Vd1277-2015Q420130200.pdf

HOI NGHI KHOA HQC CGNG NGHE SINH HOC TOAN QUOC 2013

NGHieN Cl>U DlEU KIEN L^N MEN SINH T O N G HC^P NATTOKINASE CUA BACILLUS SUBTILISJ(f KHO DAU TU'CNG

Trin Van Hien. Ph?m Thj Qujnh, Nguyen U n Huwng*

Vi0n Cdng ngh^ sinh hoc vd Cdng ngh4 thi/c phim, Trudng D§i hgc B^ch idioa Hi Ngi

T6MTAT

Natokinase 14 m^t enzym thiiy pbin fibrin c6 nhieu t i ^ vpng trong li^u phip di^u hi cac b^nh huySt kh6i. Kho d^u Mong la mgt ph\i phim pau protein ciia c8ng n ^ e p chl b i ^ diu in v i dugc sii dijng U co chit cho sinh tong h^p nattokinase (NK) tt chung Bacillus subtilis D bSne phiKmg phip l&i men chun. Trong nghiSn ciiu a&y, phucmg phSp hi m|t dip ikng (RSM) da dugc sii dyng d6 t6i uu dieu ld?n sinhtong hgp nattoldnase. Ba bien thay d6i la nong dp glucose, noog d$ peptone va pH ban dau. Kit qua phfin tich tiling M cho thay, trong khoing bien thien nghien ciiu thi nong dg glucose c6 4nh hudng nhiSu den kha nang suih tong h^p nattokinase. Ho?l luc nattokinase tnroc t6i uu dat 20,83 FO/mL. Vm dieu ki?n toi uu l i uSng do glucose 1,5%, nfing d$ peptone 1,5% vi pH ban diu 7,5, ho^t Ivc nattokinase djt ducfc 56,55 FU/mL K^t qua niy tang 2,7 iSn so v6i tnnic toi mi sinh quS trinh tSng h?p nattokinase bin cbixag D.

ra khoa: Bacillus sublilis, kh6 dau hnmg, len men, nattokinase. t6i uu bSng' RSM

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B$nh huyk kh6i di/pc hlnh thSnh do sy tlch h.i fibrin trong mgch ruAu ho§c trong cfic ngSn Mm, d3n d^n cac bfnh dOt quy vA tin mgch. Theo bao cSo cua T4 chCrc Y t^ th6 gidi hSng nam cac b#nh huy^t kh6i cufrp a\ sinh mgng cOa 17 tri$u ngu^i trSn thi gidi (Wang e( al., 2009). Hl$n nay, cSc ]l|u phSp di^u b1 cSc b|nh ndy bSng c i ti&m vk u6ng cic chlit ho^t huyit diivc nghifin ci>u rOng rai. Nattokinase (NK) diu tien du'g'c chiSt xu^t ti> d§u tu-ang I6n men theo phuwng phap Imy^n thing cua Nh§t B5n. Day la m$t lo^i enzym trong ti/ nhien c6 khii nang ti6u eye m^u dfing mgnh gSp 4 lln plasmin (enzym duy nhat trong co- the c6 kh^ nang tiSu cue m^u dong). Dong th&l nattokinase cQng ISm giSm (3$ nh<i1, dO dinh cua m^u, nhd v^y giSm thidu du^gc cac dieu ki^n gSy ra huylt kh6i vd cdc tai bien. Do dd, nattokinase diK?c khuy^n cio si> dyng trong hS tn? i3i4u trj cdc b§nh huylt khoi. Hi#n nay, trSn thj tn^o-ng cic s^n phSm chiia enzym nattokinase nhu': Nattospes, NattoK. Nattokinase... dSu dvi<?c nh$p tiF M^. Nh^t Bin, O^i Loan vd Trung Quoc, nSn gid thSnh cdc sSn phSm n^y Ik tucrng d6i dit V6\ mong mu6n chO d$ng du^c ngu6n enzym kh5ng phdi nh|p vk gldm giS thSnh sSn phim so v6'i nh$p ngo^i, c^c nghiSn ci>u hxing nude da t$p tmng nhlm c6 diKyc cfing ngh# sin xu^t nattokinase phO hgrp v6i dieu ki§n Vi§t Nam. Nghien CLPU cOa Phu'O'ng vk cOng SM" (2012) I6n men b l m|t trSn mfli trifdng h?t d^o tuo'ng v6'i quy mO 5 kg/m^ thu dupe ch6 phim c6 ho?t li/c nattokinase khoang 470 FU/g (Phupng et al., 2012). Trong cfing ngh# sdn xuSt enzym, mpt trong cdc cdng do^n quan ln?ng IS quS trinh ISn men sinh t6ng hgrp enzym, \^$c t6i tfu cdc thinh phln dinh diT&ng vk cdc dilu ki^n m6i bvfrng d6ng vai trd dSnh k l dk c6 thi nSng cao difQ'c ho^ It/c enzym. Nhl>ng n9m gin.day vifc sir dgng cdc phucng phap thing kS vh cSc ph^n mim quy ho9<^ thi/c nghi^m da glup glim thieu so thi nghi$m phAf tlln hanh nhu-ng van cung d p dSy dO thflng tin v^ dnh hif6ng cua cdc ylu t l din nhau vk din hkm mgc ti§u.

KhA d$u tu'O'ng \k mOt phg phim c^a cflng ngh$ chl biln diu an t£r d^u tipong, v6\ h^m lu'Q'ng protein tiio'ng Iv d^u tuvng nhu-ng chf dirge diing lam thi>c Sn ch3n nudi v^ gi^ thSiih r6 ho'n so vdi"d^u tucrng thi dSy \k nguySn lif u tit cho I6n men sinh tSng hgp "nattokinase. Trong nghiSn ci>u nky da kh^o s^t Snh hu-frng ciia m^t so ylu t l mSi tru-drig vk nguin dinh dw&ng din khi nang sinh ting hgp nattokinase cCia Bacillus subtilis, tCr d6 tiln h^nh t6i ipu c^c ylu t l Snh hudng theo phipgng phdp b l m|t dap Ong.

NGUYgN LI$U VA PHlfONG PHAP Nguyen ll^u

B$t dju vk bgl kho dju: d§u Urerng h^t (Bic Ninh). khi d^u tuwng (NhS m^y san xuat thipc kn gia sOc Nam Thiinh.

Thir&ng Tin. Hk Ngi) du'gc xay nhfi ttiinh t>Ot dirge gpi id bOt dgu vk bqt kha d|u Gilng vi khuin: Bacillus subtilis D vk B subtilis Ml nh|n ti> bfl su'u t$p gilng cua BO m6n Cflng nghe Sinh hpc, DH B^ch khoa Hd N^i. C^c h6a chit phSn tlch- Fibrin (MP Biomedical, Ph^p). Tris base (Merck~ Oi>c), L-Tyrosine (Junsci Chem, Nh$t Bin). Mfli tru-firng len men ran trong blnh tam giSc 250 mL: 20 g nguyfln lieu (dau tu'O'ng hogc khfl d|u). bo sung 30 mL niroc cit. Mfli tru-frng ISn men long (vtl/v): nguygn li^u (bgl d|u ho|c bOt khfl dau) 5%, peptone 0,5%. glucose 0,5%.

Phu>o>ng phdp nghl6n ciju

Hc^t h6a glong: gilng diK?c hogl h6a trong blnh tam gISc 250 mL chil'a 50 mL mfll trudng NB (Huwng et a!.. 2011) &

37°C, nufll lac 150 v/p thdi gian sau 18h, OD600 dgt 3.29.

Len men t6ng hpp nattokinase

Lfin men trSn mfli tjvflng long: cSc chung vl khuin (chCing D vk Ml) sau khi hoat hoa duvc chuyin l^n lugt vSo mfli tnrirng I6n men v6\ fi 1$ tilp gilng 5%, nuoi lie tie 3$ 150 v/p d 37''C, tlifti gian len men 24 gi&. Kit thuc qua trinh ISn men, djch sau Idn men dirge ly tam 6000 v/p, 15 phijt, * 4''C, thu djch trong. LSn rfien tron-mffi tnwng ran: c^c chCing vi khuSn (chijng D vk Ml) sau khi hogt hod di/gc chuyin lln lugt vao moi tnjdng Ifln men vdi fi l# tilp gilng 5%, nufli tTnh 6 37'C hong 24 gifi-. Tilp tgc t i n hinh chilt enzym bIng nu'dc cIt theo t! 1$ 1:1 (v/wt). lie 150 v/p trong 20 phiit, thu • djch ehiet vk dem ly tam 6000 v/p, 15 phiit, 6 A'C. thu djch trong. Djch trong di/gc dem xac dinh hogt Ivc nattokinase.

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HOrNGH! KHOA HOCTCONG N G H f SINHUpC TOSM'ODOC 2013

Khao sit m^t s6 yeu to inh hu-dng din qui trtnh len men tgo naftoJcfnase

L6n men liing tr§n moi t r u ^ g bflt khfl dau t i r o n g v 6 i chung D cr 37°C, toe do l i e 150 rpm. Cac thflng s6 khao skt d i r g e t § p h g p 6 B a n g 1 .

BAng 1- M$l 86 yeu t& va thdnh phan m6i tnrimg au^ khao sit

Glucose Peptone B9tkhdd$u pH Glong ThWgian Thfttidi

Glucose (%wt/v) i^-r' .15!&*iiS^

0.5 0.5 0.5 0.5 0,5 0,5

Peptone (%wt/v) 0,5 0,5 0,5 0,5 0.5 0.5

Bgt kha dgu- (%wt/v)

5 9 5

5 1 5 5 5

pH e,s 6,5 6,5 6.5 6,5 6,5

Gidng (%vtv) 5 5 5 5 5 5

• Thi'i gian (ngay)

1 -

Thfi tlch - [mL/250 mL)

50 50 50 50 50 1 50

I ^ ^ S ^ ^

T6I iru h6a thinh phin mdi truxmg len men b^ng phuvmg phip quy ho^ch thix nghl^m: 3 y l u to 3nh hirdng n h i l u d i n quS trinh l€n men la: h^m l i f g n g peptone bo sung, h a m Ii/gng glucose bo sung v^ pH ban d a u , dirge li/a chpn d l xay di;ng mfl hinh t i i u u . Th( nghi$m d u ^ b l h i theo ma tr$n trinh bay or BSng 2. K i t q u i d u g e xi> ly bang e h u v n g trinh Design-Expert 7.1 (Stat-Ease, I n C . Minneapolis, USA), d l ph3n tlch ckc h$ s6 h l l quy v l b l m^t dap i>ng (Deepak et al., 2008).

Phucmg phipxic dinh ho^it lipc nattokinase: bang phirang p h i p so m l u (Ngan, Huong, 2011). MOt d o n vj ho^t d$

nattokinase du'gc tinh bSng lu'gng enzym c i n t h i l t d l thuy phSn fibrin t^o skn p h i m (khong k i t tiia bfli tiiehloroacetie axft) t u v n g d u o n g vdi 1 pg tyroslne/1 mL hong 1 phiit it 37^0.

K & r Q U A V A T H A O L U A N

L i ^ c h g n n g u y e n l i f u , p h i r o n g phap ten m e n v a chOng v l k h u i n

Nguydn ll$u d i / g c si> dgng l l n men l i n g vifri d i i i n g D la b$t khfl d|iu t u g n g vk b$t h^t d$u t u c n g , Hoat Igc nattokinase trong eanh I n r a n g sau l4n men du'gc the h i | n d Hinh 1 A .

H

g i 1

Hlnh 1. H o ^ tire nattokinase tren c c c h i t b^t kha d | u , b f t h ^ d9v(A], mAI tnrimg IBn man r i n , long (B) cua 2 chung O & M l (C) Hlnh IA cho t h i y hogt Igc nattokinase (NK) trong djch sau ISn men ti> b^t khd d | u tuwng d^t 12,50 FU/mL (khoang 68%) so v6l ho?t Ige NK trong djch sau len men ti> b^t d^u hrcrng (18,33 FU/mL), dieu dfl cho t h i y r^ng hogt Igc NK thu djirgc h'nh the&gia nguySn li$u thlsu- dyng khfl d$u h r a n g kinh t l horn (vl giS chf b I n g 4 0 % so.vdn hat d ^ u hfcmg), - V l - - v | y khfl d|iu tirang du'gc l^a chgn cho cde fighlSn ciru t i l p theo. Chiing D flu-gc nufli c l y tr6n 2 mfli tru'fl'ng ISn men r i n va l i n g . K i t qua Hlnh 1.B cho t h i y hoat tire NK tinh theo 1g nguy§n Ii#u ban d i u (vcri dO I n i 12%) ciia phucrng phap ISn men loiig eao hon 12 l l n so v g i len men ran. Do do li^a chgn phircmg phap ISn men l i n g de tien hanh cac khao sat t i l p theo. T i l n hanh ISn men long sir dgng 2 chiing D vS M l . Hoat Igc NK trong djch sau ISn men du-gc the hiSn a Hlnh 1.0.

ChOng D c6 kha nang sinh t i n g hgp NK vfl'i hogt \vc cao h g n 1,4 l l n so vi>\ chung M l va diitfc su dgng trong cac nghiSn e u u t i l p theo.

K h a o sat m $ t s i d l l u k i ^ n ISn men -

Thanh phan moi tnrdng len men —•

Khfl a§u t i w n g du-gc n g h l l n nho va phan lo^i theo kfch thirtrc sau dd difgc si> dghg d l ISn men l i n g bfi-i chiing 0 . Ho?t ii,fe NK thu d u g o sau len men d u g c t h i hiSn d Hinh 2 .

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Hlnh 2. Anh huwng cua (A) kfch thirdv nguyin ll^u va (S) ham lugng b9t kha d | u d i n ho^t Iifc nattokinase

HOI NGH! KHOA HOC C O N G N G H f SINH HQC T O A N Q U 6 C 2013

Hlnh 2.A. cho I h l y , hoat lu>c nattokinase sau ISn men t i IS nghich v 6 i klch tht/dv nguySn l i f u, nguySn li$u ckng d t / g c xCr l>' n h l thi hoat li/c NK cang cao. So vdi nguyen li$u ban d i u , hoat li/e NK tang g i p dOi (tif 6,25 len 13.75 FU/mL) khi nguySn li#u c6 kfch thura-c 0.1+0,5mm K i t qua nay ggi |? r i n g , khi nguySn li#u e6 kfeh thifd'C n h l thi khS nSng h i a tan cac c h i t vao m6i t r u i n g tang vS vi smh vat d l dang si> dgng eg c h i t han. Di/a vko k i t q u i nay, lira chgn phu-gng an xi> 1;? nguyen IISu v l klch thir&e 0.1-K).5mm d l filn hanh cac khko sat sau. Ham l u ^ n g bgt khfl d | u si> dgng cung la y l u t l anh hirgng d i n ho^t Ige nattokinase (Kinh 2.6). khi tang ham l i / g n g khfl d$u tii 2,5 d i n 5% thi ho^t li/e N K tang khflng dang k l (Kr n , 6 7 d i n 12.50 FU/mL) n h i m g khi tang ham l i / g n g khd dgu tO 5 den 7,5% thi tSng m^nh (tir 12,50 d i n 15,83 FU/mL), tSng t i l p hkm li/gng khfl d$u ISn 10% thi hoat Igc tang ft. Do Gin hanh trong quy mfl b1nh tam giac nSn khao sat chf dOng l^i ir mirc ham lu^nng khfl d^u tang d i n 10%.

Ngoai bOI khfl d$u, ham lu'gng peptone va glucose b l sung cOng Snh hirorng n h i l u d i n ho^t Igc nattokinase (Hinh 3).

Hlnh 3. Anh htrimg cQa (A) hSm l i / ^ n g peptone v i (B) glucose-bl sung d i n ho^t ILPC nattokinase Hinh 3 A cho t h i y . hogt Igc NK cao h g n khi mfli trvfl-ng ISn men c d b l sung peptone ti> 0,5 d i n 1.5% va d^t CIFC d^i tal ham li/gng peptone b l sung la 1,5%(wtA/). tilp tgc tang thflm lirgng peptone b l sung thi f u ^ t li/e NK 1^' cd xu h i r d n g g i i m d i n . Theo Hinh 3.B, h o ^ tg<: NK sau ISn men d^t cao n h l t la 18,33 FU/mL khi ham lus^ng glucose b l s u n g IS 1,5%. K i t qua nay t i / a n g dong vdrl nghiSn cOu cua Ku va cgng s g (2009) da si> dgng 1,75% glucose t>6 sung trong canh tnrimg ISn men Iflng (Ku e( al., 2009).

Af^f 5 ^ yiu tS inh hirdng din ho^t lye nattokinase

ChOng D dirge ISn men Iflng trong mfli trtrdng c6 thSnh p h l n (vrt/v): b ^ khfl d | u 5%, glucose b l s u n g 0,5%, peptone b l sung 0,5%. Thay d ^ gia trj pH ban d i u cua mfli tiuimg ISn men. K i t qua hogt Igc NK thu d i r g e s a u ISn men d u " ^ t h i hISn * Hlnh 4. K i t qua * Hinh 4 eho thiy.'hogt Igc NK sau ISn men cao horn khi pH ban d i u c 6 tfnh k i l m . e g c d?! d pH ban d i u la 8. Ham lui?ng g i l n g ban d i u anh hir&ng khflng n h i l u d i n hogt l y e NK, khi hkm lu-gng g i l n g c i p vSo tSng lir 5 d i n 15% thi ho?t igc NK trong djch sau iSn men e l t i n g nhu'ng khflng dkng k l (Hlnh 5). 6 2 0 % hogt l i / c nattokinase eiia dich sau ISn men cd tang, tuy nhiSn l i e dO tang k h i n g n h i l u so vd-i s g tang hkm lirgng g i l n g .

!•

nil

Hlnh 4. Anh hufrng cua pH ban Oku d i n hoft Ig'c NK

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Htm liT^n^ tfl^9 b*M

Hlnh 5. Anh hurbng cQa ham lu'gng g i l n g d i n ho^t l y e NK

MM

Hlnh S. ^ l l l hM„s cOa t h M gran » n n » n l i n h o j t UfC NK Hlnh 7. Anh h i r o n j cm I h i Oeh d|ch ISn m . n dSn h o j l Wc NK

^ " ^ I f i ; " , " ' i ' ' \ ' r ' i l ' S . ! J - ' ^ ? ' " . " " ™" ! " S ^ ' ' ^ " 3 " - " . ™ ' ' * " ' 5 , 0 0 FU/mL khi ISn men 2 ngSy vi g l i m xu6ng 13,33 FU'mL W« kSo dSi th6, gian Un men den 3 ngay N h l / v8y, 2 ngSy IS t h « gian Ihlch twp cho ChOng D phai trl4n A 1? S K f ""15^ ' * * ' ' " ^ * " ' * ' • * " " = ^ ' ^ 1*1 «>* " * ' ' I ' * ISn men B n g dSn Ihl host lire NK sau ISn men giam Dan, host lire NK Irong d|ch sail ISn men lang manh Kl 13,75 Sin 20,83 FU/mL khi t h i K d l <)|ch ISn men g l i m tCr 50 a j o n g 25 mL. DiSu nSy cung p h u h ( r p irol nWSu nghiSn c i h i cOa LI vS cOng sf (2011) khi nSn hSnh ISn men trong blnh tam giSc 00 mL v * thS l(ch dich t£r 20 - 60 mL thi thS tich djch ISn men cSng tSng thi hoal lire NK cSng g H m vS thS tlch c h o h o a t l i r c N K c a o n h a t l S 2 0 m L ( U e ( a / . , 2 0 1 1 ) .

T i l i n i h o i mSI t n r i r n g len men b I n g p h i p m g p h i p bS m a t d i p l i n g (RSM)

2 * 3 . 1 ° / ' ' ' " ! " u u * ? " " ^ . ' " ^ ' i ' ^ I ? ' " " V • > " - " « ' ^ yi" ' 4 SnU t i i r d n g d i n q u i trinh ISn men, d i Ura c h , n c i o y i u ' 10 CO xna nang i n h h i r t n g nhieu n h i t a i n hoat lire cOa NK IS: h i m l u v n g glucose b i sung. M m l i « m g peptone h i s u n g

»a pH ban d i u v i c6 d|nh cac y i u t i khSc: ham l i / y n g k h i d i u t i r m g 10%, hSm lirimg glong 10%, Diftl gian ISn m e n 2

HOI NGH! KHOA H Q C " C O N G NGHe SINH HQC T O A N Q U 6 C 2013

ngay. t h i tlch d[ch |gn men 25/250 mL. nufli lac 150 v/p a 3 7 ° C d e t i l n hanh toi u u hoa. Hogtii/f: nattnbinatip thu rt,^f~

sau qua trinh I6n men ciia cac thi nghl#m d u v c & i l hl$n a B i n g 2.

B i n g 2. Cic thi nghi$m t i l n hanh v i ho^t lire NK thu d u v c Bang 3. K i t q u i p h i n tich h i i quy

1 3

6 7 6 9 10 11 12 13 14 15 16 17 18 19 20

Glucose (%)A 1,50 2,00 1.50 1,50 1.50 1,00 1,50 1,50 1,50 0,66 1,00 1.50 1.00 2,00 2.34 2,00 1,50 1,00 1,50 '2,00

Peptone (%)8

1,50 1,00 1,50 2,34 1.50 1,00 1.50 1,50 1,50 1,50 2,00 1.50 2,00 1,00 1,50 2,00 1,50 1,00 0.66 2,00

- pH C 7,50 9.00 7,50 7,50 7,50 6,00 10,02 7,50 4,98 7,50 6,00 7,50 9,00 6,00 7,50 6,00 7,50 9,00 7.50 9.00.

Ho^t Igc NK (FU/mL)

56,53 18,33 56,53 31.67- 56.53 5S,S3 44,17 56,53 34.17 37,50 28,33 56,53 40,00 26,67 36,67 34,17 56,53 49,17 50,83 46,67

268,66 174,99 80,00 49,45 657.21 0.0900 ' 191,79 711,52 444,54 570,06

12,86 7.80 3,56 2,20 29.29

" 4,025E-003 8,55' 31.71 19.81 25.40

<0.0002 0.0190 0.0883 0.1685 0.0003 0.9507 0,0152 0,0002 0.0012 0.0005

Theo k i t q u i BSng 2, hoat Igc nattokinase .sau \&n men dao dOng trong k h o i n g tir 1S,33 FU/mL d i n 56,53 FU/mL, dihu nky cho Oily hogi Igc enzym bi anh hirgng b6i cSc y l u t l nghiSn ci>u. D l k i l m tra s g cd nghTa ciia cae h$ so va s g tu'O'ng thfeh eua mfl hlnh th^c nghf#m, p h l n m i m c6 du-a ra b i n g phSn tich h i i quy (Bang 3). C h u i n F ciia mfl hinh la 12,86 va mOe co nghTa ciia m l hlnh p<0,05 d i l u nay cd nghTa la mi>e dg tin c^y cOa mfl hlnh IS 99%. H$ so lu'gng quan bgi cOa mfl hlnh R \k 0,92 cho t h i y m6 hinh ti/crng t h l d i sat vdri thgc nghi§m. Cac phfin tich t h i n g k l cho t h i y . khi cSc b i l n thay d l l Irong khoang b i l n t h i l n thi A (ham l u v n g glucose) cd i n h h u ^ n g n h i l u n h l t d i n ho?t lye enzym, d i l u d d t h i hi$n 6 gia tit p cOa yeu t l nky \k nhd n h l t so vd'i cae y l u t l . khae va nhd hon 0,05, d i l u nfiy cOng phO h ? p vdri k i t qua & hinh 8. Chuo'ng trinh quy hogch thi/e n g h i f m c6n di/a ra phiKnig trinh bfic 2 t h i h i | n m i l liSn h$ giOa cac y l u t l d i u vao va hogt l\FC nattokinase sau \kn men (Y).

Y = 56,55 - 3 , 5 8 A - 2,42B + 1,9C + 9.06AB T 0,11 AC + 4,9BC - 7,03A^ - 5,55B^ - 6,29C^

Hinh a. Anh huwng cCia c i c y l u t i din h o j t lv« NK Hinh 9. B 4 ni|t d i p i>ng cita ho^t I i ^ nattokinase vk m i l quan h f glQ'a pH v i h i m lirgng glucose Hinh anh khdng gian 3 c h i l u b l m H dap li-ng ciia hogt lye nattokinase d u v c bifiu d i l n d Hl'nh 9. Phfin tich b l mgt dap ij-ng eho t h i y d i l m cho hogt lire nattokinase cao n h l t chlnh Ifi tSm cOa cac hinh elip. B i l m nky cd pH (7,5), hkm l i r p n g glucose (1 5%), ham l u v n g peptone (1,5%) cho ho?t lire nattokinase dgt dvQc 56,55 FU/mL. K i t q u i nay ho^n toan Ufffng thlcli v * i k i t quk k i l m tra I9I b I n g th^re nghi^m i g i d i l m t i i u-u. Nhu- v | y e6 t h i t h i y hogl li/c nattokinase tgi d i l m t i i u-ii cao g I p 2,7 Ian so v * i hoat li/c natttAlnase dgt dirgrc khi chi/a t i i iru. Tuy nhifin hogt li/c NK dgt chu« cao va thf nghiSm mdi di>ng l^i if binh tam gific, e i n phai hoan thi$n va t i l i n i khi ISn men 6 cSc t h i l t bj ISn men. Trong m^t nghiSn c i i u ciia Deepak vk c | n g s i / (2008) da tdi unj thanh p h l n m i l tartmg sinh t i n g hpp nattokinase e blnh tam giac bl-i Chung S. suM//s bang phu'cmg phap b l m?t d i p i>ng. hogt li/e nattokinase t^i d i l m t l i u-u cao g I p 2 l l n so vdi h i r o c khi t i i ifu (Deepak et al, 2008), M^t nghifin c i h i khae eua Mahajan va c<)ng sti (2010) da t i l u'u d l l n ki#n ISn men sinh t i n g hop nattokinase ciia chiing S. natto NRRL 3666 cho t h i y Wii ISn men trong t h i l t bj len men (5L) hogt \\fc nattokinase dgt d t f ^ cao g I p 1,6 l l n d' blnh tam giac (Mahajan et al., 2010).

HOI NGH! KHOA HOC C 6 N G NGHE SINH HOC T O A N Q U O C 2013

K ^ L U A N

Khd dfiu tircrng la n g u l n nguyfin li$u ^ i e h hgp cho Bacillus subtilis D iSn men sinh t i n g hgrp nattokinase bang phuMS'ng ' phfip ISn men chim. Cae yeu t l knh h u m i g n h i l u d i n hoat li/e nattokinase la ham lugrng glucose, ham l u v n g peptone b l sung vfi pH ban d i u . Sir dung ahi/cmg phap quy ho^ch thi/c nghi$m be m | t dfip t>ng da tlm du<?c dieu kiSn t i i i f u cho vi k h u i n B.si/b67/s D len men tong h g p nattokinase nhu-sau: glucose 1,5%, peptone 1,5%, pH ban d i u 7,5 ; h o a t l i / c nattokinase d^t 56,55 FU/mL. c9.o g I p 2,7 l l n so v 6 i tnrd'c khi t i i u u .

T A I U ^ U THAM K H A O

Deepak V, K Kalishwaralai, S Ramkumarpandian, S Venkatesh Babu, and SR Senthiil(umar (2006). Optimization of media composltaon for nattokinase production by Sadffus suUiifs using response surface methodology. S/o/esou/csTechno/ogy. 99, 8170-8174

Mahajan PM, SV Gokhale, and SS Lde (2010). Production of nattokinase using Bacillus natlo NRRL 3666: media optimization, scale up. and kinetic modeling. Food Sd Biateclmol, 19.1593-1603.

HiKrng NU HT Phuong. PQ I j j i n (2011). Phan l^p v i tuyln chqin chiJng vl khuin sinh ting hi^p enzym nattokinaza tCr m$t s i thi/c phim diu Iifcmg l i n men. Tap chl Idma hgc ic&ngrtgh^dc truimg d^ifiOck$Viu$t.Zi, 175-179

Phuyng LTB, VT Hsnh, TT Phong, LT Hung, TTH V i n . LT Hirong (2012) PhSn l^p v i tuyin ch^n m9t s i chiing Backus ^nh t i n g h m nattokinase. Tap cftf s/nh hgc. 34 (SSE), 99-104.

Wang C, M Du. D Zheng. K Kong, G Zu, and Y Feng (2009) PurificaHon and Characterization of Nattokinase (torn Bacillus subtilis Natto B-12.JoMffm:FoorfC/jem.57, 9722-9729.

STUDY ON FERMENTATIVE CONDITIONS FOR NATTOKINASE PRODUCTION BY BACILLUS SUBTILfS USING SOYBEAN OIL CAKE

Tran Van Hien, Pham T h I Q u y n h , Nguyen Lan H u o n g *

School of Biotechnology and Food Technology, Hanoi Univenstiy ofScience and Technology

SUMMARY

Nattoldnase is a potent fibrinolytic enzyme that is considered to he a promising agent for thrombosis dieiapy. S c ^ e a n oil cake is protein waste from vegetable oil processing industry and was used as substrate for production of nattokinase (NIQ b y Bacillus subtilis D shain in submerged fermentatioa In this study, response surface methodology (RSM) was applied to optimize conditions for NK production. The three variables were glucose concentration, peptone concentration and initial pH. The statistical analysis of the results showed that, in the range shidies, glucose concentration had a significant effect on nattokinase production. ITie nattokinase a c t i v i ^ under unoptimized conditims was 20.83 FU/mL. The optimized conditions were found to b e con^irision o f 1.5% glucose 1.5% peptone and initial pH 7.5. The nattokinase activity was obtained 56.55 FU/mL. The optimization b y RSM resulted in about 2 7 fold increase in the production QfNK by B. subtilis D strain.

Keywords: Bacillus subtilis, soybean oil cake, fermentation, nattoldnase, optimization by RSIvl

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