Bacterial inactivation and bactericidal mechanisms of pulsed ohmic heating against foodborne pathogens in milk depending on the applied frequency

Conflicting results have been reported regarding whether the relationship between frequency and microbial inactivation in ohmic heating (OH) is direct or inverse. The main purpose of this study was to investigate the bacterial inactivation and bactericidal mechanism of pulsed ohmic heating (POH) depending on the applied frequency. Milk samples inoculated with foodborne pathogens were subjected to POH at a constant voltage of 70 V_rms with applied frequencies of 0.06, 0.1, 1, or 10 kHz. The samples were heated to 60°C using POH and held at that temperature for up to 10 min. The bacterial inactivation levels of POH at 0.06 kHz were significantly greater (P < 0.05) than those at 0.1–10 kHz. At 0.06 kHz, for instance, Salmonella Typhimurium reduced more than 2 log CFU/ml compared with that at 0.1–10 kHz. Cell membrane depolarization, analyzed using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC₄(3)), was significantly greater after POH at 0.06 kHz compared to other frequencies. The value of crystal violet uptake, which reflects cell permeability, was also highest at 0.06 kHz. Because POH at 0.06 kHz fatally damaged cell membranes and increased permeability, the largest amount of intracellular material leaked. Through a DNA integrity assessment, POH at 0.06 kHz provoked severe DNA damage to all the pathogens. It was speculated that these results occurred because POH at 0.06 kHz provided enough time for the buildup of charges around the cell membranes and DNA. This research provides a fundamental scientific basis for determining the mechanism by which low-frequency POH results in high bacterial inactivation.