Application of chlorine dioxide (ClO2) gas treatment for inactivation of foodborne pathogens

Abstract

Chlorine dioxide (ClO2) has emerged as a promising non-thermal sanitizing technology. ClO2 is a strong oxidizing agent with a broad antimicrobial spectrum. Its efficacy is largely not affected by pH and organic matter and it does no react with nitrogen compounds to form chloramines. The most widely accepted antimicrobial mechanism of ClO2 is damage to protein synthesis and increased permeability of the outer cell membrane. ClO2 gas may be more effective for inactivation of foodborne pathogens than aqueous ClO2 due to its penetration ability. Also, ClO2 gas could be applied for microbial control during transportation and storage of food. Several studies have been evaluated the antimicrobial effect of ClO2 gas against foodborne pathogens on food and food contact surfaces. However, there is little information about factors affecting the antimicrobial efficacy of ClO2 gas. Also, a few studies are available that evaluate the antimicrobial efficacy of the combination treatment of ClO2 gas with other technology. The specific objectives of this study were, (ⅰ) to investigate the effect of relative humidity, surface characteristics of samples, and temperature on the antimicrobial efficacy of ClO2 gas against Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes on produce and food contact surfaces, (ⅱ) evaluate the antimicrobial effects of the combination treatment of ClO2 gas with ultraviolet (UV) radiation, aerosolized sanitizer, and dry heat against foodborne pathogens on produce and seeds, (ⅲ) develop portable sustained release formulation of ClO2 gas for field application. Spinach leaves and tomatoes were inoculated with three foodborne pathogens and treated with ClO2 gas at different concentrations (1, 5, 10, 30, or 50 ppmv) for up to 20 min under differing conditions of RH (50, 70, and 90%). As ClO2 gas concentration and treatment time increased, significant differences (p < 0.05) were observed between inactivation levels under different RH conditions. Generally, there were no significant differences (p > 0.05) in reduction levels of the three foodborne pathogens between 50 and 70% RH on spinach leaves. Exposure to 50 ppmv of ClO2 gas for 20 min resulted in 1.25 to 1.78 (50% RH) and 2.02 to 2.54 (70% RH) log reductions of the three foodborne pathogens on spinach leaves. The levels of the three foodborne pathogens was reduced to below the detection limit (1 log CFU/g) within 15 min when treated with 50 ppmv of ClO2 gas under 90% RH. Exposure to 30 ppmv of ClO2 gas (50% RH) for 20 min resulted in 1.22 to 1.52 log reductions of the three foodborne pathogens on tomatoes. Levels of the three foodborne pathogens were reduced to below the detection limit (0.48 log CFU/cm2) within 15 min when exposed to 30 ppmv of ClO2 gas at 70% RH, and within 10 min at 90% RH. Treatment with 30 ppmv of ClO2 gas did not significantly (p > 0.05) affect the color or texture of spinach leaves and tomatoes during 7 days of storage. To evaluate the influence of surface properties of samples on the antimicrobial effect of ClO2 gas against foodborne pathogens, the hydrophobicity of the selected surfaces was evaluated by water contact angle measurements. Also, white light scanning interferometry (WLSI) was used to acquire topographic images and surface roughness values of each surface. Produce (carrots, kale, cabbage, spinach, apples, tomatoes, and paprika) and food contact surfaces (Teflon, silicon, rubber, polyvinyl chloride, type 304 stainless steel with 2B or No.4 finish, and glass) inoculated with three foodborne pathogens were treated with 20 ppmv ClO2 gas for up to 15 min. Contact angles of produce and food contact surfaces were highly and negatively correlated with the log reduction of all three pathogens. The Ra (arithmetic mean roughness) values of produce surfaces were negatively correlated with the log reductions of the three pathogens, although the correlation coefficients were quite lower than those between contact angle and the bacterial log reductions. The Ra values of food contact surfaces were not significantly (p > 0.05) correlated with the log reductions of the three pathogens. The results of this study showed that surface hydrophobicity is a more important factor relating to bacterial inactivation by ClO2 gas from the surface than surface roughness. Produce and food contact surfaces inoculated with three foodborne pathogens were treated with 20 ppmv ClO2 gas at 15 and 25 °C under same conditions of absolute humidity for up to 30 min to evaluate how treatment temperature influences the solubility of ClO2 gas and the antimicrobial effect of ClO2 gas. As treatment time increased, ClO2 gas treatment at 15 °C caused significantly more (p < 0.05) inactivation of the three pathogens than ClO2 gas treatment at 25 °C. ClO2 gas treatment at 15 °C for 30 min resulted in 0.99 to 1.65, 1.05 to 1.50, and 1.25 to 1.61 further log reductions of the three pathogens on spinach leaves, tomatoes, and stainless steel No.4, respectively, compared to 25 °C treatment. Treatment with ClO2 gas at 25 °C for 20 min resulted in 1.88 to 2.31 log reductions of the three pathogens on glass while these pathogens were reduced to below the detection limit (0.48 log CFU/cm2) within 15 min when treated with ClO2 gas at 15 °C. ClO2 concentration on sample surfaces after ClO2 gas treatment at 15 °C were significantly (p < 0.05) higher than those treated at 25 °C. The antimicrobial effect of the combined treatment of UV-C radiation (UVC) and ClO2 gas against three foodborne pathogens on spinach leaves and tomato has been evaluated. In the case of spinach leaves, as treatment time increased the combined treatments of UVC and ClO2 gas showed additive effects: the total microbial inactivation of the combined treatment was not significantly (p > 0.05) different from the sum of individual treatments. On tomatoes, synergistic effects in inactivating E. coli O157:H7 and S. Typhimurium were observed after combination treatment of UVC and ClO2 gas (10 ppmv) for 15 min or more. For both pathogens, inactivation achieved with the combination treatment was significantly (p < 0.05) higher than the sum of UVC and ClO2 gas (10 ppmv) inactivation. In the case of L. monocytogenes, the synergistic effect was observed after the combination treatment of UVC and ClO2 gas (10 ppmv) for 20 min. Measuring leakage of UV-absorbing substances and analyzing transmission electron microscopy images provide evidence that damage to the cell membrane and changes to membrane permeability are involved in the synergistic lethal effect of the combination treatment of UVC and ClO2 gas. Combined treatment of UVC and ClO2 gas (10 ppmv) did not significantly (p > 0.05) affect the color and texture of samples during 7 days of storage. As an another available hurdle combination, the efficacy of ClO2 gas combined with aerosolized sanitizer for decontaminating spinach leave and tomatoes was investigated. ClO2 gas (5 or 10 ppmv) and aerosolized peracetic acid (PAA) (80 ppm) were applied alone or in combination for 20 min. Exposure to 10 ppmv of ClO2 gas for 20 min resulted in 3.39, 3.29, and 3.36 log reductions of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on spinach leaves, respectively. Treatment with 80 ppm of aerosolized PAA for 20 min caused 2.27, 1.89, and 0.84 log reductions of E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively. Combined treatment of ClO2 gas (10 ppmv) and aerosolized PAA (80 ppm) for 20 min caused 5.36, 5.06, and 4.06 log reductions of E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively. E. coli O157:H7, S. Typhimurium, and L. monocytogenes on tomatoes experienced similar reduction patterns to those on spinach leaves. As treatment time increased, most combinations of ClO2 gas and aerosolized PAA showed additive effects in the inactivation of the three pathogens. Combined treatment of ClO2 gas and aerosolized PAA produced injured cells of three pathogens on spinach leaves while generally did not produce injured cells of these pathogens on tomatoes. Combined treatment of ClO2 gas (10 ppmv) and aerosolized PAA (80 ppm) did not significantly (p > 0.05) affect the color and texture of samples during 7 days of storage. The antimicrobial effect of sequential treatment with ClO2 gas and dry heat against foodborne pathogens on alfalfa and radish seeds was evaluated. Inoculated alfalfa and radish seeds were treated with 150 ppmv of ClO2 gas for 1 h followed by 70 or 80 °C dry heat for 0, 1, 3 or 5 h. Dry heat treatment alone at 80 °C for 5 h resulted in 3.08 and 3.23 log reductions of E. coli O157:H7 and S. Typhimurium on alfalfa seeds, respectively. ClO2 gas treatment alone for 1 h resulted in 1.22 to 1.45 and 1.58 to 1.61 log reductions of E. coli O157:H7 and S. Typhimurium, respectively. Subsequent dry heat treatment (80 °C) for 5 h caused more than 5.32 and 5.29 log reduction of E. coli O157:H7 and S. Typhimurium, respectively. On radish seeds, dry heat treatment at 80 °C for 5 h resulted in 2.49 and 2.27 log reductions of E. coli O157:H7 and S. Typhimurium, respectively, and sequential treatment with ClO2 gas and dry heat (80 °C) for 5 h caused 4.38 and 4.11 log reduction of E. coli O157:H7 and S. Typhimurium, respectively. The germination rate of seeds did not significantly decrease after sequential treatment except for radish seeds sequentially treated with ClO2 gas and dry heat (80 °C). For on-site generation of ClO2 gas without equipment, mixture composition for sustained release of ClO2 gas was developed and its antimicrobial effect against foodborne pathogens on produce was evaluated. Sodium chlorite and citric acid were used to generate ClO2 gas, and diatomaceous earth (DE) was used to induce sustained release of ClO2 gas. Also, calcium chloride is used as a hydration accelerator. ClO2 gas release profiles of various mixture compositions were observed under conditions of 50 and 90 % relative humidity (RH) for up to 36 h at 22 ± 1 °C. RH affected the ClO2 gas release profile, and the generation rate and maximum ClO2 gas concentration could be controlled using DE and CaCl2. When 9 and 12 g of DE were added to the mixture, ClO2 gas concentration remained constant at 26 ± 1 ppmv for ca. 23 h and at 18 ± 1 ppmv for ca. 28 h, respectively, under conditions of 90 % RH. At 50% RH, when 0.05 g of CaCl2 was added to mixtures containing 0.5 and 0.35 g of DE, ClO2 gas concentration remained constant at 11 ± 1 ppmv for ca. 26 h and at 16 ± 1 ppmv for ca. 24 h, respectively. E. coli O157:H7 and S. Typhimurium were inoculated onto spinach leaves and tomatoes and exposed to ClO2 gas at different concentrations (10, 20, or 30 ppmv) under 50 and 90% RH conditions for up to 20 min. More than 6.16 and 5.48, and more than 6.78 and 6.34 log reductions of E. coli O157:H7 and S. Typhimurium on spinach leaves and tomatoes were observed after treatment with 30 ppmv of ClO2 gas for 15 and 10 min, respectively, at 90% RH. In conclusion, the results of this study are helpful for the food industry to establish ClO2 gas treatment conditions for maximizing the antimicrobial efficacy of ClO2 gas. The combination treatment of ClO2 gas with other technology may suggest alternatives to currently used decontamination methods. Also, portable ClO2 gas generating mixture could facilitate the use of ClO2 gas in the food industry.