Study On The Secondary Metabolites Of A Mushroom Endophytic Bacteria Bacillus Halotolerans DMG7-2

Mushrooms are one of the important sources of natural new medicines.There are many kinds of mushrooms and the types of secondary metabolites are complex.But for some mushrooms that grow in special environments,it is difficult to obtain a large number of bacteria themselves for research and analysis.Therefore,the fermentation culture of its spores or endophytes is an important research approach.Bacillus bacteria are important biocontrol bacteria and important lipopeptide compound–producing bacteria.For many years,some compounds with excellent activities have been discovered.Adopting different culture conditions from the past has the hope of producing unique structural compounds for its fermentation.In this paper,a strain of endophytic bacteria isolated from Cortinarius lucorum（Fr.）E.Berger in the subarctic coniferous forests of the Sino–Russian border in Northeast China–Bacillus halotolerans is selected.In the previous study,it exhibited good cytotoxic activity under fungal culture conditions.In this study,the strain was cultured under the conditions of fungus culture,and the effective components in the fermentation broth were extracted and separated.The resultant product comprehensively used a variety of chromatographic techniques（Sephadex LH–20,ODS,HPLC）to separate 11 monomer compounds from the fermentation product of DMG–7–2.The structures of these compounds were identified using one–dimensional(¹H NMR,¹³C NMR),two–dimensional（¹H–¹H COSY,HSQC,HMBC,TOCSY,ROESY）nuclear magnetic resonance,Marfey analysis and calculate ¹³C NMR methods.The structures of these compounds were identified as:iso–C14[Val²,Val⁷]surfactin（1）,iso–C14[Val⁷]surfactin（2）,anteiso–C15[Val⁷]surfactin（3）,anteiso–C13 surfactin（4）,iso–C13surfactin（5）,iso–C14 surfactin（6）,n–C14 surfactin（7）,anteiso–C15 surfactin（8）,iso–C15surfactin（9）,cyclo（phenylalanine–hydroxypro）dipeptide（10）and cyclo（leucine–proline）dipeptide（11）.Among them,compound 1 is a new compound,compounds 1?9 are all lipopeptide compounds–surfactant derivatives,and compounds 10–11 are two cyclic dipeptide compounds.The biological activity of the separated surfactins was studied.In the study of antibacterial activity,the tested strains of Gram–positive and negative bacteria,including drug–resistant bacteria,have no inhibitory ability.Two strains of human cancer cells（A549,MCF–7）and mice microglia cells（BV2）were selected for cytotoxicity evaluation.The results showed that surfactin compounds have moderate cytotoxicity to these three kinds of cells,IC₅₀ values are between 8～35μM.