1. INTRODUCTION
Pullulan polysaccharide, which produced from aureobasidium pullulans, is a kind of extracellular and linear homopolysaccharide of glucose. It is a α-(1-6) linked polymer of maltotriose subunits [1]. The regular alternation of α-(1-4) and α-(1-6) linkages (in the ratio of 2:1) results in some distinctive properties, such as structure flexibility, enhanced water-solubility, and excellent filmand fiber forming properties [2]. Pullulan is also edible and biodegradable. All these characteristics make it a very useful material. Pullulan is usually used as lowcalorie food additives, oxygen impermeable film for packaging, adhesives, and thickening and extending agents. Pullulan has been in commercial production for about 30 years. It is currently sold at $25/kg. This is still too high for many of its potential applications. Recently, pullulan has been getting renewed attention as an excellent material for pharmaceutical and biomedical applications [1,3]. The high price of pullulan is attributed mainly to the difficulties in its production through fermentation such as high broth viscosity, low product yield, pigmentation, and product degradation [4]. Many factors can influence the pullulan production, including strain [5], temperature [6], pH [7], dissolved oxygen (DO) level (influenced by agitation, aeration) [8], substrate [9,10], trace element [11], fermentation model [12], mixing device [13], and so on. Lots of scientists and enterprisers in the world focus on it, but its synthetic mechanism of polysaccharide is still unknown, and there are different reports about its industrial fermentation. In the process of fermenting, aureobasidium pullulans can produce various pigments, such as black, brown, and green pigments, etc., which pollute the EPS. On the other hand, more steps of decolouration and filter of active carbon can lead to the fall of yield and the lower ability for colorless strains to produce EPS. So the report about colorless strains applied to industrial production hasn’t been reported up to now.
In this investigation, we focus on flask fermentation, in the course of which the following aspects should be stressed. 1) By adding to surface active reagent, such as tween-80, we try to influence the permeation character of cell membrane, which can promote the exchange of inside substances of cell membrane with the outside ones, and improve the profitability of mediums. Thus, there is a prominent impact on the improvement of the decentralization degree by adding it, which can solve the problem of mycelium mass; as to the application of Ca2+, we replace CaCl2 with CaCO3, which has a very low dissolvability, to release Ca2+ and balance pH of fermenting medium. 2) In order to get the best formula for sucrose, K2HPO4, (NH4)2SO4, yeast extract and CaCO3, a five-factor and four orthogonal test was designed, and a high conversion rate of 70% and pullulan yield 35.0 g/L were obtained from the medium. 3) At last, we study the magnifying for cultivation by adopting Biostate 5 L according to the optimum formula and conditions (such as initial pH, volume, inoculum concentration etc. were studied already) and find the optimum aerate speed is 1.0 vvm, and the rate of stir is 500 rpm and culture temperature is 28˚C ± 1.5˚C, so a high conversion rate of 60% was obtained from the light color medium.
2. MATERIALS AND METHODS
2.1. Microorganisms and Culture Conditions
Strain W518 (donated by Yantai University), was kept for several years. By rejuvenating and sifting, the strain 714 was gotten which conversion and pigment is suitable to following research. The inoculums were incubated at 29 C and 200 rpm for 36 h. The resulting cultures were used to inoculate the production medium at a level of 5% (v/v). The production medium used had the following composition (g/L): yeast extract 1.5, K2HPO4 2.5, MgSO4·7H2O 0.1, NaCl 0.05. In addition, 50 g/L sucrose was used as carbon and energy source. 0.3 g/L (NH4)2SO4 was used as nitrogen source. Initial pH was adjusted to 6.5 before autoclaving. Medium volume (25 mL in 300 mL cone flasks) was used to mimic aeration condition. All the flasks were incubated in an incubator-shaker (HZQ-F160) operating at 28 C and 180 rpm for 5 days. All chemicals are analytical grade and purchased from domestic Ltd.
According to the optimum formula, and aerate speed is 1.0 vvm, initial pH is 6.5 to ferment in biostate@5L fermenter.
2.2. Analytical Techniques
Samples were taken from Biostate@5L every 6 h and conserved in refrigerator and then analysised together at the ending of fermentation. We detect the content of EPS measured by the method of anthranone development process, the viscosity of fermentation medium detected by viscosity analyzer and the biomass net (bake oven use). The samples was diluted with suitable volume of distilled de-ionized (DD) water before centrifugation at 1000 rpm for 15 min. The supernatants were conserved. The cell pellets were washed with suitable DD water, and centrifuged again. The second supernatants were combined with the first ones for EPS precipitation by adding 1 volume of anhydrous ethanol. The cells were then suspended and transferred to pre-dried and preweighed aluminum dishes, and dried in an oven at 105 C overnight before weighing again.
2.3. Experimental Design
2.3.1. Replace CaCl2 with CaCO3
As to pH of medium is essential and autoclaving, we replace CaCl2 with CaCO3. CaCO3 and sucrose is individually added into the medium after sterilization. We also study the impact of NaCO3 and CaCO3 to fermentation individually.
2.3.2. The Addition of Tween-80
By adding to surface active reagent, to influence the permeation character of cell membrane, and improve the profitability of mediums. We added variable dosages of tween-80 into fermentation medium to study its impact.
2.3.3. A Five-Factors and Four-Level Orthogonal Test Designed
The five factors were examined at four levels as listed in Table 1. The levels of the factors were chosen based on information from literature and preliminary experiments. The experiment was carried out using a 16-run orthogonal array, which is derived by using of the free software design.
3. RESULTS AND DISCUSSION
3.1. Pigment and EPS Production
In Figure 1 CaCO3 is better than CaCl2 and the reason is insoluble and counteraction of CaCO3, not 
ion. From figure 2, we can find the use level of tween-80 is not important at all, but more mycelium mass and more pigment can appear without it. So we can adopt suitable amount in the following fermentation.
Table 1. Five-factors and four-levels of orthogonal tests.
Figure 1. Effect on yield of polysaccharide of different additions.
Figure 2. Effect on yield of polysaccharide of different concentration of tween-80.
The effect of five factors to EPS has showed in figure 3, we can know all of substrates has optimum value except of (NH4)2SO4.
Figure 4 is the result of A. pullulans fermentation in 5 L auto-controlling fermenter, we explore the yield of EPS, the amount of residues sugar, the viscosity of medium and biomass of mycelium.