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中文論文名稱 Pseudomonas spp.細菌發酵烏賊軟骨生產生物活性物質及其應用
英文論文名稱 Production and applications of bioactive materials by Pseudomonas spp. using squid pen as carbon/nitrogen source
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 97
學期 2
出版年 98
研究生中文姓名 徐琬涵
研究生英文姓名 WAN-HAN HSU
學號 696180222
學位類別 碩士
語文別 中文
第二語文別 英文
口試日期 2009-07-14
論文頁數 47頁
口試委員 指導教授-王三郎
委員-王全祿
委員-顏裕鴻
中文關鍵字 Pseudomonas sp.  烏賊軟骨  去蛋白  N-乙醯幾丁寡醣 
英文關鍵字 Pseudomonas spp.  squid pen  deproteinization  N-Acetyl chitooligosaccharides 
學科別分類 學科別醫學與生命科學生物學
中文摘要 本研究係利用Pseudomonas aeruginosa K-187與Pseudomonas sp. TKU015發酵烏賊軟骨生產胜肽、寡醣等生物活性物質之探討。以含1% 烏賊軟骨粉、0.1% K2HPO4 及0.05% MgSO4.7H2O之100 mL液態培養基,分別在37℃及30℃進行搖瓶培養。在烏賊軟骨去蛋白方面,培養4天後,K-187所得之蛋白質去除率為68%,而TKU015能去除SPP中的幾丁質保留部分蛋白質,與0.1%市售木瓜酵素及鳳梨酵素比較,鳳梨酵素對烏賊軟骨有較高的蛋白質去除率,反應4天後之去除率為70%。以K-187與TKU015之發酵上清液與0.1% 鳳梨酵素共同反應4天後,蛋白質去除率可高達99%,而SPP幾近被完全水解,故測其上清液,分析幾丁寡醣組成,添加0.1% 鳳梨酵素於K-187共同發酵水解SPP可得1~5醣。此外於植物生長方面,TKU015與K-187之發酵上清液富含胺基酸、胜肽等對青江菜的生長明顯有幫助。綜合上述,可應用在胜肽、胺基酸及幾丁寡醣之生產。
英文摘要 Pseudomonas aeruginosa K-187 and Pseudomonas sp. TKU015 can be used for fermentation of squid pen in production of bioactive materials. The culture condition for K-187 and TKU015 was composed of 1% squid pen powder (SPP) (w/v), 0.1 % K2HPO4 and 0.05 % MgSO4.7H2O, and incubated in 100 mL of liquid medium in shaking flasks at 37℃ and 30℃. For deproteinization tests, K-187 fermentation of squid pen powder showed protein removal of 68% after 4 days, and TKU015 could removed chitin and retained protein content in spp.
The squid pen powder was treated with 0.1% commercial papain and bromelain as a comparison, the deproteinization rate in combinations of 1% Pseudomonas spp. culture supernatant and 0.1% bromelain was all 99% while the rate was 59% and 69% in 0.1% papin with Pseudomonas spp. only. The chitinase of K-187 and TKU015 combined with 0.1% bromelain can hydrolyze squid pen powder efficiently which may apply to the productions of peptides, amino acids and chitoligosaccharides by useful for biological applications. N-Acetylglucosamine (GlcNAc) to N-acetylchitopentose (GlcNAc)5 were also produced from the culture supernatant by using K-187 strain for four days fermentation with 0.1% bromelain. Besides, The fourth day culture supernatant of Pseudomonas spp. showed maximal activity of 4-6 fold growth enhancing effect on Brassica chinensis Linn weight.
論文目次 封面內頁
授權書
簽名頁
誌謝
中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
圖目錄 Ⅴ
表目錄 Ⅵ
第一章 緒論 1
第二章 文獻回顧 3
2.1 Pseudomonas spp. 之簡介 3
2.2 水產廢棄物之微生物再利用 4
2.3 烏賊軟骨之所含主要成分 6
2.4 幾丁質與幾丁聚醣之應用 6
2.5 N-乙醯幾丁寡醣及幾丁寡醣 7
第三章 材料與方法 10
3.1 實驗菌株 10
3.2 實驗材料 10
3.3 實驗儀器 11
3.4 實驗方法 11
3.4.1 Pseudomonas spp. 之培養條件 11
3.4.2 烏賊軟骨之去蛋白 12
3.4.3 蛋白酶活性之測定 12
3.4.4 幾丁質酶之活性測定 13
3.4.5 幾丁聚醣酶之活性測定 13
3.4.6 DNS還原醣量之測定 14
3.4.7 蛋白質定量分析 14
3.4.8 游離胺基酸之測定 14
3.4.9 N-乙醯幾丁寡醣製備 15
3.4.10 N-乙醯幾丁寡醣之組成分析 15
3.4.11 利用Pseudomonas spp.發酵液進行青江菜作物生長測試16
3.4.11.1 青江菜之預培養 16
3.4.11.2 促進青江菜生長試驗 16
第四章 結果與討論 17
4.1 以Pseudomonas spp.發酵法進行烏賊軟骨之去蛋白• 17
4.2 以不同培養基與Pseudomonas spp.之去蛋白比較 17
4.3 添加木瓜酵素與鳳梨酵素對烏賊軟骨去蛋白之比較 23
4.4 添加0.1%鳳梨酵素於Pseudomonas spp.發酵烏賊軟骨所幾丁寡醣組成分析 31
4.5 Pseudomonas spp.發酵上清液對青江菜作物生長之影響 34
第五章 結論 38
參考文獻 39

圖目錄

圖4.1 不同培養基對於(A)TKU015 (B)K-187 (C)TKU015+K-187之去蛋白影響 20
圖4.2 (A)TKU015(B)K-187發酵烏賊軟骨之去蛋白率 21
圖4.3 (A)TKU015 (B)K-187添加市售酵素後烏賊軟骨去蛋白率之較 27
圖4.4 添加不同濃度鳳梨酵素於烏賊軟骨之去蛋白影響 28
圖4.5 (A)TKU015 (B)K-187添加0.1%鳳梨酵素發酵烏賊軟骨0~4天乾重、蛋白質與還原醣含量之變化 29
圖4.6 經(A) K-187+0.1% bromelain (B) TKU015+01% bromelain 發酵0-4天所剩烏賊軟骨粉殘重 30
圖4.7 利用HPLC進行幾丁寡醣組成分析(A)標準品 (B)添加0.1%鳳梨酵素於Pseudomonas aeruginosa K-187發酵烏賊軟骨 32
圖4.8 Pseudomonas sp. TKU015與K-187發酵上清液對青江菜生長之影響 37





表目錄

表2.1 幾丁質與幾丁聚醣的應用 9
表4.1 Pseudomonas aeruginosa K-187 與Pseudomonas sp. TKU015之培養條件 17
表4.2 Pseuomonus sp. TKU015發酵烏賊軟骨之去蛋白率 22
表4.3 Pseudomonus sp. K-187發酵烏賊軟骨之去蛋白率 22
表4.4 本研究與其他菌株去蛋白之比較 23
表4.5 發酵烏賊軟骨所得幾丁寡醣含量之分析 33
表4.6 Pseudomonas sp. TKU015與K-187發酵液對青江菜生長之影響 35

參考文獻 Aiba, S., 1994. Preparation of N-acetylchitooligosaccharides by hydrolysis of chitosan with chitinase followed by N-acetylation. Carbohydr Res, 265, 323-8.

Austin, P. R., Brine, C. J., Castle, J. E., Zikakis. J. P., 1981. Chitin: new facets of research. Science, 212, 749-53.

Bradford, M. M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-54.

Broussignac, P., 1968. Chitosan, a natural polymer not well known by the industry. Chimie & Industrie, Gene Chimique, 99, 1241-7.

Bustos, R. O., Michael, H., 1994. Microbial deproteinization of waste prawn shell. Institution of Chemical Engineers Symposium Series, Institution of Chemical Engineer, 13-5.

Cosio, I. G., Fisher, R. A., Carroad, P. A., 1982. Bioconversion of shellfish chitin waste: waste pretreatment, enzyme production, process design, and economic analysis. J Food Sci, 47, 901-5.

Gagne, N., Simpson, B. K., 1993. Use of proteolytic enzymes to facilitate recovery of chitin from shrimp wastes. Food Biotechnol, 7, 253-63.

Hadwiger, L., Ogawa, T., Kuyama, H., 1994. Chitosan polymer sizes effective in inducing phytoalexin accumulation and fungal suppression are verified with synthesized oligomers. Mol Plant Microbe Interact, 7, 531-3.

Hirano, S., 1996. Chitin biotechnology application. Biotechnol Ann Rev, 2, 237-58.

Hudson, S. M. 1997. Applications of chitin and chitosan as fiber and textile chemicals, Jacques Andre Publisher, 590-9.

Hung, T. H., Chang, Y. M., Sung, H. Y., Chang, C. T., 2002. Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain. J Agric Food Chem, 50, 4666-73.

Imoto, T., Yagishita, K., 1971. A simple activity measurement by lysozyme. Agric Biol Chem, 35, 1154-6.

Jeon, Y. J., Kim, S. K., 2000. Continous production of chitooligosaccharides using a dual reactor system. Process Biochem, 35, 623-32.

Jo, G. H., Jung, W. J., Kuk, J. H., Oh, K. T., Kim, Y. J., Park, R. D., 2008. Screening of protease-producing Serratia marcescens FS-3 and its application to deproteinization of crab shell waste for chitin extraction. Carbohydr Polym, 74, 504-8.

Jung, W. J., Jo, G. H., Kuk, J. H., Kim, Y. J., Oh, K. T., Park, R. D., 2007. Production of chitin from red crab shell waste by successive fermentation with Lactobacillus paracasei KCTC-3074 and Serratia marcescens FS-3. Carbohydr Polym, 68, 746-50.

Kendra, D. F., Hadwiger, L. A., 1984. Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Exp Mycol, 8, 276-81.

Kendra, D. F., Christian, D., Hadwiger, L. A., 1989. Chitosan oligomers from Fusarium solani/pea interactions, chitinase/β-glucanase digestion of sporelings and from fungal wall chitin actively inhibit fungal growth and enhance disease resistance. Physiol Mol Plant Pathol, 35, 215-30.

Kienzle-Sterzer, C. A., Rodriguez-Sanchez, D., Karalekas, D., Rha, C., 1982. Stress relaxation of a polyelectrolyte network as affected by ionic strength. Macromolecules, 15 , 631-4.

Kienzle-Sterzer, C. A., Rodriguez-Sanchez, D., Rha, C. K., 1985. Flow behavior of a cationic biopolymer : chitosan. Polymer Bulletin, 13, 1-6.

King, E. O., Ward, M. K., Raney, D. E., 1954. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med, 44, 301-7.

Knorr, D., 1984. Use of chitinous polymer in food -A challenge for food research and developent. Food Technol, 38, 85-97.

Knorr, D., 1991. Recovery and utilization of chitin and chitosan in food processing waste management. Food Technol, 45, 114-22.

Kumar, M. N. V. R., 2000. A review of chitin and chitosan applications. Reactive Functional Polym, 46, 1-27.

Liang, T. W., Chen, Y. J., Yen, Y. H., Wang, S. L., 2007. The antitumor activity of the hydrolysates of chitinous materials hydrolyzed by crude enzyme from Bacillus amyloliquefaciens V656. Process Biochem, 42, 527-34.

Muzzarelli, R. A. A., Rocchetti, R., 1985. Determination of the degree of acetylation of chitosans by first derivative ultraviolet spectrophotometry. Carbohydr Polym, 5, 461-72.

Rao, M. S., Tuyen, M. H., Stevens, W. F., Chandrkrachang, S., 2001. Deproteination by mechanical, enzymatic and Lactobacillus treatment of shrimp waste for production of chitin. Tokyo: Kodansha Scientific Ltd., 301-4.

Rosen, H. 1957. A modified ninhydrin colorimetric analysis for amion acids. Arch Biochem Biophys, 67, 10-5.

Ryan, K. J., Ray, C. G., 2004. Sherris Medical Microbiology. McGraw Hill, 4th ed.

Shen, K. T., Chen, M. H., Chan, H. Y., Jeng, J. H., Wang, Y. J., 2009. Inhibitory effects of chitooligosaccharides on tumor growth and metastasis. Food Chem Toxicol, in press.

Suzuki, K., Mikami, T., Okawa, Y., Tokoro, A., Suzuki, S., Suzuki, M., 1986. Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose. Carbohydr Res, 151, 403-8.

Takeda, M., Abe, E., 1962. Isolation of crustacean chitin deacetylation by sodium ethylenediaminetetraacetate and enzymatic hydrolysis of incidental proteins. Norinsho Suidsan Koshuso Kenkyu Hokoku, 11, 339-406.

Takeda, M., Katsuura, H., 1964. Purification of king crab chitin. Susan Daigaku Kenkyu Hokoku, 13, 109-16.

Tokoro, A., Tatewaki, N., Suzuki, K., Mikami, T., Suzuki, S., Suzuki, M., 1988. Growth-inhibitory effect of hexa-N-acetylchitohexaose and chitohexaose against Meth-A solid tumor. Chem Pharm Bull, 36, 784-90.

Tokoro, A., Kobayashi, M., Tatewaki, N., Suzuki, K., Okawa, Y., Mikami, T., Suzuki, S., Suzuki, M., 1989. Protective effect of N-acetyl chitohexaose on Listeria monocytogenes infection in mice. Microbiol Immunol, 33, 357-67.

Todd, E. W., 1949. Quantitative studies on the total plasmin and trypsin inhibitor of human blood serum. J Exp Med, 39, 295-308.

Tortora, G. J., Funke, B., Case, C., 2004. Microbiology : an introduction, Benjamin Cummings, 8th ed., 312.

Ueno, K., Yamaguchi, T., Sakairi, N., Nishi, N., Tokura , S., 1997. Antimicrobial activity by fractionated chitosan oligomers, Jacques Andre Publisher, 156-61.

Wang, S. L., Chang, W. T., Lu, M. C., 1995. Production of chitinase by Pseudomonas aeruginosa K-187 in a shrimp and crab powder as a carbon source. Proc Nat Sci Counc ROC (B), 19, 105-12.

Wang, S. L., Chang, W. T., 1997. Purification and characterization of two bifunctional chitinase/lysozmes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl Environ Microbiol, 63, 380-6.

Wang, S. L., Chio, S. H., 1998. Deproteinization of shrimp and crab shell with the protease of Pseudomonas aeruginosa K-187. Enzyme Microb Technol, 22, 629-33.

Wang, S. L., Yieh, T. C., Shih, I. L., 1999. Production of antifungal compound by Pseudomonas aeruginosa K-187 using shrimp and crab shell powder as a carbon source. Enzyme Microb Technol, 25, 142-8.

Wang, S. L., Yieh, T. C., Shih, I. L., 1999. Purification and characterization of a new antifungal compound produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Enzyme Microb Technol, 25, 439-46.

Wang, S. L., Kao, T. Y., Wang, C. L., Yen, Y. H., Chern, M. K., Chen, Y. H., 2006. A solvent stable metalloprotease produced by Bacillus sp. TKU004 and its application in the deproteinization of squid pen for β-chitin preparation. Enzyme Microb Technol , 39, 724-31.

Wang, S. L., Chen, S. J., Wang, C. L., 2008. Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate. Carbohydr Res, 343, 1171-9.

Wang, S. L., Huang, T. Y., Wang, C. Y., Liang, T. W., Yen, Y. H., Sakata, Y., 2008. Bioconversion of squid pen by Lactobacillus paracasei subsp paracasei TKU010 for the production of proteases and lettuce growth enhancing biofertilizers. Biores Technol, 99, 5436-54.

Wang, S. L., Lin, H. T., Liang, T. W., Chen, Y. J., Yen, Y. H., Guo, S. P., 2008. Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials. Biores Technol, 99, 4386-93.

Wang, S. L., Chen, H. J., Liang, T. W., Lin, Y. D., 2009. A novel nattokinase produced by Pseudomonas sp. TKU015 using shrimp shells as substrate. Process Biochem, 44, 70-6.

Wang, S. L., Lin, C. L., Liang, T. W., Liu, K. C., Kuo, Y. H., 2009. Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants. Biores Technol, 100, 316-23

Wang, S. L., Yang, C. W., Liang, T. W., Peng, J. H., Wang, C. L., 2009. Degradation of chitin and production of bioactive materials by bioconversion of squid pens. Carbohydr Polym, in press.

Wang, S. L., Yang, 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 Biochem, 44, 854-61.

Yalpani, M., Pantaleone, D., 1994, An examination of the unusual susceptibilities of aminoglycans to enzymatic hydrolysis. Carbohydr Res, 256 , 159-75.

Yang, J. K., Shih, I. L., Tzeng, Y. M., Wang, S. L., 2000. Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes. Enzyme Microb Technol, 26, 406-13.

Yen, Y. H., Li, P. L., Wang, C. L., Wang, S. L., 2006. An antifungal protease produced by Pseudomonas aeruginosa M-1001 with shrimp and crab shell powder as a carbon source. Enzyme Microb Technol, 39, 311-7.
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