Bae H D, Yanke L J, Cheng K J, Selinger L B
Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta.
J Microbiol Methods. 1999 Dec;39(1):17-22. doi: 10.1016/s0167-7012(99)00096-2.
Differential agar media for the detection of microbial phytase activity use the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. When this technique was applied to the study of ruminal bacteria, it became apparent that the method was unable to differentiate between phytase activity and acid production. Strong positive reactions (zones of clearing around microbial colonies) observed for acid producing, anaerobic bacteria, such as Streptococcus bovis, were not corroborated by subsequent quantitative assays. Experimentation revealed that acidic solutions generated false positive results on the selected differential medium. Empirical studies undertaken to find a solution to this limitation determined the false positive results could be eliminated through a two step counterstaining treatment (cobalt chloride and ammonium molybdate/ammonium vanadate) which reprecipitates acid solubilized phytate. This report discusses the application of the developed two step counterstaining treatment for the screening of phytase producing ruminal bacteria as well as its use in phytase zymogram assays.
用于检测微生物植酸酶活性的鉴别琼脂培养基利用沉淀的植酸钙或植酸钠的消失作为酶活性的指标。当将该技术应用于瘤胃细菌的研究时，很明显该方法无法区分植酸酶活性和产酸情况。对于产酸的厌氧细菌，如牛链球菌，观察到的强阳性反应（微生物菌落周围的透明圈）在随后的定量分析中并未得到证实。实验表明，酸性溶液在所选鉴别培养基上产生了假阳性结果。为解决这一局限性而进行的实证研究确定，通过两步复染处理（氯化钴和钼酸铵/钒酸铵）可以消除假阳性结果，该处理可使酸溶解的植酸重新沉淀。本报告讨论了所开发的两步复染处理在筛选产植酸酶瘤胃细菌中的应用及其在植酸酶酶谱分析中的用途。
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