| 摘要註: |
本實驗室自拳擊手套中篩選出可分泌抗生物質的菌株,自定編號為Y-1,經培養於PDA上型態及經革蘭氏染色法鑑定,粗略判定Y-1為革蘭氏陰性菌;自然界中此類細菌分泌抗細菌物質為相當罕見。經培養基探討其最適Y-1生產抗生物質之培養基組成為1%葡萄糖、0.7%黃豆粉及0.3%酵母菌萃取物,培養條件為30℃、轉速150 r.p.m.培養24小時,可獲得最高產量之抗生物質。在純化部分,粗培養液以高速離心的方式去除菌體及大分子物質後,先以氯化鈣及丙酮沉澱去除大部分黏稠物質以及蛋白質,再利用極性原理,分別進行Al2O3以及silica管柱層析,除去大部分雜質成份,最後再依據分子量原理進行Sephadex LH-20管柱層析,收得透明澄清之純化產物。經由薄膜色層分析以及結晶物質之產生,判斷此抗生物質為具有高純度之物質,最終之回收率為。此菌株針對革蘭氏陽性菌(金黃色葡萄球菌以及)及白色念珠菌具高抑制能力,特別對具有抗藥性之抗萬古黴素腸球菌及抗萬古黴素金黃色葡萄球菌亦具有強力抑制。此抗生物質以100℃水浴處理60分鐘後仍保有83%的活性,表示此物質具有良好的耐熱性。並且可清除金黃色葡萄球菌生成之生物膜的能力。經由LC-Mass分析結果顯示,此抗生物質為一小分子的化合物。 An antibiotic-secreting bacterium was isolatetd from boxing glove and named as Y-1. Based on the morphology of the bacterium on PDA plate and Gram staining, Y-1 was identified as a Gram-negative bacterium. From previous research, there are rare Gram-negative bacteria that secret antibacterial substances in natural products. After experimental research, the best fermentation media of Y-1 was 1% glucose, 0.7% soybean flour and 0.3% yeast extract, incubating at 30℃, 150r.p.m for 24 hours, then there would be highest antibacterial substance produced by Y-1. In isolation and purification part, first we used high speed centrifugation to exclude bacteria and high molecular weight substances so we can collect suspension part of crude fermentation liquid. After previous step, we can exclude proteins and most glutinous substances by precipitation of calcium chloride and acetone. Based on polarity of chemical solutions, we used Al2O3 and silica column chromatography to exclude most impurities. Finally, based on molecular weight to process Sephadex LH-20 gel filtration then we can get transparent and purified substances. By thin layer chromatography analysis and production of crystal, it showed that this antibacterial substance has high purity. The final recovery rate is 4.3%. Y-1 has high inhibition to Gram positive bacteria (Bacillus and Staphylococcus aureus) and Candida albicans, especially to Vancomycin-resistant Enterococcus (VRE) and Vancomycin-resistant Staphylococcus aureus (VRSA). Y-1 has 83% antibacterial activity after water bath treatment at 100℃ for 60 minutes which means it has great heat-resistant capability, and Y-1 also has ability to clean away biofilms produced by Staphylococcus aureus. After LC-Mass analysis, it showed that Y-1 was a compound with small molecular weight. |