KOREAN J. FOOD SCI. TECHNOL. Vol. 43, No. 2, pp. 161~168 (2011) The Korean Society of Food Science and Technology 한국에유통중인신선편이채소류의미생물품질및병원성세균의오염도조사 배영민 홍유진 강동현 1 허성기 2 이선영 * 중앙대학교자연과학대학식품공학부, 1 서울대학교농업생명과학대학식품동물생명공학부, 2 농촌진흥청국립농업과학원농산물안전성부유해생물과 Microbial and Pathogenic Contamination of Ready-to-eat Fresh Vegetables in Korea Young-Min Bae, Yu-Jin Hong, Dong-Hyun Kang 1, Sunggi Heu 2, and Sun-Young Lee* School of Food Science and Technology, Chung-Ang University 1 Department of Food and Animal Biotechnology, Seoul National University 2 Microbial Safety Division, Department of Agro-food Safety, National Academy of Agricultural Science, Rural Development Administration Abstract The purpose of this study was to evaluate microbiological contamination of fresh vegetables in Korea. Twenty types of vegetables were tested for total aerobic bacteria, coliforms, Escherichia coli, yeast and mold, and pathogenic bacteria such as Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella, E. coli O157:H7, Cronobacter sakazakii, Shigella, and Campylobacter. Levels of total aerobic bacteria and coliform on 20 vegetables were between 3.74 and 8.04 log CFU/g, and 0.16 and 5.02 log CFU/g, respectively. The highest contamination levels of total aerobic bacteria were observed on water dropwort, sprouts, mungbean sprout, and ballflower root. There was no significant difference (p>0.05) in microbial contamination levels of total aerobic count, coliform, E. coli, yeast and mold between organic and nonorganic vegetables. When isolation methods using selective agars were applied, L. monocytogenes, B. cereus, Salmonella and Campylobacter were isolated from some fresh vegetable samples. Results of API kit tests showed that L. monocytogenes was identified on Chinese cabbage, cucumber, soybean sprouts, and iceberg lettuce while Salmonella was identified on Korean leek. Furthermore, Campylobacter jejuni was also identified in more than 50 of the 100 samples. However, when positive samples from API kit were tested for real-time or 16S rrna sequencing method, only B. cereus from perilla leaf, carrot, water dropwort, and sprouts showed positive results. These results indicate that selective agar and API kit detection methods might result in false positive results for some pathogens. Therefore, studies need to improve isolation or confirmation methods for such pathogens. Keywords: vegetable, microbial contamination, foodborne pathogen, isolation, identification 서 최근식품산업의발전으로인하여국민들의건강에대한관심이높아지면서가공식품보다자연식품을선호하는추세로변화하고있다 (1,2). 이러한변화에따라신선농식품에대한수요가증가하게되었으며, 특히, 익히지않고직접섭취하는비가열즉석섭취 (ready-to-eat) 신선과채류의수요가증가하고있다 (2,3). 이런신선과채류는간편하게섭취할수있는장점이있지만, 일반적으로가열하지않고직접신선한상태로섭취되기때문에병원성미생물이오염되어있을경우식품안전성에위협이될수 *Corresponding author: Sun-Young Lee, School of Food Science and Technology, Chung-Ang University, Anseong, Gyeonggi 456-756, Korea Tel: 82-31-670-4587 Fax: 82-31-676-8741 E-mail: nina6026@cau.ac.kr Received July 28, 2010; revised November 16, 2010; accepted December 12, 2010 론 있다 (4-7). 한국의최근식중독발생통계에따르면, 급식과외식의생활화로인해 5 년전보다식중독발생건수가 3.8 배증가하였고, 환자수는 20% 증가하였으며, 2009 년에도 228 건으로많은수의식중독이보고되었다 (8). 그리고건강에대한관심의증가로샐러드등여러종류의채소를생식하는일이많아지면서새싹및양상추등의신선채소와관련된식중독사고가최근식중독발생통계에서적지않은부분을차지하게되었다 (6,9,10). 2008 년통계에의하면, 자연계및곡류, 채소류에널리존재하는 Bacillus cereus 에의한식중독건수가식자재의오염, 식품간의교차오염등으로인하여증가하고있으며 (11), 그외다른병원성세균에의한식중독도조리종사자의손이나기구의혼용에의해신선채소음식으로교차오염이일어나면서발생하고있는것으로밝혀졌다 (12). 병원성미생물은과일과채소를씻는과정에서도제거되지않아식중독사고에관여되는것으로알려져있으며 (9), 다양한종류의병원성미생물이식중독을야기할수있다. 이중 Shigella spp., Salmonella, Escherichia coli O157:H7, Campylobacter spp., Listeria monocytogenes, Staphylococcus aureus, Yersinia enterocolitica, B. cereus, Clostridium botulinum, 161
162 한국식품과학회지제 43 권제 2 호 (2011) viruses 등이과일과채소와같은신선농식품과관련하여식중독사고및국민의건강상의문제에크게상관성이있는것으로보고되었다 (13). CDC 자료중미국에서 2005-2007년사이신선농식품과관련된식중독사고의원인식품으로는콩, 양배추, 사과, 바나나, 숙주, 콩나물, 시금치, 당근, 딸기, 수박, 메론, 양파등이나타났으며, 그외에도여러채소나과일을혼합시킨샐러드종류가원인식품으로확인되었다 (14). 가장많이오염된병원성세균으로는 Salmonella spp., E. coli O157:H7로나타났으며, 이외에도포자를형성하는균인 B. cereus, Clostridium spp. 와드물게 Campylobacter jejuni, Shigella sonnei가원인균으로확인되었다 (14). 병원성미생물이과일과채소등의신선농식품에오염되었을때병원성미생물은세척시물에닿기어려운틈새에주로오염되어있으며, 표면에 biofilm을형성하여세척및살균과정에저항성을지닌다 (15-17). 최근신선농식품에오염된병원성미생물을효과적으로살균하기위한연구가증가하고있으며, 이러한신선농식품에오염된병원성세균을효과적으로살균소독하기위한기술의개발을위하여한국에유통되고있는신선농식품의미생물품질, 오염된병원성세균의종류및오염수준등에대한기초적인연구가수반되어야할것으로사료된다. 이에본연구에서는한국에유통되는신선채소의미생물품질및병원성세균의오염도를확인하기위하여총 20종의채소류를 5회에걸쳐수거하여각각의채소에오염된총균수, 대장균군, 대장균, 효모및곰팡이의수준을조사하였고, 또한병원성세균의오염도를관찰하기위하여총 8종의병원성세균 (S. aureus, L. monocytogenes, B. cereus, Salmonella, E. coli O157:H7, Cronobacter sakazakii, Shigella, Campylobacter) 을대상으로오염여부를관찰하였다. 깻잎과상추의경우유기농과비유기농의제품을선정하여유기농과비유기농의일반미생물분포를비교평가하였다. 본연구결과는한국에유통중인채소류의오염된병원성세균의종류와미생물학적안전성등에대한유용한정보를제공하며관련제품의살균기술개발연구를위한기초자료로써활용될수있다. 재료및방법 실험재료미생물분포에대한조사를위한신선채소류는 2009년 9월부터 10월까지경기도안성시소재의대형마트및일반마트에서구입하여사용하였다. 제품은가열과정이없이섭취하는채소샐러드를중심으로 20종을 5회에걸쳐총 100개의제품을구입하여사용하였으며 ( 동일 20종의상품을 5회구입 ), 구입후에는냉장상자로운반하여총균수 (total aerobic bacteria), 대장균군 (coliform), 대장균 (E. coli), 효모및곰팡이 (yeast & molds), S. aureus, L. monocytogenes, B. cereus, Salmonella, E. coli O157:H7, C. sakazakii, Shigella, Campylobacter의검출및분리실험에이용하였다. 실험에이용된 20종의채소는다음과같다 : 상추 (romain lettuce), 유기농상추 (organic romain lettuce), 깻잎 (perilla leaf) 유기농깻잎 (organic perilla leaf), 배추 (Chinese cabbage), 부추 (Korean leek), 당근 (carrot), 미나리 (water dropwort), 양배추 (cabbage), 양상추 (iceberg lettuce), 적채 (red cabbage), 오이 (cucumber), 파프리카 (paprika), 새싹 (sprouts), 양파 (onion), 마늘쫑 (garlic flower stalk), 콩나물 (soybean sprout), 숙주나물 (mungbean sprout), 도라지 (bellflower root), 고추 (red pepper). 각제품은냉장온도에서보관하면서구입후 1일이내에실험에사용되었다. 일반미생물의정량적분석시료의일반미생물의정량적분석은식품공전법 (18) 을바탕으로수행되었다. 시료 25 g 을정량한후 buffered pepton water (BPW, Difco Laboratories, Detroit, MI, U) 225 ml 이담긴멸균 stomacher bag 에넣어 10 배희석한후 stomacher(bagmixer 400, Interscience, Bretèche, France) 를이용하여 2 분간균질화하였다. 균질화된시료는 9mL 의 buffered peptone water(difco) 를이용하여 10 배씩연속희석하였다. 총균수의측정을위해서각각의희석된시료 1mL 을 petrifilm aerobic count plate(3m, Seoul, Korea) 위에분주하여 30 o C 에서 24-48 시간배양하였으며, 배양후 petrifilm 위에형성된균체 (colony) 를계수하여 colony-forming unit(cfu)/g 으로나타내었다. 또한대장균군및대장균의정량적분석을위해서는 petrifilm E. coli/coliform count plate(3m) 에위에서준비한시료 1mL 을분주하여 37 o C 에서 24-48 시간배양하였다. 배양후기포를가진파란색균체를대장균양성으로, 기포를가진붉은색균체와기포를가진파란색균체를대장균군양성으로간주하여계수하였다. 효모및곰팡이의정량적분석을위해서는 petrifilm yeast/molds count plate(3m) 에위에서준비한시료 1mL 을분주하여 37 ο C 에서 24-48 시간배양하였다. 배양후녹색의균체를효모및곰팡이의양성으로간주하여계수하였다. 유해세균오염도분석및동정각각의병원성미생물은총 8종 (S. aureus, L. monocytogenes, B. cereus, Salmonella, E. coli O157:H7, C. sakazakii, Shigella, Campylobacter) 에대하여조사되었으며, 신선농식품에서병원성미생물을분리하는방법은 E. coli O157:H7 의경우식품공전법 (18) 을이용하였고, 나머지균은 FDA BAM방법 (19) 을이용하여조사하였다. 동정은각각의선택배지에전형적인군체을선택하여 API kit(biomérieux, Marcy I Etoile, France) 를사용하여동정하였다. Table 1은각각의병원성세균의분리및동정에이용한배지및 kit를보여주고있다. 각각의병원성세균의선택배지는호기성환경에서배양되었으며, Campylobacter의경우혐기배양용기 (Difco) 와가스팩 (CampyGen TM, Oxoid, Ogdensburg, NY, U) 을이용하여미호기적환경에서배양하였다. Real-time 분석 API kit의방법에서병원성세균으로동정된균주는 Chromo4 TM Real-time system(bio-rad, Richmond, CA, U) 로확인하였다. Template를얻기위해서분리된병원성세균을 tryptic soy agar(t, Difco) 에 37 o C에서 24시간배양한뒤배양된균체는 300 µl의멸균증류수가담긴마이크로튜브에넣었다. 준비된튜브는 10분동안끓인후 12,000 g의조건으로 5분간원심분리하여상등액인 DNA 농축액을수거하였다. Real-time 를위해 real-time kit(kogenbiotech Co., Seoul, Korea) 를선택하여사용하였으며 (Table 1), 각각 kit의 primer 4 µl, probe mixture 10 µl, 그리고준비된각각의 template 6 µl을 96-well에넣어총 20 µl가되게준비한후원심분리하여 real-time 분석을하였다. 분석후 Opticon Moniter2 TM Software(Bio-RAD, Richmond, CA, U) 를이용하여확인하였다. 16S rrna Sequencing 분석 Real-time 분석에서확실하게확인되지않은 Campylobacter 양성으로나온 3 가지균주는 16S rrna sequencing 을이용하여최종확인하였으며, 16S rrna sequencing 은 Macrogen Inc. (Seoul, Korea) 에의뢰하여분석하였다.
한국에유통중인신선편이채소류의미생물품질및병원성세균의오염도조사 163 Table 1. Culture media 1) and identification methods for pathogenic bacteria isolated from fresh vegetables in this study Strain Enrichment broth Selective agar Identification method API kit Real-time 2) 16S rrna sequecing 3) Staphylococcus aureus Listeria monocytogenes Bacillus cereus Tryptic soy broth (Difco Laboratories, Detroit, MI, U) in 10% NaCl Listeria Enrichment broth (Difco) Butterfield s phosphate-buffered dilution water (Difco) Mannitol salt agar (Difco) in Egg york Emulsion (Difco) Oxford agar base (Difco) in Listeria selective supplement (Oxoid) Bacillus cereus agar base (Oxoid) in Polymyxin B Supplement (Oxoid), Egg york Emulsion (Difco) API Staphy (BioMérieux, Marcy I Etoile, France) API Listeria API 50CHB - - PowerChek TM Listeria monocytogenes Real-time kit (R0106, Kogenbiotech, Seoul, Korea) PowerChek TM Bacillus cereus Real-time kit (R0104, Kogenbiotech) - Salmonella Rappaport-Vassiliadis R10 broth (Difco) Tetrathionate broth (Difco) Buffered pepton water (Difco) Xylose lysine desoxycholate agar (Difco) API 20E PowerChek TM Salmonella spp. Real-time kit (R0108, Kogenbiotech) - Escherichia coli O157:H7 Cronobacter sakazakii E.C. broth (Oxoid, Hampshire, UK) E.E. broth (Oxoid) Sorbitol MacConkey agar (Difco) Chromogenic Enterobacter sakazakii medium (Oxoid) API 20E API 20E - - - - Shigella Buffered pepton water (Difco) in novobiocin 0.5 µg/ml MacConkey agar (Difco) API 20E - - Campylobacter Bolton broth (Oxoid) mccda agar (Oxoid) API campy PowerChek TM Campylobacter jejuni Real-time kit (R0101, Kogenbiotech) Macrogen Inc., (Seoul, Korea) 1) Culture media for E. coli O157:H7 and other pathogens quote from KFDA Food Code and FDA Bacteriological Analytical Manual (BAM), respectively. 2) Samples showing positive results on tests using API kit were subsequently subjected for real-time method. 3) The only 3 samples showing slightly positive results on real-time methods for Campylobacter were identified using 16S rrna sequencing method.
164 한국식품과학회지제 43 권제 2 호 (2011) 통계처리관찰된실험결과는 S 통계프로그램 (version 9.1, S Institute, Cary, NC, U) 의 ANOVA procedure 을이용하여분석되었다. 각각의처리군이통계적인유의차를나타내는경우에 (p 0.05) 각각의 5 반복실험에의한평균값은 Duncan s multiple range test 를이용하여산출하였다. 결과및고찰 일반미생물오염수준신선농식품의총균수와대장균군, 대장균, 효모및곰팡이수에대한검사결과는 Table 2 과같다. 총균수의경우 3.74-8.04 log CFU/g 로높은수준의균이검출되었다. 특히, 미나리, 새싹, Table 2. Averages and ranges (Log 10 CFU/g) 1) of bacterial count isolated from 20 different fresh vegetables in Korea Product Total aerobic bacteria Coliform E. coli Yeast/Molds Romain lettuce Organic romain lettuce Perilla leaf Organic perilla leaf Chinese cabbage Korean leek Carrot Water dropwort Cabbage Iceberg lettuce Red cabbage Cucumber Paprika Sprouts Onion Garlic flower stalk Soybean sprout Mungbean sprout Bellflower root Red pepper Average 6.95±0.35 bac2) 3.28±1.79 bdac 1.06±1.46 a 0.34 ±0.76 b Range 6.32-7.17 0.30-5.14 0-2.84 0-1.70 Average 6.84±0.63 bdac 2.77±1.99 ebdacf 0.00±0.00 b 0.00 ±0.00 b Range 6.05-7.70 0.60-4.99 0 0 Average 6.21±0.57 ebdgcf 1.57±1.75 ebdcf 0.00±0.00 b 0.00 ±0.00 b Range 5.60-7.11 0-3.50 0 0 Average 6.31±1.29 ebdagcf 2.95±2.21 ebdac 0.62±1.00 ba 0.00 ±0.00 b Range 5-7.81 0.69-6.03 0-2.30 0 Average 6.77±0.78 ebdac 0.97±1.23 edf 0.00±0.00 b 0.14 ±0.31 b Range 5.53-7.54 0-2.90 0 0-0.69 Average 6.35±1.50 ebdagcf 2.15±2.13 ebdcf 0.00±0.00 b 0.54 ±1.21 b Range 4.15-7.91 0-5.63 0 0-2.68 Average 5.00±0.72 hgf 1.34±1.52 edcf 0.00±0.00 b 0.00 ±0.00 b Range 4.13-5.75 0-3.43 0 0 Average 7.34±0.82 bac 3.95±1.08 ba 0.52±0.71 ba 0.96±0.88 ba Range 6.39-8.32 3.07-5.77 0-1.30 0-2.04 Average 4.82±1.99 edhgf 1.78±1.66 ebdcf 0.40±0.89 b 0.00 ±0.00 b Range 2.84-7.25 0-3.46 0-2 0 Average 5.69±1.32 edgcf 0.66±1.17 edf 0.00±0.00 b 0.00 ±0.00 b Range 4.2-7.30 0-2.60 0 0-1.85 Average 5.05±1.35 ehgf 1.16±1.09 edf 0.00±0.00 b 0.00 ±0.00 b Range 4.02-7.27 0-2.30 0 0 Average 5.14±0.75 edhgf 0.94±1.64 edf 0.00 ±0.00 b 0.00 ±0.00 b Range 3.99-5.81 0-3.78 0 0 Average 4.81±2.20 hg 1.22±1.23 edf 0.00 ±0.00 b 0.37 ±0.83 b Range 3.04-8.48 0-2.60 0 0-1.85 Average 7.78±1.00 ba 3.80±2.20 bac 0.00 ±0.00 b 1.77±1.01 a Range 6.17-8.67 1.87-6.19 0 0-2.37 Average 3.74±1.52 h 0.16±0.35 f 0.00 ±0.00 b 0.00 ±0.00 b Range 1.68-5.46 0-0.77 0 0 Average 6.07±0.75 ebdgcf 0.59±0.68 ef 0.00 ±0.00 b 0.00 ±0.00 b Range 5.34-7.30 0-1.41 0 0 Average 6.61±2.06 ebdacf 2.87±2.69 ebdac 0.00 ±0.00 b 0.10 ±0.21 b Range 3.04-8.13 0-5.38 0 0-0.477 Average 7.97±0.35 a 5.02±2.8 a 0.00 ±0.00 b 1.74±1.47 a Range 7.61-8.43 0-6.35 0 0-3.38 Average 8.04±0.33 a 2.96±2.13 ebdac 0.00 ±0.00 b 0.95±1.35 ba Range 7.67-8.41 0-5.64 0 0-2.90 Average 4.93±0.29 hgf 0.42±2.03 ef 0.00 ±0.00 b 0.00±0.00 b Range 4.50-5.27 0-2.10 0 0 1) Data represent means±standard deviations of five measurements. 2) Means with the same letter within a column are not significant different (p>0.05).
한국에유통중인신선편이채소류의미생물품질및병원성세균의오염도조사 165 Fig. 1. Levels (Log 10 CFU/g) of microbial contamination of nonorganic and organic romain lettuce and perilla leaves. There was no significant difference on levels of total aerobic count, coliform, E. coli, and yeast & mold between conventional product and organic products (n=5, p>0.05). 숙주, 도라지에서 7 log CFU/g 이상의균이검출되었으며, 도라지의경우 8.04 log CFU/g 로가장높은균이검출되었다. 상추, 유기농상추, 깻잎, 유기농깻잎, 배추, 부추, 마늘쫑, 콩나물의경우 6 log CFU/g 이상의균이검출되었다. 양파의경우총 20 종의채소중가장적은양인 3.74 log CFU/g 가검출되었다. 대장균군은 0.16-5.02 log CFU/g 수준으로검출되었으며, 상추, 유기농상추, 유기농깻잎, 부추, 미나리, 새싹, 콩나물, 숙주, 도라지에서 2 log CFU/g 이상의균이검출되었다. 미나리의경우 3.95 log CFU/g, 새싹은 3.80 log CFU/g 가검출되었으며, 숙주의경우 5.02 log CFU/g 의높은균이검출되었다. 대장균의경우 0.40-1.06 log CFU/g 수준이검출되었으며, 상추, 유기농깻잎, 미나리, 양배추에서균이검출되었다. 효모및곰팡이의경우 0.00-2.68 log CFU/g 수준으로검출되었으며, 효모및곰팡이는상추, 배추, 부추, 미나리, 파프리카, 새싹, 콩나물, 숙주, 도라지에서검출되었다. 새싹의경우 1.77 log CFU/g, 숙주는 1.74 log CFU/g 의수준으로검출되어상대적은높은수준이검출되었다. 상추, 깻잎과유기농상추와유기농깻잎의비교결과는 Fig. 1 과같다. 일반농산물과유기농농산물의미생물수준은총균수가 6 log CFU/g 수준이며, 대장균군은 1.57-3.28 log CFU/g 수준을나타냈다. 대장균은일반상추에서 1.06 log CFU/g, 유기농깻잎에서 0.62 log CFU/g 가검출되었으며, 효모및곰팡이는일반상추에서 0.34 log CFU/g 가검출되었다. 하지만전네가지미생물군에서일반농산물과유기농농산물의유의적차이는나타나지않아일반농산물과유기농농산물의일반미생물적품질에는크게차이가없음이나타났다 (p>0.05). 본연구는한국에서유통되는신선농식품의일반미생물분포를조사하였다. 총 20 종의신선농식품의총균수및대장균군의분포는비교적높은수준으로나타났으며, 이는기존의몇몇연구결과와유사성을보여주고있다. Choi 등 (21) 의신선채소류의호기성세균의조사연구에서상추는평균 7.01-7.10 log CFU/ g, 범위 6.04-8.01 log CFU/g 로관찰되었으며, 깻잎은평균 6.44-6.69 log CFU/g, 범위는 4.86-7.79 log CFU/g 이고, 오이는평균 5.27-5.37 log CFU/g, 범위는 3.48-6.57 log CFU/g 로확인되었다. 또한 Kim 등 (22) 의채소샐러드의미생물오염조사에서는즉석섭 취용샐러드제품에서 60.8% 의대장균군이검출되었으며, E. coli 가 17 건, S. aureus 가 3 건, Salmonella spp. 가 1 건검출되었다. Kim 과 Shin(23) 의연구에서는전처리채소 8 종에서대하여대장균군의오염도를조사한결과, 채를썬양배추에서 6.9 log CFU/ g, 전처리상추에서 6.0 log CFU/g, 채를썬당근에서 6.6 log CFU/g, 얇게썬당근에서 3.4 log CFU/g, 채를썬양파에서 6.4 log CFU/g, 반달썰기호박에서 5.4 log CFU/g, 채를썬오이에서 6.1 log CFU/g 로각각대장균군이오염되어있는것이확인되었다. 반면 Cho 등 (24) 의유통중인양배추의미생물수준을분석한결과에서는총균수가 9.21 log CFU/g, 대장균군이 6.60 log CFU/g 수준으로분포되어있는것을확인하였다. 다른유기농채소의미생물오염도를조사한연구결과에서는당근, 감자, 상추, 오이등의유기농채소에서총호기성세균수는 4-6 log CFU/g 수준을보였으며대장균은 82.1% 의검출률을보였다 (24). Solberg 등 (20) 은급식업소에서음식의미생물적안전기준치를총균수 5.0 log CFU/g 이하, 대장균수 3.0 log CFU/g 이하로제시하고있으며, 몇몇신선채소는본기준을초과하는것으로나타났다. 유해세균오염도분석및동정결과 Table 3 은총 20 종의신선농식품에서분리된병원성미생물에대한선택배지및 API kit, real-time, 16S rrna sequencing 법을이용한동정결과를보여주고있다. 총 8 종의병원성미생물을선택배지로분리했을때, L. monocytogenes 는배추, 양상추, 오이, 콩나물등의시료에서총 100 개중 5 개의시료에서검출되었다. B. cereus 는상추, 깻잎, 당근, 미나리, 새싹에서각각 1 개씩의시료에서발견되어 5 건의시료에서발견되었으며, Campylobacter 의경우는 20 종모두에서한번이상검출되었으며총 100 개의시료중 38 건에서확인되어선택배지를이용한분리법으로분리하였을때가장높은검출율을보였다. Salmonella 는부추 1 시료에서확인되었으며, 그외 S. aureus, E. coli O157:H7, C. sakazakii, Shigella 의경우는전체 100 개의시료에서검출되지않았다. 이렇게분리된병원성세균을 1 차동정으로 APT kit 를이용하여동정하였을때, S. aureus 의경우같은 Staphylococcus 종은많이검출되었으나위험성이높은 S. aureus 는검출되지않았으며, S. sciuri 가 90% 이상의높은확률로많이분리되었다. Campylobacter 의경우 Campylobacter jejuni 가 60% 이상의확률로많이검출되었으며, 많은종류의채소시료에서분리된 Campylobacter 가 C. jejuni, C. fetu, C. coli 로확인되었다. Listeria 의경우병원성이높은 L. monocytogenes 가낮은확률이지만배추, 오이, 콩나물, 양상추에서검출되었으며, Salmonella 의경우 Salmonella choleraesuis ssp arizonae 가부추에서검출되었다. 결과적으로 API kit 를이용하여신선채소에서분리된병원성미생물을동정한결과 Campylobacter, L. monocytogenes, B. cereus 의균이양성으로확인되었으며, 이때양성으로확인된총 48 개의시료를대상으로다음 real-time 법을활용하여 2 차동정에이용하였다. 그결과새싹, 미나리, 당근, 깻잎에분리된 B. cereus 는양성의결과가나타났으며, 반면 L. monocytogenes, Salmonella spp., Campylobacter 의경우 C. jejuni 의 3 개의시료를제외하고는모두음성의결과가나타나 API kit 를이용한동정에서잘못된양성의결과가나타났음을알수있었다. Real-time 법에서음성과양성의결과가불확실했던 C. jejuni 의 3 개의시료를 16S rrna sequencing 법을이용하여확인한결과, 희미한양성의결과를나타냈던 3 개의시료마저모두음성의결과가나타나, 앞선 API kit, realtime 법에서부정확한양성결과가나타남을확인하였다. 각각세가지시료의 16S rrna sequencing 법의확인결과, 깻잎은
166 한국식품과학회지제 43 권제 2 호 (2011) Table 3. Numbers of positive samples for pathogenic contamination of raw vegetables Product 1) Numbers of positive samples S. aureus L. monocytogenes B. cereus Salmonella E. coli O157:H7 C. sakazakii Campylobacter Shigella 2) Romain lettuce 4(0) - 3) (-) 2(0) -(-) 4(1) 0(-) 0(-) -(-) 3(0) -(-) 2(0) -(-) 4(4) 0(-) 0(-) -(-) Organic romain lettuce 5(0) -(-) 5(0) -(-) 4(0) -(-) 1(0) -(-) 4(0) -(-) 2(0) -(-) 4(2) 0(-) 0(-) -(-) Perilla leaf 3(0) -(-) 5(0) -(-) 3(1) 1(-) 2(0) -(-) 1(0) -(-) 3(0) -(-) 4(3) 1(0) 1(0) -(-) Organic perilla leaf 4(0) -(-) 5(0) -(-) 3(0) -(-) 0(-) -(-) 1(0) -(-) 1(0) -(-) 2(2) 0(-) 1(0) -(-) Chinese cabbage 4(0) -(-) 1(1) 0(-) 3(0) -(-) 0(-) -(-) 3(0) -(-) 1(0) -(-) 3(3) 1(0) 0(-) -(-) Korean leek 5(0) -(-) 5(0) -(-) 4(0) -(-) 1(1) 0(-) 3(0) -(-) 1(0) -(-) 3(2) 0(-) 0(-) -(-) Carrot 1(0) -(-) 0(-) -(-) 2(1) 1(-) 0(-) -(-) 3(0) -(-) 1(0) -(-) 1(1) 0(-) 0(-) -(-) Water dropwort 2(0) -(-) 3(0) -(-) 2(1) 1(-) 0(-) -(-) 1(0) -(-) 1(0) -(-) 3(2) 0(-) 3(0) -(-) Cabbage 5(0) -(-) 0(-) -(-) 2(0) -(-) 0(-) -(-) 2(0) -(-) 1(0) -(-) 2(1) 0(-) 1(0) -(-) Iceberg lettuce 4(0) -(-) 2(1) 0(-) 1(0) -(-) 0(-) -(-) 2(0) -(-) 0(-) -(-) 1(1) 0(-) 0(-) -(-) Red cabbage 5(0) -(-) 0(-) -(-) 1(0) -(-) 0(-) -(-) 1(0) -(-) 0(-) -(-) 2(2) 0(-) 0(-) -(-) Cucumber 5(0) -(-) 4(2) 0(-) 2(0) -(-) 0(-) -(-) 4(0) -(-) 1(0) -(-) 3(1) 0(-) 0(-) -(-) Paprika 4(0) -(-) 1(0) -(-) 1(0) -(-) 0(-) -(-) 2(0) -(-) 1(0) -(-) 1(1) 0(-) 0(-) -(-) Sprouts 3(0) -(-) 5(0) -(-) 3(1) 1(-) 1(0) -(-) 0(-) -(-) 1(0) -(-) 4(2) 1(0) 0(-) -(-) Onion 3(0) -(-) 0(-) -(-) 1(0) -(-) 0(-) -(-) 2(0) -(-) 0(-) -(-) 2(2) 0(-) 1(0) -(-) Garlic flower stalk 2(0) -(-) 2(0) -(-) 1(0) -(-) 0(-) -(-) 3(0) -(-) 0(-) -(-) 2(1) 0(-) 0(-) -(-) Soybean sprout 5(0) -(-) 4(1) 0(-) 1(0) -(-) 1(0) -(-) 3(0) -(-) 2(0) -(-) 3(3) 0(-) 0(-) -(-) Mungbean sprout 5(0) -(-) 3(0) -(-) 1(0) -(-) 3(0) -(-) 0(-) -(-) 1(0) -(-) 3(3) 0(-) 0(-) -(-) Bellflower root 1(0) -(-) 3(0) -(-) 1(0) -(-) 2(0) -(-) 1(0) -(-) 2(0) -(-) 2(2) 0(-) 0(-) -(-) Red pepper 4(0) -(-) 1(0) -(-) 0(-) -(-) 1(0) -(-) 1(0) -(-) 2(0) -(-) 1(0) -(-) 1(0) -(-) Total% 74(0) -(-) 51(5) 0(-) 40(5) 4(-) 12(1) 0(-) 40(0) -(-) 23(0) -(-) 50(38) 3(0) 8(0) -(-) 1), isolation methods using selective agar; API, identification methods using API kit. Numbers of positive sample in selective agar method among each 5 samples (numbers of positive sample in API kit methods among positive samples in selective agar method). 2), identification methods using real-time method; 16S, identification methods using 16S rrna sequencing method. Numbers of positive sample in real-time method among positive samples in API kit methods (numbers of positive sample in 16S rrna sequencing method among positive samples in real-time method). 3) Not tested.
한국에유통중인신선편이채소류의미생물품질및병원성세균의오염도조사 167 Acinetobacter baumannii, 배추는 Acinetobacter genomosp. 3, 새싹은 Escherichia coli IAI 1 로각각확인되었다. 기존의다른타연구자에의한신선채소에오염된병원성세균을조사한연구결과를보면, Kim 과 Shin(23) 의연구에서비전처리채소및전처리채소에서 B. cereus 가검출되어본연구와유사한연구결과를나타내었으며 B. cereus 오염도는당근 3.6 log CFU/g, 무 2.9 log CFU/g, 부추 2.5 log CFU/g 수준으로조사되었다. 반면이연구에서는 C. jejuni 가비전처리채소인무와채를썬양배추, 오이채, 당근채에서검출되었고, S. aureus 는비전처리부추에서검출되었다. 비가열채소류의병원성오염도를관찰한다른연구의경우치커리에서 S. aureus 와 B. cereus 가 22% 로가장많이검출되었으며, B. cereus 는쑥갓에서도많이검출되었다 (26). 또한 Kim 등 (22) 의즉석섭취채소샐러드의미생물오염조사연구에서는 S. aureus 가채소샐러드 4 건검출되었으며, Salmonella ariazona 가 1 건의채소에서검출되었다. 하지만본연구결과분리후동정에이용되는방법에따라서병원성세균의검사결과에차이가있음을알수있다. 본연구는한국에서유통되는신선채소의미생물적품질및병원성세균의오염도를조사하여신선농식품의미생물적안전성을평가하고자수행되었다. 그결과전체적으로한국에서유통되고있는신선채소에총균수및대장균군이비교적높은수준으로존재하고, 몇몇신선채소는병원성세균인 B. cereus 가오염되어있는것을확인할수있었다. 한국에유통중인신선채소의미생물적품질관리를위하여지속적인관련제품의미생물적품질평가가이루어져야할것으로사료되며, 관련제품의미생물적안전성을증진시킬수있는상용화가가능한살균소독및저감화기술의개발이이루어져야할것으로생각한다. 요 신선농식품은일반적으로가열하지않고직접신선한상태로섭취하기때문에병원성미생물에오염되어있을경우식중독을일으킬수있다. 이에본연구는한국에유통되는총 20종의채소에서총균수, 대장균군, 대장균, 효모, 곰팡이및병원성세균 (S. aureus, L. monocytogenes, B. cereus, Salmonella, E. coli O157:H7, C. sakazakii, Shigella, Campylobacter) 을측정하여신선농식품의미생물오염실태를조사하였다. 채소의총균수와대장균군의분포는각각 3.74-8.04 log CFU/g, 0.16-5.02 log CFU/g 수준이였으며이중에서미나리, 새싹, 숙주, 도라지에서가장높은수준의미생물이관찰되었다. 유기농과비유기농의상추와깻잎의오염도를관찰했을때, 두군의일반미생물수준에는유의적차이가나타나지않았다. 신선농식품에오염된병원성세균을조사하였을때, 선택배지를이용한분리법에서 L. monocytogenes가배추, 양상추, 오이, 콩나물에서검출되었고, B. cereus는상추, 깻잎, 당근, 미나리, 새싹에서검출되었으며, Campylobacter 의경우는 20종모두에서한번이상검출되었다. 이중 API kit로동정한결과총 48개의시료가양성으로판명되었으며마지막으로 real-time 법과 16S rrna sequencing법을이용하여확인한결과깻잎, 당근, 미나리, 새싹에서발견된 B. cereus만이양성으로확인되었다. 따라서본연구결과로부터몇몇신선농식품에서 B. cereus가검출되었으며비교적높은총균수와대장균군의수준을확인할수있었다. 또한본연구결과분리방법에서선택배지나 API kit를이용하였을때몇몇병원성세균에대해서는맞지않는양성결과가확인되었으므로이에신선농식품에서보다정확하게분리할수있는방법의개발이필요할것으로사료된다. 약 감사의글 본연구는농촌진흥청공동연구사업 ( 과제번호 : PJ006599) 의지원에의해이루어진것이며이에감사드립니다. 문 1. 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