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研究生:詹淑玲
研究生(外文):Shu-Lin Chan
論文名稱:低分子幾丁聚醣與幾丁寡醣對大鼠腸道菌群及生理之影響
論文名稱(外文):Effects of low molecular weight chitosan and chitooligosaccharides on the intestinal microflora and physiology of rats
指導教授:蔡國珍
指導教授(外文):Gua-Jane Tsai
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:84
中文關鍵詞:幾丁寡醣腸道菌群纖維素幾丁聚醣益生菌
外文關鍵詞:chitooligosaccharidesintestinal microfloracellulasechitosanprobiotic
相關次數:
  • 被引用被引用:6
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本研究首先利用纖維素水解不同去乙醯度之蝦殼幾丁聚醣;並藉由不同水解程度之控制,比較其水解產物之抗菌活性,以期得到水溶性且抗菌活性佳之水解產物。進一步分析此水解產物之組成,並分離此水解產物,再以體外試驗探討其對腸內菌群之影響,包含病原菌Clostridium perfringens及腸道益生菌Lactobacillus、Bifidobacterium,最後進行餵食試驗,探討其對腸內細菌以及生理之影響。由還原醣分析顯示,纖維素對幾丁聚醣的水解能力,隨著去乙醯度的增加而增加,水解液的抗菌活性以90%去乙醯度幾丁聚醣(DD90)水解18小時最強。進一步以HPLC分析水解產物醣類組成,其分子量隨著水解時間增加而逐漸降低,18小時產物分子量約為12000 Da,72小時產物分子量約為4200 Da,在寡醣組成上,以18小時水解產物中聚合度為6或6以上之比率最高,為37.21%。以甲醇區分水解液,得水溶性低分子量幾丁聚醣與幾丁寡醣,進行體外試驗,得知病原菌Clostridium pergringens對低分子量幾丁聚醣具高敏感性,在濃度25 ppm下可將此菌完全抑制,幾丁聚醣水解液濃度在50 ppm下可將此菌完全抑制,而幾丁寡醣在高濃度1000 ppm下仍不具有抗菌活性。益生菌乳桿菌屬對幾丁聚醣水解液、低分子量幾丁聚醣、幾丁寡醣的抗性則隨菌種不同而異,其中以L. acidophilus CCRC 14026, L. acidophilus CCRC 10695最為敏感。雙叉桿菌屬則對幾丁聚醣各個水解產物均有良好的抗性,於培養48小時其殘存菌數比皆在94%以上。將幾丁聚醣水解液、低分子量幾丁聚醣、幾丁寡醣以每天3 g/kg劑量管餵大鼠,共餵食四週,並同時以餵食果寡醣及生理食鹽水為正控制組及控制組。各組動物體重增加量、肝臟重量及肝體比均無明顯差異。各組每日平均排便量均較控制組為高,但其糞便水分含量上,除餵食果寡醣組大鼠之糞便水分含量較低外,其餘各組並無明顯差異。血漿三酸甘油酯及總膽固醇分析上,各組間均無明顯差異。在腸道分析方面,盲腸重量以攝食低分子量幾丁聚醣與果寡醣明顯高於其他三組。菌相組成上,除了低分子量幾丁聚醣外,其餘各組盲腸中產氣莢膜梭菌的菌數均明顯較控制組低。各組的總厭氧菌數均較控制組高,並能有效增生大鼠腸道之雙叉桿菌數。顯示果寡醣、幾丁聚醣水解液、幾丁寡醣均具有調節腸道菌叢之功能。腸道益生菌對幾丁聚醣水解產物生長利用性試驗,幾丁寡醣可以如同果寡醣般提供乳酸桿菌與雙叉桿菌生長所需之碳源,幾丁聚醣水解液則選擇性的為菌株所利用,而低分子量幾丁聚醣除無法為菌株利用,更顯示出其高度的抗菌活性。
Colloidal chitin and chitosan with degree of deacetylation of 50%, 65%, 75%, and 90% (DD50, DD65, DD75, and DD90) were chemically prepared from shrimp chitin. The aims of this research are first to investigate the effect of deacetylation degree of chitin/chitosan on the hydrolytic activity of cellulase, and then to evaluate the antibacterial activity of the neutralized chitosan hydrolysate. The hydrolytic activity of cellulase on chitin/chitosan increased with increasing of deacetylation degree of substrate, with DD95 being highest. The neutralized chitosan hydrolysate obtained from cellulase digestion on DD90 at pH 5.2 for 18 hr showed the highest antibacterial activity. The low-molecular-weight chitosan (LMWC) and chitooligosaccharide in the neutralized chitosan hydrolysate were further separated by methanol precipitation.
The antibacterial activity of the chitosan hydrolysate, LMWC and chitooligosaccharide against Clostridium perfringenes and probiotics, including 5 strains of Lactobacillus and 7 strains of Bifidobacterium were evaluated. LMWC showed stronger activity against C. perfringens than chitosan hydrolysate did, with the minimal leathal concentration being 25 ppm for the former and 50 ppm for the latter; whereas little antibacterial activity was observed for chitooligosaccharide at 1000 ppm. The antibacterial activity of these three products against Lactobacillus was various with the species tested. The two strains of L. acidophillus tested much more sensitive to LMWC and chitosan hydrolysate than L. casei. and L. plantarum. The species of Bifidobacterium were quite resistant to these products.
In animal study, Wistar rats were divided into five groups of 8 each given with saline for the control, or fructooligosaccharide, chitosan hydrolysate, LMWC and chitooligosaccharide at 3 g/kg body weight per day. After 4 weeks of feeding, the increases of body weight were similar for the experimental groups and control group. There were not significant for the liver weight, and the levels of plasma cholesterol and triglyceride among groups. A significant increase in the caecal count of Bifidobacterium was observed in each experimental group. Moreover the ingestion of fructooligosaccharide, chitosan hydrolysate and chitooligosaccharide significantly decreased the caecal count of C. perfringenes. With a similar function as fructooligosaccharide, chitooligosaccharide and chitosan hydrolysate can be utilized by some species of Bifidobacterium and Lactobacillus.
目錄
中文摘要----------------------------------------------------------------------------------------Ⅰ
英文摘要----------------------------------------------------------------------------------------Ⅲ
壹、 前言------------------------------------------------------------------------------------------1
貳、 文獻整理------------------------------------------------------------------------------------3
一、 幾丁質與幾丁聚醣------------------------------------------------------------------3
(一) 幾丁質------------------------------------------------------------------------3
(二) 幾丁聚醣---------------------------------------------------------------------3
(三) 幾丁質/幾丁聚醣之生理功能--------------------------------------------4
1. 抗菌作用與機制----------------------------------------------------------4
2. 降低膽固醇之功能-------------------------------------------------------5
3. 增強免疫活性-------------------------------------------------------------5
4. 腸內菌群之影響----------------------------------------------------------6
二、 N-乙醯幾丁寡醣與幾丁寡醣-----------------------------------------------------6
(一) N-乙醯幾丁寡醣與幾丁寡醣之抗菌活性-----------------------------6
(二) N-乙醯幾丁寡醣與幾丁寡醣之免疫與抗癌活性--------------------7
三、 腸內菌對生理之影響--------------------------------------------------------------7
(一) 腸道內微生物之組成-----------------------------------------------------7
(二) 腸道微生物之分佈--------------------------------------------------------8
(三) 腸道微生物之功能--------------------------------------------------------9
(四) 益生菌的定義--------------------------------------------------------------9
(五) 益生菌的特性--------------------------------------------------------------9
1. 耐酸性----------------------------------------------------------------------9
2. 耐膽鹽---------------------------------------------------------------------10
3. 吸附的特性---------------------------------------------------------------10
4. 抗菌能力------------------------------------------------------------------10
(六) 益生菌於腸道中的角色------------------------------------------------- 11
1. 排除致病菌、維持腸道菌群平衡------------------------------------11
2. 改善乳醣不耐症---------------------------------------------------------11
3. 膽鹽之去結合作用及降低膽固醇------------------------------------12
4. 腫瘤預防------------------------------------------------------------------12
5. 刺激及活化免疫系統---------------------------------------------------13
6. 增進營養價值------------------------------------------------------------13
7. 避免酸中毒---------------------------------------------------------------13
四、 寡醣對腸道生理之影響----------------------------------------------------------14
(一) 益生物質-------------------------------------------------------------------14
(二) 寡醣的結構與特性-------------------------------------------------------15
(三) 寡醣對腸道生理的影響-------------------------------------------------15
1. 促進益生菌生長--------------------------------------------------------15
2. 預防病原菌感染--------------------------------------------------------16
3. 產生短鏈脂肪酸--------------------------------------------------------17
參、 材料設備與方法-------------------------------------------------------------------------18
一、 研究目的與實驗設計-------------------------------------------------------------18
二、 材料----------------------------------------------------------------------------------18
(一) 原料-------------------------------------------------------------------------18
(二) 化學藥品-------------------------------------------------------------------18
三、 儀器設備----------------------------------------------------------------------------19
四、 方法----------------------------------------------------------------------------------20
(一) 幾丁聚醣之製備----------------------------------------------------------20
1. 不同去乙醯度幾丁聚醣之製備--------------------------------------20
2. 膠狀幾丁質之製備-----------------------------------------------------20
3. 幾丁聚醣去乙醯度的測定--------------------------------------------20
(二) 纖維素水解幾丁聚醣-------------------------------------------------21
1. 工業級纖維素活性確認--------------------------------------------21
2. 利用纖維素水解不同去乙醯度幾丁聚醣-----------------------21
3. 還原醣量的分析--------------------------------------------------------21
4. 抗菌活性之初步測試--------------------------------------------------22
5. 幾丁聚醣水解液醣類組成分析--------------------------------------22
6. 幾丁聚醣水解產物之分離--------------------------------------------23
(三) 幾丁聚醣水解產物對腸內各測試菌株生長情形之影響--------- 23
1. 測試菌株及其培養基--------------------------------------------------23
2. 幾丁聚醣水解產物之抗菌測試--------------------------------------24
3. 腸內益生菌對幾丁聚醣水解產物之利用性試驗-----------------25
(四) 動物試驗-------------------------------------------------------------------26
1. 實驗材料-----------------------------------------------------------------26
2. 動物飼養-----------------------------------------------------------------27
3. 動物犧牲-----------------------------------------------------------------28
4. 樣品收集與分析--------------------------------------------------------28
4.1 腸內菌叢------------------------------------------------------------ 28
4.2 血漿分析------------------------------------------------------------ 29
肆、 結果與討論------------------------------------------------------------------------------ 30
一、纖維素水解幾丁聚醣活性探討--------------------------------------------- 30
(一) 工業級纖維素活性確認--------------------------------------------- 30
(二) 纖維素水解不同去乙醯度幾丁聚醣------------------------------ 30
(三) 抗菌活性的初步測試--------------------------------------------------- 31
(四) 醣類組成分析-------------------------------------------------------------32
(五) 幾丁聚醣水解產物產率分析及其抗菌活性測試-------------------33
二、 幾丁聚醣水解產物對腸內各測試菌株生長情形之影響-------------------33
三、 動物餵食結果----------------------------------------------------------------------34
四、腸道菌群對幾丁聚醣水解產物之生長利用性-------------------------------37
伍、 結論----------------------------------------------------------------------------------------39
陸、 參考文獻----------------------------------------------------------------------------------75
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