臺灣博碩士論文加值系統

中文摘要
學號：M8936017
論文名稱：果膠酵素與硬心香蕉形成之探討 總頁數：109
學校名稱：國立屏東科技大學食品科學系碩士班
畢業時間及摘要別： 九十學年度第二學期碩士學位論文摘要
研究生：陳震學 指導教授：廖遠東 許祥純 博士
論文摘要內容：
香蕉後熟為一動態的過程，其中包括許多組成分與生化作用的改變，本研究主要在探討果膠酵素與硬心香蕉形成之關係，並藉由化學成分分析及特定酵素純化與活性測定進行試驗，進而分析果膠酵素是否為硬心香蕉形成關鍵因子。實驗結果綜合歸納如下：
1.化學性質分析
(1) 成份分析，硬心香蕉(45.81%)與正常香蕉(45.28%)兩者之澱粉含量並無顯著性差異；而鉀離子處理組(40.57%)與未用鉀離子處理組(45.31%)兩組間則有顯著性差異。硬心香蕉與正常香蕉(3.62%)總果膠含量有顯著性差異(P<0.05)，其含量分別為：輕度硬心4.31%、中度硬心4.45%及重度硬心4.89%，且各組之間的差異隨著硬心程度的增加，有上升的趨勢，但硬心組間並無顯著性差異。且鉀離子的添加對總果膠含量無顯著影響。
(2) 酯化度分析，正常黃蕉果膠酯化度65.29%，輕度硬心65.83%，中度硬心66.38%及重度硬心67.14%，發現隨著硬心程度的增加果膠酯化度有逐漸增加的趨勢，硬心組別果膠酯化度比正常黃蕉高且有顯著性差異(p<0.05)。
2.酵素純化與活性測定
(1) 果膠酯酶活性，正常香蕉與硬心香蕉並無顯著性差異，隨著硬心程度的增加有稍上升的趨勢，但仍未有顯著性差異。鉀離子處理與否對與香蕉果膠酯酶亦無顯著影響。另於聚半乳糖醛酸酶測定方面，正常香蕉與硬心香蕉亦無差異，且鉀離子的添加對於聚半乳糖醛酸酶的酵素活性並無影響。
(2) 香蕉之果膠酯酶電泳分析發現，有一30 kDa之polypeptide，隨著硬心程度的增加呈現降解的趨勢。而聚半乳糖醛酸酶電泳分析，並未發現任何蛋白質區帶具降解現象。藉陽離子交換樹脂(CM-Sepharose)及膠體過濾層析(Sephadex G-75)與採用原態電泳輔以活性染色法，確認此一酵素為果膠酯酶且其分子量為30 kDa，pI值為9.3。
關鍵字：硬心香蕉、果膠酯酶、聚半乳糖醛酸酶

Abstract
The ripening of banana is a dynamic processing which involves many changes with component and biochemical reaction. The objective of the present study is to illustrate the relationship between pectic enzymes and occurrence of rubbery banana during ripening. The study was conducted by investigating chemical properties, specific enzyme purification and activity measurement of rubbery and normal banana fruits. Furthermore, whether the pectic enzymes play a crucial role in related to rubbery banana formation were identified. The results were summeried as follows:
1. Biochemical properties
(1) The composition analysis showed that the starch content were not significantly different between rubbery and normal banana, and potassium added group (40.57%) is significantly lower than non potassium added group (45.31%).
(2) The total pectin content was significantly different between normal and rubbery banana. There were: light rubbery (LR) 4.31%, medium rubbery (MR) 4.45%, heavy rubbery (HR) 4.89% and normal banana (N) 3.62%. The total pectin content is independent on potassium addition. The DE (degree of esterification) of banana are N: 65.29 %, LR: 65.83 %, MR: 66.38 % and HR: 67.14 % respectively. It revealed that the DE value increase with degree of rubbery. DE of rubbery banana is significantly higher than normal yellow banana (p<0.05).
2. Enzyme purification and activity measurement
(1)Pectin enzymes: Both activity of pectinesterase (3.78 ± 0.22 units) and polygalacturonase (26.67 ± 2.34) of rubbery banana were not significantly different with those of normal banana fruits.
(2) A 30 kDa polypeptide disappeared gradually with the extent of rubbiness of rubbery banana by examining protein pattern of SDS-PAGE. For polygalacturonase, no significant degradation can be observed by electrophoresis between normal and rubbery banana fruits. A consecutive purification procedure, CM-sepharose, Sephadex G-75, acidic electrophoresis and activity stain, were employed for protein identification. The purified and identified enzyme with the molecular weight of 30 kDa and pI 9.3 was identified as pectinesterase.
Key words: rubbery banana, pectinesterase, polygalacturonase

目錄
頁次
中文摘要---------------------------------------------------------------- I
英文摘要---------------------------------------------------------------- II
誌謝---------------------------------------------------------------------- V
目錄--------------------------------------------------------------------- VI
圖索引----------------------------------------------------------------- XII
表索引---------------------------------------------------------------- XIV
第一章 前言--------------------------------------------------------- 1
第二章 文獻整理--------------------------------------------------- 4
2.1 香蕉果實生長及後熟-------------------------------------------- 5
2.1.1 香蕉果實生長 ----------------------------------------------- 5
2.1.2 香蕉果實後熟因子------------------------------------------ 6
2.2 硬心香蕉----------------------------------------------------------- 7
2.2.1 硬心香蕉之發生--------------------------------------------- 7
2.2.2. 硬心香蕉發生之調查-------------------------------------- 8
2.2.3 硬心發生原因之探討--------------------------------------- 9
2.3 蛋白質生合成---------------------------------------------------- 10
2.4 細胞壁崩解與質地軟化關係---------------------------------- 11
2.5 果膠之化學組成及分子結構---------------------------------- 16
2.5.1 化學組成----------------------------------------------------- 16
2.5.2 酯化度-------------------------------------------------------- 17
2.5.3 分子結構----------------------------------------------------- 17
2.5.4 香蕉後熟之調控機制-------------------------------------- 18
2.5.5 果膠酯酶(pectinesterase, PE)與聚半乳糖醛酸酶 (polygalacturonase, PG)------------------------------------- 18
2.6 酵素萃取之最佳條件------------------------------------------- 20
2.7 果膠酯酶---------------------------------------------------------- 21
2.7.1 果膠酯酶及其作用方式----------------------------------- 21
2.7.2 果膠酯酶作用方式及基質專一性--------------------- 21
2.7.2.1 作用方式------------------------------------------------ 21
2.7.2.2 基質專一性--------------------------------------------- 23
2.7.2.3 果膠酯酶的理化性質--------------------------------- 23
2.7.3 果膠酯酶活性分析及影響果膠酯酶活性之因素----- 26
2.7.3.1 果膠酯酶活性的測定方法--------------------------- 26
2.7.3.2 影響果膠酯酶活性之因素--------------------------- 26
2.7.4 果膠酯酶之多分子型態----------------------------------- 30
2.7.5 果膠酯酶在蔬果發育過程中所扮演的角色及生理意義---------------------------------------------------------------- 33
2.7.5.1 果膠酯酶在蔬果發育過程中所扮演的角色----- 33
2.7.5.2 果膠酯酶同功酶之生理意義------------------------ 35
2.7.5.3 果膠酯酶之活性染色--------------------------------- 36
第三章 材料與方法---------------------------------------------- 38
3.1 實驗架構--------------------------------------------------------- 38
3.2 樣品來源---------------------------------------------------------- 38
3.3 採樣方法---------------------------------------------------------- 40
3.4 實驗組別---------------------------------------------------------- 40
3.5 儀器設備---------------------------------------------------------- 40
3.5.1 組成份、電泳及電泳分析設備-------------------------- 40
3.6實驗藥品----------------------------------------------------------- 41
3.6.1 組成份分析試藥-------------------------------------------- 41
3.6.2 酵素分析藥品----------------------------------------------- 42
3.6.3電泳試藥------------------------------------------------------ 42
3.6.4 酵素純化材料----------------------------------------------- 43
3.7 實驗方法---------------------------------------------------------- 42
3.7.1 組成份樣品製備與分析方法----------------------------- 43
3.7.1.1 香蕉粉末製備------------------------------------------ 43
3.7.1.2 酒精不溶物製備--------------------------------------- 43
3.7.1.3 果膠含量測定------------------------------------------ 44
3.7.1.3.1 液化------------------------------------------------- 44
3.7.1.3.2 呈色------------------------------------------------- 44
3.7.1.4 甲氧基含量測定--------------------------------------- 44
3.7.1.5 澱粉含量測定------------------------------------------ 45
3.7.1.6 Nelson還原糖測定方法------------------------------- 46
3.7.1.6.1 配藥------------------------------------------------- 46
3.7.1.6.2 還原糖測定---------------------------------------- 46
3.7.2 酵素萃取與分析-------------------------------------------- 46
3.7.2.1 聚半乳糖醛酸酶粗酵素液萃取-------------------- 46
3.7.2.2 聚半乳糖醛酸酶活性測定-------------------------- 47
3.7.2.3 蛋白質定量--------------------------------------------- 48
3.7.2.4 果膠酯酶粗酵素液萃取------------------------- 48
3.7.2.5 果膠酯酶活性測定------------------------------- 48
3.7.2.5.1 酸鹼滴定法---------------------------------------- 48
3.7.2.5.2 吸光光度法(酸鹼指示法)----------------------- 49
3.7.3 果膠酯酶抑制劑(PEI)之分離純化及活性分析-------- 50
3.7.3.1果膠酯酶抑制劑之分離純化------------------------- 50
3.7.3.2 果膠酯酶抑制劑活性分析--------------------------- 50
3.7.4 分子量測定-------------------------------------------------- 50
3.7.4.1 SDS-聚丙烯醯胺膠體電泳(SDS-polyacrylamide gel electrophoresis；SDS-PAGE)-------------------------------- 50
3.7.4.1.1 樣品之處理---------------------------------------- 50
3.7.4.1.2 電泳膠片之製備---------------------------------- 51
3.7.4.1.3 電泳之進行---------------------------------------- 51
3.7.4.1.4 膠片-玻璃紙三明治組合包紮乾燥法-------- 52
3.7.4.2 酸性原態膠體電泳------------------------------------ 52
3.7.4.2.1 試劑------------------------------------------------- 52
3.7.4.2.2 酸性膠體電泳------------------------------------- 53
3.7.4.3 果膠酯酶活性染色------------------------------------ 53
3.7.4.4 等電點之測定------------------------------------ 58
3.7.4.4.1 試劑配製------------------------------------------- 58
3.7.4.4.2 樣品處理------------------------------------------- 58
3.7.4.4.3 電泳之進行---------------------------------------- 58
3.8 管柱層析---------------------------------------------------------- 58
3.8.1 陽離子(CM-Sephaose)交換層析------------------------- 57
3.8.2 膠體過濾(Sephadex G-75)層析-------------------------- 59
3.9 統計分析---------------------------------------------------------- 59
第四章 結果與討論------------------------------------------------- 60
4.1香蕉組成份分析------------------------------------------------- 60
4.1.1各組樣品採樣數之百分比-------------------------------- 60
4.1.2硬心香蕉澱粉含量----------------------------------------- 60
4.1.3硬心香蕉總果膠含量-------------------------------------- 62
4.2硬心香蕉果膠酵素活性分析---------------------------------- 61
4.2.1 果膠酯酶活性分析---------------------------------------- 65
4.2.2 聚半乳糖醛酸酶活性分析------------------------------- 68
4.2.3果膠酯化度測定-------------------------------------------- 70
4.3 果膠酯酶之純化---------------------------------------------- 71
4.3.1硫酸銨沉澱-------------------------------------------------- 71
4.3.2 陽離子交換樹脂------------------------------------------- 72
4.3.3 果膠酯酶之聚丙烯醯胺膠體電泳---------------------- 77
4.3.4 果膠酯酶活性染色---------------------------------------- 81
4.4 香蕉之果膠酯酶等電點分析--------------------------------- 83
4.5 果膠酯酶抑制劑對香蕉果膠酯酶抑制情形---------------- 84
4.6硬心香蕉形成假說----------------------------------------------- 85
第五章 結論---------------------------------------------------------- 88
第六章 參考文獻---------------------------------------------------- 90
作者簡介-------------------------------------------------------------- 109

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