Strain Screening And Submerged Culture Condition Study Of Coriolus Versicolor Fungus Producing Anticancerous Peptide

Thirteen Coriolus versicolor strains were cultivated by using submerged culture. Their mycelial biomasses were recorded and the small molecule peptide extracts were obtained from mycelia and broth using 70% ethanol respectively. The protein contents of peptide extract were measured by Lowry method. The results show that the total average biomass of 13 Coriolus versicolor strains is 1.4746g/100ml, among of which the TV-5 strain produces the highest (2.0625g/100ml) . Protein contents of peptide extracts from different strains varies greatly, which the average is 21.61% in mycelial extracts, and the TV-33 strain is at the top (30.4%). The average protein contents of peptide in broth is 23.34%, among of which the TV-42 strains has the highest (34.3%).MTT assay illustrates that the small peptide extracts from TV-42 strain mycelia and broth have a higher in vitro anti-proliferation activity against human T cell leukemia Molt-4 than the rest. The anti-proliferation rates are 22.95±1.00% (mycelial extract) and 20.43±2.09% (broth extract) respectively (2000μg/ml,72hr). Therefore, TV-42 strain was selected for the further submerged culture condition study, and time duration, pH and temperature on mycelial biomasses production, the glucose residue amount in culture medium, small peptide extract yield and in vitro anticancerous activity were studied.The results demonstrate that TV-42 strain produce the highest anti-proliferation activities at conditions as: a six-day fermentation duration, acidified medium and a temperature of 28℃. Under such circumstances, the Molt-4 inhibitory rates of peptide from mycelia and broth, respectively, are: 46.78±3.54% and 21.68±2.20% (6 days), 49.48±3.08% and 36.00±0.46%(acidified medium), 36.38±1.90% and 18.35±3.69% at 28℃.Morphologic observation and flow cytometric analysis further show that the inhibitory effect of TV-42 small peptide extracts from mycelia and broth relate to the Molt-4 cell cycle arrest. At the 72th hour onset of treatment, small peptide extracts (400μg/ml) result a statistical significant increase of the cell percentage in S phase, showing the S phase's retardation activity.Moreover, the conditions of Tricine electrophoresis for small peptide analysis were also studied. The optimal conditions are found as: 20% separating gel concentration, 5% Glutaraldehyde as fixation solution, 0.025% brilliant blue G250 in 10% acetic acid as the staining solution and 10% acetic acid as destain solution. In this condition, the molecular weights of small peptides are found in arrange between 3496Da to1060Da, with an evidence showing glyco-peptide characteristic.The data illustrates the potential of using Coriolus versicolor strains and submerged culture to manufacture the anti-cancerous peptide products.