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1 The Korean Journal of Microbiology (2011) Vol. 47, No. 3, pp Copyright c 2011, The Microbiological Society of Korea 케피어그레인으로제조한요쿠르트로부터 Enterococcus faecalis OA18 균주의분리및특성규명 유진주 김수곤 서경원 오석흥 * 우석대학교대학원생명공학과 Isolation, Identification, and Characterization of Ornithine- Producing Enterococcus faecalis OA18 from Kefir Grain Jin-Ju Yu, Su-Gon Kim, Kyoung-Won Seo, and Suk-Heung Oh * Department of Biotechnology, Woosuk University Graduate School, Jeonju , Republic of Korea (Received July 26, 2011 / Accepted September 14, 2011) Lactic acid bacteria (LAB) OA18 was isolated from yogurt prepared by using Kefir Grain as a starter. The OA18 strain was a Gram-positive, cocci-type bacterium, and able to grow anaerobically with CO 2 production. The OA18 strain grew well on MRS broth supplemented with 50 mm arginine at C and ph of The optimum temperature and ph for growth are 37 C and ph 7.0. The isolate fermented ribose, D-glucose, cellobiose, D-trehalose, but not L-xylose, D-melibiose, and inositol. The 16S rrna gene sequence of the isolate showed 99.8% homology with the Enterococcus faecalis 16S rrna gene (Access no. AB012212). Based on the biochemical characteristics and 16S rrna gene sequence analysis data, it was identified and named as E. faecalis OA18. The E. faecalis OA18 strain showed a high ornithine-producing capacity in the presence of arginine and also showed an antimicrobial activity against Streptomyces strains such as Streptomyces coelicolor subsp. Flavus, S. coeruleorubidus, S. coeruleoaurantiacus, S. coelicolor, S. coeruleoprunus. The cell growth of E. faecalis OA18 strain was maintained in MRS broth with a NaCl concentration of 0-7%. Keywords: anti-microbial activity, E. faecalis, Kefir grain, ornithine 티벳버섯은일명케피어그레인 (Kefir Grain) 이라고도하며, 전자현미경으로살펴보면원형의다당류집락에골이파여있고, 이곳에주요종균들이분포되어있는형태를하고있다. Kefir Grain에함유되어있는미생물로는 Lactobacillus caucasium, Lactococcus lactis ssp. lactis, Streptococcus salivarius subsp. thermophilus 등의젖산균과 Torula keffir와 Saccharomyces kefi 등의효모가주를이루고있는것으로알려져있다 (13, 14, 15, 21). 코카시안 (Caucasian) 산악지대에서유래된것으로알려진케피어 (Kefir) 는 Kefir Grain으로발효를시켜서만드는데산과알코올발효가함께일어나서발효가끝나면 % 의젖산과 % 정도의에탄올, 그리고다량의탄산가스가함유되어있어서독특한향취를갖는다 (9). Kefir 발효유는필수아미노산을함유하고있으며일반유산균발효유제품과달리효모와유산균에의하여생산된 biotin, niacin, pyridoxine, * For correspondence. shoh@woosuk.ac.kr; Tel.: ; Fax: folic acid와같은비타민 B군을풍부하게함유하고있다 (13, 14, 18). L-오르니틴은비단백태아미노산으로담수성쌍각류조개류 (29), 와인과치즈 (1, 22), 김치 (16) 등에서발견되고있다. 예를들면, 재첩엑기스 100 g 중에유리오르니틴함량은 mg이며 (29), 김치 100 g 중에는 mg이함유되어있는것으로조사되고있다 (16). L-오르니틴은성장호르몬분비, 근육의합성증가, 비만예방 (8, 12), 간기능개선 (23), 주름살개선 (28) 등의기능성을갖는것으로밝혀지고있다. 이에오르니틴함유식품에대한관심이증대되고있으며 (16, 31, 32), 오르니틴생산능력을갖는젖산균을이용하여영양기능성이증진된발효식품을제조하려는시도가진행되고있다 (16). 따라서, 본연구는식용식품자원인티벳버섯으로부터오르니틴생성능력이있는젖산균 Enterococcus faecalis OA18를분리하여특성을조사함으로써티벳버섯유래젖산균의정보를파악하고, 그활용가능성을제안하고자한다.

2 케피어그레인 E. faecalis OA18 균주 219 재료및방법 Enterococcus 속균주의분리 균주분리에사용한티벳버섯은경북경산시진량읍소재가정에서배양된것을인터넷을통해구매하여사용하였다. 구매한티벳버섯을요구르트제조를위한스타터로사용하였고, 제조한요쿠르트는오르니틴생산능이있는젖산균을분리하는데시료로사용하였다. 요쿠르트의제조는티벳버섯스타터 1스푼 ( 약 5 g) 을우유 100 ml에넣고통성혐기적인조건의 37 C 배양기에서 24-36시간동안정치발효하여제조하였다. 요쿠르트시료는펩톤수로 범위로희석하였고, 젖산균을선택적으로분리하기위해희석액을 0.002% bromocresol purple (BCP) (Sigma Chemical Co., USA) 가첨가된 DE MAN, ROGOSA and SHARPE (MRS) 한천배지 (Difco, USA) 에 1 ml씩분주하여 37 C에서 48시간동안배양하였다 (25, 32). 콜로니중에서단일콜로니를형성하면서노랑색을띄는것을후보균주로분리하였고, 이들후보균주의오르니틴생성능을확인하였다. 오르니틴생성능은 50 mm 아르기닌이첨가된 MRS 배지 (ph 7.0) 에균주를접종 (1%, v/v) 하고 37 C에서 48시간배양한후 TLC를이용하여조사하였다. TLC는 silica gel F 254 (Merck, Germany) 과표준오르니틴 (Merck) 을이용하여실시하였다 (32). E. faecalis OA18 균주의동정 오르니틴생성능을갖는후보균주로부터 genomic DNA를분리정제하여 PCR 증폭을시행하였다. DNA의분리는 DNeasy Blood & Tissue kit (QIAGEN, Germany) 를이용하였고, 정제는제조사의지침을따랐다. 16S rrna gene의클로닝을위한 Primer는 5'-AGAGTTTGATCMTGGCTCAG-3' (Forward) 와 5'-ACGGGCGGGTGTGTRC-3' (Reverse) 를이용하였다. 1% 아가로스겔전기영동으로확인된 PCR 산물을 PCR purification kit (Bioneer, Korea) 를이용하여정제한후, T4 ligase를이용하여 pgem T-easy vector (Promega, USA) 에 ligation하였으며, 16S rrna gene sequencing을통해 OA18의염기서열정보를얻었다. 16S rrna gene의염기서열정보는 NCBI ( 에등록하고, DNA 의상동성조사에활용하였으며, CLUSTAL X 프로그램을통해공시균주의염기서열과의상동성을비교 분석하였다. E. faecalis OA18 균주의특성 분리된균주의당발효패턴과생화학적인특성은 API 50 CHL kit (biomérieux, France) 를이용하여조사하였고제조사의지침에따라사용하였다. 카탈라아제활성은 3%(v/v) 과산화수소를이용한기포생성여부로판단하였고, 이산화탄소생성여부는 Durham 관을이용하여판단하였다 (19). 균주의성장에대한 ph의영향을측정하기위해 100 mm sodium acetate/100 mm acetate 완충액 (ph 4.0, 5.0, 6.0), 100 mm Na 2HPO 4/100 mm HCl 완충액 (ph 7.0, 8.0, 9.0) 및 100 mm Na 2HPO 4/100 mm NaOH 완충액 (ph 10) 으로 MRS 배지를 조제하였고, 균주를 37 C에서 12시간배양한후 600 nm에서의흡광도를측정하였다. 온도의영향을조사하기위하여 MRS 배지 (ph 7.0) 를이용하여 4, 10, 25, 30, 37, 40 C 범위에서 12시간동안균주를배양한후 600 nm에서흡광도를측정하였다. 내염성을측정하기위하여 0, 1, 3, 5, 6, 7, 8%(w/v) 의 NaCl이첨가된 MRS 배지 (ph 7.0) 에서균주를배양한후 (37 C, 24시간 ) 600 nm에서의흡광도를측정하였다 (31, 32). 선발된균주의오르니틴생성능을측정하기위하여 50 mm 아르기닌이첨가된 MRS 배지 (ph 7.0) 에균주를접종하고 37 C에서 48시간배양하였고, 5,000 g에서 20분간원심분리한후세포와배양액을분리하여각각오르니틴을분석하였다. E. faecalis OA18 균주의오르니틴생성분석균주가생성한오르니틴함량은 TLC와 HPLC 분석을통하여측정하였다. TLC는 Enterococcus 속균주의분리 에서와같이실시하였다. HPLC는 Baum 등 (2) 이사용한아미노산분석방법을기본으로하고 Park과 Oh(25) 의분석방법을약간수정하여사용한 Yu 등의방법 (32) 에따라서실시하였다. HPLC (Waters, Milford, USA) 분석을위해시료는 6-aminoquioly-N-hydroxysuccinimidyl carbonate (AQC) 로유도체화하였고, mm AccQ Tag TM (Nova-Pak TM C 18, Waters) 컬럼으로유도체들을분리하였다. 오르니틴함량은표준오르니틴의분석결과를토대로작성한표준곡선을이용하여산출하였다 (31, 32). E. faecalis OA18 균주의항균능측정항균능측정대상균주에대한 E. faecalis OA18 균주의항균능력은 paper disc법 (4) 으로측정하였다. 항균능측정에사용한 OA18 균주시료는 MRS 배지에배양 (37 C, 48 h) 한 OA18 균주배양액 (cultured medium), 배양액을원심분리하여얻은세포 (cell), 세포를제거한배양상등액 (spent culture) 으로하였다. 항균능측정대상균주 Vibrio parahaemolyticus (KCCM 11965), Bacillus cereus (KCCM 11204), Clostridium perfringens (KCCM 12098), Staphylococcus aureus subsp. aureus (KCCM 11335), Listeria monocytogenes (KCCM 40307), Streptomyces coelicolor subusp. Flavus (KCCM 41303), Streptomyces coeruleorubidus (KCCM 40535), Streptomyces coeruleoaurantiacus (KCCM 12609), Streptomyces coelicolor (KCCM 11394), Streptomyces coeruleoprunus (KCCM 41264) 은한국미생물보존센터 (KCCM) 에서분양받아사용하였다 (Table 2). 대상균주는 5 ml의액체배지에서 24시간 (30 C 또는 37 C) 배양하였으며, 배양된대상균주를 0.1 ml씩 1.5% agar가첨가된고체배지에도말하여항균성시험용평판배지를조제하였다. 이어멸균된 paper disc (Advantec, 10 mm, Toyo Roshi Kaisha, Japan) 를대상균주가도말된평판배지표면에놓아밀착시킨후 OA18 균주시료를 50 μl를천천히흡수시켜 30 C 또는 37 C에 24-48시간배양후 disc 주변에생성된생육저해환 (clear zone, mm)

3 220 Yu et al. Table 1. Biochemical characteristics of E. faecalis OA18 Characteristics E. faecalis E. faecalis E. faecalis OA18 K-4 a strain b Gram stain + c + + Catalase - d - - Cell shape cocci cocci cocci CO 2 producton + + ND e Spore formation D-adonitol D-Arabitol D-Fructose D-Galactose D-Glucose D-Manitol D-Manose D-Melibiose - ND - D-Melezitose D-Sorbitol D-Tagatose D-Trehalose + + ND L-Arabinose L-Sorbose L-Xylose N-Acetylglucosamine Amygdalin Arbutin Cellobiose Esculin Gentiobiose + ND + Inositol - + ND Glycerol + + ND Lactose + + ND Maltose Ribose Salicin Sucrose + ND + Growth in the presence of NaCl 1% NaCl + ND + 3% NaCl + ND + 6% NaCl + ND + 7% NaCl + ND - 8% NaCl - ND - a Eguchi et al. (7); b Du Toit et al. (6); c Positive reaction; d Negative reaction; e Not Determined 의크기를측정하여항균활성을비교 분석하였다 (17). 결과 Enterococcus 속후보균주의분리 케피어그레인을이용하여제조한요쿠르트로부터젖산균을분리하기위해 bromocresol purple이첨가된 MRS 한천배지 A O Fig. 1. TLC analysis of lactic acid bacteria cultures for ornithine production. The strains were cultured in an MRS liquid medium with 1% arginine. The strains with circles are selected as candidate lactic acid bacteria for the ornithine production. Lanes: A, spot of standard arginine; O, spot of standard ornithine; 1-11, spots of OA13-OA23 strains, respectively. The spots with circles are in the same location as the standard ornithine. 에서노랑색을띄는 25개의단일콜로니를젖산균후보균주로선발하여 OA1-OA25 균주로각각표기하였다. 후보균주들중에서오르니틴생성가능성을갖는균주를선발하기위해 TLC를이용하여조사하였으며, TLC 상에서표준오르니틴 spot과비교했을때뚜렷하게오르니틴 spot을나타낸 OA18 균주를선발하여 (Fig. 1) Enterococcus 속후보균주로삼았고, 이균주의동정과생화학적특성을조사하였다. E. faecalis OA18 균주의동정 16S rrna gene 염기서열분석을통해 OA18 균주와유전자은행에등재되어있는균주와유사성을 NCBI ( nlm.nih.gov) 에서 DNA의상동성으로조사하였다. CLUSTAL X 프로그램을통해공시균주 Enterococcus faecalis (AB012212) 의염기서열과 OA18 균주의염기서열이 99.8% 유사함을확인하였다. 형태학적및생화학적특성과염기서열분석결과를종합하고, Sharpe 등 (27) 의젖산균동정방법을참고로균주를 E. faecalis 균주로동정하고, E. faecalis OA18로명명하였으며, 분리된균주의 16S rrna gene 서열을 NCBI에등록을하였다 ( 등록번호 : JN628990). E. faecalis OA18 균주의특성 OA18 균주의형태학적및생화학적특성을조사한결과그람양성의구균이었으며, 혐기적조건에서이산화탄소를생성하였다. 분리된 OA18 젖산균주는 D-글루코오스, cellobiose, D-trehalose 등을분해하여산을생성하였고, L-xylose, D-melibiose, inositol은분해하지못하였다 (Table 1). 분리된균주는 MRS 배지에서 C 범위와 ph 범위에서자랐으며성장을위한최적온도와 ph는각각 37 C와 ph 7.0이였다 (Fig. 2). 또한비교적높은염농도 (7.0%) 에서도증식이가능한것으로조사되었다 (Table 1). 이러한특성은 Eguchi 등 (7) 과 Du Toit 등 (6) 이분리한 E. faecalis 균주들의특성과비교시매우유사하였으나, inositol의분해능, 내염성등에있어서는차이가있는것으로조사되었다 (Table 1).

케피어그레인 E. faecalis OA18 균주 221 (A) OD 600 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 4 5 6 7 8 9 10 11 ph (A) Content (mm) 60 50 40 30 20 10 0 ornithine arginine OD 0 6 12 24 36 48 Time (h) 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 OD 600 (B) OD 600 1.

4 케피어그레인 E. faecalis OA18 균주 221 (A) OD ph (A) Content (mm) ornithine arginine OD Time (h) OD 600 (B) OD Temp. ( ) Fig. 2. Effects of ph (A) and temperature (B) on the growth of E. faecalis OA18. Growth was estimated by monitoring at the OD 600 after 12 h cultivation. E. faecalis OA18 균주의오르니틴생성능분석 E. faecalis OA18의오르니틴생성능을조사하기위하여 50 mm 아르기닌이첨가된 MRS 배지에서 (ph 7.0, 37 C) 균주를 48시간동안배양하였다. E. faecalis OA18 균주의오르니틴생성정도는 TLC와 HPLC를이용하여분석하였다. E. faecalis OA18 균주는 50 mm 아르기닌이첨가된 MRS 배지에서오르니틴을생성하였고, 생성된오르니틴의함량은 mm로첨가한아르기닌을모두오르니틴으로전환하는 (B) A O Fig. 3. Production of ornithine in the cultured medium during the growth of the selected E. faecalis OA18(A). The E. faecalis OA18 was cultured in MRS medium supplemented with 50 mm arginine as described in Materials and Methods. (B) TLC analysis of ornithine production: Lanes: A, spot of standard arginine; O, spot of standard ornithine; 1-6, spots of E. faecalis OA18 culture at 0 h, 6 h, 12 h, 24 h, 36 h, 48 h cultivation, respectively. 것으로조사되었다 (Fig. 3). E. faecalis OA18 균주의항균능측정 E. faecalis OA18의항균능을조사하기위해항균능측정대상균주의평판배지 disc에 OA18 균주로부터얻은세가지 Table 2. List of test strains and culture conditions used for antimicrobial experiments Strains KCCM No. Media a Temp. ( C) Vibrio parahaemolyticus NB+3% NaCl 37 Clostridium perfringens RCM 37 Staphylococcus aureus subsp. aureus TSB 37 Listeria monocytogenes BHIB 37 Bacillus cereus NB 30 Streptomyces coelicolor subusp. Flavus YMEB 30 Streptomyces coeruleorubidus YMEB 30 Streptomyces coeruleoaurantiacus YMEB 30 Streptomyces coelicolor YMEB 30 Streptomyces coeruleoprunus YMEB 30 a NB, Nutrient broth (Difco); RCM, Reinforced clostridial medium (Difco); TSB, Tryptic soy broth (Difco); BHIB, Brain heart infusion broth (Difco); YMEB, Broth with yeast extract 0.4%, malt extract 1%, and dextrose 0.4%

5 222 Yu et al. Table 3. Antimicrobial activity of E. faecalis OA18 Streptomyces strain Cell a Spent culture Cultured medium KCCM b 18.60±0.14 c 13.20± ±0.14 KCCM ± ± ±0.12 KCCM ± ± ±0.10 KCCM ± ± ±0.19 KCCM ± ± ±0.11 a Cell, cells obtained from the cultured medium of E. faecalis OA18. Spent culture, the spent culture supernatant of E. faecalis OA18. Cultured medium, the cultured medium with cells of E. faecalis OA18. b KCCM 41303, Streptomyces coelicolor subusp. Flavus; KCCM 40535, S. coeruleorubidus; KCCM 12609, S. coeruleoaurantiacus; KCCM 11394, S. coelicolor; KCCM 41264, S. coeruleoprunus. c Activity was expressed as the diameter (mm) of inhibition zone against each strain. Data expressed as Mean±SD from three independent experiments. 시료 cell, spent culture, cultured medium을흡수시켜투명환의크기로대상균주에대한항균능을측정하였다. Enterococcus 균주는 enterocin A, B, P, Q 등다양한박테리오신을생산하여 Clostridium perfrigens, Vibrio cholera 및 Listeria monocytogenes 등의식중독균에대한항균효과를나타내는것으로알려져있다 (20). 본연구에서분리한오르니틴을생성하는 E. faecalis OA18 균주에서는이들식중독균에대한항균능이나타나지않았다. 또한 Staphylococcus aureus subsp. aureus와 Bacillus cereus에대하여도항균능을보이지않았다 ( 자료미제시 ). 이전의연구에서는 Du Toit 등 (6) 이태국의 grass silage에서분리한 E. faecalis 균주가식중독균에대한항균능이나타나지않았으나, Eguchi 등 (7) 이 pig faeces 에서분리한 E. faecalis SE-K4는박테리오신을생산해 Listeria monocytogenes 균주에대해항균능이있는것으로보고한바있어균주에따른항균특성이상이함을보여주고있다. 식중독균들이외의 Streptomyces coelicolor subusp. Flavus (KCCM 41303), Streptomyces coeruleorubidus (KCCM 40535), Streptomyces coeruleoaurantiacus (KCCM 12609), Streptomyces coelicolor (KCCM 11394), Streptomyces coeruleoprunus (KCCM 41264) 균주들에서는 Enterococcus faecalis OA18 균주가강한항균활성을보였다 (Table 3). 특히 Streptomyces coelicolor (KCCM 11394) 에대한투명환의크기는 cell, spent culture, cultured medium에서각각 18.31± 0.18 mm, 20.20±0.18 mm, 21.70±0.19 mm로나타났고, Streptomyces coeruleoprunus (KCCM 41264) 균주에대한투명환의크기는 cell, spent culture, cultured medium에서각각 20.08±0.13 mm, 16.80±0.13 mm, 22.00±0.11 mm로항균력이높게나타났다 (Table 3). 고찰 Enterococcus 균주는그람- 양성젖산균으로발효식품, 어류, 채소류, 태아의분변등다양한원료에서분리되고있다 (20, 24). Enterococcus 균주는단백질이나지질가수분해능력이뛰어나서지중해안의치즈나 black olives의독특한맛과향에영향을주는것으로알려져있으며, 사람이나동물의장내세균총의균형유지와정장작용에도효과가있어일부국가에서는 probiotic로이용되고있다 (20, 24). 또한 E. faecalis strain의투여가 diarrhea를경감시키는것으로알려지면서 chicken/pig/cattle의설사를줄이기위한 probiotics로사용되기도하였다 (10). 본연구에서는케피어그레인으로제조한요쿠르트로부터 E. faecalis OA18를분리하여동정하고 ornithine 생산능, 항균능등의특성을조사하였다. E. faecalis OA18 균주는 arginine이 50 mm 첨가된배지에서 arginine을 ornithine 으로전환하는능력이우수하였다 (Fig. 3). 젖산균중에는 arginine deiminase (ADI) pathway를가지고있어서 arginine 를분해하여 ornithine을생성하는능력을보유하고있는것들이있다. 예를들면, 포도주의 Lactobacillus hilgardii X 1B, 맥주의 L. brevis TMW1.465, 천일염의 Weissella koreensis MS1-3 균주가 ADI pathway를이용하는것으로알려져있다 (1, 3, 31). 지금까지 ornithine 생산능을갖는젖산균의효능이나이들젖산균에의해생성된 ornithine의기능에대한보고는매우미미하다. 그중에서 L. hilgardii X 1B 균주가생산한 ornithine은 yeast에의한알코올발효를저해하거나 Hansenula minuta와같은부패효모의생육을저해하여포도주에생물학적안정성을부여해줄수있는것으로제안되고있다 (1). 또한 arginine의분해에의한 ornithine의생성은 ATP의형성을수반하기때문에 arginine은세포의성장을위한에너지원으로간주되기도한다 (1). 따라서본연구를통해분리된 E. faecalis OA18 균주가티벳버섯사용요쿠르트에서 ornithine을생성하고요쿠르트의발효와기능성뿐만아니라안전성에영향을주는젖산균일가능성이있을것으로판단되어이에대한심도있는연구가필요하다. E. faecalis OA18 균주는 Streptomyces 균주에대한항균능도우수한것으로조사되었다 (Table 3). Streptomyces 균주는 bleomycin, clorobiocin 등다양한항생물질을생성하는균주로알려져있는바 (5, 30) E. faecalis OA18가이들 Streptomyces 균주에대한항균능을보인다는것은흥미로운것이다. Enterococcus 균주는 enterocin A, B, P, Q 등다양한박테리오신을생산하여 Clostridium botulinum, Clostridium perfrigens, Salmonella choleraesuis, Vibrio cholera 및 Listeria monocytogenes 등의식중독균에대한항균효과를나타내는것으로알려져있다 (20). 또한 Nam 등 (24) 은 infant feces로부터 Helicobacter pylori에대한항균성을보이는 E. faecalis PL9003 균주를분리하여보고한바있다. E. faecalis PL9003 균주는 H. pylori의성장을저지할뿐아니라 human gastric cell line MKN-45에결합하여 H. pylori가결합하는것을방해하는것으로조사되었다 (24). E. faecalis OA18 균주가어떤작용메커니즘에의해서 Streptomyces 균주에대한항균능을보이는지에대한연구와박테리오신의종류등에대한심도있는연구가향후에이루어진다면 E. faecalis OA18 균주의활용폭을넓힐수있을것이다. 박테리오신생

6 케피어그레인 E. faecalis OA18 균주 223 산능력이있는 Enterococcus 균주는비교적높은염농도에서도증식이가능한것으로알려져있어외국에서는발효소시지의리스테리아균의제어에활용되고있다 (20, 26). 우리나라에서도김치유산균이생산하는박테리오신을이용하여훈제연어등의리스테리아균을제어하려는시도가진행되고있다 (11). 따라서, 본연구를통해분리한 E. faecalis OA18 균주가비교적높은염농도에서도증식이가능한것으로확인되어 (Table 1) 염도가높은식품에도활용될수있을것으로판단되므로이에대한심도있는연구도향후과제이다. 적요 케피어그레인을이용하여제조한요쿠르트로부터젖산균 OA18을분리하여그특성을조사하였다. 분리된균주는그람양성, 구균이었으며, 혐기적조건에서이산화탄소를생성하였다. 균주는 MRS 배지에서 C 온도범위와 ph 범위에서잘자랐으며, 성장을위한최적온도와 ph는각각 37 C와 ph 7.0이었다. 분리된젖산균은리보오스, D-글루코오스, cellobiose, D-trehalose 등을분해하여산을생성하였고, L-xylose, D-melibiose, inositol은분해하지못하였다. 16S rrna gene 염기서열분석을통해 OA18 균주는유전자은행 (NCBI) 에등재되어있는 Enterococcus faecalis (AB012212) 의염기서열과 99.8% 동질성이있음을확인하였다. 이와같은생화학적특성과염기서열분석결과를토대로분리된균주를 Enterococcus faecalis OA18로명명하였다. E. faecalis OA18 균주는오르니틴생성능력과 Streptomyces coelicolor subsp. Flavus, S. coeruleorubidus, S. coeruleoaurantiacus, S. coelicolor, S. coeruleoprunus 대한항균활성을보유하고있었으며, 0-7% NaCl을함유하는 MRS 배지에서증식이가능한것으로조사되었다. 감사의말 본연구는 2011학년도우석대학교교내학술연구비지원에의하여연구되었으며지원에감사드립니다. 참고문헌 1. Arena, M.E., F.M. Saguir, M.C. Manca, and M. Nadra Arginine, citrulline and ornithine metabolism by lactic acid bacteria from wine. Int'l. J. Food Microbiol. 52, Baum, G., L.Y. Simcha, Y. Fridmann, T. Arazi, H. Katsnelson, and M. Zik Calmodulin binding to glutamate decarboxylase is required for regulation and GABA metabolism and normal development in plants. EMBO J. 15, Behr, J., M.G. Ganzle, and R.F. Vogel Characterization of a highly hop-resistant Lactobacillus brevis strain lacking hop transport. Appl. Environ. Microbiol. 72, Davidson, P.M. and M.E. Parish Method for testing the efficacy of food antimicrobials. Food Technol. 43, Demain, A.L Contribution of genetics to the production and discovery of microbial pharmaceuticals. Pure Appl. Chem. 60, Du Toit, M., C.M. Franz, L.M. Dicks, and W.H. Holzapfel Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces. J. Appl. Microbiol. 88, Eguchi, T., K. Kaminaka, J. Shima, S. Kawamoto, K. Mori, S.H. Choi, K. Doi, S. Ohmomo, and S. Ogata Isolation and characterization of enterocin SE-K4 produced by thermophilic enterococci, Enterococcus faecalis K-4. Biosci. Biotechnol. Biochem. 65, Evain-Brion, D., M. Donnadieu, M. Roger, and J. Job Simultaneous study of somatotrophic and corticotrophic pituitary secretions during ornithine infusion test. Clin. Endocrinol. 17, Garrote, G.L., A.G. Abraham, and G.L. De Antoni Characteristics of kefir prepared with different grain: milk ratios. J. Dairy Res. 65, Gilmore, M.S., D.B. Clewell, P. Courvalin, G.M. Dunny, B.E. Murray, and L.B. Rice Nonhuman reservoirs of Enterococci, pp In M.S. Gilmore (ed.), The Enterococci: pathogenesis, molecular biology, and antibiotic resistance, American Society for Microbiology Press, Washington, DC, USA. 11. Jang, S.H Control of Listeria monocytogenes on smoked salmon by antimicrobial effect of lactic acid bacteria. Food Industry Nutr. 16, Jeevanandam, M., N.I. Holaday, and S.R. Petersen Ornithine α-ketoglutarate (OKG) supplementation is more effective than its component salts in traumatized rats. J. Nutrition 126, Jeong, K.H., J.H. Choi, J.M. Lee, J.H. Lee, S.Y. Jang, and Y.J. Jeong Fermentation characteristic of kefir beverage added fruit juice. Food Ind. Nutrition 7, Kandler, O. and P. Kunath Lactobacillus kefir sp., component of microflora of kefir. Syst. Appl. Microbiol. 4, Kemp, N Kefir, the champagne of cultured dairy products. Cultured Dairy Products J. 19, Kim, M.J Preparation and characterization of kimchi using lactic acid bacteria having GABA and ornithine producing capacity and its some functional properties. MS thesis, Chonbuk Nat l. Univ., Jeonju. 17. Kim, J.Y., J.A. Lee, K.L. Kim, W.J. Yoon, W.J. Lee, and S.Y. Park Antioxidative and antimicrobial activities of Sargassum muticum extracts. J. Korean Soc. Food Sci. Nutr. 36, Kim, D.S., S.K. Park, H.S. Kwak, and K.W. Lee Isolation, identification and characterization of lactose non-fermenting yeast from kefir cultures. Korean J. Food Sci. Resour. 14, Lee, J.S., K.C. Lee, J.S. Ahn, T.I. Mheen, Y.R. Byun, and Y.H. Park Weissella. koreensis sp. nov., isolated from kimchi. Int. J. Syst. Evol. Microbiol. 52, Lim, S.M Synergistic effect of physico-chemical treatment and bacteriocin produced by Enterococcus faecium MJ-14. J. Fd. Hyg. Safety 20, Lim, Y.S., S.Y. Kim, and S.K. Lee Characteristics of lactic acid bacteria isolated from kefir made of goat milk. Korean J. Food Sci. Ani. Resour. 28, Liu, S.Q., R. Holland, and V.L. Crow The potential of dairy lactic acid bacteria to metabolise amino acids via non-transaminating reactions and endogenous transamination. Int. J. Food Microbiol. 86,

7 224 Yu et al. 23. Muting, D. and J.F. Kalk Long-term effectiveness of highdosed ornithine-aspartate on urea synthesis rate and portal hypertension in human liver cirrhosis. Amino Acids 3, Nam, H.R., M.S. Ha, E.J. Lee, and Y.H. Lee Effect of Enterococcus faecalis strain PL9003 on adherence and growth of Helicobacter pylori. J. Microbiol. Biotechnol. 12, Park, K.B. and S.H. Oh Isolation and characterization of Lactobacillus buchneri strains with high γ-aminobutyric acid producing capacity from naturally aged cheese. Food Sci. Biotechnol. 15, Sabia, C., S. Niederhausern, P. Messi, G. Manicardi, and M. Bondi Bacteriocin-producing Enterococcus casseliflavus IM 416K1, a natural antagonist for control of Listeria monocytogenes in Italian sausages ( cacciatore ). Int. J. Food Microbiol. 87, Sharpe, M.E., T.F. Fryer, and D.G. Smith Identification of lactic acid bacteria, pp In B.M. Gibbs and F.A. Skinner (eds.). Identification Methods for Microbiologists: part A. Academic Press, Inc., New York, USA. 28. Shi, H.P., R.S. Fishel, D.T. Efron, J.Z. Williams, M.H. Fishel, and A. Barbul Effect of supplemental ornithine on wound healing. J. Surgical Res. 106, Uchisawa, H., A. Sato, J. Ichita, H. Matsue, and T. Ono Influence of low-temperature processing of the brackish water bivalve, Corbicula japonica, on the ornithine content of its extract. Biosci. Biotechnol. Biochem. 68, Wolpert, M., B. Gust, B. Kammerer, and L. Heide Effects of deletions of mbth-like genes on clorobiocin biosynthesis in Streptomyces coelicolor. Microbiology 153, Yu, J.J. and S.H. Oh Isolation and characterization of lactic acid bacteria strains with ornithine producing capacity from natural sea salt. J. Microbiol. 48, Yu, J.J., H.J. Park, S.G. Kim, and S.H. Oh Isolation, identification, and characterization of Weissella strains with high ornithine producing capacity from kimchi. Korean J. Microbiol. 45,

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06_HM hwp The Korean Journal of Microbiology (2011) Vol. 47, No. 4, pp. 316-322 Copyright c 2011, The Microbiological Society of Korea 김치유래 Lactobacillus sakei OPK2-59 의 γ-aminobutyric Acid 생성및 Glutamate Decarboxylase