Vidocs.com Khao Sat Kha Nang s FF09DD4D

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 -

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 -

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 -

- - i

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

- - ii

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.

- - iii

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).

- - iv

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

- - v

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

- - vi

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

- - vii

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

- - viii

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

- - ix

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

- - 1

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

- - - 2 -

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.

- - - 3 -

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

- - - 4 -

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.

- - - 5 -

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:

- - - 6 -

- 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

- - - 7 -

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

- - - 8 -

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.

- - - 9 -

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).

- - - 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.

- - - 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 _

- - - 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)

- - - 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)

- - 16

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

- - 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.

- - 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%)..

- - 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


C nh mu bng Lugol trong 1 pht

Ra nc, thm kh

Ty cn 96C khong 15 giy

Ra nc, thm kh


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.


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


Mi trng Clark Lubs

Pepton bt 7 g

Glucose 5 g

KH2PO4 5 g

Nc ct 1000 ml

pH = 6.9 + 0.2


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


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



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


Family error rate = 0,0500

Individual error rate = 0,0500

Critical value = 3,93


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



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


Family error rate = 0.0500

Individual error rate = 0.0500

Critical value = 3.93


0.01-dc

0.01-tn -6.080

9.135

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