游 若 篍臺灣大學：食品科技研究所范繼宗Fan, Chi-TzungChi-TzungFan2007-11-272018-06-292007-11-272018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/56317本研究以溶解血纖維蛋白活性為指標自市售納豆篩選納豆菌，並進行納豆製備，探討納豆水萃物對活性氧物質抗致突變性研究。使用安氏試驗法之測試菌株Salmonella typhimurium TA102分析納豆水萃物對氧化突變劑tert-butylhydroperoxide ( t-BOOH )及過氧化氫( H2O2 )誘導突變之防禦效果。結果顯示，由6株Bacillus sp.所製備之納豆水萃物(10 mg/mL)，對t-BOOH 和 H2O2之抗致突變性分別為36.8-44.2 %和53.6-59.6 %，而納豆的抗氧化突變性可歸因於其抗氧化能力。以6株納豆菌發酵所製備之納豆水萃物(5 mg/mL )，對過氧化氫之清除能力及螫合亞鐵離子分別為74.2-84.0 %及82.7- 94.3%。在清除DPPH自由基及還原力測定中，以Bacillus sp. CN08製備之納豆水萃物(10 mg/mL)為最佳，其具有38.4 % DPPH自由基清除能力，其還原力相當於30.1μg/mL抗壞血酸。而納豆水萃物(10 mg/mL)清除超氧陰離子的能力為44.5-54.7%。In this study, several Bacillus spp. with high fibrinolytic activity were isolated from several commercial natto products. Functional natto with high fibrinolytic activity was then made by these Bacillus isolates. The antimutagenic activities of natto water extracts against reactive oxygen species were studied by a modified Ames test using Salmonella typhimurium TA102 as a test strain. Assays for the activities of natto water extracts against mutations induced by peroxide mutagens tert-butylhydroperoxide (t-BOOH, 10 μg/plate) and hydrogen peroxide (H2O2, 6 μmol/plate) toward S. typhimurium TA102 were developed. The results indicated inhibitory effects of natto water extracts (10 mg/mL) preparation from 6 Bacillus spp. on the mutagenicity induced by t-BOOH and H2O2 toward S. typhimurium TA102 were range from 36.8 to 44.2 % and 53.6 to 59.6 %, respectively. The antimutagenic activities of natto water extracts might attribute to their antioxidative activities. Results also showed that scavenging effects on H2O2 and chelating effects on ferrous ions of natto water extracts (5 mg/mL) were 74.2-84.0 % and 82.7- 94.3%, respectively. Scavenging effects on DPPH radical and reducing activity of natto water extracts (10 mg/mL) preparation from Bacillus sp. CN08 showed the highest DPPH scavenging activity and ascorbic acid activity, 38.4 % and 30.1μg/mL, respectively. The scavenging effects on superoxide anions of natto water extracts were ranged from 44.5 to 54.7%.目 錄 頁次 壹、前言------------------------------------------------1 貳、文獻整理--------------------------------------------3 一、納豆簡介--------------------------------------------3 二、納豆菌之命名----------------------------------------4 三、鹼性發酵食品----------------------------------------4 四、納豆之生理功能--------------------------------------6 (一)納豆激酶(nattokinase)預防血栓-----------------------6 (二)抗氧化----------------------------------------------9 (三)降低膽固醇預防粥狀動脈硬化--------------------------9 (四)預防骨質疏鬆症-------------------------------------10 (五)預防高血壓-----------------------------------------11 (六)促進有益菌生長-------------------------------------13 五、活性氧與自由基之特性-------------------------------13 (一)超氧陰離子-----------------------------------------18 (二)過氧化氫-------------------------------------------18 (三)羥基自由基-----------------------------------------19 (四)單重態氧-------------------------------------------19 (五)過氧化脂質-----------------------------------------20 六、自由基對生物體之傷害-------------------------------20 (一)自由基對脂質的損害---------------------------------20 (二)對核酸的影響---------------------------------------21 (三)對蛋白質的影響-------------------------------------21 (四)對醣類的影響---------------------------------------22 七、抗氧化防禦系統-------------------------------------22 (一)抗氧化酶-------------------------------------------23 (二)抗氧化劑-------------------------------------------24 八、膳食與癌症-----------------------------------------27 九、抗致突變物/抗致癌物之作用機制----------------------28 十、安氏試驗法-----------------------------------------31 (一) S. typhimurium TA102之介紹------------------------33 (二) t-butyl hydroperoxide之介紹-----------------------35 十一、實驗架構-----------------------------------------36 參、材料與方法-----------------------------------------41 一、實驗材料----------------------------------------41 (一)試驗菌株---------------------------------------41 (二)原料大豆及市售納豆---------------------------------41 (三)培養基---------------------------------------------41 (四)儀器-----------------------------------------------42 (五)藥品-----------------------------------------------42 二、實驗方法-------------------------------------------44 (一)菌種活化及保存-------------------------------------44 (二) 菌種分離及納豆製備--------------------------------45 (三) 納豆激酶活性測定----------------------------------48 (四) 納豆水萃物之製備----------------------------------48 (五) 納豆水萃物抗致突變測試----------------------------49 (六) 納豆水萃物之抗氧化活性測定------------------------56 1、還原力之測定----------------------------------------56 2、螫合亞鐵離子能力之測定------------------------------56 3、清除DPPH自由基能力之測定----------------------------57 4、超氧陰離子清除效應----------------------------------57 5、過氧化氫清除效應------------------------------------58 肆、結果與討論-----------------------------------------59 一、菌種分離及納豆激酶活性-----------------------------59 二、S. typhimurium TA102試驗菌株基因型態之確認---------59 (一) 組胺酸需求之確認----------------------------------62 (二) rfa突變之測試-------------------------------------62 (三) uvrB 突變測試-------------------------------------64 (四) R-factor之測試------------------------------------64 (五) pAQ1 plasmid之測試--------------------------------64 三、納豆水萃物對S. typhimurium TA102之毒性試驗及 致突變性試驗---------------------------------------65 (一) 毒性試驗------------------------------------------65 (二) 致突變試驗----------------------------------------65 四、納豆水萃物之抗致突變性-----------------------------65 (一) 納豆水萃物對tert-butyl hydroperoxide誘導 S.typhimurium TA102氧化突變之抑制效果-------------68 (二) 納豆水萃物對H2O2誘導S. typhimurium TA102氧化 突變之抑制效果------------------------------------68 五、納豆水萃物之抗氧化活性-----------------------------71 (一) 還原力--------------------------------------------72 (二) 螫合亞鐵離子能力----------------------------------72 (三) DPPH 自由基清除效應-------------------------------76 (四) 超氧陰離子清除效應--------------------------------76 (五) 過氧化氫清除效應----------------------------------78 伍、結論-----------------------------------------------82 參考文獻-----------------------------------------------841106866 bytesapplication/pdfen-US納豆抗致突變抗氧化性活性氧物質nattoantimutagenicityantioxidative activityreactive oxygen speciesthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56317/1/ntu-94-R92641015-1.pdf