Screening Of A High Polysaccharose-Producing Strain Of Aureobasidium Pullulans And Analysis On Fermentation

Aureobasidium pullulans is a fungus with various cellular forms.Theβ-glucan in the polysaccharides of A.pullulans has the functions of regulating immunity,antioxidation,antitumor,anticoagulation and regulating intestinal flora.Thus far,the most widely usedβ-glucan are oatβ-glucan,barleyβ-glucan and lentinanβ-glucan.There are minimal studies onβ-glucan production by A.pullulans.Pullulan is widely used in food,preservation,packaging,cosmetics and many other fields because of its strong film forming,fiber forming,viscosity and other characteristics.The use of A.pullulans to produce polysaccharose has the unique advantages such as high yield,short cycle and easy extraction,but there are still some problems,for instance,melanin probably produces during fermentation and binds closely to the polysaccharides.Therefore,in order to solve this problem,A.pullulans ACCC 30356 was selected as starting strain,and an mutant strain with high yield and no melanin production was screened by random mutagenesis method in vitro,named A.pullulans.sp-A333.Then,the physicochemical properties and antioxidant activity of polysaccharides in vitro were studied in further.Following that,the single factor experiment was used to optimize the culture conditions.Finally,the metabolomics affecting the polysaccharide synthesis was analyzed from the metabolic level.The main results of this paper are as follows:The strain A.pullulans ACCC 30356 was treated by ultraviolet and EMS mutagenesis,and the mutant strain was identified by molecular biology after subculture.The colony morphology of the original strain and excellent strain was observed before.The colony of the original strain showed loose and black or dark green color,with dry surface and mycelium form,with a diameter of 0.6-0.8cm,and the cells were mainly in mycelial form.The target strain showed viscous and white,with a smooth and wet surface,with a diameter of 0.7-0.9cm and the cells were mainly in yeast-like.By observing the cell morphology of the target strain and identifying it by ITS molecular biology method,the result showed that the mutant strain was classified as A.pullulans strain.The antioxidant activities of polysaccharides from the original strain A.pullulans ACCC 30356 and the mutant strain A.pullulans.SP-A333 were compared.The contents of protein and uronic acid in polysaccharides were determined.The contents of mutant strain were 0.098±0.0213mg/mg and the original strain was 0.207±0.0242mg/mg.The antioxidant capacity of two kinds of polysaccharides was compared by determining the scavenging capacity and total reducing power of DPPH,ABTS and hydroxyl radical.The results showed that both exopolysaccharides had good antioxidant capacity,but there were slight differences.Among them,the mutant strain A333 had a slightly higher antioxidant activity.When the highest polysaccharide concentration was 5mg/m L,the scavenging ability of DPPH,ABTS and hydroxyl radical of the mutant strain A333 was 74.55%,97.27%and97.84%.The reducing power of the mutant strain A333 was 0.39.The scavenging ability of polysaccharides to free radicals of the original strain A356 was 68.58%,76.06%and98.50%.The reducing power of the original strain A356 was 0.21.DEAE-650m ion exchange column was used to elute the polysaccharides from the mutant.Neutral polysaccharide P0 and acidic polysaccharide P2 were found.The physicochemical properties of polysaccharides were further studied by physical and chemical methods.UV spectrum showed that the contents of nucleic acid and protein in polysaccharides were very few.The results of IR spectra showed that polysaccharide characteristic absorption peak andβ-glycoside bond vibration peak existed in all polysaccharides.GC-MS was used to analyze the monosaccharide composition of polysaccharides.Both polysaccharides are composed of glucose,which may be the framework of two polysaccharides.Furthermore,culture medium composition include carbon source,nitrogen source,inorganic salt and initial p H were optimized by single factor experiment,and the optimal culture conditions were obtained as follows:Sucrose 50.0g/L,peptone 1.0g/L,NH4Cl1.0g/L,Na Cl 1.0g/L,Mg SO4·7H₂O 0.2g/L,K₂HPO₄ 6.0g/L,Na Cl 2.0g/L,the initial p H is6.5.A 5L fermenter was used to verify the optimized culture conditions.The highest polysaccharide yield of A.pullulans.sp-A333 was 24.64mg/m L,and the substrate conversion rate was up to 71.84%.GC-MS was used to analyze the intracellular small molecule metabolites in the growth period（24h）,sugar production period（48h）and late fermentation period（120h）.Compared to A.pullulans ACCC 30356,A.pullulans.sp-A333 detected 2 up-regulated metabolites including L-arabinol and succinic acid,and 9 down-regulated metabolites including D-mannose,L-lysol,proline,aspartic acid,alanine,D-fructose,D-galactose,glycine and threonine.It affects the synthesis of polysaccharides by affecting the energy supply and the synthesis of precursor UDPG.