High Production Of Pullulan Fermentation Process Optimization And Structural Analysis

Pulullan is an extracellular water-soluble polysaccharide,similar to dextran and xanthan gum,which is widely used in fields like food,cosmetics,medicine and industry due to its non-toxic,good solubility and strong stability.Now Aureobasidium pullulans is the main production strain of pullulan,but it can produce a large amount of melanin at the same time.So how to reduce or not produce melanin and increase pullulan production is the emphasis and difficulty of the study.With Aureobasidium pullulans as the original strain,this paper tries to optimize the fermentation process of pullulan and preliminarily reveal synthesis mechanism of Aureobasidium pullulans melanin,so as to make the strain not produce melanin on the basis of significantly increasing pullulan production.Meanwhile,the structure of pullulan is analyzed and the main research results are as follows:Pullulan production is significantly increased through optimizing the fermentation process of Aureobasidium pullulans.Optimized pullulan synthetic medium（g/L）is determined by using methods like response surface optimization:sucrose 109.4,yeast extract 1.465,NaCl 1,KH₂PO₄ 2.0,K₂HPO₄ 4.0,MgSO_4·7H₂O 0.2,（NH₄）₂SO₄ 0.603,Tween80 0.5mL/L.At this moment,the maximum production of pullulan is up to 31.39g/L.Further study is made on the key factors influencing the synthesis of pullulan like dissolved oxygen and temperature.Optimized culture condition:amount of bottled liquid50/250 mL,shake bed speed 220 r/min,temperature 28℃,combined with the optimized fermentation medium,the pullulan production reaches 32.84 g/L,increased by 26.31%compared with the production before optimization.Moreover,Aureobasidium pullulans produce nearly no melanin under the above-mentioned optimized culture condition.Melanin synthesis mechanism is preliminarily revealed based on the research of melanin synthesis pathway.There are mainly three melanin synthetic pathways as L-dopa melanin,DHN melanin and HGA melanin.Tricyclazole is a key enzyme inhibitor in DHN melanin pathway,Cu²⁺is a key enzyme activator in L-dopa melanin pathway,and anisaldehyde is a key enzyme inhibitor in L-dopa melanin pathway.Add individually or in combination a small amount of tricyclazole,Cu²⁺and anisaldehyde into Aureobasidium pullulans culture medium to verify the condition of melanin synthesis.Preliminary research results reveal that the synthetic pathway of Aureobasidium pullulans melanin is L-dopa melanin pathway.Sterling pullulan is obtained by methods like column chromatography to determine the structure of pullulan by using infrared spectrum and nuclear magnetic resonance（NMR）spectroscopy.Firstly,add anhydrous ethanol into fermented liquid to get crude polysaccharide which should be carried on the drying process.Secondly,remove impurities protein by using alkaline protease-Sevage combination method.Conduct further separation and purification to obtain sterling polysaccharides by using method of Sephadex A-25 column chromatography elution.The polysaccharide is determined as uniform polysaccharide without impurities by scanning with ultraviolet spectrum.Finally,analyze the polysaccharides through infrared spectrum and nuclear magnetic resonance（NMR）spectroscopy,and the result shows that the polysaccharide has the same structure with the pullulan standard,a kind of straight chain polysaccharide,which is firstly connected into malt trisaccharide byα-1,4 glycosidic bond of glucose,then connected into pullulan byα-1,6 glycosidic bond of malt trisaccharide.The above study has established the optimized synthesis process of pullulan produced by Aureobasidium pullulans,significantly increasing the production of pullulan with nearly no melanin produced,and providing better technical supports for the industrial production of pullulan.