Screening Of Exopolysaccharides-producing Strains From Antarctic Microorganisms And Research On The Application Of Exopolysaccharides

Exopolysaccharide(EPS)is a kind of bio-macromolecule that composed of a variety of monosaccharides and secreted by microorganisms to the extracellular environment during the growth process.Exopolysaccharides produced by Antarctic microorganism,have many different biological activities and novel chemical structures,making it to be of a broad application prospects in medicine,food processing,chemical engineering and other fields.In this study,a total of 132 strains of different EPS-producing strains were screened from these Antarctic strains by phenol sulfuric acid method and alcohol precipitation method.Among them,(A)7 strains yields less than 20 mg/L EPS occuping 5.3%of the EPS-produced strains;(B)25 strains of 18.94%yields 20-60 mg/L EPS;(C)79 strains yields EPS at 60-100 mg/L,that take up 59.85%of the screening bacteria;(D)and there were 21 strains with EPS yield over 100 mg/L,take up 15.91%of tested samples.9 Antarctic strains screened from different sediments with high exopolysaccharides-producing ability,named as W38-8,W29-21,W32-2,W13-13,W38-16,W32-3,W14-6,W18-3 and W20-15,were sequenced and respectively analyzed to be Loktanella salsilacus,Rheinheimera soli,Shewanella frigidimarina,Planococcus halocryophilus,Marinobacter sp.,Shewanella sp.,Pseudomonas stutzeri,Planococcus halocryophilus,Flavobacterium sp.by 16S rDNA gene sequences.According to the screening results,a W32-2 strain(Shewanella frigidimarina)with EPS yield of 109.56 mg/L was targeted for further studies on the physical and chemical properties and biological activity,nonspecific immune activity to Red Tilapia,and the development of fermentation process for EPS manufacturing.The results showed that the protein content of W32-2 was only 0.5%,the content of uronic acid of W32-2 was 8.46%,and the sulfuric acid content of EPS was 83.35%,which was proved to be acidic polysaccharide.In vitro antioxidant activity assay demonstrated that the EPS from W32-2 possessed significant scavenging activities on superoxide and DPPH radical.The antioxidant capacity was the strongest with the mass concentration 0.5 mg/mL,and the scavenging ability of DPPH was 60.43%,the scavenging ability of superoxiderate of was 70.03%.The experimental conditions for the non-specific activity of EPS produced by strain W32-2 were:a)1×PBS with EPS concentration of 3 mg/mL,b)EPS concentration of 5 mg/mL,c)pH7.4 PBS,the injection was 0.1mL.Enzyme activities were measured at 3 d,5 d,7 d,14 d,21 d and 28 d after injection.The results showed that the AKP activity of experimental group was 2.29 times as many as that of the control and reached the maximum value at day 3.The ACP activity was increased by 51.18%at day 7.At day 7,the activity of SOD was the highest,but at day 14,the experimental group increased 33.25%.At day 3,the CAT activity of the experimental group increased 57.64%.The results indicated that the EPS produced by strain W32-2 has the potential to be used as immunological additive.The optimal EPS-producing conditions of strains W32-2 were determined by single factor experiments.The results showed that:the optimal carbon and nitrogen sources for the production of EPS were determined to be fructose and yeast extract,and their optimal concentrations were 4%and 2%,respectively.The optimal pH,temperature and loading volume as well as inoculum density were 9,10?,150/500 mL and 3%,respectivily.The above results provide the research experiences on the bio-diversity of EPS-producing microorganisms in Antarctica,by screening the EPS-producing bacteria and studying on the physical and chemical properties and biological activity of the W32-2-generated EPS.This project also give a theoretical and practical direction for the application of EPS as nonspecific immune reagent on aquaculture,and the fermentation process of EPS manufacturing at bench-scale test.