臺灣博碩士論文加值系統

摘要
幾丁聚醣生產可由蝦殼幾丁質經NaOH處理獲得，去乙醯度分別為54、67、78及91﹪之幾丁聚醣（標示為ADC54、ADC67、ADC78及ADC91）；由ADC91進行醋酸酐乙醯化，可得到去乙醯度52、65及75﹪幾丁聚醣（標示為PAC52、PAC65及PAC75）。測試這些幾丁聚醣的溶解性、抗菌活性與纖維素（Cellulase）對幾丁聚醣水解力。接著探討纖維素作用幾丁聚醣所得水解液，以酒精區分分離寡醣，測試所得寡醣之抗菌活性。一般在抗菌活性方面，幾丁聚醣抗菌活性隨著去乙醯度增加而增大；在相同去乙醯度下，PAC（chitosan obtained from partially acetic anhydride acetylation）之抗菌活性低於ADC（chitosan prepared from alkaline deacetylation of chitin）。在75-100 ppm濃度下ADC91能有效的完全抑制Escherichia coli ATCC 11229、Listeria monocytogenes LM-LM、Pseudomonas aeruginosa CCRC 10944、Salmonella typhi CCRC 10746、Staphylococcus aureus CCRC 12652與 Vibrio parahaemolyticus CCRC 10806生長。在溶解性方面PAC較ADC佳，PAC在2.5 % 醋酸環境下100 % 溶解，在二次水中的溶解度為2.37 ~ 3.48 %。纖維素對ADC比PAC具有較高水解能力，依序為ADC91 > ADC78 > ADC67 > PAC75 > PAC65 > PAC52 ≈ ADC54。幾丁聚醣水解產物經95﹪酒精及70﹪的酒精區分，可去除大量的鹽類及聚合度（degree of polymerization, DP）約為1∼3的寡醣，以提高DP為4∼6的幾丁寡醣相對量。經由四次的抗菌實驗結果，我們觀察到DP為1∼6的幾丁寡醣在5,000 ppm濃度下，對 Listeria monocytogenes LM-LM 能減少1.02 log CFU/mL，對Aeromonas hydrophila CCRC 13881、Escherichia coli ATCC 11229、Candida albicans、Candida magnoliae CCRC 21703及Saccharomyces cerevisiae CCRC 22223於5,000 ppm濃度下則只有些許抗菌效果。70 % 酒精處理所得之幾丁寡醣沈澱物，在5,000 ppm濃度下，對S. cerevisiae 可降低4.80 log CFU/mL。

Abstract
Shrimp chitin was treated with NaOH to obtain the chitosan products, with deacetylation degrees of 54%, 67%, 78% and 91% (designated as ADC54, ADC67, ADC78 and ADC91). ADC91 was then acetylated with acetic anhydride to obtain the chitosan products with deacetylation degrees of 52%, 65% and 75% (designated as PAC52, PAC65 and PAC75). The solubility, antimicrobial activity and the susceptibility to cellulase digestion of these chitosan products were investigated. Then, the antimicrobial activity of the hydrolysate of these chitosans by cellulase digestion was further evaluated. In general, the antimicrobial activity of the chitosan was increased with the increasing of decaetylation degree (DD). ADC (chitosan prepared from alkaline deacetylation of chitin) showed much higher antimicrobial activity than PAC (chitosan obtained from partially acetic anhydride acetylation). No survival cell was detected for Escherichia coli ATCC 11229, Listeria monocytogenes LM-LM, Pseudomonas aeruginosa CCRC 10944, Salmonella typhi CCRC 10746, Staphylococcus aureus CCRC 12652 and Vibrio parahaemolyticus CCRC 10806 at 75 ~ 100 ppm of ADC91. The solubility of PAC was much higher than ADC, with 100 % solubility in 2.5 % acetic acid and 2.37 ~ 3.48 % in double deionized water for the PAC. Higher hydrolytic activity was obtained by cellulase digestion of ADC than PAC, which was in the order of ADC91 > ADC78 > ADC67 > PAC75 > PAC65 > PAC52 ≈ ADC54. The treatments of the chitosan hydrolysate with 95 % ethanol, followed by 70 % ethanol can remove the salt and some chitooligomers with degree of polymerization (DP) 1 ~ 3, and accordingly increase the percentages of chitooligomers with DP 4 ~ 6. Except for Listeria monocytogenes LM-LM by reduction of 1.02 log CFU/mL, little antimicrobial activity against Aeromonas hydrophila CCRC 13881, Escherichia coli ATCC 11229, Candida albicans, Candida magnoliae CCRC 21703 and Saccharomyces cerevisiae CCRC 22223 for chitooligomers with DP 1 ~ 6 at 5,000 ppm. The cell was reduced by 4.80 log CFU/mL for S. cerevisiae in the presence of chitooligomers obtained from 70 % ethanol precipitate at 5,000 ppm whereas little antimicrobial activity was observed for the rest of 4 species tested.

目 錄
中文摘要………………………………………………………………………………1
英文摘要………………………………………………………………………………3
壹、前言………………………………………………………………………………5
貳、文獻整理…………………………………………………………………………7
一、幾丁質與幾丁聚醣………………………………………………………………7
1、結構……………………..……………………………………………………7
1.1、幾丁質結構……………………………………………………………7
1.2、幾丁聚醣結構…………………………………………………………7
2、影響幾丁聚醣溶解性質因子…..……………………………………………8
2.1、氫鍵……………………………………………………………………8
2.2、分子量…………………………………………………………………8
2.3、乙醯基含量與分佈……………………………………………………9
二、N-乙醯幾丁寡醣與幾丁寡醣……………………………………………………11
1、結構………….……………..…………………………………………….…11
2、N-乙醯幾丁寡醣與幾丁寡醣之製備………..…………………………….11
2.1、化學法……………………………………………………………….11
2.2、酵素法……………………………………………………………….11
2.2.1、幾丁質……………………………………………………..…12
2.2.2、幾丁聚醣…………………………………………………..…12
2.2.3、纖維素………………………..………………………………12
2.2.4、溶菌…………………………………………………………..14
2.2.5、醣…..………………………………………………………14
3、N-乙醯幾丁寡醣與幾丁寡醣之生理活性…………………………………14
三、幾丁質與幾丁聚醣之應用………………………………………………………15
1、食品方面……………………………………………………………………15
2、化工方面……………………………………………………………………15
3、醫藥方面……………………………………………………………………16
4、生物技術方面………………………………………………………………16
四、幾丁聚醣之抑菌作用……………………………………………………………16
1、對細菌之抑菌效果…………………………………………………………16
2、對酵母菌之抑菌效果………………………………………………………17
3、對黴菌之抑菌效果…………………………………………………………18
4、抗菌作用之機制……………………………………………………………19
參、材料設備與方法…………………………………………………………………22
一、研究目的與實驗設計……………………………………………………………22
二、材料………………………………………………………………………………22
(一) 原料………………………………………………………………………22
(二) 化學藥品…………………………………………………………………22
三、儀器設備…………………………………………………………………………23
四、方法………………………………………………………………………………23
（一）、製備不同去乙醯度之幾丁聚醣…………………………………………23
1、幾丁聚醣製備…………………………………………………………23
1.1、高去乙醯度幾丁聚醣製備……………………………………23
1.2、中度去乙醯度幾丁聚醣製備…………………………………24
1.3、乙醯化反應製備不同去乙醯度幾丁聚醣製備………………24
2、幾丁聚醣去乙醯度之測定……………………………………………24
2.1、滴定法…………………………………………………………24
2.2、Fourier轉換紅外線光譜測儀…………………………………25
3、不同去乙醯度幾丁聚醣物化性質之測定……………………………25
3.1、溶解度測定……………………………………………………25
3.2、黏度測定(內生性黏度) ………………………………………26
4、不同去乙醯度幾丁聚醣分子量分析…………………………………26
（二）、幾丁寡醣之製備與分離純化……………………………………………27
1、利用工業級纖維素水解幾丁聚醣…………………………………27
2、還原醣分析……………………………………………………………27
2.1、Somogyi reagent製備…………………………………………27
2.2、Somogyi reagent製備…………………………………………27
2.3、Nelson reagent製備...…………………………………………28
3、幾丁寡醣分離與純化…………………………………………………28
4、幾丁寡醣組成分析……………………………………………………29
（三）、抗菌活性分析……………………………………………………………30
1、測試菌株及其培養基…………………………………………………30
1.1、來源……………………………………………………………30
1.2、培養基…………………………………………………………31
2、菌株活化………………………………………………………………31
3、幾丁聚醣及幾丁寡醣對測試菌株生長影響之測試…………………31
3.1、細菌……………………………………………………………31
3.2、酵母……………………………………………………………32
肆、結果與討論………………………………………………………………………33
1、不同去乙醯度幾丁聚醣的製備及其物化特性………………………………33
2、不同去乙醯度幾丁聚醣對測試菌株生長之影響……………………………35
3、利用工業級纖維素生產幾丁寡醣…………………………………………37
4、ADC78 與PAC75酵素水解產物比較………………………………………38
5、幾丁寡醣物質對測試菌株生長之影響………………………………………39
伍、結論………………………………………………………………………………41
陸、參考文獻…………………………………………………………………………42
表目錄
表一、較常見之幾丁質類物質分解酵素……………………………………………13
表二、膠體滴定及FT-IR測定幾丁聚醣之去乙醯度………….……………………51
表三、幾丁聚醣於2.5 % 醋酸與水中的溶解度…..…………..……………………51
表四、在2.5﹪醋酸溶液中不同去乙醯度幾丁聚醣測得之內生性黏度[η]……...52
表五、以高壓液相分子篩層析法測得之不同去乙醯度幾丁聚醣平均分子量……52
表六、幾丁聚醣水解產物經不同濃度酒精處理醣類組成變化……………………53
表七、ADC91、ADC78及PAC75水解液之平均分子量……….…………………54
表八、幾丁聚醣經酵素水解之產物醣類組成變化…………………………………54
圖目錄
圖一、傳統去乙醯及醋酐乙醯化之幾丁聚醣乙醯基分佈狀態示意圖.…………55
圖二、傳統去乙醯（ADC）與醋酐乙醯化法（PAC）幾丁聚醣經酵素水解（10U/ml Cellulase）還原醣量變化情形（A）還原醣標準曲線；（B）ADC78與PAC75之比較；（C）ADC67與PAC65之比較；（D）ADC54與PAC52之比較。……56
圖三、不同去乙醯度幾丁聚醣對Bacillus cereus CCRC 10250生長之影響（300 ppm）……………………………………………………………………………57
圖四、不同去乙醯度幾丁聚醣對Listeria monocytogenes LM-LM生長之影響（75 ppm）…………………………………………………………..………………58
圖五、不同去乙醯度幾丁聚醣對Staphylococcus aureus CCRC 12652生長之影響（75 ppm）……………………………………………………….……………59
圖六、不同去乙醯度幾丁聚醣對Aeromonas hydrophila CCRC 13881生長之影響（200 ppm）……………………………………………………………………60
圖七、不同去乙醯度幾丁聚醣對Escherichia coli ATCC 11229生長之影響（75 ppm）……………………………………………………………………………61
圖八、不同去乙醯度幾丁聚醣對Pseudomonas aeruginosa CCRC 10944生長之影響（100 ppm）…………………………………………………………………62
圖九、不同去乙醯度幾丁聚醣對Salmonella typhi CCRC 10746生長之影響（100 ppm）……………………………………………………………………………63
圖十、不同去乙醯度幾丁聚醣對Shigella dysenteriae CCRC 13983生長之影響（200 ppm）……………………………………………………………………………64
圖十一、不同去乙醯度幾丁聚醣對Vibrio cholerae CCRC 13860生長之影響（500 ppm）……………………………………………………………………………65
圖十二、不同去乙醯度幾丁聚醣對Vibrio parahaemolyticus CCRC 10806生長之影響（100 ppm）…………………………………………………………………66
圖十三、不同去乙醯度幾丁聚醣對Candida albicans 生長之影響（300 ppm）……………………………………………………………………………67
圖十四、不同去乙醯度幾丁聚醣對Candida magnoliae CCRC 21703生長之影響（300 ppm）……………………………………………………………………68
圖十五、不同去乙醯度幾丁聚醣對Saccharomyces cerevisiae CCRC 22223生長之影響（300 ppm）………………………………………………………………69
圖十六、不同濃度之ADC91、ADC78及PAC65酵素水解產物對Escherichia coli ATCC 11229生長之影響………………………………………………...……70
圖十七、ADC91、ADC78及PAC75水解液之高效液相分子篩層析圖譜………..71
圖十八、不同濃度之小分子幾丁寡醣對Listeria monocytogenes LM-LM, Aeromonas hydrophila CCRC 13881 與 Escherichia coli ATCC 11229生長之影響……………………………………………………….……………………72
圖十九、不同濃度之大分子幾丁寡醣對Listeria monocytogenes LM-LM, Aeromonas hydrophila CCRC 13881 與 Escherichia coli ATCC 11229生長之影響……………………………………………………………………….……73
圖二十、不同濃度之小分子幾丁寡醣對Candida albicans, Candida magnoliae CCRC 21703 及 Saccharomyces cerevisiae CCRC 22223生長之影響…...74
圖二十一、不同濃度之大分子幾丁寡醣對Candida albicans, Candida magnoliae CCRC 21703 及 Saccharomyces cerevisiae CCRC 22223生長之影響…...75
附圖目錄
附圖一、醋酸酐量對幾丁聚醣去乙醯度之影響……………………………………76
附圖二、不同去乙醯度幾丁聚醣高效液相分子篩層析圖譜………………………77
附圖三、高壓液相層析（HPLC）圖：（A）幾丁寡醣標準品（DP：1-6）；（B）ADC91酵素水解液凍乾粉末…………………………..……….…….………78
附圖四、高壓液相層析（HPLC）圖：（A）經95﹪酒精處理寡醣凍乾粉末上澄液；（B）95﹪-80﹪酒精處理寡醣上澄液……………………………………79
附圖五、高壓液相層析（HPLC）圖：（A）95﹪-70﹪酒精處理寡醣上呈液；（B）70﹪酒精處理寡醣沈澱物………………………………………….…………80
附圖六、高壓液相層析（HPLC）圖：（A）幾丁寡醣標準品（DP：1-6）；（B）經95﹪-70﹪酒精處理寡醣凍乾粉末（5,000 ppm）…………………….……81
附圖七、高壓液相層析（HPLC）圖：（A）幾丁寡醣標準品（DP：1-6）；（B）ADC91酵素水解液...……………………………………………….…………82
附圖八、高壓液相層析（HPLC）圖：（A）ADC78酵素水解液；（B）PAC75酵素水解液.………………………………………………………………………83
附表目錄
附表一、不同去乙醯度幾丁聚醣對測試菌株生長之影響…………………………84
附表二、幾丁寡醣對測試菌株生長之影響…………………………………………85

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