Characterization of Tryptamine-Producing Bacteria Isolated from Commercial Salted and Fermented Sand Lance Ammodytes personatus Sauces

792

Copyright © 2016 The Korean Society of Fisheries and Aquatic Science pISSN:0374-8111, eISSN:2287-8815

서 론

전통수산발효식품인액젓은어류또는패류에고농도의염 을가하여원료에존재하는미생물과효소작용으로발효되기 때문에원료특유의맛과풍미가있으나비위생적인환경에노 출되어유해미생물이증식하면제품에부정적인영향을미친 다

.

biogenic

amines

유리아미노산이미생물의 탈탄산효소작용에 의해생성되는 질소화합물로 화학적 구조에 따라 지방족화합물

(putrescine, cadaverine, agmatine, spermine, spermidine),

(tyramine, 2-phenylethylamine, noradrenaline, dopamine)

(histamine, tryptamine, serotonin)

(Shalaby, 1996; Gardini et al., 2016).

우에는

biogenic amines

(Fernandes-Salfuero and Mackie, 1987). Biogenic amines

·

,

,

,

,

,

,

,

(Kim et al., 2012; Biji et al., 2016).

,

,

histamine

histamine

,

,

,

,

,

,

,

,

,

,

,

,

, scombrotoxin

(Fritz and Baldwin, 2003). Tyra-

시판 까나리(Ammodytes personatus) 액젓에서 분리한 tryptamine 생성균의 특성

엄인선·김태옥·김희대

·박권삼*

군산대학교 식품생명공학과, ¹충북도립대학 바이오생명의약과

Characterization of Tryptamine-Producing Bacteria Isolated from Commercial Salted and Fermented Sand Lance

Ammodytes personatus Sauces

In-Seon Um, Tae-Ok Kim, Hee-Dai Kim

¹

Department of Food Science and Biotechnology, Kunsan National University, Gunsan 54150, Korea

1

Department of Biotechnology and Biomedicine, Chungbuk Provincial College, Cheongju 28160, Korea

We isolated seven tryptamine-producing bacteria from commercial salted and fermented sand lance ( Ammodytes personatus ) sauces using an L-tryptophan decarboxylating medium. These tryptamine-producing bacteria, identified using an API kit and 16S rRNA analysis, included Lysinibacillus xylanilyticus (one strain), Lysinibacillus fusifor- mis (four strains), and Staphylococcus epidermidis (two strains). Lysinibacillus spp. produced the highest levels of tryptamine in culture broth containing 0.5% L-tryptophan, compared with 1.0% and 2.0% preparations. After 72 h of incubation, Staphylococcus epidermidis produced the highest levels of tryptamine (60.50 μg/mL and 664.86 μg/

mL) in culture broth containing 2.0% L-tryptophan. While Lysinibacillus spp. comprised the dominant tryptamine- producing bacteria in sand lance sauces, Staphylococcus epidermidis also showed high tryptamine-producing activ- ity. This is the first report on the isolation and identification of tryptamine-producing bacteria in sand lance sauces.

Key words: Ammodytes personatus, Fish sauces, Biogenic amines, Tryptamine producing bacteria

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://dx.doi.org/10.5657/KFAS.2016.0792 Korean J Fish Aquat Sci 49(6) 792-799, December 2016

Received 24 October 2016; Revised 19 November 2016; Accepted 30 November 2016

*Corresponding author: Tel: +82. 63. 469. 1822 Fax: +82. 63. 469. 7448

E-mail address: [email protected]

mine

monoamine oxidase

MAOI (monoamine oxidase inhibitor)

‘cheese reaction’

(Blackwell, 1963;

Joostern, 1988).

biogenic amines

,

MAOI

biogenic amines

(Cho et al., 2006).

histamine

50 mg/kg

(USFDA, 2011),

hista- mine

200 mg/kg

400 mg/kg

(Yang et al., 2014),

200 mg/kg,

100 mg/kg

(Canadian Standards for Various Chemical Contaminants in Food, 2012).

,

,

,

/

histamine

200 mg/kg

(KMFDS, 2016),

biogenic amines

biogenic amines

.

biogenic amines

tryptamine

10

tryptamine

70.41-482.86 mg/kg

(Kim et al., 2011)

8

, 8

5

tryptamine

60.1-296.8, 62.0-187.2

5.3-10.6 mg/kg

(Cho et al., 2006).

biogenic amines

biogenic amines

.

biogenic amines

tryptamine

,

을검토하여

tryptamine

자한다

.

재료 및 방법

재료 및 시약

실험에사용한까나리액젓은대형마트및도매시장에서시판 되고있는제품으로

B

(

), C

(

), D

(

), G

(

), H

(

), K

(

), N

(

)

7

. Tryptamine (98%), 1,7-diaminoheptane (I.S.)

dansyl chloride

Sigma-Aldrich (Switzerland)

, acetonitrile

HPLC

, ether

.

Takara (Otsu, Japan)

Ex Taq polymerase

,

Becton Dickinson

(BBL Sensi-Disk, Sparks, MD, USA)

Sigma (St. Louis, MO, USA)

. Tryptamine 생성균의 분리

Tryptamine

Zaman et al. (2011)

1 mL

[tryptone (0.5%), yeast extract (0.5%), sodium chloride (3%), glucose (0.1%), tween 80 (0.05%), MgSO

·7H

O (0.02%), CaCO

(0.01%), MnSO

·4H

O (0.005%), FeSO

·7H

O (0.004%), bromocresol purple (0.006%), L-tryptophan (2%) and agar (2%)]

35℃

48

Luria-Bertani (tryptone 1%, yeast-extract 0.5%, NaCl 3%) broth

35℃

15%

Cryovial storage box (Simport, Canada)

-80℃

.

분리 균주의 동정

균주는형태학적

,

16S rRNA

.

(Olympus CX31, Philippines)

,

API Staph kit

API 50 CHB kit (BioMerieux,

France)

.

16S rRNA

genomic DNA

Genomic DNA extraction kit

(Qiagen, USA)

, 16S rRNA

primer (Dunbar et al., 2000)

27F primer (5'-AGAGTTT-

GATCCTGGCTCAG-3')

1492R primer (5'-TACCTTGT-

TACGACTT-3')

. PCR

0.5 µL (2.5 U)

Taq polymerase, 5.0 µL Taq polymerase buffer (10×), 4.0 µL

2.5 mM dNTP, 35.5 µL

20 pmol

primer 2.0

µL

1.0 µL

DNA

50 µL

. DNA

PCR

95℃

3

1

95℃ 30

, 55℃ 30

, 72℃ 2

30

DNA

GeneAmp PCR system 9700 (Applied Biosys-

tems, USA)

. PCR

1.5% agarose gel

(Sigma, USA)

ethidium bromide

DNA

DNA

QIAEX

II Gel Extraction kit (Qiagen, USA)

pT7Blue T

Vector (Novagen, USA)

TA cloning

Escherichia coli

DH5α

plasmid DNA

QIAprep Spin

Miniprep kit (Qiagen, USA)

. DNA

ABI310 sequencers (Applied Bio-

systems, Foster City, CA, USA),

DNASIS

MAX v3.0 program (Hitachi Software, Tokyo, Japan)

NCBI (http://www.ncbi.nim.ni h.gov/BLAST)

.

항균제 내성 검사

각종항균제에대한감수성은

Acar and Goldstein (1991)

NCCLS (National Committee for Clini- cal Laboratory Standards, 2002)

.

3%

LB broth

35℃

2

McFarland No. 0.5

0.4 mm

Muller Hinton agar (Mer- ck, Germany)

.

35℃

16-18

.

amikacin (AK; 30 µg), amoxicillin (AML; 10 µg), ampicillin (AMP; 10 µg), bacitracin (B, 10 unit), cefepime (FEP; 30 µg), cefotax- ime (CTX; 30 µg), cefotetan (CTT; 30 µg), cefoxitin (FOX;

30 µg), cefuroxime (CXM; 30 µg), ceftriaxone (CRO; 30 µg), cephalothin (KF; 30 µg), cephazolin (KZ; 30 µg), chloram- phenicol (C; 30 µg), ciprofloxacin (CIP; 5 µg), clindamycin (CC; 2 µg), erythromycin (E; 15 µg), gentamicin (GN; 10 µg), imipenem (IPM; 10 µg), kanamycin (K; 30 µg), nalidixic acid (NA; 30 µg), nitrofurantoin (F; 100 µg), norfloxacin (NOR;

10 µg), oxacillin (OX; 1 µg), penicillin (P; 10 µg), pipemidic acid (PIP; 20 µg), rifampin (RD; 5 µg), streptomycin (S; 10 µg), sulfamethoxazole/trimethoprim (SXT; 23.75/1.25 µg), tetracycline (TE; 30 µg), ticarcillin (TIC; 75 µg), trimethoprim (W; 5 µg), vancomycin (VA; 30 µg)

32

.

항균제 최소발육억제농도(Minimum Inhibitory Con- centration, MIC)의 측정

내성을 나타내는 항균제에 대한 최소발육억제농도는 미국

NCCLS (National Committee for Clinical Laboratory Stan- dards, 2002)

.

Muller- Hinton broth (Merck, Germany)

2,048 µg/mL

1 µg/mL

2 mL

3%

LB broth

2 μL

35℃

에서

16

.

효소활성 측정

Tryptamine

API ZYM kit (BioMerieux, France)

.

LB agar (NaCl 3%)

PBS (phosphate buffered saline, 137 mM NaCl, 2.7 mM KCl, 10 mM Na

HPO

·2H

O, 2.0 mM KH

PO

, pH 7.4)

Mcfarland

5-6

.

API ZYM kit

cupule

65 µL

37℃

4

.

Tryptamine 생성량의 측정

Tryptamine

Mah et al. (2003)

.

[LB broth (NaCl 3%)

tryptophan

0.5%, 1.0%

2.0%

pH

5.8

pyridoxal-HCl 0.0005%

] 5 mL

50 μL

35℃

24, 48

72

0.45 μm syringe filter

tryptamine

HPLC

. Tryptamine

0.1N HCl

500 μg/mL

,

(1,7-diaminoheptane)

0.1N HCl

100 μg/mL

.

Tryptamine

규격

(KMFDS, 2016)

.

험용액

(

) 1 mL

액

0.1 mL,

0.5 mL

1% dancyl chloride

0.8 mL

45℃

1

.

10% proline

0.5 mL

ether 5 mL

10

acetonitrile 1 mL

0.2 μm syringe filter

HPLC system (YL 9100, YoungLin Instrument Co., Ltd., Korea)

,

Table 1

.

결과 및 고찰

시판 까나리액젓에서 tryptamine 생성균의 분리 Biogenic amines

Parameter Conditions

Detector UV

Column Carbamate Column C18

(4.6×250 nm×5 μm)

Column temp 40℃

Flow rate 1 mL/min

Run time 35 min

Wavelength 254 nm

Gradient elution (min) ACN¹ (%) H₂O (%)

0 55 45

15 65 35

25 80 20

35 90 10

1ACN, acetonitrile.

판까나리액젓에서

tryptamine

. Tryptamine

L-tryptophan

amino acid decarboxylase

L-trypto- phan

tryptamine

pH

으로바뀌어분리배지에존재하는지시약에의해

colony

에는보라색또는자주색환이형성된다

. 7

1 mL

,

5.0×10

CFU/mL

1.0×10

CFU/mL

이로대체로적었으며그중

colony

하여

tryptamine

7

-80℃ deep freezer

. 분리 균주의 동정

Tryptamine

7

,

.

, 5

2

.

API 50 CHB kit

, 5

Bacillus non- reactive (65.7-87.5%

)

.

2

API Staph kit

, 2

Staphylococcus epidermidis

84.9%

97.9%

의상동성을보였다

(Table 2).

16S rRNA

Dunbar et al. (2000)

universal primer set (27F

1492R)

PCR assay

,

1,500 bp

(

). 16S rRNA

NCBI

BLAST

,

5

4

Lysinibacillus fusiformis strain L84 (98.14-99.21%

)

1

Lysinibacillus xylani- lyticus strain B85 (97.32%

)

.

Staphylococcus epidermidis

2

16S rRNA

Staphylococ- cus epidermidis strain L29

99.27

99.34%

였다

(Table 2).

tryptamine

Lysinibacillus spp.

Staphylococcus spp.

.

L. fusiformis

L. sp-

hearicus

으며

, S. epidermidis

,

(Otto, 2012).

Moon et al. (2013)

Bacillus licheniformis A7

B. coagulans SL5

,

Paenibacillus tyraminigenes

tyramine

(Mah et al., 2008)

tryptamine

.

항균제 내성 및 최소발육억제농도(Minimum Inhibi- tory Concentration, MIC)의 검토

까나리액젓에서

tryptamine

7

32

, 32

nalidixic acid

bacitracin

30

7

(

). 3

nalidixic acid

MIC

128 μg/mL

. Bacitracin

1

, 2

4

MIC

3

512 μg/mL

. Tryptamine

7

.

Tryptamine 생성 균주의 효소활성검토

Biogenic amines

,

(Species)

Characters Strain No.

1 2 3 4 5 S1 S2

Gram stain + + + + + + +

Morphology rod rod rod rod rod cocci cocci

API test API 50CHB API STAPH

BNR¹ BNR BNR BNR BNR S. epidermidis S. epidermidis

Identity (%) 65.7 65.7 65.7 87.5 65.7 84.9 97.9

16S rRNA Ly. xylanilyti-

cus² Ly. fusiformis³ Ly. fusiformis Ly. fusiformis Ly. fusiformis S. epidermi-

dis⁴ S. epidermidis

Identity (%) 97.32 99.08 98.26 99.21 98.14 99.27 99.34

1BNR, Bacillus non-reactive; ²Ly. xylanilyticus, Lysinibacillus xylanilyticus; ³Ly. fusiformis, Lysinibacillus fusiformis; ⁴S. epidermidis, Staphylococcus epidermidis.

성의연구결과가없다는점도검토의필요성으로대두되었다

.

tryptamine

7

API ZYM Kit

(Table 3). 20

3

,

6

1-3

. Lysinibacillus fusiformis (2

, 4

5

)

esterase (C4)

esterase lipase (C8)

5 nmol

, Lysinibacillus xylanilyticus (1

)

esterase (C4), esterase lipase (C8)

α-chymotrypsin

5 nmol

.

Staphylococcus epidermidis (S1

S2

)

acid phosphatase

20 nmol

(Table 3).

6

Pediococcus pen- tosaceus SH-10 (Shin et al., 2012)

Lactobacillus salivarius CPM-7 (Lim et al., 2007)

,

(Genus)

.

조건에 따른 분리 균주의 tryptamine 생성량 측정 Tryptamine

R

0.9987

, retention time (RT)

17.43

(Fig. 1A

1B). Tryptamine

7

L-tryptophan

0.5%, 1.0%

2.0%

LB broth (NaCl 3%) 5 mL

24, 48

72

tryptamine

HPLC

(Table 4).

Table 4

1

5

5

L-tryptophan

0.5%

tryptamine

L-tryptophan

trypt-

amine

.

전구체인

L-tryptophan

,

L-tryptophan

을

0.5%

(

결과미제시

).

tryptamine

24

2

(1

5

), 48

1

(4

)

72

2

(2

Enzyme Strain No.

1 2 3 4 5 S1 S2

Control 0¹ 0 0 0 0 0 0

Alkaline phosphate 0 0 0 0 0 0 0

Esterase (C4) 1 1 0 1 1 0 0

Esterase Lipase (C8) 1 1 0 1 1 0 0

Lipase (C14) 0 0 0 0 0 0 0

Leucine arylamidase 0 0 0 0 0 0 0

Valine arylamidase 0 0 0 0 0 0 0

Crystine arylamidase 0 0 0 0 0 0 0

Trypsin 0 0 0 0 0 0 0

α-chymotrypsin 1 0 0 0 0 0 0

Acid phosphatase 0 0 0 0 0 3 3

Naphtol-AS-BI-phosphohydrolase 0 0 0 0 0 0 0

α-galactosidase 0 0 0 0 0 0 0

β-glucuronidase 0 0 0 0 0 0 0

β-glucosidase 0 0 0 0 0 0 0

α-glucosidase 0 0 0 0 0 0 0

β-glucosidase 0 0 0 0 0 0 0

N-acetyl-β-glucosaminidase 0 0 0 0 0 0 0

α-mannosidase 0 0 0 0 0 0 0

α-fucosidase 0 0 0 0 0 0 0

10, 0 nmol; 1, 5 nmol; 2, 10 nmol, 3, 20 nmol.

번및

3

)

.

Staphylococcus epidermidis strain L29

S1

S2

tryptamine

(Table 4).

,

24

(

).

trypt- amine

72

11

.

biogenic amines

biogenic amines

. Lysinibacillus spp.

lysine

ornithine decar-

boxylase

(Lee et al., 2010; Coorevits et al., 2012; Duan et al., 2013), tryptophan decarboxylase

.

tryptamine

,

biogenic amines

.

사 사

이 논문은

2016

(

시판용까나리액젓의바이오제닉아민정량및생성균의분리 와동정

)

.

Fig. 1. Calibration curve for concentrated of tryptamine (A) and HPLC chromatograms of tryptamine (B).

17.43 3,000

2,000

1,000

0

0 10 20 30 40

Voltage (mV)

Time (min)

R² = 0.9987

0

0 20 40 60 80 100 120

1 2 5 4 6 7

3

Concentration (mg/L)

(A) (B)

Area

Table 4. Production of tryptamine of tryptamine producing bacteria in LB broth by incubation time and concentration of L-tryptophan

Strain No.

Incubation time

24 h 48 h 72 h

L-tryptophan concentration

0.5% 1% 2% 0.5% 1% 2% 0.5% 1% 2%

1 75.67 12.84 4.76 66.40 32.87 17.94 52.69 30.79 22.73

2 6.76 5.26 4.09 12.20 6.52 3.50 17.17 16.94 10.36

3 0.24 0.18 0.15 3.24 2.66 1.57 4.50 2.41 1.86

4 16.74 4.58 3.72 31.36 10.47 3.92 26.32 13.06 0.41

5 97.67 24.81 17.51 91.24 47.18 41.17 66.62 59.63 47.57

S1 66.85 79.42 148.01 136.58 151.09 361.44 171.98 293.20 664.86

S2 9.25 11.37 18.99 19.59 27.53 38.84 10.07 33.43 60.50

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