Curr. Top. Lact. Acid Bact. Probiotics 219;5(2):65-69 https://doi.org/1.35732/ctlabp.219.5.2.65 ISSN 2287-852X Note 추동훈 1 국무창 2,* 1 콜마비앤에이치, 2 배화여자대학교식품영양과 Physiological Activity of Lactobacillus Species Isolated from Kimchi Donghun Chu 1 and Moochang Kook 2,* 1 Kolmarbnh, 2 Dept. of Food & Nutrition, Baewha Women s University Received: Dec. 9, 219 Revised: Dec. 16, 219 Accepted: Dec. 18, 219 *Corresponding author : Moochang Kook Department of Food and Nutrition, Baewha Women s University, Seoul, 339, Korea. Tel: +82-2-399-765, E-mail: bmse153@gmail.com ORCID Donghun Chu https://orcid.org/-2-6719-6234 Moochang Kook https://orcid.org/-3-498-8298 Abstract According to the previous study, Lactobacillus pentosus A67 and L. plantarum subsp. plantarum C2 were isolated and screened. In this study, culture broths of strains A67 and C2 were performed various physiological activity assays including antimicrobial, antioxidant, cytotoxicity, cell proliferation activity. The remarkable anti-microbial activities using the culture broths of the two strains were showed by disc diffusion method, MIC tests. In the 2,2-diphenyl-1-picryl hydrazyl (DPPH) and superoxide dismutase (SOD) assays to verify antioxidant activity, culture broths of strains A67 and C2 significantly scavenged free radical maximum by 91.33% and 81.95% respectively compared with arbutin. Also, they showed equivalent activity same as 297.22 units and 255.56 units of SOD respectively. In RAW264.7 cells, culture broths of strain A67 and C2 showed no cytotoxicity. Moreover, they showed significant cell proliferative effects by 173.5% and 143.5% compared to control group respectively. Overall, this study showed that the culture broths of strain A67 and C2 had antimicrobial and antioxidant activities, which could synergically increase the physiological activities. And they had no cytotoxicity and cell proliferative effects. Therefore, it is expected that L. pentosus A67 and L. plantarum C2 could be applied to a wide variety of functional materials including food and medicine as new probiotics. Keywords Lactobacillus pentosus, Lactobacillus plantarum, physiological activity, toxicity, antimicrobial activity, antioxidant activity, probiotics 서론 유산균은발효과정동안당을분해하여유기산과박테리오신등의항균물질을생산함으로써식품의풍미 를향상시키고, 부패균의증식을억제하여식품의저장성을연장시킬뿐만아니라, 프로바이오틱스로서 장내 ph 의산성화를통한유해세균의증식을저해시켜장내균총을정상화시키는것으로알려져있다 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright Korean Society for Lactic Acid Bacteria and probiotics 65
추동훈 국무창 (Mercenier et al., 22). 또한유산균은유당분해효소를분비하여유당불내증을완화시키고, 장점막면역시스템의기능을강화시켜변비, 설사, 궤양성대장염이나과민성대장증후군을예방하는것으로알려져있으며, 면역증강, 항균, 항산화, 항염, 항암등의연구들이보고되어있다 (Mercenier et al., 22). 발효된김치는다양한유산균 (CFU/g) 을함유하고있으며, 김치내주요발효유산균은 Leuconostoc, Lactobacillus, Weissella 와 Pediococcus 속등이보고되어있다 (Hansongyi et al., 214). 그중에서 L. plantarum 은가장널리알려진김치내프로바이오틱스유산균으로, 가공및육가공산업의발효공정에대표적인유산균으로활용되고있으며, 항균활성, 과민성대장증후군과관련된설사또는변비증상완화등의장관련건강기능성에대한여러연구들이보고되고있다 (Vaughan et al., 26). 따라서본연구에서는선행된연구 (Chu and Kook, 219) 를통하여분리한 Lactobacillus pentosus A67과 L. plantarum subsp. plantarum C2의생리학적활성을확인하여우수성을검증하였다. 또한김치유래유산균의항산화활성을평가하기위하여 DPPH (2,2-diphenyl-1-picrylhydrazyl) 자유라디컬소거능시험 (Yoon et al., 214) 및 Superoxide dismutase (SOD) 유사활성시험 (Li et al., 212) 을수행하였으며, 시험균주의세포증식률에미치는영향을확인하기위해 RAW 264.7 세포주를이용하여검토하였다 (Moeslinger et al., 21). 결과및고찰 항균활성검토 L. pentosus A67과 L. plantarum C2의배양액을처리한한천배 재료및방법 세포주및지시균주식중독및염증유발균주에대한항균활성평가를위해미국세포주은행 (American Type Culture Collection (ATCC), USA) 과국립농업과학원미생물은행 (Korean Agricultural Culture Collection (KACC), Korea) 에서식중독과염증의원인이되는 8 종의지시균주 E. coli KCTC 2443, Bacillus cereus ATCC 14579, B. subtili subsp. spizizenii KCTC 375, Staphylococcus aureus KACC 1778, Salmonella enterica subsp. enterica KACC 1763, Candida tropicalis var. tropicalis KCTC 17762, C. glabata KCTC 7219, C. albicans KCTC 727을분양받았다. 각각의균은최적조건에서 24시간배양후시험에사용하였다. 또한 L. pentosus ATCC 841, L. plantarum ATCC 14917 은미국세포주은행 (American Type Culture Collection (ATCC), USA) 에서분양받아대조군으로사용하였고, 모든시험은 3회반복수행되었다. 생리활성검토김치유래유산균의항균활성을검토하기위하여식중독과염증의원인이되는 8종의지시균주에대하여한천배지확산법, 최소저해농도 (minimum inhibitory concentration; MIC) 시험법등을이용하여항균실험을수행하였다 (Clinic and Laboratory Standards Institute, 212, Fatima et al., 212). Fig. 1. Anti-microbial activity of L. pentosus A67 and L. plantarum C2 using disc diffusion test. Each of plates showed the size of clear zone after treating supernatant of culture broth for 1 day against various indicators. Left disc, L. pentosus A67; Right disc, L. plantarum C2. 66 https://doi.org/1.35732/ctlabp.219.5.2.65
지확산법실험결과 (Fig. 1), L. pentosus A67과 L. plantarum C2는 E. coli, S. enterica, B. cereus, B. subtilis, S. aureus 등 5주의지시균주에대해강한항균활성을보였으며, C. albicans, C. glabata, C. tropicalis 등진균류인 3주의지시균주에서는약한항균활성을보였다. L. pentosus A67과 L. plantarum C2는그람양성세균보다그람음성세균에서높은항균활성을보였으며, 진균류에서의항균활성은세균류에비하여낮은결과를보였다. 또한 L. pentosus A67과 L. plantarum C2의배양액에서각지시균주에대한생육저해최소농도를확인하였다 (Table 1). L. pentosus aa67과 L. plantarum C2의배양액은지시균주 8종의생육을대조균주 L. pentosus ATCC 841, L. plantarum ATCC 14917 보다각각더효과적으로저해하였다. 유산균은박테리오신, 과산화수소그리고유기산과같은항균물질을만들어낸다고보고되어있다 (Douglas et al., 23). 따라서본연구들을통해 L. pentosus A67과 L. plantarum C2는우수한항균물질을생성하여항균효과를보였고, 박테리오신이주된역할을하였을것으로판단된다. 항산화활성검토항산화활성을평가하기위한 DPPH (2,2-diphenyl-1- picrylhydrazyl) 자유라디컬소거능시험 (Yoon et al., 214) 및 Superoxide dismutase (SOD) 유사활성시험 (Li et al., 212) 을수행하였다. L. pentosus A67과 L. plantarum C2의 DPPH 자유라디컬소거능시험결과 (Fig. 2), 가장높은활성을보인 L. pentosus A67 상등액에서의 DPPH 자유라디컬소거율은 91.33% 로, 항산화물질로잘알려진 arbutin 1 µg/ml 에서의항산화활성 (67.6%) 보다 24.27% 높은활성을보였으며, L. plantarum C2에서의 DPPH 자유라디컬소거율은 81.95% 로, arbutin 1 µg/ml 에서의항산화활성 (67.6%) 보다 14.89% 더높은활성을보였다. 또한. L. plantarum C2의배양액은 297.22 units, L. plantarum A67의배양액은 255.56 units 의 SOD 활성과상응하는항산화활성을확인하였다 (Fig. 2). 따라서본연구들을통해항산화효과가뛰어난 L. plantarum A67 및 L. plantarum C2의섭취는체내산화적스트레스에대항하여활성산소의축적을막는데도움을줄것으로판단된다. 면역활성및세포독성검사시험균주의세포증식률에미치는영향을확인하기위해 RAW 264.7 세포주를이용하여검토하였다 (Moeslinger et al., 21). 실험결과, L. pentosus A67과 L. plantarum C2의배양액처리후의세포의수가대조군에비해각각유의적으로증가하였다 (Fig. 3). 배양액과동일한농도의유산균증균배지 (MRS) 는세포증식에영향을미치지않았고, 배양액의증식률은 L. pentosus A67의배양액 (B) 과 L. plantarum C2의배양액 (C) 에서각각 173.5%, 143.5% 의결과를얻었다. 유산균은면역을담당하는세포의분열 Table 1. Minimum inhibitory concentration (MIC) of L. pentosus A67 and L. plantarum C2 Indicator A67 MIC (μg/ml) L. pentosus L. plantarum B. cereus ATCC 14579 2.11 N.D 2.11 N.D B. subtilis subsp. spizienii KCTC 375 2.11 16.88 4.22 8.44 S. aureus KACC 1778 4.22 67.5 4.22 67.5 E. coli KCTC 2443 8.44 N.D 8.44 67.5 S. enterica subsp. enterica KACC 1763 16.88 N.D 16.88 N.D C. albicans KCTC 727 67.5 N.D 67.5 N.D C. glabata KCTC 7219 67.5 N.D 67.5 N.D C. tropicalis var. tropicals KCTC 17762 67.5 N.D 67.5 N.D ATCC 841 C2 ATCC 14917 The inoculation concentrations of indicators were CFU/mL from bacteria strains and CFU/mL from yeast strains. Those were cultured for 1 day at 3 C after treatment. N.D; not detected. Three independent experiments repeated. Curr. Top. Lact. Acid Bact. Probiotics 219;5(2):65-69 67
추동훈 국무창 (C) 4 (D) 3 SOD-like activity (units) 3 2 1 135 135 a b (μ g/ml) SOD-like activity (units) 2 1 25 25 a b (μ g/ml) Fig. 2. Antioxidation activity of L. pentosus A67 and L. plantarum C2. DHHP assay : (A), (B); Superoxide dismutase (SOD) like activity : (C), (D). (A), Supernatant compared with arbutin; (B), Cell debris compared with arbutin; (C), Supernatant correspond to SOD; (D), Cell debris correspond to SOD; Concentrations of sample were defined as 1, 1, 1 μg/ml of arbutin and 135 and 25 μg/ml by Bradford protein assay. Values are mean±standard deviations (SDs). a, L. pentosus A67; b, L. plantarum C2. * p<.5 indicates significant differences. Cell viability (% of Normal) 15 1 5 Nor Con.1 1 1.1 1 1 (%) D E Fig. 3. Cell proliferation and cell cytotoxicity by L. pentosus A67 and L. plantarum C2. The results were expressed as percent of proliferation rate and each bar showed the mean ± SD of the three independent experiments repeated. The RAW 264.7 cells were incubated for 24 h in each of samples as calculating increased viable cell from initial seeded 1.5 x cells/well. A-C were diluted by DMEM media. Normal, no treatment; A, supernatant of L. pentosus A67 (1 μg/ml); B, supernatant of L. plantarum C2 (1 μg/ml); C, MRS broth control (1 μg/ml). * p<.5 indicated significant differences. The RAW 264.7 cells were incubated for 24 h in the various concentration of supernatant of L. pentosus A67 and L. plantarum C2. Viable cell numbers, determined using the MTT assay, were represented as the relative percentages (viable cell number obtained from the untreated control cells is 1). Each bar showed the mean±sd of the three independent experiments repeated. Nor, LPS negative; Con, LPS positive; D, culture broth of L. pentosus A67; E, culture broth of L. plantarum C2. * p<.5 indicated significant differences. 68 https://doi.org/1.35732/ctlabp.219.5.2.65
및증식을촉진시켜서면역기능을활성화시키는기능 (Garcia 등, 214) 을갖는것으로보고되어있으며, L. pentosus A67과 L. plantarum C2의배양액이면역을담당하는 RAW 264.7 대식세포 (macrophage cell) 를활성화시킨것으로사료된다. 또한유산균배양액의안전성을확인하기위해 RAW 264.7 세포주를이용하여세포독성을확인하는 MTT 시험을수행하였다 (Sylvester 등, 211). 실험결과, L. pentsosus A67의배양액은 1% 에서도 91.3% ± 2.77% 의생존율을보였고, L. plantarum C2의배양액에서도 1% 에서 91.78% ± 3.22% 의생존율을보였다 (Figure 3). 따라서두유산균배양액을 1% 까지처리하였음에도불구하고대조군과비교하였을때 1% 이상의유의적인세포독성이없었으므로 L. pentsosus A67과 L. plantarum C2 배양액의안전성을확인할수있었다. 요약 김치에서분리한 L. pentosus A67과 L. plantarum C2는그람양성세균보다그람음성세균에서높은항균활성을보였으며, 진균류에서의항균활성은세균류에비하여낮은결과를보였고, 각지시균주에대한생육저해최소농도를확인하였다. 뿐만아니라 DPPH 시험에서두균주의배양액이각각 91.33%, 81.95% 의라디칼소거능으로양성대조군인 arbutin 보다높은항산화활성을보였으며, SOD 유사활성시험에서는두균주의배양액이각각 255.56 units, 297.22 units 의 SOD와상응하는항산화활성을보였다. 또한 L. pentosus A67과 L. plantarum C2의배양액은 RAW 264.7 세포에서독성을보이지않았다. L. pentosus A67와 L. plantarum C2는장내환경에생존하기적합하고, 같은종의균주보다우수한내산성능및내담즙성능을가진프로바이오틱스이자다양한계열의항생제에대한우수한내성을가진프로바이오틱스로보고되어있다 (Chu and Kook, 219). 따라서, L. pentosus A67과 L. plantarum C2는동일한종의균주들보다우수한항산화및항균활성등의생리학적활성을보유하고있는안전하고유용한기능성프로바이오틱스로판단되며, 추가적인연구가요구된다. 감사의글 이논문은 219 년도과학기술정보통신부의재원으로한국연구재단의지원을받아수행된기초연구사업임 (NRF-219R1F1A1 58399). References 1. Ann YG (211) [Lactic acid bacteria] Probiotic lactic acid bacteria. Korean J. Food & Nutr. 24, 817-832. 2. Casado MMC, Benomar N, Ennahar S, Horvatovich P, Lavilla LL, Knapp CW, Gálvez A, and Abriouel H (216) Comparative proteomic analysis of a potentially probiotic Lactobacillus pentosus MP-1 for the identification of key proteins involved in antibiotic resistance and biocide tolerance. Int. J. Food Microbiol. 222, 8-15. 3. Chu D and Kook MC (219) Probiotics characterization of Lactobacillus species isolated from Kimchi. Curr. Top. Lact. Acid Bact. Probiotics 5(1), 33-37. 4. Francoise B, Anna C and Daniel KO et al. (25) Lactobacillus plantarum subsp. argentoratensi subsp. nov., isolated from vegetable matrices. Int. J. Syst. Evol. Microbiol. 55, 1629-1634. 5. García RA, González LD and Esteban FA et al. (214) Assessment of probiotic properties in lactic acid bacteria isolated from wine. Food Microbiol. 44, 22-225. 6. Georgieva R, Yocheva L and Tserovska L et al. (215) Antimicrobial activity and antibiotic susceptibility of Lactobacillus and Bifidobacterium spp. intended for use as starter and probiotic cultures. Biotechnol. Biotechnol. Equip. 29, 84-91. 7. Gueimonde M and Salminen S (26) New methods for selecting and evaluating probiotics. Dig. Liver Dis. 38, S242-S247. 8. Radulović Z, Miočinović J and Mirković N et al. (217) Survival of spray-dried and free-cells of potential probiotic Lactobacillus plantarum 564 in soft goat cheese. Anim. Sci. J. 88, 1849-1854. 9. Saarela M, Mogensen G and Fonden R et al. (2) Probiotic bacteria: safety, functional and technological properties. J. Biotechnol. 84, 197-215. 1. Sahadeva RPK, Leong SF and Chua KH et al. (211) Survival of commercial probiotic strains to ph and bile. Int. Food Res. J. 18, 1515-1522. 11. Tulumoglu S, Yuksekdag ZN and Beyatli Y et al. (213) Probiotic properties of Lactobacilli species isolated from children s feces. Anaerobe. 24, 36-42. Curr. Top. Lact. Acid Bact. Probiotics 219;5(2):65-69 69