周正俊臺灣大學:食品科技研究所吳佳璇Wu, Chia-HsuanChia-HsuanWu2010-05-112018-06-292010-05-112018-06-292008U0001-0708200817113700http://ntur.lib.ntu.edu.tw//handle/246246/182158本研究以黑豆為原料，接種Bacillus subtilis BCRC 14715進行固態發酵，探討不同發酵溫度 (35, 40, 45, 50°C) 於培養期間 (0, 6, 12, 18小時) 對黑豆中一些活性成分，包括異黃酮素、納豆激酶 (nattokinase) 、維生素K2、血管收縮素轉化酶抑制劑 (angiotensin- converting enzyme inhibitor, ACE inhibitor) 、超氧歧化酶 (superoxide dismutase, SOD) 及吡啶二羧酸 (dipicolinic acid, DPA) 變化之影響。 結果顯示，40及45°C為B. subtilis BCRC 14715在黑豆基質中之最適生長溫度。無論發酵溫度為何，黑豆之總異黃酮含量在發酵過程中皆逐漸降低，其中以在35℃下發酵者下降之趨勢最為顯著。經發酵後之黑豆，其醣苷配基 (aglycone) 形式之異黃酮素含量，隨發酵時間增長而有明顯上升之趨勢 (p<0.05)，其中以在50°C下發酵之黑豆可獲得最高aglycone形式之異黃酮素。在各發酵溫度下，黑豆中β-葡萄醣苷酶 (β-glucosidase) 活性皆於發酵6小時達最大值，其後即呈下降之趨勢。此外，隨發酵時間延長，黑豆中納豆激酶活性、維生素K2含量、ACE inhibitor活性、SOD活性及DPA含量均逐漸提升。黑豆經發酵18小時後，以在35°C下發酵者可得最高之納豆激酶活性，維生素K2則是在45°C下發酵可獲得最大產量；黑豆於40及45°C下發酵可呈現較佳之ACE抑制活性及SOD活性；在50°C下發酵者，則可獲得最大之DPA產量。In the present study, fermented black soybean was prepared with Bacillus subtilis CRC 14715. The changes of some functional constituents of fermented black soybean, including isoflavone, nattokinase, vitamin K2, angiotensin- converting enzyme inhibitor (ACE), superoxide dismutase (SOD), and dipicolinic acid (DPA), during the fermentation (0, 6, 12, 18 h) with B. subtilis BCRC 14715 at various temperatures (35, 40, 45, 50°C) were investigated. The results revealed that the optimum growth temperatures of B. subtilis BCRC 14715 are 40－45°C in black soybean. The total content of isoflavone in black soybean decreased during fermentation, regardless of fermentation temperatures. The most marked change was observed at 35°C. Fermentation caused a significant increase (p<0.05) in the content of aglycone, which enhanced with fermentation time. The highest level of aglycone was obtained in black soybean fermented at 50°C. Regardless of fermentation temperatures, the activity of β-glucosidase noted in black soybean reached the highest after 6 h of fermentation and then decreased progressively. In addition, nattokinase activity, vitamin K2 content, ACE inhibitor activity, SOD activity and DPA content of black soybean increased as the fermentation time was extended. Black soybean fermented at 35°C for 18 h exhibited the highest level of nattokinase activity, while the highest content of vitamin K2 and DPA was found in black soybean fermented at 45 and 50°C, respectively. On the other hand, a higher activity of ACE inhibitor and SOD was observed in black soybean fermented at 40 and 45°C.摘要-------------------------------------------------------------------------------------------- ibstract---------------------------------------------------------------------------------------- ii錄-------------------------------------------------------------------------------------------- iii目錄----------------------------------------------------------------------------------------- vii目錄----------------------------------------------------------------------------------------- viii、前言-------------------------------------------------------------------------------------- 1、文獻整理-------------------------------------------------------------------------------- 2、黑豆----------------------------------------------------------------------------------- 21. 黑豆簡介------------------------------------------------------------------------------ 22. 黑豆之機能性------------------------------------------------------------------------ 2、納豆食品------------------------------------------------------------------------------ 3、納豆之活性成分--------------------------------------------------------------------- 41. 異黃酮--------------------------------------------------------------------------------- 4 2. 納豆激酶------------------------------------------------------------------------------ 63. 維生素K2----------------------------------------------------------------------------- 7 4. 血管收縮素轉化酶------------------------------------------------------------------ 7. 超氧歧化酶--------------------------------------------------------------------------- 8. 吡啶二羧酸--------------------------------------------------------------------------- 8. 發酵溫度對發酵製品的影響------------------------------------------------------ 9、材料與方法----------------------------------------------------------------------------- 11、實驗架構----------------------------------------------------------------------------- 11 二、實驗材料------------------------------------------------------------------------------ 11. 菌種------------------------------------------------------------------------------------ 11. 黑豆------------------------------------------------------------------------------------ 11. 培養基--------------------------------------------------------------------------------- 11. 試驗藥品------------------------------------------------------------------------------ 115. 儀器設備------------------------------------------------------------------------------ 14、B. subtilis BCRC 14715之保存活化及菌酛之製備---------------------------- 15. 菌株之保存--------------------------------------------------------------------------- 15. 菌種活化及菌酛之製備------------------------------------------------------------ 15 四、黑豆之發酵--------------------------------------------------------------------------- 16 五、分析方法------------------------------------------------------------------------------ 16. 菌數之測定--------------------------------------------------------------------------- 16 2. 異黃酮素之分析--------------------------------------------------------------------- 17 2.1. 發酵黑豆中異黃酮素之萃取方法-------------------------------------------- 17.2. 異黃酮素之分離----------------------------------------------------------------- 17.3. 異黃酮素標準曲線製作與發酵黑豆中異黃酮素之定量----------------- 17. β-glucosidase 活性之測定------------------------------------------------------- 18.1酵素液之製備---------------------------------------------------------------------- 18.2. 酵素活性之測定----------------------------------------------------------------- 18. 納豆激酶活性之測定--------------------------------------------------------------- 19.1萃取液之製備---------------------------------------------------------------------- 19 4.2 血纖維蛋白平板試驗法--------------------------------------------------------- 19 4.3 納豆激酶活性測定--------------------------------------------------------------- 19 5. 維生素K2之分析-------------------------------------------------------------------- 20 5.1維生素K2之萃取------------------------------------------------------------------ 20 5.2維生素K2之分離------------------------------------------------------------------ 20.3. 維生素K2標準曲線製作與發酵黑豆中維生素K2之定量--------------- 20. 血管收縮素轉化酶抑制劑之分析------------------------------------------------ 21.1萃取液之製備---------------------------------------------------------------------- 21.2 血管收縮素轉化酶抑制劑活性之測定--------------------------------------- 217. 超氧歧化酶之分析------------------------------------------------------------------ 22 7.1 萃取液之製備--------------------------------------------------------------------- 22 7.2 超氧歧化酶活性之測定--------------------------------------------------------- 22. 吡啶二羧酸之分析------------------------------------------------------------------ 22 8.1 萃取液之製備--------------------------------------------------------------------- 22.2 吡啶二羧酸含量之分析--------------------------------------------------------- 23. 統計分析------------------------------------------------------------------------------ 23、結果與討論----------------------------------------------------------------------------- 24、不同溫度下B. subtilis BCRC 14715於黑豆中之生長------------------------- 24、B. subtilis BCRC 14715發酵黑豆之異黃酮素含量---------------------------- 26. 不同溫度對B. subtilis BCRC 14715發酵黑豆總異黃酮含量之影響------ 26. B. subtilis BCRC 14715發酵黑豆過程中各異黃酮含量之變化------------- 28. 不同溫度對B. subtilis BCRC 14715發酵黑豆異黃酮組成之比較--------- 29 三、不同發酵溫度對B. subtilis BCRC 14715發酵黑豆β-葡萄醣苷酶活性之影響-------------------------------------------------------------------------------- 41 四、不同發酵溫度下B. subtilis BCRC 14715在黑豆中納豆激酶之產生------- 43 五、不同溫度對B. subtilis BCRC 14715發酵黑豆中維生素K2含量之影響---------------------------------------------------------------------------------------- 47、不同溫度對B. subtilis BCRC 14715發酵黑豆中血管收縮素轉化酶抑制之影響------------------------------------------------------------------------------ 49、不同溫度對B. subtilis BCRC 14715發酵黑豆中超氧歧化酶之影響------- 51、不同溫度對B. subtilis BCRC 14715發酵黑豆中吡啶二羧酸之影響------- 54、結論--------------------------------------------------------------------------------------- 56、參考文獻-------------------------------------------------------------------------------- 57錄一----------------------------------------------------------------------------------------- 69圖一、B. subtilis BCRC 14715於35°C發酵黑豆對血管收縮素轉化酶之抑制能力---------------------------------------------------------------------------- 69圖二、B. subtilis BCRC 14715於40°C發酵黑豆對血管收縮素轉化酶之抑制能力---------------------------------------------------------------------------- 70圖三、B. subtilis BCRC 14715於45°C發酵黑豆對血管收縮素轉化酶之抑制能力---------------------------------------------------------------------------- 71圖四、B. subtilis BCRC 14715於50°C發酵黑豆對血管收縮素轉化酶之抑制能力---------------------------------------------------------------------------- 72錄二、台灣農業化學與食品科學期刊格式------------------------------------------ 73application/pdf795357 bytesapplication/pdfen-USBacillus subtilis異黃酮納豆激酶維生素K2血管收縮素轉化酶抑制劑超氧歧化酶吡啶二羧酸isoflavonenattokinasevitamin K2angiotensin- converting enzyme inhibitorsuperoxide dismutasedipicolinic acidthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182158/1/ntu-97-R95641025-1.pdf