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Bacillus 속 분리주가 생산하는 박테리오신의 특성 조사
함승희(Seung-Hee Ham),최낙식(Nack-Shick Choi),문자영(Ja-Young Moon),백선화(Sun-Hwa Baek),이송민(Song-Min Lee),강대욱(Dae-Ook Kang) 한국생명과학회 2017 생명과학회지 Vol.27 No.2
토하젓에서 분리한 박테리오신 생산 균주 중 상대적으로 넓은 항균스펙트럼을 나타내는 1균주를 선발하였고 16S rRNA 유전자 염기서열 분석 결과 B. subtilis E9-1와 거의 일치하는 것으로 동정되었다. B. subtilis E9-1가 생산하는 박테리오신의 물리화학적 특성을 조사하였고, 박테리오신을 정제하였다. 이 박테리오신은 B. cereus KCCM 11204, M. luteus IAM 1056, L. monocytogenes KCCM 40307, E. faecium KCCM 12118 및 S. aureus subsp. aureus KCCM 40050 등에 대해서 항균활성을 나타내었다. pH 2.0~8.0 범위에서는 안정하였으나 8.0 이상에서는 항균활성이 감소하였다. Isopropanol, ethanol 및 methanol 등의 유기용매에서 100%까지, acetone과 acetonitrile에서는 80%까지 항균활성을 유지하였다. 내열성의 경우 40~100℃에서 60분까지는 안정하게 항균활성을 보였다. 박테리오신 농도를 증가시키면서 B. cereus, L. monocytogenes, E. faecium 및 S. aureus subsp. aureus 등 시험균 4주의 감수성을 조사한 결과 농도 의존적으로 시험균의 생육이 감소하였고 이중 L. monocytogenes 의 감수성이 가장 높게 나타났다. 박테리오신의 작용양상을 알아본 결과 B. cereus와 L. monocytogenes 배양액에 박테리오신 용액을 첨가한 후 흡광도와 CFU값이 감소하여 bactericidal임을 확인하였다. 식품에 적용 가능성을 알아보기 위해 실험한 결과 3일째부터 박테리오신을 처리하지 않은 대조구에 비해 실험구에서 생균수(CFU/ml)가 감소하였다. 아세톤을 이용한 배양상등액의 농축, superdex peptide HR 10/300 column 이용한 겔여과크로마토그래피, 역상 HPLC로써 박테리오신을 정제하였다. 역상 HPLC를 통해서 정제한 박테리오신의 분자량을 tricine SDS- PAGE로 분석한 결과 약 4 kDa이었고 질량분석법으로 측정한 정확한 분자량은 3347.6 Da로 나타났다. As an effort to find a potential biopreservative, we isolated bacterial strains producing bacteriocin from fermented foods. A strain was finally selected and characteristics of the bacteriocin were investigated. The selected strain was identified as Bacillus subtilis E9-1 based on the 16S rRNA gene analysis. The culture supernatant of B. subtilis E9-1 showed antimicrobial activity against Gram-positive bacteria. Subtilisin A, α-chymotrypsin, trypsin and proteinase K inactivated the antimicrobial activity, which means its proteinaceous nature, a bacteriocin. The bacteriocin activity was fully retained at the pH range from 2.0 to 8.0 and stable at up to 100℃ for 60 min. Solvents such as ethanol, isopropanol and methanol had no effect on the antimicrobial activity at the concentration of 100% but acetone and acetonitrile reduced the activity at up to 100% concentration. Cell growth of four indicator strains was dramatically decreased in dose-dependent manner. Listeria monocytogenes was the most sensitive, but Enterococcus faecium was the most resistant. Bacillus cereus and Staphylococcus aureus showed the medium sensitivity. The bacteriocin showed its antimicrobial activity against B. cereus and L. monocytogenes via bactericidal action. The number of viable cells of L. monocytogenes started to reduce after addition of bacteriocin to the minced beef. The bacteriocin was purified through acetone concentration, gel filtration chromatography and RP-HPLC. The whole purification step led to a 6.82 fold increase in the specific activity and 6% yield of bacteriocin activity. The molecular weight of the purified bacteriocin was determined to be 3.3 kDa by MALDI-TOF/TOF mass spectrometry.
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함승희,서영주,정은미,채선희 효성여자대학교 문리과대학 학도호국단 1984 文理大論集 Vol.4 No.-
This study was carried out to get the basic date for effective control of apple canker disease in Daegu; Chilgog, Gyeongsan, and Weolseong, which were major appleproduction districts. The relationship among occurence rate of apple canker, district, infected side of trunk, and nutrition state of apple leaf were examined. The results of the study were summarized as follows. 1. The occurence rates of apple canker were a little high in Yeongcheon and Gyeongsan district as 81.7%, 83.5% respectively, but in Chilgog district was 70.2%. 2. The occurence rates were high in Spur Early Blaze, Spur Golden and Megumi varieties as 84.8%, 87.3%, 88.6% respectively, but in Rallss Genet, Moris delicious, Starkrimson, Raritan variety were tend to lower as 69.4%-72.3%. 3. The occurence rate of free stocked tree was high as 81.8%, But in dwarf stocked tree was low as 73.8%. 4. The occurence rate was 80.7% in trunk and main blanch part, and in the east and south side of trunk. The occurence rate of apple canker was 55.7%. 5. There was significant positive correlation(r=0.516^*)between occurence rate of apple canker and N content of apple leaf, but negative correlation (r=-0.589^*) in P_2O_5 content. 6. Occurence rate of apple canker was tend to higher as the ca/Mn ratio in apple leaf was lower.
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