冬蟲夏草Cordyceps sinensis加工食品分子檢測之研究

Abstract

冬蟲夏草 (Cordyceps sinensis) 又名夏草冬蟲、蟲草、冬蟲草、中華蟲草，寄生在蝙蝠蛾科昆蟲蝙蝠蛾Hepialus armoricanus幼蟲的蟲菌複合體 ，主要分布於四川、雲南、西藏、青海等地的高山草原。近年研究發現，冬蟲夏草具有抗菌、抗腫瘤、免疫調節、治療肺氣腫、去痰作用等功效，因而市面上出現許多冬蟲夏草製成的食品，但其真偽性值得進一步確認。本研究針對蟲草屬核糖體 RNA 基因18S 區域設計專一性引子組cordyF/cordyR，可擴增出長度約為250 bp之蟲草屬專一片段，以 denaturing gradient gel electrophoresis (DGGE) 系統，檢驗十種市售冬蟲夏草食品，結果發現僅一市售產品驗出含有冬蟲夏草，六種產品含有大團囊蟲草 (Cordyceps ophioglossoides)，其餘三種尚無驗出含有任何蟲草屬成份，推測這些產品可能並無添加冬蟲夏草，或添加量很低，而在加工過程中又遭到嚴重破壞，導致無法檢測出冬蟲夏草的 DNA 片段。另又在核糖體RNA基因ITS區域設計對冬蟲夏草專一引子組149csF/149csR，擴增出149 bp之DNA片段，可對含有冬蟲夏草之市售產品進行半定量分析。而模擬冬蟲夏草萃取液經高溫高壓殺菌後，確實會使DNA受到破壞，繼而影響後續之檢驗。

Cordyceps sinensis (Berk.) Sacc., is also known as “Dong Chong Xia Cao” (herb in winter and a worm in summer) in China. This species parasitizes on dead caterpillars of the moth Hepialus armoricanus and forms a caterpillar fungus widely distributed in the alpine regions of south-west China including Sichuan, Yunnan, Tibet, Qinghai. Recently, many reports have mentioned its activities in anti-bacterial, anti-tumor, immuno-modulatory, emphysema therapy and phlegm-removal. Therefore, many processed foods added with Cordycesp sinensis could be found in the markets and its authenticity is remaining confirmation. In this study, a primer pair cordyF/cordyR is designed to specifically amplify Cordyceps spp. about 250 bp DNA fragment in the 18S ribosomal RNA gene region. Ten Cordycesp sinensis processed food products purchased from market were examined by PCR-DGGE method. The results revealed that only one of the ten products has been detected with Cordyceps sinensis ingredient and six products have been identified Cordyceps ophioglossoides. However, another three products failed to show any Cordyceps ingredients. It suggests that these products may contain little or even no Cordyceps sinensis. Besides, extensive food procedure may lead to DNA degradation and result in no DNA pattern of Cordyceps after PCR amplification. Furthermore, another primer pair 149csF/149csR is designed to specifically amplify Cordyceps sinensis about 149 bp DNA fragment in the ITS ribosomal RNA gene region. Semi-quantification analysis of previously commercial product contained Cordyceps sinensis could be carried out by this primer pair. And in self-made tonic analysis, Cordyceps sinensis extraction with sterilization would conduce to DNA degradation and affect the resolution of PCR-based examination.