臺灣博碩士論文加值系統

乳酸不僅為生物可分解材料─聚乳酸的主要原料，在製藥、食品、造紙和塑膠等工業上亦有廣泛的應用；微生物發酵為乳酸主要的生產方式，而大型藻類─綠藻石蓴中富含多醣與蛋白質，具有做為乳酸發酵基質之潛力。本研究利用乳酸菌發酵綠藻石蓴水解液並探討其乳酸產量。固定 109 CFU/mL 的 Lactobacillus rhamnosus BCRC 10940 與 BCRC 14068 於聚乳酸微管束陣列膜 (Poly-L-Lactic Acid Microtube Array Membrane, PLLA-MTAM)，可分別得到 84.0 ± 6.4% 與 83.1 ± 5.0% 之包覆率，以此固定化菌體發酵含有 3% (w/v) 葡萄糖的 MRS 培養基 72 h 後之乳酸濃度分別為 15.32 ± 0.13 g/L 與 15.33 ± 0.19 g/L、乳酸轉換率分別為 0.55 ± 0.01 g/g sugar 與 0.58 ± 0.01 g/g sugar；另外也會產生非目標產物乙醇 5.25 ± 0.08 g/L 與 5.28 ± 0.02 g/L，而乙醇轉換率分別為 0.20 ± 0.00 g/g sugar 與 0.20 ± 0.00 g/g sugar，產物莫耳數比則為分別 1 : 0.70 與 1 : 0.70 (乳酸 : 乙醇)。透過酸水解及電透析法製備石蓴酸水解液，其葡萄糖含量為 9.35 ± 0.07g/L、鼠李糖含量為 39.27 ± 0.12 g/L，並能將其鹽度降至 8% (w/w) 以下。以 Lb. rhamnosus BCRC 10940 及 BCRC 14068 發酵 4% (w/v) 鼠李糖作為替代碳源之 MRS 培養基，Lb. rhamnosus BCRC 10940 雖能利用鼠李糖產生乳酸，最終乳酸濃度為 8.79 ± 0.04 g/L，但其轉換率為 0.36 ± 0.11 g/g sugar，最高乳酸生產速率達 0.28 ± 0.01 g/L∙h；而 Lb. rhamnosus BCRC 14068 則幾乎無法利用鼠李糖產生乳酸，僅消耗 0.94 ± 0.04 g/L 的鼠李糖，產生 0.46 ± 0.01 g/L 的乳酸。上述之實驗結果說明綠藻石蓴並不適合做為 Lb. rhamnosus BCRC 10940 與 BCRC 14068 之乳酸發酵基質中的碳源，而此二菌株也不適合做為發酵生產高濃度乳酸的菌株。利用 12% (w/v) 石蓴藻粉、纖維素酶 Cellulase 15 U/mL與蛋白酶 Umamizyme 5.40 U/g Ulva sp.、水解時間 48 h 的條件製備石蓴酵素水解液，其游離胺基酸含量為 103.06 ± 1.38 mM，葡萄糖與鼠李糖含量分別為 8.20 ± 0.12 與 6.33 ± 0.06 g/L。將石蓴酵素水解液之初始葡萄糖濃度調整至約 40 g/L 並添加 0.26 M 乙酸鈉作為緩衝劑，接種 Lb. acidophilus BCRC 10695 進行乳酸發酵，其最終乳酸濃度為 24.32 ± 0.40 g/L，最高乳酸生產速率為 0.58 ± 0.04 g/L∙h，乳酸轉換率為 0.86 ± 0.02 g/g sugar，最高乳酸菌數為 9.31 ± 0.03 Log CFU/mL。此結果表示 Lb. acidophilus 於石蓴酵素水解液中的生長狀況良好，且綠藻石蓴確實可以做為良好的營養補充物，替代乳酸發酵基質中的氮源提供游離胺基酸。

Lactic acid is not only the main raw material of polylactic acid, but also widely used in industry such as pharmaceutical, food, and plastics industries. Green alga Ulva sp., which contains high amount of polysaccharides and proteins, has the potential to be an alternative substrate for lactic acid fermentation. This study intends to use green alga Ulva sp. for lactic acid fermentation. The encapsulation efficiency of immobilized Lactobacillus rhamnosus BCRC 10940 and BCRC 14068 were 84.0 ± 6.4% and 83.1 ± 5.0%, respectively, by using Poly-L-Lactic Acid Microtube Array Membrane (PLLA-MTAM). The yields of lactic acid in fermentation of MRS broth using immobilized Lb. rhamnosus BCRC 10940 and BCRC 14068 were 0.55 ± 0.01 and 0.58 ± 0.01 g/g sugar, respectively. In addition, by-product ethanol was also producted, the yields of ethanol were 0.20 ± 0.00 and 0.20 ± 0.00 g/g sugar, respectively. The Ulva sp. acid hydrolysate was prepared by acid hydrolysis and electrodialysis, in which contained 9.35 ± 0.07g/L of glucose, 39.27 ± 0.12 g/L of rhamnose. The MRS broth with rhamnose as carbon source was fermented by Lb. rhamnosus BCRC 10940 and BCRC 14068, Lb. rhamnosus BCRC 10940 was able to use rhamnose and produce lactic acid, the yields of lactic acid were 0.36 ± 0.11 g/g sugar, and the highest lactic acid production rate was 0.28 ± 0.01 g/L∙h; Lb. rhamnosus BCRC 14068 was unable to produce lactic acid from rhamnose. The results above indicated that Ulva sp. is not suitable as carbon source in the lactic acid fermentation using Lb. rhamnosus BCRC 10940 and BCRC 14068. Lb. rhamnosus BCRC 10940 and BCRC 14068 are also not suitable as strains for the fermentation aims high production of lactic acid. 12% (w/v) Ulva sp. powder, 15 U Cellulase/mL, 5.40 U Umamizyme/g Ulva sp., and hydrolysis for 48 hours were used to prepare the Ulva sp. enzymatic hydrolysate, and the free amino acid content was 103.06 ± 1.38 mM. The Ulva sp. enzymatic hydrolysate was fermented by Lb. acidophilus, the highest lactic acid production rate was 0.58 ± 0.04 g/L∙h, the yield of lactic acid was 0.86 ± 0.02 g/g sugar, and the highest count of viable Lb. acidophilus cells was 9.31 ± 0.03 Log CFU/mL. These results indicated that Lb. acidophilus grew well in the Ulva sp. enzymatic hydrolysate, and Ulva sp. could be used as a good nutrient supplementation for lactic acid fermentation, to replace the nitrogen source in the media and provide amino acids.

摘要.....................................................I
Abstract................................................II
目錄...................................................III
表目錄...................................................VI
圖目錄..................................................VII
附錄目錄...............................................VIII
壹、前言..................................................1
貳、文獻整理..............................................2
一、乳酸..................................................2
1.1. 乳酸之特性...........................................2
1.2. 乳酸之應用...........................................2
1.2.1. 聚乳酸.............................................2
1.3. 乳酸之生產...........................................3
1.3.1. 化學合成法.........................................3
1.3.2. 微生物發酵法.......................................4
1.4. 乳酸之分離純化.......................................5
二、乳酸菌................................................5
2.1. 乳酸菌之定義與特性....................................5
2.2. 乳酸菌之發酵特性與分類................................6
2.3. 乳酸菌之營養需求.....................................7
2.3.1. 醣類..............................................7
2.3.2. 胺基酸與胜肽.......................................8
2.3.3. 脂肪酸.............................................9
2.3.4. 維生素.............................................9
2.3.5. 礦物質............................................10
三、發酵基質.............................................10
3.1. 醣類來源............................................10
3.1.1. 澱粉類作物........................................10
3.1.2. 纖維素植物........................................11
3.2. 營養補充物..........................................11
3.3. 海藻...............................................11
3.3.1. 石蓴之生態特性....................................12
3.3.2. 石蓴之組成份......................................12
四、糖化.................................................13
4.1. 酸水解..............................................13
4.2. 酵素水解............................................14
五、蛋白質水解...........................................14
5.1. 蛋白質萃取..........................................14
5.1.1. 化學性萃取........................................15
5.1.2. 物理性處理........................................15
5.1.3. 酵素水解多醣......................................15
5.2. 酵素水解蛋白質......................................15
六、生產乳酸之發酵方法....................................16
6.1. 批次發酵與重複批次發酵...............................16
6.2. 饋料批次發酵........................................17
6.3. 連續式發酵..........................................17
七、固定化細胞...........................................17
7.1. 吸附或黏著於固體載體表面.............................18
7.2. 包埋在多孔洞基質中...................................18
7.3. 藉由絮凝劑或交聯劑自我凝集...........................18
7.4. 圍阻在機械屏障內....................................19
7.5. 微管束陣列膜........................................19
參、實驗設計.............................................20
肆、實驗材料與方法........................................21
一、實驗材料.............................................21
1.1. 原料...............................................21
1.2. 實驗菌株............................................21
1.3. 酵素...............................................21
1.4. 化學藥品............................................21
1.5. 儀器設備............................................22
1.6. 培養基組成..........................................23
二、實驗方法............................................24
2.1. 菌株活化、保存及生長曲線.............................24
2.1.1. Lb. rhamnosus....................................24
2.1.2. Lb. acidophilus..................................24
2.2. 微管束陣列膜固定化乳酸菌.............................25
2.2.1. 微管束陣列膜之製備.................................25
2.2.2. 乳酸菌之固定化....................................25
2.3. 乳酸菌鼠李糖利用性試驗...............................26
2.4. 乳酸發酵最適糖濃度...................................26
2.5. 石蓴之一般成分分析...................................26
2.5.1. 水分.............................................26
2.5.2. 粗蛋白............................................26
2.5.3. 粗脂肪............................................27
2.5.4. 灰分.............................................27
2.6. 石蓴酸水解液製備....................................27
2.6.1. 酸水解............................................27
2.6.2. 酸水解液之電透析處理...............................28
2.7. 石蓴酵素水解液製備...................................28
2.7.1. 纖維素酶最適添加量.................................28
2.7.2. 蛋白酶最適添加量..................................28
2.7.3. 最適水解時間......................................28
2.8. 發酵石蓴水解液製備乳酸...............................29
2.9. 游離胺基酸含量分析...................................29
2.10. 醣類及乳酸定量分析..................................29
2.11. 統計分析...........................................30
伍、結果與討論...........................................31
一、PLLA-MTAM 固定化 Lb. rhamnosus 製備..................31
1.1. Lb. rhamnosus 生長曲線..............................31
1.2. PLLA-MTAM 固定化乳酸菌之包覆率測試及乳酸菌菌數對包覆率之影響....................................................31
二、PLLA-MTAM 固定化 Lb. rhamnosus 乳酸發酵 MRS 培養基......................................................32
三、石蓴酸水解液製備......................................34
3.1. 石蓴一般成分分析....................................34
3.2. 石蓴酸水解液之製備...................................34
四、Lb. rhamnosus 之鼠李糖利用性..........................34
五、Lb. acidophilus 生長曲線.............................36
六、Lb. acidophilus 乳酸發酵最適葡萄糖添加量探討...........36
七、石蓴酵素水解液之製備..................................37
7.1. 最適纖維素酶添加量...................................37
7.2. 最適蛋白酶添加量....................................38
7.3. 最適水解時間........................................38
八、Lb. acidophilus 乳酸發酵石蓴酵素水解液................39
陸、結論.................................................42
參考文獻.................................................43
圖表....................................................58
附錄....................................................79

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