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中文論文名稱 Paenibacillus macerans TKU021發酵幾丁類物質所生產抗氧化物之特性研究
英文論文名稱 Studies on the antioxdants produced by using Paenibacillus macerans TKU021 fermentation of chitinous materials
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 98
學期 2
出版年 99
研究生中文姓名 李永鵬
研究生英文姓名 Yung-Peng Li
學號 697180320
學位類別 碩士
語文別 中文
口試日期 2010-07-08
論文頁數 44頁
口試委員 指導教授-王三郎
共同指導教授-郭曜豪
委員-郭曜豪
委員-王三郎
委員-王全祿
中文關鍵字 抗氧化  烏賊軟骨  發酵 
英文關鍵字 Antioxidation  Paneibacillus macerans  Squid pen  Fermentation 
學科別分類 學科別醫學與生命科學生物學
中文摘要 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。
英文摘要 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.
論文目次 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



參考文獻 Anraku, M., Fujii, T., Furutani, N., Kadowaki, D., Maruyama, T., Otagiri, M., Gebicki, J. M., Tomida, H. (2009). Antioxidant effects of a dietary supplement: Reduction of indices of oxidative stress in normal subjects by water-soluble chitosan. Food and Chemical Toxicology, 47, 104-109.

Aurand , L. W., Boone, N. H., Giddings, G. G. (1977). Superoxide and singlet oxygen in milk lipid peroxidation. Journal of Dairy Science, 60, 363-369.

Bhaskar, N., Suresh, P. V., Sakhare, P. Z., Sachindra, N. M.(2007). Shrimp biowaste fermentation with Pediococcus acidolactici CFR2182: Optimization of fermentation conditions by response surface methodology and effect of optimized conditions on deproteination/demineralization and carotenoid recovery. Enzyme and Microbial Technology, 40, 1427-1434

Boor, P. J., Yang, Y. Z., Yang, Y. S., Trent , M. B., He, N., Lick, S. D., Zimniak , P., Awasthi, Y. C. (2004). Glutathione-S-transferase A4-4 modulates oxidative stress in endothelium: possible role in human atherosclerosis. Atherosclerosis, 173, 211-221.

Chou, C. C., Juan, M. Y. (2010). Enhancement of antioxidant activity, total phenolic and flavonoid content of black soybeans by solid state fermentation with Bacillus subtilis BCRC 14715. Food Microbiology, 27, 586-591.

Desai, N., Sabanegh, Jr. E., Kim, T., Agarwal, A. (2010). Free radical theory of aging: Implications in male infertility. Urology, 75, 14-19.

Đorđević, T. M., Šiler-Marinković, S. S., Dimitrijević-Branković, S. I. (2010). Effect of fermentation on antioxidant properties of some cereals and pseudo cereals. Food Chemistry, 119, 957-963.

Fogliano, V. , Vitaglione, P. (2004). Use of antioxidants to minimize the human health risk associated to mutagenic/carcinogenic heterocyclic amines in food. Journal of Chromatography B, 802, 189-199.

Gabriel Hocman (1988). Chemoprevention of cancer: Phenolic antioxidants (BHT, BHA). International Journal of Biochemistry, 20, 639-651.

Getoff, N. (2007). Anti-aging and aging factors in life. The role of free radicals. Radiation Physics and Chemistry, 76, 1577-1586.

Gracy, R. W., Talent, J. M., Kong, Y., Conrad, C. C. (1999). Reactive oxygen species: the unavoidable environmental insult?. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 428, 17-22.

Halliwell, B., Gutteridge, J. M. C. (1989). Free Radicals in Biology and Medicine, 2nd edn. Clarendon Press, Oxford, UK.

Huo, M., Zhang, Y., Zhou, J., Yu, D., Wu, Y. (2009). Potential of amphiphilically modified low molecular weight chitosan as a novel carrier for hydrophobic anticancer drug: Synthesis, characterization, micellization and cytotoxicity evaluation. Carbohydrate Polymers, 77, 231-238

Ito, T., Matsutani, T., Mori, K., Nomura, T. (1992). Phagocytosis and hydrogen peroxide production by phagocytes of the sea urchin Strongylocentrotus nudus. Developmental & Comparative Immunology, 16, 287-294.

Jiang, Y. M., Yang, B., Zhao, M. M., Shi, J., Yang, N. (2008). Effect of ultrasonic treatment on the recovery and DPPH radical scavenging activity of polysaccharides from longan fruit pericarp. Food Chemistry, 106, 685-690

Johnson, B. G., Gonzales, C. A., Gill , P. M. W., Pople , J. A. (1994). A density functional study of the simplest hydrogen abstraction reaction. Effect of self-interaction correction. Chemical Physics Letters, 221, 100-108.

Jouikov, V. V., Fattakhova, D. S., Kozhevnikov, A. Yu. (2001). The potential-determining reaction of electrogenerated cation radicals of diphenylselenide: dimerization versus disproportionation. Electrochimica Acta, 46, 807-812.

Kehrer, J. P. (1993). Free radicals as mediators of tissue injury and disease. Critical Reviews in Toxicology, 23, 21-48.

King, M. D., France, J. L., Lee, J. T. (2007). Hydroxyl (OH) radical production rates in snowpacks from photolysis of hydrogen peroxide (H2O2) and nitrate (NO3−). Atmospheric Environment, 41, 5502-5509.

Kinnula, V. L., Crapo, J. D. (2004). Superoxide dismutases in malignant cells and human tumors. Free Radical Biology and Medicine, 36, 718-744.

Lee, R. F. (2003).Photo-oxidation and Photo-toxicity of Crude and Refined Oils. Spill Science & Technology Bulletin, 8, 157-162.

Leeuwenburgh, C., Hansen, P. A., Holloszy, J. O., Heinecke, J. W. (1999). Hydroxyl radical generation during exercise increases mitochondrial protein oxidation and levels of urinary dityrosine. Free Radical Biology and Medicine, 27, 186-192

Leitner,N. K. V., Berger, P., Doré, M., Legube, B. (1999). Ozone and hydroxyl radicals induced oxidation of glycine. Water Research, 33, 433-441.

Li, L. T., Zhu, Y. P., Fan, J. F., Cheng Y. Q. Improvement of the antioxidant activity of Chinese traditional fermented okara (Meitauza) using Bacillus subtilis B2. Food Control, 19, 654-661.

Masika, P. J., Adedapo, A. A., Jimoh, F. O., Koduru, S., Afolayan, A. J. (2008). Evaluation of the medicinal potentials of the methanol extracts of the leaves and stems of Halleria lucida. Bioresource Technology, 99, 4158-4163.

Matyjaszewski, K., Tsarevsky , N. V., Braunecker,W. A. (2007). Electron transfer reactions relevant to atom transfer radical polymerization. Journal of Organometallic Chemistry, 692, 3212-3222.
Michiels, C., Raes, M., Toussaint, O., Remacle, J. (1994). Importance of SE-glutathione peroxidase, catalase, and CU/ZN-SOD for cell survival against oxidative stress. Free Radical Biology and Medicine, 17, 235-248.

Murry, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C., Yolken, R. H. (1999). Manual of Clinical Microbiology, 7th ed.

Muzzarelli, R., Weckx, M., Filippini, O., Lough, C. (1989). Characteristic properties of N-Carboxybutyl chitosan. Carbohydrate Polymers, 11, 307-320.

Omaye, S. T., Zhang, P. (2001). Antioxidant and prooxidant roles for β-carotene, α-tocopherol and ascorbic acid in human lung cells. Toxicology in Vitro, 15, 13-24.

Oyaizu, M. (1986). Studies on product of browning reaction prepared from glucose amine. Japanese Journal of Nutrition, 44, 1986, 307–315.

Richardson, A., Bokov, A., Chaudhuri, A. (2004). The role of oxidative damage and stress in aging. Mechanisms of Ageing and Development, 125, 811-826.

Sachindra, N. M., Bhaskar, N. (2008). In vitro antioxidant activity of liquor from fermented shrimp biowaste. Bioresource Technology, 99, 9013-9016

Samman, S., Balasundram, N., Sundram, K. (2006). Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chemistry, 99, 191-203.

Sarkar, P. K., Moktan, B., Saha, J. (2008). Antioxidant activities of soybean as affected by Bacillus-fermentation to kinema. Food Research International, 41, 586-593

Shirai, K., Cira, L. A., Huerta, S., Hall, G. M (2002). Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery. Process Biochemistry, 37, 1359-1366.

Sies H. (1997). Oxidative stress: oxidants and antioxidants. Exp
Physiol , 82, 291–295.

Smith, D. L., Jung , W. J., Souleimanov, A., Park, R. D. (2007). Enzymatic production of N-acetyl chitooligosaccharides by crude enzyme derived from Paenibacillus illioisensis KJA-424. Carbohydrate Polymers, 67, 256-259.

Teel, A. L., Watts, R. J., Washington, D., Howsawkeng, J., Loge, F. J. (2003). Comparative toxicity of hydrogen peroxide, hydroxyl radicals, and superoxide anion to Escherichia coli. Advances in Environmental Research, 7, 961-968.

Wang, S. L., Liou, J. Y., Liang, T. W., Liu, K. C. (2009). Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides. Process Biochemistry, 44, 854-861.

Wang, S. L., Liu, K. C., Liang, T. W., Kuo, Y. H., Wang, C. Y. (2010). In vitro antioxidant activity of liquor and semi-purified fractions from fermented squid pen biowaste by Serratia ureilytica TKU013. Food Chemistry, 119, 1380-1385.

Yıldız, G., Demiryürek, A.T. (1998). Ferrous iron-induced luminol chemiluminescence: a method for hydroxyl radical study. Journal of Pharmacological and Toxicological Methods, 39, 179-184.

Yen, G. C., Hsieh, C. L. (1998). Antioxidant activity of extracts from du-zhone(Eucommia ulmoides) toward various lipid peroxidation model in vitro. Journal of Agricultural and Food Chemistry, 46, 3952–3957.

Yen, G. C., Chang, Y. C. (2003). Production of antioxidant from Aspergillus candidus broth filtrate by fermentor. Process Biochemistry, 38, 1425-1430.

Zhu, B. Z., Kitrossky, N., Chevion, M. (2000). Evidence for production of hydroxyl radicals by pentachlorophenol metabolites and hydrogen peroxide: A metal-independent organic fenton reaction. Biochemical and Biophysical Research Communications, 270,942-946.
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