Identification of bioactive substances against Candida albicans by marine Bacillus subtilis 2713
Objective To investigate the antagonistic potential against Candida albicans by marine Bacillus subtilis 2713 and to analyze the natural antimicrobial active substances produced.Methods Evaluation of antagonistic activity against Candida albicans by the combination of plate inhibition zone observation,Spot-on-lawn test,and scanning electron microscopy.The relevant antimicrobial genes were analyzed by genome sequencing and anti-SMASH online analysis.Submerged fermentation was applied to verify the ability of antimicrobials production at a spot scale.The antimicrobials were separated by macroporous resin adsorption and then further purified by HPLC.The active fractions were subjected to stability tests.Finally,the chemical nature of the antimicrobial was elucidated by LC-MC.Results It was found that strain 2713 could inhibate the growth of Candida albicans.Scanning electron microscopy revealed that it caused the crumbling of Candida albicans cells and even disrupted their integrity of their cell membrane.Bioinformatics analysis showed that it harbored a variety of gene clusters encoding secondary metabolites such as fengycin,surfactin,bacilibactin,bacillaene,macro lactin,difficidin,and bacilysin.Pilot fermentation convinced that strain 2713 could produce the anti-Candida albicans substance by submerged fermentation.The substances were recovered from the fermentation by one-step macroporous resin absorption,with a recovery rate of up to 75％.The crude extract was purified by HPLC and the activity was identified in one fraction corresponding to a single peak.The purified substance was stable at pH 2-12 and remained its activity when treated at 100 ℃ for 30 minutes.It was not sensitive to trypsin and pepsin treatment but could be inactivated by papain.Lastly,by using LC-MS,the purified anti-Candida albicans substance was confirmed to be C16-Fengycin A,composed of a 16-carbon fatty acid chain and a 10-amino acid cyclic peptide,with the molecular formula as C72H110N12O20.
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