Cong nghe sinh hgc & Giong cdy trdng
TUYEN CHON CHUNG VI KHUAN LACTIC CO TIEM NANG ITNG DUNG TAO CHE PHAM SINH HOC (PROBIOTIC)
BO SUNG VAO THlTC AN CHAN NU6l
Nguyin Thi H^ng Nhung, Le Thi Thuffng, Nguyin Thi Thu Hang, Nguyin Thi Huygn Trudng Dgi hoc Lam nghiep
TOM TAT
Probiotic la nhimg vi sinh vat s6ng khi dup-c bo sung vao ca the voi lieu luong dii lan se tao ra lgi ich d6i vm siic khoe ciia vat chti. Muc dich nghien ciru nay la phan lap dugc vi khuan lactic tir cac mlu thirc pham len men danh gia mgt so dac tinh probiotic cua chiing de ung dung tao che pham probiotic bo sung vao thiic an chan nuoi. Mirai chung vi khuan da dugc phan lap su dung m6i truang MRS (de Man, Rogosa & Sharpe). Su dung phuang phap khuech tan dia thach, 3 chung C2, LA6 va LT7 co kha nang doi khang tot nhat voi ca 3 lo^i vi khuan gay benh: E. coli, Samonella sp, Shigella sp. Nhung chung nay tiep tuc dugc danh gia kha nang sinh enzyme ngoai bao (protease, cellulase, amylase). Ket qua cho thay, chiing LT7 va C2 c6 kha nang sinh enzyme ngoai bao cao han chung LA6. Hai chung LT7 va 0 2 dirge danh %\k kha nang chiu pH thip (tir 2 din 4), chju muoi mat (0,5 - 3%), khang 3 loai khang sinh ((Tetracyclmc, Gentamycin, Streptomycin) n6ng do 10 - 50 Ug/ml, nhan thay chiing LT7 co kha nang chiu, pH thap, mu6i mat va khang sinh cao ban chung C2. Chiing LT7 da dirge lira ebon la chiing probiotic tiem nang va dirge dinh danh la Lactobacillus plantarum d^a trSn trinh tu gen 16S rRNA (1445 bp) da dugc phan tich. Nghien cum dac tinh sinh ly, sinh boa ciia chiing nay cho ket qua: tk bao hinh que dai, khong smh catalase, co kha nang len men lactose.
Tir khoa: Lactobacillus plantarum, muoi m^t, probiotics thuc an chan nuoi, thirc phSm len men.
1. DAT VAN DE
Probiotics dong mpt vai trd quan trpng, la chat thich hgp nhat de thay thk khang sinh vi nhieu lpi ich ciia nd va dugc su dung nhu chat kich thich tang trudng cho ngudi va ddng vat bao gdm gia cSm va thiiy san (Palamidi I, 2016). Probiotics dugc dinh nghia la "cac sinh vat sdng ma khi dugc dua vao ca thi vdi lugng du Idn se tao ra lgi ich v8 sue khde cho vat chu" (FAG/WHO, 2002). Probiotics cung dugc dinh nghia nhu la thiic an bd sung vi sinh vat sdng CO Igi ddi vdl vat chu thong qua tang cudng su can bang trong dudng mot va do dd nang cao hieu qua su dung thiic an, hip thu chat dinh duong, t6c dp tang trudng va mang l^i hieu qua kinh tl cho nganh chan nuoi gia c£im (Abd El-Hack ME, 2017).
Cac loai vi sinh vat pho bien dugc tim thay h-ong cac san phSm probiotics hien nay la mot sd loai vi khudn cd lgi, n4m hoac nSm men va thudng gap nhat la cac chung Bacillus subtilis, Lactobacillus, Bifidobacterium va Streptococcus, ngoai su gia tang hoat ddng cua chung ciing lam giam sd lugng vi Ichuin cd hai nhu: Salmonella typhimurium. Staphylococcus
aureus, Escherichia coli, Clostridium perfringens... (lannitti T, 2010).
Vi khudn lactic (LAB Lactic Acid Bacteria) la vi sinh vat phd biin dugc phan lap tir mdt sd ngudn chmh nhu: thuc phSm len men, dat va thuc vat. Chiing ciing la mpt phk ciia nhdm vi khuan ed lgi sdng trong dudng tieu hda cua dpng vat tren can va thuy hai san.
LAB la vi khuan Gram duong, t l bao hinh que hoac cau; Chung khdng di ddng, khong sinh bao hi, Ididng Idiii nitrate va catalase, oxidase am tinh. Nhirng vi khu4n nay sii dung carbonhydrate nhu la nang lugng chinh va tao ra axit lactic la san phim duy nhdt hoac nhu la san pham chinh khi kit thiic qua ttinh ciia qua hinh trao ddi chat. LAB gdm cac chimg Streptococcus, Enterococcus, Lactobacillus, Aerococcus, Carnobacterium, Leuconostoc, Lactococcus va Pediococcus (Ring0 E, 1998).
LAB cd the san xuat axit, hydrogen peroxide, bacteriocin va cd ti6m nang iing dung Idn lam chdt bao quan sinh hgc trong thuc phdm (Aslim, 2005). Da cd cac nghien ciiu sii dung cac chiing LAB khac nhau d l tao che pham probiotics, chu yeu la Lacobacillus 18 TAP CHI KHOA HOC VA CONG NGHE L A M NGHIEP SO 2 - 2019
Cong nghe sinh hgc & Giong cdy trong va Bifidobacteria (nhdm vi khuan hdi sinh
sdng trong dudng rugt cua ngudi va dgng vat), cho thay kha nang dieu tri benh tdt (Lavanya, 2011).
LAB dugc coi nhu la mpt nhdm vi khuan probiotic chinh cho con ngudi va ddng vat (Chen, 2005). Vi chiing an toan, cd the chiu dugc axit va mudi mat. Chung bam dinh tot vao bieu md nipt ciia cac vat chu va cd the irc che su phat trien cua vi khuan gay benh nhu Escherichia coli va Salmonella do la nhiing vi sinh vat gay benh chinh trong dudng rugt ciia ga (Murry, 2004). Chiing cd the duy tri kha nang tdn tai trong qua trinh che bien va bao quan thiic an gia siic (Lin, 2007). Vi vay, nd luc de giam khang sinh trong chan nudi bang each su dung probiotic dang ngay cang dugc quan tam, nhu mdt giai phap thay the hieu qua ve chi phi kiem soat benh dpng vat va cai tien nang suat vat nudi (Renter, 2001).
Muc tieu cua nghien ciiu nay la tuyen chpn dugc cac ddng LAB tir nguon thuc pham len men de lua chpn cac chung vi sinh vSt tiem nang cd the iing dung lam che pham vi sinh bd sung vao thiic an chan nudi, chiing tdi da thuc hien nhiing thi nghiem sang Ipc nhu: thu nghiem hoat tinh ddi khang vdi vi khuan gay benh, kha nang sinh emzyme ngoai bao, kha nang chiu axit va chiu mudi mat, kha nang khang khang smh.
2. PHl/ONG PHAP NGHIEN CUtJ 2.1. Vat lieu nghien cihi
Cac mau thuc phdm len men (dua mudi, ca mudi, mang chua, nem chua...) dugc thu mua tai khu vuc Xuan Mai, Chuang My, Ha Ndi vao thdi gian tu thang 4 - 12/2017.
Cac chiing vi khudn kiem dinh: E. coli, Samonella sp. Shigella sp nSm trong bd suu tap giong vi sinh vat cua Bd mdn Cdng nghe Vi sinh - Hda sinh, Vien Cdng nghe Sinh hgc Lam nghiep.
2.2. Phuong phap nghien cihi 2.2.1. Phdn lap vi lihudn lactic
Pha loang mau den cac ndng dp khac nhau:
10"', 10"^ 1 0 \ 10^... hiit 0,05 ml dich mlu d mdi ndng dp nhd len dia peptri chiia mdi
trudng thach MRS (pH 6,5; khu trung 1 2 1 ^ / 1 5 phiit). Nudi d 37°C frong 48 gid.
Kiim tra su xudt hien cac khuan lac fren dia petti, tach va thudn khilt cac khudn lac cd vdng trong sudt xung quanh chiing (axit phan giai CaCOg tao vdng trong).
2.2.2, Khd ndng khdng vi lihudn gdy b§nh Ba chiing vi khudn kiim dinh sii dung la: E.
coli, Salmonella sp, Shigella sp dupe nuoi qua dem d 28°C ttong mdi trudng Meat-Peptone (MP) Idng. Sau dd, vi khudn kiim dinh dugc cay ttai tten mdi trudng MP agar va due nhiing lo thach dudng kmh 9 nun. LAB dugc nudi trong 2 ml MRS long, dudi dieu kien yem khi ttanh hinh thanh H2O2, tdi giai doan pha tinh (khoang 48 - 36 gid). Dung dich dugc ly tam 10.000 vdng/phiit ttong 15 phiit d 4°C. Loai bd phan can chiia xac te bao vi khuan, lay phan nude ttong cua dung dich sau ly tam, dieu chinh pH tdi 6,5 bdng NaOH 0,1 N thu dupe dung dich bacteriocin thd. Ldy 0,1 ml dung dich bacteriocin thd nho vao mdi giSng ciia dia thach da chiia ddng vi khuan chi thi. U mau d 4''C ttong 30 phiit. Sau do, li d 37°C cho vi khudn chi thi phat triin. Ddi chiing: Gieng chiia 0,1 ml mdi trudng MRS Idng. Hoat tinh khang khuan ciia nhirng ddng phan lap dupe tinh bang viing vd khuan quanh mieng gieng tten dia (Buntin N, 2008).
2.2.3. Khd ndng sinh enzyme ngogi bdo Thu nghiem kha nang sinh mpt sd enzyme:
amylase, protease va cellulase thdng qua cac ca chat tuang iing: tinh bdt, casein, CMC (Carboxymethyl cellulose) bang phuang phap due Id thach.
Chuan bi mdi tnrdng thach (15% w/v) co bo sung 1% chat cam iing thich hop (tinh bdt, casein, CMC), due 16 thach cd dudng kinh 0,9 cm. Nudi cac chung vi LAB tren mdi trudng MRS Idng trong khoang 48 - 72 gid, ly tam 8.000 vdng/phiit thu dich enzyme thd. Nho 0,1 ml dich enzym vao cac 16 da due, de d 4''C trong vdng 30 phiit, sau dd li d 37 C trong 24 gid. Nhudra bang thudc thii lugol, coomassie brilliant blue, congo do de phat hien vdng phan giai CO chat tuang iing la: tinh bdt, casein,
TAP CHI KHOA HOC VA CONG NGHE LAM NGHIEP SO 2 - 2019
Cong nghe sinh hgc & Giong cdy trong CMC. Do vong phan giai D-d (mm), D la dudng kinh vdng ngoai, d la dudng kinh Id nho dich (Trdn Thanh Thuy, 1998).
2.2.4. Thu- nghiem hhd ndng chiu axit vd muoi mat
a. Khd ndng chiu pH thdp
Cac chung LAB dupe nudi qua dem 1 6 - 1 8 gid. Ly tam 5000 vdng/10 phiit d 4''C, thu can va huyin phu lai ttong PBS (photphatse buffered saline) pH 7,0 tao thanh huyen djch cd dp due McFarland 0,5 (0,5 ml dung djch BaCb 1% va 99,5 ml dung dich H2SO4 1%, OD625 - 0,08 - 0,1) ttrang duong 10^ CFU/mL Chuyen dich vi khuan vao binh ndn chiia nude mudi sinh ly (0,9% w/v) va chinh pH co gia tti Idn lugt la: 1,5; 2; 2,5. U d 37''C. 6 moi thdi diem 1 gid, 2 gid va 3 gid lay 1 ml dung dich mau thu trung hoa ve pH ^ 7. Tien hanh pha loang den mat dp thich hgp dem dugc ttong dung dich dem va ttai tten dia peptti chiia moi ttudng MRS agar. U d 37''C, ttong 24 - 48 gid.
Dem khuan lac va tinh sd dan vi sdng ciia vi khuan khao sat. Mdi thi nghiem tien hanh 3 lan (Cukrowska B, 2009).
b. Khd ndng chiu muoi mat
Cac chiing LAB dugc hoat hda ttong m6i trudng MRS, li qua dem khoang 16 - 18 gid.
Vi khuan dupe pha loang den dp due McFarland 0,5 tuong duong 10^ CFU/mL. Ldy 1ml dich vi khuan ly tam, cho tiep xiic vdi dich mudi mat vdi cac ndng do lan lupt 0,5%, 1%, 2%, 3%) ttong 1 gid, 2 gid, 3 gid. Sau d6 ly tam rua sach va huyen phii te bao vi khuan bang dich PBS (pH = 7). Tien hanh pha loang din mat dp thich hgp va cay trai tten dia peptti chua mdi trudng MRS agar. U d 37''c, ttong 24 - 48 gid va dem khuan lac va tinh sd dan vj
sdng cua vi khuan khao sat. Moi thi nghiem tien hanh 3 lan.
Phdn tram sdng sot ^ Ni/Nx xioo, Ni: S6 lugng khudn lac dem dugc tai thdi diem nuoi cdy (1, 2, 3 gid), Nx: Sd lugng khuan lac dim dugc tai thdi diim 0 gid (Cukrowska B, 2009).
2.2.5. Ddnh gid do nhgy cdm khdng sinh Dp nhay cam khang sinh ciia cac chiing vi khudn da phan lap dugc danh gia bang phuang phap khulch tan tten thach theo hudng din cua CLSI (Clinical and Laboratory Standards Institute, 2014). Cac khang sinh dupe sii diing bao gom: Gentamycin n6ng dp: 10 M-g/ml;
Streptomycin, Tettacylin ndng do: 10, 50
|j.g/ml. Do kich thudc vdng vd khuan va danh gia do nhay cam khang sinh (khang R, trung gian I va nhay cam S).
2.2.6. Phuffng phdp dinh danh chung vi khuan a. Binh danh bdng phuang phdp sinh hpc phdn tu:
Tach chiet DNA va khuyech dai gen 16S rRNA bang phan iing PCR (polymerase chain reaction). Viing gen 16S rRNA dupe khuech dai bang cap m6i ph6 bien:
Moi xudi 27F: 5'- AGA GTT TGA TCC T G G C T C A G - 3 ' ;
M6i ngugc 1525R: 5'-AAA GGA GOT GAT CCA GCC - 3'.
Thanh phdn phan iing PCR:
Thanh phan Th€ tich (^1) Master mix 2x
Primer xuoi (20 pmol/^tl) Primer nguac (20 pmoI/|il)
DNA khuon H2O
10 2 2 2 vira du 20 Chu ttinh nhiet cho phan iing PCR:
Biro'c 1 Bu-ffc 2: lap lai 40 chu ky
94"C:5phut 94"C:45giay 54,5T: 45 giay 72''C: 1 phut 72"C: 7 phiit ^"cT^
b. Phuong phdp nghien ciru .su dung cong cu BLAST vd phdn tich phdt sinh lodi
Dvt lieu trinh tu nucleotide 16S rRNA cua chiing vi khuan dupe gui vao ca sd dii lieu tten NCBl de xac dinh loai gdn nhdt da biet cua mpt phdn ttinh hr 16S rRNA thu duoc, tim
kiem cac trinh tu tuang ddng bdng cdng cu BLAST (SaeediM, 2015).
Cay phat sinh loai dugc xay dung bdi phuang phap thdng ke "neighbor-joining" vdi phien ban Mega 6.0 (Kumar M, 1980).
20 TAP CHI KHOA HOC VA C 6 N G NGHE LAM NGHIEP SO 2 - 2019
Cong nghe sinh hgc & Giong cdy trong c. Xdc djnh dgc tinh sinh ly, sinh hoa: Nhudm
Gram; Quan sat hinh dang khuan lac, te bao;
phan ling catalase, kha nang len men cac loai dudng (Tran Thanh Thuy, 1998).
3. K E T QUA VA T H A O LUAN 3.1. Ket qua phan lap cac chung vi khuan
Tir cac mau thuc pham len men (rau qua len men, nem chua, thit chua) da phan lap dugc 10 chung vi khuan. Mudi chung nay duoc quan sat hinh thai khuan lac va mpt sd dac tinh sinh hda, sinh ly cho ket qua: khuan lac cd mau trang siia, trdn, nhSn, mep tron, b l mat udt va xung quanh c6 vdng trong sudt do CaCO? bi phan giai bdi axit lactic, te bao hinh que dai, gram duang, khdng sinh bao hi, catalase am tinh.
3.2. K i t qua khao sat hoat tinh doi k h a n g vdi vi sinh vat Idem dinh
D I han chi su phat frien cua vi khuan gay benh, ngoai viec canh ttanh vi tri bam dinh thi kha nang ddi khang vdi vi khuan gay benh la mdt dac tinh rat quan ttgng ciia cac chiing vi sinh vat dugc chgn lam probiotics. Tu 10 chiing phan lap dugc, tuyen chpn chung cd boat tinh ddi Ichang manh vdi 3 chiing vi khuan kiem dinh gdm E. coli (gay tieu chay, viem mpt, tao dpc t6 dudng rugt d dpng vat).
Salmonella sp (sdt thuong han, gay tieu chay d ngudi va vat nudi) va Shigella sp (true khuan li gay benh dudng rugt, tieu chay d ngudi va vat nudi). Ket qua thu dugc d bang 1 va hinh 1.
Bang 1. Ket qua vong khang vi sinh vat kiem dinh STT
1 2 3 4 5 6 7 8 9 10
Ki hieu cliiing LAI LA2 C2 LA4 LAS LA6 LT7 LA8 LA9 LAIO
Duimg Idnli E. coli 10 ±0,01 13 ±0,02 20 ±0,01 10,5 ±0,01 12,5 ±0,01 19 ±0,02 22 ±0,01 15,5 ±0,01 15,5 ±0,01 14 ±0,01
vong Idiang Miuan Salmonella
11,5 ±0,01 9 ±0,01 19 ±0,01 14 ±0,02 10 ±0,01 16,5 ±0,01
18 ±0,01 13 ±0,02 12 ±0,01 13,5 ±0,02
( D-d) mm Shigella 10 ±0,01 12,5 ±0,01 17,5 ±0,01 9,5 ±0,01 6 ± 0,01 18 ±0,01 18,5 ±0,01 14,5 ± 0,01 16 ±0,01 15 ±0,01 D: Dudng kinh vong khdng khudn; d: duang kinh 16 thach; n = 3
Hinh I. Kha nang doi khang vdi vi khuan Idem dinh ciia cac chiing Lactic a. E coli b. Samonella sp c. Shigella sp Hau het cac chiing phan lap dugc dieu cd
kha nang khang cac vi sinh vat kiem dinh.
Trong dd, cd 7 chiing khang tii yeu den trung binh: vdng khang khuan do dugc tii 6 -^ 16 mm va 3 chiing C2 , LA6 va LT7 cd kha nang khang manh vdi hau het cac chiing vi sinh vat gay benh hi 16,5 -^ 22 mm. Cu the, vdng khang
khuan do dugc ciia 3 chiing C2, LT7, LA6 ddi vdi Ecoli sp la: 18 -^ 22 mm, ddi vdi 2 chung Samonella sp va Shigella sp la: 16,5 ^ 19 mm.
Nhu vay, 3 chiing vi khudn: C2, LA6, LT7 dupe chpn Ipc de thuc hien nhihig nghien cim tiep theo.
T ^ P CHI KHOA H O C VA CONG N G H E L A M N G H I E P SO 2 - 2019
Cong nghe sinh hgc & Gidng cdy trong 3.3. Kit qud xdc dinh khd ndng sinh enzyme ngogi bdo
Kha nang sinh enzyme ngoai bao ciia cac chung probiotics cd vai ttd rdt quan ttgng, nhdm hd tra tieu hda thiic an, chuyen hda cac chat khd tieu thanh cac chat d l tieu, phan buy
cac thiic an du thua ttong chuong nudi lam giam mui hdi thoi cua chudng ttai.
Tir 3 chiing lactic da dugc chpn Ipc d buoc tten, khao sat kha nang sinh enzyme ngoai bao (amylase, protease, cellulase). Ket qua thu dugc nhu bang 2 va hinh 2.
Bang 2. Hoat tinh enzyme ngoai bao cua cac chung vi sinh vat Dudng kinh vong phan giai (D-d) mm
Ki hieu chiing ^
Tinh hot CMC Casein
C2 LT7 LA6
20 ±0,01 20 ±0,01 16 ±0,01
25 ±0,01 30 ±0,01 16,5 ±0,01
19,5 ±0,01 30 ± 0,01 16,5 ±0,01 D. Dudng kinh vong phdn gidi; d- duang kinh lo thach
Hinh 2. Kha nang sinh enzyme ngoai bao ciia cac chung Lactic a, a': Amylase b. Cellulase c. Protease Ca 3 chung deu co kha nang sinh enzyme
ngoai bao d miic tir trung binh den manh. Cu the vdng phan giai ca chat ciia cac chung: LA6 tir 16 -^ 16,5 mm (kha nang sinh enzyme d miic trung binh); C2 tu 19,5 -^ 25 mm (kha nang sinh enzyme d miic cao); LT7 tu 20 -^ 30 mm (kha nang sinh enzyme d miic rat cao). Do vay, chpn lua 2 chung la: C2, LT7 de tiep tuc nghien ciiu tiep theo.
3.3. Ket qua xac dinh kha nang chiu p H thap va muoi mat
Tien hanh thu nghiem cac chiing tten d 3 miic pH 2, 3, 4 va mudi mat d cac ndng dp 0,5%, 1%., 2%, 3 % ttong khoang thdi gian tu 1 den 3 gid. Theo hinh 3, dudi tac dpng cua pH thap va mudi mat ca 2 chiing khao sat deu cd xu hudng giam ti le sdng khi keo dai thdi gian xii ly (0 den 3 gid), giam do pH (4 xuIng 2) va tang ndng dp mudi mat (tir 0,5% din 3%).
Ta thay, sau 3 gid nudi u, sd lupng te bao s6ng sot d pH 2 ciia 2 chiing la kha cao: dat >
50% (chung LT7) va 45% (chung C2), d pH 3 va 4 thi ti le nay idn lupt la: > 60% va > 70%o
ddi vdi 2 chiing. CJ ndng dp mu6i mkX 0,5%, sau 3 gid nudi li, 2 chung c6 ty le s6ng cao tten 90% (chung LT7) va gdn 90% (chung C2).
Cdn d ndng dp mudi mat cao 3%) thi sau 3 gia ty le sdng la > 50% ddi vdi ca 2 chung (Hinh 3). Nhan thdy chiing LT7 cd kha nang chiu pH thap va mudi mat tot hon chiing C2.
Theo Zhou va cong su (2007) cho rang gia tri pH 2 va pH 3 dugc xem la gidi han quyet dinh ttong sang loc cac chiing vi sinh vat co tiem nang sir dung lam probiotic. Ket qua nghien ciiu cua Kim va cdng su (2007) cho thay khi xir ly bang dich da day pH 2,5 co 3/7 chung vi khuan cd kha nang chiu mdi trudng acid, tuy nhien, ti le song ciia cac chung nay giam manh chi cdn 0,8%) din %% sau 30 phut xii ly va tiep tuc giam manh sau 2 gid xir ly (tii 0,04% din 0,2% so vdi ban ddu). Nhdm tac gia Duong Nhat Linh va cdng sir (2011) khi phan lap dugc cac chung probiotic cd ty le sdng >
60%> sau 3 gid d pH 2,5 va d ndng dp mudi mat 0,3%) sau 3 gid cac chung cd ty le sdng > 90%.
22 TAP CHI KHOA H O C VA CONG N G H E LAM N G H I E P SO 2 - 2019
Cong nghe sinh hoc & Giong cdy trong Tucmg tir ket qua nghien cuu ctia chiing toi,
nghien ciiu cua Kim va cong sir (2007), Trjn Qudc Viet va cong su (2009) cung cho thdy
cac chiing vi sinh vat thii nghiem deu co kha nang t6n tai trong moi truang chiia muoi mat voi nong dp 0,3%.
I I I i i i I i
1 1.11 •^^;.l I .L.i
._M ^
•":; I i i
Hinh 3. Ty 1^ $6ng cua cac chiing vi khuan Lactic thu nghiem theo thdi gian 1,2 va 3 gid d pH 2; 3; 4 va muoi mat 6" cac nong do 0,5%>; l%i; 2%.; 3%i
3.4. Ket qua kha nang de khang khang sinh Tien hanh thii nghiem vdi 3 loai khang sinh:
Gentamycin, Tetracycline, Streptomycin la 3 loai khang sinh dugc diing phd bien de chiia cac benh thudng gap d gia siic va gia cam (Lavanya, 2011).
Sii dung vdi lieu tdi thieu cd kha nang lie che su phat ttien cua vi khuan gay benh (5 - 30 M-g/ml): Gentamycin (Ge) ndng do 10 pg/ml;
Tettacycline (TT) ndng dp 50 pg/ml, 10 p-g/ml Streptomycin (St) ndng dp 50 pg/ml, 10 pg/ml.
Ket qua thu dugc d bang 3.
Bang 3. Ket qua khang khang sinh cua cac chung vi khuan lactic
1 2
Kl hieu mSu
C2 LT7
Tetracycline (|ig/ml) 10 R R
50 S R
Gentamycin (Mg/ml)
10 R R
Streptomycin (|ig/ml) 10 SO R S R R Ghi chu: R: Khdng: S- Nhay cam.
T4,P CHI KHOA H Q C VA C 6 N G N G H E L A M N G H I E P SO 2 - 2019 23
Cong ngfie sinh Itgc & Giong cdy trong
Hinh 4. Kha nang khang khang sinh ciia cac chiing vi khuan Lactic Hdu hit cac chiing vi sinh vat dugc tuyen
chpn diu cd kha nang khang manh ca 3 loai khang sinh khao sat: Stteptomycine, Tetramycline, Gentamycin d ndng dp 10 pg/ml. Nhu vay, trong qua trinh dieu tti benh thudng gap cho vat nudi, cd the phdi hgp sir dung cac chung vi sinh vat nay vdi cac chat khang sinh (Tetracycine, Streptomycm, Gentamycin) do cac khang sinh nay Ididng co kha nang tieu diet va lam mat di hoat tinh cua cac chung vi sinh vat hiiu ich da tuyen chpn tten trong dudng rugt ciia vat nudi.
Chung LT7 cd kha nang sinh enzyme ngoai bao cao, kha nang chiu pH thap va muoi mat va khang khang sinh tdt nhat ttong sd cac chiing phan lap dugc. Chung LT7 dupe chgn la chung probiotic tiem nang iing dung tao chi pham sinh hgc bd sung vao thiic an chan nuoi.
Chung LT7 dugc nghien ciiu dac diem sinh hgc va dinh danh den loai.
3.4. Ket qua dinh danh chung
Dac diem sinh ly, sinh hda cua chung LT7 thi hien d bang 4 va hinh 5.
Bang 4. Dac diem hinh thai, sinh ly va sinh h6a ciia chiing LT7 Hinh dang khuan lac Hinh dang te bao Gram Catal;
Khuan lac hinh tron, True Idiuan (hinh que tting siJa, be mat loi dai), dung rieng re tran, mep nhan, kich hoac thanh dam.
thuoc 1,5 - 2,5 mm.
Kha nang len men cac loai dudng Glucose
+
Manitol Fructose + +
Sucrose +
Lactose +
Hinh 5. Hinh thai te bao va khuan lac chung LT7 a: Hinh thdi te bdo; b: Hinh thdi khudn lac
Hinh 6. Ket qua dien di san pham PCR Gieng M: Thang DNA lOObp, Gieng I: Chung
LT7. Gieng 2: Mdu dm tinh vi khudn TAP CHI KHOA H O C VA CONG NGHE LAM N G H I E P SO 2 - 2019
Cong nghe sinh hgc & Giong cdy trong Ket qua d hinh 6 cho thay he gen ciia chung
LT7 da dugc tach chiet va doan gen I6S rRNA dugc khuech dai thanh cdng vdi san pham PCR thu dugc cd chat lugng tdt: mpt bang sang rd duy nhat tren gel dien di vdi kich thudc doan gen khoang 1500 bp.
San pham PCR cua chung LT7 dugc giii din Cdng ty TNHH Phat Triin Cdng Nghe LTng Dung Viet Nam, d l giai ttinh tu dinh
danh din loai. K i t qua giai ttinh tu doan gen 16S rRNA ciia LT7 cho thdy doan gen gdm 1445 bp. Cay phat sinh loai dugc xay dung (hinh 7), chiing LT7 ndm tren ciing mdt nhanh vdi nhdm Lactobacillus plantarum. Tien hanh so sanh tren Genbank ciia NCBI ttinh Ur gen 16s rRNA ciia chiing LT7 cd dp tuong ddng la 100% vdi loai Lactobacillus plantarum MH33186.1 khi tiin hanh BLAST.
Hinh 7. Cay phat sinh loai cua chung LT7 Lactobacillus plantarum, mot trong nhiing
vi khuan lactic kha phd bien dugc tim thay trong cac thuc pham len men va trong dudng rugt ciia ngudi va ddng vat. Chiing dugc sir dung lam probiotic ngay cang nhieu ttong nhiing nam gan day. Han nira, chiing dugc nghien ciiu la khdng nhiing an toan tuyet ddi cho con ngudi va dpng vat ma cdn cd nhieu im diem ttong viec dieu tn chiing rdi loan tieu hda Hen quan den khang sinh (Alba LP, 2011). Do vay, chung tuyen chpn LT7 cd tiem nang sir dung lam probiotic bd sung vao thiic an chan nudi. Tuy nhien, can phai co cac nghien ciiu them khi iing dung vao thuc tien.
4. K E T LUAN
Tir cac mau thuc pham len men (rau qua len men, nem chua, thit chua) da phan lap 10 chiing vi khuan lactic (khuan lac cd mau trang siia, trdn, nhan, m^p tron, be mat udt va xung quanh cd vdng trong sudt va cd mpt sd dac tinh sinh ly, sinh hda la: la true khuan, gram duang, Ididng sinh bao hi, catalase am tinh).
Chpn Ipc dupe 3 chiing C2, LT7 va LA6 cd hoat tinh khang khuan tdt nhat vdi ca 3 loai vi
khuan gay benh: E. coli, Samonella sp.
Shigella sp, dudng kinh vdng vd khuan tir 16,5
^ 22 mm. Ba chung nay dugc danh gia kha nang sinh enzyme ngoai bao (protease, cellulase, amylase). Ket qua cho thay, chiing LT7 va C2 (dudng kinh vong phan giai 19,5 -^
30 mm) cd kha nang sinh enzyme ngoai bao cao hon chung LA6 (dudng kinh vdng phan giai hi 16 -^ 16,5 mm).
Hai chung LT7 va C2 dugc danh gia kha nang chiu pH thap (2 -^ 4), chiu mudi mat (0,5
^ 3%)), khang 3 loai khang sinh (Tettacycline, Gentamycin, Streptomycin) ndng dp 10 -^ 50 pg/ml, nhan thay chung LT7 cd kha nang chiu pH thap, mudi mat va khang sinh cao han chung C2. Chiing LT7 da dupe lua chpn la chiing probiotic tiem nang va dugc dinh danh la Lactobacillus plantarum dua tren trinh tu gen 16S rRNA (1445 bp) da dugc phan tich.
Nghien cuu dac tinh smh ly, sinh hda cua chiing nay cho ket qua: te bao hinh que dai, khdng sinh catalase, cd kha nang len men lactose.
TAP CHI KHOA H O C VA C O N G N G H E LAM N G H I E P SO 2 - 2019
Cong nghe sinh hgc & Giong cdy trong TAI LIEU THAM KHAO
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26 TAP CHI KHOA HOC VA CONG NGHE LAM NGHIEP SO 2 - 2019
Cong nghe sinh hgc & Gidng cdy trdng
SCREENING OF LACTIC ACID BACTERLA AS POTENTL^L PROBIOTIC ADD TO ANIMAL FEED
Nguyen Thi Hong Nhung, Le Thi Thuong, Nguyen Thi Thu Hang, Nguyen Thi Huyen Vietnam National University of Forestry
SUMMARY
Probiotics are defined as "Live microorganisms which when administered in adequate amounts confer a health benefit on the host". This study aimed to isolate lactic acid bacteria from fermented foods and evaluate their probiotic properties for application as probiotic additives in animal feed. Ten bacteria strains were isolated using MRS (de Man, Rogosa & Sharpe) media. Using an agar well diffusion method, three strains, LT7, C2 and LA6, showed the best antagonistic activities against all test pathogens belonging to E.
coll. Salmonella sp. Shigella sp. These strains were evaluated the potential production of extracellular enzymes.
The results showed that LT7 and C2 strains were able to higher exfracellular enzyme production than LA6 strain. Two strains, LT7 and C2, were evaluated tolerance to low pH (2 - 4), bile salt tolerance (0.5 - 3%), resistance to 3 antibiotics (Tetracycline, Gentamycin, Streptomycin) 1 0 - 5 0 ug/ml, found that sfrain LT7 displayed higher tolerance than strain C2. Thus, strain LT7 was selected as a probiotic candidate and identified as Lactobacillus plantarum based on the sequences determined in 16S rRNA gene (1445 bp) analysis. It was observed to be ovoid in shape and to be catalase-negative, to be able to fermented lactose.
Keywords: Animal feed, bile salt, fermented food, Lactobacillus plantarum, probiotics.
Ngay nhan bai : 25/12/2018 Ngay phan bien : 18/3/2019 Ngay quyet djnh dang : 25/3/2019
TAP CHI KHOA HOC VA C 6 N G NGHE LAM NGHIEP SO 2 - 2019 27