臺灣博碩士論文加值系統

本研究目的在探討製備吳郭魚魚鱗膠原胜肽的加工條件及其鈣結合能力之功能性。首先將吳郭魚魚鱗經過洗淨、乾燥及研磨成粉末後備用。利用加熱處理、酸處理及商業酵素處理吳郭魚魚鱗粉，探討製備具有鈣結合能力的膠原胜肽水解物之最適加工條件。首先以121oC加熱及酸處理吳郭魚魚鱗，兩者所得的水解液之鈣結合能力不佳，且經人工消化液處理後鈣離子結合能力有顯著性下降。其次，使用五種市售商業酵素，(Papain、Alcalase、Flavourzyme、Protase-N及Protamax)處理吳郭魚魚鱗，以Papain與Flavourzyme獲得的胜肽之鈣結合能力較佳，將兩者進行複合酵素試驗。複合酵素的最適反應條件如下：pH 7.5; 50℃; Papain與Flavourzyme用量皆為3000 U/mL；魚鱗用量4%;反應2小時。吳郭魚魚鱗經上述最適反應條件處理得到的水解液再經人工消化液處理獲得的二次水解液仍具有良好鈣離子結合能力。將上述二次水解液放入中空纖維管柱(Hollow Fiber)分離，以<3 kDa之分劃具有較高鈣離子結合力。繼續以QHP管柱分離得到三個分劃，分別命名為Q-1、Q-2、Q-3，以Q-3具有較高鈣離子結合能力。吳郭魚魚鱗膠原蛋白水解液噴霧乾燥得到的粉末之鈣結合能力與市售酪蛋白磷酸肽(CPP)相當。最後，將魚鱗膠原胜肽水解液調製成兩種膠原胜肽飲品均獲得良好的整體接受度。

The objective of this study is to establish the process conditions for preparing collagen peptides with calcium-binding (Ca-B) activity from tilapia scales. The scales of tilapia were washed, dried, and milled to obtain a powder sample. Collagen peptides with Ca-B activity were prepared from tilapia scales treated with heating, acidic treatment and commercial proteinase (Papain,Alcalase,Flavourzyme,Protase-N and Protamax). Optimal conditions in heating, acidic and enzymatic treatment were investigated and compared for their Ca-B activity. The hydrolysates from heating and acidic treatment didn’t have high Ca-B activity, and Ca-B activity decreased significantly after artificial gastrointestinal liquid treatment. Higher Ca-B activity was obtained from tilapia scales treated with papain and flavourzyme. The tilapia scales were then treated with two enzymes system, including papain and flavourzyme. The optimal conditions in two enzymes system were listed as follows: pH 7.5; 50°C; enzyme activity, 3000 U/mL for papain and flavourzyme; the amount of tilapia scales, 4%; reaction time 2 h. The secondary digestive liquid was obtained from the tilapia scales hydrolysates followed by artificial gastrointestinal liquid treatment, and Ca-B activity remained in this liquid. The secondary digestive liquid was then fractionated with hollow fiber and QHP column separation. Three fractions (named Q1, Q2, Q3) were obtained, and Q3 showed the highest Ca-B activity. Collagen peptide powder from the spray dried hydrolysates of tilapia scale exhibited similar Ca-B activity when compared with commercial casein phosphorpeptide (CPP). Two collagen peptide drinks were prepared from fish scale hydrolysates and showed good acceptability by sensory evaluation.

目錄
中文摘要...........................................I
英文摘要...........................................Ⅱ
誌謝..............................................Ⅲ
目錄..............................................Ⅳ
表目錄............................................Ⅶ
圖目錄............................................Ⅷ
附表目錄...........................................Ⅸ
附圖目錄...........................................X

壹、前言............................................1

貳、文獻整理.........................................3
一、吳郭魚介紹........................................3
(一)吳郭魚簡介........................................3
(二)吳郭魚生態習性.....................................3
(三)養殖情形及現況.....................................4
(四)吳郭魚的營養成分...................................4
(五)魚鱗組成分........................................5
(六)魚鱗膠原胜肽......................................5
二、膠原蛋白的介紹.....................................7
(一)膠原蛋白簡介......................................7
(二)膠原蛋白來源......................................7
(三)膠原蛋白類型與組織分布..............................8
(四)膠原蛋白水解方式..................................12
(五)膠原蛋白水解物功能性...............................14
(六)膠原蛋白應用......................................14
三、鈣質的介紹........................................16
(一)鈣簡介...........................................16
(二)鈣的日常需要量.....................................16
(三)鈣質的吸收與代謝....................................18

參、材料與方法..........................................21
一、材料...............................................21
(一)吳郭魚魚鱗..........................................21
(二)商業酵素............................................21
(三)藥品...............................................22
(四)儀器...............................................22
二、實驗架構與流程........................................23
三、實驗方法與步驟........................................24
(一)魚鱗粉製備...........................................24
(二)鈣濃度測定方法........................................24
(三)鈣結合能力測定方法.....................................26
(四)魚鱗膠原胜肽水解液製備..................................27
(五)體外消化試驗(in vitro)................................31
(六)中空纖維管柱分劃.......................................31
(七)離子交換管柱層析.......................................32
(八)可溶性蛋白質含量的測定..................................32
(九)胜肽濃度測定..........................................33
(十)噴霧乾燥粉末製備.......................................35
(十一)蛋白酶活性測定.......................................35
(十二)膠原胜肽飲品製備與喜好性品評............................37
(十三)統計分析............................................40

肆、結果與討論.............................................41
一、不同加工條件對吳郭魚魚鱗水解液的鈣結合能力之影響..............39
(一)熱處理................................................41
(二)酸鹼處理...............................................42
(三)體外消化試驗(in vitro)..................................43
(四)酵素處理...............................................44
二、體外消化試驗(in vitro)..................................52
三、吳郭魚魚鱗水解物中鈣結合胜肽的部分純化.......................53
四、吳郭魚魚鱗膠原胜肽粉末與市售酪蛋白磷酸肽(CPP)的鈣結合能力比較...56
五、膠原蛋白胜肽飲品製備與喜好性品評............................58

伍、結論...................................................59

陸、參考文獻................................................60

中文文獻
中華民國水產種苗協會。2003。吳郭魚繁殖介紹。水產種苗(64)。
江福松。2002。認識台灣鯛。台灣鯛策略聯盟。
朱鴻鈞、陳葦芋、陳政忻。2009。台灣鯛產業概況及趨勢。農業生技產業季刊，19，21-23。
李武忠。1989。吳郭魚飼育及其營養需求探討(上)。飼料營養，4:94-103。
李英漢。2012。魚鱗膠原蛋白胜肽之礦物質結合功能性探討。國立高雄海洋科技大學水產食品科學研究所碩士學位論文。
林文彬。2005。骨質疏鬆與鈣。台灣中醫臨床學會雜誌， 11(3)176。
林文彬。2006。腸鈣吸收障礙之探討。中華中醫雜誌，1:11-19。
林文彬。2006。談鈣在人體內的吸收與代謝。北縣中醫會刊雜誌，9:74-86。
林文彬。2006。骨質疏鬆腸鈣代謝吸收障礙之研究。臺灣中醫臨床醫學雜誌，12:113-131。
洪雅萍。2004。膠原蛋白產品的功效。科學發展380 : 30-35。
胡興華。2004。台灣養殖漁業。遠足文化事業股份有限公司。
湯家潤。2004。組織工程中的膠原蛋白。科學發展380 : 18-23。
蔡慧君、胡燕君。2005。魚鱗的完全利用。水產試驗所水產加工組。第12期13-15。
莫長原。2010。虱目魚魚肉水解液的製備及應用。國立高雄海洋科技大學水產食品科學研究所碩士學位論文。
陳秀蓮 。1993。常用調味料中的蛋白質水解液。食品工業，25 (6):33-43
陳建誌。2011。鱸魚膠原蛋白胜肽製備及其功能性。國立海洋科技大學水產食品科學研究所碩士學位論文。
黃子瑜。2010。秋刀魚魚肉水解液之製備及應用。國立高雄海洋科技大學水產食品科學研究所碩士學位論文。
黃彥富、湯正明、徐善慧。2003。揭開膠原蛋白的神秘面紗。科學發展，362:44-47。
鄭詩亭。2012。吳郭魚頭蛋白質水解物的抗氧化特性及其螯合銅離子胜肽之純化的研究。國立高雄海洋科技大學水產食品科學研究所碩士學位論文。
廖桂玉。2012。虱目魚魚鱗膠原蛋白胜肽的製備及鈣結合能力探討。國立高雄海洋科技大學水產食品科學研究所碩士論文。
錢阜甯。1994。魚肉蛋白之酵素水解。食品工業月刊，26: 27-35。
劉柏青。2008。利用枯草桿菌水解台灣雕魚鱗膠原蛋白。國立成功大學化學研究系研究所碩士論文。
劉富光。2008。吳郭魚168。水產試驗所特刊第10 號。行政院農業委員會水產試驗所。基隆市。
英文文獻
Aaslyng, M. D., Martens, M., Poll, L., Nielsen, P. M., Flyge, H., & Larsen, L. M. (1998). Chemical and sensory characterization of hydrolyzed vegetable protein, a savory flavoring. Journal of agricultural and food chemistry, 46(2), 481-489.
Bagnasco, L., Pappalardo, V.M., Meregaglia, A., Kaewmanee,T.,Ubiali, D., Speranza, G., Cosulich, M.E.(2013) Use of food-grade proteases to recover umami protein–peptide mixtures fromrice middlings.Food Research International 50 :420–427.
Bailey, A. J. (1988). The biological diversity of collagen: a family of molecules.Adv. in Meat Research 4: 1-17.
Berdanier,C.D,(1998) Macarominerals. In: Berdanier,C.D,(Ed.),Advanced nutrition micronutrients,Boca Raton Boston London New York Washington. D. C.,pp.157-176.
Bhatia, V. (2008). Dietary calcium intake-a critical reappraisal. The Indian journal of medical research, 127(3), 269.
Bishop, P. N., Crossman, M. V., McLeod, D., & Ayad, S. (1994). Extraction and characterization of the tissue forms of collagen types II and IX from bovine vitreous. Biochem. J, 299, 497-505.
Bornstein, P., & Sage, H. (1980). Structurally distinct collagen types. Annual review of biochemistry, 49(1), 957-1003.
Bougatef, A., Nedjar-Arroume, N., Manni, L., Ravallec, R., Barkia, A., Guillochon, D., & Nasri, M. (2010). Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinella aurita) by-products proteins. Food chemistry, 118(3), 559-565.
Chen, J., Wang, Y., Zhong, Q., Wu, Y., & Xia, W. (2012). Purification and characterization of a novel angiotensin-I converting enzyme (ACE) inhibitory peptide derived from enzymatic hydrolysate of grass carp protein. Peptides,33(1), 52-58.
Cuneo, F., Costa-Paiva, L., Pinto-Neto, A. M., Morais, S. S., & Amaya-Farfan, J. (2010). Effect of dietary supplementation with collagen hydrolysates on bone metabolism of postmenopausal women with low mineral density. Maturitas,65(3), 253-257.
Deyl, Z., & Adam, M. (1989). Separation methods for the study of collagen and treatment of collagen disorders. Journal of Chromatography B: Biomedical Sciences and Applications, 488(1), 161-197.
Diniz, F. M., & Martin, A. M. (1997). Effects of the extent of enzymatic hydrolysis on functional properties of shark protein hydrolysate. LWT-Food Science and Technology, 30(3), 266-272.
El-Sayed, A. F. M. (1999). Alternative dietary protein sources for farmed tilapia,< i> Oreochromis spp. Aquaculture, 179(1), 149-168.
Fahmi, A., Morimura, S., Guo, H. C., Shigematsu, T., Kida, K., & Uemura, Y. (2004). Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales. Process Biochemistry, 39(10), 1195-1200.
Gelse, K., Pöschl, E., & Aigner, T. (2003). Collagens—structure, function, and biosynthesis. Advanced drug delivery reviews, 55(12), 1531-1546.
Gill, I., Lopez-Fandino, R., Jorba, X., & Vulfson, E. N. (1996). Biologically active peptides and enzymatic approaches to their production. Enzyme and Microbial Technology, 18(3), 162-183.
Hamada, M. and Kumagai, H. (1988). Chemical composition of sardine scale. Nippon Suisan Gakkaishi 54(11) : 1987-92
Hayashi, T., & Mizuno, K. (1999). Collagen. Encyclopedia of molecular biology.
Ikoma, T., Kobayashi, H., Tanaka, J., Walsh, D., & Mann, S. (2003). Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas. International Journal of Biological Macromolecules,32(3), 199-204.
Je, J. Y., Qian, Z. J., Byun, H. G., & Kim, S. K. (2007). Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis. Process Biochemistry, 42(5), 840-846.
Jung, W. K., Karawita, R., Heo, S. J., Lee, B. J., Kim, S. K., & Jeon, Y. J. (2006). Recovery of a novel Ca-binding peptide from Alaska Pollack (< i> Theragra chalcogramma) backbone by pepsinolytic hydrolysis. Process Biochemistry, 41(9), 2097-2100.
Jung, W. K., Park, P. J., Byun, H. G., Moon, S. H., & Kim, S. K. (2005). Preparation of hoki (Johnius belengerii) bone oligophosphopeptide with a high affinity to calcium by carnivorous intestine crude proteinase. Food chemistry,91(2), 333-340.
Klompong, V., Benjakul, S., Kantachote, D., & Shahidi, F. (2007). Antioxidative activity and functional properties of protein hydrolysate of yellow stripe trevally (< i> Selaroides leptolepis) as influenced by the degree of hydrolysis and enzyme type. Food chemistry, 102(4), 1317-1327.
Klompong, V., Benjakul, S., Kantachote, D., Hayes, K. D., & Shahidi, F. (2008). Comparative study on antioxidative activity of yellow stripe trevally protein hydrolysate produced from Alcalase and Flavourzyme. International Journal of Food Science & Technology, 43(6), 1019-1026.
Koyama, Y. I., Hirota, A., Mori, H., Takahara, H., Kuwaba, K., Kusubata, M., ... & Irie, S. (2001). Ingestion of gelatin has differential effect on bone mineral density and body weight in protein undernutrition. Journal of nutritional science and vitaminology, 47(1), 84-86.
Lahl, W. J., & Braun, S. D. (1994). Enzymatic production of protein hydrolysates for food use. Food Technology, 48(10), 68-71.
Liu Z, Dong S, Xu J, Zeng M, Song H, and Zhao Y. (2008). Production of cysteine-rich antimicrobial peptide by digestion of oyster (Crossostrea gigas) with alcalase and bromelin. Food Control, 19: 231-235.
Mamboya, E. A. F. (2012). PAPAIN, A PLANT ENZYME OF BIOLOGICAL IMPORTANCE: A REVIEW. American Journal of Biochemistry and Biotechnology, 8(2), 99-104.
Manley, C. H., & Ahmedi, S. (1995). The development of process flavors. trends in food science & technology, 6(2), 46-51.
Manley, C. H., McCann, J. S., & Swaine Jr, R. L. (1981). The chemical bases of the taste and flavor enhancing properties of hydrolyzed protein.
Miller, E. J., & Kent, R. R. (1982). 2] Preparation and characterization of the different types of collagen. Methods in enzymology, 82, 33.
Miller, E. J. (1988). Collagen types: Structure, distribution, and functions.Collagen, 1, 139-157.
Moskowitz, R. W. (2000, October). Role of collagen hydrolysate in bone and joint disease. In Seminars in arthritis and rheumatism (Vol. 30, No. 2, pp. 87-99). WB Saunders.
Murray, B. A., & FitzGerald, R. J. (2007). Angiotensin converting enzyme inhibitory peptides derived from food proteins: biochemistry, bioactivity and production. Current Pharmaceutical Design, 13(8), 773-791.
Nomura, Y., Oohashi, K., Watanabe, M., & Kasugai, S. (2005). Increase in bone mineral density through oral administration of shark gelatin to ovariectomized rats. Nutrition, 21(11), 1120-1126.
Oshima, G., Shimabukuro, H., & Nagasawa, K. (1979). Peptide inhibitors of angiotensin I-converting enzyme in digests of gelatin by bacterial collagenase.Biochimica et Biophysica Acta (BBA)-Enzymology, 566(1), 128-137.
Pihlanto-Leppälä, A. (2000). Bioactive peptides derived from bovine whey proteins: opioid and ace-inhibitory peptides. trends in Food Science & technology, 11(9), 347-356.
Pommer, k. (1995). Short introduction to enzyme Application in the protein industry. Enzyme Protein Division EM-9514346
Prockop, J. D. (1995). Collagens: molecular biology, diseases, and potentials for therapy. Annual review of biochemistry, 64(1), 403-434.
Ranathunga, S., Rajapakse, N., & Kim, S. K. (2006). Purification and characterization of antioxidative peptide derived from muscle of conger eel (Conger myriaster). European Food Research and Technology, 222(3-4), 310-315.
Samaranayaka, A. G., & Li-Chan, E. C. (2008). Autolysis-assisted production of fish protein hydrolysates with antioxidant properties from Pacific hake (Merluccius productus). Food chemistry, 107(2), 768-776.
Theodore, A. E., & Kristinsson, H. G. (2007). Angiotensin converting enzyme inhibition of fish protein hydrolysates prepared from alkaline‐aided channel catfish protein isolate. Journal of the Science of Food and Agriculture, 87(12), 2353-2357.
Trentham, D. E., Dynesius-Trentham, R. A., Orav, E. J., Combitchi, D., Lorenzo, C., Sewell, K. L., ... & Weiner, H. L. (1993). Effects of oral administration of type II collagen on rheumatoid arthritis. Science, 261(5129), 1727-1730.
Tsai, J. S., Lin, T. C., Chen, J. L., & Pan, B. S. (2006). The inhibitory effects of freshwater clam (Corbicula fluminea, Muller) muscle protein hydrolysates on angiotensin I converting enzyme. Process biochemistry, 41(11), 2276-2281.
Tsuruoka, N., Yamato, R., Sakai, Y., Yoshitake, Y.,Yonekura, H. (2007) . Promotion by Collagen Tripeptide of Type I Collagen Gene Expression in Human Osteoblastic Cells and Fracture Healing of Rat Femur.Bioscience, biotechnology, and biochemistry, 71(11), 2680-2687.
Undeland, I., Lindqvist, H., Chen-Yun, Y., Falch, E., & Luten, J. B. (2009). Seafood and health: what is the full story. Marine Functional Food. Luten, JB (Ed.). Wageningen academic press, Wageningen, 17-87.
Van der Rest, M., & Garrone, R. (1991). Collagen family of proteins. The FASEB Journal, 5(13), 2814-2823.
Yamauchi, K., Goda, T., Takeuchi, N., Einaga, H., & Tanabe, T. (2004). Preparation of collagen/calcium phosphate multilayer sheet using enzymatic mineralization. Biomaterials, 25(24), 5481-5489.
Yang, J. I., Ho, H. Y., Chu, Y. J., & Chow, C. J. (2008).Characteristic and antioxidant activity of retorted gelatin hydrolysates from cobia (Rachycentron canadum) skin. Food chemistry, 110(1), 128-136.
. Zhang,J., Duan, R..,Wang, Y., Yan .B, Xue W.(2012)Seasonal differences in the properties of gelatins extracted from skin of silver carp(Hypophthalmichthys molitrix).Food Hydrocolloids 29:100e105.