Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Combination of Hydrophobic Filtration and Enrichment Methods for Detecting Bacillus cereus in Fresh-Cut Cabbage

  • Lee, Sujung (Department of Food and Nutrition, Sookmyung Women's University) ;
  • Choi, Yukyung (Department of Food and Nutrition, Sookmyung Women's University) ;
  • Lee, Heeyoung (Risk Analysis Research Center, Sookmyung Women's University) ;
  • Kim, Sejeong (Department of Food and Nutrition, Sookmyung Women's University) ;
  • Lee, Jeeyeon (Department of Food and Nutrition, Sookmyung Women's University) ;
  • Ha, Jimyeong (Department of Food and Nutrition, Sookmyung Women's University) ;
  • Oh, Hyemin (Department of Food and Nutrition, Sookmyung Women's University) ;
  • Lee, Yewon (Department of Food and Nutrition, Sookmyung Women's University) ;
  • Kim, Yujin (Department of Food and Nutrition, Sookmyung Women's University) ;
  • Yoon, Yohan (Department of Food and Nutrition, Sookmyung Women's University) ;
  • Lee, Soomin (Risk Analysis Research Center, Sookmyung Women's University)
  • Received : 2018.04.05
  • Accepted : 2018.10.01
  • Published : 2018.10.30
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Abstract

This study developed a rapid detection method for Bacillus cereus in fresh-cut cabbages. Fresh-cut cabbage samples were inoculated at 1-, 2- and 3-Log CFU/g, and pathogens were enriched in tryptic soy broth containing 0.15% polymyxin B at $30^{\circ}C$, $37^{\circ}C$, and $42^{\circ}C$ to determine the detection limit and appropriate enrichment temperature for multiplex PCR detection. Enriched bacterial cells in enrichment broth were collected in a hydrophobic filter prior to DNA extraction for multiplex PCR. Filters were resuspended in distilled water, and DNA was extracted from the suspension. DNA samples were further analyzed by multiplex PCR. Detection limit of multiplex PCR was 5-Log CFU/mL. B. cereus cell counts were higher (P < 0.05) at $42^{\circ}C$ than other temperatures. Detection rate of 1-, 2-, and 3-Log CFU/g inoculated samples were 60%, 80%, and 100% after enrichment respectively. However, when enriched samples were filtered with hydrophobic membrane filter, detection rates became 100%, regardless of inoculation level. Results indicate a combination of enrichment with hydrophobic filtration improves rapid detection efficiency of B. cereus in fresh-cut cabbage by multiplex PCR.

본 연구에서는 신선편이 양배추 내 Bacillus cereus의 최적 증균 온도를 선정하고 증균배양액에 소수성필터를 적용하여 multiplex PCR의 검출률을 확인하였다. B. cereus 증균온도는 B. cereus 균주 5 개를 혼합하여 1 Log CFU/mL이 되도록 증균배지에 접종하고 $30^{\circ}C$, $37^{\circ}C$, $42^{\circ}C$에서 증균한 뒤 3시간 간격으로 MYP agar에 도말한 후 계수하여 선정하였다. 소수성필터 미적용 그룹은 B. cereus 균주 5 개를 혼합하여 신선편이 양배추에 접종한 뒤 최적 증균온도에서 증균하였으며, 증균배양액을 가열하여 DNA를 추출한 뒤 multiplex PCR을 진행하였다. 소수성필터 적용 그룹은 증균배양액을 소수성 필터에 적용하고 필터에 있는 균을 멸균증류수로 현탁한 뒤 가열하여 추출된 DNA로 multiplex PCR을 진행하였다. 증균온도 확인 결과, 6시간 증균 시 $42^{\circ}C$에서 증균된 샘플($5.4{\pm}0.3Log\;CFU/mL$)과 $30^{\circ}C$에서 증균된 샘플($4.6{\pm}0.6Log\;CFU/mL$) 간 유의차가 확인되었다(p < 0.05). 소수성필터 적용 유무에 따른 multiplex PCR 결과, 1 Log CFU/g 접종된 샘플의 검출률이 소수성 필터 적용 전 60%(3/5)에서 100%(5/5)로 향상되었다. 2 Log CFU/g 접종 샘플은 소수성필터 적용 전 80%(4/5)에서 소수성 필터 적용 후 100%(5/5)로 검출률이 증가하였으나, 3 Log CFU/g 접종 샘플은 소수성 필터 적용 전후 모두 100%(5/5)로 확인되었다. 이상의 결과를 통해 신선편이 양배추 내 B. cereus 검출 시 증균배양액에 소수성필터를 적용하고 multiplex PCR을 적용했을 때 신속하고 효율적인 검출이 가능할 것으로 판단된다.

Keywords

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