Analysis On Structural Characterization Of Exopolysaccharides And Transcription Of Eps Gene Cluster From Lactococcus Lactis Subsp.lactis IMAU11823

The Lactococcus lactis are widely used in fermented dairy products,and some strains can produce exopolysaccharides(EPS).B ecause of variety structures and complex biosynthesis of the lactic acid bacteria EPS,its important to understand the structure characteristic of novel EPS and explore its biosynthesis mechanism.In present study,an EPS-producing strain was focused on.L.lactis IMAU11823 which was isolated from natural fermented dairy products Jiaokou in XilinGol League.Firstly,the EPS produced by this strain was Isolated and purified to investigate the primary structure characterizations.Then,the eps gene cluster of the strain was identified by a complete genome sequencing strategy.Finaly,the transcriptional patterns of EPS genes were performed at different fermentation times by droplet digital PCR.The main results are as follows:1.The crude EPS yield could be 201.44 mg/L when the strain IMAU11823 was fermented in M17 liquid medium at the inoculation amount of 2%and cultured at 37? for 24 h.The investigation of the crude EPS anti-oxidant activities were examined in vitro.The results showed that the crude EPS exhibited strong reducing ability as well as scavenging ability of DPPH radical and hydroxyl radical,which were depended by the concentration of crude EPS.The crude EPS was purified by DEAE-Cellulose 52 and three components were obtained.They were named EPS-1,EPS-2 and EPS-3,respectively.2.Further study on the structural features of EPS-1 and EPS-2,showed that EPS-1 was composed of mannose and glucose with a molar ratio of 1:7.01,and the EPS-2 was composed of mannose,rhamnose and glucose with a molar ratio of 7.45:1:2.34.Both EPS-1 and EPS-2 exhibited the typically absorption peak of polysaccharide and presented pyranoid sugers by FT-IR spectrum analysis.Combining with results of 1D,2D NMR and the results mentioned above,the following speculation can be inferred that the EPS-1 was composed of?4)-?-D-Glcp-(1?,?4,6)-?-D-Manp-(1?and T-?-D-Glcp as the backbone chain.The backbone of EPS-2 was composed of T-?-D-Manp,T-?-D-Glcp?,2,3,4)-?-D-Manp-(1?,?4,6)-?-D-Manp-(1?,?2,3)-?-D-Manp-(1?,?2)-?-D-Map-(1?,?6)-?-D-Manp-(1?,?4)-?-D-Manp-(1?,?2)-?-D-Rhap-(1?and?4)-?-D-Glcp-(1? as the backbone chain.3.The results of complete genome sequencing of L.lactis IMAU11823 showed that this strain contained a single circular chromosome and three circular plasmids.An eps gene cluster located on the chromosome in a 16 Kb region consisting of 20 coding genes.Analysis of droplet digital PCR showed that epsR was highly expressed at the initial stage of EPS synthesis,while the expression of epsD,epsB and epsK reached the highest at the 6 h of growth,when the rate of EPS biosynthesis was the fastest.It could be inferred that EPS biosynthesis was mainly through epsR regulating the expression of epsD,epsB and epsK to accumulate EPS production.In this study,the structural characteristics of the EPS produced by L.lactis IMAU11823 were preliminarily analyzed,and its biosynthesis mechanism was discussed,which provides theoretical basis and data support for the comprehensive utilization of lactic acid bacteria and their EPS.