产胞外多糖干酪乳杆菌的筛选鉴定及galU基因克隆与分析

The Preliminary Isolation and Indentification of Lactobacillus casei ZJ-4 Producting Excellular Polysaccharide and the Cloning of Gal U

乳酸菌胞外多糖具有良好的功能特性。从泡菜中分离乳酸菌,初筛到11株产胞外多糖的乳酸菌。经黏度及EPS产量测定,复筛出1株高产EPS的乳酸菌,其产量为771.97μg/m L。采用CTAB法提取该菌株基因组,测定其16S r RNA序列,在NCBI上进行同源序列比对,建立系统发育树。结果初步确定该菌株为干酪乳杆菌ZJ-4(Lactobacillus casei ZJ-4)。以该菌株基因组为模板,参照干酪乳杆菌BL23(HM162415)的gal U序列设计引物。采用Touch-down PCR扩增得到大约900 bp的gal U序列,经TA克隆、测序,与干酪乳杆菌BL23(HM162415)的gal U序列同源率为98%,表明克隆正确。

Extracellular polysaccharide yielded from lactic acid bacteria has good functional characteristic. The 11 initial lactic acid bacteria strains, which can product exopolysaccharides, were isolated from a traditional fermented food kimchi. One lactic acid bacteria strain with the 771.97 μg/m L exopolysaccharides yield was selected through fermentation,viscosity and exopolysaccharide production determination. The 16 S r RNA sequence was amplified by PCR, its nucleotide sequence was determined and the homology and phylogenetic of the sequence was analysised after extracting the genome of Lactobacillus with CTAB method, then the strain Lactobacillus casei ZJ-4 was preliminary determined. After designing the primers of gal U sequence referring to the gal U of Lactobacillus casei BL23（HM162415）, the gal U gene about 900 bp was amplified by touch-down PCR with the genome as template. The PCR production was cloned to T vector and the homology was 98% comparing to the gal U sequence of Lactobacillus casei BL23（HM162415）.