Antioxidative and antimutagenic activities of the soybean koji extract prepared with different starters

本研究比較5株常用菌酛：Aspergillus sojae BCRC 30103、Aspergillus oryzae BCRC 30222、Aspergillus awamori、Actinomucor taiwanensis及Rhizopus sp.所製備黃豆豆麴甲醇萃出物之抗氧化 (DPPH自由基清除、亞鐵離子螯合及還原力)與對4-Nitroquinoline N-oxide (4NQO)之抗致突變活性。並進一步探討不同溫度下製備Asp. awamori-豆麴之甲醇萃取物對抗氧化活性與抗致突變性之影響。 結果顯示，黃豆對於所有試驗菌株均為一良好生長基質，且黃豆發酵後其所測得之抗氧化活性與總酚類化合物含量均提高。其中以Asp. awamori-豆麴之甲醇萃取物呈現較強之DPPH自由基清除力、亞鐵離子螯合力、還原力及抗致突變活性。此外此豆麴萃出物具有最高之總酚類化合物含量。進一步利用Asp. awamori在不同溫度 (25℃、30℃及35℃)與發酵時間 (1~5天)下製備豆麴，發現以30℃發酵3天之Asp. awamori-豆麴之甲醇萃出物所呈現之抗氧化活性最高，亦具有最高之總酚類化合物含量，顯示總酚含量與抗氧化能力有關連性。而在35℃下製備之Asp. awamori-豆麴之甲醇萃出物所呈現之抗致突變性明顯 (p < 0.05)高於在其他溫度下所製備者。In the present study, methanolic extracts of soybean koji prepared with various starters：Aspergillus sojae BCRC 30103、Aspergillus oryzae BCRC 30222、Aspergillus awamori、Actinomucor taiwanensis or Rhizopus sp. were investigated for their antioxidative activities (DPPH radicals scavenging effects, Fe+2-chelating ability and reducing power) and antimutagic avtivities against 4-Nitroquinoline N-oxide (4NQO). Effects of fermentation temperature and time on the antioxidative and antimutagenic activity of extracts of Asp. awamori-koji were also measured. The results showed soybean was good substrate for the grown of all the starters tested. The antioxidative activity, antimutagenic activity and total phenolic content of the extract of soybean were increased after fermentation. The soybean koji prepared with Asp. awamori exhibited the highest DPPH free radical scavenging activity, Fe+2-chelating ability, reducing power and contained the highest total phenolic content among the 5 kinds of koji tested. Studies on the soybean koji prepared with Asp. awamori at different temperatures (25℃、30℃ and 35℃) and fermented periods (1~5 days) further revealed that the methanolic extract of Asp. awamori-koji fermented at 30℃ for 3 days showed the highest antioxidative activity and containted the highest amount of total phenolic compounds. It suggested that the total phenolic content might be related to the antioxidative activity observed. In addition, the extract of Asp. awamori-koji fermented at 35℃ showed a higher antimutagenic activity than those prepared at the other temperatures.中文摘要------------------------------------------------------------------------------------ Ⅰ 英文摘要------------------------------------------------------------------------------------ Ⅱ 目錄------------------------------------------------------------------------------------------ Ⅲ 圖次------------------------------------------------------------------------------------------ Ⅵ 表次------------------------------------------------------------------------------------------ Ⅶ 壹、前言------------------------------------------------------------------------------------ 1 貳、文獻回顧------------------------------------------------------------------------------- 3 一、黃豆--------------------------------------------------------------------------------- 3 1. 黃豆簡介-------------------------------------------------------------------------- 3 2. 黃豆之機能性-------------------------------------------------------------------- 3 2.1 異黃酮---------------------------------------------------------------------------- 4 二、發酵食品之抗氧化性與抗致突變性------------------------------------------- 5 1. 微生物之抗氧化性與抗致突變性-------------------------------------------- 5 2. 微生物發酵食品之抗氧化性與抗致突變性-------------------------------- 7 三、氧化作用及抗氧化之機制與原理--------------------------------------------- 9 1. 自由基與活性氧----------------------------------------------------------------- 9 2. 活性氧的種類與來源----------------------------------------------------------- 10 3. 自由基對生物體之作用與傷害----------------------------------------------- 10 4. 氧化性傷害----------------------------------------------------------------------- 12 5. 抗氧化劑作用的原理與機制-------------------------------------------------- 12 (1)自由基終止型 (free radical terminator)----------------------------------- 12 (2)還原劑或氧清除劑 (reducing agent or oxygen scavengers)----------- 12 (3)單重態氧抑制劑 (singlet oxygen inhibitor)------------------------------ 14 (4)金屬螯合劑 (chelating agent)----------------------------------------------- 14 (5)抗氧化酵素 (antioxidative enzyme)--------------------------------------- 14 6.天然抗氧化劑---------------------------------------------------------------------- 15 四、癌症形成原因與抗癌機制------------------------------------------------------ 15 五、安氏實驗法------------------------------------------------------------------------ 19 參、材料與方法--------------------------------------------------------------------------- 23 一、材料--------------------------------------------------------------------------------- 23 1. 菌種-------------------------------------------------------------------------------- 23 2. 黃豆-------------------------------------------------------------------------------- 23 3. 培養基----------------------------------------------------------------------------- 23 4. 試驗藥品-------------------------------------------------------------------------- 23 5. 儀器設備-------------------------------------------------------------------------- 24 二、樣品製備--------------------------------------------------------------------------- 25 1. 菌株之保存與活化-------------------------------------------------------------- 25 2. 冷凍保存-------------------------------------------------------------------------- 25 3. 活化-------------------------------------------------------------------------------- 25 4. 接種源之製備-------------------------------------------------------------------- 25 5. 豆麴之製備----------------------------------------------------------------------- 25 6. 豆麴甲醇萃取物之製備-------------------------------------------------------- 26 三、分析方法---------------------------------------------------------------------------- 26 1. 豆麴萃取物之萃出率----------------------------------------------------------- 26 2. 菌絲含量測定-------------------------------------------------------------------- 26 3. 豆麴萃取液之總酚類化合物含量-------------------------------------------- 27 4. 抗氧化活性之檢測-------------------------------------------------------------- 27 4.1 DPPH自由基清除效力-------------------------------------------------------- 27 4.2 亞鐵離子螯合能力------------------------------------------------------------- 28 4.3 還原力測定---------------------------------------------------------------------- 28 5. 抗致突變性試驗 ---------------------------------------------------------------- 28 5.1 S. typhimurium TA 100 BCRC 12378之保存與活化---------------------- 29 5.2 S. typhimurium TA 100 BCRC 12378之基因型態確認------------------- 29 5.3 毒性試驗------------------------------------------------------------------------- 31 5.4 致突變性試驗------------------------------------------------------------------- 31 5.5 抗致突變性試驗---------------------------------------------------------------- 32 6. 統計分析-------------------------------------------------------------------------- 32 肆、結果與討論--------------------------------------------------------------------------- 33 一、不同菌酛製備豆麴之甲醇萃取物菌絲之增殖------------------------------- 33 二、不同菌酛製備豆麴甲醇萃出物之抗氧化活性------------------------------- 33 1. DPPH自由基清除效力---------------------------------------------------------- 33 2. 亞鐵離子螯合能力-------------------------------------------------------------- 37 3. 還原力----------------------------------------------------------------------------- 39 4. 不同豆麴抗氧化活性之比較-------------------------------------------------- 42 三、豆麴萃取物中總酚類化合物之含量------------------------------------------- 44 四、不同溫度下製備Asp. awamori-豆麴甲醇萃取物及菌絲之增殖---------- 46 五、不同溫度下製備Asp. awamori豆麴之甲醇萃出物之抗氧化活性-------- 46 六、不同溫度下製備Asp. awamori-豆麴中總酚類化合物之含量------------- 52 七、不同發酵時間下製備Asp. awamori-豆麴甲醇萃取物及菌絲之增殖---- 52 八、不同發酵時間下製備Asp. awamori-豆麴之甲醇萃出物之抗氧化活性 55 九、不同發酵時間下製備Asp. awamori-豆麴中總酚類化合物含量測定---- 60 十、不同菌酛製備豆麴萃取物之抗致突變性------------------------------------ 62 1. S. typhimurium TA 100 試驗菌株基因型態之確認------------------------ 62 (1)組酸酸需求性之確認------------------------------------------------------- 62 (2) rfa突變之測試---------------------------------------------------------------- 62 (3) uvrB突變之測試------------------------------------------------------------- 62 (4) R-factor之測試--------------------------------------------------------------- 62 十一、不同菌酛製備豆麴萃取物之毒性與抗致突變性------------------------ 63 十二、不同菌酛製備豆麴萃取物之抗致突變性試驗--------------------------- 63 十三、不同溫度製備豆麴萃取物其抗致突變分析------------------------------ 69 伍、結論------------------------------------------------------------------------------------- 74 陸、參考文獻------------------------------------------------------------------------------- 7