Tgp chi Khoa hgc Tru&ng Dgi hoc Cdn Tha Phdn B Ndng nghiip. Thuy san vd Cdng nghi Sinh hgc: 26 12013). I2--133
Tap chl Khoa hpc Trirdng Oai hpc Can Tha website: sj.ctu.edu.vn
ANH HlTdNG CUA CAC Ll£U IASC^G C H E F H A M SEVH HOC KHAC NHAU DEN MOI TRlTOfNG VA SU* PHAT T R I E N CL A TAG CHAETOCEROS MUELLERI Ngd Thi Thu Thao'
' Khoa Tlmy sdn, Truang Dgi hoc Cdn Tha
Thong tin chung:
Ngaynhdn: 26/12/2012 Ngdy chdp nhdn: 20/06/2013 Title:
Effects of probiotic supplementation on the environmental factors and development of Chaetoceros muelleri
Tu khda:
Chi phdm sinh hoc. mat dd tdo, Chaeloceros mulleri Keywords:
Probiotic, algae density Chaetoceros mulleri
ABSTRACT
Algae Chaetoceros muelleri was cultured by F 2 based solution and supplemented with different concentrations of probiotics based on Baccillus and Lactobacillus (0. 0.5, 0.75 and 10 mgl) .it supplemented concentration of 0.75 mg/L. algae could reach the highest density (86.56 ± 0,95 x 10^ cells/ml) after 7 days and maintained until 12 days of culture period, these results were significantly different from control or other probiotic supplemerued concentrations (p<0.05). Probiotic supplementation into algae culture increased the total bateria counts and also Bacillus but reduced the density of Vibrio from dgy 4 of culture period in algae medium (p<0.05). The findings from this study will be a useful tool in algae culture lo prevent the pathogenic bacterium from live food to aquaculture species in hatchery.
TOM TAT
Tdo khue Chaetoceros muelleri dugc nudi bdng mdi trudng ca ban F 2 vd bd sung che phdm sinh hgc cd chua vi kliudn Bacillus vd Lactobacillus vcri cdc lieu luong khdc nhau Id 0; 0,5; 0,75 vd 1,0 mg Z.
Kit qud cho thdy khi bd swig vai hdm lugng 0,75 mg L mat dg tdo dot cao (86.56 ± 0.95 x IO' tb/ml) vd duy tii duac trong 12 ngdy. kit qud ndy cd y nghia thdng ki (p<0.05j klii so sdnh vai viic khdng bd sung hogc bo sung che phdm sinh hgc vdi cdc hdm lugng khdc. Khi bd sung chi phdm sinh hgc da ldm tdng mat do vi khudn idng vd vi kliudn Bacillus nhung ldm gidm ddng ke mat do vi khudn Vibrio trong me tdo nudi tir ngdy thu! 4 (p<0.05l. Kit qud thi nghiim cd klid nang dng dung trong nudi sinh khdi tdo nham hgn che vi khudn gdy hgi.
1 GIOI THIEU
Nhilu quin the vi sinh vdt cimg tdn tai frong cung mdt he sinh thai thi se cd su canh tranh vl dinh dudng va nang lugng. Canh tranh frong gidi vi sinh vdt chu yeu la xay ra d nhdm di dudng nhu canh Uanh cdc chat htSu ca ma chii yeu ngudn carbon vd nang lugng.
Rico-Mora et al. (1998) da ciy mdt ddng vi
khuan dugc chgn Igc cd khd nang phat trien fren mdi trudng ngheo hihi ca vao be nudi tao khue Cling vdi Vibrio alginolyticus thi \i khudn Vibrio khdng phat frien. Dieu dd chiing td % i khuan dugc chgn Igc canh uanh lin at Jlbrio trong dieu kien ngheo hihi ca. Nhirng ddng vi khuan ndy cd the khdng tot ddi vdi ucmg au triing bang nudc xanh. tu> nhien se cd
Tgp chi Khoa bgc Tnr&ng Dgi hoc Cdn Tha Phdn B: Ndng nghiep. Thu\- san vd Cdng nghi Sinh hgc: 26 (2013): 12 7-133 lgi khi tao phat trien qua muc uong ao nudi.
Nghien cuu ciia Xiang-Hong et al. (1998) cung cho thdy mdt so vi khuan bun ich cd the kich thich hoac uc che su phdt Uien cua tao. Tac gid con cho biel them nhung vi khudn cd igi frong nudc se loai tru nhanh NH3, H2S, vat chat huu ca cd hai. Ngoai ra, chiing cdn cd the can bang pH trong ao nudi.
Tren the gidi, tao khue Chaetoceros sp dugc su dung rdng rai ldm thuc an frong cac trai san xuat gidng gidp xac va ddng vat than mim (Hemaiswarya et al, 2011). Qud trinh nudi sinh khdi tao se khdng trdnh khoi su lay nhiem vi khuin gay hai tir ngudn nudc, dung cu nudi va khdng khi... Han chl qud frinh lay nhiem nay bang viec phdt trien mdi quan he canh franh gi&a nhdm vi khuan huu ich va vi khuan gay hai se gdp phan nang cao fr'nh an toan vd chat lugng cua sinh khoi tdo phuc cho viec sdn xudt gidng cac doi tugng thuy san dat hieu qud cao han.
2 VAT LIEU VA PHU'ONG PHAP 2.1 Cac nghiem thirc bo tri
Thi nghiem dugc thuc hien tren tao khue Chaetoceros muelleri, vdi 4 lieu lugng chl pham sinh hgc (CPSH) dugc b6 sung vao mdi trudng nudi tao Id: 1). Ddi chung: Khdng bd sung CPSH; 2), Bd sung CPSH vdi lieu lugng 0.5 mg/L nudc tao nudi; 3). Bd sung CPSH vdi lieu lugng 0.75 mg/L vd 4). Bd sung CPSH vdi lieu lugng 1,0 mg/L.
Cac ylu Id mdi trudng (pH, TAN va NO3-) dugc kiem tra sau mdi 3 ngay, mat do tdo dugc xac dinh hang ngdy va mat do vi khuin Bacillus. Vibrio, vi khuan tdng dugc kilm tra sau mdi 2 ngay.
Thanh phin CPSH Tado-Bacillus co chua Bacillus subtilis (1.5x10" CFU), Lactobacillus acidophilus (1,5x10^ CFU), cac loai men nhu Protease (16000 UI). Amylase (4000 UI).
2.2 Cach nuoi cay va xac dinh mat dg tao Tao khue Chaetoceros muelleri dugc nudi bang moi trudng ca ban F/2 frong cdc binh ihu\ linh CO the lich 8 lit cd gin sue khi. Cac
binh nudi tdo dugc dat frong phdng cd may dieu hda nhiet do giu dn dinh d 26*'C va cudng do anh sdng la 5400 lux. Tao C. muelleri thuan dugc dat mua tir trudng Dai hgc Nha Trang vd mat do nudi ban dau la 5 x 10^ tb/mL.
Mat do tao dugc xdc dinh hang ngay bang cdch su dung budng dem Improved Neubauer va cdng thuc tinh la: N (tb/ml) = (n/64) xio^
Trong do: n la tdng sd te bao dem dugc frong 64 d nhd cua budng dem.
2 3 Xac dinh mSt dg vi khuan
Chudn hi cdc dng nghiem chua nudc muoi sinh ly (0,85%) dugc tiet friino d I 2 r c frong 20 phut, Ldy 1 mi mdu nudc can xdc djnh cho vao dng nghiem chua nudc mudi sinh iy, frgn deu dugc ndng do pha loang ID''. Tiep tuc lay 1 ml nudc d ndng do l O ' cho vao dng nghiem chua 9 ml nudc mudi sinh Iy va udn deu, dugc ndng dp pha loang 10"". Cdc budc tiep theo tuong tu s6 dugc ndng do pha loang can thiet.
Doi vdi miu xdc dinh mat do vi khuan Bacillus, sau khi pha loang dugc ndng do thich hgp dem sdy d nhiel do SO^C frong 20 phut,
Cach liy mau vao mdi trudng thach:
Dung Micropipete hut 100 pL dung djch frong dng nghiem chua vi khuan cho vao dia chua mdi trudng chuyen biet rdi dimg que tan diu den khi khd hodn loan. Cdc dTa sau khi tan diu dugc dem u trong tu 28''C vd kilm tra sau 24 gid. Sd vi khudn dugc tinh theo cdng thuc:
Dan vj hinh thanh khuin lac (CFU/ml) ^ s6 khuan lac x do pha loang x 10.
3 K E T QUA VA T H A O LUAN 3.1 Cac yeu to mdi t r u d n g
Kel qua cho thay 3 nghiem thuc dugc b6 sung CPSH cd gia tri pH dn dinh frong suot chu ky phdt frien cua tao. Tuy nhien, pH d nghiem thuc bd sung 0,75 mg/L la dn dinh nhat va nghiem thuc ddi chung cd pH biln ddng nhilu nhit (Hinh 1). Xiang-Hong et al (1998) nghien cuu va bao cdo ring mdt so vi khuan hihi i'ch cd the can bing pH trong ao nudi.
" Tap cbi Khoa hgc Tnr&ng Doi hgc Cdn Tha Phdn B: Ndng nghiep. Thuysanv, Cdng nghi Smh boc. 2612013) 12 '-133
Hinb 1: Bien dgng gia trj pH trong cac nghidm thih: theo thdi gian
J
•'^' z '- :-" / / V
\ /
: - 2 : E L I iLzL 1
Trong cdc nghidm thuc diu khdng xdc dinh dugc ham lugng TAN, cd Ihl TAN da dugc tao hdp thu hoac cung cd the da dugc chuyen hda thdnh NO3". NH4" trong nudc rdt cdn thiet cho su phdt trien ciia cac sinh vat lam thuc an tu nhien, nhung nlu ham lugng NHj^
qua cao se lam cho thuc vat phii du phat trien qua muc khdng cd lgi cho cac ddi tugng thuy sdn.
Ham lugng nifrat giam nhanh d cdc lieu lugng CPSH bd sung Id 0 vd 0,75 mg/L. Tuy nhien. khi ta bd sung 0,5 mg/L vao ngay thir 3 thi ham lugng nitrat tang so vdi ban dau (50 mg/L), cd khd ndng vi khuan chuyen hda dam phdt trien nhanh hem va qua frinh chuyen hda dam diln ra nhanh han cdc nghiem thuc khac (Hinh 2),
Hinh 2: Bi£n dpng hdm ^s.
Iugng NO3" (mg/L) trong — cac Dgfai|m tfairc z v
3.2 Bien dgng mat dp tao theo thdi gian Vdi mat dp ban ddu la 5 x 10" tb/mL, viec bd sung CPSH vdo mdi trudng nuoi lam cho tao C. muelleri dat mat do cao hem vd cd the du\ fri trong 12 ngay (Bdng I va Hinh 3).
Nghiem thuc bd sung 0,75 mg/L cd mat dp tao dat cue dai vdo ngdy thu 7 (86,56 ± 0,95 x|0'^ ib/mL) va khac biet thong ke so vdi cac nghiem thuc cdn Iai (/7<0,05). Nghien cuu ciia Nguyin Thj Thanh Mai va ctv (2009) cho
rang mat dp tdo C. calcitrans dat cao nhdt la 21 X 10^ tb/mL trong thdi gian 6 ngdy nudi uong moi trudng TT3 vdi mat dp ban dau ia 6 X 10^ tb/mL. Co khd nang tao C. muelleri cd kich thudc nhd han, qud frinh phan chia te bao se xa\ ra vdi tdc dp nhanh hem, Tuy nhien, ket qua ciia thi nghiem bd sung CPSH vao mdi trudng nudi tao C. muelleri cho thay thdi gian dat mat do cue dai cham han nhung su phdt trien cua tao dugc duy fri lau ban. Day cd t h i
Tap chi Khoa hgc Tnr&ng Dgi hoc Cdn Tha PMn B: Ndng nghiip. Thuy-san vdC&ngt^hfSirA hgc: 2612013). ^- '1^3 la mdt Uong nhirng ung dung nham dieu tiet \ a
duy fri qua uinh nudi sinh khdi tdo trong cac trai sdn xuat gidng. Grossart et al. (2006) thi nghiem tren 2 loai tao khue Skeletonema costatum va Thalassiosira rotula bdng nudc bien tiet triing vd nudc bien cd chua qudn the vi khuan tu nhien cung quan sat thay su phat
B a n g 1: Bien d g o g m a t d o l a o t r u n g binh tbeo Ifadi gian (10'^ t b / m L )
triln cua tao trong mdi trudng cd vi khuan sg cham hom nhung dien ra trong thdi gian keo dai han. Cac tac gia nhdn manh vai frd ciia \\
khuin dj dudng frong viec kiem sodt su phat triln va kit ldng cua tdo trong mdi trudng nudc bien.
1 2 3 4 5 6 7 8 9 10 11 12
0 17.40±0.36'' 20.44i0.35"'' 66.37±0.88' 69.32*0,87'' 58.51*0.87"
47.44±0.92"
26.14±0.67"
2.42±0.07"
0.88±0.03"
0.14±0.02' O.OOiO.OO"
O.OOiO.OO'
L i l n l i n n i s C P S H bd 0,5 15.53±0.32' 22.06*1,15"
27,42*0.85"
41,77*0,73"
62.88*0.81"
71.00*0.63"
75.44*0,88""
64.40*1,02"
57.89*1.52' 46.87*0.58"
26.23*0.55""
15.8iW).48''
s u n g ( m a n . ) 0,75 12,98*0.42' 21.79*0,32""
30.84*0,57"
4 8 , 4 6 * 1 , 0 4 ' 67.78*2.00'' 78.41*0.92"
8 6 , 5 6 * 0 , 9 5 ' 81,38*0,97"
73,76*1,49"
4 3 , 4 3 * 1 . 9 0 ' 24.80*1.95""
15.54*2.04'
1,0 10,94*0,54' 20,28*0,13"
25.52*0,87"
32,76*1,48' 53,44*1,07"
66.76*1,48' 74.48*2.03""
56.17*2,03' 45,79*3,19' 37.61*1,05' 33,36*2,60' 22,10*4,41' Cdc sd lieu cirng moi hdng co chu cdi khdc nhau Ihe hien khdc biel cd y nghTa thdng ke (p< 0.05)
Khi dugc bd sung ham lugng CPSH la 1,0 mg/L thi mat do tdo d nghiem thiic nay dat thap so vdi b6 simg 0,75 mg/L va ket qua khdc biet cd y nghTa thdng ke {p<0,05). Nghien ctru cua Xiang-Hong et al. (1998) cho thdy mpt sd vi khuan hiiu ich cd the kich thich hoac uc che su phdt trien cua tao. Su dung CPSH vdi lieu
lugng 1 mg/L cd the da lam can u d qua Uinh tiep nhan dnh sang va trao ddi chat ciia tao khi chting bdm vao tao nhu gia the. Ngodi ra.
thanh phdn cua CPSH cdn chua vi khuin Lactobacillus va mdt sd men lam tang khd nang tieu hda do dd cd the da uc che su phan chia te bao tao.
H i n h 3 : Bien d p n g m a t d o tao (lO^tb/mL) tbeo tfadi gian
Tap chi Khoa hgc Tnr&ng Dgi hoc Cdn Tha Phdn B. Nong nghiep. Thuy san vd Cdng ngh? Smh hoc 26 (2013): 127-133 3 3 Bien dgng mat do vi khuan troog mdi
t n r d n g audi tao
3.3.1 Mat do vi khudn long trong nu&c (CFU/mL)
Cac nghiem thuc bo sung CPSH cd mat do vi khudn tdng lang tu 2,75x10' - 9,8^
10* CFU/mL, Tu ngay thu 8 - 12 mat dd vi
khuan tdng cua 3 nghiem thuc bd sung CPSH lang khdng dang ke nhung cao hon so vdi khdng bd sung CPSH (Hinh 4), Ngd Thj Thu Thao et al. (2012) thu nghiem cdc phuong phap bd sung CPSH vdo mdi trudng uang ngheu cung ldm cho mat do vi khuan tdng tang Ien d a n g k l ( t u 4 , 8 x 1 0 ' ' - 6 , 4 x 1 0 ^ CFU/mL),
Hinh 4: Bien d^ng m^t dp vi kfauan =
ting cgng C n:^i iiJfsL n:_'==EL • : . : ^ L
miiflli
iillli.. -I. J
f l f l
E I
r i 3.3.2 Mat dp vi khudn Bacillus subtilis trong
nuac (CFU/ml)
Trong cdc nghiem thuc bd sung CPSH.
mat dd B. subtilis bat ddu tang vdo ngay thu 4 den ngay thu 6 (6,1x10^ CFU/mL) vd dudng nhu khdng tang them lu ngay thu 8-12 cua qua frinh nudi tao (Hinh 5). Nghien cuu cua Pham Thi Tuyet Ngan va Truang Qudc Phii (2011) cho thay vi khudn Bacillus phdt trien vao ngay thu 5-7 khi dugc bd sung vao mdi trudng ucmg tdm sii. Mat do vi khuan B. subtilis khdng khac . biet giua cdc nghiem thuc dugc bd sung CPSH
tuy nhien rat khac biet so vdi nghiem thuc ddi chiing (p<0,05). Dieu do chiing td trong mdi trudng nudi sinh khdi ldo, vi khuan Bacillus da phdt trien tdt vd khdng loai tru kha nang nhdm vi khuan nay da su dung tao C. muelleri lam' gia the phuc vu cho su phat trien cua chiing.
Nghien ciru ciia Grossart et al. (2006) cho thay mat dp vi khudn dinh uen tao khue 5. costatum va T rotula chi tang nhe vao ngay 9 va ngay 11 khi mat dp lao giam xudng. Cdc tac gia cho rang vi khuan da md rpng xam nhap va phat trien Uen nhdng le bao tao da hda gia.
Hinfa 5: Biln dgng mat do B. subtilis theo tfadi gian trong cac ngfaiem tfauc
Tgp chi Khoa hgc Tnr&ng Dgi hoc Cdn Tha Phdn B Nong nghiip. Thuy san vd Cong nghi Sinh hgc: 26 (2013): 127-133
3.3.3 Mat dp Vibrio trong nir&c (CFU/mL) Trong cdc nghiem thuc bd sung CPSH thi mat dp Vibrio rat thap va den ngay thu 6 thi nhdm vi khuin nay khdng cdn xuat hien (Hinh 6). Su phdt uien ciia vi khudn Bacillus tu ngay thu 5 den ngay thu 7 cd the da lan at su phdi trien cua nhdm Vibrio. Trong nghiem
thuc khdng bd sung CPSH thi mat dp Vibrio cang tang ve cudi chu ky nudi tdo, sau ngay nudi thu 4 thi mat do Vibrio tang rat nhanh tu 8.75x10' den 7,'75xlO- CFU/mL, dat cao nhit vao ngay thu 8 va cao ban so vdi cdc nghiem thuc khac (p<0,05).
Hinfa 6: Bien dpng mat do vi — kfauan Vibrio tfaeo thdi gian ^ ~
(CFU/mL) ~
Qiid 3'Js.iL Dl'iisL DLiEgL
Cdc loai vi khuan Bacillus cd khoang bien ddng Idn ve khd nang uc che vi sinh vat. Cdc nghien ciiu frudc day cho thay mgt sd lodi' vi khuan Bacillus spp. cd khd nang chdng lai vi khuan Gram dm va Gram duong (Yilamz et al., 2006). Rengpipat et al. (1998) bdo cdo v l loai vi khuan Bacillus Sl 1 phan lap tu tdm su khde manh cd kha nang giam ty le lu vong khi gay cam nhiem vdi vi khudn phdt sang V. harveyi. Ngd Thi Thu Thao vd Pham Thi Tuyet Ngan (2011) khi bd sung ddng Bacillus B37 vao be uong dc huang da ban chl su phat trien vd ldm cho mat dp vi khudn Vibrio dat thdp nhdt uong tat ca cdc nghiem thuc thi nghiem (10 - 80 CFU/ml). Ket qud bd sung CPSH vdo tdo nudi da cd tac dung han chl su phat frien cua nhdm vi khuan Vibrio nhu vdy se cd y nghTa frong viec phdng benh do vi khuan lay nhiem tix ngudn thuc an tu nhien.
4 KET LUAN VA DE XUAT 4.1 Ket luan
Viec bd sung che pham sinh hgc chtra vi khuan Bacillus vdi ham lugng 0,75 mg/L vao tao Chaetoceros muelleri da dat dugc mat dp
4 ^"Pr 3 10 12 tao cao nhit (86,56 ± 0,95 xio^ tb/mL) va duy tri mdi frudng dn djnh sau 12 ngay nudi.
Bd sung che pham sinh hpc vao mdi trudng nudi tdo da gdp phan dn dinh mdi trudng va han che su phdt frien cda vi khuan Vibrio.
4.2 De xuat
Ket hgp bd sung CPSH vao tao va true tiep vao mdi trudng nham han chl su phat triln ciia vi khuan Vibrio va nang cao ty le sdng cdc doi tugng thiiy san dugc uang nuoi.
TAI LIEU THAM K H A O
1. Grossart, H.P.. Czub G. and Simon M. 2006.
Algae-bacteria interactions and their effects on aggregation and organic matter flux in the sea Environmental Microbiology 8 (6): Pages 1074-1084,
2. Hemaiswaiya S.. Raja R., Ravi Kumar R., Ganesan v., Anbazhagan C. 2011.
Microalgae: a sustainable feed source for aquaculture. World J Microbiol Biotechnol 27:
Pages 1737-1746.
3. Ngo Thj Thu Thdo vd Pham Thi Tuyet Ngan.
2011. Anh hudng cua viec bl stmg cac lo^i che
Tap cbi Khoa bgc Tnrong Dai hoc Cdn Tha Phdn B: Ndng nghiep. Tlmy san vd Cdng ngbe Sinh hgc. 26 (2013) 127-133 pham sinh hpc chda vi khuan Bacillus frong
qua trinh uong au triing 6c huong (Babylonia areolata). Ky ylu Hgi nghi Khoa hpc Thuy san lan 4. Trudng Dai hpc Cdn Tha, ngay 26/1 /20U. Nha Xuat ban Nong nghiep. Trang 55-64.
Ngo Thi 'Thu Thdo, Dao Thi MJ Dung ya Vo Minh The. 2012. Anh hudng ciia viec b6 sung che phdm sinh hpc den sinh trudng va ty le song cua ngheu {Meretrix lyrata) giai doan gidng. Tap chi Khoa hpc Truong Dai hgc Can Thasi 21b/2012. ISSN: 1859-2333. Trang 97-107.
Nguyen Thj Thanh Mai, Trinh Hoang Khai, Dao Van Tri, Nguyin Van Hiing. 2009.
Nghien cthi phan lap, nuoi cdy in viUo tao silic nudc man Chaetoceros calcistrans Paulsen.
1905 va ling dung sinh khoi tao lam thuc an cho t6m he chan trdng (Penaeus vannamei}.
Tap chi Khoa hpc va Cong ngh?, tdp 12, so 13.
Dai hpc Quoc gia Thanh pho Ho Chi Minh.
6. Pham Thi Tuyet Ngan vd Truong Quoc Phu.
2011. Anh hudng cua vi khuan Bacillus (B8.
B37 vd B18) len chat lugng nudc bl torn su.
Ky yeu Hgi nghj Khoa hpc Thiiy san ldn 4.
Trudng Dai hoc Can Tha. Nha Xuit ban Nong nghiep TP. H6 Chi Minh: Trang 28-41.
7. Rengpipat S., Phianphak W.,
Piyativatitivorahul S. and Menasveta P.,1998.
Effect of probiotics on black tiger shrimp Penaeus monodon survival and growth.
Aquaculmre 167: Pages 301-313.
8. Rico-Mora R, Vollolina D, Villaescusa-Celaya JA, 1998. Biological conUol of Vibrio alginolyticus in Skeletonema costatum (Bacillariophyceae). Aquacult. Eng. 19: Pages 1-6.
9. Xiang-Hong W. et al 1998. Application of probiotics in aquaculture.
http://www.alkenmurray.com/China98.htm. 4 pp.
10. Yilmaz M., 2006. Antimicrobial activities of some Bacillus spp. strains isolated from the soil. Microbiol Res 161: Page 5.
