| Fuco Pol is a water-soluble polysaccharide composed of L-fucose and other monosaccharide residues,as well as acetyl groups and pyruvate groups.Microbial-derived Fuco Pol has the characteristics of high yield and easy industrial production.In the previous study,a Kosakonia strain was isolated and identified as Kosakonia sp.CCTCC M2018092.In this study,the capsular EPS produced by Kosakonia sp were partial hydrolysis by the acid,high purity of EPS were isolated and extracted by using modern separation and purification techniques such as ceramic membrane filtration,ultrafiltration membrane filtration,dialysis and ion exchange chromatography.The composition of monomer sugars and the contents of uronic acid and pyruvate in polysaccharides were analyzed by High Performance Liquid Chromatography(HPLC)and Gas Chromatography-Mass Spectrometry(GC-MS).At the same time,the main structure of EPS was identified by Nuclear Magnetic Resonance Spectroscopy and Infrared Spectroscopy.Based on the reducibility and stability of EPS,the films containing silver nanoparticles were prepared.Their antibacterial activities were also studied to explore their potential as a new antibacterial material.The main research contents and results are as follows:1.Fermentation preparation,extraction and purification process of the fucose-rich bacterial exopolysaccharide.The fermentation broth containing EPS was produced by the method of fluid feeding and fermentation in a 50 L fermenter.After purification,high purity extracellular polysaccharide(EPS)was obtained.The polysaccharide was partially hydrolyzed by sulfuric acid and then filtered by ceramic membrane and ultrafiltration membrane.After deproteinization by Sevage method and lyophilization,Partially acid hydrolyzed EPS(AH-EPS)were obtained.Studies have shown that partial acid hydrolysis extraction process is more suitable for industrial large-scale extraction of fucose-rich exopolysaccharide.Detected by Gel Permeation Chrommatograph(GPC),EPS had a molecular weight of 3.65 × 105 Da,while AH-EPS had a molecular weight of 3.47 × 104 Da2.Determination of constituent monomer components in AH-EPS.High-performance liquid chromatography(HPLC)and gas chromatography-mass spectrometry(GC-MS)were used to analyze and identify the structure of AH-EPS.After AH-EPS hydrolyzed completely by TFA,use High Performance Liquid Chromatography(HPLC)and separate them through Xtimate(Welch)Sugar-H column.And then detect it by differantial detector.Determine the monomer composition by comparing the retention time with the retention time of each monomer standard.The results showed that the monomer sugar including glucose,galactose and fucose.Using High Performance Liquid Chromatography(HPLC)and separate complete acid hydrolysate through Wonda Sil C18 column or Xtimate(Welch)Sugar-H column.Then detect it by ultraviolet detector and analyze it quantitatively through external standard method.The results showed that the content of pyruvate in AH-EPS was wt%=6.7%.Quantification of the components in AH-EPS was determined by GC-MS.After AH-EPS is completely acid-hydrolyzed and dried with nitrogen.The hydrolysate is derivatized with ethanethiol-trifluoroacetic acid and acetic anhydride-pyridine systems,using xylose as an internal standard,and quantitative analysis by GC-MS.The results show that AH-EPS is composed of fucose,glucose,galactose,and glucuronic acid units,and its molar ratio is 2.03: 1.00: 1.18: 0.64.The content of uronic acid determined by this method was consistent with that determined by m-hydroxybiphenyl colorimetry(wt%=15.33%)3.Structural characterization of AH-EPS.After the monomer components of AH-EPS were identified,the structure composition of polysaccharides was determined by the combination of periodate oxidation,Smith degradation,methylation analysis and nuclear magnetic resonance analysis.The results of the oxidation of periodate and the degradation by Smith showed that glucose,galactose and fucose had 1→3 links,and 4-substituted glycosyl group and 1,4-substituted fucose residue are existed in the polysaccharide.From the methylation analysis,it was found that AH-EPS was mainly composed of 1,4-linked fucose,1,3,4-linked fucose,1,3-linked glucose,1,3-linked galactose and terminal galactose,with a molar ratio of 1:1.02:1.63:0.33:0.68.The AH-EPS chain was composed of the only branch in fucose residue C3.Nuclear magnetic resonance(NMR) analysis shows:a →3)-β-D-Glcp-(1→4)-α-L-Fucp-(1→4)-α-L-Fucp-(1→ was determined to be the repeating unit of the backbone.The chain of AH-EPS composed of a sole branch point at C3 of fucose residue: α-D-Galp-(1→3)-α-D-Glcp A-(1→3)-α-D-Galp-(1→.The pyruvate group is connected to Galp at the end of the branch,and Galp’s O-4 and O-6 form a six-membered cyclic ketal with the pyruvate group.4.Preparation and antibacterial testing of EPS/Nano silver filmAH-EPS contains abundant hydroxyl groups that could stabilize and reduce the Ag+ to Ag0 with ultraviolet light.The ratio of reducing and stabilizing agents for synthesis have significant effects on the morphology and size distribution of silver nanoparticles.Silver nanoparticles were prepared with different concentrations of AH-EPS.It was found that when the concentration of AH-EPS was 0.05 mg/m L,the UV-vis spectrum of the solution showed the strongest absorption peak of silver nanoparticles at 423 nm.Therefore,silver nanoparticles were prepared with 0.05 mg/m L AH-EPS for further characterization and application.TEM images confirmed that the silver nanoparticles(AH-EPS @ Ag NPs)prepared by AH-EPS were uniformly spherical,with an average diameter of about 20 nm.Using the high molecular weight and film-forming properties of the original extracellular polysaccharide EPS,AH-EPS @ Ag NPs were loaded into EPS films to prepare EPS / nano-silver films.The antibacterial activity of EPS / nano silver film against Staphylococcus aureus was analyzed by disk diffusion method.A clear inhibition zone appeared around the EPS / nano-silver film,indicating that AH-EPS @ Ag NPs can effectively inhibit the growth of Staphylococcus aureus.The diameters of the inhibition zones of the AH-EPS @ Ag NP EPS / nano-silver films containing 0,0.22%,0.44% and 0.89%(mass fractions)of Staphylococcus aureus growth were 0 mm,12.3 mm,14.1 mm and 16.5 mm,respectively.The antibacterial ability shows a dose dependence. |
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