靈菌紅素(prodigiosin；PG)具有諸如抗癌、抗菌以及殺蟲等生物活性。本研究針對Serratia marcescens TKU011發酵烏賊軟骨生產靈菌紅素之條件進行探討。結果發現磷酸鹽會抑制靈菌紅素的生產。此1.5％烏賊軟骨培養基經高壓滅菌後，添加50mL蒸餾水於離心所得沉澱物作為培養基，於30℃發酵三天所得靈菌紅素產量 (2.38mg/mL)提升約2.5倍。
     添加乳酸菌、棉花或木棉花於1.5%烏賊軟骨培養基，探討藉由這些添加物之吸附作用，增加靈菌紅素產量之可行性。結果顯示添加0.1%（w/v）木棉花所得靈菌紅素產量(1.30mg/mL)為未添加（0.26mg/mL）之5倍。至於與烏賊軟骨同屬幾丁類物質的蝦頭、蟬蛻以及幾丁質之效果亦有探討，其中以乳酸菌和蟬蛻粉吸附靈菌紅素的效果最好。
    靈菌紅素屬脂溶性色素，因此亦探討上述幾丁類物質對於紅色40號（R40）或黃色4號（Y4）水溶性食用色素之吸附效果。結果顯示烏賊軟骨的效果最好，對於紅色40號及黃色40號之吸附率分別為14mg/g及12.3mg/g。此外，亦探討粒徑大小、去乙醯度、去蛋白質以及色素酸鹼度等條件對於吸附效果之影響。界面活性劑SDS可將烏賊軟骨所吸附色素幾乎完全都脫附下來，最具重複使用之潛力。

Prodigiosins are known to exert antitumor, antibacterial and insecticidal activities. Using fishery-processing wastes of squid pen power（SPP）as the sole carbon and nitrogen（C/N）source, Serratia marcescens TKU011 produced prodigiosin. The relationship between prodigiosin production and phosphate concentration was studied. The medium（1.5％SPP、0.05％MgSO4‧7H2O、0.1％K2HPO4） was sterilized and centrifuged, and the deposits were collected and supplied 50ml water. Production in the medium at 30 ℃ for 3 days showed highest level of prodigiosin production（2.38mg/mL）.
    Lactic acid bacteria, cotton, and Bombax ceiba have been tested for their enhancing effects on prodigiosin production of S. marcescensTKU011 by supplementing the cells and adsorbents into SPP-containing medium. Bombax ceiba showed best results of increasing 5-folds of prodigiosin productivity compared with the non-supplement group. Among SPP and other chitinous materials of cicada casting powder（CCP）, shrimp shell power（SSP）and chitin, lactic acid bacteria and CCP showed best adsorption on prodigiosin.
     As for the adsorbing effects of water-soluble food colorants of allura red AC（R40）and tartrazne（Y4），SPP showed the best adsorption. The capacities of SPP adsorption for R40 and Y4 were determined to be 14mg/g and 12.3 mg/g respectively. Regeneration experiments showed that the water-soluble pigments can be completely regenerated with surfactant-SDS solution.

中文摘要I
英文摘要II
目錄III
圖目錄IX
表目錄XII

第一章 緒論1
第二章 文獻回顧2
2.1 Serratia marcescens TKU011基本特性2
2.2 靈菌紅素(PG )基本特性3
2.3 培養條件對靈菌紅素（PG）之影響3
2.3.1碳源3
2.3.2 無機鹽5
2.4 幾丁質、幾丁聚醣5
2.5 食用色素、食品添加物9
2.5.1 食用色素、食品添加物之簡介9
2.5.2 食用色素與病理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實驗器材及儀器14
3.4 靈菌紅素定量方法15
3.5 PG產量計算方式	15
3.6微生物生長之分析15
3.7 培養基組成條件之探討16
3.8 磷酸鹽濃度及溫度之探討18
3.8.1 濃度對色素生產之影響18
3.8.2 溫度對靈菌紅素之影響18
3.9 培養基多次滅菌之探討18
3.10 乳酸菌添加之探討18
3.10.1 乳酸菌發酵與靈菌紅素的產率18
3.10.2 TKU012菌體添加量之探討19
3.10.3 靈菌紅素上清液之製備19
3.10.4 L. paracaseiTKU012吸附PG20
3.10.5 微生物黏附溶劑法20
3.11棉花與木棉花吸附PG之探討	21
3.11.1 棉花中的色素定量21
3.11.2 棉花重量之探討21
3.11.3 棉花與木棉花之比較22
3.11.4添加磷酸鹽、木棉花及培養溫度之探討22
3.12 幾丁類物質吸附PG22
3.13 蟬蛻表面處理對PG的影響23
3.14 幾丁類物質吸附食用色素、修飾及條件探討23
3.14.1 幾丁類物質吸附食用色素23
3.14.2 粒徑與吸附率24
3.14.3 酵素（木瓜酵素、鳳梨酵素）水解SPP 24
3.14.4 熱鹼處理24
3.14.5 SPP去除蛋白質對於吸附食用色素之影響24
3.14.6 色素的酸鹼度變化對於吸附影響24
3.14.7 不同去乙醯度之幾丁聚醣對PG的影響25
3.15 傅立葉轉換紅外線光譜的鑑定25
3.16 活性碳吸附色素25
3.17 脫附實驗25
3. 17.1 脫附實驗25
3. 17.2非離子界面活性劑脫附色素26
3. 17.3離子界面活性劑脫附色素26
3. 17.4界面活性劑對於SPP吸附色素之影響26
3. 17.5SPP循環利用對吸附色素之影響27
3.18 食用色素對乳酸菌的影響27
3.18.1 乳酸菌接種於色素環境（MRSB）下27
3. 18.2 乳酸菌接種於色素環境（MRSA）下27

第四章 結果與討論28
4.1培養基組成條件之探討28
4.2磷酸鹽濃度及溫度之探討31
4.3 培養基多次滅菌之探討34
4.4 乳酸菌添加之探討36
4.4.1 TKU012菌體添加量之探討36
4.4.2 L. paracaseiTKU012吸附PG37
4.4.3 乳酸菌表面極性對PG的影響37
4.5 棉花與木棉花吸附PG之探討	43
4.5.1 棉花重量之探討43
4.5.2 棉花與木棉花之比較43
4.5.3添加磷酸鹽、木棉花及培養溫度之探討44
4.6幾丁類物質吸附PG51
4.7 蟬蛻吸附PG51
4.8幾丁類物質之食用色素吸附效果53
4.9 粒徑對吸附率之影響53
4.10 色素酸鹼度變化對於吸附之影響53
4.11 去蛋白質處理對於SPP吸附食用色素之影響54
4.12 不同去乙醯度幾丁聚醣之色素吸附效果54
4.13 傅立葉轉換紅外線光譜的鑑定61
4.13.1 烏賊軟骨吸附紅色40號61
4.13.2 烏賊軟骨吸附黃色4號61
4.14 脫附實驗65
4.15 界面活性劑與脫附效果65
4.16 多次吸附循環對於SPP的吸附能力之影響66
4.17 乳酸菌於含色素液態或固態培養基之生長71
第五章 結論70
第六章 參考文獻71



圖目錄
圖2.1 靈菌紅素結構2
圖2.2 幾丁質、幾丁聚醣之結構（A）chitin,（B）chitosan6
圖2.3 幾丁質的三種多態性結構
（A）α-chitin（B）β-chitin（C）γ-chitin7
圖2.4 食用色素的化學結構(A) R40；(B) Y411
圖3.1 實驗流程圖17
圖4.1 滅菌後的1.5％SPP培養基再不同處理方法之S.marcescensTKU011生長情形29
圖4.2 滅菌後的1.5％SPP培養基再不同處理方法之PG濃度30
圖4.3 不同濃度的K2HPO4對菌體之生長情形32
圖4.4不同濃度的K2HPO4對PG之生產情形32
圖4.5 通氣量及磷酸鹽添加濃度對PG生產之影響33
圖4.6 滅菌次數對於PG生長情形	35
圖4.7烏賊軟骨培養基經不同乳酸菌株發酵於PG產率提升之影響38
圖4.8 不同乳酸菌株發酵上清液對烏賊軟骨培養基於PG產率提升之影響39
圖4.9 L. paracaseiTKU012添加量於PG產率提升之影響40
圖4.10 不同TKU012菌體量之PG吸附結果40
圖4.11 利用L. paracaseiTKU012吸附靈菌紅素41
圖4.12 乳酸菌之死菌，吸附PG之吸附率41
圖4.13 溶劑正十六烷、氯仿、乙酸乙酯處理對於乳酸菌吸附PG之影響(A)活菌、(B)死菌42
圖4.14 棉花吸附PG前後及脫色後之比較圖	46
圖4.15 脫脂棉吸附色素之平均濃度46
圖4.16 棉花與懸浮液之色素分布47
圖4.17 木棉花或棉花吸附PG之總平均濃度48
圖4.18 添加木棉花及改變培養溫度之PG生產情形（無添加K2HPO4）49
圖4.19 添加木棉花及改變培養溫度之PG生產情形（添加K2HPO4）50
圖4.20 幾丁類物質之PG吸附率52
圖4.21 正己烷或熱鹼處理對蟬蛻吸附PG之影響52
圖4.22不同吸附劑對於食用色素R40 (A)及Y4 (B)之吸附效果56
圖4.23 SPP與活性碳對於食用色素R40 (A)及Y4 (B)之吸附效果57
圖4.24 SPP粒徑大小對於色素Y4及R40吸附之影響58
圖4.25 酸鹼值對於SPP吸附色素R40及Y4之影響59
圖4.26 SPP的FTIR圖譜62
圖4.27 SPP吸附R40的FTIR圖譜63
圖4.28 SPP吸附Y4的FTIR圖譜64
圖4.29 以不同溶液自SPP脫附色素R40及Y4之效果67
圖4.30 以不同界面活性劑自SPP及活性碳脫附色素R40（A）及Y4之效果（A）脫附不同的吸附劑68
圖4.31 界面活性劑與色素共存對於SPP吸附色素R40及Y4之影響69
圖4.32 SPP利用SDS進行多次的循環吸附（A）R40；（B）Y470
圖4.33 L. paracasei TKU012於含色素之MRS液態培養基之生長情形72
圖4.34 培養於含色素R40或Y4之MRS液體（A）或固體培養基（B）所回收L. paracaseiTKU012菌體73



表目錄
表2.1 S. marcescens在不同碳源生產PG4
表2.2 烏賊軟骨與蝦殼的成分比較7
表2.3 微生物利用烏賊軟骨生產生物活性物質8
表2.4著色劑與食品添加物使用範圍及限量暨規格標準9
表4.1 六種幾丁類物質對於三種色素之吸附率58
表4.2 三種去蛋白法處理所得SPP對於R40與Y4吸附效果之比較60
表4.3 三種幾丁質相關吸附劑對於色素R40及Y4之吸附率60

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