蜂王浆中蛋白质特性之探讨.doc

蜂王漿中蛋白質特性之探討Characterization of protein in royal jelly研究生：李安玲Lee, An-Ling指導教授:陳景川Jiing-chuan Chern溫惠美Hwei- mei Wen【摘要】本實驗採用中國美健股份有限公司所提供之新鮮蜂王漿，進行一般成分分析、蛋白質特性探討 及由儲存實驗之蛋白質變化，找尋品質劣變指標，作為簡易、快速檢測蜂王鮮度之標準蜂王漿之 一般組成分，水分約占68.4%、粗蛋白質約占14.8%、粗脂肪約3.4%、灰分約1.3%及無氮抽出 物約18」％利用HPLC分析糖類成分，主要由葡萄糖及果糖組成，分別含6.4%及4.9%蜂王 漿含豐富之維生素，包括Bl、B2、B6、葉酸、泛酸及菸鹼酸等，但不含有維生素A、C及E ； 且含豐富之礦物質，包括N a、K、Ca、Mg、P等大量元素及Cu、Fe、Zn、Mn等微量元素蜂 王漿中游離胺基酸及水解胺基酸之含量分別為0.68%及14.63%，而總氮含量為2.5%，水解胺基 酸之氮含量為2.4%，顯示其中大部分氮化物以蛋白質的形態存在於蜂王漿中，且蛋白質分子是以 大分子的形式存在蜂王漿適量溶於0.1M磷酸鋼緩衝液(pH 7.0)，經離心、硫酸銭沉澱和析等步 驟，可將蛋白質區分成水溶性和鹼溶性，其中又以水溶性蛋白質之特性分析。

將初步純化之水溶性 蛋白質溶於0.05M 心磷酸鋪緩衝液(pH 7.0)，經DEAE-Sephacel離子交換管柱分離，所得區分物 質於SDS-PAGE電泳分析，得到四個蛋白質染色帶，其分子量分別為65000、54000、4200()及 34000 ；而經毛絢管電泳分析測定，則分離得到三種蛋白質，其分子量分別為65000、9200()及 110000，顯示不同心分析方去，所得到之蛋白質亦有所差異新鮮蜂王漿分別儲存在室溫及37C 下，每週定時取樣，連續採樣三個月蜂王漿之水溶性蛋白質含量隨儲存時間增長而有減少之趨勢 不，水不溶性蛋白質含量則隨心增加利用SDS-PAGE分析儲存之蜂王漿，觀察染色帶顏色之變 化，結果顯示分別在室溫儲存六週及37C儲存二週後才有顯著變化利用毛細管電泳分析新鮮蜂 王漿，發現在pH2.5心磷酸鋪及pH6.0之Cit/MES緩衝溶液下分離，有很好之分析效果另外， 將上述分離條件，應用於蜂王漿儲存試驗中，發現儲存一週後，各蛋白質之波峰即有明顯之變化； 但在SDS-PAGE中，則需儲存數週或更長之時間方能觀察出變化從分析時間、樣品處理、操作 步驟及分離效果等方面比較，毛細管電泳均較傳統之SDS-PAGE電泳為一更優良，更有效率之分 析方法。

關鍵字：蜂王漿蛋白質毛細管電泳儀[Abstract!The objective of this study was to analyze the nutritional composition and to characterize the protein fraction in royal jelly ・ To assure the quality of royal jelly, an index of freshness was tried to be established in this study.The proximate analysis showed the content of moisture, crude protein, fat, ash and nitrogen-free extract in royal jelly was 68.4, 14.8, 3.4, 1.3 and 18.1 %, respectively. The major components of nitrogen-free extract were mainly glucose(6.4 %) and fructose(4.9 %). Vitamin analysis revealed that fresh royal jelly was rich in vitamin B complex, but it did not have any vitamin A, C and E. Fresh royal jelly also contained abundanl minerals, such as Na, K, Ca, Mg, P, Cu, Fe and Mn.The total nitrogen con tent of fresh royal jelly was 2.5 %, which was mostly derived from protein nitroge n. Analyzed by amino acid analyzer, the con tent of free amino acid and total amino acid in protein hydrolysate was found to be 0.68 and 14.63 %, respectively.To characterize the protein, royal jelly was dissolved in ()」M phosphate buffer(pH 7.0), then proceed centrifugation, ammonia sulfate precipitation and dialysis to separate the protein into water soluble and water insoluble fractions. Water soluble fraction accounts for more than 60 % of the total protein in royal jelly, and was further investigated by DEAE-Sephacel, SDS-PAGE and capillary gel electrophoresis (CGE). By DEAE-Sephacel, two fraction peaks were identified and collected from water soluble protein, whereas, in SDS-PAGE, four protein bands were well separated of which the molecular weight was estimated to be 65000、54000、42000 and 34000, respectively. However, by CGE, only three protein peaks with molecular weight of 110000, 92000 and 65000 were detected, respectively. Apparently、further studies were required to elucidate the different results observed by SDS-PAGE and CGE to estimate the molecular weight of protein in royal jelly.To study the temperature effect on the freshness, royal jelly was stored at 25 and 37C .respectively, and was evaluated by indexes such as, appearance, water solubility and variation of protein patterns analyzed by SDS-PAGE and capillary zone electrophoresis(CZE). Obviously, the water solubility of protein was decreased, and brown color was increased as the time was increased・ Conspicuous changes of SDS-PAGE patter ns of water soluble protein in royal jelly was not observed until it was stored at 25 C for six weeks, whereas, the change of pattern was noted at the second week when it was stored at 37C. Based on the SDS-PAGE patterns, the concentrations of the bands corresponding to molecular weight of 65000 and 540(X) were decreased, while that corresponding to 34000 was decreased, as the storage time was increased.In the presence of phosphate buffer at pH 2.5, water soluble fraction of royal jelly was resolved by capillary 7one electrophoresis(CZE) into four major peaks as denoted by N1, N2, N3 and N4. Peak height ratio of N1/N2 was decreased , while that of N3/N4 was increased as the storage time was in creased ・ The cha nge of both peak height ratios was obviously observed in seve n days, when the royal jelly was stored at 37C- In the presence of Cit/MES buffer at pH 6.0, two distinct peaks (as denoted by Rland R2) were well resolved by CZE・ R2 peak diminished as storage time increased and vanished completely when it was stored at 37Cfor one week. Apparently, the peak height ratio of N1/N2, N3/N4 and R1 /R2 obtained by CZE at pH 2.5 and pH 6.0 respectively, can be used as an index to evaluate the freshness of royal jelly.Keywords: Royal Jelly , Protein 、Capillary Electrophoresis。