煅燒條件對牡蠣殼抗菌能力之影響及抗菌物種- 單線態氧的檢測

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洪瑋廷(Wei-Tung Hong) 查詢紙本館藏

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環境工程研究所

論文名稱

煅燒條件對牡蠣殼抗菌能力之影響及抗菌物種- 單線態氧的檢測
(Effects of calcination conditions on the antibacterial ability of oyster shells and the detection of antibacterial species-singlet oxygen)

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至系統瀏覽論文 (2027-1-1以後開放)

摘要(中)

根據行政院農業委員會統計資料顯示，台灣每年約有16萬噸的廢棄牡蠣殼產出量，過去大部分的牡蠣殼被隨意棄置，在日照高溫下，散發惡臭味孳生蚊蠅，不僅占空間、破壞環境及漁村景觀，同時也危害附近居民的生活品質與健康。國內近年來大力推動廢棄牡蠣殼的回收計畫，此舉雖然有效改善廢棄物的問題，但是國內對牡蠣殼的應用開發仍侷限於飼料添加劑，其附加價值及使用率並不高。此外，隨著工業技術的進步和化學合成技術的開發，化學抗菌劑的使用已經變得普遍，甚至達到了濫用的程度，對人類和環境帶來了嚴重的威脅。為了解決上述問題，近年來，有許多學者致力於天然抗菌劑的研究，雖然已有文獻證實牡蠣殼經高溫煅燒後可製成高值化的新型抗菌劑，但對於抗菌機制眾說紛紜，普遍認為活性氧物種是主要的抗菌機制，而單線態氧(1O2)被視為具有高度活性的氧化劑，在抗菌過程中至關重要。本研究為首篇探討煅燒條件對於牡蠣殼中1O2生成之研究，並評估1O2含量與牡蠣殼抗菌能力的關係，另外，本研究亦建立更完整詳細的煅燒牡蠣殼之特性分析。本研究製備三種抗菌材料，分別是經過900°C和1000°C煅燒的牡蠣殼粉(900HOS(Heated oyster shell)、1000HOS)，以及900°C下通氮氣煅燒的牡蠣殼粉(900N-HOS)，並模擬大腸桿菌在生長的條件下，三種材料的抗菌效力，結果顯示，在1 g/L的劑量下，三種材料在60分鐘抗菌效果分別是0.87-log；0.74-log；0.63-log，抗菌能力大小順序為: 1000HOS > 900N-HOS > 900HOS，證實提升煅燒溫度和通入氮氣煅燒能夠有效提升牡蠣殼的抗菌能力，煅燒後的牡蠣殼展現出卓越的抗菌特性，1000HOS經過120分鐘後幾乎達到100%的抗菌效果(7.59-log)；同樣地，900N-HOS和900HOS在240分鐘內達到完全的抗菌。為了更進一步確認抗菌的機制，本研究透過電子自旋共振光譜儀技術，檢測抗菌物種1O2之含量，分析結果顯示，1O2的含量與抗菌能力成正比，確定了1O2在抗菌機制中扮演著不可或缺的關鍵角色。綜合上述結果，期望本研究在廢棄牡蠣殼的應用上盡一份心力，同時實踐循環經濟的理念，促進廢棄牡蠣殼多元化的目標發展，並且創造漁業廢棄物的商業價值。

摘要(英)

According to statistics from the Council of Agriculture, Executive Yuan, approximately 160,000 tons of discarded oyster shells are produced in Taiwan annually. In the past, most of the shells were dumped randomly. This improper disposal not only results in the occupation of space and environmental damage to the picturesque landscape of the fishing village but also poses a significant threat to the quality of life and the health of the neighboring residents. In recent years, Taiwan has actively promoted the recycling plan for discarded oyster shells. While this approach effectively addresses the waste issue, the domestic application and development of oyster shells remain limited to feed additives.
Furthermore, their added value and utilization rate are small. Again, chemical antiseptics have become increasingly widespread with the continuous advancement of industrial technology and the rapid development of chemical synthesis technology. Unfortunately, this general use has escalated to the point of abuse, posing a significant and imminent threat to human beings and the environment. Many scholars have been researching natural antibacterial agents to address these challenges in recent years. While previous literature has confirmed that oyster shells can be transformed into high-value antibacterial agents through high-temperature calcination, there are different perspectives on their antibacterial mechanisms. Reactive oxygen species (ROS) are generally considered the primary antibacterial mechanism, while singlet oxygen (1O2) is regarded as a highly reactive oxidant critical in the antibacterial process.
This study represents the primary investigation focusing on the impact of calcination conditions on the generation of 1O2 in oyster shells. Furthermore, the study aims to assess the correlation between the concentration of 1O2 and the antibacterial efficiency of oyster shells. Additionally, a comprehensive analysis of the characteristics of calcined oyster shells was conducted. Three types of antibacterial materials were prepared: oyster shell powder calcined at 900°C and 1000°C (represented as 900HOS and 1000HOS, respectively), and oyster shell powder calcined at 900°C under a nitrogen atmosphere (900N-HOS). The antibacterial ability of these materials against Escherichia coli was evaluated at different time points. The antibacterial efficacy of these three materials was evaluated under conditions simulating the growth of Escherichia coli.
The results demonstrated that at a dosage of 1 g/L, the antibacterial effects of the materials after 60 minutes were 0.87-log, 0.74-log, and 0.63-log for 1000HOS, 900N-HOS, and 900HOS, respectively. The antibacterial capacity followed the order: 1000HOS>900N-HOS> 900HOS, confirming that increasing the calcination temperature and conducting calcination under a nitrogen atmosphere effectively enhanced the antibacterial capability of oyster shells. The calcined oyster shells exhibited remarkable antibacterial properties, with 1000HOS achieving nearly 100% (7.59-log) antibacterial effect after 120 minutes, while 900N-HOS and 900HOS reached complete antibacterial efficacy within 240 minutes.
To further validate the antibacterial mechanism, electron spin resonance (ESR) spectrometer technology was employed to detect the content of the antibacterial species 1O2. The analysis showed a direct correlation between the 1O2 content and the antibacterial efficacy, thus confirming the crucial role of 1O2 in the antibacterial mechanism.
Based on the results above, this research is expected to contribute to the application of discarded oyster shells. At the same time, practice the circular economy concept, promote the diversification of discarded oyster shells, and create commercial value of fishery waste.

關鍵字(中)

★ 廢棄牡蠣殼
★ 氧化鈣
★ 抗菌能力
★ 活性氧物種(ROS)
★ 單線態氧(1O2)

關鍵字(英)

★ waste oyster shells
★ calcium oxide
★ antibacterial ability
★ reactive oxygen species (ROS)
★ singlet oxygen (1O2)

論文目次

摘要 i
Abstract ii
致謝 iv
目錄 v
圖目錄 ix
表目錄 xii
第一章研究緣起與目的 1
1.1 研究緣起 1
1.2 研究目的 3
1.3 研究重要性與創新性 4
第二章文獻回顧 5
2.1 廢棄牡蠣殼回收現況 5
2.2 牡蠣殼的特徵及應用 8
2.2.1 牡蠣殼的結構 8
2.2.2 廢棄牡蠣殼當前和潛在的用途 8
2.3 牡蠣殼抗菌材料的製備條件 11
2.3.1 鹼洗對牡蠣殼的型態影響 11
2.3.2 煅燒溫度及方式提升氧化鈣轉化率 11
2.4 牡蠣殼製備天然抗菌劑 12
2.4.1 天然抗菌劑的優勢 12
2.4.2 牡蠣殼的抗菌機制 13
2.4.3 氧化鈣的抗菌應用 15
2.4.4 抗菌劑的體外抗菌試驗方法 16
2.5 活性氧物種的檢測方法 19
2.5.1 螢光探針檢測 19
2.5.2 化學發光法 19
2.5.3 電子自旋共振(ESR)法 19
2.5.4 1O2定量技術 23
第三章研究方法與材料 24
3.1 研究流程與架構 24
3.2 研究設備與藥品 25
3.2.1 實驗藥品 25
3.2.2 實驗儀器與設備 26
3.3 材料的製備 27
3.3.1 牡蠣殼粉的製備 27
3.3.2 不同煅燒溫度及方式製備牡蠣殼粉 27
3.4 抗菌材料的特性分析 28
3.4.1 pH 28
3.4.2 動態光散射(DLS) 28
3.4.3 比表面積及孔徑分佈分析(BET) 28
3.4.4 熱重損失(TGA) 29
3.4.5 衰減全反射傅立葉轉換紅外線光譜儀分析(ATR-FTIR) 29
3.4.6 X射線繞射儀(XRD) 29
3.4.7 X射線光電子能譜儀分析(XPS) 29
3.4.8 掃描式電子顯微鏡- X射線能譜儀分析(SEM-EDS) 30
3.5 菌種活化 30
3.6 平板計數及光學密度法(OD600)推估菌落數 32
3.7 抗菌能力測試 34
3.7.1 定性分析-瓊脂孔擴散法 34
3.7.2 定量分析-瓊脂稀釋法 34
3.8 單線態氧(1O2)的檢測方法 36
3.8.1 電子自旋共振(ESR)光譜法 36
3.8.2 ABDA法定量1O2 37
3.8.3 抗菌試驗差異性分析 37
第四章結果與討論 38
4.1 藉由TGA結果選擇實驗材料 38
4.2 抗菌前置試驗 39
4.3 牡蠣殼抗菌材料之基本特性分析 40
4.3.1 動態光散射(DLS) 41
4.3.2 比表面積及孔徑分佈分析(BET) 41
4.3.3 X射線繞射儀(XRD) 45
4.3.4 衰減全反射傅立葉轉換紅外線光譜儀分析(ATR-FTIR) 46
4.3.5 掃描式電子顯微鏡- X射線能譜儀分析(SEM-EDS) 48
4.3.6 X射線光電子能譜儀分析(XPS) 53
4.4 抗菌實驗結果 58
4.4.1 定性分析-瓊脂孔擴散法 58
4.4.2 定量分析-瓊脂稀釋法 60
4.5 單線態氧(1O2)檢測結果 69
4.5.1 ESR定性分析 69
4.5.2 ABDA法定量1O2 76
第五章結論與建議 78
5.1 結論 78
5.2 建議 80
參考文獻 81
附錄 91
附錄一牡犡殼特性分析補充數據 91
附錄二抗菌實驗結果照片(第三階段) 93
附錄三牡犡殼抗菌實驗原始數據 94

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指導教授

林伯勳(Po-Hsun Lin)

審核日期

2023-7-28

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