J Koren So Food Si Nutr 한국식품영양과학회지 50(9), 904~911(2021) https://doi.org/10.3746/jkfn.2021.50.9.904 Ltoillus prsei AO356 의항비만활성및안전성평가 이소영 1,2 신동욱 2 김승용 1 엄지은 1 남영도 1 신희순 1,2 김정은 3 우용호 3 1 한국식품연구원식품기능연구본부 2 과학기술연합대학원대학교식품생명공학 3 ( 주 ) 코스맥스엔에스 Assessment the of Anti-Oesity Effets nd Sfety of Ltoillus prsei AO356 So-Young Lee 1,2, Dong-Uk Shin 2, Seung Yong Kim 1, Ji-Eun Eom 1, Young-Do Nm 1, Hee-Soon Shin 1,2, Jeong-Eun Kim 3, nd Yong-Ho Woo 3 1 Kore Food Reserh Institute, Reserh Division of Food Funtionlity 2 University of Siene & Tehnology, Food Biotehnology 3 COSMAX NS INC. ABSTRACT The prevlene of oesity nd ssoited metoli disorders is rpidly inresing nd eoming severe helth prolem. Reent reports hve suggested tht the onsumption of proiotis n e onsidered to e n effetive pproh in the tretment nd prevention of oesity. In this study, we investigted the nti-oesity effet of Ltoillus prsei AO356 (AO356) in n oese mie model nd its sfety for use in funtionl foods. The orl dministrtion of AO356 (10 7 10 8 CFU/d for 10 weeks) to mie fed high-ft diet (HFD) signifintly suppressed ody weight gin nd ft mss. Totl plsm triglyerides, totlholesterol, nd LDL-holesterol levels were signifintly lower in mie tht were treted with AO356 s ompred with mie fed just n HFD. Also, the mie treted with AO356 exhiited signifint redution of the insulin resistne index (HOMA-IR). To evlute the sfety of AO356, we onduted n ntiiotis resistne test, hemolyti test, nd n enzymes (geltinse, β-gluuronidse) tivity test. The minimum inhiitory onentrtion (MIC) vlue for the 9 ntiiotis speified in the Europen Food Sfety Authority (EFSA) guidne ws elow the eptle ut-off vlue. In the hemolysis test, AO356 showed γ-hemolysis (non-hemolyti response) on lood gr plte. In ddition, AO356 did not produe the enzymes suh s geltinse nd β-gluuronidse. Tken together, these results indite tht AO356 ould e sfely used s helth funtionl food for the prevention of ntioesity. Key words: proiotis, Ltoillus prsei, oesity, sfety, high ft diet 서론비만은에너지의섭취와소비의불균형으로초래되는대사성질환으로소비후남은에너지가중성지방형태로체내지방조직에과도하게축적된상태를말하는데, 유전적 환경적요인이복합적으로작용하여나타난다 (Alu 등, 1997). 지방조직은잉여에너지를저장하는장소일뿐만아니라아디포카인을합성, 분비함으로써전신대사와염증에도관여하는내분비기관으로알려져있다 (Furukw 등, 2004; Prk과 Kim, 2002). 비만으로인한과도한지방축적은지방조직의내분비기능변형을초래하여인슐린저항성, 당뇨병, 대사증후군, 심혈관질환등의발생을촉진하는것으로보고되고있다 (Gregor와 Hotmisligil, 2011). 우리나라비만유병률은 2001년 29.2%, 2010년 30.9%, 2017년 34.1% 로매년지속해서증가하고있으며 (KDCA, 2019), 2018년국민건강보험공단의보고에따르면비만으로인한사회경제적손실이연간 11조원에달하는것으로알려져있다 (NHIS, 2018). 이에따라비만의예방및치료를위해소재 Reeived 13 My 2021; Revised 15 June 2021; Aepted 21 June 2021 Corresponding uthor: So-Young Lee, Kore Food Reserh Institute, Reserh Division of Food Funtionlity, 245, Nongsengmyeong-ro, Iseo-myeon, Wnju-Gun, Jeonuk 55365, Kore, E-mil: sylee09@kfri.re.kr Author informtion: So-Young Lee (Reserher), Dong-Uk Shin (Grdute student), Ji-Eun Eom (Grdute student), Hee-Soon Shin (Reserher) Copyright 2021 y The Koren Soiety of Food Siene nd Nutrition. All rights Reserved. This is Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Non-Commeril Liense (https://retiveommons.org/lienses/ y-n/4.0) whih permits unrestrited non-ommeril use, distriution, nd reprodution in ny medium, provided the originl work is properly ited.
Ltoillus prsei AO356 의항비만활성및안전성 905 개발에관한연구가꾸준히이루어지고있는데최근주목받고있는소재가프로바이오틱스이다. 프로바이오틱스는적정량을섭취하였을때숙주의건강에유익한효과를주는살아있는미생물을말하며, 식품분야에서는 Ltoillus 속, Bifidoterium 속, Pedioous 속, Ltoous 속, Enteroous 속균주가대표적인상업용프로바이오틱스로사용되고있다 (Gspr 등, 2013; Kng 등, 2017). 일반적으로프로바이오틱스는장내균총의안정화, 변비예방, 유해균생육억제등주로장건강과관련된기능성을가지는것으로알려져왔으나 (Axelsson 등, 1989; Alnder 등, 1999), 2010년이후장내미생물과질환과의상관성연구가다수보고되면서 (Clesson 등, 2012; Lynh와 Pedersen, 2016; Fn과 Pedersen, 2020) 프로바이오틱스를이용한항비만 (Lee 등, 2006), 콜레스테롤저하 (Prk 등, 2017), 항알레르기 (Corthesy 등, 2007), 면역조절 (Jouni 등, 2012), 갱년기증상완화 (Lim 등, 2018), 아토피 (Lee 등, 2008) 개선을위한소재개발연구가증가하고있다. 이중항비만활성을가지는프로바이오틱스에대한연구로 Lee 등 (2006) 은 L. rhmnosus PL60 균주가간에서지방대사를촉진하고지방생합성을억제함을보고하였으며, Kwon 등 (2020) 은 L. plntrum 균주가 SIRT1과 PCG1-α 발현을증가시켜항비만활성을가진다고보고한바있다. 또한 P. pentoseus LP28 균주는 CD 36과 steroyl-coa desturse I의발현억제를통해항비만효과를나타내는것이보고되었으며 (Zho 등, 2012), L. gsseri BNR17 균주도고지방식이로유도한비만모델에서체중증가억제활성을가진다고보고된바있다 (Kng 등, 2013). 특히, L. gsseri BNR17 균주는현재건강기능식품개별인정형균주로등록되어체지방감소에도움을줄수있는원료로상업화되어이용되고있다. 한편, 기능성이밝혀진프로바이오틱스를건강기능식품원료로이용하기위해서는안전성에대한검증이필요한데, 식품의약품안전처 ( 식약처 ) 의고시형프로바이오틱스균주나 Europen Food Sfety Authority(EFSA) 의 Qulified Presumption of Sfety(QPS) 균주들은오랜식경험을바탕으로안전하다고분류되어별도의안전성검증이요구되지않았으나, 최근프로바이오틱스의복용에따른부작용사례가증가하고 (KFSI, 2020) 프로바이오틱스균주를통한항생제내성유전자의이동이문제가되면서 (Huddleston, 2014; Verres 등, 2013; Kpoor 등, 2017) 각국에서는안전성과관련한규제를강화하고있다. EFSA에서는 2012 년 Enteroous 속유산균의식품및사료용사용을금지하거나항생제내성및병독성인자가없음을추가로입증한후제한적으로사용하도록하고있다 (EFSA, 2012). 식약처에서도건강기능식품공전에프로바이오틱스로고시한 5속 19종균주에대해서는별도의안전성자료를요구하지않았으나 2019년부터 Enteroous 속미생물에대해서는항 생제내성및독성평가를통하여안전성을입증하도록의무화하였다 (MFDS, 2017). 또한건강기능식품원료로등록하고자하는균주가고시형일경우에는항생제내성, 용혈활성등의기초적인안전성자료를요구하고있으며, 비고시형균주일경우에는이외에도독성시험자료를추가로제출하도록하고있다 (NIFDS, 2020; Kim 등, 2020). 이에본연구에서는건강한한국인여성으로부터분리한 Ltoillus prsei AO356 균주의체중증가억제활성과안전성을평가하여체지방감소에도움을줄수있는건강기능식품원료로의사용가능성을평가하였다. 재료및방법 AO356 균주준비 AO356 균주 (KCCM 12145P) 는 ( 주 ) 코스맥스엔에스 (Seoul, Kore) 로부터제공받아 -70 C의 Deep freezer (U725-86, Eppendorf, St Alns, UK) 에보관하며사용하였다. 항비만활성평가시에는제공받은균주분말을현탁하여사용하였으며, 항생제내성, 용혈성, indole 생성능, ure 분해능, geltinse 활성평가시에서는제공받은균주를활성화한후사용하였다. 활성화를위하여 MRS 고체배지에선상도말평판법으로 37 C, 24시간동안배양한후단일 olony를취하여 Ltoilli MRS roth(bd Difo TM, Sprks, MD, USA) 배지에접종하고호기적인조건하에서 37 C, 24시간배양한후실험에사용하였다. DIO 동물모델구축및균주투여 C57BL/6J 마우스 (5주령, 수컷 ) 를 ( 주 ) 지바이오 (Gwngju, Kore) 에서구입하여 1주일간순화시킨후, 체중에따라난괴법에의해 10마리씩 5군 ( 정상식이군 (Norml), 고지방식이군 (HFD), 고지방식이 +tehin 162 mg/kg 투여군 (tehin), 고지방식이 +AO356 1 10 7 CFU/ hed 투여군 (AO356 10 7 CFU), 고지방식이 +AO356 1 10 8 CFU/hed 투여군 (AO356 10 8 CFU)) 으로분류하였다. 정상식이군에는일반사료 (ND, Tekld 2018S, Envigo Reserh, Indinpolis, IN, USA) 를공급하였으며, 나머지실험군에는 60 kl% ft 사료 (D12492, Reserh Diets In., New Brunswik, NJ, USA) 를공급하였고, positive ontrol인 tehin 및 AO356 시료는생리식염수에현탁하여 100 µl씩주 5일, 10주간경구투여하였다. 사육실온도는 23±2 C, 습도는 50±5%, 명암주기는 12시간간격으로설정하고물과사료를자율급식시켰으며, 동물실험은한국식품연구원동물실험위원회에서승인 (KFRI-M-20042) 한절차에따라수행하였다. 7일마다일정한시간에체중을측정하였으며, 식이섭취량및수분섭취량을급여량과잔여량의차이로측정하였다. 식이효율 (feed effiieny rtio: FER) 은체중증가량 (g/d) 을식이섭취량 (g/d) 으로나누어계산하였다.
906 이소영 신동욱 김승용 엄지은 남영도 신희순 김정은 우용호 혈액분석시료투여기간종료후 16시간절식시킨마우스의후대정맥을통해채혈한혈액을 3,000 rpm에서 10분간원심분리하여혈청을분리하였다. Gluose, triglyeride, totl holesterol, HDL-holesterol, LDL-holesterol은 Hithi utomti nlyzer modulr nlytis(hithi, Tokyo, Jpn) 를이용하여측정하였다. Insulin 함량은 mouse insulin ELISA kit(alpco dignostis, Slem, NH, USA) 을이용하여측정하였으며, 측정된혈청 gluose 및 insulin 함량으로부터 HOMA-IR을구하였다. HOMA-IR={fsting insulin (µu/ml) fsting gluose (mg/dl)}/ 405 성능을평가하였다. β-gluuronidse 생성능확인을위해 15번째큐플의색변화를평가하였으며, 색변화가없거나미색일때음성, 파란색으로의색변화가관찰되면양성으로판정하였다. AO356 균주의 indole 생성및 ure 분해능은 indole/ure test kit(mb ell, Los Angeles, CA, USA) 을사용하여측정하였다. MRS 고체배지에활성화한 AO356 균주를 sterile loop를이용하여 ure/indole test mixture 에현탁시킨다음 vseline oil을 2~3방울첨가한후 36± 1 C에서 4시간배양하고 Kov s-regent를 2~3방울첨가한후색변화를관찰하였다 (M Fddin, 1980). 혼합액과혼합액상단에생성된링의색이노란색이면음성으로, 붉은색이면양성으로판정하였다. 항생제내성평가항생제에대한내성은 Sensititre TM Grm Positive GPALL1F AST plte(thermo fisher Sientifi, Bsingstoke, UK) 제품을이용하여최소억제농도 (Minimum inhiitory onentrtion, MIC) 를측정하였다. 실험균주는 Ltoilli MRS gr(bd Difo) 배지에서 37 C, 24시간동안배양하여활성화하고순수배양된 olony를 MFrlnd stndrd 0.5로보정한후실험을진행하였다. 배지는 Iso-Sensitest roth(oxoid, Bsingstoke, UK) 와 Ltoilli MRS roth(bd Difo) 를 9:1로혼합하여제조후 (Sho 등, 2015; Munoz 등, 2014) 사용하였으며, 제조한액체배지에 MFrlnd stndrd 0.5로탁도를보정한현탁액을넣고 50 μl씩 sensititre pnel plte에분주접종하였다. 접종한 plte는 24시간동안 37 C에서배양하였고배양이끝난뒤에는제조사의 MIC 결과판정기준에따라판독을진행하였다. 용혈성, geltinse 활성, β-gluuronidse 생성능, indole 생성능및 ure 분해능평가 AO356은 MRS 배지에서 37 C, 24시간 2회배양하여활성화된상태로준비하였다. 용혈성확인을위해활성화한균주를 sheep lood gr plte(asn phrmeutil, Seoul, Kore) 에획선으로접종하여 37 C에서 48시간배양한후용혈반응유형을확인하였다 (Argyri 등, 2013). Geltinse 활성측정을위해활성화된균주는 geltin 사면배지에 MRS roth+geltin 1.2% 를접종하여 37 C, 24시간배양한후 4 C에서약 4시간동안냉장방치하여배지의응고여부를확인하였다 (Isenerg, 1992). AO356 균주의 β- gluuronidse 생성능은 API ZYM test kit(bio-merieux, Lyon, Frne) 을사용하여평가하였다. 균주를 MRS 고체배지에서 37 C, 24시간배양한후단일 olony를취하여 API suspension medium에현탁하고, MFrlnd stndrd 5 탁도로보정한후각큐플에접종하여 37 C에서 4시간동안배양하였다. 배양후큐플에 ZYM A와 ZYM B를 1방울첨가하고 5분뒤제조사의프로토콜에따라효소생 통계처리본실험결과는 IBM SPSS Sttistis(Version 20, IBM, Armonk, NY, USA) 를이용하여평균과표준편차를나타내었다. 실험군간의유의성검증은 P<0.05 수준에서 onewy ANOVA를시행한후 Dunn s multiple rnge test를이용하여분석하였다. 결과및고찰 AO356 균주의마우스체중및지방조직증가억제활성 AO356 균주의항비만활성을평가하기위해 6주령 C57 BL/6J 마우스에고지방식이를공급하며주차별체중을특정한결과 (Fig. 1A), 고지방식이를공급한 HFD군마우스의 10주차평균체중은 42.42 g으로 0일대비 20.06 g 증가하여 6.33 g 증가한정상식이공급군에비해 3.16배증가하였다. 그러나고지방식이와함께 AO356 균주를경구투여한군에서는농도의존적인체중증가억제활성이나타났으며, 1 10 8 CFU 투여군의경우 10주간의체중증가량이 16.39 g으로 HFD군대비약 20% 감소하여 positive ontrol인 tehin을 162 mg/kg BW 농도로투여한군과유사한수준의체중증가억제활성을보였다. 식이섭취량에는실험군간유의적인차이는없어체중증가억제활성이식이섭취량감소에기인한것이아님을알수있었으며, 식이섭취효율도 tehin 및 AO356 섭취군에서 HFD군에비해감소한것을확인할수있었다 (Fig. 1C, 1D). 지방조직특히백색지방은체중증가와더불어비만의주요위험요소로알려져있다 (Bjorntorp, 1990; Despres, 1993). 지방조직은백색지방과갈색지방으로구분되는데, 체내잉여에너지를중성지방의형태로저장하는백색지방조직의비정상적인비대는지방조직의아디포카인분비조절장애를유도하고포도당과지질대사를변화시켜심각한대사증후군을유발하는것으로알려져있다 (Asselh 등, 2006; Prk과 Kim, 2002). 이에 AO356 섭취가지방조직증가에미치는영향을평가하기위해주요지방조직의무게를측정하였다. 그결과, 고지방식이에의해부고환지방, 후복막지방, 서혜부지방등백
Ltoillus prsei AO356 의항비만활성및안전성 907 A Body weight (g) 50 45 40 35 30 25 Norml Ctehin AO356 10^8 8 CFU HFD AO356 107 7 CFU d d C Food effiieny rtio. 0.12 0.10 0.08 0.06 0.04 0.02 20 0 1 2 3 4 5 6 7 8 9 10 Time (weeks) 0.00 Norml HFD Ctehin AO356 AO356 107 CFU CUF 108 CFU CUF 70 d Norml HFD Ctehin d B AO356^7 10 7 A0356^8 AO356 10 8 60 Orgn/ody weight rtio. (mg/g ody weight). 50 40 30 20 10 0 EFT RFT IFT BAT D Food intke (g/dy). 6 5 4 3 2 1 0 Norml HFD Ctehin AO356 AO356 107 CUF CFU 108 CFU CUF Fig. 1. The meliortive effet of Ltoillus prsei AO356 on oesity prmeters. (A) Body weight ws mesured weekly nd presented s men±sd (n=8). (B) The tissues were weighted nd normlized y the ody weight of eh mouse. (C) The food effiieny rtio (EFR) of eh group ws lulted y pplying the eqution; FER=ody weight gin (g/d)/ Food intke (g/d) nd showed s men±sd. (D) The food intke of eh group ws lulted nd shown s men±sd. Different letters ove rs indite signifint differene t P<0.05 y Dunn s multiple rnge test. HFD, High ft diet group; EFT, epididyml white ft tissue; RFT, retroperitonel white ft tissue; IFR, inguinl white ft tissue; BAT, intrspulr rown dipose tissue. 색지방조직의무게가정상식이군대비각각 2.75, 4.5 및 3.35배증가하였으나, AO356 투여시증가한지방조직의무게가고지방식이군대비약 10%, 5% 및 23% 감소한것으로나타났다 (Fig. 1B). 한편, 갈색지방은미토콘드리아를다량함유하고있어에너지소비촉진과관련있는지방조직이지만, 고지방식이를섭취한동물에서는비정상적인지방산의축적에의해그무게가증가하는것으로보고되고있다 (Lldo 등, 1997; Ryu 등, 2011). 본실험에서도고지방식이군에서견갑골갈색지방의무게는증가하였으나, AO356 식이시갈색지방의무게가감소한것으로관찰되었는데, 이는 AO356 균주투여가고지방식이에의해유도되는지방산의축적을억제하였기때문으로생각된다. AO356 균주의이상지질혈증및인슐린저항성개선활성고지방식이로인해증가한잉여에너지는지방으로전환되어체내에축적되고, 축적된지방의비정상적인지질대사는간내유리지방산의유입을증가시켜간에서의지방합성및초저밀도지단백 (very low density lipoprotein; VLDL) 의생성증가와 lipoprotein lipse의활성감소를유도하는 데, 이로인해혈중중성지방이증가하게된다 (Kruss, 2004). 고지방식이투여군의경우중성지방의함량이정상식이군대비 1.66배증가하였고, AO356 투여시 tehin 투여시와유사하게약 32% 감소하였다. 한편, 비만으로인해발생하는이상지질혈증의다른지표로는총콜레스테롤, LDL 및 HDL 콜레스테롤이있다. 지질은세포막, 담즙, 호르몬의중요성분이지만혈중지질함량이높으면다양한질병의위험도를높이는것으로알려져있고, 특히 LDL 콜레스테롤은간에서다른조직으로콜레스테롤을운반하고체내에축적하는것으로알려져있다 (Kolovou 등, 2005). 반면, HDL 콜레스테롤은 LDL 콜레스테롤의생성을억제하거나혈관에축적된콜레스테롤을감소시켜동맥경화발생을억제하는작용을한다 (Imno 등, 2011; Gordon 등, 1977). AO356의경구투여는고지방식이에의해증가된혈청총콜레스테롤, LDL-콜레스테롤을유의한수준으로감소시켰으며, 총콜레스테롤중 HDL-콜레스테롤의비율을유의적으로높게유지시켰다 (Tle 1). 또한고지방식이군에의해증가한동맥경화지수 (therogeni index, AI) 와심혈관위험지수 (rdi risk ftor, CRF) 도감소되어 AO356은고
908 이소영 신동욱 김승용 엄지은 남영도 신희순 김정은 우용호 Tle 1. The effet of Ltoillus prsei AO356 on HFD-indued dyslipidemi nd insulin resistne in serum isolted from mie Norml HFD 1) Ctehin AO356 10 7 CFU AO356 10 8 CFU Triglyeride (mg/dl) Totl holesterol (mg/dl) LDL 2) -holesterol (mg/dl) HDL 3) -holesterol (mg/dl) Atherogeni index 4) Crdi risk ftor 5) Fsting insulin (μu/ml) Fsting gluose (mg/dl) HOMA-IR 6) 63.71±14.34 103.43±8.92 12.14±2.27 92.14±5.64 0.12±0.04 1.12±0.04 1.41±0.41 186.43±47.99 0.32±0.25 105.86±27.70 180.86±9.97 29.14±4.30 141.43±7.52 0.28±0.05 1.28±0.05 14.50±2.53 317.86±55.46 5.48±1.27 81.29±7.70 158.33±9.18 24.83±2.98 131.67±5.47 0.20±0.02 1.20±0.02 4.81±0.75 233.29±61.72 2.30±0.89 72.38±9.86 161.63±11.19 23.75±3.06 133.00±6.30 0.21±0.03 1.21±0.03 6.99±2.72 244.88±27.28 2.46±1.21 74.63±10.38 165.63±8.96 25.00±2.62 135.88±6.29 0.22±0.03 1.22±0.03 4.55±1.16 229.00±64.85 2.41±1.10 Dt re expressed s men±sd. Mens with different letters re signifintly different t P<0.05 y Dunn s multiple rnge test. 1) HFD: High ft diet group. 2) LDL: Low-density lipoprotein. 3) HLD: High-density lipoprotein. 4) Atherogeni index (AI)=(Totl holesterol-hdl holesterol)/ HDL holesterol. 5) Crdi risk ftor (CRF)=Totl holesterol/ HDL holesterol. 6) HOMA-IR: Homeostti model ssessment of insulin resistne. HOMA-IR={fsting insulin (µu/ml) fsting gluose (mg/dl)}/ 405. 지방식이로유도되는이상지질혈증개선에효율적인소재임을확인할수있었다. 최근연구에따르면 AO356과같은 Ltoillus 속균주의이상지질개선효과는 AMPK, SIRT1 및 PGC-1α 발현증가를통한담즙산의생성증가와간으로의콜레스테롤회수증가에의한것으로보고되고있는데 (Jng 등, 2019; Kwon 등, 2020), AO356 균주도이와유사한기전으로이상지질혈증개선에효과를보일것으로예상되나추가적인연구가필요할것으로생각된다. 한편, 혈중중성지질과 LDL-콜레스테롤의양이증가되고말초조직과복부에중성지질이축적되면인슐린저항성등과같은합병증이유발되는데 (Potter, 1995; Spiegelmn, 2001), AO356 투여는고지방식이에의한인슐린저항성도개선시키는것으로확인되었다 (Tle 1). 고지방식이군의공복혈청인슐린농도는 14.50±2.53 μu/ml로 1.41±0.41 μu/ ml인정상식이군대비증가한고인슐린혈증상태였으며, 공복혈당역시 317.86±55.46 mg/dl로정상군대비 1.7배높은인슐린저항성이발생된상태였다. 그러나 AO356을 1 10 8 CFU 농도로투여시공복혈청인슐린농도가약 70% 감소하였으며, 공복혈당도 244.88~229.00 mg/dl로고지방식이군대비 23~28% 감소한것으로나타나 AO356 식이를통해인슐린저항성이개선됨을확인할수있었다. 이는공복혈당과인슐린농도를바탕으로계측하는인슐린저항성지표인 HOMA-IR 결과에서도확인되었는데, 고지방식이로인해약 16배증가한 HOMA-IR 수치가 AO356 투여시 55~56% 감소하여 58% 가량감소한 tehin 투여군과유사한수준으로인슐린민감도가향상됨을확인할수있었다. 이상의결과로부터 AO356 균주는고지방식이에의해유도되는체중증가, 지방조직무게증가, 이상지질혈증및인슐린저항성을완화하여항비만활성을나타내는것을확인할수있었으며, 현재이러한항비만활성에대한구체적 인기전연구를진행중에있다. 항생제에대한감수성항생제내성균주는항생제내성유전자를병원성또는잠재적인병원성미생물에전달시킬수있으며, 이러한균주의섭취는사람에게도심각한보건상의문제를일으킬수있기때문에 (Chon 등, 2020) 식의약품으로사용하기위한프로바이오틱스의개발에있어항생제내성에대한확인은필수적으로확인되어야하는항목이다 (FAO/WHO 2020; EFSA, 2012; KFSI, 2019). AO356 균주와같은 lti id teri(lab) 는대부분이 generlly reognized s sfe(gras) 와 QPS 상태를통해안전하다고인정받아왔으나최근발효식품에서분리된 LAB 균의일부변종이 erythromyin, tetryline, vnomyin 등에저항성을가지는유전자를보유한것이확인되면서동식물에공생하는 LAB 에대한안전성에의문이제기되고있다 (Coppol 등, 2005; De 등, 2017; EFSA, 2012). 이에항비만활성을가지는 AO356 균주의주요항생제 9종에대한감수성정도를 minimum inhiitory onentrtion(mic) 측정을통해분석하였다 (Tle 2). 그결과 MIC 수치가 mpiillin 1 µg/ ml, hlormpheniol 4 µg/ml, gentmyin 4 µg/ml, streptomyin 4 µg/ml, tetryline 2 µg/ml, erythromyin 0.25 µg/ml, lindmyin 0.5 µg/ml 및 knmyin 16 µg/ml로 EFSA의 L. prsei 종에대한한계치보다낮거나같은값을보여 (EFSA, 2012), AO356 균주는식품원료로사용할수있는안전한균주임을확인할수있었다. 용혈성, geltinse 활성, β-gluuronidse 생성능, indole 생성능및 ure 분해능평가 AO356은 L. prsei 종에속하는균주로 QPS 미생물이며식약처의고시형프로바이오틱스로서식경험이풍부하
Ltoillus prsei AO356 의항비만활성및안전성 909 Tle 2. The ntiioti suseptiility of Ltoillus prsei AO356 y MIC test MIC 1) (µg/ml) MIC-ut off vlue of EFSA 2) AO356 (for Ltoillus prsei) Ampiillin Chlormpheniol Clindmyin Erythromyin Gentmiin Knmyin Streptomyin Tetryline Erythromyin 4 4 1 1 32 64 64 4 1 Dt re men±sd of three replites. 1) MIC, Minimum inhiitory onentrtion. 2) EFSA, Europen Food Sfety Authority. 1±0.00 4±0.00 0.5±0.00 0.25±0.00 4±0.00 16±0.00 4±0.00 2±0.00 0.25±0.00 고안전하다고알려진균주이다 (EFSA, 2012; MFDS, 2017). 일반적으로프로바이오틱스의기능성은 strin 특이적인것으로알려져있는데 (Snel 등, 2011), 기능성뿐만아니라안전성또한 strin 특이적인반응을보일수있으므로항생제내성이외에도 AO356 균주의안전성을평가하기위해용혈성, geltinse 활성, β-gluuronidse 생성능, indole 생성능및 ure 분해능을확인하였다. 용혈은적혈구가파괴되어헤모글로빈이혈구밖으로용출되는현상으로혈액한천배지상에서나타나는용혈유형에따라 α, β, 및 γ-hemolysis 유형으로구분할수있다. α-hemolysis 는적혈구의부분적용해로헤모글로빈이메트헤모글로빈으로환원되어 olony 주변이녹색으로변화하며, β-hemolysis는적혈구가완전히용해되어배양후 olony 주변이투명하게변한다. γ-hemolysis는용혈이일어나지않는반응으로 olony 주변에아무런변화가없는것이특징이다 (Isenerg, 1992). AO356 균주는혈액배지를이용한용혈반응에서 γ-hemolysis 반응을보여용혈성이없는균주임을확인하였다 (Fig. 2A). Geltinse는젤라틴을분해하는단백질분해효소로서병원성미생물에의해분비되어 iofilm 형성에관여하는것으로알려져있다. 생체내콜라겐, 피브리노겐및보체등다양한종류의단백질을분해함으로써숙주의면역시스템을교란시키고, 심내막염을유발하는등의독성과관련있는것으로알려져있는데 (Thurlow 등, 2010), 본결과를통해 AO356 균주는 geltinse 활성이없는것을확인할수있었다 (Fig. 2B). 또한 AO356 균주는 tryptophnse에의해생성되어대장암과방광암유발에관여할수있는 indole 생성 (Chung 등, 1975), 유해물질인암모니아생성과관련있는 ure(ni 등, 2017) 분해능및 enzopyrene 등의지용성독성물질의무독화형태인 gluuroni id onjugte를분해하여발암원을제공하는 β-gluuronidse(nnno 등, 1986) 의활성도없는것으로나타났다 (Fig. 2C, 2D, 2E). 이상의실험결과와오랜식경험으로미루어볼때, AO356 균주는식품원료로사용할수있는안전한균주로생각되며현재전장유전체결과를바탕으로항생제내성및독성물질생성유전자의유무를파악하여안전성을확인하는추가연구를진행중이다. 요약고지방식이로비만을유도한 6주령 C57BL/6J 마우스에 AO356을 1 10 7 및 1 10 8 농도로 10주간투여하여항비만활성을평가하였다. AO356 투여시식이섭취량변화없이고지방식이에의해유도되는체중증가및지방조직무게증가가억제되었으며, 이상지질혈증및인슐린저항성이개선되었다. 특히 1 10 8 CFU 농도투여군에서는 tehin 투여군과유사한수준의항비만활성을보여체지방개선에도움을줄수있는건강기능식품으로의개발가능성을확인하였다. 또한식품원료미생물로의안전성을확인하기위해 9종항생제에대한감수성을평가한결과, AO356은 L. prsei 종에대한 EFSA의기준을충족하였으며, 용혈성평가에서는 γ-hemolysis 반응으로적혈구용혈을일으키지않았다. 또한 β-gluuronidse, geltinse, ure 분해능및 indole 생성능등의유효효소와유해물질을생산하지 A B C D E γ-hemolysis (non-hemolyti) Negtive (non-liqueftion) Negtive (very ple yellow) Negtive (yellow ring) Negtive (yellow olor) Fig. 2. The hemolyti tivity, geltinse tivity, β-gluuronidse tivity, ure hydrolysis, nd indole produing tivity of Ltoillus prsei AO356. (A) The hemolyti tivity ws mesured using sheep lood gr pltes. (B) The geltinse tivity ws mesured using geltin slnt medium. (C) The β-gluuronidse tivity ws mesured using 15th upule on API ZYM kits. (D) The indole produing tivity ws mesured using ure/indole test mixture kits. (E) The ure hydrolysis tivity ws mesured ure/indole test mixture kits. All experiments were performed in triplite.
910 이소영 신동욱 김승용 엄지은 남영도 신희순 김정은 우용호 않는것으로나타나식품원료로사용가능한안전한균주임을확인할수있었다. 감사의글 이논문은과학기술정보통신부의재원으로연구개발특구진흥재단-기술이전사업화사업 (No. 2020-JB-RD-0011, 체지방감소능을갖는천연소재인프로바이오틱스제품개발 ) 의지원을받아수행된연구입니다. REFERENCES Alnder M, Stokri R, Korpel R, Sxelin M, Vilpponen-Slmel T, Mttil-Sndholm T, et l. Persistene of oloniztion of humn oloni muos y proioti strin, Ltoillus rhmnosus GG, fter orl onsumption. Appl Environ Miroiol. 1999. 65:351-354. Alu J, Allison D, Boozer CN, Heymsfield S, Kissileff H, Kretse A, et l. Oesity solutions: report of meeting. Nutr Rev. 1997. 55:150-156. Argyri AA, Zoumpopoulou G, Krtzs KAG, Tsklidou E, Nyhs GJE, Pngou E, et l. Seletion of potentil proioti lti id teri from fermented olives y in vitro tests. Food Miroiol. 2013. 33:282-291. Asselh T, Rul-Brndt L, Mrellin P, Negro F. Stetosis in hroni heptitis C: why does ti relly mtter?. Gut. 2006. 55:123-130. Axelsson LT, Chung TC, Dorogosz WJ, Lindgren SE. Prodution of rod spetrum ntimiroil sustne y Ltoillus reuteri. 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