海藻已被研究出具有多種對人體有益的活性成分，包括活性胜肽或者是酚類化合物，本研究預使用紅葡萄藻(Botryocladia leptopoda)作為樣品，並且鑑定紅葡萄藻活性胜肽，接著以酵素水解後取得胜肽，進一步以體外試驗探討其蛋白質水解物之生理活性。紅葡萄藻經酵素及鹼處理萃取蛋白質，接著利用十二烷基硫酸鈉聚丙烯醯胺膠體電泳、膠內消化 (in-gel digestion) 以及蛋白質體學之質譜技術鑑定蛋白質，並從其中挑選兩個蛋白質進行後續潛在活性胜肽探討，挑選依據為蛋白質來源的親緣關係與紅葡萄藻較為相近者且序列覆蓋率較高者，結果以核酮糖-1,5-二磷酸羧化酶/加氧酶 (Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) 及藻紅蛋白β鏈 (Phycoerythrin beta chain)最為合適。2種蛋白質序列以BIOPEP-UWM活性胜肽資料庫進行分析，兩者皆具有ACE抑制活性胜肽、DPP-4抑制活性胜肽及抗氧化活性胜肽，根據BIOPEP-UWM之分析結果顯示紅葡萄藻蛋白質可以作為製備ACE抑制、DPP-4抑制及抗氧化保健商品之素材。紅葡萄藻蛋白質以六種不同的酵素(Papain、Pepsin、Proteinase K、Subtilisin、Pancreatic elastase、Leukocyte elastase)進行水解，並測試各酵素之最適水解條件及ACE抑制活性，結果顯示以Pepsin水解5小時之紅葡萄藻水解物 (0.5 mg/mL) 相較其他水解物具有最強的抑制活性 (97.65 ± 1.78 %)，與據BIOPEP-UWM模擬結果相符，另以分子量進行區分(低於3 kDa，3-10 kDa和高於10 kDa)，結果以高於10 kDa之ACE抑制活性最佳。本研究之初步結果顯示蛋白質體學之質譜技術搭配BIOPEP-UWM能夠作為協助製備功能性水解物或胜肽之有力工具。綜合上述結果，紅葡萄藻蛋白質具有製備血管收縮素轉換酶抑制胜肽的潛力，未來可以進一步純化分析其中的血管收縮素轉換酶抑制胜肽，並且定序其胺基酸組成。

Varieties of active ingredients from algae have been widely developed, including bioactive peptides and phenolic compounds. In this study, Botryocladia leptopoda was selected for protein identification and bioactive peptides analysis. Proteins were extracted using enzymatic and alkali treatment, and then identified using proteomics. Two proteins, such as ribuose-1, 5-bisphosphate carboxylase/oxygenase large subunit and phycoerythrin beta chain, were chosen for the analysis of potential active peptides based on the phylogenetic relationship and the higher sequence coverage. Those two protein sequences were subsequently analyzed the potential bioactive peptides using BIOPEP-UWM database. According to the result, Botryocladia leptopoda proteins were predicted to generate ACE inhibition, DPP-4 inhibition and antioxidant peptides. The proteins were then hydrolyzed with six different enzymes (Papain, Pepsin, Proteinase K, Subtilisin, Pancreatic elastase, Leukocyte elastase) to test the optimal hydrolysis conditions and the activity of ACE inhibition from each enzyme. Data showed that the 5-hour pepsin hydrolysates (0.5 mg/mL) had the strongest ACE inhibitory activity (97.65 ± 1.78 %) compared with other hydrolysates, which was consistent with the BIOPEP-UWM simulation results. The molecular weight of pepsin hydrolysates was differentiated (≧10 kDa, 3-10 kDa ,≦3 kDa), and the result showed that peptides with MW above 10 kDa gave the best ACE inhibitory activity. Overall, this study shows that proteomics combined with BIOPEP-UWM database can be a powerful tool to assist in the preparation of functional hydrolysates or peptides. This study also revealed that Botryocladia leptopoda proteins had a great potential to generate ACE inhibitory peptides. In addition, ACE inhibitory peptides obtained from Botryocladia leptopoda proteins can be purified to identify the composition of amino acids possessing the inhibitory activity in the future study.

壹、前言.................................................1
貳、文獻回顧.............................................3
一、紅葡萄藻簡介.........................................3
二、藻類蛋白質萃取.......................................3
1.酵素處理法.............................................4
2.物理處理法.............................................5
3.化學處理法.............................................5
4.其他..................................................6
三、蛋白質水解...........................................6
1.抗氧化活性.............................................7
2.抗過敏活性.............................................8
3.抗高血壓活性...........................................9
4.血糖調節活性..........................................10
四、蛋白質鑑定技術......................................11
1.蛋白質分離技術........................................11
2.蛋白質分解技術........................................13
3.質譜分析技術..........................................13
參、實驗架構............................................15
肆、實驗材料與方法......................................16
一、實驗材料............................................16
二、實驗方法............................................16
1.一般成分分析..........................................16
2.樣品前處理............................................18
3.紅葡萄藻蛋白質萃取....................................18
4.電泳分析..............................................20
5.蛋白質鑑定............................................21
6.生物資訊工具分析......................................23
7.酵素水解物製備........................................23
8.胜肽生理活性測定......................................24
9.統計分析..............................................25
伍、結果與討論...........................................26
陸、結論.................................................34
柒、未來研究方向..........................................35
捌、參考文獻..............................................36

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