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研究生:柯彥如
研究生(外文):Yen-Ju Ke
論文名稱:台灣五葉松松針精油對致齲鏈球菌及其生物膜之抑制能力評估
論文名稱(外文):Evaluation of anti-bacterial and anti-biofilm formation abilities of the essential oil of Pinus morrisonicola Hayata
指導教授:溫曉薇
指導教授(外文):Hsiao-Wei, Wen
口試委員:胡淼琳蔣育錚
口試委員(外文):Miao-Lin HuYu-Jeng Jiang
口試日期:2016-07-18
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
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:2016
畢業學年度:104
語文別:中文
論文頁數:54
中文關鍵詞:五葉松抑菌生物膜抑菌圈
外文關鍵詞:Pinus morrisonicola HayataAntibacterialBiofilmInhibitory zone
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台灣五葉松(Pinus morrisonicola Hayata)為台灣特有之松科植物,其松針發芽主要為四月和九月,業者多以此時期之嫩芽製汁販售。本研究欲探討台灣五葉松松針精油是否能抑制齲齒菌的生長和抑制其生物膜形成。研究使用蒸餾萃取法(Distillation)萃取松針中精油物質,抑菌試驗使用24微量多孔盤進行(24-well cell culture microplates)與生物膜結晶紫染色法(crystal violet assay)和抑菌圈判定法(Inhibitory zone assay)。結果顯示,松針精油會影響齲齒菌Staphylococcus aureus、Streptococcus mutans、Streptococcus sanguinis、Streptococcus sobrinus的生長,與控制組相比,精油濃度達到16200 ppm時,Sta. aureus、S. mutans、S. sanguinis、S. sobrinus菌量剩0%、16%、15%、7.24% ; 精油濃度高至18000 ppm時,對Sta. aureus、S. sanguinis、S. sobrinus有全面抑制功效,雖然S. mutans的生長未完全被抑制,但其菌量已降量8.25%。松針精油會抑制Sta. aureus、S. mutans、S. sanguinis、S. sobrinus的生物膜形成,隨著精油濃度提高,其菌膜會逐漸變少;當濃度提高至16200ppm時,Sta. aureus、S. sanguinis、S. sobrinus已無菌膜存在;濃度再提升至18000ppm後,S. mutans菌膜的生成亦可完全抑制,判定抑制Sta. aureus、S. sanguinis、S. sobrinus抑制菌膜生成的精油濃度在16200ppm,S. mutans抑制菌膜生成的精油濃度則在18000ppm。在抑菌圈試驗結果,松針精油對Sta. aureus、S. sobrinus、S. sanguinis有較佳的抑菌效菌,其抑制圈大小分別為13.00 ± 0.0 mm、11.67 ± 0.19 mm 、10.33 ± 0.58 mm,而精油對S. mutans顯示較低抑菌範圍,其直徑僅6.33 ± 0.58 mm。由於民眾近年來逐漸有崇尚自然的想法,許多以天然植物為訴求的產品廣為人們所接受,本實驗結果可作為開發牙齒保健產品之參考依據,證實了五葉松松針精油抑制齲齒菌的功效,可應用到日常生活,如製作有抑菌效果的潔牙口香糖、潔牙清涼片、防蛀糖、天然漱口水等,提高臺灣原生五葉松之商業價值。

Pinus morrisonicola Hayata belongs to the pinaceae (pine family) and it is an endemic species in Taiwan. Furthermore, P. morrisonicola Hayata has been shown to have antioxidant and anti-inflammatory abilities. For purpose of evaluating whether essential oil of P. morrisonicola Hay. needle can inhibit bacterial growth and biofilm formation or not. This study applies distillation method to extract essential oil from pine needle. Antimicrobial activity was tested by MIC assay and inhibitory zone assay. Moreover, anti-biofilm formation was measured by using the dye crystal violet. The results showed that when 16200 ppm of essential oil was used, the bacteria of Streptococcus aureus , Streptococcus mutans , Streptococcus sanguinis , Streptococcus sobrinus decreased to 0%、16%、15%、7.24%, respectively. At 18000 ppm of essential oil, the growth of Sta. aureus, S.sanguinis, S. sobrinus were inhibited. Although S. mutans was not inhibited completely, the amount of bacteria was reduced to 8.25%; Moreover, at 16200 ppm of essenctial oil, biofilm formation of S. mutans, S. sanguinis, S. sobrinus was inhibited compelety. As the concentration of essential oil increased to 18000 ppm, S. mutans was inhibited completely. The result of inhibitory zone assay showed that essential oil had better ability to inhibit the growth of Sta. aureus, S. sanguinis, and S. sobrinus, and the inhibitory zones were 13.00 ± 00, 10.33 ± 0.58, and 11.67 ± 0.19 mm. However, the essential oil had smaller inhibitory zone for S. mutans which was 6.33 ± 0.58 mm. This study shows that the essential oil of P. morrisonicola Hayata can inhibit bacteria growth and can be applied to oral products in the future.

摘要……………………………………………………………………………..……i
Abstract…..………………………………………………………………………......ii
目次…………………………….………………………………………..……..iii 、iv
圖次…..…………………………….…………….……………………………….…v
表次…..…………………………….………...….………………………………..... vi

第一章、文獻回顧
一、微生物與齲齒關係之簡介
(一) 齲齒的致病過程…………………………………………………….…….……1
(二) 牙菌斑的生成………………………………………………………….……….2
(三) 齲齒菌的型態與特性……………………………………………………....…..3
(四) 抑制牙菌斑形成的方法與相關藥劑介紹……………………………….…….4
(五) 牙齒保健產品功效評估與比較……………………………………….……….6
(六) 天然物應用在齲齒預防上的相關研究………………………………………..7
二、台灣五葉松簡介
(一) 五葉松之形態特徵與生長特性…………………………………...….………..9
(二) 五葉松相關生理功效的研究………………………………………….………11
(三) 五葉松商業化產品與其功效介紹………………………………….…………13
(四) 五葉松的安全性評估………………………………………………………….14
三、精油之簡介
(一) 精油的分類與利用…………………………………………………………….16
(二) 精油的萃取方法……………………………………………………………….16
(三) 精油的抗菌性研究…………………………………………………………….18
(四) 台灣五葉松松針精油的研究………………………………………………….19
第二章、材料與方法
一、實驗材料
(一) 試驗材料…………………………………………………………….…………20
(二) 化學試藥………………………………………………………….……………20
(三) 實驗菌株………………………………………………………….……………21
(四) 實驗菌株使用之液體培養液與固體培養基…………………….……………22
(五) 儀器設備……………………………………………………………………….22
二、實驗方法
(一) 實驗架構…………………………………………………………….………....23
(二) 五葉松松針精油之萃取…………………………………….……….………...24
(三) 五葉松松針精油溶液之配製……………………………………….……........24
(四) 菌種的活化與保存/活菌時間與其它參數……………………………...…….24
(五) 五葉松精油對菌株最小抑菌濃度(MIC)與最小殺菌濃度(MBC)測定….......26
(六) 五葉松精油對菌株生物膜抑制之測定……………….……………...……….28
(七) 五葉松精油對菌株其抑菌圈測定……….………………………….…...……28
(八) 數據整理……………………………………….………………………...…….29
第三章、結果與討論
(一) 五葉松精油對菌株最小抑菌濃度(MIC)與最小殺菌濃度(MBC)測定…......30
(二) 五葉松精油對菌株生物膜抑制之測定………………………………...….…32
(三) 五葉松精油對菌株其抑菌圈測定……………………………………………33
第四章、結論與未來展望:…………………………………………..……...……...35
參考文獻……………………………………………………………………...…..…45
附錄……………………………….…………………….……………………..….…52


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