一新分離枯草菌所產生細菌素之部分特性鑑定

Abstract

枯草桿菌 Bacillus subtilis 廣泛存在於自然環境中，被認定是 Generally Recognized As Safe (GRAS) 菌種，在細菌素相關研究中視為具有相當發展潛力。本研究室自淡水金沙灣沙灘篩選出菌株 Bacillus subtilis strain 6-1-1，為一具有抗菌活性之細菌菌株。B. subtilis strain 6-1-1 在平板培養基上能抑制格蘭氏陽性菌：Bacillus subtilis ATCC 6633、Micrococcus luteus BCRC 10449與Staphylococcus aureus BCRC 10541；以及格蘭氏陰性菌：Aeromonas hydrophila CKH-29 與 Escherichia coli BCRC 10450。在本研究中發現，B. subtilis strain 6-1-1 抗菌物質存在於培養離心上清液中，加入蛋白酶 trypsin、pepsin 與 proteinase K 能使其抗菌活性喪失，證明該抗菌物質應為蛋白質。含抗菌物質之培養上清液經測試對於溫度與 pH 值有很好的穩定性，80oC 下加熱處理 3 小時、pH 2 到 10 儲放四天都不致使抗菌活性消失，低溫 (4oC 或 -30oC) 下長時間儲存也能維持其抗菌活性。B. subtilis strain 6-1-1 培養液經離心、硫酸銨沉澱，與膠體過濾法等純化步驟後，可得到具抗菌活性之回收純化樣品。小分子電泳 tricine SDS-PAGE 分析純化樣品，可知 strain 6-1-1 產生之抗菌物質分子量小於 3.5 kDa，純化後純度約為 97%。將純化後抗菌物質與指標菌株共培養後，會造成指標菌種細胞死亡，推測 strain 6-1-1 所產生之抗菌物質進行殺菌作用。

Bacillus subtilis strain 6-1-1 was isolated from the sandy beach in Jinshawan Beach Park in Taipei County in Taiwan and was screened for its antimicrobial activity. B. subtilis strain 6-1-1, which had the broader spectrum of antibacterial activity, was active against the Gram-positive bacteria Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus; and against the Gram-negative bacteria Aeromonas hydrophila and Escherichia coli. The antimicrobial compound produced by strain 6-1-1 existed in the cell-free culture supernatant, and was determined as a proteinaceous substance, as it was sensitive to protease. It remained its antimicrobial activity under heat treatment and long-term storage at low temperature. The antimicrobial activity was stable between pH 2 and 10. The bacteriocin-like substance produced by strain 6-1-1 was purified sequentially by centrifugation, ammonium sulfate precipitation, and gel filtration. The molecular weight was determined by tricine SDS-PAGE to be about below 3.5 kDa. And the bacteriocin-like substance produced by strain 6-1-1 acts as a bacteriocidal action. B. subtilis strain 6-1-1 produces a low molecular mass bacteriocin-like compound, with a wide spectrum of activity, and potential technological properties in thermostability and pH stability for use as a bio-preservative.