Fermentation And Extraction Of Polysaccharide From Endophyti Fusarium HBG16 Of Salvia Miltiorrhiza And Their Biological Activity

ObjectiveFusarium HBG16 with anti-oxidation and anti-coagulant activity was isolated from the roots of Salvia miltiorrhiza Bge.Through optimizing the fermentation conditions and extraction technique,the production of intracellular and extracellular polysaccharides of Fusarium HBG16 was improved.Based on this,polysaccharides were separated and purified using modern biotechnology.Furthermore,the biological activity of polysaccharide was investigated with biochemical and molecular biological techniques.This study provided a theoretical foundation for utilization,clinical application,and industrial pr-oduction of fungal polysaccharides.MethodsThe single factor test and Box-Behnken response surface experiment were used to determine the optimal fermentation and extraction conditions for production of intracellular and extracellular polysaccharides from Fusarium HBG16.The lipids,proteins,and pigments in crude polysaccharides were removed using ultrasonic extraction with petroleum ether,Sevag method,and activated carbon adsorption,respectively.On this base,the obtained crude polysaccharide was further fractionated by DEAE-52 cellulose column chromatography.Then,the small molecule inorganic salt was separated through the dialysis bag with molecular weight cut off 8000-14000.The DPPH method was used to evaluate the antioxidant activity of intracellular and extracellular polysaccharides.The anticoagulation of polysaccharides with different concentration and the purified fractions was determined in vitro with APTT,PT,and TT as indicators.ResultsThis endophytic fungus of Salvia miltiorrhiza was identified as Fusarium sp.through analyzing morphology and ITS sequence.The optimal fermentation conditions of extracellular polysaccharides production were maltose 32.16 mg/mL,yeast extract 2.62 mg/mL,inoculum 6.19%,and pH 6.02.And polysaccharide production is 0.95 mg/mL under this condition.The best conditions for exopolysaccharides extraction were alcohol precipitation time 20.04 h,ethanol concentration 91.59%,filtrate pH 6.09,and the yield of extracellular polysaccharide reached 1.29 mg/mL.In addition,Fusarium HBG16 produced intracellular polysaccharide with the optimal conditions 33.88 mg/mL of corn flour,2.55 mg/mL of sodium nitrate,and pH 6.09.Under this optimized condition,the production of intracellular polysaccharide was 12.92 mg/g DCW.The optimum extraction conditions for intracellular polysaccharides of Fusarium HBG16 were extraction time 2.90 h,temperature 90.4 °C,and material-to-liquid ratio 21.48: 1.Moreover,the lipid contents in extracellular and intracellular polysaccharides were 3.41% and 2.87%,respectively.The yields of extracellular and intracellular polysaccharide were 92.97% and 94.54%.After deproteinizing,the retention rate of extracellular and intracellular polysaccharides was 56.98% and 48.11%,respectively.And five components were obtained through separating and purifing by DEAE-52 cellulose column chromate-graphy.The IC50 values of DT-1 and DT-2 were 0.38 mg/mL and 0.72 mg/mL by DPPH method.The APTT could be significantly prolonged by these two polysaccharides through anticoagulant experiments in vitro.ConclusionsThe metabolites of Salvia miltiorrhiza endophyte HBG16 had certain antioxidant and anticoagulant activities.Through the optimization of fermentation and extraction processes,polysaccharide production was greatly improved.It provided the foundation for utilization of fungal polysaccharides and drug development.