Phân lập, định danh và xác định các đặc tính có lợi của chủng bacillus SPP từ ao nuôi tôm ở tỉnh bến tre

Tap chi KHOA HOC DHSP TPHCM Si; 64 flan? 2014 PHAN LAP, DINH DANH VA XAC DINH CAC DAC TINH CO LQI CUA CHUNG BACILLUS SPP AO NUOI TOM O TiNH BEN THE NGUYEN VAN PHOC., PHAN THI PHU'O'NG TRANG.TOM TAT Bacillus la nhom vi khuan có 10 hien dien da so cac che ph am vi sinh dung cho nuoi thily hai san dac biet la nuoi torn nu& to cac che pham vi sinh clang nuoi thily hai sem deu phai nh Op ' ngoai, gib cao Trong nghien cieu nay, nham phcin lap cac cluing Bacillus tit cac ao nuoi tom o tinh Ben Tre va sang loc, dinh danh, khao sat dac diem sinh trwang caa cac Chung mang cac dac tinh cc') l07 lam ca ser cho viec (mg dung san xucit che pham vi sinh Tie kh6a: Bacillus, che pham vi sinh, enzyme ngoai bao, Vibrio parahaemolyticus, ao nuoi torn, tinh Ben Tre ABSTRACT Isolating, identifying and determining the beneficial properties of Bacillus spp strains from shrimp ponds in Ben Tre province Bacillus is a group of bacteria that presents in the majority of biological products for aquaculture, especially for shrimp In our country, microbial products used in aquaculture have to be imported and at a very high price In this study, we isolated some Bacillus strains from the shrimp ponds of Ben Tre province and screened the beneficial properties for shrimp Potential Bacillus strains were classified and growth characteristics were investigated to serve as a basis for manufacturing biological products Keywords: Bacillus, biological products, extracellular enzymes, Vibrio parahaemolyticus, shrimp ponds, Ben Tre province Mir diu Ben Tre mOt cac tinh co dien tich nuoi tom khd 18n, th8ng ke len den 32.086 chiem 73% tong dien tich nuoi thily san, dat san lugng 47.397 tar ' vac) nam 2013 Tuy nhien viec nuoi tom v8i dien tich Ian va lien tuc clan den mat can bang sinh thai, nhiem nguOn nuorc lugng thirc an thira va chAt thai tir tom nuoi din den tom nuoi de nhiem cac benh, virus va vi khuAn gay ra, lam thiet hai nang cho nguiri nuoi tom Hien che pham vi sinh chira cac vi sinh vat co lgi dugc dung nhu mOt phuang tien de kiem soat dich benh bang each ire che, canh tranh vOi nhting sinh vat c6 hal, hi!) trg he tieu h6a, can bang vi Ichuan d0mg rutt [12] NgoAi the phatil cling dugc dung xir li ao nuoi bAng each phan 114 cac chat thong tieu h6a dugc, tilde an thira, dac biet IA rat than thien voi moi toting [7] Bacillus IA nhom vi IchuAn co ThS, Trung tam Khoa hoc va Cong nghe Sinh hoc TS, TrulYng Dai hoc Khoa hqc Tyr nhian, DHQG TPHCM 94 Tap chi KHOA HOC DHSP TPHCM Nguyin Van Phtic va tgk mat chii yeu cac the pham vi sinh vi c6 nhiing dac tinh c6 lqi nhu: (i) lam sach mai truang nha kha nang sinh cac loaf enzyme protease, amylase, cellulase, lipase phan huy cac hop chat him ca, (ii) kie'm soat su phat trien qua mirc,ciia cac vi sinh vat gay b8nh nhu Vibrio co the canh tranh nguen dinh &rang (iii) tit cac chat khang khuan, giir cho mei truang luon a tang thai can bang sinh h9c [4] Do dO chiing toi lien hanh phan lap Bacillus tir cac ao torn a Ben Tre nham tuyen ch9n nhang chung co cac dac tinh co Iqi de sir dung cac the pham vi sinh cho tom V4t ligu va phuong phap nghien cuv 2.1 V(it ligu 2.1.1 Doi ttrong nghien citu Chung Bacillus phan lap tir mAu dat, mau nuerc va mL torn cac ao torn tinh Ben Tre Chung vi sinh vat kiem dinh V parahaemolyticus &roc phan lap tir ao torn 138nh chet (duqc cung cap bai Trung tam Khoa hoc va C6ng ngh8 Sinh h9c, Truang Dai h9c Khoa h9c Tu nhien TP He Chi Minh) 2.1.2 moi trtrong sit dung nghien caw MOi truang LB: tryptone 10 g, cao nam men g, NaC1 g, nuac cat vira di: lit M6i truang kich thich tao bao tir DSM: KCI g, MgSO4.7H20 0,12 g, NaOH 0,06, pH = 7,6, mrac cat vira du lit Hap khir trung a 121°C, 15 phut Lam nguei den 50°C, be sung them cac dung dich: ml Ca(NO3)2 (1 M), ml MnC12 (0,01 M), ml FeSO4 (1 mM) Mai truang TSA khao sat dei khang val V parahaemolyticus: tryticase peptone 15 g, phytone peptone g, NaC115 g, nuac cat vira du lit Mai twang thach: phan nhu ten co b6 sung them 2% agar Cac moi twang ten dm:lc hap kha tiling a 121°C, 15 phiit truck sir dung 2.2 Phtrang phdp nghien cult 2.2.1 Phtrang phap pilau 10p va lam thu0n Loai b6 to bao sinh duerng bang cach den hanh gia nhi8t mAu be en nhi8t 80°C 10 phut Pha loang mAu den 10-2, but 100 µl dich gec va dich da pha loang trai ten moi twang LB - agar U a 37°C 24 gia ChQn khuan lac dac trung cho Bacillus va tin hanh lam thuAn bang each cAy ria ten moi truang LB - agar, cho tai quan sat thAy chi c6 met clang khan lac that tr8n moi tnaing [11] 2.2.2 Phuong phap dinh danh Bacillus De xac dinh cac chimg vi khuan thuc gi6ng Bacillus, mgt s6 thir nghi8m cho sang 19c buck dau duqc thuc hien nhu: nhuOm Gram [2], quan sat hinh thai to bao, nhuem bao tir [2], thir nghi8m catalase bang phuong phap sir dung dung dich H202 3%, thir nghi8m oxidase ten dia gi'a'y c6 tam N-dimethyl-para phenylenediamine, thir nghi8m kha nang di ding tren moi truang thach mem [3] 95 Tap chi KHOA HOC DHSP TPHCM Si; 64 nit nt 2014 Sau buck sang lgc tin hanh dinh danh bang phuang phdp giai trinh to 16S rRNA: Tach chiet be gen vi khuan bang b0 kit dm QIAgen, khuech dui trinh to 16S rRNA bang phan img PCR voi cap moi c6 trinh to nhu sau: 27F (5'-AGAGITTGATCMTGGCTCAG-3) 1492R (5'-TACGGYTACCTFGTTACGACTT-3') San phAm PCR dugc tinh the va giri giai trinh tu Cdc trinh to nucleotide hoan chinh dugc so sanh vbi ngan hang du lieu gen cua NCBI bang each sir dung cong cu BLAST 2.2.3 Burang phap nghien cfru ccic cla'c tinh có lai !Chi nang sinh enzyme ngoui bio: Khao sat kha nang sinh enzyme ngoai bao tren mei tnrarng LB — agar c6 bo sung ca chat thich hgp Te bao cua chimg Bacillus dugc nuoi cay mei truong LB long a 37°C 24 gib ChAm dich Bacillus sau 24 gib nuoi cay len dia m6i truong LB — agar c6 be sung tirng loai ca chit: 0,5% CMC (carboxy methyl cellulose), 1% tinh bet, 1% gelatin, 1% casein, 1% (v/v) ddu oliu tuang img cho khao sat khd nang sinh cdc enzyme cellulase, amylase, protease va lipase U cdc dia khao sat b 37°C 12 gib va quan sat vOng phan giai teen dia [3] Khi nang khfing viti Vibrio parahaemolyticus: Sir dung phucmg phdp cay vach thang vuong goc (Cross-streak) [8], [9] va phuang phdp khuech tan qua to thach [6], [10], de khan sat ac tinh doi khdng vOi vi khudn V paraheamolyticus 2.2.4 Phtrcmg pheip khao sat dijc tinh sinh trur'mg Kha nang chit' pH: Cdy cdc chimg Bacillus tuyen ch9n dugc mei truong LB dugc dieu chinh pH bang HC1 va NaOH theo cdc gid tri pH: 4, 5, 6, 7, 8, 9, 10 Nuoi cay lac a 37°C, OD600 xac dinh mat de vi khuAn sau 18 gib [5] Khi nang chin min: Cdy caz chimg Bacillus tuyen ch9n dugc mei tnrong LB be sung them NaC1 vbi cdc neng a: 0, 1, 2, 3, 4, 5% Nuoi cay lac 37°C, OD600 de xdc dinh mat de vi khudn sau 18 gig [5] Khi nang twang thich giira cfic ching tuyen ch9n: Sir dung phuang phdp cay vach thing vuemg gOc Ket qui va thio luin 3.1 Phdn 1(ip vet sang 1pc so bp' chang Bacillus Tir caz m'Au &at, mau Junk, mdu tom c6 ki hieu tucmg Ong la BD, BN, BT lua ch9n dugc 70 chimg c6 cac dac di6'm hinh thai khuAn lac ging ved Bacillus nhu: IchuAn lac tron, r1a rang cua khong au, mau yang xam (hinh 1) Cac chimg tiep tuc dugc lam thudri tren mei truorng LB — agar va 'cep vao clang khuan lac nhu hinh 96 Tap chi KHOA HOC DHSP TPHCM Nguyin Van PI !tic va tgk Hinh Hinh dang cac khudn lac ph& kip tren WO' Throng LB Hinh clang khudn lac ddc dwc lam thucin tren mot trufmg LB Tir 70 chi:mg c6 hinh clang dd'c tnmg dm Bacillus tienhanh met so thin nghiem de sang 19c so bO thong qua cac ddc diem can thiet dm Bacillus (te bao hinh que, c6 sinh bao tir, Gram duong, catalase duong tinh, oxidase duong tinh va c6 kha nang di &Ong) ch9n duqc 61 chimg 3.2 Khdo sat cac d#c tinh a 10 caa chang Bacillus phan dwpr 3.2.1 Khci Wing sinh enzyme ngogi bao Cac vi sinh vat cac the phdm thuong co kha nang tiet cac enzyme ngoai bao de phan hOy cac chdt can bA, thirc an thira ten de'ng ao, han che kha nang gay benh cua cac chi:mg Vibrio Do vay, cac chung da ducc phan lap va sang 19c so b0 se duqc tien hanh khao sat kha nang tiet enzyme ngoai bao nhu amylase, cellulase, protease, lipase ten cac did LB — agar c6 bo sung co chat thich h9p Sir dung cac thuOc nhuOm lugol, congo, TCA tuong von co chAt tinh bet, CMC, gelatin de hien Ong phan giai tren dia thach NhUng chimg co kha nang sinh enzyme ngoai bao xuat hien yang phan giai xung quanh khudn lac (hinh 3) 97 Tap chi KHOA HOC DHSP TPHCM So 64 nain 2014 4111111k Hinh 3: Jiang phan giai tinh b5t (1), cellulose (2), gelatin (3), casein (4), lipase (5) clic: cac chUng Bacilhis phan lap dux Da s6 cac chung Oran lap dugc deu c6 kha nang sinh enzyme amylase Trong c6 chAng BT21.1, BD17, BD22.2 kha nang phan giai tinh Not manh nhAt voi ban kinh vong phan giai Ian nhAt mm Tuang to nhu tray, chAng BT21.1, BT22.1, BD23.1 c6 kha nang sinh enzyme cellulase math vOi ban kinh yang phan giai la 6,25 — mm Tat ca cac chAng deu c6 kha nang tiet gelatinase va caseinase Trong co chAng BT15.2, BT24.2, BT21.2, BT22.2, BD23.1 cho ban kinh yang phan giai gelatin lOn nhAt la - 7,75 mm VA chung BN12, BD1, BN3, BN5, BN8, BNIO c6 ban kinh yang phan giai casein lan nhAt la 6,5 — mm Rieng kha nang sinh enzyme lipase thAp, chi co chAng co kha nang tiet enzyme lipase nhtmg ban kinh yang phan giai chi mm TA ket qua tr'en cho thAy cac chAng Bacillus phan lap dugc co kha nang tiet cac loai enzyme ngoai bao b cac mAc dq khac Cac chAng tiep tic dugc khao sat kha nang doi khang vii V parahaemolyticus 3.2.2 Kha nang den khang V parahaemolyticus V parahaemolyticus la vi khuan gay benh eh& sant tren torn gay thiet hai lan cho nganh nuoi tom ca nuck not chung va tinh Ben Tre not rieng Do tien hanh thi nghiem khao sat de tuyen chon nhang chAng c6 kha nang khang V parahaemolyticus nharn huong ten Cmg ding cac the pharn vi sinh cho tom Khao sat kha nang Mi khang ctia 61 chung tuyen chon bang phuong phap vach ding vuong goc va dpc16 thach Veri phucmg phap vach thing vuong goc chung CO doi khang duAng cay ctia chiing V parahaemolyticus moc cach xa dtrong cay chung Bacillus khao sat (Hinh 4A) \Teri plurcmg 014 chic 16 thach chAng c6 d6i khang xung quanh 16' thach dugc nho dich nuoi cay vi khuan Bacillus c6 yang vo khuan (Hinh 4B) Hinh Ket qua dii khang ctia chting BN1 A Phumg phap vach thing vuong g6c;B Phutmg phap duc to thach 98 Tap chi KHOA HOC DHSP TPHCM Nguyin Van Ph tic va tgk Ke't qua khao sat 61 chang cho tha'y c6 chang c6 d8i khang vai V parahaemolyticus la BN1, BT15.3, BT21.1, BT24.2, BD23.1 Qua ket qua khao sat kha Pang sinh enzyme ngoai bao va &Si khang vai V parahaemolyticus ch9n doge 17 chang Bang CO kha nang sinh it nhat enzyme ngoai bac) Trong chang BN1 va BD23.1 ductc ch9n dal dien de dinh danh den mac la bang phucmg phap giai trinh to 16S rRNA Vi chang c6 kha nang Binh hau het cac loci enzyme ngoai bao khao sat va c6 kha nang doi khang vai V parahaemolyticus 3.3 Kit qua dinh dank bdng phwang phcip giai trinh tot 16S rRNA 138 gen oh hai chang tuyen ch9n la BN1 va BD23.1 ducic tach chiet Trinh tir 16S rRNA tren bi) gen dugc nhan ban bang phacmg phap PCR va chay dien di tren gel agarose 1% kiem tra ket qua (hinh 5) Tren hinh cho thy a gieng BN1 va BD23.1 da thu nhan ducfc doan trinh to DNA (— 1500 bp) ma h6a cho 16S rRNA caa chang BN1 va BD23.1 Wing trinh tir 16S rRNA dirge giai trinh ttr va so sanh (18 tuang d8ng di truyen vai cac loci tren ngan hang gen NCBI bang cong cu BLAST Ket qua &cm trinh bay Bang a M BN1 BD23.1 by 20000 7000 4000 2500 1500 1000 700 400 200 Hinh Kit qua dien di tren gel Agarose 1% san phdin PCR rang 16S rRNA cluing BN1 va BD23.1; M: thang DNA 99 Tpp chi KHOA HOC DHSP TPHCM So 64 nam 2014 Bang Tang hop ket qua vong phcin giai ca chat cart enzyme ngoai bao via dai khang v&i V parahaemolyticus Diii khang Vach thing Due to yang goc thach (mm) (mm) Amylase (mm) Casein (mm) Gelatin (mm) Cellulase (mm) Lipase (mm) BN12 BDI 0,5 6,5 6,5 3,25 3,5 4,5 0 0 10 11 12 13 14 15 16 BN3 BN5 BN8 BNIO BT15.2 BT15.3 BT21.1 BT21.2 BT22.1 BT22.2 BT24.2 BD17 BD22.2 3,5 2 0,5 3,5 2,25 4,5 4,5 6,5 6,5 6,5 3,75 3,5 5,5 0,25 5,5 2,25 1,5 2,75 5,5 4,25 6,25 7,25 5,5 1,5 7,5 7,75 4,5 3,25 4,5 6,25 0,75 0 1 0 0 1 0 0 0 0 0 0 0 0 Ki hi?u chfing STT Bang Tom tat Ic- t qua dinh danh hai chung BN1 va BD23.1 Max score Query Coverage E-Value Max Identities Idt loan BN1 2494 100% 0,0 100% B subtilis BD23.1 2508 100% 0,0 100% B subtilis Chung Dua tren ket qua phan tich trinh to vang 16S RNA xac dinh dugc chang BN1 va B23.1 la chang B subtilis So sanh giaa chang BN1 va BD32.1, chang BN1 la chang doi khang v6i V parahaemolyticus manh COn chang BD23.1 la chang tiet enzyme ngoai bao manh hon Nhu vay chang nen dugc khao sat tier) theo de ph6i trgn che pham vi sinh 3.4 DOc tinh sinh truiing ceta chiing Kha Jiang chit.' pH va chit' man: tir ket qua ten D6 thi chO thay chang BN1 va BD23.1 c6 kha nang chlu dugc pH tir - 9, n6ng ci(5' mmSi to - 5% Va a pH 7, 100 ATglOn Van Pinuc va tgk Tap chi KHOA HOC DHSP TPHCM n6ng d6 mu6i tir — 2% la dieu kien sinh truang t6i uu So sanh vai khoang pH trong,dieu kien ao nuoi torn nuac 1g a nu6c ta la 7,5 - 8,2 va nemg dO mue>i la 1,5 — 2,5 % [I], dieu cho they chung BN1 va BD23.1 deu co kha nang chiu khoang pH va n6ng d'6 mu6i rOng phis hop vai dieu kien ao nuoi torn nuac ta, do, thich hop de lam the pham sinh hoc nuoi torn Ket qua phir hop vai du loan vi cac chimg vi sinh vat dugc phan lap chinh tir cac ao nuoi torn 12 a pH4 • pH5 a pH6 pH7 • pH8 • pH9 BN1 A BD23.1 Chimg • pH10 BD23.1 BN Chong flo thi OD 600 nrn cue hai chting BN1 va BD23 a cac nang dO pH (A) va njng dO muoi NaCI (B) khcic sau 18 gia nuoi coy Kha nang ttrcrng thich cue hai chfing tuytn chip: Cac the pham sinh hoc thuomg dugc sir dung duai clang lion hop cac chimg vi khuan nham irng dong town b¢ cac tiem nang cue tong chimg cfing nhu dam bao tinh thich nghi va hieu qua moi diet' kien moi tnr6ng Do do, viec tien hanh kiem tra tuong thich gifra cac chimg tuyen chon nham c6 co sof phi trOn phis hqp Phuong phap dugc tien hanh nhu da mo ta phan ten Ket qua the hien Hinh Duang cay hai chung BN1 va BD23.1 IncIC sat chimg to hai chimg CO kha nang tuong thich vai Hai chung BN1 va BD23.1 co kha nang Ichang vi khuan V parahaemolyticus nhung Ichong Ichang vai nhau, chimg co kha nang tuong Su turmg thich gifia hai cluing thich vai nen chung hoan town CO the ph6i Hinh BN1 va BD23.1 sau 18 gia nuoi cay trOn vai the pham Ket Juan Tir 70 chimg phan lap dugc cac mau &At, mAu ntrac, m'au torn thu dugc tir 31 ao nuoi torn a BenTre sang loc so b6 chon dugc 61 chung co cac dac diem cira Bacillus Trong dO tuyen chon dugc 17 chung c6 kha nang sinh enzyme ngoai bac va 101 Tap chi KHOA HOC DHSP TPHCM Si) 64 nam 2014 kha rang khang vai V parahaemolyticus Rieng hai chfing BN1 va BD23.1 có kha nang khang vai V parahaemolyticus va sinh enzyme mann nen dugc dinh danh va phan tich ket qua djnh danh cho thy hai chimg thuOc B subtilis Hai chimg BN1 va BD23.1 có (lac diem sinh truemg phi" hgp vai dieu kien nuoi tom Cr nuac ta nhu khoang chili pH (5-9) va chili man (0-5%) rOng , ngoai hai chfing tu.cmg thich vai va dac tinh có 19i dm timg 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of probiotics in aquaculture", Veterinary microbiology, 114(3-4), pp.173-186 BalcAznr, J.L and Rojas-Luna, T (2007), "Inhibitory Activity of Probiotic Bacillus subtilis UTM 126 Against Vibrio Species Confers Protection Against Vibriosis in Juvenile Shrimp (Litopenaeus vannamei)", Current Microbiology, 55(5), pp.409-412 Chythanya, R., Karunasagar, I and Karunasagar, I (2002), "Inhibition of shrimp pathogenic vibrios by a marine Pseudomonas-2 strain", Aquaculture, 208(1), 1-10 Mishra, V and Prasad, D.N (2005), "Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics", International Journal of Food Microbiology, 103(1), pp.109-115 10 Purivirojkul, W., and Areechon, (2007), "Application of Bacillus spp isolated from intestine of black tiger shrimp (Penaeus monodon Fabricius) from natural habita for control pathogenic bacteria in aquacult", Kasetsart J (Nat Sci), 41, pp.125-132 11 Vaseeharan, B and Ramasamy, P (2003), "Control of pathogenic Vibrio spp by Bacillus subtilis BT23", a possible probiotic treatment for black tiger shrimp Penaeus monodon, Letters in applied microbiology, 36(2), pp.83-87 (Ngay Tda soon nh$n ettioc bat 13-8-2014; ngdy phan bin danh gia: 18-8-2014; chip nhin ding: 21-11-2014) 102 ... cfru ccic cla'c tinh có lai !Chi nang sinh enzyme ngoui bio: Khao sat kha nang sinh enzyme ngoai bao tren mei tnrarng LB — agar c6 bo sung ca chat thich hgp Te bao cua chimg Bacillus dugc nuoi... thucin tren mot trufmg LB Tir 70 chi:mg c6 hinh clang dd'c tnmg dm Bacillus tienhanh met so thin nghiem de sang 19c so bO thong qua cac ddc diem can thiet dm Bacillus (te bao hinh que, c6 sinh bao... ttrong nghien citu Chung Bacillus phan lap tir mAu dat, mau nuerc va mL torn cac ao torn tinh Ben Tre Chung vi sinh vat kiem dinh V parahaemolyticus &roc phan lap tir ao torn 138nh chet (duqc