Abstract:Pullulan is an extracellular polysaccharide produced by Aureobasidium pullulans fermentation.Due to its high viscosity，pullulan is difficult to be separated from the fermentation medium，and the Chinese national standard has a strict requirement on the protein content of its product.Therefore，it is significant to find an effective process for bacterial separation and deproteinization.Biopolymer flocculation and protease deproteinization methods were used to optimize and screen the optimal flocculation and protease from four flocculants and six proteases.The results indicated that chitosan and alkaline protease were preferable in sterilization and deproteinization，respectively.The sterilization and deproteinization techniques were optimized by the single factor experiment and response surface test.The results showed that the optimum sterilization conditions were as follows:pH3.7，flocculation time of 20 min，flocculation temperature of 40 ℃，and chitosan addition of 0.96 g/L，which resulted in the sterilization rate of 99.19% and the polysaccharide yield of 91.3% .The optimum deproteinization conditions were as follows:pH8.6，enzymolysis time of 2.5 h，enzymolysis temperature of 45 ℃，and alkaline protease addition of 83 U/mL，which resulted in the deproteinization rate of 74.70% and the polysaccharide yield of 98.42% .With the optimized sterilization and deproteinization processes，the protein content of the product was 0.045% ，meeting the requirements of the national standard.The purity of the product reached 91.5% after traditional decolorization，ultrafiltration，concentration，and drying.