Background:: Antifungal cyclic lipopeptides, bioactive metabolites produced by many species of the genus Bacillus, are promising alternatives to synthetic fungicides and antibiotics for the biocontrol of human pathogenic fungi. In a previous study, the co- production of five antifungal lipopeptides homologues (designated as AF1, AF2, AF3, AF4 and AF5) by the producer strain Bacillus subtilis RLID 12.1 using unoptimized medium was reported; though the two homologues AF3 and AF5 differed by 14 Da and in fatty acid chain length were found effective in antifungal action, the production/ yield rate of these two lipopeptides determined by High-Performance Liquid Chromatography was less in the unoptimized media. Methods:: In this study, the production/yield enhancement of the two compounds AF3 and AF5 was specifically targeted. Following the statistical optimization (Plackett-Burman and Box-Behnken designs) of media formulation, temperature and growth conditions, the production of AF3 and AF5 was improved by about 25.8- and 7.4-folds, respectively under static conditions. Results:: To boost the production of these two homologous lipopeptides in the optimized media, heat-inactivated Candida albicans cells were used as a supplement resulting in 34- and 14-fold increase of AF3 and AF5, respectively. Four clinical Candida auris isolates had AF3 and AF5 MICs (100 % inhibition) ranging between 4 and 16 μg/ml indicating the lipopeptide’s clinical potential. To determine the in vitro pharmacodynamic potential of AF3 and AF5, time-kill assays were conducted which showed that AF3 (at 4X and 8X concentrations) at 48h exhibited mean log reductions of 2.31 and 3.14 CFU/ml of C. albicans SC 5314, respectively whereas AF5 at 8X concentration showed a mean log reduction of 2.14 CFU/ml. Conclusion:: With the increasing threat of multidrug-resistant yeasts and fungi, these antifungal lipopeptides produced by optimized method promise to aid in the development of novel antifungal that targets disease-causing fungi with improved efficacy.

Antifungal compounds are produced by Bacillus species under various growth conditions. While optimizing the antifungal production by using the one-factor-at-a-time (OFAT) and response surface methodology (RSM) approaches mannose and malt extract were identified as the most suitable carbon and nitrogen sources respectively. The RSM was applied to determine the optimum conditions of the three parameters such as pH, carbon and nitrogen sources for improved production. Optimum concentrations of carbon and nitrogen sources were 0.3% and 0.6% respectively with optimum media pH of 7.0 which showed optimum value of 40 AU/ml of antifungal compound against the Candida albicans SC5314 used as an indicator strain. In the present study, the F-value was determined as 0.0034 to imply that the model is significant. The goodness of the fit of the model was tested using coefficient of determination, R2  value, that tantamounts to 0.8562. The identification of antifungal compounds with their molecular masses was accomplished by MALDI-TOF mass spectrometry after n- butanol extraction. The present study thus has provided a platform to upgrade the yield of antifungal compounds which have got immense clinical potential to tread Candidosis.