DONG HOC V 6 HOAT POLYPHENOL OXIDASE TRONG N G 6 SEN B A N G C A C
PHU GlARieNG Le *
L i Van Vui, Nguyen V i n Uiriri. Tran Thanh Tnic Truvng Dai hgc Cin Tha
T 6 M TAT
Thi nghifim du^c ti5n hanh nhim muc dich xac djnh phuong trinh dgng hpc sy vS ho?i enzyme polyphenol oxidase sau khi ti6n xii Ii ngo sen voi cic phy gia rieng le. Ket qui nghiSn cihi da xic dinh Aagc cic thong sd d§ng hgc ban ihi ciia enzyme polyphenol oxidase, cu the li K,„ = 0.246.10'^ M, V™ = 0,812.10"* OD/giay = 0,812 don vi tr€n co chat catechol vi hogt thih ciia djch tofch enzyme polyphoiol oxidase ih6 trong nguySn li^u ngfi sen ban diu \i 3274 U/ g ng6 sen khd. N^ien curu d^ng hpc ciia qua trinh tiin xil li cic loai phy ^ a len hogt tinh enzyme PPO ngo sen cho thiy, vi?c tien xu li vdi cic logi phy gia rieng Ic nhu acid citric, acid ascorbic. CaCb va NaCl cho hi§u qui lie ch£ host dOnecua enzyme PPO ttong ng6 sen ding ki. Hi§u qui lie ch^ hogt ilOo§ enzyme PPO ttong ng6 sen ciia acid citric li t6t nhit theo sau lan Itt(rt li acid ascorbic, CaClj va NaCl vdi hing s6 t6c d^ v6 hogt lin lupt li 7,55 ± 0,60; 7,01 ± 0,68; 6.82 ± 0,58; 5,08 ± 0.75 (1/C). Qua kit qua nghifin cim, cic phuong trinh dOng hpc sy v6 hogt b6i cic lo»i phv gia rifing le da dir^c thifit 1^: Acid citric: A|= 495 + 0,84.exp (-7,54.CdBic); Acid ascorbic: Ai= 703 + 0,78.exp (-7,01.C.„^;
CaClj: A, = 594 + 0,81.«^ (-6,82.Coci2); NaCl A4= 545 + 0,83 .exp ( - 5 , 0 8 . C N ^ . Tir khda: ng6 sen, phuong trinh dSng hgc, dich trich enzyme tho, phu gia, chit lic che.
MdDAU
Hi^n nay, sen \i logi cSy b^ng khd ph6 bien a ding bing s6ng Ci>u Long vi dang din mang Igi gia tri kinh t^ cao cho ngiriri n6ng d9n. Tit cS c^c bp ph$n dia sen nhu> \i, ng6 sen, hoa, cO, h^t d6u c6 th^ si> dyng du'Q'c; dgc bi§t la ngd sen vi hgt sen c6 thSnh phin dinh dir&ng rit cao, du^TC si> dyng nhu thvc pham hang ngdy trong ddi s6ng (Nguyen, 2001) MOt trong nhOng vdn d l thi/£mg xdy ra trong qud trinh t6n trir vi chS bldn ngd sen Id cdc bidn ddi sinh 1;^ vd sinh hda d^c bi§t Id phdn i>ng hda ndu do enzyme, chu yfiu Id polyphenol oxidase (PPO). Sir h6a ndu thirdng Idm gidm tlnh chSt cim quan cGa sdn phim do lien quan den sy thay dfii trong mdu s^c, hu-cng vj vd Idm m4m clu tnic (Martinez va Whitaker, 1995). Biiu ndy din din vi$c suy gidm chit lu'gng nhanh chdng vd gid thdnh thip ngay th£ri diem thu hogch ndn ddu ra cho ngd sen c6n phg thupc rit nhieu vdo thu^rg Idi. Ngd sen du'g'c tidu Uiy tren thj frudng chQ yiu dyi/c tiy tring bing hda chit khdng rd nguOn gic, xuit xtr, ttong khi dd cdc nghidn cuu chuydn sdu v i phuong thu'C tien xi> i;^ ngd sen vin chu'a duvc quan tdm, khdng cd tdi ll$u khoa hgc cho qua trlnh so' che, XLF I;^ ngd sen sau thu hogch du^c cdng bd d \/\^
Nam. Gan ddy, mdt s6 nghidn cini vd enzyme PPO frdn ngd sen da du^c t h i ^ htgn nhu-ng (^[ tdp trung & Tnjng Quic.
Rong Bao-hua (2010) da bird'c diu du'a ra mgt sd tinh chit ca bdn cOa PPO thd dirg'c trich ly tit ngd sen vd hidu qud i>c chi hogt tlnh cua PPO tO ngd sen cua acid ascorbk;, acid citric, L-cystein. Trum: d6, Liu vd dong tdc gid (2007) cOng dS khang djnh sy d n thiit cOa vide O-c chd sy hda ndu ngd sen bang acid alginic, acid ascorbic va kit hpp vdi CaCb nhim ngan cdn sy biin ddi cau tnJc cua ngd sen tLrci. Vpc nghidn ci>u d$ng hgc sy vd hoat PPO tnsng mo t i bdo rau qui thiFdng chl du^c dp dgng v6i hlnh thCrc v6 hoat nhi§t, md hlnh ddng hpc sy vo hogt PPO bang chat i>c chi chua dupi:
phd bien. Mdcdii vdy, nghidn ci>u tO sdm cua Weemaes vd ddng tdc gia (1999)daxdcnh§n, hogt tlnh PPO trong mdt^
bdo qud bo' khdng thi tiin v i 0 ma vin cdn sy hidn dl§n tCr 5 den 20% ngay khi tang nong dg phu gia xi> 1;^, ding thdi cOng d i nghj md hinh bdc mdt chuydn ddi mgt phan d i md ta ddng hpc sy vd hogt PPO trong qud ba do tdc ddng d6ng thcri cda chit i>c chi hda ndu vd ap suit cao. Ddy cOng chinh Id ccr sd cho vide nghien CLFU xdc dinh ddng hgc sy i>c chd PPO cua ngd sen bang phy gia thyc phim, Id ca sd cho vifc nghidn cd'u so' chd, bdo quan vd chi bien ngd sen.
N G U Y £ N L I ^ U V A PHlfONG P H A P NGHIDN CCrU Nguyen li^u, hda chit, thiet bi
Ngd sen dypc liy nj$ng sen d huygn Tdn Thgnh, tfnh Long An. Ngd sen sau thu hdi daiyc cho vdo bao PE, bdo quin bang nuwc dd trong thiing xdp ir nhidt dp tip 0 + 4°C vd vdn chuydn v i phdng thi nghi§m bd mdn Cdng nghd thyc phim, Dgi hgc Cin Tha. Thd'i gian vdn chuyin t i l da 5 gid'. Nguydn lidu du'pc tidn hanh xi> II sa bd, cit khOc khodng 5 + 6 cm.
Acid ascorbic. Acid citric, Calcium chloride anhydrous vd Sodium chlonde (PA, Tnjng Quic), Disodium hydrogen photphate dodecahydrate. Sodium sulfite. Catechol (Merck, Dire).
Mdy ly tam (Rotona 46 R, Oi>c), May do quang phd (Hitachi U-1500 Spectrophotometer) vd cdc dgng cy khdc.
Phuxmg phdp nghidn ci>u
Tl4n xi> ly ngd sen bing cac phu gia rieng le
Nguydn li^u ngd sen sau qua trinh xi> ly so bd du'O'c ngdm trong dung djch cdc phu gia khdc nhau, bao gim add citric, acid ascorbic, NaCl vd CaClz tuvng ipng vdi 7 mirc ndng dO sir dung, tir 0,125% den 1,5% w/v. Co djnh thdi gian ngim Id 30 phOt vd ty Ip nguyfin ligu: dich ngam Id 1:2. Mlu sau khi ngdm theo tirng nghigm thCro khdc nhau (28 nghidm thire) dirpc chuyin qua ly trich vd do hoat tlnh polyphenoloxidase cdn Igi trong mau ngd sen sau khi tidn xO ly, mdt phin mau du'pc do xdc dinh dg i m
Phircmg phap chuan bj djch trich enzyme tho PPO tu ngd sen
Phuio-ng phdp chuan bj djch trich ly enzyme thd PPO du-pc tien hanh du'a trdn phu-o'ng phdp ciia Chlkezie (2000). Cfln chlnh xdc 25 g mau ngd sen, tidn hdnh tiin xi> li ngd sen vdi cdc phy gia rieng Id khdc nhau d cdc ning dd khdc nhau.
Vdi 6 If dich ngam vd ngd sen la 2 : 1 bong thdi gian ngdm 30 phut Mau sau khi tiin w li dirpc d t nhd vd ngdm ttong
^ ^ djch sodiiHn sulfite (lOg/L) vdi ty Id nguyen ^u: dich ngdm Id 1: 2 (ddm t>ao mau duvc ngdm ngdp hodn todn) vd ben hanh gii> Ignh bdng niroc dd troiig thdi gian 20 [rfMiL sau dd v*t rao vd rOa lgi bdng n u ^ d L Ngd sen sau khi khi
^•ydirpcnghientrongcoisir, bo surigSOmL dung dich defnptwspt»atepH =7, giir Ignh khodng 3 phiiL Hon hpp dirpc oong hda sau dd kjc qua gidy Ipc vd dirpc ^ O l ^ h . ttwi dkii lpc enzyme ihd va bin hdnh ly tdm a 1500 vdng/phiit trong l^iphuL Dung dfch tinh sgch mpt phin ndy diroc dung d l xdc djrtfi boat dp cua polyphenol oxidase. Ghi nhgn Igi &ie Uch mch trich enzyme V thu duvc. sir dyng d i bnh todn hoat tinh PPO c6 bong nguyen ligu.
Phirong phap xac ^nh ho^t dd cua PPO
Hogt bnh cua PPO (U/g) dirpc tinh todn dya vdo phirvng phdp cua Ensimiger vd Vamos -Vigyazo (1995, trich din boi
^ ™ ^ ^ " ^*'**°'" ^^^ " ^ ^ " ^ " ° " 9 ^^ *^ catechol theo day tir 0.012 M. 0,006 M, 0,003 M, 0,0015 M. 0,00075 M.
0.000375 M. 0,000188 M. Moi dng nghidm dirpc thdm vdo 1 mL dung tfich catechol. Sau dd bd sung lln luvt 1 mL 0,1 M ddm i^nsphate (pH=7,Q). 3 mL nydc d t vd i*an irr^ enzyme dirpc bat diu bdng vide b i sung 0,5 mL djch trich enzyme l t » PPO sau kN duvc tinh sach. Hon hop nay duvc rtianh didng chuyin vdo cuvette va do quang phd hip thu a tnrcrc sdng 540 nm sau mdi 30 gidy phan irng. Hogt Unh PPO ty 1# thudn vcri cuong dd hip thu cu-c dgi cila tienzpquinones. Mdt don vj enzyme (U) Id luvng enzyme cin t h l ^ cho phdn img oxy hda quinin de mgt dg quang eirdng do hdp thu tgi eye dgi cda san pham tgo thdnh benzoquinone tai bircre sdng 540 nm len 0,001 dan vj ttong 1 gidy ttong dreu kipn phan img 25°C, pH = 7. Nhir vgy, 1 don vj enzyme U = lOr'.OD/ giay (Chlkezie. 2000, Phan Thanh Binh va dong tdc gia. 2009).
Cong thuv tinh ho^t Hnh cua PPO
Hogt tirtfi cua PPO dtrpc tinh dt/a ttdn khdi lirpng ngd sen khd. theo c6ng thCrc a. V. 100
vdi a: hogt tinh cda PPO ttong 1 mL djch bieh (U/mL); V: ttw tfch djch ttich thu duvc (mL) irng vdi khdi lirpng ngd sen tifcri (m, g), X Id dp I m eua ngd sen ttong miu khao sdt (%).
Phuang phap xic ajnh cic thong so d^ng hgc V«„, K_ cua Pf*0 ngd sen
Sir syng phin mdm Graphpad Prism 5.01 de xdc i^nh cdc Oidng sd K™ vd Vma, cua enzyme thd PPO ttong nguyen lidu ngd sen t)an ddu. Sa dd phan img eua polyphenol oxidase ngd sen xiic tde ehuyin hda catechol thdnh benzoquinones dirpc gii thiet theo ca che Miehealis - Menten (cho phdn img hai giai dogn). Sy phy Ihupc giO'a vln tdc ptian irng vd nong dp ca ehit duvc bidu diin bang phuvng ttinh Misehaelis-Menten:
^"*""^E^I ^"^ ~ * ^ "*"^ fm+t^]' V V;B<U"*"»WU:'[S]
Trong dd : Vmn Id tdc dp eye dai cua phan irng (U, 10"^ OD/gidy); Km Id hdng sd Michaelis-Menten (M) vdi ca chit la catechol.
Xdc dfnh phuang trinh dgng hgc sf/- vd ho#( PPO trong ngd sen
Ap di^ig phirong trinh chuydn ddi timg phin trong vide xdc ^rnh phirong blnh dpng hpc eua sy vd hogt enzyme hda ndu trong nguyen lidu ngd sen (Weemaes ef ai, 1999). Phirang trinh chuyen ddi mpt phin Id mdt tairong hpp dgc btet aia phan irng bdc mpL duvc ung dyng tiong tnrdng hpp hdm lirpng cua chit nghien ciru khdng gidm din gld tri bang khdng vd ham lirprtg cOa chat ndy sau khi giam din mdt mCrc nao dd se khdng ddi khi tiip tyc gia tang ndng dd (A^)- Phirong trinh cd dgrtgr
\i = A„ + iA„- /l„).exp(-k.C) .(V^^)
Trong dd: A: hogt h'nh cua enzyme sau tien xi> li (U/g ngo sen Bid); Ao: hogt tinh ban diu cua enzyme (U/g ngd sen khd); k: hang so tdc dd vd hoat (1/C); C: ndng dd xir li (%w/v)
Si> dyng phin mim SAS 9.1 trong phan tich xdc djnh hing sd tdc dp vd hoat (k) vd phu-ong trinh dgng hpc sy vd hogt polyphenol oxidase trong ngo sen.
K t T QUA vA T H A O LUAN
Xdc tnnh cac thdng sd ddng hpc vd hogt tinh enzyme t)an d i u ciia nguyen lidu ngd sen
Kit qua do dgc sy thay ddi mgt dd quang OD cua cde phan irng polyphenol oxidase ttong ngd sen d l chuyen hda catechol (CO ehit) thdnh benzoquinones (san pham). Tir gid tri ttiay dii mgt dd quang OD (buoc sdng 540 nm) do duvc theo thoi gian trong qua trinh xdc dinh hogt tinh cua enzyme, xac djnh du'ac toe dp ctia phan irng tuvng ung vd'i cac gia bi ndng dO co ctilt V=f([S)). Kit qua phdn tich sy thay doi mdt do quang xdc dinh hoat tinh eua polyphenol oxkJase ttong ngd sen duvc the h i ^ trong Bdng 1 Tir ket qua thu nhgn, Igp dd thj bidu diin sir phy thupc gi&a nong dp ca chat vd v$n tdc phan img ctia enzynte trong nguydn lipu ban ddu (hinh l.a). Cde thdng s6 dpng hpc Km, Vma duoc xdc djnh theo phuong phdp Uneweaver - Burii (Hinh 1.b)nhu'sau: hr cdc gid ttj nghich ddo 1/V(ddng8) va I/JS] (ddng 9) duvc ghi ttong bdng 1 ldp dd thj 1A/ = f (1/(S]) (Hlnh l.b).
T i r do thj hlnh 1 .b cho t h i y , cac t h d n g s d eua PPO nhu- Vmax va Km c d t h i d u p e x d c d j n h t h d n g q u a p h u o n g ttinh du'dng S l i n g y ( I / I S ) ) = 0,227x (1/[S]) + 1231 v o l R^= 0.947 ed d d tin c d y d m u c c h i p n h d n . N g h i d n ci>u c u a R o n g B a o h u a (2010) cung d a sir d y n g p h u o n g ttinh du'dng t h i n g ti> m d hinh Lineweaver - Burk v d i g i d ttj R^ = 0,878 d e x d c i^nh Vm v d Kmax c d a P P O n g d sen.
B i n g 1. S y thay ddl mdt d$ quang A t r o n g q u i trinh x i c djnh hoat t l n h cua polyphenol oxidase n g d sen
Stt Then gian (giay)
V, (OD/gidy) 1/V. (OD/giSy') 1/[S], M-'
0,0657 0,0852 0,0787 0.0812 0.0792 0,0700 0,00093 989,333 83,3333
N i n g d$ catechol (S], M
0,0120 0.0060 0,0015 0,00075 0,000375
0.0627 0,0617 0,0597 0,0615 0,0597 0.0607 0,00085 1192,1351
0,0595 0,0577 0,0610 0,0592 0,00081 1238,440 333.3333
0,0527 0,0460 0,0512 0,0495 0,0530 0,1357 0.00078 1291.5779 666,6667
0,0465 0.0485 0,0452 0,0460 0.0450 0,0452 0,00064
0,0375 0,032 0.03 0,032 0,0312 0.0315 0,00045 2173,913 2666,667
0,0353 0,032 0,028 0.0301 0.0302 0.0305 0,00044 2272,727 5333,333
y = 0,227j(*1231 R =0,947 1/V(00/sec')
Hlnh 1 . Md hinh x i c djnh cac ttiAng s d d&ng hpc V ^ u K„i ciia PPO trong nguyen li^u n g d sen. a. M6 hinh Miehealis - Menten.
Md hlnh Lineweaver - Burk
Qua k i t qua phdn t i c h . cdc thdng s d ddng hpc cua P P O d u v c x d c d j n h v d i h i n g s d Km Id 0.246.10"^ M v d Vmax cd gld bj t u o n g li-ng la 0,812.10"^ OD/gidy = 0,812 d o n vj (U).
D d n g thd'i, d y a trdn djnh nghTa d a ndu v d Vmox t l m du-pc, hogt t i n h P P O t u o n g i>ng trong 1 m L djch trich d y p c tlnh li a = 0,812 ( 1 0 - ' OD/giay) x ( 1 p h u t / 6 0 giay) x 1000/0,5 m l djch enzyme phan liYig = 9 7 , 4 8 U/mL
V d i t h i t i c h djch ttich V thu d u p e Id 4 6 , 5 mL, t u o n g d-ng vir'i khdi l i r p n g m d u b a n d i u Id 2 5 , 5 g v d d p i m t a i n g blnh cua ngd sen Id 94.57%, u'dc t l n h hogt t i n h e n z y m e PPO t h d nguydn lidu ngd sen ban d i u dgt d i n 3274 U/g ngd sen khd. Do d d , d d c d t h i si> d y n g ngd sen c h o c d c qud ttinh c h l bien tiep ttieo, v i ^ c x d c d j n h bidn p h d p irc chd hogt ddng PPO trong ngudn nguyen li§u ndy d n d o p e quan tarn. C d c thi nghi$m t h a m do eho t h i y , x ^ 1<^ nhidt Id bidn phdp hO'u hi$u giiip i>e chd d u v c PPO trong ngd s e n . T u y nhidn, phu'ang phap tien x i i li nhi§t d n h hu'O'ng d i n d$c tinh c i u tnic vd cim quan c i i a n g d s e n , khdng tiie sir dgng c h o cdc q u d trlnh c h i b i l n t i l p t h e o , nghidn cd'u c d a Liu vd ddng tdc g i i (2007) cung k h i n g djnh didu ndy. Do d d d d n g hpc qua trinh ben xi> ly PPO bang p h y gia n d n g le d y p c d p d y n g , O p n g h p c v o h o g t PPO t r o n g n g d s e n d o t d c d $ n g c d a t i i n x i > i i b a n g phi^ g i a rieng Id a c a c n o n g d p k h d c nhau D y a trdn s y thay ddl hogt t l n h c d a P P O (A/Ao) trong cdc m i u ngd sen d w p c tien XCP ii bang 4 logi phy gia rieng Id 6 c i c nong d d khac nhau, k i t qud do d g c v d phdn t i c h cho t h i y , d* dieu kien khao sdt, c d s y g i d m boat t l n h PPO nhanh ngay d 3 m i r c ndng d d khao sdt t h i p ( 0 , 1 2 5 % , 0,25% vd 0,5%) vd'i cd 4 loai p h u gia sir d y n g . Tuy nhien, khi tdng ndng dd cic phy gia sii' dyng len d i n h o n 0 , 5 % khdng giiip gia t d n g hidu q u d v d hoat e n z y m e ( H i n h 2). Dieu ndy chipng td v i n cdn s y hidn dldn cOa nhdm e n z y m e P P O k h d bj bien doi. n i m sau trang m d t l b d o n g u y d n lidu. K i t qua eho phdp khing (^nh, s y v d hogt PPO trong ngd sen t u d n theo phu'ang frinh bdc mdt c h u y i n ddi m p t p h i n (Hinh 2).
Hidu qud irc c h l doi v d i PPO dgt tdt nhat 6* cdc ndng d d Wide nhau c u a c d c logi p h g gia khdc nhau, Hogt t i n h ciia PPO cdn Igi trong ngd sen t h i p n h l t ddi v d i m i u d y p c tien xir li v d i acid citnc, vd'i g i n 8 0 % hogt tinh enzyme ban d i u bj v6 hogt. Acid citric ed tac dgng khdng ehi h a t h i p pH cda mdi t n j ' d n g m a c d n tao p ht r e "cdng cua" (chelating) v d i p h i n dfing cua ttung t d m enzyme. Theo Martinez & Whitaker (1995), acid cittic tgo m a t r u o n g acid m g n h vd'i pH t h i p c d tdc dgng u'C c h e hogt tinh PPO hogt ddng m g n h i> pH 5 + 7 . Ket q u a nghien c i r u d p n g h p c v d hogt PPO bang acid citric trong nghien cd'u ndy phd h p p v a i k i t q u a nghidn c i r u cua Pongsakui ef ai (2006) khi s i r d g n g acid cibic vd'i cdc m u c ndng dO 1, 2 , 5 v d 5 mH^, nham i>c c h l hogt d p n g cua PPO t r d n trdi nhdn eho hidu q u a i j c c h e t u a n g i>ng Id 16, 2 8 . vd 4 1 % , trong khi higu qua irc che thu d u v c c a o h o n khi ndng d p acid citric c a o 10 m M id 7 2 % . D d i v d i muoi NaCl. hi$u qu^ i)c c h l P P O Id t h i p n h l t v 6 i h i n g s d t d c d d v d hogt P P O t h i p h a n so v * i c d c logi p h y gia khdc, NaCI d u v c b i i t d i n nhy \i tdc nhan chdng oxi hda P P O ; Ion C r la m$t tac nhdn i>c c h l h d a ndu y l u ; mdt s o nhd khoa hgc di bio do r i n g ndng dd ion Cl" ydu c i u cho vigc LPC c h e P P O Id cao, vd c d t h i t d c d d n g d i n mCii vj c i l a san pham (Mayer & Harel. 1991). Hi$u q u a irc c h e hda ndu cua NaCl c a o khi su- dung n i n g d d cao vd trong d i l u kipn p H giam. O d l v a l acid ascorbic, khd nSng
c h d n g cde p h d n O n g hda n d u d o e n z y m e d i r p c b i l l d i n Id r i t tot ma k h d n g gdy t i n t h u o n g d i n edc m d U i y e vgt n l u d u v c s i r d y n g v d i m ^ t l i r p n g v i r a d u . T h e o P o n g s a k u i etai (2006), a n d n g d d ac^d t h i p ascoriaic 1 , 2, 5 m M , tdc d g n g i>c c h i trdn P P O c u a trdi n h d n Id 2 7 % , 4 4 % , 5 4 % t u v n g i r n g . t r o n g khi k i t q u a t h u d u p e khi n d n g d d ascorbic la 10 m M Id g i n 1 0 0 % h o g t t i n h e n z y m e P P O bj i r e c h l . C a c t i e c i i a s y iPC c h e P P O cua ascorbic acid lidn q u a n d e n s y Idm g i a m c d c h p p c h i t q u i n o n e d u v c t g o t h d n h b a i P P O ( P o n g s a k u i ef a/., 2 0 0 6 ) . T u y n h i d n , a d d ascorbic c d t h i bj p h d n hOy t h e o t h d i g i a n bd'l oxy k h d n g k h i , a n h s d n g . p H v a nhidt d p c a o cua mdi tnrd'ng ( M a y e r & Harel, 1991). V i the, higu q u d c h d n g h d a n d u c u a a i d aseortiic phg fliudc r i t n h i i u v d o d i i u ki$n cda mdi tnrd'ng xit II. Drake & S p a y d (1983) d a n g h i d n cii'u v e h i d u q u a c u a vide t i i n xi> li vi>i ion Ca^* t r o n g v i d e c a i t h i f n d d c i r n g c u a m d te b d o c u n g nhu* trong v i d e l d m g i a m h d a n a u d o e n z y m e d a c h o t h i y vai ttd c u a ton C a trong vide irc c h i hogt d d n g e i i a P P O .
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Hlnh 2. Do t h j b i i u diSn s i ^ thay ddl ho^t tlnh ciia polyphenol oxidase khi xue ly vdri cdc phu gia rigng 16
H o g t t i n h v d h i n g s o t d c d p v d hogt c i i a P P O s a u q u d ttinh t i i n xir ii v d i cac loai p h g gia khde du'pc trinh bdy d Bang 2 Bdng 2. Phu-cmg t r l n h dong hpc s y v d ho^t cda polyphenol oxidase khi xir ly vd'i cdc IQ;II phy gia khdc nhau
Logi phy gia Ao (U/g) A . (U/g) k (1/C) Phirong trinh d^ng hpc /teid citric 3274 495 ± 27 7.55 ± 0.60 A i = 495 + 0,84,exp (-7,55,Cdbk) Acid ascort)ic 3274 703 ± 34 7.01 ± 0,69 A!= 703 + 0.78.exp (-7,01 . C « m c ) CaClj 3274 594 ± 32 6.82 ± 0,58 A3= 594 + 0,81 ,exp (-6,82.Cca2) NaCI 3274 545 ± 74 5.08 ± 0.75 A,= 545 + 0,83,exp ( - 5 , 0 8 , C H ^ ) D y a v d o k i t q u a b a n g 2 n h ^ n t h i y , hogt t i n h P P O c d n lgi s a u khi tidn x u li v d i c d c logi phg gia k h d c n h a u t h i r i t k h d c nhau. H o g t t i n h P P O c d n Igi g i a m d i n tir mudi acid ascorbic. CaCla, NaCI, acid citric v d i hogt t l n h t y a n g y n g l l n lu'pt Id 703 ± 34 U/g, 5 9 4 ± 3 2 U/g, 545 ± 7 4 U/g, 4 9 5 ± 2 7 U/g, t l n h tt*n n g d sen k h d . B d n c g n h d o , h a n g sd t o e d d v d hogt k cQng cd s y k h d c bidt d a n g k i 6 cdc logi phg gia k h d c n h a u . H i n g sd tdc d g v d hogt l d n n h l t la acid d t t i c vd t h i p n h i t Id NaCl v d i k i t q u a l i n l u g l Id 7,55 ± 0,60 v d 5,08 ± 0,75 (1/C). K i t qua trdn c h u n g t d acid citnc c h o hldu qua c h d n g h d a n d u tot n h i t s o v 6 i c d c logi phy gia cdn lgi O i l u ndy c d t h i giai t h i d i Id d o acid citric t h i hign higu qua i r c c h l tdt h o n s o v d i cdc p h y g i a c d n Igi b a n g e a c h ndi v d i t d m hogt d g n g c u a enzyme t g o t h d n h t h i g p n g k i m v d d d n g t h d i t a o mdi t n r d n g acid i r c c h l hogt d p n g c u a e n z y m e hda n d u . T u y nhien, co s y khac bigt v l mdi t u a n g q u a n giO'a hogt t i n h cudi vd h d n g s d t d c d p v d hoat eua 2 phy g i a acid ascorbic v d CaClz. Hogt l i n h P P O c d n Igi khi t i i n xi> li v d ^ acid ascorbic l a n h o n trong t r u d n g h p p t i i n xir li v d i C a C l j nhu'ng h a n g sd tdc d p v d hogt c a o h o n s o v d i h i n g sd tdc d p v d hogt c u a CaCl2.
N h i n Chung, p h a n i r n g h d a ndu d o e n z y m e Id mdt b i i n d i i sinh hda r i t p h d c tgp v a d n h h y d n g r i t l d n d i n c h i t l u p n g dinh d u d n g v d gid tri c a m q u a n c i i a san p h i m , ldm g i a m m i r c d g c h i p n h d n c u a n g o d i tidu d u n g v a d o dd g d ^ ra t d c d d n g kinh te d a n g k l d e n n g y d i san xudt vd n g o d i tieu d i i n g . T u y nhien, phan u-ng hda n a u d o e n z y m e c d t h i d u a c k i i m sodt b a n g n h i l u p h u a n g phdp k h a c n h a u . Viec d p d y n g cdc tdc n h d n c h d n g hda ndu trong q u d trlnh t i l n x d li d a c h o t h i y h i ^ u q u a trong vige lam c h d m p h a n d n g h d a n a u tren n g d sen gdy d n h h u d n g d i n gia trj c d m quan va chat l u p n g ciia n g d s e n .
K ^ L U A N
Nghien c d u d d n g hpc q u d trinh tien xir II c d c logi phy gia ldn hogt tinh P P O n g d s e n cho t h i y , vige d p d y n g cac loai p h y gia ridng Id n h u a c i d d t t i c . acid a s c o r b i c . CaClz v d N a C l c h o higu qua d c c h d hogt d g n g c u a P P O b'ong ngd sen d d n g k l . Nong d d t i l u u ddi v d i t d n g logi phy gia d e t h y c h i | n trong tidn trinh t i l n x d 11 Id khde n h a u . Higu qua d c che hogt d p n g P P O t r o n g n g d s e n c u a acid citric Id tdt n h i t t h e o s a u l l n l i r a t la a d d ascortsic, CaClz v a NaCt v d i hang s i tdc d d v d hogt l l n l u p t Id 7,55 + 0 , 6 0 ; 7,01 ± 0 , 6 8 ; 5,08 ± 0.75; 6,82 ± 0,58 (1/C) v d i c o chat la catechol. Q u a ket qua p h d n t i c h , nghidn c d u t h u d o p e cdc p h u o n g trinh dOng hoc s o v d hogt P P O b d i cdc logi phy gia ridng l i l i n l o p t Id: A c i d citric: A i = 4 9 5 + 0,84,exp (-7,54.Cawc); A c i d ascoriaic: /kz= 703 + 0,78.exp (-7.01 ,Cas«.rt.te); C a C l j : A3= 594 + 0,81 .exp ( - 6 , 8 2 . C C « M ) ; NaCI: A^= 5 4 5 + 0.83.exp (-S.OS.CNSCI).
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STUDY INHIBITOR KINETIC POLYPHENOL OXIDASE IN LOTUS ROOTLET BY INDIVIDUAL ADDITIVES
Le V a n VuJ, Nguyen V a n Muoi, T r a n T h a n h True Can Tho University
SUMMARY
This research aimed lo determine kinetic equations of polyphenol oxidase's inactivation after pretreatment lotus rootlet with individual additives. Experiment's results determiaed initial kinetic parameters of polyphenol oxidase in lotus rootlet: K„, = 0.246x10"' M, V™, = 0.812x10"' OD/sec = 0.812 unit and the crude extract PPO activity was 3274 U/g of lotus sprout (dry basic).
Study kinetic of pretreatment processing with individual additives on PPO activity shows that, treatment wilh idividual additives such as acid citric, acid ascorbic, CaCl^ and NaCl made an inhibitory effect PPO acUvity significandy. The inhibitory effect PPO activity in lotus rootlet when pretreatment with citric acid was the best followed by acid ascorbic, CaCl2 v4 NaCl with inactivated rate constant follow by 7.55 ± 0.60; 7.01 ± 0.68; 6.82 ± 0.58; 5.08 ± 0.75 respectively, using catechol as a substrate. According to research's results, the inactivated kinetic equations of individual additives were established by first order fbctional conversion model: Cihic acid: A|= 495 + 0.84xexp (-7.54''Cc^y, Ascoriiic acid: Ai= 703 + 0.78xexp (-T.OIXCUCMI*); CaClz: Aj= 594 + 0 Slxexp (-6.82"CCCU);NaCI: A4= 545 + CSa^exp (-S.OSxCfjoci)-
Keywords, lotus rootlet, kinetic equation, crude extract, additives, inhibitor ' Author for conesspondence: Tei: +84-710-3833551; Email: levanvul1988@gmail,com