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研究生:吳承彥
研究生(外文):CHENG-YEN WU
論文名稱:由脫脂大豆粉提取與分離大豆異黃酮素
論文名稱(外文):Isolation and Separation of Isoflavones from Defatted Soy Flakes
指導教授:賴世明賴世明引用關係
指導教授(外文):SHIH-MING LAI
學位類別:碩士
校院名稱:國立雲林科技大學
系所名稱:工業化學與災害防治研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:139
中文關鍵詞:脫脂大豆粉大豆異黃酮提取與分離液相萃取管柱層析
外文關鍵詞:Defatted soy flakesIsoflavonesIsolation and separationLiquid phase extractionColumn chromatography
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異黃酮為大豆中的重要成份,因其具有預防癌症與骨質疏鬆症等功效,且有助於膽固醇之降低,故可作為健康食品及醫療藥品。然而,大豆異黃酮在大豆食品的含量卻甚為稀少(0.1~0.3%)。大豆異黃酮可分為葡萄糖配醣體與無葡萄糖配醣體兩種化學形式,97~98%的大豆異黃酮是以葡萄糖配醣體存在,但卻以無葡萄糖配醣體具有較高的生理活性。脫脂大豆粉為生產大豆沙拉油的副產品,本研究以脫脂大豆粉為原料進行異黃酮的製備生產,藉以提高其附加價值。本研究的目的即在於發展一經濟有效且適合大規模化的提取以及分離技術,可以自脫脂大豆粉中製備生產高純度(>50%)與高回收率(>80%)的異黃酮。
研究結果顯示,首先在初步萃取方面,採用提取比例1:5(100g脫脂大豆粉:500ml 80%乙醇水溶液)萃取3小時為粗提條件下,可得粗提率約為10%以及總異黃酮素的純度約介於2.0~2.5%。接著將所得粗提液以1.2g烘乾物轉溶於100ml純水為較佳的轉溶條件(異黃酮素濃度介於0.2~0.3mg/ml),可得總異黃酮素的純度約介於2.0~2.3%,損失率約為6~30%。在提取程序方面,本研究所採用的提取濃縮方法包括液相萃取法與吸附及脫附層析法(固相萃取法)。批次式液相萃取法操作步驟較為簡便,採用10%正丁醇與90%乙酸乙酯的混合溶劑,以萃取條件(水油比例為1:2、萃取溫度為55℃以及萃取時間為2小時)所得最佳液相萃取結果可知,總異黃酮素的純度可提升至約35%,損失率約為30%以內。連續式固相萃取法的步驟則較為繁瑣,由最佳吸附與階段脫附條件所得結果可知,中度極性材質的XAD-7HP管柱可得總異黃酮素純度約31%,回收率為85%;而中低極性吸附材質的XAD-4管柱則可將總異黃酮素純度更提昇至約58%,回收率可達89%。由兩種方法在純度與回收率的比較得知,純度的高低順序為XAD-4管柱固相萃取>液相萃取>XAD-7HP管柱固相萃取,而回收率的高低順序方面,仍為XAD-4管柱固相萃取最高>XAD-7HP管柱固相萃取>液相萃取。
Isoflavones are considered to be important fractions in soybean. They possess the functions of preventing cancer and osteoporosis and reducing the cholesterol levels in the human body, so they can be used as nutrition foods and medicines. However, isoflavones are found in very low abundance (0.1~0.3%) in soy-foods. Isoflavones can be categorized into two groups: glycosides and aglycones. 97~98% of isoflavones are glycosides, but aglycones may have more biological activity than their respective glycosides. Defatted soy flakes (DSF), the by-product from the production of soybean oil, were used as the starting material for the production of isoflavones in this study, from which their added-values can be increased. The objective of this study is to develop an efficient and easy-to-scale-up technique for the isolation and separation of isoflavones from DSF that contains 50+% purity and 80+% recovery.
The results show that, first, the DSF was extracted under an optimal condition (using 500ml 80% ethanol aqueous for 3h per 100g DSF). The extraction yield was about 10% and the purity of isoflavones was found to be about 2.0~2.5%. Then, the extracts were dissolved in water under an optimal condition (100ml water per 1.2g extracts and the concentraction range of isoflavones was about 0.2~0.3mg/ml), the purity of isoflavones was about 2.0~2.3% and the loss of isoflavones was about 6.0~30%. The isolation methods used in this study include the liquid-phase extraction method and the solid-phase extraction method. For the liquid-phase extraction method operated in a batch mode, the procedure is simple and easy to proceed. Using the mixed solvent of 10% 1-butanol and 90% ethyl acetate and operated under an optimal extraction condition(water/oil ratio as 1:2, extraction temperature as 55℃ and extraction time as 2 hours), the purity of total isoflavones was raised to be about 35% and the loss of total isoflavones was under 30%. For the solid-phase extraction method operated in a continuous mode, the procedure is more complicated and tedious. Under the optimal adsorption and desorption conditions, for the column packed with the intermediate polar XAD-7 HP resins, the purity of total isoflavones was raised to be about 31% and the recovery of total isoflavones was about 85%. For the column packed with the low polar XAD-4 adsorbents, the purity of total isoflavones was raised to be about 58% and the recoveries of total isoflavones higher than about 89%. In comparison, the order of higher purity was found to be XAD-4 solid-phase extraction > liquid-phase extraction > XAD-7HP solid-phase extraction and the order of higher recovery was found to be XAD-4 solid-phase extraction > XAD-7HP solid-phase extraction > liquid-phase extraction.
中文摘要 ------------------------------------------------------------------------ i

英文摘要 ------------------------------------------------------------------------ iii

誌謝 ------------------------------------------------------------------------ v
目次 ------------------------------------------------------------------------ vi
表目次 ------------------------------------------------------------------------ viii

圖目次 ------------------------------------------------------------------------ ix

一、 緒論------------------------------------------------------------------ 1
1.1 前言------------------------------------------------------------------ 1
1.2 研究動機----------------------------------------------------------- 3
1.3 研究目的及方法-------------------------------------------------- 3
二、 文獻回顧與理論部分--------------------------------------------- 6
2.1 文獻回顧----------------------------------------------------------- 6
2.1.1 大豆異黃酮的概述------------------------------------------------ 6
2.1.2 分析方法----------------------------------------------------------- 9
2.1.3 分離純化方法----------------------------------------------------- 11
2.2 理論部份------------------------------------------------------------ 14
2.2.1 正相/逆相分離機制----------------------------------------------- 14
2.2.2 製備級液相層析的操作模式------------------------------------ 16
2.2.3 實驗設計理論----------------------------------------------------- 21
三、 實驗部分------------------------------------------------------------ 24
3.1 實驗材料與儀器--------------------------------------------------- 24
3.2 實驗步驟------------------------------------------------------------ 29
3.2.1 異黃酮素的HPLC定量分析------------------------------------ 29
3.2.1.1 逆相HPLC分析--------------------------------------------------- 29
3.2.1.2 標準品檢量線的製作--------------------------------------------- 29
3.2.1.3 各類樣品中異黃酮素的HPLC定量分析-------------------- 31
3.2.2 初步萃取步驟----------------------------------------------------- 32
3.2.3 轉溶程序----------------------------------------------------------- 34
3.2.4 提取濃縮程序------------------------------------------------------ 36
3.2.4.1 液相萃取法--------------------------------------------------------- 36
3.2.4.1.1 單一溶劑(乙酸乙酯)的23因子加入中央點實驗設計------ 37
3.2.4.1.2 正丁醇與乙酸乙酯的不同溶劑與混合溶劑效應----------- 37
3.2.4.2 吸附與脫附層析法(固相萃取法)------------------------------ 40
3.2.4.2.1 管柱充填步驟----------------------------------------------------- 40
3.2.4.2.2 固相萃取操作流程------------------------------------------------ 40
四、 結果與討論--------------------------------------------------------- 46
4.1 異黃酮素的HPLC定量分析------------------------------------ 46
4.2 提取條件對粗提液中異黃酮素純度的影響------------------ 54
4.3 粗提液的放大提取----------------------------------------------- 54
4.4 轉溶條件對異黃酮素性能的影響------------------------------ 57
4.5 以液相萃取法進行異黃酮素的提取濃縮-------------------- 65
4.5.1 以實驗設計法探討單一溶劑(乙酸乙酯)萃取系統的水油比例、萃取時間及溫度效應------------------------------------
65
4.5.2 正丁醇與乙酸乙酯的不同溶劑與混合溶劑效應----------- 71
4.6 以固相萃取法進行異黃酮素的提取濃縮-------------------- 72
4.6.1 XAD-7HP管柱--------------------------------------------------- 72
4.6.1.1 吸附操作:吸附處理量之量測-------------------------------- 72
4.6.1.2 脫附操作:較佳階段脫附程式之建立----------------------- 75
4.6.2 XAD-4管柱-------------------------------------------------------- 88
4.6.2.1 吸附操作:吸附處理量之量測---------------------------------- 88
4.6.2.2 脫附操作:較佳階段脫附程式之建立------------------------ 90
4.6.3 酸性氧化鋁管柱-------------------------------------------------- 102
4.6.3.1 吸附操作:吸附處理量之量測--------------------------------- 102
4.6.4 管柱材質的比較--------------------------------------------------- 106
4.7 提取程序(液相萃取與固相萃取)之比較---------------------- 108
五、 結論------------------------------------------------------------------ 110
六、 參考文獻------------------------------------------------------------ 112
附錄A XAD-7 HP管柱的層析圖譜------------------------------------- 117
附錄B XAD-4管柱的層析圖譜----------------------------------------- 126
附錄C 酸性氧化鋁管柱的層析圖譜------------------------------------ 135
自述 ------------------------------------------------------------------------ 139
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