跳到主要內容

臺灣博碩士論文加值系統

(44.211.26.178) 您好!臺灣時間:2024/06/24 21:18
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:方慧萍
研究生(外文):Fang Hui Ping
論文名稱:黏質沙雷氏菌株YOR1之色素分離液的研究
論文名稱(外文):The studies on pigment extracts from Serratia marcescens YOR1
指導教授:呂誌翼
指導教授(外文):Jyh-Yih Leu
口試委員:陳建盛李宗輝
口試委員(外文):Chen, C.-SLee Tzong-Huei
口試日期:2015-07-21
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2015
畢業學年度:104
語文別:中文
論文頁數:63
中文關鍵詞:黏質沙雷氏菌色素黏康酸靈菌紅素
外文關鍵詞:Serratia marcescenspigmentmuconic acidprodigiosin
相關次數:
  • 被引用被引用:0
  • 點閱點閱:480
  • 評分評分:
  • 下載下載:45
  • 收藏至我的研究室書目清單書目收藏:0
黏質沙雷氏桿菌(Serratia marcescens)屬於腸內桿菌科(Enterobacteriaceae),常見於土壤及水等環境。Serratia菌屬中某些菌株會產生色素,主要是紅色靈菌紅素(prodigiosin)及粉紅色pyrimine;此外,文獻中研究發現不產色素黏質沙雷氏桿菌亦會產生一種黃色的muconic acid (2-hydroxy-5-carboxymethyl muconic acid semialdehyde, CHMSA)。本實驗發現篩選自產色素型黏質沙雷氏桿菌YOR1,會產生一種黃色化合物,但藉由薄層層析片、高效液相層析探討證明不是muconic acid,而是一種未知化合物,此未知黃色化合物在波長365 nm 之UV光下含有多苯環、具共軛雙鍵串聯並具有螢光的特性,高效液相層析中顯示結果可能為苯甲酸,未來將繼續探討其結構。由菌株YOR1分離出的色素化合物中,紫色與黃色色素對金黃色葡萄球菌、枯草桿菌和大腸桿菌顯示無抗菌反應,粉紅色色素能有效的抑制金黃色葡萄球菌和枯草桿菌,在對單純皰疹病毒一型(Herpes Simplex virus-1, HSV-1)之抑制率中,發現紫色及黃色色素分別有20.99%及25.93%之病毒抑制率;非洲綠猴腎元母(vero E6)細胞存活率中,在紫色及黃色色素為101.89%及101.45%,粉紅化合物為49.20% ; 細胞存活率Jurkat T細胞(急性白血病T細胞)含有NF-kB Luciferase之抑制率中,紫色、粉紅色及黃色色素都無抑制率,並含有促進率; Jurkat T細胞(急性白血病T細胞)存活率中,紫色及黃色色素存活率為81.28%及76.92%,粉紅色為33.20%細胞存活率;巨噬細胞產生一氧化氮之抑制率中,紫色及黃色色素為0%及5.93%抑制率,粉紅色素有30.54%抑制率; 巨噬細胞 (Raw 264.7)細胞毒性率中,紫色色素為17.54%、粉紅色素為39.11%及黃色色素為36.01%。根據本研究所獲得之結果,未來可以更進一步解析這些萃取色素的結構及其應用。
Serratia marcescens classified to Enterobacteriaceae is commonly found in soil and water environments. Some of Serratia produce pigments, mainly including red prodigiosin and pink pyrimine. In addition, S. marcescens was found to generate a yellow muconic acid (2-hydroxy-5-carboxymethylmuconic acid semialdehyde, CHMSA). In the study yellow compound produced from Serratia marcescens YOR1 was identified to be not muconic acid, but an unknown compound by the analyses of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The unknown yellow pigment is characteristic of benzene rings, conjugated double bonds, and fluorescence under UV light at 365 nm of wavelength. The further work to the yellow compound is to reveal its structure. Of the pigments extracted from strain YOR1, purple and yellow pigments did not show the inhibition effect on the growth of Staphylococcus aureus、Bacillus subtilis and E. coli; pink pigment can effectively inhibit Staphylococcus aureus and Bacillus subtilis. The purple and yellow pigments were found with 20.99% and 25.93% of the inhibition rate against the herpes simplex virus (Herpes Simplex virus-1, HSV-1), and 101.89% and 101.45% of survival rate of African green monkey kidney yuan mother (vero E6) cell, respectively. The survival rate of vero E6 cell for the pink pigment was 49.20%. In the test of cell viability of Jurkat T cells (T cell acute leukemia), purple, pink and yellow pigments were showed with no inhibitory rate of the NF-kB luciferase and with the promotion rate of cell proliferation. When analyzed in Jurkat T cells survival test, the purple, pink, and yellow pigments demonstrated with 81.28, 33.20%, and 76.92% of the cell survival rate, respectively. In the test of nitric oxide-generation in macrophages (Raw 264.7), purple, pink, and yellow pigments showed with 0%, 30.54%, and 5.93% of inhibition rate, respectively; in the test of cytotoxicity , purple, pink and yellow pigments was 17.54%, 39.11% and 36.01% of inhibition rate, respectively. Based on the results in the study, the structure of pigments extracted from S. marcescens YOR was further needed for the application in bio-industrial sectors.
摘要 …………………………………………………………………….…1
Abstract…………………………………………………………………….2
一、 研究前言與目的………………………………………………...8
1.1 黏質沙雷氏桿菌的介紹…………………………………………...………8
1.2 黏質沙雷氏桿菌的色素…………………………………..……….………8
1.2.1 靈菌紅素……………..…………………………………….………9
1.2.2 pyrimine……………..…………………………………..….……..10
1.2.3 muconic acid…..………………………………………....….……..11
1.3研究目的……………………………………....………………..……..12
二、材料與方法…………………………………………....……………13
2.1實驗藥品與儀器設備……………………………..…………….…13
2.1.1實驗藥品………………………………………………...……….13
2.1.2實驗材料……………………………………………………….…14
2.1.3實驗設備……………………………………………………….…15
2.1.4使用之培養基………………………………………………….…16
2.2實驗菌株與細胞……………………………..………………….…17
2.3菌種培養………………………………………………………..18
2.4萃取……………………………………………………………18
2.5.管柱層析分離(sephadex LH-20為固定相)………………....19
2.6.高校液相層析儀………………………………….……………20
2.7.薄層層析法分析…………………..……………….…………......20
2.8.管柱層析分離(silica gel為固定相)…..………….………...…21
2.9.生物活性…………………………………………………………...22
2.9.1抗菌試驗..…………….…………………………….…………..…22
2.9.2紫外/可見/近紅外光譜儀……………………………………..……22
2.9.3 病毒抑制率...…………………...……………………....……..……23
2.9.3.1單純皰疹病毒第一型病毒抑制率…………………....……..……23
2.9.3.2非洲綠猴腎元母細胞存活率……………..………....……..……23
2.9.4 免疫抑制劑……………….…………………………….……….…24
2.9.4.1急性白血病T細胞含有NF-kB Luciferase之活化率.……….…24
2.9.4.2 Jurkat細胞株(急性白血病T細胞)存活率.……………………..…24
2.9.5巨噬細胞發炎反應抑制劑…………...…………………………...25
2.9.5.1巨噬細胞產生一氧化氮抑制率…...…………………………...25
2.9.5.2巨噬細胞RAW264.7細胞毒性…...…………………………...25
3.1. YOR5與BCRC11576黃色色素差異…………………………..26
三、結果……………………………………………...……………27
3.1.管柱層析分離(sephadex LH20為固定相)…………………27
3.2.高效液相層析儀…………………...……………………………28
3.3.薄層層析分析法……………………………………….………29
3.4.管柱層析分離(silica gel為固定相)……………………29
3.5.生物活性……………………………………….………30
3.5.1抗菌試驗………………………..……………………………30
3.5.2紫外/可見/近紅外光譜儀…………………...……………….30


3.5.3病毒抑制率…………………...……………….……………31
3.5.3.1單純皰疹病毒第一型病毒抑制率..……………….……………31
3.5.3.2非洲綠猴腎元母細胞存活率..……………….……………31
3.5.4免疫活性…………………...…………………………………32
3.5.4.1急性白血病T細胞含有NF-kB Luciferase之活化率………………32
3.5.4.2 Jurkat細胞株(急性白血病T細胞)存活率………………32
3.5.5巨噬細胞發炎反應抑制劑………………...……..….………33
3.5.5.1巨噬細胞產生一氧化氮抑制率……………...……..….………33
3.5.5.2巨噬細胞RAW264.7細胞毒性……………...……..….………33
3.6. YOR5 與BCRC11576黃色色素相比較……………………....…34
四、討論……………...……………….………………………….…….35
實驗之圖……………...……………….………………………….…….38
實驗之表……………...……………….………………………….…….57
五、參考文獻……………...…………….………………………….…….58












圖目錄
圖1: 管柱層析(sephadex LH20為固定相)....……………………38
圖2 :管柱層析(sephadex LH20為固定相)經薄層層析片……….39
圖3: 高效液相層析經折射率偵測器....…………………………..40
圖4 :14.8分鐘波峰之核磁共振氫譜圖....…………………………41
圖5.驗證14.8分鐘波峰是否為苯甲酸………….…………...……42
圖6: 11.4分鐘波峰之核磁共振氫譜圖....…………….……………43
圖7:粉紅化合物之核磁共振氫譜圖....…………….………………44
圖8 : 薄層層析片,展開液:乙酸乙酯/甲醇/水=10/1/0.5.……..…45
圖 9 : 高效液相層析,展開液:乙酸乙酯/甲醇/水=10/1/0.5…….46
圖10 : 黏質沙雷氏菌YOR1非為muconic acid …….…………. 47
圖11: 管柱層析(Silica gel為固定相) …..……………………..48
圖12.核磁共振光譜:樣品15 …..…………………………………..49
圖13.核磁共振光譜:樣品20 …..…………………………………..49
圖14 : 抗菌試驗…..………………….………………………..50
圖15: 穿透率光譜圖…..………………….……………………51
圖16: 單純皰疹病毒第一型病毒抑制率和細胞存活率...….….…52


圖17: 化合物在100 μg/ml濃度下,Jurkat T 細胞抑制NF-kB Luciferase和細胞存活率………...………….……………...53
圖18:化合物在50μg/ml濃度下,RAW264.7一氧化氮抑制率和細胞存活率……………………….………………….…………54
圖19. YOR1、YOR5、BCRC11576菌株萃取液相比較:…….….…55
圖20: YOR5與BCRC11576是否為muconic acid ………………..56



林芊 (2005). 由 Serratia Marcescens 生產prodigiosin之研究."
張琇雲 (2005). 黏質沙雷氏菌幾丁質分解酵素Chia基因之選殖在枯草桿菌中表現之研究.
Antony V. Samrot ., Chandana. K., Senthilkumar. P ., G.Narendra Kumar (2011). Optimization of prodigiosin production by serratia marcescens SU-10 and evaluation of its bioactivity. Journal of Biotechnology 2(5): 128-133.
A.Khanafari ., Mahnaz M Assadi ., Fatemeh A. Fakhr (2006). Review of Prodigiosin, Pigmentation in Serratia marcescens. Journal of Biological Sciences 6(1): 1-13.
Ahmad W. A., et al. (2012). Isolation of pigment producing bacteria and characterization of the extracted pigments. SpringerBriefs in Molecular Science.
p. 25-44.
Alihosseini, F., Ju Kou-San., Lango J. ., Hammock BD. .,Sun G. (2008). Antibacterial colorants: characterization of prodiginines and their applications on textile materials. Biotechnol Prog 24(3): 742-747.
Alonso, J. M. and A. Garrido-Pertierra (1982). Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli. Biochim Biophys Acta 719(1): 165-167.
Alonso, J. M. and A. Garrido-Pertierra (1986). Kinetic properties of 5-carboxymethyl-2-hydroxymuconate semialdehyde dehydrogenase from Escherichia coli. Biochimie 68(5): 731-737.
Bharmal ,M.H ., N. J., Aruna, K(2012). Study on optimization of prodigiosin production by Serratia marcescens MSK1 isolation from air. International journal of advanced biological research 2(4): 671-680.
Castro, A. J. (1967) Antimalerial activity of prodigiosin. Nature 213:903-904
Cuskey, S. M. and R. H. Olsen (1988). Catabolism of aromatic biogenic amines by Pseudomonas aeruginosa PAO1 via meta cleavage of homoprotocatechuic acid.
Journal of Biotechnology 170(1): 393-399.
Ding, M. J. and R. P. Williams (1983). Biosynthesis of prodigiosin by white strains of Serratia marcescens isolated from patients. Journal of Clinical Microbiol 17(3): 476-480.
Francisco, R ., Perez-Tomas R ., Gimenez-Bonafe P ., Soto-Cerrato V ., Gimenez-Xavier P ., Ambrosio S (2007). Mechanisms of prodigiosin cytotoxicity in human neuroblastoma cell lines. European journal of pharmacology 572(2-3): 111-119.
Grimont, P. A. and Grimont, F. (1978). The genus Serratia. Annual review of microbiology: 219-244.
Hardjito, L., Anwar Huq ., Rita, R. Colwell (2002). The Influence of Environmental Conditions on the Production of Pigment by Serratia marcescens. Biotechnology and Bioprocess Engineering 7: 100-104.
Hollender, J ., Dott, W ., Hopp, J (1994). Regulation of chloro- and methylphenol degradation in Comamonas testosteroni JH5. Applied and environmental microbiology 60(7): 2330-2338.
Ibrahim, D., T. F. N., Kassim, J.., S.H. Lim (2014). Prodigiosin an antibacterial red pigment produced by Serratia marcescens IBRL USM 84 associated with a marine sponge Xestospongia testudinaria. Journal of Applied Pharmaceutical Science 4(10): 001-006.
Katz, D. S. and R. J. Sobieski (1979). Detection of pigment precursors in white clinical strains of Serratia marcescens. Journal of clinical microbiology 9(2): 301-303.
Kutty, R. K., Devi, N. A., Veeraswamy, M ., Ramesh, S ., Rao, P. V (1977). Degradation of (+/-)-synephrine by Arthrobacter synephrinum. Oxidation of 3,4-dihydroxyphenylacetate to 2-hydroxy-5-carboxymethyl-muconate semialdehyde. The Biochemical journal 167(1): 163-170.
Lack, A (1949).Prodigiosin ; antibiotic action on coccidioides immitis in vitro. Proceedings of the Society for Experimental Biology and Medicine 72:656-658.
Lapenda Lins JC ., M. C., Xavier HS., Alves da Silva CA ., Campos-Takaki GM (2008-2014). Production and toxicological evaluation of Prodigiosin from Serratia marcescens UCPWFCC1549 on mannitol solid medium. Journal of Applied Research in Natural Products 7(2): 32-38.
Lee, J. S., Kim Y.S ., Park S., Kim J., Kang S.J ., Lee M.H ., Ryu S ., Choi J.M .,Oh T.K., Yoon J.H (2011). Exceptional production of both prodigiosin and cycloprodigiosin as major metabolic constituents by a novel marine bacterium, Zooshikella rubidus S1-1. Applied and environmental microbiology 77(14): 4967-4973.
Liebgott, P. P., Labat M., Casalot L ., Amouric A ., Lorquin J(2007). Bioconversion of tyrosol into hydroxytyrosol and 3,4-dihydroxyphenylacetic acid under hypersaline conditions by the new Halomonas sp. strain HTB24. FEMS microbiology letters 276(1): 26-33.
Priya, K. A., S. Satheesh., B. Ashokkumar ., P. Varalakshmi., G. Selvakumar ., N. Sivakumar (2013). Antifouling activity of prodigiosin from estuarine isolate of Serratia marcescens CMST 07. Microbiological research in agroecosystem management.p.11-21.
Song, M. J., Bae J., Lee D.S ., Kim C.H., Kim J.S., Kim S.W ., Hong S.I (2006). Purification and characterization of prodigiosin produced by integrated bioreactor from Serratia sp. KH-95. Journal of bioscience and bioengineering 101(2): 157-161.
Sparnins, V. L. and P. J. Chapman (1976). Catabolism of L-tyrosine by the homoprotocatechuate pathway in gram-positive bacteria. Journal of bacteriology 127(1): 362-366.
Suzuki, K ., Matsunaga,H., Itami, C., Kimura, Y (2014). Polymyxin B enhances formation of a new pigment, a peptide-ferropyrimine complex, in Serratia marcescens. Biotechnology and Biochemistry 57(10): 1763-1765.
Trias, J., Vinas, M., Guinea, J., Loren, J. G (1987). Isolation from urine of two Serratia marcescens strains excreting a diffusible yellow pigment. Journal of general microbiology 133(3): 773-777.
Trias, J., Vinas, M ., Guinea, J ., Loren, J. G (1988). Induction of Yellow Pigmentation in Serratia marcescens. Applied and environmental microbiology 54(12): 3138-3141.
Wang, F., Luo,H ., Song, G ., Liu, C., Wang, J., Xu, J ., Su,X ., Xu Yuan Ma (2013). Prodigiosin found in Serratia marcescens y2 initiates phototoxicity in the cytomembrane. Journal of biotechnology 16(4).
W. A. Black.,L .A. Hatch., P. Binnie., J. Newberry (1973)Serratia marcescens infections in general hospitals. Journal of clinical pathology 26(12): 984–985.



QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊