Mutagenesis And Fermentation Conditions' Optimization Of A Berberine-Producing Endophytic Fungus

The strain S6 was an endophytic fungus previously separated from Amur cork and could produce the bioactive compound berberine like its host plant. The work involved in this paper is focused on the mutagenesis study of the strain S6, using different physical and chemic mutagens. in order to get mutant strain with higher berberine yield.Various mutation methods were used to mutagenize the initial strain S6 and the content of berberine produced by each mutant was subsequently detected using the ultraviolet spectrophotometer. The effects of both single-factor mutagenesis (including, UV. He-Ne laser and NaNO₂ ) and multiple mutagenesis (NaNO₂-UV, NaNO₂-laster, UV-laster. UV-LiCl. laster-LiCl, NaNO₂-LiCl) were explored in this study. The results showed that the optimal single mutagenic condition was 150s UV, 25min He-Ne laser or 30min NaNO₂. All the composite mutagenesis methods apart from NaNO₂-LiCl were able to improve berberine yield. Among those treatments, Laster(25 min) - LiCl (1g/L) multiple mutagens was the most efficient method. All the high-yielding strains derived from the above methods were then compared and the strain S-NU-3-2, mutagenized by NaNO₂-ultraviolet multiple mutagens, proved to be the best strain in this study. Growth rate of the strain S-NU-3-2 was 5.72g/L (increased by 81.7% compared with the initial strain) and the berberine yiele was 12.28mg/L (increased by 170%). Additionally, the yield of product showed no distinct changes after successive culture of 10 generations.In order to further improve the product yield, the culture conditions of the strain S-NU-3-2 were then optimized. Based on the growth rate and the yield of berberine, the optimal solid culture medium was PDA and the liquid culture medium was bean sprouts medium. The optimum carbon source was sucrose, the optimum nitrogen source was yeast extract and the optimum growth temperature was 26℃. A L16(4³) orthogonal experiment was also carried out to determine the effect of carbon concentration, nitrogen concentration ,and pH on the product yield and mycelial growth rate. The results showed that the most important influencing factors to mycelial growth and the yield of berberine were pH and carbon source concentration, respectively. The optimum culture medium (both for the growth rate of mycelia and the yield of berberine) was determined as bean sprouts medium containing sucrose 3%, yeast extract 0.2%. pH 7.0, 26℃.The berberine yield was 47.2% and the growth rate of mycelia was improved by 24%.