臺灣博碩士論文加值系統

本研究擬額外添加不同濃度羧甲基纖維素 (CMC) 或市售幾丁質之複合培養基，藉以利用Bacillus subtilis YJ1生產高活性蛋白酶與幾丁質酶。結果發現以1.5% 市售幾丁質組於37oC、150 rpm震盪培養3天後，中、鹼性蛋白酶分別為655.2及638.6 U/mLmin。藉由還原醣增量法與N-乙醯胺基葡萄糖生成法測定之幾丁質酶活性分別為438.04 U/mLmin及4.22 U/mLmin。利用該酵素液於50oC水解蝦殼溶液3或4小時，其可溶性蛋白質、胜肽、總游離胺基酸含量明顯比未水解高，顯示蝦殼溶液中之蛋白質被降解。另探討蝦殼水解物添加不同濃度之葡萄糖與調整pH，對乳酸菌Pediococcus pentosaceus S或益生菌Lactobacillus acidophilus BCRC 17010、Bifidobacterium adolescentis BCRC 14608或Lactobacillus casei BCRC 12272發酵之影響，以期得到具保健機能性成分之幾丁寡醣益生菌發酵飲品。實驗結果以2% 葡萄糖及中性pH值下，併用B. adolescentis BCRC 14608 發酵36小時，其菌數達10.68 log CFU/mL，顯示蝦殼水解物可作為益生菌生長之基質。由胜肽電泳觀察該發酵產物為小分子物質，其分子量約為2.5 kDa。薄層層析法結果顯示該發酵產物可得到N-乙醯幾丁五醣；進一步以HPLC分析確定具有N-乙醯幾丁二~五醣；抗氧化能力試驗中，TEAC值達4 mM、還原力為1.5 nm 及螯合亞鐵離子能力為80.42%。該項結果顯示採用蝦殼水解物併用益生菌B. adolescentis BCRC 14608發酵36小時之產品可作為機能性幾丁寡醣益生菌發酵飲品。

To produce high protease and chitinase activities, extra addition of chitins and CMC to the medium was conducted on the cultivation of Bacillus subtilis YJ-1. When this strain was incubated in a complex medium containing 0.5% (w/v) commercial chitin at 37oC, 150 rpm for 3 days, the highest neutral (655.2 U/mL•min) and alkaline (638.2 U/mL•min) proteases activities were obtained. The chitinases activities were 438.04 U/mL•min and 4.22 U/mL•min measured by neocuproine and DMBA methods, respectively. The shrimp shell solution was subjected to the hydrolysis by the above crude selective enzymes at 50oC for 3-4 hr, the soluble proteins, peptides, total free amino acid and reducing sugar of shrimp hydrolysates increased significantly (p<0.05), compared with those before hydrolysis. This phenomenon suggested the degradation of proteins contained in shrimp shell solution occurred. This study also investigated the effects of glucose content and pH on the fermerntation by Lactobacillus acidophilus BCRC 17010, Bifidobacterium adolescentis BCRC 14608 or Lactobacillus casei BCRC 12272. It was found that addition of 2% glucose at neutra pH was benefit for the growth of Bifidobacterium adolescentis BCRC 14608 (10.68 log CFU/mL after 36 hr fermentation). This result indicated that the shrimp shell hydrolysates were a good medium for Bifidobacterium adolescentis BCRC 14608. The molecular mass of the degraded products by electrophoresis weas approximately 2.5 kDa and the degraded chitooligosaccharides were pentaacetylchitopentaose and N-acetylchitobiose to pentaacetylchitopentaose measured by TLC and HPLC, respectively. The antioxidation ability including trolox equivalent antioxidant capacity, reducing power and Fe2+ ion chelating ability of the hydrolysate were 4 mM, 1.5 nm and 80.42% respectively. These results suggested that the combination use of selective enzymes produced by B. subtilis YJ1 with B. adolescentis BCRC 14608 fermentation could process the shrimp shell into a fermented functional chitooligosaccharide beverages.

目錄
頁碼
中文摘要 -----------------------------------------------------------1
英文摘要 -----------------------------------------------------------2
壹、研究背景與目的 ---------------------------------------------------3
貳、文獻整理 ---------------------------------------------------5
(一) 蝦殼廢棄物之組成與其功能性 -----------------------------------5
1. 蝦殼之成分 ---------------------------------------------------5
2. 幾丁質和幾丁聚醣之分子構造 ----------------------------------------5
3. 幾丁質酶之種類----------------------------------------------------7
4. 幾丁質酶活性測定方法 -------------------------------------------8
(二) N-乙醯幾丁寡醣及幾丁寡醣-----------------------------------------10
1. 簡介 -----------------------------------------------------------10
2. N-乙醯幾丁寡醣及幾丁寡醣之製備-------------------------------------11
(三) 枯草桿菌 (Bacillus subtilis) -----------------------------------15
1. Bacillus subtilis生產之胞外酵素-----------------------------------15
(四) 益生菌---------------------------------------------------------17
1. 益生菌之定義及特性------------------------------------------------17
2. 乳酸菌與雙歧桿菌對人類產生之益處------------------------------------18
3. 目前通過健康食品認證之乳酸菌產品之保健功效 ---------------------------20
4. 乳酸菌和雙歧桿菌與寡醣的關係---------------------------------------20
參、實驗材料與方法 ---------------------------------------------------23
Ι. 實驗材料 ---------------------------------------------------23
II. 主要儀器 ---------------------------------------------------25
III. 實驗流程圖 ----------------------------------------------------26
IV. 實驗方法 ---------------------------------------------------27
ㄧ、培養基組成對菌種生長與酵素活性之影響 ---------------------------27
(ㄧ) 菌酛的活化與保存 -------------------------------------------27
(二) Bacillus subtilis YJ1之培養條件 ---------------------------27
(三) 粗酵素液之製備與活性之測定 -----------------------------------27
1. 粗酵素液之製備----------------------------------------------------27
2. 粗酵素液pH之測定--------------------------------------------------27
3. 中、鹼性蛋白酶活性測定---------------------------------------------27
4. 纖維素酶活性測定--------------------------------------------------28
5. 幾丁質酶活性測定--------------------------------------------------28
二、蝦殼水解物之製備與化學分析----------------------------------------31
(ㄧ) 水解蝦殼廢棄物之粗酵素液之製備條件 --------------------------31
(二) 發酵基質之製備 ------------------------------------------32
(三) 益生菌發酵蝦殼溶液之條件 ----------------------------------32
1. 未水解蝦殼溶液之發酵條件 ----------------------------------32
2. 蝦殼水解物之發酵條件 ------------------------------------------32
3. 發酵溫度與接菌量之條件 ------------------------------------------33
4. 發酵期間益生菌數與pH值之變化--------------------------------------33
(四) 蝦殼水解物發酵過程之品質測定-------------------------------------34
1. 可溶性蛋白質含量測定 ------------------------------------------34
2. 胜胜含量測定-----------------------------------------------------34
3. 總游離胺基酸含量測定 ------------------------------------------34
4. 還原醣含量測定 --------------------------------------------------35
5. 發酵產物之N-乙醯幾丁寡醣之薄層層析 --------------------------35
6. 發酵產物之N-乙醯幾丁寡醣之高效液相層析 --------------------------36
7. 胜肽電泳分析 --------------------------------------------------37
(五) 水解物及發酵產物之抗氧化能力測定---------------------------------38
1. 還原力測定 --------------------------------------------------38
2. Trolox equivalent antioxidant capacity (TEAC)------------------38
3. 螯合亞鐵離子能力-------------------------------------------------39
肆、結果與討論------------------------------------------------------40
一、Bacillus subtilis YJ1生長曲線之變化 --------------------------40
二、培養基組成對Bacillus subtilis pH之影響---------------------------40
(一) 羧甲基纖維素 (CMC) 添加量--------------------------------------40
(二) Chitin添加量--------------------------------------------------40
三、培養基組成對Bacillus subtilis YJ1生產蛋白酶、纖維素酶或幾丁質酶活性之影響----------------------------------------------------------------41
(一) CMC添加量對Bacillus subtilis YJ1生產蛋白酶或纖維素酶活性之影響----41
(二) Chitin添加量對Bacillus subtilis YJ1生產蛋白酶活性之影響----------42
(三) 比較兩種幾丁質酶測定法------------------------------------------43
四、還原醣含量的變化------------------------------------------------44
五、探討水解溫度與時間對Bacillus subtilis YJ1蛋白酶與幾丁質酶之殘存活性影 響----------------------------------------------------------------44
六、粗酵素液水解市售幾丁質與幾丁聚醣所得N-乙醯幾丁寡醣之薄層層析--------45
七、探討MRSC培養基組成對益生菌生長之影響與評估乳酸菌與雙歧桿菌共生之可行性--45
(一) 益生菌培養條件對其pH值與菌數之影響--------------------------------45
(二) 總游離胺基酸與還原醣含量之變化 -----------------------------------46
(三) 評估乳酸菌與雙歧桿菌共生之可行性----------------------------------47
八、探討Bacillus subtilis YJ1蛋白酶與幾丁質酶水解蝦殼溶液之最適條件-----48
九、併用益生菌發酵蝦殼溶液之水解物-------------------------------------50
(一) 發酵期間pH值及益生菌數之變化-------------------------------------50
(二) 益生菌發酵蝦殼水解物之影響 -----------------------------------55
一、可溶性蛋白質含量變化---------------------------------------------55
二、胜肽含量變化----------------------------------------------------57
三、總游離胺基酸含量變化---------------------------------------------59
四、還原醣含量變化--------------------------------------------------61
五、發酵產物之N-乙醯幾丁寡醣之薄層層析---------------------- ----------62
六、發酵產物之N-乙醯幾丁寡醣之HPLC分析--------------------------------62
七、胜肽電泳 (Tricine-SDS-PAGE) 分析--------------------------------63
(三) 水解物及發酵產物之抗氧化能力 ----------------------------------63
1. 還原力----------------------------------------------------------63
2. Trolox equivalent antioxidant capacity (TEAC) 抗氧化當量---------64
3. 螯合亞鐵離子能力--------------------------------------------------64
伍、 結論 -----------------------------------------------------------66
陸、 參考文獻--------------------------------------------------------67
表目錄
頁碼
表一、培養基組成對益生菌發酵期間之總游離胺基酸含量影響 ----------------84
表二、培養基組成對益生菌發酵期間之還原醣含量影響 ----------------85
表三、B. subtilis YJ1於含1.5% chitin 之複合培養基生產之粗酵素液水解蝦殼溶液之pH值變化-------------------------------------------------------86
表四、B. subtilis YJ1於含1.5% chitin之複合培養基生產之粗酵素液水解蝦殼溶液之可溶性蛋白質、胜肽含量、總游離胺基酸含量變化 ------------------------87
表五、未水解蝦殼溶液併用乳酸菌或益生菌發酵48小時期間其pH值之變化--------88
表六、不同處理條件之蝦殼水解物併用乳酸菌或益生菌發酵48小 時期間其pH值之變化-------------------------------------------------------------89
表七、乳酸菌或益生菌發酵未水解之蝦殼溶液其菌數之變化-------------------90
表八、乳酸菌或益生菌發酵不同處理條件之蝦殼水解物之菌數變化--------------91
表九、未水解蝦殼溶液併用乳酸菌或益生菌發酵期間之可溶性蛋白質含量之變化----92
表十、不同處理條件之蝦殼水解物併用乳酸菌或益生菌發酵期間之可溶性蛋白質含量之變化---------------------------------------------------------------93
表十一、未水解蝦殼溶液併用乳酸菌或益生菌發酵期間之胜肽含量之變化---------94
表十二、不同處理條件之蝦殼水解物併用乳酸菌或益生菌發酵期間之胜肽含量之變化-95
表十三、未水解蝦殼溶液併用乳酸菌或益生菌發酵期間之總游離胺基酸含量之變化 ---96
表十四、不同處理條件之蝦殼水解物併用乳酸菌或益生菌發酵效期間之總游離胺基酸含量之變化 ----------------------------------------------------------97
表十五、未水解蝦殼溶液併用益生菌發酵期間之還原醣含量之變化---------------98
表十六、不同處理條件之蝦殼水解物併用益生菌發酵期間之還原醣含量之變化-----99
圖目錄
頁碼
圖一、Bacillus subtilis YJ1於TSB培養基培養期間pH與生菌數之變化-------100
圖二、以0.1% 或0.5% CMC額外添加於複合培養基對Bacillus subtilis YJ1培養5天pH值之影響--------------------------------------------------------101
圖三、含不同濃度chitin之複合培養基對Bacillus subtilis YJ1培養4天pH值之影響------------------------------------------------------------------102
圖四、Bacillus subtilis YJ1於TSB培養基培養期間其中、鹼性蛋白酶與纖維素酶活性之變化 ---------------------------------------------------------103
圖五、以0.1% 或0.5% CMC額外添加於複合培養基對Bacillus subtilis YJ1培養5天其中、鹼性蛋白酶與纖維素酶活性之變化 ---------------------------------104
圖六、含不同濃度chitin之複合培養基對Bacillus subtilis YJ1培養4天期間其中、鹼性蛋白酶活性之變化------------------------------------------------105
圖七、Bacillus subtilis YJ1於不同培養基組成培養5天期間其幾丁質酶活性之變化 (還原醣增量法)----------------------------------------------------106
圖八、Bacillus subtilis YJ1於不同培養基組成培養5天期間其幾丁質酶活性之變化 (N-乙醯胺基葡萄糖生成量測定法)--------------------------------------107
圖九、含不同濃度chitin之複合培養基對Bacillus subtilis YJ1培養4天期間其還原醣含量之變化 -------------------------------------------------108
圖十、粗酵素液分別於45、50及55 oC放置5小時其pH值之變化---------------109
圖十一、粗酵素液分別於45、50及55 oC放置5小時其中、鹼性蛋白酶殘存活性之變化110
圖十二、Bacillus subtilis YJ1之蛋白酶與幾丁質酶於不同溫度水解市售幾丁質與幾丁聚醣得到N-乙醯幾丁寡醣產物之薄層層析圖-------------------------------111
圖十三、培養基組成對益生菌發酵期間pH值與生菌數之影響 ------------------112
圖十四、培養基組成對混合益生菌發酵期間pH值與生菌數之影響-----------------113
圖十五、Bacillus subtilis YJ1之蛋白酶與幾丁質酶於50 oC水浴中震盪
(150 rpm) 水解蝦殼溶液1~5小時之薄層層析圖----------------------------114
圖十六、以H組併用益生菌發酵48小時期間之薄層層析圖----------------------115
圖十七、以F2組併用Lb. acidophilus BCRC17010發酵48小時期間之薄層層析圖-116
圖十八、以F2組併用B. adolescentis BCRC 14608發酵48小時期間之薄層層析圖117
圖十九、以F2組併用Lb. casei BCRC 12272發酵48小時期間之薄層層析圖------118
圖二十、以F2組併用B. adolescentis BCRC 14608發酵36小時之高效液相層析圖-119
圖二十一、以F2組併用益生菌發酵48小時期間之胜肽電泳圖譜 ------------------120
圖二十二、不同處理條件之未水解蝦殼溶液併用益生菌發酵期間之還原力之變化----121
圖二十三、不同處理條件之蝦殼水解物併用益生菌發酵期間之還原力之變化---- ---122
圖二十四、不同處理條件之未水解蝦殼溶液併用益生菌發酵期間之TEAC値之變化----123
圖二十五、不同處理條件之蝦殼水解物併用益生菌發酵期間之TEAC値之變化--------124
圖二十六、不同處理條件之未水解蝦殼溶液併用益生菌發酵期間之螯合亞鐵離子能力之變化----------------------------------------------------------------125
圖二十七、不同處理條件之蝦殼水解物併用益生菌發酵期間之螯合亞鐵離子能力之變化126

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