臺灣博碩士論文加值系統

類澱粉蛋白奈米纖維(Amyloid protein nanofibrils)，是在特定的變性條件下某些蛋白質的二級結構β-sheets會自組裝(self-assembling)所形成的不可溶的蛋白質奈米纖維。本論文第一部分是研究直接利用乳清蛋白（Whey protein isolate）作為類澱粉蛋白奈米纖維之源料，乳清蛋白含有大量的β-Lactoglobulin，可形成類澱粉蛋白奈米纖維，在pH 2及80°C下反應20 小時，經由蛋白質電泳分析可以得知乳清蛋白中β-Lactoglobulin會隨著反應的時間而逐漸減少，而由螢光分析及AFM表面結構觀察，溶液中可得到直徑為15 nm，長度達2 μm之奈米纖維；第二部分則是研究將類澱粉蛋白奈米纖維的回收並固定化於孔徑為0.2 μm之醋酸纖維膜上，此類澱粉蛋白奈米纖維所形成的濾膜可用於天然花青素之吸附濃縮，將濃度為4 mg/mL之花青素溶液濃縮約5倍，在以1,1-二苯基-2-三硝基苯肼(DPPH)抗氧化分析其抗氧化性，抗氧化率可從花青素溶液之75 %提升花青素膜之85 %。此類澱粉蛋白奈米纖維膜可用於奈米銀顆粒之過濾回收，可將顆粒大小為31 nm之奈米銀過濾回收達96 %，此含有奈米銀之纖維濾膜，亦有觸媒反應之活性，在反應30分鐘後可將4-nitrophenol還原成4-aminophenol。

Amyloids are aggregated proteins that self-assembled into fibrils. The proteins or polypeptides generally form β-sheet structure under a denaturation condition before aggregate into long fiber. β-Lactoglobulin is one of often used proteins for amyloid fibrils generation. Whey protein isolate (WPI) has rich content of β-Lactoglobulin was found in this study can be used directly for the preparation of amyloid fibrils under the condition of pH 2 and heating to 80°C for 20 h. The as-prepared fibrils have an averaged diameter of 15 nm and length of more than 2 μm as observed by AFM. The zeta potential of the fibrils was measured to be +12.9 mV at pH 2 and became zero charge at pH 5.2. The amyloid nanofibrils prepared from WPI could be easily fabricated into membrane by filtering the amyloids suspension using 0.2 μm cellulose acetate microfiltration membrane. Clitoria ternatea (butterfly pea flower) extract that has rich content of anthocyanin was easily concentrated by filtering through the WPI amyloid fibrils membrane. The anthocyanin collected on the membrane exhibited strong antioxidant activity as measured by DPPH assay. The WPI nanofibrils membrane could also work as a nanofiltration membrane for the recovery of silver nanoparticles. Silver nanoparticle (AgNP) prepared by citrate reduction method with particle size about 31 nm could be very effectively collected by the nonofibrils membrane that 96.8 % of AgNP was recovered.

摘要 I
Abstract II
致謝 III
第一章 緒論 1
1-1 前言 1
1-2 研究目的與簡介 2
第二章 理論基礎與文獻回顧 3
2-1 類澱粉蛋白奈米纖維(Amyloid protein nanofibrils) 3
2-1-1 乳清蛋白(Whey protein) 4
2-1-2 蛋白質的摺疊與錯誤摺疊(protein folding and misfolding) 6
2-1-3 類澱粉蛋白奈米纖維的形成過程(Nanofibril assembly processes) 7
2-2 類澱粉蛋白奈米纖維應用 9
2-3 蝶豆花(butterfly pea) 14
2-3-1 花青素(Anthocyanin) 16
2-4 奈米銀顆粒 17
第三章 實驗材料與方法 20
3-1 實驗架構 20
3-2 實驗藥品 21
3-3 實驗儀器 22
3-4 溶液配製 23
3-5 實驗步驟與分析方法 25
3-5-1 3 wt%Whey protein isolate(WPI)奈米纖維的製備 25
3-5-2 蛋白質電泳分析 25
3-5-3 螢光光譜分析 27
3-5-4 掃描探針顯微鏡表面分析 27
3-5-5 WPI奈米纖維濾膜製備 28
3-5-6 製備花青素水溶液 29
3-5-7 製備奈米銀顆粒 30
3-5-8 WPI奈米纖維濾膜對花青素吸附之分析 30
3-5-9 DPPH自由基清除能力測定 30
3-5-10 WPI奈米纖維濾膜對奈米銀顆粒過濾之分析 31
3-5-11 將4-nitrophenol 還原 4-aminophenol 31
第四章 結果與討論 32
4-1 乳清蛋白(Whey protein isolate, WPI)奈米纖維之製備 32
4-1-1 蛋白質電泳分析 32
4-1-2 硫黃素T螢光染色WPI奈米纖維 34
4-1-3 掃描探針顯微鏡AFM觀察WPI奈米纖維 36
4-1-4 WPI奈米纖維之Zeta界面電位測定 38
4-2 WPI奈米纖維濾膜製備及其應用 39
4-2-1 FE-SEM場發式掃描電子顯微鏡 39
4-2-2 蛋白奈米纖維膜之硫黃素T螢光染色 42
4-2-3 WPI奈米纖維膜之濾速分析 43
4-3 WPI奈米纖維濾膜應用 44
4-3-1 花青素不同酸鹼值之顏色變化 44
4-3-2 WPI奈米纖維濾膜吸附過濾花青素 46
4-3-3 花青素吸附測定 46
4-3-4 花青素最大吸附量 48
4-3-5 吸附花青素之抗氧化分析 51
4-3-6 pH值對花青素溶液之影響 54
4-4 WPI奈米纖維膜過濾奈米銀溶液 56
4-4-1 奈米銀顆粒吸附過濾 56
4-4-2 奈米銀顆粒最大吸附過分析 59
4-4-3 奈米銀在WPI奈米纖維膜表面之型態 62
4-4-4 奈米銀催化作用分析 64
第五章 結論 66

Alberts, B., Johnson, A., Lewis, J., Walter, P., Raff, M., & Roberts, K. (2002). Molecular Biology of the Cell 4th Edition: International Student Edition: Routledge.
Bolder, S. G., Hendrickx, H., Sagis, L. M., & van der Linden, E. (2006). Fibril assemblies in aqueous whey protein mixtures. Journal of agricultural and food chemistry, 54(12), 4229-4234.
Bolisetty, S., & Mezzenga, R. (2016). Amyloid–carbon hybrid membranes for universal water purification. Nature nanotechnology, 11(4), 365-371.
Cherny, I., & Gazit, E. (2008). Amyloids: not only pathological agents but also ordered nanomaterials. Angewandte Chemie International Edition, 47(22), 4062-4069.
Giusti, M. M., & Wrolstad, R. E. (2001). Characterization and measurement of anthocyanins by UV‐visible spectroscopy. Current protocols in food analytical chemistry.
Herland, A., Thomsson, D., Mirzov, O., Scheblykin, I. G., & Inganäs, O. (2008). Decoration of amyloid fibrils with luminescent conjugated polymers. Journal of Materials Chemistry, 18(1), 126-132.
Jahn, T. R., & Radford, S. E. (2008). Folding versus aggregation: polypeptide conformations on competing pathways. Archives of biochemistry and biophysics, 469(1), 100-117.
Knowles, T. P., & Buehler, M. J. (2011). Nanomechanics of functional and pathological amyloid materials. Nature nanotechnology, 6(8), 469-479.
Knowles, T. P., & Mezzenga, R. (2016). Amyloid Fibrils as Building Blocks for Natural and Artificial Functional Materials. Advanced Materials, 28(31), 6546-6561.
Kosai, P., Sirisidthi, K., Jiraungkoorskul, K., & Jiraungkoorskul, W. (2015). Review on Ethnomedicinal uses of Memory Boosting Herb, Butterfly Pea, Clitoria ternatea. Journal of Natural Remedies, 15(2), 71-76.
Kyle, R. A. (2001). Amyloidosis: a convoluted story. British journal of haematology, 114(3), 529-538.
Li, C., Adamcik, J., & Mezzenga, R. (2012). Biodegradable nanocomposites of amyloid fibrils and graphene with shape-memory and enzyme-sensing properties. Nature nanotechnology, 7(7), 421-427.
Ling, S., Li, C., Adamcik, J., Shao, Z., Chen, X., & Mezzenga, R. (2014). Modulating Materials by Orthogonally Oriented β‐Strands: Composites of Amyloid and Silk Fibroin Fibrils. Advanced Materials, 26(26), 4569-4574.
Morris, A. M., Watzky, M. A., & Finke, R. G. (2009). Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1794(3), 375-397.
Mukherjee, P. K., Kumar, V., Kumar, N. S., & Heinrich, M. (2008). The Ayurvedic medicine Clitoria ternatea—from traditional use to scientific assessment. Journal of Ethnopharmacology, 120(3), 291-301.
Mukherjee, P. K., Kumar, V., Mal, M., & Houghton, P. J. (2007). Acetylcholinesterase inhibitors from plants. Phytomedicine, 14(4), 289-300.
Nyström, G., Fernández‐Ronco, M. P., Bolisetty, S., Mazzotti, M., & Mezzenga, R. (2016). Amyloid templated gold aerogels. Advanced Materials, 28(3), 472-478.
Sagis, L., Humblet-Hua, K., & Van Kempen, S. (2014). Nonlinear stress deformation behavior of interfaces stabilized by food-based ingredients. Journal of Physics: Condensed Matter, 26(46), 464105.
Sasso, L., Suei, S., Domigan, L., Healy, J., Nock, V., Williams, M., & Gerrard, J. (2014). Versatile multi-functionalization of protein nanofibrils for biosensor applications. Nanoscale, 6(3), 1629-1634.
Tanaka, H., Herland, A., Lindgren, L. J., Tsutsui, T., & Andersson, M. R. (2008). Enhanced current efficiency from bio-organic light-emitting diodes using decorated amyloid fibrils with conjugated polymer. Nano letters, 8(9), 2858-2861.
Tanaka, M., Collins, S. R., Toyama, B. H., & Weissman, J. S. (2006). The physical basis of how prion conformations determine strain phenotypes. Nature, 442(7102), 585-589.
Taranalli, A., & Cheeramkuzhy, T. (2000). Influence of Clitoria ternatea extracts on memory and central cholinergic activity in rats. Pharmaceutical biology, 38(1), 51-56.
Tyedmers, J., Mogk, A., & Bukau, B. (2010). Cellular strategies for controlling protein aggregation. Nature reviews Molecular cell biology, 11(11), 777-788.
Uversky, V. N., Li, J., & Fink, A. L. (2001). Evidence for a partially folded intermediate in α-synuclein fibril formation. Journal of Biological Chemistry, 276(14), 10737-10744.
Wang, Y., Sarkar, M., Smith, A. E., Krois, A. S., & Pielak, G. J. (2012). Macromolecular crowding and protein stability. Journal of the American Chemical Society, 134(40), 16614-16618.
Wilson, M. R., Yerbury, J. J., & Poon, S. (2008). Potential roles of abundant extracellular chaperones in the control of amyloid formation and toxicity. Molecular Biosystems, 4(1), 42-52.
Zhong, C., Gurry, T., Cheng, A. A., Downey, J., Deng, Z., Stultz, C. M., & Lu, T. K. (2014). Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature nanotechnology, 9(10), 858-866.