한수지 49(6), , 2016 Original Article Korean J Fish Aquat Sci 49(6), ,2016 시판까나리 (Ammodytes personatus) 액젓에서분리한 tryptamine 생성균의특성 엄인선 김태옥 김희

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1 한수지 49(6), , 206 Original Article Korean J Fish Aquat Sci 49(6), ,206 시판까나리 (Ammodytes personatus) 액젓에서분리한 tryptamine 생성균의특성 엄인선 김태옥 김희대 박권삼 * 군산대학교식품생명공학과, 충북도립대학바이오생명의약과 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 and Kwon-Sam Park* Department of Food Science and Biotechnology, Kunsan National University, Gunsan 5450, Korea Department of Biotechnology and Biomedicine, Chungbuk Provincial College, Cheongju 2860, 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 6S rrna analysis, included Lysinibacillus xylanilyticus (one strain), Lysinibacillus fusiformis (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.0% and 2.0% preparations. After 72 h of incubation, Staphylococcus epidermidis produced the highest levels of tryptamine (60.50 μg/ml and μg/ ml) in culture broth containing 2.0% L-tryptophan. While Lysinibacillus spp. comprised the dominant tryptamineproducing bacteria in sand lance sauces, Staphylococcus epidermidis also showed high tryptamine-producing activity. 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 서론. biogenic amines (putrescine, cadaverine, agmatine, spermine, spermidine), (tyramine, 2-phenylethylamine, noradrenaline, dopamine) (histamine, tryptamine, serotonin) (Shalaby, 996; Gardini et al., 206). biogenic amines (Fernandes-Salfuero and Mackie, 987). Biogenic amines,,,,,,, (Kim et al., 202; Biji et al., 206).,, histamine histamine,,,,,,,,,,,,, scombrotoxin (Fritz and Baldwin, 2003). Tyra- Korean J Fish Aquat Sci 49(6) , December 206 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 24 October 206; Revised 9 November 206; Accepted 30 November 206 *Corresponding author: Tel: Fax: address: parkks@kunsan.ac.kr Copyright 206 The Korean Society of Fisheries and Aquatic Science 792 pissn:0374-8, eissn:

2 트립타민생성균의분리및동정 793 mine monoamine oxidase MAOI (monoamine oxidase inhibitor) cheese reaction (Blackwell, 963; Joostern, 988). biogenic amines, MAOI biogenic amines (Cho et al., 2006). histamine 50 mg/kg (USFDA, 20), histamine 200 mg/kg 400 mg/kg (Yang et al., 204), 200 mg/kg, 00 mg/kg (Canadian Standards for Various Chemical Contaminants in Food, 202).,,, / histamine 200 mg/kg (KMFDS, 206), biogenic amines biogenic amines. biogenic amines tryptamine 0 tryptamine mg/kg (Kim et al., 20) 8, 8 5 tryptamine , mg/kg (Cho et al., 2006). biogenic amines biogenic amines. biogenic amines tryptamine, tryptamine. 재료및시약 재료및방법 B ( ), C ( ), D ( ), G ( ), H ( ), K ( ), N ( ) 7. Tryptamine (98%),,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 생성균의분리 Tryptamine Zaman et al. (20) ml [tryptone (0.5%), yeast extract (0.5%), sodium chloride (3%), glucose (0.%), tween 80 (0.05%), MgSO 4 7H 2 O (0.02%), CaCO 3 (0.0%), MnSO 4 4H 2 O (0.005%), FeSO 4 7H 2 O (0.004%), bromocresol purple (0.006%), L-tryptophan (2%) and agar (2%)] Luria-Bertani (tryptone %, yeast-extract 0.5%, NaCl 3%) broth 35 5% Cryovial storage box (Simport, Canada) -80. 분리균주의동정, 6S rrna. (Olympus CX3, Philippines), API Staph kit API 50 CHB kit (BioMerieux, France). 6S rrna genomic DNA Genomic DNA extraction kit (Qiagen, USA), 6S rrna primer (Dunbar et al., 2000) 27F primer (5'-AGAGTTT- GATCCTGGCTCAG-3') 492R primer (5'-TACCTTGT- TACGACTT-3'). PCR 0.5 µl (2.5 U) Taq polymerase, 5.0 µl Taq polymerase buffer (0 ), 4.0 µl 2.5 mm dntp, 35.5 µl 20 pmol primer 2.0 µl.0 µl DNA 50 µl. DNA PCR , 55 30, DNA GeneAmp PCR system 9700 (Applied Biosystems, USA). PCR.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 DH5 plasmid DNA QIAprep Spin Miniprep kit (Qiagen, USA). DNA ABI30 sequencers (Applied Biosystems, Foster City, CA, USA), DNASIS MAX v3.0 program (Hitachi Software, Tokyo, Japan) NCBI ( h.gov/blast).

3 794 엄인선ㆍ김태옥ㆍ김희대ㆍ박권삼 항균제내성검사 Acar and Goldstein (99) NCCLS (National Committee for Clinical Laboratory Standards, 2002). 3% LB broth 35 2 McFarland No mm Muller Hinton agar (Merck, Germany) amikacin (AK; 30 µg), amoxicillin (AML; 0 µg), ampicillin (AMP; 0 µg), bacitracin (B, 0 unit), cefepime (FEP; 30 µg), cefotaxime (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), chloramphenicol (C; 30 µg), ciprofloxacin (CIP; 5 µg), clindamycin (CC; 2 µg), erythromycin (E; 5 µg), gentamicin (GN; 0 µg), imipenem (IPM; 0 µg), kanamycin (K; 30 µg), nalidixic acid (NA; 30 µg), nitrofurantoin (F; 00 µg), norfloxacin (NOR; 0 µg), oxacillin (OX; µg), penicillin (P; 0 µg), pipemidic acid (PIP; 20 µg), rifampin (RD; 5 µg), streptomycin (S; 0 µg), sulfamethoxazole/trimethoprim (SXT; 23.75/.25 µg), tetracycline (TE; 30 µg), ticarcillin (TIC; 75 µg), trimethoprim (W; 5 µg), vancomycin (VA; 30 µg) 32. 항균제최소발육억제농도 (Minimum Inhibitory Concentration, MIC) 의측정 NCCLS (National Committee for Clinical Laboratory Standards, 2002). Muller- Hinton broth (Merck, Germany) 2,048 µg/ml µg/ml 2 ml 3% LB broth 2 L 효소활성측정 Tryptamine API ZYM kit (BioMerieux, France). LB agar (NaCl 3%) PBS (phosphate buffered saline, 37 mm NaCl, 2.7 mm KCl, 0 mm Na 2 HPO 4 2H 2 O, 2.0 mm KH 2 PO 4, ph 7.4) Mcfarland 5-6. Table. Instrument condition for HPLC analysis of tryptamine Detector Column Parameter API ZYM kit cupule 65 µl Tryptamine 생성량의측정 Tryptamine Mah et al. (2003). [LB broth (NaCl 3%) tryptophan 0.5%,.0% 2.0% ph 5.8 pyridoxal-hcl % ] 5 ml 50 L 35 24, m syringe filter tryptamine HPLC. Tryptamine 0.N HCl 500 g/ml, (,7-diaminoheptane) 0.N HCl 00 g/ml. Tryptamine (KMFDS, 206). ( ) ml 0. ml, 0.5 ml % dancyl chloride 0.8 ml 45. 0% proline 0.5 ml ether 5 ml 0 acetonitrile ml 0.2 m syringe filter HPLC system (YL 900, YoungLin Instrument Co., Ltd., Korea), Table. 결과및고찰 Conditions UV Carbamate Column C8 ( nm 5 μm) Column temp 40 Flow rate Run time Wavelength ml/min 35 min 254 nm Gradient elution (min) ACN (%) H 2 O (%) ACN, acetonitrile. 시판까나리액젓에서 tryptamine 생성균의분리 Biogenic amines

4 트립타민생성균의분리및동정 795 Table 2. Identification of tryptamine producing bacteria Characters Strain No S S2 Gram stain Morphology rod rod rod rod rod cocci cocci API test API 50CHB API STAPH BNR BNR BNR BNR BNR S. epidermidis S. epidermidis Identity (%) Ly. xylanilyticus 2 3 Ly. fusiformis Ly. fusiformis Ly. fusiformis S. epidermidis S. epidermidis 4 6S rrna Ly. fusiformis Identity (%) BNR, Bacillus non-reactive; 2 Ly. xylanilyticus, Lysinibacillus xylanilyticus; 3 Ly. fusiformis, Lysinibacillus fusiformis; 4 S. epidermidis, Staphylococcus epidermidis. tryptamine. Tryptamine L-tryptophan amino acid decarboxylase L-tryptophan tryptamine ph colony. 7 ml, CFU/mL CFU/mL colony tryptamine 7-80 deep freezer. 분리균주의동정 Tryptamine 7,., 5 2. API 50 CHB kit, 5 Bacillus nonreactive ( % ). 2 API Staph kit, 2 Staphylococcus epidermidis 84.9% 97.9% (Table 2). 6S rrna Dunbar et al. (2000) universal primer set (27F 492R) PCR assay,,500 bp ( ). 6S rrna NCBI BLAST, 5 4 Lysinibacillus fusiformis strain L84 ( % ) Lysinibacillus xylanilyticus strain B85 (97.32% ). Staphylococcus epidermidis 2 6S rrna Staphylococcus epidermidis strain L % (Table 2). tryptamine Lysinibacillus spp. Staphylococcus spp.. L. fusiformis L. sphearicus, S. epidermidis, (Otto, 202). Moon et al. (203) Bacillus licheniformis A7 B. coagulans SL5, Paenibacillus tyraminigenes tyramine (Mah et al., 2008) tryptamine. 항균제내성및최소발육억제농도 (Minimum Inhibitory Concentration, MIC) 의검토 tryptamine 7 32, 32 nalidixic acid bacitracin 30 7 ( ). 3 nalidixic acid MIC 28 g/ml. Bacitracin, 2 4 MIC 3 52 g/ml. Tryptamine 7. Tryptamine 생성균주의효소활성검토 Biogenic amines, (Species)

5 796 엄인선ㆍ김태옥ㆍ김희대ㆍ박권삼 Table 3. Enzymatic activities of tryptamine producing bacteria by API ZYM analysis Enzyme Strain No S S2 Control Alkaline phosphate Esterase (C4) Esterase Lipase (C8) Lipase (C4) Leucine arylamidase Valine arylamidase Crystine arylamidase Trypsin α-chymotrypsin Acid phosphatase Naphtol-AS-BI-phosphohydrolase α-galactosidase β-glucuronidase β-glucosidase α-glucosidase β-glucosidase N-acetyl-β-glucosaminidase α-mannosidase α-fucosidase , 0 nmol;, 5 nmol; 2, 0 nmol, 3, 20 nmol.. tryptamine 7 API ZYM Kit (Table 3). 20 3, 6-3. Lysinibacillus fusiformis (2, 4 5 ) esterase (C4) esterase lipase (C8) 5 nmol, Lysinibacillus xylanilyticus ( ) esterase (C4), esterase lipase (C8) -chymotrypsin 5 nmol. Staphylococcus epidermidis (S S2 ) acid phosphatase 20 nmol (Table 3). 6 Pediococcus pentosaceus SH-0 (Shin et al., 202) Lactobacillus salivarius CPM-7 (Lim et al., 2007), (Genus). 조건에따른분리균주의 tryptamine 생성량측정 Tryptamine R , retention time (RT) 7.43 (Fig. A B). Tryptamine 7 L-tryptophan 0.5%,.0% 2.0% LB broth (NaCl 3%) 5 ml 24, tryptamine HPLC (Table 4). Table L-tryptophan 0.5% tryptamine L-tryptophan tryptamine. L-tryptophan, L-tryptophan 0.5% ( ). tryptamine 24 2 ( 5 ), 48 (4 ) 72 2 (2

6 트립타민생성균의분리및동정 797 (A) (B) 7 3, y = 0.078x R² = , Area 4 3 Voltage (mv), Concentration (mg/l) Time (min) Fig.. Calibration curve for concentrated of tryptamine (A) and HPLC chromatograms of tryptamine (B). Table 4. Production of tryptamine of tryptamine producing bacteria in LB broth by incubation time and concentration of L-tryptophan Incubation time Strain No. 24 h 48 h 72 h L-tryptophan concentration 0.5% % 2% 0.5% % 2% 0.5% % 2% S S ). Staphylococcus epidermidis strain L29 S S2 tryptamine (Table 4)., 24 ( ). tryptamine 72. biogenic amines biogenic amines. Lysinibacillus spp. lysine ornithine decarboxylase (Lee et al., 200; Coorevits et al., 202; Duan et al., 203), tryptophan decarboxylase. tryptamine, biogenic amines. 사사 206 ( ).

7 798 엄인선ㆍ김태옥ㆍ김희대ㆍ박권삼 References Acar JF and Goldstein FW. 99. Disk susceptibility test. In: Antibiotics in Laboratory Medicine, Lorian V, ed. Williams & Wilkins, Baltimore, U.S.A., Biji KB, Ravishankar CN, Venkateswarlu R, Mohan CO and Gopal TK Biogenic amines in seafood: a review. J Food Sci Technol 53, s x. Blackwell B Hypertensive crisis due to monoamineoxidase inhibitors. Lancet 2, org/0.06/s (63) Canadian standards for various chemical contaminants in food Health Canada. Canada. Cho TY, Han GH, Bahn KN, Son YW, Jang MR, Lee CH, Kim SH, Kim DB and Kim SB Evaluation of biogenic amines in Korean commercial fermented foods. Korean J Food Sci Technol 38, Coorevits A, Dinsdale AE, Heyrman J, Schumann P, Van Landschoot A, Logan NA and De Vos P Lysinibacillus macroides sp. nov., nom. rev. Int J Syst Evol Microbiol 62, Duan YQ, He ST, Li QQ, Wang MF, Wang WY, Zhe W, Cao YH, Mo MH, Zhai YL and Li WJ Lysinibacillus tabacifolii sp. nov., a novel endophytic bacterium isolated from Nicotiana tabacum leaves. J Microbiol 5, Dunbar J, Ticknor LO and Kuske CR Assessment of microbial diversity in four southwestern Unites States soils by 6S rrna gene terminal restriction fragment analysis. Apple Environ Microbiol 66, Fernandes-Salfuero J and Mackie IM Preliminary survey of the contents histamine and other higher amines in some samples of Spanish canned fish. Int J Food Sci Technol 22, tb00504.x. Fritz SB and Baldwin JL In: Food allergy: Adverse Reactions to Foods and Food Additives. Pharmacologic food reactions rd ed. Metcalfe DD, Sampson Ha, Simon RA(eds). Blackwell Publishing, Malden Massachusetts, USA. Gardini F, Özogul Y, Suzzi G, Tabanelli G and Özogul F Technological factors affecting biogenic amine content in foods: a review. Front Microbiol 7, org/0.3389/fmicb Joostern HMLJ The biogenic amine contents of dutch cheese and their toxicological significance. Neth Milk Dairy J 42, Kim B, Byun BY and Mah JH Biogenic amine formation and bacterial contribution in Natto products. Food Chem 35, Kim BK, Kim YH, Lee HH, Cho YJ, Kim DS, Oh SM and Shim KB. 20. Comparison of the chemical compositions and biogenic amine contents of salt-fermented fish sauces produced in Korea to evaluate the quality characteristics. J Fish Mar Sci Edu 23, KMFDS (Korea Ministry of Food and Drug Safety) Korean Food Code. KMFDS, Cheongju, Korea. Retrieved from food/foodrvlv/food Rvlv.do. on August. Lee CS, Jung YT, Park S, Oh TK and Yoon JH Lysinibacillus xylanilyticus sp. nov., a xylan- degrading bacterium isolated from forest humus. Int J Syst Evol Microbiol 60, Lim SJ, Jang SS and Kang DK Probiotic properties of Lactobacillus salivarius CPM-7 isolated from chicken feces. Kor J Microbiol Biotechnol 35, Mah JH, Ahn JB, Park JH, Sung HC and Hwang HJ Characterization of biogenic amine producing microorganisms isolated from Myeolchi-Jeot, Korean salted and fermented anchovy. J Microbiol Biotechnol 3, Mah JH, Chang YH and Hwang HJ Paenibacillus tyraminigenes sp. nov. isolated from Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy. Int J Food Microbiol 27, Moon JS, Kim SY, Cho KJ, Yang SJ, Yoon GM, Eom HJ and Han NS Isolation and characterization of histamineproducing bacteria from fermented fish products. J Microbiol 5, NCCLS (National Committee for Clinical Laboratory Standards) Performance standards for antimicrobial susceptibility testing. Twelfth informational supplement M00- S2. Wayne, Pennsylvania, U.S.A., Otto M Molecular basis of Staphylococcus epidermidis infections. Semin Immunopathol 34, org/0.007/s Shalaby AR Significance of biogenic amines to food safety and human health. Food Res Int 29, dx.doi.org/0.06/s (96)00066-x. Shin DM, Kim HD, Koo JG and Park KS Inhibition of pathogenic bacteria by Pediococcus pentosaceus strain SH- 0 isolated from hard clam Meretrix meretrix Sikhae. Korean J Fish Aquat Sci 45, KFAS USFDA (US Food and Drug Administration). 20. Fish and Fishery Products Hazards and Controls Guidance. Fourth Edition. Chapter 7. Scombrotoxin (histamine) formation, Yang J, Ding X, Qin Y and Zeng Y Safety assessment of the biogenic amines in fermented soya beans and fermented bean curd. J Agric Food Chem 62,

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한수지 51(6), , 2018 Original Article Korean J Fish Aquat Sci 51(6), ,2018 국내산시판멸치 (Engraulis japonicas) 액젓의품질평가 엄인선 서정길 김희대 1 박권삼 * 군산대학교식품

한수지 51(6), , 2018 Original Article Korean J Fish Aquat Sci 51(6), ,2018 국내산시판멸치 (Engraulis japonicas) 액젓의품질평가 엄인선 서정길 김희대 1 박권삼 * 군산대학교식품 한수지 5(6), 667-672, 208 Original Article Korean J Fish Aquat Sci 5(6),667-672,208 국내산시판멸치 (Engraulis japonicas) 액젓의품질평가 엄인선 서정길 김희대 박권삼 * 군산대학교식품생명공학과, 충북도립대학바이오생명의약과 The Quality of Commercial Salted and