The Screening Of SLP From Lactobacillus Spp. By Colony In Situ Hybridization

As probiotics, Lactobacilli confers great benefit to human.It is generally known that the adhesion to the intestinal mucosal surface is one of the most important prerequisite for them to persist in the intestinal tract and this is considered important for their function. To date, S-layer protein (SLP) is the main component that is involved in adhesion to different host tissues. SLP is crystalline arrays of proteinaceous subunits present as the outermost component of cell wall. SLP characterized from the genus Lactobacillus ranges from 25 to 71 kDa in size, the smallest one of the known for SLP, which is a basic protein.A blast analysis, based on the genus Lactobacillus SLP gene, has been done, the specific sequences were found which were used to design oligonucleotide probe labeled with DIG as follow:5'-AGACTATTAT GGACAACGCT TACTTCTACG-3'. To evaluate the specificity of this probe, colony hybridization in situ was done based on the SLP gene of genus L.brevis C, L.brevis M and L. helveticus ATCC 15009, stored in our lab, which have been confirmed possessing SLPs. The result showed that the probe above could effectively detect the SLP gene with a certain specificity.In total 45 strains were isolated by SL culture, from pickle water, sour pork, bright cheese and animal intestinal ect. By microscopic examination and catalase analysis, they were initially identified as Lactobacillus. The DNA of those were exposed on NC filter by schizolysising, fixed by virtue of roasting, the hybridization signal was detected by enzymatic catalysis.The result showed that, in the 45 strains, there are 8 strains possessing SLP encoding gene. And these 8 strains possess different Slps, according to SDS-PAGE analysis, some have single band of SLP, and it is necessary to do further study of the 8 genus Lactobacilli.Of the 8 strains,3 with different SLPs were analysed by 16S rRNA. And 2 of the 3 were Lactobacillus reuteri, the other one was Lactobacillus plantarum. Interestingly enough, SLPs have rarely been reported among genus Lactobacillus reuteri and Lactobacillus plantarum.