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研究生:杜氏潭美
研究生(外文):Do Thi Tra My
論文名稱(外文):Identify and characterize the fermenting and electrogenic skin bacteria using selective prebiotics
指導教授:黃俊銘黃俊銘引用關係
指導教授(外文):Chun-Ming Huang
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生醫科學與工程學系
學門:工程學門
學類:生醫工程學類
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:58
中文關鍵詞:表皮葡萄球菌皮膚痤瘡桿菌細胞外電子轉移益生元益生菌
外文關鍵詞:Staphylococcus epidermidis (S. epidermidis)Cutibacterium acne (C. acne)Extracellular Electron Transfer (EET)PrebioticProbiotic
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表皮葡萄球菌S. epidermidis是一種皮膚上常見的微生物能為宿主提供許多好處。在我們先前的研究中表明,表皮葡萄球菌ATCC 12228可以作為皮膚益生菌使甘油發酵,普遍認知此發酵反應能自然地發生在人類皮膚上,當發酵後產生短鏈脂肪酸Short-chain fatty acids(SCFA)後,藉由逆轉由UV-B輻射誘導所造成的氧化應激(Oxidative Stress),能阻止致病菌的生長,例如皮膚痤瘡桿菌Cutibacterium acnes或是金黃色葡萄球Staphylococcus aureus。然而益生菌的局部應用仍是一個爭議的話題。一直以來,我們實驗室專注研究從人類皮膚上所分離出的細菌,特別是能與皮膚共生的表皮葡萄球菌,當它作為一種益生菌,研究其可提供的效益以及細菌的生物電位活性,期望尋找一株細菌可用來替代目前FDA批准使用在臨床治療的ATCC 12228。
  我們已從人類皮膚中篩選分離出十多種表皮葡萄球菌菌株,並且發現其中的一種菌株U2,相較於ATCC 12228 而言U2擁有較高的發酵能力與較低的生物膜作用,此現象可降低細菌致病毒力。此外分析由益生質所介導的發酵作用後,能發現U2在通過細胞外電子轉移Extracellular Electron Transfer (EET)後,能釋放更多的電子作為次級代謝產物。
  除了益生菌篩選分析外,在實驗過程中發現一種潛在的益生質-椰油辛酸 Coco-caprylate (LCC),這是一種選擇性發酵誘導物Selective Fermentation Inducer (SFI),相比傳統的甘油發酵而言,LCC能更好地促進發酵過程。我們的研究表明,較好的皮膚益生菌以及最佳的SFI可有益於維護皮膚的微生物組成。
Staphylococcus epidermidis (S. epidermidis) in the skin microbiome provides a huge benefit to the host. Our previous studies demonstrated that S. epidermidis ATCC 12228 served as a skin probiotic that can mediate the glycerol fermentation which is known to naturally co-exists in the human skin to yield short-chain fatty acids (SCFAs) to protect against the growth of pathogenic bacteria such as Cutibacterium acnes or Staphylococcus aureus (strain USA300) and reverse the oxidative stress induced by UV-B radiation. However, the topical application of probiotic bacteria is still a debatable topic. In our study, we have been focusing on commensal bacterium especially S. epidermidis commensal strains that are isolated from human skin to study on their probiotic and electro biotic activity for clinical applications in replacement to FDA approved S. epidermidis (ATCC 12228). We have screened more than 10 different strains of S. epidermidis from human skin and found a commensal bacterium S. epidermidis (U2) which has higher fermentation and lower bio-film role in association to lower virulence than S. epidermidis ATCC 12228. Also, upon prebiotic mediated fermentation they release higher electron as their secondary metabolite via the extracellular electron transfer (EET). Besides the probiotic screening, we also figured out a potential prebiotic selective fermentation inducer (SFI) coco-caprylate (LCC) which can boost the fermentation process better than traditionally used glycerol. Our study reveals the better skin probiotic strain as well as optimum SFI for the beneficial maintenance of skin microbiome.
Abstract i
Acknowledgments ii
Table of contents iii
List of figures v
List of tables v
Abbreviation List vii
1. INTRODUCTION - 1 -
1.1. LITERATURE REVIEW - 2 -
1.1.1. Pro-prebiotic - 2 -
1.1.2. Bacterium - 3 -
1.1.3. Fermentation of bacterium - 4 -
1.1.4. Pyruvate dehydrogenase complex - 6 -
1.1.5. Phosphotransacetylase enzyme - 7 -
1.1.6. Intercellular adhesion gene cluster (ica) - 7 -
1.1.7. Electro-Fermentation (EF) - 8 -
2. RESEARCH OBJECTIVE - 9 -
3. MATERIALS AND METHODS - 10 -
3.1. Materials - 10 -
3.1.1. Apparatus or Instruments - 10 -
3.1.2. Reagents - 10 -
3.2. Methods - 11 -
3.2.1. Medium preparation - 11 -
3.2.2. Isolate and identify commensal S. epidermidis - 12 -
3.2.3. Identification of pdh, pta, and ica. - 13 -
3.2.4. Screening probiotic properties. - 14 -
3.2.5. Biofilm detection - 15 -
3.2.6. Detection of skin conductance - 15 -
3.2.7. Minimum bactericidal concentration - 16 -
3.2.8. In vitro Electricity detection. - 16 -
3.2.9. Nanopore sequencing, genome assembly, and annotation - 17 -
4. RESULT - 18 -
4.1. Isolate and identify S. epidermidis commensals - 18 -
4.3. Testing probiotic properties of S. epidermidis. - 21 -
4.4. Biofilm detection. - 22 -
4.5. S. epidermidis produced electricity - 23 -
4.6. Genomic analysis of U2 S. epidermidis. - 25 -
4.7. Identification of new prebiotic selective - 28 -
5. DISCUSSION - 35 -
6. CONCLUSION - 39 -
7. FUTURE WORK - 39 -
Reference - 40 -
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