臺灣博碩士論文加值系統

肉品之色澤安定性為目前業界與消費者所重視的問題。而肌肉之色素主要有血紅素 (Hemoglobin，Hb) 及肌紅素 (Myoglobin，Mb)，但當動物被屠宰放血後，Mb 則為形成肉色之主要物質。肌肉中還原型 Mb 為紫色，而氧合型的 oxyMb 則為鮮紅色，兩者呈平衡狀態。肉色改變主要是因 Mb 之還原型鐵被氧化為氧化型之 metMb 所致。本研究之目的在探討 metMb 還原對紅肉魚肉色安定性之影響，並藉以瞭解 metMb 還原對維持紅肉魚色澤之機制。
為瞭解鮪魚在冷藏過程中色澤安定性與肌肉中 metMb 還原之關係，新鮮藍鰭鮪 (切成 3×2×0.5 cm 大小) 以 PE 膜包裝及不包裝之樣品貯藏於4C，測定 metMb 還原活性的變化、metMb 生成速率、NADH 及 NADPH 量之變化。結果顯示兩組樣品在 120 小時貯藏中，Hunter a 值下降而L值上升，metMb 還原活性隨貯藏時間延長而下降，反之 metMb 量則隨貯藏時間之延長而上升。PE 膜包裝組，其 NADH 含量在貯藏 56 至 80 小時期間呈增加之趨勢，隨後則快速下降；然而，不包裝組，貯藏至 56 小時，其 NADH 增至最高量。在貯藏期間，雖兩組樣品之 NADPH 量均呈下降之趨勢，但貯藏 80 小時內，NADH 和 NADPH 的總量大概為一穩定值，之後始產生明顯的下降。由上述結果，顯示 metMb 還原活性與冷藏中鮪魚肉的 metMb 蓄積量呈負相關。
進一步探討 metMb 還原對肌肉色澤之影響。藍鰭鮪肌肉以磷酸緩衝液萃取得到 metMb 還原之粗酵素液，並經硫酸銨分劃、DEAE 離子交換層析、G-75 膠過濾層析及 5’-AMP-Sepharose 4B 親和性層析等步驟而獲得純化。其分子量為 100 kDa，最適酸鹼值及溫度分別為 7.3 及 25C，酸鹼值及溫度安定性為 7.0-7.3 及 4-15C。此純化酵素被 K+、Cd2+、Sr2+ 和 Ni2+ 致活，Mn2+ 和 Ca2+ 之致活能力較小，Na+ 則不影響酵素活性，但被 Li+、NH4+、Mg2+、Zn2+ 和 Co2+ 抑制，而 Cu2+ 和 Fe2+ 能完全抑制該酵素活性。 MetMb 還原對氧化肌紅蛋白及 NADH 之最大反應速率 (Vmax) 為 0.32 和 0.43 mmol/min/mg，Km 值為 2.3 X 10-5 和 24.4 X 10-5 M。
在探討 metMb 還原之還原能力，將褐變鮪魚肉片浸泡在部分純化之 metMb 還原溶液 (0.103 unit/ml)，於 4C 浸泡 120 分鐘。浸泡 10 分鐘之褐變鮪魚肉片較之對照組，酵素活性明顯增加。比較 metMb 還原活性、metMb 含量、Hunter a 值及外觀色澤，該酵素確能使褐變鮪魚肉片恢復成紅色。為進一步確定 metMb 還原對色澤之安定效應，將新鮮鮪魚肉片浸泡在部分純化之 metMb 還原溶液 (0.503 unit/ml) 及添加 NADH (1 mM) 於此酵素液中，在 4C 浸泡 10 分鐘，並貯藏在 4C、相對濕度 100﹪ 的環境下。浸泡酵素液及酵素混合 NADH 之溶液，其 metMb 還原活性及 metMb 含量沒有很大的差異性。比較控制組與浸泡組之 metMb 還原活性、metMb 含量、Hunter a 值及外觀色澤之變化，結果顯示 metMb 還原確能延長鮪魚肉色澤的安定性，而能將冷藏中的 metMb 進行酵素性還原。

The stability of meat color has long been concerned by meat industry and consumers. The major pigments in muscle are hemoglobin (Hb) and myoglobin (Mb). However, after being slaughtered, Mb would become the major pigment. In tissue, the reduced form of Mb, which is purple, exists in equilibrium with its oxygenated form of oxyMb. Browning of meat is considered to be due to the oxidation of iron from reduced- into oxidized-form. This study aims to investigate the effect of metMb reductase on color stability of red-flesh tuna, which consequently elucidate the role of metMb reductase in the color stability of red meats.
To investigate the accumulation of pigments in relation to the decrease in metMb reductase activity, bluefin tuna flesh cubes(Thunnus thynnus) (3 X 2 X 0.5 cm / piece) were placed on a polyethylene (PE) tray with/without wrapping with PE film. Both samples were then stored at 4C under a relative humidity (RH) of 100% for 120 h, during which the change in metMb reductase activity, the rate of metMb formation, and the changes in amounts of NADH and NADPH were measured.
Decrease in Hunter a value and increase in L value were observed for both samples during 120-h storage. MetMb reductase activity of both samples decreased, while the metMb increased with the duration of storage. The NADH of tuna meat increased during the first 56-80 h of storage, but decreased during the prolonged storage. Although the NADPH of both wrapped and unwrapped samples decreased, the total amount of NADH and NADPH seemed to keep a constant level during the first 80-h storage and then significantly decreased. These results suggested that metMb reductase might be an important factor affecting the metMb accumulation in refrigerated tuna.
For further investigation of the effect of metMb reductase on color stability, the reductase was purified to electrophoretical homogeneity from ordinary bluefin tuna muscle by ammonium sulfate fractionation, ion exchange and organomercurial agarose affinity chromatographies. The molecular mass (M) was estimated to be 100 kDa, while the optimal pH and temperature were 7.3 and 25C, respectively. This enzyme was stable at a pH range of 7.0-7.3 and a temperature of 4-15C. It was strongly activated by K+, Cd2+, Sr2+and Ni2+, moderately activated by Mn2+and Ca2+, but not affected by Na+. However it was moderately inhibited by Li+, NH4+, Mg2+, Zn2+ and Co2+, and completely inhibited by Cu2+and Fe2+. The Vmax for the reduction of met-Mb and NADH were 0.32 and 0.43 mmol/min/mg, while the Km were 2.3 X 10-5 and 24.4 X 10-5 M for metMb and NADH, respectively.
To investigate the metMb reducing ability of metMb reductase, the browned tuna meat was immersed with/without metMb reductase (0.103 unit/ml) solution for 120 min at 4C. The metMb reductase activity of browned tuna meat significantly increased, while no significant change was observed on control sample during the first 10 min immersion. Comparing the changes in metMb reductase activity, metMb content, Hunter a value and color, this reductase did recover the bright red color of the meat. To further confirm the role of metMb reductase in color stability, fresh tuna meat was first immersed in metMb reductase (0.503 unit/ml) solution or a mixture of the reductase and NADH (1 mM) solution at 4C for 10 min, and then stored at 4C, RH 100%. No significant change in metMb accumulation and in metMb reductase activity was observed between samples treated with enzyme alone and enzyme + NADH. The changes in metMb reductase activity, metMb, Hunter a value and color of both control and treated samples suggested that immersion of metMb reductase could extend the color stability of tuna meat, and the enzymatic reduction of metMb by its reductase occurred during refrigerated storage.

封面
名詞縮寫
中文摘要
英文摘要
第一章　前言
第二章　文獻整理
一、消費者之意識形態
二、肌肉組織中色素之組成及形態
三、影響肌肉變色之因數
1、內生性因素
1）肌肉部位間之差異性
2）硬直現象發生及最終酸檢值
2、外在因素
1) 電擊刺激
2) 溫體去骨作業
3) 冷卻速率
3、操作技術上的因素
1) 溫度
2) 氧分壓
3) 光之形態
4) 細菌污染
4、肌紅蛋白氧化還原機制
1) 肌紅蛋白之自氧化作用
2) 氧化肌紅蛋白還原?活性
四、參考文獻
五、假說
六、實驗設計
第三章 低溫貯藏之鮪魚肌肉其氧化肌紅蛋白還原?活性與肉品色澤之關連性
一、摘要
二、前言
三、材料與方法
1、原料
2、藥品
3、儀器
4、方法
1) 酸?值之測定
2) 魚肉顏色之測定
3) 氧化肌紅蛋白含量之測定
4) metMb還原?之粗酵素液製備及活性測定
5)還原態芋鹼胺腺二核?酸及還原態芋鹼醯胺腺二核?酸磷酸之測定
四、結果與討論
1、酸鹼什與Hunter L.a.b值之變化
2、氧化肌紅蛋白
3、酵素活性
4、還原態芋鹼醯胺腺二核?酸及還原態芋鹼醯胺腺二核?酸磷
五、結論
六、參考文獻
七、圖表
第四章 鮪魚肌肉中氧化肌紅蛋白還原?之純化與特性探討
一、摘要
二、前言
三、材料與方法
1、原料
2、藥品
3、儀器
4、方法
1) metMb還原?之純化
2) metMb還原?純度鑒定
3) metMb 還原?生化特性試驗
(A) 分子量測定
(B) 最適酸鹼度
(C) 酸鹼度安定性
(D) 最 適溫度
(E) 熱安定性
(F) 金屬離子的效應
(G) 金屬離子整合劑及Shblock agent等化合物之影響
(H) 動力學參數之計算
4) 蛋白質定量
5) 純化酵素量之測定
四、結果與討論
1、純化方法
2、酸鹼值及溫度效應
3、金屬離子效應
4、金屬離子整合劑及SH block agent等化合物之影響
5、分子量
6、酵素動力學
五、結論
六、參考文獻
七、圖表
第五章 鮪魚肌肉中氧化肌紅蛋白還原?對冷藏鮪魚肉色澤安定性之影響
一、摘要
二、前言
三、材料與方法
1、原料
2、藥品
3、儀器
4、方法
1)、netMb 還原?之純化
2)、低溫貯存之安定性
3)、還原劑之影響
4)、酸鹼值之測定
5)、魚肉顏色之測定
6)、氧化肌紅蛋白含量之測定
7)、netMb 還原?之粗酵素液製備及活性測定5
5、統計分析
四、結果與討論
1、低溫貯藏中metMb還原?活動之變化
2、metMb還原?對褐變鮪魚肉氧化肌紅蛋白還原之影響
3、netMb還原?對新鮮鮪魚肉色澤安定性之影響
4、討論
五、結論
六、參考文獻
七、圖表
第六章 綜合討論
未來研究方向

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