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標題: | 類澱粉纖維複合生物材料之應用:製備具有抗菌功能之與多酚分子結合的類澱粉纖維水凝膠複合材料 Applications of Amyloid Fibril-Based Hybrid Biomaterials: Preparation of Antibacterial Composite Hydrogels Consisting of Amyloid Fibrils and Polyphenolic Molecules |
作者: | Shang-Yu Shen 沈尚諭 |
指導教授: | 王勝仕 |
關鍵字: | 溶菌?,類澱粉纖維,多酚類,抗菌活性,水凝膠, lysozyme,amyloid fibrils,polyphenols,antimicrobial activity,hydrogel, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 本研究嘗試使用類澱粉纖維(amyloid fibril)結合多酚類(polyphenols),製作出具有抗菌性的水凝膠。本研究中的類澱粉纖維是以蛋白質本身即具有抗菌性的母雞蛋白溶菌酶製備而成,試圖強化所製得水凝膠之抗菌能力。多酚類則是選用了兩種亞類:黃酮類的楊梅素(myricetin)以及芪類的白藜蘆醇(resveratrol)。以上述原料製備了不同 pH 值以及不同的濃度的多酚類所製成之水凝膠,並比較了這些水凝膠的抗菌能力、外觀以及流體性質,此外還評估了不同濃度以及 pH 值環境下,此兩種多酚類對於類澱粉纖維的影響。
研究結果發現類澱粉纖維在加入多酚類之後,可以在接近中性的 pH 6 以及 pH 7.4 形成水凝膠;而在 pH 2 以及 pH 9 下則無法形成。 以革蘭氏陰性細菌 E. coli 進行抗菌測試,將樣品與菌液混合後於不同時間點取樣塗盤。實驗結果可以發現以楊梅素混合類澱粉纖維所製成的水凝膠具有良好的抗菌能力,在高濃度細菌環境下可在 8 小時內完全殺滅細菌;而以白藜蘆醇所製成之水凝膠以及單獨類澱粉纖維則不具有抗菌能力。 流變儀實驗結果可以觀察出水凝膠的黏度與 pH 值以及多酚類的濃度均有相關,且發現類澱粉纖維在加入多酚類後,黏度均有上升。然而隨著楊梅素加入的量越多,黏度的提升卻越少,是由於類澱粉纖維可能會被楊梅素所降解,造成其結構不穩定;而白藜蘆醇組別則無此現象。 從 ThT 螢光光譜分析可以發現,兩種多酚類對於類澱粉纖維的影響有巨大差異,楊梅素展現了很強的競爭 ThT 鍵結位置或是降解類澱粉纖維的能力,在高楊梅素濃度組別螢光強度比起單獨類澱粉纖維降低非常多,而白藜蘆醇則沒有很明 顯降低趨勢。 ANS 螢光光譜分析也可以觀察到加入楊梅素的組別螢光強度有急劇降低的趨勢,代表類澱粉纖維裸露的疏水區幾乎完全被楊梅素所佔據,或是楊梅素降解了類澱粉纖維結構。而白藜蘆醇的組別則同 ThT 結果沒有明顯變化。 綜上所述,在適當濃度下的楊梅素混和溶菌酶類澱粉纖維所製成的水凝膠,相較於單獨類澱粉纖維以及白藜蘆醇組別,具有更好的抗菌能力,且機械性質也較好,因此這種功能性水凝膠具有潛力成為抗菌生物材料。 In this study, we attempted to produce antimicrobial composite hydrogels consisting of amyloid fibrils and polyphenolic molecules. The amyloid fibrils were made from hen egg-white lysozyme (HEWL), and myricetin and resveratrol were chosen as the polyphenolic molecules. The antibacterial capability, morphology and fluid property of the hydrogels prepared under different conditions were analyzed. The effects of these two polyphenols on amyloid fibrils were also evaluated. Our results showed that, as opposed to the conditions at pH 2 and pH 9, HEWL amyloid fibrils were able to form hydrogels at pH 6 and pH 7.4 after the addition of polyphenols. The antibacterial activity assay was carried out with a high concentration of Gram-negative bacteria E. coli which was mixed with the hydrogel. The mixture was sampled at different times using the spread-plate method to observe for the colony formation. We found that the hydrogels incorporated with myricetin possessed a better antibacterial activity and eliminated bacteria within 8 hr. On the contrary, the amyloid fibril-based hydrogels with resveratrol and amyloid fibrils alone displayed negligible antibacterial activity. The results from the rheological analysis indicated that the viscosity of hydrogels is correlated with the pH value, concentration of polyphenol, and types of polyphenol. Moreover, it was found that the viscosity of the amyloid fibrils increased upon the addition of polyphenols. However, the more the amount of myricetin was added, the less the increase in viscosity. which might be attributed to the fact that the amyloid fibrils may be degraded by myricetin, resulting in structural instability. It should be noted that the similar trend was not seen in the groups with resveratrol. It could be found from the ThT binding analysis that the effects of myricetin and resveratrol on amyloid fibrils were significantly different. The myricetin molecule exhibited a strong competition with ThT for binding positions or ability to degrade amyloid fibrils. Therefore, in the groups of high concentrations of myricetin, the fluorescence intensity was observed to be much lower than that of amyloid fibrils alone. An analogous situation was also encountered in ANS binding analysis- showing that the occupation of exposed hydrophobic area of amyloid fibrillar structure by myricetin likely contributed to the reduction of ANS fluorescence intensity. However, this drastic reduction in ANS fluorescence emission was not perceived in the hydrogels combined with resveratrol. Moreover, hydrogels with the molar ratio of lysozyme fibril to myricetin at 0.1:1 showed better antibacterial ability and mechanical property. Therefore, this hydrogel is a promising antibacterial composite material. The outcome from this study is in support of the potential application of amyloid fibrils as functional biomaterials. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21712 |
DOI: | 10.6342/NTU201900760 |
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顯示於系所單位: | 化學工程學系 |
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