- 期刊
源自豬隻糞便與牛瘤胃液凝結芽孢桿菌的篩選、鑑定與性狀分析
Selection and identification and characterization of Bacillus coagulans from pig excrement and cattle rumen fluid
摘要
芽孢桿菌屬（Bacillus）屬於革蘭氏陽性菌，具有產生細胞外酵素與形成孢子進入休眠狀態，且較不受外界環境的影響，適合作為畜禽飼料添加物。本試驗目的由豬隻糞便與牛瘤胃液中，篩選與鑑定具耐熱、耐酸、耐膽鹽，且會形成孢子之凝結芽孢桿菌（Bacillus coagulans），供作為畜禽飼料添加物用。試驗分別由豬隻糞便與牛瘤胃液中篩選出各10株候選菌株，經加溫誘發形成孢子與90℃高溫加熱30分鐘殺死不形成孢子菌株，接續比對菌株16S rDNA核苷酸序列的相似度，挑選出6株近似於凝結芽孢桿菌候選菌株及基質輔助雷射脫附游離飛行時間質譜儀（MALDI-TOFMS）進行菌種鑑別，再測試pH 2與2%膽鹽之耐受性。結果顯示5株（編號R6、R8、R9、S7與S10）為蠟樣芽孢桿菌（Bacillus cereus），僅有源自瘤胃液編號R3屬於凝結芽孢桿菌，且對pH 2與2%膽鹽之耐受性均高於70%。凝結芽孢桿菌R3形成孢子能力經培養後每1 mL有1×10^8 cfu以上。綜上而言，篩選自瘤胃液之凝結芽孢桿菌R3有耐酸、耐膽鹽與形成孢子等特性，具有作為畜禽飼料益生菌使用之潛力。
並列摘要
Bacillus belongs to gram-positive bacteria that can secret extracellular enzymes, form of spores to progress into dormancy and less likely to receive impact from the external environment, which is also suitable a feed additive for livestock and poultry. The objective of the study is to select and identify Bacillus coagulans with acid and bile salt-resistant and sporulation ability from pig feces and cattle rumen fluid. The 10 candidate strains of Bacillus coagulans were selected by screen culture agar and medium from pig feces and cattle rumen fluid, respectively. The strains were heated to 45°C for 24 hours to induce sporulation, and further heated up to 90°C for 30 minutes to kill non-sporulation of strains and 16S rDNA nucleotides BLAST, in order to identify the DNA similar to Bacillus coagulans. Subsequently, 6 candidate strains of bacillus coagulan were selected. Then treatment with pH 2 and 2% bile salt culture medium for confirming the acid and bile salt-tolerance ability and use of MALDI-TOF MS were applied to identify microbial species. The results showed that 5 candidate strains (R6, R8, R9, S7S and S10) belonged to Bacillus cereus and only R3 strain belonged to B. coagulans. In addition, the B. coagulans sporulation ability of R3 exceeded 1 x 10^8 cfu/mL. In conclusion, the B. coagulans R3 isolated and identified from cattle rumen fluid possessed the characteristics of acid and bile salt-resistant and sporulation ability, which had the potential to be used as a feed additive in livestock ration.