CHAN NUOI DONG VAT VA CAC VAN DE KHAC
TUYEN CHPN vA TON TRU" VI KHUAN LACTOBACILLUS CO TIEM NANG PROBIOTIC l/NG DUNG TRONG CHAN NUOI GA
Trail Vdn Be Nam'. Nguyen Bd Phdc'. Trang Tlianh Gid', Le Tlii Hgi\
Nguyen Vu Trung' vd Nguyin Trgng Ngir' -' N g a y nhgn bai bao: 28/09/2019 - N g a y n h g n bai p h a n bien: 19/10/2019
N g a y bai b a o d u p c c h a p nhgn d a n g : 31/10/2019 T O M T A T
Sir dyng probiotic nham nang cao hieu qua phong l>enh cho vat nuoi la van de dupc quan tam hien nay. Nghien ciiu nay dupe thyc hien nham tim ra cac dong vi khuah Lactobacillus co tiem nang probiotic de diing trong chan nuoi gia cam, TCi 75 mau rupt ga \'a thyc phain len men, co 11 dong Lactobacillus d u p c phan lap. Kot qua co 7 dong thich nghi dupc vdi dieu kien pH 1,5 va muoi mgt 0,3% va 3 dong co kha nang khang khang sinh tetracycline (30 pg/ml) va ampiallin (10 pg/ml). Trong do dong LIN3 co mgt so cao d pH 2,0 (6,32 log cfu/ml) va tang dupc mat so'khi ii vdi muoi mgt 0,3%
trong 3 gio. Ket qua giai trinh h^ dinh danh dong nay la Lactobacillus ptantarum. Thir nghiem ton tnj dong vi khuan L. plantarunt cho tha'y co chat gom 50% ggo va 50% bpt mi la phii hpp, vi khuan giu' dupc mgt so'4,80 log cfu/ml den 60 ngay khao sat trong dieu ki^n nhiet dp phong
Tir kh6a.' Khdng khuan, Lactobacillus, muoi mat, probiotic.
ABSTRACT
Selection and storage of probioric potenHal of Lactobacillus applied for chicken production The application of probiorics to improve the effecriveness of animal disease prevention is of concern novi'adays. The current study was conducted to isolate the probiotic potential of Lactobacillus strains for using in poultry production. From 75 samples of chicken intestines and fermented foods, 11 strains of Lactobacillus were isolated. Seven strains were found to adapt to pH 1.5 and 0.3% bile salts and three isolates were resistant to tetracycline (30 pg/ml) and ampicillin (10
|ag/ml), In parricular, LIN3 strain had a high density at pH 2.0 (6,32 log cfu/ml) and increased the density when incubated with 0.3% bile salt for 3 hours. This strain was sequenced and identiried as Lactobacillus plantamm. Addionally, the combinadon of 50% rice and 50% wheat flour were appropriate where the density of L. plantarum remained stable (4.80 log cfu/ml) after 60-day storage at room temperature.
Keywords: Antibacterial activity, Lactobacillus, bile salt, probiotics.
I.BATVANDE
Hien nay, n g a n h c h a n n u d i la m g l l r o n g nhiing n g a n h k i n h t e m u i n h g n va q u a n t r p n g d Viet N a m . T h e o T d n g C u e T h d n g k e (2018), trong n h i i n g n a m g a n day, s o l u g n g d a n gia sue, gia cam n g a v m g t t h a y d d i r o ret. D a c biel ddi vdi gia cam, s o l u p n g l a n g hr 361,7 h i e u con n a m 2016 ten 385,4 trieu con v a o n a m 2017 va 408,9 h i e u con v a p n a m 2018. Q u i ' Tnrong Dai hoc Can Tho
• B^nh vivn B?nh nhi|t doi Trung irong ' Truong Dai h(,ic Y Ha Npi
• Tac gia hen hv PGS.TS. Nguy^ Trgng Ngii, Khoa Nong nghiep, Tnrdng D^i hgc Can Tho. Dien tho^in 0989828295;
Email; ninguflitrtu edu.vn
m d c h a n n u d i vdi s o l u p n g ldn va tang n h a n h lien t y e d d i hdi \'iec kich thich s i n h h u d n g , p h d n g b e n h va d i e u tri la c o n g tac het s u e can thiet. D o d d , k h a n g sinh d u p e sii' d y n g r p n g rai t r o n g c h a n n u d i d e d a p t i n g m y c dich n a y la k h d n g t h e t r a n h k h d i ( N g u y e n D a n g Vang, 2015).
Tir k h i Viet N a m gia n h a p W T O , cac q u i d m h v e sir d y n g k h a n g s i n h h e n vat n u d i n g a y c a n g chat che h o n va m d t s6' k h a n g sinh bi c a m s u d u n g - M a c dii h a u h e l cac t r u d n g h g p p h a l hien d u l u g n g ciia cac k h a n g sinh d u o c p h e p s u d u n g va n a m h o n g gidi h a n c h o p h e p . Tuy n h i e n , v a n ed n h i e u t r u d n g h g p d u l u g n g k h a n g sinh v u p t q u a gidi h a n . M g l
CHAN NUOI b p N G VAT VA CAcTv.^ - -^--^
nghien ciiu da cho thay Campylcbactcr phan lap hi ga thit ciing cd miic khang cao: 90%
khang vai nalidixic axit, S9'„ vdi tetracj-cline va 82% vdi ciprofloxacin ( \ g u \ e n \ a n Kmh, 2010). Hon nira, rinh hinh su dung khang sinh hong chan nudi chua hgp ly \'iee lira chgn khang sinh va lieu dung duge quvet dinh chii ye'u dy-a hen kinh nghiem cua chu hg (44",>), 33% theo hudng dan ciia bae ^v thu \', 17%
theo hudng dan cua nha san xuat (Nguven V3n Kinh, 2010). Do dd. can nhanh chdng rim ra giai phap hiiju qua do thay the'khang sinh chd'ng Igi cae tac nhan gay benh hogc kieh thich sinh trudng.
Hien nay axit hihi co, probiotic, prebiotic, phytogeme va zeolit la nhung lya chgn hun hipu cho \ H'c thay the khang sinh (Papatsiros va ctv, 2013). Dgc biet la probioric vi day la phucmg phap an loan va dem lgi hieu qua cao de phdng benh va nang cao siie de khang cho vat nudi- Tren thj trudng \'iet Nam hien nav cd nhieu logi ehe pham probiotic dupc lun hanh nham bd sung vao thire an chan nudi. Tuy
\'.iv. nguyen lipu cho san xua't probioric diii veil deu la'v tir nudc ngoai, hem niia hieu qua dem lgi chua that su on djnh va gia thanh cdn cao (Hd Trung Thdng va Ho Le Quynh Chau, 2009), Do dd, \'iec nghien ciiu su dyng cac vi sinh vat. dgc biet nhdm vi khuan Lactobacillus cd tiem nang de tao che pham probioric nham khae phyc tinh trgng tren la viee Iam het siic cin thiet-
2. VAT U t U VA PHUONG P H A P NGHltN CUru 2.1. Thu mau va xir Iy mau
Mau dupc thu tii rudt ga (rudt non va rudt gia), thyc pham len men (dua cai, ca phao va dua leo) tai rinh VTnh Long, Sdc Trang va Hau Giang.
Ddi vdi rudt ga, mdi tinh tien hanh thu 5 niau tir 5 ca the g^i Ndi hong giai doan sinh san thupc 5 hgi khac nhau. Diing d'ng liem hiit 1ml dich rudt, sau dd eho dich hiil vao dng luyp v6 triing. Mau dupc ghi ky hipu va tru lanh trong qua trinh van chuyen ve phdng till nghiem.
Cae m5u dua cji, ea phao, dira li"^' j ; ^^^
men. Dimg dng tiem hiit 1ml djch ''^"^ jg^
pham (gom ca phao, dua cai va '^^^ J .,^rf men), sau dd cho dich hiit \'ao ong cph . vd triing. Ghi kv hipu va dio ^ .lo thung da tni lgnh khoang 4 C dem ve phdng thi ngr . nudi cay phan lap.
2.2. Phuomg phap 2.2.1. Phdn lap vi khuan
Mau dugc pha loang den nong dp lOJ va dugc trai tren dia Petri chua moi iniong MKb, Dia dupe de khd lu nhien \'a u d 37"C trong 24-48 gid. Cac khuan lgc xuat hipn trong dia dugc cay sang dia mdi de thu dupe khuan lgc rdi rgc, cac khuan lgc dupe dupe tach rdng tren dia petri chua mdi trudng MRS agar mdi. Cac ddng vi khuan duge giir hong dng tuyp chua mdi trudng NB d -80"C cho cac thi nghipm tie'p theo,
2.2.2. Ddnh gid khd nang chju pH thap Thi nghiem dugc rieh hanh theo phuong phap danh gia chiu pH tha'p cua Erkkila va Petaja (2000), Thi nghiem duge bdtri 3 nghiem thuc (NT) vdi muc pH khac nhau: 2,5; 2,0; 1,5, 3 lan lap lgi. Chi rieu theo ddi la mat so' vi khuan sd'ng dupc tren moi trudng nudi ca'y.
2.2.3. Ddnh gid khd ndng chiu muoi mat Cac ddng ed kha nang chiu pH td't dugc tuyen ehon eho thi nghiem danh gia kha nang chiu mud'i mat theo phuang phap cita GilUland va ctv (1984). Thi nghiem duge bo tri 2 NT' NT eac ddng vi khuan duge ii trong mdi trudng NB ed bo sung 0,3% mud'i mat va NT dd'i chung (DC) dugc ticn hanh tuong ty, nhung khdng chung vi khuan va na'm men vao mdi trudng NB cd bo sung 0,3% muo'i mat. Thi nghipm duge thyc hipn vdi 3 lan lap lai. Chi rieu theo ddi la mat so vj khuan sd'n dupc tren mdi trudng nudi cay
2.2.4. Dinh danb ddng vi khudn va nam men Trich ADN ciia vi khud'n: Qui trinh trich ADN thuc hipn theo Breugelmans va Uytte- broek (2004). Sau khi tinh saeh ADN, phan ung PCR duge thuc hien vdi rap mdi IfiS ARN dugc thiet ke'theo Lane (I9K^, V,,J JJ-J -
1 song
KHKT Chdn nuoi so 254 - thdn
x tJOi D y N C VA7 VA CAC VAN DE KHAC
h^ sau: 27F: 5' AGA GTT TGA TCC TGG c r c AG 3'; 1492R: 5'GGT TAC CTT GTT ACG ACT T 3'. Cac san pham PCR cd vach dam va ro tren gel agarose dugc giri giai ma trinh tu ADN lgi cdng ly Macrogene (Han Qud'c). Tir ket qua giai trinh ty, eac trinh ty nay duoc so sanh vdi cac trinh ty ed trong ngan hang gene NCBI su dyng cdng cy BLAST. Ke't hgp cac dae diem hinh thai khuan lgc, te'bao de xac djnh ten loai ciia ddng vi khuan cd liem nang probioric cao nha't.
2.2.5. Khdo sdt dnh hudng cua thctigian ton trie den mat sdvi khuan trong che pham probiotic Ddng vi khuan LIN3 dugc nudi trong mdi Imimg NB dal mat so 9 log chi/ml, sau dd phdi trgn vdi eo cha't len men trong 48h, dich len men dugc ph6'i hgn vdi chat mang theo ty Ie Ll {the rich/khd'i lugng). Hdn hgp dupc dpng khd va danh nhuyen lhanh che' pham dgng bpt. Che'pham dugc ddng gdi 5 g/gdi va tie'n h^nh danli gia mgt so sau 0,15, 30, 45, 60 ngay, Hai NT phd'i Iron bao gom 100% bpt ggo va 50% bgt ggo+ 50% bpt mi. La'y lg che'pham hoa vdi 9mL nudc, sau dd nil 0,2ml trai deu tren dia de'm mgt sd'
2.3. Xirly sdlieu
Sd'lieu dugc tdng hgp va xu ly bang phan mem Microsoft Excel 2013 va Minitab 16.
3.K^QUAVATHAOLUAN 3.1. Phan lgp cac ddng vi khuan
Til' 75 mau thu ban dau, qua qua trinh phan lap va dinh danh sa bp bang cac phuong phap nuoi ca'y, quan sat hinli thai khuan lac va nhugm Gram, thu dugc 11 ddng vi khuan Lactobacillus (15%) dupc diing lrong cac thir nghiem tie'p theo, Cac ddng vi kluian phan lgp dugc ky hipu nhu sau: chu cai dau rien la chir cai dau rien hong ten cua vi khuan (L - Ijictobacillus); so' thii ty La Ma ta dia diem thu mau (I: Vinh Lpng, 11: Sdc Trang, III: Hau Giang); hai chu' cai hep theo la ten mau phan lgp (N: rupt non; G: rupt gia; DC: dua cai, CP:
ca phao, DL; dua leo); so thu hJ 1, 2,3 la so thii tymau thu duge.
3.2. Dac diem probiotic cua cac dong vi khuan dirge phan lap
3.2.3. Khd nang chiu pH thdp
Nhirng dae rinh quan trpng va can thiet hong vipc lua chpn cae ddng vi sinh vat san xuat che pham probioric la kha nang sd'ng sdt cua vi khuan dudi tae dung cua pH tha'p d dg day. Tai day, o cac loai gia cam, pH chi khoang 2,0-3,5 (Nguyen Diic Hung, 2006), Chinh vi vay ma nhung ddng \ i khuan dupc ehpn Iam vi sinh vat cd tiem nang probiotic duge khao sat kha nang chiu pH tha'p vdi niu'c pH:
2,5; 2; 1,5. Ket qua thi nghipm danh gia kha nang chiu pH tha'p cua nhung ddng vi khuan Lactobacillus trinh bay d Hinh 1.
Qua ket qua danh gia kha nang chiu pH a dieu kien pH=2,5 cho tha'y mat so' cua cac ddng vi khuan phan lap cd sy khac nhau va dao dpng trong khoang tii' 5,63-7,82 log chi/
ml, Ddng LllDLl cd kha nang chiu pH td't, kliac bipt cd y nghia thd'ng ke so vdi cac ddng edn lgi vdi mat sd' cao nha't la 7,82 cfu/ml. Cac ddng LIIIGl, LIIDC2 va LIDCl cd mat so tha'p nha't (5,71; 5,71 va 5,63 log cfu/ml), kliac biet cd y nghia thd'ng ke vdi cac ddng cdn lgi (P<0,001). Ddi vdi muc pH 2,0 va 1,5 lhi cac ddng LIDLl va LIIDLl cd mat so cao nha't (tutmg ling pH 2,0 la 6,58; 6,59 log cfu/ml va pH 1,5 la 5,34; 5,29 log cfti/ml).
Kdt qua hen cung tu'ong ty vdi nghien cun ciia Nguyen Thi Huyen va ctv (2014), nhdm tac gia da chi ra rang 54 ddng vi khuan probioric deu the hien su chd'ng chiu eao khi dugc u trong dich md phdng axit dg day (pH 2,0).
Ngoai ra, theo nghien cun ciia Tran Qud'e Vipt va ct\' (2009), cae ddng vi khuan lactic va nam men dupc lya chgn deu ed kha nang song trong mdi tru'dng pH thay doi tir rat tha'p (pH 2,0) deh tning tinh. Ket qua hen ding bj'ong ty vdi nghien ciin gan day cua Nguyen Manh Tua'n va ct\' (2014) cho r ^ g 4 ddng Lactobacillus LT4, NSl, BC va BB2 cd kha nang sd'ng va phat trien hong mdi trudng cd pH thap 2,0-8,0, lrong do ddng TL4 cd kha nang chd'ng chiu tha'p td't nhal len deh 95% khi pH giam edn 2,0. Nghien cun cua Nguyen Phudc Hien va Nguyen Hiiu
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C H A , .
Hiep (2014) da chiing minh hau het cac ddng
\i khuan lacrie phan Igp tir sira de va che pham men rieu hda deu cd kha nang chju dung dugc pH 3,0. Ket qua tren cao hem so vdi bao cao
cua Hoque va ct\' (2010), thec^ ^1^^ " ^ '^wrt khuan LKtobacillits phan Igp tir Bogra )"-'^^,^
x-a Khulna yoghurt co tdi: dg sinh tnnmg i' trong mdi trudng pH 2,2
I '
Hinh 1. Kha nang chiu pH thap cua nhihig dong Lactobacillus 3.2.2. Klid ttang chju mud'i mdt
Bang 1. Mgt sd vi k h u a n Lich'lv.ciliu^ (log etii/ml) theo thdi gjan trong dieu kien co muoi mat 0,3''o
Cac dong Thai pan khao sal Lncfoimr/(ills 0 gi6 (TO) IgidiTH 2gid(T2) 3gio(T3)
TB 7 4Vrli,01 7,53'±0.02 8,47<t0,02 7,82=±0,08 TB 5,974tO,02 6,99*i0.03 7,13'±0,01 ^ 10^,01 TB 6,82*±0,02 7,7r±0,03 8,68"±0,I>1 8,02''+0,02 TB 6,69^,03 7,ll'y),02 7,15'±0,01 8,10'±0,02 TB 7.10*0,04 7,9310,06 7,86'y),06 7,%'-'^,04 TB 5,90'±0,01 6,88'±0,05 7,08'-±0,02 7,88''=±0,04
100 117 120 133
TB 7,78*±0,04 6,99*±0,03 6,35'±0.02 6,07''±0,01
% 100 89.9 81,6 78,1 TB 6,95"^,06 6,99*'±0.02 5,90-4^0,06 6.00M),04
"'i. 100 101 84.9 86,2 TB 7,11^,04 7,70^10.03 8,97'±0.01 8,00*^+0,03
100 10 126 112
0,000 G'li chti- Cdc gid Iri Irung bmh trong cung cat mang chircai a, b. c. d kbac nhuu thi khuc biel co y nglna thong ke(P<U,05l
Kha nang chiu mudi mat dupc danh gia dua tren so lupng khuan lgc de'm dugc hen cac dia pehi sau khi ii d 37''C trong khoang thdi gian 0, 1, 2 va 3 gid tu'ong ung vdi thdi gian lun thiic an lrong rugt non {Kumura va ctv, 2004). Zhou va ctv (2007) da chi ra rang vi sinh vat probiotic chi phat huy tac dung cd Igi len vat chu khi ehung dinh cir va ton lai trong rudt non, day la mdi trudng chiia lhanh phan mud'i mat la cac ye'u to uc che sinh trudng ciia vi sinh vat- Theo GilUland va ctv (1984), 0,3%
dugc xem la nong do quye't dinh de sang lpc cac chung vi sinh vat cd kha nang chd'ng chiu mud'i mat.
Ke't qua Bang 1 cho tha'y dudi lac ddng ciia ndng dp mud'i mat 0,3% da anh hudng de'n kha nang sinh trudng, phat trien va lam giam ty Ip sd'ng eua cac ddng Lactobacillus. Ty le sd'ng tang til' thdi diem ban dau de'n T2 nhung giam sau dd de'n thdi diem T3 cho eae ddng sau: LIG2 LI1ICP4 va LIDCl vdi ly Ip sdhg lan lu^t chi cd 117,112 va 105%. Han nua, ty le sdhg giam d 2 ddng LIIN5 va LIIN4 tai fhdi diem Tl cho den thdi diem T3 vdi ty Ip sd'ng d T3 lan luot chi cdn 86,3 va 78,1%- Tuy nhien, sau 3 gio xu ly cac ddng edn lai deu cd ty Ip mat sd' vi khuan tane len, ty le tang cao nhat la ddng [JDl i (p^fry^
KHKT Chdn nuoi so 254 - ihui •
"iim 2020
CHAN NUOI DQNG VAT VA CAC VAN DE KHAC
va thap nhat la chung LHCPl (112%). Chung UDLl cho tha'y cd sy thich nghi cao hong mdi trudng chiia mudi mgt. Dong thdi 2 dong cdn lgi LIIDLl va L1N3 ding cho tha'y cd kha nang thich nghi trong mdi trudng chtia mudi mat vdi ty le mgt sd'sau 3 gid xir ly so vdi ban dau la 133 va 121%.
Ke't qua hien tgi luong ty vdi c6ng bo ciia Quach Diic Tinh va ctv (2014) khi nghien ciiu ve vi khuan Lactobacillus, theo dd hong 2 gid dau mgt dp vi sinh vat tang len cham vi day la giai dogn hp vi sinh vat thich nghi vdi mdi trudng cd mud'i mgt, muo'i mat tac dgng de'n cae te'bao vi sinh vgt lam han che'sy phat hien ciia chiing. Tuy nhien, hong 2 gid tie'p theo, tdc dp phat trien ciia cac vi sinh vat tang len dang ke nguyen nhan la do mgl so protein va enzyme tren mang te'bao vi khuan lactic cd kha nang trung hda tae dgng ciia mud'i mat bSng each cat dut lien ke't N-acyl giiia gd'c steroid va chudi amino axil ben cua axit mat.
Theo Tran Qud'c Vipt va ctv (2009), cac chung vi sinh vgt thiv nghipm deu cd kha nang ton tgi trong mdi tru'dng chu'a mud'i mgt vdi ndng dp 0,3%. Kel qua nghien ciiu cua Duong Nhat Linli va ctv (2011) d ndng dp mudi mat 0,3%
sau 3 gid hau he't cae chung cd ty Ip sd'ng cao tir 90 de'n 116%. Dieu nay cd y nghia ra't quan hpng ddi vdi eae vi sinh vat probiotic vi mud'n phat huy tac dung cd lgi len vgt chu khi chiing djnh cu va ton tgi trong nipt non (Havenaar va Huis, 1992). Ngoai ra, ket qua tren eao hon so vdi nghien cun ciia Nguyen Manh Tua'n va ctv (2014), 2 dong BC va BB2 khi ii vdi mud'i mat 0,3% sau 3 gio mgt sd' dgt dugc luong iing chi CO 6,45 va 4,70 log cfu/ml. Tlieo Hoque va ctv (2010), cac ddng vi khuan Lactobacillus phan lgp bi Bogra yoghurt va Khulna yoghurt cd ldc dp sinh trudng cham hong mdi tmdng pH thap 2,2 nhung cac dong nay ed kha nang stnh tmdng va phat trien duge trong mdi trudng CO bd sung 0,05-0,3% mud'i mat.
3.2.3. Kttd ndng khdng khdng sinh Qua Bang 2 cho tha'y d nhdm khang sinh uc che' qua trinh hinh thanh tc bao vi khuan lAnipidllin) thi chi cd 3 ddng (LIDCl, UDLl,
LIN3) cd kha nang khang lai chie'm t\' le 42,9%, sd cdn lai cho ke't cjua nhgy cam \'eu.
O nhdm khang sinh lic die' qua hinh dieh ma ciia vi khuah thi khang sinh Kanamycin, Chloramphenicol cho ciing ket qua 100% nhgy cam ye'u, cdn Gentamydn cho ket qua 100%
nhay cam vira. Rieng khang sinh Tehacycline cho 2 ddng (LIG2, LIN3) khang Iai khang sinh chie'm 28,6%, edn Igi la so ddng eho ket qua nhay cam ye'u. Do dd cd the sir dyng LIN3 cho gia cam khi dang su dung 2 loai khang sinh Ampidllin va Tehacycline hong chan nudi.
Bang 2. Dudng kinh vong vo khuan (mm) va tinh nhgy cam khang sinh ciia cac dong
Lactobacillus Cac dong Khang sinh
Laclabacillus KM GM TC AMP LIDCl 10,67 (MS) 23,00 (S) 7,67 (MS) 6,33 (R) LIDLl 12,00 (MS) 22,33 (S) 8,33 (MS) 6,33 (R) L1G2 11,67 (MS) 22,67 (S) 6,67 (R) 7,00 (MS) L1N3 9,67 (MS) 19,00 (S) 6,00 (R) 6,67 (R) LIICPl 11,67 (MS) 2233 (S) 7,67 (MS) 10,33 (MS) LilDLl 10.0O(MS) 18,00(5) 9,00 (MS) 10,67 (MS) L111CP4 8,67 (MS) 18,67(5) 10,33 (MS) 11,33 (MS)
KM: Kanamycin (30 pg/ml); GM: Gentamycin (W pg/
ml); TC: Tetracycline (30 pg/ml); AMP: Ampicillin (10 pg/ml). Tlieo tieu chudn cho Lactobacillus ciia Georgieva vd ctv (1966): KItdng R<7inm; Nhay cam yeu 7<MS<16mm; Nhgy cdm vira 16<S<25mm; Nhgy cdm manh 25nun<SS.
3.3. Dinh danh dong vi khuan Lactobacillus Dua hen ket qua khao sat tiem nang pro- bioric chpn ra dupe ddng Lactobacillus LIN3 ed riem nang probioric cao de Ihuc hien phan ling PCR vdi cap mdi 27F-1492R, khue'ch dai vimg 16S rRNA cua vi khuan. San pham PCR ed kich thude khoang l.SOObp, rie'n hanh giai trinh tu gen va su dung phan mem BlastN de so sanh vdi trinh ty ADN ciia eae ddng vi khuah cd hong ngan hang dii lieu NCBI.
Ket qua eho thay trinh ty doan gene cua ddng Lactobacillus LJK3 tuong ddng vdi doan gene 165 rRNA ena vi khuah Lactobacillus plantarum vdi do tuong ddng la 99,88% va dp bao phil 100% nhu duge trinh bay trong Hinh 2. Do dd, ddng Lactobacillus LIN3 dupc dinh danh nhu loai Lactobacillus plantarum.
CHAN.
Hinh 2. Ket qua dinh danh ten Ioai cua dong Lactobacillus LIN3 theo dp tuong dong dogn gen 16S rRNA
Tinh den thdi diem hipn tai, tren co so du lipu NCBI da cd hon 106.262 trinh ri^
nucleotid ciia vi khuan L. plantarum, trong dd ed 3 trinh ty bp gen hoan dunh vdi chieu dai 3.267.626bp cua ehung L. plantarum SN35\
(Noda va ch', 2018), 3.252.258 bp cua ehung L plantarum SRCM1O2022 va 3 3SS,033 cua chung L plantarum JSA22 va hon 990.(X)0 trinh hi gen 165 rRNA (NCBI, 2019).
3.4. Anh hudng ciia thdi gian ton trii den mat so vi khuan trong che phaim probioric
Sau khi san pham dugc ddng gdi bao quan, tgi ngay dau rien, kiem tra mgt so yi khuan va sau dd 15 ngay kiem tra lgi cho den khi kel thiic qua trinh khao sat. Ket qua Bang 3 cho thay, sau thdi gian tdn trii mgt sovi khuan thay ddi theo thiii gian va ed sy khac bipt giua 3 NT. Cy the, sau 15 ngay tdn tni a nhipt dp phong, mgt sd'vi khuan trong che'pham phdi hpn iO()"i. bpt ggo cao hon so vdi NT 50% bgt ggo va 50".. bgt m\ cung nhu 100% bgt mi do'i vdi L. plantarum.
Bang 3. Mat sd%^ khuan (log cln mil ciia che pham probiotic tir L. Plantamm theo thdi gian ton trii Ccr chat bao auan
10(1% bpt g<io 100% t«;)t mi 50% bijI ggo * 50",, bgi mi SEM
P
6,13' 4,05- 4,90*' 0,028 il 0(XI
15 nK^v 5,07' 3.93- 4,5.T 0,043 0,000
4,37*
3,70' 4,60*
0 023 0.000
45 neav 4,43' 3,61*' 4,53' 0,049 0,000
60 neikv 4,30'''
3,65'' 4,80- 0,162 0,007 Gin chw Cac gia tri Irung hhib Irong cdng moi
ke(P<0.05)
Ddi \'ai che'pham chiia L plantarum, khi sir dyng 100% bpt ggo lam chat m^mg, mat sd' sau ton trii vugt trpi so vdi die pham chiia 100% bpt mi va che' pham chua 50°o gao va 50% bgt mi d thdi diem 15 ngay. Tuy nhien, khi khao sat d thdi gian dai hon (60 ngay), mat sd' hong che'pham 100% bgt ggo va l>pt mi cd sy syt giam rd rpt hong khi che'pham phdi hgp 50".. bpt ggo \'a 50% bdt mi van duy tri dugc mat so te'bao dn dinh. Tgi thdi diem cud! ciing cua qua trinh khao sat, mat sd' L. plantarum trong che pham 100% bgt mi chi 3,65 log chi/
ml va che'pham chiia 100",> bpt gao dgt 4,30 log efu/ml. Trong khi dd, mat s d d che'pham phdi trgn ggo va bgt mi la (4,S log cfu/ml) cao hem so vdi che pham ehiia 100% bgt ggo va bgt mi.
M.it ^d trong cac die pham cd sy sut giam gan 30"i. khi so sanh \oi thdi diem ban dau (6,13
,(,' chu cm a. b. c, d kbac nhau lhi khdc biet co y nglna tho'ng log cfu/ml) va ed sy khae bipt y nghTa tho'ng ke vdi mii'c y nghia P<0,05. Mat so vj khuan L. plantarum trong nghien ciiu hrong ddi tha'p ban so vdi nghien ciiu ciia Vo Thi Thiiy Hue va ctv (2018) khi thir nghipm cac ty le phoi trgn tai cac NT bao gdm: 1007.. cam ggo; 95% cam hip va 5% cam ggo; 90% cam bSp va 10% cam gao;
85% cam bap va 15% cam ggo dgt mat so 11,41 log cfu/ml. Nguyen nhan cd the la do trong cac chat mang dupe phoi h-pn hen ed lhanh phan dinh dudng rirong ddi phii hgp giup vi khuan on dinh va phat hien trong thdi gian bao quan.
Kel qua phii hgp vdi nghien ciiu ciia Tran Thi My Trang (2006) cho rang, mat so khuan !ac trong hai tuan dau va khac bipl khdng y nghia d mirc 5% sau 3 hian hi 5,92.10^ chi/g 6 thdi diem ban dau giam cdn 3,95.10^ chj/g. Nhu vgy, ty Ip va thanh phan chat mang dugc phdi
KHKTChdn nuoi.sd 254 • thdng 2 ndm 2020
Ciinis NUOI DQNG VAT VA CAC VAN DE KHAC
hpn cd anh hudng de'n mat sd'vi khuan L. Plan- tarum hong qua trinh nghien cun. Mat khac, trong thi nghipm nay viec bo sung bgt mi trong thanh phain cha't mang ciing cd anh hudng lot de'n mgt sd' vi khuan sau 60 ngay ton trii.
4.K^LUAN
Tir 75 mau thu ban dau, qua qua trinh phan Igp tuyen chgn, quan sat te' bao va kiem tra dac h'nh sinh hda thu duge 11 ddng vi khuan Lictobacillus cd hogt tinh probiotic, trong dd cd 5 ddng LlDLI, L1G2, LIN3, LIIDLl, LIIICP4 mang nhung dac diem cua vi sinh vat probiotic. Tuy nhien, chi cd ddng LIN3 cd tiem ning cao nha't ve chju pH tha'p, muoi mat va kha nang khang khang sinh va ket qua dinh danh ddng LIN3 vdi ten loai la L. plantarum.
Ket qua danh gia anh hudng cua thdi gian ton trii san pham probiotic lu' L. plantarum vdi ty le phdi hgn 50% bgt gao va 50% bpt mi cho tha'y cd su gia tang mat so sau 60 ngay tdn tni' d dieu kipn nhipt dp thudng. Che pham nay dang dugc sir dyng nham danh gia hipu qua trong phdng bpnh Salmonella gay ra tren ga.
LIJICAMON
Detdi ndy dirac tdi tra bdi Du dn Nang cap Trirdng Dgi bgc Cdn Tha VN14-P6 bdng ngudn vdn vay ODA tir chinb pbii Nhdt Ban.
TAILI£UTHAMKHAD
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