尖吻鱸魚 (Lates calcarifer) 為台灣重要養殖漁業的重要魚種之一，且目前全世界對尖吻鱸魚的需求量仍穩定增加。由於亞洲國家加工尖吻鱸魚多為魚片切肉，因此在加工過程中同時會產生大量的加工副產物，其中包括魚骨。因此本研究將以尖吻鱸魚之魚骨進行蛋白質之萃取並進行鑑定及探討其水解物之生理活性。先以酒精、NaOH 去除魚骨的脂質與雜質，並透過酸鹼處理萃取蛋白質分離物，使用十二烷基硫酸鈉聚丙烯酰胺凝膠電泳 (SDS – PAGE) 分離蛋白質，再經過膠內消化與萃取後，以質譜儀技術鑑定與經過 Mascot 資料庫比對出魚骨蛋白質分離物的蛋白質，含有 α 骨骼肌 A 肌動蛋白、原肌凝蛋白 α-1、骨骼肌 3 肌球輕鏈蛋白以及骨骼肌 3 肌球輕鏈蛋白 A 型等組成。使用 BIOPEP-UWM 模擬上述蛋白質之潛在活性胜肽與選擇最適水解酵素，增加本次實驗效率。魚骨胃蛋白酶水解物及魚骨木瓜蛋白酶水解物在經過 6 個小時水解率分別為22.41 ± 0.41 % 以及14.27 ± 1.96 % 。而在最佳水解率下，魚骨胃蛋白酶水解物之抗氧化能力上升為 1,1-二苯-2-三硝苯肼 (DPPH) 自由基清除能力 39.26 ± 1.44 % 、金屬離子螯合活性 69.98 ± 1.55 % 以及2,2-聯氮-雙-3-乙基苯並噻唑啉-6-磺酸 (ABTS) 自由基清除活性 37.85 ± 2.44 % 。魚骨木瓜蛋白酶水解物之抗氧化能力上升為 DPPH 自由基清除能力 29.09 ± 0.97 % 、金屬離子螯合活性 66.09 ± 2.55 % 以及 ABTS 自由基清除活性 38.77 ± 1.24 % 。而魚骨胃蛋白酶水解物及魚骨木瓜蛋白酶水解物之血管收縮素Ⅰ轉化酶 (ACE) 抑制能力上升到 36.02 ± 0.10 % 及 13.40 ± 0.18 % 。綜上所述，尖吻鱸魚魚骨具有抗氧化以及 ACE 抑制能力之活性胜肽，有潛力作為功能性食品，未來可以經由動物實驗來佐證其功效，以增加其附加價值。

Barramundi (Lates calcarifer) is one of the important species of Taiwan's important aquaculture fisheries. And the world's current consumption of barramundi. Demand is still increasing steadily. Since the processing of barramundi in Asian countries is mostly fillets and meat, a large number of processing by-products, including fish bones, are also produced during the processing. Therefore, in this study, the fish bone of barramundi was used for protein extraction, identification and physiological activity of its hydrolysate. First, the lipids and impurities of fish bones were removed with alcohol and NaOH, and the protein isolates were extracted by acid-base treatment. The proteins were separated by SDS-PAGE, and after in-gel digestion and extraction, they were identified by mass spectrometry and compared with the Mascot database. The protein of fishbone protein isolate was composed of α skeletal muscle A actin, tropomyosin α-1, skeletal muscle myosin light chain protein 3 and skeletal muscle myosin light chain protein 2. Use BIOPEP-UWM to simulate the potential active peptides of the above proteins and select the most suitable hydrolytic enzymes to increase the efficiency of this experiment. The hydrolysis rates of fish bone pepsin hydrolysate and fish bone papain hydrolysate after 6 hours were 22.41 ± 0.41 % and 14.27 ± 1.96 %, respectively. Under the optimal hydrolysis rate, the antioxidant capacity of fish bone pepsin hydrolysate increased to 39.26 ± 1.44 % for DPPH free radical scavenging activity, 69.98 ± 1.55 % for metal ion complexing activity and 37.85 ± 2.44 % for ABTS free radical scavenging activity. The antioxidant capacity of fishbone papain hydrolysate increased to 29.09 ± 0.97 % of DPPH free radical scavenging activity, 66.09 ± 2.55 % of metal ion complex activity and 38.77 ± 1.24 % of ABTS free radical scavenging activity. The ACE inhibitory ability of fish bone pepsin hydrolysate and fish bone papain hydrolysate increased to 36.02 ± 0.10 % and 13.40 ± 0.18 %. To sum up, barramundi fish bones have active peptides with antioxidant and ACE inhibitory capabilities, and have the potential to be used as functional foods. In the future, animal experiments can be used to prove their efficacy to increase their added value

中文摘要 i
英文摘要 ii
圖文摘要 iii
壹、前言 1
貳、文獻回顧 2
一、尖吻鱸魚簡介 2
二、副產物分離蛋白 3
三、蛋白質 3
1.分離蛋白 3
2.明膠 4
3.肌原纖維蛋白 4
3.1肌動蛋白 4
3.2肌凝蛋白 4
四、蛋白質水解 5
1.化學水解 5
1.1鹼水解 5
1.2酸水解 5
1.3酵素水解 6
五、活性胜肽 6
1.抗氧化活性 7
1.1氧化壓力 7
1.2抗氧化劑 7
2.抗高血壓活性 8
2.1高血壓 8
2.2抗高血壓 8
3.抗血栓活性 9
3.1凝血作用 9
3.2血栓凝集抑制 9
六、蛋白質鑑定 10
1.胜肽質量指紋 10
2.串聯式質譜法 10
參、實驗架構 12
肆、實驗材料與方法 14
一、實驗材料與藥品 14
二、實驗方法 14
1.一般成分分析 14
2.魚骨蛋白質萃取製備 14
3.電泳分析 15
3.1膠片製作 15
3.2樣品製備 15
3.3電泳 15
4.蛋白質鑑定 16
4.1膠片裁切 16
4.2膠內消化 16
4.3胜肽萃取 16
4.4液相層析串聯質譜儀分析 17
4.5資料庫比對與蛋白質鑑定 17
5.生物資訊工具分析 18
6.蛋白質酵素水解物製備 18
6.1水解物產率 18
6.2 水解度 (DH) 18
6.3胜肽含量測定 19
7.抗氧化活性測試 19
7.1 DPPH抗自由基測試 19
7.2 ABTS自由基清除能力測試 19
7.3亞亞鐵離子螯合能力測試 20
8.抑制血管收縮轉換酵素活性測試 20
8.1 ACE抑制活性測試 20
9.統計分析 21
伍、結果與討論 22
一、尖吻鱸魚魚骨成份分析及產率 22
二、尖吻鱸魚魚骨蛋白質萃取 22
三、尖吻鱸魚魚骨蛋白質鑑定 22
四、尖吻鱸魚魚骨水解物分析 24
五、尖吻鱸魚魚骨經水解後對於生理活性之影響 25
1. DPPH自由基清除活性 25
2.金屬離子螯合活性 25
3. ABTS自由基清除活性 26
4. ACE抑制活性 26
陸、結論 28
柒、未來研究方向 29
捌、參考文獻 30
玖、圖表 3

劉汝晏。 (2009)。 探討鱸魚與吳郭魚對正常及糖尿病大鼠創傷修復之影響。 靜宜大學食品營養學系碩士論文。 臺中市，台灣。
朱衛星。 (2012)。 豬肉肌原纖維蛋白氧化其凝膠特性的變化研究。 國醫藥衛生科際。湖南省，中國。
中研院。 (2022)。台灣魚類資料庫，台北市，台灣。
Kwankijudomkul, A., Dong, H. T., Longyant, S., Sithigorngul, P., Khunrae, P., Rattanarojpong, T., & Senapin, S. (2021). Antigenicity of hypothetical protein HP33 of vibrio harveyi Y6 causing scale drop and muscle necrosis disease in asian sea bass. Fish and Shellfish Immunology, 108, 73-79.
Ahmad, M., & Benjakul, S. (2010). Extraction and characterisation of pepsinsolubilised collagen from the skin of unicorn leatherjacket (Aluterus monocerous). Food Chemistry, 120, 817-824.
Antolovich, M., Prenzler, D. P., Patsalides, E., McDonald, S., & Robards, K. (2002). Methods for testing antioxidant activity. School of Science and Technology, America.
Antonios, T. F. T., & Macgreger, G. A. (1995). Angiotensin-converting enzyme-inhibitor in hypertension- potential problems. Journal of Hypertension, 13, S11-S16.
Bruce, A., Alexander, J., Julian, L., Martin, R., Keith, R., & Peter, W. (2002). Molecular biology of the cell. Garland Science. New York, America.
Chakrabarti S., Jahandideh, F., & Wu, J. (2014). Food-derived bioactive peptides on inflammation and oxidative stress. BioMed Research International, 2014, 11.
Chen, J., Li, L., Yi, R., Xu, R., Gao, R., & Hong, B. (2016). Extraction and characterization of acid-soluble collagen from scales and skin of tilapia (Oreochromis niloticus). Food Science and Technology, 66, 453-459.
Chen, Y. J., & Liao, H. K. (2003). Mass spectrometry-based proteomics. Chemistry, 61(2), 235-247.
F. A. O. (2022). https://www.fao.org/home/en/ , Statistics, Lates Calcarifer.
Guérard, F., Dufossé, L., Broise, D., & Binet, A. (2001). Enzymatic hydrolysis of protein from yellow fin tuna (Thumus albacares) wastes with alcalase. Journal of Molecular Catalysis B: Enzymatic, 11, 1057-1059.
Gulcin, I. (2020). Antioxidants and antioxidant methods: An updated overview. Archives of Toxicology, 94(3), 651-715.
Gupta, A., Mann, B., Kumar, R., & Sangwan, B. R. (2009). Antioxidant activity of Cheddar cheeses at different stages of ripening. International Journal of Dairy Technology, 62(3), 339-347.
Huang, B., Lin, C., & Chang, W. (2015). Analysis of proteins and potential bioactive peptides from tilapia (Oreochromis spp.) processing co-products using proteomic techniques coupled with BIOPEP database. Journal of Functional Foods, 19, Part A, 629-640.
Huang, C. Y., Kuo, J. M., Wu, S. J., & Tsai, H. T. (2016). Isolation and characterization of fish scale collagen from tilapia (Oreochromis sp.) by a novel extrusion–hydro-extraction process. Food Chemistry, 190, 997-1006.
Huang, H., & Huang, G. (2020). Extraction, separation, modification, structural characterization, and antioxidant activity of plant polysaccharides. Chemical Biology & Drug Design, 96(5), 1209-1222.
Identification and release behavior of ACE-inhibitory peptides. Lebensmittel-Wissenschaft & Technologie, 139, 110502.
James, R. S. (2000). Myosins: A diverse superfamily. Biochimica et Biophysica Acta, 1496(1), 3-22.
Jongiareonrak, A., Benjakul, S., Visesssanguan, W., Nagai, T., & Tanaka, M. (2005). Isolation and characterisation of acid and pepsin-solubilised collagens from the skin of Brownstripe red snapper (Lutjanus vitta). Food Chemistry, 93, 475-484.
Kaewsahnguan, T., Noitang, S., Sangtanoo, P., Srimongkol, P., Saisavoey, T., Reamtong, O., Choowongkomon, K., & Karnchanatat, A. (2021). A novel angiotensin I-converting enzyme inhibitory peptide derived from the trypsin hydrolysates of salmon bone proteins. Plos One, 16(9), e0256595-e0256595.
Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., Nagai, T., & Tanaka, M. (2005). Characterisation of acid soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food Chemistry, 89, 363-372.
Kohama, Y., Matsumoto, S., Oka, H., Teramoto, T., Okabe, M., & Miura, T. (1988). Isolation of angiotensin-converting enzyme inhibitor from tuna muscle. Biochemical and Biophysical Research Communications, 155, 332-337.
Kuba, M., Tana, C., Twata, S., Yasuda, M. (2005). Production of anigiotensin I-converitng enzyme inhibitory peptides from soybean protein with monuascus purpureus acid proteinase. Process Biochemistry, 40, 2191-2196.
Lander, E. S. (1996). The new genomics: Global views of biology. Science, 274, 536-539.
Lin, H. C., Alashi, A. M., Aluko, R. E., Pana, B. S., & Chang Y. W. (2017). Antihypertensive properties of tilapia (Oreochromis spp.) frame and skin enzymatic protein hydrolysates. Food & Nutrition Research, 61, 1391666.
Lu, Z., Li, L. X., & Zhang, F. (2013). Study on extraction process parameters of acid soluble collagen from grass carp scales. Food Science and Technology, 38(5), 250-254.
Maruyama, S., & Suzuki, H. (1982). A peptide inhibitor of antiotensin I-converting enzyme in the triptic hydrolysate on casein. Agricultural and Biological Chemistry, 46, 1393-1394.
Masaaki, T., Mana, Oe., Karin, O., & Saki, M. (2011). Inhibition strength of short peptides derived from an ACE inhibitory peptide. Journal of Agricultural and Food Chemistry, 59, 11234-11237.
Matsufuji, H., Matsui, T., Seki, E., Osajima, K., Nakashima, M., Osajima, Y. (1994). Angiotensin I-converitng enzyme inhibitory peptides in an alkaline protease hydrolysate derived from sardine muscle. Bioscience, Biotechnology, and Biochemistry, 58, 2244–2245.
Miyoshi, S., Ishikawa, H., Kaneko, T., Fukui, F., Tanaka, H., & Maruyama, S. (1999). Structure and activity of angiotensin I-converting enzyme inhibitors in a α-zein hydrolysate. Agricultural and Biological Chemistry, 55, 1313-1318.
Mo, W. & Karger, B.L. (2002). Analytical aspects of mass spectrometry and proteomics. Current Opinion in Chemical Biology, 6(5), 666-675.
Muyonga, J. H., Cole, C. G. B., & Duodu K. G. (2004). Extraction and physico-chemical characterisation of Nile perch (Lates niloticus) skin and bone gelatin. Food Hydrocolloids, 18(4), 581-592.
Ogawa, M., Schexnayder, M., & Portier, J. R. (2004). Biochemical properties of bone and scale collagens isolated from the subtropical fish black drum (Pogonia Cromis) and sheepshead sea bream (Archosargus probatocephalus). Journal of Agricultural and Food Chemistry, 51, 8088-8092.
Owens, P., & Mackman, N. (2010). Tissue factor and thrombosis: The clot starts here. Thrombosis and Haemostasis, 104(3), 432-439.
Piazza, G., Seddighzadeh, A., & Goldhaber, SZ. (2008). Heart failure in patients with deep vein thrombosis. The American Journal of Cardiology, 101, 1056-1059.
Poulter, R. N., Prabhakaran, D., & Caulfield, M. (2015). Hypertension. The Lancet Journal, 386, 801-812.
Richard, E. C., & Mark, S. M. (1992). Unconventional myosins. Current Opinion in Cell Biology, 4(1), 27-35.
Rustad, T., Storrø, I., & Slizyte, R. (2011). Possibilities for the utilisation of marine by-products. International Journal of Food Science & Technology, 46(10), 2001-2014.
Sayer, G., & Bhat, G. (2014). The renin-angiotensin-aldosterone system and heart failure. Cardiology clinics, 32(1), 21-32.
Shahidi, F. (1994). Proteins from seafood processing discards. Seafood Proteins, 171-193.
Shahidi, F. (2000). Antioxidants in foods and food antioxidants. Molecular Nutrition and Food Research, 44(3), 158–163.
Shahidi, F., Han, X.-Q., & Synowiecki, J. (1995). Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chemistry, 53(3), 285-293.
Shen, Y., Maupetit, J., Derreumaux, P., & Tufféry, P. (2014). Improved PEP-FOLD approach for peptide and miniprotein structure prediction. Journal of Chemical Theory and Computation, 10(10), 4745-4758.
Shimizu, M., Sawashita, N., Morimatsu, F., Ichikawa, J., Taguchi, Y., Ijiri, Y., & Yamamoto, J. (2009). Antithrombotic papain-hydrolyzed peptides isolated from　pork meat. Thrombosis Research, 123, 753-757.
Shori, A. B., Ming, K. S., & Baba, A. S. (2020). The effects of Lycium barbarum water extract and fish collagen on milk proteolysis and in vitro angiotensin I-converting enzyme inhibitory activity of yogurt. Biotechnology and Applied Biochemistry, 68(2), 221-229.
Sinthusamran, S., Benjakul, S., & Kishimura, H. (2013). Comparative study on molecular characteristics of acid soluble collagens from skin and swim bladder of seabass (Lates calcarifer). Food Chemistry, 138, 2435-2441.
Steen, H., & Mann, M. (2004). The ABC’s (and XYZ’s) of peptide sequencing. Nature reviews Molecular Cell Biology, 5, 699-711.
Suleria, H. A. R., Gobe, G., Masci, P., & Osborne, S. A. (2016). Marine bioactive compounds and health promoting perspectives; innovation pathways for drug discovery. Trends in Food Science & Technology, 50, 44-55.
Sylvie, B. (2011). The collagen family. Cold Spring Harbor Perspectives in Biology, 3(1), a004978
Torres, F. C., Alaiz, M., Vioque, J. (2012). Iron-chelating activity of chickpea protein hydrolysate peptides. Food Chemistry, 134(3), 1585-1588.
Vercellotti, J. R., Angelo, St. A. J., & Spanier, M. A. (1992). Lipid Oxidation in Foods. An overview. In A. J. St. Angelo (Ed.), Lipid oxidation in food (pp. 1-11).Washington, DC: American Chemical Society.
Wallace, M., Chen, Q., Fang, M., Erickson, B., Orr, G., & Holl, B. (2010). Type I collagen exists as a distribution of nanoscale morphologies in teeth, bones, and tendons. American Chemical Society, 26(10), 7349-7354.
Wanasundara, U. N., Shahidi, F., & Jablonski, C. R. (1995). Comparison of standard and NMR methodologies for assessment of oxidative stability of canola and soybean oils. Food Chemistry, 52(3), 249-253.
Wang, C., Zhou, L., & Zhou, T. (2013). Study on the extraction of collagen protein from tilapia scale with papain. Anhui Agricultural Sciences, 41(3), 129l-1292.
Wang, X., & Ng, B. (1999). Natural products with hypoglycemic, hypotensive, hypocholesterolemic, antiatherosclerotic and antithrombotic activities. Life Science, 65(25), 2663-2677.
Wołosiak, R., Drużyńska, B., Derewiaka, D., Piecyk, M., Majewska, E., Ciecierska, M., Worobiej, E., & Pakosz, P. (2021). Verification of the conditions for determination of antioxidant activity by ABTS and DPPH assays-A practical approach. Molecules, 27(1), 50.
Wu, Lian., Kang, Bin., & Li, J. (2017). Technology for Extracting effective components from fish scale. Food Science and Engineering, 7, 351-358.
Wu, Y., Ma, Y., Li, L., & Yang, X. (2018). Preparation and antioxidant activities in vitro of a designed antioxidant peptide from pinctada fucata by recombinant escherichia coli. Journal of Microbiology and Biotechnology, 28(1), 1-11.
Xie, Z., Huang, J., Xu, X., & Jin, Z. (2008). Antioxidant activity of peptides isolated from alfalfa leaf protein hydrolysate. Food Chemistry, 111(2), 370-376.
Yarnpakdee, S., Benjakul, S., Kristinsson, H. G., & Kishimura, H. (2015). Antioxidant and sensory properties of protein hydrolysate derived from Nile tilapia (Oreochromis niloticus) by one- and two-step hydrolysis. Food Science and Technology, 52(6), 3336-3349.
Yokoyama, K., Chiba, H., Yoshikawa, M. (1992). Peptide inhibitors for angiotensin I-converting enzyme from thermolysin digest of dried bonito. Bioscience, Biotechnology, and Biochemistry, 56, 1541-1545.
Yu, J., Hu, Y. L., Xue, M. X., Dun, Y. H., Li, S. N., Peng, N., Liang, Y. X., & Zhao, S. M. (2016). Purification and identification of antioxidant peptides from enzymatic hydrolysate of spirulina platensis. Journal of Microbiology and Biotechnology, 26(7), 1216-1223.
Zhang, G., Zheng, S., Feng, Y., Shen, G., Xiong, S., & Du, H. (2018). Changes in nutrient profile and antioxidant activities of different fish soups, before and after simulated gastrointestinal digestion. Molecules, 23(8), 1965.
Zheng, L., Lin, L. Z., Su, G. W., Zhao, Q. Z., & Zhao, M. M. (2015). Pitfalls of using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay to assess the radical scavenging activity of peptides: Its susceptibility to interference and low reactivity towards peptides. Food Research International, 76, 359-365.