효소 , 대사산물 Pseudomonas sp. Aryl Acylamidase 의 정제 및 성질

황인균,방원기 ( In Gyun Hwang,Won Gi Bang ) 한국미생물생명공학회 1998 한국미생물·생명공학회지 Vol.26 No.5

국내 분유 및 영.유아식품에서 분리된 Cronobacter spp. (Enterobacter sakazakii)의 Biofilm 생성 특성 및 내산성 비교

김선애,이유미,오세욱,곽효선,황인균,강동현,우건조,이민석,Kim, Sun-Ae,Lee, Yu-Mi,Oh, Se-Wook,Gwak, Hyo-Sun,Hwang, In-Gyun,Kang, Dong-Hyun,Woo, Gun-Jo,Rhee, Min-Suk 한국축산식품학회 2009 한국축산식품학회지 Vol.29 No.6

We investigated biofilm formation in various media, growth in low pH, and the hemolytic activity of 14 strains of Cronobacter spp. (Enterobacter sakazakii) isolated from a variety of foods including powdered infant formula (n=75), infant cereal (n=100), honey (n=30), and other infant foods (n=100) in Korea. The Cronobacter spp. adhered and formed biofilms on polyethylene, and a greater extent of biofilm was observed in nutrient-rich media. No clear difference in biofilm-forming ability was noted among the media constituents and the pattern of biofilm formation was strain-dependent. Seven strains out of 14 strains (50%) grew at pH 4.1, indicating that the acid resistance of these Cronobacter spp. isolated in Korea was relatively low. Hemolytic activity was not observed in any of the strains. This study provides basic information for the physiological and biochemical characteristics of Cronobacter spp. isolated from a variety of infant foods in Korea.

분말식품에서 Cronobacter spp. 검출을 위한 Real-Time PCR과 배지배양법의 비교검증

천정환,송광영,김선영,현지연,김윤경,황인균,곽효선,서건호,Chon, Jung-Whan,Song, Kwang-Young,Kim, Sun-Young,Hyeon, Ji-Yeon,Kim, Yun-Gyeong,Hwang, In-Gyun,Kwak, Hyo-Sun,Seo, Kun-Ho 한국미생물학회 2011 미생물학회지 Vol.47 No.1

본 연구에서는 분말 식품에서 real-time PCR과 배지배양법을 사용하여 Cronobacter spp.를 검출하는 방법이 비교검증 되었다. 조제분유, 이유식, 미숫가루에 Cronobacter를 인위적으로 접종시킨 후, 식품공전의 방법에 따라 멸균증류수와 Enterobacteriaceae enrichment (EE) broth에서 각각 1, 2차 증균배양 하였으며, Druggan-Forsythe-Iversen에 선택배양하여 Cronobacter를 검출하였다. Real-time PCR은 멸균증류수 및 EE broth에서 1 ml을 채취한 후 DNA를 추출하여 시행하였다. 실험결과 모든 식품에서 배지배양법과 real-time PCR간에는 통계학적 유의차가 존재하지 않았다(p>0.05). 한편 모든 실험회차에서 real-time PCR 수행 시, 1차 증균액인 멸균증류수에서의 양성검출율이 2차 증균액인 EE broth에서보다 높았는데, 이는 2차 증균액 내의 구성성분 중 일부분이 real-time PCR의 반응을 저해했기 때문으로 사료된다. 연구결과를 종합해 볼 때, 1차 증균 후, real-time PCR을 통해 Cronobacter를 검출하는 방법은 정확한 민감도를 보이면서도 시간과 노동력을 절감할 수 있는 효과적인 방법으로 사료된다. The aim of this study was to compare the performance of conventional culture and real-time PCR for detection of Cronobacter spp. in powdered foods. Infant formula, baby food and Misugaru inoculated with Cronobacter were enriched in distilled water as first enrichment step, followed by incubating in Enterobacteriaceae enrichment (EE) broth as second enrichment step. A loopful of enriched sample was streaked onto Druggan-Forsythe-Iversen agar, followed by incubating at $37^{\circ}C$ for 24 h. One milliliter of the enriched distilled water and EE broth were used in real-time PCR assay. No statistical differences were observed in the number of positive samples between culture method and real-time PCR (p>0.05) in all types of food samples. The number of positives of real-time PCR was higher in the first enrichment media (distilled water) than the second enrichment media (EE broth), though there was no significant difference (p>0.05). It appears that some components of the second enrichment broth, EE broth, inhibit the reaction of real-time PCR. These results show that real-time PCR using a single enrichment with distilled water could be useful as an effective screening method for detection of Cronobacter while saving much time and labor compared to conventional culture method.

해산식품과 채소에서 Vibrio parahaemolyticus 검출을 위한 배지배양법과 real-time PCR의 비교검증

천정환(Jung-Whan Chon),현지연(Ji-Yeon Hyeon),황인균(In-Gyun Hwang),곽효선(Hyo-Sun Kwak),한정아(Jeong-A Han),정윤희(Yun-Hee Chung),송광영(Kwang-Young Song),서건호(Kun-Ho Seo) 한국식품과학회 2010 한국식품과학회지 Vol.42 No.3

V. parahaemolyticus는 국내에서 여름과 가을철에 걸쳐 발생되며 생선이나 패류 등의 해산식품을 날것으로 섭취하거나 불완전하게 익혀 먹을 경우 식중독을 일으키는 병원균이다. 본 연구의 목적은 표준 검출기법인 배지배양법을 이용한 V. parahaemolyticus의 검출과 real-time PCR을 이용한 V. parahaemolyticus의 검출에 있어서 그 유효성과 효율성을 검증하는 것이다. V. parahaemolyticus의 발생기록과 발생가능성이 있는 여러 해산식품과 무순에 적절한 균량을 접종하고 APW로 증균배양하였다. 증균배양이 끝난 후 TCBS선택배지에 배양액을 획선도말하고, 동시에 증균배양액에서 1 ㎖을 채취하여 real-time PCR을 실시하였다. TCBS에서 초록색이 나온 집락을 1-3개 선별하여 TSI 배지에 접종하여 screening test를 거친 후 API 20NE strip을 사용하여 확인동정하였다. 또한 정상세균총이 목적균의 성장과 검출에 어느 정도의 영향을 미치는지 평가하기 위하여 25 g의 식품 내 정상세균총의 수준을 함께 측정하였다. 실험결과, 자체 제작한 real-time PCR서열은 V. parahaemolyticus를 특이적으로 검출할 수 있었고 검출한계는 PBS에서 10³ CFU/㎖ 였다. 또한, 식품 내 정상세균총이 높을 경우 증균배양 시 목적균의 성장에 영향을 미칠 수 있다는 것을 확인하였다. Real-time PCR은 180개의 전체 샘플 중 76개의 양성 결과를 보여 66개의 양성 결과를 낸 배지배양법에 비해 더 많은 양성 검출율을 보였으나 통계학적인 유의차는 발견되지 않았다. Real-time PCR은 표준검출법인 배지배양법과 비교해 볼때 동등하거나 우수한 검출력을 지닌 것으로 보이며 이러한 realtime PCR법은 24시간 이내에 확정동정까지 가능하여 시간과 노동력의 소모가 많은 배지배양법에 앞서 선별검사로 사용할 경우 시간, 비용, 노동력 절감에 매우 유효할 것으로 판단된다. Vibrio parahaemolyticus (V. parahaemolyticus), which is commonly found in raw seafood, causes gastroenteritis in humans. Rapid and effective methods have been developed as culture methods require up to 5-7 days. In this study, real-time PCR was compared with the standard culture method for detecting V. parahaemolyticus in seafood and radish sprout samples. Five hundred grams of the samples were artificially contaminated with V. parahaemolyticus then divided into 20 samples. The samples were incubated in alkaline peptone water and then streaked onto thiosulfate-citrate-bile saltssucrose agar. Biochemical tests for suspicious colonies were performed using the API 20NE strip. In parallel, real-time PCR was performed targeting the toxR gene using the enrichment broth. The real-time PCR was sensitive in discriminating V. parahaemolyticus from other foodborne pathogens. The detection limit of the real-time PCR was 10³ CFU/㎖ in phosphate-buffered saline. Although the real-time PCR detected more positive samples (76 out of 180, 42%) than the culture method (66 out of 180, 37%), there was no significant statistical difference (p>0.05) between the two methods. In conclusion, real-time PCR assays could be an alternative to the standard culture method for detecting V. parahaemolyticus in seafood and radish sprouts, which has many advantages in terms of detection time, labor, and sensitivity.

회원논단 : 식품위해요소로서의 메치실린내성 황색포도상구 (MRSA)

황인균 ( In Gyun Hwang ),곽효선 ( Hyo Sun Kwak ),윤상현 ( Sang Hyeon Yoon ) 한국식품위생안전성학회 2010 Safe Food Vol.5 No.1

기획특집 : 국내외 식품 중 화학물질의 모니터링 데이터베이스 및 위해평가 시스템 동향

정다인 ( Da In Jeong ),강희승 ( Hui Seung Kang ),황명실 ( Myung Sil Hwang ),황인균 ( In Gyun Hwang ),민성기 ( Sung Gi Min ),이헌주 ( Hun Joo Lee ) 한국식품위생안전성학회 2015 Safe Food Vol.10 No.3

가공식품과 비가공식품에서의 황색포도상구균 검출을 위한 배지법과 Real-time PCR법의 비교

이재훈,송광영,현지연,황인균,곽효선,한정아,정윤희,서건호,Lee, Jae-Hoon,Song, Kwang-Young,Hyeon, Ji-Yeon,Hwang, In-Gyun,Kwak, Hyo-Sun,Han, Jeong-A,Chung, Yun-Hee,Seo, Kun-Ho 한국축산식품학회 2010 한국축산식품학회지 Vol.30 No.3

Staphylococcus aureus is one of the major pathogens that can cause staphylococcal infection and food poisoning. In this study, we compared conventional culture methods and real-time PCR for detection of S. aureus in artificially inoculated milk, sausage, raw pork, and vegetable salad. The performance of a coagulase test for confirming S. aureus was also compared with a colony PCR test. Bulk food samples (500 g each) were artificially inoculated with S. aureus and divided into 20 samples (25 g or mL each). All samples were added to tryptic soy broth (225 mL/sample) with 10% NaCl and incubated at $37^{\circ}C$ for 24 h. After the enrichment, broth cultures were streaked onto Baird-Parker (BP) agar with egg yolk tellulite, and incubated at $37^{\circ}C$ for 24 h. In addition, 1 mL of broth cultures was collected to perform real-time PCR. Two suspicious colonies from the BP agar were picked up and plated on nutrient agar and incubated at $37^{\circ}C$ for 24 h followed, by a coagulase confirmation test and a colony PCR analysis. There were no statistical differences between culture methods and realtime PCR in food samples with low background microflora, such as milk and sausage. However, a significant statistical difference was found between the culture methods and real-time PCR for raw pork and vegetable salad. Furthermore, the colony PCR test of the presumptive colonies on BP agar for confirming S. aureus is more accurate and efficient than the coagulase test for unprocessed foods.

국내 미등록 유기인계 농약의 수입 농식품에 대한 다성분 잔류분석법

전영환 ( Young Hwan Jeon ),황정인 ( Jeong In Hwang ),안지운 ( Ji Woon Ahn ),김효영 ( Hyo Young Kim ),도정아 ( Jung Ah Do ),오재호 ( Jae Ho Oh ),황인균 ( In Gyun Hwang ),임무혁 ( Moo Hyeog Im ),이중근 ( Joong Keun Lee ),이영득 ( Yo 한국환경농학회 2012 한국환경농학회지 Vol.31 No.3

외국으로부터 수입되는 농식품에 대하여 국내에 미등록 된 유기인계 농약 aspon, chlorthion, chlorthiophos, crotoxyphos, demeton-O, demeton-S, demeton-S-methyl, dioxathion, heptenophos, iodofenphos, leptophos, methyl-trithion, propetamphos 및 sulfotep 등 14종에 대한 안전성 평가를 위하여 다성분 잔류분석법을 확립하고자 하였다. 잔류분석법은 식품의약품안전청에서 고시한 다종농약다성분 동시분석법-제2법에 잘 적용되었다. 수입 농산물의 대표 시료로 선정된 현미와 오렌지에 대한 유기인계 농약 14종 대한 잔류분석법의 밸리데이션을 실시한 결과 특이성, 직선성, 정확성, 정밀성 및 정량한계 수준을 만족시키는 것으로 나타났다. 단지 crotoxyphos의 경우는 기기상의 정량한계가 낮아서 저농도에서의 회수율은 좋지 않은 결과를 나타내었다. 따라서 본 연구에서 확립된 다성분 잔류분석법은 수입 농산물 중 crotoxyphos를 제외한 aspon외 12종의 유기인계 농약에 대해 적용 가능한 것으로 나타났다. BACKGROUND: For safety evaluation of imported agri-food in Korea, the multiresidue analysis method was establised for unregistered organophosphorus pesticides, aspon, chlorthion, chlorthiophos, crotoxyphos, demeton-O, demeton-S, demeton-S-methyl, dioxathion, heptenophos, iodofenphos, leptophos, methyl-trithion, propetamphos and sulfotep. METHODS AND RESULTS: The used method for multiresidue analysis in brown rice and orange used as representative samples of imported agri-food was the official method of Korean Food and Drug Administration. The results of validation test of 13 organophosphorus pesticides except crotoxyphos for multiresidue analysis method are compared to the criteria such as specificity, linearity, accuracy, precision and limit of quantification. CONCLUSION: The used method for multiresidue analysis of unregistered 13 organophosphorus pesticides except crotoxyphos in Korea can surely be used as an official method for routine analysis of imported agri-food.

시판 횟감어류에서의 Vibrio parahaemolyticus 분포 및 항생제 감수성에 관한 연구

성창현 ( Chang Hyeon Sung ),천정환 ( Jeong Hwan Cheon ),현지연 ( Ji Yeon Hyeon ),황인균 ( In Gyun Hwang ),곽효선 ( Hyo Sun Kwak ),윤상현 ( Sang Hyeon Yoon ),이정수 ( Jeong Soo Lee ),정윤희 ( Yun Hee Chung ),송광영 ( Kwang Young 한국수의공중보건학회 2010 예방수의학회지 Vol.34 No.3

Vibrio parahaemolyticus (V. parahaemolyticus) has been recognized as a significant food-borne pathogen around the world. In this study, we investigated the prevalence and antimicrobial resistance of V. parahaemolyticus isolated from raw fishes. A total of 64 samples of raw fishes purchased from a traditional seafood market in Seoul, Korea. were examined for the presence of V. parahaemolyticus using intestines, gills, and fins. Twenty five grams of all samples were enriched in 225ml of alkaline peptone water at 37℃ for 24h and then streaked onto thiosulfate citrate bile sucrose agar. Suspected colonies were inoculated into triple sugar iron agar for biochemical screening test and were finally confirmed with API 20NE strip. Antimicrobial resistance tests were performed with disc diffusion method in accordance with National Committee for Clinical Laboratory Standard. Thirty three V. parahaemolyticus strains were isolated from raw fishes among 33 out of 64 (51.6%). Among 33 isolates, 16 isolates (48.5%) were resistant to ampicillin, 7 isolates (21.2%) were resistant to amikacin, and all isolates were not resist to other antibiotics such as amoxicillin & clavulanic acid, sulfamethoxazole & trimethopenem, ciprofloxacin, cefotaxime and cefepime. Although the prevalence of V. parahaemolyticus was high in raw fishes compared to other studies, antimicrobial resistance rate of the isolates was relatively low. These results could be useful information for risk assessment of V. parahaemolyticus in raw fishes.

다양한 식품에서 Campylobacter jejuni 검출을 위한 real-time PCR과 배지배양법의 비교검증

천정환(Jung-Whan Chon),현지연(Ji-Yeon Hyeon),황인균(In-Gyun Hwang),곽효선(Hyo-Sun Kwak),한정아(Jeong-A Han),김무상(Moo-Sang Kim),김종현(Jong-Hyun Kim),송광영(Kwang-Young Song),서건호(Kun-Ho Seo) 한국식품과학회 2011 한국식품과학회지 Vol.43 No.1

본 연구에서는 두 종류의 선택배지를 활용한 배지배양법과 realtime PCR의 C. jejuni 검출능력을 비교하였다. 소시지, 쇠고기 분쇄육, 무순에 C. jejuni를 접종하고 Hunt broth로 증균배양 하였으며, mCCD agar와 Preston agar에 배양액을 획선도말하여 미호기적으로 배양하였다. 동시에 증균배양액에서 1 ㎖을 채취하여 realtime PCR을 실시하였다. 실험결과, real-time PCR은 쇠고기 분쇄육과 소세지에서 두 가지 선택배지와 비교하여 동일한 검출력을 보였으나 무순에서는 훨씬 더 많은 양성을 검출하였다(p<0.05). 두 배지간의 비교에서는 Preston agar와 mCCD agar는 통계학적 유의차가 없는 민감도를 보였다(p>0.05). 결론적으로 real-time PCR 은 표준검출법인 배지배양법과 비교하여 동등하거나 우수한 민감도를 지닌 신속검출기법인 것으로 사료되며, 배지배양법에 앞서 선별검사로 사용할 경우 시간, 비용, 노동력 절감에 있어서 매우 유효한 방법이 될 것으로 판단된다. In this study, performances of culture methods using two selective media and real-time PCR were evaluated for detection of Campylobacter jejuni (C. jejuni) in various food samples. Sausage, ground beef, and radish sprouts inoculated with C. jejuni were enriched in Hunt broth and then streaked onto modified cefoperazone charcoal deoxycholate agar and Preston agar, followed by incubation under microaerobic conditions. The enriched Hunt broth (1 ㎖) was used in real-time PCR assay. No statistical differences were observed in sensitivity among the two selective media and real-time PCR for sausage and ground beef. However, the number of positives by real-time PCR in radish sprouts was much higher than the two selective media (p<0.05). It appears that real-time PCR could be used as an effective screening tool to detect C. jejuni, particularly in foods with a high number of background microflora such as fresh vegetables.