Preparation And Application Of Shiitake Polysaccharide Nanoemulsion

In this study,shiitake polysaccharide was extracted from lentinus edodes,and a W/O shiitake polysaccharide nanoemulsion was prepared to improve its stability and biological value.Using sevage-enzymatic method to deproteinize,the soluble stability of shiitake polysaccharide was determined,and the basic formulation of shiitake polysaccharide nanoemulsion was selected by pseudoternary phase diagram.On this basis,the effects of different volume fractions of Simx(surfactant +co-surfactant)and shiitake polysaccharide solution on the physicochemical properties of shiitake polysaccharide nanoemulsion were compared to obtain the optimized formulation of nanoemulsion.The specific methods and results are as follows:(1)Shiitake polysaccharide was extracted from haw by hot water,its extraction ratio was 7.8%.By comparing effects of three different methods,such as sevage method,enzymatic method and sevage-enzymatic method,on precipitating protein of shiitake polysaccharide,the results showed that the sevage-enzymatic method had the highest protein removal rate(78.88%),which proved that the composite deproteinization method could effectively improve the effect of single method.The purity of shiitake polysaccharide deproteined by sevage-enzymatic method increased from 50.7% to 82.3%.The protein content decreased from 40.2% to 8.49%.(2)In order to determine the precipitation of polysaccharides dissolved in water,the shiitake polysaccharide solution was storage at room temperature or 4°C for 15 days,and by measuring the changes of the particle size of polysaccharide droplets after 6 days,the results showed that the shiitake polysaccharide dissolved in water was unstable,which continuously precipitated from solution with the prolongation of storage time,after 15 days storage,15%and 40% shiitake polysaccharide precipitates were observed at room temperature and 4°C,respectively.The ralative soluble saccharides respectively decreased to 85.01% and 61.22%.The particle sizes of shiitake polysaccharide dissolved in water were 415 nm,564 nm and600.7 nm,with the preservation periods of 0,3 and 6 days,respectively,indicated that shiitake polysaccharide congregated in water solution during storage.(3)To determine the basic formula for preparing W/O shiitake polysaccharide nanoemulsion,apparent transparency and pseudoternary phase diagram were used as evaluation criteria,surfactant,co-surfactant,Km value,oil phase,and the concentration of shiitake polysaccharide solution were selected as the basic influence factors on the preparation of shiitake polysaccharide nanoemulsion,investigating the areas of the pseudoternary phase diagram constructed by each factor.Finally,the basic formation was:Tween80-Span85 as composite surfactant,anhydrous ethanol as co-surfactant,IPM as oil phase,shiitake polysaccharide solution(1%)asaqueous phase,of which the HLB value of Tween80-Span85 was 6,and the ratio of Tween 80-Span85 to anhydrous ethanol was 3:1.(4)On the basis of the basic formulation,the effect of the volume fraction of Smix(surfactant+co-surfactant)and shiitake polysaccharide solution of nanoemulsion on the physicochemical properties were investigated by measuring the particle size distribution,PDI,transparency and fluidity,to determine the optimized formulation.The stability test of the shiitake polysaccharide nanoemulsion was carried out to verify the reliability of the final formula.The composition of optimized formulation was: Tween80-Span85 as composite surfactant accounted for 33.75%,anhydrous ethanol as co-surfactant accounted for 11.25%,IPM as oil phase accounted for 39%,shiitake polysaccharide solution(1%)as aqueous phase accounted for 16%,of which,the HLB value of Tween80-Span85 was 6,and the ratio of tween80-span85 to anhydrous ethanol was 3:1.(5)Determine the properties and stability of the nanoemulsion prepared by the above formula,the droplet size of nanoemulsion was 144.5 nm,the PDI was 0.128 and great rheological characteristics.Moreover,the nanoemulsion remained stable after centrifugal test,freeze-thaw cycle test and high and low temperature acceleration test,and after storing for 3months,the nanoemulsion remained transparent and clear,with no delamination,turbidity and polysaccharide precipitation.(6)By determining the in vitro inhibitory effects of shiitake polysaccharide nanoemulsion on K562 cells,the results showed that the in vitro anti-tumor activity of shiitake polysaccharide nanoemulsion was significantly higher than that of shiitake polysaccharide,and the IC50 value of shiitake polysaccharide nanoemulsion was 18 times higher than shiitake polysaccharide itself,and the inhibitory rate was significantly increased.