Screening Of Exopolysaccharide-producing Lactobacilli And Separation, Structure And Bioactivities Of Exopolysaccharide

Lactic acid bacteria exopolysaccharides (LAB EPS) are extracellular polysaccharideswhich are either associated with the cell surface in the form of capsules or secreted into theextracellular environment in the form of slime. LAB are generally regarded as safe (GRAS)organisms, therefore the EPS-producing LAB strains can be directly applied to fermenteddairy products (e.g. yoghurt and cheese) with highly stable properties in the rheology, textureand mouthfeel. Moreover, LAB EPS may provide health benefits, such as antitumouractivities, immune-modulating activities, antivirus activities, antioxidant activities,antihypertensive and cholesterol-lowering activities. LAB EPS can also be utilized by humanintestine bacteria as prebiotics to promote the proliferation of other probiotics. The objectivesof this work were to isolate and identify producing-exopolysaccharides lactobacilli, to isolateand purify EPS and to investigate the bioactivity, molecular characteristics and structurecharacterization of EPS. The main results are described as follows:Combining with the appearance of “ropy” strand colony formed on the screening plateand the polysaccharide production, Lactobacillus rhamnosus KF5was screened from healthyhuman feces, producing higher EPS (230mg/L) in reconstituted skim milk.The biologic characteristics of L. rhamnosus KF5strain were studies. The strain grewwell in MRS medium, optimum growth temperature37℃, optimum growth pH6.0, could begrown in containing7%NaCl MRS. Strain grew well in0.2%bile salt concentration, and0.3%bile salts can be tolerated. KF5was good acid resistance at pH3.0environment, thenumber of viable cells maintained3h almost no decline. KF5strain had a strong adhesion ofCaco-2cell (23bacteria/Caco-2), better than the control strain LGG (9bacteria/Caco-2).Antibacterial experiments showed that the strain produced acid against pathogenic bacteria S.aureus, E. coli and B. subtilis growth.The crude EPS were prepared from the fermented milk by removing the bacteria cellsand proteins, followed by centrifugation, ethanol precipitation, dialysis and lyophilization.Thecrude EPS were then isolated by a DEAE-Sepharose Fast Flow ion-exchange chromatographycolumn and two fractions were obtained, EPS1a neutral polysaccharide and EPS2an acidicone. Two fractions S1and S2were obtained by Sephacryl HR S200, Sepharose Cl-6B gelfiltration chromatography, respectively. There was one single and symmetrical peak appearedon the elution profile for each fraction. S1was composed of glucose, arabinose, glucosamine,galactosamine and galactose in an approximate molar ratio of2.03:1.29:1.25:0.72:0.61; S2contained rhamnosus, glucose and galactose in a molar ratio of approximately2.4:1.74:1.00.Both S1and S2could significantly stimulate splenocytes proliferation in vitro. Besides,the effects of KF5EPS on the spleen cells secreting cytokines were studies. When spleen cellswere first stimulated by LPS/ConA, adding KF5EPS for co-culture could activate Th1and Th2cells to promote the expression of cytokines. With the increase of the polysaccharideconcentration, reduced Th1, upregulated Th2. KF5EPS showed some anti-inflammatoryeffects. KF5EPS could be partly utilized by human intestine bacteria as fermentablesubstrates in fecal slurry batch cultures, and increase the levels of short-chain fatty acids(SCFA) in vitro. KF5EPS also stimulate the bifidobacteria in the fecal slurry, exhibiting theputative prebiotic properties.The intrinsic viscosity [η] of EPS S2in0.1mol/L NaNO3solution was2.339dL/gmeasured by an Ubbelohde capillary viscometer. The value of K’–K"(~0.53), obtained fromthe Huggins coefficient (K’) and Kraemer coefficient (K’’), indicated that the EPS S2moleculein sodium nitrate aqueous solution was a random coil. In the SEC-MALLS chromatogram, themolecular parameters Mw, Mn, Mzand Rgof EPS S2were approximately7.50×105,5.50×105,1.04×106Da and44.3nm, respectively. The Mark-Houwink-Sakurada exponent α was foundto be0.687, indicated that the S2molecule adopted flexible coils in sodium nitrate aqueoussolution. The size distribution of EPS S2in0.1mol/L NaNO3aqueous solution measured bydynamic light scattering indicated that the small particle species represented individualmolecules and the Rhvalue calculated was about29.4nm. Combining Rgwith Rh, the structuresensitive parameter ρ (Rg/Rh) was found to be1.51, indicating that the polysaccharidemolecules adopted a flexible random coil conformation in sodium nitrate aqueous solution.The surface topography of EPS S2in aqueous solution was observed using Atomic ForceMicroscopy (AFM). The different topographic images of EPS S2were obtained in differentconcentration of polysaccharide solutions. With the decrease of the concentration ofpolysaccharide solution, the interactions between polysaccharide molecules were weakened.The surface topography of S2was changed from the spherical lump, island-shape, entanglednetwork to one/two strand chains. At a concentration of50ng/mL, the polysaccharidemolecules formed network structures, indicated S2had branched structures. When theconcentration of S2polysaccharide was10ng/mL, the polysaccharides existed as flexiblechains, formed disordered random coils in water. This further proved that the random coilconformation of this molecule obtained from light scattering and viscometry experiments,according to the theory of dilute polymer solutions. SDS, a hydrogen bonds breaker, wasadded to disperse the entangled EPS S2in water. The extended polymer chains (fibrousmorphology) were observed, and the circular side chains were also clearly observed, whichindicated that EPS S2had branched structure. It could be speculated the diameter of singlechain of S2molecule was about0.4~0.5nm.Combining with monosaccharide composity, FT-IR spectral analysis, methylationanalysis and1D/2D NMR analysis, the possible structure of S2was as follows:...