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B Y T

TRNG I HC DC H NI

PHM NGUYN PHNG

NGHIN CU KH NNG TO
BACTERIOCIN CA VI KHUN
LACTOBACILLUS ACIDOPHILUS

KHA LUN TT NGHIP DC S

H NI 2013

B Y T
TRNG I HC DC H NI

PHM NGUYN PHNG

NGHIN CU KH NNG TO
BACTERIOCIN CA VI KHUN
LACTOBACILLUS ACIDOPHILUS
KHA LUN TT NGHIP DC S

Ngi hng dn:


DS. L Ngc Khnh
Ni thc hin:
B mn Cng nghip Dc

H NI 2013

LI CM N
Trc ht, em xin gi li cm n su sc ti DS. L Ngc Khnh, ngi
trc tip hng dn v to mi iu kin thun li em c th hon
thnh kha lun ny.
Em xin chn thnh cm n TS. m Thanh Xun ng gp nhiu
kin qu bu v tn tnh gip em thc hin ti.
Em cng xin cm n s quan tm, gip ca cc thy c gio, cc anh
ch k thut vin B mn Cng nghip Dc i vi em trong sut qu trnh
nghin cu v lm thc nghim ti b mn.
Cui cng, em xin by t lng bit n gia nh, thy c, bn b, nhng
ngi lun ng vin, gip em trong hc tp v trong cuc sng.
Em xin trn trng cm n!

H Ni, ngy 17 thng 5 nm 2013


Sinh vin
Phm Nguyn Phng

MC LC

Danh mc cc k hiu, cc ch vit tt


Danh mc cc bng
Danh mc cc hnh v, th
T VN

Chng 1. TNG QUAN

1.1. Khi qut v bacteriocin

1.1.1. Vi nt v lch s

1.1.2. Phn b

1.1.3. Danh php v phn loi

1.1.4. Tnh hnh nghin cu

1.2. Bacteriocin ca vi khun lactic

1.2.1. Vi nt v vi khun lactic sinh bacteriocin

1.2.2. Phn loi

1.2.3. Tnh cht

1.2.4. Sinh tng hp, ph tc dng v c ch tc dng

1.2.5. nh gi hot tnh bacteriocin

11

1.2.6. Chit xut v tinh ch

11

1.2.7. ng dng

13

Chng 2. NGUYN LIU, THIT B, NI DUNG V PHNG

16

PHP NGHIN CU
2.1. Nguyn vt liu, thit b

16

2.1.1. Chng vi khun

16

2.1.2. Ha cht

16

2.1.3. Mi trng

16

2.1.4. My mc, dng c

17

2.2. Ni dung nghin cu

18

2.2.1. S b xc nh bacteriocin trong dch ln men v sinh khi vi

18

khun L. acidophilus ATCC 4653


2.2.2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi vi khun

18

L. acidophilus ATCC 4653


2.3. Phng php nghin cu

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2.3.1. Phng php bo qun ging vi sinh vt

18

2.3.2. Phng php nhn ging L. acidophilus

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2.3.3. Phng php x l sinh khi L. acidophilus

20

2.3.4. Phng php nh gi hot tnh bacteriocin theo c ch khuch

20

tn qua ging thch


2.3.5. Phng php kho st nh hng ca pH n hot tnh

21

bacteriocin
2.3.6. Phng php kho st nh hng ca nhit n hot tnh

22

bacteriocin
2.3.7. Phng php kho st kh nng chit bacteriocin bng

22

(NH4)2SO4
Chng 3. THC NGHIM, KT QU V BN LUN

23

3.1. S b xc nh bacteriocin trong dch ln men v sinh khi vi

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khun L. acidophilus ATCC 4653


3.1.1. Xc nh bacteriocin trong dch ln men

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3.1.2. Xc nh bacteriocin trong sinh khi

26

3.2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi vi

28

khun L. acidophilus ATCC 4653


3.2.1. Nghin cu nh hng ca pH n hot tnh bacteriocin ca L.

28

acidophilus ATCC 4653


3.2.2. Nghin cu nh hng ca nhit n hot tnh bacteriocin ca

31

L. acidophilus ATCC 4653


3.2.3. Nghin cu kh nng chit bacteriocin ca L. acidophilus ATCC

34

4653 bng phng php kt ta (NH4)2SO4


KT LUN V KIN NGH
TI LIU THAM KHO
PH LC

39

DANH MC CC K HIU, CC CH VIT TT

ATCC (American Type Culture

Bo tng ging chun vi sinh vt Hoa

Collection)

BC (Bacterial Cellulose)

Cellulose vi khun

Dha

Dehydroalanine

Dhb

Dehydrobutyrine

FDA (U.S. Food and Drug

Cc qun l Thc phm v Dc

Administration)

phm Hoa K

GC

Guanine v Cytosine

GRAS (Generally Recognized As

c cng nhn chung l an ton

Safe)
kDa

kiloDalton

LAB (Lactic Acid Bacteria)

Vi khun lactic

Lan

Lanthionine

MeLan

MethylLanthionine

MRS

de Man, Rogosa, Sharpe

MRSA (Methicillin-Resistant

T cu vng khng methicillin

Staphylococcus aureus)
PMF (Proton Motive Force)

ng lc proton

SDS (Sodium Dodecyl sulfate)

Natri dodecyl sulfat

sp. (species)

Loi

subsp. (subspecies)

Phn loi

VRE (Vancomycin-Resistant

Cu khun rut khng vancomycin

Enterococci)
UV (Ultraviolet)

Tia cc tm

DANH MC CC BNG

STT

Tn bng

Trang

1.1

Phn loi bacteriocin ca LAB

2.1

Cc ha cht s dng trong ti

16

2.2

Cc my mc s dng trong ti

17

3.1

Kt qu xc nh bacteriocin trong dch ln men ca L.

24

acidophilus ATCC 4653


3.2

Kt qu xc nh bacteriocin trong sinh khi ca L.

26

acidophilus ATCC 4653


3.3

Hot tnh bacteriocin ca L. acidophilus ATCC 4653 cc

30

gi tr pH nghin cu trn vi khun kim nh B. subtilis


ATCC 6633
3.4

Hot tnh bacteriocin ca L. acidophilus ATCC 4653 sau

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khi c x l nhit trn vi khun kim nh B. subtilis


ATCC 6633
3.5

Hot tnh bacteriocin ca phn ta v phn dch thu c


khi b sung (NH4)2SO4 vo dch ln men ca L.
acidophilus ATCC 4653 vi vi khun kim nh l B.
subtilis ATCC 6633

35

DANH MC CC HNH V, TH

STT

Tn hnh

Trang

1.1

Biu thng k s lng cc cng b lin quan n

bacteriocin trn Pubmed trong giai on t 1950 2010


3.1

Vng c ch to bi dch ln men ca L. acidophilus ATCC

26

4653 trn vi khun kim nh B. subtilis ATCC 6633


3.2

Vng c ch to bi dch ln men ca L. acidophilus ATCC

26

4653 trn vi khun kim nh E. coli ATCC 25922


3.3

nh hng ca pH n hot tnh bacteriocin ca L.

31

acidophilus ATCC 4653


3.4

nh hng ca nhit n hot tnh bacteriocin ca L.

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acidophilus ATCC 4653


3.5

S thay i hot tnh bacteriocin ca phn ta v phn dch


theo nng (NH4)2SO4 s dng

36

T VN
Vic pht hin ra cc cht khng sinh nh du mt bc tin cch mng
ca loi ngi trong cuc chin chng li cc bnh nhim khun, cng ngh
khng sinh c nh gi l mt trong nhng thnh tu khoa hc ln nht ca
th k XX [52]. Tuy nhin, bc vo th k XXI, con ngi ang phi i mt
vi k nguyn hu khng sinh vi s thiu ht cc nhm khng sinh mi trong
khi cc chng vi khun khng thuc khng ngng gia tng [13]. Trc tnh
hnh , vic nghin cu v pht trin nhng hp cht mi c tc dng khng
khun gim s ph thuc vo khng sinh l ht sc cn thit. Bacteriocin
chnh l mt trong s [52].
Bacteriocin l nhng peptid hay protein c tc dng dit khun do vi
khun sinh ra [34]. Chng c pht hin t u th k XX nhng mi
c nghin cu rng ri trong mt vi thp nin tr li y khi nhu cu v
nhng hp cht khng khun nhm thay th khng sinh v cc cht bo qun
ha hc tr nn cp thit [26]. Vi c tnh an ton c FDA xc nhn, vi
khun lactic (LAB) l mt nhm vi khun sinh bacteriocin thu ht s quan
tm c bit ca cc nh khoa hc [52]. Nhiu cng trnh nghin cu
chng minh tc dng c ch vi khun c hi ca cc bacteriocin sinh ra bi
LAB [20]. Nisin mt bacteriocin ca Lactococcus lactis spp. c s
dng rng ri trn th gii nh mt cht bo qun ngun gc sinh hc an ton
v hiu qu [23]. Thnh cng ca nisin bc u cho thy tim nng ng
dng to ln ca bacteriocin t LAB [22].
Ti Vit Nam, bacteriocin ca LAB bt u c nghin cu trong
nhng nm gn y, tuy nhin s lng nghin cu cha nhiu v tp trung
ch yu trong nhm ngnh thc phm v th y. Vi mong mun gp phn
nh vo nghin cu bacteriocin ca LAB Vit Nam, ti Nghin cu

kh nng to bacteriocin ca vi khun Lactobacillus acidophilus c


thc hin vi cc mc tiu c th nh sau:
1. S b xc nh bacteriocin trong dch ln men v sinh khi vi khun L.
acidophilus ATCC 4653.
2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi L. acidophilus
ATCC 4653.

Chng 1. TNG QUAN

1.1. Khi qut v bacteriocin


1.1.1. Vi nt v lch s
Nhng bo co u tin lin quan ti bacteriocin c cho l c t cui
th k XIX khi cc nh khoa hc nghin cu v tnh i khng gia cc vi
khun (bacterial antagonism) [26]. Nm 1925, bacteriocin chnh thc c
Gratia tm ra trn mt chng Escherichia coli [74], v sau cht ny c bit
n vi tn gi colicin V [73] v nay l microcin V [28]. Nm 1953, thut
ng bacteriocin ln u c xut v nh ngha bi Jacob v cng s,
ch yu da trn cc c tnh ca colicin [34]. Nm 1976, Tagg v cng s
ch ra s khc bit gia colicin v cc bacteriocin sinh ra bi vi khun Gram
(+) [66], t nh ngha v bacteriocin c m rng v n ngy nay, cc
peptid v protein do vi khun sinh ra c tc dng dit khun u c gi l
bacteriocin [34].
1.1.2. Phn b
Bacteriocin l sn phm ph bin ca gii vi khun [34]. Chng c
tm thy nhiu nhm vi khun tht (Eubacteria) v c trn vi khun c
(Archaebacteria) [59]. n nay, c khong 200 bacteriocin c m t
[26]. Tuy nhin s lng bacteriocin trong thc t c c tnh ln hn th
rt nhiu. Theo Klaenhammer, 99% cc loi vi khun c th sinh ra t nht
mt bacteriocin [41].
1.1.3. Danh php v phn loi
a. Danh php
Hin nay cha c h thng danh php quc t cho bacteriocin. Tn ca
bacteriocin thng xut pht t tn chi hay tn loi vi sinh vt u tin c

pht hin sinh ra bacteriocin . V d nh colicin t E. coli, pyocin t


Pseudomonas pyocynea, megacin t Bacillus megaterium [25]
b. Phn loi
H thng phn loi quc t dnh cho bacteriocin cha c chnh thc
xy dng v hin vn ang l ch tranh lun trong gii khoa hc [26].
Riley v Wertz nghin cu bacteriocin theo 3 nhm: bacteriocin ca vi khun
Gram (-), bacteriocin ca vi khun Gram (+) v bacteriocin ca vi khun c
[59]. Chi tit hn, theo Desriac v cng s, bacteriocin c phn loi cn c
nhiu tiu ch, trong cc tiu ch c bn bao gm: h vi khun sinh
bacteriocin, trng lng phn t, trnh t chui acid amin v cu trc ca
on gen (gene cluster). Cc tc gi chia bacteriocin lm 2 nhm ln: nhm
th nht l cc bacteriocin bn cht protein (protein-bacteriocin), thng c
sinh ra bi vi khun Gram (-), tiu biu l cc vi khun ng rut
(Enterobacteriae); nhm th 2 l cc bacteriocin bn cht peptid (peptidbacteriocin), thng c sinh ra bi vi khun Gram (+), tiu biu l cc vi
khun lactic (LAB). Mi nhm li c chia thnh nhiu lp v phn lp [26]
(Ph lc 1).
1.1.4. Tnh hnh nghin cu
a. Th gii
D c tm ra t kh sm (1925) nhng thi gian u s lng cc
nghin cu v bacteriocin khng nhiu v hu ht tp trung vo cc
bacteriocin ca vi khun Gram (-). Cho n nhng nm 60 ca th k XX, mi
ch c 3 chi vi khun Gram (+) c i su nghin cu v kh nng sinh
bacteriocin l Bacillus, Listeria v Staphylococcus [26]. Tuy nhin, cng
trong giai on ny, nisin v subtilin 2 bacteriocin quan trng t vi khun
Gram (+) c pht hin. Roger v cng s cng b nghin cu u tin
v nisin vo nm 1928 [60], trong khi subtilin c Jansen v Hirschmann

pht hin vo nm 1944 t vi khun Bacillus subtilis [37]. n u nhng


nm 70, trnh t chui acid amin ca 2 bacteriocin ny mi c xc nh r
[29] [75].
K t nhng nm 80, s lng cc nghin cu v bacteriocin c s gia
tng ng k, bao gm c cc nghin cu trn colicin v trn cc bacteriocin
khc [26]. Tip , vic nisin c FDA cng nhn t tiu chun GRAS vo
nm 1988 [58] thc y s quan tm ca cc nh khoa hc i vi cc
bacteriocin ca LAB bi tim nng ng dng to ln trong cng ngh thc
phm, y t, th y... Kt qu tt yu l s bng n cc nghin cu v
bacteriocin ca LAB vo nhng thp nin cui th k XX v u th k XXI
[26] (Hnh 1.1).

Hnh 1.1: Biu thng k s lng cc cng b lin quan n bacteriocin


trn Pubmed trong giai on t 1950 2010 [26]
Trong 20 nm gn y, trn ton th gii c 706 bng sng ch lin quan
ti bacteriocin ca LAB c cp, trong s c 421 bng trong lnh vc
cng ngh bo qun thc phm v 124 bng v cht tr sinh (probiotic) cho
ng vt [26]. Cc kh nng ng dng khc ca bacteriocin nh ngn nga

nhim trng khu tr [64], lm cht bo v thc vt [33], lm cht thay th


khng sinh [38] cng c xut v nghin cu bc u.
Microcin v bacteriocin t vi khun bin (marine bacteria) l 2 nhm
bacteriocin mi c a ra gn y [26]. Microcin c ngun gc t vi khun
ng rut nh colicin [28] nhng khi lng phn t nh hn v phn ln
c bin i sau dch m, tng t nhm lantibiotic vi khun Gram (+)
[35]. Cc bacteriocin t vi khun bin n nay vn cha c nghin cu
nhiu tuy nhin c d bo l s pht trin mnh trong tng lai gn [26].
b. Vit Nam
Vit Nam, bacteriocin bt u c cc nh khoa hc quan tm nghin
cu trong nhng nm gn y, c bit l bacteriocin ca LAB [8]. Bn cnh
cc nghin cu l thuyt [1] [2] [8], mt s cng trnh bc u a
bacteriocin vo ng dng trong thc tin. Nm 2008, cc tc gi Nguyn
Thy Hng v Trn Th Tng An s dng mng mng BC hp ph dch
bacteriocin ca Lactococcus lactis bo qun tht ti s ch ti thiu, gip
tng thi gian bo qun t 1 ln 3 ngy [3]. Vic ng dng cc k thut tin
tin nh c nh t bo [3], ti t hp gen [6] [7] nhm mc tiu sn xut
bacteriocin t hiu qu cao cng ang c nghin cu.
1.2. Bacteriocin ca vi khun lactic
1.2.1. Vi nt v vi khun lactic sinh bacteriocin
Vi khun lactic (LAB) c nh ngha l mt nhm vi khun Gram (+),
gm cc trc khun v cu khun khng sinh bo t, khng a kh (nonaerobic) nhng chu oxy (aerotolerant), sinh ra acid lactic l sn phm chnh
khi ln men carbohydrat. LAB gm nhiu chi, trong tiu biu nht l cc
chi Lactobacillus, Leuconostoc, Pediococcus v Streptococcus [43].
Cc LAB u khng di ng, catalase m tnh, t l GC thp di 50%
[61]. LAB a m v chu nhit, nhit sinh trng ti u ty tng loi, dao

ng t 28 n 45oC. LAB c nhu cu dinh dng phc tp, khi ln men cn


cc vitamin, acid amin v peptid ngn. Ngun carbohydrat c th l glucose,
fructose, lactose, maltose, sucrose; mt s chng vi khun c th s dng tinh
bt nh Lactobacillus amylophilus v L. amylovorus [5].
LAB phn b rng ri trong nhiu h sinh thi, thng c tm thy
trong mt s loi thc phm (cc sn phm t sa, tht v rau qu ln men,
bt nho chua), trn thc vt, trong ng tiu ha, h hp v sinh dc ca
ngi v ng vt [61].
LAB c lch s ng dng lu i trong thc phm ln men v cc tc
ng c li v mt dinh dng, cm quan v tui th sn phm, nht l vi
cc sn phm d hng nh sa, tht, c, mt s loi rau qu [43] [69]. Trong y
dc, nhiu loi LAB c s dng lm probiotic ci thin v nng cao
sc khe vi nhiu tc dng tr liu ng ch nh ngn nga nhim khun
ng rut, iu tr tiu chy, tng cng p ng min dch, gim
cholesterol mu, gim nguy c mc ung th [61]
Tc dng khng khun l mt c im rt ng ch ca LAB. LAB
sinh ra nhiu loi cht c tc dng khng khun: acid lactic, acid acetic,
ethanol, acid bo, H2O2, diacetyl v c bit l bacteriocin. Mc d
bacteriocin c th c tm thy nhiu loi vi khun khc, bacteriocin sinh
ra bi LAB vn thu ht c s ch c bit ca cc nh khoa hc bi tnh
an ton v tc dng c ch nhiu vi khun gy bnh nguy him nh
Staphylococcus aureus, Listeria monocytogenes [20] Trong s khong 200
bacteriocin c m t cho n nay, bacteriocin t LAB chim ti 90%
[26].
1.2.2. Phn loi
Bacteriocin sinh ra bi LAB rt a dng, trong vic phn loi chng
cng tn ti nhiu quan im khc nhau. Cch phn loi ph bin nht do

Klaenhammer v cng s xut vo nm 1993 [26], chia bacteriocin ca vi


khun Gram (+) ni chung, ca LAB ni ring lm 3 lp [42]. c im ca 3
lp ny c tm tt trong bng 1.1. Mt s tc gi xut lp th 4 gm
cc bacteriocin phc tp trong cu trc c carbohydrat hoc lipid; tuy nhin
d liu v cc bacteriocin loi ny cn rt hn ch nn cn c nghin cu
thm [34].

Bng 1.1: Phn loi bacteriocin ca LAB [26] [34] [71]


Lp
I

II

III

c im
- Cn gi l cc lantibiotic
- Peptid nh (thng < 5 kDa)
- Bn nhit
- Bin i mnh sau dch m, dn ti s to thnh
cc acid amin him gp: Lan, MeLan, Dha, Dhb
- Chia lm 2 typ: typ A v typ B
- Peptid nh (thng < 10 kDa)
- Bn nhit
- t bin i sau dch m, khng cha Lan
- Chia lm 4 phn lp: IIa, IIb, IIc, IId
- Protein kch thc ln (thng > 30 kDa)
- Km bn nhit

V d
Nisin, lactocin S,
epidermin

Pediocin PA-1,
lactacin F,
enterocin AS48
Helveticin J,
millericin,
enterolysin

1.2.3. Tnh cht


Tr mt s t cc bacteriocin thuc lp III l cc protein, bacteriocin ca
LAB a phn l cc peptid [26]. Chng thng mang in (+), c tnh s
nc (hydrophobic) hoc lng thn (amphiphilic) nhng a phn l lng
thn, gm t 20 n 60 acid amin [50] [71]. Ging nh nhiu bacteriocin ca
vi khun Gram (+), chng c th tn ti di dng cc kt tp c khi lng
ln (30 300 kDa) [54] [66]. Dng kt tp lm gim hot tnh ca

bacteriocin, ng thi d gy nhm ln trong vic xc nh khi lng phn


t ca bacteriocin tinh khit. ph v cc kt tp ny, c th s dng cc
tc nhn phn tch nh ur, SDS, cc bin php nh siu lc, chit loi lipid
bng dung mi hu c [54].
n nh l mt tiu ch quan trng khi nghin cu v bacteriocin, n
lin quan ti kh nng sn xut v a bacteriocin vo ng dng trong thc
t. 3 yu t nh hng thng c nh gi l nhit , pH v enzym [54];
nh hng ca cc yu t ny vi mi bacteriocin c th rt khc nhau [66].
a s bacteriocin ca LAB kh bn nhit (tr lp III), chu pH acid tt hn
pH kim [66], d b bt hot bi cc enzym phn gii protein nh proteinase
K, pronase, pepsin, trypsin [54].
Ngoi mt s nt chung, cc bacteriocin ca LAB c nhng c im
ring rt a dng. Mt s tnh cht c bn ca cc bacteriocin tiu biu t
LAB c gii thiu trong Ph lc 2.
1.2.4. Sinh tng hp, ph tc dng v c ch tc dng
a. Sinh tng hp
Bacteriocin c tng hp ti ribosom di dng tin cht khng hot
ng, chng ch tr nn c hot tnh sau khi c bin i nh cc phn ng
enzym c hiu [51]. Theo Tagg v cng s, cc bacteriocin ca vi khun
Gram (+) c th tn ti di dng ngoi bo (extracellular form) hoc gn vi
t bo (cell-associated form), t l mi dng ph thuc vo c im mi
trng [66].
Mi trng sng ca vi khun cn c nh hng ln ti lng
bacteriocin sinh ra, trong cc yu t ch yu l thnh phn mi trng,
nhit v pH. Cc yu t ny c th nh hng trc tip n s sn sinh
bacteriocin hoc gin tip thng qua s sn xut sinh khi do bacteriocin
c coi l sn phm trao i cht bc 1 ca vi khun [20].

10

b. Ph tc dng
Ph tc dng ca cc bacteriocin nhn chung rt khc nhau (Ph lc 2).
Cn c ph tc dng, c th chia bacteriocin ca LAB lm 2 nhm. Nhm th
nht l cc bacteriocin ch c tc dng trn cc loi gn gi vi loi sinh ra
n, v d nh lactobin A sinh ra bi Lactobacillus amylovorus ch c ch mt
s Lactobacillus sp. gn gi. Nhm th 2, t gp hn, c ph tc dng rng, v
d nh nisin sinh ra bi mt s dng Lactococcus lactis subsp. lactis c kh
nng c ch nhiu vi khun Gram (+). Ni chung, cc bacteriocin ca LAB
thng t th hin hot tnh trn vi khun Gram (-) [54].
Bacteriocin c th c tc dng dit khun hoc km khun trn cc chng
vi khun nhy cm, mc tc dng khc nhau do nh hng bi nhiu yu
t nh nng , mc tinh sch bacteriocin, cc iu kin thc nghim
[17]
c. C ch tc dng
C ch tc dng ca bacteriocin rt a dng, mt bacteriocin c th c
nhiu hn mt c ch tn cng t bo ch [52]. Mt s c ch c
xut: thay i hot tnh enzym ni bo, c ch bo t ny mm, to l trn
mng t bo Nhn chung, lp v t bo c coi l ch tc dng chnh ca
cc bacteriocin t LAB. Bacteriocin ph v tnh ton vn ca lp v t bo vi
khun nhy cm, gy mt cc cht ni bo, tiu hao PMF, cui cng gy cht
t bo [54].
C ch tc dng c th ca bacteriocin tng lp l khc nhau. Cc
bacteriocin lp I (lantibiotic) c ch tng hp vch t bo v / hoc to l trn
mng t bo bng cch gn vo lipid II [52]. Cc bacteriocin lp II c cu trc
xon c v lng thn nn c th xen vo mng t bo ch, t to l, kh
cc v lm cht t bo [18]. C ch tc dng ca cc bacteriocin lp III ni
chung cha r [54]; mt s tc gi cho rng l cc lysin tiu khun hay tiu

11

khun t (bacteriolysin), tc ng trc tip ln vch t bo ch gy ph hy


t bo [18].
1.2.5. nh gi hot tnh bacteriocin
C nhiu phng php nh gi hot tnh bacteriocin, v c bn chng
u da trn vic nh gi tc dng i khng ca mu th i vi cc chng
vi sinh vt ch th [66]. Mt s v d: phng php spot-on-lawn, phng
php khuch tn qua ging thch [56], phng php o c [40] (Ph
lc 3). nh hng ca cc tc nhn c ch khc khng phi l bacteriocin nh
pH, H2O2 cn c loi tr bng phng php thch hp [66].
Gn y mt s phng php mi nh gi hot tnh bacteriocin
c xut. Nm 2001, Mugochi v cng s a ra phng php nh gi
hot tnh bacteriocin bng cch o nng ion K+ gii phng t vi khun ch
th [48]. Phng php ny c cho l c hiu qu snh ngang vi phng
php khuch tn qua ging thch [34].
1.2.6. Chit xut v tinh ch
Do tnh cht ca cc bacteriocin rt a dng nn khng c phng php
hay qui trnh chit tch chung [32]. Vn thng gp trong qu trnh chit
xut v tinh ch bacteriocin l s hao ht bacteriocin v s gim hot tnh c
ch vi sinh vt [66]. Mt qui trnh sn xut bacteriocin ch c coi l l tng
khi c th p dng vi qui m ln, cho hiu sut thu nhn bacteriocin trn
50% v tinh sch khong 90% [54].
a. Chit xut
Bacteriocin c th c chit xut bng nhiu cch, tiu biu trong s
l cc phng php: kt ta bng amoni sulfat, hp ph - gii hp ph v chit
bng dung mi hu c [56].
- Kt ta bng amoni sulfat

12

Do c bn cht protein, bacteriocin c th c kt ta bng mui do


hin tng salting out (tnh tan ca protein gim mnh nng mui cao)
[56]. Amoni sulfat thng c s dng nht [56], ngoi ra cng c tc gi s
dng diatomite calcium silicate [19]. Mui c thm t t vo mu (dch ni
loi sinh khi t bo) cho n khi t nng mong mun. Nng mui
cn thit kt ta cc bacteriocin khc nhau c th khc nhau, cn kho st
tm ra nng mui thch hp vi tng trng hp c th. Ta bacteriocin
c tch ring bng cch li tm, sau ho trong lng nh (va ) m
phosphat pH 7 (gi tr pH ca dung dch m c th khc nhau gia cc
nghin cu) hoc nc ct v trng. Thng loi mui bng bng phng
php thm tch [56].
- Hp ph - gii hp ph
Phng php ny c pht trin bi Yang v cng s da trn c tnh
ca mt s bacteriocin: bacteriocin c hp ph ln b mt t bo vi khun
sinh bacteriocin pH gn trung tnh v c gii phng tr li mi trng
sau khi x l pH thp (pH khong 1,5 2,0) [70]. u im ca phng php
ny l t ko theo cc protein tp so vi phng php kt ta bng amoni
sulfat [56].
- Chit bng dung mi hu c
Phng php ny cng xut pht t bn cht protein ca bacteriocin: khi
thm dung mi hu c vo mu, hng s in mi gim xung, tan ca
protein gim v to kt ta [4]. Ethanol v aceton l hai dung mi kh thng
dng c s dng chit bacteriocin. Phng php chit bng dung mi
hu c c ng dng thnh cng vi mt s bacteriocin nh lactococcin
B, pediocin PA-1, lacticin Q [56]
b. Tinh ch

13

Bacteriocin sau khi c chit xut thng cn ln nhiu tp. thu


c bacteriocin tinh khit cn tri qua cc bc tinh ch [54]. Cc k thut
tinh ch bacteriocin rt a dng (Ph lc 4), c th da trn kch thc (v d:
sc k lc gel, siu lc) hoc in tch ring ca bacteriocin (v d: sc k
trao i ion, in di) [66]. Qu trnh tinh ch thng tri qua nhiu bc,
kt hp nhiu k thut khc nhau [54]. Tuy nhin, mt s qui trnh n gin
nhm tinh ch bacteriocin qui m cng nghip c pht trin thnh
cng. Nm 1997, Surez v cng s xut qui trnh tinh ch nisin A bng
phng php sc k i lc min dch nh khng th n dng khng nisin A,
cho kt qu tt [63]. Nm 2004, Cheigh v cng s tinh ch c nisin Z
t mi trng nui cy Lactococcus lactis subsp.lactis A164 bng phng
php sc k trao i ion nn m rng, thu c nisin Z c tinh khit gp 31
ln vi hiu sut 90% [16].
1.2.7. ng dng
Vi tc dng khng khun, bacteriocin c ng dng trong 2 lnh vc
ln: cng ngh dc phm v cng ngh thc phm [59].
a. Trong cng ngh dc phm
Bacteriocin c nghin cu ng dng trong cng ngh dc phm theo
2 hng chnh: probiotic [27] v cht thay th khng sinh [59].
- Probiotic
Probiotic c nh ngha l cc vi sinh vt sng khi c s dng vi
lng ln s em li tc ng c li cho sc khe ca vt ch [55]. Tc
dng ca cc ch phm probiotic c th l trc tip hoc gin tip thng qua
s iu chnh khu h vi sinh vt ng rut [9]. Bacteriocin c cho l ng
vai tr quan trng trong hot ng ca cc probiotic, l mt trong cc tiu ch
la chn chng vi sinh vt lm probiotic [27].

14

c nhiu gi thit c a ra v c ch bacteriocin tham gia vo


hot ng ca probiotic. Mt l, bacteriocin gip probiotic xm nhp v
chim u th trong h vi sinh vt ng rut. Hai l, bacteriocin th hin tc
dng khng khun, trc tip c ch cc vi khun gy bnh. Ba l, bacteriocin
mang tn hiu kch thch h min dch ca vt ch. Hin nay, cc nh khoa
hc vn ang tm kim nhng bng chng khng nh chnh xc vai tr v
c ch tc dng ca bacteriocin trong hot ng ca probiotic, nht l cc
bng chng in vivo [27].
- Cht thay th khng sinh
S xut hin v gia tng cc chng vi khun gy bnh a khng thuc l
mt vn ln ca y hc hin nay. gii quyt vn ny, bn cnh vic
s dng hp l cc khng sinh hin c, mt yu cu cp thit c t ra l
nghin cu pht trin cc phng php iu tr mi thay th cho s dng
khng sinh. Bacteriocin l mt trong nhng gii php c xut v
nghin cu [59].
V mt l thuyt, bacteriocin c sinh ra trong t nhin rt a dng nn
kh nng tm ra nhng cht c tc dng khng li cc vi khun gy bnh l rt
ln. Mt khc, do bacteriocin thng c tc dng c hiu trn mt s loi vi
khun nht nh, chng c th c s dng nh nhng thuc thit k ring
cho tng tc nhn gy bnh, nh gim tn sut xut hin vi khun khng
thuc [59].
Cc nghin cu thc nghim cho nhng kt qu bc u kh quan.
y ch nu ra mt vi v d: Nisin, mersacidin, mutacin 1140, lacticin
3147 c bo co v tc dng trn cc vi khun gy bnh khng thuc
nh MRSA, VRE [52]. Nisin F tim vo chut nhim Staphylococcus aureus
cho thy c tc dng kim sot s pht trin ca S. aureus [14]. Lacticin 3147
th hin tc dng c ch Clostridium difficile trn m hnh ex vivo ca rut

15

kt [57]. Mt liu duy nht mutacin B-Ny266 tim mng bng vo chut
nhim S. aureus nhy cm vi methicillin cho t l t vong l 0%, trong khi t
l t vong nhm chut i chng (khng dng mutacin B-Ny266) l t 70
n 100% [47].
Mc d vn cn thm nhiu nghin cu c th a bacteriocin vo
ng dng trn thc tin lm sng nhng nhng kt qu trn cho thy tim
nng to ln ca bacteriocin trong vic iu tr cc bnh nhim khun [27].
Trong tng lai, s dng bacteriocin ha hn s l mt liu php an ton v
hiu qu thay th cc khng sinh truyn thng [59].
b. Trong cng ngh thc phm
Bacteriocin ca LAB c nghin cu ng dng lm cht bo qun
trong cng ngh thc phm bi c nhiu u im: c ch hiu qu nhiu vi
khun gy hng v gy bnh trong thc phm (in hnh l Listeria
monocytogenes) [34], thn thin vi mi trng v sc khe con ngi [54].
Chng c s dng di 2 dng: trc tip hoc gin tip [20]. Dng trc tip
l cc bacteriocin c tinh ch, hin nay mi ch c nisin v pediocin PA1 c dng rng ri lm cht bo qun thc phm vi cc tn thng mi
nh Nisaplin, ALTA 2341 [20] [52] [54] Dng gin tip l cc LAB c
dng lm ging khi ng (starter culture) hoc b sung vo cc thc phm
ln men, bacteriocin c chng sinh ra s pht huy tc dng khng khun,
gip tng tui th v tnh an ton cho sn phm [20]. tng hiu qu khng
khun, bacteriocin c th c s dng kt hp vi cc phng php khc
nh to mi trng acid, b sung NaCl[34]

16

Chng 2. NGUYN LIU, THIT B, NI DUNG V PHNG


PHP NGHIN CU

2.1. Nguyn vt liu, thit b


2.1.1. Chng vi khun
a. Vi khun th nghim
Chng vi khun Lactobacillus acidophilus ATCC 4653
b. Vi khun kim nh (ch th)
- Chng vi khun Bacillus subtilis ATCC 6633
- Chng vi khun Escherichia coli ATCC 25922
2.1.2. Ha cht
Bng 2.1: Cc ha cht s dng trong ti
Ha cht

Xut x

Ha cht

Xut x

Acid lactic

Trung Quc

MgSO4.7H2O

Trung Quc

Amoni citrat

Trung Quc

MnSO4.H2O

Trung Quc

Cao nm men

NaCl

Trung Quc

Cao tht

NaOH

Trung Quc

Ct thy tinh

Trung Quc

Natri acetat

Trung Quc

Ch th mu

Trung Quc

(NH4)2SO4

Trung Quc

Glucose

Trung Quc

Pepton

HCl 1N

Trung Quc

Thch

Vit Nam

K2HPO4

Trung Quc

Xanh methylen

Trung Quc

KH2PO4

Trung Quc

2.1.3. Mi trng
a. Cng thc mi trng MRS lng

17

Pepton

: 1,0 g

Amoni citrat

: 0,2 g

Cao tht

: 1,0 g

Natri acetat

: 0,5 g

Cao nm men

: 0,5 g

MgSO4.7H2O

: 0,02 g

Glucose

: 2,0 g

MnSO4.H2O

: 0,004 g

K2HPO4

: 0,2 g

Nc my va

: 100 ml

b. Cng thc mi trng thch thng


Pepton

: 1,0 g

Cao tht

: 0,5 g

NaCl

: 0,5 g

Thch

: 2,3 g

Nc my va : 100 ml
2.1.4. My mc, dng c
a. My mc
Bng 2.2: Cc my mc s dng trong ti
Tn my

Xut x

Tn my

Xut x
c (Rotofix)

Bp cch thy

Trung Quc

My li tm

Cn k thut

c (Sartorius)

My li tm cao c (Universal)
tc

Cn phn tch

c (Sartorius)

L vi sng

Hn Quc (Daewoo) T m

My o vng v

Ty Ban Nha

khun

(IUL)

My khuy t

Hn Quc (Wisd) T cy v trng

Nht (Sanyo)

My lc Vortex

c (IKA)

T lnh

Hn Quc (LG)

Knh hin vi

M (Labomed)

T sy

c (Memmert)

b. Dng c

Ni hp tit trng Nht (ALP)


T m CO2

c (Memmert)
Nht (Sanyo)

18

Bnh nh mc 50, 100, 1000 ml

ng nghim

Bnh nn 100, 250, 500, 1000 ml

Pipet chia vch

Chy ci s

Pipet Pasteur

Cc c m

Pipet tip (u cn)

n cn

Pipetman (Pipet Eppendorf)

a petri ng knh 9 cm

Que cy u trn

ng ong

Tup Eppendorf 1,5 ml

2.2. Ni dung nghin cu


2.2.1. S b xc nh bacteriocin trong dch ln men v sinh khi vi khun
L. acidophilus ATCC 4653
- Xc nh bacteriocin trong dch ln men.
- Xc nh bacteriocin trong sinh khi.
2.2.2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi vi khun L.
acidophilus ATCC 4653
- nh hng ca pH n hot tnh bacteriocin.
- nh hng ca nhit n hot tnh bacteriocin.
- Kh nng chit bacteriocin bng phng php kt ta (NH4)2SO4.
2.3. Phng php nghin cu
2.3.1. Phng php bo qun ging vi sinh vt
Cc chng vi khun s dng trong ti, bao gm chng vi khun th
nghim L. acidophilus, cc chng vi khun kim nh B. subtilis v E. coli
c bo qun bng phng php cy truyn nh k ln mi trng mi. Cc
thao tc cy truyn c thc hin trong t cy vi sinh. T cy c tit trng
trc khi s dng bng cch lau sch bng cn 70o v bt n UV trong 20
pht.
a. Phng php cy truyn L. acidophilus t mi trng dch th

19

Mi trng MRS lng c pha ch theo cng thc mc 2.1.3 a, phn


phi vo cc ng nghim, mi ng 9 ml mi trng, nt kn v tit trng
trong ni hp 115oC trong 20 pht, ngui. Trn u ng ging lng ca
L. acidophilus bng my lc Vortex c hn dch t bo ng nht. Dng
pipet Eppendorf ht 1 ml hn dch t bo t ng ging cy sang ng mi
trng mi. Nui trong t m CO2 5% 37oC trong 24 gi.
Sau thi gian , loi b cc ng khng t yu cu nu c (vi sinh vt
khng mc, mc km, nghi ng b nhim). Cc ng ging c gi k, bo
qun trong t lnh nhit khong 10oC (ngn mt). Vic cy truyn c
thc hin 1 2 tun/ln.
b. Phng php cy truyn B. subtilis v E. coli t mi trng thch
Mi trng thch thng c pha ch theo cng thc mc 2.1.3 b,
phn phi vo cc ng nghim, mi ng 10 ml mi trng, nt kn v tit
trng trong ni hp 121oC trong 20 pht. Sau khi tit trng, t cc ng mi
trng nm nghing khong 15 20o trn mt bn sch thch ng li.
Dng que cy v trng ly mt vng vi sinh vt t ng ging c cy sang
ng mi trng mi, lt que cy trn b mt thch theo hnh zigzag. C B.
subtilis v E. coli u c nui trong t m 37oC trong 24 gi.
Sau thi gian , loi b cc ng khng t yu cu nu c (vi sinh vt
khng mc, mc km, nghi ng b nhim). Cc ng ging c gi k, bo
qun trong t lnh nhit khong 10oC (ngn mt). Vic cy truyn c
thc hin 1 2 thng/ln.
2.3.2. Phng php nhn ging L. acidophilus
Phn phi mi trng MRS lng vo cc bnh nn, mi bnh 100 ml mi
trng, nt kn v tit trng trong ni hp 115oC trong 20 pht, ngui.
Trong t cy v trng, tin hnh cy 1 ng ging lng 10 ml ca L.

20

acidophilus vo mi bnh nn. Nui trong t m CO2 5% 37oC trong 18 gi


[30].
2.3.3. Phng php x l sinh khi L. acidophilus
Sinh khi L. acidophilus c thu nhn t dch nui cy vi khun sau 18
gi bng cch li tm (4.000 vng/pht, 20 pht [10]). Tch ring phn dch
ni, thu c sinh khi y ng li tm. X l sinh khi thu c theo cc
bc sau:
- Ra sinh khi bng nc ct (1 ln).
- Trn sinh khi t vi ct thy tinh hp tit trng theo t l 1:1,
nghin hn hp to thnh bng chy ci s ph v t bo vi khun.
- B sung NaCl 0,9% vo hn hp thu c theo t l 1:1 (1 ml NaCl
0,9% cho 1 g hn hp), khuy u to thnh hn dch.
- Li tm hn dch thu c bng my li tm cao tc 18.000 vng/pht
trong 20 pht thu dch ni (dch ph t bo).
Kim tra mc ph v t bo ca phng php nghin bng cch lm
tiu bn sinh khi trc v sau khi x l, nhum n vi thuc nhum xanh
methylen ri soi qua knh hin vi vi vt knh du (100x).
2.3.4. Phng php nh gi hot tnh bacteriocin theo c ch khuch tn
qua ging thch
Nguyn tc:
Bacteriocin t mu th trong cc ging khuch tn vo mi trng thch
cy vi sinh vt kim nh v c ch s pht trin ca cc vi sinh vt nhy cm
to ra vng c ch [65]. o ng knh vng c ch to thnh nu c v nh
gi.
Tin hnh:

21

- Lm hn dch vi khun kim nh bng cch thm 5 ml nc ct v


trng vo ng ging c ca vi khun kim nh, g nh thnh ng
nghim vo lng bn tay vi khun trn thch phn tn vo nc.
- Mi trng thch thng c pha ch theo cng thc mc 2.1.3 b,
phn phi vo cc bnh nn, nt kn v tit trng trong ni hp 121oC
trong 20 pht.
- Khi mi trng thch thng cn m (khong 45 50oC), hn dch
vi khun kim nh vo bnh nn ng mi trng theo t l 5 ml hn
dch : 100 ml mi trng, lc trn nh vi khun phn tn u trong
mi trng ri ra cc a petri ng knh 9 cm v trng (16 ml mi
trng/a), ngui cho thch ng li.
- Khi thch ng, dng thanh kim loi v trng (ng knh 7,8mm)
khoan to cc ging trn mi trng thch trong a petri.
- Dng pipet Eppendorf nh 50 l mu th vo mi ging thch.
- Cc a petri nh mu th c trong t m 37oC trong 24 gi
cho vi khun kim nh pht trin.
- Sau thi gian , o ng knh vng c ch (nu c) bng my o
ng knh vng v khun v nh gi.
Mt th nghim c tin hnh vi 3 a th song song, ng knh
vng c ch ca mi mu th l kt qu trung bnh o c t 3 a th. Theo
Sifour v cng s, vng c ch nh nht c ngha l 1 mm [62]. Do ng
knh ging thch l 7,8mm, ng knh vng c ch ti thiu c xc nh l
8,8mm.
2.3.5. Phng php kho st nh hng ca pH n hot tnh bacteriocin
Ly 10 ml dch bacteriocin cho mi mu th, xc nh pH bng ch th
mu, t chnh pH v cc gi tr pH nghin cu bng NaOH 1N hoc HCl
1N tit trng [44]. 2 gi [1] nhit phng [62]. nh gi hot tnh

22

ca dch bacteriocin chnh pH theo phng php nu mc 2.3.4, so


snh gia cc mu v rt ra nhn xt.
2.3.6.Phng php kho st nh hng ca nhit n hot tnh
bacteriocin
Ly 10 ml dch bacteriocin cho mi mu th, un trn bp cch thy
cc gi tr nhit nghin cu trong 30 pht [44]. ngui nhit phng.
nh gi hot tnh ca dch bacteriocin x l nhit theo phng php
nu mc 2.3.4, so snh vi mu chun i chiu l dch bacteriocin khng
x l nhit v rt ra nhn xt.
2.3.7. Phng php kho st kh nng chit bacteriocin bng (NH4)2SO4
Nguyn tc:
Bacteriocin c bn cht protein nn to kt ta khi nng (NH4)2SO4
ln [56]. Tch ring phn ta bacteriocin v phn dch, nh gi hot tnh
bacteriocin ca mi phn. Bacteriocin kt ta cng nhiu th hot tnh ca
phn ta cng ln, hot tnh ca phn dch cng nh.
Tin hnh:
- Lm lnh dch bacteriocin bng cch trong t lnh 4oC [1] trong
khong 1 gi.
- Thm dn (NH4)2SO4 theo cc nng mui nghin cu, c kt hp
khuy t [1].
- Li tm dch bacteriocin ha tan mui bng my li tm cao tc
10.000 vng/pht trong 20 pht phn ring phn ta v phn dch
[56].
- Ha tan phn ta trong m phosphat pH 7,0 [56] theo t l khong 1
ml m cho phn ta ca 30 ml dch bacteriocin ban u [46].
- nh gi hot tnh bacteriocin ca phn ta v phn dch theo phng
php nu mc 2.3.4, so snh gia cc mu v rt ra nhn xt.

23

Chng 3. THC NGHIM, KT QU V BN LUN


3.1. S b xc nh bacteriocin trong dch ln men v sinh khi vi khun
L. acidophilus ATCC 4653
L. acidophilus l vi khun Gram (+), do v mt l thuyt bacteriocin
ca n c th tn ti di 2 dng: dng gn vi t bo v dng ngoi bo [66].
V vy, ni dung u tin ca ti l tin hnh xc nh s b bacteriocin
trong dch ln men v sinh khi ca chng L. acidophilus th nghim.
Mc ch:
- Xc nh v tr tn ti ca bacteriocin sinh ra bi vi khun L.
acidophilus ATCC 4653.
- Nhn nh s b v hot tnh ca bacteriocin sinh ra bi L. acidophilus
ATCC 4653 trn cc vi khun kim nh c s dng.
Cch tin hnh:
Nui cy L. acidophilus ATCC 4653 theo mc 2.3.2. Li tm dch nui
cy 4.000 vng/pht trong 20 pht tch ring phn dch ni (dch ln
men) v sinh khi [10]. X l sinh khi thu c theo mc 2.3.3 thu dch
ph t bo. Tin hnh xc nh bacteriocin trong dch ln men v dch ph t
bo theo phng php nu mc 2.3.4; trong mu trng i vi dch
ln men l dung dch acid lactic c pH 4 (tng ng pH dch ln men), i
vi dch ph t bo l dung dch NaCl 0,9%. Cc vi khun kim nh c la
chn l B. subtilis ATCC 6633, i din cho vi khun Gram (+) v E. coli
ATCC 25922, i din cho vi khun Gram (-) [44]. Th nghim c lp li 3
ln trn 3 m nui cy c lp ca L. acidophilus ATCC 4653.
Kt qu:
3.1.1. Xc nh bacteriocin trong dch ln men

24

Kt qu xc nh bacteriocin trong dch ln men ca L. acidophilus


ATCC 4653 c th hin trong bng 3.1.
Bng 3.1: Kt qu xc nh bacteriocin trong dch ln men ca L. acidophilus
ATCC 4653
ng knh vng c ch (mm)
Ln th
nghim

Trn B. subtilis ATCC 6633


Dch ln men

Acid lactic
pH 4

Trn E. coli ATCC 25922


Dch ln men

Acid lactic
pH 4

Ln 1

16,0

(-)

18,7

(-)

Ln 2

16,2

(-)

19,8

(-)

Ln 3

17,7

(-)

22,5

(-)

Trung bnh

16,6

20,3

Ghi ch: (-): khng xut hin vng c ch

Nhn xt v bn lun:
Kt qu bng 3.1 cho thy: trong c 3 ln th nghim, dch ln men ca
L. acidophilus ATCC 4653 u to c vng c ch trn cc chng vi khun
kim nh B. subtilis ATCC 6633 v E. coli ATCC 25922. Mu trng l dung
dch acid lactic c pH 4 (tng ng pH dch ln men) khng to vng c
ch trong tt c cc th nghim tng ng, iu ny cho php loi tr tc dng
ca acid hu c c trong dch ln men. T , c th rt ra 2 kt lun sau:
Mt l, bacteriocin c trong dch ln men ca L. acidophilus ATCC
4653. Kt qu ny ph hp vi nhiu nghin cu c v bacteriocin ca L.
acidophilus [24] [39] [49] [67] [72]. Trong cc nghin cu ny, cc tc gi
u s dng dch ln men ca vi khun thu nhn bacteriocin.

25

Hai l, bacteriocin trong dch ln men ca L. acidophilus ATCC 4653 c


tc dng c ch i vi c B. subtilis v E. coli, tc l th hin hot tnh trn
c vi khun Gram (+) v Gram (-). y l mt c im khng thng gp
bacteriocin ca LAB. Theo Parada v cng s, bacteriocin ca LAB thng
c tc dng trn vi khun Gram (+) nhng t tc dng trn vi khun Gram (-)
[54]. Nm 2010, Ahmed v cng s tin hnh thng k 16 bacteriocin
c tm thy L. acidophilus (Ph lc 5), trong 13 bacteriocin c ph tc
dng gii hn trn vi khun Gram (+). Trong 3 bacteriocin cn li c tc dng
c ch vi khun Gram (-), ng ch nht l acidocin AA11 vi ph tc dng
trn c B. subtilis v E. coli, ging vi bacteriocin ca L. acidophilus ATCC
4653 [11].
Tin hnh so snh vng c ch to bi bacteriocin trong dch ln men
ca L. acidophilus ATCC 4653 trn B. subtilis v trn E. coli cho thy: V
mt nh tnh, vng c ch ca bacteriocin trn B. subtilis trong v r hn so
vi trn E. coli (Hnh 3.1 v 3.2). V mt nh lng, trong c 3 ln th
nghim, ng knh vng c ch khi dng E. coli lm vi khun kim nh u
ln hn so vi khi dng B. subtilis. C th, ng knh vng c ch trn E.
coli trong 3 ln th nghim ln lt l 18,7; 19,8 v 22,5mm; trong khi ng
knh vng c ch trn B. subtilis tng ng ch l 16,0; 16,2 v 17,7mm.
ng knh vng c ch trung bnh ca 3 ln th nghim trn E. coli l
20,3mm, ln hn 3,7mm so vi gi tr tng ng trn B. subtilis (16,6mm).
Trong mt nghin cu ca Abo-Amer nm 2007 v acidocin AA11 t L.
acidophilus AA11, tc gi khng cng b s liu c th v ng knh vng
c ch to bi bacteriocin trn cc vi khun kim nh m ch cho bit ng
knh vng c ch trn B. subtilis v E. coli u ln hn 10 mm [10]. Kt qu
thc nghim thu c trong ti cng ph hp vi cng b ny.

26

Hnh 3.1: (1) Vng c ch to bi

Hnh 3.2: (1) Vng c ch to bi

dch ln men ca L. acidophilus

dch ln men ca L. acidophilus

ATCC 4653 trn vi khun kim nh

ATCC 4653 trn vi khun kim nh

B. subtilis ATCC 6633

E. coli ATCC 25922

3.1.2. Xc nh bacteriocin trong sinh khi


Kt qu xc nh bacteriocin trong sinh khi ca L. acidophilus ATCC
4653 c th hin trong bng 3.2.
Bng 3.2: Kt qu xc nh bacteriocin trong sinh khi ca L. acidophilus
ATCC 4653
ng knh vng c ch (mm)
Ln th

Trn B. subtilis ATCC 6633

Trn E. coli ATCC 25922

nghim

Dch ph t

Dch ph t

bo

NaCl 0,9%

bo

NaCl 0,9%

Ln 1

(-)

(-)

(-)

(-)

Ln 2

(-)

(-)

(-)

(-)

Ln 3

(-)

(-)

(-)

(-)

Ghi ch: (-): khng xut hin vng c ch

27

Nhn xt v bn lun:
Kt qu bng 3.2 cho thy: trong c 3 ln th nghim, dch ph t bo
ca L. acidophilus ATCC 4653 u khng to vng c ch trn cc chng vi
khun kim nh B. subtilis ATCC 6633 v E. coli ATCC 25922. Mt khc,
theo kt qu mc 3.1.1, bacteriocin ca L. acidophilus ATCC 4653 c hot
tnh trn 2 chng vi khun kim nh ny. V vy, c th s b kt lun
bacteriocin khng c trong dch ni bo ca L. acidophilus ATCC 4653.
Theo Tagg v cng s, c im mi trng l yu t quyt nh t l cc
dng tn ti ca bacteriocin vi khun Gram (+) [66]. Trong ti ny, L.
acidophilus ATCC 4653 c nui cy trong mi trng MRS lng 37oC
trong 18 gi v khng kim sot pH mi trng trong qu trnh nui cy.
Khng loi tr kh nng bacteriocin ca L. acidophilus ATCC 4653 c th
tn ti dng ni bo nu thay i iu kin nui cy vi khun.
Phng php x l sinh khi c thc hin trong ti l nghin hn
hp sinh khi t ca L. acidophilus trn vi ct thy tinh bng chy ci s
(mc 2.3.3). y l mt phng php ph t bo thch hp vi qui m phng
th nghim [4]. u im ca phng php ny l n gin, d thc hin,
khng yu cu ha cht, thit b t tin, khng c nguy c gy nh hng ti
hot tnh bacteriocin nh mt s phng php khc. Tuy nhin, vi phng
php nghin, t bo vi khun mi c ph v v mt c hc. Khng loi tr
kh nng phng php ny c th cha thch hp gii phng bacteriocin t
sinh khi.
Trong phm vi ti ny, do cha c iu kin th nghim cc iu kin
nui cy v cc phng php x l sinh khi khc, vic xc nh bacteriocin
trong sinh khi ca L. acidophilus ATCC 4653 tm dng li vic kt lun
bacteriocin khng c trong dch ni bo ca vi khun.

28

Tm li, kt qu mc 3.1 cho thy, trong cc iu kin thc nghim ca


ti, bacteriocin ca L. acidophilus ATCC 4653 khng tn ti dng ni
bo m hon ton dng ngoi bo. V vy, dch ln men ca L. acidophilus
ATCC 4653 c coi l dch bacteriocin th, c s dng thc hin cc
th nghim tip theo nghin cu v tnh cht ca bacteriocin. Bacteriocin ny
c hot tnh trn c vi khun Gram (+) (B. subtilis ATCC 6633) v vi khun
Gram (-) (E. coli ATCC 25922), trong vng c ch to bi bacteriocin trn
B. subtilis c ng knh nh hn nhng li trong v r hn so vi trn E.
coli. Do , thun tin cho vic quan st v n gin ha qu trnh th
nghim, B. subtilis c la chn lm vi khun kim nh nh gi hot
tnh bacteriocin trong cc th nghim tip theo.
3.2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi vi khun L.
acidophilus ATCC 4653
Cc bacteriocin khc nhau c th c nhng c im ring rt a dng
[32], dn n phng php sn xut v phm vi ng dng khc nhau. V vy,
ni dung tip theo ca ti l tin hnh nghin cu mt s tnh cht c bn
ca bacteriocin sinh ra bi chng L. acidophilus th nghim, bao gm nh
hng ca pH, nhit n hot tnh bacteriocin v kh nng chit
bacteriocin bng phng php kt ta (NH4)2SO4.
3.2.1. Nghin cu nh hng ca pH n hot tnh bacteriocin ca L.
acidophilus ATCC 4653
nh hng ca pH l mt ni dung quan trng khi nghin cu v n
nh ca bacteriocin [54] [66]. Khong pH hot ng ca cc bacteriocin khc
nhau c th rt khc nhau, c th rt hp nh boticin P (khong pH hot
ng 6,5 7,5), c th rt rng nh butyricin 7423 (khong pH hot ng 2
12) [66]. Xc nh khong pH hot ng ca bacteriocin cho php s dng

29

bacteriocin c hiu qu. Do , bacteriocin ca chng L. acidophilus th


nghim c nghin cu v nh hng ca pH n hot tnh bacteriocin.
Mc ch:
Xc nh s b khong pH hot ng ca bacteriocin sinh ra bi L.
acidophilus ATCC 4653.
Cch tin hnh:
Nui cy L. acidophilus ATCC 4653 theo mc 2.3.2. Li tm dch nui
cy 4.000 vng/pht trong 20 pht loi sinh khi, thu dch ni l dch
bacteriocin th (dch ln men) [10]. Kho st nh hng ca pH n hot tnh
bacteriocin theo phng php nu mc 2.3.5, trong cc gi tr pH
c nghin cu l 2, 4, 6, 8. Vi khun kim nh c s dng l B. subtilis
ATCC 6633. Th nghim c lp li 3 ln trn 3 m nui cy c lp ca L.
acidophilus ATCC 4653.
Kt qu:
Kt qu nh gi hot tnh bacteriocin ca L. acidophilus ATCC 4653
cc gi tr pH nghin cu c th hin trong bng 3.3.
Nhn xt v bn lun:
Kt qu bng 3.3 cho thy trong cc gi tr pH nghin cu, bacteriocin
ca L. acidophilus ATCC 4653 th hin hot tnh tt nht pH 2, vi ng
knh vng c ch trung bnh qua 3 ln th nghim l 18,8mm. pH cng tng
th ng knh vng c ch cng nh, hot tnh bacteriocin cng yu. Hot
tnh bacteriocin gim t ngt pH 6 (gn trung tnh), vi gim ng
knh trung bnh so vi pH 2 l 37,8%, cao hn hn so vi gim 9,0% pH
4. n gi tr pH 8 (mi trng kim nh), ng knh vng c ch trung
bnh ch cn 9,7mm, gim 48,4% so vi pH 2 v ch ln hn 0,9mm so vi
ng knh vng c ch ti thiu c ngha (8,8mm) [62]. iu ny chng t

30

bacteriocin ca L. acidophilus ATCC 4653 km hot ng trong mi trng


kim.
Bng 3.3: Hot tnh bacteriocin ca L. acidophilus ATCC 4653 cc gi tr
pH nghin cu trn vi khun kim nh B. subtilis ATCC 6633
ng knh vng c ch (mm)

Ln th
nghim

pH 2

pH 4

pH 6

pH 8

Ln 1

17,6

16,5

11,7

9,3

Ln 2

18,5

16,8

11,2

9,7

Ln 3

20,2

18,1

12,1

10,2

Trung bnh

18,8

17,1

11,7

9,7

(%)

9,0

37,8

48,4

Ghi ch: : gim ng knh trung bnh pH x so vi pH 2


(%) =
vi d(2): ng knh vng c ch trung bnh pH 2
d(x): ng knh vng c ch trung bnh pH x

Nh vy, t nhng kt qu trn, c th nhn nh bacteriocin ca L.


acidophilus ATCC 4653 ch yu hot ng pH acid v pH cng nh th
hot tnh bacteriocin cng ln. y l c im ca a s bacteriocin t LAB
m in hnh l nisin [21]. Nisin hu nh ch pht huy tc dng trong mi
trng acid (hot tnh cao nht pH 2, b bt hot pH trn 8) nn c s
dng lm cht bo qun trong cc thc phm c tnh acid [31]. S tng hot
tnh ca bacteriocin khi pH gim c Jack v cng s l gii theo mt s gi
thit nh sau [36]:
- Khi pH gim, bacteriocin t to kt tp hn, do c nhiu phn t
tham gia tn cng tiu dit t bo ch.

31

- Khi pH gim, bacteriocin t gn vi t bo hn, a s bacteriocin


dng ngoi bo pht huy tc dng dit khun.
- pH gim to iu kin cho bacteriocin d thm qua vch t bo ch.
- Phn ng tng tc gia bacteriocin vi t bo ch b c ch trong mi

ng knh vng c ch trung bnh (mm)

trng pH cao.

20

18.8
17.1

18
16
14

11.7

12

9.7

10
8
6
4
2
0
2

pH

Hnh 3.3: nh hng ca pH n hot tnh bacteriocin ca L.


acidophilus ATCC 4653
3.2.2. Nghin cu nh hng ca nhit n hot tnh bacteriocin ca L.
acidophilus ATCC 4653
Cng vi nh hng ca pH, bn nhit cng l mt ni dung quan
trng khi nghin cu v bacteriocin [54], c ngha c v l thuyt v thc
tin. V l thuyt, l mt trong nhng tiu ch c bn trong vic m t v
phn loi bacteriocin ca LAB [53]. V thc tin, n lin quan n kh nng
ng dng ca bacteriocin, nht l trong cng ngh thc phm khi nhiu qui

32

trnh ch bin thc phm yu cu phi s dng nhit [62]. V vy, bacteriocin
ca chng L. acidophilus th nghim c nghin cu v nh hng ca
nhit n hot tnh bacteriocin.
Mc ch:
nh gi bn nhit ca bacteriocin sinh ra bi L. acidophilus ATCC
4653.
Cch tin hnh:
Thu dch bacteriocin th (dch ln men) tng t mc 3.2.1. Kho st
nh hng ca nhit n hot tnh bacteriocin theo phng php nu
mc 2.3.6, trong cc gi tr nhit c nghin cu l 70oC, 80oC, 90oC
v 100oC. Vi khun kim nh c s dng l B. subtilis ATCC 6633. Th
nghim c lp li 3 ln trn 3 m nui cy c lp ca L. acidophilus
ATCC 4653.
Kt qu:
Kt qu nh gi hot tnh bacteriocin ca L. acidophilus ATCC 4653
sau khi c x l nhit c th hin trong bng 3.4.
Bng 3.4: Hot tnh bacteriocin ca L. acidophilus ATCC 4653 sau khi c
x l nhit trn vi khun kim nh B. subtilis ATCC 6633
Ln th
nghim

ng knh vng c ch (mm)


Khng x
l nhit

70oC

80oC

90oC

100oC

Ln 1

18,1

18,7

19,4

19,4

19,3

Ln 2

18,7

18,4

18,0

18,3

18,3

Ln 3

16,3

16,7

16,2

16,1

16,3

Trung bnh

17,7

17,9

17,9

17,9

18,0

ng knh vng c ch trung bnh (mm)

33

20
17.7

18

17.9

17.9

17.9

18.0

16
14
12
10
8
6
4

2
0

Khngx
xl un 70oC
70oC/ / un
un80oC
80oC// un
un 90oC
90oC// un
un100oC
100oC//
Khng
lnhit
nhit
30
30 pht
30 pht
pht
30 pht
pht
30 pht
30
Hnh 3.4: nh hng ca nhit n hot tnh bacteriocin ca L.
acidophilus ATCC 4653

Nhn xt v bn lun:
Kt qu bng 3.4 cho thy sau khi x l nhit dch ln men cc gi tr
nhit nghin cu, hot tnh bacteriocin ca L. acidophilus ATCC 4653 hu
nh khng thay i. C th, ng knh vng c ch trung bnh qua 3 ln th
nghim sau khi un dch ln men ca chng L. acidophilus th nghim cc
nhit 70oC, 80oC, 90oC, 100oC trong 30 pht u xp x nhau v xp x vi
ng knh vng c ch trung bnh khi dch ln men khng c x l nhit
(17,7mm). T , c th nhn nh bacteriocin ca L. acidophilus c tnh bn
nhit.
Trong cc bacteriocin ca LAB, tnh bn nhit l c tnh c bn ca lp
I v lp II bao gm cc bacteriocin c cu trc l cc peptid phn t lng

34

nh (mc 1.2.2). Theo De Vuyst v Vandamme, c tnh bn nhit ca cc


bacteriocin ny l do chng c cu to n gin (khng c cu trc bc 3)
hoc c cu trc hnh cu c n nh nh cc lin kt ng ha tr [21].
Oscariz v Pisabarro li nhn mnh vai tr ca cc cu ni sulfit v disulfit
ni phn t trong vic n nh cu trc, m bo tnh bn nhit ca
bacteriocin. Cc tc gi cn a ra gi thuyt rng s cu ni cng tng th
bacteriocin cng bn nhit [53].
Trong s cc bacteriocin c tm ra L. acidophilus, a s c tnh
bn nhit ging vi bacteriocin trong nghin cu, ch c mt s t c ghi
nhn l nhy cm vi nhit gm c bacteriocin ca L. acidophilus AC1 [45]
v acidophilucin A ca L. acidophilus LAPT 1060 [68].
Tm li, ging vi a s bacteriocin c tm ra L. acidophilus,
bacteriocin ca L. acidophilus ATCC 4653 c tnh bn nhit, vi nhit cao
nht c x l l 100oC trong 30 pht.
3.2.3. Nghin cu kh nng chit bacteriocin ca L. acidophilus ATCC
4653 bng phng php kt ta (NH4)2SO4
Kt ta bng (NH4)2SO4 l mt phng php thng c p dng
chit bacteriocin [20]. So vi cc phng php chit bacteriocin khc nh hp
ph - gii hp ph, chit bng dung mi hu c, phng php ny c u
im l n gin, d thc hin, ha cht r tin, khng c hi. Do ,
phng php kt ta bng (NH4)2SO4 c la chn bc u nghin cu
kh nng chit bacteriocin t dch ln men ca chng L. acidophilus th
nghim.
Mc ch:
Nhn nh s b v kh nng chit bacteriocin t dch ln men ca L.
acidophilus ATCC 4653 bng phng php kt ta (NH4)2SO4 theo cc nng
mui khc nhau.

35

Cch tin hnh:


Thu dch bacteriocin th (dch ln men) tng t mc 3.2.1. Kho st
kh nng chit bacteriocin bng (NH4)2SO4 theo phng php nu mc
2.3.7, trong cc nng (NH4)2SO4 c nghin cu l 20; 30; 40; 50; 60;
70 v 80% (khi lng/th tch). Vi khun kim nh c s dng l B.
subtilis ATCC 6633.
Kt qu:
Kt qu nh gi hot tnh bacteriocin ca phn ta v phn dch thu
c sau khi b sung (NH4)2SO4 vi cc nng khc nhau vo dch ln men
ca L. acidophilus ATCC 4653 c th hin trong bng 3.5.
Bng 3.5: Hot tnh bacteriocin ca phn ta v phn dch thu c khi b
sung (NH4)2SO4 vo dch ln men ca L. acidophilus ATCC 4653 vi vi
khun kim nh l B. subtilis ATCC 6633
ng knh vng c ch (mm)

Nng
(NH4)2SO4 (%)

Phn ta

20

Phn dch
Khng to ta

30

13,2

16,6

40

13,7

15,3

50

13,9

14,6

60

14,7

14,2

70

15,6

13,7

80

Mui khng tan ht, ln vi ta

Nhn xt v bn lun:
Kt qu bng 3.5 cho thy, trong dy nng (NH4)2SO4 nghin cu (t
20 n 80%), nng mui thp nht cho php tch c ta bacteriocin t

36

dch ln men ca L. acidophilus ATCC 4653 l 30% v cao nht l 70%.


nng (NH4)2SO4 20%, bacteriocin cha to kt ta; cn nng
(NH4)2SO4 80%, mui khng tan ht v ln vi phn ta bacteriocin.
Trong khong nng (NH4)2SO4 tch c ta bacteriocin (t 30% n
70%), nhn thy: khi tng nng (NH4)2SO4, hot tnh bacteriocin ca phn
ta tng dn, trong khi hot tnh bacteriocin ca phn dch gim dn. ng
knh vng c ch ca phn ta nng (NH4)2SO4 70% l 15,6mm, tng
2,4mm so vi nng 30% (13,2mm). Ngc li, ng knh vng c ch
ca phn dch nng (NH4)2SO4 70% l 13,7mm, gim 2,9mm so vi
nng 30% (16,6mm). T , c th kt lun lng bacteriocin kt ta tng
dn theo lng (NH4)2SO4 s dng.

ng knh vng c ch (mm)

18
16
14
12
10

Phn ta

Phn dch

6
4
2
0
30

40
50
60
Nng (NH4)2SO4 (%)

70

Hnh 3.5: S thay i hot tnh bacteriocin ca phn ta v phn dch theo
nng (NH4)2SO4 s dng

37

Trong ti ny, nng (NH4)2SO4 cao nht trc khi mui bo ha


c nghin cu l 70%. nng ny phn dch vn cn hot tnh
bacteriocin (to vng c ch c ng knh 13,7mm), chng t bacteriocin
ca L. acidophilus ATCC 4653 vn cha c chit ht. Cng nng ny,
ng knh vng c ch to bi phn ta l 15,6mm, ln hn 1,9mm so vi
vng c ch ca phn dch (13,7mm). Mt khc, phn ta c c c 30
ln so vi phn dch (mc 2.3.7). T , c th d on hiu sut chit
bacteriocin t dch ln men ca L. acidophilus ATCC 4653 bng phng
php kt ta (NH4)2SO4 khng cao.
Phng php kt ta bng (NH4)2SO4 c nhiu tc gi s dng
chit bacteriocin t LAB, hiu sut chit t c c th rt cao ln ti 95%
[46] nhng cng c khi kh thp ch gn 20% [12]. Trong ti ny, hiu sut
chit bacteriocin bng phng php kt ta (NH4)2SO4 cha c tnh ton c
th m mi ch a ra nhng nhn nh bc u. la chn phng php
chit bacteriocin thch hp cn tin hnh thm nhng kho st v nh gi
trn mt s phng php chit khc. Thc t c nhng nghin cu la
chn chit bacteriocin bng cc phng php khc nh chit bng ethanol [1],
butanol [15].
Tm li, t cc kt qu mc 3.2, c th tm lc mt s tnh cht c
bn ca bacteriocin sinh ra bi L. acidophilus ATCC 4653 nh sau:
V khong pH hot ng: bacteriocin ch yu hot ng pH acid, pH
cng nh th hot tnh bacteriocin cng ln.
V bn nhit: bacteriocin c tnh bn nhit, hot tnh bacteriocin hu
nh khng thay i sau khi x l nhit 100oC trong thi gian 30 pht.
V kh nng chit bacteriocin bng phng php kt ta (NH4)2SO4:
bacteriocin c th c chit t dch ln men vi nng (NH4)2SO4 t 30%

38

n 70%, lng bacteriocin kt ta tng dn theo lng (NH4)2SO4 s dng.


nng (NH4)2SO4 70%, bacteriocin vn cha ta ht.
Trn y mi l nhng m t mang tnh s b v bacteriocin ca L.
acidophilus ATCC 4653. m t chi tit bacteriocin ny cn c nhng
nghin cu su hn.

39

KT LUN V KIN NGH

1. Kt lun:
ti hon thnh c cc mc tiu ra v thu c mt s kt qu
nh sau:
- Trong cc iu kin thc nghim ca ti, bacteriocin ca chng L.
acidophilus ATCC 4653 c tm thy c trong dch ln men v khng
c trong sinh khi. Bacteriocin ny th hin tc dng c ch trn c vi
khun Gram (+) (B. subtilis ATCC 6633) v vi khun Gram (-) (E. coli
ATCC 25922). Trn B. subtilis, bacteriocin to vng c ch trong, r
hn v c ng knh nh hn so vi trn E. coli. ng knh vng c
ch trung bnh ca 3 ln th nghim trn B. subtilis l 16,6mm, cn trn
E. coli l 20,3mm.
- Nhn nh s b mt s tnh cht ca bacteriocin sinh ra bi chng L.
acidophilus ATCC 4653:
Bacteriocin a pH acid, hot tnh tng khi pH gim. Trong cc
gi tr pH nghin cu, bacteriocin th hin hot tnh cao nht
pH 2 vi ng knh vng c ch trung bnh qua 3 ln th
nghim l 18,8mm.
Bacteriocin c tnh bn nhit. Sau khi un 100oC trong 30 pht,
hot tnh bacteriocin hu nh khng thay i.
Bacteriocin c th c chit t dch ln men bng phng php
kt ta (NH4)2SO4 vi nng mui t 30% n 70% (khi
lng/th tch), lng bacteriocin kt ta tng dn khi tng lng
(NH4)2SO4 s dng. nng (NH4)2SO4 70%, phn ta
bacteriocin to vng c ch c ng knh 15,6mm; phn dch
sau khi loi ta bacteriocin vn to vng c ch c ng knh

40

13,7mm, cho thy bacteriocin trong dch ln men vn cha kt


ta ht.
2. Kin ngh:
ti c th c tip tc pht trin theo mt s hng nh sau:
- Kho st nh hng ca iu kin nui cy n kh nng sinh
bacteriocin ca L. acidophilus ATCC 4653 theo cc thng s nh nhit
, pH, thi gian nui cy; t la chn c iu kin ti u sao
cho thu c nhiu bacteriocin nht.
- Tip tc nghin cu cc phng php chit xut v tinh ch bacteriocin
ca L. acidophilus ATCC 4653, t xy dng qui trnh chit tch ti
u v nh danh bacteriocin nghin cu.

TI LIU THAM KHO

Ting Vit
1. Nguyn Th , Hoa Th Minh T, L Thanh Bnh (2008), Mt s tnh
cht ca bacteriocin c tng hp bi vi khun lactic phn lp t sa b
ti, Tp ch Khoa hc v Cng ngh, 46(6), 33-41.
2. Phan Th Khnh Hoa, Nguyn Vit Cng, L Thanh Bnh (2001), nh
hng ca mt s ngun khong v nit ln sinh trng v sinh tng hp
nisin ca Lactococcus lactis subsp. lactis, Tp ch Khoa hc v Cng ngh,
39(5), 37-43.
3. Nguyn Thy Hng, Trn Th Tng An (2008), Thu nhn bacteriocin
bng phng php ln men bi t bo Lactococcus lactis c nh trn cht
mang Cellulose vi khun (BC) v ng dng trong bo qun tht ti s ch ti
thiu, Tp ch Pht trin Khoa hc v Cng ngh, 11(9), 100-109.
4. Nguyn Hong Lc (2007), Gio trnh Nhp mn Cng ngh sinh hc, Nh
xut bn i hc Hu, Hu.
5. Bin Vn Minh, Kiu Hu nh, Phm Ngc Lan, Th Bch Thy
(2008), Gio trnh in t Vi sinh vt hc cng nghip, Nh xut bn i hc
Hu, Hu.
6. Trn Ngc Tn, inh Th Thu Hng, Th Huyn, Trng Nam Hi
(2009), Biu hin enterocin AS-48 ca vi khun Enterococcus faecium di
dng thng trong t bo E. coli, Tp ch Khoa hc v Cng ngh, 47(4), 4955.
7. Trn Ngc Tn, inh Th Thu Hng, Nguyn Thanh Nhn, Phm Thy
Linh, Th Huyn, L Vn Trng, Trng Nam Hi (2008), Tng hp v
biu hin gen m ha enterocin vng AS-48 ca vi khun Enterococcus

faecalis trong t bo Escherichia coli, Tp ch Cng ngh Sinh hc, 6(3),


367-373.
8. Hoa Th Minh T, Nguyn Th , L Thanh Bnh (2009), c im sinh
hc ca chng vi khun lactic sinh tng hp bacteriocin, Tp ch Khoa hc
v Cng ngh, 47(5), 17-25.
9. Phm Vn Ty, V Nguyn Thnh (2009), Cng ngh sinh hc - Tp 5:
Cng ngh vi sinh v mi trng, Nh xut bn Gio dc Vit Nam, H Ni.

Ting Anh
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compound

produced

by

Lactobacillus

acidophilus

AA11, Acta

microbiologica et immunologica hungarica, 54(2), 107-119.


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PH LC
Ph lc 1: Phn loi s b cc bacteriocin v c im ca mt s bacteriocin tiu
biu cho mi nhm [26]
(A)
Proteinbacteriocin
Vi khun Gram
(-)
Escherichia coli

Lp

Phn
lp

Tn

Khi lng
phn t
(kDa)

C ch tc
dng

Colicin

A, E1 E9,
K, L, N, S4,
U, Y
B, D, H, Ia,
Ib, M, 5, 10
Pyocin R2
Pyocin S1,
S2, AP41
Pyocin F
Alveicin A, B

40 n 80

Nuclease /
To l

40 n 80

Nuclease /
To l
To l
Ging phage

408/358 aa

Ging phage
To l

Klebicin C, D

96

Nuclease

Serracin

Serracin P

66

Ging phage

Glynericin

Glynericin A

50

Ging phage

669

Ging phage

68/76

Ging phage

B
Pseudomonas
aeruginosa

Pyocin

Hafnia alvei

Alveicin

Klebsiella
pneumonia
Serratia
plymithicum
Xanthomonas
campestris
Yersinia
enterocolitica
Erwinia
carotovora
Vi khun Gram
(+)
Lactobacillus
helveticus
Streptococcus
milleri
Enterococcus
faecalis
Staphylococcus
aureus

Klebicin

Typ R
Typ S
Typ F
Ging
colicin
Ging
colicin

Enterocoliticin
Carotovoricin

Carotovoricin
Er

270 aa
75/84/
94

Helveticin J

III

37,5

Cha r

Millericin

III

30

Enterolysin

III

34,5

Lysostaphin

III

25

Thy phn
peptidoglycan
Thy phn
peptidoglycan
Thy phn
peptidoglycan

(B)
Lp

Phn
lp

Tn

Khi lng
phn t
(kDa)

C ch tc
dng

Microcin

Microcin
B17
Microcin V
Microcin
E492

3,1

Enzym ni
bo
To l
To l

Nisin
Lacticin 481
Mersacidin
Pediocin
Plantaricin
E/F
Carnocyclin
A
Lactococcin
A

3,5
3
2
4,6
3,5/3,7

To l
To l

5,8

To l

5,8

To l

Patellamides

0,7

Peptidbacteriocin
Vi khun Gram
(-)
Escherichia coli

II

IIa
IIb

Typ A

A1
A2

8,8
7,9

Vi khun Gram
(+)
I
(Hay
lantibiotic)
II

Typ B
IIa
IIb
IIc
IId

Cyanobacteria
Prochloron
didemni

Ging
microcin

Ghi ch: aa: acid amin

To l
To l

Ph lc 2: Mt s bacteriocin ca LAB v nhng tnh cht c bn [55]


Loi sn
xut
Lactococcus
lactis subsp.
lactis

Lactococcus
lactis subsp.
cremoris
Lactobacillus
acidophilus

Bacteriocin

Ph tc dng

c im

Nisin

Vi khun Gram (+)

Lacticin 3147

Lactococcin B

Clostridium sp.
Listeria monocytogenes
Staphylococcus aureus
Streptococcus
dysgalactiae
Enterococcus faecalis
Propionibacterium acne
Streptococcus mutans
Lactobacillus

Lantibiotic, 3,5 kDa, 34


acid amin, c sn
phm thng mi
Lantibiotic, 4,2 kDa, bn
nhit, th hin hot tnh
pH acid v sinh l

Acidocin CH5

Vi khun Gram (+)

Lactacin F

Lactacin B

Lactobacillus
amylovorus

Lactobin A

Lactobacillus
casei

Lactocin 705

Leuconostoc
gelidum

Leucocin A

Leuconostoc
mesenteroides

Mesentericin
Y105

Pediococcus
acidilactici

Pediocin F

Bacteriocin lp II, xp x
5 kDa, ph tc dng hp

Bacteriocin lp II, to kt
tp khi lng ln
Lactobacillus fermentum
Bacteriocin lp II, 6,3
Enterococcus faecalis
kDa, 57 acid amin, bn
Lactobacillus delbrueckii
nhit
Lactobacillus helveticus
Lactobacillus delbrueckii
Bacteriocin lp II, 6,3
Lactabacillus helveticus kDa, bn nhit, ch c
Lactobacillus bulgaricus
pht hin khi pH mi
Lactococcus lactis
trng nui cy c gi
n nh t 5,0 6,0
Lactobacillus acidophilus
Bacteriocin lp II, 4,8
Lactobacillus delbrueckii kDa, 50 acid amin, ph
tc dng hp
Listeria monocytogenes Bacteriocin lp II, gm 2
Lactobacillus plantarum
peptid; mi peptid gm
33 acid amin, khi lng
phn t 3,4 kDa
Lactobacillus
Bacteriocin lp II, 3,9
Enterococcus faecalis
kDa, 37 acid amin, bn
Listeria monocytogenes
pH acid, bn nhit
Enterococcus faecalis
Bacteriocin lp II, 3,8
Listeria monocytogenes
kDa, 37 acid amin, bn
nhit
Vi khun Gram (+)
Bacteriocin lp II, 4,5
kDa, bn vi nhit v

Pediocin PA-1

Listeria monocytogenes

Pediocin AcH

Vi khun Gram (+)


Vi khun Gram (-) khi b
sung stress
Lactobacillus
Lactococcus
Leuconostoc
Pediococcus
Staphylococcus
Enterococcus
Listeria
Clostridium
Listeria monocytogenes
Pediococcus

Pediococcus
pentosaceous

Pediocin A

Enterococcus
faecium

Enterocin A

Lactobacillus
sake

Lactocin S

Sakacin P
Lactobacillus
curvatus
Lactobacillus
helveticus

Curvacin A
Helveticin J

Lactobacillus
Leuconostoc
Pediococcus
Listeria monocytogenes
Listeria monocytogenes
Enterococcus faecalis
Lactobacillus bulgaricus
Lactococcus lactis

dung mi hu c, hot
ng trong khong pH
rng
Bacteriocin lp II, 4,6
kDa, 44 acid amin
Bacteriocin lp II, 4,6
kDa, 44 acid amin, ph
tc dng rng
Bacteriocin lp II, 2,7
kDa, bn nhit

Bacteriocin lp II, 4,8


kDa, 47 acid amin, bn
nhit
Lantibiotic, 3,7 kDa, th
hin hot tnh trong
khong pH 4.5 7.5
Bacteriocin lp II, 4,4
kDa, bn nhit
Bacteriocin lp II, 4,3
kDa
Bacteriocin lp III, 37
kDa, ph tc dng hp,
km bn nhit

Ph lc 3: Mt s phng php nh gi hot tnh bacteriocin


Phng php

Nguyn tc

Cch tin hnh

Phng php

Bacteriocin c kh nng khuch Mu th c nh git ln mi

spot-on-lawn

tn trong mi trng c hoc

trng c (~2% thch). hn

(Spot-on-lawn

bn c, do mu th cha

hp vi sinh vt ch th trn trong

assay) [57]

bacteriocin s c ch s pht

mi trng thch mm (~0.7%

trin ca vi sinh vt ch th, to

thch) ln trn. qua m, nh

thnh vng c ch xung quanh

gi vng c ch to thnh.

mu th.

Nh mu th ln cc khoanh giy

Phng php
khuch tn qua

lc v trng, kh ri t ln b

khoanh giy lc

mt mi trng c cy vi sinh

(Disc diffusion

vt ch th. qua m, nh gi

assay) [57]

vng c ch to thnh.

Phng php

Trn mi trng c cy vi

khuch tn qua

sinh vt ch th, khoan to cc

ging thch

ging. Nh mu th vo cc

(Well diffusion

ging. qua m, nh gi vng

assay) [57]

c ch to thnh.

Phng php o Trong mi trng lng, mu

Cho mu th vo ng mi trng

th cha bacteriocin s c ch

lng cy vi sinh vt ch th.

(Turbidometric

s pht trin ca vi sinh vt ch

qua m, o mt quang (OD

assay) [41]

th, lm cho s lng t bo vi

optical density) bc sng thch

sinh vt ch th gim xung so

hp, so vi ng i chng (khng

vi khi khng c mt mu th.

cho mu th). nh gi tc dng

S lng t bo c nh gi

c ch ca mu th i vi vi sinh

thng qua c ca mi

vt ch th.

trng.

Ph lc 4: Mt s qui trnh tinh ch bacteriocin ca LAB [57]


Bacteriocin

Tinh ch

Plantaricin ST31

Kt ta bng amoni sulfat Chit pha rn (Ct


Sep-pak C18) RP HPLC
Sc k trao i cation
Sc k trao i ion nn m rng
Trao i cation Sc k phn b pha o
Hp ph / tch RP HPLC
Hp ph - Gii hp ph RP HPLC
Kt ta bng amoni sulfat Sc k phn b
(Ct C18)
Kt ta bng ethanol in di tp trung ng
in Siu lc
Sc k trao i cation Sc k phn b (Ct
C18) HPLC

Nisin Z
Enterocin AS48
Plantaricin C19
Pediocin ACM
Salivaricin
Pediocin PA-1
Lactococcin
Mesentericin
Y105
Sakacin A
Sakacin P
Enterocin A
Pediocin A-1
Divercin V41
Acidocin D20079

Kt ta bng amoni sulfat Sc k trao i


cation Sc k tng tc k nc (Ct Octyl
Sepharose)
Lacticin Q
Kt ta bng aceton Sc k trao i cation
RP HPLC
Lactobin A
Kt ta bng amoni sulfat Chit bng h dung
mi chloroform : methanol RP HPLC
Divergicin M35 Sc k trao i cation Chit pha rn (Ct Seppak C18) RP HPLC
Enterocin CRL35
Kt ta bng amoni sulfat Lc phn t (Ct
Biogel P-6) Sc k trao i cation HPLC
Bozacin 14
Kt ta bng amoni sulfat - Chit pha rn (Ct
Sep-pak C18) RP HPLC
Mutacin B-Ny266
Sc k phn b (Ct C18) HPLC (x3)
Abp118
Kt ta bng amoni sulfat Sc k tng tc k
nc Sc k trao i cation RP HPLC (Ct
C8 hoc C18)
Macedocin
Kt ta bng amoni sulfat Sc k trao i
anion Sc k trao i cation Sc k phn b
pha o Lc gel
Piscicocin CS526
Kt ta bng amoni sulfat Lc gel Sc k

S
Hiu
bc sut
(%)
3
0,8
1
1
2
2
2
2

5,9
90,0
24,3
15,0
40,4
7,3

3
3
3

29,0
41,0
60,0

3
3
3
3
3
3

10,0
10,0
66,0
25,0
10,0
16,0

64,0

0,07

10,0

2,0

4,4

4
4

1,0
6,4

1,6

7,0

Propionicin F
Pediocin PD-1

trao i cation Sc k phn b (Ct C18) RP


HPLC
Kt ta bng amoni sulfat Sc k trao i
anion Sc k phn b pha o (x3)
Kt ta bng amoni sulfat Thm tch ng
kh Chit bng methanol Chit bng
chloroform Trao i cation

0,5

34,0

Ph lc 5: Cc bacteriocin c tm thy Lactobacillus acidophilus [11]


Bacteriocin
Lactacin B
Lactacin F

Chng vi khun
L. acidophilus N
L. acidophilus
11088
Acidophilucin L. acidophilus
A
LAPT 1060
Acidocin
L. acidophilus
8912
TK8912
Acidocin B
L. acidophilus M46
Acidocin A
L. acidophilus
TK9201
Acidocin
L. acidophilus CH5
CH5
Acidocin
L. acidophilus JCM
J1229
1229
Acidocin
L. acidophilus JCM
J1132
1132
Acidophilicin L. acidophilus LA-1
LA-1
Acidocin
L. acidophilus
LF221A
LF221
Acidocin
LF221B
Acidophilin
L. acidophilus IBB
801
801
Acidocin
L. acidophilus
D20079
DSM20079
Acidocin
L. acidophilus
AA11
AA11
Acidocin 1B L. acidophilus
GP1B

Ph tc dng
Cc vi khun thuc h Lactobacillaceae
Cc lactobacilli, Enterococcus faecalis
L. delbrueckii, L. helveticus
Lactobacillus v Lactococcus sp.
L. fermentum, Clostridium sporogenes
Mt s LAB gn gi v Listeria
monocytogenes
Cc chi Lactobacillus, Bacillus, Micrococcus
v Corynebacterium
Lactobacillus v Lactococcus sp.
Lactobacillus sp.
L. delbrucki, L. casei, L. jugurti
Mt s LAB, B. cereus, Clostridium sp., L.
innocua, S. aureus
Mt s lactobacilli gn gi, E. coli v S.
panama
Lactobacillus spp.
Salmonella, Shigella, E. coli, S. aureus, B.
cereus, B. subtilis
Shigella, E. coli, P. aeruginosa, L.
monocytogenes, Yersinia enterocolitica

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