KR20120066216A - Bacillus cereus specific bacteriophage - Google Patents
Bacillus cereus specific bacteriophage Download PDFInfo
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Abstract
Description
본 발명은 식중독을 유발하고 장류 등의 발효과정에서 부패를 유발하는 유해세균인 바실러스 세레우스(Bacillus cereus)에 특이적인 신규한 박테리오파지(bacteriophage) 및 그 이용방법에 관한 것으로, 상기 박테리오파지를 이용하면 방부제나 항생제를 사용하지 않으면서 유해세균인 바실러스 세레우스와 같은 유해 미생물만을 선택적으로 제거할 수 있으므로, 식품의 보존성을 향상시키거나 청국장과 같은 장류 발효시 첨가하여 제품의 품질을 높일 수 있다. The present invention relates to a novel bacteriophage specific to Bacillus cereus , a harmful bacterium that causes food poisoning and causes decay during fermentation such as enteric, and a method of using the bacteriophage. Only harmful microorganisms such as Bacillus cereus, which is a harmful bacterium, can be selectively removed without using antibiotics. Therefore, the preservation of foods can be improved or added during fermentation of soy sauce such as Cheonggukjang.
바실러스 세레우스(Bacillus cereus)는 자연계에 널리 분포하며 특히 곡류에서 많이 존재한다. 바실러스 세레우스는 식품 원재료와 그 가공 조리식품의 식중독 원인 균주로 알려져 있어 HACCP 위해요소 관리대상 균주이며, 청국장 등의 전통 발효 장류의 발효시 장류 특유의 맛과 향취에 악영향을 끼치는 것으로 알려져 있다. Bacillus cereus is widely distributed in nature and is particularly present in cereals. Bacillus cereus is known as a strain that causes food poisoning of food ingredients and processed cooked foods, and is a strain to be managed for HACCP risk factors.
바실러스 세레우스는 다른 균주의 포자에 비해 어느 표면이든 잘 들러붙어 세척과 소독이 어려우며 페니실리나제(Penicillinase)를 생성하여 페니실린(penicillin)에 대해 내성을 가지는 항생제 내성균주이다. 또한, 열(126℃ 90분 이상 동안), 산, 알칼리, 단백질 가수분해효소에 저항력을 갖는 등의 사멸하기 까다로운 특성을 가지고 있으며, 적절한 살균 방법이 아직 개발되지 않은 실정이다. Bacillus cereus is an antibiotic-resistant strain that is resistant to penicillin by producing penicillinase because it adheres to any surface more difficult than spores of other strains, making it difficult to clean and disinfect. In addition, it is difficult to kill the heat (126 ℃ 90 minutes or more), resistant to acid, alkali, proteolytic enzymes, etc., and the proper sterilization method has not been developed yet.
최근 발효 또는 숙성시킨 식품에서 식중독을 유발하는 니트로사민과 같이 발암물질로 전환될 잠재성이 있는 바이오제닉아민이 검출되고 있어 정부 차원의 인체 위해성 평가가 진행되고 있으며, 식품내 상기 물질의 함량을 저감화하기 위한 대책이 강구되고 있지만 방부제나 항생제는 발효식품에 사용할 수 없으며 유익한 미생물인 프로바이오틱스(probiotics)에는 무해하며 유해 미생물만을 선택적으로 사멸 시킬 수 있는 방법이 고안되지 않아 어려움에 처한 상황이다. Recently, biogenic amines that are potentially converted to carcinogens, such as nitrosamines that cause food poisoning, have been detected in fermented or matured foods, and governmental risk assessments are underway to reduce the content of these substances in foods. Although precautionary measures are being taken, preservatives and antibiotics cannot be used in fermented foods, and they are in trouble because they are harmless to beneficial microorganisms (probiotics) and have not been devised to selectively kill only harmful microorganisms.
박테리오파지(Bacteriophage)는 각각의 고유 특성에 맞는 세균에만 특이적으로 감염되는 바이러스로 1915년 Twort와 1917년 d'Herelle 등이 처음 발견한 이래, 박테리오파지를 이용한 미생물의 살균 및 치료용도로서의 개발이 한동안 지속되었으나, 페니실린 계열 항생제 개발 이후 치료제로서의 본격적인 연구는 미미하였다. Bacteriophage is a virus that is specifically infected only with bacteria that meet its unique characteristics. Since its discovery by Twort in 1915 and d'Herelle in 1917, development of bacteriophage as a bacteriophage bacteriophage has been continued for a while. However, since the development of penicillin antibiotics, there has been little research as a therapeutic agent.
하지만 최근 지속적인 항생제 오남용으로 인한 다 약제 내성 세균 및 슈퍼박테리아 출현으로 박테리오파지는 항생제의 대안으로서 주목받고 있다. 일예로 강력한 항생제로 알려진 반코마이신에 내성을 지닌 Enterococcus spp.에 특이적인 박테리오파지에 대한 연구 및 바이오테러로 생화학무기로 사용될 수 있는 탄저균을 사멸할 수 있는 박테리오파지에 대한 연구가 활발히 진행되고 있다. However, the recent emergence of multi-drug resistant bacteria and superbacteria due to the continued abuse of antibiotics has attracted attention as an alternative to antibiotics. For example, studies on bacteriophages specific to Enterococcus spp. Resistant to vancomycin, known as potent antibiotics, and bacteriophages capable of killing anthrax, which can be used as biochemical weapons for bioterrorism, are being actively conducted.
WHO의 2000년 전염병 보고서에 의하면 신규 항균제 1개를 개발하는데 15년 내지 20년이 소요되며, 개발경비는 제약회사의 추계에 의하면 약 5 억$가 필요한 것으로 알려져 있으나 박테리오파지를 이용한 특이 항생제 개발은 비교적 짧은 연구개발 기간(2-5년) 및 비용(5-8억원)이 소요되므로, 상대적으로 경쟁력이 있으며 미생물 생태계의 자연 현상을 이용한 기술이므로 부작용이 없고, 다양한 세균성 질병에 적용 할 수 있어 관련 산업으로 많은 장점을 지니고 있다.According to the WHO's 2000 Epidemic Report, it takes 15 to 20 years to develop a new antimicrobial agent, and the development cost is estimated to be about $ 500 million according to the estimates of pharmaceutical companies, but the development of specific antibiotics using bacteriophages is relatively As it takes a short research and development period (2-5 years) and costs (5-8 billion won), it is relatively competitive and uses technology of natural phenomena of microbial ecosystem, so it has no side effects and can be applied to various bacterial diseases. It has many advantages.
또한 식품에 이용시 목적하는 유해 미생물만을 선택적으로 사멸시킬 수 있다는 장점을 가지고 있어 식중독 균 등의 사멸을 통한 식품의 보존성을 향상시킬 수 있으며, 장류나 김치 등의 발효과정에서 유익한 미생물인 프로바이오틱스(probiotics)에는 영향을 미치지 않으면서 유해한 미생물만을 제거할 수 있어 발효식품의 맛과 향취를 제고할 수 있다는 장점도 가지고 있다. In addition, it has the advantage of selectively killing only harmful microorganisms when used in foods, which can improve food preservation through killing of food poisoning bacteria and probiotics, which are beneficial microorganisms in fermentation process of soy sauce and kimchi. Only harmful microorganisms can be removed without affecting the taste, which has the advantage of enhancing the taste and flavor of fermented foods.
본 발명에서는 상기와 같은 종래 기술의 제반 문제점을 해결하기 위하여 식중독을 유발하는 바실러스 세레우스에 특이적인 박테리오파지를 분리?동정하였으며, 분리된 박테리오파지는 식중독 등의 예방을 위한 식품의 처리 및 전통 장류 발효시 바실러스 세레우스의 선택적 제거를 위해 이용될 수 있다.In the present invention, in order to solve the above problems of the prior art, bacteriophage specific to Bacillus cereus causing food poisoning was isolated and identified, and the isolated bacteriophage was treated during the treatment of foods and traditional fermentation for the prevention of food poisoning. It can be used for selective removal of Bacillus cereus.
상기와 같은 목적을 달성하기 위하여 본 발명은 바실러스 세레우스에 특이적인 박테리오파지를 새로이 분리?동정하였으며, 상기 박테리오파지는 식품의 보존을 위한 첨가제 및 청국장 등의 전통 장류 발효시 첨가제로 이용할 수 있다.In order to achieve the above object, the present invention newly isolates and identifies bacteriophages specific to Bacillus cereus, and the bacteriophages can be used as additives for fermentation of traditional Jangjang, such as additives for the preservation of food and Cheonggukjang.
본 발명에 따르면, 항생제 오남용 문제를 유발하지 않으면서 식중독을 유발하는 미생물인 바실러스 세레우스의 선택적 사멸이 가능하여 식품 보존성을 크게 향상시킬 수 있으며, 청국장 등의 전통 장류 발효시 첨가하여 장류 고유의 맛과 향취를 제고할 수 있는 효과가 있다.According to the present invention, the selective killing of Bacillus cereus, a microorganism causing food poisoning without causing the problem of antibiotic misuse, greatly improves food preservation, and is added during fermentation of traditional soybeans such as Cheonggukjang. There is an effect to enhance the smell and.
도 1은 본 발명에 따른 박테리오파지의 분리과정을 나타낸 개략도를 나타낸 것이고,
도 2는 본 발명에서 분리된 박테리오파지의 흡착테스트 결과를 나타낸 도면이며,
도 3은 본 발명에서 분리된 박테리오파지의 온도 안정성 테스트 결과를 나타낸 도면이고,
도 4는 본 발명에서 분리된 박테리오파지의 pH 안정성 결과를 나타낸 도면이다.Figure 1 shows a schematic diagram showing the separation process of the bacteriophage according to the present invention,
Figure 2 is a view showing the adsorption test results of the bacteriophage separated in the present invention,
3 is a view showing the temperature stability test results of the bacteriophage isolated in the present invention,
Figure 4 is a view showing the pH stability results of the bacteriophage isolated in the present invention.
이하 본 발명을 첨부된 도면을 참조하여 상세히 설명한다.
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
[실시예 1] 바실러스 세레우스 특이적 박테리오 파지의 검출Example 1 Detection of Bacillus cereus Specific Bacteriophage
박테리오파지를 검출하기 위해 박테리오파지가 존재할 것이라 예상되는 진도, 완도, 고금도등 여러 서해안 양식장 100 여군데를 통해 해수를 채취하여 표 1에 개시된 바실러스 균과 함께 37℃에서 진탕배양 후, 8000 rpm에서 20분간 원심분리하여 박테리아 부분을 제거하였다. 이후 남아 있을 박테리아 부분 제거를 위해 0.45 um 필터지를 사용하여 필터링한 후 플래크 분석(Plaque assay)를 통해 세균 특이적인 파지를 검출하였다(도 1 참조).To detect bacteriophages, seawater was collected from 100 western coastal farms, such as Jindo, Wando and Gogeum, where bacteriophages were expected to be present. Isolate to remove bacterial parts. Then, bacteria-specific phages were detected by plaque assay after filtering using 0.45 um filter paper to remove the remaining bacterial portions (see FIG. 1).
상기 방법에 따라 바실러스 세레우스 균주와 바실러스 스런지엔시스(Bacillus thuringiensis)에 특이성을 가지는 박테리오파지를 분리하였으며, 분리된 박테리오파지는 유익한 미생물로 알려진 바실러스 서브틸리스(Bacillus subtilis)에는 용균효과를 보이지 않는 것으로 확인되었다. According to the above method, bacteriophages having specificity to Bacillus cereus strain and Bacillus thuringiensis were isolated, and the isolated bacteriophages were confirmed to show no lytic effect on Bacillus subtilis , which is known as a beneficial microorganism. It became.
[실시예 2] 분리된 파지의 세균 흡착도 테스트를 통한 생화학적 특성 규명Example 2 Biochemical Characterization of Bacterial Adsorption Test of Isolated Phage
상기 실시예 1에서 분리된 박테리오파지의 세균 흡착도는 108 cfu/ml의 바실러스 세레우스와 108 pfu/ml의 박테리오파지를 1 ml 씩 혼합한 후 5분 간격으로 37℃에서 항온 방치시키고 샘플을 100 ㎕씩 채취해 박테리오파지 액체배지로 100배 희석한 후 5000 rpm 10분 원심분리한 후, 어느 시간대에 박테리오파지가 흡착하는 지를 박테리오파지수를 정량하여 확인하였다. The bacterial adsorption degree of the bacteriophage isolated in Example 1 was mixed with 10 8 cfu / ml Bacillus cereus and 10 8 pfu / ml of bacteriophage by 1 ml and allowed to stand at 37 ℃ at 5 minutes intervals and the
그 결과 5~15분 사이에 박테리오파지가 바실러스 세레우스에 흡착하는 것을 확인할 수 있었다(도 2 참조).
As a result, it was confirmed that the bacteriophage adsorbed to Bacillus cereus within 5 to 15 minutes (see FIG. 2).
[실시예 3] 온도 및 pH 안정성 테스트를 통한 생화학적 특성 규명Example 3 Biochemical Characterization through Temperature and pH Stability Tests
상기 실시예 1에서 분리된 박테리오파지의 온도 안정성 테스트를 위해 박테리오파지 샘플 10 ㎕을 기준으로 101~108까지 희석한 후 4℃, 16℃, 20℃, 36℃, 50℃로 온도를 설정하여 24시간 배양을 실시한 후 생존율을 조사하였다. 그 결과 전체적으로 50℃ 이하에서는 활성을 보였으며, 4℃에서 40℃까지는 일정하게 플라크 형성도(plaque forming unit (PFU))/㎖을 유지함을 확인할 수 있었다(도 3 참조).In order to test the temperature stability of the bacteriophage isolated in Example 1 based on 10 μl of the
상기 실시예 1에서 분리된 박테리오파지의 pH 안정성 테스트를 위해 멸균수를 각각 pH 2~10까지 설정하여 각각 박테리오파지 샘플과 101~108까지 희석하여 24시간 배양한 후 생존율을 체크하였다. 그 결과 pH 6~9에서 일정한 PFU/㎖을 유지하였고 pH 7에서 가장 좋은 생존율을 보였다(도 4 참조).
For pH stability test of the bacteriophage isolated in Example 1, sterilized water was set to pH 2 to 10, respectively, diluted with bacteriophage samples to 10 1 to 10 8 , and then cultured for 24 hours to check the survival rate. As a result, a constant PFU / mL was maintained at pH 6-9 and showed the best survival rate at pH 7 (see FIG. 4).
상술한 바와 같이, 본 발명의 바람직한 실시예를 참조하여 설명하였지만 해당 기술 분야의 숙련된 당업자라면 하기의 특허청구범위에 기재된 본 발명의 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 및 변경시킬 수 있음을 이해할 수 있을 것이다. As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated that it can be changed.
Claims (8)
상기 박테리오파지는 바실러스 스렌지엔시스(Bacillus thuringiensis)에 대해서 용균효과를 나타내지만, 바실러스 서브틸리스(Bacillus subtilis)에 대해서는 용균효과를 보이지 않음을 특징으로 하는 신규 박테리오 파지.The method of claim 1,
The bacteriophage shows a bactericidal effect against Bacillus thuringiensis , but does not show a bactericidal effect against Bacillus subtilis .
상기 식품은 발효식품, 곡류, 기능성 식품에서 선택된 하나 이상인 것을 특징으로 하는 방법.The method of claim 3,
The food is characterized in that at least one selected from fermented foods, cereals, functional foods.
상기 장류는 청국장 또는 된장인 것을 특징으로 하는 방법.The method of claim 5,
The Jangjang is characterized in that the Cheonggukjang or miso.
상기 전통 장류의 맛과 향취를 향상시키는 방법은 첨가된 박테리오파지가 유해 미생물인 바실러스 세레우스(Bacillus cereus) 또는 바실러스 스렌지엔시스(Bacillus thuringiensis)를 선택적으로 제거함으로써 전통 장류의 맛과 향취를 향상시키는 것을 특징으로 하는 방법.The method of claim 5,
The method for improving the taste and flavor of the traditional Jangjang is Bacillus cereus ( Bacillus) is added bacteriophage harmful microorganisms cereus ) or Bacillus thuringiensis , which selectively enhances the taste and flavor of the traditional Jangjang.
상기 첨가된 박테리오파지는 유익한 미생물인 바실러스 서브틸리스(Bacillus subtilis)에는 용균효과를 보이지 않는 것을 특징으로 하는 방법.The method of claim 5,
The added bacteriophage is characterized in that it does not show a lytic effect on Bacillus subtilis , a beneficial microorganism.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101443140B1 (en) * | 2011-12-14 | 2014-09-24 | 순창군 | Composition for controlling harmful bacteria comprising bacteriophage as effective component and method for controlling harmful bacteria using the same |
| CN110205306A (en) * | 2019-06-10 | 2019-09-06 | 武汉轻工大学 | Bacillus cereus bacteriophage, bacteriophage composition and inhibiting-bacteria preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101443140B1 (en) * | 2011-12-14 | 2014-09-24 | 순창군 | Composition for controlling harmful bacteria comprising bacteriophage as effective component and method for controlling harmful bacteria using the same |
| KR101443141B1 (en) * | 2011-12-14 | 2014-09-24 | 순창군 | Method for producing starter culture for traditional fermented food without harmful bacteria using bacteriophage |
| CN110205306A (en) * | 2019-06-10 | 2019-09-06 | 武汉轻工大学 | Bacillus cereus bacteriophage, bacteriophage composition and inhibiting-bacteria preparation |
| CN110205306B (en) * | 2019-06-10 | 2023-03-03 | 武汉轻工大学 | Bacillus cereus bacteriophage, bacteriophage composition and bacteriostatic preparation |
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