Đánh giá độc tính cấp của chế phẩm synbiotic và khả năng sinh bào tử của b subtilis HU58

Y HOC VIET NAM THANG 2 - SO 2/2014
DANH
GlA
DOC TINH CAP CUA CHE PHAM SYNBIOTIC
VA KHA NANG SINH
BAO
TIT
CUA B. SUBTILIS HU58
Ho Anh
Son*,
Nguyin
Linh
Toan*,
Nguyen Trong
Tai*",
Nguyen Thi Mai Phuffng***
TOM TAT
Muc tieu nghien ciru la danh gia doc tfnh cap
ciia
che pham
synbiotic
va kha nang tang
sinh
bao
ty
B.
subtilis
HU58.
Ket qua nghien ciru cho thay khong co
sy thay
doi

cong
thyc
mau,
chffe
nang gan than va hinh
anh
giai
phau benh gan than cua chuot uong synbiotic.
So
lUOng
B.
subtilis HU58
tang sinh manh trong khoang
48
gicJ
roi giam dan sau 7 ngay uong
vi
khuan.
Tu"
khoa: acute toxicity, synbiotic, mice, B.
subtilis HU58
SUMMARY
EVALUATE ACUTE TOXICITY OF SYNBIOTIC
AND PROLIFERATIVE ABILITY
OF B. SUBTILIS HU58
The research objective was to assess the acute
toxicity of synbiotic preparations and proliferative
ability of
B.
subtilis

HU58.
The study results showed no
change in. Our purpose to evaluate the acute toxicity
of synbiotic and proliferative ability of B. subtilis HU58.
The results showed there were un change in blood
cell counts, kidney and liver function and anatomical
images of mice livers and kidneys. The number of B.
subtilis
HU58
proliferated rapidly within 48 hours and
then decrease gradually after 7 days of
B.
subtilis
HU58
oral
administrabon.
I.
DAT VAN
DE
Tren the
gidi,
phan Idn eac san pham thffc
pham bo sung dffdc san xuat d dang
probiotie
hay prebiotic rieng re. Viee ket hdp hai chat nay
trong ciing mpt san pham se cd gia tri sff dung
cao hdn nhieu va tien Idi cho ngffdi tieu diing
nen dffdc gpi la synbiotic. Cae dac tinh
chii
yeu

ciia
synbiotic la kha nang khang khuan, tang
cffdng loai bd vi khuan gay benh va tang dap
ffng mien djch khang nguyen dac hieu va khong
dac
hieu;
tang kha nang thffc bao; chdng ung
thff, chong ia chay; chdng dj
ffng;
ngan chan hdi
chffng bang xffdng; giam chat beo trong huyet
thanh va dffdng trong mau. Nhd vay, synbiotic
* Hoc vien Quan y
'* 'oai
Hpc Y Vinh
'** Vien Cong
nghe Smh hoc
Phan bien khoa hoc: TS. Can Van Mao
thyc sy la san pham cd nhieu hffa hen. San
pha'm
synbiotic
(P&P)
sff dung trong
ngjiien ciry
nay tao ra tff
viec
phoi trpn giffa dan xuat tff cam
gao vdi bao tff
ciia
chung vi sinh vat sinh bao

tii"
B. subtilis HU58. Oe cd ed sd cho cac nghien cffu
tiep va dffa dffpc san pham ra thj trffdng, can
kiem
soat tfnh an toan
ciia
che pham, do vay
muc tieu
eiia
chiing toi la danh gia dpc tfnh
cOa
che pham (neu cd) va kha nang sinh bao tff sau
khi dpng vat thffc nghiem udng che pham nay.
(Day ia mpt phan san pham
ciia
de tai "Nghien
cffu san xuat che pham synbiotic tff vi khuan sinh
bao tff va cam gao" Ma so:
KC04.TN01/11-15).
II.
OOI
TUOMG
VA
PHU'aNG
PHAP NGHIEN
CU\J
2.1.
Ddi tu'dng nghien cu'u
Chuot nhat trang do ban dpng vat Hpc vien
Quan y eung cap, khong phan biet gidng, khoe

manh,
dat tieu ehuan
thi
nghiem, can nang 20,0
± 2,0 g. Chuot dffdc nuoi trong dieu kien nhiet dp
24±2°C,
anh sang ty nhien, nydc va thffc an
dffde nau
ehfn,
bao dam theo nhu cau. Qua trinh
nudi dffdc bat dau trffdc khi tien hanh thf nghiem
ft nhat 3 ngay.
2.2.
Phu'dng phap nghien cu'u
2.2.1. Nghien cu'u dgc tinh cap
Opng vat dffdc chfa thanh cac
16,
moi
16
10 cor,
Chuot eiing
16
dffdc danh so va nhot chung vao 1
chuong.
Trffdc khi cho chuot diing thudc, chuot bj bo
ddi trong 12 gid. Sau khi tim kha nang hda tan
ciia
che pham, chiing tdi
tim
dffdc lieu P&P cao nhat

cho|
chuot uong la 15 g/kg can nang chuot, tffdng ffng
viJ
0,2ml/10g the trpng. Cac lieu tiep theo dffdc tfnh
giam so vdi lieu toi da 20%, tong sd cd 6 nhom lieu
dffdc sff dung. Sau khi cho diing thudc, chuot
dffoc
theo ddi, an va uong ty do. Thdi gian theo doi lien tuc
trong 72 gid. Sd ehupt chet dddc dem theo nhdm.
Cac chi tieu theo doi: Tinh trang toan
than,
long,
tfnh chat phan (khudn, nat), thay doi hanh vi; chi so
huyet hpc, chdc nang gan than doi vdi nhdm chuot
diing lieu cao nhat.
2.2.2.
Oem quan the vi khuan trong phin
Tong so gom
^
chuot,
6iScic
nuoi rieng
tiftig
eon.
Moi ehupt dffdc cho uong mot lieu duy nhat,
Y HOC VIET NAM THANG 2 • SO
2/2014
10
CFU vi khuan dffdc hoa tan trong nffdc cat.
Lay phan vao cac thdi diem khac nhau

(toi
da sau
7 ngay). Trffdc
khi
thu thap phan, day hop thu
thap dffdc trai giay trang da dffdc say vo khuan.
Chuot dffdc dffa vao timg hop rieng le. Cae mau
phan rieng biet cua tffng
ehupt
dddc thu thap,
danh so. Can 0,lg phan pha vdi Q,9m! dem PBS,
sau dd dddc trpn (votex) ky de tan phan hoan
toan va u trong
tii
am d
65°
trong 1
gid.
Quan the vi khuan dffdc dem ngay trong
ngay. Mat do vt khuan dddc xac dinh bang
phffdng phap pha loang va cay trai tren dia moi
tru'dng
LB agar. Cac dTa thach sau
khi
eay dffdc u
d
37°C
trong 48
gid
cho den

khi
cac khuan lac
dffdc
hinh
thanh hoan
toan.
III.
KET QUA
3.1.
Ket qua danh gia dpc tinh cap
3.1.1. Tinh trang toan than
Sau khi uong lieu duy nhat che pham synbyotic, nhdm lieu cao tffdng ffng vdi 15 g/kg the trpng va
cac nhdm uong vdi lieu thap hdn, tat ea ehupt thf nghiem d cae
16
deu an uong binh thu'dng, tinh trang
phan,
quan sat hoat dpng
ciia
chuot thay chuot binh thffdng.
Bang 3.1: So Iffdng chuot song sau khi udng
lieu
duy nhat che pham symbiotic P&P
Nhom
1
2
3
4
5
6
Liieu

su'
dung
(g/kg
TLCT/24h)
2,5
5,0
7,5
10,0
12,5
15,0
n
10
10
10
10
10
10
So lu'dng dong vat chet
'
(sau'72h)
0
0
0
0
0
0
So
lu'dng
dong vat
song (sau 72h)

10
10
10
10
10
10
Theo doi sau 72 gid, khong co chuot thi nghiem bi
chit,
phau thuat
ehupt
cho thiy hinh anh cac cd quan
binh th&dng.
3.1.2.
Sit
bien doi cac
chi sd
huyet
hgc
Chuot tai' nhdm sff dung che pham synbiotic lieu eao nhat (15 g/kg) dffde lay mau, kiem tra sd Iffdng
te bao
rriau
de so sanh vdi nhdm chffng khong uong che pham.
Bang 3.2: Sd Iffdng te bao mau ngoai
vi
trong mau chuot
Hong cau
(T/l)
Bach eau (G/l)
Tieu cau (G/l)
Nhom chffng

fn=10)
7,9± 0,26
Nhom uong
Synbiotic (n=10)
9,1± 3,3
620 ±65,3
p>0,05
p>0,05
p>0,05
Sdiiiu dbang
3.2 cho thiy
sdl&dng tebao
mau ngoai vi cua nhom chuot udng Synbiotic
lieu
cao
15g/kg
khong co
s&khac
biet dang ke vdi nhom ch&ng (p>0,05). So
l&dng
hong cau dao dpng quanh khoang 8
G/i;
sdl&dng
bach
cBu
dao dpng quanh 9 G/l va so l&dng tieu cau khoang trin 700 G/l.
3.1.3.
Theo doi chu'c
nSng
gan than

Bang 3.3: Nong dp ure, creatinin, SGOT va 5GPT trong mau ehupt
Ure (mg/dl)
Creatinin (mg/dl)
SGOT
(U/l)
Nhom chu'ng
(n=10)
22,67±2,11
0,55± 0,07
32,5± 2,59
Nhom uong
Synbiotic (n=10)
p>0,05
p>0,05
p>0,05
SGPT(U/1) "1
^7,6+3,56
| 35,5 + 8,92 | p>0,05 .
Kit qua thu d&dc d bang 3.3 cho thiy ndng do cdc chit ure, creatinin trong mau ngoai vi cua chuot
nhom ch&ng khong cd s& khac biit dang ke vdi nhom uong chi pham synbiotic. Dieu nay cho thiy ch&c
nSng
than dn dinh sau khi chuot uong lieu duy nhit synbiotic 15 g/kg.
Tudng
ttf,
nong dp hai enzyme ia SGOT va SGPT cua gan khong co s& khac biet
giO^
nhom s& dung
che
pham va nhom
chuhg.

Y
HOC VIET
NAM THANG
2 - SO
2/2014
3.1.4.
Hinh anh vi the gan than
Hinh
1: Hinh anh vl the cau true than
'• ;^-
Z
,'
•,cv
•w
WOTA
2/
Hinh anh vl the cau
true
gan
A:
7
i
Hinh anh
vi
thi cau
true
than
(Hinh
1) cho thay
ciu

true
than ro vung vo va vung tuy. Vung vo
vdi cac cau than
gom
cac mao mach
xung huyet.
Bao
Bowman
mong,
khoang
Bowman
t&
do.
Sat
vdi
cau
than la
Macula
densa,
cac ong
l&dn
gan
va l&dn xa d&dc lot bieu mo khoi
vudng.
Vung
tuy vdi
cac
dng
gop.
Khe than co

cac mach
mau
xung
huyet
T&dng
tu'
hinh
anh vi the ciu
true
gan (Hinh 2)
cho thiy ciu
true
cua gan vdi cac tieu thuy ro.
Khoang c&a
co
ranh
gidi
ro.
Trong khoang c&a co
cac dng mat, tieu quan mat, dpng
mach
va tinh
mach
gan.
Co si/xam
nhiem ft te bao
lympho va
bach cau ai
toan.
Cac

ti
bao
xip thanh be
gom
hai
hang tebao canh cac xoang
mach.
Rai raced
cac
te
bao Kulfer va
te
bao
npi mo lot
long.
3.2. Danh gia kha nang tang so Iffdng quan the vi khuan B. subtilis HU58 trong
rupt
Hinh 3.3. Sff thay doi so Iffdng bao tff vi khuan
B.
subtilis
HU58 theo thdi gian
24 il 4S
Thcri oian
Kit
qua
thu
d&dc
d
hinh
3.3

cho
thiy d thdi diim
tr&dc
khi
cho
chuot uong chi pham, sdl&dng
bao
t&B.
subtilis HU58
dat
m&c Iff
CFU/g,
mat dp nay
d&dc xem
la mat dp ban dau vdn co (base line)
trong rugt
gii
Sau
khi
du^
vi
khuan dang bao
t&
vao
cd
the chuot qua d&dng
uong,
sd
Iwdng
quan the vi

khuan tang
lin m&c 5xl(f°
CFU/g,
cao
hdn
ca l&dng bao t&dwa vao ban
dau.
IV. BAN LUAN
4,1.
Sff
dung
che pham P&P khong gay doc
San pham synbyotic cd thanh phan la chat xd
tff cam gao va vi khuan Bacillus deu da dffdc
chffng minh an toan trong qua trinh sff dung nhff
mpt thyc pham
chye
nang.
Cac khia canh an toan
Y HQC VIET NAM THANG 2 - SO 2/2014
duOc
quan tam chfnh la chung vi khuan dddc bo
sung vao che pham co an toan hay khong.
Cac nha nghien ciru da eong bd che pham
synbiotic chira Bacillus khdng gay hai eho cd the.
Sorokulova (2007) cdng bo cac ket qua cho thay
sff dung che pham chffa Bacilus kh6ng gay tan
mau,
vi khuan cung khong gay cac chat dpc to,
cae gen dpc nhff

bceTva
cytK khong tim thay.
Cac tac gia cung khdng phat hien dddc lieu gay
chet tren chuot. Cac nghien cffu tru'dng dien de
tim dpc tfnh ban cap
ciia
che pham cung khong
phat hien ton thffdng d cae cd quan, mo.
Cong bd mdi day
ciia Sulemankhil
va cpng sy
(2012) cung cho rang cac vi khuan trong thye
pham
chffe
nang la an
toan.
Nghien cffu eho thay
sff dung vi khuan lactobacillus cd tac dung chong
viem,
lam giam cytokine IL-8, dong thdi khdng
phat hien dffdc bat thffdng tren ehupt udng lien
tue trong vdng 28 ngay. Khdng eo sy bien doi
nao ve sff tan mau hay cae chat sinh hpc khac
trong cd the.
4.2.
Kha nang tang so lu'dng quan the vl
khuan B. subtilis
HU58
So Iffdng bao tff thai ra qua phan chuot cao
hdn so Iffdng dffdc dffa vao cd the giai thfch la do

bao tff da
QiftSc
nay mam trong rupt sau do phat
trien va lai tai tao bao tff. Neu rupt dpng vat
khdng phai la moi trffdng
phii
hdp, bao tff sau khi
dffa vao se bj thai ra theo dffdng phan va nhffng
bao tff da nay mam se bj chet trong rupt. Mffc dp
thay doi Idn ve so Iffdng hinh thanh bao tff va sy
cd
m^t
ciia
bao
ty
trong tat ca cac mau phan
chuot thf nghiem
6a
eho thay rang rupt la mdi
trddng thfch hdp nhff la ndi cff tru tff nhien cho
no.
Nghien ciru
ciia
Cutting va cpng sff tru'dc day
CLJng
da chffng minh dffdc B. subtilis ed the sinh
trffdng,
phat trien va tao bao tff trong dieu kien
yem khf eua rupt gia. Do thj
d

hinh 3.3 cung cho
thay so Iffdng vi khuan duy tri d mffc dp cao nay
trong khoang
48
gid va bat dau giam dan theo
thdi
gian.
Sau 7 ngay thi so Iffdng vi khuan da
gl^m
xuong tdi mffc ban dau. Nhff vay bao tff vi
khuan B. subtilis
HU58
da nay mam, phat trien va
hinh thanh bao tff trong ruot
gia
cua
ehupt
tru'dc
khi dffdc
thai
ra ngoai mdi tru'dng qua phan.
V. KET LUAN
1.
Khong phat hien dffdc dpc tinh cap d lieu
tdi da 15 g/kg the trpng sau khi chudt udng lieu
duy nhat che pham Synbiotic ed nguon gdc tff
cam gao. Chudt udng ehe pham Synbiotic vdi
lieu
duy nhat khdng lam thay ddi tinh trang toan
than,

so lu'dng te bao mau, chffe nang va
hinh
anh vi the gan
than.
2.
Bao tff vi khuan B. subtilis MUSS da nay
mam,
phat trien va hinh thanh bao tff trong rupt
ket
ciia
chuot trffdc khi dffdc thai ra ngoai moi
trffdng qua phan.
TAI LIEU THAM
KHAO
1.
Collins MD, Gibson GR. Probiotics,
prebiotics,
and
synbiotics:
approaches for modulating the
microbial ecology of the gut. Am J Cim Nutr. 1999
;69{5):1052S-1057S.
2.
GabrJella,
C. and Cutting S., (2002), Bacillus
Probiotic:
Spore Germination in the
Gastrointestinal Tract. Appl Environ Microbiol
68(5):
2344-2352.

J.
Hisanga, S., (1980), Studies on the germination
of genus Bacillus spores in rabbit and canine
intestines. J.
Nagoya City
Med.
-^5S0C
30:
456-469.
t.
Hoa, TT.,
Due,
LH.,
Isticato,
R ,
Baccigalupi,
L., Ricca, E., Van, PH. and Cutting, SM.,
(2001), The fate and dissemination of Bacillus
subtilis spores
in
a murine model. Appl Environ
Microbiothl:
3819-3823,
5.
Irina
B. Sorokulova,
Iryna
V. Pinchuk, Muriel
Denayrolles, Irina G.
Osipova,

Jen M. Huang,
Simon M. Cutting, Maria C. Urdaci. The Safety
of Two Bacillus Probiotic Strains for Human Use.
Dig Dis Sci (2008) 53:
954-963.
6. Le H. D., Huynh A.
H„
Barbosa
T.M.,.
Henriques
A.O., and Cutting S.M.
(2004)Charactenzation
of Bacillus Probiotics
Available for Human Use. Applied and
Environmental Microbiology, 70(4):
2161-2171.
7. Nguyen K. M. T, Nguyen Q. U., Huynh A. H.,
Le H. D., Tran T. H., Claudia R. S., Adriano
0.
H., and Cutting S.M. (2006) The Intestinal
Life
Cycle of Bacillus subtilis and Close Relatives.
Journal of Bacteriology. 188(7): 2692-2700.