Screening Of Athelia Rolfsii Strains With High-Yield Polysaccharide By Protoplast Fusion And Optimizing Of Scale-Up Cultural Condition

Athelia rolfsii polysaccharide belong to Scleroglucan, which is widely used attributed to its excellent properties. So far, the overseas research about scleroglucan has focused mainly on its physicochemical properties, molecular conformation, antitumor activity, immune activity and drug delivery and at the same time the domestic research has focused mainly on the optimization of fermentation medium. But the research about increasing the yield of polysaccharide by screening and breeding has not been reported. In this paper, one strain with high-yield polysaccharide and low-yield oxalic acid was screened by protoplast fusion uesing Athelia rolfsii AY6657741 and composite mutagenesis mutant strains Ar-1 and Ar-2 as original strain and starting strain, respectively. And further fermentation conditions of this strain in 5L fermenter was optimized. The conclusions in this paper were as follows:1 Through the single factor experiment and response surface optimization experiment, optimal technological conditions for Athelia rolfsii protoplasts preparation was as follows: using 5.56mmol/L β-mercaptoethanol to treat fungal suspensions, 0.4 mol/L KCl as osmotic stabilizers, 3.2mg/mL compound enzyme fluid(including 2% cellulase, 2% snail enzyme, 4% soluble enzyme), pH 5.8, 32℃ enzymolysis 55 min. The protoplast formation rate and renewable rate were 94.34%, and 22.01%., respectively.2 Ultraviolet radiation was utilized to inactivate protoplast. Inactivated condition was 6min and death rate of protoplast was 100%.3 The inactivated condition of protoplast by heating was as follow: Heat treatment at 65℃ for 40 min. Death rate of protoplast was 100%.4 The fusion conditions of protoplast by polyethylene glycol 6000 were as follows: protoplast fusion was occurred in 0.4g/mL polyethylene glycol 6000 for 25 min at 30℃ and protoplast fusion rate was 6.792%. One strain named AY-3 with high-yield polysaccharide and low-yield oxalic acid was screened. Compared with Athelia rolfsii AY6657741, the polysaccharide yield of AY-3 was 24.67g/L with 54.42% increase, and oxalic acid content was 0.28g/L with 43.57% decrease.5 Screening fusion strain AY-3 was uesd as the starting strain. Using polysaccharide yield as the response variable, optimal fermentation process in 5L fermenter for AY-3 was determined by Box-Behnken design based on single factor tests. The optimal fermentation conditions were as follows: 3.2L culture volume, 2L/min ventilation volume, 180r/min rotation rate, 9% inoculum size, initial pH 4.5, fermentation period 3.5d. The polysaccharide yield was 25.14g/L with 27.41% increase.6 The spectras of Scleroglucan standard polysaccharide and the polysaccharide from this study were scanned and recorded from 197 nm to 400 nm with a Uv-vis spectrophotometer. Both of them had an absorption peak at 191 nm and it could be concluded that the proposed samples were Scleroglucan polysaccharide.