Richard H. Linton

Reduction of listeria monocytogenes on green peppers (Capsicum annuum L.) by gaseous and aqueous chlorine dioxide and water washing and its growth at 7 degrees C.

Reduction of Listeria monocytogenes Scott A on uninjured and injured surfaces of green peppers after 0.3- and 3-mg/ liter gaseous and aqueous ClO2 treatment and water washing for 10 min at 20 degrees C was studied. Growth of the L. monocytogenes untreated or treated with 0.6 mg/liter ClO2 gas for 30 min at 20 degrees C on green peppers also was investigated. A membrane-surface-plating method was used for resuscitation and enumeration of L monocytogenes treated with ClO2. The bacterial viability on pepper surfaces was visualized using confocal laser scanning microscopy (CLSM). Live and dead cells of L. monocytogenes were labeled with a fluorescein isothiocyanate-labeled antibody and propidium iodide, respectively. More than 6 log CFU/5 g L. monocytogenes on uninjured surfaces and about 3.5 log CFU/5 g on injured surfaces were inactivated by both 3-mg/liter and 0.6-mg/liter ClO2 gas treatments. The 3-mg/liter aqueous ClO2 treatment achieved 3.7- and 0.4-log reductions on uninjured and injured surfaces, respectively; whereas, water washing alone showed 1.4- and 0.4-log reductions, respectively. ClO2 gas treatment was the most effective in reducing L. monocytogenes on both uninjured and injured green pepper surfaces, when compared with aqueous ClO2 treatment and water washing. The significant difference (P < 0.05) between log reductions on uninjured and injured surfaces and the results from CLSM analysis suggested that injured surfaces protected more bacteria from sanitation treatments than did uninjured surfaces. Not only could L. monocytogenes grow on green pepper surfaces at 7 degrees C, bacteria that survived the 0.6-mg/liter ClO2 gas treatment also could grow.

Decontamination of strawberries using batch and continuous chlorine dioxide gas treatments.

Efficacy of chlorine dioxide (ClO2) gas in reducing Escherichia coli O157:H7 and Listeria monocytogenes on strawberries was determined using batch and continuous flow ClO2 gas treatment systems. Effects of continuous ClO2 gas treatment on total aerobic plate count, color, and residual ClO2 and chlorite on strawberries were also evaluated. Strawberries were spot inoculated with 7 to 8 log CFU per strawberry of each pathogen (E. coli O157:H7 and L. monocytogenes), stored for 1 day at 4 degrees C, and treated at 22 degrees C and 90 to 95% relative humidity with 0.2 to 4.0 mg/liter ClO2 gas for 15 or 30 min using a batch treatment system or with 0.6, 1.8, and 3.0 mg/liter for 10 min using a continuous treatment system. Surviving microbial populations were determined using a membrane-transfer plating recovery method. Increased ClO2 gas concentrations resulted in increased log reductions of each pathogen for both the batch and continuous systems. A batch treatment of strawberries with 4 mg/liter ClO2 for 30 min and continuous treatment with 3 mg/liter ClO2 for 10 min achieved greater than a 5-log reduction for both E. coli O157:H7 and L. monocytogenes. After continuous exposure to 3.0 mg/liter ClO2 gas for 10 min followed by 1 week of storage at 4 degrees C, no aerobic microorganisms were detected and the color of the strawberry surface did not change significantly (P > 0.05). Residues of ClO2 and chlorite on strawberries after the treatment were 0.19 +/- 0.33 mg ClO2 per kg and 1.17 +/- 2.02 mg Cl2 per kg, respectively, whereas after 1 week of storage no ClO2 residues were detected and residual chlorite levels were down to 0.07 +/- 0.12 mg Cl2 per kg. These results suggest that ClO2 gas treatment is an effective decontamination technique for improving the safety of strawberries while extending shelf life.

Inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce by chlorine dioxide gas.

The purpose of this investigation was to study inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce leaves by ClO(2) gas at different concentrations (0.5, 1.0, 1.5, 3.0, and 5.0 mg l(-1)) for 10 min and to determine the effect of ClO(2) gas on the quality and shelf life of lettuce during storage at 4 degrees C for 7 days. One hundred microliters of each targeted organism was separately spot-inoculated onto the surface (5 cm(2)) of lettuce (approximately 8-9 log CFU ml(-1)), air-dried, and treated with ClO(2) gas at 22 degrees C and 90-95% relative humidity for 10 min. Surviving bacterial populations on lettuce were determined using a membrane transferring method, which included a non-selective medium followed by a selective medium. The inactivation kinetics of E. coli O157:H7 and S. enterica was determined using first-order kinetics to establish D-values and z-values. The D-values of E. coli and S. enterica were 2.9+/-0.1 and 3.8+/-0.5 min, respectively, at 5.0 mg l(-1) ClO(2) gas. The z-values of E. coli and S. enterica were 16.2+/-2.4 and 21.4+/-0.5 mg l(-1), respectively. A 5 log CFU reduction (recommended by the United States Food and Drug Administration) for E. coli and S. enterica could be achieved with 5.0 mg l(-1) ClO(2) gas for 14.5 and 19.0 min, respectively. Treatment with ClO(2) gas significantly reduced inherent microflora on lettuce and microbial counts remained significantly (p<0.05) lower than the uninoculated control during storage at 4 degrees C for 7 days. However, treatment with ClO(2) gas had a significantly (p<0.05) negative impact on visual leaf quality. These results showed that treatment with ClO(2) gas significantly reduced selected pathogens and inherent microorganisms on lettuce; however, the processing conditions would likely need to be altered for consumer acceptance.

Response surface modeling for the inactivation of Escherichia coli O157:H7 on green peppers (Capsicum annuum L.) by chlorine dioxide gas treatments.

The effects of chlorine dioxide (ClO2) gas concentration (0.1 to 0.5 mg/liter), relative humidity (RH) (55 to 95%), treatment time (7 to 135 min), and temperature (5 to 25 degrees C) on inactivation of Escherichia coli O157:H7 on green peppers were studied using response surface methods. A four-factor, central, composite, rotatable design was used. The microbial log reduction was measured as a response. A direct membrane-surface-plating method with tryptic soy agar and sorbitol MacConkey agar was used to resuscitate and enumerate ClO2-treated E. coli O157:H7 cells. The statistical analysis and the predictive model developed in this study suggest that ClO2 gas concentration, treatment time, RH, and temperature all significantly (P < 0.01) increased the inactivation of E. coli O157:H7. ClO2 gas concentration was the most important factor, whereas temperature was the least significant. The interaction between ClO2 gas concentration and RH indicated a synergistic effect. The predictive model was validated, and it could be used to determine effective ClO2 gas treatments to achieve a 5-log reduction of E. coli O157:H7 on green peppers.

Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Poona on whole cantaloupe by chlorine dioxide gas.

The objectives of this study were to examine inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Poona inoculated onto whole cantaloupe and treated with ClO(2) gas at different concentrations (0.5, 1.0, 1.5, 3.0 and 5.0 mg l(-1)) for different times (0, 2.0, 4.0, 6.0, 8.0 and 10.0 min). The effect of ClO(2) gas on the quality and shelf life of whole cantaloupe was also evaluated during storage at 22 degrees C for 12 days. A 100 microl inoculation of each targeted organism was spotted onto the surface (5 cm(2)) of cantaloupe rind (approximately 8-9 log CFU 5 cm(-2)) separately, air dried (60 min), and then treated with ClO(2) gas at 22 degrees C and 90-95% relative humidity for 10 min. Surviving bacterial populations on cantaloupe surfaces were determined using a membrane transferring method with a non-selective medium followed by a selective medium. The inactivation kinetics of E. coli O157:H7, L. monocytogenes and S. Poona were determined using nonlinear kinetics (Weibull model). A 3 log CFU reduction of E. coli O157:H7, L. monocytogenes and S. Poona were achieved with 5.0 mg l(-1) ClO(2) gas for 5.5, 4.2 and 1.5 min, respectively. A 5l og CFU reduction of S. Poona was achieved with 5.0 and 3.0 mg l(-1) ClO(2) gas for 6 and 8 min, respectively. A 4.6 and 4.3 log reduction was achieved after treatment with 5.0 mg l(-1) ClO(2) gas at 10 min for E. coli O157:H7 and L. monocytogenes, respectively. Treatment with 5.0 mg l(-1) ClO(2) gas significantly (p<0.05) reduced the initial microflora (mesophilic bacteria, psychrotrophic bacteria, and yeasts and molds) on cantaloupe by more than 2 log CFU cm(-2) and kept them significantly (p<0.05) lower than the untreated control during storage at 22 degrees C for 12 days. Treatment with ClO(2) gas did not significantly (p>0.05) affect the color of whole cantaloupe and extended the shelf life to 9 days compared to 3 days for the untreated control, when stored at ambient temperature (22 degrees C).

Use of high-concentration-short-time chlorine dioxide gas treatments for the inactivation of Salmonella enterica spp. inoculated onto Roma tomatoes

Salmonella outbreaks have been recently linked to the consumption of fresh tomatoes. Thus, there is a need to develop systems that reduce the risk of microbial contamination to increase product shelf-life and keep fresh fruit attributes. The objectives of this study were to evaluate high-concentration-short-time chlorine dioxide gas treatments effects on Salmonella-inoculated Roma tomatoes and determine the optimal treatment conditions for microbial inactivation and shelf-life extension. Effects of ClO(2) concentration (2, 5, 8 and 10mg/l) and exposure time (10, 30, 60, 120 and 180 s) on inoculated Roma tomatoes were studied. Salmonella enterica strains, serotype Montevideo, Javiana and Baildon, were used to experimentally inoculate the food product. After ClO(2) treatments, tomatoes were stored at room temperature for 28 days. Inherent microbial population, change in tomato color, and chlorine dioxide gas residuals were evaluated. ANOVA analysis showed that both ClO(2) concentration and treatment time were significant (p<0.01) for Salmonella inactivation. Surviving Salmonella populations of 3.09, 2.17 and 1.16 logCFU/cm(2) were obtained treating tomatoes with 8 mg/l ClO(2) for 60 s, 10 mg/l ClO(2) for 120 s, and 10 mg/l for 180 s, respectively (initial Salmonella population: 6.03±0.11 log CFU/cm(2)). The selected treatments significantly reduced background microflora (p<0.05), while fruit color and residual contents were not significantly different (p>0.05), as compared to the control. Results suggest the potential for high-concentration-short-time treatments ClO(2) gas as an effective pathogen inactivation technology for large-scale produce packing operations.

Inactivation of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on spinach leaves by X-ray

Several recent foodborne disease outbreaks associated with leafy green vegetables, including spinach, have been reported. X-ray is a non-thermal technology that has shown promise for reducing pathogenic and spoilage bacteria on spinach leaves. Inactivation of inoculated Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on spinach leaves using X-ray at different doses (0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy) was studied. The effect of X-ray on color quality and microflora counts (mesophilic counts, psychrotrophic counts and yeast and mold counts) of untreated and treated spinach was also determined. A mixture of three strains of each tested organism was spot inoculated (100 microl) onto the surface of spinach leaves (approximately 8-9 log ml(-1)), separately, and air-dried, followed by treatment with X-ray at 22 degrees C and 55-60% relative humidity. Surviving bacterial populations on spinach leaves were evaluated using a nonselective medium (tryptic soy agar) with a selective medium overlay for each bacteria; E. coli O157:H7 (CT-SMAC agar), L. monocytogenes (MOA), and S. enterica and S. flexneri (XLD). More than a 5 log CFU reduction/leaf was achieved with 2.0 kGy X-ray for all tested pathogens. Furthermore, treatment with X-ray significantly reduced the initial inherent microflora on spinach leaves and inherent levels were significantly (p < 0.05) lower than the control sample throughout refrigerated storage for 30 days. Treatment with X-ray did not significantly affect the color of spinach leaves, even when the maximum dose (2.0 kGy) was used.

Evaluation of chlorine dioxide gas residues on selected food produce.

In recent years, the consumption of fresh fruits and vegetables has greatly increased, and so has its association with contamination of several foodborne pathogens (Listeria, Salmonella, and Escherichia coli). Hence, there is a need to investigate effective sanitizer systems for produce decontamination. Chlorine dioxide (ClO(2)), a strong oxidizing gas with broad spectrum and sanitizing properties, has previously been studied for use on selected fruits and vegetables. ClO(2) gas treatments show great potential for surface pathogen reduction; however its use from a residue safety standpoint has yet to be assessed. Thus, the objective of this study was to evaluate residues of ClO(2), chlorite, chlorate, and chloride on selected fresh produce surfaces after treatment with ClO(2) gas. A rinse procedure was used and water samples were analyzed by N, N-diethyl-p-phenylenediamine and ion chromatography method (300.0). Seven different foods--tomatoes, oranges, apples, strawberries, lettuce, alfalfa sprouts, and cantaloupe--were analyzed after ClO(2) treatment for surface residues. Very low residues were detectable for all the food products except lettuce and alfalfa sprouts, where the measured concentrations were significantly higher. Chlorine dioxide technology leaves minimal to no detectable chemical residues in several food products, thus result in no significant risks to consumers. Practical Application: Potential for chlorine dioxide gas treatments as an effective pathogen inactivation technology to produce with minimal risk for consumers.

A comparative study on the effectiveness of chlorine dioxide gas, ozone gas and e-beam irradiation treatments for inactivation of pathogens inoculated onto tomato, cantaloupe and lettuce seeds

The increase in reported food-borne outbreaks linked with consumption of raw fruits and vegetables has motivated new research focusing on prevention of pre-harvest produce contamination. This study evaluates and compares the effectiveness of three non-thermal technologies, chlorine dioxide gas, ozone gas and e-beam irradiation, for inactivation of Salmonella enterica and Escherichia coli O157:H7 on pre-inoculated tomato, lettuce and cantaloupe seeds, and also their corresponding effect on seeds germination percentage after treatments. Samples were treated with 10mg/l ClO(2) gas for 3 min at 75% relative humidity, with 4.3mg/l ozone gas for 5 min and with a dose of 7 kGy electron beam for 1 min. Initial load of pathogenic bacteria on seeds was ~6 log CFU/g. Results demonstrate that all treatments significantly reduce the initial load of pathogenic bacteria on seeds (p<0.05). In particular, after ozone gas treatments 4 log CFU/g reduction was always observed, despite the seeds and/or microorganisms treated. ClO(2) and e-beam treatments were noticeably more effective against Salmonella on contaminated tomato seeds, where 5.3 and 4.4 log CFU/g reduction were respectively observed. Germination percentage was not affected, except for cantaloupe seeds, where the ratio was significantly lowered after ClO(2) treatments. Overall, the results obtained show the great applicability of these non-thermal inactivation techniques to control and reduce pathogenic bacteria contamination of seeds.

Inactivation of Salmonella enterica and Listeria monocytogenes Inoculated on Hydroponic Tomatoes Using Chlorine Dioxide Gas

The main objective of this study was to determine survivability of a cocktail of three strains of Salmonella enterica (Montevideo, Javiana, and Baildon) and two strains of Listeria monocytogenes (LCDC 81-861 and F4244) on hydroponic tomatoes after treatment with chlorine dioxide (ClO(2)) gas. An initial concentration of 8-9 log cfu/mL of Salmonella and Listeria cocktails was inoculated individually, in separate experiments, on tomato skin to obtain a population of 7-8 log cfu/cm(2) after drying of the inoculums on the tomato skin. The aim was to achieve a 5 log reduction consistent with the recommendations of the National Advisory Committee on Microbiological Criteria for Foods. The tomato skins were treated with 0.1, 0.3, and 0.5 mg/L ClO(2) gas for 12 min at 22 degrees C and at the relative humidity of 90%. Untreated skin samples were processed under the same conditions. ClO(2)-gas-treated and untreated samples were recovered by an overlay method. The bottom layer contains tryptic soy agar, and the top layer consists of xylose-lysine-desoxycholate agar or modified Oxford antimicrobial supplement agar for Salmonella and Listeria, respectively. More than a 5 log reduction in Salmonella and Listeria was observed on the tomato skin surfaces after treatment with 0.5 mg/L ClO(2) gas for 12 min. Treatment with 0.5 mg/L ClO(2) gas for 12 min also delayed the growth of natural microflora on tomato surfaces and extended the shelf life of tomatoes by 7 days during storage at 22 degrees C, compared with the untreated control. These results revealed that ClO(2) gas is a promising antimicrobial technology for fresh tomato skin surfaces.