부산대학교 도서관

Diverse bacteria were isolated from various fermented foods and their culture supernatants were tested for antibacterial activity by agar well diffusion assay. One isolate producing bacteriocin was finally selected and identified Bacillus vallismortis by 16S rRNA gene sequence analysis. And it was named as B. vallismortis MCBL 1012. SEM photograph showed the morphology of B. vallismortis MCBL 1012 was rod-shape. Additionally, it was observed that the treatment of bacteriocin caused the leakage of cellular contents in Listeria monocytogenes KCCM 40307, Enterococcus faecium KCCM 12118 and Streptococcus mutans KCTC 3065. The culture supernatant of B. vallismortis MCBL 1012 showed antibacterial activity against Gram (+) bacteria such as L. monocytogenes KCCM 40307, E. faecium KCCM 12118, Staphylococcus aureus supsp. aureus KCCM 40050, Micrococcus luteus IAM 1056, Bacillus cereus KCCM 11204, S. mutans KCTC 3065 and Gram negative Salmonella enteritidis KCCM 12021. PCR analysis showed B. vallismortis MCBL 1012 harbored subtiloisin A gene (sbo A), surfactin genes (srfA, Srf/lch, sfP), nonhemolytic enterotoxin C gene (nhe C) and hemolysin BL A, C, D (hbl A, C, D) genes. The highest bacteriocin activity was obtained when B. vallismortis MCBL 1012 was grown in LB medium at pH 7.0 and 37℃ for 24 hours. Antibacterial activity was decreased by proteinase K, subtilisin A and α-chymotrypsin. The bacteriocin was stable at up to 80℃ for 60 min and at pH range from 4.0 to 8.0. Antibacterial activity was not affected up to 100% organic solvents like ethanol, methanol acetonitrile and tetrahydrofuran but was decreased at more than 40% isopropanol and 80% acetone. It was resistant to 0.1% Triton X-100, Triton X-114, SDS, and 1% Tween 20, Tween 80, 0.05M EDTA, NaCl, MgCl2 and ZnCl2. The approximate half minimal inhibitory concentration (MIC50) of culture supernatant against L. monocytogenes KCCM 40307, M. luteus IAM 1056 and E. faecium KCCM 12118 was 0.427 ㎎/㎖, 0.444 ㎎/㎖, 0.643 ㎎/㎖ respectively. The bacteriocin exhibited its antibacterial activity through bactericidal action against L. monocytogenes KCCM 40307. The number of viable cells of L. monocytogenes KCCM 40307 was decreased after addition of bacteriocin to the blended pork and soft tofu. The concentrated bacteriocin was stable at –22℃ and 4℃ for 10 weeks and 3 weeks respectively. The bacteriocin was purified by ammonium sulfate concentration, DEAE anion exchange chromatography, and RP-HPLC. The purification resulted in a final yield of 0.03% and 283.8-fold increase in the specific activity. The molecular weight of purified bacteriocin was estimated to be about 3.0-4.0 kDa by SDS-PAGE and gel overlay assay. The exact molecular weight was determined to be 3,326.136 Da by MALDI-TOF MS analysis.