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研究生:蔡世芬
研究生(外文):Shih-Fen Tsai
論文名稱:維生素E及鈣離子對鵝隻屠後骨骼肌肌原纖維蛋白質降解之影響
論文名稱(外文):Effect of vitamin E and calcium ions on postmortem changes in myofibrillar proteins from goose skeletal muscles
指導教授:周榮吉周榮吉引用關係
指導教授(外文):Rong-Ghi R. Chou Ph. D.
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:動物科學系研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:107
中文關鍵詞:維生素E鈣離子
外文關鍵詞:vitamin Ecalcium ions
相關次數:
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本研究之目的旨在探討,維生素E及鈣離子對鵝隻屠後骨骼肌肌原纖維蛋白質降解之影響。本實驗共分3個試驗( 試驗I、II及III ),每個試驗組重複3次。試驗I:約100日齡之華鵝及白羅曼鵝各20隻,於屠宰後將屠體真空包裝置於5℃貯存。試驗II:白羅曼鵝屠前4週(自72日齡至100日齡)餵飼補充1200 ppm維生素E [(+)-α-tocopherol acetate, 1360 IU /g ] 之飼糧;對照組則餵飼基礎飼糧(含25 ppm 維生素E之NRC推薦量);每組各20隻。鵝隻屠宰後,屠體真空包裝置於5℃貯存。試驗I及II之屠體,每隔1、3、7及14日逢機採下胸肌及腿肌樣品;第0日樣品則於屠後2-4小時採集。試驗III:白羅曼鵝飼養條件如試驗II,鵝隻於屠後2-4小時內,取下個別屠體右側之腿肌,浸漬於5℃之0.1 M乳酸鈣溶液;左側腿肌則為對照組。乳酸鈣浸漬組及對照組樣品,置於5℃經0、1、3、7及14日逢機採樣。試驗I結果顯示,華鵝及白羅曼鵝胸肌之pH均較腿肌為低( P < 0.05 )。SDS-PAGE顯示,胸肌之30 kDa裂解成分之出現及titin之消失均較腿肌為快。Western blot亦顯示,胸肌desmin及vinculin之消失遠快於腿肌;但α-actinin則殆無變化。試驗II結果顯示,維生素E組與對照組之胸肌或腿肌之pH無顯著差異( P > 0.05)。SDS-PAGE顯示,維生素E組之胸肌及腿肌之30 kDa裂解成分之出現及titin之消失均較對照組為慢。Western blot顯示,desmin之消失以維生素E組之胸肌及腿肌較對照組為慢。試驗III之SDS-PAGE顯示,腿肌之30 kDa裂解成分之出現及titin之消失,均以乳酸鈣組最快而維生素E組最慢。Western blot顯示,desmin之消失亦以乳酸鈣組最快而維生素E組最慢。綜上所述,華鵝及白羅曼鵝胸肌肌原纖維蛋白質降解速率快於腿肌;而飼糧補充維生素E可減緩屠後骨骼肌肌原纖維蛋白質之降解;但是乳酸鈣浸漬則可加快之。
Abstract

There were three studies in this master thesis. The first study was to compare the postmortem changes between breast (BM) and leg (LM) muscles from Chinese and White roman geese. Myofibrils prepared from BM and LM that were randomly taken from the carcasses of two goose breeds at 5°C after 0, 1, 3, 7 and 14 days of storage. Changes in pH and degradation of myofibrillar proteins were analyzed. The results showed that pH was lower in BM than in LM samples from both goose breeds (P < 0.05). Appearance of 30 kDa components and disappearance of titin were more rapidly as seen on SDS-PAGE in BM than in LM samples. Western blots labeled with a monoclonal antibody either to desmin or to vinculin also demonstrated that desmin and vinculin degraded more quickly in BM samples. α-Actinin, however, remained no change in all samples. Our data might suggest that postmortem proteolysis occurred more rapidly in BM than in LM from both Chinese geese and White roman geese.
The second study is to examine the effect of vitamin E supplementation on the postmortem changes in BM and LM muscles from White roman geese. Twenty geese were fed a standard diet with minimal level of vitamine E [ 25ppm, (+)-α-tocopherol acetate, 1360 IU/g ] (CON). Another 20 geese were fed with inclusion of 1200 ppm vitamin E supplementation on the last 28 days (VitE). The diets and water of both groups were provided ad libitum. The geese were sacrificed at 100 days old. Myofibrils prepared from BM and LM that were randomly taken from the carcasses of both VitE and CON at 5°C after 0, 1, 3, 7 and 14 days of storage. The results showed that no significant difference in pH of BM and LM was found between VitE and CON. However, appearance of 30 kDa components and disappearance of titin of BM and LM were more slowly in VitE than in control samples. Western blots labeled with a monoclonal antibody to desmin also demonstrated that desmin degraded more slowly both in BM and LM of VitE samples. Our data suggested that vitamin E supplementation might reduced the rate of postmortem proteolysis in goose BM and LM muscles.
The third study was to examine the effect of 0.1 M calcium lactate incubation at 5℃ on postmortem changes in LM from the geese that was fed with inclusion of 1200 ppm vitamin E supplementation on the last 28 days. Twenty geese were fed a standard diet with minimal level of vitamine E [ 25ppm, (+)-α-tocopherol acetate, 1360 IU/g ] (CON). Another 20 geese were fed with inclusion of 1200 ppm vitamin E supplementation on the last 28 days (VitE). The diets and water of both groups were provided ad libitum. The geese were sacrificed at 100 days old. LM was taken from the carcasses of both VitE and CON after 2-4 h postmortem. Each right side of LM from VitE or CON was separately incubated in 0.1 M calcium lactate at 5°C (VitE/Ca or CON/Ca). The left side of LM from VitE or CON was non-incubated, directly vaccum-packed and stored at 5°C. Myofibrils prepared from LM that were randomly taken from VitE , CON, VitE/Ca and CON/Ca after 0, 1, 3, 7 and 14 days of storage. The results indicated that appearance of 30 and 32 kDa components and disappearance of titin and desmin were the most rapidly in CON/Ca and the slowest in VitE samples. Our data suggested that 0.1 M calcium lactate incubation could enhanced the postmortem proteolysis in LM muscles from geese fed with inclusion of 1200 ppm vitamin E supplementation on the last 28 days.
目 錄
壹、前言 .............................................1
貳、文獻檢討...........................................3
一、屠後肌肉細胞之變化.................................3
(一) pH值之變化........................................3
(二) 細胞內外鈣離子濃度之穩衡狀態......................4
1. 細胞內外鈣離子濃度之變化 ............................4
2. 屠後肌肉細胞內鈣離子濃度之變化......................9
(三) 氧化壓力(oxidative stress)........................10
1. 氧化壓力對細胞之損害................................10
二、維生素E ( Vitamin E )..............................14
(一) 維生素E之起源及結構...............................14
(二) 維生素E之生理功能.................................16
三、Calpain system.....................................18
(一) Calpains活化的調節機制............................24
(二) Calpains與屠後肌肉肌原纖維蛋白質降解之關係........24
四、添加外源性鈣離子對屠後肌肉肌原纖維蛋白質降解之影響.25
參、材料與方法.........................................27
一、試驗材料之製備 .....................................27
試驗I:華鵝及白羅曼鵝屠後胸肌及腿肌肌原纖維蛋白質降解之變化 ..............................................27
試驗II:日糧補充維生素E對白羅曼鵝屠後胸肌及腿肌肌原纖維蛋白質降解之影響...........................................27
驗III:乳酸鈣浸漬(5˚C)對日糧補充維生素E之白羅曼鵝屠後腿肌之影響...................................................28
二、試驗方法...........................................29
(一)pH值測定..........................................29
(二)肌原纖維製備.......................................29
(三)蛋白質濃度之測定...................................29
(四)肌原纖維之電泳( SDS-PAGE )測定.....................30
(五)Western blotting...................................31
(六) 還原及氧化態麩胱甘肽(glutathione, GSH/GSSG)含量之分析.....................................................31
肆、結果與討論.........................................34
試驗I:華鵝及白羅曼鵝屠後胸肌及腿肌肌原纖維蛋白質降解之變化.....................................................34
一、華鵝及白羅曼鵝屠後胸肌及腿肌 pH 之變化.............34
二、華鵝及白羅曼鵝屠後胸肌及腿肌肌原纖維蛋白質之變化...34
三、華鵝及白羅曼鵝屠後胸肌及腿肌Titin之變化............39
四、華鵝及白羅曼鵝屠後胸肌及腿肌Desmin之變化...........42
五、華鵝及白羅曼鵝屠後胸肌及腿肌Vinculin之變化.........45
六、華鵝及白羅曼鵝屠後胸肌及腿肌α-actinin之變化........48

試驗II:日糧補充維生素E對白羅曼鵝屠後胸肌及腿肌肌原纖維蛋白質降解之影響...........................................52
一、飼糧補充維生素E對白羅曼鵝屠後胸肌及腿肌 pH 之影響..52
二、飼糧補充維生素E對白羅曼鵝屠後胸肌及腿肌還原態麩胱甘肽(GSH)與氧化態麩胱甘肽(GSSG)比之影響....................52

三、飼糧補充維生素E對白羅曼鵝屠後胸肌及腿肌肌原纖維蛋白質降解之影響................................................55
四、飼糧補充維生素E對白羅曼鵝屠後胸肌及腿肌Titin之影響..55
五、飼糧補充維生素E對白羅曼鵝屠後胸肌及腿肌Desmin之影響.60
試驗III:乳酸鈣浸漬(5˚C)對日糧補充維生素E之白羅曼鵝屠後腿肌肌原纖維蛋白質降解之影響................................65
一、乳酸鈣浸漬對日糧補充維生素E之白羅曼鵝屠後腿肌pH 之影響......................................................65
二、乳酸鈣浸漬(5˚C)對日糧補充維生素E之白羅曼鵝屠後腿肌肌原纖維蛋白質降解之影響....................................65
三、乳酸鈣浸漬(5˚C)對日糧補充維生素E之白羅曼鵝屠後腿肌Titin之影響..................................................69
四、乳酸鈣浸漬(5˚C)對日糧補充維生素E之白羅曼鵝屠後腿肌 Desmin之影響............................................69
伍、結綸................................................76
陸、參考文獻............................................77
附錄一.................................................107










圖 次
頁次
圖1、胞內鈣離子穩衡機制 5
圖2、維生素E之基本結構 15
圖3、α-tocopherol與過氧化自由基之反應 17
圖4、抗壞血酸鹽對α-tocopherol之再生作用 17
圖5、μ及m-Calpains之胺基酸序列簡圖 19
圖6、m-Calapin之晶體結構圖 20
圖7、華鵝及白羅曼鵝屠後胸肌及腿肌 pH 之變化 36
圖8、華鵝屠後胸肌及腿肌肌原纖維蛋白質之電泳分析 37
圖9、白羅曼鵝屠後胸肌及腿肌肌原纖維蛋白質之電泳分析 38
圖10、華鵝屠後胸肌及腿肌之Titin之變化 40
圖11、白羅曼鵝屠後胸肌及腿肌之Titin之變化 41
圖12、華鵝屠後胸肌及腿肌Desmin之變化 43
圖13、白羅曼鵝屠後胸肌及腿肌Desmin之變化 44
圖14、華鵝屠後胸肌及腿肌Vinculin之變化 46
圖15、白羅曼鵝屠後胸肌及腿肌Vinculin之變化 47
圖16、華鵝屠後胸肌及腿肌α-Actinin之變化 49
圖17、白羅曼鵝屠後胸肌及腿肌α-Actinin之變化 50
圖18、飼糧補充維生素E對白羅曼鵝屠後胸肌及腿肌 pH 之影響
…………………………………………………………………53
圖19、飼糧補充維生素E對白羅曼鵝屠後胸肌及腿肌 GSH與GSSG 比之影響 54

圖20、飼糧補充維生素E對白羅曼鵝屠後胸肌肌原纖維蛋白質之影響 56
圖21、飼糧補充維生素E對白羅曼鵝屠後腿肌肌原纖維蛋白質之影響 57
圖22、飼糧補充維生素E對白羅曼鵝屠後胸肌Titin之影響 58
圖23、飼糧補充維生素E對白羅曼鵝屠後腿肌Titin之影響 59
圖24、飼糧補充維生素E對白羅曼鵝屠後胸肌Desmin之影響 61
圖25、飼糧補充維生素E對白羅曼鵝屠後腿肌Desmin之影響 62
圖26、鈣浸漬對日糧補充維生素E之白羅曼鵝屠後腿肌pH 之影響 66
圖27、乳酸鈣浸漬(5℃)對屠後腿肌肌原纖維蛋白質降解之影響 67
圖28、乳酸鈣浸漬(5℃)對日糧補充維生素E之屠後腿肌肌原纖維蛋白質降解之影響 68
圖29、乳酸鈣浸漬(5℃)對屠後腿肌Titin之影響 70
圖30、乳酸鈣浸漬對日糧補充維生素E之屠後腿肌Titin之影響
71
圖31、乳酸鈣浸漬對屠後鵝腿肌Desmin之影響 72
圖32、乳酸鈣浸漬對日糧補充維生素E之屠後鵝腿肌Desmin之影響 73
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