Breeding Of Aureobasidium Pullulans AP-35 And Preliminary Study On The Fermentation Process Of Pullulan From Xylose

The xylose content of cellulose is very considerable.It is very important to convert xylose into high value-added products.Pullulan has non-toxic and good film-forming properties,is widely used in food and medicine industry.Aureobasidium pullulans is capable of producing a high-value exopolysaccharide pullulan from xylose,In this study,a dual wavelength method was established for simultaneous determentation of the amount of polysaccharide and xylose in fermentation broth.A NTG mutant strain AP-35 which can producing high content of pullulan from xylose was selected.Then we constructed fermentation kinetics model and the metabolic flux model.The main experimental results are as follows:Phloroglucinol-sulfuric acid was used as coloring agent.The wavelength of 444 nm and 405 nm,420 nm and 465 nm were selected as the measuring wavelength for xylose and Pullulan respectively.The color reagent solution was prepared by dissolving 2 grams of phloroglucinol in 100mL anhydrous alcohol,followed by adding 100mL sulfuric acid.The optimal determination conditions are 1mL substrates with 5 mL coloring agent,boiling water bath for 10 min,cooling to room temperature,add 10 mL of distilled water,then mixed evenly.Results show that the concentration of glucose and xylose in 10?g/mL-300?g/mL obey beer's law.Optimal water bath time is 10min.The repetitive experiment indicated that the RSD%of Pullulan and xylose was 1.56%and 2.42%respectively.Recovery rate of polysaccharide and xylose was 92.53%-100.34%and 96.95%-103.84%,relative standard deviation were 3.61%and 3.46%respectively.A NTG mutant strain AP-35 which can producing high content of pullulan from xylose was selected,resulting in a 16.55%of increase compared with AY-82.The optimized kinds and contents of nitrogen of medium were selected by single factor experiment.The optimized medium were:NH₄NO₃ of 0.42g/L,Corn steep liquor of 3.33g/L,Under the optimized conditions,resulting in a 26.51%of increase compared with the result before optimization.Aureobasidium pullulans AP-35 fermentation metabolic flux model was built.Through the analysis of the actual conditions and assumed conditions of the metabolic flux distribution,two key nodes of polysaccharide synthesis was obtained,One is Fructose-6-phosphate and the other one is pyruvic acid.DO disturbance was introduced to verify the two key nodes.Through the analysis of Fructose-6-phosphate node we found the flow used in synthesis Fructose-1,6-diphosphate more the used in synthesis of pullulan when DO was lower.Through the analysis of pyruvic acid node we found more of the flow was used in synthesis OAA when DO was lower,and then for the synthesis of malic acid.While malic acid and pullulan is two metabolites of Aureobasidium pullulans,there is competition between them.So increase the level of DO can promote the pullulan production.In addition,We can reduce their production by weaken the enzyme activities associated with Fructose-1,6-diphosphate and OAA,in order to increase the yield of polysaccharide.Logistic and Luedeking-Piret model was used to construct the strain Aureobasidium pullulans AP-35 fermentation kinetics model.The fermentation kinetics fitting equation was obtained.Verifying test experiment indicates the fitting equation can predict the parameters in the fermentating process well.