本研究篩選自淡水紅樹林生物界面活性劑生產菌TKU029，經由16SrDNA序列比對及API試驗鑑定為Paenibacillus macerans 。
     P. macerans TKU029 生產生物界面活性劑之較適培養條件為，將含有2%烏賊軟骨粉、0.1% K2HPO4 及0.05% MgSO4．7H2O之100mL液態培養基(pH 7.21)充填於250 mL之錐形瓶，經滅菌20分鐘後進行接菌，並於30℃搖瓶(150rpm)培養3天。醱酵所得離心上清液，經鹼沈澱、甲醇萃取等步驟，進行生物界面活性劑之純化，其產量為1.76 g/L。
     P. macerans TKU029所生產生物界面活性劑能將水的表面張力從72降至36.34 mN/m，其乳化活性為52～56%且具熱穩定性。TKU029生物界面活性劑之pH安定性為4～10、鹽度安定性為1%～5%。微生物抑制測試顯示，TKU029生物界面活性劑對Aspergillus fumigatus、Fusarium oxysporum、E. coli、Bacillus subtilis TKU007、Staphylococcus aureus和Pseudomonas aeruginosa K187皆有抑制效果，且可應用在草莓之保存。

The biosurfactant producing strain, TKU029, was isolated from Taiwanese soil and identified as Paenibacillus macerans by 16S rDNA gene sequencing and API (Analyitcal Profile Index) test.
     The optimized culture condition for biosurfactant production was composed of 2% squid pen powder (SPP), 0.1% K2HPO4, 0.05% MgSO4．7H2O (pH 7.21), with autoclave treatment for 20 min, afterward, P. macerans TKU029 was incubated in 100mL of above liquid medium in a flask (250 mL) and kept shaking at 30℃ for three days. The biosurfactant was extracted from the culture supernament by alkaline precipitation, extraction of methanol. The yields of biosurfactant was 1.76 g/L.
     P. macerans TKU029 biosurfactant could reduce surface tension of water from 72 to 36.34 mN/m. The emulsification index was 52～56%. And the biosurfactant had good thermal staility and was resistant to higher temperatures. The pH stability was 4～10, and the salinity stability was 

1%～5%. The P. macerans TKU029 biosurfactant showed antimicrobial activity against A. fumigatus、F. oxysporum、E. coli、B. subtilis TKU007、S. aureus and P. aeruginosa K187. Besides, P. macerans TKU029 biosurfactant could preserve strawberry.

目錄
頁次
封面內頁
授權書
簽名頁
中文摘要	I
英文摘要	II
誌謝	IV
目錄	V
圖目錄	IX
表目錄	XII

第一章	 緒論	1
第二章	 文獻回顧	3
2.1 多黏芽孢桿菌 (Paenibacillus) 之簡介	3
2.2 界面活性劑	3
2.3 界面活性劑之種類	4
2.3.1 陰離子界面活性劑 (Anionic surfactants)	4
2.3.2 陽離子界面活性劑 (Cationic surfactants)	5
2.3.3 非離子界面活性劑 (Nonionic surfactants)	5
2.3.4 兩性離子界面活性劑 (Amphoteric surfactants)	5
2.4 生物界面活性劑	7
2.4.1 生物界面活性劑之種類	7
2.4.2 生物界面活性劑之性質	9
2.4.3 生物界面活性劑之應用	11
第三章	材料與方法	13
3.1 實驗菌株	13
3.2 實驗材料	13
3.3 實驗儀器	14
3.4生物界面活性劑生產菌之篩選	15
3.5表面張力測量	15
3.6生物界面活性劑較適生產條件探討	15
3.6.1碳/氮源濃度	15
3.6.2培養體積	16
3.6.3 培養溫度	16
3.6.4培養基pH值	16
3.6.5 培養時間	17
3.7生物界面活性劑之分離純化	17
3.8生物界面活性劑之特性分析	17
3.8.1熱穩定性	17
3.8.2 鹽安定性	18
3.8.3 pH安定性	18
3.8.4 臨界微胞濃度	18
3.8.5 乳化指數	19
3.9 微生物生長抑制試驗	20
3.9.1 黴菌抑制試驗	20
3.9.2 細菌抑制試驗	20
3.10 生物界面活性劑對草苺之保存	21
第四章  結果與討論	22
4.1 生物界面活性劑生產菌之篩選	22
4.2菌株TKU029之鑑定	22
4.3 生物界面活性劑較適生產條件探討	23
4.3.1 碳/氮源濃度探討	23
4.3.2 培養液體積探討	23
4.3.3 培養溫度探討	24
4.3.4 培養pH值探討	24
4.3.5 較適培養條件探討	25
4.4 生物界面活性劑製成研究	33
4.4.1 生物界面活性劑純化程序	34
4.4.2 生物界面活性劑結構分析	34
4.5 生物界面活性劑特性分析	40
4.5.1 熱穩定性分析	40
4.5.2 鹽安定性分析	44
4.5.3 pH值安定性分析	50
4.5.4 臨界微胞濃度分析	55
4.5.5 乳化能力分析	57
4.6 生物界面活性劑對微生物生長抑制之影響	62
4.6.1 黴菌抑制率之探討	62
4.6.2 細菌抑制率之探討	67
4.7 生物界面活性劑對草苺保存之影響	75
第五章  結論	78
參考文獻	79





圖目錄
頁次

圖 3.1 乳化指數計算之示意圖	19
圖 4.1 TKU029之API 菌種鑑定	22
圖 4.2 烏賊軟骨添加濃度對P. macerans TKU029生物界面
       活性劑生產之影響	26
圖 4.3 培養液體積對P. macerans TKU029 生物界面活性劑
       生產之影響	27
圖 4.4 培養溫度對P. macerans TKU029 生物界面活性劑生
       產影響	28
圖 4.5 培養基的pH值對P. macerans TKU029 生物界面活
       性劑生產之影響	29
圖 4.6 發酵液之表面張力及pH值變化	29
圖 4.7 P. macerans TKU029於SPP培養基生產生物界面活
      性劑之生長曲線圖	30
圖 4.8 P. macerans TKU029 生物界面活性劑分離程序	35
圖 4.9 P. macerans TKU029 所生產生物界面活性劑之純化
      流程圖	36
圖4.10 P. macerans TKU029生物界面活性劑之MALD-TOF 
      MS圖譜	37
圖 4.11 P. macerans TKU029 生物界面活性劑之質量光譜	38
圖 4.12 P. macerans TKU029 生物界面活性劑之1H NMR	39
圖 4.13 高溫高壓處理對P. macerans TKU029生物界面活
        性劑的表面張力及乳化作用之影響	41
圖 4.14 高溫高壓處理對不同界面活性劑表面張力之影響	42
圖 4.15 高溫高壓對不同界面活性劑乳化活性之影響	43
圖 4.16 鹽濃度對於P. macerans TKU029生物界面活性劑
        表面張力與乳化作用之影響	46
圖 4.17 鹽濃度對於P. macerans TKU029生物界面活性劑
        乳化作用之影響	47
圖 4.18 鹽濃度對於不同界面活性劑表面張力之影響	48
圖 4.19 鹽濃度對於不同界面活性劑乳化作用之影響	49
圖 4.20 pH對於P. macerans TKU029生物界面活性劑表面
        張力與乳化作用之影響	52
圖 4.21 pH對於不同界面活性劑表面張力之影響	53
圖 4.22 pH對於不同界面活性劑乳化活性之影響	54
圖 4.23 臨界微胞濃度值。(a) P. macerans TKU029 生物界
        面活性劑 (b) SDS	56
圖 4.24 不同界面活性劑之乳化能力 (a)不同濃度之比較 
        (b)濃度為5mg/L的乳化能力	59
圖 4.25 P. macerans TKU029生物界面活性劑之Aspergillus 
        fumigatus抑制活性	64
圖 4.26 P. macerans TKU029生物界面活性劑之Fusarium 
        oxysporum 抑制活性	65
圖 4.27 P. macerans TKU029生物界面活性劑之E. coli抑制
        活性。(a) 12小時，(b) 24小時	70
圖 4.28 P. macerans TKU029生物界面活性劑之P. aeruginosa 
        K187 抑制活性。(a) 12小時，(b) 24小時	71
圖 4.29 P. macerans TKU029生物界面活性劑之S. aureus抑
        制活性。(a) 12小時，(b) 24小時	72
圖 4.30 P. macerans TKU029生物界面活性劑之B. subtilis 
        TKU007抑制活性。(a) 12小時，(b) 24小時	73
圖 4.31 P. macerans TKU029生物界面活性劑對草苺重量損
        失率之影響	76
圖 4.32 P. macerans TKU029生物界面活性劑對草苺生長之
        影響	77
表目錄
頁次

表 2.1 界面活性劑之種類	6
表 2.2 微生物及其生產的界面活性劑	12
表 4.1 P. macerans TKU029生物界面活性劑之較適生產條
       件	30
表 4.2 微生物生產生物界面活性劑條件之比較	31
表 4.3 微生物生產之生物界面活性劑特性比較	60
表 4.4 P. macerans TKU029生物界面活性劑與其他界面活
       性劑對真菌半抑制率之比較	66
表 4.5 P. macerans TKU029生物界面活性劑與其他界面活
       性劑對細菌半抑制率之比較	74

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