臺灣博碩士論文加值系統

本研究擬探討Bacillus subtilis生產protease、amylase、chitosanase及cellulase之最適培養基組成，再利用該類酵素液對鯖魚及龍鬚菜(Gracilaria spp.)進行水解，評估所得的水解物之抗氧化活性、對類人類B細胞株 (human hybridoma HB4C5) 之增生效果以及動物細胞生理活性。結果顯示該Bacillus subtilis在複合培養基 (含1 % skim milk、1 % soya meal、0.25 % starch、0.25% K2HPO4、0.5% NaCl、0.05 % MgCl2.6H2O) 於37℃培養三天後中性蛋白酶、澱粉酶及纖維素酶活性分別達238.6、166.0及1131.1 unit/mL。利用該酵素液於50℃水解鯖魚肉漿0∼8小時，結果發現水解7小時後產物之抗氧化力達最高∼總抗氧化力(TEAC)為2.3 mM；清除��,��-diphenyl-��-picrylhydrazyl (DPPH)自由基效果為61.5%；還原力為0.75；螯合亞鐵離子能力為75.8%。但經8小時水解後產物之抗氧化能力均下降。在水解過程中，總氮含量均無顯著差異(p<0.05)，揮發性鹽基態氮 (VBN)＜25 mg/100 g。由SDS-PAGE電泳分析與非蛋白質態氮的結果顯示蛋白質降解快速且明顯，但對類人類B細胞增生效果不明顯。該酵素液於50℃水解龍鬚菜0∼8小時，其水解產物對清除DPPH自由基的效果不顯著，但隨著水解時間延長TEAC值則有增加之趨勢，未經酵素水解之龍鬚菜之TEAC值為0.28mM，經8小時水解後增加為0.89mM。水解前後還原醣、可溶性蛋白及胜肽量變化結果顯示該酵素液可有效水解龍鬚菜。

This study aimed to investigate the best medium for Bacillus subtilis to produce protease, amylase, chitosanase and cellulase. The produced enzymes were selectively collected and employed to hydrolyze mackerel and asparagus (Gracilaria spp.). The antioxidation ability, proliferation of human hybridoma HB4C5 and physiological activation ability on animal cells of the hydrolysates were analyzed. According to the results obtained in this study, the best complex medium for Bacillus subtilis to produce protease, amylase, chitosanase and cellulase was the mixture containing 1% skim milk, 1% soya meal, 0.25% starch, 0.25% K2HPO4, 0.5% NaCl and 0.05% MgCl2. After 3 days incubation in this complex medium at 37oC, the neutral protease, amylase and cellulase activities were 238.6, 166.0 and 1131.1 unit/mL, respectively. During 8 h hydrolysis using the selected enzymes solution, the antioxidant ability of hydrolysate increased gradually and got the highest values after 7 h hydrolysis at 50oC [total antioxidation ability (TEAC): 2.3 mM, α, α-diphenyl-β-picrylhydrazyl (DPPH) scavenging ability: 61.5%, reducing power: 0.75, Fe2+ chelating ability: 75.8%]. However, the antioxidation ability significantly decreased after 8 h hydrolysis (p<0.05). No significant changes in total nitrogen and volatile basic nitrogen (VBN, <25 mg/100 g) was observed during 8 h hydrolysis. Changes in SDS-PAGE and nonproteinous nitrogen suggested that obvious degradation of muscle proteins occurred during hydrolysis. However, the hydrolyzed mackerel could not proliferate the human hybridoma HB4C5 cell and physiologically activate the animal cells. During 8 h hydrolysis of Gracilaria spp. at 50oC, although no increase in DPPH scavenging ability was obtained, the TEAC increased with the duration of hydrolysis (from 0.28 to 0.89mM). According to the changes in reducing sugar, soluble protein and peptides before/after hydrolysis, the selected enzymes from Bacillus subtilis incubated in the mentioned complex medium could effectively hydrolyze the asparagus (Gracilaria spp.).

目 錄
謝辭 --------------------------------------------------------------------Ⅰ
中文摘要 --------------------------------------------------------------------Ⅱ
英文摘要 --------------------------------------------------------------------Ⅲ
目錄 --------------------------------------------------------------------Ⅴ
壹、研究背景與目的 -----------------------------------------------------1
貳、文獻整理 --------------------------------------------------------------3
一、枯草桿菌--------------------------------------------------------------3
(一)簡介 ---------------------------------------------------------------3
(二)Bacillus subtilis之酵素 ------------------------------------------3
1、蛋白酶 -------------------------------------------------------------3
2、α-澱粉酶------------------------------------------------------------5
3、植酸酶 -------------------------------------------------------------6
4、幾丁質酶 ----------------------------------------------------------6
5、其他酵素 -------------------------------------------------------------------7
二、微生物生長之營養需求 -----------------------------------------------------7
1、碳源--------------------------------------------------------------8
2、氮源-----------------------------------------------------------------8
3、礦物質--------------------------------------------------------------8
4、維生素--------------------------------------------------------------8
三、蛋白質水解產物 ---------------------------------------------------9
(一)水解方式 -------------------------------------------------------9
1、化學水解法 ---------------------------------------------------------9
2、酵素水解法 --------------------------------------------------------9
(二)蛋白質水解物之機能性 -----------------------------------------10
1、植物來源 -----------------------------------------------------------10
2、動物來源 -----------------------------------------------------------10
四、抗氧化活性 ---------------------------------------------------------11
1、抑制亞麻油酸自氧化 ---------------------------------------------11
2、金屬離子螯合能力--------------------------------------------------12
3、清除DPPH自由基--------------------------------------------------12
4、還原力------------------------------------------------------------13
5、Trolox equivalent antioxidant capacity (TEAC) ----------------13
參、材料方法-----------------------------------------------------------------15
Ⅰ. 實驗材料 -----------------------------------------------------------------15
Ⅱ. 實驗方法 -----------------------------------------------------------------17
一、 菌株之活化與保存 --------------------------------------17
二、 菌株培養基組成對菌種生長之影響 ---------------------------- 17
1、菌數之計數 -----------------------------------------------------17
2、pH值之測定 ------------------------------------------------------17
3、酵素活性測定--------------------------------------------------17
三、Bacillus subtilis 之粗酵素液製備------------------------------18
四、 酵素水解物之製備 --------------------------------------------19
五、品質檢測 ---------------------------------------------------------19
1、揮發性鹽基態氮(VBN)測定 -----------------------------------19
2、Hunter L、a、b 值之測定 -----------------------------------------19
六、水解物之成份變化----------------------------------------------19
1. 總含氮量測定---------------------------------------------------19
2. 非蛋白質態氮測定--------------------------------------------20
3. 可溶性蛋白質含量測定-----------------------------------20
4. 胜肽含量測定-------------------------------------------------20
5. 電泳分析--------------------------------------------------------20
6. 還原醣之測定----------------------------------------------------21
七、水解物之抗氧化探討 --------------------------------------21
1. 清除DPPH自由基能力之測定----------------------------------21
2. 還原力測定-----------------------------------------------------21
3. Trolox equivalent antioxidant capacity (TEAC)------------------21
4. 螯合亞鐵離子能力-------------------------------------------------22
八、動物細胞活性試驗-----------------------------------------------22
肆、結果與討論 ------------------------------------------------------------24
一、菌株培養基組成對菌種生長之影響 ----------------------24
二、培養基組成對生產之酵素種類及活性的影響----------------24
1. 幾丁聚醣酶--------------------------------------------------------24
2. 蛋白酶-------------------------------------------------------------25
3. 纖維素酶 -------------------------------------------------------------25
4. 澱粉酶 -------------------------------------------------------------26
三、鯖魚水解物品質檢測-------------------------------------------------27
1、揮發性鹽基態氮(VBN)測定 ----------------------------------------27
2、Hunter L、a、b 值之測定 -----------------------------------------27
四、水解物之成份變化--------------------------------------------------27
1. 鯖魚水解物--------------------------------------------------------27
(1) 總含氮量及非蛋白質態氮含量測定 -------------------------28
(2)可溶性蛋白及胜肽含量之變化---------------------------------28
(3)電泳分析-------------------------------------------------29
2. 龍鬚菜水解物--------------------------------------------------29
(1) 還原醣含量變化 -------------------------------------------------29
(2)可溶性蛋白及胜肽含量之變化------------------------------29
五、抗氧化活性試驗--------------------------------------------------30
1. 鯖魚水解物-----------------------------------------------------30
(1)清除DPPH自由基能力之測定-------------------------------30
(2)還原力測定-------------------------------------------------30
(3)Trolox equivalent antioxidant capacity (TEAC)---------------31
(4)螯合亞鐵離子能力-------------------------------------------31
2.龍鬚菜水解物----------------------------------------------------32
(1)清除DPPH自由基能力之測定-------------------------------32
(2)Trolox equivalent antioxidant capacity (TEAC)-------------------32
六、動物細胞活性試驗------------------------------------------32
1. 鯖魚水解物------------------------------------------------------32
2.龍鬚菜水解物-------------------------------------------------------33
伍、結論 ---------------------------------------------------------------------34
陸、參考文獻---------------------------------------------------------------35
圖表--------------------------------------------------------------------------------47

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