Bacterial strain. GA strain was provided by College of Life Science and Agronomy, Zhoukou Normal University.

Culture medium. Agarslantculture-medium(PDA medium) was composed of 20% boiled juice filtering of peeled potatos, 2% sucrose, 0.3% KH₂PO₄, 0.15% MgSO₄, 2% agar, natural pH. Culture medium of liquid strain was composed of 1% glucose, 2% sucrose, 1% peptone, 0.5% beef extract, 0.1% KH₂PO₄, 0.15% MgSO₄, natural pH.

Preparation of GA strain. GA strain, which was propagated on PDA medium for 5 d at 28℃, was grown for 4 d at 28℃ with the shaking speed of 100 r/min prior to inoculation, according to the user’s manual instructions. Then, the strain had been obtained and stored at 4℃ in the refrigerator for further sutdy.

Determination of mycelial biomass. Mycelium was obtained by removing supernatant of fermentation broth centrifuged at 3000 r/min for 15 min. Mycelium which was dried at 50℃ after washing with distilled water several times was weighed.

Determination of raw exopolysaccharides. The supernatant, which was decanted after the fermentation broth centrifuged, was added 3 times volume of 95% ethanol and was set aside for 24 h at 50℃. After centrifuging at 4000 r/min for 20 min, the precipitate dried to constant weight was weighed.

Optimization of fermentation conditions. Carbon source, nitrogen source, initial pH, inoculums size, fermentation temperature, fermentation time were investigated respectively to study their effect on shake flash fermentation of GA by measuring mycelial biomass and raw exopolysaccharides.

Determination of antioxidant activity of exopolysaccharides. The scavenging effect of exopolysaccharides on HRswas measured with the reaction system of fenton [9]. The scavenging effect of exopolysaccharides on SARs was measured with the methods of pyrogallol autoxidation [10].

Effect of carbon source on shake flash fermentation of GA. Fermentation broth was prepared with 1% peptone, 0.5% beef extract, 0.1%KH₂PO₄, 0.15%MgSO₄ and 3% carbon source changed as glucose, maltose, sucrose, corn powder, soluble starch. 8％ of GA strain were added and cultured at 28℃ for 5 d. Then, Mycelial biomass and raw exopolysaccharides were determined (Table 1).

It was shown in Table 1 that using corn powder as the sole carbon source, mycelial biomass and raw exopolysaccharides produced by GA were significantly higher than those of the other carbon sources produced. And corn powder with relatively low cost, wide source, was suitable for use in production. Thus the optimal carbon source was corn powder.

Effect of nitrogen source on shake flash fermentation of GA strain. Fermentation broth was prepared with 3% corn powder, 0.1%KH₂PO₄, 0.15%MgSO₄ and 1.5% nitrogen source changed as peptone, yeast extract, beef extract, bran, soy powder.. 8％ of GA strain were added and cultured at 28℃ for 5 d. Then, Mycelial biomass and raw exopolysaccharides were determined. The results were shown at Table 2.

Table 2 showed that the effect of bran and soy powder utilized by GA was significantly higher than that of yeast extract, beef extract and peptone, while the difference between the soy powder and bran was significant. Both soy powder and bran are easily available raw materials. They could be selected according to desired product fermented by GA strain in practical production. If the desired product was mycelium, soy powder was the best nitrogen source; If the desired product was exopolysaccharides, bran was the best nitrogen source.

Effect of initial pH on shake flash fermentation of GA strain. Fermentation broth was prepared with 3% corn powder, 1.5% soy powder 0.1%KH₂PO₄, 0.15%MgSO₄ and initial pH changed as 4.0、5.0、6.0、7.0、8.0. 8％of GA strain were added and cultured at 28℃ for 5 d. Then, Mycelial biomass and raw exopolysaccharides were determined (Table 3).

It was shown in Table 3 that, with the rise of pH value, the mycelial biomass and raw exopolysaccharides increased gradually. At pH 6.0, both mycelial biomass and raw exopolysaccharides reached to the peak, and then decreased correspondingly with pH value rise. Therefore, GA strain is suitable to grow in acidic environment. The most suitable pH value for GA was 6.0.

Effect of inoculum size on shake flash fermentation of GA strain. Fermentation broth was prepared with 3% corn powder, 1.5% soy powder 0.1%KH₂PO₄, 0.15%MgSO₄ and initial pH6.0. Inoculum size of GA strain changed as 4％, 6％, 8％, 10％and 12％were added and cultured at 28℃ for 5 d. Then, Mycelial biomass and raw exopolysaccharides were determined（Table 4）.

Table 4 showed that when the inoculums size was 8% or 10%, mycelial biomass fermented by GA strain was significantly higher than the other. While the difference between 8% and 10% was not significant. When the inoculum size was 6% or 8%, the content of raw exopolysaccharides fermented by GA strain was significantly higher than the other. While the difference between 6% and 8% was not significant. All things considered, the suitable inoculum size was 8%.

Effect of fermentation time on shake flash fermentation of GA strain. Fermentation broth was prepared with 3% corn powder, 1.5% soy powder 0.1%KH₂PO₄, 0.15%MgSO₄ and initial pH6.0. 8% of GA strain were added and cultured at 28℃, and fermentation time was changed as 1 d, 3 d, 5 d, 7 d and 9 d. Then, Mycelial biomass and raw exopolysaccharides were determined. The results were shown at Table 5.

Table 5 showed that the longer the fermentation time, the higher was the mycelial biomass. The mycelial biomass increased to a peak value at 5 d, but then did not changed with the extension of time, which meant the growth curve of GA strain reached the stationary phase after fermenting for 5 d; The content of raw exopolysaccharides increased significantly with the increase of fermentation time. The content increased to a peak value at 7 d, but then lowered down significantly with the extension of time. The time taken to reach the top amount of mycelial biomass was not consistent with the time taken to reach the top amount of exopolysaccharides. Therefore, the growth of mycelia and secretion of exopolysaccharides was not synchronized, and the secretion of exopolysaccharides lagged some time. All things considered, the suitable fermentation time was 7 d.

Effect of fermentation temperature on shake flash fermentation of GA strain. Fermentation broth was prepared with 3% corn powder, 1.5% soy powder 0.1%KH₂PO₄, 0.15%MgSO₄ and initial pH6.0. 8% of GA strain were added and cultured for 7 d, and fermentation temperature was changed as 22℃, 25℃, 28℃, 32℃ and 35℃. Then, Mycelial biomass and raw exopolysaccharides were determined (Table 6).

It was shown in Table 6 that, effect of fermentation temperature on shake flash fermentation of GA was significant. Mycelial biomass and raw exopolysaccharides increased significantly with the increase of fermentation temperature. The content increased to a peak value at 32℃, but then lowered down with the rise of temperature. Its reason might be that high temperature caused the strain premature aging, and metabolites can also be reduced accordingly. Therefore, the optimum fermentation temperature was 32℃.

It was shown in Table 7 that, the exopolysaccharides could scavenge HRs and SARs, and the more radicals were scavenged with the increase of concentration of raw exopolysaccharides. It indicated that its scavenging activity might be related to exopolysaccharides. When the concentration of exopolysaccharides was 1.2 mg/mL, the ratio of HRs scavenged had exceeded 50%; When the concentration of exopolysaccharides was 1.8 mg/mL, the ratio of SARs scavenged had reached 50%, too.