청국장으로부터 Bacillus cereus에 대한 길항 균주 분리 및 길항 효과

이남근(Nam Keun Lee),박정완(Joung Whan Park),조일재(Il Jae Cho),김병용(Byung-Yong Kim),권기옥(Ki-Ok Kwon),함영태(Young Tae Hahm) 한국식품과학회 2008 한국식품과학회지 Vol.40 No.6

청국장 내에 B. cereus을 효과적으로 저지하기 위한 생물학적 제어 방법의 개발을 위하여 전통적 방법으로 제조한 백태 및 흑태 청국장으로부터 총 20종의 Bacillus 속 균주를 분리하였고, 이중에서 24시간 배양액에서 B. cereus에 대한 길항활성이 가장 높은 Bacillus sp. SC-8 균주를 선별하였다. 이 균주를 B. cereus와 혼합하여 청국장을 제조한 후 길항능을 분석한 결과 청국장 발효 중에는 B. cereus에 대한 길항효과가 있었으나, 발효 후 4℃ 저장 중에는 균체수가 감소하여 억제효과가 감소하는 것으로 분석되었다. 또한 대조군으로 사용하였던 Bacillus sp. SC-15 균주에서는 배양액에서는 B. cereus에 대한 길항 활성이 낮았으나 청국장 내에서는 길항효과를 보임에 따라 청국장 발효에 관여하는 균들은 배양 환경에 따라 길항물질의 생성에 차이가 있음을 보였다. For the development of a biological control method against B. cereus in cheonggukjang, 20 Bacillus spp. were isolated from the naturally fermented baektae and heuktae cheonggukjang, identified by using 16S rDNA sequences. Among the isolated strains, Bacillus sp. SC-8 was selected using the B. cereus lawn cell assay as an antagonistic microorganism against B. cereus. The culture medium of Bacillus sp. SC-8 after 24 hr of incubation at 37℃ also evidenced a high level of antagonistic activity. In cheonggukjang fermented with the mixed culture of Bacillus sp. SC-8 and B. cereus, antagonistic effect against B. cereus was maintained during the fermentation of cheonggukjang, while its effect was reduced during storage at 4℃ due to the decrement of cell population of Bacillus sp. SC-8. In Bacillus sp. SC-15, which was utilized a control, antagonistic activity against B. cereus was not demonstrated on the lawn cell plate assay and culture medium, but its effects were detected in cheonggukjang. Therefore, the production of antagonistic substances of Bacillus spp. depends on the fermentative environment.

선식에서 Bacillus cereus의 분리 및 특성 조사와 열에 대한 사멸률 연구

조용선,정은영,이명기,양철영,신동빈 한국식품위생안전성학회 2008 한국식품위생안전성학회지 Vol.23 No.4

Recently, Sunshik has been issued because of easy-cook and well-being food. Sunshik basically was made of the heated cereals. Amount of spore-forming Bacillus cereus was detected and it has been caused some problem of food safety. B. cereus was isolate from 57 out of 161 Sunshik samples resulting in the isolation rate of 35.4 %. Quantitative analysis of 57 samples showed that 21 samples were less than 100 CFU/g, 33 samples were between 100 and 1,000 CFU/g and distinctively even 3 (1.9 %) samples had over 1,000 CFU/g. Typical morphology of B. cereus isolated from Sunshik was observed on MYP agar and then further characteristics was identified by using VITEK 2 (Biomeriux, France). 53 strains out of 57 strains isolated from Sunshik (about 93.0 %) produced diarrheal enterotoxin in brain heart infusion broth which was detected by the Bacillus cereus enterotoxin reversed passive latex agglutination test kit (Oxoid England). The D-values of the B. cereus spores were 75 ℃ (37.1mim), 80 ℃ (22.5mim), 85 ℃ (4.9mim), and 90 ℃ (3.1mim) respectively. The Z-value was calculated 12.8 ℃ in Sunshik sample inoculated with B. cereus. Therefore, the management of B. cereus in Sunshik is required for the food-safety. Recently, Sunshik has been issued because of easy-cook and well-being food. Sunshik basically was made of the heated cereals. Amount of spore-forming Bacillus cereus was detected and it has been caused some problem of food safety. B. cereus was isolate from 57 out of 161 Sunshik samples resulting in the isolation rate of 35.4 %. Quantitative analysis of 57 samples showed that 21 samples were less than 100 CFU/g, 33 samples were between 100 and 1,000 CFU/g and distinctively even 3 (1.9 %) samples had over 1,000 CFU/g. Typical morphology of B. cereus isolated from Sunshik was observed on MYP agar and then further characteristics was identified by using VITEK 2 (Biomeriux, France). 53 strains out of 57 strains isolated from Sunshik (about 93.0 %) produced diarrheal enterotoxin in brain heart infusion broth which was detected by the Bacillus cereus enterotoxin reversed passive latex agglutination test kit (Oxoid England). The D-values of the B. cereus spores were 75 ℃ (37.1mim), 80 ℃ (22.5mim), 85 ℃ (4.9mim), and 90 ℃ (3.1mim) respectively. The Z-value was calculated 12.8 ℃ in Sunshik sample inoculated with B. cereus. Therefore, the management of B. cereus in Sunshik is required for the food-safety.

KHT5 마커를 사용한 Bacillus cereus 그룹에서 Bacillus anthracis의 구별

김형태,김성주,채영규 한국미생물학회 2003 미생물학회지 Vol.39 No.1

탄저균은 그람양성 아포형성세균으로 탄저를 일으키는 원인균이다. Bacillus cereus그룹에 속하는 22종을 포함하여 Bacillus 속의 29종에서 탄저균을 검증할 수 있는 DNA 마커를 개발하고 이를 이용하여 B. cereus 그룹에서 탄저균만을 구분하였다. 한국산 탄저균 경주로부터 709 bp마커(KHTS)를 확보하였다. KHTS분절로부터 얻어진 internal primer set의 PCR 산물은 B. cereus 그룹의 다른 종으로부터 탄저균만을 구별하였다. Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. In order to develop a DNA marker specific for Bacillus anthracis and to discriminate this species from Bacillus cereus group, we applied the randomly amplified polymorphic DNA (RAPD)-PCR technique to a collection of 29 strains of the genus Bacillus, including 22 species of the B. cereus group. A 709-bp RAPD marker (KHT5) specific for B. anthracis was obtained from B. anthracis BAK. The PCR product of internal primer set from the KHT5 fragment distinguished B. anthracis from the other species of the B. cereus group.

장수지역 전통된장의 미생물 군집 및 바실러스 세레우스 길항 효과

전두영(Doo-Young Jeon),윤기복(Gi-Bok Yoon),윤연희(Yeon-Hee Yoon),양수인(Soo-In Yang),김중범(Jung-Beom Kim) 한국식품영양과학회 2016 한국식품영양과학회지 Vol.45 No.7

본 연구는 우리나라 장수지역에서 제조된 전통된장을 수거하여 식중독세균 오염도와 미생물 군집 현황을 분석하고, 분리된 미생물 군집의 Bacillus cereus 길항 효과를 평가함으로써 전통된장의 미생물학적 안전성을 평가하고자 하였다. 전통된장의 미생물학적 안전성을 평가하기 위해 일반세균, 대장균군, 식중독세균을 실험한 결과 일반세균수는 10<SUP>6</SUP> ~10<SUP>9</SUP> CFU/g의 분포를 나타내었고 대장균군은 불검출 되었다. 식중독세균 중 B. cereus는 전통된장 10개 중 7개에서 검출되었으나 기타 식중독균은 불검출 되었다. 실험 결과 장수지역 전통된장에 가장 문제가 되는 미생물은 B. cereus로 나타나 전통된장의 안전성 확보를 위해 B. cereus 제어가 중요한 것으로 판단되었다. 전통된장의 미생물 군집 분석결과 Bacillus subtilis가 155균주(47.4%)로 가장 높은 분포를 나타내었으며, B. licheniformis 68균주(20.8%), B. amyloliquefaciens 46균주(14.1%)로 높은 분포를 나타내는 등 총 327균주가 동정되었다. B. cereus에 대한 길항 효과를 평가한 결과 분리균주 327균주 중 20균주(6.1%)에서 9~12 mm 생육저지환이 관찰되었으며, B. subtilis가 12균주로 가장 많았고, B. amyloliquefaciens 5균주, B. licheniformis 3균주 순으로 분포하였다. 전통된장의 경우 B. cereus 오염도가 식품공장 제조 된장에 비해 높지만 B. cereus 길항효과를 나타내는 균주가 많은 것으로 판단되었다. 이러한 결과를 종합해 볼 때 전통된장에 오염된 B. cereus 위험성은 기존의 생각보다 낮을 것으로 판단되며, 향후 다수의 전통된장을 대상으로 한 B. cereus 길항 효과 실험이 필요한 것으로 판단되었다. This study investigated the prevalence of foodborne pathogens and the bacterial community of traditional Doenjang collected from a longevity area in Korea as well as the antagonistic effect of traditional Doenjang isolates against Bacillus cereus to estimate the microbiological safety of traditional Doenjang. Aerobic bacteria showed 10<SUP>6</SUP>∼10<SUP>9</SUP> CFU/g, whereas coliform bacteria was not detected. Foodborne pathogens were not detected except B. cereus, which was detected in seven samples out of 10 Doenjang samples. A total of 327 isolates were identified from traditional Doenjang. The isolates consisted of Bacillus subtilis 155 (47.4%), Bacillus licheniformis 68 (20.8%), Bacillus amyloliquefaciens 46 (14.1%), and Bacillus pumilus 18 (5.5%). Antagonistic effect against B. cereus was detected in 20 (6.1%) of 327 isolates, which consisted of B. subtilis (12 strains), B. amyloliquefaciens (5 strains), and B. licheniformis (3 strains). The inhibitory zone for the antagonistic effect was 9.0∼12.0 mm in diameter. Although a small amount of traditional Doenjang was tested in this study, these results indicated that the potential risk of B. cereus in traditional Doenjang is lower than generally presumed. It is necessary to monitor the antagonistic effect of traditional Doenjang isolates against B. cereus.

Mannitol-Egg York-Polymyxin B 선택 배지에서 Bacillus cereus 계수 방법의 재평가

윤숙현,김용상,정도연,한금수,엄태붕,Yun, Suk-Hyun,Kim, Yong-Sang,Jeong, Do-Yeon,Hahn, Kum-Su,Uhm, Tai-Boong 한국미생물학회 2009 미생물학회지 Vol.45 No.2

장류에서 식중독 미생물인 Bacillus cereus의 계수 방법상 문제점을 해결하기 위하여, B. cereus 선택 배지인 mannitol-egg york-polymyxin B (MYP)에서 자란 약 1,500개의 집락들을 형태적 차이에 따라 12종으로 분류하였다. 생화학 특성 및 16S rRNA 유전자의 염기서열 분석 결과 집락 주위로 환을 형성하는 직경 11~16 mm 크기의 집락들은 B. cereus 와 B. cereus subsp. cytotoxis로 동정되었고, 환을 형성하지 않아 B. cereus 계수에 포함시키지 않았던 직경 5 mm 크기의 분홍색 집락은 B. cereus로, 6.5 mm 크기의 이중환을 형성하여 B. cereus로 계수하기가 모호하였던 붉은색 집락은 B. cereus로 동정되었다. 나머지 세 종들은 Enterococcus sp., Brevibacillus sp., B. subtilis로 밝혀졌다. B. cereus로 동정된 9종 모두에서 적어도 한 종류 이상의 B. cereus 독소 유전자들을 함유하고 있었지만 다른 형태들에서는 발견되지 않았다. To avoid ambiguity in counting the number of colony, about 1,500 of colonies grown on B. cereus selective agar plates were grouped into 12 types by morphological difference and then identified by biochemical and 16S rDNA nucleotide sequence. Among them, seven colony types with 11 to 15 mm diameters of halo were identified as B. cereus or B. cereus subsp. cytotoxis. Five mm sized colonies with no halo, which have not been considered as B. cereus according to the manufacturer's manual, were identified as B. cereus. A colony type with double halos of only 6 mm in diameter was also B. cereus. Other three types were proven to be Enterococcus sp., Brevibacillus sp., and B. subtilis, respectively. PCR results showed that only 9 types that are identified as B. cereus strains harbor at least one of B. cereus toxin genes.

Bacillus cereus에 대한 actinonin의 항균 효과

정동윤(Dongyun Jung),염수진(Su-Jin Yum),유연철(Yeon-Cheol Yu),김종헌(Jong-Heon Kim),이병휘(Byung-Hwi Lee),장훈녕(Hoon-Nyung Jang),정희곤(Hee Gon Jeong) 한국식품과학회 2016 한국식품과학회지 Vol.48 No.6

본 연구에서는 지금까지 알려진 바와 달리 항균활성의 적용범위가 모호한 actinonin을 대표적 식중독균인 B. cereus에 적용함으로써 식중독균에 대한 항균 효과에 대해 연구하였다. 계대배양된 대수기의 B. cereus에 actinonin을 처리하여 생장 저해 여부를 확인하였고, 96-well plate를 이용한 broth micro-dilution 방법과 agar disk diffusion 방법을 통해 actinonin의 B. cereus에 대한 항균활성을 측정해본 결과, actinonin의 농도에 비례하여 B. cereus의 생장이 저해되는 것으로 나타났다. 또한, actinonin의 처리에 따른 B. cereus의 세포 독성 측정 결과 actinonin이 B. cereus의 세포 독성 또한 억제하는 것을 확인하였다. 따라서, actinonin은 천연항균물질로서 B. cereus에 의한 식품 오염을 억제하고 식중독 예방 및 잠재적 치료제로서 적용 될 가능성이 있는 것으로 관찰되었으며, 나아가 다른 식중독균들에 대한 적용 가능성 탐색과 관련한 연구가 필요할 것으로 생각된다. The objective of this study was to investigate the anti-Bacillus cereus activity of actinonin. Actinonin inhibited the growth of B. cereus in a dose dependent manner. The growth-inhibitory activity of actinonin was evaluated using a broth micro-dilution method, and minimum inhibitory concentration (MIC) and agar disk diffusion tests. B. cereus showed high susceptibility to actinonin in a concentration-dependent manner and MIC was determined to be 0.192 μg/mL. Additionally, 1 and 2 mM actinonin induced formation of B. cereus inhibition zones. In addition, as compared to B. cereus alone, B. cereus added with 10 μM actinonin showed a lower level of cytotoxicity in HeLa cells in vitro. Thus, this study revealed that actinonin could be a potential source of a natural antimicrobial agent or a pharmaceutical component against B. cereus.

Inhibition of Bacillus cereus Growth and Toxin Production by Bacillus amyloliquefaciens RD7-7 in Fermented Soybean Products

( Jeong Seon Eom ),( Hye Sun Choi ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.1

Bacillus cereus is a gram-positive, rod-shaped, spore-forming bacterium that has been isolated from contaminated fermented soybean food products and from the environment. B. cereus produces diarrheal and emetic toxins and has caused many outbreaks of foodborne diseases. In this study, we investigated whether B. amyloliquefaciens RD7-7, isolated from rice doenjang (Korean fermented soybean paste), a traditional Korean fermented soybean food, shows antimicrobial activity against B. cereus and regulates its toxin gene expression. B. amyloliquefaciens RD7-7 exhibited strong antibacterial activity against B. cereus and inhibited the expression of B. cereus toxin-related genes (groEL, nheA, nheC, and entFM). We also found that addition of water extracts of soybean and buckwheat soksungjang (Korean fermented soybean paste made in a short time) fermented with B. amyloliquefaciens RD7-7 significantly reduced the growth and toxin expression of B. cereus. These results indicate that B. amyloliquefaciens RD7-7 could be used to control B. cereus growth and toxin production in the fermented soybean food industry. Our findings also provide a basis for the development of candidate biological control agents against B. cereus to improve the safety of fermented soybean food products.

즉석조리식품에서의 Bacillus cereus오염실태조사 및 생육 pattern 분석

김순한,김미경,강민철,손영욱,이창희,김인복,이영자,최수영 한국생명과학회 2004 생명과학회지 Vol.14 No.4

국민 다소비 식품인 생선회, 패류 등의 횟감류 118건, 김밥, 햄버거 등의 도시락류 82건 및 기타 ready-to-eat(RTE) 식품 40건, 총 240건을 대상으로 Bacillus cereus의 오염실태를 조사한 결과, 횟감류 16건(13.6%), 도시락류 17건(20.7%), 기타 RTE 식품에서 3건(7.5%), 총 36건(15.0%)에서 B. cereus가 검출되었다. B. cereus의 검출은 polymyxin B를 첨가한 TSB배지를 사용한 증균배양 후, MYP agar에서 전형적인 집락을 선별하였으며, 선별된 균주들을 대상으로 그람염색, oxidase test, 혈액한천배지에서의 rhizoid growth, $\beta$-용혈성 유ㆍ무등을 통해 추정 균주들을 분리하였다. API 50CHB/20E system을 통한 생화학적 분석과 motility시험 등을 행하여 최종적으로 B. cereus로서 동정ㆍ확인하였다. 확인된 36균주 중 28균주(77.8%)가 BCET-RPLA 시험을 통해 BHI broth에서 설사형 독소를 생성하는 것으로 나타났다. 아울러 항생제 감수성 시험 결과 분리균들은 ampicillin (100%), penicillin G (100%) 및 rifampicin (69.5%)에 내성을 보였으며, gentamicin (100%), vancomycin (100%), bacitracin (86.1%), chloram-phenicol (97.2%), erythromycin (69.4%), kanamycin (86.1%) 및 streptomycin (97.2%)에 감수성을 나타내었다. B. cereus의 생육은 온도와 시간에 밀접한 관련성을 보였는데, 냉장온도인 1$0^{\circ}C$이하에서는 균의 생육이 상당히 저해된 반면, 2$0^{\circ}C$ 및 3$0^{\circ}C$에 보관시에는 급격한 증식을 보여 기온이 상승하는 하절기에 특히 많은 주의가 필요하다고 판단되었다. The contamination of Bacillus cereus was investigated in 240 RTE (ready-to-eat) food samples including 118 seafoods, 82 Korean packaged meals and 40 other RTE foods. Many B. cereus presumptive strains were isolated from the enrichment culture in Tryptic Soy Broth (TSB) added polymyxin, followed by selective culture in Mannitol Egg Yolk Polymyxin (MYP) agar and Gram staining. A total of 36 strains (16 in seafoods, 17 in Korean pack-aged meals and 3 in other RTE foods) were identified as B. cereus by the analysis of 61 biochemical tests of the API 50CHB/20E system test and supplementary tests of $\beta$-hemolysis, rhizoid growth, motility and oxidase activity. The 28 strains out of 36 B. cereus isolates produced diarrhoeal enter-otoxin in Brain Heart Infusion (BHI) broth. All isolates were resistant to ampicillin and penicillin antibiotics, and most of them were susceptible to gentamicin, vancomycin, bacitracin, chloram-phenicol, kanamycin and streptomycin. The growth of B. cereus was affected by environmental temperature and incubation time. Culture with temperature under 1$0^{\circ}C$ effectively restricted the growth of B. cereus.

영ㆍ유아용 식품원료의 Bacillus cereus와 일반세균 모니터링 및 제조공정 중 미생물 품질제어

정우영(Woo-Young Jung),엄준호(Joon-Ho Eom),김병조(Byeong-Jo Kim),주인선(In-Sun Ju),김창수(Chang-Soo Kim),김미라(Mi-Ra Kim),변정아(Jung-A Byun),박유경(You-Gyoung Park),손상혁(Sang-Hyuck Son),이은미(Eun-Mi Lee),정래석(Rae-Seok Jung),나미 한국식품과학회 2010 한국식품과학회지 Vol.42 No.4

영ㆍ유아용 식품에 주로 사용되는 원료에 대한 미생물 오염도를 조사한 결과, 총 10종(n=20)의 원료 중 B. cereus가 검출된 원료는 2종(n=4, 20%) 이었고, 1.02±1.36 log CFU/g으로 나타났으며, B. cereus가 검출된 원료로는 organic brown rice powder(C사)와 mixed orgarnic vegetable powder(C사) 이었다. 미생물이 검출되지 않거나 낮게 검출된 원료는 제조공정에 살균, 분무건조, 팽화 및 압출 등의 열처리 공정이 있었다. 각 원료에 대한 일반세균수를 알아보기 위해 10종(n=20) 검사결과 4종(n=8) 검출(40%) 되었으며, 3.21±3.64 log CFU/g으로 나타났다. 원료에서 분리한 미생물 분포를 조사한 결과 총 11종이 분리 되었으며, 분리된 일반세균 중 76%를 차지하는 우점종은 S. paucimobilis, P. fluorescens, R. radiobactor, St. maltophilia 순으로 나타났다. 본 연구에서는 영ㆍ유아식의 미생물 안전성 확보를 위해 원료 생산공정에 살균 등의 열처리 공정이 필요하며, 미생물에 오염된 원료를 사용할 경우에도 생산공정에 드럼건조(drum surface temperature: 100-135℃), 분무건조(inlet air temperature: 135-204℃), 살균(pasteurization, UHT 130-150℃/1-4 sec), 미생물 저감화 방안의 제조 공정을 거쳐 생산된 제품은 일반세균, 대장균군, B. cereus가 검출되지 않아 안전한 제품의 생산이 가능하였다. 또한, 영ㆍ유아용 식품을 제조할 경우 명확한 살균조건을 설정하고, 공정품의 품질평가를 거쳐야만 제품의 안전성을 확보할 수 있을 것으로 보인다. The purpose of this study was to examine the presence of Bacillus cereus, aerobic bacteria and coliforms in the raw material of infant formulas and investigate the manufacturing process in terms of microbial safety. Among ten kinds of raw infant formula material samples (n=20), Bacillus cereus appeared in two (n=4). Aerobic bacteria were not detected in raw infant formula material or maximum 4.15 log CFU/g. Eleven species of aerobic bacteria were isolated and 76% of them were Sphingomonas paucimobilis, Pseudomonas fluorescens, Rhizobium radiobactor, or Stenotrophomonas maltophilia. A Pearson’s correlation analysis revealed that the most influential factors for detecting Bacillus cereus were aerobic bacteria and coliforms. In other words, when the measured values of aerobic bacteria and coliforms were higher, the possibility that Bacillus cereus would appear increased. In a regression model to predict Bacillus cereus, the rate of appearance was correlated with aerobic bacteria and coliforms, and its contribution rate for effectiveness was 86%. Improving microbial quality control by pasteurization, spray dry, popping and extrusion resulted in a decrease in the numbers of Bacillus cereus, aerobic bacteria and coliforms in the raw materials. The results suggest that a hazard analysis and critical control point system might be effective for reducing microbiological contamination.

Usefulness of a Chromogenic Selective Agar for the Identification of Bacillus cereus Isolated from Blood Cultures

정은선,신종희,신명근,서순팔,양동욱 대한진단검사의학회 2010 Annals of Laboratory Medicine Vol.30 No.4

Background : The incidence of Bacillus cereus bacteremia is increasing, but the identification of Bacillus species remains difficult. Brilliance Bacillus cereus agar (BBC agar; Oxoid, UK) is a new CHROMagar medium that allows selective isolation and identification of B. cereus; however, its clinical usefulness is seldom studied. We evaluated the usefulness of BBC agar to identify B. cereus isolates recovered from blood cultures. Methods : We analyzed a total of 53 blood isolates that showed a Bacillus-like morphology on Gram staining. All isolates were identified by using both the API Coryne (bioMérieux, France) and API 50CH/B (bioMérieux) systems. They were subsequently subcultured on BBC agar, incubated for 24 hr, and then examined for characteristic blue-green colonies. The clinical characteristics of patients whose isolates were identified as B. cereus were assessed. Results : Of the 53 isolates, 18 were identified as B. cereus by API 50CH/B. With the API 50CH/B system used as gold standard, the sensitivity and specificity for the identification of B. cereus were 100% (18/18) and 100% (35/35), respectively, using BBC agar, and 67% (12/18) and 100% (35/35),respectively, using the API Coryne system. Of the 18 patients with B. cereus bacteremia, 15 showed infectious signs, and 3 had more than 2 blood cultures positive for B. cereus on separate days. Conclusions : Our study shows, for the first time, that BBC agar, with its good agreement and ease of use, is a valuable alternative to the API 50CH/B system for the presumptive identification of B. cereus isolates from blood cultures. (Korean J Lab Med 2010;30:394-9)