Nghiên cứu khả năng ức chế Staphylococcus aureus của Lactobacillus acidophilus trên hai môi trường sữa đậu nành và nước chiết thịt – peptone để bảo quản thực

ii

LI C
 xin gi li cc n Th  Dung  B 
 sinh hi h H  t nhiu
tht,  nhng l t thi gian thc
hi 
 i li cn  Nguy
Ti  ThK  Hunh Nguyn Anh Khoa  B 
ngh sinh hi h  H  ng ging vi sinh
vt s t cn thi  s d 
 n  m 117  B 
 sinh hi h H  o nhu
kin thun li nht   s dng trang thit b ng c m.
Xin c  t thc.
t nhng kin th
B  sinh hi h H 
li nghim. Xin gn mi
i nhng li c




iii



T
Staphylococcus aureus nh, ng ln sc khe

i. Bng t , lung c ch
ca L. acidophilus  n ca S. aureus by S. aureus
L. acidophilus    ng dch th: s   c chit tht. 
30
0
C, L. acidophilus    c ch S. aureus vi t l c ch t   S.
aureus/L. acidophilus = 1/100.  30
0
C, m S. aureus   l 1/100, 1/50, 1/10
 i mc 810
7
t          c ch S.
aureus   c ch  n sau c
.

ABSTRACT
Staphylococcus aureus is a opportunistic pathogen and a major concern for
human health. By determining the amount of staphylococci, the growth inhibitory
effect produced by the antimicrobial activity of the Lactobacillus acidophilus strain on
the Staphylococcus aureus strain was tested on liquid media, soya milk and meat
matrix, that contained a mixture of S. aureus and L. acidophilus. The optimum ratio of
S. aureus to effector was 1/100 at 30
0
C. The population of S. aureus at ratios of 1/100,
1/50, 1/10 never attain 810
7
cell per milliliter at 30
0
C. Production of acid lactic
appears to inhibit growth of S. aureus in the early but not the late stages of incubation.

iv

MC LC
LI C ii
T iii
MC LC iv
 VIT TT vi
DANH MC BNG vii
DANH M viii
M U 1
U 2
1.1 Gii thiu chung v L. acidophilus S. aureus 2
1.1.1 Vi khun Lactobacillus acidophilus 2
1.1.2 Vi khun Staphylococcus aureus 4
1.2 ng da vi khun lactic 8
1.2.1 Acid lactic ca vi khun lactic 8
1.2.2 Bacteriocin ca vi khun lactic 8
1.3 S S. aureus trong thc phm 8
1.3.1 S  9
1.3.2 S t 11
1.3.3 S troxide (H
2
O
2
) 13
1.3.4 S cng 13

1.3.5 ng ca t l LAB/S. aureus 14
2T LIU 16
2.1 Vt liu 16
ng ging 16
2.1.2 Nguu 16
2.2 Tim 16
2.2.1  nm 16


v

2.2.2 Ni dung b m 18
m s dng 19
i th 19
m Gram 19
2.3.3 Kh ng th 19
 catalase 19
ng chun 20
ng vi sinh vt bm khun lc 21
2.3.7 Kh  22
2.3.8  22
 3: KT QU M 23
3.1 S i pH cng si gian 23
3.2 Kt qu khh c ch S. aureus ca L. acidophilus 23
3.2.1 L. acidophilus  c ch S. aureus 23
3.2.2 S c ch c tht mng su
 26
3.2.3 T l c ch tS. aureus/L. acidophilus = 1/100 27
3.2.4 T l p  nh 
nhau 29

3.2.5 pH thp  t trong nhng yu t c ch kh   n ca S.
aureus. 32
3.2.6 Kh c ch ca L. acidophilus ch n nhng
 nhnh 35
N NGH 37
T LUN 44
U THAM KHO 45
PH LC



vi


 VIT TT

LAB Lactic Acid Bacteria - vi khun sinh acid lactic
CPS Coagulase Positive Staphylococci - stap



vii

DANH MC BNG

Bng 1.1  hong 12
Bng 3.1 Log (t S. aureus/ml) theo thi gian  ng s
c chit tht vi m S. aureus u 10
5
t  27

Bng 3.2 ng c ch ca L. acidophilus trong 26 gi ng su
 S. aureus u 10
5
t  28
Bng 3.3 ng c ch ca L. acidophilus trong 26 gi c
chit tht - peptone, M S. aureus 10
5
t  28
Bng 3.4 ng c ch ca L. acidophilus trong 48 gi c
chit tht - peptone, M S. aureus 2.510
6
t  29
Bng 4.1 M t S. aureus ng sc chit
tht - peptone theo thi gian 39
 4.2 S. aureus 
0
C
40
Bng 4.3 T l vi khun lactic cn thi S. aureus  n 810
7
t
 41
Bng 4.4 ng ca s b        n ca S.
aureus ng APT, 30
0
C) 42

viii

DANH M


 Lactobacillus acidophilus 4
 Staphylococcus aureus 5
nh 1.3 S. aureus ng th 7
2.1 m 17
  th biu din s i pH si gian khi mt
 L. acidophilus 
7
t  23
  th biu din s i m t S. aureus theo thi gian 
nhng t l S. aureus/L. acidophilus  24
  th biu din s i pH cng theo thi gian  nhng
t l S. aureus/L. acidophilus  30
4  th th hin m gi t ng
s S. aureus 
5
t  32
5  th th hin m gi t ng
c chit th S. aureus 
5
t  33
6  th th hin m gi t ng
c chit th S. aureus 10
5
t  34

1

M U
S. aureus  u ki
vi sinh v c thc phm. Thc phm ch bin sn ho
sn phm s ng lo nhic do S. aureus
nht.
Ti Ving nhim c S. aureus  mc cao. Mt kho
 cho thy 25% mu thng  
ph H m S. aureus nh ca B Y t [5,
57]. o qun ch yu hi d
cht ph gia.  dit khun, mt s sn xut  - m
ch - thc phm [57].
Vi khumt thay th ticho lot bo qu.
sinh ra   c ch s n ca
S. aureus     n sc kh  i. Mt trong s   L.
acidophilus, vi khun c bin rv n xut
n phm s    n ca L. acidophilus  
u  nhic ng dn xut bacteriocin
[45].
Trong u kin   thy rng, viu kh  c ch
Staphylococcus aureus ca Lactobacillus acidophilus    ng sa
c chit tht  peptone  bo qun thc phm  ht sc
cn thit.
Lu c thc hin vi mu kin tc
c ch Staphylococcus aureus bng Lactobacillus acidophilus, to ti  cho

vic ng dng bo qun thc phu t
nh:
1. T l tL. acidophilus c ch S. aureus mnh nht.
2. T l L. acidophilus c ch S. aureus  m chp nhc trong
ng gn vt.

Chương 1: Tổng quan tài liệu
2

: TU
1.1 Gii thiu chung v Lactobacillus acidophilus Staphylococcus aureus
1.1.1 Vi khun Lactobacillus acidophilus
1.1.1.1 Lactobacillus acidophilus

 L. acidophilus 

[27, 33].

L. acidophilus
.


L. acidophilus.
1.1.1.2 
L. acidophilus:






Lactobacillus acidophilus [28]
1.1.1.3 
L. acidophilus      , 
  0.5 - 1      
-

45
0
C [28].
1.1.1.4 

Chương 1: Tổng quan tài liệu
3

L. acidophilus
    . L. acidophilus  
            ,
saccharose, maltose, ma     ol, arabinose,
xylose, rhamnose, sorbitol, glycerin.       

L. acidophilus        , lactacin B,
acid[2, 28].
1.1.1.5 
L. acidophilus
(5% CO
2
, 10% H
2

85% N

2
37
0

3.8 - 6.5. L. acidophilus

acid, tryptone
, monooleate (Tween-80), sodium acetat
magnesium
.

L. acidophilus
-0.15-1% oxgall) [4].
1.1.1.6 L. acidophilus






,L. acidophilus

,.
      L. acidophilus    
.

Chương 1: Tổng quan tài liệu
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 L. acidophilus 


Lactobacillus acidophilus 
acidophilin, 


 [45].
L. acidophilus 
Candida albicans 
L. acidophilus 

L. acidophilus
Staphylococcus aureus, Salmonella typhimuriumE. coli.



 1.1 Lactobacillus acidophilus [59]
1.1.2 Vi khun Staphylococcus aureus
Staphylococcus aureus p t m ung
nh         u t m  
Staphylococcus thuc h Micrococcaceae [1].
1.1.2.1 i
Ging Staphylococcus  
 [1].
1.1.2.2 

Chương 1: Tổng quan tài liệu
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Staphylococcus    .8-1) x (0.5-1.  p xp
 t nhng t l  canh

y hoc t m ng thy chui ngng t
hay tng con mi ng ca yu t 
sinh), ta t cu khu xut hin th u hoc
 cu khun Gram 
t s ch (capsule).  nhng t 
 br [1].

 1.2 Staphylococcus aureus [58]
1.1.2.3 
T cu khui hiu, yn t
ng  nhi 37
0
7.2 - 7.4 (gii hn
nhi t 10 - 45
0
C).  nhi  cu khun sinh ra sc t
c t c t trng, sc t ng, t
cu mc vi khun l    -   n vi soi
khun lc thy nhng hc  trunn lc
 to sc t  S, t cu khu to ra
khun lc th  canh thang, cu khun mn 
ng h u khun mo 
 mt. Cu khun tt thanh
 th cng cu th, cu) [1].
T cu khuc chng cao vu kii vi
  t tri    c.  u ki   cu s  
  bi 50 -   tan lp lt t
ct tri chiu trc tip trong nhiu gi c t cu.

0

C, t cu st gi, 
0
C cht sau 10 - t

Chương 1: Tổng quan tài liệu
6

trong chch phenol 5% dit t cu sau 15 -  cu khun
rt nhy cm vi mt s thuc nhum anilin, ch yng
 u tr   cu khu 
v - i 4 - 5 loi
thuc bi ci methicillin.
T cu tng  
kha, kh 
catalase, phosphatase, sinh ammoniac, H
2
  ng glucose, levulose,
 c
 y arginin. N    n mi quan h gia ho  
 sinh alpha-toxin [1].
1.1.2.4 Kh c t
 cu tng hc t 
b        ng
hoi t           
n ca t chc
t.
 cy lecith
chng cu.   cc cht chng

 ca bch cu,

c t rung.
c tc rut 6 loc t c t rut
thuc loi chu nhit (tn tn vng vng
c ruc phm  c t ruti b 
chy [1].

Chương 1: Tổng quan tài liệu
7


 S. aureus ng th58]
1.1.2.5. Kh nh
Gây bệnh cho động vật
ng vn nhy cm vi t cu khun. N
ch th cht l cy xut hin hing nhim
c cng vt s chng ming ho
bc t rut t ct b
ng cc t y chng [1].
Gây bệnh cho người
T cu khu t nh      c bng git
c b c c
bt, hch bch huy        
(osteomyelitt tht m
  tr 
ng hp bnh t cu khun do th  bu
 m khun v    u thu  ng nh 
nhim khun huyi th  cu khun  tr nh
c ph
nhim t cnh s xut hin nhic thp nht
n phm ca sa, th

T cu khu          i hp,
         m khun vi

Chương 1: Tổng quan tài liệu
8

Streptococcus, C.diphteriae, M. tuberculosis, Actinomycosis, virus   
bnh cp.
Vio st tr li
 chuyn np yu t R-plasmide gi cu. N thuc tp
trung cao   i dn s y qun th vi khun hoi sinh,
ng thi to ra nhng chc [1].
1.2 ng da vi khun lactic
1.2.1 Acid lactic ca vi khun lactic
Acid lactic do vi khun lactic tc s dng rtrong bo qu
mui chua rau quc gi chua th

Vi khu h tr i trong vic hng
n phm t s
1.2.2 Bacteriocin ca vi khun lactic
  n cnh
ng thc phListeria, Clostridium, Staphylococcus, Bacillus sp.,
Enterococcus sp [45].
V mt cu tn chein do vi khun sinh tng hp
 c ch s n cng vi khu gn vi
ging sn xu t nhng chng vi khu
gn vi chng sn xu  t nhiu ging vi khun sinh tng hp bacteriocin,
 u nht do bacteriocin ca
 n rt bo qun thc
phm.

Hi n ph
Nisaplin (sn phm t n phm t pediocin PA-1) [22].
1.3 S S. aureus trong thc phm
S. aureus  u ki
sinh vc phm. Nhic do S. aureus 
t thc ph bing sn xut hoc do hong ci.
Thc phm ch bin sn hon phm sng lo
b nhic do S. aureus nht [35].

Chương 1: Tổng quan tài liệu
9

n kh c ch cS. aureus t rt
sm. Nhu kin n s n ca S. aureus bc ngh
cu t nh [12]c ph 
kiu kiu gi thuyu kin c
ch (s  t t
2
O
2
c ki
u khoa hc.
u tc phc bin phm t
st).
1.3.1 S 
S. aureus  t n 10 v pH
tc c t hp vi
nhng yu t  S. aureus  nhy ci acid khi n
mui cao [30]ng minh rng c ca oxy
n kh ng chu ca S. aureus v

[11].
 c ch ging nhaung vi m pH
    a S. aureus s      
thc phn l
ng pha sn hong sm pH xung khong 4.4  4.5,
s n ca S. aureus s b c ch [13, 26, 52], b
m pH sa xung minh rng
sinh khi S. aureus gim 99% so vi ch [42]. S
c ch  y ra v dng acid acetic, pH 4.5 vi
acid citric, pH 4.1 vi acid hydrochloric. S c ch do
ng xy ra v [42], ch nhng lo
m  t. Khi pH trong t t
c               
propionic, v pKa l c ch nhi
v
c bi
 gim pH n i cn s suy
yu trong hong ca nhiu enzyme nhy cm vi pH. S n c
khu i mnh do phn ln trong t c s

Chương 1: Tổng quan tài liệu
10

d kh acid t t bo ra m
t t [15].
G biu hin gen ca S. aureus c kiu kin acid
yu [55]. Kt qu cho thy s biu hin ca nhiu gen chu ng b
tr  n 5.5. Loc g
Stimulon). Bn sao ci din cho s a S. aureus vi
acid y n vi

n     ure    aperon n clpB 
nhin s i chn chuy
Stress acid y     n biu hin c  u t   
protein b m n phn ng ca S. aureus v
[55].
   t qu c     u t
n s c ch S. aureus. ng c n ca
S. aureus  u cho thy
rt mc tip gi c ch
S. aureus  n hoc kh ng
y hn hp vi LAB [11, 31, 39]. Nhn mt kt
lu c ch 
Trong mng (TSBng LAB (E.
faecium L. mesenteroides t S. aureus. Khi pH c
6.3, kh  c ch  vn tip
din [31]y hn hp ca S. aureus n
phc gi la) duy t qu cho thy
rng trong nhu kii), S. aureus vn mn cm vi
hong c c n 210
4
cfu/ml sau khi  6
gi  32
0
C [23]. Do v kt lun rng pH n kh n
ca S. aureus c ch  b mt mt phn hou
u kicng s ng minh kh c ch ca
L. lactis ssp. diacetylactis  n ca S. aureus y hn hp
c th c ch bin my trong
ng pha sn  i (pH 6.8) [18].
  quan trng. Nhi

thy rng S. aureus th hin din ngay t u trong sua

Chương 1: Tổng quan tài liệu
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n xu  n
xut mi tht s quynh s n ca S. aureus. Mu thc hin
hy m ca S. aureus  u
   n xu   thu    nhim khu  u.
 sn xu acid th 
th S. aureus n. M S. aureus sau 6 gi sn xut s i
ph thung th c gim m S.
aureus p [19].   
thy  s n ca S. aureus trong sua n
xut t khi  p mui [40] li
cn ng cn m S. aureus 
n phm th 
gm Lactobacillus sakei Lactobacillus curvatusp
c Lb. plantarum, Lb. casei, Lb. brevis Lb. alimentarius [46]. M 
chS. xylosus hoc S. carnosus c
sn phm thn
n vic sinh ra acid. S b 
 acid dn gim nhim khun do S. aureus [11, 32, 39,
44, 47].
 ng s dng pha sn, tht hoc s
ch bin thc phacid sinh ra t hong c ng nh
ng  n ca S. aureus [12].
1.3.2 S t
S n ca LAB, ch yn s tbacteriocin
u lc vi S. aureus [15]. Nhiu LAB t

vi ph n r u bacteriocin c c ng d
thc phm [22].  c ch hoc tht mt ch
    nhn bi  a
bacteriocin tc bi p t thc pha
hoc th  kt h vo qun [38, 50, 51, 54]. Vi
hon mnh, lactacin ca L. acidophilus c chng minh
 c ch S. aureus [56].


Chương 1: Tổng quan tài liệu
12

Bng 1.1  hong [12]
Bacteriocin
Ph/ hoS. aureus
Lactococcus sp.

Nisin
Ph rng (+)
Lacticin 3147
Ph rng (+)
Lacticin 481
Ph 
Lactococcin A, B et M
Ph hp (-)
Lactobacillus sp

Lactocin 27
Ph hp (nd)
Sakacin A

Ph hp (nd)
Sakacin B
Ph hp (nd)
Plantaricin C
Ph rng (nd)
Curvaticin 13
Ph rng (nd)
Pediococcus sp

Pediocin A
Ph rng (+)
Pediocin AcH (PA-1)
Ph rng (+)
Leuconostoc sp.

Leucocin A-UAL187
Ph rng (nd)
Enterococcus sp.

Enterocin A
Ph hp (-)
Enterocin P
Ph rng (+)
Carnobacterium sp.

Carnocin H
Ph rng (+)
Piscicolin 126
Ph rng (-)
Divercin V41

Ph rng (nd)
(+) : S. aureus b c ch
(-) : S. aureus  c ch
(nd) nh


Chương 1: Tổng quan tài liệu
13

Nhng ging trong vic bo qun thc ph
kh o ra bacteriocin c ch s n ca nhng vi khu
Listeria monocytogenes S. aureus. Sn phm bacteriocin t
th c ch c S. aureus.
1.3.3 S t
2
O
2
)
S tH
2
O
2
bc bi i
S. aureus. S t
2
O
2
bt yu c
c bip t h o. M cn vai
a H

2
O
2
c phm [17, 21, 23, 26]. M
c ch s n ca S. aureus bo ra H
2
O
2
.  n 0.18 mmol/l,
H
2
O
2
  n t n 1.0 mmol/l  t vi khun.
1.3.4 S cng
Nhu cng ca S. aureus u, mu
cng sng,
 m bo s n
c [10, 14, 16, 36, 41, 49].
S. aureus cng t nhng hp cht hS. aureus s
d          
u (mannitol), acid hng h
(glutamine, arginine).
Nhu c thuu kiu kin
hi S. aureus i nhng ngu
 y s hin din
ca h enzyme lactose phosphotransferase [34] S. aureus 
th ng sa [48].
nh chng trong y hn hp  cn trong
mu [9, 29]u cho thm

s ng S. aureus do cnh tranh chng. cng s y
s cn kit chng trong nhy pha sc bit
 cn kin s c ch S. aureus bi L. lactis [29].

Chương 1: Tổng quan tài liệu
14

 ng minh s c ch cht dinh
ng thit yu, chng hn s c ch S. aureus
y chung vi L. lactis  30
0
ng pha sn [26].
y s dng pha sn
trong khi nhn ra trong cht nn thc phm, nhng ch
th phc tu so vng pha sn. Chng hng c
ch ca L. lactis   n ca S. aureus    
ng s      tn ti     t dinh
ng   u kin (pH ging nhau) khnh m u kh  c ch
 thun cy [13].
1.3.5 ng ca t l LAB/S. aureus
T l S. aureus ng quynh kh c ch.
c kh nh trong nhiu nghi u s d  ng pha sn,
trong sa, hoc trong cht nt. Kt qu c
cho thy,  ng (APT), khi m S. aureus nhiL.
lactis (t l 10/1, S. aureus 10
5
S. lactis 10
4
cfu/ml), m ca S. aureus
          bng v   i

chng (10
10
cfu/ml, m s i t l (S.
aureus: L. lactisc 1/10, m cao nhS. aureus  c
ng ch 
6

5
cfu/ml [25]. S ng ca t l 
 thung h
thung cnh tranh, bt qu  
ng tng hy vi E. coli [53].
Nhi ra rng t l S. aureus
quynh kh c ch, in situt nt. Trong
mt s lo     st  staphylococci coagulase
 bng vi s  CPS
trong mu l y, m sau 24 gi 
ph thun sau 6 gi n xut [40, 19].
m (Camembert) sn xut t sng S.
aureus  u (t 10
2
n 10
6
cfu/ml) mt phn s quy n s ng S.
aureus khi chun b p mui (khong 22 gi sau khi 
 ng S. aureus i gian  thuc

Chương 1: Tổng quan tài liệu
15

ng S. aureus p mu
bo qun [40]. Kt qu  v 
Nha) [24].
Trong cht nn thcng s ng S. aureus ban
u (10
4

5
t i lactobacilli (0, 10
5

6
t 
t qu cho thy rng t l LAB/S. aureus t l trc tip vi s c
ch S. aureus [39].
Nhi  ng ln s c ch. ng sa, s c
ch ty ra  nhi th
0
C [10, 14, 16, 36, 41, 49].

Chương 2: Nguyên vật liệu và phương pháp nghiên cứu
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2T LIU
U

2.1 t liu
2.1.1 ng ging
 ng gi    m do B    sinh hc 
i h H p, bao gm:

 Ging vi khun Staphylococcus aureus  Vi H 
Minh.
 Ging vi khun Lactobacillus acidophilus VTCC2156.
ng gic cy chuyn sau mi 2 tun.
2.1.2 Nguu
c nghim:
 ng gi ging vi khun Staphylococcus aureus:
ng Nutrient Broth (NB) .
  ng gi gi    ng vi khun Lactobacillus
acidophilusng MRS .
 ng sc chit tht  peptone  kho
 c ch Staphylococcus aureus ca Lactobacillus acidophilus.
  p Staphylococcus aureus.
n  Ph lc 7.
2.2 Tim
2.2.1  nm





Chương 2: Nguyên vật liệu và phương pháp nghiên cứu
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2.1 Qui m
     nghim nhm hai m  t   l  y
Staphylococcus aureus/Lactobacillus acidophilus tL. acidophilus c
ch S. aureus mnh nht; kh l y c ch Staphyloccocus aureus sao
n vnh  
nghic ti 2.1.
Khging S. aureus
ng chun quan
h gi t 
Khng L. acidophilus
ng chun quan h
gi t 

Kh    i pH c 
ng s
m p

Kh ng ca S.
aureus   
  p
Kh ng ca nhng t l 
cy Staphylococcus aureus/Lactobacillus
acidophilus      
ng ca S. aureus.

ng s
   c
chit tht - peptone
Kh    i
   ng
y theo thi gian

Chương 2: Nguyên vật liệu và phương pháp nghiên cứu
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2.2.2 Ni dung b m
n 1
Ging vi khun Staphylococcus aureus c cy chuy    
ng NA. Ging vi khun Lactobacillus acidophilus c cy chuy 
ng MRS. Kii th b. Kim tra vi th b
nhum Gram  n vi.
Giai n 2
 Dng chun ca Staphylococcus aureus ng NB.
 Dng chun ca Lactobacillus acidophilus  ng MRS
dch th.
 Kht s a Lactobacillus acidophilus  
 sa, kh 

n 3
Kh    i pH c  ng s     sung L.
acidophilus theo thn khi sa bng L. acidophilus ban
u 10
7
t m
Kh ng ca S. aureus c chit tht 
      : 510
5
, 10
6
, 2.510
6
 10
6
t  nh
ng S. aureus b   p s khun l   ng
Mannitol Salt Agar. Ghi nhng t i  mm.
n 4
Nghiên cứu khả năng ức chế S. aureus của L. acidophilus
Kh c ch S. aureus ca L. acidophilus ng sa
   c chit tht  peptone   l S. aureus/L. acidophilus =
1/100, 1/50, 1/10, 1/1. Ly mnh s S. aureus sng  theo thi
ng S. aureus bp s khun lc tr
ng Mannitol Salt Agar (ch S. aureus m       
L. acidophilus 