B Y T TRNG I HC DC H NI NGUYN NGC NH KHO ST NH HNG CA MI TRNG DINH DNG LN KH NNG TO ENZYM CA VI KHUN Bacillus subtilis natto KHA LUN TT NGHIP DC S

H NI 2013 B Y T TRNG I HC DC H NI NGUYN NGC NH KHO ST NH HNG CA MI TRNG DINH DNG LN KH NNG TO ENZYM CA VI KHUN Bacillus subtilis natto KHA LUN TT NGHIP DC S Ngi hng dn:TS. m Thanh Xun Ni thc hin: B mn Cng nghip dc H NI - 2013 LI CM N Vi s knh trng v lng bit n su sc ti xin gi li cm n n c gio,TS. m Thanh Xunv DS.L Ngc Khnh,nhngngithy tn tnh hng dn v ch bo cho ti trong thi gian lm kha lun. ngthi,tixingi licmnticcthyc gio,ccanhchk thut vin trong b mn Cng Nghip Dc to mi iu kin gip ti trong sut thi gian nghin cu hon thnh kha lun. Ti cng xin gi li cm n n Ban gim hiu cng ton th cc thy c gio trng i hc Dc H Ni dy d v to mi iu kin thun li cho ti trong thi gian ti hc tp ti trng. Cui cng, ti xin by t lng bit n su sc ti gia nh, ngi thn, bn b lun gip , ng vin ti trong sut thi gian hc tp va qua. H Ni, ngy 21 thng 5 nm 2013. Sinh vinNguyn Ngc nh MC LC T VN .........................................................................................................1 CHNG I: TNG QUAN.................................................................................... 2 1.1. Vi khun Bacillus subtilis natto ........................................................................2 1.1.1. Phn loi khoa hc.................................................................................2 1.1.2.Ngun gc ............................................................................................. 2 1.1.3. c im hnh thi .................................................................................2 1.1.4.c im sinh dng.............................................................................3 1.1.5.c im sinh l.................................................................................... 3 1.1.6.Sn phm trao i cht ca B. subtilis natto..........................................3 1.2. Natto v Nattokinase......................................................................................... 4 1.2.1. Natto.......................................................................................................4 1.2.2. Cc enzym c kh nng tiu huyt khi trc Nattokinase...................... 5 1.2.3. Nattokinase............................................................................................. 6 1.3. Qu trnh ln men sn xut enzym.................................................................. 10 1.3.1.Cc phng php ln men sn xut enzym t vi sinh vt...................... 10 1.3.2. Thu nhn ch phm enzym.................................................................... 11 1.3.3. Cc yu t nh hng ti s tng hp enzym........................................ 11 1.4. Cc nghin cu v nh hng ca mi trng dinh dng n kh nng sinh enzym Nattokinase ca vi khun ........................................................................... 12 1.4.1. Ngoi nc........................................................................................... 13 1.4.2. Trong nc........................................................................................... 13 CHNG II: I TNG V PHNG PHP NGHIN CU...................... 14 2.1. Nguyn vt liu v thit b s dng................................................................. 14 2.1.1. Vi sinh vt s dng ............................................................................... 14 2.1.2. Nguyn liu, ha cht s dng:............................................................. 14 2.1.3. Cc mi trng s dng: ...................................................................... 14 2.1.4. Thit b ................................................................................................. 15 2.1.5. Dng c................................................................................................ 16 2.2. Ni dung nghin cu ...................................................................................... 16 2.2.1. Phn lp vi khun B. subtilis natto t sn phm Natto v xc nh enzym do Bacillus subtilis natto to ra. ..................................................................... 16 2.2.2. Kho st nh hng ca iu kin dinh dng ln kh nng to enzymca B. subtilis natto. ....................................................................................... 16 2.3. PHNG PHP NGHIN CU:.................................................................. 16 2.3.1. Phng php phn lp vi khun t sn phm Natto.............................. 16 2.3.2. Phng php nhum Ogiestka.............................................................. 17 2.3.3. Phng php gi ging trn thch nghing.......................................... 17 2.3.4. Phng php nhn ging trong mi trng lng.................................. 17 2.3.5. Phng php ln men chm vi khun .................................................... 18 2.3.6. Phng php kho st nh hng ca mi trng dinh dng ln kh nng sinh enzym............................................................................................. 18 2.3.7. Phng php xc nh s c mt ca enzym trong sn phm Natto...... 19 2.3.8. Phng php xc nh enzym trong dch trong v trong sinh khi ca dch ln men vi khun Bacillus subtilis natto .................................................. 19 2.3.9. Phng php th hot tnh protease:.................................................... 19 2.3.10. Phng php th hot tnh amylase.................................................... 20 2.3.11. Phng php th hot tnh cellulase: ................................................. 202.3.12. Phng php th hot tnh fibrinolytic (tiu fibrin) ............................ 21 CHNG BA: THC NGHIM, KT QU V BN LUN ........................... 22 3.1. Phn lp vi khun B. subtilis natto t ch phm Natto v xc nh enzym do vi khun sinh ra......................................................................................................... 22 3.1.1. Phn lp vi khun Bacillus subtilis natto t ch phm Natto................. 22 3.1.2. Xc nh mt s enzym do vi khun sinh ra........................................... 23 3.1.3. Xc nh enzymtrong sn phm Natto................................................. 25 3.2.Kho st nh hng ca mi trng dinh dng n kh nng sinh enzym ca vi khun B. subtilis natto ....................................................................................... 26 3.2.1. Kho st nh hng ca ngun hydratcarbon ...................................... 26 3.2.2. La chn nng maltose thch hp nht ............................................ 30 3.2.3. Kho st nh hng ca u tng v cht cm ng............................ 32 3.2.4. Kho st nh hng ca mt s mui v c.......................................... 34 3.2.5. Xy dng mi trng tng hp cho vi khun Bacillus subtilis natto sinh protease ......................................................................................................... 35 KT LUN V XUT ............................................................................................ 39 DANH MC CC BNG Tn bngTrang Bng 1.1: Thnh phn chnh trong 100g Natto4 Bng 2.2: Thc phm chc nng cha Nattokinase9 Bng 2.1 . Cc nguyn liu, ha cht s dng14 Bng 2.2. Cc thit b s dng15 Bng 3.1. Kt qu nh tnh enzym trong dch ly tm v trong sinh khi 24 Bng3.2.Hottnhenzymth hin bng ngknhvngphn giicchtcadchenzymchittphnhtuvphndch nht ca Natto 25 Bng 3.3. Hot tnh protease ca dch ln men khi nui cy trong mitrngMT2vmitrngMT2cbsungccngun hydratcarbon khc nhau th hin bng ng knh vng phn gii casein 27 Bng3.4.Hottnhamylasevcellulasecadchlnmenkhi nuicytrongmitrngMT2vmitrngMT2cbsung ccngunhydratcarbonkhcnhauthhinbngngknh vng phn gii c cht 27 Bng 3.5. Thi gian tiu fibrin ca dch ln men thu c khi nui cytrongmitrngcbsungccngunhydratcarbonkhc nhau 29 Bng 3.6. Hot tnh protease ca dch ln men khi nui cy trong30 mitrngMT2vmitrngMT2cbsungmaltosecc nngkhcnhauthhinbngngknhvngphngii casein Bng3.7.Hottnhamylasevcellulasecadchlnmenkhi nui cy trong mi trng MT2 c b sung maltose cc nng khc nhau th hin bng ng knh vng phn gii c cht 31 Bng 3.8. Hot tnh protease ca dch ln men khi nui cy trong mitrngMT2vmitrngMT2cbsungutngv caseinth hin bng ng knh vng phn gii c cht 32 Bng 3.9.Hottnhenzymeca dchlnmenkhi nuicytrong mi trng MT2 v MT2 c b sung mt s mui v c, th hin bng ng knh vng phn gii c cht 34 Bng 3.10. Hot tnh enzyme ca dch ln men khi nui cy trong mitrngMT2vmitrngtnghp,thhinbngng knh vng phn gii c cht 36 Bng3.11.Thigiantiufibrincadchlnmenthuckhi nui cy trong mi trng canh thang v mi trng tng hp37 DANH MC CC HNH V, BIU Tn hnhTrang Hnh 1.1: T bo vi khun Bacillus subtilis natto2 Hnh 1.2. Cu trc bc 3 ca Nattokinase7 Hnh 1.3. C ch tiu fibrin ca Nattokinase8 Hnh3.1.nhkhunlcphnlpcvhnhnhtbovi khun khi soi trn knh hin vi phng i 1.000 ln 22 Hnh3.2.Ktquthhottnhprotease,amylase,cellulase trong dch ly tm v dch chit t sinh khi 24 Hnh3.3.Biuthhinnhhngcangunhydratcarbon n hot tnh enzym ca vi khun 28 Hnh3.4.BiusosnhkhnngsinhenzymcaBacillus subtilis natto khi nui cy trong mi trng MT2 v mi trng tng hp 37 DANH MC CH VIT TT STTCh vit ttTn y 1B. subtilis nattoBacillus subtilis natto 2E. coliEscherichia coli 3CUCaseinolytic units 4FUFibrinolytic units 5UUnit 6t- PATissue plasminogen activator 7 PGA - polyglutamat 8Na CMCNatri carboxymethylcellulose 1 T VN Hin nay, t l mc bnh nghn ng mch do cc cc mu ng nh chng nhimuctimhaynhimunoangtngcaoVitNamcngnhtrnth gii.Ccloithucangdnghinnaynhurokinase,streptokinase,...cs dng ch yu trong cp cu tai bin v c nhiu tc dng ph. Nattokinase l enzym c ngun gc t Natto, mt mn n truyn thng ca Nht Bn. Nattokinase l mt enzym protease do vi khun Bacillus subtilis natto sinh ra, c s dng phng v ph cc cc mu ng do c tc dng tiu fibrin mnh, an ton khi s dng.Trongiukinlnmenchm,vikhunBacillussubtilisnattocnckh nng sinh nhiu enzym ngoi bo nh protease, amylase, cellulase... Vic thay i mi trng dinh dng c th nh hng n s sinh enzym ca vi khun. V th, lachn mt mitrng tt nhtcho enzymsinhlnglnproteasevgim bt ccenzymkhcngvaitrquantrngtrongvictchchitprotease,saul Nattokinase.Do,khalunKhostnh hngcamitrngdinh dngnkh nng sinh enzym ca vi khun Bacillus subtilis natto c thc hin vi hai mc tiu: 1. Phn lp vi khun Bacillus subtilis natto t sn phm Natto trn th trng v xc nh mt s enzym do vi khun sinh ra. 2.BculachnmitrngnuicychoBacillussubtilisnattosnxut protease tiu fibrin. 2 CHNG I: TNG QUAN 1.1. Vi khun Bacillus subtilis natto B. subtilis natto (cn c gi l Bacillus subtilis var. natto) l mt chng thuc loiBacillussubtilis,cgiosSawamuraphnlpvnhdanhlnutin vo nm 1905 [ 21]. 1.1.1. Phn loi khoa hc B. subtilis natto l mt vi khun tht (Eurobacteria) thuc: H (Family): Bacillaceae Chi (Genus): Bacillus Loi (Species): Bacillus subtilis Phn loi (Subspecies): Bacillus subtilis natto [32]. 1.1.2.Ngun gc B. subtilis natto c gio s Sawamura tm thy trong Natto, mt mn n truyn thngct hngngn nmtrcca ngi Nht.B.subtilis nattoc nhiutrong rm r, c kh... v pht trin tt trong cc loi u, c bit l u tng; vi khun ny cng pht trin trn thc phm c ngun gc ng vt v thc vt khc nh: to bin, c, tht, tm, cua...[13], [22]. 1.1.3. c im hnh thi Hnh 1.1: T bo vi khun Bacillus subtilis natto [27], [30] B. subtilis natto l trc khun hiu kh, bt mu Gram dng, ni bo t hnh que c kch thc di 1m. Cc t bo vi khun ng ring r hay ni vi nhau thnh 3 chui.Khunlckh,khngmuhocmu xmnht,ckchthclnvhnh dngbtnh.Bmthinhnhoctoralpmngmnlanrngtrnbmt thch, c mp nhn bm cht vo mi trng thch [20],[ 22]. 1.1.4.c im sinh dng B.subtilisnattolsinhvtddng,ckhnngsdngnhiungun hydratcarbon: ng nnh glucose,fructose..., ng i nh maltose,lactose, saccharose...,polysaccharidnhtinhbt...Ngunnitcnthitchosphttrin ca vi khun l protein v aminoacid. B. subtilis natto c th s dng d dng acid glutamic, arginine, acid aspartic... nhng khng s dng c threonine, trypophan, phenylalanine v methionine [13],[22]. B.subtilisnattocnbiotinchosphttrin, trong mitrngkhngcbiotin cctbosinhdngkhngthphttrinvbotcachngkhngnymm c.Nng biotin tithiu cnthitchos nymmca botl 0,1%[13], [22]. 1.1.5.c im sinh l B.subtilis nattolsinhvt hiukh bt buc, cn oxysinhtrng vcth pht trin nng oxy ln hn 3%. B. subtilis natto pht trin trong khong nhit rng. Nhit thch hp cho vi khun pht trin l t 30- 50C, nhit ti u l 37C. Nhit ti u cho s ny mm ca bo t l 40C. Bo t B. subtilis natto vn c th ny mm sau khi bo qun 0C. 55C, vi khun ngng pht trin v s ly gii t bo sinh dng din ra. B. subtilis natto pht trin pH trung tnh hoc hi kim, khong pH ti u cho s sinh trng l 7,2- 7,6. S ny chi v pht trin b c ch pH 4,5 [ 13], [22]. 1.1.6.Sn phm trao i cht ca B. subtilis natto CcsnphmtraoichtcaB.subtilisnattort adng, nhng quantrng nht l h enzym. Vi khun ny sn xut mt lng ln cc enzym c ng dng nhiutrongcngnghipnh:protease,amylase,phytase...[22],trongc Nattokinase,mt proteasengoibolc nghanhtvangc nghincu, ng dng rng ri. 4 1.2. Natto v Nattokinase 1.2.1. Natto Natto l mt mn n truyn thng ca Nht Bn, l nhng ht u nnh luc chn vln menvivikhunB.subtilis nattonhit khong 40oCtrongvng 14-18 gi [13],[19]. Natto cha nhiu cht b dng cho sc khe trong enzym NattokinaselmthotchtsnsinhtrongqutrnhlnmenNattocxeml hot cht c hiu qu trong vic ngn nga cc chng bnh tim mch [19]. Bng 1.1: Thnh phn chnh trong 100g natto: [15], [24] Thnh phnHmlng Calorie200 Kcal Protein16,5 g Lipid10,0 g ng9,8 g Nattokinase7100 CU Vitamin B10,07 mg Vitamin B20,56 mg Vitamin B31,1 mg Vitamin K2870 g Calci90 mg T hng ngn nmtrc,Natto tr thnh mn nkhngththiuca ngi Nht do n cha nhiu cht b dng cho sc khe, c li cho tiu ha, tim mch vlmttrongnhngmnnlmtngtuithcangiNht.Vth,nhiu nghin cu v Natto c tin hnh. Nm 1905, tin s Shin Sawamura (i hc Tokyo) tch c hai loi vi khun tNattogmvikhunB.subtilisnattocvaitrlnmenhtu,gyrami hngcbitvvikhunBacillusmesentericusvulgaristochtchtnhtc trng v v ngt [21]. Nm 1913, Sawamura cng b kt qu nghin cu ca mnh 5 qua nhiu mu Natto v kt lun vi khun ch yu trong cc mu chnh l B. subtilis natto. ng a ra m t chi tit v c im vi khun, enzyme v nhu cu dinh dngca n.Ngynay,ccsnphmNattotrnthtrng cln menvivi khun B. subtilis natto [21]. Nm1986,tinsSumiHiroyukicngbtonbktqunghincuvtc dng ca Natto sau khi th nghim gn 200 loi thc phm khc nhau so snh, xc nh Natto c kh nng phn hy huyt khi mt cch hu hiu v mnh nht trongccloithcphm.ngxcnhenzymNattokinase,mthotchtsn sinhtrongqutrnhlnmenNattocvaitrphnhyhuytkhimtcchhu hiu, mnh gp 4 ln enzym ni sinh lm tan mu ng l plasmin [21, [23]. TihinghquctvdinhdngtchctiNewZealand(30/4-3/5/2006) giosMasafumiKitakazekhngnhNattoctc dnglmgimrrtcc chngm mucao(triglyceridevcholesterol).ngkugicngngthgii ci thin np sng sinh hot v tiu th Natto trc nguy c bo ph do tha dinh dng v cc chng bnh him ngho, nan y (tiu ng, ung th) ngy cng tng khp ni trn th gii [31].1.2.2. Cc enzym c kh nng tiu huyt khi trc Nattokinase TrckhiNattokinasecnghincurngri,cnhiuenzymcng dng trong tr liu v huyt khi nh Streptokinase, Urokinase, Alteplase... C ch chung ca cc enzym ny l: hot ha plasminogen thnh plasmin c tc dng phn giifibrin,fibrinogencngccyutcaqutrnhngmu(yutV,VIII, XIIIa), ng thi cc sn phm ging ha ca fibrin v fibrinogen cng c tc dng khng thrombin, c ch kt tp tiu cu ngn qu trnh ng mu. V th plasmin c tc dng lm tan huyt khi, gip ti lp li s ti mu cho cc m [1], [2]. Cn c vo tnh chn lc trn fibrin m ngi ta chia cc enzyme tiu huyt khi thnh hai nhm:a.Enzym tiu huyt khi khng tc dng chnlc trn fibrin: l nhng thuc tc ng n c plasminogen gn hoc khng gn vi fibrin v th gy tc dng khng mong mun l chy mu ton thn. Mt s enzym in hnh ca nhm ny nh: 6 - Streptokinase (SK): chit xut t mi trng nui cy cc chng Streptococcus haemolyticus tan huyt nhm C. Streptokinase l protein l nn khi vo c th s kch thch c th sinh khng th lm mt hot tnh v gy d ng. -Urokinase(UK):chitxuttnctiu ngi,tmitrngnuicytbo thn bo thai, hoc c th sn xut bng cng ngh gen. -Anistreplase(APSAC):lstreptokinasethayicutrcphntbngcng ngh sinh hc [1], [2]. b. Enzym tiu huyt khi tc dng chn lc trn fibrin: l nhng enzym hot ha plasminogen gn trn fibrin nn c u im l t gy chy mu ton thn, hiu lc tiu fibrin mnh hn v khng gy d ng khi s dng. Tuy nhin, do sn xut bng cng ngh cao nn gi thnh ca chng kh t. Mt s enzym in hnh ca nhm ny nh: - Alteplase (t-PA): c ngun gc t cc t bo ni mc ca thnh mch, c tit ratronghuhtdchngoibocacthnhhuyttng,nctiu,ncbt, nc mt... Alteplase trong lm sng c sn xut bng cng ngh ti t hp ADN t t bo bung trng chut ng Trung Quc v gi l rt- PA. -Pro-urokinase:ltinchtcaUrokinase,ctrongnctiu.Pro-urokinase c sn xut bng cng ngh ti t hp t vi khun E. Coli. Mcd mtsenzym cptrn cs dngrngritrong iutr huyt khi hin nay, chng vn c nhc im, chng hn nh chi ph cao, thi gian bn thingn,nguycchymutonthn,ringStreptokinasecngyphnngd ng [9]. Hin nay, cc tc nhn lm tan huyt khi c trong to, nm v thc phm ln men angc nghincu cungcp ngun thuctr huytkhiantonv gi r [9]. 1.2.3. Nattokinase a. Ngun gc Nattokinase(khiuEC3.4.21.62)lmtenzymchottnhtiufibrinrt mnhcngungctmnntruynthngcaNhtBnctnlNatto[23]. 7 Nattokinasecncgil:SubtilisinNAT,SubtilisinBSP,enzymetrongNatto [32]. b. c im cu to Nattokinase c cu trc l mt chui polypeptid n gm 275 gc acid amin v khng c cu ni disulfid trong phn t. Trng lng phn t l 27,728 Da v pI= 8,6 0,3 [12]. L enzym thuc h subtilisin ca protease serin, Nattokinase cng c trung tm hot ng bao gm nhm hydroxyl ca Ser-221, imidazol ca His-64 v nhm carboxyl ca Asp-32. C cht c hiu ca Nattokinase l: Suc-Ala-Ala- Pro-Phe-pNA [12]. Hnh 1.2 . Cu trc bc 3 ca Nattokinase [32] c. Tnh cht Nattokinase bn vng pH t 6,0 10,0, bt hot pH nh hn 5 v pH ln hn 11, hot tnh ti u pH =8- 9. Nattokinase bn vng nhit di 40C, b gim hot tnh khi nhit tng ln 50C v b phn hy 70C [12], [13]. Hot tnh ca Nattokinasebcch1phnbiHgCl2,H2O2vbcchhontonbi phenylmethylsulfonylfluorides(PMSF),ethylendiamintetraaceticacid(EDTA), ccionFe3+vAl3+cngcchenzymny[9],[13],[25].Hottnhca Nattokinase tng ln khi c mt MgSO4, CaCl2, MnCl2 [ 9]. d. C ch tiu fibrin Nattokinase phn hu fibrin trc tip hay gin tip thng qua ba con ng khc nhau: 8 Hnh 1.3 . C ch tiu fibrin ca Nattokinase [19] A. Nattokinase trc tip ging ho fibrin trong cc mu ng, lm bin i fibrin t dng polymer khng tan thnh cc sn phm ging ho c trng lng phn t thp, ho tan. Nh vy, Nattokinase c c ch tiu fibrin ging vi plasmin. B.Nattokinase ging ho fibringin tipbngcchhot ho Pro-urokinase thnh Urokinase,dnnthcyqutrnhbiniplasminogenthnhplasmin,lm tng plasmin ni sinh l enzyme phn gii fibrin t nhin trong c th. C. Nattokinase ging ho fibrin gin tip bng cch hot ho qu trnh tng hp t- PA (Alteplase) trong huyt tng. Kt qu l tng cng to thnh plasmin. cbit,Nattokinasekhngnhhngnqutrnhhnhthnhfibrint fibrinogen, do khng lm suy gim c ch ng mu bnh thng ca c th. V vy, Nattokinase khng gy ra bin chng chy mu nh cc thuc chng ng mu bnh thng [19]. e. Cng dng Nattokinasectcdngphngvphccccmungdockhnngtiu fibrin mt cch hu hiu (cao gp 4 ln plasmin), ng thi rt an ton cho c th khihpthquangung[23].Nattokinasecthhatanccmungkhi ung 100mg/ ngy v tc dng ca n ko di 8- 12 gi. Do u im an ton, hiu lccao,girhnrtnhiusoviccthuctanhuytkhithngthng, 9 Nattokinasectimnnglntrongtrliuhuytkhi[21],[28].Ngoira, Nattokinase cn c tc dngtt trong cc bnh tim mch nh lm gim huyt p, gim lipid mu, ngn x va ng mch...[ 28] f. Nattokinase dng trong thc phm chc nng v thuc Nattokinase ln u tin c phn lp v bn trn th trng di tn NSK-SD vonm1998biPhngthnghimsinhhcNhtBn,loibvitaminK2. Nattokinaseikhicsdngtrongyhcnhlmttcnhnlmgimcc mu ng, thay th cho iu tr bng aspirin hng ngy. Tuy nhin, s thay th ny khng c khuyn co [28]. Hin nay, c rt nhiu cc sn phm cha Nattokinase c sn xut di dng thc phm chc nng vi mc ch h tr phng nga v iu tr cc bnh l v tim mch [28]. Bng 2.2: Thc phm chc nng cha Nattokinase [28], [29] Sn phmHmlng Nattokinase (FU) Dngbo ch Nh sn xut Nattopes300Vin nangCng ty IMC, Vit Nam S- Nattokinase5.000Vin nangUnited Biomedical, M Nattokinase1.000Vin nangCNSPHARMKOREACo.,Ltd, Hn Quc Nattokinase plus 3.000Vin nangFoodScience ofVermontdbaDa Vinci Laboratories, M Nattokinase plus Fucoidan 2.000Vin nangUmeken, Nht Bn 10 1.3. Qu trnh ln men sn xut enzym 1.3.1.Cc phng php ln men sn xut enzym t vi sinh vt a. Phng php ln men b mt ylphngphpchyusdngcchtlccloicmvbt:cmm, cm go, bt u, bt ng...c hp chn v lm m sau trn thm tru v mn ca tng xp. Vi sinh vt c nui nhit khong 28- 30C, m 55-65%, trong thi gian nui t 36- 48 gi. u im ca phng php ln men b mt l : -Nng enzym cao hn ln men chm. -D sy kh v t hao tn hot tnh enzym. -Ch phm th d vn chuyn. -Thit b n gin, tn t nng lng. -C th x l cc b khi b nhim. Tuy nhin, phng php ny cng c nhc im l kh kim sot chnh xc m,sinhkhi,lngO2,CO2 nn chtlngchphm cc mkhng ng u,tcsnxutsn phmthphnlnmenchm,khcgiiha,tng ha,cndintchnuiln...[4],[5].Hinnay,phngphplnmenbmtt c s dng trong sn xut khng sinh v cc ch phm sinh hc nhng vn c s dng trong ln men sn xut enzym [4]. b. Phng php ln men chmVi sinh vt c nui cy trong mi trng lng vi c cht l tinh bt, nit, cht khong...Phngphpnuicychmihiphicvtrngtuyticc khuvsinhtnghp,thanhtrngmitrngdinhdng,thaotcnuicy, khng kh cung cp cho qu trnh nui cy. u im ca phng php ln men chm l: -D kim sot qu trnh do c th sn xut cng nghip, c gii ha, t ng ha. -Nng sut cao v cht lng ng u. 11 -C th nui cy d dng cc chng vi sinh vt t bin c kh nng sinh enzym cao v la chn ti u thnh phn mi trng, cc iu kin nui cy. -Enzym thu c tinh khit hn ln men b mt, m bo v sinh, v trng. Tuy nhin do nng enzym thu c thp nn enzym thu c sau khi tch v thu hi s c gi thnh cao, nu khng m bo v trng tt c cc bc th s b nhim hng lot, gy hao ph ln [4], [5]. 1.3.2. Thu nhn ch phm enzym Enzym thng phm c sn xut di hai dng: dng bt kh v dng lng, c th l dng th hoc dng tinh khit. i vi ln men b mt c th ng ha nu cn,sau dngdungdch mhayncctchitrtenzymtacdchchit enzym. i vi ln men chm ch cn lc b sinh khi l c dch chit nh trn sau c th dng tc nhn kt ta thun nghch nh aceton, ethanol, mui trung tnh c ch phm enzym dng sch hn. T ch phm sch ny, bng k thut in di, lc gel... ta c th tch tng phn c enzym tinh khit hn [4], [5]. 1.3.3. Cc yu t nh hng ti s tng hp enzym a. nh hng ca thnh phn mi trng dinh dng Dinh dng l yu t quan trng c nh hng ln n s pht trin v tng hp enzym ca vi sinh vt. Cng mt loi vi sinh vt nhng nu nui cy trn cc mi trng c thnh phn dinh dng khc nhau th mc to thnh enzym v thnh phn enzym c to thnh cng khc nhau. Vd: nuicyAspergillus oryzaetrnmitrngcm i mchthth cth tocnhiuenzym,trongcccloienzymhottnhcaonhamylase, protease, maltase, pectinase... nhng nu nui cytrn mi trng Czapek ci tin vi nitrat v tinh bt th ch to thnh ch yu l amylase, cn cc enzym khc to thnh vi lng khng ng k. Hintngnychthyvisinhvtchkhngthyngvthcvt.Khi trong mi trng c nhng cht kh ng ha, vi sinh vt phi tit vo mi trng nhngenzymtngng phn hychng thnhnhngcht ngha c.V 12 vy khichochtcm ngvo mitrng dinh dngthkh nngsinhtnghp enzym ca vi sinh vt c th tng ln gp nhiu ln so vi trng hp bnh thng. Trongcng nghsnxutenzymcn philachnchtcm ngthch hp v xc nh nng ti u ca n trong mi trng c hiu sut sinh tng hp cao nht [4], [5]. b. nh hng ca pH ca mi trng pH mi trng bin i kh r rt trong qu trnh nui cy v gy nh hng ln n s tng hp enzym. Do ngi ta thng iu chnh pH thu c enzym vi hiu sut cao nht. Mi loi enzym thng c tng hp mnh trong mt vng pH xc nh i vi tng loi vi sinh vt. V d: amylase v pectinase ca nm mc c tng hp mnh trong mi trng c pH= 4,5 - 5,0 cn protease th pH=6 - 6,5 [4], [6]. c. nh hng ca nhit Phn ln cc vi sinh vt c kh nng tng hp cc enzym u thuc loi a nhit trung bnh, c nhit ti thch 22- 32oC, mt s vi khun thch hp nhit cao hn (35-55 oC) v chng c kh nng tng hp cc enzym c bn nhit cao. Khi nng nhit mi trng ti qu mc thch hp th kh nng sinh tng hp enzym b gim r rt [4], [6]. d. nh hng ca thi gian nui cy Thigiannuicythchhpcamiloivisinhvtcxcnhbngthi gian cho php tch t enzym ti a. Thi gian ph thuc vo tng chng vi sinh vt v t ph thuc vo enzym chng tng hp. Ngoi ra, ty tng loi vi khun m ta la chn iu kin cp kh v m cho ph hp [4], [5]. 1.4.Ccnghincuvnh hngcamitrngdinhdngnkhnng sinh enzym Nattokinase ca vi khunCng vi s quan tm ngy cng nhiu i vi Nattokinase, nhng nm gn y cnhiu nghincuti u hamitrngnuicychovikhunsnxutlng Nattokinase ln nht. 13 1.4.1.Ngoi nc:JunguoLiuv cngs(2006)nghincuti u hacciu kindinhdngchosinhtnghpNattokinasesdngchngBacillussubtilis nattoNLSSE.Khostccngunnitrogen:(NH4)2SO4,NaNO3,natriglutamat, casein, pepton t u nnh v kho st cc ngun carbon: glucose, sucrose, maltose, xylosevglycerol.Ktqunguncarbonttnhtlmaltose2%,ngunnittt nht l pepton u nnh, hot tnh Nattokinase t 1.300 U/ml vi thnh phn mi trng ti u (g/l): pepton u nnh: 8,28, CaCl2: 0,64 v cao nm men: 0,74 [18]. Deepakvcngs(2008)nghincutiuhamitrngchosinhtnghp Nattokinase t chng Bacillus subtilis. Cc nhn t c chn kho st: glucose, pepton, MgSO4, v CaCl2 5 mc . Hot tnh t 3.194 U/ml vi thnh phn mi trng(%):glucose1%,pepton5,5%,MgSO4 0,2%vCaCl20,5%[10].LiHui Rong(2011)xcnhmitrngtiuchosnxutNattokinasetvikhun Bacillus subtilis natto gm: MgSO4 0,02%, K2HPO4 0,02%, KH2PO4 0,01%, CaCl2 0,05%, maltose 3%, peptone t u nnh: 3% [16].1.4.2. Trong nc LThBchPhngvcngs(2012)phnlpchaichngBacillus sp.7.2vBacillus sp.NP3ckh nngsinhtng hp mnhenzymelmtan huyt khi(Nattokinase)t mtschngBacillusspp lytbsutpgingcaVin SinhhcnhitivphnlptthcphmNatto;hottnhNattokinasev proteasecamuNP3v7.2tngngvimtsthcphmchcnnghin angbntrnthtrng[7].HongThKhnhHng(2012)nghincukh nngbiuhinvtitNattokinasenhADNbgenBacillussppphnlptsn phmNatto[3].HinVireak(2012)xc nh mitrngnuicyBacillussubtilis natto cho hot tnh protease tt nht l canh thang b sung casein 0,5 % [8]. 14 CHNG II: I TNG V PHNG PHP NGHIN CU 2.1. Nguyn vt liu v thit b s dng 2.1.1.Visinhvts dng:VikhunB.subtilisnatto c phnlptsn phm Natto ca Nht Bn. 2.1.2. Nguyn liu, ha cht s dng: Bng 2.1 . Cc nguyn liu, ha cht s dng TTNguyn liuNgun gc 1Cao thtc 2Peptonc 3Caseinc 4Thch btVit Nam 5GlucoseTrung Quc 6LactoseTrung Quc 7SaccharoseTrung Quc 8MaltoseTrung Quc 9Tinh btTrung Quc 10 NaClTrung Quc 11Na CMCTrung Quc 12Dung dch lysozym 1mg/mlVin kim nghim 13Dung dch xanh methylenTrung Quc 14MgSO4 Trung Quc 15CaCl2 Trung Quc 16MnCl2 Trung Quc 17Dung dch LugolTrung Quc 2.1.3. Cc mi trng s dng: Mi trng thch thng (MT1): 15 Pepton 1,0 g Cao tht0,5 g NaCl 0,5 g Thch bt 2 g Nc my v 100ml Mi trng canh thang (MT2): Pepton 1,0 g Cao tht0,5 g NaCl 0,5 g Nc my v 100ml 2.1.4. Thit b Bng 2.2. Cc thit b s dng Thit bNgun gc T cy UV BioairItaly T m Memmertc T sy Memmertc My ly tm Universalc My lc Bioshakerc Cn k thut Sartoriusc Knh hin vi LabomedM T lnh ToshibaNht Bn L vi sng DaewooHn Quc Ni hp ALPNht Bn My khuy t Heidolphc Thc o ng knh vng phn gii Trung Quc 16 2.1.5. Dng c: bnh nn (100ml, 250 ml), cc c m, ng nghim, pipet, a petri, que cy, ng ong 2.2.Ni dung nghin cu 2.2.1. Phn lp vi khun B. subtilis natto t sn phm Natto v xc nh enzym do Bacillus subtilis natto to ra. - Phn lp vi khun B. subtilis natto t ch phm Natto. -XcnhenzymdoB.subtilisnattotorakhilnmenchmtrongmitrng MT2. - Xc nh enzym trong sn phm Natto. 2.2.2.Khostnhhngcaiukindinhdnglnkhnngtoenzymca B. subtilis natto -Khostnhhngcangunhydratcarbon:glucose,saccharose,maltose, lactose. -Kho st nh hng ca u tng (dng bt v dng ht) v ca cht cm ng (casein). -Kho st nh hng ca mt s mui v c: MgSO4, MnCl2, CaCl2. -TomitrngtnghpvsosnhkhnngtoenzymcaBacillussubtilis natto khi nui cy trong mi trng tng hp vi trong mi trng ban u. 2.3. Phng php nghin cu 2.3.1. Phng php phn lp vi khun t sn phm Natto [6]. Cn 10 g Natto cho vo bnh nn 250ml c cha 90ml nc ct v trng v lc u,cnngphalong10-1.Chunb 8ngnghimcha 9mlncctv trng, nh s th t t 1 n 8. Dng micropipete ht 1ml t bnh nn cha mu cnngphalong10-1chovongnghim1vlcu,cnngpha long10-2.Tiptclmchonngnghimcuicng.Ttcccthaotcu c tin hnh trong t cy v trng. Pha 100ml mi trng MT1 trong cc c m. un mi trng trong l vi sng cho thch tan hon ton, hp tit khun 115C; 0,6 atm trong 20 pht. Sau , chia u mi trng vo khong 6 a petri hp tit trng. Dng pipet ht 0,1ml t mi nng pha long 10-7, 10-8, 10-9 cho ln a 17 mitrngthchchunb(minnglpli2ln)vtrangubngque trang v trng, cho nhng a thch ny vo t m 37C trong 24 gi. Tt c cc thao tc cy ging u c tin hnh trong t cy v trng. Sau quan st khun lchnhthnhtrna,nhumOgiestkarisoiknhhinvixcnhhnhthivi khun, dng que cy vng cy gi ging trn ng thch nghing.2.3.2. Phng php nhum Ogiestka [6]. - Lm vt bi vi vi khun phn lp c v vt bi kh t nhin. - Nh vi git HCl 0,5% ln vt bi, h nng trn ngn n cn cho bc hi, gi 2 pht ri ra li bng nc ct.-tlnvtbiminggiylcrinhvigitdungdchFuchsinZiehl,h nng cho n khi bc hi. B giy ra v ra li vt bi bng nc ct.- Tymubngcch nhng phin knhvo dungdchH2SO4 1%trong2 pht. Ra li vt bi bng nc ct.- Nhum vt bi bng dung dch xanh methylen trong 5 - 15 pht.- Ra nc ct , kh t nhin vt bi.- Quan st tiu bn trn knh hin vi vi vt knh du. Bo t vi khun bt mu , t bo cht bt mu xanh. 2.3.3. Phng php gi ging trn thch nghing [6]. Pha 100ml mi trngMT1 trong cc c m. un mi trng trong l vi sng chothchtanhonton.Chiaumitrngvotrong10ngnghim.Sau, hp tit khun 115C; 0,6 atm trong 20 pht, t nghing cc ng nghim sao cho chiucaomitrngnkhong2/3ng,nguichothchngli.Tng ging, cy truyn ging sang mi trng mi, cy theo hnh ziczac. Tt c cc thao tc cy ging u c tin hnh trong t cy v trng. em cc ng cy ging vo t m, 37C trong 24 gi. Sau , bo qun ging trong t lnh 4-5C.2.3.4. Phng php nhn ging trong mi trng lng [6]. Pha 100 ml mi trng MT2 vo bnh nn 250 ml, nt bng, hp tit trng 18 115C; 0,6 atm trong 20 pht. ngui nhit phng. Cy ging t ng ging vo bnh nn, tin hnh cy ging trong t cy v trng, sau tin hnh nui trn my lc 37C trong 24 gi, tc lc 150 vng /pht. 2.3.5. Phng php ln men chm vi khun [6]. Pha50mlmitrngMT2vobnhnn100ml,ntbng,hptittrng 115C; 0,6 atm trong 20 pht. ngui nhit phng. Cy 5 ml dch nhn ging vo bnh nn,sau tin hnh nuitrnmylc 37 Ctrong 24gi,tc lc 150 vng /pht. 2.3.6.Phngphpkhostnhhngcamitrngdinhdnglnkh nng sinh enzym Nguyn tc: So snh kh nng sinh enzym ca cng mt vi khun khi c nui cy trong cc mi trng khc nhau cn cc iu kin nui cy khc l nh nhau. -Kho st nh hng ca ngun hydratcarbon:Pha 50 ml mitrngMT2 v mitrng MT2cbsungring 2%tngloi ngkhcnhau:glucose,saccharose,maltose,lactose, hptittrng.Tinhnh nuicyvikhun nh phng php mtphng php 2.3.5.Sau , em thvsosnh hottnhenzymcadchln mentccmitrngtheo phng php mt mc 2.3.9, 2.3.10, 2.3.11.Lm 3l 3thi imkhc nhauvly kt qa trung bnh. -Khostnhhngcautng(dngbtvdnght)vcachtcm ng to protease (casein): Pha 50ml cc mi trng nh sau: mi trng MT2, mi trng MT2 b sungring tng thnh phn: 2% u tng ht, 2% bt u tng; 0,5% casein vo cc bnh nn 100 ml, hp tit trng. Tin hnh nui cy vi khun nh phng php m t mc 2.3.5. Sau , em th v so snh hot tnh enzym ca dchln mentccmitrngtheo phng phpmt mc2.3.9, 2.3.10, 2.3.11. Lm 3 l 3 thi im khc nhau v ly kt qa trung bnh. - Kho st nh hng ca mt s mui v c: Pha 50 ml mi trng MT2 v mi trng MT2 b sung ring 0,05 % tng loi mui v c CaCl2, MnCl2, MgSO4, hp tit trng. Tin hnh nui cy vi khun nh phng php m t mc 2.3.5. Sau 19 , emthvsosnhhottnhenzymcadchln mentccmitrngtheo phng php m t mc 2.3.9, 2.3.10, 2.3.11. Lm 3 l 3 thi im khc nhau v ly kt qa trung bnh. 2.3.7. Phng php xc nh s c mt ca enzymtrong sn phm Natto Ly khong 5g Natto ha vo 20ml dung dch m phosphat pH=7,6. em khuy bng mykhuyttc 200vng /phtkhong 15 pht nkhi phn nhttch khi phn u. Phn dch nht gi li th hot tnh enzym. Lc ly phn ht u, nghin nh, ha trong 20ml dung dch m phosphat pH=7,6 ri em ly tm 4.000 vng/ pht trong 15 pht, thu phn dch trong th hot tnh enzym theo phng php m t mc 2.3.9, 2.3.10, 2.3.11 2.3.8. Phng php xc nh enzym trong dch trong v trong sinh khi ca dch ln men vi khun Bacillus subtilis natto [8] - Ly tm dch sau khi ln men vi tc 5.000 vng/ pht trong 10-15 pht. Thu sinh khi v dch trong, dch trong c gi li th hot tnh enzym theo phng php m t mc 2.3.9, 2.3.10, 2.3.11 - Sinh khi c ha trong 10ml dung dch m phosphat pH = 7,6; thm khong 15-20 ml dung dch lysozym (1 mg/ml), 37 C trong 30 pht ph v v bc t bo, gii phng enzym.Ly tm dch vi tc 6.000 vng/ pht trong 10- 15 pht, thu c cn v dch ly tm, dch ly tm c gi li th hot tnh enzym theo phng php m t mc 2.3.9, 2.3.10, 2.3.11. 2.3.9. Phng php th hot tnh protease [6]. Nguyntc:Khiproteasetcdnglncchtcaseintrongmitrngthch, caseinbphngiilmchocmitrnggimi,mitrngtrnntrong sut. Tin hnh: Pha 100ml mi trng cha 2% thch, 1% casein trong nc ct vo cccm200ml,thm10gitxanhmethylen,khuyu.unnngtronglvi sng cho thch v casein tan ht. Phn phi u vo 5 a petri c ng khnh 9cm chun b trc. Khi thch ng, c cc l thch c ng knh 0,6cm. Nh ln 20 lt 0,05 ml dch nui cy vo cc l thch, trong t m 37C trong 24 gi. em o ng knh vng phn gii, tnh kt qu trung bnh . - Phn ng (+): Mi trng xung quanh l thch trong sut, l vng phn gii casein. - Phn ng (-): Mi trng xung quanh l thch khng c vng phn gii casein2.3.10. Phng php th hot tnh enzyme amylase [6]. Nguyn tc: Khi amylase tc dng ln c cht tinh bt trong mi trng thch s lmtinhbtbphngii.Khinhdungdchlugolvothphntinhbtchab thyphncmuxanhtmdobt mulugol,cnphntinhbtbthyphnto thnh vng trong sut. Tin hnh: Pha 100 ml mitrngcha 1%tinh bt, 2%thch trongncct, un trong l vi sng cho thch tan. Sau phn phi u vo 5 a petri c ng knh9cmchun btrc.Khithchng,ccclthchcngknh 0,6 cm. Nh ln lt 0,05 ml dch nui cy vo cc l thch, yn khong 30 pht nhitphngchodungdchkhuchtnutrongccl.Sautrongtm 37C trong 24 gi. em o ng knh vng phn gii, tnh kt qu trung bnh. - Phn ng (+): Mi trng xung quanh l thch trong sut, l vng phn gii tinh bt. -Phnng(-):Mitrngxungquanhlthchkhngcvngphngiitinh bt.2.3.11. Phng php th hot tnhcellulase [6]. Nguyn tc: Khi cellulase tc dng ln c cht Na CMC trong mi trng thch s lm Na CMC b phn gii. Khi nh dung dch lugol vo th phn Na CMC cha bthyphncmunuvng,cnphnNaCMCbthyphntothnhvng trong sut. Tin hnh: Pha 100 ml mitrngcha 2%thch, 1%tinh bttrongncct, un trong l vi sng cho thch tan. Sau phn phi u vo 5 a petri c ng knh9cmchun btrc.Khithchng,ccclthchcngknh 0,6 cm. Nh ln lt 0,05 ml dch nui cy vo cc l thch, yn khong 30 pht 21 nhitphngchodungdchkhuchtnutrongccl.Sautrongtm 37C trong 24 gi. em o ng knh vng phn gii, tnh kt qu trung bnh. - Phn ng (+): Mi trng xung quanh l thch trong sut, l vng phn gii Na CMC. -Phnng(-):Mitrngxungquanhlthchkhngcvngphngii NaCMC. 2.3.12. Phng php th hot tnh fibrinolytic (tiu fibrin) [2], [8]. Nguyn tc: Nattokinase phn hy fibrin dng polyme khng ha tan to ra cc sn phm ging ha c trng lng phn t thp, ha tan. Tin hnh: - Chun b fibrin th: Ly tm mu ln khe mnh c chng ng 1.500 vng/ pht trong 5 pht thu c huyt tng ngho tiu cu, dng ngay. -Tofibrin:chunbcccmcha:50mldungdchNaCl0,9%,1mldung dch huyt tng ngho tiu cu, 0,8 ml dung dch CaCl2 0,1M. Khuy k bng a thy tinh mt u mi rp. cc c a thy tinh cm trong vo t m 2 - 3 gi37C.Sauthmvocckhong20mldung dchNaCl0,9%chocc fibrin tch ra, n nh a thy tinh vo thnh cc, va n va cun cho fibrin bm vothnh athnh mt mngmng.Ramngfibrin dnh u a bng dung dch NaCl 0,9% v bng nc ct. -Thkhnngtiufibrin:Cmathytinhcfibrinbmvongnghim cha10mldungdchthritvotm37C.Xcnhthigianmng fibrin a thy tinh tan hon ton. 22 CHNG BA: THC NGHIM, KT QU V BN LUN 3.1. Phn lp vi khun B. subtilis natto t ch phm Natto v xc nh enzyme do vi khun sinh ra 3.1.1. Phn lp vi khun Bacillus subtilis natto t ch phm Natto Mc ch: Tch ring vi khun B. subtilis natto t ch phm Natto. Tin hnh: Ly mu v phn lp theo phng php m t mc 2.3.1. Sau khi 24 gi 37C, quan st hnh thi, c im khun lc to thnh. Nhum Gram, nhum Ogiestkanhphngphp mtmc 2.3.2vquanstcimcavikhun di knh hin vi in t c phng i 1.000 ln. Kt qu c th hin trong hnh 3.1: Hnh 3.1. Hnh nh khun lc phn lp c v hnh nh t bo vi khun khi soi trn knh hin vi phng i 1.000 ln Nhnxt:Quansthnhthikhunlccavikhunphnlpctrnmi trng MT1 cho thy: khun lc phn lp c ging nhau, khng pht hin khun l,ctrngchokhunlcBacillussubtilis, hnhtrn hockhngxc nh,kch thc ln, mu trng hoc hi xm. B mt khun lc hi nhn, c mp nhn bm chc vo mi trng thch. Vi khun khi soi trn knh hin vi c hnh dng ging vi vi khun B. subtilis natto thun khit ca Nht Bn: l vi khun Gram dng, t bo hnh que, t li thnh m hoc ng ring l, bo t bt mu , t bo sinh 23 dngbtmuxanh.Nhvy,cthsbkhngnhvikhunphnlpc chnh l B. subtilis natto. Bn lun: Cc nghin cu v phn lp vi khun t Natto c nghin cu t kh lu. Nm 1905, gio s Shin Sawamura phn lp c 2 loi vi khun t sn phm Natto truyn thng, l: B. subtilis natto v B. mesentericus vulgatus, trong B.subtilisnattolvikhuncvaitrchnhtrongqutrnhlnmenhtu tng. Nm 1913, sau nhiu nghin cu v cc mu Natto nhiu vng khc nhau, ng khng nh ch c vi khun B. subtilis natto tn ti trong tt c cc mu Natto. Ngynay,snphmNattothngmicsnxutbngcchlnmenvivi khun B. subtilis natto [21], [28]. S phn bit B. subtilis natto vi cc vi khun khc thuc loi Bacillus subtilis da vo s khc nhau gia b gene, nhu cu bt buc s dng biotin pht trin, kh nngsnxutcht nhtl polyglutamat [20], [24],[27].Kh nngsnxut- PGA l mt c im quan trng phn bit B. subtilis natto vi cc chng khc. B. subtilis natto c kh nng sinh - PGA trong mi trng khng c L- glutamat, trongkhiccchngvikhunkhccn phi cL-glutamat mi snxut - PGA [24]. Do iu kin khng cho php nn kha lun khng thc hin ht c tt c cc th nghim ny, tuy nhin sn phm dng phn lp l Natto thng mi s dng B. subtilis natto sn xut vqua phn lp, quan st vi khun c c im hnh thi ging vi vi khun c m t nn c th s b khng nh vi khun phn lp c l B. subtilis natto. 3.1.2. Xc nh mt s enzym do vi khun sinh ra Mc ch: nh tnh enzym do B. subtilis natto sinh ra trong qu trnh nui cy. Tin hnh: -Ln men chm vi khun nh phng php m t mc 2.3.5 -Tinhnhthudchlytmvdchchitsinhkhitheophngphpmt mc 2.3.8 24 -Tin hnh xc nh protease, amylase, cellulase trong dch ly tm v trongsinh khi theo phng php khuch tn trn cc c cht: casein, tinh bt, Na CMC m t mc 2.3.9, 2.3.10, 2.3.11. Kt qu c trnh by trong bng 3.1 Bng 3.1. Kt qu nh tnh enzym trong dch ly tm v trong sinh khi ProteaseAmylaseCellulase Dch ly tm(+)(+)(+) Sinh khi(-)(-)(-)

(Ghi ch: 1: dch ly tm, 2: sinh khi) Hnh 3.2. Kt qu th hot tnh protease, amylase, cellulase trong dch ly tm v dch chit t sinh khi Nhnxt:Ktquthucchothytrongdchlytmcprotease,amylase, cellulasecntrongsinhkhikhngc.VyvikhunBacillussubtilisnattosinh amylase 1 2 1 cellulase 2 25 enzym ngoi bo. Do , cc th nghim tip theo s s dng dch ly tm ca mi trng sau khi nui cy th hot tnh enzym. 3.1.3. Xc nh enzymtrong sn phm Natto Mcch:XcnhccenzymnmtrongsnphmNattovenzymnmtrong phn ht u hay phn dch nht ca sn phm Natto. Tin hnh: -Thu enzym t dch nht v t ht u ca sn phm Natto nh phng php m t mc 2.3.7 -Tinhnhthhottnhenzymnhphngphpmtmc2.3.9,2.3.10, 2.3.11 Kt qu c trnh by trong bng 3.2 Bng 3.2. Hot tnh enzym th hin bng ng knh vng phn gii c cht ca dch enzym chit t phn ht u v phn dch nht ca Natto Hot tnh enzym th hin bng ng knh vng phn gii c cht (mm)Mu th Dp Da DcPhn ht u15,510,314,8 Phn dch nht18,517,617,0 (Ghich:Dp :ngknhvngphngiicaseintrungbnh,Da :ngknhvngphngiitinhbt trung bnh,Dc : ng knh vng phn gii NaCMC trung bnh). Nhn xt: Kt qu thu c t bng 3.2 cho thy c phn dch nht v phn ht ucasn phmNatto ucvng phngiicasein,tinh bt,NaCMC nghal protease, amylase, cellulase u c mt trong c phn dch nht v phn ht u ca ch phm. Mt khc, ng knh vng phn gii casein, tinh bt, Na CMC ca dch nhtlnhn dchchitt phn ht u nn c3enzymc nhiutrong phn dch nht hn. 26 Bn lun: S c mt ca c ba enzym trong Natto c gii thch nh sau: c kh nng ng ha ngun tinh bt trong ht u, vi khun cn phi sinh ra cellulase ph v lp v ngoi ca ht u v amylase phn gii tinh bt. V th cc th nghimtiptheocthitktheohngkhostccthnhphnthamgiavo mitrng nui cyB.subtilis nattosaochohnchvicsinhamylase,cellulase v tng sinh protease. Bn lun chung phn 3.1: Vi khun B. subtilis natto c phn lp t Natto c khnngsinhc3enzym:protease,amylase,cellulase,tuynhinccenzymch yunmtrongphndchnuicychkhngnmtrongphnsinhkhi.Ktqu ny ph hpviktquxcnhthnh phnenzymectrongdch nhtvphn ht u ca sn phm Natto. Cc th nghim tip theo c thit k kho st mi trng dinh dng nhm la chn mi trng gip vi khun tng sinh protease v hn ch sinh amylase, cellulase. 3.2.Kho st nh hng ca mi trng dinh dng n kh nng sinh enzym ca vi khun B. subtilis natto 3.2.1. Kho st nh hng ca ngun hydratcarbon Mc ch: Tm ngun hydratcarbon thch hp cho vi khun B. subtilis natto sinh ra nhiu protease nht . Tin hnh: -Tinhnh nuicyvikhuntrong mitrngMT2vmitrngMT2cb sung mt s hydratcarbon nh phng php m t mc 2.3.6 -Tinhnhthhottnhccenzymcaccdchlytmthuctccmi trng nui cy trn nh phng php m t mc 2.3.9, 2.3.10, 2.3.11 -Tinhnhthhottnhfibrinolyticcaccdchlytmthuctccmi trng nui cy trn nh phng php m t mc 2.3.12 Th nghim c tin hnh 3 ln, ly kt qu trung bnh. Kt qu c trnh by bng 3.3, 3.4, 3.5 27 Bng 3.3. Hot tnh protease ca dch ln men khi nui cy vi khun trong mi trng MT2 v mi trng MT2 c b sung cc ngun hydratcarbon khc nhau, th hin bng ng knh vng phn gii casein (mm) Mu th Dp p% MT217,50% MT2 + 2% glucose18,55,7 % MT2 + 2% saccharose17,50% MT2 + 2% maltose19,29,7 % MT2 + 2% lactose17,71,1 % (Ghi ch: Dp : ng knh vng phn gii casein trung bnh, p%: % tng gimDp so vi MT2, cch tnh p%nhsau:numitrngAcDp =a,MT2cDp =bth p%= bb a x100%,p% 0:mi trng A c Dp ln hn p% so vi MT2) Bng 3.4. Hot tnh amylase v cellulase ca dch ln men khi nui cy trong mi trng MT2 v mi trng MT2 c b sung cc ngun hydratcarbon khc nhau th hin bng ng knh vng phn gii c cht (mm) AmylaseCellulase Mu th Da a% Dc c% MT216,10%18,70% MT2 + 2% glucose19,319,9 %18,80,5 % MT2 + 2% saccharose19,118,6 %19,54,3 % MT2 + 2% maltose17,911,2 %17,1 8,5 % MT2 + 2% lactose16,73,7 %19,01,6 % (Ghi ch:Da , Dc : ng knh vng phn gii tinh bt, NaCMC trung bnh; a%, c%:% tng gim Da ,Dc so vi MT2, tnh tng t nh p% trn) Nhnxt:Ktqubng3.3v3.4chothy:khibsungngunhydratcarbon vo mi trng MT2 th ng knh vng phn gii casein, tinh bt, Na CMC u tng,nghalhottnhprotease,amylase,cellulaseutng,ngoitrbsung 28 maltose th ng knh vng phn gii Na CMC gim so vi MT2. Tuy nhin khi sosnhmctnggiaccngunhydratcarbonkhcnhauththybsung maltose cho kch thc vng phn gii casein ln nht (19,2 mm), cao hn 9,7% so vi mi trng MT2, ng thi b sung maltose li c tc dng lm gim kh nng tit enzyme cellulase, th hin kch thc vng phn gii Na CMC nh nht, nh hn8,5%sovimitrngMT2vkchthcvngphngiitinhbttng nhng khng nhiu (11,2%) nn kh nng tit amylase tng t. 0510152025Protease Amylase CellulaseCSLGM Hnh 3.3. Biu th hin nh hng ca ngun hydratcarbon n hot tnh enzym ca vi khun (Ghi ch: C: mi trng canh thang, G, S, M, L:mi trng canh thang b sung glucose, saccharose, maltose, lactose, d: ng knh vng phn gii c cht) Ktquthhintrnbiuhnh3.3chothybsungmaltosevomi trng canh thang th hot tnh protease cao nht trong khi hot tnh cellulase thp nht. V th nhm mc tiu thu nhiu protease v gim bt cc enzym khc, maltose c la chn lm ngun hydratcarbon cho mi trng nui cy. Nh vy, vic b sung maltose vo mi trng nui cy B. subtilis natto c tc dnglmtngkhnngtitproteasesovimitrngMT2.Tuynhin,cha khngnhclngproteasetnglnnyclmkhnngtiufibrinhay khng.Vth,thnghimtiptheonhmkhostkhnngtiufibringiadch Hot tnh enzym th hin bng d Enzyme 29 lnmenB.subtilisnattoca mitrngMT2soviccmitrngMT2c b sung cc ngun hydratcarbon khc nhau. Bng 3.5. Thi gian tiu fibrin ca dch ln men thu c khi nui cy trong mi trng c b sung cc ngun hydratcarbon khc nhau: Mi trngThi gian tiu fibrin (pht) MT2>300 MT2 +2%lactose>300 MT2 + 2% saccharose250 MT2 + 2% glucose230 MT2 + 2% maltose210 Nhn xt: Kt qu bng 3.5 cho thy thi gian tiu fibrin tng dn theo th t: MT2+2%maltose