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研究生:謝睿文
研究生(外文):Jui-Wen Hsieh
論文名稱:以微生物生產葡萄糖胺之動力學研究
論文名稱(外文):Production Kinetic of Glucosamine by Microorganism
指導教授:吳和生吳和生引用關係王孝憲
指導教授(外文):Ho-Shing WuShaw S. Wang
學位類別:碩士
校院名稱:元智大學
系所名稱:化學工程與材料科學學系
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:111
中文關鍵詞:鹽酸化葡萄糖胺微生物發酵動力學策略產率生產力
外文關鍵詞:Glucosamine hydrochloridemicrobial fermentationkinetics and strategyyieldproductivity
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葡萄糖胺(glucosamine)為一種單胺醣類物質,是構成幾丁質(chitin)和幾丁聚醣(chitosan)之單糖;為人類關節間潤滑結締組織之主要成分,具修復、補充、滋潤、再造受損的軟骨,可治療退化性關節炎。本研究主要是利用微生物醱酵生產葡萄糖胺,討論製備葡萄糖胺的可行性以及分離純化葡萄糖胺。使用四種菌株: Rhizopus oligosorus BCRC 31996; Monascus purpures BCRC 31499; Monascus pilosus BCRC31527; Aspergillus sp. BCRC31742,實驗主要分成三部份討論:(1)分析純化,分析方式為利用1-naphthyl isothiocyanate為衍生試劑,衍生反應在50°C下反應1小時,以吡啶為反應溶劑,衍生物利用逆向液相層析分離管柱分離,以230 nm 紫外光檢出器偵測生產之葡萄糖胺含量,其精密度可達1.69%、準確度可達2.73%; (2)以搖瓶培養探討培養條件,如菌種種類、培養基、酸鹼度、碳氮比等等,菌種產量最高為Aspergillus sp. BCRC31742培養於葡萄糖胺(glucose)和蛋白質(peptone)複合培養基(3430 mg/L),在培養條件下以酸鹼度影響最大;(3)以4公升醱酵槽進行培養,探討變因有酸鹼度、轉速、碳氮比,實驗中使用Aspergillus sp. BCRC31742培養於葡萄糖胺和蛋白質複合培養基中,可獲得葡萄糖胺濃度為2310 mg/L、生物量為10.1g/L、含量為229mg/g biomass、產率為92.4mg/g carbon source、生產力13.8mg/L×h;(4)純化方式使用Aspergillus sp. BCRC31742醱酵所得之菌塊經6N HCl在100oC下反應24小時,以NaOH中和至pH 7,使用活性碳除色,再由減壓濃縮機濃縮成葡萄糖胺鹽酸鹽濃縮液,經烘箱烘乾後,其純度由原來2.29 %提升至13.4 %。
Glucosamine is an amino-monosaccharide and one of the basic constituents of Chitin and Chitosan. Glucosamine is the major component of human inter-articular lubricant connective tissue. It can repair and rebuild the damaged cartilage and it also can cure the disease of osteoarthritis. The objective of this work is to study the parameters affecting the production of glucosamine hydrochloride by microbial fermentation and to obtain the method of separation and purification of glucosamine hydrochloride.This study uses four funguses Rhizopus oligosorus BCRC 31996, Monascus purpures BCRC 31499, Monascus pilosus BCRC31527 and Aspergillus sp. BCRC31742 to product glucosamine hydrochloride by using fermentation.Three parts were discussed in this study.First is the kinetic and strategy by flask culture which conditions include kinds of fungus, mediums, pH value, and carbon and nitrogen source.The experimental result shows that the glucosamine concentration had an optimum value and was 3428mg/L by using Aspergillus sp. BCRC31742 culture in GP medium, the pH controlled an important rate in culture. Second, the kinetics and strategoy by fermenter culture that the factors were pH value, incubation time and carbon and nitrogen source in the 4L fermentor fermentation.The result shown the glucosamine concentration was 2311mg/L; biomass, 10.1g/L; content, 229(mg/g biomass); yield, 92.4mg/g of carbon source; productivity, 13.8mg/L×h-1 that Aspergillus sp. BCRC31742 was incubated in GP medium. Third is the part of analysis and purification of glucosamine. The glucosamine was analyzed with 1-naphthyl isothiocyanate (NITC) as derivatizing agent. The reaction was carried out in pyridine at 50oC for 1 h. The derivative was analyzed of High Performance Liquid Chromatography. The precision of glucosamine is below to 1.69% and the accuracy is to 2.73%. Purify method is the dry cell reacting with 6N HCl at 100oC for 24 h, then neutralization with NaOH to pH 7 to obtain glucosamine hydrochloride aqueous solution. The purity of glucosamine is from 2.29% to 13.4% after the purification process containing depigmentation and condensing by Rotary Evaporator.
Chinese Abstract Ⅰ
English Abstract Ⅱ
誌謝 Ⅲ
Contents Ⅳ
List of Captions of Figures Ⅷ
List of Captions of Tables ⅩⅣ
Notations ⅩⅤ

Chapter 1 Introduction 1
1.1 Prolegomenon 1
1.2 Glucosamine 2
1.2.1 Structure, Quality and Source of Glucosamine 2
1.2.2 Physiological Effects of Glucosamine 3
1.2.3 Safety of Glucosamine 4
1.2.4 Applications of Glucosamine 5
1.3 Analysis of Glucosamine Hydrochloride 6
1.4 Mechanism of Microbial Fermentation 13
1.4.1 Microbial Metabolism- Primary Metabolism 13
1.4.2 Microbial Metabolism- Secondary Metabolism 13
1.4.3 The Relationship between Microbial Growth and Form of Production 14
1.5 Production of Glucosamine by Microbial Fermentation 15
1.5.1 Monascus 19
1.5.2 Rhizopus oligosporus 21
1.5.3 Aspergillus nigher 21
1.5.4 Ganoderma tsugae 22
1.5.5 Transformation of Escherichia Coli 24
1.6 Motivation and Purpose of this Study 26
Chapter 2 Experimental Section 27
2.1 Material 27
2.2 Apparatus 28
2.3 Source of Bacteria 29
2.4 Recovering Culture and Preservation of Bacteria 29
2.4.1 Opening a Double-Vial 29
2.4.2 Recovering Culture from Lyophilized Specimen 29
2.4.3 Fungal Pre-Culture 29
2.5 Medium for Experiment 30
2.6 Preparing a Prescription of Standard Solution for HPLC 31
2.6.1 Glucosamine Hydrochloride Standard Aqueous Solution 31
2.6.2 Derivatizing Agent of 1-Naphthyl Isothiocyanate Pyridine Solution 31
2.6.3 Internal Standard Solution 32
2.7 Analytical Apparatus 32
2.8 Conditions for Derivatization 32
2.8.1 Discussion of Derivatizing Agent Concentration 33
2.8.2 Discussion of Derivatizing Temperature and Time 33
2.8.3 Calibration Curve 33
2.8.4 Precision and Accuracy of Calibration Curve 34
2.8.5 Certification of GluN Concentration 35
2.9 Condition of Hydrochlorination 35
2.10 Yield for Glucosamine Production by Microorganism 36
2.10.1 Flack Culture 36
2.10.2 Fermenter Fermentation 36
2.10.3 Analysis of Yield for Different Generation Time 37
2.11 Analysis of Content for Commercial Glucosamine 38
2.12 Flow Draft of Experiment 39
2.13 Kinetic Model for Glucosamine Production 40
2.13.1 Microbial Growth 40
2.13.2 Spore Germination Kinetic 41
Chapter 3 Analysis and Purification of Glucosamine Hydrochloride 42
3.1 Analysis of Glucosamine Hydrochloride Standard 44
3.1.1 The Effect of Derivatizing Agent Concentrations 44
3.1.2 The Effect of Derivatization Time and Temperature 45
3.1.3 Calibration Curve of Glucosamine Hydrochloride 46
3.1.4 Precision and Accuracy of Calibration Curve 47
3.2 Hydrochlorination Condition of Glucosamine Hydrochloride in Fungus 49
3.2.1 The Effect of Hydrochlorination Time 50
3.2.2 The Effect of Hydrochlorination Cell Dry Weight 52
3.3 Analysis of Content for Commercial Glucosamine 52
Chapter 4 Kinetics and Strategy of Producing Glucosamine by Flask Culture 53
4.1 Monascus pilosus BCRC31527 54
4.2 Monascus purpures BCRC31499 58
4.3 Rhizopus oilgosporus BCRC 31996 61
4.4 Aspergillus sp. BCRC 31742 65
4.5 Generation Time of Aspergillus sp. BCRC31742 72
4.6 Production Kinetic of Aspergillus sp. BCRC 31742 73
Chapter 5 Production Kinetic of Glucosamine and Cost Evaluation of Glucosamine in a Fermenter 76
5.1 Monascus pilosus BCRC31527 Fermentation 76
5.2 Monascus purpures BCRC31499 Fermentation 76
5.3 Rhizopus oligosporus BCRC31996 Fermentation 77
5.4 Aspergillus sp. BCRC 31742 Fermentation 77
5.5 Cost Evaluation 79
Chapter 6 Conclusions and Future Outlook 81
6.1 Conclusions 81
6.2 Future Outlook 82
References 83
Appendix A: HPLC Chromatography for Commercial Glucosamine 90
Appendix B: Incubation Aspect of Fermentation Using Aspergillus sp. BCRC 31742 93
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