臺灣博碩士論文加值系統

麻油豆腐乳與陳年豆腐乳發酵熟成期間，發酵液 pH 值約為 pH 5.4∼5.8。陳年豆腐乳發酵過程中第50天的蛋白質水解&;#37238;活性為最高(343 U, pH 5.5)，而麻油豆腐乳蛋白質水解&;#37238;活性皆維持在50∼100 U範圍左右。陳年豆腐乳所含蛋白質水解&;#37238;總活性為麻油豆腐乳總活性3倍以上。利用四種蛋白質水解&;#37238;抑制劑(EDTA、PMSF、IAA 與 DANMEL)來區分各種蛋白質水&;#37238;活性所佔百分比，陳年豆腐乳中以金屬性蛋白質水解&;#37238;與絲胺酸蛋白質水解&;#37238;為主(>80 %)，天門冬胺酸蛋白質水解&;#37238;在 pH 5.5時活性較高，約佔總活性20 %，隨著 pH 上升其活性隨之下降。麻油豆腐乳中，以金屬性蛋白質水解&;#37238;活性為主(>50 %)，隨著 pH 值上升絲胺酸蛋白質水解&;#37238;活性也隨之上升。透過 reverse zymography分析發現陳年豆腐乳在酸性下測得分子量174~290 kDa 金屬性與絲胺酸蛋白質水解&;#37238;；麻油豆腐乳在酸性下測得分子量225~290 kDa 絲胺酸蛋白質水解&;#37238;及分子量135~174 kDa 金屬性蛋白質水解&;#37238;活性，在鹼性下測得分子量48~81 kDa 金屬性蛋白質水解&;#37238;。透過SDS-PAGE gel slice分析發現陳年豆腐乳在酸性下測得分子量81~290 kDa絲胺酸蛋白質水解&;#37238;活性，在中性下測得分子量48~290 kDa 絲胺酸蛋白質水解&;#37238;活性，在鹼性下測得分子量81~104 kDa與135~290 kDa絲胺酸蛋白質水解&;#37238;活性；在酸性與中性下測得大範圍分子量37~290 kDa區間金屬性蛋白質水解&;#37238;活性。金屬離子對陳年豆腐乳蛋白質水解&;#37238;活性的影響，其中 Ca2+ 與 Mg2+ 離子可回復分子量174~290 kDa 區間蛋白質水解&;#37238;約70 %活性，Mn2+ 離子分別可回復174∼290 kDa與134∼174 kDa區間202 %與98 %蛋白質水解&;#37238;活性。

The pH of fermentation broth keep at 5.4~5.8 during white and aged sufu fermentation. The maximm protease activity was 343U, it appeared at the 50th day in aged sufu fermetation, but it shows constantly about 50~100 U in white sufu fermetation. The accumulated protease activity of the former is three-fold high than of the latter. The types of protease were classified with specific proteas inhibitors EDTA, PMSF, IAA and DANMEL. The fermentation broth of aged sufu shows more than 80 % protease activity from metallo- and serine protease activity, and about 20 % from aspartic protease, which activity decreases with increasing pH. The fermentation broth of white sufu has more than 50 % protease activity from metallo protease. The serine protease activity increases with increasing pH value. Using reverse zymography, the broth of aged sufu shows protease activity of serine protease in the size range of 174~290 kDa and metallo protease in the size range of 174~290 kDa at pH 5.5. The broth of white sufu shows protease activity of serine protease in the size range of 225~290 kDa and metallo protease in the size range of 135~174 kDa at pH 5.5, and metallo protease in the size range of 48~81 kDa at pH 9.0. Alternatively, using SDS-PAGE slicing about aged sufu, the serine protease in the size range of 81~290 kDa at pH 5.5, in the size range of 48~290 kDa at pH 7.5, and in the size range of 135~290 kDa at pH 9.0 show enzymatic activities. The metallo protease at size range of 37~290 kDa shown activity at pH 5.5 and 7.5. The metal ions Ca2+ and Mg2+ ions recovered 70 % activity of metallo protease in the size range of 174~290 kDa. Mn2+ ion ions recovered 202 % and 98 % activity of metallo protease in the size range of 174~290 kDa and 134~174 kDa, respectively.

目錄.......................................................i
中文摘要.................................................iii
英文摘要...................................................v
圖目錄....................................................vi
附錄....................................................viii
示意圖....................................................ix
第一章 前言................................................1
1.1 豆腐乳介紹.............................................1
1.2 菌種介紹...............................................5
1.3 蛋白質水解酶之簡介.....................................7
1.4 蛋白質水解酶抑制劑....................................13
1.5 研究目標..............................................16
第二章 實驗材料...........................................18
2.1 實驗試劑..............................................18
2.2 實驗儀器..............................................20
第三章 實驗方法...........................................21
3.1 豆腐乳來源............................................21
3.2 樣品的製備............................................21
3.3 蛋白質濃度測定........................................21
3.4 酵素活性測定..........................................22
3.5 蛋白質水解酶活性電泳分析..............................24
3.6 估測陳年豆腐乳蛋白質水解酶活性區分子量分佈............26
3.7 蛋白質銀染色法........................................28
3.8不同金屬離子對陳年豆腐乳蛋白質水解酶的活性影響.........28
第四章 結果...............................................31
4.1豆腐乳熟成期間 pH 值變化...............................31
4.2豆腐乳發酵過程蛋白質水解酶活性曲線.....................31
4.3豆腐乳中蛋白質水解酶種類分析...........................32
4.4SDS-PAGE Gel蛋白質水解酶活性區分子量影響...............34
4.5不同金屬離子對陳年豆腐乳蛋白質水解酶活性回復的影響.....36
第五章 討論...............................................37
第六章 參考文獻...........................................44
附圖......................................................50
附錄......................................................69
示意圖....................................................76

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