供飲用的牛乳需經過加熱殺菌或滅菌。本研究在模式系統下觀察牛乳蛋白質與乳糖混合溶液經不同加熱處理，對其結構、免疫反應性及抗氧化性的影響；並探討加熱處理之生乳及市售乳品其蛋白質組成與免疫反應性的變化。利用pH 4.6沉澱、Native-PAGE、還原性與非還原性SDS-PAGE、免疫轉漬法、酵素連結免疫吸附法分析牛乳蛋白質之變化與相互作用之情形。並在模式系統下以ABTS清除自由基的能力、螯合亞鐵離子能力及還原力測其抗氧化性。模式系統的電泳與免疫轉漬法結果顯示，隨著溫度上升會增加β-Lg、α-La、酪蛋白間的交互作用與聚合反應的程度。酵素連結免疫吸附法以β-Lg和α-La抗體偵測的結果，免疫反應性隨溫度上升而增加，這代表結構的舒展與暴露出更多的抗原決定區。加熱至80 ℃以上時，抗氧化性質有顯著增加，但含酪蛋白的模式系統中，僅還原力結果顯示如此。生乳的結果與模式系統相似；市售乳在電泳與免疫檢測法相互搭配下，可分辨出不同加熱程度的牛乳。

Milk products have to be pasteurized or sterilized. In this study, milk proteins-lactose model systems were used to investigate the effects of heat treatments on the structure, immunoreactivity, and antioxidant capicity of proteins. The changes of protein composition and immunoreactivity on heated raw milk and commercial milk products were also investigated. Precipitation at pH 4.6, Native-PAGE, reducing and non-reducing SDS-PAGE, Western blot analysis, and ELISA were used to elucidate the changes and the interactions among milk proteins. In model systems, the antioxidant capacity was analyzed by ABTS radical scavenging ability, ferrous ion chelating ability and reducing power methods. The results of electrophoresis and Western blot showed that the interactions and degree of polymerization increased among β-Lg, α-La and casein as the heating temperature increased. The immunoreactivity of β-Lg and α-La increased, measured with ELISA using β-Lg and α-La antibodies, as the temperature increased, indicating the protein unfolding and exposure of more epitopes. At temperatures above 80 ℃, the antioxidant properties increased significantly. However, only reducing power followed the above trend for model systems containing casein. The changes of protein composition and immunoreactivity for heated raw milk were similar to those in model systems. Combining electrophoresis and immunoassay could differentiate different heat-treated commercial milk products.

摘要 I
ABSTRACT II
誌謝 III
縮寫對照表 IV
目錄 V
表目錄 VIII
圖目錄 IX
第一章、前言 1
第二章、文獻回顧 3
2.1 牛乳之組成 3
2.2 牛乳蛋白質 5
2.3 乳糖 8
2.4 牛乳之加熱殺菌 8
2.5 加熱處理對牛乳蛋白質的影響 10
2.6 牛乳的加熱指標 13
2.7 電泳與免疫檢測法 18
2.8 抗氧化 24
第三章、研究目的 26
第四章、實驗架構 27
第五章、材料與方法 29
5.1 實驗材料 29
5.2 化學試藥 30
5.3 實驗方法 32
5.3.1 蛋白質與牛乳樣品溶液配製 32
5.3.2 原態聚丙烯醯胺膠體電泳 (Native-PAGE) 36
5.3.3 十二烷基硫酸鈉-聚丙烯醯胺膠體電泳 (SDS-PAGE) 38
5.3.4 競爭型酵素連結免疫吸附分析 42
5.3.5 西方轉漬法 (Western Blot) 47
5.3.6 清除ABTS自由基的能力 51
5.3.7 還原力測定 53
5.3.8 螯合亞鐵離子能力 55
5.4 統計分析 58
第六章、結果與討論 59
第七章、結論 100
第八章、參考文獻 102

林文慶：乳品加工學。華香園出版社。台北，臺灣 (2008)。
賴滋漢、金安兒：食品加工學 加工篇。富林出版社。191-197 (2003)。
Anese, M., Nicoli, M. C., & Lerici, C. R. (1994). Influence of pH on the oxygen scavenging properties of heat-treated glucose-glycine system. Italian Journal of Food Science, 3, 339-343.
Bramanti, E., Quigley, W. W. C., Sortino, C., Beni, F., Onor, M., Raspi, G., & Synovec, R. E. (2004). Multidimensional analysis of denatured milk proteins by hydrophobic interaction chromatography coupled to a dynamic surface tension detector. Journal of Chromatography A, 1023, 79-91.
Bu, G., Luo, Y., Zheng, Z., & Zheng, H. (2009). Effect of heat treatment on the antigenicity of bovine α-lactalbumin and β-lactoglobulin in whey protein isolate. Food and Agricultural Immunology, 20, 195-206.
Cervato, G., Cazzola, R., & Cestaro, B. (1999). Studies on the antioxidant activity ofccaseins. International Journal of Food Science and Nutrition, 50, 291-296.
Chen, W. L., Huang, M. T., Liu, H. C., Li, C. W., & Mao, S. J. T. (2004). Distinction between dry and raw milk using monoclonal antibodies prepared against dry milk proteins. Journal of Dairy Science, 87, 2720-2729.
Chen, W. L., Liu, W. T., Yang, M. C., Hwang, M. T., Tsao, J. H. & Mao, S. J. T. (2006). A Novel Conformation-Dependent Monoclonal Antibody Specific to the Native Structure of β-Lactoglobulin and Its Application. Journal of Dairy Science, 89, 912-921.
Davis, P.J., & Williams, S.C. (1998). Protein modification by thermal processing. Allergy, 53, 102-105.
Decker, E. A., & Welch, B. (1990). Role of ferritin as a lipid oxidation
catalyst in muscle food. Journal of Agricultural and Food Chemistry, 38 , 674-677.
Díaz, M., & Decker, E.A. (2004). Antioxidant mechanisms of caseinophosphopeptides and casein hydrosylates and their application in ground beef. Journal of Agricultural and Food Chemistry, 52, 8208-8213.
Dupont, D., Rolet-Répécaud, O., & Muller-Renaud, S. (2004). Determination of the heat treatment undergone by milk by following the denaturation of α-lactalbumin with a biosensor. Journal of Agricultural and Food Chemistry, 52, 677-681.
Elliott, A. J., Datta, N., Amenu, B., & Deeth, H. C. (2005) Heat-induced and other chemical changes in commercial UHT milks. Journal of Dairy Research, 72, 442-446.
Feinberg, M., Dupont, D., Efstathiou, T., Louapre, V., & Guyonnet, J.P. (2006). Evaluation of tracer for the authentication of thermal treatments of milks. Food Chemistry, 98,188–194.
Hernández-Ledesma, B., Dávalos, A., Bartolomé , B., & Amigo, L. (2005). Preparation of antioxidant enzymatic hydrosylates from α-lactalbumin and β-lactoglobulin. Identification of active peptides by HPLC-MS/MS. Journal of Agricultural and Food Chemistry, 53, 588-593.
Hinrichs, J., & Rademacher, B. (2005). Kinetics of combined thermal and pressure-induced whey protein denaturation in bovine skim milk. International Dairy Journal, 15, 315-323.
ISO 13875/IDF 178 standard. (1996). Liquid milk – determination of acid-soluble beta-lactoglobulin content – reverse-phase HPLC method. International Standardisation Organisation, Geneva.
Kaminarides, S. E., & Koukiassa, P. (2002). Detection of bovine milk in ovine yoghurt by electrophoresis of para-κ-casein. Food Chemistry, 78, 53-55.
Kinsella, J. E., & Whitehead, D. M. (1989). Proteins in whey: Chemical, physical, and functional properties. Advances in food and nutrition research, 33, 343-438.
Kleber, N., Maier, S., & Hinrichs, J. (2007). Antigenic response of bovine β-lactoglobulin influenced by ultra-high pressure treatment and temperature. Innovative Food Science and Emerging Technologies, 8, 39-45.
Lan, X. Y., Wang, J. Q., Bu, D. P., Shen, J. S., Zheng, N., & Sun, P. (2010). Effects of heating temperatures and addition of reconstituted milk on the heat indicators in milk. Journal of Food Science, 75, 653-658.
Lin, S., Sun, J., Cao, D., Cao, J., & Jiang, W. (2010). Distinction of different heat-treated bovine milks by native-PAGE fingerprinting of their whey proteins. Food Chemistry, 121, 803-808.
Lindmark-Månsson, H., Timgren, A., Aldén, G., & Paulsson, M. (2005). Two-dimensional gel electrophoresis of proteins and peptides in bovine milk. International Dairy Journal, 15, 111-121.
Liu, Q., Kong, B., Han, J., Sun, C., & Li, P. (2013). Structure and antioxidant activity of whey protein isolate conjugated with glucose via the Maillard reaction under dry-heating conditions. Food structure.
Lozano, J. M., Giraldo, G. I., & Romero, C. M. (2008). An improved method for isolation of β-lactoglobulin. International Dairy Journal, 18, 55-63.
Morales, F. J., & Babbel, M. B. (2002). Antiradical efficiency of Maillard reaction mixtures in a hydrophilic media. Journal of Agricultural and Food Chemistry, 50, 2788-2792.
Pesic, M. B., Barac, M. B., Stanojevic, S. P., Ristic, N. M., Macej, O. D. & Vrvic, M. M. (2012). Heat induced casein–whey protein interactions at natural pH of milk: A comparison between caprine and bovine milk. Small Ruminant Research, 108, 77-86.
Rada-Mendoza, M., Villamiel, M., Molina, E., & Olano, A. (2006). Effects of heat treatment and high pressure on the subsequent lactosylation of β-lactoglobulin. Food Chemisty, 99, 651–655.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation recolorization assay. Free Radical Biology & Medicine, 26, 1231-1237.
Rival, S.G., Boeriu, C.G., & Wichers, H.J. (2001a). Caseins and casein hydrolysates. 2 Antioxidative properties and relevance to lipoxygenase inhibition. Journal of Agricultural and Food Chemistry, 49, 295-302.
Sawyer, L., Barlow, P.N., Boland, M.J., Creamer, L.K., Denton, H., Edwards, P.J.B., et al. (2002). Milk protein structure-what can it tell the dairy industry? International Dairy Journal, 12, 299-310.
Shimada , K., & Cheftel, J. C. (1988). Sulfhydryl group/disulfide bond interchange reactions during heat-induced gelation of whey protein isolates. Journal of Agricultural and Food Chemistry, 37, 161-168.
Suetsuna, K., Ukeda, H., & Ochi, H. (2000). Isolation and characterization of free radical scavenging activities peptides derived from casein. Journal of Nutritional Biochemistry, 11, 128-131.
Sun, L., & Wang, D. (2009). A new method to estimate the heat treatment of milk and milk-like systems. International Journal of Dairy Technology, 62(3), 321-325.
Veloso, A. C. A., Teixeira, N., Peres, A. M., Mendonca, A., & Ferreira, I. M. P. L. V. O. (2004). Evaluation of cheese authenticity and proteolysis by HPLC and urea–polyacrylamide gel electrophoresis. Food Chemistry, 87, 289-295.
Wal, J. M. (2001). Structure and function of milk allergens. Allergy, 56, 35-38.
Wang, C. H., Abouzied, M. M., Pestka, J. J., & Smith, D. M. (1992). Antibody development and enzyme-linked immunosorbent assay for the protein marker lactate dehydrogenase to determine safe cooking end-point temperatures of turkey rolls. Journal of Agricultural and Food Chemistry, 40, 1671-1676.
Wedholm, A., Hellén, E., Larsen, L. B., Lindmark-Månsson, H., Karlsson, A. H., & Allmere, T. (2006). Comparison of milk protein composition in a Swedish and a Danish dairy herd using reversed phase HPLC. Acta Agriculturae Scandinavica A-An, 56, 8-15.
Whitney, R. M., Brunner, J. R., Ebner, K. E., Farrell, H. M., Josephson, R. V., Morr, C. V., & Swaisgood, H. E. (1976). Nomenclature of the Proteins of Cow's Milk: Fourth Revision1. Journal of Dairy Science, 59, 795-815.
Wu, H. C., Chen, H. M., & Shiau, C. Y. (2003). Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus). Food Reserch International, 36, 949-957.
Yen, G. C., & Hsieh, P. P. (1995). Antioxidative activity and scavenging effects in native oxygen of xtlose-lysine Maillard reaction products. Journal Science of Food Chemistry , 67, 415-420.
Yoshimura, Y., Iijima, T., Watanabe, T., & Nakazawa, H. (1997). Antioxidative effect of Maillard reaction products using glucose-glycine model system. Journal of Agricultural and Food Chemistry, 45, 4106-4109.
Zulueta, A., Maurizi A., Frígola., A., Esteve, M.J., Coli, R., & Burini, G. (2009). International Dairy Journal, 19, 380-385.