젓갈에서 분리한 Bacillus vallismortis BK6가 생산하는 항균 및 계면활성물질의 특성

Abstract

젓갈은 우리나라전통 발효음식으로 유용미생물이 분리가 되어 Probiotics, 항균제 및 발효식품에 첨가되는 starter로 이용 되고 있다. 젓갈에서 분리된 Bacillus sp.는 bacteriocin 유사 물질을 분비하게 되는데 합성기작에 따라 ribosomal 물질과 nonribosomal 물질로 나뉘게 되며, subtilin, coagulin 이 전자에 속하며 fengycin, surfactin, bacitracin, iturin group, gramicidins 등이 후자에 속한다. 또한 Bacillus sp는 생물계면활성(Biosurfactant) 물질을 효율적으로 생산한다는 특징을 가지고 있다. 따라서 본 연구는 bacteriocin 작용에 의한 항균활성이 우수한 균주를 젓갈에서 분리하여 항균물질을 추출하여 생리활성을 조사하였으며, 또한 공시균주의 biosurfactant을 추출하여 특성을 연구하였다. 젓갈에서 분리된 균주는 6종으로 분리균주 BK6가 빠른 균생육과 항균활성을 보여 실험대상 공시균주로 선정하였으며 항균활성 감수성이 높은 Streptococcus parauberis를 피검균으로 선택하였다. 공시균주 BK6를 동정하여 본 결과 Bacillus vallismortis로 동정이 되어 B. vallismortis BK6로 명명하였다. 항균물질을 최대로 생산하기위한 최적배양조건을 검토한 결과 탄소원은 soluble starch 1%, 질소원은 yeast extract 0.5%, NaNO3 0.5%, 무기염은 K₂HPO₄ 0.05%, NaCl 0.05%로 조사되었으며, 배양온도는 25℃, 배지의 초기 pH는 8∼9, 배양시간은 36시간으로 조사되었다. 항균물질의 열, pH 및 효소안정성 결과 효소가 첨가되지 않은 대조구에 비해 효소 첨가구에서 15∼20% 낮은 항균활성을 보였으며 pepsin 처리구에서 항균활성을 보이지 않았다. 또한 pH에 대체적으로 안정하며 열에 항균활성을 나타내지 않았기 때문에 열에 불안정하며 pepsin에만 선택적으로 불활성화 되는 구조를 가진 peptide성 물질이라고 사료된다. 항균물질을 용매 fraction을 통해 추출한 결과 hexane, chloroform 층에서는 물질추출이 이루어 지지 않았으며, ethyl acetate, butanol 층에서 추출된 물질을 가지고 생리활성을 측정하였다. 그 결과 최소억제농도는 1㎎/㎖로 조사되었으며, 피검균에 처리하였을 떄 세포벽이 붕괴되는 현상이 나타났다. 또한 항산화활성을 측정하여 1∼5 ㎎/㎖ 범위에서 50% Inhibition concentration이 조사되었다. 따라서 공시균주 BK6는 항균활성 이외에 항산화활성을 나타내는 균주라고 생각되어진다. 공시균주 BK6의 Biosurfactant의 생산을 조사하여 유화활성과 유화안정성을 검토하여본 결과 tributyrin을 기질로 사용 하였을 때, 가장 높은 유화활성을 보였으며 olive oil, squalene, kerosene 순으로 양호한 유화활성을 나타내었다. 유화 안정성은 olive oil이 가장 높은 안정성을 보였다. 따라서 본 실험의 공시균주는 생리활성 물질로서의 이용가능성과 생물계면활성제가 유류오염방제 및 화장품산업에 유용하게 적용될 것이라 사료되며, 앞으로 실질적으로 적용되는 추가실험을 실시하여야 한다고 생각 된다.

Jeot-kal, a Korean traditional fermentation food in Korea, is usually produced using fish and shellfish. This product is usually fermented using certain types of microorganisms and enzymes. This is not only as a source of protein supply, due to its peculiar flavor, and relative abundance of free amino acid and taste related components, but also a subsidiary material of Kimchi or spices. Six bacterial strains were isolated from several Jeot-kal using MRS agar medium. A bacterium which has high activities such as protease and pathogens bacterial species, was isolated from Myungran Jeot-kal, which was recorded as Bacillus valismortis BK6. It was identified as Bacillus vallismortis BK6 as based on the biochemical properties and 16S rRNA gene sequence. The bacterium was Gram-positive, catalase-negative, oxidase-positive, hydrosis of esculin ferric citrate, was rod-shaped, and had dimensions of 0.6∼0.7 ㎛ and 1.5∼2.0 ㎛. The strains showed characteristics secrete substance of gooey. When the material was rubbed it was slippery. Nutritional and cultural conditions for the production of antibacterial substance by this organism under shake-flask conditions have optimized. The optimal medium compositions for production of antibacterial substance in the B. vallismortis BK6 were 1%(v/w) of soluble starch, 0.5%(v/w) yeast extract, 0.5%(v/w) NaNO₃, 0.05%(v/w) K₂HPO₄ and 0.05%(v/w) NaCl. The optimum temperature and pH of the growth of the B. vallismortis BK6 was 25℃ and pH 8, respectively. The optimal culture time for antibacterial activities of the bacterium were shown to be in the range of 24∼36 hour. The antibacterial activity was found to be stable after heat and various enzyme treatment, and in the pH range of 3.5∼10.5. The antibacterial activity was stable in the pH range of 3.5∼10.5, but about all of temperature was lost after 5 min, so it assume that is inactive protein cause by heat. The antibacterial compound was only inactivated by pepsin treatment, its suggesting competitive inhibition of proteinaceous nature. This study investigated the effect of the antibacterial and antioxidant activity of solvents(ethyl acetate, butanol) from Strain BK6 supernatant. The minimum inhibitory concentrations of solvent extracts 1 ㎎/㎖ against S. parauberis. The extracts exhibited high scavenging activity on DPPH, alkyl and Hydrogen peroxide radical, and the activity increased with increment of concentration of the extracts. Scanning electron micrographs observation of S. parauberis treated with butanol extract(1 ㎎/㎖) revealed apparent lysis of the cell wall. Biosurfactants are biological surface-active compounds produced by bacteria and fungi that have a broad range of industrial applications as well as several advantages over chemical surfactants. The genus Bacillus is a bacterial group most often studied for production mechanisms of lipopeptides including biosurfactants and antibiotics. To further advance the study of biosurfactant production in the BK6 strain we investigated emulsification activity and stability. The crude biosurfactant was obtained from supernatant by acid precipitation, solvent extraction and evaporation. The yield of the crude biosurfactant was about 6 g/ℓ by optimum medium. In addition, the crude biosurfactant show high emulsification activity when tributyrin, soybean oil, squalene and kerosene were used as a substrate. Thus, these studies could contribute to the development of biosurfactant beneficial to the environment.