Simultaneous combination treatment with superheated steam and nebulized organic acid to inactivate Bacillus cereus endospores on stainless steel surfaces

Superheated steam is a practical thermal-inactivation technology used in the food industry; however, temperatures above 200 ? are required to reduce the number of endospore cells on surfaces. The purpose of this study was to develop a system combining treatments with superheated steam (SHS) (below 200 ?) and nebulized organic acids (lactic, malic, and citric acid) and to evaluate its sporicidal effects against B. cereus endospores. The results showed that the sporicidal effects of SHS at 150 ? and 200 ? were significantly (P < 0.05) enhanced when 10% of the organic acids were nebulized into SHS. The combination treatment with SHS at 150 ? and 10% lactic, malic, and citric acids for 5 min reduced the endospores on stainless steel by 3.2, 2.6, and 1.9 log CFU/cm(2) respectively. To investigate the mechanisms underlying the combination treatment-mediated inactivation of the spores, dipicolinic acid release, diphenyl-1-pyrenylphosphine oxide (DPPP=O) value, superoxide dismutase activity, intracellular reactive oxygen species (ROS) amount, and DNA integrity were measured. The synergistic effects of the combination treatment were determined to be a consequence of increased inner membrane and DNA damage induced by membrane peroxidation and accumulation of intracellular ROS, respectively. However, only inner membrane damage contributed to the difference in the sporicidal effects between the three organic acid combination treatments. Our findings can be used for surface sterilization in the food industry or as basic information for further studies on eliminating endospore contamination.