KR101188611B1 - Virulent Phages inhibiting the Growth of Cronobacter sakazakii - Google Patents
Virulent Phages inhibiting the Growth of Cronobacter sakazakii Download PDFInfo
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Abstract
본 발명은 Cronobacter sakazakii 생육을 저해하는 효과를 가지는 독성파아지에 관한 것으로서, 가축 분변 유래의 ES2(수탁번호 KCTC11614BP) 또는 EI-1(수탁번호 KCTC11613BP)에 관한 것이다.
본 발명에 따르면, Cronobacter sakazakii 생육을 인체에 유해하지 않으며, 간단한 방법으로 제어할 수 있게 된다. Cronobacter sakazakii The present invention relates to toxic phages having an effect of inhibiting growth, and to ES2 (Accession No. KCTC11614BP) or EI-1 (Accession No. KCTC11613BP) derived from livestock feces.
According to the present invention, Cronobacter sakazakii Growth is not harmful to the human body, it can be controlled in a simple way.
Description
본 발명은 Cronobacter sakazakii 생육을 저해하는 효과를 가지는 독성파아지에 관한 것으로서, 가축 분변 유래의 ES2(수탁번호 KCTC11614BP) 또는 EI-1(수탁번호 KCTC11613BP)에 관한 것이다. Cronobacter sakazakii The present invention relates to toxic phages having an effect of inhibiting growth, and to ES2 (Accession No. KCTC11614BP) or EI-1 (Accession No. KCTC11613BP) derived from livestock feces.
사카자키균은 주요 매개체로서 건조 조제분유가 보고되며 장염을 일으키기도 하고 피로 들어가면 패혈증, 뇌로 침입하면 뇌수막염을 일으킬 수 있으며 한 살 미만인 아기가 패혈증에 걸릴 경우 10 내지 20%의 사망률을, 뇌수막염일 경우 20 내지 30%의 사망률을 나타낸다. 이는 정상적인 면역력을 가진 사람에게는 크게 문제가 되지 않으나 신생아, 특히 면역력이 약한 신생아에게 치명적인 위험을 줄 수 있다.As a major mediator, SAKAZAKI is reported as a dry formula and can cause enteritis, sepsis when entering the blood, and meningitis when invaded into the brain, and 10-20% mortality when babies younger than 1 year have sepsis. Mortality from to 30%. This is not a big problem for people with normal immunity but can pose a fatal risk for newborns, especially those with weak immunity.
또한, 선진국의 식중독 관련 통계자료를 살펴보면 전체 식중독 사고의 10% 미만만이 보고되고 나머지 90%는 정확히 집계되지 못하고 있는 실정이며 이러한 식중독 사고의 미흡한 보고는 일반 병원이나 실험실에서 기술 설비가 부족하여 원인 식중독세균을 규명하지 못하는 것이 주요 원인으로 작용한다. In addition, statistics on food poisoning in developed countries show that less than 10% of all food poisoning incidents are reported and the remaining 90% are not accurately counted. The insufficient reporting of such food poisoning accidents is due to lack of technical facilities in general hospitals and laboratories. Failure to identify food poisoning bacteria is a major cause.
본 발명은 식중독의 예방, 식중독의 원인 파악 및 식품이나 음식재료의 오염 현황 파악 등을 위하여 식품 등에서 Cronobacter sakazakii를 효율적으로 제어하는 기술을 제공하고자 한다. The present invention is to provide a technique for efficiently controlling Cronobacter sakazakii in food, etc. for the prevention of food poisoning, the cause of food poisoning, and the status of contamination of food or food ingredients.
상기 과제를 해결하기 위하여 본 발명은 Cronobacter sakazakii 생육을 저해하는 효과를 가지는 독성파아지 ES2(수탁번호 KCTC11614BP) 또는 EI-1(수탁번호 KCTC11613BP)을 제공한다. The present invention to solve the above problems Cronobacter sakazakii Toxic phage ES2 (Accession No. KCTC11614BP) or EI-1 (Accession No. KCTC11613BP) having an effect of inhibiting growth is provided.
또한, 본 발명은 Cronobacter sakazakii 생육 제어 방법으로서, Cronobacter sakazakii가 포함된 시료에 독성파아지 ES2(수탁번호 KCTC11614BP) 및 EI-1(수탁번호 KCTC11613BP)을 함께 처리하는 것을 특징으로 하는 Cronobacter sakazakii 생육 제어 방법을 제공한다. In addition, the present invention Cronobacter sakazakii As a growth control method, Cronobacter sakazakii is treated with toxic phage ES2 (Accession No. KCTC11614BP) and EI-1 (Accession No. KCTC11613BP) to a sample containing Cronobacter sakazakii. Provide growth control methods.
본 발명에 따르면, Cronobacter sakazakii 생육을 인체에 유해하지 않으며, 간단한 방법으로 제어할 수 있게 된다. According to the present invention, Cronobacter sakazakii Growth is not harmful to the human body, it can be controlled in a simple way.
도 1은 ES2 및 EI-1의 접종에 따른 Cronobacter sakazakii 생장 저해 효과를 나타낸다.
도 2는 분유 내에서 ES2 및 EI-1의 접종에 따른 Cronobacter sakazakii 생장 저해 효과를 나타낸다.
도 3은 또한 분리된 ES2와 EI-1 bacteriophage를 혼합하여 cocktail 형태로 제조 후 식품으로부터 분리된 Cronobacter sakazakii KYU48, KYU50, KYU51, KYU60, KYU98을 각각 배양 후 5개의 분리주를 혼합하여 co-culture를 했을 때 ES2와 EI-1 phage cocktail에 의한 감소 효과를 나타낸다. 1 shows Cronobacter sakazakii following inoculation of ES2 and EI-1 It has a growth inhibitory effect.
Figure 2 Cronobacter sakazakii following the inoculation of ES2 and EI-1 in milk powder It has a growth inhibitory effect.
Figure 3 is also prepared by mixing the isolated ES2 and EI-1 bacteriophage cocktail form and then cultured Cronobacter sakazakii KYU48, KYU50, KYU51, KYU60, KYU98 isolated from food and then mixed five isolates were co-culture When the effect is reduced by ES2 and EI-1 phage cocktail.
경기도 소재의 도축장에서 수집된 소와 돼지의 분변 20종으로부터 Cronobacter sakazakii의 제어를 위한 bacteriophage의 분리를 시도하였다. An attempt was made to isolate bacteriophage for control of Cronobacter sakazakii from 20 cow and pig feces collected at a slaughterhouse in Gyeonggi-do.
샘플을 채취하여 냉장 상태로 실험실에 이동한 후 24시간 이내에 실험을 수행하였으며, 실험 후 나머지 샘플은 -70도 deep freezer에 보관하였다. Samples were taken and moved to the laboratory in a refrigerated state, and the experiment was performed within 24 hours. After the experiment, the remaining samples were stored in a -70 degree deep freezer.
고체의 샘플일 경우, Cronobacter sakazakii가 배양된 10mM CaCl2가 첨가된 Luria-bertani(LBC) broth 50mL에 50gram을 첨가한 후 37도에서 200rpm으로 추가로 배양하였다. 그리고 배양 후 배양액에 chloroform을 1% 첨가한 후 혼합하고 10,000g에서 10분 동안 원심분리 하였다. 원심분리 후 상등액을 취해 0.22um syringe filter를 사용하여 제균하여 Cronobacter sakazakii의 virulent phage 분리를 위해 4도에서 보관하여 사용하였다. In the case of a solid sample, 50 grams of Luria-bertani (LBC) broth to which 10 mM CaCl 2 was added was incubated with Cronobacter sakazakii, and further cultured at 200 rpm at 37 degrees. After culturing, 1% of chloroform was added to the culture solution, mixed, and centrifuged at 10,000 g for 10 minutes. After centrifugation, the supernatant was taken and sterilized using a 0.22um syringe filter. The supernatant was stored at 4 degrees for separation of virulent phage of Cronobacter sakazakii.
또한, 액체 샘플의 경우, 5XLBC broth를 제조하고 샘플(4):5XLBC broth(1)로 혼합 후 배양된 Cronobacter sakazakii를 첨가한 후 37도에서 200rpm으로 배양한 후 고체시료와 동일하게 수행하였다. In addition, in the case of a liquid sample, 5XLBC broth was prepared, mixed with sample (4): 5XLBC broth (1), and then cultured at 200 rpm at 37 ° C., followed by incubation with a solid sample.
Cronobacter sakazakii를 LBC broth에서 37도, 200rpm으로 배양한 후 제균액과 배양액을 1:1의 비율로 혼합하고 동일한 배양 조건에서 1시간 동안 추가 배양하였다. 이를 LBC soft agar에 접종하여 vortex 한 후 LBC agar에 lawn으로 분주하고 37℃에서 24~48시간 동안 배양하면서 bacteriophage에 의한 plaque 형성 유무를 확인하였다. 형성된 plaque의 형태, size, 혼탁 정도 등을 비교하여 서로 다른 plaque morphology를 분리하였다. Plaque를 tooth-picking 하고 SM buffer에 현탁한 후 현탁액을 SM buffer를 이용하여 10진 희석하여 plaque assay를 수행하고 single plaque를 분리하였다. 확인된 single plaque들로부터 plate lysate를 추출한 후 이를 이용하여 plaque assay를 통해 virulent phage의 양을 확인한 후 이를 이용하여 liquid lysate를 얻은 후 plaque assay를 통해 virulent phage의 양을 개수하여 실험에 사용하였다. Cronobacter sakazakii was incubated at 37 ° C. and 200 rpm in LBC broth, and then the fungicide and culture medium were mixed at a ratio of 1: 1 and further cultured for 1 hour under the same culture conditions. This was inoculated with LBC soft agar and vortex, and then aliquoted to LBC agar with a lawn and incubated at 37 ° C. for 24 to 48 hours to confirm plaque formation by bacteriophage. Different plaque morphologies were isolated by comparing the shape, size, and turbidity of the formed plaques. Plaque was tooth-picked and suspended in SM buffer, and then the suspension was diluted 10 with SM buffer to carry out plaque assay and single plaque was isolated. After extracting plate lysate from the identified single plaques, the amount of virulent phage was confirmed by using the plaque assay. After that, the liquid lysate was obtained, and the amount of virulent phage was counted by plaque assay.
Cronobacter sakazakii의 virulent phage 분리를 위해 다양한 환경 샘플, 경기도 소재의 도축장에서 수집된 소와 돼지의 분변 20종으로부터 분리한 결과 총 11주의 virulent phage를 분리할 수 있었다. The virulent phage of Cronobacter sakazakii was isolated from 20 samples of feces and pigs collected from various environmental samples and slaughterhouses in Gyeonggi-do.
또한, 분리된 virulent phage들의 host spectrum을 확인한 결과 Cronobacter sakazakii ATCC51329, ATCC29544의 표준 균주에 작용하였으며, 식품으로부터 분리된 Cronobacter sakazakii들 중 Cronobacter sakazakii KYU46, KYU58, KYU60, KYU129는 분리된 virulent phage 모두 plaque를 형성하는 것을 확인하였으며, Cronobacter sakazakii KYU50은 ES2, ESSI-2, ESSI-3, Cronobacter sakazakii KYU51은 EI-1, EI-2, EI-3만이 plaque를 형성하는 것으로 확인되었다.In addition, as a result of confirming the host spectrum of the virulent phages isolated, it acted on the standard strains of Cronobacter sakazakii ATCC51329 and ATCC29544. Cronobacter sakazakii KYU50 is ES2, ESSI-2, ESSI-3, Cronobacter sakazakii KYU51 was confirmed that only EI-1, EI-2, and EI-3 form plaque.
Host spectrum을 통해 확인된 결과 분리된 virulent phage 중 ES2와 EI-1 phage을 이용하여 Cronobacter sakazakii의 제어에 사용하였다. The host spectrum was used to control Cronobacter sakazakii using ES2 and EI-1 phages.
분리된 ES2, EI-1을 각각 LBC broth에 접종 후 Cronobacter sakazakii ATCC29544를 접종하고 37도에서 200rpm으로 배양하면서 시간마다 흡광도를 측정하여 균 감소 효과를 확인하였다. 그 결과 시간이 흐름에 따라 virulent phage를 접종하지 않은 Cronobacter sakazakii의 경우는 흡광도 값이 지속적으로 증가하는 반면, virulent phage와 함께 배양하였을 경우 흡광도 값이 감소하는 것을 확인할 수 있었다. 또한, 분리된 ES2, EI-1 phage를 시판 중인 분유를 제조 방법에 따라 조제한 후 Cronobacter sakazakii ATCC29544를 약 2 log CFU/mL로 접종 후 약 6 log PFU/mL로 접종하여 시간이 지남에 따라 Cronobacter sakazakii의 감소 정도를 비교하였다. 그 결과 virulent phage를 infection하지 않았을 경우는 시간이 지나감에 따라 계속 증가하는 양상을 나타내고 있으나, ES2와 EI-1을 각각 함께 배양하였을 경우에는 Cronobacter sakazakii가 증가하지 않는 것으로 확인할 수 있었다(도 1 및 도 2 참조). 이들 독성파아지는 2010년 1월 6일자로 한국생명공학연구원 생물자원센터에 ES2(수탁번호 KCTC11614BP) 및 EI-1(수탁번호 KCTC11613BP)로 기탁하였다. After inoculation of the isolated ES2, EI-1 to LBC broth, inoculated with Cronobacter sakazakii ATCC29544 and incubated at 37 ° C at 200 rpm, the absorbance was measured every hour to confirm the bacterial reduction effect. As a result, the absorbance value of Cronobacter sakazakii, which was not inoculated with virulent phage, increased continuously over time, whereas the absorbance value was decreased when incubated with virulent phage. In addition, after formulating commercially available powdered ES2, EI-1 phage according to the production method Cronobacter sakazakii ATCC29544 was inoculated at about 2 log CFU / mL and then at about 6 log PFU / mL to compare the reduction of Cronobacter sakazakii over time. As a result, when the virulent phage was not infected, it continued to increase with time, but when ES2 and EI-1 were cultured together, Cronobacter sakazakii did not increase (FIG. 1 and 2). These toxic phages were sent to the Korea Institute of Bioscience and Biotechnology, January 6, 2010. It was deposited with ES2 (Accession No. KCTC11614BP) and EI-1 (Accession No. KCTC11613BP).
또한, 분리된 ES2와 EI-1 bacteriophage를 혼합하여 cocktail 형태로 제조 후 식품으로부터 분리된 Cronobacter sakazakii KYU48, KYU50, KYU51, KYU60, KYU98을 각각 배양 후 5개의 분리주를 혼합하여 co-culture를 했을 때 ES2와 EI-1 phage cocktail에 의한 감소 효과를 확인하고자 하였다. 그 결과 5주의 Cronobacter sakazakii의 생육 억제 효과는 ES2와 EI-1을 각각 처리했을 경우, phage를 처리하지 않았을 경우에 비해 생육을 억제하는 효과가 있었으며, phage cocktail 형태로 처리하였을 경우 균의 감소를 확인할 수 있었다. 따라서 단독으로 phage를 처리할 경우보다 혼합 형태로 처리하였을 때 Cronobacter sakazakii에 대한 제어가 효과적이었다(도 3 참조). In addition, ES2 and EI-1 bacteriophage were mixed to prepare a cocktail form, and then cultured Cronobacter sakazakii KYU48, KYU50, KYU51, KYU60, KYU98 isolated from food, and then mixed five isolates to co-culture ES2. And EI-1 phage cocktail to reduce the effect. As a result, the growth inhibition effect of Cronobacter sakazakii for 5 weeks had the effect of inhibiting the growth of ES2 and EI-1, compared to the case of no treatment of phage. Could. Therefore, the control for Cronobacter sakazakii was more effective when treated in mixed form than when treated with phage alone (see FIG. 3).
Claims (2)
Cronobacter sakazakii가 포함된 시료에 가축 분변 유래의 독성파아지 ES2(수탁번호 KCTC11614BP) 및 EI-1(수탁번호 KCTC11613BP)을 함께 처리하는 것을 특징으로 하는 Cronobacter sakazakii 생육 제어 방법.Cronobacter sakazakii As a growth control method,
Cronobacter sakazakii characterized in that the sample containing Cronobacter sakazakii is treated with toxic phage ES2 (Accession No. KCTC11614BP) and EI-1 (Accession No. KCTC11613BP) derived from livestock feces. Growth control method.
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| EP1533369A1 (en) * | 2003-11-19 | 2005-05-25 | Nestec S.A. | Isolated phages and their use as disinfectant in food or for sanitation of factory environment |
| US20090246336A1 (en) | 2008-03-25 | 2009-10-01 | Ecolab Inc. | Bacteriophage treatment for reducing and preventing bacterial contamination |
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| EP1533369A1 (en) * | 2003-11-19 | 2005-05-25 | Nestec S.A. | Isolated phages and their use as disinfectant in food or for sanitation of factory environment |
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| US11058131B2 (en) | 2015-04-16 | 2021-07-13 | Kennesaw State University Research And Service Foundation, Inc. | Escherichia coli O157:H7 bacteriophage Φ241 |
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