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研究生:蔡明謙
研究生(外文):Ming-Chien Tsai
論文名稱:奈米銀載體粒徑大小與製成溫度對真菌孢子殺菌效率影響之研究
論文名稱(外文):The Effect of Support and Heat Treatment Temperature on The Antifungal Spores Efficiency of Nano-Silver
指導教授:余國賓余國賓引用關係
指導教授(外文):Kuo-Pin Yu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:環境與職業衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:79
中文關鍵詞:奈米銀黑麴菌抑菌圈銀抗性
外文關鍵詞:nano-silverAspergillus nigerzone of inhibitionsilver-resistance
相關次數:
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摘要
麴菌屬(Aspergillus)是室內常見的菌種之一,對於人體健康會產生負面影響,如經由呼吸道進入體內引起過敏性呼吸症狀和造成免疫能力低落者感染等。另一方面,由於抗生素的濫用,使得多種微生物對現有的抗生素產生抗藥性,因此近年許多研究著重於研發新的抗菌劑,其中奈米銀是常見應用之一。本研究選擇黑麴菌(Aspergillus niger )作為奈米銀之殺菌試驗菌株,探討奈米銀在不同製成條件下對黑麴菌孢子的殺菌效率之影響。
本研究使用之奈米銀以兩種不同粒徑大小之二氧化鈦作為載體(30nm/200nm),於不同製成溫度(120oC/200oC)以初濕含浸法(Incipient Wetness Impregnation, IWI)製成。奈米銀的殺菌能力試實驗分三方向探討,1.抑菌圈試驗: 將黑麴菌孢子懸浮液接種在Malt Extra Agar(MEA)培養基上後,於培養基表面挖空一個圓洞作為奈米銀儲槽,再將預測奈米銀滴入,放入25oC的環境培養箱兩天後量測抑菌圈大小,並得到臨界濃度(Critical Concentration)。2. 殺菌實驗: 將奈米銀塗佈於石英片上再滴上菌液,實驗環境條件為未照光(Dark)及有未照光暴露臭氧(Dark-O3),實驗時間分別為8小時與6小時,實驗結束之後進行存活率分析。3.奈米銀抗性實驗: 將殺菌實驗未照光條件下之暴露8小時存活之黑麴菌孢子,再進行第二次殺菌實驗,觀察其存活率是否提升產生抗性。
抑菌圈試驗結果發現,200oC之奈米銀之載體每單位面積奈米銀含量與臨界關係呈現一個『U』形的趨勢,載體每單位面積奈米銀含量在0.25-0.625 mg/m2區域具有良好之殺菌能力。不同奈米銀製成溫度比較發現200 oC之奈米銀具有較佳的殺菌效果,此外,暴露奈米銀的黑麴菌孢子,經抗藥性試驗結果發現沒有明顯的抑菌圈,且暴露8小時後之存活率仍有60%以上,表示黑麴菌孢子產生了抗藥性。
綜合以上實驗結果,本研究歸納出以下結果:不同溫度合成之奈米銀因為高溫製成加強了其與載體之間的作用力使得銀離子更容易釋出因而使高溫和成奈米銀具有較好的殺菌效率;奈米載體每單位表面積之銀含量在0.25-0.625mg/m2具有最佳的殺菌能力;此外,奈米銀的殺菌效率不論其載體粒徑大小皆因環境中含臭氧而加強其效率。

關鍵字:奈米銀、黑麴菌、抑菌圈、銀抗性

Abstract
The Aspergillus genus is one of the most common fungi found on the indoor environment. It would cause respiration allergy syndrome through air track inhalation and cause hypoimmunity-persons infection. On the other hands, due to the abuse of antibiotic, causing a variety of microorganisms resistant to existing antibiotics; therefore, in recent years, many studies have focused on the research of new antifungal agents, and one of the most common among them is the nano-silver.
This study utilized two different particle size of TiO2 (P25 TiO2 30nm/Merck TiO2 200nm) as the support of AgNPs and two different heat temperatures (120oC/200oC) to synthesize the nano-silver particle by Incipient Wetness Impregnation (IWI), and choosing Aspergillus niger as the nano-silver antifungal property testing strain.
There were three antifungal properties testing experiments: (1)The disc diffusion assay ; At first, we incubated the A. niger spores suspension on the Malt Extract Agar, then hollowed a well as the reservoir for AgNPs. After filling the well with the 100μL AgNPs we analyzed the critical concentration (Cc) after incubating at 25oC for 2days. (2)Inactivation experiment; we coated the AgNPs on the quartz glass then dropped A. niger spores suspension onto the quartz glass. The experiment was in two different conditions: non-ozone and ozone, and the exposure time were for 8 and 6 hours, at the end of the experiment we proceeded to analyze the survival ratio. (3) The nano-silver resistance experiment ; we took the A. niger spores that survived from the 8hours exposure of AgNPs under non-ozone conditions to conduct, for the second time, the disc diffusion assay and inactivation experiment and observed whether the survival ratio increased or not.
The results of the disc diffusion assay showed that, under 200oC, the nano-silver content per unit of the support’s surface and the critical concentration’s relationship forms a U-shaped trend. When the content of nano-silver’s coverage of unit surface is within 0.25-0.625 mg/m2, it has a good antifungal property.
After 8 hours of AgNPs exposure, it revealed that the support particle size didn’t have much effect on the antifungal property, and comparing the two different heat treatment temperatures we learned that Ag NPs treated with 200oC had a better antifungal property. Furthermore the results of the A. niger spores that survived after exposure showed no significant inhibition zone and its survival ratio after 8 hours of Ag NPs exposure is still more than 60%, meaning that the A. Niger spores have produced resistance.
Based on the result from the summaries above, we conclude that the particle size of the AgNPs support has no significantly effect on the antifungal property. And with higher heat treatment temperature it would enhance the interaction between the metal and support, which makes the silver ion easier to reveal and enhance a better antifungal property. In addition, no matter of the support particle sizes, the antifungal property would enhance by adding ozone.
Keyword: nano-silver, Aspergillus niger, zone of inhibition, silver-resistance

目錄 V
摘要 I
ABSTRACT III
表目錄 VII
圖目錄 IX
第一章 前言 1
1.1 研究背景與動機 1
1.2研究目的 2
第二章 文獻探討 3
2.1奈米銀金屬 3
2.1.1奈米銀的特性 3
2.1.2奈米銀抗菌原理 5
2.1.3奈米銀的風險危害 6
2.2臭氧抗菌原理 7
2.3黑麴菌(Aspergillus niger)與健康效應關係 10
2.3.1真菌對人體生理作用 11
2.3.2流行病學研究 12
2.4.真菌對銀抗性機制 13
第三章 材料與方法 16
3.1奈米銀粉體製備 16
3.2黑麴菌之培養方法 18
3.3抑菌圈(Zone of inhibition) 20
3.4殺菌實驗(Inactivation Experiment) 22
3.5奈米銀抗性檢測(Resistance test)實驗 28
3.6資料處理與統計分析 29
3.7藥品與設備 30
第四章 實驗結果 32
4.1奈米銀粒徑形態 32
4.2奈米銀製備條件與抑菌效果的關係 43
4.3殺菌實驗結果 46
4.3.1奈米銀的殺菌效果 46
4.3.2臭氧(Ozone)與奈米銀的協同殺菌效果 47
4.3.3小結 48
4.4 Aspergillus niger對奈米銀的抗性結果 51
第五章 討論 55
5.1奈米銀載體粒徑大小對真菌孢子殺菌效率之影響 55
5.2奈米銀合成溫度對真菌孢子殺菌效率之影響 56
5.3奈米銀重量百分比對真菌孢子殺菌效率之影響 56
5.4 Aspergillus niger產生奈米銀抗性的可能原因 57
第六章 結論與建議 59
6.1結論 59
6.2後續研究建議 60
REFERENCE 61
附錄 70

表目錄
表2.1 奈米銀毒性影響因素 4
表2.2 氧化劑與其氧化力 7
表2.3 不同菌種對人體健康的影響 12
表3.1 奈米金屬粉末條件 17
表3.2 Merck TiO2為載體之奈米銀粉體抑菌圈試驗條件 21
表3.3 P25 TiO2為載體之奈米銀粉體抑菌圈試驗條件 21
表3.4 未照光(Dark)及未照光暴露臭氧(O3)試驗之試驗艙通氣條件 25
表3.5 殺菌實驗測試之奈米銀粉體 25
表4.1 不同載體大小之奈米銀粒徑分布 33
表4.2 不同條件之奈米銀在未照光(Dark)和未照光暴露臭氧(Ozone)環境下對Aspergillus niger孢子之存活率衰減常數(k0) (附載量為2mg/cm2) 50
表4.3 不同條件之奈米銀在未照光(Dark)和未照光暴露臭氧(Ozone)環境下抑制90% Aspergillus niger孢子所需時間(hour) (D-value=ln10/k) 50
表4.4 奈米銀抗性殺菌試驗比較 52

圖目錄
圖2.1 奈米銀的抗菌機制 5
圖2.2 Ozone molecule 7
圖3.1 殺菌實驗(Inactivation Expriment)流程圖 23
圖3.2 殺菌實驗系統圖 27
圖3.3 抗性檢測(Resistance test)實驗流程圖 28
圖4.1 5wt% Ag-P25-200 TEM 34
圖4.2 2wt% Ag-P25-200 TEM 34
圖4.3 1wt% Ag-P25-200 TEM 35
圖4.4 0.5w Ag-P25-200 TEM 35
圖4.5 0.2wt% Ag-P25-200 TEM 36
圖4.6 5wt% Ag-Merck-200 TEM 36
圖4.7 2wt% Ag-Merck-200 TEM 37
圖4.8 1wt% Ag-Merck-200 TEM 37
圖4.9 0.5wt% Ag-Merck-200 TEM 38
圖4.10 0.2wt% Ag-Merck-200 TEM 38
圖4.11 奈米銀於掃描式顯微鏡(SEM)的型態 39
圖4.12 奈米銀於掃描式顯微鏡(SEM)的型態 40
圖4.13 奈米銀於掃描式顯微鏡(SEM)的型態 41
圖4.14 奈米銀於掃描式顯微鏡(SEM)的型態 42
圖4.15 不同製備條件下奈米銀之抑菌圈效果 43
圖4.16 不同載體表面奈米銀之含量與抑菌圈效果 45
圖4.17 未照光(Dark)條件下奈米銀對Aspergillus niger孢子不同時間之存活率 49
圖4.18 臭氧(ozone)與未照光(Dark)環境下奈米銀對Aspergillus niger孢子不同時間之存活率比較 49
圖4.19 0.5wt% Ag-P25-200奈米銀抗性抑菌圈測試A. niger 之生長情形 53
圖4.20 0.5wt% Ag-P25-200 奈米銀抗性實驗(紅色實線)的存活率與未照光(綠色虛線)存活率之比較 54


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