Research On The Fermentation Technology Of Efficient Production For Low Molecular Weight Pullulan By Aureobasidium Spp.

Microbial polysaccharides are high-molecular compounds produced by microorganisms in the growth and metabolism process.Compared with plant polysaccharides,it has the advantages of shorter production cycle,not restricted by seasonal,regional pests and diseases.Simultaneously it has excellent characteristics,such as safety,nontoxicity,biodegradability and biocompatibility.It has vast potential for development prospects.Pullulan is a microbial linear-chain polysaccharide connected byα-1,6 glycosidic bonds with maltotriose as a unit.Pullulan containedα-glycosidic bonds and hydroxyl groups determine its unique properties,such as structural flexibility,good solubility and excellent barrier capacity.Therefore,it has been widely used in food,medicine,cosmetics,environmental protection and so on.At present,pullulan is mainly produced from the fermentation of wild type Aureobasidium spp,and most molecular weight of pullulan are greater than 5×10~4Da.But the batch fermentation titer is mostly less than 50 g/L,and the problem of various fermentation by-products.Therefore,it is necessary to continue screening novel low molecular weight pullulan producing strains with excellent fermentation traits and establish a fermentation process for produce pullulan for practical significance.The object of this study is GXZ-6 obtained by the laboratory’s preliminary screening.GXZ-6 identified as A.melanogenium GXZ-6,according to systematic morphological observation,physiological and biochemical characteristic,sequence analysis identification.Characterize the extracellular polysaccharide obtained by fermentation of GXZ-6 by enzymolysis with Thin-layer chromatography,Fourier transform infrared spectroscopy,Nuclear magnetic resonance spectroscopy confirm that it is pullulan.Use thermogravimetry analysis and differential scanning calorimetry,the glass transition temperature of pullulan is 126℃,the melting temperature is 218℃.The molecular weight of pullulan is 8729 Da by Gel permeation chromatography.The results show that it is a low molecular weight pullulan.Aim at GXZ-6 high-efficiency production of pullulan,single-factor and orthogonal experiments were used to optimize the fermentation medium components and shake flask fermentation conditions.The results show that both glucose and sucrose can effectively promote the production of pullulan.And the cheap inorganic nitrogen source ammonium chloride has the same effect on GXZ-6 cell growth and pullulan synthesis with the organic nitrogen of yeast extract.As a metabolic intermediate product,succinic acid can significantly increase the titer of pullulan.The optimal fermentation medium components of GXZ-6:120 g/L glucose,7 g/L ammonium chloride,0.2 g/L potassium dihydrogen phosphate,7.5 g/L succinic acid,20 g/L calcium carbonate.The optimal fermentation conditions in shake flasks with pullulan titer as an indicator,the optimum liquid volume is 50 m L in 250 m L shake flask,the optimum incubation time is 24 h,the optimum incubation level is 12%(v/v),the optimum temperature is 30℃,the optimum rotation speed 180 rpm,fermentation period 5 days.Through single factor and orthogonal experiments to optimize the fermentation medium and shake flask fermentation conditions,the titer of pullulan was increased to 76±1.15 g/L,which was an increase of 26.67%compared with basic fermentation medium.Taking the 5 L fermenter as the research level,Optimal of fermentation process:aeration volume is 0.5 vvm;rotation speed is 250 rpm;temperature is30℃;inoculum volume is 6.25%(v/v),pullulan titer can reach 96 g/L,which is45.45%higher than basic medium and culture conditions,the yield and productivity are 0.80 g/g and 0.44 g/L/h,respectively.In 5 L fermentor,the yield of pullulan produced by fermentation with sucrose as the substrate is 70 g/L,the yield is 0.5 g/g,and the production intensity is 0.37 g/L/h,which are lower than the results of fermentation with glucose as the substrate.In addition,established a fermentation kinetic model of strain growth,product synthesis and substrate consumption in a 5 L fermentor.These satisfactory models laid the foundation for the subsequent optimization of fermentation strategy.In order to improve the pullulan titer,yield and productivity,proposing a two-stage controlled of p H,aeration,rotation speed,temperature and batch fed fermentation strategy.Among the titer,yield and productivity obtained by the two-stage controlled aeration strategy are 76 g/L,0.54 g/g and 0.32 g/L/h,respectively.Compared with the pullulan titer of 66 g/L under the basic fermentation conditions,they increased by 15.15%.The titer of pullulan obtained by the two-stage controlled rotation speed,p H and temperature fermentation strategy was 42 g/L,4 g/L and 22 g/L,respectively,and lower than the titer under basic fermentation conditions.The rotation speed controlled strategy delayed the time when Plu began to synthesize a large amount of time,so that the Plu titer did not reach the peak at the end of the fermentation.The p H controlled strategy promotes the strain to enter the decline phase prematurely,which is not conducive to pullulan synthesis.Although the temperature control strategy is helpful to the accumulation of the amount of strain in the early stage,it is easy to cause the strain to consume the pullulan produced in the later stage of fermentation for secondary growth,thereby reducing the titer of pullulan.The titer,yield and productivity obtained by the fed-batch fermentation strategy were92 g/L,0.66 g/g and 0.40 g/L/h,respectively.Under the basic fermentation conditions,the titer increased by 39.39%,which laid the foundation for the industrialized batch fed fermentation of pullulan.