Study On Breeding Of Spirolaxines-producing Strain Phanerochaete Chrysosporium SIIA-F1802 And Optimization Of Fermentation Conditions

Studies have found that Spirolaxine,the fermentation product of the P.chrysosporium,has good anti-Helicobacter pylori activity and the pharmacological effect of lowering blood lipids.In order to improve the fermentation unit of Spirolaxine,this paper mainly studies the Spirolaxine producing strain SIIA-F1802through the selection of strains and the optimization of fermentation conditions.The main research contents of this paper are as follows:1.Based on ITS sequencing and phylogenetic tree analysis,the strain SIIA-F1802 was finally determined to be P.chrysosporium.2.The metabolites in the mycelium were studied by LC-MS,and the chromatographic peak consistent with the target product was initially determined;according to the development of the crude sample in different developing reagents,it was determined that the developing reagent of TLC was methanol:dichloromethane=1:20,after methanol extraction fermentation broth is separated and purified by C18column chromatography and crystallization methods,the crystal sample 0.7235 g is obtained,with a purity of more than 95.0%;it is confirmed by UV spectrum,infrared spectrum,high resolution mass spectrometry,specific rotation and nuclear magnetic resonance spectrum analysis.Target substance is the same as Spirolaxine,and its molecular formula is C₂₃H₃₂O₆.3.After natural isolation,the strain F1802-07（yield 712.648 mg/L）with higher yield was selected as the starting strain for subsequent mutagenesis.According to the comparison of the positive mutation rates,it was determined that the UV mutagenesis irradiation time should be 120 s.The concentration of NTG mutagenesis should be100.0μg/m L;the high-yielding mutant strain F1802-07-48 obtained by UV mutagenesis screening,with a yield of 1060.558 mg/L;F1802-07-48 was used as the starting strain for NTG mutagenesis,The strain F1802-07-48-15 has a yield of1536.357 mg/L and stable genetic performance.4.Through single factor screening,it is determined that the carbon source is maltose and glucose,the organic nitrogen source is cottonseed meal,the rapid utilization of nitrogen source is yeast extract powder,the inorganic salt is potassium dihydrogen phosphate,the trace element is copper sulfate,and the type of amino acid is Arginine.Then through response surface optimization method,the optimal formula of each component of the medium was determined to be maltose 135.0 g/L,yeast extract 9.0 g/L,arginine 4.5 g/L,cottonseed meal powder 30.0 g/L,and glucose 20.0g/L and potassium dihydrogen phosphate 1.0 g/L;through the optimization of the culture conditions,the optimal culture conditions are determined to be the culture temperature of 28±0.3℃,the seed culture for 2 days,the fermentation period of 9days,the initial p H of 5.5,and the amount of liquid in the shaker bottle is 25 m L,the inoculation volume is 10%,and the rotating speed of the shaker is 250 r/min.The output of the target substance reaches 3597.438 mg/L,which was an increase of 134.2%compared with the initial yield.In this study,the fungus SIIA-F1802 was identified as P.chrysosporium;its secondary metabolites were separated and purified,and the structure confirmed that the product was homogenous with Spirolaxine;it was isolated naturally,UV mutagenesis and NTG mutagenesis,the mutations strain F1802-07-48-15 was obtained,the yield of spirolaxine is 1536.357 mg/L,and the genetic performance is stable.Through the optimization of the culture medium and fermentation conditions,the yield of Spirolaxine producing strain P.chrysosporium SIIA-F1802 reached3597.438 mg/L,which was an increase of 405.5%compared with the initial yield of naturally isolated strains（712.648 mg/L）.It has industrial prospects and provides a basis for further drug research.