studies on lactobacillus spp. and artemisia extract ... · rhamonosus or l. casei (klb 298) by 16s...
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Studies on Lactobacillus spp. and Artemisia extract
inhibiting Staphylococcus aureus
2005 2
()
Studies on Lactobacillus spp. and Artemisia extract
inhibiting Staphylococcus aureus
2005 2
()
Studies on Lactobacillus spp. and Artemisia extract
inhibiting Staphylococcus aureus
by
Hyun-Soo Jung
A THESIS
Submitted to the faculty of
INHA UNIVERSITY
in partial fulfillment of the requirements
For the degree of
MASTER OF SCIENCE
Department of Biological Engineering
February 2005
.
2005 2
:
:
:
i
.
.
105 .
7 . , Lactobacillus KLB
298 L. rhamonosus or L. casei .
, KLB 298 pH 4.5
. KLB 298
, catalase, lipase, -amylase
proteases (pepsin, proteinase K, trypsin, -chymotrypsin)
, . ,
KLB 298
. ,
.
(ANOVA)
.
.
. Artemisia mongolia fishcher ,
,
. 3 17%
.
.
. ,
.
ii
ABSTRACT
Lactobacilli have been considered to play important roles in the
health of human. They secrete inhibitory substance to prevent
infection by pathogenic organism. In a previous study we have
newly isolated 105 strains of Lactobacillus spp. from human
samples. In this study, using the plate diffusion method 7 strains
showing antagonistic activity against pathogenic Staphylococcus
aureus were selected among them. One strain which had shown
the highest level of antimicrobial activity was identified as L.
rhamonosus or L. casei (KLB 298) by 16S rDNA sequencing. In
mixed culture experiments growth of S. aureus was inhibited by
KLB 298 at the early stationary phase when the culture pH
dropped to 4.5. To characterize the antimicrobial activity cell-
free culture supernatant was treated with catalase, lipase, -
amylase and various proteases (pepsin, proteinase K, trypsin, -
chymotrypsin), none of which inactivated the inhibitory activity
of the supernatant. Therefore, it is likely that the inhibitory
substance of KLB 298 is organic acid by which the culture
becomes acidic so that S. aureus is inhibited. In addition, as
H2O2 produced by lactobacilli has been suggested to be
antimicrobial substance we have examined the correlation
between H2O2 production and antagonistic activity. Based on
both qualitative and quantitative measurement there seems to be
no correlation between H2O2 production and antagonistic activity
of Lactobacillus spp. using ANOVA.
Antimicrobial substances originated from plants have been
investigated to obtain natural sources of antimicrobial activity. In
iii
this study, we performed antimicrobial activity test by using the
extract of Artemisia Mongolia Fishcher on S. aureus along with
lactic acid bacteria, Lactobacillus spp. isolated from human
vagina to determine the possibility of using the extract as vaginal
antiseptics. The Artemisia extract which was extracted with
solvents such a acetone, ethanol, and methanol showed
selective antimicrobial activity against S. aureus. The optimal
anti-staphylococcal activity was observed when the Artemisia
extract (17%) was added. By using broth dilution method, the
Artemisia extract inhibited the growth of S. aureus effectively
while did not inhibit the growth of Lactobacillus spp.. Moreover,
change of cell morphology was observed by scanning electron
microscopy (SEM). As a result, cell morphology of S. aureus was
highly damaged but Lactobacillus spp. was not.
iv
i
ABSTRACTiii
LIST OF FIGURESviii
LIST OF TABLESxii
. 1
1. 1
1.1 1
1.2 MRSA2
(meth ic i l l i n res is tan t S t rep tococcus aureus )
2. 3
2.1 3
2.2 4
3. 5
. 7
. 8
1. 8
2. 8
2.1 8
2.2 9
2.3 9
2.4 pH 9
v
3. 12
3.1 Chromosomal DNA 12
3.2 PCR 16S rDNA 12
3.3 Ligation and transformation13
3.4 Plasmid 16
3.5 16
3.6 Data 17
4. 17
4.1 17
4.2 17
4.3 18
5. 18
5.1 18
5.2 19
5.3 19
. 20
1. 20
1.1 20
1 . 2
20
1.3
20
1 . 4
pH 21
2. 35
3. 40
3.1 40
3.2 40
3.3 51
vi
4. 65
4.1 65
4.2 Acetone, Methanol, Ethanol
65
4.3 Acetone, Methanol, Ethanol 17%
71
. 90
. 93
vii
LIST OF FIGURES
Figure 1. Antagonistic activity of Lactobacillus spp. isolates
against S. aureus27
Figure 2. Mixed and pure culture of Lactobacillus KLB 298 and
S. aureus28
Figure 3. Mixed and pure culture of Lactobacillus KLB 270 and
S. aureus29
Figure 4. Distinguishing S. aureus from Lactobacillus KLB 298
on X-gal plate30
Figure 5. Anti-staphylococcal activity of KLB 298 SCS after
catalase treatment and pH neutralization32
Figure 6. Culture pH changes of Lactobacillus KLB 270, 298
and S. aureus33
Figure 7. Culture pH changes after mixed culture34
Figure 8. Cell type and colony type of Lactobacillus KLB 298
36
Figure 9. 16S rDNA sequence of Lactobacillus KLB 298
38
viii
Figure 10. Sequence homology of Lactobacillus KLB 298
39
Figure 11. Colony color by H2O2 production in Lactobacillus
spp.41
Figure 12. Standard curve for quantitative analysis of H2O2
42
Figure 13. Correlation between quantitative of H2O2 production
and antagonistic activity in Lactobacillus KLB 298
55
Figure 14. Correlation between quantitative of H2O2 production
and antagonistic activity in Lactobacillus KLB 288
56
Figure 15. Correlation between quantitative of H2O2 production
and antagonistic activity in Lactobacillus KLB 271
57
Figure 16. Correlation between quantitative of H2O2 production
and antagonistic activity in Lactobacillus KLB 227
58
Figure 17. Correlation between quantitative of H2O2 production
and antagonistic activity in Lactobacillus KLB 274
ix
59
Figure 18. Correlation between quantitative of H2O2 production
and antagonistic activity in Lactobacillus KLB 233
60
Figure 19. Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of
S. aureus72
Figure 20. Effect of Artemisia 17% extracted with various
so lvents for 48 hours on the surv iva l o f
S. aureus73
Figure 21. Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of
Lactobacillus KLB 21274
Figure 22. Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of
Lactobacillus KLB 21275
Figure 23. Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of
Lactobacillus KLB 22476
Figure 24. Effect of Artemisia 17% extracted with various
x
so lvents for 48 hours on the surv iva l o f
Lactobacillus KLB 22477
Figure 25. Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of
Lactobacillus KLB 23978
Figure 26. Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of
Lactobacillus KLB 23979
Figure 27. Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of
Lactobacillus KLB 26080
Figure 28. Effect of Artemisia 17% extracted with various
so lvents for 48 hours on the surv iva l o f
Lactobacillus KLB 26081
Figure 29. Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival
of Lactobacillus KLB 28682
Figure 30. Effect of Artemisia 17% extracted with various
so lvents for 48 hours on the surv iva l of
Lactobacillus KLB 28683
xi
Figure 31. Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival
of Lactobacillus KLB 28884
Figure 32. Effect of Artemisia 17% extracted with various
so lvents for 48 hours on the surv iva l of
Lactobacillus KLB 28885
Figure 33. Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival
of Lactobacillus KLB 29886
Figure 34. Effect of Artemisia 17% extracted with various
so lvents for 48 hours on the surv iva l of
Lactobacillus KLB 29887
Figure 35. Analysis of morphological changes when Lactobaci
-llus KLB 224 was cultured for 48 hours after
addition of 17% (v/v) Artemisia extracted with
various solvents 100% for 3 days88
Figure 36. Analysis of morphological changes when S. aurues
was cultured for 48 hours after addition of 17%
(v/v) Artemisia extracted with various solvents
100% for 3 days89
xii
LIST OF TABLES
Table 1. Media used in this study10
Table 2. Media used in this study11
Table 3. PCR condition for 16S rDNA amplification14
Table 4. PCR reaction mixture14
Table 5. Ligation mixture component15
Table 6. Screening of Lactobacillus spp. inhibiting S. aureus22
Table 7. Screening of Lactobacillus spp. inhibiting S. aureus23
Table 8. Screening of Lactobacillus spp. inhibiting S. aureus24
Table 9. Screening of Lactobacillus spp. inhibiting S. aureus25
Table 10. Screening of Lactobacillus spp. inhibiting S. aureus26
Table 11. Anti-staphylococcal activity of KLB 298 SCS (spent
culture supernatant) after various enzyme treatment
31
Table 12. Qualitative analysis of H2O2 production43
Table 13. Qualitative analysis of H2O2 production44
Table 14. Qualitative analysis of H2O2 production45
Table 15. Qualitative analysis of H2O2 production46
xiii
Table 16. Quantitative analysis of H2O2 production47
Table 17. Quantitative analysis of H2O2 production48
Table 18. Quantitative analysis of H2O2 production49
Table 19. Quantitative analysis of H2O2 production50
Table 20. Correlation between qualitative of H2O2 production and
antagonistic activity52
Table 21. Correlation between qualitative of H2O2 production and
antagonistic activity53
Table 22. Correlation between qualitative of H2O2 production and
antagonistic activity54
Table 23. Correlation between quantitative of H2O2 production
and antagonistic activity61
Table 24. Correlation between quantitative of H2O2 production
and antagonistic activity62
Table 25. Correlation between quantitative of H2O2 production
and antagonistic activity63
Table 26. Correlation between quantitative of H2O2 production
and antagonistic activity64
- 1 -
.
1.
1.1
Micrococaceae ,
0.8~1.0 . .
.
.
,
,
. (, , ,
), ()
(
) . ,
.
.
(toxic shock syndrome, TSS)
F, (
. , . ).
- 2 -
1.2 Methicillin-resistant Stphylococcus aureus (MRSA)
(Staphylococcus aureus)
40%
,
,
(Francis et al.
1995, Han et al. 1999). , , ,
.
(Hiramatsu et al. 1997). Penicillin,
methicillin
, 1940
penicillin
1950 tetracycline,
chloramphenicol erythromycin
penicillin
methicilin 1961
methicillin
(Lencastre et al. 2000). ,
, penicillin
methicillin( oxacillin, nafcillin)
MRSA(methicillin resistant staphylococcus aureus) .
MRSA
,
. penicillin G beta-
lactam (cephalosporin, ampicillin )
- 3 -
outbreak .
MRSA
teicoplanin vancomycin
. , , ,
.
MRSA (airborne transmission)
(contact transmission) .
. MRSA
.
, , , MRSA
.
MRSA
(. .
. 1996).
2.
2.1
, ,
(Elmer et al. 1996, Pavlova et al. 1997) lactic
acid pH ,
(Eschenbach et al. 1989, Kim et al.
- 4 -
1994). bacteriocin
(Ahn and Stiles.
1990).
probiotics (Jo et al. 1997,
Verellen et al. 1998).
,
. pH 6~8
.
. glycogen ,
lactic acid .
pH 4.5 . glycogen
lactic acid , lactic
acid .
(H2O2) bacteriocin
(Eschenbach et al. 1989).
2.2
(Aroutcheva et al. 2001). Dderlein
acid
(Thomas et al. 1928).
acidolin, lactacin B
(Aroutcheva et al. 2001, Eschenbach
et al. 1989).
(nonacidophilic organism) ,
(acidophilic organism)
- 5 -
(Hillier et al. 1993). heme
cytochrome system
flavoprotein
. peroxidase-
halide catalase peroxidase
.
halide ion enzyme peroxidase
enzyme (lactoperoxidase),
(myeloperoxidase), (eosinophil
peroxidase), (rat uterine fluid, , ,
) (Dahiya et al. 1968, Klebanoff
et al. 1970, Wheater et al. 1952). ,
, ,
(Usui et al. 2002,
Thomas et al. 1928, Mcgroarry et al. 1992).
hydrogen peroxidase-halide antibacterial system
(Aroutcheva et al. 2001).
.
3.
(Artemisia) , , ,
, ,
, ,
,
(. 1980).
- 6 -
, ,
, ,
2,000
,
300
(. 1977).
,
A, B1, B2, C (Fe), (K), (Ca), (P)
, (81.4%), (6.9%),
(3.7%), .
, , (Marrif et al. 1995),
(. 1991, Chihara et al. 1970), ,
( . 1973)
.
, , , ,
, , ,
( . 1989). thujone,
cineol, camphor, limonene, mycene, terpineol ,
, ,
,
( . 1988).
,
,
( . 1994, . 1988).
( . 1991).
- 7 -
.
.
.
, penicillin methicillin
( oxacillin, nafcillin) MRSA(methicillin
resistant staphylococcus aureus)
.
105
.
.
.
.
- 8 -
.
1.
105
.
E. coli JM 109 , S.
aureus KTCC 25175 . Lactobacillus spp., S. aureus,
E. coli JM 109 Modified-MRS, BHI, LB
(Table. 1, 2).
(Artemisia magnolia Fischer) . 100 g
Methanol, Ethanol, Acetone 90%, 95%, 100%
400 1, 2, 3 .
200 .
Evaporator (EYELA rotary vacuum Evaporator) 70
.
2.
2.1
stock 1% 16~18 ,
2 . 1.5% MRS agar plate
5 20 , 1.5% MRS
coating agar 1~2 20 ,
- 9 -
37 5 . , plate 0.7% BHI soft agar
(20 ) 1% 6 20
, 12
.
2.2
1% 37 , 0, 2, 4, 7, 10
CFU/ .
, X-gal 10 (50 /), 0.85% NaCl 70 , cell
100 1.5% MRS agar plate colony
.
2.3
15 .
, , , ,
pH . ,
catalase (1 /), proteinase K (2 /), lipase (0.1 /),
pepsin (1 /), trypsin (1 /, pH 7.5), -amylase (1 /),
-chymotrypsin (1 /, pH 7.5) 37
3 . 5 N NaOH 1 N HCl pH
. , control pH 7.0 catalase
(Miteva et al.,1998).
2.4 pH
Lactobacillus spp. S. aureus pH
.
- 10 -
Table. 1 Media used in this study
Ingredient Modified MRS (g/L)
Lactose
Tryptose
Tryptic soy broth
Yease extract
L-cystein HCl
Ammonium citrate 2H2O
K2HPO4
KH2PO4
Tween 80
FeSO47H2O
MgSO4
MnSO47H2O
10.0 g
3.0 g
10.0 g
5.0 g
0.3 g
3.0 g
3.0 g
2.0 g
1.0 g
35 mg
575 mg
120 mg
Ingredient SDM (g/L)
Lactose
Beef extract
Ammonium citrate 2H2O
K2HPO4
Sodium acetate
Tween 80
MgSO47H2O
MnSO4
20.0 g
5.0 g
2.0 g
2.0 g
5.0 g
1.0 g
0.1 g
0.05 g
- 11 -
Table. 2 Media used in this study
Ingredient SOC
(Amount / 100mL)
Bacto-Tryptone
Bacto-Yeast Extract
NaCl
KCl
2 M Mg2+ stock
(1 M MgCl26H2O,
1 M MgSO47H2O)
2 M Glucose
20 g
5 g
0.58 g
0.19 g
10 mL (Filtration)
10 mL (Filtration)
Ingredient LB
(Amount / 100mL)
Bacto-Tryptone
Yeast Extract
NaCl
1 N NaOH
1 g
0.5 g
0.5 g
0.1 mL
Ingredient Benzidine
(Amount / 100mL)
Brucella broth
Benzidine
Horseradish peroxidase
Hemin
Vitamin K1
Starch
4.3 g
20 mg
1 mg (Filtration)
0.5 g (Filtration)
0.1 g (Filtration)
1 g
- 12 -
3.
3.1 Chromosome DNA
Genomic DNA broth 5
15000 rpm 5 cell pellet PBS
buffer , cell
pellet TE buffer(50 mM Tris-HCl, 20 mM EDTA, pH 8.0)
. 0.4 glass beads (425-600 microns,
Sigma Chemical Co., USA) 3 voltexing.
phenol : chloroform : isoamylalcohol (25 : 24 : 1) 600
30 voltexing 10 .
(400 ) chloroform 500 voltexing
10 (400 ) Ice EtOH 100% 1
3 M sodium acetate 50 1
. , 10 70% EtOH 1
10 dry speed
vacuum 30 EtOH .
10 RNase (option) 50 TE buffer (10 mM Tris-HCl, 1
mM EDTA, pH 8.0) voltexing , .
3.2 PCR (polymer chain reaction) 16S rDNA
PCR 16S rDNA(1.6 kb) universal primer
UNI primer(5CCCAAGCTTAGAGTTTGATCCTGGCTCAG3)
UNI primer(5ACGCGTCGACAAGGAGGTGATCCAGCC3)
. PCR reaction mixture template DNA(1
), UNIprimer(1 ), UNIprimer(1 ), ddNTP(1 ), Taq
polymerase(1 ), Taq polymerase buffer(5 ), ddH2O(40 )
- 13 -
(Table. 3). PCR 94 3
preheating, 94 1 denaturation, 56 40
primer annealing, 72 2 30 polymerase extension,
72 5 final elongation (Table. 4).
3.3 Ligation and transformation
Ligation mixture pGEM-T easy vector(1 ), PCR product(3
), ligase(0.5 ), ligase buffer(5 ), ddH2O(0.5 )
water bath 2 ligation(Table. 5).
E. coli JM 109 fresh LB 5 10%
shaking incubator (37, 250 rpm) . E.
coli JM 109 7000 rpm 10 .
0.1 M MgCl2 5 , voltexing 7000 rpm
10 0.1 M CaCl2 5
voltexing. 4 20 , 7000 rpm
10 , 0.1 M CaCl2 200
microtube competent cell
. Ligation 10 competent cell 100
, 4 1 . ,
42 water bath 1 30, ice water 1 heat-shock
microtube SOC 500 rotary
incubator 1 .
, pellet IPTG
(isopropylthiogalactoside) 10 mM 20 X-gal (5Br-4-Cl-
3-Indoyl-beta-D-galactoside) 15 LB (Amp+) agar
plate 37 ,
transformation cell white colony .
- 14 -
Table. 3 PCR condition for 16S rDNA amplification
Table. 4 PCR reaction mixture
PCR reaction mixture component Composition
Template DNA 1
UNI primer 1
UNI primer 1
dd NTP 1
Taq polymerase 1
Taq polymerase buffer 5
ddH2O 40
Thermal condition for PCR
Procedure Temperature Time
Pre-denaturation 94
Denaturation 94 3 min
Annealing 56
Extension 72
1 min
40 sec
2 min 30 sec
30 cycle
Final extension 72 5 min
- 15 -
Table. 5 Ligation mixture
Ligation mixture component Composition
pGEM-T easy vector 1
PCR product 3
Ligase 0.5
Ligase buffer 5
ddH2O 0.5
- 16 -
3.4 Plasmid
Transformation white colony LB 5 Amp+ (50 /
) 5 rotary incubator E. coli JM 109
2 microtube 1.5 15000 rpm 3
. sol 100
, sol 200 5~6
5 . sol 150 ,
10 15000 rpm 10
. microtube phenol : chloroform :
isoamylalcohol (25 : 24: 1) (450 ) (450 )
voltexing , 15000 rpm 3
(350 ) (*). RNase 5
30 37 (*)
. 100% EtOH ( 2) 400 3 M
sodium acetate ( 1/10) 20 microtube
voltexing , -20 15
150000 rpm 10 .
70% EtOH 1 10
15000 rpm . DNA pellet speed-vacuum
10~15 , TE buffer (1) 50
voltexing 4 . ,
plasmid .
3.5
Cloning vector EcoR (1 ) 10 buffer (1 )
plasmid (8 ) 37 2
. Vector band (3 kb) insert (1.6 kb) UV
illuminator . Sequencing
- 17 -
Bionex .
3.6 Data
BLAST (http://www. ncbi. nlm, nih. gov) 16S rDNA
sequences homology .
4.
4.1
H2O2 Benzidine
(Table. 2). H2O2 Hoseradish peroxidase
benzidine ,
(), (+), (++), (+++)
.
1.5% Benzidine agar plate 5 spotting ,
37 .
(Aroutcheva et al., 2001).
, colony color pH, O.D. (600 nm)
.
4.2
H2O2 SDM
(Table. 1). H2O2 330 nm absorption peak
peroxomolybdic acid complex molybdate
, molybdate
ion 350 nm
- 18 -
. 2.4 mmol/L molybdate 0.5 mol/L H2SO4 (250 )
0.10 g (NH4)Mo7O244H2O 3%
hydrogen peroxide solution Standard curve
(Chai et al., 2004).
SDM 30 2% (v/v) shaking
incubator (37, 250 rpm) . 0 blank
dH2O(1 ) (100 )
O.D. , sample molydate(1 ) (100
) O.D. . blank
dH2O(1 ) (100 )
, sample 0 O.D. .
6 pH, CFU/,
. standard curve
(Figure. 12).
4.3
ANOVA (analysis of variance)
5% (Juarez
Tomas et al., 2003).
5.
5.1
S. aureus 0.7% BHI soft agar 6 agar plate
, (Acetone, Methanol,
Ethanol), (90%, 95%, 100%) (1, 2, 3)
- 19 -
plate spotting 12
( ., 1997, ., 2003).
5.2
MRS broth 10 1%
, 100% (Acetone, Methanol, Ethanol) 3
8.3%, 12.5%, 17% (v/v) 0, 24, 48
. S. aureus BHI broth
10 1%
. , control
100% (Acetone, Methanol, Ethanol) 8.3%, 12.5%, 17% (v/v)
.
5.3
Lactobacillus sp. S. aureus
, 1% 3
100% 17% 48
. cell
pellet . coverglass 70% EtOH
, PB buffer cell pellet
. 2.5% glutaraldehyde PB buffer 1:4(v/v)
4 3~4 , 0.01 M PB
buffer 3 PB buffer .
50%, 70%, 90%, 95%, 100% (2) Ethanol 10
, . SEM(Scanning
Electron Microscopy, Hitach-4200, Inha univ., Incheon, Korea)
(Jan et al., 2001).
- 20 -
.
1.
1.1
101 Lactobacillus spp. S. aureus
, 7
(Table. 6~10). 7 KLB 212, KLB
224, KLB 239, KLB 260, KLB 286, KLB 288, KLB 298,
2.1~2.4 cm
(Figure. 1).
1.2
Lactobacillus KLB 298
, S. aureus KLB
298 S. aureus
7 15
(Figure. 2). Lactobacillus KLB 270 S.
aureus S. aureus
(Figure. 3). colony .
, S. aureus , KLB 298
(Figure. 4).
- 21 -
1.3
KLB 298 ,
, proteinase K (2 /),
lipase (0.1 /), pepsin (1 /), trypsin (1 /, pH 7.5), -
amylase (1 /), -chymotrypsin (1 /, pH 7.5)
S. aureus (Table.
11).
, catalase (1 /)
. pH 7.0
. pH 7.0 catalase
pH 7.0 (Figure.
5).
1.4 pH
Lactobacillus KLB 298 KLB 270 S. aureus
, 0~15 pH KLB 270 S.
aureus pH KLB 298
(Figure. 6). KLB 270 S. aureus
pH KLB 298 S. aureus
pH 10
(Figure. 7).
- 22 -
Table. 6 Screening of Lactobacillus spp. inhibiting S. aureus
Lactobacillus spp. Inhibition zone (cm)
KLB 201 1.7
KLB 202 1.8
KLB 203 0.0
KLB 204 1.1
KLB 205 1.4
KLB 206 1.4
KLB 207 0.0
KLB 208 0.0
KLB 209 1.7
KLB 210 1.0
KLB 211 1.3
KLB 212 2.4
KLB 213 1.7
KLB 214 1.8
KLB 215 1.4
KLB 216 1.9
KLB 217 1.0
KLB 218 1.5
KLB 219 1.3
KLB 220 1.7
KLB 221 1.4
KLB 222 2.0
KLB 224 2.2
KLB 225 2.0
- 23 -
Table. 7 Screening of Lactobacillus spp. inhibiting S. aureus
Lactobacillus spp. Inhibition zone (cm)
KLB 226 1.0
KLB 227 1.2
KLB 228 1.8
KLB 229 1.8
KLB 230 1.7
KLB 231 1.4
KLB 232 1.9
KLB 233 0.0
KLB 235 1.0
KLB 236 1.4
KLB 237 1.4
KLB 238 1.0
KLB 239 2.4
KLB 240 1.5
KLB 241 1.8
KLB 242 1.9
KLB 243 1.2
KLB 244 1.8
KLB 245 1.4
KLB 246 1.0
KLB 247 1.3
KLB 248 1.7
KLB 249 2.0
- 24 -
Table. 8 Screening of Lactobacillus spp. inhibiting S. aureus
Lactobacillus spp. Inhibition zone (cm)
KLB 250 1.1
KLB 251 1.5
KLB 252 1.0
KLB 253 1.3
KLB 254 1.4
KLB 255 1.5
KLB 256 1.0
KLB 257 0.0
KLB 258 1.7
KLB 259 0.0
KLB 260 2.2
KLB 261 2.0
KLB 262 1.5
KLB 263 1.4
KLB 264 1.9
KLB 265 1.8
KLB 266 1.4
KLB 267 1.8
KLB 268 1.8
KLB 269 1.4
KLB 270 0.0
KLB 271 1.2
KLB 272 1.8
KLB 273 1.8
- 25 -
Table. 9 Screening of Lactobacillus spp. inhibiting S. aureus
Lactobacillus spp. Inhibition zone (cm)
KLB 274 0.0
KLB 275 1.1
KLB 276 1.1
KLB 277 1.4
KLB 278 1.1
KLB 279 0.0
KLB 280 1.4
KLB 282 1.8
KLB 283 1.8
KLB 284 0.0
KLB 285 2.0
KLB 286 2.1
KLB 287 0.0
KLB 288 2.2
KLB 289 1.7
KLB 290 1.0
KLB 292 1.8
KLB 293 1.7
KLB 294 1.1
KLB 295 1.9
KLB 296 2.0
KLB 297 0.0
KLB 298 2.1
KLB 299 0.0
- 26 -
Table. 10 Screening of Lactobacillus spp. inhibiting S. aureus
Lactobacillus spp. Inhibition zone (cm)
KLB 300 0.0
KLB 301 1.2
KLB 302 1.0
KLB 304 1.2
KLB 305 0.0
KLB 306 1.1
- 27 -
Figure. 1 Antagonistic activity of Lactobacillus spp. isolates
against S. aureus ; (A) Control (KLB 270), (B) KLB 298, (C)
KLB 288
(A)
(B) (C)
- 28 -
Figure. 2 Mixed and pure culture of Lactobacillus KLB 298 and
S. aureus
Time (hours)
0 2 4 6 8 10 12 14 16
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
S. aureus ( pure culture )Lactobacillus sp. KLB 298 ( pure culture )S. aureus ( mixed culture )Lactobacillus sp. KLB 298 ( mixed culture )
- 29 -
Figure. 3 Mixed and pure culture of Lactobacillus KLB 270 and
S. aureus
Time (hours)
0 2 4 6 8 10 12 14 16
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
S. aureus ( pure culture )L. fermentum KLB 270 ( pure culture )S. aureus ( mixed culture )L. fermentum KLB 270 ( mixed culture )
- 30 -
(B)
(A)
Figure. 4 Distinguishing S. aureus (A) and Lactobacillus KLB
298 (B) on X-gal plate ; (A) Yellow colonies are S. aureus, (B)
Blue colonies are Lactobacillus KLB 298
- 31 -
Table. 11 Anti-staphylococcal activity of KLB 298 SCS (spent
culture supernatant) after various enzyme treatment
Enzyme treatment Activity change
Pepsin (1 mg/mL) -
- chymotrypsin (1 mg/mL) -
Trypsin (1 mg/mL) -
Proteinase K (2 mg/mL) -
Lipase (0.1 mg/mL) +/-
- amylase (1 mg/mL) +/-
- 32 -
Figure. 5 Anti-staphylococcal activity of KLB 298 SCS after
catalase treatment and pH neutralization
Time (hours)
0 2 4 6 8 10 12
CFU
/ m
L
1e+6
1e+7
1e+8
1e+9
1e+10
with catalasewith pH 7.0controlwith catalase and pH 7.0
- 33 -
Figure. 6 Culture pH changes of Lactobacillus KLB 270, 298
and S. aureus
Time (hours)
0 5 10 15
pH
3.5
4.0
4.5
5.0
5.5
6.0
6.5
L. fermentum KLB 270L. crispatus KLB 298S. aureus
- 34 -
Figure. 7 pH change after mixed culture
Time (hours)
0 5 10 15 20 25
pH
3.5
4.0
4.5
5.0
5.5
6.0
6.5
L. fermentum KLB 270 + S. aureusL. crispatus KLB 298 + S. aureus
- 35 -
2.
KLB 298 cell type
colony type (Figure. 8).
16S rDNA L.
rhamnosus L. casei (Figure. 9, 10).
- 36 -
Figure. 8 Cell type (A) and colony type (B) of Lactobacillus KLB
298
- 37 -
ORIGIN
1 GATGAACGCT GGCGGCGTGC CTAATACATG CAAGTCGAAC GAGTTCTGAT
51 TATTGAAAGG TGCTTGCATC TTGATTTAAT TTTGAACGAG TGGCGGACGG
101 GTGAGTAACA CGTGGGTAAC CTGCCCTTAA GTGGGGGATA ACATTTGGAA
151 ACAGATGCTA ATACCGCATA AATCCAAGAA CCGCATGGTT CTTGGCTGAA
201 AGATGGCGTA AGCTATCGCT TTTGGATGGA CCCGCGGCGT ATTAGCTAGT
251 TGGTGAGGTA ACGGCTCACC AAGGCAATGA TACGTAGCCG AACTGAGAGG
301 TTGATCGGCC ACATTGGGAC TGAGACACGG CCCAAACTCC TACGGGAGGC
351 AGCAGTAGGG AATCTTCCAC AATGGACGCA AGTCTGATGG AGCAACGCCG
401 CGTGAGTGAA GAAGGCTTTC GGGTCGTAAA ACTCTGTTGT TGGAGAAGAA
451 TGGTCGGCAG AGTAACTGTT GTCGGCGTGA CGGTATCCAA CCAGAAAGCC
601 ACGGCTAACT ACGTGCCAGC AGCCGCGGTA ATACGTAGGT GGCAAGCGTT
651 ATCCGGATTT ATTGGGCGTA AAGCGAGCGC AGGCGGTTTT TTAAGTCTGA
701 TGTGAAAGCC CTCGGCTTAA CCGAGGAAGT GCATCGGAAA CTGGAAAACT
751 TGAGTGCAGA AGAGGACAGT GGAACTCCAT GTGTAGCGGT GAAATGCGTA
801 GATATATGGA AGAACACCAG TGGCGAAGGC GGCTGTCTGG TCTGTAACTG
851 ACGCTGAGGC TCGAAAGCAT GGGTAGCGAA CAGGATTAGA TACCCTGGTA
901 GTCCATGCCG TAAACGATGA ATGCTAAGGT GTTGGAGGGT TTCCGCCCTT
951 CAGTGCCGCA GCTAACGCAT TAAGCATTCC GCCTGGGGAG TACGACCGCA
1001 AGGTTGAAAC TCAAAGGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT
1051 GTGGTTTAAT TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCTT
1101 TTGATCACCT GAGAGATCGG GTTTTCCCTT CGGGGGCAAA ATGACAGGTG
1151 GTGCATGGTT GTCGTCAGCT CGTGTCGTGA GATGTTGGGT TAAGTCCCGC
1201 AACGAGCGCA ACCCTTACGA CTAGTTGCCA GCATTTAGTT GGGCACTCTA
1251 GTAAGACTGC CGTGACAAAC CGGAGGAAGG TGGGGATGAC GTCAAATCAT
1301 CATGCCCCTT ATGACCTGGG CTACACACGT GCTACAATGG ATGGTACAAC
1351 GAGTTGCGAG ACCGCGAGGT CAAGCTAATC TCTTAAAGCC ATTCTCAGTT
1041 CGGACTGTAG GCTGCAACTC GCCTACACGA AGTCGGAATC GCTAGTAATC
1091 GCGGATCAGC ACGCCGCGGT GAATACGTTC CCGGGCCTTG TACACACCGC
1141 CCGTCACACC ATGAGAGTTT GTAACACCCG AAGCCGGTGG CGTAACCCTT
- 38 -
1191 TTAGGGAGCG AGCCGTCTAA GGTGGGACAA ATGATTAGGG TGAAGTCGTA
1213 ACAAGGTAGC CGTAGGAGAA CCTGC
Figure. 9 16S rDNA sequences of Lactobacillus KLB 298
- 39 -
>gi|27464420|gb|AF526388.1|
Lactobacillus casei 16S ribosomal RNA gene, partial sequence
Length = 1562
Score = 2946 bits (1486), Expect = 0.0
Identities = 1518/1526 (99%), Gaps = 2/1526 (0%)
Strand = Plus / Plus
>gi|38455821|gb|AY370680.1|
Lactobacillus rhamnosus strain LCG SHARDINGER 16S
ribosomal RNA gene, partial sequence; 16S-23S intergenic
spacer, complete sequence; and 23S ribosomal RNA gene, partial
sequence
Length = 1814
Score = 2930 bits (1478), Expect = 0.0
Identities = 1507/1514 (99%), Gaps = 2/1514 (0%)
Strand = Plus / Plus
Figure. 10 Sequence homology of Lactobacillus KLB 298
- 40 -
3.
3.1
101
(), (+), (++), (+++)
colony color (Figure. 11).
29 (Table.
12~15). pH O.D. (600 nm)
4.42 1.416.
3.2
83 6 pH
(Table. 16~19). pH
5.98, 1.94108 CFU/,
1.6510-4 mM.
- 41 -
Figure. 11 Colony color by H2O2 production in Lactobacillus
spp. ; from left to right ++, +, -, +++
- 42 -
Figure. 12 Standard curve for quantitative analysis of H2O2
Hydrogen peroxide (mM)
0.000 0.005 0.010 0.015 0.020
Abs
orba
nce
at 3
50 n
m
0.0
0.2
0.4
0.6
0.8
1.0
1.2
In 1mL 2.4 mmol/L Ammonium molybdate solutioiny = 52.0864*x + 0.0093 , r = 0.9996
- 43 -
Table. 12 Qualitative analysis of H2O2 production
Lactobacillus spp. H2O2 pH O.D. (600 nm)
KLB 201 - 3.88 1.822
KLB 202 - 3.87 1.857
KLB 203 + 5.97 0.183
KLB 204 +++ 5.32 0.759
KLB 205 - 3.79 1.503
KLB 206 ++ 4.23 1.527
KLB 207 - 3.87 1.913
KLB 208 ++ 3.23 2.011
KLB 209 +++ 5.62 0.803
KLB 210 - 3.95 1.907
KLB 211 ++ 3.29 1.925
KLB 212 + 3.37 2.174
KLB 213 - 3.16 2.208
KLB 214 - 3.17 2.219
KLB 215 - 3.18 2.111
KLB 216 - 3.17 2.198
KLB 217 ++ 3.21 2.043
KLB 218 - 3.16 2.213
KLB 219 - 2.89 2.175
KLB 220 - 2.88 2.182
KLB 221 - 2.87 2.145
KLB 222 + 3.18 2.024
KLB 223 + 3.53 1.484
KLB 224 + 3.37 2.174
KLB 225 - 3.09 2.023
KLB 226 +++ 5.43 0.915
- 44 -
Table. 13 Qualitative analysis of H2O2 production
Lactobacillus spp. H2O2 pH O.D. (600 nm)
KLB 227 +++ 4.19 1.472
KLB 230 +++ 3.85 1.688
KLB 231 +++ 3.59 1.692
KLB 232 + 3.13 1.388
KLB 233 +++ 3.09 1.355
KLB 235 +++ 3.18 1.678
KLB 236 +++ 3.91 1.442
KLB 237 +++ 3.34 1.779
KLB 238 +++ 3.02 2.019
KLB 239 - 3.80 1.839
KLB 240 ++ 3.41 1.215
KLB 241 +++ 2.92 1.924
KLB 242 ++ 3.25 1.815
KLB 243 ++ 2.88 1.903
KLB 244 - 3.37 0.753
KLB 245 ++ 3.83 2.133
KLB 246 ++ 4.94 0.773
KLB 247 - 3.60 1.663
KLB 248 ++ 5.44 0.805
KLB 249 - 3.81 2.185
KLB 250 - 5.34 1.416
KLB 251 ++ 3.97 2.025
KLB 252 +++ 3.95 1.653
KLB 253 +++ 3.94 1.968
KLB 254 +++ 5.45 0.797
KLB 255 +++ 5.45 0.950
- 45 -
Table. 14 Qualitative analysis of H2O2 production
Lactobacillus spp. H2O2 pH O.D. (600 nm)
KLB 256 + 4.04 2.120
KLB 257 +++ 5.43 0.915
KLB 258 - 3.76 2.118
KLB 259 +++ 5.42 0.658
KLB 260 - 3.80 1.867
KLB 261 - 4.34 1.833
KLB 262 +++ 3.92 1.993
KLB 263 ++ 4.51 1.431
KLB 264 - 4.03 1.953
KLB 265 + 3.93 1.933
KLB 266 +++ 5.44 0.921
KLB 267 - 3.79 1.989
KLB 268 +++ 6.18 0.325
KLB 269 - 3.67 2.051
KLB 270 ++ 5.40 1.065
KLB 271 - 3.78 2.173
KLB 272 +++ 3.73 1.983
KLB 273 +++ 3.73 2.012
KLB 274 - 3.91 1.903
KLB 275 +++ 3.96 2.032
KLB 276 + 4.24 1.775
KLB 277 + 3.81 2.113
KLB 278 + 5.68 0.875
KLB 279 +++ 4.20 1.740
KLB 280 + 4.13 2.058
KLB 281 - 4.19 2.119
- 46 -
Table. 15 Qualitative analysis of H2O2 production
Lactobacillus spp. H2O2 pH O.D. (600 nm)
KLB 282 - 4.28 2.084
KLB 283 +++ 4.35 2.015
KLB 284 +++ 5.76 1.136
KLB 285 - 4.32 2.040
KLB 286 - 3.70 1.792
KLB 287 - 4.26 1.875
KLB 288 ++ 5.31 1.049
KLB 289 - 5.64 1.510
KLB 290 ++ 5.87 1.047
KLB 292 - 4.54 1.985
KLB 293 - 3.89 2.207
KLB 294 - 3.88 2.289
KLB 295 - 4.14 1.984
KLB 296 ++ 4.04 2.065
KLB 297 - 4.15 2.065
KLB 298 - 3.79 1.850
KLB 299 + 4.02 2.005
KLB 300 ++ 4.03 2.060
KLB 301 + 3.75 2.077
KLB 302 +++ 5.69 0.522
KLB 304 + 3.98 2.001
KLB 305 ++ 3.28 2.104
KLB 306 +++ 4.26 1.932
- 47 -
Table. 16 Quantitative analysis of H2O2 production
Lactobacillus spp. H2O2 pH CFU / mL
KLB 201 3.5910-4 5.74 1.80108
KLB 202 1.6710-4 5.73 2.40108
KLB 203 1.2910-4 5.58 1.53108
KLB 204 1.2910-4 5.62 1.73108
KLB 205 2.4410-4 5.68 2.20108
KLB 206 1.2910-4 6.25 2.60107
KLB 207 0.0 5.69 1.87108
KLB 208 0.0 6.18 1.67108
KLB 209 1.0910-4 5.59 4.07108
KLB 210 2.6310-4 5.87 1.93108
KLB 211 1.2910-4 5.69 1.47108
KLB 212 0.0 6.00 1.87108
KLB 213 1.3410-5 5.52 3.07108
KLB 214 1.8610-4 6.14 2.27108
KLB 215 3.6010-4 6.33 5.33107
KLB 216 2.0510-4 6.29 4.00107
KLB 217 1.0910-4 6.23 1.60108
KLB 218 0.0 6.09 1.33108
KLB 219 1.8610-4 6.39 2.47108
KLB 220 3.2110-4 5.57 2.13108
KLB 221 0.0 6.58 1.20108
KLB 224 4.5510-4 6.16 1.80108
KLB 225 2.8210-4 5.85 2.93108
KLB 226 0.0 6.17 2.67107
KLB 227 3.2110-4 5.80 2.93108
KLB 230 5.7010-4 6.66 1.93108
- 48 -
Table. 17 Quantitative analysis of H2O2 production
Lactobacillus spp. H2O2 pH CFU / mL
KLB 231 1.0910-4 5.42 4.20108
KLB 232 9.0210-4 6.07 2.40108
KLB 233 0.0 5.58 3.60108
KLB 235 1.3410-4 6.19 9.33107
KLB 236 9.0210-5 6.26 7.33107
KLB 237 0.0 5.84 3.73108
KLB 238 7.1010-5 6.12 1.33108
KLB 239 7.1010-5 5.49 3.47108
KLB 240 3.0110-4 5.33 8.00108
KLB 242 3.0110-4 6.34 9.33107
KLB 243 2.0510-4 5.65 4.13108
KLB 244 2.2510-4 6.07 1.20108
KLB 247 3.2010-4 6.33 9.33107
KLB 248 2.0510-4 5.97 1.27109
KLB 249 1.6710-4 6.00 2.93108
KLB 250 0.0 6.38 7.33107
KLB 251 1.0910-4 4.99 4.67108
KLB 253 2.6310-4 6.22 6.87108
KLB 254 9.0210-4 6.12 1.00108
KLB 255 1.3010-3 5.74 2.60108
KLB 256 0.0 6.35 4.00107
KLB 257 3.2610-5 6.27 4.20107
KLB 258 0.0 6.25 1.00108
KLB 260 2.4410-4 6.28 5.20107
KLB 261 0.0 5.74 1.67108
KLB 263 2.4410-4 5.97 1.27108
- 49 -
Table. 18 Quantitative analysis of H2O2 production
Lactobacillus spp. H2O2 pH CFU / mL
KLB 264 0.0 5.89 3.27107
KLB 265 7.1010-5 4.92 1.40108
KLB 266 5.7010-4 5.91 3.67107
KLB 267 7.1010-5 6.19 3.07107
KLB 268 0.0 6.46 1.27107
KLB 270 0.0 6.46 1.93107
KLB 271 0.0 6.49 2.47107
KLB 274 0.0 5.57 2.53108
KLB 275 1.1510-3 6.47 5.47107
KLB 276 0.0 6.48 5.33106
KLB 277 3.2610-5 5.49 2.13108
KLB 278 3.5910-4 6.07 2.67107
KLB 282 2.0510-4 6.6 1.13107
KLB 283 0.0 5.52 5.40108
KLB 285 2.4410-4 6.73 6.00107
KLB 286 5.1810-5 5.75 5.13108
KLB 288 1.3410-5 5.57 3.73108
KLB 290 1.6710-4 6.27 3.13107
KLB 292 3.2610-5 5.35 2.73108
KLB 293 3.5910-4 5.96 2.53108
KLB 294 0.0 5.79 3.80108
KLB 295 3.2610-5 5.46 8.67107
KLB 296 7.1010-5 6.15 3.93107
KLB 297 2.0510-4 5.46 3.73108
KLB 298 1.4810-4 5.59 3.80108
KLB 299 0.0 6.45 5.33106
- 50 -
Table. 19 Quantitative analysis of H2O2 production
Lactobacillus spp. H2O2 pH CFU / mL
KLB 300 0.0 6.48 6.67106
KLB 301 1.0910-4 6.37 1.53107
KLB 302 4.5510-4 6.1 1.40108
KLB 305 2.2510-4 6.37 8.07107
KLB 306 0.0 5.22 2.67108
- 51 -
3.3.
(0.0 cm) 15 9
KLB 208, 233, 257, 259, 270, 279, 284, 300, 305
(++, +++) (Table. 20).
(1.2 cm) 5 2 KLB 227,
243 (++, +++) (Table. 21).
(2.1~2.4 cm) 7 1 KLB 288
(++) (Table. 22).
, KLB
298(2.1 cm) 6 H2O2 3.8910-13 mM/CFU,
pH 5.59 3.8108 CFU/, KLB
288(2.2 cm) 6 H2O2 3.6010-14 mM/CFU,
pH 5.57 3.73108 CFU/
(Figure. 13, 14).
, KLB 271(1.2 cm)
H2O2 0 mM/CFU , KLB 227(1.2 cm) 6
H2O2 1.010-12 mM/CFU, pH 5.57
2.93108 CFU/ (Figure. 15, 16).
, KLB 274(0.0 cm) 3
H2O2 2.0510-13 mM/CFU, pH 6.49
1.07107 CFU/, KLB 233(0.0 cm) 10
H2O2 3.0310-13 mM/CFU, pH 5.57
4.67108 CFU/ (Figure. 17, 18). 83
6
(Table. 23~26).
- 52 -
Table. 20 Correlation between qualitative of H2O2 production
and antagonistic activity
Lactobacillus spp. No
antimicrobial activity (cm)Colony color
KLB 203 0.0 +
KLB 207 0.0 -
KLB 208 0.0 ++
KLB 233 0.0 +++
KLB 257 0.0 +++
KLB 259 0.0 +++
KLB 270 0.0 ++
KLB 274 0.0 -
KLB 279 0.0 +++
KLB 284 0.0 +++
KLB 287 0.0 -
KLB 297 0.0 -
KLB 299 0.0 +
KLB 300 0.0 ++
KLB 305 0.0 ++
- 53 -
Table. 21 Correlation between qualitative of H2O2 production
and antagonistic activity
Lactobacillus spp. Medium
antimicrobial activity (cm)Colony color
KLB 227 1.2 +++
KLB 243 1.2 ++
KLB 271 1.2 -
KLB 301 1.2 +
KLB 304 1.2 +
- 54 -
Table. 22 Correlation between qualitative of H2O2 production
and antagonistic activity
Lactobacillus spp. Highest
antimicrobial activity (cm)Colony color
KLB 212 2.2 +
KLB 224 2.2 +
KLB 239 2.4 -
KLB 260 2.1 -
KLB 286 2.2 -
KLB 288 2.2 ++
KLB 298 2.3 -
- 55 -
Figure. 13 Correlation between quantitative of H2O2 production
and antagonistic activity (2.3 cm) in Lactobacillus KLB 298 ; pH
() , Hydrogen peroxide (), CFU ()
Time (hours)
0 5 10 15 20 25 30
Hyd
roge
n pe
roxi
de (m
M)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
pH
3
4
5
6
7
CFU
1e+6
1e+7
1e+8
1e+9
1e+10
- 56 -
Figure. 14 Correlation between quantitative of H2O2 production
and antagonistic activity (2.2 cm) in Lactobacillus KLB 288 ; pH
() , Hydrogen peroxide (), CFU ()
Time (hours)
0 5 10 15 20 25 30
Hyd
roge
n pe
roxi
de (m
M)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
pH
3
4
5
6
7
CFU
1e+6
1e+7
1e+8
1e+9
1e+10
- 57 -
Figure. 15 Correlation between quantitative of H2O2 production
and antagonistic activity (1.2 cm) in Lactobacillus KLB 271 ; pH
() , Hydrogen peroxide (), CFU ()
Time (hours)
0 5 10 15 20 25 30
Hyd
roge
n pe
roxi
de (m
M)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
pH
3
4
5
6
7
CFU
1e+6
1e+7
1e+8
1e+9
1e+10
- 58 -
Figure. 16 Correlation between quantitative of H2O2 production
and antagonistic activity (1.2 cm) in Lactobacillus KLB 227 ; pH
() , Hydrogen peroxide (), CFU ()
Time (hours)
0 5 10 15 20 25 30
Hyd
roge
n pe
roxi
de (m
M)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
pH
3
4
5
6
7
CFU
1e+6
1e+7
1e+8
1e+9
1e+10
- 59 -
Figure. 17 Correlation between quantitative of H2O2 production
and antagonistic activity (0.0 cm) in Lactobacillus KLB 274 ; pH
() , Hydrogen peroxide (), CFU ()
Time (hours)
0 5 10 15 20 25 30
Hyd
roge
n pe
roxi
de (m
M)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
pH
3
4
5
6
7
CFU
1e+6
1e+7
1e+8
1e+9
1e+10
- 60 -
Figure. 18 Correlation between quantitative of H2O2 production
and antagonistic activity (0.0 cm) in Lactobacillus KLB 233 ; pH
() , Hydrogen peroxide (), CFU ()
Time (hours)
0 5 10 15 20 25 30
Hyd
roge
n pe
roxi
de (m
M)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
pH
3
4
5
6
7
CFU
1e+6
1e+7
1e+8
1e+9
1e+10
- 61 -
Table. 23 Correlation between quantitative of H2O2 production
and antagonistic activity
Lactobacillus
spp.
H2O2
(mmol/ 10-12 CFU)
Inhibition zone
(cm)
KLB 201 1.99 1.7
KLB 202 0.70 1.8
KLB 203 0.84 0.0
KLB 204 0.74 1.1
KLB 205 0.11 1.4
KLB 206 0.50 1.4
KLB 207 0.0 0.0
KLB 208 0.0 0.0
KLB 209 0.27 1.7
KLB 210 1.36 1.0
KLB 211 0.88 1.3
KLB 212 0.0 2.4
KLB 213 0.04 1.7
KLB 214 0.82 1.8
KLB 215 6.75 1.4
KLB 216 5.13 1.9
KLB 217 0.68 1.0
KLB 218 0.0 1.5
KLB 219 0.75 1.3
KLB 220 1.51 1.7
KLB 221 0.0 1.4
KLB 224 2.53 2.2
KLB 225 0.96 2.0
KLB 226 0.0 1.0
- 62 -
Table. 24 Correlation between quantitative of H2O2 production
and antagonistic activity
Lactobacillus
spp.
H2O2 / CFU
(mmol/ 10-12 CFU)
Inhibition zone
(cm)
KLB 227 1.10 1.2
KLB 232 0.38 1.9
KLB 233 0.0 0.0
KLB 235 0.14 1.0
KLB 236 1.23 1.4
KLB 237 0.0 1.4
KLB 238 0.53 1.0
KLB 239 0.20 2.4
KLB 240 0.38 1.5
KLB 242 3.23 1.9
KLB 243 0.50 1.2
KLB 244 1.88 1.8
KLB 247 3.43 1.3
KLB 248 0.16 1.7
KLB 249 0.57 2.0
KLB 250 0.0 1.1
KLB 251 0.23 1.5
KLB 253 3.83 1.3
KLB 254 0.90 1.4
KLB 255 5.00 1.5
KLB 256 0.0 1.0
KLB 257 0.78 0.0
KLB 258 0 1.7
KLB 260 4.69 2.2
- 63 -
Table. 25 Correlation between quantitative of H2O2 production
and antagonistic activity
Lactobacillus
spp.
H2O2 / CFU
(mmol/ 10-12 CFU)
Inhibition zone
(cm)
KLB 261 0.0 2.0
KLB 264 0.0 1.9
KLB 265 0.51 1.8
KLB 266 15.55 1.4
KLB 267 2.32 1.8
KLB 268 0.0 1.8
KLB 270 0.0 0.0
KLB 271 0.0 1.2
KLB 274 0.0 0.0
KLB 275 21.04 1.1
KLB 276 0.0 1.1
KLB 277 0.15 1.4
KLB 278 13.47 1.1
KLB 282 18.09 1.8
KLB 283 0.0 1.8
KLB 285 4.07 2.0
KLB 286 0.10 2.1
KLB 288 0.04 2.2
KLB 290 5.33 1.0
KLB 292 0.12 1.8
KLB 293 1.42 1.7
KLB 294 0.0 1.1
KLB 295 0.38 1.9
KLB 296 1.81 2.0
- 64 -
Table. 26 Correlation between quantitative of H2O2 production
and antagonistic activity
Lactobacillus
spp.
H2O2 / CFU
(mmol/ 10-12 CFU)
Inhibition zone
(cm)
KLB 297 0.55 0.0
KLB 298 0.39 2.1
KLB 300 0.0 0.0
KLB 301 7.11 1.2
KLB 302 3.25 1.0
KLB 305 2.79 0.0
KLB 306 0.0 1.1
- 65 -
4.
4.1
Acetone, Methanol, Ethanol
(90%, 95%, 100%) (1, 2, 3)
100%,
3 .
.
4.2 Acetone, Methanol, Ethanol
KLB 212, 224, 239, 260, 286, 288, 298
Acetone, Methanol, Ethanol (100%) 3
8.3%, 12.5%, 17% 0, 24, 48
.
3.8107 CFU/ 24, 48
6.73109, 5.6109 CFU/. Methanol 8.3%
24, 48 9.3107, 3.5107
CFU/ 102 . Acetone
6.7104, 1.1105 CFU/ 105, 104
, Ethanol 6.0104, 2.7104 CFU/
105 (Figure. 19(A)). Acetone, Methanol,
Ethanol 12.5% 24, 48
Acetone 6.0104, 2.7104 CFU/ 105
, Ethanol 6.7105, 1.9
104 CFU/ 104, 105 . Methanol
4.0106, 3.6105 CFU/ 103, 104
- 66 -
(Figure. 19(B)). Acetone, Methanol, Ethanol
17% 24
106, 106, 107 48
(Figure. 20).
KLB 212 4.3107 CFU/ 24, 48
3.4109, 1.9108 CFU/. Acetone, Methanol,
Ethanol 8.3% , 24, 48
Acetone 8.7108, 1.0109 CFU/ 101
, Methanol 4.0109,
3.1109 CFU/ 100, 101 .
Ethanol 2.5109, 2.5109 CFU/ 100
(Figure. 21(A)). Ethanol
12.5% 24, 48 1.1
109, 8.9108 CFU/ 100 , Acetone
6.5106, 4.0105 CFU/ 103
. Methanol 2.1106,
8.7103 CFU/ 103, 105 (Figure.
21(B)). Acetone, Methanol, Ethanol 17%
24 Acetone
, Methanol 104
, Ethanol 107 .
48 Acetone, Ethanol ,
Methanol 107 (Figure. 22).
KLB 224 6.5107 CFU/ 24,
48 3.4109, 1.7108 CFU/. Acetone, Methanol,
Ethanol 8.3% 24, 48
Acetone 2.1109, 2.1109 CFU/ 100,
101 , Methanol
3.6109, 1.1109 CFU/ 100, 101
. Ethanol 3.3109, 1.0109
- 67 -
CFU/ 100, 101 (Figure.
23(A)). Acetone, Methanol, Ethanol 12.5%
24, 48 Acetone 9.0
108, 7.8109 CFU/ 101
, Methanol 8.2108, 2.3107 CFU/
101 . Ethanol
4.7108, 7.1108 CFU/ 101, 100
(Figure. 23(B)). Acetone, Methanol, Ethanol
17% 24
101 , 48
Acetone, Ethanol
Methanol 101
(Figure. 24).
KLB 239 1.73109 CFU/ 24,
48 2.0109, 3.3108 CFU/. Acetone, Methanol,
Ethanol 8.3% , 24, 48
Acetone 3.3104, 0 CFU/ 105, 0
, Methanol
2.0106, 1.8109 CFU/ 103, 101
. Ethanol 2.7105, 1.1107
CFU/ 104, 101 (Figure. 25(A)). 12.5%
,
1.1108 CFU/ 24, 48 1.7
1010, 5.3109 CFU/. Acetone
12.5% 24, 48 8.0104, 3.3103
CFU/ 106 , Methanol
3.2106, 8.7104 CFU/ 104, 105
. Ethanol 1.5107, 2.1
105 CFU/ 103, 104 (Figure. 25(B)).
Acetone, Methanol, Ethanol 17%
- 68 -
, 24 Acetone
104 Methanol, Ethanol
103 . 48 Acetone
Methanol, Ethanol 104
(Figure. 26).
KLB 260 4.0107 CFU/ 24, 48
7.5108, 1.9109 CFU/. Acetone, Methanol,
Ethanol 8.3% , 24, 48
Acetone 8.7105, 2.7104 CFU/ 103,
105 , Methanol 1.6
107, 2.7108 CFU/ 101 . Ethanol
6.7108, 1.4109 CFU/ 100
(Figure. 27(A)). Ethanol 12.5%
24, 48 2.3108, 1.1108 CFU/
100, 101 , Acetone
8.0104, 6.7102 CFU/ 104, 107
. Methanol 4.1107,
4.7107 CFU/ 101, 102 (Figure.
27(B)). Acetone, Methanol, Ethanol 17%
, 24 Acetone
105 Methanol
102 , Ethanol 103
. 48 Acetone
Methanol, Ethanol 103, 106
(Figure. 28).
KLB 286 1.5108 CFU/ 24,
48 1.81010, 1.9109 CFU/. Acetone, Methanol,
Ethanol 8.3% 24, 48
Acetone 6.0108, 2.1108 CFU/ 102
, Methanol 3.0109,
- 69 -
3.7109 CFU/ 101, 100 .
Ethanol 1.8109, 4.1108 CFU/ 101
(Figure. 29(A)). Acetone, Methanol,
Ethanol 12.5% 24, 48
Acetone 1.8107, 1.8107 CFU/ 103,
102 , Methanol 1.8
107, 4.4106 CFU/ 103 .
Ethanol 2.7107, 2.7106 CFU/ 103
(Figure. 29(B)). Acetone, Methanol,
Ethanol 17%
24 105, 104, 105 ,
48 104, 104, 105 (Figure. 30).
KLB 288 5.4107 CFU/ 24,
48 6.0109, 2.7109 CFU/. Acetone, Methanol,
Ethanol 8.3% , 24, 48
Acetone 3.4108, 7.3108 CFU/ 101,
101 , Methanol 1.7
108, 1.1109 CFU/ 101, 100
. Ethanol 2.6107, 5.3108 CFU/
102, 101 (Figure. 31(A)). Ethanol
12.5% 24, 48 6.0
106, 5.3106 CFU/ 103 , Acetone
7.3106, 1.7108 CFU/ 103,
101 . Methanol
8.0106, 4.0106 CFU/ 103 (Figure.
31(B)). Acetone, Methanol, Ethanol 17%
24 106, 104, 105
, 48 24
(Figure. 32).
KLB 298 8.7106 CFU/ 24,
- 70 -
48 2.21010, 4.0109 CFU/. Acetone, Methanol,
Ethanol 8.3% , 24, 48
Acetone 5.3104, 3.3104 CFU/ 106,
105 , Methanol 1.1
107, 4.9109 CFU/ 103, 100
. Ethanol 6.7105, 1.3104 CFU/
105 (Figure. 33(A)). Acetone
12.5% 24, 48 4.7105, 0
CFU/ 105 , Methanol
1.5106, 5.3104 CFU/ 104,
105 . Ethanol
7.3105, 4.0104 CFU/ 105 (Figure.
33(B)). Acetone, Methanol, Ethanol 17%
, 24 Acetone
106 Methanol
104 , Ethanol 104
. 48 Acetone
Methanol, Ethanol 105, 104
(Figure. 34).
- 71 -
4.3 Acetone, Methanol, Ethanol 17%
Lactobacillus KLB 224 3
Acetone, Methanol, Ethanol (100%)
17% 48 SEM
(Figure. 35).
Methanol
Acetone, Ethanol
(Figure. 36).
- 72 -
Figure. 19 Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of S. aureus
(A)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
(B)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 73 -
Figure. 20 Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of S. aureus
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 74 -
Figure. 21 Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of Lactobacillus
KLB 212
(A)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
(B)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 75 -
Figure. 22 Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of Lactobacillus KLB 212
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 76 -
Figure. 23 Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of Lactobacillus
KLB 224
(A)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
(B)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 77 -
Figure. 24 Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of Lactobacillus KLB 224
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%Wtih ethanol 100%
- 78 -
Figure. 25 Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of Lactobacillus
KLB 239
(A)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
ControlWith acetone 100%With methanol 100%With ethanol 100%
(B)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 79 -
Figure. 26 Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of Lactobacillus KLB 239
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 80 -
Figure. 27 Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of Lactobacillus
KLB 260
(A)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
ControlWith acetone 100%With methanol 100%With ethanol 100%
(B)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 81 -
Figure. 28 Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of Lactobacillus KLB 260
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 82 -
Figure. 29 Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of Lactobacillus
KLB 286
(A)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
(B)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 83 -
Figure. 30 Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of Lactobacillus KLB 286
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 84 -
Figure. 31 Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of Lactobacillus
KLB 288
(A)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
(B)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 85 -
Figure. 32 Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of Lactobacillus KLB 288
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 86 -
Figure. 33 Effect of Artemisia (A) 8.3%, (B) 12.5% extracted
with various solvents for 48 hours on the survival of Lactobacillus
KLB 298
(A)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
(B)
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 87 -
Figure. 34 Effect of Artemisia 17% extracted with various
solvents for 48 hours on the survival of Lactobacillus KLB 298
Time (hours)
0 10 20 30 40 50 60
CFU
/ m
L
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
ControlWith acetone 100%With methanol 100%With ethanol 100%
- 88 -
Figure. 35 Analysis of morphological changes when Lactobacillus
KLB 224 was cultured for 48 hours after addition of 17% (v/v)
Artemisia extracted with various solvents 100% for 3 days ; (A)
Nontreated cells, (B) Cell treated with acetone extract, (C) Cell
treated with methanol extract, (D) Cell treated with ethanol
extract
(A)
(C) (D)
(B) (A)
- 89 -
Figure. 36 Analysis of morphological changes when S. aurues was
cultured for 48 hours after addition of 17% (v/v) Artemisia
extracted with various solvents 100% for 3 days ; (A) Nontreated
cell, (B) Cell treated with acetone extract, (C) Cell treated with
methanol extract, (D) Cell treated with ethanol extract
(A) (B)
(C) (D)
- 90 -
.
105
7 ,
KLB 298 Lactobacillus
rhamnosus or casei . KLB
298 pH ,
, ,
.
Chai et
al. (2004) .
reference .
(Collins et al. 1980, Dahiya et al.
1968, Price et al. 1969).
7 KLB 288
, 15
KLB 208, 233, 257, 259, 270, 279, 284, 300, 305
.
.
.
. 3 Acetone, Methanol, Ethanol
(100%)
17%.
Acetone Ethanol
- 91 -
. (100%) 3
17% (v/v) 48
KLB 224
. SEM
. KLB 224
. Kwun
C.
perfringens E. coli
, Han 60
Bifidobacterum sp. C. perfringens
,
( . 1997, . 1994).
,
200 ppm
( . 1999),
Acetone, Methanol, Ethanol 17%
48
.
100% ,
KLB 224
. (data
not shown).
.
KLB 224
- 92 -
.
.
metronidazole
1 6070% ,
metronidazole 30%
3 (Holmes et al. 1999, Hillier et al.
1990). Hillier 83%
1 65%
.
(Hillier et al. 1993).
.
.
.
- 93 -
.
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. 1. 1.1 1.2 MRSA(methicillin resistant Streptococcus aureus)
2. 2.1 2.2
3.
. . 1. 2. 2.1 2.2 2.3 2.4 pH
3. 3.1 Chromosomal DNA 3.2 PCR 16S rDNA 3.3 Ligation and transformation3.4 Plasmid 3.5 3.6 Data
4. 4.1 4.2 4.3
5. 5.1 5.2 5.3
. 1. 1.1 1.2 1.3 1.4 pH
2. 3. 3.1 3.2 3.3
4. 4.1 4.2 Acetone, Methanol, Ethanol 4.3 Acetone, Methanol, Ethanol 17%
. .
[-1] Media used in this study[-2] Media used in this study[-3] PCR condition for 16S rDNA amplification[-4] PCR reaction mixture[-5] Ligation mixture component[-6] Screening of Lactobacillus spp. inhibiting S. aureus[-7] Screening of Lactobacillus spp. inhibiting S. aureus[-8] Screening of Lactobacillus spp. inhibiting S. aureus[-9] Screening of Lactobacillus spp. inhibiting S. aureus[-10] Screening of Lactobacillus spp. inhibiting S. aureus[-11] Anti-staphylococcal activity of KLB 298 SCS (spent culture supernatant) after various enzyme treatment[-12] Qualitative analysis of H2O2 production[-13] Qualitative analysis of H2O2 production[-14] Qualitative analysis of H2O2 production[-15] Qualitative analysis of H2O2 production[-16] Quantitative analysis of H2O2 production[-17] Quantitative analysis of H2O2 production[-18] Quantitative analysis of H2O2 production[-19] Quantitative analysis of H2O2 production[-20] Correlation between qualitative of H2O2 production and antagonistic activity[-21] Correlation between qualitative of H2O2 production and antagonistic activity[-22] Correlation between qualitative of H2O2 production and antagonistic activity[-23] Correlation between quantitative of H2O2 production and antagonistic activity[-24] Correlation between quantitative of H2O2 production and antagonistic activity[-25] Correlation between quantitative of H2O2 production and antagonistic activity[-26] Correlation between quantitative of H2O2 production and antagonistic activity
[-1] Antagonistic activity of Lactobacillus spp. Isolates against S. aureus[-2] Mixed and pure culture of Lactobacillus KLB 298 and S. aureus[-3] Mixed and pure culture of Lactobacillus KLB 270 and S. aureus[-4] Distinguishing S. aureus from Lactobacillus KLB 298 on X-gal plate[-5] Anti-staphylococcal activity of KLB 298 SCS after catalase treatment and pH neutralization[-6] Culture pH changes of Lactobacillus KLB 270, 298 and S. aureus[-7] Culture pH changes after mixed culture[-8] Cell type and colony type of Lactobacillus KLB 298[-9] 16S rDNA sequence of Lactobacillus KLB 298[-10] Sequence homology of Lactobacillus KLB 298[-11]Colony color by H2O2 production in Lactobacillus spp[-12] Standard curve for quantitative analysis of H2O2[-13] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 298[-14] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 288[-15] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 271[-16] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 227[-17] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 274[-18] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 233[-19] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of S. aureus[-20] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of S. aureus[-21] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 212[-22] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 212[-23] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 224[-24] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 224[-25] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 239[-26] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 239[-27] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 260[-28] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 260[-29] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 286[-30] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 286[-31] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 288[-32] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 288[-33] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 298[-34] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 298[-35] Analysis of morphological changes when Lactobaci -llus KLB 224 was cultured for 48 hours after addition of 17% (v/v) Artemisia extracted with various solvents 100% for 3 days[-36] Analysis of morphological changes when S. aurues was cultured for 48 hours after addition of 17% (v/v) Artemisia extracted with various solvents 100% for 3 days
. 1 1. 1 1.1 1 1.2 MRSA(methicillin resistant Streptococcus aureus) 2 2. 3 2.1 3 2.2 4 3. 5. 7. 8 1. 8 2. 8 2.1 8 2.2 9 2.3 9 2.4 pH 9 3. 12 3.1 Chromosomal DNA 12 3.2 PCR 16S rDNA 12 3.3 Ligation and transformation 13 3.4 Plasmid 16 3.5 16 3.6 Data 17 4. 17 4.1 17 4.2 17 4.3 18 5. 18 5.1 18 5.2 19 5.3 19. 20 1. 20 1.1 20 1.2 20 1.3 20 1.4 pH 21 2. 35 3. 40 3.1 40 3.2 40 3.3 51 4. 65 4.1 65 4.2 Acetone, Methanol, Ethanol 65 4.3 Acetone, Methanol, Ethanol 17% 71. 90. 93
[-1] Media used in this study 10[-2] Media used in this study 11[-3] PCR condition for 16S rDNA amplification 14[-4] PCR reaction mixture 14[-5] Ligation mixture component 15[-6] Screening of Lactobacillus spp. inhibiting S. aureus 22[-7] Screening of Lactobacillus spp. inhibiting S. aureus 23[-8] Screening of Lactobacillus spp. inhibiting S. aureus 24[-9] Screening of Lactobacillus spp. inhibiting S. aureus 25[-10] Screening of Lactobacillus spp. inhibiting S. aureus 26[-11] Anti-staphylococcal activity of KLB 298 SCS (spent culture supernatant) after various enzyme treatment 31[-12] Qualitative analysis of H2O2 production 43[-13] Qualitative analysis of H2O2 production 44[-14] Qualitative analysis of H2O2 production 45[-15] Qualitative analysis of H2O2 production 46[-16] Quantitative analysis of H2O2 production 47[-17] Quantitative analysis of H2O2 production 48[-18] Quantitative analysis of H2O2 production 49[-19] Quantitative analysis of H2O2 production 50[-20] Correlation between qualitative of H2O2 production and antagonistic activity 52[-21] Correlation between qualitative of H2O2 production and antagonistic activity 53[-22] Correlation between qualitative of H2O2 production and antagonistic activity 54[-23] Correlation between quantitative of H2O2 production and antagonistic activity 61[-24] Correlation between quantitative of H2O2 production and antagonistic activity 62[-25] Correlation between quantitative of H2O2 production and antagonistic activity 63[-26] Correlation between quantitative of H2O2 production and antagonistic activity 64
[-1] Antagonistic activity of Lactobacillus spp. Isolates against S. aureus 27[-2] Mixed and pure culture of Lactobacillus KLB 298 and S. aureus 28[-3] Mixed and pure culture of Lactobacillus KLB 270 and S. aureus 29[-4] Distinguishing S. aureus from Lactobacillus KLB 298 on X-gal plate 30[-5] Anti-staphylococcal activity of KLB 298 SCS after catalase treatment and pH neutralization 32[-6] Culture pH changes of Lactobacillus KLB 270, 298 and S. aureus 33[-7] Culture pH changes after mixed culture 34[-8] Cell type and colony type of Lactobacillus KLB 298 36[-9] 16S rDNA sequence of Lactobacillus KLB 298 38[-10] Sequence homology of Lactobacillus KLB 298 39[-11]Colony color by H2O2 production in Lactobacillus spp. 41[-12] Standard curve for quantitative analysis of H2O2 42[-13] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 298 55[-14] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 288 56[-15] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 271 57[-16] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 227 58[-17] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 274 59[-18] Correlation between quantitative of H2O2 production and antagonistic activity in Lactobacillus KLB 233 60[-19] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of S. aureus 72[-20] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of S. aureus 73[-21] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 212 74[-22] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 212 75[-23] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 224 76[-24] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 224 77[-25] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 239 78[-26] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 239 79[-27] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 260 80[-28] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 260 81[-29] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 286 82[-30] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 286 83[-31] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 288 84[-32] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 288 85[-33] Effect of Artemisia (A) 8.3%, (B) 12.5% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 298 86[-34] Effect of Artemisia 17% extracted with various solvents for 48 hours on the survival of Lactobacillus KLB 298 87[-35] Analysis of morphological changes when Lactobaci -llus KLB 224 was cultured for 48 hours after addition of 17% (v/v) Artemisia extracted with various solvents 100% for 3 days 88[-36] Analysis of morphological changes when S. aurues was cultured for 48 hours after addition of 17% (v/v) Artemisia extracted with various solvents 100% for 3 days 89