臺灣博碩士論文加值系統

臺灣藜 (Chenopodium formosanum Koidz.) 具豐富營養價值及無麩質之植物性蛋白質，是患有麩質不耐症敏感族群之良好飲食攝取來源。本研究主要分析台灣藜種子，以鹼萃取-等電點沉澱法及酸萃取-等電點沉澱法得到之蛋白質分離物，探討其於十二烷基硫酸鈉聚丙烯醯胺膠體電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 之蛋白質分子量分佈，並以蛋白質體學之質譜技術鑑定其主要蛋白質之序列，利用生物資訊工具 BIOPEP 資料庫預測台灣藜蛋白潛在之活性胜肽。以鹼萃取方式得到的台灣藜鹼萃取蛋白質分離物 (Djulis alkaline-extracted protein isolates, DBPI) 之粗蛋白含量 (94.25%) 顯著高於酸萃取蛋白質分離物 (Djulis acid-extracted protein isolates, DAPI)，顯示利用鹼萃取方式可以提高蛋白質含量，產率為 32.29 %。將電泳分析條帶 (band) 進行膠內消化 (in-gel digestion) 以及質譜分析，鑑定出四種蛋白質：11S 種子儲存球蛋白 (11S seed storage globulin)、GBSSIb 蛋白、乙醯羥酸合成酶 (acetohydroxyacid synthase）、甜菜鹼醛去氫酶 (betaine aldehyde dehydrogenase)。4 種蛋白質序列以 BIOPEP 活性胜肽資料庫進行分析，發現主要有：抑制二肽基胜肽酶-IV (dipeptidyl peptidase-IV, DPP-IV) 活性及抑制血管收縮轉換酵素 (angiotensin converting enzyme, ACE) 活性，其中 DPP-IV 抑制活性之胜肽頻率為 0.6396－0.6562，ACE 抑制活性之胜肽頻率為 0.4000－0.4580。BIOPEP 的分析結果顯示，Proteinase K、Chymotrypsin A、Chymotrypsin C、Pancreatic elastase、Thermolysin、Papain 適合用於從台灣藜蛋白中製備 DPP-IV 抑制活性胜肽及 ACE 抑制活性胜肽，且模擬經人體腸胃道酵素消化後所釋放之活性胜肽數量亦增加，顯示台灣藜蛋白質具有良好的潛力生產 DPP-IV 抑制活性胜肽及 ACE 抑制活性胜肽，是有前景之食品保健素材來源。

Taiwanese quinoa, Djulis (Chenopodium formosanum Koidz.), is rich in nutritional value and contains gluten-free proteins which can be a good source of dietary intake for susceptible populations with gluten intolerance.The major purpose of this study was to analysis the proteins from Djulis seeds extracted by acid and alkaline pretreatment followed by isoelectric point precipitation method, and to identify extracted proteins using proteomic approaches including SDS-PAGE, in gel digestion, mass spectrometry techniques and BIOPEP analysis..The protein content (94.25%) of Djulis alkaline-extracted protein isolates (DBPI) was significantly higher than that of Djulis acid-extracted protein isolates (DAPI). The method of alkaline-extracted can improve the protein content with 32.29% yield. A total of 4 proteins were identified as 11S seed storage globulin, GBSSIb, acetohydroxyacid synthase, betaine aldehyde dehydrogenase. Sequences of identified proteins were further analyzed using BIOPEP database. Frequency of DPP-IV inhibitory peptides embedded in identified Djulis proteins are 0.6396-0.6562, and ACE inhibitory peptides are 0.4000-0.4580. In silico results from BIOPEP recommended that Djulis proteins can be great precursor to produce DPP-IV inhibitory and ACE inhibitory peptides.The results of BIOPEP analysis showed that Proteinase K, Chymotrypsin A, Chymotrypsin C, Pancreatic elastase, Thermolysin and Papain were suitable for the preparation of DPP-IV inhibitory peptides and ACE inhibitory peptides from Djulis protein isolates. Simulation of the human gastrointestinal tract indicates the numbers of released active peptides after gastrointestinal digestion are increased. In silico prediction suggested that Djulis proteins can be a good potential to produce DPP-IV inhibitory and ACE inhibitory peptides. It’s a promising source of food-grade nutraceutical ingredients.

摘要....................................................I
Abstract...............................................II
目次..................................................III
圖次....................................................V
表次...................................................VI
縮寫對照表.............................................VII
壹、前言.................................................1
貳、文獻回顧.............................................2
一、台灣藜簡介...........................................2
二、台灣藜營養成分.......................................3
(一)一般營養成分.........................................3
(二)機能性營養成分.......................................4
三、麩質不耐症...........................................4
四、蛋白質鑑定技術.......................................5
(一)蛋白質之純化分離.....................................5
(二)蛋白質分解消化.......................................5
(三)質譜分析鑑定及資料庫比對..............................5
五、蛋白質水解物.........................................7
(一)簡介................................................7
(二)生理活性.............................................7
2.1 抗高血壓活性.........................................7
2.2 抗氧化活性...........................................8
2.3 降血糖活性...........................................8
六、活性胜肽資料庫.......................................9
參、實驗架構............................................11
肆、實驗材料與方法......................................12
一、實驗材料與試藥......................................12
(一)台灣藜.............................................12
(二)實驗藥品............................................12
(三)儀器...............................................12
二、實驗方法............................................13
(一)一般成份分析........................................13
1.1水分................................................13
1.2粗蛋白質.............................................13
1.3粗脂肪..............................................13
1.4灰分................................................14
1.5碳水化合物...........................................14
(二)蛋白質分離物製備....................................14
2.1臺灣藜蛋白質分離物製備................................14
2.2產率計算.............................................16
(三)十二烷基硫酸鈉聚丙烯醯胺膠體電泳分析..................16
3.1膠片製作.............................................16
3.2樣品製備.............................................16
3.3電泳................................................16
(四)蛋白質鑑定..........................................16
4.1溶液配製.............................................16
4.2膠片裁切.............................................17
4.3膠內消化.............................................17
4.4胜肽萃取.............................................17
4.5液相層析串聯質譜儀分析................................17
4.6資料庫比對與蛋白質鑑定................................18
(五)生物資訊工具分析....................................18
伍、結果與討論..........................................19
一、台灣藜種子及其蛋白質分離物之一般成分分析...............19
二、台灣藜種子蛋白質鑑定.................................19
三、以BIOPEP分析鑑定出的台灣藜蛋白質......................21
陸、結論................................................22
柒、參考文獻............................................23
捌、附表與附圖..........................................27
附錄一、20種胺基酸列表...................................40

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