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This is to certify that the

thesis entitled

Influence of Chlorine Dioxide Ice on
Pathogen Survival and Recovery on
Chilled Pork and Turkey Subprimals

presented by

Justin Robert Ransom

has been accepted towards fulfillment
of the requirements for

Master's degree in Animal Science

Mahm- W

Jlajor professor

Date _May__8_._2_Q_QD__

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

PLACE IN REI'URN BOX to remove this checkout from your record.
TO AVOID FINE return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

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11m comma.“

Influence of Chlorine Dioxide Ice on Pathogen Survival and Recovery
on Chilled Pork and Turkey Subprimals

By

Justin Robert Ransom

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE
Department of Animal Science

2000

ABSTRACT

Influence of Chlorine Dioxide Ice on Pathogen Survival and Recovery on Chilled
Pork and Poultry Subprimals

By
Justin Robert Ransom

Chlorine dioxide (ClOz) has been investigated as an antimicrobial in meat
washing systems and brine chillers. This study was designed to observe the
antimicrobials effectiveness of ClOz applied in an ice form. An ice machine with a
ClOz generator produced ice containing 0 or 20 ppm inactivated ClOz and 5 or 10
ppm activated CiOz. Subprimals were inoculated with E. coli 0157:H7, S.
typhimurium, L. monocytogenes or Y. entercolitica. Inoculated subprimals were
placed in ice treatments for 24 hours, then stored (2°C) for 7 days. Samples
were taken for bacterial enumeration before and after ice and during storage.
The main effect of CD; concentration did not reduce (P>0.05) the microbial load
of pathogens on subprimals. The interaction of temperature during ice
application and day of storage did reduce (P<0.05) the microbial load. Ice applied
to pork at 26°C reduced L. monocytogenes, and at 2°C, E. coli O157:H7 and S.
typhimurium were reduced after 7 days of storage. Neither temperature during
ice application reduced Y. entercolitica. In turkey, ice treatments applied at the
environmental temperature of 26°C reduced E. coli01571H7 and S. typhimurium.
This reduction may be attributed to the release of more ClOz during melting. The
largely activated ice-melt in the bottom of the cooler contained 2.5-3.0
longFU/ml fewer pathogens compared to the control ice-melt. The use of ClOz
ice as an antimicrobial should be further investigated for potential use in retail

meat display cases or in ice-packed meat transportation systems.

Copyright by
Justin Robert Ransom
2000

This thesis is dedicated to everyone who has encouraged me to go beyond
my perceived limits.

iv

Acknowledgments

Special thanks to. . .

Dr. Wes Osbum for serving as my major advisor. l have grown
exponentially as a research scientist since my first day at Michigan State. I
am grateful for everything I have learned not only in Animal Science/Meat
Science, but in life in general.

Dr. Harlan Ritchie for encouraging me to attend MSU. I appreciate
the way he has taught me to approach research. . .thinking outside of the
box. His willingness to help others excel can be matched by no one.

Dr. Al Booren for being on my committee and giving constant
encouragement.

Dr. Laura M. Cheney for her economic perspective on my committee.

Dr. Jamie Prater for all of her assistance in the microbiology
laboratory. She has taught me everything I know about micro lab
techniques. In addition, she worked tirelessly. I consider her to be a great
friend.

Dr. Rob Tempelman for his assistance, and more importantly patience
in SAS programming.

Drs. Hogberg, Hawkins, Doumit and Banks for all of their support and

encouragement when I needed it over the last two and a half years.

Additional Acknowledgements. . .

Ms. Nicole Zarb, Mr. John Heller and Mr. Charles Farber for all of their
help in the lab during my research project.

Mr. Tom Forton for his help with meats judging programs, extension
activities and research.

Ms. Debi Beeseuwart for her assistance in generating travel
authorizations and ordering supplies.

1998 and 1999 MSU Meats Judging Teams, I learned as much from
them as they did from me.

Dr. Mark Miller and Mr. Aaron Alejandro for all of their encouragement
over the years.

Mr. Ken Guens and family for all of their hospitality.

My friends and roommates. . . I will never forget all of the great times
we have had.

Mr. Bob Ransom, Ms. Linda Gray, Ms. Cleone Ransom and Mr. and
Mrs. Chris Sosebee and family, thank you for making me the young man I
am today. Thank you for all of the thoughts and prayers a thousand miles
away.

Thank you to the Lord above, there have been times that l have

strayed, but I couldn’t have done any of this without you.

vi

Table of Contents

List of Tables ............................................................................... x

List of Figures .............................................................................. xi
Introduction .................................................................................. 1
Chapter 1 ...................................................................................... 3
Review of Literature ......................................................................... 3
I. Overview of Food Safety ............................................................... 3

1. Current Status of Food Safety
2. Federal Regulation

II. Food Safety Regulations .............................................................. 6
1. Pathogen Reduction
2. HACCP System Final Rule
3. Impact of HACCP

lll. Organisms and symptoms associated with food borne pathogens ...... 10
1. Escherichia coli 0157:H7
2. Samonella typhimun'um
3. Yersinia entercolitica
4. Listeria monocytogenes

lV. Pathogen Intervention Strategies ................................................. 18
1. Overview
a) Antimicrobials
b) Delivery Systems
Acid Spray
Acid Dip
Quick Chill
Brine Chill .
Hurdle Technology

99:59!“

V. Chlorine Dioxide ..................................................................... 28
1. Overview '
2. Chemical Mechanisms
3. Delivery Systems

Vl. Economic Implications .............................................................. 31
1. Costs Associated with Food Poisoning
2. Costs Associated with Food Safety

vii

VII. Summary of Literature ............................................................. 33

VIII. References ........................................................................... 35
Chapter 2 .................................................................................... 42
Materials and Methods .................................................................... 42

I. Development of Ice Machine
II. Chlorine Dioxide Concentrations
III. Titration of Chlorine Dioxide
IV. Product Procurement

V. Test Microorganisms

VI. Inoculation

VII. Sampling Method

VIII. Bacterial Enumeration

IX. Meat Storage Containers

X. Vacuum Packaging

XI. Determination of pH

XII. TBA Analysis

XIII. Statistical Analysis

Chapter 3 .................................................................................... 48
Development and Evaluation of an Ice Machine that Generates Chlorine
Dioxide Ice at Targeted Levels of Concentration ................................... 48
I Abstract

II. Introduction

III. Materials and Methods
IV. Results and Discussion
V. References

VI. Figure

Chapter4....................... .............................................................. 59

Influence of Chlorine Dioxide Ice on Pathogen Survival and Recovery on
Pork and Turkey Subprimals

I. Abstract
II. Introduction
III. Materials and Methods
IV. Results and Discussion
V. References
VI. Tables
VII. Figures
viii

Appendices ................................................................................. 77

Recommendations for Future Research .......................................... 95

List of Tables

Table 1 Survival of pathogens on pork subprimals submerged in various ice
treatments for 24 hours at refrigerated (2°C) or room (26°C) temperature,
vacuum packaged and stored for 1 and 7 days at 2°C ........................... 68

Table 2 Survival of pathogens on turkey subprimals submerged in various
ice treatments for 24 hours at refrigerated (2°C) or room (26°C) temperature,
vacuum packaged and stored for 1 and 7 days at 2°C ............................ 69

Table 3 Milligrams of malonaldehydel1000 9 sample in pork and turkey
subprimals submerged in various ice treatments for 24 hours at refrigerated
(2°C) or room (26°C) temperature, at day 1 and day 7 (vacuum packaged
storage at 2°C) .............................................................................. 70

List of Figures

Figure 1.1 Chlorine Dioxide Ice generator ......................................... 55

Figure 1.2 Ratio of Phosphoric Acid and Water used to generate targeted
levels of activated or inactivate CIOz Ice ............................................ 56

Figure 1.3 Linear correlation between PAO and total available CIOz ....... 56
Figure 1.4 Ice pH decline over 4-hour observation period ..................... 57

Figure 1. Two-way interaction (P<0.05) of the growth of E. coli 0157:H7 on
pork sub-primal submerged in various ice treatments for 24 hours at
refrigerated (2°C) or room (26°C) temperature, vacuum packaged and stored
for 1 and 7 days at 2°C .................................................................. 71

Figure 2. Two-way interaction (P<0.05) of the growth of Salmonella
typhimurium on pork sub-primal submerged in various ice treatments for 24
hours at refrigerated (2°C) or room (26°C) temperature, vacuum packaged
and stored for 1 and 7 days at 2°C .................................................... 71

Figure 3. Two-way interaction (P<0.05) of the growth of Listeria
monocytogenes on pork sub-primal submerged in various ice treatments for
24 hours at refrigerated (2°C) or room (26°C) temperature, vacuum
packaged and stored for 1 and 7 days at 2°C. ...................................... 72

Figure 4. Two-way interaction (P<0.05) of the growth of Yersinia
entercolitica on pork sub-primal submerged in various ice treatments for 24
hours at refrigerated (2°C) or room (26°C) temperature, vacuum packaged
and stored for 1 and 7 days at 2°C. ................................................... 72

Figure 5. Two-way interaction (P<0.05) of the growth of E. coli 0157:H7 on
turkey sub-primal submerged in various ice treatments for 24 hours at
refrigerated (2°C) or room (26°C) temperature, vacuum packaged and stored
for1 and 7days at 2°C....... ............................................................ 73

Figure 6. Two-way interaction (P<0.05) of the growth of Salmonella
entercolitica on turkey sub-primal submerged in various ice treatments for 24
hours at refrigerated (2°C) or room (26°C) temperature, vacuum packaged
and stored for 1 and 7 days at 2°C ..................................................... 73

Figure 7. Two-way interaction of the growth of Listeria monocytogenes on
turkey sub-primal submerged in various ice treatments for 24 hours at
refrigerated (2°C) or room (26°C) temperature, vacuum packaged and stored
for 1 and 7 days at 2°C .................................................................... 74

xi

Figure 8. Effect of Chlorine Dioxide ice-melt on the survival of pathogens in
the bottom of the ice chests. ............................................................ 74

Figure 9 Differences in the Iogm CFU/cm2 of E. coli 0157:H7 that were
recovered after ice application 2, 4, 6, 8, 10 and 12 hours after
inoculation .................................................................................... 75

xii

INTRODUCTION

Recent illnesses associated with new strains of microorganisms, such as
Salmonella typhimurium and virulent strains of Escherichia coli 0157:H7 have
increased interest in pathogen intervention strategies and technologies within
the food industry. The Centers for Disease Control and Prevention consider
Escherichia coli 0157:H7, Salmonella and Listeria monocytogenes to be of
greatest concern because of the severity and number of illnesses they cause
(Richardson, et al., 1998). To address these concerns, pathogen intervention
strategies such as meat and poultry carcass washing systems have been tested
extensively in laboratories and in processing plants. Incorporation of various
organic acids and chlorine compounds as part of these washing systems are
critical to effectively reduce these pathogenic organisms.

Chlorine is a disinfectant that has been used for the last 100 years, but
has drawn concern because of the toxic mutagens (chlorofonns) generated
during the reaction with organic matter (Tsai et al., 1997). Lillard (1979) found
chlorine dioxide (ClOz) to be seven times more effective than chlorine and it did
not generate toxic chloroform upon reaction with organic matter. Several studies
have reported the effectiveness of CD; as an antimicrobial applied as a liquid
(Cutter and Dorsa, 1995). Coupling the bactericidal effectiveness of CIOz and
the temperature reducing/stabilizing ability of ice could offer a unique pathogen
intervention strategy to reduce the growth and survivability of pathogenic

organisms.

The purpose of this study was to observe the effectiveness of CD;
applied in an ice form on the survival of pathogens on fresh pork and poultry
subprimals. The hypothesis was that the bactericidal effect of the ClOz and the
low temperature of the ice would result in multiple hurdles, averting pathogen
survival and growth on the meat product.

There were two primary objectives in this study. The first objective was
the development of an ice machine prototype that accurately generated CIOz ice
at 20 ppm inactivated CI02 ice, 20 ppm marginally activated CIOz ice (5 ppm
activated CIOz, 15 ppm inactivated CIOz), 20 ppm largely activated CIOz ice (10
ppm activated ClOz, 10 ppm inactivated CIOz) or control (no CIOZ). The second
objective was the determination of the CD; ice application, concentrations and
storage parameters that reduced pathogen survival and growth during retail
storage. The Iong-tenn objective was to transfer knowledge gained into

recommendations for the pork and poultry industries.

CHAPTER 1

Review of Literature

I. Overview of Food Safety:
Current Status of Food Safety

Meat and meat by-products are a significant part of the diet for much of
the world’s population. Meat is an important contribution to the economy of
domestic and global agriculture. Due to the “presumed” increased incidence of
virulent pathogens in the food supply, foodbome illness has become a major
topic of both public and scientific debate. Despite the high level of concern,
there is much to be learned about the nature of food poisoning. The safety of
meat can be compromised in various ways, including chemical residues
(pesticides, antibiotics), physical hazards (glass, metal) and most importantly,
microbiological contamination. Although researchers have made significant
advances in food safety in recent years, it continues to be an evasive problem.

Food safety is defined as those measures that are taken to ensure that
foods can be eaten without adversely affecting the consumer (IDEX, 1998).
This includes minimizing the contamination of foods, killing microbial
contaminates, denaturing toxins and inhibiting the growth of pathogenic
organisms on food sources. In order for this to be accomplished, researchers,
producers, packers, and consumers must work together to minimize

microbiological contamination from “farm to fork” (Smith, 1999).

Despite current efforts being made, more than half of the current
foodbome outbreaks reported remain of unknown origin. The World Health
Organization predicts that there are 300 to 350 percent more outbreaks than
what are reported (Saucier, 1999). Some estimates suggest that hundreds of
millions of people around the world suffer due to the incidence of foodborne
illnesses. In addition, it is predicted that at least five to ten percent of the
population is affected annually (Saucier, 1999).

Although the estimates of cases of microbial foodbome disease vary, it is
suggested that world wide, 6.5 to 33 million annually become sick and 525 to

9,000 die (CAST Report, 1994).

Federal Regulation

Unsanitary conditions in meat-packing plants and the use of poisonous
preservatives and dyes in foods were the major problems leading to the
enactment of food laws (FSIS, 1999). By the end of the 19‘" century many
countries had created laws that prevented the sale of diseased and spoiled
meats; however, the USA fell far behind. Because of adulteration,
misrepresentation and threat to consumers, European countries prohibited the
import of American food into their domestic markets. The result of this caused

US. officials to rethink their lack of regulation.

In 1906, the United States Congress passed two laws: 1.) The Pure Food
and Drug Act (1906) and 2.) The Meat Inspection Act (1906). These laws were

administrated by the United States Department of Agriculture's Food Safety and

Inspection Service (USDA-FSIS) and extended to all segments of the food and
beverage industries. FSIS has jurisdiction over food that contains more than 2-3
percent meat or poultry. The Department of Health, Education and Welfare
through its agency, the Food and Drug Administration (FDA), has jurisdiction
over all food products other than those inspected by FSIS (FDA Backgrounder,
1999)

Many multinational agreements for international transport and marketing
of food and beverages are based on the 1906 food laws. The problems with
these laws were insufficient standards and lack of strict enforcement. This was
due to inadequate inspection. In 1938, the 1906 Pure Food and Drug Act and
the Meat Inspection Act were revised. The law is presently called the Food,
Drug and Cosmetic Act of 1938. This serves as the foundation for the laws that
regulate the food and beverage industry today. In 1954, the Food, Drug and
Cosmetic Act of 1938 was amended to include the regulation of pesticides in
food crops. This new law was the Miller Pesticide Amendment. The Food
Additives Amendment of 1958 specifies the kinds and quantities of additives
industry can add to food without compromising human safety. The 1959 Poultry
Inspection Act and the 1972 Fair Packaging and Labeling Act were two of the
last regulations administered (FDA Consumer, 1981).

Secretary of Agriculture Espy directed FSIS in 1993 to initiate rulemaking
to require all inspected-meat and poultry slaughter facilities to develop and
maintain a Hazard Analysis and Critical Control Point (HACCP) system (Manis,

1995). HACCP would supplement, not replace FSIS inspection. FSIS

inspectors would maintain a daily presence in these establishments to
determine whether products were being produced under the direction of the
HACCP system and in adherence to regulations set forth by the Food, Drug and
Cosmetic Act. It was not until July of 1996, that FSIS handed down the final rule
of the long-awaited modernization of the meat and poultry inspection system

(Manis, 1995).

ll. Food Safety Regulations
Pathogen Reduction

The FSIS proposal to require the implementation of HACCP in meat and
poultry establishments was supported by both large and small businesses,
scientific and public health officials, consumers and public interest
organizations. Supporters believed the system should systematically build
science-based food safety measures into their production systems (Federal
Register, 1996). FSIS stated the goal of its food safety strategy and proposed
Pathogen Reduction] HACCP regulations as follows:

”FSIS believes its food safety goal should be to reduce the risk of foodbome
illness associated with the consumption of meat and poultry products to the
maximum extent possible by ensuring that appropriate and feasible
measures are taken at each step in the food production process where

hazards can enter and where procedures and technologies exist or can be

developed to prevent the hazard or reduce the likelihood it will occur (60 FR

6785) (Federal Register, 1996).”

Through establishing this goal, FSIS realized no single solution or technology
could solve the problems facing the food industry. However, FSIS believed
through continuous efforts to improve hazard identification and prevention, this
goal could be attained.

From a food safety standpoint, the most important objective is to create
effective measures to reduce and control harmful bacteria on raw meat and
poultry products. HACCP, combined with appropriate food safety performance
standards, and science based technology, is the most effective means available
for controlling and reducing harmful bacteria on raw meat and poultry products

(Manis, 1996).

HACCP System Final Rule

The USDA/FSIS mandated the requirements to reduce the occurrence
and numbers of pathogens on meat and poultry products, to reduce the
incidence of foodborne illness associated with consuming these products, and to
provide a framework for modernization of the meat and poultry inspection
service on July 25, 1996 (IDEXX, 1998). Four new programs were the basis of
new regulations. The first of these programs required the establishment of
Sanitation Standard Operation Procedures (SSOPs). Secondly, a regular

microbial testing program for slaughter establishments for the removal of fecal

contamination and associated bacteria. The third program required slaughter
and grinding plants to meet pathogen reduction performance standards for
Salmonella. The fourth and final program mandated all meat and poultry plants
to develop and implement HACCP programs.

By January 27, 1997 all federally inspected establishments were
expected to implement SSOPs and test for generic E. coli as an indicator of the
adequacy of the plant's process control for fecal contamination. At the same
time, tests for generic Salmonella were mandated because it is the leading
cause of foodborne illness among enteric pathogens (FSIS Backgrounders,
1996). Plants that employed 500 people or more had to have HACCP program
implementation by January 26, 1998. Smaller establishments with 10 to 499
employees had to comply by January 25, 1999. Finally, plants with fewer than
10 employees or less than $2.5 MM were forced to comply by January 25, 2000.
Salmonella pathogen reduction performance standards had to begin at the

same time HACCP implementation took place (Federal Register, 1996).

Impact of HACCP

HACCP is not an inspection system; it is an industry process control
system. The FSIS inspector’s role changed from a command and control
regulator, to a performance standard monitor. Inspectors determine if each
plant’s SSOPs and HACCP plan conforms to regulatory requirements. Some of
the duties include microbial verification, preparation of written material to

document failure and to meet regulatory requirements and enforcement when a

plant is not in conformance with the established requirement (Nunes, 1997)
FSIS is now working on the development of measures that can be used on the
farm to reduce food safety hazards, although it does not mandate production
practices at this stage.

Six studies have been planned over the next three years to evaluate
HACCP Systems (FSIS, Nov 1999). Some of the questions that these studies
hope to answer are: Do HACCP processing control systems reduce foodborne
illness? Do HACCP systems make inspection more effective? Do HACCP
systems increase consumer confidence? Do HACCP systems control production
safety hazards? The final question is, Do HACCP systems provide an
opportunity for increased productivity?

In addition to these studies, the lntemational HACCP Alliance has been
created as a proactive step to assist the meat and poultry industry in preparing
and regulating HACCP. The mission of the alliance is to promote international
public health and food safety by facilitating uniform development and
implementation of HACCP programs from farm to table. The founders of the
HACCP Alliance realized the need to standardize the training for those
implementing HACCP systems. The Alliance works with USDA’s FSIS and
other government agencies worldwide to enhance communication between
industry and government (International HACCP Alliance Homepage)

In addition, other programs such as the Food Safety Virtual University, the State
HACCP Network, USDNFDA HACCP Training Program and Resource

Database, the USDA/FDA Foodbome Illness Education Information Center and

the FDA Bad Bug Book were set up to help industry establish HACCP systems.

III. Organisms and symptoms associated with food borne pathogens

Escherichia coli 0157:H7

Escherichia is of the Enterobacteriaceae family of which it shares
common characteristics. E. coli is one of six species in the genus Escherichia.
Most of the Escherichia species ferment lactose and produce gas (Vamam,
1991). In addition, they are facultative, non-spore forming, rod-shaped
anaerobes (Kraft, 1992). Currently there are five known strains of E. coli:
Enteropathogenic (EPEC), Enterotoxigenic (ETEC), Enteroinvasive (EIEC),
Enteroadherent-aggregative (EA-AggEC) and Enterohemoraggic (EHEC). The
four main types, EPEC, ETEC, EIEC and EHEC are distinctly different in
epidemiology, pathogenicity and O:H serovar (Vamam, 1991).

MacConkey and violet red-bile agars are selective agars used when
isolating E. coli, however, these media will not differentiate pathogenic
serogroups from non-pathogenic serogroups. Serological and genetic methods
have been developed for differentiating E. coli strains. Reliable methods such
as polymerase chain reaCtion (PCR) and pulsed field gel electrophoresis
(PFGE) have been devised to distinguish different enteroinvasive and
enterohaemorrhagic E. coli strains (Buchanan and Doyle, 1997)

In the past 20 years E. coli 0157:H7 has emerged as a pathogen of public

health concern (Riemann and Cliver, 1998). Although most E. coli are normal

IO

flora and reside in the colons of healthy people and other warm-blooded
animals, several strains are capable of causing significant human threat. EHEC
has been transmitted via food and caused disease ranging from hemorrhagic
colitis (bloody diarrhea) to Hemolytic Uremic Syndrome (HUS) (Buchanan and
Doyle, 1997). EHEC exhibit disease symptoms similar to Shigella dysentery,
and is most commonly found in developed countries (Riemann and Cliver 1998).
In 1975, E. coli 0157:H7 was first isolated from an infected woman in
California (Merritt 1998); however, it was not until 1982 that it was considered a
human pathogen following two hemorrhagic colitis outbreaks. The first outbreak
took place in Oregon, where of 26 cases reported, 19 were hospitalized. In
Michigan a second outbreak occured, 21 cases were reported and 14 were
hospitalized. In both cases, undercooked hamburgers testing positive for E. coli
0157:H7 were the cause (Buchanan and Doyle, 1997). Many food items have
been found to be a vehicle for E. coli. These foods include unpasteurized milk
and apple juice; ham, turkey and cheese sandwiches; ground beef patties; dry
fermented sausage; salad; and non-chlorinated water (Riemann & Cliver 1998).
Fortunately, there are several factors that prevent, hinder and stop the
growth of E. coli. A pH less than 4.4 or greater than 9.0, a water activcity (a...) of
less than 0.95 and a 3KGy dose of irradiation are sufficient for the inhibition or
destruction of E. coli (Vamam, 1991). In addition, the pathogen is unable to
grow at temperatures less than 7 °C or greater than 48°C. E. coli cannot

withstand temperatures at 55°C for 5 minutes or 60°C for 0.1 minute. There is

I]

no evidence that E. coli can survive after pasteurization or after proper cooking
has been achieved (Vamam, 1991).
Salmonella typhimurium

The genus Salmonella is a member of the Entembacteriaceae, and are
classified on the basis of being gram-negative, non-sporeforming, motile bacilli.
They are facultative anaerobes, consisting of three kinds of antigens (Ekperigin
and Nagaraja, 1998). There are more than 2200 serovars in the genus, of
which 50 to 150 have been implicated in disease outbreaks (Merritt, 1998).

From the late 1800’s to 1949, typhoid fever caused by Salmonella typhi
was the predominant strain to cause infections in humans (T auxe, 1991).
However as technology and antimicrobials advanced, typhoid fever was nearly
eliminated in many industrialized nations. At the same time the non-typhoid
(Salmonella typhimurium) strain began to become reported more frequently as a
leading cause of gastroenteritis (T auxe, 1991).

Salmonella typhimurium, known as Definitive Type 104 (S. typhimurium
DT104) can affect not only man, but also a wide range of warm-blooded animals
(Merritt, 1998). In 1888, it was first isolated from a man who had consumed a
moribund cow, however, the strain did not appear to become prevalent until the
middle of the 20'" century. (Tauxe, 1991). Today, it is the leading cause of
Salmonellosis in the United States (T auxe, 1991 and Vela, 1997).

S. typhimurium may be present in the gastrointestinal tract of poultry and
large meat animals that show no symptoms of sickness. Healthy animals may

carry the deadly pathogen in their feces, lymph nodes, spleen, liver, kidneys and

12

gall bladder (Merritt, 1998). In pork processing, Saide-Albonoz, et. al., (1995)
noted the stress of transportation and feed deprivation from farm to
slaughterhouse increases the likelihood for pathogen shedding via fecal
material, which ultimately passes S. typhimurium onto the slaughter floor and
throughout the packing plant. Ekperigin and Nagaraja, (1998) suggest that when
beef, pork or poultry are infected with S. typhimurium prior to slaughter their
carcasses test positive for the pathogen 0% to 90% of the time.

One of the factors that make S. typhimurium so unique is the ability to
withstand antibiotic treatments of Ampicilin, Chloramphenicol, Streptomycin,
Sulfonamides and Tetracyclines and more recently Trimethoprim and
Fluoroquinolones (Ekperigin and Nagaraja, 1998). In recent years, there has
been an observed association between antibiotic therapy and increased
susceptibility to infection by the antibiotic-resistant strain. In a clinical study,
patients who had been on antibiotic therapy, then exposed to S. typhimurium
were fivefold more likely to show symptoms of infection. It was hypothesized
that the antimicrobials increased host susceptibility by depleting the host’s
normal microflora and creating favorable conditions for S. typhimurium (Vamam,
1991)

Although S. typhimurium is resistant against several antimicrobial agents
that other pathogens are not, there are ways the pathogen can be eliminated.
Salmonella grows over .a range of temperatures from 5°C to 47°C, with an
optimum temperature of 37°C. Growth rates at below 10°C are very slow, but

are significant if the shelf life of the product is prolonged. Salmonella is

13

destroyed at temperatures of 74°C. Irradiation at 3KGy has proven to be
effective in controlling Salmonella in poultry, shellfish and frog legs. In addition,
it grows over a range of pH from 4.5 to 9.0, with an optimum pH of 6.5 to 7.5. At
pH levels lower than 4.1, inactivation and death will occur. Succinic acid and
chlorine have proven to be effective in eliminating Salmonella loads in chicken
carcasses (Vamam, 1991). Using multiple steps to eliminate the pathogen load,

S. typhimurium can be controlled.

Yersinia entercolitica

The genus Yersinia is composed of a collection of gram-negative,
facultatively anaerobic bacilli that share morphological, biochemical and
serological features with other genera in the Enterobacteriaceae family. There
are three species that are recognized as human pathogens: Y. pestis, Y.
pseudotuberculosis and Y. entercolitica (Karib et. al., 1995). Y. entercolitica are
psychrotrophic in nature, which presents a unique problem in the food industry.
They can multiply at temperatures from -2° to 45°C (Vamam, 1991).

Y. entercolitica was first recognized as a human pathogen in 1939;
however, its significance to foodbome illness was not realized until the mid
1970's (Kraft, 1992) Y. ent'ercolitica causes a variety of human infections. It is
the cause of 1-3% of the gastroenteritis cases reported annually. The
foodbome transmission of Y. entercolitica was established in 1976, when 222

people became sick after consuming contaminated chocolate milk. An outbreak

l4

of 87 cases occurred in 1981, when tofu was packed in untreated spring water,
which contained the same serotype as the infected patients (Kraft, 1992).

The incidence of Y. entercolitica in fresh meat is very high, yet few
foodbome outbreaks of yersiniosis have been reported due to the high number
(millions) of cells needed to cause disease (Smulders, 1987). The symptoms of
the infection include abdominal pain and diarrhea, which may vary from loose
stools to ulcerated lesions. When the bacteria invade the intestinal wall and
enter the blood system, secondary pathological effects such as arthritis or
meningitis can result (Frank, 1992).

Swine are the major reservoir for the pathogen, which results in human
infections. Although isolations of Yersinia have been made from poultry, the
importance of yersiniosis spreading in poultry is questionable (Kraft, 1992). The
epidemiology of yersiniosis is complex and has not been fully established.
Modes of transmission are contact with infected animals, person-to-person
transmission within infected families or consumption of contaminated foods
(Karib et. al., 1995).

Y. entercolitica will not survive heat treatments of 628°C for 0.7 to 17.0
seconds. Irradiation has proven to be effective in eliminating the pathogen load
at 0.7 to 1.2 KGy. In addition, it is considered to be sensitive to a low pH range.
However, the pH value alone has little effect on its growth and survival. Y.
entercolitica can be controlled by 0.1 to 0.2% sorbate at pH 5.5 (Vamam, 1991).

Listeria monocytogenes

15

The genus Listeria is composed of small motile, nonspore-forming,
facultative anaerobic, gram-positive rods. These organisms are catalase
positive and oxidase negative and ferment glucose (Cooper and Walker, 1998).
There are seven species of Listeria. L. monocytogenes is the only one
associated with human foodbome disease (Vela, 1997). It is most commonly
found in the soil, but can also be found in animal food and organs as well as on
the skin of infected animals (Frank, 1992).

L. monocytogenes is capable of growing at temperatures from 3° to 45°C.
It is resistant to freezing, thawing and drying. It can survive for years at a neutral
(7.0) pH. The organism resides in the intestinal tract of vertebrae and
invertebrate animals (Cooper and Walker, 1998). It is estimated that 5 to 10%
of the general population are carriers of L. monocytogenes (Notermans, et. al.,
1998).

L. monocytogenes is capable of existing as both a plant and animal
pathogen. It enters the body by penetrating the epithelial barrier in the intestine
and multiplies in the hepatic and splenic macrophages. It also enters through
damaged mucosal surfaces, by inhalation or conjuctival contamination, and then
invades the central nervous system via the neural sheath of peripheral nerve
endings (Cooper and Walker, 1998).

Even with the frequency of human exposure to L. monocytogenes,
research suggests that listeriosis is a relatively rare disease. Studies conducted
by the Center for Disease Control have established a incidence of listeriosis of

7.1 cases per one million people (Notermans, et. al, 1998). Historically,

l6

listeriosis is not contagious and only occurs in sporadic cases worldwide
(Cooper and Walker, 1998).

In 1929, it was first reported as a human pathogen, but had been known
as an animal pathogen since 1911 (Kraft, 1992). The first major outbreak of
listeriosis occurred in Boston MA, in 1979. Although the total number of cases
was not definitely established, 5 people died after consuming raw vegetables
that had been fertilized with fecal material from infected dairy cattle (Vela, 1997).
In December 1998, L. monocytogenes was responsible for the largest meat
recall ever. Thirty-five million pounds of ready to eat meat were recalled;
ultimately, 101 people were sickened and 21 people died (Young, 1999). The
people who are the most susceptible to listeriosis are the immunocompromised,
elderly and infants (Kraft, 1992). Some scientists suggest that the unique
characteristics of L. monocytogenes make it a problem of great concern, but
most healthy people are not at risk to listeriosis, thus outbreaks have not been
more widespread (Doyle, 1988).

There are three main clinical disease symptoms associated with L.
monocytogenes infections in human: (1) neural (circling disease), (2) visceral
(flu-like symptoms followed by neural or reproductive tract infections), and (3)
reproductive (causing abortions, stillbirths or pre-mature births). The onset of
serious complications depends upon the health of the person and the amount of
bacteria ingested. Penicillins such as ampicillin or amoxicillin in combination with
an aminoglycoside have been shown to be effective in the treatment of infected

peOpIe (Cooper and Walker, 1998).

I7

 

In 1983 it was thought that pasteurization was adequate for the
destruction of the pathogen, but when the milk was harvested from cows
infected with L. monocytogenes, pasteurization did not kill all of the deadly
bacteria (Vela, 1997). It was determined that the bacteria were being protected
by the bovine leukocytes in the milk. In view of this, pasteurization procedures
became scrutinized and thermal processing parameters for milk were
reconsidered. New requirements were established to modify the pasteurization
process to include thermal processing at 717°C for 15 s, resulting in a 5.2D
reduction to inactivate L. monocytogenes in milk. The safety margin is greater in
low-temperature short—time pasteurization (628°C for 30 min, resulting in a 39D
reduction) (Varnam, 1991). Previous research concluded that L.
monocytogenes growth is compromised at a pH of 5.6 or less; however,
scientists have found that a pH of 4.42 is considerably more limiting. As with
other pathogens, L. monocytogenes is vulnerable at irradiation doses of 3KGy
(Vamam, 1991).

IV. Pathogen Intervention Strategies
Overview

The muscle tissue of healthy animals is usually free from microorganisms
(Sammarco et al., 1997). It has been well documented that microbial
contamination of animal carcasses during the slaughter process is both
undesirable and yet unavoidable (Dickinson and Anderson, 1991, Podolak et al.,
1996, Sirgusa, 1996, Tamblyn and Conner, 1997). Advances in engineering

technology have allowed the meat industry to become modernized, streamlined,

18

and more efficient (Saucier, 1999). Although technology enhances productivity,
the delicate balance between efficiency and food safety cannot be
compromised.

Carcass decontamination refers to the ability to reduce pathogen loads
by sprays, washes, steam-vacuuming, hot-fat-trimming and quick-chilling, or a
combination of treatments. It is not possible to reduce the level of microbial
contamination on a carcass to zero using a single decontamination method
(Siragusa, 1996).
Antimicmbials

As part of the FSIS Pathogen Reduction/HACCP regulation, FSIS
recommended that all beef, pork, lamb and poultry slaughter establishments
apply at least one antimicrobial treatment prior to carcass chilling. Proposed
treatment recommendations included any antimicrobial compounds previously
approved by FSIS, as well as hot water and chlorine compounds (Federal
Register, July, 1996). There have been a wide variety of antimicrobials used to
reduce the pathogen load on meat and poultry carcasses. Short chain organic
acids have been identified as the most logical agents to spray on carcasses.
Acetic, lactic, citric, formic and propionic acids have all been used for this
purpose (Siragusa, 1996). Acetic and lactic acids are inexpensive,
environmentally friendly and occur naturally in nature. In general, organic acids
reduce aerobic plate counts by 1 to 2 log CF Ularea of tissue surface, regardless
of the acid type (Siragusa, 1996). The acid concentration appears to be an

important factor in the degree of pH decline and antimicrobial effect (Podolak et

19

al., 1996, Tamblyn & Conner, 1997, Dorsa et al, 1997, Cutter and Siragusa,
1994). However it is important to consider that high acid concentrations could
have adverse affects on meat quality (color and flavor).

Chlorine (Clz) water has also been used to decontaminate carcasses.
Most researchers agree that chlorinated water has little or no effect unless
sprayed frequently on the carcass over a long period of time (2- 4 hours) (Yang
et al., 1998, Skelly et al., 1985, Cutter and Siragusa 1995, Siragusa, 1996). It is
assumed that greater pathogen reductions did not occur because of the
inactivation of chlorine by the organic and nitrogenous compounds associated
with meat (Cutter and Dorsa, 1995).

Chlorine Dioxide (CIOz) is a chlorinated compound that has been widely
used throughout Europe for the disinfection of public water supplies (Latshaw,
1994). Lilliard (1979) was the first to use 0102 in the reduction of pathogens in
poultry chiller water. Results from his studies showed that 0102 was effective at
reducing pathogens at C102 of 20 parts per million (ppm). Other studies showed
similar differences when used as a soaking solution or brine chiller (Cutter and
Dorsa, 1995, Theissen et al., 1984, Villarreal et al., 1990). However when used
as a wash or spray, it was proven to be ineffective (Cutter and Dorsa, 1995).
Delivery Systems

A variety of antimicrobial delivery systems have been used to reduce the
levels of pathogens on meat surfaces (Cutter et. al., 1997). Inconsistencies in
the literature suggest the terms “acid sprays”, “acid washes” and “acid rinses”

are synonymous and are used interchangeably. These systems utilize

20

antimicrobial agents to spray the meat surface in order to reduce microbial loads
(Yang, et al., 1998, Dickens, 1995, Brackett, et al., 1994, Dorsa, et al., 1997a
1997b, Conner, et al.,1997, Woolthuis, et al., 1985, Prasai, et al., 1997). “Acid
spray” will be the term used throughout this literature review to signify the use of
these technologies.

Another way of applying acids and chlorine compounds to the meat
surface is to place the meat product in a container containing the antimicrobial
(Dickens, et al., 1994; El-Khatteib, et al., 1992; Fratamico, et al., 1996; Tamblyn,
et al., 1997; and Podolak, 1996) . “Acid dip”, “acid bath”, “acid rinse” are terms
used to describe this method. “Acid dip” will be the term used throughout this
literature review to signify the use of these technologies.

Treating the brine chiller water for poultry carcasses is another way of
utilizing antimicrobials agents. Organic acids, chlorine and chlorine dioxide
have all been added to poultry chilling systems to aid in the reduction of
pathogens (Tsai, et al., 1991; Lillard, 1979, 1980; Dickens and Whittemore,
1995; Rathgeber and Waldroup, 1995; \fillarreal, et al., 1990).

Acid Spray

Numerous studies have investigated spraying organic acids and chlorine
compounds on carcasses and meat products in order to reduce undesirable
bacteria (Cutter et. al., 1997). Organic acids and chorine compounds are
effective on those organisms that are in contact with the acid for a period of time
(> 60 s) that is sufficient to be lethal to that organism. However, the acid does

not affect organisms that have become entrapped or protected by the carcass

21

surface, because the acid is not able to penetrate the carcass surface (Cutter
and Siragusa, 1994). Dickens, (1995) found that spraying the carcass
preevisceration not only sanitized the carcass, but also reduced the stickiness of
the carcass. Wetting the carcass surface reduces the stickiness; therefore,
pathogens have less opportunity to adhere to the carcass. Marshal et al. (1977)
observed acid spray systems were most effective in reducing the bacterial load
when application of acid was at the highest pressure, and when the highest
volume of acid was applied for the longest period of time. Therefore, in order to
decontaminate a carcass, a combination of pressure, volume and concentration
must be used. The high-pressure spray will cause detachment of the bacteria
and the acid will then cause inactivation (Siragusa, 1996).

Woolthuis and Smolders (1995) used lactic acid (LAC) in a pilot model
washing system. They noted that LAC in concentrations of 2.0% discolored the
beef carcasses. Nonetheless, LAC was effective in reducing the aerobic plate
counts (APC) by 0.8 and 1.3 log") CFUlcmz. Cutter and Siragusa (1994) used
acetic acid (AAC), citric acid (CAC) and LAC at concentrations of 1, 3, and 5%
to inoculate beef carcass tissue with E. coli 0157:H7. The researchers observed
no pathogen reduction differences in the types of acids used; however, the 5%
concentrations were the most effective in reducing the pathogen load (1 to 2
IogIo CFU/cmz). It was noted that use of 5% acid concentration did reduce the
pathogen load to zero levels. Three years later, Cutter and Siragusa (1997)
performed another study analyzing the effectiveness of spraying LAC, AAC and

trisodium phosphate (TSP) acid on beef carcass tissue. The product was

22

vacuum packaged and stored at 5°C. Samples were removed from vacuum
packaging at 0, 7, 14, and 21 days for bacterial enumeration. The acids proved
to be effective in reducing the pathogen load by 1.3 to 2.0 IOQIo CFU/cmz. Yang
et al. (1998), observed a 1.7 to 2.0 IOQIo CFUlcm2 reduction in S. typhimurium
when poultry carcasses were sprayed with either LAC, TSP, cetylpyridinium
chloride (CPC) or sodium bisulfate (SBS). Although no reduction greater than
2.0 10910 CFU/cm2 were observed, other researchers found similar results
(Prasai et al., 1997, Conner et al., 1997).

Brackett and Doyle (1994) and Cutter et al. (1997) heated LAC AAC to
approximately 55°C and observed heat treatments had no significant
advantages over acids held at. room temperature. They noted that this method
was not effective for practical use.

Acid Dip

Acid dip is another method used by researchers to reduce the pathogen
load on meat products. Instead of spraying the product with nozzles, they dip
the product in antimicrobial agents. Dickens et al. (1994) observed less than 1
IOQIo CFUlcm2 reductions in the poultry carcasses dipped in 0.6% AAC.
Podolak, et al. (1996) reported using a 1% concentration of fumaric acid as a
bactericidal dip was effective in reducing the pathogen load of L.
monocytogenes and E. coli 0157:H7 by 1 and 1.3 logIo units respectively.
Tamblyn and Conner (1997) showed that the bactericidal activity of organic

acids increased linearly with increased acid concentrations. They noted that in

23

general, greater than 4% acid is need to kill greater than 2 IOQIo number of cells
of S. typhimurium attached to broiler skin.

Fratamico, et al. (1996) found that when TSP is used as a dip, TSP
reduced the levels of E. coli attached to the tissue up to 3.2 logIo units. These
researchers suggested that TSP could be effective for reducing E. coli on beef
carcass tissue. Although not all organic acids are effective in reducing the
pathogen levels, it is suggested that greater pathogen reduction is observed at
higher the acid concentrations.

Quick Chill

Quick chilling is used throughout the US. in pork and beef processing
plants. The carcasses come off of the kill floor and go straight into chilling
rooms at temperatures ranging from -1 to 2°C. The air velocity is 2 to 4
meters/second (Bem and Hechelmann, 1995).

The rate of chilling meat product has a significant effect on bacteria. The
leading factor in the cause of foodborne illness is improper holding temperatures
for food (ASHRAE, 1994).

The growth of bacteria takes place in four phases: latent phase, (lag
phase), log phase (exponential growth), stationary phase and organism
reduction phase (Bern andHechelmann, 1995). The lag phase is the period of
time when the organisms are adapting to the environment. In the food industry,
it is most ideal to lengthen the lag phase as long as possible, not allowing
organisms to enter the log phase. The log phase is when the organisms

experience the most growth. Once the organisms enter this phase, it is difficult

24

to control the growth without cooking or irradiation. At this point the organisms
can double every 20 minutes or less. Toxin production usually occurs at the end
of this phase. The stationary phase is the phase where the organisms begin to
run out of nutrients to continue to reproduce. Finally, the reduction phase is the
phase where organisms die off (ASHRAE, 1994).

Control of pathogens in the food industry has typically depended on
temperatures below 5°C (Palumbo, et al., 1995). Fung et al., (1981) found that
the initial chilling rate of beef carcasses is important in ensuring the
microbiological quality of hot-boned beef. Lee, et al. (1985) showed that hot-
boned beef chilled to 21°C in 6 hours had less pathogen growth than beef
chilled to 21°C in 11.3 h. The microflora at the time of packaging ultimately
affected the number of microflora isolated after storage. Proper chilling of meat
will significantly reduce pathogen propagation and increase the shelf life of meat

products (Smith, 1976).

Brine Chill

There are a variety of quick chilling methods that are implemented by the
poultry industry. Brine chilling or “continuous immersion chilling”, is the most
common method. Carcasses are submerged in water and tumbled by
mechanical agitation through several chill tanks. Ice is placed in the first tank;
thus, the ice and water flow continuously from one tank to another (Kraft, 1992).

Although it is a commonly used method, it has drawn criticism because of the

25

bacterial load sometimes associated with the chiller water. Lillard (1979) showed
that fecal coliform counts in the water were as high as 3.4 logIo CFU/ml.

Although the primary goal is to reduce the temperature of the poultry
carcasses, the secondary goal became to reduce the microbial load in the brine
chillers (Lillard, 1979). The use of chlorine was earlier suggested by Gorselin et
al. (1951) and chlorine and chlorine compounds are now used to control
spoilage bacteria in the brine chillers. Lillard was the first to try and generate
chlorine dioxide (0'02) on-site. Lillard hypothesized that because CIOz did not
react with nitrogenous compounds, including simple amino acids, it would be
effective in reducing the bacterial load in the brine chiller. Five ppm of CIOz was
proven to be as equally as effective and less corrosive than 34 ppm of Clz
(Lillard, 1979). Lillard (1980) published work that showed the ClOz and Clz
extended the shelflife on 95% of the poultry carcasses in excess of 20 days.

Villarreal et al. (1990) reported that 1% slow release 0102 ice-slurry in the
brine eliminated any recoverable Salmonella from turkey carcasses. Tsai et al.
(1992) found in laboratory tests that Cl; at 100 to 150 ppm would reduce the
bacterial load of the brine chiller water by 99% in 3 to 5 minutes. Increased
exposure resulted in increased reductions.

In less confounding. evidence, Dickens and Whitmore (1995) showed
AAC added to the brine water reduced the Salmonella incidence by 0.32 to 1.4
10910 CFU/cmz. Rathgeber and Waldroup (1995) used up to 1.5% Brifisol KTM

(a commercial blend of sodium acid pyrophosphate and orthophosphoric acid)

26

during the chilling of poultry carcasses in a brine chiller. The solution was
effective in reducing the bacterial load by approximately 1 logo.
Hurdle Technology

A combination of the previously mentioned prevention strategies in food
processing could result in the inhibition or destruction of microorganisms. VWth
this in mind, the implementation of these strategies may prevent negative effects
of processing the product as well as decrease the likelihood of foodbome
disease (Frank, 1992).

There are four basic methods used to inhibit or destroy microorganisms
(Leistner, 1978). Those methods are sterilization, freezing, chilling and
adjustment of pH and a... The hurdle effect is the process of controlling growth,
metabolic activity, resistance and survival of microorganisms in food. Each
hurdle represents a method used for controlling microbial growth (Frank, 1992).
In 1991 the idea of combining the hurdle effect with HACCP systems offered a
new dimension to food safety. The implementation of this technology has
become known as hurdle technology (Leistner, 1994).

Until 1998, few studies had attempted to determine the long-tenn viability
of bacterial populations found in ground beef when the beef carcasses were
subjected to hurdle technolOgy. Dorsa et al (1998) found that meat treated with
antimicrobial compounds in a hurdle technology system had residual efficacy

and resulted in lower or no detectable levels of pathogenic organisms 21 days

later.

27

Dickson and Anderson (1991) found that washing with distilled water
before evisceration and using 2% AAC to sanitize after washing decreased the
pH of the carcass surface and ultimately reduced the microbial population by 2
logm. Although not significant, slight reduction trends were observed when AAC
was heated to 55°C prior to washing.

The combined treatments of a water wash, trimming, hot water washes
and acid washes reduced the pathogen load on beef carcass tissue by 4.0 to
4.9 log CFUlcmz. Individual treatments alone observed less reduction than the
combined treatments (Castillo et al., 1998).

Castillo et al. (1999) studied the magnitude of microbial reduction when
inoculated carcasses were steam vacuumed or steam vacuumed combined with
sanitizing hot water or lactic acid sprays. The results showed that a combination
of the treatments were more effective in reducing the pathogen load. The
combination treatment reduced the pathogen load by 3 log").

V. Chlorine Dioxide

Overview

The bacteriacidal properties of chlorine dioxide (ClOz) have been known since
the early 1900’s (Wei, et al., 1985). The first commercial use of C102 as a
biocide was in the early 1940’s when the city of Niagara Falls added it to the
drinking water to control the odor and taste of the water (Latshaw, 1994).

Chlorine dioxide is soluble in water (Latshaw, 1994), it is a yellow-green
gas at low concentrations, and is similar in appearance and odor to chlorine

(C12). (Kim, 1997). Due to its highly explosive nature, ClOz cannot be

28

compressed and bottled, therefore it must be generated on site, (Latshaw, 1994,
Kim, 1997, Lillard, 1979).

Some of the attributes that make CIOz unique is its effectiveness in killing
microorganisms by interrupting their protein synthesis at pH levels from 4-10
(Latshaw, 1994). Unlike chlorine, CIOz does not react with ammonia or organic
nitrogen, therefore there is no harmful chloramine or trihalomehtane formation
(Kim, 1997). When CID; reacts with organics it is reduced to chlorite anions
(CIOz'): ClOz + e' —> CIOz'. In addition, CIOz has a greater oxidizing capacity
than Cl; (Kim et al., 1998).

The bactericidal effect of 0102 has proven to be seven times more
effective than CI; in poultry chiller water (Lillard, 1979). In addition, C102
maintains it bactericidal activity longer than Clz (Wei, et al., 1985). C102 kills
microorganisms that are resistant to 01;» treatment. Also, 0'02 has been
effective in killing microorganisms and extending shelflife for fresh produce, fish,
beef and poultry (Richardson, et al., 1998).

In Europe, chlorine dioxide is widely used as an alternative to chlorine for
drinking water disinfection (Kim, 1997). CI02 is used for treating drinking water
in more than 500 US. cities (Richardson, et al., 1998), including such cities as

Philadelphia, PA, Shreveport, LA, El Paso, TX, and Galveston, TX (Kim, 1997).

Chemical Mechanisms
Theoretically, C102 is a mixed anhydride of chlorus acid (HCIOz) and

chloric acid (H0103). It can be produced by the following reduction process
(Kim, 1997):

29

2 CI02 + H20 -+ HCIOz + HCIO3
Most commercial operations utilize this process of reduction to optimize CIOz
production. (Kim, 1997).

To date, little is known about the mechanisms CIOz has on
microorganisms. Bemarde et al. (1967) showed how 0'02 disrupts protein
synthesis, resulting in the inactivation of the bacteria. Roller et al., (1980)
suggested that C102 caused damage to the cell membrane of the bacteria
resulting in the inhibition of key enzymes that ultimately change the permeability
of the cell. More recently, Berg et al. (1986) noted cell membrane damage
resulting in massive leakage of intracellular macromolecules does not occur.
Berg et al., (1996) believed ClOz caused the efflux of potassium, caused the loss
of permeability control and was the primary cause of cell death.

Delivery Systems

C102 has been proven to be effective in the reduction of microorganisms
on a variety of foods (Richardson, et al., 1998). The effect of 0102 on the
organic matter and microorganisms in poultry chiller water has proven to be
successful. Lillard (1980) observed that ClOz was seven times more effective
than CI; in reducing the aerobic bacteria in the poultry chiller water. In addition,
Lillard (1980) found ClOz was less corrosive on processing equipment. Thiessen
et al., (1984), Tsai, et al., (1995) found CD; was effective in reducing the
microbial load in poultry chiller water. In 1996, FDA and the USDA approved its

use for poultry chiller water (T sai, et al., 1997).

30

Villarreal, et al., (1990) showed when chlorine dioxide was slowly
released as an additive to turkey rinse and chiller water, the incidence of
Salmonella contaminated carcasses was reduced an average of 70% after
evisceration to 25% after chilling.

Cutter and Dorsa (1995) observed that CIOz spray-treatments were no
more effective than water spray treatments in reducing the microbial load on
contaminated beef carcass tissue. It was suggested that 0102 could be more
effective if the meat was: (a) exposed to the CIOz spray-treatments for more
than one minute or (b) sprayed with ClOz intermittently during the 24 hour
chilling period.

Murano, et al., (1999) showed polyethylene film could be impregnated
with up to 1.8% CIOz. Meat was wrapped in the ClOz impregnated polyethylene
film. The film proved to be effective in reducing the microbial load on beef cuts
by 90%. Unfortunately, the C102 film had deleterious effects on the meat color,
causing it to turn from red to a dark green. This would ultimately result in
decreased consumer appeal.

VI. Economic Implications
Costs Associated with Food Poisoning

Although difficult to estimate total direct and indirect costs, foodbome
poisoning causes significant economic loss to all involved in the food processing
chain (Vamam, 1991). For the people directly affected by foodbome poisoning,

there are economic impacts associated with loss of earnings and job

31

productivity, as well as possible loss of human life or life-long side effects. In
addition, there are medical costs associated with treatments for recuperation.

USDA’s Economic Research Service (ERS) estimated the value of
lifetime medical costs and lost productivity costs to generate the estimated direct
annual costs of illness caused by pathogens. FSIS took this figure, and then
multiplied it by the percentage of illnesses attributed to meat and poultry. It was
estimated that foodborne illness in meat and poultry costs consumers $1.1 to
$4.1 billion annually (Roberts, et al., 1996).

Vamam (1991) suggests the company involved in the foodbome
poisoning will incur economic losses as well. Destruction of stock, loss of
production, loss of sales, and brand rehabilitation are some of the more obvious
losses. In addition, the company may have to retrain staff, perform an in-house
investigation, renovate facilities and possibly compensate victims.

The economic impact to the company varies from situation to situation.
In 1998, BiIMar Foods of Zeeland, MI encountered the largest meat (processed)
recall ever (35 million pounds), resulting in a direct economic loss of $76 million.
After the recall, sales dropped for the following six months, resulting in an
estimated $200 million economic loss (Young, 1999).

Costs Associated with Food Safety

The Food Safety Research Workgroup reports 21 federal agencies spend

$200 million per year on food safety research. State and industry officials match

those funds, resulting in a total of at least $400 million spent on food safety

32

research each year. This amount is less than 0.1% of the $647 billion spent on
food in the US. each year (Forsythe, 1996, 1997).

The ability to put a dollar amount on food safety is difficult, but can be
helpful in identifying critical control points that are both risk-effective and cost-
effective. (Morales, 1998). Food safety has been given a value by estimating
consumers' willingness to pay, behavior costs and costs incurred for medical
care and labor productivity (Roberts, et al., 1996).

The ERS estimates the cost of the federally mandated HACCP
regulations will cost the meat and poultry industry $1.0 to $1.2 billion per year
($0.18 per pound) over the next twenty years. However, it is anticipated that
HACCP implementation will reduce the prevalence of pathogens in meat and
poultry products, resulting in public health savings/benefit of $7.13 to $26.59

billion over the next twenty years. (Roberts, et al., 1996).

VII. Summam

Food safety is not a new problem. The establishment of HACCP is proving to
be effective from both a food safety and an economic perspective. Through
science, pathogen intervention strategies have been established in meat
production systems to effectively minimize the threat of foodbome disease.
Pathogen intervention strategies such as carcass washing/rinsing, steam
vacuuming, and rapid chilling are proving to be effective in reducing the
microbial load on fresh meat and poultry products. Scientists continue to search

for new antimicrobial compounds and intervention technologies that offer

33

maximum bactericidal effects. Realistically, more research is needed to
determine all possible pathogen intervention strategies to ensure the safety of
the food supply. The findings of this research address the efficacy of ClOz in the
form of ice, and its application as a new intervention strategy that may be
implemented in the food industry to prevent the proliferation of pathogens

commonly found in meat and meat products.

34

VIII. References

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35

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36

Dreesen, D.W. 1998. Hazard Analysis and Critical Control Point systems as a
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Tissues and Removal Using Selected Sanitizing Rinses. J. of Food Prot.
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Fung, D. K. C., C. L. Kastner, C. Y. Lee, M. C. Hunt, M. E. Dikeman, D. H.

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37

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41

CHAPTER 2
Materials and Methods

Development of Ice Machine

A Scottsman® ice machine (Fairfax, SC) was assembled in the meat science
laboratory at Michigan State University. An Aqua Pure (Meriden, CT) water filter
was used to filter the tap water used for the ice production. Two Valcor
Scientific (Springfield, NJ) pumps were attached to the ice machine to pump the
Oxine° and phosphoric acid (J.T. Baker, Pittsburgh, NJ) into the water reservoir
of the ice machine. Two ldec (Sunnyvale, CA) timers were used to regulate the
rate (time) and amount (volume) of Oxine® and phosphoric acid pumped. The
ice machine drained the contents of the water reservoir into the ice-mixing
chamber where ice was made.

Concentrations

Oxine° is 2% ClOz and 98% inert ingredients. It was not further diluted for this
study. Eighty-six percent phosphoric acid (PA) was diluted and then used for
the activation of marginally and largely activated CIOz ice. Activating the C102
generates “free” ClOz, which is the most likely to reduce the pathogen load. A
ratio of one part PA to two parts water was used in the activation of marginally
activated (5 ppm activated ClOz, 15 ppm inactivated CIOz) ClOz ice. The largely
activated (10 ppm activated ClOz, 10 ppm inactivated CIOz) CIOz ice was
generated using a one to one ratio (PAszO). When making either marginally or

largely activated 0102 ice, PA and C102 are pumped into the water reservoir

42

every 41 s. The ice machine drains the contents of the water reservoir into the
ice-mixing chamber where the ice is made. When making inactivated CIOz (20
ppm), only 0102 is pumped into the water reservoir. Control ice is made using
no ClOz or PA.
Titration

The Burette Titration Method was used for the determination of total
available chlorine dioxide (Bio-Cide lntemational, 1999). The reagent 0.6N
hydrochloric acid (HCI) solution was prepared by slowly adding 60 ml of 31.45%
hydrochloric acid to 1 l of deionized water. The 6% potassium iodide (Kl)
solution was made by adding 65 grams of Kl to 1 liter of deionized water, while
mixing on a magnetic stir plate. After the KI has dissolved in the water, 20 g of
sodium bicarbonate was added to adjust the pH of the solution to between 7.5
and 8.5. The reagents 0.00564N Phenylarsine Oxide (PAO) (Fischer,
Pittsburgh, PA) and 0.5% Starch Solution (SS) (Lab Chem Inc., Pittsburgh, PA)
were purchased.

The 6% Kl solution (25 ml) and 50 ml of the 0.6N HCI were mixed in a
125 ml Erlenmeyer flask on a magnetic stir plate. Ice (25 g) was added to the KI
and HCI mixture. After the ice completely melted in the mixture, it was titrated
with PAO until the mixture turned pale yellow. At this point, 1 ml of the SS was
added to the mixture, turning the color to dark blue. Slowly, PAO was added to
result in a clear end point. The milliliters of PAO used was recorded. The
following equation was used to determine total available C102:

(ml of PAO) X 76.083

 

= total ppm available ClOz (wt/wt)

43

sample weight in grams

Product Procurement

In the pork study, Boston Butts (BB) (406, NAMP Meat Buyers Guide) were
purchased from DeVries Meats Inc., Coopersville, MI. The BB were taken from
the right side of the pork carcasses 24 h after slaughter. The BB were split in
half (laterally) at the slaughter plant then kept together in a plastic bag. The BB
were placed in an insulated ice chest for transport to the meat microbiology lab
at MSU. Subsamples were excised from each BB, and bacterial enumeration
was conducted to determine the microbial quality prior to inoculation. In the
turkey study, birds were slaughtered at the MSU meat laboratory in the morning
then chilled to 4°C. Once properly chilled, the turkeys were fabricated. Leg-
thigh combinations were removed from the birds then transported in an ice chest
to the meat microbiology lab. Because of the surface area needed (350 cm2) to
collect adequate samples throughout the study, four leg-thigh combinations
were required for one sample. Subsamples were excised from the leg-thigh
combinations to determine initial baseline microflora.

Test Microorganisms

For inoculation of the BB, isolates of E. coli 0157:H7 (AB317), Salmonella
typhimurium (601074), Listeria monocytogenes (DUP1907-1 1038) and Yersinia
entercolitica (Michigan State University isolate) were used. E. coli 0157:H7,
Salmonella typhimurium. and Listeria monocytogenes were transferred from
stock cultures to 10 ml sterile tryptic soy broth (Difco, Detroit, MI). Yersinia

entercolitica was transferred into brain-heart infusion broth (Difco, Detroit, MI).

44

All pathogens were incubated at 35°C for 20 to 22 hours. For the turkey study
E. coli 0157:H7 (AB317), Salmonella typhimurium (G01074), Listeria
monocytogenes (DUP1907-1 1038) were used.

Inoculation of Test Microorganisms

Each subprimal (pork) or quad of subprimals (turkey) were placed in a sterile tin-
foil tray to prevent cross contamination and to minimize handling of inoculated
product. On each subprimal, 12 5 cm2 areas were inoculated with either
Escherichia coli 0157:H7, Salmonella typhimurium, Listeria monocytogenes or
Yersinia entercolitica. Immediately after inoculation, each 5 cm2 area was
marked with green food coloring to assist in the identification of the inoculated
area. During the 12 to14 h attachment time, the tinfoil trays were wrapped in
tinfoil then placed in a 2°C cooler .

Sampling Method

Using a sterilized scalpel, 5 cm x 5 cm x 2 mm subsamples were excised for
bacterial enumeration. A total of 14 subsamples were excised from the
subprimals (2 excised prior to inoculation, 4 excised after inoculation, 4 excised
after ice treatment and 4 excised after 7 d of 2°C storage).

Bacterial Enumeration

Subsamples were placedin a 400 ml Stomacher® bag (Seward Medical,

London, UK) and weighed. The subsamples were serially diluted using 1%
buffered peptone water (Difco, Detroit, MI). The subsamples were stomached
for 90 s in a 400 ml masticator (IUL Instruments, Cincinnati, OH). From the bag,

0.1 ml was extracted then serially diluted to 103, 10'5 and 10:7. The dilutions

45

were plated in duplicate for determination of most probable number. E. coli
0157:H7 was serially diluted and plated on MacConkey Sorbitol Agar (Difco
Laboratories, Detroit, MI) and 3M“ coliform and aerobic plates (3M, St. Paul,
MN). Salmonella typhimurium was serially diluted and plated on Bismuth Sulf'lte
Agar (Difco Laboratories, Detroit, MI) and 3MTM coliform and aerobic plates (3M,
St. Paul, MN). Yersinia entercolitica was plated on Yersinia Selective Agar
(Difco Laboratories, Detroit, MI) and 3M?" coliform and aerobic plates (3M, St.
Paul, MN). Listeria monocytogenes was plated on Oxford medium with
antimicrobic supplement added (Difco Laboratories, Detroit, MI) and 3MTM
aerobic plates (3M, St. Paul, MN).

Meat Storage Containers

Eight 96-quart ice chests were used to store meat and ice treatments in one of
two temperatures (2° or 26°C). A 120 cm x 44 cm x 4 mm sheet of expanded
steel was placed 9.5 cm from the bottom of the cooler to allow draining of the
melted ice. Each ice chest was partitioned into five sections, using 4 mm-thick
plexi-glass. To prevent cross contamination between samples, the plexi-glass
was stabilized by commercial sealing caulk.

Vacuum Packaging

After subsamples were excised following ice treatment, they were vacuum
packaged. They were packaged using 4-mil thick heat seal polyethylene bags
(Diagger, Lincolinshire, IL). The bags were sealed using an impulse heat sealer
(Diagger, Lincolinshire, IL). A sterile 20 gauge needle was attached to a 15

mm-diameter rubber hose that was attached to a Welch vacuum pump (Welch

46

Vaccum, Skokie, IL). The vacuum pump was used to extract the air from the
bag. After air extraction, the bag was sealed again to prevent airflow from the
needle insertion point.

pH Determination

The pH of the ice was measured with a Fischer Scientific pH meter (Pittsburgh,
PA). Ice (250 ml) was placed in a 300 ml beaker. The pH was measured by
inserting the pH probe into the middle of the ice filled beaker. In order to observe
pH decline over time, 22 readings were taken over a 4-hour period.

TBA Analysis

Thiobarbituric acid analysis was conducted to measure the effect of Ice
treatments on the rate of rancidity. Only samples that were not inoculated, but
exposed to ice treatments were analyzed. TBA analysis was conducted on
samples after ice treatments and after 7 d of storage according to the methods
established by Tarladgis et al., (1960) and Zipser et al., (1962)

Statistical Analysis

Statistical analysis was conducted using SAS, Version 7.0 (SAS Institute Inc.,
Cary, NC,) For all meat studies, the mixed procedure to analyze least square
means was used for each ice x temperature x day with all possible interactions.

All treatments in the study were replicated twice.

47

CHAPTER 3

Development and Evaluation of an Ice Machine that Generates Chlorine Dioxide
Ice at Various Levels of Activation

Abstract

Chlorine Dioxide (CIOz) ice was generated by injecting varying amounts
of Oxine® (Bio-Cide International) and phosphoric acid (PA) into the water
reservoir of a commercial ice machine. The ice machine accurately (i 3 ppm)
generated inactivated CIOz ice (20 ppm), marginally activated CIOz ice (5 ppm
activated CIOz, 15 ppm inactivated 0102), largely activated C102 Ice (10 ppm
activated C102, 10 ppm inactivated CIOz) and a control (no 0102). Burette
titration method was used for the determination of total available CIOz, When
titrated, there was a linear correlation (R2 = .99) between the amount of
Phenylarsine Oxide (PAO) needed to titrate and the amount of Clog present in
the ice. Over time, the pH of the ice declined from 4.8 to 2.6 as the CI02 and PA
was released from the ice crystal during melting and tended to stabilized after 4

houn

Introduction

Leistner (1978, 1994, 1995) has shown the effectiveness of hurdle
technology in food safety. Hurdles such as low pH and temperature inhibit
pathogen growth and possibly kill pathogens. The Federal Register (1996)
reported that as part of the FSIS Pathogen Reduction/HACCP regulation, all
beef, pork, lamb and poultry slaughter establishments should apply at least one

antimicrobial treatment prior to carcass chilling. Proposed treatment

48

recommendations included any antimicrobial compounds previously approved
by FSIS, as well as hot water and chlorine compounds

Chlorine dioxide (ClOz) is a chlorinated compound that has been widely
used throughout Europe for the disinfection of public water supplies (Latshaw,
1994). In 1979 Lilliard was the first to use C102 in the reduction of pathogens in
poultry chiller water. Results from his studies showed that CIOz at 5 ppm was
effective at reducing pathogens. Other studies showed similar differences when
used as a soaking solution or brine chiller (Cutter and Dorsa, 1995, Theissen et
al., 1984, Villarreal et al., 1990).

Phebus et. al., (1997) showed the most effective reduction of bacteria on
beef carcass tissue was observed when multiple hurdles in the pathogen
intervention strategy were introduced. When using a combination of steam
pasteurization, knife trimming, water washing, hot water/vacuum spot cleaning
and spraying with 2% vol/vol lactic acid, pathogens were reduced by 4.2 to 5.3
logIo CFU/cmz. When used individually, knife trimming, steam vacuuming or
steam pasteurization reduced pathogen load from 2.5 to 3.7 '0910 CFU/cmz.

The purpose of this research was to develop an ice machine that could
generate CIOz ice at various targeted concentrations that could be used as an
added pathogen interventiOn strategy to ultimately decrease the likelihood of
foodborne illness. The antimicrobial properties of the 0102 ice offer a new
dimension in pathogen intervention strategies for the reduction of harmful
bacteria. The bactericidal effect of CIOz in the ice could prevent the growth and

possibly kill pathogens on the meat surface. Bern and Hechelmann (1995)

49

suggested that the simplest application of refrigeration is the direct contact
between the substance being chilled and melting ice. Coupling the antimicrobial
effectiveness of Cl02 with the temperature reduction ability of ice could be an
effective pathogen intervention strategy. When using C102 ice prior to and
during product shipment, the pathogens on the product would be exposed to the
treated ice for an extended period of time. During treated ice application, the
C102 would be released from the ice and could inhibit pathogen survival. Also,
during ice application, the ClOz ice would lower the temperature and pH,
diminishing the ideal environment for pathogen survival and growth. When
produced, this ice could be proven beneficial as an antimicrobial control

mechanism in the pork and poultry industries.

Materials and Methods
Ice Machine and Pump Assembly

A Scottsman® ice machine (Fairfax, SC) was assembled in the meat
science laboratory at Michigan State University. An Aqua Pure (Meriden, CT)
water filter was used to filter the tap water that was used for the ice production.
Two Valcor Scientific (Springfield, NJ) pumps were attached to the ice machine
to pump the Oxine® and phosphoric acid (J.T Baker, Pittsburg, NJ) into the
water reservoir of the ice machine. The two separate pumps were used in order
to vary the concentration of each chemical. Two Idec® (Sunnyvale, CA) timers
were used to regulate the rate(time) and amount(volume) of Oxine® and

phosphoric acid pumped. The ice machine drained the contents of the water

50

reservoir into the ice-mixing chamber, where the ice was made. Figure 1.1
demonstrates the development of the chlorine dioxide generator that is used to
pump the Oxine and PA into the water reservoir. The set up of the ice machine

and pumps was relatively easy and took 10 hours.

Concentrations

Oxine® is 2% ClOz and 98% inert ingredients. It was not further diluted
for this study. 86% phosphoric acid (PA) was diluted and then used for the
activation of marginally and largely activated CIOz ice. Activating the CD;
generates “free” ClOz, which is the most effective in the reduction of the
pathogen load. A ratio of one part PA to two parts water was used in the
activation of marginally activated (5 ppm free CIOz, 15 ppm inactivated ClOz)
C102 ice. The largely activated (10 ppm free C102, 10 ppm inactivated CIOz)
CIOz ice was generated using a one to one ratio (PAzHZO). When making either
marginally or largely activated CIOz ice, PA and ClOz are pumped into the water
reservoir every 41 seconds. The ice machine drains the contents of the water
reservoir into the ice-mixing chamber where the ice is made. When making
inactivated CD; (20 ppm), only Oxine® (ClOz) is pumped into the water

reservoir. Control ice is made using no Oxine® or PA.
Titration

The burette titration method (Bio-Cide lntemational, 1999) was used for

the determination of total available chlorine dioxide. The reagents, 0.6N

51

 

hydrochloric acid (HCI) solution, and 6% potassium iodide (Kl) solution were
prepared. The 0.00564N Phenylarsine Oxide (PAO) (Fischer, Pittsburg, PA)
and 0.5% Starch Solution (SS) (Lab Chem Inc., Pittsburg, PA) were purchased.

Twenty-five ml of the 6% KI solution and 50 ml of the 0.6N HCI were
mixed in a 125 ml Erlenmeyer flask on a magnetic stir plate. Ice (25 g) was
added to the KI and HCI. After the ice melted in the mixture, it was titrated with
PAO until the mixture turned pale yellow. At this point, 1 ml of the SS was
added to the mixture, turning the color to dark blue. Slowly, PAO was added
until a clear end point was reached. The milliliters of PAO used were recorded.
The following equation was used to determine total available ClOzI

(ml of PAO) x 76.083

 

= Total ppm available 0102 (wt/wt)
sample weight in grams

To determine amount of activated C102 the following equation is used:

(ml PAO) X 320 = ppm of activated C102 (wt/wt)

 

sample weight in grams
Results and Discussion

The results of this study showed that chlorine dioxide ice could be
produced at various targeted concentration levels. The difficult part of
generating ClOz ice was determining the frequency and length of pump stroke,
which regulated the amount of Oxine® and PA that was injected into the water
reservoir. The first goal was to establish the most ideal length of pump stroke.
Through trial and error, using only half of the possible pump stroke length

proved to be ideal. It is important that the Oxine® and PA are pumped into the

52

water reservoir continuously to attain maximum consistency. It was found that a
pump stroke every 41 s was adequate to maintain the appropriate level of

Oxine® and PA in the water reservoir for consistent generation of targeted

concentrations of ClOz. The ClOz ice contains approximately 0.002 - 0.0025%
CIOz. The level of activation of CD; was controlled by the (PA:H20) ratio that
was injected. A ratio of one part PA to two parts water was used in the
activation of marginally activated CIOz ice. The largely activated ClOz ice was

generated using a (PAzHZO) ratio. Only Oxine® (C102) was used in the
generation of inactivated C102. For the generation of control ice, no Oxine®

(CIOZ) or PA were used (Figure 1.2). One of the drawbacks of the ice generator
was that only one ice treatment could be made at a time. In order to generate
different kinds of ice for this pathogen reduction study, 10 h was necessary for
manufacturing of each ice treatment to produce sufficient quantities of ice.
Figure 1.3 shows the linear correlation between PAO and total ppm of
0102, which predicts the accuracy that 0102 ice can be made. A R2 value of
0.99 indicates the ability to predict the ppm of CIOZ to be generated at a certain
volume (ml) of PAO. Increasing the mls of PAO used to titrate the dark blue
color that was activated by the starch indicator solution, indicated there was
more ppm of ClOz in the melted ice. One thing to be considered with each
titration is that there are slight differences in levels of total ppm of CIOZ, due to
differences in ice crystal shape and size. Therefore, the ability to acheive the

targeted ClOz ppm varied :t 3 ppm (Figure 1.2).

53

The pH decline (Figure 1.4) in the ice over time explains that as the ice
melts, the C102 and PA is released from the ice crystal, which causes a
significant (P > 0.05) drop in pH from 4.89 to 2.56. Ultimately, the pH returned to
2.97. Due to the fact that many pathogens cannot withstand a low pH or low
temperatures, applying this treated ice in a pathogen intervention strategy could
prove to be effective in the reduction of pathogens.

When an economic analysis was conducted after the initial fixed
(depending on size and kind of ice machine and C102 generator) cost of
approximately $4,000.00, the cost of producing the CIOz ice is approximately $
0.0017 per kg ($0.38 per 100 pounds) of ice. This antimicrobial ice delivery
system could possibly serve as a potential intervention strategy for poultry
slaughter facilities in brine chillers. Due to lack of refrigeration in some meat
transportation and holding facilities in third-world countries, the 0102 ice could
serve as both an antimicrobial and as a temperature control mechanism. The ice
also could be used in ice-packed meat transportation systems or in fresh poultry

and seafood retail case displays.

54

Figure 1.1 Chlorine Dioxide Ice generator

 

 

 

 

 

      
 

"16" plastic hose

Q

 

 

55

 

Figure 1.2 Ratio of Phosphoric Acid and Water used to generate targeted levels of activated or
inactivate CIO2 Ice

 

 

 

 

 

Ice Ratio Targeted ppm Actual ppm
Phosphoric Acid H20

Control 0 0 0 0

Inactivated 0 0 20 23

Marginally activated 1 2 20 20

Largely Activated 1 1 20 21

 

Figure 1.3 Linear correlation between PAO and total available CIO2

 

 

 

 

 

14
12 R2=0.9912
o 10-
g 3 ,_
“6 6 - :99ch
'5 4 -
2 4-
o
o 10 20 30 40

ppm of CI02

56

 

Flgure 1.4 Ice pH decline over 4-hour observation period

 

I

 

 

 

 

 

 

 

 

 

 

 

5.00
4.00

o

3

‘9 3.00 c

> b

I

D.
2.00 _
1-00.r-.......-fi..........

2 8 I4 20 26 35 50 240

 

Time (minutes)

i
l
I
l
i
g;

" °' ° Means with the same letter are not significantly different (P > 0.05)

57

 

References

Federal Register. 1996. Pathogen Reduction Hazard Analysis Critical Control
Point (HACCP) Systems Final Rule. Vol. 61 No. 144. National Technical
lnforrnation Service (NTIS) US. Department of Commerce.

Latshaw, C.L. 1994. Chlorine dioxide: effective, broad-spectrum biocide for
white-water systems. Tappi J. 78:163-166.

Leistner, L. 1978. Microbiology of Ready-to-serve Foods. Die
F Iieschwirtschaft. 12:2008-201 1.

Leistner, L. 1994. Further Developments in the Utilization of Hurdle Technology
for Food Preservation. J. of Food Eng. 22:421-432.

Lillard, HS. 1979. Levels of Chlorine Dioxide of Equivalent Bactericidal Effect
in Poultry Processing Water. J. of Food Science. 44:1594-1597.

Phebus, R.K., A.L. Nutsch, D.E. Schafer, R.C. Wilson, M.J. Riemann, J.D.
Leising, C.L. Kastner, J.R. Wolf, and R.K. Prasai. 1997. Comparison of Steam
Pasteurization and Other Methods for Reduction of Pathogens on Surfaces of
Freshly Slaughtered Beef. J. of Food Prot. 60:476-484.

Thiessen, G.P., W.R. Usborne, and H.L. Orr. 1984. The Efficacy of Chlorine
Dioxide in Controlling Salmonella Contamination and its Effect on Product
Quality of Chicken Broiler Carcasses. Poultry Science. 63:647-653.

Villarreal, M.E., R.C. Baker, and J.M. Regenstein. 1990. The Incidence of

Salmonella on Poultry Carcasses Following the Use of Slow Release Chlorine
Dioxide (Alcide). J. of Food Prot. 53:465-467.

58

CHAPTER 4

Influence of Chlorine Dioxide Ice on Pathogen Survival and Recovery on Chilled
Pork and Turkey Subprimals

Abstract

Chlorine dioxide (CIO2) solutions have been investigated as an
antimicrobial to reduce pathogens in meat washing/dipping systems and brine
chillers. This study investigated various concentrations of CIO2 solutions
incorporated as an antimicrobial ice treatment for pork and turkey subprimals.
An ice machine with a CIO2 generator produced ice containing 0 or 20 ppm
inactivated CIO2 and 5 or 10 ppm activated CIO2. Subprimals were inoculated
with Escherichia coli 0157:H7, Salmonella typhimurium, Listeria monocytogenes
and Yersinia entercolitica (pork) or Escherichia coli 0157:H7, Salmonella
typhimurium, and Listeria monocytogenes (turkey). Inoculated subprimals were
placed in partitioned ice chests with one of four ice treatments for 24 h at either
refrigerated (2°C) or room temperature (26°C). Subprimals were removed from
ice, vacuum packaged, and stored (2°C) for 7 days. For bacterial enumeration,
samples were excised from the subprimals prior to and after ice treatment and
during storage (2°C; 7 days). The main effect of CIO2 concentration did not
reduce (P>0.05) the microbial load of pathogens on subprimals. The interaction
of temperature during treated ice application (2° and 26°C) and day of storage
(day 1 and 7) did significantly reduce (P<0.05) the microbial load. Treated ice
applied to pork at 26°C reduced Listeria monocytogenes, and at 2°C, E. coli

0157:H7 and Salmonella typhimurium were reduced after 7 days of storage.

59

Neither temperature during ice application reduced Yersinia entercolitica. In
turkey, ice treatments applied at the environmental temperature of 26°C reduced
E. coli 0157:H7 and Salmonella typhimurium. This reduction may be attributed
to the release of more CIO2 during melting. The largely activated ice-melt in the
bottom of the cooler contained 2.5-3.0 IOQIoCFUImI fewer pathogens compared
to the control ice-melt.
Introduction

Chlorine is a disinfectant that has been used for the last 100 years, but
has drawn concern because of the toxic mutagens generated during the
reaction with organic matter (T sai et al., 1997). Another chlorinated compound
that Is currently used in more than 600 water treatment facilities in the US is
chlorine dioxide (CIO2) (Latshaw, 1994). Lillard (1979) found CIO2 to be seven
times more effective than chlorine and did not generate the toxic chloroform
when it reacted with organic matter. Several studies have reported the
effectiveness of CIO2 as an antimicrobial applied as a liquid (Cutter and Dorsa,
1995). Bem and Hechelmann (1995) suggested that the simplest application of
refrigeration is the direct contact between the substance being chilled and
melting ice. Coupling the bactericidal effectiveness of CIO2 and the
temperature reducing/stabilizing ability of ice would offer a pathogen intervention
strategy that has never been investigated.

The purpose of this study was to observe the effectiveness of CIO2
applied in an ice form on the survival of pathogens on fresh pork and turkey

subprimals. The hypothesis was that the bactericidal effect of the CIO2, the low

60

temperature and the low pH of the ice would result in multiple hurdles,
preventing pathogen survival and growth on the meat product.
Materials and Methods

A Scottsman® ice machine (Fairfax, SC) was assembled in the meat
science laboratory at Michigan State University. An Aqua Pure (Meriden, CT)
water filter was used to filter the chlorinated tap water that was used for the ice
production. Two Valcor Scientific (Springfield, NJ) pumps were attached to the
ice machine to pump Oxine® and phosphoric acid (J.T Baker, Pittsburg, NJ) into
the water reservoir of the ice machine. The ice machine accurately (within 3
ppm) generated inactivated CIO2 ice (20 ppm), marginally activated CIO2 ice (5
ppm free CIO2, 15 ppm inactivated CIO2), largely activated CIO2 ice (10 ppm
free CIO2, 10 ppm inactivated CIO2) or control (no CIO2). Burette titration method
(Bio-Cide International, 1999) was used for the determination of total available
CIO2,

Fresh pork and turkey subprimals were used in the model ice application.
Twelve 25 cm2 areas were inoculated on each subprimal with approximately 5
logto of Escherichia coli 0157:H7, Salmonella typhimurium, Listeria
monocytOgenes and Yersinia entercolitica (pork, Study I) or Escherichia coli
0157:H7, Salmonella typhimurium, and Listeria monocytogenes (turkey, Study
ll). Each subprimal was inoculated with a different pathogen. To allow
attachment, inoculated subprimals were stored at 2°C for 12 to 14 h prior to ice

treatment. Samples were excised after inoculation and prior to ice treatments

61

for baseline bacterial enumeration. Methods described by the USDA’s
Bacteriological Analytical Manual (BAM, 1995) were used.

Eight Commercial sized (96-quart) ice chests were partitioned into five
(Study l) or four (Study II) sections with 4 mm-thick sheets of plexiglass.
Expanded steel was placed 9.5 cm from the bottom of the ice chest to allow the
melted ice to drain off of the product. Each ice chest was assigned one of four
ice treatments and an environmental temperature of either 2°C (refrigerated) or
26°C (room temperature). In every ice chest, 1 Kg of one of the ice treatments
was placed in each partitioned section. Next, inoculated subprimals were
placed on top of the ice, then an additional 2 Kg of ice was placed on top of the
subprimal (3 Kg ice total).

After 24 h of ice application, the subprimals were removed and
subsamples were excised for bacterial enumeration and TBA analysis. To test
the effect of ice treatments on shelflife, the subprimals were vacuum packaged
and stored in refrigeration (2°C) for 7 days. Subsamples were then removed
and bacterial enumeration and TBA analysis (Tarladgis, G. G. et. al., 1960 and
Zipser, M. W. et. al., 1962) were conducted.

Results and Discussion

The ice was successfully generated at targeted ppm of CIO2 for each ice
treatment, In both studies, the main effect of CIO2 concentration did not
significantly reduce (P>0.05) the microbial load of pathogens on either pork or
turkey subprimals (Tables 1 and 2). This could be due to a number of reasons.
If the pathOQens were exposed to CIO2 for a longer period of time, it could have

had more of a detrimental effect on the pathogen load. Cutter and Dorsa, (1995)

62

suggested the longer the exposure time (> 60 seconds), a higher pathogen
reduction would occur. Also, if the total ppm of activated CIO2 was increased,
the CIO2 ice could prove to have a greater antimicrobials effect.

The interaction of temperature during treated ice application (2° and
26°C) and day of storage (day 1 and 7) did significantly reduce (P<0.05) the
microbial load. The increase in the room temperature (26°C) environment could
have caused more ice melt, resulting in an increase of CIO2 being released from
the ice as well as a decrease in the pH of the ice melt. When using similar
concentrations of CIO2, Lillard (1980) found greater reductions of total aerobic
bacteria on chicken carcasses (1.2 logto CFUIg) when chilled in CIO2 treated
versus untreated poultry chiller water. No significant difference was observed
between 34 ppm of CI2 and 5ppm of CIO2 treatments on the poultry carcasses or
the chiller water in that study. In all observations, the chicken breast skin had a
higher total aerobic plate count than the chiller water they were in. In correlation
with Lillard’s findings, in this study, the pathogen load was higher on the meat
product than in the ice melt.

In this study, E. coli 0157:H7 and Salmonella typhimurium on pork
subprimals were slightly reduced after 7 days of storage when the ice treatment
was applied at an envirOnmental temperature of 2°C (Figures 1 and 2
respectively). Although both pathogens are facultative anaerobes, the vacuum
packaged storage at 2°C could have caused the reduction. In addition,
maintaining the environmental temperature at 2°C immediately after inoculation,

during ice application and through the 7 day storage period, could have

63

prevented the proliferation of pathogens. In past research, Lillard et al., (1980),
showed that CI2 and CIO2 had a residual effect which extended the shelf-life of a
product by more than 20 days.

In both the pork and turkey studies, treated ice applied to subprimals at
the environmental temperature of 26°C reduced Listeria monocytogenes,
(Figures 3 and 7). Considering the fact that L. monocytogenes is
psychrotrophic, the increased temperature (26°C) during ice application could
have contributed to this observed reduction in numbers.

Neither temperature during ice applications reduced Yersinia entercolitica
(Figure 4). Y. entercolitica was tested in this study because of its prevalence in
the pork industry. The ineffectiveness of CIO2 on Y. entercolitica is not well
documented. It could be postulated that Y. entercolitica contains a cellular-
barrier mechanism that inhibits the antibacterial effectiveness of CIO2,

In turkey, ice treatments applied at the environmental temperature of
26°C reduced E. coli 0157:H7 and Salmonella typhimurium (Figures 5 and 6).
This reduction may be attributed to the release of more CIO2 due to increased
ice melt during exposure at 26°C. Another theory suggested by Wei et al.,
(1985) was that the bactericidal activity of CIO2 decreases as application
temperatures decreases, thus, when the CIO2 is at room temperature it is more
effective.

To observe the viability of pathogens that could have been rinsed off the
product during ice melt, samples were taken from the bottom of the ice chests.

The largely activated ice-melt in the bottom of the ice chests contained a mean

average of 1.8 log1o CFU/ml fewer (P<0.05) microorganisms compared to the
control ice-melt (Figure 8). In a follow-up study, the largely activated ice-melt
contained 3.6 Iogto CFU/ml fewer (P<0.05) total aerobic counts than the control
ice. The ice melt in the bottom of the cooler contained up to 100% higher ppm
than the original ice melt. Biocide lntemational engineers (Khanna, 2000)
speculated that the ions in the ice crystal leach out the CIO2. The CIO2 then
becomes heterogeneous and localizes in the bottom of the ice chest, which
causes a higher reduction of pathogens. In additon, during ice melt, the pH
declines and could aid in the reduction of pathogenic orgaisms.

Using 5 ppm of CIO2, Lillard (1979) found less of a reduction in the
poultry chiller water than what was found in the 20 ppm CIO2 ice melt in this
study. Although exact ppm of CIO2 were not reported, Thiessen et al., (1983)
observed Salmonella and coliforrns could be reduced in the chiller water from
3.14 '0910 to 0 CFUs when CIO2 was added at 1.39 mg of CIO2/liter of water.
However, on the poultry carcasses, coliforrns were only slightly reduced (<0.5
logto). When using CI2, Tsai, et al., (1991) showed that 100 to 150 ppm was
needed to reduce poultry chiller water by 2 IOQIo.

On beef carcass tissue, Cutter and Dorsa, (1995) showed that bacterial
populations were not reduced by more than 0.93 log CFU/cmz, regardless of the
liquid CIO2 concentration. In addition, they did observe CIO2 length of application
period (15, 30 or 60 seconds) did not affect amount of reduction. They

concluded that CIO2 was no more effective than water when used as a spray.

65

Alter data analysis in this study, it was hypothesized that the extended
time (12 to 14 hour) that was allowed for the pathogens to attach to the meat
surface was to long. An attachment study was conducted to determine if a
shorter attachment time would increase the effectiveness of the ice treatments.
Poultry subprimals were inoculated with E. coli 0157:H7, and then ice was
applied at either 2, 4, 6, 8, 10 or 12 hours after inoculation. Figure 9 shows
there were significant differences (P<0.05) between attachment times. However,
it is important to note that the maximum differences in pathogen reduction over
different time periods were no more than 0.20 IOQ1OCFU/Cm2. It can be
postulated that the attachment time in the initial study had little to no affect on
the effectiveness of the ice treatments.

In addition to studying the effect of CIO2 ice on the survival of pathogenic
organisms, the effect of CIO2 on the product shelf life was observed as well. A
thiobarbituric acid (TBA) test was conducted to determine if the application of
CIO2 ice treatments had an affect on the shelf life of the pork and turkey
subprimals. In both the pork and turkey study (T able 3 ) there was a significant
main effect (P<0.05) of day. In the pork study, none of the samples contained
more than 0.33 mg of malonaldehydel1000 9 sample. In the turkey study,
greater lipid oxidation was observed, especially on day 7. This increase was
likely due to the higher level of polyunsaturated fatty acids in the turkey skin. In
the both the pork and turkey studies, no significant differences were observed
between CIO2 ice treatments or ice application at either environmental

temperature (2°C or 26°C).

66

Conclusions

A model CIO2 ice application protocol was developed to simulate ice-
packed meat transportation systems or meat retail case displays. On fresh pork
and turkey subprimals, the interaction of temperature during treated ice
application (2° and 26°C) and day of storage (day 1 and 7) significantly reduced
(P<0.05) the microbial load on E. coli 0157:H7, S. typhimurium and Listeria
monocytogenes. Y. entercolitica was not reduced. In this study, relatively low
concentrations of CIO2 were used to prevent the leaching of the chlorine odor
associated with chlorine compounds onto the meat product and to prevent
discoloration of the meat product. It is also important to note that the ice
treatments used had no deleterious effects on the shelf life of the subprimals.
Increasing the ppm of CIO2 in the ice would likely enhance the effectiveness of
the ice in a pathogen reduction system. Furthermore, increasing the ice melt by
using warm (body temperature immediately after slaughter) meat will increase
the CIO2 released from the ice, which will offer more of an antimicrobial effect.
The CIO2 in the ice melt in the bottom of the ice chest was more localized and
thus was more effective at reducing the pathogens in the melted ice water. In
addition, the pH of the ice declines as it melts, potentially decreasing pathogen
numbers. Due to lack of refrigeration in some meat transportation and holding
facilities in developing and third-world countries, the CIO2 ice could serve as
both an antimicrobial and as a temperature control mechanism. This CIO2 ice
could also be used in ice-packed meat transportation systems or in a meat retail

case.

67

Table 1 Survival of pathogens on pork subprimals submerged in various ice treatments for 24
hours at refrigerated (2°C) or room (26°C) temperature, vacuum packaged and stored for 1 and
f 7 days at 2°C.

 

 

 

 

Plates Baseline 2°c 26°C SEM°
Day Day
Eschericia coli 0157:H7 1 7 1 7
APC' 4.76“ 4.50“ 4.31“ 3.93“ . 4.30“ 0.34
Coliformz 4.61“ 3.97“ 4.20“ 3.94“ 3.92“ 0.38
MaConkeys° 4.56“ 4.01“ 3.94“ 3.99“ 4.13“ 0.35
Salmonella typhinnirium
APC' 5.01“ 4.96“ 4.40“ 4.71“ 4.80“ 0.15
Coliformz 4.62“ 4.77“ 3.92“ 4.36“ 4.56“ 0.21
Bismuth3 4.80“ 4.65“ 4.13“ 4.30“ 4.51“ 0.20
Listeria monocytogenes
APC‘ 3.87“ 4.13“ 4.28“ 3.79“ 4.25“ 0.27
Oxford° 3.15“ 3.07“ 3.05“ 3.21“ 2.74“ 0.42
Yersinia entercolitica
APC‘ 5.39“ 5.41“ 6.87“ 5.34“ 6.90“ 0.23
Conform“ 5.46“ 5.37“ 6.73“ 5.18“ 6.67“ 0.23
Yersinia“ 5.45“ 5.40“ 6.76“ 5.24“ 6.74“ 0.22

 

 

 

" 3M® Aerobic Plates Count petri film

“ 3M® Coliform perti film

3 Selective Agar specific to the pathogen

" ° Means having the same superscript within columns are not significantly different (P>0.05)
° Standard Error of the Mean

68

Table 2 Survival of pathogens on turkey subprimals submerged in various ice treatments for 24
hours at refrigerated (2°C) or room (26°C) temperature, vacuum packaged and stored for 1 and

7 days at 2°C.

 

 

 

 

 

Plates Baseline 2°C 26°C SEMd
Day Day
Eschericia coli 0157:H7 1 7 1 7
APC' 4.07“ 4.94“ 3.86“ 5.40“ 3.55“ 0.39
Coliformz 4.30“ 4.81“ 3.38“ 5.16“ 2.75““ 0.45
Masonkeys“ 3.70“ 4.98“ 3.56“ 5.72“ 2.91“ 0.52
Salmonella typhinnirium
APC' 4.97“ 5.82“ 4.74“ 6.35“ 4.23“ 0.27
Coliformz 4.90“ 5.50“ 4.58“ 6.19“ 4.48“ 0.30
Bismuth?“ 4.29“ 5.80“ 4.70“ 5.43“ 3.95“ 0.44
'steria monocytogenes
APC‘ 4.46“ 5.29“ 4.90““ 5.38“ 4.89““ 0.15
Oxforda 3.54“ 3.95“ 4.17“ 4.17“ 3.76“ 0.60

 

 

' 3M® Aerobic Plates Count petri film
2 3M® Coliforrn perti film
3 Selective Agar specific to the pathogen

" °' ° Means having the same superscript within columns are not significantly different (P>0.05)
° Standard Error of the Mean

69

Table 3 Milligrams of malonaldehydel1000 9 sample in pork and turkey subprimals submerged
in various ice treatments for 24 hours at refrigerated (2°C) or room (26°C) temperature, at day 1
and day 7 (vacuum packaged storage at 2°C).

 

 

 

 

Study Day SEM°

1 7
Pork 0.231“ 0.283“ 0.02
Turkey 0.685“ 2.096“ 0.20

 

 

"° Means having the same superscript within columns are not significantly different (P> 0.05)
° Standard Error of the Mean

70

Flgure 1. Two-way interaction (P<0.05) of the growth of E. coli 0157:H7 on pork sub-primal
submerged in various ice treatments for 24 hours at refrigerated (2°C) or room (26°C)
temperature, vacuum packaged and stored for 1 and 7 days at 2 C.

I
4.8 .

4.6 .

 

_ A ._ Refrigeration
+ Room Temp

 

 

 

 

 

 

Baseline Day 1 Day 7
Microbial Analysis

 

 

 

 

Figure 2. Two-way interaction (P<0.05) of the growth of Salmonella typhimurium on pork sub-
primal submerged in various ice treatments for 24 hours at refrigerated (2°C) or room (26°C)

temperature, vacuum packaged and stored for 1 and 7 days at 2°C.

 

 

._ A _ Refrigeration
+ Room Temp

 

 

 

 

 

Baseline Day 1 Day 7
Microbial Analysis

 

 

 

 

71

Figure 3. Two-way interaction (P<0.05) of the growth of Listeria monocytogenes on pork sub-
primal submerged in various ice treatments for 24 hours at refrigerated (2°C) or room (26°C)
temperature, vacuum packaged and stored for 1 and 7 days at 2°C.

l
I 3:3 1
I 3.1

 

 

... -A _ Refrigeration
+ Room Temp

 

 

 

Log CFUIg
N
on (O r»

 

Baseline Day 1 Day 7
Microbial Analysts

 

 

Figure 4. Two-way interaction (P<0.05) of the growth of Yersinia entercolitica on pork sub-primal
submerged in various ice treatments for 24 hours at refrigerated (2°C) or room (26°C)
temperature, vacuum packaged and stored for 1 and 7 days at 2°C.

.— —____....."/.

 

l

l

 

 

1

_ ¢ .. Refrigeration
+ Room Temp

 

J

 

L J

LogCFUIg
OANwémONm

 

Baseline Day 1 Day 7
Microbial Analysis

 

 

 

72

Figure 5. Two-way interaction (P<0.05) of the growth of E. coli 0157:H7 on turkey sub-primal
submerged in various ice treatments for 24 hours at refrigerated (2°C) or room (26°C)
temperature, vacuum packaged and stored for 1 and 7 days at 2 C.

 

 

 

7

6
9 5
a 4 _ A _. Refrigeration
‘3, 3 +Room Temp
° 2
J .

1 I

0 I I 1

Baseline Day 1 Day 7
Microbial Analysis

 

 

 

Figure 6. Two-way interaction (P<0.05) of the growth of Salmonella entercolitica on turkey sub-
primal submerged in various ice treatments for 24 hours at refrigerated (2°C) or room (26°C)
temperature, vacuum packaged and stored for 1 and 7 days at 2°C.

 

I 7 .

6 .5 ‘
g i 1 ”’ \~“-A _—————. .
ll. 1 I— i — Refrigeration
o 3 I +Room Temp
§ 2 I

1 I

0 L7 .LL,,L L , . L LLL L

Baseline Day 1 Day 7
Microbial Analysis

 

 

 

73

Figure 7. Two-way interaction of the growth of Listeria monocytogenes on turkey sub-primal
submerged in various ice treatments for 24 hours at refrigerated (2°C) or room (26°C)
temperature, vacuum packaged and stored for 1 and 7 days at 2 C.

4.4 i I
4.2 a
4 4
3.8 a
3.6 a
3.4 L
3.2 . , , L
I Baseline Day 1 Day 7
' Microbial Analysis

 

_ A _ Refrigeration
+ Room Temp

 

 

 

Log CFUIg

 

 

 

 

 

 

 

Figure 8. Effect of chlorine dioxide ice-melt on pathogen survival in the bottom of the
ice chests.

 

 

 

 

 

 

 

 

 

g

5?

3 I Control

g I Inactivated

E El Marginally Activated
to” I Largely Activated

.i

 

 

Total Plate Counts

 

 

 

" °' ° ° Means having the same superscript are not significantly different (P>0.05)

74

Figure 9 Differences in the log", CFUlcm2 of E. coli 0157:H7 that were recovered after ice
application 2, 4, 6, 8, 10 and 12 hours after inoculation

 

 

5.5.
a ab a

Q)

 

5.

4.5.r-I — — — —I —
4.4‘ '—'I — — —

3.5.2— — — — —

 

Log CFUIcm2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2hr ' 4hr ' 6hr ' 8hr '10nr'12hr'
Time

 

 

 

"’ Means having the same superscript are not significantly different (P>0.05)

75

References

Bacteriological Analytical Manual. 1995. FDA

Bern, Z, and H. Hechelmann. 1995. Chilling and refrigerated storage of meat.
Fleischwirtsch. 2:25-33.

Cutter, ON. and W.J. Dorsa. 1995. Chlorine Dioxide Spray Washes for
Reducing Fecal Contamination on Beef. J. of Food Prot. 58:1294-1296.

Latshaw, C.L. 1994. Chlorine dioxide: effective, broad-spectrum biocide for
white-water systems. Tappi J. 78:163-166.

Lillard, HS. 1979. Levels of Chlorine Dioxide of Equivalent Bactericidal Effect
in Poultry Processing Water. J. of Food Science. 44:1594-1597.

Lillard, HS. 1980. Effect on Broiler Carcasses and Water of Treating Chiller
Water with Chlorine or Chlorine Dioxide. Poultry Science. 59:1761-1766.

Tarladgis, G. G> Watts, B. M. Youthathan, M. T., and Dugan, L. Jr., J. Am. Oil
Chemists, 37:44-48 (1960).

Thiessen, G.P., W.R. Usborne, and H.L. Orr. 1984. The Efficacy of Chlorine
Dioxide in Controlling Salmonella Contaminaton and its Effect on Product
Quality of Chicken Broiler Carcasses. Poultry Science. 632647-653.

Tsai, L-S., J.E. Schade, and B.T. Molyneux. 1991. Chlorination of Poultry

Chiller Water: Chlorine Demand and Disinfection Efficiency. Poultry Science.
71 :188-196.

Tsai, L-S., R. Wilson, and V. Randall. 1997. Mutagenicity of Poultry Chiller
Water Treated with either Chlorine Dioxide or Chlorine. J. Agric. Food Chem.
45:2267—2272.

Wei, C-l., D.L. Cook, and JR. Kirk. 1985. Use of Chlorine Compounds in the
Food Industry. Food Tech. ~ January:107-1 15.

76

APPENDICES

77

APPENDIX

Procedures and Protocols

1. Development of Ice Machine ........................................................ 79
2. Chlorine Dioxide Concentrations ................................................... 80
3. Titration of Chlorine Dioxide ......................................................... 81
4. Product Procurement .................................................................. 83
5. Test Microorganisms .................................................................. 84
6. Inoculation ................................................................................ 85
7. Sampling Method ....................................................................... 86
8. Bacterial Enumeration ................................................................. 87
9. Meat Storage Containers ............................................................. 88
10.Vacuum Packaging ..................................................................... 89
11.Determination of pH .................................................................... 90
12. TBA Analysis ............................................................................. 91
13. Structure and Reaction of Chlorine Dioxide ...................................... 94

78

APPENDIX 1: Development of Chlorine Dioxide Generator

 

1.

A Scottsman® ice machine (Fairfax, SC) was assembled in the meat
science laboratory at Michigan State University. An Aqua Pure (Meriden,
CT) water filter was used to filter the tap water used for the ice production.

Two Valcor Scientific (Springfield, NJ) pumps were attached to the ice
machine to pump the Oxine® and phosphoric acid (J.T. Baker, Pittsburgh,
NJ) into the water reservoir of the ice machine.

Two ldec (Sunnyvale, CA) timers were used to regulate the rate (time) and
amount (volume) of Oxine® and phosphoric acid pumped. The ice

machine drained the contents of the water reservoir into the ice-mixing
chamber where ice was made.

79

APPENDIX 2: Concentrations of Chlorine Dioxide

 

1.

Oxine® (Bio-Cide International, Norman, OK) is 2% CIO2 and 98% inert
ingredients. It was not further diluted for this study.

Phosphoric acid (PA) (86%) was diluted and then used for the activation of
marginally and largely activated CIO2 ice.

A ratio of one part PA to two parts water was used in the activation of
marginally activated CIO2 ice.

Oxine® and PA were each in 500 ml Erlenmeyer flasks attached to the
side of the ice machine.

Using the Chlorine Dioxide Generator, the Oxine® and PA were pumped
into the water reservoir, which was then drained into the ice-mixing
chamber where the ice was made.

When making inactivated or marginally/largely activated CIO2 ice, PA and
Oxine® are pumped into the water reservoir every 41 seconds, using only
half of a pump stroke each time.

The largely activated CIO2 ice was generated using a PA ratio of one to
one.

When making inactivated CIO2 ice only Oxine® was pumped in the water
reservoir. If PA were added, it would cause the CIO2 to become activated.

Control ice is made using no CIO2 or PA.

80

APPENDIX 3: Burette Titration Method for Determination of Total

Available Chlorine Dioxide

 

9.

10.

The Burette Titration Method was used for the determination of total
available chlorine dioxide (Bio-Cide lntemational, 1999).

The reagent 0.6N hydrochloric acid (HCI) solution was prepared by slowly
adding 60 ml of 31.45% hydrochloric acid to 1 liter of deionized water.

The 6% potassium iodide (KI) solution were made by adding 65 grams of
Kl to 1 liter of deionized water, while mixing on a magnetic stir plate. After
the KI has dissolved in the water, 20 grams of sodium bicarbonate was
added to adjust the pH of the solution to between 7.5 and 8.5.

The reagents 0.00564N Phenylarsine Oxide (PAO) (Fischer, Pittsburgh,

PA) and 0.5% Starch Solution (SS) (Lab Chem Inc., Pittsburgh, PA) were
purchased.

The 6% KI solution (25 ml) and 50 ml of the 0.6N HCI were mixed in a 125
ml Erlenmeyer flask on a magnetic stir plate.

Ice (25 g) was added to the KI and HCI mixture.

After the ice completely melted in the mixture, it was titrated with PAO until
the mixture turned pale yellow.

At this point, 1 ml of the SS was added to the mixture, turning the color to
dark blue.

Slowly, PAO was added to result in a clear end point.

The milliliters of PAO used was recorded.

The following equation was used to determine total available CIO2:
(ml of PAO) X 76.083

= total ppm available CIO2 (wt/wt)

 

sample weight in grams

The following equation was used to determine activated CIO2:

(ml of PAO) X 320 ' = activated 0102 (wt/wt)

sample weight in grams

81

Theory behind calculation:

mlxNxex10°

 

= ppm available CIOZ (wt/wt)
sw

Where:

ml = PAO volume in millimeters

N = normality of PAO (0.00564N)

e = Milliequvalent weight of chlorine dioxide (0.01349)
sw = sample weght

10° = factor to convert milligrams/ml to ppm

82

APPENDIX 4: Product Procurement

 

In the pork study, Boston Butts (BB) (406, NAMP Meat Buyers Guide)
were purchased from DeVries Meats lnc., (Coopersville, MI).

The BB were taken from the right side of the pork carcasses 24 hours
after slaughter.

The BB were split in half (laterally) at the slaughter plant then kept
together in a plastic bag.

The BB were placed in an insulated ice chest for transport to the meat
microbiology lab at MSU.

Subsamples were excised from each BB and bacterial enumeration was
conducted to determine the microbial quality prior to inoculation.

In the turkey study, birds were slaughtered at the MSU meat laboratory in
the morning then chilled to 4°C.

Once properly chilled, the turkeys were fabricated. Leg-thigh
combinations were removed from the birds then transported in an ice
chest to the meat microbiology lab.

Because of the surface area needed (350 cm2) to collect adequate
samples throughout the study, four leg-thigh combinations were required
for one sample.

Subsamples were excised from the leg-thigh combinations to determine
initial baseline microflora.

83

APPENDIX 5: Test Microorganisms

 

1. For inoculation of the BB, isolates of E. coli 0157:H7 (AB317), Salmonella
typhimurium (601074), Listeria monocytogenes (DUP1907-1 1038) and
Yersinia entercolitica (Michigan State University Isolate) were used.

2. For inoculation of the turkey subprimals, isolates of E. coli 0157:H7
(AB317), Salmonella typhimurium (601074), Listeria monocytogenes
(DUP1907-11038) were used.

3. E. coli 0157:H7, Salmonella typhimurium and Listeria monocytogenes
were transferred from stock cultures to 10 ml sterile tryptic soy broth
(Difco, Detroit, MI).

4. Yersinia entercolitica was transferred into brain-heart infusion broth (Difco,
Detroit, MI).

5. All pathogens were incubated at 35°C for 20 to 22 hours.

84

APPENDIX 6: Inoculation of Test Microorganisms

 

1.

Each subprimal (pork) or quad of subprimals (turkey) were placed in a
sterile tin-foil tray to prevent cross contamination and to minimize handling
of inoculated product.

On each subprimal, 12 5cm2 areas were inoculated with either Escherichia
coli 0157:H7, Salmonella typhimurium, Listeria monocytogenes or Yersinia
entercolitica.

Immediately after inoculation, each 5cm2 area was marked with green
food coloring to assist in the identification of the inoculated area.

During the 12-14 hour attachment time, the tin-foil trays were wrapped in
tin-foil then placed in a 2°C cooler.

85

APPENDIX 7: Sampling Method

1. Using a sterilized scalpel, 5cm x 5cm x 2mm subsamples were excised for
bacterial enumeration.

2. A total of 14 subsamples were excised from the subprimals (2 excised
prior to inoculation, 4 excised after inoculation, 4 excised after ice
treatment and 4 excised after 7 days of 2°C storage).

86

APPENDIX 8: Bacterial Enumeration

 

1.

Subsamples were placed in a 400 ml Stomacher® bag (Seward Medical,
London, UK) and weighed.

The subsamples were serially diluted using 1% buffered peptone water
(Difco, Detroit, MI).

The subsamples were stomached for 90 seconds in a 400 ml masticator
(IUL Instruments, Cincinnati, OH).

Fro7m the bag, 0.1ml was extracted then serially diluted to 10"“, 10'5 and
10‘ .

The dilutions were plated in duplicate for determination of most probable
number.

E. coli 0157:H7 was serially diluted and plated on MacConkey Sorbitol
Agar (Difco Laboratories, Detroit, MI) and 3M?" coliform and aerobic plates
(3M, St. Paul, MN).

Salmonella typhimurium was serially diluted and plated on Bismuth Sulfite
Agar (Difco Laboratories, Detroit, MI) and 3M” coliform and aerobic plates
(3M, St. Paul, MN).

Yersinia entercolitica was plated on Yersinia Selective Agar (Difco
Laboratories, Detroit, MI) and 3M“ coliform and aerobic plates (3M, St.
Paul, MN).

Listeria monocytogenes was plated on Oxford medium with antimicrobic
supplement added (Difco Laboratories, Detroit, MI) and 3M” aerobic
plates (3M, St. Paul, MN).

87

APPENDIX 9: Meat Storage Containers

 

1.

Eight 96-quart ice chests were used to store meat and ice treatments in
one of two temperatures (2° or 26°C).

A 120 cm x 45 cm x 4mm sheet of expanded steel was placed three
inches from the bottom of the cooler to allow draining of the melted ice.

Each ice chest was partitioned into five sections, using 4 mm-thick plexi-
glass.

To prevent cross contamination between samples, the plexi-glass was
stabilized by commercial sealing caulk.

88

APPENDIX 10: Vacuum Packaging

 

1.

After subsamples were excised after ice treatment, they were vacuum
packaged.

They were packaged using 4 mil thick 51 cm x 76.5 cm heat seal
polyethylene bags (Diagger, Lincolinshire, IL).

The bags were sealed using an impulse heat sealer (Diagger, Lincolinshire,
IL).

A sterile 20 gauge needle was attached to a 1.5 cm-diameter rubber hose
that was attached to a Welch vacuum pump (Welch Vaccum, Skokie, IL).

The vacuum pump was used to extract the air from the bag.

After air extraction, the bag was sealed again to prevent airflow from the
needle insertion point.

89

APPENDIX 11: pH Determination

 

1. The pH of the ice was measured with a Fischer Scientific pH meter
(Pittsburgh, PA).

2. Ice (250 ml) was placed in a 300 ml beaker.

3. The pH was measured by inserting the pH probe into the middle of the ice
filled beaker.

4. In order to observe pH decline over time, 22 readings were taken over a 4-
houn

90

APPENDIX 12: TBA Analysis

 

Tarladgis, G. 6., Watts, B. M., Younathan, M. T., and Dugan, J. Jr., J. Am. Oil

Chemists, 37:44-48, (1960).

Zipser, M. W., Watts, B.M., Food Technology, 16 (7): 102, (1962).

1. TBA Reagent

Prepare the amount of TBA Reagent needed for your samples according to the

 

 

 

 

table below.
Thiobarbituric Acid Distilled Water Total VolumeH20 with
Acid
1.4416 g 50 ml 500 ml
0.7208 g 25 ml 250 ml
0.5766 g 20 ml 200 ml
0.2883 g 10 ml 100 ml
0.1442 g 5 ml 50 ml

 

 

Dissolve the Thiobarbituric Acid (Sigma Chemical Co., St. Louis, MO) in the

distilled. Place the flask in sonic cleaner (5 minutes) and shake

occasionally until TBA is dissolved. Allow reagent to come to room

temperature then bring to volume. Store in cooler, may be kept for two

days. Reclaim unused reagent as described above.

2. HCI Solution

Make volume as needed; 1:2, HCI:H2O (vlv).

3. Antifoam (Thomas®, Swedeboro, NJ)

91

 

The use of antifoam may or may not be needed, depending on the product.

Fish and eggs require antifoam. In this study, antifoam was used.

Procedure:

_L

10.

11.

Assemble connecting tube (spouts) and graduated cylinders
Turn on condenser water

Into 500 ml extraction flasks, add 2 4mm glass beads (Fisher Scientific,
Pittsburgh, PA) 2.5 ml HCI solution and antifoam.

Weigh 10 9 sample directly into homogenizer flasks
Add 50 ml distilled water plus antioxidant solution
Homogenize 1 minute

Quantitatively transfer homogenate into 500 ml extracting flask and rinse
with distilled water to bring total volume to 100 ml

Connect extraction flasks to distilling tubes and tighten heating mantels in
place

Turn power on and heat flasks rapidly.
Distill and collect 50 ml of the distillate.

Transfer distillate to culture tubes, cap and hold in refrigeration for TBA
reaction.

TBA Reaction / Spechtrophotometric Determination

12.

13.

14.

15.

Invert each test tube containing the 50 ml distillate and pipet 5 ml into
each of 2 blanks. Pipet 5 ml of distilled water into another blank.

Add 5ml of TBA Reagent into each tube containing 5 ml of sample.
Thoroughly mix each tube on the vortex Genie shaker.

Immerse tube support containing tubes into boiling water bath for 35
minutes.

Turn spectrophotometer on to warm up (10 minutes).

92

16.

17.

18.

19.

When the tubes are done heating in the water bath, cool them in cold
water for 10 minutes.

Read with spectrophotometer within 1 hour.

Fill spec cell and read % transmittance of the sample against the Blank.
Using 1 ml volumetric pipet, transfer 1 ml of solution to 5 ml volumetric
flask. Bring to volume with distilled water. Mix and Read sample.
Calculation: 7.8 x 5 = 39; 39 is the constant to use in the calculations to
get the TBA number.

Convert % T to optical density and multiply by the constant 7.8 to convert
to mg malondehydel1000 g of sample, i. e. TBA number

93

 

APPENDIX 13: Structure and Reaction of Chlorine Dioxide

 

CI Cl
'//\\ H //\\°
0 0 0 0

2 CIO2 + H20 —> H0102 + H0103 (2H+ + Cl02' + 0103')

94

Recommendations for Future Research

The results of this microbial project have indicated that CIO2 ice can be
accurately generated at various ppm. After the initial fixed costs of the ice
machine and generator, the production of CIO2 ice is relatively inexpensive. In
this research, a model CIO2 ice application protocol was developed to simulate
ice-packed meat transportation systems or meat retail case displays. The CIO2
ice could be used as a new hurdle in a pathogen intervention strategy already in
place in meat processing, packaging and transportation systems. Some
developing and third-world countries lack proper refrigeration and pathogen
intervention strategies to guard against foodbome illness. Using CIO2 ice as
antimicrobial and as a temperature control mechanism in unrefrigerated meat
transportation and temporary storage systems could prove to be beneficial.

Chlorine dioxide ice was proven to be effective in the reduction of E. coli
0157:H7, Salmonella typhimurium and Listeria monocytogenes on fresh pork
and turkey subprimals by up to 0.5 10910 CFUIcm2. Although not effective in the
reduction of Yersinia entercolititca, future research could investigate the
mechanisms that allows Y. entercolitica to be resistant to CIO2 treatment.
Results of this research indicated that increasing the level of CIO2 and more
importantly activated CIO2 up to 100 to 200 ppm could prove to be effective in
the reduction if not elimination of the pathogen load. Currently, USDA has
approved 1200 ppm of CIO2 in meat washing pathogen reduction systems.
When exposure times up to 24 hours are used, excessive levels (> 200 ppm) of

CIO2 ice should be used with extreme caution to prevent discoloration or off-

95

odors due to the leaching of CIO2 onfinto the meat product. In addition, when
trying to maximize the effectiveness of CIO2 ice, increasing the ice melt by using
warm (body temperature immediately after slaughter) meat will increase the
CIO2 released from the ice, which will offer more of an antimicrobial effect. Also,
increasing the exposure time in an environmental temperature of 26°C could
allow more CIO2 to negatively affect the pathogen viability on the meat product.
This research showed that the melted largely activated CIO2 ice contained up to
3.6 IOQIo CF Ulml fewer microorganisms than the melted control ice. Future
research could focus on implementing this new pathogen intervention strategy in
food production systems from immediately after slaughter to time of purchase in
the retail display case.

In a final thought, CIO2 ice is not the cure-all to food safety in the meat
industry. It is merely one step of a pathogen intervention strategy that

minimizes the risk of a foodbome disease outbreak.

96

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