本研究係利用Pseudomonas aeruginosa K-187與Pseudomonas sp. TKU015發酵烏賊軟骨生產胜肽、寡醣等生物活性物質之探討。以含1％ 烏賊軟骨粉、0.1％ K2HPO4 及0.05％ MgSO4．7H2O之100 mL液態培養基，分別在37℃及30℃進行搖瓶培養。在烏賊軟骨去蛋白方面，培養4天後，K-187所得之蛋白質去除率為68％，而TKU015能去除SPP中的幾丁質保留部分蛋白質，與0.1％市售木瓜酵素及鳳梨酵素比較，鳳梨酵素對烏賊軟骨有較高的蛋白質去除率，反應4天後之去除率為70％。以K-187與TKU015之發酵上清液與0.1％ 鳳梨酵素共同反應4天後，蛋白質去除率可高達99％，而SPP幾近被完全水解，故測其上清液，分析幾丁寡醣組成，添加0.1％ 鳳梨酵素於K-187共同發酵水解SPP可得1～5醣。此外於植物生長方面，TKU015與K-187之發酵上清液富含胺基酸、胜肽等對青江菜的生長明顯有幫助。綜合上述，可應用在胜肽、胺基酸及幾丁寡醣之生產。

Pseudomonas aeruginosa K-187 and Pseudomonas sp. TKU015 can be used for fermentation of squid pen in production of bioactive materials. The culture condition for K-187 and TKU015 was composed of 1% squid pen powder (SPP) (w/v), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O, and incubated in 100 mL of  liquid medium in shaking flasks at 37℃ and 30℃. For deproteinization tests, K-187 fermentation of squid pen powder showed protein removal of 68% after 4 days, and TKU015 could removed chitin and retained protein content in spp.
The squid pen powder was treated with 0.1% commercial papain and bromelain as a comparison, the deproteinization rate in combinations of 1% Pseudomonas spp. culture supernatant and 0.1% bromelain was all 99% while the rate was 59% and 69% in 0.1% papin with Pseudomonas spp. only. The chitinase of K-187 and TKU015 combined with 0.1% bromelain can hydrolyze squid pen powder efficiently which may apply to the productions of peptides, amino acids and chitoligosaccharides by useful for biological applications. N-Acetylglucosamine (GlcNAc) to N-acetylchitopentose (GlcNAc)5 were also produced from the culture supernatant by using K-187 strain for four days fermentation with 0.1% bromelain. Besides, The fourth day culture supernatant of Pseudomonas spp. showed maximal activity of 4-6 fold growth enhancing effect on Brassica chinensis Linn weight.

封面內頁	
授權書	
簽名頁	
誌謝	
中文摘要	Ⅰ
英文摘要	Ⅱ
目錄	Ⅲ
圖目錄	Ⅴ
表目錄	Ⅵ
第一章 緒論	1
第二章 文獻回顧	3
2.1  Pseudomonas spp. 之簡介	3
2.2  水產廢棄物之微生物再利用	4
2.3  烏賊軟骨之所含主要成分	6
2.4  幾丁質與幾丁聚醣之應用	6
2.5  N-乙醯幾丁寡醣及幾丁寡醣	7
第三章  材料與方法	10
3.1  實驗菌株	10
3.2  實驗材料	10
  3.3  實驗儀器	11
  3.4  實驗方法	11
    3.4.1   Pseudomonas spp. 之培養條件	11
    3.4.2   烏賊軟骨之去蛋白	12
    3.4.3   蛋白酶活性之測定	12
    3.4.4   幾丁質酶之活性測定	13
    3.4.5   幾丁聚醣酶之活性測定	13
    3.4.6   DNS還原醣量之測定	14
    3.4.7   蛋白質定量分析	14
    3.4.8   游離胺基酸之測定	14
    3.4.9   N-乙醯幾丁寡醣製備	15
    3.4.10  N-乙醯幾丁寡醣之組成分析	15
    3.4.11  利用Pseudomonas spp.發酵液進行青江菜作物生長測試16
      3.4.11.1  青江菜之預培養	16
      3.4.11.2  促進青江菜生長試驗	16
第四章   結果與討論	17
  4.1  以Pseudomonas spp.發酵法進行烏賊軟骨之去蛋白•	17
  4.2  以不同培養基與Pseudomonas spp.之去蛋白比較	17
  4.3  添加木瓜酵素與鳳梨酵素對烏賊軟骨去蛋白之比較	23
  4.4  添加0.1%鳳梨酵素於Pseudomonas spp.發酵烏賊軟骨所幾丁寡醣組成分析	    31
4.5  Pseudomonas spp.發酵上清液對青江菜作物生長之影響	34
第五章   結論	38
參考文獻	39

圖目錄	

圖4.1  不同培養基對於(A)TKU015 (B)K-187 (C)TKU015+K-187之去蛋白影響 20
圖4.2  (A)TKU015(B)K-187發酵烏賊軟骨之去蛋白率 21
圖4.3  (A)TKU015 (B)K-187添加市售酵素後烏賊軟骨去蛋白率之較 27
圖4.4  添加不同濃度鳳梨酵素於烏賊軟骨之去蛋白影響 28
圖4.5  (A)TKU015 (B)K-187添加0.1％鳳梨酵素發酵烏賊軟骨0~4天乾重、蛋白質與還原醣含量之變化 29
圖4.6  經(A) K-187+0.1% bromelain (B) TKU015+01% bromelain 發酵0-4天所剩烏賊軟骨粉殘重 30
圖4.7  利用HPLC進行幾丁寡醣組成分析(A)標準品 (B)添加0.1%鳳梨酵素於Pseudomonas aeruginosa K-187發酵烏賊軟骨 32
圖4.8  Pseudomonas sp. TKU015與K-187發酵上清液對青江菜生長之影響 37





表目錄
	
表2.1  幾丁質與幾丁聚醣的應用 9
表4.1  Pseudomonas aeruginosa K-187 與Pseudomonas sp. TKU015之培養條件 17
表4.2  Pseuomonus sp. TKU015發酵烏賊軟骨之去蛋白率 22
表4.3  Pseudomonus sp. K-187發酵烏賊軟骨之去蛋白率 22
表4.4  本研究與其他菌株去蛋白之比較 23
表4.5  發酵烏賊軟骨所得幾丁寡醣含量之分析 33
表4.6  Pseudomonas sp. TKU015與K-187發酵液對青江菜生長之影響 35

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