臺灣博碩士論文加值系統

乳酸及其衍生物被廣泛的應用在食品、醫藥衛生、紡織、皮革、包裝材料等工業上，而乳酸的聚合物因擁有良好的生物相容性和降解性，其應用性與日俱增，因此如何提升乳酸的產量是一個重要的課題。乳酸的主要來源為微生物發酵法，常用之發酵微生物有乳酸菌及米根黴菌(Rhizopus oryzae)。利用米根黴菌發酵生產乳酸的優點為菌體之營養需求簡單、所產之L(+)-乳酸純度高、菌體有高含量之多醣體可當副產物等。因此本論文研究以米根黴發酵生產L(+)乳酸、探討發酵槽的擋板效應、不同鹼液饋料控制酸鹼值、以及不同碳源對米根黴發酵生產L(+)乳酸之影響。最佳之發酵條件為前培養以孢子濃度106 / ml進行前培養 16小時後，接種至無擋板之發酵槽以35℃、300 rpm、2 vvm、40 % CaCO3泥漿溶液控制pH在4.3進行發酵時，L(+)乳酸產量可達最高100.61 g /L，葡萄糖轉化率為83.84 %。就菌體生長形態來看，棉絮狀的L(+)乳酸產量＞顆粒狀＞團塊狀，主要是因為團塊狀菌體其營養和氧氣的質傳效率最差所致。無擋板之發酵槽其氧氣質傳係數雖然比有擋板的發酵槽低，但是其L(+)乳酸產量反而比較好，主要因為有擋板之發酵槽易使菌體在擋板和槽壁間糾結成團塊。利用碳酸鈣饋料控制pH值的L(+)乳酸產量(89.27 g /L)會比氫氧化鈉(78.52 g /L)和銨水(70.46 g /L)高。而在不同碳源下發酵，使用葡萄糖為碳源的轉化率(83.84 %)會遠高於水解後的玉米澱粉(61.53 %)和玉米粉(52.92 %)。發酵完後的菌體濃度約為3-4 g / L，在經過鹼液處理後可得到3到4成鹼不可溶物質(AIM)，其中幾丁質含量約有49-88 wt %。

Lactic acid is already extensively applied in food, pharmaceutical, cosmetic, and textile industries. Recently, its polymer, polylactic acid has been used as a biocompatibile and biodegradabile materials. Because of its wide application, the efficient production process of lactic acid is critically needed. The purpose of this research is (1) to find the optimal conditions for Rhizopus oryzae fermentation to achieve high L(+)-lactic acid productivity (2) to investigate the effect of carbon source, alkali liquid (NaOH, NH4OH ,CaCO3 solution) for pH control and the baffle in agitated fermentor on Rhizopus oryzae for the production of L(+)-lactic acid. L(+)-lactic acid of 100.61 g / L could be obtained by fermentation at 35℃, 300 rpm, 2 vvm, using 40 % CaCO3 for pH controlled at 4.3 in a agitated fermentor without baffle. In contrast, the existence of baffle in fermentor will decrease L(+)-lactic acid production. Using 40 % CaCO3 for pH control, L(+)-lactic acid about 89.27 g / L was obtained. In contrast, NaOH and NH4OH could obtain 78.52 g / L and 70.46 g / L lactic acid, respectively. Furthermore, using glucose as carbon source could achieve the highest lactic acid yield 83.84%, much higher than 52.92% of corn powder and 61.53% of corn starch. Biomass conc. of 3 - 4 g / L could be obtained at the end of fermentation. About 30 - 40% of the biomass is alkali insoluble materials (AIM) and 49 - 88% of AIM was analyzed to be chitin.

中文摘要 I
Abstract II
目錄 III
圖目錄 VI
表目錄 VIII

第一章 緒論 1

1.1 研究目的 1
1.2 研究內容簡介 2

第二章 文獻回顧 3

2.1 乳酸 3
2.1.1 基本性質介紹 3
2.1.2 應用 4
2.1.3 乳酸來源 5
2.1.3.1 化學合成法 5
2.1.3.2 微生物發酵法 6
2.1.4 乳酸的回收與純化 6
2.1.5 聚乳酸的性質及合成原理與方法 8
2.1.5.1 開環聚合法 9
2.1.5.2 縮合聚合法 9
2.1.6 聚乳酸的應用 11
2.2 微生物發酵生產乳酸 12
2.2.1 乳酸菌 12
2.2.2 米根黴菌 14
2.2.3 乳酸菌與米根黴菌之比較 15
2.3 影響乳酸生成產量的因素 16
2.3.1 溫度 16
2.3.2 溶氧值 16
2.3.3 酸鹼值 17
2.3.4 生長形態 17
2.3.5 培養基成分 18
2.4 菌種改良與篩選方法 21
2.5 乳酸分析方法 23
2.5.1 高效能液相層析儀(HPLC) 23
2.5.2 酵素電極分析法 24
2.5.3 EDTA定鈣分析法 25
2.6 氧氣質傳係數與氧氣吸收速率 26
2.6.1 化學法(氧化還源法) 27
2.6.2 動態法(Gas out – Gas in Method) 29
2.7 菌體幾丁質含量分析 30

第三章 實驗材料與方法 32

3.1 實驗藥品 32
3.2 實驗儀器及設備 34
3.3 實驗菌株 36
3.4 實驗方法 36
3.4.1 培養基成分 39
3.4.2 孢子培養方法 41
3.4.3 孢子收集方法 42
3.4.4 計算孢子濃度方法 43
3.4.5 發酵前培養程序 45
3.4.6 發酵程序 45
3.4.7 樣品處理方法 46
3.4.8 菌株篩選方法 46
3.4.9 孢子甘油低溫保存 47
3.4.10 孢子冷凍乾燥保存 48
3.4.11 玉米粉酵素水解 48
3.4.12 玉米澱粉酵素水解 49
3.4.13 菌體回收 49
3.5 電漿親水性改質保存孢子用之多孔性聚乙烯膜 50
3.6 氧氣質傳係數測定 51
3.6.1 動態法(Gas out – Gas in method) 51
3.6.2 化學法(氧化還原法) 53
3.7 分析方法 54
3.7.1 葡萄醣分析儀 54
3.7.2 高效能液相層析儀 54
3.7.2.1 步驟 54
3.7.3 還原糖測定法(DNS法) 56
3.7.4 玉米粉還原糖含量分析 57
3.7.5 菌體幾丁質含量分析 58
第四章 結果與討論 60
4.1 HPLC有機酸分析 60
4.2 孢子濃度對乳酸產量之影響 62
4.3 前培養時間對發酵之影響 64
4.4 低溫保存之孢子對米根黴發酵之影響 66
4.5 菌種篩選實驗結果 68
4.6 電漿改質多孔性聚乙烯膜並冷凍乾燥保存孢子之結果 70
4.7 氧氣質傳係數測定 73
4.8 發酵槽之擋板對米根黴發酵之影響 77
4.9 不同鹼液控制酸鹼值對發酵之影響 80
4.10 不同碳源對米根黴發酵之影響 82
4.11 菌體幾丁質含量分析 85

第五章 結論與建議 87

附錄一 89
附錄二 90
附錄三 91

參考文獻 94

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