臺灣博碩士論文加值系統

鹹鴨蛋蛋白液經電透析脫鹽處理後，添加0.5%蛋白質分解酵素[Protease N(PN)、Protease A(PA)或Prozyme 6(P6)]，置於45℃反應槽中水解24小時後，其水解液凍乾成粉末，進行抗氧化能力及抗氧化機制試驗。鹹鴨蛋蛋白液水解後明顯提升總抗氧化能力，當水解物濃度為5mg/ml時，未電透析組以Protease N抗氧化能力最高，可達73.03%，電透析組則是Protease A，可達70.71%、Prozyme 6組不管電透析與否，其總抗氧化能力則約60~62%。分析水解物在清除氫氧自由基、捕捉超氧陰離子、螯合鐵離子、清除DPPH(1,1-Diphenyl-2- picryl-hydrazyl)自由基及清除過氧化氫與還原力等不同抗氧化機制分析試驗中，以電透析經Protease N、Protease A與Prozyme 6處理之水解物，清除氫氧自由基之能力為最強分別為68.8%、60.7%與65.5%；捕捉超氧陰離子能力分別為58.1%、52.1%與58.5%；螯合鐵離子能力與清除DPPH自由基能力分別為17.7~21.7%、與31.8~45.0%；而水解物之還原力最低。將各水解物經膜過濾後探討區分物分子量是否影響抗氧化機制之能力。經酵素處理之水解物(Protease N、Protease A與Prozyme 6)其分子量在10-30kDa、3-10kDa、1-3kDa與＜1kDa之劃分物，皆比原水解物提高清除氫氧自由基之能力。Protease N與Prozyme 6之水解物其分子量在10-30kDa之劃分物，亦可以提高捕捉超氧陰離子之能力，Protease N與Prozyme 6之水解物其分子量為＞30kDa之劃分物，則可以略為提高螯合鐵離子能力。水解物分子量為10kDa以下，對於清除DPPH自由基有提升的結果。除Protease A之水解物其分子量為1-3kDa之劃分物可提高清除過氧化氫達14.8%外，其餘並無顯著關係，而在還原力方面，分子量大小並無任何提升之效果。將水解物粉末應用於肉品加工，探討其對TBARS生成量以表示其抗氧化性，在冷凍儲藏方面，鮮肉加鹽會促進TBARS的生成量，鮮肉加水解物未加鹽組其TBARS生成量經儲藏5個月仍然很低，但添加食鹽與水解物組，則TBARS皆有明顯上升的趨勢，但其中以經電透析Protease A處理之鹹鴨蛋蛋白水解物有較低TBARS生成量。在冷藏試驗方面，鮮肉混合食鹽加熱處理後，會促進TBARS生成量迅速增加，其中以90℃處理組之生成量與生成趨勢為最高；而未電透析經Protease A、Prozyme 6及電透析經Protease N處理組則可有效降低經160℃加熱處理的加鹽鮮肉之TBARS生成量，其量分別為未添加水解物組的30%、31.3%及44%。進一步將電透析組抗氧化力最強的水解物─Protease N及Prozyme 6處理組，與市售之抗氧化劑BHA及Carnosine併用，結果顯示任何一種組合對抑制TBARS生成量的效果，都比單獨添加組來的好，但其效果並沒有大於兩者單獨添加時的總合，故其抗氧化能力僅具相加作用。

In this study, the salted duck egg white was treated by electrodialysis desalination and hydrolyzed with the addition of 0.5% (w/w) protease (Protease N, Protease A or Prozyme 6) for 24 hours at 45℃ in a reactor. The hydrolysates were then freeze-dried into powder, and the antioxidative activity and antioxidative mechanism of freeze-dried hydrolysates were investigated. When the concentration of the hydrolysates was 5 mg/ml, for the samples without treatment of electrodialysis desalination, Protease N had the strongest antioxidative activity up to 73.0% but, for the samples treated by electrodialysis desalination, Protease A showed a higher antioxidative ability up to 70.7%. However, wheather treated or untreated by electrodialysis desalination, the total antioxidative ability of hydrolysates with Prozyme 6 was about 60-62%. The analysis of different antioxidative mechanism of hydrolysates, the scavenging effects on H2O2, superoxide anion, DPPH radical, hydroxyl radical and chelating effects on Fe2+. Results of the antioxidative mechanism on hydrolysates which hydrolyzed with Protease N, Protease A and Prozyme 6 were followed: the scavenging effectson hydroxyl radical were 68.8%, 60.7% and 65.5%; the scavenging effects on superoxide anion were 58.1%, 52.1% and 58.5%; the chelating effects on Fe2+ were 17.7-21.7%; the scavenging effects on DPPH radical were 31.8-45.0%; reducing power of hydrolysates was weakest. The hydrolysates were further screened by membranes of different pore size and then to confer the effect of antioxidative mechanism between all kinds of membrane fractionation. Membrane fractionations hydrolyzed with Protease N, Protease A and Prozyme 6 with the molecular weight range at 10-30kDa, 3-10kDa, 1-3kDa and <1kDa could raise the scavenging effects on hydroxyl radical and had a better effect than original hydrolysates. The membrane fractionations of Protease N and Prozyme 6 with molecular weight range between 10-30kDa could raise the scavenging effects on superoxide anion. The membrane fractionations of Protease N and Prozyme 6 with molecular weight range >30kDa could raise chelating effects on ferrous ion slightly. The membrane fractionations of Protease N, Protease A and Prozyme 6 with molecular weight <10kDa could raise the scavenging effects on DPPH radical. The molecular weight range between 1-3kDa of the hydrolysates with Protease A could raise the scavenging effects on H2O2 up to 14.8%, but the others were not effective. The change of molecular weight did not raise reducing power at all. Further application of hydrosate powders can be made to investigate the TBARS amount produced during storage. On storage at -18℃, fresh meat with added salt could promote TBARS production. After 5 months storage, fresh meat with added hydrolysates but no salt addition had a fewer TBARS content. But fresh meat with added salt and hydrolysates had raised the content of TBARS to a certain degree, only the hydrolysates treated with Protease A by electrodialysis desalination showing fewer TBARS content. If stored at 4℃, fresh meat with added salt and hydrolysates and treated with heat could promote the increase of TBARS immediately. Among all groups, the content of TBARS with meat heated at 90℃ was the highest. The hydrolysates treated with Protease A and Prozyme 6 by electrodialysis desalination and Protease N treated by non-electrodialysis desalination could reduce TBARS content effectively when heated at 160℃. Their contents were about 30.0%, 31.3% and 44.0% respectively if compare with cases without hydrolysates addition. Furthermore, the hydrolysates treated with Protease N and Prozyme 6 by electrodialysis desalination had the highest antioxidative ability and combined with commercial antioxidants — BHA and Carnosine. Results showed that any of the combinations could inhibit TBARS production and had better effects than individual one but, not better than the sum of two individual effects. Therefore, they only demonstrated synergistic effects.

目　錄
頁次
目錄----------------------------------------------------------------------------------I
表目錄-----------------------------------------------------------------------------X
圖目錄----------------------------------------------------------------------------XI
中文摘要-----------------------------------------------------------------------XIII
英文摘要-----------------------------------------------------------------------XV
第一章　前言---------------------------------------------------------------------1
第二章　文獻回顧---------------------------------------------------------------3
一、鹹鴨蛋(Salted duck egg) 與鹹鴨蛋蛋白液的特性---------------------3
(一)鹹鴨蛋蛋白液的特性------------------------------------------------3
(二)鹹鴨蛋蛋白液之應用-----------------------------------------------4
二、脫鹽技術-----------------------------------------------------------------------5
(一)膜過濾方法---------------------------------------------------------------5
1.逆滲透法 (Reverse osmosis) ---------------------------------------------5
2.超過濾法(Ultrafiltration)--------------------------------------------------6
3.薄膜蒸餾法(Membrane distillation)-------------------------------------6
4.電透析(Electrodialysis, ED)分離技術-----------------------------------8
(二)膜過濾技術之應用---------------------------------------------------10
三、蛋白質分解酵素的特性---------------------------------------------------12
(一)來源-----------------------------------------------------------------------12
(二)種類-----------------------------------------------------------------------12
1.絲胺酸型蛋白質分解酵素(Serine protease)--------------------------13
(1)胰凝乳蛋白酶與胰蛋白--------------------------------------------13
(2)麴菌屬(Aspergillus)之蛋白酶------------------------------------------14
2.硫醇型蛋白質分解酵素(Thiol proteases)-----------------------------14
(1)木瓜蛋白分解酶(Papain)-----------------------------------------------15
(2)鳳梨蛋白分解酶(Bromelin)--------------------------------------------15
3.金屬型蛋白質分解酵素(Metalloproteases)---------------------------16
4.酸性蛋白質分解酵素(Acid proteases)---------------------------------16
四、影響蛋白質水解之因素---------------------------------------------------17
(一)水解方式-----------------------------------------------------------------17
(二)酵素的選擇--------------------------------------------------------------18
(三)酵素與受質比例--------------------------------------------------------19
(四)pH值與溫度-------------------------------------------------------------20
(五)食鹽濃度與抑制劑-----------------------------------------------------20
五、蛋白質水解物之應用------------------------------------------------------21
(一)機能性--------------------------------------------------------------------21
1.抗菌--------------------------------------------------------------------21
2.神經活性--------------------------------------------------------------22
3.酵素調節及抑制-----------------------------------------------------22
4.荷爾蒙及荷爾蒙調節-----------------------------------------------22
5.免疫活性--------------------------------------------------------------23
6.其他生物活性--------------------------------------------------------23
7.抗氧化-----------------------------------------------------------------23
（二）其他功能特性--------------------------------------------------------24
1.調味利用-------------------------------------------------------------------24
2.營養利用-------------------------------------------------------------------25
3.其它-------------------------------------------------------------------------26
六、脂質氧化作用---------------------------------------------------------------26
1.自氧化作用(Autoxidaton)-----------------------------------------------27
2.光氧化作用(Photosensitized oxidation)-------------------------------28
3.熱氧化作用(Thermal oxidation)----------------------------------------28
4.酵素性氧化作用(Enzymatic oxidation)-------------------------------29
七、抗氧化作用------------------------------------------------------------------29
(一)作用機制-----------------------------------------------------------------29
1.自由基終止劑(Free radical terminators)-------------------------------29
2.還原劑和氧的清除劑(Reducants or oxygen scavengers)-----------30
3.金屬螯合劑(Metal chelators)--------------------------------------------30
4.單重態氧抑制劑(Singlet oxygen inhibitor)---------------------------30
(二) 抗氧化劑---------------------------------------------------------------31
1.特性--------------------------------------------------------------------------31
2.來源--------------------------------------------------------------------------31
(1)天然抗氧化劑------------------------------------------------------------31
(2)合成抗氧化劑------------------------------------------------------------32
3.抗氧化活性之相乘、相加效應------------------------------------------32
4.生物體內之抗氧化作用--------------------------------------------------33
5.食品中之抗氧化作用-----------------------------------------------------34
八、蛋白質相關物質的抗氧化性---------------------------------------------35
1.蛋白質類--------------------------------------------------------------------35
2.類-----------------------------------------------------------------------36
3.胺基酸類--------------------------------------------------------------------37
九、畜產品儲藏過程之氧化及防止氧化之方法---------------------------38
(一)畜產品之氧化現象-----------------------------------------------------38
(二)防止畜產品氧化之方法-----------------------------------------------39
1.加工中產生之抗氧化物質-----------------------------------------------39
2.包裝--------------------------------------------------------------------------40
3.冷藏或凍藏-----------------------------------------------------------------40
4.添加抗氧化劑--------------------------------------------------------------41
第三章　材料與方法-----------------------------------------------------------43
一、實驗流程---------------------------------------------------------------------43
二、實驗材料---------------------------------------------------------------------43
1.鴨蛋--------------------------------------------------------------------------43
2.鹹鴨蛋白液-----------------------------------------------------------------43
3.電透析脫鹽之鹹鴨蛋蛋白粉末-----------------------------------------43
4.蛋白質分解酵素-----------------------------------------------------------44
5.水解物之膜過濾劃分與收集--------------------------------------------44
6.豬肉--------------------------------------------------------------------------45
7.原料肉製備-----------------------------------------------------------------45
(1)鮮肉------------------------------------------------------------------------45
(2)90℃水煮處理組--------------------------------------------------------45
(3)160℃烘烤處理組------------------------------------------------------46
8.化學藥品--------------------------------------------------------------------46
三、實驗方法---------------------------------------------------------------------52
1.鹹鴨蛋蛋白液酵素水解--------------------------------------------------52
2.鹹鴨蛋蛋白水解物於亞麻油酸(linoleic acid)乳化系統之抗氧化活性-----------------------------------------------------------------------------52
3.捕捉氫氧自由基能力測定-----------------------------------------------53
(A)去氧核糖氧化傷害試驗----------------------------------------------53
(B) Electron Paramagentic Resonance (EPR)測定--------------------54
4.捕捉超氧陰離子能力測定-----------------------------------------------54
5.螯合亞鐵離子能力測定--------------------------------------------------55
6.清除α,α-diphenyl-β-picrylhydrazyl (DPPH)自由基能力測定------------------------------------------------------------------------------55
7.捕捉過氧化氫能力測定--------------------------------------------------56
8.還原力測定-----------------------------------------------------------------56
9.Thiobarbituric acid reactive substances (TBARS)--------------------57
10.統計分析(Statistical analysis)------------------------------------------57
第四章 結果與討論-----------------------------------------------------------58
一、鹹鴨蛋蛋白水解物之凍乾粉末的抗氧化能力及機制---------------58
(一)總抗氧化能力-----------------------------------------------------------58
(二)清除氫氧自由基能力--------------------------------------------------62
1.去氧核糖氧化傷害試驗--------------------------------------------------62
2.清除氫氧自由基能力─EPR測定--------------------------------------64
(三)捕捉超氧陰離子能力--------------------------------------------------67
(四)螯合亞鐵離子能力-----------------------------------------------------69
(五)清除過氧化氫能力-----------------------------------------------------72
(六)清除1,1-diphenyl-2-picrylhydrazyl (DPPH)自由基能力---------74
(七)還原力--------------------------------------------------------------------77
二、鹹鴨蛋蛋白水解物劃分物之凍乾粉末的抗氧化機制---------------80
(A)各膜區分物之產率------------------------------------------------------80
(B)膜區分物之抗氧化機制------------------------------------------------82
(一)清除氫氧自由基能力--------------------------------------------------82
(二)捕捉超氧陰離子能力--------------------------------------------------84
(三)螯合亞鐵離子能力-----------------------------------------------------86
(四)清除過氧化氫能力-----------------------------------------------------88
(五)清除1,1-diphenyl-2-picrylhydrazyl (DPPH)自由基能力---------88
(六) 還原力------------------------------------------------------------------92
抗氧化機制總結-----------------------------------------------------------------94
三、鹹鴨蛋蛋白水解物凍乾粉末在加工肉品之抗氧化試驗------------96
(一)鮮肉混合食鹽與水解物之冷凍儲藏試驗--------------------------96
(二)鮮肉混合食鹽與水解物後經不同溫度處理之冷藏試驗------102
1.未加熱處理---------------------------------------------------------------102
2.90℃加熱水煮處理------------------------------------------------------104
3.160℃烤箱加熱處理-----------------------------------------------------104
四、鹹鴨蛋蛋白水解物凍乾粉末與抗氧化劑之相乘、相加效應------109
第五章 結論--------------------------------------------------------------------113
六、參考文獻--------------------------------------------------------------------115

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