冠突散囊菌Ec-12上清抑制小鼠的伤寒沙门菌机制的初步分析

Preliminary Study of the Mechanism of Inhibition of Salmonella Typhimurium by Ec-12 Supernatant

本研究旨在筛选黑茶中有益的冠突散囊菌(Eurotium cristatum,EC)并揭示该菌上清抑制鼠伤寒沙门菌(Salmonella Typhimurium)的初步机制。采用稀释涂布法分离冠突散囊菌;通过形态学和ITS rRNA基因序列分析其分类地位;采用生长速率法分析其增殖条件及增殖的稳定性;采用琼脂扩散抑菌试验、耐酸碱、耐热及紫外光稳定性试验、电镜观察及小鼠动物试验等以评估该Ec-12上清抑制鼠伤寒沙门菌的初步机制。结果显示:筛选到一株有较强抑制鼠伤寒沙门菌作用的冠突散囊菌Ec-12,抑菌圈分别为10.4 mm±1.5 mm。根据形态学、生长特征及ITS rRNA基因序列分析,将其鉴定为冠突散囊菌,并命名菌株Ec-12。粗分离的菌株Ec-12上清在pH近中性条件产生较好的抑菌效果,抑菌稳定好。经电镜观察可见粗分离菌株Ec-12上清导致鼠伤寒沙门菌的菌体变形,表层粗糙等抑菌现象。并明显降低小鼠感染鼠伤寒沙门菌引起的腹泻率和死亡率,提高小鼠绒毛的完整度和增加黏液蛋白的分泌(P<0.05),减少结肠细胞的凋亡(P<0.05),抑制鼠伤寒沙门菌对小鼠的危害。冠突散囊菌Ec-12菌株上清作用于鼠胃肠道内的鼠伤寒沙门菌,破坏了鼠伤寒沙门菌结构完整性,同时又作用于鼠的肠道,提高肠绒毛完整性,促进结肠黏液蛋白的分泌,减少结肠细胞的凋亡,进而降低鼠伤寒沙门菌引起的腹泻及死亡,它具备一定益生的潜力。

To investigated the probiotic function of Eurotium cristatum(EC)in black tea and reveal the preliminary mechanism of inhibit Salmonella Typhimurium,we used dilution coating method to isolate EC,and analyzed its taxonomic status by morphology and ITS rRNA gene sequence.And then the agar diffusion inhibition test,thermal acid-base and UV stability test,electron microscopy and mouse animal test were used to evaluate the preliminary mechanism of inhibition of S.Typhimurium bacteria by this Ec-12 supernatant.A strain of Ec-12 was identified as E.cristatum based on morphology,growth characteristics and ITS rRNA gene sequence analysis,and named strain Ec-12.The supernatant of the crude isolate Ec-12 produced good inhibition at pH near neutral conditions with good stability.The supernatant of tEc-12 showed good bacterial inhibition at pH near neutral and good stability of inhibition.The inhibition of S.Typhimurium was observed by electron microscopy,which showed that the supernatant of the crude isolate Ec-12 caused deformation of the body and roughness of the surface layer of the bacterium.It also significantly reduced the rate of diarrhea and mortality caused by S.Typhimurium infection in mice,improved the integrity of mouse villi and increased the secretion of mucin(P<0.05),reduced the apoptosis of colonic cells(P<0.05),and inhibited the harm of S.Typhimurium to mice.The supernatant of Ec-12 strain acted on S.Typhimurium in the murine gastrointestinal tract,disrupting the structural integrity of S.Typhimurium,while acting on the murine intestine,improving the integrity of intestinal villi,promoting the secretion of colonic mucin,reducing the apoptosis of colonic cells,and thus reducing the diarrhea and death caused by S.Typhimurium.It has a probiotic potential.