臺灣博碩士論文加值系統

動物腸道菌相為健康的指標，其易受到宿主年齡及飼糧組成等因子所影響，而菌相組成、菌數的改變及菌種的分類在微生物學的研究已有許多方法可以量化及質化。在環境微生物相的研究上常採用PCR-DGGE （polymerase chain reaction-denaturing gradient gel electrophoresis）電泳技術，以一種較簡便及巨觀的方式探討環境中主要菌群的組成及改變。因此，本研究目的為（1）建立腸道中乳酸桿菌相變化之PCR-DGGE電泳條件，（2）使用此技術監測年齡改變及益生菌對肉雞消化道中乳酸桿菌相之影響。結果顯示，雞隻腸道內容物樣品選用乳酸桿菌專一性引子對（LabtufGC及LabtufR），經巢式PCR （nested PCR）擴增並純化後，於含10％丙烯醯胺且變性梯度為35-47.5％之膠體，在60℃下以80V進行16小時之電泳後經銀染方式進行染色，可獲得條帶分離效果較佳及解析度較高之電泳圖譜。而初生雛口服三天乳酸桿菌液後犧牲並蒐集消化道內容物進行PCR-DGGE，在嗉囊及迴腸內容物DGGE圖譜上均可檢測到對照組未出現之乳酸桿菌條帶，顯示PCR-DGGE技術於探討家禽消化道乳酸桿菌相之變化有一定的可行性。進一步於雞隻飼糧中添加乳酸桿菌粉，於不同日齡犧牲後蒐集消化道內容物進行PCR-DGGE電泳，結果顯示，雞隻隨著年齡增長，迴腸內容物DGGE圖譜上條帶數目隨著日齡增加逐漸減少，乳酸桿菌種歧異度（Shannon’s index）亦隨之降低，於1日齡時為3.262，至35日齡時降低為2.237，但在盲腸反之，隨年齡增長則乳酸桿菌相歧異度增加，由1日齡之2.753至35日齡時提高為3.738。另外，由相似度指數結果可得知，不同消化道部位乳酸桿菌的發育程度均有所不同，嗉囊在1日齡以後菌相趨於穩定，而迴腸菌相則在7日齡後較無改變，至於盲腸部分菌相隨年齡增長改變，至採樣後期（35日齡）仍持續變化。另外，添加益生菌不會影響嗉囊中乳酸桿菌的種數，但可能提高菌種間的均勻度。益生菌可增加21日齡時盲腸乳酸桿菌條帶數，進而提升菌相歧異度之效果。益生菌和抗生素對促進宿主生長性狀之機制不完全相同，抗生素主要藉由減少腸道中互相競爭之細菌種類，降低腸道菌相之歧異度而達到減少能量浪費之作用，而給予益生菌處理可提高嗉囊及迴腸乳酸桿菌相之歧異度，藉由有益菌的均勻度增加，使消化道菌相趨於正相平衡，進一步達到促進生長之效果。綜上所述，PCR-DGGE技術可應用於動物腸道乳酸桿菌相之監測，未來可配合分子選殖技術進一步判定圖譜條帶所代表之菌種，並藉由不同引子對的組合，更清楚的了解腸道中複雜菌相的改變。

An understanding of the dynamics of bacterial community structure in the chicken intestine is essential for selection of diets for optimal nutrition, effective treatment of enteric pathogens, and the development of competitive exclusion products. The microbial diversity, count of bacteria and classification of bacteria in microbiology are many ways to quantitative and qualitative. It’s used in research on PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis), that a more convenient way to explore the changes in the composition of flora. In the present study, establishing the optimal condition of PCR-DGGE, and investigate the transition of the bacterial community structure and the predominant bacteria in the intestine of chicks from hatching to 5 wk of age or diet supplement antibiotic and probiotic were investigated using PCR-DGGE and phylogenetic analysis. The results demonstrated that samples of intestinal contents used lactobacillus-specific primers (LabtufGC / LabtufR), by nested PCR amplification and purification, with 10% acrylamide and denaturing gradient of 35-47.5% of the gel, in order to 80V at 60 ℃ for 16 hours after electrophoresis were stained by the silver staining method, obtain a better separation bands and higher resolution of DGGE profile. Newborn chicks after oral lactobacillus broth three days, digestive tract contents collected for PCR-DGGE, the crop and ileal contents in the DGGE patterns can be detected the bands of lactic acid bacteria that not appear in the control group. With age, the bands number of ileal contents DGGE profiles were decreased, and the biodiversity of Lactobacillus species (Shannon''s index) also decreases. However, the opposite in the cecum, the biodiversity of Lactobacillus species were increase with age. Index of similarity results that the development of lactic acid bacteria were different on disparity parts of the gastrointestinal tract. Crop microflora stabilized after 1-day-old, and ileum microflora in no change after 7 days, as cecal microflora changes due to age, to the late sampling (35 days) continued to vary. Probiotics and antibiotics to promote growth performent of the host mechanisms are not identical, that antibiotics to decrease intestinal bacterial species and reducing the biodiversity of intestinal microflora and to reduce energy waste, the probiotic can improve the biodiversity of lactobacillus on crop and ileum, and the digestive tract microflora to become positive equilibrium, and further to improve the growth performent. Application of PCR-DGGE to monitor the changes of Lactobacillus population in gastrointestinal tract of broiler is feasible. The species of Lactobacillus can be determined by PCR-DGGE combine with molecular cloning technique in the further study.

目次
第一章、 文獻探討…………………………………………………………1
壹、 消化道生態系簡介…………………………………………………1
一、 動物消化道微生物組成…………………………………………1
二、 消化道微生物與宿主之關係……………………………………5
三、 影響消化道微生物之因素………………………………………7
貳、 消化道微生物相研究方法…………………………………………9
一、 傳統培養法………………………………………………………11
二、 分子遺傳法………………………………………………………12
參、 變性梯度膠體電泳（DGGE）……………………………………18
一、 DGGE之原理……………………………………………………18
二、 DGGE於複雜微生物相分析之應用……………………………20
第二章、 肉雞消化道乳酸桿菌相PCR-DGGE條件建立與應用……21
壹、 前言…………………………………………………………………21
貳、 材料與方法…………………………………………………………21
一、 材料………………………………………………………………21
二、 試驗設計…………………………………………………………24
參、 結果…………………………………………………………………27
一、 引子專一性評估…………………………………………………27
二、 膠體染色方法……………………………………………………28
三、 巢式PCR及PCR產物純化……………………………………29
四、 變性梯度區間、電壓及電泳時間………………………………31
五、 利用PCR-DGGE追蹤肉雞消化道中菌群變化之可行性………34
肆、 討論…………………………………………………………………35

第三章、 年齡及益生菌對肉雞消化道乳酸桿菌相之影響…………38
壹、 前言…………………………………………………………………38
貳、 材料與方法…………………………………………………………38
一、 材料……………………………………………………………….38
二、 試驗方法………………………………………………………..39
三、 測定項目及分析方法…………………………………………..39
四、 統計分析…………………………………………………………40
參、 結果………………………………………………………………….41
一、 年齡對肉雞消化道乳酸桿菌相之影響…………………………41
（一） 以平板培養法計算消化道乳酸菌及大腸桿菌群菌數……41
（二） 以DGGE探討年齡對肉雞消化道乳酸桿菌相之影響………43
二、 益生菌對肉雞消化道乳酸桿菌相之影響………………………53
（一） 以平板培養法計算消化道乳酸菌及大腸桿菌群菌數……53
（二） 以DGGE探討年齡對肉雞消化道乳酸桿菌相之影響…………56
肆、 討論……………………………………………………………..….60
一、 年齡對肉雞消化道乳酸桿菌相之影響…………………………60
二、 益生菌對肉雞消化道乳酸桿菌相之影響………………………64
第四章、 參考文獻………………..……………………………………….66

表目次
表 1. 肉雞迴腸及盲腸菌相16S rDNA序列分析結果………………………4
表2. 影響胃腸道菌相之因子…………………………...……………………7
表 3. 目前用於檢測複雜微生物之研究方法…………….…………………10
表 4. 變性膠體溶液…...…………………………………………………….23
表 5. 年齡變化對肉雞腸道乳酸菌、大腸桿菌群菌數及兩者比值之影響42
表 6. 不同年齡雞隻飼糧中添加益生菌或抗生素之消化道乳酸桿菌相DGGE圖譜條帶數…………………….………………………………………44
表 7. 不同年齡雞隻飼糧中添加益生菌或抗生素之消化道乳酸桿菌相歧異度指數………………………………..…………………………………45
表 8. 混合型益生菌及抗生素對肉雞嗉囊乳酸菌、大腸桿菌群菌數及兩者比值之影響………………………………………………………………53
表 9. 混合型益生菌及抗生素對肉雞迴腸乳酸菌、大腸桿菌群菌數及兩者比值之影響…………..……………………………………………………54
表 10. 混合型益生菌及抗生素對肉雞盲腸乳酸菌、大腸桿菌群菌數及兩者比值之影響……...………………………………………………………55

圖目次
圖1. 以16S rDNA定序技術分析肉雞迴腸及盲腸菌相組成…………………3
圖2 . 宿主及細菌對碳水化合物利用之概述…………………………………6
圖3. 應用rRNA定序方法之免培養技術探討微生物群落組成……………12
圖4. 已定義菌門及準菌門之可培養比例……………………………………13
圖5. 垂直型DGGE電泳原理…………………………………………………19
圖6. 垂直（A）及水平式（B）變性梯度膠體電泳………………………19
圖7. 變性梯度膠體電泳設備…………………………………………………21
圖 8. LabtufGC/LabtufR引子組專一性試驗之電泳圖………………………27
圖9. 染色方法對腸道內容物乳酸桿菌相分析之影響……………………28
圖10. 不同PCR方法對腸道內容物乳酸桿菌相分析之影響………………29
圖11. PCR產物純化與否對腸道內容物乳酸桿菌相分析之影響……………30
圖12.變性梯度區間對腸道內容物乳酸桿菌相分析之影響…………………31
圖13.電壓及電泳時間對腸道內容物乳酸菌相分析之影響…………………32
圖14.電壓及電泳時間對腸道內容物乳酸桿菌相分析之影響………………33
圖15. 應用PCR-DGGE分析雞隻口服乳酸桿菌液後腸道乳酸桿菌相變化34
圖16. 不同年齡雞隻飼糧中添加益生菌或抗生素之嗉囊乳酸桿菌相PCR-DGGE圖譜………………………………………………………46
圖16（續）. 不同年齡雞隻飼糧中添加益生菌或抗生素之嗉囊乳酸桿菌相PCR-DGGE圖譜………………………………………………………47
圖17. 不同年齡雞隻飼糧中添加益生菌或抗生素之迴腸乳酸桿菌相PCR-DGGE圖譜………………………………………………………48
圖17（續）. 不同年齡雞隻飼糧中添加益生菌或抗生素之迴腸乳酸桿菌相PCR-DGGE圖譜………………………………………………………49
圖18. 不同年齡雞隻飼糧中添加益生菌或抗生素之盲腸乳酸桿菌相PCR-DGGE圖譜………………………………………………………50
圖18（續）. 不同年齡雞隻飼糧中添加益生菌或抗生素之盲腸乳酸桿菌相PCR-DGGE圖譜………………………………………………………51
圖19. 不同年齡雞隻嗉囊（A）、迴腸（B）及盲腸（C）乳酸桿菌相相似度指數UPGMA歸群樹狀圖………………………………………………52


圖20. 不同年齡雞隻飼糧中添加益生菌或抗生素之嗉囊乳酸桿菌相相似度指數UPGMA歸群樹狀圖………………………………………………57
圖21. 不同年齡雞隻飼糧中添加益生菌或抗生素之迴腸乳酸桿菌相相似度指數UPGMA歸群樹狀圖………………………………………………58
圖22. 不同年齡雞隻飼糧中添加益生菌或抗生素之盲腸乳酸桿菌相相似度指數UPGMA歸群樹狀圖………………………………………………59

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