B GIO DC V O TO TRNG I HC NNG LM THNH PH H CH MINH B MN CNG NGH SINH HC

NGUYN DUY KHNH

KHO ST IU KIN NUI CY V SINH BO T VI KHUN Bacillus subtilis

LUN VN K S CHUYN NGNH CNG NGH SINH HC

Thnh ph H Ch Minh Thng 9/2006

B GIO DC V O TO TRNG I HC NNG LM THNH PH H CH MINH B MN CNG NGH SINH HC

KHO ST IU KIN NUI CY V SINH BO T VI KHUN Bacillus subtilis

LUN VN K S CHUYN NGNH CNG NGH SINH HC

Gio vin hng dn TS. NGUYN NGC HI

Sinh vin thc hin NGUYN DUY KHNH KHA: 2002 2006

Thnh ph H Ch Minh Thng 9/2006

MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY, HCMC FACULTY OF BIOTECHNOLOGY

EXAMINE CULTURE CONDITION AND SPORULATION OF Bacillus subtilis

GRADUATION THESIS MAJOR: BIOTECHNOLOGY

Professor Dr.NGUYEN NGOC HAI

Student NGUYEN DUY KHANH TERM: 2002 - 2006

HCMC, 09/2006

LI CM NVi tt c lng knh trng, em xin chn thnh cm n Ban Gim hiu trng i hc Nng Lm thnh ph H Ch Minh, Ban ch nhim B Mn Cng ngh sinh hc, cng tt c qu Thy C truyn t kin thc cho em trong sut qu trnh hc ti trng. Em xin chn thnh cm n TS. Nguyn Ngc Hi ht lng hng dn dy d, ng vin, quan tm, ng h em hon thnh kho lun. Em xin chn thnh cm n TS. L Anh Phng, BSTY. Nguyn Th Kim Loan gip , to iu kin thun li cho em trong sut qu trnh hon thnh kho lun. Em xin chn thnh cm n Phng vi sinh, Khoa Chn nui Th Y cho php v to mi iu kin thun li cho em hc tp v nghin cu ti phng. Ti xin cm n cc bn lp CNSH 28 chia x cng ti nhng vui bun trong thi gian hc cng nh ht lng h tr, gip ti trong thi gian thc tp. Sinh vin thc hin Nguyn Duy Khnh

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TM TTNGUYN DUY KHNH, H Nng Lm Tp. H Ch Minh. Thng 8/2006. KHO ST IU KIN NUI CY V SINH BO T VI KHUN Bacillus subtilis. Hi ng hng dn: TS. NGUYN NGC HI Vit nam l mt nc nng nghip c nghnh chn nui rt pht trin v c ng gp rt ln vo s pht trin kinh t ca t nc. V vy, vn nng cao nng sut, cht lng sn phm v ci thin mi trng chn nui rt c quan tm nc ta hin nay. Xut pht t vn ny, chng ti tin hnh nghin cu kho st c im ca vi khun Bacillus subtils, tm hiu iu kin nui cy thch hp v x l bo t sn xut ch phm sinh hc nhm cung cp nhng thng tin chn la nhng iu kin nui cy vi khun Bacillus subtilis thch hp, t sn xut ch phm sinh hc cung cp cho nghnh chn nui. Qua qu trnh thc hin ti chng ti c nhng ghi nhn sau: Kho st nh hng ca ch nui cy tnh v nui cy lc (15 pht lc, 45 pht ngh) th ch nui cy lc cho s lng vi khun cao hn. Kho st nh hng ca 4 loi mi trng khc nhau (TSB, TSB + 1% glucose, TSB + 1% cao nm men, TSB + 1% glucose + 1% cao nm men) th mi trng TSB cho s lng vi khun thp nht, 3 mi trng cn li l nhng mi trng ph hp cho Bacillus subtilis pht trin. Kho st nh hng ca pH mi trng (pH 7 v 7,5), thi gian (24,36 v 48 gi) v nhit nui cy (nhit phng, 37oC) th pH 7, thi gian 48 gi v nhit nui cy 37oC cho s lng vi khun ln nht. Kho st nh hng ca nhit (50, 70), pH (6, 9) v thi gian x l (3, 5 v 7 gi) n s hnh thnh bo t th khi x l cc nhit v pH ny c s nh hng n qu trnh hnh thnh bo t ca vi khun Bacillus subtilis.

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MC LCTrang ta Li cm t ........................................................................................................... i Tm tt ............................................................................................................... ii Mc lc ............................................................................................................. iii Danh sch ch vit tt ...................................................................................... iv Danh sch cc hnh ............................................................................................ v Danh sch cc bng .......................................................................................... vi Phn 1. M U ............................................................................................. 1 1.1. t vn ......................................................................................... 1 1.2. Mc ch Yu cu ........................................................................... 1 Phn 2. TNG QUAN ..................................................................................... 2 2.1. i cng v vi khun Bacilus subtilis ........................................... 2 2.1.1. Lch s pht trin .................................................................... 2 2.1.2. c im phn loi ................................................................. 2 2.1.3. c im phn b ................................................................... 2 2.1.4. c im hnh thi .................................................................. 2 2.1.5. c im nui cy................................................................... 3 2.1.6. c im sinh ho ................................................................... 3 2.1.7. Cu trc khng nguyn ........................................................... 4 2.1.8. Tnh cht i khng ca B. subtilis vi mt s vi sinh vt gy bnh ....................................................................................... 4 2.2. Bo t ca vi khun Bacillus subtilis............................................... 5 2.2.1. Kh nng sinh bo t................................................................ 6 2.2.2. Cu to ca bo t .................................................................... 7 2.2.3. Thnh phn ho hc ca bo t ................................................ 8 2.2.4. S ny mm ca bo t ............................................................ 9 2.2.5. Sc khng ca bo t .......................................................... 9 2.3. H vi sinh vt ng rut v s lon khun ................................... 10 2.3.1. H vi sinh vt ng rut ....................................................... 10 2.3.2. Vai tr ca h vi sinh vt ng rut ...................................... 11iii

2.3.3. S lon khun ......................................................................... 12 2.4. Gii thiu chung v probiotic ......................................................... 13 2.4.1. nh ngha .............................................................................. 13 2.4.2. Chc nng sinh hc ca probiotic .......................................... 13 2.5. Tnh hnh nghin cu v ng dng ch phm cha vi khun Bacillus subtilis .............................................................. 14 PHN 3. NI DUNG V PHNG PHP NGHIN CU ................... 16 3.1. Thi gian v a im thc hin ti ............................................ 16 3.2. Vt liu th nghim .......................................................................... 16 3.2.1. Ging vi khun ....................................................................... 16 3.2.2. Mi trng nui cy............................................................... 16 3.2.3. Ho cht .................................................................................. 16 3.2.4. Thit b v dng c.................................................................. 16 3.3. Ni dung nghin cu ....................................................................... 17 3.4. Phng php thc hin ti ......................................................... 17 3.4.1. Kho st c im sinh hc ca Bacillus subtilis .................. 17 3.4.2. Cc th nghim v Bacillus subtilis ........................................ 17 3.4.2.1. nh hng ca ch nui cy (nui cy tnh, nui cy lc) v thi gian nui cy n s lng vi khun ............................. 17 3.4.2.2. Kho st mi trng v thi gian nui cy thch hp cho vi khun Bacillus subtilis pht trin to sinh khi .................... 18 3.4.2.3. Kho st pH mi trng thch hp cho nui cy vi khun Bacilus subtilis .......................................................... 19 3.4.3. Cc th nghim v bo t Bacillus subtilis ............................. 20 3.4.3.1. Kho st nh hng ca nhit n s to bo t ca vi khun Bacillus subtilis ........................................................ 20 3.4.3.2. Kho st nh hng ca pH n s to bo t ca vi khun Bacillus subtilis ....................................................... 21 PHN 4. KT QU V THO LUN ...................................................... 22 4.1. Kho st c im sinh hc ca Bacillus subtilis .................................... 22 4.1.1. c im hnh thi ca Bacillus sutilis .......................................... 22

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4.1.2. Quan st c im khun lc ca Bacillus subtilis ......................... 22 4.1.3. Quan st dc im nui cy Bacillus subtilis trn mi trng canh23 4.1.4. Tnh cht sinh ho ........................................................................... 23 4.2. Cc th nghim v Bacillus subtils ......................................................... 25 4.2.1. Kho st ch (nui cy tnh, nui cy lc) v thi gian nui cy thch hp ................................................................................. 25 4.2.2. Kho st mi trng v thi gian nui cy thch hp .................... 27 4.2.3. Kho st pH mi trng nui cy vi khun thch hp .................... 29 4.2.4. Cc th nghim v bo t Bacillus subtilis ..................................... 30 4.2.4.1. Kho st nh hng ca nhit n s hnh thnh bo t ca Bacillus subtilis ......................................................................... 30 4.2.4.2. Kho st nh hng ca pH n s hnh thnh bo t ca vi khun Bacillus subtilis .......................................................... 32 Phn 5. KT LUN V NGH ............................................................. 34 5.1. Kt lun.................................................................................................... 34 5.2. ngh .................................................................................................... 34 Phn 6. TI LIU THAM KHO .............................................................. 36 Phn 7. PH LC ........................................................................................ 38

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DANH SCH CC HNHHnh 2.1. Hnh thi vi khun Bacillus subtilis .................................................. 3 Hnh 2.2. Qu trnh to bo t ........................................................................... 6 Hnh 2.3. Cu to bo t Bacillus sutilis ........................................................... 7 Hnh 4.1 T bo vi khun Bacillus subtilis di knh hin vi phng i 1000 ln ..................................................................... 22 Hnh 4.2. c im khun lc ca Bacillus subtilis trn mi trng TSA ..... 23 Hnh 4.3. Khun lc Bacillus subtilis trn mi trng thch tinh bt ............. 23 Hnh 4.4. Phn ng ln men mt s loi ng ca vi khun Bacillus subtilis................................................................ 25

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DANH SCH CC BNGBng 2.1. Cc phn ng sinh ho ca Bacillus subtilis ..................................... 4 Bng 2.2. S khc nhau gia bo t v t bo sinh dng ca Bacillus subtilis 8 Bng 3.1. B tr th nghim 1 .......................................................................... 18 Bng 3.2. B tr th nghim 2 .......................................................................... 19 Bng 3.3. B tr th nghim 3 .......................................................................... 20 Bng 3.4. B tr th nghim 4 .......................................................................... 21 Bng 3.5. B tr th nghim 5 .......................................................................... 21 Bng 4.1. nh hng ca thi gian v ch nui cy n s lng vi khun Bacillus subtilis................................................................... 25 Bng 4.2. nh hng ca mi trng v thi gian nui cy n s lng vi khun Bacillus subtilis................................................................... 27 Bng 43. nh hng ca pH, nhit v thi gian nui cy n s lng vi khun Bacillus subtilis.................................................................... 29 Bng 4.4. nh hng ca nhit n s hnh thnh bo t ca vi khun Bacillus subtilis.................................................................... 30 Bng 4.6. nh hng ca pH n s hnh thnh bo t ca vi khun Bacillus subtilis ................................................................................... 32

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DANH SCH CC BIU Biu 4.1. nh hng ca ch nui cy v thi gian n s lng vi khun Bacillus subtilis.............................................................. 26 Biu 4.2. nh hng ca thi gian v mi trng nui cy n s lng vi khun Bacillus subtilis............................................................. 28 Biu 4.3. nh hng pH, nhit , thi gian nui cy n s lng vi khun Bacillus subtilis............................................................ 29 Biu 4.4. nh hng ca nhit n s hnh thnh bo t ca vi khun Bacillus subtilis............................................................ 31 Biu 4.5. nh hng ca pH n s hnh thnh bo t ca vi khun Bacillus subtilis .......................................................................... 33

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Phn 1. M U 1.1. t vn Hin nay, cc loi ch phm sinh hc t vi sinh vt v ang c s dng ngy cng ph bin. c bit l cc ch phm t vi khun Bacillus subtilis. Nc ta l mt nc nng nghip, ngnh chn nui rt pht trin. Vi mc ch tng nng sut cht lng sn phm chn nui, bo v sc kho ngi tiu dng, bo v mi trng sng khng b nhim bi cc loi ho cht c hi v c bit l nhm hn ch dn vic s dng khng sinh trong chn nui, ch phm sinh hc l mt gii php ti u thc hin mc ch ny. V vy, cc nh chn nui rt ch n vn s dng ch phm sinh hc. Ch phm sinh hc cha cc vi sinh vt sng c li, ngi ta chn lc cc vi sinh vt c li c tnh i khng cao a vo ng rut to s cn bng c li cho h vi sinh vt ng rut, khi phc li hot ng bnh thng, c ch vi sinh vt c hi cho vt nui. Ngoi ra, ch phm sinh hc cn ci thin lng thc n n vo v kh nng tiu ho, cung cp cht dinh dng Xut pht t nhu cu thc tin trn di s hng dn ca TS. Nguyn Ngc Hi, chng ti tin hnh nghin cu ti Kho st iu kin nui cy v sinh bo t vi khun Bacillus subtilis. 1.2. Mc ch Yu cu Mc ch Tm hiu iu kin: nhit , thi gian, pH, mi trng nui cy thch hp v nhit , pH x l bo t ng dng sn xut th nghim ch phm sinh hc. Yu cu Kho st c im ca Bacillus subtilis: v hnh thi khun lc, t bo vi khun Tm iu kin: nhit , pH, mi trng v thi gian nui cy thch hp. Tm iu kin: nhit , pH x l to bo t.

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Phn 2. TNG QUAN 2.1. i cng v vi khun Bacillus subtilis 2.1.1. Lch s pht trin Bacillus subtilis c pht hin ln u tin trong phn nga (1941) bi T chc y hc Nazi ca c. Lc u, ch yu c s dng phng bnh l cho cc bnh s c chin u Bc Phi. Vic s dng iu tr bnh phi i n nhng nm 1949 - 1957 khi Henry, Albot v cc cng s tch c cc chng thun khit ca Bacillus subtilis. T , subtilistherapie c ngha l thuc subtilin ra i tr cc chng vim rut, vim i trng, chng tiu chy do ri lon tiu ho. Ngy nay, vi khun Bacillus subtilis tr nn ph bin v c s dng rng ri trong chn nui, y hc, thc phm. 2.1.2. c im phn loi Theo kho phn loi ca Bergey, vi khun Bacillus subtilis thuc B: H: Ging: Loi: 2.1.3. c im phn b Vi khun Bacillus subtilis thuc nhm vi sinh vt bt buc ng rut, chng c phn b hu ht trong t nhin nh: c kh, bi, t nc. Phn ln chng tn ti trong t, thng thng t trng trt cha khong 10 - 100 triu CFU/g. t ngho dinh dng sa mc, t hoang th Bacillus subtilis rt him. Nc v bn ca sng cng nh nc bin c s tn ti ca bo t v t bo sinh dng Bacillus subtilis (V Th Th, 1996). 2.1.4. c im hnh thi Bacillus subtilis l vi khun nh, hai u trn, G+, kch thc 0,5 - 0,8 m x 1,8 3 m, ng thnh chui ngn hoc n l, di ng, 8 - 12 lng. Sinh bo t nh hn vi khun v nm gia t bo, kch thc t 0,8 - 1,8 m. Eubacteriales Bacillaceae Bacillus Bacillus subtilis

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Pht trin bng cch ny mm do s nt bo t, khng khng acid, c kh nng chu nhit, chu m, tia t ngoi, tia phng x(T Minh Chu, 2000).

Hnh 2.1. Hnh thi vi khun Bacillus subtilis(www.microscopyconsulting.com/ Gallery/pages/Ba..._)

2.1.5. c im nui cy iu kin pht trin: hiu kh, nhit ti u 37oC. Nhu cu O2: Bacillus subtilis l vi khun hiu kh nhng c kh nng pht trin trong mi trng thiu oxy. pH: Bacillus subtilis thch hp nht vi pH = 7,0 7,4. Mi trng thch a TSA: khun lc c dng hnh trn, ra rng ca khng u, c tm sm mu, pht trin chm, mu vng xm, ng knh 3 - 5mm. Sau 1- 4 ngy b mt nhn nheo mu hi sm. Mi trng thch nghing TSA: d mc, to thnh mu xm, ra gn sng. Mi trng canh TSB: Bacillus subtilis pht trin lm c mi trng, to mng nhn, lng cn kt li nh vn my y, kh tan u khi lc ln. Dinh dng cn cc nguyn t C, H, O, N v cc nguyn t khc. 2.1.6. c im sinh ho Ln men khng sinh hi cc loi ng: glucose, maltose, manitol, saccharose, xylose, arabinosse. Indol (-), nitrate (-), VP (+), H2S (-), NH3 (+), catalase (+), amylase (+), casein (+), citrate (+), di ng (+), hiu kh (+). Dung huyt: mt s dng gy dung huyt dng trn thch mu nga v th do tc ng ca hemolysine.

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Bng 2.1. Cc phn ng sinh ho ca Bacillus subtilis Phn ng sinh ho Hot tnh catalase Sinh Indol MR VP S dng citrate Kh Nitrate Tan chy Gelatin Di ng Phn gii tinh bt Arabinose Xylose Saccharose Mannitol Glucose Lactose Maltose Kt qu + + + + + + + + + + + + + +

(Theo Holt, 1992) 2.1.7. Cu trc khng nguyn Bacillus subtilis c khng nguyn H v O, cu trc khng nguyn dng D v L acid glutamic. Sn sinh khng sinh subtilin v bacitracin c tc dng c ch vi khun G+ v G-. Bnh hc: a s chng Bacillus subtilis khng gy bnh. 2.1.8. Tnh cht i khng ca Bacillus subtilis vi mt s vi sinh vt gy bnh Do Bacillus subtilis l vi khun bt buc ng rut nn ngoi kh nng chu ng c acid d dy , cc cht dch tiu ho trong ng rut. Chng cn c kh nng u tranh li vi cc vi sinh vt gy bnh ng rut.

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Vi cc vi sinh vt gy bnh Mi trng nui cy nm bnh c s hin din ca Bacillus subtilis vi mt s lng ln s gy ra s cnh tranh dinh dng, cnh trnh khng gian sng gia vi khun v nm. Do vi khun pht trin nhanh hn (trong 24h) s s dng phn ln cc cht dinh dng trong mi trng, ng thi to ra khng sinh subtilin nn s sinh trng ca nm b c ch. Vi ng loi Cc chuyn gia ti i Hc Havard, M cho bit: khi cht dinh dng bt u cn kit, cc vi sinh vt i ph bng cch chuyn sang tnh trng ng ng, hay ngh ngi trong mt thi gian di. Bacillus subtilis thc hin iu bng cch to ra bo t, c th duy tr trng thi sng tim tng trong nhiu nm, thm ch hng th k. Tuy nhin trong th nghim ca mnh, nhm nghin cu nhn thy giai on rt sm ca s hnh thnh bo t, mt vi t bo Bacillus to ra khng sinh git cht nhng t bo vi khun bn cnh cha bt u qu trnh ny. Cht khng sinh s ph v mng t bo vi khun b tn cng, gii phng cht dinh dng v c t bo ang hnh thnh bo t tiu th. Theo cc nh nghin cu trn, qu trnh to bo t tiu tn mt lng ln nng lng, phi mt vi gi v khi bt u th khng th o ngc. Do , vi khun s c gng trnh thi im cng lu cng tt. c bit, khi dinh dng trong mi trng cn kit, vi khun s tiu dit nhng k xung quanh ht cht dinh dng v ko di thi k ch i ny, cho n khi phi chuyn sang sng tim sinh (Nguyn Th Cng Dung, 2004). 2.2. Bo t ca vi khun Bacillus subtilis Bo t l mt hnh thc tim sinh ca vi khun, n gip cho vi khun vt qua nhng iu kin bt li nh: mi trng ngho dinh dng, nhit , pH khng thch hp, mi trng tch lu nhiu sn phm trao i cht bt li. Mi vi khun ch to c mt bo t. Khi gp iu kin thun li, bo t s ny mm tr v dng t bo sinh dng.

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2.2.1. S hnh thnh bo t Mt trong nhng c im quan trng ca Bacillus subtilis l kh nng to bo t trong nhng iu kin nht nh. Bacillus subtilis c kh nng hnh thnh bo t trong chu trnh pht trin t nhin hoc khi vi khun gp iu kin bt li (dinh dng trong mi trng b kit qu, nhit ) (T Minh Chu, 2000). Qu trnh hnh thnh bo t gm cc bc 1. Hnh thnh vch ngn. 2. S to tin bo t. 3. To lp v bo t. 4. S tng hp cc lp v bo t. 5. S gii phng bo t.

Hnh 2.2. Qu trnh to bo twww.biol.lu.se/cellorgbiol/ membprot/pop_sv.html

Lc u lp nguyn sinh cht trong t bo c s dng. T bo cht v nhn tp trung ti mt v tr nht nh trong t bo. T bo cht tip tc c c v to thnh tin bo t (prospore). Tin bo t dn c bao bc bi cc lp mng. Tin bo t pht trin v tr thnh bo t. Khi bo t trng thnh, t bo sinh dng phn gii v bo t c gii phng ra khi t bo m. Khi gp iu kin thun li th bo t ht nc v b trng ra. Sau v ca chng b ph hu v bo t ny mm pht trin thnh t bo mi. Mi t bo sinh dng ch to ra mt bo t (L Mai Phng, 2004).

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2.2.2.Cu to ca bo t Bo t l mt khi nguyn sinh cht c, c cha cc thnh phn ho hc c bn nh t bo sinh dng nhng c mt vi im khc v t l gia cc thnh phn v c thm mt s thnh phn mi. Pha ngoi ca nguyn sinh cht c bao bc bi nhiu lp mng.

Hnh 2.3. Cu to bo t Bacillus sutiliswww.biol.lu.se/cellorgbiol/ membprot/pop_sv.html

Ngoi cng ca bo t l mt lp mng, rt mng nhng khng thm nc, cu to ch yu l lipoprotein. Di lp mng l v, v bo t c nhiu lp, b mt ca cc lp ny x x, thnh phn ho hc l protein v c s tham gia ca keratin y l nhng lp c kh nng ngn chn s thm thu ca nc v cc cht ho tan trong nc, chng c tc dng tng cng kh nng bo v bo t trc cc iu kin bt li. Di lp v l lp mng trong ca bo t v trong cng l mt khi t bo cht ng nht. Trong cc bo t t do khng tn ti s trao i cht, v vy c th gi trng thi tim sinh trong nhiu nm (L Mai Phng. 2004). Bo t khc t bo sinh dng v cu trc, thnh phn ho hc, tnh cht sinh l.

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Bng 2.2. S khc nhau gia bo t v t bo sinh dng ca B. subtilis c tnh Cu trc Thnh phn ho hc Canxi Protein Hot tnh enzyme c tnh chu nhit c tnh chu bc x c tnh chu cc cht ho hc v acid Kh nng bt mu cht nhum Thp Thp hn Cao Yu Km Yu D nhum Cao Cao hn Thp Cao Mnh Cao Phi s dng phng php c bit T bo sinh dng T bo G+, in hnh Bo t V bo t dy, kh thm nc

2.2.3. Thnh phn ho hc ca bo t Cc lp bao v mng ca bo t c cu to c bn l protein c cha nhiu glyxin, tyroxin v c bit l cystein, ngoi ra cn c s tham gia ca keratin. Nguyn sinh cht ca bo t c cha nhim sc th, ribosome v enzyme chuyn ho trng thi khng hot ng. Khi bo t ny mm th nhng enzyme ny bt u hot ng. Bo t c cha mt lng ln canxi, magie v acid dipicolinic. Acid ny chim t 5- 12% khi lng kh ca bo t (acid ny khng bao gi c trong t bo sinh dng, n c hnh thnh trong qu trnh hnh thnh bo t v mt i khi ny mm). Lng nc trong bo t rt thp v tn ti dng lin kt. 2.2.4. S ny mm ca bo t (Nguyn Ln Dng v cc cng s, 1998) Qu trnh chuyn bo t t trng thi ngh sang t bo sinh dng ca vi khun c gi l qu trnh ny mm ca bo t. Qu trnh ny gm 3 giai on: hot ho, ny mm v sinh trng:

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Hot ho Sau khi cho bo t Bacillus subtilis tn ti trng thi ngh 7 ngy, ta x l 60oC trong 5 pht c th xc tin qu trnh ny mm. Sau khi x l nhit, ta chuyn vo mi trng nui cy thch hp.. C mt s ho cht c bit c th xc tin qu trnh ny mm ca bo t. V d: L-alanine, Mn2+, cht hot ng b mt, glucose,. Cng c nhng cht li c tc dng c ch qu trnh ny mm: D-alanine, natri bicarbonate Ny mm Protein c cha nhiu cystein trong o bo t ho xp ln lm tng tnh thm, xc tin s hot ng ca enzyme protease. Khi lng protein trong bo t o gim xung. Cc cation bn ngoi co th xm nhp vo lp v bo t v lm trng lp v bo t ln, sau lm tan ra v tiu i. Khi , nc bn ngoi s xm nhp vo lp li ca bo t, lm cho li trng to ln, cc loi enzyme bt u c hot ho, bt u qu trnh tng hp thnh t bo. Trong qu trnh ny mm cc c tnh chu nhit, tnh chit quangbt u gim dn; lng dipicolinate-canxi, acid amin, polipeptide dn dn mt i; bt u vic tng hp DNA, RNA v protein trong v bo t. Bo t chuyn thnh t bo sinh dng. Khi ny mm, bo t c th m ra theo pha cc hoc m ngang. Lc thnh t bo cn rt mng v cha hon chnh, do nng cao kh nng tip nhn thm DNA ngoi lai thc hin qu trnh bin np. 2.2.5. Sc khng ca bo t Bo t c sc khng cao i vi cc yu t vt l v ho hc nh: nhit , tia cc tm, p sut v cht st trng. S d bo t c sc khng cao v sng lu l do cc yu t sau: Nc trong bo t phn ln trng thi lin kt, do khng c kh nng lm bin tnh protein khi tng nhit . Do bo t c khi lng ln ion Ca2+ v acid dipicolinic, protein ca bo t kt hp vi dipicolinate canxi thnh mt phc cht c tnh cht n nh cao i vi nhit . Cc enzyme v cc hot cht sinh hc khc cha trong bo t u tn ti di dng khng hot ng, hn ch s trao i cht ca bo t i vi t bo bn ngoi.

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Vi cu trc c nhiu mng bao bc v tnh t thm thu ca cc lp mng lm cho cc cht ho hc v cht st trng kh c th tc ng ti bo t. 2.3. H vi sinh vt ng rut v s lon khun 2.3.1.H vi sinh vt ng rut Theo Nguyn Vnh Phc (1987), rut v d dy ng vt khi mi sinh ra hon ton khng c vi khun, sau vi gi t khi chng sinh ra mi thy c s xut hin ca mt vi vi khun v t chng bt u sinh sn dn. Sau mt vi vi khun khc s theo thc n, nc ung vo ng tiu ho, sng v sinh si ny n , chng s bin i t nhiu nhng c bn chng vn tn ti cho n khi con vt cht. Thnh phn, s lng ca vi sinh vt ng tiu ho ph thuc vo tui tc ca gia sc, cch nui dng, chm scngoi ra cn c cc iu kin vt l, ho hc ca mi trng ng tiu ha. Theo Nguyn Vnh Phc (1970) v nhiu tc gi khc, c th chia h vi sinh vt ng tiu ho ra lm 2 nhm: Nhm vi sinh vt tu nghi Mt s nhng vi sinh vt ny l nhng vi sinh vt c hi, chng thay i theo iu kin thc n, mi trng, ng tiu ho, kh nng khng ca c thnh: Samonella, Klebsiella, E. coli, Clostridium, Shigella, Staphylococus. a s chng thch nghi vi mi trng pH trung tnh n kim. Di nhng iu kin mi trng thch hp chng pht trin, sn sinh c t xm nhp ph v t bo ng rut, gy tn thng thnh ng rut nguy hi cho gia sc gia cm. Nhm vi sinh vt bt buc y l nhng vi sinh vt chu c pH thp, chng pht trin tt trong ng rut ca gia sc, gia cm v nh c vnh vin . a s chng c th gip c th ng vt tiu ho thc n tt hn nh vo h thng enzyme ca chng v gip c th phng chng mt s bnh do vi sinh vt c hi gy ra. Nhm vi sinh vt bt buc gm c: Vi khun: Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococus lactic (hin nay gi l Lactococus lactis), Bacillus subtilis, Bifidobacterium, Cellulosemonas Nm men: Saccharomyces cerevisiae, Saccharomyces bouladii

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Nm mc: Aspergillus niger, Aspergillus oryzae, Mucor spp Protozoa: Entodinium, Diploinium Ngoi ra, ngi ta cng chia h vi sinh vt ng rut ra lm 3 nhm da vo s lng ca chng c trong ng rut nh Nhm h ph chnh: chim tng s trn 90% s lng vi sinh vt ng rut nh: Bifidobacterium, Lactobacillus, Bacterioides Nhm h ph v tinh chim di 10% bao gm: nm men, Clostridium, Proteus, Samonella Nhm tu nghi: chim 0,1% gm: Clostridium, Samonella (T Minh Chu, 2000) Hai nhm vi sinh vt trn lun trng thi cn bng ng v cng l iu kin cn thit bo m cho sc kho vt ch. Cng c th hai nhm vi khun khc nhau tranh chp cng mt cht dinh dng c khi lng rt t trong ng tiu ho. Vi khun no ng ho cht dinh dng ny tt th s loi tr vi khun kia. Mt s vi khun c th lm thay i mt s c im l ho ca h thng tiu ho nh: pH, tim nng oxy ho kh, nng cc cht chuyn ho lm cho mt s vi sinh vt khc pht trin hoc khng pht trin c (Nguyn Kiu Uyn Vi, 2004). 2.3.2. Vai tr ca h vi sinh vt ng rut Gip cho qu trnh tiu ho tt hn nh vo h enzyme. a s cc vi khun c li trong ng rut tham gia vo qu trnh tiu ho, phn gii tinh bt, ng, m v cht x thnh nhng sn phm d hp thu hn. Nhm ny gm nhng vi sinh vt sinh mt s enzyme c kh nng phn hu cc cht trn: amylase, protease, glucanase, cellulase gm cc vi khun Lactic, Bacillus subtilis, Isotrichs, Aspergillus oryzae, Aspergillus niger Theo Nguyn Th Vn Hng (1990), b sung ch phm Saccharomyces boulardii vo khu phn g tht lm gim c h s chuyn bin thc n trn 1 kg tng trng, lm tng sc khng bnh ca g lm gim t l cht, t gp phn lm tng hiu qu kinh t trong chn nui. i vi th trng thnh tiu ho ch yu nh enzyme th s xut hin ca vi sinh vt sn sinh enzyme cng gip th tiu ho thc n tt hn. So vi enzyme tng

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hp, ngun enzyme ca cc vi sinh vt tng i l tng, do c nhiu u im hn so vi cc ngun t ng vt v thc vt nh: Tc sinh sn ca vi sinh vt rt nhanh. Ngun nguyn liu d nui cy, d kim v r tin. Cc enzyme ca vi sinh vt c hot tnh cao. Ngoi ra, c th iu khin cc iu kin ti u trong qu trnh nui cy t c hiu qu cao trong sn xut (Trn Th Minh Chin, 2002). 2.3.3. S lon khun T lon khun c Nissle a ra vo nm 1916. ng cho rng cc vi khun c trong rut ngi v gia sc lun th qun bnh m bo cho s tiu ho bnh thng ca vt ch. Th qun bnh ny da vo 2 c ch: (1) Cng tranh ginh cht dinh dng no cn cho s sinh trng ca chng. (2) Chng c th tit ra cht c tnh khng i vi cc vi khun khc. Cc nguyn nhn gy lon khun: Theo T Minh Chu (2004) th c 3 nguyn nhn: Do khng sinh c ph rng lu ngy lm mt i s cn bng ca h vi sinh vt ng rut. Do yu t ngoi cnh: thc n km phm cht, th b stress, thay i thi tit, v sinh km Do yu t sinh l ca th con: Sc khng ca c th cn yu C ch ca hin tng lon khun Theo Nguyn Vnh Phc (1977), khi c th kho mnh, hai nhm vi sinh vt bt buc v tu nghi lun th cn bng c li nh c ch cnh tranh sinh hc. Khi mt trong hai h ny mt cn bng th d gy ra s ri lon h vi khun ng rut. Thng thng, h vi khun tu nghi ln t h vi khun bt buc. Di tc ng ca mt vi iu kin bt li no , lm cho sc khng ca c th gim hoc do dng khng sinh lu ngy, trng clm cho h vi sinh vt ng rut b thay i v s lng, v cc dng vi khun cng nh v tr c tr ca chng. T , gy gim tiu ho, to iu kin cho vi khun gy thi hot ng, gy ra qu trnh thi ra, phn gii cc cht trong rut sinh ra kh CO2, H2S, CH4 Nhng

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cht ny s ph hu cc phn ng bnh thng trong d dy, rut ph hoi qu trnh to men ca thc n, gy ra nhng phn ng trn ng tiu ho nh: biu m nim mc b phng ln, rp ra v trc i to iu kin cho thun li cho s xm nhp ca nhng vi khun gy hi. Do n nh h vi sinh vt c li, ng thi gip cho vt nui sm tr li trng thi ban u th vic b sung ch phm vi sinh vt c li cho ng rut l mt vn rt thit thc cn c quan tm. 2.4. Gii thiu chung v probiotic 2.4.1. nh ngha Thut ng probiotic c a ra ln u tin bi Lilly v Stillwel (1965) m t nhng yu t kch thch sinh trng c sn sinh bi vi sinh vt. Probiotic c bt ngun t ngun gc Hy Lp vi ngha tin s sng (prolife) Nm 1989, Fuller nh ngha probiotic nh l thc n b sung vi sinh vt sng c tc ng c li n ng vt ch thng qua ci tin cn bng vi sinh vt ca n. Nm 1992, Havenar v cng s ch ra rng nh ngha probiotic ca Fuller b hn ch nhng thc n b sung cho ng vt qua ng rut. ng m rng nh ngha probiotic ca Fuller nh l mt la cy n hoc hn hp cc vi sinh vt sng m chng c nh hng c li i vi vt ch bng cch ci thin nhng tnh cht ca h vi sinh vt bn x. 2.4.2.Chc nng sinh hc ca probiotic Trung ho c t, kh c v phn hu mt s thuc c c tnh cao. Kch thch h thng min dch. Gip n nh h vi sinh vt ng rut Lm tng thc n n vo v lm tng kh nng tiu ho nh h thng enzyme Tng hp vitamine nhm B v K manh trng. 2.5. Tnh hnh nghin cu v ng dng ch phm cha vi khun Bacillus subtilis Trong y hc v chn nui Trong khng chin chng Php Bacillus subtilis c cc gio s ng c Trch, Hong Thu Nguyn (l cc bc s qun y) nghin cu sn xut ch phm Bacillus subtilis a ra chin trng nhm gii quyt dch tiu chy.

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Nm 1949, ti Php lu hnh thuc ung dng ng cha vi khun Bacillus subtilis chng IB 5832, n nm 1955 c thm thuc dng bt ng ng v vin nang. Nm 1962,Guy Albot pht hin Bacillus subtilis c tc dng trong iu tr tiu chy do lm dng khng sinh v vim i trng mn, trn thm vi cc vi khun ln men lactic khc cha lon khun ng rut rt hiu qu. 1958 - 1960 bc s Phm Ngc Thch sn xut ng lot ch phm Bacillus subtilis dng tr bnh ng rut. Khoa v sinh y hc bnh vin Bch Mai H Ni nghin cu v sn xut ch phm Bacillus subtilis dng iu tr bnh tiu chy ngi. Vin bo ch Pharimex Tp.HCM, Vin Pasteur Nha Trang nghin cu sn xut ch phm Bacillus subtilis. 1971, Trn Minh Hng, L Th Ba, Nguyn Vn Hng nghin cu sn xut ch phm Bacillus subtilis dng vin nui cy trn mi trng u tng, cua nghp thu bng tinh bt tan. Ch phm ny dng cho heo ung t 0,5 - 1g/kg th trng. Kt qu heo sau khi s dng ch phm Bacillus subtilis tng trng nhanh. 1982 V Vn Ng v cc cng s sn xut th nghim ch phm coli_subtly (Escherichia coli v Bacillus subtilis) lm gim ti pht do bnh tiu chy gy ra ln so vi phng php iu tr bng khng sinh, kt qu heo tng trng tt. Nghin cu ca Baker, Braude v Kon (1925), Hauser (1957) khi b sung khng sinh liu lng thp vo thc n th nhn thy: acid folic, acid patothenic, vitamin A, vitamin B12 tch t trong gan cao. Ngoi ra Bacillus subtilis cn c phi trn vi mt s chng nm mc, nm men v mt s vi khun khc dng trong ch phm EM, probiotic Trong nng nghip Ch phm Bacillus subtilis c dng phng tr vi sinh vt gy bnh nh nm Rhizoetonia solani, Fusarium sp, Pyriculariaoryaze v trong bo v nng sn sau thu hoch. Trung tm sinh hc thuc lin hip sn xut ho cht thuc b cng nghip nng Tp.HCM, nghin cu sn xut ch phm Bactophyl t Bacillus subtilis phng tr cc loi nm gy bnh trn rau ci.

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H Th M Hng, Nguyn Thanh Bnh Trung tm ng dng sinh hc H Ni sn xut ch phm Bacillus subtilis phng tr nm gy bnh bp Ostrinia furnacalis. 1940 Norio Kimura Yokohamo s dng ch phm Bacillus subtilis ngn chn s pht trin v sinh c t ca chng nm mc Aspergilus flavus, Aspergillus paraciticus. Roman v cc cng s nghin cu B. subtilis (ATCC_6633, ATCC_9372) lm gim i 40 - 50% aflatoxin trong dch cha aflatoxin trong vng 20 ngy.

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PHN 3. NI DUNG V PHNG PHP NGHIN CU 3.1.Thi gian v a im thc hin ti ti c thc hin ti Phng Vi sinh Khoa Chn nui Th Y - Trng i hc Nng Lm Tp.HCM. Thi gian thc hin: t 3/2006 n 7/2006. 3.2. Vt liu th nghim 3.2.1. Ging vi khun Ging vi khun Bacillus subtilis c cung cp t phng vi sinh khoa Chn nui Th y trng i hc Nng Lm Tp H Ch Minh. 3.2.2. Mi trng nui cy Mi trng gi ging, m s lng t bo: TSA (Trypticase Soya Agar). Mi trng kho st c im sinh hc: TSB (Tripticase Soya Broth), Simmon Citrate, mi trng ln men cc loi ng Cc mi trng u c v trng trc khi s dng. 3.2.3. Ho cht Ho cht c bn: NaOH 1N, HCl 1%, NaCl, Ho cht dng kho st c tnh sinh hc ca vi khun: du soi, xylen, Thuc th Kowacs, Methyl-Red, Thuc nhum Gram: Thuc nhum bo t. 3.2.4. Thit b v dng c Thit b Knh hin vi, t m, t sy, ni hp autoclave, t lnh, cn in t, ni cch thu, microwave Dng c a petri, ng nghim, bnh tam gic, lame, a thu tinh, pipette, ng Durham, u tip v trng

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3.3. Ni dung nghin cu Kho st c im ca Bacillus subtilis: hnh thi khun lc, t bo vi khun, tnh cht nui cy trn mt s mi trng, th c tnh sinh ho. So snh phng php m s lng vi khun bng phng php a v phng php m trn knh hin vi. nh hng ca hnh thc v thi gian nui cy n s lng t bo vi khun. nh hng ca mi trng v thi gian nui cy n s lng t bo vi khun. 3.4. Phng php thc hin ti 3.4.1.Kho st c im sinh hc ca Bacillus subtilis Quan st c im hnh thi ca Bacillus subtilis Vi khun Bacillus subtilis c nui trong mi trng thch nghing TSA . Sau 24 gi em nhum Gram v xem di knh hin vi phng i 1000 ln (Xem phng php nhum Gram phn ph lc). Quan st c im nhm ca Bacillus subtilis Phn phi 15ml mi trng TSA c hp tit trng vo a petri v trng ri ngui. Ho vi khun vo nc mui sinh l c hp tit trng, ly 10 dch pha long trang ln mi trng to khun lc n ca vi khun Bacillus subtilis. Sau , gi a petri v nhit 37oC trong 24 gi. Quan st c im nui cy ca Bacillus subtilis trong mi trng canh Mi trng TSB c phn phi 4 - 5 ml vo ng nghim v trng, em hp tit trng v lm ngui. Cy ging vi khun vo mi trng v nhit 37oC trong 24 gi. Sau quan st v xem s thay i ca mi trng nui cy. 3.4.2. Cc th nghim v Bacillus subtilis 3.4.2.1. nh hng ca ch nui cy (nui cy tnh, nui cy lc) v thi gian nui cy n s lng vi khun Nguyn tc Khi nui cy vi sinh vt trong cng mt loi mi trng nhng khc nhau v thi gian v ch nui cy (Nui cy tnh v nui cy lc) th cho lng sinh khi khc nhau. Mc ch Xc nh s lng vi khun thi gian v ch nui cy no l ph hp.

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Bng 3.1. B tr th nghim 1 Mi trng Thi gian 24h 36h 48h Nui cy tnh Nui cy lc

Cch lm Cy vi khun Bacillus subtilis t ng ging vo ng nghim cha 4 - 5 ml mi trng TSB c hp tit trng (121oC/15 pht), em i 37oC trong 24 gi. Sau , cy vi khun t ng nghim vo bnh tam gic cha 50ml mi trng TSB vi t l 2%, nui cy trong 2 ch khc nhau: nui cy tnh v nui cy lc (lc 15 pht, ngh 45 pht) iu kin 37oC. Kim tra s lng vi khun sau thi gian 24, 36, 48 gi bng phng php m khun lc. T kho st trn chng ti chn c ch nui cy vi khun Bacillus subtiis thch hp tip tc kho st thi nghim tip theo. 3.4.2.2. Kho st mi trng v thi gian nui cy thch hp cho vi khun Bacillus subtilis pht trin to sinh khi Nguyn tc Khi nui cy vi khun trong cc loi mi trng khc nhau iu kin nui cy ging nhau (pH, nhit , thi gian nui cy) th s lng vi khun s khc nhau. Mc ch Xc nh mi trng nui cy no l thch hp. Cch lm Cy vi khun t ng ging vo ng nghim cha 4 5 ml mi trng TSB, 37oC trong 24 gi.

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Sau , chuyn vi khun t ng nghim vo 4 loi mi trng khc nhau: TSB, TSB + 1% glucose, TSB + 1% cao nm men, TSB + 1% glucose + 1% cao nm men (vi t l 2%). Sau em nui cy ch c chn ra t th nghim 1, m s lng vi khun sau 24, 36, 48 gi nui cy bng phng php m khun lc. T kho st trn chng ti chn c loi mi trng nui cy thch hp tip tc kho st th nghim tip theo. Bng 3.2. B tr th nghim 2 Mi trng Thi gian (gi) 24 36 48 TSB TSB+1%Glucose TSB + 1%Cao nm men TSB + 1%Glucose + 1%Cao nm men

3.4.2.3. Kho st pH, mi trng thch hp nui cy vi khun Bacillus subtilis Nguyn tc Mi loi vi khun thch hp vi mt pH khc nhau, v vy khi nui cy vi khun trong cng mt loi mi trng nhng cc pH khc nhau th s lng vi khun pht trin sau cng mt khong thi gian l khc nhau. Bng 3.3. B tr th nghim 4 pH Thi gian (gi) 24 36 48 7,0 Nhit phng 37oC 7,5 Nhit phng 37oC

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Mc ch Xc nh pH mi trng nui cy thch hp. Cch lm Cy vi khun t ng ging vo ng nghim cha 4 -5 ml mi trng TSB, 37oC trong 24 gi. Sau , chuyn vi khun t ng nghim vo mi trng c chn t th nghim 2 cc pH khc nhau vi t l 2% v nui cy ch nui cy (1). Sau 48 gi m s lng vi khun bng phng php m khun lc. T th nghim chng ti chn ra pH mi trng nui cy vi khun Bacillus subtilis thch hp. 3.4.3. Cc th nghim v bo t Bacillus subtilis 3.4.3.1. Kho st nh hng ca nhit n s to bo t ca vi khun Bacillus subtilis Cch lm Cy vi khun t ng ging vo ng nghim cha 4 5 ml mi trng TSB, 37oC trong 24 gi. Sau , chuyn vi khun vo mi trng c chn t cc th nghim trn. Sau thi gian nui cy thch hp chng ti x l nhit . X l 50oC v 70oC, m s lng bo t hnh thnh sau 3, 5, 7 gi x bng phng php m khun lc (xem phn ph lc) Bng 3.4. B tr th nghim 4 Thi gian (gi) 3 5 7 50oC Nhit 70oC

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3.4.3.2 Kho st nh hng ca pH n s to bo t ca vi khun Bacillus subtilis Cch lm Cy vi khun t ng ging vo ng nghim cha 4 5 ml mi trng TSB, 37oC trong 24 gi. Sau , chuyn vi khun vo mi trng c chn t cc th nghim trn. Sau thi gian nui cy thch hp chng ti x l pH. m s lng bo t hnh thnh sau 3, 5, 7 gi x l bng phng php m khun lc. Bng 3.5: B tr th nghim 5 Thi gian (gi) 3 5 7 6

pH 9

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PHN 4: KT QU V THO LUN 4.1 Kho st c im sinh hc ca Bacillus subtilis 4.1.1. c im hnh thi ca Bacillus sutilis Sau khi tiu bn vi khun c nhum Gram v c quan st trn knh hin vi phng i 1000 ln, chng ti nhn thy vi khun Bacillus subtilis l : Trc khun bt mu Gram dng, ngn v nh. Kch thc t 0,5 - 0,8 m x 1,5 -3 m, hai u trn, i khi ni thnh chui di, c to bo t khng lm phnh t bo.

Hnh 4.1. T bo vi khun B. subtilis di knh hin vi phng i 1000 ln: www.answers.com/ topic/bacillus-1

4.1.2. Quan st c im khun lc ca Bacillus subtilis Vi khun c cy trn mi trng TSA, sau 24 gi cho thy hnh dng ca khun lc: khun lc kh, c mu xm nht trng, to ra lp mng mn, lan trn b mt thch, c mp nhn, mp li lm, bm cht vo mi trng thch.

Hnh 4.2. c im khun lc ca Bacillus subtilis trn mi trng TSA

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4.1.3. Quan st dc im nui cy Bacillus subtilis trn mi trng canh Sau khi cy vi khun Bacillus subtilis vo mi trng canh TSB, em 37oC: Sau 12 gi th vi khun lm c mi trng. Nui cy n 24 gi th thy vi khun to vng trn b mt ca mi trng v lm cho mi trng pha di trong hn. 4.1.3. Tnh cht sinh ho Bng 4.1. Cc phn ng sinh ho khc ca Bacillus subtilis Th nghim Hot tnh catalase Sinh Indol MR VP S dng citrate Kh Nitrate Saccharose Mannitol Glucose Lactose Maltose Kt qu + + + + + + + + +

Ghi ch: (-) l kt qu m tnh (+) l kt qu dng tnh Qua bng cho thy kt qu kho st ca chng ti ph hp vi tnh cht sinh ho ca Bacillus subtilis (Nguyn Vnh Phc, 1976). Ngoi ra chung ti cn kho st thy c rng Bacillus subtilis c kh nng s dng cc cht nh: glucose, citrate, hp cht cha nitrogen v c kh nng di ng. Tuy nhin, Bacillus subtilis li khng c enzyme tryptophase phn gii tryptophan to Indol.

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Hnh 4.4. Phn ng ln men mt s loi ng ca vi khun Bacillus subtilis Kh nng phn gii tinh bt Chng ti tin hnh kho st kh nng s dng tinh bt bng cch o ng knh vng phn gii trn mi trng thch tinh bt (Starch agar) v thu c kt qu ng knh vng phn gii trung bnh l 2,9 cm.

Hnh 4.3. Khun lc Bacillus subtilis trn mi trng thch tinh bt. 4.2. Cc th nghim v Bacillus subtilis 4.2.1. Kho st ch (nui cy tnh, lc) v thi gian nui cy thch hp Chng ti tin hnh: Nui cy vi khun trong mi trng TSB hai ch nui cy khc nhau: nui cy tnh v nui cy lc (15 pht lc, 45 pht ngh). m s lng vi khun 3 thi im: 24gi, 36gi, 48 gi bng phng php a (th nghim c lp li 2 ln). Kt qu c trnh by bng 4.1.

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Bng 4.1. nh hng ca thi gian v ch nui cy n s lng vi khun Bacillus subtilis Thi gian (gi) 12 18 24 X Ch nui cy Nui cy lc 10,15 11,07 11,16 10,79 Nui cy tnh 8,79 9,92 9,95 9,55

(S lng vi khun/ml tnh theo logarit)

S lng vi k hun (tnh theo logarit)

12 10 8 6 4 2 012 18 24

Thi gian (gi)

Nui cy lc

Nui cy tnh

Biu 4.1 nh hng ca ch v thi gian nui cy n s lng vi khun B. subtilis Qua kt qu bng 4.1 cho thy s lng vi khun trung bnh trong 1 ml canh khun ch nui cy lc l 10,79 (gi tr logarit) (s thc t l 6,17.1010) cao hn so vi s lng vi khun trung bnh ch nui cy tnh 9,55 (gi tr logarit) (s thc t l 35,48.108). Kt qu x l thng k cho thy rng c s khc bit v s lng vi khun gia hai ch nui cy tnh v lc, s khc bit ny c ngha v mt thng k (vi P