Preliminary Study On The Antifungal Activity Of The Secondary Metabolites Of Gordonia WA8-44 From The Intestinal Tract Of Periplaneta Americana

Gordonia is a kind of rare actinomycete.In the early stage,our group isolated 15 Gordonias from the intestinal tract of Periplaneta americana.In this paper,Candida albican ATCC 10231 was used as the indicator for the preliminary screening of the antifungal activities of these 15Gordonias.Finally,strain WA8-44 was selected as the research target.On this basis,the antifungal spectra of WA8-44 were determined by Candida albican ATCC 10231,Aspergillus niger ATCC 16404,Aspergillus fumigatus ATCC 96918 and Trichophyton rubrum ATCC 60836.We investigated species characterization of Gordonia strain WA8-44 and optimation of its fermentation,and the chemical constituents of its fermented broth.Morphological identification,molecular biological identification and phylogenetic tree analysis were performed.Single factor analysis and orthogonal experiment were used to optimize the liquid fermentation conditions of strain WA8-44,and then the fermentation scale was expanded.The secondary metabolites of the strain WA8-44 were studied by the combination of activity tracking and chemical separation.The main experimental results of this paper are as follows:1.Screening of antifungal activity of 15 Gordonias:Candida albicanATCC 10231 was used as the indicator for the preliminary screeningof the antifungal activities of these 15 Gordonias which isolated fromthe intestinal tract of Periplaneta americana.Experimental resultsshow that 6 Gordonias had different inhibition on Candida albican,and the strain WA8-44 had the strongest activity,strain WA8-44 wasselected as the research target.2.Antifungal spectrum and phylogenetic tree analysis of strain WA8-44:strain WA8-44 showed antifungal activity against Candida albicanATCC 10231,Aspergillus niger ATCC 16404,Aspergillusfumigatus ATCC 96918 and Trichophyton rubrum ATCC 60836.Byinvestigation on the the bacterial colony morphology of the strain wasidentified by gram staining and scanning electron microscope.Usingthe phylogenetic tree analysis method of 16S rDNA sequenceconstruction,the strain WA8-44 was identified to have 100%homology with Gordonia didemni,and the gene sequence wassubmitted to GenBank for registration,and the accession number wasMH605445.3.Optimization of fermentation conditions of strain WA8-44 andexpansion of fermentation scale:the fermentation conditions wereoptimized by single factor and orthogonal experiments.Candidaalbican was used as indicator bacteria,and the antifungal activity of thefermentation broth activity test using agar diffusion method with Candida albican.The antifungal activity was determined by filter paper method.It was finally determined that the optimal fermentation conditions for strain WA8-44 were ISP₂ medium,60L of fermentation-scale fermentation was expanded to obtain enough crude extract.4.Isolation,purification and structure identification of compounds:thefermentation broth of strain WA8-44 was extracted three times withethyl acetate and n-butanol,respectively,and the mycelium wasextracted with acetone.The biological activity of Candida albicanwas used as an indicator to trace the antifungal activity of the strainWA8-44,activity test results showed the ethyl acetate site hadantifungal activity.We have performed multiple chromatographicapproaches including TLC,ODS,MCI,silica gel and SephadexLH-20 column chromatography,preparative HPLC,etc.Finally,thepreparation was carried out by semi-preparative HPLC.The structureof the isolated compounds was analyzed and identified by modernNMR,MS,IR,UV and so on,and a total of eight compounds wereobtained:actinomycin D,actinomycin X₂,collismycin A,dibutylphthalate,cyclo?leucine-leucine?,cyclo?proline-glycine?,and sinapic acid.All these seven compounds were isolated fromGordonia for the first time.5.Evaluation of antibacterial activity:The antibacterial activity of eightisolated compounds was determined respectively.Actinomycin D,actinomycin X₂ and collismycin A showed strong inhibitory activityagainst Candida albican.The MIC values for Candida albican were4?g/mL,32?g/mL and 2?g/mL.But the other five compounds had noantifungal activity.In addition,the three compounds also showeddifferent inhibitory activities against MRSA ATCC 25923,Staphylococcus Aureus ATCC 25213,Bacillus Subtilis ATCC 6633and Escherichia coli ATCC 25922.