Paneibacillus macerans TKU021係篩選自台灣北部土壤，能發酵烏賊軟骨生產抗氧化物質。本研究以烏賊軟骨作為唯一碳/氮源，調整添加濃度(0.5~2%)，於37℃培養0~6天，測定其發酵上清液之DPPH自由基清除率，發現以烏賊軟骨濃度2%，培養5天的發酵液有最佳的清除率。以此濃度為基準，而後進行了不同pH值的發酵上清液之DPPH自由基清除率測定。本研究亦對其抗氧化物質做了熱安定性以及酸鹼耐受性的實驗，發現其抗氧化物質有很好的熱安定性以及酸鹼耐受性。此外本研究也對Paneibacillus macerans TKU021的發酵上清液做了還原力以及總酚含量測試，同時也和本實驗室保存菌株Bacillus subtilis TKU004做了抗氧化力比較的實驗。

In this study, Paneibacillus macerans TKU021 produced antioxidants by ferment squid pen powder as the sole carbon / nitrogen source. We incubated Paneibacillus macerans TKU021 by different concentrations of squid pen powder (0.5~2%) for 1~6 days at 37℃.We found that the fermented supernatant liquid has best DPPH radical scavenging activity when medium contain 2% squid pen powder and culture for five days. We measuremented the DPPH radical scavenging activity in different pH value of culture supernatant. This study also made its antioxidant thermal stability and pH tolerance experiments and found that antioxidants have a good thermal stability and pH tolerance. 
In addition, this study also determined the reducing power and total phenol of supernatant liquid which fermented by Paneibacillus macerans TKU021. And we also done a comparison of antioxidant with Bacillus subtilis TKU004 which keep in our laboratory.

目錄                                                   頁次
中文摘要…………………………………………………………………I
英文摘要…………………………………………………………………II
目錄……………………………………………………………………III
圖目錄……………………………………………………………………VI
表目錄…………………………………………………………………VIII

第一章	緒論……………………………………………………………1
第二章	文獻回顧………………………………………………………4
2.1	多粘芽孢桿菌之特性…………………………………………4
2.2	幾丁質…………………………………………………………4
2.3	自由基…………………………………………………………5
2.3.1	自由基的定義…………………………………………………5
2.3.2	自由基的危害…………………………………………………5
2.4	自由基的種類…………………………………………………6
2.4.1	超氧陰離子……………………………………………………6
2.4.2	過氧化氫………………………………………………………6
2.4.3	氫氧自由基……………………………………………………7
2.4.4	單態氧…………………………………………………………8
2.4.5	過氧化自由基及烷氧自由基…………………………………8
2.5	抗氧化劑………………………………………………………8
2.6	抗氧化活性測定………………………………………………10
2.6.1	DPPH自由基清除率測定………………………………………10
2.6.2	總酚含量測定…………………………………………………10
2.6.3	還原力測定……………………………………………………10
第三章	材料與方法……………………………………………………12
3.1	材料與試劑……………………………………………………12
3.2	實驗儀器………………………………………………………13
3.3	實驗方法………………………………………………………13
3.3.1	菌種篩選………………………………………………………13
3.3.2	菌種鑑定………………………………………………………13
3.3.3	DPPH自由基清除率測定………………………………………14
3.3.4	還原力測定……………………………………………………14
3.3.5	總酚含量測定…………………………………………………14
第四章	結果與討論……………………………………………………16
4.1	菌種鑑定………………………………………………………16
4.1.1	16 SrRNA基因定序分析………………………………………16
4.1.2	API 50 CHB鑑定套組鑑定……………………………………18
4.2	DPPH自由基清除能力之測定…………………………………19
4.2.1	濃度……………………………………………………………20
4.2.2	pH值……………………………………………………………21
4.2.3	pH值變化………………………………………………………22
4.2.4	生菌數量………………………………………………………23
4.2.5	菌數和pH值對DPPH自由基清除率的影響……………………24
4.2.6	pH值安定性測試………………………………………………27
4.2.7	熱安定性測試…………………………………………………28
4.2.8	與其他研究的比較……………………………………………30
4.3	還原力測定……………………………………………………33
4.4	總酚含量………………………………………………………36
第五章	結論……………………………………………………………38
參考文獻…………………………………………………………………39
圖目錄                                                 頁次
圖2.1 幾丁質結構圖……………………………………………………5
圖2.2 幾丁聚醣結構圖…………………………………………………5
圖2.3 超氧陰離子的路易斯結構式……………………………………6
圖2.4 過氧化氫結構式…………………………………………………7
圖4.1 NCBI Blast基因序列比對圖……………………………………17
圖4.2 鑑定TKU021的結果………………………………………………18
圖4.3 不同濃度及發酵時間對DPPH自由基清除率的影響……………20
圖4.4 不同pH值對DPPH自由基清除率的影響…………………………21
圖4.5 發酵上清液pH值隨時間的變化…………………………………22
圖4.6 不同的pH值隨發酵時間不同的菌數……………………………23
圖4.7 培養液pH 3的DPPH自由基清除率、pH 變化以及生菌數量…………………………………………………………………………24
圖4.8 培養液pH 5的DPPH自由基清除率、pH 變化以及生菌數量…………………………………………………………………………25
圖4.9 培養液pH 7的DPPH自由基清除率、pH 變化以及生菌數量…………………………………………………………………………25
圖4.10 培養液pH 9的DPPH自由基清除率、pH 變化以及生菌數量…………………………………………………………………………26
圖4.11 培養液pH 11的DPPH自由基清除率、pH 變化以及生菌數量…………………………………………………………………………26
圖4.12 TKU021在不同pH值的DPPH自由基清除率……………………27
圖4.13 加熱時間與DPPH自由基清除率的變化………………………29
圖4.14 TKU021和CFR2182的自由基清除率與濃度的關係比較………30
圖4.15 TKU021與TKU004 DPPH自由基清除率比較圖…………………32
圖4.16 TKU021與TKU004的還原力……………………………………35
圖4.17 TKU021與TKU004的總酚含量…………………………………37
表目錄                                                 頁次
表2.1 國際癌症研究中心對致癌風險的分類…………………………9
表4.1 TKU021 16S rDNA基因序列……………………………………16
表4.2 TKU021基因序列比對基因資料庫的結果………………………18
表4.3 加熱時間與EC50的變化………………..………………………29
表4.4 不同微生物發酵液之DPPH自由基清除率比較表………………33
表4.5 不同微生物發酵液之還原力比較表……………………………36

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