1 Chng 1 M U 1.1. t vn Thcphmcbsungvisinhvtsng(probiotic)cnhiunghincu xcnhlclichosckheconngivngvtvhinvnangccc chuyn gia trn nhiu lnh vc c lin quan nghin cu to ra nhiu ch phm sinh hc c li (Ross v ctv, 2005). V vy thc phm cha probioic ang c ngi tiu dng v c nh sn xut quan tm. Hin nay, cc sn phm cha probiotic khng dng li vic ch tn ti mt ging vi sinh vt no m cn l hn hp ca nhiu ging vi sinh vt probiotic nhm to ra li ch rng ln hn trn mt liu dng. Lactobacillusacidophiluslvikhunclithngtrtrongng rutgipgicnbngchohvisinhvtngrut,cchsphttrin caccvisinhvtchi(KliglervCohrssen,2008),htriutrmtschng bnhlinquanntiuha(FrankvRoelof,1993),sinhraenzymelactasegipc th hp thu ng sa (Monatalto v ctv, 2006). Bacillus subtilis l mt trong nhng vi sinh vt quan trng trong vic kch thch hthngmindchtnhin,cdngiutrmtschngbnhlnquann tiuha(Kahl,1999),sinhramtsenzymeclichohtiuhanhamylase, protease, Saccharomycescerevisiaegipcithintltiuhaccchtdinhdng (Ghasemivctv,2006),tngkhnngpngmindch(Aslivctv,2007),cnh tranhvtrbmdnh,cnhtranhdinhdngvivisinhvtgybnhtrongng rut. Snuicybagingvisinhvtktrntrncngmtmitrnglcn thit, gip n gin ha qu trnh nui cy, thun tin chovic thu sinh khi to sn phm probiotic c mt c L. acidophilus, B. subtilis, S. cerevisiae v sn phm ny c c tc dng l tc dng cng gp ca c ba ging probiotic trn. 2 Trn c s chng ti thc hin ti: Nghin cu nui cy ng thi Lactobacillusacidophilus, Bacillus subtilis, Saccharomyces cerevisiae trn canh trng MRS ci tin. 1.2.Mc ch Mc ch: nui cy ng thi L. acidophilus, B. subtilis, S. cerevisiae trn canh trngMRScitinnhmtmtmichng>106CFU/mldchnuicyng dng thu sinh khi sn xut sn phm probiotic cha c ba loi vi sinh vt c li trn. t c mc ch trn, ti thc hin nhng ni dung sau: 1.Xcnhngcongsinhtrngcabachngvisinhvtthnghim khi tng sinh chung trn canh trng MRS. 2.Thnghimsnglcccyutnhhngnmtvisinhvtnui cy theo thit k Plackett Burman. 3.Ti u ha qu trnh nui cy s dng phng php RSM-CCD. 4.nh gi mc tng thch ca gii php ti u bng thc nghim. 3 Chng 2 TNG QUAN TI LIU 2.1.Probiotic Probiotic c FAO/ WTO (2001) nh ngha l: Cc vi sinh vt sng m khi dng vi s lng s to ra li ch v sc khe trn vt ch. Hin naythut ng probioticcsdngchnhngchphmvisinhvtsnghuchkhic avocthngvtvconngithngquathcnhocncungtonn nhng nh hng c li cho vt ch. Vic con ngi s dng vi sinh vt sng nhm tng cng sc khe khng phi l mi. T lu, con ngi bit tiu th cc thc phm cha vi sinh vt sng c li chng hn nh cc sn phm ln men: sa chua, chao, rau qu ln men cha chng y bng, n khng tiu, to bn, au bng tiu chy d khng bit n tc ng ca h vi sinh vt c li trong sn phm. Nm 1930 cc nh khoa hc i hc Havard chng minh rng cc vi khun ng rut ng mt vai tr quyt nh trong qu trnh tiu ha, gip tiu ha thc n, cung cp mt s vitamin v cc cht dinh dng khc nhau m c th vt ch khng t sn xut ra c (Fuller, 1992). Trong nhng nm gn y, cc nh nghin cu quan tm n kh nng khng vi khun gy bnh ca vi sinh vt probiotic thay th cho vic s dng khng sinh v h tr vic s dng khng sinh trong iu tr bnh, phng nga v lm chm qu trnh pht trin ca bnh ung th, gim cholesterol mu. Cnhiugingvisinhvtcnuicyvsdngvivaitrlprobiotic nhng ch yu thuc chng Lactobacillus v Bifidobacterium; ngoi ra cn c mt s loithucEnterococcus,Streptococus,Saccharomyces,Bacillus,(Tannock,2001). Trong hin nay L. acidophilus, B. subtilis, S. cerevisiae l cc dng chnh c p dng (Chen v ctv, 2009).Vamanu(2002) cho rng cc snphmprobiotic thu c vi sinh khi hn hp (vi khun v nm men) c th ng mt vai tr quan trng trong 4 iu tr cc bnh khc nhau, li th ca cc sn phm ny l khng cn s h tr ca thuc v khng h c tc dng ph. Hiuqucaprobioticphthucvoloivisinhvtsdng,liudng, tui, tnh trng sc khe ca ngi dng. Liu dng thng c khuyn co l 5 - 10 ttbosngtrnmtliudnghngngychotremv10-20tchongiln (KliglervCohsen,2008).Hinnay,khoahccngchngminhmclm sng l probiotic c dng vi liu lng thch hp c tc dng tch cc i vi sc khenhtngcngsvisinhvtclitrongngrut,tngkhnngtiuha thcndosinhvitaminvemzymngoibo,cbitlenzymphngiing lactose.TheoDesmondvctvnm2000thprobioticcngiptngcnghmin dch t nhin chng li nhng bnh nhim trng ng tiu ha v ri lon tiu ha do s dng khng sinh. Cn theo cng b ca Kligler v ctv nm 2008 th probiotic cn chiuqutrnhngvnlinquanntiuhanh:tiuchy,tobn,dng thcn;iunytrngvinghincucaKimvonm2006.Nm2000Brady cng cng s cho rng ngoi tc dng tt n h tiu ha probiotic cn c hiu qu trong ngn nga v lm chm s pht trin ca mt s bnh ung th nh: ung th rut kt,bnhvimrut,vimitrng,TamayocngnhLiongcngcccngsca mnhtrongnm2008,OKeefetrongnm2009cngnghincuvccngkt lunnhtrn.LiongvShahtrongnghincuvonm2005v2008chorng probiotic cn c hiuqu gim cholesterolhuyt thanh v gim huyt pmu khi c s hin din ca prebiotic - cht kch thch s sinh trng ca li khun. Hin nay, ngoi vic dng probiotic nh ch phm h tr tiu ha b sung vo ch n ung, probiotic cn c dng nh mt thuc cha bnh, tuy nhin cha c bng chng xc thc no v hiu qu ca cch dng ny nn vn c WHO v FDA quan tm khuyn kch nghin cu. Khi ni v tc ng ca probiotic, Kligler (2008) cho rng tc dng c li ca probiotic ch th hin khi chng vn cn sng khi vo n rut non, cc ging vi sinh vt khc nhau th c nhng li ch v kiu tc ng khc nhau nhng chyu c cc tc ng nh: Cnh tranh v tr bm dnh v cnh tranh dinh dng vi cc vi sinh vt c hi haycnhtranhloitr.Ccvisinhvtprobioticcngvtngdnstrongrut, 5 khachtccvtrthcmvngncnsbmdnhcaccvisinhvtkhcnh Escherichiacoli,Salmonella,...Mtsnmmenprobiotic(S.cerevisiae;S. boulardii) khng ch tranh v tr bm dnh vi cc vi khun khc m cn c th y cc vi khun c roi ra khi v tr bm dnh trn nim mc rut (Kligler v Cohsen, 2008). Tuy nhin, cnh tranh dinh dng l phng thc cnh tranh khc lit nht, v s sinh si vi s lng ln ca mt loi vi sinh vt no l mt mi e da nghim trng i vi cc loi khc v ngun c cht cho s pht trin. Vi sinh vt probiotic sinh ra cht khng khun sinh hc, l cc cht km hm vi khun nh lactoferrin, lysozym, hydrogen peroxide cng nh mt s axit hu c khc. Cc cht ny gy tc ng bt li ln vi khun c hi ch yu l do s gim thp pH trong rut (Conway, 1996), ng thi cng kch thch h thng min dch t nhin ca vt ch. Ngoi ra, cc vi sinh vt probiotic cn c th sinh ra cc cht c li cho vt ch nhenzyme,vitamin.Enzymeclichotiuhathngcvisinhvttoral amylase, protease, c bit l enzyme lactase hy ng lactose gip tr chng khng dung np lactose (Gilliland, 1990); cc vitamin thng l vitamin nhm B v K (Rossi vctv,2011)vmtschtkhcgiptnghshpththcncavtch,ci thin kh nng gim hp thu khong. Tuy vy, khng phi vi sinh vt c li no cng c th lm probiotic, vic la chn cc chng vi sinh vt c chc nng probiotic l rt phc tp. Tiu chun u tin ivivisinhvtlmprobioticlphiantonchovtch,ckhnngsngst trong sut qu trnh sn xut, bo qun v tr nn chim u th trong ng tiu ha vtch.Cctiuchunlachnnychplhathngquaccthnghimin vitro,tstuynchncccchngctimnngnhlngunprobiotic.Cc tiu chun la chn nh sau: Mt l khng gy bnh, c li i vi vt ch, d nui cy, c th sng st v n nh trong sut qu trnh sn xut ch phm. Hailctnhbmdnhtrnbmtngtiuhahoccctbobium. Cc chng probiotic phi bm dnh c vo thnh rut non v sinh si ny n y. y l mt trong nhng ch tiu quan trng v vi sinh vt c bm dnh th mi tn ti, 6 pht trin trong ng tiu ha v c thi gian phi lu pht huy tc dngca mt probiotic. Balchottnhkhngkhunchngliccvikhungybnh.Lachn cccchngckhnngsnsinhccchtkhngkhunlctnhquantrng trongphttrinprobiotic.Ccchngprobioticcnchottnhcchvikhungy bnh nh E. coli, Salmonella v Campylobacteria (Klaenhammer v Kullen, 1999). Bn l c t l sng cao khi qua h tiu ha vn c axit thp, enzyme v mui mt. Khoang ming v d dy ca vt ch l ni c mi trng axit pH t 2 - 3 v c mtccenzymtiuho(amylase,protease,lysozym,).Ngoira,ccchng probioticphickhnngtntivphttrinvinngmuimt2%,hayc mtsgingnmmen,BacillusvLactobacillusckhnngkhnglimuimt (Klaenhammer v Kullen, 1999). Hin nay th trng a vo s dng cc b bc bo v hayb sung cc cht prebiotic tng kh nng sng xt ca vi sinh vt lm probiotic. Cui cng l c kh nng khng li khng sinh v khng sinhcng l mt vn nhhngnkhnngsngcavisinhvtprobiotic(KlaenhammervKullen, 1999). 2.2.Lactobacillus acidophilus Phn loi: B:Bacilli H:Lactobacillaceae Ging:Lactobacillus Loi:Lactobacillusacidophilus L.acidophiluscxemlvikhunthnthinthngtrtrongng rut,mingvmocangi;thucnhmvikhunGram+,khngsinhbo t, l loi vi sinh vt d dng, k kh hoc vi hiu kh; lnmen c 15 loi ng khcnhautrongcglucose,galactose,lactose,fructose,sucrose,(Wheater, 1955). L vi khun ln men lactic ng hnh theo EMP, sinh ra cc cht hu c c vai tr l khng sinh. Sinh sn bng cch nhn i. L.acidophiluslvikhunvihiukhnnphttrinctrnmitrng khng c hoc c t oxy (Klaenhammer v Kullen, 1999); pH tng trng tt l t 4,5 7 n 7, ti u pH 6 (Riaz, 2010).Nhit pht trin trong khong 20- 48 0C, pht trin ti u 370C. L.acidophiluslloicnhucudinhdngrtphctp,ngoinguncabon vnitL.acidophiluscncnhucutuytiivimtsaxitaminvcht khong.TrongmitrngMRSAkkhsau48gi370Cxuthinkhunlc nh, hnh cu ra dp c mu vng nht. Trn mt mi trng PDA L. acidophilus pht trin rt yu, ch to ra cc chm nh li ti mu trng. Theo nghin cu ca Nguyn V Tng Vy v ctv nm 2007 th L. acidophilus chu c mi trng axit trong 2 gi pH 1 v 2 vi t l rt thp nhng pH 3 v 4 l41-65%;khngvitnhtl3khngsinh,cthchucampicillin(32 g/ml),streptomycin(32g/ml)hn10gi.HoodvZottola(1988)chorngsinh khi L. acidophilus pH 4,0 khng gim ng k sau 2 gi. L.acidophiluscxemlvikhunthnthintrongngrut,thnglin quan n s c ch ca cc loi vi sinh vt gy bnh, gip h tr tiu ha, c bit l vinhngngikhngdungnpcnglactose(Hovevctv,1999;Vresev ctv,2001;Monataltovctv,2006).L.acidophilusclinquannvicngnnga tiu chy c lin quan n s dng khng sinh, nhim virut hay tiu chy du lch do n phi thc phm nhim khun, ci thin chng nhim trng m o, gim kh nng ungthrutkt(Gillilandvctv,1990;Havenaarvctv,1992;FrankvRoelof, 1993). iu tr triu chng ca hi chng rut kch thch v bnh vim rut (Bhatia v ctv,1989;KliglervCohrssen,2008)tngpngmindch(Perdigon,vctv, 1990),gimmccholesteroltronghuytthanhbngcchphngiimuimt (Gilliland, 1990; Frank v Roelof, 1993), iu tr vim da d ng tr em (Kligler v Cohrssen, 2008). Theo Sanders v Klaenhammer (2001) L. acidophilus c bit n lvikhunsnxutkhngsinhtnhinvcthhnchc27loivikhun, trongc11loigybnhinhnhnhStaphylococcusaureu,Salmonella, Clostridium, E. coli (Kligler v Cohrssen, 2008). Liudngckhuynco100triun35ttbosng/ngy(Kliglerv Cohsen, 2008) 8 2.3.Bacillus subtilis Phn loi: B:Bacillales H:Bacillaceae Ging:Bacillus Loi:Bacillus subtilis VikhunB.subtilisthucnhmvikhunbtbuctrongngrut,chng c phn bhu httrong t nhin: t,nc, bi, ckh, nhng chyunhiu nht l trong t. B. subtilis thuc nhm trc khun,Gram+, c kh nng sinh ni bo t, hiu kh hoc k kh ty nghi (Dahl, 1999); ln men c cc loi ng: glucose, maltose, manittol, saccharose, xylose, arabinosse. B. subtilis c khng nguyn H v O, cu trc khng nguyn dng D v L - glutamic axit; sn sinh khng sinh subtitracin c tc dng c ch vi khun G+, G-. Sinh sn bng cch nhn i hoc ny mm do s nt bo t, c kh nng chu nhit, chu m (T Minh Chu, 2000). B.subtilislvikhunhiukhnhngcngcthphttrintrongmitrng thiu oxy. pH pht trin t 7 n 7,4; ti u pH 7,2. Nhit pht trin t 30 45oC, tiu370C.DinhdngcnC,H,O,Nvmtsnguyntvilngkhc.B. subtiliskhngphttrinctrongmitrngMRSAymkh;trnbmtmi trng PDAto vt loanmu trng, rng ca khng u trnmt thch, ra dpnht mu xung quanh.B. subtilis l vi khun chu c axit d dy v cht dch tiu ha trong ng rut. Nguyn V Tng Vy v ctv (2007) ch ra rng B. subtilis chu c mi trng axit trong 2 gi pH 1; 2; 3 v 4 ln lt l 3; 9; 70 v 85%, chu c mi trng c 0,5 - 1% v 1,5 - 2% mui mt trong 10 gi ln lt l 2% v 1%. B. subtilis khng vitnhtl4khngsinh,cthchucampicillin(32g/ml)hn10gi, streptomycin (2 mg/ml) hn 5 gi. ylloivisinhvtchenzymephongphnhngquantrngnhtl amylase v protease, l hai loi enzyme thuc h thng men tiu ha nn c tc dng h tr tiu ha thc n. B. subtilis c th c ch s pht trin caE. coli, Salmonella v c dng trong iu tr tiu chy, vim rut cp v mn tnh, ri lon tiu ha, tr em i ngoi phn sng, vim i trng. 9 B. subtilis t lu c s dng sn xut nhiu loi enzyme v s dng c t bo sng trong nhiu qu trnh sn xut v ch bin thc phm nh mt vi sinh vt an ton (Kahl, 1999). 2.4.Saccharomyces cerevisiae Phn loi: B:Saccharomycetales H:Saccharomycetaceae Ging:Saccharomyces Loi:Saccharomyces cerevisiae S. cerevisiae thuc nhm vi sinh vt khng thng tr trong ng rut nhng rt ph bin trong t nhin. L loi nm men hu dng nht c s dng t lu i ln men sinh ra cn v sn xut bnh m. S. cerevisiae ln men c hu ht cc loi ng tr lactose v cellobiose. Tuy nhin,khnngtngtrngcaS.cerevisiaetrnccloingkhcnhaulkhc nhau (Chen, 2009). H enzyme ca nm men cng kh phong ph bao gm: saccarase, catalase, invertase, amylase, nhng khng c enzym phn hy protein. S. cerevisiae sinh sn ch yu bng cch ny chi nhng cng c th sinh sn hu tnh. S. cerevisiae l loi c th pht trin trong iu kin hiu kh hay k kh, iu kin hiu kh sinh trng tng sinh khi nhanh, iu kin ym kh ch yu ln men to cn;pHphttrintrongkhong4-6,8,tiupH5,8;nhitphttrintrong khong t 27 35oC, ti u 320C. Ngun dinh dng carbohydrate chnh l cc loi ng, thch hp nht l ng fructose, c th dng ngun nit v c ln hu c, s c mt ca biotin vmt s nguyn t vi lng nh st, canxi, magi kch thch pht trin ca S. cerevisiae. S. cerevisiae trong mi trng MRSA ym kh khng pht trin c, trn b mt mi trng PDA to khun lc mu trng c, trn nh cao, b mt bng. Theo Nguyn V Tng Vy v ctv (2009), S. cerevisiae c th sng trong mi trng axit ca d dy trong 3 gi, mt s chng thuc S. cerevisiae c kh nng thch nghi tt trong mi trng cha pancreatine ca rut non v mi trng c cha mui mt. Kh nng bm dnh rut non ca S. cerevisiae t ti a 80,88% (H L Qunh Chu v ctv, 2010). Khi sng trong h tiu haS. cerevisiae kch thch tiu ha, hp 10 thu cht c v cnh tranh v tr bm dnh, cnh tranh dinh dng vi vi sinh vt gy bnhnhE.coli,Shigella,Salmonella(Ghasemivctv,2006;Aslivctv,2007), Sthaphylococcusaureus(Vamanu,2002).cbit,S.cerevisiaephttrinnhanh trong mt thi gian ngn, tuy nhin khng c tr lu di v d loi b khi ng rut. 2.5.Cc nghin cu c lin quan NguynThanhThong(2007)trongnghincucamnhchrarngkhi nui phi hp B. subtilis v S. cerevisiae trn mi trng r ng ti u th vn gi c tc tng trng tng ng vi trn mi trng tt u ca mi ging. Hosoi v ctv (2000) cho rng B. subtilis gip tng kh nng tng trng v kh nng tn ti ca Lactobacilli. Benjamasvctv(2003)thchorngS.cerevisiaecnhngtcngtchcc nvicgimlngaxitlacticdnnhiungtchcclnstngtrngca Lactobacillus,bilngaxitdoLactobacillussinhrakhisinhtrnglmgimpH, n mt lc no s l tc nhn km hm i vi chnh n, cn do S. cerevisiae sinh ra s trung ha mt phn axit gi pH mi trng nui cy khng gim qu nhanh nn ko di thi gian tng trng ca Lactobacillus. Chng ti chn mi trng MRS lm mi trng nui cy th nghim v mi trngnycychtdinhdngcnthit,pHnmtrongkhongsinhtrng tng sinh khi ca c ba chng trn; chng ti thc hin ti u ha mi trng t mt t bo ca mi chng l ti a v tng ng gia ba chng. 11 Chng 3 VT LIU V PHNG PHP NGHIN CU 3.1.Thi gian v a im nghin cu Thi gian tin hnh: 3/2011 - 7/2011 a im thc hin: Phng th nghim vi sinh thuc Vin Cng ngh Sinh hc - Thc phm, Trng i hc Cng Nghip TP. H Ch Minh. 3.2.Vt liu v phng tin th nghim ChngvisinhL.acidophilus,B.subtilis,S.cerevisiaecphnlpvbo quntiVinCngnghSinhhc-Thcphm,TrngihcCngNghip TP.HCM. Mi trng nui cy vi sinh: Mi trng gi ging, hot ha ging v m vi sinh vt c trnh by bng 3.1. Thnh phn mi trng c trnh by ti ph lc 1. Bng 3.1: Mi trng nui cy vi sinh vt Mi trng Vi sinh vt Gi gingHot ha gingm L. acidophilusMRSAMRSBMRSA B. subtilisNANBPDA S. cerevisiaeThch SabouraudSabouraudPDA Ha cht s dng: cn 900, pepton, D - glucose, HCl 10%, NaOH 10%. Thic b: Ni hp tit trng BK75 T m hiu SHELAB T cy v trng ESCO pH k Sartorius - Docu 12 Cn k thut 2 s l Sartorius TE 612 T sy hiu SHELAB Dngc:nhitk,pipettman,apetri,ngnghim,quecytrn,quetrang, n cn, erlen 150 ml, erlen 250 ml, ng ong, u tp v cc iu kin thng thng khc ca ca phng th nghim vi sinh. tithchinquyhochthcnghimbngphngphppngbmt- thit k cu trc c tm (RSM - CCD), phn mm Design - Expert 7.0.0 c s dng thit k th nghim v s l s liu. 3.3.Phng php th nghim 3.3.1.K thut nui cy T mi trng gi ging sau 24 gi dng que cy trn ly mt khun lc ring l cy vo erlen 150 ml cha 50 ml mi trng hot ha ging, L. acidophilus c 370C; B. subtilis, S. cerevisiae nui cy lc 90 vng/ pht nhit phng 30 20C. Dngquecyvnglymtvngvikhuntmitrnghothagingcy vo erlen 150 ml cha 50 ml mi trng th nghim MRS. Th nghim c nhit 300C c iu chnh bi t m, nhit di 300C c t trong phng my lnh c nhit k theo di. 3.3.3. K thut m s lng vi sinh vt Vi tt c cc nng pha long c dng m khun lc, dng pipettman vi u tp v trng ht 0,1 ml dch nui cy cy ln a petri chun b sn mi trng. Vi L. acidophilus s dng phng php a ym kh, dch mu ln a trc ri mi mt lp 5 ml mi trng thch lng ln, sau khi lp mi trng ny ng hn th tip mt lp 5 ml mi trng thch lng ln tip to mi trng k kh,ngui.Vihaigingcnlidngphngphptranga,dchnuicyln mi trng chun b sn mt ngy trc v trang u bng que trang v trng. nhng a petri ny trong t m 37 0C trong 24 gi em ra m, 24 gi sau em ra kim tra li. Chs dng s liu ca nhng a c s lng t 25 n 300 khun lc ca mi loi vi sinh vt. Khun lc c trng trn mi trng thch m c th hin hnh 3.1. 13 h Hnh 3.1: A: mi trng PDA, (a) - khun lc B. subtilis; (b) - khun lc S. cerevisiaeB: khun lc L. acidophilus trn MRS Agar. Cng thc tnh mt t bo:

Trong : A l mt t bo (CFU/ ml) N l tng s khun lc m c ni l s a c m nng pha long i V l s ml dch mu cy vo mi a fi l nng pha long c s khun lc c chn m. 3.4.Ni dung thc hin 3.4.1.Th nghim 1:Kho st ngcong sinh trng ca ba chngvi sinh vt khi tng sinh chung trn canh trng MRS Mc ch: lp ng cong sinh trng ca ba chng vi sinh vt nghin cu c c nhn nh s b v s sinh trng ca ba chng khi nui cy chung trn canh trng MRS. y l c s chn yu t th nghim v phm vi nghin cu cc th nghim tip theo. BA 14 T mitrng hot ha ging dng que cyvng cymt vngvikhun vo mitrngthnghim,thnghimcthchintrn50mlmitrng,nhit 370C vi ba ln lp li. 3.4.2.Th nghim 2: Thit k sng lc yu t th nghim theo Plackett - Burman Mc ch: tm ra yu t th nghim nh hng nht n ch tiu theo di, qua loi b bt cc yu t th nghim. Kt qu thu c th nghim 2 s c dng lm yu t th nghim th nghim 3 tip theo. Plackett-Burmanlthitkchuynbitchothnghimt2-31yut,m miyutlkhcnhauhnhaicp.Thitknycsdngkhiginhs vngmtcahaiyuttngtc,kimtrachcchnchosloibyuttc dng rt t hockhng c tc ng n ch tiu theo di. Kt qu s a ra 3 hoc4 yu t c mc nh hng nht n ch tiu theo di. Th nghim c thc nghim mc mt yu t, tc l lm cc th nghim ch cmtyutthayinhmxcnhnghacaccyutnghincu,xcnh phng hng v mc nh hng ca mi yu t trong cc th nghim tip theo ca qu trnh ti u ha. Sau khi thc hin th nghim thm d, xc nh mc nh hng n mt visinhvtnuicy,chngtichnccyutthnghimviphmvikhostca ccyuttheodinhbng3.2.Matrnquyhochthcnghimtheothitk Plackett - Burman vi t hp 12 th nghim khc nhau c thit lp bi 5 yu t th nghimmc thp (1) vmc cao (1).Btr th nghim c thc hin nhbng 3.4; yu t th nghim v ch tiu theo di c trnh by trong bng 3.2 v 3.3. Bng 3.2: Cc yu t s dng trong Plackett - Burman Yu t Tn yu tn v Phm vi nghin cu Mc -11 X1pH5,8 - 6,55,86,5 X2Nhit 0C30 - 373037 X3Thi gian gi12 - 181218 X4Pepton b sung% (w/v)0 - 10010 X5D - glucose b sung% (w/v)0 - 20020 15 Bng 3.3: Ch tiu theo di trong ti Ch tiu theo diTnn v Y1L. acidophilusCFU /ml Y2B. subtilisCFU /ml Y3S. cerevisiaeCFU /ml Bng 3.4: Bng ma trn theo thit k Plackett - Burman Nghim thcX1X2X3X4X5 111-111 2-111-11 31-111-1 4-11-111 5-1-11-11 6-1-1-11-1 71-1-1-11 811-1-1-1 9111-1-1 10-1111-1 111-1111 12-1-1-1-1-1 o= 0,05 3.4.3.Th nghim 3: Thit k ti u ha mi trng nui cy theo RSM - CCD Mc ch: trn c s kt qu thc hin ca th nghim 2 mc 3.4.2, chng ti tin hnh quy hoch thc nghim, kt qu thu c l phng n sn xut ti u nht tha cc iu kin a ra trc. Phng php quy hoch thc nghim c s dng l phng php hm p ng b mt - thit k cu trc c tm (RSM CCD). i vi vic ti u mi trng nui cy vi sinh vt bng RMS c miu t bi Giang Th Kim Lin (2009), qu trnh gm hai giai on c bn: Giai on u: Thc nghim mt yu t. Tc l lm cc th nghim ch c mt yutthayinhmxcnhnghacaccyutnghincu,xcnhphng hngvmcnhhngcamiyuttrongccthnghimtiptheocaqu trnh ti u ha. Giai on th hai: Tm mt tng quan ti u cho ccyu t quan trng nht (c ngha nht) trn c s c nh cc yu t cn li gim bt chi ph th nghim. 16 Phng n cutrc c tm (CCD)lmt trng hp c bit trong nhm cc m hnh quy hoch thc nghim. N c cu thnh t ba thnh phn: -Nhn:lmtphngntuyntnhvi2knhcamthnhkhiu trong khng gian k chiu. Nu k > 5, c th gim bt s th nghim bng cch s dng phng n yu t tng phn 2k-1. -2k im sao ( * ) nm trn cc trc ta ca khng gian yu t. Cc ta ca cc im sao l ( o, 0, 0,...,0), (0, o, 0,...,0),..., (0, 0,...,0, o). o l khong cch t tm phng n n im sao gi l cnh tay n sao. Cc im sao l cn thit m rng khng gian nghin cu kho st tc ng ca mt yu t n c th tm c cc c lng ca h s bii trong phng trnh hi quy bc 2. -n0 th nghim tm phng n tm phng sai ti hin. Cnhtaynsaoovsthnghimnotmcchnphthucvotiu chun ti u, thng l phng n trc giao hay phng n quay. Phng trnh hi quy ( m hnh ha) c biu din bng phng trnh sau: y = b0 + b1x +b2x2 + b3x3 + b12x1x2 + b23x2x3 + b13x1x3+ b11x12 + b22x22 + b33x32 Trong : b0:l h s t do b1, b2, b3: l h s bc 1 b11, b22, b33 : l h s bc 2 b12, b23, b13 :

h s ca tng cp yu t. x1, x2, x3: l cc bin y : l ch tiu theo di Ta c: ==Nii ij jy xNb11 Nu dng phng trnh hi quy y hn c k n cc hiu ng tng tc hai bin: 3 2 23 3 1 13 2 1 12 3 3 2 2 1 1 0 x x b x x b x x b x b x b x b b y + + + + + + =Th: ==Nii ij jy xNb11 17 ==Nii i l j jly x xNb1) (1 Phng sai ca cc h s c tnh nh sau: NSSSSPEbj=Tin hnh: th nghim c b tr theo thit k Central Composite. Th nghim c b tr nh bng 3.6; yu t X5 c dng mc 10% trong tt c cc th nghim. CentralCompositelthitkthnghimmtrongmiyutsadng hn 5 cp : cng alpha (+o) v tr alpha (-o), cng mt (1) v tr mt (- 1) v im trungtm(0).Nuyutphnloicthmvo,thitktrungtmtnghps c nhn i cho mi s kt hp ca cc cp yu t phn loi. Kt qu sng lc cc yu t th nghim theo thit k Plackett Burman thc hin mc 3.4.2 cho thy phm vi nghin cu ccyu t th nghim thc hin l hp l. Cc yu t th nghim v ch tiu theo di dng trong RSM - CCD c trnh by bng 3.5 v bng 3.3. Bng 3.5: Cc yu t s dng trong RSM - CCD Yu t Tnn v Phm vi nghin cu Mc -o-101o X1pH5,45 - 6,855,455,86,156,56,85 X2Nhit 0C26,5 - 40,526,53033,53740,5 X3Thi gian gi9 - 21912151821 X4 Pepton b sung% (w/v) - 5 - 15-5 051015 Saukhislphntchsliu,chngtrnhsaraccthngschotng bin hm t gi tr cc i, sau s phn tch a ra phng n sn xut ti u theo yu cu t ra. 3.4.4.Chn phng n sn xut ti u - kim tra bng thc nghim Sau khiphn tch phnmm s a ra cc phng n thayucu t ra bao gmgitrcachtiutheoditcci,ccyuttheodinmtrongphmvi 18 nghim cu. Sau khi chn c phng n sn xut ti u chng ti tin hnh kim tra bng thc nghim trn 1000 ml mi trng vi 3 ln lp li, mc ch kim tra la nh gi li s tng thch gia l thuyt m hnh vi thc t sn xut. Cc s liu thc hin theo thit k Plackett - Burman v RSM - CCD c nhp vo chng trnh Design Expert 7.0.0 v tin hnh phn tch. Bng 3.6: K hoch b tr th nghim ti u ha mi trng nui cy theo thit k Central Composite Nghim thc X1X2X3X5 Nghim thc X1X2X3X5 1-1-1-1-1161111 21-1-1-117-o000 3-11-1-118o000 411-1-1190-o00 5-1-11-1200o00 61-11-12100-o0 7-111-12200o0 8111-123000-o 9-1-1-1124000o 101-1-11250000 11-11-11260000 1211-11270000 13-1-111280000 141-111290000 15-1111300000 o= 0,05 19 Chng 4 KT QU V THO LUN 4.1.Kt qu th nghim 1 - Kho st ng cong sinh trng ng congsinh trng ca L. acidophilus,B. subtilis, S. cerevisiae c th hin trn hnh 4.1. Khi nui cy kt hp, L. acidophilus khng c pha tim pht v mi trng hot ha ging cng l MRSB, t cc i gi th mi tm; B. subtilis vo pha tng trng t gi nui cy th su, t cc i gi th mi lm; S. cerevisiae vophatngtrngtginuicythsu,tccigithmitm.Tuy nhin,S.cerevisiaeimtngtrngccilthp,khngcstbinvs lng trong pha tng trng, im cc i ch t 5,1 (log CFU/ml). Hnh 4.1: th din t ng cong sinh trng ca L. acidophilus, B. subtilis, S. cerevisiae khi nui cy kt hp trn canh trng MRS. Khi nui cy trn mi trng MRS nhit 370C l iu kin tng trng ti ucaL.acidophilusnnvikhunnyphttrinmnh.Tuynhin,vinhngiu kin nh trn khng l iu kin cc thun cho s pht trin ca B. subtilis nhng vi 0123456789100 5 10 15 20 25 30L. acidophilusB. subtilisS.cerevisiaelog cell (CFU/ml) hour 20 khun ny vn tng trng tt, iu ny c th gii thch l do s tc ng qua li ca B. subtilis vi hai chng cn li. S. cerevisiae pht trin chm, dc ca ng cong sinh trng thi im pha tng sinh khng ln c th gii thch nh sau: iu kin mitrng(pH=6,5;nhit370C)lkhngphhpchosphttrincaS. cerevisiae, cng vi s pht trin nhanh chng ca L. acidophilus v B. subtilis khin cho s cnh tranh v dinh dng ca S. cerevisiae km i. Nh vy c th thy rng: pH mi trng, nhit nui cy v dinh dng u l nhngyu t nh hng n s pht trin ca cc vi sinh vt th nghim. Tmcchtcmtvisinhvtlnnhtvtngnggiaccvi sinh vt nui cy, chng ti xc nh nhim v ca ti u ha mi trng nui cy l phitngmttbocaS.cerevisiaebngcchcitinmitrng,iuchnh iukinnuicy;ngthi,phiduytrsphttrinttcaL.acidophilusvB. subtilis. 4.2.Kt qu th nghim 2 - Thit k sng lc yu t th nghim theo Plackett - Burman KtqumslngmtvisinhvtthchintheothitkPlackett Burmanctnhbybng4.1,ktqunycnhpvophnmmDesign Expertphntch.Ktquphntchlmcnhhngcayutthnghimn chtiutheodivphngsaimtyuttheothitkPlackettBurmansai bit o = 0,05%. Kt qu phn tch mc nh hng ca yu t th nghim n ch tiu theo di c th hin bng 4.2, kt qu phn tch ANOVA c th hin ph lc 3.1. Tiktquphntchmcnhhngcachtiutheodinyutth nghim, nu kt qu cho mc nh hng c gi tr dng c ngha l yu t th nghim lm tng mc ca ch tiu theo di, ngc li, nu mc nh hng cho gi tr m c ngha l yu t th nghim lm gim mc ca ch tiu theo di. Kt qu bng 4.2 cho thy cc yu t th nghim X1, X2, X3, X4 cho mc nh hng dng i vi c ba ch tiu theo di Y1, Y2, Y3 vi gi tr p < 0,05; yu t X5 cho mc nh hng m i vi c ba ch tiu theo di vi gi tr p < 0,05, iu ny c ngha l ng glucose b sung lm gim mt t bo ca vi sinh vt nui cy th nghim. Trong bng phn 21 tch ANOVA, chng ti tr trng n mc ngha v mt thng k (gi tr p) i vi tng yu t th nghim, gi tr ny c th hin bng 4.2. Bng 4.1: Kt qu th nghim sng lc theo Plackett-Burman Nghim thc Yu t th nghimCh tiu theo di X 1 X 2 X 3 X 4 X 5Y1Y2Y3 1111-114,89 x 10117,23 x 1074,754 x 108 2-1111-19,758 x 10101,193 x 1071,548 x 108 31-1-1111,99 x 1071,815 x 1071,193 x 105 4-111-116,327 x 10105,236 x 1072,609 x 108 5-1-111-15,086 x 1084,9 x 1052,45 x 106 6-1-1-1-113,761 x 1061,906 x 1071,545 x 106 71-11-1-13,636 x 1094,012 x 1062,7 x 105 811-1-1-11,834 x 1077,5 x 1071,572 x 105 911-11-13,182 x 1063,12 x 1068,35 x 105 10-11-1111,996 x 1061,9 x 1051,868 x 106 111-11115,154 x 10102,413 x 1062,031 x 106 12-1-1-1-1-13,039 x 1062,4 x 1054,654 x 105 Bng 4.2: Kt qu phn tch mc nh hng ca cc yu t th nghim n ch tiu theo di theo Plackett Burman Yu t Y1Y2Y3 Mc nh hng p Mc nh hng p Mc nh hng p X16,382 x 10100,0121,513 x 1070,00219,196 x 1060,0024 X29,902 x 10100,0052,843 x 1070,0271,467 x 1080,003 X31,176 x 10110,0064,635 x 1060,00051,482 x 1080,0002 X48,369 x 10100,021,160 x 1070,00339,603 x 1070,0028 X5-6,771 x 1010 0,026-3,110 x 1070,039-9,722 x 1070,0012 22 T kt qu phn tch ANOVA v kt qu nh gi mc nh hng ca tng yu t th nghim ln ch tiu theo di chngtinh giyu t th nghim vi phm vi nghin cu nh trn l ph hp v c ngha v mt thng k (p < 0,001) (xem ph lc3.1).Vimctiulchtiutheodi(mtvisinhvt)tiannchngti chn cc yu t c mc tng tc dng v gi tr tng tc ln, gi tr p nh (s liu c ngha v mt thng k) thit lp cc th nghim tip theo. Nh vy cc yu t thnghimcchnsdngthnghiptiptheolpH(X1),nhit(X2),thi gian (X3) v pepton b sung (X4); D - glucose (X5) c c nh mc trung tm l 10% do c mc nh hng m ln c ba ch tiu theo di nhng khng th loi b do s gy thiu ht ngun carbohydrates trong mi trng. 4.3.K qu th nghim 3 - Thit k ti u ha mi trng nui cy theo RSM - CCD Kt qu m mt vi sinh vt khi thc hin th nghim theo thit k RSM CCD c th hin bng 4.3, kt qu ny c nhp vo phn mm Design Expert tin hnh phn tch. Trong phn nh gi ca chng trnh a ra, phng trnh bc ba khng s liu thc hin, phng trnh bt hai hon ton tng thch. Do chng ti chn phng trnh bc hai tin hnh phn tch. 23 Bng 4.3: Kt qu ma trn quy hoch thc nghim theo RSM - CCD Th nghim Yu t th nghimCh tiu theo di X 1X 2 X 3 X 4Y1Y2Y3 1-1-1-1-12,037 x 106 1,541x 1068,714 x 105 21-1-1-13,041 x 1061,026 x 1065,067 x 105 3-11-1-13,067 x 1061,134 x 1072,941 x 105 411-1-18,136 x 1065,395 x 1071,47 x 104 5-1-11-14,343 x 1074,133 x 1072,97 x 108 61-11-17,545 x 1075,189 x 1074,852 x 107 7-111-18,656 x 1071,346 x 1089,727 x 107 8111-13,084 x 1084,09 x 1082,975 x 107 9-1-1-113,561 x 1061,907 x 1062,766 x 106 101-1-115,479 x 1062,4 x 1067,687 x 105 11-11-113,545 x 1061,968 x 1065,517 x 105 1211-111,233 x 1076,880 x 1072,436 x 105 13-1-1111,118 x 1087,809 x 1076,245 x 108 141-1113,068 x 1083,232 x 1087,095 x 107 15-11112,794 x 1086,798 x 1075,609 x 108 1611115,294 x 1074,023 x 1085,663 x 107 17-o0007,092 x 1063,37 x 1048,348 x 107 18o0003,324 x 1071,324 x 1082,286 x 105 190-o002,855 x 1073,404 x 1042,091 x 107 200o004,767 x 1071,11 x 1082,114 x 105 2100-o08,51 x 1055,673 x 1065,858 x 104 2200o04,914 x 1085,219 x 1085,136 x 108 23000-o1,263 x 1074,095 x 1061,224 x 108 24000o1,099 x 1073,616 x 1067,246 x 106 2500004,045 x 1072,586 x 1081,038 x 108 2600003,809 x 1072,042 x 1081,418 x 108 2700002,677 x 1071,436 x 1087,543 x 107 2800002,655 x 1073,456 x 1087,654 x 107 2900004,908 x 1073,501 x 1081,054 x 107 3000001,91 x 1083,431 x 1081,535 x 107 24 4.3.1.Kt qu phn tch thc nghim theo cu trc c tm cho L. acidophilusKtquphntchANOVActrnhbybng4.4.Saukhiphntch ANOVA, phng trnh hi quy c dng nh l mt m hnh tin on s lng vi sinh vt. Phng trnh tin on mt t bo L. acidophilus c th hin nh sau:

(

)

Bng 4.4: Kt qu phn tch ANOVA thc nghim cho L. acidophilus SV SSdfMSFTNP Nghim thc 5,3428 x 101786,679 x 101619,58< 0,0001 pH 2,4568 x 101612457 x 10167,203**0,014 Nhit 2,1446 x 101612,145 x 10166,288**0,02 Thi gian 2,995 x 101712,995 x 101787,81***< 0,0001 Pepton b sung 2,1539 x 101612,154 x 10166,315**0,02 pH x Thi gian 2,9072 x 101612,907 x 10168,524**0,008 Nhit x Thi gian 2,6672 x 101612,667 x 10167,82**0,011 Thi gian x Pepton b sung 3,1062 x 101613,106 x 10169,107**0,007 Thi gian x Thi gian 8,0422 x 101618,042 x 101623,58***< 0,0001 Sai bit 5,1286 x 1016213,411 x 1015 Tng cng 6,059 x 1017294,068 x 1015 R hi quy0,8818 R hiu chnh0,8368 R tin on0,7705 o = 0,05. S khc bit:

rt c ngha (*** ), kh c ngha (** ), c ngha (* ), khng c ngha (NS). H s hi quy (R2) tnh c l 0,8818. iu ny th hin rng c88,18 % s liu thc nghim tng thch vi s liu tin on theo m hnh. Thng thng, gi tr R2 ln hn 0,75 th hin m hnh tng thch vi thc nghim. Gi tr R2 tin on l 0,7705 ph hp vi R2 iu chnh l 0,8368 ( lch 0,0663 < 0,2).T l tn hiu so vi nhiu l 16,548 > 4 ch ra rng tn hiu y . thbmtpng(xemhnh4.2vhnh4.3)thhinstngtcca tng cp yu t, t th ny c th xc nh c gi tr ti u ca tng yu t lm cho hm p ng cc i. M hnh d on s lng L. acidophilus ti a t c 25 l 4,354 x 108 CFU/ml ( v mt l thuyt vi 88,18 %) gi tr cc yu t: pH = 6,5; nhit 370C; thi gian 18 gi; pepton b sung 10 %. T th chng ti nhn thy trongphmviccyutkhost,slngL.acidophilustngkhipH,thigian, nhit , lng pepton b sung tng; c s tng tc gia pH v nhit , nhit v thi gian, thi gian v pepton b sung. Hnh 4.2: th biu din nh hng ca cc yu t th nghim n mt ca L. acidophilus. 26 Hnh 4.3: th 3D biu din nh hng ca nhit v pH n mt caL. acidophilus. 4.3.2.Kt qu phn tch thc nghim cu trc c tm cho B. subtilisKtquphntchANOVActrnhbybng4.5.Saukhiphntch ANOVA, phng trnh hi quy c dng nh l mt m hnh tin on s lng vi sinh vt. Phng trnh tin on mt t bo B. subtilis c th hin nh sau:

Hshiquy(R2)tnhcl0,8358.iunythhinrngc83,58%s liu thc nghim tng thch vi s liu tin on theo m hnh. Thng thng, gi tr R2 ln hn 0,75 th hin m hnh tng thch vi thc nghim. Gi tr R2 tin on l 0,7013 ph hp vi R2 iu chnh l 0,7726 ( lch 0,0713 < 0,2).T l tn hiu so vi nhiu l 12,729 > 4 ch ra rng tn hiu y . th b mt p ng (xem hnh 4.4 v hnh 4.5)th hin s tng tc ca tng cpyu t, t thny c th xc nh c gi tr ti u ca tngyu t lm cho hm p ng cc i. M hnh d on s lng B. subtilis ti a t c l 4,268 x 108 CFU/ml ( v mt l thuyt vi 83,58%) gi tr cc yu t: pH = 6,49; nhit 34,550C; thi gian 18 gi; pepton b sung 5,41%. T th chng ti nhn thy trong phm vi cc yu t kho st, s lng B. subtilis t l thun vi thi gian; cn khi pH, 27 nhit , lng pepton b sung tng th s lng B. subtilis ch tng n mt mc no th bt u gim; c s tng tc gia pH v thi gian nui cy. Bng 4.5: Kt qu phn tch ANOVA thc nghim cho B. subtilisSVSSdfMSFTNp Nghim thc 5,9497 x 101787,4372 x 101613,3129< 0,0001 pH 6,3931 x 101616,3931 x 101611,4439**0,0028 Nhit 3,1577 x 101613,1577 x 10165,65235*0,027 Thi gian 2,396 x 101712,396 x 101742,8896***< 0,0001 Pepton b sung 2,4193 x 101512,4193 x 10150,43306NS0,5176 pH x Thi gian 3,5638 x 101613,5638 x 10166,37934*0,0197 pH x pH 7,2213 x 101617,2213 x 101612,9265**0,0017 Nhit x Nhit 8,0026 x 101618,0026 x 101614,3251**0,0011 Pepton x Pepton 1,2335 x 101711,2335 x 101722,0809*0,0001 Sai bit 1,1732 x 1017215,5864 x 1015 Tng cng 7,1229 x 101729 R hi quy0,8358 R hiu chnh0,7726 R tin on0,7013 o= 0,05. S khc bit:

rt c ngha (*** ), kh c ngha (** ), c ngha (* ), khng c ngha (NS). Hnh 4.4: th biu din nh hng ca nhit v pHn s lng ca B. subtilis. 28 Hnh 4.5: th 3D biu din nh hng ca nhit v pH n s lng ca B. subtilis. 4.3.3.Kt qu phn tch thc nghim cu trc c tm cho S. cerevisiaeKtquphntchANOVActrnhbybng4.6.Saukhiphntch ANOVA, phng trnh hi quy c dng nh l mt m hnh tin on s lng vi sinh vt. Phng trnh tin on mt t bo S. cerevisiae c th hin nh sau:

Hshiquy(R2)tnhcl0,8406.iunythhinrngc84,06%s liu thc nghim tng thch vi s liu tin on theo m hnh. Thng thng, gi tr R2 ln hn 0,75 th hin m hnh tng thch vi thc nghim. Gi tr R2 tin on l 0,6811 ph hp vi R2 iu chnh l0,7898 ( lch 0,1087 < 0,2).T l tn hiu so vi nhiu l 14,091 > 4 ch ra rng tn hiu y . thbmtpng(xemhnh4.6vhnh4.7)thhinstngtcca tng cp yu t v t th ny c th xc nh c gi tr ti u ca tng yu t lm cho hm p ng cc i. M hnh d on s lng S. cerevisiae ti a t c l5,003x108CFU/ml(vmtlthuytvi84,06%)gitrccyut:pH=5,8; nhit 370C hoc 300C; thi gian 18 gi; pepton b sung 10%. T th chng ti 29 nhn thy trong phm vi cc yu t kho st, s lng S. cerevisiae t l thun vi thi gian, lng pepton b sung; t l nghch vi pH, c s tng tc gia pH v thi gian, pH v pepton b sung, thi gian v pepton b sung. Bng 4.6: Kt qu phn tch ANOVA thc nghim cho S. cerevisiae SVSSdfMSFTNp Nghim thc 7,146 x 101771,021 x 101716,5707< 0,0001 pH 9,923 x 101619,923 x 101616,1083***0,00064 Thi gian 3,282 x 101713,282 x 101753,2762***< 0,0001 Pepton b sung 1,565 x 101611,565 x 10162,54017NS0,125 pH x Thi gian 1,174 x 101711,174 x 101719,0653***0,0002 pH x pepton b sung 3,455 x 101613,455 x 10165,60895*0,027 Thi gian x pepton b sung 4,387 x 101614,387 x 10167,12217*0,014 Thi gian x thi gian 7,561 x 101617,561 x 101612,2739**0,002 Sai bit 1,355 x 1017226,16 x 1015 Tng cng 8,501 x 101729 R hi quy0,8406 R hiu chnh0,7898 R tin on0,6811 o = 0,05. S khc bit:

rt c ngha (*** ), kh c ngha (** ), c ngha (* ), khng c ngha (NS). Hnh 4.6: th 3D biu din nh hng ca Pepton b sung v thi gian n mt ca S. cerevisiae. 30 Hnh 4.7: th biu din nh hng ca yu t th nghim n mt ca S. cerevisiae. 4.4.Chn phng n sn xut ti u - kim tra bng thc nghim Kt qu phntch RSM CCD ca Design- Expert c 29gii php c tm thy(xembng4.7)thayucumtvisinhvtnuicytccivicba chng, ccyu t th nghim trong phm vi kho st. Trong 29 gii php ny, xt v mt ca mi chng th gii php th 29 cho gi tr hm cc i c L. acidophilus v B. subtilis l ln hn c, gii php th 20 cho gi tr hm cc i cho S. cerevisiae 31 l ln hn nhng gii php cn li. Xt v tng mt vi sinh vt th t gii php th 1ngiiphpth15tngmtvisinhvt>9,6x108(CFU/ml)visailch gia cc gii php l 0,024 x 108, mt ny l ln hn so vi cc gii php cn li. Phng n ti u nht c chn phi tha mn hai yu cu, th nht l tng mt vi sinh vt phi ln m bo li ch kinh t, th hai l sai lch mt phi nh mborngmttbogiabachngtngngnhtvinhau.ichiuvi yu cu trn, chng ti nhn thy gii php th 7 v 8 tha mn vi tng mt t bo l 9,619 x 108 (CFU/ml), sai lch mt l 0,193 x 108. Tuy nhin gii php th 8 c lng pepton b sung l 9,69% t hn gii php th 7 (10%) hay gii php th 8 t tn km mi trng hn gii php th 7. Vy gii php ti u nht chng ti chn a vo sn xut thc t l gii php th 8 vi cc thng s nh sau: pH = 6,11; nhit 36,840C~370C;thigiannuicy18gi;9,69%peptonbsung;10%D glucose b sung. Sau khi chn c phng n sn xut chng ti tin hnh kin tra bng thcnghimvibalnlplitrn1000mlmitrng.Vickimtrabngthc nghim ny nhm nh gi xem s d on ca m hnh c ph hp vi thc t hay khng. Kt qu c trnh by bng 4.8, th b mt p ng c th hin hnh 4.8 v hnh 4.9. So snh mt vi sinh vt thc nghim m c vi gii php sn xut th 8, chng ti nhn thy mt t bo vi sinh vt ca mi chng v tng mt t bo vi sinh vt u ln hn so vi gii php th 8, nh vy chng t phng n cchnphhpvithcnghim.Mttbocamichngutmc108 (CFU/ml), tng cng c ba chng t 109 (CFU/ml) nh vy t mc yu cu ti t ra. Phng n sn xut c chn c th a vo sn xut thc t. Bng 4.8: Kt qu kim tra thc nghim Vi sinh vt Mt (CFU/ml) T l vi m hnhLn 1Ln 2Ln 3 Trung bnh SD L. acidophilus 4,906x1084,636x1084,489x1084,667x1082,115x107136,038% B. subtilis3,672x1083,577x1083,473x1083,574x1080,995x107120,094% S. cerevisiae 3,941x1083,227x1083,673x1083,614x1083,607x107112,751% Tng11,864x108 123,339% 32 Kt qu thc nghim cho kt qu mt L. acidophilus t 4,667 x 108 (CFU/ml) nh vy l thp hn so vi mt trn mi trng MRS ca nhng nghin cu trc y, nghincucaSivakumar(2010)l8,3x108 (CFU/ml),Rooj(2010)l8x108 (CFU/ml).KtquthcnghimchoktqumtB.subtilist3,574x108 (CFU/ml) sau 18 gi nui cy l cao hn so vi nghin cu ca Korsten (1996) l 1,8 x108 (CFU/ml)sau24ginuicylctrnmitrngSTDvnghincuca Th Bch Thy (2006) l 2,51,8 x 108 (CFU/ml) trn mi trng c hot protase cao sau24gi.KtquthcnghimchoktqumtS.cerevisiaet3,614x108 (CFU/ml)sau18ginuicylthphnsovinghincucaPasternakiewicz (2006) l 2,04x 109 (CFU/ml) trnmitrng c b sung canxi sau 36 ginui cy lc v nghin cu ca Donev (2002) l 1,8 x 109 (CFU/ml) sau 30 gi nui cy. Tng mt vi sinh vt l 1,186 x 109 (CFU/ml) tuy khng cao nhng vn mc chp nhn c.Mttchcccaphngphpnyltitkimmitrngcngnhdintch mui cy, ngoi vic thun tin cho vic thu sinh khi cng lc c ba chng, gim thi gian thu sinh khi ca B. subtilis v S. cerevisiae, hay ni cch khc, vic nui cy kt hp ny gip n gin qu trnh nui cy v tit kim chi ph sn xut. Hnh 4.8: th biu din nh hng ca cc ch tiu theo di n mt ti a ca ba chng vi sinh vt. 33 Hnh 4.9: th 3D biu din s tng tc ca cc yu t th nghim n mt ti a ca c ba chng vi sinh vt. Bng 4.7: Cc gii php ti u theo RSM - CCD TnMc tiu Gii hn di Gii hn trn pHtrong phm vi nghin cu5,86,5 Nhit trong phm vi nghin cu3037 Thi giantrong phm vi nghin cu1218 Pepton b sungtrong phm vi nghin cu010 L. acidophilusti a8,509 x 1055,294 x 108 B. subtilisti a3,370 x 1045,219 x 108 S. cerevisiaeti a1,470 x 1046,245 x 108 Gii php pH Nhit (0C) Thi gian (gi) Pepton b sung (%) Mt (CFU/ml) Tng mt (CFU/ml) SD mt (x108)L. acidophilus B. subtilis S. cerevisiae 16,1136,6618103,426x1082,904x1083,312x1089,642x108 0,274 26,1136,7418103,455x1082,909x1083,279x1089,643x108 0,278 36,1136,68189,993,445x1082,925x1083,279x1089,649x108 0,265 34 46,1136,81189,993,471x1082,898x1083,274x1089,643x108 0,291 56,1136,67189,933,435x1082,946x1083,253x1089,634x108 0,247 66,1136,321810 3,482x1082,895x1083,249x1089,626x108 0,296 76,1236,341810 3,409x1083,023x1083,187x1089,619x108 0,193 86,1136,84189,69 3,347x1083,036x1083,236x1089,619x108 0,193 96,1136,1218103,326x1082,996x1083,29x1089,612x108 0,268 106,0836,97189,833,424x1082,822x1083,391x1089,637x108 0,338 116,1436,86189,993,552x1082,997x1083,074x1089,623x108 0,300 126,0536,37189,913,245x1082,796x1083,583x1089,624x108 0,394 136,0735,7718103,172x1082,904x1083,509x1089,585x108 0,303 146,0535,8818103,166x1082,847x1083,582x1089,595x108 0,368 156,1136,5617,97103,387x1082,931x1083,241x1089,559x108 0,232 166,1735,6518103,374x1083,241x1082,913x1089,528x108 0,237 176,2436,7918103,746x1083,245x1082,541x1089,532x108 0,605 186,1134,69189,943,034x1083,113x1083,252x1089,399x108 0,189 196,223717,9893,577x1083,375x1082,496x1089,448x108 0,574 205,9535,7518102,916x1082,431x1084,173x1089,52 108 0,899 216,234,418103,178x1083,344x1082,766x1089,288x108 0,297 226,0837187,233,042x1083,248x1082,944x1089,234x108 0,155 236,234,12189,993,127x1083,346x1082,743x1089,216x108 0,305 246,2233,9118103,12x1083,367x1082,659x1089,146x108 0,359 256,1137186,452,983x1083,397x1082,683x1089,063x108 0,358 26637186,482,763x1083,017x1083,179x1088,959x108 0,209 276,2233,3318102,999x1083,309x1082,666 1088,974x108 0,321 285,8437187,212,526x1082,194x1084,112x1088,832 108 1,025 296,4835,8188,843,885x1083,895x1081,118 1088,898x108 1,600 35 Chng 5 KT LUN V KIN NGH 5.1.Kt lun Sau khi hon thnh cc th nghim, chng ti rt ra c cc kt lun v nhn xt nh sau: Khi nui cy ng thi trn canh trng MRS nhit 370C s pht trin ca L. acidophilus v B. subtilis l tt nhng s pht trin ca S. cerevisiae l thp, khng tng ng vi hai chng cn li. KtqusnglctheomatrnPlackettBurmanxcnhcsnh hng ca cc yu t pH, nhit , thi gian nui cy v lng pepton b sung ln mt vi sinh vt nghin cu. QutrnhnuicytiutheothitkRSM-CCDc29phngnt c mt vi sinh vt ln nht v tng ng gia ba chng, phng n mi trng ti u c chn ngoi thnh phn mi trng MRS cn c thnh phn nh sau: Pepton b sung:9,69% D glucose b sung10%Vi iu kin nui cy: pH:6,11 Nhit :36,84 ~ 37 0C Thi gian:18gi Mt vi sinh vt (CFU/ml) thc hin theo phng n t c vi cc chng L.acidophilus,B.subtilis,S.cerevisiaelnltl4,667x108;3,574x108;3,614x 108, tng mt ba chng trong mi trng nui cy t 11,684 x 108 (CFU/ml). 36 5.2.Kin ngh tinguyn cu ca chng ti gp phn n gin ha qutrnhnui cyvi sinhvtthusinhkhisdngvosnxutsnphmprobiotic.Tuynhin,trong khunkhcathigiantinhnhti,ccktqucachngtimichlbc u, cn c nhiu hn ch. Chng ti c mt s ngh sau: Nghincunhhngcaccnguyntvilngnstngtrng ca cc chng vi sinh vt nhm tng s lng vi sinh vt nui cy. Kim tra tnh i khng vi cc vi sinh vt gy bnh ch th. Kim tra hot tnh enzyme ca cc vi sinh vt . 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Truy cp ngy 24/6/2011. < http://ebooks.unibuc.ro/biologie/RBL/Archive/2002/nr1/art5.doc > 41 PH LC Ph lc 1: Thnh phn mi trng nui cy vi sinh vt. 1.NB (Nutrient Borth) Peptone5,0 g Cao tht3,0 g Nc ct1000 ml pH sau tit trng 7,2 0,2; hp tit trng 121 0C /15 pht 2.NA (Nutrient Agar) Mi trng NB thm 15 20 gram agar cho 1000 ml mi trng. 3.Sabouraud D-glucose40,0g Pepton20,0g Nc ct1000ml Hp tit trng 121 0C/ 20 pht 4.MRSB (Man, Rogosa and Sharpe) Pepton10g Cao nm men10g Cao tht5g Glucose20g Tween 801ml Phosphate dipotassique2g Natri acetate5g Citrate triammonique2g Magie sulfate200mg Mangan sulfate500mg Nc ct1000ml Agar (nu cn)15 20g pH sau tit trng 6,5 0,2; hp tit trng 121 0C/ 15 pht 42 5.PDA (Potato D - glucose Agar) Khoai ty200g D - glucose20g Agar15 20g Khoai ty ct 2 3 cm, un si 30 pht, lc v nh mc cho th tch; hp tit trng 121 0C /30 pht Ph lc 2: Kt qu m s lng vi sinh vtBng s 1: S lng vi sinh vt kho st theo thi gian Thi gian S lng trung bnh (CFU/ ml) log10 trung bnh s lng L. acidophilus B. subtilisS. cerevisiae L. acidophilus B. subtilis S. cerevisiae 33158,33334836,3636166,19053,49953,68452,2206 617000,016000,0260,95234,23044,20412,4166 9322727,272747333,33331204,76195,50884,67523,0809 124690909,091238636,36363571,42856,67135,37773,5528 15346363636,410330000011904,76198,53958,04,0757 18187727272717095238175238,09529,27358,23294,8764 212761904762228181818,21575009,44128,35835,1972 242445454545208636363,6141363,63649,38838,31945,1503 43 Ph lc 3: Kt qu phn tch ANOVA Ph lc 3.1.Kt qu phn tch phng sai ANOVA ca ma trn Plackeet Burman Bng 1: Kt qu phn tch phng sai ANOVA ca ma trn Plackeet Burman cho L. acidophilus Bng 2: Kt qu phn tch phng sai ANOVA ca ma trn Plackeet Burman cho B. subtilis 44 Bng 3: Kt qu phn tch phng sai ANOVA ca ma trn Plackeet Burman cho S. cerevisiae 45 Ph lc 3.2.Kt qu phn tch phng sai ANOVA ca thit k RSM CCD Bng 4: Kt qu phn tch phng sai ANOVA ca thit k RSM - CCD cho L. acidophilus 46 Bng 5: Kt qu phn tch phng sai ANOVA ca thit k RSM - CCD cho B. subtilis 47 48 Bng 6: Kt qu phn tch phng sai ANOVA ca thit k RSM - CCD cho S. cerevisiae 49