HQI NGH! KHOA HQC COA HQI HOA SINH Y Dl/OC HA NQI VA CAC TINH PHIA B A C L A N THifXVII
NGHIEN cdu THUY PHAN PROTEIN SUA
B A N GPAPAIN
Nguyen Van Thiet*, Tran Dinh Toai^ Golovach T.N., Gavrilenko N.V., Kurtrenko V.P^.
TOM T A T
Dj li'ng thiTc pham (FA) la hien tu'dng kha phd bien, nguyen nhan bdi sy" phat trien cam Li'ng ciia cd the vdi cac allergent thii'c pham. Mdt trong nhQ'ng ngudn allergent thi/c pham quan trgng la cac protein siia bd, trong dd p- lactoglobulin (piG) la allergent chinh. Trong cdng trinh nay papain da du'dc sii dung de nghien cu'u lam giam hoat tmh khang nguyen cua pLG. Da nghien cu\i chi tiet ddng hgc thiiy phan pLG va cac protein khac cua sQ'a bd bang papain. Ket qua nghien cu\i cho thay papain cd tfnh dac hieu cd chat vdi piG cao hdn nhieu so vdi a- lactalbumin (aLA): trong cac dieu kien khac nhau protease nay thiiy phan 60 - 85% pLG, nhu'ng chi thiiy phan khoang 20 - 30% aLA va lam giam manh hoat tinh khang nguyen ciia piG.
Tu'khda: Papain, p-lactoglobulin (pLG), a- lactalbumin (aLA), dj ufhg thii'c pham (FA).
Cac chO" Viet tSt FA - Dj uYig thi/c pham;
CMS - djch cd dac huyet thanh sffa; pLG - p- lactoglobulin; aLA - a-lacta!bumin; BSA - protein huyet thanh bd; OC - ddi chihng.
SUMMARY
Studying hydrolysis of milk proteins catalysed by papain
Food allergy is rather widespread phenomenon, caused by development of sensibilization of organism to food allergents.
One of food allergents source is cow milk contained p-lactoglobulin (pLG) as the main allergent. In this paper papain was used to decrease antigenic activity of pLG. The kinetics of papaain hydrolysis for pLG and other cow milk proteins was studied in details. The obtained ' Vi^n Cong ngh^ sinh hgc, Vi$n KH&CN Vi$t Nam
^Vi?n Hoa hgc, Vien KH&CN Vi?t Nam
^Trucmg DH Tong hap Quoc gia Belarus, Minsk
results showed that papain has much more higher substrate specificity with pLG than that of a-lactalbumin (aLA): in different conditions this protease hydrolysed up to 60 - 85% of pLG, but only about 20 - 30% of aLA was cleaved, and strongly decreased antigenic activity of pLG.
Key wordR Papain, p-lactoglobulin (pLG), a-lactalbumin (aLA), food allergy (FA).
I. Md oAu
Theo quan didm hidn dai, trong phat tridn cac bdnh di umg nhiing roi loan mien dich gay ra bdi cac allergent cd y nghia quyet dinh, trong dd cd cac allergent thuc pham.
Bidu hidn lam sang ciia FA thdng thudng ban ca la vidm da man cam - vidm da di umg kinh nidn. Nguyen nhan chinh ciia bdnh la cac rdi loan thuc hidn churc nang cua he thdng mien dich [3]. Vidm da di umg gap d mgi nai, d moi lira tuoi, nhung vdi tdn sd Idn han d phu nii. Theo cac sd lieu mdi nhat, cd tdi 10% tre nhd tudi bi di umg thirc an. C) 80% sd bgnh nhan vdi bidu hidn vidm da man cam da xac dinh dugc mdi lidn he cua bdnh nay vdi FA [6]. FA cd nguydn nhan bdi su phat tridn cam ling cua ca thd vdi cac allergent thuc pham
[5].
Theo cac ket qua nghidn ciim tre em vdi FA, da xac dinh dugc tdn sd cao phat hign cac IgE d^c hieu allergent vdi cac protein sfta bd (68,9%) va cac phan doan cua siia: casein (70,6%) va pLG (66,3%), va ca vdi protein d%\x tuong (68,9%). Tdn so phat hign IgE dac hieu vdi siia dd thdp han 2 idn (35,9%) [2].
Y HOC VIET NAM THANG 8 - S6' 2/2011
Nhu vay la su phat tridn vidm da min cam chii ydu dugc nguydn nhan bdi di umg vdi cac protein sfta bd. Vi vay ndi dung chinh cua cdng trinh nay la nghidn cuu kha nang thiiy phan cac protein sira bdng protease thuc vat - papain tach chidt tir nhua qua du du, thdng qua dd danh gia kha nang su dimg cac protease tir ngudn thuc vat nhiet ddi phong phii ciia nude ta trong cdng nghiep chd bien sira, nhdm tao cac san pham siia it gay di ling.
II. Odi TUQN6 VA PHUONG PHAP NGHIEN COU
Dich cd dac huydt thanh sfta (CMS) nhan dugc bdng phuang phap sieu Igc, vdi ti Id khdi lugng protein la 72,8%. Ndng do protein dugc do bang phuang phap Lowry.
Papain dugc tach chidt tir nhua du du tuai vdi boat tinh enzyme dac tnmg la 20 dan vi/mg protein (20 U/mg). Khang huydt thanh chdng PLG cua hang Sigma. Qua trinh thuy phan protein CMS dugc th\rc hign trong thdi gian 2-3 gid d nhigt do 2a-60°C, pH 5-10, ndng do cac protein sfta la 1,5% va vimg ndng do papain la 0,7-1,8 U/mL, ti Id enzyme/ca chdt la 0,4-1,0%. Enzyme bi bdt boat bdi dun ndng dich thuy phan d 90°C hong 10 phiit, sau dd lam lanh ngay va giir hong khoang da ciia tu lanh d -20°C.
100 T
80
.S a 60
«2 2
>>40
Phuang phap didn di SDS dugc su dung dd danh gia lugng protein bi thuy phan (sau khi didn di cac bang protein dugc nhugm vdi coomassie brilliant blue R250 va scan dd xac dinh ham lugng protein trong moi bang).
III. KET QUA NGHIEN CQU
Trong cdng trinh nay protein trong CMS dugc thuy phan bang protease thuc vat papain, lugng protein trong CMS bi phan buy dugc danh gia theo lugng a- aLA va pLG bi thuy phan bdng phuang phap dien di SDS trdn gel polyacrylamide. Da khao sat miic do thuy phan cac protein sira trong mdi phu thudc vao ndng do cua papain, pH cua hon hgp phan ling, thdi gian va nhiet do tidn hanh phan ling thuy phan.
Trdn hinh 1 bidu dien mdi phu thudc cua miic do thuy phan protein sGa vao ndng do papain va thdi gian tidn hanh phan umg thuy phan va cac didn di dd tuang ling cua cac thi nghidm nay. Cac kdt qua nhan dugc cho thdy khi tang ndng do papain trong viing 0,7-1,8 U/mL (0,41-1,03%) lugng pLG bi phan buy tang tir 50 ddn 95%, va lugng aLA - tang tir 10 ddn 70% (cac dd thi hinh lA va cac dudng 2-8 hinh IC).
S? ^'
20
100 •alfa-LA
•beta-LG
t, mm
0.5 1 1,5
[Papain], U/ml B
HQI NGH! KHOA HQC C O A HQI HOA SINH Y DlfOC HA NQI VA CAC TJNH PHIA BAC LAN THlJ XVII
A
BSAV fe'j*
- - . - 6 6 kDa
B S A A ' 66 I kDa
PLG'' aLA^,^**
^ 1 ^ 6 : kDa 27 i kDa
* I'^^kDa 13 kDa teS^'^kDa
PLG Wi
a L A ^ * ' 4i^& .V*?
18 I,'' kDa kDa
3 4 5 6 7 8 7 8 9
C - D Hinh 1. D6 thj bieu dien mdi phu thugc mii-c do thuy phan protein cua CMS vao nong do papain
(A, dieu kien phan irng: ndng do cac protein huyet thanh sua 1,5%, nhiet dg 37°C, pH 6,3, 2 gid phan img) va vao thai gian thuy phan (B, dieu kien thuy phan: nong do enzyme: 0,92 U/ml, thai gian thuy phan 3 gia, cac dieu kien khac nhu thi nghiem dau); duong cong 1 - PLC; duong cong 2 - aLA; va dign di do cac san pham thuy phan cua cac thi nghiem nay (C va D, tuang img). C: Nong dg papain tuomg ling vai cac dudng dign di hi 1 den 8 la: 0 (DC); 0,7; 0,8; 0,9; 1,0; 1,2; 1,4; 1,8 U/ml va dudng 9 la marker chudn; D: Thdi gian thuy phan tuang img vai cac dudng dign di tir 1 den 9 la: 0 (DC); 15;
30; 45; 60; 75; 90; 105 va 120 phiit
Cdn trong cac didu kign ndng do protein cua CMS la 1,5%, ndng do papain bdng 0,92 U/mL (0,51%), pH 6,3, thuy phan PLG dugc thuc hidn chu ydu trong 15 phiit dau tidn cua phan ling (dudng cong 1 hinh 1B va dudng 2 didn di dd trdn hinh ID), chidm gan 60%
protein bi phan huy, trong khi aLA bi phan hiiy lien tuc, tuy cham, trong 2 h dau cua phan ling (dudng cong 2 hinh IB). Theo ket qua didn di SDS, sau 3 h phan umg gan 70%
PLG va 25% aLA bi phan huy thanh peptide.
Kdt qua nghidn ciiu mdi phu thudc tdc dg ciia qua trinh thuy phan protein CMS vao pH (didu kien phan ling: ndng do protem huydt thanh sfta: 1,5%, ndng do papain: 0,92 U/mL (0,51%), nhiet do 37°C, pH 5-8, thai gian thiiy phan: 3 h) va vao nhiet do (didu kign phan irng:
ndng do protein huydt thanh sua: 1,5%, ndng do papain: 0,92 U/mL (0,51 %), nhiet do ttong vung 20-60°C, pH 6,3 va thdi gian thuy phan: 2 h) dugc trinh bay trdn hinh 2.
100
80
* 1
1 ,^
J. 60 ,e
1
4020
01
^_„„-.——*->^
y"""^ 2
r
1 1 1
alfa-LA beta-LG
100 •
nil '
i^ r
* • j l '
hydro
E a.
20 .
0 ;
alfa-LA beta-LG
B
BSA-V-*' (kDa BBSAI 66 K/ kDa
i k D a ikDa
« *
*-,*
(1 PLCJp
„ a L A | p « i , ^ , ^ | | | i
W M 6 K/ kDa 2 7 K/) kDa 19 K; kDa U K/kDa
•»^6K/lkDa
'•*'
C D /R/l/l 2. Do thj bieu diln mdi phy thugc cua lugng cac protein huyet thanh aLA (duong cong 1)
va PLC (dudng cong 2) bj phan huy vao pH (A), vao nhigt dg (B) va cac dign di do tuong irng (C va D). Tren dign di do 2C: Dudng 1 irng vdi ^C (nhng do papain bdng 0), cac dudng tir 2 den 7 tuong ling vdi cac gia trj pH 5, 6, 7, 8, 9 va 10; tren dign di &h 2D: duong 1 - DC (n6ng dg papain bdng 0);
cdc dudng tir 2 den 6 tuang iirng vai nhigt dg thuy phan la 20, 30,40, 50 va 60°C; duomg 7 - marker.
Cac dd thi va didn di dd trdn hinh 2 cho thay d pH 5 trdn thuc te la khdng xay ra thiiy phan cac protein huydt thanh sua bd, d pH 6 lugng PLG va aLA bj thuy phan tuomg ling la 60% va 20%. Khi tang pH > 6 ti Id cac protein nay bi thuy phan thay ddi khdng dang ke. Cac dd thj bieu dien mdi phu thudc chidu sau ciia qua trinh thuy phan cac protein nay v^o nhigt d^ cd dang d§c trung, miic do thuy
phan thay ddi khdng dang kd trong vimg nhiet do 20 - 50°C, nhung tang manh d 60''C:
tdi 85% PLG va 100% aLA bi thuy phan sau 2 h phan ling d dO^C.
Kdt qua phan tich boat tinh khang nguydn cua cac san pham thuy phan protein ciia CMS vdi ddi huydt thanh chdng pLG bang phuang phap khudch tan mien dich kep trong gel agarose dugc trinh bay trdn hinh 3.
HQI NGH! KHOA HQC CCiA HQI HOA SINH Y DlJOC HA NQI VA CAC TJNH PHIA BAC L A N THifXVII
Hinh 3. Anh khuech tan mien djch kep trong gel agarose cua cac dich thuy phan protein CMS vai cac ddi huyet thanh chong lai PLG. 1 -DC (CMS), ndng do protein 0,2 mg/mL, cac 16 tii 2 den 6 - djch thuy phan a cac thai didm 15, 30, 60, 90 va 120 phiit; 7 - ddi thuydt thanh chong PLG.
Vach tua cua complex khang thd khang nguydn pLG h-en hinh 3 cho thdy boat tinh khang nguydn cua PLG giam ddn cimg vdi tang thdi gian thuy phan, hie la cimg vdi tang miic do phan hiiy khang nguydn nay trong phan ling thiiy phan do papain xiic tac. Biic tranh khuydch tan mien dich kep tuang tu ciing nhan dugc vdi dich thuy phan protein CMS vdi cac lugng papain khac nhau, d cac gia tri pH va nhigt do khac nhau.
IV. BAN LUAN
Nhu chiing ta biet rdng trong sira ndi chung cd 2 phan doan protein: casein (chidm
~80%) va cac protein huydt thanh (chiem
~20%) va trong siia bd cd >20 protein cd kha nang gay ra FA [1], trong dd cazein va pLG la nhumg khang nguydn chinh gay ra FA [2].
Sir phat tridn FA d lira tudi nhd lidn quan tdi tinh thdm tang cudng cua dudmg tidu hda ddi vdi cac protein trong thiic an [4]. Nhidu kdt qua nghidn ciiu da chi ra rdng cd khoang 10% protein dugc v$n chuydn qua thanh rudt d dang khdng bi bidn ddi [7], day chinh la ngudn khang nguyen gay FA. Trong cac protein cd trong siia nude, pLG la protein cd d lugng nhidu nhdt (chidm -10%, tiic mdt niia lugng protein CMS), tidp ddn la aLA ('-'5%), cac immunoglobulin (-3%) va BSA (-1%) [8], trong dd pLG la khang nguydn chinh ciia CMS, aLA cd boat tinh khang nguydn thdp va hoat tinh khang nguydn cua BSA la khdng dang kd. Vi vay, mdt h-ong cac
muc tidu chinh cua nghidn ciiu nay la tim didu kign lam giam hoat tinh khang nguydn cua pLG.
Cac kdt qua nhan dugc trdn day cho thdy tinh dac higu ca chdt cua papain ddi vdi pLG cao hom nhidu doi vdi aLA: trong tat ca cac thi nghidm dugc tidn hanh cd tir 60 den 95%
PLG bi thiiy phan bdi papain, trong khi chi khoang 20 - 30% aLA bi phan huy, trir trudng hgp tang ndng do papain trong dich thuy phan, lugng aLA bi thuy phan cd the dat 70% va thuy phan d dO^C la 100%. Ddng hgc thuy phan theo thdi gian cho thay pLG bi thiiy phan rdt nhanh, trong 15 phiit ddu tidn ciia phan umg cd tdi 60% pLG bi thuy phan, trong khi aLA hdu nhu khdng bi thuy phan, khi tang thdi gian phan limg Idn 120 phiit ciing chi cd -22% aLA bi thiiy phan. Tuomg tu, d pH 5 cac protein CMS khdng bi thuy phan, nhung d pH 6 ti Id thiiy phan cua 2 proteui nay la -60 va 20%, tuomg ling; khi tang tidp pH Idn ddn pH 10 ti Id phan hiiy cua chiing tang Idn khdng nhidu. Miic do thiiy phan ciia PLG gdn nhu khdng phu thudc vao nhigt do trong vung tir 20 ddn 50°C, deit khoang 60%
va tang len -80% d 60°C. Mdi ph\i thudc miic do thiiy phan ciia aLA vao nhiet do cd dang phiic tap hom nhidu: d 20°C khoang 30% aLA bi thuy phan, tang Idn -40% d 40°C, sau dd giam xudng cdn -25% d 40 va 50°C, rdi tang manh Idn 100% khi tang nhiet dd Idn 60°C.
Y HQC VIET NAM THANG 8 - SO 2/2011
Trong tat ca cac trudng hgp miic do thuy phan cac protein CMS ddu dugc kidm duydt bdng didn di SDS. Didn di dd trdn hinh IC cho thay cimg vdi tang ndng do papain trong hon hgp phan ung khdng chi lugng PLG giam manh, ma giam ca lugmg cac peptide kich thudc Idn (ndm phia dudi vach aLA trdn didn di dd). C) ndng do papain 1,4-1,8 U/mL bang protein tuomg umg vdi PLG cdn rdt md. Sau 15 phiit thuy phan bang protein cua PLG giam manh va cudng do ciia bang nay hdu nhu khdng bidn ddi sau 2 h phan img (didn di dd hinh ID). 6 pH 5 bang PLG dam va rdng tuomg duomg vdi trong DC, nhumg d pH 6 cudng do bang nay giam rdt manh, sau do giam rdt cham khi tang tidp pH Idn ddn gia tri pH 10 (didn di dd hinh 2C). Tuomg tu, cudng do ciia bang pLG cung rat nhat va hau nhu khdng thay ddi khi thuy phan protein CMS trong vimg 20-50°C va d 60°C khdng con nhin thdy bang pLG trdn didn di dd niia (hinh 2D). Cac dan lieu nay cho thdy ddng hgc thuy phan pLG bdi papain cd mdt sd net dac bigt: 1) thuy phan xay ra rdt nhanh trong 15 phiit ddu cua phan ung, sau dd tdc do thuy phan giam rdt manh; 2) phan ling thuy phan khdng xay ra d pH 5, xay ra rat manh d pH 6 va tdc do thuy phan it phu thudc vao pH hong vimg pH 6-10; 3) toe do thuy phan hdu nhu khdng phu thudc vao nhigt do trong viing 20-50°C.
V. KET LUAN
Tir cac kdt qua trinh bay trdn day cd thd kdt luan nhu sau:
1. Papain cd tinh dac hieu ca chat vdi PLG cao ban nhidu so vdi aLA;
2. Phan ling thuy phan PLG bdi papain xay ra rdt nhanh trong 15 phiit ddu cua phan ling, sau dd tidn tridn cham. Tdc do thuy phan PLG it phu thudc vao pH trong vimg pH 6-10 va hau nhu khdng phu thudc vao nhiet do trong viing 20-50°C;
3. Papain lam giam manh boat tinh khang nguydn cua pLG.
TAI U E U THAM K H A O
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Chir.,Vol. 16, J^2 5, p. 413^19.
2. Denisova S.N. (2008). RAMS, Moscow. Dr.
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Proc, Vol. 31, J^2 5, p. 392-397.
4. Holt P.G. et al. (1995). Pediatr. Allergy Immunol., Vol. 6, p. 85-90.
5. Kulig M. et aL (1999). J. Allergy Clin.
Immunol., Vol. 103, p. 1173-1179.
6. Revyakina V.A. (2004). Pediatrics, >r2 3, p. 3-7 (Tieng Nga).
7. Terpend K. et al. (1998). Gut., Vol.42, p. 538-545.
8. Wal, J.M. (2002) Ann. Allergy Asthma Immunol., Vol. 89, J^e i, p. 3-10.