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研究生:鄭宇婷
研究生(外文):CHENG, YU-TING
論文名稱:乳鐵蛋白水解物複合乳酸鏈球菌素 對皮脂腺毛囊致病菌之功效評估
論文名稱(外文):Studies on the Antimicrobial Function of Lactoferrin Hydrolysates, Nisin and Their Synergistic Effects on Skin Lesion Pathogens
指導教授:林世斌林世斌引用關係陳莉臻陳莉臻引用關係
指導教授(外文):LIN, SHIH-BINCHEN, LI-CHEN
口試委員:林恩仕曹博宏林仲聖孫舜國林明靜
口試委員(外文):LIN, EN-SHYHTSAO, PO-HUNGLIN, CHUNG-SAINTSUN, SHUN-KUOLIN, MING-CHING
口試日期:2017-07-28
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:食品科學系碩士班
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:75
中文關鍵詞:尋常性痤瘡複合抗菌療法乳鐵蛋白水解物乳酸鏈球菌素槲皮素
外文關鍵詞:acne vulgariscombination antibacterial therapylactoferrin hydrolysatesnisinquercetin
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中文摘要 I
英文摘要 II
謝誌 IV
目錄 IV
表目錄 VIII
圖目錄 IX
附錄目錄 X
縮寫表 XI
壹、前言 1
貳、文獻整理 2
一、尋常性痤瘡 (acne vulgaris) 2
(一) 致病機理 2
(二) 痤瘡致病菌及其相關研究 2
1. 痤瘡致病菌 2
(1)痤瘡丙酸桿菌 (Propionibacterium acnes, PA) 2
(2)表皮葡萄球菌 (Staphylococcus epidermidis, SE) 3
(3)皮屑芽孢菌 (Malassezia furfur, MF) 3
2. 生物膜 (biofilm) 3
3. 脂解酶 (lipase) 4
4. 發炎反應 (inflammation) 4
5. 活性氧 (reactive oxygen species, ROS) 4
(三) 現行療法及其瓶頸 5
1. 抗生素 (antibiotic) 5
2. 類視黃醇 (retinoid) 5
3. 過氧化苯 (benzoyl peroxide) 6
4. 壬二酸 (azelaic acid) 6
5. 荷爾蒙療法 (hormonal therapy) 6
6. 有機酸類 (organic acid) 6
7. 複合療法 (combination therapy) 7
二、乳鐵蛋白及其水解物 7
(ㄧ) 乳鐵蛋白 (lactoferrin, Lf) 7
1. 結構及特性 7
2. 抗微生物活性 8
(二) 乳鐵蛋白水解物 (lactoferrin hygrolysates, LfH) 8
(三) 乳鐵蛋白素 (lactoferricin, Lfcin) 9
1. 結構及特性 9
2. 抗微生物活性 9
三、乳酸鏈球菌素 (nisin) 9
(一) 乳酸鏈球菌素之介紹 9
(二) 結構與特性 10
(三) 抗菌機制 10
(四) 應用與限制 10
四、抗菌胜肽 (antimicrobial peptide, AMP) 11
(一) 抗菌胜肽之介紹 11
(二) 結構與特性 11
(三) 抗菌機制 11
(四) AMP 與尋常性痤瘡 12
五、槲皮素 (quercetin) 12
(一) 槲皮素之介紹 12
(二) 結構與特性 12
(三) 生理活性 13
參、實驗架構 14
肆、材料與方法 15
一、實驗材料 15
(一) 試驗物質 15
(二) 試驗菌株 15
(三) 試驗細胞 15
二、藥品、試劑及其配製方法 15
(一) 藥品及試劑 15
(二) 藥品配製方法 16
1. 緩衝溶液 16
2. 十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳 (sodium dodecyl sulfate-polyacryamide del electrophoresis, SDSPAGE) 相關試劑 17
3. 紅黴素溶液 18
4. 電顯固定液 (fixative solution) 18
5. 0.1% 結晶紫染劑 (crystal violet dye) 18
(三) 培養基及其製備方法 18
1. 培養基相關成份 18
2. MF 培養相關培養基之製備 19
(四) 細胞實驗相關成份之配製 19
1. 藥品及試劑 19
2. DMEM細胞培養液 20
3. 10 mM 磷酸鹽緩衝溶液 20
4. MTT 試劑 20
5. Griess 試劑 20
三、實驗儀器 21
四、實驗方法 22
(一) 乳鐵蛋白水解物之製備 22
(二) SDS-PAGE 膠體電泳 22
1. 樣品製備 22
2. 膠體電泳 22
3. 膠片染色與退染 22
(三) 試驗菌株之活化與保存 22
1. 菌株活化與培養 22
2. 菌株之保存 23
(四) 最低抑制濃度 (minimum inhibitory concentration, MIC) 之測試 23
1. 試驗菌之菌數測定 23
2. 菌體遲滯期之測定 23
3. MIC定義與測定方法 23
(五) 協同抗菌試驗 24
(六) 鉀離子流失試驗 (potassium efflux assay) 24
(七) 掃描式電子顯微鏡 (scanning electron microscope, SEM) 之觀察 24
1. 樣品製備 24
2. SEM之拍攝 25
(八) 抗生物膜試驗 25
1. 生物膜形成時間之測定 25
2. 抑制生物膜之試驗 25
(九) 氧自由基吸收能力 (oxygen radical absorption capacity, ORAC) 25
(十) 試驗細胞株之活化與培養 26
1. 細胞保存凍管之解凍 26
2. 細胞繼代與培養 26
(十一) 細胞存活率測定 26
(十二) 一氧化氮分泌量測定 26
(十三) 統計方法 27
伍、結果 28
一、LfH 之分子量分佈 28
二、抗痤瘡致病菌評估 28
(一) PA 28
1. 抗菌能力試驗 28
2. 鉀離子流失試驗 28
3. SEM之菌體型態變化觀察 28
(二) SE 29
1. 抗菌能力試驗 29
2. 鉀離子流失試驗 29
3. SEM之菌體型態變化觀察 29
(三) MF 29
1. 抗菌能力試驗 29
2. 鉀離子流失試驗 29
3. SEM之菌體型態變化觀察 30
三、抗生物膜能力評估 30
(一) 預防生物膜形成之能力測定 (prevention) 30
(二) 清除已形成之生物膜能力測定 (scavenging) 30
四、氧自由基吸收能力評估 31
五、抑制 NO 生成能力評估 31
陸、討論 32
一、LfH 之分子量分佈 32
二、抗痤瘡致病菌評估 32
(一) LfH 之抗菌效果 32
(二) Nisin 之抗菌效果 32
(三) Quercetin 之抗菌效果 33
(四) LfH、nisin 及 quercetin 協同抗菌效果 33
1. 鉀離子流失試驗 33
2. SEM 之菌體型態觀察 34
(五) LfH、nisin 及 quercetin 協同作用機制之探討 35
三、抗生物膜能力評估 36
(一) Lf 之抗生物膜效果 36
(二) LfH 之抗生物膜效果 36
四、氧自由基吸收能力評估 36
(一) LfH 之氧自由基吸收能力 37
(二) Quercetin 之氧自由基吸收能力 37
五、抑制 NO 生成能力評估 37
柒、結論 38
捌、參考文獻 39
玖、表 50
拾、圖 51
拾壹、附錄 62
表一、試驗物質對PA、SE 及 MF 的抗菌活性 50
圖一、乳鐵蛋白及其水解物之分子量分布 51
圖二、PA 經不同試驗物質作用後的鉀離子流失情形 52
圖三、SE 經不同試驗物質作用後的鉀離子流失情形 53
圖四、MF 經不同試驗物質作用後的鉀離子流失情形 54
圖五、PA 施予試驗物質處理之 SEM 圖 55
圖六、SE 施予試驗物質處理之 SEM 圖 56
圖七、MF 施予試驗物質處理之 SEM 圖 57
圖八、Lf 與 LfH 預防 PA 及 SE 生物膜形成之能力測定 58
圖九、Lf 與 LfH 清除 PA 及 SE 已形成之生物膜能力測定 59
圖十、LfH、nisin、quercetin 及三者合併施用對 RAW 264.7 細胞存活率
之影響 60
圖十一、RAW 264.7 經熱失活之 PA 處理後,以 LfH、nisin、quercetin 及三者合併施用對 RAW 264.7 其 NO生成之影響 61
附表一、乳鐵蛋白經胃蛋白酶水解所釋出之胜肽片段 62
附表二、乳鐵蛋白之胺基酸組成比例 65
附圖一、Lfcin 胺基酸組成結構圖 66
附圖二、Nisin 胺基酸組成結構圖 67
附圖三、Nisin 形成孔洞之模型 68
附圖四、抗菌胜肽藉由桶壁模式進行細胞膜的破壞 69
附圖五、抗菌胜肽藉由環狀孔洞模式進行細胞膜的破壞 70
附圖六、抗菌胜肽藉由地毯模式進行細胞膜的破壞 71
附圖七、槲皮素之化學結構 72
附圖八、合成Lfcin之檢測報告 73
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