1. Use of Chlorine Dioxide Gas as an Antimicrobial Agent for Foods and Food Contact Surfaces Richard H. Linton, Professor of Food Safety Valentina Trinetta, Postdoctoral Research Associate Mark T. Morgan, Professor of Food Engineering October 25, 2010

2. Impact of Fresh Produce Safety “Of 110 recent outbreaks, 35% were associated with fresh fruits and vegetables ” - CDC “ Fresh fruit and vegetables are responsible for more illnesses, by far, than any other commodity we regulate” - FDA

3. PATHOGENFOOD VEHICLE Salmonella spp. Alfalfa sprouts, cantaloupe, lettuce, orange juice, salads items, tomatoes, peppers, spinach, bean sprouts E. coli O157:H7 Apple cider/juice, cabbage, celery, coleslaw, cucumber, lettuce, mushroom, potatoes, radishes, salad items, spinach, onion Shigella spp. Cantaloupe, scallions, sprouts, tomatoes, watermelon, L. monocytogenesColeslaw, green onion, lettuce, milk, chicken CyclosporaRaspberries Hepatitis A virus and Norovirus Green onions, leafy greens, strawberries Produce Associated Outbreaks (Data taken from Beuchat et al. 1996; CDC 2010; NACMCF 2009)

4. Aqueous Chemical Use for Produce ANTIMICROBIAL AGENTUSESEFFECTIVNESS Hypochlorous acid, hypochlorites (200 ppm, 20,000 ppm sprouts) Whole & fresh cut, processing waters 1-2 log reduction Ozone (1-4 ppm)Whole & fresh cut, processing waters 1-3 log reduction Chlorine dioxide – aqueous (1-5 ppm)Whole & fresh cut, processing waters 1 log reduction Hydrogen peroxide (5%)Whole & fresh cut,3 log reduction Peroxyacetic acid (200 ppm)Whole & fresh cut, processing waters 2 log reduction Organic acidsWhole & fresh cut,1-2 log reduction OUR GOAL (ClO 2 gas)Fresh produce>5 log reduction (Cherry, J. Food Technology Vol. 53, No. 11)

5. An EPA laboratory bus circles the Hart building to monitor the air for gas leaks Clean-up postal office at Hamilton Township, NJ Chlorine Dioxide? 2001

6. A Promising Antimicrobial Alternative Greenish-yellow gas, smells like chlorine Highly soluble in water Higher oxidation capacity than chlorine (2.5 x) Oxidizes rather than chlorinates Works over broad pH range (2 – 10) Wide spectrum of antimicrobial capabilities Low dosage required Aqueous and gaseous forms can be used Chlorine Dioxide (ClO 2 )

7. History of uses of aqueous ClO 2 - 1811 First generated by Davy (acidification of KClO 3 ) - 1930’s Industrial scale preparation with sodium chlorite - 1940’s Bleaching agent in pulp and paper industry - 1967 Registered with EPA as disinfectant/sanitizer - 1977 Used for water treatment plants in USA and Europe - 1990’s 500-900 water utilities in USA - 1995 Approved by FDA for poultry processing water - 1998 Approved by FDA for washing produce (<3ppm) Aqueous ClO 2

8. History of uses of gaseous ClO 2 - 1988 Registered by EPA as a sterilant (for hard surfaces and implements) - 1990’s Research on pathogen reduction in produce - 2000 GRAS status for ClO 2 releasing LDPE films (Bernard Technologies, Inc., Chicago, IL) - 2001 Approved by EPA for emergency use in anthrax decontamination - 2002 Approved by EPA for storage of potatoes Gaseous ClO 2

9. 2 % Chlorine Gas Sodium Chlorite ClO 2 Monitor %RH Controller Cl 2 + 2 NaClO 2 → 2 ClO 2 + 2 NaCl Generation of ClO 2 Gas

10. 4 HA + 5 NaClO 2  4 ClO 2 + NaCl + 4 Na + + 2 H 2 O Other Methods to Generate ClO 2 Gas (Courtesy of ICA TriNova, LLC, Atlanta, GA)

11. Our Journey… Can we commercially sterilize aseptic holding tanks? Can we kill pathogens on food contact surfaces? Produce surfaces? What are the optimum conditions? What impact is there on quality (microbial, chemical, physical) and chemical safety? How can we build systems that the industry can use? How do we gain approval from FDA, EPA?

12. ClO 2 on Epoxy Aseptic Tank Surfaces for Inactivation of Spoilage Organisms

13. ClO 2 on Epoxy Aseptic Tank Surfaces for Inactivation of Spoilage Organisms

14. Batch ClO 2 Gas Treatment System Cl 2 /N 2 gas Generator Chamber Monitor Humidifier Cl 2 + 2 NaClO 2 2 ClO 2 + 2 NaCl

15. Treated with 3 mg/l ClO 2 for 30 min and stored for 6 weeks at 4 o C Untreated and stored for 6 weeks at 4 o C ClO 2 Gas Treatment (Batch) on Strawberries

16. Inactivation of E. coli O157:H7 by ClO 2 Gas on Peppers Treatments for 30 min at 20 o C under 90-95%RH Significant differences (P<0.05) (Han et al., 2001)

17. ClO 2 Gas Treatment (Batch) on Strawberries 0.2- 4 mg/l ClO 2 gas treatments (batch system) for 30 min at 22 o C and 90-95% relative humidity. Initial inoculation levels ~ 8.0 log cfu

18. Work Summary: ClO 2 Gas Treatment (Batch System) ORGANISMSURFACECONC.TIMEREDUCTION Lactobacillus spp. Penicillium spp. S. cervasia Epoxy tank surfaces (Han et al. 1999) 10 mg/l30 min6 log cfu Bacillus sporesPaper, plastic, wood (Han et al. 2002) 15 mg/l 30 min5 log cfu E. coli O157:H7 L. monocytogenes Green peppers (Han et al. 2000, 2001) 0.6 mg/l 30 min7.3 log cfu 6.3 log cfu E. coli O157:H7Strawberries (Han & Linton 2002) 0.6 mg/l15 min5.6 log cfu E. coli O157:H7 L. monocytogenes Apples (Du et al. 2002) 4.0 mg/l 4.8 mg/l 10 min 5.5 log cfu 4.8 log cfu Salmonella spp.Oranges (Wong et. Al,, 2004) 0.3 mg/l13 min5 log cfu L. monocytogenesLettuce (D’lima & Linton 2002) 0.2 mg/l 30 min1.3 log cfu** E. coli O157:H7 and Salmonella spp. Cantaloupes (Han et al. 2003) 2.8 mg/l 8.6 mg/l 10 min 2 log cfu** 3 log cfu**

19. 3 1 2 45 6 7 8 9 1. Chlorine gas 2. ClO2 gas generator 3. Chamber 4. Alternative door 5. Sample in and out 5. Operating gloves 7. ClO 2 gas monitor 8. Gas mixer 9. ClO 2 gas and humidity monitor Continuous ClO 2 Gas Treatment System

20. Microbiological Studies: D-values for several ClO 2 concentrations Z-values across ClO 2 concentrations Quality studies: Visual color test Instrumental (Hunter) color test Microbial Shelf-life studies Total aerobic plate counts Psychrotrophic plate counts Yeast/Mold counts Surfaces Sprouts, berries, leafy greens, melons, tomatoes Experimental Protocol

21. Spot inoculated 100μl Air-dried (1 h) ClO 2 treatments at 0.5, 1.0, 1.5, 3.0 and 5.0 mg/l for 0, 2, 4, 6, 8 and 10 min Membrane transferring method TSA CT-SMAC E. coli O157: H7 MOX L. monocytogenes XLD S. enterica

22. D-and z-value determination

23. D-value and Z-value “D”-value “Z”-value D Z

24. E.coli O157:H7Listeria sppSalmonella spp FOOD SURFACEClO 2 (mg/l) D-Value (min) Z-value (min) D-Value (min) Z-value (min) D-Value (min) Z-value (min) CANTALOUPE (Mahmoud et al, 2008) 0.54.228.83.327.63.47.9 14.03.43.1 1.53.93.22.2 3.03.63.01.4 5.02.72.50.9 LETTUCE (Mahmoud and Linton, 2008) 0.57.216.26.916.2 13.45.8 3.03.24.2 5.02.93.8 ORANGE (Bhagat et al, 2010) 0.13.8 0.32.8 0.52.3 TOMATOES (Bhagat et al, 2010) 0.17.51.26.81.3 0.36.95.9 0.53.23.6 STRAWBERRIES (Mahmoud et al, 2007) 0.54.716.84.615.84.223.3 14.24.73.8 1.53.93.13.5 3.02.72.52.9 5.02.52.32.7

25. PVC film Experimental Protocol Quality and shelf-life evaluation (after 0,4,8,12 and 16 days) ClO 2 treatments at 0.5 and 5.0 mg/l for 0, 2 and 10 min Analysis of appearance and color (Visual and by Hunter) Microbiological analysis (Mesophilic, Psycotrophic, Yeast & Molds counts)

26. Color Evaluation

27. Storage time (days) Control0.5 mg l -1 2min 0.5 mg l -1 10min 5.0 mg l -1 2min 5.0 mg l -1 10min 05.7±0.5 5.3±0.6 45.3±0.5 5.6±0.55.3±0.5 85.3±0.6 5.6±0.65.7±0.6 125.6±0.55.3±0.5 5.7±0.55.5±0.7 165.3±0.5 No Changes in Visual Color During Storage at 4 o C Numerical values from 1 (light) -10 (dark red)

28. No Changes in Hunter Values (L) During Storage at 4 o C Day 0 Day 4 Day 8 Day 12 Day 16 0 mg/l.5 mg/l, 2 min..5 mg/l, 10 min. 5.0 mg/l, 2 min. 5.0 mg/l, 10 min.

29. 0 Days Storage of Treated/Untreated Strawberries Untreated 0.5 mg l -1 /2min 0.5 mg l -1 /10min 5 mg l -1 /2min5 mg l -1 /10min

30. 16 days Storage of Treated/Untreated Strawberries Untreated 0.5 mg l -1 /2min 5 mg l -1 /2min 5 mg l -1 /10min 0.5 mg l -1 /10min

31. Day 0 of Treated/Untreated Cantaloupes Untreated 5 mg l -1 /2min 5 mg l -1 /10min

32. 9 Days Storage of Treated/Untreated Cantaloupes 5 mg l -1 /2min Untreated 5 mg l -1 /10min

33. Day 0 Treated/Untreated Tomatoes Untreated 0.5 mg l -1 /10min

34. Day 21 for Tomatoes Untreated 0.2 mg l -1 /30min

35. Day 0 Storage of Treated/Untreated Lettuce 0.5 mg l -1 /2min 5 mg l -1 /2min 0.5 mg l -1 /10min 5 mg l -1 /10min Untreated

36. 7 Days Storage of Treated/Untreated Lettuce 0.5 mg l -1 /2min Untreated 5 mg l -1 /2min 0.5 mg l -1 /10min 5 mg l -1 /10min

37. Day 0 for Treated Lettuce Leaves Untreated 0.2 mg l -1 /30min

38. Microbial Shelf-life

39. Strawberries – Microbial Shelf-life Early = 0 days; Middle = 8 days; Late = 16 days Control = No treatment; Low = 0.5mg/l for 2 min.; High = 5 mg/l for 10 min.

40. Strawberries – Microbial Shelf-life Early = 0 days; Middle = 8 days; Late = 16 days Control = No treatment; Low = 0.5mg/l for 2 min.; High = 5 mg/l for 10 min.

41. Strawberries – Microbial Shelf-life Early = 0 days; Middle = 8 days; Late = 16 days Control = No treatment; Low = 0.5mg/l for 2 min.; High = 5 mg/l for 10 min.

42. Strawberries – Microbial Shelf-life Early = 0 days; Middle = 8 days; Late = 16 days Control = No treatment; Low = 0.5mg/l for 2 min.; High = 5 mg/l for 10 min.

43. Cantaloupes – Microbial Shelf-life Early = 0 days; Middle = 6 days; Late = 12 days Control = No treatment; Low = 5 mg/l for 2 min.; High = 5 mg/l for 10 min.

44. New Continuous System for Produce

47. Chemical By-Products

48. Residual Oxidative By-Products Inorganic oxidative by-products: ClO 2, chlorite, and chlorate are of interest - measured using. amperometric titration methods (EPA approved for drinking water) National Primary Drinking Water Regulations: Disinfectants and Disinfection Byproducts <0.8 mg/l ClO 2 maximum residual disinfectant level goal (MRDLG) <1.0 mg/l chlorite maximum contaminant level goal (MCLG)

49. Potential Applications for Military Operations Viable antimicrobial treatment option to improve safety and extends shelf-life of fruit and vegetables – during shipment and/or storage Viable antimicrobial treatment option to sanitize and/or commercially sterilize food contact surfaces

50. Acknowledgements PEOPLE Dr. Yingchan Han Dr. Philip Nelson Barakat Mahmoud Arpan Bhagat Jay Kim Les Bourquin Richa Vaid All the undergraduate helpers! FUNDING USDA/CSREES FDA ClorDiSys Solutions, Inc. Enerfab Inc.

51. Questions ?