大貓熊腸道微生物於纖維之利用

Abstract

本研究之目的旨在探討大貓熊腸道微生物對於纖維食材的利用。依試驗方向可分為兩部分，試驗一透過纖維分解菌株的篩選，了解大貓熊腸道纖維分解優勢菌之種別、活性及菌群大小；試驗二則利用體外發酵法評估微生物體 （microbiota）對於高纖維竹材的利用。兩試驗皆以臺北市立動物園圈養之大貓熊糞樣作為腸道菌源。試驗一根據以下兩篩選程序進行菌株篩選：（A）待測菌株以不同的纖維基質為碳源，於液態培養基培養6及24 小時，依據釋出的還原糖進行篩選；（B）疊覆含剛果紅-羧甲基纖維素之洋菜膠薄層於平板培養之菌落，經短暫復培養後根據水解圈的形成篩選菌株。由A、B兩程序分別篩得四株（A1、A8、A13及A15） 及六株 （B1∼6）纖維分解菌，其定序結果除了A15為Enterococcus hirae外，其餘皆為Escherichia coli。試驗二以大貓熊的單一或混合日糧組成下列五處理組：LV（孟宗竹葉）、CM（黃金竹桿）、BM（LV及CM以1：1混合）、WT（窩窩頭）及 MX（LV、CM及WT以2：2：1混合）。為模擬進入後腸之消化物，各基質均以商用消化酵素進行預處理，其後與微生物培養6、12、24及48 小時，以了解微生物對基質利用之時序變化，結果顯示基質的消化速率以及短鏈脂肪酸的生成效率都以6 小時前較佳，且微生物於6∼24 小時間持續增殖，顯示若基質停留時間較短，腸道微生物供給宿主能量產物的效率會較高；而在五基質處理組中，CM及WT組均有較高的累積產氣量，但唯有中洗纖維含量最高的CM組有最高的短鏈脂肪酸產出，顯示大貓熊之腸道微生物對纖維成分的利用。綜合以上，大貓熊腸道存在具纖維分解之潛力菌株，且與大貓熊腸道之優勢菌一致，若以優勢菌之比例推算，其纖維分解菌群極可能遠大於以往之估計，是以在整體菌群的作用下能有效地將纖維基質代謝成能量產物供宿主所用。

The objective of this study was to evaluate the fiber utilization of microorganisms in giant pandas’ digestive tract. It included two parts：（1）Clarify the species, activity and group size of predominant cellulolytic bacteria by bacteria screening. （2）Evaluate the whole scheme of microbial effect in fiber utilization via in vitro fermentation technique. Fecal samples collected from captive giant pandas in Taipei Zoo were used as sources of microorganisms in both parts of trails. In the first part, there were two screening procedures involved：（A）Four candidatus strains were selected according to the reducing sugar released from fiber content after short term culturing（6 and 24 hr）. Three of which were confirmed as Escherichia coli（A1, A8 and A13）, and one as Enterococcus hirae（A15）. （B）Congo Red- carboxymethyl cellulose was used as agar slim to culture plate. After short term culturing, there were 6∼7% colonies grown with clear zones. Six strains were selected accordingly and all the strains were confirmed identities as E. coli. In the second part, daily feed of giant pandas were used as substrates for microbial fermentation, the following five groups were included：LV（leaves of moso bamboo）、CM（culms of golden bamboo）、BM（mixture of LV and CM with ratio of 1：1）、WT（wowotou） and MX（mixture of LV, CM and WT with ratio of 2：2：1）. All substrates were pretreated with commercial enzyme and then cultured with microorganisms for 6, 12, 24 and 48 hr. The time profile of digestibility of substrates, formation of short chain fatty acids and microbial synthesis showed that bacterial fermentation would efficiently contribute to their host in short term（0∼6 hr） gut transit. Furthermore, among substrates, CM and WT group both showed higher volume of gas production but only CM group, the one held the highest fiber content, had the highest short chain fatty acid production. To sum up, the cellulolytic bacteria were confirmed to exist in giant pandas’ digestive tract. As the isolated strains were identical to the predominant strains, a large group of fiber utilization bacteria can be pictured. That is, the framework of microorganisms was able to contribute the energy sources via the metabolism of fiber.