The Study On The Active Metabolites Of Anti-candida Albicans By Siderophlic Bacteria Bacillus Altitudinis AS19 From The Rhizosphere Of Paris Polyphylla Var.yunnanensis

Objective:The purpose of this study was to isolate and identify the siderophilic bacteria in rhizosphere soil of the traditional chinese medicine Paris polyphylla var.yunnanensis,analyse the antimicrobial activity and purify active metabolites with anti-Candida albicans effect from the secondary metabolites of Bacillus altitudinis AS19.Methods:1.Isolation and identification of rhizosphere bacteria.The gradient dilution method was used to gradient dilute the rhizosphere soil samples of Paris polyphylla var.yunnanensis and cultured on NA plates respectively.The samples were incubated at 37℃for 1-2 days,and different monoclone was selected on the plates.The rhizosphere soil strains were identified by morphological,physiological and biochemical,Gran staining and molecular biological methods.2.Detection of the relative content and types of siderophore produced by rhizosphere bacteria.The CAS plate and CAS reagent method were used to detect the siderophore production of rhizobacteria in iron-low medium（SA medium）.The Arnow’s test,Fe Cl₃ test and Shenker’s test were used to determin different types of siderophore respectively.3.The bacteriostatic activity of siderophore bacteria.The isolates was cultured in LB and SA medium respectively in 30℃for 48 h.The gram-positive Staphylococcus aureus,gram-negative Escherichia coli and fungus Candida albicans were used as indicator microbes.The agar diffusion method（the punch method）was used to investigate the antimicrobial activity of the secondary metabolites of isolates.4.Cluster mining of secondary metabolite synthesis gene of Bacillus altitudinis AS19 strain.Combined with bioinformatics methods,anti-SMASH 6.0 and PRISM4.0 platforms were used to analyze the genes that might produce antibacterial active substances.5.Culture medium screening for Bacillus altitudinis AS19 metabolites with antibacterial activity.SA medium contains sucrose（Z）,asparagine（T）,dipotassium hydrogen phosphate（K）and magnesium sulfate（M）.The SA media which could produce active metabolites against Candida albicans were recombined and arranged to 15 types of medium,including Z,M,K,T,ZM,ZK,ZT,MK,MT,KT,ZMK,ZMT,ZKT,MKT and ZMKT（namely SA）.The growth,siderophore production and bacteriostasis of AS19 at specific time points in different medium were detected.6.Stability evaluation of antibacterial active metabolites produced by Bacillus altitudinis AS19.The fermentation supernatants were treated with 0.22μm,0.45μm filter membrane and different temperature,p H,protease to evaluate the physicochemical properties of active metabolites.7.Purification of anti-Candida albicans metabolites from Bacillus altitudinis AS19 in ZM and MT medium.The active compounds were separated and determinated by the chemical extraction,TLC,column chromatography,HPLC,UPLC-IT-TOF,NMR and SC-XRD.8.MIC determination and toxicity evaluation of ZM17 samples.Gradient dilution method was used to determine the Minimum Inhibitory Concentration（MIC）and Concentration Killing Curve（CKC）of active substances against Candida albicans.The biotoxicity of active metabolites was evaluated in the larvae of Hermetia illucens L.Results:1.Through morphological,physiological and biochemical characteristics and 16S r RNA sequence analysis,22 strains of rhizobacteria were isolated,including Bacillus,Enterobacter and Stenostomonas.The dominant genus in the rhizobacteria of PPVY was Bacillus.2.21 strains were identified as siderophlic bacteria and the relative amount of siderophores ranged from 4%to 41%.Most of the isolates produced hydroxamate siderophores,and four isolates belonging to Enterobacter produced the catechol type.None of them produced carboxylate siderophores.3.The fermentation supernatant of 22 rhizosphere bacteria strains in LB medium had no antimicrobial activity against Escherichia coli,Staphylococcus aureus and Candida albicans.16 strains could produce anti-Candida albicans active substances only in iron-limited medium（SA medium）.4.Genomic analysis of selected Bacillus altitudinis AS19 strain（with high siderophores yield and strong antibacterial activity）revealed 6 and 9 secondary metabolite synthesis gene clusters,respectively.The active metabolites were involved in NRPS and PKS synthesis pathways.5.In addation to SA medium,Bacillus altitudinis AS19 strain could also produce bacteriostatic substances in Z,T,ZM,ZK,ZT,MT,ZMT and ZMK medium.The growth of AS19 strain,the production of siderophore and the determination of antibacterial activity in different medium also suggested that there were a variety of active metabolites in the culture supernatant of AS19.6.The active metabolite could pass through 0.22μm microporous membrane,not sensitive to heat,not affected by protease treatment,unstable in strong alkali environment7.The active substance ZM17 was isolated from the supernatant of ZM culture with sucrose as the main component,which contained a very small amount of compounds and had antibacterial effect on Candida albicans.After column chromatography and Prep-HPLC separation,11 possible active metabolites of Bacillus were obtained by UPLC-MS analysis,and 32 compounds corresponding to molecular weight were not found in the existing Bacillus database.8.The MIC of ZM17 was 220μg/m L,and the bactericidal curve showed that the inhibition of active metabolites on the growth of Candida albicans started from the time of administration.ZM17 has no biological toxicity to the larvae of Hermetia illucens L.Conclusions:1、In this study,22 strains of rhizosphere bacteria were firstly isolated and identified from the rhizosphere soil of Paris polyphylla var.yunnanensis,among which 21 were siderophilic bacteria,and 16 strains of siderophilic bacteria were screened out for their anti-candidal activity.2、In this study,the active substance ZM17 with sucrose as the main component was purified from the supernatant of ZM culture.The active substance ZM17exhibited growth inhibition against Candida albicans with MIC of 220μg/m L,nearly100 times higher than the crude extract of 20 mg/m L.Moreover,ZM17 had no biological toxicity to Hermetia illucens L.larvae.11 possible active metabolites of Bacillus were obtained by UPLC-MS analysis in the supernatant of purified MT culture.