In the present study, an attempt was made to biochemically characterize the antimicrobial substance from the soil isolate designated as RLID 12.1 and explore its potential applications in biocontrol of drug-resistant pathogens. The antimicrobial potential of the wild-type isolate belonging to the genus Bacillus was determined by the cut-well agar assay. The production of antimicrobial compound was recorded maximum at late exponential growth phase. The ultrafiltered concentrate was insensitive to organic solvents, metal salts, surfactants, and proteolytic and nonproteolytic enzymes. The concentrate was highly heat stable and active over a wide range of pH values. Partial purification, zymogram analysis, and TLC were performed to determine the preliminary biochemical nature. The molecular weight of the antimicrobial peptide was determined to be less than 2.5 kDa in 15% SDS-PAGE and in zymogram analysis against Streptococcus pyogenes. The N-terminal amino acid sequence by Edman degradation was partially determined to be T-P-P-Q-S-X-L-X-X-G, which shows very insignificant identity to other antimicrobial peptides from bacteria. The minimum inhibitory concentrations of dialysed and partially purified ion exchange fractions were determined against some selected gram-positive and gram-negative bacteria and some pathogenic yeasts. The presence of three important antimicrobial peptide biosynthesis genes ituc, fend, and bmyb was determined by PCR.

New lipopeptide homologues (AF, AF, and AF) with antifungal activities against and spp. were purified from a cell-free supernatant of RLID 12.1. The lipopeptides AF, AF, and AF were identified with the same peptide sequence Asn-Pro-Tyr-Asn-Gln-Thr-Ser with variations in the fatty acid branching type and chain length (-C, -C, and -C, respectively). Upon comparing the three homologues for MICs against 81 ( = 64) and ( = 17) clinical isolates and their cytotoxicities, we found that AF was the most promising antifungal lipopeptide, since it demonstrated 100% inhibition at geometric mean MICs of 3.31, 3.41, 3.48, and 2.83 μg/ml against ,, , and, respectively, with low hemolysis values (<6%) and 50% inhibitory concentrations (13.31 μg/ml). The additive effects among the homologues AF, AF, and AF were evaluated against three species, along with the cytotoxicity studies. Five combinations exhibited good additive interaction effects: AF/AF (at corresponding concentrations of 4 and 4 μg/ml [4/4 μg/ml]), AF/AF (4/4 μg/ml), AF/AF (2/4 μg/ml), AF/AF (4/4 μg/ml), and AF/AF (2/4 μg/ml) in planktonic cell inhibition and AF/AF (4/4 μg/ml), AF/AF (4/4 μg/ml), and AF/AF (2/4 μg/ml) in the inhibition of biofilm formation. However, combinations AF/AF and AF/AF, which showed >70% cell survival with low hemolysis (<5%), were found to be comparatively effective. We describe here the additive effects of lipopeptide homologues showing reduced cytotoxicity against mammalian cells; these combinations might serve as a potent antibiofilm-forming substitute.

In the pursuit of new antifungal compounds, five coproduced lipopeptide variants (AF to AF) from wild-type Bacillus subtilis RLID 12.1 were identified in our previous study. Out of five, AF was identified as a novel lead molecule belonging to the bacillomycin family showing less cytotoxicity at its respective minimum inhibitory concentrations (MIC) evaluated against 81 strains of Candida and Cryptococcus species (including clinical isolates); besides this, AF purified in the present study exhibited encouraging MIC values against 10 clinical mycelial fungi. Aiming for a selective production augmentation of AF lipopeptide variant, a new fermentation media comprising malt extract (1.01%), dextrose (0.55%), peptone (1.79%), MnSO (2 mM), and NaCl (0.5%) was formulated. Maximum production of 954.8 ± 10.8 mg/L was achieved with 44% selectivity at 30 °C compared to unoptimized conditions (186.4 ± 6.1 mg/L). Use of calcium alginate beads in the formulated media during the onset of lipopeptide production resulted in an augmentation in the selectivity of the most efficacious AF variant to about 72% presumably due to attenuation of other coproduced lipopeptide variants AF and AF. Difference in yield of lipopeptides varied with bead size, bead preparation ratios, and sodium alginate concentrations. Use of Ca-alginate beads in the upstream production process of the lead AF variant may be considered as a novel strategy to address the potential challenge that may arise during the scale-up and downstream processing steps. Another significant finding derived from the study is that the proportion of bacillomycin variants of B. subtilis RLID 12.1 could be controlled by temperature and metal ions under static and shaking conditions.

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.

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