耐胆汁型牛黄转化菌的分离、鉴定及其牛黄转化活力研究

Isolation and identification of a bile-tolerant Bovis Calculus transformingbacteria and its transformation activity

目的分离和鉴定能够耐受牛胆汁的牛黄转化细菌,并应用到体外培育牛黄的制备。方法从形成牛黄的牛胆囊胆汁中分离到可在牛胆汁中长期存活且具有较强牛黄转化能力的菌株(NDZNM-01),观测其外部形态、理化性状、分类学地位、牛胆汁耐受力,以及牛黄转化活力。结果根据对NDZNM-01菌株外部形态与生理生化特征的检测结果,判断为柠檬酸杆菌属细菌。进一步根据其16S rDNA序列的聚类分析结果,显示该菌株与其他3种柠檬酸杆菌属的细菌聚在一个分支上,但形成单系,属于新发现的菌株。应用菌株NDZNM-01发酵新鲜牛胆汁,其中的胆红素含量增加了2.35倍,胆酸含量增加了2.13倍。结论综合对NDZNM-01菌株的形态观察、生理生化鉴定,以及分子鉴定结果,NDZNM-01属于一种能够耐受牛胆汁的柠檬酸杆菌属的肠道细菌,具有高效转化牛胆汁为牛黄的活力。该牛黄转化细菌最终命名为Citrobacter bovis NDZNM-01 Wang Houwei,菌种收藏编号为CGMCC24893。

Objective To isolate and identify a Niuhuang(Bovis Calculus)transformed bacterium that can tolerate bovine bile,and apply it to the transformation of Bovis Calculus cultivated in vitro.Methods The strain(NDZNM-01)that could survive in bovine bile for a long time with high Bovis Calculus transformation ability was screened and isolated from bovine bile,which external morphology,physicochemical properties,taxonomic status,ability to tolerate bovine bile,and transformation activity of Bovis Calculus were observed and determined.Results According to the detection results of the external morphology and physiological and biochemical characteristics of NDZNM-01,it was preliminarily determined to be a bacterium of the genus Citrobacter.According to the results of cluster analysis of 16S rDNA sequence,it was shown that NDZNM-01 and the other three Citrobacter bacteria clustered into one branch,but formed a single line,belonging to a newly discovered strain.Using strain NDZNM-01 to ferment fresh bovine bile,the content of bilirubin increased by 2.35 times and the content of bile acid increased by 2.13 times.Conclusion Based on results of the morphological observation,physiological and biochemical identification,and molecular identification,it can be judged that strain NDZNM-01 belongs to an intestinal bacterium of the genus Citrobacter that can tolerate bovine bile,and has the activity of efficiently converting bovine bile into Bovis Calculus.The Bovis Calculus transformed bacterium was finally named Citrobacter bovis NDZNM-01 Wang Houwei,and the collection number was CGMCC24893.