两歧双歧杆菌ATCC 29521在小鼠肠道的定植分析

Colonization of B.bifidum ATCC 29521 In Mice Intestinal Tract

目的 :为了解两歧双歧杆菌ATCC 29521在小鼠肠道中的定植情况,应用实时荧光定量PCR技术分析小鼠粪便中双歧杆菌的菌体量。方法 :对C57BL/6雌性小鼠分组灌胃,单次灌胃组予1×10~9CFU灌胃一次,灌胃后0、2、4、6、8、10、12、16、20、24 h收取粪便;连续灌胃组同等剂量连续灌胃3周,灌胃的0、7、14、21、24、28 d取粪便,提取DNA后检测菌体情况。结果 :整体菌量变化呈先升高后降低趋势,单次灌胃组在0 h、灌胃2h后未检测到,4 h后开始逐渐升高,10 h菌量达到最高峰为6.0×10~7 CFU/g粪便,之后菌量逐渐下降,灌胃12至16 h区间下降幅度最大;连续连续灌胃组灌胃1周后菌体量达2.0×10~7 CFU/g,2周后上升至1.0×10~8CFU/g,3周后菌量增加不明显,说明灌胃2周后达到平台期,两组分别于24 h和停止灌胃1周后菌量明显下降。结论 :两歧双歧杆菌ATCC 29521的给药周期至少需要2周,如果要维持此菌的治疗作用,需要持续给药,使体内的有效菌量维持在平台期。

Objective： Effect of gut microbiota on human health has become a hotspot in recent years with the emergence of new microbial technology.B.bifidum is a traditional probiotics and has been widely used in food and medicine. But the metabolism and function of B.bifidum ATCC 29521 has not been well documented. To investigate colonization and growth of B.bifidum ATCC 29521 in mice intestine, fecal B.bifidum concentrations were analyzed by real-time fluorescent quantitative PCR. Methods： Female C57 BL/6 mice were orally gavaged with eigher a single dose of 1×109 CFU B.bifidum ATCC 29521 or continuous doses of 1×109/day for 3 weeks. Feces were collected at 0, 2, 4, 6, 8, 10, 12, 16, 20, 24 h and at 10, 7, 14, 21, 24, 28 d, respectively. Total DNA was isolated from the feces using the Qiagen DNA stool extraction kit according to the manufacturer＇s instructions. The linear standard curve was established by each dilution degrees of B.bifidum ATCC 29521 and their corresponding CT values.The curve equation was y = 0.1835 x ＋ 9.0628 and R2 was 0.9994. The concentration of B.bifidum ATCC 29521 in mice feces were calculated by substituted CT values obtained by RT-PCR into the curve equation. Results： The curves of B.bifidum ATCC 29521 concentration rose at first and then reduced gradually. In single dose group, the concentration of B.bifidum ATCC 29521 began to increase at 4 h after oral gavage, reaching its peak 6.0 × 107 CFU/g at 10 h and then decreased gradually. The biggest drop occurred at the period between 12 h and 16 h after B.bifidum treatment. In successive administration group, the concentration rose at the highest rate in the first week when it achieved 2.0×107 CFU/g and kept on inceasing to 1.0×108 CFU/g in the next week. However, the concentration did not rose up significantly in the third week. It means that the concentration of B.bifidum ATCC 29521 in mice intestine reached platform in the second week after oral gavage. The concentration of B.bifidum ATCC 29521 significantly decreased at 24 h and one week after B.bifidum treatment course in two group repectively and was still higher than baseline before oral gavage. Conclusion： Once the B.bifidum ATCC 29521 supplement was discontinued, the concentration fell down in short time. B.bifidum ATCC 29521 could not sustain colonization and growth in mice intestine without external supplement. It is necessary to provide daily supplemention for at least two weeks and to keep on in order to maintain sufficient concentration.