Methylene blue active substances in plaque of Bacillus subtilis subsp. subtilis and enrichment by supplemental calcium in culture media

A series of experiments was conducted to identify the molecular species responsible for surface active emulsification (surfactant) bioactivity in Bacillus subtilis subsp. subtilis strain ATCC PTA‐125135, and to describe culture conditions to support the enriched production of said bioactivity in cultured plaque of the strain. The assay for methylene blue active substances (MBAS) was found to be suitable for describing surfactant activity, where a solvent‐extracted molecular fraction from the biofilm was found to retain surfactant activity and positively quantified as MBAS. Furthermore, an HPLC‐refined protein fraction was found to quantify as MBAS with approximately 1·36‐fold or greater surfactant activity per mol than sodium dodecyl sulphate, and a proteomic analysis of solvent extracted residues confirmed that biofilm surface layer protein BslA was a primary constituent of extracted residues. Surfactant bioactivity, quantified as MBAS, was enriched in cultured plaque by the supplementation of culture media with calcium chloride or calcium nitrate. Significance and Impact of the Study: Surfactants with emulsifying bioactivity are known to be produced by Bacillus subtilis. Here, a colorimetric assay for methylene blue active substances is adapted for use in bacterial plaque to describe surfactant bioactivity, and supplemental salts of calcium during culture are shown to enrich cultured plaque for said bioactivity. Where B. subtilis is utilized as a feed additive to food producing animals, the manufacture of bacterial plaque with controlled and enriched concentration of surfactant bioactivity is desirable, especially where the dose amount is limited by mass, total colony forming units, enzymatic activities or other limiting qualities.