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研究生:侯秀儀
研究生(外文):Hsiu Yi Hou
論文名稱:利用固定化脂氧合修飾雞油氣味之可行性
論文名稱(外文):Aroma modification of chicken fat using immobilized lipoxygenase
指導教授:孫寶年孫寶年引用關係
指導教授(外文):Bonnie Sun Pan
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系
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:2002
畢業學年度:90
語文別:中文
論文頁數:88
中文關鍵詞:脂氧合固定化雞油氣味
外文關鍵詞:lipoxygenaesimmobilizationChicken oilodor
相關次數:
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中文摘要
雞油的特殊氣味使其在食品上的應用受到限制。以游離態海藻脂氧合修飾其氣味可明顯改善。游離態酵素於4℃貯藏八天,活性即喪失50%,且無法回收使用,本研究將酵素固定化以增加重覆使用及酵素安定性。
以褐藻膠( 2及4 % )包埋脂氧合,以褐藻膠4 % 包埋脂氧合,4 % 之膠粒較2 % 之膠粒圓潤且堅硬,與亞麻油酸作用平衡時間為200分鐘,較2 % 的60分鐘長。包埋時添加Ca2+ (0.2 - 0.8 M),濃度越高時,膠粒越堅實。
,以oxirane acrylic beads在0.05 M磷酸緩衝液( pH 7.5 )固定黃豆脂氧合,於25 ℃、以75 rpm震盪24小時,蛋白質與載體結合率最高達75 %。固定化脂氧合與0.5μmole亞麻油酸作用於25℃、以75 rpm震盪,反應平衡需20分鐘,反應速率為9.04 ± 0.32 nmole/min-mg protein。固定於oxirane acrylic beads之脂氧合可反覆使用8批次以上,活性並無損失,儲藏一個月,活性仍保有65﹪,半衰期42天。
游離態及固定化黃豆脂氧合Km分別為60.61及100.00 M,Vmax為358.42及246.27 nmole/mg protein-min。以methyl cinnamate作為內部標準品定量,游離態與固定化黃豆脂氧合作用C18:2之產物,以13-HODE為主,分別佔兩種型態LOX總產物的77.42 % 及80.25 %,各為0.35±0.05、0.16±0.01 mmole methyl cinnamate /mg protein,而9-HODE分別佔游離及固定化黃豆LOX總產物的22.58 % 及19.75 %。C18:3之產物,以13-HOTE為主,分別佔游離及固定化黃豆LOX總產物的93.81 % 及90.89 %,以花生四烯酸(C20:4)為基質,有12-及15-異構,產物比例分別為9:1與10:1,因此以oxirane acrylic beads固定黃豆脂氧合,並不影響其催化基質的特異性。
固定化黃豆脂氧合於25℃與雞油反應30分鐘,仍為清澈流體且顏色不變,多元不飽和脂肪酸由7.48 % 減少為1.77 %。修飾後之雞油氣味,以氣相層析嗅聞法分析得水果味、青草味及茶葉味為主要氣味(odor note),脂氧合與雞油作用時間由30分鐘延長至120分鐘,雞油味、油味減弱,令人喜好的氣味越強。
Abstract
The odor of chicken oil is the factor limiting its food use. This study was to modify the aroma of chicken oil with immobilized lipoxygenase (LOX). A significant loss of LOX activity was found during process and storage of the enzyme in free form. Free LOX cannot use repeatedly. The objective of this study was to immobilize to stabilize LOX and to reduce loss of LOX after each treatment.
LOX was entrapped using 2 and 4 % alginate-silicate sol-gel matrix. The concentration of alginate and calcium affected reaction time and firmness of the gel beads. The higher concentration resulted in;more circular beads of firmer texture. The entrapment of LOX at 2 and 4 % alginate-silicate sol-gel beads need 60 and 200 minutes to reach max. catalytic activity respectively.
Soybean LOX immobilized on oxirane acrylic beads yielded higher efficiency and catalytic activity in 0.05 M phosphate buffer, pH 7.5 at 25℃, 75 rpm than that obtained at other pH ranged 6.0 to 8.0, and temperature between 4-50℃, shaking rate of 0 and 100. To reach maximal efficiency, the shaking time was 24 hours, and 20 minutes for max. catalytic activity 9.04± 0.32 nmole/min-mg protein. The immobilized LOX was used repeatedly at least for 8 times without losing activity. The residual activity of the immobilized LOX was 65 % after one month storage at 4℃. The half-life was 42 days.
Km of free and immobilized LOX using C18:2 as substrate were 60.61 and 100.00 M,Vmax were 358.42 and 246.27 nmole/mg protein-min. Using methyl cinnamate as an internal standard, the ratio of 9-: 13-hydroperoxy derivative was not changed by immobilization in comparison to the free enzyme.
Treatment of chicken oil with immobilized LOX at 25℃ for 60 min resulted in 5.71 % decrease in total polyenoic acids. After LOX modification a noticeable improvement in odor of chicken oil was observed by sensory evaluation.
Using a GC-sniffing technique, LOX modified chicken oil produced odor notes of apple, melon, fruit, grass and tea-leave. The longer the reaction time with LOX;the stronger the desirable odor was, in addition to a significant reduction in “chicken fat odor” and oily odor.
目 錄
中文摘要 Ⅰ
英文摘要 III
壹、 前言 1
貳、 文獻整理 2
一、雞油組成與貯藏安定性 2
1. 脂肪酸組成 2
2. vitamin E 及 β- carotene 3
3. 貯藏安定性 3
二、油脂與氣味形成之關係 4
1. 種類與特徵…..………………………………………………...4
2. 形成機制……………………………………………………….5
三、脂氧合對於氣味的影響 6
1. 脂氧合作用機制……………………………………………6
2. 植物……………………...…………………………..………...7
3. 水產品……………...………………………………………….8
4. 油脂...………………………………………………………….9
四、界面活性劑及有機溶劑對於脂氧合反應速率的影響………10
1. 非離子性界面活性劑………………………………………...10
2. 有機溶劑……………………………………………………...11
五、脂氧合不穩定之因素…………………………………………13
1. 植物與動物結構之區別……………………………………..13
2. 活性中心鐵離子( Fe3+ )的還原……………………………...14
3. 氫過氧化物氧化活性部位之histidine……………………..14
4. 結構組成胺基酸氧化………………………………………..14
六、酵素固定…………………………………………………………15
1. 固定化酵素之功能…………………………………………..15
2. 固定化方法…………………………………………………..15
(1) 物理方式-褐藻膠包埋……………………………………..16
(2) 化學方式-oxirane acrylic beads共價鍵結 ……………..18
參、材料與方法……………………………………………………19
一、材料…...……………………………………………………….19
1. 雞脂肪組織………………………………………………...19
2. 海藻(石蓴)……………………………………………...19
3. 黃豆脂氧合…………….………………………………..19
4. 褐藻酸鹽……………………………………...……………...19
5. Oxirane acrylic beads…….. …………………...…………20
二、分析方法………………………………………………………21
1. 脂氧合萃取方法…………………………………...…….21
(1) 原粗酵素液製備方式…………………………………….21
(2) 粗酵素液萃取方法修正……………………………. 21
2. 硫酸銨分劃………………………………………………… 22
3. 脂氧合產物測定…………………………………………. 22
(1) 吸光度法 (LOOH 產量)……………………………...22
(2) 高效性液相層析………………………………………… 23
(3) 蛋白質定量-染料結合法(dye binding)………. 24
4. 固定化酵素的製備……………………………...………….. 24
(1) 物理方式-包覆( Entrapment ) 24
(2) 化學方式- 共價鍵結 25
5. 固定化脂氧合活性測定 25
(1) Oxirane immobilized soybean LOX 25
(2) Soybean LOX entrapped in alginate gel beads 26
6. 製備條件對於固定化酵素活性影響之測定 26
(1) pH及時間測定 26
(2) 震盪速率 27
7. 酵素固定化反應條件之測定 27
(1) 震盪速率 27
(2) 震盪速率與固定化酵素之貯藏安定性 27
(3) 反應時間 28
8. 黃豆脂氧合特性之測定 28
(1) 最適溫度 …28
(2) Km及Vmax 28
(3) 重覆利用率 29
(4) 貯存安定性 29
(5) Glutathione對於脂氧合活性影響 29
9. 雞油之製備 30
10. 雞油脂肪酸分析 30
11. 雞油修飾前後香氣之官能品評 31
12. 雞油與固定化黃豆脂氧合揮發性成份之萃取條件 31
13. 揮發性成分萃取 31
(1) 揮發性化合物之收集 31
(2) 揮發性成分萃取液之濃縮 32
14. 揮發性化合物之分析鑑定 32
(1) 氣相層析 32
(2) 氣相層析嗅聞法(GC-Sniffing) 33
(3) 滯留指數 (retention index, R.I.)的測定 33
(4) 氣相層析-質譜儀之分析鑑定 34
肆、結果與討論 35
一.萃取條件對於海藻(Ulva spp.)脂氧合活性之影響 35
(1) Ca 2+ 35
(2) 界面活性劑 Triton X-100 35
(3) 吸附劑 polyvinyl — pyrrolidone(PVP) 36
(4) 蛋白抑制劑 Phenylmethylsulfonyl fluoride (PMSF) 36
二. 硫酸銨分劃海藻脂氧合 38
三. 褐藻酸包埋黃豆脂氧合 40
1. 褐藻膠粒外型……………………………………………… 40
2. 褐藻酸鈉濃度對於脂氧合包埋於 alginate gel beads 反應
活性之影響 42
3. 褐藻酸鈉濃度對於脂氧合於alginate gel beads分佈之影 44
四. 黃豆脂氧合固定於oxirane acrylic beads之最適條件 45
1. 最適固定化之pH、時間 45
2. 震盪與攪拌對載體影響oxirane acrylic beads之影響 47
3. 震盪速率對於黃豆脂氧合與載體結合之影響 48五. 反應條件對於固定化脂氧合活性之影響 47
1. 震盪速率 50
2. 震盪速率對於固定化脂氧合安定性的影響 51
3. 反應時間 52
六. 固定黃豆脂氧合之特性 53
1. 最適溫度 53
2. 酵素動力學 55
3. 重複利用率 57
4. 貯藏安定性 58
5. 異構種類 59
6. 雞油脂肪酸的變化 67
七. 作用時間對雞油氣味之影響 69
八. 修飾後雞油揮發性成份 70
伍、結論 72
陸、參考文獻 75
七、參考文獻
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