TAP CHI KHOA HOC & CONG NGHE CAC TRUOTNG OAI HOC KY THUAT * SO 92 - 2013
NANG C A O KHA NANG SINH TONG H O P NATTOKINASE CUA CHUNG BACILLUS SUBTILIS D BANG PHU^OTVG PHAP LEN MEN FED-BATCH
IMPROVEMENT OF NATTOKINASE PRODUCTION BY BACILLUS SUBTILIS D USING FED-BATCH FERMENTATION
Nguyen Lan HiroTtg, Pham Tudn Anh, Ld Thj Quynh Nhu, Duong Vdn Hd, To Kim Anh Tnr&ng Dgt hoc Bdch khoa Hd Noi
Bin Toa soan 14-10-2012, chdp nhan dang 27-12-2012 TOM T A T
Enzym nattokinase duoc biit din la enzym thuy phan fibrin co nhiiu triin vong trong lieu phap diiu tp eae benh vi tim mach Trong nghien eiru nay. chung Bacillus subtilis D dwae len men theo me CO bd sung ea chit (fed-bateh) voi mong mudn nang cao hoat iwc sinh tdng hop nattokinase Khi tiin hanh len men fed-batch, thdi diim bd sung ca chit la sau 5 gia len men Hat ky thuat fed-batch duuc sif dung trong nghien ciru nay (i) fed-batch bo sung dmh dwang glucose vol tie do khong ddt 1 hoac 2 g/Uh, (ll) fed-batch dwa tren duy tri pH khong doi vai dich bd sung hoac hdn hop glucose va HCI ttoac eh! bd sung glucose Kit qua cho thiy hoat Iwc nattokinase trong canh trwang fed-bateh theo pH vdl viec ehi sir dung glucose c6 hoat Iwc eao gap 4 lan so hoat Iwc trong canh trwiyng vdi fed-batch glucose lien tuc va 4,6 lin so vdt hoat Iwc trong canh trwang fed-bateh theo pH vai dich b6 sung la hdn hop glucose va HCI vi gip 7 lin so vdt hoat Iwc trong canh truang len men theo me.
Tir khda nattokinase, len men fed-batch, Bacillus subtilis ABSTRACT
Nattokinase is a novel fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy In this study. Bacillus subtilis strain D was cultured in fed-batch fermentation for impmving the production of nattokinase In fed-batch culture, feeding was started after 5 hour of batch culture. The feeding strategies for nutrient were glucose supplying with 1or2 g/L/h and pH-stat using mixture of glucose and HCI, and only glucose. The nattokinase activity in fed-batch culture with pH- stat using only glucose feeding was 4, 4 6, and 7 times higher than in fed-batch with constant glucose feeding, pH-stat using mixture of glucose and HCI, and batch culture, respectively
I . M O D A U fed-batch thuong cho hieu suat tao san pham ,., , . , , , cao ban so vdi len men gian doan [2,3, 5], Vdi Nattokmase la mpt loa, enzym proteaza _^^^ ,..^ ^^^ 1^^, ^-^^ ^.^^ ,j„g ^ ^ CO tac dung lam tar, nhung sg, fibrm trong „,tt„ki„^,e trong moi trucmg long chung toi da huj-a thanh nguo,. Enzym nay da duoc t,m _j, ^.^ .^.^^ ^..^ j_.j^l^ |.^ ^^^ f^^_^^,^,, thay trong cac thuc iSn truyen thong cua nguo, ^. ^^^ j . nattokinase ti, Bacillus chau A, nhu Natto, Tofufo cua Nhat ban, nuoc ^_^j, .,.^ ,^^^ 2 ^^ j„. i, ^ j . ^ , , , , , , „ .^jt tuong Chungkook-Jang, nuoc mam Jeot-Oal |.._^ ^^^ ^.^ ,^„„g ^„. (^,„|, ,.„ ,^^„
cua Han quoc, Douclii cua Trung quoc. Cac v, ^^. ^.^ _,. , ,^^5^ ^ ^y^^._ ..^ f^j.^^,^^ j„y ,^i s,nh vat CO kha nang s,nh tong hop enzym da ^^. ^..^ |^. ^^.^, ^g„ | , ^ g|„^,„^^ ^j
% c t,m ra trong do cac lighten cuu vc v, j ^ ^ , |^^.^"^i,. ^. ^^^^^^ ^j„ (^„„g ^^„l, Khuan, dac biet la loai Bacillus la mgt trong
nhflng vi khuan cd kha nang sinh tdng hap
enzymnattokinasecao[4]. 2. NGUYEN VAT LIEU VA P H U O ^ N G
„ , , . , , . , , ,. , , . PHAP NGHIEN CUU Fed-batch la phuang phap len men co bo
sung CO chat theo thdi gian. U'u diem cua 2.1 Nguyen vat lieu
phirong phap nay la co chat dugc bd sung din _ ^ . ^ ^ 3^^ ^i„l^^ ^^^ ^^^^^ ,g„g ^^^g ^^
•ian trong qua trinh len men nen khong uc che ,^^^ ^.^ ^^^^^ j , . ^ Vj sinh vat, qua trinh len men co the dieu khien
toe do phat trien cua vi sinh vat, do do len men
I \ P C H i KHOA HQC A t ONG N < , F I K ( U TRUCJNG DAI HQC KY THUAT * SO 92 - 2013 - Chung gidng Bucdlus subtilis D (bd suu tap
gidng ciia lid mdn Cdng nghe Sinh hgc, DH B:ichkhoallN)[l],
Ciic hda chk phan lich: (Ibrin (MP Buuiicdical, Phap), Tris base (Merck, Dire), L- l\rnsinc(.luiisci Chem. Nhat ban).
- Mdj Irirdng NB (g/l) glucose- 10, cao niim men: 10. Kd IPO., 31 LO I. MgSO., J l LO: 0,5.
- Mdi Irudng lOn men (g I) bdt d;iu: 50, glucose, 10. KdlPOiJlJ.O- L MgS()|.71l-0 0..S - Dich dudng bd sung \'ao qua iriuli len men UiS) 60gizhicoscpha trong450ml.
Mdi irudng duac thanh iriing llO't trong 30 pluil.
2.2 PliuDiig pli:i[i nghien ciiu
2 2 I lloal hdu gii'ug. gidiig dugc boat hda iioiii! binh taiii giac 250 mL chira 50 ml. mdi trudng NB o nhict dd J 7 " C . liic 150 v/p thdi gian tir I4-I8h.
2 2 2 Len men trong he thdng len men 2 L: qua liinh len men dugc thirc hien trong he thdng len men 2L (hinh 1) vdi IL mdi trudng len men.
lliuh I Slur Kir I IIS
He ihdng len men 21 (Bio.siat B.
. Dire)
Cac thdng sd dugc dieu kien nlur sau:
nhiet dg len men 37"C. pH 7 diroc dicu chinh tir ddng bSng bom axit HCI 2N hoac NaOH 2N vao liuct hi len men, khuay trgn (200-800 rpm) va luu lugng khi (l-3vvp) d£ khong che ham luong oxi du trong dich 20 %. Sau 5 h len men djcii GS dugc bd sung vao thief bi len men, khi pH cua dich len men hon 7,0, djch GS se dugc tu dgng bam vao thiet bj len men. Trong suot qua trinh len men. djch len men dugc dinh k\
lay mau xac djnh hoat luc nattokinase \a ham lugng dudng khu.
2 2 3 Hogt lire nattokinase phuang phap so mau [1].
2.2 4 Himi hrang dirdng khir. dich len men dirpc Iy tam 10000 rpm, trong 10 phiit d 4"C, thu djch trong. LSy 200 jxl dich trong cho vao ong r.ppcndort, them 600 pi DNS (dinitrocalicylic axit) vao, trdn deu va tien hanh phan ting cr 95"C, trong 5 phiit. Sau dd dugc lam mat trong da 5 phiit Tien hanh do OD d budc sdng 540 nm. Dudng chuan dugc xay dyng vdi glucose la chiil chuan. Dua tren dd thj ducrng chuan se xac dinh dugc ham Iugng dudng khu trong djch len men.
3. KRT QI \ VA THAO LL.AN J.I Xac dinh thoi diem bd sun[> ca chat
Trong qua trinh Ien men fed-batch, thdi dicm bd xung co chat thudng la thdi diem co chat trong mdi trudng bat dau can kiet. Do va\.
dc xac dinh thdi diem fed-batch co chat, chiing toi lien hanh xac dinh lugng dudng khu trong dich theo thdi gian, dong thdi lugng axit tieu tdn de duy tri pH 7,0 trong thiet bi Ien men (hinh 2).
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Ifuih 2 Hdm lirang dic&ng khir va hrong axil bo sung theo thai gian len men
Theo hinh 2, lugng duong trong dich len men se giam nhanh va can kiet sau 5h len men. \ay de duy tri lugng dudng trong djch len men. qua trinh fed-batch se dugc khdi dgng vao gid thli 5, Iugng dudng tieu tdn tir I - 2 g/L/h, day se la can cii de bd sung Iugng dudng vao trong thiet bi.
Trong 5 h dau qua trinh len men pH luon duoc duy tri 7, tuy nhien bat dau tu gid thiJ 5.
axit HCI bat dau dugc bd xung de duy tri pH 7.
Day ciing chinh thdi diem Iugng glucose trong binh Ien men can kiet, do do cd the su dung pH
T^P CHI KHOA HOC & CONG NGHE CAC TRUONG DAI HQC kV THUAT * S 6 92 2013 nhir tin hieu ve sir thieu hut glucose trong qua
trinh len men. Day la ca sd de thuc hien qua trinh len men fed-batch theo pH (pH-stat), trung binh Ih se can bd xung 2 mL axil HCI 2 N/1 L canh trudng de du\ tri pH 7.
Nhu vay trong cac thi nghiem tiep theo chung ldi da tien hanh qua trinh Ien men fed- batch tir gid thii' 5, fed-batch theo co chat va fed-batch theo pH.
3.2 Len men fed-batch theo co' chai Qua trinh len men dugc bat dau vdi cac thong so nhu tren (muc 2.2.2). Sau 5h len men, qua trinh fed-batch bat dau, bom se ty ddng cung cap mdi gid Ig dudng glucose vao Irong binh len men. Thiet bj len men dugc ket ndi vdi
may linh va loan bg dir lieu nhu su thay ddi ciia ham luong oxi hda tan trong dich (p02), pH va lugng axit theo thdi gian len men dugc tu dong ghi lai (hinh 3).
KQ{ qua d hinh 3 cho llia\ sau hon 5h axit bat dau dugc dua vao trong binh len men di duy tri pH tai 7, Chung vi sinh vat phat trien manh sau I 1 h len men the hien d ham luang 0X1 hda tan ludn 0 % mac dii toe do khuay da len tdl da 800 rpm va da md van khi vdi tdc dd 2 vvp. Ddng Ihdi mdt luong ldn axil ciing dugc cap vao d^ duy tri pH 7 tir 13-24h Sau 24li axil khdng dugc cap vao den tan 28h. Hoat luc nattokinase trong dich len men sau 22, 24. 26 va 28 il dugc the hien d hinh 4
.„-\::;:. ,ij^
CZrj ig glucose/Ui I ! 2g glocose/L/h i H Duong + Axil I IDuang
iU
Htnh 3. Str lhay doi pO?, pH vd luang axil .sir dmg theo th&i gian Irong thiet bi 2L vdi qud trinh fed-batch Ig glucose/L/h
Ket qua d hinh 4 cho thay khi bd sung I g glucose/L/h hoat lye nattokinase dat cao nhat la 207 FU/mL sau 26 gid len men va hoat luc giam manh d 28 gid. So vdi len men theo me d ciing dieu kien hoat luc dat cue dai da tang dugc 1,7 lan (sd lieu khdng dua ra).
Tiep tuc tang Iugng co chat bd sung vao la 2 g glucose/L/h, qua trinh Ien men dugc tien hanh tuang ty nhu khi bd sung 1 g glucose/L/li.
Chiing phat trien nhanh sau 10 gid, tieu thu lirong ldn oxi va pH bat dau tang dan, tuy nhien lugng axit bd xung thap hon gan 3 lan so vdi trong trudng hgp fed-batch Ig glucose/L/h (sd lieu khdng dua ra). Hoat luc nattokinase trong djch Ien men d cac thdi diem khac nhau dugc the hien d hinh 4, Theo ket qua d hinh 4, hoat Iyc nattokinase trong dich len men thap han so v6i trudng hgp bd sung 1 g glucose/L/h, dieu nay CO the giai thich khi tang lugng co chat bd
Hinh 4 Hogt live nattokinase trong canh tru&ng len men & cdc th&i diem khdc nhau
sung len 2 g glucose/L/h da gay ra du thira ca chat (sd lieu khdng dua ra), iam cho qua trinh sinh tdng hop nattokinase chain lai, 3.3 Len men fed-batch theo pH
Trong len men fed-batch theo pH (pH- stat), pH duoc sir dung nhu chi thi de xac dinh thdt diem ngudn glucose trong binh len men can kiet qua dd de bd sung ihem ca chat. Theo nghien ciru da dugc cdng bd thi pH thich hop d^ Bacillus .subtilis phat trien tdt thay ddi tir 6,0 den 8,5 va pH tdi uu la 7,0 [4]. Trong cac thi nghiem d£ luon duy tri pH la 7,0 chiing tdi da X\kn hanh i) thay hoan toan axit bang dudng glucose, ii) bd sung hdn hap dudng va axit vao dich len men.
pH-.\lat bo sung du&ng
Trong 5 gid dau qua trinh len men dugc tiln hanh nhu len men theo me, bat dau tir gid
I M'CHi KHOA HQC & CONG NGHE CAC TRUdNG DAI HQC KV THUAT •SO 92-2013 thir 5, hinh chua axit dugc thay the bang binh
chira dirimg glucose. Tai gid thir 6 khi pH djch
^7.0, bam se tu dgng bat dua glucose vao trong ihict bi len men \di tdc do •- 2 g glucose/L/h.
Muc dich ciia vice khdng che nay la han die Iuong glucose duoc cap qua nhicu vao Irong canh Irudng, ddng ihdi lao dg irc ve thai gian di' \ I sinh vai chuyen lir Irang thai sir dyng cac chai du trir sang su dung glucose san sinh ra cac axil hini ca giam pH nidi Irudng. Trong qua trinh ien men do ludn co dg tre khi vi sinh vat chuyen tir trang thai sir dung axit huu ca du Iru sang trang ihai sir dung dudng glucose nen pH ludn Idn hon 7. Tai mdt sd thdi dicm chimg phat Irien nianii pll ien tdi 7,2 den 7,5. Do iugng dudng glucose duoc bd sung theo nhu can sii' dung ciia chiing nen chung phai trien manh. pO: ludn duy iri tai miic 0 sau gan 10 h Ien men (hinh 5),
\ A i l , HCI
1 1 1./
/ /
1
B 5
..
75
r
Hinh 5. Sir lhay dot cita pO^. pH vd lurmg axit sir dimg theo that gian trong thiet bi 2L vdi (pid trinh pH-stat ho sung dirang
lioat luc nattokinase trong dich len men dugc the hien trong hinh 4, tir 24h trd di hoat luc cua enzym tang dan chimg phat tnen manh ma dien hinh la den gid thu 30h thi hoat luc cua enzym dat d miic rat cao (832 FU/ml) so vdi cac thdi diem khac, gap 5 Ian so vdi thdi di^ni iuc24h (158,4 FU/ml).
pH-slal bo sung hdn hap dir&ng vd axil Dc khac phue nhugc diem du glucose trong dich len men ciia thi nghiem Iren, dich glucose dugc phoi trdn them axit HCI 2 N, luong axit dugc tinh toan sao cho mdt gid khong qua 2 mL/L (lugng axit tieu thu Ih tai len men theo me). Khi pH thay ddi, may cung ty dpng bo sung hdn hgp dudng va axit vao
dich len men de dua pH 7,0. Su bien ddi ndng do oxi hda tan, pH va luang axit bd sung vao dich len men hau nhu khdng thay ddi so vcri phuong an pH-stal bd sung dudng (hinh anh khdng dua vao). Tuy nhien hoat luc nattokinase trong dich len men khdng cd sir bien dgng ldn nhu trudng hgp bd sung dudng thay cho axit nhu d tren Sau 24h thi boat luc da dat cao nhat (179 FU/ml) trong khoang thdi gian khao sat va giam nhe khi keo dai thdi gian len men (hinh 4) Khi so sanh hoat Iyc nattokinase trong dich len men khi chi bo sung dudng cao gap 4,6 lan khi bd sung hdn hap dudng va axit. Ket qua lir binh 4 cung cho thay khi bd sung theo pH ket qua cao han han so vdi bd sung deu dan glucose theo thdi gian nhung khdng duy tri pH.
Khi bd sung vdi tdc dd glucose khdng ddi, kha nang thira hoac thieu dudng cd the xay ra trong cac giai doan phat tnen manh yeu khac nhau cua chiing trong qua trinh len men. Trong khi dd bd sung theo pH cd kha nang cung cap co chat theo nhu cau cua chiing trong qua trinh len men va lugng dudng dua vao thap hon nhu cau cua vi sinh vat. Hoat luc nattokinase trong djch len men khi bd sung dudng theo pH cao gap 4 lan so vdi len men bd sung vdi ca chat dn djnh va tang 7 lan so vdi len men theo me (sd lieu khong dua ra).
Ket qua tuang ty cung chi ra trong nghien ciru cua Cho \a cdng su (2010) khi sii dung phuang phap pH stat de Ien men fed-batch chiing Bacillus sublilus. hoat luc nattokinase thu dugc cung tang 2,1 Ian so vdi Ien men theo me [2]. Kwon VA cgng su (2011) su dung phuong phap pH stat fed-batch, glucose va pepton duoc ddng thdi dua vao mdi trudng len men vdi ti le thay ddi tir 0,2 den 5 g glucose/g pepton. Tac gia da nhan thay d ti le 0,33 giu'a glucose \a pepton dugc cung cap vao canh trudng len men theo pH boat Iyc nattokinase tang 4,3 Ian so vdi qua trinh len men theo me [3]. Nam 2012, Unrean va cgng sy su dung phuang phap bo sung co chat de Ien men chung Bacillus subtilis K-C3, glycerol dugc dua vao moi trucmg theo ham sd mil, hoat Iyc nattokinase thu dugc tang 20 Ian so vdi qua trinh len men theo me [5].
TAP CHI KHOA HOC & CONG NGHf CAC TRUONG DAI HOC KY THUAT • s 6 92 - 2013
4, KET LUAN Hoat lire nattokinase trong dich len men Len men theo me c6 b6 sung theo co chit ''''' ' * ^^^Pi ' ™ " 5 <*« «" ''i"!' P " '=.''° f P \ ^^
ho^c pH deu cho hoat luc nattokinase trong dich len men cao han so vdi Ien men theo me
so vdi Ien men bd sung dudng vdi toe dp co djnh nhung khdng on djnh pH va tang 7 lan so vai Ien men theo me
TAI LIEU THAM KHAO
1. Nguyen Lan Huong, Ha Thj Phuang, Pham Quoc Luan; Phan lap va tuyen chgn chiing vi khuan sinh tdng hgp enzym nattokinaza tir mgt sd thuc pham dau tuang Ien men; Tap chi khoa hgc &
cong nghe cac trudng dai hgc ky thu^t, 81, 175-179, (20! I).
2. Cho Y. H., Song J. Y., Kim K. M., Kim M. K., Lee I.Y., Kim S. B., Kim H, S., Han N. S., Lee B.
H., and B. S. Kim; Production of nattokinase by batch and fed-batch culture of Bacillus subtilis;
New Biotechnology, 27 (4), 341 -346, (2010)
3. Kwon E. Y., Kim K. M., Kim M K , Lee 1. Y., and B. S. Kiin; Production of nattokinase by high cell density fed-batch culture o(Bacillus sublili.K; Bioprocess Biosyst. Eng , 34(7), 789-793, (2011).
4. Peng, Y., X. Yang, and Y. Zhang; Miocrobial fibrinolytic enzim: an overview of source, production, properties, and thrombolytic in vivo; Microbiol. Biotechnol, 69, 126- 132, (2005), 5. Unrean P., Nguyen H. A. N., Visessanguan W., and P. Kitsubun, Improvement of nattokinase
production by Bacillus sublilis K-C3 using an exponential feeding fed-batch strategy. KKURes. J., 17(5), 769-777, (2012).
Dja chi lien he: Nguyen Lan Huang - Tel: (04)38682453- Email: [email protected] Vien Cdng nghe Sinh hgc va Cdng nghe Thuc pham
Trudng Dai hgc Bach khoa Ha Ndi
