臺灣博碩士論文加值系統

酚酸化合物已被證實對人體具有抗發炎(anti-inflammatory)、抗氧化(anti-oxidant)等功效。自然界大量存在之咖啡酸(caffeic acid)為水溶性多酚類，其酯類衍生物的親油性高，可強化其抗氧化能力，並提高其應用價值。利用脂解酵素(lipase)合成酚酸酯類，藉此改變酚酸的親油特性，使抗氧化及自由基清除力提高的功能更穩定。脂解酵素可催化咖啡酸及苯乙醇(phenyl ethanol)合成咖啡酸苯乙酯(caffeic acid phenethyl ester, CAPE)，酵素合成比起化學合成方式不僅更天然，且反應條件溫和，製程對環境較友善，所合成出來的產物亦偏向天然，在食品或化妝品的應用上，對於消費者之接受度較高。為了縮短催化酚酸酯化物(phenolic acid ester)的合成時間，本研究藉由生物催化 (biocatalysis)技術並配合超音波(ultrasound)系統及連續式酵素填充床生物反應器(continuous packed-bed bioreactor)進行催化合成反應，以符合未來工業量產之應用。
將基質咖啡酸與苯乙醇(phenyl ethanol)於異辛烷(isooctane)溶劑中，利用固定化脂解酵素(Novozym® 435)，經由中心混層實驗設計(central composite rotatable design, CCRD)或Box-Behnken實驗設計(Box-Behnken design)，以直接酯化(direct esterification)方式合成咖啡酸苯乙酯，再以反應曲面法(response surface methodology, RSM)進行分析，分別探討合成反應參數對莫耳轉換率的影響，最後，利用脊型分析(analysis of ridge max)詳細探討酵素合成咖啡酸苯乙酯之最優化反應條件。
本研究分成三階段進行，第一階段為利用固定化酵素Novozym® 435合成咖啡酸苯乙酯，使提高咖啡酸在親油性環境下之穩定性，增加其有效利用率。將咖啡酸及苯乙醇以直接酯化方式，並利用五階層四變數之中心混層實驗設計，探討合成咖啡酸苯乙酯之最優化條件。實驗結果顯示，反應時間59小時、反應溫度69 °C、咖啡酸與苯乙醇莫耳比1:72及酵素用量351 PLU (propyl laurate unit)，咖啡酸苯乙酯莫耳轉換率之理論值與實驗值分別為91.36%及91.65±0.66%。
第二階段藉由超音波輔助系統合成咖啡酸苯乙酯。超音波於液相溶液中形成空穴現象(cavitation)，產生許多微小氣泡及超臨界區域，提供了化學介質力量，使增加了酵素與基質間作用的接觸機會，進而提高合成酯化作用，縮短咖啡酸苯乙酯合成時間。結果顯示，超音波輔助系統利用Novozym® 435於反應溫度70 °C，合成咖啡酸苯乙酯之最優化條件為：反應時間9.6 小時、咖啡酸與苯乙醇莫耳比1:71、酵素用量2938 PLU及超音波功率為2 W/cm2，咖啡酸苯乙酯莫耳轉換率之理論值與實驗值分別為96.03%及93.08±0.42%。
第三階段為超音波輔助系統結合連續式酵素填充床生物反應器合成咖啡酸苯乙酯，利用超音波加速酵素作用並在連續式生物反應器下，模擬工業連續化生產流程，提供業界大量製備之依據。結果顯示，當咖啡酸與苯乙醇莫耳比1:100，填充15,000 PLU固定化酵素Novozym® 435於連續式酵素填充床生物反應器，合成咖啡酸苯乙酯之最優化條件為：反應溫度72.66 °C、體積流量0.046 mL/min及超音波功率為1.64 W/cm2，莫耳轉換率之理論值與實驗值分別為97.84%及92.11±0.75%。
綜合以上實驗結果，利用固定化酵素Novozym® 435合成咖啡酸苯乙酯，並在超音波輔助系統下，克服了酚酸酯類合成耗時的障礙。藉由連續式生物反應器，達到連續生產咖啡酸苯乙酯之目的，探討合成咖啡酸苯乙酯之最優化條件，找出最佳莫耳轉換率，節省操作時間及能源，進一步提供予業界量化的參考。

Phenolic acids are good radical scavengers for anti-inflammatory and anti-oxidant performances. Caffeic acid, one kind of phenolic acid, increase the solubility in oil-based formulas and emulsions is to esterify the compounds with alcohols and enhance anti-oxidant ability in the food and cosmetics applications. However, the reagents used in chemical synthesis of caffeic acid phenethyl ester (CAPE) are harmful to natural environmental. In contrast, enzymatic synthesis offers the advantages of specificity, milder reaction conditions, and minimization of side reactions and byproduct formation. Therefore, the value of using continuous ultrasound-assisted packed-bed bioreactor for the lipase-catalyzed processing should also permit an easier approach to producing commercial amount of CAPE.
In this study, optimum conditions for the enzymatic synthesis of CAPE, catalyzed by immobilized lipase (Novozym® 435) were investigated. Novozym® 435 was used to catalyze caffeic acid and 2-phenyl ethanol in an isooctane system. 5-level-4-factor central-composite rotatable design (CCRD), Box-Behnken experiment design and response surface methodology (RSM) were employed to evaluate the effects of synthesis parameters on percentage conversion of CAPE by esterification for three part experiments.
In the first part, immobilized enzymes were used to catalyze the esterification of caffeic acid with phenyl ethanol. The esterification improved the stability and hydrophobicity of phenolic acid. On the basis of ridge max analysis, the optimum conditions for synthesis were: reaction time 59 h, reaction temperature 69 ºC, substrate molar ratio1:72, and enzyme amount 351 PLU. The molar conversion of predicted value was 91.86% and actual experimental value was 91.65±0.66%, respectively.
In the second part, ultrasonication causes cavitations in the liquid medium. Subsequent collapses of the cavitation bubbles appear to cause a thorough mixing and stirring of the liquid solution, and the energy thus released should accelerate the enzymatic reactions. Ultrasound provides a very effective mixing and stirring in the reaction solution and increases the contacts between substrates and enzyme. The optimum condition for CAPE synthesis were reaction time 9.6 h, substrate molar ratio 1:71, enzyme amount 2938 PLU, and ultrasonic power 2 W/cm2. The molar conversion of predicted values and actual experimental values were 96.03% and 93.08±0.42%, respectively.
In the third part, the ultrasound-acceleration synthesis of CAPE in a continuous packed-bed bioreactor was investigated. A three-level-three-factor Box-Behnken and RSM were employed on percent molar conversion of CAPE. The optimum conditions for synthesis CAPE were: reaction temperature of 72.66 ˚C, flow rate of 0.046 mL/min, and ultrasonic power of 1.64 W/cm2. The molar conversion of predicted values and actual experimental values were 97.84% and 92.11±0.75%, respectively. This work demonstrates of lipase in a continuous ultrasound-acceleration packed-bed bioreactor for industrial production of CAPE.
The use of continuous ultrasound-acceleration packed-bed bioreactor in Novozym® 435-catalyzed synthesis of CAPE from caffeic acid and 2-phenyl ethanol in isooctane was investigated. Compared with chemical synthesis was more natural and milder synthesis process reduced the environmental damage, while the synthesized product of CAPE was also relatively safe for food or cosmetic applications. According to our results, used the natural enzyme catalysis and ultrasound to accelerate improve time-consuming for synthesis CAPE. The value of using packed-bed bioreactors for the lipase-catalyzed processing should also permit an easier approach to producing commercial amount of the product.

封面內頁
簽名頁
授權書iii
中文摘要iv
英文摘要vii
誌謝ix
目錄x
圖目錄xiv
表目錄xvii
1.緒論1
2.文獻回顧5
2.1酚酸酯類之酵素合成5
2.1.1酚類化合物的定義及抗氧化作用5
2.1.2蜂膠與咖啡酸苯乙酯8
2.1.3脂解酵素10
2.1.3.1酵素之優點10
2.1.3.2酵素之固定化優點及應用11
2.1.3.3Novozym® 435介紹12
2.1.4酚酸酯化物之合成13
2.1.4.1酚酸酯類化學合成14
2.1.4.2酚酸酯類酵素合成15
2.1.5反應曲面法之應用17
2.2超音波於酵素合成酯類之研究18
2.2.1超音波的定義及特點18
2.2.2超音波應用型態簡介19
2.2.3超音波水解及合成酯類22
2.3連續式酵素填充床生物反應器於酵素合成酯類之研究24
2.3.1生物反應器介紹24
2.3.2連續式酵素填充床生物反應器之酯類合成26
3.以反應曲面法探討酵素合成咖啡酸苯乙酯之最優化反應條件28
3.1摘要28
3.2前言29
3.3實驗材料33
3.3.1藥品33
3.3.2儀器設備33
3.4實驗設計與方法34
3.4.1實驗設計34
3.4.2咖啡酸苯乙酯之合成方法34
3.4.3高效能液相層析之分析方法34
3.4.4咖啡酸苯乙酯之莫耳轉換率35
3.5結果與討論37
3.5.1反應時間對Novozym® 435催化合成咖啡酸苯乙酯莫耳轉換率之影響37
3.5.2Novozym® 435合成咖啡酸苯乙酯之變數分析39
3.5.3Novozym® 435合成咖啡酸苯乙酯之最優化探討48
4.以反應曲面法探討超音波輔助合成咖啡酸苯乙酯之最優化反應條件52
4.1摘要52
4.2前言53
4.3實驗材料56
4.3.1藥品56
4.3.2儀器設備56
4.4實驗設計與方法57
4.4.1實驗設計57
4.4.2咖啡酸苯乙酯之合成方法57
4.4.3高效能液相層析之方法分析57
4.5結果與討論59
4.5.1超音波輔助對酵素催化合成咖啡酸苯乙酯莫耳轉換率之影響59
4.5.2反應時間及酵素用量對酵素催化合成咖啡酸苯乙酯莫耳轉換率之影響59
4.5.3超音波功率對酵素合成咖啡酸苯乙酯莫耳轉換率之影響60
4.5.4超音波輔助合成咖啡酸苯乙酯之變數分析64
4.5.5超音波輔助合成咖啡酸苯乙酯之最優化探討73
5.以反應曲面法探討連續式酵素填充床生物反應器合成咖啡酸苯乙酯之最優化反應條件77
5.1摘要77
5.2前言78
5.3實驗材料82
5.3.1藥品82
5.3.2儀器設備82
5.4實驗設計與方法83
5.4.1實驗設計83
5.4.2咖啡酸苯乙酯合成方法83
5.4.3高效能液相層析之分析方法84
5.5結果與討論86
5.5.1流速及超音波功率對合成咖啡酸苯乙酯莫耳轉換率之影響86
5.5.2連續式酵素填充床生物反應器合成咖啡酸苯乙酯之變數分析89
5.5.3連續式酵素填充床生物反應器合成咖啡酸苯乙酯之最優化探討97
5.5.4酵素再利用率98
6.結論105
參考文獻108
附錄121
圖1.1整體研究架構圖4
圖2.1酚類化合物之分類6
圖2.2抗氧化之酚酸結構7
圖2.3咖啡酸苯乙酯之化學結構式9
圖2.4超音波生物反應器之型態21
圖3.1Novozym® 435催化咖啡酸與苯乙醇生成咖啡酸苯乙酯之酯化反應31
圖3.2Novozym® 435催化咖啡酸及苯乙醇合成咖啡酸苯乙酯之實驗架構圖32
圖3.3反應時間對酵素合成咖啡酸苯乙酯莫耳轉換率之影響38
圖3.4Novozym® 435合成咖啡酸苯乙酯之莫耳轉換率實驗值與預測值之線性關係43
圖3.5反應時間及基質莫耳比對酵素合成咖啡酸苯乙酯莫耳轉換率之反應曲面圖45
圖3.6反應時間及酵素用量對酵素合成咖啡酸苯乙酯莫耳轉換率之反應曲面圖46
圖3.7基質莫耳比及酵素用量對酵素合成咖啡酸苯乙酯莫耳轉換率之反應曲面圖47
圖3.8Novozym® 435合成咖啡酸苯乙酯莫耳轉換率之等高線曲面圖50
圖4.1超音波簡要裝置54
圖4.2超音波輔助咖啡酸及苯乙醇合成咖啡酸苯乙酯之實驗架構圖55
圖4.3比較超音波輔助及機械式振盪對於酵素催化合成咖啡酸苯乙酯之影響61
圖4.4反應時間及酵素用量對超音波輔助合成咖啡酸苯乙酯莫耳轉換率之影響62
圖4.5超音波功率對合成咖啡酸苯乙酯莫耳轉換率之影響63
圖4.6超音波輔助合成咖啡酸苯乙酯莫耳轉換實驗值與預測值之線性關係68
圖4.7反應時間及基質莫耳比對超音波輔助合成咖啡酸苯乙酯莫耳轉換率之反應曲面圖70
圖4.8基質莫耳比及酵素用量對超音波輔助合成咖啡酸苯乙酯莫耳轉換率之反應曲面圖71
圖4.9基質莫耳比及超音波功率對超音波輔助合成咖啡酸苯乙酯莫耳轉換率之反應曲面圖72
圖4.10超音波輔助合成咖啡酸苯乙酯莫耳轉換率之等高線曲線圖75
圖5.1連續式酵素填充床生物反應器裝置80
圖5.2連續式酵素填充床生物反應器催化咖啡酸及苯乙醇合成咖啡酸苯乙酯之實驗架構圖81
圖5.3流速及超音波對連續式酵素填充床生物反應器合成咖啡酸苯乙酯莫耳轉換率之影響87
圖5.4超音波功率對連續式酵素填充床生物反應器合成咖啡酸苯乙酯莫耳轉換率之影響88
圖5.5連續式酵素填充床生物反應器對合成咖啡酸苯乙酯莫耳轉換率之實驗值與預測值之線性關係93
圖5.6反應溫度及流速對連續式酵素填充床生物反應器合成咖啡酸苯乙酯莫耳轉換率之反應曲面圖95
圖5.7反應溫度及超音波功率對連續式酵素填充床生物反應器合成咖啡酸苯乙酯莫耳轉換率之反應曲面圖96
圖5.8不同反應溫度對連續式酵素填充床生物反應器合成咖啡酸苯乙酯莫耳轉換率之等高線曲線圖100
圖5.9不同流速對連續式酵素填充床生物反應器合成咖啡酸苯乙酯莫耳轉換率之等高線曲線圖101
圖5.10不同功率對連續式酵素填充床生物反應器合成咖啡酸苯乙酯莫耳轉換率之等高線曲線圖102
圖5.11酵素在超音波系統及連續式酵素填充床生物反應器合成咖啡酸苯乙酯之再用104
表3.1酵素合成咖啡酸苯乙酯實驗設計反應參數實驗值之範圍36
表3.2Novozym® 435合成咖啡酸苯乙酯之五階層四變數中心混層實驗設計及其莫耳轉換率41
表3.3Novozym® 435合成咖啡酸苯乙酯莫耳轉換率之變異分析42
表3.4Novozym® 435合成咖啡酸苯乙酯變數之聯合檢測分析44
表3.5脊型分析評估Novozym® 435合成咖啡酸苯乙酯之莫耳轉換最大值51
表4.1超音波輔助合成咖啡酸苯乙酯之實驗設計反應參數實驗值範圍58
表4.2超音波輔助合成咖啡酸苯乙酯之五階層四變數中心混層實驗設計及其莫耳轉換率66
表4.3超音波輔助合成咖啡酸苯乙酯莫耳轉換率變數之變異分析67
表4.4超音波輔助合成咖啡酸苯乙酯變數之聯合檢測分析69
表4.5脊型分析評估超音波輔助合成咖啡酸苯乙酯之莫耳轉換率最大值76
表5.1連續式酵素填充床生物反應器合成咖啡酸苯乙酯三階層三變數之實驗設計反應參數實驗值範圍85
表5.2連續式酵素填充床生物反應器合成咖啡酸苯乙酯之三階層三變數Box-Behnken實驗設計及其莫耳轉換率91
表5.3連續式酵素填充床生物反應器合成咖啡酸苯乙酯莫耳轉換率對合成變數之變異分析92
表5.4連續式酵素填充床生物反應器合成咖啡酸苯乙酯變數之聯合檢測分析94
表5.5脊型分析評估使用連續式酵素填充床生物反應器合成咖啡酸苯乙酯之莫耳轉換率最大值103

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