The Study On Compound Mutation Of Pullulan Production Strain And Fermentation Conditions

Pullulan is a kind of extracellular homopolysaccharide mostly produced by strains of Aureobasidium pullulans in saccharide culture during growth and metablism .It can be applied in many fields including pharmaceutical industry,food packaging,fruits and seafood preservation, cosmetics,tabacoo manufacturing ,seeds protection,disposal of industrial sewage etc.Pullulan is a kind of multi-function creature material with great value and prospect to develop.Original strain P53 is one of wild Aureobasidium pullulans.P53 does not accord with the requirements of pullulan industrialized production by fermentation because its polysaccharide yield is relatively low. With a view of pullulan industrialized production by fermentation ,the study breeds the strain that pullulan yield is high and fit for pullulan industrial fermentation.Meanwhile, fermentation conditions of the mutant including the optimal culture medium and culture conditions are determined. And the purification technology of pullulan on the large-scale industrial production is explored.On the base of original strain's shape and fermentation kinetic diagram,this study screen the strain by compound mutation using UV and NTG in order to increase the yield of pullulan and improve the fermentation characteristics.By two times UV and two times NTG, a mutant U₂N₂-326 is obtained which polysaccharide yield is high and melanin basicly is not produced. Genetic stability experimentation of the mutant strain U₂N₂-326 show that polysaccharide throughput is relatively stable.In course of ten subcultures,the polysaccharide yield changed little and maintained in higher level.Study on fermentation conditions of mutant strain U₂N₂-326 including culture medium and culture conditions was investigated. Finally the optimal fermentation conditions of U₂N₂-326 was confirmed: granulatedsugar7%,peptone0.4%,(NH₄)₂SO₄0.06%,KH₂PO₄0.3%,MgSO₄ ? 7H₂O0.01%,NaC10. 2%,culture temperature 30℃, initial pH7.0,seed age 36h,liquid medium volumes 80ml/500ml,inoculated volumes 5%, rotate speed 180rpm,fermentation time 5d.Under the optimal fermental conditions,a high polysaccharide yield of 43.2g/L was obtained and pigment produced little.In addition the crude polysaccharide that produced from the original strain P53 was purified using industry grade DEAE-cellulose ,composite pillar with D001-CC weakly acidic cation exchanger-sulfonic acid functionality and D296R weakly basic anion exchanger-primary secondary and tertiary amine functionality. The results testified that deprotein and depigment preferred DEAE-cellulose over D001-CC weakly acidic cation exchanger-sulfonic acid functionality and D296R weakly basic anion exchanger-primary secondary and tertiary amine functionality.Pigment produced by P53 was elementary confirmed that is anion pigment.Purified polysaccharide recycle rate was 93.7% and 97.7 using DEAE-cellulose and composite pillar.