The Research Of Exopolysaccharide Produced By Pesudoalteromonas Issachenkonii HZ

With the rapid development of marine industry,marine active substances come to be known,marine bacteria and their polysaccharides have also caused more researchers' attention.In this paper,the composition of culture medium and fermentation conditions of exopolysaccharide named EPS-P? produced by Pseudoalteromonas issachenkonii HZ were optimized.Except this,we studied the separation,purification,structure identification and related functions of EPS-P?.The main results were as follow:First,the fermentation medium,such as carbon,nitrogen,sea salt and the other fermentation factors were going on the single factor experiment.Further with Plackett-Burman designed out three significant factors affecting the yield of EPS-P?,they were inoculation quantity,incubation time and shaking speed.Finally,the linear fitting equation,response surface graphics and optimum fermentation condition were obtained through Box-Behnken experiment.Eventually determined the fermentation medium composition for: 30 g/L glucose,4.5 g/L yeast extract,35 g/L sea salt;The best fermentation conditions for: 33.8 h of incubation time,inoculation quantity of 8.8%,shaking speed of 170 r/min.The average yield of EPS-P? could reach 2.603±0.022 g/L.The crude polysaccharide in the fermented liquid was extracted by water extraction and alcohol precipitation.Sevage method to remove the protein of EPS-P?,EPS-P? was detected by UV scanning,found that EPS-P? with special absorption peaks at 220 nm,and no nucleic acid and protein absorption peaks at 260 and 280 nm,then using DEAE-52 anion exchange column and Sephadex G-100 column to purify and remove impurity of EPS-P?,by high performance liquid chromatography?HPGPC?determined the number average molecular weight?Mn?and weight average molecular weight?Mw?of EPS-P? were 2.26×105 g/mol and 2.81×105 g/mol,the dispersion coefficient was 1.24,indicated the purity of EPS-P? was relatively homogenous.The purified EPS-P? was gray powder,and easily soluble in water after freeze drying.EPS was measured through the analysis of the elements,the percentage composition of C,H,O,N,S were 3.901,2.584,25.56,0.485,0.402 respectively;EPS-P? was analyzed by GC-MS after hydrolysis,the main monosaccharide composition of EPS-P? were D-xylose,arabinose,D-glucopyranose,mannose and fucose,xylose was accounted for more than 58%;by Fourier transform infrared spectroscopy?FT-IR?,NMR and methylation reaction analyzed that the glycosidic bond was ? configuration,EPS-P? was contained oxygenated methyl,methyl sugar,but almost no uronic acid and glycoprotein,suggesting that the main chain of EPS-P? was: D-xylose by ?-1,4 glycosidic linkage.Finally,we researched the function of crude EPS-P?,including its moisture absorption and resistance,oxidation resistance and adsorption of heavy metal ions.In the desiccator of 73% relative humidity,EPS-P? absorbent capacity slightly was less than the sodium hyaluronate and chitosan.In the desiccator of 73% relative humidity,EPS-P? absorbent capacity was low.In a desiccator of silica gel,EPS-P? moisturizing property were better than chitosan and sodium hyaluronate.At a humidity of 32%,the moisturizing property was slightly higher than sodium hyaluronate but slightly lower than chitosan.Antioxidant Activity of EPS-P? showed that,at a concentration of 0.5 mg/m L,the ?OH scavenging rate was 11.89%,the clearance of O2.-experiment showed,the IC50 of EPS-P? and Vc were 0.43 mg/m L and 0.258 mg/m L,the scavenging of O2-? was better.Pb²⁺ and Cu²⁺ adsorption experiments showed,the adsorption rate of EPS-P? on Pb²⁺ was greater than Cu²⁺,the maximum adsorption rates were 82.46% and 69.79% respectively.