Ferme Ntation Condition Optimization And Metabolites Separation Of Strain Streptomyces Galilaeus AF1

The strain Streptomyces galilaeus AF1 was separated by ourteam,preliminary research found that the metabolites of Streptommyces galilaeus AF1 have agood sterilization and antibacterial activity against gram-positive bacterium,and the minimum inhibitory concentration?MIC?against methicillin-resistant Staphylococcus aureus?MRSA?is 68?g/mL,But there is not sensitive to Gram-negative bacteria.The metabolites of Streptommyces galilaeus AF1 have some antifungal activity,The inhibitory rate was94.1%of crude extract with 0.3mg/mL concentration toPathogen ofBanana Fusarium Wilt,The metabolites of Streptommyces galilaeus AF1may breakdown cell wall of Gram-positive bacteria resting cells,such as Staphylococcus aureus,which indicate that its mechanism of actionis different from other antibiotics which be used as medicine obviously,Based on the preliminary research results of the antimicrobial metabolitesdistribution in cell and culture mediumoptimization,differentinoculantsand additives of surfactant were be studied for improve the fermentation efficiency of S.galilaeus AF1.The other way,the separation of metabolites were studied.Using microscope to observe the cell morphology,DNS to determine the total sugar and reducing sugar in fermentation liquid.The p H value and antibacterial activity of fermentation liquid were determined.The results showed that cell mycelium morphology,total sugar,reducing sugar and bacteriostasis diameters are different by various inoculation methods.The spore inoculated mycelium was small and compactness,the surface was smooth and growth phase synchronization.Although the delayed phase was longer,the utilization of starch was more sufficient.When the spore inoculations are 5×10⁶/100mL,the antibacterial circle diameter is 1.17 times of mycelia inoculation,which indicated that the inoculation of spores was more beneficial to the production of antibiot ics.The results of single factor test showed that the surfactants such as Tween-80,Triton X-100 and Sorbitol could increase the accumulation of antimicrobial activity in fermentation broth.The best adding time is 120 hours after the inoculation.The Box-Behnken design expert and response surface analysis were used to choose the best appropriate concentrations of surfactants.The regression equation for the response value?Y?of the bacteriostatic circle diameter of S.galilaeus AF1 fermentation broth was obtained:Y=19.07+0.94X₁+0.52X₂-0.49X₃+1.15X₁X₂+0.17X₁X₃+0.56X₂X₃-2.56X₁²-2.83X₂²-3.03X₃²,the optimum conditions of surfactants addition were:tween-80:8.85g/L,triton X-100:2.14 g/L,sorbitol:3.84g/L.After the combination of the three surfactants,the antibacterial activity of the fermentation broth increased by 25.45%.Finally,flow cytometer instrument was used to measure the cell fluorescence intensity to explore the surfactant mechanism on the antibacterial bioactivity.The fluorescence intensit y of S.galilaeus AF1 was significantly higher than that the blank control,which resulted in the increase of the efflux of intracellular active substance and the biosynthesis of the antibacterial substance.In this paper,a new compound was isolated from the fermentation broth of S.galilaeus AF1 by normal phase silica gel column chromatography,Sephadex LH-20 column chromatography and thin layer chromatography.It was determined to be anthraquinone compound 4-deoxy-?-pyrrolycinone by NMR?nuclear magnetic resonance?.