Effect of gaseous ozone treatment on cells and biofilm of dairy Bacillus spp. isolates

<i>Bacillus</i> spp. can produce biofilms and cause recurrent contamination in the food industry. The common clean-in-place (CIP) method is usually employed in sanitizing processing equipment. However, CIP is not always effective in removing biofilms. Ozone represents a promising "green" alternative to control biofilms. In this study, the effect of gaseous ozone (50 ppm) was evaluated <i>in vitro</i> against planktonic and sessile <i>B. cereus</i> and <i>B. subtilis</i> isolates collected from the dairy sector. Planktonic cells were enumerated by plate counts after 10 min, 1 h, and 6 h of ozone treatment. After a short-term (10 min) exposure, a slight reduction in microbial loads (0.66-2.27 ± 0.15 Log₁₀ CFU/mL) was observed for <i>B. cereus</i> strains, whereas a more pronounced reduction (2.90-3.81 ± 0.12 Log₁₀ CFU/mL) was noted in <i>B. subtilis</i> isolates. The microbial load further decreased after 1 h-treatments, around 1.5-3.46 ± 0.11 Log₁₀ CFU/mL for <i>B. cereus</i> strains, and 4.0-5.6 ± 0.11 Log₁₀ CFU/mL for <i>B. subtilis</i> isolates, until complete inactivation of bacterial cells after 6 h of exposure. Moreover, the effect of gaseous ozone treatment (50 ppm, 6 h) was evaluated for its ability to inhibit and eradicate biofilms formed on two common food-contact materials (polystyrene and stainless steel). Sessile <i>B. subtilis</i> cells were the more sensitive to the action of ozone, while a weak effect was highlighted on <i>B. cereus</i> isolates on both surface types. These results were further confirmed by scanning microscopy analysis. The number of cells in the biofilm state was also assessed, showing a not-complete correlation with a decrease in Biofilm Production Indices (BPIs). These findings highlighted the effectiveness of the sanitizing protocol using gaseous ozone in contrasting Bacillus free-living cells, but a not completely counteraction in biofilm formation (inhibition) or eradication of pre-formed biofilm. Thus, the application of ozone could be thought of not alone, but in combination with common sanitization practices to improve their effectiveness.