臺灣博碩士論文加值系統

火龍果是近幾年風行我國的熱帶水果，屬於仙人掌科三角柱屬(Hylocereus sp.)植物，原產於中美洲。紅肉的火龍果含有高濃度色素，此色素經初步分析含大量的betalains。色素萃取纯化後，以高效液相層析法(HPLC)和電灑法質譜(ESI-mass)分析鑑定，並評估果汁色素在不同溫度、pH值和存在不同二價離子和醇類中的安定性。
火龍果果汁色素經由半製備的HPLC-ODS管柱分離纯化，並和甜菜苷的標準品(Beta vulgaris)比較，區分帶一和帶二初步確定為betanin和isobetanin。利用專一性的酵素(β-glucosidase)水解果汁色素，水解物經HPLC測定，火龍果果汁色素色素的區分帶一為betanin，並進一步由串聯質譜法(mass-mass)測定其分子量及斷裂圖譜加以證實。
果汁色素HPLC分析，主要可區分成為六個區分帶，而其中的四個區分帶由質譜分析，區分帶一、二，分析確定為betanin和isobetanin，區分帶三、五，亦經由質譜儀測定分子量及斷裂圖譜而推論為phyllocactin (6''-O-malonylbetanin) 和isophyllocactin。
Betacyanin為水溶性，pH對其呈色的影響很小，適用於pH 2~9的食品中。加熱時對pH的安定性為pH 5＞pH 3和pH 7；添加BaCl2和存在於低溫時，具有延緩色澤降解的作用；果汁色素對於光和加熱並不安定，且色素存在於含有醇類的溶液時，並不安定因此不適合於酒精性食品。火龍果果汁色素較適合應用於低溫食品的製造，如奶製品及冷藏飲品。

Fruit of a new hybrides of Hylocereus polyrhizus, which grows in the deserts of Middle America, contains a red pigment, betalanin, on both outside peel and inside fruit pulp. The pigments were extracted, and compounds of interest were isolated and characterized by HPLC and ESI-Mass techniques. The stability of pigment extracts were evaluated at different temperatures and pH values and in the presence of different divalent ion and alcohols.
Betacyanins had been ftractionated by semipreparative HPLC-
ODS. By comparing to standard betanin (Beta vulgaris), fraction 1 and 2 were identified initially to be betanin and isobetanin respectivly. The conclusion were further confirmed by HPLC analysis of β-glycosidase of betanin and fraction 1.
Betacyanins has been fractionated by HPLC into six fractions. Among them, four major betacyanins and their fragments has been identified by positive ion electrospray mass spectrometry. Fraction 1 and 2 were unequivocally assigned to be betanin, isobetanin, and fraction 3 and 5 could be assigned to be phyllocactin (6''-O-malonylbetanin) and isophyllocactin respectively.
The betacyanin is water-soluble and quite consistant in deep purple-red color in pH range of 2-9. The order of thermalstability to acidity was : pH 5＞pH 3, pH 7. The pigment stability was enhansed in the presence of BaCl2 and are quite stable at low temperatures. The pigment was light sensitive and unstable in the presence of alcohol.
Hylocereus polyrhizus pigment has the potential for use in food processing at low temperatures such as in the dairy industry for the production of ice cream and cold drinks.

目錄
壹、 中文摘要..........................................................................................................1
貳、 英文摘要..........................................................................................................2
參、 研究動機..........................................................................................................3
肆、 文獻整理..........................................................................................................4
一、火龍果.......................................................................................................4
二、甜菜苷(betalain)簡介................................................................................7
三、Betalain生合成........................................................................................11
四、Betalain之安全性...................................................................................14
五、Betalain之抑制腫瘤研究.......................................................................15
六、Betalain安定性.......................................................................................16
七、分離及鑑定.............................................................................................21
八、毛細管電泳.............................................................................................24
伍、實驗材料與方法.............................................................................................30
一、實驗材料.........................................................................30
二、實驗方法.........................................................................31
1.紅龍果(Hylocereus polyrhizus)可溶性固形物測定及果肉中色素之
萃取、分離............................................................................................31
2.圓仔花(Gomphrena globosa) 花序、九重葛(Bougainvillea glabra)
花序莧菜(Amaranthus tricolor) 葉子，色素液的萃取及純化.............31
3.紅龍果(Hylocereus polyrhizus)之betacyanin及其異構物之分離.......32
4.分子量測定.............................................................................................33
三、利用HPLC分析betacyanine..................................................................33
四、火龍果果汁的安定性.............................................................................35
1.火龍果果汁色素在不同pH值下的呈色安定性........................35
2.添加不同金屬離子的安定性...............................................35
3.熱安定性.........................................................................36
4.添加醇類對色素安定性的影響..........................................................36
五、甜菜苷在不同二價離子存在下的毛細管電泳分析........................36
1.毛細管柱.........................................................................38
2.實驗方法.........................................................................38
3.實驗操作條件.......................................................................................40
陸、結果與討論......................................................................................................41
一、HPLC分析...............................................................................................41
二、光譜分析..................................................................................................44
三、火龍果betacyanin的化學結構鑑定.........................................................46
1. Aglycone測定.......................................................................................46
2. 質譜分析...............................................................................................48
四、安定性分析.............................................................................................53
1.火龍果果汁色素在不同pH值下的呈色安定性................................53
2.添加不同金屬離子的安定性...............................................................54
3.熱安定性...............................................................................................57
4.添加醇類對色素安定性的影響............................................................65
五、甜菜苷在不同二價離子的毛細管電泳分析...........................................68
柒、結論..................................................................................................................73
捌、參考文獻..........................................................................................................74
玖、附錄..................................................................................................................83
圖目錄
圖一、紅皮紅肉和紅皮白肉火龍果之外觀..........................................6
圖二、(1)Betanin,(2)isobetanin,(3)betanidin和(4)isobetanidin的
化學構造......................................................................................9
圖三、Dopa經由tyrosine以形成betanidin途徑....................................12
圖四、Betalain的生合成......................................................................13
圖五、Betanin之降解機制...................................................................17
圖六、涉及betanin酵素性降解的反應第一步驟................................19
圖七、Cyclo dopa 6-O-β-D-glucoside radical的共振結構(上)，可
能的cyclo dopa 5-O-β-D-glucoside dimer的結構.....................20
圖八、Betanin 酵素性降解的可能機制..............................................20
圖九、Betanin在鹼性條件下的平衡...........................................22
圖十、Betanin在鹼性條件下的降解...........................................22
圖十一、利用甲基化反應決定甜菜苷醣鍵結位置反應式................24
圖十二、毛細管壁和電解質溶液形成的電雙層及則塔電位簡圖....27
圖十三、電滲流的產生(a)石英表面負離子 (b)水合陽離子在表面
積聚(c)電場作用下指向負極的體相流動.............................29
圖十四、毛細管電泳裝置圖...............................................................38
圖十五、熔矽(fused silica)毛細管之結構..........................................39
圖十六、控制燒去毛細管表面柱的polyimide長度之簡圖............39
圖十七、解析度的計算.......................................................................40
圖十八、利用HPLC(isocratic elution)分析火龍果中的betacyanins
之層析圖................................................................................41
圖十九、利用HPLC(gradient elution)分析火龍果中的betacyanins
之層析圖...............................................................................42
圖二十、火龍果及其區分帶一和區分帶三之紫外-可見光譜圖......43
圖二十一、火龍果、甜菜及圓仔花萃取液之紫外-可見光譜圖......45
圖二十二、甜菜和火龍果色素區分帶一經 β-glucosidase水解前後
的HPLC分析比較圖..........................................................47
圖二十三、火龍果汁色素第一帶之mass及mass-mass圖..............49
圖二十四、火龍果汁色素第二帶之mass-mass圖............................50
圖二十五、火龍果汁色素第三帶之mass及mass-mass圖..............51
圖二十六、火龍果汁色素第五帶之mass及mass-mass圖..............52
圖二十七、在不同pH值時火龍果果汁色素其紫外光-可見光光
譜圖....................................................................................55
圖二十八、在不同pH值時火龍果果汁色素呈色之影響.................56
圖二十九、添加不同金屬離子於火龍果果汁色素中紫外光-可見
光光譜圖............................................................................58
圖三十、添加不同金屬離子對火龍果果汁色素安定性之影響........59
圖三十一、在不同pH值和時間下加熱，溫度對火龍果果汁色素
，其紫外光-可見光光譜之變化....................................60
圖三十二、在不同pH值和時間下加熱，溫度對火龍果果汁色素
安定性之影響....................................................................61
圖三十三、Betanin在酸性或在加熱條件下降解機制......................65
圖三十四、添加不同醇類對火龍果果汁色素安定性之影響............66
圖三十五、添加不同濃度BaCl2在毛細管電泳液中對分離甜菜苷
的影響................................................................................70
圖三十六、添加不同濃度CaCl2在毛細管電泳液中對分離甜菜苷
的影響................................................................................71
圖三十七、添加不同濃度MgCl2在毛細管電泳液中對分離甜菜苷
的影響................................................................................72
表目錄
表一、仙蜜果之不同稱謂...............................................................5
表二、歐洲允許使用的天然色素..........................................................8
表三、一些已知的betacyanins結構....................................................10
表四、分離火龍果(H. polyrhizus)、甜菜(B. vulgaris) betacyanin
色素所使用移動相梯度............................................................34
表五、分離圓仔花(G. globosa) 花序、九重葛(B. glabra)花序、
莧菜(A. tricolor) 葉子中的betacyanin色素所使用移動相
梯度...........................................................................................34
表六、火龍果各區分帶的滯留時間及相對濃度...............................44
表七、火龍果各區分帶的滯留時間及利用電灑法質譜所分析的
各個分子峰及其斷裂峰...........................................................53
表八、添加不同金屬離子對火龍果果汁色素呈色之影響...............59
表九、在pH 3.10不同時間下加熱，溫度對火龍果果汁色素呈色
之影響........................................................................................62
表十、在pH 5.02不同時間下加熱，溫度對火龍果果汁色素呈色
之影響........................................................................................63
表十一、在pH 7.05不同時間下加熱，溫度對火龍果果汁色素呈
色之影響.....................................................................................64
表十二、添加不同醇類對火龍果果汁色素安定性之影響.................67
表十三、甜菜苷在不同二價離子的毛細管電泳分析其解析度.........69
附錄
附圖一、利用HPLC(gradient elution) 甜菜色素水解產物中的
betacyanins..............................................................................84
附圖二、利用HPLC(gradient elution) 圓仔花中的betacyanins..........85
附圖三、Cai 等人(2001)利用HPLC分析G. globosa inflorescences
之層析圖..................................................................................86
附圖四、添加不同金屬離子對火龍果果汁色素呈色之影響..............87
附圖五、在不同pH值和時間下加熱，溫度對火龍果果汁色素
呈色之.....................................................................................88
附圖六、添加不同醇類對火龍果果汁色素呈色之影響.....................89
附表一、甜菜色素水解產物各區分帶的滯留時間及相對濃度.......90
附表二、圓仔花色素各區分帶的滯留時間及相對濃度.....................90
附表三、Citrate-phosphate buffer配製法.............................................91
附表四、Hydrochloric-potassium chloride buffer配製法....................92
附表五、Glycine-sodium hydroxide buffer配製法..............................92

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