产苦马豆素疯草内生真菌U.oxytropis的发酵培养及苦马豆素检测方法的建立

Fermentation of locoweed endophytic (U.oxytropis) producing swainsonsine and the establishment of swainsonine detection method

通过对生长初期疯草内生真菌U.oxytropis菌丝复苏后连续培养32d,每隔4d取样1次,并设3个平行培养组,测定其不同培养时期菌丝干重变化,利用超声减压旋蒸法提取菌丝和发酵液中苦马豆素(SW),并分别利用薄层层析法、气相色谱法和α-甘露糖苷酶抑制法对菌丝、发酵液中SW含量进行测定。结果显示,经测定疯草内生真菌U.oxytropis菌丝干重质量,确定疯草内生真菌U.oxytropis生长周期为24d;薄层层析法、气相色谱法和α-甘露糖苷酶抑制法都可快速对菌丝和发酵液中提取的SW进行定性、定量检测,并比较3种检测方法,发现气相色谱法检测限低、灵敏度更高。本试验完成了疯草内生真菌U.oxytropis生长周期的测定,并建立了菌丝、发酵液中SW的有效、快速提取及检测方法,为下一步产苦马豆素疯草内生真菌U.oxytropis生物合成机理的研究奠定基础。

The sample was collected at 4days intervals for 32days after resuscitated the early growth of U.oxytropis mycelium.Three parallel culture groups were set up to determine the dry weight of mycelium in different culture periods.The swainsonine of mycelium and fermentation broth was extracted through ultrasonic vacuum rotary method.The content of swainsonine inmycelia and fermentation broth was determined through thin layer chromatography,gas chromatography and inhibition ofα-mannosidase.The results showed that the growth cycle of U.oxytropis was 24days.The swainsonine,extracted from mycelium and fermentation broth,can be quickly determined through the method of thin layer chromatography,gas chromatography andα-mannosidase inhibition,and gas chromatography method has lower detection limit and higher sensitivity than other methods.The experiment provides the theoretical basis for the next research of swainsonsine producing endophytic fungi biosynthesis mechanism of locoweed.