【摘要】 为研究地衣芽孢杆菌A1（Bacillus Licheniformis, Bli）在斑点叉尾鮰（Ictalurus punctatus）肠道的定植规律,通过每周更换100%的循环水量以减少水体中的芽孢杆菌对实验的干扰,通过Co⁶⁰辐照灭菌杀灭饲料中的芽孢杆菌以消除其对实验的干扰。采用52℃高温选择性培养肠道Bli,研究外源Bli在肠道内的消长规律。使用嗜热脂肪地芽孢杆菌（Geobacillus stearothermo philus）的芽孢作为惰性对照,通过洗涤肠壁去除非黏附菌,研究Bli对肠粘膜的黏附。使用灭菌处理的肠内容物体外培养Bli,研究其在肠道的增殖潜能。结果显示,每周100%换水量使水体Bli含量<1 cfu/mL。辐照灭菌使饲料Bli含量由1.4×10⁴ cfu/g降至检出限以下。停止外源Bli摄入后,在持续投喂无菌饲料的情况下, Bli在斑点叉尾鮰肠内容物中的含量可维持3.3×10² cfu/g左右至少42天;在禁食情况下, 14天后肠内容物中便无Bli检出。Bli对肠粘膜的黏附能力弱于惰性对照,但Bli可以在肠内容物中生长至2.0×10⁷ cfu/g。以上结果提示:在持续喂食情况下,地衣芽孢杆菌可以在斑点叉尾鮰肠道内长期定植,而禁食情况不能定植。其原因是由于地衣芽孢杆菌不能有效黏附于肠粘膜,但可以在肠内容物中增殖。更多还原

【Abstract】 Bacillus licheniformis（Bli） has been widely used as a probiotic and has positive effects on host health of different fish species. The ability to colonize the gastrointestinal tract is an important criterion for probiotic screening, but little attention has been paid to this character. To investigate the colonization characteristics of Bli-A1, full water exchange and feed sterilization with Co⁶⁰ irradiation were employed to avoid their influence on the gastrointestinal microbiome of Ictalurus punctatus. A high temperature selective culture method for Bli was assessed for its sensitivity and specificity, and then it was employed to investigate the dynamic characters of exogenous Bli-A1 in the intestine for 42 days with or without fasting. The spores of Geobacillus stearothermophilus were used as an inert control to investigate the adhesion ability of Bli-A1 to mucosa in a feeding trial with Bli/Gs=186.4% in feed, and non-adhesion bacteria were removed by washing the intestinal wall. Then, Bil-A1/Gs on the intestinal content and wall were calculated. At last, the potential of Bli-A1 multiplying in the intestine was assessed with a sterile intestinal content culturing assay in vitro. With a full water exchange and feed sterilization, the concentration of Bli was reduced to <1 cfu/mL in water and not detected in feed（1.4×10⁴ cfu/g before irradiation）. Selective culture for Bli-A1 was successful at 52℃ with a growth rate at 91.5±13.7% in water, 65.9±26.7% in 20% intestinal content homogenate, and 72.5±19.7% in 20% intestinal wall homogenate. It took 21 days for the inert Gs control to be completely excreted from the intestine after withdrawing exogenous supplement（n=3, <1 cfu/intestine）. When exogenous Bli-A1 supplement was withdrawn, the concentration of Bli-A1 in the intestinal content was sustained at ～3.3×10² cfu/g for at least 42 days with continuous sterile feed supplement, whereas no Bli-A1 was detected at 14 days under fasting conditions. In the Bli-A1/Gs feeding trial, Bli-A1 in intestinal content was detectable to at least 28 days after exogenous Bli-A1 withdrawal. Adhesion-related Bli-A1 on the intestinal wall was undetectable at 7 days. At day 0, Bli-A1/Gs was equal to 197.9% in intestinal content, which was similar with the 186.4% in feed. Bli-A1/Gs was equal to 23.7% on the intestinal wall. Although Bli-A1 failed to adhere to mucosa and its adhesion ability was even weaker than that of the inert control, it grew well in intestinal content and reached a concentration of 2.0×10⁷ cfu/g in vitro. In conclusion, Bli-A1 can colonize the intestine of Ictalurus punctatus long-term, unless the fish are fasted. That is because Bli-A1 possessed the ability to grow in intestinal content but fails to effectively adhere to mucosa.更多还原