圈養食葉性哺乳類動物腸道健康監控及糞源乳酸菌篩選之研究

Abstract

本研究以兩種食性較為特殊的食葉性哺乳類動物為研究對象，分別探討大貓熊（Ailuropoda melanoleuca）糞源乳酸菌篩選應用之可能性，並以過去大貓熊之腸道健康探討模式，嘗試建立無尾熊（Phascolarctos cinereus）腸道健康評估之可能指標。 大貓熊部分參考選擇性糞菌移殖（selective microbiota transplantation, SMT）的概念，利用選擇性培養基中加入竹葉萃取物，從腸道健康狀況較佳的大貓熊個體四種類型糞便中，分離出可以耐受竹葉萃取物的優勢乳酸菌菌株，以作為將來具有消化障礙之大貓熊改善腸道健康時使用。經過乳酸菌選擇性培養基與竹葉萃取物篩選後，再以API 50 CHL進行初步菌種鑑定，並配合16s rDNA定序進行確認菌株。大貓熊菌株篩選結果顯示，在此篩選條件下，從大貓熊糞便所分離出的乳酸菌主要為Weissella confusa。推估此種菌可能為於較高濃度竹葉萃取物環境下較為強勢的乳酸菌。 無尾熊部分參照過去大貓熊腸道健康評估的建立方法，以台北市立動物園內四隻個體為對象，連續收集一整年的菌相、糞便狀況、採食評分等資料作基礎，並參考園內個體的採食習慣及採食評分標準，分成三級採集其所食之桉樹葉進行成分分析，以評估其採食狀況與葉子成分變化間之關聯性。由無尾熊菌相監測結果發現，Enterococcus、E. coli、Coliform佔總好氧菌的比例隨著時間變化較大，Clostridium與乳酸菌佔總厭氧菌的比例則較穩定。而當菌相波動較大時，糞便狀況也較容易出現不正常。因10到11月為秋冬季節轉換時期，糞便不正常狀況較多，糞便菌相波動明顯，且糞便與桉樹葉樣本收集較完整，故進一步取其中兩個時間區段分析葉子成分。分析結果發現，園內無尾熊對具有較高水分、粗蛋白及較低總酚單寧的葉子採食意願有較高的趨勢，桉樹葉中的總酚及單寧含量較高時對園內無尾熊採食偏好可能仍具有負面影響。 本研究分離出的W. confusa菌株於大貓熊利用上，由於此菌種目前並不在動物益生菌可添加的菌種正面列表上，實際使用仍需要進行體外模擬試驗及安全性評估等程序。而所建立的無尾熊採食偏好與全年期間食物來源對採食與糞便菌相狀況資料及監控指標，可有效提供長期圈養無尾熊之腸道健康監控使用。

Two mammalian folivores, the giant panda and the koala, were used as research subjects in this study, to explore the application possibility of isolating the fecal lactic acid bacteria (LAB) from giant panda, and referring to previous research, to establish the possible methods and index for intestinal health assessment of koala. The first part of this study referred to the concept of selective microbiota transplantation (SMT) and used bamboo leaf extracts in a selective medium to isolate LAB from four types of feces from a healthy giant panda in the Taipei Zoo. The dominant LAB species that could tolerate the bamboo leaf extract were selected for future use, to help other giant pandas with digestive tract disorders improve their intestinal health. After the selection, API 50 CHL was used for the preliminary strain identification, while 16s rDNA sequencing was used for confirmation. The second part referred to the method of intestinal health assessment established in the past with giant pandas as research subjects, taking four individual koalas in the Taipei Zoo as subjects of research, continuously collecting the fecal microbiota, fecal conditions, dietary score and other data as basis information for a whole year, while collecting Eucalyptus leaves for nutritional analysis, to assess the correlation between dietary conditions of the koalas and changes in Eucalyptus leaf composition. The results showed that under this selecting condition, the LAB species isolated from feces of the giant panda were mainly Weissella confusa. It’s estimated that W. confusa might be a dominant LAB species in the environment with a higher concentration of bamboo leaf extracts. The microbial monitoring results of the koalas showed that the ratio of Enterococcus, E. coli and Coliform in aerobes varied widely over time, but the ratios of Clostridium and LAB in anaerobes were relatively stable. When the fluctuations of the fecal microbiota were larger, more abnormal fecal conditions were observed. The four main species of Eucalyptus leaves that were consumed by the subjects, were collected in 3 gradations corresponding to the dietary score for evaluations. Considering that October and November marks the change from Autumn to winter, more abnormal fecal conditions and significant fluctuations in fecal microbiota were observed. The collection of feces and eucalyptus leaf samples during this period was also more complete, therefore, the samples collected were then used for further analysis. The results showed that koalas in this research had a higher preference to consume Eucalyptus leaves containing higher water, crude protein, lower total phenols and tannins content. Higher total phenols and tannins content in the leaves could had a negative effect on the preferences of the koalas in this study. Since W. confusa is currently not regarded as a safe species of animal probiotic, the strains isolated during this study still require further investigations for their characteristics and safety assessments in regards to the giant panda. The established data and index of koala’s dietary preference and conditions, fecal conditions and microbiota collected throughout the year can effectively provide a long-term basis on health monitoring work for captive koalas in the Taipei Zoo.