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B GIO DC V O TO TRNG I HC NNG LM TP.H CH MINH
B MN CNG NGH SINH HC
KHA LUN TT NGHIP
KHO ST KH NNG SINH AXT LACTIC V TNH
KHNG CA LACTOBACILLUS ACIDOPHILUS
I VI VI KHUN E. COLI DNG
SN XUT CH PHM
PROBIOTIC
Ngnh: CNG NGH SINH HC
Nin kho: 2003 - 2007
Sinh vin thc hin: PHM NH TRC LINH
Thnh ph H Ch Minh - 2007 -
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B GIO DC V O TO TRNG I HC NNG LM TP.H CH MINH
B MN CNG NGH SINH HC
KHO LUN TT NGHIP
KHO ST KH NNG SINH AXT LACTIC V TNH
KHNG CA LACTOBACILLUS ACIDOPHILUS
I VI VI KHUN E. COLI DNG
SN XUT CH PHM
PROBIOTIC
NGNH: CNG NGH SINH HC
NIN KHO: 2003 - 2007
GVHD: TS. NGUYN NGC HI SVTH: PHM NH TRC LINH
Thnh ph H Ch Minh - 2007 -
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MINISTRY OF EDUCATION AND TRAINING
NONG LAM UNIVERSITY, HCMC
FACULTY OF BIOTECHNOLOGY
Graduation thesis
STUDYING THE ABILITY OF ACID LACTIC PRODUCE
AND THE ANTAGONISM WITH E. COLI
OF LACTOBACILLUS ACIDOPHILUS
FOR PROBIOTIC
Major: Biotechnology
Term: 2003 - 2007
Student: PHAM INH TRUC LINH
Ho Chi Minh City
- 2007 -
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LI CM T
Xin chn thnh cm n:
Ban Gim Hiu Trng i Hc Nng Lm Tp. HCM, ban Ch Nhim b
mn Cng Ngh Sinh Hc to iu kin cho ti thc hin thnh cng kha lun.
TS. Nguyn Ngc Hi, ngi thy ng knh tn tnh hng dn, gip
cng nh ng vin ti lc ti gp kh khn.
Ton th Thy, C trang b cho ti nhng kin thc qu bu.
Cc Thy, C ti phng thc tp vi sinh ht lng gip v cho ti
nhng kinh nghim qu bu ti thc hin thnh cng kha lun ny.
Cc anh ch, cc bn cng thc tp trong phng vi sinh khuyn khch ,
ng h v gip ti thc hin tt kha lun ny.
Cng ton th lp CNSH29 h tr, gip v ng vin ti trong sut
thi gian lm ti.
Cng n ca cha m sinh thnh, dng dc v hy sinh tt c cho con
n hc nn ngi. Con xin cm n gia nh l ch da vng chc cho con vng
bc qua mi kh khn.
Tp H Ch Minh, ngy 30 thng 07 nm 2007
Phm nh Trc Linh
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TM TT LUN VN
PHM NH TRC LINH, i hc Nng Lm TP HCM, thng 9/2007.
KHO ST KH NNG SINH AXT LACTIC V TNH KHNG CA
LACTOBACILLUS ACIDOPHILUS I VI VI KHUN E. COLI DNG
SN XUT CH PHM PROBIOTIC . ti c thc hin ti phng Vi sinh
Khoa CNTY, i hc Nng Lm TP HCM t thng 3/2007- 7/2007.
Gio vin hng dn: TS. NGUYN NGC HI
Qua phn lp, kho st cc c im sinh ho, kh nng sinh axt lactic v
tnh khng ca vi khun Lactobacillus acidophilus i vi E. coli. Chng ti c
nhng ghi nhn sau y :
Chng ti phn lp tt c 10 chng vi khun L. acidophilus t ch phm
Antibio ca Hn Quc sn xut.
Kho st kh nng sinh axt lactic ca 10 chng phn lp c th kt qu l
chng s 7 cho hm lng axt lactic cao nht : 0,8055 g/100 ml (tng ng
89,50T) sau khi nui cy 24 gi.
Th nghim tip theo l b sung saccharose vo mi trng sa ti v o
hm lng axt lactic. Kt qu l axt lactic c tng nhng khng ng k. Hm
lng axt lactic dao ng trong khong 0,558 - 1,088 g/100 ml khi b sung 5%
saccharose (24 gi) v 0,63 - 0,9 g/100 ml khi b sung 6% saccharose (24 gi).
Chn 3 chng L. acidophilus sinh axt lactic cao nht em th i khng vi
E. coli. Kt qu L. acidophilus khng vi E. coli rt c ngha v phng din
thng k hc.
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SUMMARY
PHAM INH TRUC LINH, Nong Lam University, 9/2007. STUDYING THE
ABILITY OF ACID LACTIC PRODUCE AND THE ANTAGONISM WITH E.
COLI OF LACTOBACILLUS ACIDOPHILUS FOR PROBIOTIC . Thesis was
carried out at Microorganism Lab, Faculty of Husbandry and Veterinary, Nong
Lam University from March to July in 2007 under the suppervisor of Dr.
NGUYEN NGOC HAI.
10 isolates of Lactobacillus acidophilus was collected from Antibio products
manufactured in Korea.
Surveying the ability of acid lactic produce of 10 isolates, the result showed
that these strains could produce acid lactic. Among of them, the seventh strain
produce highest acid lactic that was about 0,8055 g per 100 ml (eviqualent 89,50T).
Adding with 5% and 6% saccharose in fresh milk culture showed an
negligible increased of acid lactic production. After 24 hours incubation, acid lactic
concentration fluctuated from 0,558g/100ml to 1,088g/100ml and from 0,63
g/100ml to 0,9 g/100 ml, respectively.
The result of antaganism experiment in co-culture L. acidophilus and E. coli
in fresh milk showed that L. acidophilus could strongly inhibite the development of
E. coli. The amounts of E. coli reduce 203 times (9th
strain) and 667 times (7th
strain).
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MC LC Trang
Li cm t ................................................................................................... i
Tm tt lun vn ......................................................................................... ii
Summary .................................................................................................... iii
Mc lc ....................................................................................................... iv
Danh sch cc ch vit tt .......................................................................... vii
Danh sch cc bng .................................................................................... viii
Danh sch cc hnh ..................................................................................... ix
Danh sch cc s ................................................................................... ix
Chng 1. M U ................................................................................ 1
1.1 t vn ....................................................................................... 1
1.2 Mc tiu nghin cu ............................................................................. 2
1.2.1 Mc ch.......................................................................................... 2
1.2.2 Yu cu............................................................................................ 2
Chng 2. TNG QUAN ........................................................................ 3
2.1 Vi khun lactic ..................................................................................... 3
2.2 Tng quan v Lactobacillus ................................................................. 4
2.2.1 c im ........................................................................................... 4
2.2.2 c tnh v chc nng sinh hc ca Lactobacillus ........................... 5
2.2.3 ng dng ca vi khun Lactobacillus .............................................. 7
2.3 S lc v Lactobacillus acidophilus .................................................. 9
2.3.1 c im v phn loi ....................................................................... 9
2.3.2 c tnh nui cy .............................................................................. 10
2.2.3 c tnh sinh ho ............................................................................... 11
2.2.3.1 Phn ng ln men ng ca Lactobacillus acidophilus .............. 11
2.2.3.2 Cc phn ng sinh ho khc........................................................... 12
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2.3.4 Li ch sc kho ................................................................................ 13
2.3.5 Tnh cht i khng ca Lactobacillus acidophilus.......................... 14
2.3.6 Mt s ch phm c sn xut t vi khun ..................................... 15
Chng 3. NI DUNG V PHNG PHP NGHIN CU ............ 16
3.1 Thi gian v a im thc hin ti ................................................ 16
3.1.1 Thi gian ........................................................................................... 16
3.1.2 a im ............................................................................................ 16
3.2 Vt liu th nghim ............................................................................... 16
3.2.1 Mu kho st ..................................................................................... 16
3.2.2 Mi trng ........................................................................................ 16
3.2.3 Ho cht ............................................................................................ 16
3.2.4 Thit b v dng c ............................................................................ 16
3.3 Ni dung ti ..................................................................................... 17
3.4 Phng php thc hin ti ............................................................... 17
3.4.1 Phn lp v nh danh Lactobacillus acidophilus ............................. 17
3.4.1.1 Ly mu .......................................................................................... 17
3.4.1.2 Phn lp .......................................................................................... 17
3.4.1.3 Kho st cc phn ng sinh ho ..................................................... 18
3.4.2 Kho st kh nng sinh axt lactic ca L. acidophilus ...................... 18
3.4.3 Kho st kh nng sinh axt lactic ca cc chng ............................. 19
3.4.4 Th i khng Latobacillus acidophilus vi E. coli ......................... 19
Chng 4. KT QU V THO LUN .............................................. 22
4.1 Kt qu phn lp v nh danh Lactobacillus acidophilus .................. 22
4.1.1 Kt qu phn lp vi khun Lactobacillus acidophilus ...................... 22
4.1.2 c im nui cy v hnh thi vi khun L. acidophilus .................. 23
4.1.2.1 Quan st i th .............................................................................. 23
4.1.2.2. Quan st vi th ............................................................................... 24
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4.2 c im sinh ho ca cc chng phn lp c ................................ 25
4.2.1 Kh nng ln men cc loi ng ..................................................... 25
4.2.2 Cc phn ng sinh ho khc .............................................................. 26
4.2.3 Kh nng sinh axt lactic ca vi khun L. acidophilus .................... 27
4.3 Kt qu o hm lng acid lactic trong mi trng ............................ 28
4.4 Kh nng khng ca vi khun Lactobacillus acidophilus ................... 30
Chng 5. KT LUN V NGH ................................................... 34
5.1 Kt lun ................................................................................................ 34
5.2 ngh ................................................................................................. 34
TI LIU THAM KHO .......................................................................... 35
PH LC
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DANH SCH CC CH VIT TT
MRSA: De Man, Rogaso, Sharpe, Agar
MRSB: De Man, Rogaso, Sharpe, Broth
TSB : Trypticase Soy Agar
EC : Enrichement E. coli Broth
EMB : Eosin Methylene Blue Agar
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DANH SCH CC BNG
Trang
Bng 2. 1: Mt vi loi bacteriocin t vi khun Lactobacillus .................. 7
Bng 2. 2: Cc phn ng ln men ng ca L. acidophilus .................... 12
Bng 2. 3: Phn ng sinh ho ca Lactobacillus acidophilus ................... 12
Bng 3. 1: Kho st kh nng sinh axt lactic ca cc chng phn lp c
sau khi b sung saccharose vo mi trng sa. ....................................... 19
Bng 4.1: Kt qu phn ng ln men cc loi ng ca cc chng
phn lp c ............................................................................................. 25
Bng 4 .1: Kt qu phn ng sinh ho khc ca vi khun ......................... 26
Bng 4 .2: Kt qu o hm lng axt lactic ca vi khun L. acidophilus
trong mi trng sa sau 24 gi v 48 gi nui cy.................................. 23
Bng 4 .3: Kt qu o hm lng axt lactic ca vi khun L. acidophilus
sau khi b sung saccharose vo mi trng sa ti trong 24 gi
v 48 gi ..................................................................................................... 29
Bng 4. 4: c kt qu trn mi trng EC...............................................
Bng 4 .5: Kt qu th nghim IMViC ......................................................
Bng 4 .6: S lng vi khun E. coli trong th nghim i khng
vi Lactobacillus acidophilus trn mi trng sa ................................... 33
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DANH SCH CC HNH
Trang
Hnh 2. 1: Hai loi ng phn axt lactic ................................................... 6
Hnh 4. 1: Lactobacillus acidophilus c nui trong mi trng
MRSB ........................................................................................................ 23
Hnh 4.2 : Lactobacillus acidophilus c nui trong mi trng
sa ti ....................................................................................................... 24
Hnh 4.3: S ng nghim dng tnh trn mi trng EC ........................ 31
Hnh 4.4: Kt qu th nghim IMViC trn chng s 7 .............................. 32
Hnh 4.5: Khun lc E. coli trn mi trng EMB .................................... 34
DANH SCH CC S
S 2.1: Hai kiu ln men axt lactic ...................................................... 4
S 3. 1: Quy trnh phn lp Lactobacillus acidophilus ......................... 17
S 3. 2: m s lng vi khun E. coli bng phng php MPN........ 20
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Chng 1
M U
1.1 t vn
Vit Nam l mt nc nng nghip, trong ngnh chn nui gi mt vai
tr quan trng. Mc tiu ca cc nh chn nui l t c kinh t ti a trong sn
xut chn nui vi chi ph thp nht. cng l mc tiu ca cc nh khoa hc
nhm a cc thnh qu nghin cu khoa hc vo trong chn nui.
Cng vi s pht trin ca ngnh chn nui, bnh trn gia sc ngy cng
phc tp hn. c bit l cc bnh v ng tiu ho. iu tr bnh trn ng
tiu ho, cc nh chn nui thng dng cc loi khng sinh c ph khng khun
rng. Cng pht trin chn nui theo hng sn xut ln, nng cao nng
sutngi ta cng l thuc nhiu vo vic s dng khng sinh nh l yu t kch
thch sinh trng v cha bnh cho vt nui. Khng sinh trong nhiu thp nin qua
chng t vai tr quan trng ca chng trong vic m bo thnh cng trong chn
nui. Tuy nhin, vic s dng khng sinh trong thi gian di s gy ra hin tng
khng thuc i vi mt s vi khun ng rut. Ngoi ra, vic s dng khng sinh
lin tc cn lm thay i h vi sinh vt ng rut to iu kin cho cc mm bnh
xm nhp. Kt qu l bnh ti pht nng hn v kh iu tr hn.
Hin nay, vic phng v iu tr bnh trn ng tiu ho gia sc hn ch s
dng khng sinh l mt vn ang c cc nh khoa hc v cc nh chn nui
quan tm. Ngi ta v ang y mnh vic ng dng nhng sn phm sinh hc
vo chn nui. Cc ch phm sinh hc (probiotic) ngy cng chng t hiu qu ca
chng trong vic thay th khng sinh gii quyt cc vn v tng trng v sc
kho vt nui. Probiotic l nhng ch phm sinh hc cha vi sinh vt sng c li
khi a vo trong c th ca vt ch s gip cn bng h vi sinh vt ng rut, to
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iu kin ci thin tng trng v tng cng sc khng ca vt ch. Probiotic
v ang c s dng thay th khng sinh trong phng v tr cc bnh trn
ng tiu ho.
V th, chng ti thc hin ti: Kho st kh nng sinh axt lactic v
tnh khng ca Lactobacillus acidophilus i vi vi khun E. coli dng sn
xut ch phm probiotic.
1.2 Mc tiu nghin cu
1.2.1 Mc tiu
Kho st kh nng sinh axt lactic ca vi khun Lactobacillus acidophilus v
tm hiu tnh khng ca chng i vi vi khun E. coli nhm ng dng vo sn xut
ch phm probiotic.
1.2.2 Yu cu
- Phn lp cc chng Lactobacillus acidophilus t ch phm Antibio, sa chua
Vinamilk.
- nh gi kh nng sinh axt lactic ca cc chng Lactobacillus acidophilus phn
lp c.
- Kho st kh nng sinh axt lactic ca cc chng phn lp c trong iu kin
b sung thm ng saccharose vo mi trng sa tui.
- Tm hiu tnh khng ca Lactobacillus acidophilus i vi vi khun E. coli.
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Chng 2
TNG QUAN TI LIU
2.1 Vi khun lactic
Nhng vi khun gy ln men sinh axt lactic c gi l vi khun lactic. Vi
khun lactic c c trng bi kh nng sinh axt lactic rt mnh t cc loi ng
khc nhau, c bit l ng lactose. Hu ht cc vi sinh vt sinh axt lactic u
thuc v h Lactobacillaceae v c xp bn chi: Streptococcus, Pediococcus,
Lactobacillus v Leuconostoc. Chng c dng hnh cu hoc hnh que, Gram
dng, khng bo t, khng di ng. Tuy nhin, hin nay ngi ta tm thy mt s
ging trong h vi khun lactic c kh nng to bo t. Vi khun lactic khng kh
nitrate, phn ng catalase m tnh, k kh tu , mt vi loi k kh sng trong h
tiu ho ca con ngi.
Vi khun lactic l nhng vi sinh vt c nhu cu dinh dng cao. sinh
trng bnh thng, ngoi ngun cacbon, chng cn nit, mt phn di dng cc
axt amin, mt s vitamin, cc cht sinh trng, v cc cht khongChng khng
th pht trin c trong mi trng c thnh phn n gin nh glucose v NH4+
nh mt s loi khc. V th ngi ta phi cho vo mi trng mt s cht giu
dinh dng: cao nm men, cao tht, cc loi ng chng c th ln men.
Vi khun lactic c th tn ti trong mi trng kh, c th chu c hm
lng cn t 10 15% v c th chu c nng CO2 cao. Chng c tm thy
khp ni trong t nhin, di da, trong h tiu ho
Lactobacillus c vai tr quan trng nht trong ln men lactic.
C hai kiu ln men lactic l ln men lactic in hnh v len men lactic
khng in hnh. Hai kiu ln men ny khc nhau ch: ln men lactic in hnh
ch to ra axt lactic cn ln men lactic khng in hnh ngoi to ra axt lactic cn
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to ra ethanol, axt acetic, glycerol v mt s cht khc nhng ch c ln men lactic
in hnh l c ngha v mt ng dng trong sn xut cng nghip (T Minh Chu,
2000).
Lactose (ng i)
Glucose Galactose
(ng n) (ng n)
Axt Pyruvic
+O2 Acetadehyt
Axt lactic A. lactic CO2 + a. Acetic Ru ethylic Diacetyl
CH3CHOCOOH
Ln men lactic Ln men lactic khng in hnh
in hnh
S 2.1 Hai kiu ln men axt lactic
(Ngun: T Minh Chu, 2000)
2.2 Tng quan v Lactobacillus
2.2.1 c im
Lactobacillus l vi khun k kh tu Gram dng. Chng l nhng vi
khun c dng hnh que di, khng sinh bo t, t bo thng xp i hoc thnh
chui, khng di ng. Chng l nhm chnh ca vi khun axt lactic, hu ht cc
chng ca chng bin i ng lactose v nhng ng khc thnh axt lactic.
Chng l vi khun rt ph bin v thng l lnh tnh. ngi, chng c mt m
o v rut. Nhiu loi c thc vt ang phn r. S sn xut axt lactic lm
ngn cn s pht trin ca mt vi loi vi khun c hi khc.
Lactobacillus c s dng sn xut cc ch phm cng nghip nh
yaourt, ph mai, da ci, da chua, ru bia, kim chi v nhng thc n len men
khc, cng nh thc n chua cho ng vt.
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Vi khun Lactobacillus l nhm vi khun c s dng nhiu nht trong cc
ch phm probiotic.
(http://www.umm.edu/altmed/articles/lactobacillus_000310.htm)
2.2.2 c tnh v chc nng sinh hc ca Lactobacillus
Nhng c tnh v chc nng ny u c ngha trong ng dng sn xut
probiotic.
Qu trnh trao i cht ca Lactobacillus c vai tr rt quan trng trong kh
nng cha bnh. Cc nghin cu nui cy Lactobacillus trong mi trng sa
th hin r rng nhng hot tnh ng ch sau:
Phn gii protein
Lactobacillus sn sinh enzyme proteinase phn gii protein thnh cc
polypeptide mch ngn.
Hot tnh ny ca vi khun gip cho protein c c th vt ch tiu ho d
dng. V vy, cc ch phm t hot ng ln men ca Lactobacillus c nh gi
l ngun dinh dng c gi tr cao cho cc i tng: tr s sinh, ngi ang dng
bnh, ngi gi hay gia sc non.
Phn gii lipid
Nh c enzyme lipase, Lactobacillus c kh nng phn ct cht bo dng
triglyceride thnh cc axt bo v glycerol. iu ny cng c ngha v mt dinh
dng i vi ngi v vt nui.
C nhng nghin cu lm sng v tin lm sng cho rng Lactobacillus phn
gii c cholesterol trong lipid huyt thanh v mui mt. C hai kh nng ny iu
c ngha v mt lm sng.
Phn gii ng lactose
Lactobacillus mang enzyme beta galactosidase, glycolase v lactic
dehyrogenase c tc dng chuyn ho ng lactose thnh axt lactic. y l mt
axt hu c c nhng c tnh sinh hc c bit.
Vai tr ca axt lactic:
V mt sinh l hc, axt lactic c nhng u im sau:
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- Tng cng kh nng tiu ho protein sa thng qua s ng vn.
- Tng cng hot tnh Ca, P, Fe.
- Kch thch s tit dch v.
- Tng nhanh c ng y nhanh thc n i xung d dy.
- L ngun nng lng cho qu trnh h hp.
Chnh nhng u im trn phn no chng minh hiu qu ca vic ng
dng Lactobacillus lm probiotic. Tu thuc vo loi v iu kin nui cy,
Lactobacillus sn xut hai loi ng phn quang hc: D (-) v L (+). ngi, c hai
loi ng phn ny u c hp thu trong ng rut.
COOH COOH
H C OH OH C H
CH3 CH3
D (-) axt lactic L (+) axt lactic
Hnh 2.1 Hai loi ng phn axt lactic
L (+) axt lactic: c chuyn ho hon ton v nhanh chng trong qu trnh
tng hp glycogen.
D (-) axt lactic: c chuyn ho t hn v phn khng chuyn ho s c
bi tit di dng urine. S hin din ca axt khng c chuyn ho trong ng
tiu ho s gy tnh trng nhim axt trong trao i cht tr s sinh.
Ngoi ra, axt lactic cn lm h pH ng rut cn 4 5. Do , s pht
trin ca vi sinh vt gy thi v E. coli (thch nghi pH 6 7) b c ch.
Sn xut bacteriocin v cc c cht khng khun
Bacteriocin l protein hay hp cht protein do vi khun sn xut c hot tnh
dit khun trc tip. C cht ny gip vi khun Lactobacillus th hin hot tnh c
ch i vi cc vi sinh vt gy thi trong h tiu ho.
CH3 D (-) axt lactic
CCCH3
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Bng 2.1 Mt vi loi bacteriocin t vi khun Lactobacillus
Tn bacteriocin Loi sn xut
Acidolin
Acidophilin
Lactacin B
Lactacin F
Bulgarin
Plantaricin A
Lactolin
Plantaricin B
Lactolin 27
Helveticin J
Reuterin
Lactobrevin
Lactobacillin
L. acidophilus
L. acidophilus
L. acidophilus
L. acidophilus
L. bulgaricus
L. plantarum
L. plantarum
L. plantarum
L. plantarum
L. herveticus
L. reuteri
L. brevis
L. brevis
Vi khun Lactobacillus cn c th c ch s pht trin ca cc vi sinh vt
gy thi nh vo nhng sn phm trao i cht khc nh: H2O2, CO2 v diacetyl
(Trn Hnh Trit, 2005).
2.2.3 ng dng ca vi khun Lactobacillus
Vi khun lactic c ng dng rng ri trong nhiu lnh vc khc nhau nh
cng nghip, nng nghip, mi trng, y dc v nhiu nht l trong ch bin bo
qun thc phm.
Trong chn nui th y
Lactobacillus c hiu qu trong phc hi s cn bng h vi sinh vt ng
rut v gip hnh thnh h vi sinh vt d c. Nh vo s gim nng NH3 v hn
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ch vi sinh vt gy thi nhim vo ng rut, Lactobacillus c hiu qu kch thch
tng trng th nui.
Ngoi ra, ngi ta cn dng Lactobacterium casei v Lactobacterium
plantarium rm, rau, c cho gia sc n. Trong qu trnh ln men vi khun
lactic sn sinh ra mt s sn phm c gi tr nh vitamin, cht thm, khng sinh lm
cho thc n gia sc chua c gi tr dinh dng cao lm tng nng sut vt nui.
Trong y hc
- V mt tr liu:
Nh vo kh nng sn xut axt lactic v bacteriocin trong ng rut,
Lactobacillus ci thin c tnh trng tiu chy, tng nhu ng rut, cha c
chng to bn.
Lactobacillus duy tr pH m o khong 4 4,5 nh vo hot ng ln men
glycogen thnh axt lactic. Mi trng ny khng thch hp cho mm bnh pht
trin nh Trichomoncisvaginaleic (protozoa k sinh) v Candida albicans (nm
men)...
Trong nha khoa c hai ch phm c s dng nhiu l Puramex v Puracal.
Puramex gm c: almulinium lactat, Fe-lactat (c s dng iu tr bnh thiu
mu), Mg-lactat, Zn-lactat. Cn Purical ch c lactat canxi. Cc ch phm ny
thng lm cho rng kho hn.
Cc ch phm cha Lactobacillus u cho thy hiu qu trong cha tr
nhng ri lon v vim nhim bao gm: vim rut kt, y hi, ung bu, lm h
cholesterol trong mu, au u, vim m o khng in hnh v ci thin c tnh
trng khng s dng c lactose.
- V mt dinh dng:
B sung lactat canxi vo thnh phn sa bt dinh dng, bnh nng hay
bnh ngt tng hm lng canxi cho c th. Thiu canxi nh hng n hot
ng c tim, s to huyt v ng mu, l nguyn nhn gy ci xng tr em v
gin xng xp ngi gi.
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Trong cng nghip
Vi khun lactic c s dng ln men thu axt lactic. C v chua d chu
v c c tnh bo qun nn c th lm gia v i vi cc loi nc ung nh, tinh
du, dch qu, mt. Chng c dng axt ha ru vang v hoa qu ngho axt,
ngoi ra cn c s dng trong cng nghip thuc da, dt, nhum, sn v cht
do.
Trong nng nghip v mi trng
Vi khun lactic c kh nng hn ch s pht trin ca Fusarium- loi nm
gy bnh quan trng trong nng nghip. Nm Fusarium khi pht trin s lm cy
yu i v y l c hi gy bnh cho cy trng.
Ch phm EM (effective microorganism) hay ch phm vi sinh hu hiu, n
bao gm 80 chng vi sinh trong c s gp phn ca vi khun lactic. Hiu qu
ca ch phm ny l ci to t, tng nng sut cy trng v gii quyt vn
nhim mi trng.
Trong bo qun v ch bin thc phm
Trong bo qun v ch bin thc phm vi khun lactic c s dng lm
da chua, lm chua qu m khng lm mt mu t nhin ca qu. Dng sn xut
tng, u ph hay ln men sa chua (Nguyn c Duy Anh, 2005).
2.3 S lc v Lactobacillus acidophilus
2.3.1 c im v phn loi
Gii : Bacteria
Ngnh : Firmicutes
Lp : Bacilli
B : Lactobacillales
H : Lactobacillaceae
Ging : Lactobacillus
Loi : Lactobacillus acidophilus
( Moro, 1900 ; Hansen v Mocquoc, 1970)
(http://en.wikipedia.org/wiki/Lactobacillus_acidophilus).
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- - - 10 -
Lactobacillus acidophilus ln u tin c phn lp bi Moro (1900) t phn
ca tr s sinh qua phu thut. ng m t c cc c im trao i cht,
phn loi cng nh chc nng ca vi khun ny.
Lactobacillus acidophilus l mt trong nhng vi khun ph bin thuc ging
Lactobacillus. mt vi quc gia, n c s dng thng mi cng vi
Streptococcus salivarius v Lactobacillus delbrueckii spp. Bulgaricus trong sn
xut yaourt.
Tuy nhin khng phi tt c cc chng vi khun L. acidophilus u c tc
dng cha tr, do ngi ta ch chn nhng chng c tc dng tt sn xut ra
cc ch phm vi sinh probiotic m thnh phn thng ch c vi khun L.
acidophilus hay phi hp chung vi vi khun khc nh Bacillus subtilis, Echerichia
coli...hoc nm men Saccharomyces boulardii... (Nguyn Vnh Phc, 1976).
L. acidophilus c rut ngi v ng vt, ming, m o. L. acidophilus
ln men lactose thnh axt lactic. Ging nh nhiu loi vi khun, L. acidophilus c
th b cht nhit cao, m cao, hoc nh sng trc tip.
Trong t nhin, vi khun L. acidophilus thng c trong ng rut, trong
phn v sa ca hu ht cc loi ng vt c v v ng vt khng xng sng
khc.
2.3.2 c tnh nui cy
Theo ti liu T Minh chu (2000):
Vi khun Lactobacillus acidophilus thng c c tnh vi hiu kh khi va
mi phn lp. Vi khun pht trin tt 37 40C (khng pht trin hay pht trin
rt yu nhit thp hn 20C), khng sinh sc t hay c t, pH thch hp l 5,5
6 (c th pht trin pH 5).
Vi khun pht trin tt trong mi trng c glucose, nc chit nm men. Vi
khun cng c th mc c trong mi trng c 2% mui NaCl (khng mc trong
mi trng c 4% NaCl) hay c 2% mui mt.
Trn mi trng thch MRSA c cha 2% glucose th vi khun pht trin tt.
Sau 48 gi nui cy 37C xut hin nhng khun lc nh, hnh cu ra dt, u,
-
- - - 11 -
ng knh khong 0,25 mm. Sau 72 96 gi khun lc khong 1 mm, c mu
vng nht, chnh gia c tm sm mu.
Trong mi trng canh dinh dng MRSB, sau 48 gi 37C, vi khun
pht trin rt tt. Mi trng tr nn c, c cn lng y, i khi thy bm vo
thnh ng nghim.
Vi khun khng pht trin c trn mi trng khoai ty.
Trn mi trng b sung gelatin: khng ho lng gelatin 20C do men
phn gii lipid v protic yu.
Trong mi trng sa: vi khun lm ng vn sa thnh khi, khng ln
cn, ln men lactose sinh axt lactic dng D L hay dng L.
Sc khng: vi khun Lactobacillus acidophilus khng c sc khng
c bit. Vi khun d dng b tiu dit trong hi nc nng 56C trong 30 pht.
Nhng vi khun c sc khng mnh vi axt nn chng c th sng trong mi
trng canh c cha 0,5 1% axt lactic hay axt acetic trong khong 1 3 tun.
Trong mi trng canh b sung 2% glucose 37C vi khun c th sng c 15
ngy.
L. acidophilus c kh nng sng 2 ngy trong dch v, 5 ngy trong dch mt
tinh khit, 8 ngy trong dch trng.
2.3.3 c tnh sinh ho
2.3.3.1 Phn ng ln men ng ca Lactobacillus acidophilus
Thng thng vi khun c enzyme phn gii mt s loi ng v to ra cc
axt hu c lm gim pH ca mi trng, ngoi ra cn c th to ra cc cht kh nh
H2 v CO2.
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- - - 12 -
Bng 2.2 Cc phn ng ln men ng ca Lactobacillus acidophilus
Loi ng Ln men sinh axt Sinh hi
Arabinose _ _
Dextrin _
Glucose + _
Lactose + _
Maltose + _
Mannitol _ _
Sacharose + _
Salicin + _
Raffinose _
Xylose _ _
2.3.3.2 Cc phn ng sinh ho khc
Bng 2.3 Phn ng sinh ho ca Lactobacillus acidophilus
Phn ng sinh ho khc Kt qu
Sinh indol _
MR +
VP _
Citrat _
Arginin _
Nitrat _
Gelatin _
Di ng _
ng vn sa +
Catalase _
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- - - 13 -
2.3.4 Li ch sc kho
Mt vi dng ca L. acidophilus c th c s dng lm probiotic hoc vi
khun thn thin. Nhng vi khun c li cho sc kho ny sng rut v m o,
c kh nng chng li mt vi loi vi sinh vt c hi cho sc kho. L. acidophilus
sn xut axt lactic, hydrogen peroxide v nhng sn phm khc chng li vi sinh
vt c hi. Trong qu trnh ln men, L. acidophilus sn xut niacin, axt folic v
pyridoxine.
Mt vi nghin cu c bo co l L. acidophilus c li cho sc kho,
bao gm ci thin chc nng d dy v bnh tiu chy. Nghin cu ca i hc
Nebraska dng L. acidophilus b sung vo thc n v cho gia sc n, kt qu l lm
gim 61% Escherichia coli 0157:H7. Mt bo co khc, L. acidophilus c th lm
gim cholesterol trong mu.
L. acidophilus l phn ca h vi sinh vt m o. Axt lactic c sn xut
bi L. acidophilus m o hn ch s pht trin ca nm Candida albicans, gip
ngn cn s xm nhp ca nhng nm khc vo m o. Cht dit tinh trng v
nga thai c th lm cht L. acidophilus m o.
Sau mt kho iu tr bng khng sinh, bnh nhn thng c cung cp
thm L. acidophilus n nh li h tiu ho.
Lactobacillus acidophilus l vi khun quan trng trong ln men thc phm,
t sn phm sa n tri cy v rau qu.
(http://en.wikipedia.org/wiki/Lactobacillus_acidophilus)
L. acidophilus sn xut axt lactic v cc cht dit khun nh lactocidin, ngn
cn s xm nhp v c ch s tng sinh ca cc vi khun gy bnh, gip cho c th
khng vi nhim khun ng rut.
L. acidophilus ng vai tr sinh l quan trng nh tng hp cc vitamin.
L. acidophilus c kh nng bn vng vi 40 loi khng sinh.
(http://www.ivac.com.vn/dsp_product.aspx?CaterogyID=3&ProductID=23)
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- - - 14 -
2.3.5 Tnh cht i khng ca Lactobacillus acidophilus
Nhng nghin cu ca i hc Nebraska ch ra rng vic chn nhng dng
Lactobacillus acidophilus, mt vi khun ph bin c s dng trong ln men sa
chua, c thm vo lm probiotic lm gim vi khun E. coli O157:H7. Kt qu
th nghim Lactobacillus lm gim 80% E. coli O157:H7 trong h tiu ho ca gia
sc. Chin lc pht trin ca cc nh khoa hc ti i hc Nabraska l lm gim s
lng E. coli gia sc trc khi em git m. Vic s dng Lactobacillus lm
probiotic cha nhng ha hn to ln trong vic kim sot s pht trin ca E. coli
(http://ard.unl.edu/rn/0902/ecoli.html).
Mt nghin cu khc ti i hc Texas cho thy vic phn lp nhng vi
khun c trong sa chua, ph mai, tht ln men lm probiotic gip b chng li E.
coli O157:H7. Vic trn Lactobacillus acidophilus vo thc n gip chng li trn
60% vi khun E. coli gia sc, gim tc hi ca Salmonela.
(http://www.fass.org/FASStrack/news_item.asp?news_id=338)
Hot ng i khng bi vi khun lactic c lin quan cht ch vi sn phm
cui ca qu trnh trao i cht. Hng lot cc sn phm ph ca qu trnh trao i
cht do Lactobacillus c kh nng c hot ng i khng (trong phng th
nghim). Cc sn phm ph c bit ti nhiu nht l cc axt hu c nh axt
lactic, axt acetic v hydroperoxid. Cc axt lactic, axt acetic c ch s pht trin
ca nhiu vi sinh vt gy bnh Gram m. Hot ng ca cc axt ny ph thuc vo
pH. Nu pH thp s tng mc axt dng khng ha tan.
Tuy nhin c nhiu yu t cn phi lu nu mun nhn c kt qu tt khi
s dng probiotic. Trong a s cc trng hp cn phi bit chc chn rng cc vi
sinh vt cn phi sng st v pht trin trong ng rut phi c kh nng sng trong
mi trng pH thp v c kh nng chng li tc dng ca mt. sng c trong
ng rut, cc chng vi sinh vt cn c kh nng nh vo v sinh si ny n trn
b mt ca rut non. Mc d mt vi tc gi a ra mt s c ch gii thch ti sao vi
sinh vt c li trong ng rut c th c ch s xm nhp ca vi sinh vt c hi
nhng c ch chnh xc ca s loi tr cnh tranh ca vi sinh vt gy bnh bng
-
- - 15
probiotic vn cha c khng nh. Trong s cc c ch ny c s cnh tranh v v
tr, cnh tranh cht dinh dng, cnh tranh v khi lng cc cht sinh ra bi vi sinh
vt (Nguyn c Duy Anh, 2005).
2.3.6 Mt s ch phm c sn xut t vi khun Lactobacillus acidophilus
Hin nay c nhiu dc phm c bo ch t cc chng vi sinh vt sng
hoc cht, hoc cht chuyn ho ca chng, dch cy vi khun, nm menc
trnh by di nhiu dng khc nhau: vin nn, vin bc ng, ng nc, ng
kh.
Ch phm t vi khun sng:
- Mt loi vi khun:
Lactobacillus acidophilus: c Bacid, Dofus (M); Antibioplus, Proflor,
Vivacidol (Php); Antibio granules (Hn Quc).
- Hai loi vi khun:
Lactobacillus acidophilus, Lactobacillus bulgaricus: c Lactines (M).
Lactobacillus acidophilus, Escherichia coli: c Coliphilus (M).
- Ba loi vi khun:
Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus lactic: c
Biolactyl (Php).
Lactobacillus acidophilus, Streptococcus feacalis, Bacillus subtilis: c
Biofermin (Nht).
(T Minh Chu, 2000)
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Chng 3
NI DUNG V PHNG PHP NGHIN CU
3.1 Thi gian v a im thc hin ti
3.1.1 Thi gian
ti c thc hin t thng 3/2007 n thng 7/2007
3.1.2 a im
Ti phng Vi sinh , khoa Chn nui Th y, Trng i hc Nng Lm TP.
HCM.
3.2 Vt liu th nghim
3.2.1 Mu kho st
Ch phm sinh hc Antibio do Hn Quc sn xut: 3 mu.
Sa chua Vinamilk: 5 mu.
3.2.2 Mi trng
Mi trng canh tng sinh chn lc MRSB.
Mi trng phn lp MRSA.
Mi trng nui cy v th sinh ho.
Mi trng gi ging: mi trng MRSA, mi trng MRSB, mi trng
sa ti tit trng.
3.2.3 Ho cht
Thuc nhum Gram.
Thuc th Kowacs; Methyl Red; NaOH 40%; H2O2 30%...
3.2.4 Thit b v dng c
Thit b: Knh hin vi, t m, t lnh, cn in t, t sy, ni hp cao p
autolave
-
- - 17
Dng c: Gi ng nghim, ng nghim, n cn, a petri, pipette, u tp
v trng, a thu tinh, ng Durham
3.3 Ni dung ti
ti c thc hin vi cc ni dung ln lt nh sau:
- Phn lp v nh danh vi khun Lactobacillus acidophilus.
- Kho st kh nng sinh axt lactic ca cc chng phn lp c.
- Kho st kh nng sinh axt lactic ca cc chng phn lp c sau
khi b sung saccharose vo mi trng sa ti.
- Tm hiu tnh khng ca Lactobacillus acidophilus i vi vi khun
E. coli.
3.4 Phng php thc hin
3.4.1 Phn lp v nh danh Lactobacillus acidophilus
3.4.1.1 Ly mu
Mu Antibio do Hn Quc sn xut v sa chua Vinamilk c mua cc
ca hng trong khu vc Th c.
3.4.1.2 Phn lp
Mu
Pha long vi NaCl 9
Mi trng MRSA, 370C/48-72 gi
Chn khun lc c trng Nhum Gram
Th cc phn ng sinh ho
Mi trng MRSB, 370C/24 gi
Mi trng sa gi ging
S 3.3 Quy trnh phn lp Lactobacillus acidophilus
Cch thc hin
Cho 1g ch phm Antibio (hay 1 ml sa chua Vinamilk) vo ng nghim c
9 ml nc mui sinh l v trng, pha long mu t c nng l 10-6.
-
- - 18
Sau khi pha long, dng micropipette vi u tp v trng ht 0,2 ml dch
khun cc nng 10-6, 10-5 v 10-4 c pha long cho vo a petri v trng
(mi nng ht vo 2 a). Tip theo mi trng MRSA c hp kh
trng vo cc a petri trn. Sau cc a vo t m 37C trong 48 - 72 gi.
Quan st hnh thi khun lc v t bo vi khun
Quan st c im hnh thi khun lc vi khun trn mi trng thch
MRSA c CaCO3 nui cy 37C trong 48 gi.
Sau thi gian 48 - 72 gi trong t m 37C: khun lc trn, mu vng
nht, c vng sng phn gii CaCO3.
Quan st c im hnh thi ca t bo vi khun trn mi trng canh
MRSB, nui cy 37C/24 gi.
Sau khi chn c khun lc c trng, dng que cy vng ly mt khun
lc cho vo mi trng canh MRSB c cht ch th bromocresol. Sau 24 gi quan
st s i mu ca mi trng MRSB. Tin hnh nhum Gram xem hnh thi chung
ca vi khun : vi khun hnh que ng ring l hay xp thnh chui, bt mu tm
(Gram dng).
3.4.1.3 Kho st cc phn ng sinh ho
1. Kh nng ln men cc loi ng
2. Phn ng methyl red
3. Phn ng Voges-Proskauer
4. Kh nng s dng citrat
5. Kh nng kh nitrat
6. Kh nng to indol
7. Phn ng catalase
(Xem phn ph lc)
3.4.2 Kho st kh nng sinh axt lactic ca Lactobacillus acidophilus
Xc nh chua Therner :
Sau khi cy ging vi khun Lactobacillus acidophilus vo mi trng sa v
trng (vi t l 10% th tch mi trng), yn v 37C/24 gi. Sau ly
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- - 19
10 ml mi trng sa nui cy vi khun + 90 ml nc mui sinh l + 2-3 git
phenolphthalein cho vo bnh tam gic 250 ml ri lc u. Dng burret chun
NaOH 0,1N cn thit dng chun 100 ml sa.
Cng thc tnh chua Therner:
T = (n x 100)/V
Cng thc tnh hm lng axt lactic trong 100 ml dch mu:
A = (n x 100 x 0,09)/V
T: chua Therner cn xc nh
A: s gam axt lactic trong 100 ml
n: s ml dung dch NaOH dng chun
V: th tch dch mu
3.4.3 Kho st kh nng sinh axt lactic ca cc chng phn lp c sau khi
b sung saccharose vo mi trng sa
B tr th nghim: theo bng 3.1
Bng 3.1 Kho st kh nng sinh axt lactic ca cc chng phn lp c sau
khi b sung saccharose vo mi trng sa
Sau khi cho cc chng phn lp c vo mi trng sa b sung
saccharose, ta em hai khong thi gian trn v em o hm lng axt lactic.
3.4.4 Th i khng Lactobacillus acidophilus vi E. coli trn mi trng sa
Chn ra 3 chng L. acidophilus phn lp c sinh axt lactic cao nht th
i khng vi E. coli.
Chun b: Mi trng sa ti v trng.
Nui cy E. coli trong mi trng TSB, 370C/ 24 gi.
Nui cy L. acidophilus trong mi trng sa 370C/24 gi.
Thi gian nui cy Saccharose b sung vo mi trng sa ti
5% 6%
24 gi
48 gi
-
- - 20
Ht ln lt 1 ml dch sa nui cy L. acidophilus 370C/24 gi cho
vo 3 ng nghim cha mi trng sa (mi ng nghim 15 ml sa). Ht tip ln
lt 1 ml, 0,1 ml v 0,01 ml dch canh khun E. coli nui cy 370C/24 gi cho
vo 3 ng nghim trn. em 3 ng nghim ny 370C/24 gi. Tin hnh m s
lng vi khun E. coli bng phng php MPN (Most Probable Number).
ng nghim nui cy L. acidophilus v E. coli 370C/24 gi
Pha long mu c 3 nng pha long lin tip
Ht mi nng pha long cy vo 3 ng nghim cha mi trng EC, 44,50C/24
gi
Chn ng nghim dng tnh (c bt kh trong ng durham)
Cy ln thch EMB, 37C/24 gi (mi ng nghim dng tnh cy 1 a)
Chn 3 khun lc in hnh
Th nghim IMViC
m s ng canh EC (+) v IMViC ++--, tra bng MPN
S 3.2 m s lng vi khun E. coli bng phng php MPN
Mu i chng khng c L. acidophilus c thc hin nh sau :
Cy E. coli vo mi trng TSB 37C/24 gi. Sau dng micropipette
vi u tp v trng ht ln lt 1 ml, 0,1 ml v 0,01 ml dch canh khun E. coli vo
cc ng nghim cha mi trng sa ti (15 ml) em 37C/24 gi .Tin hnh
-
- - 21
pha long mu trong nc mui sinh l c cc nng pha long thch hp. T
cc nng pha long ny, dng micropipette ht 0,1 ml cho vo a cha mi
trng EMB (mi nng ht vo 2 a). Tin hnh trang a v em 37C/24
gi. p dng cng thc tnh s lng t bo kim tra s lng vi khun E. coli.
Cng thc tnh s lng t bo trong 1g hay 1 ml dch mu mi nng
pha long :
X = A x 1/h x 1/V
Trong :
X: s lng khun lc trong 1g hay 1 ml dch mu.
A: s khun lc trung bnh c trong a (trong tng s 2 a cng nng
pha long)
h: pha long (10-6, 10-7, 10-8)
V: th tch dch cy trn 1 a
S lng t bo trung bnh trong 1g mu hay 1 ml dch mu 3 nng pha
long lin tip:
Y = (Xi + Xj + Xk)/3
Trong :
Y: s lng t bo trung bnh cc nng pha long
Xi, Xj, Xk: s lng khun lc trung bnh c trong 1g hay 1 ml dich mu
cc nng pha long lin tip nhau.
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- - 22
Chng 4
KT QU V THO LUN
4.1 Kt qu phn lp v nh danh Lactobacillus acidophilus
4.1.1 Kt qu phn lp vi khun Lactobacillus acidophilus
Sau thi gian thc hin ti, chng ti phn lp c tt c 10 chng L.
acidophilus.
thun tin trnh by chng ti tm thi gi tn cc chng phn lp c
ln lt theo th t t 1,2,...10.
Vic phn lp L. acidophilus t mu sa chua Vinamilk khng t yu cu
nh mong mun nguyn nhn c th do :
- Trong mt s mu sa chua chng ti phn lp c rt t vi khun L.
acidophilus v hu ht trong cc mu sa chua m chng ti em phn lp khng
ch c vi khun L. acidophilus m cn c s hin din ca rt nhiu loi vi khun
ln men lactic khc nh : L. bulgaricus, L. casei, Strep. lactic,...Theo Nguyn Ln
Dng v cc nh nghin cu khc (1976) cho bit : trong cc sn phm ln men
lactic nh sa chua...th s lng loi L. bulgaricus lun chim u th. Loi vi
khun ny pht trin v sinh ra lng axt lactic rt nhanh lm gim pH ca mi
trng, s st gim nhanh pH ca mi trng kim hm s pht trin ca cc
loi vi khun ln men lactic khc cng tn ti, trong c loi L. acidophilus.
- Mt s vi khun v nm men khc mc gy kh khn cho chng ti trong
qu trnh bt khun lc ca L. acidophilus.
i vi mu ch phm Antibio do Hn Quc sn xut th t l phn lp c
vi khun L. acidophilus ca chng ti l rt cao (100%)..
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- - 23
4.1.2 c im nui cy v hnh thi vi khun Lactobacillus acidophilus
T cc khun lc nghi ng vi khun Lactobacillus acidophilus phn lp
c, chng ti tin hnh quan st cc c im nui cy, hnh thi ca vi khun v
kho st cc c im sinh ho ca chng.
4.1.2.1 Quan st bng mt trn
- Trn mi trng thch MRSA : vi khun L. acidophilus c cy trn
thch a MRSA (c CaCO3) nui cy 37oC cho thy :
Sau 48 gi : to khun lc c, nh li ti, mi trng nui cy xung quanh
khun lc bt u chuyn sang mu vng.
Sau 72 gi : ton b mi trng trong a chuyn sang mu vng, khun lc
r hn, c mu vng nht, hnh trn, ng knh khong 0,5 - 1 mm, b mt lng,
xung quanh khun lc c vng sng phn gii CaCO3.
- Trn mi trng canh MRSB, vi khun Lactobacillus acidophilus c
nui 37C, quan st thy :
Sau 24 gi : mi trng chuyn sang mu nu vng, c cn mu trng
lng di y ng nghim.
Sau 48 gi : mi trng chuyn sang mu vng, lp cn dui y
ng nghim dy hn, c khi bm vo thnh ng nghim.
Hnh 4.1 Lactobacillus acidophilus c nui trong mi trng MRSB
sau 24 gi
Hnh 4. 2: Lactobacillus acidophilus c nui trong mi trng MRSB
-
- - 24
ng nghim c mi tn l ng cha Lactobacillus acidophilus c nui
trong mi trng MRSB/24 gi.
ng nghim khng c mi tn l ng i chng trn MRSB.
Trn mi trng sa, sau 24 - 48 gi nui cy 37C, mi trng sa c,
tr nn ln cn, c lp nh thanh trong mu vng nht nm tch bit pha trn.
Hnh 4.2 Lactobacillus acidophilus c nui trong mi trng sa sau
24 gi.
ng nghim nh s (1) l ng i chng trn mi trng sa ti.
Hai ng nghim cn li l Lactobacillus acidophilus c nui trong mi
trng sa trong 24 gi.
4.1.2.2 Quan st di knh hin vi quang hc
Trn mi trng MRSB sau khi nui cy 24 gi, vi khun c nhum
Gram, quan st di knh hin vi quang hc ( phng i 1000 ln) cho thy vi
khun L. acidophilus bt mu tm (Gram dng ), t bo vi khun dng trc di,
mnh, khng c bo t, thng xp thnh tng i hay chui di, kch thc
khong 0,5 0,8 m x 1 3 m.
Lp nh thanh
(1)
-
- - 25
4.2 c im sinh ho ca cc chng Lactobacillus acidophilus phn lp
c
4.2.1 Kh nng ln men cc loi ng
Kh nng ln men ng l mt trong nhng phn ng quan trng nh
gi nhm vi khun ln men lactic. Chng ti kho st kh nng ln men ng
ca tt c 10 chng vi khun phn lp c.
Bng 4.7 Kt qu phn ng ln men cc loi ng ca cc chng
phn lp c
Ghi ch : +: phn ng ln men ng dng tnh.
-: phn ng ln men ng m tnh.
Tt c 10 chng vi khun nghi ng L. acidophilus chng ti phn lp c
u ln men khng sinh hi ng glucose v khng ln men ng mannitol.
So vi kt qu kho st Chu Vit Cng (2005) v Nguyn c Duy Anh
(2005) kt qu kho st ca chng ti cho thy ph hp.
Chng phn lp Glucose Mannitol
1 + -
2 + -
3 + -
4 + -
5 + -
6 + -
7 + -
8 + -
9 + -
10 + -
-
- - 26
4.2.2 Cc phn ng sinh ho khc
khng nh cc chng vi khun phn lp c l vi khun L. acidophilus
sau khi kho st kh nng ln men ng, chng ti tin hnh th thm mt s phn
ng sinh ho khc. Kt qu c trnh by qua bng 4.2.
Bng 4 8 Kt qu phn ng sinh ho khc ca vi khun
Chng
Indol
MR VP S dng citrat
Kh nitrat
Catalase
1 - - + - - -
2 - - + - - -
3 - - + - - -
4 - - + - - -
5 - - + - - -
6 - - + - - -
7 - - + - - -
8 - - + - - -
9 - - + - - -
10 - - + - - -
Ghi ch : +: phn ng dng tnh
- : phn ng m tnh
MR: phn ng Methyl-Red
VP: phn ng Voges-Proskauer
Kt qu trnh by bng 4.2 cho thy tt c cc chng vi khun phn lp
c u cho cc phn ng sinh ho ging nhau. Kt qu phn ng VP dng tnh,
tt c cc phn ng khc u m tnh.
Vi cc kt qu kho st c im hnh thi khun lc, t bo, kh nng ln
men cc loi ng v mt s phn ng sinh ho cn thit khc, chng ti c th
-
- - 27
kt lun rng tt c 10 chng phn lp c u l chng vi khun Lactobacillus
acidophilus.
4.2.3 Kh nng sinh axt lactic ca vi khun Lactobacillus acidophilus
Chng ti tin hnh o hm lng axt lactic ca 10 chng vi khun L.
acidophilus phn lp c sau 24 gi v 48 gi nui cy trong mi trng sa.
Kt qu c trnh by qua bng 4.3.
Bng 4.9 Kt qu o hm lng axt lactic ca vi khun L. acidophilus
trong mi trng sa sau 24 gi v 48 gi nui cy
Qua kt qu trnh by bng 4.3, tt c cc chng vi khun L. acidophilus
phn lp c t ch phm Antibio sau 24 gi v 48 gi nui cy trong mi trng
sa u c kh nng sinh axt lactic cao. Chng s 7 sinh axt lactic cao nht:
0,8055 g axt lactic/100 ml (24 gi) v 1,404 g axt lactic/100 ml (48 gi), chng
sinh axt lactic thp nht l chng s 3: 0,513 g axt lactic/100 ml (24 gi) v 0,9315
Chng
24 gi 48 gi
chua
Therner (T)
Hm lng
axt lactic
(g/100 ml)
chua
Therner (T)
Hm lng
axt lactic
(g/100 ml)
1 76 0,711 128,5 1,1565
2 75,5 0,6795 133 1,197
3 57 0,5130 103,5 0,9315
4 81,5 0,7335 135 1,215
5 74,5 0,6705 127 1,143
6 66 0,594 133 1,197
7 89,5 0,8055 156 1,404
8 69,5 0,6255 132 1,188
9 79 0,711 151 1,359
10 70,5 0,6345 165,1 1,4859
-
- - 28
g axt lactic/100 ml (48 gi).
Trong mi trng sa, vi khun L. acidophilus chuyn ho ngun ng
lactose v saccharose sn c trong sa thnh axt lactic. ng lactose trong sa
c thu phn thnh 2 ng n l glucose v galactose, ng saccharose thnh
glucose v fructose. Vi khun L.acidophilus chuyn ho cc loi ng ny theo
chu trnh EMP (Embden-Mayerhoff-Parnas) to thnh axt pyruvic, gii phng nng
lng ATP v enzyme NAD.H2 dng kh. Enzyme NAD.H2 s c hon nguyn
tr li thnh NAD dng oxi ho. Do , gii phng H2 kt hp vi axt pyruvic to
ra axt lactic (T Minh Chu, 2000).
Theo ghi nhn ca Nguyn Vnh Phc (1976): vi khun L. acidophilus
thuc loi sn sinh axt lactic rt cao, c th chua t ti 300T (tng ng
2,7 g axt lactic/100 ml).
i vi cc chng vi khun L. acidphilus phn lp t ch phm Antibio c
kh nng sinh axt lactic cao l hon ton hp l v cc chng vi khun ny c
kim tra v c chn lc trc khi a vo sn xut.
4.3 Kt qu o hm lng axt lactic trong mi trng sa ti c b sung 5%
v 6% saccharose
Chng ti tin hnh kho st kh nng sinh axt lactic ca 10 chng vi khun
L. acidophilus phn lp c sau khi b sung thm saccharose vo mi trng sa
ti sau 24 gi v 48 gi nui cy. Kt qu c trnh by bng 4.4.
-
- - 29
Bng 4.4 Kt qu o hm lng axt lactic ca vi khun L. acidophilus sau khi
b sung saccharose vo mi trng sa ti trong 24 gi v 48 gi.
n v axt lactic l g/100 ml.
Hm lng axt lactic c sinh ra t 10 chng vi khun L. acidophilus phn
lp c khi khng b sung ng saccharose dao ng trong khong 0,6705
0,8055 g/100 ml (nui cy 24 gi). Trong khi , sau khi b sung 5% saccharose
vo mi trng sa ti hm lng axt lactic sinh ra dao ng trong khong 0,576
1,008 g/100 ml (nui cy 24 gi) v b sung 6% saccharose l 0,63 0,9
g/100 ml (nui cy 24 gi). Nhn chung kh nng sinh axt lactic sau khi b sung
saccharose c gia tng nhng khng ng k.
Sau khi b sung saccharose, hm lng axt lactic c sinh ra thay i theo
tng chng. Chng s 5 cho hm lng axt lactic cao nht khi b sung 5%
saccharose 1,008 g/100 ml (112T). Chng s 4 cho hm lng axt lactic cao nht
Chng
B sung 5% saccharose B sung 6% saccharose
24 gi 48 gi 24 gi 48 gi
Therner
(T)
Axt
lactic
Therner
(T)
Axt
lactic
Therner
(T)
Axt
lactic
Therner
(T)
Axt
lactic
1 62 0,558 86 0,774 83 0,747 130 1,17
2 86 0,774 141 1,269 93 0,837 120 1,08
3 100 0,9 148 1,132 84 0,756 115 1,035
4 91,5 0,8235 124 1,116 100 0,9 160 1,44
5 112 1,008 165 1,485 85 0,765 127 1,143
6 65 0,585 106 0,954 70 0,63 138 1,242
7 74 0,666 134 1,206 85 0,765 143 1,287
8 85 0,765 90 0,81 88 0,792 150 1,35
9 64 0,576 110 0,99 80 0,72 119 1,071
10 64 0,576 108 0,972 70 0,63 106 0,954
-
- - 30
khi b sung 6% saccharose 0,9 g/100 ml (100T). Trong khi khi khng b sung
saccharose th chng s 7 sinh axt lactic cao nht 0,8055 g/100 ml (89,5T).
Nguyn nhn c th l do mi chng thch hp vi nhng nng ng khc
nhau.
Kt qu cng cho thy rng c s khc bit gia hai mc thi gian o hm
lng axt lactic (24 gi v 48 gi). Theo kt qu ca chng ti, khi o hm lng
axt lactic ca vi khun L. acidophilus sau 48 gi nui cy cao hn khi o mc
thi gian l 24 gi. Sau 24 gi s lng vi khun tng sinh cha chuyn ho
ht ng lactose. Sau 48 gi s lng vi khun s tng nhiu hn. Do , lng
axt lactic sinh ra s cao hn.
Theo ghi nhn ca Nguyn Ln Dng v ctv (1976): yu t thi gian nui
cy cng s c nh hng n vic tng s lng t bo vi khun L. acidophilus.
chuyn ho c ht ngun dinh dng thnh axt lactic vi khun cn phi c mt
khong thi gian nht nh nhng khong thi gian ny qu di s tc ng ngc
tr li lm gim s lng vi khun v lng axt lactic c sinh ra.
Tm li, khi b sung saccharose vo mi trng sa ti tng kh nng
sinh axt lactic ca vi khun L. acidophilus. Tuy nhin s gia tng ny l khng ln.
4.4 Kh nng khng ca vi khun Latobacillus acidophilus i vi E. coli trn
mi trng sa ti
Chn 3 chng s 4,7 v 9 em th i khng vi E. coli do 3 chng ny sinh
axt lactic cao.
Chng ti tin hnh nui chung vi khun Lactobacillus acidophilus v E. coli
trong mi trng sa ti, 37C/24 gi. Sau em pha long mu c cc
nng 10-8, 10-7, 10-6 v cy vo mi trng EC (ht 0,1 ml dch mu).
Kt qu s ng nghim dng tnh trong mi trng EC c trnh by
bng 4.5 (xem phn ph lc).
Qua bng 4.5 ta thy a s cc ng nghim c phn ng dng tnh (c hin
tng bt kh trong ng Durham).
-
- - 31
Hnh 4.3 S ng nghim dng tnh trn mi trng EC
T cc ng nghim dng tnh ny, ta cy trn mi trng EMB, 37C/24
gi. Chn cc khun lc nghi ng l E. coli em th nghim IMViC. Kt qu th
nghim IMViC c trnh by bng 4.6 (xem phn ph lc).
Da vo s ng EC dng tnh v kt qu th nghim IMViC ++-- tra bng
MPN.
-
- - 32
Bng 4.7 S lng vi khun E. coli trong th nghim i khng vi L.
acidophilus trn mi trng sa
T kt qu ca bng 4.7, ta thy:
- S lng vi khun E. coli c trong dch mu i khng thay i theo tng chng
v tng th tch dch mu.
S lng vi khun E. coli thay i theo tng chng nguyn nhn c th
l do cc chng c tnh khng E. coli khc nhau.
Trong cng mt chng, s lng vi khun E. coli cng thay i theo
tng th tch dch mu. Chng s 4 th tch dch mu l 0,01 th s
lng E. coli l 9.107 (CFU/ml), trong khi th tch dch mu 1 ml
th s lng vi khun E. coli l 4.104 (CFU/ml). Chng ti tht s khng
th hiu v sao kt qu li nh th.
Chng
S ml dch canh
khun E. coli
cho vo mi
trng sa c 1
ml dch sa L.
acidophilus
MPN
S lng E. coli
(t bo/ml) c
trong dch mu
sa i khng sau
24 gi nui cy
S lng vi
khun E. coli
(CFU/ml) trong
mu sa khng
c
L. acidophilus
4
1 4 4.104
4672.107
0,1 4 4.107
65.107
0,01 9 9.107
1.107
7
1 7 7.107
4672.107
0,1 9 9.107
65.107
0,01 29 29.107
1.107
9
1 23 23.107
4672.107
0,1 15 15.107
65.107
0,01 0 0
1.107
-
- - 33
- i vi mu i chng: mi trng sa khng c L. acidophilus, s lng vi
khun E. coli rt cao.
So snh mu i chng vi mu i khng ca cc chng, s lng vi khun
E. coli cao hn rt nhiu ln, cao hn gp 203 ln so vi s lng vi khun chng
s 9 cng th tch dch mu l 1 ml (4672.107 so vi 23.107) v gp 667 ln so vi
chng s 7 (7.107 CFU/ml). S lng vi khun E. coli mu i chng 0,1 ml cao
gp 4,2; 7; 14 ln ln lt cc chng s 9, 7, 4 (65.107 so vi ln lt 15.107,
9.107, 4.10
7).
Chng ti tin hnh x l s liu trn vi phn mm Minitab. Kt qu l
th tch dch mu 1 ml v 0,1 ml (bao gm c phn th nghim v phn i chng)
c s khc bit rt c ngha v phng din thng k hc (P = 0,034< 0,05 v P =
0,006 < 0,05). Cn dch mu 0,01 ml khng c s khc bit v phng din thng
k hc (xem phn ph lc).
Nh vy, th tch dch mu 1 ml v 0,1 ml L. acidophilus khng rt tt i
vi vi khun E. coli. Cn th tch dch mu l 0,01 ml th L. acidophilus c khng
nhng khng r rng.
Tm li, Lactobacillus acidophilus khng rt mnh i vi vi khun E. coli.
Hnh 4.5 Khun lc E. coli trn mi trng EMB (mu i chng) sau
24 gi nui cy
-
- - 34
Chng 5
KT LUN V NGH
Qua phn lp, kho st cc c im hnh thi, sinh ho, kh nng sinh axt
lactic v tnh khng ca Lactobacillus acidophilus i vi vi khun E. coli. Chng
ti c nhng kt lun v ngh nh sau:
5.1 Kt lun
- T 10 chng phn lp, chng ti kho st c chng s 7 c kh nng
sinh axt lactic cao nht l 0,8055 g/100 ml (tng ng 89,5T).
- Hm lng axt lactic tng khng ng k khi b sung saccharose vo mi
trng sa ti. Chng s 5 sinh axt lactic cao nht khi b sung 5% saccharose
1,008 g/100 ml (sau 24 gi nui cy).Chng s 4 sinh axt lactic cao nht khi b
sung 6% saccharose 0,9 g/100 ml (sau 24 gi nui cy).
- Lactobacillus acidophilus khng vi E. coli rt c ngha v phng din
thng k hc (P = 0,034 v P = 0,006).
5.2 ngh
- Tm hiu cc iu kin gia tng s sinh trng, pht trin, kh nng sinh
axt lactic v tnh khng ca vi khun Lactobacillus acidophilus i vi vi khun E.
coli.
- Sn xut th ch phm probiotic c cha vi khun L. acidophilus nhm ng
dng vo chn nui th y.
-
TI LIU THAM KHO
TI LIU TRONG NC
1. Nguyn c Duy Anh, 2005. Xc nh mi trng ti u thu sinh khi v enzyme ca vi khun Bacilulus subtilis, Lactobacillus acidophilus v th nghim sn xut ch phm sinh hc. LVTN B mn CNSH. T sch trng i hc Nng Lm.
2. T Minh Chu, 2000. Vi sinh vt ng dng trong chn nui. T sch trng i hc Nng Lm.
3. Chu Vn Cng, 2005. Kho st mt s c im ca vi khun Lactobacillus acidophilus dng sn xut ch phm probiotic. LVTN Khoa Chn nui th y. T sch trng i hc Nng Lm.
4. Nguyn Ln Dng, Nguyn ng c, ng Hng Nguyn, Phm Vn Ty, Nguyn Vnh Phc, Nguyn Phng Tin, 1976. Mt s phng php nghin cu vi sinh vt hc tp 1, 2, 3. NXB Khoa hc k thut.
5. Nguyn Ln Dng, Nguyn nh Quyn, Phm Vn Ty, 1998. Vi sinh vt hc. NXB Gio Dc.
6. Nguyn Thnh t, 2005. C s sinh hc vi sinh vt, tp 2. T sch trng i hc Nng Lm.
7. Nguyn Ngc Hi, 2007. Cng ngh sinh hc trong th y. NXB Nng Nghip.
8. Nguyn Vnh Phc, 1976. Vi sinh vt gy bnh th y, tp 1, 2, 3, NXB Kim ng.
9. Phm Th Trc Phng, 2005. Nghin cu c im ca vi khun Lactobacillus sporogenes nhm ng dng trong sn xut probiotic. LVTN Khoa Chn nui th y. T sch trng i hc Nng Lm.
10. Trn Linh Thc, 2005. Phng php phn tch vi sinh vt trong nc, thc phm v m phm. NXB Gio Dc.
-
TI LIU NC NGOI
11. W.I.Li, Benjamin G.Brackett and Jaroslava Halper. Culture Supernatant of Lactobacillus acidophilus Stimulates Proliferation of Embryonic Cell.
12. Lievin-Le Moal, V., R. Amsellem, A. L. Servin, and M.-H. Coconnier. 2002. Lactobacillus acidophilus (strain LB) from the resident adult
gastrointestinal microflora exerts activity against brush border damage
promoted by a diarrhoeagenic Escherichia coli in human enterocyte-like
cells. Gut 50:803-811.
13. Bernet-Camard, M.-F., V. Lievin, D. Brassart, J.-R. Neeser, A. L. Servin, and S. Hudault. 1997. The human Lactobacillus acidophilus strain LA1
secretes a non-bacteriocin antibacterial substance(s) active in vitro and in
vivo. Appl. Environ. Microbiol. 63:2747-2753.
14. Wagner R.D; Pierson Carey ; Warnet Thomas, 2000.Probiotic effects of feeding heat-killer Lactobacillus acidophilus and Lactobacillus casei to
Candida albicans-conolized immunodeficient mice. Joural of Food
Protection.
CC TRANG WEB
15. http://en.wikipedia.org/wiki/Lactobacillus_acidophilus
16. http://www.umm.edu/altmed/articles/lactobacillus_000310.htm
17. http://www.ivac.com.vn/dsp_product.aspx?CaterogyID=3&ProductID=23
18. http://clwb.unl.edu/Techer/NSF/C11/C11Links/www.bact.wis C.edu/scienceed/Lactobacillus acidophilus.html
19. http://www.fass.org/FASStrack/news_item.asp?news_id=338
20. http://ard.unl.edu/rn/0902/ecoli.html
21. http://www.biopharmachemie.vnn.vn
22. http://ianrnews.unl.edu/static/0310210.shtml
23. http://www.nlm.nih.gov/medlineplus/druginfo/natural/patient-acidophilus.html
-
PH LC
1. Cch thc hin cc phn ng sinh ho
Kim tra kh nng ln men ng
- Nguyn tc: Cc vi sinh vt s dng v len men mt s loi ng sinh ra
axt lm cho pH ca mi trng gim. Khi , cht ch th mu phenol red hin din
trong mi trng t mu s chuyn sang mu vng.
- Chun b: Mi trng cc loi ng cn kim tra (glucose, saccharose,
mannitol, maltose, lactose) v ng ging vi khun.
- Cch tin hnh: Phn mi trng c cc loi ng vo cc ng nghim
(khong 5ml) c ng Durham. Hp kh trng 1150C/10 pht. Sau cy dch vi
khun vo mi trng, 370C/24 gi. Quan st s i mu v sinh hi.
- Cch c kt qu:
Ln men ng c sinh hi: mi trng chuyn t mu sang mu vng,
ng Durham c hi.
Ln men ng khng sinh hi: mi trng chuyn t mu sang mu
vng, ng Durham khng sinh hi.
Khng ln men ng: mi trng vn gi nguyn mu , ng Durham
khng c hi.
Vi khun Lactobacillus acidophilus ln men khng sinh hi cc loi ng
glucose, saccharose, maltose, lactose v khng ln men ung mannitol.
Kim tra kh nng sinh Indol
- Nguyn tc: Mt s vi sinh vt c enzyme tryptophanase lm chuyn ho
tryptophan thnh indol. Indol s kt hp vi cht para-dimethylaminobenzaldehyde
(c trong thuc th Kowacc) s to thnh phc cht roindol mu .
- Chun b: ng ging vi khun, mi trng NB.
-
- Cch tin hnh: Cy vi khun vo mi trng canh NB, nui cy 370C/24
gi, sau thm vi git thuc th Kowacc vo. yn, quan st hin tng ri
c kt qu.
Phn ng dng tnh: lp mt mi trng c mu .
Phn ng m tnh: mi trng c mu vng (khng i mu).
Phn ng MR (Methyl-Red)
- Nguyn tc: Mt s vi sinh vt s dng v ln men ng glucose c trong
mi trng lm cho pH ca mi trng gim (sinh axt). Khi , thuc th methyl-
red vn gi nguyn mu (MR dng tnh). Ngc li, nu vi sinh vt khng s
dng ngun glucose m ch s dng ngun nitrogen trong mi trng lm cho pH
mi trng tr nn kim v thuc th methyl-red chuyn t mu sang mu vng
(MR m tnh).
- Chun b: Mi trng Clark Lubs, thuc th methyl-red, ng ging vi
khun.
- Cch tin hnh: Dng que cy ly mt vng canh khun cy vo trong ng
nghim cha mi trng Clark Lubs, nui nhit 370C/24 gi. Sau , nh 2-3
git thuc th methyl-red vo ng nghim v c kt qu.
Phn ng MR dng tnh: mi trng c mu .
Phn ng MR m tnh: mi trng mu vng.
Kh nng kh nitrat
- Nguyn tc: Mt s vi sinh vt c kh nng sinh enzyme nitrat-reductase c
tc ng kh nitrat thnh nitrit (NO3- thnh NO2-), ri tip tc kh thnh NH3 hay
N2.
- Chun b: Mi trng thch nitrat bn lng
Dung dch thuc th Griess A (axt sulfanilic).
Dung dch thuc th Griess B (-naphthylamine).
ng ging vi khun.
- Cch tin hnh: Dng que cy thng ly huyn dch vi khun cy su vo
trong mi trng thch nitrat bn lng, nui 370C/24 gi. Sau 24 gi nh vo mi
-
rng 2 - 3 git thuc th Griess A, sau nh tip 2 - 3 git thuc th
Griess B. c kt qu.
Kim tra hot tnh catalase
- Nguyn tc: Mt s vi khun hiu kh tuyt i hay hiu kh tu nghi c
kh nng sinh ra enzyme catalase c th phn gii thnh H2O2 thnh CO2 v [O].
- Chun b: ng nghim thch nghing cha mi trng MRSA cy vi
khun 24 gi, 370C.
Lam sch.
L H2O2 30% kn.
- Cch tin hnh: Dng que cy vng ly mt t vi khun, pht ln gia lame
knh. Sau nh git H2O2 30% ln vt vi khun. c kt qu sau khong 15 giy.
Phn ng catalase dng tnh: c hin tng si bt.
Phn ng VP (Voges_Proskauer)
- Nguyn tc: Mt s vi khun ln men ng to ra axt pyruvic, sau tip
tc chuyn ha thnh acetyl metyl carbinol (ACM). Trong mi trng kim ACM
b oxi ha thnh diacetyl. Diacetyl s kt hp vi nhm guadinin cha trong acid
amin arginin ca thuc th -napton to thnh hp cht mu cam (VP dng
tnh).
- Chun b: Mi trng Clark Lubs
Thuc th -napton 10%
NaOH 40%
- Cch tin hnh: Cy vi khun vo ng nghim cha khong 3 ml mi
trng Clark Lubs, nui nhit 37C/24 gi. Sau nh 3 - 5 git NaOH 40%
v 3 - 5 git thuc th -napton 10%.
Sau 15 pht c kt qu.
Phn ng VP dng tnh: mi trng chuyn sang mu cam.
Phn ng VP m tnh: mi trng mu vng.
-
Kh nng s dng citrat
- Nguyn tc: Mt s vi sinh vt c kh nng s dng citrat nh mt ngun
carbon duy nht, to ra ion Na+ lm cho pH mi trng tng. S thay i pH mi
trng c nhn bit nh cht ch th mu bromothylmol blue.
- Chun b: Mi trng tng hp Simmons citrate
ng ging vi khun.
- Cch tin hnh: Dng que cy vng ly vi khun trong ng ging cy sang
ng nghim thch nghing Simmons citrate c cht ch th mu bromothymol blue.
Cho vo t m 370C/24 gi. Sau c kt qu.
Phn ng citrat dng tnh: mi trng chuyn t mu xanh lc sang mu
xanh dng.
Phn ng citrat m tnh: mi trng vn gi mu xanh lc (khng i).
2. Phng php nhum Gram
K thut nhum: Cc bc tin hnh
Pht canh khun ln phin knh
C nh mu bng cch hi qua ngn la n cn 3 ln
t giy lc ln vt pht vi khun
Nhum bng crystal violet trong 1 pht
Ra nc, thm kh
t giy lc ln vt pht vi khun
C nh mu bng Lugol trong 1 pht
Ra nc, thm kh
Ty cn 96C khong 15 giy
Ra nc, thm kh
t giy lc ln vt pht vi khun
Nhum mu bng dung dch fuchsine kim long
Ra nc, thm kh
Xem knh hin vi phng i x100 (vt knh du).
-
3. Mi trng
Mi trng tng sinh chn lc MRSB (De Man, Rogaso, Sharpe)
Cao tht 8 g
Pepton bt 10 g
Cao nm men 4 g
Acetat natri 5 g
K2HPO4 2 g
Triamonium citrat 2 g
MgSO4 0.2 g
MnSO4 0.2 g
Twen 80 1 ml
Bromocresol 1 ml
Nc ct 1000 ml
iu chnh pH = 6.2 + 0.2. un nh ho tan cc thnh phn mi trng.
Sau phn vo mi ng nghim khong 10ml.
Hp kh trng bng autoclave 121C/20 pht.
Mi trng tng sinh chn lc MRSA
Mi trng MRSB 1000 ml
CaCO3 32 g
Agar 16 g
pH = 6.2 + 0.2
un si ho tan cc thnh phn mi trng.
Hp kh trng bng autoclave 121C/20 pht.
Mi trng ln men cc loi ng
Cao tht 5 g
Pepton bt 10 g
ng 10 g
Phenol red 0.01 g
Nc ct 1000 ml
-
pH = 7.4
Hp kh trng bng autoclave 121C/10 pht.
Mi trng Clark Lubs
Pepton bt 7 g
Glucose 5 g
KH2PO4 5 g
Nc ct 1000 ml
pH = 6.9 + 0.2
Hp kh trng bng autoclave 121C/20 pht.
Mi trng thch nitrat bn lng
Cao tht 3 g
Pepton bt 5 g
KNO3 1 g
Agar 5 g
Nc ct 1000 ml
pH = 7.2 +0.2
Hp kh trng bng autoclave 121C/20 pht.
Mi trng Simmons Citrate Agar
Sodium citrate 2g
NaCl 5g
K2HPO4 1g
NH4H2PO4 1g
MgSO4 0,2g
Bromothymol blue 0,08g
Agar 15g
Nc ct 1000 ml
un nng nh v thnh thong lc. un si 1 - 2 pht cho n khi ho tan.
Hp 121C/15 pht.
-
Trypticase Soy Agar (TSA)
Trypticase pepton 15g
Phytone pepton 5g
NaCl 5g
Agar 15g
Nc ct 1000 ml
un nng ho tan agar. Hp kh trng 121C/15 pht. pH 7,3 0,2.
EC Broth (canh EC)
Trypticase hoc tryptose 20g
Mui mt No. 3 1,5g
Lactose 5g
K2HPO4 4g
KH2PO4 1,5g
NaCl 5g
Nc ct 1000 ml
Rt mi trng vo ng nghim c cha ng durham. Hp kh trng
121C/15 pht. pH 6,9 0,2.
-
4. Cc bng
Bng 4.5 c kt qu trn mi trng EC
Chng
S ml E.coli
cho vo mi
trng sa
c L.
acidophilus
Pha long mu v cho vo mi trng EC
10-3
10-4
10-5
10-6
10-7
10-8
4
1 + - - - - - - - - - - - - - - - - -
0,1 + + + + + + + + +
0,01 + + + + + + + + +
7
1 + + + + + + + + +
0,1 + + + + + + + + +
0,01 + + + + + + + + +
9
1 + + + + + + + + +
0,1 + + + + + + + + +
0,01 - - + + + + - + +
Ghi ch: 10-3, 10-4, 10-5 l cc nng pha long.
+ phn ng dng tnh
- phn ng m tnh
-
Bng 4 10 Kt qu th nghim IMViC
Chng
S ml E. coli
cho vo mi
trng sa c
L. acidophilus
IMViC
10-3
10-4
10-5
10-6
10-7
10-8
4
1 + - - - - - - - -
0,1 - - + - - - - - -
0,01 - - - + + - + - -
7
1 - - + - - - - - +
0,1 - - - - - - + + +
0,01 + - + + + + - - -
9
1 + + + - - - - - -
0,1 + - - - + + - - +
0,01 - - - - - -
Ghi ch: + kt qu th nghim IMViC l ++--.
- kt qu th nghim IMViC khng phi l ++--.
-
5. Kt qu x l thng k One-way ANOVA: So luong 1 versus Lo 1 Analysis of Variance for So luong
Source DF SS MS F P
Lo 1 1 20.91 20.91 10.07 0.034
Error 4 8.31 2.08
Total 5 29.21
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev --+---------+---------+---------+----
1-dc 3 10.670 0.000 (---------*--------)
1-tn 3 6.936 2.038 (--------*--------)
--+---------+---------+---------+----
Pooled StDev = 1.441 5.0 7.5 10.0 12.5
Tukey's pairwise comparisons
Family error rate = 0.0500
Individual error rate = 0.0500
Critical value = 3.93
Intervals for (column level mean) - (row level mean)
1-dc
1-tn 0.466
7.000
-
One-way ANOVA: So luong 2 versus Lo 2 Analysis of Variance for So luong
Source DF SS MS F P
Lo 2 1 1,2207 1,2207 29,14 0,006
Error 4 0,1676 0,0419
Total 5 1,3883
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev ---------+---------+---------+-------
0.1-dc 3 8,8129 0,0000 (-----*------)
0.1-tn 3 7,9108 0,2895 (-----*------)
---------+---------+---------+-------
Pooled StDev = 0,2047 8,00 8,50 9,00
Tukey's pairwise comparisons
Family error rate = 0,0500
Individual error rate = 0,0500
Critical value = 3,93
Intervals for (column level mean) - (row level mean)
0.1-dc
0.1-tn 0,4381
1,3661
-
One-way ANOVA: So luong 3 versus Lo 3 Analysis of Variance for So luong
Source DF SS MS F P
Lo 3 1 3.5 3.5 0.31 0.607
Error 4 45.0 11.3
Total 5 48.5
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev ----------+---------+---------+------
0.01-dc 3 7.000 0.000 (--------------*--------------)
0.01-tn 3 5.472 4.746 (---------------*--------------)
----------+---------+---------+------
Pooled StDev = 3.356 3.5 7.0 10.5
Tukey's pairwise comparisons
Family error rate = 0.0500
Individual error rate = 0.0500
Critical value = 3.93
Intervals for (column level mean) - (row level mean)
0.01-dc
0.01-tn -6.080
9.135