메주에서 분리한 항 Bacillus cereus 활성을 지닌 Bacillus subtilis의 분리, 특성 규명과 그 항균물질의 분리정제

Abstract

Bacillus cereus is foodborne pathogenic bacteria. It causes diarrhea, nausea and vomiting. It is commonly present in food production environments by adherence endospores spreading to all kinds of foods. In this study, the one bacterial strain which anti-B. cereus activity was isolated from Meju, a Korea soy bean fermentation starter, was investigated. It was identified using morphological and biochemical properties, and nucleotide sequence determination of 16S rRNA gene. It was identified as Bacillus subtilis SN7. B. subtilis SN7 was proved not to carry toxin genes such as Hbl, Nhe, bceT, entFM, cytK and ces through PCR analysis. The absence of the toxin production was confirmed using Oxoid RPLA and Tecra BDE kits. Antibacterial activity of B. subtilis SN7 was stable in the pH range of 2.0∼9.0. But it was unstable at 70℃ for 24 hr and proteolytic enzyme treatments such as proteinase K, protease and α-chymotrypsin. The anti-B. cereus compound indicated proteineous nature. The growth inhibitory effect of B. subtilis SN7 on four B. cereus (KCTC 1012, KCTC 1013, KCTC 1092 and KCTC 3624) observed. When B. subtilis SN7 was co-cultured with four B. cereus, it completely inhibited growth of all tested B. cereus strains within 4∼24 hr. Except B. subtilis SN7 was not inhibited when B. cereus KCTC 1012, B. cereus KCTC 3624 inoculated of 6.2∼6.3 log CFU/mL. When B. cereus KCTC 3624 was cultured with supernatant of B. subtilis SN7, it completely inhibited growth of B. cereus KCTC 3624 with 13∼22 hr. Antibacterial activity of B. subtilis SN7 against B. cereus was confirmed by observation of cell morphology using a scanning electron microscopy. To investigate of the potential of the anti-B. cereus compound as a biopreservative, B. subtilis SN7 and B. cereus KCTC 3624 were adapted in Cheongkukjang. None of B. .cereus cell counts were not detected in Cheongkukjang after 24 hr incubation. B. subtilis SN7 was detected over 8.3∼8.7 log CFU/g. The antibacterial compound from the isolated B. subtilis SN7 was purified using C18 Sep-pak cartridge, prep-HPLC, and recycling HPLC. Antibacterial compound was separated into one fraction through three steps HPLC. The purified compound was confirmed antibacterial activity against B. cereus KCTC 3624, and it was destroyed completely with proteolytic enzyme treatment.