untitled


2005 2



2005 2
Eun-Suk Lee
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF MASTER OF
NATURAL SCIENCE
2005. 2.
2. Marin Broth Streptomyces sp. JR1 ··············································· 7
3. Streptomyces sp. JR1 ····················································································· 7
1) Marin Broth 2216 ···································································································· 7
2) ±MgCl2 broth ············································································································· 8
4. MgCl2 Streptomyces sp. JR1 ··························· 12
5. pH red-brown pigmentation ····························································· 12
6. ························································································································· 13
1) ··················································································································· 13
2) ··················································································································· 13
7. DPPH ············································································ 14
8. Melanin ········································································································ 14
9. Viability (MTT assay) ·········································································· 15
III. ············································································································ 16
1. Streptomyces sp. JR1 ··········· 16
2. Streptomyces sp. JR1 growth rate pigmentation ··········· 18
3. pH pigmentation ····················································· 20
4. ························································································································· 21
5. DPPH radical ··················································································· 23
6. melanin ··································································· 25
7. Cell viability (MTT assay) ················································································· 28
IV. ····························································································································· 31
- i -
ABSTRACT
However, They are true bacteria-prokaryotic cells-unlike eukaryotic fungal
cells. As Actinomycetes grow, they form branching filaments of cells which
become a network of strands called a mycelium, similar in appearance to the
mycelium of some fungi.
Actinomycetes are the source of several useful antibiotics that used not only
in the treatment of various human and animal diseases but also in agriculture
as metabolic poisons. At least 70 of the approximately 100 marketed
antibiotics used for the treatment of infections in human are derived from
substances produces by Streptomyces sp..
A marin Streptomyces sp. JR1 which produced red-brown pigment was
isolated from coastal area in Jeju.
We studied that red-brown pigmentation condition of Streptomyces sp. JR1
and antibacterial activities of organic extract of cultured cell and broth.
The optimal pH of medium and fermentation temperature were pH 7.0~7.5
and 27, respectively. The presence of magnesium chloride on Marin Broth
improved the production of the pigment. The maximum absorption wavelength
(λmax)of red-brown pigment was 510 .
The organic extract of cultured cell and broth inhibited the growth of
Staphylococcus aureus and Bacilus subtilis, Bacilus cereus, but the inhibitory
action was weak compared to the ampicillin. The organic extract of cultured
cell and broth inhibited the melanin synthesis of B16F10 melanoma cell.
- ii -
Table 2. Whole Cell Sugar Patterns of Actinomycete ··············································· 2
Table 3. Antibiotics produced by Streptomyces ·························································· 4
Table 4. Chemical dependence of medium color of Streptomyces sp. JR1. ·· 16
Table 5. Results of test of antibacterial activity ······················································ 22
Table 6. DPPH free radical scavenging effects of extracts of
Marin Broth ············································································································ 23
List of Schemes
Scheme 1. Flow chart for the extraction and separation of Marin Broth ·· 8,9
Scheme 2. Flow chart for the extraction and separation of Marin Broth added
MgCl2 ·············································································································· 10
Scheme 3. Flow chart for the extraction and separation of Marin Broth removed
MgCl2 ·············································································································· 11
List of Figures
Figure 1. Streptomyces sp. JR1 on the agar plate added MgCl2 (left) or
removed MgCl2 (right) ··················································································· 17
Figure 2. Streptomyces sp. JR1 on the added MgCl2 (left) or removed
MgCl2 (right) Broth ························································································· 17
Figure 3. Streptomyces sp. JR1 growth rate on ±MgCl2 broth ······················ 18
Figure 4. Time course of pigmentation produced by Streptomyces sp. JR1 19
Figure 5. Difference Visible Spectrum of pigmentation at ±MgCl2 broth ··· 19
Figure 6. Effect of initial pH on the pigment production ···································· 20
Figure 7. Time dependence of pigmentation at different Inhitial pH of
culture medium ································································································· 21
Figure 8. DPPH free radical scavenging effects of Marin Broth extracts ···· 24
Figure 9. Comparison of DPPH free radical scavenging effects between added
and removed MgCl2 Broth extracts ·························································· 24
Figure 10. Melanin contents Inhibition of Marin Broth extracts on B16F10
melanoma cell ·································································································· 26
Figure 11. Melanin contents Inhibition of added and removed MgCl2 Broth
extracts on B16F10 melanoma cell ························································ 27
Figure 12. Cell viability Inhibition of Marin Broth extracts on B16F10
melanoma cell ································································································ 29
Figure 13. Cell viability Inhibition of Marin Broth extracts on Detroit-551
fibroblast cell ································································································· 30
, ,
, , , ,
.1),2),3)
75% ,

. 20
64% , 25.6%
10% , 82%
.1)
Actinomycetales order
gram
, . bacteria fungi

, .4)
, solid agar plate ,
agar plate
(substrate mycelium) , (septa)
(nucleoid) . (thallus)
. plate
(condidia) (aerial mycelium) .
(condiospore) , (sporangium)
(sporangiospore) .
.5)
, tetrapeptide side chain 3
, interpeptide bridge glycine
peptidoglycan 4 (Table 1).
- 2 -
type ,
(Table 2). ,
, , DNA G+C,
. 5)
T a b l e 1 . C e l l wa l l t y p e s o f A c t i n o m y c e t e s
Cell wall
glactose Saccharmonospora
T a b l e 2 . Wh o l e C e l l Su g a r Pa t t e r n s o f A c t i n o m y c e s t e s
Sugar Pattern Typesa Characteristic Sugars Repressentative Genera
A Arabinose, glactose Novcadia,
Actionplanes, Frankia
aCharacteristic sugar patterns are present only in wall types-IV, those actinomycetes
with meso-diaminopimelic acid bMadurose is 3-O-methyl-D-galactose.
- 3 -
Berger's Mannal of Determinative Bacteriology 8 group
, Nocardioform Matilocular Sporangia Actinoplatenes
Streptomycetes Maduromycetes Thermomonospora Thermoactinomycetes
Others . 16s rDNA/rRNA
.
streptomyces
,
. 0.50.1 ,
. Filament .5),6)7)
Streptomyces conidia endospore()
. Streptomyces
, .
Streptomyces . Streptomyces
. , , ,
, , .
Streptomyces . 500
, 50 , , (Table 3).
,
. 6)
Steptomyces

. ,
.8)

.
, ,
,

,
- 4 -
.
.8),9)
T a b l e 3 . A n t i b i o t i c s p r o d u c e d b y Streptomyces
Chemical
Classes
Common
Aminoglycosides Streptomycin S. griseus Most Gram negatives
Spectinomycin Streptomyces
topical applications due to toxicity
Tetracyclines Tetracycline S.aureofaciens
Macrolides Erythromycin S.erytherus Most Gram positives, frequently used
in place of penicilin, Legionella
Clindamycin S.lincolnesis Effective against obligate anaerobes,
especially Bacteroides fragilis.
Amphocetin B S.nodosus Fungi
typhoid fever.
* Most antibiotics are effective against several different bacteria. The entries in this
column refer to the most frequent clinical application of a given antibiotic.
,
Streptomyces sp. JR1
.
Streptomyces sp. JR1
Marin Broth red-brown pigment
, pigmentation ,
- 5 -
Streptomyces sp. JR1
.
- 6 -
1 .
Streptomyces sp. JR1 Marin Broth 2216 (Difco co. USA)
. Streptomyces sp. JR1 pigment
Bacto Agar, Bacto Peptone (BD, USA), Bacto Yeast Extract (Difco
co. USA), Sodium Chloride, Potassium Chloride, Sodium phosphate, Dibasic (Sigma,
USA), Strontium Chloride hexahydrate, Sodium metasilicate, Sodium Flouride
(Sigma-Aldrich, USA), Magnessium Chloride hexahydrate, Boric Acid (Merck,
Gemany) Sodium sulfate, Calcium Chloride, Potassium Bromide (Shinyo, Japan)
Sodium Bicarbonate (Amresco. USA) .
LB medium ,
gram Escherichia Coli (KCTC 1682) gram Staphylococcus
aureus (KCTC 1621), Bacillus cereus (KCTC 1012), Bacillus subtilis (KCTC
1021), Streptococcus pyogenes (KCTC 3096) KCTC (Korean Collection for
Type Cultures) , ()
Incubator(JS/IN/180) Shaking Incubator (JS-SKI-1000) .
Melanin contents viability B16F10 melanoma cell KCLB
(Korean Cell Line Bank) , 100 units/ penicillin-streptomycin,
10% fetal bovine serum(FBS) DMEM 37, 5% CO2
. 3 , 47
. melanin 3-(4,5-dimethylthiazol-2-gel)-2,5-diphenyltetrazolium
bromide (MTT) Sigma(USA) .
DPPH free radical DPPH
ascorbic acid, BHT(Butylated Hydroxytoluene) Sigma(USA)
.
- 7 -
2 . M a r i n B r o t h Streptomyces s p . J R 1
Marin Broth 2216 , Difco manual
Marin Broth . Difco manual
Streptomyces sp. JR1
28 .
3 . Streptomyces s p . J R 1
Streptomyces sp. JR1 Marin Broth 2216 MgCl2
27
(12,000 rpm, 4, 30 min) filtering()
.
1) Marin Broth 2216
acetone
powder .
filtering pH paper pH 8.0
Ethyl acetate(EtOAc), Chloroform(CHCl3), Buthanol(BuOH)
powder , HCl
pH 4.5 , EtOAC, CHCl3, BuOH
7 sample (Scheme 1-b).
acetone , acetic acid EtOAC
, CHCl3, BuOH
, 4 sample (Scheme 1-a).
- 8 -
11 sample test
DPPH free radical test , B16F10 melanoma cell
melanin contents viability (MTT assay) .
2) ±MgCl2
pH paper pH 8.0
EtOAc, CHCl3, BuOH powder
, HCl pH 4.5 , EtOAc,
CHCl3, BuOH 6 12
sample (Scheme 2, 3).
12 sample test DPPH free radical
test , B16F10 melanoma cell melanin contents
.
+ Acetone + EtOAc
Residue BuOH ext. (b-3)
Scheme 1. Flow chart for the extraction and separation of Marin Broth.
- 9 -
b.
Streptomyces sp. JR1 Marin Broth (pH>7.5)
(mycelium) + EtOAc
Residue BuOH ext. (b-2)
Scheme 1. Flow chart for the extraction and separation of Marin Broth.
- 10 -
Streptomyces sp. JR1 Broth (pH>7.5)
(mycelium) + EtOAc
Residue BuOH ext. (PB2)
Scheme 2. Flow chart for the extraction and separation of Marin Broth added
MgCl2 .
- 11 -
Streptomyces sp. JR1 Broth (pH>7.5)
(mycelium) + EtOAc
Residue BuOH ext. (NB2)
Scheme 3. Flow chart for the extraction and separation of Marin Broth
removed MgCl2 .
- 12 -
4 . M g C l 2 Streptomyces s p . J R 1
Marin broth MgCl2 (negative broth) (positive
broth) 50 .
Streptomyces sp. JR1 single colony PBS (pH 7.4)
200 27 shaking incubator 8 .
1 , 8
6 5 filtering
110 dry oven , 1 450
( 450 melanin ).
,
pigmentation .
5 . p H r e d - b r o wn p i g m e n t a t i o n
Streptomyces sp. JR1 red-brown pigmentation pH
pH 5.0 pH 9.0 0.5 pH Marin Broth
. Streptomyces sp. JR1 Marin Broth ( pigment )
pH2 , 27 shaking incubator (200 rpm)
.
red-brown pigment 510
pH pigmentation .
Filtration ,
110 , 510 ,
pH pigment pH
.
gram Bacillus cereus, Bacillus subtilis,
Micrococcus luteus, Staphylococcus aureus, Streptococcus pyogenes gram
Escherichia coli .
37 , colony
3 37 shaking incubator
. 10-1, 10-2, 10-3 , 37
, .
2 )
23 sample ±MgCl2 broth BuOH sample 4
(PB1, PB2, NB1, NB2) sample Dimethyl sulfoxide(DMSO)
2 /, 1 /, 500 /, 250 / 4
, 4 sample .
disc sample 30
. , DMSO 30 (BuOH 4 sample 30 )
, Ampicilin sample
.
3 )
50 top Agar 2 LB plate
sample 30 disc 37
. 2448 sample
disc clear zone .
- 14 -
7 . D PPH
23 sample 2 /, 1 /, 500 /, 250 /, 125 /
, 62.5 / 6 , ascorbic acid BHT
.
515 8.91.1 DPPH solution 180
sample 20 10 ELISA Reader 515
, DPPH . , sample
, Ethanol(EtOH) 180 sample 20 sample ,
ELISA Reader 515 .
DPPH - {(DPPH+sample ) -(sample )}
In h i b i t i o n = x 100
( DPPH )
8 . M e l a n i n
B16F10 melanoma cell 2x105 cells/dish 60 culture dish
CO2 incubator 37, 10 . 10 dish
sample 2 / 20 / , CO2 incubator
37, 3 .
Sample 0.25% Trypsin-EDTA ,
200 1N NaOH (95, 5).
(Sigma Chemical Co.) 10mg 1N NaOH stock solution(1 /)
700 /, 300 /, 100 /, 70 /, 30 /, 10 /, 7 /, 3 /, 1 /
, 0.1 /, 0 / standard solution . Sample
standard solution 96well plate 450
- 15 -

.
9 . V i a b i l i t y ( M T T a s s a y )
B16F10 melanoma cell 5x104 cells/well 96 well-plate CO2
incubator 37, 10 . , well
sample 2 / 20 / CO2 Incubator
37, 3 .
3 MTT solution (50/) well 50 , 4 37
, formazan DMSO ELISA Reader 540
.
, Detroit-551 fibroblast cell
2x104 cells/ml MTT assay
viability .
III.
1 . Streptomyces s p . J R 1

Difco manual Marin Broth
Streptomyces sp. JR1
27 , Marin Broth
, MgCl2
(negative)
(Table 4).
T a b l e 4 . C h e m i c a l s d e p e n d e n c e o f m e d i u m c o l o r o f streptomyces s p . J R 1
R e m o v e d c h e m i c a l s C o l o r c h a n g e R e m o v e d c h e m i c a l s C o l o r c h a n g e
Ferric Citrate + Potassium Bromide +
Sodium Chloride + Strontium Chloride +
Magnessium Chloride - Boric Acid +
Sodium Sulfate + Sodium silicate +
Calcium Chloride + Sodium fluoride +
Potassium chloride + Ammonium Nitrate +
Sodium Bicarbonate + Disodium phosphate +
** + : color change - : no color change
Streptomyces sp. JR1 Marin Broth (positive broth) MgCl2
Marin Broth (negative broth) 27 ,
MgCl2 Streptomyces sp. JR1
- 17 -
, MgCl2 negative broth
(Fig. 1, 2).
F i g . 1 . Streptomyces sp. JR1 on the agar plate added MgCl2 (left) or removed
MgCl2 (right)
F i g . 2 . Streptomyces s p . J R 1 o n t h e M a r i n b r o t h a d d e d M g C l 2 ( l e f t ) a n d
r e m o v e d M g C l 2 ( r i g h t ) .
- 18 -
2 . Streptomyces s p . J R 1 g r o wt h r a t e p i g m e n t a t i o n

Streptomyces sp. JR1 MgCl2 positive negative 27
24 5 , positive
negative (Fig 3). MgCl2
.
, 450
, positive , 2 ,
negative 4 (Fig 4).
MgCl2 ,
red-brown pigment .
7 positive negative ,
pigmentation positive , 510 , pigmentation
negative 420 (Fig 5).
F i g . 3 . Streptomyces sp. J R 1 g r o wt h r a t e o n ± M g C l 2 b r o t h
0
10
20
w e ig
Positive
negative
* Growth rate of Streptomyces sp. JR1 was determined at positive or negative
broth everyday. Amount of bacteria was measured after drying precipitated 5
cells at 110.
- 19 -
F i g . 4 . T i m e c o u r s e o f Pi g m e n t a t i o n p r o d u c e d b y Streptomyces s p . J R 1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
days
>
positive
negative
* Pigmentation of Streptomyces sp. JR1 was measured at positive or negative
broth everyday. Pigment was determined with UV/VIS spectrophotometer at 450.
F i g . 5 . Visible Difference Spectrum of pigmentation at ±MgCl2 broth
*Visible difference spectrum of pigments produced by Streptomyces sp. JR1
against marine broth after 7 days culture. λmax of pigment at negative broth was
420 , while that at positive broth was 510 .
- 20 -
3 . p H p i g m e n t a t i o n
Red-brown pigmentation Marin Broth pH 5.0 pH 9.0 0.5
Streptomyces sp. JR1 27
pigmentation
red-brown pigmentation (Fig 6).
pH pigmentation
, , 5
(Fig 7).
, 7 pH pH , pH
7.88.2 .
pH 8.0 pH
, pH 8.0 pigmentation .
F i g . 6 . E f f e c t o f i n i t i a l p H o n t h e p i g m e n t p r o d u c t i o n
* Streptomyces sp. JR1 was cultured at different initial pH for 6 days at 27.
- 21 -
F i g . 7 . Time dependence of pigmentation at different Inhitial pH of
culture medium
A b
s o
m
pH8.5
pH9.0
*Streptomyces sp. JR1 was cultured at different initial pH for 7 days at 27. Five
milliliter of broth was taken and amount of pigment produced at each pH was
measured at 510 .
4 .
1 ) G r a m
Streptomyces sp. JR1 acetone Marin Broth
Gram Staphylococus aures, Bacillus cereus, Bacillus subtilis,
Streptococcus pyogenes Table 5
. MgCl2 data
, Bacillus cereus, Bacillus subtilis EtOAc CHCl3
.
- 22 -
2 ) G r a m
Streptomyces sp. JR1 acetone Marin Broth
Gram Escherichia coli
.
T a b l e 5 . R e s u l t s o f t e s t s o f a n t i b a c t e r i a l a c t i v i t y ( u n i t s : )
sample
(2/)
Ampr 1620 78 24 1620
a-1 - - - - -
e-3 (0.1) - (2) -
a: acetone ext., b: BuOH ext., c: CHCl3 ext., e: EtOAc ext.
- 23 -
5 . D PPH r a d i c a l
DPPH free radical
, ascorbic acid
. BHT e-3
sample . (Table 6, Fig 8)
, MgCl2 , MgCl2
, pigment ,
ascorbic acid BHT
(Fig 9).
T a b l e 6 . D PPH f r e e r a d i c a l s c a v e n g i n g e f f e c t s o f e x t r a c t s o f M a r i n B r o t h
( u n i t s : % )
sample Inhibition of sample concentration
200/ 100/ 50/ 25/ 12.5/ 6.25/
ascorbic acid 95.89 95.31 94.63 93.30 90.67 61.55
BHT 45.89 30.73 24.21 18.29 15.11 12.43
a-1 31.85 20.28 17.09 13.68 12.72 13.10
b-1 18.76 16.23 13.44 12.86 11.37 11.50
b-2 16.73 13.79 11.54 11.39 11.99 12.55
c-1 16.78 15.22 13.29 11.25 9.62 10.49
c-2 19.40 14.11 11.55 11.42 10.16 10.53
e-1 16.00 11.64 10.97 10.45 9.99 9.21
e-2 16.77 11.98 11.75 10.79 9.66 10.52
a-3 17.15 16.28 14.77 13.00 11.53 12.21
b-3 12.27 13.23 11.77 10.46 11.27 10.72
c-3 14.04 12.29 11.04 10.30 10.80 10.25
e-3 43.09 47.37 30.26 20.16 15.05 13.77
a : acetone ext., b : BuOH ext., c : CHCl3 ext., e : EtOAc ext.
- 24 -
F i g . 8 . D PPH f r e e r a d i c a l s c a v e n g i n g e f f e c t s o f M a r i n B r o t h e x t r a c t s .
DPPH free radical scavenging effect
0
20
40
60
80
100
120
vit.C BHT a-1 b-1 b-2 c-1 c-2 e-1 e-2 a-3 b-3 c-3 e-3
sample (100ug/ml)
)
F i g . 9 . C o m p a r i s o n o f D PPH f r e e r a d i c a l s c a v e n g i n g e f f e c t s b e t we e n a d d e d
a n d r e m o v e d M g C l 2 B r o t h e x t r a c t s .
0
20
40
60
80
100
120
sample (100ug/ml)
Positive
Negative
- 25 -
6 . m e l a n i n
sample melanin ( )
B16F10 melanoma cell melanin contrents control
Fig 10, 11 . (2 /) sample
a-3 c-1 melanin
, Arbutin , b-1
b-2 sample melanin
(Fig. 10-a.).
, (20 /) sample Arbutin
c-1 melanin , b-1, b-2 melanin
Arbutin (Fig. 10-b.).
MgCl2 , MgCl2
MgCl2 melanin
, , CHCl3 EtOAc melanin
(Fig. 11.).
- 26 -
a.
-40
-20
0
20
40
60
80
Arbutin a-3 b-3 c-3 e-3 a-1 b-1 b-2 c-1 c-2 e-1 e-2
sample(2ug/ml)
sample (20ug/ml)
)
F i g . 1 0 . M e l a n i n c o n t e n t s In h i b i t i o n o f M a r i n B r o t h e x t r a c t s o n B 1 6 F 1 0
m e l a n o m a c e l l ( a : 2 / , b : 2 0 / ) .
- 27 -
a.
-10
-5
0
5
10
15
20
Ar PB1 PB2 PC1 PC2 PE1 PE2 NB1 NB2 NC1 NC2 NE1 NE2
sample(2ug/ml)
b.
-40
-20
0
20
40
60
80
100
Ar PB1 PB2 PC1 PC2 PE1 PE2 NB1 NB2 NC1 NC2 NE1 NE2
sample(20ug/ml)
)
F i g . 1 1 . M e l a n i n c o n t e n t s In h i b i t i o n o f a d d a n d r e m o v e d M g C l 2 B r o t h
e x t r a c t s o n B 1 6 F 1 0 m e l a n o m a c e l l ( a : 2 / , b : 2 0 / ) .
- 28 -
7 . C e l l v i a b i l i t y ( M T T a s s a y )
B16F10 melanoma cell sample melanin
MTT assay, melanin contents ,
control Fig. 12 .
(2 /) sample a-3, e-3, c-2
, a-1, b-1, e-1 b-3, c-3, b-2, c-1
, , (Fig. 12-a).
, (20 /) sample a-1, b-1, c-2
, Arbutin . e-2 ,
cell (Fig. 12-b).
B16F10 melanoma cell Detroit-551 fibroblast
cell line sample MTT assay
control Fig. 13 .
c-1, e-1, e-3 , , 50% ,
.
- 29 -
a.
-80
-60
-40
-20
0
20
40
60
80
Arbutin a-3 b-3 c-3 e-3 a-1 b-1 b-2 c-1 c-2 e-1 e-2
sample (2ug/ml)
sample (20ug/ml)
)
F i g . 1 2 . C e l l v i a b i l i t y In h i b i t i o n o f M a r i n B r o t h e x t r a c t s o n B 1 6 F 1 0
m e l a n o m a c e l l ( a : 2 / , b : 2 0 / ) .
- 30 -
a.
-10
0
10
20
30
40
50
arbutin a-1 b-1 b-2 c-1 c-2 e-1 e-2 a-3 b-3 c-3 e-3
sample (2ug/ml)
sample (20ug/ ml)
% )
F i g . 1 3 . C e l l v i a b i l i t y In h i b i t i o n o f M a r i n B r o t h e x t r a c t s o n D e t r o i t - 5 5 1
f i b r o b l a s t c e l l ( a : 2 / , b : 2 0 / ) .
- 31 -

Streptomyces Streptomyces sp. JR1 ,
pigmentation .
Streptomyces sp. JR1 . Marin Broth
pigment ,
, MgCl2 pigmentation
. MgCl2 Streptomyces sp.
JR1 , pigmentation
450
, MgCl2
, pigmentation
. pigmentation MgCl2
, pigmentation MgCl2
, pigment
510 , 420
.
pigment Marin Broth pH5.0 pH9.0 0.5
pH Streptomyces sp. JR1
pigmentation 510 ,
pigmentation ,
5 .
pH , pH 7.88.2
,
pH 8.0 ,
pH , pigmentation ,
pH 7.08.0 .
- 32 -
, Streptomyces sp. JR1 pigment Marin Broth MgCl2
, pH pigment
.
pigment Streptomyces sp. JR1
,
pigment , acetone
, CHCl3, EtOAc, BuOH .
acetone CHCl3, EtOAc, BuOH gram
Escherichia coli gram Staphylococus aures, Bacillus cereus,
Bacillus subtilis, Streptococcus pyogenes test ,
gram , gram EtOAC
CHCl3 .
Sample 100 / DPPH free radical
ascorbic acid , BHT
e-3 (EtOAc ) . e-3 sample
in vivo
.
pigment B16F10 melanoma cell
sample melanin MTT
. , melanin , (2 /)
sample a-3 c-1 melanin
, Arbutin ,
b-1 b-2 sample melanin
. sample MTT assay
, b-3, c-3, c-1 , cell
.
, (20 /) sample Arbutin c-1
melanin , sample
MTT assay .
- 33 -
sample Detroit-551 cell
MTT assay . c-1, e-1
50% .
, melanin ,
b-3, c-3, c-1 sample in
vivo .
, Streptomyces sp. JR1 , ,

.
V .
1. : -
, ( () ). Life Science &
Biotechnology.
2. Moustafa Y. El-Naggar Maha A. Hassan and Wafa Y. Said. 2 0 0 1 ., Isolation
and characterisation of an antimicrobial substance produced by Streptomyces
violatus., Egyptial Jaurnal of Biology. vol. 3. p11-21.
3. Maha A. Hassan Moustafa Y. El-Naggar and Wafa Y. Said. 2 0 0 1 .,
Physiological factors affecting the production of an antimicrobial substance by
Streptomyces violatus in batch cultures., Egyptial Jaurnal of Biology. vol. 3. p
1-10.
4. , 1 9 9 3 , -, .,
Kor. J. Appl. Microbiol. Biotechnol. Vol.21, No.1 ; 88-94
5. .,
., ., 1 9 9 7 . : p100-107
6. ., ., ., 1 9 9 4 .,
7. Dong-A Encyclopedia ( ), , 1 9 9 2 : vol.13 : p444,
vol.18 : p387
8. ., 1 9 9 3 ., - I.
No. 101 ., Bull. Korean
Fish Soc., 26(4) : 305311
9. Yong Li, Xifeng Li, . ., 2 0 0 3 ., (Screening
of Antimicrobial Activity from the Marin-Derived Fungus)., Kor. J. pharmacogn.,
34(2) : 142144
10. . 1 9 9 7 ., : (IV) -
., vol. 10, No.2 : 34-43
11. , 1 9 9 4 ,
- 35 -
., Kor. J. Biotechnol. Bioeng. Vol.9. No.5 : 464-468
12. ., 1 9 9 5 . Angiotensin Converting
Enzyme(ACE) ., Kor. J.
Appl. Microbiol. Biotechnol., vol.23. No.4 : 439-445
13. ., 1 9 9 4 .,
., Kor. J. Appl. Microbiol. Biotechnol., vol.22, No.3 : 329-331
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YBE-316 ., Kor. J. Appl. Microbiol. Biotechnol. vol.23. No.3 :
297-303

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ABSTRACT
3. Streptomyces sp. JR1
1) Marin Broth 2216
2) ±MgCl broth
4. MgCl Streptomyces sp. JR1
5. pH red-brown pigmentation
6.
1)
2)
8. Melanin
III.
1. Streptomyces sp. JR1
2. Streptomyces sp. JR1 growth rate pigmentation
3. pH pigmentation
4.
5. DPPH radical
6. melanin
7. Cell viability (MTT assay)
IV.
V.