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研究生:邱于珍
研究生(外文):Yu-Chen Chiu
論文名稱:超微細化研磨技術對竹葉萃出物類黃酮含量及抗菌活性之影響
論文名稱(外文):Effect of ultrafine milling on total flavonoids content and antimicrobial activity of bamboo leaf extracts
指導教授:賴喜美賴喜美引用關係
指導教授(外文):Hsi-Mei Lai
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:76
中文關鍵詞:竹葉粗萃液超微細化研磨類黃酮抑菌活性
外文關鍵詞:bamboo leaf extractultrafine-millingflavonoidsantimicrobial activity
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中文摘要
本研究利用超微細化研磨技術進行竹葉之微細化及萃取,探討研磨後的竹葉濾渣之粒子粒徑、粗萃物之化學成分與生物活性物質,以及竹葉粗萃液對細菌生長抑制能力之評估,並針對竹葉中抑菌物質進行初步判定。試驗中所使用之研磨萃取溶劑包括蒸餾水及60%乙醇。結果顯示,在水或60%乙醇中研磨竹葉1 hr後,其平均粒徑及d90顯著下降,延長研磨時間,其粒徑雖持續降低但趨勢漸緩,4 hr研磨後,竹葉樣品平均粒徑為12 μm,d90為25 μm,已達細胞破壁之目標。以水為溶劑,經研磨2 hr之竹葉,其水粗萃液之可溶性糖及總類黃酮含量顯著提高,但總酚類含量並無顯著差異。當以60%乙醇為溶劑進行研磨萃取時,可溶性糖含量減少,但總類黃酮含量顯著提高為1.76 mg catechin equivalent /g竹葉粗粉,顯示60%乙醇為總類黃酮之良好萃取溶劑。乙醇粗萃液之DPPH自由基清除力高於水粗萃液。每克竹葉粗粉含有總氰氫酸含量相當於2.5 μg HCN,經超微細化處理後,竹葉濾渣顆粒之總氰氫酸含量提高為3-4 μg HCN;而竹葉粗萃液之總氰氫酸含量則降低為0.17-0.21 μg HCN,且不受超微細化處理之影響。利用矽膠管柱進行粗萃液初步分離,以高效液相層析進行70%甲醇沖提液之組成分析,結果顯示有三個主要吸收峰,依200-400 nm之UV掃描吸收光譜圖,可能為類黃酮化合物,經質譜儀判定其中兩個吸收峰所代表之化合物可能為異葒草苷或葒草苷及異牡荊苷或牡荊苷。竹葉粗萃物進行抑菌試驗之結果顯示,於濾紙擴散法、孔盤擴散法及序列稀釋法試驗中,以60%乙醇研磨萃取之竹葉粗萃液10倍稀釋倍率對Bacillus cereus及Staphylococcus aureus的生長有顯著的抑制效果,但對Escherichia coli及Salmonella typhimuriun菌之生長卻無顯著抑制效果。在12 hr內,10倍稀釋倍率之乙醇粗萃液便可抑制菌落數增加。以菌落之大小能比較出竹葉乙醇萃液對S. aureus之抑制濃度。而以水為研磨萃取溶劑之竹葉粗萃物,即使以原粗萃液之含量,對本實驗4種測試菌種則均無抑制生長之效果。整體而言,臺灣具有豐富的竹類資源,而竹葉以60%乙醇作為萃取溶劑及超微細化研磨處理有助於提升其類黃酮含量及其對Bacillus cereus 及 Staphylococcus aureus的抑菌活性,因此,竹葉粗萃液作為有抑菌作用之食品添加物之潛力。
Abstract
This study applied ultrafine-milling technology on bamboo leaf. The post-milled particle size of bamboo leaf was examined. The chemical content, bioactive material of bamboo leaf extract, and its ability to suppress bacterial growth were investigated. The antimicrobial material in bamboo leaf was also preliminarily determined. The extracting solvent used in this study included water and 60% ethanol. After a 1 hour ultrafine-milling treatment, using either water or 60% ethanol, the mean particle size and d90 of bamboo leaf decreased obviously. With even longer ultrafine-milling treatment, the mean particle size continued to decrease but changed more slowly. After a 4 hour ultrafine-milling treatment, the mean particle size and d90 were 12 μm and 25 μm, reaching the goal of destructed cell wall. After a 2 hour ultrafine-milling treatment with water, the total soluble carbohydrates and total flavonoids of bamboo leaf extract elevated obviously, but the total phenolics showed no apparent differences. Under the same milling condition except using 60% ethanol, the total soluble carbohydrates of bamboo leaf extract decreased, but the total flavonoids increased to 1.76 mg catechin equivalent /g ground bamboo leaf powder(GP), suggesting that 60% ethanol is a good extracting solvent of flavonoids. The DPPH scavenging capacity of ethanol extract was the highest than that of water extract under all the conditions in this study. The result of total cyanide content analysis showed that dried ground bamboo leaf powder contained 2.5 μg HCN. After ultrafine-milling treatment, the total cyanide content of residue of bamboo leaf increased to 3-4 μg HCN/g GP. But the total cyanide content of bamboo leaf extract decreased obviously after rotary evaporation and cetrifuge treatment. Bamboo leaf extract composition analysis performed by silica gel column, 70% methanol elution and High-performance liquid chromatography (HPLC) demonstrated that there were three major peaks on the HPLC chromatograms of bamboo leaf extract. According to the UV scanning spectra of bamboo leaf extract from 200 to 400 nm wavelength, the components were very likely to be flavonoids, while the Mass spectrometry (MS) was determined as isoorientin (or orientin) and isovitexin (or vitexin). Antimicrobial assay was performed by conducting agar diffusion assay, serial dilution method, and hole-plate diffusion method. The results showed that bamboo leaf ethanol extract can apparently suppress the growth of Bacillus cereus and Staphylococcus aureus at the dilution of 10, but it was ineffective for Escherichia coli and Salmonella typhimuriun. Time-kill method showed that bamboo leaf ethanol extract at the dilution of 10 could persist antimicrobial effects within 12 hours. The inhibitory concentration of bamboo leaf ethanol extract for S. aureus was compared by using colony size method. On the contrary, bamboo leaf water extract, even at the original concentration, had no antimicrobial effect for all the 4 kinds of bacteria in this study.
Bamboo leaf treated with ultrafine-milling using 60% ethanol as extracting solvent could achieve higher flavonoids content and antimicrobial activity for Bacillus cereus and Staphylococcus aureus. According to this study and considering the fact that there are abundant bamboo resources in Taiwan, bamboo leaf extract has great potential to be excellent food additives as antimicrobial agent.
目 錄
中文摘要 ------------------------------------------------------------------------------------------- I
Abstract ------------------------------------------------------------------------------------------- II
目錄 ------------------------------------------------------------------------------------------------ IV
圖目錄 -------------------------------------------------------------------------------------------- VII
表目錄 ----------------------------------------------------------------------------------------------IX
第一章、文獻整理 -------------------------------------------------------------------------------- 1
一、天然的竹類資源 ----------------------------------------------------------------------- 1
二、竹葉萃出物 ----------------------------------------------------------------------------- 3
(一) 化學組成 --------------------------------------------------------------------------- 3
(二) 生理與藥理活性 ------------------------------------------------------------------ 9
(三) 安全評估 -------------------------------------------------------------------------- 17
(四) 食品及藥妝上之應用------------------------------------------------------------ 18
三、微細化研磨技術 ---------------------------------------------------------------------- 21
(一) 球磨研磨之原理 ---------------------------------------------------------------- 21
(二) 微細化研磨技術於食品領域之應用 ---------------------------------------- 25
四、研究目的 ------------------------------------------------------------------------------ 26
第二章、材料與方法 --------------------------------------------------------------------------- 29
一、試驗架構 ------------------------------------------------------------------------------ 29
二、試驗材料 ------------------------------------------------------------------------------ 30
(一) 竹葉原料 ------------------------------------------------------------------------- 30
(二) 菌種與培養基配製 ------------------------------------------------------------- 30
(三) 試藥 ------------------------------------------------------------------------------- 30
三、試驗方法 ------------------------------------------------------------------------------- 31
(一) 一般成分分析 ------------------------------------------------------------------- 31
(二) 微細化研磨之樣品製備 ------------------------------------------------------- 32
(三) 樣品粒徑分析 ------------------------------------------------------------------- 33
(四) 化學成分測定 ------------------------------------------------------------------- 33
(五) 總氰化物測定 ------------------------------------------------------------------- 34
(六) 竹葉粗萃液之液相層析分析 --------------------------------------------- ----36
(七) 抑菌試驗 ------------------------------------------------------------------------- 37
(八) 統計分析 ------------------------------------------------------------------------- 39
第三章、結果與討論 ---------------------------------------------------------------------------- 40
一、竹葉之一般成分分析 ------------------------------------------------------------------ 40
二、超微細化研磨對竹葉粒徑及竹葉萃出物之生物活性成分之影響 ----------- 41
(一) 超微細化研磨對竹葉樣品之顆粒形態及粒徑分布 ---------------------- 41
(二) 超微細化研磨對竹葉粗萃液之化學成分的影響 ------------------------- 44
(三) 超微細化研磨對竹葉濾渣及粗萃液之總氰化物的影響 ---------------- 48
(四) 竹葉粗萃液之高效液相層析管柱分析 ------------------------------------- 51
(五) 結語 ------------------------------------------------------------------------------- 55
三、綠竹葉萃出物抑菌活性之評估------------------------------------------------------- 55
(一) 竹葉水萃液及乙醇萃液之抑菌表現 ---------------------------------------- 56
(二) 竹葉乙醇萃液對S. aureus及 B. cereus的生長抑制 -------------------- 60
(三) 竹葉粗萃液之最低抑制濃度(MIC)測試 ----------------------------------- 62
(四) 竹葉粗萃液之化學成分與其抑菌活性之比較 ---------------------------- 65
(四) 結語 ------------------------------------------------------------------------------- 66

第四章、結論 ------------------------------------------------------------------------------------ 67
第五章、參考文獻 -------------------------------------------------------------------------- 68
圖 目 錄
圖一、黃酮類化合物基本結構圖 -------------------------------------------------------------------- 4
圖二、黃酮類化合物的主要類型及結構圖 ------------------------------------------------------- 5
圖三、毛金竹之竹葉萃出物中四種主要碳苷黃酮之結構 ------------------------------------ 6
圖四、行星式球磨機之結構圖 ---------------------------------------------------------------------- 22
圖五、行星式球磨機中球磨罐公轉軸轉動方向與自轉軸轉動方向相反及相同
時,研磨球相對速度向量的差異 ------------------------------------------------------- 23
圖六、衝擊式碾碎機之結構示意圖 --------------------------------------------------------------- 24
圖七、氣流粉碎機之結構示意圖 ------------------------------------------------------------------- 25
圖八、超微細化竹葉樣品之掃瞄式電子顯微照相圖 ---------------------------------------- 42
圖九、竹葉樣品在水中超微細化處理後之平均體積粒徑分布及累計體積粒徑
分布 ---------------------------------------------------------------------------------------------- 43
圖十、含氰醣苷之植物降解氰醣苷而生成氰氫酸之途徑 ---------------------------------- 49
圖十一、竹葉水粗萃液及乙醇粗萃液的高效液相層析圖 ---------------------------------- 52
圖十二、竹葉乙醇粗萃液之HPLC分析圖中主要吸收峰之掃描吸收圖譜
(200-400 nm) ------------------------------------------------------------------------------- 53
圖十三、不同研磨時間之水粗液及乙醇粗萃,經HPLC分析所得三個主要
吸收峰之相對信號強度百分比圖 --------------------------------------------------- 54
圖十四、不同研磨時間之竹葉乙醇粗萃液對金黃色葡萄球菌及仙人掌桿菌之
抑菌結果 ---------------------------------------------------------------------------------- 56
圖十五、以孔盤擴散法觀察不同研磨時間及不同稀釋倍率之竹葉乙醇粗萃液
對金黃色葡萄球菌及仙人掌桿菌之抑菌結果 ---------------------------------- 57
圖十六、以序列稀釋法觀察不同研磨時間及不同稀釋倍率之竹葉乙醇粗萃
液對金黃色葡萄球菌、仙人掌桿菌、大腸桿菌及沙門氏桿菌之抑
菌結果 ---------------------------------------------------------------------------------------- 58
圖十七、不同濃度之竹葉乙醇粗萃液對金黃色葡萄球菌及仙人掌桿菌生長
抑制之影響 -------------------------------------------------------------------------------- 61
圖十八、金黃色葡萄球菌及仙人掌桿菌在不同稀釋倍率之竹葉乙醇粗萃液
的生長曲線 ------------------------------------------------------------------------------- 62
圖十九、不同濃度之竹葉乙醇粗萃液對金黃色葡萄球菌之菌落大小的影響 ------- 64
圖二十、金黃色葡萄球菌在不同濃度之竹葉乙醇粗萃液之菌落大小 ----------------- 64

表 目 錄
表一、臺灣六種主要經濟竹種的資料整理 ------------------------------------------------------- 1
表二、不同竹種中以不同溶劑萃取出來的竹葉萃出物之抑菌結果 ---------------------- 13
表三、4種常見引發食品中毒之汙染菌 --------------------------------------------------------- 28
表四、HPLC-PDA之流洗梯度條件 ----------------------------------------------------------------- 36
表五、綠竹竹葉粗粉之一般成分分析 ------------------------------------------------------------ 40
表六、竹葉粗粉於水及60%乙醇中研磨0至4 hr後之體積粒徑分布 ----------------- 44
表七、不同研磨時間對竹葉水粗萃液及乙醇粗萃液中可溶性糖、總酚類、總
類黃酮含量及DPPH自由基清除能力之影響 -------------------------------------- 46
表八、研磨2小時對竹葉水粗萃液及乙醇粗萃液之可溶性糖、總酚類、總類
黃酮含量及DPPH自由基清除能力相較於未研磨者之提升程度 ------------- 47
表九、竹葉粗粉與經超微細化之竹葉濾渣及竹葉粗萃液的總氰氫酸含量 ----------- 50
表十、不同研磨時間之水粗萃液及乙醇粗萃液於HPLC分析圖譜中三個主要
吸收峰之相對信號強度百分比 ---------------------------------------------------------- 54
表十一、竹葉水粗萃液與乙醇萃液之總酚類、總類黃酮與總氰氫酸含量及其
抑菌活性 ----------------------------------------------------------------------------------- 65
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