Screening Of Antibiotic-resistant Marine Actinomycetes And Study On The Activity Of Their Secondary Metabolites

Because of the special ecological environment,marine actinomycetes may produce various of bioactive substances different from terrestrial microorganisms,new effective drug molecules can be found to inhibit a variety of pathogenic microorganisms and to treat malignant diseases such as tumors.In this study,marine actinomycetes from marine sediments in Rizhao were isolated and purified,and a highly active marine actinomycete was screened for optimization of conditions and metabolite activity,which provided a theoretical basis for the subsequent isolation and purification of secondary metabolites with novel structure,unique function and low toxicity and side effects.?1?105 marine actinomycetes were isolated from sunshine marine sediments by five different treatment methods and five different screening media.By screening of antagonistic activity against Aspergillus flavus,antibiotic strain with strong inhibitory activity against Aspergillus flavus was selected.The strain SSRRZ-B11 was identified as Streptomyces rubiginosohelvolus by 16S rDNA sequence analysis,morphological observation and physiological and biochemical tests.?2?Fermentation conditions for the production of antimicrobial active substances by Streptomyces rubiginosohelvolus SSRRZ-B11 strain were optimized by single factor experiments.The results showed that the optimum medium was improved ISP2 medium.The culture conditions were as follows:5%inoculation,the best culture temperature is 30?,the best initial pH is 7.0,the optimum time is 11 days,the best liquid loading is 90 mL/300 mL.Through P-B experiment design,the main influencing factors of temperature,incubation time and inoculation amount were determined.The fermentation conditions of antimicrobial substances were studied by steepest climbing experiment and response surface experiment.The optimum culture conditions were determined as follows:the best temperature is 29?,the optimum incubation time is 11 d,the optimum inoculation amount is 5%.Under these conditions,the diameter of the antimicrobial circle of the actual antimicrobial substances reached?29±0.1?mm.Compared with the original fermentation conditions,the activity of antimicrobial substances increased by 51.72%.?3?Antagonistic Aspergillus flavus active substances produced by Streptomyces rubiginosohelvolus SSRRZ-B11 strain were preliminarily extracted by precipitation method.The results showed that the inhibition activity of ammonium sulfate precipitation on Aspergillus flavus reached 82.1%at 80%saturation.Therefore,the active substances against Aspergillus flavus can be preliminarily extracted by ammonium sulfate fractional precipitation method.On this basis,the stability of antimicrobial active substances to temperature,acidity,alkalinity storage time and genetic stability was discussed,the results showed that the antibacterial active substances produced by SSRRZ-B11 strain are sensitive to temperature and have high stability below 60?,and pH remained stable in the range of 4¹0.During 5¹5 days,its antibacterial activity was not obviously decreased,but it still had strong activity,and it had good genetic stability.These studies indicated that SSRRZ-B11 strain had strong antimicrobial activity in the future,so it provided a theoretical basis for separation and purification of substances.?4?The cytotoxic activities of the screened antibiotic strain SSRRZ-B11 were investigated.It was found that it could significantly inhibit the growth of human lung cancer A549 cells,and the ID₅₀0 value of the fermentation broth was as high as 460.Polarity experiments on antineoplastic metabolites produced by antibiotic strains showed that most of the antineoplastic active substances could be extracted by ethyl acetate,indicating that most of the antineoplastic active substances were moderately polar metabolites.The results of temperature and acid-base stability of the antineoplastic active substance showed that the active substance had good stability at 40⁸0?and pH 7¹1.The cell cycle assay showed that the number of S-phase cells in A549 cells increased in a multiple-dependent manner,suggesting that human lung cancer A549 cells were mainly blocked to S-phase.The results of apoptotic experiment showed that the crude extracts of antineoplastic metabolites could significantly induce apoptosis of human lung cancer A549 cells when diluted to 1.6×10⁴,3.2×10⁴ and 6.4×10⁴.