Korean Journal of Microbiology (2019) Vol. 55, No. 2, pp pissn DOI eissn Copyright

Transcription

1 Korean Journal of Microbiology (2019) Vol. 55, No. 2, pp pissn DOI eissn Copyright c 2019, The Microbiological Society of Korea 발효된장의바이오제닉아민함량에영향을미치는바실러스균의분리동정및프로바이오틱특성 임은서 * 동명대학교식품영양학과 Isolation, identification, and probiotic characteristics of Bacillus strains affecting the biogenic amine content in fermented soybean paste Eun-Seo Lim* Department of Food Science & Nutrition, Tongmyong University, Busan 48520, Republic of Korea (Received February 11, 2019; Revised March 25, 2019; Accepted March 27, 2019) The primary objective of this study was to determine the content of biogenic amines in Korean traditional fermented soybean pastes (doenjang) and to isolate potential probiotic Bacillus sp. with the ability to inhibit biogenic amines accumulation. There were significant differences in the bacterial cell counts, ph value, titratable acidity, salinity, and biogenic amine content between the samples. Among Bacillus strains isolated from doenjang, Bacillus (B.) licheniformis DB102, B. subtilis DB203, B. stearothermophilus DB206, Bacillus sp. DB209, Bacillus sp. DB310, B. coagulans DB311, B. cereus DB313, B. amyloliquefaciens DB714, Bacillus sp. DB917, B. cereus DB 915, B. subtilis DB1020, and Bacillus sp. DB1022 were found to be able to produce biogenic amines. On the other hand, biogenic aminedegrading strains were identified as Bacillus sp. DB403, Bacillus sp. DB407, B. subtilis DB517, B. licheniformis DB612, and B. subtilis DB821. In particular, Bacillus sp. DB407 and B. subtilis DB821 showed probiotic properties including tolerance to artificial digestive juices, adherence to intestinal epithelial cells, resistance to antibiotics, and antibacterial activity against biogenic amineproducing strains. In conclusion, the two probiotic Bacillus strains may be considered as the suitable starter for manufacture of fermented soybean foods with low biogenic amines content. Keywords: Bacillus, biogenic amine, probiotic *For correspondence. limsm020@tu.ac.kr; Tel.: ; Fax: 바이오제닉아민 (biogenic amine) 은생체내생리기능을유지하는데관여하는물질로서세포증식및분화, 핵산기능조절, 단백질합성, 두뇌발달, 신경세포성장과조절등살아있는세포에있어내재적필수불가결한성분이다 (Kalač and Krausová, 2005). 하지만과량의티라민이나페닐에틸아민등은고혈압의위험과식이성편두통을야기하는것으로보고되고있으며, 히스타민은알레르기식중독유발원인물질로알려져있고, 푸트레신, 스페르민, 스페르미딘및카다베린등은자체적으로는인체에해가적으나, 아질산염과반응하면발암물질인니트로자민을생성하게된다 (Hernandez-Jover et al., 1997). 특히히스타민의독성은소장내에있는히스타민대사효소를저해하는푸트레신이나카다베린등혼재하는다른종류의바이오제닉아민에의해유의하게증가된다 (Lehane and Olley, 2000). 게다가일부바이오제닉아민은장관에흡수되어대사기능을방해하고항영양물질 (anti-nutritional compound) 로서작용하며, 아드레날린, 노르아드레날린및위산의분비를자극하고심장병발병위험을높일뿐만아니라, 부정맥, 혈당상승및혈압을높이기도한다 (Shalabhy, 1996). 바이오제닉아민은세균, 효모및곰팡이등다양한미생물에의한주로생성되는저분자질소화합물로서탈탄산효소에의해특정아미노산의카르복시기를제거하여아민과이산화탄소를생성하게되는데 (Gardini et al., 2016), 독성을유발할정도의바이오제닉아민은탈탄산화를유발하는미생물의

2 132 Eun-Seo Lim 생균수가적어도 7 log CFU/g 이상이어야하므로유해한미생물의증식여부지표로써바이오제닉아민함량을측정하기도한다 (Al Bulushi et al., 2009). 식품내에생성되는가장흔한바이오제닉아민으로는히스타민, 티라민, 페닐에틸아민, 트립타민, 푸트레신및카다베린등이있다 (Wunderlichová et al., 2014). 사실상거의대부분의식품은단백질이나유리아미노산을함유하고있기때문에미생물학적활성에의해바이오제닉아민이생성될수있는데, 주로생선및가공품, 육류및가공품, 난류, 치즈, 발효야채, 과일, 너트, 초콜릿, 와인등단백질이풍부한음료나유제품및장류등의발효식품에서흔히검출된다 (Shalaby, 1996). 특히두류가공품내다량의유해아민은숙성에관여하는그람양성균및음성균, 곰팡이등의과도한증식에따른것으로알려져있다 (Shalaby, 1996). 일본식발효된장인미소 (miso) 내에는히스타민 (462 mg/100 g), 푸트레신 (1,234 mg/100 g), 카다베린 (634 mg/100 g) 및티라민 (3,568 mg/100 g) 등의바이오제닉아민이다량검출되었다고보고된바있다 (Shalaby, 1996). 한편 Cho 등 (2006) 은우리나라전통발효된장내에서히스타민과티라민이각각 952 mg/kg과 1,430.7 mg/kg 정도검출되었다고확인한바있다. 이외에도간장이나춘장및 sufu 등아시아각국의전통콩발효식품내에서도바이오제닉아민의함량이높게측정되었다 (Guan et al., 2013; Mah, 2015). 바이오제닉아민함량은식품을구성하는성분, 제조공정, 원료의품질, 원료내콩의비율, 발효스타터의종류및발효조건과기간에따라차이가있다 (Brink et al., 1990; Eerola et al., 1998). 따라서단백질함량이높고숙성과정을거쳐제조하는콩발효식품들의유해아민으로인한중독위험을최소화하기위해선미생물오염도가낮은신선한원료사용, 바이오제닉아민생성능이없는발효스타터이용및탈탄산효소생성미생물의증식을억제할수있는제조공정개선등저감화할수있는최적조건설정에관한연구가활발히진행되고있다. 최근연구에따르면, 유기산이나박테리오신과같은항균물질을생산하는세균들이바이오제닉아민생성균의증식을억제시킴으로써발효식품내유해아민의함량을낮출수있다는결과가보고된바있다 (Zhang et al., 2013). 유산균이나바실러스균은프로바이오틱 (probiotic) 균주로서숙주의장내미생물의균형을유지하여장건강에이로운역할을하는인체유익균이므로이들을발효스타터로이용하여발효식품을제조할경우유해아민생성위험을낮추고생리활성물질을생성함에따라건강향상에도도움을줄수있어유용하게이용될수있다 (Fuller, 1991). 지금까지보고된연구들은주로유산균을대상으로바이오제닉아민생성균을제 어하는내용이주를이루고있으므로본연구에서는시판되고있는발효된장의바이오제닉아민함량에영향을미치는바실러스균을분리동정하였고, 이중에서바이오제닉아민분해능을비롯하여인공소화액과항생제에대한내성, 용혈능및항균물질생성능등프로바이오틱활성을나타내는균주를최종선발하였다. 재료및방법 시판된장의미생물학적및물리화학적특성부산일대전통시장과마트에서된장 10종을구입한후시료 30 g에멸균된인산완충용액 (phosphate buffer saline, PBS) 270 ml를가한다음약 2분간스토마커 (3M Center) 로분쇄하였다. 균질화된시료용액은십진희석한후 Plate Count Agar (BD Difco Co.) 를사용하여표준한천평판배양법으로생균수를측정하였다. 한편, 시료용액은 80 C에서 15분간가열처리직후냉각한다음 Luria-Bertani (BD Difco Co.) 평판배지상에서내열성포자형성균수를측정하였고, 독립집락을 Brain Heart Infusion agar (BHI, BD Difco Co.) 상에서순수분리배양한다음배양액을 20% (v/v) glycerol stock으로제조하여 -80 C에서보관하면서실험하였다. 시료용액의 ph 및염도는 ph meter (Fisher Scientific) 와염도계 (CAS salt-free 2500) 로각각측정하였다. 한편, 시료 5 g에동량의증류수를가하고 1% (w/v) 페놀프탈레인지시약을첨가한후에 0.1 N NaOH 용액으로적정하여적정산도를계산하였다. 시료의바이오제닉아민함량은 Li 등 (2018) 의방법을일부변형하여측정하였다. 즉, 시료 (0.5 g) 에 1.5 ml 0.4 M HClO 4 를가한다음 1시간진탕하여추출한후원심분리 (12,000 g, 10분, 4 C) 해서얻은상등액 (250 μl) 에 25 μl 2 M NaOH와 75 μl 포화 NaHCO 3 를첨가하고 500 μl dansyl chloride를넣어 50 C, 45분간반응시켰다. 반응물은 25 μl 25% NH 4OH와혼합하고잔존하는 dansyl chloride을제거하기위해 50 C에서 15분배양한다음 1.5 ml acetonitrile를가하고원심분리 (2,500 g, 5분 ) 하여얻은상등액을 membrane filter (0.22 μm, Millipore Corp.) 로여과하여 dansyl 유도체를제조하였다. 시료내바이오제닉아민은 High pressure liquid chromatography (HPLC, Shimadzu) 의 Nova-Pak C 18 컬럼 ( mm, Waters) 을사용하여 30 C에서분석하였다. 이동상 ammonium acetate (0.1 M, solvent A) 와 acetonitrile (solvent B) 을선형구배로하여유속은 1 ml/min 하에서흡광도 (254 nm) 를측정함으로써시료내바이오제닉아민함량을구하였다. 미생물학회지제 55 권제 2 호

3 Biogenic amine-degrading ability of probiotic Bacillus strain 133 바실러스균주동정분리된내열성포자형성균주는그람염색하여그람양성균만을바실러스균으로간주한다음 DNA purification kit (Promega) 로 genomic DNA를추출한후 universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') 와 1492R (5'-GGTTA CCTTGTTACGACTT-3') 을이용하여 polymerase chain reaction (PCR) 으로 16S rrna 유전자를증폭시켰다. PCR 조건으로는주형 DNA 변성 (97 C에서 5분 ) 시킨다음 94 C에서 1분, 56 C 에서 1분, 72 C에서 1분 30초동안 35회반복수행하여 DNA 증폭시켰고최종적으로 72 C에서 5분간반응시켰다. PCR 산물은전기영동으로증폭여부를확인하였고, QIAquick PCR purification kit (Qiagen) 로정제한후염기서열을분석하였다. 16S rrna sequencing 결과는 National Center for Biotechnology Institute (NCBI) 의 Basic Local Alignment Search Tool (BLAST) analysis ( 를이용하여 GenBank database를통해분리균주와의상동성을비교하여동정하였다. 분리균주의바이오제닉아민생성및분해능동정된바실러스균주의바이오제닉아민생성능은효소유도를촉진시키기위해전구체아미노산 (L-histidine monohydrochloride monohydrate, L-tyrosine disodium salt, L-lysine monohydrochloride 및 L-ornithine monohydrochloride, Sigma- Aldrich, 1 g/l) 과 1 mg/l pyridoxal 5-phosphate를첨가한탈카르복시화액체배지 (decarboxylating broth) 에서 37 C. 24시간동안 5회전배양하였다. 각각의아미노산 (2%, w/v) 이첨가된탈카르복시화액체배지 (1 ml) 에전배양액 (0.5 ml) 을접종한후혐기적인조건 (Anoxomat 8000 system, MART Co.) 에서 37 C, 72시간동안배양한후앞서설명한방법에따라 HPLC를이용하여바이오제닉아민생성량을측정하였다. 바이오제닉아민분해능은 Lee 등 (2015) 의방법을일부변형하여측정하였다. 즉, 실험균주는 BHI broth에접종하여 37 C, 24시간동안배양한후원심분리 (7,000 g, 10분, 4 C) 해서모은세포를 PBS (ph 7.0) 로 2회세척하였다. 세포현탁액 (1 ml, CFU/ml) 은바이오제닉아민 (histamine dihydrochloride, tyramine hydrochloride, cadaverine dihydrochloride, putrescine dihydrochloride, 0.1%, w/v) 을첨가한액체배지 [glucose 0.1% (w/v), yeast extract 0.2% (w/v), NaCl 0.5% (w/v), K 2HPO % (w/v); ph 7.0, 10 ml] 에접종하고난다음 35 C, 5일간배양한후에얻은배양액 (0.1 ml) 은바이오제닉아민 (2%, w/v) 평판배지에도말접종하여 30 C, 5일간배양하였다. 평판배 지상에자란독립된집락을선택하여 trypticase soy agar (TSA, BD Difco Co.) 상에서순수분리한다음바이오제닉아민분해능을조사하기위해바이오제닉아민 (50 ppm) 이첨가된 trypticase soy broth (TSB, BD Difco Co.) 에접종하고 35 C에서 24시간동안배양하였다. 바이오제닉아민혼합표준용액 (500 ppm) 및세포배양액 1 ml에 0.4 M perchloric acid (Merck) 9 ml를가하고진탕혼합한후원심분리 (3,000 g, 10분 ) 하여얻은상등액은 Whatman paper No. 1로여과하였다. 시험용액은 dansyl chloride로유도체화한후에앞서언급에조건과같이 HPLC 를이용하여잔존하는바이오제닉아민함량을측정하여계산식 (M = [(A-B)/A] 100, M: 바이오제닉아민분해능 (%), A: 초기바이오제닉아민함량, B: 잔존하는바이오제닉아민함량 ) 에따라분해능을조사하였다. 프로바이오틱활성측정인공위액및담즙액에대한저항성 : 인공소화액에대한저항성은 Lee 등 (2017) 의방법을일부변형하여측정하였다. 즉, 분리균주를 BHI broth에접종한후 37 C, 24시간배양하여얻은배양액으로부터원심분리 (7,000 g, 10분, 4 C) 을통해모은세포침전물을 PBS (ph 7.0) 로 2회세척후세포수를 CFU/ml에맞춰현탁액을제조하였다. PBS (ph 2.5) 에 125 mm NaCl, 7 mm KCl, 45 mm NaHCO 3 및 1 mg/ml pepsin (Sigma-Aldrich) 을첨가하여제조한인공위액에바실러스균현탁액을접종한후 37 C에서 2시간배양한다음잔존하는균수를측정하여인공위액상에서의생존율 (%) 을조사하였다. 한편, BHI broth에 3.0% (w/v) bile salts (Sigma-Aldrich) 를첨가하여인공담즙액 (10 ml) 을제조한다음인공위액에서잔존하는균수로조정한균현탁액 (1 ml) 을접종하고 37 C에서 3 시간배양하였다. BHI agar 상에서평판배양후잔존하는균수를측정하여대조구 (bile salts대신 PBS 첨가 ) 상의균수와비교하여인공담즙액상에서의생존율 (%) 을조사하였다. 장내상피세포에대한부착능 : 선발균주의장관상피세포에대한부착능은 Kim 등 (2009) 의방법을일부변형하여측정하였다. Korean Cell Line Bank (KCLB) 로부터분양받은 Caco-2 세포는 56 C에서 30분간가열처리한 10% (v/v) fetal bovine serum (FSB, Gibco), 2 mm L-glutamine, 1 mm sodium pyruvate, 100 U/ml penicillin 및 0.1 mg/ml streptomycin을첨가한 Dulbecco s modified Eagle s minimal essential medium (DMEM, Sigma-Aldrich) 에접종하고 37 C, 5% CO 2 조건하에서배양하여 monolayer를형성하도록하였다. FBS와항생제가첨가 Korean Journal of Microbiology, Vol. 55, No. 2

4 134 Eun-Seo Lim 되지않은 DMEM 배지를 12-well culture plate (Falcon) 에분주하고난다음 Caco-2 세포 ( cells/ml) 현탁액을접종한후 37 C, 5% CO 2 조건하에서 2시간동안전배양하였다. 한편, BHI broth에서 37 C, 24시간동안배양한바실러스균배양액은원심분리 (7,000 g, 10분, 4 C) 를통해세포만을모아 PBS (ph 7.0) 로세척한다음 DMEM 배지 (1.6 ml) 내에현탁시켜앞서인공담즙액에서배양한후잔존하는균수로조정하였다. Caco-2 세포현탁액 (0.2 ml) 을접종한 plate의 well에바실러스균현탁액 (0.2 ml) 을접종하고 37 C에서 2시간동안배양한후 Caco-2 세포에부착되지않은바실러스균을제거하였다. 부착된세균은 trypsin-edta 용액을처리하여탈착시킨다음 PBS (ph 7.0) 로세척하고 BHI agar에서평판배양하여바실러스균수를측정하여부착율 (%) 로나타내었다. 항생제에대한저항성 : 항생제에대한저항성은 Jeon 등 (2016) 과 Wang과 Su (2016) 의방법을일부변형하여최소증식억제농도 (minimum inhibitory concentration, MIC) 를측정하였다. 선발균주는 BHI broth에접종하여 37 C, 24시간배양한후원심분리 (7,000 g, 10분, 4 C) 해서모은세포침전물을 PBS (ph 7.0) 로 2회세척한다음세포수를 CFU/ml로조정하였다. 세포현탁액 (1%, v/v) 은 BHI agar (agar 1.0%, w/v) 에접종하고난후 BHI 평판배지위에중층하여응고시켰다. 항생제 (ampicillin, erythromycin, kanamycin, penicillin G, streptomycin, tetracycline 및 vancomycin; Sigma-Aldrich) 의 stock solution (1 mg/ml) 을 2배씩연속적으로희석한다음 paper disk (φ 8 mm) 에 loading (50 μl) 한후균현탁액이접종된 BHI 평판배지위에올리고 37 C에서 24시간배양하여 disk 주변에저해환을생성한최소농도를측정하였다. 용혈능실험균주는 BHI agar 사면배지에서 3회계대배양하여활성을높인후 5% (w/v) human blood가함유된 BHI agar 평판배지에획선접종후 37 C에서 48시간배양한후 α-haemolysis ( 집락주변녹색환생성 ), β-haemolysis( 집락주변황색투명환생성 ) 및 γ-haemolysis( 집락주변환생성없음 ) 를조사하였다. 항균활성측정바실러스균의박테리오신활성은 Savitha 등 (2016) 의방법을일부변형하여측정하였는데, 즉 BHI broth에접종하여 37 C에서 24시간배양한전배양액을 0.1% (w/v) 포도당이첨 가된 BHI broth상에서 35 C, 22시간동안 150 rpm으로진탕배양하였다. 배양액은원심분리 (7,000 g, 10분, 4 C) 해서얻은배양상등액 6 N NaOH를이용하여 ph 6.5로조정하고 1 mg/ml catalase (Sigma-Aldrich) 를처리한다음 40% (w/v) 황산암모늄을첨가하여 4 C에서 overnight 동안교반해서단백질을침전시켰다. 원심분리 (12,000 g, 30분, 4 C) 해서모은침전물은 20 mm PBS (ph 6.5) 에현탁시키고 4 C에서 24시간동안동일한 buffer 내에서투석막 (molecular weight cut-off = 1,000 Da; Spectrum Medical Industries, Inc.) 으로투석시켰다. 시료용액 (100 ml) 에클로로포름과메탄올 1:1의비율로만든혼합용액 (100 ml) 을첨가하여격렬하게진탕시킨후 4 C에서 1시간동안방치시킨다음분액깔대기로옮겨수상과유기상사이박테리오신이함유된계면층을회수하고남은클로로포름은고속진공기로제거하여조박테리오신용액을제조한것을 microtiter plate method (Holo et al., 1991) 로항균활성을측정하였다. 항균력측정에사용된지시균주는된장에서분리한바이오제닉아민생성바실러스균주로서이들을 BHI broth에접종하여 37 C에서 24시간배양한후원심분리 (7,000 g, 10분, 4 C) 하여세포를모으고 PBS (ph 7.0) 로 2회세척한다음세포수를 CFU/ml로조정하였다. Plate well에 BHI broth를분주하고 2진희석법으로농도를맞춘박테리오신용액을첨가한다음지시균주 ( CFU/ml) 의세포현탁액 (1%) 을접종하였다. 37 C에서 24시간배양한후 microplate reader (BioTek, Inc.) 를이용하여흡광도 (600 nm) 를측정하고박테리오신용액대신 PBS (ph 7.0) 를처리한대조구와비교하여배양액의혼탁도가 50% 저해된최대희석배수의역수를박테리오신활성 (arbitrary units, AU) 으로나타내었다. 결과및고찰 시판된장의미생물학적및물리화학적특성시료 10종에대한호기성일반세균수, 바실러스균수, ph, 적정산도, 염도및바이오제닉아민의함량을측정한결과는 Table 1과같다. 일반세균수는 8.8 ± ~5.4 ± CFU/g, 바실러스균수는 4.0 ± ~1.8 ± CFU/g 으로나타났다. Lee 등 (2009) 은재래된장과시판된장으로부터일반세균수는 6.02 ± 0.57~7.96 ± 0.03 Log CFU/g의분포를보여시판개량식된장보다재래식된장에서유의하게높은균수가검출되었다고보고하였는데본연구결과에서는이보다다소높은균수가측정되었으며, 시료별균수에차이가나는것은원료, 제조방법및환경의차이에서기인한다고보 미생물학회지제 55 권제 2 호

5 Biogenic amine-degrading ability of probiotic Bacillus strain 135 Table 1. Microbiological and physicochemical characteristics of traditional fermented soybean paste Sample Viable cell counts (CFU/g) Titratable Salinity Biogenic amine contents (mg/kg) ph No. Aerobic Bacillus sp. acidity (%) (%) Cadaverine Histamine Putrescine Tyramine 1 5.0± ± ± ± ±0.1 1,616.3± ±10.7 ND 130.5± ± ± ± ± ± ±22.5 1,123.3±0.2 ND 1,008.3± ± ± ± ± ± ± ±20.4 1,128.0± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±5.8 ND 205.4± ± ± ± ± ± ± ±22.9 ND 90.1± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.2 ND 127.7± ±8.7 ND 9 7.1± ± ± ± ± ± ± ± ± ± ± ± ± ±0.0 1,951.4± ± ±3.3 1,156.3±0.3 ND, not detected (biogenic amine level less than 1 ppm). 고하였다. 또한된장에서분리되는주요미생물로는누룩곰팡이및효모와같은진균류를비롯하여바실러스균, 유산균, Micrococcus sp. 등과같은각종세균들이주를이루고, 우점종인바실러스균주들은주로메주의발효과정에사용되는볏짚이나발효환경으로부터오염된것으로추정되었다고보고 (Lee et al., 2009) 되었다. Shim 등 (2016) 은된장제조직후바실러스균수가 7.59 ± 0.10~9.36 ± 0.59 log CFU/g에이르렀고발효가진행될수록균수는서서히증가하다가일정기간후에다시감소하는경향을나타내었다고하였는데본연구에서도일부의시료에서비슷한수준의균수가검출되었다. 한편, ph (4.91 ± 0.22~6.30 ± 0.08), 적정산도 (0.35 ± 0.01~ 0.88 ± 0.01), 염도 (12.7 ±0.0~17.2 ± 0.2%) 등도시료마다측정값이다양하게나타났다. Shukla 등 (2010) 은우리나라전통발효된장 23종의 ph를측정한결과 4.8~6.0 정도로발효가진행될수록산도가높아졌다고하였는데이는유산균의증식에따른것이라고하였는데본연구에서도이와유사한 ph값이측정되었다. Kim 등 (2003a) 도된장의낮은 ph (3.0~6.0) 가탈탄산효소활성을증가시켜바이오제닉아민의생성량을증가시킨다고보고하였다. 카다베린은시료 8에서는검출되지않았으나그외시료에서는 58.8 ± 8.6~1,951.4 ±13.7 mg/kg, 히스타민은시료 5와 6에서불검출되었으나, 나머지시료에서는 ± 6.6~1,123.3 ± 0.8 mg/kg, 푸트레신은시료 1과 2에서는검출되지않았으나, 그외시료에서는 56.1 ± 3.3~1,128.0 ± 15.8 mg/kg, 티라민은시료 8 이외에서 41.8 ± 2.8~1,008.3 ± 26.9 mg/kg으로시료마다상이하게측정되었다. Lee 등 (2009) 은재래식및개량식된장으로부터검출된바이오제닉아민의함량은시료별로유의한차이 ( 푸트레신 : 28.8 ± 9.6~1,076.6 ± 9.5 mg/kg, 티라민 : 12.5 ± 4.1~967.6 ± 7.4 mg/kg, 소량의히스타민과카다베린 ) 가있었는데재래식된장보다개량식된장에서푸트레신, 티라민, 히스타민의함량이훨씬높게나타났다고하였다. Cho 등 (2006) 도재래식및개량식된장에서검출된바이오제닉아민으로는푸트레신, 카다베린, 히스타민, 티라민, 스페르미딘및스페르민등이있었는데개량식 (180~295.5 mg/kg) 보다는재래식된장 (234.1~1,453.7 mg/kg) 에서더많은양의아민이검출되었다고하여이들을본연구결과와비교해볼때원료, 제조방법및상재하는미생물의종류에따라된장내바이오제닉아민의함량에큰차이가있음을알수있었다. 또한된장시료별바이오제닉아민함량의차이는원료내단백질의비율, 제조과정 ( 콩의침지시간이나가열온도 ) 및미생물오염도에기인하는것으로알려져있다 (Shukla et al., 2010). 한편, Lee 등 (2009) 은시료내일반세균수와바이오제닉아민함량과는상관성이크지않았고, 반면식품내아민의함량은내재된미생물의아미노산이용능, 아미노산탈탄산효소를생성하는미생물수, 발효및숙성기간, 제조공정, 식염첨가량, 주정등의기타원료사용여부에따라차이가나는것으로고찰하였다 (Kim et al., 2003b). 게다가낫또 (natto) 의 ph (4.9~7.3), 호기성세균수 (7.8~11.2 Log CFU/g) 및히스타민의함량사이에는상관관계가없다고확인되었으며, 본연구결과도이와유사하게호기성세균수, 바실러스균수, ph, 산도및염도와바이오제닉아민함량간에는뚜렷한상관관계가없는것으로확인되었다. 두반장, 된장이나낫또와같은두류발효식품에는특히베타-페닐에틸아민, 티라민및히스타민의함량이다른유해아민들에비해높다고알려져있으며, 티라민은 100~800 mg/kg, 베타-페닐에틸아민은 30 mg/kg 검출되었고, 히스타민은 100 mg/kg 이상섭취시독성을유발한다고보고 Korean Journal of Microbiology, Vol. 55, No. 2

6 136 Eun-Seo Lim 하였는데 (Mah, 2015), 본연구결과에서도우리나라대표적인콩발효식품인된장의특정시료내에서는위험수준의아민이검출되었다. 바실러스균분리동정및바이오제닉아민생성과분해능전통발효된장으로부터분리된바실러스균의동정결과는 Table 2와같다. DB102 (99.9%), DB203 (99.0%), DB206 (99.8%), DB311 (100.0%), DB313 (99.7%), DB513 (99.2%), DB517 (98.7%), DB605 (99.0%), DB612 (100.0%), DB618 (98.9%), DB714 (99.9%), DB804 (99.1%), DB809 (99.6%), DB821 (100.0%), DB1019 (99.9%), DB1020 (100.0%) 등은상동성이높게나타나각각 Bacillus (B.) licheniformis, B. subtilis, B. stearothermophilus, B. coagulans, B. cereus, B. thuringiensis, B. subtilis, B. thermoamylovorans, B. licheniformis, B. pumilus, B. amyloliquefaciens, B. circulans, B. amyloliquefaciens, B. subtilis, B. cereus 및 B. subtilis로동정되었다. 하지만 DB108 (98.2%), DB209 (98.1%), DB310 (96.3%), DB403 (95.1%), DB407 (97.0%), DB520 (96.0%), DB730 (97.0%), DB814 (96.8%), DB915 (97.2%), DB917 (98.0%), DB1022 (98.4%) 등은신종여부의판단기준인 98.65% (Kim et al., 2014) 보다상동성낮아이들은 Bacillus sp. 로명명하였다. 이상의결과와같이시료마다분리된바실러스균종은다양하였으며, B. subtilis 가검출된시료수가가장많았고, 식중독을유발하는 B. cereus 도몇몇시료로부터분리되었다. 한편, 본연구에서사용된된장내아민의함량이높은이유는시료내에함유된아미노산탈탄산효소생성능이있는미생물에기인하는것으로사료되므로된장내우점종중하나인바실러스균을대상으로바이오제닉아민생성능을조사한결과 Table 2. Identification of Bacillus sp. isolated from traditional fermented soybean paste Sample No. Isolated strain Related strain in NCBI GenBank access Percentage identity (%) 1 DB102 Bacillus licheniformis PB1 MH DB108 Bacillus megaterium DS-4 MF DB203 Bacillus subtilis PS01 MG DB206 Bacillus stearothermophilus N1233 KY DB209 Bacillus pumilus PM2 KX DB310 Bacillus subtilis GS1 MG DB311 Bacillus coagulans LA204 KM DB313 Bacillus cereus B4 KM DB403 Bacillus licheniformis SMR6 KU DB407 Bacillus subtilis N3 KU DB513 Bacillus thuringiensis B17 JQ DB517 Bacillus subtilis J1 KT DB520 Bacillus cereus 16L KF DB605 Bacillus thermoamylovorans JNTUH16 FR DB612 Bacillus licheniformis SCTB104 JN DB618 Bacillus pumilus A586 AF DB714 Bacillus amyloliquefaciens PB3 MH DB730 Bacillus circulans EGK6 MG DB804 Bacillus circulans AMJ245 KY DB809 Bacillus amyloliquefaciens LH23 JQ DB814 Bacillus licheniformis LZ049 JQ DB821 Bacillus subtilis DMS KR DB915 Bacillus amyloliquefaciens SML1161 MG DB917 Bacillus licheniformis KKR2017 MF DB1019 Bacillus cereus WS-1 MF DB1020 Bacillus subtilis SPB18 KY DB1022 Bacillus megaterium SAK KM 미생물학회지제 55 권제 2 호

7 Biogenic amine-degrading ability of probiotic Bacillus strain 137 는 Table 3과같다. 실험균주중에서가장많은양의바이오제닉아민생성균을조사한결과, 카다베린은 B. licheniformis DB102 (1,051.0 ± 33.5 mg/l), 히스타민은 Bacillus sp. DB310 (1,345.5 ± 37.2 mg/l), 푸트레신은 B. amyloliquefaciens DB714 (1,069.5 ± 31.1 mg/l), 티라민은 B. subtilis DB203 (2,018.4 ± 45.3 mg/l) 에의해생산되었다. 이외에도 B. stearothermophilus DB206은카다베린 (804.2 ± 25.1 mg/l), B. coagulans DB311 은히스타민 (778.2 ± 10.2 mg/l) 과푸트레신 (809.1 ± 40.5 mg/l), Bacillus sp. DB915는과량의티라민 (925.4 ± 22.5 mg/l) 을생산하는것으로확인되었다. 이상의결과에서볼때, 동일한균 종일지라도균주에따라생성하는바이오제닉아민의종류가다르고생성량에도큰차이가있음을확인하였다. 반면, Bacillus sp. DB403은카다베린 (33.4 ± 0.7%) 을분해하였고, Bacillus sp. DB407은푸트레신 (9.8 ± 0.9%) 과티라민 (26.7 ± 1.3%) 을분해하는것으로확인되었다. 또한 B. licheniformis DB612는히스타민 (30.4 ± 0.9%), B. subtilis DB821은히스타민 (29.6 ± 1.1%) 과푸트레신 (55.0 ± 0.2%) 에대한분해능이나타났다. 하지만그이외의균주들로부터는아민분해능이확인되지않았고균주에따라분해능이상이하였다 (Table 4). Kim 등 (2012) 도된장내주된미생물인바실러스균은종에따라아 Table 3. Biogenic amine-production ability of Bacillus sp. isolated from traditional fermented soybean paste Strain Cadaverine (mg/l) Histamine (mg/l) Putrescine (mg/l) Tyramine (mg/l) Bacillus licheniformis DB102 1,051.0±33.5 ND ND ND Bacillus subtilis DB203 ND ND ND 2,018.4±45.3 Bacillus stearothermophilus DB ± ±19.5 ND ND Bacillus sp. DB ±13.4 ND ND ND Bacillus sp. DB310 ND 1,345.5±37.2 ND ND Bacillus coagulans DB311 ND 778.2± ±40.5 ND Bacillus cereus DB ±30.2 ND 462.0±5.9 ND Bacillus amyloliquefaciens DB714 ND ND 1,069.5±31.1 ND Bacillus sp. DB ±11.5 ND ND 925.4±22.5 Bacillus sp. DB917 ND 582.3±29.6 ND ND Bacillus cereus DB1019 ND ND ND 391.2±11.3 Bacillus subtilis DB ±8.9 ND ND 489.1±21.5 Bacillus sp. DB ±2.3 ND ND ND ND, not detected. Table 4. Biogenic amine-degradation ability of Bacillus sp. isolated from traditional fermented soybean paste Strain Cadaverine (%) Histamine (%) Putrescine (%) Tyramine (%) Bacillus sp. DB108 ND ND ND ND Bacillus sp. DB ±0.7 ND ND ND Bacillus sp. DB407 ND ND 9.8± ±1.3 Bacillus thuringiensis DB513 ND ND ND ND Bacillus subtilis DB517 ND 44.1± ±2.7 ND Bacillus sp. DB520 ND ND ND ND Bacillus thermoamylovorans DB605 ND ND ND ND Bacillus licheniformis DB612 ND 30.4±0.9 ND ND Bacillus pumilus DB618 ND ND ND ND Bacillus sp. DB730 ND ND ND ND Bacillus circulans DB804 ND ND ND ND Bacillus amyloliquefaciens DB809 ND ND ND ND Bacillus sp. DB814 ND ND ND ND Bacillus subtilis DB821 ND 29.6± ±0.2 ND ND, not detected. Korean Journal of Microbiology, Vol. 55, No. 2

8 138 Eun-Seo Lim 민을생성하기도하지만일부 B. subtilis, B. amyloliquefaciens 및 B. licheniformis 등은바이오제닉아민의함량을감소시키는것으로보고한바있는데본연구와일부균종에서동일한결과가나타났다. 콩을삶은후볏짚내상재균을이용하여발효시킨메주가된장의주원료이므로된장에서분리된주요미생물들은주로메주발효과정중의유입균에기인하는것으로알려져있다. 메주로부터분리된주요바실러스균으로는 B. citreus, B. circulans, B. licheniformis, B. megaterium, B. mesentricus, B. subtilis, B. pumilus 등이있다고보고 (Shin and Jeong, 2015) 된바있는데이와동일한균종일부가된장에서검출되었다. 대두발효식품내우점종인바실러스균종은매우다양하며이들의바이오제닉아민생성능및분해능도균종에따라상이한것으로나타났다. Han 등 (2007) 에따르면청국장에서분리된 B. subtilis, B. amyloliquefaciens, B. licheniformis 등이바이오제닉아민을생성하였고, Choi 등 (2012) 도전통발효청국장으로부터 B. subtilis, B. amyloliquefaciens, B. licheniformis 등이탈탄산효소를생성하여바이오제닉아민을생성한다고보고하였는데본연구의전통발효된장에서도이와유사한균종들이바이오제닉아민을생성하는것으로밝혀졌다. 전통장류로부터분리된 B. subtilis SCC B1110, SCK B1108, SCK B1109, SCS B1106과 B. amyloliquefaciens SCB B1307 은 27~92% 의바이오제닉아민을분해하였는데이중에서도 B. subtilis은히스타민과티라민보다는푸트레신과카다베린의분해율이더높게나타났으며, 아민의분해정도는동일한균종이라도균주에따라상이한것으로밝혀졌다 (Kim et al., 2012). 또한전통발효방식으로제조한청국장으로부터분리한 B. subtilis subsp. subtilis NCIB 3610과상동성이 99% 이상인 YD-6, KJ-21, 2RL2-3, PJ-7, KJ-10 및 Mpul-4 등의균주들은비교적높은티라민분해능을보여주었는데 (Moon et al., 2015) 이와같이아민분해메커니즘으로는아민산화효소 (amines oxidase) 에의한산화적탈아미노화반응이나, 항균물 질을생산함으로써유해아민생성균의증식을억제하거나사멸시켜아민생성량을감소시키는것으로밝혀졌다 (Tabanelli et al., 2014). 바이오제닉아민분해능이있는바실러스균의프로바이오틱활성 바이오제닉아민분해능이있는프로바이오틱바실러스균을발효스타터로이용하고자인공소화액에대한내성, 장관상피세포에대한부착능및병원성을조사한결과는 Table 5와같다. 실험균주중에서 B. subtilis DB517 (4.5 ± 1.4%) 과 Bacillus sp. DB403 (2.7 ± 0.2%) 은인공위액에대한저항성이높았던반면, Bacillus sp. DB407, B. licheniformis DB612 및 B. subtilis DB821의생존율은 1% 미만으로저항성이낮게나타났다. 한편, 3% bile salts가첨가된인공담즙액내에서 B. licheniformis DB612 (90.7 ± 4.0%) 와 B. subtilis DB821 (93.8 ± 0.7%) 은인공담즙액에대한저항성이높게나타났으나, Bacillus sp. DB403, Bacillus sp. DB407 및 B. subtilis DB517은이들보다낮은생존율을나타내었다. 체내로유입된세균의많은생균수가대장에도달한후상피세포에부착한다음기능성을발휘해야만하므로산도가높은위액과담즙액에대한저항성은프로바이오틱균주가되기위한필수적인요건이다 (Jena et al., 2013). Hanifi 등 (2015) 에따르면 B. subtilis R0179는위장관을통과하는동안높은생존율을나타내었고인체에무해하여다양한형태로이용가능하다고설명하였다. Argyri 등 (2014) 에따르면 ph 2.5의인공위액내에서 3시간동안 Lactobacillus 속보다포자형성균인바실러스균이인공소화액에대한저항성이크다고보고하였고 B. amyloliquefaciens AMS1은 0.3% pepsin (ph2.0) 이첨가된배양액에서 3시간동안 70.07% 의생존율을보였다 (Manhar et al., 2015). 전통간장으로부터분리된바실러스속균주는위액에대해 91% 이상의높은저항성을나타내었고, 특히 3% 의 bile salts 하에서도 90.31~99.31% 에이르렀으나 (Lee et al., 2017), 본연구에서분리된바실러스균들의저 Table 5. Probiotic characteristics of Bacillus sp. isolated from traditional fermented soybean paste Strain Survival (%) Adhesion Antibiotics resistance (MIC, μg/ml) Gastric juice Intestinal juice (%) A E K P S T V Haemolysis Bacillus sp. DB ± ± ± α Bacillus sp. DB ± ± ± α Bacillus subtilis DB ± ± ± α Bacillus licheniformis DB ± ± ± γ Bacillus subtilis DB ± ± ± α A, ampicillin; E, erythromycin; K, kanamycin; P, penicillin; S, streptomycin; T, tetracycline; V, vancomycin. 미생물학회지제 55 권제 2 호

9 Biogenic amine-degrading ability of probiotic Bacillus strain 139 항성은이들보다저항성이낮았으나, 일부분리균들은사람이나동물의장관내에서상당수생존할것으로추정되었다. Caco-2 세포에대한부착능을측정한결과, B. subtilis DB517 (2.2 ± 0.2%) 이가장낮았고, Bacillus sp. DB403 (20.5 ± 1.1%) 과 B. licheniformis DB612 (11.8 ± 1.8%) 의부착능도비교적높았으며, B. subtilis DB821 (30.5 ± 3.5%) 은가장높은부착능을보였다. 간장에서분리된 Bacillus sp. MKSK-MI, E1 및 J1 균주들은강한세포소수성과자기응집성을나타내었으므로세포부착력이높은것으로나타났으며 (Lee et al., 2017), B. amyloliquefaciens SRCM 의인체상피세포에대한부착율도 72.2% 에이르렀다고보고하였는데 (Ryu et al., 2017), 본연구에서분리된바실러스균주들은이들보다는다소낮은부착능을보여주었다. 자일렌, 클로로포름혹은에틸아세테이트와같은용제로인한세포소수성은세균의세포부착능지표에이용되며, 부착능이높을수록연동운동에의한세포의탈락을막고상피세포에부착하도록하여장내환경의균형을맞춰기능성을발휘하게된다고보고하였다 (Pedersen and Tannock, 1989; Alander et al., 1997). 체내상피조직에대한부착과집락형성을통해프로바이오틱미생물은세포수용체에입체적상호작용이나특이적봉쇄에의해병원균의접근을막을수있다 (Otero et al., 2004). Bacillus sp. DB403은 erythromycin (128 μg/ml), kanamycin (256 μg/ml) 및 streptomycin (128 μg/ml), Bacillus sp. DB407 은 ampicillin (128 μg/ml), kanamycin (128 μg/ml), penicillin G (256 μg/ml) 및 tetracycline (512 μg/ml), B. subtilis DB517은 ampicillin (256 μg/ml), erythromycin (128 μg/ml) 및 tetracycline (256 μg/ml), B. licheniformis DB612은 streptomycin (256 μg/ml) 및 tetracycline (512 μg/ml), B. subtilis DB821은 erythromycin (256 μg/ml), penicillin G (256 μg/ml) 및 vancomycin (128 μg/ml) 등의항생제에대한저항성이강한것으로나타나균주마다저항할수있는항생제의종류가다르고, MIC도상이하였다. B. subtilis DET6은 B. megaterium JHT3과 B. thuringiensis DET9 균주보다 erythromycin과 novobiocin에대해감수성이높았다. 반면 JHT3은 DET6과 DET9 균주에비해 tetracycline과 lincomycin에대해서는감수성이높게나타났으나, ampicillin 과 penicillin에대해서는감수성이낮은것으로확인되었고 (Patel et al., 2009), B. amyloliquefaciens AMS1은 ampicillin 과 penicillin에대해강한저항성을보였다 (Manhar et al., 2015). 간장으로부터분리된 MKSK-E1, J1 및 M1 바실러스균주는세포벽을저해시키는항생제인 ampicillin, cephalexin, penicillin G 및 vancomycin 등과단백질합성저해제인 chloramphenicol, erythromycin, gentamicin 및 tetracyclin 등에감수성이높았다 (Lee et al., 2017). B. licheniformis DB612만이 γ-haemolysis를나타내었으나, 그이외의균주들은모두 α-haemolysis를생산하는것으로확인되었다. B. megaterium JHT3은 γ-haemolysin을생산한반면, B. subtilis DET6과 B. thuringiensis DET9는 β-haemolysin 을생산하였다 (Patel et al., 2009). B. cereus는 β-haemolysin 현상을나타내었는데이는세균의세포독성인지질분해효소에의하며, 용혈인자는숙주를위한철원자의공급원인헤모글로빈의양을감소시킨다 (Seker, 2010). 이미보고된결과와비교해볼때바실러스균의항생제에대한저항성과용혈성은균주에따라차이가있음을알수있었다. 바이오제닉아민생성능이있는바실러스균주에대한프로바이오틱활성이있는바실러스균의항균활성을측정한결과는 Table 6과같다. Bacillus sp. DB407은 B. subtilis DB203 (256 AU/ml), Bacillus sp. DB310 (128 AU/ml), B. cereus DB313 (128 AU/ml), B. cereus DB1019 (64 AU/ml), B. subtilis DB1020 (64 AU/ml) 에대해항균활성을나타내었다. B. subtilis DB821은 B. subtilis DB203 (64 AU/ml), Bacillus sp. DB209 (128 AU/ml) 와 Bacillus sp. DB1022 (32 AU/ml) 의증식억제효과가있는것으로확인되었으므로이들을된장발효스타터로이용한다면유해아민의함량을낮추는데효과적일것으로판단된다. 하지만 Bacillus sp. DB403과 B. licheniformis DB612 및 B. subtilis DB517은지시균주의증식을억제할수있는박테리오신을생산하지못하는것으로나타났다. 간장에서분리된바실러스속 MKSK-MI, E1 및 J1 균주들은 S. aureus, B. cereus, E. coli, Shigella sonnei, Shigella flexneri, Pseudomonase fluorescens 및 Klebsiella pneumoniae에대해항균활성을나타내었는데특히전통발효장류식품의안전을위협하는대표적인 B. cereus의증식을가장강하게억제하는데효과적임을확인하였다 (Lee et al., 2017). 바실러스균은대사활성이높기때문에인체장관내에서중요한역할을하는것으로알려졌는데이는항균물질생산에따른것이며항균스펙트럼과항균물질의구조는매우다양하다. 특히, B. subtilis 는다양한병원균 (S. aureus, L. monocytogenes, B. cereus, Streptococcus pyogenes, Candida albicans 등 ) 에대해특이적이고신속하게사멸시킬수있는항균물질을생산하였고, 이물질은인체항미생물의방어시스템의일부분처럼작용하므로병원균의내성과부작용발생가능성이극히낮다고하였다 (Olmos and Paniagua-Michel, 2014). B. subtilis가생산한항균물질로는 subtilin, ericin, mersacidin, subtilosin, bacillocin, surfactin, bacilysin 등매우다양하며이들은세포막에구멍을뚫어서세포내물질을유출시키고, 세포벽합성을저해하고 Korean Journal of Microbiology, Vol. 55, No. 2

10 140 Eun-Seo Lim Table 6. Antibacterial activity of probiotic Bacillus strain against biogenic amine-producing Bacillus sp. isolated from traditional fermented soybean paste Indicator Bacillus sp. DB403 Bacillus sp. DB407 Bacteriocin activity (AU/ml) Bacillus subtilis DB517 Bacillus licheniformis DB612 Bacillus subtilis DB821 Bacillus licheniformis DB102 ND ND ND ND ND Bacillus subtilis DB203 ND 256 ND ND 64 Bacillus stearothermophilus DB206 ND ND ND ND ND Bacillus sp. DB209 ND ND ND ND 128 Bacillus sp. DB310 ND 128 ND ND ND Bacillus coagulans DB311 ND ND ND ND ND Bacillus cereus DB313 ND 128 ND ND ND Bacillus amyloliquefaciens DB714 ND ND ND ND ND Bacillus sp. DB915 ND ND ND ND ND Bacillus sp. DB917 ND ND ND ND ND Bacillus cereus DB1019 ND 64 ND ND ND Bacillus subtilis DB1020 ND 64 ND ND ND Bacillus sp. DB1022 ND ND ND ND 32 ND, not detected. 세포막수용체에결합하여기능을방해하기도하며, 핵산, 아미노산, 조효소합성에관여하는효소를불활성화시켜결국세포를용해시킨다고알려져있다 (Suva et al., 2018). 유해아민저감화를위해방사선조사, 진공포장, 고압처리및염장과같은삼투압을이용한가공법이보고된바있으나 (Dapkevicius et al., 2000), 이런물리적처리방법들은식품의조직감이나영양학적가치손상및독성을유발하는단점이있으므로바이오제닉아민생성능이없거나분해능이있는미생물을이용하는생물학적제어방법을이용한연구가활발히이루어지고있다 (Mah, 2015). 생물학적보존제인박테리오신이나과산화수소등과같은항균물질을생산하는유산균을발효스타터로이용하거나혼용함으로써발효식품내아민축적을억제시키는데효과적인것으로보고되었다 (Herrero-Fresco et al., 2012). 프로바이오틱균주로는주로 Lactobacillus 속및 Bifidobacterium 속들이이에해당되는것으로많이알려져왔으나, 포자형성균인바실러스균도그활성이보고되고있는데이들은위액에대한강한저항성및가열처리공정과저온하에서도활성이안정할뿐만아니라장내상피세포에대한병원성균의부착억제, 항산화활성및면역조절기능등이확인된바있다 (Elshaghabee et al., 2017). 바실러스균은포자를형성함으로써외부환경에대한저항성이강하므로프로바이오틱균으로서의이용가치가높으며독성유발이나위해발생가능성이낮으므로안전성이확보되어 generally recognized as safe (GRAS) 의요구조건을충족하는것으로확인되었다 (Patel et al., 2009). 프로바이오틱활성을나타내는바실러스속균주로는 B. coagulans, B. subtilis, B. licheniformis, B. cereus, B. cereus var. toyoi, B. natto (subtilis), B. clausii, B. pumilus, B. clausii, B. amyloliquefaciens 및 B. polyfermenticus 등이보고된바있는데 (Manhar et al., 2015; Suva et al., 2018), 본연구결과발효된장에서분리된 Bacillus sp. DB407과 DB821도프로바이오틱활성및항균물질생산에따라바이오제닉아민생성균에대한항균활성도확인되었으므로향후에는분리균주들을발효스타터로이용하여된장을제조한후바이오제닉아민함량측정및다양한생리활성검색에관한연구를계속이어갈것이다. 한편, 바이오제닉아민의함량이높음에도불구하고발효장류제품들에대한허용가능한유해아민기준치가마련되어있지않아소비자들의건강을위협하고있으므로유해아민의만성독성시험을통해 1일섭취허용량결정하고유해아민생성을최소화할수있는발효조건설정에관한연구도시급하다. 적요 본연구에서는우리나라전통발효된장내바이오제닉아민함량을측정하고이들아민의축적을억제할수있는프로바이오틱바실러스균을분리하였다. 된장내세균수, ph, 적정산도, 염도및바이오제닉아민함량은시료마다유의한차이 미생물학회지제 55 권제 2 호

11 Biogenic amine-degrading ability of probiotic Bacillus strain 141 가있었다. 된장에서분리된바실러스균중에서 Bacillus (B.) licheniformis DB102, B. subtilis DB203, B. stearothermophilus DB206, Bacillus sp. DB209, Bacillus sp. DB310, B. coagulans DB311, B. cereus DB313, B. amyloliquefaciens DB714, Bacillus sp. DB917, B. cereus DB 915, B. subtilis DB1020 및 Bacillus sp. DB1022는바이오제닉아민생성능이있는것으로확인되었다. 반면, 바이오제닉아민분해균은 Bacillus sp. DB403, Bacillus sp. DB407, B. subtilis DB517, B. licheniformis DB612 및 B. subtilis DB821로동정되었다. 특히, Bacillus sp. DB407 과 B. subtilis DB821은인공소화액에대한저항성, 장관상피세포에대한부착능, 항생제에대한내성및바이오제닉아민생성균에대한항균활성등의프로바이오틱특성을나타내었다. 결론적으로이들두프로바이오틱바실러스균은바이오제닉아민이낮은대두발효식품제조에적합한스타터로사료된다. References Al Bulushi I, Poole S, Deeth HC, and Dykes GA Biogenic amines in fish: roles in intoxication, spoilage, and nitrosamine formation. A review. Crit. Rev. Food Sci. Nutr. 49, Alander M, Korpela R, Saxelin M, Vilpponen-Salmela T, Matilla- Sandholm T, and Wright A Recovery of Lactobacillus rhamnosus GG from human colonic biopsies. Lett. Appl. Microbiol. 24, Argyri AA, Nisiotou AA, Malauchos A, Panagou EZ, and Tassou CC Performance of two potential probiotic Lactobacillus strains from the olive microbiota as starters in the fermentation of heat shocked green olives. Int. J. Food Microbiol. 171, Brink B, Damink C, Joosten HMLJ, and Huis In t Veld JHJ Occurrence and formation of biologically active amines in foods. Int. J. Food Microbiol. 11, Cho TY, Han GH, Bahn KN, Son YW, Jan 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, Choi JY, Hong SW, and Chung KS Selection of biogenic amine-reducing microorganisms from a traditional Korean-style fermented food, Cheonggukjang. Korean J. Food Sci. Technol. 44, Dapkevicius MLNE, Nout MJR, Rombouts FM, Houben JH, and Wymenga W Biogenic amine formation and degradation by potential fish silage starter microorganisms. Int. J. Food Microbiol. 191, Eerola S, Roig-Sagues AX, and Hirvi TK Biogenic amines in Finnish dry sausages. J. Food Saf. 18, Elshaghabee FMF, Rokana N, Gulhane RD, Sharma C, and Panwar H Bacillus as potential probiotics: Status, concerns, and future perspectives. Front. Microbiol. 8, Fuller R Probiotics in human medicine. Gut 32, Gardini F, Özogul Y, Suzzi G, Tabanelli G, and Özogul F Technological factors affecting biogenic amine content in foods: A review. Front. Microbiol. 7, Guan RF, Liu ZF, Zhang JJ, Wei YX, Wahab S, Liu DH, and Ye XQ Investigation of biogenic amines in sufu(furu): A Chinese traditional fermented soybean food product. Food Cont. 31, Han GH, Cho TY, Yoo MS, Kim CS, Kim JM, Kim HA, Kim MO, Kim SC, Lee SA, Ko YS, et al Biogenic amines formation and content in fermented soybean paste (Cheonggukjang). Korean J. Food Sci. Technol. 39, Hanifi A, Culpepper T, Mai V, Anand A, Ford AL, and Ukhanova M Evaluation of Bacillus subtilis R0179 on gastrointestinal viability and general wellness: A randomized, double-blind, placebo-controlled trial in healthy adults. Benef. Microbes 6, Hernandez-Jover T, Izquierdo-Pulido M, Vechiana-Nogues MT, Marine-Font A, and Vidal-Carou MC Biogenic amines and polyamine contents in meat and meat products. J. Ag. Food Chem. 45, Herrero-Fresno A, Martinez N, Sanhez-Llana E, Diaz M, Ferrandez M, Martin MC, Ladero V, and Alvarez MA Lactobacillus casei strains isolated from cheese reduce biogenic amines accumulation in an experimental model. Int. J. Food Microbiol. 157, Holo H, Nilssen O, and Nes IF Lactococcin A. a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene. J. Bacteriol. 173, Jena PK, Trivedi D, Thakore K, Chaudhary H, Giri SS, and Seshadri S Isolation and characterization of probiotic properties of lactobacilli isolated from rat fecal microbiota. Microbiol. Immunol. 57, Jeon HH, Jung JY, Chun BH, Kim MD, Baek SY, Moon JY, Yeo SH, and Jeon CO Screening and characterization of potential Bacillus starter cultures for fermenting low-salt soybean paste (doenjang). J. Microbiol. Biotechnol. 26, Kalac P and Krausová P A review of dietary polyamines: Formation, implications for growth and health and occurrence in foods. Food Chem. 77, Kim JH, Ahn HJ, Kim DH, Jo C, Yook HS, Park HJ, and Byun MW. 2003a. Irradiation effects on biogenic amines in Korean fermented soybean paste during fermentation. J. Food Sci. 68, Kim YS, Cho SH, Jeong DY, and Uhm TB Isolation of biogenic amines-degrading strains of Bacillus subtilis and Bacillus amyloliquefaciens from traditionally fermented soybean products. Korean J. Microbiol. 48, Kim KM, Kim MJ, Kim DH, Park YS, and Kang JS Characterization of Bacillus polyfermenticus KJS-2 as a probiotic. J. Microbiol. Biotechnol. 19, Korean Journal of Microbiology, Vol. 55, No. 2

12 142 Eun-Seo Lim Kim M, Oh HS, Park SC, and Chun J Towards a taxonomic coherence between average nucleotide identity and 16S rrna gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 64, Kim JH, Park HJ, Kim MJ, Ahn HJ, and Byun MW. 2003b. Survey of biogenic amine contents in commercial soy sauce. Korean J. Food Sci. Technol. 35, Lee HT, Kim JH, and Lee SS Analysis of microbiological contamination and biogenic amines content in traditional and commercial doenjang. J. Fd. Hyg. Safety 24, Lee SK, Lee JJ, Jin YI, Jeong JC, Chang YH, Lee YS, Jeong YH, and Kim MS Probiotic characteristics of Bacillus strains isolated from Korean traditional soy sauce. LWT-Food Sci. Technol. 79, Lee YC, Lin CS, Liu FL, Huang TC, and Tsai YH Degradation of histamine by Bacillus polymyxa isolated from salted fish products. J. Food Drug Anal. 23, Lehane L and Olley J Histamine fish poisoning revisited. Int. J. Food Microbiol. 58, Li L, Wen X, Wen Z, Chen S, Wang L, and Wei X Evaluation of the biogenic amines formation and degradation abilities of Lactobacillus curvatus from Chinese bacon. Front. Microbiol. 9, 1 9. Mah JH Fermented soybean foods: Significance of biogenic amines. Austin J. Nutri. Food Sci. 3, Manhar AK, Saikia D, Bashir Y, Mech RK, Nath D, and Konwar BK In vitro evaluation of celluloytic Bacillus amyloliquefaciens AMS1 isolated from traditional fermented soybean (Churpi) as an animal probiotic. Res. Veterinary Sci. 99, Moon JY, Kwon SW, Hong SB, Seok SJ, Kim JS, and Kim SJ Characteristics and functional analysis of Bacillus strains from the fermented soybean products, Cheonggukjang. Korean J. Microbiol. 51, Olmos J and Paniagua-Michel J Bacillus subtilis a potential probiotic bacterium to formulate functional feeds for aquaculture. J. Microb. Biochem. Technol. 6, Otero MC, Ocana VS, and Macias ENM Bacterial surface characteristics applied to selection of probiotic microorganisms. Methods Mol. Biol. 268, Patel AK, Ahire JJ, Pawar SP, Chaudhari BL, and Chincholkar SB Comparative accounts of probiotic characteristics of Bacillus spp. isolated from food wastes. Food Res. Int. 42, Pedersen K and Tannock GW Colonization of the porcine gastrointestinal tract by lactobacilli. Appl. Environ. Microbiol. 55, Ryu MS, Yang HJ, Kim JW, Jeong SJ, Jeong SY, Eom JS, and Jeong DY Potential probiotics activity of Bacillus spp. from traditional soybean pastes and fermentation characteristics of Cheonggukjang. Korean J. Food Preserv. 24, Savitha K, Srinivas M, and Dhanalakshmi K Isolation and characterization of bacteriocin from Bacillus cereus MTCC Int. J. Appl. Pure Sci. Agric. 2, Seker E Identification of Candida species isolated from bovine mastitic milk and their in vitro hemolytic activity in Western Turkey. Mycopathologia 169, Shalaby AR Significance of biogenic amines to food safety and human health. Food Res. Int. 29, Shim JM, Lee KW, Yao Z, Kim HJ, and Kim JH Properties of doenjang (soybean paste) prepared with different types of salts. J. Microbiol. Biotechnol. 26, Shin DH and Jeong DY Korean traditional fermented soybean products: Jang. J. Ethnic Food 2, 2 7. Shukla S, Park HK, Kim JK, and Kim MH Determination of biogenic amines in Korean traditional fermented soybean paste (Doenjang). Food Chem. Toxicol. 48, Suva MA, Sureja VP, and Kheni DB Novel insight on probiotic Bacillus subtilis: Mechanism of action and clinical applications. J. Curr. Res. Sci. Med. 2, Tabanelli G, Montanari C, Bargossi E, Lanciotti R, Gatto V, Felis G, Torriani S, and Gardini F Control of tyramine and histamine accumulation by lactic acid bacteria using bacteriocin forming lactococci. Int. J. Food Microbiol. 190, Wang T and Su J Bacillus subtilis from soybean food shows antimicrobial activity for multidrug-resistant Acinetobacter baumannii by affecting the ades gene. J. Microbiol. Biotechnol. 26, Wunderlichova L, Bunkova L, Koutny M, Jancova P, and Bunka F Formation, degradation, and detoxification of putrescine by foodborne bacteria: a review. Comp. Rev. Food Sci. Food Safety 13, Zhang Q, Lin S, and Nie X Reduction of biogenic amine accumulation in silver carp sausage by an amine-negative Lactobacillus plantarum. Food Control 32, 미생물학회지제 55 권제 2 호