利用層析法探討不同Aspergillus terreus菌株於米中產生土震素B之能力   全文下載

Determination of Territtrem B in Rice Media with Different Strains of Aspergillus terreus by Chromatography

土震素B（territrem B）為土麴菌（Aspergillus terreus）產生之震顫性黴菌毒素，可導致動物全身性顫抖、流涎、抽搐、甚至導致死亡，其作用機制為抑制乙醯膽鹼酵素（acetylcholinesterase）的活性。本研究目的在於探討食品工業發展研究所菌種保存中心之17株A. terreus於米基中產生土震素B的能力。首先從薄膜層析法及高效能液相層析法進行分析，發現僅編號32111與32664之菌株可產生與土震素B類似之藍色螢光物質，進一步將此藍色螢光物質以質譜儀鑑定為土震素B。土震素B的含量以高效能液相層析法配合C18逆向層析管柱及於335nm波長下測定。土震素B之標準線性公式為Y=1428.5X+306.0 （R2= 0.9998），其中Y為土震素B於高效能液相層析儀之面積，X為土震素B的濃度，顯示良好的線性關係；同日間及異日間之相對標準偏差分別為1.6～2.8%及0.6～2.3%，顯示具有良好之準確性及精確性；添加回收率在91.5～95.2%之間，顯示氯仿可作為米基萃取液，利用此方法可知A. terreus 32111 及32664 產生土震素B 含量分別為0.2 及0.3 mg／100g 米。從地源性而言，產生土震素B之A. terreus 32111及32664兩株菌為台灣本土之菌株，其它15株分別來自不同國家之A. terreus，無法產生土震素B。

Territrem B is a tremogenic mycotoxin produced by Aspergillus terreus. Animal studies have demonstrated that territrem B caused whole body tremor, salivation, convulsion and death. Territrem B is also a potent irreversible inhibitor of acetylcholinesterase (AchE, E. C. 3.1.1.7). The aim of the present study is to assess the ability of strains of Aspergillus terreuss to produce territrem B in the rice media, which were obtained from Food Industry Research and Development Institute in Taiwan. In the 17 strains of A. terreus, only No. 32111 and 32664 strains produced territrem B-like blue fluorescence compound by TLC and HPLC analyses. Later on, this compound was identified as territrem B by mass spectroscopy. The amount of territrem B was determined by reverse phase HPLC on a C18 column with UV detection at 335 nm. The linear regression coefficient of the standard curve (Y=1428.5X+306.0) for territrem B at concentration ranging from 0 to 250 ng/L is 0.9998. In rice media, the recovery rates for territrem B at 50, 100 and 200 ng/L ranged from 91.5% to 95.2% with a coefficient of variation less than 2.1%. The relative standard of territrem B ranged between 1.6-2.8% (intra-day) and 0.6-2.3% (interday). In rice culture of A. terreus 32111 and 32664 strains, the contents of territrem B were 0.20 and 0.31 mg/100 g rice, respectively. From the above results, it can be concluded that this HPLC method for determination of territrem B in rice media is suitable and only domestic strains can produce territrem B. 土震素B（territrem B）為土麴菌（Aspergillus terreus）產生之震顫性黴菌毒素，可導致動物全身性顫抖、流涎、抽搐、甚至導致死亡，其作用機制為抑制乙醯膽鹼酵素（acetylcholinesterase）的活性。本研究目的在於探討食品工業發展研究所菌種保存中心之17株A. terreus於米基中產生土震素B的能力。首先從薄膜層析法及高效能液相層析法進行分析，發現僅編號32111與32664之菌株可產生與土震素B類似之藍色螢光物質，進一步將此藍色螢光物質以質譜儀鑑定為土震素B。土震素B的含量以高效能液相層析法配合C18逆向層析管柱及於335nm波長下測定。土震素B之標準線性公式為Y=1428.5X+306.0 （R2= 0.9998），其中Y為土震素B於高效能液相層析儀之面積，X為土震素B的濃度，顯示良好的線性關係；同日間及異日間之相對標準偏差分別為1.6～2.8%及0.6～2.3%，顯示具有良好之準確性及精確性；添加回收率在91.5～95.2%之間，顯示氯仿可作為米基萃取液，利用此方法可知A. terreus 32111 及32664 產生土震素B 含量分別為0.2 及0.3 mg／100g 米。從地源性而言，產生土震素B之A. terreus 32111及32664兩株菌為台灣本土之菌株，其它15株分別來自不同國家之A. terreus，無法產生土震素B。