Use of Chlorine Dioxide Gas as an Antimicrobial Agent for Foods and

Food Contact Surfaces

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

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

PATHOGEN FOOD 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. monocytogenes Coleslaw, green onion, lettuce , milk, chicken

Cyclospora Raspberries

Hepatitis A virus and Norovirus

Green onions, leafy greens, strawberries

Produce Associated Outbreaks

(Data taken from Beuchat et al. 1996; CDC 2010; NACMCF 2009)

Aqueous Chemical Use for Produce

ANTIMICROBIAL AGENT USES EFFECTIVNESS

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 acids Whole & fresh cut, 1-2 log reduction

OUR GOAL (ClO2 gas) Fresh produce >5 log reduction

(Cherry, J. Food Technology Vol. 53, No. 11)

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

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 (ClO2)

• History of uses of aqueous ClO2

- 1811 First generated by Davy (acidification of KClO3)

- 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 ClO2

• History of uses of gaseous ClO2

- 1988 Registered by EPA as a sterilant (for hard surfaces and implements)

- 1990’s Research on pathogen reduction in produce - 2000 GRAS status for ClO2 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 ClO2

2 %

Ch

lori

ne

Gas

So

diu

m C

hlo

rite

ClO2 Monitor

%RH Controller

Cl2 + 2 NaClO2 → 2 ClO2 + 2 NaCl

Generation of ClO2 Gas

4 HA + 5 NaClO2 4 ClO2 + NaCl + 4 Na+ + 2 H2O

Other Methods to Generate ClO2 Gas

(Courtesy of ICA TriNova, LLC, Atlanta, GA)

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?

ClO2 on Epoxy Aseptic Tank Surfaces for Inactivation of Spoilage Organisms

Microbes Initial levels (log cfu)

ClO2 gas sterilization conditions

Lactobacillus buchneri 6.6 6.8

6 mg/l – 30 min, 25-26oC, 87% RH 8 mg/l – 30 min, 10-11oC, 89% RH

Lactobacillus mesenteroides

6.4 6.5

8 mg/l – 30 min, 27oC, 75% RH 8 mg/l – 30 min, 11oC, 93% RH

Candida spp. and Saccharomyces cerevisiae

5.6 5.6

10 mg/l –10 min, 26oC, 91% RH 8 mg/l – 30 min, 11oC, 89% RH

Eurotium spp. and Penicillium spp

5.4 5.4

10 mg/l –10 min, 26oC, 91% RH 8 mg/l – 30 min, 11oC, 89% RH

ClO2 on Epoxy Aseptic Tank Surfaces for Inactivation of Spoilage Organisms

Batch ClO2 Gas Treatment System

Cl2/N2 gas Generator Chamber Monitor Humidifier

Cl2 + 2 NaClO2 2 ClO2 + 2 NaCl

Treated with 3 mg/l ClO2 for 30 min and stored

for 6 weeks at 4oC

Untreated and stored for 6 weeks

at 4oC

ClO2 Gas Treatment (Batch) on Strawberries

Inactivation of E. coli O157:H7 by ClO2 Gas on Peppers

0

2

4

6

8

10L

og

Red

uct

ion

/sam

ple Uninjured

Surfaces

InjuredSurfaces

0.1 0.3 0.6 1.2ClO2 Gas Concentration (mg/l)

AaAb

Bx

AcAd By

BzBz

• Treatments for 30 min at 20oC under 90-95%RH • Significant differences (P<0.05)

(Han et al., 2001)

ClO2 Gas Treatment (Batch) on Strawberries

0

1

2

3

4

5

6

7

0.2 0.6 1.2 1.8 2.4 3 4Chlorine dioxide gas concentration (mg/l)

Bac

teri

al

log

re

du

cti

on

(lo

g c

fu)

E. coli O157:H7

Listeria monocytogenes

0.2- 4 mg/l ClO2 gas treatments (batch system) for 30 min at 22oC and 90-95% relative

humidity. Initial inoculation levels ~ 8.0 log cfu

Work Summary: ClO2 Gas Treatment (Batch System)

ORGANISM SURFACE CONC. TIME REDUCTION

Lactobacillus spp.Penicillium spp.S. cervasia

Epoxy tank surfaces (Han et al. 1999)

10 mg/l 30 min 6 log cfu

Bacillus spores Paper, plastic, wood (Han et al. 2002)

15 mg/l 30 min 5 log cfu

E. coli O157:H7L. monocytogenes

Green peppers (Han et al. 2000, 2001)

0.6 mg/l 30 min 7.3 log cfu6.3 log cfu

E. coli O157:H7 Strawberries (Han & Linton 2002)

0.6 mg/l 15 min 5.6 log cfu

E. coli O157:H7L. monocytogenes

Apples (Du et al. 2002)

4.0 mg/l 4.8 mg/l

10 min 10 min

5.5 log cfu4.8 log cfu

Salmonella spp. Oranges(Wong et. Al,, 2004)

0.3 mg/l 13 min 5 log cfu

L. monocytogenes Lettuce(D’lima & Linton 2002)

0.2 mg/l 30 min 1.3 log cfu**

E. coli O157:H7 and Salmonella spp.

Cantaloupes(Han et al. 2003)

2.8 mg/l8.6 mg/l

10 min10 min

2 log cfu**3 log cfu**

3

1

24 5

6

7

8

9

1. Chlorine gas 2. ClO2 gas generator3. Chamber4. Alternative door5. Sample in and out5. Operating gloves7. ClO2 gas monitor

8. Gas mixer9. ClO2 gas and humidity monitor

Continuous ClO2 Gas Treatment System

• Microbiological Studies: • D-values for several ClO2 concentrations• Z-values across ClO2 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

Experimental Protocol

Spot inoculatedSpot inoculated

100μl100μl

Air-dried(1 h)

ClO2 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-SMACE. coli O157: H7

MOXL. monocytogenes

XLDS. enterica

D-and z-value determination

D-value and Z-value

8.5

5.8

4.2

1.8

0123456789

10

0 10 20 30 40

Lo

g s

urv

ivo

rs

Time (minutes) at Constant Concentration

2 . 5

1 . 1

0 . 3

0

1

2

3

54 62 70

Log D

-valu

eDifferent Concentrations

“D”-value

“Z”-value

D

Z

E.coli O157:H7 Listeria spp Salmonella sppFOOD SURFACE ClO2

(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)

0.5 4.2 28.8 3.3 27.6 3.4 7.91 4.0 3.4 3.1

1.5 3.9 3.2 2.23.0 3.6 3.0 1.45.0 2.7 2.5 0.9

LETTUCE(Mahmoud and Linton, 2008)

0.5 7.2 16.2 6.9 16.21 3.4 5.8

3.0 3.2 4.25.0 2.9 3.8

ORANGE(Bhagat et al, 2010)

0.1 3.80.3 2.80.5 2.3

TOMATOES(Bhagat et al, 2010)

0.1 7.5 1.2 6.8 1.30.3 6.9 5.90.5 3.2 3.6

STRAWBERRIES(Mahmoud et al, 2007)

0.5 4.7 16.8 4.6 15.8 4.2 23.31 4.2 4.7 3.8

1.5 3.9 3.1 3.53.0 2.7 2.5 2.95.0 2.5 2.3 2.7

PVC filmPVC film

Experimental Protocol

Quality and shelf-life evaluation(after 0,4,8,12 and 16 days)

ClO2 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)

Color Evaluation

Storage time(days)

Control 0.5 mg l-1

2min0.5 mg l-1

10min5.0 mg l-1

2min5.0 mg l-1

10min

0 5.7±0.5 5.7±0.5 5.3±0.6 5.3±0.6 5.3±0.6

4 5.3±0.5 5.3±0.5 5.6±0.5 5.3±0.5 5.3±0.5

8 5.3±0.6 5.3±0.6 5.3±0.6 5.6±0.6 5.7±0.6

12 5.6±0.5 5.3±0.5 5.3±0.5 5.7±0.5 5.5±0.7

16 5.3±0.5 5.3±0.5 5.3±0.5 5.3±0.5 5.3±0.5

No Changes in Visual Color During Storage at 4oC

Numerical values from 1 (light) -10 (dark red)

No Changes in Hunter Values (L) During Storage at 4oC

0 10 20 30 40

L

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.

0 Days Storage of Treated/Untreated Strawberries

Untreated

0.5 mg l-1/2min 0.5 mg l-1/10min

5 mg l-1/2min 5 mg l-1/10min

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

Day 0 of Treated/Untreated Cantaloupes

Untreated

5 mg l-1/2min 5 mg l-1/10min

9 Days Storage of Treated/Untreated Cantaloupes

5 mg l-1/2min

Untreated

5 mg l-1/10min

Day 0 Treated/Untreated Tomatoes

Untreated

0.5 mg l-1/10min

Day 21 for Tomatoes

Untreated

0.2 mg l-1/30min

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

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

Day 0 for Treated Lettuce Leaves

Untreated 0.2 mg l-1/30min

Microbial Shelf-life

0

1

2

3

4

5

6

APCControl

APC(Low)

APC(High)

PPCControl

PPC(Low)

PPC(High)

Y/MControl

Y/M(Low)

Y/M(High)

Lo

g C

FU

Early Middle Late

Strawberries – Microbial Shelf-life

Early = 0 days; Middle = 8 days; Late = 16 daysControl = No treatment; Low = 0.5mg/l for 2 min.; High = 5 mg/l for 10 min.

0

1

2

3

4

5

6

APCControl

APC(Low)

APC(High)

PPCControl

PPC(Low)

PPC(High)

Y/MControl

Y/M(Low)

Y/M(High)

Lo

g C

FU

Early Middle Late

Strawberries – Microbial Shelf-life

Early = 0 days; Middle = 8 days; Late = 16 daysControl = No treatment; Low = 0.5mg/l for 2 min.; High = 5 mg/l for 10 min.

0

1

2

3

4

5

6

APCControl

APC(Low)

APC(High)

PPCControl

PPC(Low)

PPC(High)

Y/MControl

Y/M(Low)

Y/M(High)

Lo

g C

FU

Early Middle Late

Strawberries – Microbial Shelf-life

Early = 0 days; Middle = 8 days; Late = 16 daysControl = No treatment; Low = 0.5mg/l for 2 min.; High = 5 mg/l for 10 min.

0

1

2

3

4

5

6

APCControl

APC(Low)

APC(High)

PPCControl

PPC(Low)

PPC(High)

Y/MControl

Y/M(Low)

Y/M(High)

Lo

g C

FU

Early Middle Late

Strawberries – Microbial Shelf-life

Early = 0 days; Middle = 8 days; Late = 16 daysControl = No treatment; Low = 0.5mg/l for 2 min.; High = 5 mg/l for 10 min.

0123456789

APCControl

APC(Low)

APC(High)

PPCControl

PPC(Low)

PPC(High)

Y/MControl

Y/M(Low)

Y/M(High)

Lo

g C

FU

Early Middle Late

Cantaloupes – Microbial Shelf-life

Early = 0 days; Middle = 6 days; Late = 12 daysControl = No treatment; Low = 5 mg/l for 2 min.; High = 5 mg/l for 10 min.

New Continuous System for Produce

New Continuous System for Produce

New Continuous System for Produce

Chemical By-Products

Residual Oxidative By-Products

• Inorganic oxidative by-products:

ClO2, 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 ClO2 maximum residual disinfectant level goal (MRDLG)

<1.0 mg/l chlorite maximum contaminant level goal (MCLG)

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

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.

Questions ?