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1 Journal of Bacteriology and Virology Vol. 44, No. 3 p Original Article The Growth Inhibition Effect on the Causative Bacteria of Bacterial Vaginosis by Bacterial Strains Isolated from the Vagina of a Healthy Woman Wan-Jin Sihn 1 and Nam-Woong Yang 2 * 1 Department of Alternative Complementary Medicine, Graduate School Chosun University, Gwangju; 2 Department of Microbiology, College of Medicine, Chosun University, Gwangju, Korea Two Gram-positive rod strains isolated from the healthy vagina of a woman were tested for the possibility as probiotics. One strain was identified as Steroidobacter denitrificans (YH1) and the other as Lactobacillus crispatus (YH2) by 16S rrna partial sequencing. The Casman agar and Man-Rogosa-Sharpe (MRS) agar were mixed in same quantity, supplemented with 5% human rbc lysate (CMB agar). The Wilkins-Chalgren agar and MRS agar were mixed in same quantity (WCM agar). Gardnerella vaginalis was cultured in Casman broth, supplemented with 5% human rbc lysate and 1,000 x-diluted with normal saline. Bacteroides fragilis, Mobiluncus mulieris and Peptostreptococcus asaccharolyticus were cultured in Wilkins-Chalgren anaerobe broth and 2,000x-diluted. S. denitrificans YH1 and L. crispatus YH2 were cultured in MRS broth anaerobically and 100x-diluted. The diluted suspensions of B. fragilis, M. mulieris and P. asaccharolyticus were inoculated on WCM agar and G. vaginalis on CMB agar by cotton swabs. Ten μl aliquots of YH1 and YH2 were inoculated on the center of WCM agar and CMB agar. The growth inhibition zone diameters of B. fragilis, G. vaginalis, M. mulieris and P. asaccharolyticus by YH1 were 35 mm, 35 mm, 25 mm and 60 mm. The inhibition diameters by YH2 were 25 mm, 30 mm, 20 mm and 40 mm, respectively. These results implicate that S. denitrificans YH1 can be the stronger probiotics for the treatment of bacterial vaginosis than L. crispatus, compared inhibition zone diameters by YH1 and YH2. Key Words: Bacterial vaginosis, Lactobacillus crispatus, Probiotics, Steroidobacter denitrificans INTRODUCTION Lactobacillus는현재 140여종에이르며, 미국식품의약국에의해서중요한 GRAS (generally recognized as safe) food lactic acid bacteria로인정되고있으며아직까지주로산업적인용도로사용되고있다 (1). 세균성질증은질내의정상균무리인 Lactic acid bacteria들이 Gardnerella vaginalis와무산소세균들 (Mobiluncus spp., Peptostreptococcus spp., Prevotella spp., Bacteroides spp. 등 ) 로교체되면서무산소세균특유의악취를내는질분비물의증가를초래하는흔한질염이다 (2). 임상에서세균성질증을치료하기위해 metronidazole, clindamycin 등무산소세균에효과적인항생제나요오드계통살균제혹은 1% 유산용액등을사용하여왔으나완치가어렵고재발이흔하여뚜렷한효과를내는약제의개발이아쉬운실정이다. 이 Received: May 15, 2014/ Revised: July 22, 2014/ Accepted: July 24, 2014 * Corresponding author: Nam-Woong Yang. Department of Microbiology, College of Medicine, Chosun University, 309 Pilmun-daero Dong-gu Gwangju , Korea. Phone: , Fax: , nwyang@chosun.ac.kr ** This study was supported by research fund from Chosun University, CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( 244

2 The Growth Inhibition on Bacterial Vaginosis Bacteria 245 에새롭게주목을받고있는것은 probiotics인유산생균을사용하는방법이다 (3). 저자들은과거에질염을앓은적이없는건강한질을유지하고있는여성들에서분리된균주들이 probiotics로사용될수있는가능성을염두에두고, 출산경험이있고병력상질염을앓은적이없는 43세의건강한여성의질에서 Man-Rogosa-Sharpe (MRS) agar를사용하여 2가지균주를분리하였다. 이균주들의일반적인배양특성, 그람염색성, 생화학적성상등을파악하고 16s rrna partial sequencing을이용하여동정한다음, 이균주들이세균성질증에서흔히분리되는균종들중에서대표적인 4가지표준균주들에대하여성장억제효과를나타내는지알기위하여항생제원판확산법과혼합액체배양법을원용하여실험하였다. MATERIALS AND METHODS 건강한여성에서실험균주의분리세균성질증의진단기준인, Amsel's composite clinical criteria (4) 와 Yang 등 (5) 의결과를적용하여세균성질증이없는것으로확인된 43세의건강한여성의질에서채취한샘플을 MRS agar (MERCK, Germany) 에접종하고 5% 이산화탄소와함께일회용무산소배양기 (Quick anaerosystem, Sindo Co., Gwangju, Korea) 에서 48시간무산소배양을하였다 (6). 분리된균주들의분자생물학적동정분리된균주들을무산소상태로한국미생물보존센터 (KCCM) 에보내어 16S rrna partial sequencing을의뢰한결과, 각각 Steroidobacter denitrificans와 Lactobacillus crispatus로동정되었다 (Gene Bank Data homology search results: 99%). 상기균주들을분리한여성의이름을사용하여, 잠정적으로전자는 S. denitrificans YH1, 후자는 L. crispatus YH2로명명하였다. 분리균주의 16S rrna partial sequencing은다음과같은방법으로실행하였다. 상기균주들의 chromosomal DNA를 Wizard genomic DNA purification kit (Promega, USA) 를이용해분리한다음, 16S rrna sequencing에사용하는 universal primer인 27F (5'-AGAGT- TTGATCATGGCTCAG-3') primer를사용하여 MyCycler Thermal Cycler system (Bio-Rad) 을이용해 PCR 증폭하였다. 증폭된 PCR 산물은 Wizard SV Gel and PCR clean-up system (Promega) 을이용하여정제하였고, 정제된산물은 ABI PRISM BigDye TM Terminator Cycle Sequencing kits (Applied Biosystems Co.) 를사용하여 ABI PRISM 3730XL Analyzer (96 capillary type) 를통해염기서열을분석하였다. 그결과를 BLASTN program을이용하여 GENEBANK 의 ribosomal DNA sequence와비교하였으며, sequence의상동성은 Clustal X와 Mega 6 program을이용하여비교분석하였다. 분리된균주들의생화학적성상분리된두균주를대상으로 API 50 CHL kit (BIOMÉRIUX, France) 시험을실시하였다 (Table 1, 2). S. denitrificans YH1에대하여효소활성상태를알기위하여 API ZYM kit (BIOMÉRIUX, FRANCE) 시험을추가로실시하였다 (Table 3). 두균주모두무산소상태에서배양하였다. 사람적혈구용해액의제조농축적혈구혈액에생리식염수를첨가하여이것을냉장원심분리기 (Vision Scientific Co., VS-21SR, Seoul, Korea) 에서 3,000 rpm으로 3차례원침하여세척한다음, 상청액을제거한적혈구에증류수를부가하여사람전혈용량으로환원하였다. 이적혈구액을유연한플라스틱용기에넣고 -80 냉동고에 30분동안동결하고다시꺼내어즉시해동하는과정을 3회반복하였다. 용혈된적혈구액을다시증류수로 2.5배희석한다음, 이를냉장원심분리기에서 15,000 rpm으로원침하여상청액만수합하고 0.45 μm Millipore 일회용여과기로여과멸균하여 -80 에서냉동보관하면서필요할때해동하여사용하였다 (7). 실험에사용된세균성질증의원인균주들분리된두세균의 probiotics 효과측정은 Garderella vaginalis (ATCC 14018), Bacteroides fragilis (ATCC 25285), Mobiluncus mulieris (ATCC 35239), Peptostreptococcus asaccharolyticus (KCTC 3321) 균주를사용하여측정하였다. G. vaginalis에대한 S. denitrificans YH1과 L. crispatus YH2의성장억제효과 Casman agar base (BBL, USA) 와 MRS agar를동량혼합하고사람적혈구용해액을 5% 첨가하여 ph를 6.3으로조정하고이배지를 CMB medium이라고약칭하였다.

3 246 W-J Sihn and N-W Yang 사람적혈구용해액 5% 를첨가한 Casman broth (BBL) 에서 5% 이산화탄소와함께 48시간무산소배양한 G. vaginalis를생리식염수로 1,000배희석한균액을만들었다. 동시에 MRS broth (MERCK) 에서 24시간무산소배양한 S. denitrificans YH1과 L. crispatus YH2를생리식염수로 100배희석한균액을만들었다. G. vaginalis 의희석균액을면봉으로취하여 CMB agar 평판들에도포접종하였다. 이어서그위에 S. denitrificans YH1과 L. crispatus YH2의희석균액 10 μl씩을평판들정중앙에각각투하접종하였다. B. fragilis, M. mulieris 및 P. asaccharolyticus에대한 S. denitrificans YH1과 L. crispatus YH2의성장억제효과 Wilkins-Chalgren agar (DIFCO, USA) 와 MRS agar를동량혼합하고 ph를 6.3으로조정하여이배지를 WCM medium 이라고약칭하였다. Wilkins-Chalgren anaerobe broth (OXOID, England) 에서 48시간무산소배양한 B. fragilis, M. mulieris 및 P. asaccharolyticus를생리식염수로 2,000배희석한균액을만들었다. 동시에상기와동일하게배양한 Figure 1. Sample A shows the 99% similarity to Steroidobacter denitrificans accession No. EF in the unrooted neighbour-joining phylogenetic tree based on 16s rrna gene sequence comparison. Figure 2. Sample B shows the 99% similarity to Lactobacillus crispatus accession No. AF in the unrooted neighbour-joining phylogenetic tree based on 16s rrna gene sequence comparison.

4 The Growth Inhibition on Bacterial Vaginosis Bacteria 247 No Table 1. Carbohydrate biochemical test results of L. crispatus YH2 by API CHL kit Lactobacillus crispatus YH2 API CHL No Lactobacillus crispatus YH2 0 Control - 25 Esculine + API CHL 1 Glycerol - 26 Salicine - 2 Erythritol - 27 Cellobiose - 3 D-Arabinose - 28 Maltose + 4 L-Arabinose - 29 Lactose + 5 Ribose - 30 Melibiose - 6 D-Xylose - 31 Saccharose + 7 L-Xylose - 32 Trehalose - 8 Adonitol - 33 Inuline - 9 β-methyl-xyloside - 34 Melezitose - 10 Galactose + 35 D-Raffinose - 11 D-Glucose + 36 Amidon - 12 D-Fructose + 37 Glycogen + 13 D-Mannose + 38 Xylitol - 14 L-sorbose - 39 β-gentiobiose - 15 Rhamnose - 40 D-Turanose - 16 Dulcitol - 41 D-Lyxose - 17 Inositol - 42 D-Tagatose - 18 Mannitol - 43 D-Fucose - 19 Sorbitol - 44 L-Fucose - 20 α-ethyl-d-mannoside - 45 D-Arabitol - 21 α-methyl-d-glucoside - 46 L-Arabitol - 22 N-acetyl glucosamine + 47 Gluconate - 23 Amygdaline Keto-gluconate - 24 Arbutine Keto-gluconate - S. denitrificans YH1과 L. crispatus YH2를생리식염수로 100배희석한균액을만들었다. B. fragilis, M. mulieris 및 P. asaccharolyticus의희석균액들을면봉으로취하여 WCM agar 평판들에도포접종하고그위에 S. denitrificans YH1 과 L. crispatus YH2의희석균액 10 μl씩을평판들정중앙에각각투하접종하고 48시간무산소배양을하였다. 각균주에대하여평판 3개를사용하였다. G. vaginalis를 S. denitrificans YH1 및 L. crispatus YH2와혼합액체배양 2개의 CMB broth에희석하지않은 G. vaginalis 배양원액 100 μl와 S. denitrificans YH1과 L. crispatus YH2의배양원액 20 μl를각각 48시간혼합무산소배양한후, 자동피펫으로 100 μl씩취하여 G. vaginalis를분리하기위하여 Casman's blood agar에선상도말하고, S. denitrificans YH1과 L. crispatus YH2를분리하기위하여 MRS agar에

5 248 W-J Sihn and N-W Yang 선상도말한다음, 5% 이산화탄소와함께 48시간무산소배양을하였다. RESULTS 건강한여성의질에서분리된균주들의동정결과세균성질증이나다른성인성질환이없는 43세여성의질에서모양과크기가다른 2가지집락이분리되어그람염색을시행한결과, 둘다그람양성막대균이었으 며두균주의현미경상형태는서로달랐다. 분리된두균주는한국미생물보존센터 (KCCM) 에보내어 16S rrna partial sequencing에의해서각각 Steroidobacter denitrificans (Fig. 1) 와 Lactobacillus crispatus (Fig. 2) 로동정되었으며 (Gene Bank Data homology search results: 99%), 분리한여성이름의약자를사용하여잠정적으로전자는 S. denitrificans YH1으로, 후자는 L. crispatus YH2로균주들의명칭을정하였다. 두균주들은무산소상태에서최적성장을하였으며, 5~10% 산소의존재하에서도성장을하였으나무 No Table 2. Carbohydrate biochemical test results of S. denitrificans YH1 by API CHL kit Steroidobacter denitrificans YH1 API CHL No Steroidobacter denitrificans YH1 0 Control - 25 Esculine + API CHL 1 Glycerol - 26 Salicine + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 Maltose + 4 L-Arabinose - 29 Lactose + 5 Ribose - 30 Melibiose - 6 D-Xylose - 31 Saccharose + 7 L-Xylose - 32 Trehalose + 8 Adonitol - 33 Inuline - 9 β-methyl-xyloside - 34 Melezitose - 10 Galactose + 35 D-Raffinose - 11 D-Glucose + 36 Amidon + 12 D-Fructose + 37 Glycogen + 13 D-Mannose + 38 Xylitol - 14 L-sorbose - 39 β-gentiobiose - 15 Rhamnose - 40 D-Turanose - 16 Dulcitol - 41 D-Lyxose - 17 Inositol - 42 D-Tagatose - 18 Mannitol + 43 D-Fucose - 19 Sorbitol - 44 L-Fucose - 20 α-ethyl-d-mannoside - 45 D-Arabitol - 21 α-methyl-d-glucoside - 46 L-Arabitol - 22 N-acetyl glucosamine + 47 Gluconate - 23 Amygdaline Keto-gluconate - 24 Arbutine Keto-gluconate -

6 The Growth Inhibition on Bacterial Vaginosis Bacteria 249 산소상태보다는느리게성장하였다. 두균주는최적성장에이산화탄소를필요로하지는않았으며, 5% 저산소상태에서냉장보관하였을경우, 5일후에균수가감소하여 12일후에는모두사멸하였다. 반면에무산소상태에서냉장보관하였을경우, 24일후까지도균수의감소가없었다. 이러한결과로미루어두균주는호무산소, 내기성, 비호이산화탄소상태에서성장하는균주들임을알수있었다. S. denitrificans YH1과 L. crispatus YH2의생화학적검사결과 API CHL kit 검사에서 S. denitrificans는 galactose, D- glucose, D-fructose, D-mannose, mannitol, N-acetyl glucosamine, esculine, salicine, cellobiose, maltose, lactose, saccharose, trehalose, amidon, glycogen을가수분해하였으며, L. crispatus 는 galactose, D-glucose, D-fructose, D-mannose, N-acetyl glucosamine, esculine, maltose, Lactose, saccharose, glycogen 을가수분해하였으나 salicine, cellobiose, trehalose, amidon 은분해하지못하였다 (Table 1, 2). API zym kit 효소활성검사에서 S. denitrificans는 alkaline phosphatase, trypsin, β-galactosidase, α-glucosidase, β-glucosidase가활성을나타내었다 (Table 3). 세균성질증원인균들에대한 S. denitrificans YH1과 L. crispatus YH2의성장억제효과 S. denitrificans YH1의 B. fragilis, G. vaginalis, M. mulieris 및 P. asaccharolyticus에대한평판 3개의억제대의크기를평균한결과, 각각 35 mm, 35 mm, 25 mm, 60 mm 이었다 (Fig. 3). L. crispatus YH2의 B. fragilis, G. vaginalis, M. mulieris 및 P. asaccharolyticus에대한평판 3개의억제대의크기를평균한결과각각 25 mm, 30 mm, 20 mm, 40 mm 이었다 (Fig. 4). S. denitrificans YH1과 L. crispatus YH2를 G. vaginalis와각각혼합액체배양 G. vaginalis와 S. denitrificans YH1을 CMB broth에혼합배양한후, 선상도말법에의해서 Casman's blood agar에서 G. vaginalis를분리배양하였으나 G. vaginalis의집락은전혀분리되지않았고, MRS agar에서는 S. denitrificans YH1이대량으로배양되었다. G. vaginalis와 L. crispatus YH2를 CMB broth에혼합배양한후, 동일한방법으로 Table 3. Enzyme activity results of Steroidobacter denitrificans YH1 by API ZYM kit No Analysis items Results 0 Control - 1 Alkaline phosphatase + 2 Esterase (C4) - 3 Esterase Lipase (C8) - 4 Lipase (C14) - 5 Leucine arylamidase - 6 Valine arylamidase - 7 Crystine arylamidase - 8 Trypsin + 9 α-chymotrypsin - 10 Acid phosphatase - 11 Naphthol-AS-B1-phosphohydrolase - 12 α-galactosidase - 13 β-galactosidase + 14 β-glucuronidase - 15 α-glucosidase + 16 β-glucosidase + 17 N-acetyl-β-glucosaminidase - 18 α-mannosidase - 19 α-fucosidase - 분리배양하였으나, 마찬가지로 G. vaginalis의집락은전혀분리되지않았으며, MRS agar에서 L. crispatus YH2가대량으로배양되었다. DISCUSSION 세균성질증은질내의유산생산균들이감소또는사멸하고조건무산소혹은절대무산소세균들이생태학적우위를점하여서오는현대여성들의가장흔한질염이다 (8). 세균성질증을유발하는유인들에대해서는논란이많으나조건무산소균인 G. vaginalis의증가로인하여질내 ph의상승에따른절대무산소세균의증가유발이주된이유라고하였다 (9). 최근에는 L. crispatus가 biofilm에이미부착되어있는상태에서도세균성질증관련균종들중에서 G. vaginalis가가장큰부

250 W-J Sihn and N-W Yang A B C D Figure 3. Growth inhibition zones by S. denitrificans YH1. A, B, C, and D is B. fragilis, G. vaginalis, M. mulieris, and P. asaccharolyticus in regular order.

착능력을갖고있으며, 다른세균성질증관련균종들의부착을증진시킨다는사실이입증되었다 (10). 이와같이 G. vaginalis가세균성질증의중요한유인이라는사실은여러연구자들의보고들에의하여거의확실시되고있다. 따라서세균성질증의치료를위해서는 G.

7 250 W-J Sihn and N-W Yang A B C D Figure 3. Growth inhibition zones by S. denitrificans YH1. A, B, C, and D is B. fragilis, G. vaginalis, M. mulieris, and P. asaccharolyticus in regular order. A B C D Figure 4. Growth inhibition zones by L. crispatus YH2. A, B, C, and D is B. fragilis, G. vaginalis, M. mulieris, and P. asaccharolyticus in regular order. 착능력을갖고있으며, 다른세균성질증관련균종들의부착을증진시킨다는사실이입증되었다 (10). 이와같이 G. vaginalis가세균성질증의중요한유인이라는사실은여러연구자들의보고들에의하여거의확실시되고있다. 따라서세균성질증의치료를위해서는 G. vaginalis의수적우위를감소시켜서세균성질증관련균종들의증가를억제하고, 세균성질증관련균종들을직접선택적으로억제하는 probiotics의개발이세균성질증의궁극적인치료방법이라는것을시사하고있다. G. vaginalis는과거에 Hemophilus vaginalis로명명된적이있는호혈성박테리아로최적성장을위해서는혈액이필요하다. 그러나액체배양의경우에는적혈구자체를사용하면배양액의혼탁도를관찰하기어렵고균의희석등에서불편하기때문에적혈구용해액을사용하는것이편리하다. 따라서본연구는저자의이전보고의방법으로적혈구용해액을제조하여일관되게사용하였다 (7). 현재임상에서세균성질증을치료하기위해주로사용하는방법 은여전히무산소세균에효과적인항생제를질좌제형태로처방하거나, 살균소독제혹은질내 ph를증가시키기위해 1% 유산용액을처방하는정도에머무르고있다. 세균성질증을치료하기위하여항생제를사용하는것은세균성질증관련균종들의항생제내성을증가시킬뿐, 근본적인완치를기대하기어렵기때문에바람직하지않다고생각한다. 또한지역약국에서쉽게구입하여자가치료하는요오드화합물계열의살균제는유익한균까지모두사멸시킨다는점에서역시바람직하지않다. 2003년의보고에의하면 G. vaginalis에대한 clindamycin 의 MIC 평균은 0.16 μg/ml로감수성이있었으나, metronidazole의 MIC 평균은 25.8 μg/ml로높은내성을보였다 (7). 현재 G. vaginalis와무산소세균들에대한두항생제의내성은더증가하였을것으로생각한다. 따라서세균성질증의치료를위해서는 G. vaginalis의수적우위를감소시켜서세균성질증관련균종들의증가를억제하고, 이들균종을직접선택적으로억제하는 probiotics의개발이세균성

8 The Growth Inhibition on Bacterial Vaginosis Bacteria 251 질증의궁극적인치료방법이라는것을시사하고있다. 이러한시도의예로여성의질에서분리된 L. crispatus가질내정상무리균을회복하여세균성질증을치료할수있다고보고된바있다 (11). Antonio 등은과산화수소를생산하는 L. crispatus, L. jensenii와 L. gasseri 중에서세균성질증을예방하는균은 L. crispatus이며과산화수소의생산능력과무관한다른요인들에의한것이라는견해를보고하였다 (3). 본연구에서분리한 L. crispatus YH2도세균성질증관련세균종들의성장을억제시킨다는사실을확인하였다 (Fig. 4). 한편동시에분리된 S. denitrificans YH1의세균성질증관련세균종들에대한성장억제능력은 L. crispatus YH2보다더우세하였다. S. denitrificans는자연생태계에버려지는스테로이드화합물들에의한환경오염이동물들의내분비계를교란하여생태계에큰위협이되고있다는생태학적연구들로인하여새롭게주목을받게되었다. 스테로이드화합물을생태계에서제거하는여러세균종들이발견되었으며그중에서 denitrifying γ-proteobacterium이새로운속과종의지위를받아 Steroidobacter denitrificans gen. nov., sp. nov. 로명명되었다 (12). S. denitrificans는특히무산소상태에서 testosterone 및그와관련된 steroid 호르몬들을유일한탄소원및에너지원으로사용할수있다는사실이보고되었다 (13, 14). 저자들에의하여분리된 S. denitrificans YH1이 testosterone의무산소분해와어떤관계가있는지는현재로서는알수없으나세균성질증관련세균종들에대하여강력한성장억제효과를나타낸다는사실은분명하다 (Fig. 3). S. denitrificans YH1의이러한효과에대해서향후더구체적인연구가필요하지만, S. denitrificans YH1이 L. crispatus YH2보다세균성질증의치료에더효과적인 probiotics로활용될가능성이있다는사실은분명하다고생각한다. REFERENCES 1) Singh S, Goswami P, Singh R, Heller KJ. Application of molecular identification tools for Lactobacillus, with a focus on discrimination between closely related species: A review. Lwt-Food Sci Tech 2009;42: ) Joesoef MR, Schmid G. Bacterial vaginosis. Clin Evid 2005; 13: ) Antonio M, Petrina M, Meyn L, Hillier S. Lactobacillus crispatus colonisation reduces risk of bacterial vaginosis (BV) acquisition. Sex Transmit Infect 2011;87:A ) Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983; 74: ) Yang NW, Sin SH, Chang JS. Diagnosis of bacterial vaginosis with relation to isolation of Gardnerella vaginalis. J Bactriol Virol 2002;32: ) Yang NW, Kim JM, Choi GJ, Jang SJ. Development and evaluation of the quick anaero-system-a new disposable anaerobic culture system. Korean J Lab Med 2010;30: ) Yang NW, Lim Y, Sin SH. Drug-resistant profiles of clinical isolates of Gardnerella vaginalis on Columbia agar base supplemented with human erythrocyte lysate and horse serum. Infect Chemother 2003;35: ) Dawson SG, Harris JR. Gardnerella vaginalis and nonspecific vaginitis. Br J Hosp Med 1983;29: ) Borchardt KA, Adly BS, Smith RF, Eapen J, Beal CB. Importance of Gardnerella vaginalis as an aetiological agent in bacterial vaginosis. Genitourin Med 1989;65: ) Machado A, Jefferson KK, Cerca N. Interactions between Lactobacillus crispatus and bacterial vaginosis (BV)-associated bacterial species in initial attachment and biofilm formation. Int J Mol Sci 2013;14: ) Chang CE, Kim SC, So JS, Yun HS. Cultivation of Lactobacillus crispatus KLB46 isolated from human vagina. Biotechnol Bioprocess Eng 2001;6: ) Fahrbach M, Kuever J, Remesch M, Huber BE, Kämpfer P, Dott W, et al. Steroidobacter denitrificans gen. nov., sp. nov., a steroidal hormone-degrading γ-proteobacterium. Int J Syst Evol Microbiol 2008;58: ) Chiang YR, Fang JY, Ismail W, Wang PH. Initial steps in anoxic testosterone degradation by Steroidobacter denitrificans. Microbiology 2010;156: ) Wang PH, Leu YL, Ismail W, Tang SL, Tsai CY, Chen HJ, et al. Anaerobic and aerobic cleavage of the steroid core ring structure by Steroidobacter denitrificans. J Lipid Res 2013;54:

한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 α-galactosidase 를생산하는고온성 Bacillus coagulans KM-1 균주의생화학적특성 남기호 * 장미순

한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 α-galactosidase 를생산하는고온성 Bacillus coagulans KM-1 균주의생화학적특성 남기호 * 장미순 한수지 47(5), 516-521, 2014 Original Article Kor J Fish Aquat Sci 47(5),516-521,2014 α-galactosidase 를생산하는고온성 Bacillus coagulans KM-1 균주의생화학적특성 남기호 * 장미순 박희연 이레나코네바 1 국립수산과학원식품안전과, 1 태평양수산연구소틴로센터 Biochemical