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研究生:陳宜孜
研究生(外文):Yi-Tzu Chen
論文名稱:溫度與pH對不同品種山藥抗氧化特性及其儲存性蛋白質dioscorin安定性之影響
論文名稱(外文):Effect of Heating Temperature and pH on the Antioxidative Properties and the Stability of Dioscorin of Various Yam Cultivars
指導教授:林國維林國維引用關係
指導教授(外文):Kou-Wei Lin
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2005/04/
畢業學年度:93
語文別:中文
論文頁數:164
中文關鍵詞:抗氧化能力儲存性蛋白質山藥膠體過濾層析螯合亞鐵離子DPPH自由基清除能力表面疏水性
外文關鍵詞:DPPH free radical scavenging effectFerrous ion chelating capacityGel filtration chromatographySurface hydrophobicityAntioxidative activityStorage proteinDioscorinYam
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本研究比較不同加熱溫度(50~100˚C)及pH (pH 4~10)對名間長紅(MC)、台農2號(TN2)及基隆山藥(KL)抗氧化能力及其儲存性蛋白質特性之影響。結果顯示未經加熱的山藥處理之總酚類化合物含量、DPPH自由基清除能力及螯合亞鐵離子能力皆較加熱處理試驗組高,而隨著加熱溫度上升其抗氧化能力有隨之下降的趨勢。經萃取純化後發現,三種山藥之膠體過濾層析皆具有三個較明顯波峰,且在20~40管間具有一明顯蛋白質帶,經由SDS-PAGE發現其分子量為31 kDa。而隨著加熱溫度升高,其膠體過濾層析圖之波峰逐漸降低,且在80˚C以上因加熱破壞其儲存性蛋白質,所以無法萃出任何蛋白質。儲存性蛋白質因加熱而結構改變,其表面疏水性隨著加熱溫度上升而有明顯的下降。
山藥皆在pH 5酸性環境下有最高的總酚類化合物含量,名間長紅與台農2號且在pH 5以上即隨著pH上升而逐漸下降,而MC與TN2在pH 8時,總酚類化合物含量又有上升的趨勢,KL則無此現象。三種山藥亦在酸性環境下有較好的DPPH自由基清除能力,且分別在不同的pH值下,其DPPH自由基清除能力之趨勢與總酚類化合物含量的變化相似。三種山藥之螯合亞鐵離子能力在pH 8時為最好。MC、TN2及KL於 pH 4緩衝溶液萃取純化下,並無發現dioscorin蛋白質存在。在pH 5~10,膠體過濾層析結果在約第25 ~ 40管數間僅有一明顯之蛋白質,其分子量約為31 kDa。三種山藥儲存性蛋白質dioscorin之相對表面疏水性RS0受pH之影響,MC及TN2皆在pH 6時之RS0最高,但當pH > 8時三種山藥之RS0皆隨著pH上升而逐漸下降。
The purposes of this study were to investigate the effects of different heating temperatures and pH on the antioxidative activity and the stability of dioscorin of various yam cultivars ( Mingchien, Tainung No.2 and Keelung ). Results from crude yam extracts of all varieties showed decreases in total phenolic and dioscorin contents, DPPH radical scavenging effect, and ferrous ion chelating capacity with increasing heating temperatures. Following separation and purification, only one protein band was collected and electrophoresized from three major peaks ranging from 20-40 fraction numbers of all yam varieties, and had a molecular weight of 31 kDa. The fraction numbers of peaks were reduced and the peak area narrowed with increasing heating temperatures and no protein was extracted at temperature above 80˚C. The structure of storage protein dioscorin was affected due to severe heating, and the protein surface hydrophobicity declined with increasing heating temperatures.
The contents of total phenolic compounds from all yam varieties were the highest at pH 5, but gradually decreased with elevated pH. The DPPH scavenging effects of all yam varieties at acidic pH environment were superior to other pH, and paralleled to the trend of total phenolic compounds. The ferrous ion chelating capacity was found to be the highest for all yams at pH 8. No dioscorin protein could be extracted at pH 4. At pH 5-10, a single dioscorin protein band with molecular weight of 31 kDa was observed and appeared between the fraction numbers of 25-40. The relative protein surface hydrophobicity (RS0) of all yams was influenced by pH, and MC and TN2 showed the highest RS0 at pH 6. At pH > 8, the RS0 tended to decline with increasing pH.
目 錄
頁次
中文摘要.................................................. Ⅰ
英文摘要.................................................. Ⅱ
謝誌...................................................... Ⅳ
目錄...................................................... Ⅴ
表目錄.................................................... Ⅸ
圖目錄.................................................... Ⅹ
第一章 前言.............................................. 1
第二章 文獻回顧.......................................... 3
壹、山藥介紹........................................... 3
一、簡介........................................... 3
二、山藥品種....................................... 3
三、山藥成份....................................... 4
(一)、營養成分.................................. 4
(二)、保健功能.................................. 5
四、加工上之應用................................... 8
貳、植物儲存性蛋白質................................... 9
參、自由基與抗氧化物質................................. 15
一、自由基之介紹................................... 15
二、自由基種類..................................... 15
三、自由基與活性氧所造成的傷害..................... 15
肆、生物體內抗氧化防禦..................................... 17
一、酵素性抗氧化系統............................... 17
(一)、超氧歧化...................................... 17
(二)、觸............................................ 18
(三)、麩胱過氧化................................ 18
二、非酵素性抗氧化系統............................. 18
(一)、麩胱.......................................... 18
(二)、維生素C.......................................... 21
(三)、維生素E.......................................... 21
伍、抗氧化劑........................................... 21
一、天然抗氧化物................................... 22
(一)、類黃酮............................................ 24
(二)、花青素............................................ 24
三、人工抗氧化劑................................... 29
陸、植物抗氧化特性..................................... 29
一、山藥之抗氧化特性............................... 29
二、其他植物之抗氧化特性........................... 32
柒、加工處理對於抗氧化性質之影響....................... 33
一、熱處理對抗氧化性質之影響....................... 34
二、pH對抗氧化性質之影響.......................... 36
捌、蛋白質表面疏水性................................... 38
第三章 研究目的................................................ 41
第四章 材料與方法.............................................. 42
壹、實驗材料........................................... 42
貳、實驗方法........................................... 42
一、基本成分分析................................... 42
(一)、水分...................................... 42
(二)、粗脂肪.................................... 42
(三)、粗蛋白.................................... 42
(四)、灰份...................................... 44
(五)、總澱粉含量................................ 44
二、粗山藥萃取液製備............................... 45
(一)、不同溫度處理.............................. 45
(二)、不同pH處理............................... 47
三、抗氧化特性..................................... 48
(一)、總酚類化合物.............................. 48
(二)、DPPH自由基清除能力之測定................. 48
(三)、亞鐵離子螯合能力之測定.................... 48
四、蛋白質定量..................................... 49
(一)、蛋白質濃度校正曲線........................ 49
(二)、蛋白質濃度測定............................ 49
五、山藥儲存性蛋白之萃取與純化..................... 50
(一)、硫酸銨劃分................................ 50
(二)、DEAE Sephadex A-25批式法離子交換層析..... 50
(三)、SephadexTM G-75膠體過濾層析............... 50
(四)、蛋白質濃縮................................ 51
(五)、SDS-聚丙烯醯胺膠體電泳.................... 51
1、試藥配置................................. 51
2、鑄膠操作................................. 52
3、樣品處理................................ 53
六、蛋白質表面疏水性............................... 54
(一)、ANSA溶液之配製.............................. 54
(二)、蛋白質樣品................................ 54
(三)、測定蛋白質表面疏水性...................... 54
(四)、數據分析.................................. 54
七、等電點pI值之測定............................... 55
(一)、樣品處理.................................. 55
(二)、實驗步驟.................................. 55
參、統計分析........................................... 56
第五章 結果與討論.................................................. 57
壹、不同品種山藥之基本成分分析.......... .............. 57
貳、不同溫度加熱處理後其理化特性之影響................. 57
一、加熱對山藥抗氧化性質之影響..................... 57
(一)、總酚類化合物含量.......................... 57
(二)、DPPH自由基清除能力之測定................ 61
(三)、螯合亞鐵離子能力.......................... 61
二、加熱對山藥儲存性蛋白之影響..................... 63
三、加熱對山藥儲存性蛋白質表面疏水性之影響......... 82
參、山藥儲存性蛋白質等電點之測定....................... 90
肆、不同pH處理對山藥之理化特性影響................... 93
一、pH對山藥抗氧化性質之影響...................... 93
(一)、總酚類化合物含量.......................... 93
(二)、DPPH自由基清除能力之測定................ 95
(三)、螯合亞鐵離子能力.......................... 97
二、pH對山藥儲存性蛋白之影響...................... 99
三、pH對山藥儲存性蛋白質表面疏水性之影響.......... 114
伍、各測定值間之相關性................................. 128
第六章 結論............................................... 131
第七章 參考文獻........................................... 133
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