Purification and Characterization of an Antimicrobial Substance from Bacillus subtilis HH28 Antagonistic to Bacillus cereus

Bacillus cereus를 억제하는 Bacillus subtilis HH28의 항균물질 정제와 특성규명

차현아^1,2, 정다은³, 홍성욱⁴, 정건섭¹*

1연세대학교생명과학기술학부

2농촌진흥청국립축산과학원

3연세대학교원주의과대학

4세계김치연구소

Received: November 25, 2014 / Revised: December 9, 2014 / Accepted: December 10, 2014

서 론

Bacillus cereus

^,

^.

^,

^,

^[1].

B. cereus

enterotoxin

소를생성하여설사형

^,

^[28].

한미생물때문에발생하는식품부패와식중독을예방하기 위해식품보존제를첨가한다

^.

^.

^,

^,

^.

미생물이생산하는항균물질은대부분이박테리아가생산

하는단백질또는

^peptide

소화효소에의해분해되므로식품에서안전한천연보존제로 사용가능하다

^.

¹⁹²⁸

Lactococcus lactis

^nisin

bacteriocin

¹⁹⁹⁸

^FDA

^.

bacteriocin

Purification and Characterization of an Antimicrobial Substance from Bacillus subtilis HH28 Antagonistic to Bacillus cereus

Hyun A Cha

, Dawn Chung

, Sung Wook Hong

, and Kun Sub Chung

*

1

Division of Biological Science and Technology, Yonsei University, Wonju 220-710, Republic of Korea

2

National Institute of Animal Science, RDA, Suwon 441-706, Republic of Korea

3

Department of Obstetrics and Gynecology, Wonju College of Medicine, Yonsei University, Wonju 220-701, Republic of Korea

4

World Institute of Kimchi, Gwangju 503-360, Republic of Korea

A bacterium producing antimicrobial substance was isolated from cheonggukjang. The bacterium was identified as a strain of Bacillus subtilis by 16S rDNA sequencing and designated as Bacillus subtilis HH28. The antimicrobial substance produced from Bacillus subtilis HH28 was purified by 0-80% ammonium sulfate precipitation, DEAE-sepharose FF column chromatogra- phy, and Sephacryl S-200 HR gel chromatography. The molecular weight of the purified antimicrobial substance was estimated to be approximately 3,500 Da using Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and direct detection analysis. Antimicrobial substance from B. subtilis HH28 not only inhibited B. cereus, but also Listeria monocytogenes and Vib- rio parahaemolyticus. The purified antimicrobial substance was stable at 40-80

C, and between pH 2 and 8. Antimicrobial activ- ity of the purified substance was completely destroyed by treatment of protease, proteinase K, and pronase E, indicating that it is proteinaceous.

Keywords: Purification, antimicrobial substance, Bacillus subtilis, Bacillus cereus

*Corresponding author

Tel _: +82-33-760-2252, Fax: +82-33-760-2183 E-mail: [email protected]

© 2014, The Korean Society for Microbiology and Biotechnology

가활발히이루어지는발단이되었다

^[12].

bacteriocin

Bacillus species

^[2].

Bacillus species

^{. Class I}

subtilin, sublancin 168, subtilosin A

^,

(post-translational modification)

^{. Class II}

13 kDa

^pH

에서합성되며번역후구조변화가없는선형펩타이드로 서

coagulin, thurincin H, lichenin

. Class III

^{30 kDa}

bacteriocin

^, megacin A group

^[2].

antimicrobial peptides

^DNA

bacteriocin - like inhibitory substances (BLIS)

. B. subtilis LFB112

^BLIS

^[30], B. subtilis MJP1

^BLIS

^[32].

Bacillus species

^.

첫번째의약품산업으로적용할수있다

^.

관련질병에

bacteriocin

있다

[13]. Staphylococcus aureus

mersacidin [20]

subtilosin A [25]

^.

두번째로축산산업에적용할수있다

^.

^,

^.

^.

. Pattnaik

^[19]

Bacillus licheniformis

가생산하는

^lichenin

¹

Streptococcus bovis

^,

^.

세번째로식품산업에적용할수있다

^.

‘fresh foods’

^.

bacteriocin

^.

antimicrobial peptides P45 [26, 27], paenibacillin [6]

Bacillus brevis

항진균활성

^peptide

^[7].

네번째로환경산업에대한적용이다

. Bacillus species

^,

bacteriocin

‘biocontrol agents’

^.

Bacillus thuringiensis NEB17

bacteriocin

^,

^[9].

따라서본연구에서는청국장에서분리한

Bacillus subtilis HH28

^.

재료 및 방법

미생물 분리 및 배양

본실험의미생물은전통적으로제조한청국장및이를제 조할때사용한볏짚을이용하여분리하였다

^.

[0.85% (w/v) NaCl]

^1:9

, homogenizer (Stomacher 400, Seward, England)

¹⁰

^.

tryptic soy agar (TSA; Difco, Detroit, MI, USA)

100

^l

³⁷

^C

¹⁴

^.

항균물질 활성 측정

실험에사용된피검균

B. cereus KCCM 12667

^tryptic soy broth (TSB, Difco, MI, USA)

^{1% (v/v)}

37

C

²⁴

^.

B. cereus

paper disc

^[29]

. B. cereus

0.4% (v/v)

tryptic soy soft agar

^{8 mm}

paper disc (Advantec, Toyo Roshi Kaisha Ltd., Japan)

50

^l

³⁷

^C

¹²

^.

분리균주의 동정

항균활성을나타내는분리균주를동정하기위해서형태 학적

^,

^.

^.

API 50 CHB kit (bioMerieux, Marcy IEtoile,

France)

⁴⁹

고

, API 50 CHB database V3.0(http://apiweeb.biomerieux.

com)

^.

^16S

ribosomal DNA

chromosomal DNA

^template

universal primer

²⁷

forward primer (5'-AGAGTTTGATCATGGCTCAG-3')

1492 reverse primer (5'-GGATACCTTGTTACGACTT-3')

16S rDNA gene

^.

1,400 bp

^fragment

T-vector (Invitrogen, Carlsbad, CA, USA)

T-vector sequencing primer

, BLAST search program

GenBank (NCBI)

ribosomal DNA gene sequence

^.

생육시기에 따른 항균 활성조사

분리 미생물을

glucose minimal medium (0.002%

ammonium molybdate, 0.003% CaCl

·2H

O, 0.0002%

FeSO

·7H

O, 1% Glucose, 0.7% K

HPO

, 0.15% KH

PO

, 0.05% MgSO

·7H

O, 0.004% MnSO

·4H

O, 0.5% yeast extract) 500 ml

^{1% (v/v)}

⁴⁰

^C

^{180 rpm}

^{. 0-144}

spectrophotometer (VersaMax ELISA Microplate Reader, Molecular Devices Sunnyvale, CA, USA)

^{660 nm}

^,

^.

^ml

AU (activity unit)

^{. AU}

²

^,

^{1 ml}

AU/ml

^.

항균물질의 분리 및 정제

선발 미생물을

glucose minimal medium 500 ml

^1%

(v/v)

⁴⁰

^C

³

^.

^80%

^{, 4}

^C

¹²

^(20,000

^g, 4

C, 30

⁾

10 mM Tris·HCl

(pH 8.0)

^.

(MWCO 6-8 kDa cut- off, Spectrum Laboratories, Inc.)

⁴

^C

¹²

DEAE-sepharose fast flow (Pharmacia, Biotech. AB Uppsala, Sweden) column (25

^{400 mm)}

Sephacryl S-200HR (Pharmacia) column (15

^{960 mm)}

^.

^{280 nm}

^,

^.

⁴

^C

^.

단백질 함량 측정

항균물질단백질정량은

^Lowry

였다

^[17].

⁵⁰

^l

biuret reagent (0.75 mmol/l cupric sulfate, 94 mmol/l sodium hydroxide) 550

^l

²⁵

^C

¹⁰

, Folin and Ciocalteu’s phenol reagent (Sigma Co., St. Louis, MO, USA) 25

^l

²⁵

^C

³⁰

^.

^spectro- photometer

^{725 nm}

^,

bovine serum albumin (BSA; Sigma Co.)

^.

항균물질 분자량 측정

항균물질의분자량을측정하기위해

Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS- PAGE)

[21]. 20% polyacrylamide gel

^{100 V}

⁵

silver staining kit (Amersham Biosciences AB Inc., Sweden)

^.

^marker

ultra-low range molecular weight marker (1,060-26,600 Da, Sigma)

. Tricine SDS-PAGE

^direct detection

[3]. Tricine SDS-PAGE

polyacrylamide gel

25% (v/v) isopropanol

10% (v/v) glacial acetic acid

²

⁶

^wash

B. cereus KCCM 12667

⁴⁰

^C

⁶

^.

항균 spectrum 조사

Bacillus subtilis, B. licheniformis, B. cereus

TSB, Staphylococcus aureus, Salmonella Typhimurium, Pseudomonas aeruginosa, Shigella flexineri, S. sonnei, S. boydii, Escherichia coli O157:H7

^nutrient broth (NB, Difco, MI, USA), Vibrio parahaemolyticus

2.5 % (w/v)

^TSB

^. Streptococcus mutans, Listeria monocytogenes

^Brain Heart Infusion broth (BHI, Difco), Candida albicans

Yeast Mold broth (YM, Difco, MI, USA), Aspergillus flavus, A. fumigatus, Rhizopus oryzae, Mucor circinelloides, Penicillum camemberti

potato dextrose agar (PDA,

Difco)

^.

주들은생명자원센터

^(KCTC),

^(KACC)

^(KCCM)

^(Table

2).

C. albicans

³⁰

C, A. flavus, A. fumigatus,

R. oryzae, M. circinelloides, P. camemberti

²⁵

^C,

³⁷

^C

측정하였고

paper disc

^.

항균작용기전은

^Nicolas

^[16]

^. B. cereus KCCM 12667

^TSB

^{5% (v/v)}

²

진탕배양한배양액에항균물질

^{(32 AU/ml)}

^,

항균물질을첨가하지않은

B. cereus

^.

¹²

2

.

항균물질의 안정성

항균물질의

^pH

pH 2.0-4.0

0.1 M glycine-HCl

, pH 4.0-6.0

0.1 M sodium acetate

, pH 6.0-8.0

0.1 M sodium phosphate

, pH 8.0-10.0

^Tris-HCl

^.

^pH

^1:15

⁴⁰

^C

¹²

^.

^pH

^.

^40-80

^C

¹²

^{, 100}

^C

¹

^.

lysozyme (EC 3.2.1.17, Sigma),

-amylase (EC 3.2.1.1, Sigma), lipase (EC 3.1.1.3, Type VII, Sigma), trypsin (EC 3.4.21.4, Sigma), protease (EC 3.4.24.31, Sigma), Proteinase K (EC 3.4.21.64, Sigma), pronase E (EC 3.4.24.4, Merck)

10 mM sodium phosphate

^{(pH 7.0)}

^{4 mg/ml}

^.

^{2 mg/ml}

³⁷

^C

³⁰

^.

결과 및 고찰

항균활성 균주의 분리 및 동정

전통적인방법으로제조한청국장과제조할때사용한볏 짚을멸균생리식염수로희석한후

^{, TSA}

¹⁴

^,

. B. cereus

^,

¹

^.

분리한미생물은포자를형성하는그람양성간균이며

^{, API}

50 CHB system

는

Bacillus subtilis

^.

^{16S rDNA}

^{, GenBenk}

B. subtilis

^98%

의상동성을보였다

^.

^,

B. subtilis

, B. subtilis HH28

^.

분리균주의 생육시기에 따른 항균활성

B. subtilis HH28

^{(Fig. 1).}

^,

¹²

²⁴

. B. cereus

⁹

⁶⁰

^{(80 AU/ml)}

^.

많은항균물질이영양원이고갈되는정체기에축적되는것 과같이본연구의항균물질또한정체기인

¹²

^[22].

⁶⁰

¹⁴⁴

⁽⁶

⁾

^.

^Guo

^[5]

B. licheniformis B11

bacteriocin - like substance (BLIS)

²⁰

^,

^Yang

^[32]

^.

항균물질의 분리 및 정제

B. subtilis HH28

^{500 ml}

^0-80%

^,

^.

DEAE-sepharose fast flow

^{, 0-1 M}

^{(Fig. 2).}

^143-156

sephacryl S-200HR

다

^{(Fig. 3).}

^46- 56

^.

^{Table 1}

^{, 19.7}

^38.4%

^.

Fig. 1. Growth and antimicrobial activity of Bacillus subtilis

HH28 in the glucose minimal medium.

항균물질의 분자량 측정

정제한항균물질의분자량을측정하기위해

Tricine SDS-

PAGE

^,

^direct

detection

^.

과

direct detection

^{(Fig. 4).}

^marker

^{3,500 Da}

^.

B. subtilis

^{3,500 Da}

lantibiotic

subtilin, subtilosin A, sublancin 168, ericin S

^{. Subtilin}

pentacyclic peptide

^{3,319 Da}

[24], ericin S

^{3,442 Da}

subtilin

⁴

^[23]

. subtilosin

^{3,399 Da}

^post- translational modification

macrocyclic

[15], sublancin 168

^{3,877 Da}

Fig. 2. Ion exchange chromatography of the antimicrobial

substance from Bacillus subtilis HH28 on the DEAE-sephar- ose FF. The DEAE-sepharose FF ion exchange column (25 × 400 mm) was equilibrated with 10 mM Tris·HCl buffer (pH 8). The column was eluted with a linear gradient of 0-1 M NaCl in 10 mM Tris·HCl buffer (pH 8) at a flow rate of 1 ml/min. The volume of each fraction was 5 ml.

Fig. 3. Gel chromatography of the antimicrobial substance from Bacillus subtilis HH28 on the sephacryl S-200HR. Seph- acryl S-200HR column (15 × 960 mm) equilibrated with 10mM Tris·HCl buffer (pH 8). The column was eluted with 10 mM Tris·HCl buffer (pH 8) at a flow rate of 0.5 ml/min. The volume of each frac- tion was 2 ml.

Table 1. Purification summary of antimicrobial substance produced by Bacillus subtilis HH28.

Steps

Volume

(ml)

Total protein (mg)

Total activity (AU/ml)

Specific activity (AU/mg)

Purification (fold)

Yield (%)

Culture broth 500.0 1,159.6 40,000 34.5 1 100.0

AS 3.0 101.1 30,720 303.8 8.8 76.8

IEX 2.0 49.2 20,480 416.3 12.1 51.2

GF 1.5 22.6 15,360 678.5 19.7 38.4

*AS, ammonium sulfate precipitation; IEX, ion exchange chromatography; GF, gel chromatography

Fig. 4. Tricine SDS-PAGE and direct detection of purified anti- microbial substance from Bacillus subtilis HH28. Lane 1, Ultra-low range molecular weight marker (26,600 Da, Triosephos- phate isomerase from rabbit muscle; 17,000 Da, Myoglobin from horse heart; 14,200 Da, α-Lactalbumin from bovine milk; 6,500 Da, aprotinin from bovine lung; 3,496 Da, insulin chain B.); Lane 2, Purified antimicrobial substance eluted by sephacryl S-200HR;

Lane 3, Gel overlayed with Bacillus cereus KCCM 12667.

로연결된

lanthionine

^[18]

^.

본연구의항균물질의정확한구조를파악하기위해아미 노산서열을분석방법인

^Edman

^[4]

^.

N-terminal

^block

^,

^.

항균 spectrum

B. subtilis HH28

^,

^{Table 2}

^. Paper disk method

^,

B. cereus KCCM 12667, KCCM 11204, KCCM 40168, KCCM 40935, B. licheniformis KCCM 11237, B. subtilis KCCM 11314, Listeria monocytogenes KCCM 40307

^,

^B.

cereus

L. monocytogenes

^.

Shigella boydii KCCM 41649

, Vibrio parahaemolyticus

KCCM 11965

^.

이와효모에서는활성이없었다

^.

대부분의

Bacillus spp.

bacteriocin

^[8].

B. subtilis W42

^B.

cereus

L. monocytogenes

^[11].

V. parahaemolyticus

^,

^B.

subtilis NT-6

^[31].

B. cereus

L. monocytogenes

^.

B. subtilis

B. licheniformis

^.

항균 작용 기전

2

^{B. cereus}

Table 2. Inhibitory spectrum of antimicrobial substance from Bacillus subtilis HH28.

Microorganisms Indicator species Antimicrobial activity

Gram-positive bacteria Bacillus cereus KCCM 12667 +++

Bacillus cereus KCCM 11204 ++

Bacillus cereus KCCM 40168 +++

Bacillus cereus KCCM 40935 +++

Bacillus licheniformis KCCM 11237 ±

Bacillus subtilis KCCM 11314 ±

Listeria monocytogenes KCCM 40307 +++

Staphylococcus aureus KCCM 12214 −

Streptococcus mutans KCTC 3065 −

Gram-negative bacteria Escherichia coli O157 _: H7 ATCC 35150 −

Pseudomonas aeruginosa ATCC 27853 −

Salmonella typhimurium ATCC 19855 −

Shigella boydii KCCM 41649 ±

Shigella sonnei KCCM 11903 −

Shigella flexneri KCTC 2008 −

Vibrio parahaemolyticus KCCM 11965 +++

Mold Aspergillus flavus KACC 41809 −

Aspergillus fumigatus KACC 42590 −

Rhizopus oryzae KACC 40256 −

Mucor circinelloides KACC 40245 −

Yeast Candida albicans KCCM 11282 −

−, no inhibition zone; ±, below 10 mm; +, below 13 mm; ++, 13-15 mm; +++, above 15 mm.

한후

²

^{B. cereus}

(Fig. 5). 2

B. cereus

^{8.4 log} (CFU/ml)

B. subtilis HH28

²

^{B. cereus}

^.

B. cereus

(bactericidal action)

^. Kindoli

^[10]

B. subtilis H27

, 1,600 AU/ml

12

L. monocytogenes

^.

^{32 AU/ml}

²

B. cereus

¹²

^.

항균물질의 안정성

pH

^{pH 2-10}

으로정제된항균물질을희석하여배양온도인

⁴⁰

^C

¹²

^.

^{pH 2-8}

^100%

^{, pH 9}

^50%

^{pH 10}

^25%

(Table 3). pH

^{B. cereus}

^{B. cereus}

^.

산성일때안정성이더높다는것을확인할수있었다

^{. Lee}

등

^[14]

B. subtilis SC-8

^{pH 3}

항균활성이감소하고

^{pH 4-10}

^.

^.

온도안정성측정은정제한항균물질을

^40-80

^C

¹²

^{, 100}

^C

¹

^{. 40-60}

^C

¹²

^{, 100%}

의잔존항균활성을유지하였고

^{, 80}

^C

¹²

^{, 12.5%}

^{(Table 3).}

Lee

^[14]

⁸⁰

^C

¹⁰⁰

^C

¹

^B.

cereus

^{. Kindoli}

^[11]

^60-80

^C

¹⁵

^50%

가 감소하였고

^{, 100}

^{C 10}

^20%

^L.

monocytogenes

^.

^.

단백질분해효소인

^trypsin

^50%

, protease, proteinase K, pronase E

. Lysozyme,

-amylase, lipase

리하였을경우

^,

^100%

(Table 3).

본연구의항균물질을단백질성물질임을확인하였다

^. Fig. 5. Inhibition mode of antimicrobial substance from Bacil-

lus subtilis HH28. Opened circle, Bacillus cereus KCCM12667 culture with purified antimicrobial substance; closed circle, Bacillus cereus KCCM12667 culture without purified antimicrobial sub- stance.

Table 3. Effect of enzyme treatment, heat and pH on the anti- microbial activity of B. subtilis HH28.

Treatment Residual activity (%) pH

2 100

4 100

6 100

8 100

10 25

Heat

40

C 100

60

C 100

80

C 12.5

100

C 25

Enzyme

lysozyme 100

α

^{-amylase 100}

lipase 100

trypsin 50

protease 0

proteinase K 0

pronase E 0

요 약

청국장으로부터

Bacillus cereus

높은균주를분리하여형태학적

^,

^{16S rDNA}

염기서열결정을통해

Bacillus subtilis HH28

. B. subilis HH28

^,

⁹

⁶⁰

^{(80 AU/ml)}

¹⁴⁴

⁽⁶

⁾

^.

DEAE-sepharose fast flow, sephacryl S-200HR

^{, 19.7}

38.4%

. Tricine SDS-PAGE

^directed detection

^{3,500 Da}

^.

B. cereus

Listeria monocytogenes, Vibrio parahaemolyticus

^,

^.

^40-80

^C

^{, pH}

^{pH 2-9}

^pH

^.

^.

B. subtilis HH28

^,

^,

^.

Acknowledgments

This study was supported by the 2012 Korea Institute of Plan- ning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET), Ministry of Agriculture, Food and Rural Affairs (MAFRA).

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