Progress 09/15/04 to 08/31/09

Outputs
OUTPUTS: In this study, we evaluated the use of CLO2 gas on inactivation and survivability of Salmonella and Listeria monocytogenes on surfaces of tomatoes, oranges, and sprouts. The overall goal was to achieve a 5 log reduction (cfu/cm2) consistent with the recommendations of the National Advisory Committee on Microbiological Criteria for Foods (NACMCF). Foodborne outbreaks associated with raw tomatoes have promoted the industry and regulatory agencies to seek alternative intervention treatments. 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 (ClO2) gas, The mixed culture of Salmonella was inoculated onto tomato skin, stem, and stem scar areas. The tomato skin surfaces then were treated with 0.0, 0.3, 0.5, 0.7, 0.9 and 2.0 mg/l ClO2 gas for 10 min at 21C and 85-90% relative humidity. Stem and stem scar areas were treated with higher concentrations (5.0 and 8.0 mg/l ClO2 gas) since the bacteria are more difficult to reduce/eliminate in these confined areas. The results obtained from these preliminary studies were used to plan experiments to inactivate Salmonella enterica and Listeria monocytogenes on tomato skin surfaces using 0.1, 0.3, and 0.5 mg/l ClO2 gas. To resuscitate and enumerate the surviving Salmonella enterica and Listeria monocytogenes cells on tomato surfaces after ClO2 gas treatment, thin agar layer of Xylose-Lysine-Desoxycholate (XLD) agar and Modified Oxford agar (MOX) agars, respectively, were used as a top layer over Tryptic soy agar (TSA) in the plating procedure. Microorganisms including pathogens of public health significance have been shown to contaminate orange juice through washed oranges during the mechanical extraction of juice from oranges. A mixed culture of 4 Salmonella strains, isolated from orange juice outbreaks was spot inoculated onto orange skin surface areas. The oranges were then treated with 0.1, 0.3 and 0.5 mg/l ClO2 gas for 2-8 minutes at 21C and 90-95% relative humidity. Surviving bacteria on treated areas were recovered and enumerated over storage time by growing on a non-selective medium followed by a selective medium, Xylose Lysine Deoxycholate (XLD) agar. Salmonella has been the primary pathogen of concern in fresh, raw sprouts and 26 outbreaks of alfalfa sprouts and more than 1636 cases of illnesses have been reported by Center for Disease Control and Prevention (CDC) from 1995 to 2004. This indicates that the current practices are not adequate to prevent Salmonellosis from the consumption of fresh, raw sprouts. A mixed culture of 3 Salmonella strains (Salmonella Mbandaka, Salmonella Newport and Salmonella Stanley), recovered from alfalfa sprouts outbreaks was spot inoculated onto sprouts surfaces. The sprouts were then treated with 3.0 and 5.0 mg/l ClO2 gas for 6-20 minutes at 21C and 90% relative humidity. Surviving bacteria on treated areas were resuscitated and enumerated using a non-selective medium (Trypticase soy agar) with selective medium overlay (Xylose Lysine Deoxycholate). PARTICIPANTS: Purdue University (Richard Linton, Philip Nelson, Arpan Bhagat, Barakat Mahmoud, Yinchange Han) Michigan State University (Les Bourquin) Enerfab (Cinncinati,Ohio) Chlor Di Sys TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
For the stem and stem scar areas of tomatoes, pathogen reduction was challenging. Even at high concentrations of ClO2 (up to 8.0 mg/l) less than a 3 log reduction was observed. However a > 8 log reduction in Salmonella enterica and Listeria monocytogenes was observed on the tomato skin surface after treatment with 0.1, 0.3 and 0.5 mg/l ClO2 gas for 10 min. The results from pathogen inactivation studies were further validated in the laboratory as well as in a miniaturized industrial tunnel system using a bacterial surrogate. Also, bacterial inactivation studies were correlated with the effect on quality on the hydroponic tomatoes and no adverse effect was observed on the hydroponic tomatoes at the concentrations of ClO2 gas used in the experiment. A greater than 5 log reduction of Salmonella per in2 of orange surface was observed with 0.1 mg/l ClO2 and 0.3 mg/l ClO2 gas treatments after 8 minutes and a similar log reduction was observed at 0.5 mg/l ClO2 gas after 6 minutes. This result demonstrates that the treatment of oranges with ClO2 gas is a promising technology which could be successfully employed for the treatment of whole oranges to reduce the pathogens on orange surfaces, therefore reducing the risk of outbreaks due to contaminated orange juice. A greater than 3 log reduction of Salmonella per gram of sprouts was observed with 3.0 or 5.0 mg/l ClO2 gas treatment after 20 minutes. Similar results were obtained in a miniaturized industrial sized ClO2 gas tunnel system using H. alvei as a surrogate organism to validate the laboratory studies. These results demonstrate that treatment of sprouts with ClO2 gas has a potential to be successfully employed for the treatment of contaminated alfalfa sprouts to reduce the risk of Salmonella-related outbreaks. ClO2 gas is a promising technology for use as an antimicrobial for fresh tomato skin surface. The research has demonstrated the potential industrial application for this use of this novel technology. Information about this project and the potential use of this technology has resulted in 7 peer reviewd manuscripts and has been presented in several national and international food safety meetings.

Publications

• Bhagat, A., Mahmoud, B. and R. Linton 2009. Inactivation of Salmonella enterica and Listeria monocytogenes inoculated on hydroponic tomatoes using chlorine dioxide gas. Foodborne Pathogens and Disease (Accepted and in Press).

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica on strawberries by chlorine dioxide gas at different concentrations (0.5, 1, 1.5, 3 and 5 mg/l) for 10 min were studied. Approximately a 4.3-4.7 log CFU reduction per strawberry of all examined bacteria was achieved by treatment with 5 mg/l ClO2 for 10 min. The inactivation kinetics of E. coli O157:H7, L. monocytogenes and Salmonella enterica were determined using first-order kinetic models to establish D-values and z-values. The mean D-values of E. coli, L. monocytogenes and Salmonella enterica were 2.6, 2.3 and 2.7 min, respectively at 5 mg/l ClO2. The mean z-values of E. coli, L. monocytogenes and Salmonella enterica were 16.8, 15.8 and 23.3 mg/l, respectively. A surrogate organism was also identified so that studies could be carried out within a food processing plant environment. Thirty two strains of pathogenic and non-pathogenic microorganisms and 7 isolates from mushroom, tomatoes, and strawberries were studied. Among the 39 strains tested, MR1 (mushroom isolate), E. coli O157:H7 C7927 and 204P, STB2 (strawberry isolate), and Bacillus cereus 232 were most resistant to gaseous ClO2 treatment and D-values were 3.53, 1.95, 1.72, 1.68, and 1.57 min, respectively. MR1 and STB2 strains were evaluated with RobotyperTM with the EcoRI restriction enzyme of 16S rDNA sequence. The MR1 was identified as a Hafnia alvei both with a similarities value of 0.94 by ribotype pattern and with 93.6% similarity by API 20E. STB2 was identified as Enterococcus faecalis by ribotyping. PARTICIPANTS: Parternship with Michigan State University Partnership with produce industry, EnerFab (equipment manufacuterer), and chlorine dioxide gas manufacturer. TARGET AUDIENCES: Produce industry (on farm and manufacturing) PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Treatment with ClO2 gas significantly reduced inoculated foodborne pathogens and inherent microflora (mesophilic, psychrotrophic bacteria, yeasts and molds) on strawberries. Treatment with ClO2 gas did not affect the color of strawberries and extended the shelf-life to 16 days compared to 8 days for the untreated control. Collectively, this would provide an effective alternative to produce decontamination and would lead to safer and higher quality products. Development of surrogate microorganisms is very desirable to determine and validate the efficacy of ClO2 for the decontamination process of fresh produce because it is not prudent to introduce pathogens into a processing facility. Inactivation characteristics and kinetics of surrogate were used to predict those of the target pathogen when exposed to ClO2. The, MR1 surrogate strain would be a good non-pathogenic surrogate to evaluate ClO2 gas decontamination systems.

Publications

• Mahmoud, B. S. M. and R. H. Linton. 2008. Inactivation kinetics of inoculated Escherichia coli O157:H7, and Salmonella enterica on lettuce by chlorine dioxide gas. Food Microbiology 25 244-252.
• Mahmoud, B. S. M. and R. H. Linton. 2008. Inactivation kinetics of inoculated Escherichia coli O157:H7, and Salmonella Poona on whole cantaloupe by chlorine dioxide gas. Food Microbiology 25(7) 857-865.
• Kim, J. and R. H. Linton. 2008. Identification of a Non-Pathogenic Surrogate Organism for Pathogenic Bacteria Treated with Chlorine Dioxide Gas. Food Microbiology 25 597-606.

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica on strawberries by chlorine dioxide gas at different concentrations (0.5, 1, 1.5, 3 and 5 mg l-1) for 10 min were studied. The D-values of E. coli, L. monocytogenes and Salmonella enterica were 2.6, 2.3 and 2.7 min, respectively at 5 mg l-1 ClO2. The z-values of E. coli, L. monocytogenes and Salmonella enterica were 16.8, 15.8 and 23.3 mg l-1, respectively. The inactivation kinetics of inoculated E. coli O157:H7 and Salmonella enterica on lettuce leaves by ClO2 at different concentrations (0.5, 1, 1.5, 3 and 5 mg l-1) for 10 min was also studied. The D-values of E. coli and Salmonella enterica were 2.9 and 3.8 min, respectively at 5 mg l-1 ClO2 gas. The z-values of E. coli and Salmonella enterica were 16.2 and 21.4 mg l-1, respectively. The identification of non-pathogenic surrogate microorganisms is beneficial for determining and validating the efficacy of antimicrobial treatments used to reduce pathogens during food processing. In this study, a surrogate organism was identified to aid in the decontamination process of fresh produce when treated with ClO2 gas. Thirty two known strains of pathogenic and non-pathogenic microorganisms and 7 unknown microbial isolates from mushroom, tomatoes, and strawberries were evaluated. The main goal was to find alternative non-pathogenic organisms that had an equal to or higher resistance compared to E. coli O157:H7, Salmonella spp., and Listeria monocytognes. Among the strains tested, MR1 (mushroom isolate), E. coli O157:H7 C7927 and 204P, STB2 (strawberry isolate), and Bacillus cereus 232 were found to be the most resistant to gaseous ClO2 treatment and D-values were 3.53, 1.95, 1.72, 1.68, and 1.57 min, respectively. For identification, the MR1 and STB2 strains were evaluated using a RibotyperTM with the EcoRI restriction enzyme of 16S rDNA sequence. MR1 was identified as Hafnia alvei with a similarity value of 0.94 using the ribotype pattern and with a 93.6% similarity using a API 20E strip. STB2 was identified as Enterococcus faecalis by ribotyping. The Ped-2E9-based cytotoxicity assay was conducted for the extracellular toxin and whole cell toxicity. Both isolated strains did not show any cytotoxicity. PARTICIPANTS: Barakat Mahmoud, Purdue University Arpan Bhaghat, Purdue University Les Bourquin, Purdue University Jay Kim, University of Korea Bruce Applegate, Purdue University TARGET AUDIENCES: Academia Fruit and Vegetable Industry State, local, and federal government

Impacts
Treatment with ClO2 gas significantly (p&#8804;0.05) reduced the initial microflora (mesophilic, psychrotrophic bacteria, yeasts and molds) on strawberries. Treatment with ClO2 gas did not affect the color of strawberries and extended the shelf-life to 16 days compared to 8 days for the untreated control. Treatment with ClO2 gas significantly reduced inherent microflora on lettuce and microbial counts remained significantly (p<0.05) lower than the un-inoculated control during storage at 4oC for 7 days. However, treatment with ClO2 had a significantly (p< 0.05) negative impact on visual leaf quality. These results showed that treatment with ClO2 gas significantly reduced selected pathogens and, inherent microorganisms on lettuce, however, the processing conditions would likely need to be altered for consumer acceptance. Inactivation characteristics and kinetics of surrogate organisms can be used to predict processing conditions needed to inactivate targeted levels of foodborne pathogens exposed to ClO2. Hafnia alvei is a suitable non-pathogenic surrogate, with slightly higher resistance to ClO2 gas compared to pathogens studied, that may be useful to establish optimum conditions of ClO2 gas decontamination systems for produce.

Publications

• Mahmoud, B. S. M. and R. H. Linton. 2007. Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. on strawberries by chlorine dioxide gas. Food Microbiology: 24: 736-744.

Progress 10/01/05 to 09/12/06

Outputs
Consumers and restaurateurs are increasingly purchasing raw fruit and vegetable products. Recent foodborne outbreaks associated with these products have promoted the industry and regulatory agencies to seek alternative intervention treatments. The purpose of our studies this year was to evaluate the inactivation of common foodborne pathogens exposed to chlorine dioxide gas treatments on selected fruit and vegetable surfaces (tomatoes and strawberries). Inactivation of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. on strawberries by chlorine dioxide gas at different concentrations (0.5, 1.0, 1.5, 3.0 and 5 mg per liter) for 10 min were studied. Approximately a 4.3-4.7 log CFU reduction per strawberry of all examined bacteria was achieved by treatment with 5 mg per liter chlorine dioxide for 10 min. More than a 3 log CFU reduction per strawberry was achieved by treatment with 1.5 and 3 mg per liter chlorine dioxide for 10 min. The bactericidal effect of ClO2 against these bacteria was increased by increasing chlorine dioxide concentration and/or treatment time. The inactivation kinetics of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. were also determined. The D-values of E. coli, L. monocytogenes and Salmonella were 4.7, 4.6 and 4.2 min, respectively at 0.5 mg per liter chlorine dioxide after 10 min. While, the D-values of E. coli, L. monocytogenes and Salmonella spp. were 2.6, 2.3 and 2.7 min, respectively at 5 mg per liter chlorine dioxide after 10 min. A 5 log CFU reduction of E. coli, L. monocytogenes and Salmonella spp. could be achieved with 5 mg per liter ClO2 gas for 12.5, 11.5 and 13.5 min, respectively. The z-values of E. coli, L. monocytogenes and Salmonella spp. were 16.8, 15.8 and 23.3 mg per liter, respectively. Our next objective was to determine survivability of Salmonella spp. and Listeria spp. on hydroponic tomatoes after treatment with chlorine dioxide gas. Our goal was to achieve a 5 log reduction consistent with the recommendations of the National Advisory Committee on Microbiological Criteria for Foods (NACMCF). The mixed culture of Salmonella was inoculated onto tomato skin, stem, and stem scar areas. The tomatoes were then treated with 0.0, 0.3, 0.5, 0.7, 0.9 and 2.0 mg per liter chlorine dioxide gas for 10 minutes at 22 C and 85% relative humidity. Stem and stem scar areas were also treated with higher concentrations (5 and 8 mg/l chlorine dioxide gas). A greater than 8 log reduction in Salmonella spp. (p< 0.05) was observed on the tomato skin surface after treatment with 0.3mg/l chlorine dioxide gas for 10 minutes. For the stem and stem scar areas, pathogen reduction was far more challenging. Even at high concentrations of chlorine dioxide (up to 8mg/l) less than 3 log reduction was observed.

Impacts
These results imply that the treatment with chlorine dioxide gas had a very powerful antimicrobial effect against tested pathogenic bacteria on produce. This relatively new technology could be a viable alternative for pathogen reduction methods used to make produce safer. It is our hope in the next year to use this in-laboratory information to scale up to our pilot plant tunnel system.

Publications

• Linton, R. H., Han, Y., Selby, T. L., and Nelson, P. E. (2005) Gas/vapor-phase decontamination treatments for produce. In Microbiology of Fruits and Vegetables. Ed. G. M. Sapers, J. R. Gorny, and A. E. Yousef. CRC Press, LLC.

Progress 10/01/04 to 09/30/05

Outputs
Fresh fruits and vegetables are seen as important components of a healthy diet and there has been an increased demand for citrus products by consumers seeking convenient and healthy food products. Orange juice is the most popular fruit beverage in the United States and in many other countries. The number of foodborne illness outbreaks linked to fresh produce has increased in the last few years. This is often attributed to inherent contamination and the minimum processing that is often used for fresh and fresh-cut produce. Newer processing steps that are effective in reducing pathogens should be studied and developed to improve safety of these products. The primary objective this year was to establish a mathematical model for reducing Salmonella spp. on orange surfaces using chlorine dioxide (ClO2) gas treatments within a newly developed tunnel system. Naval organic oranges were spot inoculated at the calyx, stem, and side areas with 100 &#61617; 1 &#61549;l of a mixed culture of Salmonella spp. and air dried for 1 h at 23C. Treated areas were aseptically cut and remaining bacteria enumerated using a membrane transfer plating method on Xylose Lysine Desoxycholate agar. A central composite rotatable design was used to determine the effect of time and concentration of chlorine dioxide on the reduction of Salmonella spp. Concentrations ranged from 0.09 to 0.51 mg/l and times from 1 to 14 min. After analyzing the data with response surface methodology, a recommended treatment was chosen that resulted in a 5 log10 reduction for Salmonella spp. Information from this model was then used as a basis to test the efficacy of a selected treatment on the reduction of Escherichia coli O157:H7, Listeria spp. and Shigella spp. Salmonella spp. was significantly reduced by the ClO2 treatments studied (2 to &#8805; 6.0 log. A 5 log reduction for Salmonella spp. for a treatment of 0.24 mg/l for 10 min also led to high microbial reductions for the other pathogens tested. Reductions of 5.4 log for E. coli O157:H7 and greater than 6.0 log reductions for Shigella spp and Listeria spp. were observed. The recommended chlorine dioxide (0.24 mg/l for 10 min) can be successfully used to reduce pathogens on orange surfaces therefore reducing the risks associated with derived products.

Impacts
The use of chlorine dioxide as an alternative antimicrobial treatment for orange surfaces could have a significant public health, food quality, and economic impact for the juice industry. The new technology, once optimized, will effectively reduce foodborne pathogens which minimizes the risk of foodborne illness. Reduction of spoilage organisms will enhance quality and lengthen product shelf-life.

Publications

• None in 2005 - project has recently started.

Progress 10/01/03 to 09/29/04

Outputs
This project has just started. No progress to report.

Impacts
This project has just started. No impact to report.

Publications

• This project has just started. No publications to report in 2004.