利用 Agrobacterium sp. 菌株生產 Curdlan 之研究 余靜宜、吳建一
E-mail: 9511124@mail.dyu.edu.tw
摘 要
Curdlan為β-1,3鍵結的葡萄糖所構成之非水溶性多醣體，大多由Agrobacterium sp.及Alcaligenes faecalis於氮源限制條件下合 成。而本研究利用Agrobacterium sp.發酵來生產curdlan。探討五種不同碳源(葡萄糖、蔗糖、果糖、乳糖與麥芽糖)及四種不 同氮源(氯化銨、硝酸鈉、尿素與酵母萃取物)。實驗結果顯示蔗糖和尿素各自為最有效生產curdlan之碳源和氮源。另外，
也探討溶氧量對curdlan生產之影響。當曝氣量從0.5 L/min增加到2.0 L/min，其結果顯示菌體濃度及curdlan產量皆會增加
。同時，並研究調節pH值對curdlan生產之重要性。當pH值為6.6時有最高的比細胞生長率，pH值為5.5時有最高的
比curdlan生產率。由此可知，控制培養條件來增加curdlan生產率與降低curdlan生產成本是相當重要的。最後，將純化之產 物進行FT-IR(fourier transform infrared)與NMR(nuclear magnetic chromatography)分析來確定產物之結構，結果顯示已知此產 物確定為curdlan。 在單一因子實驗中，已知培養基組成中蔗糖濃度、尿素濃度及pH值為影響Agrobacterium sp.生產curdlan 最重要的因子。因此，本研究主要利用回應曲面實驗設計法進行Agrobacterium sp.生產curdlan之培養基最適化。使用中心 混層設計來探討蔗糖濃度( 56- 224 g/L )、尿素濃度( 0.05- 4.75 g/L )及pH(4-11 )之間複雜的關係。於最佳的培養基組成中，
蔗糖濃度為142.9 g/L，尿素濃度為0.52 g/L及pH為7.5磷酸緩衝溶液，其預測curdlan產量為5.22 g/L，比未最適化之培養基 高9.6﹪。
關鍵詞 : Agrobacterium sp.，curdlan，中心混層設計，回應曲面法 目錄
封面內頁 簽名頁 授權書 iii 中文摘要 iv 英文摘要 vi 誌謝 viii 目 錄 ix 圖目錄 xiv 表目錄 xix 第一章 緒論 1 第二章 文獻回顧 4 2.1多醣體（polysaccharide）之簡介 4 2.1.1微生物生產的胞外多醣體(Microbial exopolysaccharides) 5 2.1.2胞外多醣體的種類 5 2.1.3胞外多醣體之應用範圍 11 2.2 Curdlan 15 2.2.1 Curdlan之結構 15 2.2.2 Curdlan膠體之結構 18 2.2.3 Curdlan之物化特 性 19 2.2.4生產Curdlan之微生物種類 21 2.2.5影響Curdlan生產之環境因子與培養基組成 25 2.3 Curdlan之生合成機制 29 2.4 Curdlan之應用 32 2.4.1食品工業上之應用 32 2.4.2農業生物技術上之應用 35 2.4.3醫療上之應用 36 2.4.4抗腫瘤之應用 37 2.4.5環境上之應用 41 2.4.6固定化上之應用 41 2.5 回應曲面法（Response surface methodology） 50 2.5.1 回應曲面法之介紹 50 2.5.2 回應曲面法之原理 50 2.5.3 回應曲面法之應用 54 第三章 材料與方法 56 3.1實驗材料 56 3.1.1菌株 56 3.1.2藥品 56 3.1.3儀器設備 57 3.2菌株培養 59 3.2.1菌株保存與更新 59 3.2.2菌株活化 59 3.3菌體SEM觀察 59 3.4 Curdlan生產培養 60 3.5 不同培養基組成對Agrobacterium sp.生產curdlan之探討 61 3.5.1 不同碳源對菌體生長與curdlan生產之影響 61 3.5.2 蔗糖濃度 對菌體生長與curdlan生產之影響 61 3.5.3 不同氮源對菌體生長與curdlan生產之影響 61 3.5.4 尿素濃度對菌體生長與curdlan 生產之影響 62 3.5.5 不同pH值對菌體生長與curdlan生產之影響 62 3.5.6 不同溶氧量對菌體生長與curdlan生產之影響 62 3.6 不生產培養基中之分析 63 3.6.1 微生物之生長分析 63 3.6.2 pH測定 64 3.6.3 培養期間培養液之黏度分析 64 3.6.4 Curdlan之 分析 64 3.6.5 胞外多醣體分析（酚硫酸法） 65 3.7 Curdlan之純化 66 3.8 Curdlan之結構鑑定與分析 67 3.8.1 核磁共
振(Nuclear Magnetic Resonance，NMR)分析 67 3.8.2 傅立葉轉換紅外線(Fourier Transform Infrared spectrometry，FT-IR)光譜 分析 67 3.8.3 示差掃瞄熱分析(Differential Scanning Calorimetry，DSC)68 3.8.4 X光單晶繞射儀分析（X-ray diffractometer）
68 3.9 回應曲面法之實驗設計 68 3.9.1 統計分析 70 3.10 固定化顆粒製備 70 3.10.1 固定化顆粒基本性質 70 3.10.2 顆粒內菌 體濃度之測量 71 3.10.3 吸附實驗 73 3.10.4 掃瞄式電子顯微鏡(SEM)觀察顆粒菌相 73 第四章 結果與討論 75 4.1 菌株外觀型 態觀察 75 4.1.1 掃瞄式電子顯微鏡下（SEM）之型態 75 4.2 環境因子之探討 77 4.2.1 不同曝氣量對curdlan生產的影響 77 4.3 培養基成分之探討 81 4.3.1 最適碳源探討 81 4.3.2 Sucrose對curdlan生產的影響 86 4.3.3 最適氮源探討 89 4.3.4 Urea 對curdlan生產的影響 93 4.3.5 pH值對curdlan生產的影響 96 4.3.6 Urea濃度固定，不同Sucrose濃度對curdlan生產的影響 101 4.4 回應曲面法實驗設計 106 4.4.1 中心混成實驗之產curdlan情形 106 4.4.2 最適化之培養基組成 113 4.4.3 培養基產curdlan 之最適化 117 4.4.4 產curdlan之生產速率ν(g/L/h)最適化 125 4.4.5 產curdlan之td最適化 133 4.4.6 RSM設計預測結果之驗證 141 4.5 Curdlan產物分析 143 4.5.1 Curdlan產物之NMR分析 143 4.5.2 Curdlan產物之FT-IR分析 147 4.5.3 Curdlan產物 之DSC分析 149 4.5.4 Curdlan產物之X-ray分析 152 4.5.5 Curdlan產物之黏度分析 154 4.5.6 Curdlan產物之SEM分析 156 4.6 固定化實驗 158 4.6.1 吸附實驗 158 4.6.2 懸浮菌體與固定化菌體系統生產curdlan之比較 160 4.6.3 固定化顆粒的基本性質 162 第五章 結論 167 參考文獻 168 圖目錄 Figure 1-1 Schematic of this study procedure 3 Figure 2-1 Structure of curdlan. 16 Figure 2-2 The photograph of curdlan 16 Figure 2-3 Formating of curdlen high-setand low-set gel. 19 Figure 2-4 Lineage of representative strains for curdlan production23 Figure 2-5 Metabolic pathway for the synthesis of curdlan. 31 Figure 2-6 The
polysaccharides structure of antitumor activity. 39 Figure 2-7 The antitumor mechanish of polysaccharide 40 Figure 2-8 Process flowchart of response surface 53 Figure 3-1 The Standard curve of the cell concentration of the Agrobacterium sp63 Figure 3-2 Standard curve of concentration of curdlan 65 Figure 3-3 Standard curve of concentration of glucose. 66 Figure 3-4 Calibration curve of Protein and Absorbance 72 Figure 3-5 Calibration curve of Protein and Biomass. 73 Figure 4-1 Photograph of
Agrobacterium sp. by SEM. 76 Figure 4-2 Time course of biomass and curdlan by Agrobacterium sp. at various agition and aeration rate. 79 Figure 4-3 Effect of agitation and aeration on curdlan products and cell growth by Agrobacterium sp. 80 Figure 4-4 Time course of biomass and curdlan by Agrobacterium sp.84 Figure 4-5 Effect of carbon sources by Agrobacterium sp. 85 Figure 4-6 Time course of bimass and curdlan by Agrobacterium sp. at various carbon amount. 87 Figure 4-7 Effect of sucrose by
Agrobacterium sp. 88 Figure 4-8 Time course of biomass and curdlan by Agrobacterium sp. at various nitrogen source 91 Figure 4-9 Effect of nitrogen sources by Agrobacterium sp. 92 Figure 4-10 Time course of biomass and curdlan by Agrobacterium sp. at various urea concentration 94 Figure 4-11 Effect of urea concentration by Agrobacterium sp. 95 Figure 4-12 Time course of biomass and curdlan by Agrobacterium sp. at various pH 99 Figure 4-13 Effect of pH on curdlan and EPS products by Agrobacterium sp. after 50 h and 100 h. 100 Figure 4-14 Time course of biomass and curdlan by Agrobacterium sp. at various carbon concentration 103 Figure 4-15 Effect of sucrose concentration(urea:1.8 g/L) on curdlan and EPS products by Agrobacterium sp.104 Figure 4-16 Viscosity as function of speed for Agrobacterium sp.on curdlan products. 105 Figure 4-17 Experimental results of central composite design for running NO 1 to NO 20 111 Figure 4-18 Main effect analysis of curdlan production,ν(g/L h)and td 116 Figure 4-19 Contour plot and response surfaces of curdlan production (g/L)：the effect of urea and pH on curdlan production 121 Figure 4-20 Contour plot and response surfaces of curdlan production (g/L)：the effect of sucrose and pH on curdlan production 122 Figure 4-21 Contour plot and response surfaces of curdlan production (g/L)：the effect of sucrose and urea on curdlan production 123 Figure 4-22 Contour plot and response surfaces of curdlan product rate (g/L/h)：the effect of urea and pH on the product rate of curdlan. 129 Figure 4-23 Contour plot and response surfaces of curdlan Product rate (g/L/h)：the effect of sucrose and pH on the product rate of curdlan.130 Figure 4-24 Contour plot and response surfaces of curdlan Product rate (g/L/h)：the effect of sucrose and urea on the product rate of curdlan.131 Figure 4-25 Contour plot and response surfaces of the lag time of curdlan
production(day)：the effect of urea and pH on the lag time of curdlan production 137 Figure 4-26 Contour plot and response surfaces of the lag time of curdlan production(day)：the effect of sucrose and pH on the lag time of curdlan production 138 Figure 4-27 Contour plot and response surfaces of the lag time of curdlan production(day)：the effect of sucrose and urea on the lag time of curdlan production 139 Figure 4-28 Comparsion of the calculate values from RSM of the work and experimental values of curdlan production 142 Figure 4-29 13C-NMR spectrum of curdlan standard and purified curdlan 144 Figure 4-30 1H-NMR spectrum of curdlan standard and purified curdlan 145 Figure 4-31 FTIR spectrum of purified curdlan 148 Figure 4-32 Differential scaning calorimetry thermogram of curdlan standard and purified curdlan 151 Figure 4-33 X-ray diffraction spectra for curdlan 153 Figure 4-34 Shear rate versus shear stress and viscosity for purified curdlan from Agrobacterium sp. 155 Figure 4-35 Photograph of purified curdlan by SEM 157 Figure 4-36 The time course of adsoption by immobilized-free cell bead 159 Figure 4-37 Comparison of curdlan products of suspended cell and immiobilized beads during repeated-batch fermentation 161 Figure 4-38 The photograph of immobilized beads 164 Figure 4-39 Microbial population development and distribution of the surface of the gel beads in the immobilized cell system during continuous operation 165 Figure 4-40 Microbial population development and distribution of the inside section of the gel beads in the immobilized cell system during continuous operation 166 表目錄 Table 2-1 A variety of glucans having β-(1,3) linkage in their backbones. 8 Table 2-2 Established application of microbial expolysaccharides.14 Table 2-3
Comparison various Alcaligenes sp. with Agrobacterium sp. of culture condition and curdlan production. 24 Table 2-4 Application of curdlan as food additives and essential ingredient 34 Table 2-5 The application of curdlan. 44 Table 2-6 Patent holders and publication year. 46 Table 2-7 A list of the patent of curdlan application.. 48 Table 3-1 Seed culture medium 60 Table 3-2 Variables and their levels for CCRD. 69 Table 3-3 Treatment schedule for a three-factor CCRD.. 69 Table 4-1 Experimental results of central composite design. 112 Table 4-2 Definition and trial level of factor inexperiment. 114 Table 4-3 Design and experimental results of experiment. 115 Table 4-4 The least-squares fit and parameter estimaters(significance of regression coefficients) of curdlan
production. 120 Table 4-5 ANOVA for the quadratic model 124 Table 4-6 The least-squares fit and parameter
estimaters(significance of regression coefficients) of curdlan product rat 128 Table 4-7 ANOVA for the quadratic model 132 Table 4-8 The least-squares fit and parameter estimaters(significance of regression coefficients) of the lag time of curdlan production 136 Table 4-9 ANOVA for the quadratic model 140 Table 4-10 13C-NMR chemical shifts of water-insolubleβ-1.3-D-glucan extracted from Saccharomyces cerevisiae and Agrobacterium sp. with various protic acid、water-soluble β-1.3-D-glucan phosphates derived from the corresponding microparticulate β-1.3-D-glucana 146 Table 4-11 The physical characteristics of PVA immobilized-cell 163
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