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1 The Korean Journal of Microbiology (2010) Vol. 46, No. 4, pp Copyright c 2010, The Microbiological Society of Korea 갓김치에서분리된유산균의활성산소종에대한저항성과항산화활성 임성미 * 동명대학교식품공학과 Resistance to Reactive Oxygen Species and Antioxidant Activities of Some s of Lactic Acid Bacteria from the Mustard Leaf Kimchi Sung-Mee Lim Department of Food Science and Technology, Tongmyong University, Busan , Republic of Korea (Received November 10, 2010/Accepted December 21, 2010) In present study, five strains of Lactobacillus acidophilus 20, Lactobacillus brevis 55, Lactobacillus paracasei 74, Lactobacillus plantarum 81, and Leuconostoc mesenteroides 104 isolated from the mustard leaf kimchi were investigated for resistance to reactive oxygen species (ROS) and antioxidant activity. L. acidophilus 20, L. brevis 55, L. paracasei 74, and L. plantarum 81 were resistant to hydrogen peroxide (0.5 mm), showing a survival rate of 50% or more. In particular, L. acidophilus 20 and L. paracasei 74 were the most superoxide anions-resistant and L. paracasei 74 and L. plantarum 81 were most likely survive hydroxyl radicals. Meanwhile, the intracellular cell-free extract (ICFE) from L. plantarum 81 exhibited significantly higher DPPH radical scavenging values (96.4±2.8%) than the intact cells (IC). The ICFE of L. plantarum 81 showed the highest superoxide radical scavenging ability and chelating activity for Fe 2+ ions among the 5 lactic acid bacteria (LAB) tested, and IC and ICFE from L. plantarum 81 demonstrated excellent reducing activity, which was higher than those of BHA and vitamin C as a positive control. Keywords: Fe 2+ -chelating activity, Lactobacillus plantarum, radical scavenging ability, reactive oxygen species, reducing activity 산화적스트레스 (oxidative stress) 는살아있는생명체에유해한생화학반응을유발하고죽상동맥경화증을비롯한각종혈관계질환, 돌연변이, 암, 신경퇴행성질환, 면역기능장애및노화의원인으로알려져있다 (7). 생체의에너지생산을위한호흡대사과정중일정량의산소는반응성이높은활성산소종 (reactive oxygen species, ROS) 이라는유해물질을생성하게되는데그종류로는 hydroxyl radical (OH ), superoxide anion radical(o 2 ), peroxyl radical (RO 2 ) 과같이쌍을이루지못한전자를가지는 free radical과과산화수소 (H 2O 2), 일중항산소 (singlet oxygen) 및오존 (O 3) 등이있다 (30). Free radical에의해생성된단백질산화생성물은세포내촉매기능을담당하고있는효소의기능을상실케하며 (26), 또한다가불포화지방산과콜레스테롤의과산화를유발하여 2차산물인 malondial- * For correspondence. limsm020@tu.ac.kr; Tel: ; Fax: dehyde나 4-hydroxynonenal 등의지질과산화물을축적하여세포막투과성의변화와단백질합성능력을감소시킨다 (12). 또한 ROS는세포의 DNA 가닥을분리시키고자매염색분체의교환을유도하여악성형질전환을유발하여발암의요인이된다 (29). 활성산소를소거하는기능이있는항산화물질로는 vitamin C, glutathione, uric acid와같은수용성항산화제, tocopherol, carotenoids, flavonoid/flavonoids 등의지용성항산화제, tertbutylhydroxytoluene (BHT), tert-butylhydroxyanisol (BHA) 등의합성항산화제, superoxide dismutase (SOD), catalase, glutathione peroxidase 등의항산화효소가있으며 (7) 이들은활성산소중간물질을제거하여산화적손상으로부터세포를보호하고세포의자연사 (apoptosis) 에대하여저항할수있다 (3, 35). 합성항산화제는산화억제효과는뛰어나지만, 독성에의한여러부작용을초래할수있으므로최근천연물을이

2 376 Sung-Mee Lim 용한항산화력측정에관한연구가활발히진행되고있다. 가자, 소목, 지유, 복분자및초두구등다양한한약재들은토코페롤보다더높은항산화력이있으며 (20), lemon vervena, chamomile 등의허브류및오레가노, 육두구, 계피등의향신료들도높은항산화력이있는것으로보고되고있다 (8, 33). 한편일부미생물의 Mn-SOD, Cu-SOD는활성산소를비독성화시키고 (39), NADH, NADPH, glutathione 및 uric acid 같은항산화제를생산하여 hydroxyl radical을분해할수있다고알려져있다 (1). Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium adolescentis 및 Bifidobacterium longum는 NADH peroxidase에의해 hydrogen peroxide를분해할수있었다 (32). 김치에서분리된 Lactobacillus plantarum의세포와세포를파쇄하여얻은세포추출물은지질과산화를저해하였는데이는 SOD 활성에기인하기보다는금속이온을 chelating 하는활성이있어높은항산화력을나타내었는데이들은 ROS 에대한저항력도강한것으로밝혀졌다 (16). 한편, Lactobacillus lactis 및 L. plantarum와같이 SOD를생산하는유산균은대장의염증개선에도효과가있는것으로알려졌다 (37). 본연구는 probiotic bacteria의선정기준의하나인항산화활성을조사하기위해갓김치에서분리된유산균 5종을대상으로 hydrogen peroxide, superoxide anions 및 hydroxyl radical 등의 ROS에대한저항력과 DPPH radical, superoxide radical 소거능, 환원력및 Fe 2+ chelating 활성등을측정하여산화억제능력을실험한결과이다. 사용배지및시약 재료및방법 유산균분리및배양에사용된배지 Lactobacilli MRS agar 및 broth는 Difco (USA) 사제품, 분리된유산균의동정에사용된 API 50 CHL kit는 biomériux (France) 사제품, ROS에대한저항성측정과 radical 소거능, 환원력및 Fe 2+ -chelating 활성측정에사용된시약은모두 Sigma Co. (USA) 에서구입하여사용하였다. 유산균분리및동정 가정에서담궈 3개월가량숙성시킨갓김치 (ph ) 8 종을수집하여채취한시료 50 g과멸균생리식염수 (0.85% NaCl 용액 ) 450 ml을혼합하여 2분간균질화한후시료용액 1 ml를 1% CaCO 3 가첨가된 Lactobacilli MRS agar 배지에접종하여 37 C, 24-48시간동안미호기성조건 (10% O 2, Anoxomat system, MART Co., Netherland) 으로배양한후투명한환을생성하는독립된집락을사면배지에 3회계대배양하여활성을높인후사용하였다. 선발된 5종의유산균을대상으로 Bergey s Manual of Determinative Bacteriology (27) 에따라배양학적및생화학적특성과 API 50 CHL를이용하여당발효능을조사하여추정되는유산균명을확인하였다. ROS 에대한저항성 Hydrogen peroxide에대한저항성 : H 2O 2 에대한유산균의저항성은 Fu 등 (11) 의방법을일부변형하여측정하였다. 분리된유산균은 Lactobacilli MRS broth 배지에접종하여 37 C, 18시간미호기적조건에서배양한후원심분리 (7,000 g, 20분, 4 C) 한다음 phosphate buffered saline (PBS; 0.85% NaCl, 2.68 mm KCl, 10 mm Na 2HPO 4 및 1.76 mm KH 2PO 4) 으로 3회세척하여세포수약 10 8 CFU/ml로맞춰동일한 buffer에서현탁시켰다. 0.5 mm H 2O 2 용액 (35% H 2O ml/dw 1,000 ml) 500 μl와동량의세포현탁액을즉시혼합하여 60분간격으로총 5시간반응시킨후잔존하는 H 2O 2 을제거하기위해다시원심분리 (20,000 g, 1분, 4 C) 하여 pellet을회수하였다. Pellet을 buffer에재현탁시킨다음단계별로희석하고 Lactobacilli MRS agar에평판배양 (37 C, 24시간 ) 하여생성된집락수를측정하여초기균수에대한감소율을나타내었다. Superoxide anions에대한저항성 : Paraquat(1,1 -dimethyl- 4,4 -bipyridinium) 에의해유도된 superoxide anions에대한유산균의저항성은 Bauer 등 (5) 의 diffusion assay법에따라측정하였다. 37 C에서 18시간배양한유산균은원심분리 (7,000 g, 20분, 4 C) 하여세포를모으고 PBS로 3회세척한현탁세포액 ( 약 10 8 CFU/ml) 100 μl를 Lactobacilli MRS agar 평판배지위에도말한후멸균수에용해시킨 10 mm paraquat의 10 μl 를균접종한평판배지위에 spot하여 37 C에서 24시간배양하여생성된저해환을측정하였다. Hydroxyl radical에대한저항성 : Fenton 반응을통해발생된 hydroxyl radical에대한유산균의저항성은 Barreto 등 (4) 에따라일부변형하여측정하였다. PBS에용해시킨 10 mm terephthalic acid (1,4-benzenedicarboxylic acid), 유산균세포 (10 8 CFU/ml) 및 0.01 mm CuSO 4 5H 2O가포함된용액내에서 hydroxyl radical을발생시킨다음 1 mm H 2O 2 를첨가하여반응시켜 15분간격으로용액을채취한다음 Lactobacilli MRS agar를이용하여생균수를측정하여저해율을계산하였다. 항산화활성측정 유산균의항산화력측정에사용된유산균의세포는 Lin과 Chang (22) 의방법에따라준비하였다. 즉, Lactobacilli MRS broth 배지에접종하여 37 C, 18시간배양한배양액을원심분리 (7,000 g, 20분, 4 C) 하여회수한 pellet (intact cell, IC) 은 PBS로 3회세척후 PBS에재현탁하였다. 한편 intracellular cell-free extract (ICFE) 는 IC를탈이온수로 2회세척한후 icebath상에서 10분간세포를초음파 (Qsonical, USA) 파쇄한후 10,000 g, 10분간원심분리하여세포파편을제거하고상등액만을회수하여 membrane filter (0.45 μm, Millipore Corp., USA) 로여과하여얻었다. 유산균의 IC와 ICFE 항산화활성은 DPPH (1,1-diphenyl-2-picryl hydrazyl), superoxide radical 소거능및환원력을측정하여평가하였다. 양성대조구로서 100 mg/l vitamin C 및 BHA을사용하여활성을비교하였다.

3 유산균의항산화활성 377 DPPH radical 소거능측정 : Brand-Williams 등 (6) 의방법을일부변형하여 DPPH radical 소거능을측정하였다. 유산균 IC 혹은 ICFE 100 μl에에탄올에녹인 M DPPH 용액 100 μl를 96 well plate에 loading한다음빛을차단하여 20 C에서 30분간반응시킨후 517 nm에서 microplate reader (Spectrocount; Packard Instruments, USA) 로흡광도를측정하여다음식에따라 DPPH radical 소거능을계산하였다. DPPH radical 소거능 (%) = 1-( 시험구흡광도 / 대조구흡광도 ) 100 Superoxide radical 소거능측정 : Superoxide radical 소거능은 Kim 등 (15) 방법에따라측정하였다. 즉, 96 well plate 에 sodium carbonate buffer (ph 10.0), xanthine, ethylenediaminetetracetic acid (EDTA), bovine serum albumin 및 nitro blue tetrazolium 혼합용액 100 μl와유산균의 IC 혹은 ICFE 100 μl를가하고, xanthine oxidase를최종적으로 12 mu가되도록첨가하여 25 C에서 20분간반응시킨후 CuCl로반응을정지시킨후 560 nm에서흡광도를측정하여다음식에따라 superoxide radical 소거능을계산하였다. Superoxide radical 소거능 (%) = 1-( 시험구흡광도 / 대조구흡광도 ) 100 환원력측정 : 유산균의환원력은 Oyaizu (24) 의방법을일부변형하여측정하였다. 즉, 유산균 IC 혹은 ICFE (100 μl) 은 1% potassium ferric cyanide (100 μl) 와 sodium phosphate buffer (0.2 M, ph 7.0)(100 μl) 를혼합하여 50 C에서 20분간반응시켰다. 반응후 10% trichloroacetic acid를첨가하여 3,000 g에서 5분간원심분리하여얻은상등액은 0.1% ferric chloride 200 μl와혼합하여 700 nm에서흡광도를측정하였다. Fe 2+ -chelating 활성측정 : 유산균의 Fe 2+ -chelating 능력측정은 Yu 등 (38) 방법에따라측정하였다. 유산균의 ICFE 0.2 ml, Tris-HCl buffer (ph 7.4) 0.8 ml, 1.8 mm FeSO ml, 10% hydroxylamine-hcl 0.32 ml 및 0.2 N HCl에용해한 0.1% 2,2 -bipyridyl 0.8 ml을혼합하여 522 nm에서흡광도를측정하여 Fe 2+ -chelating 활성을측정하여다음식에따라계산하였고, 이때양성대조구로서는 100 mg/l EDTA를사용하였다. Chelating 활성 (%) = 1-( 시험구흡광도 / 대조구흡광도 ) 100 성, lysine과 ornithine 가수분해및 45 C 하에서증식하며 arabinose, ribose, mannose 및 raffinose 등을분해하고동정확률 (ID) 99.9% 를나타내어 Lactobacillus acidophilus로확인되었다. 55는 D형 lactic acid 생산, catalase 양성, arginine 가수분해하고혐기적조건하에서증식하지못하고 xylose, maltose, melibiose 및 gluconate 등을이용하여 Lactobacillus brevis (ID=97.2%) 로추정된다. 또한 74는 glucose로부터가스생산, L형 lactic acid 생산, catalase 음성, 1-5% NaCl 하에서증식하며 sorbose, sorbitol, tagatose 및 arabitol 등을발효하는능력이있어 Lactobacillus paracasei (ID=99.2%) 인것으로추정된다. 81은 L형 lactic acid 생산, catalase 양성, ornithine 가수분해, 호기적조건하에서만증식하며 mannitol, α -Methyl-D-mannoside, melezitose 및 gentiobiose 등의당을분해하는능력이있으므로 L. plantarum (ID=99.9%) 으로확인되었다. 104의세포는구형이며 D형 lactic acid 생산, catalase 음성, 45 C에서증식할수없고, 혐기적조건하에서만증식하며 α-methyl-d-glucoside, N-Acetyl glucosamine, salicine 및 turanose 등을이용하는능력이있어 Leuconostoc mesenteroides (ID=99.8%) 로추정된다. Park 등 (25) 에따르면갓김치담금직후유산균수는약 4.5 log cycle에서숙성이진행됨에따라서서히증가하여숙성 14 일만에 7 log cycle에이르렀다고보고하였으며, Shim과 Lee (31) 에따르면, reca 유전자를특이적으로증폭하는 PCR을이용하여갓김치내에있는유산균을조사한결과, L. plantarum 및 L. mesenteroides가검출되었다고하여본실험에서분리된유산균의종류와일부일치하였다. ROS 에대한유산균의저항성 Hydrogen peroxide, superoxide anions 및 hydroxyl radical 에대한분리유산균의저항성을알아본결과는각각 Fig. 1, 2 및 3과같다. 0.5 mm hydrogen peroxide에대한유산균의저항성결과, 1시간반응후대부분균들의저해율은 10% 내외정도였으나, 반응 3시간만에는 L. brevis 55의저해율이 통계처리실험은모두 3회반복하여실시하였으며, 유산균주간의차이는 SPSS (version 12.0, USA) 프로그램의분산분석 (ANOVA) 을이용하여비교하였고, 평균간유의성은 Duncan s multiple range test를사용하여 p<0.05 수준에서시험구들과대조구간의유의적차이를검정하였다. Inhibition(%) 결과및고찰 갓김치로부터유산균분리가정에서담궈숙성시킨갓김치 8종을수집하여 Lactobacilli MRS agar 평판배지에배양하여전형적인유산균으로추정되는균을대상으로생화학적특성및당발효능을조사한결과는 Table 1과같다. 20은 L형 lactic acid 생산, catalase 음 Incubation time with H 2 O 2 (h) Fig. 1. Resistance to hydrogen peroxide (0.5 mm) of various lactic acid bacteria isolated from the mustard leaf Kimchi. Data represent the means of three experiments±standard deviation (error bars). ( ), L. acidophilus 20; ( ), L. brevis 55; ( ), L. paracasei 74; ( ), L. plantarum 81; ( ), L. mesenteroides 104.

4 378 Sung-Mee Lim Table 1. Differentiating characteristics and carbohydrate reactions of lactic acid bacteria from the mustard leaf Kimchi Contents Sugar Sugar Cell shape Rods Rods Rods Rods Cocci Glycerol Salicine Gram staining Erythritol Cellobiose Spores staining D Arabinose Maltose Acid fast staining L Arabinose Lactose Motility Ribose Melibiose Gas from glucose + + D Xylose + + Saccharose H2S production L Xylose Trehalose Lactic acid L D L L D Adonitol Inuline Methyl red β Methyl xyloside Melezitose + + Voges Proskauer Galactose D Raffinose + + Horse blood hemolysis D Glucose Amidon Sheep blood hemolysis D Fructose Glycogene Catalase + + D Mannose Xylitol Oxidase L Sorbose + β Gentiobiose + + Urease Rhamnose D Turanose + + Arginine hydrolysis + + Dulcitol D Lyxose Lysine Inositol D Tagatose + Ornithine Mannitol D Fucose Growth in aerobic condition Sorbitol + + L Fucose anaerobic α Methyl D mannoside + D Arabitol + Growth at α Methyl D glucoside + L Arabitol Growth at ph N Acetyl glucosamine Gluconate + + Growth in 1 5% NaCl Amygdaline Ceto gluconate 10% NaCl Arbutine Ceto gluconate Esculine

5 유산균의항산화활성 Clear zone (mm) Inhibition(%) Fig. 2. Resistance to superoxide anions of various lactic acid bacteria isolated from the mustard leaf Kimchi. Data represent the means of three experiments±sd (error bars). 12.2% 로가장낮은반면, L. mesenteroides 104의저해율은 42.4% 로가장높았다. 또한 5시간후에도 L. brevis 55 의생존율은 83.6% 로가장높았고, 그다음으로 L. acidophilus 20, L. paracasei 74 및 L. plantarum 81도 50% 이상의생존율을보여비교적안정하였으나, L. mesenteroides 104의생존율은약 30.1% 에불과하여실험균주들중가장낮은저항성을나타내었다. 일반적으로유산균은 catalase 음성반응을나타내는것으로알려져있는데, 본실험에사용된 L. brevis 55와 L. plantarum 81는특이하게 catalase 를생성하는것으로확인되었으며, 이들이 H 2O 2 에대한저항성이높은것은 catalase 생성능력에기인하는것으로추정된다. 한편, 유산균을접종한평판배지에 superoxide anions을유도하는 paraquat을 spot하여배양한후생성된저해환의크기를측정하였다. Paraquat는확산을통해세포막을통과하여산소와반응한결과 superoxide anions을생성하여세포에유해성분으로작용한다 (34). L. acidophilus 20와 L. paracasei 74의저해정도가가장낮았으며, 반면 L. mesenteroides 104에대한저해환의크기가가장크게나타났으므로 superoxide anions에대한저항성도균주의종류에따라다소차이가있었다. Hydroxyl radical에대한저항성을조사한결과, 반응 15분만에 L. acidophilus 20, L. plantarum 81 및 L. mesenteroides 104들의생균감소율은약 20-25% 정도로이들균주간에는유의적인차이가없었으며, 이는 L. brevis 55에비해유의적으로다소낮았다. 모든유산균들은반응시간이경과함에따라저해율이서서히증가하는경향을나타내었는데, 반응 60분후에는 L. acidophilus 20, L. brevis 55 및 L. mesenteroides 104는 95% 이상의높은사멸율을보인반면, L. paracasei 74는약 24.4%, L. plantarum 81는약 33.7% 정도생존한것으로나타났다. Kullisaar 등 (17) 에따르면, 항산화력이없는 Lactobacillus fermentum E 균주는 0.4 mm hydrogen peroxide와반응시켜 240분동안에만생존이가능하였으나, 항산화력이있는 L. fermentum E-3과 E-18 균주는각각 450과 390분후에 Incubation time with hydroxyl radicals (min) Fig. 3. Resistance to hydroxyl radicals of various lactic acid bacteria isolated from the mustard leaf Kimchi. Data represent the means of three experiments±standard deviation (error bars). ( ), L. acidophilus 20; ( ), L. brevis 55; ( ), L. paracasei 74; ( ), L. plantarum 81; ( ), L. mesenteroides 104. 도생존하였으며, 1 mm 농도하에서 E-3와 E-18 균주는 180 및 150분동안생존하였다고보고하였다. 또한 E 은 hydroxyl radicals 존재하에서약 15분정도에만생존하였으나, E-3과 E-18은 34분동안생존가능하였으며, 또한이들균주는 superoxide anions에대한저항성도있는것으로보고하였다. Lee 등 (19) 은김치에서분리된 L. plantarum KCTC 3099 는 1 mm hydrogen peroxide와 0.4 mm hydroxyl radical의존재하에 8시간반응시킨후에도생존하였고, paraquat에의해발생된 superoxide anion에의해영향을받지않는것으로나타났다. 유산균의항산화활성 갓김치에서분리된유산균들의 IC와 ICFE에대한 DPPH radical 소거활성을측정한결과는 Table 2와같다. IC에의한 DPPH radical 소거능은 L. acidophilus 20, L. brevis 55 및 L. mesenteroides 104들은 5% 유의수준에서차이가없이다른 2종의유산균에비해낮은활성을보인반면, L. paracasei 74의소거능은 47.6±6.3% 에이르렀고, L. plantarum 81은실험균주중가장높은소거능 (70.8± 10.1%) 을나타내었다. 하지만이들의활성은양성대조구인 BHA나 vitamin C보다는낮은수준이었다. 한편, ICFE의소거능을측정한결과, 가장낮은소거능은 L. brevis 55와 L. mesenteroides 104에서나타났고, 가장높은소거능은 L. plantarum 81에서나타났는데이들의소거활성은 BHA와 vitamin C와유의적인차이가없을정도로높게나타났다. 또한동일한균주의 IC와 ICFE들간의활성을비교한결과, 20, 74 및 81은 ICFE에서더높은활성을보인반면, 55와 104의경우는 IC에서더높은소거능을나타내었다. Superoxide radical의소거능을측정한결과는 Table 3과같다. IC의 superoxide radical 소거능은 L. mesenteroides 104

6 380 Sung-Mee Lim Table 2. DPPH radical scavenging activity of various lactic acid bacteria isolated from the mustard leaf Kimchi DPPH radical scavenging activity (%) Intracellular Intact cells cell free extracts L. acidophilus ±8.6 a 55.6±8.7 a L. brevis ±7.4 ab 26.0±3.9 b L. paracasei ±6.3 b 61.5±5.6 a L. plantarum ±10.1 c 96.4±2.8 c L. mesenteroides ±1.8 a 24.3±4.0 b BHA 89.1±2.9 d 89.1±2.9 c Vitamin C 95.5±0.5 d 95.5±0.5 c DPPH radical scavenging activity was defined as [1 A 517(sample)/ A 517(control)] 100%. Data represent the means of three experiments±sd (error bars). a d Means with different letters in a column are significantly different at p<0.05 by Duncan s multiple range test. 에서가장낮은 (19.9±1.3%) 반면, L. brevis 55와 L. plantarum 81은각각 66.2±2.4% 와 62.1±8.8% 로높은활성을나타내었다. ICFE의경우에도 L. mesenteroides 104 에서가장낮은 (18.0±0.5%) 반면, L. plantarum 81의소거활성이가장높게나타났으나 (80.6±4.7%), 이는 BHA와 vitamin C의활성과는유의적인차이가있었다. 산화과정에생성된 free radical을비롯한각종 ROS 및독성생성물들은생물학적분자를공격하여생체내세포를손상시킨다. DPPH radical은화학적으로유도되는비교적안정한 radical로서다른원자나분자로부터전자를공여받아비가역적으로결합하면보라색이엷어지면서흡광도가감소하게되는원리를이용하여 radical 소거활성을측정할수있다. DPPH radical 소거능이높으면 free radical을환원시키거나상쇄시키는능력이높아항산화활성이있다는것을의미한다 (2). Cho 등 (9) 은김치에서분리한 L. plantarum YS712 균주가 95.8% 로가장높은 radical 소거능을나타내었으며, L. plantarum ATCC 8014 (95.3%), L. plantarum L155 (95.2%), Enterococcus sp. 01 (94.9%), Lactococcus sp. KU107 Table 3. Superoxide radical scavenging activity of various lactic acid bacteria isolated from the mustard leaf Kimchi Superoxide radical scavenging activity (%) Intact cells Intracellular cell free extracts L. acidophilus ±7.9 a 65.6±9.0 a L. brevis ±2.4 b 50.0±5.6 b L. paracasei ±3.3 c 69.8±0.2 a L. plantarum ±8.8 b 80.6±4.7 c L. mesenteroides ±1.3 d 18.0±0.5 d BHA 90.3±1.7 e 90.3±1.7 e Vitamin C 97.9±0.8 e 97.9±0.8 e Superoxide radical scavenging activity was defined as [1 A 560(sample)/ A 560(control)] 100%. Data represent the means of three experiments±sd (error bars). a e Means with different letters in a column are significantly different at p<0.05 by Duncan s multiple range test. (94.6%) 순으로나타났다고보고한바있다. Kaizu 등 (14) 의보고에따르면, 7종의 lactobacilli 균주는 70% 의산화억제효과를나타내었고, 이중에서헤테로발효형인 Lactobacillus sp. SBT 2038의활성이가장높았으며, 특히 Lactobacillus sp. SBT 2028와 L. casei ssp. rhamnosus SBT 2257의 ICFE는비타민 E가결핍된식이를투여한 rat에게서도항산화효과가뚜렷하게나타났다고보고하였다. Lee 등 (18) 은 γ-aminobutyric acid를생산하는 L. brevis BJ20에의해발효된다시마는양성대조구 BHA보다더높은 DPPH, superoxide 소거활성을나타내었고, xanthine oxidase 저해능도뛰어났다고보고하였다. 한편, 결장암유발원인의하나인결장내의산화적스트레스억제효과를조사한결과, L. casei strain Shirota로발효시킨탈지유에의해 2,2-diphenyl-1-picrylhydrazyl radical 소거활성이증가되었다고하였고, hamster에게 14일간발효탈지유를투여했을때가열처리한탈지유에비해 radical 소거능이높게나타났다고하였다 (23). L. casei, L. acidophilus, L. lactis는강력한 2,2-diphenyl-1-picrylhydrazyl, malonaldialdehyde, hydrogen peroxide radical 소거능이있고, 또한 linoeic acid 과산화억제효과는 L. casei 활성이가장높고, L. acidophilus 와 L. lactis 순으로항산화효과가나타났다고하였다 (13). 또한 L. mesenteroides ssp. cremoris, Lactobacillus jensenii ATCC 및 L. acidophilus ATCC 4356도높은 radical 소거능을보였는데, 이들의활성은단백질분해와관련이있으며, 높은항산화활성을가지는부분은 4-20 kda의분자량범위내에서많은펩타이드를가지며, 특히소수성아미노산의함량이더높게나타났다고알려져있다 (36). 유산균의 IC와 ICFE의환원력측정을위해 700 nm에서흡광도를측정한결과는 Table 4와같다. IC에의한환원력은 L. acidophilus 20와 L. mesenteroides 104에의해가장낮게나타난반면, L. plantarum 81의환원력은가장높게나타났는데이는 BHA와 vitamin C보다유의적으로더높게나타났으며, L. paracasei 74의환원력도 vitamin C와비슷한수준인것으로확인되었다. 한편, L. plantarum 81의 ICFE 환원력도실험균주중가장높았으며, 양성대조구보다도유의적으로높았다. 또한 L. acidophilus 20, L. paracasei Table 4. Reducing activity of various lactic acid bacteria isolated from the mustard leaf Kimchi Absorbance at 700 nm Intracellular Intact cells cell free extracts L. acidophilus ±0.050 a 0.80±0.023 a L. brevis ±0.015 b 0.49±0.009 b L. paracasei ±0.008 c 0.83±0.020 a L. plantarum ±0.011 d 1.66±0.031 c L. mesenteroides ±0.026 a 0.47±0.028 b BHA 1.09±0.045 e 1.09±0.045 d Vitamin C 0.81±0.010 c 0.81±0.010 a Data represent the means of three experiments±sd (error bars). a e Means with different letters in a column are significantly different at p<0.05 by Duncan s multiple range test.

7 유산균의항산화활성 381 Table 5. Fe 2+ chelating activity of various lactic acid bacteria isolated from the mustard leaf Kimchi Fe 2+ chelating activity (%) Intracellular Intact cells cell free extracts L. acidophilus ±2.7 a 20.6±1.3 a L. brevis ±7.8 b 18.9±3.6 a L. paracasei ±5.1 b 22.3±2.2 ab L. plantarum ±3.0 a 45.6±6.5 c L. mesenteroides ±4.3 b 27.4±1.9 b EDTA 92.9±2.4 c 92.9±2.4 d Fe 2+ chelating activity was defined as [1 A 522(sample)/A 522(control)] 100%. Data represent the means of three experiments±sd (error bars). a d Means with different letters in a column are significantly different at p<0.05 by Duncan s multiple range test. 74 및 L. plantarum 81들의환원력은 IC보다 ICFE에서더높았으나, L. brevis 55와 L. mesenteroides 104는 IC에서좀더높은환원력을나타내었다. Saide와 Gilliland (28) 에따르면, Lactobacillus delbrueckii ssp. lactis, L. delbrueckii ssp. bulgaricus, L. acidophilus 및 L. casei의항산화력은유산균의세포보다는세포추출물에서더높게나타났다고보고하였고, 이들세포추출물의항산화력과환원력사이에는명백한상관관계는없는것으로보고하였다. 하지만 Lee 등 (19) 이보고한 L. plantarum KCTC 3099는세포와세포추출물모두에서높은항산화력을보였다고하여본실험에서도다른균주에비해 L. plantarum의항산화력이다소높았다. 한편, L. acidophilus 606 세포에서얻어진수용성다당류로부터강력한항산화활성을나타내었다고보고하였다 (10). 유산균 IC와 ICFE의 Fe 2+ -chelating 활성을측정한결과는 Table 5와같다. L. acidophilus 20과 L. plantarum 81 IC의 Fe 2+ -chelating 활성은약 20-23% 정도였으나, L. brevis 55, L. paracasei 74 및 L. mesenteroides 104 IC는 30% 이상을나타내었다. 한편 ICFE의 chelating 활성은 L. acidophilus 20, L. paracasei 74 및 L. mesenteroides 104는모두 20% 이상이었으며, L. plantarum 81은 45% 이상의활성을나타내었다. 하지만실험균주모두양성대조구인 EDTA에비해 Fe 2+ -chelating 활성은유의적으로낮은수준이었다. Lin과 Yen (21) 는 L. acidophilus, L. bulgaricus, S. thermophilus 및 B. longum의 ICFE의항산화활성을조사한결과, S. thermophilus 821가가장높은 Fe 2+ chelating 활성을나타내었고, B. longum 15708은가장높은 Cu 2+ chelating 활성을보였다. 또한 L. plantarum KCTC 3099는 SOD 활성을거의나타나지않은반면, Fe 2+ (13.6 mg/l) 와 Cu 2+ (23.9 mg/l) 에대해선높은 chelating 활성을나타내었다고보고하였다 (19). Fe 2+, Cu 2+ 등의금속이온은생체내지질과산화물이나과산화수소생성을촉진하므로이들에대한 chelator는 Fe 2+ 와 hydrogen peroxide 시스템으로부터 hydroxyl radical 생성을 감소시키므로 chelating 활성이높을수록지방산화반응의촉매작용을감소시킬수있다 (33). 이상의결과에서미루어볼때, 갓김치에서분리된 5종의유산균모두 radical 소거능, 환원력및금속이온 chelating 활성을가지고있는것으로확인되었으나, 항산화력의크기는균주의종류에따라다소차이가있었으며, 실험균주중 L. plantarum 81은다른균주에비해 ROS에대한저항성이크고, radical 소거능및환원력이크므로이를이용한지방산화방지제혹은노화억제제로서의개발가치가있는것으로판단된다. 적요 갓김치로부터분리된유산균 5종 (Lactobacillus acidophilus 20, Lactobacillus brevis 55, Lactobacillus paracasei 74, Lactobacillus plantarum 81, 및 Leuconostoc mesenteroides 104) 의활성산소종에대한저항성및항산화활성을조사하였다. Hydrogen peroxide (0.5 mm) 와반응 5시간후 L. acidophilus 20, L. brevis 55, L. paracasei 74 및 L. plantarum 81들은 50% 이상의생존율을보였으며, superoxide anions에의한저해율은 L. acidophilus 20와 L. paracasei 74가가장낮았고, hydroxyl radical과의반응에서는 L. paracasei 74와 L. plantarum 81이가장안정하였다. 한편, L. plantarum 81는실험균주중가장높은 DPPH 소거능 (70.8±10.1%) 을나타내었는데, 세포 (IC) 보다는세포추출물 (ICFE) 의활성이더높았다. 또한 L. plantarum 81의 ICFE는다른균주들에비해높은 superoxide radical 소거능을보였고, 이균주의 IC와 ICFE에의한환원력은 BHA 와 vitamin C보다유의적으로더높게나타났으며, L. paracasei 74 IC의환원력도 vitamin C와비슷한수준으로나타났다. L. brevis 55, L. paracasei 74 및 L. mesenteroides 104 IC의 Fe 2+ -chelating 활성은 30% 이상이었고, L. plantarum 81의 ICFE는 45% 이상의활성을나타내었다. 따라서실험균주중 L. plantarum 81은다른균들에비해 ROS에대해비교적안정하고, 항산화활성도가장높은것으로확인되었다. 참고문헌 1. Akaike, T., K. Sato, S. Ijiri, Y. Miyamoto, M. Kondo, M. Ando, and H. Maeda Bactericidal activity of alkyl peroxyl radicals generated by heme-iron-catalyzed decomposition of organic peroxides. Arch. Biochem. Biophys. 294, Aoshima, H., H. Tsunoue, H. Koda, and Y. Kiso Aging of whisky increased 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. J. Agr. Food Chem. 52, Bai, J., A.M. Rodriguez, J.A. Melendez, and A.I. Cederbaum Over-expression of catalase in cytosolic or mitochondrial compartment protests Hep G2 cells against oxidative injury. J. Bio. Chem. 274, Barreto, J.C., G.S. Smith, N.H.P. Strobel, P.A. McQuillin, and

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