Study On The Diversity Of Extracellular Polysaccharide Producing Bacteria Isolated From The Nearshore Of Beibu Gulf And Its Bioactivities

Marine bacteria are the most widely distributed organisms in the marine environment,and many strains have ability to produce Extracellular Polysaccharide(EPS).There are still many marine bacteria which have not been discovered,and these bacteria have become the potential producers of EPS.The production of EPS is closely related to the living environment of bacteria.The EPS produced by bacteria from different environments have great differences in structural composition,which makes the specificity of bioactivities of the EPS,such as antioxidant activity,antitumor activity,and immune regulation.In order to lay a foundation for the comprehensive utilization of bacterial resources in the Beibu Gulf,the purpose of this study is to clarify the marine bacterial species and biological diversity of the EPS produced in the northwestern Guangxi Bay(Qinzhou,Beihai,Fangchenggang).Firstly,marine bacterial enrichment and cultivation from the seawater,sand and mangrove soil samples collected from the coast of Guangxi Beibu Gulf was carried out with LB medium.Sticky colonies were selected as potential EPS producing strains and were further isolated and purified.After fermentation culture,the EPS-producing strain was further determined by alcohol precipitation and phenol sulfuric acid method.Finally,the EPS-secreting strain was further identified by 16 S rDNA.As a result,205 strains of EPS-producing bacteria were screened,and further 16 S rDNA identification of the 28 representative strains showed that the isolates belonged to 2 families,4 orders,4 families,6genera and 15 species of bacteria.The two classes are Gammaproteobacteria and Bacilli,each of them accounting for 21.43%and 78.57% of the total identified strains.The six genus are Acinetobacter,Vibrio,Photobacterium,Proteus,Bacillus and Staphylococcu.There are 20 strains belong to Bacillus which accountingfor the 71.43% of the total identification number.There are 2 strains each belong to Vibrio,Photobacterium and Staphylococcu,and accounted for7.14% of the total number of identification,respectively.There is only 1strain belong to Acinetobacter and Proteus,and each accounted for3.57% of the total number of identifications,respectively.In addition,the mainly EPS-producing strains are Bacillus which included 8 species,followed by Vibrio and Staphylococcu genus(2 species each),and the lowest number of the genus are Acinetobacter,Proteus and Photobacterium which only has 1 species.Secondly,the EPS fermentation conditions of some isolated strains,Vibrio B1208,Proteus 528103 and Acinetobacter Q24,were optimized.The optimum fermentation conditions for Vibrio B1208 were: 9.5 g/L lactose,5 g/L yeast powder,0.4 g/L calcium chloride,pH 7.8-8.0,fermentation time 60 h,and the optimized yield was 3.26 mg/mL,which was 2.75 times higher than the initial one.The culture condition of Proteus 528103 were : soluble starch 4.5 g/L,sodium nitrate 20 g/L,calcium chloride 3 g/L,pH 5.2-5.4,fermentation time 60 h,optimized yield reached 2.919 mg/mL,which was 11.14 times higher than the initial one.Acinetobacter genus Q24 were : sucrose 20 g/L,sodium nitrate 7 g/L,calcium chloride 0.2 g/L,temperature 35 ° C,pH 7.0,fermentation time72 h,the optimized yield reached 0.838 mg / mL,which was 3.10 times higher than the initial one.Finally,some biological activities of EPS of the representative strains of different species were tested.The antioxidant activities of EPS of different strains were evaluated by the ability to scavenge DPPH.As a result,the clearance rate of DPPH detected by EPS was varied from9.43% to 71.10%,and the DPPH clearance rates of Vibrio 528102 and B1208 were 71.10% and 65.00%,respectively.The clearance rate of DPPH by Bacillus altitudinis and Bacillus subtilis in Bacillus is between55% and 65%;The EPS clearance rate of Bacillus cereus and Bacillus halosaccharovorans is between 25% and 40%.The EPS of Photobacterium B1211 and Proteus 528103 were 38.07% and 45.15%,respectively.The EPS clearance rate of Staphylococcus 528091 was less than 10%.In the detection of antiviral activity,the EPS concentration ofVibrio isolate BHc-09 is 25 mg/mL,which is safe to chicken embryos and can effectively reduce the IBDV viral load of chicken embryo chorioallantoic membrane,however,BHc-09 EPS couldn't directly inactivate the virus.All in all,a total of 15 kinds of EPS-producing marine bacteria of 2classes,4 orders,4 families,6 genera were successfully determined,and the optimal EPS production conditions of Vibrio B1208,Acinetobacter genus Q24 and Proteus 528103 were obtained.The EPS of some representative strains of different species showed different antioxidant activities,and the isolate BHc-09 EPS exhibited anti-IBDV virus activity.The results suggested that there were species diversity of EPS producing bacteria in the coastal area of Guangxi Beibu Gulf.The EPS of some strains showed the antioxidant activity and reduced viral replication activity,which laid a material foundation for the further development and utilization of Beibu Gulf bacteria EPS.