周正俊臺灣大學：食品科技研究所錢香伶Chien, Hsian-LingHsian-LingChien2007-11-272018-06-292007-11-272018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/56319摘 要 異黃酮為天然存在於大豆之植物雌激素，共有十二種衍生物。其中以醣苷配基 (aglycone) 形式存在之異黃酮素較易被人體吸收且具較高之生理活性。本研究以豆奶為基質，利用乳酸菌 (Streptococcus salivarius subsp. thermophilus BCRC 14085, Lactobacillus acidophilus BCRC 14079) 及雙叉桿菌 (Bifidobacterium infantis BCRC 14633, B. longum B6) 單獨或共同進行發酵，探討發酵過程中異黃酮素含量與β-葡萄糖苷酶活性之變化情形，並比較發酵豆奶製品中之異黃酮素在不同貯存條件下含量之變化。 結果顯示，豆奶發酵後，醣苷配基 (aglycone) 形式之異黃酮素 (daidzein, genistein 及 glycitein) 含量顯著增加，在所測試不同菌酛發酵之豆奶中，以 S. thermophilus 單獨發酵者醣苷配基之增加最多，其次為 S. thermophilus 與 B. longum 之共同發酵者。此外，隨著發酵時間之延長，豆奶中之β-葡萄糖苷酶 (β-glucosidase) 活性逐漸提高，且與醣苷配基形式之異黃酮素含量增加有相同的趨勢，而含葡萄糖苷之異黃酮素 (β-glucosides, malonylglucosides 及acetylglucosides) 之含量則逐漸減少。 發酵豆奶飲料經 4℃ 貯存期間中，以避光包材貯存者之異黃酮素含量保存效果較佳。此外，發酵豆奶乾燥製品之異黃酮素含量以添加有乾燥劑及脫氧劑且於 4℃ 下貯存者之保存效果最佳，於 25℃ 下不添加任何食品保存劑者異黃酮素之保存效果最差。Abstract Isoflavone is an important category of flavonoids and belongs to the phytoestrogen class. There are twelve major isoflavone isomers found in soybean. Isoflavone aglycones (daidzein, genistein and glycitein) are thought to be the most bioactive isomers among the isoflavones. This study investigated the changes of isoflavone composition and activity of β-glucosidase during the fermentation of soymilk with lactic acid bacteria (Streptococcus salivarius subsp. thermophilus BCRC 14085, Lactobacillus acidophilus BCRC 14079) and bifidobacteria (Bifidobacterium infantis BCRC 14633, B. longum B6) alone or simultaneously. On the other hand, the changes of isoflavone contents of the fermented soymilk products under different packaging and storage conditions were investigated. The results showed that soymilk after fermentation, the contents of isoflavone malonylglucosides, acetylglucosides and β-glucosides decreased significantly. On the contrary, the amount of aglycones, (daidzein, genistein and glycitein) increased significantly. Among all the fermented soymilks tested, the greatest increase in the contents of aglycones and the highest β-glucosidase activity were noted in soymilk fermented with Streptococcus thermophilus, followed by that fermented with S. thermophilus and Bifidobacterium longum simultaneously. It was also noted that as the fermentation time was extended, the contents of aglycones and activity of β-glucosidase in soymilk was raised. Fermented soymilks kept in dark glass bottles retained isoflavone contents much more than those kept in glass bottles exposure to light during the storage period. A better retention of isoflavone was noted in dried fermented soymilk that was held in dessicant and deoxidant containing bottle and stored at 4℃. On the other hand, retention of isoflavone in dried fermented soymilk was the poorest which was held in bottle without dessicant and deoxident and stored at 25℃.目 錄 頁次 摘要 -------------------------------------------------------------------------- I Abstract ---------------------------------------------------------------------- II 目錄 -------------------------------------------------------------------------- III 圖次 -------------------------------------------------------------------------- V 表次 -------------------------------------------------------------------------- VII 壹、前言 -------------------------------------------------------------------- 1 貳、文獻整理 --------------------------------------------------------------- 2 一、 黃豆及其加工製品 ---------------------------------------------- 2 1. 黃豆之簡介 ------------------------------------------------------- 2 2. 黃豆之化學組成 ------------------------------------------------- 3 3. 黃豆之加工利用及食用限制 ---------------------------------- 4 二、 異黃酮素 ---------------------------------------------------------- 6 1. 異黃酮素之簡介 ------------------------------------------------- 6 2. 異黃酮素之吸收代謝 ------------------------------------------- 6 3. 異黃酮素之生理活性 ------------------------------------------- 10 3-1異黃酮素與癌症 ------------------------------------------- 10 3-2異黃酮素與心血管疾病 ---------------------------------- 12 3-3異黃酮素與骨質疏鬆症 ---------------------------------- 12 3-4異黃酮素之抗氧化活性 ---------------------------------- 13 4. 黃豆及其製品中異黃酮素之含量 ---------------------------- 13 5. 異黃酮素之分析方法 ------------------------------------------- 17 三、 益生菌 ------------------------------------------------------------- 26 1. 益生菌之簡介 ---------------------------------------------------- 26 1.1益生菌之定義 ---------------------------------------------- 26 1-2理想益生菌之特性 ---------------------------------------- 27 1-3益生菌之營養機能與健康效益 ------------------------- 27 2. 乳酸菌之簡介 ---------------------------------------------------- 32 2-1乳酸菌之定義與特性 ------------------------------------- 32 2-2乳酸菌之分類 ---------------------------------------------- 32 3. 雙叉桿菌之簡介 ------------------------------------------------- 33 參、材料與方法 ------------------------------------------------------------ 35 一、實驗材料 ----------------------------------------------------------- 35 1.菌種 ---------------------------------------------------------------- 35 2.黃豆 ---------------------------------------------------------------- 35 3.藥品 ---------------------------------------------------------------- 35 4.儀器設備與器材 ------------------------------------------------- 36 二、實驗方法 ----------------------------------------------------------- 38 1.菌種之保存 ------------------------------------------------------- 38 2.接種源之製備 ---------------------------------------------------- 38 3.豆奶基質之製備 ------------------------------------------------- 38 4.發酵豆奶之製備 ------------------------------------------------- 38 5.異黃酮素之分析方法 ------------------------------------------- 39 5-1 異黃酮素之分離 ------------------------------------------ 39 5-2 HPLC分析效果之評估 ---------------------------------- 39 5-3異黃酮素標準曲線製作 ---------------------------------- 40 5-4豆奶中異黃酮素之定量 ---------------------------------- 41 6.異黃酮素之萃取方法 ------------------------------------------- 41 7.豆奶中β-葡萄糖苷酶活性之測定 --------------------------- 41 8.貯存試驗 ---------------------------------------------------------- 42 8-1 發酵豆奶飲料之貯存試驗 ------------------------------ 42 8-2 發酵豆奶乾燥製品於貯存過程中異黃酮素含量變化之情形 -------------------------------------------------- 42 9.統計分析 ---------------------------------------------------------- 42 肆、結果與討論 ------------------------------------------------------------ 44 一、分析方法之選擇 -------------------------------------------------- 44 二、HPLC異黃酮素之分離 ------------------------------------------ 44 三、HPLC分析效果之評估 ------------------------------------------ 47 四、異黃酮素標準曲線之製作 -------------------------------------- 49 五、發酵豆奶之異黃酮素含量 -------------------------------------- 52 六、發酵豆奶中 β-glucosidase 活性 ----------------------------- 54 七、豆奶發酵過程中異黃酮素含量與β-glucosidase活性之變化 -------------------------------------------------------------------- 56 八、發酵豆奶飲料於貯存過程中異黃酮素含量變化之情形 -- 62 九、發酵豆奶乾燥製品於貯存過程中異黃酮素含量變化之情形 ------------------------------------------------------------------- 74 伍、結論 --------------------------------------------------------------------- 82 陸、參考文獻 --------------------------------------------------------------- 84 圖 次 頁次 圖一、異黃酮素之化學結構式 ------------------------------------------ 7 圖二、異黃酮素之代謝與排泄途徑以及腸道微生物之影響 ------ 9 圖三、黃豆食品中genistein之化學形式 ------------------------------ 16 圖四、由烘烤去脂大豆薄片中萃出的黃豆雌激素之層析圖 ------ 18 圖五、組織黃豆粉以80%甲醇萃取後之高效率液相層析圖 ------ 19 圖六、利用C18半製備型管柱分析黃豆萃取物之逆相層析圖 --- 21 圖七、豆奶 (A)、豆腐 (B) 及味噌 (C) 萃取物之逆相層析圖 --- 22 圖八、組織性植物蛋白中異黃酮素之逆相高效率液相層析圖 --- 23 圖九、組織性植物蛋白中異黃酮素之高效率液相層析圖 --------- 24 圖十、一般黃豆食品中異黃酮素之高效率液相層析圖 ------------ 25 圖十一、十二種異黃酮標準品及內部標準品之逆向HPLC層析圖譜 -------------------------------------------------------------- 46 圖十二、發酵豆奶之1683590 bytesapplication/pdfen-US豆奶乳酸菌異黃酮素發酵豆奶fermented soymilksoymilkisoflavonebifidobacterialactic acid bacteriathesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56319/1/ntu-93-R91641015-1.pdf