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B GIO DC V O TO

B Y T

TRNG I HC DC H NI

TRNH TH PHNG DUNG

XY DNG QUY TRNH XC NH (NH


TNH, NH LNG) BIFIDOBACTERIUM
LONGUM TRONG CC CH PHM
PROBIOTICS

LUN VN THC S DC HC

H NI 2014

B GIO DC V O TO

B Y T

TRNG I HC DC H NI

TRNH TH PHNG DUNG

XY DNG QUY TRNH XC NH (NH


TNH, NH LNG) BIFIDOBACTERIUM
LONGUM TRONG CC CH PHM
PROBIOTICS
LUN VN THC S DC HC

CHUYN NGNH KIM NGHIM THUC V C CHT


M S 60 72 04 10

Ngi hng dn khoa hc: TS. Trn Vit Hng

H NI 2014

LI CM N
Lun vn ny c hon thnh ti khoa Vi Sinh Vin Kim nghim Thuc
Trung ng B Y t. c c bn lun vn tt nghip ny ti xin by t lng
bit n chn thnh v su sc ti TS. Trn Vit Hng, ngi thy trc tip hng
dn, du dt ti trong sut thi gian thc hin nghin cu ny.
Xin chn thnh cm n cc thy c trong Ban Gim hiu, Phng o to sau
i hc, B mn Kim Nghim Thuc v c cht v cc B mn khc ca trng
i Hc Dc H Ni gip ti trang b tri thc, to mi trng, iu kin thun
li cho ti trong sut qu trnh hc tp ti trng cng nh trong qu trnh hon
thnh lun vn.
Ti xin chn thnh cm n TS. on Cao Sn Vin trng Vin kim
Nghim Thuc Trung ng, Ban Gim c Vin Kim Nghim Thuc Trung
ng, cc ng nghip trong khoa v Th s. Khng Th Minh Hu h tr, gip
ti hon thnh tt lun vn thc s v hon thnh kha hc theo ng thi hn.
Cui cng, ti xin gi li tri n su sc n gia nh v bn b lun bn
ti ng vin, chia s, gip ti trong thi gian va qua

H Ni 27 thng 8 nm 2014
Dc s Trnh Th Phng Dung

MC LC
CC CH VIT TT
DANH MC BNG
DANH MC HNH
T VN ....................................................................................................... 1
CHNG 1. TNG QUAN ................................................................................ 3
1.1. TNG QUAN V PROBIOTICS ............................................................... 3
1.1.1. nh ngha ................................................................................................ 3
1.1.2. ng dng ca probiotics trong ngnh Dc ............................................ 3
1.1.3. Cc vi sinh vt thng c s dng trong ch phm probiotics............ 4
1.1.4. Tng quan v tiu chun cht lng probiotics ....................................... 4
1.2. TNG QUAN V CHI BIFIDOBACTERIUM .......................................... 6
1.2.1. c im chung ca chi Bifidobacterium [30], [33],............................... 6
1.2.2. Bifidobacteria vi vai tr l probiotics. ................................................... 6
1.3. TNG QUAN V B. LONGUM .................................................................. 7
1.3.1. c im hnh thi ................................................................................... 7
1.3.2. Kh nng chuyn ha carbohydrat [31]. .................................................. 8
1.3.3. Cu trc gen cu B. longum [24].............................................................. 8
1.3.4. Vai tr ca B. longum [9], [11], [24], [26] , [15], [33], [35]. ................... 9
1.3.5. Phng php kim nghim B. longum trong ch phm probiotics ........10
1.3.6. Tnh hnh nghin cu nh tnh v nh lng B. longum trong cc ch
phm probiotics trong nhng nm gn y ...........................................17
CHNG 2. I TNG, NGUYN VT LIU, TRANG THIT B
PHNG PHP NGHIN CU .............................................................20
2.1. PHNG TIN DNG TRONG NGHIN CU ..................................20
2.1.1. Mi trng, ho cht, dung mi, thit b ...............................................20
2.1.1.1. Mi trng ......................................................................................20
2.1.1.2. Ho cht, dung mi .........................................................................22
2.1.1.3. Thit b, dng c .............................................................................23
2.1.2. Phn mm x l kt qu: Apiweb ca hng BioMrieux. ......................24
2.1.3. Chng vi sinh vt chun .........................................................................24
2.2. I TNG NGHIN CU ....................................................................25

2.3. PHNG PHP NGHIN CU ..............................................................26


2.3.1. Phng php nh lng B. longum trong ch phm ............................26
2.3.2. Phng php nh danh vi sinh vt bng kit API 20A ..........................27
2.3.2.1. Nguyn tc chung ............................................................................27
2.3.2.2. Tin hnh .........................................................................................28
2.3.3. nh danh n loi bng k thut PCR v gii trnh t .........................31
2.3.3.1. S chung nh tnh B. longum trong cc ch phm probiotics
bng k thut PCR v gii trnh t .................................................31
2.3.3.2. Tch DNA vi khun bng WizardR Genomic DNA Purification Kit
.........................................................................................................32
2.3.3.3. nh lng DNA bng phng php o hp th nh sng t
ngoi bc sng 260 nm (A260) ...................................................33
2.3.3.4. Nghin cu, thit k mi c hiu ................................................33
2.3.3.5. Kim tra kh nng tch DNA ..........................................................34
2.3.3.6. Nhn bn on gen c hiu ca cc i tng nghin cu bng k
thut PCR ........................................................................................35
2.3.3.7. Nhn dng v gii trnh t ..............................................................36
2.3.4. Thm nh quy trnh xy dng ..........................................................39
2.3.4.1. Thm nh phng php nh lng B. longum .............................39
2.3.4.2. Thm nh qui trnh nh danh bng kit API 20A ..........................41
2.3.4.3. Thm nh qui trnh nh danh bng k thut PCR v gii trnh t41
2.3.5. ng dng quy trnh chun nh lng v nh tnh B. longum trong
cc ch phm probiotics ang lu hnh trn th trng ........................44
CHNG 3. KT QU NGHIN CU .........................................................45
3.1. XC NH S LNG VI SINH VT TRONG CH PHM
PROBIOTICS .............................................................................................45
3.2. NH DANH VI SINH VT BNG KIT API 20A .................................46
3.3. KT QU NH DANH VI SINH VT TRONG CC MU BNG
K THUT PCR V GII TRNH T .................................................51
3.3.1. Kt qu tch DNA ..................................................................................51
3.3.1.1 Tch DNA bng WizardR Genomic DNA Purification kit ca hng
Promega ..........................................................................................51
3.3.1.2. Kim tra kh nng tch DNA ..........................................................51
3.3.2. Nhn bn cc on gen c hiu ca cc i tng vi sinh vt thuc i
tng nghin cu ...................................................................................52

3.3.3. Nhn dng v gii trnh t .....................................................................55


3.3.3.1.Tinh sch sn phm PCR nhn bn cc on gen c hiu ca cc vi
sinh vt phn lp t cc mu nghin cu bng b kit Fermentas ...55
3.3.3.2. Nhn dng cc on gen c hiu vo vector nhn dng v bin np
vo t bo E. coli chng DH5 .......................................................56
3.3.3.3. Kim tra s c mt ca cc on gen c hiu trong plasmid ti t
hp ..................................................................................................57
3.3.3.4. Gii trnh t mu cha phn on 831 bp .......................................60
3.4. NH GI PHNG PHP ....................................................................62
3.4.1. Kt qu kim tra tnh chn lc ca mi trng nh lng BSM agar .62
3.4.2. lp li ca php nh lng ..............................................................63
3.4.3. Thm nh c hiu ca quy trnh nh danh vi sinh vt bng kit API
20A ........................................................................................................64
3.4.4. nh gi c hiu ca quy trnh nh danh vi sinh vt bng k thut
PCR........................................................................................................65
3.4.5. Xc nh ngng pht hin ca quy trnh nhn bn on gen c hiu
ca B. longum bng cp mi c hiu. ..................................................68
CHNG 4. BN LUN ..................................................................................71
KT LUN V KIN NGH ...........................................................................73
TI LIU THAM KHO .................................................................................74
PH LC

CC CH VIT TT
AA:

Acid acetic

AL:

Acid lactic

ATCC:

American Type Culture Collection

B. longum:

Bifidobacterium longum

dATP:

Deoxy adenosine Triphosphat

dCTP:

Deoxy cytosineTriphosphat

dGTP:

Deoxy guanine Triphosphat

DNA:

Deoxyribo nucleic acid

dNTP:

Deoxy nucleic Triphosphat

dTTP:

Deoxy thymin Triphosphat

E. coli :

Escherichia coli

EDTA

Ethylenediaminetetraacetic acid

FAO

Food and Agriculture Organization of the United Nations

FISH:

fluorescence in situ hydridization

JCM:

Japan Colletion of Microorganisms

LAB:

Lactic acid bacteria

LB

Luria Bertani

PCR:

Polymerase Chain Reaction

rRNA:

Ribosome Ribonucleic acid

VSV:

Vi sinh vt

DANH MC BNG
Trang
Bng 2.1

Mi c hiu

22

Bng 2.2

Ha cht, dung mi

22

Bng 2.3

Cc vi sinh vt chun c s dng

24

Bng 2.4

Cch c kt qu kit Api 20A

30

Bng 2.5

Trnh t cc cp mi dng cho phn ng PCR

34

Bng 2.6

Thnh phn ca phn ng PCR kim tra DNA mi tch

35

Bng 2.7

Thnh phn ca phn ng PCR n mi

36

Bng 2.8

Thnh phn phn ng gn sn phm PCR vo vector

38

Bng 2.9

Cc chng chun kim tra tnh chn lc ca mi trng BSM

40

agar
Bng 2.10

Cng thc tnh c hiu

42

Bng 2.11

Tiu chun nh gi cht lng ca sn phm PCR

43

Bng 3.1

S lng vi sinh vt trong ch phm

45

Bng 3.2

Theo di phn ng nh danh vi sinh vt bng kit API 20A

48

Bng 3.3

Kt qu nh danh vi sinh vt bng phn mm Apiweb

50

Bng 3.4

Kt qu o quang cc dung dch DNA mi tch

51

Bng 3.5

Thnh phn ca phn ng PCR

53

Bng 3.6

Kt qu kim tra tnh chn lc ca mi trng BSM agar

62

Bng 3.7

Kt qu m s lng vi khun trong mu A

64

Bng 3.8

Kt qu kim tra c hiu ca cp mi BlonF/BlonR trn

66

20 chng
Bng 3.9

Kt qu m s lng vi khun B. longum

68

Bng 3.10

Kt qu xc nh ngng pht hin

69

DANH MC HNH
Trang
Hnh 1.1

Hnh nh B. longum trn knh hin vi in t

Hnh 1.2

Hnh nh genom B. longum

Hnh 1.3

S phn ng chui polymerase

14

Hnh 1.4

Hnh minh ha k thut PCR

14

Hnh 2.1

S nh tnh B. longum bng k thut PCR v gii trnh t

31

Hnh 3.1

nh danh vi sinh vt trong trong mu A bng kit API 20A

46

Hnh 3.2

nh danh vi sinh vt trong trong mu B bng kit API 20A

46

Hnh 3.3

nh danh vi sinh vt trong trong mu C bng kit API 20A

47

Hnh 3.4

nh danh vi sinh vt trong trong mu D bng kit API 20A

47

Hnh 3.5

nh danh vi sinh vt trong trong mu E bng kit API 20A

47

Hnh 3.6

Quan st ging ESC di n UV bc sng 365nm

48

Hnh 3.7

Kt qu in di sn phm PCR nhn bn on gen 16S rDNA

52

bng cp mi ID16R08F v IDL16R09R


Hnh 3.8

Kt qu in di sn phm PCR nhn bn on gen c hiu

54

ca B. longum bng cp mi BlonF/BlonR


Hnh 3.9

Kt qu in di mu tinh sch sn phm PCR nhn bn cc

55

on gen c hiu phn lp t cc mu nghin cu trn gel


agarose 1%
Hnh 3.10

S vector nhn dng pGEMT-eseay

56

Hnh 3.11

Kt qu bin np sn phm gn vo vi khun E. Coli DH5. v

57

cy tri trn mi trng nui cy c b sung ampicillin


100g/ml
Hnh 3.12

Kt qu in di sn phm PCR kim tra plasmid ti t hp

58

mang cc on gen c kch thc 831bp


Hnh 3.13

Kt qu in di sn phm ct gii hn vector ti t hp mang


cc phn on 831 bp bng enzym EcoRI

59

Hnh 3.14

Hnh thi khun lc B. longum trn mi trng BSM agar

63

Hnh 3.15

nh danh B. longum JCM 1217 bng kit Api 20A

65

Hnh 3.16

Kt qu in di sn phm PCR kim tra c hiu ca cp

67

mi nhn bn on gen c hiu ca B. longum


Hnh 3.17

Kt qu in di sn phm PCR xc nh ngng pht hin ca


quy trnh nhn bn on gen c hiu ca B. longum

70

T VN
Vi khun lactic (LAB) c vai tr rt quan trng trong cuc sng ca chng ta.
Chng to ra cc thc phm ln men v bo qun thc phm khi b h hng. T
u th k 20, Elie Metchnikoff (1845-1916) xut s dng cc LAB cho mc
ch cha bnh. T , lnh vc nghin cu probiotics ngy cng c quan tm v
pht trin. n nay, nhng nghin cu v probiotics khng ngng cung cp
nhng bng chng c tnh khoa hc v hiu qu thc s ca probiotics i vi sc
khe con ngi. Bn cnh , cc sn phm chc nng s dng cc vi khun
probiotics xut hin ngy cng nhiu Chu u, Nht, M Nm 2008, doanh thu
t cc ch phm ny l 15,9 t USD trn ton cu [10].
Probiotics bt ngun t ngn ng Hy Lp c ngha l v s sng (for life).
Nm 2002 t chc Nng Lng Lin hip quc v t chc Y t th gii nu r:
probiotics l nhng vi sinh vt sng m khi s dng vi mt lng thch hp, s
to nn nhng hiu qu tt i vi sc khe ca c th ch [32]. Cc probiotics
thng c trong cc ch phm l vi khun sinh acid lactic (LAB) trong bao gm
chi Lactobacillus v chi Bifidobacterium, vi khun sinh bo t Bacillus, nm
Saccharomyces cerevisiae
Theo Robin Temmerman (2001) trong s 55 mu kho st ch c khong 20%
ch phm c cha vi khun ng nh trn nhn, 16% ch phm khng cha mt
trong cc chng probiotics c lit k trn nhn [28]. Mt khc s lng vi sinh
vt trong cc ch phm probiotics gim nhiu trong thi gian bo qun. Nh vy,
vic nh tnh, nh lng cc loi vi khun probiotics trong cc ch phm l rt
quan trng, m bo cht lng ca ch phm, mang li hiu qu cho ngi dng.
Th trng thuc Vit Nam hin c hng trm ch phm probiotics di dng
thuc v thc phm chc nng, trong c rt nhiu ch phm c cha
Bifidobacterium longum (B. longum l mt vi khun thuc nhm LAB) di dng
n hoc a thnh phn. Mc d trn th gii c rt nhiu cng trnh nghin cu
nh tnh v nh lng B. longum trong cc ch phm a thnh phn, tuy nhin
Vit Nam vic kim sot cht lng ca cc ch phm probiotics cn ang b

ng. C th l Vit Nam cha c mt ti liu chnh thc no hng dn kim sot
cht lng cc ch phm probiotics bao gm c cc ch phm cha B. longum. a
s cc tiu chun c s ca cc thuc v thc phm chc nng trong nc cng nh
nhp khu cha nh lng ring cng nh cha nh tnh c B. longum trong
cc ch phm c cha hn hp nhiu vi sinh vt.
Xut pht t nhu cu thc t v tnh cp thit v yu cu kim tra cht lng
ca cc ch phm probiotics, chng ti tin hnh ti: Xy dng quy trnh xc
nh (nh tnh, nh lng) Bifidobacterium longum trong cc ch phm
probiotics vi cc mc tiu:
- Xy dng quy trnh chun nh tnh, nh lng B. longum trong cc ch
phm a thnh phn.
- ng dng quy trnh thit lp kho st cht lng ca mt s ch
phm probiotics c cha B. longum ang lu hnh trn th trng.

CHNG 1. TNG QUAN


1.1. TNG QUAN V PROBIOTICS
1.1.1. nh ngha
Hng ngn nm trc y, con ngi bit s dng cc ch phm sa ln
men vi mc ch tng cng sc khe. Tuy nhin, phi n u th k 19 nh
khoa hc ngi Nga Elie Metchnikoff mi thc s nghin cu v vn ny. T
n nay, lnh vc nghin cu v probiotics ngy cng c quan tm v pht trin.
C rt nhiu nh ngha khc nhau v probiotics, tuy nhin nh ngha ca t chc
Nng Lng Lin hip quc v t chc Y t th gii nm 2002 l c s dng
rng ri ph bin nht: probiotics l nhng vi sinh vt sng m khi s dng vi
mt lng thch hp, s to nn nhng hiu qu tt i vi sc khe ca c th
ch [32].

1.1.2. ng dng ca probiotics trong ngnh Dc


Probiotics c cc tc dng sau:
- Tiu dit hoc km hm s pht trin ca cc vi sinh vt c hi bng cch
tit ra cc cht ging khng sinh nh acidolin, lactocidin v acidophilin.
- Sinh ra cc vitamin bao gm niacin, acid folic, biotin, vitamin B6...
- H tr cho qu trnh tiu ha, iu chnh nng enzym.
- Gim pH bng cch sinh ra acid lactic, hydrogen peroxyd... do c ch s
pht trin ca cc vi sinh vt v nm c hi.
- Gii c v bo v nim mc rut: Lactobacillus c kh nng lin kt vi
cc cht c nh kim loi nng, t bo ung th, cc vi sinh vt ny s cht
cng vi cht c v c loi tr ra khi c th cng vi cht c di
dng cht thi rn.
- Gim nng cholesterol.
- H tr hp thu cc khong cht, c bit l calci do tc dng lm gim pH
rut ca chng.
- Gim nguy c b ung th, khi u [3], [6].

Vi nhng tc dng trn, Probiotics c s dng vi mc ch chnh l lm


cn bng h vi sinh vt ng rut trong hoc sau khi iu tr bng khng sinh do
khng sinh lm thay i h vi sinh vt trong ng tiu ha, gim s lng vi
sinh vt c li v thng gy a chy, ri lon tiu ha [34]. Ngoi ra, Probiotics
cn c s dng iu tr vim ng tiu ha thng gp tr s sinh do b
nhim Rota virus, iu tr tiu chy, ung th rut kt, au d dy do nhim
Helicobacteria pylori, bnh v d ng, ngn cn nhim trng m o do Candida
albicans...[3], [6].

1.1.3. Cc vi sinh vt thng c s dng trong ch phm probiotics


Nu trc kia s la chn vi sinh vt cho ch phm Probiotics dng lm thuc
ch yu da trn kinh nghim th hin nay, cc nh khoa hc a ra nhng tiu
ch rt r rng, l:
- C kh nng chu c pH thp d dy v acid mt rut non
- C kh nng bm dnh vo nim mc ng tiu ha
- Khng gy bnh, khng sinh c t
- C kh nng sng v c tr trong rut.
Bn nhm vi sinh vt thng c s dng trong cc ch phm probiotics l
Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus. Ngoi ra, cn mt
s nhm vi sinh vt khc c s dng nh Bacillus, Streptococcus [3], [6], [29].

1.1.4. Tng quan v tiu chun cht lng probiotics


Hin nay trn th gii c rt nhiu hng dn xy dng tiu chun cht lng
ca probiotics. Theo khuyn co ca FAO (2002), tiu chun cht lng ca cc
probiotics bao gm cc ch tiu [32]:
- nh danh vi sinh vt: yu cu phi nh danh n loi bao gm c kiu
hnh v kiu gen
- Cc th nghim in vitro v hot tnh ca probiotics: khng khng sinh,
khng dch v, khng mui mt
- Cc bng chng v s an ton
- Th in vivo trn ng vt v trn ngi

- Cc yu cu v nhn sn phm
Nm 2009, B y t Canada cng ban hnh chuyn mc v probiotics, trong
cng yu cu nh danh vi sinh vt n loi bao gm c kiu hnh v kiu gen [10].
n nm 2011, B sc khe v gia nh cng vi B Cng ngh sinh hc ca
n ban hnh hng dn nh gi cht lng probiotics kh y bao gm
9 ch tiu [16]:
- nh danh vi sinh vt: yu cu phi nh danh n loi bao gm c kiu
hnh v kiu gen. Trong ch ra k thut PCR l k thut sinh hc phn t
nh danh vi sinh vt.
- Cc th nghim in vitro v hot tnh ca probiotics: khng khng sinh,
khng dch v, khng mui mt
- Cc bng chng v s an ton trn ng vt
- Cc th nghim v tc dng ca probiotics trn ng vt
- Cc bng chng v s an ton trn ngi
- Th tc dng trn ngi
- Cc yu cu v nhn sn phm
- Cc th nghim xc nh liu
- Yu cu ghi nhn
- Yu cu trong sn xut v th tc ng k
Hin nay Vit Nam cha c vn bn chnh thc no v vic nh gi cht
lng ca cc ch phm probiotics. Ch c duy nht TCVN 7849:2008 a ra
phng php m L. acidophilus trong sa v cc ch phm sa trn mi trng
c hiu [5]. Vic kim sot cht lng mi ch dng li xc nh s lng vi sinh
vt, nh tnh da trn kiu hnh, nhim khun v cc ch tiu v dng bo ch
theo tiu chun nh sn xut. Trong rt nhiu trng hp, tiu chun cht lng cn
rt s si, c bit cha nh danh c tt c cc vi sinh vt trong ch phm.

1.2. TNG QUAN V CHI BIFIDOBACTERIUM


1.2.1. c im chung ca chi Bifidobacterium [30], [33],
Cc vi khun thuc chi Bifidobacterium trc y thng c gi l
Lactobacillus bifidus v c phn loi thnh 29 loi khc nhau. Ngy nay, ngi
ta pht hin ra hn 33 loi thuc chi Bifidobacterium, trong s c khong 12
loi c mt ng tiu ha ca ngi.
Bifidobacteria l cc trc khun gram dng, k kh bt buc. Chng thuc
loi vi khun ln men khng ng nht, khng di ng v khng hnh thnh bo t.
Bifidobacteria cho phn ng catalase m tnh, phn ng indol m tnh v phn ng
fructose6-phosphate phosphoketolase dng tnh. Chng pht trin khong nhit
thch hp t 37-41oC, khng pht trin nhit di 20oC v trn 46oC, Tuy
nhin, c mt s loi c th pht trin nhit trn 46oC nh B.
thermacidophilum. Khong pH ti u cho s pht trin ca Bifidobacteria l 6,57,0. Bifidobacteria khng pht trin c pH di 4,5 v trn 8,5 ngoi tr B.
thermacidophilum c th pht trin c pH 4,0.
Cc vi khun Bifidobacterium l nhng vi sinh vt quan trng trong h ng
rut ca ngi v chim 3,5 n 10% h sinh vt rut kt. Chng chim u th
trong phn ca tr b m, cn trong phn ca tr b bnh chng chim khong 40%
h vi sinh vt. Chng l mt trong nm loi chim u th trong h thng ng tiu
ha ca ngi, ngoi ra cn c tm thy m o v khoang ming. Ngoi ra
Bifidobacteria cng c tm thy trong h thng ng tiu ha ca nhiu ng
vt nh b ci, cu, ln, g, th, chut v ong mt.

1.2.2. Bifidobacteria vi vai tr l probiotics.


Bifidobacteria l nhng probitics ph bin v l mt phn quan trng ca h vi
khun trong ng tiu ha. Chng thuc nhm vi khun sinh acid lactic (LAB), c
kh nng tng hp amino acid (acid glutamic v acid aspartic), sn xut mt s
vitamin nh riboflavin, thiamin, vitamin B2, vitamin B6 v biotin.

Ngoi ra Bifidobacteria cn c kh nng sn xut bacteriocin (ha cht khng


khun) v cc cht ging khng sinh do chng rt c li i vi h thng tiu
ha v min dch [33].
Tuy nhin, khng ging nh cc vi khun Lactobacillus, cc vi khun
Bifidobacterium cn sn sinh ra acid acetic, l acid bo chui ngn. Acid acetic hn
ch nm pht trin hiu qu hn acid lactic v n cng c xem l ngun nng
lng cho c th. Do to ra ng thi acid acetic v acid lactic cng nh mt s
cht c ch khc nn cc vi khun Bifidobacterium rt cn thit cho ng m o
v ng niu sinh dc, ni m nm v cc vi khun c hi c th gy bnh, cng
nh trong h tiu ha [36].
Mt s li khun Bifidobacterium c tc dng tt i vi c th ngi do
chng c th lm thay i h min dch. Trong mt s trng hp chng gip tng
cng h min dch cng nh c tc ng chng vim.
Mt s Bifidobacteria hay c dng trong cc ch phm thuc, thc phm
chc nng vi vai tr l probiotics l Bifidobacterium longum, Bifidobacterium
infantis, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium breve

1.3. TNG QUAN V B. LONGUM


1.3.1. c im hnh thi
B. longum thuc nhm vi sinh vt sinh acid lactic v thuc chi
Bifidobacterium.
B. longum l vi khun Gram dng, hnh que phn nhnh, k kh bt buc,
khng di ng, khng sinh bo t v cho phn ng catalase m tnh. B. longum tng
trng ti u 38 - 39C, ti thiu 19-20C, ti a 44,5-45C. B. longum tng
trng ti u pH 7-8, khng tng trng pH 5,0 hoc 9,5 [12].

Hnh 1.1. Hnh nh B. longum trn knh hin vi in t


1.3.2. Kh nng chuyn ha carbohydrat [31].
B. longum c kh nng ln men cc ng: arabinose, xylose, glucose,
fructose, mannose, galactose, maltose, sucrose, lactose, melibiose, raffinose. Sn
phm cui cng ca qu trnh ln men ng l acid lactic (AL) v acid acetic
(AA), vi t l t 1,0 (AL): 1,7 (AA) n 1,0 (AL): 2.0 (AA).
B. longum khng ln men cc ng: rhamnose, melezitose, cellobiose,
trehalose, dextrin, tinh bt, inulin, sorbitol, manitol, glycerol, salicin, gluconate v
lactate.

1.3.3. Cu trc gen cu B. longum [24]


Nhim sc th ca B. longum c chiu di khong 2,26 Mb, t l Guanine Cytosine l 65%.

Hnh 1.2. Hnh nh genom B. longum

Trong :
A: Kch thc gen biu din trn thang Mb
B: Chui m ha: gm 2 si b sung, cc mi tn ch phn chuyn tip GC
C: Thang G-C
D: Cu to nguyn vn genom ca B. longum.Vng hnh trn biu din cc
rARN operon, hnh vung l vng gen ca prophage, tam gic biu din v
tr ca 3 vng gen ging nhau ca B. longum, hnh ch nht l vng gen
plasmid tch hp.

1.3.4. Vai tr ca B. longum [9], [11], [24], [26] , [15], [33], [35].
B. longum l mt loi vi khun rt cn thit vi c th con ngi, thng gp
rut v m o do n c vai tr quan trng trong vic gi cho h tiu ha v h
min dch con ngi khe mnh. B. longum c bit quan trng i vi tr s sinh
do n gip tng cng h min dch ca tr s sinh. B. longum c nhiu trong h
tiu ha ca tr s sinh v chim u th hn tr b m so vi tr b bnh. Mt
iu d thy l tr b m t b tiu chy v d ng hn so vi tr b bnh. ngi
ln, B. longum gi cho h thng min dch khe mnh, h tr cn bng vi khun
ng rut v c th ngn nga ung th nh ung th rut kt. B. longum sn sinh
acid lactic, do vy lm gim nguy c vim m o.
Mt nghin cu lin quan n gim cn, bo ph v to bn ch ra rng nhng
ngi s dng B. longum t c kt qu gim cn v ci thin to bn tt hn so
vi nhng ngi dng thuc nhun trng [11].
Mt nghin cu khc hin c thc hin ti Nht Bn nhm xc nh liu B.
longum c th c s dng nh l vector chuyn gen iu tr ung th hay
khng. Tin hnh thc nghim trn chut cho thy B. longum c th xm nhp vo
khi u v tch ly trong khi u. y l mt nghin cu rt tch cc v hy vng n s
gip cho cc nh nghin cu tm ra cch mi a thuc trc tip vo khi u [15].
Do c vai tr rt quan trng i vi sc khe con ngi nn B. longum c
s dng rng ri trong cc ch phm thuc, thc phm chc nng, sa...

1.3.5. Phng php kim nghim B. longum trong ch phm probiotics


1.3.5.1. Phng php nh lng vi sinh vt trong ch phm [14], [19], [21].
a. Phng php m trn a thch
Phng php m trn a thch c s dng nhiu nht nh lng B.
longum trong ch phm Probiotics do c chnh xc cao, n gin, d thc hin
quy m phng th nghim, ng thi c th nh lng ring c B. longum trong
hn hp. Vi sinh vt trong ch phm ban u c pha long n nng thch
hp, sau c cy trn vo mi trng dinh dng trong a petri v nui cy
nhit , thi gian thch hp. Sau thi gian nui cy, m s khun lc v tnh s
lng vi sinh vt c trong ch phm ban u
Tuy nhin, nhc im ca phng php ny l tn thi gian nui cy, kh
khn trong vic la chn mi trng c hiu cho vi sinh vt cn nh lng trong
hn hp gm nhiu vi sinh vt khc nhau.
b. nh lng bng phng php real-time PCR
Real-time PCR l mt trong nhng k thut cho php nh lng chnh xc
nht s lng trnh t DNA trong mt mu th nghim. Khc vi vic nh lng
khi qu trnh khuch i hon tt, k thut ny cho php kim sot v nh lng
s lng DNA ngay c khi phn ng ang xy ra. Trong k thut ny, mi chu
k, lng DNA c khuch i s pht ra mt lng tn hiu hunh quang nht
nh. Ni cch khc, lng tn hiu hunh quang s tng t l thun vi mi chu k
khuch i thnh cng ca phn ng real-time PCR. Nh vy bng cch ghi nhn
lng tn hiu hunh quang pht ra sau mi chu k, ta c th kim sot phn ng
PCR ngay trong giai on khuch i tuyn tnh ca chng. V s gia tng u tin
ca tn hiu hunh quang s tng ng vi lng DNA ban u ca mu
Trong phn ng real-time PCR, ngi ta thng s dng hai tc nhn pht
hunh quang bao gm tc nhn gn vo DNA mch i (Ethidium Bromide, SYBR
Green, EvaGreen...) hoc tc nhn dng nh du mu d c hiu (FAM, HEX ...).
Tc nhn lin kt vi mch i DNA. Trong phn ng, khi c s hin din ca
cc sn phm DNA mch i do qu trnh nhn bn th cc tc nhn lin kt vi
DNA mch i nh SYBR Green s lin kt vi cc sn phm va c to ra ny

10

v pht tn hiu hunh quang mnh m hn (so vi trng thi t do trong dung
dch). Loi tc nhn hunh quang ny c th s dng c cho mi trnh t ch
nn chi ph s thp; nhng c hiu ca sn phm phi c kim tra cht ch
thng qua mt bc phn tch b sung sau khi phn ng nhn bn kt thc phn
tch ng cong nng chy (Melting curve analysis).
Tc nhn dng nh du mu d c hiu rt a dng nh FAM, TAMRA,
Texas Red, Cy3, Cy5 c s dng vi nhiu loi mu d khc nhau nh mu d
lai, mu d thy gii, mu d dng kp tc.
Khi s dng vi cc mu d khc nhau, cng tn hiu hunh quang u t
l thun vi hm lng sn phm ch ang c nhn bn trong phn ng. Cc tn
hiu hunh quang pht ra s c cc u d ca my lun nhit real-time PCR thu
nhn v x l. Khi cng tn hiu hunh quang ca mu vt qua ng tn hiu
hunh quang nn (base line) ca phn ng th mu c xem l dng tnh v
ngi ta ly thi im vt qua (biu hin qua gi tr chu k ngng Ct, Cycle
Threshhold) so snh vi mt ng cong chun (standard curve) bit suy
ra nng trnh t DNA ch trong mu th nghim ban u. ng cong chun
c xy dng t chu k ngng ca cc mu chun c cha sn phm ch bit
trc nng .
c. Phng php lai hunh quang ti ch (FISH-fluorescent insitu
hybridization).
FISH d tm v pht hin ra cc trnh t acid nucleic ca vi sinh vt cn nh
lng bng cch dng u d c nh du hunh quang lai c hiu vi cc trnh
t c hiu b sung vi n bn trong t bo nguyn vn (khng cn ph v t bo)
Quy trnh thc hin bao gm cc bc:
- Chun b mu v u
- C nh mu
- Lai gia u d v trnh t ch c hiu nm trong t bo
- Ra mu loi b cc u d khng c lai
- D tm mu (bc ny c th b qua nu s dng u d pht hunh quang
trc tip)

11

- Quan st, hin th v lu tr kt qu bng knh hin vi qut laze ng tiu


hoc knh hin vi gn thm b lc di hp nhiu tn cng vi mt my nh
v phn mm x l hnh nh.
Cc u d thng c s dng l cc oligonucleotide rRNA 16S m
bo s lin kt b sung t hiu qu cao nht trong qu trnh lai.
u d thng c nh du bng cch gn trc tip cht nhum hunh quang ln
u d hoc kt hp u d vi mt s cht ch th nh Digoxygenin, sau chng
s c d tm thng qua mt cht phn hunh quang
u im ca 2 phng php Realtime PCR v FISH l nhanh, cho chnh
xc cao. Tuy nhin nhc im l i hi u t thit b t tin, yu cu trnh
k thut cao v khng phn bit c vi khun sng hay vi khun cht.
1.3.5.2. nh danh
a. nh danh kiu hnh
Da theo kha phn loi c cng nhn quc t: Da vo c im hnh thi,
phn ng sinh ha. tin hnh kim tra cc c im sinh ha th phng php s
dng ph bin hin nay l s dng cc kit sinh ha c sn (V d Api kit ca hng
BioMerieux-Php). nh danh vi sinh vt thuc chi Bifidobacterium c th s
dng kit API 20A hoc Rapid ID 32A.
b. nh danh kiu gen.
nh danh n loi, k thut ph bin c s dng hin nay l k thut
nhn gen v gii trnh t
K thut ny bao gm cc bc:
Tch chit DNA ca vi sinh vt nghin cu: Thnh t bo vi sinh vt c
ph v nh lysozyme v cc cht ty ra mnh nh SDS-tris-hydroclorid
gip gii phng ton b DNA ca vi sinh vt. Protein v RNA c tch
khi DNA nh cc enzym RNAse, protease v isopropanol. Cui cng DNA
c ta bng ethanol tuyt i.
Tin hnh phn ng PCR: Vi vic s dng DNA polymerase v cc
oligonucleotid tng hp nhn to, mt on DNA dng lm khun c
nhn ln nhanh chng vi s bn sao gp hng t ln m khng cn n

12

nhiu t bo vi sinh vt. Nh phn ng ny, mt on gen bt k s c


khuch i khi bit trnh t ca hai im t u 5 n u 3 ca si b
sung v nh c enzym DNA polymerase m DNA khun c tng hp khi
c sn dNTP.
Kim tra sn phm bng in di trn gel agarose. Phn tch trnh t 16S
rDNA theo phng php Sanger. Cui cng, chui trnh t ny c so
snh vi tt c cc chui nucleotid 16S rDNA c cng b trn ngn
hng gen quc t v t a ra kt lun chnh xc tn ca vi sinh vt.
Phng php ny c chnh xc rt cao. Cho n nay, cc chuyn gia phn
loi vi sinh vt trn th gii u cho rng phng php tt nht nh danh vi sinh
vt v thnh lp cy pht sinh chng l da trn trnh t rDNA kt hp vi phng
php phn loi kinh in. Tuy nhin, phng php ny i hi u t trang thit b
rt ln m khng phi phng th nghim no cng c kh nng trang b c.
- K thut nhn gen PCR (Polymerase Chain Reaction) [8].
PCR l mt k thut c s dng ph bin trong cng ngh sinh hc hin i
v ng gp rt ln cho nhng tin b v sinh hc phn t.
PCR da trn c s phn ng ko di primer nh enzym Taq DNA
polymerase khuch i in vitro cc acid nucleic c hiu trong thit b iu nhit
tun hon (thermocycler) hay cn gi l my PCR. PCR cho php khuch i theo
hm m ln n hng triu ln cc on DNA c chiu di t 200- 3000 bp. on
DNA c khuch i (DNA ch) c nhn dng nh cp primer c hiu
(oligonucleotid) thng c chiu di khong 20 nucleotide.
Taq DNA polymerase l mt loi enzym DNA polymerase chu nhit (c vi
khun chu nhit cao Thermus aquaticus), c dng tng hp cc on DNA
mi trong mi trng c bn loi deoxyribonucleotid (dATP, dCTP, dGTP v
dTTP) v hai primer, trn c s khun mu ca mt on DNA nht nh bit
hoc cha bit trnh t. Cc on DNA mi hnh thnh li c s dng lm khun
mu. Sau nhiu chu k, s lng on DNA ni trn c nhn ln gp nhiu ln,
nh vy c th s lng tch ra, phn tch trnh t hoc to dng...

13

Nguyn tc ca phn ng khuch i gen c trnh by tng qut trong Hnh


1.3 v Hnh 1.4

Hnh 1.3. S phn ng chui polymerase

Hnh 1.4.

14

Nguyn tc ca PCR c trnh by trong Hnh 1.3 v Hnh 1.4. Theo , t


chu k th hai, Taq polymerase (Taq pol) bt u to ra cc on DNA c chiu di
xc nh. Cc primer thng l mt oligonucleotid tng hp, c khong 10 - 20
nucleotid hoc hn. Nu bit trnh t ca on gen cn khuch i th c th tng
hp nhn to cc primer tng ng thc hin PCR v tch chng ra bng k
thut in di. Trong k thut PCR ngi ta thng tin hnh khong 25 - 35 chu k.
Mi chu k PCR bao gm ba bc c nhit khc nhau (xem hnh v minh ho):
Bc 1 l bin tnh DNA ( 90- 95oC), c tc dng tch hai si n t si khun
xon kp. Bc 2 l bt cp hai mi vo hai si n ca khun ( 40-65oC). Bc 3
l ko di chui theo mi, ( nhit 70-72oC) chiu 5-3 vi qu trnh trng hp
gn cc dNTP dc theo si khun to thnh phin bn mi theo nguyn tc b
sung. Tnh c hiu ca phn ng c quy nh bi mi v s sao chp trc tip
theo trnh t si khun gia hai mi. Nh vy, kt qu to ra hng triu phin bn
si khun trong vi gi.
- in di DNA trn gel agarose
Nguyn l in di: cc phn t DNA l cc polyanion nn trong mi trng
trung tnh v kim, di tc dng ca in trng, chng s di chuyn v cc
dng. Mc di chuyn ca cc phn t DNA trong in trng ph thuc ch
yu vo kch thc (hay khi lng phn t) ca chng. Phn t c kch thc nh
s di chuyn nhanh cn phn t c kch thc ln s di chuyn chm. S dng
thang chun DNA vi kch thc bit c th d dng xc nh c kch thc
tng i ca DNA mu.
Tin hnh: chun b gel agarose X%, tin hnh tra mu gm 10 l mu DNA
c trn vi 2 l m mu (c cha: glycerol 20%, cht ch th mu bromophenol
xanh, xylene cyanol) v tra vo cc ging trn gel. Chy in di vi hiu in th n
nh l 10 volt/cm gel n khi vch mu bromophenol xanh cch mp gel khong 2
cm th dng li. Nhum bn gel trong dung dch ethidium bromide (EtBr) nng
50 g/ l trong 15-30 pht. Sau , gel c ly ra v soi di nh sng t ngoi
ca my soi chp gel, cc bng gn vi EtBr xut hin di dng cc bng mu
sng trn nn gel mu en.

15

- Nhn dng
Nhn dng l s tch lp v thu nhn nhiu bn sao ng nht ca mt gen
hay ca mt mnh on DNA.
Nguyn tc:
K thut nhn dng bao gm vic phi ci mt mnh (chui) DNA l vo mt
vector (plasmide hoc phage ) bng phng php ha sinh. Sau , a phn t
lai nay vo t bo ch chn la bng phng php bin np hoc ti np. Trng
hp mun to dng tng hp enzym th mnh DNA nh ci phi m ha cho gen
cu trc ca mt enzyme no .
+ Cc bc c bn ca phng php nhn dng:
Tch lp cc DNA l cn nhn dng:
Chn v x l vector nhn dng
To DNA ti t hp (vector ti t hp).
Chuyn DNA ti t hp vo t bo ch bng phng php bin np hoc ti
np.
Pht hin dng cn tm
Kim tra thu nhn sn phm ca gen ti t hp.
- Gii trnh t
Phng php chnh xc v a li nhiu thng tin nht cho nh danh v nh
typ vi sinh vt l xc nh chnh xc trnh t chui DNA ca mt vng quy nh
trn nhim sc th. Phng php gii trnh t DNA pht trin nhanh chng, kt qu
so snh s tng ng ca trnh t gen nghin cu tr thnh phng php phn loi
chun theo sinh hc phn t trong nghin cu h thng hc v cy ph h di truyn
gia cc i tng nghin cu. Cc gen bo th c gii trnh t lm c s xc
nh v tr ca sinh vt nghin cu, trong khi cc trnh t khng tng ng
(khc bit) li lm c s cho s bit ha cng nh xc nh mi quan h gia cc c
th gn vi nhau.
T nhng nm 1977 mt s phng php gii trnh t gen c pht minh
nh phng php gii trnh t gen theo phng php ha hc (Alan

16

Maxam v Walter Gilbert), phng php gii trnh t gen bng enzyme (do
Frederick Sanger v cng s )
C hai phng php ny nh du bc ngot ln trong lch s pht trin
ca b mn sinh hc hin i. Ngy nay, rt nhiu my gii trnh t gen t ng u
da trn nguyn tc chnh l phng php gii trnh t gen ca Sanger.
Phng php ny da vo s tng hp mch b sung cho trnh t cn xc nh
nh DNA polymerase. Vi vic s dng thm cc dideoxynucleotide cng vi cc
deoxynucleotid thng thng, kt qu tng hp cng l s hnh thnh mt tp hp
nhiu on DNA c kch thc khc nhau.
Cc phn on DNA s c phn tch bng in di trn gel polyacrylamid c
kh nng phn tch hai trnh t DNA ch chnh nhau mt nucleotid. Vi vic s
dng mt loi nucleotide c nh du ng v phng x, kt qu trnh t DNA cn
xc nh c c trn bn phng x t ghi t bn in di.
thc hin c gii trnh t bng my t ng th cc mch DNA n sn
sinh trong ng phn ng gii trnh t phi c nh du hunh quang cc vch
in di ca cc mch n ny pht sng khi i qua mt chm tia sng laser. Nguyn
tc hot ng ca my l trong sut qu trnh in di, mi khi c mt vch in di
i qua chm tia laser th vch in di s pht sng ln v s pht sng ny s c
con mt cn quang ghi nhn v lu li thnh mt nh cng sng trong biu .
T biu ca cc nh cng sng ny, my s so dng ca cc nh tng ng
vi cc mu cui cng phn tch thnh trnh t ca on DNA.

1.3.6. Tnh hnh nghin cu nh tnh v nh lng B. longum trong cc


ch phm probiotics trong nhng nm gn y
- Trn th gii
Phng php nh lng B. longum ph bin vn l phng php m trn a
thch. Bn cnh cc nh khoa hc nghin cu thm mt s phng php mi
l phng php FISH, phng php real-time PCR v k thut nhum hunh
quang.

17

nh lng B. longum trong cc ch phm gm nhiu vi sinh vt bng


phng php m trn a thch, vic kh khn nht l la chn c mi trng
ph hp. Mi trng ny phi p ng yu cu l khuyn khch s pht trin ca B.
longum v c ch hon ton s pht trin ca cc vi sinh vt cn li trong hn hp.
t c yu cu ny, ngi ta thng da vo kh nng nhy cm hoc khng
li khng sinh ca vi sinh vt trong hn hp. Do ngi ta s thm vo mi
trng c bn ban u mt hoc nhiu khng sinh khc nhau vi mc ch l cc
khng sinh ny s c ch cc vi sinh vt khc trong hn hp, s nh lng v phn
lp ring c vi sinh vt cn nghin cu l vi sinh vt khng li cc khng sinh
thm vo mi trng. Da trn nhiu cng trnh nghin cu ca nhiu tc gi,
Janet E.L.Corry v cng s tng quan li mt s mi trng nh lng
Bifidobacteria trong chng 10 cu quyn Handbook of culture Media for Food
and Water Microbiology, 3rd edition [19]. Theo vic la chn mi trng
nh lng ty thuc vo s c mt ca nhng vi sinh vt no trong hn hp cn
nh lng, t cho php nh lng ring c tng vi sinh vt trong hn hp.
nh danh B. longum trong hn hp, phng php ph bin thng c
dng l nh danh bng k thut PCR [27], [22].
- Trong nc
Hin nay, th trng thuc Vit Nam c rt nhiu thuc v thc phm chc
nng c cha B. longum nh LACTIBIO (Cng ty TNHH DP UNIVN), LAZEBIO
(Cng ty INTECHPHARM), LACCLEAN GOLD (Cell Biotech CO, Ltd),
YBIOPLUS (Cng ty Dc Hu Giang), Safikid BIO (Hc vin Qun Y), L-Bio3D (Cng ty Lin Doanh Dc phm Mebiphar Austrapharm)
Tuy nhin cha c nghin cu no v vic nh lng v phn lp ring B.
longum trong thuc v thc phm chc nng. Vic nh lng ch dng li nh
lng hn hp vi sinh vt trong ch phm. ng thi vic nh danh B. longum
cng cha c nghin cu thu o. Trong mt s tiu chun c s ca cc nh
sn xut trong nc s dng kt Api 20A nh danh B. longum trong ch
phm [1], [2]. Tuy nhin, vic s dng kit Api 20A mi ch cho php nh danh n
chi Bifidobacterium, ng thi cc tiu chun ny cng cha ch ra c cch phn

18

lp khun lc thun khit tin hnh phn ng sinh ha. iu ny s d gy ra sai


s trong vic nh danh. Cha c cng trnh nghin cu no ng dng k thut nhn
gen v gii trnh t nh danh B. longum.
Do hng nghin cu ca ti ny l nghin cu la chn mi trng c
hiu nh lng ring c B. longum trong hn hp, t phn lp c khun
lc thun khit nh tnh bng kt sinh ha v nh danh bng k thut sinh hc
phn t. Thm nh qui trnh thit lp p dng vo vic kim nghim B.
longum trong mt s ch phm thuc v thc phm chc nng lu hnh trn th
trng Vit Nam.

19

CHNG 2. I TNG, NGUYN VT LIU,


TRANG THIT B PHNG PHP NGHIN CU
2.1. PHNG TIN DNG TRONG NGHIN CU
2.1.1. Mi trng, ho cht, dung mi, thit b
2.1.1.1. Mi trng
- Mi trng BSM c (Fluka) l BCBK6505V.
Hn hp khng sinh thm vo mi trng BSM c, l BCBK 1651V
- Mi trng MRS c (Merck)
Cng thc
Pepton

10,0g

Cao tht

8,0g

Cao men bia

4,0g

Glucose

20,0g

Dikali hydrophosphat

2,0g

Tween 80

1,0g

Amoni citrat

2,0g

Natri acetat

5,0g

Mangan sulfat

0,04g

Magnesi sulfat

0,2g

Thch

14,0g

Nc ct va

1000ml

pH sau khi tit trng: 5,7 0,2


- Mi trng MRS lng (Merck) c cng thc ging mi trng MRS c
ch khc l khng c thch.

20

- Mi trng API 20A (BioMrieux, Php): ng 4ml. S l: 1002717340.


Hn dng: 5/5/2015
Cng thc
Trypticase

5,0g

Cao men bia

5,0g

Natri clorid

2,5g

L-tryptophan

0,2g

L-cystine

0,4g

Hemin

0,005g

Vitamin K1

0,01g

Natri sulfit

0,1g

Nc ct va

1000ml

pH sau khi tit trng: 6,9 7,3.


- Mi trng thch mu
Cng thc
Pepton

10,0g

Natri clorid

5,0g

Cao tht

2,5g

Thch

20,0g

Nc ct va

1000ml
7,4 7,6

pH sau khi tit trng:


Mu th

50,0ml ( mu 5%)

21

2.1.1.2. Ho cht, dung mi


- Ha cht, dung mi c ghi Bng 2.1 v Bng 2.2
Bng 2.1. Mi c hiu
Loi
on 16S rDNA

B. longum

Tn mi

S l

Hng sn xut

ID16R08F

208918A07

IDL16R09R

208918A08

BlonF

208918B05

BlonR

208918B05

Invitrogen

Invitrogen
Promega

VectorpGemT easy nguyn


bn
Bng 2.2. Ha cht, dung mi
Tn

S l

Hng sn xut

Lysozym

061M13281V

Sigma-Aldorich

WizardR Genomic DNA

297885

Promega

Taq DNA polymerase

1130853

Life technologies

dNTPs (100mM)

1139131

Life technologies

Thang DNA chun

668361

Invitrogen

DNA loading Dye (6X)

00056239

Fermentas

m PCR MgCl2 (10X)

1113366

Invitrogen

Nc ct siu tinh khit

764952

Invitrogen

Agarose siu tinh khit

0000144547

Invitrogen

Tris siu tinh khit

90920S196

Biobasic Inc

EDTA, free acid

1004SHLS0125B0210J

Biobasic Inc

Kit API 20A

1002998430

BioMrieux

Thuc th BCP

1003101450

BioMrieux

Purification Kit

Thuc th XYL
Thuc th HER

Merck
1003059340

22

BioMrieux

- c chun McFarland s 2: Ht chnh xc 2,0ml dung dch bari clorid 1,0%


vo bnh nh mc dung tch 100ml v pha long bng dung dch acid sulfuric 1,0%
va n vch, trn u.
- c chun McFarland s 3: Ht chnh xc 3,0ml dung dch bari clorid 1,0%
vo bnh nh mc dung tch 100ml v pha long bng dung dch acid sulfuric 1,0%
va n vch, trn u.
o hp th ca cc c chun bc sng 600nm, cc o dy 1cm, s
dng dung dch acid sulfuric 1,0% lm mu trng th hp th ca c chun
s 2 khong 0,451, hp th ca c chun s 3 khong 0,582. c chun
ch dng trong vng 3 thng k t ngy pha.

2.1.1.3. Thit b, dng c


a. Thit b.
c hiu chun theo ISO/IEC-17025 v GLP.
- Bung thi kh sch Kebo (Thu in).
- T m CO2 T 305-F (Thu in)
- T sy Memmert ULE 600 (c)
- Ni hp Tomy SS-325 (Nht)
- Cn k thut in t Sartorius TE 412, chnh xc 0,01g (c)
- My quang ph t ngoi kh kin Shimadzu (Nht Bn)
- T lnh su Kebo 2951 (Thu in)
- My li tm lnh Mikro-200R (c)
- My in di gel XL-ULTRA-V-2 (M)
- My chp nh gel UVDI- 312 (i Loan)
- My khuych i gen RealTime Step one Plus (Singapore)
- My lc trn Vortex Genie 2 (M)
- n soi UV bc sng 365nm
b. Dng c
Cc dng c thu tinh nh pipet, bnh nh mc, bnh nn... Class A.
Micropipet (Eppendorf - c) 10 l, 100 l 1000 l, 5ml v 10ml. ng nha 1,5ml,
2,0ml; ng thy tinh kch thc 16x120mm.

23

2.1.2. Phn mm x l kt qu: Apiweb ca hng BioMrieux.


2.1.3. Chng vi sinh vt chun
Cc chng vi sinh vt chun c s dng trong ti ny c trnh by
Bng 2.3
Bng 2.3. Cc vi sinh vt chun c s dng
TT

Tn chng

K hiu

TT

Tn chng

K hiu

Bacillus

ATCC 11778

11

Lactobacillus

ATCC

rhamnosus

9595

Bifidobacterium

ATCC

breve

15700

Bifidobacterium

ATCC

bifidum

11863

Streptococcus

ATCC

faecalis

33186

Streptococcus

ATCC

pyogenes

19615

Pseudomonas

ATCC

aeruginosa

9027

Escherichia coli

ATCC

cereus
2

Bacillus

ATCC 14884

12

pumilus
3

Bacillus

ATCC 795

13

stearothermo
philis
4

Bacillus

ATCC 6333

14

subtilis

Lactobacillus

ATCC

acidophilus

4356

Lactobacillus

ATCC 7830

15

16

leichmannii
7

Bacilus

ATCC 7050

17

coagulans
8

Lactobacillus

8739
ATCC 334

18

casei
9

Lactobacillus

ATCC 9338

19

fermentum
10

Lactobacillus

ATCC 8014

20

plantarum

Staphylococcus

ATCC653

aureus

Streptococcus

ATCC

thermophilus

19258

Bifidobacterium

JCM 1217

longum

24

2.2. I TNG NGHIN CU


L mt s ch phm probiotics trn th trng
- Gi bt SamubioTHYMO (k hiu mu A)
Cng ty sn xut: Cng ty c phn Dc, vt t y t Hi Dng
S ng k: 12735/2011/YT-CNTC
S l: 010112. Hn dng: 300115
Cng thc: Mi gi 3g bt cha
+ Lactobacillus acidophilus

108CFU

+ Bifidobacterium longum

108CFU

+ Streptococcus faecalis

108CFU

- Gi bt safikid BIO (k hiu mu B)


Cng ty sn xut: Trung tm nghin cu ng dng sn xut thc phm chc
nng- Hc vin Qun Y.
S ng k: 1757/2010/YT-CNTC
S l: 1113. Hn dng: 11/2015
Cng thc: Mi gi 3g bt cha
+ Lactobacillus acidophilus

108CFU

+ Bifidobacterium longum

108CFU

+ Streptococcus thermophilus

108CFU

- Gi bt L-Bio-3D (k hiu mu C)
Cng ty sn xut: Cng ty lin doanh dc phm MEBIPHARAUSTRAPHARM
S ng k: QLSP -0746-13
S l: 061213. Hn dng: 25/12/2015
Cng thc: Mi gi 1g bt cha hn hp cc vi khun sinh acid lactic
108CFU
+ Lactobacillus acidophilus
+ Bifidobacterium longum
+ Lactobacillus rhamnosus
- Gi bt Lackid (K hiu mu D)

25

Cng ty sn xut: Novarex, Hn Quc


S ng k: 3056/2013/ATTP - XNCB
S l: 4002. Hn dng: 08.04.2017
Cng thc: Mi gi 3g bt cha hn hp cc vi khun sinh acid lactic
108CFU
+ Lactobacillus acidophilus
+ Lactobacillus rhamnosus
+ Lactobacillus plantarum
+ Bifidobacterium longum
+ Streptococcus faecalis
- Gi bt Probiotics Lactomin plus (k hiu mu E)
Cng ty sn xut: Novarex, Hn Quc
S ng k: 1160/2014/XNQC - ATTP
S l: 4002. Hn dng: 04.02.2017
Cng thc: Mi gi 3g bt cha hn hp cc vi khun sinh acid lactic
108CFU
+ Lactobacillus acidophilus
+ Bifidobacterium longum
+ Streptococcus faecalis

2.3. PHNG PHP NGHIN CU


2.3.1. Phng php nh lng B. longum trong ch phm
S dng phng php m trn a thch. Trn c s tham kho cc cng
trnh nghin cu c lin quan, chng ti la chn mi trng BSM agar nh
lng B. longum trong hn hp [19], [25].
Chun b hn dch th: Cn v xc nh khi lng trung bnh ca 20 gi ch
phm. Cn chnh xc khong 10,0g bt thuc vo bnh nn v trng, thm dung mi
pha long vi th tch tnh theo mililit bng 9 ln khi lng bt thuc cn v vi
vin bi thu tinh v trng. Lc mnh trong 30 pht vi sinh vt phn tn u trong
dung dch, thu c hn dch c pha long 10-1. Ly 1000 l hn dch trn cho

26

vo ng nghim cha sn 9,0ml dung mi pha long, lc u bng my lc trn


Vortex c hn dch c pha long 10-2. Tip tc pha long 10 ln nh trn
thu c hn dch c c pha long 10-5, 10-6 v 10-7.
Tin hnh: Chun b ba a petri v trng, chuyn vo mi a chnh xc
1000 l hn dch c pha long 10-5. Tip tc lm nh trn vi hn dch c pha
long 10-6 v 10-7. Cy trn vi mi trng BSM c ( c tit trng v ngui
xung khong 45oC), mi trng trong a ng t nhin. Lt ngc a petri v
1oC trong 48 - 72 gi trong iu kin k kh. Sau thi gian , m s lng

37

khun lc trong cc a petri (ch m cc a petri c s khun lc nm trong


khong 25 - 250).
Tnh kt qu: S lng vi sinh vt trong 1 gi ch phm c tnh theo cng
thc:
A = nTB

MTB

Trong :
A

: s lng vi sinh vt trong 1 gi ch phm (CFU/gi),

nTB

: s lng khun lc trung bnh trong 3 a petri (CFU),

MTB

: khi lng trung bnh ca bt thuc trong gi ch phm (g/gi),

: h s pha long ca hn dch th.

2.3.2. Phng php nh danh vi sinh vt bng kit API 20A


2.3.2.1. Nguyn tc chung
Kit API 20A l mt h thng c tiu chun ha ca hng BioMrieux
(Php). Kit API 20A c dng cng vi mi trng API 20A nh danh vi sinh
vt k kh, trong c chi Bifidobacterium.
Kit API 20A gm 20 ging cha cc cht nn khc nhau. Vi sinh vt cn xc
nh s c cy vo mi trng API 20A v nh mi trng c vi sinh vt vo
tng ging, lc ny cc cht nn trong ging s c ho tan vo trong mi trng.
Trong qu trnh nui cy sn phm chuyn ha nu c s t lm thay i mu mi
trng hoc thay i mu sau khi thm thuc th. Tn ca cc cht nn s c
cp n trong mc 2.3.2.2 13 .

27

2.3.2.2. Tin hnh


Sau khi nui cy vi sinh vt nh m t trong mc nh lng 2.3.1, la chn
mt khun lc c trng, tch bit v cy chuyn sang 5,0ml mi trng MRS lng,
c b sung khong 0,5ml du khong lp b mt v trong iu kin k kh 37
1oC trong 24 gi.
a. Nhum Gram: Tin hnh nhum t bo theo phng php nhum Gram v
soi di knh hin vi vi vt knh du c phng i 1000 ln.
Nu vi sinh vt bt mu tm, hnh que, khng c bo t th kt lun vi sinh
vt l trc khun Gram dng, khng sinh bo t.
b. nh danh vi sinh vt bng kit API 20A:
Sau khi xc nh vi sinh vt l trc khun Gram dng, khng sinh bo t
th tip tc cy mt quai cy canh chng t ng MRS lng trn ln mi trng
thch mu, trong iu kin k kh 37

1oC trong 18-24 gi. Gt vi sinh vt

trn b mt a thch v phn tn u trong ng nghim c sn 1ml nc ct v


trng thu c hn dch gc.
Nh t t tng git hn dch gc trn vo mt ng nghim cha sn 4ml
nc ct v trng n khi c trong ng tng ng vi c chun
McFarland s 3. Ghi li s git hn dch gc cho vo ng nghim trn.
Cho vo 4ml mi trng API 20A s git hn dch gc bng s git cho
vo ng nghim trn v lc u. Nh vy, nng vi sinh vt trong mi trng
API 20A tng ng vi c chun McFarland s 3 (so snh c bng
mt thng).
Nh mi trng API 20 cy vi sinh vt vo cc ging ca b kit API 20A
bt u t ging s 0 n ging 20, ch trnh to bt kh trong ging, ch ch
nh phn tube. Ring i vi ging IND nh dung dch du khong v trng ln
trn b mt ging to iu kin k kh cho vi sinh vt pht trin. i vi ging
GEL nh c phn tube v phn ming ging. 36 2oC trong iu kin k kh
trong vng 24 2h.
Cch c kt qu: Sau 24h nui cy, quan st v c kt qu.

28

- i vi ging IND: Thm 1 git thuc th XYL vo lp du khong pha


trn, trn u v 2-3 pht. Sau thm 1 git thuc th EHR. c kt
qu trong vng 5 pht. Kt qu l dng tnh nu xut hin mu .
- i vi cc ging c cha cc ng khc nhau: Thm vo mi ging mt
git thuc th BCP. Kt qu c ghi l dng tnh nu xut hin mu
vng hoc vng-xanh.
- Phn ng CAT: kit tip xc vi khng kh khong 30 pht, sau thm
2 git thuc th H2O2 3% vo mt ging dng tnh bt k. Kt qu c
ghi l dng tnh nu thy xut hin bt kh.
Cch c ging dng tnh v ging m tnh c trnh by Bng 2.4

29

Bng 2.4. Cch c kt qu kit Api 20A

Hm
Ging

Cht nn

lng(mg/gi

Kt qu
Phn ng

Dng tnh

m tnh

ng)

XYL-trn u /2-3 pht + HER/5 pht


IND

L-tryptophan

0,98

S hnh thnh indol

Vng

URE

Urea

0,648

Urease

Vng-cam

BCP

GLU

D-glucose

1,96

Acid ha

MAN

D-manitol

1,96

Acid ha

LAC

D-lactose

1,96

Acid ha

SAC

D-saccharose

1,86

Acid ha

MAL

D-maltose

1,96

Acid ha

SAL

Salicin

1,64

Acid ha

XYL

D-xylose

1,64

Acid ha

ARA

L-arabinose

1,64

Acid ha

GEL

gelatin

0,6

ESC

Esculin

0,36

St (III) citrat

0,11

S thy phn gelatin

Tm

Vng/ vng-xanh

Khng c s khuch tn cht

C s khuch tn

mu

cht mu en

Vng

Nu-en

Soi di n UV 365nm
S thy phn Esculin
Pht quang

Khng pht quang


BCP

GLY

Glycerol

1,82

Acid ha

CEL

D-cellobiose

1,86

Acid ha

MNE

D-mannose

1,96

Acid ha

MLZ

D-melezitose

1,96

Acid ha

RAF

D-raffinose

2,18

Acid ha

SOR

D-sorbitol

2,18

Acid ha

RHA

L-rhamnose

1,96

Acid ha

TRE

D-trehalose

1,96

Acid ha

Tm

Vng/ Vng-xanh

Sau khi tip xc vi khng kh 30 pht, thm H2O2


vo 1 ging dng tnh
CAT

Catalase

Khng c bt kh

Xut hin bt kh

SPOR

Bo t

Khng c

GRAM

Hng

Tm

COCC

Que

Cu

Hnh thi

30

Ghi kt qu vo phiu theo di. Nhp s liu vo phn mm Apiweb nh


danh chnh xc vi sinh vt trong ch phm.

2.3.3. nh danh n loi bng k thut PCR v gii trnh t


2.3.3.1. S chung nh tnh B. longum trong cc ch phm probiotics bng k
thut PCR v gii trnh t
Sau khi la chn khun lc c trng tch bit, tin hnh tng sinh trong mi
trng MRS lng c b sung 0,5ml du khong v c nh danh n chi
Bifidobacterium bng kit API 20A nh m t trong mc 2.3.2, tip tc cy tng sinh
VSV t phn sinh khi cn li sang mi trng MRS lng, b sung thm 0,5ml du
khong v khun, 3710C trong iu kin k kh trong 24 gi. Sau VSV
trong ng mi trng MRS lng ny s c tch chit DNA v nh danh bng k
thut PCR v gii trnh t theo s sau

VSV trong mi
trng MRS lng

Tch chit DNA

Kim tra sn phm


tch

Tin hnh phn ng


PCR vi mi c hiu

Nhn dng v gii


trnh t
Hnh 2.1. S nh tnh B. longum bng k thut PCR v gii trnh t

31

2.3.3.2. Tch DNA vi khun bng WizardR Genomic DNA Purification Kit
Nguyn tc:
Tch chit DNA ca vi sinh vt nghin cu: Thnh t bo vi sinh vt c ph
v nh lysozyme v dung dch ly gii t bo gip gii phng ton b DNA ca vi
sinh vt. Protein v RNA c tch khi hn hp nh enzym RNAse v dung mi
kt ta protein. Cui cng DNA c ta bng Isopropanol.
DNA ca cc chng nghin cu v ca cc vi sinh vt phn lp trong cc mu
c tch v tinh sch bng kit ca Promega c ci tin. Quy trnh thc hin nh
sau:
- Cc chng chun c tng sinh t cc ng gc bng mi trng MRS
lng, 370C trong 48 gi. i vi cc mu nghin cu, tip tc tng sinh
VSV trong mi trng MRS lng t phn sinh khi ca VSV c phn lp
sau khi nh danh n chi Bifidobacterium (mc 2.3.2 )
- Ly 1,0 1,5ml mi trng tng sinh chng trn vo ng nha 1,5ml.
- Mang ly tm 10000-12000 vng/pht trong 2 pht loi dch ni thu ly cn.
- Thm 480l dung dch EDTA 50mM vo ng cn, lc u.
- Thm tip 120l dung dch lysozyme (nng 20mg/ml) trn u.
- 370C trong 30-60 pht, sau ly tm 10000-12000 vng/pht trong 2
pht. B dch ni thu ly cn.
- Thm 600l dung dch Nuclei Lysis vo ng cn va thu c. Dng pipet
o nh to thnh hn dch.
- hn dch trn 800C trong 5 pht ly gii t bo, sau ngui n
nhit phng
- Thm 3l dung dch RNAse vo dch ly gii t bo, trn u bng cch o
ng t 5-7 pht.
- 370C trong 15-60 pht, ngui n nhit phng.
- Thm 200l dung dch Protein Precipitation (dung dch kt ta protein)
vo hn hp trn, lc mnh trong 20 giy.
- trong lnh 5 pht.
- em ly tm 10000-12000 vng/pht trong 3 pht.

32

- Chuyn dch ni cha DNA vo mt ng nha 1,5ml sch khc cha 600l
isopropanol. Trn u bng cch o ngc ng t 5-7 ln.
- Ly tm 10000-12000 vng/pht trong 2 pht.
- Loi b dch ni, thu ly cn. Thm 600l dung dch ethanol 70% vo ng,
o nh nhng ra cn DNA.
- Ly tm 10000-12000 vng/pht trong 2 pht. Cn thn loi ht ethanol
tha, kh t nhin trong 10-15 pht.
- Thm 100l dung dch DNA Rehydration (dung dch ha tan DNA) vo
ng cha cn DNA, 650C trong 1 gi.
- Bo qun dung dch DNA va tch c -200C.

2.3.3.3. nh lng DNA bng phng php o hp th nh sng t ngoi


bc sng 260 nm (A260)
Nguyn tc: Da vo kh nng hp th nh sng t ngoi bc sng 260 nm
(A260) ca cc phn t DNA. T gi tr mt quang bc sng 260 nm ca cc
mu, vi h s A260 = 1 tng ng vi 50 ng/l, s dng cng thc C = A260 x 50
x n (trong n l s ln pha long) nh lng DNA c trong mu tch chit.
Tin hnh:
Ly 20l dung dch DNA trn, thm 1,98 ml nc ct siu tinh khit v
khun. Tin hnh o quang bc sng 260nm thu c A260. Da vo kt qu o
tnh ton nng dung dch DNA gc ri pha long dung dch DNA gc n nng
50 ng/l.

2.3.3.4. Nghin cu, thit k mi c hiu


Trn c s tham kho cc cng trnh nghin cu c lin quan, tham kho
ngun gen ca cc loi vi sinh vt trn h thng ngn hng gen th gii (Genbank)
v phn mm Primer 3, NCBIblast. T nghin cu thit k cp mi c hiu
cho i tng nghin cu.

33

Bng 2.5. Trnh t cc cp mi dung cho phn ng PCR


Tn mi

Loi

Trnh t

Kch

Ti

thc

liu

gen ch

tham
kho

on

ID16R08

16S

rDNA

IDL16R0

5-GGGGGATCCAGAGTTTGATCCTGGCTCA-3
5-GGGCTCGAGTACCTTGTTACGACTTCACC-3

1500 bp

[20]

831 bp

[27],

9R
B.

BlonF

5-TTC CAG TTG ATC GCA TGG TC-3

longum

BlonR

5-GGG AAG CCG TAT CTC TAC GA-3

[22]
Vector

T7-Fw

5 AATACGACTCACTATAG 3

pGemT
nguyn
bn

[23]

SP6-Rv

5 TATTTAGGTGACACTATAG 3

186 bp

2.3.3.5. Kim tra kh nng tch DNA


16S ribosome RNA l mt thnh phn ca cc tiu n v 30s nh trong
ribosome vi khun. Cc gen m ha cho thnh phn ny c gi l 16S rDNA c
tnh c th cho vi khun. Da trn tnh cht ny tin hnh phn ng nhn bn oan
gen 16s rDNA bng mi c hiu chng minh s c mt ca DNA vi khun.
* Phn ng PCR
Kim tra sn phm DNA tch c bng phn ng nhn bn on gen c
hiu 16S rDNA ca vi khun bng k thut PCR.

34

Bng 2.6. Thnh phn ca phn ng PCR kim tra DNA mi tch
STT

Thnh phn

Th tch (l)

m PCR (10X)

2,5

dNTPs (2mM)

2,5

Dung dch MgCl2 (50mM)

0,75

ID16R08F (10 pmol)

IDL16R09R (10 pmol)

Tag DNA polymerase (5u/l)

0,1

DNA khun (50ng/l)

Nc ct

14,15

Tng th tch phn ng

25l

Chu trnh nhit cho phn ng PCR nhn bn on gen 16S rDNA: 940C trong
5 pht bin tnh hon ton DNA khun, tip theo l 30 chu k ca 3 bc: 940C
trong 45 giy lm bin tnh DNA, 560C trong 45 giy gn mi v 720C trong 1
pht 30 giy ko di chui. Mu sau c tip 720C trong 5 pht v gi
40C cho n khi phn tch.
* in di trn gel agarose
Chun b bn gel c nng 1% (kl/tt) trong m TBE (pH 8,0) (Tris-Boric
acid-EDTA), nhum sn phm theo k thut in di trn gel gii thiu mc
1.3.5.2 b, km theo mt ging nh cha thang kch thc DNA chun. in di bng
my in di gel XL-ULTRA-V-2 100V trong 30 pht. Nhum sn phm bng
dung dch ethidium bromide 0,1% trong 15-30 pht. Ghi li kt qu bng my chp
nh bn gel UVDI- 312.

2.3.3.6. Nhn bn on gen c hiu ca cc i tng nghin cu bng k


thut PCR
Tin hnh phn ng PCR vi cc thnh phn ghi trong Bng 2.7

35

Bng 2.7. Thnh phn phn ng PCR n mi


STT

Thnh phn

Th tch (l)

m PCR (10X)

2,5

dNTPs (2mM)

2,5

Dung dch MgCl2 (50mM)

0,75

BlonF (10pmol)

BlonR (10pmol)

Tag DNA polymerase (5u/l)

0,1

DNA khun (50ng/l)

Nc ct

14,15

Tng th tch phn ng

25l

Chu trnh nhit cho phn ng PCR nhn bn on gen c hiu: : 94oC trong
5 pht bin tnh hon ton DNA khun, tip theo l 30 chu k ca 3 bc: 940C
trong 45 giy lm bin tnh DNA, 56oC trong 45 giy gn mi v 720C trong
30 giy ko di chui. Mu sau c tip 72oC trong 5 pht v gi 4oC
cho n khi phn tch.

2.3.3.7. Nhn dng v gii trnh t


a. Tinh sch DNA/dsRNA t gel agarose bng b kit Fermentas.
B kt GenJETTM Gel Extraction c thit k tinh sch cc phn on
DNA t gel agarose chy trong m TAE hoc TBE vi hiu sut cao trong thi
gian ngn. B kt s dng tinh sch cc phn on DNA/dsRNA si i c kch
thc t 25 bp n 20 kb, hiu sut c th ln n 95% i vi cc phn on t
100 bp n 10 kb. Quy trnh c thc hin nhit phng bao gm nhng bc
nh sau:
- Bng DNA/dsRNA quan tm c ct chnh xc bng li dao sch t gel
agarose v a vo ng eppendorf 1,5 ml.

36

- B sung m bm (binding buffer) vi 1 th tch bng khi lng ming


gel (1l : 1 mg) v trong b n nhit 60C trong khong 10 pht n khi
gel tan hon ton.
- Dung dch gel agarose ha tan c chuyn ln ct tinh sch v ly tm
10000 vng/pht trong 1 pht. Sau phn dch i qua mng c gn b.
- B sung 500 l m ra (wash buffer) vo ct v ly tm 1 pht vi vn tc
10000 vng/pht. Phn dch i qua mng c gn b. Lp li bc ny
mt ln na.
- Ct tinh sch c cha DNA/dsRNA c chuyn sang ng eppendorf 1,5
mi v b sung 100 l elution buffer vo chnh gia lp mng silica ca
ct.
- Sau DNA/dsRNA c thu bng cch ly tm 1 pht vi tc 10000
vng/pht.
Lng DNA/dsRNA tinh sch c gi -20C phc v cho th nghim
tip theo.
b. Nhn dng sn phm PCR bng b kit pGEMT-Easy cloning.
- Phn ng gn sn phm PCR vo vector pGEMT- Easy.
Cc on gen c hiu ca cc i tng nghin cu c nhn bn bng
phn ng PCR nh enzyme Taq polymerase, do sn phm PCR c to ra l
cc on DNA c sn u A. Cc on DNA ny d dng ghp ni vo vector
pGEMT-Easy c u T nh tc dng ca enzyme T4 ligase. Phn ng gn sn phm
PCR vo vector pGEMT-Easy u T c thc hin theo quy trnh km theo ca b
kit, vi cc thnh phn nh trong Bng 2.8
Hn hp phn ng gn sau c trong b n nhit 16C qua m.

37

Bng 2.8. Thnh phn phn ng gn sn phm PCR vo vector.


TT

Thnh phn

Th tch (l)

m phn ng 2X

10 l

Sn phm PCR

3 l

Vector u T pGEMT-T

1 l

T4 DNA ligase (5 u/l)

1 l

H2O ct kh ion v trng

5 l

Tng th tch

20 L

- Bin np DNA vo t bo kh bin E.coli chng DH5.


T bo kh bin E.coli DH5 (bo qun trong t lnh su -80C) c lm tan
trn trong 10 pht. Tip , b sung 10l hn hp phn ng gn sn phm PCR
vo vector pGEMT-easy vo dung dch t bo r ng v trn 20 pht
to iu kin cho vector bm vo thnh t bo. T bo c sc nhit bng cch
chuyn sang b n nhit 42C trong 45 giy, sau c chuyn li trn trong
2 pht. Tip theo, b sung 450l LB lng vo hn hp bin np v nhit
37C trong vng 20 pht trc khi c nui lc vi tc 220 vng/pht trong
thi gian 30 pht. Hn hp t bo bin np c cy tri trn a mi trng LB
c c b sung ampicillin 100 g/ml, ph trn b mt c cht X-gal (20 l, nng
20 mg/ml) v IPTG (Isopropyl -D-1-thiogalactopyranoside, 20

l, nng

100mM), 37C qua m.


c. Tinh sch plasmid t vi khun E. coli bng b kit GenJETTM Plasmid Miniprep.
DNA ti t hp c tinh sch t t bo vi khun E. coli bng b kit
GenJETTM Plasmid Miniprep (Fermentas). Quy trnh c thc hin nh sau:
- Mt khun lc dng tnh c nui lc trong 5ml mi trng LB lng c
b sung khng sinh ampicillin 100 g/ml vi tc 220 vng/pht 37C
qua m.

38

- T bo c thu bng cch ly tm vn tc 6000 vng/pht trong 10 pht,


4oC. T bo c ha tr li trong 250 l m P1 (Resuspension
solution) b sung enzyme RNase A.
- B sung 250 l m P2 (lysis solution) vo dch t bo v o nh 3-5 ln,
nhit phng 5 pht ph v t bo gii phng plasmid.
- Hn hp c trung ha bi 350 l m P3 (neutralization solution), o
u 4-6 ln. Mnh v t bo v protein bin tnh c loi b bng cch ly
tm hn hp vi vn tc 13000 vng/pht trong 10 pht 4C.
- Dch ni sau khi ly tm c a ln ct tinh sch plasmid v ly tm 10000
vng/pht trong 1 pht. Plasmid c gi li trn lp mng silica cn dch
qua ct c gn b.
- B sung 500 l m ra (Wash buffer), ly tm 30- 60 giy, sau gn b
dch qua ct. Bc ny c lp li mt ln na.
- Ct tinh sch cha plasmid c chuyn sang ng eppendorf 1,5 ml mi.
B sung 50 l m y (Elution buffer) vo chnh gia mng silica ca ct
v nhit phng 2 pht.
- Plasmid c thu bng cch ly tm vi vn tc 10000 vng/pht trong 1
pht.
Mu plasmid tinh sch c bo qun -20C.

2.3.4. Thm nh quy trnh xy dng


La chn mu A lm i tng nghin cu tin hnh thm nh phng
php.

2.3.4.1. Thm nh phng php nh lng B. longum


a. Kim tra tnh chn lc ca mi trng nh lng BSM agar
Tng sinh cc chng chun c ghi trong Bng 2.9 trong mi trng MRS
lng, c thm lp du khong b mt, 35-37oC trong 48 gi trong iu kin k
kh. Sau mang hn dch li tm vi tc 3000-4000 vng/pht, loi b dch ni,
thu ly sinh khi. Thm t t vo ng nghim mt lng dung dch nc mui sinh

39

l 0,9 % v khun, trn u cho n khi thu c hn dch c c tng ng


vi c Mc Farland s 2 (khong 6,0 x108 CFU/ml).
Chun b mt dy ng nghim thy tinh v trng, mi ng cha 9 ml nc
mui sinh l 0,9 % v khun. Ly 1ml hn dch chng trn thm vo mt ng
nghim trn, lc u thu c hn ch c pha long 10-1. Tip tc pha long n
pha long 10-7.
- Ly cc ng nghim c pha long 10-5, 10-6, 10-7, vi mi pha long
tin hnh cy vi khun trn 4 a. Cy vo mi a petri v trng 1ml hn dch
chng pha long 10-5, 10-6, 10-7, thm vo 2 a cng mt pha long 1520ml mi trng BSM agar v trng lm ngui n 45-500C. Thm vo 2 a
cng mt pha long cn li 15-20ml mi trng MRS agar. 35-37oC trong
iu kin k kh. Sau 48 gi em ra m s lng khun lc trn a (ch m a
c 25-250 khun lc) v so snh s lng khun lc thu c trn 2 mi trng.
Bng 2.9. Cc chng chun c dng kim tra tnh chn lc ca mi trng
BSM agar
Tn chng

K hiu

Lactobacillus rhamnosus

ATCC9595

Lactobacillus acidophilus

ATCC 4356

Lactobacillus plantarum

ATCC 8014

Lactobacillus casei

ATCC 334

Lactobacillus fermentum

ATCC 9338

Streptococcus faecalis

ATCC 33186

Bifidobacterium longum

JCM 1217

Streptococcus thermophilus

ATCC 19258

STT

40

b. nh gi lp li ca phng php [7], [17], [18].


Tin hnh php th nh lng nh m t mc 2.3.1. Tin hnh nh
lng lp li 10 ln.
nh gi kt qu
Tt c cc kt qu nh lng c chuyn sang dng log10 ( thu c mu
c phn b tng i chun) trc khi phn tch thng k.
Yu cu: Sr 0,25
Tnh lch chun Sr theo cng thc:
2

xi
S ri

i 1

n 1

Trong :
- xi: Kt qu ca mi ln lp li
- x : Kt qu trung bnh ca cc ln lp li
- n: S ln lp li (n=10)

2.3.4.2. Thm nh qui trnh nh danh bng kit API 20A


Tin hnh tng sinh chng chun B. longum trong mi trng MRS lng,
iu kin k kh 36-38oC trong 48 gi. Sau thi gian nui cy, cy mt quai cy t
hn dch ny ln mi trng thch mu, 36-38oC trong iu kin k kh trong 24
gi.
Sau tin hnh nh tnh chng vi sinh vt chun B. longum bng kit API 20A
theo phng php ghi trong mc 2.3.2. Ghi li kt qu phn ng. Nhp kt qu vo
phn mm ApiwebTM
- Yu cu:
+ Kt qu nh tnh chng chun l thuc chi Bifidobacterium vi %ID > 80%

2.3.4.3. Thm nh qui trnh nh danh bng k thut PCR v gii trnh t
a. nh gi c hiu

41

Nguyn tc: Trong cng mt phn ng PCR tin hnh nhn bn on gen c
hiu ca i tng nghin cu vi s c mt DNA ca nhiu loi khc.
Bng 2.10. Cng thc tnh c hiu
Chng chun

Php th
K thut PCR

Cng

Dng tnh

m tnh

Dng tnh

a+b

m tnh

c+d

a+c

b+d

a+b+c+d

Cng

* c hiu = s trng hp m tnh tht/(s trng hp m tnh tht +s trng


hp dng tnh gi)=d/(b+d)
Quy trnh c tin hnh nh sau:
- Tch DNA t chng chun B. longum JCM 1217 v cc chng kim tra
c hiu nh quy trnh ghi mc 2.3.3.2.
- Kim tra kt qu tch DNA nh trong mc 2.3.3.5.
- Tin hnh th nghim kim tra c hiu bng phn ng PCR vi thnh
phn cc ng th nghim c ghi Bng 2.7 ngoi tr khun ln lt l
DNA ca 20 chng vi sinh vt chun c ghi Bng 2.3. Cc iu kin
ca phn ng c ghi trong mc 2.3.3.5.
- in di DNA trn gel agarose
Tin hnh: Nh m t trong mc 2.3.3.5
b. nh gi nhy ca phng php
Nguyn tc: Tin hnh nh gi ngng pht hin ca ton b quy trnh phn
tch bao gm c phn x l mu (tch DNA) v phn tch mu (phn ng PCR)
bng cch pha mt lot hn dch chng c nng gim dn. Tin hnh quy trnh
phn tch v xc nh nng hn dch nh nht cn ln kt qu phn ng PCR.
Quy trnh c tin hnh nh sau:
- Tng sinh chng chun B. longum JCM 1217 trong mi trng MRS lng
sau 48 gi trong iu kin k kh, em ly tm 3000 - 4000 vng/pht trong

42

15 pht. B dch ni thu ly cn. Thm vo ng nghim 2ml dung dch


nc mui sinh l 0,9 % v khun trn u.
- Chun b mt dy ng nghim thy tinh v trng, mi ng cha 9 ml nc
mui sinh l 0,9 % v khun. Ly 1ml hn dch chng trn thm vo mt
ng nghim trn, lc u thu c hn ch c pha long 10-1. Tip tc
pha long nh vy n pha long 10-8.
- Ly cc ng nghim c pha long 10-6, 10-7, 10-8, vi mi pha long
m s lng vi khun trn 2 a. Cy vo mi a petri v trng 1ml hn
dch chng pha long 10-6, 10-7, 10-8, thm vo tng a 15-20ml mi
trng BSM agar v trng lm ngui n 45-500C. 36-38oC trong
iu kin k kh. Sau 48 gi em ra m s lng khun lc trn a (ch
m a c 25-250 khun lc).
- Cc ng hn dch chng trn c bo qun 2-80C.
- Xc nh s lng vi khun trong cc ng pha long
X= ((N1 + N2)/2) x k
Trong :
N1, N2: s khun lc trn tng a.
k: h s pha long tng ng.
- Sau khi xc nh c nng ca cc chng trong cc ng hn dch, tin
hnh tch DNA theo quy trnh ghi trong mc 2.3.3.2, thc hin phn ng
PCR vi thnh phn v chu trnh nhit nh trong mc 2.3.3.6. in di sn
phm trn gel agarose, xc nh ng pha long cui cng cn ln phn ng
PCR chnh l nhy ca phng php.
+ nh gi cht lng ca sn phm PCR

43

Bng 2.11. Tiu chun nh gi cht lng ca sn phm PCR


Tnh cht bng in di trn bn gel chy in di

nh gi kt qu

Khng c bng

(-)

Bng DNA m hoc chm ht thng hng

(+)

Bng DNA nh hoc va, quan st r

(++)

Bng DNA ln, m, quan st rt r

(+++)

2.3.5. ng dng quy trnh chun nh lng v nh tnh B. longum


trong cc ch phm probiotics ang lu hnh trn th trng
Tin hnh nh lng, nh tnh B. longum trong cc ch phm probiotics
c lit k trong phn i tng nghin cu theo quy trnh xy dng (Ph lc
12). Khi tin hnh phn ng PCR th nghim c b tr nh sau:
- Mt ng phn ng PCR vi DNA khun tch t mu.
- Mt ng phn ng PCR vi DNA khun ca loi vi khun chun tng ng
(chng dng tnh)
- Mt ng phn ng PCR vi nc ct siu tinh khit thay th cho phn dung
dch ADN khun (chng m tnh)

44

CHNG 3. KT QU NGHIN CU
3.1. XC NH S LNG VI SINH VT TRONG CH PHM
PROBIOTICS
nh lng vi sinh vt trong cc mu A, B, C, D, E bng phng php ghi
mc 2.3.1. Kt qu c ghi trong Bng 3.1.
Bng 3.1. S lng vi sinh vt trong ch phm
Kt qu
Stt

MU

(CFU/gi)
1, 17

108CFU/gi

(117,0% so vi lng ghi trn nhn)


0,86

108 CFU/gi

(86,0% so vi lng ghi trn nhn)

7,32 X 106 CFU/gi

2,83 x 108CFU/gi

3,22 x 108CFU/gi

T kt qu bng trn cho thy, mu A hm lng B. longum so vi lng


ghi trn nhn l 117,0% do t ch tiu v nh lng, mu B s lng B.
longum ch c 86,0% so vi lng ghi trn nhn. Cc mu C, D, E do khng c
yu cu v s lng B. longum m nh sn xut ch cng b tng s lng cc vi
sinh vt trong hn hp l 108CFU, do khng kt lun. Trong trng hp ny vic
s dng mi trng BSM agar nhm mc ch phn lp khun lc thun khit
nh danh B. longum.

45

3.2. NH DANH VI SINH VT BNG KIT API 20A


Tin hnh nh danh vi sinh vt bng kit API 20A trong cc nu trong phn
i tng nghin cu bng phng php ghi mc 2.3.2.
Kt qu nh danh bng kit API 20A c ghi li trong Hnh 3.1, Hnh 3.2,
Hnh 3.3, Hnh 3.4, Hnh 3.5, Hnh 3.6, Ph lc 1, Ph lc 2, Ph lc 3, Ph lc
4, Ph lc 5, Bng 3.2 v Bng 3.3

Hnh 3.1. nh danh vi sinh vt trong mu A bng kit API 20A

Hnh 3.2. nh danh vi sinh vt trong mu B bng kit API 20A

46

Hnh 3.3. nh danh vi sinh vt trong mu C bng kit API 20A

Hnh 3.4. nh danh vi sinh vt trong mu D bng kit API 20A

Hnh 3.5. nh danh vi sinh vt trong mu E bng kit API 20A

47

Hnh 3.6. Quan st ging ESC di n UV bc sng 365nm.


Bng 3.2. Theo di phn ng nh danh vi sinh vt bng kit API 20A

TT

Mu A

Mu B

Mu C

Mu D

Mu E

Ging

Cht nn

IND

L-tryptophane

URE

Urea

GLU

D-glucose

MAN

D-mannitol

LAC

D-lactose

SAC

D-saccharose

MAL

D-maltose

48

SAL

Salicin

XYL

D-xylose

10

ARA

L-arabinose

11

GEL

Gelatin

12

ESC

Esculin ferric

citrate
13

GLY

Glycerol

14

CEL

D-cellobiose

15

MNE

D-mannose

16

MLZ

D-melezitose

17

RAF

D-raffinose

18

SOR

D-sorbitol

19

RHA

L-rhamnose

20

TRE

D-trehalose

CAT (catalase)

Bo t

Gram

Hnh cu

49

Bng 3.3. Kt qu nh danh vi sinh vt bng phn mm Apiweb


Stt

Tn mu

Kt qu nh danh

tin cy

Phn ng nghi ng

Bifidobacterium spp1

94,3%

RAF 91%

Actinomyces israelii

4,5%

XYL

A
99%,

RAF

82%, TRE 90%


Bifidobacterium spp1
2

Actinomyces israelii

97,1%
XYL
2,2%

Bifidobacterium spp1

97,1%

Actinomyces israelii

2,2%

99%,

RAF

99%,

RAF

82%,

C
XYL
82%,

Bifidobacterium spp1

94,3%

RAF 91%

Actinomyces israelii

4,5%

XYL

D
99%,

RAF

82%, TRE 90%


5

Bifidobacterium spp1

97,1%

Actinomyces israelii

2,2%

XYL

99%,

RAF

82%,

T kt qu trn cho thy cc vi sinh vt phn lp t cc mu A, B, C, D, E u


thuc chi Bifidobacterium vi % ID cao (>90%).

50

3.3. KT QU NH DANH VI SINH VT TRONG CC MU BNG


K THUT PCR V GII TRNH T
3.3.1. Kt qu tch DNA
3.3.1.1 Tch DNA bng WizardR Genomic DNA Purification kit ca

hng

Promega
Tin hnh tng sinh chng B. longum chun v cc vi sinh vt trong cc mu
probiotics trong phn i tng nghin cu phn lp c mc 3.1, tin hnh tch
DNA theo hng dn ti mc 2.3.3.2. Tin hnh o quang cc dung dch DNA mi
tch theo m t trong mc 2.3.3.3 v pha long cc dung dch DNA bng nc ct
siu tinh khit v khun thu c dung dch DNA c nng 50 ng/l.
Bng 3.4. Kt qu o quang cc dung dch DNA mi tch
i tng

Atn

Nng dung dch


DNA (ng/l)

B. longum

0,1125

562,5

Mu A

0,0911

455,5

Mu B

0,0956

478

Mu C

0,1174

587

Mu D

0,0851

425,5

Mu E

0,1052

526

3.3.1.2. Kim tra kh nng tch DNA


Sau khi tch DNA, cc dung dch DNA c pha long ti nng thch hp.
Kim tra sn phm DNA tch bng bng phn ng PCR nhn bn on gen 16S
rDNA ca vi khun, vi thnh phn v chu trnh nh trong mc 2.3.3.5. Sn phm
PCR c in di kim tra trn bn gel agarose 1%. Kt qu c trnh by Hnh
3.7

51

1500bp
bp

Hnh 3.7. Kt qu in di sn phm PCR nhn bn on gen 16S rDNA bng


cp mi ID16R08F & IDL16R09R
M: Marker 100 bp (Invitrogen)
Ging 1: Khun l DNA tch chit t hn dch B. longum JCM 1217
Ging 2: Chng m ( Khun l nc ct)
Ging 3-7: Khun l DNA tch chit t hn dch tng sinh t khun lc phn
lp t cc mu A, B, C, D v E.

T Hnh 3.7 cho thy, phn ng PCR nhn bn on gen 16S rDNA c hiu
u cho cc bng nhn bn kch thc 1500 bp, trong khi mu chng m (khng
c DNA) khng xut hin bng nhn bn no. Kt qu ny cho thy thu c cc
khun DNA ca cc i tng nghin cu. DNA sau khi tch chit c bo qun
-200C dng cho cc th nghim tip theo.

3.3.2. Nhn bn cc on gen c hiu ca cc i tng vi sinh vt thuc


i tng nghin cu
S dng cp mi BlonF /BlonR nhn bn cc on gen ca cc i tng
nghin cu tch c trn (mc 3.3.1.1).
Thnh phn ca phn ng PCR c ghi li ti Bng 3.5

52

Bng 3.5. Thnh phn ca phn ng PCR


STT

Thnh phn

Th tch ( l)

m PCR(10X)

2,5

dNTPs (2mM)

2,5

Dung dch MgCl2 (50mM)

0,75

Mi xui (10 pmol)

Mi ngc (10 pmol)

Taq DNA polymerase

0,1

(5u/ l)

DNA khun (50ng/l)

Nc ct

14,15

Tng th tch phn ng

25

Vi thnh phn nh trn tin hnh phn ng PCR vi chu trnh nhit:
94oC 45 giy
94oC 5 pht

56oC 45 giy

x 30 chu k

72oC - 5 pht

4oC

72oC 30 giy
Kt qu phn ng PCR c in di trn gel agarose 1% v c ghi li kt
qu bng my chp nh bn gel UVDI-312 (Hnh 3.8)

53

M 1 2

6 7

800 bp

Hnh 3.8. Kt qu in di sn phm PCR nhn bn on gen c hiu ca


B. longum bng cp mi BlonF/BlonR
M: Marker 100 bp ( Invitrogen)
Ging 1: Khun l DNA tch chit t hn dch B. longum JCM 1217
Ging 2: Chng m ( Khun l nc ct khng cha DNA)
Ging 3-7: Khun l DNA tch chit t hn dch tng sinh t khun lc phn
lp c t cc mu A, B, C, D v E.
Kt qu thu c Hnh 3.8 cho thy vi chu trnh nhit v thnh phn phn
ng nh trn, vi phn ng PCR s dng cp mi BlonF/BlonR, chng ti thu
c bng nhn bn c kch thc khong hn 800 bp ging vi khun l DNA
tch chit t chng chun B. longum JCM1217 (ging 1). Kch thc bng ny hon
ton ph hp vi kch thc tnh ton l thuyt ca B. longum l 831 bp. Trong khi
, chng ti khng thu c bng nhn bn no mu khng cha DNA (chng
m-ging 2).
Nh vy, kt qu th nghim thu c cho php khng nh vi cp mi
BlonF/BlonR chng ti nhn bn thnh cng on gen c hiu tng ng ca B.
longum.
Vi cc ging t 3-7 c khun l DNA tch t cc vi sinh vt phn lp c
t cc mu nu trong phn i tng nghin cu, cng u cho bng nhn bn c
kch thc khong 831 bp, tng ng vi vi bng nhn bn vi khun l DNA ca
B. longum JCM 1217. iu ny bc u khng nh cc vi sinh vt phn lp t
cc mu ny l B. longum.

54

3.3.3. Nhn dng v gii trnh t


3.3.3.1.Tinh sch sn phm PCR nhn bn cc on gen c hiu ca cc vi
sinh vt phn lp t cc mu nghin cu bng b kit Fermentas
S dng 5 mu DNA l sn phm nhn bn on gen c hiu ca cc i
tng nghin cu bng k thut PCR lm nguyn liu cho th nghim nhn dng
gen.
Sn phm PCR nhn bn cc on gen c hiu c in di trn gel agarose
1%, kt qu chng ti thu c bng DNA c kch thc 831 bp. Cc bng DNA
ny c ct chnh xc khi bn gel agarose v tinh sch bng b kit GenJET TM Gel
Extraction (Fermentas) theo quy trnh ca nh sn xut nhm loi b ton b cc
thnh phn d tha ca phn ng PCR. Sn phm DNA tinh sch tip tc c in
di kim tra trn gel agarose 1%, kt qu chng ti thu c trn Hnh 3.9 cho thy
chng ti thu c sn phm DNA hon ton tinh sch, c kch thc 831 bp.

831 bp

Hnh 3.9. Kt qu in di mu tinh sch sn phm PCR nhn bn cc on gen c


hiu phn lp t cc mu nghin cu trn gel agarose 1%.
M: Marker 100 bp ( Promega )
Ging 1-5: Sn phm PCR tinh sch ca cc mu nghin cu
Cc sn phm PCR tinh sch nhn bn 5 on gen c hiu c bo qun 20oC s dng cho th nghim nhn dng tip theo.

55

3.3.3.2. Nhn dng cc on gen c hiu vo vector nhn dng v bin np vo


t bo E. coli chng DH5
Trong nghin cu ny, chng ti dng ha 5 on gen c hiu trn vo
vector nhn dng pGEMT-easy. Vector nhn dng ny c thit k c cha im
khi u sao chp nn c kh nng sao chp c lp vi h gen ca t bo ch E.
coli, ng thi cha gen ch th khng khng sinh Ampicillin, cho php chn lc cc
th bin np mang vector ny trn mi trng nui cy c Ampicillin. Ngoi ra,
vector nhn dng cn cha gen chn lc gip phn bit cc th bin np mang
plasmid ti t hp v plasmid t ng vng.

Hnh 3.10. S vector nhn dng pGEMT- Easy


Phn ng gn sn phm PCR vo vector nhn dng c thc hin theo quy
trnh km theo ca nh sn xut. Hn hp phn ng gn u bng sau c
chng ti bin np vo t bo kh bin E. coli chng DH5 bng phng php sc
nhit. Kt qu bin np sn phm PCR vo t bo E. coli trn Hnh 3.11 cho thy
chng ti thu c nhiu khun lc trng (c kh nng mang vector ti t hp)
trn mi trng chn lc c b sung ampiciline 100 g/ml, ph trn b mt c cht
X-gal (20 l, nng 20 mg/ml) v IPTG (20 l, nng 100mM )

56

Hnh 3.11. Kt qu bin np sn phm gn vo vi khun E. coli DH 5 v


cy tri trn mi trng nui cy c b sung ampicillin 100 g/ml, ph trn b mt
c cht X-gal (20 l, nng 20 mg/ml) v IPTG (20 l, nng 100mM )

3.3.3.3. Kim tra s c mt ca cc on gen c hiu trong plasmid ti t hp


tip tc khng nh s c mt ca cc on gen c hiu trong vector nhn
dng, i vi mi mu, chng ti chn 1 khun lc dng tnh (c mu trng)
tinh sch plasmid v kim tra plasmid ny bng PCR v phn ng ct enzyme gii
hn.
a/ Kim tra s c mt cc phn on
Khun lc dng tnh c nui cy v tch chit plasmid theo quy trnh tch
chit plasmid nh m t mc 2.3.3.7c. Sn phm DNA tinh sch c chng ti
s dng lm khun cho phn ng PCR vi hai cp mi khc nhau: cp mi c hiu
Blon F/Blon R v cp mi c hiu ca vector. Trong , cp mi vector l hai trnh
t nm trn vector nhn dng, c khong cch khong 186 bp.

57

M 1 2 3 4 5 6 7

M 8 9 10 11 12 13 14

1017bp

831 bp

Hnh 3.12. Kt qu in di sn phm PCR kim tra plasmid ti t hp mang cc


on gen c kch thc 831 bp
M: Marker 100 bp (Invitrogen)
Ging 1-7 : sn phm PCR vi cp mi c hiu BlonF/BlonR.
Ging 8-14: sn phm PCR vi cp mi vector
Ging 1: sn phm PCR vi khun l DNA ca B. longum JCM 1217
Ging 2,9: i chng m khng s dng DNA khun.
Ging 3-7; 10-14: sn phm PCR vi khun l DNA ca cc plasmid ti t hp
mang cc on gen c kch thc 831bp.
Ging 8: sn phm PCR vi khun l vector nhn dng nguyn bn tch t khun
lc xanh
Kt qu in di sn phm PCR trn gel agarose 1% Hnh 3.12 cho thy sn
phm PCR vi cp mi c hiu c kch thc khong 831 bp ( ging 1, 3-7), tng
ng vi kch thc tnh ton l thuyt ca on gen c hiu ca cc loi B. longum.
Sn phm PCR vi cp mi vector v khun l plasmid tch t khun lc xanh cho
mt bng in di vi kch thc khong 186 bp, tng ng vi kch thc theo tnh
ton l thuyt (ging 8). Sn phm PCR vi cp mi vector khi in di trn gel
agarose 1% cho mt bng c kch thc khong 1017bp ( ging 10-14), kch thc
ny ph hp vi kch thc on DNA theo tnh ton l thuyt cng vi 186 bp ca
plasmid. i vi cc phn ng i chng m khng s dng DNA khun, chng ti

58

khng thu c bng DNA no (ging 2, 9). Kt qu ny cho php chng ti khng
nh plasmid tinh sch c l plasmid ti t hp mang cc on gen phn lp c
t genome ca loi B. longum

vi enzyme ct gii hn
khng nh chc chn on DNA c chn vo vector nhn dng l
on gen c kch thc 831bp, chng ti tin hnh th nghim ct gii hn plasmid
ti t hp c tinh sch t khun lc dng tnh. Theo l thuyt, trn vector
pGEMT Easy (Hnh 3.10) c hai v tr ct gii hn ca EcoRI nm trong vng a
im ct. Nh vy, khi c x l bng EcoRI vector ti t hp s b ct thnh 2
on DNA, mt on chnh l cc on gen c chn vo v mt on l b khung
vector c kch thc 3 kb.

M1 M2 1 2 M2 3 4 M2 5 6 M2 M2 7 8 M2 9 10 M2 11 12 M1

3000
KL xanh
831

831

831

831

831

3000

Hnh 3.13. Kt qu in di sn phm ct gii hn vector ti t hp mang cc


phn on 831 bp bng enzyme EcoRI.
M1:Marker 1000 bp (Fermentas)

M2: Marker 100 bp (Promega)


Ging 1, 3, 5, 7, 9, 11, 12: vector ti t hp nguyn bn;
Ging 2, 4, 6,8,10: sn phm ct gii hn.

59

Kt qu thu c trn Hnh 3.13 cho thy cc sn phm ct vector ti t hp


mang cc on gen ch ca cc i tng nghin cu xut hin 2 bng DNA
(ging 2, 4, 6, 8, 10): trong bng DNA 3 kb l b khung nguyn bn ca vector,
v cc on DNA kch thc 831bp l kch thc tng ng ca on gen c hiu
nhn bn t h gen B. longum theo tnh ton l thuyt. Kt qu thu c trn cho
php chng ti khng nh chc chn hn vic nhn dng thnh cng cc on gen
phn lp c t genome ca cc loi phn lp t i tng nghin cu ca ti.
Cc vector nhn dng mang cc on gen c hiu ca cc i tng nghin
cu c tinh sch v bo qun -20oC. Cc dng t bo E. coli mang cc vector
ti hp cha cc on gen ny c gi trong glycerol 25% v bo qun -80oC.

3.3.3.4. Gii trnh t mu cha phn on 831 bp


khng nh DNA tch chit c t cc mu trong phn i tng nghin
cu c ng l ca loi B. longum hay khng, chng ti tin hnh nhn bn
on gen 831 bp bng quy trnh thit lp, nhn dng on gen ny vo vector
nhn dng. Sn phm vector nhn dng mang on gen c hiu 831 bp c gi i
gii trnh t.
Kt qu gii trnh t ca mu C nh sau:
GAAGGAATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCA
GGCGGCCGCGAATTCACTAGTGATTTTCCAGTTGATCGCATGGTCT
TCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGC
TTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGG
CCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGA
CTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAA
GCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGT
AAACCTCTTTTATCGGGGAGCAAGCGAGAGTGAGTTTACCCGTTGA
ATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG
GGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGC
GGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCC
GCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAAT
TCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAA

60

TGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAA
AGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCG
TATACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGT
CGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAA
GGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAGCGGGCGGA
GCATGCGGAATTATTCGATGCAACGCGAAGAACCCTTACCCTGGGG
CTTGACATGTTTCCCGGACGGTTGGTTAGAAGAATAACGGGC
Kt qu gii trnh t trn c chng ti tin hnh so snh trnh t vi cc
trnh t khc trn ngn hng gen th gii bng ng dng BLAST (Basic Local
Alignmen CSearch Tool) trn trang web ca NCBI (National Center for
Biotechnology Information-http://blast.ncbi.nlm.nih.gov/Blast.cgi) [37].
Kt qu blast khng nh on gen nhn bn c chnh l on gen c
phn lp t h gen ca Bifidobacterium longum.

61

Kt qu gii trnh t u cho thy c 5 mu nghin cu u cha B. longum.


Kt qu gii trnh t ca cc mu A, B, D v E c trnh by Ph lc 7, Ph lc
8, Ph lc 9 v Ph lc 10.

3.4. NH GI PHNG PHP


3.4.1. Kt qu kim tra tnh chn lc ca mi trng nh lng BSM agar
Tin hnh kim tra tnh chn lc c m t trong mc 2.3.4.1a. Kt qu th
nghim c trnh by Bng 3.6.
Bng 3.6. Kt qu kim tra tnh chn lc ca mi trng BSM agar

Mi trng BSM agar (s


STT

Tn chng

lng khun lc trn a)

Mi trng MRS agar


(s lng khun lc trn
a)

10-5

10-6

10-7

10-5

10-6

10-7

Lactobacillus

Khng

Khng

Khng

Nhiu

45 +37

4 +3

rhamnosus ATCC 9595

mc

mc

mc

Lactobacillus ATCC

Khng

Khng

Khng

Nhiu

40+ 50

2+4

4356

mc

mc

mc

Khng

Khng

Nhiu

35+29

2+2

Nhiu

48 +39

3+5

Nhiu

37+ 46

3+3

Nhiu

50 +57

5+4

Nhiu

48+ 38

4+4

Lactobacillus plantarum Khng


ATCC 8014

mc

mc

mc

Lactobacillus casei

Khng

Khng

Khng

ATCC 334

mc

mc

mc

Lactobacillus

Khng

Khng

Khng

fermentum ATCC 9338

mc

mc

mc

Streptococcus faecalis

Khng

Khng

Khng

ATCC 33186

mc

mc

mc

45 +34

3 +4

Bifidobacterium longum Nhiu


JCM 1217

62

T kt qu Bng 3.6 cho thy cc chng thuc chi Lactobacillus v


Streptococcus faecalis khng pht trin c trn mi trng BSM agar, trong khi
vn mc tt trn mi trng MRS agar. B. longum mc tt trn c hai mi
trng MRS agar v BSM agar. Nh vy chng t mi trng BSM agar c tnh
chn lc cao, thch hp nh lng v phn lp ring B. longum trong hn hp vi
sinh vt c nu trong phn i tng nghin cu. Trn mi trng MRS agar
khun lc ca B. longum c mu trng, cn trn mi trng BSM agar khun lc
ca B. longum c mu tm rt thun tin cho vic nhn bit v m khun lc

Hnh 3.14. Hnh thi khun lc B. longum trn mi trng BSM agar

3.4.2. lp li ca php nh lng


Tin hnh php th nh lng nh m t mc 2.3.1. Tin hnh nh
lng lp li 10 ln. Kt qu c trnh by Bng 3.7

63

Bng 3. 7. Kt qu m s lng vi khun trong mu A


Ln

Nng 10-6
a 1 a 2 a 3 TB CFU/gi

Ln 1

30

36

27

31

96x106

Ln 2

29

40

42

37

114x106

Ln 3

34

38

47

40

124x106

Ln 4

30

35

42

36

111x106

Ln 5

41

31

31

34

105x106

Ln 6

48

33

36

39

120x106

Ln 7

40

30

36

35

108x106

Ln 8

29

39

31

33

102x106

Ln 9

31

35

43

36

111x106

Ln 10

37

30

28

32

99x106

p dng cng thc tnh trnh by mc 2.3.4.1 ta c Sr = 0,04.


Vy Sr < 0,25.
Nh vy phng php nh lng B. longum trong ti ny c lp li
chp nhn c i vi phng php m vi sinh vt.

3.4.3. Thm nh c hiu ca quy trnh nh danh vi sinh vt bng kit


API 20A
Tin hnh nh tnh chng vi sinh vt chun B. longum bng API kit theo
phng php ghi trong mc 2.3.2. Kt qu nh danh c trnh by Hnh 3.15 v
Ph lc 6 .

64

Hnh 3.15. nh danh B. longum JCM 1217 bng kit Api 20A
Kt qu nh danh cho thy vi sinh vt thuc chi Bifidobacterium vi % ID l
97,1%
Nh vy phng php nh tnh vi sinh vt n chi Bifidobacterium bng kit
Api 20A c c hiu cao.

3.4.4. nh gi c hiu ca quy trnh nh danh vi sinh vt bng k


thut PCR
Tin hnh quy trnh nh gi c hiu nh trong mc 2.3.4.3 a. Kt qu
kim tra c hiu ca cc cp mi trn 20 loi chng vi sinh vt chun c trnh
by Bng 3.8 v Hnh 3.16

65

Bng 3.8. Kt qu kim tra c hiu ca cp mi Blon F/Blon R trn 20 chng


TT

Tn chng

Kt qu

TT

Tn chng

Kt
qu

PCR

PCR
1

Bacillus cereus ATCC

11

11778
2

Bacillus pumilus ATCC

Bacillus

ATCC 9338
-

12

14884
3

Lactobacillus fermentum

Lactobacillus plantarum

ATCC 8014
-

13

stearothermophilis

Bifidobacterium breve

ATCC 15700

ATCC 795
4

Bacillus subtilis ATCC

14

6633
5

Lactobacillus rhamnosus

Lactobacillus casei

15

Lactobacillus

16

Bacillus coagulans

17

Bifidobacterium longum

18

Lactobacillus

Pseudomonas

Escherichia coli

ATCC8739
+

19

JCM 1217
10

Streptococcus pyogenes

aeruginosa ATCC 9027

ATCC 7050
9

ATCC 19615

acidophilus ATCC 4356


8

Streptococcus faecalis
ATCC 33186

ATCC 334
7

ATCC11863

ATCC 9595
6

Bifidobacterium bifidum

Staphylococcus aureus

ATCC6538
-

20

leichmannii ATCC 7830

Salmonella typhimurium
ATCC 14028

66

Ghi ch: (-): Kt qu PCR m tnh: khng xut hin bng nhn bn.
(+): Kt qu PCR dng tnh: xut hin bng nhn bn
M

1 2 3 4 5

6 7 8

9 10 11 12 13

M 14 15 16 17 18 19 20

831bp

Hnh 3.16. Kt qu in di sn phm PCR kim tra c hiu ca cp


mi nhn bn on gen c hiu ca B.longum
M: Marker 100 bp
Ging 1,14: sn phm PCR vi khun l DNA ca B. longum JCM 1217
Ging 2-13,15-20: sn phm PCR vi khun l DNA ca 19 loi vi khun
khc c trnh by Bng 3.8
Kt qu trn cho thy, vi cp mi Blon F/ Blon R quy trnh PCR nhn bn
on gen c hiu ch cho kt qu PCR dng tnh vi khun l DNA ca loi
B. longum trong khi 19 loi vi sinh vt dng kim tra c hiu cn li u c
kt qu PCR m tnh.
*Tnh c hiu:
p dng vo cng thc
Chng chun

Php th
K thut PCR

Cng

Dng tnh

m tnh

Dng tnh

a (1)

b (0)

a+b

m tnh

c (0)

d (19)

c+d

a+c

b+d

a+b+c+d

Cng

* c hiu = s trng hp m tnh tht/(s trng hp m tnh tht +s trng


hp dng tnh gi) = d/(d+b)

67

Trong : d: s trng hp m tnh tht = 19 (19 mu khng phi l loi B.


longum u c kt qu PCR m tnh).
b: s trng hp dng tnh gi = 0 ( cc chng vi sinh vt u l chng
chun, c chng nhn c ngun gc r rng. Khng c chng vi sinh
vt khc ngoi B. longum JCM 1217 c kt qu PCR dng tnh)
Nh vy, c hiu ca cp mi BlonF/BlonR cng nh quy trnh thit
lp nhn bn on gen c hiu cho loi B. longum l: 19/(19+0)= 1
(100%)
Kt lun: Vi cc thnh phn phn ng, quy trnh thit lp v cp mi
thit k, chng ti nhn bn thnh cng on gen c hiu cho i tng nghin
cu. Trong khun kh ca ti nghin cu, quy trnh t c hiu rt cao
(100%).

3.4.5. Xc nh ngng pht hin ca quy trnh nhn bn on gen c hiu


ca B. longum bng cp mi c hiu.
Tin hnh quy trnh xc nh ngng pht hin nh m t mc 2.3.4.3b. Kt
qu m s lng hn dch B. longum chun c ghi ti bng nh sau.
Bng 3.9. Kt qu m s lng vi khun B. longum
10-6

10-7

10-8

a 1(CFU/a)

Nhiu

115

a 2 (CFU/a)

Nhiu

127

pha long

Trung bnh

121
1,21 x 109

Dd D (CFU/ml)

T hn dch D trn pha long thnh mt dy cc dung dch c nng


109CFU/ml, 108 CFU/ml, 107 CFU/ml, 106 CFU/ml, 105 CFU/ml v 104CFU/ml.
Tch chit DNA t cc hn dch ny theo quy trnh c trnh by mc
2.3.3.2. Tin hnh phn ng PCR n mi vi khun l cc DNA tch c t hn
dch trn. Sau in di trn gel agarose 1%. Kt qu thu c nh sau

68

Bng 3.10. Kt qu xc nh ngng pht hin


Nng vi khun
Trong hn

Trong dch

dch trc khi

chit DNA

tch chit

(copy/l)(*)

Kt qu PCR trong cc ln th
1

(CFU/ml)
109

107

++

++

++

108

106

++

++

++

107

105

++

++

++

106

104

++

++

105

103

104

102

(*) V DNA sau khi tch chit c ha tr li trong 100l DNA Rehydration
Solution, nn s lng bn copy ca phn t DNA trong dung dch sau khi tch
chit s gim 100 ln so vi s lng vi khun bt u trong hn dch trc khi
tch chit.

69

Kt qu cho thy:
- Ngng pht hin ca quy trnh nhn bn on gen c hiu cho B. longum
l 105 CFU/ml vi khun (tng ng vi khong 103copy/l dng lm khun
cho phn ng PCR). Kt qu c th hin nh Hnh 3.17

3 4

831 bp

Hnh 3.17. Kt qu in di sn phm PCR xc nh ngng pht hin ca


quy trnh nhn bn on gen c hiu ca B.longum
M: Marker 100 bp (Invitrogen)

Ging 1-6: Sn phm PCR vi khun l DNA tch chit t nng chng t
109 CFU/ml vi khun n 104 CFU/ml vi khun (tng ng vi khong 107
copy/ l n 102 copy/ l dng lm khun cho phn ng PCR)

70

CHNG 4. BN LUN
Hin nay, cc ch phm probiotics lu hnh trn th trng hu ht l cc sn
phm hn hp ca nhiu loi vi khun khc nhau. Tuy nhin cc tiu chun c s
do cc nh sn xut a ra cn rt nhiu thiu st. Vic nh lng ch dng li
vic m tng s vi sinh vt trong mu. Trong a s cc trng hp, nh danh
cn rt s si, ch dng li phn ng nhum soi v mt vi phn ng sinh ha.
Do , nu thc hin theo tiu chun, rt kh kt lun mu t hay khng v s
nh hng n quyn li ca bnh nhn. Xut pht t thc t, nhu cu cp thit
t ra l phi nghin cu nh lng, nh danh n loi i vi cc vi sinh vt
c ghi trn nhn ca ch phm. Cng vi s pht trin ca khoa hc k thut,
vic nh danh vi sinh vt khng ch bao gm kiu hnh m cn phi nh danh
kiu gen.
Nghin cu trc y ca Nguyn Th Thu Hng [4] ng dng k thut
PCR nh danh mt s vi sinh vt thuc chi Lactobacillus trong hn hp nhiu
vi sinh vt bng cch tch chit DNA trc tip t mu. Trong nghin cu ny, tc
gi thnh cng trong vic nh danh n loi mt s Lactobacillus trong hn
hp gm nhiu vi sinh vt thuc chi Lactobaccillus m bng cc phn ng sinh
ho khng th thc hin c. Tuy nhin, nhc im ca k thut ny l khng
phn bit c vi khun sng hay vi khun cht. Vi quy trnh xy dng c
trong ti ny, chng ti nh lng ring c B. longum trong hn hp
ng thi phn lp c khun lc thun khit tin hnh nh danh kiu hnh
bng kit sinh ha v nh danh kiu gen bng k thut PCR v gii trnh t. Do
s khng nh c chc chn s lng B. longum c mt trong hn hp nng
l bao nhiu. iu ny rt c ngha trong vic kim tra gim st cht lng
thuc.
ng dng quy trnh thit lp, chng ti kim tra c mt s mu lu
hnh trn th trng, khng nh c chc chn cc sn phm ny u c cha B.
longum nh trn nhn do nh sn xut cng b. Vi nhng mu probiotics m nh
sn xut ch cng b tng s lng vi sinh vt trong hn hp (L-Bio-3D, Lackid,

71

Lactominplus ), vic s dng mi trng BSM agar nh lng ring B.longum


vn c ngha trong vic phn lp c khun lc thun khit nh danh B.
longum .
S dng kit Api 20A ch cho php nh danh n chi Bifidobacterium, nhng
theo quy nh gn y v tiu chun cht lng probiotics [16], [10], [32] vic nh
danh kiu hnh vn rt cn thit. ng thi, nghin cu s dng kit Api 20A cng
cho php nh danh n loi i vi Bifidobacterium breve, Bifidobacterium
bifidum, Bifidobacterium adolescentis, v Bifidobacterium dentium bng cch lm
thm cc test b sung. Cc vi khun ny cng hay c dng trong cc ch phm
probiotic. Hn na do khng yu cu k thut cao, chi ph r nn vic s dng kit
Api 20A nh danh mt s probiotics thuc chi Bifidobacterium rt d p dng
cc trung tm kim nghim thuc v thc phm chc nng, nhm gp phn vo
cng tc kim tra cht lng cc ch phm probiotic a phng.
nh danh n loi B. longum, cng c hiu qu nht vn l k thut PCR
v gii trnh t. u im ln nht ca phng php ny l c nhy v chnh
xc rt cao, vt tri so vi cc phng php thng thng. Tuy nhin, y cng
khng phi l mt phng php hon ho v vn c mt s hn ch: yu cu k
thut vin c trnh cao, trang thit b hin i, t tin, quy trnh thc hin phc
tp khng d p dng mi phng kim nghim. Mc d vy, vi nhng u im
vt tri v nhy, tnh c hiu cng nh kh nng xc nh chnh xc n loi,
k thut PCR v gii trnh t ngy cng c ng dng rng ri trong vic nh
danh B. longum cng nh nhiu vi khn khc trong cc ch phm probiotics, c
bit l i vi nhng ch phm c cha nhiu vi sinh vt thuc chi Bifidobacterium.

72

KT LUN V KIN NGH


KT LUN:
T nhng kt qu thu c trong qu trnh nghin cu, chng ti t c
mc tiu ra:
1. xy dng c quy trnh nh tnh, nh lng B. longum trong hn hp
gm nhiu vi sinh vt bng vic s dng mi trng BSM agar, kit API
20A, k thut PCR v gii trnh t. Vi qui trnh ny, chng ti s khng
nh c chnh xc n loi B. longum vi s lng l bao nhiu. iu ny
rt c ngha trong cng tc kim tra gim st cht lng thuc. ng dng
c quy trnh nghin cu nh tnh, nh lng B. longum trong mt s
ch phm lu hnh trn th trng nh: Samubiothymo, Safikid BIO, LBio-3D, Lackid v Lactomin plus.
2. thm nh c lp li ca phng php nh lng, c hiu ca
k thut nh danh bng kit sinh ha, nhy, c hiu ca k thut
nh danh vi khun bng k thut PCR n mi v gii trnh t.
KIN NGH:
Chng ti c mt s kin ngh sau:
1. Do gp kh khn trong vic mua c hn dch B. longum chun c s
lng bit trc, nn chng ti cha thm nh c ng ca php nh
lng. Do tip theo y chng ti s tip tc tra cu v t mua hn dch
chun ny thm nh ng.
2. Tip tc nghin cu quy trnh nh danh mt s loi khc thuc chi
Bifidobacterium cng hay c s dng trong cc ch phm probiotics nh
Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium infantis...
bng vic s dng kit API 20A cng vi k thut PCR v gii trnh t.
3. Tip tc nghin cu nh tnh, nh lng mt s vi khun probiotic ang
gp nhiu kh khn trong cng tc kim tra, gim st cht lng nh
streptococcus faecalis, Enterococcus faecium...

73

TI LIU THAM KHO


TING VIT
1. Cng ty lin doanh Dc phm Mebiphar-Austrapharm (2007), Tiu chun
nh sn xut: Thuc L-BIO-3D.
2. Cng ty lin doanh Dc phm Mebiphar-Austrapharm (2007), Tiu chun
nh sn xut: Thuc Lactomin plus.
3. Nguyn Trng Hip, Bi Tng Hip (2008), "Vi sinh vt trong ch phm
Probiotics", Tp ch Dc hc, s 390, B Y t, H ni.
4. Nguyn Th Thu Hng (2012), nh tnh mt s loi thuc chi Lactobacillus
trong ch phm probiotics lu hnh trn th trng bng k thut PCR, H
Ni
5. Tng c o lng Vit Nam (2008), TCVN 7849 :2008 Sa v sn phm .
sa. nh lng Lactobacillus acidophilus gi nh trn mi trng chn
lc, k thut m khun lc 37 0C.
6. Phm Vn Ty, V Nguyn Thnh (2006), Cng ngh sinh hc, Nh xut bn
Gio dc, tp 5, tr. 129-154.
7. Trung tm k thut tiu chun o lng cht lng 3 (2013), nh tr v tnh
ton khng m o kt qu th nghim vi sinh., H Ch Minh.
8. Nguyn Hng Vn (2009), PCR v Realtime PCR. Cc vn c bn v cc
p dng thng gp. Nh xut bn Y hc chi nhnh thnh ph H Ch Minh.

TING ANH
9. Akatsu H, Iwabuchi N, Xiao JZ, Matsuyama Z, Kurihara R, Okuda K,
Yamamoto T, Maruyama M (2012), Clinical Effects of Probiotic
Bifidobacterium longum BB536 on Immune Function and Intestinal
Microbiota in Elderly Patients Receiving Enteral Tube Feeding, JPEN J
Parenter Enteral Nutr, 37(5), p. 631-640.

74

10. Alschuler Lise (2011), Ensuring Probiotics quality, Natural medicine


journal, 3(10).
11. Amenta M, Cascio MT, Di Fiore P, Venturini I (2006), Diet and chronic
constipation. Benefits of oral supplementation with symbiotic zir fos
(Bifidobacteriumlongum W11 + FOS Actilight), Actabiomed, 77(3), p.157162.
12. Bahaka D, Neut C, Khattabi A, Monget D, Gavini F (1993), Phenotypic and
genomic analyses of human strains belonging or related to Bifidobacterium
longum, Bifidobacterium infantis and Bifidobacterium breve, Int J Syst
Bacteriol, 43(3), p. 565-73.
13. BioMerieux, Inc (2011), API 20A, France.
14. Ethiopian standard ES ISO 29981 (2012), Milk products Enumeration of
presumptive bifidobacteria Colonycount technique at 37 C.
15. Hamaji Y, Fujimori M, Sasaki T, Matsuhashi H, Matsui-Seki K, ShimataniShibata Y, Kano Y, Amano J and Taniguchi S (2007), Strong Enhancement
of Recombinant Cytosine Deaminase Activity in Bifidobacterium longum for
Tumor-Targeting Enzyme/Prodrug Therapy, Biosci. Biotechnol. Biochem,
71, p. 874-883.
16. Indian council of medical research (2011), Guidelines for the Evalution of
Probiotics in Food, Indian.
17. IOS/TS 19036 (2006), Microbiology of food and animal feeding stuffs
Guidelines for the estimation of measurement uncertainty for quantitative
determinations.
18. IOS/TS 4833 (2003), Microbiology of food and animal feeding stuffs
Horizontal method for the enumeration of microorganisms Colony count
technique at 30oC.
19. Janet E L Corry, Gordon D W Curtis, Baird.R.M (2012), Handbook of
Culture Media for Food and Water Microbiology, 3rd Edition, Royal Society
of Chemistry, p. 199-221.

75

20. Kwon HS, Yang EH, Yeon SW, Kang BH, Kim TY (2004), Rapid
identification of probiotic Lactobacillus species by multiplex PCR using
species-specific primers based on the region extending from 16S rRNA
through 23S rRNA., FEMS Microbiology Letters, 239, p 267-275.
21. Lahtinen SJ, Gueimonde M, Ouwehand AC (2006), Comparison of four
methods to enumerate probiotic bifidobacteria in a fermented food product,
Food microbiology, 23, p. 571-577.
22. Matsuki T, Watanabe K, Tanaka R, Fukuda M and Oyaizu H (1999),
Distribution of Bifidobacterial Species in Human Intestinal Microflora
Examined with 16S rRNA-Gene-Tareted Species Specifics Primers,
Applied and Enviroment Microbiology, 65 (10), p. 4506-4512.
23. Promega Technical manual, pGEM-T and pGEM-T easy vector system, USA,
6/2009.
24. Schell

MA, Karmirantzou

M, Snel

B, Vilanova

D, Berger

B, Pessi

G, Zwahlen MC, Desiere F, Bork P, Delley M, Pridmore RD, Arigoni F


(2002), The genome sequence of Bifidobacterium longum reflects its
adaptation to the human gastrointestinal tract, Proc Natl Acad Sci U SA
99(22), p. 14422-14427.
25. Simpson. P.J, Fitzgerald.G.F, Stanton.C, Ross.R.P (2004), The evaluation
of a mupirocin-based selective medium for the enumeration of bifidobacteria
from probiotic animal feed, Journal of Microbiological Methods, 57(1), p. 916.
26. Singh J, Rivenson. A, Tomita. M, Shimamura. S, Ishibashi. N, Reddy. B.S
(1997), Bifidobacterium longum, a lactic acid-producing intestinal bacterium
inhibits colon cancer and modulates the intermediate biomarkers of colon
carcinogenesis, Carcinogenesis, 18(4), p.833841.
27. Sul Sy, Kim HJ, Kim TW v Kim KY (2007), Rapid Identification of
Lactobacillus and Bifidobacterium in Probiotic Products Using Multiplex
PCR, J. Microbiol. Biotechnol, 17 (3), p.490-495.

76

28. Temmerman R, Pot B, Huys G, Swing J (2001), A quality analysis of


commercial probiotic products, BiomedExperts 66 (3b), p. 535, 537-542.
29. Tuomola E, Crittenden R, Playne M, Isolauri E, and Salminen S(2001),
"Quality assurance criteria for probiotic bacteria", The American Journal of
Clinical Nutrition, 73(2), p.393-398.
30. UELI VON AH (2006), Identification of Bifidobacterium thermophilum
RBL67 isolated from baby faeces and partial purification of its bacteriocin,
Zurich.
31. Wasilewska Ewa, Bielecka Maria, Markiewicz Lidia(2003), Numerical
analaysis of biochemical and morphological features of bifidobacteria as a
tool for species characteristic and identification , Polish jounal of food and
nutrition sciences, 12(53), p 149-156.
32. World Health Organization (2002), Guidelines for the Evalution of
Probiotics in Food, FAO of the United Nations.
33. Zinedine. A and Faid. M (2007), Isolation and Characterization of Strains
of Bifidobacteria with Probiotic Proprieties In vitro, World Journal of Dairy
& Food Sciences, 2(1), p. 28-34.
34. Zoppi G, Cinquetti M, Benini A, Bonamini and Minelli EB (2001),
"Modulation of the Intestinal Ecosystem by Probiotics and Lactulose in
children during treatment with Ceftiazone", Current Therapeutic Research,
62(5), p. 418 - 435.
35. http://www.questhealthlibrary.com/news/bifidobacterium-longum
36. http://www.powerofprobiotc.com/Bifidobacterium.html
37. http://blast.ncbi.nlm.nih.gov/Blast.cgi

77

PH LC
Ph lc 1.

X l kt qu nh danh vi sinh vt trong ch phm


SamubioTHYMO (mu A) bng phn mm Apiweb

Ph lc 2.

X l kt qu nh danh vi sinh vt trong ch phm Safikid


BIO (mu B) bng phn mm Apiweb

Ph lc 3.

X l kt qu nh danh vi sinh vt trong ch phm L-Bio-3D


(mu C) bng phn mm Apiweb

Ph lc 4.

X l kt qu nh danh vi sinh vt trong ch phm Lackid


(mu D) bng phn mm Apiweb

Ph lc 5.

X l kt qu nh danh vi sinh vt trong ch phm Probiotic


Lactomin Plus (mu E) bng phn mm Apiweb

Ph lc 6.

X l kt qu nh danh B. longum JCM 1217 bng phn mm


Apiweb

Ph lc 7.

Kt qu gii trnh t vi sinh vt trong ch phm


SamubioTHYMO (mu A)

Ph lc 8.

Kt qu gii trnh t vi sinh vt trong ch phm Safikid BIO


(mu B)

Ph lc 9.

Kt qu gii trnh t vi sinh vt trong ch phm Lackid (mu


D)

Ph lc 10.

Kt qu gii trnh t vi sinh vt trong ch phm Probiotics


Lactomin Plus (mu E)

Ph lc 11.

Quy trnh nh lng, nh tnh B. longum trong cc ch phm


probiotics

Ph lc 1

Ph lc 2

Ph lc 3

Ph lc 4

Ph lc 5

Ph lc 6

Ph lc 7
Kt qu gii trnh t ca VSV phn lp t mu SamubioTHYMO
CGGTTTGCATCCAACGCGTTGGTGAGCTCTCCCATATGGTCGACCTGCA
GGCGGCCGCGAATTCACTAGTGATTTTCCAGTTGATCGCATGGTCTTCTG
GGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCCGCTTGACGG
CGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGG
CGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGG
CAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACG
CCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGA
GCAAGCGAGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTG
CCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTG
GGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCAT
CGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAG
GGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGA
AGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAG
GAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAC
GCCGTAGACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTG
TCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAGGGC
TAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGC
GGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTC
CCGACGGTCGTAGAGATACGGCTTCCCAATCGAATTCCCGCGGCCGCCA
TGGCGGCCGGGAGCATGCGACGTCAGGCCCAATTCGCCCTATATGGAGG
TCGTATTAAT

Kt qu so snh trnh t gii vi cc trnh t trong ngn hng gen th gii


bng ng dng BLAST

Ph lc 8
Kt qu gii trnh t ca VSV phn lp t mu Safikid BIO
CGCGGGTCTATGTGATCTTACAGTGCTCCCGGCCGCCATGGCGGCCGCG
GGAATTCGTATCTGGGAAGCCGTATCTCTACGACCGTCGGGAACATGTC
AAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCC
GCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCC
GTACTCCCCAGGCGGGATGCTTAACGCGTTAGCTCCGACACGGAACCCG
TGGAACGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCA
GGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTA
ACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACAC
ATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCC
GCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGG
ACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAATTCCGGATAAC
GCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTG
CTTATTCAACGGGTAAACTCACTCTCGCTTGCTCCCCGATAAAAGAGGTT
TACAACCCGAAGGCCTCCATCCCTCACGCGGCGTCGCTGCATCAGGCTT
GCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGC
CGTATCTCAGTCCCAATGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGT
CGAAGCCACGGTGGGCCGTTACCCCGCCGTCAAGCGGATAGGACGCGA
CCCCATCCCATACCGCGAAAGCTTTCCCAGAAGACCATGCGATCAACTG
GAAAATCACTAGTGAATTCGCGGCCGCCTGCAGGTCGACCATATGGGAG
AGCTCCCAACGCGTTGGATGCATAGCTTGAGTATCTATAGTAGTCACCT
AAAT

Kt qu so snh trnh t gii vi cc trnh t trong ngn hng gen th gii


bng ng dng BLAST.

Ph lc 9
Kt qu gii trnh t ca VSV phn lp t mu Lackid
TGGGTGGGTACACGCATTGGGAGGGTCCCACATGGCGACCTGCAGGCGT
CCGCGAATTCACAAATGATTTTCCAGTTGATCTTATGGTCTTCTGGGAAA
GCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCCGCTTGACGGCGGGG
TAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACC
GGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCA
GTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCG
TGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAG
CGAGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTGCCAGC
AGCCGCGGTAATACGTAGGGTGGAAGCGTTATCCGGAATTATTGGGCGT
AAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTT
AACGGTGGATCCGCGCCGGGTAAGGGCGGGCTTGAGTGCGGTAGGGGA
GACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAAC
ACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCG
AAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGT
AAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGA
GCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCGAGGCTAAA
ACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGAT
TAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGA
CGGTCGTAGAGATACGGCTTCCCAATCGAATTCCCGCGACCGCCATGGC
GGCCGGGAGCATGCGACGTCGGGCCCATTTCGCCCTATAGTGAGTCGTA
TTACAATTCACTGGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCC
TGGCGTTACCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTG
GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCA
GCCTGAATGGCGAATGGACGCGGCCTGTAGCGGCGCATTAAGCGCGGC
GGGGGGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCAGGGCCTAG
CGCCGCTCCTTTCCCTTTCTTCCCTTCTTTCTCGCCCGTTCGCGGGTTCCC
GTCAAGTTTAATCGGGGGCTCCCTTTAGGTTCGATTATGGCTTTACGGAC
CTCAACCCCAAAACTTGATAAGGGGGTGGTCAGGGTTTG

Kt qu so snh trnh t gii vi cc trnh t trong ngn hng gen th gii


bng ng dng BLAST.

Ph lc 10
Kt qu gii trnh t ca VSV phn lp t mu Probiotics Lactomin Plus
CCGATGTTCTCTGTACTCTCGTCGAGTCGTTCTCTGCGCTCCGGCCGCCT
GGCGGCCGCGGGAATTCGTTCTGGGAAGCCGTATCTCTACGACCGTCGG
GAATTGCCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCG
CATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCC
TTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTAGCTCCGACAC
GGAACCCGTGGAACGGGCCCCACATCCAGCATCCACCGTTTACGGCGTG
GACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAG
CGTCAGTAACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGAT
ATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCAC
TCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTT
CACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCGAATAATTC
CGGATAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTG
AGCCGGTGCTTATTCAACGGGTAAACTCACTCTCGCTTGCTCCCCGATAA
AAGAGGTTTACAACCCGAAGGCCTCCATCCCTCACGCGGGTCGCTGCTC
AGGCTTGCGCCCATTGTACAATATTCCCCACTGCTGCCTCCCGTAGGATC
TGGGCCGTATCTCATCCCAATGTGGCCGGTCGCCCTCTCAGGCCGGTAC
CCGTCAAAGCCCGGGGGGGCCGTTACCCCGCCGTCAAGCGGATAGGAC
GCGACCCCATCCATACCGGGAAAGCTTTCCCAGAAGACCATGCGTTCAA
CTGGAAAAATCACTAATGAATTCGGGGGCCGCCTGTAGGCGACCATATG
GGAA

Kt qu so snh trnh t gii vi cc trnh t trong ngn hng gen th gii bng
ng dng BLAST

Ph lc 11
1. nh lng B. longum trong cc ch phm probiotics
Cn v xc nh khi lng trung bnh ca 20 gi ch phm. Cn chnh xc
khong 10,0g bt thuc vo bnh nn v trng, thm dung mi pha long vi th
tch tnh theo mililit bng 9 ln khi lng bt thuc cn v vi vin bi thu
tinh v trng. Lc mnh trong 30 pht vi sinh vt phn tn u trong dung dch,
thu c hn dch c pha long 10-1. Ly 1000 l hn dch trn cho vo ng
nghim cha sn 9,0ml dung mi pha long, lc u bng my lc trn Vortex
c hn dch c pha long 10-2. Tip tc pha long 10 ln nh trn thu
c hn dch c c pha long 10-5, 10-6 v 10-7.
Tin hnh: Chun b ba a petri v trng, chuyn vo mi a chnh xc
1000 l hn dch c pha long 10-5. Tip tc lm nh trn vi hn dch c pha
long 10-6 v 10-7. Cy trn vi mi trng BSM c ( c tit trng v ngui
xung khong 45oC), mi trng trong a ng t nhin. Lt ngc a petri v
1oC trong 48 - 72 gi trong iu kin k kh. Sau thi gian , m s lng

37

khun lc trong cc a petri (ch m cc a petri c s khun lc nm trong


khong 25 - 250).
Tnh kt qu: S lng vi sinh vt trong 1 gi ch phm c tnh theo cng
thc:
A = nTB

MTB

Trong :
A

: s lng vi sinh vt trong 1 gi ch phm (CFU/gi),

nTB

: s lng khun lc trung bnh trong 3 a petri (CFU),

MTB

: khi lng trung bnh ca bt thuc trong gi ch phm (g/gi),

: h s pha long ca hn dch th.

2. nh danh n chi Bifidobacterium bng kit APi 20A


Sau khi nui cy vi sinh vt nh m t trong mc nh lng (mc 1), la
chn mt khun lc c trng, tch bit v cy chuyn sang 5,0ml mi trng MRS
lng, c b sung khong 0,5ml du khong lp b mt v trong iu kin k kh
37

1oC trong 24 gi.

2.1. Nhum Gram


Tin hnh nhum t bo theo phng php nhum Gram v soi di knh hin
vi vi vt knh du c phng i 1000 ln.
Nu vi sinh vt bt mu tm, hnh que, khng c bo t th kt lun vi sinh v l
trc khun Gram dng, khng sinh bo t.
2.2. nh danh vi sinh vt bng kit API 20A
Sau khi xc nh vi sinh vt l trc khun Gram dng, khng sinh bo t th
tip tc cy mt quai cy canh chng t ng MRS lng trn ln mi trng thch
mu, trong iu kin k kh 37

1oC trong 18-24 gi. Gt vi sinh vt trn b

mt a thch v phn tn u trong ng nghim c sn 1ml nc ct v trng thu


c hn dch gc.
Nh t t tng git hn dch gc trn vo mt ng nghim cha sn 4ml
nc ct v trng n khi c trong ng tng ng vi c chun
McFarland s 3. Ghi li s git hn dch gc cho vo ng nghim trn.
Cho vo 4ml mi trng API 20A s git hn dch gc bng s git cho vo
ng nghim trn v lc u. Nh vy, nng vi sinh vt trong mi trng API
20A tng ng vi c chun McFarland s 3 (so snh c bng mt
thng).
Nh mi trng API 20 cy vi sinh vt vo cc ging ca b kit API 20A bt
u t ging s 0 n ging 20, ch trnh to bt kh trong ging, ch ch nh
phn tube. Ring i vi ging IND nh dung dch du khong v trng ln trn b
mt ging to iu kin k kh cho vi sinh vt pht trin. i vi ging GEL nh
c phn tube v phn ming ging. 36 2oC trong iu kin k kh trong vng
24 2h.
Cch c kt qu: Sau 24h nui cy, quan st v c kt qu.
-

i vi ging IND: Thm 1 git thuc th XYL vo lp du khong pha trn, trn
u v 2-3 pht. Sau thm 1 git thuc th EHR. c kt qu trong vng 5
pht. Kt qu l dng tnh nu xut hin mu .

i vi cc ging c cha cc ng khc nhau: Thm vo mi ging mt git


thuc th BCP. Kt qu c ghi l dng tnh nu xut hin mu vng hoc vngxanh.

Phn ng CAT: kit tip xc vi khng kh khong 30 pht, sau thm 2 git
thuc th H2O2 3% vo mt ging dng tnh bt k. Kt qu c ghi l dng
tnh nu thy xut hin bt kh.
Cch c ging dng tnh v ging m tnh c trnh by Bng 1

Bng 1. Cch c kt qu kit Api 20A

Hm
Ging

Cht nn

lng(mg/gi

Kt qu
Phn ng

ng)

Dng tnh

m tnh

XYL-trn u /2-3 pht + HER/5 pht


IND

L-tryptophan

0,98

S hnh thnh indol

Vng

URE

Urea

0,648

Urease

Vng-cam

BCP

GLU

D-glucose

1,96

Acid ha

MAN

D-manitol

1,96

Acid ha

LAC

D-lactose

1,96

Acid ha

SAC

D-saccharose

1,86

Acid ha

MAL

D-maltose

1,96

Acid ha

SAL

Salicin

1,64

Acid ha

XYL

D-xylose

1,64

Acid ha

ARA

L-arabinose

1,64

Acid ha

GEL

gelatin

0,6

ESC

Esculin

0,36

St (III) citrat

0,11

S thy phn gelatin

Tm

Vng/ vng-xanh

Khng c s khuch tn cht

C s khuch tn

mu

cht mu en

Vng

Nu-en

Soi di n UV 365nm
S thy phn Esculin
Pht quang

Khng pht quang


BCP

GLY

Glycerol

1,82

Acid ha

CEL

D-cellobiose

1,86

Acid ha

MNE

D-mannose

1,96

Acid ha

MLZ

D-melezitose

1,96

Acid ha

RAF

D-raffinose

2,18

Acid ha

SOR

D-sorbitol

2,18

Acid ha

RHA

L-rhamnose

1,96

Acid ha

TRE

D-trehalose

1,96

Acid ha

Tm

Vng/ Vng-xanh

Sau khi tip xc vi khng kh 30 pht, thm H2O2


vo 1 ging dng tnh
CAT

Catalase

Khng c bt kh

Xut hin bt kh

SPOR

Bo t

Khng c

GRAM

Hng

Tm

COCC

Que

Cu

Hnh thi

Ghi kt qu vo phiu theo di. Nhp s liu vo phn mm Apiweb nh


danh chnh xc vi sinh vt trong ch phm.
3. nh danh n loi B. longum bng k thut PCR v gii trnh t

3.1. S chung nh tnh B. longum trong cc ch phm probiotics bng k


thut PCR v gii trnh t
Sau khi la chn khun lc c trng tch bit, tin hnh tng sinh trong mi
trng MRS lng c b sung 0,5ml du khong v c nh danh n chi
Bifidobacterium bng kit API 20A nh m t trong mc 2, tip tc cy tng sinh
VSV t phn sinh khi cn li sang mi trng MRS lng, b sung thm 0,5ml du
khong v khun, 3710C trong iu kin k kh trong 24 gi. Sau VSV
trong ng mi trng MRS lng ny s c tch chit DNA v nh danh bng k
thut PCR v gii trnh t theo s sau

VSV trong mi
trng MRS lng

Tch chit DNA

Kim tra sn phm


tch

Tin hnh phn ng


PCR vi mi c hiu

Nhn dng v gii


trnh t

3.2. Tch DNA vi khun bng WizardR Genomic DNA Purification Kit
DNA ca cc chng nghin cu v ca cc vi sinh vt phn lp trong cc
mu c tch v tinh sch bng kit ca Promega c ci tin. Quy trnh thc hin
nh sau:
Cc chng chun c tng sinh t cc ng gc bng mi trng MRS lng,
370C trong 48 gi. i vi cc mu nghin cu, tip tc tng sinh VSV trong mi
trng MRS lng t phn sinh khi ca VSV c phn lp sau khi nh danh
n chi Bifidobacterium (mc 2 )
-

Ly 1,0 1,5ml mi trng tng sinh chng trn vo ng nha 1,5ml. Mang ly
tm 10000-12000 vng/pht trong 2 pht loi dch ni thu ly cn.

Thm 480l dung dch EDTA 50mM vo ng cn, lc u.

Thm tip 120l dung dch lysozyme (nng 20mg/ml) trn u.


370C trong 30-60 pht, sau ly tm 10000-12000 vng/pht trong 2 pht.
B dch ni thu ly cn.

Thm 600l dung dch Nuclei Lysis vo ng cn va thu c. Dng pipet o


nh to thnh hn dch. hn dch trn 800C trong 5 pht ly gii t bo, sau
ngui n nhit phng

Thm 3l dung dch RNAse vo dch ly gii t bo, trn u bng cch o ng t
5-7 pht. 370C trong 15-60 pht, ngui n nhit phng.

Thm 200l dung dch Protein Precipitation (dung dch kt ta protein) vo hn


hp trn, lc mnh trong 20 giy. trong lnh 5 pht. em ly tm 10000-12000
vng/pht trong 3 pht.

Chuyn dch ni cha DNA vo mt ng nha 1,5ml sch khc cha 600l
isopropanol. Trn u bng cch o ngc ng t 5-7 ln. Ly tm 10000-12000
vng/pht trong 2 pht. Loi b dch ni, thu ly cn.

Thm 600l dung dch ethanol 70% vo ng, o nh nhng ra cn DNA. Ly


tm 10000-12000 vng/pht trong 2 pht. Cn thn loi ht ethanol tha, kh t
nhin trong 10-15 pht.

Thm 100l dung dch DNA Rehydration (dung dch ha tan DNA) vo ng cha
cn DNA, 650C trong 1 gi.

Bo qun dung dch DNA va tch c -200C.

3.3. nh lng DNA bng phng php o hp th nh sng t ngoi


bc sng 260 nm (A260)
Ly 20l dung dch DNA trn, thm 1,98 ml nc ct siu tinh khit v
khun. Tin hnh o quang bc sng 260nm thu c A260. Da vo kt qu o
tnh ton nng dung dch DNA gc ri pha long dung dch DNA gc n nng
50 ng/l.

- Kim tra kh nng tch DNA


Kim tra sn phm DNA tch c bng phn ng nhn bn on gen c
hiu 16S rDNA ca vi khun bng k thut PCR. Thnh phn phn ng c trnh
by Bng 2.
Bng 2. Thnh phn ca phn ng PCR kim tra DNA mi tch
STT

Thnh phn

Th tch (l)

m PCR (10X)

2,5

dNTPs (2mM)

2,5

Dung dch MgCl2 (50mM)

0,75

ID16R08F (10 pmol)

IDL16R09R (10 pmol)

Tag DNA polymerase (5u/l)

0,1

DNA khun (50ng/l)

Nc ct

14,15

Tng th tch phn ng

25l

Chu trnh nhit cho phn ng PCR nhn bn on gen 16S rDNA: 940C trong
5 pht bin tnh hon ton DNA khun, tip theo l 30 chu k ca 3 bc: 940C
trong 45 giy lm bin tnh DNA, 560C trong 45 giy gn mi v 720C trong 1
pht 30 giy ko di chui. Mu sau c tip 720C trong 5 pht v gi
40C cho n khi phn tch.
* in di trn gel agarose

Chun b bn gel c nng 1% (kl/tt) trong m TBE (pH 8,0) (Tris-Boric


acid-EDTA), tin hnh in di sn phm bng my in di gel XL-ULTRA-V-2
100V trong 30 pht. Nhum sn phm bng dung dch ethidium bromide 0,1%
trong 15-30 pht. Ghi li kt qu bng my chp nh bn gel UVDI- 312.

3.4. Nhn bn on gen c hiu ca cc i tng nghin cu bng k thut


PCR
Tin hnh phn ng PCR vi cc thnh phn ghi trong Bng 3
Bng 3. Thnh phn phn ng PCR n mi
STT

Thnh phn

Th tch (l)

m PCR (10X)

2,5

dNTPs (2mM)

2,5

Dung dch MgCl2 (50mM)

0,75

BlonF (10pmol)

BlonR (10pmol)

Tag DNA polymerase (5u/l)

0,1

DNA khun (50ng/l)

Nc ct

14,15

Tng th tch phn ng

25l

Chu trnh nhit cho phn ng PCR nhn bn on gen c hiu: : 94oC trong
5 pht bin tnh hon ton DNA khun, tip theo l 30 chu k ca 3 bc: 94 0C
trong 45 giy lm bin tnh DNA, 56oC trong 45 giy gn mi v 720C trong
30 giy ko di chui. Mu sau c tip 72oC trong 5 pht v gi 4oC
cho n khi phn tch.

3.5. Nhn dng v gii trnh t


3.5.1. Tinh sch DNA/dsRNA t gel agarose bng b kit Fermentas.
Quy trnh c thc hin nhit phng bao gm nhng bc nh sau:
-

Bng DNA/dsRNA quan tm c ct chnh xc bng li dao sch t gel agarose


v a vo ng eppendorf 1,5 ml. B sung m bm (binding buffer) vi 1 th

tch bng khi lng ming gel (1l : 1 mg) v trong b n nhit 60C trong
khong 10 pht n khi gel tan hon ton.
-

Dung dch gel agarose ha tan c chuyn ln ct tinh sch v ly tm 10000


vng/pht trong 1 pht. Sau phn dch i qua mng c gn b.

B sung 500 l m ra (wash buffer) vo ct v ly tm 1 pht vi vn tc 10000


vng/pht. Phn dch i qua mng c gn b. Lp li bc ny mt ln na.

Ct tinh sch c cha DNA/dsRNA c chuyn sang ng eppendorf 1,5 mi v


b sung 100 l elution buffer vo chnh gia lp mng silica ca ct. Sau
DNA/dsRNA c thu bng cch ly tm 1 pht vi tc 10000 vng/pht.
Lng DNA/dsRNA tinh sch c gi -20C phc v cho th nghim tip
theo.
3.5.2. Nhn dng sn phm PCR bng b kit pGEMT-Easy cloning.
- Phn ng gn sn phm PCR vo vector pGEMT- Easy.
Phn ng gn sn phm PCR vo vector pGEMT-Easy u T c thc hin
theo quy trnh km theo ca b kit, vi cc thnh phn nh trong Bng 4
Hn hp phn ng gn sau c trong b n nhit 16C qua m.
Bng 4. Thnh phn phn ng gn sn phm PCR vo vector.
TT

Thnh phn

Th tch (l)

m phn ng 2X

10 l

Sn phm PCR

3 l

Vector u T pGEMT-T

1 l

T4 DNA ligase (5 u/l)

1 l

H2O ct kh ion v trng

5 l

Tng th tch

20 L

Bin np DNA vo t bo kh bin E.coli chng DH5.


T bo kh bin E.coli DH5 (bo qun trong t lnh su -80C) c lm tan
trn trong 10 pht. Tip , b sung 10l hn hp phn ng gn sn phm PCR
vo vector pGEMT-easy vo dung dch t bo r ng v trn 20 pht
to iu kin cho vector bm vo thnh t bo. T bo c sc nhit bng cch
chuyn sang b n nhit 42C trong 45 giy, sau c chuyn li trn trong

2 pht. Tip theo, b sung 450l LB lng vo hn hp bin np v nhit


37C trong vng 20 pht trc khi c nui lc vi tc 220 vng/pht trong
thi gian 30 pht. Hn hp t bo bin np c cy tri trn a mi trng LB
c c b sung ampicillin 100 g/ml, ph trn b mt c cht X-gal (20 l, nng
20 mg/ml) v IPTG (Isopropyl -D-1-thiogalactopyranoside, 20

l, nng

100mM), 37C qua m.


3.5.3. Tinh sch plasmid t vi khun E. coli bng b kit GenJETTM Plasmid Miniprep.
DNA ti t hp c tinh sch t t bo vi khun E. coli bng b kit
GenJETTM Plasmid Miniprep (Fermentas). Quy trnh c thc hin nh sau:
-

Mt khun lc dng tnh c nui lc trong 5ml mi trng LB lng c b sung


khng sinh ampicillin 100 g/ml vi tc 220 vng/pht 37C qua m.

T bo c thu bng cch ly tm vn tc 6000 vng/pht trong 10 pht, 4oC.


T bo c ha tr li trong 250 l m P1 (Resuspension solution) b sung
enzyme RNase A.

B sung 250 l m P2 (lysis solution) vo dch t bo v o nh 3-5 ln,


nhit phng 5 pht ph v t bo gii phng plasmid.

Hn hp c trung ha bi 350 l m P3 (neutralization solution), o u 4-6


ln. Mnh v t bo v protein bin tnh c loi b bng cch ly tm hn hp vi
vn tc 13000 vng/pht trong 10 pht 4C.

Dch ni sau khi ly tm c a ln ct tinh sch plasmid v ly tm 10000


vng/pht trong 1 pht. Plasmid c gi li trn lp mng silica cn dch qua ct
c gn b.

B sung 500 l m ra (Wash buffer), ly tm 30- 60 giy, sau gn b dch qua


ct. Bc ny c lp li mt ln na.

Ct tinh sch cha plasmid c chuyn sang ng eppendorf 1,5 ml mi. B sung
50 l m y (Elution buffer) vo chnh gia mng silica ca ct v nhit
phng 2 pht.

Plasmid c thu bng cch ly tm vi vn tc 10000 vng/pht trong 1 pht.


Mu plasmid tinh sch c bo qun -20C.
Sn phm vector nhn dng c mang i gii trnh t nh danh n loi.