中华鳖肠道益生菌的筛选、鉴定与生长特性研究

THE SELECTION, IDENTIFICATION, AND CHARACTERIZATION OF THE GROWTH OF PROBIOTICS FROM INTESTINAL TRACT OF CHINESE SOFTSHELL TURTLE(TRIONYX SINENSIS)

通过点种法初筛和牛津杯法复筛,从中华鳖肠道筛选到1株对嗜水气单胞菌(Aeromonas hydrophila)、温和气单胞菌(A.sobria)以及豚鼠气单胞菌(A.caviae)具有拮抗作用的益生菌株Dec-43。对该菌株进行形态、生理生化特征分析,结果与《常见细菌系统鉴定手册》中屎肠球菌(Enterococcus faecium)的生理生化特征一致。提取细菌基因组DNA,通过16S r RNA基因通用引物进行PCR扩增,从该菌株的16S r RNA基因中克隆到了一个长度1448 bp的片段,经测序和序列比对,该片段与Genbank中屎肠球菌的序列相似性达99%,因此,该菌株确定为屎肠球菌。通过单因素试验确定了该菌株的最佳生长温度、盐度、初始p H、接种量等参数分别为:36℃、盐度0.6%、培养基初始p H 7、接种量10%;通过正交试验,确定了该菌株培养基中碳源(葡萄糖)、氮源(蛋白胨)最适含量分别为15和10 g/L。研究发现了一株中华鳖肠道潜在益生菌,并研究了其生长特性,为中华鳖养殖行业益生菌的应用提供了基础。

The aim of this study was to isolate the potential probiotics and to identify their effects. Using spot inoculation as the first screening and the Oxford cup method as the second screening, we isolated a strain of bacterium, namely Dec-43, from the intestinal tract of Chinese softshell turtle （Trionyx sinensis）. This strain had antagonistic effects on bacterial strains in-cluding Aeromonas hydrophila, A. sobria and A. caviae. The analysis in bacterial morphology, physiology, and biochemistry indicated that Dec-43 shared the same physiological and biochemical properties with Enterococcus faecium. We used universal primers to amplify the 16S rRNA gene with PCR, and obtained a segment of 1448 bp from the total genomic DNA of Dec-43. After the sequencing and the BLAST analysis of this segment we found that the homology between Dec-43 and E. faecium was 99%. These results suggested that the Dec-43 should be E. faecium. By conducting the single factor test we determined that the optimal growth temperature of Dec-43 was 36℃; the optimal salinity was 0.6%; the optimal initial pH was 7; the optimal growth inoculum size was 10%. Through the orthogonal experiment we also determined the proper carbon （glucose） and ni-trogen （peptone） contents should be 15 and 10 g/L respectively.