枇杷種子中總氫氰酸檢驗方法之探討   全文下載

Study on Method of Test for Total Hydrocyanic Acid in Loquat Seeds

枇杷種子含有苦杏仁苷（Amygdalin），其經酵素或酸的作用會反應生成具毒性的氫氰酸。因應日本國民生活中心發布由枇杷種子製成，宣稱具抗腫瘤功效之保健茶產品檢出高量氫氰酸之訊息，本研究參考衛生福利部食品藥物管理署（以下稱食藥署）公開之「木薯製品中總氫氰酸之檢驗方法」進行枇杷種子中氫氰酸之檢驗方法開發。由於枇杷種子中之氰醣苷為苦杏仁苷，故以β-葡萄糖苷酶（β-Glucosidase from almonds）取代亞麻苦苷酶進行水解，針對酵素水解條件中pH值、反應溫度、作用時間及酵素作用量等4個方向進行探討。本研究取均質樣品0.5 g，添加10 U之β-葡萄糖苷酶於pH值6.6進行水解（38℃作用4小時），水解後經蒸餾收集氫氰酸，再以離子層析儀分析。以所建立之方法執行添加回收測試，於枇杷種子粉中分別添加相當於100及200 mg/kg氫氰酸之苦杏仁苷，其總氫氰酸之平均回收率介於78.4-86.4%，變異係數介於0.38-3.65%。檢驗市售2件含枇杷種子之茶包，總氫氰酸含量分別為119.6 mg/kg及80.6 mg/kg，本研究提供枇杷種子中總氫氰酸檢驗之流程架構，未來進行總氫氰酸相關檢驗方法精進時，可朝多重酵素反應之最適pH值或減少反應過程中的起泡反應進行探討以擴增檢驗方法適用性。

Loquat seeds contain amygdalin, which further reacts with enzymes or acids to generate toxic hydrocyanic acid. In a recent report announced by the Japan National Life Center, various health tea products claimed anti-tumor effects were found high levels of hydrocyanic acid. Therefore, an analytical method for the determination of total hydrocyanic acid content in loquat seed tea is on demand. This study evaluated and modified the “Test method for total hydrocyanic acid in cassava products” (published by TFDA) to develop a test method for hydrocyanic acid in loquat seeds. Because the cyanogenic glycoside in loquat seeds was amygdalin, the β-glucosidase (from almonds) enzyme was used in the developed method to replace linamarinase in the original method. Various parameters of enzyme were optimized as well including pH value, reaction temperature, reaction time and enzyme action amount. The established method applied 0.5 g of homogeneous sample each, added 10 U of β-glucosidase enzyme for hydrolysis at pH 6.6 for 4 hours at 38°C, collected hydrocyanic acid by distillation after hydrolysis, and then performed ion chromatographic instrument analysis. Recovery studys were conducted by spiking amygdalin equivalent to 100 and 200 mg/kg hydrocyanic acid to loquat seed powder. The average recoveries of total hydrocyanic acid were 78.4-86.4%, and the coefficients of variation were 0.38-3.65%. In addition, 2 commercial tea bags containing loquat seeds were tested, and the total hydrocyanic acid content were found 119.6 mg/kg and 80.6 mg/kg, respectively. This study provided a process for the test of total hydrocyanic acid in loquat seeds. To make the analytical method more robust, future studies will focus on the pH value modification, multiple enzymes application, and foaming reduction in the reaction to expand the applicability of the testing method.