG2 c khi lng phn t 44kD v c khong 50% b bt
hot 57oC. PG1 c n nh ti a pH 4,3, tri li PG2 n nh ti a
pH 5,6.
Exo-PG thy phn cc u khng kh ca chui polygalacturonic, to ra
galacturonic acid l sn phm thy phn chim u th.S thy phn polymer
ny b gin on ca s tn ti ca cc mch nhnh trong c cht. Mc thu
phn tng t l vi kch thc c cht, t ti a vi mc polymer ho 20 i
vi cc exo-PG c rt v o. Hot ng ca cc exo-enzyme lm tng nhanh
Nhm 20Trang 10

GVHD: Th.S Nguyn Th Thu Sang

s to thnh cc nhm kh v lm tng chm nht ca dung dch c cht.
S phn hy polyuronide trong qu trnh chn khng gy ra s tch ly
galacturonic acid, v ch c endo-enzyme PG l c lin quan.
1.4.2. Enzym pectinase t vi sinh vt
Ngun giu enzyme pectinase l nm mc, nm men, vi khun.
Nm mc: penicillium glaucum, p.ehrlichii, p.chrysogenum, p.expanam,
p.cilrimim, aspergillus awamori, a.foetidus, a.niger, a.terrus, a.saitoi, a.aureus,
a.oryzae, a.wentii, fusarium moniliforme,
Nm men: saccharomyces fragilis
Vi khun: bacillus polymyxa, flavobacterium pectinovorum, klebsiella
aerogenes
Cc loi vi sinh vt ny thng c trong b mt tt c cc loi qu, cc
b phn khc ca thc vt. khi qu b h hng, hoc thc vt cht, chng s
cng cc loi vi sinh vt khc ph hu nhanh qu v cc b phn ca thc vt.
Ngi ta thng thu pectinase t canh trng b mt hoc t canh trng b
su ca nm mc.
Cc vi khun v nm men cng tng hp c enzime ny a.niger ch
yu tng hp ra pectinnesterase. con.diplodiella, pen.citrimin to ra ch yu l
polygalacturonase.
Nm men sacch.fragilis dng nh ch to ra endopectintraseliminase.
Vi khun bac.polymyxa, bac.species li ch yu to ra transeliminase.

Pectinesterase:
Pectinesterase thu c t cc ngun khc nhau c gi tr PH ti u
khc nhau: PH ti u ca pectinesterase t ngun nm mc l 4,5 n 5,5 cn
ca ch phm loi b enzime polygalacturonase s c PH ti u t 2,0 n

Nhm 20Trang 11

GVHD: Th.S Nguyn Th Thu Sang

6,5. Tri li ph ti u ca pectinesterase t ngun thc vt thng ng l t 6
n 7,5 8.
Nhit ti u ca pectinesterase t nm mc l 40 n 45 0C.t 55 n 620C
th enzime b v hot, trong khi nhit ti u ca enzime pectinesterase t
thc vt thng ng cao hn: t 55 600C
Pectinesterase c th nhn c t canh trng nm mc A.niger c
nhit ti u l 30 45oC, PHopt= 4,5-5,5 v b v hot 55- 62 oC v t thc
vt (phopt=7,5-8, toopt= 55-60oC). kh nng hot ng ca chng ph thuc vo
ngun thu nhn, mc ester ho ca pectin. Ion natri v c bit l ion canxi,
cng nh chlorua ca Na, K v Ca s hot ha pectinesterase t nm mc
conithyrium diplodiella v t a.niger.tri li cc cation ha tr 3 v 4 (thy
ngn nitrat, ch nitrat, nhm sunfat v st clorua) s km hm tc dng ca
pectinesterase
Ngoi ra, ngi ta thu c enzime pectinesterase trng thi ng
th. v ngi ta cng xc lp c rng n-axit cui trong phn t enzime l
phenylalanin.
Pectinesterase ca nm mc s thy phn trc nht l nhm
methylester nm gia hai nhm carboxyl t do. v enzime s thy phn ln
lt ct lin kt ester dc theo phn t pectin. hot ng ca pectinesterase ph
thuc nhiu vo mc ester ha ca pectin v t l thun vo mc ester
ha. Chng hn, i vi tc ng ca pectinesterase t nm mc a.niger, cn
thit phi c pectin ester ha mc cao khng t hn 70%.

Polygalacturonase:
Hu ht cc nghin cu v Polygalacturonase u trn c s cc ngun
Vi sinh vt. Polygalacturonase thng c tm thy trong cc phn tit ngoi
bo ca cc loi nm v vi khun gy bnh, chng hn nh: saccharomyces
fragilis, asperigillus niger, lactobacillus plantarum, cochlibolus carbonum,
neurosrora crassa, cc loi ascomycete, rhizopus arrchizus v fusarium
oxysrorum.
Nhm 20Trang 12

GVHD: Th.S Nguyn Th Thu Sang

PH ti u ca cc polygalacturonase cng khc nhau, ph thuc vo
ngun

thu

cht.

Chng

hn

polygalacturonase

dch

ha

(endopolygalacturonase) khi tc dng trn acid pectinic th ph ti u nm trong

khong t 4,0 5,5. cng enzime nhng khi tc dng trn pectin th li c
ph ti u trong khong 5,5 6. cn polygalacturonase ng ha khi tc dng
trn pectin th ph ti u t 3 4 nhng khi tc dng trn acid pectinic th ph ti
u cao hn mt t vng 4,4- 6
Cc polygalacturonase ch yu bn vng vng pH t 4 6
Polygalacturonase ng ha ch yu t A.niger nu c hot ha
bng thy ngn th c th bn vng khi pH= 2,5
Nhit ti u ca a s polygalacturonase nm trong khong t 40
-450C.trong khong nht , chng thng bn vng, nhng s b v hot
ha khi nhit 50 v 55 - 650C.
1.5.

Trung tm hot ng ca pectinase

Enzyme pectinase cha vng c 8 10 vng xon kp v pha phi vi
2 vng to thnh khe lin kt vi c cht.
Trung tm hot ng ca enzyme ny cha axit amin Aspartate v
Lysine. C 1 Histidine nm gn trung tm hot ng s nh hng n kh
nng xc tc ca enzyme
Nhit ti u ca enzyme pectinase khong t 45 550C.

1.6.

C ch tc dng ca enzyme pectinase

Trong ch bin nc qu, ngi ta s dng cc ch phm enzym nhm hai mc
ch c bn.
-

Ph v thnh t bo thc vt nhm nng cao hiu sut thu nc qu.

Lm trong v n nh cht lng nc.

Ph v thnh t bo. T bo thc vt c cu to bng v t bo

(thnh t bo). V t bo nh mt lp thnh bo v rt hu hiu v to hnh cho

Nhm 20Trang 13

GVHD: Th.S Nguyn Th Thu Sang

t bo. v t bo thc vt c nhiu cht pectin, cc cht pectin c xem nh
cht ciment gn cc t bo vi nhau. Ph v s gn kt ny s to iu kin cho
cc vt cht trong t bo thot ra khi t bo. Cc ch phm enzym c cha
khng ch pectinase m cn cha cc enzym trong nhm cellulase. Cc loi
enzym ny s lm ph v thnh t bo v gip qu trnh thu nhn dch t bo
tt hn.
Lm trong nc qu. Nc qu sau khi c tch khi t bo

thng cha nhiu cht khc nhau. Trong cht pectin chim lng ng k
v pectin thng gy hin tng nht cao v gy c nc qu.
Cc cht protein c trong bo tng, mng t bo v gian bo. Pectin cha
polygalacturonic acid, araban v galactan.Trong lng polygalacturonic
acid chim ti 40-60%. Khi b thy phn, pectin tch thnh hai phn:
-

Phn trung tnh phc cht galactanoraban

Phn acid acid pectic.

Trong bo tng, pectin nm dng ha tan. Trong mng t bo v gian

bo, chng nm dng khng ha tan gi l protopectin. Protopectin mng

gian bo c cha lng kim loi kh ln v mt lng nhm metocyl
lm protopectin bn vng. Cn protopectin mng t bo cha mt lng kim
loi khng nhiu, c metocyl ha cao. V th, t bo thc vt c kh nng
trng n tt.
Nu enzym tham gia phn gii pectin gian bo s lm cc t bo kh lin kt
vi nhau v tht qu d dng b mm ra.Pectin thng c mi lin kt hydro v
lin kt nguyn t yu hn so vi cellulose. Tham gia phn hy pectin gm
nhiu loi enzym : cellulose, pectinase,
1.7.

Li ch khi s dng enzyme pectinase

T khi pht hin ra enzym v kh nng chuyn ha ca enzym loi ngi
tng nhanh qu trnh sn xut v ng dng enzym trong cng nghip. S
lng enzym pht hin ngy cng nhiu v s lng enzym c ng dng vo
cng nghip cng ngy cng nhiu.

Nhm 20Trang 14

GVHD: Th.S Nguyn Th Thu Sang

Trong sn xut thc phm, ngi ta thng s dng cc ch phm
pectinase di dng tinh khit. Ngi ta khng dng ch phm di dng canh
trng nm mc sy kh. T lng ch phm pectinase c c trn lng
nguyn liu em ch bin vo khong t 0.03 0.05 n 0.10%.
Pectinase thng c s dng trong cc ngnh cng nghip thc phm
sau:
-

Sn xut ru vang.

Sn xut nc qu v nc ung khng c cn;

Sn xut cc mt hng t qu: nc qu c c, mt nh, mt ng,..

Sn xut nc gii kht.

Sn xut c ph v c ph ha tan.

Trong sn xut ru vang, cng nh trong sn xut nc qu v cc

nc ung khng ru, u c th s dng pectinase mt cch rt hiu qu.
Nh tc dng ca pectinase m cc qu trnh p, lm trong v lc dch qu rt
d dng, do lm tng hiu sut sn phm. Chng hn a pectinase vo khu
nghin qu, s lm tng hiu sut nc qu sau khi p ln ti 15 25%. Bi l
khi c pectin th khi qu nghin s c trng thi keo, do khi p dch qu
khng thot ra c. Nh pectinase phn gii cc c cht pectin, lm cc cht
chit trong dch bo d thot ra ngoi hn, lm tng hiu sut cht chit, hn
na dch qu trong sut khng b vn c v lc rt d dng. Khng nhng vy,
enzym pectinase cn gp phn chit rt c cc cht mu, tanin

v nhng

cht ha tan na, do lm tng thm cht lng ca thnh phm.

Cc ch phm pectinase dng trong sn xut phi c nhng yu cu
sau:
-

Ch phm khng c lm gim cht lng ca vang, khng c gy nh

hng xu n hng v v mu sc ca sn phm; ngha l ch phm phi
c lm sch ti mc ti a khi cc tp cht c nh hng xu n cht

lng vang.
Ch phm c a vo dch hay b nghin tng cng qu trnh s ch
qu, tng nhanh v lm trong dch, nng cao tc lc, tng hiu sut chung v

Nhm 20Trang 15

GVHD: Th.S Nguyn Th Thu Sang

c bit l hiu sut ca phn t chy c cht lng cao. tng nhanh v tt
qu trnh lm trong dch th tng quan hot ca cc enzym chnh nh endo
PMG l 55,0.102 n v/mg protein cha trong ch phm, cn enzym Cx l
57,5 n v/mg protein trong 1 lt dung dch. Trong trng hp ny khng cn
c mt PE. Nhng nu hot ca endo PMG trong ch phm l 31,0.10 2
n v/mg protein th cn c mt PE vi hot l 4,1 n v/mg protein.
Trong sn xut vang nho gia hm lng enzym endo PMG va Cx phi c s
tng quan nht nh. Khi ch phm c hot endo PMG l 28,2.10 2 n
v/mg protein v Cx l 28,7 n v/mgP qu trnh lm trong s xy ra nhanh
hn khi c hot endo PMG l 25,4.10 2 n vi/mg P v Cx l 55,0 n
v/mgP.
-

Ch phm pectinase cng phi tng n nh ca vang ngha l phi cha

enzym proteinase vi hot khng thp hn 120 n v/g theo globulin v
140 n v/g theo anbumin.

trnh gy tn tht cc cht mu ca vang v trnh xut hin mu ti

trong vang trng th hot ca cc enzym oxy ho trong ch phm khng
c vt qu 0,1 n v/mg axit ascorbic trong mt pht trn mt gam ch

phm.
Ch phm pectinase dng trong vang qu phi bo ton c hot trong
iu kin c cha ru (10-12%) v phi tc dng c hiu qu trong iu kin

c pH nht nh.
x l dch hoc b nghin trng thi dng c th dng ch phm pectinase
c hot cao (12000v/g) hoc dng ch phm pectinase khng tan.
Chng hn trong sn xut vang nho, khi s dng ch phm
pectawamorin PM10x, ngi ta thy c th tng hiu sut dch t chy ln 32%.
Trung bnh th tch ca phn dch t chy c th tng c 10%, cn hiu sut
chung ca dch tng ln 1 2%. Hoc khi p b nghin nho trng c x l
bng pectinol th thy qu trnh p tin hnh nhanh hn. Hiu sut dch khi
tng ln 9,6%. Vy dng ch phm pectinase s lm tng tc lm trong v
tc lc ca dch.

Nhm 20Trang 16

GVHD: Th.S Nguyn Th Thu Sang

Khi s dng ch phm pectinase, cc polime ca dch nh protein,
pectin s thy phn lm gim nht do lm tng tc lc. Trong 1 gi
dch thu c t b nghin ca nho c x l bng ch phm pectinase s qua
lc 7 ln nhanh hn t b khng c x l.
Khi dch khng c x l bng enzym th trong qu trnh lng, cc ht
vn c ln v nh s b kt ta xung. Khi s dng enzym th cc cht cao
phn t ca dch s b thy phn tng phn, nht b gim, kt qu l tc
lm trong u tng.
S dng ch phm pectinase s lm tng cht lng ca dch qu v ca
vang.
Trong qu trnh x l b nghin bng ch phm pectinase, thnh phn
ha hc ca b thay i rt ng k m trc tin l hm lng cht phenol.
Ngi ta thy rng khi x l b nghin bng ch phm pectinase s lm tng
hm lng catesin trong dch. Khi qu trnh trch ly s vt ln trc qu
trnh oxy ho catesin ngay c khi nhit tng. iu ny rt quan trng, v cc
hp cht phenol c bit l catesin c cc nhm hydroxil v tr octo thng c
hat tnh ca vitamin P. Nh vy x l b nghin bng ch phm pectinase s
lm tng gi tr sinh hc ca dch qu v vang.
Khng nhng vy vang c pha ch t dch v b nghin c x l b
bng ch phm pectinase s chn nhanh hn do cn chit sm hn. Ngi ta
cho thy rng ch phm thu c t nm mc, Botrylis cinerea gy nh hng
rt tt n cht lng ca vang, vang c hng thm mnh v du do hm
lng glyxerin v ester trong vang cao.
Khi ch bin nc qu c tht qu, ngi ta thng s dng ch phm
enzym bao gm pectintrancelinutase, hemicellulase, cellulase. Trong , enzym
pectintrancelinutase ng vai tr quan trng nht. Trong hn hp ch phm
enzym trn, tuyt i khng c c mt enzym polygalacturonase, c bit
khng c cha enzym endopolygalacturonase. Nhng enzym ny thng
lm gim nht ca nc qu v ph v ng nht ca nc qu.
Nhm 20Trang 17

GVHD: Th.S Nguyn Th Thu Sang

CHNG 2: CNG NGH SN XUT ENZYME

PECTINEASE
2.1.

Ngun gc thu nhn

T vi sinh vt : Nu thc vt ngi ta ch thy c mt loi enzyme th

vi sinh vt l mt loi enzyme rt phong ph.Vi sinh vt phn gii pectine

hu nh c mt cc i din nm mc, nm men, vi khun nh:
Nm

mc:

Asp.niger,

Asp.oryase,

Asp.terreus,

Asp.saito,

Asp.japonicus
Nm men: sac.ellipsoideus, sac.fragilis, sac.ludwigii
Vi khun: Bac.polymixa, Bac.felseneus, Clostridium roseum
2.2.

Phng php nui cy b mt

Quy trnh sn xut

Thu nhn enzy

Nui cy tron

Nhm 20Trang 18

GVHD: Th.S Nguyn Th Thu Sang

Thuyt minh quy trnh:

Mi trng nui cy
L mi trng rn, gm cc thnh phn t nhin: cm m, cm go, ng

mnh, bt u tng pha thm mui khong. Mi trng rn thng dng

nht l cm, t bit trong cm m c tng i y cc cht dinh dng v
khi lm m s to ra cu trc cn thit cho nm mc pht trin, sinh ra nhiu
enzyme. C th b sung nhng thnh phn giu pectin nh b c ci ng,
bt c rt lm cht cm ng thu c hm lng pectinase cao. Bn cnh
, chng ta cn b sung thm tru to mi trng thong kh tt, gip cho
s pht trin ca nm. Vy mi trng hon chnh nui cy gm c: 70%
cm m + 23% tru + 5% cht cm ng + cht dd khc ( mm m, nc khoai
ty, cung cp thm ngun Nit, Photphat, Kali, ). Mi trng rn cn
c lm m n khong 60%, nu m qu cao th nhiu loi vi khun pht
trin gy tp nhim, nu m qu thp s lm mi trng nhanh kh, sinh
bo t mnh.

Nui cy: Cc khay c mi trng cy mc c t vo phng nui

c sn cc gi.

Dng c: Dng c v mi trng cn c kh trng 121 0C/1atm/30

pht trnh tp nhim.

ngui, phi vo dng c: Mi trng c tri mng ra cc khay

c thanh trng vi lp dy khong 2 2,5cm v ngui n 30 0C th tin

hnh cy ging.

Ging

S dng nm mc.Nm mc c nhn v nui cy trong mi trng v trng

trnh nhim tp.ging c nhn theo phng php b mt v cng trn mi
trng trn ging quen vi iu kin sn xut. Khi nhn ging thng
cho nm mc mc gi n khi sinh ra nhiu bo t, bo t c thu theo
Nhm 20Trang 19

GVHD: Th.S Nguyn Th Thu Sang

phng php tch bo t khi mi trng v cha trong cc bnh c nt kn. T
l nhn ging vo khong 0,2 2%. Mi gam bo t mc c th cy vo 10kg
mi trng.

Nui cy
Cc khay c mi trng cy mc c t vo phng nui c sn cc

gi.Phng nui c th iu chnh c nhit , m v c thi gi.Nhit

thch hp vi a s mc l 30-320C.Nu nhit xung di 240C th nm
mc pht trin chm, sinh bo t yu, thi gian nui cy di nh vy s lm
gim kh nng sinh tng hp Enzyme.
Qa trnh nui cy nm mc trn b mt mi trung c chia thnh 3 giai
on:

Giai on 1: Khong 10 14h u: giai on ny c nhng thay i sau:

Bo t nm bt u hnh thnh c mu trng hoc mu sa.
Bt u hnh thnh enzym, khng i hi phi thng kh nhiu ch cn
lm thong khong 2 3 th tch khng kh / th tch phng / gi.
Khi mi trng cn ri rc, cc thnh phn dinh dng bt u c s
thay i.

Giai on 2: ko di khong 14 18h: giai on ny c nhng thay i

sau:
Mc pht trin nhanh, h hp mnh, si nm c th quan st bng mt thng,
lc u l lp lng t mu trng xm v ngy cng r, lm mi trng kt
bnh li, m mi trng gim dn.Cc cht dinh dng trong mi trng
tiu hao nhanh phc v cho cc qu trnh trao i cht trong t bo v ging
h hp mnh ta ra mi trng chung quanh 80 90kcal/gi. Lm nhit mi
trng tng ln 400 - 450C. Thi k ny cn phi thng kh mnh ti 60 th tch
kh/1th tch phng/gi cung cp O2 cho mc th v ui CO2 ra khi mi
trng, ng thi lm gim nhit bung nui. Nhit bung nui giai
on ny cn gi 28 - 300 C v m trong phng khong 90%.
Nhm 20Trang 20

GVHD: Th.S Nguyn Th Thu Sang

Cc loi Enzyme c hnh thnh trong giai on ny, pectinase c hnh
thnh nhiu nht v c c cht cm ng.

Giai on 3: ko di trong khong 10 20h: giai on ny c nhng

bin i
Qu trnh trao i cht yu dn v do mc gim cht dinh dng s chm
li. Lng nhit to ra gim, khong 15 30kcal/kg/gi.Thng kh khng qu
20 25 th tch khng kh/th tch phng/gi, gi nhit bung nui duy tr
300 C.
Ty thuc vo c tnh sinh l ca tng ging mc, thi gian nui cy c th
kt thc ti im m lng enzyme to thnh ti a, Enzyme vn c hnh
thnh giai on ny.
Mi trng sau khi nui cy c sy kh ti m di 12%, nghin nh,
ng trong cc bao polyetylen hoc giy chng m.Sn phm ny l ch phm
enzyme th.
2.3.

Tch v lm sch
2.3.1. Phng php thu nhn
C 2 loi enzyme: ni bo v ngoi bo, mi enzyme i hi phi c phng
php tch v thu nhn ring:

Enzyme ngoi bo: (gm pectinase) do vi sinh vt tit ra trong mi

trng nui cy ngoi t bo, thng ho tan trong nc do d trch ly v

tinh sch.

Enzyme ni bo: l enzyme c sn xut ra bn trong t bo vi sinh vt

v khng c tit ra mi trng bn ngoi. Nhng t bo vi sinh vt sau khi

c nui cy s c tch ra v ph v t bo. Mc ch ca vic ny l gii
phng ton b cc cht c trong t bo gm c enzyme ni bo. Hn hp thu
c s em ly tm tch tp cht v nhng cht c phn t lng ln; cn
enzyme s c kt ta bng cn hay mui. Enzyme sau s c em tinh
sch.
Nhm 20Trang 21

GVHD: Th.S Nguyn Th Thu Sang

Enzyme ni bo

Enzyme ngoi bo

Kh tch

D tch

Phi ph v thnh t bo

Khng cn ph v thnh t bo

Thng ln chung zi cc cht

KHng ln chung vi cc thnh

khc ca t bo sau khi b ph v

phn ni bo, nu c ch l mt

(acid nucleic, cht nguyn sinh,

vi enzyme ngoi bo khac

2.3.2.
T

lipid)
Ch bn vng trong mi trng

Bn cng hn

ni bo
Phng php tinh sch kh thc

Phng php tinh sch d v r

hin, quy trnh cng ngh phc

hn

tp, gi thnh t
mi trng nui cy b mt
chit rt enzyme t mi trng rn ngi ta dng nc, cc dung
dch mui trung tnh, cc dung mi hu c (cn, axeton).Nhiu kt qu th
nghim cho thy dng nc trong mc ch ny c kt qu tt v d c dng
rng ri trong sn xut.Theo phng php khuch tn bng nc c th chit
c lng enzyme trn 90 95% v trong nc chit khng cha cc tp cht
khng tan. Nc thng dung khuch tn nhit 25 28 0C. trnh tp
nhim nn thm vo nc mt t focmalin hoc cht st trng khc. Dch chit
thu c c mu nu sm, kh trong, cha 10 15% cht kh ha tan v c
lm lnh kp thi xung 10 120C.
Phng php tch chit v lm sach enzyme c s dng rng ri nht
hin nay l phng php kt ta enzyme bng dung mi hu c (etanol,
izopropanol v axeton). Cc dung mi hu c ny lm gim hng c in mi
ca mi trng. nh ta bit, lc ht tnh in t l nghch vi hng s in
mi. V vy cc enzyme, cc cht protein cng nh cc cht c phn t thp
trong h dung dch nc dung mi hu c s ta v lng xung.
ha tan ca enzyme vo dung dch cn nc ph thuc vo nng cn,
nhit , lc ht ion ca dung dch v tnh cht protein ca enzyme. trnh
Nhm 20Trang 22

GVHD: Th.S Nguyn Th Thu Sang

mt hat tnh ca enzyme tt c phi c lm lnh xung 3 5*C. Khi trn
phi khuy mnh, khi cc enzyme kt ta v lng xung di cn tch ly ngay.

S tinh sch Enzyme t canh trng nm mc.

2.3.3. T mi trng nui cy b su

Phng php hiu kh

S tch t enzyme trong mi trng c bt u khi s pht trin ca vi sinh

vt gn t n pha n nh, khi mi trng b acid ho mnh v khi lng
phospho v c c s dng hon ton, pH ca mi trng nui cy thng
t t 6-7.2 l thch hp. i vi nm mc, pH km hm s tng hp sinh khi
v s tch lu enzyme pectinase.Khi pH dch v pha acid, ngay c khi pH nm
trong khong 4.5-5.0, tuy s to thnh sinh khi khng b nh hng nhng s
to thnh enzyme pectinase b km hm.
Vt liu gieo cy c th l si nm 24, 32 v 48h tui v vi hm lng t 210%.i vi A.niger v A.awamori, vt liu gieo cy l si nm c s b
trong mi trng dinh dng cho n khi bt u nt nanh bo t, thi gian
s b thng l 38-42h.Lng si nm em gieo cy thng l 2%. Trong qu
trnh nui cy, hm lng cc cht ho tan trong mi trng gim t 6% xung
cn 1.5-1.8%
thu ch phm kh, cn tch si nm ra khi canh trng lng. C c chn
khng canh trng lng n khi hm lng cht kh t 5-8% ri sy kh trn
thit b sy phun. iu kin sy phun l nhit cht ti nhit i vo phi t
165-180 C v i ra t 60-70 C. Thi gian lu ca ch phm enzyme
trong thit b sy phun phi khng qu 7 giy v nhit ch phm sau khi sy
phi khng qu 40 C. Ch phm thu c cn phi ng gi kn trnh
ht m.
Phng php ym kh

Nhm 20Trang 23

GVHD: Th.S Nguyn Th Thu Sang

Mi trng: b c ci 2%; (NH4)2HPO4 0.75%; KH2PO4 0.1%; CaCO3 0.3%;
nc chit ng 0.5%
Clostridium pectinofermentants 15 c kh nng tng hp pectinase mt cch
mnh m pha tng trng ca qu trnh sinh trng v tng ng thi vi s
tch lu sinh khi. S tch lu enzyme s ti a tng ng vi pha n nh ca
s sinh trng qua 55-60h, pH ban u ca mi trng dinh dng l 6.5-7.0.
Vt liu gieo cy ban u c chun b trng thi canh trng cha bo t
v c cy vi lng 4% theo th tch. Qu trnh nui cy c tin hnh
nhit 35%
Cl.felsineum cng c th c nui cy ym kh thu pectinase. Thnh phn
mi trng gm c: lactose 2%; pectin c ci 1%; (NH4)2HPO4 0.4%; K2HPO4
0.7%; KH2PO4 0.3%; NaCl 0.1%; MgSO4 0.025%; FeSO4 dng vt; CaCO3
0.5%; dch nm men t phn 0.05%; ascorbic acid 0.5%
C th tin hnh thu ch phm t dch lc canh trng bng cch kt ta
enzyme vi dung mi hu c hoc vi mui ammonium sulfat. Nu kt ta
bng dung mi hu c, pH ca dung dch x l l 6.5-6.8.Nu kt ta bng
2-2.5 th tch aceton th hot enzyme trong kt ta t 93-95% so vi hot
ban u.
Khi kt ta ammonium sulfat c bo ho bng 0.2 th s thu c ch phm
ch cha pectinesterase v pectintranseliminase, khi bo ho l 0.9-1.0 th s
thu c ch phm ch cha pectintrenseliminase v exopolygalacturonase.
2.4.

Phng php tinh sch

C rt nhiu k thut v phng php tinh sch khc nhau tinh sch enzyme.
Cc k thut ny c p dng ch yu da vo tnh cht ca phn t protein
nh kch thc, khi lng phn t, kh nng tch in, ... Mi k thut tinh
sch u c u v nhc im ring trong vic chit tch cc loi proteinenzyme khc nhau. Trong khun kh ca lun vn ny, chng ti xin gii thiu
mt s k thut c bn v thng c p dng trong tinh sch pectinase
2.4.1. Gii thiu chung
Cc dng ch phm enzyme:

Nhm 20Trang 24

GVHD: Th.S Nguyn Th Thu Sang

Ch phm th: ch phm th t canh trng lng v ch phm th t

canh trng b mt.

Ch phm k thut: l ch phm c tinh ch s b, trong mt s

protein v enzyme tp c tch ra. Trong ch phm k thut thng cha

mt vi enzyme ch yu.

Ch phm tinh khit: l ch phm trong ch cha mt loi enzyme

nht nh vi hm lng cao.

Thng tinh sch enzyme t cc ch phm enzyme th. Ch phm th thng
cha nhng thnh phn c bn sau:

Nc chim khi lng ln nht.

Protein khng c hot tnh sinh hc.

Cc tp cht t t bo (nu l ngun ng, thc vt), t mi trng nui

cy (nu l ngun VSV).

Enzyme (hay protein c hot tnh sinh hc cao).

Qu trnh tinh sch enzyme l qu trnh loi b ba phn u, ch nhn

sn phm cui l enzyme mong mun (trong phn ln enzyme tp c

loi b). Vic loi ba thnh phn u khng th cng thc hin trong mt giai
on m phi thc hin qua nhiu giai on khc nhau, mi giai on, ta s
loi b c mt phn khng phi l enzyme mong mun ra khi hn hp,
khi

lng

ch

phm

gim

theo.

ngha ca k thut tinh sch trong cng ngh enzyme:

Khi lng ch phm enzyme c gim dn qua tng bc tinh sch.

Tng hot tnh enzyme c ngha quan trng trong qu trnh s dng

sau ny.

Tng gi tr kinh t thng qua gi thnh sn phm.

To nhng ch phm tinh khit nhm mc ch nghin cu hoc ng

dng trong y hc..

Nhm 20Trang 25

GVHD: Th.S Nguyn Th Thu Sang

Tm tt qu trnh tinh sch
2.4.2. Phng php kt ta

Kt ta bng cch thay i lc ion.

Nguyn tc

Enzyme ha tan trong dung dch da vo s cn bng gia lc tnh in v

tng tc k nc. S kt ta enzyme xy ra khi ta thm mui trung tnh vo
dung dch vi nng thch hp. Khi , enzyme kt ta thun nghch v c
th thu li dung dch enzyme bng cch tch (ly tm) v ha tan li kt ta.
Ngoi ra, trong mt s trng hp, khi b sung mui cn c tc dng lm bn
enzyme, chng li s bin tnh v phn hy hoc nhim vi sinh vt

Tc nhn kt ta

kt ta enzyme c th s dng nhiu loi mui khc nhau. Trong thc t,

ammonium sulfate c dng ph bin nht v mui ny c ha tan cao
trong nc, t c lc ion cao v gi thnh r. T trng ca dung dch bo
ho khong 1.235 g/mL, thun li cho qu trnh ly tm tch kt ta.

Cc yu t nh hng
Nng mui: nng thp, s c mt ca mui lm bn nhm mang
in trong phn t enzyme v lm tng tnh tan ca enzyme. Khi nng
mui tng th tan ca protein cng tng. Nu tip tc tng nng
mui th tan ca enzyme s gim v enzyme bt u kt ta v khi
thm mui vo dung dch, mt s phn t nc s solvat phn t mui
Tnh cht ion: s nh hng ca mui n qu trnh kt ta c xc
nh qua tnh cht ca cc ion, trong in tch ion l yu t nh
hng nhiu nht. Hiu qu nh hng ca cc ion m gim dn theo
trt t sau: phosphate, sulphate, acetate, chloride Nhng ion dng
ha tr mt c nh hng mnh hn ion dng ha tr hai. Hiu qu nh
hng xp theo th t gim dn ca cc ion dng nh sau: NH 4+ , K+ ,
Na+.

Nhm 20Trang 26

GVHD: Th.S Nguyn Th Thu Sang

Thnh phn, nng dch protein, nhit qu trnh cng c nh hng
n s kt ta protein. Nhit cng cao th tnh tan ca protein cng
gim

Phng php tin hnh

Nng mui ti u cn s dng thay i ty theo tng trng hp v c

xc nh bng thc nghim. Khi thc hin th nghim, cn tin hnh b sung
ammonium sulphate chm v khuy u. Kt ta c th tch ra bng ly tm hay
lc. Phng php lc ch c p dng khi t trng ca protein kt ta gn
bng vi t trng trong dung dch.
Trong mt s trng hp, c th s dng phng php kt ta phn on
tinh sch protein. Ngi ta b sung ammonium sulfate vo dch trch n nng
m protein mong mun khng ta, cc kt ta xut hin s c tch b.
Sau b sung mui vo dch t n nng m protein mong mun kt
ta. Cui cng, tch v thu nhn kt ta.

ng dng trong nghin cu tinh sch pectinase

Ammonium sulphate l tc nhn thng gp nht trong nghin cu tinh sch

enzyme ni chung v c bit trong tinh sch enzyme pectinase (Afifi A.F. v
cng s, 1988; Miyazaki Y., 1990; Jaffar M. B. v cng s, 1993, Guo ChungTeng v cng s, 2001). Ammonium sulphate c u im ni bt l nguyn liu
r tin, ph bin v t c kh nng gy bin tch bt thun nghch enzyme. V
th, y thng l tc nhn kt ta c nh sn xut u tin la chn. Nng
ammonium sulphate ti u cho tinh sch enzyme thng nm trong khong
t 60-80% tu vo tng loi enzyme c th.

Kt ta enzyme bng dung mi hu c

Nguyn tc

Qu trnh kt ta enzyme da trn s thay i hng s in mi ca mi

trng. Lc y v lc ht t l nghch vi hng s in mi ca mi trng.
V vy, khi b sung cc dung mi c hng s in mi nh (ethanol, acetone...),
Nhm 20Trang 27

GVHD: Th.S Nguyn Th Thu Sang

chng s lm gim hng s in mi ca mi trng, t lm gim kh nng
solvat ha ca cc phn t nc i vi phn t enzyme trong dung dch. Do
vy, tnh tan ca enzyme gim v enzyme b kt ta. Hin tng kt ta xy ra
tt hn v cn t dung mi hn khi pH dung dch gn vi pI ca enzyme.

Dung mi

Protein c th kt ta bng dung mi hu c tan trong nc, nh ethanol,

acetone, methanol, isopropanol. Trong , acetone v ethanol l hai dung mi
ph bin nht trong vic kt ta protein. Dung mi s dng phi an ton, khng
c v c nhit bc chy ln hn 20oC.

Cc yu t nh hng
Kch thc ca protein cng nh hng n s kt ta. Nhng protein
c kch thc ln s ta nng dung mi thp hn so vi protein c
kch thc nh
S ta nhit ca dung mi: khi b sung dung mi vo dung dch
enzyme, nhit ca hn hp tng v y l mt qu trnh ta nhit. Khi
nhit tng cao, cu hnh khng gian ca enzyme b thay i v
enzyme c th b kt ta bt thun nghch
T trng ca dung mi cng nh th nht cng thp, kt ta cng d
tch
di mch carbon ca dung mi cng di th kh nng gy bin tnh
bt thun nghch cng tng.

Phng php thc hin

Thng thng, ngi ta b sung dung mi vo dung dch enzyme vi mt t l

th tch nht nh. Sau , dung dch enzyme cn c khuy u v yn
trong mt thi gian nhm to iu kin kt ta thun li. Khi cc enzyme
kt ta ht th cn phi tch ra ngay bng qu trnh ly tm lnh. Trong kt ta
vn cn mt phn dung mi. Lng dung mi ny c th tch ra bng phng
php bc hi p sut chn khng
Nhm 20Trang 28

GVHD: Th.S Nguyn Th Thu Sang

Kt ta enzyme s dng polymer hu c

Nguyn tc:

Cc polymer hu c l dung mi c tnh ho nc ln. khi b sung vo dung

dch cha protein, chng c th tng tc vi lp v hydrate bao quanh phn t
protein v lm gin kh nng ha tan ca protein. Nh , cc phn t protein
v enzyme s kt ta v tch ra khi dung dch.

Cc dung mi polymer hu c:
Thng dng l: polyethylene glycol (PEG 6000) v polyethylene glyco
(4000).

Nhc im:

Tng nht dch enzyme, gy kh khn cho vic tch kt ta ra khi dung
dch. Nu s dng phng php lc phn ring, thi gian lc s ko di, cn
nu s dng phng php ly tm s tc ng ln dung dch 1 lc ly tm ln.
iu ny rt kh thc hin khi trin khai quy m sn xut ln. Nu lc ly tm
nh th c th khng thu hi ht lng kt ta. Theo thc nghim th tinh
sch ca ch phm tng ch dao ng t 1.16 n 1.57 ln. Nh vy, nhn
chung th hiu qu ca phuong php l khng cao.
2.4.3. K thut siu lc

Nguyn tc:

K thut siu lc (Ultrafiltration) l qu trnh phn ring chn lc cc hp cht

bng mng lc c kch thc siu nh vi p sut lm vic vo khong 0,5
5bar. ng knh mao qun trung bnh t 2 n 50 nm.

Vt liu lc

Trong phng php ny, vt ngn phn ring cc cu t l membrane. p

lc l ng lc duy nht ca qu trnh. Membrane c th c sn xut t
nhiu loi vt liu khc nhau nh: cellulose acetate, polyamide, polysulfone
hay ceramic.
Nhm 20Trang 29

GVHD: Th.S Nguyn Th Thu Sang

ng dng trong nghin cu tinh sch pectinase

Trong lnh vc tinh sch enzyme ni chung, c bit l pectinase, qu

trnh siu lc thng c p dng trong giai on u ca quy trnh tinh sch.
Mc ch ca qu trnh l tch nc v cc tp cht c phn t lng nh trong
canh trng lng nh cc ion kim loi, ng st, acid amin, peptide (dng
permeate). Trong hu ht cc trng hp, enzyme tinh sch phi c gi li
trn b mt vt liu lc (dng retentate). V th, vt liu lc cn phi c kch
thc mao qun nh hn kch thc enzyme.

Phn ln cc enzyme polygalacturonase c khi lng phn t trong

khong 30-80kDa (Jayani R. S. v cng s, 2005). V vy, tinh sch canh

trng nui cy thu nhn loi enzyme ny th cn vt liu c kch thc nh
hn 30kDa. Kch thc ny khng tch c cc protein tp (c khi lng
phn t ln hn 30kD) b ln trong canh trng.

Kt qu nghin cu ca Manachini P.L. v cng s (1987) khi tinh sch

endo-PGase bng k thut siu lc s dng si Diaflo (kch thc 10kDa) t

canh trng nui cy nm si Rhizopus Stolonifer cho thy, tinh sch ca
ch phm sau tinh sch tng ln 2,1 ln vi hiu sut thu hi l 89%. Mt
nghin cu khc ca Gainvors A. v cng s (2000) c th nng cao tinh
sch pectinase ln n 10,4 ln vi hiu sut thu hi khong 83% bng k thut
siu lc vi kch thc l 30kDa. Tuy nhin, trong mt s trng hp, vic p
dng k thut siu lc khng t hiu qu cao do la chn kch thc l mng
khng thch hp (Dinu D. v cng s, 2007).
2.4.4. Phng php sc k
Thut ng sc k dng ch cc k thut phn tch v iu ch cho php tch
bit cc hp cht khc nhau ca mt hn hp. Php phn tch sc k ny da
vo s di chuyn khc nhau trong mt pha ng ca cc cht ho tan c
gn trn mt pha tnh trng thi rn. Ngi ta thng chn cc cht c kh
nng kt gn c vi cc cht nh phn tch lm pha tnh. Tng tc gia
cht ho tan v pha tnh c th l tng tc hp ph, tng tc trao i ion,
Nhm 20Trang 30

GVHD: Th.S Nguyn Th Thu Sang

tng tc k nc, tng tc kiu ry phn t hoc tng tc c hiu sinh hc.
Da vo cc kiu lin kt ngi ta chia ra thnh cc kiu sc k sau:

Sc k lc gel: (sc k loi tr phn t) phn on protein theo klg.

Sc k lc (sc k loi tr khng gian, loi tr khuch tn hay phng php

loi tr phn t) cho php phn on mt s hn hp protein theo khi lng
phn t ca chng. Thc t ngoi kch thc ra, hnh dng ca phn t cng c
nh hng.Tuy nhin thng s ny c th b qua nu protein trong hn hp l
hnh cu. Theo phng php ny, cc loi gel c np vo trong ct dng
tch enzyme, cc phn t c tch ra da theo kch thc v hnh dng ca
chng.
Pha tnh: cc ht gel xp cha y trong ct.
Khi cho dung dch cc protein chy qua ct th cc phn t ln nht ca hn
hp c ng knh ln hn l ca pha tnh, khng th khuch tn vo bn trong
ht nn b loi tr v chng s i ra khi ct cng vi th tch dch ra bng th
tch gia cc ht gel (Vo).
Cc phn t b, ngc li, s khuch tn ti a vo torng ht gel v th tch
dch ra ca chng s bng tng th tch pha lng ca ct (V1).
Cn cc phn t c kch thc trung gian s khuch tn hn ch vo ht, th
tch dch ra ca chng cng ln khi qung ng i (ca chng) vo bn trong
ht cng di th kch thc ca chng cng nh.
Phng php lc gel thng c ng dng tch cc phn t nh nh mui
v c v cc phn t nc thng qua cu trc mng li ca sephadex. Thng
phng php ny ch lm tng c c nhng gim hiu sut thu hi sn
phm do cc phn t protein b kt li gia cc ht gel.

Sc k trao i ion: lin kt ion.

Nguyn tc:

Da trn s tch in ca cc phn t protein. Phng php trao i ion c

s dng rng ri trong sn xut ezyme trong quy m cng nghip.
Tnh cht tch in ca phn t protein chu nh hng rt nhiu bi gi tr pH,
Nhm 20Trang 31

GVHD: Th.S Nguyn Th Thu Sang

t

ngi

ta

chn

cht

trao

anion

hay

cation.

Trong sc k trao i ion, vic gn mt protein enzyme vo nha trao i ion

s ph thuc vo trng thi ion ho ca potein, cng nh trng thi ion ho ca
nha trao i, pH, lc ion v nhit .
Qu trnh phn tch trn bao gm hai giai on:
Hp th thun nghch protein-enzyme cn tinh sch (v cc protein c in tch
gn ging nhau) vo nha trao i ion.
Kh hp ph cc ion c hp th.
Bng cch thay i pH ca dch ra s dn ti s thay i ion ho v do
thay i in tch tng ca protein. Hoc bng cch tng lc ion v tng nng
ion i cnh tranh. Cc protein enzyme no c i lc vi nha trao i ion
yu nht s b y ra trc tin v ngc li.
Sc k i lc: lin kt k nc.
Sc k hp ph: lin kt yu nh lin kt k, nc, lin kt Van der Waals.

2.4.5. Phng php thm tch

Nguyn l: trong qu trnh tinh sch cc enzyme, loi b cc phn t
ho tan nh khng mong mun khi dch trch ly enzyme nh l amoni sulfat
sau kt ta, dng mt mui kh hp th enzyme trong sc k trao i ion,
hoc mt phi t cnh tranh trong sc k i lc,... ngi ta thng dng
phng php thm tch.
Thi gian nui

Hot ng pectinase
(dvhd/1g canh trng)

30

361

32

740

34

1150

Nhm 20Trang 32

GVHD: Th.S Nguyn Th Thu Sang

36

1300

38

887

40

288

Ti thm tch c cu to bng mt mng bn thm (thng bng solephan).

Ti thm tch c cha dch chit enzyme, c t vo trong mt dung dch
m khng c cha cht ho tan cn loi b.
Cht ho tan khuch tn ra khi mng theo chiu Gradient nng , trng thi
cn bng th nng cht ho tan bn trong ti v bn ngoi dch m s cn
bng nhau do phi dng mt th tch ln dung dch m v thnh thong
phi thay dung dch mi.
(NH4)2SO4 c cho vo ti thm tch, ri t ti vo dung dch c cha
polymer
cacboxyl methyl cellulose, polyetylenglycol (PEG) l nhng polymer c tnh
ho nc.

2.4.6. Kt ta i lc
Kt ta i lc l phng php phn tch da trn s to phc gia mt phi t
i lc vi protein cn tch nm trong dch enzyme th, sau phc ny s
c

kt

ta

bng

cch

thm

vo

mt

tc

nhn

hp

l.

Alginate l copolymer ca acid guluronic v acid mannuronic, to kt ta vi

ion Ca 2+, c dng lin kt vi pectinase trong qu trnh tinh sch (i t
A.niger). Nu x l alginate bng vi sng nhit 75 C s lm tng kh
nng chn lc, ch phm enzyme thu c c t l tinh sch 20 ln v gi c
83% hot tnh.
2.5.

Thu ch phm t ASP.NIGER 72-32

Thnh phn vi mi trng thch hp chng Asp.Niger cho pectinase cao.

2.5.1. nh hng thi gian nui

Nhm 20Trang 33

GVHD: Th.S Nguyn Th Thu Sang

nghin cu nh hng ny Asp.niger c nui cy trn mi trng c
m 50% v nui nhit 30oC trong khang t 30-40 gi.Sau 2 gi xc nh
hat pectinase ca canh trng.Kt qu thu c ghi bng 3.
Qua bng 3 thy s sinh tng hp pectinase ca chng nm mc ny xy ra
mnh m t gi th 32 tr i v t cc i vo gi th 36.Sau gim dn.
2.5.2. nh hng nhit nui
Asp.niger 72-32 c nui cy trn mi trng c bn c m 50% nhit
khc nhau.Sau 2 gi xc nh hat pectinase ca canh trng.
Nhit nui (0C)

Hot ng pectinase (dvhd/1g canh trng)

28

803

30

1134

32

1351

34

1688

36

764

48

520

Bng: nh hng nhit nui n sinh tng hp pectinase Asp.niger 72-73

Ta thynhit t 30-32oC l thch hp nht cho sinh tng hp pectinase ca
Asp.niger 72-32.
2.5.3. nh hng m ban u ca mi trng nui cy
Asp.niger 72-32 c nui cy trn mi trng c m ban u tahy i t
40-65% v nui 32oC. Sau 36 gi xc nh hat pectinase ca canh
trng
m ban u Hot ng pectinase
40

(dvhd/1g canh trng)

481

45

731

50

957

Nhm 20Trang 34

GVHD: Th.S Nguyn Th Thu Sang

55

1355

60

897

65

638

Bng: nh hng m ban u n nui n sinh tng hp

pectinase Asp.niger 72 -73
Qua ta thy m ban u ca mi trng c nh hng r rt n s hnh
thnh pectinase ca Asp.niger 72-32.Hat pectinase mnh nht khi mi
trng nui cy c m ban u l 55%.Khi m hi tng hat pectinase
gim i 1 cch nhanh chng, ch cn xp x 2/3 hc khi m ban u 6065%.

Lng nguyn Hot ng pectinase (dvhd/1g canh trng)

MT c c rt
MT c c ci
MT c b to
liu
cha
pectin
0

142

142

142

302

296

202

621

532

496

980

820

600

1290

1050

726

11

1650

1265

850

12

1400

1500

970

13

1150

1300

1100

14

930

1180

900

15

650

870

800

Nhm 20Trang 35

GVHD: Th.S Nguyn Th Thu Sang

nh hng ca hm lng v ngun nguyn liu cha pectin khc nhau n sinh
tng hp pectinase ca Asp.niger &72-32 (hng php ng)
Hot ng pectinase (dvhd/1g canh trng)

Lng
nguyn liu
cha pectin
(%)
0

0.39

0.39

0.39

0.65

0.59

0.49

0.72

0.62

0.59

11

0.89

0.65

0.6

12

0.79

0.79

0.65

13

0.62

0.62

0.68

14

0.58

0.58

0.52

15

0.5

0.5

0.46

nh hng ca hm lng v ngun nguyn liu cha pectin khc nhau n sinh
tng hp pectinase ca Asp.niger &72-32
Theo tnh tan s b cho thy mi trng k trn hi thiu nit.V vy nghin
c nh hng ca vic cho thm mui (NH4)2SO4 vo mi trng. Asp.niger 72-32
c nui cy trn mi trng c bn c cha 11% c rt vi hm lng mui amon
sunfat thay i, nui 320C v sau 36 gi em xc nh hat pectinase ca canh
trng.

kt

qu

thu

bng

Qua bng 8 nhn thy mui amon sunfat trong mi trng c nh hng n tng hp
pectinase ca nm mc trn mi trng c c rt 11%. Khi thm t 1-2.5%amon sufat
hat pectinase ca chng Asp.niger c th tng t 7- 10
2.6.

Xc nh hot tnh enzyme

Xc nh hot tnh pectinase c th tin hnh bi mt trong nhng
phng php sau y:
2.6.1. Phng php nht k
nht ca dung dch cha pectine s thay i sau mt thi gian b
pectinase tc dng. nht c o bng nht k, thng qua thi gian dch

Nhm 20Trang 36

GVHD: Th.S Nguyn Th Thu Sang

chuyn

mt

th

tch

nht

nh

dung

dch

cha

pectinase.

S dng 9 bnh tam gic, mi bnh cho vo 50ml dung dch pectin 0.6% vo t
m 50 C, thi gian 15 pht, sau cho enzyme vo vi lng t 0.1-0.9 ml
v b sung nc ct cho 1ml. Duy tr 9 mu ny nhit 50 C, thi
gian 1h, sau lm lnh xung 2-3 C, lc qua vi 2 ln.
Ly 5ml dung dch th nghim ny cho qua nht k o nht thng qua
thi gian chy ca 9 mu dung dch cha pectine em th nghim ni trn.
2.6.2. Phng php ng chung(Cu-pectat)

Nguyn tc:
Thy phn pectin di tc dng pectinase.Sau dng CuS04 5% kt

ta axit pectin di dng ng pectat(Cu-pectat).Ha tan kt ta bng NH 4OH

25%. Xc ng lng ng nh it. Mt n v hat ng pectinase theo
phng php ny l mt lng emzyme c kh nng xc thy phn ht 1 mg
pectin trong thi gian 1 gi iu kin tiu chun.
2.6.3. Phng php so mu:
Theo phng php ny: Mt n v hat ng pectinase l mt lng
emzyme c kh nng xc tc thy phn ht 1 g pectine trong thi gian 1gi
thnh acid galacturonic iu kin tiu chun.

Nhm 20Trang 37

GVHD: Th.S Nguyn Th Thu Sang

CHNG 3: NG DNG ENZYME PECTINEASA TRONG LM

NC QU
3.1.

Gii thiu v ng dng enzyme pectinase

Trong sn xut thc phm, ngi ta s dng cc ch phm pectinase dui dng
tinh khit. Ngi ta khng dung ch phm di dng canh trng nm mc sy
kh. T l ch phm pectinase c c trn lng nguyn liu em ch bin vo
khong t 0,03- 0,05 n 0,10%.
Trong sn xut ru vang, cng nh trong sn xut nc qu v cc nc ung
khng ru, u c th s dng pectinase mt cch hiu qu. Nh tc dng ca
pectinase m cc qu trnh p, lm trong v dch lc qu rt d dng, do lm
tng hiu sut sn phm. Chng hn a pectinase vo khu nghin qu, s lm
tng hiu sut nc qu sau khi p ti 15 25%. Bi l khi c pectinase phn
gii cc cht pectinase m dch qu trong sut khng b c v lc rt d dng.
Khng nhng vy. Enzyme pectinase cn gp phn chit rt c cc cht
mu, tannin v nhng cht ho tan na, do lm tng thm cht lng thnh
phm.
Tn ti 3 nhm enzyme pectinase phn gii pectin.
-

Pectin methyl esterase

Depolymease
Exoenzym pectinase

Pectin methyl esterase (EC.3.1,1,11): Enzyme ny tham gia phn gii pectin
thnh acid pectic v methanol. Enzyme ny cn c tn khc l pectiestenase
(PE).Hiu sut thu phn pectin ca enzyme ny rt l cao, c th t c
98%.
Nhm 20Trang 38

GVHD: Th.S Nguyn Th Thu Sang

Pectinesterase ca nm si hot ng mnh pH 3-5.
C hai loi pectinase ny b bin tnh nhit 800C.
Ezyme pectin methyl esterase tham gia tc ng vo COOH3theo c ch sau:
Pectin depolymease: Ezyme ny c trong t bo thc vt. Tuy nhin hin nay
ngi ta sn xut enzyme ny theo quy m cng nghip t nm si v t vi
khun. Chng c phn ra lm 4 loi nh sau:
1.
2.
3.
4.

Endo pectin traseliminase (EC,4.2.2.3), vit tt l PTE pectiliase.

Endo petin acid traseliminase (EC, 4.2.2.1), vit tt l PATE.
Endo poly galacturonase (EC, 3.2.1.15), vit tt PMG.
Endo poly methyl galacturonase

.
Vic ng dng pectinase thu nhn nc qu c p dng ln u tin vo
nm 1930. T n nay vic p dng enzyme ny tr nn rt ph bin
nhiu nc trn th gii. Vic thu nhn nc qu t trc n nay ch yu
bng phng php p. Nu pectin cn nhiu s theo nc qu v gy ra hin
tng nc qu b c, c keo cao v rt kh lc trong.
Trong t bo ca qu nc chim khong 90-95%. Nu ta ch nghin sau p
th ta ch c th thu nhn c khong 60-70% l ti a. Khi ta cho enzyme
pectinase vo, hiu sut p s tng 15-30%. Nhiu trng hp, hiu sut p
tng n 50%. Liu lng ch phm enzyme tinh khit cho vo l 0,03- 0,05%
hoc ch phm th l 0,5 2%. Nhit duy tr cho qu trnh thu phn l 43
45%.Thi gian thu phn l 4 8h.dch qu thu c bng pectinase s tronng
hn, kh nng lc s tt hn v hiu qu kinh t thy r.
3.2. ng dng enzyme pectinase sn xut nc qu
3.2.1. Cc ch phm enzyme
Trong sn xut nc qu v ru vang, vic s dng enzym l mt tin b khoa
hc rt ln.Cc ch phm enzyme c s dng trong sn xut nc qu v
ru vang c th l mt hn hp nhiu loi enzyme v cng c th ch l mt
loi enzyme ring bit.vic s dng hn hp enzyme hay tng loi enzyme
ring bit ph thuc vp nguyn liu v sn phm cn t ti. Ngoi ra, vic s
dng cc loi ch phm enzyme nh trnh by cn ph thuc vo cng ngh
sn xut ( cc yu t k thut).
Nhm 20Trang 39

GVHD: Th.S Nguyn Th Thu Sang

Trong sn xut nc qu v ru vang, ngi ta thng s dng mt trong su
nhm enzyme sau:
Nhm ch phm enzyme dng sn xut nc qu c. Mc ch s dng
nhm ch phm enzyme ny l lm tng hiu sut trch ly thu c lung
sn phm ln.
Nhm ch phm enzyme dng sn xut nc qu trong, khng cha pectin.
Mc ch s dng nhm ny l lm tng hiu sut trch ly v thu phn hon
ton cc cht protein, pectin, lm gim nht v lm trit tiu nguyn nhn
lm c nc qu.
Nhm ch phm enzyme dng lm tng kh nng ng ho nc qu v tht
qu, lm tng kh nng trch ly nc qu.
Nhm ch phm enzyme dng sn sut bn sn phm ru vang nhm lm
tng hiu sut trch ly ca bn sn phm.
Nhm ch phm enzyme dng ngn cn qu trnh oxy ho v lm cn tr s
pht trin ca vi sinh vt hiu kh pht trin trong nc qu, trong ru vang.
Nhm ch phm enzyme dng vo mc ch chng li ng trong sn sut
siro thnh phm.
Vic s dng cc ch phm enzyme phi c xem xt k lng trn c
s c im nguyn liu tri cy cn x l.Trong cc c im ca tri cy,
ngi ta quan tm rt nhiu n c im mu sc t nhin ca sn phm.
Trong nhiu trng hp, vic s dng enzyme phi m bo gi c mu sc
t nhin ban u hoc to ra nhng mu sc theo mun bng cch iu khin
nhng phn ng enzyme. Theo mu sc t nhin, cc loi tri cy c chia
lm hai loi:
Tri cy c mu nht: to, l, nho trng, chui, cam, xoi, bi..
Tri cy c mu sm do cha nhiu antian: anh o, mn , nho ,
mn en, du..
x l qu nghin c mu nht, ngi ta thng s dng mt lot cc
enzyme sau:
Enzym endo-PmG lm gim nht ca dch qu.
Enzyme PE lm tng hiu sut trch ly
Enzyme khc nh cellulose, hemicelluase, protease khng bt buc.
3.2.2. ng dng ch phm pectinase trong sn xut nc qu
Nhm 20Trang 40

GVHD: Th.S Nguyn Th Thu Sang

T cc loi qu khc nhau, ngi ta thng ch bin thnh cc loi nc qu
trong, nc qu c, trc tin phi chit rt c dch qu t m c bn, m
b i khi m c hc na. Do , hiu sut chit rt dch qu s ph thuc vo
kh nng thm ca t bo, cu to gii phu v tnh cht c l ca nguyn liu,
nht ca chnh dch qu, chc ca tht qu, cng nh thnh phn nh
tnh v nh lng ca pectin cha trong dch bo v trong thnh t bo ca m
qu.
s dng vo vic ch bin nc qu trong th ch phm th pectinase pha
c enzym endo v exo-polygalacturonse, enzym pectinestenaza.Trong ch
phm loi ny th nhng enzym va k trn l nhng enzym quyt nh hiu
qu ca ch phm.V endo v exo-polygalacturonse s lm gim nht ca
dch qu, cn enzymPE cng gp phn vap tc dng ca enzym ny.Enzym
proteinase ca ch phm s thu phn v protein ca v nguyn sinh t bo, kt
qu l lm cho s thot ca dch qu d dng.S c mt ca xenluase v
hemixenlulase trong ch phm cng tt nhng khng bt buc. khi s dng
cho cc loi qu nh to, l th trong ch phm khng c php c enzyme
pectinaseliminase v enzym ny s phn hu protopectin vn gn cc t bo
ring bit ca m qu li vi nhau, do gy mn ho m qu, lm tng
nht ca dch qu v kt qu l lm gim hiu sut dch qu. Ngc li, khi s
dng cho cc loi qu mng nh du tyth s c mt trong ch phm enzym
PTE li cn thit v enzym ny s lm mn ho t bo lm cho tnh cht thot
nc ca t bo tng ln cn nht ca dch qu li gim xung.
Ngoi ra, i vi cc ch phm pectinase dng trong sn xut nc qu trong
cng khng c php c cc enzym oxydase( ascohatoxydase,
poliphenoloxydase).
Trong ch bin nc qu c tht th cc enzym PTE, hemixenlulase v
xenlulase li l nhng enzym quyt nh hiu qu tc dng ca ch phm.trong
vai tr dn ng l enzym PTE c tc dng phn hu protopectin ca m
qu. Hemixenlulase v xenlulase s cng vi PTE lm cho ng th ca
nc qu c mu sang th trong ch phm khng cha enzym oxi ho, cn cho
cc nguyn liu c carotenoid v antian th khng cha enzym ph hu cc
cht ny.
Nhm 20Trang 41

GVHD: Th.S Nguyn Th Thu Sang

thu c nc qu, trong trng hp c s dng enzym pectinase, ngi ta
cng phi nghin nh nguyn liu qu. B nghin nhnh c em x l bng
enzym, sau mi em p hoc ly tm.Trong trng hp c s dng enzyme,
ngi ta c th nghin nh ti mc ti a, ct sao thun li cho tc dng ca
enzym m khng s b tc nghn knh dn dch qu trong qu trnh p sau ny.
Dng pectinase khng ch lm tng hiu sut dch qu m cn lm trong
dch qu. Phng php s dng enzym c hiu qu hn c.Bi l nh tc dng
ca cc enzyme m cc h keo ca nc qu s b ph hu hon ton.Nu nh
khi lm trong bng cc phng php khc pectin b kt ta mt phn th khi x
l bng pectinase, pectin b phn gii hon ton thnh cc cht ho tan.
Vi phng php enzym, khi mang mt lng tha ch phm s loi tr c
trng hp lm trong khng hon ton.
Dch nc qu c x l bng ch phm pectinase thng c v hon ton
hn v t c khuynh hng b c hn.
Qu trnh lm trong dch qu di tc dng ca pectinase c th chia lm 3
giai on:
Giai on bt n nh ho c tiu biu bng s gim nht mt
cch su sc v thng c gi l trng thi ph v
Giai on kt lng bt u t trng thi ph v v kt thc khi kt
ta hon ton.
Giai on cui cng thng c xc nh bng s vng mt cc pectin
kt ta bi ion canxi.
S dng enzym sn xut nc qu thanh trng ung trc tip t
qu to v qu vi
Ngy nay, nc qu trong hoc nc qu c c sn xut nhiu nc trn
th gii. Ty theo hm lng ng v acid c trong nc qu, ngi ta ch
bin nc qu c hm lng ng v acid thp, ngi ta b sung ng v
acid iu chnh cht lng cui cng ca sn phm. Hm lng pectin cao sn
phm thng khng c li v khi nht ca sn phm rt cao. Vic thy
phn pectin c trong dch qu cn lm cho cc sn phm dch qu khng b
c.Tuy nhin, vic x l pectin bng pectinase khng nn tin hnh phn gii
pectin n cng m cn phi gi mt lng nht nh trong sn phm nc
qu.Chnh lng pectin ny s gip cho cht lng nc qu tt hn.

Nhm 20Trang 42

GVHD: Th.S Nguyn Th Thu Sang

Trong sn xut nc qu to v nc qu vi, ngi ta s dng ch phm
pectinase cho hiu qu rt cao.Khi x l nc qu 500C trong 2 gi, hiu sut
tch nc qu tng 20%, 37- 40% sau2 -4 gi th tng 20 25%.Cc nghin
cu v thc t sn xut cho thy hiu qu x l nc to rt cao.Chnh v th,
vic ng dng enzyme pectinase c p dung tt c cc nh my sn xut
nc to trn th gii.Trong khi x l nc qu bng enzyme pectinase, ngi
ta thng cho thm ascorbic acid vo chng qu trnh oxy ha ca nc qu
to.
Bng 3.1. Hiu qu x l nc qu v bng ch phm pectinas
Stt

Loi nc qu

Hiu

Cht

ng

Pectinase

Cht

im

sut

kh

acid

(%)

(%)

cht

cm

(%)

(%)

(%)

(%)

quan

71,2

10,2

0,53

6,7

0,33

0,04

4,5

78,8

11,5

0,62

8,0

0,15

0,02

4,8

49,2

8,5

0,91

5,62

0,26

0,09

3,8

72,4

10,2

1,16

7,4

0,12

0,16

4,3

Nc qu t to
a- Khng cho
ch phm
enzyme
b- C ch phm

enzyme
Nc qu vi
a- Khng cho
ch phm
enzyme
b- C cho ch
phm
enzyme
Sn xut nc qu ung ngay t du ty, anh o v phc bn t
Ngi ta thng sn xut c dng nc qu trong v nc qu c t
cc loi tri cy trn. Trong qu trnh sn xut, ngi ta thng cho thm
ng v iu chnh li lng acid cho ph hp.

Nhm 20Trang 43

GVHD: Th.S Nguyn Th Thu Sang

Cc loi nc qu ny thng cha nhiu pectin. Do , vic s dng
cc ch phm enzyme pectinase d loi b kh nng gy c cho nc qu
cng nh n nh v nng cao cht lng dch qu l iu cn thit.
Ngi ta ra sch cc loi qu trn bng nc ca vi hoa sen. Sau ,
cc loi nc qu c em ch thnh dng pure. Qu nghin c un nng
nhit 40-500C c cho vo my trn. Ti y nc qu c phi trn vi
t l mt phn nc qu v 10 phn nc enzyme c lng enzyme 0,03%.
Ring i vi nc du ty, ngi ta cho thm 0,03% Vitamin C lm cht
chng oxy ha. Hn hp ny c gi 40C.Thi gian lu nhit 400C l
4-8 gi. Nc qu sau c cho qua my p v c un nng n 450C
.Tip , ngi ta em nc qu ly tm v cui cng l lc qua my p lc
khung bn.
Bng 3.2:Hiu qu vic x l nc qu du ty, anh o bng enzyme
Stt
1

Loi nc qu
Nc qu
du ty
a- Khng
cho ch
phm
enzyme
b- C cho
ch
phm
enzyme
Nc qu mm
si
a- Khng
cho ch
phm
enzyme
b- C cho
ch
phm
enzyme

Nhm 20Trang 44

Hiu
sut
(%)
71,2

Cht
Kh
(%)
10,2

Acid
(%)
0,53

ng Pectin
(%)
(%)
0,33

Cht
cht
(%)
0,04

im
cm
quan
4,5

6,7

78,8

11,5

0,62

8,0

0,15

0,02

4,8

49,2

8,5

0,91

5,62

0,26

0,09

3,8

72,4

10,2

1,16

7,4

0,12

0,16

4,3

GVHD: Th.S Nguyn Th Thu Sang

Sn xut nc qu t nho
Nc ung t tri nho l loi nc ung trong ch khng thuc loi
nc ung c.Loi nc ung t nho dng trong thng c sn xut nhiu
hu ht cc nc chu u v chu M. Tuy nhin hin nay, ngi ta cng sn
xut nc ung t nh dng c (ch yu l mu nho c mu ).Trong ,
l nc sn xut nc ung dng c nhiu nht, sau l Php, Rumani.Nho
l loi qu cho nhiu dch v cha nhiu ng.Tuy nhin, nc nho thng
cha nhiu tht qu. Trong tht qu c nhiu pectin, d gy c hoc nu thu
nhn dch qu trong thng khng cho hiu sut cao.Chnh v th, ngi ta
phi x l nc nho bng ch phm enzyme pectinase. Lng enzyme
pectinase c s dng trong trng hp lm nt nho l 0,2% so vi khi
lng nho. Thi gian x l bng enzyme l 3 gi nhit 450C, bng phng
php x l ny ngi ta lm tng hiu sut thu nhn dch nho t 65,9 ln
77,3% i vi nho trng v t 66-82,2% i vi nho .
i vi nho trng nghin k, khi x l enzyme pectinase trong thi gian
2,5-3 gi nhit 40-450C, hiu sut tng 14-18%. Nu tip tc x l sau
giai on x l trn thm 4-5 gi 40 500C, nc to s hon ton trong.
giai on lm trong dch qu ny, ngi ta un nng dch qu ln 900C, sau
h nhit xung 500C v khi mi s dng enzyme lm trong nc qu.
Trong khi nhng mu dch khng x l enzyme, chu trnh lm trong nc
nho phi ko di 2 - 4 thng. Hiu sut thu dch khng s dng enzyme ch t
trung bnh 65%.
Bng 3.2: Hiu qu vic x l nc nho bng enzyme
Stt

Nc nho

Cht
kh
(%)

Hiu
sut
(%)

acid
(%)

ng
(%)

Cht
cht
(%)

Mu i
chng

17,5

65,0

0,54

15,1

0,125 3,0

Mu x l
bng ch
phm
pectinase

19,0

70,1

0,68

16,85

0,135 C
4,5
mt t
capecta
t

Nhm 20Trang 45

Cn
(%)

i
m
came
quan
4,2

GVHD: Th.S Nguyn Th Thu Sang

Qu trnh sn xut nc nho trng c x l enzyme pectinase

Nho c ra sch, tch cung, loi ht bng my x v cha chng vo cc
thng kn trong thi gian 6 8 gi hoc cc thng ln men lin tc trong thi
gian 3 4 gi. Trong thi gian lu tr ny, ngi ta cho 0,02% ascorbic acid,
sau p thu nhn dch nho. Dch nho c lm nng 800C, sau lm
ngui nhanh n 400C, nc nho cho thm 0,01% enzyme pectinase.
Tin hnh qu trnh thy phn cho nht ca nc nho trong sut.Thi gian
thy phn ny khong 6 8 gi.Kt thc qu trnh thy phn, li un nng dch
qu ln th hai n 800C v lm ngui n 200C v lc nc qu qua my lc
p, tin hnh rt chai v em thanh trng.

Qu trnh sn xut nc nho c s dng pectinase

Nho ra sch, tch cung v un nng 85 900C trong my gia nhit v
ngay lp tc lm ngui n 45 500C, gi dch nho nhit ny trong thi
gian 4 6 gi trong thng kn hoc 3 4 gi trong nhng thit b ln men lin
tc. Thng cho enzyme pectinase trong giai on ny vi liu lng 0,03%.
Sua thi gian thy phn trn, ngi ta tin hnh lc p. Dch lc c cho thm
0,03% enzyme pectinase lm trong dch qu. Qu trnh lm trong dch qu
c thc hin trong 6 8 gi.Sau nc qu c lc, rt chai v em i
thanh trng.
Hiu sut v cht lng nc qu nho sn xu theo phng php x l
pectinase.
Sn xut nc mn
Ngi ta thng sn xut nc qu pha ng t mn. Mn l loi qu
cho hiu sut thu nhn nc p rt thp. thu nhn nc qu t mn, ngi ta
phi p dng phng php nhit v enzyme. Qu trnh cng ngh c thc
hin nh sau:
Mn c ra sch, un nng vi nc.Nc c cho vo khong 15
20% so vi khi lng mn.nhit c gi 85 900C trong 5 20 pht
ty thuc vo chn ca mn. Sau c khi qu qua h thng p v x l
enzyme pectinase 40 450C trong 3 6 gi. Dch qu c thu nhn bng
my lc p ln na, ng chai v em thanh trng.
Phng php x l nhit v enzyme kt hp cho ta hiu sut t ti
78,9% so vi hiu sut 58,2% khi khng x l enzyme.
Nhm 20Trang 46

GVHD: Th.S Nguyn Th Thu Sang

TI LIU THAM KHO

1. ng Dng Cng Ngh Sinh Hc Trong Cng Ngh Thc Phm, gio trnh Trng Cng
Nghip Thc Phm TP.HCM.
2. Lng c phm, Cng ngh vi sinh vt, 1998, nh xut bn Nng Nghip.
3. Tailieu.vn

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