乳酸菌破壁方法的比较研究

Comparision research on the cell wall-breaking methods of lactic acid bacteria

本实验旨在探索一种快速、高效、经济的乳酸菌破壁方法。通过甘氨酸破壁法、反复冻融破壁法、溶菌酶破壁法、氧化锆珠破壁法、超声波破壁法以及3种复合破壁法对植物乳杆菌Lactobacillus.plantarum 1.557进行破壁处理,并以革兰氏染色和十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)为评价方法,比较破壁效果和破壁时间的差异。此外,通过β-葡萄糖醛酸酶(GUS)活力的测定对氧化锆珠破壁法所提蛋白进行活性检测,通过琼脂糖凝胶电泳对氧化锆珠破壁法所提RNA的质量进行了检测。结果表明:氧化锆珠破壁法优胜于其他几种处理方法,该法所提取的GUS蛋白具有蛋白活性,提取的RNA的OD_(260)/OD_(280)的比值在1.8~2.1之间,符合标准的比值,纯度较高,降解程度低。因此,氧化锆珠破壁法适用于快速提取乳酸菌菌体蛋白和总RNA,它是一种快速、高效、经济的乳酸菌破壁方法。

This study aims to explore a rapid, efficient and economical method for breaking lactic acid bacteria cell wall. Five different methods including glycine treatment, repeated freezing and thawing, lysozyme digestion, zirconia beads disruption, uhrasonic treatment and three composite wall-breaking methods were used to treat Lactobacillus.plantarum 1.557 cells, respectively.And gram staining and sodium dodecyl sulfate polyaerylamide gel electrophoresis （SDS-PAGE）were used as evaluation method to analyze the difference of cell wall-breaking effects and tittle costs. In addition, the activity of protein extracted by zirconia beads method was tested by the β-glucuronidase（GUS） assays.The quality of RNA extracted by zirconia heads method was tested by agarose gel electrophoresis. Results showed that zireonia beads method was better than other methods,the GUS protein extracted by this method had the activity of protein.The OD260/OD280 ratio of RNA extracted by this method was between 1.8 and 2.1 ,which was in accordance with the standard ratio,and the purity was high and the degradation degree was low.Therefore,the zirconia beads method was a fast, efficient and economical method for rapid extraction of lactic acid bacteria bacterial protein and total RNA.