bacillus subtilis pda0426鉴定及抑菌活性产物研究word格式论文.docxVIP

山东轻工业学院硕士学位论文摘要微生物产生的抑菌活性物质是筛选抗菌新药的重要来源，为新药的开发提供 了大量的原材料。随着耐药性菌株的大量出现，新型抗菌药开发显得尤为重要。 本文以抑菌活性为导向，分离到一株具有强烈抑制金黄色葡萄球菌的细菌菌株 pda0426，并对该菌株进行了形态学及分子生物学的鉴定，确定 pda0426 菌株 为一株枯草芽孢杆菌，命名为 Bacillus subtilis pda0426。以发酵液的抑菌活性为指标，对 Bacillus subtilis pda0426 的发酵条件进行了 初步优化，确定其发酵的最佳培养基组成为蛋白胨、葡萄糖、NaCl，最佳培养条 件：最初 pH 为 8.0，培养温度为 30 °C，转速为 180 r/min，培养时间为 3 天。同 时，以组分回收率为指标，研究了发酵液的最佳粗分离方法，采用 LH-20 葡聚糖 凝胶柱层析与反相柱层析进行活性产物的粗分离。经过发酵液的粗分离以及活性组分的纯化与精制，得到了 9 个主要的组分。 经过 1H-NMR，13C-NMR、二维核磁数据、质谱等数据进行分析，其中确定结构 的组分有 4-11-1、4-11-2、R6-5-13.14-1 与 R7-1.2.3-13.18-5，4-11-2 与 R6-5-13.14-1为同一种化合物，属于大环内酯类，4-11-1 为环二肽。采用高通量及 MTT 法，对确定结构的化合物进行生物活性研究，主要测定 了其对供试菌株的最小抑制浓度（MIC）以及对肿瘤细胞的半数抑制浓度（IC50）。 结果表明化合物 4-11-2 与 R6-5-13.14-1 对金黄色葡萄球菌的 MIC 为 2.0 μg/mL， 对 PC3 的 IC50 为 41.0 μg/mL，对真菌没有非常明显的活性，是发酵液中抑制金黄 色葡萄球菌的主要有效成分。由于发酵液粗提物对一些植物致病真菌具有一定的 抑制活性，具有抗真菌活性的化合物仍然需要继续分离。关键词：抑菌；枯草芽孢杆菌；活性产物；鉴定；活性IABSTRACTIt is important to exploit new antimicrobial drugs, as the strains with drug resistance appear. The important sources of selecting new antimicrobial drugs are the active antibiotics produced by microbial.In this thesis, one bacteria named pda0426 was separated based on activity of inhibiting staphylococcus aureus. The strain was identified to be Bacillus subtilis by morphology and 16S rDNA sequence and phylogenetic analysis. It was named Bacillus subtilis pda0426.The conditions of fermentation of Bacillus subtilis pda0426 were optimized according to activity of broth. The best constitutes of medium were peptone, glucose and NaCl. The best condition of fermentation was described as below: initiative pH is 8.0, temperature is 30 °C, rotate speed is 180 r/min and it has been cultured for three days. At the same time, the method of crude separation has been builded based on the recovery rate of fraction, and confirmed to be LH-20 sephadex gel chromatography and reverse phase gel chromatography.Nine main compounds have been obtained by crude separation and purification of active fraction. The compounds, 4-11-1, 4-11-2, R6-5-13.