Nghiên cứu sản xuất probiotic và enzym tiêu hoá dùng trong chăn nuôi phân lập, tuyển chọn, định danh và đánh giá các đặc tính probiotic của các chủng vi sinh vật hữu ích

BO KHOA
HOC
VA CONG NGHE BO NONG NGHIEP VA PTNT
CHirONG TRINH
TRONG DIEM PHAT
TRIEN
VA
ITNG
DUNG CONG NGHE
SINH
HOC TRONG
LINH VlTC
NONG NGHIEP VA PHAT TRIEN NONG THON DEN NAM 2020
BAO CAO
CHUYEN
DE
KET QUA NGHIEN
ClTU
KHOA HOC CONG NGHE DE
TAI
Ten
chuyen de
PHAN LAP,
TUY6N
CHON,
DINH
DANH VA
DANH
GIA
CAC

OAC TINH
PROBIOTIC CUA CAC
CHUNG VI SINH
VAT
HOU
ICH
Chu
nhiem de
tai:
TS. Tran
Quoc
Viet
Co"
quan
chu tri de tai: Vien Chan
nuoi
Ha
Noi
- 2008
A
/\
BO KHOA HOC VA CONG NGHE BO NONG NGHIEP VA PTNT
CHirONG
TRINH TRONG DIEM PHAT TRIEN
VA
iTNG
DUNG CONG NGHE SINH HOC TRONG LINH
VUC
NONG NGHIEP VA PHAT TRIEN NONG THON DEN NAM 2020
BAO CAO CHUYEN DE

KET QUA NGHIEN
ClTU
KHOA HOC CONG NGHE DE TAI
Ten chuyen de
PHAN LAP, TUYEN CHON, DINH DANH VA
DANH GIA CAC DAC TINH PROBIOTIC CUA CAC
•
CHUNG VI SINH VAT
H&U
ICH
Chu nhiem de tai Xac nhan nghiem thu cua
co*
quan chu
tri
TS.
Tran Quoc Viet
Ha Noi - 2010
Phanl
DAT VAN DE
Tu
thai
CO
dai,
con
nguai
da
biet Igi
dung nhung vi sinh vat c6
ich
>

7 f
trong
nhieu ITnh virc
de phuc vu dai song dan sinh. Hien nay, cong nghe vi
sinh
vin
la mot trong bon
ITnh
vuc quan trong cua cong nghe sinh hoc hien
dai (Cong nghe di truyen- Genetic
engineering;
cong nghe te bao -Cell
engineering; cong nghe vi sinh vat - microbial engineering; cong nghe
enzym/protein
- enzym/Protein
engineering)
(Nguyen Lan
Diing,
2005). Tuy
nhien, viec
su
dung cac vi sinh vat
huu ich
de lam thay doi quan he can bang
cua khu he vi sinh vat
duang
tieu hoa, thong qua do cai thien
dugfc sue
khoe,
nang cao tuoi

thg
a con nguai va cai thien nang suat a vat nuoi mai chi
dugc
biet den trong mot vai thap ky tra
lai
day. Cac vi sinh vat
htJu
ich
dugc sii
dimg
cho muc dich nay dugc ggi la cac vi sinh vat probiotic va cac che pham
gom cac vi sinh vat huu ich dugc su dung nhu
nhiJng
chat bo sung trong thuc
an hoac nuac uong dung cho con nguai va vat nuoi dugc ggi la cac che pham
probiotic.
De san xuat dugc cac san pham probiotic, nha san xuat can c6 cac
vi
sinh vat huu ich, c6 day du cac dac tinh probiotic nhu c6 kha nang ton tai va
phat
triln
trong duang tieu hoa cua vat nuoi va con nguai, kim ham su phat
t
trien cua cac vi sinh vat c6 hai trong duang tieu hoa thong qua viec san
sinh
cac bacteriocin, axit huu ca, canh tranh duang chat va vi tri bam dinh tren
niem mac rugt vv. De c6 dugc cac vi sinh vat probiotic, can phai phan lap
chung tu cac
ngu6n
khac nhau trong tu nhien, sau do danh gia cac dac tinh

sinh hgc va cac dac tinh probiotic cua chung. Nhung vi sinh vat (VSV) nao
hoi du cac
di6u
kien cua cac VSV probiotic se dugc phan
loai,
dinh danh de
dam bao rang chung thuc su la
nhOng
vi sinh vat hoan toan v6 hai
d6i
vai
sue
khoe cua vat nuoi va con nguai.
Muc
tieu
cua chuyen de nay nham tong ket mot
each
c6 he thong cac
ket qua nghien cuu ve phan lap, tuyen chgn,
djnh
danh va danh gia cac dac
tinh probiotic
ciia
cac chung vi sinh vat huu ich, mot noi dung ca ban cua de
tai "Nghien cuu san xuat probiotic va enzyme tieu hoa dung trong chan
nuoi", thugc
"Chuang trlnh
trong diem phat trien va ung dung cong nghe
sinh hoc trong
ITnh

vuc nong
nghiep va
phat
trien
nong
thon "
giai doan 2006-
2009
Phan II
TONG QUAN TAI LIEU
2.1.
Khai niem probiotic
r r
Theo ngon ngu Hi Lap, probiotic c6
nghia
la "vi su song" Trong tieng
Anh,
CO
mot thuat ngu kha quen thugc, ngugc
nghia
vai tu probiotic do la tu
f
r
"antibiotic", tu nay c6 nghia la chong lai su song hay thuang ggi la khang
sinh.
Thuat ngu probiotic dugc Parker de nghi su dung lan dau tien vao nam
1974 de chi
"nhijng
vi sinh vat va nhung chat lam can bang he vi sinh vat
rugt" (Fuller, 1989).

Tir
do den nay thuat ngu probiotic da dugc ca the giai su
dung de chi nhung che pham VSV song huu ich khi dugc dua vao ca
thi
dgng vat thong qua thuc an hoac nuac uong tao nen nhung anh huang c6
Igi
cho vat chu. Ke tu khi xuat hien, khai niem probiotic
vIn
chua c6 mot dinh
nghia
thdng
nh§t.
Tuy nhien, hien c6 hai dinh nghia dugc cho la phan anh kha
diy du ban chat cua probiotic va dugc su dung nhieu trong cac an pham khoa
hgc:
(i) theo Fuller (1989), probiotic la "chat bo sung vi sinh vat song vao
thuc an giup cai thien can
bSng
cua he vi sinh vat duang tieu hoa theo huang
CO Igi
cho vat chu"; (ii) theo
t6
chuc Y
td th6
giai (WHO,
2001),
probiotic la
"cac vi sinh vat song khi dua vao ca the theo duang tieu hoa vai mot
s6 lugng
du se dem lai

sue
khoe
t6t
cho vat chu"
2.2.
He vi sinh vat ruot va
siic
khoe ciia he thong tieu hoa
6'
vat nuoi.
Hien nay, trong dinh duang gia
sue
hien dai,
sue
khoe cua he tieu hoa
dang la van de dugc ca the giai quan tam. Mot khau phan an
ly
tuang se
khong
CO
nhieu y nghia doi vai mot he tieu hoa khong
lanh
manh.
Sue
khoe
cua he tieu hoa a vat nuoi phu thugc vao rat nhieu yeu to, trong do su can
bang cua he VSV cong sinh c6 y nghia quyet dinh.
Khi con a trong bao thai, duang tieu hoa
ciia
vat nuoi a trang thai v6

trung, nhung chi vai gia sau khi sinh, cac VSV da bat dau cu tni va tra thanh
nhiing
"cu dan" binh thuang trong duang tieu hoa (WHO,
2001).
Theo thai
r r
gian, do tiep xuc true tiep vai moi truang, dac biet la qua thuc an va nuac
r r
uong, so lugng va tinh da dang sinh hgc cua cac VSV cong sinh khong ngung
tang
len.
So lugng te bao VSV cu tru trong duang tieu hoa cua vat nuoi c6 the
cao gap 10 lan so lugng te bao cau tao nen ca the chung (Fonty, 1995). So
lugng loai c6 the len tai tu 400-500 (Tannock, 1999). Tuy nhien, mat do VSV
a cac phan doan khac nhau cua duang tieu hoa (da day; ta trang; rugt non va
rugt gia) a loai dgng vat da day dan rat khac nhau (khoang
lO'-lO^; lO'-lO'*;
10^-10^
va
10^-10'^
cfu/ml chit
chua tuang ung) (Jans, 2005).
Sue
khoe cua he thong tieu hoa a vat nuoi phu thugc vao 3 yeu to chinh:
7
X
r f
trang thai sinh ly cua vat
chii,
khau phan thuc an va he

VSV
Cac yeu to nay
chiu tac dgng cua moi truang, cua cac stress va tac dgng qua lai
lln
nhau.
Trong s6 cac nhan t6 tren, he VSV duang tieu hoa dong vai tro trung tam, chi
mot bien dgng bat
Igi
cua mot trong hai yeu to con lai cung anh huang xau tai
he VSV (Conway, 1994). Su cong sinh cua cac loai VSV trong duang tieu
hoa cua vat nuoi (chu yeu la trong rugt) tao nen mot he sinh thai ma va moi
can bang cua quan the VSV dugc xac lap chi mot
thofi
gian rat ngan sau khi
sinh (Jans, 2005).
Co nhieu quan diem khac nhau ve moi tuang quan can bang cua he
VSV rugt. Theo Jans (2005), de danh gia trang thai can bang, cac VSV rugt
dugc chia thanh 3 nhom (1) nhom chu yeu (main flora) gom cac loai vi khuan
ki khi
{Clostridia;
Lactobacilli;
Bifidobacteria;
Bacteroides,
Eubacteria);
(2)
nhom ve tinh (Satellite flora), gom chu yeu la Enterococci va
E.
Coli,
va (3)
nhom con lai (Residual flora) gom cac VSV c6 hai nhu Proteus,

Staphylococci va
Pseudomonas.
Mot quan the VSV dugc coi la can bang khi
ty
le
cua cac nhom dao dgng trong
khoang
90; 1,0 va
0,001%
tuang ung.
Trang thai ma cac nhom nay hinh thanh mot ty le
90:1:0,001
dugc ggi la trang
thai "eubiosis" (tieng Hy Lap c6 nghia la su chung song c6
Igi
giua cac vi
khuan vai nhau va vai vat chu). O trang thai "eubiosis", vat chu cung cap cac
>
A
7
dieu kien song ly tuang nhu nhiet do on dinh, pH trung tinh, dinh duang va
SU
dao thai cac chat chuyen hoa. Doi lai, he VSV se mang lai
Igi
ich cho vat
chu thong qua tang cuang tieu hoa cac chat dinh duang, giai doc, tong hgp
cac vitamin nhom B va vitamin K, loai tru cac VSV c6 hai, tang cuang dap
ung mien dich cua vat chu. Su can bang cua he VSV trong duang tieu hoa bi
tac dgng bai mot so nhan to v6 sinh va
hilu

sinh nhu: sinh ly vat chu, khau
phan thuc an va ca cau noi tai cua ban than he VSV. Thuc an la
n8n
dinh
7
\
7
X
duang ca ban cua
vi
sinh vat, bai vay su thay doi thanh phan khau phan, thuc
an khong dam bao ve sinh, phuang phap cho an khong hgp ly vv deu lam
ton hai den trang thai can bang he VSV rugt. Tuang tu nhu vay, cac chat bai
tiet cua he tieu hoa (dich mat, cac enzyme, chat dem va chat nhay vv)
cQng
nhu kieu va tan so nhu dgng rugt cung tac dgng
true
tiep den he vi sinh vat.
Kilu
va tin so nhu dgng rugt bi tac dgng rat lan bai cac stress (sinh de, cai
sfta,
don chuong, van
chuyen w).
Khi quan he can bang cua he VSV rugt bi
/
7
pha va se tao nen trang thai "dysbiosis" (trang thai "chung song c6 hai"). Bieu
7
hien cua trang thai "dysbiosis" a vat
chii

thuang la the tang kem, sinh truang
cham va mac cac benh duang tieu hoa nhu tieu chay, viem rugt hoai tu vv.
De cai thien quan he can bang cua he VSV rugt a vat nuoi, mot phuang phap
thuang dugc ap dung la bo sung vao khau phan thuc an mot so loai khang
sinh lieu thap nhu nhung chat kich thich sinh truang. Tuy nhien, viec su dung
khang sinh trong thuc an chan nuoi mot each khong c6 kiem soat da va dang
gay ra nhu'ng hau qua
ddng lo
ngai ve ve sinh an toan thuc
phim
va dac biet la
gay nen tinh trang khang thuoc ngay cang gia tang cua cac vi khuan gay benh
tren nguai va vat nuoi. Hien nay, khoi lien minh chau Au (EU) da cam su
dimg
khang sinh de bo sung vao thuc an nhu chat kich thich sinh truang tu
ngay
01
thang
01
nam 2006. Viec cam
sir
dung khang sinh trong thuc an chan
nuoi cung dat ra
nhOng
thach thuc
Ion
ve ky thuat, dac biet doi
vai
chan
nuoi

gia
sue,
gia cam non hoac trong dieu kien ve sinh kem va vat
nuoi chiu
nhieu
Stress. De vugt qua nhiing thach thuc do, da c6 rat nhieu nhung nghien cuu
nham tim ra
nhiJng
chat thay the khang sinh an toan vai vat nuoi. Mot trong
nhung chat do la probiotic.
2.3.
Cac vi sinh vat probiotic, vai tro va co che tac dong cua probiotic.
2.3.1.
Cac vi sinh vat probiotic.
Cac vi sinh vat probiotic thuang dugc su dung trong chan nuoi gom 3
nhom chinh: vi khuan (VK) lactic; VK Bacillus va nam men. Khi vao duang
tieu hoa cua vat nuoi thong qua thuc an hoac nuac uong, tuy theo loai ma ca
che tac dgng c6
Igi
cua chung
d8n
vat chu theo cac phuang thuc khac nhau.
Cac VK lactic (cac vi khuin chuyen hoa mot so loai duang nha qua trinh
f
r r
len men tao axit lactic) c6 kha nang san xuat cac chat (bacteriocin) uc che su
phat
triln
cua cac cac vi
khuin

c6 hai va axit
hiJu
ca lam giam pH moi truang
duang chip trong rugt, tao dieu kien thuan
Igi
cho cac VSV huu ich phat
triSn.
Chung sinh
soi,
phat trien nhanh chong, chiem vi tri bam dinh tren niem
mac duang tieu hoa, canh tranh cac chit dinh duang
d6i
vai cac vi khuan gay
benh, tang cuang trao doi mu6i mat, tang kha nang hip thu cac chat dinh
duang (Servin, 2004).
Cac vi khuan Bacillus (nhom c6 kha nang hinh thanh bao tu) khi dugc
dua vao duang tieu hoa (thuang dual dang cac bao tu), chung nay mam va
sinh
truofng
nhu nhung te bao thuc vat va san sinh cac enzyme tieu hoa ho trg
cho cac enzyme noi sinh, ngoai ra chung con kich thich he thong mien dich a
rugt (Sander va
ctv,
2001).
Khac vai cac VK Lactic va bacillus, cac nam men probiotic
(chii
yeu la
cac
chiing
thugc Saccharomyces cerevisiae va

Saccharomyces
boulardii) c6
kha nang
tmng
hoa doc to cua vi khuan (Castagluiolo va ctv,
1999),
gan ket
7
>
cac vi khuan c6 roi (phan lan la cac VK gay benh) nha su hien dien cua cac
1
t r
ca quan cam thu mannose va day chung ra khoi co the qua phan, nha do so
lugng cua cac VSV co ich tang len (Czerucka va Rampal, 2002).
Cac vi khuan va nam men thuang dugc su dung nhu
nhtjng
probiotic la
Bifidobacterium longum; B breve; B infantis; B
bifidum;
B adolescetis;
Lactococcus cremoris; L lactis; Streptococcus thermophilus; Enterococcus
feacium; Lactobacilus rhamnosus; L acidophilus; L casei; L bulgaricus; L
gasseri; Saccharomyces
boulardii;
S
cerevisiae.
2.3.2.
Vai
tro
cua probiotic

doi vai vat chu
Tu khi khang sinh bi cim su dung nhu chit kich thich sinh truang trong
thuc an chan nuoi a mot
s6
nuac thugc khoi lien minh chau Au (bat dau la
7
\
Thuy Dien vao nam 1986) thi probiotic dugc coi la mot trong nhung nguon
thay
th§
co
triln
vgng nhit vi co nhieu dac tinh uu viet. Tren co sa cac ket
qua nghien cuu cua
nhihu
tac gia, Patterson (2003) da
t6ng
ket cac anh huang
CO Igi
cua probiotic
d6i
vai dai
s6ng
dgng vat the hien a cac khia canh sau:
(1) lam thay
d6i
ciu
true
quin
thI

vi sinh vat duang rugt theo chieu huang co
Igi
cho vat chu; (2) tang cuang kha nang
miln
dich; (3) giam phan ung viem;
(4) ngan can su xam nhap va uc
ch8
su phat
triln
cua vi khuan gay benh; (5)
f
7
tang san xuat cac axit beo bay hai; (6) tang cuang qua trinh sinh tong hgp cac
vitamin nhom B; (7) tang hap thu chat khoang; (8) lam giam cholesterol huyet
thanh; (9) lam tang nang suat vat nuoi; (10) lam giam ham lugng amoniac va
r r
ure trong chat thai. Ngoai ra probiotic con rat an toan
v6i
dgng vat va than
thien vai moi
truofng.
Vi la chat bo sung VSV song
hOu
ich, viec
sii
dung
probiotic se khong tao ra cac chat ton du trong cac san pham chan nuoi co hai
cho sue khoe
ngucxi
tieu dung.

2.3.3.
Co
che
tac dong
ciia cac probiotic
Bang
each
nao ma
cdc
probiotic tao ra nhirng anh huang co
Igi doi
vai
vat
chii
?,
CO
rat nhieu
each
giai thich khac nhau ve ca che tac dgng, nhung
phan lan cac tai lieu ve probiotic de cap den ba khia canh sau: (i) canh tranh
loai
trir;
(ii)
d6i
khang vi khuan va (iii) dieu chinh mien dich (Steiner, 2006).
Canh tranh loai tru la dac tinh dau tranh sinh ton dien hinh cua cac vi
sinh vat. Hinh thuc canh tranh loai tru thuang thay a cac VSV rugt la canh
tranh vi tri bam dinh. Cac VSV probiotic cu ngu va nhan len trong rugt, khoa
chat cac vi tri thu cam va ngan can su bam dinh cua cac vi sinh vat khac nhu
E. coli,

Salmonella w.
Mot so nam men probiotic (Saccharomyces
cereviese; S
Boulardii)
khong chi tranh vi tri bam dinh cua cac vi khuan khac
ma con gin
kSt
cac vi khuan co roi (phan lan la nhung vi khuan co hai) thong
qua cac ca quan thu cam mannose va day chung ra khoi vi tri bam dinh a
niem mac rugt (Czerucka va Rampal, 2002). Tuy nhien, canh tranh dinh
duang la phuang thuc canh tranh khoe
liet
nhat. Su sinh soi vai so lugng lan
r
cua mot loai vi sinh vat nao do la mot de dga nghiem trgng doi vai cac loai
khac
v6
nguon ca chit cho phat trien.
D6ng
thai vai canh tranh loai
trir,
cac VSV probiotic con san sinh cac
chit kim ham vi khuin nhu lactoferrin, lysozyme, hydrogen peroxide cung
nhu mot
s6
axit
hiJu
ca khac. Cac chit nay gay tac dgng bat
Igi
len vi khuin

CO
hai chu
y6u
la do su giam thip pH trong rugt (Kelly va King, 2001;
Conw^ay,
1996).
Rugt la ca quan mien dich lan nhat
6f
dgng vat c6
vii.
Giua he VSV
rugt va he thong miln dich co moi tuang tac dac thu. Nang
luc
miln dich the
dich va miln dich te bao cua he thong mien dich duang rugt bi anh huang rat
lan bai su can bing
ciia
he vi sinh vat rugt (Cebra, 1999). Thong qua tuang
tac vai he thong mien dich rugt, cac probiotic co the dieu chinh ca mien
djch
thu dgng va
chii
dgng hoac
c^
hai. Tac dgng dieu chinh miln djch dac hieu
ciia
probiotic phu thugc vao giong hoac cac loai vi khuan probiotic (Dugas va
r r
Ctv, 1999). Tuy nhien, ca che tac dgng
ciia

probiotic doi vai viec nang cao
chuc nang miln dich van con chua dugc hieu biet day
dii.
2.4.
NhCrng
dac tinh probiotic cua cac chung vi sinh vat huu
ich.
7 7
> >
De
CO
the dugc su dung nhu nguon probiotic, cac VSV can hoi
dii
cac
dieu kien sau: (i) phai la cac vi sinh vat
hiJu
ich, v6 hai doi vai vat nuoi va
con nguai; (ii) song dugc trong duang tieu hoa
ciia
vat nuoi vai cac dieu kien
kha bat
Igi
(pH thay doi tu rat toan den kiem, muoi mat va cac dieu kien sinh
hoa trong lumen rugt w); (iii) co kha nang uc che cac VSV co hai bang
nhieu
each
khac nhau (canh tranh loai tru, san sinh ra cac chat uc che, tang
cuang dap ung mien dich
w);
(iv) c6 the sinh truang va phat trien trong cac

dieu kien moi truang khac nhau (khong hoac it ken chgn ca chat cho sinh
truang, giai pH va nhiet do
rgng
.vv);
(v) tuang thich cao vai cac thanh phan
CO
hoat tinh trong khiu phan thuc an (mot so muoi kim loai, cac chat axit
h6a w) va (vi) c6 kha nang song sot cao trong cac dieu kien che bien va bao
quan.
Theo
t6 churc
y
t8 thd
giai (WHO, 2002), cac chiing VSV dugc
lira
chgn
nhu
ngudn
probiotic nhat thiet phai dugc chung minh nguon goc ro rang tu
gidng
(genus), loai (species) va chiing (strain) bing cac phuang phap phan
loai thong thuang va sinh hgc phan tu. Nhieu nghien cuu cho thay, anh huang
8
probiotic
ciia
cac VSV co tinh dac hieu rit cao
d6i
vai
mli
chiing

(ciing
giong,
cung loai nhung dac tinh probiotic khac nhau rat lan giua cac chiing).
Phuang phap xac dinh danh phap
ciia
cac chiing VSV probiotic phai dugc
chung thuc bing ten khoa hgc dugc cac
t6
chuc chuyen mon co uy tin chip
nhan va mang tinh
ph6
biln.
Mudn phat buy tac dung, cac VSV probiotic phai ton tai dugc trong
duang tieu hoa
ciia
vat nuoi,
kh^
nSng
nay khong
hi dl
dang
d6i v6i nhilu
loai VSV ma moi
tru5fng
cu tru thuang xuyen
ciia
chiing khong phai la trong
he tieu hoa
ciia
dgng vat cap cao. Bai vay, dl chgn

Igc
dugc cac chiing VSV
probiotic co kha
nSng
song dugc trong moi
trud^ng
da day rugt
ciia
dgng vat,
nguai ta thuang phan lap chiing
tir
cac chat
chiia
trong duang tieu hoa. Tuy
nhien, he tieu hoa
ciia
nguai va dgng vat la mot he thong ma, theo do cac
tilu
phan thuc an dugc xem la cac "phuang tien di cu"
ciia
cac VSV, nen mac du
dugc phan lap tu lumen rugt, ciing khong the khang dinh chac chan kha nang
7
\
f
CO
the ton tai va phat trien
ciia
chiing trong duang tieu hoa
ciia

vat nuoi. Mat
khac quan he can bang
ciia
he vi sinh vat rugt a nguai va dgng vat la mot he
\ \
7
can bang dgng, nen viec danh gia kha nang ton tai va phat trien
ciia
cac chiing
dugc
lira
chgn trong duang tieu hoa
ciia
vat nuoi cang tra nen can thiet (Jan,
2005).
Kha nang ton tai
ciia
cac chiing VSV probiotic trong duang tieu hoa
ciia
vat nuoi dugc danh gia thong qua cac thi nghiem in-vitro nham khao sat
kha nang song trong moi truang co do pH khac nhau, kha nang bam dinh vao
niem mac rugt (Misha va ctv, 2005) va kha nang chiu muoi mat (Dora va ctv,
2003).
Cac
thii
nghiem in-vitro rat huu ich de thong qua do hilu dugc khong
chi nhung dac tinh sinh hgc ma con cac ca che tac dgng
ciia
cac chiing VSV
dugc

lira
chgn (FAO/WHO, 2002). Ton tai va phat trien dugc trong duang
tieu hoa
ciia
vat nuoi
la
mot dieu kien can, nhung chua
dii ciia
cac VSV
probiotic. De hoi
dii
cac dieu kien, cac VSV probiotic phai co cac dac tinh
quan trgng khac nhu kha nang san sinh cac chat uc che cac vi sinh vat c6 hai
(nhung dac tinh nay dl dang dugc khing dinh thong qua cac phep
thii
trong
nghien
ciiu
VSV thong thuang),
Idia
nang tuang thich cao vai cac thanh phin
CO
hoat tinh
ciia
khiu phin. Phin lan cac san phim probiotic dugc dua vao ca
7
the thong qua thuc an. Bai vay,
sir
tuang tac giua cac chiing VSV probiotic
vai cac thanh phin khiu phin la mot tra ngai dang

kl d6i
vai hoat tinh
ciia
chiing. Rit may min la chi c6 mot
s6
thanh phin co hoat tinh nhu mot s6
mu6i kim loai, cac chit axit hoa, mot
s6
chit
b6
trg khac la co tac dgng tieu
cue
den
sue s6ng
cua cac chiing VSV probiotic, con phin lan cac thanh phin
khac
ciia
khiu phin
diu
v6 hai
d6i
vai chiing.
Vl
nguyen tic, gia
trj
probiotic
ciia
mot chiing VSV probiotic ty le thuan vai tinh tuang thich cua chiing
d6i
vai cac chung khac vai cac thanh phin

ciia
khiu phin.
Phan III
TONG QUAN VE MUC TIEU, NOI DUNG
VA SAN PHAM KHOA HOC CUA DE TAI
3.1.
Muc tieu cua de tai
San xuat va dua vao
sii
dung mot so che pham sinh hgc gop phan nang cao
hieu qua chan nuoi:
F
1
- Cac che pham probiotic dang long va dang bgt
- Che pham da enzyme (amylase, protease, cellulase,
beta-glucanase
va
xylanase)
3.2.
Noi dung nghien
ciru
cua de tai
- Nghien cuu san xuat cac che pham probiotic dung trong chan nuoi
F
Ft
- Nghien
ciiru
san xuat cac che pham da enzyme tieu hoa diing trong chan nuoi
10
3.3.

TT
1
2
3
4
5
6
San pham khoa hoc cua
dS
tai
Ten san pham
Che pham
probiotic cho
Ign
va ga
Che pham da
enzyme tieu
hoa
Qui trinh san
F
Ft
xuat che pham
probiotic, che
t
pham da
enzyme
Mo hinh sur
dung probiotic
va che pham da
enzyme

Bai bao khoa
hoc
Dao tao DH,
SDH
So lugng
20-50
lit.
25-50kg
25-50 kg
02
2-4
3-4
2-3
Chi tieu kinh
t^
- ky thuat
hoac yeu cau khoa hgc
-Mat do VSV huu ich :
lO'-lO'
cfu/g
-T^ng
sinh
tru'ang:
5-
10%
-Giam tieu
t6n
thuc an: 5-
10%
-Gi^m

ty le benh duang tieu hoa:
8-15%
+Hoat
tinh
-Amylaza: 1000
lU/g
Proteaza: 100
lU/g
-
p-Glucanaza
:
200
BGX/g
Xylanaza:
1000
lU/g
-Xellulaza: 1000
lU/g
+Tang sinh truang: 6-
10%
F
w
+Giam tieu ton TA: 5-
10%
Qui mo san xuat: 20-50 lit/me;
25-50 kg /me
- Dugc ap dung tai ca sa san xuat
-
1 -2
mo hinh chan nuoi

Ign,
qui
mo 100-200
Ign
thit
-
1 -2
mo hinh chan nuoi ga qui mo
500-1000 ga
thit
Cong bo tren tap chi/hgi nghi khoa
hgc trong nuac hoac quoc te
Bao ve thanh cong
Thai
gian
hoan
thanh
12/2008
11/2009
12/2008
11/2009
12/2008
11/2009
11
3.4.
Vi tri va nhiem vu cua chuyen de trong de tai
Muc tieu
ciia dh
tai la san
xuk

dugc hai san
ph§m
sinh hgc (probiotic va
enzyme tieu hoa) dung trong
chan
nuoi.
M6i chi phIm
co nhung dac
dilm
khac nhau
v6
thanh phin, hieu qua tac dgng doi vai vat nuoi, phuang
thiic
san
F
xuat va su dung. Bai vay, ngi dung va phuang phap nghien cuu
ciia
de tai
dugc chia thanh hai
ph^n
rieng biet. Chuyen de nay he
thdng
cac ket qua
nghien
ciiu
lien quan
d8n
viec phan lap, danh gia va tuyen chgn cac vi sinh
vat huu ich nhu ngudn probiotic phuc vu cho viec san xuk cac
chi phIm

probiotic dung trong chan nuoi.
Phan IV
MUC TIEU, NOI DUNG VA
PHl/ONG
PHAP
NGHIEN ClTU CUA CHUYEN DE
4.1.
Muc tieu cu the
7
Phan lap, danh gia va tuyen chgn dugc cac chiing vi sinh vat
hiJu
ich nhu
>
F F
t
nguon probiotic phuc vu cho viec san xuat cac che pham probiotic dung trong
chan nuoi.
4.2.
Noi dung nghien
ciiu
- Phan lap cac chiing vi sinh vat
hiJu
ich
tir
cac
ngu6n
khac nhau
Danh gia va
tuyin
chgn cac chiing vi sinh vat da dugc phan lap.

4.3.
Vat lieu va
phu-ong
phap nghien
cihi
4.3.1.
Vat lieu nghien
cwu.
- Cac vat lieu phuc vu cho viec phan lap
tuyin
chgn cac vi sinh vat probiotic:
Phan, chat
chiia
ducmg tieu hoa (rugt non, manh trang)
ciia Ign
va gia cim
12
(ga, ngan, vit). Mot
s6
san phim probiotic thuang mai (Lacto-Sac
ciia
hang
Alltech-Canada; EM
ciia
Nhat; E-Lac
ciia
Han qu6c vv)
- Cac vi sinh vat
kilm
dinh {Salmonella enteritidis, E. coli va Shigella

flexneri);
cac vi sinh vat probiotic da dugc phan lap,
tuyln
chgn va danh gia
dac tinh probiotic dugc bao quan tai bao tang
gidng
chuan vi sinh vat thugc
Vien Vi Sinh Vat va Cong nghe sinh hgc-Dai hgc
Qu6c
gia Ha ngi.
- Cac hoa chit dung dl tach ADN va phan giai trinh tu: EDTA, Tris- SDS,
chlorofom-isoamyl alcohol, dNTP, Taq polymeraza, Ethidium Bromit, kit
QIAgen w.
Cac vat lieu khac: Gia
d6,
duang
gluco,
K2HPO4,
MgS04,
natri glutamat,
pepton, cao nim men; dextrin, tinh bgt sin, bgt sua whey.
4.3.2.
Phifffngphap
nghien cuu
4.3.2.1.
Phan lap, tuyen chon cac chung vi sinh vat huu ich.
-
MIU
dugc pha
loang

a
n6ng
do tu
10'^-10"^
va ciy gat tren moi truang MRS
(d6i vai vi khuin Lactic) va YM
(d6i
vai nim men). Sau khi ciy cac
mlu
dugc giu trong
tii
im a nhiet do
37°C
cho vi khuin (VK) va 30
°C
cho nim
men trong 48 gia.
- Sau 48 gia tren moi truang MRS chgn cac khuin lac tao vong phan giai
CaCOs;
tren moi truang (Yeast Malt- YM) chgn cac khuin lac nim men tron
deu, tran nhan soi kinh co hinh trung va nay choi.
4.3.2.2. Danh gia dac diem hinh thai, phan loai vd dinh danh cac chung VSV
hitu
ich.
-
Hinh
thai t6' bao dugc quan sat sau khi nu6i
c^'y
tir
24 den 48

gid
tren cac
m6i
trucmg
MRS
(vdri
VK Lactic) va YM
(vdi na'm
men). Hoat
tinh
catalaza va
sinh
khi CO2 tir
cac ngu6n
dudng
khac nhau co n6ng
d6
1% (raffinoza
=
2%)
dugc xac dinh theo phuang phap cua Kozaki va ctv (1992). Ngoai ra, cac
chung
na'm
men con dugc
thir
phan
ling
DBB va hoat
tinh
ureaza.

13
Viec phan loai cac chung vi khuin dugc thuc
hi6n
dua tren ca
sd
khoa phan
loai cua Bergeys (Holt va ctv, 2000) (theo cac
chi
tieu chii yeu nhu:
hinh
thai
te'
bao, khuin lac,
nhu6m
Gram, phan
liiig
catalaza va
len
men mot so loai
dudng)
va phan
tich,
giai trinh tu 16S rARN theo phuang phap dugc
gi6i
thieu
bc^i
Lane (1991).
- Xac dinh trinh tu rADN 16s
ciia
cac chiing vi khuin lactic theo phuang phap

ciia Saito va Miura (1963) va trinh tu
lADN
ITSl,
ITS4 cua cac chung nam
men theo phuang phap ciia Manitis va ctv
(1982).
4.3.2.3. Danh gia mot
so
dac tinh probiotic cua cac chung VSV
hi/u
ich.
Danh gia kha nang nang chiu nhiet: Cac chiing vi sinh vat dugc nuoi
cay
tren m6i trucmg MRS dich
th^
(VK Lactic) va m6i trucmg YM (nam men) (pH
=7.0),
trong cac may
lie 6n
nhiet (200
vong/phiit)
v6i cac giai nhiet do khac
nhau 30; 37; 45;
55°C.
Danh gia kha nang sinh truang trong moi
trudng
co do pH khac nhau:
Tuang tu nhu tren, cac chung vi khuan va
na'm
men cung dugc nuoi ca'y tren

m6i trucmg dich the trong dem acetat co pH khac nhau (2,2; 3,0; 4,0; 5,0; 6,0;
7,0), tren may
lie
6n nhiet (200 vong/ phut tai 37°C cho vi khuan Lactic va
30°C cho na'm men),
- Danh gia kha nang sinh truang trong moi trudng co nong do muoi mat khac
nhau: Cac chung nghien
ciiu
cGng dugc nuoi
c^y
tren m6i trucmg dich the nhu
tren
CO
nong
d6
muoi mat (0,2; 0,5;
1;1,5;
2; 3%). Nu6i cay
lie
(200
vong/phut tai 37°C doi
v6i
vi khuan va 30°C d6'i v6i
na'm
men).
De'm
mat do te' bao sau 48h nu6i ca'y gat tren
dia
Petri c6
chiia

m6i trucmg
thich
hgp.
Sau 48
gior,
td'c d6
sinh trucmg cua cac chung vi khuin dugc danh gia
thdng
qua so lugng VSV
tinh
bang cfu/ml va kha nang san sinh axit lactic. Kha nang
san sinh axit lactic cua cac VK Lactic dugc danh gia theo phuang phap cua
Themerva
ctv (1989).
14
- Kha nang khang cac vi khuan kiem dinh {Salmonella
enteritidis,
E.
coli.
Shigella
flexneri):
dugc tien hanh theo phuang phap do vong khang khuin.
Tinh doi khang cua cac vi sinh vat: Dugc thii bing ky thuat cay vach tren
dia
thach.
- Danh gia kha nang bam dinh vao
niem
mac duang tieu hod.
Chuin bi vi sinh vat va vat lieu bam
dinh:

Vi sinh vat (vi khuin va na'm men) dugc nu6i ca'y
thu^n
khiet trong m6i
trucmg thich hgp cho
tirng
chiing.
Chuin bi cac mau ruot ga tuai:
Rua cac doan
ru6t
non (tii ga broiler manh khoe) 3 lan vai dem PBS sao cho
ta't ca cac vi sinh vat
khdng
con tren hi mat niem mac
ru6t.
Rua 1 lan vai moi
trucmg (MRS cho vi khuin va YM cho na'm men).
Tien hanh thu: bam
dinh:
-
Phii
dich te bao da chuin bi a tren len tren be mat niem mac
ru6t.
- U 90 phut trong tii am
(37°C).
- Rua
mau ru6t
\in 1 bing
nudfc
muoi sinh
lyl%.

- Rua
mau ru6t
3
lin
bang dem PBS.
F
^ X
- Thu lay dich nia cua ca 3 lan va
tr6n
deu.
- De'm s6' lugng VSV trong dich nia
di
xac dinh kha nang bam
dinh
ciia cac
VSV nghien
ciiu.
2.2.5. Danh gia tinh tuang thich
ciia
cac
chiing
vi sinh vat vai cac thanh phan
CO
hoat tinh trong
khau
phan an cho lan vd gia cam.
Cac chung nghien
ciJu
dugc nu6i ca'y tren m6i trucmg dich the tuang
ling

(MRS cho VK Lactic; thach
thudng
cho VK Bacillus va YM cho na'm men)
15
nhung
CO
b6 sung cac thanh
phSn
co hoat
tinh
thucmg co trong cac khiu
ph^n
an cho
Ign
va gia
cSm
gom
m6t
s6' loai khang sinh: Bacitracin Methylene
Disalicylate (BMD) (50 ppm); Saigon Nox (100 ppm), Colistine 98% (100
ppm),
chlotetracyclin (CTC) 15% (100 ppm);
m6t
s6' loai khoang CuS04 (250
ppm Cu); ZnS04 (100 ppm Zn) va h6n hgp axit
hiJu
ca (g6m axit lactic axit,
axit formic, axit citric.)
v6i li^u
200mg/lit

Nu6i
ca'y
lie
(200 vong/phiit)
trong tii a'm
(37°C).
Da^m
mat
d6
te'
bao tren m6i trucmg
dia
thach sau 48h.
Phan V
KET QUA VA THAO
LUAN
5.1.
Ket qua phan
lap,
tuyen chon, phan
loai
va djnh danh cac chung vi
sinh vat.
Tiif
cac ngu6n khac nhau (cha't
chiia duong
rugt cua
Ign,
ga va cac
mSu

san
phim probiotic thuang mai), 64 chiing vi sinh vat da dugc phan lap, trong do
CO
27 chung vi khuan lactic (42,2%) va 39 chung na'm men (57,8%) (bang 1).
Bang
1:
Ke't
qua phan lap cac vi sinh vat tii cac nguon khac nhau
Ngu6n phan lap
Chat
chiia ru6t
non va
ru6t
gia cua
Ign.
Cha't
chijfa ru6t
non,
ru6t
gia cua ga
B6'n
(4)
mau
san phim probiotic thucmg
mai
T6ng
Vi sinh vat
VK Lactic
10
15

2
27
Na'm men
4
28
7
39
T6ng
s6'
66
Trong do
tir
cha't
chiia
ducmg rugt cua
Ign
va gia
c^m
da phan lap dugc 25
chung vi khuin lactic, 32 chung na'm men. Tii
m6t
s6'
che' pham probiotic hien
dang
luu
hanh tren thi trucmg, da phan lap dugc 2 chung vi khuin lactic va 7
16
chiing na'm men. Ke't qua phan lap cac
mSu
san phim probiotic thuang mai

cho tha'y, ta't ca cac san phim nay diu la cac san phim probiotic da chung. De
lam ca
sd
cho viec tuyen chgn, cac chiing VSV tren tie'p tuc dugc
thu^n
khie't
tren
m6i
trucmg
dia
thach va chgn dugc 4 chiing (2 chung VK lactic va 2
chiing na'm men) de danh gia cac dac
tinh hinh
thai, sinh ly, sinh hoa quan
trgng lam ca
sd
cho viec phan loai. Cac ke't qua dugc trinh bay
d
cac bang 2 va
3.
Bang 2: Dac dilm
hinh
thai, sinh ly, sinh hoa ciia 2 chiing vi khuin lactic
TT
1
2
3
4
5
6

7
8
9
10
11
12
13
14
15
16
17
18
Dac
dilm
Hinh
dang
te'
bao
Nhu6m
Gram
Phan ling catalaza
Kl hieu chiing
NCI
Que ngin
Gram +
NC2
Que ngin
Gram +
-
Kha nang dong hod nguon cacbohydrat

Riboza
Xyloza
Arabinoza
Rhamnoza
Trehaloza
Lactoza
Mannitol
Sucroza
Cellobioza
Raffinoza
Galactoza
Tinh bgt
Gluconat
Fructoza
Thanh
te'
bao co meso-DAP
+
-1-
+
+++
-I-+
+++
+++
++
+
-1-
++
+
-1-

+
+
+
-f-
++
++
++
+++
+++
++
++
+++
++
+
Hai chiing vi khuin Lactic dugc
lua
chgn
(NCI
va NC2) deu la nhung
VK
hinh
que ngan, Gram duang va phan ling katalaza
am
tinh.
Day la nhung
dac dilm ra't dac trung ciia cac VK Lactic. Tuy nhien, kha nang d6ng hoa cac
ngu6n carbohydrate ciia 2 chiing nay co nhirng khac biet. Chiing NC2 co kha
nang dong hoa
hdu he't
cac ngu6n carbohydrate nghien

ciiu trir
tinh
b6t,
trong
do chiing
NCI
co kha nang d6ng hoa tinh
b6t or miic
th^p,
nhung lai khong co
17
kha nang d6ng hoa riboza va xyloza. Ca 2 chiing deu co kha nang dong hoa
t6't 2 ngu6n carbohydrate ca ban la Sucroza va Cellobioza.
Hai chung na'm men
d^u
co
hinh
tiling,
elip,
nhung SC nay ch6i 1,2,3
phia,
trong khi do SB chi nay ch6i
1
phia.
Ca 2 chiing diu co
hinh
thai khuin
lac nhu nhau (tring siia, tron nhan bong va c6 vong d6ng tam) (bang 3).
Bang 3:
M6t

s6' dac
tinh hinh
thai
vk
sinh ly sinh hoa ciia hai chung na'm men
SC va SB
TT
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Dac diem
Hinh
dang
t€

bao
Hinh thdi khudn
lac
Sorboza
Riboza
Xyloza
Arabinoza
Rhamnoza
Trehaloza
Lactoza
Sucroza
Cellobioza
Raffinoza
Galactoza
Tinh b6t
Melibioza
Glucoza
Sinh axit
Phan ling DBB
Phan ling thuy
phan ure
Chiing nam men
Saccharomyces
cerevisiae
(SO
Hinh
trumg,
eh'p, nay ch6i
1,2,3
phia

Trdng
sua tron
nhSn
bong
l6i
CO
vong
d6ng
tam
Khd
nang
dSng hod
nguSn
cacbohydrat
+
-
+
-
+
+
-
-
+
Khd nang
len men
nguon
cacbohydrat
-
-
-

+
-
+
-
+
+
-
-
+
-
Saccharomyces boulardii
(SB)
Hinh
trutig,
eh'p, nay ch6i 1
phia
TrSng
sua tron nhan bong
loi
CO
vong d6ng tam
Khd nang
dong hod
nguon
cacbohydrat
-
-
-
-
+

-
+
-
+
+
-
+
Khd nang len
men nguon
cacbohydrat
+
+
-
+
+
+
-
-
-
18
Nhin
chung, dac diem d6ng hoa va len men cac ngu6n carbohydrate ciia
2 chiing na'm men tuang tu nhau, ca hai
diu
co phan
ling am tinh v6i
DBB va
thuy phan ure. Can
cii
vao khoa phan loai

ciia
Bergeys va cac du lieu
b
cac
bang 2 va 3
thi
chiing
NCI thu6c
nhom Lactobacillus fermentum, chiing NC2
thu6c
nhom Lactobacillus casei, chiing SC
thu6c
Saccharomyces cerevisiae
va chung SB
thuOc
nhom Saccharomyces nhung chua ro loai. Tuy nhien, de
ggi ten chinh
xdc,
cac chiing tren da dugc giai trinh tu ARN va ADN
16S.
Cac
ke't qua dinh danh dugc trinh bay nhu sau:
Trinh tu rADN
16S
cua chiing
NCI
>NC1
GCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCG
GGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAGCGTTGTTCGCAT
GAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGT

GCArrAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGA
GrrGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACG
GGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAAC
ACCGCGTGAGTGAAGAAGGGrrTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGA
ACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCAC
GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCG
GATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAGC
CrrCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAG
AGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAAATATATGGAAGAAC
ACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGC
ATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAG
TGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGC
ACTCCGCCTGGGGAGTACGACCGCGAGGTTGAAACTCAAAGGAATTGACGGG
GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACC
TTACCAGGTCrTGACATCTTGCGCCAACCCTAGAGATAGGGCGTTTCCTTCGGG
AACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTG
GGrTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTT
GGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACG
TCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGG
TACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCA
GTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATC
GCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG
TCCCACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAG
CCAGCCGCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCC
GTAGGAGAACCTGCGGCTGGATCACCTCCTTT
19
Trinh
tu
rADN
16S cua

chiing NCI tuang d6ng vofi trinh
tu
rADN
16S
ciia
Lactobacillus fermentum
99,7 % (1446/1450).
Trinh
tu
rADN
16S
ciia chiing
NC2
>NC2
ACGCTGGCGGCGTGCCTAATACATGCAAGGCGAACGAGTTCTCGTTGATGATCGG
TGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTG
GGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCA
TAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTT
GGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGA
TGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGC
CCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCT
GATGGAGCAACGCCGCGTGAGTGAAGAAGGCTGTCGGGTCGTAAAACTCTGTTG
TTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCA
GAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGC
GTTATCCGGATTTATrGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATG
TGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTG
CAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGA
AGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGA
AAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGAT
GAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAG

CATrCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGG
GCCCGCACAAGCGGTGGAGCATGTGGTTTAATrCGAAGCAACGCGAAGAACCTT
ACCAGGTCTTGACATCnTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGC
AAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA
AGTCCCGCAACGAGCGCAACCCTTATGACTAGTrGCCAGCATTTAGTTGGGCACT
CTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCAT
CATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTT
GCGAGACCGCGAGGTCAAGCTAATCrCTTAAAGCCAITCTCAGTTCGGACTGTAG
GCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCC
GCGGTGAATACGTTCCCGGGCCITGTACACACCGCCCGTCACACCATGAGAGTTT
GTAACACCCGAAGTCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAAGGTG
GGACAAATGATTAGGGG
Trinh
tu
rADN
16S
ciia chiing
NC2
tuang dong v6i trinh
tu
rADN
16S
ciia
Lactobacillus casei
99,7 % (
1483/1486
).
Trinh
tu ADN 26S cua
chiing

SC
>SC-I1
TTAAAGAAATTTAATAATTTTGAAAATGGATITrTrTGTTrTGGCAAGAGCATGA
GAGCmTACTGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCT
TAAGTGCGCGGTCTrGCTAGGCTTGTAAGnTCnTCrTGCTArrCCAAACGGTGA
GAGATrTCTGTGCTrTTGTTATAGGACAArrAAAACCGTTTCAATACAACACACT
GTGGAGTnTCATATCnTGCAACi-lTnClllGGGCATTCGAGCAATCGGGGCCC
20
AGAGGTAACAAACACAAACAATTTTATTTATTCATTAAATTnTGTCAAAAACAA
GAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACnTCAACAACGGATCTCT
TGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGC
AGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCA
GGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAACATTCTGTTTGGTAGTGAG
TGATACTCITTGGAGTTAACnTGAAATTGCTGGCCTTTTCATTGGATGTTTITnTC
CAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGG
TTTTACCAACTGCGGCTAATCnTTTTATACTGAGCGTATTGGAACGTTATCGATA
AGAAGAGAGCGTCTAGGCGAACAATGTTCTTAAAGTTTGACCTCAAATCAGGTA
GGAGTACCCGCTGACTTAGCATATCAATAGCGAAGAAAGAAAGCATATCAATAA
GCGGAGGAAAAGAAACCAACCGGGATTGCCTTAGTAACGGCGAGTGAAGCGGC
AAAAGCTCAAATTTGAAATCTGGTACCTTCGGTGCCCGAGTTGTAATTTGGAGAG
GGCAACnTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGG
GTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTGTAAAGTGCCTTCGAAGAGT
CGAGTTGTTTGGGAATGCAGCrCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAA
TATTGGCG
AGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAA
AGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGAC
ATGGTGTnTGTGCCCTCTGCrCCTTGTGGGTAGGGGAATCTCGCATTTCACTGGG
CCAGCATCAGnTTGGTGGCAGGATAAATCCATAGGAATGTAGCTTGCCTCGGTA
AGTATTATAGCCTGTGGGAATACTGCCAGCTGGGACTGAGGACTGCGACGTAAG
TCAAGGATGCTGGCATAATGGTTATATGCCGCCGTCTTGAAACAGGACC

Trinh
tu gen
ciia chiing
SC
tuang dong
100% vai
Saccharomyces cerevisiae
>SB-I1
GCCGGGCCTGCGCTTAAGTGCGCGGTCrrGCTAGGCTTGTAAGTTTCITrCnTGCr
ATTCCAAACGGTGAGAGATITCTGTGCTTrTGTTATAGGACAATTAAAACCGTTT
CAATACAACACA(n^GTGGAGTTITCATATCITrGCAACITrTTCTTTGGGCATTCG
AGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATCTATTCATTAAAT
TriTGTCAAAAACAAGAArrrrCGTAACrGGAAATTTTAAAATArrAAAAACTTr
CAACAACGGATCTCrTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATA
CGTAATGTGAATTGCAGAATrCCGTGAATCATCGAATCTTTGAACGCACATTGCG
CCCCnTGGTATTCCAGGGGGCATGCCrGTTTGAGCGTCATTTCCrrCTCAAACATT
CTGTTrGGTAGTGAGTGATACTCTTrGGAGTTAACITGAAATTGCTGGCC'TTTrCA
TTGGATGri'l'ril'irCCAAAGAGAGGTTTCrCTGCGTGCTTGAGGTATAATGCAAG
TACGGTCGTTITAGGTITrACCAACTGCGGCTAATCITTTTTTATACTGAGCGTAT
TGGAACGTTATCGATAAGAAGAGAGCGTCTAGGCGAACAATGTTCTTAAAGTTT
ACCTCTCAAATCAGGTAGGAGTACCCGCTGAACTTAAGCATAC
Trinh
tu gen cua SB
tuang dong
100%
v6i Saccharomyces
boulardii,
sai
khac
12 /707 bp

v6i Saccharomyces cerevisiae
Cac ke't qua
phan loai theo phuang phap
c6
diln
va dua vao
phan tich trinh
tu
ADN va ARN
dugc trinh
bay
b bang
4.
21
Bang 4:
Kit
qua phan loai va dinh danh cac chung dugc lua chgn
STT
1
2
3
4
Kihieu
NCI
NC2
SC
SB
Dua vao phan tich trinh tu
16S
rARN

Lactobacillus
fermentum
Lactobacillus casei
Saccharomyces cerevisiae
Saccharomyces boulardii
Theo phuang phap c6
diln
Lactobacillus fermentum
Lactobacillus casei
Saccharomyces cerevisiae
Saccharomyces sp.
Cac ke't qua
d
bang 4 cho tha'y co su
phd
hgp ra't cao cua viec phan loai
giGa
phuang phap c6 diln va phan loai dura tren ca
scf
phan
tich
trinh tu AND
va ARN riboxom.
Tir
ke't qua phan loai tren cho tha'y, viec phan lap va
tuy
In
chgn cac chiing VSV hiru ich da chgn dugc hai chung VK Lactic:
Lactobacillus
fermentum-NCI

va Lactobacillus
casei-NC2.
5.2.
Dac tinh probiotic ciia cac chiing VSV
hiifu
ich.
Cac chiing vi khuin Lactic
thu6c
nhom L. fermentum va L. casei cung
nhu cac chung na'm men
thu6c
cac nhom S. cerevisiae vd S. boulardii la
nhiing chiing VSV lanh tinh (khong phai la nhiing VSV gay benh), thucmg
dugc
sir
dung nhu nguon VSV Probiotic (Sanders va ctv, 2001). Tuy nhien,
m6i m6t
chung thugc cac nhom nay co cac dac
tinh
probiotic manh yeu khac
nhau.
Bcri
vay,
tru6c
khi dugc su dung, cac chung nay
cdn
dugc danh gia cac
dac tmh probiotic. Ke't qua danh gia dugc trinh bay
6
cac bang 5; 6; 7; 8 va 9.

Ke't qua
d
bang 5 cho tha'y 2 chiing vi khuan lactic
(NCI
va NC2) phat
triln t6't
c^
pham vi nhiet
d6
tii
30-37°C.
Khi nu6i ca'y
or
nhiet
d6
cao han (45
va
55°C)
sinh
khd'i
va kha nang san sinh axit lactic giam ro ret
(tir
5 de'n 8 x
10^
b 37°C xud'ng
2,8-5 x
10'
cfu/ml
b 45°C
va

tiT
2-4 x
10'
cfu/ml or
55°C).
Tuang ling
vdi
su giam sinh khd'i VSV, ham lugng axit lactic cung giam
manh. Dap
ling
vl sinh trucmg doi vdri nhiet
d6
nu6i ca'y cua 2 chung na'm men
(SC va SB) ciing co xu
hu^g
tuomg tu
dd'i
vdri nhom VK lactic, nhung 2 chiing
nam men to ra sinh trucmg tot han khi nu6i ca'y or nhiet
d6
30°C.
Ngoai
tr^
nhihig
VSV dugc phan lap tii m6i truomg ben ngoai, con hiu het cac VSV
22