Study of methods and factors on phytase activity determination

植酸&;#37238;(phytase)常添加於單胃動物飼糧中作用植酸(phytic acid)，釋出植酸所鍵結之磷(phytic acid-phosphorous)，供動物體吸收利用。然而，飼糧中之預拌劑含兩價或三價陽離子，易與植酸螯合形成不可溶複合物，進而抑制植酸&;#37238;活性。市售植酸&;#37238;產品琳瑯滿目，卻無統一之活性單位，故活性檢測多依循製造廠商提供之檢驗法，易造成不同酵素力價間檢測誤差。本試驗目的為進行影響植酸&;#37238;活性測定之因子探討及建立擴散法測定植酸&;#37238;活性。結果顯示，去離子水添加Tween 20進行酵素萃取所檢測之活性高於以緩衝液萃取。以去離子水配製KH2PO4之檢量線，所測得植酸&;#37238;活性顯著低於以緩衝液配製者。以不同分析方法進行不同產品之活性檢測，Ronozyme&;reg;之產品活性穩定性優於Natuphos&;reg;與Phyzyme&;reg;之產品。除了各種濃度之無機Mn與有機Mn-100 ppm外，所有不同濃度之礦物質均顯著(P &;lt; 0.05)抑制植酸&;#37238;之活性。除高濃度之Cu (500、700 ppm)及Fe (700 ppm)外，無機型態之Fe2+, Zn2+, Cu2+ 和Mn2+ 抑制植酸&;#37238;活性的能力，皆高於有機型態之影響。無機型態之礦物質，抑制植酸&;#37238;活性能力依序為Cu2+> Fe2+> Zn2+> Mn2+。有機型態之礦物質，抑制植酸&;#37238;活性能力依序為Mn2+> Fe2+> Cu2+> Zn2+。商業飼糧配方組成不同，產生之吸光背景值差異甚大，易造成飼糧植酸&;#37238;活性分析值誤差，而以酵素擴散圈法可改善以吸光值測定植酸&;#37238;活性檢測之缺點。綜上所述，檢測條件與飼糧組成均會顯著影響植酸&;#37238;之活性，酵素擴散圈法可檢測不同植酸&;#37238;之活性。Phytase are supplemented in feed to offer hydrolyze phytic acid, releasing phytic acid-phosphorous to meet the P requirement of the monogastric animals. However, premix of feed containing divalent and trivalent cations forms insoluble complexes with phytic acid, causing inhibit phytase activity. There is a variety of commercial phytase products, but hasn't unified activity unit. Therefore, most of measure steps are to follow the ways provided by manufacturers, causing different enzyme activity inaccuracy. The objective of this study is to determine the factors on phytase activity and build radial enzyme diffusion method to measure phytase activity. The results of this study showed that adding Tween 20 to deionized water for extraction conditions had higher phytase activity compared with buffer. KH2PO4 in deionized water for standard curve had significantly lower than buffer. Using different analysis methods to measure varied phytase products activity, we can find out that the stability of Ronozyme® activity was better than Natuphos® and Phyzyme®. Four minerals significantly influenced phytase activity significance (P < 0.05) at different concentration, expect Mn in all inorganic type and organic type at 100 ppm. However, Fe2+, Zn2+, Cu2+ and Mn2+ of inorganic type had higher ability of inhibition compared with organic type, expect Cu (500 ppm,700 ppm) and Fe (700 ppm). In inorganic mineral type, the order of inhibition at phytase was Cu2+> Fe2+> Zn2+> Mn2+. In organic mineral type, the order of inhibition at phytase was Mn2+> Cu2+> Fe2+> Zn2+. The background data of colorimetric methods vary with the ingredients of feed, which causes phytase activity inaccuracy. We can analyze phytase by using radial enzyme diffusion method to improve the disadvantages of colorimetric methods. In conclusion, both conditions of assay and ingredients of food can influence phytase activity significantly while radial enzyme diffusion can analyze different phytase. Key word: phytase, premix, radial enzyme diffusion method目 次 第一章 文獻探討................................................................................................1 壹、植酸...........................................................................................................1 一、定義.................................................................................................1 二、抗營養特性.....................................................................................1 三、植酸在消化道之作用......................................................................1 貳、植酸酶.......................................................................................................9 一、植酸酶命名......................................................................................9 二、植酸酶分類依據..............................................................................9 三、植酸酶應用...................................................................................10 四、植酸酶活性單位...........................................................................11 五、植酸酶檢測方法...........................................................................12 六、影響植酸酶活性之因子...............................................................12 参、飼料添加物-礦物質型態......................................................................18 飼、擴散圈檢測酵素活性...........................................................................18 第二章 不同萃取溶液與分析條件對植酸酶活性之影響.............................19 壹、前言......................................................................................................19 貳、試驗設計..............................................................................................19 参、力價定義..............................................................................................19 肆、材料與方法..........................................................................................20 一、酵素來源.....................................................................................20 二、酵素萃取之溶液與方法.............................................................20 三、測定方法與試劑配製.................................................................20 四、KH2PO4標準溶液配製...............................................................22 五、試藥.............................................................................................22 六、統計分析.....................................................................................22 伍、結果與討論.......................................................................................26 一、萃取溶液對植酸酶活性之影響................................................26 二、分析條件對植酸酶活性之影響................................................26 第三章 礦物質型態與濃度對植酸酶活性之影響........................................28 壹、前言...................................................................................................28 貳、試驗設計...........................................................................................28 一、高濃度金屬離子........................................................................28 二、現場使用之金屬離子濃度........................................................28 三、商業預拌劑產品........................................................................28 参、材料與方法.......................................................................................28 一、材料來源....................................................................................28 二、酵素萃取....................................................................................29 三、試劑與檢量線溶液配製............................................................30 四、檢測方法....................................................................................30 五、試藥............................................................................................30 六、統計分析....................................................................................30 飼、結果與討論.......................................................................................30 一、高濃度金屬離子.........................................................................30 二、現場使用之金屬離子濃度.........................................................36 三、商業預拌劑產品.........................................................................39 第四章 商業飼糧添加植酸酶活性之檢測....................................................41 壹、前言.....................................................................................................41 貳、試驗架構.............................................................................................41 一、比色計法....................................................................................41 二、擴散法........................................................................................41 参、材料與方法.........................................................................................41 一、材料來源....................................................................................41 二、酵素萃取溶液與方法................................................................41 三、試劑與檢量線溶液配製............................................................42 四、瓊脂平皿製備............................................................................42 五、檢測流程...................................................................................42 六、試藥...........................................................................................43 七、統計分析...................................................................................43 伍、結果與討論......................................................................................45 一、比色計法...................................................................................45 二、擴散法.......................................................................................45 第五章 結論..................................................................................................53 第六章 參考文獻..........................................................................................54