Structural Characterization,Chemical Modification And Bio Activity Of Exopolysaccharides From Lactobacilus Plantarum

The exopolysaccharides(EPS)generally exist in two forms depending on their locations:cell-bound exopolysaccharides(c-EPS)which closely adhere to the bacterial surface,and released exopolysaccharides(r-EPS)that release into the surrounding medium.Most EPS-producing strains reported to date produced r-EPS,whereas some LAB strains can simultaneously produce both c-EPS and r-EPS.Although there have been many reports about the polysaccharides from animals,plants and fungi,EPS from lactic acid bacteria(LAB)have particularly received intensive interests,mainly because of their "generally recognized as safe"(GRAS)status and their beneficial effects to human beings.Compared to the synthetic antitumor and antioxidant agents,EPS from LAB would likely possess beneficial effects and further avoid the severe side effects.Hence,it is attracting to develop the EPS of LAB as functional food supplements of agents.Lactobacilus plantarum 70810(L.plantarum 70810)was a high-producing sticky lactic acid bacteria isolated from Chinese paocai by our laboratory.In this study,we studied the isolation,purification,structure,chemical modification and bioactivity of EPS from L.plantarum 70810.The main research results are listed as follows:1.In order to get an effective extract method of c-EPS,we compared the extracting effects of three common extract protocols,ultrasonic,EDTA and Phenol treatment.Ultrasonic treatment gave the highest c-EPS yield of 64.17 mg/L compared to the other two extraction protocols.The yields of r-EPS isolated by ethanol precipitation were 512.50 mg/L.After purified by anion exchange and gel filtration chromatography,two released exopolysaccharides(i.e.,r-EPS 1 and r-EPS2)and one cell-bound exopolysaccharide(i.e.,c-EPS)were obtained.The Mw were 204.6 kDa,202.8 kDa and 169.6 kDa,respectively.Composition analysis revealed the crude r-EPS contained 2.98%of protein,while the purified fractions had no protein.The total carbohydrate contents in all of purified fractions were higher than 90%.And the sulfated group content of r-EPS2 and c-EPS was 1.88%and 0.55%,respectively.Additionally,the uronic acid contents of c-EPS were 1.28%.It had a negative response to the Bradford test and no absorption at 260 nm or 280 nm in the UV spectrum,indicating the absence of protein or nucleic acid in all of samples.2.GC analysis demonstrated that both r-EPS 1 and r-EPS2 were composed of glucose,mannose and galactose,while c-EPS were mainly composed of galactose.The FTIR spectra showed all the peaks detected were in agreement with the typical absorption peaks of the polysaccharides.Methylation reaction,GC-MS and NMR analysis indicated r-EPS 1 were consisted by 1,6-linked-a-D-Glcp,1,2-linked-a-D-Manp,1,3-linked-a-D-Manp,1.4-linked-a-D-Galp,1,3,4-linked-?-D-Manp,1,6-linked-a-D-Manp and a tail-?-D-Manp-1.r-EPS2 were consisted by 1,6-linked-a-D-Glcp,1,4-linked-?-D-Galp,1,3,6-linked-?-D-Galp,1,6-linked-a-D-Galp,1,3,4-linked-a-D-Galp and a tail-a-D-Manp-1.And c-EPS were consisted by 1,6-linked-?-D-Galp,1,4-linked-?-D-Galp,1,2,3-linked-?-D-Galp,1.4-linked-?-D-Galp and a tail-?-D-Galp-1.3.SEM analysis indicated the surface of three polysaccharide samples had significant differences in size and shape.Congo red reaction indicated that all polysaccharides had no triple helical structure.4.Preliminary tests in vitro indicated the EPS had strong hydroxyl radicals scavenging activity,moderate DPPH radical scavenging activity and relatively weak reducing power.The effects of c-EPS on Caenorhabditis elegans were also evaluated.After treated by different concentrations of c-EPS,the mean lifespan,thermotolerance and oxygen tolerance of C.elegans were significantly enhanced.5.MTT assay revealed EPS had significant inhibition effects on the proliferation of HepG-2 cells,and the inhibition activities depend on polysaccharide concentrations.Further experiments indicated that the c-EPS could cause cell shrinkage and the ROS increase.Cell cycle analysis indicated that the c-EPS induces G2/M phase arrest and apoptosis in HepG-2 cells.Finally,the kit assay suggested the apoptosis induced by c-EPS occurred through the activation of Caspase-3 and Caspase-9.6.FTIR and NMR analysis demonstrated the acetylated,carboxylated,phosphorylated and sulfated polysaccharides were successfully prepared.The degrees of substitution were 0.41,0.08,1.1 and 0.37,respectively.SEM and TGA analysis showed r-EPS 1 and its derivatives had different surface morphology and thermal behavior.Compared with r-EPS 1,the derivatives exhibited stronger antioxidant and antitumor activities.As a whole,the acetylated and sulfated derivatives exhibited stronger antioxidant activity,whereas the phosphorylated and carboxymethylated derivatives showed a better promoting effect on antitumor activity.