Sublethal Damage Caused by Cold Plasma on Bacillus cereus Cells: Impact on Cell Viability and Biofilm-Forming Capacity

The <i>Bacillus cereus</i> group represents a serious risk in powdered and amylaceous foodstuffs. Cold plasma (the fourth state of matter) is emerging as an alternative effective nonthermal technology for pasteurizing a wide range of matrices in solid, liquid, and powder form. The present study aims to evaluate the mechanisms involved in <i>Bacillus cereus</i> inactivation via cold plasma, focusing on (i) the technology's ability to generate damage in cells (at the morphological and molecular levels) and (ii) studying the effectiveness of cold plasma in biofilm mitigation through the direct effect and inhibition of the biofilm-forming capacity of sublethally damaged cells post-treatment. Dielectric barrier discharge cold plasma (DBD-CP) technology was used to inactivate <i>B. cereus</i>, <i>B. thuringiensis</i>, and <i>B. mycoides</i> under plasma power settings of 100, 200, and 300 W and treatment times ranging from 1 to 10 min. Inactivation levels were achieved in 2-7 log₁₀ cycles under the studied conditions. Percentages of sublethally damaged cells were observed in a range of 45-98%, specifically at treatment times below 7 min. The sublethally damaged cells showed poration, erosion, and loss of integrity at the superficial level. At the molecular level, proteins and DNA leakage were also observed for <i>B. cereus</i> but were minimal for <i>B. mycoides</i>. Biofilms formed by <i>B. cereus</i> were progressively disintegrated under the DBD-CP treatment. The greater the CP treatment intensity, the greater the tearing of the bacteria's biofilm network. Additionally, cells sublethally damaged by DBD-CP were evaluated in terms of their biofilm-forming capacity. Significant losses in the damaged cells' biofilm network density and aggregation capacity were observed when <i>B. cereus</i> was recovered after inactivation at 300 W for 7.5 min, compared with the untreated cells. These results provide new insights into the future of tailored DBD-CP design conditions for both the inactivation and biofilm reduction capacity of <i>B. cereus</i> sensu lato species, demonstrating the effectiveness of cold plasma and the risks associated with sublethal damage generation.