1株广谱抗菌活性菌株的筛选鉴定及其抗菌蛋白的分离纯化

从湖南传统腊肉中分离筛选得到1株具有广谱抑菌活性的菌株HLR13,该菌株发酵液对蜡样芽孢杆菌、荧光假单胞菌、藤黄微球菌和肠炎沙门菌等食品中常见的致病菌具有较强的抑制活性。通过形态学特征、生理生化特征及16S rRNA序列分析,该菌株被鉴定为枯草芽孢杆菌斯皮兹仁亚种(Bacillus subtilis subsp.spizienii)。采用硫酸铵分级沉淀、离子交换层析(Q Sepharose FF)和凝胶过滤层析(Sephadex G-75)等方法对该菌株所产抗菌蛋白进行分离纯化,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测显示纯化后的蛋白为单一条带,通过基质辅助激光解吸电离飞行时间质谱分析和NCBI/nr蛋白数据库比对初步确定该抗菌蛋白分子质量为31 494 Da,是枯草芽孢杆菌产生的一种假定蛋白。本实验可为新型天然防腐剂的开发提供一定理论依据。

Screening and Identification of a Strain with Broad-Spectrum Antimicrobial Activity and Isolation and Purification of Antimicrobial Protein

A new strain with broad-spectrum antimicrobial activity, named as HLR-13, was isolated from traditional smoked bacon from Hunan province. The cell-free supernatant produced by the strain HLR-13 showed a high inhibitory activity towards many foodborne pathogenic bacteria and fungi such as Bacillus cereus, Pseudomonas fluorescens, Mirococcus luteus, and Salmonella enteritidis. Based on its morphological, physiological and biochemical characteristics as well as 16S rDNA sequence analysis, the strain HLR-13 was identified as Bacillus subtilis subsp. spizienii. Then the antimicrobial protein produced by the strain was isolated and purified through ammonium sulfate precipitation, ion exchange chromatography (Q Sepharose FF) and gel filtration chromatography (Sephadex G-75). Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band and the antimicrobial protein was preliminary identified as a hypothetical protein with molecular weight of 31 494 Da by matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis through comparison with NCBI non-redundant (NR) protein database. This research can provide a theoretical basis for developing new biological preservatives.