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1 Journal of Life Science 0 Vol.. No.. 3~ ISSN (Print) 5998 ISSN (Online) DOI : Probiotic Properties of Lactobacillus strains Isolated from Kimchi Hye Jung Choi, Bo Ram Lim, Dong Wan Kim, GiSeok Kwon 3 and Woo Hong Joo, * Department of Biology, Changwon National University, Changwon 60, Korea Department of Microbiology, Changwon National University, Changwon 60, Korea 3 School of Bioresource Science, Andong National University, Andong 76079, Korea Received September, 0 /Revised November, 0 /Accepted November 7, 0 The objective of this study was to evaluate the safety and functional properties of four potential probiotic strains isolated from Kimchi, traditional Korean fermented vegetables. Based on being higher tolerance to bile salts and showing higher acid resistance or hydrophobic properties, one Lactobacillus arizonensis strain () and three L. brevis strains (, and BCNU 90) were selected in the screening experiment. All strains can survived up to 99% after 3h culture in ph.5 and resistant to % bile salts. These strains also showed good antimicrobial activities against a number of food borne pathogens, especially against Escherichia coli and Shigella sonnei. The ability to lower cholesterol levels of L. arizonensis and L. brevis 90 were demonstrated by bile salt hydrolytic activity and cholesterol assimilation tests. Moreover, L. brevis and BCNU 90 showed higher adherence to Caco cells (.76 and.86%, respectively) than Lactobacillus rhamnosus GG, a commercial probiotic strain used worldwide. The results suggest that these strains could be used as probiotics. Key words : Adhesion abilities, cholesterol assimilation, Lactobacillus strains, potential probiotics 서 한국의대표적인전통발효식품으로자리매김한김치는최 근국외에서도건강식품으로서그기능성이인정되어세계 5 대건강식품중에하나로꼽혀미국 health magazine 에소개 된바가있다 ( healthiest foods KimchiKorea, Feb, 008). 배추, 양배추무, 무청및 파등소금에절인채소에마늘, 고춧가루, 생강, 소금및젓갈 등을양념으로첨가하여발효시킨김치는항산화, 면역자극 활성, 항돌연변이 [5], 항종양 [3], 항혈전, 항노화 [3], 항동맥경 화 [] 및항균활성 [] 등다양한기능을가지고있음이보고 되면서관심이고조되고있다. 이러한기능성은각종비타민, 무기질, 식이섬유및 phytochemical 등원재료자체의생리활 성과젖산균에의한발효산물그리고상존하는미생물군집의 활성에서기여되고있는것으로보고되고있다 [5]. Leuconostoc (Leuc.), Lactobacillus, Weissella, Pediococcus 그리고 Lactococcus 속종등을포함하고있는 lactic acid bacteria (LAB) 는김치 발효에서맛과질뿐만아니라인체건강에도중요한역할을 담당하고있으며, 최근에는김치발효표준화를위해 Leuc. *Corresponding author *Tel : , Fax : * whjoo@changwon.ac.kr This is an OpenAccess article distributed under the terms of the Creative Commons Attribution NonCommercial License ( which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 론 mesenteroides, Leuc. citreum 및 L. plantarum을 starter 균주로적용한제품이다수출시되고있다. 일반적으로 LAB는미국과유럽의안전성평가기준인 Generally Recongnized as Safe (GRAS) 와 Qualified Presumption of Safety (QPS) 에해당되는균주로인체위해성이없는것으로간주되고있지만, Lactobacilli와 bifidobacteria는매우드물게인체에기회감염을일으켜감염성심내막염또는균혈증등의감염병변 [] 에서발견되기도하여프로바이오틱스연구에있어서기본적으로안전성이확보된균주의개발이절실히필요한실정이다. Lactobacillus는대표적인김치유산균가운데하나로 L. acidophilus LA, L. casei Shirota, L. plantarum LP0, L. rhamnosus GG 및 L. fermentum RC 등은현재프로바이오틱스로서상업적으로이용되고있다 [9]. 특히 Lactobacillus는장내균총의안정화, 과민성대장증후군및유당불내증개선효과가뛰어나며 [, 7], 유산균및장내유해균에의해생성되어인체에위해를줄수있는발효식품에존재하는 biogenic amine을생산하지않거나분해하는등인체건강에도움을주는것으로보고되고있다. 따라서본연구에서는안전성확보및향상된기능성을가진잠재적인프로바이오틱스를분리하기위해일반가정에서담근다양한김치로부터 Lactobacillus sp. 을분리하였으며, 항생제내성, 용혈성및유해효소생산및기본적인안전성, 내산성및내담즙산등의안정성과식중독원인균에대한항균활성, bile salt hydrolase 활성, 콜레스테롤동화작용및 Caco cell을이용한장상피세포부착능등의기능성검증을통해다기능성프로바이오틱스균주를개발하고자본연구를수행

2 3 생명과학회지 0, Vol.. No. 하였다. 재료및방법프로바이오틱스의분리및동정일반가정에서담근배추김치, 열무김치, 물김치, 총각김치 5종의다양한김치를수집하여프로바이오틱스균주를분리하는데이용하였다. ml의시료를 PBS buffer (ph 6) 에 0배희석하여이를 nutrient agar (NA), 0.5% (w/v) CaCO 3 을첨가한 lactobacilli de ManRogosaSharpe (MRS) agar 및 RA 배지에접종하여 C에서 일배양한후균주를순수분리하였다. 순수분리된균주는 PBS (ph.0) 에현탁하고 시간정치배양후, 내산성이뛰어난균주 0 colonies를 차선별하였고, Bergey's Manual of determinative bacteriology를참고하여생리 생화학적특성을조사하였다 []. 또한 S 리보솜 RNA 염기서열을분석한뒤 Bioedit (USA), Clustal X.0 (CLC bio, Denmark) 그리고 Mega 를이용하여계통학적인위치를확인하였다 []. 용혈성및안전성검사대상균주를면양혈액한천배지 (Asan Pharmacy Co., Seoul, Korea) 에접종하여 C에서 8시간배양한뒤, 균주의용혈성여부를확인하였고, Scientific Committee for Animal Nutrition (SCAN, 00) 와 European Food Safety Authority (EFSA, 008) 지침을참고하여 ampicillin, chloramphenicol, erythromycin, gentamicin, streptomycin 및 tetracycline에대한항생제감수성을조사하였다 [8, 0]. 또한 βglucoronidase, tryptophanse 및 urease와같은효소생성여부를대조군으로 Escherichia coli ATCC 0798과 L. acidophilus ATCC 356을사용하여조사하였다. 내산성및내담즙산활성조사 MRS 배지에전배양한실험균주 ml을원심분리하여 (8,000 g, 5 min, ), PBS (ph 6.0) 로두번세척한뒤버퍼및배지 ml에현탁하였다. 내산성조사는 9 ml의 PBS (ph.5) 에접종하여 에서 3시간동안진탕배양한뒤 0시간과 3시간후의생균수를측정하였고, 담즙산염에대한저항성은 0.3%, 0.6% 및 % 의 bile salt가각각첨가된 9 ml MRS broth에서 0시간과 시간배양후의생균수를비교함으로써생존률을조사하였다. 항균활성조사 6종의식중독원인균 (Bacillus cereus ATCC 579, Listeria monocytogenes ATCC 533, Staphylococcus aureus ATCC 6538, E. coli ATCC 0798, Shigella sonnei ATCC 593 및 Salmonella typhimurium ATCC 08) 에대한항균활성은 agar diffusion 법으로조사하였다 [8]. MRS 배지에서전배양한균주를원심분리하여 (8,000 g, 5 min, C) 배양상등액을 ph 6.5로중화시킨뒤, 여과하여 (0. μm membrane; Millipore) 항균력측정을위한시료로사용하였다. Bile salt hydrolase (BSH) 활성및콜레스테롤동화작용 BSH 활성은 0.5% taurodeoxycholic acid와 0. g/l CaCl 이함유된 MRS agar에실험균주배양액을 0 μl 점적하여, C에서 8시간배양한뒤흰색환의생성여부로판단하였다. 또한콜레스테롤동화능의측정은 0.5% (w/v) TDCA와 0. g/l 수용성 cholesterol을첨가한 MRS broth에실험균주 (0 8 cells/ml) 를접종하여 C에서 시간배양하였다. 배양액을원심분리한뒤 (0,000 g, 0 min, C) 배양상등액에 95% 에탄올과 0.5 M potassium hydroxide을첨가하여 60 C에서반응시켰다. 0분후 hexane을첨가하여실온에서추가반응시킨후용매층을회수하여액체질소로증발시키고 ophthaladehyde 용액과 H SO 를가하고 0분간반응시킨후흡광도 (OD 550) 를측정하여잔존콜레스테롤양을정량적으로측정하였다 [, 7]. 세포표면소수성균주의세포표면소수성은탄화수소로서 nhexadecane과 chloroform에부착하는정도를측정하였다 [7]. 전배양한균주는원심분리후, 균체를모아 PBS (ph 6.0) 로두번세척하였고 0 5 cells/ml 농도로현탁한뒤 nhexadecane 또는 chloroform 을 : 비율로첨가하였다. 분간혼합하고실온에서 30분간정치배양을한뒤에배지층의흡광도 (OD 580) 를측정하여초기흡광도와비교함으로써세포표면의소수성정도를확인하였다. Caco cell에대한부착능측정실험에사용한 colon cancer cells (Caco cells, KCLS) 은 0% FBS (fetal bovine serum), 00 units/ml penicillin과 00 μg/ml streptomycin이첨가된 Dulvecco's Modified Eagle Media (DMEM, Gibco) 배지를사용하여, C, 5% CO 조건에서배양하였다. 실험균주를 MRS 배지에서 8시간배양한후, 원심분리 (3,000 rpm, 0 min, C) 로균체를회수하여 PBS (ph 7.) 로 회세척한후에 serum free DMEM 에 0 8 CFU/ml 농도로현탁하고 caco 세포가 0 6 cells/well 농도로접종되어있는 well plates에 0.5 ml/well를첨가하였다. C, 5% CO 조건하에서 시간배양한후에 ml의 0.% tritonx00 을첨가하여 5분간고정시키고 PBS로세척을통해부착되지않은세포를제거한뒤생균수를측정함으로써대조구와비교하여 Caco cell에대한부착능을측정하였다 [5].

3 Journal of Life Science 0, Vol.. No. 33 결과및고찰 Lactobacillus 균주들의선발및동정 차선별된 50여개의콜로니는형태및생리학적특성조사를통해그람양성의장간균으로낮은 ph 범위에서생존가능한 종의균주를선별하였고 (Table ), S 리보솜 RNA 염기서열분석을통해최종적으로 L. arizonensis DSM 3 T (GenBank accession number: AJ96587) 와근연종인 BCNU 903와 L. brevis DSM 005 T (GenBank accession number: M5880) 의근연종인, 그리고 BCNU 90 균주를선발하였다. 안전성조사를위한 in vitro 시험프로바이오틱스로서섭취시인체에유해성을나타낼수있는여러가지인자를조사한결과, 종의균주는 sheep blood가포함된한천배지에서용혈현상이없는 γ형으로나타났고, 장내세균이생산하는유해효소인 βglucoronidase, tryptophanase 및 urease를분비하지않는것으로조사되었다 (Table ). 섭취하는음식과장내세균은밀접한관계가있으며, 유해하지않은식품도대사과정에서장내세균이생산하는유해효소 (βglucoronidase와 tryptophanase 등 ) 로인해발암물질로전환될수있다는연구가보고된바있다 []. 항생제감수성조사에서 가 streptomycin에대해 EFSA와 SCAN의기준보다높은저항성을가지고있었으며, 종의균주가 gentamicin에대해 SCAN 기준에비해저항성이있는것으로나타났다 (Table 3). 그러나 Danielsen & Wind는많은 Lactobacillus 속균주가 aminoglycoside계항생제에저항성이높은것으로보고하였으며 [6], 김치에서분리된다수의 L. sakei와 L. plantarum이선발균주와비슷한경향의항생제감수성을가진것으로보고되었다 [5]. 그러므로선발된균주모두안전한것으로판단된다. ph 및담즙산염에대한안정성섭취시균체가장내에도달하여기능성을나타내기위해서는낮은 ph와담즙산에대한내성능력이필수적이다. 선발된균주의 ph에대한내성은 ph.5에서초기균수와 3시간후의 Table. Physiological properties of selected Lactobacillus strains Characteristics BCNU 90 Gram's reaction Optimum growth temperature ( ) Growth ph Growth NaCl (%) Assimilation of carbohydrate Arabinose Cellobiose Fructose Galactose Glucose Lactose Mannose Mannitol Raffinose Sucrose Xylose ) ; Positive, ; negative ) Table. Haemolytic activity and production of enzyme in LAB strains Characteristics BCNU 90 E. c L. a Heamolysis Gelatinase βglucosidase βglucuronidase Tryptophanase Urease γhemolysis ) γhemolysis γhemolysis ) E.c: Escherichia coli ATCC 0798; L.a: Latobacillus. acidophilus ATCC 356 ) : positive, : negative γhemolysis γhemolysis

4 3 생명과학회지 0, Vol.. No. Table 3. Minimum inhibitoy concentration (MIC) μg/ml of antibiotics to Lactobacillus strains BCNU 90 SCAN Danielsen & Wind EFSA Em ) Gm Am Te Ch Sm 8 ) Em: erythromycin; Gm: gentamicin; Am: ampicillin; Te: tetracycline; Ch: chloramphenicol; Sm: streptomycin. 8 >6 3 >56 6 Table. Acid resistance of Lactobacillus sp. strains BCNU 90 Initial mean counts (log CFU/ml) 8.8± ± ± ±0.0 Survival after 3 hr at ph.5 8.8± ± ± ±0.00 Survival rate (%) 생존력을비교함으로써확인하였다 (Table ). 와 BCNU 90은낮은산성환경에전혀영향을받지않았으며, 과 도 99.6% 이상생존률을보임으로써산에대한안정성은매우높은것으로확인되었다. 또한 종의선발균주는 % 의고농도의담즙산에서 9900% 생존률을보임으로써담즙산에대한영향을받지않는것으로나타났으며 (Table 5), 이는김치에서분리한 0여종의 L. sakei가 3.5% oxgall에서생존률이 0% 내외인것으로보고된바있으나 [5] 이에비해 개의선발균주가본실험을통해담즙산 에대한뛰어난안정성을보유한균주임이확인되었다. 항균활성 Paper disc법에따라 8 mm의 disc에 50 μl의균배양상등액을접종하여그람양성세균 3종과그람음성세균 3종의식중독원인균에대해항균활성을조사하였다. 와 의배양상등액은대체로넓은항균스펙트럼을가지고있는것으로나타났다 (Table 6). 특히 E. coli에대해저해환 9 mm의높은항균활성을보였고, List. monocytogenes와 S. sonnei에대해서도우수한항균활성을가지고있음이확인되었다. 과 BCNU 90의배양상등액또한 E. coli 에대한항균활성이가장높은것으로나타남으로써 종의균주가생산하는이차대사산물들이전반적으로그람음성세균에대한높은항균활성을나타내는것으로조사되었다. 기존의연구된 Lactobacillus sp. 균주가대부분 E. coli에대한항균활성이없거나 S. aureus에비해상대적으로낮은항균활성을가진것과비교하여다소차이가있었으며 [, 7], 실험균 Table 5. Bile resistance of Lactobacillus sp. strains BCNU 90 Control (log CFU/ml) 7.87± ± ± ± % 7.87± ± ± ±0.0 Survival rate (%) % 7.85± ± ± ±0.0 Survival rate (%) % 7.78± ± ± ±0.0 Survival rate (%) Survival of LAB strains was compared by plate counting after exposure with bile salt 0.3%, 0.6%, % in MRS broth for 0 and hr. Table 6. Antimicrobial activities of Lactobacillus sp. strains against foodborne pathogens BCNU 90 B. c ) L. m S. a E. c S. s S. t.5±0.5 ) ±.0 ±.0.5±. 5.5±3.5 3).5± ±. 9 9±.0.5±0.5 7±.0 ±. 5.5±0.7 ±. 3.5±0.7.5±0.7 ) B.c: Bacillus cereus; L.m: Listeria monocytogenes; S.a: Staphylococcus aureus; E.c: Escherichia coli; S.s: Shigella sonnei; S.t: Salmonella typhimurium. ) Each value represents a diameter of clear zone (mm; mean ± SD, n=3). 3) ; negative

5 Journal of Life Science 0, Vol.. No. 35 Table 7. Cholesterolrelated activities and hydrophobicity of selected probiotic Lactobacillus sp. strains Strains BSH activity Cholesterol assimilation (%) BCNU 90 ) 5.8 ± ± ± ± 3.5 ) ; positive for BSH activity, :negative for BSH activity Hydrophobicity (%) nhexadecane chloroform ± ± ± ± ± ± ± ±.6 이대부분의식중독원인균인그람음성세균에대해활성이매우우수함을확인할수있었다. 콜레스테롤저하효능과소수성 BSH 활성은 BSH agar 배지에서흰색침전환이생성되는것으로확인할수있었으며, 와 은모두 BSH 활성을가지고있는효소를분비하는것으로확인되었다 (Table 7). 그리고 TDCA와수용성 cholesterol을배지에첨가하고배양하여잔존콜레스테롤양을측정하는동화능실험에서도 와 은 50% 이상의활성을보임으로써콜레스테롤저하효능이뛰어남을확인할수있었다. 김치에서분리한 L. sakei는 060%, L. plantarum NR7는 7.8% 그리고상업균주인 L. rhamnosus GG가 8% 의콜레스테롤저하효능이있음이보고되어있다 [5]. 그러므로실험에사용한균주에서이들균주보다다소뛰어난콜레스테롤저하효능이확인되었다. 세포표면의소수성은병원성세균과의부착및응집현상, 숙주세포의방어등다양한현상들과연관되어있으며 [3, 0], 본연구에서는미생물의탄화수소에대한부착능을측정함으로써소수성정도를측정하였다. 은 nhexadecane 첨가시 99.6% 이상의높은부착능을보였으며, 과 BCNU 90도 90% 이상의부착능을가짐으로써세포표면의소수성이매우높은것으로나타났다 (Table 7). 발효코코아에서분리한 L. brevis FFC99와 SAU05는 xylene 첨가시각각 0.6%, 60.98% 의부착능을보였으며, L. rhamnosus GG는 nhexadecane 처리시 00% 의부착능을가진것으로나타남으로써콜레스테롤동화능과세포표면소수성면에서 BCNU 90과유사함을확인할수있었다. Caco cell에대한부착능프로바이오틱스균주의장관상피세포유래의 Caco cell 에대한부착능력은 과 BCNU 90이각각.8%,.9% 로 의 8.%, 의 5.8% 보다상대적으로높게나타났다 (Fig. ). 세포표면특성은장내세포와의응집력및부착능과연관성이높은것으로알려져있으며 [6], 소수성이높은 과 BCNU 90이상대적으로 Caco cell에대한부착능이높은것으로본실험에서도확인되었다. 낙농제품에서분리한 9종의 Lactobacillus sp. 균주의 Caco 부착능은 0.5.5% 범위로평균 5% 내외인것으로보고되고있으며 [], L. plantarum SAU96, L. brevis FFC99 및상업균주인 L. rhamnosus GG의 Caco cell 부착능은각각.8%, 0.9% 그리고.5% 로보고되고있으므로 [9] 결과를참고로할때이들균주들은인체장관의상피세포에의부착능력도우수할것으로사료되며차후에이를실험으로증명할필요가있다. 김치로부터분리한 종의 Lactobacillus sp. 는 GRAS 균주로식품첨가물로써사용가능한안전한균주이며, 본연구에서도인체에유해할수있는효소생산을하지않는것으로확인되었다. 프로바이오틱스로서기본적인산성과담즙산에대해우수한안정성을보였으며, 와 90은 E. coli와 S. sonnei에대해특히뛰어난항균활성을가진것으로나타났다. 또한 와 90은 BSH 활성및콜레스테롤수준을 50% 이상감소시켰으며, 소수성실험을통해섭취시장내부착능이우수할것으로판단되어프로바이오틱스로서섭취시장내환경개선에크게도움을줄수있을것으로사료된다. 감사의글 이논문은 03~0년도창원대학교연구비에의하여연구되었음. Fig.. Adhesion ability of Lactobacillus sp. strains to Caco cell. The adherence percentage of LAB strains to Caco cells was calculated by comparing the plate counts between initial and adhered bacteria. Each value represents mean ± SD, n=3. References. Alander, M., Satokari, R., Korpela, R., Saxelin, M., Vilppo

6 36 생명과학회지 0, Vol.. No. nensalmela, T., MattilaSandholm, T. and Von Wright, A Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption. Appl Environ Microbiol 65, Allain, C. C., Poon, L. S., Chen, C. G., Richmond, W. and Paul, C. 97. Enzymatic determination of total serum cholesterol. Clin Chem 0, BarNess, R., Avrabamy, N., Matsuyama, T. and Rosenberg, M Increased cell surface hydrophobicity of a Serraia marcescens NS 38 mutant lacking wetting activity. J Bacteriol 70, Borriello, S. P., Hammes, W. P., Holzapfel, W., Marteau, P., Schrezenmeir, J., Vaara, M. and Valtonen, V Safety of probiotics that contain lactobacilli or bifidobacteria. Clin Infect Dis 36, Cho, E. J., Rhee, S. H., Lee, S. M. and Park, K. Y In vitro antimutagenic and anticancer effects of kimchi fractions. J Korean Ass Cancer Prev, Danielsen, M. and Wind, A. A Susceptibility of Lactobacillus spp. to antimicrobial agents. Int J Food Microbiol 8,. 7. 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7 Journal of Life Science 0, Vol.. No. 초록 : 김치로부터분리된 Lactobacillus strains의 probiotic 특성최혜정 임보람 김동완 권기석 3 주우홍 * ( 창원대학교생물학과, 창원대학교미생물학과, 3 안동대학교생명자원과학부 ) 본연구의목적은한국의전통발효식품인김치로부터분리된 개의잠재적인프로바이오틱스균주의안전성과기능적특성을측정하기위한것이다. 담즙산염에대한높은내성과내산성그리고소수성특성에기초하여한균주의 Lactobacillus arizonensis () 와 3 균주의 L. brevis (, and BCNU 90) 는선택되었다. 모든균주는 ph.5에서 3시간후에도 99% 이상생존가능했고, % 의담즙산염에내성을보였다. 이들균주는특히 Echerichia coli와 Shigella sonnei 같은다수의식중독원인균에대해우수한항균활성을보였다. L. arizonensis 와 L. brevis 90의콜레스테롤수준을감소시키는능력은시험관내담즙염가수분해활성과콜레스테롤동화작용시험을통해검증되었다. 게다가, L. brevis 과 BCNU 90은 Caco cells 에대해 ( 각각.76,.86%) 상업균주인 Lactobacillus rhamnosus GG 보다높은부착능을가진것으로확인되었다. 이러한결과들을이들균주를프로바이오틱스로서사용할수있음을제시하고있다.

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untitled 理葉 CNFH-88-04 行 87 年 9 1 88 年 6 30 類葉 Staphylococcus aureus Staphylococcus epidermidis Streptococcus mutans Streptococcus sanguis Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Candida