INACTIVATION OF ESCHERICHIA COLI O157:H7 ON BABY SPINACH USING LOWENERGY X-RAY IRRADIATION
By
Scott Moosekian

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Food Science
2012

ABSTRACT
INACTIVATION OF ESCHERICHIA COLI O157:H7 ON BABY SPINACH USING LOWENERGY X-RAY IRRADIATION
By
Scott Moosekian
Low-energy X-ray irradiation was assessed as a means of inactivating Escherichia coli
O157:H7 on baby spinach. Round-cut (2.54 cm diameter) baby spinach samples were dipinoculated in a 3-strain E. coli O157:H7 cocktail, and irradiated on each side of the leaf using a
prototype low-energy (70 kV) X-ray irradiator to achieve X-ray doses of up to 0.176 kGy per
side. The D10 value for E. coli O157:H7 on baby spinach was 0.033 ± 0.001 kGy. Irradiated
bags of baby spinach (225 g) yielded a dose rate distribution of 1.93 ± 0.75 Gy/s at the mid-plane
to 7.18 ± 3.85 Gy/s on the bag’s surface. Predicted log reduction for E. coli O157:H7 in the bag
was 4.10 log CFU, while the observed mass averaged value was 3.99 log CFU. Next, preirradiated, round-cut baby spinach leaves were inoculated with an E. coli O157:H7 cocktail preexposed to a peracetic acid-based (PAB), chlorine-based (CB) or quaternary ammonium
compound-based (QACB) sanitizer to obtain 90 to 99% injury, and subjected to X-ray doses of
up to 0.063 kGy. Compared to control values of 0.021 ± 0.001 kGy, D10 values for PAB-, CB-,
and QACB-injured E. coli O157:H7 on baby spinach were 0.0136 ± 0.000, 0.0223 ± 0.001, and
0.0242 ± 0.001 kGy respectively, with exposure to PAB significantly (P < 0.05) enhancing E.
coli O157:H7 susceptibility to X-ray irradiation, while exposure to CB significantly (P < 0.05)
reduced susceptibility. Based on these findings, low-energy X-ray irradiation appears to be most
effective against E. coli O157:H7 when spinach is flume tank-washed using a PAB sanitizer
prior to irradiation.

TABLE OF CONTENTS

LIST OF TABLES

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LIST OF FIGURES

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1. INTRODUCTION

1

2. CHAPTER 1
LITERATURE REVIEW
2.1. Introduction
2.2. Potential Sources of Contamination
2.2.1. Irrigation practices
2.2.2. Interaction with wildlife and animal excrement
2.2.3. Transfer of E. coli O157:H7 from contaminated water and soil to the plant
2.2.4. Washing/bagging
2.3. Stress and Sublethal Injury
2.3.1. Chemical sanitizers
2.3.2. Oxidizing sanitizers
2.3.3. Peracetic acid-based compounds
2.3.4. Chlorine-based compounds
2.3.5. Quaternary ammonium compounds
2.3.6. Sanitizer limitations
2.4. Irradiation
2.4.1. History of X-ray
2.4.2. X-rays as a form of ionizing irradiation
2.4.3. Mechanism of action
2.4.4. Dose measurement
2.4.5. The need for alternative microbial reduction strategies
2.4.6. X-ray as a viable alternative
2.4.7. Advances in X-ray technology
2.4.8. X-ray irradiation process control and validation
2.4.9. Consumer acceptance
2.5. Cross-protection
2.5.1. Hurdle approach for pathogen control
2.5.2. Development of cross-protection
2.6. Summary

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3. CHAPTER 2
INACTIVATION OF ESCHERICHIA COLI O157:H7 ON ROUND-CUT AND
COMMERCIAL BAGGED BABY SPINACH
3.1. Abstract

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3.2. Introduction
3.3. Materials and Methods
3.3.1. Bacterial strains
3.3.2. X-ray irradiator
3.3.3. D10 value determination for E. coli O157:H7 on spinach
3.3.4. Determination of dose rate and dose in bags of baby spinach
3.3.5. Reduction of E. coli O157:H7 in bags of baby spinach
3.3.6. Statistical analysis
3.4. Results and Discussion
3.4.1. Uninoculated controls
3.4.2. D10 value for E. coli O157:H7 on baby spinach
3.4.3. Determination of dose rate and dose
3.4.4. Reduction of E. coli O157:H7 in commercial bags of baby spinach

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4. CHAPTER 3
INACTIVATION OF SANITIZER-INJURED ESCHERICHIA COLI O157:H7 ON
BABY SPINACH USING X-RAY IRRADIATION
4.1. Abstract
4.2. Introduction
4.3. Materials and Methods
4.3.1. Bacterial strains
4.3.2. Peroxyacetic acid-based sanitizer (PABS) injury
4.3.3. Quaternary ammonium compound-based sanitizer (QACBS) injury
4.3.4. Chlorine-based sanitizer (CBS) injury
4.3.5. Quantification of injury
4.3.6. X-ray irradiator
4.3.7. Baby spinach preparation
4.3.8. X-ray irradiation
4.3.9. Enumeration
4.3.10. Statistical analysis
4.4. Results and Discussion

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5. CHAPTER 4
SUGGESTIONS FOR FUTURE WORK WITH LOW-ENERGY X-RAY
IRRADIATION
5.1. Determine the Cost Associated with X-ray Irradiation
5.2. Application to Commercial Processing
5.3. Sensory Analysis
5.4. Enhanced Growth from Elimination of Background Microflora
5.5. Sanitizer Mechanisms for Microbial Inactivation

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6. APPENDIX A
INACTIVATION OF ESCHERICHIA COLI O157:H7 ON FLAT LEAF PARSLEY
USING X-RAY IRRADIATION
6.1. Purpose
6.2. Materials and Methods

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6.2.1. Strain preparation
6.2.2. Sample preparation
6.2.3. Inoculation
6.2.4. X-ray irradiation
6.2.5. Enumeration
6.2.6. Statistics
6.3. Results and Discussion
6.4. Raw Data

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7. APPENDIX B
INACTIVATION OF ESCHERICHIA COLI O157:H7 IN BAGGED ICEBERG
LETTUCE USING X-RAY IRRADIATION
7.1. Purpose
7.2. Materials and Methods
7.2.1. Strain preparation
7.2.2. Sample preparation
7.2.3. X-ray irradiation
7.2.4. Enumeration
7.3. Results
7.3.1. Dose determination
7.4. Raw Data

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8. APPENDIX C
EVALUATING LISTERIA MONOCYTOGENES GROWTH ON IRRADIATED
SPINACH
8.1. Purpose
8.2. Materials and Methods
8.2.1. Sample preparation
8.2.2. Culture preparation
8.2.3. Inoculation
8.2.4. Enumeration
8.3. Results
8.4. Interpretation/Limitations
8.5. Raw Data

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9. APPENDIX D
INACTIVATION OF BIOFILM-ASSOCIATED ESCHERICHIA COLI O157:H7 ON
LABORATORY GROWN BABY SPINACH
9.1. Purpose
9.2. Design/Objectives
9.3. Materials and Methods
9.3.1. Bacterial collection and characterization
9.3.1.1.
Field sampling
9.3.1.2.
Enumeration and isolation
9.3.1.3.
Biofilm analysis
9.3.2. Spinach growth and inoculation
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9.3.2.1.
Planting instructions
9.3.2.2.
Growth conditions
9.3.2.3.
Inoculation
9.3.2.4.
Spinach sampling
9.3.3. X-ray efficacy
9.3.3.1.
X-ray on surface-inoculated E. coli O157:H7
9.3.3.2.
X-ray on biofilm-incorporated E. coli O157:H7
9.4. Results
9.4.1. Bacterial collection and characterization
9.4.1.1.
Enumeration and isolation
9.4.1.2.
Biofilm analysis
9.4.2. Persistence on E. coli O157:H7 during spinach growth
9.4.3. X-ray efficacy on surface-inoculated vs. biofilm-incorporated cells of E. coli
O157:H7

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10. APPENDIX E
COMPARISON OF SELECTIVE AND NON-SELECTIVE PLATING FOR THE
DETERMINATION OF D10 VALUES FOR SANITIZER-TREATED AND X-RAY
IRRADIATED ESCHERICHIA COLI O157:H7 ON BABY SPINACH
10.1. Purpose
10.2. Materials and Methods
10.3. Results
10.4. Conclusion

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11. REFERENCES

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LIST OF TABLES

Table 1 Approved uses for food irradiation (USFDA, 2010)

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Table 2 Properties of X-ray at energy levels of 10.0, 7.5, and 5.0 MeV (Meissner et al.,
2000)

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Table 3 Characteristics of the RS 2400 Irradiator (Mehta and Parker, 2011)

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Table 4 Microbial efficacy (D10 value) of X-ray irradiation on various products

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Table 5 Predicted and observed average reductions of E. coli O157:H7 in bagged baby
spinach

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Table 6 E. coli O157:H7 cell injury following sanitizer treatment

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Table 7 D10 values for sanitizer-treated inoculum

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Table 8 Reduction of E. coli O157:H7 on the surface of flat-leaf parsley leaves

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Table 9 Reduction of E. coli O157:H7 on the surface of flat-leaf parsley stems

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Table 10 Dose rate distribution at the mid-plane region in bags of iceberg lettuce

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Table 11 Log reduction values for E. coli O157:H7 in bags of iceberg lettuce on
10/19/2010

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Table 12 Log reduction values for E. coli O157:H7 in bags of iceberg lettuce on 12/7/2010

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Table 13 Growth of total aerobic bacteria and L. monocytogenes on irradiated and nonirradiated baby spinach

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Table 14 Characterization of organisms isolated from field-sampled spinach

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Table 15 Biofilm-forming ability of EI, Ps, and EC

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Table 16 Growth of Total Aerobic Bacteria, E. coli O157:H7, Pseudomonas, and Lactic
Acid Bacteria over the growth period

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Table 17 X-ray inactivation of surface inoculated laboratory grown spinach- Batch 1

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Table 18 X-ray inactivation of surface inoculated laboratory grown spinach- Batch 2

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LIST OF FIGURES

Figure 1. Diagram illustrating the positioning of radiochromic film dosimeters on a
cardboard segment.

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Figure 2. Reduction of E. coli O157:H7 on baby spinach using X-ray irradiation (95%
confidence limit).

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Figure 3. Dose rate distribution at the mid-plane region of a 225 g bag of baby spinach.

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Figure 4. Dose rate distribution at the surface of a 225 g bag of baby spinach.

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Figure 5. X-ray reduction of Control, PABS-, CBS-, and QACBS-treated cells of E. coli
O157:H7.

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Figure 6. E. coli O157:H7 cell injury following X-ray irradiation.

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Figure 7. D10 values for E. coli O157:H7 on the leaves and stems of flat-leaf parsley.

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Figure 8. Growth of total aerobic bacteria and L. monocytogenes on irradiated and nonirradiated baby spinach.

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Figure 9. Growth of total aerobic bacteria, E. coli O157:H7, Pseudomonad, and lactic acid
bacteria over the growth period.

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Figure 10. D10 values for selective and non-selective control samples of baby spinach.

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Figure 11. D10 values for selective and non-selective XY-12-treated samples of baby
spinach.

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Figure 12. D10 values for selective and non-selective Tsunami-treated samples of baby
spinach.

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Figure 13. D10 values for selective and non-selective QUAT-treated samples of baby
spinach.

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viii

1. INTRODUCTION

Leafy green vegetables are among the top ten riskiest foods regulated by the U.S. Food
and Drug Administration (CSPI, 2009a). This is due in large part to the potential for
contamination to occur throughout all facets of production. Leafy greens are prone to
contamination when grown in an open environment and undergo minimal processing before
consumption. Evidence of contamination has mounted in recent years, as the number of
documented outbreaks of E. coli O157:H7 associated with the consumption of leafy greens has
increased (Soderstrom et al., 2005; Ackers et al., 1998; Hilborn et al., 1999; Ethelberg et al.,
2010). Heightened consumer concerns regarding the safety of leafy greens have warranted a
reassessment of the production practices for ready-to-eat leafy greens.
Ensuring the safety of leafy greens is a process that must also be balanced with
maintaining product quality. Current microbial reduction practices have relied solely on
chemical sanitizers during washing of leafy greens. However, this process has remained largely
ineffective, demonstrating pathogen reductions of < 2 logs (Sapers, 2001; Beuchat et al., 2004;
Keskinen et al., 2009; Lopez-Galvez et al., 2009). Considering the low infectious dose
associated with E. coli O157:H7 and the increasing number of outbreaks, chemical sanitizers
alone are insufficient to ensure the safety of leafy greens. In response to three nationwide
outbreaks linked to bagged baby spinach and shredded iceberg lettuce in 2006, the FDA issued a
rule allowing the use of ionizing irradiation as an alternative strategy to ensure the safety of fresh
and bagged iceberg lettuce and baby spinach (FDA, 2008).
Ionizing radiation, in the form of X-rays, E-beam, and gamma ray, has found many food
applications, including the control of insects, inhibition of sprouting, and pathogen reduction. In

1

controlling pathogens associated with leafy greens, ionizing irradiation has been particularly
effective. Using gamma irradiation, D10 values for E. coli O157:H7 on surface-inoculated
lettuce, in internally inoculated lettuce, and in homogenized lettuce suspensions were 0.1190.140, 0.30-0.45, and 0.092-0.339 kGy, respectively (Niemira et al., 2002; Niemira, 2008). Ebeam irradiation (10 MeV) has shown to be equally effective, with a D10 value of 0.186 kGy for
E. coli O157:H7 on spinach (Gomes et al., 2008).
Until recently, X-rays have been undervalued as an alternative source of ionizing
irradiation. The technology was considered to be inefficient and less effective compared to
gamma radiation and E-beam; however, recent advances in the efficiency and versatility of
machine sources have been working to change perception. X-ray radiation is now being
recognized for its low shielding requirement, small machine footprint, and the ability to be
incorporated into an existing processing system.
Using a newly developed low-energy X-ray irradiator (Rayfresh Technologies, Ann
Arbor, MI), the efficacy of X-rays as been demonstrated for ground beef, lettuce, asparagus,
blueberries, almonds, walnuts, and chestnuts. X-ray irradiation has been particularly effective
for inactivating E. coli O157:H7 on lettuce, with a D10 value of 0.040 kGy (Jeong et al., 2010b),
which is 3.4 times lower than the previously reported value of 0.136 kGy using gamma radiation
(Niemira et al., 2002).
Sublethal injury and cross-protection from one stressor to the next is always a concern
when multiple microbial reduction strategies are used. Chemical sanitizers and ionizing
irradiation often target similar components of an organism, with varying degrees of injury seen
depending on the concentration and length of exposure to the stressor. Given similarities in the
inactivation mechanisms for many chemical sanitizers and ionizing irradiation, the potential for
2

enhanced microbial resistance from sublethal sanitizer stress needs to be addressed before
implementing low-energy X-ray technology as a microbial reduction strategy in the processing
of leafy greens.
This thesis aims to investigate the efficacy of low-energy X-ray irradiation for
inactivating E. coli O157:H7 on baby spinach, with a focus on the commercialization of this
technology within existing leafy green processing facilities. Assuming that X-ray radiation is
used as the final kill step, pathogens on leafy greens will be exposed to X-rays following
sanitizer treatment of the product, which will remain a standard industry practice. Objectives
outlined in this thesis include establishing the D10 value for E. coli O157:H7 on baby spinach,
determining the dose required to result in a 5-log reduction of E. coli O157:H7 in commercial
bags of baby spinach, and to evaluate the potential for cross-protection from chemical sanitizers
during production. Lastly this thesis will address further considerations for commercial
applications and safety concerns of X-ray technology.

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2. CHAPTER 1
LITERATURE REVIEW

2.1. Introduction
Escherichia coli O157:H7 first appeared in the public headlines in 1993 following an
outbreak at Jack-in-the-Box restaurants from consumption of improperly cooked hamburgers
(CDC, 1993). Since its emergence, this pathogen has been routinely associated with the
gastrointestinal tract of ruminant animals, and consequently as a potential hazard during
slaughter and beef production. Other outbreaks involving E. coli O157:H7 have been traced to
apple juice (Cody et al., 1999), sprouts (Breuer et al., 2001), cucumbers (Duffell et al., 2003),
and leafy vegetables (Soderstrom et al., 2005; Ackers et al., 1998; Hilborn et al., 1999; Ethelberg
et al., 2010).
Initial symptoms from E. coli O157:H7 infections are generally quite mild and selflimiting, resulting in diarrhea and stomach cramps. Complications of the infection may occur in
some cases and can be severe, beginning with bloody diarrhea (hemorrhagic colitis) and severe
dehydration, with the potential to progress further into conditions such as hemolytic uremic
syndrome (HUS) and thrombocytopenic purpura (TTP) (FDA, 2009). HUS is of particular
concern in children under the age of 5. It is a condition that develops in up to 15% of cases and
can lead to permanent loss of kidney function, with a mortality rate of 3-5% (Buchanan and
Doyle, 1997). TTP, a neurological illness more common in the elderly, can have a fatality rate
of up to 50%. In 2010, the hospitalization and fatality rates for E. coli O157:H7 infections were
41.6% and 0.45%, respectively (CDC, 2010a; CDC, 2010b). It is estimated that on average, E.

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coli O157:H7 causes approximately 63,000 illnesses, 2,100 hospitalizations, and 20 deaths each
year (Scallan et al., 2011).
From 1973 to 1997, produce accounted for a total of 190 outbreaks from all causes,
16,058 illnesses, 598 hospitalizations, and 8 deaths, with produce-associated outbreaks
increasing in proportion from 0.7% in the 1970’s to 6% in the 1990’s (Sivapalasingam, 2004).
From 1998 to 2007, the Center for Science in the Public Interest (CSPI) reported that produce as
a category had accounted for 684 outbreaks, and resulted in the largest number of illnesses in the
United States compared to any other food group (CSPI, 2009b). CSPI reports further identified
particular risks associated with a number of produce-pathogen combinations, namely Salmonella,
E. coli O157:H7 and Norovirus contamination of leafy greens. These three pathogens combined
for a total of 265 outbreaks and 9,001 illnesses from consumption of lettuce and leafy greenbased salads.
Concern for E. coli O157:H7 contamination of leafy greens came to a head in 2006
following three prominent outbreaks: one in September associated with baby spinach resulting in
205 confirmed cases in 28 states and 3 deaths (CFERT, 2007), and another two in December
associated with shredded lettuce that resulted in a total of 80 confirmed cases in 5 northeastern
states (U.S. FDA, 2007a; U.S. FDA, 2007b). In light of the heightened severity of illness and
symptoms associated with E. coli O157:H7 infections, the pathogen has presented itself as the
target for control on leafy green vegetables, with the primary focus on spinach and iceberg
lettuce.
Control of pathogens in the processing of leafy greens has been reliant on the application
of chemical sanitizers in the wash water; however, the efficacy of these agents has been less than
ideal with bacterial populations decreasing only 1 to 2 logs on the surface of the produce during

5

commercial processing (Sapers, 2001; Beuchat et al., 2004; Keskinen et al., 2009; Lopez-Galvez
et al., 2009). In light of the poor performance of sanitizers and the continued outbreaks
associated with leafy greens, alternative solutions such as X-ray irradiation need to be
investigated.
X-ray irradiation has proven to be a highly effective means of bacterial inactivation,
particularly in the treatment of leafy greens (Jeong et al., 2010b, Moosekian et al., 2010a;
Mahmoud, 2010a; Mahmoud et al., 2010). The technology also provides the capability of
implementation into a processing line as a final kill step. Given similarities in the inactivation
mechanisms for many chemical sanitizers and ionizing irradiation, the potential for enhanced
microbial resistance from sublethal sanitizer stress should be addressed before implementing
low-energy X-ray technology as a microbial reduction strategy in the processing of leafy greens.
The implications of cross-protection for the food industry and public health, begins with
an understanding of the actions of chemical sanitizers and their limitations, and how the bacteria
use their environment (produce surface, organic load) and defense mechanisms (gene expression,
protection from reactive oxygen species (ROS)) to overcome, adapt and thrive under such
conditions.

2.2. Potential Sources of Contamination
Measures to prevent the interaction of E. coli O157:H7 with leafy green vegetables have
proven to be difficult, due to the manner in which the leafy greens are grown and the uncertain
origin of the initial contamination. Plants grow in the open environment, exposed to the
elements of nature, and low to the ground. Leafy greens also demand plenty of water for growth,
and depending on the variety and fragility of the leaf tissue, cultivation by hand may be

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necessary. Measures to amplify the cultivation of leafy greens have helped to elevate the
potential risk of bacterial contamination by creating a process that requires larger plots of land,
unskilled workers, and irrigation water to further expand growing seasons and locations.
Furthermore, the raw manner in which most leafy greens are consumed adds another level of
difficulty in the assessment of contamination routes. In order to maintain the leaf’s visual and
organoleptic qualities, leafy greens cannot be heat-treated to inactivate pathogens. Such
difficulties in either preventing or reducing the contamination of leafy greens with E. coli
O157:H7 have culminated in a reassessment of established field environment and processing
conditions in a search for reliable strategies to better ensure end product safety. Furthering the
understanding of these key components in the process and their role in a contamination event has
been a primary focus in determining the overall risk from the field and future outbreak
prevention.

2.2.1. Irrigation practices
Water is a bare necessity to a plant’s growth and survival, and may be considered a
limiting factor to the location and growing season of certain crops. Leafy greens in particular
have a relatively high water requirement, and are therefore not traditionally grown in certain
areas of the country due to water limitations and temperature extremes. However, demand for
fresh produce is consistent year-round, driving the necessity for their growth in greenhouses or in
the outdoors under less-than-favorable conditions. Irrigation, or artificially supplying land with
water, is a necessary practice in locations where rainfall may be limited. By transferring water
from sources where it is plentiful to locations where it is lacking, crop growth may be possible
under even the driest conditions.

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Irrigation has found widespread use in the U.S. and abroad, including the expansion of
the growing season and growing locations. In 2008, the Farm and Ranch Irrigation Survey
showed that U.S. farmers and ranchers irrigated 54.9 million acres of land, with the amount of
water applied totaling 91.2 million acre-feet from 2003 to 2008 (USDA, 2008). The source of
this irrigation has originated primarily from surface waters such as lakes, rivers, ponds, and
streams located in either on-farm water supplies or off-farm reservoirs. Other less common
sources include treated wastewater, ground water and well water. Similar trends have also been
recorded in the United Kingdom, where the total irrigated area reported in 2003 was about
12,700 acres, with surface waters accounting for approximately 71% of the water used for leafy
green irrigation (Tyrrel et al., 2006).
Considering the close contact of irrigation water with the edible portions of the growing
plant, the quality of the water has been identified as a primary source of microbial concern.
Consequently, guidelines for irrigation water quality have been set by the EPA and WHO for
treated waste water (Hespanhol and Prost, 1994) and surface waters (National Research Council,
1974), requiring fewer than 1,000 fecal coliforms per 100 ml.
Testing of surface waters is difficult and unreliable, as the quality of surface water may
fluctuate from day to day depending on how it is supplied and collected from the water source,
which may in turn vary due to any number of interactions that the water may encounter en route
to the farm. Pollution of surface waters may occur from industrial dumping or run-off from
areas such as feedlots, waste piles, or known sources of fecal contamination, during events of
flooding (Wing et al., 2002) or the sudden rise in river flow (Fremaux et al., 2009).
Not all methods for applying irrigation water are equal. It is estimated that the majority
of farms and ranches in the United States prefer spray irrigation over other methods such as drip,

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hand-watered, subirrigation or gravity irrigation (USDA, 2008). Irrigation by spraying, however,
potentially enhances the interaction of the growing crop with contaminated water by increasing
the dissemination and survival of E. coli O157:H7 across the crop (Moyne et al., 2011; Fonseca
et al., 2011; Solomon et al., 2002a; Solomon et al., 2003).

2.2.2. Interaction with wildlife and animal excrement
Plant contact with wildlife can occur directly through interactions that take place in the
field, or indirectly by means of contaminated water, manure, or other items that may be used in
growing crops. Direct interaction in the field from uncontrolled wild animals or insects has
shown to be an uncommon and sporadic route of contamination, while indirect sources have
been the focus of greater scrutiny due to their unpredictability and potential to amplify and
spread contamination throughout an entire lot of product.
Animals that graze on nearby fields are seen as the greatest contributors to surface water
contamination. E. coli O157:H7 has consistently been isolated from the hides and feces of
livestock including pigs (Gill and Jones, 1998), cattle (Fegan et al., 2004; Smith et al., 2009),
sheep (Ogden et al., 2005), deer (Rice et al., 2003), and wild rabbits (Scaife et al., 2006). E. coli
O157:H7 has also been shown to survive for more than two months in feces (Beuchat, 1999;
Wang et al., 1996) and manure-amended soils (Mukherjee, et al., 2006a; Duffy, 2003;
Johannessen et al., 2005; Islam et al., 2004), and up to 28 days on equipment that has come into
contact with feces (Williams et al., 2005), with the ability to leach out from ruminant feces
(Williams et al., 2008) under rainy conditions.
Minimizing the spread of pathogens from the water source can be achieved through good
grazing management practices, such as rotational grazing, portable water supply, portable shade

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source, and fencing animals from water access (Hubbard et al., 2004). During investigations into
the spinach outbreak of 2006, E. coli O157:H7 was recovered from cattle feces, wild pig feces,
soil, and water, with 58% of the isolates being similar to the outbreak strain by PFGE analysis
(CFERT, 2007). In a report from the California Food Emergency Response Team potential risk
factors included the presence of wild pigs in and around spinach fields and the close proximity of
irrigation wells to surface water exposed to feces from cattle and wildlife. In a rural Virginia
watershed, with the installation of fencing and in-pasture watering stations to restrict the range of
cattle on the farm, fecal coliforms were reduced by an average of 94%, from pre-fencing average
populations of 15,900 per 100 ml to post-fencing average populations of 960 per 100 ml
(Hagedorn et al., 1999).
Air contamination by bioaerosols can also occur from nearby fields. When sprayed onto
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a neighboring field, E. coli K12-contaminated pig slurry (10 CFU/ml) contributed to bacterial
levels recovered as far as 125 m into the field (Hutchison et al., 2008). Further reports on the
spread of pathogens by bioaerosols have been collected in a review by Pillai and Ricke (2002).
Potential for insects to act as vectors of pathogen transmission, particularly from local
sources of ruminant feces to the fields of edible plants, has also been raised as an area of
concern. In an investigation of leafy green fields in the Salinas Valley of California, 11 of 18
flies collected from one field tested positive for E. coli O157:H7 (Talley et al., 2009). Further
research by this group also determined experimental transmission of the pathogen to the crop, in
which contaminated flies successfully delivered GFP-labeled E. coli O157:H7 to leaves (Talley
et al., 2009). Previous reports of flies as vectors for E. coli O157:H7 transmission has also been
documented for apples (Janisiewicz et al., 1999).

10

Slugs have also been considered to be potential vectors. E. coli O157:H7 can survive on
the surface of a slug for up to 14 days and within its feces for up to 3 weeks. In a field
investigation, E. coli O157:H7 was isolated from 0.21% of field slugs from an Aberdeenshire
sheep farm (Sproston et al., 2006).

2.2.3. Transfer of E. coli O157:H7 from contaminated water and soil to the plant
Screening for E. coli O157:H7 on leafy greens in actual production fields has shown the
presence of avirulent E. coli on various leafy greens across the United States (Johnston et al.,
2005; Mukherjee et al., 2006b), Canada (Arthur et al., 2007), and Europe (Oliveira et al., 2010),
but has not revealed the presence of pathogenic E. coli. These studies indicated the presence of
fecal material, suggesting the possibility for E. coli to follow a similar route to the field.
Considering the intentional application of water and manure to fields, E. coli and coliform levels
may not be the best indicator of contamination.
Contamination of leafy greens with E. coli O157:H7 has been accomplished
experimentally through the use of contaminated manure-amended soils (Mootian et al., 2009;
Islam et al., 2004; Franz et al., 2007) and water irrigation (Ibekwe et al. 2004; Mootian et al.,
2009; Wachtel et al., 2002). Damage to the plant’s leaf structure can also significantly increase
E. coli O157:H7 persistence and survival in the field (Harapas et al., 2010; Barker-Reid et al.,
2009), with potential for internalization of the pathogen (Solomon et al., 2002). Such conditions
used for experimental research purposes are often exaggerated, using unrealistically high
inoculation levels and may not reflect actual events as they occur in nature. Overall,
internalization of E. coli O157:H7 into lettuce and spinach leaf tissue would require greater than

11

4.4 log CFU/leaf, and is therefore unlikely to occur in the field at low levels (Erickson et al.,
2010).

2.2.4. Washing/bagging
The final steps of adding value to leafy greens, including shredding, washing, drying and
bagging, have the potential to not only introduce contamination but also to spread incoming
pathogens to otherwise clean product. Contamination can be introduced into the process by the
incoming product, wash water (Hilborn et al., 1999), food handlers, and the equipment surfaces.
A contaminated batch of lettuce can in turn contaminate the processing equipment, allowing the
contamination to spread to subsequent product (Buchholz et al, 2009). Cross-contamination
between leaves of fresh-cut lettuce may occur from contaminated water regardless of treatment
with sanitized water (Lopez-Galvez et al., 2009). Furthermore, populations of E. coli O157:H7
can increase 4, 4.5, and 11-fold on lettuce leaves that are mechanically bruised, cut into large
pieces, or shredded into multiple pieces, respectively, compared to only 2-fold increase on intact
leaves under the same conditions (Brandl, 2008).
Once inside the bag, E. coli O157:H7 populations will decrease at proper refrigeration
temperatures <5°C, while logarithmic growth will likely occur at higher temperatures >8°C (Luo
et al. 2010; Abdul-Raouf et al., 1993; Francis and O’Beirne, 2001; Li et al., 2001; Koseki and
Isobe, 2005). Such elevated temperatures may be reached during transport of the leafy greens to
or from the market, and in storage at the market or home.

12

2.3. Stress and Sublethal Injury
Microorganisms encounter physical, chemical, and nutritional stressors throughout all
phases of processing, challenging the organism’s ability to survive and persist under diverse
conditions. In the growth of leafy greens, bacteria may be stressed with temperature extremes,
radiating energy from the sun, and a limited supply of nutrients in the field. Under processing
conditions, the bacteria may be further exposed to agents such as chemical sanitizers, acidic
environments, high pressure, and/or radiation. Under each agent and condition, the bacteria act
to protect themselves against damage to their cellular barriers, metabolic processes, and
reproductive functions.
Injured microorganisms are characterized by a greater sensitivity to chemical compounds
used in selective environments, due to structural or metabolic damage (Brashears et al., 2001).
Sublethal injury can be further explained as damage to an organism without complete kill,
implying loss of function that may be either transient or permanent (Gilbert, 1984). Injury may
occur from low levels of an applied stressor, with predictable and reproducible injury occurring
rapidly, and is reversible under nonselective conditions (Camper and McFeters, 1979). Such
attacks often leave the organism in a viable but not culturable state, void of detection using
traditional methods (Hurst, 1977). The use of nonselective conditions is often used as a basis for
quantifications of microbial injury, utilizing an organism’s ability to proliferate on non-selective
compared to a selective media (Wu et al., 2001). Often enrichment of the medium with added
nutrients may further facilitate the recovery. Chlorine-injured cells of E. coli recover better in
environments where ROS are neutralized (Tandon et al., 2007).

13

2.3.1. Chemical sanitizers
Sanitizers are agents that work to inactivate microorganisms through chemical
interactions with the cell and the components therein. Such agents are used commercially, and
vary in application depending on the primary mechanism of action, desired target organism, and
physical properties of the material to which the sanitizer is applied. Based on these properties,
sanitizers are commonly grouped into the following categories: alcohols, aldehydes, anilides,
biguanides, diamidines, halogen-releasing agents, silver compounds, peroxygens, phenols, bisphenols, halophenols, and quaternary ammonium compounds.
Chemical sanitizers have been used as the primary reduction strategy throughout the
produce industry, especially in the treatment of leafy green vegetables, because of their ease of
use and relatively low negative impact on product quality. Peroxygens and chlorine-based
compounds (a halogen-releasing agent) are used in flume water during produce washing, while
quaternary ammonium compounds are more commonly used to sanitize processing equipment
surfaces between production runs.
Correlating the properties of sanitizers to the control of bacteria in flume water has
proven to be problematic. During the processing of fresh produce, many variables act to inhibit
optimum sanitizer function which leads to difficulties in reducing bacterial populations to
acceptable levels. Considering the role of organic debris, bacterial defense strategies, and the
structural composition of the product making up the organic load, there is a cumulative
protective effect offered to the microorganism and a subsequent decrease in sanitizer efficacy.

14

2.3.2. Oxidizing sanitizers
Oxidants are a broad category of chemical sanitizers that function through the generation
of reactive oxygen species (ROS) as charged particles that elicit deleterious effects on the
composition of the bacterial cell. Such stress is known to damage proteins, membranes, and
DNA (Dowds, 1994). The bulk of the research within this category, and the primary focus of
this review has fallen within the subset of peroxygens, namely hydrogen peroxide and
peroxyacetic acid; and the halogen-releasing agents, that of the chlorine-based compounds.
Using hydrogen peroxide as a model for oxidative stress, Imlay and Linn (1988) have
attributed a major portion of oxidant toxicity towards E. coli to DNA damage mediated by a
Fenton reaction that generates active forms of hydroxyl radicals, DNA-bound iron, and a
constant source of reducing equivalents (Imlay and Linn, 1988). Cellular DNA was affected
directly by a derivative of hydrogen peroxide, suggesting that the oxidant may be a ferryl radical
as opposed to a hydroxyl radical (Imlay et al., 1988). Imlay and Linn (1987) have further
characterized the killing of E. coli by H2O2 as a two-step mechanism: (1) DNA damage,
requiring active metabolism during exposure; (2) uncharacterized damage, occurring in the
absence of metabolism (Imlay and Linn, 1987). Increases in free-iron concentration have the
effect of accelerating DNA oxidation (Keyer et al., 1995).
The action of oxidants has shown multiple stages of activity depending on the contact
time and sanitizer concentration. Upon initial contact, sublethal injury is incurred by the cell,
beginning with the cellular membrane and acting further on the physiological functions of the
cell following uptake of the applied sanitizer. Lisle et al. (1999) quantified the progression of
sublethal attack through a series of physiological indicators, suggesting that the site and extent of
injury of bacterial cells can be determined following the order: viable plate counts > substrate
15

responsiveness > membrane potential > respiratory activity > membrane integrity (Lisle et al.,
1999). An increase in O2- of more than two-fold above wild-type levels substantially
diminished the activity of labile dehydratases, a four-fold or greater increase in O2- measurably
impaired growth, and a five-fold increase in O2- sensitized the cells to DNA damage (Gort and
Imlay, 1998).

2.3.3. Peracetic acid-based compounds
Peracetic acid, as it is commercially available, is prepared as a mixture of acetic acid,
hydrogen peroxide, peracetic acid, and water according to the formula:

CH3CO2H + H2O2 -> CH3CO3H + H2O
Where:
CH3CO2H = acetic acid
CH3CO3H = peracetic acid
H2O2 = hydrogen peroxide (Kitis, 2004)

These sanitizers, which function similar to hydrogen peroxide, act against DNA (Tutumi
et al., 1974), oxidize essential enzymes (Baldry and Fraser, 1988), denature proteins and
enzymes and increase cell wall permeability by disrupting sulfhydryl and sulfur bonds (JolivetGougeon et al., 1996).

16

2.3.4. Chlorine-based compounds
Chlorine-based sanitizers have an extensive history compared to other forms of oxidizing
agents and disinfectants. First discovered in 1774 by Carl Wilhelm Scheele, chlorine gas was
found to be water soluble and capable of bleaching paper, vegetables and flowers (White, 1999).
Some of the first recorded uses of chlorine as a disinfectant occurred in the early 1800’s, when
Guyton de Morveau in France and Cruikshank in England outlined its use as a common
household cleaner by mixing together table salt, manganese, water, and sulfuric acid (Rideal,
1895). When added to water, chlorine gas almost completely hydrolyzes to form hypochlorous
acid, the active form in common household bleach, which reversibly dissociates into hydrogen
ions and hypochlorite ions (WHO, 1995).
The mechanism by which chlorine-based compounds inactivate bacterial cells is a topic
of debate, one that has consisted of numerous research studies with inconclusive results.
Chlorine-based compounds have been found to disrupt cell surface components (Ingols et al.,
1953; Venkobachar et al., 1977; Haas and Engelbrecht, 1980; Virto et al., 2005), inhibit key
cellular functions (Knox et al., 1948; Pereira et al., 1973; Barrette et al., 1989), and damage
DNA (Patton et al., 1972; Hoyano et al., 1973).
Hypochlorous acid is generally considered to be a highly destructive, nonselective
oxidant which strongly reacts with various subcellular compounds and affects metabolic
processes, and therefore is unlikely itself to reach the DNA (Dukan and Touati, 1996). The
progression of chlorine-based sanitizers to the interior components of the cell is therefore likely
carried through by derivatives of chlorine generated through chemical reactions with the cell;
hypochlorous acid formed by hydrolysis of the compound may be responsible for such reactivity.
Marks et al. (1945) first described the binding of hypochlorous acid to nitrogen, and the

17

production of the N-chloro compound. The reactivity of the N-chloro compound in this research
was found to be dependent on the ratio of chlorine to nitrogen, and also on the nature of the
nitrogen compound (Marks et al., 1945).
The primary focus of further reactive compounds is the generation of chloramines, the
products of the reaction of hypochlorous acid or other chlorinating agents with primary and
secondary amines (Thomas et al., 1986). Chloramines can effectively break both single- and
double-stranded DNA in vitro and in vivo (Shih and Lederberg, 1976). Cho et al. (2010)
determined that because of chloramines and other chlorine derivatives, chlorine, as a weaker
oxidant, is actually a more effective bactericidal agent in comparison to stronger oxidants like
chlorine dioxide. Chlorine action involves continuous penetration into the cell without
significant surface damage or cell permeability changes as evidenced by limited protein release,
and lipid peroxidation (Cho et al., 2010).

2.3.5. Quaternary ammonium compounds
In contrast to chlorine and peracetic acid, quaternary ammonium compounds (QACs)
operate as surface-active agents or surfactants. Instead of using ROS to disrupt chemical bonds
of the organism, these agents use a bipolar design consisting of two regions in their molecular
structures, one a hydrocarbon, water-repellent (hydrophobic) group and the other a waterattracting (hydrophilic) group (McDonnell and Russell, 1999).
th

Research into the antimicrobial properties of QACs dates to the early 20 century, and
has been captured in a detailed review by Rahn and Van Eseltine (1947). In general, QACs act
against the cell membrane (Przestalski et al., 2000; Ioannou et al., 2007) through modifications
to bacterial cell surface components.
18

Individual characteristics and composition of the bacterial cell surface are responsible for
the efficacy of quaternary ammonium compounds, allowing the compounds to bind by
chemisorption to the cell surface of bacteria because it is a negatively charged physiological pH
(Neu, 1996). This property of QACs varies from one organism to the next depending on the
genetic make-up of the individual species. Russel et al. (1987) found that higher levels of LPS,
shown on rough strains of E. coli, exhibited greater inactivation in comparison to the smooth
strains.
Another function provided by QACs, due to the bipolar nature of the compound, is to
reduce bacterial attachment to food contact surfaces. QACs alter the hydrophobicity of the
surface, resulting in a decrease of the contact angles (and an increase of the surface free energy),
accompanied by a reduction in the number of adhered bacteria in comparison with the standard
conditions (Sinde and Carballo, 2000). This has been the justification for using QACs in
sanitation programs; in their application to clean surfaces of processing equipment as opposed to
as an additive during production.
The ability to limit attachment has again varied between organisms. QACs were more
effective in reducing attachment of Salmonella spp. than L. monocytogenes (Sinde and Carballo,
2000). Quaternary ammonium compounds have shown to inhibit and reduce the attachment of
Salmonella to poultry tissues (Breen et al., 1995).

2.3.6. Sanitizer limitations
A primary limitation to the efficacy of chemical sanitizers in the processing of leafy
greens has been the presence of organic matter and the inherent defense mechanisms of the plant.
Organic matter may present itself in many forms in food processing systems, from noticeable

19

residual soil or debris, to minute bacterial biofilms on the surface of equipment or food surfaces.
Leafy greens in comparison to other produce may contain high levels of debris because of their
growth very close to the ground. Greens also generally have high microbial populations on the
leaf surface. In addition, the plant itself, composed of water, cellulose, vitamins, nutrients and
phytochemicals, produces alternative sources to accept reactive oxygen species that are intended
for microorganisms (Foyer and Halliwell, 1976; Gillham and Dodge, 1986; Luwe et al., 1993).
The disruption of leaf cells during washing and cutting of leafy greens allows for high levels of
these organic materials to be deposited into the processing water and onto equipment surfaces.
The concern with organic interference has been most apparent with chlorine-based
sanitizers as opposed to peracetic acid or QACs. Small amounts of organic matter can drastically
reduce chlorine efficacy. Trypticase Soy Broth at a concentration of 150 ppm in distilled water
resulted in less leakage of UV-absorbing substances from cells of E. coli, L. monocytogenes, B.
subtilis and Y. enterocolitica and no uptake of propidium iodide, an indicator of cytoplasmic
membrane damage, compared to control cells in distilled water (Virto et al., 2005). The presence
of food residues in the form of flour, whole-milk or egg yolk resulted in a decline in the
sporicidal efficacy of sodium hypochlorite against B. anthracis, while the dose-response
relationships for peroxyacetic acid and hydrogen peroxide showed little variation when
challenged with the same residues (Hilgren et al., 2007).

2.4. Irradiation
X-ray irradiation as an inactivation strategy for fresh produce has been discussed in detail
in a review paper by Moosekian et al (2012). A segment of this paper is included in this section.

20

Ionizing radiation is attracting renewed attention as a potential non-thermal microbial
intervention strategy to ensure the safety of fresh fruits and vegetables. One of the newest
strategies involves the development of machines that generate low-energy X-ray electromagnetic
waves for in-line processing with these units likely to receive greater consumer acceptance than
gamma irradiators that rely on a radioactive source.
Electromagnetic waves are inherent to the world in which we live, with human exposure
coming from many diverse sources, ranging from the sun to cellular phones. Typical
2

electromagnetic wavelengths/frequencies vary in size from 10 m (AM radio signals) to 10

10

m

(X-rays). Based on the duality of matter as hypothesized by de Broglie in 1925 (Khan, 2003),
electromagnetic waves also can be referred to as particle radiation. Depending on the
wavelength/frequency, electromagnetic waves will yield different effects, with the particles or
photons from shorter wavelengths able to more easily penetrate solid materials. Radiation defined as the process by which electromagnetic waves or particles pass through a medium - is
classified as either ionizing or nonionizing. If the electromagnetic wave energy is greater than
the minimum required for ionization of atoms, it is categorized as ionizing radiation and if not it
is nonionizing radiation. Ionizing radiation includes both direct (electrons, protons, alpha
particles, heavy ions) and indirect radiation (X-ray, gamma rays) (Podgorsak, 2006).
Based on particle mass and speed, varying amounts of energy are transmitted to a food
product or any other material upon impact. The amount of energy deposited in a unit mass (J kg
1

) is measured using a standard unit called a gray (Gy), which is named in honor of the British

physicist Louis Harold Gray, the father of modern-day radiobiology. Typical ionizing radiation
doses for treating food products range from 1 to 44 kGy. The dose required to reduce a

21

-

microbial population by 90% (i.e., 1 log) is termed the D10 value (kGy) (Molins, 2001).
Another convenient energy unit in atomic and nuclear physics is the electron volt (eV), which is
19

the kinetic energy, equal to 1.602  10

J, needed to accelerate an electron across a one volt

electric potential difference (Khan, 2003). The energy level for gamma rays is approximately 1
6

 10 eV or (1 MeV), with X-ray being in the range of a few keV to MeV.
Radioactive materials and particle accelerators are the two main sources for ionizing
radiation. Gamma rays are a natural byproduct from the decay of radioisotopes (e.g.,

60

Co or

137

Cs) and are emitted in all directions, whereas electron beams (E-beams) generated from

particle accelerators can be aimed at specific targets. Consequently, each type of radiation has
its inherent advantages and disadvantages in food, medical, and other types of applications.

2.4.1. History of X-ray
Ionizing irradiation - including gamma ray, E-beam, and X-ray - has long been
recognized as a viable cold pasteurization strategy for reducing the levels of both pathogenic and
spoilage microorganisms in a wide range of foods for the purpose of enhancing food safety and
product shelf life. The introduction of X-rays as a source of ionizing irradiation dates back to the
late nineteenth century, when German physicist W. C. Roentgen first observed the generation of
radiation during his experiments with Hittorf-Crookes tubes, also known as modified cathode ray
tubes. In his 1895 paper, Eine neue Art von Strahlen (A New Kind of Ray), Roentgen described
how rays exiting the device generated transparencies of several objects, including paper, tinfoil,
wood, rubber, and flesh. Roentgen noted that “if one holds a hand between the discharge
apparatus and the screen, one sees the darker shadow of the bones within the slightly fainter
22

shadow image of the hand itself” (Roentgen, 1895). Roentgen termed the unknown light rays
that were emitted from the tube “X”-rays, forming the basis behind the function of medical Xrays.
Further understanding of X-rays and their properties as a source of ionizing irradiation
developed in accordance with the discovery of radioactive elements. In 1896, A. H. Becquerel
observed a form of radiation similar to that of Roentgen, stemming from elemental uranium,
finding that when a photographic plate was exposed to salts of the element, a distinct impression
was left on the plate (Strutt, 1904). Marie and Pierre Curie expanded upon the work of
Becquerel with experimentation into understanding the source of energy and radioactivity of
elements. Marie Curie further clarified the radiation emitted from radioactive materials,
describing the similarities between Roentgen’s rays and alpha, beta, and gamma rays, as
penetrating rays that are unaffected by a magnetic field (Curie, 1961).

2.4.2. X-rays as a form of ionizing irradiation
X-rays, or Roentgen rays, appear next to gamma rays in the electromagnetic spectrum at
frequencies of 10

16

to 10

19

Hz. The somewhat lower energy photons emitted by X-rays are

formed from the interaction of a charged particle with matter, either from replacing displaced
electrons from a low-lying orbit or through bremsstrahlung, also known as braking radiation
(Newton, 1963). Machine sources of X-rays primarily use bremsstrahlung, where active photons
emitted when high-velocity electrons strike a dense metal target, such as tungsten, tantalum, or
gold, are directed toward the desired object. These high energy particles alone may also generate
a lower level of ionization, the technology of which has been used in the development of highenergy E-beams.
23

Research into the ionizing effects of X-ray radiation did not begin until several years after
its initial discovery, when researchers began to explore the power and effect of X-rays on various
targets. At this time, researchers were still relatively unaware of the potential mechanism of
action. Much of the work with X-rays during the early 1900s focused on food preservation and
the control of insects or pests that negatively impact the quality of food and other consumer
goods.
Through the early part of the twentieth century, it was generally concluded that the
technology and efficiency of X-rays was simply too expensive for large-scale industrial use. In
1912, W. D. Hunter documented some of the first effects of Roentgen rays on insect control. In
his review, he concluded that for the insects being tested with the technology of the time, there
were no indications of any practical applications of X-rays for the destruction of insect pests
(Hunter, 1912). A few years later, Morgan and Runner (1913) detailed the use of Roentgen rays
in controlling the tobacco or cigarette beetle’s infestation of tobacco products. Their work
coincided with an attempt by the American Tobacco Company, under the assistance of Hunter, to
commercialize X-rays for this particular purpose. The commercial design used two seven-inch
X-ray tubes operating at 64,000 to 70,000 volts, with a current of 1.5 to 2.5 mA passing through
the tubes. However, this treatment was ineffective in sterilizing tobacco because of variable Xray penetration.
Using a subsequently modified Roentgen ray that allowed the operator to control both the
intensity and penetrating power, higher doses applied at 600 mA (15 mA x 40 min) to infected
tobacco yielded more promising results, showing signs of beetle egg infertility (Runner, 1916).
The American Tobacco Company later implemented an in-line conveyor system for irradiating
boxes of cigars in 1929, using a water-cooled X-ray at a maximum power of 30 mA at 200 kV.

24

However, the equipment still proved to be unsuitable for continuous use, as too much time and
energy were wasted in machine maintenance and operation (Diehl, 1995).
In 1918, a U.S. patent was granted to D. C. Gillett for his design of an “Apparatus for
Preserving Organic Materials by the Use of X-rays” (Gillett, 1918). Gillett’s design was
intended to “destroy utterly any destructive insects or other animal life that would tend to destroy
perishable articles, or to sterilize these insects and prevent the further propagation of their
species.” In 1921, based on a design similar to Gillett’s, the U.S. Department of Agriculture
began research through the Zoological Division of the Bureau of Animal Industry on X-rayinitiated inactivation of Trichinella, the parasite in pork responsible for human trichinosis. The
end results, however, were inconclusive and did not justify X-ray as a feasible means of trichinae
destruction. X-rays appeared to injure the organism and/or disrupt reproduction, but the
trichinae exhibited considerable variation in susceptibility (Schwartz, 1921).
The impact of X-rays and other forms of ionizing irradiation on matter had a monumental
impact in the United States throughout the 1940s and 1950s. During the 1940s, interest focused
on the application of irradiation toward preserving foods in an effort to extend the food supply.
In 1953, President Eisenhower proposed the Atoms for Peace Program in a speech to the United
Nations, describing and advocating the use of ionizing irradiation of food as a means to decrease
world hunger by limiting the need for preservation and reducing pests. However, as the United
States entered the Cold War, interest shifted toward the negative effects of ionizing irradiation on
the nutritional properties of food and the side-effects of consuming irradiated foods.
Breakthroughs in the actual mechanism of X-rays and ionizing irradiation on cells and
microorganisms took place toward the middle of the past century in conjunction with
developments in DNA research. During that time, Bergonie and Tribondeau (1959) reported that

25

X-rays were more effective against rapidly growing cells, noting that they destroyed tumors as
opposed to the surrounding tissue. Further information on the historical developments of X-rays
and ionizing irradiation can be found in reviews by Josephson (1983) and Whitmore (1995), and
greater detail pertaining to research done by the U.S. Army can be found in reviews by
Josephson et al. (1978) and Brynjolfsson (1979).

2.4.3. Mechanism of action
When an atom is exposed to X-rays, energy transactions occur between the projected
photons and the orbiting electrons. These interactions result in a net transfer of energy from Xrays to electrons in the absorbing material, raising the electron excitation level (Newton, 1963).
Excitation, resulting from a low level of energy, moves an electron further out in its atomic orbit,
thereby increasing the net energy. Ionization then occurs when the energy level sufficiently
increases to produce highly reactive positive and negative ions by the removal of an orbiting
electron (Wilkinson and Gould, 1998).
The extent to which ionization occurs in irradiated matter depends on the energy of the
photon and the physical properties of the matter. Activity generated by the photons of X-rays
may result in one of two energy absorption processes in the irradiated material: compton
scattering or photoelectric absorption (Miller, 2005). When using low-energy photons,
photoelectric absorption is generally seen with all of the photon’s energy transferred to the
electron, which then goes on to interact with other atoms. Compton scattering occurs at higher
energy levels where photon interactions are confined to outer, loosely bound electrons, causing
only a portion of the photon energy to be absorbed by the encountered electron (Pizzarello and
Witcofski, 1975). Under these conditions, the initial photon and any excited or ionized particles

26

may continue to react. It is estimated that an electron may produce 30,000 - 40,000 additional
ionization processes and 45,000 - 80,000 excitations (Nawar, 1986).
The impact of charged particles on matter is classified according to the type of contact.
Ionized or excited molecules may exert their effect by either direct contact of the photon with
their target, i.e., the direct effect, or by the formation of cations created from target components
such as water, i.e., the indirect effect (Podgorsak, 2006). Direct effects occur randomly and are
dependent upon the electron density of the biological material. Water, a major component of
foods and biological material, is the primary target for energy coming from the X-ray source.
Reactions with photons and water lead to the generation of highly reactive free hydrogen and
hydroxide radicals that are split from the hydrogen bond. Excited water molecules can further
react to produce hydrogen and hydrogen peroxide, the only stable end products of water
radiolysis (Miller, 2005).
The effects of ionizing irradiation can be seen throughout each component of an
organism; however, the primary target remains the DNA. Approximately 20% of this attack is
on DNA sugars and 80% on bases, with thymine being the most sensitive, followed by cytosine,
adenine, and guanine (Moseley, 1990). Pollard (1966) attributed this observation to the relative
mass of DNA compared with the organelles of the cell, stating that radiation sensitivity of
organic substances is proportional to their molecular weight. He estimated that a dose of 0.1
kGy would damage 0.005% of the amino acids, 0.14% of the enzymes, and 2.8% of the DNA
within a given cell. Within DNA, most strand breaks occur from the scission of the C-3
phosphate ester bond, producing 5-PO4 and 3-PO4 termini in a 3:1 ratio (Johnston and
Stevenson, 1990). Both single- and double-strand breaks can occur in DNA based on the
random action of ionized particles with the latter occurring far less frequently.
27

2.4.4. Dose measurements
Radiation doses are measured using ionizing radiation sensitive materials that can be
classified according to their accuracy and range. Based on accuracy of the measurement, the
following four categories are now recognized: (a) primary standards (1%~2% uncertainty)
maintained by national standards laboratories, (b) reference standards (~3% uncertainty) for
calibrating radiation environments and routine dosimeters, (c) transfer standards for establishing
traceability of an irradiation facility, and (d) routine standards (5%~10% uncertainty) for
radiation process quality control, absorbed-dose monitoring, and mapping (ISO/ASTM, 2005).
5

4

A water calorimeter (10 ~10 Gy) or ionization chamber is used for primary standards, an
1

4

alanine dosimeter (10 ~10 Gy) is used for for reference standards, and a clear PMMA
2

5

(polymethylmethacrylate) or radiochromic film (10 ~10 Gy) is used for routine dosimeters
(Molins, 2001). Of these, alanine-EPR (electron paramagnetic resonance) and radiochromic
dosimeters are the most popular. The alanine-EPR dosimetry system uses an alanine dosimeter
(film or pill type) and EPR spectroscopy to measure free radicals (ISO/ASTM 2004a; MaltarStrmecki and Ravkin, 2004). In radiochromic dosimetry (film type), a spectrophotometer is used
to measure radiation-induced color changes on a film as a series of absorption bands
(ISO/ASTM, 2004b; Mehta and Parker, 2001). Alanine dosimeters are stable over long periods
of time, whereas calibration curves must be continually established for radiochromic films.

2.4.5. The need for alternative microbial reduction strategies
Across the globe, interest in ionizing irradiation has increased steadily since the
beginning of the millennium, with the market for irradiation equipment increasing from 19

28

billion to over 25 billion U.S. dollars. The United States alone claims roughly one quarter of this
spending (Parker, 2005). Worldwide, various irradiation technologies are now being used in at
least 55 countries to treat food products (IAEA, 2009). Renewed interest in ionizing irradiation
has developed in response to continued outbreaks traced to fresh produce, including lettuce
(Ethelberg et al., 2010; Irvine et al., 2009, Nygard et al., 2008; Sodha et al., 2001), spinach
(Grant et al., 2008; Wendel et al., 2009), and raw nuts (Danyluk et al., 2007; Isaacs et al., 2005;
Kirk et al., 2004) since these products are are adversely affected by thermal processing. From
1998 to 2007, a total of 1,999 outbreaks and 35,554 illnesses were associated with consuming
meat, poultry, and seafood, with 684 outbreaks and 26,735 cases of illness from produce (CSPI,
2009b).
Microbial reduction strategies for fresh fruits and vegetables have remained largely
ineffective because of current growing/harvest/processing practices and the nature of the
material. Contamination of leafy greens and other produce, as discussed by Doyle and Erickson
(2008), can occur in the field from irrigation water and animals as well as during processing
from contaminated flume water. Because many produce items are traditionally consumed raw,
to offer some degree of protection to the consumer, processing of fresh-cut produce typically
includes one or more washing steps using chemical sanitizers. However, these means are largely
ineffective, as evidenced from recent outbreaks, given that bacterial levels are typically reduced
only 1 - 2 logs on the product (Sapers, 2001).
U.S. Food and Drug Administration’s approval of irradiation began in 1963, with doses
of up to 0.5 kGy being allowed for controlling insect infestation of wheat and flour (Table 1).
This list has since expanded with 12 approved uses for irradiation, the most recent addition
coming in response to the 2006 outbreaks that were traced to Escherichia coli O157:H7 on

29

spinach and lettuce. In a report issued by the joint FAO/IAEA/WHO study group on the
nutritional and safety impact of food irradiation, foods irradiated at doses below 10 kGy were
deemed to be wholesome (WHO, 1997).

Table 1 Approved uses for food irradiation (USFDA, 2010)
Use
Control of insects in wheat and flour
Inhibiting spouting in potatoes
Pork carcasses for Trichinella spiralis
Culinary herbs, seeds, spices, vegetable seasonings
Delay ripening of fruit and disinfecting fruits and
vegetables of insects
Fresh or frozen, uncooked poultry products
Frozen, packaged meats used solely in NASA space
flight programs
Refrigerated or frozen, uncooked meat products

Year Dose
1963 ≤ 0.5 kGy
1964 ≤ 0.15 kGy
1986 ≤ 1 kGy
1986 ≤ 30 kGy
1986 ≤ 1.0 kGy

Fresh shell eggs for Salmonella
Seeds for sprouting
Fresh or frozen molluscan shellfish for Vibrio bacteria
Iceberg lettuce and spinach

2000
2000
2006
2008

1990
1995

≤ 3 kGy
Minimum dose 44 kGy

1997

≤ 4.5 kGy (refrigerated);
≤ 7.0 kGy (frozen)
≤ 3.0 kGy
≤ 8.0 kGy
≤ 5.5 kGy
≤ 4.0 kGy

2.4.6. X-ray as a viable alternative
Until very recently, food irradiation as a microbial reduction strategy focused almost
exclusively on gamma rays and E-beams, with gamma rays identified as the only energy efficient
means for cold pasteurization. This large body of literature on food irradiation has been
reviewed elsewhere with respect to meat and poultry (O’Bryan et al., 2008), fish and seafood
(Arvanitoyannis et al., 2009a; Venugopal et al., 1999), and fruits and vegetables (Arvanitoyannis
et al., 2009b). It is only within the past decade that X-ray irradiation has garnered some attention
as a viable microbial reduction strategy based on its now proven efficacy, minimal
30

environmental impact, and potential for direct installation in commercial processing lines. Given
the extensive shielding and other hazards associated with the radioactive sources for gamma
radiation (e.g.,

60

Co or

137

Cs), X-ray and E-beam are becoming more practical alternatives. In

addition, these nonradionuclide machine-source irradiators can be turned on and off by a switch,
allowing for more efficient commercial processing and greater operator control.

2.4.7. Advances in X-ray technology
Reinvention of X-ray machines with increased efficiency, combined with recent
developments in legislation and engineering, is now allowing X-ray to actively compete with
gamma irradiation and E-beam as a microbial reduction strategy for foods. In the generation of
bremsstrahlung, one of the unfortunate outcomes is the inadequate conversion of energy from
integrated photons to integrated electrons, which decreases process efficiency. This has been
viewed by some as the primary limitation to X-ray use for commercial applications, but is also
an area of debate. Initially, the approved maximum energy level permitted for X-rays was set at
5.0 MeV; however, at an October 16 - 18, 1995 meeting of the FAO/IAEC/WHO in Vienna,
Austria, it was concluded that X-ray machines producing up to 7.5 MeV “can be used without
any concern about induced radioactivity but would be a satisfactory, efficient and cost effective
addition to other radiation sources available for food processing” (ICGFI, 1995). In light of this
conclusion, FDA amended their food additive regulations in December 2004 by establishing a
new maximum permitted energy level for X-rays of 7.5 MeV, provided that the X-rays are
generated from machine sources that use tantalum or gold as the target material (Federal
Resister, 2004).

31

The effective increase in energy from 5.0 to 7.5 MeV is not substantial, yet it is still
important, as the emission efficiency increases proportionately with an increase in electron
energy, as indicated in Table 2. Using Monte Carlo simulation, Meissner et al. (2000) found that
bremsstrahlung yield - a measure of the emission efficiency - doubled when the energy level
increased from 5.0 to 10.0 MeV. They also estimated a greater degree of penetration when an
equivalent uniform dose was applied. Consequently, more powerful X-ray machines for food
irradiation are now being designed based on these new rules. One manufacturer (L3
Communications Titan Pulse Sciences Division, San Leandro, CA) is preparing to develop an
irradiator that incorporates both a 7.5 MeV electron linear accelerator capable of generating 100
kW of average power and a tantalum converter.

Table 2 Properties of X-ray at energy levels of 10.0, 7.5, and 5.0 MeV (Meissner et al., 2000)
Energy (MeV)

Emission efficiency
a
(%)

Double-sided
2 -1 b
treatment (g cm )

Dose uniformity
c
ratio (Dmax/Dmin)

10
7.5
5.0

16.2
13.3
8.23

43
38
34

1.54
1.54
1.54

a

Efficiency of the bremsstrahlung yield (X-ray generation) in the forward direction assuming

normal electron incidence on to tantalum converter (conversion efficiency)
b

Aerial density (g cm

2 -1

) of stacked polyethylene plates (density 0.96 g cm

80  40 cm height)
c

Max to min dose ratio for the stacked polyethylene plates

32

3 -1

, dimension 49 

In 2009, these L3 devices were used as an 18 kW E-beam linear accelerator and a 15 kW,
10 MeV X-ray linear accelerator, and then compared with a hydrostatic pressure treatment for
3

-1

inactivation of E. coli O157:H7 in ground beef inoculated at 10 CFU g (Schilling et al., 2009).
Using X-ray and E-beam doses of 2 kGy, the E. coli O157:H7 population decreased below the
limit of detection, whereas hydrostatic pressure (300 mPa) did not completely eliminate the
pathogen. Furthermore, sensory panelists found the irradiated and nonirradiated control samples
to be comparable and overall more acceptable than ground beef treated with hydrostatic pressure.
No potentially hazardous volatile compounds were detected in irradiated or hydrostatic pressuretreated samples.
Further technological advancements in X-ray technology have focused on improved
efficiency. Rad Source Technologies (Suwanee, GA) led these developments with their RS 2000
X-ray design. This machine uses a patented Rad Source RAD+ Chamber and a point source Xray tube (Rad Source Technologies, 2008) to deliver a uniform irradiation dose over a large area.
Additional energy is provided from a reflector of low Z (atomic number), high-density material
that is positioned within the chamber to reflect radiation back to the product (Gueorguiev, 2002).
The RS 2000 X-ray irradiator has been tested at several institutions across the United
States, with the bulk of this work having been done at Mississippi State University. Using this
irradiator at a maximum dose of 2.0 kGy (dose rate of 1 kGy per 50 min at 145 kV and 19 mA),
Robertson et al. (2006) were able to reduce initial Listeria monocytogenes populations of 4.4 log
-1

CFU g in vacuum-packaged smoked mullet to undetectable levels with no recovery seen when
the fillets were held for 90 or 17 days at 3C or 10C, respectively. Sensory panelists were also
unable to detect any differences between the treated and untreated samples (Robertson et al.,

33

2006). However, the 100 min exposure time to achieve these results was cited as being very
problematic for the industry.
In 2009, Collins et al. (2009) expanded on this research by evaluating the same system
and operating conditions in treating fresh channel catfish fillets for the reduction of L.
monocytogenes, mesophilic aerobic bacteria, psychrotrophic bacteria, and total coliforms. Their
findings were similar to those of Robertson et al. (2006), with 40%, 27%, 0%, and 7% of
samples yielding L. monocytogenes after receiving irradiation doses of 0, 0.5, 1.0, and 1.5 kGy,
respectively. They also reported the development of an off aroma in unirradiated control
samples during storage at 5°C over 17 days, which was not detected on the irradiated fillets, with
these changes attributed to fewer inherent bacteria and spoilage organisms on the treated fillets.
In other developments, Rad Source Technologies received a U.S. patent (number
7,346,147) for 4pi X-ray emitters (Kirk and Gorzen, 2008). The manufacturer claims that this
device delivers higher dose rates, comparable to gamma irradiators, through the use of an
extended anode design in which X-rays are generated from a cylindrical rather than a point
source, using all of the photons produced (Rad Source Technologies, 2008). Their design results
in both the creation of X-rays proceeding through the anode along its length and X-rays that are
reflected back through the length of the anode and throughout the circumference of the
cylindrical anode (Kirk and Gorzen, 2008).
This 4pi technology has been used by Rad Source (RS) Technology in the construction of
their RS 2400 and RS 2500 irradiators. Specifications for the RS 2400 X-ray irradiator in Table
3 illustrate how high dose rates can be achieved under low-energy conditions (< 1 MeV).
However, the operating capabilities of the cabinet remain a limitation to the design, allowing

34

only small batches of product to be treated within an exposure chamber measuring 91.4  60.0 
63.5 cm.

Table 3 Characteristics of the RS 2400 Irradiator (Mehta and Parker, 2011)
Characteristic
Maximum tube voltage
Maximum tube current
Maximum power
X-ray converter
Dose rate (to water) in center of rice-filled
canister
Dose energy ratio at this location

Value
150 kV
45 mA
6.75 kW
12 ìm gold
14.1 Gy min

1

0.0374 Gy (kW s)

1

Efficacy of this improved RS 2400 irradiator (dose rate of 1.0 kGy per 16 min at 145 kV
and 45 mA) has been demonstrated through a series of reports on the treatment of shellfish. In
2009, whole live and half-shell oysters were irradiated to inactivate Vibrio parahaemolyticus
(Mahmoud and Burrage, 2009) and later Vibrio vulnificus (Mahmoud, 2009a). In this work, V.
parahaemolyticus populations decreased more than 6 logs to levels below the limit of detection
using X-ray irradiation doses of 0.75, 2.0, and 5.0 kGy for pure-culture, half-shell, and wholeshell oysters, respectively. Under the same conditions, V. vulnificus was more susceptible, with
reductions of greater than 6 logs seen after exposing the pure-culture, half-shell, and whole-shell
oysters to doses of 0.75, 1.0, and 3.0 kGy, respectively. In addition, the shelf life of whole
oysters could be extended using a dose of 5.0 kGy, as evidenced by oyster survival at the high
dose, and inherent microorganisms were reduced to levels below the limit of detection.
Mahmoud (2009b) reported equally effective results using the same system on ready-to-eat
shrimp, demonstrating more than a 6 log CFU reduction for E. coli O157: H7, Salmonella

35

enterica, Shigella flexneri, and V. parahaemolyticus using doses of 2.0, 4.0, 3.0, and 3.0 kGy Xray, respectively. Lower X-ray doses of 0.75 kGy also significantly reduced the initial
-1

microflora in ready-to-eat shrimp samples from 3.8  0.2 to < 1.0 log CFU g .
Mahmoud later went on to demonstrate the efficacy of this same X-ray irradiator for a
series of produce items, including spinach (Mahmoud et al., 2010), iceberg lettuce (Mahmoud,
2010a), and Roma tomatoes (Mahmoud, 2010b). In these experiments, results indicated a
greater than 5 log reduction for E. coli O157:H7, L. monocytogenes, S. enterica, and S. flexneri
at 2.0, 1.0, and1.5 kGy for iceberg lettuce, spinach, and Roma tomatoes, respectively. In
addition, treatment at these dose levels significantly decreased inherent microflora, which in turn
enhanced product shelf life during refrigerated storage. Results obtained on leafy greens were
similar to those reported for both gamma (Niemmira et al., 2002; Niemira, 2008) and E-beam
(Gomes et al., 2008) radiation (Table 4).

Table 4 Microbial efficacy (D10 value) of X-ray irradiation on various products
Products

Microorganisms

Inoculation
method

Oysters
(whole live
and half
shell)

Vibrio
parahaemolyticus

Immersion

D10 value
(kGy)
2.0 log
reduction
kGy-1
(whole
a
shell)
4.9 log
reduction
-1
kGy (half

Reference

X-ray
irradiator

(Mahmnoud
and Burrage,
2009)

150 kV
-1
6.75 kW
(RS 2400,
Rad Source
Technologi
es Inc.,
Alphretta,
GA)

a

Soy broth
(TSB)
Skim milk
Low-fat
milk (1%)

Enterobacter
sakazakii

shell)
0.41  0.1

Mix

0.54  0.04
0.65  0.02
36

(Mahmoud,
2009b)

Table 4 (cont’d)
Low-fat
milk (2%)
Whole-fat
milk (3.5%)
Spinach
(leaves)

Iceberg
lettuce
(shredded)

0.71  0.3
0.74  0.03
Escherichia coli
O157:H7
Listeria
monocytogenes
Salmonella
enterica
Shigella flexneri
E. coli O157:H7

Spot

1.1
1.0
1.2

Spot

L. monocytogenes

S. enterica

S. flexneri

Roma
tomatoes
(whole)
Shrimp
(frozen,
cooked,
peeled ready
to eat)
Mullet
(vacuumpackaged
smoked)

(Mahmoud et
al., 2010)

E. coli O157:H7
L. monocytogenes
S. enterica
S. flexneri
E. coli O157:H7
S. enterica
S. flexneri

Spot

L. monocytogenes

Immersion

Immersion

37

0.96
4.4 log
reduction
-1 (a)
kGy
4.1 log
reduction
-1 (a)
kGy
4.8 log
reduction
-1 (a)
kGy
4.4 log
reduction
-1 (a)
kGy
0.39  0.5
0.66  0.1
0.56  0.1
0.98  0.3
1.1  0.01
1.3  0.03
1.2  0.01
1.6 log
reduction
-1 (a)
kGy

(Mahmoud,
2010a)

(Mahmoud,
2010b)

(Mahmoud,
2009a)

(Robertson et 145 kV
-1
al., 2006)
2.76 kW
(RS 2,000,
Rad Source
Tech, Boca
Raton, FL)

Table 4 (cont’d)
E. coli O157:H7

Immersion

0.040 
0.001

spot

Iceberg
lettuce
(leaves)

0.078 
0.008

a

(Jeong et al.,
2010b)

70 kV 4
-1
kW
(Rayfresh
Foods Inc.,
Ann Arbor,
MI)

D10 values were not reported.
The RS 2400 irradiator was also evaluated for inactivation of Enterobacter sakazakii in

milk (Mahmoud, 2009c). X-ray doses of 5.0 and 6.0 kGy reduced (P < 0.05) E. sakazakii
-1

populations to < 1 log CFU ml in skim milk and milk containing > 1% fat, respectively.
However, this study did not investigate the possible negative impact of X-ray doses on product
quality and rancidity, which is an important consideration in high-fat products.
Rayfresh Foods (Ann Arbor, MI) also developed a patent-pending process termed The
Rainbow Process, which is based on low-energy (< 1 MeV) X-rays (Rayfresh Foods, 2009).
This technology has been evaluated at Michigan State University over the past five years using a
wide range of products, including lettuce, spinach, parsley, asparagus, blueberries, almonds,
walnuts, chestnuts, and ground beef. Research to date on this system has been conducted using a
pilot-scale irradiator (maximum power 4 kW, operating energy 70 kV) comparable in design to
the RS 2400 irradiator. Rayfresh Foods currently is focused on development of a commercial
scale irradiator for in-line processing.
Experiments assessing the efficacy of this new technology against E. coli O157:H7 in
ground beef yielded a D10 value of 0.100 kGy (Jeong et al., 2007), which was significantly
lower than previously published D10 values for gamma or E-beam irradiation (~0.270 kGy)
38

(Thayer and Boyd, 1993). This system also has proven to be highly effective against E. coli
O157:H7 on leafy green vegetables. Treatment of iceberg lettuce yielded a D10 value of 0.040
kGy (Jeong et al., 2010b), which is 3.4 times lower than the previously reported value of 0.136
kGy using gamma radiation (Niemira et al., 2002) (Table 4). When ten stacked leaves were
irradiated from both sides, a dose of 0.2 kGy was achieved at the center of the stack, with a
surface dose of 1 kGy corresponding to a 5-log reduction for E. coli O157:H7 at the center of the
stack. Lower D10 values were also observed when spinach (0.035 kGy), parsley leaves (0.0522
kGy), and parsley stems (0.067 kGy) were surface inoculated with E. coli O157:H7 (Moosekian
et al., 2010a).
Efficacy against Salmonella on tree nuts (almonds and walnuts) has also been
demonstrated (Jeong et al., 2010a). Nuts were inoculated with Salmonella Enteritidis PT30 or
Salmonella Tennessee, and conditioned to different water activities (0.23 - 0.84). The efficacy
was significantly (P < 0.05) affected by both nut type and water activity, with greater
inactivation on the surface of almonds than on walnuts, for equivalent doses, and maximum
resistance at a water activity ~0.6. Also, irradiation of uninoculated samples to a dose that would
achieve a 5-log reduction (1.1 and 2.4 kGy for almonds and walnuts, respectively) resulted in no
perceivable sensory changes in almond quality, as determined by a consumer panel, but did
result in a decrease in acceptability for the walnuts, indicating the importance of product-specific
data in evaluating the technology.
The Palletron system is another proposed commercial design for implementing X-ray
technology in food-processing facilities. This irradiator increases dose uniformity through a
rotational pallet system comprising a radiation source, adjustable collimator, turntable, and
control system (Kotler and Borsa, 2003), with the goal of reducing the dose uniformity ratio

39

(DUR) or the ratio between the maximum and minimum dose absorbed by different areas of the
food or within a food container (Grandison, 2006). According to Lazurik et al. (2007),
application of X-ray beams from multiple angles and orientations increases both dose uniformity
and efficiency. When applied from four sides, beam efficiency was more than 60%, which
allowed large objects to be processed at a DUR < 1 (Lazurik et al., 2001). Monte Carlo
simulation studies with the Palletron further demonstrated that it is possible to reach a DUR >
1.5 for all densities up to 0.8 g cm

3 -1

while preserving a high treatment capacity (Stichelbaut et

al., 2004a).

2.4.8. X-ray irradiation process control and validation
Irradiation doses need to be uniformly delivered to the product to avoid an overdose
because the energy from X-ray (unlike E-beam) is attenuated exponentially along the projection
axis of the material. Therefore, processing models are needed to predict both X-ray dose
distribution in the food matrix and microbial inactivation efficacy. Because of the complexities
in generating ionizing radiation, transport, and interaction with matter, construction of a purely
analytical model is clearly challenging. Unlike mono-energetic gamma rays and E-beam, Xray’s multi-energetic energy spectrum results in angular dependence of the radiation, energy
dependence of X-ray absorption, and product particle scattering (Miller, 2003).
For food irradiation, the dose estimation methods and modeling techniques resemble the
radiation treatment plans for medical patients (Ay and Zaidi, 2005; Borsa et al., 2002; Reynaert
et al., 2007). Given recent computer advances, simulations of particle transport and interactions
are now being used to estimate dose distributions within the intended target. A typical stochastic
process suited for this type of simulation is the Monte Carlo method, in which the history of each
40

particle (trajectory, interactions) is traced in detail (Cantone and Hoeschen, 2011). A detailed
comparison among several Monte Carlo particle transport codes, including MCNPX, GEANT4,
FLUKA, MARS, and PHITS, is available (Los Alamos National Laboratory, 2011). Typically,
the composition of the food matrix is mapped using computer tomography images (CT scan),
after which a three-dimensional image of the object is reconstructed with embedded
density/composition information (Borsa et al., 2002). This material composition model is then
coupled with Monte Carlo particle transport codes to predict the radiation dose received at a
specific location (kGy), with this dose then converted into a process lethality based on the D10
value for the target organism.
Some readily available Monte Carlo codes include MCNPX (McKinney, 2011) (license
needed from the Radiation Safety Information Computation Center, Oak Ridge National
Laboratory, Oak Ridge, TN) and GEANT4 (Agostinelli et al., 2003) (free and downloadable
from http://geant4.web.cern.ch/geant4/index.shtml). The main difference is that GEANT4 can
handle electromagnetic field problems, which enables the modeling of accelerated electrons in an
electromagnetic field.
Using these Monte Carlo codes, Stichelbaut et al. (2004a; 2004b) successfully modeled
®

the Rhodotron TT300 (X-ray from 5, 7, and 10 MeV) and Palletron™ using the GEANT
Monte Carlo simulation toolkit. For complex foods, including chicken carcass/broccoli (Kim et
al., 2007; Kim et al., 2006), apple (Brescia et al., 2003; Kim et al., 2006), an apple surrogate
(Rivadeneira et al., 2007), and bagged spinach (Gomes et al., 2008), this modeling procedure
was successful using machine source irradiation. Even though Monte Carlo simulation provides
an accurate dose-distribution map of the product, this is not an ordinary task. Therefore, Miller
(2003) developed a faster and more convenient analytical/empirical model for X-ray irradiation
41

that can be validated using integrated TIGER series Monte Carlo codes. After model validation,
an economic analysis is then needed to assess industry feasibility. Based on available economic
models, all three available irradiation technologies, gamma, E-beam, and X-ray, have their own
benefits and hindrances that must be specifically addressed by each user (Kunstadt, 2001; Sadat,
2004).

2.4.9. Consumer acceptance
The efficacy and safety of food irradiation have been recognized for more than a century,
especially in recent decades, by the scientific community as well as numerous health
organizations and governmental agencies. Given this consensus, food irradiation is now
approved in more than 50 countries, 30 of which are irradiating multiple commodities (Mostafavi
et al., 2010). However, some hurdles to further expansion of food irradiation remain because of
negative consumer perception of irradiated foods, industry costs associated with adopting the
technology; and the labeling requirement (e.g., Radura symbol) by regulatory agencies.
Despite increased efficacy and continued research on the safety of irradiated foods, some
consumers remain skeptical (Lyndhurst, 2009). Therefore, the issues are how much negative
perception can be tolerated and can consumer perceptions of food irradiation be improved
through education? Consumer acceptance, perception, and attitude are influenced by many
factors including socio-demographic/economic status, risk-benefit perceptions, knowledge, trust
in the source of information, and labeling (Rollins et al., 2011). In the United States (Bruhn,
2001), Europe (Rollin et al., 2011), and other countries, including Brazil (Behrens et al., 2009;
Martins et al., 2008), Chile (Junqueira-Goncalves et al., 2011), South Korea (Byun et al., 2009),
Argentina (Curzio and Croci, 1998), Africa (Mostafavi et al., 2010), Turkey (Gunes and Tekin,

42

2006), Egypt (El-Fouly et al., 2002), consumer attitudes and negative perceptions (or fear) of
food irradiation improved after consumer education. Commodity-specific consumer acceptance
of various irradiated products, including ground beef (Lorenzen and Heymann, 2003; Spaulding
et al., 2006; Wheeler et al., 1999), fruits (Deliza et al., 2010; Martins et al., 2008), pork (Fox et
al., 2002; Wolfe et al., 2005), fish (Aworh et al., 2002), meat (Eustice and Bruhn, 2010), onions
(Curzio and Croci, 1998), apple cider (Yulianti et al., 2004), and turkey meat (Lee et al., 2003),
also has been assessed. Although most of this work focused on consumer acceptance, adoptive
behavior of irradiation by retailers was also assessed (Jaenicke et al., 2006). A recent trend has
been seen toward improved consumer acceptance of certain health-promoting food additives,
such as antioxidants, which can also reduce radiation byproducts and increase product value
(Over et al., 2010; Yan et al., 2006). One approach to improving consumer acceptance involved
the public display and advertisement of irradiated foods (Furuta, 2004). After consumer
acceptance improves to the level of other technologies, food irradiation is likely to become far
more widely adopted by the food industry.

2.5. Cross-protection
2.5.1. Hurdle approach for pathogen control
Combining irradiation with other treatments, including chemical preservatives and
growth inhibitors in a hurdle approach, has been proposed as an additional option for enhancing
product safety and quality. Thayer et al. (2006) found that irradiation and chlorination acted
synergistically in the inactivation of Salmonella, E. coli O157:H7, and L. monocytogenes on
fresh produce. In a separate report, Foley et al. (2004) determined that although water, chlorine

43

(200 ppm), and irradiation (1.05 kGy) significantly reduced levels of E. coli O157:H7 on
cilantro, combined use of irradiation with a wash treatment was superior to irradiation alone.

2.5.2. Development of cross-protection
Cross-protection can be described as an innate means of protection from destructive
conditions or treatments (Sykes, 1963). This infers that microorganisms have the ability to adapt
to adverse situations for their temporary survival. Cross-resistance has the potential to occur
when different antimicrobial agents attack the same target, initiate a common pathway to cell
death, or share a common route of access to their respective targets (Chapman, 2003).
Treatment with sublethal levels of stress has been found to spur cross-protection by
inducing proteins which protect the cells against high levels of the same stress and often against
additional, unrelated stressors (Browne and Dowds, 2001). When utilizing multiple barriers at
sublethal levels to achieve bacterial inactivation, there is the concern for bacterial adaptation to
the applied stressor and the possibility that a hurdle approach may be creating more harm than
good.
Cross-protection has been investigated across many distinct categories of stressors;
however, oxidizing sanitizers have been a primary focus of scrutiny in this area, considering their
inefficacy in processing and the subsequent demand for a combined approach to bacterial
inactivation. Research has demonstrated that sanitizer damage can infer protection against
further oxidative damage. Jenkins et al. (1988) found that starvation or prior adaptation of E.
coli (K-12) to heat, hydrogen peroxide, or ethanol was protective against further H2O2 oxidative
damage. Zook et al. (2001) later observed that E. coli O157:H7 sublethally injured by low levels
of peroxyacetic acid sanitizer induced peroxidative adaptation, but not thermal resistance.

44

The occurrence of cross-protection from oxidizing agents has primarily been seen when
using low levels of the primary stressor. This is often attributed to the theory that at a sublethal
dose the cellular components react with the oxidant, while at higher concentrations the ability for
these components to scavenge the oxidants is overwhelmed. In 1983, Demple and Halbrook
reported that prior treatment with peroxide doubled the survival rate for E. coli (AB1157) that
was further exposed to gamma irradiation, with the survival greatest for radiation doses <10 krad
(0.1 kGy) (Demple and Halbrook, 1983). Dukan and Touati (1996) found that pretreatment with
a low concentration of HOCl protected cells from H2O2; however, at higher concentrations this
was not seen (Dukan and Touati, 1996).
Cross-protection may also develop in nature from repeated exposure to environmental
stress and expression of inherent systems to survive. Superoxide dismutase (SOD) acts to reduce
the affects of oxidation and ROS (Keyer et al., 1995). Induction of microbial defense systems in
response to oxygen radicals from exposure to peroxides results in the synthesis of proteins such
as catalases, superoxide dismutase, and alkyl hydroperoxidases (Chapman, 2003). Exposure of
E. coli to hydrogen peroxide also resulted in a 10-fold increase in catalase levels and enhanced
ROS scavenging (Imlay and Linn, 1987).
Resistance can be achieved by mutation, acquisition of new genetic information by
horizontal gene transfer, expression of previously silent genes, growth in a biofilm, and other
poorly defined phenotypic alterations which can give rise to a transiently resistant phenotype
(Chapman, 2003). Lisle et al. (1998) found that E. coli O157:H7 chlorine resistance
progressively increased through the starvation period, demonstrating that the organism adapts to
starvation conditions by developing a chlorine resistant phenotype.

45

Some clades of E. coli O157:H7 have demonstrated greater resistance to chlorine
treatment than others. Wang et al. (2009) found that strains in clade 8, including the strain
implicated in the 2006 spinach outbreak, showed significantly (P < 0.05) higher resistance to
chlorine than strains from other clades of E. coli O157:H7.
Gene analysis has shown that the response of bacteria, in regards to gene activation, is
similar regardless of the stressor. Imlay and Linn (1988) found that exposing E. coli to H202
also resulted in the induction of functions under control of the OxyR regulon, which enhances
the scavenging of active oxygen species. Similarly, Dukan and Touati (1996) found resistance to
HOCl to be largely mediated by genes involved in resistance to hydrogen peroxide. In their
research, rpoS was the gene responsible for protection in stationary phase, while in exponential
phase, induction of the OxyR regulon protected against HOCl exposure (Dukan and Touati,
1996).
Stressors have been found to activate similar sets of genes in the bacterial defense
response. In investigating the changes in gene expression after treatment of E. coli cultures with
mitomycin C, over 1000 genes were induced during the damage response including those
involved in the SOS response as well as other stress response pathways, such as those of
oxidative stress and osmotic protection (Khil and Camerini-Otero, 2002). In a study of the
transcriptional changes in Pseudomonas aeruginosa following exposure to sodium hypochlorite,
peracetic acid and hydrogen peroxide, 40 common genes were upregulated and 23 common
genes were downregulated, suggesting similar response mechanisms to oxidative stress (Small et
al., 2007). More recently, Wang et al. (2009) demonstrated a similar response to chlorinated
water. Here it was found that over 380 genes were expressed in response to low levels of

46

chlorine or hydrogen peroxide, in particular several regulatory genes responsive to oxidative
stress such as the OxyR regulons and genes encoding putative oxidoreductases.

2.6. Summary
Contamination of leafy greens may occur at any stage throughout processing due to the
extensive use of poor irrigation practices and the uncontrollable interactions that the growing
plants will have with wildlife in the field. Increased demand for leafy greens, including
conveniently packaged fresh-cut leafy greens, has amplified these concerns by allowing the
spread of pathogens throughout entire lots of food during processing. Chemical sanitizers, the
current microbial control strategy, have their limitations for use in produce processing in that the
high organic load allow for the target organisms to receive lower than optimum contact time with
the sanitizer. The result of sanitizer treatment of leafy greens has been minimal inactivation and
sublethal injury of the target pathogens.
Ionizing irradiation, including X-ray, has received greater attention in the inactivation of
pathogens on produce following the approval by the FDA allowing the use of up to 4 kGy on
fresh and bagged iceberg lettuce and spinach. X-ray irradiation has proven to be a viable
pathogen reduction strategy for several food commodities including, iceberg lettuce, roma
tomatoes, oysters, shrimp, mullet, and milk; however, information is still lacking on the efficacy
of X-rays for pathogen reduction on baby spinach. Of concern however, is that X-ray irradiation
has a similar inactivation mechanism to that of chemical sanitizers, with the primary target being
cellular DNA. Considering the continual use of chemical sanitizers in produce processing, there
is a legitimate concern for cross-protection if and when X-ray technology is implemented into
the current processing conditions.

47

3. CHAPTER 2
INACTIVATION OF ESCHERICHIA COLI O157:H7 ON BABY SPINACH USING LOWENERGY X-RAY IRRADIATION

3.1. Abstract
Low-energy X-ray irradiation was assessed as a means of eliminating Escherichia coli
O157:H7 on baby spinach. Round-cut baby spinach leaves (2.54 cm diameter) were dipinoculated in a 3-strain cocktail of E. coli O157:H7, and then irradiated at four different dose
levels up to 0.176 kGy using a prototype low-energy (70 kV) X-ray irradiator. Radiochromic
film dosimeters were then used to map the X-ray dose rate distribution on the surface and at the
mid-plane region of 225-g bags of baby spinach. D10 values for round-cut leaf pieces were used
to estimate inactivation of E. coli O157:H7 in commercial-sized bags of baby spinach.
Inoculated bags were irradiated for a total duration of up to 480 s per treatment to simulate 120 s
of processing in a four tube commercial scale irradiator to determine actual reduction values
within the bag. The D10 value for E. coli O157:H7 on baby spinach was 0.033 ± 0.001 kGy.
Dose rate distribution in the bags of baby spinach ranged from 1.93 ± 0.75 Gy/s at the mid-plane
to 7.18 ± 3.85 Gy/s on the bag’s surface. The predicted minimum log reduction for E. coli
O157:H7 in bagged baby spinach using a simulated exposure time of 120 s was 4.10 log CFU,
while the maximum dose received by the surface was 1.32 kGy. The observed reduction value,
determined as an average throughout the bag, was 3.99 log CFU. Based on these findings, lowenergy X-ray irradiation appears to be a promising means to inactivate E. coli O157:H7 on baby
spinach, with a 4 – log reduction achievable without exceeding the maximum allowable dose of
4 kGy at the surface of the bag.

48

3.2. Introduction
Increased interest in convenience, healthy eating, and unprocessed, raw fruits and
vegetables has greatly escalated the market for fresh-cut, pre-washed bagged salad greens in the
United States (Mintel, 2008). However, meeting this demand through expansion in farming and
mechanization of production practices has not come without costs. The industry has been
plagued with product recalls and occasional outbreaks. From 1998 to 2007, the Center for
Science in the Public Interest (CSPI) reported that three pathogens - Salmonella, E. coli
O157:H7, and Norovirus - combined for a total of 265 outbreaks and 9,001 illnesses from
consumption of lettuce and leafy green-based salads (CSPI, 2009b). Baby spinach moved to the
forefront of consumer concerns following an E. coli O157:H7 outbreak in September 2006 that
resulted in 205 confirmed cases in 28 states and 3 deaths (CDC, 2006).
Considering the numerous and still undefined contamination routes for baby spinach
(CFERT, 2007), pathogen control has been targeted at value-added processing. Chemical
sanitizers, including chlorine- and peracetic acid-based sanitizers, used in commercial flume
tanks are the only current means for decreasing microbial contamination. However, these
methods have proven to be marginally effective, generally reducing pathogens < 2 logs (Sapers,
2001; Beuchat et al., 2004; Keskinen et al., 2009; Lopez-Galvez et al., 2009).
In response, the FDA has allowed the use of ionizing irradiation at levels up to 4 kGy for
bagged spinach (FDA, 2008). Ionizing irradiation is commonly produced using either
radioactive sources, such as

60

Co or

137

Cs to generate gamma rays, or through machine sources

in the form of electron beams (E-beam) and X-rays, the latter of which has been gaining
attention given the hazards and negative public perception associated with radioactive sources.
E-beam has proven effective for spinach. Gomes et al. (2008) established a D10 value of 0.186
49

kGy for E. coli O157:H7 on spinach using E-beam (10 MeV) as a form of ionizing irradiation
(Gomes et al., 2008). A dose of 0.70 kGy was also shown to decrease E. coli O157:H7 in
bagged spinach by 4 logs (Neal et al., 2008).
Using a newly developed low-energy X-ray irradiator (Rayfresh Technologies, Ann
Arbor, MI), we obtained a D10 value of 0.040 kGy for E. coli O157:H7 on the surface of iceberg
lettuce (Jeong et al., 2012) which is 3.4 times lower than the previously reported value of 0.136
kGy using gamma radiation (Niemira et al., 2002). Low-energy X-ray technology can be
incorporated into existing processing lines due to the low shielding requirement and small
machine footprint. X-rays can also be used to treat packaged products, thereby minimizing the
risk of post-treatment contamination. Consequently, the objectives of this study were to (1)
establish the D10 value for E. coli O157:H7 on baby spinach using low-energy X-ray irradiation,
and (2) determine the time required to inactivate E. coli O157:H7 in commercial bags of baby
spinach using simulated processing conditions.

3.3. Materials and Methods
3.3.1. Bacterial strains
Three E. coli O157:H7 outbreak strains - K3995 from a 2006 spinach outbreak, K4830
from a 2006 bagged lettuce outbreak, and K4492 from a different 2006 bagged lettuce outbreak
were obtained from Dr. Michael Doyle at the Center for Food Safety, University of Georgia,
Griffin, GA and stored at -80°C in trypticase soy broth containing 0.6% (w/v) yeast extract
(TSBYE) (Becton Dickinson, Sparks, MD) and 10% (v/v) glycerol. Each strain was subjected to
two consecutive 37ºC/24 h transfers – the first in 10 ml of TSBYE, and the second in 15 ml of
9

TSBYE, and then combined to obtain 45 ml of a 3-strain cocktail containing ~10 CFU/ ml as
50

determined by optical density at 600 nm using a Genesys 20 ThermoSpectronic
spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA) and plating in duplicate on
trypticase soy agar containing 0.6% (w/v) yeast extract (TSAYE) (Becton Dickinson, Sparks,
MD).

3.3.2. X-ray irradiator
A pilot-scale, custom designed, low-energy X-ray food irradiator (Rayfresh Foods Inc.,
Ann Arbor, MI) housed in the biosafety level 2 pilot plant at Michigan State University was used
to irradiate the samples. This irradiator, which contained a 53 × 53 × 58 cm treatment chamber
shielded by 25 mm-thick lead, generated 70 kV with a 4 kW maximum capacity. X-rays were
filtered using a thin beryllium window (30 mm in diameter and 0.127 mm in thickness). The
typical dose rate from this irradiator was 17 Gy/s in air at 10 cm from the source, as calibrated
using an ion chamber dosimeter.

3.3.3. D10 value determination for E. coli O157:H7 on baby spinach
Baby spinach was purchased from a local supermarket and then stored at 4°C for < 24 h
before use. Baby spinach leaves were round-cut (2.54 cm diameter) in a biosafety hood using a
sterilized cork borer to allow for uniform X-ray dose distribution across the leaf, and acting to
simulate actual tissue damage incurred during leafy green processing. The 3-strain E. coli
O157:H7 cocktail was diluted 1:1 in sterile, pre-chilled (4oC) 0.1% phosphate buffer solution
(PBS), after which triplicate sets of 20 round-cut spinach samples were submerged in the
inoculum for 3 min, and then partially spin-dried for 1 min in a biosafety hood using a salad
spinner (Oxo Intl., New York, NY). Two additional samples served as uninoculated controls.

51

®

Each sample was aseptically inserted into a 4 oz sterile Whirl-Pak bag (NASCO, Fort Atkinson,
WI) using sterile tongs and a sterile spatula. Trapped air in the bag was purged by hand to
prevent the sample from moving inside the bag during subsequent handling. All bagged samples
were then held 24 h at 4°C until irradiated.
Round-cut baby spinach samples were irradiated at room temperature (~20°C) on one
®

side inside the Whirl-pak bag, after which the bag was flipped to achieve X-ray doses of 0,
0.050, 0.101, 0.126, 0.151, or 0.176 kGy per side (maximum treatment time = 7 s per side).
Irradiation dose was measured using radiochromic film dosimeters (GAF3001DS; maximum
dose of 3 kGy; GEX Corporation, Centennial, CO) which were read 24 h after exposure at 500 or
®

550 nm using a Spectronic Genesys 20 spectrophotometer (Thermo Electron Corporation,
Madison, WI).
For quantification of E. coli O157:H7, 15 ml of PBS was added to each bagged sample.
After homogenizing in a stomacher for 3 min, the samples were serially diluted in PBS or
filtered using 0.45 µm membranes and then plated in duplicate on Sorbitol MacConkey Agar
containing 0.005% (w/v) cefixime and 0.25% (w/v) tellurite (CT-SMAC, Becton Dickinson).
Preliminary tests showed no significant difference in recovery for treated samples plated on CTSMAC and CT-SMAC overlayed with TSAYE, indicating negligible sublethal injury as a result
of X-ray exposure. All E. coli O157:H7 colonies were counted after 18 – 24 h of incubation at 37
2

± 2ºC, with these counts converted to log CFU/cm . Log reductions were then calculated by
subtracting the log survivor counts from those of the unirradiated controls. Negative control
samples were also spread-plated onto CT-SMAC, with any suspect colonies further evaluated
®

using Reveal test strips for E. coli O157:H7 (Neogen, Lansing, MI).
52

3.3.4. Determination of dose rate and dose in bags of baby spinach
Dose rate (Gy/s) at the mid-plane region of the bag was determined by inserting a
2

cardboard section (236 cm ) - mounted with nine radiochromic film dosimeters positioned 7.6
cm apart (figure 1) - into the center of an uninoculated bag. The bag was then irradiated for 60 s
upon on a miniature conveyor belt system within the X-ray irradiation chamber, programmed to
oscillate at 8 – 10 cm/s. To evaluate the dose rate (Gy/s) at the surface of the bag the cardboard
section was irradiated directly for 30 s as it oscillated upon the conveyor belt within the X-ray
chamber. Dosimeters were read 24 h after exposure as previously described. Dose distribution
mapping was performed using the values generated by the dosimeters at each position to
illustrate the variations throughout the bag.

X

X

0 X

X

X

7.6 X
7.6

Position (cm)

7.6 X

X
0
Position (cm)

X
7.6

Figure 1. Diagram illustrating the positioning of radiochromic film dosimeters on a cardboard
segment.

53

3.3.5. Reduction of E. coli O157:H7 in bags of baby spinach
Bags of baby spinach (225 g) were purchased locally on the day of inoculation. Spinach
bags were inoculated with 2.25 ml of inoculum by inserting a sterile syringe into one end of the
7

bag to achieve a final population of 10 CFU/g. After covering the syringe holes with tape, the
bags were gently agitated by hand for 1 min to evenly distribute the inoculum. A prior trial
®

using Glo-germ was done to demonstrate the efficacy of this syringe inoculation method. A
®

visual inspection using a black light showed even distribution of the Glo-germ across the entire
bag using these procedures. Following agitation, the bags were held for 24 h at 4°C.
Commercial bags of baby spinach were irradiated using a miniature conveyor belt system
within the X-ray irradiation chamber, programmed to oscillate at 8 – 10 cm/s. Triplicate sets of
bags were irradiated twice on each side of the bag (4 exposures) for a total duration of 0, 120,
240, 360, and 480 s per treatment to simulate 0, 30, 60, 90, and 120 s of processing in a four tube
commercial scale irradiator.
After irradiation, the bags were again agitated for 1 min to account for the uneven dose
distribution across the bag. Subsequently, two 25 g samples were aseptically taken from each
®

bag using sterile tongs and placed into 10 oz Whirl-pak bags. E. coli O157:H7 populations
were determined by homogenizing samples in 100 ml of sterile PBS in a stomacher for 3 min,
and then plating appropriate serial dilutions on CT-SMAC. Survivors were counted after 24 h of
incubation at 37°C.

54

3.3.6. Statistical analysis
The X-ray radiation D10 value for the 3-strain E. coli O157:H7 cocktail on round-cut
baby spinach was calculated as the absolute value of the reciprocal slope for the linear regression
of log reduction vs. dose. The maximum dose (kGy) received by the bag was determined as the
maximum average dose rate seen at the surface of the bag throughout the maximum exposure
time using the equation:
Max. Dose = (Max. dose rate surface) x t
Where: t = time
Log reduction values for E. coli O157:H7 in bags of baby spinach were predicted as the
dose rate exposure over time at the mid-plane region and at the surface of the bag using the
equations:
RMin = (Dose Rate mid-plane x t)/Dv
RMax = (Dose Rate surface x t)/Dv
Where: RMin = Predicted minimum reduction value
RMax = Predicted maximum reduction value
t = time
Dv = Calculated D10 value for E. coli O157:H7 on baby spinach

55

3.4. Results and Discussion
3.4.1. Uninoculated controls
None of the uninoculated baby spinach leaves had quantifiable numbers of E. coli
O157:H7 as determined by spread-plating on CT-SMAC and subsequent testing of suspect
®

colonies using Reveal test strips specific for E. coli O157:H7.

3.4.2. D10 value for E. coli O157:H7 on baby spinach
Unirradiated, dip-inoculated baby spinach contained an E. coli O157:H7 population of
2

2

~6.00 log CFU/cm , with a maximum reduction of ~4.91 log CFU/cm seen after irradiation.
The D10 value for baby spinach was 0.033 ± 0.001 kGy (Figure 2), which is comparable to our
previously reported value of 0.040 ± 0.001 kGy for iceberg lettuce (Jeong et al., 2010b), 5.3
times lower than the previously reported value of 0.186 kGy using E-beam irradiation (Gomes et
al., 2008), and 33 times lower than the value of 1.1 kGy reported by Mahmoud using X-ray
irradiation (Mahmoud et al., 2010).

56

1.0
Dose (kGy)
0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Log 10 (N/N0)

0.0
0.00
-1.0
-2.0
-3.0
-4.0
-5.0
-6.0

Figure 2. Reduction of E. coli O157:H7 on baby spinach using X-ray irradiation (95%
confidence limit).

The observed differences in D10 values between inoculated and irradiated leafy green
varieties is consistent with the observations of Niemira et al. (2002), who found that E. coli
O157:H7 was significantly more sensitive to gamma radiation on red leaf (D10 value = 0.119 ±
0.004) and green leaf lettuce (D10 value = 0.123 ± 0.003) than on iceberg (D10 value = 0.136 ±
0.004) or Boston lettuce (D10 value = 0.140 ± 0.003). Mahmoud also observed similar
differences in reduction of E. coli O157:H7 on lettuce as compared to spinach using X-ray
irradiation (Mahmoud, 2010a; Mahmoud et al., 2010). The reason for the sensitivity differences
between leaf varieties was not explored in these experiments. These slight variations in D10
values for different leafy greens will likely impact the exposure times needed for commercial
processing of bagged salad greens with a standardized process needed to ensure equivalency
between leaf varieties.
57

3.4.3. Determination of dose rate and dose
Average dose rates across the mid-plane region and surface of the bags containing baby
spinach were 1.93 ± 0.75 and 7.18 ± 3.85 Gy/s, respectively (Figures 3 and 4).

7.6

Dose Rate
[Gy/s]
3.0-4.0
0

2.0-3.0
1.0-2.0
0.0-1.0

-7.6

-7.6
7.6

0
Position [cm]

Figure 3. Dose rate distribution at the mid-plane region of a 225 g bag of baby spinach.

58

7.6

Dose Rate
[Gy/s]
15.0-20.0
0

10.0-15.0
5.0-10.0
0.0-5.0

-7.6

-7.6
7.6

0
Position [cm]

Figure 4. Dose rate distribution at the surface of a 225 g bag of baby spinach.

Differences in D10 values seen between X-ray technologies may be associated with
differences in dose rate. Rad Source technology’s RS 2400 generates X-rays at a rate of 0.235
Gy/s (Mehta and Parker, 2011), which is 5 times lower than the minimum dose rate of 1.18 Gy/s
established within a bag of baby spinach using the Rayfresh technology. Longer exposure times
at lower dose rate may allow time for the target organism to repair, with a higher dose needed to
achieve inactivation.
3

E-beam dose rates are generally much higher than those for X-rays, at a rate of 10 – 10
Gy/sec, while a typical

60

Co irradiator generates 1 – 100 Gy/min (Moreira, 2006). After

conversion to X-rays the dose rate of electrons generated by E-beams is significantly reduced,
while the penetrating power of the resulting photon exceeds that of the electrons alone.
59

6

Differences in efficacy between the sources of ionizing irradiation may therefore be explained by
the beam characteristics and energy levels of the technology, specifically the relationship
between linear energy transfer (LET) and relative biological effectiveness (RBE) of the targeted
cell. Barendsen (1968) quantified this relationship and reported that the RBE needed to kill
mammalian cells increases with LET up to about 100 keV per µm and then decreases.
Consequently, low LET sources produce a more mutations compared to high LET sources
(Stapleton et al., 1952; Mortimer et al., 1965).
Varying levels of LET also contribute to the bystander effect (BE) on neighboring cells.
BE accounts for indirect actions of ionizing irradiation on intracellular communication,
providing another route for cell death (Prise and O’Sullivan, 2009). The cellular response to
bystander signals is considered to be non-linear and may involve greater involvement from the
development of reactive oxygen species (Kundrat and Friedland, 2012).
This concept of further reactivity indirectly supports the results for lower dose reduction
values achieved using X-ray irradiation in comparison to other sources. However, direct
comparisons between sources of ionizing irradiation using similar methods need to be performed

3.4.4. Reduction of E. coli O157:H7 in commercial bags of baby spinach
The predicted log reduction for E. coli O157:H7 in a bag of baby spinach ranged from
4.10 to 38.5 log CFU using a simulated exposure time of 120 s. However, the observed log
reduction at 120 s, calculated as the average log reduction in a bag, was 3.99 log CFU (Table 5).

60

Table 5. Predicted and observed average reductions for E. coli O157:H7 in bagged baby spinach

Simulated Time (s)
0
30
60
90
120

Max. Dose
Received at
Surface (kGy)
0.00
0.331
0.662
0.993
1.32

Min.
Predicted
Mid-plane
0.00
-1.03
-2.05
-3.08
-4.10

Log10(N/N0)
Max.
Predicted
Surface
0.00
-9.63
-19.30
-28.90
-38.50

Tested Avg.
0.00
-1.63
-2.82
-3.81
-3.99

The predicted mid-plane dose rate appears to provide a better indication of actual E. coli
O157:H7 reduction in the bag of spinach, with the observed average reductions more closely
resembling the predicted minimum value at the mid-plane region of the bag. The observed E.
coli O157:H7 reductions were expected to lie between the predicted values, considering that
higher dose levels at the bag surface would balance the overall average. Some limitations of the
predicted values should be considered in this regard. Dosimeters, while able to measure the dose
received, do not fully account for the dose received by the target organism. Dosimetry also does
not factor the recovery of injured cells following X-ray treatment. The predicted reductions also
assumed that E. coli O157:H7 inactivation follows first-order kinetics.
These results clearly demonstrate that low-energy X-ray can be an effective strategy for
inactivating E. coli O157:H7 in commercial bags of baby spinach, achieving approximately a 4
log reduction within the bag. The maximum dose of 1.32 kGy received by the bag under these
conditions was also significantly lower than the limit of 4.0 kGy set by the FDA. In addition,
there were no obvious visual changes in the product quality at these dose levels; however,
sensory evaluation will need to be performed under these conditions to fully evaluate product
acceptability.

61

These small-scale experiments cannot express the full potential of this technology, since
many modifications were made to simulate actual processing conditions. Full scale X-ray
irradiators will likely contain multiple X-ray tubes, expanding the exposure range of the
instrument and shortening exposure duration.

62

4. CHAPTER 3
INACTIVATION OF SANITIZER-INJURED ESCHERICHIA COLI O157:H7 ON BABY
SPINACH USING X-RAY IRRADIATION

4.1. Abstract
This study assessed the potential of sanitizer-induced cell injury to enhance resistance of
E. coli O157:H7 to X-ray irradiation on baby spinach. A 3-strain E. coli O157:H7 cocktail was
exposed to two different sanitizers used in flume washing systems - 5.2 ppm peroxyacetic acid®

®

based (PABS, Tsunami 100) and 22 ppm chlorine-based (CBS, XY-12 ) sanitizer, and 18 ppm
quaternary ammonium compound-based sanitizer used in equipment sanitation (QACBS,
TM

Whisper

) - to obtain 90 to 99% injury. Pre-irradiated, round-cut (2.54 cm diameter) baby
®

spinach leaves were immersed for 5 min in the injured cocktail and irradiated in Whirl-pak

bags at doses of up to 0.063 kGy using a low-energy X-ray irradiator (Rayfresh Foods, Ann
Arbor, MI). Healthy and injured survivors were respectively quantified by plating appropriate
dilutions on SMAC overlayed with TSAYE and SMAC. E. coli O157:H7 injury on inoculated
spinach decreased from 90-99 to 66, 63, and 1% for PABS-, CBS-, and QACBS-treated cells,
respectively. D10 values for PABS-, QACBS-, and CBS-injured E. coli O157:H7 on baby
spinach were 0.0136 ± 0.000, 0.0223 ± 0.001, and 0.0242 ± 0.001 kGy, respectively. Prior
exposure to PABS significantly (P < 0.05) enhanced E. coli O157:H7 susceptibility to X-ray
irradiation, while exposure to CBS significantly (P < 0.05) reduced susceptibility. Results
suggest that PABS may be preferred for irradiated baby spinach.

63

4.2. Introduction
Consumption of baby spinach and other leafy greens continues to be a topical issue
among nutritionists and food safety experts. However, concerns have been raised regarding the
increasing number of leafy green-associated outbreaks due to Escherichia coli O157:H7. During
1998 to 2007, E. coli O157:H7 was implicated in 13 mixed green salad and12 lettuce outbreaks,
resulting in a total of 652 total illnesses (CSPI, 2009b). Leafy green concerns climaxed in 2006
with three prominent E. coli O157:H7 outbreaks: the first in September associated with baby
spinach resulting in 205 confirmed cases in 11 states, with 31 cases of hemolytic uremic
syndrome (HUS) and 3 deaths (CFERT, 2007), followed by two in December traced to shredded
lettuce that included a total of 152 confirmed cases in 5 northeastern states, and 10 cases of HUS
(U.S. FDA, 2007a; U.S. FDA, 2007b). These outbreaks confirm that currently used chemical
sanitizers are not sufficiently effective, with bacterial populations decreasing only 1 to 2 logs on
leafy greens during commercial processing (Sapers, 2001; Beuchat et al., 2004; Keskinen et al.,
2009; Lopez-Galvez et al., 2009). Consequently, alternative microbial reduction strategies
including irradiation need to be explored.
In 2008, the US Food and Drug Administration issued a rule allowing the use of
irradiation at doses up to 4 kGy for loose and bagged iceberg lettuce and spinach (FDA, 2008).
Previous work focused on gamma rays and E-beams as irradiation sources, with X-ray
technology, until very recently, considered being unfeasible economically. Using gamma
radiation, E. coli O157:H7 internalized in spinach and on the surface of lettuce yielded D10
values of 0.27 and 0.14 kGy, respectively (Niemira, 2002; Niemira, 2007). Similar E. coli
O157:H7 inactivation values have been observed using E-beam, with a D10 value of 0.20 kGy
for baby spinach (Neal, 2008).

64

Given several recent technological advances, X-ray irradiation has reemerged as a viable
non-thermal pathogen reduction strategy for a wide range of foods, including ground beef (Jeong
et al., 2007; Schilling, 2009), almonds (Jeong et al., 2008; Jeong and Marks, 2010), milk
(Mahmoud, 2009b) various types of seafood (Mahmoud, 2009a; Robertson et al., 2006;
Mahmoud and Burrage, 2009; Mahmoud, 2009c) and fresh produce (Mahmoud, 2010a;
Mahmoud, 2010b; Jeong et al., 2010; Mahmoud et al., 2010). X-rays inactivate microorganims
primarily through single- or double-strand DNA breaks (Pollard, 1966), which can occur both
directly through high energy photons and indirectly through the excitation and ionization of
electrons within a food (Podgoršak 2006). In our most recent work (Jeong et al., 2010), E. coli
O157:H7 populations decreased 5 logs when fresh-cut iceberg lettuce was exposed to an X-ray
irradiation dose of 0.20 kGy. Further studies demonstrated that X-ray doses of 0.18 and 0.26
kGy decreased E. coli O157:H7 populations 5 logs on baby spinach and flat-leaf parsley,
respectively (Moosekian et al., 2010). Regardless as to whether or not X-ray irradiation can
eventually be commercialized as a final kill step in leafy green processing, prior washing of the
product in sanitizer-treated flume water will still remain an integral step in the process.
Several previous reports have raised concerns regarding enhanced protection of E. coli
following sublethal exposure to chemical sanitizers. Zook et al. (2001) reported that E. coli
O157:H7 cultures treated with peroxyacetic acid exhibited substantially increased tolerance to
further peroxidative stress, while Dukan and Touati (1996) found that pre-treating E. coli
O157:H7 with hydrochloric acid conferred resistance to hydrogen peroxide. Similarly, prior
exposure to an oxidizing agent may also increase resistance to ionizing irradiation. Demple and
Halbrook (1983) reported that prior treatment with peroxide doubled the survival rate of E. coli

65

K-12 that was further exposed to gamma irradiation, with enhanced survival greatest at radiation
doses <0.1 kGy.
Inactivation of bacterial cells by chlorine and peroxyacetic acid is well documented but
still poorly understood with their oxidative ability directed towards the cell membrane (Baldry
and Fraser, 1988; Venkobachar et al., 1977), functional processes of the cell (Barrette et al.,
1989; Benarde et al., 1967; Bloomfield 1996; Winter et al., 2008), and potentially DNA.
Quaternary ammonium compounds (QACs) also target the bacterial cell membrane (Hotchkiss,
1946; Ioannou et al., 2007; Przestalski et al., 2000).
Given similarities in the inactivation mechanisms for many chemical sanitizers and
ionizing irradiation, the potential for enhanced microbial resistance from sublethal sanitizer
stress should be addressed before implementing low-energy X-ray technology as a microbial
reduction strategy in the processing of leafy greens. Hence, in this study three commonly used
commercial sanitizers - one chlorine- and one peroxyacetic acid-based sanitizer commonly used
in flume washing systems and one quaternary ammonium-based sanitizer used in postproduction clean-up - were investigated for their potential to enhance the resistance of E. coli
O157:H7 on baby spinach to X-ray irradiation.

4.3. Materials and Methods
4.3.1. Bacterial strains
Three E. coli O157:H7 strains - K3995 (2006 spinach outbreak), K4830 (2006 lettuce
outbreak A), and K4492 (2006 lettuce outbreak B) were obtained from Dr. Michael Doyle at the
Center for Food Safety, University of Georgia, Griffin and maintained at -80°C in trypticase soy
broth containing 0.6% (w/v) yeast extract (TSBYE; Becton Dickinson, Sparks, MD) and 10%

66

(v/v) glycerol (Mallinckrodt Baker, Phillipsburg, NJ). Each strain was transferred from the
frozen stock cultures and grown in TSBYE for 24 h at 37°C. Following a second transfer in 200
ml of TSBYE, the strains were pelleted by centrifugation for 15 min at 2200 x g, resuspended in
phosphate buffer solution (PBS), combined in equal volumes and adjusted to 1 L with PBS to
obtain a 3-strain cocktail containing ~ 9.7 log CFU/ml E. coli O157:H7 as determined by plating
on trypticase soy agar containing 0.6% (w/v) yeast extract (TSAYE; Becton Dickinson).

4.3.2. Peroxyacetic acid-based sanitizer (PABS) injury
Five 250 ml Erlenmeyer flasks, each containing 200 ml of the E. coli O157:H7 cocktail,
were agitated at 200 rpm on a Gyrotory Shaker (Model G2; New Brunswick Scientific Co.,
®

Edison, NJ) during exposure to 5.2 ppm of PABS (Tsunami 100, Ecolab, St. Paul, MN). After
2 min, the reaction was stopped by adding 1 ml of 38.5x neutralizing buffer (Difco

TM

neutralizing buffer, Becton Dickinson) to obtain ~90% injury. The injured cocktail was then
pelleted by centrifugation at 2200 x g for 15 min and resuspended in PBS to obtain a population
of ~8.0 log CFU/ml.

4.3.3. Quaternary ammonium compound-based sanitizer (QACBS) injury
Four 2.8 l Fernbach flasks, each containing 250 ml of the E. coli O157:H7 cocktail, were
agitated at 200 rpm on a Gyrotory Shaker during exposure to 18 ppm QACBS (Whisper

TM

,

Ecolab). After 2 min, the reaction was stopped by adding 1 ml of 38.5x neutralizing buffer to
obtain ~86% injury. The injured cocktail was then pelleted by centrifugation and resuspended in
PBS to obtain a population of ~8.4 log CFU/ml.

67

4.3.4. Chlorine-based sanitizer (CBS) injury
Four 2.8 l Fernbach flasks, each containing 250 ml of the E. coli O157:H7 cocktail, were
®

agitated at 200 rpm on a Gyrotory Shaker during exposure to 22 ppm CBS (XY-12 , Ecolab).
After 4 min, the inoculum was transferred to a sterile 400 ml beaker containing 10 ml of 77x
neutralizing buffer to halt the reaction and obtain ~99% injury. The injured cocktail was then
pelleted by centrifugation and resuspended in PBS to obtain a population of ~8.5 log CFU/ml.

4.3.5. Quantification of injury
Initial cell injury was determined by plating each E. coli O157:H7 suspension on
TSAYE and Sorbitol MacConkey Agar (SMAC; Becton Dickinson). After 48 h of incubation at
37°C, percent injury was determined following the equation:

% Injury = [(NTSAYE – NSMAC) / NTSAYE] x 100
Where: NTSAYE = CFU/ml recorded from TSAYE plates
NSMAC = CFU/ml recorded from SMAC plates

4.3.6. X-ray irradiator
A pilot-scale, custom designed, low-energy X-ray food irradiator (Rayfresh Foods Inc.,
Ann Arbor, MI) housed in the biosafety level 2 pilot plant at Michigan State University was used
to irradiate the samples. This irradiator, which contained a 53 × 53 × 58 cm treatment chamber
shielded by 25 mm-thick lead, generated 70 kV with a 4 kW maximum capacity. X-rays were
filtered using a thin beryllium window (30 mm diameter, 0.127 mm thick). The typical dose rate
68

from this irradiator was 17 Gy/s in air at 10 cm from the source, as calibrated using an ion
chamber dosimeter.

4.3.7. Baby spinach preparation
Leaves of baby spinach, originating from California and purchased in 5 oz clamshell
containers from a local supermarket on the day of use, were round-cut (2.54 cm diameter) using
®

a sterile cork borer and placed in a Whirl-pak bag. Prior to inoculation, all leaves were
irradiated for 10 s on each side for a combined dose of 0.167 kGy to reduce background
microflora confirmed using radiochromic film dosimeters (GAF3001DS, GEX Corporation,
Centennial, CO). The spinach samples were aseptically removed from the bag, immersed in the
injured E. coli O157:H7 cocktail for 5 min, spin-dried in a salad spinner (Oxo Intl., New York)
®

for 1 min in a biosafety cabinet, placed in a clean Whirl-pak bag and irradiated within 30 min
of inoculation.

4.3.8. X-ray irradiation
Fifteen round-cut leaves (three replicates at each dose) were irradiated on each side for
combined doses of 0.012, 0.028, 0.043, 0.054 and 0.063 kGy. The irradiation dose was again
confirmed using radiochromic film dosimeters.

4.3.9. Enumeration
Following irradiation, spinach samples were diluted in 20 ml of PBS and homogenized in
a Seward Stomacher 400 circulator for 3 min at 260 rpm. E. coli O157:H7 survivors were
enumerated by plating appropriate dilutions on SMAC and SMAC overlayed with approximately
69

10 ml TSA-YE less than one hour prior to plating for recovery of healthy and healthy plus
sublethally injured cells, respectively. Survivors were counted after 48 h of incubation at 37°C.

4.3.10. Statistical analysis
All sanitizer experiments were performed in triplicate. A Wilcoxon signed-rank test was
used as a non-parametric alternative to the Student’s t-test to analyze the injury results (α =
0.05), since there was an abnormal distribution of the matched population. D10 values were
determined from the absolute value of the inverse slope of the linear regression for the log
reduction values, using Microsoft Office Excel 2007. D10 values between treatments were then
®

compared by Analysis of CoVariance (α = 0.05), using Minitab 14 statistical software.

4.4. Results and Discussion
Combined use of multiple microbial reduction strategies, including X-ray irradiation as
one such strategy, during leafy green processing will likely remain the most effective approach
for maintaining both end product quality and safety. However, some concern is warranted
regarding the efficacy of ionizing irradiation for leafy greens given the sublethal concentrations
of sanitizers currently used in the industry. This study aimed to address this concern, with the
particular focus on evaluating the efficacy of X-ray irradiation against cells that have been
sublethally injured by chemical sanitizers and have become attached to baby spinach.
Prior treatment of E. coli O157:H7 cell suspensions reduced the initial levels present on
the leaf following inoculation from 8.27 cfu/g to approximately 4.84, 5.09, and 5.79 cfu/g, for
PABS-, CBS-, and QACBS-treated inoculum, respectively. All cell populations exposed to Xray irradiation decreased linearly regardless of prior sanitizer exposure (Figure 5).
70

9.0

Control

8.0

PABS

CBS

QACBS

7.0
Log 10 (N/N0)

6.0
R² = 0.96

5.0
4.0
3.0

R² = 0.78

2.0

R² = 0.83

1.0
R² = 0.89

0.0
-1.0
0.00

0.01

0.02

0.03
0.04
Dose (kGy)

0.05

0.06

0.07

Figure 5. X-ray reduction of Control, PABS-, CBS-, and QACBS-treated cells of E. coli
O157:H7.

Significant (P < 0.05) repair from sublethal injury was observed for each of the three
sanitizer-treated E. coli O157:H7 cocktails after inoculation onto baby spinach leaves. The
extent of injury, however, varied for the different sanitizer treatments, with QAC-treated cells
undergoing virtually complete recovery from an initial injury level of 88%. In contrast, PABand CB-treated cells exhibited less repair with percent injury decreasing from 89 to 66% and 95
to 63%, respectively (Table 6).

71

Table 6 E. coli O157:H7 cell injury following sanitizer treatment
Sanitizer Treatment
Peroxyacetic acid based

Inoculum
Injury (%)*
a
89 ± 4

Quaternary ammonium compound
based
Chlorine based

88 ± 4

None (Control)

a

10 ± 10

95 ± 3

a
b

30 min Post-inoculation
Injury (%)*
c
66 ± 11
b

1 ± 10
63 ± 4
8±6

c

b

*Letters indicate significant difference between treatments (P < 0.05).

Recovery of E. coli O157:H7 on baby spinach 30 minutes after exposure to CB- and
PAB-based sanitizer injury offers insight into the similarities in the mechanism of action of these
two sanitizers and the cellular response to stress. This is to be expected, as both sanitizers have
some of the same targets for inactivation. The action of oxidants has shown multiple stages of
activity depending on the contact time and sanitizer concentration. Upon initial contact,
sublethal injury is incurred by the cell, beginning with the cellular membrane and acting further
on the physiological functions of the cell following sanitizer uptake. Lisle et al. (1999)
quantified the progression of sublethal attack from chlorine through a series of physiological
indicators, suggesting that the site and extent of injury of bacterial cells can be determined
following the order: viable plate counts > substrate responsiveness > membrane potential >
respiratory activity > membrane integrity.
Depending on the type of prior sanitizer exposure, sublethally injured cells of E. coli
O157:H7 exhibited different degrees of X-ray resistance on baby spinach leaves. D10 values for
PABS-, QACBS-, and CBS-injured E. coli O157:H7 on baby spinach were 0.014, 0.022, and
0.024 kGy, respectively (Table 7). Prior exposure to PABS significantly (P < 0.05) decreased E.

72

coli O157:H7 resistance to X-ray irradiation, while exposure to CBS significantly (P < 0.05)
increased resistance. Significant cell injury after irradiation was not seen (P > 0.05) for cells
pretreated with either the PABS or QACBS; however, CBS-treated cells demonstrated an initial
decline in percent injury at low doses of irradiation with a significant (P < 0.05) increase in
injury seen at higher doses (Figure 6). Graphical comparisons illustrating the recovery of cells
between each sanitizer can be found in Appendix E. These results can be attributed to the
physiological state of the organism before X-ray exposure and the mechanism of sanitizer action
as it interacts with the spinach leaf.
Table 7 D10 values for sanitizer-treated inoculum
Sanitizer Exposure
None (Control)

D10 values (kGy)*
a
0.021 ± 0.001

PABS

0.014 ± 0.000

CBS

0.023 ± 0.001

QACBS

b
c

ac

0.022 ± 0.001
*Letters indicate a significant difference between treatments (P < 0.05).

73

80%
PABS

QACBS

CBS

Control

% Injury

60%

40%

20%

0%

-20%
30 min postinoculation

0.012

0.028

0.043

0.054

0.063

Dose (kGy)

Figure 6. E. coli O157:H7 cell injury following X-ray irradiation.

The mechanism of action for oxidizing chemical sanitizers may again help explain the
differences in D10 values seen between PABS- and CBS-treated cultures. Commercial chlorineand peroxyacetic acid-based sanitizers exhibit different rates of oxidation. Strong oxidizing
agents such as peroxyacetic acid primarily target cell surface components, while weaker
oxidizing agents such as chlorine diffuse through the protective cell barrier to react with inner
cellular components (Cho et al., 2010; Kitis, 2003).
Dukan and Touati’s research (1996) demonstrated how the further reactivity of chlorine
compounds impacts the efficacy of chlorine-based sanitizers. In their experiments, hypochlorous
acid (HOCl) reacted with many cellular components and was unlikely to reach the DNA;
however, mutants in recombinational repair genes exhibited increased sensitivity to HOCl,
indicating DNA damage. This result was attributed to hypochlorous acid derivatives including
74

chloramines, which are less reactive and thus more diffusible and efficient at breaching cellular
barriers. Chloramines are formed when HOCI or other chlorinating agents react with primary
and secondary amines (Thomas, 1986), which make up the structure of bacterial cells. The
concept of further reactivity offers a mechanistic separation between chlorine- and peroxyacetic
acid-based sanitizers, suggesting that chlorine compounds can penetrate to the core of the cell.
Consequently, chlorine and ionizing irradiation attack similar targets.
The effect of X-ray irradiation on QAC-injured cells of E. coli O157:H7 could not be
assessed due to almost total repair on the spinach leaves prior to X-ray exposure. This may be
explained by the additional role of QACs as a surfactant, which binds by chemisorption to the
cell surface to influence the zeta potential of the cell (Neu, 1996). On equipment surfaces, QACs
provide a barrier to prevent bacterial attachment by decreasing the contact angle, and increasing
surface free energy, which in turn reduces bacterial adherence (Sinde and Carballo, 2000).
Pretreatment with QAC was also shown to completely inhibit S. Typhimurium attachment to
chicken skin (Breen et al., 1995). Consequently, pre-treating E. coli O157:H7 with a QAC-based
sanitizer may have consequently decreased the organism’s ability to attach to the leaves of baby
spinach, preferentially allowing for the attachment of healthy, unaffected cells.
Properties inherent to baby spinach may also have lead to the repair of sublethally injured
cells of E. coli O157:H7 or increased attachment to the leaf surface during contact with the
leaves prior to X-ray exposure. In this study, sanitizer-injured cells of E. coli O157:H7 had two
extended 30 min periods of contact with the spinach – the first after inoculation to allow for
attachment to the spinach surface and transport to the X-ray irradiator, and the second after
irradiation as the spinach leaves were crushed to fully homogenize the samples. A high
concentration of naturally present vitamins and antioxidants including ascorbate in spinach

75

leaves aide in neutralizing reactive oxygen species (Foyer and Halliwell, 1976). Ascorbate
fluctuates between the reduced ascorbate and the oxidized form, dehydroascorbate to prevent
oxidative leaf damage from environmental ozone (Luwe et al. 1993) and hydrogen peroxide
(Nakano and Asada, 1981).
Overall, these findings suggest that peroxyacetic acid-based sanitizers may be preferred
for X-ray irradiated baby spinach, because exposure to a chlorine-based sanitizer resulted in
cross-protection to X-ray irradiation. Hence, combined use of PAB sanitizers with X-ray
irradiation can allow for shortened X-ray exposure times, with fewer negative impacts on
spinach quality from high levels of sanitizer and/or irradiation alone. While these results do
indicate that sublethal injury of E. coli O157:H7 due to prior treatment with a chlorine-based
sanitizer may reduce the efficacy of X-ray irradiation compared to those cells that did not receive
prior sanitizer exposure, this research does not suggest that combined treatment of a CBS and Xray will not be more effective. To this end, it is recommended for those processers using
chlorine in the wash water to carefully monitor levels of sanitizer, and to frequently clean
equipment and reduce the build-up of product in an effort to minimize the presence of
sublethally injured cells during production.

76

5. CHAPTER 4
SUGGESTIONS FOR FUTURE WORK WITH LOW-ENERGY X-RAY
IRRADIATION

Several questions and concerns need to be addressed prior to full implementation of lowenergy X-ray irradiation for leafy green processing. As with the implementation of any new
technology, one must consider both the cost and impact on the product.
Consumers eat spinach and other leafy greens for the crisp texture and high nutrient
quality. Additional processing using ionizing irradiation, heat, or chemical treatments, may
potentially disrupt the quality attributes of the product. This thesis focused on the ability of lowenergy X-ray irradiation to reduce levels of E. coli O157:H7 on spinach, while past work using
the Rayfresh irradiator was concerned with pathogen reduction levels on iceberg lettuce. In this
research, other attributes, such as sensory or quality changes, were not considered. In other
published research, gamma ray and E-beam irradiation doses of up to 3.85 kGy did not adversely
affect the nutritional value or texture of leafy greens (Denise et al., 2004; Gomes et al., 2008;
Han et al., 2004; Likui et al., 2006; Niemira and Fan, 2006; Prakash et al., 2000). X-ray’s impact
on nutritional composition should likewise be fully evaluated considering the treatment times
and conditions used for a 5-log reduction in commercial bags of baby spinach and other leafy
greens.
Results of this thesis have also suggested that the efficacy of X-ray irradiation may be
commodity-specific. X-ray dose levels to reduce E. coli O157:H7 differed for spinach, lettuce,
and parsley, with similar findings seen using gamma ray irradiation (Niemira et al., 2002).
Previous studies also reported dose differences between pathogens (Mahmoud, 2010a; Mahmoud
77

et al., 2010). Such variations highlight the need to further explore additional leafy greens and
potential pathogens, and identify target levels for industry.
Scale-up of the data for commercial processing must also be considered. D10 values are
valuable tools for demonstrating the efficacy of X-rays at their most basic level, but not under
actual processing conditions. This topic was touched upon in Chapter 2, with the irradiation of
bagged spinach; however, it should be expanded to include industry scale equipment as well as
consideration for distribution of the product within the bag.
Lastly, this research has highlighted the need for expanding the understanding of the
interaction between chemical sanitizers, ionizing irradiation and components of the bacterial cell.
Prior treatment of the cell with peracetic acid-based sanitizers in conjunction with X-ray
enhanced inactivation of E. coli O157:H7, while prior exposure to chlorine-based sanitizers
reduced the efficacy of X-ray irradiation. Genetic analysis has suggested similar routes of
bacterial inactivation by oxidizing agents (Dukan and Touati, 1996; Khil and Camerini-Otero,
2002; Wang et al., 2009); however, the details of inactivation are still not clearly understood, and
minor differences in the genetic response of the cell may have significant implications.
Exploring the reaction of the cell following exposure to chemical sanitizers and ionizing
irradiation will be useful in determining the route of inactivation and the ideal sanitizer to be
used when processing leafy greens with X-ray.
Given the limitations of this thesis, future research objectives using low-energy X-ray
irradiation should focus on expanding our current understanding of the technology, with special
consideration given to the economics and commercialization of the technology, the impact of Xrays on the sensory attributes of the leaf, the reduction of spoilage microorganisms, and the
mechanisms of bacterial inactivation.

78

5.1. Determine the Cost Associated with X-ray Irradiation
Cost of any food safety intervention is critically important to a business. An in-depth
cost analysis must be done to determine whether produce companies are capable of absorbing the
added cost of implementing the technology with the potential for a contamination response.
Implementing X-ray technology into an existing processing line will include a modification of
the production process, retraining of employees, and the upfront cost of the technology itself. In
addition, there will be the maintenance of the equipment, including specially designed parts and
experienced technicians. On the other hand, total retail sales for bagged spinach declined $201.9
million during the first 68 weeks after the FDA announced the 2006 outbreak of E. coli O157:H7
(Arnade et al., 2009). The cost of this outbreak included untold millions in lost product, recalls,
and lawsuits. The final considerations will be the shelf-life extension of the product and
consumers’ view of irradiation.
5.2. Application to Commercial Processing
The D10 values generated in this thesis must be translated to real-life processing
conditions and quantities of product. Key concepts to evaluate will therefore include dosemapping of irradiated packages, and applying the D10 values determined in this thesis to that
dose information. Tests will then need to be performed to evaluate whether the calculated doses
are in fact effective and practical for commercial processing.

5.3. Sensory Analysis
When attempting to sell a product, two of the most important factors include the taste and
the willingness to purchase. Consumer acceptance is therefore another key component in the

79

implementation of X-ray into the processing of leafy greens. A sensory study should be
conducted to evaluate consumer response to irradiation of spinach, lettuce, and parsley, using
doses determined to be lethal to E. coli O157:H7. This study should also aim to address
consumer interest in purchasing X-ray irradiated produce in addition to their taste perceptions of
the treated product.

5.4. Enhanced Growth from Elimination of Background Microflora
In addition to cost and customer considerations, the implementation of X-ray irradiation
must be fully integrated with the safety of the technology as the primary focus. This thesis
focused solely on the reduction of pathogens specifically applied to the surface of the leaf, but
had no regard to the safety of the treated product, including how it may fair in the household and
after opening. A risk analysis should be conducted to evaluate the potential for consumer
mishandling and to assess whether the dose applied to reduce pathogen levels does not also allow
for other, unseen concerns.
Ionizing irradiation is a very effective strategy for reducing microorganisms on produce,
including background and spoilage microflora. In the absence of competitive or spoilage
organisms, opportunistic organisms or pathogens, such as Listeria monocytogenes, could grow
uncontrolled on the surface of irradiated foods. Given the extended shelf-life of irradiated foods,
growth of these pathogens in comparison to product spoilage should be investigated. Appendix
C outlines the beginning of this process.

80

5.5. Sanitizer Mechanisms for Microbial Inactivation
Developing the optimal sanitizer to be used in combination with X-ray irradiation
requires a greater understanding of the role that sanitizers play in bacterial inactivation and how
the sanitizer alters the target organism prior to X-ray exposure. Individual chemical sanitizers
should be investigated for their role in eliciting a genetic response by the bacterial cell, based on
exposure time and sanitizer concentration. These results can be built to track the genetic markers
of cell exposure, provide an indication of inactivation, and help to identify key components of
the sanitizer that make the cell vulnerable to X-ray irradiation.

81

6. APPENDIX A
INACTIVATION OF ESCHERICHIA COLI O157:H7 ON FLAT LEAF PARSLEY
USING X-RAY IRRADIATION

82

6.1. Purpose
This study was conducted to determine the dose of X-ray irradiation to create a 1-log
decrease in the population (D10 value) of Escherichia coli O157:H7 on the surface of flat-leaf
parsley. The D10 value can vary between different organisms, across different food
commodities, and between technologies. This research has been a part of a greater goal to apply
X-ray irradiation to the safe production of bagged, cut leafy green vegetables. The first step to
understanding how X-ray interacts with flat-leaf parsley is to determine the D10 value for the
destruction of E. coli O157:H7. This value then needs to be scalable to larger quantities of
product.

6.2. Materials and Methods
6.2.1. Strain preparation
Three E. coli O157:H7 strains - K3995 (2006 spinach outbreak), K4830 (2006 lettuce
outbreak A), and K4492 (2006 lettuce outbreak B) preserved at -80°C were grown in trypticase
soy broth containing 0.6% (w/v) yeast extract (TSBYE) for 24 h at 37°C. Each individual strain
was then transferred to 20 ml TSBYE in centrifuge tubes. Following overnight growth at 37°C,
the strains were pelleted by centrifugation for 15 min at 4500 rpm, resuspended in phosphate
buffer solution (PBS), and combined in equal volume.

6.2.2. Sample preparation
Flat-leaf parsley leaves, purchased from the local supermarket on the day of
experimentation, were aseptically removed from the stem and digitally imaged with these

83

pixilated images factored into a computer algorithm to determine the leaf surface area. Parsley
stems were aseptically removed from the leaf using sterile scissors.

6.2.3. Inoculation
Prepared samples of parsley leaves and parsley stems were dip-inoculated in the 3-strain
cocktail of E. coli O157:H7 for 3 minutes, and spun dry in a salad spinner for 1 minute. Leaves
TM

of parsley were placed directly within a 4 oz Whirl-pak

bag, while the parsley stems were cut

into 2 cm segments, of which 3 segments were arranged side-by-side in the corner of the Whirlpak bag. All samples were then held 24 h at 4°C.

6.2.4. X-ray irradiation
Three replicates of 15 samples each were irradiated on each side of the leaf or stem to
achieve five combined surface doses of up to 0.205 kGy for flat-leaf parsley leaves and stems
using a prototype low-energy X-ray irradiator (Rayfresh Foods, Ann Arbor, MI). Irradiation
dose was confirmed using radiochromic film dosimeters (GAF3001DS, GEX Corporation,
Centennial, CO).

6.2.5. Enumeration
Numbers of E. coli O157:H7 in homogenized samples were determined by dilution or
filtering and plating on Sorbitol MacConkey Agar with cefixime and tellurite (CT-SMAC). The
highly selective media was used to prevent the growth of background bacteria present on the
spinach and parsley, after determining that there had been no evidence of injury using this
medium in previous work. Survivors were counted after 24 h incubation at 37°C.
84

6.2.6. Statistics
D10 values were determined from the absolute value of the inverse slope of the linear
regression for the log reduction values.

6.3. Results and Discussion
D10 values for flat-leaf parsley and parsley stems were 0.052, and 0.067 kGy,

Log10(N/N0)

respectively (Figure 7).

1.0
0.5
0.0
-0.5 0
-1.0
-1.5
-2.0
-2.5
-3.0
-3.5
-4.0

Dose (kGy)
0.05

0.1

0.15

0.2

Parsley Leaf
D10 = 0.052

Parsley Stem
D10 = 0.067

Figure 7. D10 values for E. coli O157:H7 on the leaves and stems of flat-leaf parsley.
This research suggests that X-ray irradiation needs to be tailored to the commodity being
treated, and consideration must be made to the additional, inedible portions of product,
specifically the stems of flat-leaf parsley if it is intended to be packaged along with the leaves.

85

6.4. Raw Data
Table 8 Reduction of E. coli O157:H7 on the surface of flat-leaf parsley leaves
Date
Initial Conc.
(CFU)
Control Avg.
(log CFU)
Dose [kGy]

0

0.059

0.117

0.147

0.176

0.205

7/17/2009

3/3/2009

2.00*10^9 2.30*10^9

3/18/2009
2.30*10^9

5.8873

5.70230

5.7285

#1
0.192148479
-0.045469482
-0.210850396
-0.089897273
-0.01641547
1.253038016
1.001787521
0.807514275
1.955636981
2.155460313
2.21183976
2.869958769
1.879187759
2.788211004
3.41366791
3.584788866
3.453787258
3.997871985
3.952483914
4.043642486

#2
0.042946
-0.85274
-0.84689
-0.88273
-1.05127
0.004884
-0.09041
-0.01728
0.977047
0.770461
0.768892
1.537592
1.620674
0.423361
2.082121
2.710039
2.823199
3.163645
3.816698
3.251514

#3
-0.031394
-0.718641
-1.368647
-0.856825
-1.10573

86

-0.24191
-0.072445
0.7001719
0.3316571
0.9106054
1.5149451
1.4655402
1.1153441
1.9234307
2.5167755
2.0308585
3.4548041
2.9917274
3.3437066

Log
Reduction

Std. Dev.

0.522827

-0.51197

-0.33065

-0.58839

-1.19797

-0.70846

-1.69053

-0.76468

-2.72652

-0.6457

-3.55734

-0.39977

Table 9 Reduction of E. coli O157:H7 on the surface of flat-leaf parsley stems
Date
Initial Conc.
(CFU)
Control Avg.
(log CFU)
Dose [kGy]
0
0.059
0.117
0.147
0.176
0.205

7/17/2009

3/3/2009

2.00*10^9 2.30*10^9

3/18/2009
2.30*10^9

5.4549

4.73197

4.9776244

#1
-0.2830
0.3580
-0.0750
1.2525
0.3966
1.5656
1.9897
2.0724
2.1512
1.5976
3.2134
3.4743
2.4917

#2
0.16524
-0.40236
0.23712
0.67364
0.91906
1.32225
1.48431
1.97705
2.13675
1.92749
3.26399
2.50796
3.37979

#3
-0.372139
0.2907648
0.0813738
1.797929
0.0883227
2.3244119
1.8238095
1.2689911
3.4035931
3.2086165
3.8393217
2.6711994
2.7701247

87

Log
Reduction

Std. Dev.

-2E-16

-0.29346

-0.85466

-0.61352

-1.75168

-0.36889

-2.16833

-0.69018

-2.84173

-0.87522

-2.88251

-0.43539

7. APPENDIX B

INACTIVATION OF ESCHERICHIA COLI O157:H7 IN BAGGED ICEBERG LETTUCE
USING X-RAY IRRADIATION

88

7.1. Purpose
The purpose of this research is to determine the time of X-ray irradiation required to
result in the inactivation of Escherichia coli O157:H7 in commercial bags of iceberg lettuce.
The results of this research can be used to determine processing conditions considering the
implementation of X-ray irradiation within a commercial processing line.

7.2. Materials and Methods
7.2.1. Strain preparation
Three E. coli O157:H7 strains - K3995 (2006 spinach outbreak), K4830 (2006 lettuce
outbreak A), and K4492 (2006 lettuce outbreak B) preserved at -80°C were grown in trypticase
soy broth containing 0.6% (w/v) yeast extract TSBYE for 24 h at 37°C. Each individual strain
was then transferred to 20 ml TSBYE in centrifuge tubes. Following overnight growth at 37°C,
the strains were pelleted by centrifugation for 15 min at 4500 rpm, resuspended in phosphate
buffer solution (PBS), and combined in equal volume.

7.2.2. Sample preparation
Bags of iceberg lettuce/romaine blend (312 g) were purchased from the local supermarket
at the day of inoculation. Lettuce was inoculated with 3.15 ml of prepared inoculum to achieve a
7

final concentration of 10 CFU/g using a sterile 2 in syringe. Syringe holes were then covered
with tape, and the bags were agitated gently by hand for approximately 1 min. Following
agitation, the bags were held for 24 h at 4°C.

89

7.2.3. X-ray irradiation
Samples bags were placed onto a conveyor system within the prototype low-energy X-ray
irradiator (Rayfresh Foods, Ann Arbor, MI) to simulate processing conditions as outlined in
section 3.3.4.

7.2.4. Enumeration
Following irradiation, 2 – 25 g samples were aseptically taken from each bag and placed
TM

within a 10 oz Whirl-pak

bag. Numbers of E. coli O157:H7 in homogenized samples were

determined by dilution or filtering and plating on Sorbitol MacConkey Agar with cefixime and
tellurite (CT-SMAC). Survivors were counted after 24 h incubation at 37°C.

7.3. Results
7.3.1. Dose determination
Table 10 Dose rate distribution at the mid-plane region in bags of iceberg lettuce
Product Position Absorbance Exp Time (s) Dose [Gy]
Lettuce
0
0.241
60
54.1
1
0.281
60
73.3
2
0.23
60
48.9
3
0.241
60
54.1
4
0.257
60
61.8
5
0.211
60
39.9
6
0.247
60
57
7
0.232
60
49.8
8
0.199
60
34.2

7.4. Raw Data

90

Table 11 Log reduction values for E. coli O157:H7 in bags of iceberg lettuce on 10/19/2010

Treatment
Control
Control
Control
1-log
1-log
1-log
3-log
3-log
3-log
5-log
5-log
5-log

Sample
A
1
B
A
2
B
A
3
B
A
4
B
A
5
B
A
6
B
A
7
B
A
8
B
A
9
B
A
10
B
A
11
B
A
12
B

Plate
Counts
(CFU)
104 126
160 166
136 130
84
84
136 118
18
24
105 95
167 198
40
55
161 144
26
23
37
34
12
13
31
38
48
41
30
40
110 80
135 143
190 191
86
94
92
97
38
43
9
15
1
1

Plate
Dilution
10000
10000
10000
10000
10000
100000
1000
1000
1000
100
1000
1000
10
10
100
1000
100
100
100
10
10
100
10
10

Bag
Dilution
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

CFU/25 g
1.15E+08
1.63E+08
1.33E+08
8.40E+07
1.27E+08
2.10E+08
1.00E+07
1.83E+07
4.75E+06
1.53E+06
2.45E+06
3.55E+06
1.25E+04
3.45E+04
4.45E+05
3.50E+06
9.50E+05
1.39E+06
1.91E+06
9.00E+04
9.45E+04
4.05E+05
1.20E+04
1.00E+03
91

Avg.
CFU/25 g

Weight
(g)

Avg.
CFU/Bag

Avg. log
CFU/Bag

Log
Reduction

1.39E+08

312

1.73E+09

9.24

0.00

6.75E+06

312

8.43E+07

7.93

1.31

1.06E+06

312

1.32E+07

7.12

2.12

4.18E+05

312

5.22E+06

6.72

2.52

Table 12 Log reduction values for E. coli O157:H7 in bags of iceberg lettuce on 12/7/2010

Treatment
Control
Control
Control
1-log
1-log
1-log
3-log
3-log
3-log
5-log
5-log
5-log

Sample
A
1
B
A
2
B
A
3
B
A
4
B
A
5
B
A
6
B
A
7
B
A
8
B
A
9
B
A
10
B
A
11
B
A
12
B

Plate
Avg.
Counts
Plate
Bag
CFU/25 CFU/25 Weight
Avg.
Avg. log
(CFU)
Dilution Dilution
g
g
(g)
CFU/Bag CFU/Bag
183 195
10000
100
1.89E+08
359 322
10000
100
3.41E+08
98
97
10000
100
9.75E+07
482 580
10000
100
5.31E+08
348 326
10000
100
3.37E+08
448 406
10000
100
4.27E+08 3.20E+08
312
4.00E+09
9.60
108 107
1000
100
1.08E+07
55
41
1000
100
4.80E+06
162 150
1000
100
1.56E+07
680 384
1000
100
5.32E+07
332 328
1000
100
3.30E+07
1500 1500
1000
100
1.50E+08 4.46E+07
312
5.56E+08
8.75
0
0
10
100
0.00E+00
4
1
10
100
2.50E+03
53
72
100
100
6.25E+05
540 452
100
100
4.96E+06
2
2
10
100
2.00E+03
0
0
10
100
0.00E+00 9.32E+05
312
1.16E+07
7.07
0
0
0.1
100
0.00E+00
4
1
0.1
100
2.50E+01
86
70
1
100
7.80E+03
35
27
1
100
3.10E+03
0
2
0.1
100
1.00E+01
0
0
0.1
100
0.00E+00 1.82E+03
312
2.27E+04
4.36

92

Log
Reduction

0.00

0.86

2.54

5.24

8. APPENDIX C

EVALUATING LISTERIA MONOCYTOGENES GROWTH ON IRRADIATED SPINACH

93

8.1. Purpose
In the absence of competitive or spoilage organisms, opportunistic organisms or
pathogens such as Listeria monocytogenes could grow uncontrolled on the surface of irradiated
foods. This concern should be addressed when implementing X-ray irradiation as a pathogen
control strategy for packaged leafy green vegetables. X-ray has demonstrated 5-log reduction of
several pathogens on a variety of produce, as well as a decrease in the levels of inherent
microbes and an overall extension in the shelf-life of the produce.

8.2. Materials and Methods
8.2.1. Sample preparation
Spinach samples were round-cut (2.54 cm diameter) using a sterile cork borer, and
divided into 4 groups based on the following treatments:
1. Irradiated/non-inoculated
2. Irradiated/inoculated
3. Non-irradiated/inoculated
4. Non-irradiated/non-inoculated

Samples that received a pre-irradiation treatment were transferred to Whirl-pak bags and
irradiated for 10 seconds on each side of the leaf, for a combined dose of 0.167 kGy as
confirmed using radiochromatic film dosimeters.

94

8.2.2. Culture preparation
Four strains of Listeria monocytogenes, preserved at -80°C, were grown in trypticase soy
broth containing 0.6% (w/v) yeast extract (TSBYE) for 24 h at 37°C. Each individual strain was
then transferred to 9 ml TSBYE. Following overnight growth at 37°C, individual strains were
combined and adjusted to an optical density of 0.967 and an initial concentration of 1.6 x 10^8
CFU.

8.2.3. Inoculation
The prepared inoculum was serially diluted through 6 dilutions in sterile phosphate buffer
solution (PBS), and 50µl was then spot inoculated onto treatments 2 and 3. Inoculated samples
were then placed into Whirl-pak bags and held 12 days at 4°C.

8.2.4. Enumeration
At 2 day intervals, two samples of each treatment were removed from refrigeration,
homogenized in 10 ml of PBS, and appropriate dilutions were plated in duplicate to either
trypticase soy agar containing 0.6% (w/v) yeast extract (TSAYE) (non-inoculated spinach) for
enumeration of total aerobic plate count (APC), or Modified Oxford Agar (MOX) (inoculated
spinach) for enumeration of L. monocytogenes. Colonies were counted following incubation for
24 – 48 hr at 37°C.

8.3. Results
2

Initial background levels of bacteria decreased from 5.33 to 5.01 log CFU/cm spinach.
2

Following 12 days storage at 4°C, total aerobic growth increased to 6.12 and 5.73 CFU/ cm on
95

irradiated and non-irradiated spinach, respectively. L. monocytogenes grew better on pre2

irradiated spinach than on untreated spinach, with levels reaching 4.13 and 2.75 CFU/cm on
pre-irradiated and non-irradiated spinach, respectively following 12 days at 4°C.

7.00
6.00

Log CFU/g

5.00

Irradiated APC
y = 0.0607x + 5.1184

4.00
Irradiated LM
y = 0.3598x

3.00
2.00

Non-irradiated LM
y = 0.2609x

1.00
Non-irradiated APC
y = 0.0886x + 5.256

0.00
0

2

4

6

8

10

12

14

Time (days)

Figure 8. Growth of total aerobic bacteria and L. monocytogenes on irradiated and non-irradiated
baby spinach.
8.4. Interpretation/Limitations
L. monocytogenes can grow to a higher level on X-ray irradiated spinach compared to
non-irradiated spinach. Further work with this study should include a comparison with higher
initial irradiation levels, extended storage times, and also at a variety of storage temperatures.

8.5. Raw Data
96

Table 13 Growth of total aerobic bacteria and L. monocytogenes on irradiated and non-irradiated baby spinach
Treatment

Irradiated; non-inoculated

Time
(day)

TSAYE Count (CFU)

0
2
4
6
8
10
12

193000
183000
280000
420000
870000
580000
720000

0
2
4
6
8
10
12

0
0
14
120
1150
4500
21000

0
2
4
6
8
10
12

30
10
16
108
180
350
750

Irradiated; inoculated

Non-irradiated; inoculated

215000
3200
189000 198000
280000 210000
330000 210000
570000 380000
570000 540000
580000 400000
MOX Count (CFU)
0
0
10
10
23
26
96
100
770
11240
2000
1680
22000
5800
MOX Count (CFU)
80
30
0
0
11
30
95
36
140
160
400
580
800
340

97

Avg.
(CFU)

Dilutio
n (ml)

2500
205000
240000
300000
380000
480000
480000

103425
193750
252500
315000
550000
542500
545000

10
10
10
10
10
10
10

Surfac
e area
2
(cm )
10.12
10.12
10.12
10.12
10.12
10.12
10.12

CFU/cm

0
20
19
80
930
1890
5300

0
10
20.5
99
3522.5
2517.5
13525

10
10
10
10
10
10
10

10.12
10.12
10.12
10.12
10.12
10.12
10.12

0
9.881423
20.25692
97.82609
3480.731
2487.648
13364.62

0.00
0.99
1.31
1.99
3.54
3.40
4.13

10
40
31
35
100
370
370

37.5
12.5
22
68.5
145
425
565

10
10
10
10
10
10
10

10.12
10.12
10.12
10.12
10.12
10.12
10.12

37.05534
12.35178
21.73913
67.68775
143.2806
419.9605
558.3004

1.57
1.09
1.34
1.83
2.16
2.62
2.75

2

Log
CFU/cm
2

102198.6
191452.6
249505.9
311264.8
543478.3
536067.2
538537.5

5.01
5.28
5.40
5.49
5.74
5.73
5.73

Table 13 (cont’d)
Non-irradiated; noninoculated

TSAYE Count (CFU)
0
2
4
6
8

200000
228000
320000
710000
910000

140000
216000
370000
560000
900000

10

420000
0
190000
0

390000
0
230000
0

12

180000
193000
690000
370000
111000
0
132000
0
630000

98

340000
160000
460000
410000
105000
0
118000
0
520000

215000
199250
460000
512500
992500

10
10
10
10
10

10.12
10.12
10.12
10.12
10.12

212450.6
196887.4
454545.5
506422.9
980731.2

5.33
5.29
5.66
5.70
5.99

265000
0
133750
0

10

10.12

2618577

6.42

10

10.12

1321640

6.12

9. APPENDIX D

INACTIVATION OF BIOFILM-ASSOCIATED ESCHERICHIA COLI O157:H7 ON
LABORATORY GROWN BABY SPINACH

99

9.1. Purpose
Contamination of leafy greens is thought to occur in the field by any number of
interactions that the plant has with the environment, with the primary source thought to be by
means of irrigation water. Contamination of irrigation can occur through close contact with
wildlife and manure, and has shown the capability of transfer to growing plants.
Biofilms have also demonstrated the ability to resist means of disinfection and
irradiation. The plant leaf surface has been shown to be a highly unfavorable environment for
the growth of bacteria. Therefore in an attempt to encourage survival, bacteria form biofilms. It
is likely that in the field environment, this biofilm will consist of many other bacteria common to
the field. Considering that E. coli in general are relatively poor biofilm formers, many others
may aid in the development of the film. In previous research there has been no consideration for
the natural microflora present on the leaf or the potential benefits of advanced biofilms in the
protection of pathogens.
This study aims to determine the persistence of E. coli O157:H7 on the surface of baby
spinach within an established biofilm, and to further assess whether a natural biofilm provides a
means of protection from exposure to X-ray irradiation.

9.2. Design/Objectives


Collect and characterize the microflora of field-grown spinach.



Determine biofilm-forming ability of chosen bacteria strains and compatibility with E. coli
O157:H7.



Establish laboratory growing conditions suitable for spinach



Grow spinach with inoculation procedures replicating contaminated irrigation practices.

100



Evaluate the growth/survival of E. coli O157:H7, pseudomonad, lactic acid bacteria, and total
aerobic bacteria throughout the growth period.



Evaluate the efficacy of X-ray irradiation on the reduction of E. coli O157:H7.

9.3. Materials and Methods
9.3.1. Bacterial collection and characterization
9.3.1.1.

Field sampling

Baby spinach was aseptically collected from two different growth conditions at the
Michigan State University Student Organic Farm (Holt, MI) - inside of a greenhouse and from an
th

outdoor plot, on August 8 , with outdoor conditions approximately 70°F, cloudy, with a light
rain. Organic spinach was collected in order to determine naturally occurring microorganisms
that may reside on the surface of the plants and that have not been exposed to common
processing conditions such as sanitizer treatment and employee handling.

9.3.1.2.

Enumeration and isolation

Two 25 g samples from each location were placed in Whirl-pak bags to which 100 ml
phosphate buffer solution (PBS) was added and homogenized for 60 s at 260 rpm. Appropriately
diluted samples were placed on Trypticase Soy Agar with 0.6% yeast extract (TSAYE) and
incubated at 37°C for 24 hr. Plates were evaluated for total growth, and then individual colonies
of differing morphologies were selected for isolation and identification.

101

9.3.1.3.

Biofilm analysis

Biofilm analysis was conducted on organisms isolated from field-grown spinach, GFPlabeled E. coli O157:H7, and laboratory Pseudomonad strains in order to determine a suitable
cocktail for inoculation. Pseudomonas were selected because of their known ability to form a
biofilms. Testing procedures were based on a modification of the assay described by Stepanovic
et al. (2000). All strains ere grown in TSBYE at 37°C for 18 h, and then streaked onto TSAYE
to obtain confluent growth after incubation at 37°C for 18 h. Cells were then harvested from the
plate by flooding the agar surface with 10 ml of 0.1% sterile PBS. Cell concentrations were then
estimated using MacFarland Turbidity Standards, and adjusted to a final O.D. of 1.0 ± 0.5.
2

Cultures were then serially diluted to a final concentration of 10 CFU/ml in TSBYE. 200 µl of
each diluted cell suspension was then transferred in triplicate to wells of a 96-well untreated
polystyrene microtiter tissue culture plate (BD Falcon Microtest Flat Bottom; Becton Dickinson,
Franklin Lakes, NJ), with sterile TSBYE serving as a negative control. Plates were incubated for
4 days at 22 ± 2°C. Following incubation, microtiter plate wells were emptied, rinsed three
times with 0.85% physiological saline, while being gently shaken to remove unattached cells,
and allowed to air-dry. Remaining cells were fixed to the well by the addition of 200 µl 99%
methanol (Fisher Chemicals, Fair Lawn, NJ) with the methanol decanted 15 min later. After
allowing the plates to air dry, the microtiter wells were stained using 200 µl of 2% crystal violet
(Remel, Lenexa, KS) for 5 min. After decanting the crystal violet, the wells were rinsed five
times with deionized water and air-dried. The remaining dye was resolubilized in 160 µl of 33%
(v/v) glacial acetic acid (Sigma Chemical Company, St. Louis, MO) and optical densities were
read at 570 nm using a Synergy HT Microplate Reader (Bio-Tek Instruments Inc., Winooski,
VT).
102

9.3.2. Spinach growth and inoculation
9.3.2.1.

Planting instructions

Soil (Scott’s Premium Potting Soil) (0.07% total nitrogen, 0.01% phosphate, 0.03%
soluble potash) was appropriately portioned into each of 32 cells in a 2 in flat (Jiffy seed starter)
and pressed in lightly. Distilled water was then applied to moisten the soil. Three spinach seeds
(Spinach double choice hybrid, W. Atlee Burpee and Co., Warminster, PA) were added per cell
and a covered with a light layer of soil (1-2 cm). After the plants were 1 – 2 in tall, the plants
were thinned to 1 plant per cell.

9.3.2.2.

Growth conditions
®

Plants were grown under a grow lamp (Sun System 10) equipped with a 400 W metal
®

halide bulb (Econo Gro ). The grow system provided an 11.5 hr light cycle, and the
temperature was consistent at 25 – 28 °C with approximately 70% relative humidity.

9.3.2.3.

Inoculation

Individual colonies of 4-environmental isolate (EI) bacterial strains (determined to be
better biofilm formers), 5-Pseudomonad (Ps) strains, and 4-avirulent GFP-labeled Escherichia
coli O157:H7 (EC) strains are selected from TSAYE and transferred to 9 ml TSBYE and grown
for 48 h at 25 °C. Following incubation, EI and Ps strains were combined in a 250 ml centrifuge
container and centrifuged at 4500 rpm for 15 min. The supernatant is discarded and the culture is
resuspended in 100 ml DI water. (Initial concentration determined by spread plating following
appropriate dilutions.) EC strains were combined in TSBYE. Spray inoculum consisted of 100
ml EI mixture in DI water, 5 ml EC in TSBYE, 100 µl fertilizer, and 400 ml DI water. 300 ml
103

was then evenly applied to the sheet of spinach within a biosafety cabinet. Spinach inoculation
was conducted daily over a three week span following three weeks of growth.

9.3.2.4.

Spinach sampling

At 3-day intervals for 3 wks, three spinach leaves were aseptically cut from mature
spinach plants prior to watering and daily inoculation. Each leaf was weighed, transferred to a 2
oz Whirl-pak bag, and hand-homogenized in 5 ml PBS. Appropriate dilutions were plated onto
TSAYE, TSAYE w/ampicillin, MRS, and PIA for enumeration of total aerobic bacteria, GFPlabeled E. coli, lactic acid bacteria, and Pseudomonas, respectively. Plates were incubated for 24
h at 37°C.

9.3.3. X-ray efficacy
Tests were conducted to compare the efficacy of X-ray on E. coli O157:H7 on the surface
of spinach and ingrained within a natural biofilm on the leaf.

9.3.3.1.

X-ray on surface-inoculated E. coli O157:H7

Spinach was grown following the procedures outlined above, with the E. coli O157:H7
omitted from the routine inoculum. Four strains of GFP-labeled E. coli O157:H7, preserved at 80°C, were grown in TSBYE for 24 h at 37°C. Each individual strain was then transferred to 20
ml TSBYE in centrifuge tubes. Following overnight growth at 37°C, the strains were pelleted by
centrifugation for 15 min at 4500 rpm, resuspended in PBS, and combined in equal volume.
Following plant maturity, spinach leaves were round-cut using a size 10 sterile corkborer, dipped inoculated in a 1:10 dilution of the prepared culture for 5 min, and spun dry for 1

104

min in a salad spinner. Spinach rounds were X-ray irradiated in triplicate at 5 dose levels of 1 –
5 seconds. Experiment was done in duplicate from two successive batches of grown spinach.

9.3.3.2.

X-ray on biofilm-incorporated E. coli O157:H7

This portion of the experiment was not conducted. Levels of E. coli O157:H7 did not
reach high enough levels on the growing spinach to be irradiated and demonstrate logarithmic
inactivation.

9.4. Results
9.4.1. Bacterial collection and characterization
9.4.1.1.

Enumeration and isolation

Total aerobic growth was 5.52 and 5.76 log CFU/g on spinach taken from the inside of
the greenhouse and outside of the greenhouse, respectively.

Table 14 Characterization of organisms isolated from field-sampled spinach
Isolate Location
1

Outside

2

Outside

3
4
5

Outside
Greenhouse
Outside

6

Greenhouse

Colony Morphology
Yellow, circular, raised, sticky,
medium sized
Yellow, circular, raised, fragile,
medium sized
white, circular, cratered, medium
yellow w/white clearing, raised, large
Yellow, divot in center, circular, medlarge
Pale yellow, irregular shape, flat,
large

105

Gram
Stain
neg

Organism
Description
short rod, ~1-2 μm

neg

short rod, ~1-2 μm

pos
neg
pos

rod, ~4-5 μm
rod, ~1 μm
diplococci, 2-3 μm

pos

rod, ~5-6 μm

Table 14 (cont’d)
7

Greenhouse

pale white, slightly raised, rough
edges, medium sized

pos

rod, ~10 μm, chained

8

Outside

pos

rod, ~10-15 μm

9
10
11

Greenhouse
Greenhouse
Greenhouse

glossy white, raised, circular, large
size
irregular shaped, large
white, circular, cratered, large
white, convex, circular, very small

pos
pos
pos

rod, ~6 μm
rod, ~10-12 μm
cocci, ~1 μm diameter

12

Outside

pos

rod, ~10-12 μm

13

Outside

pale white, flat, circular, rough edges,
large size
cream colored, very irregular shaped,
raised, mucoid

pos

rod, ~4-5 μm

9.4.1.2.

Biofilm analysis

Of the organisms tested, 4 of the environmental isolates (strains 1, 2, 4, and 11) were
found to be acceptable biofilm formers, having an absorbance >0.10 in the microtiter well assay.
All of the pseudomad strains tested had acceptable biofilm-forming ability as did one of the
GFP-labeled E. coli O157:H7.

Table 15 Biofilm-forming ability of EI, Ps, and EC
Strain
Env.
Isolates

1
2
3
4
5
6
7
8
9
10
11
12

Absorbance
0.13
0.57
0.02
0.26
0.01
0.03
0.01
0.01
0.00
0.01
0.26
0.00
106

Table 15 (cont’d)

GFP- E.C.

Ps. Strains

13
ATCC 43888
CV2b7
6980-2
6982-2
20
21
22
23
24

0.00
0.20
0.01
0.08
0.04
0.17
0.15
0.28
0.24
0.19

9.4.2. Persistence of E. coli O157:H7 during spinach growth
Levels of E. coli O157:H7 remained consistent during inoculation of spinach plants,
ranging from 3.07 – 3.43 log CFU/g. At the last time point, following 6 days without
inoculation, levels dropped to approximately 1 CFU/g.

Table 16 Growth of Total Aerobic Bacteria, E. coli O157:H7, Pseudomonas, and Lactic Acid
Bacteria over the growth period
Day Total Aerobic Growth E. coli O157:H7 Pseudomonad Lactic acid bacteria
(log CFU/g)
(log CFU/g)
(log CFU/g)
(log CFU/g)
0
2.58
0.00
0.00
1.92
3
5.48
3.26
3.09
5.44
7
5.69
3.21
3.60
5.40
10
5.61
3.07
3.43
5.40
14
5.57
3.29
3.34
5.31
17*
5.53
3.43
4.08
5.07
21
4.37
1.06
1.16
3.32
*Day 17 was the last day of inoculation

107

6.00
5.00
4.00

Total Aerobic Growth

3.00

E. coli O157:H7
Pseudomonad

2.00

Lactic acid bacteria
1.00
0.00
0

5

10

15

20

25

Figure 9. Growth of total aerobic bacteria, E. coli O157:H7, Pseudomonad, and lactic acid
bacteria over the growth period.
9.4.3. X-ray efficacy on surface-inoculated vs. biofilm-incorporated cells of E. coli
O157:H7.

108

Table 17 X-ray inactivation of surface inoculated laboratory grown spinach- Batch 1
Sample

Dose
Time
(s)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

0
0
0
0
0
0
0
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5

Plate
Count
A
(CFU)
2
0
0
10200
10900
9700
12900
5500
6700
4600
1700
2390
4000
810
1050
1130
550
650
700
370
510
470

Plate
Count
B
(CFU)
0
0
0
10700
8300
7000
10000
4800
4200
4000
1610
2170
4000
820
930
1150
590
530
660
280
460
270

Average
Plate
Count
(CFU)
1
0
0
10450
9600
8350
11450
5150
5450
4300
1655
2280
4000
815
990
1140
570
590
680
325
485
370

Amount
Plated

Initial
Dilution

1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00

15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15

109

Sur
Area
(cm^2)
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08

Log
CFU/cm^2

Avg Log
CFU/cm^2

Log
Reduction

0.0000
0.0000
0.0000
4.7071
4.6702
4.6096
4.7467
4.3997
4.4243
4.3214
3.9067
4.0459
4.2900
3.5991
3.6836
3.7448
3.4438
3.4588
3.5204
3.1998
3.3737
3.2561

0.00

0.00

4.68

0.00

4.38

0.30

4.08

0.60

3.68

1.01

3.47

1.21

3.28

1.41

Table 18 X-ray inactivation of surface inoculated laboratory grown spinach- Batch 2
Sample

Dose
Time
(s)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

0
0
0
0
0
0
0
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5

Plate
Count
A
(CFU)
0
0
0
6100
5700
6100
6100
3000
3900
44500
1330
1890
1740
1350
1940
950
920
840
720
540
590
590

Plate
Count
B
(CFU)
0
0
0
5500
5500
6400
5300
2100
3200
5200
1680
1380
1710
1720
1370
1030
750
870
660
520
440
700

Average
Plate
Count
(CFU)
0
0
0
5800
5600
6250
5700
2550
3550
24850
1505
1635
1725
1535
1655
990
835
855
690
530
515
645

Amount
Plated

Initial
Dilution

1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00

15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15

110

Sur
Area
(cm^2)
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08
3.08

Log
CFU/cm^2

Avg Log
CFU/cm^2

Log
Reduction

0.0000
0.0000
0.0000
4.4514
4.4361
4.4838
4.4438
4.0945
4.2382
5.0833
3.8655
3.9015
3.9247
3.8740
3.9067
3.6836
3.6096
3.6199
3.5268
3.4122
3.3997
3.4975

0.00

0.00

4.45

0.00

4.47

-0.02

3.90

0.56

3.82

0.63

3.59

0.87

3.44

1.02

10. APPENDIX E

COMPARISON OF SELECTIVE AND NON-SELECTIVE PLATING FOR THE
DETERMINATION OF D10 VALUES FOR SANITIZER-TREATED AND X-RAY
IRRADIATED ESCHERICHIA COLI O157:H7 ON BABY SPINACH

111

10.1. Purpose
A direct comparison between selective and non-selecting plating of X-ray irradiated and
sanitizer-injured Escherichia coli O157:H7 illustrates the impact of the treatment on the injury of
the cell, as well as the affect of the medium on the determination of dose-reduction values.

10.2. Materials and Methods
Methods for this comparison are detailed in section 3.2.

10.3. Results

9
8
Log CFU/cm2

7
6

Overlay
D10 = 0.021

5
4

SMAC

3

D10 = 0.021

2
1
0
0

0.01

0.02

0.03
0.04
Dose (kGy)

0.05

0.06

0.07

Figure 10. D10 values for selective and non-selective control samples of baby spinach.

112

6

Log CFU/cm2

5
4
Overlay
D10 = 0.024

3
2

SMAC
D10 = 0.027

1
0
0

0.01

0.02

0.03
0.04
Dose (kGy)

0.05

0.06

0.07

Figure 11. D10 values for selective and non-selective XY-12-treated samples of baby spinach.

6

Log CFU/cm2

5
4
Overlay
D10 = 0.014

3
2

SMAC
D10 = 0.015

1
0
0

0.01

0.02

0.03
0.04
Dose (kGy)

0.05

0.06

0.07

Figure 12. D10 values for selective and non-selective Tsunami-treated samples of baby spinach.

113

7

Log CFU/cm2

6
5
Overlay
D10 = 0.022

4
3

SMAC
D10 = 0.022

2
1
0

0

0.01

0.02

0.03
0.04
Dose (kGy)

0.05

0.06

0.07

Figure 13. D10 values for selective and non-selective QUAT-treated samples of baby spinach.
10.4. Conclusion
Results of the selective vs. non-selective plating show that the plating medium is an
important consideration in assessing the recovery of injured microorganisms and in accurately
reporting dose-reduction values. These results clearly illustrate that there is no clear indication
of injury obtained from X-ray treatment alone (figure 10) or from sanitizer exposure using a
quaternary ammonium compound-based sanitizer (figure 13), while treatment with a chlorinebased (figure 11) and peracetic acid-based (figure 12) sanitizer did show signs of injury from the
sanitizer treatment.
By evaluating the data with the injured microorganisms in mind through the use of a
selective medium with a non-selective overlay, the D10 value is significantly reduced compared
to the D10 value of the selective medium alone. Without a selective medium, the D10 value may
be over-reported.

114

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