臺灣博碩士論文加值系統

根據世界衛生組織指出，全世界每年約有6億人口，因食用受汙染的食品而生病，並且有42萬人因食源性疾病而死亡，尤其是兒童和老人。這些遭到細菌、病毒等有害物質汙染的食物，造成了超過兩百多種的疾病，範圍從腹瀉到癌症都有。其中金黃色葡萄球菌和仙人掌桿菌是常見的食源性病原菌，以生肉、生鮮蔬菜、米飯和未煮熟的食物，較常受到這些細菌的汙染。在食品工業中，會利用有很多方法來控制食品中的微生物，包括加熱、冷凍、輻照、高壓和添加化學防腐劑等。儘管這些方法可以抑制病原菌和腐敗菌的生長，但它們對食物甚至人類的健康都可能造成不良影響，尤其是化學防腐劑。因此，越來越多人在探討天然抗菌劑是否能替代食品中添加的化學防腐劑。植物來源的天然抗菌劑，如酚類化合物，不但對健康有許多益處，也具有抗菌，抗病毒活性。芪類化合物，包括紫檀芪、白皮杉醇和松芪，都是酚類化合物的一種。它們具有抗氧化，抗菌，抗發炎等多種功能，是理想的天然抗菌成份，但其抗菌活性及機轉仍不清楚。另外，奈米銀由於其良好的抗菌特性，已被許多領域拿來當作抗微生物劑使用，而在食品工業中，奈米銀較常被拿來使用在食品的包裝材料上，雖然已知奈米銀具有良好的抗菌特性，但其抗菌機轉目前還不完全清楚。因此本研究的目的是了解芪類化合物和奈米銀對於食源性病源菌的抗菌活性，並進一步探討單獨及合併處理對食源性病源菌可能的作用機轉。
本研究使用的食源性病原菌為金黃色葡萄球菌和仙人掌桿菌。藉由最低抑制濃度、最低殺菌濃度、抗菌協同作用、抑菌圈和時間殺菌曲線測試等方法進行抗菌實驗，測試紫檀芪、白皮杉醇、松芪和奈米銀的抗菌活性。通過掃描式電子顯微鏡和流式細胞儀觀察細胞形態，分析細菌存活力、活性氧累積、細胞凋亡以及DNA損傷，並以定量聚合酶連鎖反應分析基因表現量。另外藉由次世代定序檢測基因表現量的變化情形，推測可能的抗菌機制。
在抗菌實驗中，三種芪類化合物與奈米銀均具有抗菌活性。在紙錠擴散測試中，紫檀芪及奈米銀對金黃色葡萄球菌和仙人掌桿菌的抑制環約為1到1.4公分。紫檀芪、白皮杉醇、松芪和奈米銀對金黃色葡萄球菌和仙人掌桿菌的MIC和MBC範圍為25－100 μg/ml。在時間殺菌曲線測試中，以50 μg/ml紫檀芪處理4小時後，仙人掌桿菌減少5個log cfu。FICI值顯示紫檀芪和奈米銀之間具有部分協同作用。以紫檀芪及奈米銀處理後，細菌大小和細胞膜結構完整性改變。在仙人掌桿菌中，以紫檀芪處理後細胞存活及細胞凋亡的程度呈現劑量依賴性關係，且可觀察到細胞內ROS增加。在金黃色葡萄球菌中，經紫檀芪和奈米銀處理後，細胞存活力逐漸下降、ROS產生，且細胞凋亡的程度逐漸增加，此外由qPCR的結果得知，紫檀芪和奈米銀單獨及合併處理會誘導與凋亡樣細胞死亡相關的基因改變。如Rec A、Uvr A的表現量上升，Lex A的表現量下降。經由上述結果總結顯示，紫檀芪與奈米銀會藉由誘導細菌細胞內產生ROS，使Rec A蛋白活化，並導致Lex A蛋白水解，最後造成細菌凋亡樣死亡。

According to the report of WHO in 2015 that there are 420,000 people die for foodborne disease every year. Most of foodborne diseases were caused by bacteria, viruses, and parasites. Thus, controlling the foodborne pathogens in food is very important. Stilbenoid compounds are kinds of phenolic compounds with many functions, including the antioxidant, antibacterial and anti-inflammation effects. It’s an ideal natural antibacterial ingredient. In addition, silver nanoparticle has been used as antimicrobial agent in many fields owing to its good antibacterial properties. However, the antibacterial effects and the mechanisms of those compounds are not clear. Therefore, the purpose of this study was to understand the antibacterial activity of stilbenoid compounds and silver nanoparticle against foodborne pathogens, and study the possible antibacterial mechanisms of stilbenoid compounds in combination with silver nanoparticles. Inhibition zone, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), fractional inhibitory concentration index (FICI) and time-kill curve test are used to test the antibacterial activity of the stilbenoid compounds and silver nanoparticles. Three kinds of stilbenoid compounds and silver nanoparticles show antibacterial effects against foodborne pathogens. Among them, pterostilbene against Bacillus cereus and pterostilbene combined with silver nanoparticles against Staphylococcus aureus have better antibacterial effects. After treated with pterostilbene and silver nanoparticles, the morphology of B. cereus and S. aureus change. The viability of B. cereus and S. aureus decreased while ROS production and apoptosis increased. And the expression of related gene of apoptosis like death, including Rec A, Uvr A were upregulated, and Lex A was downregulated. Taken together, treatment of pterostilbene and silver nanoparticles may cause ROS accumulation and induce apoptosis like death.

第一章、 序論 1
第二章、 文獻回顧 2
第一節、 食源性疾病的發展 2
第二節、 食品保存方法及可能產生的問題 3
第三節、 天然抗菌劑 5
第四節、 芪類化合物(Stilbenoid)及其類似物的抗菌作用和機制 6
第五節、 奈米銀抗菌機轉 10
第六節、 細菌死亡機制 12
第七節、 腸道微生物 15
第八節、 次世代定序(Next Generation Sequencing；NGS) 16
第三章、 研究目的 18
第四章、 研究材料與方法 19
第一節、 研究材料 19
(一) 菌株 19
(二) 儀器 19
(三) 試劑與耗材 20
第二節、研究方法與實驗步驟 22
第五章、 研究架構 28
第六章、 研究結果 29
第一節、 奈米銀之物理化學特性 29
第二節、 芪類化合物與奈米銀對食源性病原菌的抗菌活性 29
第三節、 芪類化合物與奈米銀對食源性病原菌的最小抑菌濃度(MIC)和最小殺菌濃度(MBC) 29
第四節、 紫檀芪對食源性病原菌的殺菌效果 30
第五節、 紫檀芪結合奈米銀對食源性病原菌的抗菌協同作用 30
第六節、 紫檀芪與奈米銀對食源性病原菌形態的影響 31
第七節、 紫檀芪與奈米銀對食源性病原菌存活力、活性氧化物質(ROS)產生以及細胞凋亡的影響 32
第八節、 紫檀芪與奈米銀誘導食源性病原菌產生凋亡樣細胞死亡(Apoptosis-like cell death；ALD) 34
第九節、 紫檀芪對腸道微生物的影響 34
第十節、 紫檀芪與奈米銀對食源性病原菌基因之影響 35
第七章、 討論 36
第八章、 結論與建議 41
第九章、 參考文獻 42

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