臺灣博碩士論文加值系統

牛乳為高營養價值食品，必須經殺菌處理，消滅病原菌以確保飲用安全，其殺菌條件愈高，乳中殘留酵素活性及生菌數愈低，保存期愈長，然而，不同的殺菌條件造成牛乳不同程度之理化學性質變化。本研究主要藉不同殺菌條件之鮮乳，依據蛋白酶水解後胜肽生成量，應用反應曲面法，評估鮮乳之適宜殺菌條件。首先尋找反應曲面法二因子中心複合設計之第一因子X1取得殺菌溫度；接著以其第二因子X2取得殺菌時間；再以X1與X2二因子之最高點，作為二因子旋轉中心複合設計試驗之中心點，找出最適殺菌條件；進而分析最適殺菌條件鮮乳之理化學性質，並與市售低溫鮮乳及保久乳作比較。結果顯示，第一因子中心點為81℃，第二因子中心點為20秒，以81℃，20秒作二因子旋轉中心複合設計試驗，經蛋白酶水解之胜肽生成量，利用SAS統計軟體系統分析，得81℃，20秒殺菌鮮乳之胜肽生成量落在反應曲面圖之最高點與等高線圖之中心點；且81℃，20秒殺菌鮮乳之鹼性磷酶及乳過氧化氫酶均呈陰性反應，其乳成分與市乳比較並無特別差異（P>0.05），色澤L*、b*值及滴定酸度較市乳低（P<0.05），pH值、有效離胺酸含量及未變性乳清蛋白含量均高於市乳（P<0.05），經SDS-PAGE電泳分析顯示，81℃，20秒殺菌鮮乳較市乳保有較多未變性乳清蛋白，經蛋白酶水解後，其胜肽生成量較市乳高（P < 0.05），官能品評之總接受度介於各種市乳間。所以藉體外蛋白水解試驗之胜肽生成量，建立鮮乳最適殺菌條件為81℃維持20秒。

Milk is a highly nutritious food, and must be pasteurized to destroy all the pathogenic and microorganisms in order to extend shelf life. However, the different pasteurization conditions could induce physicochemical changes in milk. This study was intended to establish an optimal pasteurization condition of milk using response surface methodology by the peptide content from protein hydrolysis in vitro. The first factor temperature (X1) and the second factor holding time (X2) of response surface were located through preliminary study. Then X1 and X2 were used as the center point of central composite rotatable design in order to establish an optimal pasteurization condition of fresh milk. The physicochemical properties of fresh milk processed from the optimal pasteurization condition were then compared with market milk. Results suggested that temperature (X1) of 81℃ and holding time (X2) of 20 seconds, were used as center point for central composite rotatable design. Fresh milk pasteurized by 81℃ for 20 seconds had the highest peptide content. The alkaline phosphatase and lactoperoxidase activity tests were negative of fresh milk pasteurized by 81℃ for 20 seconds. The L*, b* value and titratable acidity of fresh milk pasteurized by 81℃ for 20 seconds were significantly lower (P<0.05) than market milk. In addition, pH value, available lysine content, undenatured whey protein content and peptide content were significantly (P<0.05) higher than market milk. The electrophoresis showed that fresh milk pasteurized by 81℃ for 20 seconds had more undenatured whey protein than market milk. The overall acceptability of fresh milk pasteurized by 81℃ for 20 seconds in sensory evaluation was among different market milk. Therefore, temperature of 81℃ and holding time of 20 seconds was the optimal pasteurization condition of fresh milk.

壹、摘要 1
貳、前言 3
參、文獻檢討 5
一、牛乳之殺菌 5
二、熱處理對乳成分之影響 5
（一）酪蛋白（Casein） 6
（二）乳清蛋白質（Whey protein） 7
（三）酵素（Enzyme） 9
（四）礦物質（Mineral） 10
（五）維生素（Vitamin） 11
（六）糖類（Carbohydrate） 12
三、熱處理對牛乳理化學性質之影響 13
（一）風味（Flavor） 13
（二）色澤（Color） 14
（三）有效離胺酸（Available lysine） 14
四、熱處理對消化率之影響 15
五、牛乳之加熱程度評估 18
肆、材料與方法 20
一、材料 20
二、試驗方法 21
（一）反應曲面法二因子中心複合設計之第一因子中心點X1
（殺菌溫度） 21
（二）反應曲面法二因子中心複合設計之第二因子中心點X2
（維持時間） 22
（三）反應曲面法二因子（溫度與維持時間）中心旋轉複合
設計 22
（四）依反應曲面法取得之最適殺菌條件鮮乳之理化學性質 23三、分析項目 26
（一）蛋白質水解與胜肽生成量（Protein hydrolysis and peptide content） 26
（二）鹼性磷酶活性（Alkline phosphatase activity） 31
（三）過氧化氫酶活性（Lactoperoxidase activity） 32
（四）乳成分（Milk composition） 33
（五）滴定酸度（Titratable acidity） 33
（六）pH值（pH value） 34
（七）有效離胺酸量（Available lysine content） 34
（八）色澤（Color difference test） 38
（九）乳清蛋白變性程度分析（Denature whey protein analysis） 38
（十）牛乳蛋白SDS-PAGE電泳分析（Milk protein SDS-PAGE electrophoresis） 43
（十一）感官品評（Sensory evaluation） 45
四、統計分析 46
伍、結果與討論 47
一、二因子中心複合設計之第一中心點X1（殺菌溫度） 47
二、反應曲面法二因子中心複合設計之第二中心點X2
（殺菌時間） 49
三、反應曲面法二因子（溫度與維持時間）旋轉中心複合設計 51
四、分析最適殺菌條件處理鮮乳之理化學性質 62
陸、結論 76
柒、參考文獻 77
捌、英文摘要 84
玖、小傳 86
拾、附錄 87

中國國家標準。1970。CNS 3447。N 6063。乳品檢驗法－磷酶之試驗。經濟部中央標準局。台北市。
中國國家標準。1995。CNS 3441。N 6057。乳品檢驗法－酸度之滴定。經濟部中央標準局。台北市。
沈明來。2004。試驗設計學，第537- 594頁。九州圖書文物有限公司，台北市。
林慶文。1993。乳品加工學，第41-141頁。華香園出版社，台北市。
張勝善。1989。牛乳與乳製品，第263-293頁。長河出版社。台北市。
Adriana, S. G., B. N. Gabriela, S. M. Laura, and S. U. Maria. 2003. Available lysine, protein digestibility and lactulose in commercial infant formulas. Int. Dairy J. 13: 95-99.
AlKanhal, H. A., A. A. Al-Othman, and F. M. Hewedi. 2001. Changes in protein nutritional quality in fresh and recombined ultra high temperature treated milk during storage. Int. J. Food Sci. Nutr. 52: 509-514.
Ames, J. M. 1998. Applications of the Maillard reaction in the food industry. Food Chem. 62(4): 431-439.
Andrews, G. R. 1986. Formation and occurrence of lactulose in heated milk. J. Dairy Res. 53: 665-680.
Angela, C. M. and F. X. Malcata. 1997. Secondary proteolysis in Serra cheese during ripening and throughout the cheese-making season. Z Lebensm Unters Forsch A. 204: 173-179.
Atherton, H. V. and J. A. Newlander. 1977. Tests for milk quality. & Acidity of milk and its products. In Chemistry and testing of dairy products. pp. 173-199, 264-267. AVI Publ. Co. Inc. Westport, Connecticut.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram qntities of protein utilizing the principle of protein-die binding. Anal. Biochem. 72: 248-254.
Burton, H. 1988. Chemical and physical changes in milk at high temperatures. In Ultra-high-temperature processing of milk and milk products. pp. 44-76. Elsevier applied science publishers LTD. England.
Carbonaro, M., F. Bonomi, S. Iametti, M. Cappelloni, and E. Carnovale. 1998. Aggregation of proteins in whey from raw and heat-processed milk: Formation of soluble macroaggregates and nutritional consequences. Lebensm.-eiss. U.-Techonl. 31: 522-529.
Carbonaro, M., M. Lucarini, and G. Dilullo. 2000. Composition and calcium status of acid whey from pasteurized, UHT-treated and in-bottle sterilized milks. Nahrung 44: 422-425.
Church, F. C., G. L. Catignani, and H. E. Swaisgood. 1981. Hydrolysis of milk protein by Streptomyces griseus pronase. J. Dairy Sci. 64: 724.
De Wit, J. N. and G. Klarenbeek. 1984. Effects of various heat treatments on structure and solubility of whey proteins. J. Dairy Sci. 67: 2701-2710.
Erbersdobler, H. F. and B. Dehn-Muller. 1989. Formation of early Maillard products during UHT treatment of milk. Bull IDF 238: 62-67.
Estelle, P. C., Y. Kakuda, and D. Irvine. 1988. Heat-induced protein changes in milk processed by vat and continuous heating systems. J. Dairy Sci. 77: 1473-1483.
Farkye, N. Y. and G. I. Imafidon. 1995. Thermal denaturation of indigenous milk enzymes. In Heat-induced changes in milk. 2nd. pp. 331-348. Int. Dairy Federation. Ireland.
Greenbank, G. R. and M. J. Pallansch. 1962. Inactivation and reactivation of xanthine oxidase in dairy products. J. Dairy Sci. 45: 958-961.
Griffiths, M. W. 1986. Use of milk enzymes as indices of heat treatment. J. Food Prot. 49: 696-705.
Haque, Z. and J. E. Kinsella. 1988. Interaction between heated κ-casein and β-lactoglobulin: predominance of hydrophobic interactions in the initial stages of complex formation. J. Dairy Res. 55: 67-80.
Hashizume, K. and T. Sato. 1988a. Gel-forming characteristics of milk proteins. I. Effects of heat treatmen. J. Dairy Sci. 71(6): 1439.
Hashizume, K. and T. Sato. 1988b. Gel-forming characteristics of milk proteins. II. Roles of sulfhydryl groups and disulfide bonds. J. Dairy Sci. 71(6): 1447.
Holt, C. 1995. Effect of heating and cooling on the milk salts and their interaction with casein. In Heat-induced changes in milk. 2nd. pp. 105-133. Int. Dairy Federation. Ireland.
Ito, O. and R. Akuzawa. 1983. Purification, crystallization and properties of bovine milk catalase. J. Dairy Sci. 66: 967-973.
Jelen, P. and W. Rattray. 1995. Thermal denaturation of whey proteins. Chapter 4. pp. 66-80. In: Heat-induced changes in milk. edited by. Brussels, Belgium: Int. Dairy Federation.
Kazmierski, M. and M. Corredig. 2003. Characterization of soluble aggregates from whey protein isolate. Food Hydro. 17: 685-692.
Laemmli, U. K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature. 227: 681.
Law, A. J. R. and J. Leaver. 1999. Factors affecting the heat denaturation of whey proteins in cow’s milk. Int. Dairy J. 9: 407-408.
Li-chan, E., A. dummer, J. N. Losso, D. D. kitts, and S. Nakai. 1995. Stability of bovine immunoglobulins to thermal treatment and processing. Food Res. Int. 28 (1): 9-16
Mansel, W. G. 1986. Use of milk enzymes as indices of heat treatment. J. Food Prot. 49(9): 696-705.
Maria, E., B. Povoa, and M. S. Tasso. 1997. Effect of heat treatment on the nutritional quality of milk proteins. Int. Dairy J. 7: 609-612.
Milena, C. and D. G. Dalgleish. 1996. Effect of temperature and pH on the interactions of whey proteins with casein micelles in skim milk. Food Res. Int. 29 (1): 49-55.
Milena, C. and D. G. Dalgleish. 1999. The mechanisms of the heat-induced interaction of whey proteins with casein micelles in milk. Int. Dairy J. 9: 233-236.
Moore, S. and W. H. Stein. 1948. Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176: 367-388.
Morales, F. J., C. Romero, and J. P. Salvio. 2000. Characterization of industrial processed milk by analysis of heat-induced changes. Int. J. Food Sci. Technol. 35: 193-200.
Moreno, P. and V. Salvadó. 2000. Determination of eight water- and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by high-performance liquid chromatography. J. Chromatogr. 870: 207-215.
Morgan, F. and P. Gaborit. 2001. The typical flavour of goat milk products: technological aspects. Int. J. Dairy Technol. 54(1): 38-40.
Mulvihill, D. M. and J. E. Kinsella. 1987. Gelation characteristics of whey proteins and β-lactoglobulin. Food Technol. 41(9): 102.
Mustapha, B., M. F. Guingamp, J. L. Gaillard, and G. Humbert. 2001. Improvement of a method for the measurement of lactoperoxidase activity in milk. Int. Dairy J. 11: 795-799.
Naranjo, G. B., L. S. Malec, and M. S. Vigo. 1998. Reducing sugars effect on available lysine loss of casein by moderate heat treatment. Food Chem. 62: 309-313.
Panouill’e, M., T. Nicolai, and D. Durand. 2004. Heat induced aggregation and gelation of casein submicelles. Int. Dairy J. (14): 297-303.
Panouill’e, M., D. Durand, T. Nicolai, E. Larquet, and B. Nicolas. 2005. Aggregation and gelation of micellar casein particles. J. Colloid and Interface Sci. 287: 85-93.
Parnell-Clunies, E., Y. Kakuda, and A. Irvine. 1988. Heat-induced protein changes in milk processed by vat and continuous heating systems. J. Dairy Sci. 71(6): 1472.
Pearce, K. N., D. Karahalios, and M. Friedman. 1988. Ninhydrin assay for proteolysis in ripening cheese. J. Food Sci. 53(2): 432-438.
Pellegrino, L., P. Resmini, and W. Luf. 1995. Assesment (indices) of heat treatment. In: Heat induced changes in milk. 2nd. pp. 409-453. Int. Dairy Federation. Ireland.
Porter, D. H., H. E. Swaisgood, and G. L. Catignani. 1984. Characterization of an immobilized digestive enzyme system for determination of protein digestibility. J. Agric. Food. Chem. 32: 334.
Reddy, I. M., N. K. D. Kella, and J. E. Kinsella. 1988. Structural and conformational basis of the resistance of β-lactoglobulin to peptic and chymotryptic digestions. J. Agric. Food. Chem. 36: 737.
Rodriguez-Otero, J. L., M. Hermida, and J. Centeno. 1997. Analysis of dairy products by near-infrared spectroscopy: a review. J. Agric. Food. Chem. 45: 2815-2819.
SAS. 1999. SAS procedure guide for personal computers. 6th ed. SAS Institute Inc. Cary, NC. USA.
Schmidt, R. H. and H. A. Morris. 1984. Gelation properties of milk proteins, soyproteins, and blended protein systems. Food Technol. 38(5): 85.
Sensidoni, A., D. Peressini, and C. M. Pollini. 1999. Study of the Maillard reaction in model systems under conditions related to the industrial process of pasta thermal VHT treatment. J. Sci. Food Agric. 79: 317-322.
Singh, H. and P. F. Fox. 1987. Heat stability of milk: role of β-lactoglobulin in the pH-dependent dissociation of micellar κ-casein. J. Dairy Res. 54: 509.
Singh, H. S. and L. K. Creamer. 1993. In vitro digestibility of whey protein-κ-casein complexes isolated from heated concentrated milk. J. Food Sci. 58: 299-302.
Soledad, A. H., V. N. Teresa, and I. P. María. 1997. Determination of water-soluble vitamins in infant milk by high-performance liquid chromatography. J Chromatogra. A. 778: 247-253.
Swaisgood, H. E. and G. L. Catignani. 1991. Protein digestibility: in vitro methods of assessment. Adv. Food Nutr. Fes. 35: 185.
van Boekel, M. S. J. S. 1998. Effect of heating on Maillard reactions in milk. Food Chem. 62(4): 403-414.
Vasbinder, A. J. and C. G. de Kruif. 2003. Casein-whey protein interactions in heated milk: the influence of pH. Int. Dairy J. 13: 669-677.
Villamiel, M., R. Lopez-Fandino, N. Corzo, and A. Olano. 1997. Denaturation of β-lactoglobulin and native enzymes in the plate exchanger and holding tube section during continuous flow pasteurization of milk. Food Chem. 58: 49-52.
Villamiel, M., M. Arias, N. Corzo, and A. Olano. 1999. Use of different thermal indices to assess the quality of pasteurized milks. Z Lebensm Unters Forsch A 208: 169-171.
Wilbey, R. A. 1996. Estimating the degree of heat treatment given to milk. J. Soc. Dairy Technol. 49: 109-112.
Yvon, M., S. Beucher, P. Scanff, S. Thirouin, and J. P. Pélissier. 1992. In vitro simulation of gastric digestion of milk proteins: comparison between in vitro and in vivo data. J. Agric. Food Chem. 40: 239-244.