4.2 以 Bottom-up 方法製備微/奈米級 BC
4.2.1 纖維水解酵素濃度對發酵期間 BC 生成之影響
4.2.1.5 醣類分析
Oikawa 等(1995)嘗試以不同的碳源來發酵生產細菌性纖維素，發 現在發酵50 小時之後，添加 D- mannitol 的實驗組比其他醣類碳源下降 幅度來的少，顯示不能即時利用糖醇作為發酵時的碳源。本實驗的發 酵基質碳源為甘露糖醇，當醋酸菌以甘露糖醇作為碳源發酵生產細菌
性纖維素時，由於甘露糖醇為果糖酮基還原之衍生物，醋酸菌須將糖 醇轉換後才可利用進行發酵(Oikawa et al., 1995)，因此進一步以 HPLC 分析發酵過程中醣類的變化，評估發酵過程中甘露糖醇消耗量及轉換 成其它醣類間的消長關係，以推導發酵液中添加纖維水解酵素，以 bottom-up 方式製備微/奈米級纖維素時調控醋酸菌的合成低聚合度纖 維素的機制。發現甘露糖醇消耗量隨著發酵天數而增加，甘露糖醇原 始含量為2.80 g/100 mL，實驗組和空白組的甘露糖醇分別於 5 天和 4 天後消耗殆盡；由甘露糖醇轉換成的醣類以果糖、葡萄糖為主，其含 量皆隨發酵天數而增加，空白組果糖生成量在發酵過程中持續增高，
至5－6 天後維持在 2.04 g/100 mL 左右，實驗組發酵液中的果糖濃度 則在2 天後不再增加，維持在 1.2 g/100 mL 附近；空白組在發酵期間 葡萄糖被偵測到的量非常低，都在 0.06 g/100 mL 以內，實驗組部份則 在2 天後維持在 0.4 g/100 mL 附近(圖 4.17)。
醋酸菌在發酵過程中，糖類代謝經由 glyoxylate cycle 進入 TCA cycle 後利用糖質新生作用(gluconeogensis)獲得菌體生長所需的能量，
在 這 些 過 程 中 會 利 用 甘 露 糖 醇 轉 換 為 果 糖 ， 提 供 代 謝 所 需(Asia, 1968)。在 bottom-up 的實驗中，實驗組發酵液中甘露糖醇消耗量較空 白組慢，但此時期實驗組菌數高於空白組(圖 4.14)，理論上甘露糖醇應 被利用較快，推論是纖維水解酵素可以將G. xylinus 分泌出的聚葡萄糖
鏈水解成寡醣及單醣，而降低對轉換甘露糖醇的依賴。實驗組甘露糖 醇轉換成果糖的殘留量比空白組少，但葡萄糖的殘留量卻比空白組 高，推測可能是由於實驗組菌數較高，使果糖迅速被G. xylinus 利用，
再轉變成葡萄糖；另ㄧ路徑為果糖迅速被G. xylinus 利用，進ㄧ步合成 纖維素，使纖維水解酵素可以作用的位置增加，水解效率高，造成葡 萄糖量較空白組高。而且這些反應應該是由G. xylinus 分泌酵素於在胞 外進行甘露糖醇-果糖-葡萄糖的轉換，才能在發酵液中偵測到果糖。
由以上結果顯示，發酵期間添加纖維水解酵素者，G. xylinus 在利 用碳源及代謝速率皆有幫助，因而使發酵過程菌數提高，有利纖維素 大量生成，且纖維水解酵素可避免纖維聚集，因此利用G. xylinus 產生 聚葡萄鏈在未聚集纏繞成大分子纖維時，中止醋酸菌發酵形成較低聚 合度的微纖維，聚集成體積較小的纖維束，可得到較多次微米及奈米 級纖維素。纖維水解酵素調控奈米/次微米纖維合成的假設模式如(圖 4.18)。
伍、結論
2. Bottom-up(1) 纖維水解酵素可有效的在醋酸菌分泌的聚葡萄鏈未聚集纏繞成
(3) 以Bottom-up方式製備微/奈米級纖維素，可避免傳統Top-down 方式必須耗費較多人力、物力、時間的缺點，唯目前產量不高，
若能進一步將調控BC合成機制的條件最適化及規模化，將具有 開發並量化生產生物性奈米纖維的潛力。
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柒、圖
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (μm) 0
1 2 3 4 5 6 7 8 9
Volume (%)
均質未球磨 年 月 日 下午
圖4.1 細菌性纖維素均質後之體積平均粒徑分佈。
Fig. 4.1 Volume mean diameter of homogenized BC.
P a rt i c l e S i z e Di st ri but i on
0.01 0.1 1 10 100 1000 10000
Particle Size (μm) 0
Particle Size (μm) 0
Particle Size (μm) 0
Fig. 4.2 Volume mean diameter of BC milled sequentially with 10mm (a), 3mm (b), and 0.3mm (c), Zirconia balls, respectively.
a
c
b
Pa rtic le Siz e Distribution
Pa rtic le Siz e Distribution