studies on lactobacillus spp. and artemisia extract ... · rhamonosus or l. casei (klb 298) by 16s...

Studies on Lactobacillus spp. and Artemisia extract

inhibiting Staphylococcus aureus

2005 2

()



2005 2

()



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



56



57



58



ix

59



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



Lactobacillus KLB 21274


solvents for 48 hours on the survival of






x







solvents for 48 hours on the survival of









with various solvents for 48 hours on the survival

of Lactobacillus KLB 28682


so lvents for 48 hours on the surv iva l of


xi













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 11. Anti-staphylococcal activity of KLB 298 SCS (spent

culture supernatant) after various enzyme treatment

31

Table 12. Qualitative analysis of H2O2 production43




xiii

Table 16. Quantitative analysis of H2O2 production47




Table 20. Correlation between qualitative of H2O2 production and

antagonistic activity52





Table 23. Correlation between quantitative of H2O2 production

and antagonistic activity61







- 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


, 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 -



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 -



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 -



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 -



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 -



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 -



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 -



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 -



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 -



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 -



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 -



Lactobacillus spp. Medium


KLB 227 1.2 +++

KLB 243 1.2 ++

KLB 271 1.2 -

KLB 301 1.2 +

KLB 304 1.2 +

- 54 -



Lactobacillus spp. Highest


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 -




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 -




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 -




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 -




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 -




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


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 -



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 -



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 -



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,


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.


, 24 Acetone

105 Methanol

102 , Ethanol 103

. 48 Acetone

Methanol, Ethanol 103, 106

(Figure. 28).

KLB 286 1.5108 CFU/ 24,


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,


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.


24 106, 104, 105

, 48 24

(Figure. 32).

KLB 298 8.7106 CFU/ 24,

- 70 -


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.


, 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


- 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


- 74 -


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


(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


- 75 -


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


- 76 -



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


(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


- 77 -



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 -



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


(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


- 79 -



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


- 80 -



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


(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


- 81 -



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


- 82 -



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


(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


- 83 -



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


- 84 -



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


(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


- 85 -



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


- 86 -



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


(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


- 87 -



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


- 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

studies on lactobacillus spp. and artemisia extract ... · rhamonosus or l. casei (klb 298) by 16s...

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