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研究生中文姓名:陳亭屹
研究生英文姓名:Chen, Ting-Yi
中文論文名稱:鯖魚蒸煮液蛋白質鑑定及其潛在活性胜肽之特徵化分析
英文論文名稱:Characterization of Proteins and Potential Bioactive Peptides from Mackerel (Scomber australasicus) Steaming Juice
指導教授姓名:張祐維
口試委員中文姓名:教授︰蘇南維
副教授︰陳泰源
副教授︰宋文杰
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學號:10532026
請選擇論文為:學術型
畢業年度:106
畢業學年度:105
學期:
語文別:中文
論文頁數:78
中文關鍵詞:鯖魚蒸煮液蛋白質鑑定BIOPEP活性胜肽
英文關鍵字:Mackerel steaming juiceProtein identificationBIOPEPBioactive peptides
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鯖魚罐頭在生產的過程會經過蒸煮的步驟,蒸煮的目的為使魚肉軟化。平均一公噸的鯖魚會產生約 150公斤的鯖魚蒸煮液。這些大量的蒸煮液一般被當作廢水排出。然而鯖魚蒸煮液中含有豐富的蛋白質值得被研究和利用。本研究目的為尋找鯖魚蒸煮液副產物經濟價值,利用蛋白質體學技術鑑定鯖魚蒸煮液中的蛋白質,並且藉由酵素水解的方式探討鯖魚蒸煮液中的潛在活性胜肽。經過前處理的鯖魚蒸煮液凍乾粉末,以十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 分析分子量分佈。其次以蛋白質體學技術進行蛋白質鑑定,鯖魚蒸煮液中含有肌凝蛋白重鏈、肌凝蛋白輕鏈、原肌球蛋白 α-1 鏈、肌動蛋白、膠原蛋白、β - 烯醇化酶、果糖二磷酸醛縮酶、甘油醛-3-磷酸氫化酶等蛋白質。將被鑑定出來的蛋白質,以 BIOPEP 進行潛在活性胜肽分析和模擬酵素水解。結果顯示鯖魚蒸煮液可能具有降血糖、抗高血壓、抗氧化、抗血栓、抗健忘等活性胜肽,其中以抗高血壓和降血糖的機率較高。鯖魚蒸煮液以六種不同的酵素 (Papain, Pepsin, Thermolysin, Proteinase K, Bromelain, Alcalase) 在其最適條件下進行水解,並測定 1,1-二苯基-2-三硝基苯肼 (2,2-diphenyl-1-picrylhydrazyl, DPPH) 自由基清除能力、亞鐵螯合能力、還原力和血管收縮素Ⅰ轉化酶 (Angiotensin-converting enzyme, ACE) 抑制活性。Pepsin 水解物 (5 mg/mL) 具有最高的 DPPH 自由基清除力 (61.54%);Alcalase 水解物 (5 mg/mL) 具有最高亞鐵螯合能力 (89.76%);Proteinase K 水解物 (5 mg/mL) 具有最高還原力 (1.52 abs);Pepsin 水解物 (0.1 mg/mL) 具有最高 ACE 抑制率 (86.15%)。綜合上述結果,鯖魚蒸煮液具有製備抗氧化胜肽和血管收縮素轉換酶抑制胜肽的潛力,未來可以進一步純化分析其中的抗氧化胜肽和血管收縮素轉換酶抑制胜肽,並且定序其胺基酸組成。
The production of canned mackerel accounts for 80% of canned fishery products in Taiwan. Furthermore, steaming is an important process in canning fish to soften fish meats and bones. Generally, one ton of mackerel will produce roughly 150 kg of mackerel steaming juice (MSJ). MSJ can be a good source of soluble proteins; however, it is often regarded as a waste product. Therefore, the objective of this study was to investigate the potential bioactive peptides from MSJ and to increase its added value. Proteins obtained from MSJ were identified by proteomic techniques, followed by sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in-gel digestion and tandem mass spectrometry. Myosin heavy chain (fast skeletal muscle), actin, myosin light chain 1 (skeletal muscle isoform), collagen alpha-2(I) chain, tropomyosin alpha-1 chain, beta-enolase, fructose-bisphosphate aldolase A, and glyceraldehyde-3- phosphate dehydrogenase were identified by proteomic analysis. Identified protein sequences were further analyzed using BIOPEP database. The results obtained from BIOPEP database showed that MSJ had potential activities as dipeptidyl peptidase IV inhibitor, ACE inhibitor, antioxidant, antithrombosis, and anamnesis. MSJ was then hydrolyzed using six proteases (Papain, Pepsin, Proteinase k, Alcalase, Bromelain, Thermolysin). In particular, Pepsin hydrolysate (5 mg/mL) showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (61.54%) among others. Alcalase hydrolysate (5 mg/mL) exhibited the highest metal chelating activity (89.76%) and Proteinase K hydrolysate (5 mg/mL) indicated the highest reducing power (1.52 abs), respectively.Moreover, Pepsin hydrolysate (0.1 mg/mL) possessed the highest Angiotensin-converting enzyme (ACE) inhibitory activity (86.15%). The results showed that MSJ hydrolysates had potential to produce antioxidant and ACE inhibitory peptides.
壹、前言.................................................1
貳、文獻回顧.............................................2
一、鯖魚簡介.............................................2
二、水產罐頭加工.........................................2
1.鯖魚罐頭...............................................2
三、蒸煮液簡介...........................................3
1.蒸煮液成分成...........................................3
2.蒸煮液的利用...........................................4
四、蛋白質水解物.........................................7
1.抗氧化活性.............................................7
2.抗高血壓活性...........................................7
3.抗菌活性..............................................8
五、蛋白質鑑定技術.......................................8
1.蛋白質分離技術.........................................8
2.蛋白質分解.............................................9
3.質譜儀分析與資料庫比對.................................10
參、實驗架構............................................11
肆、實驗材料與方法......................................12
一、實驗材料與藥品......................................12
1.材料.................................................12
2.酵素.................................................12
二、實驗方法............................................12
1.一般成分分析 (A.O.A.C., 2003).........................12
2.pH 值及鹽度測定.......................................14
3.樣品前處理............................................14
4.蛋白質萃取............................................14
5.電泳分析..............................................15
6.蛋白質鑑定............................................15
7.生物資訊工具分析......................................17
8.酵素水解物製備........................................17
9.抗氧化活性分析........................................18
10.血管收縮素轉換酶 (ACE) 抑制能力.......................19
11.統計分析.............................................19
伍、實驗結果............................................20
一、鯖魚蒸煮液成分分析...................................20
二、鯖魚蒸煮液蛋白質鑑定.................................20
三、鯖魚蒸煮液 BIOPEP 資料庫分析.........................22
四、鯖魚蒸煮液酵素解物抗氧化和血管收素轉換酶抑制能力分析....23
1.水解物特徵分析........................................23
2.抗氧化能力測定........................................24
3.抗高血壓能力測定......................................25
陸、結論................................................27
柒、參考文獻............................................28
附錄...................................................69

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