C12 | Study and evaluations on the in vitro inactivation of Escherichia coli using Plasma Activated Fog technology

A. Pandiscia¹, D. Aceto², P. Ambrico², P. Lorusso¹, A. Manfredi¹, V. Terio¹. | ¹ Dip. di Medicina Veterinaria, Università degli studi di Bari Aldo Moro, strada provinciale per Casamassima Km 3, Valenzano (BA); ²Consiglio Nazionale delle Ricerche, Istituto per la Scienza e Tecnologia dei Plasmi, CNR-ISTP, c/o Dip. Interateneo di Fisica “M. Merlin”, via Amendola 173, Bari.

Purpose. Ready-to-eat (RTE) foods are at high microbiological risk due to the possible presence of pathogens responsible for infections/food poisoning, such as Escherichia coli, Salmonella spp., Listeria monocytogenes and foodborne viruses. The production of RTE is expanding due to the convenience it offers consumers, and companies need innovative technologies to reduce microbiological risk. Among the emerging non-thermal technologies for food decontamination, plasma-activated water (PAW) is a sustainable and effective alternative for microbiological risk control. The antimicrobial efficacy of PAW is attributable to reactive oxygen and nitrogen species (RONS), which are generated during treatment. Plasma Activated Fog (PAF) technology is not limited to simply spraying PAW in a confined environment, but uses RONS produced by electrical discharge, at high concentrations and dispersed in an aerosol, for microbial decontamination. This study investigated the effectiveness of PAF for the in vitro inactivation of E. coli. Methods. A plasma source, powered by ambient air and operating at atmospheric pressure with different energy levels, was used to evaluate the most suitable parameters for the in vitro decontamination of E. coli. Two types of PAF were used: PAF_A with an average energy level of 40 mJ and PAF_B with an average energy level of 12 mJ. The E. coli CECT 9198 strain at 3 different concentrations (10², 10³ and 10⁴ CFU/100µL) was distributed evenly until complete adsorption on Plate Count Agar (PCA, Oxoid) plates. The plates were then exposed to PAF for 3 minutes and incubated for 24 hours at 37 °C. The qualitative and quantitative concentration of the products obtained by plasma activation of the two types of PAF used in the study (RONS) was monitored using commercial kits. All analyses were performed in duplicate. Results. Treatment with PAF_A demonstrated significantly higher antimicrobial efficacy than PAF_B. With PAF_A, a reduction of 2 log CFU/mL of E. coli was achieved in all dilutions tested, while with PAF_B, a smaller reduction was achieved (approximately 1.4 log CFU/mL). These results are closely related to the amount of RONS (nitrite ions (NO₂^-), nitrate ions (NO₃^-) and hydrogen peroxide (H₂O₂)) found in the two different PAFs. In particular, 135 mg/L, 1.00 mg/L, and 1.14 mg/L of NO₃^-, NO₂^-, and H₂O₂, respectively, were measured in PAF_A. Conclusions. PAF allowed for good inactivation of E. coli growth on PCA medium at the different concentrations examined, expressing an efficacy proportional to the energy used in the treatment and the relative presence of RONS. The results obtained allow us to assert that the use of PAF technology can be considered an innovative and promising approach to the microbial decontamination of RTE foods. Therefore, in the future, investigations will focus on evaluating the antimicrobial activity of PAF on other pathogens with investigations carried out directly on food matrices at risk.