A combined hurdle approach of organic acid simultaneous with X-ray irradiation to inactivate Bacillus cereus spores: synergistic effect and mechanisms

초록

Some Bacillus species are associated with food spoilage and various diseases. Their ability to form highly heat-resistant spores presents a significant challenge, necessitating the development of more effective inactivation strategies. The purpose of this study was to evaluate the sporicidal effects of combined treatments using X-ray and organic acids (phenyllactic, acetic, gallic, fumaric, caprylic, tartaric, citric, and malic acid) against Bacillus cereus spores. Among the organic acids tested, malic acid (MA) exhibited the greatest synergistic effect. MA was evaluated at concentrations 2–4%, with the maximum synergistic effect occurring at 4%. Treatment with a combination of 0.5 kGy X-ray irradiation and 4% MA resulted in a 5.78 log CFU/mL reduction in B. cereus spore counts. The synergistic effect of 4% MA corresponded to an additional 1.28 log CFU/mL reduction in B. cereus spore counts beyond the sum of the reductions achieved by the individual X-ray and MA treatments. To elucidate the inactivation mechanisms, intracellular reactive oxygen species (ROS) levels, spore membrane damage (propidium iodide uptake, scanning electron microscopy analysis, and dipicolinic acid release), intracellular esterase activity, and DNA integrity were evaluated following individual X-ray or MA treatment, and combined X-MA treatment. Mechanistic investigations suggested that the synergistic sporicidal effect is associated with a notable increase in ROS levels and concomitant partial membrane damage. These findings indicate that the combined X-MA treatment may serve as a promising non-thermal approach for controlling Bacillus spores in the food industry applications. © 2026 Elsevier Ltd

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