臺灣博碩士論文加值系統

本研究主要探討不同品種絲瓜之多元酚氧化之酵素特性及所含之多元酚成分、含量與褐變間之關係。以夏作絲瓜東光、東光2號、東光3號、七喜、七星、七美2號、F85-568、美菱、三喜等品種作為實驗材料，經萃取、離心後所取得之粗酵素液，進行不同品種間絲瓜之多元酚氧化特性研究及多元酚成分、含量分析。實驗結果顯示，絲瓜多元酚氧化若以L-DOPA為基質，其最適反應pH值在3.0∼6.0之間，最適反應溫度則在30∼35℃之間。在酵素活性方面以七喜為最高，其次依序為東光3號、七美2號、F85-568、七星、東光2號、美菱、三喜、東光。絲瓜多元酚氧化在熱安定性試驗中則顯示出高熱穩定之性質，七美2號為所有品種中對熱最不安定者，而三喜於60℃時之酵素活性達最高，其餘品種之多元酚氧化在50℃時活性均有逐漸上升之趨勢，但於60∼90℃時，則活性趨於穩定，而無明顯之變化。抑制劑試驗方面，則以抗壞血酸（ascorbic acid）的抑制效果優於半胱胺酸（cysteine）、重亞硫酸鈉（sodium metabisulfide）及苯甲酸（benzoic acid）。絲瓜之褐化以各品種之切片及液狀樣品培養在30℃下0、10、30、60、180分鐘後，做色差L、a、b值之檢測，結果發現切片樣品之褐化程度以東光最為顯著，其次依序為七美2號、東光2號、F85-568、美菱、七星、七喜、東光3號、三喜；而液狀樣品之褐化程度則以七美2號最為顯著，其次為七星、七喜、東光、三喜、美菱、東光2號、東光3號、F85-568。絲瓜切片樣品及液狀樣品色差結果不盡相同的原因推測為切片樣品與破碎樣品中，多元酚氧化之分佈和受空氣、基質影響的面積不同，而造成此等差異。將L、a、b值與酵素活性比對，發現酵素活性和絲瓜褐變程度並未呈現一定之趨勢，故影響絲瓜褐變之因素除酵素活性外，亦可能與多元酚含量、種類有關。由HPLC分析各品種絲瓜之多元酚類試驗中發現以含有漂木酸（chlorogenic acid）為主的品種居多數，其次為兒茶酚（catechol）及表兒茶酚（epicatechin），另在30℃烘箱培養0、10、30、60、180分鐘後之多元酚含量分析結果發現，不易褐變之品種如東光3號及F85-568，其漂木酸、表兒茶酚及兒茶素（catechin）含量多為穩定的狀態，而較易褐變之品種如東光及七美2號，其兒茶酚、表兒茶酚及兒茶素之含量多隨培養時間增長，而有明顯下降之趨勢，因此可推論漂木酸、表兒茶酚及兒茶素為影響絲瓜褐變之主要多元酚類，至於總酚類化合物含量則與褐變並無相關性。

The objectives of this research are focused on two aspects. The first one is to study the enzyme characteristics of polyphenol oxidase of the samples chosen. The second one is to study the relationship among the polyphenol composition, the contents, and tissue browning in various cultivars of vegetable sponge and angled luffa. Summer-harvested of vegetable sponges and angled luffas, including Dong Guang, Dong Guang No. 2, Dong Guang No. 3, Chi Shi, Chi Shing, Chi Mei No. 2, F85-568, Mei Ling, and San Shi were used for this study. Crude enzyme extracts were obtained through extraction and centrifugation. When L-DOPA is used as the substrate for the determination of polyphenol oxidase, it is found that the optimum temperature for the reaction is between 30-35℃, and the optimum pH value is between 3.0-6.0. Among the tested cultivars, Chi Shi has the highest enzyme activity, followed by Dong Guang No. 3, Chi Mei No. 2, F85-568, Chi Shing, Dong Guang No.2, Mei Ling, San Shi, and Dong Guang. Chi Mei No.2 is the most unstable one in thermal stability tests by heating to 50℃. When temperature is raised above 50℃ thermal stabilities of all samples become stable between 70-90℃. In terms of inhibitors, the research results show that the inhibition of ascorbic acid is more effective than cysteine, sodium metabisulfide, and benzoic acid. The enzymatic browning of each cultivar was tested by using both slice samples and liquid samples, both incubated in 30℃ oven for 0, 10, 30, 60, 180 minutes. Differences were noted when the L, a, and b values were examined by the color difference meter. For slice samples the degrees of browning are in the descending order are: Dong Guang, Chi Mei No. 2, Dong Guang No. 2, F85-568, Mei Ling, Chi Shing, Chi Shi, Dong Guang No. 3, and San Shi, while in liquid samples the order are:Chi Mei No. 2, Chi Shing, Chi Shi, Dong Guang, San Shi, Mei Ling, Dong Guang No. 2, Dong Guang No. 3, and F85-568 in descent. The differences between slice samples and liquid samples should be attributed to different destroyed methods applied to vegetable sponge and angled luffa texture, different contact areas exposed to oxygen and substrates for polyphenol oxidase. Experiment results fail to show a significant correlation between the tendencies of L, a, b values and enzyme activities. It is proposed that the enzymatic browning of vegetable sponge and angled luffa could also be affected by the contents and composition of polyphenols. Results from the analyses of nine different vegetable sponge and angled luffa cultivars on HPLC indicate that chlorogenic acid is the most aboundent one, catechol is the second, and then epicatechin. Cultivars of vegetable sponge and angled luffa were incubated for 0, 10, 30, 60, 180 minutes in a 30℃ oven to see if there were difference in polyphenol contents by HPLC analysis it was shown that the content of chlorogenic acid, epicatechin and catechin were stable in Dong Guang No. 3 and F85-568, decreasing along with time in Dong Guang and Chi Mei No. 2, and nonsignificant changes in Dong Guang No. 2, Chi Shi, Chi Shing, Mei Ling, and San Shi. From this study, it is suggested that the changes in the magnitudes of chlorogenic acid, epicatechin and catechin contained should be the major factors attributing to the enzymatic browning of vegetable sponge and angled luffa, while total contents of phenolic compounds do not play a significant role.

目 錄 頁次表目錄圖目錄中文摘要．．．．．．．．．．．．．．．．．．．． Ⅰ英文摘要．．．．．．．．．．．．．．．．．．．． Ⅲ第一章 前言．．．．．．．．．．．．．．．．．． 1第二章 文獻回顧．．．．．．．．．．．．．．．． 3 第一節 多元酚氧化之介紹．．．．．．．．．．　 3 一、命名．．．．．．．．．．．．．．．．．．　 3 二、分佈．．．．．．．．．．．．．．．．．． 3 三、分析及純化．．．．．．．．．．．．．．．　 4 四、多元酚氧化的特性．．．．．．．．．．．　 5 （一）分子量及四級結構．．．．．．．．．　 5 （二）胺基酸組成及一級結構．．．．．．． 6 （三）銅離子．．．．．．．．．．．．．．　 6 （四）基質的特異性．．．．．．．．．．．　 7 （五）最適ｐＨ值．．．．．．．．．．．．　10 （六）活化．．．．．．．．．．．．．．．　10 五、反應機制．．．．．．．．．．．．．．．．　10 六、生理功能．．．．．．．．．．．．．．．．　13 第二節　多元酚氧化與蔬果褐變之關係．．．．．　14 第三節　多元酚類的種類及與褐變之關係．．．．．　14 第四節　酵素性褐變之控制．．．．．．．．．．．　15 一、抑制酵素活性．．．．．．．．．．．．．．　15 二、ＰＰＯ抑制劑的使用．．．．．．．．．．．　18第三章　試驗材料與方法．．．．．．．．．．．．．　25 第一節 材料．．．．．．．．．．．．．．．．．　25 　一、原料．．．．．．．．．．．．．．．．．．　25　　二、化學試劑．．．．．．．．．．．．．．．．　25 第二節 方法．．．．．．．．．．．．．．．．．　26 一、多元酚氧化粗酵素萃取液．．．．．．．．　26 二、多元酚氧化活性測定．．．．．．．．．．　26 三、多元酚氧化蛋白質含量測定．．．．．．．　27 四、多元酚氧化特性測定．．．．．．．．．．　27 （一） 最適酸鹼度．．．．．．．．．．．．　27 （二） 最適溫度．．．．．．．．．．．．．　28 （三） 熱安定性．．．．．．．．．．．．．　28 （四） 不同抑制劑對多元酚氧化的影響．．　28 五、電泳分析法．．．．．．．．．．．．．．．　29 （一） 試劑．．．．．．．．．．．．．．． 29 （二） ＳＤＳ－ＰＡＧＥ膠體溶液配製法．．　30 （三） 膠體之製備．．．．．．．．．．．． 31 （四） 試樣之製備及泳動的條件．．．．．．　32 （五） 標準蛋白質之製備與泳動的條件．．．　32 （六） 電泳膠體染色及保存．．．．．．．． 33 １．標準蛋白染色法．．．．．．．．．． 33 ２．ＰＰＯ染色法．．．．．．．．．．． 33 （七） 分子量的測定．．．．．．．．．．．　33 六、多元酚的萃取及高效能層析儀分析．．．．． 34 七、褐變與色澤及多元酚含量關係之測定．．．． 35 （一） 不同絲瓜品種褐變之色澤差異測定．．　35　　　　 １．液狀樣品 ．．．．．．．．．．．．　35 ２．固狀樣品 ．．．．．．．．．．．．　36 （二）褐變後多元酚含量變化之測定 ．．．． 36 八、統計分析 ．．．．．．．．．．．．．．．　36 九、實驗架構流程圖．．．．．．．．．．．．． 37第四章 結果與討論．．．．．．．．．．．．．．．　38 一、多元酚氧化之萃取 ．．．．．．．．．． 38 （一）絲瓜品種之選取 ．．．．．．．．．　38 （二）多元酚氧化之萃取 ．．．．．．．　38 二、多元酚氧化之特性測定 ．．．．．．．．　40　　　　 （一） 最適酸鹼度．．．．．．．．．．．　40 （二）最適溫度 ．．．．．．．．．．．．　40 （三）熱安定性 ．．．．．．．．．．．．　43 （四）不同抑制劑對ＰＰＯ的影響 ．．．．　49 （五）電泳分析 ．．．．．．．．．．．．　52 三、多元酚類之HPLC分析及總酚類化合物含量． 59 四、褐變與色澤及多元酚含量關係之測定．．．　67 （一）不同絲瓜品種褐變之色澤差異測定 ．　67 １． 液狀樣品 ．．．．．．．．．．．　67 ２． 固狀樣品 ．．．．．．．．．．．　70 （二）褐變後多元酚含量變化之ＨＰＬＣ測定 75第五章 結論．．．．．．．．．．．．．．．．．．　82第六章 參考文獻．．．．．．．．．．．．．．．．　85表 目 錄表1 天然果汁中的多元酚氧化之加熱抑制作用．．． 16表2 九測試絲瓜品系多元酚氧化萃取後之活性情形． 39表3 不同抑制劑對各品種絲瓜ＰＰＯ之影響．．．．． 50表4 各品種絲瓜間之多元酚含量及總酚類化合物含量． 63表5 各品種絲瓜間之酵素總活性與多元酚含量及總酚類 化合物含量之比較．．．．．．．．．．．．． 66表6 各液狀樣品L、a、b值之樣品數、平均值、最小值、　　 最大值及變異係數．．．．．．．．．．．．．． 68表7 液狀樣品之L、a、b值在各品種、各時間、品種與 時間交感作用之效應．．．．．．．．．．．．． 68表8 各品種間液狀樣品之Ｌ、ａ、ｂ值．．．．．．． 69表9 各作用時間液狀樣品之Ｌ、ａ、ｂ值．．．．．． 69表10 固狀樣品之Ｌ、ａ、ｂ值在各品種、各時間、品種 與時間交感作用之效應．．．．．．．．．．．． 72表11 各固狀樣品Ｌ、ａ、ｂ值之樣品數、平均值、最小 值、最大值及變異係數．．．．．．．．．．．． 72表12 各品種間固狀樣品之Ｌ、ａ、ｂ值．．．．．．． 73表13 各品種絲瓜間之酵素總活性與多元酚含量、總酚類 化合物含量及液狀樣品Ｌ值之比較．．．．．．． 74表14 各品種褐變後總酚類化合物之含量．．．．．．． 76圖 目 錄圖1 ＰＰＯ作用之反應式．．．．．．．．．．．．． 8圖2 蔬果中常見的酚類化合物．．．．．．．．．．． 9圖3 ＰＰＯ的氧化機制．．．．．．．．．．．．．． 12圖4 ＰＰＯ催化反應中的中間產物．．．．．．．．． 12圖5 影響於桃泥多酚氧化酵素之加熱時間與溫度．．． 17圖6 不同品種絲瓜ＰＰＯ之最適ｐＨ值．．．．．．． 41圖7 不同品種絲瓜ＰＰＯ之最適溫度．．．．．．．． 42圖8（A） 圓筒絲瓜東光ＰＰＯ之熱安定性．．．．．． 44圖8（B） 圓筒絲瓜東光２號ＰＰＯ之熱安定性．．．． 44圖8（C） 圓筒絲瓜東光３號ＰＰＯ之熱安定性．．．． 45圖8（D） 圓筒絲瓜七喜ＰＰＯ之熱安定性．．．．．． 45圖8（E） 圓筒絲瓜七星ＰＰＯ之熱安定性．．．．．． 46圖8（F） 圓筒絲瓜七美２號ＰＰＯ之熱安定性．．．． 46圖8（G） 圓筒絲瓜Ｆ８５－５６８ＰＰＯ之熱安定性． 47圖8（H） 菱角絲瓜美菱ＰＰＯ之熱安定性．．．．．． 47圖8（I） 菱角絲瓜三喜ＰＰＯ之熱安定性．．．．．． 48圖9 各品種絲瓜ＰＰＯ之電泳膠片圖．．．．．．．． 53圖10（A）圓筒絲瓜東光多元酚類之ＨＰＬＣ圖譜．．． 54圖10（B）圓筒絲瓜東光２號多元酚類之ＨＰＬＣ圖譜． 55圖10（C）圓筒絲瓜東光３號多元酚類之ＨＰＬＣ圖譜． 56圖10（D）圓筒絲瓜七喜多元酚類之ＨＰＬＣ圖譜．．． 57圖10（E）圓筒絲瓜七星多元酚類之ＨＰＬＣ圖譜．．． 58圖10（F）圓筒絲瓜七美２號多元酚類之ＨＰＬＣ圖譜． 59圖10（G）圓筒絲瓜Ｆ８５－５６８多元酚類之ＨＰＬＣ 　　　　圖譜．．．．．．．．．．．．．．．．．． 60圖10（H）菱角絲瓜美菱多元酚類之ＨＰＬＣ圖譜．．． 61圖10（I）菱角絲瓜三喜多元酚類之ＨＰＬＣ圖譜．．． 62圖11 各品種間絲瓜所含多元酚類之比較圖．．．．．． 64圖12（A）圓筒絲瓜東光多元酚類褐變後之變化．．．． 77圖12（B）圓筒絲瓜東光２號之多元酚類褐變後之變化． 77圖12（C）圓筒絲瓜東光３號之多元酚類褐變後之變化． 78圖12（D）圓筒絲瓜七喜之多元酚類褐變後之變化．．． 78圖12（E）圓筒絲瓜七星之多元酚類褐變後之變化．．． 79圖12（F）圓筒絲瓜七美２號之多元酚類褐變後之變化． 79圖12（G）圓筒絲瓜Ｆ８５－５６８之多元酚類褐變後之　　　　 變化．．．．．．．．．．．．．．．．．． 80圖12（H）菱角絲瓜美菱之多元酚類褐變後之變化．．． 80圖12（I）菱角絲瓜三喜之多元酚類褐變後之變化．．． 81

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