Structure Analysis And Biological Activities Evaluation Of Exopolysaccharide Produced By P. Fluorescens

Polysaccharides have increasing market demand in recent years because of theirbiological activities. Bacterial extracellular polysaccharides (EPS) come from liquidfermentation of bacteria, the advantages of bacterial fermentation are short incubationperiod, high yield, stable quality which present a kind of high efficiency, energysaving industrial producing method of polysaccharides. The culture conditions ofbacterial strains differ from each other, and the yields of extracellular polysaccharidesof directional screened strains could be improved to different levels by optimizing theculture conditions, these extracellular polysaccharides usually have potentialbiological activities. The exopolysaccharide producing strain in this paper wasisolated from beer barley and preserved in our lab, which was found with relativelystable yield and character. The liquid fermentation culture conditions were optimizedusing the extracellular polysaccharide production as an index. The methods of highperformance gel permeation chromatography (HPGPC), gas chromatography (GC),enzyme hydrolysis, liquid mass spectrometry (LC-MS), and nuclear magneticresonance (NMR) were employed to analyze the structure of the extracellularpolysaccharide. Preliminary evaluation experiments of in vitro moisture retention andabsorption abilities as well as antioxidant activities of the EPS were conducted. Themain research contents and results are as follows:1. The exopolysaccharide producing strain was identified as Pseudomonasfluorescens according to the morphological characteristics and16SrDNA sequenceanalysis, which was named Pseudomonas fluorescens PGM37, and currentlypreserved in China Center for Type Culture Collection with a serial number CCTCCNO. M2012183.2. The Fermentation conditions of EPS were optimized using statistical methodssingle factor experiments and response surface methodology (RSM) design. As aresult, the optimum cultivation conditions initial pH value, medium volume, inoculum size, temperature and rotate speed were7.5,100mL/250mL,5%,28℃and180rpmrespectively. The optimized media: sucrose36.2g/L, yeast extract3.3g/L, sodiumchloride1.1g/L and calcium chloride0.2g/L. The maximum yield of EPS was10.1163g/L after36h under these conditions. The strain fermentation curve showedthe highest yield11.37g/L appeared at48h.3. The polysaccharide extraction technology was optimized, fermentation liquorpH was adjusted to8,2.5times of ethanol was used to precipitate polysaccharide,then the precipitate was dissolved with deionized water, precipitate polysaccharideagain with ethanol, then freeze dried to polysaccharide samples. Pure polysaccharidewas obtained using Sephacryl S-300HR gel filtration chromatography, the molecularweight of the EPS was determined to be367.6kDa through high gel permeationchromatography method.4. Gas Chromatography analysis revealed that the polymer was made up ofmannose and glucose in the ratio of1:1. Infrared spectroscopy and1D NMR showedthat the polysaccharide was-D-pyranoid configuration and contained no othersubstituents. Graded by different multiples of ethanol after specific degradation byenzyme, then detected by ESI-LC-MS, the repeating unit of EPS structure wasdetermined to be-D-Glcp-(1,4)--D-Manp-(1,4)--D-Glcp-(1,4)--D-Manpconsidering the results of1D NMR,2D NMR and infrared spectroscopy.5. In vitro moisture retention and absorption abilities as well as antioxidantactivities of the EPS were studied. The moisture retention ability of the EPS wasfound to be superior to glycerol and only a little inferior to hyaluronic acid (HA). Itcould reach a dynamic equilibrium moisture content in a certain concentration range.The effects of EPS to remove hydroxyl free radical, DPPH free radical and resistanceto lipid peroxidation were significant. The results showed a dose-responserelationship between EPS scavenging capacity and EPS concentration. The EC50ofscavenging free radicals were7.44mg/mL,16.76mg/mL,17.18mg/mL, respectively.P. fluorescens PGM37EPS has significant moisture retention and absorption abilitiesas well as antioxidant activities, which present potential application value incosmetics and clinical medicine fields. The structure of P. fluorescens PGM37extracellular polysaccharide has greatsimilarities to konjac glucan-mannan (KGM), a kind of plant polysaccharide, whichhas been confirmed by many scholars both at home and abroad to have good moistureabsorption and retention capacities, antioxidation as well as antitumor activities.Exploring experiments proved that the P. fluorescens PGM37extracellularpolysaccharide also has certain biological activities such as antioxidation, moistureabsorption and retention capacities. There was no microorganism producing this typeof glucomannan reported, currently glucomannan could only be obtained from plantkonjac. P. fluorescens PGM37is suitable for large-scale industrial production whichhas great development prospects because of simple medium composition, stableproduction, low cost and so on. The study of this paper is to provide theoreticalfoundation to the industrial production, the relationship between structure andactivities as well as potential development and application for microbialglucomannan.