Listeria Inactivation with Ozone
Table of Contents

Implementation of Ozone

Resolution Concerning the Use of Ozone in Food Processing

Compiled Research
Listeria is a common term to refer to a specific strain of bacteria species. There are seven (7)
known species of Listeria. The specific species L. monocytogenes is the cause of Listeriosis, a
serious infection caused by eating food contaminated with this strain of bacteria. This disease can
be deadly and will pose a greater risk to those with weakened immune system. Both L.
monocytogenes, and Liseteriosis, are commonly known only as Listeria, and Listeria illness.
Listeria is found in soils, this can lead to fruit and vegetable contamination. Listeria can also be
found in all types of meat products, milk, and eggs. Higher risk foods are any uncooked or
undercooked foods, unpasteurized milk, raw vegetables, and some ready-to-eat foods.
Click here to learn more about Listeria from the CDC (Center for Disease Control and Prevention).
Ozone can be used for the reduction, or elimination of L. monocytogenes on food products. Since
achieving GRAS approval for the use of ozone for direct contact with food in 2001 the use of
ozone for the elimination of L. monocytogenes has increased significantly.
To eliminate Listeria or any other bacteria with ozone successful implementation of ozone is
necessary. While every application is different, there are a few fundamentals that will apply in
most applications.
Implementation of Ozone
Aqueous Ozone
The most common method of using ozone for pathogen reduction is by dissolving ozone into
water. Aqueous ozone is very stable, safe, and easy to manage. Typically, ozone is dissolved into
water using an Ozone Injection System and then sprayed onto the surface requiring disinfection.
This surface may be a hard equipment surface, or the surface of a food product.
In 2000, the Journal of Food Science published a paper by Kim & Yousef showing the effect of
dissolved ozone in a batch reactor on Listeria monocytogenes. Dissolved ozone at 0.4 and 0.8
ppm inactivated 4.6 and 5.7 log CFU/ml within 30 seconds. Additional tests were run at higher
dissolved ozone levels. Higher dissolved ozone levels did show faster (immediate) inactivation of
Listeria monocytogenes.
Dissolved ozone can be sprayed on food and produce using spray bars, or other spraying
methods. Conveyers work well to allow the sufficient contact time, and offer full coverage of the
aqueous ozone. It is important that all of the produce is contacted by the aqueous ozone to
achieve desired antimicrobial intervention. Contact times can be varied by altering conveyer
speeds, spray tip design, and spray bar design/quantity. If water is already used in an application
to wash produce it is very simple to add ozone to this water and achieve an antimicrobial
intervention step without any major changes to the current processes.
Gaseous ozone
The use of gaseous ozone for the elimination of pathogens is less common. There is less
research showing the effects of gaseous ozone on bacteria. The application of gaseous ozone is
dependent upon the temperature, humidity, contact time, and ozone levels. Research has been
conducted to determine that gaseous ozone will reduce and inactivate L. monocytogenes;
however, more research is necessary to determine the effectiveness of ozone within different
variables.
Produce in need of disinfection can be placed in chambers, rooms, or even cargo containers for
ozone treatment. A sealed area that can contain the produce and ozone gas while maintaining
human safety will work. It is necessary to assure sufficient air movement past each piece of
produce. Ozone levels from 1.0 - 100 ppm are used in this application with contact times from 20
minutes to 10 hours. For more information on the potential of using gaseous ozone in your
application contact our application engineers today.
Resolution Concerning the Use of Ozone in Food Processing
Source: Dee Graham, "Ozone as an Antimicrobial Agent for the Treatment, Storage and
Processing of Foods in Gas and Aqueous Phases", August 2, 2000.
The Use of Ozone as an effective sanitizer and disinfectant worldwide beginning in France in
1902 and has been documented in an Expert Panel Report entitled "Evaluation of the History and
Safety of Ozone in Processing Food for Human Consumption". This Declaration of GRAS Status
for Use of Ozone in Food Processing was presented to FDA on April 10, 1997 and published
thereafter in the scientific literature and the trade press.
Numerous ozone applications have been installed throughout the food industry in the United
States during the past two years. The benefits to public food safety are major, especially related
to the food hazards identified in the President's Food Safety Initiative. These include newer
pathogens such as E. coli 0157:H7, Listeria, and resistant cyst formers such as Cryptosporidium
and Giardia, all of which are inactivated effectively by ozonation.
We urge the responsible Federal Agencies, particularly USDA-FSIS and FDA-CSAN to
proactively support the adoption of ozonation for application broadly in food and agriculture as
described in the Expert Panel Report Declaration of GRAS Status for ozone on April 10, 1997
and in this Petition.
Resolution Signers
Charles D. Sopher, Ph.D. Director, EPRI Food & Agricultural
Technology Alliances 2000 L Street, Suite 805 Washington, DC
20036
Nari Nayini, Ph.D. Senior Development Associate Food
William P. Roenigk Senior Vice President, National Chicken Council
Applications R&D Praxair, Inc. 7000 High Grove Boulevard Burr
1015 Fifteen Street, NW — Ste. 930 Washington, DC 20005-2605
Ridge, Illinois 60521-7595
James T. C. Yuan, Ph.D. Head, Food & Biochemical Research Air Robert E. Smith, Ph. D., President R. E. Smith Consulting, Inc. 222Liquide 5230 South East Avenue Countryside, Illinois 60525
B Eagle Point Road Newport, Vermont 05855
Caleb L. Gilchrist, Ph.D. Director, Scientific Affairs American Meat
Jurgen Strasser, Ph.D., President Process & Equipment
Institute 1700 North Moore Street, Ste. 1600 Arlington, VA 22209
Technology 3312 Las Huertas Road Lafayette, CA 94549-5109
Richard Forsythe, Ph.D. Professor Emeritus -- Poultry Science
Dee M. Graham, Ph.D., President R and D Enterprises 2747
Dept. University of Arkansas Fayetteville, Arkansas 72701
Hutchinson Court Walnut Creek, CA 94598
Frank Busta, Ph.D. Professor and Emeritus Dept. Head Food
Charles W. Pearsall Vice President RGF Environmental Group 3875
Sciences & Nutrition University of Minnesota 1334 Eckles Avenue,
Fiscal Court, Suite 100 West Palm Beach Florida 33404
Room 258 St. Paul, MN 55108-6099
Barbara Blakistone Senior Specialist Food Chemistry & Packaging
Abit Massey Georgia Poultry Federation P. O. Box 763 Gainesville,
Dept. National Food Processors Association. 1350 I Street, NW,
Georgia 30501
Ste. 300 Washington, DC 20005
Stuart Proctor, Jr. National Turkey Federation 1225 New York Ave Don Dalton U.S. Poultry & Egg Association 1530 Cooledge Road
NW-Ste 400 Washington, DC 20005
Tucker, GA 30084
Michael W. Pariza, Ph. D. University of Wisconsin -- Madison Food
Rip G. Rice, Ph. D. President Rice International Consulting
Research Institute 1925 Willow Drive Madison, WI 53706
Enterprises 1331 Patuxent Drive Ashton, MD 20861
S. R. Tatini, Ph.D. Food Sciences & Nutrition Dept. University of Sharon P. Shoemaker, Ph.D. Executive Director California Institute
Minnesota 1334 Eckles Avenue St. Paul, MN 55108-6099
of Food & Agricultural Research 250 Cruess Hall Davis, CA 94516
Lee C. Ditzler President Novazone 346 Earhart Way Livermore, CA
Don Dalton U. S. Poultry and Egg Associ 1530 Cooledge Road
94550
Dennis Lavelle, President Dell Industries 3428 Bullock Lane San
Luis Obispo, CA 93401
Compiled Research
We have assembled some research on the use of ozone specifically for L. monocytogenes. This
research is below, we have provided the white paper title, author, and abstract for your review,
along with a link to the full paper for your use.
If you have any further questions on the use of ozone for the inactivation of L. monocytogenes, or
any other pathogen, please contact our application engineers today.
Efficacy of Ozone in Killing Listeria monocytogenes on Alfalfa
Seeds and Sprouts and Effects on Sensory Quality of Sprouts
Source: Journal of Food Protection: Vol. 66, No. 1, pp. 44-51.
Authors: W. N. Wade (a, b); A. J. Scouten (a, b); K. H. McWatters (b); R. L. Wick (c); A. Demirci
(d); W. F. Fett; and L. R. Beuchata (b)

Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin,
Georgia 30223-1797[PARA]

Department of Food Science and Technology, University of Georgia, 1109
Experiment Street, Griffin, Georgia 30223-1797[PARA]

Department of Microbiology, 639 Pleasant Street, Morrill Science Center IV-N203,
University of Massachusetts, Amherst, Massachusetts 01003-9298[PARA]

Department of Agricultural and Biological Engineering, Life Sciences Consortium,
Pennsylvania State University, University Park, Pennsylvania 16802[PARA] U.S.
Department of Agriculture, Agricultural Research Service, Eastern Regional
Research Center, Food Intervention and Technology Research Unit, 600 East
Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA
Abstract
A study was done to determine the efficacy of aqueous ozone treatment in killing Listeria
monocytogenes on inoculated alfalfa seeds and sprouts. Reductions in populations of naturally
occurring aerobic microorganisms on sprouts and changes in the sensory quality of sprouts were
also determined. The treatment (10 or 20 min) of seeds in water (4°C) containing an initial
concentration of 21.8 ± 0.1 g/ml of ozone failed to cause a significant (P 0.05) reduction in
populations of L. monocytogenes. The continuous sparging of seeds with ozonated water (initial
ozone concentration of 21.3 ± 0.2 g/ml) for 20 min significantly reduced the population by 1.48
log10 CFU/g. The treatment (2 min) of inoculated alfalfa sprouts with water containing 5.0 ± 0.5,
9.0 ± 0.5, or 23.2 ± 1.6 g/ml of ozone resulted in significant (P 0.05) reductions of 0.78, 0.81, and
0.91 log10 CFU/g, respectively, compared to populations detected on sprouts treated with water.
Treatments (2 min) with up to 23.3 ± 1.6 g/ml of ozone did not significantly (P > 0.05) reduce
populations of aerobic naturally occurring microorganisms. The continuous sparging of sprouts
with ozonated water for 5 to 20 min caused significant reductions in L. monocytogenes and
natural microbiota compared to soaking in water (control) but did not enhance the lethality
compared to the sprouts not treated with continuous sparging. The treatment of sprouts with
Ozonated water (20.0 g/ml) for 5 or 10 min caused a significant deterioration in the sensory
quality during subsequent storage at 4°C for 7 to 11 days. Scanning electron microscopy of
uninoculated alfalfa seeds and sprouts showed physical damage, fungal and bacterial growth,
and biofilm formation that provide evidence of factors contributing to the difficulty of killing
microorganisms by treatment with ozone and other sanitizers.
http://www.sproutnet.com/Research/efficacy_of_ozone.htm
Inactivation of Escherichia coli O1 57:H7, Listeria
monocytogenes, and Lactobacillus leichmannii by
combinations of ozone and pulsed electric field.
Authors: Unal R , Kim JG , Yousef AE .
Source: J Food Prot. 2001 Jun;64(6):777-82.
Publisher: Department of Food Science and Technology, The Ohio State University, Columbus
43210, USA.
Abstract
Pulsed electric field (PEF) and ozone technologies are nonthermal processing methods with
potential applications in the food industry. This research was performed to explore the potential
synergy between ozone and PEF treatments against selected foodborne bacteria. Cells of
Lactobacillus leichmannii ATCC 4797, Escherichia coli O157:H7 ATCC 35150, and Listeria
monocytogenes Scott A were suspended in 0.1% NaCl and treated with ozone, PEF, and ozone
plus PEE Cells were treated with 0.25 to 1.00 microg of ozone per ml of cell suspension, PEF at
10 to 30 kV/cm, and selected combinations of ozone and PEF. Synergy between ozone and PEF
varied with the treatment level and the bacterium treated. L. leichmannii treated with PEF (20
kV/cm) after exposure to 0.75 and 1.00 microg/ml of ozone was inactivated by 7.1 and 7.2 log10
CFU/ml, respectively; however, ozone at 0.75 and 1.00 microg/ml and PEF at 20 kV/cm
inactivated 2.2, 3.6, and 1.3 log10 CFU/ml, respectively. Similarly, ozone at 0.5 and 0.75
microg/ml inactivated 0.5 and 1.8 log10 CFU/ml of E. coli, PEF at 15 kV/cm inactivated 1.8 log10
CFU/ml, and ozone at 0.5 and 0.75 microg/ml followed by PEF (15 kV/cm) inactivated 2.9 and 3.6
log10 CFU/ml, respectively. Populations of L. monocytogenes decreased 0.1, 0.5, 3.0, 3.9, and
0.8 log10 CFU/ml when treated with 0.25, 0.5, 0.75, and 1.0 microg/ml of ozone and PEF (15
kV/cm), respectively; however, when the bacterium was treated with 15 kV/cm, after exposure to
0.25, 0.5, and 0.75 microg/ml of ozone, 1.7, 2.0, and 3.9 log10 CFU/ml were killed, respectively.
In conclusion, exposure of L. leichmannii, E. coli, and L. monocytogenes to ozone followed by the
PEF treatment showed a synergistic bactericidal effect. This synergy was most apparent with mild
doses of ozone against L. leichmannii.
http://www.ncbi.nlm.nih.gov/pubmed/11403125
Elimination of Listeria monocytogenes Biofilms by Ozone,
Chlorine, and Hydrogen Peroxide
Authors: Robbins Justin B.; Fisher Christopher W.; Moltz Andrew G.; Martin Scott E.
Source: Journal of Food Protection , Volume 68, Number 3, March 2005 , pp. 494-498(5)
Publisher: International Association for Food Protection
Abstract
This study evaluated the efficacy of ozone, chlorine, and hydrogen peroxide to destroy Listeria
monocytogenes planktonic cells and biofilms of two test strains, Scott A and 10403S. L.
monocytogenes was sensitive to ozone (O3)), chlorine, and hydrogen peroxide (H2O2). Planktonic
cells of strain Scott A were completely destroyed by exposure to 0.25 ppm O3) (8.29-log reduction,
CFU per milliliter). Ozone's destruction of Scott A increased when the concentration was
increased, with complete elimination at 4.00 ppm O3 (8.07-log reduction, CFU per chip). A 16-fold
increase in sanitizer concentration was required to destroy biofilm cells of L. monocytogenes
versus planktonic cells of strain Scott A. Strain 10403S required an ozone concentration of 1.00
ppm to eliminate planktonic cells (8.16-log reduction, CFU per milliliter). Attached cells of the
same strain were eliminated at a concentration of 4.00 ppm O2 (7.47-log reduction, CFU per chip).
At 100 ppm chlorine at 20°C, the number of planktonic cells of L. monocytogenes 10403S was
reduced by 5.77 log CFU/ml after 5 min of exposure and by 6.49 log CFU/ml after 10 min of
exposure. Biofilm cells were reduced by 5.79 log CFU per chip following exposure to 100 ppm
chlorine at 20°C for 5 min, with complete elimination (6.27 log CFU per chip) after exposure to
150 ppm at 20°C for 1 min. A 3% H2O2 solution reduced the initial concentration of L.
monocytogenes Scott A planktonic cells by 6.0 log CFU/ml after 10 min of exposure at 20°C, and
a 3.5% H2O2 solution reduced the planktonic population by 5.4 and 8.7 log CFU/ml (complete
elimination) after 5 and 10 min of exposure at 20°C, respectively. Exposure of cells grown as
biofilms to 5% H2O2 resulted in a 4.14-log CFU per chip reduction after 10 min of exposure at
20°C and in a 5.58-log CFU per chip reduction (complete elimination) after 15 min of exposure.
the potential synergy between ozone and PEF treatments against selected foodborne bacteria.
Cells of Lactobacillus leichmannii ATCC 4797, Escherichia coli O157:H7 ATCC 35150, and
Listeria monocytogenes Scott A were suspended in 0.1% NaCl and treated with ozone, PEF, and
ozone plus PEE Cells were treated with 0.25 to 1.00 microg of ozone per ml of cell suspension,
PEF at 10 to 30 kV/cm, and selected combinations of ozone and PEF. Synergy between ozone
and PEF varied with the treatment level and the bacterium treated. L. leichmannii treated with
PEF (20 kV/cm) after exposure to 0.75 and 1.00 microg/ml of ozone was inactivated by 7.1 and
7.2 log10 CFU/ml, respectively; however, ozone at 0.75 and 1.00 microg/ml and PEF at 20 kV/cm
inactivated 2.2, 3.6, and 1.3 log10 CFU/ml, respectively. Similarly, ozone at 0.5 and 0.75
microg/ml inactivated 0.5 and 1.8 log10 CFU/ml of E. coli, PEF at 15 kV/cm inactivated 1.8 log10
CFU/ml, and ozone at 0.5 and 0.75 microg/ml followed by PEF (15 kV/cm) inactivated 2.9 and 3.6
log10 CFU/ml, respectively. Populations of L. monocytogenes decreased 0.1, 0.5, 3.0, 3.9, and
0.8 log10 CFU/ml when treated with 0.25, 0.5, 0.75, and 1.0 microg/ml of ozone and PEF (15
kV/cm), respectively; however, when the bacterium was treated with 15 kV/cm, after exposure to
0.25, 0.5, and 0.75 microg/ml of ozone, 1.7, 2.0, and 3.9 log10 CFU/ml were killed, respectively.
In conclusion, exposure of L. leichmannii, E. coli, and L. monocytogenes to ozone followed by the
PEF treatment showed a synergistic bactericidal effect. This synergy was most apparent with mild
doses of ozone against L. leichmannii.
http://www.ncbi.nlm.nih.gov/pubmed/15771172
Effect of Ozone and Ultraviolet Irradiation Treatments on
Listeria monocytogenes Populations in Chill Brines
Author: Govindaraj Dev Kumar
Date Created: November 19, 2008
Abstract
The efficacy of ozone and ultraviolet light, used in combination, to inactivate Listeria
monocytogenes in fresh (9% NaCl, 91.86% transmittance at 254 nm) and spent chill brines
(20.5% NaCl, 0.01% transmittance at 254 nm) was determined. Preliminary studies were
conducted to optimize parameters for the ozonation of "fresh" and "spent" brines. These include
diffuser design, comparison of kit to standard methods to measure residual ozone, studying the
effect of ozone on uridine absorbance and determining presence of residual listericidal activity
post ozonation. An ozone diffuser was designed using 3/16 inch PVC tubing for the ozonation of
brines. The sparger was designed to facilitate better diffusion and its efficiency was tested. The
modified sparger diffused 1.44 ppm of ozone after 30 minutes of ozonation and the solution had
an excess of 1 ppm in 10 minutes of ozonating fresh brine solution (200ml). Population levels of L.
monocytogenes were determined at various time intervals post-ozonation (0, 10, 20, 60 min) to
determine the presence of residual listericidal activity. The population post ozonation (0 minutes)
was 5.31 Log CFU/ml and was 5.08 Log CFU/ml after a 60 minute interval. Therefore, residual
antimicrobial effect was weak. Accuracy of the Vacu-vial Ozone analysis kit was evaluated by
comparing the performance of the kit to the standard indigo colorimetric method for measuring
residual ozone. The kit was inaccurate in determining residual ozone levels of spent brines and
1% peptone water. Uridine was evaluated as a UV actinometric tool for brine solutions iii that
were ozonated before UV treatment. The absorbance of uridine (A262) decreased after ozonation
from 0.1329 to 0.0512 for standard 10 minutes UV exposure duration. Absorbance of uridine was
influenced by ozone indicating that the presence of ozone may hamper UV fluence determination
accuracy in ozone-treated solutions. Upon completion of diffuser design and ozone/UV analysis
studies, the effect of ozone-UV combination on L. monocytogenes in fresh and spent brines was
evaluated. Ozonation, when applied for 5 minutes, caused a 5.29 mean Log reduction while 5
minutes of UV exposure resulted in a 1.09 mean Log reduction of L. monocytogenes cells in fresh
brines. Ten minutes of ozonation led to a 7.44 mean Log reduction and 10 minutes of UV
radiation caused a 1.95 mean Log reduction of Listeria in fresh brine. Spent brines required 60
minutes of ozonation for a 4.97 mean Log reduction in L. monocytogenes counts, while 45
minutes resulted in a 4.04 mean Log reduction. Ten minutes of UV exposure of the spent brines
resulted in 0.30 mean Log reduction in Listeria cells. A combination of 60 minutes ozonation and
10 minute UV exposure resulted in an excess of 5 log reduction in cell counts. Ozonation did not
cause a sufficient increase in the transmittance of the spent brine to aid UV penetration but
resulted in apparent color change as indicated by change in L*a*b* values. Ozonation for
sufficient time had considerable listericidal activity in fresh brines and spent brines and when
combined with UV treatment, is effective reducing L. monocytogenes to undetectable levels in
fresh brines.
Click here for the paper.
Effectiveness of Ozone in Inactivating Listeria monocytogenes
from Milk Samples
Authors: Mariyaselvam Sheelamary, Muthusamy Muthukumar
Affiliations: Division of Environmental Engineering and Technology Department of
Environmental Sciences Bharathiar University, Coimbatore, Tamil Nadu, INDIA
Accepted: June, 2011
Publisher: World Journal of Life Sciences and Medical Research 2011;1(3):40-4.
Abstract
Inactivation of Listeria monocytogenes using ozonation was studied in raw milk and various
branded milk samples in and around Coimbatore city. Total of 20 milk samples were obtained
from super markets and other places. The PALCAM agar was used in the study to enumerate L.
monocytogenes from raw milk and various branded milk samples. Results indicate that all the
samples are positive prior to the ozonation process. A controlled flow rate 0.5 m/l of oxygen was
used to produce 0.2g/h of ozone. The milk samples were ozonated at 0, 5, 10, and 15 minutes.
After treatment the samples are inoculated and L. monocytogenes were enumerated by using
listeria PALCAM agar. After 15 minutes ozonation L. monocytogenes were completely eliminated
from milk samples. Before and after ozonation the samples were analyzed for protein,
carbohydrate, and calcium content. After treatment the nutritional values were slightly different in
the milk samples..
Click here for the paper.
Inactivation Kinetics of Foodborne Spoilage and Pathogenic
Bacteria by Ozone
Authors: J.G. Kim and A.E. Yousef
Keywords: fluorescens, L. mesenteroides, and L. monocytoge
Abstract
Ozone was tested against Pseudomonas fluorescens, Escherichia coli O157:H7, Leuconostoc
mesenteroides, and Listeria monocytogenes. When kinetic data from a batch reactor were fitted
to a dose-response model, a 2-phased linear relationship was observed. A continuous ozone
reactor was developed to ensure a uniform exposure of bacterial cells to ozone and a constant
concentration of ozone during the treatment. Survivors plots in the continuous system were linear
initially, followed by a concave downward pattern. Exposure of bacteria to ozone at 2.5 ppm for
40 s caused 5 to 6 log decrease in count. Resistance of tested bacteria to ozone followed this
descending order: E. coli O157:H7, P.
Click here for the paper.
Influence of Catalase and Superoxide Dismutase on Ozone
Inactivation of Listeria monocytogenes
Authors: Christopher W. Fisher, Dongha Lee, Beth-Anne Dodge, Kristen M. Hamman, Justin B.
Robbins, and Scott E. Martin
Publication Details: Department of Food Science and Human Nutrition, University of Illinois,
Urbana, Illinois. Received 20 September 1999/Accepted 6 January 2000
Abstract
The effects of ozone at 0.25, 0.40, and 1.00 ppm on Listeria monocytogenes were evaluated in
distilled water and phosphate-buffered saline. Differences in sensitivity to ozone were found to
exist among the six strains examined. Greater cell death was found following exposure at lower
temperatures. Early stationary-phase cells were less sensitive to ozone than mid-exponentialand late stationary-phase cells. Ozonation at 1.00 ppm of cabbage inoculated with L.
monocytogenes effectively inactivated all cells after 5 min. The abilities of in vivo catalase and
superoxide dismutase to protect the cells from ozone were also examined. Three listerial test
strains were inactivated rapidly upon exposure to ozone. Both catalase and superoxide
dismutase were found to protect listerial cells from ozone attack, with superoxide dismutase being
more important than catalase in this protection.
Related Information
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USDA, FDA, and Ozone
Regulations change.
Last Updated: October 22, 2012