Studies On Screening And Identification Of Short Dense Penicillium Strains And Submerged Fermentation

Penicillium brevicomarum belongs to the class of sac.It is a filamentous fungus.The secondary metabolites of the submerged fermentation of Penicillium brevicomarum have different biological activities and represent mycophenolic acid and have important medicinal value.This study was screened,purified and identified from the fruiting body of Betula birch.HPLC analysis of its secondary metabolite mycophenolic acid was carried out.The single factor test was used to optimize the submerged fermentation conditions of Penicillium brevicomarum,and the orthogonal analysis was used to study the bi-directional fermentation conditions of Penicillium brevicomarum and extract from the Fructus Swietenia Macrophylla.The findings are as follows:Firstly,the pure strain was isolated from the fruiting body of Bai Hua antler,and its genomic DNA was extracted and amplified by PCR,and the 18 S rDNA sequence was analyzed to construct the evolutionary tree.According to the results of Blast comparison and morphological characteristics,the strain MC-4 was identified as Penicillium brevicomarum.The deep fermentation product(sample)and mycophenolic acid standard product of Penicillium brevicomarum were analyzed by HPLC.The results showed that the retention time of mycophenolic acid standard product and sample was 4.763 min,and the concentration of mycophenolic acid in the sample was 65.40 %.Then,on the basis of PDB medium,the optimum liquid fermentation conditions of Penicillium brevicomarum were determined by single factor test: the content of Chinese yam(polysaccharide)100 g,the temperature 24 ?,the rotation speed 130 r / min,and the initial pH=6.According to the optimized liquid submerged fermentation conditions of Penicillium brevicomarum,the growth cycle of Penicillium brevicomarum was studied.The results showed that the growth cycle of Penicillium brevicomarum was adjusted by 0~2 d as the adjustment period,and 2~6 d by the logarithmic growth period,and 6~8 d by the stable growth period,and 8~12 d of decay.On the 6 d,the content of mycophenolic acid,the product of deep fermentation of Penicillium brevicomarum,was the highest,reaching 1.415 g / L.Finally,in order to reduce the cytotoxicity of Penicillium brevicomarum,the bidirectional fermentation of Penicillium brevicomarum and the extract from the Fructus Swietenia Macrophylla,The ultrasonic extraction conditions were determined by single factor experimental method: methanol concentration 80,ratio of extraction material to liquid 1: 15 m/ v,extraction time 30 min and extraction temperature 70 ?,extraction time 30 min,extraction temperature 70 ?,extraction time 30 min.The extraction amount of alcohol-soluble substances from the Fructus Swietenia Macrophylla was 1:15(m/v).The bidirectional fermentation conditions of Penicillium brevicomarum and its extract were optimized by the orthogonal experiment with reference to the submerged fermentation conditions of Penicillium brevicomarum.HPLC analysis showed that the secondary metabolite of Penicillium brevicomarum was mycophenolic acid.Mycophenolic acid was a good antitumor drug.Bi-directional fermentation of Penicillium brevicomarum and extract could improve the efficacy,increase safety and reduce toxic and side effects.The optimization of submerged fermentation conditions can increase the yield of mycophenolic acid from the secondary metabolite of Penicillium brevicomarum,which can reach up to 1.537 g/L.