Aims To determine the mechanism of action of antimicrobial protein, lactosporin, against Gardnerella vaginalis and to evaluate its safety in vitro. Methods and Results Bacillus coagulans ATCC 7050 was grown at 37°C for 18 h. The cell‐free supernatant was concentrated 10‐fold and screened for antimicrobial activity against indicator strain Micrococcus luteus. The mode of action of lactosporin was determined by measuring the potassium release and monitoring the changes in transmembrane potential (Δψ) and transmembrane pH (ΔpH) of the sensitive cells. Lactosporin caused the efflux of potassium ions from M. luteus cells and dissipation of ΔpH in G. vaginalis, while it had no effect on the Δψ. The safety of lactosporin was evaluated by using EpiVaginal™ ectocervical (VEC‐100) tissue model. Over 80% of the cells in the vaginal tissue remained viable after exposure to lactosporin for 24 h. Conclusions Lactosporin potentially exerts its antimicrobial activity by selective dissipation of ΔpH and/or by causing leakage of ions from the sensitive cells. Safety studies suggest that lactosporin is a noncytotoxic antimicrobial for vaginal application. Significance and Impact of the Study This study revealed that lactosporin is an effective and safe antimicrobial preparation with potential application for the control of bacterial vaginosis.

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Aims:  To characterize the antimicrobial protein produced by Bacillus coagulans used in the probiotic dietary supplement (Lactospore® Probiotic, Sabinsa Corp., Piscataway, NJ, USA). Methods and Results:  Bacillus coagulans ATCC 7050 was grown at 37°C for 18 h. The cell free supernatant was concentrated 10‐fold (lactosporin preparation, LP). The antimicrobial activity of LP was confirmed against Micrococcus luteus ATCC 10420 in a well diffusion assay. The proteinaceous nature of LP was determined following exposure to different enzymes. The activity of LP was pH‐dependent but stable to heat. The isoelectric point of LP was determined to be 3·5–4·0. PCR analyses showed no similarity between lactosporin and known antimicrobial proteins produced by the Bacillus spp. Conclusions:  Lactosporin is a novel antimicrobial protein. Initial characterization indicates that it may fall outside of the conventional classification of class I and II bacteriocins. Loss of activity after exposure to a number of proteolytic enzymes and lipase suggest that lactosporin may posses a lipid moiety which contributes to its inhibitory activity. Significance and Impact of the Study:  The unique characteristics of lactosporin, including its antimicrobial activity against pathogenic micro‐organisms, indicate that it may have potential for application in foods and personal care products.

BioRad's Rotofor ® system has been frequently used for the purification of proteins and smaller peptides such as bacteriocins. In this study, we report that some commercially available ampholytes used with the Rotofor ® isoelectric focusing system possess antimicrobial activity, which may interfere with the purification of bacteriocins and bacteriocin-like substances.

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