The Study On The Culture Optimization And Active Substances Of Antitumor Activite Microbes

Due to a series of environmental problems caused by the unreasonable development and utilization of animal and plant resources, people have realized the importance of microbial resources, especially in the field of bioactive natural products development. In this case, the development of microbial agents, especially of antitumor agents, has become one of the hottest topics these years. In this paper, we had optimized the fermentation medium of two antitumor isolates—Aspergillus oryzae YX-5and Streptomyces parvulus HY026. After that, the large-scale fermentation of these two bacteria was carried out using the optimized fermentation medium. Then the active substances were separated from metabolites under the guidance of antitumor activity test, and the structure of the active substances was finally confirmed by using NMR and HRESI-MS. The results of the study might provide a theoretical basis and potential application for the development of environmental microbial resources.The main results of this study are as followings:(1) In this study, the Czapek’s medium was used as basic medium, and the dry weight of mycelia, weight and antitumor activity of crude extract were used as standards to screen out the best carbon source and nitrogen source of A. oryzae YX-5through single factor experiment. Then the culture medium was further optimized by the orthogonal test in five factors and four levels. The optimal culture medium was:glucose45g/L, peptone8g/L, K2HPO40.5g/L, MgSO4·7H2O0.2g/L, KC11g/L, FeSO40.01g/L. After optimization, the dry weight of mycelia, weight and antitumor activity of crude extract were increased by41.88%,226.52%and19.31%respectively.(2) Gause1medium was used as basic medium to screen out the best carbon source and nitrogen source of S. parvulus HY026through single factor experiment. The optimal culture medium was:soluble starch20g/L, soy flour20g/L, K2HPO40.5g/L, MgSO4·7H2O0.5g/L, NaCl0.5g/L, FeSO4·7H2O0.01g/L. After optimization, the dry weight of mycelia, weight and antitumor activity of crude extract were increased by299.22%,65.31%and27.58%respectively. The production and antitumor activity of crude extract was significantly increased after optimization, which could be beneficial to reduce the fermentation scale and workload in scale up fermentation later.(3) A. oryzae YX-5was mass cultured and in total,22.91g crude extract was obtained from60L liquid cultures. Based on the polarity, the crude extract was separated using flash silica gel column chromatography elucidated with dichlorometheane and methanol solvent system in different gradient and most of the active components were contained in three fractions which were eluted with,dichlorometheane to methnol30:1,20:1and15:1respectively. Since these active components shared the similar TLC profile, they were mixed and further separated with flash ODS column chromatography. Two highly active components, which were eluted with50%and60%methanol aqueous solution, were obtained. Due to the similar polarity of these two components, they were mixed and further separated by using Sephadex LH20column chromatography based on the molecular size. The active substance was found in one pure fraction and identified as hyhroxyaspergillic acid finally.(4) S. parvulus HY026was mass cultured (80L), and5.013g of crude extract was obtained from the cultures. Based on the polarity, the crude extract was separated using flash silica gel column chromatography twice, and the eluent was dichlorometheane and methyl alcohol solvent system in different gradient. By further prification on HPLC, one pure active component was obtained and identified as actinomycin-D by using NMR and HRESI-MS.