KHO ST MT S C TNH CHNG BACILLUS SUBTILIS B20.

1 LM C S CHO VIC SN XUT PROBIOTIC PHNG BNH GAN THN M DO EDWARDSEILLA ICTALUTI TRN C TRA (PANGASIUS HYPOPHTHALMUS) NUI THM CANH
INVESTIGATE SOME CHARACTERISTICS OF BACILLUS SUBTILIS B20.1 FOR PROBIOTIC PRODUCT TO PREVENT DISEASE CAUSED BY EDWARDSEILLA ICTALURI ON STRIPE CATFISH (PANGASIUS HYPOPHTHALMUS) CULTURED IN INTENSIVE FARMS
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H Th Trng Thy1, Nguyn N Trang Thy1, V Minh Sn2 B Mn Bnh hc Thy Sn, Khoa Thy Sn Trng i hc Nng Lm TPHCM 2, Vin nghin cu Nui Trng Thy Sn 2

TM TT Nhm to sn phm probiotic tt nht cho vic phng bnh gan thn m trn c tra mt cch hiu qu, chng ti chn chng Bacillus subtilis B20.1 v tin hnh cc th nghim nh sau: kho st iu kin nui cy thch hp pH (4, 5, 6, 7, 8, 9, 10), nhit (250, 30 0, 350, 40 0C), nng mui (0.5%, 1%, 2%, 3%, 4%, 5%, 6%); kh nng chu pH thp ca d dy (pH= 2, 3), v mui mt (0.5%, 1%, 2%); kho st kh nng sinh enzyme ngoi bo (amylase, lipase, protease, cellulase); kho st kh nng phn hy phn t tn hiu HHL; v kh nng c ch vi khun Ewardseilla ictaluri trong iu kin nui chung. Kt qu chng ti thu c nh sau: chng vi khun Bacillus subtilis B 20.1 pht trin tt nht v cho i khng mnh nht trong iu kin pH=6, nng mui 3%, nhit 350C; c kh nng chu pH thp (2 v 3) d dy, v nng mui mt 2%; c kh nng sinh cc loi enzyme ngoi bo amylase, lipase, v cellulase; c kh nng phn hy phn t tn hiu HHL trong qu trnh quorum sensing ca vi khun; v cnh tranh sinh dng mnh m cng nh c ch s pht trin ca E. ictaluri. ABTRACT To produce the best probiotic product for prevention of disease caused by Ewardseilla ictaluri on stripe catfish, the investigation was conducted on Bacillus subtilis B20.1 with the experiments on pH (4, 5, 6, 7, 8, 9, 10), temperature (250, 30 0, 350, 400C), salt concentrations (0.5%, 1%, 2%, 3%, 4%, 5%, 6%); low pH in condition of digestion (pH=2, 3), and bile salt (0.5%, 1%, 2%); the ability to produce digestive enzyme (amylase, lipase, protease, and cellulose), destroy the signal molecule HHL; and inhibit the growth of E. ictaluri in the shared culture media. For the result, B. subtilis B20.1 grew in the best conditions including pH= 6, salt concentration 3%, temperature 350C; resisted in low pH (2, 3) and bile salt concentration 2%; produced digestive enzyme amylase, lipase, and cellulase; destroyed signal molecule HHL in quorum sensing of bacteria; and nutritionally competed to inhibit strongly the growth of E. ictaluri. T VN Ngy nay, vic nui thm canh c tra vi quy m ln ngy cng pht trin ti cc tnh thnh trong c nc nhm p ng nhu cu xut khu ra th trng th gii ca nc ta hin nay. Chnh v th khng th trnh khi nhng ri ro xy ra dch bnh trong mi trng nui. Bn cnh cc loi thuc khng sinh dng tr bnh, cc nh khoa hc ang tm ra cc sn phm thay th nhm nng cao hiu qu trong vic phng bnh vi mc ch hn ch cc tc hi mi trng do thuc v ha cht gy ra. S dng liu php sinh hc v thn thin vi mi trng, probiotic hay cung cp cc vi sinh vt c li c xem l gii php cn hng n ngn chn nhng nguy c dch bnh xy ra v t gim thiu thit hi cho ngi nui c tra ti cc tnh ng bng song Cu Long. Cc vi sinh vt c li c kh nng ci thin mi
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trng nc v c ch vi sinh vt gy bnh trong vi khun Edwardseilla ictaluri l mt nguyn nhn. Ngoi ra, cc vi sinh vt c li cn c kh nng phn hy phn t tn hiu ca cc vi sinh vt gy bnh nhm gim c lc v kh nng pht trin ca chng. Mc tiu nghin cu nhm th nghim mt s c tnh ca chng Bacillus subtilis B20.1 cho vic ch to probiotic trong cng tc phng bnh gan thn m do Edwardseilla ictaluri gy ra trn c tra nui thm canh ti cc tnh ng bng sng Cu Long. VT LIU V PHNG PHP NGHIN CU Nghin cu c tin hnh ti phng vi sinh-phng sinh hc thc nghim ti Vin Nui Trng Thy Sn 2 t thng 3 n thng 5 nm 2011. Chng vi khun c chn gm Bacillus subtilis B20.1 c thu ti ao nui c tra tht ti ng Thp; Edwardseilla ictaluri GLy09 t phng lu gi mu ca Trung Tm Quc Gia Quan Trc v Cnh bo Mi Trng v Phng Nga Dch Bnh Thy Sn khu vc Nam B. Cc th nghim trn Bacillus subtilis B20.1 Kh nng sinh enzyme ngoi bo: cc chng vi sinh vt c dng lm probiotic trong thc n thy sn s tit ra cc enzyme ngoi bo gip h tr tiu ha v phn hy cc thc n d tha trong mi trng nc. Cc enzyme th nghim gm amylase, lipase, protease, v cellulase. Cc phn ng s c thc hin bng mt s ha cht chuyn bit nh vo mi trng nui cy nhn bit vi sinh vt c sn sinh ra enzyme hay khng sau khi chng phn gii cc hp cht nh tinh bt, cht bo, gelatine, casein, v cellulose. Kho st iu kin nui cy thch hp cho i khng mnh vi E. ictaluri: Bacillus subtilis B20.1 c cho nui cy trong mi trng dinh dng vi cc iu kin khc nhau pH (4, 5, 6, 7, 8, 9,10), nhit (250, 30 0, 350, 400), v nng mui (0%, 1%, 2%, 3%, 4%, 5%, 6%) trong 24h. Sau , dch vi khun c ly tm v ly ra nui chung cng E. ictaluri theo phng php ging khuch tn kho st tnh i khng.

S 1: Khi qut phng php tin hnh theo ging khuch tn (Chythanya et al., 2002) Kho st kh nng chu ng pH thp Vi khun Bacillus subtilis B20.1 nui tng sinh trong mi trng DSM, sau chuyn vo nui trong mi trng BHIB c pH thp 2 v 3 c xem nh pH acid ca d dy. Tip tc tin hnh thu sinh khi v m s vi khun cn sng trn mi trng LB (Jacobsen et al., 1999)
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S 2: Th nghim kh nng chu pH thp trong d dy Kho st kh nng chu ng mui mt Vi khun Bacillus subtilis B20.1 c nui tng sinh trong mi trng DMS v tng t chuyn vo mi trng nui c pha hm lng mui mt 0.5%, 1%, 2%. Sau thu sinh khi v m s khun lc cn li trn mi trng LB (Verdenelli et al., 2006).

S 3: Th nghim kh nng chu mui mt Kho st kh nng phn hy phn t tn hiu HHL-Hexanoyl Hemoserine Lactone S dng vi khun ch th cho HHL Chromobacterium violaceum v dung dch C6-HHL kho st kh nng phn hy Hexanoyl Hemoserine Lactone ca vi khun Bacillus subtilis Kho st kh nng c ch ca Bacillus subtilis vi vi khun Ewardsiella ictaluri trong mi trng nui chung: Tin hnh nui chung 2 chng vi khun trong mi trng dinh dng BHIB vi mt ban u c hai l 2107 cfu/ml trong 24h. Tip theo, tri u m s lng vi khun cn li trn mi trng BHIA. ng thi tin hnh kim tra s hin din dch tit i khng ca Bacillus subtilis bng phng php ging khuch tn.
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S 4: Kho st kh nng phn hy phn t tn hiu HHL KT QU V THO LUN Kt qu kho st kh nng sinh enzyme ngoi bo Bng 1: Kt qu kho st enzyme ngoi bo ca vi khun Bacillus subtilis B20.1 Chng vi khun B20.1 LIPASE CELLULASE + AMYLASE + GELATINASE CASEINASE +

T bng 1, vi khun Bacillus subtilis B20.1 c kh nng tit cellulase, amylase, v caseinase nhm phn hy cellulose, tinh bt v ng trong rut c gip tng cng tiu ha thc n. So vi nghin cu ca Ng T Thnh (2007) trn 236 chng Bacillus ch c 2 chng T20 v M27 th hin y cc hot tnh nh phn hy c gelatine v cht bo trong sa, iu ny Bacillus subtilis B20.1 khng th thc hin c. Cc chng cn li u ch th hin tnh nng phn hy protein v tinh bt. Nh vy, so vi a s chng Bacillus ca cc nghin cu th Bacillus subtilis B20.1 c kh nng tit enzyme ngoi bo tng i tt. Kt qu kho st cc iu kin nui thch hp nh hng ln kh nng i khng ca Baccilus subtilis B20.1 ln E. ictaluri Kt qu trong iu kin nui vi cc mn khc nhau Kt qu t biu 1 cho thy, vi khun Bacillus subtilis B20.1 l loi a mui, chng c kh nng chu ng v sng xt tt c cc nng mui 0.5% v 6% vi t l mt vi khun dao ng khng ng k. Ngoi ra, Bacillus subtilis B20.1 pht trin tt nht v cho kh nng i khng mnh nht vi E. ictaluri nng mui 3% trong mi trng nui.
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Biu 1: nh hng ca mn ln tng trng v kh nng i khng ca Bacillus subtilis B20.1 vi E. ictaluri Kt qu trong iu kin nui vi cc nhit khc nhau

Biu 2: nh hng ca nhit ln kh nng tng trng v i khng vi E. ictaluri ca vi khun Bacillus subtilis B20.1 Qua biu 2, vi khun Bacillus subtilis B20.1 thch hp v pht trin mnh nht nhit 350C ng thi cho kh nng i khng mnh nht vi E. ictaluri, th hin qua vng v khun ln nht 17.5cm so vi cc iu kin nhit cn li. Kt qu trong iu kin pH khc nhau T biu 3, iu kin pH 4, 9, 10 cho sc sng v kh nng chu ng ca Bacillus subtilis B20.1 l rt thp v iu kin ti u duy nht pH 6. Trong iu kin ny, vi khun pht trin mnh v tng v s lng ng thi cho kch thc vng v khun ln nht v kh nng i khng vi E. ictaluri.

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Biu 3: nh hng ca pH ln kh nng tng trng v i khng vi E. ictaluri ca Bacillus subtilis B20.1 Kt qu nh gi kh nng chu pH thp ca d dy

Biu 4: Kh nng chu pH thp ca vi khun Bacillus subtilis B20.1 Qua biu 4, sau khong thi gian 24h nui cc gi tr pH 2 3 4, mt vi khun dao ng trong khon t 100% gim xung gi tr 85-90%. Nh vy, tuy c gim nhng t l ny vn mc kh cao. Trong nghin cu ca B. Hyronimus v ctv (2000), hu ht cc vi khun thuc nhm lactic u nhy vi acid. Chng B. laevolacticus v B.racemilacticus nhy cm vi pH 2 v 3, ch c th sng xt trong khon 6h th nghim. Cc chng Sporolactobacillus c nghin cu nhy cm vi pH thp cho t l sng xt ch 11-26% sau 3 gi, 0.2-15% sau 6 gi. Nh vy so vi cc chng vi khun khc Bacillus subtilis B20.1 c kh nng chu ng pH thp kh mnh, cho tim nng sn xut probiotic trong tng lai. Kt qu nh gi kh nng chu ng mui mt Theo Gilliland v ctv (1984), nng 0.3% c coi l nng quan trng sn lc cc chng khng. c 4 nng sau cc gi kho st, t l sng ca vi khun dao ng t 80-100%. Nhn chung t l ny vn l kh cao so vi cc chng Lactobacillus plantarum ch vi 25% sng xt trong nng mui mt 0.3% (Harutoshi Tsuda v ctv, 2000). Theo nghin cu ca Mariam v Mirlohi v ctv, khng mt vi khun no thuc chng L. plantarum v L. rhamnosus tn ti trong nng mui mt 2%. Nh vy, so vi cc chng khc, kh nng
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chu ng mui mt ca Bacillus subtilis B20.1 l rt tt, v nng mui mt c th tit ra trong qu trnh tiu ha gi u khon 1.5-2%. Bacillus subtilis B20.1 hon ton c tim nng trong vic sn xut probiotic.

Biu 5: Kh nng chu ng mui mt ca Bacillus subtilis B20.1 Kt qu kho st kh nng phn hy phn t tn hiu HHL
6 5
Nng HHL (mg/L)

Bacillus B20.1 i chng

3 2 1 0 0 6 12 18 Thi gian phn hy (gi) 24

Biu 6: Kh nng phn hy HHL ca Bacillus subtilis B20.1 Nhn vo biu 6, vi s hin din ca vi khun th nng HHL gim i mt cch r rt. Sau 24h, nng HHL l i chng cn 3mg/l, trong khi nng HHL l th nghim cha Bacillus subtilis B20.1 th lng cn li thp hn 1mg/l. iu ny chng t, vi khun Bacillus subtilis B20.1 c kh nng tit ra enzyme AHL-acylase v AHL-lactonase phn hy HHL, b gy qu trnh quorum sensing ca vi khun. Da vo nghin cu ca Nguyn Th Ngc Tnh Vin Nghin Cu NTTS 2 (2001), Bacillus subtilis B20.1 l vi khun c kh nng phn hy HHL thuc nhm trung bnh, sau 12h nng HHL cn li < 2ppm. Kt qu kho st kh nng c ch ca Bacillus subtilis B20.1 ln s pht trin ca E. ictaluri bng phng php nui chung T bng 2, ta thy rng vi khun Bacillus subtilis B20.1 c ch hon ton s pht trin ca E. ictaluri sau 24h v tng sinh v s lng. Ngoi ra, lng Bacillus subtilis khi nui ring tng rt cao 1.910 9 cfu/ml so vi trng hp nui chung. iu ny chng t khi nui chung gia hai loi vi khun c s c ch ln nhau, nhng Bacillus subtilis mnh hn nn c kh nng tiu dit hon ton E. ictaluri.
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Bng 2: Kho st kh nng c ch ca Bacillus subtilis B20.1 i vi s pht trin ca E. ictaluri bng phng php nui chung Bacillus subtilis B20.1 + E. ictaluri Bacillus subtilis Chng Bacillus subtilis B20.1 E. ictaluri B20.1 Mt ban u (cfu/ml) 2x107 2x106 2x10 7 9 Mt sau 24h (cfu/ml) 1.9x10 0 4.45x10 8 Kt qu bng 3, sau khi nui chung, thu dch treo v thc hin phng php ging khuch tn, ta thy hon ton khng c s xut hin vng v khun. iu , chng t vi khun Bacillus subtilis B20.1 khng h tit ra cht i khng vi E. ictaluri. Nh vy, s c ch ln nhau din ra l do cnh tranh dinh dng vi s tng sinh mnh hn ca Bacillus subtilis B20.1 so vi E. ictaluri. Bng 3: Kho st tnh i khng ca Bacillus subtilis B20.1 i vi E. ictaluri ng knh vng khng khun (mm) Phng php nui Dch treo Dch khun Bacillus subtilis B20.1 + E. ictaluri 0 22.0 1.3 (nui chung) KT LUN Vi khun Bacillus subtilis B20.1 rt c tim nng trong vic s dng sn xut probiotic, v chng c th tit ra cc enzyme ngoi bo nh cellulase, amylase, caseniase gip ci thin tiu ha cho c nui. nui sinh khi Bacillus subtilis dng cho vic sn xut probiotic phng bnh do E. ictaluri gy ra, ta nn nui trong iu kin pH 6, nhit 350C, nng mui 3%. Ngoi ra, Bacillus subtilis B20.1 c kh nng chu c pH thp ca d dy, v nng mui mt cao. Khi gp cc vi sinh vt khc trong cng mt mi trng sng, chng tham gia vo vic b gy phn t tn hiu HHL, v thuc nhm vi khun c phn hy HHL trung bnh. Gia Bacillus subtilis B20.1 c th tiu dit c E. ictaluri thng qua s cnh tranh sinh dng v c ch ln nhau. TI LIU THAM KHO Ti liu ting Vit Ng T Thnh, Bi Th Vit H (2007). Nghin cu hot tnh enzyme ngoi bo ca mt s chng Bacillus mi phn lp v kh nng ng dng chng trong x l nc thi. Tp ch khoa hc HQGHN, Khoa t nhin v cng ngh 25(2009) 101-106. Ti liu ting Anh Andrew E. Goodwin, J. Spencer Roy, Jr, John M. Grizzle, M. Terrell Goldsby, Jr (1994), Bacillus mycoides: a bacterial pathogen of channel catfish, Diseases of Aquatic Organisms, Vol. 18, pp 173-179. Balcazar, J.L., de Blas, I., Ruiz-Zarzuela, I., Vendrell, D., Muzquiz, J.L., 2004. Probiotics: a tool for the future of sh and shellsh health management. J. Aquacult. Trop. 19, 239242 Bengmark, S. (1998) Immunonutrition: role of biosurfactants, fiber, and probiotic bacteria. Nutrition 14:585-594. Bergeys manual determative bacteriology

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