应用变性梯度凝胶电泳和16S rDNA序列分析对kefir粒中细菌多样性的研究

以开菲尔(Kefir)粒为材料,经过DNA抽提和16S rDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析。结果表明,DGGE图谱中可检测到的8条带的16S rDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98%,余下的1个基因序列的相似性也大于96%。相似性大于98%的7个克隆中,有3个属于鞘氨醇杆菌属(Sphingobacterium),2个属于乳杆菌属(Lactobacillus),其它2个分别属于肠杆菌属(Enterobacter)和不动杆菌属(Acinetobacter)。首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中。

Analysis of bacterial diversity of kefir grains by denaturing gradient gel electrophoresis and 16S rDNA sequencing

Kefir is an acidic,mildly alcoholic dairy beverage produced by the fermentation of milk with a grain-like starter culture.These grains usually contain a relatively stable and specific balance of microbes that exist in a complex symbiotic relationship.Kefir grains can be considered a probiotic source as it presents anti-bacterial,anti-mycotic,anti-neoplasic and immunomodulatory properties.The microorganisms in Kefir grains are currently identified by traditional methods such as growth on selective media,morphological and biochemical characteristics.However,the microorganisms that isolate by these methods can not revert to Kefir grains which indicate that there are some other bacteria that are not isolate from it.In this study,PCR-based Denaturing gradient gel electrophoresis(DGGE) and sequence analysis of 16S ribosomal RNA gene((16S rDNA)) clone libraries was used for the rapid and accurate identification of microorganisms from Kefir grains.The PCR primers were designed from conserved nucleotide sequences on region V3 of(16S rDNA) with GC rich clamp at the 5'-end.PCR was performed using the primers and genomic DNAs of Kefir grains bacteria.The generated region V3 of(16S rDNA) fragments were separated by denaturing gel,and the dominant(16S rDNA) bands were cloned,sequenced and subjected to an online similarity search.Research has shown that regions V3 of(16S rDNA)s have eight evident bands on the DGGE gel.The sequence analysis of these eight bands has indicated that they belong to different four genera,among them three sequences are similar to Sphingobacterium sp.whose similarities with database sequences are over 98%,three sequences are similar to Lactobacillus sp.whose similarities with database sequences are over 96%,the other two sequence are similar to Enterobacter sp.,and Acinetobacter sp.whose similarities with database sequences are over 99% respectively.Although the DGGE method may have a lower sensitivity than the ordinary PCR methods,because when universal bacterial PCR primers are used, only the dominant microbiota of an ecosystem will be visualized on a DGGE gel,producing complex banding patterns.However,it could visualize the bacterial qualitative compositions and reveal the major species of the Kefir grains.Among them Sphingobacterium can be found in Kefir grains as the predominant flora which is reported for the first time.PCR-based DGGE and sequence analysis of(16S rDNA) proved to be a valuable culture-independent approach for the rapid and specific identification of the microbial species present in microecosystem and probiotic products.