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1 Microbiol. Biotechnol. Lett. (2015), 43(3), pissn X eissn Microbiology and Biotechnology Letters 세균배양액과반응한 Lactobacillus plantarum 의생장속도및항균활성에대한연구 하은미 * 대구가톨릭대학교약학대학 Received: May 8, 2015 / Revised: June 9, 2015 / Accepted: June 9, 2015 Growth Rate and Antimicrobial Activity of Lactobacillus plantarum Reacting with Bacterial Culture Supernatant Eun-Mi Ha* Department of Pharmacology, College of Pharmacy, Catholic University of Daegu, Daegu , Republic of Korea In this study, L. plantarum, when reacting with the culture media of potential pathogenic bacteria, exhibited an increase in growth rate and antimicrobial activity. In order to examine the characteristics and the nature of the reaction with the bacteria, this study carried out experiments involving culturing the test bacteria in M9 minimal media. Subsequently, the supernatant was incrassated by the decompression-drying method. Through colony forming unit assay, it was confirmed that L. plantarum had the function of growth inhibition to various bacteria. After culturing L. plantarum with bacterial media, the growth rate of L. plantarum was measured by absorbance (OD 600 ), the results showed that the growth rate (E. coli treatment group: OD 600 = 0.848, S. typhimurium treatment group: OD 600 = 0.848) increased, as compared with the non-treated control group (OD 600 = 0.48). In contrast, the concentrate itself did not induce the growth of L. plantarum. These results were observed as a universal phenomenon of the Lactobacillus species. Moreover, the increase in antimicrobial activity was observed in L. plantarum, which reacted with the culture media of E. coli and S. typhimurium, through a disc diffusion assay, and the result of growth inhibition against various bacteria was induced. Finally, based on the analysis results of the characteristics of bacteria culture media, which increased the growth rate of L. plantarum and antibacterial activity, the bacterial media had a tolerance for catabolic enzymes, ph 2 8 and heat. Therefore, this substance can be said to be a small molecule which is highly stable under various conditions. Keywords: Lactobacillus plantarum, growth rate, antimicrobial activity, pathogen, M9 minimal media 서 론 인간의위장관 (gastrointestinal tract) 은매우복잡한장내균총 (gut microbiota) 으로이루어져있으며 [6, 32], 장내균총은영양소합성, 소화, 병원균정착저해및항생능력, 숙주의선천성및후천성면역시스템의성숙과증진등과같은인간의생리기능에있어매우중요한역할을담당하고있다 [13, 14, 37]. 또한장내균총은조절 T 세포 (regulatory T cells) 를유도함으로써 T helper 1 (Th1) cells과 T helper 2 (Th2) cells의균형을조절하여면역학적관용과면역활성에기여한다 [5, 33]. *Corresponding author Tel: , Fax: haem@cu.ac.kr 2015, The Korean Society for Microbiology and Biotechnology 그런데장내균총의정착및발달은유아기 (infancy) 부터시작되고태아기의환경, 출산시기, 출산방법 ( 자연분만혹은제왕절개 ), 수유방법 ( 모유혹은분유 ), 프로바이오틱스 (probiotics) 및항생제의복용에따른환경적요인과유전자유형등에따라결정된다 [8, 15]. 정상적인분만과정을겪은유아와건강한영 유아의장 (gut) 에는유익균으로알려진 Bifidobacterium과 Lactobacillus가우세하게발견되며, 병원성균은적거나발견하기가어렵다 [4]. 하지만반대로출생과정중에겪게되는조기분만, 제왕절개, 또는분유섭취와항생제처방과같은환경적요인은병원균의감염을초래한다 [2, 25, 35, 38]. 이때유아기의장내에가장먼저침입하는잠재적병원성세균은 Escherichia coli, Streptococcus, Enterococcus, Staphylococcus, Klebsiella와 Clostridium 속에해당하는균들이며 [11, 39], 이러한세균에감염된장에서는 Bifidobacterium과 Lactobacillus가극히적게발견 September 2015 Vol. 43 No. 3

2 188 Ha, Eun-Mi 된다 [3, 11]. 특히초년시기에정착한균총의유형은유년기병원균의감염여부와환경적위험요인 (environmental antigen) 에따라그감수성에영향을미치고 [16, 17], 궁극적으로성인시기의장내균총을결정하게된다 [23, 29]. 장내균총의불균형은유아기의면역연관질병 ( 알레르기관련질환, 아토피피부질환, 염증성장질환, 과민성대장증후군, 신생아괴사성장염, 제1형당뇨병등 ) 을일으키는요인 [1, 9, 17, 18, 31] 이되기도하는데, 따라서유아기의장내균총형성과정은매우중요하며, 궁극적으로는생애전반의건강과삶의질과밀접하다고할것이다 [20]. 이러한이유로최근에는면역연관질병의예방및치료를위해 probiotics 가유용하게활용되는경향을보이고있다 [21]. 또한 in vitro 와 in vivo로진행된최근연구는유아기에 Lactobacillus plantarum (L. plantarum) 을이용한 probiotics 복용이장점막장벽의안정화, 병원균으로부터의방어능력향상, 항생물질분비및알레르기질환완화에대한효과를보고하고있다 [10, 24, 30, 34]. 최근수년간소아염증질환의치료목적으로사용한 probiotics 및 synbiotics의주요균종은 Bifidobacterium와 Lactobacillus인데, 특히 L. plantarum 은 probiotics 처방후, 영유아의장에 10일내에안정적으로정착하지만, Bifidobacterium spp. 은 30일이상소요된다는결과가보고되고있다 [12]. 따라서 Lactobacillus은유아기에병원균보다먼저장내에우점종으로서정착해야면역시스템의형성과발달을결정짓고항생물질의분비를통한방어시스템이가능하지만 [28, 36], 아직 Lactobacillus가장의우점종으로정착하기위한환경적요인과항생물질분비조절에대한이해는부족한실정이다. 이에본연구는 Lactobacillus spp. 를병원성세균의배양배지와반응시켰을때 Lactobacillus spp. 의생장속도를알아보고자한다. 이를통하여증가된생장속도가항생물질의활성을유도함으로써항균효과가증대시킬것이라는연구문제를증명하고자하며, 동시에 L. plantarum의생장속도및항균물질활성의증가를유도하는병원균의배양액물질에대한특성을검토하고, 분해효소, ph와열에강한소분자물질이라는가능성을확인하고자한다. 재료및방법 사용균주의배양조건본실험에서사용한 Lactobacillus는항생제내성균주은행 (CCARM, Seoul Women s University) 에서분양을받았으며, 종류는 L. plantarum (CCARM 0067), Lactobacillus brevis (CCARM 0061), Lactobacillus delbrueckii subsp. Lactis (CCARM 0106), Lactobacillus acidophilus (CCARM 0059) 이다. 이들 Lactobacillus는 Difco TM Lactobacilli MRS broth (Becton Dickinson, USA) 배지를사용하여 37 o C 에서 24 시간동안정치배양하였다. Lactobacillus spp. 의생장속도와항균능력을관찰하기위해사용된잠재적병원성세균은 Escherichia coli (ATCC 23736), Pseudomonas aeruginosa (ATCC 29336), Salmonella typhimurium (CCARM 0125), Staphylococcus aureus (CCARM 3709) 이며, Difco TM Nutrient broth (Becton Dickinson, USA) 의배지를사용하여 37 o C에서 24시간동안배양하였다. 이병원성세균들은항생제내성균주은행과한국미생물보존센터 (KCCM, Seoul, Korea) 에서분양받았다. 균배양액의농축균의정상배지에는많은영양요소가함유되어있으며, 균의생장에필요한환경을제공하기때문에, 이러한환경적요인을배제하기위해최소배지인 M9 minimal media를선택하고균을배양하였다. 뿐만아니라실험을통해균이분비한물질에서효과가있음을확인하였고, 정상배지의요소를배재하고균이분비한물질의효과에초점을두고실험하기위해 M9 minimal media를선택했다. 시험균주가배양된상층액은균일한농도로실험하기위해농축하여분말화한후수용성인부분만취하였고자세한방법은아래에기술하였다. Lactobacillus M9: MRS 배지에키운 OD 600 =8의 Lactobacillus spp. 균은 M9 minimal broth(mb cell) 의 1L에 5% (v/v) 로접종하여 37 o C에서 36 48시간동안정치배양한다. OD 600 =1의 Lactobacillus를 10,000 g, 10분간원심분리하여 pellet을제거한후상층액을취한다. 이상층액은 10 N NaOH를이용하여 M9 배지의 ph를 사이로맞춘다 μm filter (Millipore, USA) 로잔여의균을제거하고감압건조하여분말화한다. 이분말의무게 (Dry cell weight, DCW) 는 0.5 g/l로서 ultrapure water 5 ml에녹여최종농도를 0.1 mg/ml로준비한다. 이농축액을 Lactobacillus (spp.) M9이라한다. 세균 M9: Nutrient broth 배지에서키운병원성세균들은 OD 600 =1까지키우고, M9 minimal Broth의 1 L에 1% (v/v) 로접종하여 37 o C에서 24시간동안배양한다. 이와같은조건에서 OD 600 =1의균을 10,000 g, 10분간원심분리하여 pellet을제거한후상층액을취한다. 이상층액은 0.22 μm filter (Millipore, USA) 로제균후감압건조하여분말화한다. 이분말의무게는 1 g/l로서 ultrapure water 10 ml에녹여최종농도를 0.1 mg/ml로준비한다. 이농축액을세균 M9 bacterial M9) 이라한다. 세균 M9과반응시킨 Lactobacillus M9: 농축된세균 M9이처리된 Lactobacillus (spp.) M9으로서 MRS에서배양된 Lactobacillus 균주를 M9에접종할때세균 M9의 40 μg

3 Biological Activity of L. plantarum Induced by the Bacteria 189 을동시첨가하여 37 o C에서 36 48시간동안정치배양하고위와설명한동일한방법으로분리한다. 이농축액을세균 M9 + Lactobacillus M9이라고표현한다. 대조군 M9: M9 minimal broth의 1 L를감압건조하여 ultrapure water의 10 ml에녹여대조군처리시약으로사용한다. 이농축액을 M9이라고한다. 세균 M9 과반응시킨 Lactobacillus spp. 의생장속도측정 Lactobacillus 의 OD 600 = 0.1 (M9, 1 ml) 에각세균 M9 을 40 μg 씩처리한후 37 o C 에서 3, 6, 12 시간동안배양하는동안 spectrophotometer (Optizen nano bio, Mecasys, Korea) 로 600 nm 에서흡광도를측정하여생육곡선을도출하였다. 실험은 3 회이상실시하고, 평균값을분석하였다. 항균활성측정 Colony Forming Unit (CFU) assay: Lactobacillus (spp.) M9을이용하여항균활성을측정하였다. 측정은시험균주의 OD 600 = 0.1(Nutrient broth의 1 ml) 에 Lactobacillus spp. M9을 10, 25, 50 μg의농도로처리하여 37 o C에서 24 시간동안반응시킨후 Nutrient agar plate에도말하여병원균의생균수 (CFU/ml) 를측정하여확인하였다. 실험은 3회평가평균값을결과로도출하고, 대조군의 CFU를 100% 으로하여상대적인값을분석하였다. Disc diffusion assay: Lactobacillus M9의항균활성은시험세균을대상으로 disc diffusion assay[19] 로측정하였다. 병원균은 Nutrient broth에접종하여 37 o C에서 24시간동안배양한후, 각각의균은 OD 600 = 0.1, 100 μl를 Nutrient agar 평판배지에도말한다. Lactobacillus M9의 25 mg가함유된멸균 disc paper( 지름 5 mm, Whatsman No.2) 를시험세균이도말된평판배지에올린후 37 o C에서 24시간동안배양하였다. 결과는생육저해환의크기를측정하여항균활성을평가하였다. 대조군으로서는 ampicillin의 5 mg/disc를사용하였으며, 생육저해환의크기는지름 mm 단위로측정하여, 3회평가평균값을결과로도출하였다. 세균 M9 의특성분석 L. plantarum의생장속도와항균활성의증가를유도하는세균 M9 유래물질의특성을분석하기위해 S. typhimurium M9 (St M9) 을준비하고아래와같은조건으로반응시켰다. (1) 각종효소에대한영향을측정하기위해 St M9의 40 μg에 α-amylase (EC Type VIII, Sigma), lipase (EC Type VII, Sigma), lysozyme (EC , Sigma), proteinase K (EC , Sigma) 를 2 mg/ml의농도로첨가하여 37 o C에서 30분동안반응시켰다. (2) St M9의 ph 안정성을측정하기위해 ph 은 0.1 M glycine-hcl 완충용액, ph 은 0.1 M sodium acetate 완충용액, ph 은 0.1 M sodium phosphate 완충용액, ph 은 Tris-HCl 완충용액을사용하여 St M9의 ph를조절하고, 37 o C에서 12시간동안반응하였다. (3) 온도안정성에대한측정은 St M9를 95 o C에서 30분간반응한후항균물질의잔존활성을측정하였다. 각각의요소로처리한 St M9는배양중인 L. plantarum에첨가하여반응시키고 L. plantarum M9를분리하여시험균주에대한항균활성을생균수를측정하여확인하였다. 통계분석모든실험의측정값은평균 ± 표준편차로나타냈고, SPSS 20.0 (SPSS, Chicago, IL, USA) 프로그램으로통계처리하였다. 유의한차이 (p <0.05) 는일원배치분산분석법 (one-way ANOVA) 의 Turkey 다중범위검정 (Turkey s multiple range test) 으로결정하였다. 결과및고찰 Lactobacillus spp. 의항균활성 Lactobacillus spp. 균주로부터 Lactobacillus spp. M9 농축액을확보하고 25 μg을 Escherichia coli (E. coli) 에처리하여 CFU assay를시행하였다. 사용한 Lactobacillus spp. 는 Lactobacillus plantarum (L. plantarum), Lactobacillus brevis (L. brevis), Lactobacillus delbrueckii subsp. Lactis (L. lactis), Lactobacillus acidophilus (L. acidophilus) 이다. 실험결과는대조군 M9을처리한 E. coli의 CFU를 100% 로설정하고, 상대적인값을측정하여비교하였다. L. brevis M9가처리된 E. coli의평균 CFU는 42.98%, L. lactis M9 처리군은 33.37%, L. acidophilus M9 처리군은 61.14% 였고, L. plantarum M9 처리군의평균값은 12.14% 였다. 그러므로본실험으로확보한 Lactobacillus spp. M9 에는 Lactobacillus spp. 유래항균물질이존재함을확인할수있었으며, L. plantarum M9은다른 Lactobacillus spp. M9과비교하여항균활성이가장높은것으로확인되었다 (Fig. 1). 이러한결과를바탕으로본연구는항균활성이가장높은 L. plantarum M9를선택하여그특성을연구하였다. 다양한세균에대한 L. plantarum M9의항균능력 L. plantarum M9의농도에따른살균능력을확인하기위하여사용된표적시험균주는 Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Salmonella typhimurium (S. typhimurium), Staphylococcus aureus (S. aureus) 이다. 실험에적용된 L. plantarum M9의농도는각각 10, 25, September 2015 Vol. 43 No. 3

4 190 Ha, Eun-Mi Fig. 1. Antimicrobial activity of Lactobacillus spp. against E. coli. M9: M9 only treatment, Lp: Lactobacillus plantarum M9, Lb: Lactobacillus brevis M9, Ll Lactobacillus delbrueckii subsp. lactis M9, La: Lactobacillus acidophilus M9. CFU of Escherichia coli (E. coli) is expressed as a relative value based on 100% of the treated M9 only into the each sample. Data are mean ± SD (Standard Deviation) from triplicate determinations and the significant difference of means was estimated by Turkey s multiple range test (p < 0.05). 처와스트레스를유발하고염증을일으키는 endo-, entero-, exo-toixin 및 enzyme 등을분비한다 [7, 22, 26, 27]. 본연구에서는 L. plantarum 의항균능력에대항하는병원균의독성활성을검토하였다. 즉, L. plantarum 에대한세균 M9 의살균능력을확인하였다. 이실험을위해 E. coli, P. aeruginosa, S. typhimurium, S. aureus의 M9을확보하고각세균 M9 의 40 μg을 L. plantarum에처리하여함께배양한후, L. plantarum의생장속도를확인하였다. 흥미롭게도모든세균의 M9은 L. plantarum의생장속도를증가시켰다. 세부적으로는, M9 대조군 (OD 600 = 0.48, 6시간배양기준 ) 과비교하여, E. coli M9 처리군 (OD 600 = 0.848), S. typhimurium M9 처리군 (OD 600 = 0.85), P. aeruginosa M9 처리군 (OD 600 = 0.654) 과 S. aureus M9 처리군 (OD 600 = 0.721) 이모두 L. plantarum 의확연한생장증가를유도하였다 (Fig. 3). 반면 L. plantarum M9 를처리한실험군에서는 L. plantarum (OD 600 =0.47) 의생육증가가관찰되지않았다. 그러므로 L. plantarum 은세균배양액을특이적으로인지하고생장속도를증가시킴으로써병원균에대한방어를준비하는것으로추측할수있다. 이결과를바탕으로본연구에서는세균 M9 의처리실험에서가장확연한결과를보였던 E. coli 와 S. typhimurium 의 M9 을사용하여추후실험을진행하였다. S. typhimurium M9 와반응한 Lactobacillus spp. 의생장증가유도 S. typhimurium M9 이처리된 L. plantarum 의생장증가효과가 Lactobacillus spp. 의보편적현상인지를확인하기위하여 Lactobacillus spp. 의생육증가를확인하였다. 즉, S. Fig. 2. Concentration-dependent antibacterial activity of L. plantarum M9 against the various bacteria. CFU of bacteria is expressed as a relative value based on 100% of the treated M9 only into the each sample. Ec: Escherichia coli, Pa: Pseudomonas aeruginosa, St: Salmonella typhimurium, Sa: Staphylococcus aureus. Data are mean ± SD from triplicate determinations and the significant difference of means was estimated by Turkey s multiple range test (p <0.05). 50 μg 으로선정하였다. 실험을통하여농도의존적인항균능력이확인되었으며 (Fig. 2), 다양한균에대한 L. plantarum M9 의생장억제기능을확인하였다. 이결과를바탕으로 L. plantarum M9 의항균활성관련실험은그농도를 25 μg 으로고정하여연구를진행하였다. 세균 M9 이처리된 L. plantarum 의생장속도증가일반적으로장내침입한유해세균은숙주의장세포에상 Fig. 3. Increase the growth rate of L. plantarum by bacterial M9. Each M9 was treated to L. plantarum and the absorbance (OD 600 ) of L. plantarum was measured after 3, 6 and 12 h. M9: M9 only treatment, Ec: Escherichia coli M9, Pa: Pseudomonas aeruginosa M9, St: Salmonella typhimurium M9, Sa: Staphylococcus aureus M9, Lp: Lactobacillus plantarum M9. The data represent average values of three independent determinations.

5 Biological Activity of L. plantarum Induced by the Bacteria 191 Table 1. Increase the antimicrobial activity of L. plantarum through the stimulation of bacterial M9. Growth inhibition zone (mm) Condition Sample Target strain Ec Pa St Sa Bacterial M9 + Lp M9 Cont M9 + Lp Ec + Lp St + Lp Antibiotics Ampicillin Fig. 4. Increase the growth rate of the Lactobacillus spp. through the reaction of S. typhimurium M9. M9: M9 only treatment, St M9: S. typhimurium M9 treatment to the Lactobacillus spp. Lp: Lactobacillus plantarum, Lb: Lactobacillus brevis, Ll: Lactobacillus delbrueckii subsp. lactis M9. La: Lactobacillus acidophilus. CFU of Lactobacillus spp. is expressed as a relative value based on 100% of the treated M9 only into the each sample. Data are mean ± SD from triplicate determinations and the significant difference of means was estimated by Turkey s multiple range test (p < 0.05). typhimurium M9의 40 μg을 Lactobacillus spp. 에처리하여함께배양한후 Lactobacillus spp. 의 CFU를분석하였다. 실험결과, M9이처리된대조군을 100% 라고할때, S. typhimurium M9을처리한 L. plantarum의 CFU는 503%, L. brevis는 266%, L. lactis는 275%, L. acidophilus는 206% 로서균주모두 S. typhimurium M9에의해생장속도가증가한것으로관찰되었고, 특히 L. plantarum의증가량이가장큰것으로나타났다 (Fig. 4). Cont.: M9 only treatment, M9 + LP: L. plantarum M9 incubated with 40 µg of M9 only, Ec + Lp: L. plantarum M9 incubated with 40 µg of E. coli M9, St + Lp: L. plantarum M9 incubated with 40 µg of S. typhimurium M9. Ec: Escherichia coli, Pa: Pseudomonas aeruginosa, St: Salmonella typhimurium, Sa: Staphylococcus aureus. : No inhibition. The concentration of the M9 sample used was 25 µg/disc and ampicillin was 5 mg/disc, respectively. The growth inhibition zone was included of size of disc paper. The data represent average values of three independent determinations. plantarum M9 의항균활성은 L. plantarum M9 자체와비교하여큰생육저해환이관찰되었다 (Table 1). 이실험을통해세균 M9 의자극은 L. plantarum 의생장과항균활성을촉진한다는것을관찰할수있었다. 이결과를통해세균은 L. plantarum 의생장및항균능력을자극하는유도물질이있음을암시하며, Lactobacillus 는병원균유래물질을인지함으로써항균능력이향상되는것으로추정된다. 그러므로본연구에서는세균 M9 내에존재하는 L. plantarum 의자극유도물질에관심을갖고그특성을분석하였다. 세균 M9과반응한 L. plantarum의항균활성증가이상의결과를종합해볼때, 다양한세균을비롯한 S. typhimurium M9은 L. plantarum의생장속도를증가시키는것으로관찰되었다 (Fig. 3, 4). 이를바탕으로본연구는 S. typhimurium M9의자극이 L. plantarum 생장증가뿐만아니라항균활성에도영향을주는지확인하기위해아래와같은실험을진행했다. M9만세균에처리한대조군 (Cont.), M9을처리한 L. plantarum M9 (M9+Lp), E. coli M9과반응시킨 L. plantarum M9 (Ec + Lp), S. typhimurium M9과반응시킨 L. plantarum M9 (St + Lp) 을시험균인 E. coli, P. aeruginosa, S. typhimurium, S. aureus 에처리하여각균의생육저해환정도를측정하였다. L. plantarum M9 은모든시험세균에대한항균활성을보였고, E. coli M9 과 S. typhimurium M9 와반응시킨 L. 세균 M9 의특성분석 L. plantarum 의생장과항균능력을유도하는세균 M9 의특성을파악하기위해분해효소, ph 와열에대한안정성을조사하였다. 실험에사용된세균 M9 은 S. typhimurium 이며, 본연구에서사용되었던다른세균의 M9 도유사한특성이관찰되었다 (data not shown). M9 과반응시킨 L. plantarum M9 (M9 + Lp), S. typhimurium M9 와반응시킨 L. plantarum M9 (St + Lp), 분해효소, ph 와열을각각 S. typhimurium M9과반응시킨후, 이 S. typhimurium M9을처리한 L. plantarum M9 (Treated St + Lp) 를준비하고, 이들 M9을 E. coli에처리하여항균활성을 CFU로확인하였다 (Fig. 5). 결과는다음과같다. 첫번째, 세균 M9의활성이분해효소 (α-amylase, lipase, lysozyme, proteinase K) 에저해되는지확인하였다. M9 + Lp (E. coli CFU: 100%) 보다 St + Lp (E. coli CFU: 19.07%) September 2015 Vol. 43 No. 3

6 192 Ha, Eun-Mi Fig. 5. Characteristics of bacterial M9 as the potential growth and antimicrobial inducer for L. plantarum. Antibacterial activity (CFU) of L. plantarum M9 was referred only against E. coli. M9 + Lp: L. plantarum M9 incubated with 40 µg of M9 only, St + Lp: L. plantarum M9 induced with 40 µg of S. typhimurium M9, Treated St + Lp: L. plantarum M9 induced with 40 µg of pre- treated S. typhimurium M9 by enzymes, ph and heat. +: The sample is added, : The sample-free. Data are mean ± SD from triplicate determinations and the significant difference of means was estimated by Turkey s multiple range test (p < 0.05). 의항균능력이높은것으로관찰되며, 각종효소를처리한 St + Lp (Treated St + Lp 처리군의 CFU; α-amylase 처리 : 22%, lipase 처리 : 18%, lysozyme 처리 : 24%, proteinase K 처리 : 18.2%) 의항균활성도 St + Lp와유사하게보존되었다. 그러므로세균 M9은다양한분해효소에대해안정한것으로나타났다. 두번째, ph의영향을알아보기위해다양한 ph 범위에서반응시킨 S. typhimurium M9의특성을분석하였다. ph 2 8의산도에서 St + Lp와비슷한생육저해현상 (CFU: 22%) 이관찰된것으로미루어, 세균 M9은넓은 ph 구간에서매우안정함을알수있었다. 세번째, 온도에대한안정성을검토하기위해 95 o C에서 30분간반응시킨후항균실험을실시했다. 열처리된 S. typhimurium M9 역시 L. plantarum의항균능력 (CFU: 20%) 을유도하였으며, 이결과는 S. typhimurium M9의내열성을제시한다. 추가로다양한세균 M9의첨가시증가된항균활성을생육저지환의결과로도출하고그결과사진은 Fig. 6에나타내었다. 따라서이상의결과는세균 M9에존재하는분비대사체중에서 L. plantarum의생장과항균능력에영향을주는물질이존재하며, 이물질은다양한조건에서도매우안정성을보유한 small molecule일가능성을제시한다. Lactobacillus 는유아기에병원균보다먼저장내에우점종으로서정착하는것이중요한만큼, 병원균의침입을빠르게인지하여생 Fig. 6. Photography of cell growth inhibition in L. plantarum M9 incubated with bacterial M9 against E. coli and S. typhimurium. 1: M9 control, 2: L. plantarum M9 incubated with 40 µg of M9 only, 3: L. plantarum M9 incubated with 40 µg of E. coli M9, 4: L. plantarum M9 incubated with 40 µg of S. typhimurium M9, 5: L. plantarum M9 induced with 40 µg of heat-treated S. typhimurium M9. The concentration of the sample used was 25 µg/ disc, respectively. The antimicrobial activity was demonstrated by clear zone of growth inhibition around the paper disc. 장속도를조절함으로서항생물질의분비를통한방어시스템을가동해야한다. 아직까지 Lactobacillus 가장의우점종으로정착하기위한환경적요인과항생물질분비조절에대한이해는부족한실정이므로본연구는이질문에대한대답을제공했다고사료된다. 뿐만아니라 probiotics 는항생제의기능을가짐과동시에항생제의단점인잔류량및내성문제가없기때문에병원성미생물을효과적으로억제할수있다면현재로서는가장바람직한대안이다. 그러므로본연

7 Biological Activity of L. plantarum Induced by the Bacteria 193 구를통해관찰된세균감염에의존적인 Lactobacillus 의항균활성증가는관련산업개발연구에도움을줄수있을것으로사료된다. 또한향후 Lactobacillus 의생장속도와항균능력을유도하는세균유래물질의발굴은 probiotics 의활용효과를기대할수있을것이다. 요 약 인간의위장관은매우복잡한장내균총으로이루어져있으며, 장내균총은인간의생리기능에있어매우중요한역할을담당하고있다. 특히유아기의장내균총의불균형은면역연관질병을일으키는요인이며치료목적으로 probiotics 가유용하게활용되고있다. Probiotics 의대표균종은 L. plantarum 으로서영유아기에병원균보다가장먼저정착해야면역질환을예방할수있다. 그러나아직 Lactobacillus 가장의우점종으로정착하기위한환경적요인과항생물질분비조절에대한이해는부족한실정이다. 이에본연구는세균의배양액과반응한 L. plantarum 은생장속도와항균활성이증가되는것을관찰하였다. 본연구는균의특성과반응을살펴보기위해시험균을 M9 minimal media 에배양한후상층액을감압건조방법으로농축하여실험을진행하였다. Colony forming unit assay 방법을통해, 다양한잠재적병원성세균에대한 L. plantarum 의생장억제기능을확인하였다. 세균을 L. plantarum 에처리하여함께배양한후, L. plantarum 의생장속도를흡광도 (OD 600 ) 로측정한결과, 처리하지않은대조군 (OD 600 =0.48) 과비교하여생장속도 (E. coli 처리군 : OD 600 = 0.848, S. typhimurium 처리군 : OD 600 = 0.848) 가증가되었다. 반면 L. plantarum 농축액자체는 L. plantarum 의생육증가를유도하지않았다. 이러한결과는 Lactrobacillus species 의보편적현상으로관찰되었다. 뿐만아니라 E. coli 와 S. typhimurium 의배양액과반응한 L. plantarum 은항균활성이증가되었으며, disc diffusion assay 를통해다양한세균에대한생육저해결과가관찰되었다. 마지막으로, L. plantarum 의생장속도와항균활성을증가시킨세균배양액의특성을분석해본결과, 병원균은분해효소 (a-amylase, lipase, lysozyme, proteinase K), ph 2 8 와열 (95 o C) 에내성이있음이관찰되었으므로, 이물질은다양한조건에서도매우안정한 small molecule 일가능성을제시한다. Acknowledgments This work was supported by research grants from the Catholic University of Daegu in 2013 (Project No ). References 1. Bjorksten B, Naaber P, Sepp E, Mikelsaar M The intestinal microflora in allergic Estonian and Swedish 2-year-old children. Clinical and experimental allergy. J. Allergy Clin. Immunol. 29: Blakey JL, Lubitz L, Barnes GL, Bishop RF, Campbell NT, Gillam GL Development of gut colonisation in pre-term neonates. J. Med. Microbiol. 15: Butel MJ, Suau A, Campeotto F, Magne F, Aires J, Ferraris L, et al Conditions of bifidobacterial colonization in preterm infants: a prospective analysis. J. Pediatr Gastroenterol. Nutr. 44: Duncan SH, Flint HJ Probiotics and prebiotics and health in ageing populations. Maturitas 75: Dupaul-Chicoine J, Dagenais M, Saleh M Crosstalk between the intestinal microbiota and the innate immune system in intestinal homeostasis and inflammatory bowel disease. Inflam. Bowel Dis. 19: Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, et al Diversity of the human intestinal microbial flora. Science 308: Endt K, Stecher B, Chaffron S, Slack E, Tchitchek N, Benecke A, et al The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea. PLoS Pathog. 6: e Fallani M, Amarri S, Uusijarvi A, Adam R, Khanna S, Aguilera M, et al Determinants of the human infant intestinal microbiota after the introduction of first complementary foods in infant samples from five European centres. Microbiology 157: Fukuda S, Ishikawa H, Koga Y, Aiba Y, Nakashima K, Cheng L, et al Allergic symptoms and microflora in schoolchildren. J. Adolesc. Health : official publication of the Society for Adolescent Medicine 35: Garcia-Lafuente A, Antolin M, Guarner F, Crespo E, Malagelada JR Modulation of colonic barrier function by the composition of the commensal flora in the rat. Gut 48: Gewolb IH, Schwalbe RS, Taciak VL, Harrison TS, Panigrahi P Stool microflora in extremely low birthweight infants. Arch. Dis. Child. Fetal Neonatal. Ed. 80: F Gronlund MM, Lehtonen OP, Eerola E, Kero P Fecal microflora in healthy infants born by different methods of delivery: permanent changes in intestinal flora after cesarean delivery. J. Pediatr. Gastroenterol. Nutr. 28: Hooper LV, Macpherson AJ Immune adaptations that maintain homeostasis with the intestinal microbiota. Nature reviews. Immunology 10: Hooper LV, Midtvedt T, Gordon JI How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu. Rev. Nutr. 22: Iebba V, Aloi M, Civitelli F, Cucchiara S Gut microbiota September 2015 Vol. 43 No. 3

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