B GIO DC V O TO

TRNG I HC NNG LM THNH PH H CH MINH

KHA LUN TT NGHIP

KHO ST KH NNG SINH BACTERIOCIN CA VI

KHUN LACTIC PHN LP TRN NEM CHUA

H v tn sinh vin: TRN TH THANH THO

Ngnh: BO QUN CH BIN NNG SN V VI SINH THC PHM
Nin kha: 2006 - 2010

Thng 08/2010

KHO ST KH NNG SINH BACTERIOCIN CA VI KHUN

LACTIC PHN LP TRN NEM CHUA

Tc gi

TRN TH THANH THO

Kha lun c trnh p ng yu cu

cp bng K s ngnh
Bo qun ch bin nng sn v vi sinh thc phm

Gio vin hng dn:

Tin s H Th Nguyt Thu

Thng 08/2010

ii

LI CM T
Vi tm lng tn s trng o, em xin chn thnh cm n Qy Thy C Trng
i hc Nng Lm, c bit l cc Thy C Khoa Cng ngh Thc phm tn tnh
dy bo trong nhng nm va qua, truyn t nhng kin thc qu bu lm hnh
trang cho em bc vo i.
Xin chn thnh cm n C H Th Nguyt Thu tn tnh hng dn, truyn
th kinh nghim, cng nh to nhng iu kin tt nht cho em trong sut qu trnh
hc tp v thc hin ti.
Xin ghi nh cng n ca Cha M khng qun mi gian lao kh nhc v s
hy sinh cao c cho con c c ngy hm nay.
Xin gi n nhng ngi thn yu, cc anh ch v cc bn lp DH06VT li cm
n su sc.
Sinh vin
Trn Th Thanh Tho

-1-

TM TT
Nem chua l mt sn phm ln men truyn thng rt ph bin ca Vit Nam,
c a thch bi v chua t nhin v cu trc gin, dai c trng. Tuy nhin, bn cnh
, vn cht lng v sinh ang l mi quan tm hng u ca ngi tiu dng ln
ngi sn xut.
Vi nguyn vng ci thin hn na cht lng ca sn phm v mt v sinh v
nh hng pht trin nem chua thnh thc phm chc nng, chng ti thc hin ti
Kho st kh nng sinh bacteriocin ca vi khun lactic phn lp trn nem chua nhm
tm ra cc chng vi khun lactic c kh nng to ra cc hp cht khng li vi khun
gy bnh, n nh cht lng sn phm.
Chng ti tin hnh nghin cu trn cc mu nem 5 c s khc nhau. Trong
, 3 c s (B Chn, Ninh Ha v Nm Thu) s dng ln men t nhin trong khi 2 c
s cn li (Vissan v Gio Th) thc hin ln men c nh hng. pH bt tht c o
lng vo ngy ln men th t ca nem chua. Vi khun lactic trong bt tht nem ngy
ny cng c m tng lng, phn lp, kho st cc c tnh sinh ha v tm hiu
kh nng sinh bacteriocin ca chng.
Kt qu o lng cho thy, pH bt tht ngy th 4 ba c s ln men t nhin
khc nhau khng c ngha (p > 0,05); trong , nem c s Nm Thu c gi tr pH
thp nht (4,34 0,03). i vi hai c s ln men c nh hng, phn tch thng k
cho thy pH bt tht ngy 4 ca chng khc nhau mt cch c ngha (p 0,05); trong
, Vissan c gi tr pH thp hn (4,15 0,03).
Tng lng vi khun lactic ngy 4 ca cc mu nem 3 c s ln men t nhin
khc nhau mt cch c ngha (p 0,05). Nem ngy 4 ca c s Nm Thu (1,62.108
CFU/g) cao hn hai c s cn li (ln lt l 1,43.108 v 1,29.108 CFU/g). i vi 2
c s ln men c nh hng, Vissan v Gio Th, tng lng vi khun lactic (ln lt
l 1,15.109 v 3,08.108 CFU/g) cao hn rt nhiu so vi 3 c s ln men t nhin. Gia
hai c s ln men nh hng, s khc bit v tng lng vi khun lactic nem ngy 4
l rt c ngha (p 0,001).
Chng ti ghi nhn c 7 dng vi khun lactic ti 5 c s kho st; trong ,
a phn c dng que (chim 81,82% cc chng vi khun kim tra).
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Kho st kh nng khng khun ca cc chng vi khun lactic, chng ti nhn

thy c 01 chng vi khun thuc dng VII nem Ninh Ha c kh nng khng li
Staphylococcus aureus ssp. aureus CIP 20256 v 01 chng dng I ca nem Ninh Ha
khng li Salmonella indiana LMBA.

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MC LC
Trang
Trang ta........................................................................................................................... i
Li cm t ........................................................................................................................ ii
Tm tt............................................................................................................................iii
Mc lc ............................................................................................................................ v
Danh sch ch vit tt ..................................................................................................viii
Danh sc cc hnh ...........................................................................................................ix
Danh sch cc bng ........................................................................................................ ix
CHNG 1. M U .................................................................................................1
CHNG 2. TNG QUAN .........................................................................................3
2.1 Thc phm ln men v trin vng pht trin.............................................................3
2.2 Nem chua Sn phm tht ln men ca Vit Nam ...................................................4
2.3 Qu trnh ln men lactic.............................................................................................5
2.4 Khi qut v vi khun lactic ......................................................................................5
2.5 Mt s vi khun lactic thng dng trong thc phm ..............................................6
2.5.1 Lactobacillus ....................................................................................................6
2.5.2 Lactococcus ......................................................................................................7
2.5.3 Leuconostoc ......................................................................................................7
2.5.4 Streptococcus....................................................................................................7
2.5.5 Pediococcus ......................................................................................................8
2.6 Bacteriocin.................................................................................................................8
2.6.1 Khi nim chung...............................................................................................8
2.6.2 Phn loi ...........................................................................................................9
2.6.2.1 Lp I ........................................................................................................9
2.6.2.2 Lp II .....................................................................................................10
2.6.2.3 Lp III....................................................................................................11
2.6.3 C ch hot ng ............................................................................................11
2.6.4 Li ch v hn ch ca bacteriocin .................................................................12
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2.6.4.1 Li ch ca bacteriocin ..........................................................................12

2.6.4.2 Hn ch ca bacteriocin.........................................................................13
CHNG 3. VT LIU V PHNG PHP NGHIN CU............................14
3.1 Thi gian v a im nghin cu ...........................................................................14
3.2 Vt liu v phng php nghin cu.......................................................................14
3.2.1 Mi trng v thit b phn tch .....................................................................14
3.2.2 Nguyn vt liu th nghim ............................................................................14
3.3 Ni dung nghin cu ...............................................................................................15
3.4 Phng php nghin cu .........................................................................................15
3.4.1 Kho st h vi khun lactic trn nem ngy bn ca nm c
s kho st ...............................................................................................................17
3.4.1.1 Phng php ly mu ............................................................................17
3.4.1.2 ng nht mu ......................................................................................17
3.4.1.3 K thut o pH ......................................................................................17
3.4.1.4 Kho st h vi khun lactic....................................................................17
3.4.1.5 m khun lc v cy chuyn ...............................................................18
3.4.2 nh danh s b vi khun lactic phn lp ......................................................19
3.4.2.1 Nhum Gram .........................................................................................19
3.4.2.2 Phn ng catalase ..................................................................................19
3.4.2.3 Kho st kh nng di ng ...................................................................19
3.4.2.4 Kim tra kiu ln men ...........................................................................19
3.4.2.5 Kho st kh nng sinh acid lactic ........................................................20
3.4.3 Kho st kh nng sinh bacteriocin ca cc chng vi khun
lactic phn lp..........................................................................................................20
3.4.3.1 Thu hoch dch ly tm ...........................................................................20
3.4.3.2 Kho st s b kh nng sinh bateriocin ca cc
chng vi khun phn lp....................................................................................20
3.4.3.3 Kho st kh nng sinh bacteriocin ca cc chng
lactic phn lp bng phng php khuch tn trn thch .................................21
3.5 X l thng k s liu thu thp ...............................................................................22
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CHNG 4. KT QU V THO LUN..............................................................23

4.1 Kho st h vi khun lactic ca nem chua sau 4 ngy ln men
nm c s kho st .....................................................................................................23
4.1.1 Gi tr pH v tng lng vi khun lactic ca nem chua ln men
ngy 4 5 c s kho st ........................................................................................23
4.1.2 Phn trm cc dng khun lc ca nem chua ln men
ngy 4 5 s s kho st.........................................................................................25
4.2 Tnh cht sinh ha ca nhng dng khun lc phn lp..........................................31
4.3 Kho st kh nng sinh bacteriocin ca cc chng vi khun lactic
phn lp..........................................................................................................................33
CHNG 5. KT LUN V NGH ..................................................................36
5.1 Kt lun....................................................................................................................36
5.2 ngh ....................................................................................................................37
TI LIU THAM KHO...........................................................................................38
PH LC .....................................................................................................................41

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DANH SCH CH VIT TT

CFU: Colony Forming Unit
LAB: Lactic Acid Bacteria
EMP: Embden - Meyerhof - Parnas pathway
MRS
NB
MRD
TSA

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DANH SCH CC HNH

Trang
Hnh 2.1: Cu trc ca nisin .........................................................................................10
Hnh 3.1: Tin trnh thc hin cc kho st..................................................................16
Hnh 3.2: Trnh t pha long cho vic m vi khun lactic .........................................18
Hnh 3.3: Vng khng khun ca dch bacteriocin trn a petri .................................22
Hnh 4.1: Dng v hnh thi t bo ca khun lc dng I.............................................26
Hnh 4.2: Dng v hnh thi t bo ca khun lc dng II ...........................................26
Hnh 4.3: Dng v hnh thi t bo ca khun lc dng III ..........................................27
Hnh 4.4: Dng v hnh thi t bo ca khun lc dng IV ..........................................27
Hnh 4.5: Dng v hnh thi t bo ca khun lc dng V ...........................................28
Hnh 4.6: Dng v hnh thi t bo ca khun lc dng VI ..........................................28
Hnh 4.7: Dng v hnh thi t bo ca khun lc dng VII ........................................28
Hnh 4.8: S chuyn mu ca dung dch trong kho st kh nng
sinh acid lactic ca vi khun..........................................................................................33
Hnh 4.9: Vng khng khun ca chng vi khun dng VII i vi
Staphylococcus aureus ssp. aureus CIP ........................................................................34
Hnh 4.10: Vng khng khun ca chng vi khun dng I i vi
Salmonella indiana LMBA ...........................................................................................35

DANH SCH CC BNG

Trang
Bng 4.1: Gi tr pH v tng lng vi khun lactic ca nem chua ngy 4
nm c s kho st.........................................................................................................23
Bng 4.2: Phn trm cc dng khun lc ca nem chua ngy 4
nm s s kho st.........................................................................................................29
Bng 4.3 Tnh cht sinh ha ca nhng dng khun lc phn lp
.......................................................................................................................................32
-8-

Chng 1
M U
1.1 T VN
Nem chua l mt trong nhng sn phm ln men truyn thng ni ting ca
Vit Nam c lm t tht heo, b v mt s ph liu khc. Ngy nay, nem chua c
sn xut ti nhiu vng ca t nc vi nhng thng hiu ni ting nh: nem Bnh
Dng, nem Th c, nem Lai Vung Phn ln nem chua c sn xut qui m
h gia nh theo phng thc ln men t nhin nn cht lng sn phm thng
khng n nh. Mt khc, nem chua l sn phm thng c tiu th sng sau 3 - 4
ngy ln men m khng qua ch bin. Do , vn v v sinh hin l mi quan tm
cp bch ca ngi tiu dng ln nh sn xut. Ngy nay, vic s dng nhng cht
khng khun ha hc trong thc phm b ngi tiu dng t chi v nhng l do sc
khe. Do , liu c th tm ra cc chng vi sinh vt khi ng va c kh nng nh
hng qu trnh ln men, m bo tnh n nh v ci thin cht lng sn phm va
c kh nng sinh ra nhng cht c kh nng khng li cc vi khun gy bnh?
Trc nhng yu cu trn, vi khun lactic (LAB) l nhm vi khun c ch
nhiu nht. Nhng vi khun ny c s dng lu i vi mc ch acid ha nguyn
liu thng qua vic sn sinh cc acid hu c, c bit l acid lactic; mt khc, chng
cn c kh nng sinh ra cc hp cht c kh nng khng khun quan trng trong vic
bo qun thc phm nh: hydrogen peroxide, carbon dioxide, diacetyl, bacteriocin,
reuterin v reutericyclin (Ouwehand v Vesterlund, 2004). Trong , bacteriocin l
hp cht khng khun c quan tm nhiu nht. Nhiu cng trnh nghin cu
chng minh mt vi ging vi khun lactic c kh nng sn sinh bacteriocin khng li
cc vi sinh vt gy h hng trong thc phm nh: Listeria monocytogenes,
Staphylococcus aureus, Escherichia coli, Salmonella
ti Kho st kh nng sinh bacteriocin ca vi khun lactic phn lp trn
nem chua c thc hin nhm tm ra cc chng vi khun lactic c kh nng
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khng vi cc vi sinh vt gy bnh, to tin cho ch bin nem chua c cht lng
n nh cng nh an ton v mt vi sinh.
1.2 MC CH V YU CU
ti ny c thc hin nhm mc ch kho st kh nng to bacteriocin ca
mt s vi khun lactic hin din trong nem chua. Qua hi vng s ci thin nem chua
thnh mt thc phm chc nng c li cho sc khe.
Yu cu thc hin:
Phn lp vi khun lactic trn 5 loi nem ln men ngy 4.
Thc hin cc phn ng sinh ha tin nh danh vi khun lactic.
Kho st kh nng khng khun ca cc vi khun lactic phn lp c.

-3-

Chng 2
TNG QUAN
2.1 THC PHM LN MEN V TRIN VNG PHT TRIN
Ln men l mt trong nhng cng ngh lu i nht c s dng bo qun
thc phm. Trong lch s, vic ln men thc phm c khi xng cch y t
nht 6000 nm (Holzapfef, 2000). Bt ngun t cc nc ang pht trin, qu trnh ln
men khng ch nhm mc ch bo qun m cn gp phn a dng ha thc phm vi
chi ph phi chng p ng cho ngi tiu dng c thu nhp thp (Kalui v ctv, 2010).
Hin nay, nhiu loi thc phm ln men c sn xut trn ton th gii qui
m h gia nh cng nh qui m cng nghip. S ph bin rng ri ca thc phm
ln men cho thy tm quan trng cng nh s a chung ngy cng tng ca cc sn
phm ny trn th gii. Trong mt s vng, c bit chu Phi, nhng thc phm ny
cn c s dng lm thc phm cai sa cho tr nh (Lei v Jakobsen, 2004; trch dn
bi Kalui v ctv, 2010).
Cc nghin cu c thc hin cho thy nhng thuc tnh v gi ca sn
phm ln men. Qu trnh ln men to ra cc hp cht chuyn ha gp phn ci thin
mi v v cht lng dinh dng ca sn phm. Bn cnh , mt s sn phm ca
qu trnh sinh tng hp nh acid lactic, ethanol, acetaldehyd, acid pyruvic, lm thay
i pH v gip c ch hiu qu cc vi sinh vt gy bnh. iu ny tng cng tnh an
ton v ko di tui th cho sn phm mt cch ng k (Motarjemi, 2002). Thm vo
, cc enzyme ngoi sinh trong nguyn liu ln men cng gp phn ci thin nhng
c tnh trn (Holzapfel, 2000).
Vic s dng ging khi ng vo vic sn xut cc sn phm ln men ngy
cng c p dng rng ri nh mt phng php rt ngn thi gian sn xut, nng
cp sn phm ln men v mt cht lng, tnh n nh cng nh tui th cho sn phm
(Rolle v Satin, 2002). Mt s vi sinh vt c li trong qu trnh ln men c nghin
cu nh vi khun lactic cho thy rng chng c kh nng sn sinh ra cc cht khng

-4-

sinh c li cho sc khe, to tnh an ton v bo qun lu di cho sn phm (Kalui v

ctv, 2010).
Thc phm ln men ngy nay rt a dng, ch bin t cc dng nguyn liu
khc nhau nh tht, tri cy, rau qu, ng cc, Vi nhng tin b hin nay v khoa
hc k thut, qu trnh ln men khng cn sn xut qui m th cng v tin hnh t
nhin nh trc y na m pht trin qui m cng nghip vi s ln men ch
ng nh vic s dng cc ging khi ng p ng khng ch v cht lng m
cn v s lng cho ngi tiu dng (Nguyn c Lng, 2006).
2.2 NEM CHUA - SN PHM THT LN MEN CA VIT NAM
Nem chua l mt sn phm tht ln men truyn thng Vit Nam. Ty theo
tng vng min sn xut m nem chua gn vi nhng tn gi ni ting nh nem Th
c, nem Lai Vung, nem Bnh Dng Phn ln sn phm c sn xut th cng,
ty theo kinh nghim ca tng h gia nh m cht lng v kh nng bo qun nem
chua rt khc nhau. Bn cht ca qu trnh ln men l mt qu trnh chuyn ha ng
thnh acid lactic nh hot ng ca vi khun Lactobacillus, Pediococcus v
Micrococcus. Trong , ng vai tr quan trng l cc Lactobacillus (Nguyn c
Lng, 2006; H Th Nguyt Thu v ctv, 2008).
Theo kinh nghim ca cc nh ch bin, tht dng trong sn xut nem chua phi
l tht nng, tht c t th mi va git m. Tht c lm nhuyn bng cch xay
hoc gi; sau , trn vi da heo c luc chn v ct thnh si nhuyn, cng cc
cht ph gia v gia v. Hn hp tht s c to thnh vin c trng lng khong 15 20 g, k tip c gi bng l vng, l chm rut hay l i, v c bao ph bn ngoi
bng nhiu lp chui dy (Nguyn Th Minh Uyn, 2008). Nhn chung, qu trnh ln
men c thc hin nhit phng (khong 28 - 30oC) trong thi gian t 3 - 4 ngy.
Trong thi gian ny, cc vi khun lactic hot ng mnh v chuyn ha ng thnh
acid lactic. Acid lactic c to thnh s lm gim pH ca bt tht, lm thay i cu
trc ca bt tht, bt tht tr nn chc hn, to v chua cn thit cho sn phm v lm
c ch s pht trin ca nhng vi sinh vt gy thi. Nem chua c n ti m khng
cn nu hay lm nng. Vi kiu s dng ny, ngi tiu dng tn hng c nhng

- 14 -

li ch cho sc khe do cc vi khun lactic cn sng trong sn phm mang li (Nguyen

v ctv, 2010).
Sn phm nem c nh gi l t cht lng khi b mt sn phm khng b
nht hoc nm mc, bt tht dai, khng nho, c v chua ngt va phi. Nem chua c
thi hn s dng ngn, ti a l 3 ngy khi bo qun nhit mi trng. Nu mun
ko di thi hn s dng, sn phm cn c bo qun nhit lnh (khong 1
thng nhit t 4 - 10oC).
2.3 QU TRNH LN MEN LACTIC
Ln men lactic l qu trnh chuyn ha ng trong iu kin k kh (bt buc
hay ty nghi) thnh acid lactic. Ngi ta tm thy cc vi khun (vi khun lactic,
Bacillus), cc to (Chlorella), mt vi loi nm mc thy sinh, cc protozoa, cng nh
cc c bp ca ng vt u c kh nng ln men lactic (Vng Th Vit Hoa, 2006).
Da vo sn phm sinh ra trong qu trnh ln men, ngi ta chia vi khun lactic
thnh 2 nhm l ln men ng hnh v ln men d hnh.
Ln men ng hnh
Phn gii glucose theo con ng EMP chuyn thnh pyruvat, sau kh
trc tip hu ht cc pyruvat thnh lactat nh vo enzyme lactat dehydrogenase.
Sn phm chnh l acid lactic chim 90%. Ngoi ra cn c cc sn phm ph
chim t l khng ng k nh acid acetic, ethanol, glycerin, CO2,
Cc vi khun tham gia nh Streptococcus lactis, Lactobacillus bulgaricus, Lb.
delbruikii, Lb. acidophilus, (Vng Th Vit Hoa, 2006).
Ln men d hnh
Cc vi khun ln men lactic d hnh thiu cc enzyme ch yu ca con ng
EMP. Chng phn gii k kh ng glucose ch yu theo con ng pentophosphat
(PP).
Ngoi sn phm l acid lactic (40%) cn to ra mt lng ln cc sn phm
khc nh acid succunic v ethanol (20%), acid acetic (10%), CO2 v H2 (20%).
Cc vi khun tham gia bao gm Bacterium pentoaceticum, Lactobacterium
casei, Lb. plantarum, Lb. bifrementans, Leuconostoc casei, Le. cremoris.

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2.4 KHI QUT V VI KHUN LACTIC

Vi khun lactic (LAB) l nhm vi khun Gram (+), c dng trc khun, cu
khun hay cu trc khun, nhn chung khng di ng, khng sinh bao t v cc c
tnh sinh ha m tnh khc nh catalase, nitrate reductase v cytorome oxydase (L
Ngc Qu, 2006). Chng c th h hp k kh hoc k kh ty . Khong nhit
thch hp cho cc vi khun lactic hot ng kh rng. Cc vi khun lactic a m c
nhit ti thch t 25 - 37oC, cc loi a nhit c nhit ti thch t 50 - 65oC,
nhm a lnh pht trin nhit 5oC hoc thp hn (Nguyn Th Minh Uyn, 2008).
Vi khun lactic c ng dng lu i trong nhiu loi thc phm ln men c
ngun gc ng hoc thc vt v cc tc ng c li ca chng v dinh dng, cm
quan v ci thin tui th sn phm.
Vi khun lactic c kh nng to ra cc hp cht khng khun. Trong , vic
sn sinh acid lactic v acid acetic l quan trng nht. Tuy nhin mt s chng vi khun
lactic cn c bit n bi vic sn sinh cc phn t c hot tnh sinh hc nh
ethanol, acid formic, cc acid bo, hydrogen peroxide, diacetyl, reuterin,
reutericyclin, Nhiu ging cng sn sinh cc bacteriocin v nhng phn t th hin
hot tnh khng khun tng t bacteriocin (De Vuyst v Leroy, 2007).
2.5 MT S VI KHUN LACTIC THNG DNG TRONG THC PHM
Mt s ging vi khun lactic nng ct lin quan n qu trnh ln men thc
phm nh Lactobacillus, Lactococcus, Leuconostoc, Pediococcus v Streptococcus.
2.5.1 Lactobacillus
Lactobacillus l ging ln nht trong LAB vi hn 80 loi v phn loi c
cng nhn (Limsowtin v ctv, 2002). y cng l nhm loi rt hn tp vi s a dng
v cc c tnh l ha, sinh ha v kiu hnh nhng ch yu l dng que (Axelsson,
2004). Cc Lactobacillus l nhng vi khun Gram (+), khng sinh bo t, thnh thong
cp i hoc to thnh chui, khng di dng v catalsae m tnh.
Phn b rt nhiu trong t nhin v nhiu loi c ng dng trong thc
phm, nhm vi khun ny c ghi nhn l chu acid cao nht, c mt trong c th
ngi (khoang ming, ng tiu ha,...), v trong mt s thc phm ln men nh tht,
sa, rau qu, ng cc,... (Axelsson, 2004). Mt s loi nh Lb. casei, Lb. plantarum,
- 16 -

Lb. brevis, tham gia trong qu trnh lm chn ph mai; Lb. delbrueckii subsp.
bulgaricus l mt trong hai loi vi khun cho php s dng trong ln men yogurt
(Limsowtin v ctv, 2002).
2.5.2 Lactococcus
Lactococcus c dng hnh cu hoc hnh trng, thng bt cp hoc thnh
chui. C 5 loi Lactococcus (Lactococcus lactis, Lc. garvieae, Lc. plantarum, Lc.
piscium v Lc. raffinolactis) hin ang c cng nhn. Tuy nhin, ch c Lc. lactis
subsp. lactis v Lc. lactis subsp. cremoris mi c s dng trong qu trnh ln men
thc phm (Limsowtin v ctv, 2002).
Lactococcus lactis c phn lp t sa v cc sn phm ch bin t sa. C
kh nng vi khun ny nhim vo sa trong qu trnh vt sa v cho n. Lc.
raffinolactis c phn lp t sa cha tit trng, t ng rut ng vt v t cc
mi trng trang tri nui b. Lc. garvieae v Lc. piscium gy bnh cc loi c, Lc.
garvieae cng lin quan ti bnh vim v b v nhim trng ngi.
Mt s chng Lc. lactis. subsp. lactis c kh nng chuyn ha citrate, sn sinh
CO2, diacetyl v cc cht trung gian khc to hng thm cho sn phm (v d nh b
v ph mai Gouda). Nisin, mt bacteriocin peptide sn sinh bi cc ging Lactococcus
c sn trong thng mi c coi nh l mt ph gia bn acid, c s dng ko
di thi gian bo qun thng qua vic c ch h vi sinh vt Gram dng trong thc
phm (Limsowtin v ctv, 2002).
2.5.3 Leuconostoc
Leuconostoc c dng hnh cu xp chui hoc bt cp, ln men d hnh. Chng
thng c tm thy trn l, ng cc, nho v cc cm hoa. Nhng vi khun ny pht
trin 20 - 30oC v khng pht trin 45oC. Leuconostoc (c bit l Ln. lactis v Ln.
mesenteroides) c vai tr quan trng trong vic ln men sa do chng c kh nng
sinh CO2 v mt lng ng k diacetyl t citrate trong sa (Axelsson, 2004). Diacetyl
l hp cht chnh to hng cho b chua, kem chua v ph mai ti, cn CO2 th ng
vai tr quan trng trong vic hnh thnh mt ph mai. Ngoi ra, nhm vi khun ny
cn ng vai tr chng khi ng trong vic ln men t nhin ca rau qu (v d nh
da ci).
- 17 -

2.5.4 Streptococcus
Nhm vi khun ny c dng hnh cu hoc hnh trng, kt i hay dng chui,
ln men ng hnh. Hin c trn 50 loi Streptococus c nhn bit, nhng vi
khun ny phn b ch yu trong xoang ming v ng h hp. Mt s
Streptococcus nh Sp. mutans l tc nhn gy bnh su rng v vim mng tim
(Axelsson, 2004).
V c bn, ch c Sp. thermophilus l c s dng trong sn xut yogurt (cng
vi Lb. delbrueckii. subsp. bulgaricus) v ph mai. Vi khun ny c s dng nh
mt tc nhn acid ha ng t lactic.
2.5.5 Pediococcus
Pediococcus c dng t cu, vi hiu kh, pht trin t 20 - 30oC v khng pht
trin 45oC, a acid v ln men ng hnh. Hin c 7 loi Pediococcus c nhn
bit. Nhng vi khun ny thng c tm thy vi mt lng ln trn l nho, da bp
ci ln men, tri oliu v bia.
Pediococcus quan trng trong thc phm v c hai mt tiu cc v tch cc. Pe.
damnosus l vi sinh vt gy h hng chnh trong sn xut bia, v s sinh trng ca
chng c th dn n vic hnh thnh diacetyl/acetoin to ra v b. Pe. acidilactici v
Pe. pentosaceus c s dng nh cc chng khi ng cho vic lm xc xch ln
men. Pediococcus cng tham gia vo qu trnh lm chn ph mai gp phn lm tng
hng v cho ph mai (Axelsson, 2004).
2.6 BACTERIOCIN
2.6.1 Khi nim chung
Bacteriocin l cc peptide c sn sinh t nhin bi mt vi vi khun c
ch s pht trin ca cc vi sinh vt cnh tranh khc, chng c ph khng khun hp,
ch yu l c ch cc loi gn vi cc chng sn sinh bacteriocin (Parada v ctv,
2007). Nhng hp cht ny c tm thy trong hu ht cc loi vi khun nhng ch c
mt s loi trong chng c nghin cu k. Hin nay cc bacteriocin sn sinh bi
LAB c quan tm nhiu nht v LAB c coi l vi khun an ton cho ngi tiu
dng do chng v cc cht chuyn ha ca chng khng to nn nhng tc dng ph
khi c s dng trong cc thc phm ln men (Ruiz-Larrea v ctv, 2005). Cc
- 18 -

bacteriocin sn sinh bi LAB c nhng ng dng quan trng trong bo qun thc
phm. Nhng vi khun lactic ny to ra cc protein min dch c hiu t bo v,
chng li tc ng khng khun ca bacteriocin ca chng (Cintas v ctv, 2001).
C th ni nghin cu u tin v lu i nht v bacteriocin l cng trnh
nghin cu ca Gratia v ctv vo nm 1925 v kh nng khng khun ca Escherichia
coli (colicin V) (Desriac v ctv, 2010; Ruiz-Larrea v ctv, 2005). Thut ng
bacteriocin khng xut hin cho n nhng nm 1950. nh ngha v bacteriocin u
tin da trn c tnh ca colicin, l mt cht sinh tng hp gy t vong, ph
hot ng hp b gii hn nhng loi tng t nh vi khun sn xut. Ba chng vi
khun Gram (+) c nghin cu cho vic sn sinh bacteriocin lc by gi l: Bacillus
sp.; Listeria sp. v Staphylococcus sp. Cc nghin cu trong nhng nm 1980 cho
thy c s gia tng ng k v s lng cc cng b trn bacteriocin. T thi im ny
bt u bng n nhng nghin cu v bacteriocin, nh hng nh mt cht khng
khun an ton trong lnh vc cng ngh thc phm (Desriac v ctv, 2010).
2.6.2 Phn loi
Cho n nay c khong 200 loi bacteriocin c xc nh, tuy nhin vic phn
loi cc bacteriocin vn cha c xc nh r rng v n vn ang l vn tranh ci.
Cc bacteriocin thng c phn loi kt hp vi cc tiu ch khc nhau. Nhng tiu
ch chnh l h vi khun sn xut, trng lng phn t ca chng v cui cng l trnh
t chui amino acid (Desriac v ctv, 2010). Mt cch tng qut, bacteriocin c chia
thnh 3 lp: lp I, lp II v lp III.
2.6.2.1 Lp I
Bacteriocin lp I hay cn c gi l Lanbiotic l nhng peptide nh (<5 kDa),
bn nhit v tc ng ln cu trc mng. Mt thnh vin ni ting ca nhm ny l
nisin (Parada v ctv, 2007). Cc Lanbiotic c chia thnh 2 phn lp l Ia v Ib da
trn s tng ng cu trc.
a) Phn lp Ia
Phn lp Ia gm cc peptide dng thun di, linh hot v tch in dng,
chng hot ng bng cch hnh thnh cc l trong mng t bo cht ca cc loi vi
khun nhm n (Ouwehand v Vesterlund, 2004).
- 19 -

Nisin thuc nhm ny. y l mt peptide c hnh thnh bi 34 amino acid.

C 2 bin th ca nisin c ghi nhn l nisin A v nisin Z, chng ch khc nhau bi
amino acid th 27: histidine trong nisin A c thay th bi asparagin trong nisin Z.
Cu trc ca nisin c th hin Hnh 2.1.

Hnh 2.1: Cu trc ca nisin (Ngun: Ruiz-Larrea v ctv, 2005)

Loi bacteriocin ny, c sn xut bi mt vi chng Lactococcus lactic, c
s dng nh mt cht ph gia trong thc phm. N c ph khng khun Gram (+)
rng, E. coli v cc vi khun Gram (-) khc ch b nh hng bi nisin khi mng ngoi
ca chng b ph hng. Nisin c cho l c hot tnh khng khun hiu qu i vi
Staphylococcus aureus, Listeria monocytogenes, cc t bo sinh dng ca Bacillus
spp. v Clostridium spp. (Ouwehand v Vesterlund, 2004). Chng c s dng ch
yu trong cc thc phm ng hp v cc sn phm t sa, c bit trong sn xut ph
mai, nhm chng li cc vi sinh vt chu nhit nh Bacillus v Clostridium (Deegan v
ctv, 2006; trch dn bi Parada v ctv, 2007). Nisin cng c ghi nhn c hiu qu
chng li cc bnh vim v do vi khun Gram (+) gy ra (Broadbent v ctv, 1989;
trch dn bi Parada v ctv, 2007).
b) Phn lp Ib
Phn lp Ib gm cc peptide c dng hnh cu, cu trc khng linh ng, tch
in m hoc khng tch in. Chng th hin hot ng bng cch gy nhiu vi cc
phn t enzyme thit yu ca vi khun nhy cm (Parada v ctv, 2007).
2.6.2.2 Lp II
Cn c gi l lp Non-Lanbiotic, bao gm cc bacteriocin c trng lng
phn t nh hn 10 kDa, bn nhit v khng cha lanthionine. Cc bacteriocin nhm
II c th chia thnh 3 phn lp, bao gm IIa, IIb v IIc.

- 20 -

a) Lp IIa
Lp IIa l lp ln nht, gm cc peptide hot ng chng Listeria, i din c
trng cho nhm ny l pediocin PA-1 v sakacin P. Cc bacteriocin nhm ny ha hn
cho nhiu ng dng cng nghip nh vo hot ng khng Listeria mnh ca chng.
Thm ch chng cn l cc tc nhn khng Listeria c ch nhiu hn l
bacteriocin lp I (nisin) v chng khng c ph c ch rng v v vy, chng khng
tiu dit cc ging khi ng (Ouwehand v Vesterlund, 2004).
b) Lp IIb
Lp IIb hnh thnh bi mt phc hp ca 2 peptide ring bit, nhng peptide
ny t hoc khng hot ng. Cc bacteriocin c trng cho nhm ny l lactococcin
G, plantaricin EF v plantaricin JK (Parada v ctv, 2007).
c) Lp IIc
Lp IIc l nhng peptide nh, bn nhit, gm nhng bacteriocin khng ng
nht nn phng thc hot ng ca chng cng khc nhau. Trong phn lp ny ch
tm thy cc bacteriocin nh divergicin A v acidocin B (Parada v ctv, 2007).
2.6.2.3 Lp III
Lp ny bao gm nhng peptide ln, c trng lng phn t ln hn 30 kDa,
khng bn nhit. Nhm ny c th bao gm cc enzyme ngoi bo khng li cc vi
khun c th bt chc cc hot ng sinh l ca bacteriocin. Cc bacteriocin lp III
cho n nay ch c phn lp t cc thnh vin ca ging Lactobacillus (Ouwehand
v Vesterlund, 2004).
2.6.3 C ch hot ng
Cc bacteriocin thng c hiu qu chng li cc vi khun Gram (+) nh
Bacillus, Listeria monocytogenes, Salmonella typhimurium. Cc bacteriocin ca LAB
c th khng hiu qu khi c ch vi khun Gram (-) v mng ngoi ca chng gy tr
ngi cho hot ng ca bacteriocin. Nhng mng ny ngn cn cc phn t nh cht
khng sinh, cht ty ra v thuc nhum xm nhp t bo cht (De Martinis v ctv,
2001). Tuy nhin, c mt vi nghin cu cng b hot ng ca bacteriocin chng
li cc vi khun nhm ny, v d nh plantaricin 35d sn xut bi Lactobacillus
plantarum chng li Aeromonas hydrophila (Messi v ctv, 2001), thermophylin sn
- 21 -

xut bi Streptococcus thermophillus hot ng chng li E. coli v Yersinia

enterocolitica (Ivanova v ctv, 1998).
Cc c ch hot ng khc nhau c ra cho cc bacteriocin nh thay i
hot ng enzyme, c ch s ny mm ca bo t v ngng hot ng ca cc cht
mang anion thng qua s hnh thnh ca cc l (Abee, 1995).
Cc bacteriocin t LAB c th hot ng thng qua cc c ch khc nhau
pht huy tc dng khng khun nhng mng t bo thng l ch c nhm n. S
tng tc tnh in ban u gia mng t bo v peptide ca bacteriocin c cho l
ng lc cho cc chuyn bin tip theo (Deegan, 2006)
Cc bacteriocin c th c tnh dit khun hoc nh khun, tc ng ny chu
nh hng rt nhiu bi mt s yu t nh lng bacteriocin v tinh khit ca n,
tnh trng sinh l ca cc t bo ch th v cc iu kin th nghim (Cintas, 2001)
C kh nng l lp I v II s dng cng cc c ch hot ng ging nhau. Cc
peptide lin kt mng huyt tng thng qua cc tng tc tnh in vi cc
phospholipid tch in m. V vy, vic thm nhp ca bacteriocin ngang qua mng t
bo ph thuc vo in th mng, c iu khin bi pH v phospholipid. Cc n
phn t ca bacteriocin hnh thnh cc khi proteic dn n vic hnh thnh l kt qu
l dn n s tht thot ca cc ion (ch yu l K v Mg), tn tht lng proton trong
t bo cht, tht thot ATP v acid amin. Lng proton trong t bo cht c vai tr c
bn trong s tng hp ATP v trong s di chuyn ca vi khun; v vy, vic tng hp
ca cc phn t ln cng nh sn xut nng lng b c ch, dn n cht t bo ch
(Bruno v Montville, 1993).
Phng thc hot ng ca cc bacteriocin nhm III khng c bit n. Do
, i hi phi nghin cu nhiu hn lm sng t n (Parada v ctv, 2007).
2.6.4 Li ch v hn ch ca bacteriocin
2.6.4.1 Li ch ca bacteriocin
Ngy nay, p dng rng ri cc bacteriocin trong lnh vc thc phm mang
n nhng li ch nht nh so vi vic s dng nhng cht bo qun ha hc truyn
thng nh:

- 22 -

 Bacteriocin chng minh tnh an ton ca chng trong chui thc phm
dnh cho ngi. Chng c t hn ch hn so vi nhng cht bo qun
ha hc v l cc phn t c sn sinh t nhin bi vi sinh vt ln men
trong thc phm ln men truyn thng (Ruiz-Larrea, 2005).
Khng gy tc ng n mi trng v chng b thoi bin nhanh chng.
Cc bacteriocin c s dng nh ngun thc n ch yu i vi cc tc
nhn gy bnh m khng lm nh hng n vi khun c li (Ouwehand
v Vesterlund, 2004).
Bacteriocin khng lm thay i cc tnh cht cm quan ca thc phm
(Ruiz-Larrea, 2005).
Chng c ph hot ng r rng.
C tc dng b sung cho cc tc nhn khng khun.
2.6.4.2 Hn ch ca bacteriocin
Vi nhng li ch trn bacteriocin ngy cng c ng dng rng ri trong
cng nghip nh mt loi khng sinh an ton. Tuy nhin, chng cng c mt s mt
bt li sau:
t c bit n hn so vi cc cht bo qun ha hc.
B thoi bin nhanh chng bi cc enzyme phn gii protein.
Chi ph cao trong vic p ng cc tnh nng k thut.
Ch c hiu qu chng li mt s loi vi khun no .
Trn thc t, c nhng ro cn php l yu cu s cng nhn v chp thun c
th i vi vic s dng chng dng tinh khit v bn tinh khit (Ruiz-Larrea, 2005).

- 23 -

Chng 3
VT LIU V PHNG PHP NGHIN CU
3.1 THI GIAN V A IM NGHIN CU
ti c thc hin t 5/4/2010 n 10/7/2010 ti phng th nghim Vi sinh
v Ha sinh thuc khoa Cng Ngh Thc Phm trng i hc Nng Lm, thnh ph
H Ch Minh.
3.2 VT LIU V PHNG PHP NGHIN CU
3.2.1 Mi trng v thit b phn tch
Mi trng v thuc th:
-

Thch v canh MRS (Merck, pH= 5,7 0,2) phn lp, tng sinh v gi
ging vi sinh vt.

Mi trng NB (Difco) tng sinh v gi ging vi sinh vt gy bnh.

Mi trng MRD dng pha long.

Mi trng TSA (Difco) dng thc hin th nghim khuch tn trn

a thch.

Mi trng carbohydrat dng kho st kh nng ln men ng ca cc

chng vi khun lactic phn lp.

Thuc th Uphenmen dng kho st s hin din ca acid lactic.

Thuc th peroxyde hydrogene 10% th phn ng catalase.

Thuc nhum Gram: gential violet, lugol v fuschine.

Dng c: a petri, ng nghim, pipet, micropipet, que cy, n cn

Thit b: My dp mu (Seward, Anh), my li tm (MSE Mistral 1000,
Anh), autoclave (Sanyo, Nht), cn in t (Ohaus, M), vortex (Velp, ), my o pH
(Metrohm, M), t m, t sy, knh hin vi,
3.2.2 Nguyn vt liu th nghim
Nem kho st c ly sau 4 ngy ln men ca 5 c s: B Chn (Th c, tp.
H Ch Minh), Ninh Ha (Nha Trang, Khnh Ha), Gio Th (Lai Vung, ng Thp),

- 24 -

Vissan (tp. H Ch Minh) v Nm Thu (Bnh nh). Trong , nem B Chn, nem
Ninh Ha v nem Nm Thu l sn phm ca qu trnh ln men t nhin cn qu trnh
ln men ca nem Gio Th v nem Vissan l c s dng ging khi ng.
Nm chng gy bnh c bo qun ng lnh (-20oC) c s dng lm
chng ch th, bao gm:
Staphylococcus aureus ssp. aureus CIP 20256
E. coli ATCC 25922
Bacillus cereus CIP 6624T
Listeria monocytogenes CIP 82110 T
Salmonella indiana LMBA
Thuc khng sinh Ofloxacin 200 mg c bn trn th trng c s dng
lm mu i chng cho th nghim khuch tn trn thch.
3.3 NI DUNG NGHIN CU
Chng ti thc hin cc cng vic nh sau:
Kho st h vi khun lactic ca nem sau 4 ngy ln men ca nm c s.
Thc hin cc phn ng sinh ha tin nh danh vi khun lactic.
Kho st kh nng sinh bacteriocin ca cc chng vi khun lactic phn
lp c.
3.4 PHNG PHP NGHIN CU
Cc thao tc tin hnh nghin cu c tm lc trong Hnh 3.1.

- 25 -

Ly mu
ng nht mu
Huyn ph m
o pH
Pha long mu cc
nng t 10-2 n 10-7

Cy ln mi trng thch MRS

30oC / 48 gi
- m s lng khun lc
- Tnh phn trm dng khun lc
- Gi ging.
- Cy chuyn.

Kim tra cc phn ng sinh ha

- Nhum Gram
- Catalase
- Di ng
- Kiu ln men
- Kh nng sinh acid lactic
Kho st s b
Kho st kh nng khng khun
Khuch tn trn thch
Hnh 3.1: Tin trnh thc hin cc kho st

- 23 -

3.4.1 Kho st h vi khun lactic trn nem ngy bn ca nm c s kho st

3.4.1.1 Phng php ly mu
i vi mi c s ch bin nem kho st, chng ti tin hnh ly mu lp li 3
ln nhng l sn xut khc nhau. Cc mu nem chua c vn chuyn ngay n
phng th nghim tin hnh phn tch trong thi gian sm nht.
3.4.1.2 ng nht mu
Sau khi tit trng dao k bng cn, dao c h trn ngn la n cn cho bay
ht hi cn v ngui. Dng dao ct nh mu nem v ly khong 10 g mu nem cho
vo bao PE v trng. Sau cho thm dung dch MRD vo bao theo t l
9 MRD:1 nem. Vic ng nht mu c thc hin vi my Stomacher trong 2 pht.
Sau khi ng nht, thu c dung dch huyn ph m (S1).
3.4.1.3 K thut o pH
Dung dch S1 s c em i o pH. Trc khi o, u pH k c lau sch;
sau , c cm vo dung dch mu sao cho dung dch ngp qua u d ca pH k.
Mi mu c o lp li 3 ln v gi tr pH ghi nhn cho mi mu l trung bnh ca 3
ln o lp li.
3.4.1.4 Kho st h vi khun lactic
Pha long mu v cy a
Tin hnh pha long thp phn dung dch S1 n nng 10-7 bng dung dch
MRD. Dng micropipet ht 0,1 mL dung dch cc nng 10-5, 10-6 v 10-7 cho vo
a petri. Dng que cy trang kh trng trang u ln b mt thch cho n khi thy
b mt thch kh. mi nng pha long chng ti tin hnh lp li 2 ln.
Trnh t cc thao tc c trnh by qua Hnh 3.2.

Hnh 3.2: Trnh t pha long cho vic m vi khun lactic

- 24 -

 Phng php
Cc a petri sau khi cy xong s c k kh 30oC trong 48 gi. Vic k
kh c tin hnh bng cch cc a petri vo trong cc bnh ht m, t vo
ngn n cn c thp sng, y cht np, khi ngn la n cn tt ng ngha
vi vic oxy trong bnh c tiu th ht.
3.4.1.5 m khun lc v cy chuyn
Sau khi 30oC trong 48 gi, vic m khun lc c thc hin trn cc a
c s lng khun lc t 15 n 300.
S lng khun lc m c trong hai a hai nng lin tip c tnh
bi cng thc sau:
N

V .d .(n1 0,1.n 2 )

(CFU/g)

Trong : N: s t bo (n v hnh thnh khun lc) vi khun trong 1g mu.

C: tng s khun lc m c trn cc a chn
n1: s lng a cy ti nng pha long th 1
n2: s lng a cy ti nng pha long th 2
V: th tch dch mu (mL) cy vo trong mi a
d: nng pha long u tin c nui cy.
S lng trung bnh ca khun lc c m cho 3 ln lp li ca 1 mu th
nghim:
N

N1 N 2 N 3
(CFU/g)
3

Vi N1, N2, N3 l tng s khun lc c tnh trong ln lp li th nht, th hai

v th ba.
Sau khi m khun lc, vi quan st bng mt thng, cc dng khun lc hin
din c ghi nhn da trn hnh dng v kch thc ca chng.
i vi mi dng khun lc chng ti tin hnh cy chuyn v gi ging bng
cch ly khun lc cho vo ng eppendorf v trng c cha 1 mL canh MRS v
37oC trong 24 gi trong t m. Sau , cy chuyn ln mi trng thch MRS v
37oC trong 48 gi tin hnh cc phn ng sinh ha tip theo. Nhng ng eppendorf
c b sung glycerol tit trng hai ln vi th tch 10% th tch dch khun, sau
c bo qun ng lnh gi ging.
- 25 -

3.4.2 nh danh s b vi khun lactic phn lp

3.4.2.1 Nhum Gram
Sau khi 37oC trong 48 gi, cc chng vi khun phn lp, i din cho cc
dng khun lc c nhum Gram. Vic quan st di knh hin vi sau cho chng
ti khi nim ban u v hnh thi t bo vi sinh vt nh kh nng bt mu (Gram (+)
hay Gram (-)); dng vi khun (hnh que, cu hay hnh trng) cng nh cch sp xp
ca chng (xp chui, bt cp hay ring l).
3.4.2.2 Phn ng catalase
Ly khun lc cho ln trn ming lame sch, sau nh mt git H202 10% ln.
Nu thy xut hin bt trng th phn ng l dng tnh cn khng xut hin bt trng
th phn ng l m tnh.
3.4.2.3 Kho st kh nng di ng
Dng que cy v trng ly khun lc v cy su vo trong ng nghim c
cha 10 mL mi trng thch mm MRS (4 g agar/L mi trng) v em 37oC
trong 48 gi trong iu kin k kh, tnh di ng ca vi sinh vt c quan st bi s
phn tn vi sinh vt trong mi trng so vi ng cy: nu vi khun mc lan ra khi
ng cy th vi khun c kh nng di ng, nu vi khun mc theo ng cy th
chng khng c kh nng di ng.
3.4.2.4 Kim tra kiu ln men
Cho khun lc vo trong ng nghim c cha mi trng carbohydrat thm
bromocresol ta. cc ng nghim ny 37oC trong 24 gi. Tin hnh kim tra kh
nng sinh kh ca vi sinh vt bng cch dng que cy h tht nng trn ngn la
n cn ri nhng nhanh vo trong ng nghim. Nu thy xut hin nhng bt kh nh
th chng t vi khun c kh nng sinh kh; ngc li, vi khun khng c kh nng
sinh kh.
3.4.2.5 Kho st kh nng sinh acid lactic
Mi trng carbohydrat sau kho st kh nng ln men (phn 3.4.2.4) c
trung ha bng dung dch NaOH 0,1N mi trng chuyn li thnh mu tm. Thm
vo 2 mL dung dch Uphenmen, s tn ti ca acid lactic s c th hin bi s
chuyn mu cht ch th t ta sang vng.
3.4.3 Kho st kh nng sinh bacteriocin ca cc chng vi khun lactic phn lp
3.4.3.1 Thu hoch dch ly tm
- 26 -

Sau khi tin hnh cc phn ng sinh ha, chng ti chn lc ra nhng chng vi
khun c cho l vi khun lactic tin hnh cc th nghim tip theo.
Dng que cy tit trng ly khun lc cho vo trong ng ly tm v trng cha
10 mL MRS b sung 2% cao nm men (Elmoualdi v ctv, 2008). cc ng ly tm ny
37oC trong 16 - 18 gi (Cheikhyoussef v ctv, 2008).
Sau , cc ng ly tm ny s c em ly tm 6000 vng trong 15 pht
tch cn khun. Nhm trnh nh hng n kt qu th nghim bi s c ch gy ra t
cc acid hu c c mt trong mi trng, sau khi ly tm xong chng ti iu chnh pH
ca dch khun bng dung dch NaOH 10N t n pH = 6,5 (Poncea v ctv, 2007).
Sau lc ly phn dch ni trn b mt bng cch bm dch qua mng lc Minisart
(ng knh l lc l 0,2 m) tch cn khun cn st li. Phn dch lng thu c
s c s dng cho cc th nghim tip theo.
3.4.3.2 Kho st s b kh nng sinh bateriocin ca cc chng vi khun phn lp
Tin hnh tng sinh nm chng ch th gy bnh trong 10 mL mi trng NB
(37oC / 24 gi). Sau tin hnh pha long nm chng ch th n nng 10-4 bng
dung dch MRD.
Dng micropipet ht 0,5 mL dch ly tm cho vo ng eppendorf v trng, sau
cho tip 0,5 mL dch khun ch th nng 10-4. Mi dch ly tm u c kho
st vi nm chng gy bnh nghin cu.
cc ng eppendorf 37oC trong 24 gi. Sau so snh bng mt thng
trong, c ca chng vi ng i chng (khng cho dch khun). Nu ng trong v
khng c sinh khi iu ny chng t dch ly tm trong ng c kh nng khng li
chng ch th gy bnh v c ch s pht trin ca chng. Ngc li, nu ng c v
c sinh khi, iu ny ng ngha vi vic vi khun gy bnh vn tip tc pht trin
trong dch ly tm v nh vy, dch ly tm khng c cha cht khng khun.
Vic kho st s b ny cho php chng ti tuyn lc cc chng vi khun
lactic c kh nng sinh cht khng khun cho kho st vi phng php khuch tn
trn thch tip theo.
3.4.3.3 Kho st kh nng sinh bacteriocin ca cc chng lactic phn lp bng
phng php khuch tn trn thch
Trong kho st ny, chng ti cy vi khun theo hai cch: trn b mt thch v
cy su trong thch: 0,1 mL dch canh khun ch th nng 10-4 c trang u ln
- 27 -

b mt thch TSA hoc c trn u vi 30 mL mi trng TSA v sau ra a

petri. Sau khi thch kh, c 6 - 7 l, vi ng knh l khong 8mm trn b mt a
thch (Hata v ctv, 2009). Cc l c c cch thnh a petri khong 1cm c th
quan st c vng khng khun sau ny. K n, nh khong mt git mi trng
TSA vo l bt kn y l. Dng micropipet ht 150 L dch khng khun cho vo
mi l. Bn cnh cng tin hnh th nghim vi dch khng sinh pha long 10-4
lm mu i chng. Tip theo, bo qun cc a nhit 2 - 4oC trong 2 gi vi mc
ch ngn cn s pht trin ca chng ch th v to iu kin cho bacteriocin c thi
gian thm thu vo bn trong thch. Sau , cc a petri c 37oC.
Vic c kt qu c thc hin sau 16 gi . Kh nng khng khun ca vi
khun c xc nh bng s hin din ca vng khng khun xung quanh l. Nu
ng knh vng khng khun ln hn hoc bng 6 mm (Cheikhyoussef v ctv, 2008)
th c xem l c kh nng c ch tch cc.
ng knh vng khng khun c tnh nh sau:
dkk = d dl
Trong : dkk: ng knh vng khng khun xung quanh l
d: ng knh vng c ch (bao gm ng knh l)
dl: ng knh l.

Hnh 3.3: Vng khng khun ca dch bacteriocin trn a petri

- 28 -

3.5 X L THNG K S LIU THU THP

Vic tnh cc gi tr trung bnh ( pH , N ), tng lng khun lc v phn trm
cc dng khun lc c thc hin vi phn mm Excel 2003.
Vic tnh ton SD, s khc bit gia cc l th nghim c tnh vi trc
nghim Fisher bng phn mm Minitab 12.21.

- 29 -

Chng 4
KT QU V THO LUN
4.1 KHO ST H VI KHUN LACTIC CA NEM CHUA SAU 4 NGY LN
MEN NM C S KHO ST
4.1.1 Gi tr pH v tng lng vi khun lactic ca nem chua ln men ngy 4

c s kho st
Kt qu theo di v gi tr pH v tng lng vi khun lactic ca nem chua sau 4
ngy ln men 5 c s kho st c trnh by Bng 4.1
Bng 4.1: Gi tr pH v tng lng vi khun lactic ca nem chua ngy 4 5 c s
kho st
Kiu ln men

Tham s thng k (n=3)

C s

pH SD

SD (CFU/g)

B Chn

4,39 0,03

1,43.108 7.106

Ninh Ha

4,41 0,03

1,29.108 1,2.107

Nm Thu

4,34 0,03

1,62.108 1,5.107

Ln men c

Vissan

4,15 0,03

1,15.109 8,5.107

nh hng

Gio Th

4,23 0,03

3,08.108 7.106

Ln men t nhin

(n: tng s mu kho st, pH : gi tr pH trung bnh ca mu, N : s lng vi khun

trung bnh ca mu, SD: lch chun)
Theo nghin cu trn nem chua ln men ngy 4 cc c s sn xut theo kiu
ln men t nhin, kt qu phn tch thng k ch ra rng khng c s khc bit c
ngha gia pH ca cc mu nem 3 c s kho st vi tin cy 95% (ph lc 3).
Qua Bng 4.1, nem c s Nm Thu c gi tr pH thp nht (vo khong 4,34 0,03)
so vi 2 c s cn li l B Chn (pH = 4,39 0,03) v Ninh Ha (pH = 4,41 0,03).
S khc bit ny l khng ng k v mt thng k (p > 0,05). Theo nghin cu ca
L Ngc Qu (2006) trn sn phm nem chua c s t Thng, pH ca nem chua
trong ngy ln men u tin l 6,33 v ngy th 4 l 3,99. S d c s st gim pH l
do qu trnh
- 30 -

chuyn ha ng trong nguyn liu thnh cc acid hu c, c bit l acid lactic lm

gim pH ca nguyn liu.
cc c s sn xut nem theo kiu ln men c nh hng, phn tch thng k
ch ra rng pH ca nem chua hai c s Vissan v Gio Th khc bit nhau c ngha
(p 0,05) (ph lc 3). Gi tr pH ca nem chua Gio Th l 4,23 0,03 cao hn so vi
nem Vissan (pH = 4,15 0,03). Gi tr pH 2 c s ny thp hn so vi 3 c s ln
men t nhin. iu ny c l l do qu trnh ln men nem c nh hng ti cc c s
ny. c s Gio Th ngi ta s dng mt phn tht ln men ngy hm trc cho
vic sn xut nem chua ngy hm sau (80 g bt tht ngy 2 v 10 kg bt tht ngy hm
trc c cho thm vo 40 kg tht xay ca ngy sn xut) (L Ngc Qu, 2006). Khi
ln men c nh hng, vi khun lactic chim u th v lng ngay giai on u
pht trin v sn sinh nhiu aicd hn so vi sn phm ln men t nhin (Nguyn Th
Minh Uyn, 2008). Trong ln men xc xch kh, vic s dng men vi sinh vi nhng
vi khun phn lp t chnh sn phm (men vi sinh ni ti) dn n s hnh thnh nhanh
chng acid lactic ngay t ban u ca tin trnh ln men lm gim nhanh pH ca qu
trnh ln men (Ammor, 2007; trch dn bi H Th Nguyt Thu, 2008). Bn cnh ,
pH ca sn phm tht ph thuc vo pH ban u ca bt tht, vo lng v loi gia v
v ph gia s dng, vo h vi khun ban u v vo nhng yu t c lin quan n s
tng trng ca chng nh nhit , hot tnh nc, th nng oxy ha - kh... (H Th
Nguyt Thu, 2008).
Trong sn xut nem chua, cc acid hu c, c bit l acid lactic, sinh ra trong
qu trnh ln men lm gim gi tr pH, to v chua t nhin cho sn phm v lm
cho cu trc tht sn chc, to nn kt cu dai chc c trng cho sn phm. Bn cnh
, pH thp cn c tc dng cn tr s pht trin ca mt s vi sinh vt khng mong
mun trong sn phm, gip cho vic bo qun sn phm tt hn.
Kt qu x l thng k v tng lng vi khun lactic cho thy s khc bit gia
cc mu nem 3 c s ln men t nhin l c ngha (p 0,05). Tng lng vi khun
lactic ca nem Nm Thu 1,62.108 CFU/g l cao nht, k n l nem c s B Chn vi
tng lng vi khun lactic l 1,43.108 CFU/g (thp hn 1,9.107 CFU/g) v nem Ninh
Ha c tng lng vi khun thp nht (1,29.108 CFU/g thp hn 3,3.107 CFU/g so vi
nem Nm Thu). iu ny ph hp vi cc gi tr pH m chng ti o lng, tng
- 31 -

lng vi khun lactic cng nhiu s sn sinh ra nhiu acid hn v lm gim pH sn

phm. S khc nhau v tng lng vi khun gia 3 c s ln men t nhin c th l do
h vi sinh vt trong bt tht ban u, l gi, cc nguyn ph liu, iu kin mi trng
nh nhit , m ca cc c s kho st.
i vi 2 c s Vissan v Gio Th, tng lng vi khun cao hn rt nhiu so
vi 3 c s ln men t nhin, tng lng vi khun lactic ca nem Gio Th l 3,08.108
CFU/g v Vissan l 1,15.109 CFU/g (ln lt cao hn 1,46.108 CFU/g v 9,88.108
CFU/g so vi Nm Thu). S khc bit ny c l l do cc c s Gio Th v Vissan s
dng phng php ln men nh hng, b sung vi sinh vt ch ng, bng cch s
dng men kh (Vissan) hoc bt tht c ln men ca nhng ngy trc (Gio
Th), khin chng pht trin chim u th, c ch cc vi khun cnh tranh khc, to
iu kin cho chng pht trin mnh. Phn tch thng k cho thy tng lng vi khun
lactic ca 2 c s ny khc nhau rt c ngha (p 0,001). S khc bit ny c th l
do phng php nh hng ln men ca 2 c s. C s Vissan sn xut nem theo qui
m cng nghip ln v s dng men vi cc ging thun, cc chng vi khun lactic
khi ng tt v c kh nng pht trin mnh, lm xc tin nhanh qu trnh ln men.
4.1.2 Phn trm cc dng khun lc ca nem chua ln men ngy 4 nm s s
kho st
Kt qu ghi nhn v cc dng khun lc hin din trn nem chua ngy 4 nm
c s cho thy tng cng c 7 dng khun lc c ghi nhn trong cc mu nem kho
st. Vic phn loi cc dng khun lc c da vo mu sc, hnh dng v kch thc
ca khun lc. Cc dng khun lc bao gm:
Dng I: khun lc trn, vun nh, mu trng sa, c vin sng xung quanh,
ra gn, kch thc thay i t 1- 2 mm.
Dng II: khun lc trn, vun nh, mu trng sa, kch thc thay i t 1- 2
mm.
Dng III: khun lc trn, vun nh, mu trng sa, vin sng, ra khng gn,
kch thc thay i t 1- 2 mm.
Dng IV: khun lc trn, vun nh, mu trng c, trong, kch thc 2 mm.
Dng V: khun lc trn, vun cao, mu trng sa, kch thc 1 mm.

- 32 -

 Dng VI:: khun

khu lc trn, vun nh, mu trng
ng c, rra sng, kch thc
2 mm.
Dng VII: khun
n lc trn,
tr
vun cao, mu trng hi
i vng, kch th
thc 2 mm.

a) Khun lc dng I

b) Nm
m men

c) Trc
c khun

Hnh 4.1: Dng v hnh thi t bo ca

a khun lc dng I

a) Khun
n lc dng II

b) Nm men

c) Trc khun

Hnh 4.2: Dng v hnh thi t bo ca

a khun lc dng II
- 33 -

a) Khun lc dng III

b) Trc
c khun

c) Nm men

Hnh 4.3: Dng v hnh thi t bo ca

a khun lc dng III

a) Khun
n lc dng IV

b) Trc khun

Hnh 4.4: Dng v hnh thi t bo ca

a khun lc dng IV

- 34 -

a) Khun
n lc dng V

b) Cu trc

Hnh 4.5: Dng v hnh thi t bo ca

a khun lc dng V

a) Khun
n lc dng VI

b) Trc khun

Hnh 4.6: Dng v hnh thi t bo ca

a khun lc dng VI

a) Khun
n lc dng VII

b) Trc
c khun

Hnh 4.7: Dng

D
v hnh thi t bo ca
a khun lc dng VII
Phn
n trm mi dng khun lc ca cc mu nem ln
ln men ngy 4 5 c s c
trnh by Bng 4.2
- 35 -

Bng 4.2: Phn trm cc dng khun lc ca nem chua ngy 4 5 s s kho st
Kiu ln
men

C s

Dng khun lc ( X SD) (%)

(%)
I

II

B Chn

100

55,08
6,62

35,99
8,95

Ninh Ha

100

28,76
9,53

11,14
3,51

Nm Thu

100

32,40
6,49

Vissan

100

25,19
9,51

Gio Th

100

63,8
9,29

III

IV

VI

VII
8,93
7,79

Ln men
t nhin
(n = 3)

Ln men c

2,05
3,56

10,59
9,53

47,46
5,36

16,26
8,87

51,34
6,64
24,08
2,96

50,73
6,66

nh hng
(n = 3)

36,16
9,29

(n: tng s mu kho st, X : phn trm khun lc, SD: lch chun)

- 29 -

 c s B Chn, c 3 dng khun lc c ghi nhn trong , dng I v II c

tn s xut hin cao hn nhiu so vi dng VII.
i vi c s nem Ninh Ha, chng ti ghi nhn c 5 dng khun lc, trong
dng IV c tn s xut hin cao nht (47,46%) v k n l dng I (28,76%).
C s Nm Thu c 3 dng khun lc, tn s xut hin ca dng V v dng I l
cao nht (ln lt l 51,34% v 32,40%).
c s Vissan, chng ti ghi nhn c 3 dng khun lc, trong dng VI c
tn s xut hin cao nht (50,73%).
i vi c s Gio Th c 2 dng khun lc c ghi nhn, trong dng I c
tn s xut hin cao hn dng V (ln lt chim t l l 63,84% v 36,16%).
Trong 7 dng khun lc c ghi nhn, chng ti quan st thy c 2 dng ging
vi khun lc c nhn nh ca L Ngc Qu (2006): Khun lc dng I v dng II
m chng ti ghi nhn c ging vi m t khun lc dng VI v dng I.2 trong
nghin cu ca tc gi ny. Theo ghi nhn ca chng ti, dng I xut hin tt c cc
mu nem 5 c s vi tn s kh cao (25,19% - 63,8%). Cn cc dng cn li ch xut
hin mt s c s, v d nh c s Vissan v Gio Th khng c dng II, III v VII;
c s B Chn v Ninh Ha khng c dng V v VI. Ring dng VI ch xut hin c
s Vissan.
S khc bit v nhng dng khun lc cc c s c th l do h vi sinh vt
trong tht ban u, cc nguyn ph liu (ng, l gi,), cc thao tc trong qu trnh
sn xut, iu kin mi trng ch bin, phng php ln men Theo H Th Nguyt
Thu (2008), l gi cng tham gia vo qu trnh ln men, do khi kho st h vi khun
lactic ca l chm rut tc gi ghi nhn cc vi khun lactic hin din vi lng
khng t. Ngoi ra, ng ma s dng cng b sung cc khong cht, to thun li
cho s pht trin vi khun lactic ca nem chua (Trng Thanh Long, 2004; trch dn
bi H Th Nguyt Thu, 2008)
cc c s ln men theo kiu nh hng, dng khun lc c xu hng t hn
so vi cc c s ln men t nhin do khi ln men nh hng b sung cc cc sinh vt
mong mun lm chng pht trin chim u th trong h vi sinh vt ngay t lc u, to
iu kin cho chng pht trin mnh cnh tranh vi cc h vi sinh vt khc trong bt
tht.
- 30 -

4.2 TNH CHT SINH HA CA NHNG DNG KHUN LC PHN LP

Kt qu nghin cu v cc c tnh sinh ha ca nhng dng khun lc phn lp
trn cc mu nem 5 c s c trnh by Bng 4.3.

- 31 -

Bng 4.3 Tnh cht sinh ha ca nhng dng khun lc phn lp.
Kiu ln men

Nghin cu

C s
Dng
khun
lc

Vissan

Gio Th
(%)

II

III

IV

VII

III

IV

VI

(-)

(+)

6,82

6,82

4,55

6,82

6,82

2,27

6,82

4,55

6,82

6,82

6,82

6,82

6,82

6,82

6,82

6,82

100

0,76

0,76

0,76

0,76

0,76

0,76

4,55

6,06

6,06

4,55

6,06

6,82

1,52

6,82

4,55

6,06

6,06

6,82

6,82

6,82

6,82

81,82

6,82

(-)

6,35

6,35

4,76

6,35

7,14

1,59

7,14

4,76

6,35

6,35

7,14

7,14

7,14

7,14

7,14

7,14

100

(+)

(-)

6,35

6,35

4,76

6,35

7,14

1,59

7,14

4,76

6,35

6,35

7,14

7,14

7,14

7,14

7,14

7,14

100

(+)

Dng

ng hnh

6,35

4,76

7,14

1,59

7,14

4,76

6,35

7,14

7,14

ln men

D hnh

6,35

6,35

6,35

7,14

7,14

7,14

40,48

Sinh

(-)

acid lactic

(+)

6,35

6,35

4,76

6,35

7,14

1,59

7,14

4,76

6,35

6,35

7,14

7,14

7,14

7,14

7,14

7,14

100

Nm men
(n = 6)
Hnh thi Trc khun
(n = 108)
Cu trc
(n = 18)
Catalase

(n= 126)

Nm Thu

VII

vi khun

sinh ha

Ninh Ha

Tng

II

Vi th

Phn ng

B Chn

Ln men nh hng (%)

Gram

(n= 132)

Ln men t nhin (%)

Di ng

(n: tng s mu kho st)

- 32 -

6,82 13,63

7,14 59,52

Theo kt qu c trnh by Bng 4.3, trong 132 khun lc kho st v hnh

thi vi khun, kt qu cho thy a s cc khun lc c dng trc khun (81,82%), k
n l dng cu trc (13,63%) tp trung ch yu dng V. Ch c mt s t l nm
men (4,55%), vi tn s xut hin thp cc dng I, II v III.
Sau khi kim tra v loi b cc chng vi khun l nm men, chng ti thc hin
cc phn ng sinh ha vi 126 chng vi khun phn lp xc nh vi khun lactic.
Kt qu cc phn ng catalase, kho st kh nng di ng v sinh acid lactic cho thy
100% cc chng phn lp tha mn yu cu v c tnh sinh ha ca vi khun lactic.
Theo kho st v dng ln men, cc sn xut nem chua theo kiu ln men t nhin,
70% vi khun kim tra c kh nng ln men ng hnh; trong khi cc c s sn xut
theo phng php ln men c nh hng th 60% cc chng vi khun kho st c c
tnh ln men d hnh.

Hnh 4.8: S chuyn mu ca dung dch trong kho st kh nng sinh acid lactic ca
vi khun.
4.3 KHO ST KH NNG SINH BACTERIOCIN CA CC CHNG VI
KHUN LACTIC PHN LP
Kho st s b i vi 126 chng vi khun, c xem l vi khun lactic, v kh
nng khng khun ca chng i vi 5 chng gy bnh (Staphylococcus aureus ssp.
- 36 -

aureus CIP 20256, E. coli ATCC 25922, Bacillus cereus CIP 6624T, Listeria
monocytogenes CIP 82110 T v Salmonella indiana LMBA) chothy c 28 chng c
kh nng khng c 5 chng gy bnh ni trn chim 22,22% tng s chng vi khun
kho st. Trong , c 5 chng dng I nem B Chn, Ninh Ha (3,97%); 6 chng
dng III nem Nm Thu (4,76%); 5 chng dng V nem Gio Th (3,97%); 5 chng
dng VI nem Vissan (3,97%) v 7 chng dng VII nem B Chn v Ninh Ha
(5,55%).
Kt qu kho st bng phng php khuch tn trn thch ca 28 chng ny
cho thy ch c 01 chng dng VII nem Ninh Ha c kh nng khng li
Staphylococcus aureus ssp. aureus CIP 20256 v 01 chng dng I ca nem Ninh Ha
khng li Salmonella indiana LMBA. iu ny c th l do cc chng vi khun cn li
to ra lng bacteriocin vi nng thp nn khng th hin c kh nng khng
khun ca chng.
ng knh khng khun ca chng vi khun dng VII l 15mm trong khi
dng I l 7mm. Nh vy kh nng khng Staphylococcus aureus ssp. aureus CIP ca
chng vi khun dng VII mnh hn so vi chng dng I khng Salmonella indiana
LMBA. C v nh i vi vi khun lactic, cc bacteriocin thng c hiu qu chng
li cc vi khun Gram (+) nhng c th khng hiu qu khi c ch vi khun Gram (-).
iu ny c l l do mng ngoi ca vi khun Gram (-) kh dy, gy tr ngi cho hot
ng ca bacteriocin. Nhng mng ny ngn cn cc phn t nh cht khng sinh,
cht ty ra v thuc nhum xm nhp t bo cht (De Martinis v ctv, 2001). Theo
nghin cu ca Nguyen (2010) trong 44 chng vi khun lactic kim tra, ch c mt vi
chng th hin kh nng khng khun yu i vi Staphylococcus aureus 1976 (Sa
1976) v khng c chng no c kh nng khng li Salmonella typhimurium 1826 (St
1826).
Vng khng khun ca chng vi khun lactic dng VII i vi Staphylococcus
aureus ssp. aureus CIP v dng I i vi Salmonella indiana LMBA c trnh by
Hnh 4.9 v Hnh 4.10.
- 37 -

Hnh 4.9: Vng khng khun ca chng vi khun dng VII i vi Staphylococcus
aureus ssp. aureus CIP.

Hnh 4.10: Vng khng khun ca chng vi khun dng I i vi Salmonella indiana
LMBA.

- 38 -

Chng 5
KT LUN V NGH
5.1 KT LUN
i vi cc mu nem ngy 4 ca 3 c s ln men t nhin, s khc bit v
pH sn phm l c ngha vi tin cy 95%. i vi cc mu nem 2 c s ln
men c nh hng, pH ca chng khc nhau c ngha (p 0,05) v thp hn so vi
cc gi tr pH ca cc mu nem 3 c s ln men t nhin.Tng lng vi khun lactic
gia cc mu nem kho st 3 c s ln men t nhin khc nhau c ngha (p
0,05). Tng lng vi khun lactic c s Nm Thu (1,62.108 CFU/g) cao hn tng
lng vi khun ca hai c s B Chn (1,43.108 CFU/g) v Ninh Ha (1,29.108
CFU/g). i vi 2 c s ln men c nh hng l Vissan v Gio Th, tng lng vi
khun cao hn rt nhiu so vi 3 c s ln men t nhin. Tng lng vi khun lactic
c s Vissan (1,15.109 CFU/g) cao hn Gio Th (3,08.108 CFU/g) v s khc bit
ny l rt c ngha v mt thng k (p 0,001).
C 7 dng khun lc c ghi nhn trong cc mu nem 5 c s. Kim tra
vi th cho thy 81,82% vi khun c dng trc khun, 13,63% vi khun c dng cu
trc khun v 4,55% vi khun c dng nm men. Sau khi kim tra cc phn ng sinh
ha, c 126 chng vi khun c xem l vi khun lactic (chim 95,45 % tng s chng
vi khun phn lp).
Kim tra kh nng khng khun cho thy ch c 01 chng dng VII (chim
0,79% vi khun kim tra) nem Ninh Ha c kh nng khng li Staphylococcus
aureus ssp. aureus CIP 20256 v 01 chng dng I (chim 0,79% vi khun kim tra)
ca nem Ninh Ha khng li Salmonella indiana LMBA. Trong , chng vi khun
dng VII c kh nng khng khun mnh hn.

- 39 -

5.2 NGH
Lp li nghin cu trn cc mu nem khc nhau vi nhng thi im ln
men khc nhau tm cc chng vi khun lactic c kh nng sinh
bacteriocin.
nh danh cc vi khun lactic c kh nng sinh bacteriocin.
Chun ha v tm ra cc phng php thch hp nhm thu c bacteriocin
tt nht.
ng dng cc chng lactic c kh nng sinh bacteriocin vo trong sn
phm nem chua nhm pht trin nem chua thnh thc phm chc nng.

- 33 -