弧菌屬微生物代謝產物之分析、活性測試及其固定化方法之研發與改進 (I)

Abstract

計畫目標: 本計畫之總目標為擬利用微生物對溫度及酸鹼值之適應範圍較廣, 生命週期較長, 與成本較低之特性, 以發展具特定專一性之感測器, 偵測水產品中特定致病微生物在代謝過程之中間代謝產物, 以作為檢測其衛生品質之標準.分年度工作目標: 在第一年將以弧菌屬微生物之成份中可能之致病因子成份背景進行探討, 致病因子之收集與分析, 並將對受質進行活性測試以確認其代謝產物.在第二年將繼續對所確認之致病因子成份進行活性之測試, 以確定其功能或可能之作用機制.在第三年將針對微生物之固定化方法進行研發.研究重點將探討包括吸附法、共價鍵、膠囊化、陷入法、與交叉結合法等固定化方法對微生物之穩定性、生命週期, 及活性的影響.第四年則將著重於將微生物固定與檢測之相互結合, 以試圖發展具專一性之快速、簡易微生物感測器、以達到對衛生品質的線上監測, 並有效降低檢測成本, 增進飲食衛生與安全.架構( 重要工作項目 ): 本年度之重要工作項目將包括: 1.可能致病因子之成份探討: 將針對弧菌屬細菌之胞表面相關物質及細胞外產物成分, 如蛋白質分解酵素( protease ), 溶血素( hemolysin ), 血凝集素( hemagglutinin ), 細胞毒素( cytotoxin ), 殺白血球毒素( leukocidin ), 和脂多糖( lipopolysaccharide )等成分進行探討, 尋找具特異性之可能致病因子.2.致病因子之收集: 將利用生物化學之方法, 以弧菌屬細菌外膜蛋白質( outer membrane protein )為目標, 收集與溶血素相關之致病因子.3.致病因子之分析: 將利用SDS-PAGE電泳, 對所收集得到之致病因子進行分析, 並估算其可能之分子量.4.致病因子之活性測試: 將利用所純化之外膜蛋白質進行對受質活性之測試, 以確定其功能或可能之作用機制.預期效益: 本計畫預期將發展出一有效檢測水產品中致病弧菌屬之檢測技術以取代傳統費時且緩不濟急之方法.本計畫可預見將產生以下數項預期效益: 1.可實際應用於現有國內生產與國外進口之水產品品質衛生管制制度.2.將可建立一制度化之衛生品質認證制度, 以提高國人消費水產品之衛生安全保障, 確保國人健康, 再配合適當之消費者教育, 必能提高水產食品之消費量; 並可鞏固外銷市場.3.將可加強業者自主品管之能力, 加速產業升級, 提升水產產業之國際競爭力.

Specific Aims of the Proposal: This proposal outlines a four-year program designed to develop a microbial biosensor specific to detect the metabolites of certain pathogens occurred in aquaculture.For the first year, we will focus on the collection, analysis, and identification of the specific pathogens via the activity assay of the intermediates of the metabolic pathway.For the second year, effort will focus on the characterization of the function and/or reaction mechanism of the pathogenic factor.The study and improvement of the immobilization techniques of microbial on the glass or membrane will be emphasized during the third year study.Various methods of immobilization including adsorption, covalent binding, encapsulation, entrapment, and cross-linking will be examined to study the effect on stability, life cycle and activity of microbial.For the fourth year, the combination and immobilization will be tried to generate a stable, easy-to-use microbial sensor in order to on-line monitor the quality of aquaculture.The major direction of the research within the first year will be outlined as following: ( 1 )The background study, collection, analysis, and identification of the possible metabolites such as protease, hemolysin, hemagglutinin, cytotoxin, leukocidin, and lipopolysaccharide; ( 2 )The collection of the pathogenic factor such as outer membrane protein of vibrio spp.; ( 3 )The analysis of pathogenic factor through the application of SDS-PAGE technique; and ( 4 )The activity assay of outer membrane protein on substrate to characterize the possible function or study the reaction mechanism.Expected results and accomplishments: This proposal is designed to replace the conventional, time-consuming diagnostic technique to characterize the contamination of vibrio spp..With the completion of this research program, we will generate a stable, easy-to-use microbial sensor for the on-line monitoring of the aquaculture quality.In parallel, we will improve the standard of the HACCP system as well as ensure the health of the consumers.Finally, we can upgrade the level of the aquaculture industry and promote the capability for international competition.