孫璐西臺灣大學:食品科技研究所陳姿虹Chen, Tzu-HungTzu-HungChen2010-05-112018-06-292010-05-112018-06-292009U0001-0508200923012800http://ntur.lib.ntu.edu.tw//handle/246246/182201綠茶中因含有豐富的兒茶素，但兒茶素水溶性不高，故市售綠茶飲料的加工過程無法將茶葉之兒茶素完全萃取出，同時亦會產生大量廢棄物，如：茶葉渣與茶梗等。據文獻指出經介質研磨的奈米級藥物，可有效提高其於水中的溶解度，並可完全利用物料而無廢棄物之產生。本研究將比較微奈米綠茶、傳統水萃綠茶與微奈米茶渣製成之綠茶產品在理化特性、化學成分與抗氧化活性之差異 。當研磨條件為0.3 mm釔鋯珠、3500 rpm轉速，水懸浮液之綠茶粉濃度為0.38% (w/v)，研磨時間90分鐘，粒數平均粒徑降至108 nm、體積平均粒徑降至7.83 μm；以相同操作條件研磨水萃綠茶之茶渣，微奈米茶渣之粒數平均粒徑降至114 nm、體積平均粒徑降至16.87 μm，顯示介質研磨降低綠茶粉與綠茶渣之粒徑均達到微奈米等級。理化性質方面，綠茶粉懸浮液、綠茶渣懸浮液經微奈米化後，pH值下降，可能由於酸性物質於研磨過程中釋放；色澤則不若水萃綠茶明亮與黃綠。以每克原料綠茶粉換算有效成分的釋放量，微奈米綠茶可釋放較大量兒茶素與茶胺酸，且介質研磨可能將EGCG轉變為EGC與gallic acid；微奈米綠茶可釋放111.43 mg總酚，顯著較水萃綠茶(80.31 mg)與微奈米茶渣(38.82 mg)為高；微奈米綠茶可利用之總膳食纖維含量為66.14 g/100 g綠茶粉乾重，顯著較水萃綠茶與微奈米茶渣加總(53.49 g/100 g綠茶粉乾重)為高。顯示微奈米綠茶可較傳統水萃綠茶與微奈米茶渣有效利用每克的綠茶粉原料。每克綠茶粉可提供之抗氧化能力作為比較基準，微奈米綠茶的抗氧化能力較水萃綠茶佳，其抗氧化能力主要來自酚類化合物，總酚含量與ORAC或Tlag之間的相關性(R2)均達0.88以上。於細胞抗氧化試驗，微奈米綠茶組有無PBS wash處理並不顯著影響防禦細胞被自由基侵襲的能力，顯示微奈米綠茶中化學成分組成較易進入細胞或與細胞膜表面有緊密連接，或是小粒徑的粒子較多，較容易與細胞作用而提供抗氧化能力。研究發現介質研磨可增加綠茶粉中有效成分的利用，使微奈米綠茶之總兒茶素、茶胺酸與總酚收率較水萃綠茶為高，而膳食纖維含量亦大幅提高；微奈米綠茶可開發為新型保健產品，作為國人補充抗氧化物質與膳食纖維來源之一。介質研磨方式可有效利用原料綠茶粉，或許將來可發展作為新式提取兒茶素或多酚類物質的方法。Green tea contains abundant catechins, but the extraction yield of catechins is low due to its poor water-solubility. Large amounts of wastes including tea leaves and stems are produced during the commercial processing of green tea beverages. Recently, nanotechnology via media milling has been reported to improve some poorly water-soluble drugs into stable, biologically active nanoparticlulate dispersion and enable the use of whole raw materials. This thesis aims at preparing micron/nano-sized green tea by media milling and finding the appropriate media-milling conditions. Physicochemical properties, chemical components and antioxidant activities will be compared among micron/nano-sized green tea suspension, green tea extract and micron/nano-sized green tea waste. he appropriate milling condition was to use 0.3 mm Y.Z.T. (yttria-stabilized zirconia) beads and 3500 rpm to mill green tea powder suspension (0.38%, w/v) for 90 min. The particle size of micron/nano-sized green tea was 108 nm of number mean diameter (Dnm) and 7.83 μm of volume mean diameter (Dvm); the particle size of micron/nano-sized green tea waste was 114 nm of Dnm and 16.87 μm of Dvm. The green tea powder suspension and waste suspension were ground to micron/nanoscales. or physicochemical properties, the Hunter L, a, b color results showed that green tea extract was more bright and yellow-green than the media-milled products; the media-milled products had lower pH that might be caused by the release of acidic substances. Comparison of the chemical components (on one-gram basis of dry green tea powder) among the micron/nano-sized green tea suspension, green tea extract and micron/nano-sized green tea waste showed that micron/nano-sized green tea could release more catechins and L-theanine. Some components such as EGCG would be converted into EGC and gallic acid. Micron/nano-sized green tea released 111.43 mg of total phenollics that was significantly higher than those of the green tea extract (80.31 mg) and micron/nano-sized green tea waste (38.82 mg). Total dietary fiber of micron/nano-sized green tea was found to be 66.14 g/100 g of dry green tea powder that was higher than the sum of those from green tea extract and micron/nano-sized green tea waste (53.49 g/100 g green tea powder). These results showed that micron/nano-sized green tea could release higher amounts of effective components than green tea extract.omparison of antioxidant activities among micron/nano-sized green tea suspension, green tea extract and micron/nano-sized green tea waste on one-gram basis of dry green tea powder showed that micron/nano-sized green tea had higher antioxidant activity such as ORAC and LDL oxidation. The presence of abundant phenolic components might lead to a high antioxidant activity, while the correlations (R2) between ORAC and total phenolics as well as Tlag and total phenolics were above 0.88. In CAA assay, with or without PBS wash treatment did not affect the antioxidant activity of micron/nano-sized green tea significantly. It showed that the chemical components in micron/nano-sized green tea were easily uptake by cells or bound to the surface of cell membrane, or there might be some other smaller particles interacting with cells to present antioxidant activity.his study showed that media-milling process could release high amounts of effective components. The yields of total catechins, L-theanine, total phenolics and dietary fiber from micron/nano-sized green tea were higher than those from green tea extract and therefore, micron/nano-sized green tea was expected to be a new green tea product with rich source of antioxidant and dietary fiber. Media-milling process could help collect effective components from green tea powder, it is also possible develop a new method to collect catechins and other phenolic components.口試委員審定書 i誌 ii文摘要 iii文摘要 iv錄 vi次 xi次 xiii、前言 1、文獻整理 2、綠茶的簡介 2一) 茶的分類與命名 2二) 綠茶的製程與原理 2.綠茶的製程 2.綠茶粉的加工方法 4 三) 台灣茶業的現況與特色 5.台灣茶業面臨國際化競爭 5.台灣茶飲料的現況 5.製作茶飲料之廢棄物產量 5、綠茶茶菁的化學成分 8一) 多酚類化合物 10二) 植物鹼 12三) 蛋白質與游離胺基酸 12四) 碳水化合物 13五) 植物色素 13六) 脂質與脂肪酸 14七) 微量成分 14、綠茶的生理功效 15一) 降低心血管疾病罹患率 15二) 預防癌症 16三) 控制體重 16四) 口腔健康 16、奈米科技 20一) 奈米科技之定義 20二) 奈米粒子之特性 21.表面效應 22.尺寸效應 22.量子效應 22.交互作用 23三) 奈米材料之製備方式 23.由上而下 23.由下而上 27四) 奈米材料之量測方式 27.掃描式電子顯微鏡 28.穿透式電子顯微鏡 28.原子力顯微鏡 28.雷射粒徑分析儀 28.表面電位分析儀 31五) 奈米科技於食品與藥物之應用 32.奈米科技於食品之應用 32.奈米科技於藥物之應用 33、氧化壓力與常見抗氧化物質 33一) 氧化壓力與疾病 33.脂質 34.蛋白質 34.DNA 34二) 自由基來源 35三) 氧化壓力之測定 35.偵測物質被氧化的程度 35.偵測物質的電子自旋共振 (Electron spin resonance) 37.偵測物質的螢光 (Fluorescent) 或冷光 (Luminescent) 變化 37.偵測物質的抗氧化能力 37、研究目的與實驗架構 40、研究目的 40、實驗架構 41、材料與方法 42、實驗材料 42、實驗細胞株 42、實驗藥品與溶劑 42一) 化學藥品 42二) 溶劑 44、細胞實驗培養基配方 44一) 毒性實驗 44.Phosphate-buffered saline (PBS) 44.Serum free WME (SF-WME) 45.Complete WME (c-WME) 45.MTT溶液 45二) 抗氧化實驗 45.Phosphate-buffered saline (PBS) 45.Antioxidant treatment medium 45.Complete WME (c-WME) 45.Oxidant treatment medium 46、儀器設備 46一) 一般儀器設備 46二) 介質研磨相關儀器設備 47三) 化學分析相關儀器設備 47四) 細胞實驗相關儀器設備 48、實驗方法 49一) 綠茶粉原料之一般成分分析 49.粗蛋白 (Crude protein) 49.粗脂肪 (Crude fat) 50.膳食纖維 (Dietary fiber) 51.1非水溶性膳食纖維 (Insoluble dietary fiber, IDF) 51.2水溶性膳食纖維 (Soluble dietary fiber, SDF) 51.水分 (Moisture) 52.灰分(Ash) 52.無氮抽出物含量 (Nitrogen free extract, NFE) 53二) 樣品製備 53.水萃綠茶 53.介質研磨 54三) 粒徑分析 55.粒徑分布測定 55.形態觀察 56四) 穩定性分析 56.表面電位之測定 56.濁度分析 57五) 物性分析 57.pH值 57.茶湯水色測定 57六) 化性分析 57.兒茶素含量之測定 57.茶胺酸之測定 58.總酚類化合物含量之測定 59. 膳食纖維含量之測定 59七) 抗氧化活性分析 60.氧自由基吸收能力之測定 60.1 樣品前處理 60.2 ORAC 測定 60.抑制銅離子誘導人類低密度脂蛋白氧化反應 61.1樣品前處理 61.2以超高速離心方法分離取得LDL 61.3透析LDL 61.4膽固醇濃度之測定 61.5抑制銅離子誘導LDL氧化之測定 62.細胞抗氧化活性之測定 63.1樣品前處理 63.2HepG2細胞培養條件與繼代培養 63.3細胞保存 63.4細胞解凍 63.5細胞存活率分析⎯MTT assay 64.6細胞抗氧化活性之測定 64、統計分析 65、結果與討論 66、水萃綠茶之製備與一般成分分析 66一) 水萃綠茶之製備 66二) 一般成分分析 66、綠茶粉之適當研磨條件 67一) 操作條件之探討 67.轉速之影響 67.固形物含量之影響 69.鋯珠大小之影響 70.研磨時間之影響 70二) 平均粒徑分布 72.綠茶粉水懸浮液與微奈米綠茶 72.綠茶茶渣水懸浮液與微奈米茶渣 72.離心後之微奈米綠茶與微奈米茶渣 73三) 形態觀察 77.肉眼觀察 77.光學顯微鏡觀察 77.穿透式電子顯微鏡觀察 79、穩定度分析 82一) 濁度 82二) 表面電位 84、理化性質 86一) pH值 86二) Hunter L, a, b值 86、化學成分 88一) 兒茶素與茶胺酸含量 88二) 總酚含量 93三) 膳食纖維含量 95、抗氧化活性 97一) 氧自由基吸收能力分析(ORAC) 97二) 抑制銅離子誘導人類LDL氧化分析 101三) 細胞抗氧化活性分析 (CAA) 104.細胞存活率分析⎯MTT assay 104.細胞抗氧化能力測定⎯CAA assay 107、結論 113、參考文獻 115、中文文獻 115、西文文獻 116、附錄 123application/pdf23563839 bytesapplication/pdfen-US微奈米綠茶介質研磨粒徑分布兒茶素膳食纖維抗氧化活性Micron/nano-sized green teamedia millingparticle size distributioncatechinsdietary fiberantioxidant activitythesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182201/1/ntu-98-R96641022-1.pdf