臺灣博碩士論文加值系統

此研究目的為鑑定澎湖仙人掌果汁色素之結構，與分析其安定性以應用在加工應用上，並發展毛細管電泳方法以分離仙人掌果汁色素。
在色素構造之鑑定方面：以分析型HPLC測定仙人掌果汁色素，移動相以CH3OH: KH2PO4(50mM，pH2.75)比例為18:82的條件下測定，發現與紅甜菜色素相似，且經過共注射有相同的滯留時間(retention time)；以分析型HPLC測定，使用具有專一性的β-glucosidase 酵素水解仙人掌果汁色素，水解前主要為betanin及isobetanin，水解後，色素主要為betanidin及isobetanidin。而betanin水解後為betanidin，isobetanin水解後為isobetanidin。以質譜儀(FAB- Mass)測定仙人掌果汁色素第一帶及第二帶，再以質譜串聯法(Mass-Mass)做進一步鑑定，在551 Da/e有明顯分子離子峰及389 Da/e有明顯的斷裂峰，第一帶主要結構為含glucose的betanin，第二帶主要結構為含glucose的isobetanin；利用毛細管電泳測定時，仙人掌果色素以偵測長度60cm，全長為67cm長的毛細管分離，電解緩衝液(running buffer)為 10mM Citrate-K2HPO4 buffer (pH6.0)內含6mM DTAB (Dodecyltrimethyl-ammonium bromide)及4mM CaCl2，應用電壓為-20kV，電動注射時間1秒鐘，在室溫(25℃)的條件下，可測得仙人掌果汁色素與紅甜菜色素有相同的滯留時間(migration time)，能進一步鑑定仙人掌果汁色素為具有相同betanin與isobetanin成份。
在安定度分析方面：仙人掌果汁色素為水溶性色素。此色素在pH值2~8時為紫紅色，適用的食品著色酸鹼範圍廣，呈色值較安定，遠較花青素可應用的範圍更為廣泛。仙人掌果汁色素中betanin與isobetanin為兩主要色素，兩者可見光之吸收光譜類似，皆呈紫紅色。仙人掌果汁色素對酸鹼安定性：pH5、pH4＞pH6、pH3＞pH7、pH2，而在強酸及中性下，較不安定。仙人掌果汁色素，在pH3、pH5及pH7下，不同溫度的熱安定度：25℃＞60℃＞80℃＞95℃。此色素對熱敏感，在最適pH時，80℃、95℃下的半衰期分別為30分鐘、10分鐘；對於熱加工食品，須在很短時間內完成加熱殺菌，以保持固定的色澤。空氣對此色素的影響只有些微差異，光線對此色素安定度影響較大。故存放此樣品時，以不透光容器較佳。以β-glucosidase水解，在水解20 min時，isobetanin水解90％，而betanin水解25%，故betanin遠較isobetanin安定。

Two major pigment in Opuntia dillenii fruit juice were found to be beta-cyanin and had identical retention time to betanin and isobetanin from Beta vulgaris in analytic HPLC condition. Both pigments were subject toβ-gluco-sidase hydrolysis and were converted into betanidin and isobetanidin. By using FAB-mass, both pigments showed molecular ion of 551 Da/e and fragment ion of 389 Da/e, identical to betanin and its hydrolyzed product, betanidin respec-tively. Separation of pigments was also achieved by reversed electroosmotic flow capillary electrophoresis in the presence of 4 mM Ca++. Pigments from Opuntia dillenii were found to have identical migration time to those of betanin and isobetanin from Beta vulgaris. Results from experiments above indicated that two major pigment in Opuntia dillenii fruit were betanin and isobetanin.
Opuntia dillenii fruit juice pigments are water-soluble pigment. It is purple red between pH2~8. Two major pigments in Opuntia dillenii fruit juice, betanin and isobetanin, are similar in visible absorption spectrum, and in color of purple red. The stability of juice pigment at different pH value are in following order: pH5、pH4＞pH6、pH3＞pH7、pH2. Its thermal stability order is 25℃＞60℃＞80℃＞95℃. At optimal pH, half life of the pigment at 80℃ and 95℃ is 30 min and 10 min respectively. Therefore heat processing of the juice should be finished in short time to minimize the thermal degration of pigment. Air showed little effect on storage stability. Whereas light exhibited some detrimental to stability. Betanin exhi-bited more resistant than isobetanin toβ-glucosidase hydrolysis. The degree of betanin and isobetanin hydrolysis byβ-glucosidase in 20 min is about 25% and 90% respectively.

目錄
壹、 中文摘要-----------------------------------------------------------------------------1
貳、 英文摘要-----------------------------------------------------------------------------4
參、 前言-----------------------------------------------------------------------------------7
肆、 文獻整理-----------------------------------------------------------------------------9
一、仙人掌果簡介-----------------------------------------------------------------10
二、甜菜(Betalain)簡介--------------------------------------------------------10
1.一般性質--------------------------------------------------------------------10
2.betalains生合成------------------------------------------------------------11
三、betanin之藥效----------------------------------------------------------------15
1. 止痛及抗發炎性質--------------------------------------------------------15
2. betanin抗腫瘤研究--------------------------------------------------------20
四、分離方法--------------------------------------------------------23
五、betanin安定度-----------------------------------------------------------------23
1. 降解機制-----------------------------------------------------23
2. 食品添加物之影響------------------------------------------------------24
3.噴霧乾燥之影響-----------------------------------------------------------24
六、毛細管電泳--------------------------------------------------------------------25
1.演進--------------------------------------------------------------------------25
2.原理--------------------------------------------------------------------------30
3.以毛細管電泳分離betanins----------------------------------------------32
伍、實驗材料與方法--------------------------------------------------------------------34
一、實驗材料--------------------------------------------------------35
二、實驗方法--------------------------------------------------------35
1. 仙人掌果汁色素之萃取及純化----------------------------------------35
2.Betacyanin及其異構物之分離------------------------------------------36
3.Betacyanin的化學結構鑑定---------------------------------------------36
(1) Aglycone之鑑定-----------------------------------------------------36
(2) 分子量測定-----------------------------------------------------------38
(3)毛細管電泳檢測------------------------------------------------------40
4.果汁色澤安定度試驗----------------------------------------40
(1)pH安定度------------------------------------------------40
(2)pH儲藏安定度-------------------------------------------43
(3)溫度儲藏安定度-----------------------------------------43
(4)光線和空氣-----------------------------------------------43
(5)β-glucosidase之水解作用-----------------------------44
陸、結果與討論--------------------------------------------------------------------------45
一、 一般性質之測定-------------------------------------------------------------46
二、 HPLC分離與純化-----------------------------------------------------------46
三、 色素構造之鑑定-------------------------------------------------------------47
1. HPLC測定------------------------------------------------------------------47
2. Aglycone測定--------------------------------------------------------------47
3. 分子量測定-----------------------------------------------------------------53
4. 毛細管電泳測定-----------------------------------------------------------58
(1)分離條件之改善研究-------------------------------------------------58
(2)毛細管電泳鑑定-------------------------------------------------------60
四、在安定度之分析--------------------------------------------------------------64
1. 仙人掌果汁色素光譜分析及呈色測定-------------------64
2. 仙人掌果汁色素各成份之安定性-------------------------66
3. 酸鹼度對仙人掌果汁色素安定性之影響----------------69
4. 溫度對仙人掌果汁色素安定性之影響-------------------71
5. 光線與空氣對仙人掌果汁色素安定性之影響-----------73
6. 對β-glucosidase的水解作用安定度---------------------------------76
柒、結論-----------------------------------------------------------------------------------78
捌、參考文獻-----------------------------------------------------------------------------81
玖、附錄-----------------------------------------------------------------------------------89
圖目錄
圖一、Betanin (1)，isobetanin (2)，betanidin (3)和isobetanidin (4)的化學構造-----------------------------------------------------------------------------------12
圖二、betalains的生合成---------------------------------------------------------------14
圖三、仙人掌果對carrageenan誘導物引起的老鼠背爪浮腫之影響----------19
圖四、以betanin加入飲水中抑制誘導皮膚癌之DMBA-TPA------------------22
圖五、betanin之降解機制----------------------------------------------27
圖六、食品添加劑對仙人掌果實紅色素儲存穩定性之效應--------28
圖七、以區帶毛細管電泳分離betalains------------------------------33
圖八、毛細管電泳檢測裝置圖--------------------------------------------------------41
圖九、濾紙電泳測定(A)花青素peonidin 3-glucose及(B)仙人掌果汁色素---48
圖十、仙人掌果汁色素在不同pH下之紫外可見光譜圖------------------------49
圖十一、betanin及其水解物之HPLC分析圖------------------------50
圖十二、以HPLC鑑定仙人掌果與紅甜菜-----------------------------------------51
圖十三、仙人掌果汁色素對β-glucosidase之水解作用-------------------------52
圖十四、仙人掌果汁色素第一帶之FAB-Mass圖---------------------------------54
圖十五、仙人掌果汁色素第一帶之Mass-Mass圖--------------------------------55
圖十六、仙人掌果汁色素第二帶之FAB-Mass圖---------------------------------56
圖十七、仙人掌果汁色素第二帶之Mass-Mass圖--------------------------------57
圖十八、添加不同Ca++濃度在毛細管電泳分離仙人掌果汁色素之影響-----62
圖十九、仙人掌果汁色素與紅甜菜色素毛細管電泳共注射比較圖-----------63
圖二十、仙人掌果汁色素在不同pH中的呈色-----------------------65
圖二十一、仙人掌果汁色素HPLC分離圖---------------------------67
圖二十二、仙人掌果汁色素可見光吸光光譜圖----------------------68
圖二十三、仙人掌果汁色素溫度之影響安定度圖-------------------------------72
圖二十四、仙人掌果汁色素在pH5時，於95℃加熱不同時間可見光吸收光譜------------------------------------------------74
圖二十五、仙人掌果汁色素在pH5時，於95℃加熱不同時間之呈色變化-----------------------------------------------------75
圖二十六、光線與空氣對仙人掌果汁色素安定性之影響---------------------77
表目錄
表一、一些已知的betacyanin結構------------------------------------13
表二、仙人掌果萃取物使用醋酸誘導物以扭曲測試在老鼠實驗對止痛的影響----------------------------------------------------------------------------------16
表三、仙人掌果萃取物在老鼠以熱平板測試的影響-----------------------------17
表四、仙人掌果抑制以carrageenan誘導物引起的老鼠背爪浮腫-------------18
表五、以甜菜(betanin)抑制老鼠肺腫瘤------------------------------------------21
表六、仙人掌果實紅色素之噴霧乾燥-----------------------------------------------29
表七、仙人掌果汁色素HPLC半製備分離條件-----------------------------------37
表八、仙人掌果及紅甜菜之HPLC分析條件--------------------------------------39
表九、仙人掌果及紅甜菜之毛細管電泳分析條件--------------------------------42
表十、DTAB (Dodecyltrimethylammonium bromide)濃度及電壓對betanin及betanidine毛細管中泳動時間之影響----------------------------------------61
表十一、不同pH值下仙人掌果汁色素儲存之安定性---------------------------70

林欣助、蔡正宗(1997)茄子花色素之鑑定與應用毛細管電泳在分離及鑑定花色素及花色素之研究。東海大學碩士論文。
林秀芳、吳淑珍、葉東柏(1983) 澎湖產仙人掌果肉中天然色素之研究。嘉南學報9:49-58.
黃喜玲、莊允當、劉曜東(1992) 仙人掌果實紅色素之特性與穩定性改善之研究。台灣糖業研究所研究彙報138:37-45.
黃士安，孫璐西(1981) 莧菜紅色素之研究，第一報：應用高效能液相層析法分離及鑑定莧菜紅色素。食品科學8(1):13-25.
葉碧櫻、蔡正宗(1993) 甘藷花色素之鑑定和三種天然花色素萃取物安定性之比較研究。東海大學碩士論文。
鄭元春(1997) 神奇的多用途植物圖鍵。綠生活雜誌股份有限公司。台北市。
鄭元春(1998) 台灣的海濱植物。渡假出版社。台北市。
蔡正宗(1999) 仙人掌果紅色色素之研究。國科會報告。
Adams,J.P. and von Elbe,J.H.(1977) Betanine separation and quantification by chromatography on gels. Journal of Food Science. 42(2):410-414.
Bosh-Millares,J.(1967) Siglo XVIII. la ciudad de las palmas en este siglo: otros remedios caseros en el siglo XVIII. In: Historia de la medicina en gran canaria. Excmo cabildo insular de gran canaria. Las Palmas. 1:270-271.
Ferreira,S.H.(1972) Prostaglandins, aspirin-like drugs and analgesia. Nature New Biology. 240(102):200-203.
Ferreira,S.H., Nakamura,M.(1979) I-Prostaglandin hiperalgesia, a cAMP/Ca2+ dependent process. Prostaglandins. 18(2):179-190.
Ferreira,S.H., Moncada,S., and Vane,J.R.(1973) Further experiments to establish that the analgesic action of aspirinlike drugs depends on the inhibition of prostaglandin biosynthesis. British Journal of Pharmacology. 47:48-58.
Francis,M.(1985) "Pigments and Other Colorants" in Food chemistry ed. by Fennema,O.R.
Garcialeme,J., Nakamura, L., Leite,M.P., and Rocha e silva,M.(1973) Pharma-cological analysis of the acute inflammatory process induced in rat''s paw by local injection of carrageenan and by heating. British Journal of Pharma-cology. 48(1):88-96.
Jackman,R.L. and Smith,J.L.(1992) Anthocyanins and betalains. In: Natural Food Colorants. Ch.6. Hendry,G.A.F. and Houghton,J.D. ed. Blackie Academic & Professional. London, New York.
Jackman,R.L. and Smith,J.L.(1996) Anthocyanins and betalains. In: Natural Food Colorants. Ch.8. Hendry, G.A.F. and Houghton,J.D. 2nd ed. Blackie Academic & Pro-fessional. London, New York.
Jorgen son, J.W. and Lukacs, K.D.(1981) High resolution separations based on electrophoresis and electroosmosis. Journal of Chromatography. 281: 209-211.
Kapadia,G.J., Tokuda,H., Konoshima,T. and Nishino,H.(1996) Chemopreven-tion of lung and skin cancer by Beta vulgaris (beet) root extract. Cancer Letters. 100:211-214.
Kapadia,G.J., Balasubramanian,V., Tokuda,H., Iwashima,A. and Nishino,H. (1997) Inhibition of 12-O-tetradecanoylphorbol-13-acetate induced Epstein-Barr virus early antigen activation by natural colorants. Cancer Letters. 115:173-178.
Koster,R., Anderson,M., and De beer,E.J.(1959) Acetic acid for analgesic screening. Federation Proceedings. 18:412-416.
Kuhn,R. and Hoffstetter-Kuhn,S.(1993) Capillary electrophoresis: principle and practice. Spring-Verlag, New York.
Li,S.F.Y.(1992) Capillary electrophoresis: principle and application. Elsevier, Amsterdam.
Loro,J.F.(1994) Estudios farmacologicos y toxicologicos de un extracto acuoso de frutos de Opuntia dillenii Haw. Masters Thesis. Faculty of medicine, universidad de Las palmas, Las Palmas de Gran Canaria.
Loro J.F. and Delrio,I.(1999) Preliminary studies of analgesic and antiinflam-matory properties of Opuntia dillenii aqueous extract. Journal of Ethno-pharmacology. 67:213-218.
McIlvaine,T.C.(1921) Preparation of buffers for use in enzyme studies. Journal of Biology and Chemistry. 49:183-184.
Mueller,L.A., Hinz,U. and Zryd,J.P.(1996) Charcterization of a tyrosinase from Amanita muscaria involved in betalain biosynthesis. Phytochemistry. 42(6): 1511-1515.
Perez de paz, P.L., and Medina,M.(1988) Catalogo de plantas medicinales de la flora canaria (aplicaciones populares). Viceconsejeria de cultura y deportes del gobierno de canarias e instituto de estudios canarios, La Laguna, Tene-rife.
Piatteli,M. and Minale,L.(1964) Pigments of centrospermae-I. betacyanin form Phyllocactus hybridus hort and Opuntia ficus-Indica Mill. Phytochemistry. 3:307-311.
Powie,W.D. and Fennema,O.(1963) Electrophoretic separation of beet pigments. Journal of Food Science. 28:214-220.
Reynoso,R.C. Giner,T.V. and Gonzalez de mejia.(1999) Safety of a filtrate of fermented garambullo fruit: biotransfermation and toxicity studies. Food and Chemical Toxicology. 37:825-830.
Schwartz, S.J. and von Elbe, J.H.(1980) Quantitative determination of individual betacyanin pigment by high performance liquid chromatography. Journal of Agriculture and Food Chemistry. 28:540-543.
Stuppner H. and Egger R.(1996) Application of capillary zone electrophoresis to the analysis of betalains from Beta vulgaris. Journal of Chromatography A. 735: 409-413.
Tsuda,T., Mizuno,T. and Ewing,A.G.(1987) Capillary zone electrophoresis with electrochemical detection. Analytical Chemistry. 59:678-681.
Vincent,K.R. and Scholz, R.G.(1978) Separation and quantification of red beet betacyanins and betacyanins by high performance liquid chrometography. Journal of Agriculture and Food Chemistry. 26(4):812-815.
Von Elbe,J.H., Maing,I.Y. and Amundson,C.H.(1974) Color stability of betanin. Journal of Food Science. 39:334-337.
Von Elbe,J.H., Maing,I.Y. and Gabelman,W.H.(1972) Quantitative analysis of betacyanins in red table beets(beta vulgaris). Journal of Food Science. 37:932-934.
Winter,C.A., Risley,E.A., Nuss,G.W.(1962) Carrageenan-induced edema in hind paw of the rat as an assay for antiinflammatory drugs. Proceedings of the Society of Experimental Biology and Medicine. 111:544-547.
Woolfe,G., MacDonald,A.D.(1944) The evaluation of the analgesic action of pethidine hydrochloride (demerol). Journal of Pharmacology and Experi-mental Therapeutics. 43:300-309.
Ying-mei,L. and Shuenn-jyi,S.(1994) Separation of aromatic acids by reversed electroosmotic flow capillary electrophotrsis. Journal of Chromatography A. 663: 239-243.