Screening of High-yield Pullulan Strains by UV Mutagenesis and Optimization of the Fermentation Conditions

Abstract: Pullulan, an extracellular water-soluble microbial polysaccharide produced by strains of Aureobasidium pullulans, is a linear -D-glucan made mainly of maltotriose repeating units interconnected via-(1,6)-linkages. It is a kind of characteristic polysaccharide, which was first found by R.Bauer in 1938.Pullulan is a non-ionic exopolysaccharide of fungal origin and is currently exploited in the food industry due to its many unique characteristics such as good film-forming properties, oil resistant and impermeable to oxygen. Due to its non-toxic, non-immunogenic, non-mutagenic and non-carcinogenic nature there is an attempt to explore this polysaccharide for various biomedical applications including targeted drug and gene delivery and surface modification recently.UV mutagenesis was used to obtain a mutant strain of Aureobasidium pullulans which could produce high yield of pullulan and have low secretion of black pigment, then the composition of medium and fermentation conditions were optimized based on the yield of pullulan, and a fermentation technique of pullulan was built. At last, the characteristic of pullulan capsules was studied.A mutant strain of high yield of pullulan and low secretion of pigment was obtained and the method of RSM was used to optimize the medium composition of pullulan fermentation in the shaking flask, the content of Sucrose, NaCl and (NH_4)_2 SO_4 which had important effects on pullulan production were studied especially. After optimization, the medium composition was as follows: 10.5 % sucrose, 0.2 % yeast extract, 0. 098 % NaCl, 0.02 % MgSO_4·7H_2O, 0.63 % K_2HPO_4·3H_2O, 0.06 % (NH_4)_2 SO_4 and initial pH value 6.5.10L fermentor was used to optimize the condition of fermentation, the suitable condition was as follows: the initial pH was 6.5 and temperature was maintained at 28℃; the initial agitation speed was 200r/min and the aeration rate is 1.5wm, after about 24h the agitation speed and aeration rate should be adjusted according to the dissolved oxygen which should be above 30%. After 60h's fermentation, the yield of plluluan was about 67g/L.Then the downstream processing was studied, a extraction procedure was as follows: plate and frame filter was used to remove bacteria from the fermentation broth, and then separate pullulan solutions of different molecular weights by ultra filtration, at last ethanol was used to extract the polysaccharide.The characteristic of disintegration time limited and the capacity of oxygen resistance of pullulan capsule were also studied. After temperature accelerated test, the disintegration time limited of pullulan capsule was accord with Chinese Pharmacopoeia. The capacity of pullulan capsule of oxygen resistance was studied by comparison the content of vitamin C. After accelerated test, the result showed that the capacity of oxygen resistance of pullulan capsule was better than gelatin capsule…
Key words: pullulan; UV mutagenesis; fermentation; downstream extraction technology; pullulan capsule

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