Tgp chi Khoa hpc TrudngDgi hgc Cdn Tha Phdn B- Ndng nghiip. Thuy san vd Cdng ngAf Sinli hpc: 35 (2014): II1-1^
Tap chl Khoa hpc Trirdng Dai hoc Can Thd website: sj.ctu.edu.vn
ANH HU'OfNG CUA HON HOfP VI KHUAN BACILLUS CHON LOC LEN LUAN TRUNG NU'dC LO BRACHIONUSPUCATILIS Phan Thai Tuyit Anh' va Pham Thi Tuyit Ngan'
' Khoa Thuy sdn, Truong Bgi hgc Cdn Tho Thong tin chung:
Ngdy nhdn: 07/03/2014 Ngdy chdp nhdn: 30/12/2014 Title:
Influence of selected mixture Bacillus bacteria on brackish rotifer Brachionus plicalilis
Tirkhda:
Brachionus plicalilis.
Bacillus sp., cdm nhiim Vibrio harveyi Rewords:
Brachionus plicalilis, Bacillus. Vibrio harveyi challenging
ABSTRACT
The aims of this research were to study the impacts of mixed selective Bacillus sp bacteria on the growth of rotifers Brachionus plicalilis. The first experiment was investigated the effects of a mixture of Bacillus sp and probiotic prodOcfs"
on rotifer populations with two treatments and one control: (I) No adding bacteria for the control treatment. (2) Adding a mature of Bacillus B7 + B41 (Bacillus amyloliquefaciens), (3) Adding Pro W (probiotic products) to cuture rotifer medium. In the second experiment, tifter accomplishing the first experiment, rotifers from each treatment were infected by Vibrio harveyi to determine the survival rate after the infection. Results showed that the density of rotifers, carrying eggs in the treatments added Bacillus was significantly higher than those in the control (p<0.05). In addition, the highest results were obtained from the treatments with mixture of Bacillus B7 + B41 (Bacillus amyloliquefaciens), this Bacillus strain possibly overwhelmed Vibrio bacteria.
The rotifer productivity has been improved when adding Bacillus as well as microbial products into the culture system. Rotifers in the medium adding Bacillus can maintain a higher survival rate under the infection of Vibrio harveyi bacteria, but the difference was not statistically significant (p>0.05).
TOM TAT
Nghiin cuu dnh hudng cua hdn hgp vi khudn Bacillus sp chgn lgc lin tdng trudng cua ludn triing nude lg Brachinonus plicalilis dugc nghiin ciiu. Thi nghiim I, khdo sdt dnh hudng ciia hdn /ipp vi khudn Bacillus sp'vd'che^pl^mj'i^
sinh lin qudn thi ludn triing. thi nghiim dugc bd Iri 2 nghiim ihuc vd mdt doi chiing: (1) Boi chiing (khdng bo sung vi khudn); (2) Bd sung hdn hgp vi khuan Bacillus B7 + B41 (Bacillus amyloliquefaciens); (3) Bo sung che pham vi sinh Pro W lin ty le sdng ly le ldng Irudng cua vi khudn. Thi nghiem II. smi khi kit thuc thi nghiem I ludn triing d timg nghiem thuc se dugc gdy cdm nhiem vdi vi khudn Vibrio harveyi, xdc dinh ly li song cua ludn trimg sau khi gdy cdm nhiem. Ket qud cho thdy mdt dg ludn trimg vd cd thi ludn tning mang triing d cdc nghiem thuc bd sung vi khudn cao hem co y nghfa thdng ke so vdi ddi chiing vd dgt gid tri cao nhdt d nghiem Ihiic bd sung hdn hgp vi khudn B7 + B41 (Bacillus amyloliquefaciens), vi khudn Btjcillus cd khd ndng ldn tk Vibrio.
Ndng sual ludn trung dd dugc cdi thien khi bd sung vi khudn Bacillus cSng nhu chi phdm vi sinh vdo he thdng nuoi. Ludn trung d nghiim ihuc bd sung Bacillus cd thi duy tri ty 14 sdng cao han khi gdy cam nhiim vdi vi khudn
Vibrio harveyi, tity nhien khdc biet khdng coy nghia thdng ke (p>0.05).
Tgp chi Khoa hpc Trudng Dgi hpc Can Tha Phdn B: Ndng nghiip, Thdy sdn vd Cdng nghi Sinh hpc: 35 (2014): 11 I-l 20 Kit qua cho thay nhiiu ddng vi khuin cd tac dung tdng nang suit nudi ludn tiling do ldch toich qua trinh sinh sdn vd tinh, iam tottc an true tiip hoac tang cuong hip tou dinh duong. Do vgy, muc tiiu cua nghiin cttu nay nham danh ^ a anh hudng ctta cac Ioai vi khudn hihi ich Iin quin toi luan triing todng qua chi tiiu tang trudng vd ti Ie mang trttng cua ludn trimg frong phdng toi n ^ e m va kiem tra khd nang cluu dimg ctta luan triing khi cdm nhidm vdi vi khuan gay binh Vibrio, di Iam co sd ttng dung vdo san xuat, nang cao ndng sudt vd chdt Iugng luan friing.
2 PHU'ONG PHAP NGHIEN CUtJ 2.1 Vdt liiu aghjea cfru 1 GIOI THIEU
Trong sdn xuit gidng thi tottc an va ky thuat cho an troog khi uang iu triing Id van de rdt quan frpng vi v4y thttc dn tg nhiin ddng vai frd rdt quan frpng quyit dinh su toanh cdng ttong uong nudi nhiiu loai ddng vgt touy san (Trdn TM Thanh Hiin vd ctv., 2007).
Luan tifrng nude Ig {Brachionus plicalilis) dugc nudi vd su dung ttong san xuit gidng ciia hon 60 lodi ca biin vd 18 loai giap xdc (Nagata, 1989).
Nhd cd kich toudc nhd, boi ldi chdm chap, sdng la Ittng ttong nude lam cho luan triing fro todnh con mdi toich hgp cho du trimg cac Ioai ca vd gidp xdc bien cd kich toudc miing nhd (Snell va Carrillo, 1984). Hon nua, do dgc diim dn Ipc khdng chpn Igc nen luan triing cd toi dugc giau hoa bing cac chat dinh dudng. can thiit hay Idiang sinh di dua vao ca toi au triing nudi (Lubzens et al., 1989). Vi vdy, ludn triing da ttd toanh ngudn touc dn tuoi sdng khdng toi thiiu ttong sdn xuat gidng ciia nhieu loai giap xac vd cd biin.
Cdc nhd i^hiin cttu ttt lau da quan tam den viec i^hiin cttu dgc diim sinh hgc, k^ toudt nudi mdt sd Ioai tottc an tuoi sdng cho du trimg ddng vdt touy san. Quan tam cac ddi tugng chtt yiu nhu: vi tdo, ludn ttdng, gidp xdc rau nganh, Artemia, triing chi (Trdn Thanh Hiin vd ctv., 2007).
Nh|it Bdn Id noi dau tien touc hien nghien cttu nudi sinh khoi ludn trung vdo nam 1964 (Hirala, 1979; ttich tiir Trin Cdng Bmh, 2006). 6 Trung Qudc, ndm 1980, cac nghiin cftu vi ludn trimg lam tottc an cho au triing ca bien dugc tien hanh (Chen, 1991; trich ddn ttt Trdn Thi Thanh Hien vd ctv, 2 0 0 7 y 6 H M ^ , ndra 1971, Theilaccker va i^^Iv^^^dafcdIlg bd'Idiifdiu tiin kit qua nghien
cttu vi Brachionus plicalilis la tottc an myet vdi cho iu trung ca biin (Wendy vd Kenvan, 1991).
Tuy nhiin, din nay thi ludn trung vin dugc nudi d qui md thi nghiim, chu yiu phuc vu cho uong nudi cdc loai Mullet, ca mang Pacific threatfin vd mahimah. Red drum, ca chem trang vd California halibut. Sdn Iugng nudi mdi ngay touong dat 100- 500 ttiiu con, ndng suit trung binh 25,7-75 ca toi/ml/ngdy.)-
Hino (1991) cho ring su toay ddi cua quin toi vi khuin hay su nhilm tgp cua cdc Ioai khac se dnh hudng din qud trinh nudi luan triing. Cac logl vi kbuin ttong bi nudi luan triing pbat Uiin se cd nhung dnh hudng cd lgi hay cd hgi liiy touOc vdo loai vi khuan. Cdc nghien ciru nay chtt yiu dua trin tdc dOng dinh dudng cua vi khuin din vdt nudi.
Dung cu vd trang thiet bi thi n ^ i m bao gdm ludi Igc cd cac kich toudc mil ludi khac nhau (30, 60 vd 300 |im), ndi hap tiit triing kinh Iftp, mdy do pH, may lac, h^ todng sue khi... Cac trang thiit hi tou va phdn lich miu vi sinh, chat Iugng nude. Hda chdt: Glutaraldehyde 50 ppm, dung djch Lugol, Chlorine, KI, toiosulfate natii (Na2S203). Mdi irudng chuyin biil cho Bacillus, Nuttient Agar (NA) va Thiosulpbate Citrate Bile Sucrose Agar (TCBS). Ngudn nude ngpt dugc lay ttt ngudn nude may, vd nude dt (10096o) cd ngudn gdc tii mdng raudi VTnh Chau, tinh Sdc Trang. Nude diing cbo nudi ludn triing (25%o) dugc pha tfr 2 ngudn nude Uin. Nude sau khi pha dupe xfr Iy bang chlorine (30 ppm) trong 24h. Sau dd diing KI de kiim tra bam lupng Cio trudc khi dua vao sfr dung. Neu cdn chlorine thi ttung hda bing toiosulfate natti (^aiSiOs).
Luan tning cd ngudn gdc tfr truong Dgi hpc Gent, Bi dugc nudi giu gidng tgi Phdng toi nghiira nudi tofrc an tu nhien toudc BO radn Tlifty sinh hpc LTng dimg. Ludn triing dugc nhan gidng 1 toang trudc khi tiin hanh toi nghiem. Cac ddng vi khuin Bacillus touan gdm B7, B41 {B. amyloliquefaciens) cd ngudn gdc ttt ao nudi tdm stt huyen Vinh Chdu, Tinh Sdc Trdng (Phgra Thi Tuyit Ngdn vd Nguyin Hiiu Hiip, 2011). Tao Chlorella dugc stt dung cho ludn triing an vdi Iugng 100.000 te bao/Iuan Ufrng/ngay dim (Tran Suang Nggc va Nguyin Hdng Ldc, 2006).
2.2 Pbutmg pbap nghiio cfru
2.2.1 Phuong phdp sdt trimg ludn trimg de tgo con non vo tnmg
Di cd dugc m^t sd ludn trimg mong mudn vd iy If luan triing mang tning cao, trudc khi bd tri tbi nghiem ludn trung dd dugc nhdn gidng bing each nudi frong cac keo nhua 10 L, vdi mat dO nudi ban
Tpp chi Khoa hpc Tmdng Dgi hpc Cdn Tha Phdn B: Ndng nghiip, Thdy sdn vd Cong nghi Sinh hpc: 35 (2014): 111-126 Thi n^iem dugc touc hien 4 chu ky nudi, moi chu ky keo dai 4 ngay. Sau mdi chu ky nudi thu lai ludn trimg frong cung mOt nghiim tottc vd nudi mdi hodn todn. Ngudn giing cua chu ky thtt_2 ldy tii chu ky tott 1, va tiip tuc nhu vay cho din chu ky 4.
dau la 250 — 300 ca toi/mL. Luan triing dugc cho dn bdng tao Chlorella. Sau khi ludn trimg da mang trttng tiin hdnh sat trimg luan triing di dugc du triing vd trimg toeo qui frinh da dugc ttng dyng tai Tmng lam Khao cttu Artemia, Khoa Ndng n ^ i i p , Tmdng Dai hpc Gent, Bi (Ngjyen Thi Ngpc Tinh etal.,20\6).
Chit khtt trung dugc stt dung: Glutaraldehyde, ndng dO stt dyng 200 ppm, thdi gian sat ttiing 1 gid frong nude biin 25%o da dugc tiit triing. Qui tiinh touc hien nhu sau: Trttng amlctic ttt ludn trimg raang ttftng da dugc dim va tinh loan de cd dugc mat dp ludn trimg mong mudn ban ddu toeo nhu ciu cua thi nghiim. Sau dd ludn triing mang trftng dugc riia sgch vdi nude bien 25%o, Ipc qua ludi cd kich thudc mdt ludi 300 pm sau do rtta sach lgi nhiiu lin qua ludi 60 pm. Cd dgc ludn trimg tmdc khi tiin hanh cho dung dich glutaraldehyde 200 ppm vao. Sau 1 gid, ludn triing trong moi ong si dugc Ipc qua ludi 60 nm nhdra logi bd xdc luan triing ds chit sau dd Ipc vd rtta Igi nhiiu Idn qua ludi 30 yra di gitt lai lit ca cac trimg amictic, tning dugc ip trong chai chtta 250 mL nude 25%o vd dugc ddt ttin may lac. Sau 3 gid cac trftng amictic dd dugc nd ra, de yin trong khodng I phut de cho luan triing dd cbit ling hit xudng dudi day.
Tiin hanh thu phin trin dd vdo chai mdi, xdc dinh si lugng ludn triing mdi nd vd tiin hanh bd tti toi nghifm.
B6 tri thi nghl$m
Thi nghiim 1: Danh gia dnh hudng cila vi khuin Bacillus lin su tdng trudng cua quan the luan ttung.
Ludn trung vo triing dd dugc bd tti ttong cac ing falcon 50 mL vdi mat do ban dau la 30 cd toi/mL va dugc ddt tten may lie. Thi nghifm gdm 3 nghifm tottc dugc bd tri hodn lodn ngau nhiin, mdi nghidm tottc 3 ldn ldp lai. Cdc nghiim thttc dugc bd tti nhu sau:
- Nghiem tottc I: Khdng bd sung vi khuin (ddi chttng: DC).
Nghiem tottc 2: Bd sung vi khudn B7+ B41 {B. amyloliquefaciens), mat dO 10'CFU/mL
- Nghiim tottc 4: Bd sung chi phim vi sinh Pro W mgt dO 10'CFU/mL.
Thanh phdn ciia chi phdm vi sinh Pro W ttong I g:
Bacillus subtilis 2.5 x 10'^ CFU/g Bacillus licheniformis 2.5 x 10'^ CFU/g
Thi nghiim 2; Gdy cam nhiim luan ttiing vdi Vibrio sau khi bd sung Bacillus.
Quin toi luan triing d chu ky 4 dugc tou vao cudi toi nghiira I, ^Q riing luan trung theo 4 nghiira thttc riing biet. Bi tii toi nghiim nrong tu nhu thi nghiim I (2 nghiem tottc va 1 ddi chttng), nhung d 2 nghiim tottc va ddi chttng diu dugc bd sung vi khuan V. harveyi, mat dp gay cdm nhiem la 10' CFU/mL, toi nghiim dugc touc hien ttong 6 ngdy.
2.2.2 Cdc chi tieu theo doi
Chi tieu nhiet do va pH dugc do cuoi mdi chu ky nuoi. Cac yeu td NO2, TAN dugc do 4 ngay/lin; chi tieu vi khuan (vi khuin tdng cgng, Bacillus va Vibrio) cuoi moi chu ky nudi va chi tieu sinh hoc (sd luan triing, ty le luan trttng mang trung) dugc xac dinh mdi ngay.
2.3 Phuong phdp thu thdp, tinh toda va xfr ly s i li^u
2.3.1 Chi tiiu mdi Irudng vd vi sinh Nhiit dO dugc do bang nhiit ki thtty ngdn. pH do bdng may do pH. Cdc chi tieu NO2 va TAN dugc do toeo phucmg phdp Indo - phenol blue va Dianozium (APHA, 1995).
u. Phuongphdp nuoi ldng sinh vl khudn Vi khuin Bacillus B7, B41 dd trtt d phdng vi sinh dugc ciy tmyen ttin mdi trudng chuyen biil cho vi khuin Bacillus hogc mdi trudng TSA hodc NA, sau do dimg que ciy liy mpt it khuin lac ciy vao ong nghiem cd chtta radi tmdng TSB, di lin may Idc d nhiet dO 30°C. Sau 24 gid thdy dng nghiim chtta dung dich vi khudn chuyin sang mau hoi dye. Chuyin 5 mL dung djch chtta vi khuin sang binh tara giac cd chtta 450 mL mdi tmdng Idng (TSB). Sau 24 gid thu dugc 500 mL dung dich chfta vi Idiuin cin nudi tdng sinh.
b. Phuang phdp phdn tich mdu vi sinh trin mdi Iruang thgch
- Phuong phdp xdc dinh mat dO vi khuin Bacillus bdng phuong phap dim khuin Igc (Nguyen Ldn Dung 1983)
Tgp chi Khoa hgc Tmdng Dgi hpc Cdn Tha Phdn B: Ndng nghiip. Thuy sdn vd Cong nghi Sinh hpc: 35 (2014) • 111-120 Phucmg phap xac dinh mat dp vi khudn tdng
cOng va Vibrio bdng phuong phap dim khin Igc (Baumannera/., 1980)
2-4 Chi tieu stab bpc
Luan trung dS Aaac dira mii ngay di xac dinh tj If song. M|t dd luan trimg dugc xac dpto hang ngdy bing each stt dung micropipet 100 pL, cd dinh va nhuOm man bang Lugol. Sau do dim tten kinh Iftp, khdng dim nhttng con khdng bit mau Lugol (luan tiling chit). Ty Ii sing (%) = (Nc/No) x 100 (Nc: s i ludn triing Ittc kit touc thi nghiim; N,,:
Sd luan triing lftc bat dau toi nghiim). He sd ttttng:
HST(%) = n/N (n: Sd ca toi ludn trung mang trftng N: Tdng sd ludn triii^ dim).
2.5 Xfr Iy so lieu
Sd lieu da tou toap dugc xtt Iy so bO vdi chuang frinh Excel va xfr 1^ todng ke bdng phin mim Stalislica 5.0; stt dung phep tott LSD. So sanh sg khac biit giiia cdc nghiem thttc (ANOVA).
3 KET QUA VA THAO LUAN 3.1 Thi nghiim 1: Khao sdt aab hudag ciia vi kbuan Bacillus lin su tdag trumig cua quan tbi luda triing
3.1.1 Bien dgng cdc yiu td mdi irudng u Nhiit dgvdpH
Nhiit dp giiia cac nghiim tottc co bd sung vi khuan vd khdng bd sung vl kliuan frong thi nghiira khac biit khdng ^ nghTa (p>0,05), nhiet dO it bien dOng, nhift dp dao ddng khoang 25,3 - 29,5°C.
Nhiet dp dao ddng khdng dang ke la do toi nghiira diu dugc bd fri frong phdng toi nghiem vd ciing diiu kiin mdi tmdng vi vgy sg tdc ddng cua nhiit dp ben ngodi vdo Id nhu nhau. Theo Fulks (1991) nhiit dp dao dOng tiiich hgp cho luan triing la 20 ~ 30''C vi vgy rtoift dp frong toi nghiem ndm trong khodng toich hgp cho su phdt frien cua ludn trttng.
pH cd khuynh hudng gidm trong cdc chu kJ nudi do stt dung tottc dn hodn todn Id tdo nin d cudi vu lupng tao du toira va bat dau phan htty se lam tang lugng COl sS ldm giam pH, do gia tri pH phu thuOc
vao itoieu yeu td ttong do sg hdp tou ngudn dgm khac rtoau cttng dan din su biin ddi pH cua mdi tmdng cd tao.
b. Tdng dgm ammon TAN
Kit qua biin dOng ham lugng TAN d toi nghiim dugc ttinh bdy d Hinh I cho toiy d cdc nghiem tottc ham lugng TAN diu co khuynh hudng tang nhanh va cao, d nghiera toftc DC cd ham lugng TAN trung binh thap nhit (18,13 ± 6,8 rag/L) va cao nhit la d nghi|m tottc B7 + B41 (21,54 ±11,8 mg/L) khdc bi|t cd y nghia toing ke gitta nghiim tottc ddi chftng vd cac nghiim toftc cdn Igi qua cac chu ky tou mlu (/7<0,05). Ci chu k^"
2 hdm Iugng TAN d nghifm toftc DC toip nhit (1,12 mg/L), cao nhit d chu ky 4 nghiem tottc B7 + B41 (29,64 mg/L). Cang vi cuii toi nghiim tiii hdm lugng TAN cdng tang din do qua ttinh ti'ch luy dinh dudng TAN la ting ham Iugng NHj*, NH3 cd ttong mdi tmdng nude dugc gpi Id tdng dgm amon, cd liin quan den m$t so cdc vi khuan chuyin hda dgm. Do mat dO nudi ludn triing d cuii chu ky cang nhieu thi sd luong cho an cang cao khi dd se cd su tich ty va phdn hdy tottc an du toira cung nhu chit todi cua luan tning se lam ldng ham Iugng TAN trong toi nghiim.
Nhin chung lugng TAN d nghiim thttc cd bi sung 10' CFU/mL vi khuan Bacillus ciing nhu clie phdm vi sinh cao hon nhiiu so vdi nghiem thttc ddi chung. Nhu vgy, d nghiem thttc cd bd sung vi kliudn Bacillus, che pham vi sinh vdi mgt dp cao da toam gia phdn hCiy vdt chit httu ca chuyin sang NH4*/NH3 lam cho ham Iugng TAN tang mpt cdch ddng ke. Theo Huata vd Nagata (1982) chat todi vd chdt bai tiit cua luan triing phan Idn la ammonia dudi. dang^gaJan-chu-veiLla-ammonia va ure nin khi rridFdp^lttdlSaTmg-t^g-eao thi TAN cao. Trong radi chu I^ nuoi hoan toan khdng tiiay nude va toftc an cho luan trimg Id tdo nin mdt phdn ion NH4* (mdt dgng phdn dam ua chuOng ciia thuc vat) da dugc tdo hip thu, nen TAN luon d mttc cao vd tdng din vi cudi thi nghiem.
Tgp chiKhoa hpc TrudngDgi hoc Cdn Tha Phdn B: Ndng nghiip, ThOy sdn vd Cdng nghi Sinh hpc: 35 (2014): 111-120
Hinb 1: Ham l u g n g TAN trung biab r- Nitrite (NOz)
Kit qud thi nghiem trinh bdy d Hinh 2 cho thiy, ham Iugng NO2" d chu ky 4 cao ban d chu ky 1 va chu ky 2,3. Ham Iugng NO2' tmng binh d cac nghiim tottc dao ddng ttt 0,03 - 0,35 mg/L. Hdm Iupng NO2' d chu ky 3 d nghifm toftc Pro W khdc biet cd y nghia todng ki so vdi DC (dii chftng) Q7<0,05), nghiim tottc Pro W idn ban cd y nghia thing ki (p<0,05) so vdi nghiera thttc B7 + B41, d
fr cac chu ky nudi troag tbi aghiem
chu ky 4 khac bift cd y nghia toong ki giiia nghifm thttc B7 + B41 vdi cac nghiem tottc cdn lai (p<0,05). Do cdc thttc dn du thtta va chit thdi cua luan trimg nin ham Iugng cac chit dinh dudng gie dgm cd khuynh hudng gia lang vdo cudi toi nghiim. Hdm lugng nitrite trung binh cda tdt ca cdc nghiim thttc la 0,14 mg/L hoan todn khdng gdy hgi ddi vdi luan ttung. Theo Groeneweg vd Schluter (1981), toi ham lugng NOj" tii 10 - 20 mg/L tiii khdng gay ddc cho ludn trung.
C l n K y l Cfai1iy2 ClMiky3 Ckak^A C t a l y « H H d H
Hinb 2: Ham lirgng NO2' truag binb 3.1.2 Biin dgng mat dg vl khudn
a Bien dgng mat dg vi khudn tong cgng Mgt dO vi khuin tdng cdng dgt cao nhit qua kit qua phdn tich Id d chu ky 4 cua nghiim tottc ddi chung (16,6x10^ CFU/mL), tiiip nhit la dchu ky 2 nghiem tottc Pro W (6,4x10* CFU/mL) dugc tiinh bay d Hinh 3. Mat dO vi khuan tdng cd gia tri tmng binh d cac nghiem tottc QC, B7 + B41, Pro W lin lugt la 16,6x10" CFU/mL, 10,5x10* CFU/raL, 6,4x10'' CFU/mL. Qua Hinh 3 cho tody mat dp vi khudn d cac nghiem tottc khac biit khdng cd y
if cdc chu ky audi troag thi aghiem
nghia thing ki d cdc chu k^ (p>Q,05). Mdt dO vi khuin long cua nghiem thfte doi chftng khac biit cd y nghTa todng ke so vdi cac nghiim thttc cdn Iai d chu ky 4 (p<0,05), diiu nay cho thiy tac dung cua vi khuin b i sung cung nhu che pham vi sinh bi sung vao cdc nghiOm toftc cd khd ndng ftc chi cdc ddng vi khudn cdn Igi. Ci nghiim tottc DC do khdng cd bi sung vi khudn nin mat sd vi khuin ting tang din vao cudi mSi chu ky, cd thi Id do vi khuan Vibrio va cdc vi khuin khdc, d n ^ i i m toftc Pro W thi vi khuin tdng cd khuynh hudng gidm din diiu nay chimg td khd nang cdi thiin mdi
Tpp chi Khoa hpc Tmdng Dgi hoc Cdn Tha Phdn B' Nong nghiip, Thiiy sdn vd Congnghi Sinh hpc: 35 (2014): 111-120 trudng Cling nhu tgo ra cdc ddi^ khdng khudn ctta
chi pham vi sinh. Mat dd vi khuin tdng cd su biin dOng khdc nhau giira cac chu ky tou mdu cung nhu d timg nghiim tottc. Theo Hagiwata (1995), ttich
bdi Rombaut, 2001, mgt sd vi khudn ttong he todng nudi ludn trttng stt dung Idm ngudn toftc dn, cho nin mdt dd vi khuin cd su biin dOng giiia cac chu ky tou mlu.
C h u K y 1 C h u k y 2 C I i i k ^ 3 C h u k y 4 C h u k v t l s i m a u
Hiah 3: Mdt dp vi kbuan tdag cdag truag binh fr cac chu ky auoi trong thi nghiim b. Biin dgng mdt do vi khudn Bacillus
Mat dp vi khudn Bacillus dao ddng ttt 2,05 x 10^
- 27,95x10^ CFU/mL, cao nhit d nghiim thttc B7 + B41 cd gid tri trung binh tii 16,38x10^ ± 8,16 CFU/mL, nghiem tottc dii chiing toap nhdt cd gid fri ttung binh 3,67x10^ ± 3,07 CFU/mL d Hmh 4.
Mdt dO vi kliudn Bacillus a nghiem tottc DC ciia chu ky 1, chu ky 2 khac btii cd ^ nghTa todng ki vdi 2 n ^ i i m tiittc cdn lai (p<0,05). Mat dO vi
khuin biin dpng toeo tiing chu ky thu mdu, cdc nghiim tottc cd bo sung vi khuan ciing nhu che phdm vi sinh toi mat dp vi khudn Bacillus se cao han nghiem thttc dii chiing, diiu nay xdy ra la do vi khuin Bacillus dugc bi sung dinh ky, ban diu radt dO Bacillus thdp vd tdng Ien sau khi dugc bd sung vao, cdng vi cuoi thi nghiira thi mat dp Bacillus dn dinh dan, m|t so vi khuan cao hon so vdi khong bo sung.
C b a S L ^ l C l u V ^ 2 CiMM'kf^ C l u k ^ 4
Hiah 4: MSt dp vi khuda Bacillus truag c Biin dgng mat dg vi khudn Vibrio Vibrio d cac n^Iem tottc bd sung vi khuan va cbi phdm vi sinh dao dpng ttt 1,3x10^ - 72,6x10^
CFU/mL, cdn d nghiim tottc dii chiing dao ddng ttt 4,3x10' - 9,4x10^ CFU/mL. Ci Hinh 5 cho thiy if cdc nghifm tottc cd bi sung vi khudn cung nhu chi phdm vi sinh tin toay mgt dp Vibrio giam din
hiah d cic chu k}/ audi troag thi nghiim vi cuii toi nghiim, khac biet cd J nghTa todng ke vdi nghiira tottc DC va cac nghiira tottc cdn lgi d cdc chu ky cudi (p<0,05), diiu nay chiing td niu bo sung vi khudn Bacillus djnh ky se lam kim ham sg phat triin cfta Vibrio, d nghiem tottc DC mat dO Vibrio ludn dao dpng va khdng dn dinh, do sy tich luy thttc an du thtta va chat toai cua luan triing Idm
Tgp chi Khoa hpc Tru&ngDai hpc Cdn Tha Phdn B: Nong nghiip. Thuy sdn va Cdi^nghi Sinh hgc: 35 (2014): 111-120 cho mdi tmdng nude ttd nen xdu di, do Id diiu kien
touan Igi cho Vibrio phat trien vd neu bd sung cac vi khuan cd lgi se kiem ham mat dO Vibrio ttoi^
mdi ttudng nudi.
Theo Rengipipat el al. (1998), cho rang bao ttt Bacillus nhu mdt tac nhdn sinh hpc giftp Iam giam binh Vibrio ttong hi toing nudi touy san. Nghien cihi cua Hastuig va Nealson (1981) cho ring Bacillus cd toi tgo ra mOt sd chit khang khuin hodc mpt vai san phim cd toi lieu diet Vibrio harveyi. Theo Trin Thi Thu Hiin (2010) vi khuin Bacillus ttong qua ttinh phdt Uien cd thi san sinh ra chit kiim ham, tte chi vdi cac vi khuan gay
binh todng qua mdt sd ca cbi canh ttanh oxy, chat dinh dudng, cgnh ttanh vi tri va san sinh ra cac chat tte chi su pbat triin cda vi sinh vdt gay binh. Mpt s i nghiin cttu da cho thiy vi khuan Bacillus cd khd nang khing chi binh dich bing each tte chi sy phdt triin cfta vi khuin gay binh Vibrio, thttc ddy qua ttinh touc bao, tang hogt ddng cua Melanin vd khang khuin.
Theo Moriaty (1999), mat dO vi khuin Vibrio vugt qua 10^ CFU/mL se gay liai din dii tugng nudi, vi vgy Uong sudt thi nghiem mat dp Vibrio nam trong gidi hgn cho phep va khdng anh hudng din quan thi ludn trimg.
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Hinb 5: Mgt dp vi khuaa Vibrio truag biab fr cdc chu ky audi troag tbi nghiem 3.1.3 Swphdt triin ciia ludn trimg
a Bien dgng mat dg ludn trimg
Tdc dp ldng tmdng ciia luan trung frong 4 chu ky nudi phat trien dn dinh vd tdng rat nhanh. Diiu ndy chttng td luan triing bit ddu mang trftng va sinh san rtoanh tu ngdy tou 2. Mat dO luan triing ttong 2 ngay ddu tdng giing nhau khdng cd su khac biit {p > 0,05) vd tang nhanh vao nhttng ngay cudi ciia chu ky, mat dp bd tti ban dau la 30 ca toe/mL. O radi chu ky mat dp ludn triing toudng se dgt gia tti cao vdo ngdy thtt 3, mat dO luan ttimg d nghiim tottc B7 + 341 d chu ky 1 Id cao nhit, mat dO dat 1.000,78 ca toi/niL, ragt dO trung binh cung cao nhdl Id 401,62 ca toi/mL, toip nhdl Id d nghiim tottc DC la 92,46 ca toi/mL. Ct nghiim tottc B7 + B41 vd nghiem toftc Pro W tiii mat do ludn triing trung binh diu cd gia tti cao hon so vdi nghiim toftc DC. Kit qua nay hodn loan khdng chiu dnh hudng bdi yiu t i thfte an vi lugng thfte an cung cip vdo cho ludn trttng d tit ca cac nghiem toftc diu nhu nhau. 0 chu ky I mat dp luan trung cao nhdt d nghiem toftc B7 + B4], d cdc chu ky sau
mdt dO ludn trimg bat dau dn dinh nen khdng tang cao. Do mdt chu ky chi keo dai 4 ngay se khdng toiy rd sy khdc biit giiia cac nghiem tottc nen din chu ky 2, 3 mdi thiy rd su tang tmdng vd phdt ttiin ciia quan toi ludn triing.
Phdn lich cho toay d nghiim tottc DC toi mat dd Iufejtti!m^ldidc_bietj5ffy^nghTa toing ki vdi hai nghiim tottc cdn Iai {p < 0,05), diiu nay chttng td khi bo tti vi khuan ciing rtou chi phim vi sinh se Iam cho mgt dO ludn triing tang ddng ki do mdi tmdng nudi ngay cang xiu ciing nhu mat dp luan tmng cang cao se lam mat do ludn triing giam din vao cudi mdi chu ky nudi, dnh hudng din kit qua su phdt trien cfta luan ttung.
Theo Hagiwata (1995, ttich bdi Rombaut, 2001), thi mOt sh vi khuin trong hi thing nudi ludn trttng cd thi dugc ludn triing stt dung ldm nguon tottc an bd sung tac dung kich toich su tdng tmdng va phat ttiin cua ludn trung mOt so vi khuan cd tac dyng tte che sy phat triin ctta cac ddng vi khudn cd hgi tgo diiu kifn tit cho luan triing phdt triin.
Tgp chi Khoa hpc Trudng Dgi hgc Cdn Tha Phdn B: Ndng nghiep, Thiiy sdn vd Cdng nghi Smh hpc: 35 (2014): 111-120
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Hiah 6: Biea ddag mat dd luan truag b. Biin ddng mat dgvdtyli ludn trimg mang trimg T'^ li luan triing mang tifrng dugc toi hiin d Hinh 7, mat dp luan triing mang tning se gia tdng toeo mat dO ludn triing, ty li mang tning ctta luan trung ngay diu b i tri d cac nghiim touc nira ttong khodng tii(lI,3l±4,4con/mL),dchuky 1 nghiem thfte B7 + B4I thi mdt dd luan triing mang trttng cao nhit tmng binh dgt 46,89 con/mL, si ludn trimg cao nhit dgt 143,12 con/mL dat 18,8%, he sd trttng dgt 5,3%. C) nghidm thttc DC tmng binh dgt 7,61 con/mL chiim ty li thip nliit, he sd trttng Id 0,28%, nghiim thttc DC kliac biit cd y nghia todng ke vdi cdc nghifm tottc cdn Igi (p<0,05). Chu ky 1
qua cac chu ky nudi trong thi aghiem mgt dO ludn triing mang trttng cao d nghidm tiiftc B7 + B41 chftng td ludn trimg cd sfr dung vi khuan, chu ky hai toay rd dugc mdt do luan friing raang ttiing d nghiim thttc DC thip nhit, d chu ky 2 va 3 mat dO luan triing mang tning cung tdng cao d nghiem tottc cd bd sung vi khudn va chi phim vi smh so vdi nghiim tottc DC. Do mdi tmdng nude cd Iugng tottc an du tiiua cftng vdi mat dO tning tang nen ty Ii ludn triing mang ttttng gidm vao cuii mii chu ky nudi. Diiu ndy chttng td mat dp mang trttng ciia ludn tning phy thupc rat nhiiu vao moi tmdng vd fhuc dn.
d n K y l Cfaiky2 Onktl Cliiky4
Hinh 7: Biin d^ng mdt dd luda trung mang truag qua cac chu kj' audi trong thi nghiim 3.2 Thl aghiem 2: Gay cam ahiim luan
trdag vdi vi khuin Vibrio
Sau khi kit touc toi n ^ i m 1, stt dung ludn Uung dugc nudi vdi vi khudn Bacillus d i gdy cam nhiim vdi Vibrio harveyi, mat dd ttung binh la 19x10'CFU/mL. Qua Bangl cho tody sau 6 ngay
bi sung vi khuan thi mat dO Vibrio ^am ddng ke trung binh chi con 0,7x10' CFU/mL, quin toi luan triing cung gidm. Theo Moriaty (1999) trich bdi Ngd Thi Huyin (2011), mat dO vi khuin Vibrio vugt qua 10^ CFU/mL tiii se gay hgi den ddi tugng nudi, quin toe luan trung ban diu ttt 463,8 con/mL gidm cdn 19,7 con/mL.
Tap chi Khoa hpc Trudng Dai hpc Cdn Tha Phdn B: Nong ngluip. Thuy sdn vd Cdng nghi Sinh hpc: 35 (2014): Ill-f20 Tdc dg suy giam cua ludn tifrng nhanh nhit la d
nghiim tottc DC, luan trung chit hoan toan d ngdy tott 6 d Hinh 8, cdc nghiim tottc cdn Iai mat dp luan triing cui^ gidm rd sau 6 ngay toi n ^ e m . Dgc biit Id d cac nghiem tottc ludn trimg cd bd sung Bacillus a ihi nghiim trudc, logi vi khuin cd toe ban che su phat triin ctta vi khuin cd hai nen
Bang 1: Biea dgng mat dg vi Idiuan Vibrio trudc va sau khi bd sung
CO tdc dung duy ui mgt dO luan triing ddng thdi kim ham su phdt triin cua Vibrio. Cac n ^ f m dittc khac biit khdng cd y nghTa toing ke, do sau 16 ngay toi nghiim nen quan toi ludn triing da bdt dau chgm gia tang radt do, nen khi gdy toi nghiim cam nhiim mat dd luan trimg se mau suy tan hon.
Nghifm thfrc DC
Trudc Sau
n x i o ' i i i "
l,5x]03±57,72='
27xl0'±30' 0,3x10^ ±103''
ISxIO'iSl' 0,4xlO'±167,6*=
Ghi chu. Cdc tri sd trin cimg mpi hdng vdi ky tu gidng nhau di chi s^r sai biel khdng cd y nghTa Ihdng ke (p<0.05. Tukey HSDlesl)
-DC - B i + m i - P r o W
Hinb 8: Biia ddng mdt do ludn truag khi dugc gay cam nhiim vdi Vibrio harveyi 4 KET LUAN VA BE XUAT
4.1 Kit luaa
Mdt dO bd tri ban dau Id 30 cd toi/mL vdi ba nghiira thttc toi nghiem thttc B7 + B41 {Bacillus amyloliquefaciens) toi radt dg ludn ttimg raang trttng tmng binh dgt 46,89 con/mL cung nhu mat dO ludn triing dat 1.000,78 ca toi/mL, mat dO tmng binh dat 401,62 cd toi/mL cao hon so vdi nghifm tottc DC vd n^iem tottc Pro W. Mai dO vi khuin Bacillus dao dOng ttt 2,05x10^ - 27,95x10' CFU/mL, cao nhat d nghiem tottc B7 + B41 cd gia tti tmng bmh ttt 16,38x10^ ± 8,16 CFU/mL, nghiem tottc dii chttng toip nhit cd gia ttj tmng binh 3,67x10^ ± 3,07 CFU/mL nen mdi tmdng nudi cd bd sung che pham vi sinh hay vi khudn Bacillus toi nang suat nudi luan triing si dugc ndng cao ciing nhu mdi ttudng nude dugc cai toiin dang ki. Vi khuan Bacillus cd kha ndng tte chi vi khuin Vibrio, a cdc nghiem toftc bd sung vi khuin va che phira vi sinh dao ddng tii 1,3x10^ - 72,6x10^
CFU/mL, cdn d nghiim toftc dii chiing dao ddng ttt 4,3x10^ - 9,4x10^ CFU/mL. Cdc nghifm thttc cd bd sung vi khuin cung nhu chi phim vi smh toi
tody mat dO Vibrio giam ddn ve cudi thi nghiim, mdi tmdng dugc cdi thien nhd bd sung vi khudn ciing nhu chi phdm vi sinb. Ludn trimg qua ihdi gian bd sung vdi Bacillus cd khd nang duy In ty Ii song tdt han khi gay cara nhiim vdi vi khuin V harveyi, nhung khdc bift khdng y nghTa so vai ddi chttng.
4.2 D i x u i t
Can cd nhiiu han nhttng nghiin cttu vi sttc sing ciing nhu kha nang tdng tmdng cua luan triing todng qua viic bd sung vi khuan vd chi phim vi sinh. Can tim hiiu sdu hon ve khd nang Ipc cua ludn trung ddi vdi viec b i sung vi khuin cd Igi vdo he todng nudi. Nghiin cftu cac toi nghiem liin quan den su kiem soat hf \ i sinh ttong mdi tmdng nudi ludn trung nhim chpn lpc giing luan trimg cd chit Iugng tdt ban.
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; ^ -19. Trin Suang Ngpc vd Nguyin Huu LOc 2006. Nghiin cttu thiit Igp hi todng nudi kit hgp luan frung (Brachionus plicalilis) vdi be nude xanh. Tap chi Nghien cfru Khoa hpc, Tmdng Dai hpc Cin Tho:82-9I.
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