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pissn 1229-1153 J. Fd Hyg. Safety Vol. 28, No. 3, pp. 234~240 (2013) http://dx.doi.org/10.13103/jfhs.2013.28.3.234 Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr 훈증소독제, Fumagari OPP 의 Escherichia coli 와 Salmonella typhimurium 에대한살균효과 박은기 1 김용팔 2 유은아 3 유창열 4 최현주 5 김석 이후장 * 경상대학교수의과대학생명과학연구원, 1 고신대학교의과대학, 2 엘캄코바이오 ( 주 ), 3 보건복지부통영검역소, 4 경남도립남해대학인터넷정보학과, 5 인제대학교임상병리학과 Bactericidal Efficacy of Fumagari OPP, Fumigant Against Escherichia coli and Salmonella typhimurium Eun-Kee Park 1, Yongpal Kim 2, Eun-Ah Yu 3, Chang-Yeol Yoo 4, Hyunju Choi 5, Suk Kim, and Hu-Jang Lee* Research Institute of Life Sciences, College of Veterinary Medicine, Gyeongsang National University, Chinju 600-701, Korea 1 Department of Medical Humanities and Social Medicine, College of Medicine, Kosin University, Busan 602-703, Korea 2 Elkahmco Bio Co., Ltd., Seoul 143-802, Korea 3 Tongyeong National Quarantine Station, Ministry of Health & Welfare, Tongyeong 650-110, Korea 4 Department of Computer Information, Gyeongnam Provincial Namhae College, Namhae 668-801, Korea 5 Elderly Life Redesign Institute, Department of Biomedical Laboratory Science, Inje University, Gimhae 621-749, Korea (Received July 3, 2013/Revised July 12, 2013/Accepted July 23, 2013) ABSTRACT - This test was performed to evaluate the bactericidal efficacy of Fumagari OPP, fumigation disinfectant, containing 20% ortho-phenylphenol against Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium). In preliminary tests, both E. coli and S. typhimurium working culture suspension number (N value) was 4.0 10 8 CFU/mL. And all of the colony numbers on the carriers exposed the fumigant (n1, n2, n3) were higher than 0.5N1 (the number of bacterial test suspentions by pour plate method), 0.5N2 (the number of bacterial test suspentions by filter membrane method) and 0.5N1, respectively. In addition, the mean number of bacteria recovered on the control-carriers (T value) was 3.4 10 6 CFU/mL. In the bactericidal effect of the fumigant, the reduction number of S. typhimurium and E. coli (d value) was 5.26 and 5.64 logcfu/ml, respectively. According to the French standard for the fumigant, the d value for the effective bactericidal fumigant should be over than 5 logcfu/ml. With the results of this study, Fumagari OPP has an effective bactericidal activity, then the fumigant can be applied to disinfect food materials and kitchen appliances contaminated with pathogenic bacteria. Key words: Fumigant, disinfectant, ortho-phenylphenol, Escherichia coli, Salmonella typhimurium 대장균은사람과동물의장관에일반적으로서식하는균으로, 식품에있어서분변오염지표로사용되고있다. 대장균은먹이사슬을통해서로다른생태계에쉽게전파될수있다 1). 대장균에의한질병발생은, 채소, 원유, 설익은쇠고기패티, 음용수등과같은다양한식품들과관련되어있다 2). 병원성대장균은오염된식품의섭취를통해설사, 장염, 용혈성요독증후군, 그리고출혈성대장염등을 *Correspondence to: Hu-Jang Lee, Research Institute of Life Sciences, College of Veterinary Medicine, Gyeongsang National University, Chinju 600-701, Korea. Tel: 82-55-772-2352, Fax: 82-55-772-2308 E-mail: hujang@gsnu.ac.kr 일으킬수있는것으로보고되어있다 3). 살모넬라균은동물에서장염과치명적인감염을일으키며, 사람에서는식중독과장티푸스를일으킨다 4-7). 살모넬라증은인수공통전염병으로사람과동물사이에전파되며, 많은살모넬라균감염이오염된식품의섭취에의해발생한다 7). 살모넬라증의원인균은 Salmonella spp. 이며, Salmonella spp. 는운동성이있는그람음성의간균이며, 탐식세포내에서증식할수있는선택적세포내기생세균이다 7). 또한, 살모넬라균은생물체외부환경에서도수주이상생존할수있으며, 영하의조건에서도사멸하지않는것으로알려져있다 8,9). Salmonella typhimurium (S. typhimurium) 은돼지농장, 도 234

Bactericidal Efficacy of Fumagari OPP, Fumigant Against Escherichia coli and Salmonella typhimurium 235 축돈그리고식중독등에서가장빈번하게분리되는혈청형중의하나이다 10,11). 또한, S. typhimurium은다양한숙주와환경에서생존할수있으며, 음용수와가금육을통해사람에게쉽게전파될수있다 12,13). 살모넬라균과대장균감염은일반적으로사용되는항생제에대한내성으로인해제어하기가점점어려워지고있어서, 효과적인소독제사용이균의감염과질병발생의예방을위한기본적인요소이다 14-16). 가축농장, 식품공장, 병원, 가정등에서살균제의사용이증가함에따라, 항생제내성균출현기전과동일하게, 살균제내성균의출현이증가하고있는것에대해공중보건학적관심이집중되고있다 17). 그러나살균제는주로세균의대사기전에광범위하게작용하는성분들의혼합으로조성되어있으며, 이러한조성물들은살균제에대한세균의내성형성을어렵게만드는것으로알려져있다 18). 수세기동안, 미생물, 유해해충, 그리고다른병원균으로부터식품을보호하고보존하기위한방법을찾기위해많은노력을경주해왔으며, 최근, 보존제의사용을포함한식품보존기술은, 식품공급에있어서식품의질과안전성의개선과, 부패, 미생물오염, 영양소파괴등으로부터식품을보호하는데있어서, 중요한역할을담당해왔다 19). 식품저장기간동안소독을위해, phosphine 가스, 이산화염소, 인화수소, ortho-phenylphenol (OPP) 등과같은많은훈증소독제가전세계적으로광범위하게사용되고있다 20-22). 특히, OPP는가정, 식품산업, 병원등에서기구나표면재의소독을위해사용되고있다 20). 현재까지, 식중독관련병원성세균에대해 OPP를주성분으로하는훈증소독제의효능에관한연구는거의없는실정이다. 따라서본연구에서는 Association Francaise de Normalisation (AFNOR) 23) 의 훈증소독제의살균, 살곰팡이, 살효모, 그리고살아포효력을결정하기위한기준법 에따라, 식중독관련주요세균인대장균과살모넬라에대해, OPP를주성분으로하는훈증소독제의살균효과를확인하기위해수행하였다. 재료및방법 실험물질본시험에사용된실험물질은엘캄코바이오 ( 서울 ) 에서공급받은 OPP를주성분으로하는훈증소독제, Fumagari OPP (1 캔 (20 g), OPP 4 g) 를사용하였다. 물질은흰색의분말로서사용기간동안실온에보관하여실험에사용하였다. 균주배양 Salmonella typhimurium (KVCC-BA 1200171) (S. typhimurium) 은한국수의유전자원은행 ( 안양 ) 에서, Escherichia coli (KCTC 1682) (E. coli) 는한국생명공학연구원생명자 원센터 ( 대전 ) 에서각각분양받아실험에사용하였다. S. typhimurium과 E. coli를각각고압멸균된 nutrient broth (Difco, Detroit, MI) 와 tryptone soy agar (TSA, Difco, Detroit, MI) 에심어 37 o C에서 24시간동안계대배양한후, McFaland 등의방법 24) 에따라배양액의탁도를측정하고, 희석액 ( 증류수 1L 중, tryptone 1.0 g, sodium chloride 8.5 g) 을사용하여, S. typhimurium과 E. coli의농도가 10 9 CFU/mL 이상이되도록조정하였다. 이어서, 환원유 ( 증류수 1L 중, 탈지분유 100 g) 를가하여 20배로희석하여, 각각 10 8-10 9 CFU/mL로조정하여시험에사용하였다. 환원유혼합현탁액의균수계산환원유혼합 S. typhimurium과 E. coli 현탁액을각각희석액으로 10 7 과 10 8 배로희석한다음, 각각의희석액 1mL 를취하여페트리접시에넣고, S. typhimurium과 E. coli 희석액이들어있는페트리접시에각각 45 o C와 37 o C의 TSA배지 20 ml을넣었다. 또한, 10 7 과 10 8 배로희석한균주현탁액 1mL를각각취하여분리막여과장치에넣고여과한후, 세척액으로씻어준다음, 각각의여과막을 TSA 배지위에올려놓았다. 각각의배지를 37 o C에서 24시간동안배양한다음, 균수계산이불가능한배지는폐기하고, 다시 24시간동안더배양한후, 균수를계산하였다. 평판배지법 25) 과여과법 26) 으로배양한균수를각각 N1과 N2로하였다. 균주의담체도포및소독제적용각각의 S. typhimurium과 E. coli 현탁액 0.05 ml을각각 5개의비다공성의스테인리스담체 ( 지름 3cm, 높이 1.2 mm) 에도포하고, 도포된담체들을멸균페트리접시에담아, 37 o C 배양기에서건조시켰다. 이때, 건조시간은 45 분을초과하지않도록하였다. 21 ± 0.5 o C, 습도 60 ± 10% 조건의밀폐된방 (25 m 3 ) 에건조된담체 2개를페트리접시에담아, 훈증소독제노출없이 15시간동안놓아두었다. 나머지 3개의건조된담체는 2개의건조된담체와동일한환경에서, 훈증소독제와담체를담은페트리접시와의거리를 m로하고, 바닥으로부터 1.2 m 높이에페트리접시를수직으로세워훈증소독제와반대방향이되도록한다음, 훈증소독제에불을붙여 15시간동안놓아두었다. 소독제잔류효과 Fig. 1은훈증소독제잔류에의한균증식억제효과를확인하기위한실험을수행하는과정을나타낸것이다. S. typhimurium과 E. coli를도포 건조시킨각각의담체를훈증소독제에 15시간노출시킨직후, 각각의담체를 100 ml 회복액 ( 증류수 1L 중, tryptone 1.0 g, NaCl 8.5 g) 이들어있는삼각플라스크에넣고, 수초동안흔들어준

236 Eun-Kee Park, Yongpal Kim, Eun-Ah Yu, Chang-Yeol Yoo, Hyunju Choi, Suk Kim, and Hu-Jang Lee Fig. 1. Schematic diagram of the preliminary screening test for an inhibitory effect of fumigant residues. 다음, 1 ml을취하여페트리접시에넣고, 각각의균주희석액 (10 7 과 10 8 배 ) 1 ml를섞이지않게넣은다음, 배양배지에서잘혼합한후, 37 o C에서 48시간동안배양하였다. 배양후에, 자란집락수의평균값을 n1로하였다. 담체를넣은회복액 98 ml를막여과하고, 50 ml 희석액으로 3번세정한후, 희석액에 4번침지시킨여과막을배지에넣고, 희석한각각의시험균주현탁액 (10 7 과 10 8 배 ) 1mL을가하여, 혼합한다음, 배양한후, 형성된집락수의평균값을 n2로하였다. 희석한각각의시험균주현탁액 (10 7 과 10 8 배 ) 1 ml과, 회복액에서잔류물이제거된담체를넣은회복액 1mL을페트리접시에넣고, 배지를가하여혼합하여배양한후, 자란집락수의평균값을 n3으로하였다. 이렇게얻은 n1, n2, n3를각각 N1, N2, N1과비교하였다. 훈증소독제적용 S. typhimurium과 E. coli를도포 건조시킨각각의담체를훈증소독제에 15시간노출시킨직후, 각각의담체를 100 ml 회복액이있는삼각플라스크에넣고, 수초동안흔들어준다음, 유리봉을이용하여, 담체에부착된잔류물을제거하고, 1분동안초음파진동기를이용하여남은잔류물을제거하였다. 이어서, 담체잔류물이들어있는 회복액으로부터 1mL을취하여 9mL 새로운회복액에넣고, 연속적으로두번더희석시켜 10 3 배로희석하였다. 담체잔류물이들어있는회복액과, 10 2 와 10 3 배희석액을각각 1mL을취하여배양배지가들어있는평판배지에접종하여, 37 o C에서 48시간동안배양한다음, 각각의집락수를계산하였다. 담체로부터부착된잔류물을제거한회복액현탁액 87 ml을분리막여과장치에넣고여과한후, 여과막을영양배지위에올려놓고 37 o C에서 48시간동안배양한다음, 각각의집락수의평균값을 n'1로하였다. 잔류물을제거한담체를넣은회복액현탁액을배지에넣고배양하여, 자란집락수의평균을 n'2로하였다. 실험결과의계산 14이하 300 이상인배지의집락수는세지않았으며, 실험균주현탁액중균수 (N), 대조담체의회복균수 (T), 그리고훈증소독제노출담체의균수감소 log값 (d) 의계산은아래각각의식에의해산출하였다. N ( CFU ml) x+ y+ z+ w = -------------------------- 10 7 (10 7 배로희석하여얻은값 : x, y; 10 8 배로희석하여얻은값 : z,

Bactericidal Efficacy of Fumagari OPP, Fumigant Against Escherichia coli and Salmonella typhimurium 237 T ( CFU carrier) x+ y+ z+ w = -------------------------- 10 2 100 (10 2 배회복액에서얻은값 : x, y; 10 3 배회복액에서얻은값 : z, d (logcfu/ml) = logt log(n'1 + n'2) = log[t/(n'1 + n'2)] (n'1: 담체부착물함유회복액여과막에서자란균주집락수의평균, n'2: 담체부착물함유회복액을심은배지에서자란균주집락수의평균 ) 훈증소독제살균효과판정 AFNOR 23) 의기준에따라, S. typhimurium 과 E. coli 에대 한훈증소독제의효과는 실험결과의계산 에의해얻은 d값이 5 logcfu/ml 이상인경우로하였다. 결과및고찰 소독제잔류효과 Table 1과 2는각각환원유혼합 S. typhimurium과 E. coli 현탁액의균수와, S. typhimurium과 E. coli를도포 건조시킨담체에소독제를노출시킨후, 산출한균수를나타낸것이다. AFNOR 23) 에따르면, 실험균주현탁액배양균수로부터산출된 N값은 10 8 에서 5 10 9 사이에있어야하며, 소독제에노출되지않은대조군-담체로부터배양된균수로부 1) Table 1. Viable counts (CFU/mL) of S. typhimurium in milk-to-suspensions and in the carriers exposed to disinfectant Milk-to-suspensions Exposure to disinfectant N2 2) Exposure-carriers 4) Control-carriers N1 N value 3) 10 7 10 8 10 7 10 8 No. n1 n2 n3 DT 5) 1 2 1 19 16 17 35 29 15 13 33 29 15 11 10 2 293 298 2 21 20 20 4.0 10 8 3 23 21 23 32 14 31 13 10 3 73 92 21 19 20 1) N1, the number of bacterial test suspentions by pour plate method. 2) N2, the number of bacterial test suspentions by filter membrane method. 3) N, the number of bacteria of working culture suspension. N = ------------------------------- 10 7 (x, y: 10 7 dilution colony; z, w: 10 8 dilution colony) 4) n1, n2, n3, the colony numbers on the carriers exposed the fumigant. 5) DT, dilution time. 6) T, the mean number of bacteria recovered on the control-carriers. T = ------------------------------- 10 2 100 (x, y: 10 2 dilution colony; z, w: 10 3 dilution colony) T value 6) 3.4 10 6 1) 2) Table 2. Viable counts (CFU/mL) of E. coli in milk-to-suspensions and in the carriers exposed to disinfectant Milk-to-suspensions Exposure to disinfectant N2 Exposure-carriers 4) Control-carriers N1 N value 3) 10 7 10 8 10 7 10 8 No. n1 n2 n3 DT 5) 1 2 1 19 22 15 22 20 21 17 25 27 24 22 10 2 236 287 2 17 18 17 4.0 10 8 3 18 23 16 21 19 26 23 10 3 61 83 18 21 16 1) The number of bacterial test suspentions by pour plate method. 2) N2, the number of bacterial test suspentions by filter membrane method. 3) N, the number of bacteria of working culture suspension. N = ------------------------------- 10 7 (x, y: 10 7 dilution colony; z, w: 10 8 dilution colony) 4) n1, n2, n3, the colony numbers on the carriers exposed the fumigant. 5) DT, dilution time. 6) T, the mean number of bacteria recovered on the control-carriers. T = ------------------------------- 10 2 100 (x, y: 10 2 dilution colony; z, w: 10 3 dilution colony) T value 6) 2.9 10 6

238 Eun-Kee Park, Yongpal Kim, Eun-Ah Yu, Chang-Yeol Yoo, Hyunju Choi, Suk Kim, and Hu-Jang Lee 터산출된 T값은 10 6 CFU/mL이상이어야한다고규정하고있다. Table 1과 2에서, S. typhimurium과 E. coli로부터구한 N값은모두 4.0 10 8 CFU/mL이었으며, S. typhimurium과 E. coli로부터구한 T값은각각 3.4 10 6 과 2.9 10 6 CFU/ ml이었다. 따라서 S. typhimurium과 E. coli의 N값과 T값은모두 AFNOR 23) 에서규정한기준을만족하였다. AFNOR 23) 에따르면, 소독제노출담체로부터증식한균수 n1, n2, n3값이각각 0.5N1, 0.5N2, 0.5N1값보다모두커야훈증소독제실험을수행할수있는조건을만족하는것으로규정하고있다. 만일, n1, n2, n3값이각각 0.5N1, 0.5N2, 0.5N1값과같거나작으면, 배지나여과막에균의증식을충분히억제할수있는많은양의소독제가잔류하고있다는것을의미하므로, 배지의조성을조정하거나, 담체회복액에중화제를첨가하거나, 여과막의세정횟수를증가시켜실험을다시수행하여조건을만족시켜야한다. 훈증소독제의살균효과 Table 3과 4는훈증소독제를노출시킨담체로부터회복된균수와훈증소독제의살균효과를나타낸것이다. Table 3과 4에서, S. typhimurium과 E. coli 도포담체에소독제를노출시킨후, 배양을통해확인된각각균수의 logcfu/ml 감소값, 즉, d 값은각각 5.26과 5.64 logcfu/ ml로나타나, OPP를주성분으로하는훈증소독제, Fumagari OPP 은 S. typhimurium 보다 E. coli에서더높은살균효과를나타내었다. AFNOR 23) 의기준에따르면, 훈증소독제가효과적인살균력을갖기위해서는 d 값이 5 logcfu/ml 이상이어야한다고, 규정하고있다. 본연구에서, 훈증소독제를적용한후, S. typhimurium과 E. coli의 d 값은각각 5.26과 5.64 logcfu/ml로나타나, 훈증소독제, Fumagari OPP 은 S. typhimurium과 E. coli에대해효과적인살균력을갖는것으로확인되었다. Mahmoud와 Linton 27) 의연구에따르면, 이산화염소가스를 5.0 mg/l의농도로 E. coli와 Salmonella enterica에각각 14.5분과 19분동안적용할결과, 5 logcfu/g 만큼감소하였다고보고하였으며, Himathongkham 등 28) 은알파파씨앗을유리병에넣고, S. typhimurium을 10 8-10 9 CFU/g의농도로접종시켜오염시킨후, 암모니아가스를 180 mg/l 의농도로주입하고, 밀봉한상태로 20 o C에서 22시간동안놓아둔결과, S. typhimurium 수가 2-3 logcfu/g 만큼감소하였다고보고하였다. 또한, Sapers 등 29) 은사과에 E. coli를접종하고이산화염소가스를 0.3 mg/l의농도로처리한결과, 4.5 logcfu/g가감소하였다고보고하였다. 처리대상, 처리농도, 그리고처리시간등을고려할경우, 본연구에서사용한 Fumagari OPP 의 S. typhimurium 과 E. coli에대한살균효과는 Mahmoud와 Linton 27) 의연구 Table 3. Viable counts (CFU/mL) of S. typhimurium in the carriers exposed to disinfectant and antibacterial effect of the fumigant Dilution time Colony number in carriers 1) n'1 2) n'2 3) d value (log) 4) C1 C2 C3 10 2 0 0 0 23 0 15 0 5.26 10 3 0 0 0 18 0 1) C1, C2, C3, test-carriers. 2) n'1, the mean number of bacteria in recovery solution. 3) n'2, the mean number of bacteria on carriers plated in agar. 4) d, the reduction of bacterial number. d = logt log(n'1 + n'2) = log[t/(n'1 + n'2)] (T, the mean number of bacteria recovered on the carriers) Table 4. Viable counts (CFU/mL) of E. coli in the carriers exposed to disinfectant and antibacterial effect of the fumigant Dilution time Colony number in carriers 1) n'1 2) n'2 3) d value (log) 4) C1 C2 C3 10 2 0 0 0 6 0 9 0 5.64 10 3 0 0 0 5 0 1) C1, C2, C3, test-carriers. 2) n'1, the mean number of bacteria in recovery solution. 3) n'2, the mean number of bacteria on carriers plated in agar. 4) d, the reduction of bacterial number. d = logt log(n'1 + n'2) = log[t/(n'1 + n'2)] (T, the mean number of bacteria recovered on the carriers) 결과보다는낮았으나, Himathongkham 등 28) 과 Sapers 등 29) 의연구결과보다는높았던것으로사료된다. 이상의연구결과로부터, OPP를주성분으로하는훈증소독제, Fumagari OPP 은비다공성의표면에부착되어있는 S. typhimurium과 E. coli에적용할경우, 5.0 logcfu 이상의감소결과를나타내어, S. typhimurium과 E. coli의살균을목적으로사용할수있는훈증살균소독제로사료된다. 감사의글 본연구는 ( 주 ) 엘캄코바이오 ( 서울 ) 의연구용역으로수행되었으며, 이에감사드립니다. 요약 본연구는 E. coli와 S. typhimurium을대상으로 orthophenylphenol 20% 를함유한훈증소독제, Fumagari OPP 의살균효과를평가하기위해수행되었다. 예비시험에서, E. coli와 S. typhimurium의현탁액균수는모두 4.0 10 8 CFU/ ml이었으며, 모든훈증소독제에노출시킨담체의균수는모두평판배지법과여과법으로배양한시험균주현탁액의

Bactericidal Efficacy of Fumagari OPP, Fumigant Against Escherichia coli and Salmonella typhimurium 239 균수의 50% 보다많았다. 또한, 대조담체로부터회복된 E. coli와 S. typhimurium 균수는모두 3.4 10 6 CFU/mL이었다. 훈증소독제의살균효과시험에서는, 훈증소독제를처리한담체의 E. coli와 S. typhimurium의감소균수는각각 5.64와 5.26 logcfu/ml로나타났다. 이상의결과로부터, 훈증소독제, Fumagari OPP 은 E. coli와 S. typhimurium에대해효과적인살균력을갖는것으로확인되었으며, 병원성세균에오염된식품재료및주방용품의소독에적용할수있을것으로사료된다. 참고문헌 1. Ryu, S.H., Lee, J.H., Park, S.H., Song, M.O., Park, S.H., Jung, H.W., Park, G.Y., Choi, S.M., Kim, M.S., Chae, Y.Z., Park, S.G. and Lee, Y.K.: Antimicrobial resistance profiles among Escherichia coli strains isolated from commercial and cooked foods. Int. J. Food Microbiol. 159, 263-266 (2012). 2. Zhao, T., Doyle, M.P., Zhao, P., Blake, P. and Wu, F.M.: Chlorine inactivation of Escherichia coli 0157:H7 in water. J. Food Prot. 64, 1607-1609 (2001). 3. Erkmen, O.: Antimicrobial effects of hypochlorite on Escherichia coli in water and selected vegetables. Foodborne Pathog. 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